Molecular Mechanisms Of Resistance To EGFR TKIs Driven By Aryl Hydrocarbon Receptor In Non-small Cell Lung Cancer

Lung cancer is the leading cause of cancer-related mortality world wide.Non-small cell lung cancer(NSCLC)is the most common type of lung cancer,which accounts for over85% of all cases,and tremendous efforts has been made to understand the molecular mechanisms of NSCLC tumorigenesis and progression.In the light of rapidly development of cancer cell biology,we began to realize that NSCLC is no longer a single disease,instead,it is a group of heterogeneous malignancies driven by oncogenes,and the all-in-one way treatment strategy is no longer applicable.The identification of activating mutations in epidermal growth factor receptor(EGFR)and rearrangement in anaplastic lymphoma kinase(ALK)and ROS1 kinase has led a new era of precision medicine,in which gefitinib-,erlotinib-and crizotinib-based targeted therapy significantly improved the prognosis and quality of life in NSCLC patients with distinct clinicopathological features.Unfortunately,resistance to these tyrosine kinase inhibitors(TKIs)inevitably occurs within years after treatment and severely impaires the long-term clinical efficacy of targeted therapy.The underlying mechanism of resistance in 20% EGFR TKIs resistantpatients is not fully understood.Therefore,the elucidation of unknown resistance mechanism and the development of alternative strategies are urgently needed.The aryl hydrocarbon receptor(Ah R)is a ligand-activated intracellular receptor that transcriptionally activates downstream metabolic enzymes expression.For the past four decades,the transcriptional activity of Ah R is believed to mediate all the biological effects of Ah R signaling,while recent studies revealed that Ah R signaling in cell preliferation,differentiation,apoptosis and cell cycle control did not require its transcriptional activity.Ah R is ubiquitiously overexpressed in various solid tumors and cancer cell lines.Retrospective analysis showed that Ah R expression level is associated with disease stage and progression.Our preliminary study also indicates Ah R expression is increased in TKIs resistant tumors,but its association and significance with TKIs sensitivity is not clear.In the present study,we showed that Ah R protein negatively regulates sensitivity to TKIs in NSCLC.Ah R acts as an adaptor protein to recruit Src and Jak2 kinases that resembles Jak2-Ah R-Src complex to bypass EGFR and activates downstream PI3K/Akt and MEK/Erk signaling,leading to resistance to EGFR TKIs.Inhibition of Ah R by pharmacological and molecular approaches overcomes TKIs resistance in NSCLC cells expressing high endogenous level of Ah R,whereas activation of Ah R signaling in sensitive cells promotes TKIs resistance.The croos-talk between Ah R and Src,Ah R and Jak2,does not dependent on its transcriptional activity,in which the liganded Ah R protein transiently translocate to the cell membrane and provides docking sites for Jak2 and Src.Simultaneous inhibition of Jak2 or Src kinases abolishes the bypass track and overcomes resistance to EGFR TKIs.Our study highlighted a transcriptional-independent function of Ah R protein and underlies a novel TKIs resistant mechanism,potentially pointing out a new strategy to overcome resistance to targeted therapy.Methods(1)The prognostic correlation of Ah R expression at m RNA and protein levels were analyzed in TCGA database and tissue array.(2)Expression vectors for Ah R WT,Ah R CA and Ah R sh RNA were generated.Cells stably overexpressing Ah R or Ah R sh RNA were tested for TKIs sensitivity by MTT,colonies formation,Western blot,IHC and TUNEL assays.(3)To determine effect of transcriptional activity of Ah R on TKIs sensitivity,Ah R mutant lacking nuclear localization sequence(NLS)were generated.sh RNAs were also used to inhibit ARNT,cyp1a1 and cyp1b1 expression.(4)Src was stably overexpressed or down-regulated by lentivirus.The effect of Src on TKIs sensitivity was assessed by MTT,Western blot and IHC,respectively.(5)Phospho-protein kinase array was used to determine the kinase upstream of Src.Expression vectors for Jak2,Src and Ah R were expressed in HEK293 cells,and their interactions were assessed by IP and Western blot.Result(1)Ah R at m RNA level did not correlated with NSCLC grade and patient survival.In contrast,high expression of Ah R at protein level indicated unfavorable prognosis.Western blot analysis also indicated increased Ah R protein expression in TKIs resistant cell lines.(2)Inhibition of Ah R by Ah R inhibitor α-NF or Ah R sh RNA sensitized resistant cells to TKIs.Simultaneous inhibition of EGFR and Ah R signaling abolished PI3K/Akt and MEK/Erk signaling and induced tumor shrinkage and apoptosis in vivo.(3)Activation of Ah R signaling by Ah R WT overexpression or Ah R ligands in sensitive cells led to a TKIs resistant phenotype,in which Akt and Erk were constitutively phosphorylated despite EGFR TKIs treatment.In contrast,overexpression of Ah R CA mutant failed to recaptulate a similar effect.(4)Mutation in Ah R NLS or sh RNA-mediated down-regulation of ARNT,cyp1a1 and cyp1b1 did not affect TKIs sensitivity.(5)Bioinformatic analysis and functional experiments indicated increased Src phosphorylation was crucial for Ah R-induced TKIs resistance.Ah R recruited Src through its SH2 binding motif and promoted Src Y416 phosphorylation and restored downstream PI3K/Akt and MEK/Erk pathways.Simultaneous inhibition of Src and EGFR abrogated Akt and Erk phosphorylation,inhibited cell proliferation and Ki67-positive cell proportion,and overcame resistance to EGFR TKIs.(6)Phospho-protein kinase array strongly indicated Jak2 as an immediate upstream kinase of Src.IP experiment suggested that Ah R transiently translocated into the cell membrane and provided docking sites for Jak2 and Src interaction.Inhibition of Jak2 activity by Jak2 TKIs or Jak2 sh RNA also restored EGFR TKIs sensitivity.Conclusion Collectively,we identified a novel EGFR TKIs resistant mechanism,in which activated Ah R increases Src phosphorylation and restores downstream PI3K/Akt and MEK/Erk signaling.Although Ah R is not a protein kinase,Ah R may act as an adaptor to recruit other kinases to lead to TKIs resistance.Of noted,this effect did not depend on its transcriptional activity and Ah R serves as a bridge linking Src to Jak2 kinase.The Jak2-Ah R-Src comples bypass EGFR to phosphorylate Akt and Erk in resistant cells.Therefore,Jak2,Ah R and Src are promising therapeutic targetes for overcoming TKIs resistance.