Rab25 And TAM M2 Polarization Promote Erlotinib Resistance In NSCLC

Chapter 1: Rab25 promotes erlotinib resistance by activating the β1 integrin/ AKT/β-catenin pathway in NSCLCBackground:Small molecule inhibitors of receptor tyrosine kinases have produced encouraging therapeutic results in non-small cell lung cancer（NSCLC）.Erlotinib,an epidermal growth factor receptor（EGFR）tyrosine kinase inhibitor（TKI）,is a first-line treatment for advanced NSCLC in patients harbouring activated EGFR mutations According to a randomized phase 3trial,the median PFS of advanced NSCLC patients was 11 months in the erlotinib group versus 5.5 months in the chemotherapy（gemcitabine/cisplatin）group.However,almost all patients develop acquired resistance to erlotinib after 9-13 months of treatment.Although substantial acquired resistance mechanisms,such as the T790M mutation,c-MET amplification,and IGF-1 receptor activation,have been discovered,almost30%of the required resistance cases remain unexplainable.Therefore,appropriate preclinical tumour markers that correlate with EGFR-TKI acquired resistance without the T790M mutation are urgently needed for better prognostic and therapeutic strategies in advanced NSCLC patients.Ras-associated binding protein 25（Rab25）,a member of the Rab small GTPase family,regulates intracellular vesicle trafficking.Membrane trafficking is involved in many cellular pathways related to carcinogenesis,such as cell proliferation,invasion,metastasis,and polarity loss.Rab25 upregulatedβ1 integrin levels and induced snail,fascin expression,leading to epithelial-mesenchymal transition（EMT）in breast and ovarian cancer cells.Furthermore,Rab25 interacts withα5β1 integrin and regulates the trafficking of this molecule,which can promote tumour progression in ovarian cancer cells.In our previous study,Rab25was a candidate oncogene in NSCLC,and its driving role in EGFR-TKI resistance was investigated.We established erlotinib-resistant cell lines by exposing the cells to increasing doses of erlotinib and did not detect the T790M mutation in the clones.The protein level of Rab25was increased in the erlotinib-resistant cell lines and high Rab25 was associated with poor response of EGFR-TKI in NSCLC patients.We speculated that Rab25 was involved in T790M-independent resistance to EGFR-TKIs and could be another pathway for lung cancer cells to survive in the presence of an EGFR-TKI.Methods:1.Expression of EGFR-related proteins and Rab25 in NSCLC cells western blot analyzed protein levels of EGFR,Rab25 in erlotinib-sensitive PC9,HCC827 cells and the resistant PC9/ER and HCC827/ER subclones.Construct Rab25-knockdown cells and Rab25-overexpression cells by lentivirus infection.CCK-8assasy was used to detect the IC50 of erlotinib,and Annexin V/7-AAD kit was applied to analyze cell apoptosis in these cells.2.Rab25 mediates wnt signalling pathway and promotes cell proliferation The key proteins of wnt signalling pathway—p-GSK-3βandβ-catenin,and the downstream target gene cycling D1 were detected by western blot.Cell cycle was detected by PI staining and the percentage of proliferated cell was measured by EdU assay.3.Rab25 interacts toβ1 integrin and then activates wnt signalling pathway The interaction between Rab25 andβ1 integrin was verified by co-immunoprecipitaion and the location of the two proteins was observed by immunofluorescence in NSCLC cells.siRNA was used to knock outβ1 integrin gene.The effects ofβ1 integrin-knockdown on p-GSK-3βandβ-catenin proteins were detected by western blot.4.Rab25 regulates wnt signalling pathway and induces erlotinib resistance in vivo Establish xenograft model in nude mice by injecting subcutaneously NSCLC cells.The therapeutic effect of erlotinib was assessed by xenograft volumn.Immunohistochemical staining detected the expression of Rab25,β1 integrin,β-catenin and ki-67 in the tumor sections.5.The relationship between Rab25 and EGFR-TKI response Immunohistochemical staining was used to detect Rab25 expression in tumor sections of NSCLC patients who have received EGFR-TKI treatment.The response of EGFR-TKI was evaluated according to RECIST 1.1 criteria.Kaplan-Meier curve analyzed the relationship between Rab25 expression and PFS,OS in NSCLC patients.Results:1.Rab25 is involved in erlotinib resistance We examined the expression of Rab25 and EGFR signalling in the EGFR-TKI-sensitive PC9 and HCC827 cells and the resistant PC9/ER and HCC827/ER subclones.Erlotinib only inhibited Rab25 in the sensitive cells but not the resistant cells.Then we constructed PC9/Rab25 and HCC827/Rab25 cells by transfecting the parental cell lines with Rab25cDNA,and these cells showed obvious resistance to erlotinib.Moreover,Rab25-knockdown cells exhibited increased sensitivity to erlotinib.2.Wnt signalling is correlated with Rab25-mediated erlotinib resistanc We found that Rab25 overexpression induced the expression of crucial molecules in the Wnt pathway（p-GSK-3βandβ-catenin）.In addition,Rab25 overexpression increased the percentage of cells in S phase and the percentage of EdU-positive cells.However,Rab25knockdown had the opposite effect.Moreover,LiCl,a Wnt signalling agonist,abolished the Wnt signalling inhibition induced by Rab25 knockdown.The above data suggested that Rab25 activated the Wnt signalling pathway to induce erlotinib resistance.3.β1 integrin-mediated activation of the Wnt signalling pathway Rab25 overexpression inducedβ1 integrin and p-AKT expression and Rab25knockdown led to a decrease in these proteins.In addition,silencingβ1 integrin reduced p-AKT andβ-catenin expression.A co-IP assay confirmed the direct interaction between Rab25 andβ1 integrin.Upon examination by confocal microscopy,we found that Rab25colocalized withβ1 integrin in the cytoplasm of the PC9 and HCC827 parental cells.However,increased recycling ofβ1integrin from the cytosol to the cell surface was observed in the PC9/ER and HCC827/ER cells.In combination,these results suggested that theβ1integrin recycling regulated by Rab25 was responsible for activating the Wnt pathway and inducing erlotinib resistance.4.Rab25 induces erlotinib resistance in vivo Rab25-overexpressing,Rab25-knockdown and the parental cells were subcutaneously injected into nude mice,and erlotinib treatment was delivered.Compared to the control group,Rab25-depleted xenografts were smaller and Rab25-overexpressing xenografts were larger.Furthermore,immunohistochemical analysis showed Rab25-overexpression inducedβ1integrin,β-catenin and ki-67 expression in vivo.Therefore,these results demonstrated that Rab25/β1 integrin/β-catenin signalling was critical for erlotinib resistance in vitro and in vivo.5.Elevated Rab25 expression is associated with EGFR-TKI resistance Finally,We retrospectively collected specimens from 37 NSCLC patients who had received EGFR-TKI therapy and detected Rab25 expression by IHC staining.Most patients who had poor response to EGFR-TKI treatment expressed high Rab25 protein,and this group of patients had shorter PFS,OS compared to their counterparts with low level of Rab25(PFS of 10 months vs.16months,OS of 28 months vs.49 months,respectively.These findings imply that Rab25 might be involved in EGFR-TKI in NSCLC.Conclusions:1.Rab25 expression is associated with a poor response to EGFR-TKI treatment and predicts shorter PFS and OS in NSCLC patients.2.Rab25-overexpression impairs the inhibition of erlotinib in vitro and in vivo,while Rab25-knockdown rescues the inhibition of erlotinib.3.Rab25 interacts toβ1 integrin and promotes the trafficking ofβ1 integrin to the cytoplasmic membrane.4.Theβ1 integrin on the cell membrane induces AKT phosphorylation,activates wnt/β-catenin signalling pathway,and then increases cyclin D1 expression,promotes cell proliferation at last.Chapter 2:Anlotinib reverses erlotinib resistance by inhibiting M2polarization of tumor associated macrophageBackground:Tumour microenvironment（TME）is closely related to tumour pathological characteristics.When tumor volume reaches to 2-3mm³,neovascularization is necessary to tumour progress.However,unlike normal vessels,the tumor vasculature has high permeability and poor perfusion,which lead to high osmotic pressure and hypoxia in tumor stroma.The specific environment results in less immune cells infiltration,immunosuppression and tumor metastasis.In addition,during hypoxia environment,tumor cells excrete some chemokines such as CCL22,CCL28,etc.Various chemokines recruit some immunosuppressive cells,i.e.regulatory T cells,macrophages,and result in a immunosuppressive TME.However,some anti-angiogenesis drugs lead to“vascular normalization”in a short term,which improves poor perfusion,high vascular permeability,hypoxia and increases anti-tumor drugs delivery,and induces immune cells infiltration.As a result,anti-angiogenesis combined other anti-tumor drugs therapy are confirmed to have synergistic effect and has been approved to be used in clinic.Moreover,EGFR-TKI-resistant tumor cells and stromal cells secrete more VEGF,which activates VEGFR signalling pathway,occurs“cross-talk”with EGFR,and promotes cell proliferation at last.Anti-angiogenesis combined with EGFR-TKI therapy block the effect and reverse EGFR-TKI resistance.The blood monocytes are recruited into TME by chemokines and differentiate into tumor associated macrophage（TAM）.The TAM is the largest quantity of immune cells in TME and exhibits M2 polarization.Macrophage can be divided into classically activated macrophages（M1）and alternatively macrophages（M2）.The M1 macrophage expresses MHC II,CD86,NO,iNOS and plays pro-inflammatory and anti-tumor roles,but the M2macrophage secretes IL-10,arg-1,CD206,CD163,TGF-β,expresses immunosuppressive effect and promotes tumor progression.During hypoxia environment,TAM secretes some angiogenic factor,such as VEGFA,VEGFC,PDGF,and promotes angiogenesis.Except for targeting vascularity,the anti-angiogenesis therapy has effects on some other cells,including TAM.An engineered endostation,an anti-angiogenesis drug,can internalize into TAM with the help of nucleolin and integrinα5β1,inhibit M2 polarization and suppress tumor cell proliferation and angiogenic activity of TAM.Anlotinib is a small molecule inhibitor,which can inhibits VEGFR,PDGFR,FGFR,c-Kit and has anti-angiogenesis effect.According to CSCO guidelines for lung cancer（2018）,anlotinib is approved to the third-line therapy in advanced NSCLC patients.The mechanisms of EGFR-TKI resistance contain activation of some signalling pathways in tumor cells and aberrant regulation of TME.TAM participates in immunosuppression,angiogenesis and vascular remodeling,and the M1/M2 transition is important in regulating function of TAM.We try to explore the influence of anlotinib on TAM,and look forward to provide a therapeutic choice for EGFR-TKI resistant patients.Methods:1.Establishment of TAM model The macrophage was obtained from monocyte with the PMA stimulation and the macrophage markers CD45,CD68 were confirmed by flow cytometry.The tumor associated macrophage model was established by con-culturing macrophage with NSCLC cells.2.Analysis of M1,M2 TAM Flow cytometry was used to detect the M1 marker HLA-DR and the M2 marker CD206in TAM,which co-cultured with erlotinib-sensitive or erlotinib-resistant NSCLC cells.CCK-8 assay analyzed the IC50 of erlotinib in NSCLC cells,which co-cultured with M1TAM or M2 TAM.3.Analysis of M2 polarization-related proteins The levels of VEGF in the cellular supernatant of erlotinib-sensitive and erlotinib-resistant NSCLC cells were detected by ELISA.Western blot was used to analyze the protein expression of VEGFR2,AKT,mTOR in TAM with or without anlotinib treatment.Results:1.M2 polarization of TAM mediates erlotinib resistance The macrophages were obtained by human monocyte cell lines THP-1,U937 with PMA stimulation.The proportion of CD45⁺/CD68⁺cells accounted for 90%in the THP-1,U937-derived macrophages.The TAM model was constructed by con-culturing macrophage with NSCLC cells.The TAM exhibited M2 polarization(CD45⁺/CD68⁺/CD206^high),and erlotinib inhibited the CD206 expression in TAM of erlotinib-sensitive PC9,HCC827 cells,but no inhibiting effect in TAM of erlotinib-resistant PC9/ER,HCC827/ER cells.In addition,the IC50 of erlotinib in PC9,HCC827 cells were significantly increased after co-culturing with M2 TAM.2.Anlotinib induces M1 polarization and reverses erlotinib resistance The TAM of PC9/ER,HCC827/ER cells exhibited M1 polarization(CD45⁺/CD68⁺/HLA-DR^high)after anlotinib treatment.Moreover,the IC50 of erlotinib in PC9/ER,HCC827/ER cells were significantly decreased after co-culturing with M1 TAM.3.Anlotinib inhibits M2 polarization by VEGF/VEGFR2/AKT signalling Compared to PC9,HCC827 cells,PC9/ER,HCC827/ER cells secreted more VEGF into the cellular supernatant.The VEGFR2 proteins in TAM of PC9/ER,HCC827/ER cells were increased compared with TAM of PC9,HCC827 cells,and anlotinib inhibited the p-VEGFR2expression.In addition,M2 polarization-related signalling molecules,the p-AKT/mTOR,were also increased in TAM of PC9/ER,HCC827/ER cells,and the two proteins were inhibited by anlotinib treatment as well.Conclusions:1.The erlotinib-sensitive NSCLC cells emerge resistance to erlotinib after con-culturing with M2 TAM.2.Anlotinib reverse erlotinib resistance in erlotinib-resistant NSCLC cells,which con-cultured with TAM.3.Erlotinib-resistant NSCLC cells secreted abundant VEGF,and then induced VEGFR2 expression in TAM.4.Anlotinib inhibited p-VEGFR2 and AKT/mTOR signalling pathway and inhibit M2 polarization of TAM.