PTEN Deficiency Facilitates The Sensitivity Of Gemcitabine In Cholangiocarcinoma

Background and objectiveCholangiocarcinoma is a type of epithelial malignancies that originates from different locations in the biliary tree.Past decades have witnessed the global increase in incidence and mortality rate of cholangiocarcinoma.Because of atypical symptoms at an early stage,cholangiocarcinoma is mostly diagnosed at advanced stages.The restricted objective response rate of the first-line chemotherapy leads to dismal 5-year survival and poor prognosis.Therefore,it is warranted to develop drugs that improve prognosis.Improving the efficacy of existing drugs and expanding the range of sensitive population is less time-consuming and economically costly and much safer than developing new drugs from scratch.A phase Ⅱclinical trial that our group has participated in evaluated the efficacy of bortezomib in patients with PTEN-deficient advanced cholangiocarcinoma.Patients with advanced cholangiocarcinoma who failed the first-line chemotherapy with disease progression and PTEN deficiency were recruited.The retrospective evaluation revealed that patients with PTEN-deficient advanced cholangiocarcinoma were more sensitive to the first-line chemotherapy(gemcitabine plus oxaliplatin)than patients with PTEN-positive cholangiocarcinoma.Based on these results,we aimed to investigate the mechanisms that regulate the sensitivity of differential PTEN expression to the first-line chemotherapeutic agents(gemcitabine and oxaliplatin)for cholangiocarcinoma.We used clinical samples,cell lines of cholangiocarcinoma,and mouse models of patient-derived tumor xenograft PDX)and subcutaneous cholangiocarcinoma for validation,and combined molecular biology and animal experimental techniques to explore the specific mechanisms by which PTEN positive and PTEN deficiency affect the sensitivity of first-line chemotherapeutic agents,providing evidence for treatment stratification of cholangiocarcinoma.Research methods1.Collect the clinically resected or punctured samples of cholangiocarcinoma and detect PTEN expression by immunohistochemistry.Analyze the difference in efficacy and survival between patients with positive and negative PTEN expression treated with the first-line chemotherapy,as well as the difference in survival in surgery cohorts.2.Verify the sensitivity of PTEN-positive and PTEN-deficient cell lines of cholangiocarcinoma to gemcitabine and oxaliplatin by immunoblotting,drug sensitivity assay,and flow cytometry.3.Apply recombinant plasmid transfection,siRNA transfection,shRNA lentivirus,and Cas9-sgRNA lentivirus infection to establish PTEN differentially expressed cholangiocarcinoma cell lines,including PTEN overexpression,PTEN knockdown,and PTEN knockout.Isolate and culture embryonic fibroblasts from PTEN conditional knockout mice.Use Cre or PTEN recombinant adenovirus to construct PTEN knockdown or overexpression cells.Verify the effect of differential PTEN expression at the cellular level on the sensitivity of gemcitabine and oxaliplatin by immunoblotting,immunofluorescence,cell viability assay,and immunohistochemistry.4.Establish subcutaneous tumor mouse models with PTEN differentially expressed cells and mouse PDX models with positive and negative PTEN expression.Assess the in vivo effect of PTEN deficiency on gemcitabine sensitivity.5.Detect the content of deoxycytidine(dC),gemcitabine,and phosphorylated gemcitabine in cholangiocarcinoma cell lines differentially expressing PTEN by quantitative mass spectrometry.Examine the mRNA and protein expression of genes related to gemcitabine transport,activation,and pyrimidine synthesis,as well as the transcriptional activity of the promoter region in cholangiocarcinoma cell lines differentially expressing PTEN by RNA-sequencing,immunoblotting,and dual-luciferase reporter gene assay.6.Examine the effects of overexpression and knockdown of deoxycytidine kinase(DCK)in cholangiocarcinoma cells on gemcitabine-induced DNA damage and cell cycle arrest by plasmids,siRNA transfection,immunoblotting,drug sensitivity assay and flow cytometry.Assess whether DCK is involved in the regulation of gemcitabine sensitivity by PTEN.7.Prepare and screen the DCK S74 site phosphorylation antibody.Examine the effect of differential PTEN expression on DCK S74 site phosphorylation by immunoblotting and immunohistochemistry at the cellular and animal levels.8.Detect the expression of phosphorylated DCK S74 in clinical cholangiocarcinoma samples by immunohistochemistry.Analyze the relationship between DCK S74 phosphorylation expression and chemotherapeutic efficacy or PTEN expression.9.Verify the effect of PTEN on DCK phosphorylation using AKT inhibitors and mTOR inhibitors,and different mutants of PTEN on DCK phosphorylation expression by immunoblotting.10.Detect the binding of PTEN to DCK protein by immunoprecipitation and surface plasmon resonance;eukaryotic expression of purified PTEN and DCK protein,construct in vitro dephosphorylation reaction and detect dephosphorylation of DCK by PTEN through immunoblotting.11.Identify proteins that bind to PTEN and DCK by immunoprecipitation and mass spectrometry,focusing on protein phosphatases that bind to them.Purify PTEN,DCK,phosphoglycerate mutase family member 5(PGAM5),PP1,and PP2A proteins,construct in vitro dephosphorylation reactions and verify the dephosphorylation effect of bound proteins on DCK by malachite green reaction and immunoblotting.12.Verify the effects of overexpression and knockdown of PP2Ac in cholangiocarcinoma cell lines on DCK phosphorylation and gemcitabine sensitivity by immunoblotting,flow cytometry,and drug sensitivity assays.Assess whether PP2Ac is involved in the effects of PTEN on DCK phosphorylation and gemcitabine sensitivity.Evaluate the effects of PP2A inhibitor LB100 on gemcitabine sensitivity by mouse subcutaneous tumor models.13.Construct the in vitro dephosphorylation reaction by immunoblotting to detect the effect of PTEN deficiency on C-terminal Flag-tagged PP2Ac(CF-PP2Ac)and N-terminal Flag-tagged PP2Ac(NF-PP2Ac)enzymatic activities.Examine the dephosphorylation effect of PTEN on CF-PP2Ac and NF-PP2Ac proteins.14.Verify the effects of differential PTEN expression and different enzyme activity mutants on PP2Ac phosphorylation and enzymatic activity by immunoblotting,enzyme activity assay,and dephosphorylation reaction.15.Verify the binding of PP2Ac,PTEN,and DCK by immunoprecipitation,GST-pull down assay,and surface plasmon resonance.16.Analyze the effect of PTEN and DCK expression on gemcitabine sensitivity in tumor cell lines in the Genomics of Drug Sensitivity in Cancer(GDSC)and Cancer Cell Line Encyclopedia(CCLE)Database.17.Validate the pan-tumor generalizability of PTEN deficiency promoting gemcitabine sensitivity in pancreatic cancer samples,pancreatic cancer cells,and cervical cancer cells.Research results1.A total of 98 samples of advanced cholangiocarcinoma receiving gemcitabine-based first-line chemotherapy were collected,including 30 cases with PTEN-negative expression.The efficacy of first-line chemotherapy regimens was superior in patients with PTEN-negative expression cholangiocarcinoma.Although no significant difference was observed in survival,PFS and OS were significantly longer in the PTEN-negative group than in the PTEN-positive group at the early stage of first-line chemotherapy(first six months).A total of 127 surgical-only cohorts were enrolled,with significantly shorter DFS and OS in the PTEN-negative group than those in the PTEN-positive group:2.RBE,TFK1,and CC143476 were PTEN-positive cholangiocarcinoma cell lines,while HCCC-9810 and SK-CHA-1 were PTEN-deficient cholangiocarcinoma cell lines.Compared with PTEN-positive cells,PTEN-deficient cells HCCC-9810 and SK-CHA-1 were more sensitive to gemcitabine,while there was no significant difference in the responses of both groups to oxaliplatin.PTEN deficiency in cholangiocarcinoma cells promoted the sensitivity of gemcitabine without affecting the sensitivity of oxaliplatin.3.PTEN deficiency in cholangiocarcinoma cells promoted cell cycle arrest and apoptosis induced by gemcitabine and elevated the sensitivity of gemcitabine in combination with oxaliplatin.4.PTEN deficiency in mouse subcutaneous tumor models promoted the sensitivity of gemcitabine;similarly,mouse PDX tumors with PTEN deficiency were more sensitive to gemcitabine.5.Differential expression of PTEN in cholangiocarcinoma cells did not affect the intracellular content of dC and gemcitabine,but significantly affected the activation of phosphorylated-gemcitabine;differential expression of PTEN did not affect gemcitabine uptake and deoxycytidine biosynthesis.6.In cholangiocarcinoma cells,DCK promoted gemcitabine-induced DNA damage and cell cycle arrest.7.The DCK S74 site phosphorylated antibody obtained after preparation and screening were specific and with high antibody potency.PTEN deficiency in cholangiocarcinoma promoted phosphorylation of the DCK S74 site.8.High p-DCK S74 expression in patients with cholangiocarcinoma predicted a better response to gemcitabine;PTEN expression showed a negative correlation with p-DCK S74 expression.9.The regulation of DCK phosphorylation by PTEN did not depend on the PI3K/AKT/mTOR pathway.10.PTEN can bind to DCK,but cannot dephosphorylate DCK.11.PP2Ac can bind to DCK and dephosphorylate DCK.12.Knockdown of PP2Ac promoted phosphorylation of DCK and the sensitivity of gemcitabine.PP2Ac was involved in the regulation of DCK by PTEN.The PP2Ac inhibitor promoted the sensitivity of gemcitabine.13.PTEN dephosphorylated PP2Ac at the Y307 site and promoted PP2Ac activity.14.PTEN dephosphorylated PP2Ac depending on its protein phosphatase activity.15.PTEN,DCK,and PP2Ac bound to each other and formed a complex.16.In the GDSC and CCLE Database,PTEN mutation and high mRNA expression of DCK were positively correlated with higher sensitivity of gemcitabine in tumor cells.17.First-line chemotherapy based on gemcitabine was effective in patients with PTEN-deficient pancreatic cancer.Interference with PTEN expression in pancreatic cancer cells promoted the sensitivity of gemcitabine.Meanwhile,the knockdown of PTEN in cervical cancer cells(HeLa)promoted the sensitivity of gemcitabine.ConclusionWe retrospectively analyzed the efficacy assessment of a phase Ⅱ clinical trial(NCT03345303)and found that patients with advanced cholangiocarcinoma with PTEN deficiency were more sensitive to first-line chemotherapy(gemcitabine plus oxaliplatin)than patients with PTEN-positive expression of cholangiocarcinoma.Based on that,we found that PTEN deficiency in cholangiocarcinoma increased PP2Ac phosphorylation and decreased PP2Ac enzyme activity,which promoted DCK phosphorylation and gemcitabine sensitivity.By analyses of the GDSC and CCLE Database,PTEN deficiency promoted the sensitivity of gemcitabine in various tumors,suggesting a pan-tumor generalizability of our findings.