Differential expression of microRNA501-5p affects the aggressiveness of clear cell renal carcinoma

AbstractRenal cell carcinoma is a common neoplasia of the adult kidney that accounts for about 3% of adult malignancies. Clear cell renal carcinoma is the most frequent subtype of kidney cancer and 20–40% of patients develop metastases. The absence of appropriate biomarkers complicates diagnosis and prognosis of this disease. In this regard, small noncoding RNAs (microRNAs), which are mutated in several neoplastic diseases including kidney carcinoma, may be optimal candidates as biomarkers for diagnosis and prognosis of this kind of cancer. Here we show that patients with clear cell kidney carcinoma that express low levels of miR501-5p exhibited a good prognosis compared with patients with unchanged or high levels of this microRNA. Consistently, in kidney carcinoma cells the downregulation of miR501-5p induced an increased caspase-3 activity, p53 expression as well as decreased mTOR activation, leading to stimulation of the apoptotic pathway. Conversely, miR501-5p upregulation enhanced the activity of mTOR and promoted both cell proliferation and survival. These biological processes occurred through p53 inactivation by proteasome degradation in a mechanism involving MDM2-mediated p53 ubiquitination. Our results support a role for miR501-5p in balancing apoptosis and cell survival in clear cell renal carcinoma. In particular, the downregulation of microRNA501-5p promotes a good prognosis, while its upregulation contributes to a poor prognosis, in particular, if associated with p53 and MDM2 overexpression and mTOR activation. Thus, the expression of miR501-5p is a possible biomarker for the prognosis of clear cell renal carcinoma

SelectMDx and Multiparametric Magnetic Resonance Imaging of the Prostate for Men Undergoing Primary Prostate Biopsy: A Prospective Assessment in a Multi-Institutional Study

Prostate-specific antigen (PSA) testing as the sole indication for prostate biopsy lacks specificity, resulting in overdiagnosis of indolent prostate cancer (PCa) and missing clinically significant PCa (csPCa). SelectMDx is a biomarker-based risk score to assess urinary HOXC6 and DLX1 mRNA expression combined with traditional clinical risk factors. The aim of this prospective multi-institutional study was to evaluate the diagnostic accuracy of SelectMDx and its association with multiparametric magnetic resonance (mpMRI) when predicting PCa in prostate biopsies. Overall, 310 consecutive subjects were included. All patients underwent mpMRI and SelectMDx prior to prostate biopsy. SelectMDx and mpMRI showed sensitivity and specificity of 86.5% vs. 51.9%, and 73.8% vs. 88.3%, respectively, in predicting PCa at biopsy, and 87.1% vs. 61.3%, and 63.7% vs. 83.9%, respectively, in predicting csPCa at biopsy. SelectMDx was revealed to be a good predictor of PCa, while with regards to csPCa detection, it was demonstrated to be less effective, showing results similar to mpMRI. With analysis of strategies assessed to define the best diagnostic strategy to avoid unnecessary biopsy, SelectMDx appeared to be a reliable pathway after an initial negative mpMRI. Thus, biopsy could be proposed for all cases of mpMRI PI-RADS 4-5 score, and to those with Prostate Imaging-Reporting and Data System (PI-RADS) 1-3 score followed by a positive SelectMDx

MIR-501 DEPLETION INDUCES CELL CYCLE INHIBITION BY MTOR AND P53 MODULATION IN RENAL CARCINOMA

INTRODUCTION: MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression at post-transcriptional level. The abnormal expression and mutation of miRs has been observed in most urologic cancers including renal cancer, thus they may contribute to development and progression of kidney carcinoma. In fact, their impaired function could trigger a series of altered signalling resulting in abnormal differentiation, proliferation and apoptosis. In the last years, is emerging the necessity to use miRs as biological biomarkers in order to improve diagnosis, prognosis and therapy response in renal carcinomas. Furthermore, miRNAs might be potential targets for novel therapeutic strategies, especially in patients with tumour subtypes that do not respond to currently available therapies (1-2). Here, we have focalized our study on the role of miR501-5p in kidney carcinomas because it has been found differently expressed in kidney cancer tissues compared with normals of the same patients. MATERIAL AND METHODS: Analysis of miR501-5p expression was performed by real time RT-PCR. Depletion or enrichment of this miR was conducted by specific antagomiRs and plasmid expressing miR-501-5p specific sequences, respectively. Protein activity was analyzed by immunological and cell imaging techniques. Apoptosis was studied through caspase-3 activity and cell cycle analysis was performed by propidium iodide staining. RESULTS: We have analyzed the expression of miR501-5p in 36 clear cell (ccRCC) and 11 papillary (pRCC) kidney carcinomas. The expression of miR501-5p was higher in ccRCC (3.72 fold) and lower (3.76 fold) in pRCC tissues compared with normal kidneys derived from the same subjects, respectively. However, the distribution of miR501-5p expression values in ccRCC was found strongly variable. Follow up data of 25 ccRCC and 5 pRCC patients suggest that subjects with showed lower expression of miR-501 in cancer tissues respect to control (normal kidney), exhibited a good prognosis compared with patients with unchanged or high levels of this small RNA. In order to evaluate the role of miR501-5p in renal cancer, we have depleted it by a specific antagomiR in KJ29 kidney cancer cell line (3). KJ29 cells expressed higher levels of miR501-5p than normal immortalized tubular kidney cells. The transfection of KJ29 cells with antagomiR caused a 50% reduction of miR501-5p expression compared with untransfected cells. Furthermore, the reduction of miR501-5p induced an increase in G0/G1 phase of cell cycle and a decrease of mTOR activity in KJ29 cells. In addition, the treatment with antagomiR caused an increase in caspase-3 activity, suggesting that this miR may regulate apoptosis. Moreover, miR-501-5p depletion enhanced the expression of p53, data also observed in kidney cancer tissues expressing lower levels of this miR than controls. The activation of p53 was also observed by its nuclear translocation in KJ29 treated with antagomiR. KJ29 cells were also transfected with a plasmid expressing miR-501-5p sequences and these cells showed an increased level of miR-501 compared with untranfected cells. CONCLUSIONS: Our findings show that miR501-5p was differentially expressed in ccRCC. High or unchanged levels of miR501-5p seem not related with grading and metastasis in ccRCC, however, when it is downregulated could promote a good prognosis. Data reported suggest an anti apoptotic role for miR501-5p, making it a likely risk factor for a poor prognosis in renal carcinoma. Therefore, the expression of miR501-5p could be considered as potential biomarker for the prognosis of clear cell kidney carcinoma. 1. Schaefer A, Stephan C, Busch J, Yousef GM, Jung K: Diagnostic, prognostic and therapeutic implications of microRNAs in urologic tumors. Nat Rev Urol;7(5):286-97, 2010. 2. Cairns P: Renal cell carcinoma. Cancer Biomark; 9(1-6):461-73, 2010. 3. Del Senno et al. Cell Biology International Reports. 1986; 10:195

MicroRNA501 may affect the aggressiveness of clear cell renal carcinoma

INTRODUCTION & OBJECTIVES: MicroRNAs (miR) are small, noncoding RNAs that regulate gene expression and are involved in different biological processes including differentiation, proliferation and apoptosis. Mutations or altered expression of miRs might cause several diseases including cancer. Since a variable expression of miR501 not related with the patient age or sex in 63 pairs of normal and clear cell renal carcinoma (ccRCC) tissues has been found, we have analysed the possible function of this miR in ccRCC. MATERIAL & METHODS: Analysis of miR501 expression was performed by microarray and real time RT-PCR. miR501 up or downregulation was performed by cell transfection with a specific plasmid expressing miR501 sequences (PL-501) and antagomiR, respectively. Apoptosis was studied through caspase-3 activity and cell cycle analysis. Cell proliferation was evaluated by direct cell count and with the CellTiter method. Protein levels and kinase activity were calculated by using immunological techniques and cell imaging. RESULTS: Follow up analysis at least 5 years in 35 ccRCC subjects showed a good prognosis for patients with a lower expression (<1) of miR501 in ccRCC tissues compared with normal renal parenchyma. Conversely, 50% of patients with unchanged or higher levels of miR501 exhibited a poor prognosis with a 25% of deaths. In order to evaluate the role of miR501 in renal cancer, we have modified its expression transfecting kidney carcinoma cells KJ29 with a specific antagomiR and with the PL-501 plasmid. MiR501 downregulation caused a reduction of mTOR activity, the increase of G0/G1 phase of cell cycle and induced apoptosis by enhancing the activity of caspase-3. Activation of apoptosis occurred in a p53-dependent manner without affecting the expression of the mTOR-related MDM2 protein, an inhibitor of p53, which results overexpressed in metastatic kidney carcinoma. On the contrary, miR501 upregulation caused mTOR activation, increased expression of MDM2 and enhanced cell proliferation and survival. CONCLUSIONS: These findings suggest for the miR501 a role of kingmaker among apoptosis and cell survival in ccRCC patients. When this miR is downregulated it stimulates apoptosis, but if shows unchanged or higher levels compared with control it promotes the cell growth. This hypothesis is also consistent with follow up data, therefore, the expression of miR501-5p could be considered as a new biomarker for the prognosis of clear cell renal carcinoma

MICRORNA501 UP-REGULATION MAY INCREASE THE AGGRESSIVENESS OF CLEAR CELL RENAL CARCINOMA THROUGH MTOR ACTIVATION AND P53 DEGRADATION

Introduction: Many biological processes as gene expression, cell proliferation, differentiation and apoptosis are affected by microRNAs (miR) which are small noncoding RNAs that act at post transcriptional level. In fact, the impaired function of these short RNAs might cause several diseases including cancer (1). We have found a variable expression of miR501 in 63 pairs of normal and clear cell renal carcinoma (ccRCC) tissues, therefore, a possible function of this miR in ccRCC was investigated. Material and Methods: Analysis of miR501 expression was carried out by microarray and real time RT-PCR. MiR501 up or downregulation was performed by cell transfection with a specific plasmid expressing miR501 sequences (PL-501) and antagomiR, respectively. Apoptosis was analysed through caspase-3 activity, Hoechst method and cell cycle analysis. Cell proliferation was evaluated by direct cell count and with the CellTiter method. Gene expression, protein ubiquitination and kinase activity were analysed by immunological techniques and cell imaging. Results: Follow up analysis of 35 ccRCC subjects showed a good prognosis for patients which a lower expression of miR501 in ccRCC tissues compared with normal renal parenchyma. Conversely, about the 50% of patients with unchanged or higher levels of miR501 exhibited a poor prognosis. In order to evaluate the role of miR501 in renal cancer, we have modified its expression transfecting kidney carcinoma cells KJ29 (2) with a specific antagomiR and with the PL-501 plasmid. MiR501 downregulation caused a reduction of mTOR activity, the increase of G0/G1 phase of cell cycle and induced apoptosis by enhancing the activity of caspase-3. Activation of apoptosis occurred in a p53-dependent manner without affecting the expression of the mTOR-related MDM2 protein, an inhibitor of p53, which results overexpressed in metastatic kidney carcinoma (3). On the other hand, miR501 upregulation caused mTOR activation that stimulated cell proliferation as well as cell survival. The latter biological processes were associated with an increased expression of MDM2 which induced p53 degradation activating the proteasome by p53 poly-ubiquitination. Discussion and conclusion: MiR501 seems to act as a molecular switch able to turn on or off mTOR signalling. In fact, the downregulation of miR501 led to sequential mTOR kinase inhibition, p53 activation and increased apoptosis. Conversely, miR501 upregulation caused mTOR activation, increased expression of MDM2 and p53 degradation, promoting cell survival, as already observed for follow up data. These findings support the role of kingmaker for the miR501 among apoptosis and cell survival in ccRCC patients, therefore miR501 expression could be used to evaluate the prognosis of patients with clear cell renal carcinoma. 1. Di Leva G, Croce CM: Roles of small RNAs in tumor formation. Trends Mol Med. 16(6):257-67, 2010. Review. 2. Barletta C et al.: Cytogenetic, molecular and phenotypic characterization of the newly established renal carcinoma cell line KJ29. Evidence of translocations for chromosomes 1 and 3. Anticancer Res. 15(5B):2129-36, 1995. 3. Noon AP et al: Combined p53 and MDM2 biomarker analysis shows a unique pattern of expression associated with poor prognosis in patients with renal cell carcinoma undergoing radical nephrectomy. BJU Int. 109(8):1250-7, 2012

MiR501-5p stimulating the activity of mToR may result a risk factor for a poor prognosis of clear cell kidney carcinoma

INTRODUCTION & OBJECTIVESS: MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression and are involved in the control of several biological processes such as proliferation differentiation and apoptosis. Abnormal expression and mutation of miRs is implicated in many hereditary and neoplastic diseases including kidney carcinomas. In the last years, is emerging the necessity to use microRNA as biological biomarkers in order to improve diagnosis, prognosis and therapy response in renal carcinomas. Furthermore, miRNAs might be potential targets for novel therapeutic strategies, especially in patients that are resistant to conventional treatments. MATERIAL & METHODS: Analysis of miR501-5p expression was performed by real time RT-PCR. Depletion or enrichment of this miR was conducted by specific antagomiRs and by a plasmid expressing miR501-5p specific sequences, respectively. Analysis of proteins was performed by immunological techniques and cell imaging. Apoptosis and cell cycle were studied by analysis of caspase-3 activity and flow cytometry after DNA staining with propidium iodide, respectively. RESULTS: Follow up data of 25 ccRCC and 5 pRCC patients showed that subjects with low expression of miR501-5p exhibited a good prognosis compared with patients with unchanged or high levels of this microRNA. In order to evaluate the role of miR501-5p in renal cancer, we have depleted it by a specific antagomiR in KJ29 kidney cancer cell line. The transfection of KJ29 cells with antagomiR caused a 50% reduction of miR501-5p expression compared with untransfected cells. Furthermore, the reduction of miR501-5p induced an increase in G0/G1 phase of cell cycle and a decrease of mTOR activity. Consistently, the upregulation of miR501-5p by transfection of KJ29 cells with a plasmid expressing miR501-5p sequences, caused an increase of mTOR activity compared with untransfected cells. The reduction of miR501-5p expression also induced an increased activity of caspase-3, likely in a p53-dependent manner. In fact, miR501-5p depletion enhanced the expression and nuclear translocation of p53 in KJ29 cells. CONCLUSIONS: Our findings suggest that miR501-5p overexpression could induce a resistance to apoptosis and therefore it could be a risk factor for a poor prognosis of renal carcinoma. In fact, lower expression of miR501-5p seems promote a good prognosis in clear cell kidney carcinoma patients. Furthermore, the expression of miR501-5p in combination with mTOR activity could be used as new potential biomarker for the prognosis of renal carcinomas

Role of the miR-501 in Renal Carcinomas

MicroRNAs (miR) are small, noncoding RNAs that regulate gene expression. In particular, miRs are involved in many biological processes, including differentiation, proliferation and cell death. In addition to their physiological functions, miRs are found to be aberrantly expressed in many carcinomas including renal tumors and to play oncogenic or tumor suppressive roles in cancer cells (1-2). Because, we have observed by microarray analysis that some miRs (miR 142-5p, 601, 362, 196b, 202 and 501) were differentially expressed in autosomal dominant polycystic kidney disease (ADPKD) which is an hyperproliferative disease, we have analyzed the expression of these miRs in kidney carcinomas to better understand their role in renal tumorigenesis. In particular, we have studied the most common renal neoplasms as clear cell (ccRCC), papillary (pRCC), chromophobe (chRCC), with respect to normal kidney tissues. Materials and Methods: We selected and analyzed 39 post-nephrectomy fresh frozen tissues (including 23 neoplastic samples: 14 ccRCC, 6 pRCC, 3 chRCC), and 13 paraffin-embedded tumors samples (7 ccRCC, 4 pRCC, 2 chRCC) with matched normal tissues. Total RNA was extracted with TRIZOL (fresh frozen tissues) or the RecoverAll® Total Nucleic Acid Isolation kit (paraffin-embedded tissues). Microarray analysis was performed in normal kidney and ADPKD cell lines following the manufacturer’s protocols of microarray facility (Lab of Dr. Negrini, University of Ferrara). Quantitative real time PCR for mature miRs was performed with TaqMan method. MicroRNA levels were expressed as ΔΔCt relative to reference gene U6 snRNA. MiR RNA levels in tumor sample were compared with ΔΔ those of normal tissue as fold increase calculated by 2-Ct method (3). Results: By real time RT-PCR analysis of indicated miRs, only the miR-501 was found differentially expressed in kidney carcinomas compared with normal renal tissues. In particular miR-501 expression was 5.35 fold increased in ccRCC with respect to normal tissues. Conversely, in pRCC this miR was found 2.56 fold decreased compared with normal kidney. Because, the expression levels of miR-501 in ccRCC showed a extremely variable distribution, this miR may not be used as a marker for ccRCC. Moreover, no correlation between miR-501 expression and tumor grading was observed. However, the low expression of miR-501 in ccRCC as well as in pRCC samples correlated with a good prognosis, thus the downregulation of miR501 could be considered as a marker for positive prognosis in ccRCC and pRCC kidney carcinomas. Conclusion: Our results demonstrate that in renal carcinomas the miR-501 was significantly over-expressed in ccRCC, while was downregulated in pRCC. High levels of miR-501 are not related with grading and metastasis in ccRCC, however, when it is down-regulated could promote a good prognosis