AKT3在非小細胞肺癌EGFR-TKI抗藥性之研究

Abstract

肺癌高居全球癌症死亡之首位。在肺癌治療中，EGFR-TKI為非小細胞肺癌有EGFR活化突變之第一線治療，然而病人在使用9-14個月後會產生acquired resistance，因此了解EGFR-TKI之抗藥性機轉是改善臨床療效之重要課題。為了深入研究抗藥性細胞特性及機轉，我們培養出第一代EGFR-TKI抗藥性細胞HCC827/IR (IRESSA resistance)及第三代EGFR-TKI抗藥性細胞H1975/AR (AZD9291 resistance)，均具有EMT之特性。 在這篇研究中，抗藥性細胞之EGFR表現及活性均降低，其他RTK如AXL、FGFR1之表現在HCC827/IR亦高度上升。HCC827/IR (非T790M突變) 及H1975/AR (非C797S突變) 之抗藥性方式為“off target resistance”。EGFR-TKI在parental細胞抑制AKT活性 (p-AKT) 之效果比resistant細胞好。RNA-seq之分析發現，AKT3在抗藥性細胞之表現均高度上升，Kaplan Meier plotter分析亦發現，AKT3高度表現之肺腺癌病人預後較差。在HCC827/IR knockdown AKT3可降低細胞之爬行且增加E-cadherin之表現，亦經由抑制之細胞S週期降低細胞之生長；在H1975/AR knockdown AKT3，可增加AZD9291對AKT活性 (p-AKT) 之抑制作用。利用免疫沉澱法證明AKT3對AKT之活性有貢獻，gefitinib及AZD9291皆無法抑制AKT3之活性 (p-AKT3)。HCC827/IR處理gefitinib或H1975/AR處理AZD9291皆會增加AKT3蛋白。因此，AKT3可能可成為預測EGFR-TKI療效之生物標記，其表現量之上升是EGFR-TKI之抗藥性機轉之一。此外，合併AZD9291與AKT inhibitor對於抑制H1975/AR之生長具有協同作用。

Lung cancer is the leading cause of cancer deaths worldwide, and EGFR-TKI is the first-line treatment for non-small cell lung cancer (NSCLC) harbouring EGFR activation mutation. However, most patients develop acquired resistance within 9–14 months. Therefore, it is critical to explore the mechanisms of drug resistance to improve treatment efficacy. Two resistant cells, HCC827/IR (IRESSA resistance) and H1975/AR (AZD9291 resistance) exhibiting EMT phenotypes were generated to investigate the molecular and cellular characteristics of the EGFR-TKI acquired resistance. The expression and activation of EGFR were reduced in both resistant cells. Upregulation of AXL and FGFR1 were found in HCC827/IR but not H1975/AR cells. HCC827/IR (without T790M) and H1975/AR (without C797S) showed “off target resistance”. Activation of AKT (p-AKT) in both parental cells was more sensitive to EGFR-TKI than that in resistant cells. RNA-seq revealed that AKT3 was upregulated in both resistant cells. High expression of AKT3 was predicted to correlate with the poor survival of lung adenocarcinoma patients. Knockdown of AKT3 in HCC827/IR cells inhibited cell migration and increased the protein expression of E-cadherin, as well as inhibited S phase population to reduce cell proliferation. Knockdown of AKT3 in H1975/AR cells enhanced AZD9291-induced inhibition on AKT activation (p-AKT). Immunoprecipitation of AKT3 in both resistant cells demonstrated its involvement in AKT activation, and AKT3 activation (p-AKT3) was not sensitive to gefitinib and AZD9291 inhibition in resistant cells. We also found that protein but not mRNA of AKT3 was upregulated in gefitinib-treated HCC827/IR cells and AZD9291-treated H1975/AR cells. Therefore, AKT3 might serve as a predictive biomarker for EGFR-TKIs therapy, and its upregulation might be one of the mechanisms for EGFR-TKIs acquired resistance. Besides, combination of AZD9291 with AKT inhibitor elicited synergistic inhibition on cell viability of H1975/AR cells.