Restoration Of PCK2 Suppresses Tumor Progression And Resistance To Sunitinib In Renal Cell Carcinoma By Promoting Endoplasmic Reticulum Stress

Renal cell carcinoma(RCC)is a malignant neoplasm of high prevalence in urinary system,and the incidence of RCC continues to increase in the population.Patients with early localized RCC have a good outcome after surgical treatment,even curable.However,the treatment of advanced or metastatic RCC still faces many clinical challenges.because of the unconspicuous early symptom and the lack of diagnostic biomarkers,many patients present with advanced or metastatic RCC at diagnosis.Targeted agent has benefited a lot of patients with metastatic RCC,but the expensive expenditure,potential adverse side effects and acquired resistance of the targeted drugs for RCC limit the application of target therapy in patients.Therefore,studies on the progression and targeted drug resistance of RCC are of great significance.Phos phoenolpyruvate carboxykinase 2(PCK2)is a mitochondrial isoform of the key gluconeogenesis enzyme phosphoenolpyruvate carboxykinase(PEPCK)and is widely involved in cell metabolism.An increasing number of studies have shown that PCK2 plays a role in the development and progression of a series of cancers but reports about RCC are rare.In this study,we demonstrated the expression of PCK2 was declined in RCC,and the lower PCK2 expression was associated with worse survival status of RCC patients.The downregulated expression of PCK2 in RCC is a result of DNA methylation of promoter region of PCK2,restoration of PCK2 suppresses tumor progression and resistance to Sunitinib in RCC by promoting endoplasmic reticulum stress.The content of this study is divided into three parts.Part Ⅰ: The expression changes of PCK2 in RCC and the correlation between PCK2 expression and clinical features of RCC patients Objective: Explore the expression alteration of PCK2 in RCC comparing with normal tissues and the correlations between PCK2 expression and clinical features of RCC patients.Methods: 1.Download the m RNA expression data of PCK2 in cc RCC and clinical information of cc RCC patients from online databases(TCGA database and Ocomine database)to investigate the expression alteration of PCK2 in RCC comparing with normal tissues and the correlations between PCK2 expression and clinical features of RCC patients;univariate and multivariate Cox regression analysis based on the TCGA database was used to evaluate whether PCK2 expression is an independent prognostic factor for RCC patients.2.Western blot,q RT-PCR and immunohistochemical staining analysis was used to verify the expression alteration of PCK2 in RCC tissues and cells.Results: 1.The results of statistical analysis indicated that the m RNA expression of PCK2 is declined in RCC tissues comparing with normal kidney tissues both in paired test and in unpaired test,and the lower PCK2 m RNA expression is associated with worse survival status of RCC patients.univariate and multivariate Cox regression analysis based on the TCGA database indicated that PCK2 m RNA expression is an independent prognostic factor for RCC patients.The ROC curve analysis indicated that PCK2 m RNA expression is correlated with the diagnosis of RCC.2.The results of western blot,q RT-PCR and immunohistochemistry staining analysis revealed that the expression of PCK2 is declined in m RNA and protein level in RCC tissues and cell lines.Conclusion: The expression of PCK2 is declined in RCC,and the lower PCK2 expression is associated with worse survival status of RCC patients;PCK2 expression is correlated with the diagnosis of RCC and is an independent prognostic factor for RCC patients.Part Ⅱ: The regulation effect of PCK2 expression alteration on biological function of RCC cells in vitro and in vivo Objective: Explore the effect of PCK2 expression alteration on biological function of RCC cells.Methods: 1.Establish the RCC cell lines with stable PCK2 overexpression and verify the overexpression efficiency of PCK2 by western blot and q RT-PCR;Cell proliferation assays,plate clone formation assays,wound healing assays and transwell migration and invasion assays were conducted in RCC cells with stable PCK2 overexpression to investigate the effect of PCK2 expression alteration on biological function of RCC cells.2.Establish sunitinib-resistant RCC cell lines to investigate the PCK2 expression alteration between sunitinib-resistant RCC cells and corresponding parental cells;Western blot was conducted to analyze the alteration of PCK2 expression when RCC cells was treated with increasing concentrations of sunitinib;CCK-8 assays were performed to analyze the sensitivity of sunitinib-resistant RCC cells and parental RCC cells to sunitinib when PCK2 was overexpressed.3.Rondomly divide the BALB/C nude mice of 5-6 weeks into two groups(each group with 5 mice): control group and PCK2 overexpression group.The CAKI cells with stable PCK2 overexpression and the control cells were subcutaneously injected into the axilla of these mice to construct a xenograft tumor model or injected into the tail vein to construct a metastasis model.the effect of PCK2 overexpression on biological function of RCC cells was evaluated through tumor growth and matastasis in mice.Results: 1.The expression of PCK2 is notably elevated in RCC cells after lentivirus transfection,which confirmed the effectiveness of stable PCK2 overexpression in RCC cells;the results of cell proliferation assays and plate clone formation assays showed PCK2 overexpression significantly suppressed the proliferation of RCC cells,the results of wound healing assays and transwell assays indicated PCK2 overexpression could significantly inhibit the migration and invasion of RCC cells.2.the expression of PCK2 was significantly decreased in sunitinib-resistant RCC cells comparing with parental cells,and it gradually increased as RCC cells were treated with increasing concentrations of sunitinib;the results of CCK-8 assays showed PCK2 overexpression enhances the sensitivity to sunitinib in resistant RCC cells or in parental RCC cells.3.The results of xenograft tumor models and metastasis models revealed that PCK2 overexpression suppresses tumor growth and metastasis in vivo.Conclusion: PCK2 overexpression significantly suppresses cell proliferation,migration and invasion of RCC,and it enhances the sensitivity to sunitinib in resistant RCC cells or in parental RCC cells.Part Ⅲ: The molecular mechanisms involved in declined PCK2 expression in RCC cells and the effect of PCK2 expression alteration on biological function of RCC cells Objective: To investigate the molecular mechanisms involved in declined PCK2 expression in RCC cells and the effect of PCK2 expression alteration on biological function of RCC cells Methods: 1.Analyze the correlations between declined PCK2 expression and DNA methylation of promoter region of PCK2 in RCC cells through online databases.Bisulfite Sequencing PCR(BSP)and Methylation-Specific Polymerase Chain Reaction(MSP)were conducted in 5 pairs of RCC clinical samples(cancer tissues and paracancerous tissue)to investigate the methylation changes of promoter region of PCK2 in RCC tissues and cells;RCC cells were treated with the demethylating drug 5-AZA to demonstrate the effect of methylation on the expression of PCK2,and a CRISPR/d Cas9-mediated editing system for PCK2 specific demethylation was constructed to further demonstrate the effect of methylation on the expression of PCK2.2.bioinformatics analysis including Gene Set Enrichment Analysis(GSEA)and Gene Ontology(GO)analysis were used to investigate PCK2-related pathways and protein interaction network.q RT-PCR,western blot and ER Tracker imaging were conducted to research the changes of endoplasmic reticulum stress in RCC cells and in sunitinib-resistant RCC cells after PCK2 overexpression.Functional rescue experiments were carried out by using the endoplasmic reticulum stress-specific inhibitor TUDCA to illustrate the role of endoplasmic reticulum stress in the PCK2-mediated biological functions in RCC.Results: 1.Obvious Cp G islands were found in the PCK2 promoter region through analysis of an online database(http://www.urogene.org/);the results of BSP and MSP indicated the methylation level of PCK2 promoter region was significantly higher in RCC than in its corresponding para-cancerous tissues;PCK2 experession was significantly elevated in RCC cells in m RNA and protein level after they are treated with the demethylating drug 5-AZA,a CRISPR/d Cas9-mediated editing system for PCK2 specific demethylation proved the similar results.2.The GSEA analysis showed that PCK2 is highly correlated with three basal metabolisms in RCC,including glucose metabolism,lipid metabolism,amino acid metabolism and glutathione metabolism;further analysis indicated that PCK2 was closely related to protein folding and unfolded protein response related genes;the results of q RTPCR,western blot showed the sensors of endoplasmic reticulum stress were greatly increased in RCC cells or sunitinib-resistant RCC cells with stable PCK2 overexpression;ER Tracker imaging revealed that ER tracker staining was significantly enhanced in RCC cells with stable PCK2 overexpression;the results of functional rescue experiments indicated that inhibition of endoplasmic reticulum stress significantly attenuated the weakened proliferation,migration and invasion capacities of RCC cells induced by PCK2 overexpression or PCK2 specific demethylation.Conclusion: The downregulated expression of PCK2 in RCC is a result of DNA methylation of promoter region of PCK2;overexpression of PCK2 suppressed the progression of RCC by promoting endoplasmic reticulum stress in RCC cells.