Molecular Mechanism Of Sunitinib - Induced Cell Senescence And Klotho Inhibition Of Epithelial - Mesenchymal Transition In Molecular Targeted Therapy Of Renal Clear Cell Carcinoma

Renal cell carcinoma (RCC), referred as kidney cancer, is malignant tumors originated in the renal tubular epithelial cells accounting for about90%of the adult kidney malignacy. Histologically, the RCC can be classified into a series of subtypes having unique morphology and genetic features. Clear cell renal cell carcinoma (CCRCC), originated from proximal tubule epithelial, is the most common type, accounting for70to80%of RCC. The carcinogenesis of CCRCC is associated with von Hippel-Lindau (VHL) tumor suppressor gene defects. pVHL mutation leads to enhanced hypoxia-inducible factor-a (HIF-a) stability. HIF-a translocates into nucleus to form a complex with HIF-0to transcriptionally active a series of hypoxia related genes promoting tumor angiogenesis (via VEGF, EPO), tumor cell growth (via TGF-a, PDGF-β, Cyclin D) and glucose metabolism (via GLUT1). As a result, it provides a strong theoretical basis for the targeted therapy of VHL pathway.Sunitinib (SUO11248) is a well-known multitargeted inhibitor of receptor tyrosine kinases (RTK) that selectively block several growth factor receptors downstreaming of the VHL pathway. Previous study showed that sunitinib mainly targets at vascular endothelial growth factor receptor (VEGFR), mainly expressed in the turnor vascular endothelial cells and pericytes, plays an important role in the inhibition of angiogenesis. Along with increased widespread sunitinib administration against mRCC, functional significance and molecular mechanism for sunitinib treatment performance as well as acquired therapeutic resistance await urgent elucidation for personalized molecular-targeted therapeutics to fortify efficacy and overcome drug resistance. The first part of the study is focused on functional significance and potential mechanisms for sunitinib treatment performance in RCC.Molecular targeted therapy against VHL pathway has made great strides, prolonged the overall survival of RCC patients, although20to30%of the overall efficiency accompanied with a high incidence of disease progression and adverse reactions during treatment. Therefore, a deeper study of RCC tumor pathology and molecular biology will help us to improve the existing treatments and move towards a newer and more effective therapeutic target. Klotho is an aging suppressor protein, expressed predominantly in distal renal tubular epithelial cells, mainly involved in the modulation of calcium and phosphorus metabolism along with energy metabolism. Despite tumor suppressive function of Klotho has been indicated in various cancers recently, function roles and molecular events for Klotho in RCC remain poorly understood. This study was elucidates the crucial role and molecular mechanism of Klotho during renal carcinogenesis.In part one of this thesis, although sunitinib has been used as a first-line therapy against metastatic renal cell carcinoma (mRCC), the bona fide therapeutic target cell and underlying mechanism of sunitinib treatment remain controversial.We reported that sunitinib-treated RCC cells exhibit senescence characteristics including SA-β-gal activity, DcR2and Decl expression, and senescence-associated secretary phenotype (SASP) such as secretion of pro inflammatory cytokines IL-la, IL-6and IL-8etc. Moreover, sunitinib administration also led to cell growth repression, Gl-S phase cell cycle arrest and DNA damage response in RCC cells, suggesting therapeutic of sunitinib-induced RCC cellular senescence. Mechanistic investigations indicated that therapy-induced senescence (TIS) following sunitinib treatment were mainly attributed to p53/Decl signaling activation mediated by Raf-1/NF-κB sinhibition in vitro. Importantly, in vivo study showed tumor growth inhibition and prolonged overall survival associated with increased p53and Decl expression, decreased Raf-1and Ki67staining, and upregulated SA-P-gal activity after sunitinib treatment. Immunohistochemistry analysis of tumor tissues from RCC patients receive sunitinib neoadjuvant therapy confirmed the similar treating phenotype. Conclusion:Our findings suggested that sunitinib treatment performance could be attributable to TIS, depended on p53/Decl activation via Raf-1/NF-κB activity repression, and indicated potential insights into therapeutic improvement with reinforcing TIS-related performance or overcoming SASP-induced resistance.In part two of this thesis, Klotho is an anti-aging protein predominantly expressed in renal tubular epithelial cells. Although Klotho is recently identified as a tumor suppressor gene in a variety of cancers, the potential role and molecular events for Klotho in renal cell carcinoma (RCC) remains obscure. In our present study, immunohistochemical staining in tissue microarray containing125RCC samples showed that intratumoral Klotho levels were negatively correlated with tumor size, TNM stage and nuclear grade. The overall survival rate of RCC patients with high Klotho expression was significantly higher than those patients with low Klotho expression. We found that Klotho expression could induce change of cell morphology from mesenchymal-like phenotype to epithelial-like phenotype, upregulation of epithelial marker expression (E-cadherin), downregulation of mesenchymal marker expression (N-cadherin, vimentin), which indicates Klotho expression could induce mesenchymal-epithelial transition (MET) in RCC cells. Besides, no alteration of a-2,6sialidase activity was found after Klotho overexpression in RCC. Furhter molecular analysis with gain-and lose-function of Klotho in this work implicates that Klotho blunts RCC cellular EMT phenotypic switch through PI3K/Akt/GSK3β/Snail signaling suppression. PI3K inhibition with LY294002and GSK3β knockdown with LiCl imitate similar phenomenon in RCC cells after Klotho overexpression and Knockdown, respectively. Moreover, Klotho expression in RCC cells could impair cell migration and invasion ability in vitro, which could be reversed by constitutive activated Snail cotransfection, through wound healing assay, transwell cell migration assay, and collagen cell invasion assay. Klotho expression may be involved in the invasion and metastasis of renal cell carcinoma by suppression renal cancer EMT.Conclusion:These results indicate Klotho as a tumor suppressor via inhibiting PI3K/Akt/GSK3β/Snail signaling, thus suppressing EMT and tumor migration and invasion during RCC progression, which might be used as a potential therapeutic approach for advanced RCC treatment.