探討合併抑制第一型纖維母細胞生長因子受體及聚腺苷二
磷酸核糖聚合酶治療胰管腺癌之轉譯研究

胰臟癌始終為預後最差的癌症之一，舉凡化學治療、標靶藥物、免疫治療甚至細胞療法，均不見有明顯的成效。在致病機轉的探討上，K-RAS 致癌基因的活化具有重要的角色，而第一型纖維母細胞生長因子受體(FGFR1)在訊息傳導路徑上位於K-RAS 的上游，因此為了在胰臟癌治療中找尋新的可能治療標的，我們嘗試找出第一型纖維母細胞生長因子受體對胰臟癌生成的影響機制，並利用「合成致死」(synthetic lethality)的概念，併用藥物後達到毒殺癌細胞的效果。研究方法：首先利用過去胰臟癌病患資料，及使用過去診斷時所留下之剩餘組織切片，利用免疫組織染色法檢測FGFR1 表現量，並從中分析受體表現量與預後的關係。之後利用FGFR1 抑制劑評估作用在胰臟癌細胞株間的影響，並探討在癌細胞株形成、遷移性及侵入性及細胞密度變化的變化。另外嘗試併用聚腺苷二磷酸核糖聚合酶(PARP)抑制劑Olaparib 合併治療胰臟癌細胞株及觀察動物實驗上的結果是否一致研究結果：我們使用免疫組織化學和原位雜交分析發現胰臟癌中胰管腺癌患者中第一型纖維母細胞生長因子受體表達與疾病進展之間的強相關性。具高度乙醛脫氫酶活性的胰管腺癌細胞株可表現出似腫瘤幹細胞的表現型，以及較強的具相關幹細胞特性標誌，說明胰臟癌幹細胞中FGFR1/原癌基因酪氨酸激酶/核因子活化B 细胞kappa 輕鏈增?子信號傳遞在癌變發生時，可能會被優先激活。此外，使用FGFR1 抑制劑PD173074 抑制胰臟腫瘤幹細胞的效果可從八聚體結合轉錄因子4、SRY 盒-2、同源框蛋白質和編碼轉錄因子的調節基因的表現量降低以及X-連鎖的細胞凋亡抑制蛋白、细胞凋亡促進基因和生存素表達量受抑制觀察出來，且此現象隨著藥物劑量增加，抑制效果會更加明顯。經過生物信息學的基因表達分析後發現，FGFR1 與PARP 間有相互排斥及分子對接的關聯性，而在使用藥物治療細胞株時可發現，併用PD173074及Olaparib 都比單獨使用單一藥物來的效果顯著，最後在動物實驗上，不論是提早若延後合併使用藥物均可以有效延緩腫瘤生長。結論：本研究顯示，若藉由藥物抑制FGFR1 的信號傳遞，可以顯著減緩胰臟癌幹細胞的存活與增殖，並且可降低幹細胞具有的多能性轉錄因子活性。此外併用FGFR1 抑制劑及PAPR 抑制劑對難以治療或已產生抗藥性的胰臟癌患者，相信是個可行的治療策略，未來也許可以發展相關的臨床試驗而期待能早期付諸臨床應用。

關鍵字

胰臟癌、抗藥性、第一型纖維母細胞生長因子受體、FGFR1 抑制劑 (PD173074)、聚腺苷二磷酸核糖聚合酶、PARP 抑制劑(Olaparib)

並列摘要

Although pancreatic ductal adenocarcinoma (PDAC) may develop over 20 years, it is usually diagnosed at advanced or metastatic stages. Therefore, even under gemcitabine monotherapy, the 1-year overall survival of patients with advanced disease is less than 20%, with a median survival of less than 6 months. KRAS is mutated in approximately 90% of PDACs, and the fibroblast growth factor receptor (FGFR) pathway is the third most frequently altered pathway in cancer, after the p53 and KRAS pathways. Many pancreatic cancers overexpress a number of growth factors and their receptors, including FGF and FGFR. Alterations in FGFR1 signaling modulate growth in pancreatic cancer cells. However, the effects of FGFR1 on apoptosis and DNA damage in PDAC have not been widely surveyed. We investigated the antitumor and anti-metastasis efficacy of the selective FGFR1 inhibitor, PD173074 in PDAC. We performed extensive immunohistochemical work and employed in situ hybridization techniques to identify a strong correlation between FGFR1 amplification and/or expression and disease progression. We demonstrated that pancreatic cancer cells with high aldehyde dehydrogenase activity (ALDHhigh) exhibited higher expression of FGFR1, Src and NF-κB and higher activity of stemness markers of Oct4 and Sox2 compared with control groups with null or low ALDH activity (ALDHlow/null). The FGFR1/Src/NF-κB signaling axis may play a role in pancreatic cancer stem cells (panCSCs) activation. Under treatment with PD173074, pancreatic cancer cell lines with ALDHhigh/FGFR1-rich phenotype exhibited proliferation and suppressed self-renewal ability of panCSCs. Apoptosis was induced by caspase-3 and poly (ADP ribose) polymerase (PARP) activation. The efficacy of the anti-CSCs effect of PD173074 was dose-dependently related to decreased expression of Oct4,Sox-2, Nanog, and c-Myc, and suppression of antiapoptotic expression of proteins such as XIAP, Bcl2, and survivin. Activation of cMet, Src, ERK 1/2 and NFκB (p65) was also inhibited by PD173074. Another clinical relevant finding is that the disruption of the FGFR1/Src/NF-κB signaling axis positively correlated with poor clinical prognosis. We concluded that PD173074 suppresses the tumorigenesis and CSCs-like phenotype of PDAC cells. “Synthetic lethality” may provide a therapeutic paradigm. The concept describes a process wherein mutations in two genes together result in cell death but independently do not affect viability. Because of the innovation of genetic interaction networks in model organisms, synthetic dosaglethality and conditional synthetic lethality could expand the scope of studies on conventional synthetic lethality. Resistance to FGFR1 inhibitors is required. The caspase-3/PARP-mediated antitumor and antimetastatic efficacy of PD173074 against ALDHhigh/FGFR1-rich PDAC cells was partially demonstrated. We investigated the probable synthetic lethality and therapeutic efficacy of targeted PARP inhibition combined with FGFR1 blockade in patients with PDAC. Using bioinformatics-based analyses of gene expression profiles, co-occurrence and mutual exclusivity, molecular docking, immunofluorescence staining, clonogenicity, Western blotting, cell viability or cytotoxicity screening, and tumorsphere formation assays, we demonstrated that FGFR1 and PARP co-occur, form a complex, and reduce survival. Furthermore, FGFR1 and PARP expression was upregulated in multi-targeted kinase inhibitor (dasatinib)-resistant PDAC cell lines SU8686, MiaPaCa2, and PANC-1 compared with sensitive cell lines Panc0403, Panc0504, Panc1005, and SUIT-2. Compared with the limited effect of single-agent olaparib (PARP inhibitor) or PD173074 on PANC-1 and SUIT-2 cells, low-dose combination (olaparib + PD173074) treatment significantly, dose-dependently, and synergistically reduced cell viability；upregulated cleaved PARP, pro-caspase (CASP)-9, cleaved-CASP9, and cleaved-CASP3 protein expression；and downregulated Bcl-xL protein expression. Furthermore, the combination treatment markedly suppressed the clonogenicity and tumorsphere formation efficiency of PDAC cells, regardless of FGFR1 inhibitor-resistance status, and enhanced RAD51 and γ-H2AX immunoreactivity. In vivo studies have demonstrated that both early and late initiation of combination therapy markedly suppressed tumor xenograft growth and increase in weight. Thus, FGFR1 inhibitor-resistant PDAC cells exhibited sensitivity to PD173074 after olaparib-mediated loss of PARP signaling. The present FGFR1/PARP-mediated synthetic lethality proof-of-concept study provides preclinical evidence of the feasibility and therapeutic efficacy of combinatorial FGFR1/PARP1 inhibition in human PDAC cell lines. The combination therapy may be feasible for patients with advanced PDAC or with developed drug resistance. Future clinical trials may be developed for early clinical application.