Expression And Clinical Implication Of CMTM6,a PD-L1 Regulatory Protein In Gastric Cancer

Background and objective:Gastric cancer(GC)ranks third in incidence and mortality rate in China across all cancer types.Existing treatment options are limited,including chemotherapy and target therapy against Her-2 and VEGF.The clinical benefit and survival improvement is unsatisfactory considering the concomitant toxicity and future drug resistance.Anti-PD 1/PD-L1 therapy,on the other hand,is approved by FDA in the third-line setting for heavily-treated advanced gastric cancer because of its promising efficacy,controllable toxicity and durable response.However,the objective response rate of monotherapy is around 12%.Recent negative results of KEYNOTE-061、KEYNOTE-062 require new strategies for patient stratification.PD-L1 combined positive score(CPS)based on immunohistochemistry(IHC)is a widely accepted but imperfect biomarker,given that the inconsistency between PD-L1 positivity and treatment efficacy remains to be elucidated.Membrane PD-L1 on tumor cells is part of the main target for anti-PD-1/L1 immunotherapy,while CMTM6/4 is reported for maintaining membrane PD-L1 expression and reducing its degradation at post translation level,thus might relate to cancer immunotherapy.The objective of this research is to explore the expression feature,function,regulatory role against PD-L1 as well as the clinical implication,especially in terms of immune checkpoint blockade efficacy of CMTM6 in gastric cancer,with additional attention to CMTM4 status as well.Methods:To explore the PD-L1 regulatory function of CMTM6,we performed gain/loss of function assays of CMTM6 in gastric cancer cell lines followed by western blot and flow cytometry detection of cellular and membrane expression of PD-L1.Using 7-color multiplexed immunofluorescence staining(MIS)in 180-case tissue microarray and pre-immunotherapy tumor tissues sections of 48 patients with gastric adenocarcinoma,we evaluated the CMTM6/4 and PD-L1 expression profile in gastric cancer and its clinical relevance to clinicopathological parameters,prognosis and patient selection in anti PD-1/L1 therapy.In addition,we explored the oncogenic function of CMTM6 in vitro by proliferation and transwell assays,as well as the CMTM6 inducing function of several stimulators including glucose,fructose and linolenic acid.Results:1.According to MIS,epithelial expression of CMTM6 is upregulated in GC tissues compared with adjacent normal tissues.Co-expression of CMTM6/4 with PD-L1 on the epithelial membrane can be identified in gastric cancer tissues.CMTM6 is positively correlated with PD-L1 level,tumor burden and proliferation marker Ki-67.CMTM6 is an independent prognostic indicator of GC while the predictive efficacy is even higher for CMTM6+PD-L1+to separate patients with different survival outcome.In vitro experiments using CCK8,colony formation and transwell assays show that CMTM6 knock-down inhibits the proliferation and migration of GC cell lines.These results indicate the tumor promoting function of CMTM6 in GC,which may serve as a potential therapeutic target in GC.2.By multiplexed immunofluorescence staining of GC cell lines,we identified co-localization of CMTM6/4 with PD-L1 on cell membrane.By knocking down or overexpression of CMTM6 in GC cells lines,we found that PD-L1 membrane expression is decreased or upregulated accordingly,with or without the presence of IFN y.Besides,CMTM6 expression can be influenced with stimulation of glucose,fructose and linolenic acid,while the expression of PD-L1 changes synchronously with CMTM6,indicating nutrients could be a negative interference during anti-PD-l/L1 immunotherapy by inducing persistent expression of both CMTM6 and PD-L1 in gastric cancer cell.3.Co-expression of CMTM6 with PD-L1 is not only a valuable predictor of cancer progression and worse survival,but also a pivotal predictor of favorable outcome undergoing anti-PD-1/L1 immunotherapy in gastric cancer according to the discovery that epithelial CMTM6 and CMTM6+CMTM4+levels are significantly higher in PR(partial response)group than PD(progressive disease)group in the subpopulation of relatively higher PD-L1 expression.Furthermore,by classification of expression pattern of CMTM6/4、PD-L1,we identified CMTM6+PD-L1+ embrane co-localization in tumor epithelial region may be a biomarker for anti PD-1/L1 efficacy.Together,evaluation of membrane co-expression of CMTM6 with PD-L1 may provide a novel method for precision pre-immunotherapy patients screening and critical indicator for dietary control during anti-PD-1/L1 therapy.Besides,by stratified evaluation of EBV negative,pMMR(proficient mismatch repair system)as well as anti-PD1 subsets,we found a trend of correlation between higher CMTM6 with prolonged survival,which is worth further investigation considering the lack of treatment choices within pMMR and EBV negative subgroups currently.Conclusions:CMTM6 regulates PD-L1 membrane expression in gastric cancer cell lines and co-localizes with PD-L1 on tumor cell membrane of GC.CMTM6 promotes cancer progression and poses as an independent prognostic risk factor.CMTM6 can be regulated by nutrients and cytokines.Membrane expression of CMTM6+PD-L1+ on tumor cells might be a stratification biomarker for anti-PD1/L1 treatment in GC.