探討14-3-3σ調控肝細胞癌之細胞遷移與侵入所扮演的角色

Abstract

14-3-3σ (也稱為 stratifin, SFN) 是14-3-3家族成員之一，其調控功能在腫瘤之癌化過程目前仍然具有爭議。過去的研究結果顯示，14-3-3σ在許多惡性腫瘤發展中可能扮演著腫瘤抑制者的角色或促進腫瘤生長。此外，也有證據顯示14-3-3σ 能促進多種惡性腫瘤的發生，其中包含了肝細胞癌 (hepatocellular carcinoma, HCC)。然而，14-3-3σ 如何導致肝癌細胞的細胞增生、細胞遷移、細胞侵入及腫癌細胞轉移之分子機制目前仍未完全清楚。本論文之要著重在研究 (i) 14-3-3σ可誘導下游基因進而影響肝癌之發展; (ii) 進一步探討腫瘤微環境中 (tumor microenvironment) 14-3-3σ與腫瘤細胞及間質細胞之相互用對肝癌細胞癌進程之影響。14-3-3σ可大量表現在人體肝腫瘤組織並調控下游重要基因，熱休克因子-1 (Heat shock factor-1, HSF-1) 及熱休克蛋白70 (Heat shock protein 70, HSP70)。若以穩定表達或是短暫轉染14-3-3σ 之肝癌細胞株可增加HSF-1和HSP70表現。此外，HSF-1及HSP70亦大量表現在肝腫組織且由統計分析其三者之間有顯著之關連性。並進一步由臨床病理特性中發現，14-3-3σ及HSP70 與細胞遷移或侵入有顯著關聯性。利用穩定表達14-3-3σ肝癌細胞株證實，14-3-3σ誘導 HSF-1 及 HSP70 表現增加進而促進肝癌細胞遷移 ; 反之，若以小分子干擾核醣核酸降低細胞內HSP70表現量時，亦能有效抑制此現象。更進一步抑制β-catenin表現或活化GSK-3β訊息路徑普可顯著減少14-3-3σ促進之細胞遷移。收集穩定表現14-3-3σ 肝癌細胞株之培養液 (14-3-3σ-conditioned media, CM)，將此培養液個別培養 H68 fibroblasts (纖維母細胞)， THP-1 (人類單核球細胞株) and phorbol-12-myristate-13-acetate (PMA)-treated THP-1 (PMA-THP-1，PMA誘導之巨噬細胞) 三種間質細胞後，再將此間質細胞與親代肝癌細胞株 (Parental Huh-7 cell line) 同時培養在Transwell細胞培養盤上進行細胞侵入能力之分析。結果發現，14-3-3σ-CM培養之三種間質細胞皆可有增加肝癌細胞侵入能力。此外，14-3-3σ-CM培養液可以誘導多種基質轉移蛋白酶 (matrix metalloproteinases, MMPs) 大量表現。以纖維母細胞為例，14-3-3σ-CM可增加MMP-1，MMP-2， MMP-9， MMP-12，MMP-14大量表現; 在人類單核球細胞株可增加MMP-1及MMP-12; 在巨噬細胞株可增加MMP-2, MMP-12和MMP-14 。反之，若以小分子干擾核醣核酸干擾14-3-3σ分泌到胞外亦可顯著減少經14-3-3σ-CM誘發產生之MMPs表現。不論以穩定表達14-3-3肝細胞株或人工純化14-3-3σ重組蛋白 (r14-3-3σ protein) 方式，皆可證實14-3-3σ可分泌至胞外並增加周圍間質細胞產生MMPs。進一步探討其分子機轉發現，14-3-3σ-CM或r14-3-3σ蛋白誘導產生的MMPs表現是透過影響胺肽酶N (aminopeptidase N, APN) 之表現量。使用MMP抑制劑 (GM6001)或以小分子干擾核醣核酸干擾 APN 皆可有效抑制14-3-3σ-CM誘導癌細胞侵入能力。 促進細胞侵入之能力。綜合結果，本研究證實14-3-3σ調控肝癌的進程不只透過促進肝癌細胞遷移作用，亦可增加分泌型14-3-3σ (HCC-secreted 14-3-3σ) 表現誘導周圍間質細胞產生MMPs蛋白，進而幫助癌細胞增加其侵入的能力速肝癌惡化。

14-3-3σ (also known as stratifin, SFN) is a member of 14-3-3 family and its role in regulating tumor progression remains controversial. Results from earlier studies suggest 14-3-3σ plays as a potential tumor suppressor or promoter in various human malignancy including hepatocellular carcinoma (HCC). However, the molecular mechanism by which 14-3-3σ confers HCC cell proliferation, migration, invasion as well as tumor metastasis remains unclear. In this study, we investigated the potential downstream targets of 14-3-3σ in modulating HCC development and the interaction with surrounding tumor associated stromal cells. We found that 14-3-3σ is abundantly expressed in HCC tumors. Both heat shock factor-1α (HSF-1) and heat shock protein 70 (HSP70) are major downstream factors of 14-3-3σ. Stable or transient over-expression of 14-3-3σ induces the expression of HSF-1 and HSP70 in HCC cells. 14-3-3σ mediated cell migration is impaired by siRNA knockdown of HSP70. 14-3-3σ-induced HSF-1α/HSP70 expression and cell migration is also abolished by knockdown of β-catenin or activation of GSK-3β. Moreover, both 14-3-3σ and HSP70 overexpression associated with micro-vascular thrombin in HCC patients. The results suggested that both 14-3-3σ/HSP70 expression are potentially involved in cell migration/invasion. In addition, HCC cells co-cultured with 14-3-3σ-conditioned media (CM) treated stromal cells (H68 fibroblasts, THP-1 and phorbol-12-myristate-13-acetate (PMA)-treated THP-1 (PMA-THP-1) significantly enhanced their invasive ability compared with control-CM treated cells. Incubation with 14-3-3σ-CM induced differential expression profiles of matrix metalloproteinases (MMPs) in fibroblasts (MMP-1, MMP-2, MMP-9, MMP-12 and MMP-14), THP-1 (MMP-1 and MMP-12) and PMA-THP-1 cells (MMP-2, MMP-12 and MMP-14). In contrast, silencing of 14-3-3σ by siRNA significantly abolished 14-3-3σ-CM induced MMPs. Treatment of all stromal cells (HS68, THP-1 and PMA-THP-1) with recombinant human 14-3-3σ (r14-3-3σ) protein exhibits a similar expression profile of MMPs induced by 14-3-3σ-CM. Knockdown of aminopeptidase N (APN) significantly abrogated 14-3-3σ-CM or r14-3-3σ induced expression of MMPs in HS68 fibroblasts. Finally, HCC-secreted 14-3-3 promotes cell invasion was significantly abolished by APN siRNA or treated with GM6001 (an inhibitor of MMPs). Taken together, our findings suggest that 14-3-3σ regulates HCC tumor progression not only by promoting cancer cell migration but also educating stromal cells in the tumor-associated microenvironment. 14-3-3σ is thus a potential biomarker and therapeutic target of HCC.