Study On The Application Of Proteasome Inhibitors In PTEN-deficient Cholangiocarcinoma Treatment

Background and objectiveCholangiocarcinoma（CCA）is an epithelial malignancy arising from the biliary tree,and its morbidity and mortality have been increasing in recent decades.Long incubation period,no obvious early symptoms,and lack of effective drugs,all lead to the very dismal prognosis of CCA.Therefore,novel drug development for CCA to improve the prognosis is urgently needed.At present,drug repurposing is a prevalent strategy to develop anti-tumor drugs.Comparing with the de novo drug development,drug repurposing can save more time and economic cost by reevaluating the“old drugs”or exploring combined use to broaden the indications.The anti-tumor drug development highly relys on cellular and animal experimental models,and screening models derived from patients show profound advantages.Of note,patient-derived xenograft models（PDX）and isolated PDC cells,which inherit the genetic characteristics of parental tumors,have been widely used in preclinical drug development.In order to explore eligible drug for CCA treatment,we have established a series of PDX models for CCA and isolated PDC cells to screen an anti-tumor drug library approved by FDA.By multi-omics technology,we are trying to profile the mechanism of drug sensitivity.Our study may provide experimental evidence for precise medicine of CCA,as the proteasome inhibitors and tumor suppressor gene PTEN are enriched.Research methods1.Collect fresh CCA samples and establish PDX model subcutaneously in immunodeficient mice.Isolate and culture primary PDC cells.2.The cytotoxic effect of anti-tumor drug library was evaluated in 10 primary PDC cells at a dose of 10μM.Drugs with profound cytotoxic effects were selected and tested to determine IC₅₀ values.Cell viability real-time monitoring system was used to evaluate the sensitivity of the above-mentioned drugs.3.CCA cell lines were treated with a series concentrations of proteasome inhibitor bortezomib.Cell lines were grouped according to the bortezomib sensitivity.The differences in protein synthesis,proteasome activity and cell proliferation between bortezomib sensitive and insensitive cells were analyzed.4.Transcriptome analysis was conducted to determine the differences in proteasome and PI3K-Akt signaling pathway between bortezomib sensitive and insensitive cells.Whole genome sequencing（WGS）was performed to detect genetic alterations of two groups.Focus on the mutation and expression of tumor suppressor gene PTEN.5.Manipulate PTEN expression in CCA cell lines by recombinant plasmid transfection,si RNA transfection,sh RNA and Cas9-sg RNA lentivirus infection.Evaluate the effects of PTEN differential expression on proteasome activity,subunits gene expression,and the bortezomib sesitivity in vitro.6.Evaluate the effects of PTEN differential expression on bortezomib sesitivity in vivo,using CDX,PDX and CCA spontaneous mouse models.Clinical trials were conducted to evaluate the efficacy of bortezomib in PTEN-deficient CCA patients.7.Explore the mechanism that PTEN regulating proteasome subunits transcription through antioxidant response element（ARE）related transcription factors by si RNA screening,reporter gene and chromatin immunoprecipitation assay.Focus on the role of BACH1 and MAFF.Evaluate the PTEN effects on the expression of BACH1 and MAFF by immunoblotting,real-time PCR and reporter gene assay.8.Evaluate the effects of BACH1 and MAFF on proteasome activity and bortezomib sensitivity.Determine whether BACH1 and MAFF involved in PTEN regulating bortezomib sensitivity.9.Verify whether FOXO1 binding in BACH1 and MAFF promoter region by chromatin immunoprecipitation assay.Explore the influence of PTEN-PI3K-AKT-FOXO1 pathway on BACH1 and MAFF expression using immunoblotting,real-time PCR and reporter gene assay.10.Analyze the GDSC,CTRP,CCLE and TCGA databases to explore the relationship between PTEN alteration and drug sensitivity of proteasome inhibitors.Verify the generality that PTEN status regulating the sensitivity to proteasome inhibitors in pan-cancer.Research results1.34 cases of CCA PDX models were successfully established,and 10 cases of primary PDC cells were isolated and cultured for drug screening2.The proteasome inhibitors bortezomib and cafezomib showed profound cytotoxic effect,superior to the common first-line drugs for CCA.3.According to cell survival curves,the cell lines could be divided into two groups:bortezomib-sensitive（including RBE,Huh28,FRH201 and QBC939）and bortezomib-insensitive（including Hu CCT1,TFK1,MA-CHA-1,HCCC-9810 and SK-CHA-1）.Bortezomib-sensitive cells possessed high protein synthesis rate and proteasome activity,and increased cell proliferation4.The PI3K-AKT and ubiquitin-mediated proteolysis pathways were up-regulated in bortezomib-sensitive cell line.Moreover,WGS and immunoblotting revealed PTEN deficiency in bortezomib-sensitive cell lines.5.PTEN deficiency or PTEN knockdown increased the proteasome proteolytic activity and facilitated the therapeutic effect of bortezomib in vitro.6.PTEN deficiency increased the bortezomib sensitivity in CDX,PDX and autochthonous CCA mouse models,as well as in CCA patients.7.PTEN regulated the expression of proteasome subunits through ARE negative transcription factors BACH1 and MAFF,independent of NRF1 and NRF2.8.BACH1 and MAFF inhibited the proteasome activity,the expression of proteasome subunits and the bortezomib sensitivity in CCA cells.In addition,BACH1 and MAFF participated in the regulation of PTEN on the bortezomib sensitivity.9.FOXO1 regulated the transcription of BACH1 and MAFF.As a result,PTEN regulated the expression of BACH1 and MAFF through PI3K-AKT-FOXO1 pathway.10.In multiple human cancers,PTEN status regulated proteasome activity and the sensitivity to proteasome inhibitorsConclusionWe performed a drug-screening assay using PDXs and PDCs,and identified proteasome inhibitors as promising drugs for CCA treatment.The proteasome inhibitors showed selective sensitivity in CCA cells,which was associated with PTEN deficiency.Furthermore,we determined that PTEN deficiency promoted the protein synthesis and proteasome subunit expression and proteolytic activity,creating a dependency on the proteasome for cancer cell growth and survival.Mechanistically,we found that PTEN promoted the nuclear translocation of FOXO1,resulting in the increased expression of BACH1 and MAFF.BACH1 and MAFF are transcriptional regulators that recognize the antioxidant response element（ARE）,which is present in genes encoding proteasome subunits.PTEN induced the accumulation and nuclear translocation of these proteins,which directly repressed the transcription of genes encoding proteasome subunits.By analyzing the GDSC,CTRP,CCLE and TCGA databases,we confirmed the generality that PTEN status regulating the sensitivity to proteasome inhibitors in multiple human cancers.