多環芳香烴受體於人類肺癌細胞株中透過Smad4降解引發TGF-β訊號傳遞路徑抑制之機制探討

Abstract

多環芳香烴受體 (Aryl Hydrocarbon Receptor, AhR)為細胞當中一配體活化之轉錄因子，當細胞接觸到多環芳香烴 (Polycyclic Aromatic Hydrocarbon, PAHs)時，PAHs會與AhR結合並使其受到活化而進入細胞核中，進而啟動目標基因生合成Cytochrome P450 (CYP1A1、CYP1B1)，並將外來物質氧化還原以利進行酵素代謝反應。上皮細胞間質轉換 (Epithelial- Mesenchymal Transition, EMT)是指細胞從上皮細胞型態轉變為間質細胞型態的一種過程，而在癌細胞當中則被認為與腫瘤轉移 (Metastasis)能力有關。過去研究顯示，PAHs會透過活化AhR，引起癌細胞發生EMT的現象，目前僅有少數文獻探討未活化的AhR是否與EMT有相關，機制仍舊尚未明朗，因此本篇研究目的旨在探討，不存在配體的情形之下，透過in vitro Transfection方式於肺癌細胞株中大量表現AhR，觀察是否影響EMT現象以及細胞轉移能力改變並探討其調控機制。實驗結果顯示，在大量表現AhR後，細胞中的Epithelial marker增加且Mesenchymal markers表現量會減少，顯示細胞EMT現象可能受到抑制，並且可能原因為EMT相關轉錄因子snail/slug、ID1表現減少，進一步探討其調節機制發現，細胞內的Smad4蛋白表現量發生改變，而Smad4是參與TGF-β訊號傳遞中重要的伴隨蛋白，能夠幫助受活化的調節型smad共同進核，且過去研究指出TGF-β能夠誘使細胞進行EMT，故一旦受到抑制，會造成細胞呈現MET發展，此時細胞的轉移能力也會受到抑制，過去研究發現，Smad4的蛋白表現受Jun活化結合蛋白 (Jun-Activation Domain-Binding Protein, Jab1)的影響，Jab1為一種E3-ubiquitin lagase，會使Smad4 ubiquitination後送至26s proteasome 進行降解，而本實驗結果也證實，AhR會造成Jab1與Smad4間的蛋白結合能力增強，進而導致Smad4受到降解的程度強化，最終造成TGF-β訊號傳遞減弱。最後，在體外的細胞轉移試驗 (Migration assay)中也可以發現細胞的遷移以及侵襲 (Invasion)能力明顯降低，顯示AhR的大量表現確實能夠抑制癌細胞轉移能力。並且利用shAhR進行靜默 (silence)作為反證，細胞有促進EMT的現象。綜合以上實驗結果得知，在肺癌細胞株中，未活化的AhR能夠透過Jab1調控smad4的蛋白表現量，達到抑制TGF-β的訊號傳遞，降低EMT相關轉錄因子的生合成，進而導致癌細胞無法進行EMT，而使得癌細胞的遷移能力受到干擾。

Backgrounds: The aryl hydrocarbon receptor (AhR) is a ligand-dependent-activated transcriptional factor that forms a complex with its ligand such as TCDD to activate target genes Epithelial to mesenchymal transition (EMT) is a process that epithelial cells are converted to invasive mesenchymal cells, and this process involved tumor metastasis from original cancer to distal. The role of ligand-independent AhR function in cell EMT was investigated in this study. Results: Epithelial markers were induced and mesenchymal markers were inhibited in AhR-overexpressed cell. At the same time, the expression of EMT-related transcriptional factors Snail/slug were also decreased. Interestingly, Smad4 degradation was enhanced in AhR-overexpressed lung cancer cells. Smad4 itself is a co-translocational factor from cytoplasma to nuclear when regulated-smad phosphorylate by TGF-β. TGF-β plays as a potent factor of cancer progression through the induction of EMT. If TGF-β pathway has be inhibited by suppression of smad4, the cancer cell migration will decrease via mesenchymal to epithelial transition (MET). Preliminary studies have shown that Jab1 interacts directly with smad4 and induces its ubiquitination for degradation via 26s proteasome. Our data show that AhR able to increase interaction between Jab1 and smad4, which might increase degradation of smad4 though 26s proteasome pathway. In addition, Cell migration and cell invasion ability were decreased in AhR overexpressed cells. Conclusion: Our data suggest that AhR affects TGF-β function through Jab1-induced Smad4 degradation by proteasome and resulted in tumor metastasis suppression via EMT reduction in lung cancer cells.