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

Diagnostic and prognostic microRNAs in the serum of breast cancer patients measured by droplet digital PCR

Background: Breast cancer circulating biomarkers include carcinoembryonic antigen and carbohydrate antigen 15-3, which are used for patient follow-up. Since sensitivity and specificity are low, novel and more useful biomarkers are needed. The presence of stable circulating microRNAs (miRNAs) in serum or plasma suggested a promising role for these tiny RNAs as cancer biomarkers. To acquire an absolute concentration of circulating miRNAs and reduce the impact of preanalytical and analytical variables, we used the droplet digital PCR (ddPCR) technique. Results: We investigated a panel of five miRNAs in the sera of two independent cohorts of breast cancer patients and disease-free controls. The study showed that miR-148b-3p and miR-652-3p levels were significantly lower in the serum of breast cancer patients than that in controls in both cohorts. For these two miRNAs, the stratification of breast cancer patients versus controls was confirmed by receiver operating characteristic curve analyses. In addition, we showed that higher levels of serum miR-10b-5p were associated with clinicobiological markers of poor prognosis. Conclusions: The study revealed the usefulness of the ddPCR approach for the quantification of circulating miRNAs. The use of the ddPCR quantitative approach revealed very good agreement between two independent cohorts in terms of comparable absolute miRNA concentrations and consistent trends of dysregulation in breast cancer patients versus controls. Overall, this study supports the use of the quantitative ddPCR approach for monitoring the absolute levels of diagnostic and prognostic tumor-specific circulating miRNAs

A combination of miR501-5p and mTOR as molecular markers for the prognosis of renal carcinomas

In the last years, there is great impetus to discover new biomarkers in RCC in order to address the patients toward an appropriate adjuvant therapy. In this regard, miR501-5p that is differentially expressed in RCC and mTOR which in many tumours correlates with a poor prognosis, could represent possible biomarkers. Therefore, miR501-5p expression and mTOR activity will be studied in normal and RCC tissues as well as in kidney cell lines depleted or enriched in miR501-5p sequences. Cell growth, apoptosis, autophagy and related signaling pathways will be analyzed in kidney cells and tissues expressing different levels of miR501-5p in order to evaluate the role of miR501-5p in RCC. High and low miR501-5p expression levels combined with mTOR activity of different RCC tissues will be matched with the outcome of RCC patients to evaluate their possible prognostic role

Role of calcium in polycystic kidney disease: From signaling to pathology

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited monogenic kidney disease. Characterized by the development and growth of cysts that cause progressive kidney enlargement, it ultimately leads to end-stage renal disease. Approximately 85% of ADPKD cases are caused by mutations in the PKD1 gene, while mutations in the PKD2 gene account for the remaining 15% of cases. The PKD1 gene encodes for polycystin-1 (PC1), a large multi-functional membrane receptor protein able to regulate ion channel complexes, whereas polycystin-2 (PC2), encoded by the PKD2 gene, is an integral membrane protein that functions as a calcium-permeable cation channel, located mainly in the endoplasmic reticulum (ER). In the primary cilia of the epithelial cells, PC1 interacts with PC2 to form a polycystin complex that acts as a mechanosensor, regulating signaling pathways involved in the differentiation of kidney tubular epithelial cells. Despite progress in understanding the function of these proteins, the molecular mechanisms associated with the pathogenesis of ADPKD remain unclear. In this review we discuss how an imbalance between functional PC1 and PC2 proteins may disrupt calcium channel activities in the cilium, plasma membrane and ER, thereby altering intracellular calcium signaling and leading to the aberrant cell proliferation and apoptosis associated with the development and growth of renal cysts. Research in this field could lead to the discovery of new molecules able to rebalance intracellular calcium, thereby normalizing cell proliferation and reducing kidney cyst progression

Circulating non-coding RNA as biomarkers in colorectal cancer

Recent studies suggested that colorectal cancer influences the types and quantity of nucleic acids - especially microRNAs – detected in the bloodstream. Concentration of circulating (cell-free) microRNAs, and possibly of other non-coding RNAs, could therefore serve as valuable colorectal cancer biomarker and could deliver insight into the disease process. This chapter addresses the recent discoveries on circulating microRNA and long non-coding RNA as diagnostic or prognostic biomarkers in colorectal cancer

Detection of DNA mutations in urine of patients with prostate cancer by NGS technology

Introduction: Prostatic carcinoma (PCa) is the most common cancer in males. Approximately 16% of patients develop the disease with about 3-4% of deaths (1). PCa diagnosis is based on the detection of serum prostate-specific antigen (PSA), which is the only serum biomarker for the diagnosis of prostatic carcinoma. However, at low levels, PSA is inaccurate, resulting in a high negative biopsy rate (2). Currently, no specific standard protocols to identify low-form high-risk patients are available. Therefore, direct DNA analysis from urine may represent a non-invasive test to detect gene mutations associated with poor prognosis (3). Materials and Methods: Cell-free DNA from urine samples and nuclear DNA from paraffin-embedded tumor tissues, derived from PCa patients, was extracted by using specific DNA isolation kits. Cell-free urine DNA, as well as DNA extracted from prostatic cancer tissue, was quantified by Alu007 realtime and used to generate DNA libraries containing Ion Torrent sequencing adapters and barcodes. The library fragments were clonally amplified, applied to the Ion Torrent chip containing a panel of cancer genes and sequenced using the ION PGM sequencer. Raw sequencing data were processed using the Torrent Suite program and exon sequences data were analyzed in order to identify germinal and somatic mutations. Statistical analyses were performed by ANOVA or T-test. Results: Cell-free DNA and nuclear DNA derived from urine and prostatic carcinoma tissue of the same patient, respectively, as well as from urine of normal subjects, was sequenced for gene mutations by next-generation sequencing (NGS). Specific panels containing exon sequences of genes involved in cancer development and progression were used. NGS analysis of DNA isolated from urine of PCa patients was able to identify germinal and somatic mutations in different genes including MET oncogene, mTOR kinase and KDR gene that codifies for the VEGFR-2 tyrosine kinase receptor. Interestingly, an inactivating stop mutation in the SMAD4 tumor suppressor gene, which was mutated in advanced cancers, including prostatic carcinoma, was detected. These mutations were also detected in the paired prostate cancer tissues but not found in urine DNA from normal samples. Discussion: NGS analysis is an innovative and powerful approach to detect gene mutations also starting from low amounts of DNA. Therefore, this analysis is suitable for the research of DNA mutations in biological fluids like urine. Mutated genes, discovered thanks to this approach, could lead to the identification of high-risk patients who should be subjected to pharmacological therapy. Consistently, some of identified gene mutations were already found in prostatic cancer tissues suggesting their important role in this disease. The linkage between DNA mutations and patient outcome could lead to the identification of low and high risk patients, detecting new prognostic markers for the PCa. Conclusion: These data indicate how this novel approach is able to detect gene mutations related to disease analysing DNA directly from urine of PCa patients. The analysis of DNA mutations in PCa patients could lead to the detection of new molecules able to correct altered signals associated with the disease, thus improving the pharmacological therapy

Low levels of ALS-linked Cu/Zn superoxide dismutase increase the production of reactive oxygen species and cause mitochondrial damage and death in motor neuron-like cells

Mutations of Cu/Zn superoxide dismutase (SOD1) are found in patients with familial amyotrophic lateral sclerosis (FALS). A cellular model of FALS was developed by stably transfecting the motor neuron-like cell line NSC-34 with human wild type (wt) or mutant (G93A) SOD1. Expression levels of G93ASOD1 were close to those seen in the human disease. The presence of G93ASOD1 did not alter cell proliferation but toxicity was evident when the cells were in the growth plateau phase. Flow cytometry analysis indicated that, in this phase, G93ASOD1 significantly lowered viability and that the level of reactive oxygen species was significantly higher in living G93ASOD1 cells compared to wt SOD1 cells. Biparametric analysis of mitochondrial membrane potential and viability of transfected cells highlighted a peculiar population of damaged cells with strong mitochondrial depolarization in the G93ASOD1 cells. Mitochondrial function seemed related to the level of the mutant protein since MTT conversion decreased when expression of G93ASOD1 doubled after treating cells with sodium butyrate. The mutant protein rendered G93ASOD1 cells more sensitive to mitochondrial dysfunction induced by stimuli that alter cellular free radical homeostasis, like serum withdrawal, depletion of glutathione by ethacrynic acid or rotenone-mediated inhibition of complex I of the mitochondrial electron transport chain. In conclusion, even a small amount of mutant SOD1 put motor neurons in a condition of oxidative stress and mitochondrial damage that causes cell vulnerability and death

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

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