Antagonism Or Loss Of Tumor Intrinsic PD-L1 Confers Resistance To Drug-induced Apoptosis Induction In Human Colon Cancer

Objective Colorectal cancers(CRC)with BRAF(V600E)are associated with microsatellite instability(MSI)that predicts response to immune checkpoint inhibitors.Recent clinical studies showed that anti PD-L1 antibody is promising for treating MSI colon cancer.Little evidence showed BRAF can module immune adaptation in colon cancer.DNA mismatch repair can be found in BRAF mutation,and anti PD-1 antibody is an option for the treatment.Recent studies mainly focused on the mechanism of interaction between tumor PD-L1 and T cell surface PD-1.However,a recent study demonstrated that melanoma intrinsic PD-L1 can regulate cell apoptosis by activating cell signaling.As for colon cancer PD-L1,we focused on the intrinsic factors for tumor cell signaling and drug resistance.Method 1.TCGA RNA-Seq of human colorectal cancers and associated somatic mutation data(in VCF format)together with the metadata for 478 samples and 41 normal colonic tissue samples were downloaded using GDC data portal.The somatic mutation data were utilized to classify CRC cases into mutation for BRAF or KRAS or wild-type for both genes.The Kaplan–Meier survival plot was generated for PD-L1 and PD-1 genes in human colon cancer,using “survminer” package.The survival curves of samples with high gene expression and low/medium gene expression were compared by log rank test.2.Using Western Blot and RT-PCR techniques,we analyze the PD-L1 expression in human colon cancer cells with or without MEK inhibitors for observing the PD-L1 expression.Meantime,using si RNA transfection,YAP or/and c-JUN knockdown will regulate PD-L1 expression.3.Using CRISPR-Cas9,we subcloned the knockout PD-L1 colon cancer cells treated with anti PD-L1 antibody,cobimetinib,oxaliplatin or irinotecan.With different doses and time,we examine the p H2 AX and cleaved caspase 3 for drug resistance.4.Immunodeficient SCID mice at 4-6 weeks of age were purchased.Cultured murine colorectal adenocarcinoma cell lines MC38,PD-L1 knockout with re-expression of wild-type PD-L1 or an empty vector,were injected subcutaneously into the right flank of each mouse.For IHC,primary antibodies used included rabbit anti-cleaved caspase-3,and anti-Ki-67.Results 1.Interrogation of TCGA RNA-seq human datasets revealed that BRAFV600 E tumors have significantly higher PD-L1 m RNA compared to non mutated BRAF CRCs.Using TCGA datasets,optimized cutoffs for low vs high PD-L1 m RNA expression in human colon cancers was significantly associated with inferior survival.2.Cells with an increase in BRAFV600 E or KRAS mutant alleles or with their ectopic expression showed increased cell surface PD-L1 expression and PD-L1 transcripts.Inhibition of MEK/ERK by cobimetinib was shown to attenuate mutant BRAF or KRAS-induced PD-L1 coincident with reduced c-JUN and Yap expression whose combined knockdown reduced PD-L1.3.Analysis of the role of PD-L1 as a mediator of chemosensitivity was then performed.Knockout of PD-L1 was shown to attenuate induction of DNA double strand breaks(p H2AX)and caspase-3 cleavage by oxaliplatin,irinotecan or cobimetinib compared to parental RKO cells.Results were confirmed in PD-L1 knockout MC38 murine CRC cells where re-expression of wild-type PD-L1,but not deletion mutants of extracellular or intracellular PD-L1 domains,promoted DNA damage and apoptosis.Mechanistically,knockout of PD-L1 reduced chemosensitivity in association with reductions in p-AKT and in BH3-only proteins BIM and BIK(independent of transcription),and introduction of wild-type PD-L1 into MC38 cells restored p-AKT,BIM and BIK levels.Furthermore,anti-PD-L1 antibody treatment of RKO and MC38 cells reduced p-AKT,BIM and BIK expression and attenuated apoptosis induction by oxaliplatin or irinotecan.In cells with PD-L1 knockout or treated with an anti-PD-L1 antibody,BIM and BIK induction were attenuated.Re-expression of BIM in PD-L1 knockout cells restored apoptosis induced by oxaliplatin or irinotecan.4.Using a murine xenograft model,tumors generated from MC38 cells with knockout of PD-L1 displayed resistance to oxaliplatin or irinotecan whereas re-expression of PD-L1 sensitized these cells to chemotherapy.Conclusion It is demonstrated that a novel role for PD-L1 can be regarded as a regulator of chemotherapy-induced apoptosis whose deletion or suppression confers chemoresistance.These findings expand the understanding of PD-L1 functions to include nonimmune mechanisms and suggest the potential use of PD-L1 as a biomarker of response to cytotoxic chemotherapy.