PPARγ在非小細胞肺癌(H460)抗藥性(TGFβ)生成過程中所扮演的角色:細胞暨動物實驗層次的探討

Abstract

此篇論文的研究目的在於利用細胞暨動物模式來確認原具有多重抗藥性的非小細胞肺癌細胞株(H460)對TGFβ(transforming growth factor β；轉型生長因子β是否亦具抗藥性，及其抗藥性生成的作用機轉。論文中特別著重於探討PPARγ (peroxisome proliferator-activated receptor γ)；過氧化物酶增生因子活化受體γ在其中所扮演的角色。以PPARγ是否會干擾TGFβ抑制腫瘤細胞生長的功能，卻促成TGFβ所誘發的腫瘤轉移，做為主要的研究議題，並深入分析參與調控的訊號傳遞因子或路徑。論文中更以建立一個腫瘤可以生長於其中的動物模式做為另一研究目標，於是將人類的H460細胞接種到中年鼠齡的BALB/c小白鼠體內，希望藉此動物模式模擬並進一步了解腫瘤細胞在人體內成長的需求性及其抗藥性的高低變化，更重要的是藉此研究平台於動物體內確認PPARγ對腫瘤生長及其抗藥性生成的重要性，並進一步篩選出有效的標靶藥物(例如PPARγ抑制劑 GW9662)來對抗抗藥性高的非小細胞肺癌 (H460)。研究結果發現H460細胞的確對TGFβ具有抗藥性；TGFβ所誘發的PPARγ可以經由與Smad3或p-Smad3的相互作用來抑制Smad3的磷酸化及其聚集至細胞核的程度，並藉此干擾TGFβ/Smad3對H460細胞生長的抑制作用，進而使H460細胞具有抵抗TGFβ的特質。另一方面，TGFβ可以藉由活化P38或β-catenin來促進PPARγ的蛋白表現；PPARγ則藉由活化下游不同的訊息傳遞路徑，進一步促成TGFβ所誘發的H460細胞的轉移。例如，PPARγ可活化epidermal growth factor receptor (EGFR)/c-mesenchymal-epithelial transition factor (c-MET)來降低E-cadhein的蛋白表現，或經由和β-catenin的相互作用促進β-catenin進入細胞核內，兩者均有助於TGFβ所誘發之EMT (epithelial to mesenchymal transition；表皮細胞至間葉幹細胞之轉型)及H460細胞的轉移。研究中同時驗證利用PPARγ的化學抑制劑(GW9662)或具專一性的shRNA可有效破壞PPARγ對H460細胞生長之保護及TGFβ誘發/PPARγ促成的H460細胞的轉移，至此H460細胞的抗藥性(TGFβ)業已完全被破壞。GW9662的抗癌(H460)療效更進一步在新建立的腫瘤動物模式中獲得確認。GW9662的用藥亦同時被發現能明顯抑制腫瘤組老鼠所具有的肺組織發炎現象。然而，GW9662的發炎抑制性可能是間接－因為GW9662將能促進肺發炎的H460腫瘤的生長抑制所致。此腫瘤動物模式的特點，在於中年鼠齡的小白鼠在保有其免疫力的狀況下仍然可容許人類異種移植瘤的生長及轉移，更接近人類腫瘤患者之體況，這是其它腫瘤動物模式(例如裸鼠及免疫不全鼠)所沒有的特性。初步推論，中年鼠齡小白鼠體內嗜中性白血球(Neutrophil)活性的下降可能是H460異種移植瘤可以生長於該鼠肺部的主因，而巨嗜細胞(macrophage) 活性的提升可能與該鼠肺部原生瘤的生長有關。事實上，H460異種移植瘤本身可能會進一步抑制嗜中性白血球的活性幫助腫瘤之生長，同時亦可能是造成腫瘤鼠肺組織發炎的主因。整體而言，本論文首次將PPARγ的功能與H460腫瘤細胞的生長、轉移、及抗藥性做聯繫，PPARγ對三者之重要及促進性之影響顯示日後在以標靶治療對抗高抗藥性的腫瘤(例如H460腫瘤)時，PPARγ可能是一個理想的標靶分子。GW9662不論在體外或是體內實驗中，均被證實具有極佳的抗腫瘤(H460)療效，可進一步支持上述之推論。

The primary goal of the study was to understand the molecular mechanism responsible for the development of drug resistance of non-small cell lung cancer (NSCLC) cell, H460, against transforming growth factor β (TGFβ). The role of peroxisome proliferator-activated receptor γ (PPARγ) in disabling the tumor suppressing effect whereas promoting the metastasizing effect of TGFβ on H460 cells were the first two major issues to be approached followed by signaling pathways identification. A novel tumor mouse model using a group of middle aged BALB/c mice implanted with H460 xenograft tumor cells has also been established to further examine the development and metastasis of H460 cells within the animal body and to screen for anti-cancer drug for control of H460 cells in vivo. Our results showed the H460 cells revealed a drug resistance to TGFβ after the short or long term drug exposure. TGFβ-induced PPARγ was able to interact with and prevent the nuclear infiltration of Smad3/p-Smad3 that subsequently disrupt TGFβ-induced mitoinhibition in H460 cells and led to TGFβ resistance. TGFβ was later found can act through P38 and/or β-catenin signaling pathway to trigger the expression of PPARγ, which was also critical for TGFβ-induced epithelial to mesenchymal transition (EMT) and metastasis of H460 cells. PPARγ could activate the epidermal growth factor receptor (EGFR)/c-mesenchymal-epithelial transition factor (c-MET) pathway to decrease the expression of E-cadherin, or interact with β-catenin and promote its nuclear infiltration, either way might contribute to the EMT and metastasis of H460 cells induced by TGFβ. PPARγ was shown to have a positive reciprocal interaction with β-catenin. GW9662 (PPARγ inhibitor) and PPARγ-specific shRNA both revealed a therapeutic value in the control of H460 cells by breaking of PPARγ-protected cell growth and TGFβ-induced/PPARγ-mediated metastasis of H460 cells. The therapeutic value of GW9662 in the control of H460 cells-derived tumor was further confirmed in vivo by using the tumor mouse model has just described above. In addition to the tumor suppressive effect, GW9662 also inhibited the lung inflammation in H460 cells-implanted mice. This anti-inflammatory effect, however, might be due to GW9662-caused tumor inhibition of H460 cells. The novelty of our newly-developed tumor mouse model was that, human H460 cells were able to grow in immunocompetent mice instead of nude or SCID mice (immunodeficient). In brief, the decline of neutrophil activity in middle aged mouse was critical for the development of H460 xenograft in lung whereas the increase of macrophage activity was critical for the growth of murine-derived tumor. H460 xenograft tumor, on the other hand, might further decrease neutrophil activity to favor the tumor growth, and be the reason to cause pulmonary inflammation in H460-bearing mice. In overall, PPARγ was for the first time being addressed in H460 tumorigenesis and its drug resistance to TGFβ. The growth protecting and metastasis promoting effects of PPARγ in H460 cells have suggested that PPARγ may be a good molecule target to be approached in target therapy for many drug resistant-cancer cells such as H460. The therapeutic value of GW9662 in the control of H460 cells both in vitro and in vivo further supported this notion.