水解磷酸脂促進PC-3前列腺癌細胞株之鈣網蛋白表現

Abstract

鈣網蛋白 (Calreticulin, CRT) 為一種多功能之伴護蛋白 (Chaperone protein)，在許多種類的癌症組織切片中，皆發現CRT蛋白質的表現量相對高於正常組織。而在我們實驗室先前的研究中，已證實CRT會促進胃癌和膀胱癌之腫瘤生長及轉移。但在攝護腺癌中，CRT所扮演的角色及其調控機制並不清楚。本研究中，我們首先針對早期攝護腺癌細胞株LNCaP與晚期攝護腺癌細胞株PC-3進行比較，發現PC-3的細胞貼附能力以及細胞增生能力皆顯著高於LNCaP，並且PC-3比LNCaP表現更多量的CRT與血管內皮生長因子A型（Vascular endothelial growth factor A, VEGF-A）。為了進一步研究CRT在攝護腺癌細胞癌化中扮演的角色，我們建立了穩定降低CRT表現之PC-3細胞株，結果顯示抑制CRT之表現會顯著地降低PC-3之細胞貼附能力以及細胞增生能力，此外VEGF-A之表現量顯著下降。由這些結果可推論CRT為影響攝護腺癌細胞癌化之重要調控因子。接著我們試著找出造成CRT於晚期攝護腺癌細胞株增量表現的上游調控分子。水解磷酸脂 (Lysophosphatidic acid, LPA) 是一種多功能的信息傳遞分子，已知會影響攝護腺癌細胞株之增殖 (proliferation)、轉移 (migration) 以及侵入 (invasion)。先前已有研究顯示，在小鼠D3 ES (D3 embryo stem cells) 細胞中，LPA會經由磷脂酶C（phospholipase C, PLC）的路徑調控內質網 (endoplasmic reticulum, ER) 的鈣離子 (Ca2+) 流失。另一方面，在小鼠纖維母細胞NIH/3T3細胞株中，ER的Ca2+流失會活化CRT的啟動子。根據這些研究，我們假設在攝護腺癌細胞株中，LPA會促進CRT的表現。實驗結果證實，在晚期攝護腺癌細胞株PC-3中，LPA會促進CRT之RNA和protein表現。但在早期攝護腺癌細胞株LNCaP中，LPA卻無促進CRT表現之作用。而利用LPA受器之拮抗劑 (antagonist) 或促進劑 (agonist) 以及基因敲落 (gene knockdown) 或基因大量表達 (gene overexpression) 的方式來對LPA受器進行操作的實驗中，我們發現在晚期攝護腺癌細胞株PC-3中，LPA可經由細胞表面受器LPA1和LPA3促進CRT的表現; 但同時LPA亦可經由細胞表面受器LPA2抑制CRT的表現。這些結果顯示，透過調整細胞表面的LPA受器表達模式 (LPA receptor expression pattern) ，或可有效調控CRT之表現並進一步控制攝護腺癌細胞之癌化。

Calreticulin (CRT), a multifunctional Ca2+-binding chaperone, has been shown to associate with poor prognosis in gastric cancer and bladder cancer. However, the roles of CRT in prostate cancer remain elusive. Prostate cancer is one of the most frequently diagnosed cancers in males and PC-3 is a popular cell model for investigating late stage prostate cancer. By comparing early stage prostate cancer cell line LNCaP with late stage prostate cancer cell line PC-3, we found that PC-3 showed higher cell adherent ability, cell proliferation ability and higher expression of vascular endothelial growth factor-A (VEGF-A), which is an important regulator for angiogenesis and tumor growth. Furthermore, knockdown of CRT in PC-3 caused lower cell adherent ability , cell proliferation ability and VEGF-A expression. These results indicate that CRT may be a poor prognosis factor in prostate cancer. Subsequently we further investigate the upstream regulation mechanism for calreticulin expression. Lysophosphatidic acid (LPA), a low molecular weight lipid, has been proved to stimulate cell migration, invasion and proliferation in prostate cancer cells. It has been demonstrated that LPA evoked Ca2+ mobilization from the lumen of the endoplasmic reticulum (ER) via phospholipase C (PLC) pathway. On the other hand, depletion of Ca2+ from ER activated CRT promoter activity in NIH/3T3 cells. Based on these evidences, we hypothesized that LPA regulate CRT expression in prostate cancer cells. By using RT-qPCR and Western Blotting, we found that CRT expression is up-regulated both in mRNA and protein level after LPA treatment. Pharmacological blockade by LPA1-specific antagonist AM966 or LPA1/3-selective antagonist Ki16425 inhibits the enhancement effect of LPA on CRT expression. In addition, LPA-dependent CRT expression was abolished in LPA1 and LPA3 stable knockdown PC-3 cells. Furthermore, activation of LPA3 by LPA3-specific agonist OMPT enhances CRT expression. These results indicated that activation of LPA1 and LPA3 up-regulate the expression of CRT. On the contrary, activation of LPA2 by LPA2-selective agonist MDP and LPA2-specific agonist GRI977143 impaired CRT expression. In conclusion, our findings suggested that, LPA1/3 and LPA2 inversely regulate CRT expression and subsequently regulate cell adhesion, cell proliferation and VEGF-A expression in PC-3 cells.