Aberrant Activation Of TGF-β1/Snail/SPARC Signaling Pathway Promotes Renal Cell Carcinoma Invasion And Metastasis

BackgroundRenal cell carcinoma(RCC)is one of the most common malignancies,and also one of the most aggressive urologic cancers.There are about 330,000 new RCC cases and 144000 deaths globally each year.Noticeably,the incidence of RCC is still increasing.Therefore,renal cancer has become one of the diseases that are severly threatening the public health.RCC arises from the renal tubules with unclear direct causes.It has been reported that genetic factors,smoking,obesity,heypertension and anti-heypertension treatment are risk factors of RCC.With the wildly usage of screening examination like type B ultrasound,the development of surgical techniques,and the application of targeted drugs,great progress has been made in clinical treatment of RCC.Localized RCC can be treated with partial or radical nephrectomy with favorable outcome.However,due to a late diagnosis of the tumor or disease progresses after surgical treatment,about 20-30%of the patients eventually develop metastasis.The involved target organ in metastatic RCC has a significant impact on patient’s prognosis,bone,brain and liver involvement resulting in adverse survival compared to lung and lymph nodal disease.The outcome of metastatic RCC is very poor,the median overall survival of un-treated metastatic disease is 5 months with 1-year survival of only 29%.Therefore,RCC metastasis has a big influence on patient’s prognosis,prevention or inhibition of the metastasis will significantly improve RCC patient’s outcome.Thus,exploring the biological basis and detailed molecular mechanisms driving RCC metastasis to identify druggable targets and block metastasis has become a priority of great significance.TGF-β signaling pathway plays an important role in many biological process including cell growth,differenciation,appotosis,migration,tumorigenesis and progression.TGF-β is closely associated with RCC development.Previous studies found that the expression of TGF-β is higher in RCC tissues when compared with that in benign kidney tissue.Noticeably,TGF-β is able to enhance the invasiness of RCC cells and further promote the bone metastasis of RCC.The detailed molecular mechanisms involved in TGF-β-induced metastasis-promoting effect are still not very clear.In general,we believe TGF-β promotes tumor invasion and metastasis by inducing the transcription and expression of its downstream target genes associated with metastasis,these genes includes parathyroid hormone related protein(PTHrP),Interleukin 11(IL11),connective-tissue growth factor(CTGF)and chemokine receptor type 4(CXCR4).However,it’s still unknown which downstream target gene of TGF-β is involved in RCC invasion and metastasis.Many previous studies reported that Snail is a downstream target gene of TGF-βsignaling pathway.Snail is a zinc finger transcription factor which is able to bind to the specific secquances of the upstream of a gene’s 5’end and ensure the expression of a gene in a specific degree at right place and right time.Previous studies discovered that Snail could modulate the transcription of many genes;Snail can repress the transcription of E-Cadherin,claudin,occluding,mucin and cytokeratin 18,and promote the transcription of Vimenti and Fibronectin.Noticeably,Snail also plays an important role in RCC development.Several studies reported overexpression of Snail in RCC tissues,there was a positive association between Snail expression level and RCC clinical stage and pathological grade.Furthermore,higher Snail expression predicted poor outcome of RCC patients.Secreted protein acidic and rich in cysteine(SPARC),also known as osteonectin,belongs to the family of matricellular proteins.SPARC contains three distinct domains,an acidic N-terminus domain,a follistatin-like domain and an extracellular domain which can bind to calcium.SPARC is able to bind to collagen and calcium,it involves in extracellular matrix deposition and tissue remodeling.In recent years,the important role of SPARC in cancer development has received much attention.Many studies reported that SPARC was a tumor suppressor.On the contrary,several studies pointed out that SPARC could promote cancer development.Thus,the function of SPARC in cancer is still controversial;its role in different cancers could be very different.The function of SPARC in RCC is still unknown and the related published articles are very few.However,overexpression of SPARC in sarcomatoid RCC with aggressive phenotype has been reported,implying the potential role of SPARC in RCC development.What is the role of SPARC in RCC development?What is the molecular mechanism modulating the expression of SPARC in RCC?What is the downstream network regulated by SPARC?The answer to these questions will help to discover the molecular mechanisms driving RCC progression.The molecular mechanisms modulating SPARC expression in RCC is still unknown.However,we find several Snail binding sites(CAGGTG)in SPARC gene promoter after we carefully analyzed the secquance of SPARC promoter,indicating that Snail as a transcriptin factor may regulate the transcription of SPARC.Therefore,based on our data and the current research advance,we propose this hypothesis for the first time:SPARC could be a downstream target of TGF-β/Snail signaling pathway;TGF-β1/Snail/SPARC signaling pathway may play an important role in RCC invasion and metastasis.Our study will reveal the role of TGF-β1/Snail/SPARC signaling pathway in RCC invasion and metastasis and the underlying molecular mechanisms,and will deepen and expand our understanding of the biological function of TGF-β,Snail and SPARC.This study will provide theoretical and experimental evidences for targeting TGF-β1/Snail/SPARC signaling pathway to diagnose,treat and evaluate RCC,and offer new ideas and methods for clinical treatment of RCC.MethodsWe identified TGF-β1 was closely associated with RCC progression and prognosis by analyzing the RNA-secquancing data from human RCC tissues.Then,we investigated the biological effect of TGF-β1 on RCC cells by using transwell invasion assay.SPARC was identified as a TGF-β-induced protein in RCC cells by applying microarray analysis,qRT-PCR and Western blot.We subsequently downregulated the expression of SPARC by using ShRNA and SPARC monoclonal antibody and in vitro and in vivo assays were performed to validate the role of SPARC in RCC development.Luciferase reporter gene assay revealed that TGF-β1 enhanced the transcription of SPARC and further identified the key transcription factor involved in this regulation.We further explored the related molecular mechanisms behind the metastasis-promoting effect of SPARC by using RNA interference,qRT-PCR and Western blot.Finally,SPARC expression level was examined in large sample size of RCC tissues;the correlation between SPARC expression and RCC progression as well as patient outcome were also examined by using the clinical data from RCC patients.ResultsBased on all above experiments,we got the following novel findings:1.TGF-β1 is highly associated with RCC progression.2.SPARC is a key mediator in TGF-β1 induced RCC invasion.3.TGF-β1 increases SPARC gene transcription via Snail.4.SPARC is a key mediator in TGF-β1 regulated MMP 2.5.Reduced SPARC expression innibits EMT.6.Loss of SPARC inhibits the metastasis of RCC cell in a mouse model.7.SPARC expression predicts poor outcome of RCC patients.ConclusionsThis study explores the regulatory mechanism of SPARC expression and the function of SPARC in RCC invasion and metastasis as well as the underlying molecular mechanisms.TGF-β1 induced SPARC expression in RCC cells,in vitro and in vivo assays discovered that SPARC could significantly promote RCC invasion and metastasis.Mechanistically,SPARC transcription can be induced by Snail that is a TGF-β signaling pathway downstream target gene.The presence of SPARC can enhance RCC invasion by not only increasing matrix metalloproteinase-2(MMP 2)expression and activity but also promoting EMT.Clinically,TGF-β1,Snail and SPARC were highly elevated in RCC tissues,and SPARC expression was positively correlated with RCC stage.Noticeably,survival analysis of a large cohort of RCC samples indicated that SPARC expression could predict RCC patient’s outcome.We conclude that SPARC is not only a new prognostic marker but a potential therapeutic target to prevent RCC metastasis.