新穎蛋白嵌合體VEGF121-VEGF165調控腫瘤血管新生及缺氧誘導因子訊號路徑之機制研究

Abstract

抗血管新生治療是現今癌症治療的方法之一，很遺憾的是，針對抗VEGF-A的抗血管新生療法，則出現抗藥性且增加癌症轉移等問題。抗藥性來自腫瘤內無血管供應氧氣造成嚴重「缺氧」，癌細胞為求生存，本身會分泌VEGF-A，不斷地回饋刺激VEGFR2-HIF-1α-VEGF路徑，形成自泌回饋環(autocrine loop)，讓癌細胞儘快逃離目前惡劣的環境因而造成轉移。為了獲得更有效的癌症治療藥物及降低藥物抗藥性，我們在六種VEGF-A異構物 (isoform)中，測試挑選出缺乏Heparin binding domain的VEGF121及VEGF165的兩個單體(monomer)為基本單元，透過連接一段人類抗體IgG1的Fc固定區，利用多胜肽鏈連接的方式，建構出VEGF121-VEGF165二聚蛋白嵌合體。連接Fc區域不僅可增強二聚體的結合、增強蛋白嵌合體的穩定度、還可增強免疫細胞的活性。我們試圖以拮抗弱化的理論，用VEGF121-VEGF165來競爭取代，分別由自泌 (autocrine)及旁泌(paracrine)兩大方面來抑制，弱化下游的訊號傳遞，以避免癌細胞中心出現嚴重缺氧的情況。由in vitro及in vivo實驗證實，VEGF121-VEGF165可競爭VEGF165二聚體，使VEGF165旁泌和自泌的現象降低，且使得在內皮細胞或是癌細胞的增殖、遷移、侵襲或形成管狀結構的能力也大幅度的下降。機制方面，嵌合蛋白質會拮抗癌細胞所產生抗藥性的路徑: 透過 PI3K-AKT-mTOR途徑來抑制HIF-1α-VEGF165/Lon。總結，VEGF121-VEGF165，它透過競爭VEGFR2接受體，調控細胞內訊號傳導訊息來降低細胞外過量的VEGF-A。因此VEGF121-VEGF165不僅能阻斷內皮細胞進行血管新生外，亦能解決癌細胞的HIF-1α-Lon過度表現產生抗藥性的問題，這將是一個具有發展潛力的拮抗血管生成之癌症治療蛋白質藥物，有機會成為癌症病人的一道新曙光。

Anti-angiogenesis therapy is one major approach of cancer therapies nowadays. Unfortunately, anti-angiogenesis therapy targeting VEGF-A was recently stumbled by the drug-resistance that results from adaptive responses. Accumulating study suggested that intratumor hypoxia has a critical role in many adaptive mechanisms. To obtain a more efficient therapeutic response and attenuate the drug-resistance, we created and identified a novel chimeric fusion of VEGF121 and VEGF165, which was connected by Fc region of human IgG1 to enhance dimerization. We found that the treatment of VEGF121-VEGF165 chimeric protein reduces proliferation, migration, invasion, and tube formation of endothelial and/or cancer cells through competing VEGF165 homodimer in a paracrine and an autocrine manner. Furthermore, the fusion protein attenuated autocrine VEGFR2-HIF-1α-VEGF165/Lon signaling through PI3K-AKT-mTOR pathway in cancer cells. In conclusion, our data demonstrated that the chimeric VEGF121-VEGF165 arrests the tube formation of endothelial cells and interferes with tumor cell growth, migration and invasion, suggesting that it could be a potential drug as an angiogenesis antagonist in cancer therapy. The VEGF121-VEGF165 targets not only paracrine angiogenic cascade of endothelial cells but also autocrine PI3K-AKT-mTOR mediated VEGFR2-HIF-1α-VEGF165/Lon signaling that drives drug resistance in tumor cells. Our study will open up the patient opportunities to combat drug resistance to antiangiogenic therapy.