Smad4,the Key Factor Of Valproic Acid (VPA),Inhibits Epithelial-Mesenchymal Transition In Renal Cell Carcinoma

[Purpose]Accounting for 2-3%of all human malignancies,renal cell carcinoma(RCC)is the most common kidney malignancy worldwide,with clear-cell RCC(ccRCC)being the most common subtype.Despite advances in diagnosis and treatment,particularly improved imaging techniques,patients with RCC still have an extremely poor prognosis,making RCC a serious problem for oncologic healthcare around the world.Indeed,the 5-year cancer-specific survival of metastatic ccRCC is less than 27.1%,much lower than that of nonmetastatic ccRCC,which is 70%;and ccRCC is prone to metastasis.Regrettably,the mechanism of cancer metastasis and the cause of treatment resistance are currently poorly understood and deserve more attention and research.Epithelial-mesenchymal transition(EMT),a program that transforms epithelial cell phenotypes into cells with mesenchymal phenotype,has been shown to play a pivotal role in various steps of metastatic progression in tumors.In EMT,epithelial cells lose cell polarity,disassemble cell-cell junctions and gain a more mesenchymal and motile property,endowing cancer cells with greater capabilities to invade and disseminate to distant sites.This phenomenon is triggered by a series of complex and multi-layered growth factors recruited from tumor microenvironments.TGF-P is a pluripotent cytokine with bidirectional roles in cancer progression.The natural factor determining whether TGF-β acts as a tumor suppressor or promoter has been the subject of intense research.TGF-β has also been reported to play a crucial role in initiating EMT in various cancers[.However,a striking gap still exists in our understanding of the function of early response transcription factors.Smad4 protein is recognized as a central mediator of TGF-βand/or bone morphogenetic protein(BMP)signaling pathways.Initially,studies showed that the loss of Smad4 leads to the dysfunction of the canonical TGF-βsignaling pathway.However,in many cancers,TGF-β switches from a tumor suppressor to a tumor promoter,thereby driving invasion and metastasis.By activating both Smad-dependent and Smad-independent pathways,TGF-β acts as a potent inducer of EMT,and Smad4 has been considered an independent prognostic factor in ccRCC.Epigenetic modification such as histone acetylation is one mechanism mediating gene expression.Histone acetylation is mediated by the counteracting activity of histone acetyltransferases(HATs)and histone deacetylases(HDACs).The reversible acetylation and deacetylation of histones is always accompanied with the activation and silencing of gene expression.Histone post-translational modifications(PTMs)play a fundamental role in the control of DNA-templated processes in the cell.Valproic acid(VPA),a classic anticonvulsant drug used for decades,has recently been shown to be a potent class I histone deacetylase(HDAC)inhibitor.VPA potentially induces several anticancer effects,especially inhibition of cancer cell proliferation,growth,and differentiation.Cell cycle,growth and apoptosis were also influenced by VPA in RCC.We present evidence that VPA negatively regulates Smad4 expression in RCC cells,thereby inhibiting cancer cell metastasis and invasion.In this study,we aimed to identify the role played by Smad4 in RCC progression and metastasis by administering VPA to RCC cell lines and analyzing the relation among VPA,Smad4 and EMT regulation in RCC.According to previous studies,epithelial-mesenchymal transition(EMT)is responsible for cancer metastasis.Moreover,valproic acid(VPA),an anticonvulsant drug,can suppress tumor metastasis and decrease the expression level of Smad4.Thus,VPA may suppress cancer metastasis by inhibiting epithelial-mesenchymal transition.However,the relation among VPA,EMT and Smad4 in RCC metastasis is still obscure.[Methods]The expression of Smad4 and EMT markers in RCC cell lines treated with VPA was analyzed using quantitative real-time PCR and western blot.Small interfering RNA technology was used with the human renal carcinoma cell line 786-0.We tested the invasion and metastatic ability of 786-0 cells using Transwell invasion assays.Smad4,TIF1-γ and TGF were also detected by immunohistochemistry(IHC)in 39 primary RCC samples.Results.In 786-0 and Caki-1 cell lines treated with VP A,N-cadherin,vimentin,and Smad4 protein and Smad4 mRNA were significantly reduced,while E-cadherin expression increased.Smad4 silencing strongly decreased expression of EMT markers,such as N-cadherin,and simultaneously upregulated E-cadherin protein in RCC cell lines.Smad4 overexpression counteracted the VPA-mediated EMT-inhibitory effect(p<0.05).[Conclusions]Based on its regulatory effect on EMT-related transcription factors,our study demonstrates that VPA inhibits EMT in RCC cells by altering Smad4 expression.Smad4,which can form a Smad3/Smad4 complex induced by TGF-β1,could be a potential anti-cancer drug target inhibiting tumor invasion and metastasis in RCC.