網格重鏈蛋白透過調控缺氧誘發因子-1α及血管新生因子之訊息傳遞以促進腫瘤生長及血管的新生

Abstract

胰腺癌是一種惡性以及高度致死的疾病，目前尚未有療效的治療方式提供選擇，為了提升胰腺癌的治療成效，本研究利用單株抗體技術生產Pa65-2抗體，其能專一的辨認胰腺癌細胞及腫瘤血管，並具有不辨認正常細胞的特性。Pa65-2的標靶蛋白經鑑定為人類網格重鏈蛋白 (human clathrin heavy chain, CHC)。我們發現使用小髮夾型核醣核酸(Short hairpin RNA，shRNA) 減少CHC表現的胰腺癌細胞，不僅可以減少了腫瘤細胞的增殖 (proliferation)、集落形成 (colony formation) 和侵襲 (invasion) 能力，同時也會誘導腫瘤細胞凋亡的產生。過去研究證實CHC主要功能是與細胞內吞作用有關,而透過Pa65-2處理後的胰腺癌細胞，可以抑制表皮生長因子(epidermal growth factor, EGF) 及運鐵蛋白 (transferrin, Tf) 的吞入作用，進而抑制腫瘤細胞的生長作用。在異體移植人類腫瘤的動物實驗也顯示，透過shRNA減少CHC的表現或利用Pa65-2治療，可以抑制腫瘤的生長和血管新生。本研究我們證實CHC在調控血管新生扮演了重要的角色，當細胞於缺氧情況下,CHC會與缺氧誘導因子（HIF）-1alpha蛋白結合，並幫助HIF-1alpha蛋白的穩定,促進其核轉位與其下游基因的調控子缺氧反應元件結合 (hypoxia responsive element , HRE) 進而調控血管內皮生長因子（vascular endothelial growth factor , VEGF）的表達。當透過shRNA減少CHC表現的結果，可以觀察減少了HIF-1alpha的蛋白以及下游基因，例如:VEGF, 紅血球生成素 (erythropoietin, EPO) 以及血小板生長因子-B(platelet-derived growth factor-B,PDGF-B)的表現量。綜合上述研究證實, CHC在腫瘤血管新生及腫瘤生成的過程扮演重要的角色。利用Pa65-2單株抗體或是CHC shRNA治療，未來有潛力發展為胰臟癌的治療策略。

Pancreatic adenocarcinoma is an aggressive disease with a high mortality rate. Currently, treatment options are limited. In an effort to improve the efficacy of treatments for pancreatic adenocarcinoma, we have newly generated a monoclonal antibody (mAb), Pa65-2, which specifically binds to pancreatic cancer cells and tumor blood vessels but not to normal cells. The target protein of Pa65-2 is identified as human clathrin heavy chain (CHC). We found that knockdown of CHC not only reduced cancer cell proliferation, colony formation and invasion, but it also induced cancer cell apoptosis. Additionally, treatment of Pa65-2 blocked cancer cells’ uptake of epidermal growth factor (EGF) and transferrin (Tf), following the inhibition of cancer cell growth. In vivo study showed that suppression of CHC either by shRNA or by Pa65-2 inhibited tumor growth and angiogenesis. In this study, we demonstrated that CHC plays an important role in the regulation of angiogenesis. Under hypoxia, CHC was to bind with the hypoxia-inducing factor (HIF)-1alpha protein, increasing the stability of this protein, facilitating its nuclear translocation and hypoxia responsive element (HRE) promoter binding and thereby regulating the expression of vascular endothelial growth factor (VEGF). Knockdown of CHC results in downregulations of both HIF-1alpha