| Background and purposeHepatocellular carcinoma(HCC)is one of the most common malignant tumors with poor prognosis,and it is the third leading cause of cancer death in the world.Hepatocellular carcinoma has a hidden onset and high rate of recurrence and metastasis due to its insensitivity to radiotherapy and chemotherapy.Surgical treatment is preferred in clinic,but the 5-year survival rate is extremely low and the prognosis is really poor.The lack of suitable biomarkers for early detection and limited treatment strategies are the main reasons for the high mortality rate.The current understanding of the pathogenesis of hepatocellular carcinoma is still in its infancy.Due to the high drug resistance rate and tumor recurrence rate,the overall survival benefit of multi-kinase inhibitors such as sorafenib in the treatment of advanced patients is less than 3 months.Therefore,it is urgent to further study the molecular mechanism of drug resistance and develop new therapeutic drugs.CLDN6 is a complete multi-transmembrane protein,which is one of the important components involved in the tight junction structure.Studies have found that CLDN6 is abnormally expressed in a variety of epithelial-derived malignancies.CLDN6 is one of the important members of the Claudins family.Claudins belongs to the large family of transmembrane tight junction proteins,which play a key role in a variety of biological processes,including tumor plasticity and embryonic stem cell pluripotency.In previous studies,we analyzed the transcriptome data of different developmental stages and found that the expression pattern of Claudins family genes have changed significantlyduring liver cell differentiation,while CLDN6 is highly expressed in embryonic stem cells and liver progenitor cells but has no expression in mature hepatocytes.Through bioinformatics analysis and public database mining,we found that CLDN6 expression was up-regulated in hepatocellular carcinoma and low-regulated or has no expression in other normal tissues.The above results suggest that CLDN6 may be closely related to the tumor lineage plasticity regulation of hepatocellular carcinoma,which may become a new molecular target for the treatment of liver cancer.In this study,considering the unique expression pattern of CLDN6,we developed an anti-CLDN6 antibody coupled to the cytotoxic drug DM1 to target highly plastic liver cancer tumor cells.This article intends to explore the mechanism of CLDN6 in promoting the plasticity of liver cancer cells and the experimental verification and clinical application of the anti-CLDN6 antibody conjugated drugs.Methods1.We used RT-qPCR quantitative nucleic acid amplification detection system to detect the m RNA level of CLDN6 in hepatocellular carcinoma tissue and its adjacent tumor tissue and corresponding normal liver tissue.CLDN6 was observed in hepatocellular carcinoma tissues by immunohistochemical staining in protein expression levels.2.Liver cancer cell lines overexpressing CLDN6 and knockdown CLDN6 were established through the lentiviral packaging system.By detecting the self-renewal ability of corresponding liver cancer cell lines,the expression changes of hepatic progenitor cell markers and bile duct epithelial cell markers were observed.The effect of CLDN6 on the plasticity of liver cancer cell lineage was observed by subcutaneous tumor formation in nude mice.3.We detected the relationship between CLDN6 and sorafenib resistance through drug sensitivity test in vitro.In order to investigate the relationship between CLDN6 and resistance to sorafenib in hepatocellular carcinoma and its lineage plasticity,we detected the expression change of resistance-related markers,hepatic progenitor cell markers and bile duct epithelial cell markers on liver cancer cell lines resistant to sorafenib treatment.4.We explored the downstream targets and enrichment pathways of CLDN6 by high-throughput transcriptome sequencing.In order to clarify the molecular mechanism of CLDN6 in liver cancer,we applied immunofluorescence,Western Blot,and Co-IP to study the mechanism of lineage plasticity regulation on liver cancer cell lines of CLDN6.5.We developed an anti-CLDN6 antibody-conjugated drug coupled with the cytotoxic drug DM1 and verified the effect of targeting the plasticity of liver cancer cells through experiments in vitro and in vivo.Results1.CLDN6 is mainly expressed on the cell membrane.CLDN6 is highly expressed in embryonic stem cells and hepatic progenitor cells,but has no expression in mature hepatocytes.It is significantly up-regulated in hepatocellular carcinoma tissues and its adjacent tumor tissues,but not in normal liver tissues.The expression of CLDN6 was also up-regulated in residual tissues of liver cancer cells after sorafenib resistance.2.In liver cancer cell lines,over-expressing CLDN6 can significantly enhance the ability of proliferation,clone forming,self-renewal and carcinogenicity of liver cancer cells.Conversely,knocking down CLDN6 can significantly reduce the above malignant phenotype of liver cancer cells.GSEA analysis of transcriptome sequencing showed that genes regulated by CLDN6 are closely related to embryonic stem cells and bile duct epithelial cells.RT-qPCR and Western Blot experiments also showed that mature liver cell markers(ALB,TTR,APOA1,etc.)were negatively correlated with CLDN6 expression,and bile duct epithelial cell markers(EPCAM,KRT19,SOX9,etc.)were positively correlated with CLDN6 expression.3.In liver cancer cells,CLDN6 overexpression can lead to increased resistance to sorafenib,and knockdown of CLDN6 can lead to increased sensitivity.The expression of CLDN6 and bile duct epithelial cell markers was up-regulated in liver cancer cells after sorafenib resistance.4.It can also be found in the GSEA analysis of the above transcriptome sequencing that the genes regulated by CLDN6 are closely related to the Hippo signaling pathway.In liver cancer cell lines,the expression of YAP1 and its downstream activated genes(CTGF,AREG,ID1,etc.)were up-regulated after CLDN6 overexpression.Conversely,the expression of YAP1 and its downstream activated genes were down-regulated after CLDN6 knockdown.In CLDN6 overexpressing liver cancer cell lines,YAP1 protein has stronger stability and increased nuclear translocation.The results of Co-IP experiments show that CLDN6 indirectly promotes YAP1 into the nucleus through TJP2,thereby further activating the Hippo signaling pathway.5.The expression data of CLDN6 in GTEx database showed that CLDN6 was basically not expressed in human normal tissues and organs(except for the reproductive system).Subsequently,we developed an anti-CLDN6 antibody-conjugated drug that targets CLDN6 as an antigen target and verified it in vitro and in vivo.CLDN6 is highly expressed in the liver cancer cell line Hep G2 and lowly expressed in PLC-8024.Flow cytometry and drug sensitivity experiments in vitro showed that CLDN6-positive Hep G2 had a strong binding to the anti-CLDN6 antibody conjugate drug and had significant killing effect.The antibody-drug conjugate did not bind to CLDN6-negative PLC-8024 and had almost no killing effect.In vivo animal experiments we have also shown that anti-CLDN6 antibody-conjugated drugs have a significant killing effect on liver cancer cells,and also have a significant killing effect on drug-resistant liver cancer cells treated with sorafenib.Conclusions1.Three different expression patterns of CLDN6 suggest that it may be a potential therapeutic target for patients with hepatocellular carcinoma after sorafenib resistance.2.In liver cancer cell lines,CLDN6 can promote carcinogenicity.CLDN6 can promote the plasticity of tumor lineage and promote the transdifferentiation of liver cancer cells towards bile duct epithelial cells.3.Sorafenib resistance leads to the enrichment of CLDN6 and bile duct epithelial cell markers.Overexpression of CLDN6 can lead to the enhancement of sorafenib resistance.4.CLDN6 stabilizes YAP1 proteins and promotes nuclear translocation of YAP1,which further activates the Hippo signaling pathway.5.Anti-CLDN6 antibody conjugated drugs have certain clinical application potential in targeting highly plastic,sorafenib refractory liver cancer tumor cells. |
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