Protocadherin γA-12(PCDH21)基因表現於乳癌細胞株MDA-MB-231之腫瘤抑制相關功能性分析

Abstract

鈣黏附蛋白分子 (cadherins)在細胞與細胞之間扮演著細胞吸附作用之重要角色。同時鈣黏附蛋白也調控著細胞型態、細胞接觸型抑制，以及在腫瘤演進過程中扮演訊息傳遞的功能。至今有至少80多個鈣黏附蛋白家族成員被發現，包含典型鈣黏附蛋白分子 (classical cadherins)、橋粒鈣黏附蛋白 (desmosomal cadherins)、Fat-like 鈣黏附蛋白 ( Fat-like cadherins)，以及原鈣黏附蛋白 (protocadherin)等。過去十年來的研究，大約有超過60多個protocaddherins 成員被鑑定出，同時protocaddherins也是組成鈣黏附蛋白家族成員的最龐大子群。迄今，關於protocaddherins的許多研究均是看重關於參與中樞神經系統發育的功能，而在癌症研究，該基因於腫瘤細胞中所扮演的功能仍是非常少被了解。在先前的研究中，本實驗室發現在具有腫瘤轉移能力的MDA-MB-231 乳癌細胞株中 protocadherin γA-12(PCDH21) 表現量低於不具轉移能力的MCF7乳癌細胞株，同時PCDH21的基因表現受到DNA甲基化的調控。所以為了探究PCDH21 於乳癌細胞中扮演的功能性角色，我們將PCDH21基因大量表現於MDA-MB-231細胞株。於細胞體外的研究分析裡，我們分別檢測PCDH21的表現在乳癌細胞中的細胞增生、細胞移動、細胞侵犯，以及形成細胞群落的能力。首先，於細胞的形態與細胞生長試驗裡，我們發現大量表現PCDH21的MDA-MB-231細胞株能轉變成上皮細胞的形態，同時在這些轉染的MDA-MB-231細胞，其細胞生長的情況也受到抑制。在傷口癒合的實驗裡，我們發現大量轉染表現PCDH21的MDA-MB-231，其細胞移動的能力比起未轉染的MDA-MB-231來得慢。此外，大量轉染表現PCDH21的MDA-MB-231，其細胞侵犯能力與在軟瓊膠 (Soft agar)形成細胞群落的能力也受到抑制。我們進一步將上述的乳癌細胞株以皮下注射的方式施打於 BALB/cAnN.Cg-Foxn1nu/CrlNarl裸鼠的乳腺組織附近之下腹位置，發現在注射施打具有PCDH21表現的MDA-MB-231，其裸鼠體內形成腫瘤的大小明顯小於未轉染的 MDA-MB-231 細胞。所以從上述的實驗結果證實，PCDH21的大量表現能抑制MDA-MB-231乳癌細胞的增生、移動、侵犯，以及形成細胞群落的能力。此外，在裸鼠活體的動物模式裡，PCDH21的大量表現也抑制了MDA-MB-231腫瘤生成的能力。總結本研究之結果，我們首次發現PCDH21於乳癌細胞中扮演著腫瘤抑制的功能性角色，同時該基因之腫瘤抑制於乳癌癌症演進上也相當地重要。

Cadherins play an important role in the cell-cell adhesion between adjacent cells. Meanwhile, cadherins also mediate cell morphology, contact inhibition, and signal transduction during tumorigenesis. There are at least 80 members of the cadherin superfamily in mammalians including classic cadherins, desmosomal cadherins, Fat like cadherins, and protocadherins. Over the past ten years, more than 60 members of protocadherins have been identified and constituted the largest subgroup in the cadherin superfamily. Up to now, many research reported that protocadherins are involved in the development of the central nervous system, but its cell functions in tumor cells of cancer research are poorly understood. In the previous study, we found that metastatic breast cancer cell line MDA-MB-231 expressed a lower level of PCDH21 (protocadherin γA-12) than nonmetastatic breat cancer cell line MCF7 in our lab. Addtionally, PCDH21 gene expression was regulated by DNA methylation. To investigate the functions of PCDH21 expressed in breast cells, we overexpressed the PCDH21 gene in the breast cancer cell line MDA-MB-231. In in vitro study, we examined the physiologic effects of PCDH21 on cell proliferation, cell migration, cell invasion and clonogenicity of breast cancer cell lines. In the cell morphology and cell proliferation assays, we found that overexpression of PCDH21 in the MDA-MB-231 changed cells into epithelial- type morphology. Addtionally, the cell proliferation of transfected MDA-MB-231 was suppressed. In the wound-healing assay, we found that overexpression of PCDH21 in the MDA-MB-231 was slower motility than the control of untransfected MDA-MB-231. Moreover, cell invasion and colony formation in soft agar culture were suppressed after overexpression of PCDH21 in the MDA-MB-231 cells. Furthermore, the cells has been injected subcutaneously into the abdominal region around the mammary site of BALB/cAnN.Cg-Foxn1nu/CrlNarl nude mice. We observed that the nude mice which were injected the PCDH21- overexpressed MDA-MB-231 (No.5 and No.6 clones) had smaller tumor size than those were injected the untransfected MDA-MB-231 cells. Among these studies, our results suggest that exogenous expression of PCDH21 suppressed the tumor cell growth, migration, invasion, and colony formation in MDA-MB-231 cells. Moreover, overexpression of PCDH21 also inhibited tumor formation of MDA-MB-231 in the nude mice model. Therefore, among our study, we first found that PCDH21 may play tumor suppressor roles in breast cells, and its role is critical and involved in the breast cancer tumorigenesis.