Study On The Activity And Molecular Mechanism Of Gefitinib Combined With YC-1 In Vitro Against Non-small Cell Lung Cancer

Lung cancer is the leading cause of cancer related deaths in China and worldwide.Non-small-cell lung cancer（NSCLC）accounts for approximately 85%of lung cancer cases.Aberrant expression of EGF receptor（EGFR）is identified to cause aggressive phenotypes and predict poor prognosis.Gefitinib is a well-known first-generation EGFR tyrosine kinase inhibitor（EGFR-TKI）that exhibits good antitumor activity in molecularly selected NSCLC patients with L858R/del19 mutations of EGFR.However,despite the initial response and excellent disease control with EGFR-TKI therapy,acquired resistance is inevitable and raises an immense challenge in NSCLC therapy.Moreover,about 20%to 30%of NSCLC patients with the EGFR activating mutations and many NSCLC patients who overexpress wild-type EGFR respond poorly to EGFR-TKI treatment.This evidence highlights that innovative strategies are urgently needed to overcome both primary and acquired resistance to EGFR-TKIs in NSCLC patients.Accumulating evidence suggests that EGFR has many other functions beyond kinase activity that have been known to play an essential role in cancer pathology.These non-canonical（ligand-and kinase-independent）EGFR signaling pathways are also responsible for the primary and acquired resistance of EGFR-TKIs.Currently,a model of two functional statuses of EGFR in EGFR-dependent cancer cells was revealed.The kinase activatable EGFRs are mainly involved in promoting cell growth,and the kinase unactivatable EGFRs（mainly acting as a scaffold protein）,which are blocked from autocross-phosphorylation by interacting proteins,are mainly in charge of promoting oncogenic cell survival.In cancer cells harboring kinase-activating mutations,the function of EGFR is mainly dependent on it’s kinase activity,which sensitizes these cancers cells to EGFR-TKIs;In cancer cells over-expressing wild type EGFR,the role of EGFR is shifted toward its kinase-independent functions,which display innate resistance to EGFR-TKIs treatment despite the efficient inhibition of tyrosine kinase activity;In addition,long-term EGFR-TKIs treatment can not only induce mutations of target molecular,but tilte the role of EGFR toward its kinase-independent functions that provoked noncanonical pathways of EGFR trafficking and signaling,for example,elicited abnormal endosomal accumulation of EGFR,prolonged abnormal oncogenic signaling,initiated protective autophagy and inhibited apoptosis,which provides cancer cells with a survival advantage and confers resistance to TKIs treatment.Inspired by these observations,we believe that cotargeting EGFR kinase-dependent and-independent functions may hold new promise for treating EGFR-TKI resistant cancers.In our present study,a commercial LOPAC library from Sigma of 1280pharmacologically active compounds was screened in combination with Gefitinib through cell proliferation assay.We found for the first time that YC-1 exhibited an unexpected ability to sensitize NSCLC cells to Gefitinib treatment.Since 1994,YC-1（Lificiguat,3-（5’-hydroxymethyl-2’-furyl）-1-benzylindazole）has been synthesized and it was initially described as an activator of soluble guanylyl cyclase（s GC）,prompting antiplatelet aggregation and vascular relaxation.More recent studies revealed the ability of YC-1 to inhibit tumor growth,suppress angiogenesis and enhance antitumor effects of radiation;these abilities have been associated with its activity to inhibit hypoxia-inducible factor 1α（HIF-1α）.We found the synergistic anti-tumor effect of YC-1 plus Gefitinib was attributed to cotargeting EGFR kinase-dependent and-independent functions.The important roles of HIF-1αand Fox O1pathways in influencing EGFR kinase activity-independent signaling and improving Gefitinib resistance were also identified.Collectively,we can concluded that:i)YC-1has an ability to sensitize NSCLC cells with different EGFR gene status to Gefitinib treatment;ii)YC-1 improves the primary and acquired resistance to Gefitinib by accelerating the endocytic trafficking and degradation of EGFR^WT and EGFR ^L858R/T790Mdelayed by Gefitinib,quenching the EGFR kinase-independent signaling pathway,and this effect also depended on the inhibition of HIF-1αsignaling pathway;iii)YC-1 can significantly block the autophagy induced by Gefitinib by disrupting the fusion of autophagosomes and lysosomes,and this effect is also an important mechanism to improve primary and acquired resistance to Gefitinib;iv)The combinational anti-autophagic and pro-apoptotic effects of Gefitinib and YC-1 is due to the enhancement of Fox O1 transcriptional activity by downregulating p-Fox O1（ser-256）levels.In summary,our study provided new evidence for the important role of EGFR kinase-independent signaling pathways in the primary and acquired resistance of NSCLC cells to EGFR-TKIs,new approachs for targeting EGFR kinase-independent signaling pathway,and a new insight into understanding the mechanisms responsible for NSCLC resistance.It also provides new ideas and new treatment strategies for patients with resistant NSCLC.