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消化器神経内分泌がんの細胞株を使用した前臨床モデルとPARP阻害剤、EZH2阻害剤の有用性に関する検討

大本, 晃弘 東京大学 DOI:10.15083/0002002332

2021.10.13

概要

消化器原発神経内分泌がん(NEC)は希少がんかつ難治がんである。実地診療においては小細胞肺がんに準じてシスプラチン(CDDP)併用療法が行われるが、予後不良であり臨床効果を示した新規薬剤も存在しない。消化器NECの遺伝学的背景が十分に明らかとなっていない現状のもとで、肺原発の小細胞型神経内分泌がんである小細胞肺がんに関する知見を参考にして前臨床的研究を行うことは有効と考えられる。小細胞肺がんにおいてはpoly(ADP-ribose)polymerase(PARP)1ならびにenhancer of zeste homolog 2(EZH2)の高発現が明らかとなっており、これらの標的を阻害する薬剤の有効性が期待される。PARP阻害薬はPARylationを抑制することでDNA二本鎖切断を誘導すると共に、DNA損傷部位でPARP-DNA複合体を形成することによって細胞毒性を示す。奏効のバイオマーカーとしては、BRCA1/2、ATMをはじめとする相同組み換え修復機構に関連する遺伝子変異の存在が知られており、PARP阻害作用と組み合わさることにより細胞に致死的な影響をもたらす(合成致死)。また、EZH2阻害薬はヒストンH3リシン27トリメチル(H3K27me3)を脱メチル化することによりエピジェネティックに抗腫瘍効果を示し、Y646変異を始めとしたEZH2活性型変異を有する腫瘍での効果が報告されている。PARP阻害薬は主として卵巣がん、乳がん、EZH2阻害剤はB細胞性リンパ腫に対する臨床開発が進行している。消化器原発NECにおける前臨床的検討はこれまで少数である。我々は膵原発株(A99)、十二指腸原発株(TCC-NECT-2)、食道原発株(TYUC-1)の小細胞型消化器NEC細胞株を用いて、はじめに殺細胞性抗がん剤の感受性を前臨床的に評価し、この結果を基礎としてPARP阻害薬(Veliparib)、EZH2阻害薬(Tazemetostat)の抗腫瘍効果ならびに作用メカニズムに関する検討を行った。

 3種のNEC細胞株に対するCDDPの薬剤感受性をviability assayで評価したところ、化学療法歴のない手術標本から樹立されたTYUC-1の感受性が最も高く、細胞株樹立前にCDDPをはじめとした多種にわたる化学療法歴のあるA99は不良であった(IC50A99: 3.74μg/ml、TCC-NECT-2: 1.06μg/ml、TYUC-1: 0.26μg/ml)。抗がん剤の薬剤耐性との関連が指摘されているATP binding cassette subfamily Bmember1(ABCB1)の遺伝子発現をリアルタイム定量PCRにより評価したところ、A99での発現が顕著であるのに対して、TYUC-1では発現を認めなかった。ウェスタンブロッティングによるABCB1タンパクの比較を行ったところ、同様にA99におけるシグナルが最も強く、TYUC-1ではタンパクが欠損していた。3種の細胞株をBALB/cヌードマウスに移植したゼノグラフトを作成し、腫瘍の病理組織学的評価を行った。いずれのゼノグラフトにおいても小型のN/C比が高い均一な形態の腫瘍細胞がsolidもしくはnested patternを呈して増殖しており、クロモグラニンA陽性、シナプトフィジン陽性、Ki-67index高値と併せて小細胞型NECに合致する所見であった。

 上記の知見に基づいて、PARP阻害薬ならびにEZH2阻害薬の前臨床的検討を行った。はじめに原発臓器の正常細胞由来不死化細胞株である膵管細胞株と十二指腸細胞株をコントロールとして、NEC細胞株におけるPARP1遺伝子発現をリアルタイム定量PCRにより評価したところ、A99、TYUC-1において有意な発現上昇を認めた(P=0.0008; P=0.014)。A99での遺伝子発現が最も上昇しており、細胞株ならびに細胞株を移植したマウスゼノグラフトにおいてPARP1タンパクは高発現であった。Veliparibに対する感受性はCDDP低感受性株であるA99で最も高く(IC50A99: 9.7μg/ml、TCC-NECT-2: 15.2μg/ml、TYUC-1: 26.1μg/ml)、CDDPとの併用効果を示した。Veliparib投与によるPARP1ならびにポリADP-リボシル化により生成されるpoly(ADP-ribose)(PAR)のタンパク発現については、3種の細胞株においてPARP1の発現が維持されるのに対してPARは濃度依存的に抑制された。本薬剤の作用メカニズムを明らかにするため、DNA二本鎖切断を反映するγ-H2AXを蛍光染色により評価したところ、A99においてはVeliparib投与により染色陽性細胞比率が増加し、CDDPとの併用による更なる陽性率の上昇が観察された。ウェスタンブロッティングにおいてもVeliparib投与によりA99のγ-H2AXシグナルが増強したが、他2つの細胞株ではシグナルの増強は認めなかった。セルサイクルアッセイを行ったところA99ではVeliparib投与によりS期細胞比率の上昇を認め、S期の進行に必要な細胞周期制御調節因子であるCyclin AならびにCDK2の遺伝子発現が上昇した。次にA99をBALB/cヌードマウスの皮下に移植し、コントロール群、Veliparib単独群の2群に分けて21日間の1コース投与を行った。実験終了時の治療開始時に対する相対腫瘍量(平均値 ± 標準誤差)はコントロール群: 11.1 ± 1.3、Veliparib群: 6.5 ± 1.4であり、Veliparib群で有意に腫瘍の増大が抑制された(P=0.045)。毒性の点からは体重減少は明らかでなかった。

 PARP1と同様にEZH2の遺伝子発現を評価したところ、TYUC-1における発現が有意に亢進しており、A99についても上昇傾向であった(P=0.037; P=0.071)。TYUC-1ではH3K27me3のタンパク発現はEZH2の遺伝子発現レベルに応じて高く、A99ではEZH2遺伝子発現を認めるにも関わらずH3K27me3は欠損していた。TCC-NECT-2、TYUC-1共に0.1μg/mlのTazemetostat投与によりH3K27me3のシグナルは消失した。Viability assayにおいては、TYUC-1は他の細胞株に比較しTazemetostatに対する40倍程度の感受性を有していた(IC50A99: 17.8μg/ml、TCC-NECT-2: 17.9μg/ml、TYUC-1: 0.47μg/ml)。TYUC-1を使用しセルサイクルアッセイを行うと、sub-G1期の細胞比率が薬剤の投与期間・濃度依存的に上昇し、cleaved PARP1シグナルの増強を認めた。薬剤感受性との関連を明らかにするため、A99を樹立した腫瘍組織と対照の正常組織を用いた全ゲノムシーケンス、TCC-NECT-2細胞株を用いた114のがん関連遺伝子に関するターゲットシーケンスを行った。TYUC-1については細胞株と対照の正常組織を用いて、EZH2遺伝子変異に関するサンガーシーケンスを行った。PARP阻害薬の奏効に関わる相同組み替え修復関連の遺伝子変異ならびにEZH2阻害薬の奏効に関わるEZH2の変異はA99、TCC-NECT-2で検出されず、TYUC-1においてもhotspotであるEZH2Y646変異は認めなかった。

 本研究においては、消化器NEC細胞株のうちA99とTYUC-1においてPARP1、EZH2の高発現を認めた。消化器NECに対する同様の検討はこれまでにないが、メルケル細胞がん、卵巣小細胞がんにおけるPARP1、EZH2高発現の報告がある。In vitroでのVeliparib単剤の薬剤感受性ならびにCDDPとの併用による抗腫瘍効果の増強はPARP1発現レベルが最も高いA99で顕著であり、in vivoモデルにおいてもVeliparib投与により腫瘍増大が有意に抑制された。明らかな毒性はなく、CDDP治療後再発・不応症例に対してPARP阻害薬が有効な可能性を示唆した。DNA一本鎖切断はPARPの阻害により塩基除去修復が起こらない状況下では未修復のまま蓄積し、S期で行われるDNA複製を通して複製フォークでDNA二本鎖切断を生じる。本研究においてA99ではγ-H2AX陽性細胞とS期細胞の蓄積が同時期に認められ、S期の進行に関与するCyclin A、CDK2の発現上昇を認めた。TCC-NECT-2、TYUC-1においてTazemetostatはH3K27me3の脱メチル化を引きおこしたが、アポトーシスを伴う細胞増殖抑制効果はTYUC-1でのみ認めた。この結果からEZH2阻害薬の抗腫瘍効果はエピジェネティックな機序のみではなく、腫瘍の遺伝子変異を含めた複合的な要因により決定されることが示唆される。NEC細胞株のゲノム解析において両薬剤の奏効に関わる明らかな遺伝子変異は検出されなかったが、網羅的な解析により本薬剤の感受性に関わるバイオマーカーが同定されることが期待される。

 我々は小細胞型消化器NEC細胞株を用いた前臨床的検討を行い、PARP阻害薬、EZH2阻害薬が一部の消化器NECに有効な可能性を示した。同時に各NECにより薬剤の感受性や作用メカニズムが異なることを明らかにした。

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