肺癌治療之嶄新方法：以磷酸酶為標的

Targeting phosphatases as novel approach to anti-lung-cancer therapy

王誠一(Cheng-Yi Wang)

指導教授 ： 余忠仁 ； 陳昆鋒

國立臺灣大學/醫學院/臨床醫學研究所/博士(2015年)

https://doi.org/10.6342/NTU.2015.02214

摘要

蛋白激酶在很多細胞基礎功能例如細胞增殖､細胞周期､代謝､存活､細胞凋亡等都扮演關鍵的調節角色。一些蛋白激酶例如轉錄訊號傳導子和激活子3 (signal transducers and activators of transcription 3, STAT3), c-Src, c-Abl, mitogen activated protein kinase, phosphotidylinositol-3-kinase/Akt, and epidermal growth factor receptor在癌細胞經常是過度活化，而這些重要的致癌蛋白訊息路徑也常是新的抗癌藥物的重要標靶。這些致癌蛋白激酶活性以及它的上游或下游激酶常常是受到磷酸酶的去磷酸化的負調控。磷酸酶的重要角色主要是因很多細胞重要的功能及訊息皆受到磷酸化及去磷酸化的調控，這使得磷酸酶可能可以作為新的抗癌標的。一些已知的磷酸酶例如protein phosphatase 2A (PP2A), Src homology 2-domain containing tyrosine phosphatase (SHP)等功能被認為是腫瘤抑制者。在此提出兩個假說模型支持以增強磷酸酶活性為方針的抗癌策略的可行性。
第一個假說模型顯示以erlotinib藉由抑制cancerous inhibitor of PP2A (CIP2A)蛋白來增強PP2A活性作為新的抗非小細胞肺癌（non-small cell lung cancer，NSCLC）策略。我們以erlotinib治療4株表皮生長因子受體（EGFR）野生型非小細胞肺癌細胞株（H358、H441、H460和A549），以測定這些細胞株對erlotinib所誘發之細胞死亡和細胞凋亡的感受性。試驗中分析了CIP2A, PP2A及下游AKT的表現狀況。我們在具有感受性和具有抗藥性的細胞中分別使CIP2A過度表現及剔除CIP2A基因表現，藉此確認PP2A對erlotinib誘發之細胞凋亡所造成的影響。另外也在裸鼠中測定erlotinib對H358異種移植腫瘤的體內療效。結果erlotinib在H358和H441細胞中誘發出顯著的細胞死亡和細胞凋亡（證據為caspase 3活性上升，且pro-caspase 9和PARP的裂解作用上升），但在H460或A549細胞則無此效果。Erlotinib在具有感受性之H358細胞中引起的細胞凋亡作用，會伴隨CIP2A的負向調節、PP2A活性的上升，以及AKT磷酸化作用的下降。CIP2A和AKT的過度表現可使具有感受性之H358細胞免於發生erlotinib誘發之細胞凋亡。在與具有感受性之H358細胞類似、但具有抗藥性的H460細胞中，以siRNA剔除CIP2A基因表現的做法可增強erlotinib誘發之細胞凋亡。Erlotinib亦可抑制裸鼠體內H358腫瘤的生長。藉由抑制CIP2A來增強PP2A活性促成了erlotinib在體內和體內，對不帶有EGFR突變之NSCLC細胞所造成的腫瘤消滅效果。在往後的藥物發展上，PP2A可能可以作為對抗EGFR野生型NSCLC的一項新型分子標的。
第二個假說模型呈現在EGFR野生型NSCLC以sorafenib及其結構衍生物顯示增強SHP-1活性來抑制p-STAT3訊息路徑為可行的抗癌方針。STAT3在EGFR野生型NSCLC中經常持續受到活化。試驗的目的為判定sorafenib及其衍生物是否可藉由去活化STAT3抑制EGFR野生型NSCLC。本試驗讓EGFR野生型NSCLC細胞株（A549、H292、H322、H358和H460）接受sorafenib或SC-1（一種sorafenib的衍生物，結構上與sorafenib十分近似但缺少激酶抑制活性）的治療，接著分析細胞凋亡（apoptosis）和訊號傳導作用。體內療效則是在植入H460和A549異體移植物的裸鼠身上測定。在所有受測的EGFR野生型NSCLC細胞株中，SC-1對於生長抑制和細胞凋亡的影響均優於sorafenib。在所有受測的EGFR野生型NSCLC細胞株中，SC-1均可降低705號酪胺酸上發生的STAT3磷酸化作用。STAT3所驅動之基因（包含cyclin D1和survivin）的表現程度也受到SC-1壓抑。STAT3在H460細胞中的異位性表現，消除了SC-1治療組細胞中的細胞凋亡作用。Sorafenib和SC-1增強了SHP-1的活性，而以小干擾RNA剔除SHP-1時可降低SC-1所誘發之細胞凋亡（但剔除SHP-2或PTP-1B時則無此效果）。在體內環境中，SC-1可透過SHP-1/STAT3路徑顯著減弱H460和A549腫瘤的生長。SC-1提供了證據，證明以STAT3傳訊路徑作為作用標的，有機會成為EGFR野生型NSCLC的一項新療法。未來應可應用增強磷酸酶活性為方針來研發新的抗癌藥物。

關鍵字

肺癌 ； 磷酸酶

並列摘要

Protein kinases play key roles in regulating many essential cellular functions, such as proliferation, cell cycle, cell metabolism, survival, apoptosis, and etc. Kinases such as signal transducers and activators of transcription 3 (STAT3); c-Src, c-Abl, mitogen activated protein kinase, phosphotidylinositol-3-kinase/Akt, and epidermal growth factor receptor are commonly activated in cancer cells, and these important oncogenic pathways have been hot targets of novel targeted anti-cancer agents. The activities of protein kinases and their upstream or down-stream kinases are negatively regulated by protein phosphatase, which are responsible for the dephosphorylation of these kinases. This role of protein phosphatases is extremely important since protein phosphorylation and dephosphorylation is required for the regulation of a large number of cellular activities. The important role of protein phosphatases in regulating oncokinase implicates that phosphatase may be a potential promising novel target in anti-cancer strategy. Several known protein phosphatases such as protein phosphatase 2A (PP2A) and Src homology 2-domain containing tyrosine phosphatase (SHP) have been shown to function as tumor suppressors. Here 2 proof-of-concept models are provided to support that targeting oncoprotein kinase through enhancing phosphatase activity can be a novel anti-cancer strategy for developing novel anti-cancer agents.
The first model demonstrates that enhancing PP2A activity by inhibiting cancerous inhibitor of PP2A (CIP2A) by erlotinib is a potential anti-lung-cancer strategy. Four EGFR wild type NSCLC cell lines (H358 H441 H460 and A549) were treated with erlotinib to determine their sensitivity to erlotinib-induced cell death and apoptosis, Expression of CIP2A, PP2A and the downstream AKT were analyzed. The effects of PP2A on erlotinib-induced apoptosis were confirmed by overexpression of CIP2A and knockdown of CIP2A gene expression in the sensitive cells and resistant cells respectively. In vivo efficacy of erlotinib against H358 xenograft tumor was also determined in nude mice. Erlotinib induced significant cell death and apoptosis in H358 and H441 cells, as evidenced by increased caspase 3 activity and cleavage of pro-caspase 9 and PARP, but not in H460 or A549 cells. The apoptotic effect of erlotinib in the sensitive H358 cells was associated with downregulation of CIP2A, increase in PP2A activity and further decrease in AKT phosphorylation. Overexpression of CIP2A and AKT protected the sensitive H358 cells from erlotinib-induced apoptosis. Knockdown of CIP2A gene expression by siRNA enhanced the erlotinib-induced apoptotic in the resistant H460 cells that resembled the sensitive H358 cells. Erlotinib also inhibited the growth of H358 tumors in nude mice. The CIP2A/PP2A-dependent pathway mediates the tumoricidal effects of erlotinib on NSCLC cells without EGFR mutations in vitro and in vivo. PP2A may be a novel molecular target against NSCLC for future drug development.
The second model shows that targeting phosphorylated STAT3 by enhancing SHP-1 activity by sorafenib and its analogue is another attractive anti-cancer strategy for EGFR wild type NSCLC. STAT3 is often persistently activated in EGFR wild-type non-small cell lung cancer (NSCLC). The aim of this model was to determine whether sorafenib and its derivative can inhibit EGFR wild-type NSCLC via STAT3 nactivation. EGFR wild-type NSCLC cell lines (A549 H292 H322 H358 and H460) were treated with sorafenib or SC-1, a sorafenib derivative that closely resembled sorafenib structurally but devoid of kinase inhibitory activity. Apoptosis and signal transduction were analyzed. In vivo efficacy was determined in nude mice with H460 and A549 xenograft. SC-1 had better effects than sorafenib on growth inhibition and apoptosis in all tested EGFR wild-type NSCLC lines. SC-1 reduced STAT3 phosphorylation at tyrosine 705 in all tested EGFR wild-type NSCLC cells. The expression of STAT3-driven genes, including cylcin D1 and survivin, was also repressed by SC-1. Ectopic expression of STAT3 in H460 cells abolished apoptosis in SC-1-treated cells. Sorafenib and SC-1 enhaced SHP-1 activity, while knockdown of SHP-1, but not SHP-2 or PTP-1B, by small interference RNA reduced SC-1-induced apoptosis. SC-1 significantly reduced H460 and A549 tumor growth in vivo through SHP-1/STAT3 pathway. SC-1 provides proof that targeting STAT3 signaling pathway may be a novel approach for the treatment of EGFR wild-type NSCLC. It is promising and feasible to develop novel anti-cancer agents by enhancing phosphatase activity in the future.

並列關鍵字

lung cancer ； phosphatase ； CIP2A ； PP2A ； AKT ； SHP-1 ； STAT3

參考文獻

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