研究のあゆみと業績
2012
まえがき
昨年，遅れに遅れた研究室開設 10 周年記念誌｢金沢大学がん研究所腫瘍制御研究分野：研究の
あゆみと業績 2001－2010 年｣を“Downstream, Present & Upstream”と称して発刊しました．厚くも
なく薄くもなく微妙なできあがりでした．それでも，それはとりもなおさず当研究室が 10 年経過した証であ
り，通過点でもあります．10 年を区切りにやめようと思っていた紙媒体の本報告書を昨年も発行し，皆
さまに紹介しました．電子媒体ではうまくあらわせない私たちの生身の思いとあゆみをお伝えしたく，今年
も編集しました．
私の敬愛するお一人の山本悦秀先生は本学をご退任されたあと，東京で歯科クリニックを開院され，
地域医療にご尽力されています．いつものように気楽な気持ちで，本誌と 10 周年記念誌を差し上げま
したところ，つぎのようなご感想と励ましのおことばを頂戴しました．以下は原文：
源 教授： 拝復、貴研究分野の 10 周年記念誌、他を御恵贈いただき誠にありがとうございました。質
量共に立派な業績の敬意を表します。よく頑張られたと思います。
おそらく殆どおひとりで、この記念誌を作られたのではないでしょうか。実は不肖・私の退職記念誌も依頼
原稿以外は全て自分でやりました。・・・（中略）・・・でしたが、何か突き動かすものがありました。私の恩
師は「書き記すことの大切さ」を教室員に説き続けてこられました。すなわち、私の退職記念誌のあとが
きにも書きましたが、業績集については、「教室の運営費が国の税金で賄われている以上、教授が在任
中に何をしたかを記録として残しておくことは国民に対する義務である」といった類のことを我々弟子達は
聞いて育ってきました。従って、先生のこういった研究者としての姿勢には大変、共感するところがあります。
先生のさらなる御活躍を東京の地より祈念しております。ちなみに・・・（中略）・・・ことに感謝の日々です。
【2012 年９月 11 日】
たしかに毎年，本誌の編集には余分のエネルギーを費やしてしまいます．それでも，小さいながらも研究
室を担当する者の責務のようにも感じます．このようなお便りを頂いたあとですので，今回はとくに，皆さま
が「研究のあゆみと業績 2012 年」をどのように受けとめてくださるのかなあ・・と想像しながら，お届けする
ことにします．皆さまの忌憚のないご意見，ご批評やご助言をいただければ幸いです．
今年の 11 月に当研究所外部評価が実施されました【附記１】．前回（2008 年 11 月 27 日；研究の
歩みと業績 2008 参照）から早や５年が経過し，この間にどれほどのことができたかは自分でも評価は困
難です．それでも，上西紀夫（かみにし みちお）先生と勝田省吾先生のお二方はとても暖かくご評価くだ
さいました．ありがたいことです．研究の質量は軽視できませんが，やはり大切なのは師，先輩，同僚や
仲間であることをあらためて感じています．一昨年の春に発生した東日本大震災といまも続く余震は国
家を揺るがす災害となったものの，災い転じて「ひとの絆」の大切さを私たちに問い続けています．これとは
次元が違いますが，研究室でも皆が楽しく過ごせるよう心がけたいと思います．
来年の初秋に第 24 回日本消化器癌発生学会総会を担当します．身に余る責務です．準備は大変
ですが，おかげさまで助けてくれる仲間がたくさんいます．感謝の気持ちで一杯です．日常の診療や研究
のアクティビティを落とすことなく努めたいと思います．これからもご指導とご支援をお願いします．
2012 年１2 月
源 利成
〒920-0934 石川県金沢市宝町 13 番 1 号
進展
制御
金沢大学がん 研究所・腫瘍制御
金沢大学附属病院がん高度先進治療センター（併任）
金沢医科大学病院内視鏡科（非常勤）
電話 076-265-2792 (直通)，2798 (事務，研究室)
FAX 076-234-4529
e-mail：minamoto@staff.kanazawa-u.ac.jp
Division of Translational and Clinical Oncology
Cancer Research Institute and Cancer Center,
Kanazawa University and Hospital
13-1 Takara-machi
Kanazawa 920-0934, Japan
Phone 81-76-265-2792 (office), 2798 (secretary)
Fax 81-76-234-4523
e-mail：minamoto@staff.kanazawa-u.ac.jp
HP（更新中）: http://www.kanazawa-u.ac.jp/~ganken/shuyoseigyo/index.html
-1-
金沢大学がん進展制御研究所 腫瘍制御（旧：遺伝子診断）研究分野
【目次】
研究スタッフ
共同研究者
……………………………………………………………………… 3
………………………………………………………………………… 4
１年のあゆみとできごと
研究分野と活動の概要
研究費
………………………………………………………… 5
…………………………………………………………… 6
……………………………………………………………………………… 8
研究業績 …………………………………………………………………………… 9
論文発表
学会発表
その他（地域・社会貢献など）
知的財産，関連活動など ………………………………………………………… 12
メディア・新聞報道など ………………………………………………………… 12
【附記１】研究分野外部評価資料
…………………………………………… 13
上西紀夫 氏（日本消化器内視鏡学会理事長；公立昭和病院院長）
勝田省吾 氏（金沢医科大学 学長）
研究分野評価資料：2008 年～2012 年７月
【附記２】報道記録 ……………………………………………………………… 47
【附記３】発表論文の抜粋 ……………………………………………………… 48
-2-
研究のあゆみと業績
2012
【研究スタッフ】 （2012 年現在の在籍者）
教授
源 利成
准教授
川上和之
大学院
松之木愛香
金沢大学大学院医学系研究科 心肺総合外科学
金子真美
金沢大学大学院医学系研究科 心肺総合外科学
北村祥貴
金沢大学大学院医学系研究科 心肺総合外科学
富田泰斗
金沢医科大学大学院医学系研究科 一般・消化器外科学
下崎真吾
金沢大学大学院医学系研究科 整形外科学
伊藤有美
金沢大学大学院医学修士課程（2012 年 4 月～）
（博士課程）
（修士課程）
博士研究員
金沢大学附属病院がん高度先進治療センター（併任）
廣瀬まゆみ
堂本貴寛
2012 年 4 月～
共同研究員
小竹優範
石川県立中央病院・消化器外科
卒業研究生
樺木陽子
金沢大学医薬保健学域・保健学類４年（～2012 年 8 月）
辰巳暁哉
金沢大学医薬保健学域・保健学類４年（～2012 年 8 月）
柴田莉穂
金沢大学医薬保健学域・保健学類３年（2012 年 10 月～）
佐々木規雄
金沢大学医薬保健学域・保健学類３年（2012 年 10 月～）
浅香敦子
研究支援推進員
枡井亜希子
技能補佐員（ヒトがん組織バンク）
中みぎわ
技能補佐員（ヒトがん組織バンク；～2012 年 9 月）
旭井亮一
（株）凸版印刷
川島篤弘
独立行政法人国立病院機構金沢医療センター 臨床検査科
藤沢弘範
福井県立病院・脳神経外科
横井健二
金沢大学 心肺総合外科学；（現）米国メソジスト病院研究所
島崎猛夫
金沢医科大学総合医学研究所，集学的がん治療センター
東 朋美
金沢大学大学院医学系研究科環境分子応答学／衛生学
笠島里美
独立行政法人国立病院機構金沢医療センター 臨床検査科
宮下勝吉
金沢大学附属病院 脳神経外科
中島日出夫
上尾中央総合病院 腫瘍内科（2012 年 6 月～）
山下 要
金沢大学がん進展制御研究所 腫瘍外科
塚 正彦
金沢大学大学院医学系研究科法・社会医学（法医学）
研究支援員
研究協力員
共同研究者
-3-
金沢大学がん進展制御研究所 腫瘍制御（旧：遺伝子診断）研究分野
【共同研究者】 （2012 年現在で共同研究が稼動および予定しているもの．敬称略）
教授
渡邊 剛
金沢大学大学院医学系研究科 心肺病態制御学／心肺総合外科
准教授
小田 誠
金沢大学大学院医学系研究科 心肺病態制御学／心肺総合外科
教授
濵田潤一郎
金沢大学医学系研究科 脳機能制御学／脳神経外科学
講師
中田光俊
金沢大学医学系研究科 脳機能制御学／脳神経外科学
教授
佐藤 博
金沢大学がん進展制御研究所 細胞機能統御学
教授
須田貴司
金沢大学がん進展制御研究所免疫炎症制御
教授
太田哲生
金沢大学大学院医学系研究科 がん局所制御学／消化器外科学
准教授
藤村 隆
金沢大学大学院医学系研究科 がん局所制御学／消化器外科学
助教
宮下知治
金沢大学大学院医学系研究科 がん局所制御学／消化器外科学
教授
土屋弘行
金沢大学大学院医学系研究科 機能再建学／整形外科学
特任教授
山本憲男
金沢大学大学院医学系研究科 機能再建学／整形外科学
教授
Richard Wong
金沢大学理工学域 自然システム学系
教授
松永 司
金沢大学医薬保健研究域 薬学系
教授
元雄良治
金沢医科大学腫瘍内科学
教授
小坂健夫
金沢医科大学一般・消化器外科学
准教授
表 和彦
金沢医科大学一般・消化器外科学
講師
藤田秀人
金沢医科大学一般・消化器外科学
臨床教授
伊藤 透
金沢医科大学病院内視鏡科
准教授
石垣靖人
金沢医科大学総合医学研究所
病院長
江角浩安
国立がん研究センター東病院
教授
曽我朋義
慶應義塾大学先端生命科学研究所
特任助教
杉山直幸
慶應義塾大学先端生命科学研究所
特任助教
増田 豪
慶應義塾大学先端生命科学研究所
リーダー
大野博司
理化学研究所免疫アレルギー科学総合研究センター(RCAI)
副院長
西村元一
金沢赤十字病院外科
外科部長
伴登宏行
石川県立中央病院消化器外科
准教授
吉田 優
神戸大学大学院消化器内科学
助教
富田弘之
岐阜大学大学院病理学
教授
Peter V. Danenberg
南カリフォルニア大学生化学
教授
Andy Giraud
オーストラリア王立小児病院
准教授
Serge Y. Fuchs
ペンシルヴェニア大学生物学
准教授
Vladimir Spiegelman ウィスコンシン大学皮膚科学
准教授
Barry Iacopetta
西オーストラリア大学腫瘍学
科長
Francesco Graziano
ウルビーノ病院臨床腫瘍科
-4-
研究のあゆみと業績
2012
【2012 年のあゆみとできごと】
2012 年 1 月 18 日
・源 利成：富山大学和漢医薬学総合研究所ジョイントセミナーで講演
課題：がんを含む慢性進行性疾患の創薬標的 GSK3β
・源 利成：「がんにおける質の高い看護師育成研修会」で講演（本学附属病
院） 課題：がん医科学とがん医療－消化器がんを中心に－
2012 年 1 月 21 日
・源 利成：第 5 回金沢脳腫瘍セミナー（ホ テル 日航金沢）で講演
課題：大腸がん研究から開発した新しいがん治療法
－分子基盤と膠芽腫治療への橋渡し－
2012 年 1 月 27 日
・源 利成：金沢医科大学大学院第 29 回医学研究セミナーで講演
課題：がん細胞の代謝特性と治療
2012 年 4 月 01 日
・堂本貴寛君が博士研究員に採用され，研究開始
・伊藤有美さんが大学院医学系研究科修士課程に入学
2012 年 6 月 10 日
・宮下勝吉君（研究協力員；本学脳神経外科学）がご結婚：横浜元町で挙式
2012 年 6 月 16 日
・源 利成：金沢大学公開講座：がん研究の最前線で講演
課題：難治がんの新しい治療法－膵がんと脳腫瘍への取り組み－
2012 年 7 月 05 日
・源 利成が日本癌病態治療研究会 世話人を委託される．
2012 年 7 月 14 日
・金沢医科大学腫瘍内科学との合同親睦会（七夕の会）
：べに屋（金沢駅前）
2012 年 8 月 02 日
・金沢大学保健学類卒業研究発表会：樺木陽子さん，辰巳暁哉君
課題：異なる検出法による大腸癌の K-ras と B-raf 遺伝子変異検出の比較解析
2012 年 9 月 21 日
・2011 年度北国がん基金助成の成果が北國新聞日刊に掲載
【附記２】
受賞者：小竹優範，伴登宏行（石川県立中央病院消化器外科）
西村元一（金沢赤十字病院外科）
，川上和之，源 利成
北國がん基金 27 日に助成金贈呈式．解明，治療，確かな歩み．昨年の助
成対象者の成果：組織のデータを蓄積
2012 年 9 月 30 日
・中みぎわさんが退職（技能補佐員：ヒトがん組織バンク担当）
2012 年 10 月 01 日
・本学保健学類の佐々木規雄君と柴田莉穂さんが卒業研修を開始
2012 年 10 月 10 日
・源 利成が日本消化器病学会評議員に内定
2012 年 10 月 16 日
・佐々木琢磨先生（当研究所名誉教授）ご逝去
2012 年 10 月 18 日
・源 利成：
「がんにおける質の高い看護師育成研修会」で講演（本学附属病
院） 課題：がん医科学とがん医療－消化器がんを中心に－
2012 年 11 月 01 日 ・金沢大学がん進展制御研究所外部評価委員会が開催
まえがき
当研究分野は上西紀夫氏（かみにし みちお；日本消化器内視鏡学会理事長，
【附記１】
公立昭和病院 院長）と勝田省吾氏（金沢医科大学 学長）の評価を受けた．
2012 年 11 月 22 日
・源 利成：STOP 学術講演会（福井パレスホテ ル）で講演
課題：大腸がん研究から発見したがん治療標的－金沢発膠芽腫治療法への展開
2012 年 11 月 23 日
・金沢大学がん研究所外科同門会総会・懇親会：ホテル日航金沢
2012 年 12 月 21 日
・腫瘍制御・金沢医科大学腫瘍内科合同忘年会：もんぜん（ 金沢駅前）
-5-
金沢大学がん進展制御研究所 腫瘍制御（旧：遺伝子診断）研究分野
【研究分野と活動の概要】
本研究分野は 1998 年４月に遺伝子診断の名称で開設されてから一貫して，消化器がん
と呼吸器がんを中心に，がんの多様な分子細胞病態と腫瘍外科的特性の解明を目指して，
基礎・臨床橋渡し研究を実施している．なかでも，膵がん，膠芽腫，骨軟部肉腫と再発や
転移を含む難治性がんと希少がんの病態解明と制御に重点をおいている．
１．がん化シグナル誘導の分子細胞機構とがん制御への応用
(1) Wnt 経路に関わる新しい分子細胞機構の検討
Wnt 経路の制御破綻が固有のがん化シグナルを誘発する仕組みと，それを修飾する分子
細胞機構について -カテニンを中心に研究を進めている．そして，大腸がんの腫瘍－宿
主境界の腫瘍環境で活性化される -カテニンを機軸とするがん化シグナル回路の病理作
用を明らかにしてきた．そのなかで，-カテニンが RNA 安定化因子 CRD-BP (coding
region determinant-binding protein)を転写誘導することを同定した．今年度は，大腸がん病
巣における CRD-BP の発現と各種がん関連分子の mRNA 安定化による病的作用や臨床所
見との関連について研究を継続している．
がん細胞における -カテニン活性化の仕組みについて，これまでは，その分解複合体
の構成因子とユビキチン経路による蛋白質安定性の調節異常に焦点が当てられてきた．
一方，-カテニン活性化の必須要件である核移送の仕組みやその細胞内微細構造の研究は
未開の領域である．-カテニンの通過経路は核膜孔であり，30 種類の核膜孔複合体
(nuclear pore complex: NPC)因子 (nucleoporins: Nups)から構成される．今年度は，-カテニ
ンの核移送に作用する Nup(s)を同定するために，複数の大腸がん細胞株と少数例の大腸
がん組織を対象に両分子群の発現と局在の比較解析を開始した．
(2) glycogen synthase kinase (GSK) 3 を標的とするがん治療法の開発と臨床応用
消化器がんと脳膠芽腫を中心に異常活性を示す GSK3 が腫瘍細胞の生存，増殖，遊
走・浸潤を推進し，治療（抗がん剤，放射線）抵抗性を賦与することを発見した．そし
て，その阻害によるがん治療効果の分子機構の検討と難治がん治療の臨床研究を進めて
いる．2009 年から GSK3 阻害医薬品の併用による再発膠芽腫治療の医師主導型第Ⅰ/Ⅱ
相臨床研究 (UMIN: 000005111)を本学附属病院脳神経外科で実施し，今年度までにその安
全性と治療効果を確認した．今後，本学を中心に多施設臨床研究を構想し，厚労省科学
研究費に応募する．進行膵がんに対する同様の臨床研究 (UMIN: 000005095)は 2011 年 4
月から金沢医科大学病院集学的がん治療センターで開始され，現在も症例を登録，集積
中で，その成果にもとづく新しい膵がん治療法として共同出願を予定している．今年度
は研究対象を，希少かつ難治性の骨肉腫の治療と消化管実験発がん動物モデルにひろげ，
GSK3 阻害によるがん治療とがん（化学）予防効果についてそれぞれ本学整形外科（機
能再建学），消化器外科（がん局所制御学）と共同研究を開始した．
本酵素阻害によるがん治療効果の分子機構として，がん細胞の糖エネルギー代謝に及
ぼす作用を検討している．これまでのメタボローム解析から，がん細胞特有の代謝
Warburg 効果における GSK3 の責任作用を示唆する予備成果がえられている．現在，そ
の中心的役割を担うと考えられる代謝酵素と GSK3 の相互作用について機能解析を進め
ている．創薬への応用を考え，cell-based ELISA による新規 GSK3 阻害剤のスクリーニン
グ法の開発のため，GSK3 の代表的な基質であるグリコーゲン合成酵素とその第 641 セ
リンリン酸化ペプチド特異抗体を作成中である．
２．がんの分子生物学的分類によるオーダーメイドがん化学療法
抗がん剤の感受性・有害事象予測に臨床応用できる分子生物学的診断法を開発し，オ
ーダーメイド治療を実現させることを目的に研究を進めている．大腸がんで常用される
5-FU の個別化を実現した後，放射線治療や免疫治療を含めた集学的治療の個別化につな
-6-
研究のあゆみと業績
2012
げることを目標にしている．本年度は，がんのクロマチン構造が 5-FU の DNA 取り込み
と修復過程に関与することを大腸がん細胞で解析した．この機構は thymidylate synthase
(TS) 阻害による 5-FU の効果発現機構とは異なるため，がん細胞が両機構のいずれに反応
性が高いかにより 5-FU の個別化が可能である．現在，臨床試験での検証を計画中である．
３．エピジェネティクスを標的にするがん診断・治療法の開発
大腸がんをモデルとして，発がん径路をジェネティック，エピジェネティックな変化に
より説明，細分類し，診断や治療に応用することを直近の目的としている．エピジェネテ
ィックな変化のうち，とくに DNA メチル化を解析対象として，がん表現型である CpG
island methylator phenotype (CIMP)やゲノム全体のメチル化状態の代用マーカーである LINE1 を解析している．また，microsatellite instability (MSI)，chromosomal instability などの表現
型や K-ras，B-raf，APC 遺伝子変異などのジェネティック解析を順次追加し，大腸発がん
経路の詳細を構築中である．本年度は，LINE-1 のメチル化が大腸がんの進行に伴い変化す
る様式を，各臨床ステージのがん組織検体を用いて解析した．その結果，LINE-1 の低メチ
ル化はがん進展の早期に確定し，がん浸潤や転移に伴っての変化は乏しいと考えられた．
この結果から LINE-1 の低メチル化をターゲットしてがんの早期診断が可能と考えられる．
現在，がん早期診断法開発を目的として，血液や便中の DNA メチル化解析を行っている．
４．ヒト消化管がん組織検体資源化プロジェクト
がんの分子・細胞レベルの変化，代謝変動やがん動物モデルの解析から得られる結果を
実際のがん病巣で具現化してはじめて, がんの臨床に導入することができる．医科学研究
に共通する時代の要請である．この目的で，消化管がんの研究や臨床研究の基盤資源とし
て，2008 年から本学附属病院，金沢医科大学病院と市中の基幹病院（金沢赤十字病院，
石川県立中央病院）外科と連携して，本事業を開始した．2010 年にこの事業を当研究所
ヒトがん組織バンクに継承し，大腸がんと胃がん患者さまの手術検体から正常と病巣組織
を系統的に集積している．本事業の概要は昨年までの研究のあゆみと業績を参照されたい．
これらの組織・バイオバンクを利用して複数の共同研究が学内外で進行している．
2012 年 7 月 14 日 七夕の会：研究室スタッフと研究協力員
-7-
金沢大学がん進展制御研究所 腫瘍制御（旧：遺伝子診断）研究分野
【研究費】（2012 年１月以降の新規，継続，分担と連携を含む外部資金の獲得状況）
研究種目・期間
研究代表者
（課題番号）
研究分担者
研究課題
2010 － 2012 年 度
科学研究費補助
源 利成
金（基盤研究 A）
(20890086)
川上和之
太田哲生
基幹的細胞調節経路の異常に起因
する消化器がんの病態解明とがん
制御への応用
41,990,000 円
2011 － 2013 年 度
科学研究費補助
川上和之
金（基盤研究 B）
(23390321)
源 利成
ゲノムの低メチル化とレトロポゾ
ンの活性化を特徴とする大腸がん
の診断・治療開発
16,340,000 円
2011 － 2012 年 度
科学研究費補助
源 利成
金（挑戦的萌芽
研究）(23659643)
川上和之
がん特異的ネルギー代謝を標的と
する消化器がん治療法の開発
3,340,000 円
2011 － 2013 年 度
源 利成
科学研究費補助
廣瀬まゆみ
川上和之
金（基盤研究 C）
(23591955)
GSK3阻害による消化器がん治療
法の開発と分子機構の解明
4,550,000 円
2011 － 2013 年 度
科学研究費補助
中田光俊
金（基盤研究 C）
(23591955)
悪性グリオーマの浸潤シグナルを
狙った分子標的療法の確立
4,550,000 円
2011 － 2013 年 度
源 利成，
科学研究費補助
中島日出夫
ほか
金（基盤研究 C）
(23590898)
がん温熱療法の新規分子マーカー
候補 FAM107 ファミリー蛋白質の
発現・機能解析
5,200,000 円
2011 － 2013 年 度
科学研究費補助
島崎猛夫
金（基盤研究 C）
(23590898)
源 利成，
ほか
化学療法により誘発される EMT 誘
導因子の同定とその制御による膵
がん治療法の開発
5,200,000 円
2012 年 度 金 沢 大
松本邦夫
学重点研究経費
源 利成，
ほか
拠点形成型：アカデミアがん創薬
拠点形成のための人材と知の集
約・循環プログラム
2,000,000 円
2012 年 度 金 沢 大 Richard
Wong
学重点研究経費
源 利成，
ほか
細胞核輸送分子機構の機能的およ
び構造的動態の解析
2,000,000 円
2012 年 度 金 沢 大
学がん研究所共同 元雄良治
研究（一般）
源 利成，
ほか
GSK3阻害による新規膵がん化学
療法の開発と臨床試験
500,000 円
2012 年 度 金 沢 大
学がん研究所共同 小坂健夫
研究（一般）
源 利成，
川上和之，
ほか
大腸がんの分子病理学的特性の解
析と診断，治療のための分子指標
の解明
750,000 円
奨学寄附金
源 利成
2012 年 3 月
臨床検査関連会社（A）
147,000 円
奨学寄附金
源 利成
2012 年 3 月
予防医学関連財団（I）
670,860 円
奨学寄附金
源 利成
2012 年 3 月
予防医学関連財団（I）
400,000 円
源 利成
（連携）
研究経費
期間の総額
-8-
87,637,860 円
研究のあゆみと業績
2012
【研究業績】
Ⅰ．論文発表
・英文総説，著書
1. Shimasaki T, Kitano A, Motoo Y, Minamoto T. Aberrant glycogen synthase kinase 3β
in pancreatic cancer development, progression and resistance to therapy. J Carcinog
11: 15, 2012.
2. Nakada M, Furuta T, Hayashi Y, Minamoto T, Hamada JI. The strategy for enhancing
temozolomide against malignant glioma. Front Oncol 2: 98, 2012.
3. Minamoto T, Kotake M, Nakada M, Shimasaki T, Motoo Y, Kawakami K. Distinct
pathologic role for glycogen synthase kinase 3β in colorectal cancer progression. In:
Colorectal Cancer Biology - From Genes to Tumor, Rajunor Ettarh (Ed.), ISBN: 978953-51-0062-1, pp. 107-34, 2012; InTech.
・英文原著
4. Kitano A, Shimasaki T, Chikano Y, Nakada M, Hirose M, Higashi T, Ishigaki Y, Endo
Y, Takino T, Sato H, Sai Y, Miyamoto KI, Motoo Y, Kawakami K, Minamoto T.
Aberrant glycogen synthase kinase 3β is involved in pancreatic cancer cell invasion
and resistance to therapy. PLoS ONE, in press.
5. Matsunoki A, Kawakami K, Kotake M, Kaneko M, Kitamura H, Ooi A, Watanabe G,
Minamoto T. LINE-1 methylation shows little intra-patient heterogeneity in primary
and synchronous metastatic colorectal cancer. BMC Cancer, in press.
6. Nakajima H, Koizumi K, Tanaka T, Ishigaki Y, Yoshitake Y, Yonekura H, Sakuma T,
Fukushima T, Umehara H, Ueno S, Minamoto T, Motoo Y. Loss of HITS (FAM107B)
expression in cancer of the multiple organs: a tissue microarray analysis. Int J Oncol,
2012. Jul 10. doi: 10.3892/ijo.2012.1550. [Epub ahead of print]
7. Loh M, Chua D, Yao Y, Soo RA, Zeps N, Platell C, Kawakami K, Minamoto T,
Iacopetta B, Soong R. Can population differences in chemotherapy outcomes be
inferred from differences in pharmacogenetic frequencies? Pharmacogenomics J, 2012
Jun 26. doi: 10.1038/tpj.2012.26. [Epub ahead of print].
8. Kong D, Piao YS, Oshima H, Oguma K, Minamoto T, Yamada Y, Sato K, Yamashita
S, Ushijima T, Ishikawa T, Oshima M. Inflammation-induced repression of tumor
suppressor miR-7 in gastric epithelial cells. Oncogene 31(35): 3949-60, 2012.
9. Shimasaki T, Ishigaki Y, Nakamura Y, Takata T, Nakaya N, Nakajima H, Sato I, Zhao
X, Kitano A, Kawakami K, Tanaka T, Takegami T, Tomosugi N, Minamoto T, Motoo
Y. Glycogen synthase kinase 3β inhibition sensitizes pancreatic cancer cells to
gemcitabine. J Gastroenterol 47(3): 321-33, 2012.
10. Mimura M, Masuda A, Nishiumi S, Kawakami K, Fujishima Y, Yoshie T, Mizuno S,
Miki I, Ohno H, Hase K, Minamoto T, Azuma T, Yoshida M. AP1B plays an
important role in intestinal tumorigenesis with the truncating mutation of an APC gene.
Int J Cancer 130(5): 1011-20, 2012.
-9-
金沢大学がん進展制御研究所 腫瘍制御（旧：遺伝子診断）研究分野
Ⅱ．学会発表
・国際学会
1. Minamoto T, Mai W, Kyo S, Shakoori A, Miyashita K, Yokoi K, Shimasaki T, Motoo Y,
Kawakami K. Deregulated GSK3β sustains gastrointestinal cancer cells by modulating
hTERT and telomerase. 103rd Annual Meeting of American Association for Cancer
Research (AACR 2012), March 31-April 4, 2012, Chicago, Illinois, U.S.A.
2. Matsunoki A, Kawakami K, Kotake M, Kaneko M, Watanabe G, Minamoto T. LINE-1
methylation level is a molecular marker with little intra-patient heterogeneity in
primary and synchronous metastatic colorectal cancer. 103rd Annual Meeting of
American Association for Cancer Research (AACR 2012), March 31-April 4, 2012,
Chicago, Illinois, U.S.A.
3. Nakada M, Chikano Y, Sabit H, Furuta T, Miyashita K, Hayashi Y, Sato H, Kawakami
K, Minamoto T, Hamada JI. Aberrant glycogen synthase kinase 3β is involved in
glioma invasion. 9th Meeting for the Asian Society for Neuro-Oncology, April 20-22,
2012, Taipei, Taiwan.
4. Domoto T. Cleavage of hepatocyte growth factor activator inhibitor-1 by membranetype MMP-1 stimulates tumor invasive growth. The Joint Symposium of the 7th
International Symposium of Institute Network (第 6 回附置研究所ネットワーク国際
シンポジウム) and the 45th IDAC Symposium, the 2nd Symposium for Joint Usage/
Research Center of Aging, June 14-15, 2012, Smart Ageing International Research
Center, Institute of Development, Ageing and Cancer, Tohoku University, Sendai,
Japan.
5. Kawakami K, Matsunoki A, Kotake M, Kaneko M, Kitamura H, Watanabe G,
Minamoto T. Knockdown of LINE-1 enhances sensitivity to 5-FU in LINE-1hypomethylated colorectal cancer cell. 22nd Biennial Congress of the European
Association for Cancer Research (EACR 22), July 7-10, 2012, Centre Convencions
International Barcelona, Barcelona, Spain.
6. Nakada M, Hayashi Y, Miyashita K, Kinoshita M, Furuta T, Sabit H, Kita D, Hayashi
Y, Uchiyama N, Kawakami K, Minamoto T, Hamada JI. Phase I/II study for recurrent
glioblastoma with the drugs inhibiting GSK3β. Society for Neuro-Onology 17th Annual
Meeting 2011, November 15-18, 2012, Washington DC, U.S.A.
7. Pyko VI, Nakada M, Furuyama N, Lei T, Hayashi Y, Kawakami K, Minamoto T,
Fedulau AS, Hamada JI. Glycogen synthase kinase 3β inhibition sensitize human
glioma cells to temozolomide by means of c-myc signaling. Society for NeuroOnology 17th Annual Meeting 2011, November 15-18, 2012, Washington DC, U.S.A.
・国内（全国）学会
8. 島崎猛夫，石垣靖人，高田尊信，川上和之，上田順彦，友杉直久，小坂健夫，源 利
成，元雄良治．GSK3β 標的治療と化学療法を併用する膵がんの新規治療戦略と分子
基盤．第 43 回日本膵臓学会大会，2012 年 6 月 28－29 日，ホテルメトロポリタン山形，
山形市．
9. Shimasaki T, Kitano A, Minamoto T, Motoo Y. Aberrant glycogen synthase kinase 3β
is involved in pancreatic cancer cell invasion and resistance to therapy（島崎猛夫，北
野綾子，源 利成，元雄良治．GSK3β の異常活性に起因する膵がん細胞の浸潤と治療
抵抗性）．第 10 回日本臨床腫瘍学会学術集会： IS1: Biomarker & Developmental
- 10 -
研究のあゆみと業績
2012
Therapeutics (English Session)，2012 年 7 月 26－28 日，大阪国際会議場，大阪．
10. Kaneko M, Kawakami K, Kotake M, Kitamura H, Watanabe G, Minamoto T. LINE-1
methylation level is a potential marker of the sensitivity to 5-FU plus oxaliplatin in
colorectal cancer cells（金子真美，川上和之，小竹優範，北村祥貴，渡邊 剛，源 利
成．LINE-1 メチル化は大腸がん細胞のオキザリプラチン・5−FU 併用処理への感受性
と相関する）
．第 71 回日本癌学会学術総会，2012 年 9 月 19－21 日，ロイトン札幌，
さっぽろ芸文館，札幌市教育文化会館，札幌．
11. Shimasaki T, Kitano A, Ishigaki Y, Takata T, Kawakami K, Takegami T, Tomosugi N,
Minamoto T, Motoo Y. GSK3β is an emerging therapeutic target in pancreatic cancer:
its implication for cancer cell migration and invasion（島崎猛夫，北野綾子，石垣靖人，
高田尊信，川上和之，竹上 勉，友杉直久，源 利成，元雄良治．膵癌の新規治療標的
．第 71 回日本
としての glycogen synthase kinase (GSK) 3β：がん浸潤に対する作用）
癌学会学術総会，2012 年 9 月 19－21 日，ロイトン札幌，さっぽろ芸文館，札幌市教
育文化会館，札幌．
12. ピコ イリア，中田光俊，古山奈月，滕 雷，林 裕，川上和之，源 利成， Fedulau
Аliaksandr S ， 濵 田 潤 一 郎 ． Sensitizing human glioma cells to temozolomide by
glycogen synthase kinase 3β inhibition. 第 13 回日本分子脳神経外科学会，2012 年 9 月
20－21 日，熊本．
13. 島崎猛夫，川上和之，上田順彦，小坂健夫，友杉直久，源 利成，元雄良治．GSK3β
標的治療を併用した膵癌の新規治療戦略と分子基盤．第 20 回 JDDW 2012／第 54 回日
本消化器病学会大会，2012 年 10 月 10－13 日，神戸国際展示場・ポートピアホテル・
神戸国際会議場，神戸．
14. 小竹優範，川上和之，金子真美，北村祥貴，伴登宏行，山田哲司，源 利成．【優秀
演題】大腸がんにおける microsatellite instability と CpG island methylator phenotype の
解析．第 20 回 JDDW 2012／第 10 回日本消化器外科学会大会，2012 年 10 月 10－13
日，神戸国際展示場・ポートピアホテル・神戸国際会議場，神戸．
15. 宮下勝吉，中田光俊，林 裕，渡邉卓也，木下雅史，古田拓也，淑瑠ヘムラサビット，
喜多大輔，林 康彦，内山尚之，川上和之，源 利成，濵田潤一郎．再発神経膠芽腫に
対して GSK3β 阻害作用を有する既存薬剤を用いた第 I・II 相臨床試験における剖検例
の免疫組織学的検討．第 71 回日本脳神経外科学会総会，2012 年 10 月 17－19 日，大
阪．
16. 中田光俊，林 裕，宮下勝吉，木下雅史，古田拓也，淑瑠ヘムラサビット，喜多大輔，
林 康彦，内山尚之，川上和之，源 利成，濵田潤一郎．GSK3β 阻害作用を有する既
存薬剤を用いた再発膠芽腫治療の第 I・II 相臨床試験，第 50 回日本癌治療学会学術集
会，2012 年 10 月 25－27 日，パシフィコ横浜，横浜．
17. 下﨑真吾，山本憲男，林 克洋，西田英司，武内章彦，丹沢義一，木村浩明，五十嵐
健太郎，稲谷弘幸，源 利成，土屋弘行．GSK-3β 阻害にもとづく骨肉腫に対する分子
標的治療の可能性．第 27 回日本整形外科学会基礎学術集会，2012 年 10 月 26－27 日，
名古屋国際会議場，名古屋．
18. 島崎猛夫，北野綾子，友杉直久，川上和之，源 利成．【優秀演題】GSK3β異常活性
による膵がんの浸潤と治療抵抗性．第23回日本消化器癌発生学会総会，2012年11月15
－16日，ルネッサンスリゾートナルト，鳴門市．
19. 宮下勝吉，中田光俊，林 裕，木下雅史，古田拓也，淑瑠ヘムラサビット，渡邉卓也，
喜多大輔，林 康彦，内山尚之，川上和之，源 利成，濵田潤一郎．再発膠芽腫に対し
- 11 -
金沢大学がん進展制御研究所 腫瘍制御（旧：遺伝子診断）研究分野
て GSK3β 阻害作用を有する既存薬剤を用いた単施設第 I・II 相臨床試験．第 30 回日
本脳腫瘍学会，2012 年 11 月 25 日－27 日，広島
・司会・座長など
20. 深町博史，源 利成．シグナル伝達阻害剤（３）／Signal transduction inhibitors (3)．第
71 回日本癌学会学術総会，2012 年 9 月 19－21 日，ロイトン札幌，さっぽろ芸文館，
札幌市教育文化会館，札幌．
21. 源 利成，今野雅允．ワークショップ３：消化器癌とプロテアソーム・オートファジ
ー．第 23 回日本消化器癌発生学会，2012 年 11 月 15－16 日，ルネッサンスリゾート
ナルト，鳴門市．
・その他（講演，社会・地域貢献を含む）
22. 源 利成．がんを含む慢性進行性疾患の創薬標的 GSK3β．第１回富山大学和漢医薬学
総合研究所・金沢大学がん進展制御研究所ジョイントセミナー，2012 年 1 月 18 日，
富山大学和漢医薬学総合研究所民族薬物資料館，富山．
23. 源 利成．発がん学，がん医科学とがん医療―消化器がんを中心に―．がんにおける
質の高い看護師育成研修会，2012 年 1 月 18 日，金沢大学附属病院，金沢．
24. 源 利成．大腸がん研究から開発した新しいがん治療法－分子基盤と膠芽腫治療への
橋渡し－．第 5 回金沢脳腫瘍セミナー，2012 年 1 月 21 日，ホテル日航金沢，金沢．
25. 源 利成．がん細胞の代謝特性と治療．金沢医科大学大学院第 29 回医学研究セミナー，
2012 年 1 月 27 日，金沢医科大学，内灘町．
26. 源 利成．難治がんの新しい治療法－膵がんと脳腫瘍への取り組み－．金沢大学公開
講座：がん研究の最前線，2012 年 6 月 16 日，金沢大学サテライト・プラザ（西町），
金沢．
27. 源 利成．がん医科学とがん医療―消化器がんを中心に―．がんにおける質の高い看
護師育成研修会，2012 年 10 月 18 日，金沢大学附属病院，金沢．
28. 源 利成．大腸がん研究から発見したがん治療標的－金沢発膠芽腫治療法への展開－．
STOP 学術講演会，2012 年 11 月 22 日，福井パレスホテル，福井．
Ⅲ．研究成果による知的財産権の出願状況
1. 特許出願
なし
Ⅳ．新聞，報道など
1. 小竹優範，伴登宏行，西村元一，川上和之，源 利成．北國がん基金 27 日に助成金
贈呈式．解明，治療，確かな歩み．昨年の助成対象者の成果：組織のデータを蓄積．
北國新聞 日刊，2012 年９月 21 日．
- 12 -
2008～2012 年
【附記１】がん進展制御研究所外部評価：2008 年～2012 年７月
腫瘍制御研究分野 外部評価資料一覧
・評価報告書
上西紀夫 氏
日本消化器内視鏡学会 理事長
公立昭和病院 病院長
勝田省吾 氏
金沢医科大学 学長
・腫瘍制御研究分野 評価資料
・報道資料
- 13 -
金沢大学がん進展制御研究所 腫瘍制御研究分野
分野別評価報告書
対 象 分 野 名
腫瘍制御研究分野
評 価 委 員 氏 名
上西 紀夫
評価委員所属・職
評 価 実 施 日
印
公立昭和病院・病院長
２０１２年
９月
７日
１．研究活動に関する評価（a. 研究の方向性、b. 独自性、c. 進捗状況（研究発表
状況）、d. 国際的な位置づけ、e. 将来の貢献、f. 資金獲得状況、g. その他）
a. 研究の方向性
最終目的であるがん制御のためには、まず癌の発生、進展、転移に関わる癌化
のプロセスの解明が必須であり、源 利成教授は、急速に増加しつつある大腸癌を
取り上げ、癌化シグナル誘導の分子機構の一つである Wnt シグナル、とくにその
中心となる β-catenin に着目して継続的な研究を行い、それを他の臓器における腫
瘍制御の研究に応用、発展させてきました。それが、消化器外科、腫瘍内科、脳
外科、肺外科、整形外科などの臨床教室との共同研究の推進に繋がっています。
さらに、がん制御の立場から、癌化のプロセスの中で近年注目されている
epigenetic な変化、とくにメチル化にも注目し、抗がん剤の増強作用の検討、ある
いは、がん診断への応用を目指しています。
以上のごとく、一貫した研究の中から、常に臨床応用を念頭に置いた研究を行
っており高く評価されます。
b. 独自性
β-catenin シグナルの研究の中から新規の転写標的分子である CRD-BP を同定し、
これが大腸がんでの複数の細胞増殖経路に深く関与している可能性があり、今後
の研究の成果がきたされます。
一方、Wnt シグナルの研究の中から、逆にがんに抑制的に働く分子として
glycogen synthase kinase 3β（GSK3β）に着目し、その機能破綻ががん細胞の増殖を
促進することから、その阻害によるがんの治療について臨床研究を開始しており、
成果が大いに期待されます。
以上のように、源 利成教授の研究は、いわば特殊な細胞であるがん細胞の生き
るための基本的な仕組みを研究し、その仕組みを壊すことでがんの制御を目指す、
という新しい発想に基づいており、従って、その研究成果は多くのがんに応用が
可能であり、大いに評価されます。
c. 進捗状況（研究発表状況）
研究発表については、上記の成果が Gastroenterology, Cancer Research, Clinical
Cancer Research などの極めて高い Impact Factor を有するジャーナルに掲載されて
おり、さらに、数多くの国内、国際学会で発表を行い学会賞を受賞するなど、着
実な歩みを見せており高く評価されます。
- 14 -
2008～2012 年
d. 国際的な位置づけ
海外の著明な腫瘍学者、機関との共同研究を推進し、成果を挙げています。今
後、さらなる海外への発信が必要と思われます。
e. 将来の貢献
GSK３β 阻害による制がん効果について、難治性のがんに対して第 I・Ⅱ相臨床
研究が開始されており、その成果は多くのがんに対して有効である可能性があり、
がんで苦しんでいる患者への福音をもたらすことが期待されます。
f. 資金獲得状況
文科省、厚労省の科研費、金沢大学、金沢医科大学からの共同研究費、さらに
は様々な団体、企業から奨学寄付金を獲得、十分な研究費を獲得してきています。
g. その他
10 年前は厳しい研究環境であったが、その中から地道で懸命な努力をし、目覚
ましい研究業績を挙げてきたことは大いに評価されます。
２．共同利用・共同研究拠点としての役割に関連する活動に関する評価
消化管がん組織検体資源化事業を立ち上げ、今後のがん制御研究、そして共同
研究の促進に大いに貢献することが見込まれます。
３．その他の活動に関する評価
過去 8 年間で 12 の特許出願をしている。また、多くの大学院生を教育、指導し、
今後の研究の担い手を育成している。
４．改善を必要とする課題・提言
現時点では早急に改善すべき点は見当たらない。
５．将来に対する指針、その他全般的な提言など
がんの発生、進展、転移には様々なプロセスがあり、例えば Wnt シグナルの解
析だけではそのプロセスの全貌に迫ることは無理があると思われます。しかしな
がら、そのシグナル経路にはがん化のプロセスにおける共通事項もあると思われ、
そのシグナル経路を体系化することにより、がん化の本質に迫る可能性があり、
その中からがん制御、そして創薬への道が拓かれるかもしれません。すなわち、
個々の分析をする中から体系を構築し、成果を挙げていくことを期待しています。
- 15 -
金沢大学がん進展制御研究所 腫瘍制御研究分野
分野別評価報告書
対 象 分 野 名
腫瘍制御研究分野
評 価 委 員 氏 名
勝田 省吾
評価委員所属・職
金沢医科大学・学長
評 価 実 施 日
２０１２年 ９ 月１８日
印
１．研究活動に関する評価（a. 研究の方向性、b. 独自性、c. 進捗状況（研究発表
状況）、d. 国際的な位置づけ、e. 将来の貢献、f. 資金獲得状況、g. その他）
a. 研究の方向性
源 利成教授は、研究分野の開設以来、消化器がんと呼吸器がんを中心に、がん
の多様な分子細胞病態と腫瘍外科的特性の解明を目指して、(1) がん化シグナル誘
導の分子細胞機構とがん制御への応用、(2) がんの分子生物学的分類によるオーダ
ーメードがん化学療法、(3) エピジェネティクスを標的にするがん診断・治療法の
開発に関わる基礎・臨床橋渡し研究を展開してきた。最近、膵がんや悪性脳腫瘍
などの難治性がんの分子病態の解明と制がんへの応用を視野にいれた研究も進め
ており、本研究分野が標榜する「腫瘍制御」に相応しい方向性を示していると評
価される。GSK3β 阻害研究も開始されており、期待が持たれる。
b. 独自性
がん化シグナル誘導の分子細胞機構とがん制御への応用を目指して、Wnt/βcatenin シグナルと GSK3β を中心に研究を進めてきた。β-catenin 分解系に作用する
ユビキチン連結酵素 β-TrCP や β-catenin シグナルの新規の転写標的分子 CRD-BP
を同定した。また、GSK3β の過剰発現やそのリン酸化による酵素活性調節の破綻
ががん細胞の生存や増殖を促進するという、Wnt 経路抑制機能とは異なる病的作
用を発見した。さらに、GSK3β 阻害の制がん効果を消化器がん細胞と担がん動物
で実証し、本酵素が新しいがん治療標的であると提唱し、独自性の高い成果を上
げており、大いに評価される。
c. 進捗状況（研究発表状況）
腫瘍制御研究分野の研究成果は欧文誌原著論文として、2008 年度以後 18 編報
告されており、研究スタッフ数を考慮すると十分に評価できるものである。高い
Impact Factor を有する Clin Cancer Res 誌、Cancer Res 誌、Gastroenterology 誌,
Oncogene 誌など有力科学誌に掲載された研究は世界的に見てインパクトのおおき
いものであり、内容的にも高く評価される。また、原著論文に加えて、優れた英
文の著書・総説論文を発表しており、この点も評価に値する。
- 16 -
2008～2012 年
d. 国際的な位置づけ
一流の国際科学誌に多く論文を発表しており、原著論文の数、質とも国際的評
価は高い。また、多くの国際学会で発表している点も評価される。
e. 将来の貢献
これまで将来性のあるがん研究をすすめてきている。とくに、近年、膵がん、
脳悪性腫瘍や骨軟部肉腫などの難治性がんの分子病態の解明と制がんへの応用を
視野に入れた研究を目指しており、研究成果が期待される。
f. 資金獲得状況
文科省科学研究費（基盤研究、特定領域研究）や他の外部資金、学内資金など
をコンスタントに得ており、資金獲得への努力がうかがわれる。これにより大学
院生、研究生、研究協力員を常時受けいれ、研究の推進・活性化を図っているの
は評価できる。
g. その他
これまで国内および国際特許を出願してきている。今後の研究の実用化、社会
への貢献の面で評価できる。
２．共同利用・共同研究拠点としての役割に関連する活動に関する評価
他機関の研究者とがん分子標的探索プログラム「GSK3β 阻害による新規膵がん
化学療法の開発と臨床試験」および「大腸がん個別化医療のためのバイオマーカ
ー探索」について、2011 年より共同研究を実施しているのは評価できる。
また、2008 年末から他施設と連携して、消化管がん組織検体資源化事業を創出
した。集積したヒトがん組織を共同利用に供することによって、がん研究の発展
に貢献できる。
３．その他の活動に関する評価
新聞・メディア（TV）を活用して研究成果を社会に公表しており、評価できる。
また、2008 年より毎年、高校生を対象に「がんの科学と医療」について講義して
おり、未来の研究者育成に貢献している。
４． 改善を必要とする課題・提言
常勤の研究スタッフが少ないように思われる。この点は、組織機構の問題かも
しれないが、常勤スタッフを含めて研究者が集まる仕組み作りに一層の努力が求
められる。
５．将来に対する指針、その他全般的な提言など
現在の方向で進めていけば良いと思う。検討中・構想中の研究もタイムリーな
課題である。本研究分野は着実に発展しているが、今後、人事の流動化、研究員
の拡充、若い人材の獲得や外部資金のさらなる獲得に取り組んでいただきたい。
- 17 -
金沢大学がん進展制御研究所 腫瘍制御研究分野
分子標的探索プログラム
腫瘍制御研究分野
１．研究スタッフ
常
勤
教授
准教授
2001 年 7 月〜現在
2006 年 4 月〜現在
源 利成
川上和之
非常勤
非常勤研究員
金 明姫
廣瀬まゆみ
堂本貴寛
2008 年 4 月～2010 年 2 月
2010 年 4 月〜現在
2012 年 4 月〜現在
大学院
博士課程
（医学）
修士課程
（医学）
（薬学）
研究生
宮下勝吉（金沢大学脳神経外科学） 2005 年 6 月～2009 年 3 月
麦 威（Mai, Wei：私費留学） 2006 年 4 月～2009 年 3 月
斉藤健一郎（金沢大学心肺総合外科学） 2007 年 11 月～2010 年 9 月
松之木愛香（金沢大学心肺総合外科学） 2008 年 7 月～現在
王 利明（国費留学） 2009 年 10 月～2010 年 9 月（退学）
金子真美（金沢大学心肺総合外科学） 2010 年 4 月～現在
富田泰斗（金沢医科大学消化器外科学） 2011 年 7 月～現在
北村祥貴（金沢大学心肺総合外科学） 2011 年 8 月～現在
下崎真吾（金沢大学整形外科学） 2011 年 10 月～現在
伊藤有美
北野綾子
近野祐里
2012 年 4 月～現在
2008 年 4 月～2010 年 3 月
2009 年 4 月～2011 年 3 月
小竹優範（石川県立中央病院消化器外科）
卒業研究生
（保健学）樺木陽子（金沢大学保健学類第 4 学年）
辰巳暁哉（金沢大学保健学類第 4 学年）
2009 年 12 月～現在
2011 年 10 月～現在
2011 年 10 月～現在
研究協力員
旭井亮一（株式会社 凸版印刷ライフサイエンス事業推進部） 2002 年 9 月～現在
小田惠夫（株式会社アルプ 病理研究所） 2004 年 4 月～2009 年 3 月
川島篤弘（国立病院機構金沢医療センター） 2004 年 4 月～現在
藤沢弘範（福井県立病院脳神経外科／福井大学脳神経外科学） 2005 年 4 月～現在
横井健二（米国メソジスト病院研究所ナノ医学分野） 2007 年 4 月～現在
島崎猛夫（金沢医科大学総合医学研究所） 2007 年 4 月～現在
東 朋美（金沢大学環境分子応答学／衛生学） 2007 年 4 月～現在
笠島里美（国立病院機構金沢医療センター） 2007 年 4 月～現在
宮下勝吉（金沢大学脳神経外科学） 2009 年 4 月～現在
中島日出夫（上尾中央総合病院腫瘍内科） 2012 年 6 月～現在
- 18 -
2008～2012 年
２．研究の概要，成果と今後の課題
当研究分野は 1998 年 4 月当初，遺伝子診断 (Division of Diagnostic Molecular Oncology)
として臨床研究部門に設置された．そして，2001 年７月に源が研究分野主任に選任され，
独立した研究分野として始動した．2006 年 4 月の当研究所改組にともない，分子標的が
ん医療研究開発センター (Molecular & Cellular Targeting Translational Oncology Center) が開
設され，その中核的研究分野として，がんの分子細胞特性，シグナル生物学や腫瘍外科
学に基づく探索的がん医療を指向する基礎・臨床橋渡し研究に軌道修正した．これにと
もない，腫瘍制御研究分野 (Division of Translational and Clinical Oncology) と改名し，現在
に至っている．
研究分野の開設以来，消化器がんと呼吸器がんを中心に，がんの多様な分子細胞病態
と腫瘍外科的特性の解明を目指して，以下の基礎・臨床研究を実施している．
(1) がん化シグナル誘導の分子細胞機構とがん制御への応用
(2) 遺伝薬理学的解析によるオーダーメイドがん化学療法
(3) エピジェネティクスを標的にするがん診断・治療法の開発
(4) がん組織検体資源の構築
とくに，膵がん，脳悪性腫瘍や骨軟部肉腫などの難治性がんの分子病態の理解と制がん
への応用を視野にいれた研究を目指している．がん組織検体資源化事業は，後述の共同
利用・共同研究拠点としての研究資源の一翼を担っている．
（１）がん化シグナル誘導の分子細胞機構とがん制御への応用
(i) Wnt シグナル制御破綻に関わる新しい分子細胞機構
Wnt 経路の制御破綻が固有のがん化シグナルを誘発する仕組みと，それを修飾する分
子細胞機構を解明するために -catenin を中心に研究を進めてきた．大腸がんのがん腫－
宿主境界の腫瘍環境において -catenin が活性化されると，固有のがん化シグナルネット
ワークが形成され，がんの病態やがん患者の生命予後を悪くすることを明らかにした．
そのメカニズムの一端として，-catenin 分解系に作用するユビキチン連結酵素 (-TrCP)
や，-catenin シグナルの新規の転写標的分子 CRD-BP (coding region determinant-binding
protein) を同定した．また，CRD-BP を介する Wnt と Hedgehog 経路の交差応答や，腸上
皮細胞の極性輸送の異常と -catenin 活性化の関連についても明らかにしてきた．現在，
がんにおけるこれらの分子の制御異常と病的作用を検討している．とくに CRD-BP は
IκBα，c-myc や IGF-II の RNA トランス因子であり，大腸がんで複数の細胞増殖経路 (Wnt,
NF-κB, c-Myc, IGF- II)を機能的に結びつけると仮定し，臨床がんの解析を進める．Wnt シ
グナル伝達の根本となる -catenin の核移行の分子機構について，核孔複合体との相互作
用の点から系統的な解明を試みることを計画している．
(ii) 慢性進行性疾患の創薬標的 GSK3 の消化器がんにおける発現，活性，機能解析
正常細胞の Wnt 経路制御作用からがん抑制的に働く機能分子と認識されている glycogen
synthase kinase 3 (GSK3)の大腸がんへの関与に着目した．そして，GSK3 の過剰発現や
そのリン酸化による酵素活性調節の破綻ががん細胞の生存や増殖を促進するという，Wnt
経路抑制機能とは異なる病的作用を発見した．つぎに，GSK3 阻害の制がん効果を消化
器がん細胞と担がん動物で実証し，本酵素が新しいがん治療標的であると提唱した．その
制がん効果の分子メカニズムは細胞周期やががん抑制分子経路や細胞不死化の制御による
ものであることを明らかにした．これらの成果をもとに，GSK3 阻害効果を示す医薬品
の適応外併用による再発膠芽腫と進行膵がん治療の医師主導型第Ⅰ・Ⅱ相臨床研究を本学
附属病院脳神経外科と金沢医科大学病院集学的がん治療センターで開始した．現在，これ
らの難治性がんを中心に，GSK3 が制御するがん細胞の形態特性と運動・浸潤性，治療
- 19 -
金沢大学がん進展制御研究所 腫瘍制御研究分野
（抗がん剤，放射線）抵抗性やがん細胞固有の代謝特性（Warburg 効果）に着目して機能
解析を進めている．また今後は，本酵素阻害のがん（化学）予防効果を検討したい．
（２）遺伝薬理学的解析によるオーダーメイドがん化学療法
抗がん剤の感受性・有害事象予測に臨床応用できる分子生物学的診断法を開発し，オ
ーダーメイド化学療法を実現させることを目的に研究を進めている．5-FU のターゲット
酵素であるチミジル酸合成酵素 (TS) の遺伝子発現，遺伝子型，LOH の存在に加え，複数
の核酸・葉酸代謝酵素遺伝子発現・遺伝子型と抗がん剤感受性の関連を消化器がん，肺
がんを対象に解析してきた．本年度は，次項の DNA メチル化に関する研究から偶然発見
された，LINE-1 メチル化と 5-FU の感受性が相関するメカニズムを解析し，そのメカニズ
ムに基づき新規の 5-FU 効果増強法を探索している．
（３）エピジェネティクスを標的にするがん診断・治療法の開発
がん細胞におけるエピジェネティックな変化とその背景にある代謝変動の理解を進め，
これをがん予防・診断・治療の新たな戦略構築に応用することを目指している．エピジ
ェネティックな変化のうち，とくに DNA メチル化を解析対象として，がん表現型である
CpG island methylator phenotype (CIMP)，microsatellite DNA instability (MSI)，chromosomal
instability 相互の関連を観察し，大腸がんをモデルに発がん径路をジェネティック・エピ
ジェネティックな変化により説明することを試みている．次項のがん組織検体資源を使
用して，CIMP，MSI の診断に加え，K-ras，B-raf，APC 遺伝子変異等の解析を順次追加
し，大腸がん発癌経路の詳細を構築中である．また，ゲノム全体のメチル化を高感度に
解析するため，多重蛍光を使用して LINE-1 のメチル化アッセイ法を改良し，laser capture
microdissection により採取された微量臨床検体の解析を行っている．さらに，葉酸代謝と
DNA メチル化の関連を詳細に検討する目的で，葉酸代謝酵素の強制発現細胞を作成し，
葉酸投与量・葉酸代謝酵素発現性・DNA メチル化間の相互関係を解析している．
３．共同利用・共同研究拠点としての役割を果たすことに関連する活動について
がんの分子・細胞レベルの変化，代謝変動やがん動物モデルの解析から得られる結果
を実際のがん病巣で具現化してはじめて, がんの臨床に導入することができる．医科学研
究に共通する時代の要請である．そのためにはヒトのがん検体は必須である．この目的
で，消化管がんの研究や臨床研究の基盤資源として，2008 年末から本学附属病院外科，
金沢医科大学病院外科と市中の基幹病院（金沢赤十字病院，石川県立中央病院，など）
外科と連携して，消化管がん組織検体資源化事業を創出した．2010 年にこの事業を当研
究所ヒトがん組織バンクに継承した時点で，大腸がんと胃がんを合わせて約一千例の患
者様から検体（正常と病巣組織）を集積している．本事業の所期の目的は，はからずし
も拠点としての役割に合致した．
(1) 消化管がんの腫瘍外科学と分子腫瘍学研究の基盤資源とデータベースの構築
(2) 消化管がん治療の臨床研究，臨床試験：(1)とともに共同利用と共同研究を推進する．
(3) がん医療とがん研究の産学官・地域連携への働きかけ
先進医療の共有，地域における均てん化，自治体との連携
大学や地域医療機関から社会人大学院のリクルート：がん研究の活性化
(4) 地域の GI オンコロジストの交流，コミュニティの形成
- 20 -
2008～2012 年
４．研究業績
[Impact factor (IF) 2011]
英文原著
1. Loh M, Chua D, Yao Y, Soo RA, Zeps N, Platell C, Kawakami K, Minamoto T, Iacopetta B,
Soong R. Can population differences in chemotherapy outcomes be inferred from differences
in pharmacogenetic frequencies? Pharmacogenomics J, 2012. Jun 26. doi: 10.1038/tpj.2012.
26. [Epub ahead of print] [IF: 4.536]
2. Nakajima H, Koizumi K, Tanaka T, Ishigaki Y, Yoshitake Y, Yonekura H, Sakuma T,
Fukushima T, Umehara H, Ueno S, Minamoto T, Motoo Y. Loss of HITS (FAM107B) in
cancers of multiple organs: tissue microarray analysis. Int J Oncol, 2012, in press. [IF: 2.399]
3. Kong D, Piao YS, Oshima H, Oguma K, Minamoto T, Yamada Y, Sato K, Yamashita S,
Ushijima T, Ishikawa T, Oshima M. Inflammation-induced repression of tumor suppressor
miR-7 in gastric epithelial cells. Oncogene, 2012. Epub 2011 Dec 5. doi: 10.1038/onc.2011.
558. [IF: 6.373]
4. Shimasaki T, Ishigaki Y, Nakamura Y, Takata T, Nakaya N, Nakajima H, Sato I, Zhao X,
Kitano A, Kawakami K, Tanaka T, Takegami T, Tomosugi N, Minamoto T, Motoo Y.
Glycogen synthase kinase 3β inhibition sensitizes pancreatic cancer cells to gemcitabine. J
Gastroenterol 47 (3): 321-33, 2012. Epub 2011 Nov 1. [IF: 4.160]
5. Mimura M, Masuda A, Nishiumi S, Kawakami K, Fujishima Y, Yoshie T, Mizuno S, Miki I,
Ohno H, Hase K, Minamoto T, Azuma T, Yoshida M. AP1B plays an important role in
intestinal tumorigenesis with the truncating mutation of an APC gene. Int J Cancer 130 (5):
1011-20, 2012. doi: 10.1002/ijc.26131. Epub 2011 May 30. [IF: 5.444]
6. Tomita H, Takaishi S, Menheniott TR, Yang X, Shibata W, Jin G, Betz KS, Kawakami K,
Minamoto T, Tomasetto C, Rio MC, Lerkowit N, Varro A, Giraud AS, Wang TC. Inhibition
of gastric carcinogenesis by the hormone, gastrin, is mediated by suppression of TFF1
epigenetic silencing. Gastroenterology 140 (3): 879-91, 2011. 2010 Nov 24. [Epub ahead of
print]. [IF: 11.675]
7. Kawakami K, Matsunoki A, Kaneko M, Saito K, Watanabe G, Minamoto T. Long
interspersed nuclear element-1 hypomethylation is a potential biomarker for the prediction of
response to oral fluoropyrimidines in microsatellite stable and CpG island methylator
phenotype-negative colorectal cancer. Cancer Sci 102 (1): 166-74, 2011 Jan; 2010 Oct 18.
doi: 10.1111/j.1349-7006.2010.01776.x. [Epub ahead of print]. [IF: 3.325]
8. Peterson AJ, Menheniott TR, O'Connor L, Walduck AK, Fox JG, Kawakami K, Minamoto T,
Ong EK, Wang TC, Judd LM, Giraud AS. Helicobacter pylori infection promotes methylation
and silencing of trefoil factor 2, leading to gastric tumor development in mice and humans.
Gastroenterology 139 (6): 2005-17, 2010. Epub 2010 Aug 27. [Epub ahead of print] [IF:
11.675]
9. Nakajima H, Ishigaki Y, Xia Q, Ikeda T, Yoshitake Y, Yonekura H, Nojima T, Tanaka T,
Umehara H, Tomosugi N, Takata T, Shimasaki T, Nakaya N, Sato I, Kawakami K, Koizumi
K, Minamoto T, Motoo Y. Induction of HITS, a newly identified family with sequence
similarity 107 protein (FAM107B), in cancer cells by heat shock stimulation. Int J Oncol 37
(3): 583-93, 2010. [IF: 2.399]
10. Saito K, Kawakami K, Matsumoto I, Oda M, Watanabe G, Minamoto T. Long interspersed
nuclear element 1 hypomethylation is a marker of poor prognosis in stage IA non–small cell
lung cancer. Clin Cancer Res 16 (8): 2418–26, 2010, Published online first on April 6, 2010.
[IF: 7.742]
11. Jin MJ, Kawakami K, Fukui Y, Tsukioka S, Oda M, Watanabe G, Takechi T, Oka T,
Minamoto T. Different histological types of non-small cell lung cancer have distinct folate
and DNA methylation levels. Cancer Sci 100 (12): 2325-30, 2009. Epub ahead of print Aug
25, 2009. [IF: 3.325]
- 21 -
金沢大学がん進展制御研究所 腫瘍制御研究分野
12. Noubissi FK, Sanek NA, Kawakami K, Minamoto T, Moser A, Grinblat Y, Spiegelman VS.
Wnt signaling stimulates transcriptional outcome of the Hedgehog pathway by stabilizing
GLI1 mRNA. Cancer Res 69 (22): 8572-8, 2009; Epub ahead of print Nov 3, 2009. [IF:
7.856]
13. Mai W, Kawakami K, Shakoori A, Kyo S, Miyashita K, Yokoi K, Jin MJ, Shimasaki T,
Motoo Y, Minamoto T. Deregulated glycogen synthase kinase 3 sustains gastrointestinal
cancer cells survival by modulating human telomerase reverse transcriptase and telomerase.
Clin Cancer Res 15 (22): 6810-9, 2009. Epub 2009 Nov 10. [IF: 7.742]
14. Du YC, Oshima H, Oguma K, Kitamura T, Itadani H, Fujimura T, Piao YS, Yoshimoto T,
Minamoto T, Kotani H, Taketo MM, Oshima M. Induction and downregulation of Sox17 and
its possible roles during the course of gastrointestinal tumorigenesis. Gastroenterology 137
(4): 1346-57, 2009 [Epub ahead of print, Jun 20, 2009]. [IF: 11.675]
15. Howlett M, Giraud AS, Lescesen H, Jackson CB, Kalantzis A, van Driel IR, Robb L, Van der
Hoek M, Ernst M, Minamoto T, Boussioutas A, Oshima H, Oshima M, Judd LM. The IL-6
family cytokine IL-11 regulates homeostatic epithelial cell turnover and promotes gastric
tumor development. Gastroenterology 136 (3): 967-77, 2009. [Epub ahead of print, Dec 3,
2008] [IF: 11.675]
16. Miyashita K, Kawakami K, Mai W, Shakoori A, Fujisawa H, Nakada M, Hayashi Y, Hamada
J, Minamoto T. Potential therapeutic effect of glycogen synthase kinase 3 inhibition against
human glioblastoma. Clin Cancer Res 15 (3): 887-897, 2009. [IF: 7.742]
17. Ohno S, Kinoshita T, Ohno Y, Minamoto T, Suzuki N, Inoue M, Suda T. Expression of
NLRP7 (PYPAF3, NALP7) protein in endometrial cancer tissues. Anticancer Res 28 (4C):
2493-7, 2008. [IF: 1.725]
18. Kawakami K, Ooyama A, Ruszkiewicz A, Jin M, Watanabe G, Moore J, Oka T, Iacopetta B,
Minamoto T. Low expression of γ-glutamyl hydrolase mRNA in primary colorectal cancer
with the CpG island methylator phenotype. Brit J Cancer 98 (9): 1555-61. Epub 2008 Apr 15.
[Epub ahead of print] [IF: 5.042]
著書総説
19. Minamoto T, Kotake M, Nakada M, Shimasaki T, Motoo Y, Kawakami K. Distinct pathologic
role for glycogen synthase kinase 3β in colorectal cancer progression. In: Colorectal Cancer
Biology - From Genes to Tumor, Rajunor Ettarh (Ed.), ISBN: 978-953-51-0062-1, pp. 107134, 2012; InTech.
20. Nakada M, Minamoto T, Pyko IV, Hayashi Y, Hamada JI. The pivotal role of GSK3β in
glioma biology. In: Molecular Targets of CNS Tumors, ISBN 978-953-307-736-9, Miklos
Garami (Ed.), pp. 567-590, 2011; InTech.
21. Motoo Y, Shimasaki T, Ishigaki Y, Nakajima H, Kawakami K, Minamoto T. Metabolic
disorder, inflammation and deregulated molecular pathways converging in pancreatic cancer
development: implications for new therapeutic strategies. Cancers 3 (1): 446-60, 2011.
22. Nakada M, Kita D, Hayashi Y, Kawakami K, Hamada J, Minamoto T. RNAi in malignant
brain tumors: relevance to molecular and translational research. In; Erdmann VA, Barciszewski
J, eds., RNA Technologies and Their Applications, Springer Verlag, 2010, pp. 107-129.
23. Motoo Y, Shimasaki T, Minamoto T. Gemcitabine changes the gene expression in human
pancreatic cancer cells: search for new therapeutic molecular targets. Iovanna J, Ismailov U,
eds., Pancreatology, From Bench to Bedside, Springer Verlag, Berlin, Heidelberg, 2009, pp.
33-8.
24. Miyashita K, Nakada M, Shakoori A, Ishigaki Y, Shimasaki T, Motoo Y, Kawakami K,
Minamoto T. An emerging strategy for cancer treatment targeting aberrant glycogen synthase
kinase 3. Anti-Cancer Agents Med Chem 9 (10): 1114-22, 2009. [IF: 2.862]
- 22 -
2008～2012 年
和文総説
25. 源 利成，川上和之．がんのエネルギー代謝と GSK3．実験医学 30 (15), 2012，印刷中
26. 川上和之，源 利成．DNA メチル化とがん転移．癌と化学療法 37 (11): 2042-46, 2010.
27. 島崎猛夫，石垣靖人，源 利成，元雄良治．膵癌治療への分子標的薬の応用．膵臓 25
(1): 35-45, 2010.
28. 山下 要，川上和之，源 利成．Wnt/β-カテニンシグナル制御破綻の新しい分子メカニ
ズム ―大腸がん医療との関連― Biotherapy 22 (5): 287-95, 2008.
29. 源 利成，魚谷知佳，川上和之．大腸がんの診断と検診を目指す便の細胞・分子マー
カー検出の試み．臨床消化器内科 23 (2): 183-89, 2008.
学会発表
2012 年
1. Aika Matsunoki, Kazuyuki Kawakami, Masanori Kotake, Mami Kaneko, Go Watanabe,
Toshinari Minamoto. LINE-1 methylation level is a molecular marker with little intra-patient
heterogeneity in primary and synchronous metastatic colorectal cancer. 103rd AACR Annual
Meeting, March 31-April 4, 2012, Chicago, IL, U.S.A.
2. Toshinari Minamoto, Wei Mai, Satoru Kyo, Abbas Shakoori, Katsuyoshi Miyashita, Kenji
Yokoi, Takeo Shimasaki, Yoshiharu Motoo, Kazuyuki Kawakami. Deregulated GSK3β
sustains gastrointestinal cancer cells by modulating hTERT and telomerase. 103rd AACR
Annual Meeting, March 31-April 4, 2012, Chicago, IL, U.S.A.
3. Mitsutoshi Nakada, Yuri Chikano, Hemragul Sabit, Takuya Furuta, Katsuyoshi Miyashita,
Yutaka Hayashi, Hiroshi Sato, Kazuyuki Kawakami, Toshinari Minamoto, Jun-ichiro Hamada.
Aberrant glycogen synthase kinase 3β is involved in glioma invasion. 9th Meeting of Asian
Society for Neuro-Oncology, April 20-22, 2012 – Taipei International Convention Center,
Taipei, Taiwan.
4. Takahiro Domoto. Cleavage of hepatocyte growth factor activator inhibitor-1 by membranetype MMP-1 stimulates tumor invasive growth. Joint Symposium of the 7th International
Symposium of the Institute Network and the 45th IDAC Symposium, the 2nd Symposium
for Joins Usage/Research Center of Aging, June 14-15, 2012, Smart Ageing International
Research Center, Institute of Development, Aging and Cancer, Tohoku University, Sendai,
Japan.
5. 島崎猛夫，石垣靖人，高田尊信，川上和之，上田順彦，友杉直久，小坂健夫，源 利成，
元雄良治．GSK3β 標的治療と化学療法を併用する膵がんの新規治療戦略と分子基盤．
第 43 回日本膵臓学会大会 2012 年 6 月 28-29 日，ホテルメトロポリタン山形，山形．
2011 年
6. Menheniott TR, O'Connor L, Minamoto T, Kawakami K, Tomita H, Fox JG, Wang TC, Judd
LM, Giraud AS. Interleukin-1β is a signal for Trefoil factor 2 promoter methylation in gastric
preneoplasia. DDW 2011, May 7-10, 2011, Chicago, Illinois, U.S.A.
7. Chikano Y, Hirose M, Nakada M, Kitano A, Miyashita K, Takino T, Sato H, Sai Y, Miyamoto
K, Hamada J, Kawakami K, Minamoto T. Deregulated glycogen synthase kinase (GSK) 3
participates in invasion of glioblastoma. The 6th International Symposium of Institute
Network (第６回附置研究所ネットワーク国際シンポジウム). June 09-10, 2011, Tokyo,
Japan.
8. Nakajima H, Minamoto T, Motoo Y. HITS (FAM107B): novel heat-shock induced protein as
a maker for cancer progression and diagnosis. 16th World Congress on Advances in
- 23 -
金沢大学がん進展制御研究所 腫瘍制御研究分野
Oncology and 14th International Symposium on Molecular Medicine, October 6-8, 2011;
Hotel Rodos Palace, Rhodes Island, Greece.
9. 大井章史，鈴木潮人，田尻亮輔，土橋 洋，源 利成．FISH 法を用いた大腸癌におけ
る EGFR 遺伝子の増幅の検討．第 100 回日本病理学会総会，2010 年 4 月 28－30 日，
パシフィコ横浜，横浜．
10. 廣瀬まゆみ，川島博人，村井 稔幸，川上和之，源 利成．Elevation of rat plasma Pselectin in acute lung injury. 日本分子生物学会・第 11 回春季シンポジウム，金沢国際
がん生物学シンポジウム，2011 年 5 月 25－26 日，石川県立音楽堂，金沢．
11. 廣瀬まゆみ，川島博人，村井稔幸，源 利成，岡田雅人，末次志郎，竹縄忠臣，中川
敦史．各種 SH3 ドメインと N-WASP プロリンリッチ部位の相互作用の相違により NWASP のアクチンフィラメント形成と細胞運動の相違が生じる．第 63 回日本細胞生物
学会大会，2011 年 6 月 27－29 日，北海道大学・クラーク会館，学術交流会館，札幌．
12. 島崎猛夫，石垣靖人，高田尊信，中村有香，川上和之，上田順彦，小坂健夫，源 利
成，友杉直久，元雄良治．ゲムシタビン単剤療法の壁への挑戦：新規標的分子の同
定と膵癌化学療法への展開．第 42 回日本膵臓学会大会；特別企画「進行膵がん治療
の新展開」
；2011 年 7 月 29－30 日，ホテルニューキャッスル，弘前．
13. Yuri Chikano, Mitsutoshi Nakada, Mayumi Hirose, Ayako Kitano, Katsuyoshi Miyashita,
Hironori Fujisawa, Yutaka Hayashi, Takahisa Takino, Hiroshi Sato, Yoshimichi Sai, Ken-ichi
Miyamoto, Kazuyuki Kawakami, Toshinari Minamoto (近野祐里，中田光俊，廣瀬まゆみ，
北野綾子，宮下勝吉，藤沢弘範，林 裕，滝野隆久，佐藤 博，崔 吉道，宮本謙一，
川 上 和 之 ， 源 利 成 ). Deregulated glycogen synthase kinase (GSK) 3 participates in
invasion of glioblastoma (Glycogen synthase kinase (GSK) 3β は神経膠芽腫の浸潤を促進
する). 第 70 回日本癌学会総会学術集会，2011 年 10 月 3 日－5 日，名古屋国際会議場，
名古屋．
14. Masanori Kotake, Kazuyuki Kawakami, Mayumi Hirose, Hiroyuki Bandou, Tetsuji Yamada,
Go Watanabe, Toshinari Minamoto (小竹優範，川上和之，廣瀬まゆみ，伴登宏行，山田
哲司，渡邊 剛，源 利成). Frequent hypomethylation of LINE-1 in colorectal cancer with
lymph node and distant metastases (リンパ節および遠隔転移を伴う大腸がんにおける高
頻度の LINE-1 低メチル化). 第 70 回日本癌学会総会学術集会，2011 年 10 月 3 日－5
日，名古屋国際会議場，名古屋．
15. Takeo Shimasaki, Yasuhito Ishigaki, Takanobu Takata, Yuka Nakamura, Kazuyuki
Kawakami, Tsutomu Takegami, Naohisa Tomosugi, Toshinari Minamoto, Yoshiahru Motoo
(島崎猛夫，石垣靖人，高田尊信，中村有香，川上和之，竹上 勉，友杉直久，源 利
成，元雄良治). GSK3 is a new therapeutic target in pancreatic cancer: inplications for
chemotherapy with new strategy (膵癌に新規治療標的としての glycogen synthase kinase
(GSK) 3：化学療法戦略の新展開). 第 70 回日本癌学会総会学術集会，2011 年 10 月 3
日－5 日，名古屋国際会議場，名古屋．
16. 中田光俊，林 裕，Pyko Ilya，近野祐里，淑瑠ヘムラサビット，宮下勝吉，渡邉卓也，
木下雅史，喜多大輔，林 康彦，内山尚之，川上和之，源 利成，濵田潤一郎
(Mitsutoshi Nakada, Yutaka Hayashi, Pyko Ilya, Yuri Chikano, Hemragul Sabit, Katsuyoshi
Miyashita, Takuya Watanabe, Masashi Kinoshita, Daisuke Kita, Yasuhiko Hayashi, Naoyuki
Uchiyama, Kazuyuki Kawakami, Toshinari Minamoto, Jun-ichiro Hamada)．再発膠芽腫に
対する GSK3β を治療標的とした分子標的療法の現状 (Translational research focusing
GSK3β as a new therapeutic target for glioblastoma)．第 70 回日本脳神経外科学会学術総
会，2011 年 10 月 12－14 日，パシフィコ横浜，横浜．
- 24 -
2008～2012 年
17. 島崎猛夫，川上和之，上田順彦，小坂健夫，源 利成，元雄良治．切除不能進行膵癌
に対する GSK3β を標的とした新規治療戦略．JDDW 2011，2011 年 10 月 20－23 日，
福岡国際センター/福岡サンパレス/福岡国際会議場/マリンメッセ福岡，福岡．
18. 中田光俊，林 裕，近野祐里，Pyko Ilya，淑瑠ヘムラサビット，宮下勝吉，渡辺卓也，
木下雅史，喜多大輔，林 康彦，内山尚之，川上和之，源 利成，濱田潤一郎
(Mitsutoshi Nakada, Yutaka Hayashi, Yuri Chikano, Pyko Ilya, Hemragul Sabit, Katsuyoshi
Miyashita, Takuya Watanabe, Masashi Kinoshita, Daisuke Kita, Yasuhiko Hayashi, Naoyuki
Uchiyama, Kazuyuki Kawakami, Toshinari Minamoto, Jun-ichiro Hamada)．膠芽腫の新規
治療標的 GSK3β に関する基礎研究とトランスレーショナルリサーチ (Basic and
translational research focusing GSK3β as a new therapeutic target for glioblastoma)．第 49
回日本癌治療学会学術集会，2011 年 10 月 27－29 日，名古屋国際会議場，名古屋．
19. 松之木愛香，川上和之，小竹優範，金子真美，源 利成．多重蛍光 MethyLight による
LINE-1 のメチル化解析と大腸がんにおけるその臨床的意義．第 22 回日本消化器癌発
生学会；ワークショップ７：癌個別化治療の現状と今後の展望．2011 年 11 月 25－
26 日，ホテルニューオータニ佐賀，佐賀．
20. 中島日出夫，源 利成，元雄良治．新規熱ショック誘導性蛋白質（HITS）の発現を利
用した癌の診断．第 22 回日本消化器癌発生学会；ワークショップ４：癌分子診断の
臨床応用．2011 年 11 月 25－26 日，ホテルニューオータニ佐賀，佐賀．
21. 島崎猛夫，石垣靖人，高田尊信，中村有香，川上和之，舟木 洋，上田順彦，小坂健
夫，友杉直久，源 利成，元雄良治．膵癌細胞における gemcitabine 誘導性 EMT に関
連する新規分子の同定．第 22 回日本消化器癌発生学会；シンポジウム２：消化器癌
の浸潤・転移．2011 年 11 月 25－26 日，ホテルニューオータニ佐賀，佐賀．
2010 年
22. 藤田秀人，中村慶史，石黒 要，小竹優範，山下 要，川上和之，伴登宏行，西村元一，
藤村 隆，萱原正都，太田哲生，源 利成．消化管がん組織検体資源化による進行再
発大腸がんに対する抗 EGFR 分子標的医療の均てん化への取り組み．第 110 回日本
外科学会定期学術集会 パネルディスカッション６．進行再発大腸癌に対する化学
療法の均てん化：外科医の立場から．2010 年 4 月 8－10 日，名古屋．
23. Trevelyan Menheniott, Anthony J. Peterson, Louise O'Connor, Timothy C. Wang, James G.
Fox, Toshinari Minamoto, Kazuyuki Kawakami, Louise M. Judd, Andrew S. Giraud. TFF2
inhibits tumour growth and is a target for epigenetic silencing in gastric cancer. DDW 2010,
May 1-6, 2010, New Orleans, Louisiana, U.S.A.
24. Katsuyoshi Miyashita, Kazuyuki Kawakami, Mitsutoshi Nakada, Abbas Shakoori, Hironori
Fujisawa, Yutaka Hayashi, Jun-ichiro Hamada, Toshinari Minamoto. Potential therapeutic
effect of glycogen synthase kinase 3β inhibition against human glioblastoma. 101st Annual
Meeting of American Association for Cancer Research 2010, April 17-21, 2010,
Washington DC, U.S.A.
25. Kenichiro Saito, Kazuyuki Kawakami, Isao Matsumoto, Makoto Oda, Go Watanabe,
Toshinari Minamoto. LINE-1 hypomethylation is a marker of poor prognosis in stage IA nonsmall cell lung cancer. 101st Annual Meeting of American Association for Cancer
Research 2010, April 17-21, 2010, Washington DC, U.S.A.
26. Mitsutoshi Nakada, Yutaka Hayashi, Katsuyoshi Miyashita, Daisuke Kita, Yasuhiko Hayashi,
Naoyuki Uchiyama, Kazuyuki Kawakami, Toshinari Minamoto, Jun-ichiro Hamada.
Targeting glycogen synthase kinase 3 in adult recurrent glioblastomas. 7th Meeting of Asian
Society for Neuro-Oncology, June 10-12, Seoul, Korea.
- 25 -
金沢大学がん進展制御研究所 腫瘍制御研究分野
27. Ayako Kitano, Takeo Shimasaki, Yuri Chikano, Mitsutoshi Nakada, Tomomi Higashi,
Yasuhito Ishigaki, Yoshimichi Sai, Ken-ichi Miyamoto, Yoshiharu Motoo, Kazuyuki
Kawakami, Toshinari Minamoto. Pathological role for deregulated GSK3 in pancreatic
cancer proliferation and invasion. The Joint Symposium of the 5th International
Symposium of Institute Network and the International Symposium of Commemorating
Inauguration of Kanazawa University Cancer Research Institute. June 24-25, 2010,
Kanazawa, Japan.
28. Takeo Shimasaki, Yasuhito Ishigaki, Takanobu Takata, Yuka Nakamura, Ayako Kitano,
Kazuyuki Kawakami, Naohisa Tomosugi, Toshinari Minamoto, Yoshiharu Motoo. Effect of
GSK3 inhibition agaisnt gemcitabine-induced epithelial-mesenchymal transition and invasive
ability of pancreatic cancer cells: its therapuetic implication. Joint Meeting of the
International Association of Pancreatology and the Japan Pancreas Society 2010（第 41
回日本膵臓学会大会／第 14 回国際膵臓学会合同学会），2010 年 7 月 11－13 日，福岡．
29. 小竹優範，石黒 要，伴登宏行，山田哲司，西村元一，中村慶史，藤田秀人，山下
要，川上和之，太田哲生，源 利成．ヒト消化管がん組織検体資源化によるがん研究
と個別化医療への応用：Project K の試み．第 65 回日本消化器外科学会総会：シンポ
ジウム．消化器癌化学療法にける個別化医療の最前線．2010 年 7 月 14－16 日，山口．
30. Ilya V. Pyko，中田光俊，古山奈月，Teng Lei，林 裕，川上和之．源 利成．濵田潤一
郎 ． Glycogen synthase kinase 3 inhibition affects methylation status of the O6methylguanine DNA methyltransferase (MGMT) promoter and MGMT gene expression in
human glioma cells. 第 11 回日本分子脳神経外科学会，2010 年 8 月 27-28 日，仙台．
31. 小竹優範，川上和之，廣瀬まゆみ，伴登宏行，山田哲司，渡邊 剛，源 利成．
Distinct DNA methylation profiles and genetic signatures in human colorectal cancer. 大腸癌
における DNA メチル化パターンと遺伝子変異の関連．第 69 回日本癌学会学術集会，
2010 年 9 月 22-24 日，大阪．
32. 松之木愛香，川上和之，廣瀬まゆみ，斎藤健一郎，渡邊 剛，源 利成．LINE-1
methylation is stable in primary and synchronous metastatic colorectal cancer tissues. 大腸癌
原発巣および転移巣における LINE-1 メチル化の検討．第 69 回日本癌学会学術集会，
2010 年 9 月 22-24 日，大阪．
33. 中田光俊，林 裕，宮下勝吉，喜多大輔，林 康彦，内山尚之，川上和之，源 利成，
濵 田 潤 一 郎 ． Clinical trial for treatment of recurrent glioblastoma targeting deregulated
GSK3 in combination with temozolomide. Glycogen synthase kinase (GSK) 3 を分子標的
とした再発神経膠芽腫に対する化学療法．第 69 回日本癌学会学術集会，2010 年 9 月
22-24 日，大阪．
34. 北野綾子，島崎猛夫，近野祐里，中田光俊，東 朋美，石垣靖人，遠藤良夫，廣瀬ま
ゆみ，崔 吉道，宮本謙一，元雄良治，川上和之，源 利成．Pathological role for
deregulated glycogen synthase kinase (GSK) 3 in pancreatic cancer proliferation and
invasion. 膵がん細胞の増殖と浸潤におよぼす glycogen synthase kinase (GSK) 3 の病的
作用. 第 69 回日本癌学会学術集会，2010 年 9 月 22－24 日，大阪．
35. 近野祐里，中田光俊，北野綾子，宮下勝吉，藤沢弘範，廣瀬まゆみ，林 裕，濵田潤
一郎，崔 吉道，宮本謙一，川上和之，源 利成．Pathological role for deregulated
glycogen synthase kinase (GSK) 3 in glioblastoma proliferation and invasion. 神経膠芽腫の
増殖と浸潤におよぼす glycogen synthase kinase (GSK) 3 の病的作用. 第 69 回日本癌学
会学術集会，2010 年 9 月 22－24 日，大阪．
36. 島崎猛夫，石垣靖人，北野綾子，高田尊信，中村由香，川上和之，寺田光宏，友杉
直 久 ， 源 利 成 ， 元 雄 良 治 ． Role and regulation of glycogen synthase kinase 3 in
- 26 -
2008～2012 年
gemcitabine-induced EMT of pancreatic cancer cells. Gemcitabine による膵癌細胞の EMT
における glycogen synthase kinase (GSK) 3 の役割とその制御．第 69 回日本癌学会学
術集会：English Workshop: Gastrointestinal cancer (2)，2010 年 9 月 22－24 日，大阪．
37. 中田光俊，林 裕，宮下勝吉，渡邉卓也，Pyko Ilya，喜多大輔，近野祐里，林 康彦，内
山尚之，川上和之，源 利成，濵田潤一郎．膠芽腫における GSK3 研究の進展とその阻
害薬剤を使用した再発膠芽腫に対する第 I/II 相臨床試験．第 69 回日本脳神経外科学会学
術総会：シンポジウム：悪性神経膠腫－分子病態から治療まで－，2010 年 10 月 27－29
日，福岡．
38. 小竹優範，山本大輔，伴登宏行，山田哲司，西村元一，中村慶史，藤田秀人，山下
要，川上和之，太田哲生，源 利成．ヒト消化管がん組織検体資源化によるがん研究
と個別化医療への応用．第 21 回日本消化器癌発生学会：ワークショップ２．がんの
個別化治療，2010 年 11 月 18－19 日，軽井沢．
39. 島崎猛夫，石垣靖人，高田尊信，北野綾子，川上和之，友杉直久，源 利成，元雄良
治．膵癌細胞の抗がん剤誘導性上皮－間葉移行と GSK3 阻害による制御機構．第 21
回日本消化器癌発生学会：シンポジウム２．浸潤・転移の多様性，2010 年 11 月 18－
19 日，軽井沢．
40. 中島日出夫，石垣靖人，高田尊信，小泉惠太，川上和之，源 利成，元雄良治．熱シ
ョック誘導性新規がん抑制遺伝子の解析．21 回日本消化器癌発生学会，2010 年 11
月 18－19 日，軽井沢（優秀ポスター賞）．
41. 中田光俊，林 裕，渡邉卓也，喜多大輔，林 康彦，川上和之，源 利成，濵田潤一郎．
再発膠芽腫に対する GSK3 阻害薬剤を使用した第 I/II 相臨床試験（Phase I/II clinical
trial targeting GSK3 for the recurrent glioblastoma）．第 28 回日本脳腫瘍学会学術集会，
2010 年 11 月 28－30 日，軽井沢．
42. 廣瀬まゆみ，川島博人，村井稔幸，竹縄忠臣，末次志郎，中川敦史．CRIB ドメイン
と WH1 ドメインを介した特定の N-WASP 多量体がアクチン骨格形成および細胞運
動に寄与している．第 33 回日本分子生物学会年会・第 83 回日本生化学会大会，
2010 年 12 月 7 日－10 日，神戸．
2009 年
43. Kazuyuki Kawakami, MingJi Jin, Kenichiro Saito, Aika Matsunoki, Wei Mai, Go Watanabe,
Toshinari Minamoto. Allele-specific inhibition of thymidylate synthase expression by small
interfering RNA. Annual Meeting 2009 of the American Association for Cancer Research,
April 18-22, 2009, Denver, CO.
44. 島崎猛夫，石垣靖人，夏 啓勝，中谷直喜，友杉直久，田中卓二，川上和之，源 利
成，元雄良治．GSK3 阻害剤と塩酸ゲムシタビンの併用による膵癌の新規治療戦略と
分子基盤．第 40 回日本膵臓病学会大会，シンポジウム２：膵癌に対するトランスレ
ーショナルリサーチの展望 ―bench to bed, bed to bench―，2009 年 7 月 30－31 日，東京．
45. 中田光俊，林 裕，喜多大輔，宮下勝吉，玉瀬 玲，上出智也，田中慎吾，林 康彦，
内山尚之，源 利成，濵田潤一郎．GSK3 を分子標的とした再発 GBM に対する第
I/II 相臨床試験．第 10 回日本分子脳神経外科学会，2009 年 9 月 19－20 日，岡山．
46. Iacopetta B, Kawakami K. The CPG islamd methylator phenotype as a predictor of response
to 5FU-based chemotherapy in colon cancer. 第 68 回 日 本 癌 学 会 総 会 学 術 集 会
International Session 1，2009 年 10 月 1 日－3 日，横浜．
47. Kazuyuki Kawakami, Aika Matsunoki, MingJi Jin, Kenichiro Saito, Go Watanabe, Toshinari
Minamoto（川上和之，松之木愛香，金 明姫，斉藤健一郎，渡邊 剛，源 利成）．
- 27 -
金沢大学がん進展制御研究所 腫瘍制御研究分野
Augmentation of LINE-1 expression is a possible mechanism underling cytotoxic effect of 5-FU
in colorectal cancer（LINE-1 の発現増強は大腸がんにおける 5-FU の抗腫瘍効果発現メ
カニズムに関与する）
．第 68 回日本癌学会総会学術集会，2009 年 10 月 1－3 日，横浜．
48. Takeo Shimasaki, Yasuhito Ishigaki, Qisheng Xia, Takanobu Takata, Ayako Kitano, Hideo
Nakajima, Naohisa Tomosugi, Kazuyuki Kawakami, Toshinari Minamoto, Yoshiharu Motoo
（島崎猛夫，石垣靖人，夏 啓勝，高田尊信，北野綾子，中島日出夫，友杉直久，川
上 和 之 ， 源 利 成 ， 元 雄 良 治 ）． Chemotherapy-induced changes in morphology and
invasion ability of pancreatic cancer cells（化学療法剤により誘導される膵癌細胞の形態
と浸潤性の変化）．第 68 回日本癌学会総会学術集会，2009 年 10 月 1－3 日，横浜．
49. Ayako Kitano, Takeo Shimasaki, Yasuhito Ishigaki, Yuri Chikano, Mingji Jin, Ken-ichi Miyamoto,
Yoshiharu Motoo, Kazuyuki Kawakami, Toshinari Minamoto（北野綾子，島崎猛夫，石垣靖
人，近野祐里，金 明姫，宮本謙一，元雄良治，川上和之，源 利成）．Pathological
roles for glycogen synthase kinase (GSK) 3 in proliferation and motility of pancreatic cancer
cells（膵がん細胞の増殖と細胞運動におよぼす glycogen synthase kinase (GSK) 3 の病
的作用）
．第 68 回日本癌学会総会学術集会，2009 年 10 月 1－3 日，横浜．
50. MingJi Jin, Kazuyuki Kawakami, Yasuhito Ishigaki, Abbas Shakoori, Ayako Kitano, Yuri
Chikano, Takeo Shimasaki, Yoshiharu Motoo, Toshinari Minamoto（金 明姫，川上和之，石
垣靖人，シャクーリ アッバス，北野綾子，近野祐里，島崎猛夫，元雄良治，源 利
成）
．Glycogen synthase kinase (GSK) 3 sustains colon cancer cells survival by modulating
JNK-mediated pathway. 第 68 回日本癌学会総会学術集会，2009 年 10 月 1－3 日，横浜．
51. 川上和之，源 利成．DNA メチル化マーカーによる大腸がんの予後・抗癌剤感受性診
断．第 17 回日本消化器関連学会週間（JDDW 2009）／第 51 回日本消化器病学会大
会：シンポジウム６．消化器癌におけるエピジェネティクス，2009 年 10 月 14－17
日，京都．
52. 中田光俊，林 裕，喜多大輔，宮下勝吉，玉瀬 玲，上出智也，田中慎吾，林 康彦，
内山尚之，源 利成，濵田潤一郎．再発神経膠芽腫に対する GSK3 を分子標的とし
た Phase I/II 臨床試験．第 68 回日本脳神経外科学会総会：シンポジウム，2009 年 10 月
14－16 日，東京．
53. 山下 要（優秀賞受賞），藤田秀人，伴登宏行，川上和之，西村元一，源 利成．ヒ
ト消化管がん組織検体資源化の試み：がん研究とがんの個別化医療への応用．第 47
回日本がん治療学会総会学術集会：優秀演題，2009 年 10 月 22－24 日，横浜．
54. 川上和之，源 利成．LINE-1 メチル化解析による大腸がんの予後・抗癌剤感受性診断．
第 20 回消化器癌発生学会総会：シンポジウム（３）消化器癌診断の新展開，2009
年 11 月 26－27 日，広島．
55. 島崎猛夫（最優秀賞受賞），石垣靖人，高田尊信，北野綾子，夏 啓勝，友杉直久，
川上和之，源 利成，元雄良治．GSK3 阻害による抗がん剤誘導性上皮－間葉移行
の制御に基づく新規膵癌治療ストラテジー．第 20 回消化器癌発生学会総会：シンポ
ジウム（２）消化器癌治療の新展開，2009 年 11 月 26－27 日，広島．
56. 北野綾子，島崎猛夫，東 朋美，近野祐里，石垣靖人，元雄良治，宮本謙一，川上和
之，源 利成．GSK3 による膵がん細胞の増殖と浸潤の制御．第 20 回消化器癌発生
学会総会：ミニシンポジウム（５）－３消化器癌の分子基盤：浸潤・転移－３，
2009 年 11 月 26－27 日，広島．
57. Minamoto T, Kawakami K. Glycogen synthase kinase (GSK)-3 inhibition for cancer
treatment. 第 14 回日韓がん研究ワークショップ，2009 年 12 月 18－19 日，金沢．
- 28 -
2008～2012 年
2008 年
58. 島崎猛夫，石垣靖人，中谷直喜，中島日出夫，友杉直久，田中卓二，麦 威，川上和
之，源 利成，元雄良治．膵癌における新しい分子標的としての GSK3とその関連遺
伝子群の解析．第 6 回日本臨床腫瘍学会学術集会，2008 年 3 月 20－21 日，福岡．
59. Mai W, Shakoori A, Miyashita K, Zhang B, Motoo Y, Kawakami K, Takahashi Y, Minamoto
T. Detection of active fraction of GSK3 in cancer cells by non-radioisotopic in vitro kinase
assay. Annual Meeting 2008 of the American Association for Cancer Research, April 1216, 2008, San Diego, CA.
60. Minamoto T, Shakoori A, Mai W, Miyashita K, Yasumoto K, Takahashi Y, Ooi A, Kawakami
K. Inhibition of GSK3 activity attenuates proliferation of human colon cancer cells in
rodents. Annual Meeting 2008 of the American Association for Cancer Research, April
12-16, 2008, San Diego, CA.
61. Kawakami K, Jin MJ, Saito K, Miyashita K, Mai W, Watanabe G, Minamoto T. Methylation
level of LINE-1 repeats as a prognostic factor for the patients with primary colorectal
cancer. Annual Meeting 2008 of the American Association for Cancer Research, April 1216, 2008, San Diego, CA.
62. 宮下勝吉，中田光俊，吉田優也，藤沢弘範，林 裕，川上和之，源 利成，濵田潤一
郎．神経膠芽腫における GSK3の発現・活性と機能解析．第 26 回日本脳腫瘍病理
学会，2008 年 5 月 23－24 日，東京．
63. 川上和之，金 明姫，斉藤健一郎，麦 威，宮下勝吉，源 利成．遺伝子多型とアレル
欠失を利用した癌特異的治療開発．第 19 回日本消化器癌発生学会総会：ミニシンポ
ジウム (8) 癌の診断と治療，2008 年 8 月 28－29 日，別府．
64. 島崎猛夫，石垣靖人，夏 啓勝，中谷直喜，友杉直久，田中卓二，麦 威，川上和之，
源 利成，元雄良治．膵癌の臨床試験を目指した GSK3阻害剤と塩酸ゲムシタビン
の併用療法に関する基礎的解析．第 19 回日本消化器癌発生学会総会：ミニシンポジ
ウム (4) 化学療法，2008 年 8 月 28－29 日，別府．
65. 川上和之，島崎猛夫，源 利成．慢性進行性疾患の創薬標的 GSK3の消化器がんにお
ける発現，活性と機能解析．第 16 回日本消化器関連学会週間（JDDW 2008）／第
50 回日本消化器病学会大会：ワークショップ 13．消化器疾患の分子遺伝学的病態，
2008 年 10 月 1－4 日，東京．
66. Toshinari Minamoto, Kaname Yamashita, Kazuyuki Kawakami. Novel molecular mechanism
and clinical relevance of deregulated Wnt/-catenin signaling in human colorectal cancer. 第
67 回日本癌学会学術総会，2008 年 10 月 28 日－30 日，名古屋．
67. Wei Mai, Katsuyoshi Miyashita, Abbas Shakoori, Takeo Shimasaki, Kazuo Yasumoto,
Yoshiharu Motoo, Kazuyuki Kawakami, Toshinari Minamoto. Distinct pathologic property
of glycogen synthase kinase 3 (GSK3) in gastrointestinal, pancreatic and liver cancers. 胃
がん，大腸がん，膵がんと肝がんに共通する glycogen synthase kinase 3 (GSK3)の
病的作用．第 67 回日本癌学会学術総会，2008 年 10 月 28 日－30 日，名古屋．
68. Katsuyoshi Miyashita, Mitsutoshi Nakada, Hironori Fujisawa, Wei Mai, Kazuyuki Kawakami,
Jun-ichiro Hamada, Toshinari Minamoto. GSK3 inhibition sensitizes glioblastoma cells to
chemotherapy and radiation by modulating p53 and Rb pathways. GSK3阻害による p53 と
Rb 経路を介した神経膠芽腫細胞の抗がん剤および放射線感受性の修飾．第 67 回日本
癌学会学術総会，2008 年 10 月 28 日－30 日，名古屋．
- 29 -
金沢大学がん進展制御研究所 腫瘍制御研究分野
69. Takeo Shimasaki, Yasuhito Ishigaki, Naoki Nakaya, Hideo Nakajima, Naohisa Tomosugi,
Takuji Tanaka, Wei Mai, Kazuyuki Kawakami, Toshinari Minamoto, Yoshiharu Motoo.
Combined effect of gemcitabine and GSK3 inhibitor against pancreatic cancer: basic
analysis for future clinical trial. 第 67 回日本癌学会学術総会，2008 年 10 月 28 日－30 日，
名古屋．
70. Kenichiro Saito, Kazuyuki Kawakami, Isao Matsumoto, Makoto Oda, Go Watanabe,
Toshinari Minamoto. LINE-1 hypomethylation is a novel prognostic factor in non-small cell
lung cancer. 非小細胞肺癌における LINE-1 低メチル化は新規の予後因子である．第
67 回日本癌学会学術総会，2008 年 10 月 28 日－30 日，名古屋．
特許出願
(1) 国内特許出願
出願番号：特願 2008-264695 号
発明者：川上和之，源 利成
出願者：国立大学法人金沢大学
出願日：2008 年 10 月 11 日
名 称：癌患者の外科的手術後の治療選択方法及び予後診断
(2) 国内特許出願
出願番号：特願 2010-185691 号
発明者：中田光俊，源 利成，林 裕，濵田潤一郎
出願者：国立大学法人金沢大学
出願日：2010 年 8 月 22 日
名 称：脳腫瘍治療用キット及び脳腫瘍治療方法
５．外部資金受け入れ状況
【まとめ】
区
代
分
分
表
担
種 類
科学研究費
その他
小
計
小
中
計
計
総
計
科学研究費
その他
代表＋分担
奨学寄附金
件 数
１０
８
１８
６
５
１１
２９
２１
５０
研究経費
１１７，５９０千円
４，１００千円
１２１，６９０千円
３１，２９０千円
１３，２５０千円
４４，５４０千円
１６６，２３０千円
１２，１６４千円
１７８，３９４千円
2008 年度が含まれるすべての課題を列記する．
[1] 2012 年度 金沢大学がん進展制御研究所共同研究（一般）
元雄良治（代表）源 利成，ほか（分担）
課題：GSK3阻害による新規膵がん化学療法の開発と臨床試験
研究経費：500,000 円
- 30 -
2008～2012 年
[2] 2012 年度 金沢大学がん進展制御研究所共同研究（一般）
小坂健夫（代表）源 利成，川上和之，ほか（分担）
課題：大腸がんの分子病理学的特性の解析と診断，治療のための分子指標の解明
研究経費：750,000 円
[3] 2011－2013 年度 科学研究費補助金（基盤研究 B）：課題番号 23390321
川上和之（代表）源 利成（分担）曽我朋義（連携）
課題：ゲノムの低メチル化とレトロポゾンの活性化を特徴とする大腸がんの診断・治
療開発
研究経費：16,340,000 円
[4] 2011－2013 年度 科学研究費補助金（基盤研究 C）：課題番号 23591955
廣瀬まゆみ（代表）源 利成，川上和之（分担）
課題：GSK3阻害による消化器がん治療法の開発と分子機構の解明
研究経費：4,550,000 円（交付当初額）
[5] 2011－2012 年度 科学研究費補助金（挑戦的萌芽研究）
：課題番号 23659643
源 利成（代表）川上和之（分担）曽我朋義（連携）
課題：がん特異的ネルギー代謝を標的とする消化器がん治療法の開発
研究経費：3,340,000 円（交付当初額）
[6] 2011－2013 年度 科学研究費補助金（基盤研究 C）：課題番号
中田光俊（代表）源 利成（連携）
課題：悪性グリオーマの浸潤シグナルを狙った分子標的療法の確立
研究経費：4,550,000 円（交付当初額）
[7] 2011－2013 年度 科学研究費補助金（基盤研究 C）：課題番号 23590898
中島日出夫（代表）源 利成，ほか（分担）
課題：がん温熱療法の新規分子マーカー候補 FAM107 ファミリー蛋白質の発現・機能
解析
研究経費：5,200,000 円（交付当初額）
[8] 2011－2013 年度 科学研究費補助金（基盤研究 C）：課題番号 23591016
島崎猛夫（代表）源 利成，ほか（分担）
課題：化学療法により誘発される EMT 誘導因子の同定とその制御による膵がん治療
法の開発
研究経費：5,200,000 円（交付当初額）
[9] 2011 年度 金沢大学がん進展制御研究所共同研究（一般） 整理番号：12
元雄良治（代表）源 利成，ほか（分担）
課題：GSK3阻害による新規膵がん化学療法の開発と臨床試験
研究経費：1,000,000 円
[10] 2011 年度 金沢大学がん進展制御研究所共同研究（一般） 整理番号：12
小坂健夫（代表）源 利成，川上和之，ほか（分担）
課題：大腸がん個別化医療のためのバイオマーカー探索
研究経費：1,000,000 円
[11] 2010 年度 財団法人金沢総合技術研究センター研究助成金
源 利成，川上和之，ほか
- 31 -
金沢大学がん進展制御研究所 腫瘍制御研究分野
課題：がん細胞の代謝特性を標的とするがん治療法の開発
研究経費：500,000 円
[12] 2010－2012 年度 科学研究費補助金（基盤研究 A）：課題番号 20890086
源 利成（代表）川上和之，太田哲生（分担）大野博司（連携）
課題：基幹的細胞調節経路の異常に起因する消化器がんの病態解明とがん制御への応用
研究経費：41,990,000 円
[13] 2010 年度 研究成果最適展開支援事業 A-STEP フィージビリティスタディ【FS】ステ
ージ探索タイプ AS221Z02353G
源 利成（代表）川上和之，中田光俊（分担）
課題：GSK3阻害薬剤のがん抑制効果の検証と神経膠芽腫治療の臨床研究
研究経費：1,300,000 円
[14] 2010 年（平成 22）年度 財団法人金沢総合技術研究センター研究助成金
源 利成，川上和之
課題：GSK3阻害効果に基づく新しいがん治療薬開発のための基礎研究
研究経費：500,000 円
[15] 2009－2010 年度 科学研究費補助金（若手研究 B）：課題番号
麦 威（代表）
課題：消化器がんの GSK3阻害効果に基づく新しいがん治療法と分子標的治療薬の開発
研究経費：3,930,000 円（採択後辞退）
[16] 2009 年（平成 21）年度 財団法人金沢総合技術研究センター研究助成金
源 利成，川上和之
課題：GSK3阻害効果に基づく新しいがん治療薬開発のための基礎研究
研究経費：500,000 円
[17] 2008－2010 年度 科学研究費補助金（基盤研究 B）：課題番号 20390353
川上和之（代表），源 利成（分担）
，曽我朋義，石垣靖人（連携）
課題：大腸癌における DNA メチル化の調節機構解明と遺伝子診断・治療への応用
研究経費：10,660,000 円．
[18] 2008－2009 年度 科学研究費補助金（特定領域研究・がん治療）
：課題番号 20015018
源 利成（代表）
，川上和之，横井健二，島崎猛夫（分担）
，安本和生（連携）
課題：GSK3のがん促進機能の分子機構解明とその阻害に基づく消化器がん制御法
の開発
研究経費：10,600,000 円
[19] 2008－2009 年度 科学研究費補助金（若手研究：スタートアップ：課題番号 20890086
金 明姫
課題：GSK3阻害に基づく新しいがん治療法と抗がん剤，放射線感受性の修飾効果
研究経費：3,320,000 円
[20] 2008－2009 年度 金沢医科大学共同研究費
元雄良治（代表），島崎猛夫，友杉直久，石垣靖人，源 利成
課題：抗がん剤感受性に関わる新しい分子細胞機構の解明とその耐性克服への応用
研究経費：10,000,000 円
- 32 -
2008～2012 年
[21] 2008 年（平成 20）年度 財団法人金沢総合技術研究センター研究助成金
源 利成，川上和之
課題：GSK3阻害効果に基づく新しいがん治療薬開発のための基礎研究：新規阻害
剤の開発と効果検証
研究経費：500,000 円
[22] 2008 年度 金沢大学学長戦略経費（重点研究経費）
：若手育成
麦 威（代表），稲垣冬彦（分担）
課題：GSK3阻害効果に基づく新しいがん治療薬の開発
研究経費：400,000 円
[23] 2008 年度 金沢大学学長戦略経費（重点研究経費）
：若手育成
金 明姫（代表）
課題：リン酸化シグナル阻害剤と誘導加温の併用による新しいがん温熱治療法の開発
研究経費：400,000 円
[24] 2007（平成 19）年度 財団法人金沢総合技術研究センター研究助成金（2008 年 1 月）
源 利成
課題：Wnt がん化シグナルの新規転写標的分子の同定と大腸癌における機能解析
研究経費：500,000 円
[25] 2007－2008 年度 科学研究費補助金（萌芽研究）：課題番号 19659190 整理番号 0005
島崎猛夫（代表），元雄良治，源 利成，石垣靖人，友杉直久（分担）
課題：塩酸ゲムシタビンによる膵がん細胞の EMT 誘導の検証と分子細胞機構の解明
研究経費：3,200,000 円
[26] 2007－2008 年度 科学研究費補助金（若手研究 B）
：課題番号 19790938 整理番号 0002
麦 威（代表）
課題：GSK3のがん細胞生存・増殖維持機能の解明と分子標的がん治療法の開発
研究経費：3,490,000 円
[27] 2007－2008 年度 科学研究費補助金（基盤研究 C）
：課題番号 19591536 整理番号 0011
高橋 豊（代表）
，岡本正人，源 利成（分担）
課題：樹状細胞による自己癌標的免疫療法を増強させる，各種抗癌剤の適量に関す
る検討
研究経費：4,440,000 円
[28] 2006－2008 年度 科学研究費補助金（基盤研究 B）
：課題番号 18390363 整理番号 0004
源 利成（代表）
，高橋 豊（分担）
，川上和之（分担）
課題：Wnt を中心とする基幹的細胞シグナル破綻機構の解明と大腸がん制御への応用
研究経費：19,370,000 円
[29] 2004－2008 年度 厚生労働省がん研究助成金：課題番号 15-2
「がん生物学に基づく新しい治療法に関する研究」
江角浩安（代表），源 利成（分担）
，ほか
課題：細胞調節システム破綻の解析と大腸がん制御への応用
研究経費：1,800,000 円（2004 年），1,800,000 円（2005 年），1,700,000 円（2006
年），1,700,000 円（2007 年），1,700,000 円（2008 年） 計 8,700,000 円
- 33 -
金沢大学がん進展制御研究所 腫瘍制御研究分野
奨学寄附金
[30] 源 利成（受入れ）2012 年 3 月
臨床検査関連企業 A
147,000 円
[31] 源 利成（受入れ）2012 年 3 月
予防医学関連財団 I
670,860 円
[32] 源 利成（受入れ）2012 年 3 月
予防医学関連財団 I
400,000 円
[33] 源 利成（受入れ）2011 年 3 月
臨床検査関連企業 A
252,000 円
[34] 源 利成（受入れ）2011 年 3 月
予防医学関連財団 I
1,033,960 円
[35] 源 利成（受入れ）2011 年 3 月
予防医学関連財団 I
400,000 円
[36] 源 利成（受入れ）2011 年 2 月
財団法人金沢総合技術研究センター
[37] 源 利成（受入れ）2010 年 3 月
予防医学関連財団 I
1,015,700 円
[38] 源 利成（受入れ）2010 年 3 月
予防医学関連財団 I
400,000 円
[39] 源 利成（受入れ）2010 年 3 月
臨床検査関連企業 A
283,500 円
[40] 源 利成（受入れ）2010 年 2 月
財団法人金沢総合技術研究センター
[41] 源 利成（受入れ）2009 年 9 月
製薬企業 BM
[42] 源 利成（受入れ）2009 年 3 月
予防医学関連財団 I
1,085,940 円
[43] 源 利成（受入れ）2009 年 3 月
予防医学関連財団 I
400,000 円
[44] 源 利成（受入れ）2009 年 3 月
臨床検査関連企業 A
525,000 円
[45] 源 利成（受入れ）2009 年 2 月
財団法人金沢総合技術研究センター
[46] 源 利成（受入れ）2009 年 1 月
製薬企業 M
[47] 源 利成（受入れ）2008 年 3 月
臨床検査関連企業 A
[48] 源 利成（受入れ）2008 年 2 月
予防医学関連財団 I
800,000 円
[49] 源 利成（受入れ）2008 年 2 月
予防医学関連財団 I
400,000 円
[50] 源 利成（受入れ）2008 年 2 月
財団法人金沢総合技術研究センター
500,000 円
500,000 円
500,000 円
500,000 円
1,000,000 円
850,000 円
500,000 円
６．特記事項
受賞
1. 北國がん基金研究活動助成 2011 年 7 月 27 日
受賞者：小竹優範，伴登宏行（石川県立中央病院 消化器外科）西村元一（金沢赤十字
病院 外科）
，川上和之，源 利成
課題：大腸がん組織検体資源化によるがん生物学的特性の大規模解析とがん医療への展開
2. 2010 年度日本脳神経外科学会 学会奨励賞 2010 年 10 月 27 日
受賞者：宮下勝吉（研究協力員，金沢大学脳神経外科学／横浜栄共済病院脳神経外科）
受賞論文：Miyashita K, Kawakami K, Mai W, Shakoori A, Fujisawa H, Nakada M, Hayashi Y,
Hamada J, Minamoto T. Potential therapeutic effect of glycogen synthase kinase 3
inhibition against human glioblastoma. Clin Cancer Res 15 (3): 887-897, 2009. [IF:
7.742]
- 34 -
2008～2012 年
3. 第 21 回日本消化器癌発生学会総会 優秀演題賞 2010 年 11 月 18 日
受賞者：中島日出夫（研究協力員，金沢医科大学腫瘍内科学／上尾中央総合病院腫瘍内
科）
演 題：熱ショック誘導性新規がん抑制遺伝子の解析
4. 第 47 回日本癌治療学会学術総会 優秀演題賞 2009 年 10 月 22-24 日
受賞者：山下 要，藤田秀人，伴登宏行，川上和之，西村元一，源 利成
演 題：ヒト消化管がん組織検体資源化の試み：がん研究とがんの個別化医療への応用
5. 第 20 回日本消化器癌発生学会総会 最優秀賞 2009 年 11 月 26-27 日
受賞者：島崎猛夫（研究協力員，金沢医科大学腫瘍内科学／同総合医学研究所）
課 題：GSK3阻害による抗がん剤誘導性上皮－間葉移行の制御に基づく新規膵癌治
療ストラテジー
社会活動
日本学術振興会 審査委員
日本外科学会専門医試験委員会 委員
石川県予防医学協会集団検診精度管理委員会 胃がん部会 委員
同
大腸がん部会 委員
石川県教育委員会 SSH 推進事業指導委員会 委員（石川県立金沢泉丘高等学校担当）
学会活動
2013 年 9 月：第 24 回日本消化器癌発生学会総会・会長
所属学会，役職と資格： 源 利成
基礎系 日本癌学会（評議員）
日本消化器癌発生学会（理事，評議員)
日本病理学会（死体解剖認定医・病理解剖）
American Association for Cancer Research (AACR): Corresponding Member
International Society of Preventive Oncology (ISPO): Scientific Advisory Committee
International Society of Gastroenterological Carcinogenesis
日本人類遺伝学会
日本がん転移学会
日本家族性腫瘍学会
臨床系
日本外科学会（指導医，専門医，認定医，専門医試験委員）
日本消化器外科学会（指導医，専門医，認定医）
日本消化器外科学会 消化器がん外科治療認定医
日本臨床外科学会
日本消化器内視鏡学会（学術評議員，北陸支部評議員，指導医，専門医）
日本消化器病学会（本部評議員申請中，北陸支部評議員，指導医，専門医）
日本消化器がん検診学会（評議員，東海北陸支部評議員，指導医，認定医）
日本癌治療学会
日本胃癌学会
日本膵臓学会／国際膵臓学会
日本がん治療認定機構（暫定教育医，認定医）
日本癌病態治療研究会（世話人）
大腸癌研究会（施設代表者）
北陸大腸癌研究会（幹事）
World Society of Gastroenterology
- 35 -
金沢大学がん進展制御研究所 腫瘍制御研究分野
新聞・メディア（TV）報道
2011 年
1. 源 利成，ほか．北陸がんプロ養成プログラム，治療法開発の最前線．北國新聞 日刊，
2011 年１月 24 日．
2. 源 利成，ほか．北陸がんプロフェッショナル養成プログラム市民公開講座：みんな
で拓こう新しいがん治療への道～最近注目の高度・先進治療および臨床試験・治験～．
北國新聞 日刊，2011 年２月 27 日．
3. 小竹優範，川上和之，源 利成，ほか．北國がん基金
に贈呈式．北國新聞 日刊，2011 年７月 28 日．
研究助成など 11 件，９月 21 日
2010 年
4. NHK ニュース報道：脳腫瘍の新しい治療法
5. 北國新聞 2010 年９月 19 日（日） 日刊
脳腫瘍に新治療法 金大グループ 臨床試験で延命成功
6. 北陸中日新聞
既存の薬４種で増殖抑制
2010 年９月 19 日（日） 日刊
根治法ない難病脳腫瘍 新治療薬を金沢大が開発
がる
浜田教授らグループ
延命効果上
7. 北國新聞 2010 年９月 27 日 （月）日刊
悪性脳腫瘍の新治療法 「不治の病」治療に光
8. 日本経済新聞 2010 年 10 月 19 日（火） 日刊
４種薬剤転用，がん新薬．金沢大が開発着手 悪性脳腫瘍向け
すい臓・胃・肝臓にも
9. 日経産業新聞 2010 年 10 月 19 日（火） 日刊
難治がん治療薬開発へ ３年内に承認申請 脳腫瘍にも有効
その他（講演など）
2012 年
1. 源 利成．金沢大学公開講座セミナー，2012 年 6 月 16 日，金沢
2. 源 利成．金沢医科大学大学院医学研究セミナー，2012 年 1 月 27 日，金沢医科大学，
内灘．
3. 源 利成．大腸がん研究から同定した．第 5 回金沢脳腫瘍セミナー，2012 年 1 月 21 日，
ホテル日航金沢，金沢．
4. 源 利成．発がん学，がん医科学とがん医療―消化器がんを中心に―．がんにおける
質の高い看護師育成研修会，2012 年 1 月 18 日，金沢大学附属病院，金沢．
5. 源 利成．第１回富山大学和漢医薬学総合研究所・金沢大学がん進展制御研究所ジョ
イントセミナー，2012 年 1 月 18 日，富山大学和漢医薬学総合研究所（杉谷キャンパ
ス），富山．
2011 年
6. 源 利成．がんの科学と医療．石川県立金沢泉丘高等学校 SSH（Super Science High
School）模擬講義，2011 年 12 月 16 日，金沢大学医学類，金沢．
- 36 -
2008～2012 年
7. 源 利成．第２回専門医セミナー：Cetuximab の奏効した切除不能進行再発大腸癌の２例．
第 113 回日本消化器病学会北陸支部例会，2011 年 11 月 13 日，金沢大学医学類，金沢．
8. 源 利成．難治がんに対する新たながん治療薬開発への道．がんプロ市民公開講座：
みんなで拓こう新しいがん治療への道～最近注目の高度・先進治療および臨床試験・
治験～．2011 年 1 月 23 日，北國新聞交流ホール（赤羽ホール），金沢．
9. 源 利成．発がん学，がん医科学とがん医療 ―消化器がんを中心に―．がんにおける
質の高い看護師育成研修会，2011 年 1 月 19 日，金沢大学附属病院，金沢．
2010 年
10. 源 利成．がんの科学と医療．石川県立金沢泉丘高等学校 SSH（Super Science High
School）模擬講義，2010 年 11 月 26 日，金沢医科大学医学部，内灘．
11. 源 利成．GSK3：大腸がんの Wnt 経路研究から同定したがん標的．第 22 回臨床外科
フォーラム，2010 年 11 月 13 日，金沢．
12. 源 利成．大腸がんの Wnt 経路研究から同定したがん標的 GSK3．第 4 回基礎・臨床
交流セミナー，2010 年 10 月 6 日，金沢大学．
13. 源 利成．がんの医学と研究．石川県立金沢泉丘高等学校 SSH（Super Science High
School）模擬講義，2010 年 1 月 22 日，金沢大学医学部，金沢．
14. 源 利成．発がん学，がん医科学とがん医療 ―消化器がんを中心に―．がんにおける
質の高い看護師育成研修会，2010 年 1 月 20 日，金沢大学附属病院，金沢．
2009 年
15. 源 利成．Glycogen synthase kinase 3：糖尿病，精神神経疾患とがんを繋ぐ疾患マーカ
ー．第 7 回福岡外科セミナー，2009 年 10 月 16 日，福岡．
16. 源 利成．糖尿病，精神神経疾患とがんを繋ぐ疾患マーカーの同定．第 34 回北陸臨床
病理集談会第 17 回セミナー，2009 年 9 月 26 日，金沢医療センター，金沢．
17. 源 利成．がんの医学と研究．石川県立金沢泉丘高等学校 SSH（Super Science High
School）模擬講義，2009 年 1 月 30 日，金沢大学医学部，金沢．
18. 源 利成．発がん学，がん医科学とがん医療―消化器がんを中心に―．がんにおける
質の高い看護師育成研修会，2009 年 1 月 20 日，金沢大学附属病院，金沢．
2008 年
19. 源 利成．大腸がんの遺伝子医療．一腫瘍外科医の視点．金沢医科大学第 23 回遺伝子
関連勉強会，2008 年 9 月 26 日，金沢医科大学，内灘町．
20. 源 利成．Glycogen synthase kinase 3：糖尿病，精神神経疾患とがんを繋ぐ疾患マーカ
ー．金沢大学大学院医学系研究科脳情報病態学（精神医学）セミナー，2008 年 7 月 22
日，金沢大学医学類，金沢．
21. 源 利成．がん医療と分子細胞研究－大腸がんを中心に－．金二会 学術講演会，2008
年 5 月 24 日，ANA クラウンプラザホテル金沢，金沢．
22. 源 利成．Wnt/-カテニンがん化シグナルの新しい分子細胞機構と大腸がん病態．金沢
医科大学大学院医学研究セミナー，2008 年 5 月 20 日，金沢医科大学，内灘町．
23. 源 利成．がんの医学と医療．石川県立金沢泉丘高等学校 SSH（Super Science High
School）模擬講義，2008 年 1 月 25 日，金沢大学医学部，金沢．
- 37 -
金沢大学がん進展制御研究所 腫瘍制御研究分野
報道資料
2008 年～2012 年
- 38 -
2008～2012 年
- 39 -
金沢大学がん進展制御研究所 腫瘍制御研究分野
- 40 -
2008～2012 年
- 41 -
金沢大学がん進展制御研究所 腫瘍制御研究分野
- 42 -
2008～2012 年
- 43 -
金沢大学がん進展制御研究所 腫瘍制御研究分野
- 44 -
2008～2012 年
- 45 -
金沢大学がん進展制御研究所 腫瘍制御研究分野
〔北國新聞
2011 年 7 月 28 日〕
- 46 -
研究のあゆみと業績 2012
【附記２】
- 47 -
金沢大学がん進展制御研究所 腫瘍制御（旧：遺伝子診断）研究分野
【附記３】
発表論文 2012
（抜粋）
＊＊＊＊＊＊＊＊＊＊
＊＊
Representative Publications
2012
- 48 -
研究のあゆみと業績 2012
Shimasaki T, Ishigaki Y, Nakamura Y, Takata T, Nakaya N, Nakajima H,
Sato I, Zhao X, Kitano A, Kawakami K, Tanaka T, Takegami T, Tomosugi
N, Minamoto T, Motoo Y. Glycogen synthase kinase 3β inhibition
sensitizes pancreatic cancer cells to gemcitabine. J Gastroenterol 47(3):
321-33, 2012.
BACKGROUND: Pancreatic cancer is obstinate and resistant to gemcitabine,
a standard chemotherapeutic agent for the disease. We previously showed a therapeutic
effect of glycogen synthase kinase-3β (GSK3β) inhibition against gastrointestinal cancer and
glioblastoma. Here, we investigated the effect of GSK3β inhibition on pancreatic cancer cell
sensitivity to gemcitabine and the underlying molecular mechanism. METHODS:
Expression, phosphorylation, and activity of GSK3β in pancreatic cancer cells (PANC-1)
were examined by Western immunoblotting and in vitro kinase assay. The combined effect
of gemcitabine and a GSK3β inhibitor (AR-A014418) against PANC-1 cells was examined
by isobologram and PANC-1 xenografts in mice. Changes in gene expression in PANC-1
cells following GSK3β inhibition were studied by cDNA microarray and reverse
transcription (RT)-PCR. RESULTS: PANC-1 cells showed increased GSK3β expression,
phosphorylation at tyrosine 216 (active form), and activity compared with non-neoplastic
HEK293 cells. Administration of AR-A014418 at pharmacological doses attenuated
proliferation of PANC-1 cells and xenografts, and significantly sensitized them to
gemcitabine. Isobologram analysis determined that the combined effect was synergistic.
DNA microarray analysis detected GSK3β inhibition-associated changes in gene expression
in gemcitabine-treated PANC-1 cells. Among these changes, RT-PCR and Western blotting
showed that expression of tumor protein 53-induced nuclear protein 1, a gene regulating cell
death and DNA repair, was increased by gemcitabine treatment and substantially decreased
by GSK3β inhibition. CONCLUSIONS: The results indicate that GSK3β inhibition
sensitizes pancreatic cancer cells to gemcitabine with altered expression of genes involved in
DNA repair. This study provides insight into the molecular mechanism of gemcitabine
resistance and thus a new strategy for pancreatic cancer chemotherapy.
Shimasaki T, Kitano A, Motoo Y, Minamoto T. Aberrant glycogen
synthase kinase 3β in pancreatic cancer development, progression and
resistance to therapy. J Carcinog 11: 15, 2012.
Development and progression of pancreatic cancer involves general
metabolic disorder, local chronic inflammation, and multistep activation
of distinct oncogenic molecular pathways. These pathologic processes
result in a highly invasive and metastatic tumor phenotype that is a major obstacle to curative
surgical intervention, infusional gemcitabine-based chemotherapy, and radiation therapy.
Many clinical trials with chemical compounds and therapeutic antibodies targeting growth
factors, angiogenic factors, and matrix metalloproteinases have failed to demonstrate
definitive therapeutic benefits to refractory pancreatic cancer patients. Glycogen synthase
kinase 3β (GSK3β), a serine/threonine protein kinase, has emerged as a therapeutic target in
common chronic and progressive diseases, including cancer. Here we review accumulating
evidence for a pathologic role of GSK3β in promoting tumor cell survival, proliferation,
invasion, and resistance to chemotherapy and radiation in pancreatic cancer. We also discuss
the putative involvement of GSK3β in mediating metabolic disorder, local inflammation, and
molecular alteration leading to pancreatic cancer development. Taken together, we highlight
potential therapeutic as well as preventive effects of GSK3β inhibition in pancreatic cancer.
- 49 -
金沢大学がん進展制御研究所 腫瘍制御（旧：遺伝子診断）研究分野
Minamoto T, Kotake M, Nakada M, Shimasaki T, Motoo Y, Kawakami K.
Distinct pathologic role for glycogen synthase kinase 3β in colorectal
cancer progression. In: Colorectal Cancer Biology - From Genes to
Tumor, Rajunor Ettarh (Ed.), ISBN: 978-953-51-0062-1, pp. 107-34,
2012; InTech.
Colorectal cancer (CRC) is the third most frequent cancer type and the second leading
cause of cancer-related deaths worldwide (Cunningham et al., 2010; Jemal et al., 2010). This
is despite the recent trend of stabilizing or declining rates for CRC incidence and mortality in
economically developed countries (Center et al., 2009; Edwards et al., 2010; Umar &
Greenwald, 2009). Surgical intervention is the initial treatment for most CRC patients.
Continuous efforts to optimize surgery for patients with localized CRC has resulted in
markedly improved 5-year and 10-year survival rates (Cunningham et al., 2010; Wu &
Fazio, 2000). Given the large number of CRC patients who undergo curative surgery, there is
now a substantial number who are susceptible to recurrent or metastatic tumors and could
therefore benefit from additional systemic therapies. An increasing array of options and
protocols for chemotherapies and biologically targeted therapies is now available for use in
the adjuvant setting and for the treatment of recurrent and metastatic CRC.
Based on a more detailed knowledge of the molecular characteristics of CRC (Markowitz
et al., 2009; Walther et al., 2009), biologically-based therapeutics have been developed for
the treatment of advanced stage CRC patients. Currently approved agents for the treatment of
advanced and metastatic CRC include therapeutic monoclonal antibodies that target vascular
endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR). Despite a
substantial biological rationale for the use of these new classes of therapeutic agents, largescale clinical trials have observed only incremental clinical benefits for overall patient
populations. Clearly, not all patients with recurrent and metastatic CRC benefit from these
therapies. This is due to inherent and acquired resistance of tumors to the chemotherapeutic
and biologically-based agents. Moreover, there are few reliable markers for predicting the
therapeutic and adverse effects of these agents and that would allow patients who benefit
from these systemic treatments to be identified. Therefore, new therapeutic targets are
urgently required to further improve the survival of patients with recurrent and metastatic
CRC. One such target may be glycogen synthase kinase 3β (GSK3β), a serine/threonine
protein kinase that has recently been implicated in various human cancers.
In this Chapter, we briefly summarize the scientific basis and current status of systemic
treatments for CRC, including combinations of surgery, chemotherapy and molecular targetdirected therapy. Based on our published and ongoing studies, we then focus on GSK3β as
an emerging therapeutic target in CRC and other cancer types. We describe the underlying
biological mechanism that allows exploration of a novel therapeutic strategy for CRC
involving the targeting of aberrant GSK3β.
- 50 -
J Gastroenterol (2012) 47:321–333
DOI 10.1007/s00535-011-0484-9
ORIGINAL ARTICLE—LIVER, PANCREAS, AND BILIARY TRACT
Glycogen synthase kinase 3b inhibition sensitizes pancreatic
cancer cells to gemcitabine
Takeo Shimasaki • Yasuhito Ishigaki • Yuka Nakamura • Takanobu Takata • Naoki Nakaya •
Hideo Nakajima • Itaru Sato • Xia Zhao • Ayako Kitano • Kazuyuki Kawakami • Takuji Tanaka
Tsutomu Takegami • Naohisa Tomosugi • Toshinari Minamoto • Yoshiharu Motoo
•
Received: 1 March 2011 / Accepted: 16 September 2011 / Published online: 1 November 2011
Ó Springer 2011
Abstract
Background Pancreatic cancer is obstinate and resistant
to gemcitabine, a standard chemotherapeutic agent for the
disease. We previously showed a therapeutic effect of
glycogen synthase kinase-3b (GSK3b) inhibition against
gastrointestinal cancer and glioblastoma. Here, we investigated the effect of GSK3b inhibition on pancreatic cancer
cell sensitivity to gemcitabine and the underlying molecular mechanism.
Methods Expression, phosphorylation, and activity of
GSK3b in pancreatic cancer cells (PANC-1) were
Electronic supplementary material The online version of this
article (doi:10.1007/s00535-011-0484-9) contains supplementary
material, which is available to authorized users.
T. Shimasaki (&) N. Nakaya H. Nakajima I. Sato Y. Motoo
Department of Medical Oncology, Kanazawa Medical
University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293, Japan
e-mail: takeo@kanazawa-med.ac.jp
T. Shimasaki A. Kitano K. Kawakami T. Minamoto
Division of Translational and Clinical Oncology, Cancer
Research Institute, Kanazawa University, 13-1 Takara-machi,
Kanazawa, Ishikawa 920-0934, Japan
Y. Ishigaki Y. Nakamura T. Takata T. Takegami N. Tomosugi
Medical Research Institute, Kanazawa Medical University,
1-1 Daigaku, Uchinada, Ishikawa 920-0293, Japan
examined by Western immunoblotting and in vitro kinase
assay. The combined effect of gemcitabine and a GSK3b
inhibitor (AR-A014418) against PANC-1 cells was examined by isobologram and PANC-1 xenografts in mice.
Changes in gene expression in PANC-1 cells following
GSK3b inhibition were studied by cDNA microarray and
reverse transcription (RT)-PCR.
Results PANC-1 cells showed increased GSK3b expression, phosphorylation at tyrosine 216 (active form), and
activity compared with non-neoplastic HEK293 cells.
Administration of AR-A014418 at pharmacological doses
attenuated proliferation of PANC-1 cells and xenografts,
and significantly sensitized them to gemcitabine. Isobologram analysis determined that the combined effect was
synergistic. DNA microarray analysis detected GSK3b
inhibition-associated changes in gene expression in
gemcitabine-treated PANC-1 cells. Among these changes,
RT-PCR and Western blotting showed that expression of
tumor protein 53-induced nuclear protein 1, a gene regulating cell death and DNA repair, was increased by gemcitabine treatment and substantially decreased by GSK3b
inhibition.
Conclusions The results indicate that GSK3b inhibition
sensitizes pancreatic cancer cells to gemcitabine with
altered expression of genes involved in DNA repair. This
study provides insight into the molecular mechanism of
gemcitabine resistance and thus a new strategy for pancreatic cancer chemotherapy.
X. Zhao
Institute of Pathology, Tongji Hospital, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan,
People’s Republic of China
Keywords Pancreatic cancer Gemcitabine Glycogen synthase kinase 3b TP53INP1
T. Tanaka
Department of Pathology, Kanazawa Medical University,
1-1 Daigaku, Uchinada, Ishikawa 920-0293, Japan
Abbreviations
ABC
Avidin–biotin peroxidase complex
ATP
Adenosine triphosphate
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BSA
cDNA
cRNA
DMSO
EGFR
FBS
GSK3b
IC50
MAPK
mTOR
NF-jB
NRIKA
p53DINP1
PBS
PCNA
PDGF
PI3K
RNAi
RR
RT-PCR
SD(s)
SIP
siRNA
TEAP
TK
TP53INP1
TS
TUNEL
VEGF
VEGFR
WST-8
J Gastroenterol (2012) 47:321–333
Bovine serum albumin
Complementary DNA
Complementary RNA
Dimethyl sulfoxide
Epidermal growth factor receptor
Fetal bovine serum
Glycogen synthase kinase 3b
50% inhibitory concentration
Mitogen-activated protein kinase
Mammalian target of rapamycin
Nuclear factor-jB
Non-radioisotopic in vitro kinase assay
p53-dependent damage-inducible nuclear
protein 1
Phosphate buffered saline
Proliferating cell nuclear antigen
Platelet-derived growth factor receptor
Phosphatidylinositol 3-kinase
RNA interference
Ribonucleotide reductase
Reverse transcription-polymerase chain
reaction
Standard deviation(s)
Stress-induced protein
Small interfering RNA
Thymus-expressed acidic protein
Thymidine kinase
Tumor protein 53-induced nuclear protein 1
Thymidylate synthase
Terminal deoxynucleotidyl transferasemediated dUTP nick end labeling
Vascular endothelial growth factor
VEGF receptor
4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5tetrazolio]-1,3-benzene disulfonate
Introduction
Pancreatic cancer is the fourth and fifth leading cause of
cancer death in the USA and Japan, respectively [1–4].
Early diagnosis is difficult and metastasis is frequently
found during primary tumor diagnosis. Biologically, this
tumor is characterized by highly proliferative and invasive
activity, and its aggressive nature is an obstacle to early
diagnosis and curative surgical intervention [5, 6]. Gemcitabine is a deoxycytidine analog widely used as a standard anticancer drug in the treatment of pancreatic cancer,
but it is effective in less than 20% of patients [3, 4, 7].
Radiation therapy alone or in combination with gemcitabine is insufficient for improving survival of pancreatic
cancer patients [5, 6]. For this reason, there is an urgent
need for new treatment modalities for pancreatic cancer,
such as molecular-target directed therapies.
Accumulating molecular studies have elucidated a
complex genetic mechanism of cancer that involves multidirectional signal transduction pathways [8]. The major
axis of signal transduction includes the RAS/mitogenactivated protein kinase (MAPK), phosphatidylinositol
3-kinase (PI3K)/Akt/mammalian target of rapamycin
(mTOR), and hedgehog pathways, and plays an important
role in the development and progression of pancreatic
cancer [9]. Tyrosine kinases such as the epidermal growth
factor receptor (EGFR), vascular endothelial growth factor
receptor (VEGFR), and platelet-derived growth factor
receptor (PDGFR) are targeted in cancer treatment because
they are overexpressed in many tumors, including pancreatic cancer [10]. Currently available agents targeting
these molecules are anti-EGFR antibodies (cetuximab,
panitumumab), small-molecule EGFR inhibitors (gefitinib,
erlotinib), an anti-VEGF antibody (bevacizumab), and a
small-molecule VEGFR inhibitor (axitinib). A number of
phase III clinical trials have been conducted using either
kinase inhibitors alone or in combination with gemcitabine
for treating pancreatic cancer patients. Other than a combination of erlotinib and gemcitabine [11], these trials have
shown little therapeutic benefit to the patients enrolled
(reviewed in Ref. [12]). Therefore, identification of new
molecular target(s) is necessary for developing a therapeutic strategy that enhances the effect of gemcitabine and
thus improves patient survival.
Among serine/threonine protein kinases, glycogen synthase kinase 3b (GSK3b) has emerged as a clue to
understanding the molecular mechanism of chronic, progressive diseases, and thus as a potential therapeutic target
[13]. On the basis of its known biological properties and
functions [14–16] as well as its involvement in primary
pathologies [13], GSK3b has been a target of drug development for non-insulin-dependent diabetes mellitus, Alzheimer’s disease, osteoporosis, and inflammation [13, 17,
18]. We recently demonstrated that deregulated GSK3b
expression and activity are characteristic of gastrointestinal
cancer (including pancreatic cancer) and glioblastoma, and
that GSK3b maintains survival and proliferation of these
tumor cells. The pathologic role of GSK3b in cancer is
supported by our findings that inhibition of this kinase
attenuates survival and proliferation, and induces apoptosis
of the tumor cells and their xenografts [19–22]. The antitumor effect of GSK3b inhibition is associated with
(re)activation of p53- and Rb-mediated pathways and
attenuated cell immortality activity [21, 22].
Concurrent with and following our studies on the antitumor effects of GSK3b inhibition, similar observations
were reported in various cancer types (reviewed in
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323
Ref. [23]). Based on studies indicating possible involvement of GSK3b in the nuclear factor-jB (NF-jB)-mediated cell survival pathway [24, 25], a number of studies
have shown that GSK3b participates in pancreatic cancer
cell survival via the NF-jB pathway [26–28]. Few studies
had focused on whether GSK3b inhibition sensitizes cancer
cells to chemotherapeutic agents until we found that
GSK3b inhibition sensitizes glioblastoma cells to chemotherapeutic agents (e.g., temozolomide, ACNU) and ionizing radiation [23]. In this study, we investigated the effect
of GSK3b inhibition on pancreatic cancer cell sensitivity to
gemcitabine and the underlying molecular mechanism.
Materials and methods
Cell lines
Human pancreatic cancer cells (PANC-1) and human
embryonic kidney cells (HEK293) were obtained from
American Type Culture Collection (Manassas, VA, USA).
Since HEK293 cells stop growing due to contact inhibition,
we considered these to be the non-neoplastic cell line in our
experiments. Cells were cultured in Dulbecco’s Modified
Eagle’s Medium containing 10% fetal bovine serum (FBS;
GIBCO, Carlsbad, CA, USA) at 37°C under 5% CO2 and
harvested for extraction of cellular protein and nucleic acids.
Expression and phosphorylation of cellular GSK3b
We extracted cellular protein from a frozen pellet of cells
using lysis buffer (CelLyticTM-MT, Sigma-Aldrich, St. Louis,
MO, USA) containing a mixture of protease and phosphatase
inhibitors (both from Sigma-Aldrich). A 50-lg aliquot of
protein extract was subjected to Western immunoblotting
analysis as described previously [19, 29] to examine expression and phosphorylation of GSK3b using primary antibodies
(at the dilution shown) against total GSK3 (GSK3a and b;
1:1,000; Upstate Biotechnology, Lake Placid, NY, USA),
GSK3b (1:2,500; BD Biosciences, Lexington, KY, USA),
GSK3b phosphorylated at serine 9 (pGSK3bS9:1,000; Cell
Signaling Technology, Beverly, MA, USA) and at tyrosine
216 (pGSK3bY216; 1:1,000; BD Biosciences). In each case,
the signal was developed using an enhanced chemiluminescent detection reagent (ECLÒ, Amersham, Little Chalfont,
UK). The amount of protein in each sample was monitored by
expression of b-actin (1:1,000; Ambion, Woodward-Austin,
TX, USA).
Non-radioisotopic in vitro kinase assay
We detected cellular GSK3b activity using the non-radioisotopic in vitro kinase assay (NRIKA) according to the
method in our previous report [30]. GSK3b was isolated
from 1-mg aliquots of each sample cell lysate by immunoprecipitation, and then used in the in vitro kinase reaction in the presence of its substrate, recombinant human
b-catenin protein (b-cateninHis), and non-radioisotopic
adenosine triphosphate (ATP). We analyzed the products
by Western immunoblotting for phosphorylated b-cateninHis using an antibody to b-catenin phosphorylated at
serine 33 and 37 and/or threonine 41 (p-b-cateninS33/37/T41;
Cell Signaling Technology). As a negative control (NC) for
each cell line, the mouse monoclonal antibody to GSK3b
was replaced by an equal amount of non-immune mouse
IgG in the immunoprecipitation step. Cellular GSK3b
activity is demonstrated by p-b-cateninS33/37/T41 expression
in the test lanes (T), and little or no expression of p-bcateninS33/37/T41 in the NC. The amount of GSK3b and the
presence of b-cateninHis in the kinase reaction were monitored by immunoblotting with mouse monoclonal antibodies to GSK3b and b-catenin (1:1,000; BD Biosciences),
respectively.
Effects of gemcitabine and GSK3b inhibition on cancer
cell survival
Cells seeded in 96-well culture plates at a density of
1 9 104 cells/mL were treated with dimethyl sulfoxide
(DMSO), gemcitabine (generously provided by Eli Lilly
Co., Ltd., Tokyo, Japan), or a small-molecule GSK3b
inhibitor (AR-A014418; CalbiochemÒ, EMD Bioscience,
San Diego, CA, USA) dissolved in DMSO at the indicated
final concentrations in the medium. Concentrations of
AR-A014418 used in this study (10 and 25 lM) are within
the pharmacologically relevant range previously reported
[31]. We tested each concentration or combination of
agents in four wells, and performed the experiments three
times. At designated time points, relative numbers of viable
cells were determined using a WST-8 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene
disulfonate) assay kit (Wako, Japan) and a spectrophotometer (ELx800; BioTek Instruments, Winooski, VT,
USA). We used these numbers to calculate 50% inhibitory
concentrations (IC50) of gemcitabine and AR-A014418,
and to generate an isobologram that allows evaluation of
whether the effect of the two drugs in combination is
additive, synergistic, or antagonistic [32].
RNA interference for GSK3b and tumor protein
53-induced nuclear protein 1 (TP53INP1)
The effect of depletion of GSK3b or TP53INP1 on PANC-1
cell viability and sensitivity to gemcitabine was examined
by RNA interference (RNAi) using the small interfering
RNA (siRNA) specific to GSK3b (Validated Stealth RNAi,
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J Gastroenterol (2012) 47:321–333
OligoID VHS40271, Invitrogen, Carlsbad, CA, USA) or
TP53INP1 (Oligo ID HSS150786, Invitrogen). Specific
effects of RNAi on expression of mRNA and protein of
these molecules were confirmed respectively by quantitative reverse transcription (RT)-PCR (data not shown) and
Western immunoblotting by using antibodies to total GSK3
(GSK3a and b; 1:1,000; Upstate Biotechnology) and
TP53INP1 (1:1,000; abcam, Cambridge, MA, USA).
PANC-1 cells were treated by transfection with either
GSK3b-specific (10 nM) or TP53INP1-specific siRNA
(10 nM) alone, or in combination with gemcitabine (50 mg/
mL, 190 lM). As controls, the cells were treated with PBS
and the nonspecific siRNA (Stealth RNAiTM siRNA Negative Control Med GC [12935-300], Invitrogen). At 48 h
after the respective treatment, relative numbers of viable
cells were measured by a WST-8 assay. Each assay was
done in four wells three times.
Experimental animal study
Pathogen-free 6-week-old female athymic nude mice
(BALB/c) were supplied by JAPAN SLC (Hamamatsu,
Japan). After quarantine for 2–3 weeks in pathogen-free
conditions at the Animal Experiment Facility in the
Advanced Science Research Center of the Kanazawa
Medical University, these mice were inoculated with
PANC-1 cells and subsequently treated according to the
design and protocol shown in Fig. 1a. Four weeks after
inoculation, 32 mice with PANC-1 xenografts were
assigned to four treatment groups. These groups were given
intraperitoneal injections of 200 lL 75% DMSO, gemcitabine (20 mg/kg body weight), AR-A014418 (2 mg/kg
body weight), or a mixture of both agents dissolved in
200 lL DMSO, respectively, two times a week for
8 weeks. Doses of AR-A014418 and gemcitabine corresponded to concentrations of 10 and 120 lM, respectively,
in culture media used in the in vitro treatment of cells
[20, 22]. Our pilot study using SW480 and HT29 colon
cancer cell xenografts [20, 22] estimated the AR-A014418
dose corresponding to a concentration used previously in
the in vitro treatment of cells [19, 21, 22]. Assuming that
total body fluid accounts for approximately 60% of a
mouse’s body weight, an inhibitor dose of 2 mg/kg body
weight corresponds to a concentration of approximately
10 lM in culture media [20], which is known to be a
pharmacological dose for AR-A014418 [31]. Throughout
the experiment, we carefully observed all mice daily for
adverse events and measured tumors every week. Tumor
volume (cm3) was calculated using the formula
0.5 9 S2 9 L, where S is the smallest tumor diameter (cm)
and L is the largest (cm) [20]. Body weights of animals
were monitored during the treatment (Fig. 1b). All mice
were euthanized at 12 weeks after completion of treatment.
At necropsy, tumor xenografts and the major vital organs
(lungs, liver, gastrointestinal tract, pancreas, kidneys, and
spleen) were removed, fixed in 10% neutral buffered formalin and embedded in paraffin for histopathologic, histochemical, and immunohistochemical examination.
Paraffin sections of these tumors were stained with hematoxylin and eosin (HE) for histopathologic examination.
All experiments were conducted according to the Guidelines
for the Care and Use of Laboratory Animals in Kanazawa
Medical University, and in accordance with national guidelines for animal use in research in Japan (http://www.
lifescience.mext.go.jp/policies/pdf/an_material011.pdf).
Immunohistochemical and histochemical examinations
We examined the expression of proliferating cell nuclear
antigen (PCNA) in tumor xenografts from the mice by
using a rabbit polyclonal antibody to PCNA (1:100; clone
ID SC-7907, Santa Cruz Biotechnology, Santa Cruz, CA,
USA) detected by the avidin–biotin–peroxidase complex
(ABC) method, with modifications [20, 29]. For the negative control, the primary antibody was replaced by nonimmune rabbit IgG (DakoCytomation, Glostrup, Denmark). Apoptosis was detected in tumor xenografts by
terminal deoxynucleotidyl transferase-mediated dUTP nick
end labeling (TUNEL) using an in situ apoptosis detection
TUNEL kit (Takara, Kusatsu, Japan). Frequency of proliferating cells and of apoptosis in the tumors was calculated as described previously [20]. Six optical fields,
approximately 500–700 cells, were counted in each group
by light microscopy under high-power magnification
(4009). The number of positive cells was calculated per
500 cells.
cDNA microarray analysis
We treated PANC-1 cancer cells with gemcitabine
(190 lM) and AR-A014418 (10 lM) alone or in combination, or with PBS as a control for 24, 48, and 72 h. Total
RNA was isolated from these cells using the RNeasy Mini
Kit (QIAGEN GmbH, Hilden, Germany). Labeled cRNA
was synthesized from sample RNA using a MessageAmpÒ
II-Biotin Enhanced Kit (Ambion) according to the manufacturer’s instructions. Target hybridizations were performed on a Human Genome U133 plus 2.0 GeneChip
microarray system (Affymetrix, High Wycombe, UK) in a
GeneChipÒ Hybridization Oven 640 for 16 h at 45°C. The
hybridized cRNAs were washed and stained in a GeneChipÒ Fluidics Station 450, and the resulting signal was
detected using a GeneChipÒ Scanner 3000. Digitalized
image data were processed using the GeneChipÒ Operating
Software (GCOS) version 1.4. Amounts of probe-specific
transcripts were determined on the basis of the average of
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325
a
DMSO
(n=8)
gemcitabine
20 mg/kg
(n=8)
AR-A014418
2 mg/kg
(n=8)
Gemcitabine (n=8)
+AR-A014418
2
4
6
8
10
12
2
4
6
8
10
12
2
4
6
8
10
12
2
4
6
8
10
12
Weeks
b
30
Body weight (gr)
Inoculation of
1 x 106 PANC-1 cells/nude mouse
25
20
0
1
2
3
4
euthanasia
& autopsy
Intraperitoneal injection
5
6
7
8
9
10
11
12
Week after inoculation
Fig. 1 Design and protocol of animal experiments, and changes in
body weight of animals during treatment. a Pathogen-free 6-week-old
female athymic mice (BALB/c, nu/nu) were quarantined for 3 weeks
in pathogen-free conditions. At week 0, 1 9 106 PANC-1 human
pancreatic cancer cells suspended in 50 lL of phosphate buffered
saline (PBS) were inoculated subcutaneously into each of 32 mice.
Four weeks after inoculation, visible subcutaneous tumors formed in
all mice and were size-matched. Mice were randomly assigned to four
treatment groups: DMSO (a solvent for the GSK3b inhibitor),
gemcitabine, the small-molecule GSK3b inhibitor AR-A014418, and
a combination of gemcitabine and the inhibitor, as described in
‘‘Materials and methods’’. b Effects of intraperitoneal injection of the
agents on body weights of mice during the course of treatment.
Results are shown for the groups of mice treated with the agents
described in a. DMSO, closed circle; gemcitabine, open square; ARA014418, open circle; gemcitabine and AR-A014418 in combination,
open triangle
the differences between the perfect-match and mismatch
intensities. Signal intensities of selected genes that were
upregulated or downregulated fourfold compared with the
control group were extracted by the GeneSpring GX software package version 7.3.1 (Agilent Technologies, Santa
Clara, CA, USA).
Gene Expression assays (assay ID: Hs01374570_m1,
Applied Biosystems). Human ACTB (P/N: 4333762T,
Applied Biosystems) was used as an endogenous control.
Relative quantitation was carried out using the DDCt method
with human ACTB as an endogenous control.
Statistical analysis
RNA purification and quantitative real-time RT-PCR
Total RNA was extracted using the miRNeasy Kit (Qiagen)
following the manufacturer’s instructions. Reverse transcription of RNA to cDNA was carried out using the Taqman
Reverse Transcription Reagents Kit (Applied Biosystems).
Expression levels of TP53INP1 and b-actin (ACTB) were
quantified using Taqman FAM/MGB probes on an ABI
7900HT Fast Real-Time PCR System (Applied Biosystems).
TP53INP1 PCR products were detected using the Taqman
The Student’s t test was used to determine statistical differences in cell survival between cells treated with DMSO,
AR-A014418, and gemcitabine. In the microarray analysis,
we used Fisher’s exact test to calculate p values to determine the probabilities that the biological functions assigned
to the different networks could be explained by chance
alone. Body weights of mice and tumor volumes in each
treatment group were expressed as mean ± standard
deviation (SD). The statistical significance of differences
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among the data was determined with one-way ANOVA
followed by Fisher’s PLSD post hoc test.
a
- pGSK3βS9
Results
- pGSK3βY216
Expression, phosphorylation, and activity of GSK3b
in cancer cells
- total GSK3β
- β-Actin
Western immunoblotting showed that PANC-1 cells had
higher basal levels of GSK3b and pGSK3bY216 (active
form), and a lower level of pGSK3bS9 (inactive form) than
HEK293 cells (Fig. 2a). PANC-1 cells also showed a
higher level of GSK3b expression than HEK293 cells in
the immunoprecipitated material, and the NRIKA showed
that PANC-1 cell-derived GSK3b was active for phosphorylating b-catenin (Fig. 2b). These results are consistent with our previous findings in gastrointestinal cancer
cells and tissues [19, 22]. The above characteristics of
GSK3b suggested it plays a pathologic role in pancreatic
cancer cells. Therefore, we investigated possible effects of
inhibiting GSK3b activity on pancreatic cancer cell viability and gemcitabine sensitivity.
Effect of gemcitabine and a GSK3b inhibitor on cancer
cell survival
Treatment with increasing concentrations of either gemcitabine (38, 190, and 380 lM corresponding to 10, 50, and
100 mg/mL, respectively) or AR-A014418 (10 and 25 lM)
suppressed viability of PANC-1 cells in a dose- and timedependent manner (Fig. 3). The IC50 of gemcitabine and
AR-A014418 was 225 and 12 lM, respectively, at 48 h
after treatment. We then addressed whether GSK3b inhibition could enhance the effect of gemcitabine, presently
recognized as the standard treatment for pancreatic cancer.
As shown in Fig. 3, AR-A014418 alone at a dose of
25 lM had a therapeutic effect against PANC-1 cell survival that was equivalent to or higher than that of gemcitabine alone at a dose of 380 lM (100 mg/mL). Then, we
tested for possible effects of 10 lM AR-A014418, a dose
similar to its IC50, in combination with gemcitabine at
different concentrations. We observed significantly
reduced cell viability at 72 and/or 48 h when PANC-1 cells
were treated with a combination of 10 lM AR-A014418
and gemcitabine at a dose of 38 lM (10 mg/L), 190 lM
(50 mg/L), or 380 lM (100 mg/L) compared with treatment with gemcitabine or 10 lM AR-A014418 alone
(Fig. 4a–c). Isobologram analysis [32] was used to evaluate
whether 10 lM AR-A014418 enhances the effect of
gemcitabine against PANC-1 cells at 48 h after treatment.
Analysis determined that the combined effect of 10 lM
AR-A014418 and 190 lM (50 mg/L) gemcitabine was
b
NC T NC T
- pβ-cateninS33/37/T41
- IgG
- GSK3β
- β-cateninHis
Fig. 2 Expression, phosphorylation, and activity of GSK3b in the two
cell lines. a Protein extracts from HEK293 and PANC-1 cells were
analyzed by Western immunoblotting for GSK3b expression and
levels of pGSK3bS9, pGSK3bY216, and b-actin. b GSK3b activity was
detected by NRIKA [30] in HEK293 and PANC-1 cells. An in vitro
kinase reaction was carried out in the presence of immunoprecipitated
GSK3b from cell lysate, b-cateninHis, and non-radioisotopic ATP. The
resultant products were analyzed by Western immunoblotting for
phosphorylation of b-cateninHis (p-b-cateninS33/37/T41). As a negative
control (NC) for each cell line, the antibody to GSK3b was replaced by
non-immune mouse IgG in the immunoprecipitation. GSK3b activity
was shown in the cells by presence of p-b-cateninS33/37/T41 in the test
lanes (T) and by little or no pb-cateninS33/37/T41 in NC lanes.
The amount of immunoprecipitated GSK3b and the presence of
b-cateninHis were evaluated by immunoblotting with the respective
specific antibodies
synergistic (Fig. 4d). In addition, the combination of
gemcitabine (50 mg/L, 190 lM) with GSK3b RNAi yielded a lower rate of PANC-1 cell survival than either
treatment alone (Fig. 5).
Effects of gemcitabine and GSK3b inhibitor
on PANC-1 xenografts
The four groups of mice with PANC-1 xenografts were
treated with intraperitoneal injections of DMSO, gemcitabine (20 mg/kg), AR-A014418 (2 mg/kg), and a mixture of
the latter two agents, respectively, twice a week for
8 weeks (Figs. 1a, 6a). All mice tolerated the respective
agents well for the 8-week treatment. Beginning 4 weeks
into treatment (from week 8 in Fig. 6a), mice treated with
AR-A014418 alone and in combination with gemcitabine
showed significantly decreased tumor proliferation. During
weeks 4–8 of treatment (weeks 8–12 in Fig. 6a), Fischer’s
PLSD post hoc test showed significant differences in tumor
volume between the four groups of mice. Compared with
mice treated with DMSO, significant antitumor effects
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327
increase in TUNEL-positive (apoptotic) cells and a
decrease in PCNA-positive (proliferating) cells in tumors
treated with gemcitabine, AR-A014418, and their combination (Fig. 6b, c).
a
gemcitabine
1.2
Optical density
1.0
0.8
Changes in gene expression in PANC-1 cells treated
with gemcitabine and a GSK3b inhibitor
*
0.6
*
0.4
0.2
0
b
1.2
AR-A014418
Optical density
1.0
*
0.8
0.6
0.4
*
0.2
0
0
24
48
72
Hours after treatment
Fig. 3 Effects of gemcitabine and AR-A014418 on PANC-1 cell
survival. a PANC-1 cells treated with PBS (closed circle) and
gemcitabine at 10 mg/mL (38 lM; open circle), 50 mg/mL (190 lM;
open triangle), and 100 mg/mL (380 lM; open square). b PANC-1
cells treated with DMSO (closed circle) and AR-A014418 at 10 lM
(open circle) and 25 lM (open triangle). a, b The relative number of
viable cells at each time point was measured by WST-8 assay. Each
assay was done in six wells and performed three times. All values
shown are means with SDs. Asterisks indicate statistically significant
differences (p \ 0.01) between cells treated with PBS and gemcitabine, and between cells treated with DMSO and AR-A014418
were found in mice treated with the following agents in
order of decreasing effect: a combination of the two agents,
AR-A014418 alone, and gemcitabine alone.
We observed no serious adverse events in the four
groups of mice during treatment, and there were no statistically significant differences in mean body weight
between the groups (Fig. 1b). At necropsy, gross observation and histological examination showed no lesions,
primary tumors, or metastatic PANC-1 tumors in the major
vital organs of any of the mice.
Histological examination of the tumors showed medullary proliferation of cancer cells in all cases (Fig. 6b).
Histochemical (TUNEL) and immunohistochemical
(PCNA) examinations of the tumor xenografts showed an
To find a molecular mechanism by which GSK3b inhibition enhances the antitumor effect of gemcitabine, we
generated and compared gene expression profiles of
PANC-1 cells sham (PBS)-treated and those treated with
gemcitabine alone (Electronic Supplementary Material: a
[24 h], b [48 h], c [72 h]) and in combination with
AR-A014418 for 72 h (Electronic Supplementary Material:
d [72 h]). Transcriptome comparison by cDNA microarray
analysis showed significant changes in expression of
numerous genes in gemcitabine-treated PANC-1 cells
compared with sham-treated cells. Interestingly, the degree
of change in the transcriptome profile of cells treated with a
combination of gemcitabine and AR-A014418 was less
marked than that of cells treated with gemcitabine alone
for 72 h (Fig. 7a). This suggests that treatment with
AR-A014418 counteracts gemcitabine-induced changes in
gene expression in PANC-1 cells, and allows us to
hypothesize that the gemcitabine-induced genes are associated with the cancer cells’ resistance to this drug.
We detected three groups of genes. Group A included
84 genes that were upregulated more than fourfold at 24,
48, or 72 h after treatment with gemcitabine and downregulated less than onefold at 72 h after treatment with
gemcitabine in combination with AR-A014418. Group B
included 267 genes downregulated less than onefold with
gemcitabine treatment and upregulated more than fourfold
in combination with AR-A014418 at the same time points.
Group C included 112 genes upregulated one- to fourfold
with gemcitabine treatment and more than eightfold in
combination with AR-A014418 at the same time points
(Table 1, Supplementary Table 1). The 10 genes in each
group with the most marked change in expression are
shown in Table 1. With respect to gene ontology, many of
these genes function in cell cycle regulation, growth,
death, and cell signaling. Group A genes are likely associated with or responsible for increased sensitivity
of PANC-1 cells to gemcitabine in combination with
AR-A014418.
Among the representative genes in group A, we focused
on tumor protein p53-induced nuclear protein 1
(TP53INP1), since our previous study demonstrated an
increase in TP53INP1 protein in pancreatic cancer cells
that have acquired resistance to gemcitabine [33]. Quantitative RT-PCR analysis showed that, in comparison with
sham-treated PANC-1 cells, TP53INP1 mRNA expression
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Optical density
1.2
b
gemcitabine 10 mg/mL
+ AR-A014418
1.0
*
*
0.8
0.6
gemcitabine 50 mg/mL
+ AR-A014418
1.2
Optical density
a
0.4
0.2
1.0
*
*
**
0.8
0.6
0.4
0.2
0
0
0
24
48
0
72
Hours after treatment
Optical density
1.2
1.0
*
0.8
0.6
*
*
0.4
0.2
48
72
d
gemcitabine 100 mg/mL
+ AR-A014418
Gemcitabine ( μM)
c
24
Hours after treatment
800
600
400
200
0
0
24
48
72
0
Hours after treatment
5
10
15
20
AR-A014418 (μM)
Fig. 4 Combined effect of gemcitabine and low-dose AR-A014418
on pancreatic cancer cells (PANC-1). a–c The relative number
of viable cells at each time point was measured by WST-8 assay
for PANC-1 cells treated with DMSO (closed circle), 10 lM
AR-A014418 (open circle), gemcitabine at the indicated concentrations alone (open square) and in combination with 10 lM
AR-A014418 (open triangle). Each assay was done in six wells and
performed three times. All values shown are mean with SDs. Asterisks
indicate statistically significant differences (*p \ 0.01, **p \ 0.001)
between cells treated with DMSO and either gemcitabine, ARA014418 or the two agents in combination. d The influence of 10 lM
AR-A014418 on the effect of gemcitabine against pancreatic cancer
cells was analyzed using an isobologram [32] where the IC50 of the
combination therapy was plotted (filled triangle). The analysis
showed that 10 lM AR-A014418 (closed square) synergistically
enhanced the effect of gemcitabine against PANC-1 cells
increased in cells treated with gemcitabine alone and
decreased in cells treated with gemcitabine in combination
with AR-A014418 (Fig. 7b). This result suggests a putative
role for TP53INP1 in the GSK3b inhibition-mediated
enhancement of gemcitabine’s effect against pancreatic
cancer cells. Consistent with this observation, depletion of
TP53INP1 by RNAi (Fig. 7c) significantly enhanced the
effect of gemcitabine (50 mg/mL, 190 lM) in PANC-1
cell survival (Fig. 7d).
gemcitabine [10, 12]. Recent studies have found that
GSK3b inhibition exerts therapeutic effects against various cancers, including pancreatic cancer, with little
adverse effect (reviewed in Ref. [23]). Among them, only
our study showed that inhibition of GSK3b sensitizes
human glioblastoma cells to temozolomide and ionizing
radiation in vitro [21]. In the present study, we demonstrated both in vitro (cell culture) and in vivo (tumor
xenografts) that inhibition of deregulated GSK3b activity
by its pharmacological inhibitor AR-A014418 not only
attenuates survival and proliferation of pancreatic cancer
cells, but also sensitizes them to gemcitabine. Consistent
with our previous studies on the effects of GSK3b inhibition on non-neoplastic cells and rodents [19–22], we
observed no detrimental effects of treating PANC-1
xenograft-bearing athymic mice with a pharmacological
dose of AR-A014418 for 8 weeks. These results indicate
that GSK3b inhibition combined with chemotherapy is a
novel and promising strategy for sensitizing pancreatic
cancer cells to gemcitabine.
Discussion
While gemcitabine remains a standard chemotherapeutic
agent for treating pancreatic cancer, the development of
strategies for enhancing its antitumor effect is an urgent
and important issue for improving response rate. Several
trials using agents targeting oncogenic signaling pathways mediated by receptor-type tyrosine kinases have
failed to improve the experimental or clinical effect of
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J Gastroenterol (2012) 47:321–333
1.2
***
1.0
Optical density
329
**
0.8
0.6
*
0.4
0.2
0.0
C
a
8
b
Tumor Volume (cm3 )
Fig. 5 Effect of gemcitabine, GSK3b-specific siRNA, and the two
agents in combination in PANC-1 cells. Cells were treated with
gemcitabine (50 mg/mL, 190 lM), GSK3b-specific siRNA (final
concentration, 10 nM), and the two agents in combination. As a
control, cells were treated with PBS and non-specific siRNA (10 nM).
The relative number of viable cells at 48 h after each treatment was
measured by the WST-8 assay. Each assay was done in four wells
three times. Data are presented as mean ± SD. Asterisks indicate
statistically significant differences (*p \ 0.05; **p \ 0.01;
***p \ 0.001) between cells treated with control (C; PBS and nonspecific siRNA), gemcitabine (GEM), and GSK3b-specific siRNA
(siRNA), and the two agents in combination (GEM ? siRNA)
There have been many studies investigating molecular
and biological mechanisms by which pancreatic cancer
cells resist or acquire resistance to chemotherapy and
radiation [34–36]. These studies found no decisively efficient means for overcoming pancreatic cancer cell resistance to gemcitabine and radiation. It was previously
reported that GSK3b sustains pancreatic cancer cell
survival by maintaining transcriptional activity of NF-jB
[26, 27]. However, a recent study failed to demonstrate that
disruption of NF-jB activity by inhibiting GSK3b sensitizes PANC-1 cells to gemcitabine [28]. We previously
found no effect of GSK3b inhibition on endogenous
NF-jB transcriptional activity in gastrointestinal cancers
(including pancreatic cancer) and glioblastoma [21, 22].
Therefore, a role for GSK3b in regulating NF-jB activity
is controversial.
We observed in a preliminary study that short-term
gemcitabine treatment induces mesenchymal cell-like
phenotypic change in pancreatic cancer cells. This change
in phenotype is responsible for resistance of cancer cells to
conventional cancer treatment [37, 38]. Our unpublished
observation allowed us to hypothesize that molecules
induced by gemcitabine treatment would render cancer
cells resistant to the drug. Using cDNA microarray
7
control
HE
PCNA
TUNEL
6
5
4
GEM
3
2
1
AR
0
0
1
2
3
4
5
6
7
8
9 10 11 12
Week after inoculation
% positive cells
c
50
40
*
*** ***
TUNEL
30
60
***
50
40
PCNA
**
GEM
+ AR
30
20
20
10
10
0
0
C
C
Fig. 6 Effect of gemcitabine, AR-A014418, and a combination of
both agents against human pancreatic cancer cell xenografts in
athymic mice, as detailed in Fig. 1a. a Time course of the effects of
DMSO (closed circle), gemcitabine (20 mg/kg body weight; open
square), AR-A014418 (2 mg/kg body weight; open circle), and a
combination of the latter two agents (open triangle) on PANC-1
xenografts in mice. b Representative histological findings and
frequency of PCNA- and TUNEL-positive tumor cells in the
PANC-1 xenografts removed from mice at necropsy after 8-week
treatment with the respective agents. c Relative number of PCNAand TUNEL-positive tumor cells in PANC-1 xenografts removed
from mice. Values are expressed as mean ± SDs. *p \ 0.05,
**p \ 0.01, ***p \ 0.001 (the Mann–Whitney U test). b, c C,
control; GEM, gemcitabine; AR, AR-A014418
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J Gastroenterol (2012) 47:321–333
a
Gemcitabine + AR-A014418
After treatment
Gemcitabine
Before treatment
d
2.5
Absorbance ( 450nm)
Relative expression level
b
Before treatment
2.0
1.5
1.0
*
*
0.5
0
24
0
***
**
1.6
1.4
1.2
***
1
0.8
0.6
0.4
0.2
0
C
48
Hours after treatment
c
C
TP53INP1
-
β-actin
-
Fig. 7 a Changes in transcriptome profiles of PANC-1 cells treated
with gemcitabine (190 lM) alone and in combination with ARA014418 (10 lM) for 72 h. The scatter graphs were generated by the
GeneSpring GX software package version 7.3.1. b Quantitative RTPCR analysis of changes in TP53INP1 expression in PANC-1 cells
treated with gemcitabine (190 lM; open square), AR-A014418
(10 lM; open circle), and the two agents in combination (open
triangle) for 24 and 48 h. TP53INP1 expression levels in PANC-1
cells were normalized to levels in DMSO-treated cells at each time
point. *p \ 0.05. c Western blotting analysis of changes in TP35INP1
protein expression in PANC-1 cells treated with control (C; PBS and
non-specific siRNA), gemcitabine (GEM), TP53INP1-specific siRNA
(siRNA), and the two agents in combination (GEM ? siRNA). b-actin
expression was monitored as a loading control in each sample.
d Effect of gemcitabine (50 mg/mL, 190 lM), TP53INP1-specific
siRNA (10 nM), and the two agents in combination on PANC-1 cell
survival. As a control, cells were treated with PBS and non-specific
siRNA (10 nM). The relative number of viable cells after 48 h of
treatment was measured by the WST-8 assay. Each assay was done in
four wells three times. Data are presented as mean ± SD. Asterisks
indicate statistically significant differences (**p \ 0.01; ***p \ 0.001)
between cells treated with control (C, PBS and non-specific siRNA),
gemcitabine (GEM), TP53INP1-specific siRNA (siRNA), and the two
agents in combination (GEM ? siRNA)
analysis, we detected 84 genes that were significantly
upregulated in PANC-1 cells by gemcitabine treatment and
downregulated when treated in combination with a GSK3b
inhibitor. Gene ontology analysis suggested involvement of
these molecules in the pathways mediating cell cycle, cell
proliferation, and cell death. We believe that some of them
may participate in the mechanism by which GSK3b inhibition modifies PANC-1 cell sensitivity to gemcitabine.
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331
Table 1 Representative gene expression changes in PANC-1 cells treated with gemcitabine and a GSK3b inhibitor
Group
GEM ? AR
GEM
Number of genes
A
[4-fold
\1
84 genes
B
\1
[4
267 genes
C
1 \ GEM \ 4
[8
112 genes
Total 463 genes
Top 10 molecules
Fold change (GEM ? AR)
Fold change (GEM)
Affymetrix ID
Group A
GDF11
4.058
0.0578
226234_at
FGD4
7.130
0.0649
230559_x_at
SLC1A4
4.257
0.1420
212810_s_at
FAM129A
5.767
0.1429
217967_s_at
APOBEC3F
4.734
0.1962
214995_s_at
SLFN13
4.465
0.2534
1558217_at
UCN
4.168
0.2518
206072_at
TP53INP1
5.959
0.2631
225912_at
HSPB11
4.311
0.2938
214163_at
C2ORF72
4.061
0.3067
213143_at
Group B
MET
0.9747
44.209
213816_s_at
FNIP1
0.6262
36.832
1559060_a_at
ATP11B
0.3406
36.384
238811_at
ZNF33A
0.5910
26.291
224276_at
MED13
0.6793
25.170
244611_at
MAP4K5
0.7294
24.273
211081_s_at
TMEM87A
0.7342
24.100
223772_s_at
EIF1
0.8787
23.810
228967_at
RBM33
0.7305
20.929
1554096_a_at
BRAF
0.4713
19.362
240463_at
APC
1.173
92.673
216933_x_at
ACDCP1
1.185
83.170
1554110_at
ATG7
1.094
48.946
1569827_at
MPHOSPH6
1.881
30.896
1554906_a_at
ZBTB11
1.326
29.302
230082_at
PMS1
1.749
26.298
1554742_at
ATOH7
2.123
25.071
1552879_a_at
HELLS
1.803
23.928
234040_at
OSBPL10
1.342
23.480
231656_x_at
MBNL
1.145
22.277
1558111_at
Group C
GEM gemcitabine, AR AR-A014418
Group A: GDF11, growth differentiation factor 11; FGD4, FYVE RhoGEF and PH domain containing 4; SLC1A4, solute carrier family 1 (glutamate/
neutral amino acid transporter) member 4; FAM129A, family with sequence similarity 129 member A; APOBEC3F, apolipoprotein B mRNA editing
enzyme catalytic polypeptide-like 3F; SLFN13, schlafen family member 13; UCN, urocortin; TP53INP1, tumor protein p53 inducible nuclear protein 1;
HSPB11, heat shock protein family B (small) member 11; C2ORF72, chromosome 2 open reading frame 72
Group B: MET, met proto-oncogene (hepatocyte growth factor receptor); FNIP1, folliculin interacting protein 1; ATP11B, ATPase class VI type 11B;
ZNF33A, zinc finger protein 33A; MED13, mediator complex subunit 13; MAP4K5, mitogen-activated protein kinase 5; TMEM87A, transmembrane
protein 87A; EIF1, eukaryotic translation initiation factor 1; RBM33, RNA binding motif protein 33; BRAF, v-raf murine sarcoma viral oncogene
homolog B1
Group C: APC, adenomatous polyposis coli; ACDCP1, cyclin M1; ATG7, autophagy related 7 homolog; MPHOSPH6, M-phase phosphoprotein 6;
ZBTB11, zinc finger and BTB domain containing 11; PMS1, postmeiotic segregation increased 1; ATOH7, atonal homolog 7; HELLS, helicase lymphoidspecific; OSBPL10, oxysterol binding protein-like 10; MBNL, muscleblind-like
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Among these molecules, we are interested in TP53INP1
because its expression is altered during the process in
which pancreatic cancer cells acquire gemcitabine resistance [33]. RT-PCR and Western blotting analyses confirmed changes in its expression in PANC-1 cells treated
with gemcitabine and a GSK3b inhibitor.
TP53INP1, also known as thymus-expressed acidic protein (TEAP), stress-induced protein (SIP), and p53-dependent damage-inducible nuclear protein 1 (p53DINP1), is a
p53 target gene [39–41] that encodes the critical tumor
suppressor in pancreatic carcinogenesis [33, 42]. DNA repair
genes have an inhibitory effect on tumor development in the
course of carcinogenesis. On the other hand, in our opinion,
DNA repair genes may be protective for cancer cells against
anticancer drugs after tumor development. TP53INP1 regulates p53-mediated apoptosis and cell cycle arrest in the G1
phase in response to cellular stresses. TP53INP1 expression
is induced by various agents that cause cellular stress (adriamycin, cisplatin, ethanol, heat shock, oxidants, UV light)
[39]. Gemcitabine treatment also causes cellular stress, and if
cell cycle arrest occurs, the cell begins the process of
repairing DNA damage induced by gemcitabine. Such a
repair process might lead cancer cells to become resistant to
gemcitabine. In this study, GSK3b inhibition decreased
TP53INP1 expression, which may prevent DNA repair in
response to gemcitabine and induce apoptosis in pancreatic
cancer cells. Indeed, TP53INP1 knockout cells are very
sensitive to gemcitabine (unpublished observation). The
precise mechanism of decreased TP53INP1 mRNA expression by GSK3b inhibition needs to be studied further.
We previously showed that inhibition of GSK3b in
gastrointestinal cancer and glioblastoma cells was associated with increased p53 and p21 expression in tumor cells
with wild-type p53, and with decreased Rb phosphorylation and cyclin-dependent kinase 6 expression in tumor
cells irrespective of p53 status [21, 22]. Rb is known to
function as a tumor suppressor by inducing cell cycle
arrest at the G1 phase and by inhibiting activity of the E2F
transcription factor [43]. Transcriptional targets of E2F
include ribonucleotide reductase (RR), thymidylate synthase (TS), and thymidine kinase (TK) [44]. RR plays a
critical role in cell cycle progression to the S phase and its
increased expression renders pancreatic cancer cells less
sensitive to gemcitabine [45]. Therefore, inhibition of
GSK3b may sensitize pancreatic cancer cells to gemcitabine via sequential modification of the Rb/E2F pathway. It
was recently reported that TS, a target of E2F transcription
and fluoropyrimidine-derived chemotherapeutic agents, is
frequently activated in primary pancreatic cancer tissues
[46]. This suggests that inhibition of GSK3b also sensitizes
pancreatic cancer cells to fluoropyrimidines, such as
5-fluorouracil and S-1, which are prescribed for patients
with advanced and/or recurrent pancreatic cancer.
Acknowledgments We thank Ms. Yoshie Yoshida for technical
assistance. This work was supported in part by Grants-in-Aids for
Scientific Research from the Japanese Ministry of Education, Science,
Sports, Technology and Culture (to T.S., Y.M, T.M.) and the Ministry
of Health, Labor and Welfare (to T.M.), by grants from the Kanazawa
Medical University Research Foundation (S2006-10 to T.S.) and the
Pancreas Research Foundation of Japan (to T.S.), by a Grant for
Collaborative Research from Kanazawa Medical University (C2008-3
to Y.M., T.S., T.M.) and by a Project Research Grant from the HighTech Research Center of Kanazawa Medical University (H2010-11 to
Y.M.).
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6
Distinct Pathologic Roles for
Glycogen Synthase Kinase 3E
in Colorectal Cancer Progression
Toshinari Minamoto1, Masanori Kotake1,3, Mitsutoshi Nakada2,
Takeo Shimasaki4, Yoshiharu Motoo4 and Kazuyuki Kawakami1
1Division
of Translational and Clinical Oncology, Cancer Research Institute,
of Neurosurgery, Graduate School of Medical Science,
Kanazawa University, Kanazawa,
3Department of Surgery, Ishikawa Prefectural Central Hospital, Kanazawa,
4Department of Medical Oncology, Kanazawa Medical University, Uchinada, Ishikawa,
Japan
2Department
1. Introduction
Colorectal cancer (CRC) is the third most frequent cancer type and the second leading cause
of cancer-related deaths worldwide (Cunningham et al., 2010; Jemal et al., 2010). This is
despite the recent trend of stabilizing or declining rates for CRC incidence and mortality in
economically developed countries (Center et al., 2009; Edwards et al., 2010; Umar &
Greenwald, 2009). Surgical intervention is the initial treatment for most CRC patients.
Continuous efforts to optimize surgery for patients with localized CRC has resulted in
markedly improved 5-year and 10-year survival rates (Cunningham et al., 2010; Wu & Fazio,
2000). Given the large number of CRC patients who undergo curative surgery, there is now
a substantial number who are susceptible to recurrent or metastatic tumors and could
therefore benefit from additional systemic therapies. An increasing array of options and
protocols for chemotherapies and biologically targeted therapies is now available for use in
the adjuvant setting and for the treatment of recurrent and metastatic CRC.
Based on a more detailed knowledge of the molecular characteristics of CRC (Markowitz et
al., 2009; Walther et al., 2009), biologically-based therapeutics have been developed for the
treatment of advanced stage CRC patients. Currently approved agents for the treatment of
advanced and metastatic CRC include therapeutic monoclonal antibodies that target
vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR).
Despite a substantial biological rationale for the use of these new classes of therapeutic
agents, large-scale clinical trials have observed only incremental clinical benefits for overall
patient populations. Clearly, not all patients with recurrent and metastatic CRC benefit from
these therapies. This is due to inherent and acquired resistance of tumors to the
chemotherapeutic and biologically-based agents. Moreover, there are few reliable markers
for predicting the therapeutic and adverse effects of these agents and that would allow
patients who benefit from these systemic treatments to be identified. Therefore, new
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Colorectal Cancer Biology – From Genes to Tumor
therapeutic targets are urgently required to further improve the survival of patients with
recurrent and metastatic CRC. One such target may be glycogen synthase kinase 3
(GSK3), a serine/threonine protein kinase that has recently been implicated in various
human cancers.
In this Chapter, we briefly summarize the scientific basis and current status of systemic
treatments for CRC, including combinations of surgery, chemotherapy and molecular
target-directed therapy. Based on our published and ongoing studies, we then focus on
GSK3 as an emerging therapeutic target in CRC and other cancer types. We describe the
underlying biological mechanism that allows exploration of a novel therapeutic strategy for
CRC involving the targeting of aberrant GSK3.
2. Molecular basis of colorectal cancer
2.1 Multistep and multiple molecular alterations
Colorectal carcinogenesis displays all the major biological hallmarks of cancer (Hanahan &
Weinberg, 2011). CRC evolves and develops through orchestrated, multistep genetic and
epigenetic alterations in oncogenes, tumor suppressor genes and DNA mismatch repair
genes. These include frequent aberrations in certain chromosomes, such as allelic imbalance
at several chromosomal loci (e.g., chromosome 5q, 8p, 17p, 18q) and chromosome
amplification and translocation. Various combinations of somatic and germ-line alterations
in these genes and chromosomes characterize the different genotypes and phenotypes of
sporadic and hereditary forms of CRC (Cunningham et al., 2010; Markowitz & Bertagnolli,
2009; Walther et al., 2009). Among the genes involved in the molecular process of CRC
development, several genetic markers have been reported to harbor diagnostic and
prognostic information and to predict the benefit from or resistance to systemic therapy
(Ellis & Hicklin, 2009; Markowitz & Bertagnolli, 2009; Walther et al., 2009).
Recent advances in DNA sequencing technology have allowed sequencing of the entire
coding genome of human cancer to become a reality. The high throughput, next-generation
sequencing of 18,000 genes in the Reference Sequence data-base of the National Center for
Biotechnology Information in the USA has identified cancer-associated somatic mutations in
848 genes. Amongst these, 140 are considered as candidate genes responsible for the
development and phenotype of CRC (Sjblom et al., 2006; Wood et al., 2007).
2.2 Oncogene addiction
The unrestrained survival and proliferation of cancer cells relies on distinct oncogenic
signalling pathways in which various oncoproteins, growth factor receptors and their
ligands are aberrantly activated, leading to the concept of “oncogene addiction” (Sharma &
Settleman, 2007; Weinstein, 2002; Weinstein & Joe, 2006). In theory, acute ablation of
oncogene function should lead to the rapid dissipation of its pro-survival signal in cancer
cells, thus resulting in apoptotic cell death. This “oncogenic shock” concept underlies the
strategy of molecular targeting in cancer therapy (Sharma et al., 2006). The scientific
rationale behind the development and application of therapeutic monoclonal antibodies
targeting VEGF and EGFR for the treatment of CRC is based on these concepts. Intriguingly,
however, the EGFR expression level in primary CRC determined by immunohistochemistry
was not observed to correlate with the efficacy of therapeutic anti-EGFR antibodies in
clinical trials of metastatic CRC (Hecht et al., 2010; commented by Grothey, 2010).
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3. Systemic treatment: An overview
Surgery remains the cornerstone for the cure of localized CRC (Cunningham et al., 2010; Wu
& Fazio, 2000). For colon cancer, total resection of the primary tumor with ample surgical
margins and regional lymphadenectomy are the requisites for curative surgery. For rectal
cancer, curative resection includes total excision of the mesorectum with adequate
circumferential and distal surgical margins (R0) and lymphadenectomy along the inferior
mesenteric vessels. Laparoscopic surgery has now become prevalent and safe, with longterm oncological outcomes of CRC patients undergoing this surgery reported as comparable
to those treated by the open surgical approach (Lacy et al., 2008; The Clinical Outcomes of
Surgical Therapy Study Group, 2004). Within 5 years after curative surgical resection,
disease relapse (tumor recurrence or metastasis) occurs in 40 to 50% of patients with stage III
CRC and in 20% of those with stage II CRC (Midgley & Kerr, 1999). Systemic therapy with
Chemotherapeutic agents
5-FU
Capecitabine Irinotecan
Target
TS
Indication
PO adjuvant metastatic
metastatic
Therapeutic monoclonal antibodies
Oxaliplatin
Bevacizumab Cetuximab Panitumumab
TopoDNA cross- VEGF
isomerase I link
PO adjuvant metastatic
metastatic
EGFR
EGFR
metastatic metastatic
Combination
FOLFOX
+ LV
FOLFIRI
+ LV
+
FOLFOXIRI
+ LV
+
Predictive
markers
+
TS, DPD, TP UGT1A1*
combined
combined combined
combined
combined combined
VEGF?
Tumor
microvessels?
K-ras, B-raf,
EGFR
PI3CA
copy
number**,
K-ras,
B-raf,
PI3CA,
AREG,
EREG
+
ERCC-1
Table 1. Key agents and their combinations presently used for the treatment of CRC
Abbreviations: AREG, amphyregulin; DPD, dihydropyrimidine dehydrogenase; EGFR, epidermal
growth factor receptor; ERCC-1, excision-repair cross-complementing-1; EREG, epiregulin; 5-FU, 5fluorouracil; FOLFIRI, folinate, 5-FU and irinotecan; FOLFOX, folinate, 5-FU and oxaliplatin;
FOLFOXIRI, FOLFOX and irinotecan; LV, leukovorin; PI3KCA, phosphoinositide 3 kinase (PI3K) p110
catalytic subunit gene; PO, postoperative; TP, thymidine phosphorylase; TS, thymidylate synthase;
UGT1A1, uridine diphosphate (UDP)-glucuronosyltransferase 1A1; VEGF, vascular endothelial growth
factor.
* The number of TA repeats in the TATA element in UGT1A1 gene predicts the drug toxicity and
resultant adverse effects.
** EGFR copy number is measured by fluorescence in-situ hybridization (FISH).
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Colorectal Cancer Biology – From Genes to Tumor
either chemotherapy and/or targeted therapies have been demonstrated to provide benefit
to these CRC patients in both the post-operative adjuvant and advanced disease settings
(Inoue et al., 2006; Midgley et al., 2009). Table 1 summarizes the key chemotherapeutic
agents and therapeutic monoclonal antibodies targeting VEGF and EGFR and the
combinations currently prescribed as adjuvant therapy for relapse-prone CRC patients and
patients with metastatic tumors (reviewed in Cunningham et al., 2010; Meyerhardt &
Mayer, 2005; Midgley et al., 2009; Wolpin et al., 2007; Wolpin & Mayer, 2008). The putative
predictive markers for response to the respective agents are also shown in Table 1 (Walther
et al., 2009).
3.1 Adjuvant chemotherapy
The purpose of postoperative adjuvant chemotherapy for stage II or III CRC is to destroy
residual tumor cells and/or micrometastatic foci that are latent at the time of curative
surgery. The chemotherapeutic mainstay for CRC, 5-fluorouracil (5-FU), exerts its antitumor effect by inhibiting thymidylate synthase (TS), a critical enzyme for nucleic acid
synthesis. Folinic acid (leucovorin: LV) is frequently used to enhance the anti-tumor effect of
5-FU. The clinical and pharmacological rationale for this combination derives from the
biological role of LV in stabilizing the ternary complex between TS and fluoro-deoxyuridine
monophosphate (dUMP), an active metabolite of 5-FU, thereby enhancing TS inhibition.
Adjuvant treatment regimens consist of oral (capecitabine) or infusional fluoropyrimidinebased chemotherapy as a single agent with LV, or in combination with irinotecan (a
topoisomerase I inhibitor), oxaliplatin (a DNA cross-linker) or both (Table 1) (Midgley et al.,
2009; Wolpin et al., 2007; Wolpin & Mayer, 2008).
Adjuvant fluoropyrimidine-based chemotherapy reduces the risk of cancer-related mortality
by 30% and increases the 5-year survival rate by 5-12% in patients with stage III (nodepositive) CRC. Adjuvant chemotherapy for stage II (node-negative) CRC patients is
controversial because it increases the 5-year survival rate by just 3-4%. It has been proposed
that “high-risk” stage II CRC patients characterized by T4 tumor, luminal stenosis or
obstruction, poor histological differentiation, extramural vessel invasion, inadequate
lymphadenectomy or surgical margins (R1) should preferentially undergo adjuvant
chemotherapy (Cunningham et al., 2010; Midgley et al., 2009). Tumor relapse after curative
resection occurs mostly within 3 years, irrespective of adjuvant chemotherapy (Sargent et
al., 2007). Several clinical trials have failed to show a survival benefit from combining
molecular target-directed agents (e.g., bevacizumab, cetuximab) with adjuvant
chemotherapy (reviewed in Cunningham et al., 2010). Improvement in the survival of
patients at high risk of tumor relapse therefore depends on intensive surveillance for early
diagnosis of metastatic lesions, as well as identification of patients who are susceptible to
tumor recurrence and who could thus benefit from more aggressive adjuvant treatment.
3.2 Treatment of metastatic CRC
A series of systemic, fluoropyrimidine-based combinatorial chemotherapies (Table 1) has
substantially improved tumor response to treatment and increased the duration of
progression-free and overall survival in patients with metastatic CRC. The remarkable
advance in treating metastatic CRC in recent years has been due to the emergence and
clinical application of molecular targeted therapeutics (Cunningham et al., 2010; Midgley et
al., 2009). As stated above, a number of therapeutic monoclonal antibodies that target
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111
relevant oncogenic pathways have been tested in clinical trials for CRC. Among them, the
most widely used agents are bevacizumab, a recombinant humanized monoclonal antibody
against VEGF (Ellis & Hicklin, 2008a; Li & Saif, 2009), cetuximab, a chimeric monoclonal
antibody against EGFR (Balko et al, 2010) and panitumumab, a fully humanized monoclonal
antibody against EGFR (Davis & Jimeno, 2010). These therapeutic antibodies have been used
as monotherapy for the treatment of patients with metastatic CRC, or in combination with
systemic chemotherapy (Table 1). Many clinical trials have demonstrated the additive effect
of these antibodies on tumor response rate and progression-free survival (reviewed in
Cunningham et al., 2010; Midgley et al., 2009). However, the combination of each
therapeutic antibody with systemic chemotherapy regimens produced incremental but not
always robust benefits to overall survival when compared to chemotherapy alone (Fojo &
Parkinson, 2010).
3.3 Obstacles to systemic therapy
3.3.1 Drug resistance and predictive markers
The major obstacles to systemic therapy for CRC include drug resistance (both inherent and
acquired) and the lack of reliable biomarkers for predicting response or resistance to drugs
in clinical use (Ellis & Hicklin, 2009). This has led to the recent trend of using intensive
combinatorial regimens for advanced CRC patients. Surprisingly, some recent clinical trials
have shown that combinatorial target-directed therapies resulted in decreased survival,
inferior quality of life and unexpected detrimental effects (Douillard et al., 2010; Hecht et al.,
2009; Li & Saif, 2009; Tol et al., 2009).
Understanding the molecular mechanisms that underlie drug resistance and identifying
predictive markers for drug sensitivity are one and the same thing. Pharmacogenomic
approaches (Furuta et al., 2009; Walther et al., 2009) have identified a number of factors
involved in drug metabolism and secretion, some of which (e.g., UGT1A1 polymorphism)
have been tested in clinical practice (Table 1). Several studies have suggested various
biological mechanisms of resistance to VEGF-targeted cancer therapies (Bergers & Hanahan,
2008; Ebos et al., 2008; Ellis & Hicklin, 2008b), but to date there are no clinically useful
predictive markers. Mutational activation of oncogenic pathways that lie downstream of
EGFR signaling is known to cause intrinsic resistance to therapies that target this receptor.
This has led to the identification of predictive markers (e.g., K-ras, B-raf, PIK3CA) that allow
better patient selection for such treatments (Banck & Grothey, 2009; Cantwell-Dorris et al.,
2011; De Roock et al., 2010a; Sartore-Bianchi et al., 2009). However, the complex pathways
involved in tumor progression are often intercalated and therefore single markers cannot
accurately predict the efficacy or outcome of CRC patients undergoing molecular targeted
therapies (Baldus et al., 2010; De Roock et al., 2010b; Hecht et al., 2010).
Research into the mechanisms of acquired resistance to molecular targeted agents has
generated new therapeutic strategies and agents aimed at counteracting the resistance
mechanism (Bowles & Jimeno, 2011; Cidón, 2010; Dasari & Messersmith, 2010; Presen et al.,
2010). Thus, improving the anti-tumor effects of molecular targeted therapies will depend
on the identification of novel molecular pathways, development of new classes of rationally
designed biological agents, and identification of predictive markers for response and
resistance.
3.3.2 Economic issues
The high cost of developing the biologically-based therapeutic agents shown in Table 1 is a
major issue in light of the modest clinical benefits, acquired drug resistance and lack of
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Colorectal Cancer Biology – From Genes to Tumor
suitable predictive markers. A recent study reported significantly higher hospital costs for
CRC patients with recurrence compared to those without (Macafee et al., 2009). Outside of
the United States, the high cost of molecular targeted drugs has restricted their use to
patients with sufficient income and/or health insurance. This issue highlights the
importance of accurate predictive markers that allow identification of patients who are most
likely to benefit from targeted agents, thus improving the cost effectiveness.
4. GSK3E as an emerging therapeutic target
4.1 GSK3E biology
GSK3 was identified as a serine/threonine protein kinase that phosphorylates and inhibits
glycogen synthase (GS), a rate-limiting enzyme in the regulation of glucose/glycogen
metabolism in response to insulin-mediated signaling (Embi et al., 1980). In contrast to its
original name and depending on its substrates and binding partners (Table 2) (Medina &
Wandosell, 2011; Xu et al., 2009), GSK3 has been found to participate in many fundamental
cellular pathways including proliferation, differentiation, motility, cell cycle and apoptosis
(Doble & Woodgett, 2003; Harwood, 2001; Jope & Johnson, 2004; Nakada et al., 2011). The
two isoforms of this kinase, GSK3 and GSK3, are encoded by their respective genes. Their
functions do not always overlap (Rayasam et al., 2009) and much recent attention has been
directed towards the function of GSK3.
Unlike most protein kinases, GSK3 is active in normal cells and this activity is controlled
by its subcellular localization, differential phosphorylation at serine 9 (S9) and tyrosine 216
(Y216) residues, and different binding partners. A consensus motif and context-based
computational analysis of in vivo protein phosphorylation sites indicate that GSK3 is one of
the kinases with the most substrates (Linding et al., 2007). In normal cells, multiple signaling
pathways mediated by phosphoinositide 3 kinase (PI3K)-Akt, Wnt and mitogen-activated
protein kinase (MAPK) are known to negatively regulate the activity of GSK3 via S9
phosphorylation (Medina & Wandosell, 2011). The molecular structure and details of the
functional and regulatory machinery of GSK3 have been thoroughly described in many
excellent reviews cited in this section and are not the focus of this Chapter.
4.2 GSK3E in common chronic diseases
Accumulating evidence suggests pathological roles for GSK3 in glucose intolerance due to
inhibition of GS and other signaling cascades involved in the regulation of glucose homeostasis (Frame & Zheleva, 2006; Lee & Kim, 2007) and in neurodegenerative changes through
accumulation of the neurotoxic substances amyloid A and tau protein (Annaert & De
Strooper, 2002; Bhat & Budd, 2002). Recognition that GSK3 promotes inflammation also
implicates this molecule in a broad spectrum of common diseases including type 2 diabetes
mellitus and neuropsychiatric disorders involving an inflammatory reaction (Jope et al.,
2007). GSK3 has therefore emerged as a therapeutic target in these prevalent diseases
(Cohen & Goedert, 2004; Kypta, 2005; Meijer et al., 2004; Phukan et al., 2010). Another line of
studies has demonstrated an osteogenic function for the Wnt/-catenin signaling pathway
(Hartman, 2006; Krishnan et al., 2006; Ralston & de Crombrugghe, 2006). This suggests that
GSK3 may be a putative therapeutic target for osteoporotic bone disease, since under
physiological conditions it is a well established member of a complex that destroys -catenin
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Distinct Pathologic Roles for Glycogen Synthase Kinase 3E in Colorectal Cancer Progression
113
Categories
Substrates
Metabolism
glycogen synthase, ATP citrate lyase, PKA, PDH, acetyl-CoA,
carboxylase, PP1, PP2A, PP2A inhibitor, cyclin D1, eIF2B, NGF
receptor, axin, APP, Bax, VDAC, hexokinase II, presenilin, LRP5/6
tau, MAP1B, NCAM, neurofilament, CRMP2, dynein, dynein-like
protein, maltose binding protein, APC, kinesin light chain
Cell structure
Signaling & Transcription
Wnt
Akt
PI3K-Akt
Ras-PI3K-Akt
TNF
Hedgehog
hypoxia
insulin
undetermined &
others
-catenin, snail, smad1, Hath1, smad 3
SRC-3, B-cell lymphoma (BCL)-3, p21
Mcl-1, c-Jun, phosphatase and tensin homologue (PTEN)
c-Myc, cyclin D1
nuclear factor (NF)-B
Ci (citrus interruptus), Gli-2
hypoxia inducible factor (HIF)-1
glycogen synthase, SREBP
cyclin E, AP-1, CREB, C/EBP, cdc25A, Notch, p53, p27Kip1, NFAT,
GR, HSF-1, FGD-1, FGD-3, c-Myb, mCRY2, NAC, MafA,
IPF1/PDX1, presenilin 1 C-terminal fragment
Table 2. Known substrates for phosphorylation by GSK3
Abbreviations: AP-1, activator protein 1; APC, adenomatous polyposis coli; APP, amyloid
precursor protein; ATP, adenosine triphosphate; C/EBP, CCAAT (cytidine-cytidineadenosine-adenosine-thymidine)-enhancer-binding protein; CREB, cyclic adenosine
monophosphate (cAMP) response element binding protein; CRMP2, collapsin response
mediator protein 2; eIF2B, eukaryotic protein synthesis initiation factor-2B; FGD, FYVE,
RhoGEF and PH domain-containing protein; GR, glucocorticoid receptor; HSF-1, heat shock
factor protein 1; IPF1, insulin promoter factor 1; LRP5/6, low-density lipoprotein (LDL)
receptor-related protein 5/6; MafA, musculoaponeurotic fibrosarcoma oncogene homolog
A: MAP1B, microtubule-associated protein 1B; mCRY2, mouse cryptochrome 2; NAC,
nascent polypeptide-associated complex subunit; NCAM, neural cell adhesion molecule;
NFAT, nuclear factor of activated T-cells; NGF, nerve growth factor; PDH, pyruvate
dehydrogenase; PDX1, pancreatic and duodenal homeobox 1; PKA, protein kinase A; PP,
protein phosphatase; SREBP, sterol regulatory element-binding protein; TNF, tumor
necrosis factor ; VDAC, voltage-dependent anion channel.
(Fuchs et al., 2005). In this context, an orally bioavailable GSK3/ dual inhibitor was
generated and tested as a new drug for the treatment of osteoporosis (Kulkarni et al., 2006).
4.3 GSK3E in cancer
An increasing number of cellular structural and functional proteins have been identified as
targets for GSK3 phosphorylation-dependent regulation (Table 2). However, this has also
generated results that show conflicting roles for the signaling pathways regulated by GSK3
in either suppressing or promoting cancer.
4.3.1 GSK3E suppresses cancer
In physiologically normal cells, many of the substrates for GSK3-mediated phosphorylation and subsequent ubiquitin-mediated degradation include oncogenic signaling and
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Colorectal Cancer Biology – From Genes to Tumor
transcription factors, cell cycle regulators and proto-oncoproteins (Table 2). A previous
study showed that GSK3 phosphorylates and stabilizes a major cell cycle regulator, p27Kip1
(Surjit & Lal, 2007). Recent studies have shown that inhibition of GSK3 stabilizes snail and
induces epithelial-mesenchymal transition (EMT), a morphological and phenotypic change
closely associated with tumor cell invasion and metastasis (Bachelder et al., 2005; Zhou et
al., 2004; reviewed in Doble & Woodgett, 2007; Schlessinger & Hall, 2004; Zhou & Hung,
2005). These findings are mostly observed in normal but not neoplastic cells and have led to
the hypothesis that GSK3 functions as a tumor suppressor (reviewed in Luo, 2009;
Manoukian & Woodgett, 2003; Patel & Woodgett, 2008).
Consistent with this hypothesis, a number of studies in breast, lung and non-melanoma skin
cancers have shown that GSK3 is inactivated in tumor cells, but that its activation induces
apoptosis (reviewed in Luo, 2009; Patel & Woodgett, 2008). It has been reported in several
studies that GSK3 renders cancer cells resistant to chemotherapeutic agents (reviewed in
Luo, 2009). However, in contrast to the observations described in the next section (4.3.2),
including our own, none of these studies addressed differences in the expression, activity
and biological properties of GSK3 between tumor cells and their normal cell counterparts.
Furthermore, these studies did not investigate the direct consequences of GSK3 inhibition
for tumor cell survival, proliferation and chemotactic migration and invasion.
4.3.2 Deregulated GSK3E promotes cancer
Wnt signaling plays a crucial role in embryonic development, the regeneration of adult
tissues and in many other cellular processes. Aberrant activation of the Wnt pathway due to
mutation or deregulated expression of its components mediates the multistep process of
colorectal tumorigenesis (Kikuchi, 2007; Klaus & Birchmeier, 2008; Lustig & Behrens, 2003;
Willert & Jones, 2006). Over 90% of CRC develops following activation of the Wnt signaling
pathway in which -catenin plays a central role (Fuchs et al., 2005; Giles et al., 2003). GSK3
interrupts activation of the canonical Wnt pathway by phosphorylating -catenin and
recruiting it to ubiquitin-mediated degradation. GSK3 is therefore believed to antagonize
tumorigenesis that involves active Wnt signaling (Bienz & Clevers, 2000; Manoukian &
Woodgett, 2002; Polakis P, 1999), as represented for example by CRC development. This
notion is also supported by the frequent mutational activation of Ras and PI3K-Akt
signaling (Markowitz & Bertagnolli, 2009; Parsons et al., 2005), since it is well established
that Akt kinase phosphorylates the S9 residue of GSK3 and inhibits its activity (Medina &
Wandosell, 2011). However, few studies had focused on the biological properties of GSK3
in cancer until we investigated a putative pathological role for this kinase in CRC, as
described below.
Most CRC cell lines and primary CRC tumors in our studies have shown increased
expression and activity of GSK3 and deregulation of its activity due to imbalance in the
differential phosphorylation of S9 (inactive) and Y216 (active) residues. This is in
comparison to non-neoplastic cells (e.g., HEK293) and normal colon mucosa in which
GSK3 activity appears to be regulated by the differential phosphorylation. These tumor cell
features are unrelated to the activation of -catenin or Akt (Mai et al., 2009; Shakoori et al.,
2005). A non-radioisotopic, in vitro kinase assay demonstrated an increased ability of GSK3
derived from most CRC cell lines and primary CRC tumors to phosphorylate its substrate,
as compared to non-neoplastic counterparts (Mai et al., 2006, 2009). These observations
suggest that, in contrast to having hypothetical tumor suppressor function, GSK3 may
actually promote cancer.
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Distinct Pathologic Roles for Glycogen Synthase Kinase 3E in Colorectal Cancer Progression
115
A putative pathological role for GSK3 in cancer was demonstrated by subsequent
observations that inhibition of GSK3 activity using pharmacological (small-molecule)
agents and of its expression by RNA interference reduced the survival and proliferation of
CRC cells. Such inhibition also predisposed the cells to apoptosis in vitro and in tumor
xenografts, suggesting that CRC cells depend on aberrant GSK3 for their survival and
proliferation (Mai et al., 2006, 2009; Shakoori et al., 2005, 2007). A series of studies by our
group led us to propose that aberrant GSK3 is a novel and potentially important
therapeutic target in cancer (Miyashita et al., 2009b; Motoo et al., 2011; Nakada et al., 2011),
thus allowing us to apply for domestic and international patents in this field (Minamoto).
Following our studies on the antitumor effects of GSK3 inhibition, similar observations
were reported for CRC by other groups (Ghosh & Altieri, 2005; Rottmann et al., 2005; Tan et
al., 2005; Tsuchiya et al., 2007) (Table 3). Similar results were also published for other cancer
types with underlying biological mechanisms that included GSK3 inhibition of several
pathways involved in tumorigenesis (reviewed in Miyashita et al., 2009b; Nakada et al.,
2011). A putative role for GSK3 in cancer is still being debated (Luo, 2009; Manoukian &
Woodgett, 2003; Patel & Woodgett, 2008) and was discussed in section 4.3.1. However, the
overall results to date indicate that aberrant expression and activity of GSK3 is likely to be
a common and fundamental characteristic of a broad spectrum of human cancers.
4.3.3 Oncogene addiction and the effect of GSK3E inhibition against cancer
As stated in section 2.2, the hypothesis of oncogene addiction has been proposed as a
rationale for molecular targeting in cancer treatment. It refers to the observation that a
cancer cell, despite its plethora of genetic alterations, seemingly exhibits dependence on a
single oncoprotein or oncogenic pathway for its sustained survival and/or proliferation
(Sharma & Settleman, 2007; Weinstein, 2002; Weinstein & Joe, 2006). This unique state of
dependence by cancer cells is highlighted by the fact that inactivation of the normal
counterpart of such proto-oncogene products in non-neoplastic cells is tolerated without
obvious consequence. A profound implication of this hypothesis is that acute interruption of
the critical oncogenic pathways upon which cancer cells are dependent should have a major
detrimental effect (oncogene shock), while sparing normal cells that are not similarly
addicted to these pathways (Sharma et al, 2006). In our series of studies, inhibition of GSK3E
had little effect on cell survival, growth, apoptosis or senescence in non-neoplastic cells (e.g.,
HEK293) and on major vital organs in rodents (Mai et al., 2006, 2009; Shakoori et al., 2005,
2007). This concurs with previous reports showing that GSK3E inhibition does not influence
the survival or growth of human mammary epithelial cells, embryonic lung fibroblasts
(WI38) and mouse embryonic fibroblasts (NIH-3T3) (Kunnimalaiyaan et al., 2007; Ougolkov
et al., 2005). With respect to the oncogene addiction hypothesis (Sharma & Settleman, 2007;
Weinstein, 2002; Weinstein & Joe, 2006), the selective therapeutic effect of GSK3E inhibition
against cancer can be explained by differences in biological properties of GSK3 between
neoplastic and non-neoplastic cells (Mai et al., 2006, 2009; Shakoori et al., 2005).
5. GSK3E and the hallmarks of colorectal cancer
Understanding the molecular mechanism behind a pathogenic role for GSK3E in cancer is
important for the development of treatment strategies that target this kinase. A current
review highlights 8 hallmarks of cancer in which phenotypic properties are progressively
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Colorectal Cancer Biology – From Genes to Tumor
Types of
GSK3
inhibitors
Pathological roles of GSK3 and underlying
mechanism
Shakoori et in vitro
al, 2005
AR-A014418,
SB-216763,
siRNA
Deregulated GSK3 expression and activity are
associated with CRC cell survival and
proliferation by mechanism independent of
activation of Wnt/-catenin signaling and Akt.
GSK3 inhibition attenuates survival and
proliferation of colon cancer cells.
in vitro
AR-A014418,
SB-216763,
siRNA
NRIKA detected higher activity of GSK3 for
phosphorylating its substrate (-catenin) in
gastrointestinal cancer cells including CRC cells
than non-neoplastic HEK293 cells.
Shakoori et tumor
al, 2007
xenograft
AR-A014418,
SB-216763
GSK3 inhibition attenuates survival and
proliferation of SW480 colon cancer cell
xenografts with no detrimental effects on the
major vital organs in the rodents.
Mai et al,
2009
in vitro,
tumor
xenograft
AR-A014418,
SB-216763,
siRNA
GSK3 inhibition attenuates survival and
proliferation of colon cancer cells by decreasing
hTERT expression and telomerase activity and
inducing cell senescence.
Ghosh et
al, 2005
in vitro
LiCl, TDZD8,
SB-216763,
siRNA
GSK3 functions against activation of p53dependent apoptosis in colon cancer cells.
Tan et al,
2005
in vitro
LiCl, SB216763,
SB415286,
LY2119301
GSK3 functions against activation of p53dependent apoptosis through a direct Baxmediated mitochondrial pathway in colon
cancer cells.
Rottmann
et al, 2005
in vitro,
tumor
xenograft
LiCl, siRNA
GSK3 functions against colon cancer cell
apoptosis by inhibiting a TRAIL receptordependent synthetic lethal relationship between
Myc activation and FBW7 loss of function.
Tsuchiya
et al, 2007
in vitro
BIO, LiCl,
keupaullone
GSK3 inhibits colonocyte differentiation by
destabilizing the transcription factor, Hath1.
Authors
Mai et al,
2006
Study
design
Table 3. Pathological roles and functions of GSK3 in colorectal cancer
Abbreviations: hTERT, human telomerase reverse transcriptase; NRIKA, non-radioisotopic
in vitro kinase assay; siRNA, small interfering RNA; TRAIL, tumor necrosis factor (TNF)related apoptosis-inducing ligand.
acquired during multistep pathogenesis, thus allowing cancer cells to become tumorigenic
and ultimately malignant (Hanahan & Weinberg, 2011). These hallmarks are sustained
proliferative signaling, evasion of growth suppressors, resistance to cell death, enabling of
replicative immortality, induction of angiogenesis, activation of invasion and metastasis,
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reprogramming of energy metabolism and evasion of immune destruction. The
development of each hallmark involves multiple signaling pathways. In this section, we
address how GSK3E modulates some of these hallmark characteristics of CRC by referring
to the studies shown in Table 3, including our own work.
5.1 Cell proliferation
Unrestrained cell proliferation is the most prominent feature of cancer. Our previous study
showed that the effect of GSK3E inhibition against the proliferative capacity of CRC cells
was associated with decreased expression of cyclin D1 and cyclin-dependent kinase (CDK) 6
and phosphorylation of the Rb protein (Mai et al., 2009). These observations suggest that Rb
function was restored, leading to the binding and inhibition of E2F transcription factor
(reviewed in Classon & Harlow, 2002; Knudsen & Knudsen, 2008). This is consistent with a
subsequent report that forced expression of exogenous GSK3E promotes the proliferation of
ovarian cancer cells by inducing cyclin D1 expression (Cao et al., 2006). Together, the results
suggest that suppression of excess cancer cell proliferation via the inhibition of GSK3E is
partly due to negative regulation of cell cycling by cyclin D1. In normal or non-neoplastic
cells, cyclin D1 is one of the primary targets of GSK3 for phosphorylation and subsequent
degradation in the ubiquitin-proteasome system (Diehl et al., 1998) (Table 2). The opposing
role of GSK3 in cyclin D1 expression may explain the lack of effect of GSK3 inhibition on
cell survival and growth of non-neoplastic cells found in earlier studies (Kunnimalaiyaan et
al., 2007; Mai et al., 2009; Ougolkov et al., 2005; Shakoori et al., 2005).
5.2 Resistance to cell death via tumor suppressor pathways
A major mechanism by which cancer cells evade cell death is via the inactivation of tumor
suppressor pathways mediated by p53 (Royds & Iacopetta B, 2006; Vousden & Lane, 2007;
Zilfou & Lowe, 2009) and Rb (Classon & Harlow, 2002; Knudsen & Knudsen, 2008). The
studies listed in Table 3 showed that inhibition of GSK3 induced apoptosis in human CRC
cell lines. This effect was associated with increased expression of p53 and of p21 in colon
cancer cells with wild-type p53, and decreased Rb phosphorylation in colon cancer cells
irrespective of their p53 status (Ghosh & Altieri, 2005; Mai et al., 2009; Tan et al., 2005).
Another study showed that GSK3 suppresses the apoptosis of colon cancer cells by
inhibiting a tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)
receptor-dependent synthetic lethal relationship between c-Myc activation and FBW7 (a
gene encoding a ubiquitin ligase receptor) loss of function (Rottmann et al., 2005). These
studies suggest a putative pathological role for aberrant GSK3 in mediating CRC cell
resistance to apoptosis induced by a pathway involving tumor suppressor proteins, TRAIL
and c-Myc.
One of the representative pathways for cell survival is mediated by nuclear factor-B (NFB) (Inoue et al., 2007; Karin, 2006, 2009). Based on previous studies showing the potential
involvement of GSK3 in NF-B-mediated cell survival during mouse embryonic
development (Hoeflich et al., 2000; Schwabe & Brenner, 2002), it was reported that GSK3
sustains pancreatic cancer cell survival by maintaining transcriptional activity of NF-B
(Ougolkov et al., 2005; Wilson & Baldwin, 2008). While these studies examined the activity
of exogenous (transfected) NF-B, we previously observed no effect of GSK3 inhibition on
endogenous NF-B transcriptional activity in gastrointestinal cancer cells (including CRC)
and glioblastoma cells (Mai et al., 2009; Miyashita et al., 2009a). Therefore, a role for GSK3
in regulating NF-B activity in cancer is controversial.
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5.3 Replicative cell immortality
Another critical mechanism used by cancer cells to evade cell death is replicative cell
immortality. A close relationship exists in cancer cells between the molecular mechanisms
for immortality and escape from replicative senescence (Finkel et al., 2007). Cancer cells
acquire constitutive expression and activity of human telomerase reverse transcriptase
(hTERT) and telomerase in order to circumvent telomere-dependent pathways of cell
mortality (Harley, 2008).
We recently observed a decreased level of hTERT mRNA in colon cancer cells following
inhibition of either the activity or expression of GSK3. Inhibition of GSK3 attenuates
telomerase activity and increases the -galactosidase-positive (senescent) population in
colon cancer cells. These effects were associated with increased expression of p53, p21 and cJun N-terminal kinase 1 (JNK1) and decreases in CDK6 expression and Rb phosphorylation
(Mai et al., 2009). The findings are consistent with the known relationship between these
proteins and cell senescence (reviewed in Kiyono, 2007) and with GSK3 activity (Ghosh &
Altieri, 2005; Kulikov et al., 2005; Liu et al., 2004; Mai et al., 2009; Qu et al., 2004; Rössig et al.,
2002). Consistent with our observation, a recent study found that inhibition of GSK3
suppressed hTERT expression and telomerase activity and shortened the telomere length in
various cancer cell lines including HCT116 colon cancer cells, and attenuated cell
proliferation and hTERT expression in ovarian cancer xenografts (Bilsland et al., 2009). The
putative role for GSK3 in protecting cancer cells from telomere-dependent senescence and
mortality is attributed to its effects on hTERT expression and telomerase activity.
5.4 Influence on the cancer microenvironment and tumor invasion
In cancer, various events are orchestrated to produce a distinct tumor microenvironment
that dictates the malignant potential. These include depletion of nutrients involved in cell
proliferation, tumor cell invasion, tumor neovascularization in response to hypoxic
condition, as well as stromal, inflammatory and immune reactions in the host (Joyce, 2005).
The promotion of inflammation and immune response by GSK3 (Jope et al, 2007) suggests
a broad pathological role for this kinase in the cancer microenvironment.
The pro-invasive phenotype of cancer cells is characterized by EMT, enhanced cell motility
and their ability to induce neovascularization. As discussed in section 4.3.1, inhibition of
GSK3 stabilizes snail and induces EMT (Bachelder et al., 2005; Zhou et al., 2004; reviewed
in Doble & Woodgett, 2007; Schlessinger & Hall, 2004; Zhou & Hung, 2005). A hypoxic
tumor microenvironment induces the expression of hypoxia-inducible factor-1 (HIF-1), a
transcription factor that controls oxygen homeostasis by regulating target genes involved in
angiogenesis, glycolysis and cell proliferation (reviewed in Semenza, 2009). A previous
study showed that under physiological conditions, GSK3 inhibits angiogenesis by
negatively regulating endothelial cell survival and migration in response to PI3K-, MAPKand protein kinase A (PKA)-dependent signaling pathways (Kim et al., 2002). Another study
demonstrated that hypoxia induces a biphasic effect on HIF-1 stabilization in liver cancer
cells. Accumulation of HIF-1 occurs in early hypoxia and is dependent on an active
PI3K/Akt pathway and inactive GSK3. In contrast, prolonged hypoxia results in the
inactivation of Akt and activation of GSK3. This negatively regulates HIF-1 activity by
inhibiting its accumulation (Mottet et al., 2003). Collectively, it thus appears unlikely that
GSK3 participates in cancer cell EMT and in tumor angiogenesis.
Formation of lamellipodia, the characteristic cellular microarchitecture, is responsible not
only for cell migration under physiological conditions (e.g., embryonic development,
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119
wound healing) but also for cancer cell migration and invasion (Machesky, 2008; Small et al.,
2002; Yilmaz & Christofori, 2009). A member of the Rho-GTPase family, Rac1, is known to
participate in the formation of lamellipodia and may thus play an important role in cancer
progression (Raftpoulou & Hall, 2004; Sahai & Marshall, 2002). It has been reported that
GSK3 participates in cell motility by facilitating the formation of lamellipodia (Koivisto et
al., 2003) and by activating Rac1 (Farooqui et al., 2006; Kobayashi et al., 2006; Vaidya et al.,
2006). Focal adhesion kinase (FAK) is also known to play a key role in regulating cell
motility and migration and to be deregulated in cancer (McLean et al., 2005). Earlier studies
reported that FAK is one of the downstream effectors in GSK3-mediated pathways
(Kobayashi et al., 2006) and also regulates Rac1 (McLean et al., 2005). Consistent with a recent
study for glioblastoma (Nowicki et al., 2008), our preliminary study has shown that inhibition
of GSK3 attenuates pancreatic cancer cell migration and invasion by negatively regulating
FAK and Rac1 activities (unpublished observation). Therefore, in regard to cancer treatments
that target GSK3, it is important to explore a possible role for GSK3 in CRC cell invasion by
investigating its effects on cellular microarchitecture, motility and migration.
5.5 Cancer cell stemness and metabolic traits
Cell stemness and the reprogramming of energy metabolism are primary cell characteristics
that share distinct molecular pathways and allow cancer cells to survive, proliferate, invade
their host tissues, metastasize and resist treatment. Here, we address future directions in our
approach towards ascertaining the potential of GSK3 as a therapeutic target in cancers
including CRC.
5.5.1 Cancer cell stemness and GSK3E
Arising from the concept of tissue stem cells, the notion of cancer stem cells has emerged
and proposes that cancer initiating cells are a distinct subpopulation within a tumor that
have the ability to self-renew and differentiate (Clarke et al., 2006; O’Brien et al., 2010).
Similar to other cancer types, a small population of cancer initiating cells has been identified
and characterized in CRC (Dalerba et al., 2007; O’Brien et al., 2007; Ricci-Vitiani et al., 2007;
reviewed in Yeung & Mortensen, 2009). Current cancer treatments assume that all cancer
cells in tumors are homogeneous and have a similar capacity to proliferate, invade and
metastasize, as well as having similar susceptibility to chemotherapy and radiation.
However, accumulating evidence suggests that cancer stem cells and cancer cells that are
undergoing EMT share various biological traits (Polyak & Weinberg, 2009). These cells are
also strongly resistant to current forms of therapeutics, thereby identifying this
subpopulation of cancer cells as the ultimate target for cancer treatment (Lou & Dean, 2007).
Consistent with the physiological roles of GSK3E in Wnt, Hedgehog and Notch signaling
(Foltz et al., 2002; Manoukian & Woodgett, 2002; Takenaka et al., 2007), GSK3 inhibition by
pharmacological means promotes embryonic stem cell pluripotency (Sato et al., 2004) and
hematopoietic stem cell reconstitution (Trowbridge et al., 2006). Conversely, recent studies
have demonstrated that GSK3 sustains the respective molecular pathways leading to
tumor cell stemness in a specific type of leukemia and in glioblastoma (Korur et al., 2009;
Wang et al., 2008). Although the underlying molecular mechanisms are not well understood,
these differential roles for GSK3E in normal and cancer stem cells could ultimately benefit
cancer treatment strategies by allowing this kinase to be targeted with little harm to patients.
As addressed in the next section (5.5.2), anaerobic glycolysis and the presence of a distinct
niche are thought to be characteristics of cancer stem cells, in addition to their extreme
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resistance to drug treatment. Therefore, clarification of a putative role for GSK3E in
regulating CRC cell stemness is of great interest. This could lead to a new strategy for
treatment that targets the biology of cancer cell stemness.
5.5.2 Distinct metabolic traits of cancer cells and GSK3E
Production of excess energy is thought to provide an intrinsic and selective pressure that
allows cancer cells to expand clonally and to acquire immortalized and destructive
phenotypes. Even under normoxic conditions, most cancer cells depend on increased
glucose uptake and aerobic glycolysis to produce their energy source, adenosine
triphosphates (ATP) (Kim & Dang, 2006; Vander Heiden & Cantley, 2010). This is known as
the Warburg effect and involves truncated oxidative phosphorylation in the tricarboxylic
acid (TCA) cycle, thus resulting in mitochondrial uncoupling (Samudio et al., 2009). These
properties allow cancer cells to survive and invade host tissues in a microenvironment
where the supply of both oxygen and nutrients is deficient, as well as conferring resistance
to apoptosis-inducing therapeutic stimuli (Smallbone et al., 2007). Therefore, the glycolytic
phenotype of cancer cells is a potential target for cancer diagnosis and treatment (Gatenby &
Gillies, 2007; Kroemer & Pouyssegur, 2008). For example, enhanced glucose uptake by
cancer cells can be used to visualize cancer by positron emission tomography (PET) using
the radioisotope-labeled glucose analogue 2-[18F]-fluoro-2-deoxy-D-glucose (FDG). FDGPET in combination with computed tomography (PET-CT) enables the detection of
metastatic lesions for most cancers with sensitivity and specificity both greater than 90%
(Mankoff et al., 2007).
The association between a glycolytic phenotype (i.e., TCA cycle defects) and resistance to
apoptosis is attributed to decreased mitochondrial hydrogen peroxide production and
cytochrome C release (Samudio et al., 2009; Vander Heiden & Cantley, 2010). Pyruvate
dehydrogenase (PDH) plays a crucial role in triggering the TCA cycle by converting
pyruvate to citric acid. PDH kinase 1 (PDK1), which phosphorylates and inactivates PDH, is
frequently over-activated in cancer cells, resulting in an impaired TCA cycle and
mitochondrial hyperpolarization. Thus, inhibition of PDK1 would re-activate PDH and
restore mitochondrial membrane polarity, thereby facilitating cancer cell apoptosis in
response to chemotherapeutic agents and radiation. Dichloroacetate (DCA), an orally bioavailable small molecule, is a well characterized PDK1 inhibitor. The ability of DCA to
inhibit lactate production by stimulating PDH and the TCA cycle has long been used to treat
lactic acidosis, which is a complication of inherited mitochondrial disorders (Stacpoole,
2003, 2006). A recent study demonstrated that DCA induces cancer cell apoptosis by
selectively inhibiting PDK1 in cancer cells, leading to metabolic remodeling from glycolysis
to glucose oxidation and the normalization of mitochondrial function (Bonnet et al., 2008). A
clinical trial of oral DCA in children with congenital lactic acidosis reported that DCA was
well tolerated and safe (Stacpoole, 2006). Thus, orally administered DCA is a promising and
selective anticancer agent.
The primary role of GSK3 is to control GS activity (Table 2). It thus acts as a checkpoint at
the bifurcation between glycogenesis and glycolysis, the two major pathways of
glucose/glycogen metabolism (Lee & Kim, 2007). We recently found that inhibition of
GSK3 in colon cancer cells increased GS expression and decreased its S640 phosphorylation
(unpublished observation), suggesting that GSK3 inhibition may switch cancer cells from a
glycolytic to a glycogenic phenotype. It was previously reported that GSK3 phosphorylates
and inactivates PDH (Hoshi et al., 1996) (Table 2), a key enzyme for the TCA cycle in
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121
mitochondria. This suggests that deregulated GSK3 contributes to truncation of the TCA
cycle and mitochondrial uncoupling in cancer cells, resulting in resistance to chemotherapy
and radiation. It has also been reported that the distinct metabolism of cancer cells involves
not only anaerobic glycolysis but also other metabolic pathways such as the pentose
phosphate pathway, amino acid and nucleic acid synthesis and glutaminolysis
(DeBerardinis et al., 2008). GSK3 has a number of key metabolic enzymes as substrates
(Table 2), suggesting this molecule could have broad control over various pathological
metabolic pathways in cancer cells.
6. Perspectives
Biologically-based therapy of cancer holds great promise, particularly for patients who are
refractory to existing forms of therapy. Current paradigms reviewed in the earlier part of
this Chapter (3. Systemic treatment: an overview) include the targeting of growth factor
receptor-type protein tyrosine kinases and angiogenic factors. Such therapies are directed
against cancer cell survival, proliferation and tumor angiogenesis; however they are unable
to completely eradicate cancer, as demonstrated by most large-scale clinical trials.
Fig. 1. GSK3 promotes cell stemness, invasive capacity and excess glucose metabolism that
interact to produce a distinct cancer microenvironment.
The distinct pathologic properties of GSK3 in cancer described here highlight its potential to
be an innovative target for the radical treatment of this disease, including CRC. GSK3 can
potentially promote cancer cell stemness, invasive capacity and glucose metabolism, thus
creating the selective pressures that allow cancer cells to persist in a distinct microenvironment
(Figure 1). Understanding the complex biological mechanisms for the multiple roles of GSK3
in promoting cancer should allow elucidation of novel molecular pathways that lead to cancer
development and progression. This will also provide a detailed scientific basis for the
development of cancer treatment strategies that target aberrant GSK3.
Concerns regarding the therapeutic use of GSK3 inhibitors remain because these may
activate oncogenic (e.g., Wnt) signaling, thus promoting cell proliferation (Manoukian &
Woodgett, 2003). However, this issue has not deterred preclinical studies of GSK3
inhibitors for the treatment of many cancer types (reviewed in Miyashita et al., 2009b) or
Phase II clinical trials for the treatment of neurological diseases (Chico et al., 2010).
Currently, two clinical trials are being undertaken to test a pharmacological GSK3 inhibitor
(LY2090314) for enhancement of the anti-tumor effect of chemotherapeutic agents for
advanced solid cancer (phase I: http://clinicaltrials.gov/ct2/show/study/NCT01287520)
and leukemia (phase II: http://clinicaltrials.gov/ct2/show/study/NCT01214603). Such
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trials targeting GSK3 should complement, enhance or substitute the current front line
therapies that target growth factor receptors and angiogenic factors in refractory colorectal
cancer.
7. Acknowledgments
This study was supported in part by Grants-in-Aid for Scientific Research from the Japanese
Ministry of Education, Science, Sports, Technology and Culture (to KK, TM); from the
Ministry of Health, Labour and Welfare (to TM); from the Japan Society for the Promotion of
Science (to KK, TM); and from the Japan Society for Technology (to KK and TM).
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