鑑尋參與攝護腺癌轉移的重要蛋白

Abstract

攝護腺癌是全球男性第二常見的癌症。同時，攝護腺癌也高居我國國民第六大癌症死因。由於癌轉移是造成攝護腺癌病患死亡的主因，了解其背後的分子機制對於鑑尋新的生物標靶及治療方式相當重要。為了尋找調控攝護腺癌轉移的重要因子，我們利用攝護腺癌轉移模型 CWR22Rv1-M4 進行研究。在此，我的實驗結果顯示，M4 細胞的移動、侵襲及非貼附性生長能力皆高於 CWR22Rv1 細胞。次世代定序的結果揭示了許多 CWR22Rv1 與 M4 細胞間差異表現的基因。我們挑選出在細胞周圍作用者，包括 EPHA7, CASP4, MMP14, PAI3, PN1, PRSS8, DPP4, PRSS21 及 PRSS23。我更進一步使用 q-RT-PCR 驗證這些基因的差異表現。七個顯著高表現於 M4 細胞的基因包含 EPHA7, CASP4, MMP14, PAI3, PN1, PRSS8 及 DPP4；而兩個於 M4 細胞中顯著低表現的基因為 PRSS21 及 PRSS23。首先，我藉由文獻查詢及西方墨點法排除三個基因 (EPHA7, PN1及PRSS21)。為了探討以上基因的差異表現是否在攝護腺癌轉移過程中扮演角色，我使用基因敲落的方式，發現 PAI3 及 PRSS23 對於癌細胞的移動及侵襲能力無影響。同時，在 M4 細胞中敲落 MMP14 可降低細胞的移動及侵襲能力，而 DPP4 的敲落則會提升 M4 細胞的移動及侵襲能力。此外，DPP4 競爭型抑制劑 Diprotin A 可以提升 M4 細胞的移動及侵襲力。有趣的是，DPP4 的敲落會降低 M4 細胞的非貼附性生長能力，而在 CWR22Rv1 細胞中敲落 PRSS23 的表現可同時降低細胞的非貼附性生長及群落生成能力。以上結果為這些蛋白參與攝護腺癌轉移的可能提供新的見解。

Prostate cancer (PCa) is the second most common cancer among men worldwide. In Taiwan, PCa is the sixth cause of cancer-related mortality. In fact, metastasis contributes to most PCa casualties. Therefore, understanding the molecular mechanisms behind PCa metastasis is important for identifying novel biomarkers as well as developing new therapeutic approaches. In order to identify important factors regulating PCa metastasis, we applied a CWR22Rv1-M4 PCa metastatic progression model for the study. In this study, I showed that M4 cells had increased cell motility and anchorage-independent growth capacity when compared with parental CWR22Rv1 cells. Meanwhile, many genes with differential expressions between CWR22Rv1 and M4 cells were identified via next-generation sequencing (NGS) approach. We focused on genes functioning around pericellular surface, including EPHA7, CASP4, MMP14, PAI3, PN1, PRSS8, DPP4, PRSS21 and PRSS23. The differential expressions of the above genes were further validated with q-RT-PCR. Seven genes with significantly higher expressions in metastatic M4 cells were EPHA7, CASP4, MMP14, PAI3, PN1, PRSS8 and DPP4, while two genes with significantly lower expressions in M4 cells were PRSS21 and PRSS23. Three genes were excluded from my study after literature mining (EPHA7 and PN1) or western blot analysis (PRSS21). To explore if the rest of the genes participate in PCa metastatic progression, I applied short hairpin (sh) RNA knockdown approaches and found that PAI3 and PRSS23 had no significant impacts on PCa cell migration and invasion, suggesting that these two genes were not involved in the regulation of PCa cell motility. Knockdown of MMP14 decreased M4 cell migration and invasion, while DPP4 knockdown resulted in enhanced M4 cell motility. Furthermore, inhibiting DPP4 enzymatic activity with competitive inhibitor Diprotin A suppressed M4 cell motility. Interestingly, knockdown of DPP4 resulted in decreased anchorage-independent growth of M4 cells, whereas knockdown of PRSS23 in CWR22Rv1 cells resulted in decreased clonogenic and anchorage-independent growth. These results provided insights into potential involvements of these proteins in PCa metastatic progression.