The enhancer of zeste homolog 2 (EZH2), a potential therapeutic target, is regulated by miR-101 in renal cancer cells

We investigated a prognostic significance and the mechanism of aberrant nuclear expression of EZH2, a histone methyltransferase, in human renal cell carcinoma (RCC). We found nuclear EZH2 in 48 of 100 RCCs and it was significantly correlated with worse survival in RCC patients. We detected a decreased expression of miR-101 in 15 of 54 RCCs. We found that re-expression of miR-101 resulted in EZH2 depletion and decreased renal cancer cell proliferation. Our results show nuclear EZH2 as a prognostic marker of worse survival in human RCC, and identify miR-101 as a negative regulator of EZH2 expression and renal cancer cell proliferation.     

Prognostic significance of survivin expression in renal cell cancer and its correlation with radioresistance

Survivin, an important inhibitor of apoptosis, has been found to play an important role in the initiation, progression, and chemoradioresistance of human malignancies. Previously, we have reported that upregulation of survivin in oral squamous cell carcinoma correlates with poor prognosis and chemoresistance. The aim of this study was to assess prognostic significance of survivin protein expression in RCC and analyze its correlation with radiosensitivity of RCC cells. RT-PCR and Western blot assays were performed to detect survivin mRNA and protein expression in normal human kidney epithelial cell line (HKEC) or RCC cell lines. The expression of survivin mRNA in RCC and corresponding nontumor kidney tissues was also detected by RT-PCR. Immunohistochemistry was performed to determine survivin protein expression in 75 cases of RCC tissue samples. Moreover, the association of survivin protein expression with clinicopathogical factors and prognosis of RCC patients was statistically analyzed. Small interfering RNA was used to knockdown the endogenous survivin expression in RCC cell line (ACHN) and evaluate the effects of survivin knockdown on proliferation, apoptosis, and radiosensitivity of RCC cell line. RCC cells showed sufficient expression of survivin mRNA and protein, but the expression of survivin gene was not detected in normal HKEC. Moreover, the expression level of survivin mRNA in RCC tissues was significantly higher than that in corresponding nontumor kidney tissues. The immunostaining of survivin protein was mainly located in cytoplasm of RCC tumor cells. Tumor pathological stage (P = 0.028), grade (P = 0.004), and lymph node metastasis (P = 0.017) of RCC patients were significantly correlated with survivin protein expression. In addition, patients with high survivin levels had a significantly shorter overall survival than those with low levels (P < 0.001), and the expression of survivin protein was an independent prognostic factor for RCC patients (P = 0.008). The expression of survivin gene could be reduced in RCC cell line and survivin knockdown could inhibit growth and enhance in vivo radiosensitivity of RCC cell line by inducing apoptosis enhancement. Taken together, the status of survivin protein expression may be an independent factor for predicting the prognosis of RCC patients and tumor-specific survivin knockdown combined with radiotherapy will be a potential strategy for RCC therapy.     

Uncoupling of PUMA Expression and Apoptosis Contributes to Functional Heterogeneity in Renal Cell Carcinoma — Prognostic and Translational Implications

Renal cell carcinoma (RCC) is characterized by a profound disruption of proapoptotic signaling networks leading to chemo- and radioresistance. A key mediator of DNA damage-induced apoptosis is the BH3-only protein PUMA. Given its central role in proapoptotic signaling, we analyzed a series of more than 600 precision-annotated primary RCC specimens for PUMA protein expression. We found a reduced expression of PUMA in 22.6% of RCCs analyzed. Unexpectedly, however, PUMA deficiency was not associated with more aggressive tumor characteristic as expected. Instead, a reduced PUMA expression was associated with a lower TNM stage, lower histopathologic grade, and more favorable cancer-specific patient survival. A direct correlation in a separate patient cohort revealed a profound disconnection between PUMA expression and apoptosis as exemplified by the fact that the tumor with the highest level of apoptotic cells was PUMA deficient. In a series of in vitro studies, we corroborated these results and discovered the highest propensity to undergo apoptosis in an RCC cell line with virtually undetectable PUMA expression. At the same time, PUMA expression was not necessarily associated with stronger apoptosis induction, which underscores the striking functional heterogeneity of PUMA expression and apoptosis in RCC. Collectively, our findings suggest that PUMA-independent mechanisms of cell death exist and may play an important role in suppressing malignant progression. They underscore the functional heterogeneity of RCCs and suggest that PUMA expression alone may not be a suitable predictive biomarker. A better understanding of alternative proapoptotic pathways, however, may help to design novel therapeutic strategies for patients with advanced RCC.     

Stage-specific roles of fibulin-5 during oxidative stress-induced renal carcinogenesis in rats

By using a rat model of renal cell carcinoma (RCC) induced by ferric nitrilotriacetate (Fe-NTA), this study performed genome-wide analysis to identify target genes during carcinogenesis. It screened for genes with decreased expression in RCCs, with simultaneous loss of heterozygosity, eventually to focus on the fibulin-5 (fbln5) gene. Oxidative damage via Fe-NTA markedly increased Fbln5 in the proximal tubules. RCCs presented lower levels of Fbln5. However, a fraction of RCCs presenting pulmonary metastasis revealed significantly higher levels of Fbln5 than those without metastasis, accompanied by immunopositivity of RCC cells and myofibroblast proliferation. Experiments revealed that RCC cell lines showed lower expression of fbln5 than its non-transformed counterpart NRK52E, but that fbln5 transfection to RCC cell lines changed neither proliferation nor migration/invasion. The data suggest that Fbln5 plays a role not only in the tissue repair and remodelling after renal tubular oxidative damage but also in RCC metastasis, presumably as a cytokine.     

Prognostic Value of EZH2 Expression and Activity in Renal Cell Carcinoma: A Prospective Study

Increased expression of EZH2 correlates with aggressive clinical behavior in various malignancies. In this study, we aim to investigate the clinical and prognostic values of EZH2 expression and activity in tumor tissues and improve the risk stratification in patients with renal cell carcinoma after surgery. We analyzed EZH2 expression and its activity as indicated by H3K27me3 levels comprising 373 patients with renal cell carcinoma in our institute. Outcome was assessed as overall survival and disease free survival using Kaplan-Meier analysis. Prognostic values of EZH2 and H3K27me3 expression for clinical outcomes were evaluated by Cox regression analysis. We used receiver operating characteristic to calculate diagnostic accuracy. High EZH2 expression correlates with poor overall survival in all patients, especially in advanced RCC, which is an independent prognostic factor in disease free survival and overall survival. Compared with EZH2, H3K27me3 expression is not an independent prognostic factor. The expressions of H3K27me3 and EZH2 are not completely consistent, which might be due to complicated interaction of Polycomb Repressor Complex 2. A combination of EZH2 expression and TNM stage could have better prognostic value than do TNM stage or EZH2 expression alone in both sets for disease free survival and overall survival. These results imply that evaluating intratumoral EZH2 density might improve prognostic value to the TNM staging system and inform treatment decisions for patients with late-stage renal cell carcinoma.     

Expression of heat shock protein 70 in renal cell carcinoma and its relation to tumor progression and prognosis

Heat shock proteins (HSPs) play an important role in the cellular response to environmental stress and exert a cytoprotective effect. Especially HSP70 is an effective inhibitor of apoptosis, suggesting a role of HSP70 in carcinogenesis and tumor progression. To explore the relevance of HSP70 in renal cell carcinomas (RCCs), we analyzed nuclear and cytoplasmic HSP70 protein expression in formalin-fixed tissue from 145 clear cell RCCs by immunohistochemistry as well as Western blot analysis. Nuclear HSP70 expression was found in all RCCs and 75% of the tumors also exhibited a cytoplasmic HSP70 staining. Importantly, RCCs showed significantly reduced cytoplasmic (p=0.001) and combined nuclear/cytoplasmic (p=0.0022) HSP70 expression when compared with their cells of origin. A significant (p=0.0176) decrease of nuclear HSP70 expression became evident from well to poorly differentiated clear cell RCCs. Quite similarly, a trend (p=0.0558) for reduced combined nuclear/cytoplasmic HSP70 expression was shown from early (pT1) to advanced (pT3) tumor stages. Nevertheless, no correlation between HSP70 expression and patients survival became evident. In conclusion, our investigation demonstrates a significant decrease of antiapoptotic HSP70 protein expression during carcinogenesis and during progression from well (G1) to poorly (G3) differentiated clear cell RCCs. Our results suggest that HSP70-mediated inhibition of apoptosis seems to be of minor importance for carcinogenesis and tumor progression in RCCs.     

FKBP51 promotes invasion and migration by increasing the autophagic degradation of TIMP3 in clear cell renal cell carcinoma

The occurrence of metastasis is a serious risk for renal cell carcinoma (RCC) patients. In order to develop novel therapeutic approaches to control the progression of metastatic RCC, it is of urgent need to understand the molecular mechanisms underlying RCC metastasis and identify prognostic markers of metastatic risk. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) have been known to be closely associated with extracellular matrix (ECM) turnover, which plays a highly active role in tumor metastasis. Recent studies have shown that immunophilin FK-506-binding protein 51 (FKBP51) may be important for the regulation of ECM function, and exert effects on the invasion and migration of tumor cells. However, the mechanisms underlying these activities remain unclear. The present study detected the role of FKBP51 in clear cell renal cell carcinoma (ccRCC), the most common subtype of RCC, and found that FKBP51 significantly promotes ccRCC invasion and migration by binding with the TIMP3, connecting TIMP3 with Beclin1 complex and increasing autophagic degradation of TIMP3. Given the important roles that TIMPs/MMPs play in ECM regulation and remodeling, our findings will provide new perspective for future investigation of the regulation of metastasis of kidney cancer and other types of cancer.Subject terms: Renal cell carcinoma, Extracellular matrix     

Identification of a universal 6-lncRNA prognostic signature for three pathologic subtypes of renal cell carcinoma

Renal cell carcinoma (RCC) is the most common adult renal epithelial cancer susceptible to metastasis and patients with irresectable RCC always have a poor prognosis. Long noncoding RNAs (lncRNAs) have recently been documented as having critical roles in the etiology of RCC. Nevertheless, the prognostic significance of lncRNA-based signature for outcome prediction in patients with RCC has not been well investigated. Therefore, it is essential to identify a lncRNA-based signature for predicting RCC prognosis. In the current study, we comprehensively analyzed the RNA sequencing data of the three main pathological subtypes of RCC (kidney renal clear cell carcinoma [KIRC], kidney renal papillary cell carcinoma [KIRP], and kidney chromophobe carcinoma [KICH]) from The Cancer Genome Atlas (TCGA) database, and identified a 6-lncRNA prognostic signature with the help of a step-wise multivariate Cox regression model. The 6-lncRNA signature stratified the patients into low- and high-risk groups with significantly different prognosis. Multivariate Cox regression analysis showed that predictive value of the 6-lncRNA signature was independent of other clinical or pathological factors in the entire cohort and in each cohort of RCC subtypes. In addition, the three independent prognostic clinical factors (including age, pathologic stage III, and stage IV) was also stratified into low- and high-risk groups basis on the risk score, and the stratification analyses demonstrated that the high-risk score was a poor prognostic factor. In conclusion, these findings indicate that the 6-lncRNA signature is a novel prognostic biomarker for all three subtypes of RCC, and can increase the accuracy of predicting overall survival.     

Inhibition of PTEN Gene Expression by Oncogenic miR-23b-3p in Renal Cancer

Background

miR-23b is located on chromosome number 9 and plays different roles in different organs especially with regards to cancer development. However, the functional significance of miR-23b-3p in renal cell carcinoma (RCC) has not been reported.

Methods and Results

We measured miR-23b-3p levels in 29 pairs of renal cell carcinoma and their normal matched tissues using real-time PCR. The expression level of miR-23b-3p was correlated with the 5 year survival rate of renal cancer patients. In 15 cases (52%), miR-23b-3p expression was found to be high. All patients with moderate to low miR-23b-3p expression survived 5 years, while those with high miR-23b-3p expression, only 50% survived. After knocking down miRNA-23b-3p expression in RCC cell lines, there was an induction of apoptosis and reduced invasive capabilities. MiR-23b-3p was shown to directly target PTEN gene through 3′UTR reporter assays. Inhibition of miR-23b-3p induces PTEN gene expression with a concomitant reduction in PI3-kinase, total Akt and IL-32. Immunohistochemistry showed the lack of PTEN protein expression in cancerous regions of tissue samples where the expression of miR-23b-3p was high. We studied the in vitro effects of the dietary chemo preventive agent genistein on miR-23b-3p expression and found that it inhibited expression of miR-23b-3p in RCC cell lines.

Conclusions

The current study shows that miR-23b-3p is an oncogenic miRNA and inhibits PTEN tumor suppressor gene in RCC. Therefore, inhibition of miR-23b-3p may be a useful therapeutic target for the treatment of renal cell carcinoma.     

Pinin acts as a poor prognostic indicator for renal cell carcinoma by reducing apoptosis and promoting cell migration and invasion

Pinin (PNN) was originally characterized as a desmosome-associated molecule. Its function and the mechanism of its regulation in renal cell carcinoma (RCC) are still undefined. Data on PNN expression, clinicopathological features, and prognosis of patients with RCC were obtained from The Cancer Genome Atlas (TCGA) database. Immunohistochemistry revealed high PNN expression in tumour cells. PNN expression showed negative correlation with survival in patients with RCC, acting as an independent prognostic factor in RCC. PNN up-regulation might be attributed to epigenetic alterations in RCC. Immunofluorescence revealed PNN expression mainly in the nucleus of RCC cells. The transfection of siRNA targeting the PNN gene resulted in enhanced apoptosis, which was detected by flow cytometry, and reduced cell migration and invasion, which were assessed using wound healing and transwell migration assay. Gene set enrichment analysis revealed associations between PNN expression and several signalling pathways involved in cancer progression, as a potential mechanism underlying the carcinogenicity of PNN. The analyses of the Tumor Immune Estimation Resource platform showed significant positive associations between high PNN expression and tumour immune infiltrating cells. PNN may function as an oncogenic factor by reducing apoptosis and promoting cell migration and invasion in RCC.     

UBE3C Promotes Growth and Metastasis of Renal Cell Carcinoma via Activating Wnt/β-Catenin Pathway

Renal cell carcinoma (RCC) is the most common primary malignancy of the kidney and one of the most lethal genitourinary malignancies. Clear-cell renal cell carcinoma (ccRCC) has an extremely poor prognosis because of a high potential for tumor growth, vascular invasion, metastasis and recurrence. Unfortunately, the mechanism of RCC growth and metastasis is not well understood. In this report, we for the first time demonstrated ubiquitin protein ligase E3C (UBE3C) as a driving factor for RCC growth and metastasis. UBE3C expression was increased in ccRCC tissues compared with adjacent normal tissues. ccRCC patients with high UBE3C protein expression in tumors were associated with significantly worse postoperative survival. Knockdown of UBE3C expression in ACHN cells inhibited cell proliferation, migrations and invasiveness in vitro while overexpression of UBE3C in 786-O cells exerted the opposite effects. UBE3C up-regulated β-catenin protein levels and promoted β-catenin nuclear accumulation, leading to the activation of the Wnt/β-catenin signal pathway in RCC cells. Collectively, these observations suggest that UBE3C plays an important role in RCC development and progression, and UBE3C may be a novel target for prevention and treatment of ccRCC.     

Lack of Ephrin Receptor A1 Is a Favorable Independent Prognostic Factor in Clear Cell Renal Cell Carcinoma

The EPH receptor tyrosine kinases and their cell-bound ligands, the ephrins, have been shown to be associated with cancer development and progression. In this study, mRNA and protein expression of the receptors EPHA1 and EPHA2 as well as of their ligand EFNA1 and their prognostic relevance in clear cell renal cell carcinoma was evaluated. Gene expression was measured in 75 cryo-preserved primary tumors and matched non-malignant renal specimens by quantitative PCR. Protein expression was analyzed by immunohistochemistry on tissue microarrays comprising non-malignant, primary tumors and metastatic renal tissues of 241 patients. Gene and protein expression of all three factors was altered in tumor specimens with EPHA1 and EPHA2 being generally diminished in tumors compared to normal renal tissue, whereas EFNA1 was commonly elevated. A positive EPHA1 and EPHA2 protein staining as well as a low EFNA1 protein level were significantly linked to more aggressive tumor features, but only a positive EPHA1 immunoreactivity was significantly associated with poor survival. In subgroup analyses, EPHA1 and EPHA2 protein levels were significantly higher in metastatic than in primary lesions. Patients with EPHA1/EPHA2-positive tumors or with tumors with positive EPHA1 and low EFNA1 immunoreactivity had the shortest survival rates compared to the respective other combinations. In a multivariate model, EPHA1 was an independent prognostic marker for different survival endpoints. In conclusion, an impaired EPH-ephrin signaling could contribute to the pathogenesis and progression of clear cell renal cell carcinoma.     

Enhanced IMP3 Expression Activates NF-кB Pathway and Promotes Renal Cell Carcinoma Progression

Background

Insulin-like growth factor 2 mRNA binding protein 3 (IMP3) is expressed in metastatic and a subset of primary renal cell carcinoma (RCC). However, the role of IMP3 in RCC progression was poorly understood. We aim to uncover the mechanism of IMP3 in regulating clear cell RCC (CCRCC) progression and validate the prognostic significance of IMP3 in localized CCRCC.

Methods

Caki-1 cells stably overexpressing IMP3 and Achn cells with knockdown of IMP3 were analyzed for cell migration and invasion by Transwell assay. RNA-seq was used to profile gene expression in IMP3-expressing Caki-1 cells. A cohort of 469 localized CCRCC patients were examined for IMP3 expression by immunohistochemistry using tumor tissue array.

Results

IMP3 promoted Caki-1 cell migration and invasion, whereas knockdown of IMP3 by RNAi inhibited Achn cell migration and invasion. Enhanced IMP3 expression activated NF-кB pathway and through which, it functioned in promoting the RCC cell migration. IMP3 expression in localized CCRCC was found to be associated with higher nuclear grade, higher T stage, necrosis and sarcomatoid differentiation (p< 0.001). Enhanced IMP3 expression was correlated with shorter recurrence-free and overall survivals. Multivariable analysis validated IMP3 as an independent prognostic factor for localized CCRCC patients.

Conclusion

IMP3 promotes RCC cell migration and invasion by activation of NF-кB pathway. IMP3 is validated to be an independent prognostic marker for localized CCRCC.     

Differential Expression of Prognostic Proteomic Markers in Primary Tumour,Venous Tumour Thrombus and Metastatic Renal Cell Cancer Tissue and Correlation with Patient Outcome

Renal cell carcinoma (RCC) is the most deadly of urological malignancies. Metastatic disease affects one third of patients at diagnosis with a further third developing metastatic disease after extirpative surgery. Heterogeneity in the clinical course ensures predicting metastasis is notoriously difficult, despite the routine use of prognostic clinico-pathological parameters in risk stratification. With greater understanding of pathways involved in disease pathogenesis, a number of biomarkers have been shown to have prognostic significance, including Ki67, p53, vascular endothelial growth factor receptor 1 (VEGFR1) and ligand D (VEGFD), SNAIL and SLUG. Previous pathway analysis has been from study of the primary tumour, with little attention to the metastatic tumours which are the focus of targeted molecular therapies. As such, in this study a tissue microarray from 177 patients with primary renal tumour, renal vein tumour thrombus and/or RCC metastasis has been created and used with Automated Quantitative Analysis (AQUA) of immunofluorescence to study the prognostic significance of these markers in locally advanced and metastatic disease. Furthermore, this has allowed assessment of differential protein expression between the primary tumours, renal vein tumour thrombi and metastases. The results demonstrate that clinico-pathological parameters remain the most significant predictors of cancer specific survival; however, high VEGFR1 or VEGFD can predict poor cancer specific survival on univariate analysis for locally advanced and metastatic disease. There was significantly greater expression of Ki67, p53, VEGFR1, SLUG and SNAIL in the metastases compared with the primary tumours and renal vein tumour thrombi. With the exception of p53, these differences in protein expression have not been shown previously in RCC. This confirms the importance of proliferation, angiogenesis and epithelial to mesenchymal transition in the pathogenesis and metastasis of RCC. Importantly, this work highlights the need for further pathway analysis of metastatic tumours for overcoming drug resistance and developing new therapies.     

VEGF和Survivin在肾癌中的表达及其相关性研究

目的：探讨肾癌组织中血管内皮生长因子VEGF与凋亡抑制蛋白Survivin表达的相关性及其之间的关系,研究Survivin和VEGF在肾癌发生发展中的作用机制。方法：应用免疫组织化学方法检测70例肾癌组织和70例癌旁正常肾脏组织中VEGF和Survivin的表达,并将检测结果与临床病理特征进行综合分析。结果：VEGF和Survivin在肾癌中表达均高于癌旁正常肾脏组织;Survivin和VEGF在肾癌中的阳性表达率分别为75.71%（53/70）和72.86%（51/70）,在癌旁肾脏组织中的表达率分别为0%（0/70）、17.14%（12/70）,差异均有显著性意义（P〈0.05）;VEGF和Survivin的表达与患者的性别、年龄、肿瘤大小、病理分级均无相关性;VEGF和Survivin表达呈正相关性。结论：VEGF和Survivin在肾癌组织中表达率较高,为肾癌的分子靶向治疗提供了新的靶点。Survivin和VEGF在RCC中的表达关系密切,测定RCC中Survivin、VEGF蛋白的表达,有助于临床判断病人预后。     

Cytoplasmic Accumulation of Heterogeneous Nuclear Ribonucleoprotein K Strongly Promotes Tumor Invasion in Renal Cell Carcinoma Cells

Heterogeneous nuclear ribonucleoprotein (hnRNP) K is a part of the ribonucleoprotein complex which regulates diverse biological events. While overexpression of hnRNP K has been shown to be related to tumorigenesis in several cancers, both the expression patterns and biological mechanisms of hnRNP K in renal cell carcinoma (RCC) cells remain unclear. In this study, we showed that hnRNP K protein was strongly expressed in selected RCC cell lines (ACHN, A498, Caki-1, 786–0), and knock-down of hnRNP K expression by siRNA induced cell growth inhibition and apoptosis. Based on immunohistochemical (IHC) analysis of hnRNP K expression in human clear cell RCC specimens, we demonstrated that there was a significant positive correlation between hnRNP K staining score and tumor aggressiveness (e.g., Fuhrman grade, metastasis). Particularly, the rate of cytoplasmic localization of hnRNP K in primary RCC with distant metastasis was significantly higher than that in RCC without metastasis. Additionally, our results indicated that the cytoplasmic distribution of hnRNP K induced by TGF-β stimulus mainly contributed to TGF-β-triggered tumor cell invasion in RCC cells. Dominant cytoplasmic expression of ectopic hnRNP K markedly suppressed the inhibition of invasion by knock-down of endogenous hnRNP K. The expression level of matrix metalloproteinase protein-2 was decreased by endogenous hnRNP K knock-down, and restored by ectopic hnRNP K. Therefore, hnRNP K may be a key molecule involved in cell motility in RCC cells, and molecular mechanism associated with the subcellular localization of hnRNP K may be a novel target in the treatment of metastatic RCC.     

Elevated expression of stromal palladin predicts poor clinical outcome in renal cell carcinoma

The role that stromal renal cell carcinoma (RCC) plays in support of tumor progression is unclear. Here we sought to determine the predictive value on patient survival of several markers of stromal activation and the feasibility of a fibroblast-derived extracellular matrix (ECM) based three-dimensional (3D) culture stemming from clinical specimens to recapitulate stromal behavior in vitro. The clinical relevance of selected stromal markers was assessed using a well annotated tumor microarray where stromal-marker levels of expression were evaluated and compared to patient outcomes. Also, an in vitro 3D system derived from fibroblasts harvested from patient matched normal kidney, primary RCC and metastatic tumors was employed to evaluate levels and localizations of known stromal markers such as the actin binding proteins palladin, alpha-smooth muscle actin (α-SMA), fibronectin and its spliced form EDA. Results suggested that RCCs exhibiting high levels of stromal palladin correlate with a poor prognosis, as demonstrated by overall survival time. Conversely, cases of RCCs where stroma presents low levels of palladin expression indicate increased survival times and, hence, better outcomes. Fibroblast-derived 3D cultures, which facilitate the categorization of stromal RCCs into discrete progressive stromal stages, also show increased levels of expression and stress fiber localization of α-SMA and palladin, as well as topographical organization of fibronectin and its splice variant EDA. These observations are concordant with expression levels of these markers in vivo. The study proposes that palladin constitutes a useful marker of poor prognosis in non-metastatic RCCs, while in vitro 3D cultures accurately represent the specific patient's tumor-associated stromal compartment. Our observations support the belief that stromal palladin assessments have clinical relevance thus validating the use of these 3D cultures to study both progressive RCC-associated stroma and stroma-dependent mechanisms affecting tumorigenesis. The clinical value of assessing RCC stromal activation merits further study.