Mechanism Of Aryl Hydrocarbon Receptor Regulating Stemness Of Non-small Cell Lung Cancer

Lung cancer is the leading cause of cancer incidence and mortality worldwide.More than 85% of lung cancer cases are NSCLC,which can be histologically divided into adenocarcinoma(～50%)and squamous cell carcinoma(～40%).Despite major advances in progression-free survival and overall survival has been made by targeted therapy and immunological therapy,development of drug resistance and disease relapse are still on the rise.The major cause for this is the inherent and acquired resistance to conventional therapies demonstrated by the CSC,which mediate the sustained growth,maintenance,and relapse of the tumor.The CSC theory states that tumor growth is promoted by a small number of tumor stem cells with the ability of expansion and self-renewal.Recently,several studies have indicated that CSCs are not necessarily rare or quiescent,but can be abundant and can proliferate vigorously instructed by niche signals.Differentiated cells and transient amplifying cells can be reprogrammed into CSCs through plasticity.However,further elucidations are still required to explore how these regenerative responses are regulated.The AhR,a transcription factors belonging to the basic helix-loophelix/Per/ARNT/Sim family,was originally found to mediate the toxic responses to environmental pollutants such as TCDD and Bap.It has been demonstrated to be function actively in carcinogenesis through multiple mechanisms involving disruption of the cell cycle,escape of apoptosis,modulation of inflammation and angiogenesis,and suppression of immune surveillance.Endogenous AhR ligands such as kynurenine(Kyn)and 6-formylindolo[3,2-b] carbazole(FICZ),have also been identified.Inactive AhR complexed to 90-k Da heat shock protein,cochaperone p23,AhR-interacting protein,and c-Src is primarily localized in the cytoplasm.Upon ligand binding,the complex translocated to the nucleus and the formation of AhR-AhR nuclear translocator heterodimer leads to the transcription of dioxin-responsive element-containing genes.In addition to the canonical pathway characterized by AhR nuclear translocation through which AhR regulates target gene expression,AhR influences many biological processes through non-genomic signaling mechanisms.AhR is overexpressed in 40% to 50% of NSCLC cases,which is considerably correlated with poor prognosis of NSCLC.Lung adenocarcinoma cells require elevated AhR levels for sustained proliferation,and activation of AhR signaling upregulates inflammatory mediators and growth factors.Lately,several studies have indicated new functions for this receptor to be involved in modulating cellular differentiation and pluripotency in tumor cells in a cell-specific manner.Moreover,extensive cross-talks exist among AhR and cell cycle,as well as EMT signaling,indicating that AhR may regulate cancer stem-like properties.Our early study suggested that a high AhR protein level indicated an unfavorable prognosis for NSCLC,and increased AhR protein was associated with high risk of cancer relapse.Nevertheless,the function and mechanism of AhR in maintaining of lung cancer stemness are still not fully understood.In the present study,we demonstrated that AhR promotes proliferation,migration and invasion of NSCLC cells,and upregulation of AhR in NSCLC cells led to increased anchorage independent-growth coupled with enhanced tumor sphere formation ability.In addition,aldehyde dehydrogenase(ALDH)activity,as well as a panel of genes involved in cancer stemness were elevated by the activation of AhR not through the genomic pathway but mediated by the Jak2/STAT3 signaling pathway.These results suggested that AhRtargeted therapy might have the potential to restrain the generation and development of NSCLC.Methods(1)Tissue chip was used to analyze the correlation between AhR protein level and NSCLC disease stage,histopathological grade,and lymph node invasion.(2)Construct sh AhR and lentivirus-infected NSCLC cell lines with AhR overexpression,using the apoptosis test,Ed U cell proliferation test,plate cloning test,scratch test,transwell migration and invasion test to study the impact of AhR on the malignant biological behavior of NSCLC.Nude mice tumor formation experiments further verified the role of AhR in mediating the characteristics of lung cancer stem cells.(3)Using soft agar plate cloning test,tumorsphere formation test,ALDH activity test,Western blot and RT-q PCR to study the effects of up-and down-regulation of AhR on the stemness of NSCLC.(4)Western blot and RT-q PCR to study the role of Jak2/STAT3 signaling pathway in AhR-mediated lung cancer stem cell regulation.Results(1)Increased AhR protein levels are significantly associated with NSCLC stage,histopathological grade and lymph node invasion.(2)AhR inhibitor CH-223191 or sh RNA down-regulates AhR promotes NSCLC cell apoptosis,inhibits cell proliferation,migration and metastasis,while lentivirus overexpression of AhR promotes cell migration and metastasis.(3)Sh RNA down-regulates AhR and inhibits NSCLC stem cell characteristics and expression of stem molecules.Lentivirus overexpression of AhR enhances NSCLC stem cell characteristics and expression of stem molecules.Lentivirus overexpression of AhR promotes tumor formation of NSCLC cells in nude mice.(4)Lentivirus overexpression of AhR or AhR ligand Kyn can cause Jak2/STAT3 phosphorylation,while sh RNA down-regulation of AhR inhibits Jak2/STAT3phosphorylation;sh RNA down-regulation of AhR also abrogate Jak2/STAT3 phosphorylation caused by AhR ligand activation.(5)Jak2/STAT3 signaling pathway inhibitor Ruxolitinib inhibits AhR-mediated NSCLC stem characteristics and expression of stemness associated genes.ConclusionIn summary,we found a new mechanism that does not depend on the classic transcriptional activity of AhR.Activated AhR in NSCLC cells activates the Jak2 / STAT3 signaling pathway to induce stem gene expression and confer NSCLC tumor stem-like characteristics.Our research shows that AhR induces and maintains NSCLC stem cell characteristics through a Jak2/STAT3 signaling-dependent manner,and targeting AhR could be a new method to prevent the progression and recurrence of NSCLC.