小型G蛋白訊息調節因子2調控雌激素受體陽性乳癌細胞的黏附和遷移作用之分子機轉

The Molecular Mechanism of Small G Protein Signalling Modulator 2 Regulating The Cell Adhesion and Migration of Oestrogen Receptor-Positive Breast Cancer

林若涵(Juo-Han Lin)

指導教授 ： 何元順

共同指導教授 ： 吳漢忠(Han-Chung Wu)

臺北醫學大學/醫學科技學院/癌症生物學與藥物研發博士學位學程/博士(2019年)

摘要

乳癌轉移會增加死亡的風險; 因此，尋找新的預後標記對於預防癌症轉移及癌症治療相當重要。於2007年，三個新型小型G蛋白訊息調節因子（SGSM1/2/3）首次被報導會參與調節小型GTP酶（RAP和RAB）所介導的訊息傳遞路徑。目前SGSM1/2/3蛋白的功能在癌症中仍然未知。為了釐清SGSM1/2/3蛋白是否參與乳癌的致癌機轉，我們先利用RT-PCR確認SGSM1/2/3基因在乳癌細胞株和53例乳癌患者中的表達情形。根據我們的篩選結果，我們將研究重點放在SGSM2，並進一步證實它在乳癌中的作用。
由基因表現量分析結果顯示，SGSM2顯著表現於管狀A型的乳癌中，且其基因表現在轉移的雌激素受體陽性(ER+)乳癌腫瘤中比轉移的雌激素受體陰性(ER-)患者表現更高。我們新的發現也證實，SGSM2是屬於膜蛋白，且會和細胞膜上的E-鈣粘蛋白(E-cadherin)/β-連環蛋白(β-catenin)交互作用，即使在EGTA（4 mM）誘導的E-鈣粘蛋白內吞作用期間也是如此。當我們抑制SGSM2的蛋白表現，會增加粘著斑激酶(FAK; Y576 / 577)的磷酸化 ，降低樁蛋白(Paxillin)的表現及上皮標記如E-鈣粘蛋白和β-連環蛋白的表現，並增加Snail和Twist-1蛋白的表現，進而減少細胞附著並促進癌細胞遷移。
在雌激素(oestrogen)和纖連蛋白(fibronectin)刺激之下，會發現SGSM2蛋白和磷酸化的粘著斑激酶(FAK; Y397）共同位於細胞前端的位置，說明SGSM2會參與調節細胞運動性的變化。 BioGRID數據庫也顯示，SGSM2可能會與細胞骨架重塑蛋白和細胞連接蛋白交互作用，其中包含FNBP1L、MPP1、WASL、細胞分裂週期蛋白42（CDC42）和鈣粘蛋白 1（CDH1），此預測也同時支持我們的結果。所以我們認為SGSM2在癌症遷移的剛開始，可能扮演著調控細胞粘附及調控細胞骨架動態的角色。

關鍵字

小型G蛋白信號調節因子 ； 乳癌 ； 細胞粘附 ； E-鈣粘蛋白 ； 雌激素受體 ； 上皮細胞間質轉化

並列摘要

Metastasis increases a high risk of death in breast cancer. Finding a new prognostic marker is important for the prevention of cancer metastasis and cancer therapy. In 2007, three novel small G protein signalling modulators (SGSM1/2/3) were first reported to modulate the small GTPases (RAP and RAB)-mediated signalling pathway. However, the function of SGSM1/2/3 in cancer remains unknown. To clarify whether SGSM1/2/3 proteins participate in breast cancer carcinogenesis, we used RT-PCR to determine SGSM1/2/3 gene expression in breast cancer cell lines and breast cancer patients (n=53). According to our screening results, we consequentially narrowed our focus to SGSM2 and investigated its function in breast cancer.
Gene expression profile analysis revealed that SGSM2 was significantly expressed in luminal A type breast cancer, and there was higher SGSM2 expression in metastatic ER-positive breast tumours than in ER-negative patients. Our novel findings demonstrated that SGSM2 is a plasma membrane protein that strongly interacted with E-cadherin/?-catenin, even during ethylene glycol tetraacetic acid (EGTA, 4 mM)-induced E-cadherin endocytosis. Moreover, SGSM2 downregulation enhanced the phosphorylation of focal adhesion kinase (FAK; Y576/577), decreased the expression of Paxillin and the expression of epithelial markers such as E-cadherin and ?-catenin, and increased the expression of Snail and Twist-1, which reduced cell adhesion and promoted cancer cell migration.
Oestrogen and fibronectin treatment promoted the colocalization of SGSM2 at the leading edge with phospho-FAK (Y397), indicating that SGSM2 is involved in the regulation of changes in cell motility. The BioGRID database showed that SGSM2 potentially interacts with cytoskeleton remodelling and cell-cell junction proteins, including formin-binding protein 1-like (FNBP1L), membrane palmitoylated protein 1 (MPP1), Wiskott-Aldrich syndrome-like (WASL), cell division cycle 42 (CDC42), and cadherin 1 (CDH1), which verified our results. This evidence suggests that SGSM2 plays a role in modulating cell adhesion and cytoskeleton dynamics during the initial stage of cancer migration.

並列關鍵字

small G protein signaling modulator ； breast cancer ； cell adhesion ； E-cadherin ； oestrogen receptor ； epithelial-mesenchymal transition

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