Role of microRNA-21 on tumor progression and chemoresistance of renal clear cell

Le carcinome rénal à cellules claires (cRCC) est le principal type histologique de carcinome rénal et l'une des tumeurs les plus résistantes à la chimio et à la radiothérapie. L'absence de biomarqueurs pour la détection précoce et pour le suivi des patients est responsable d'un mauvais pronostic. Il est nécessaire d'identifier de nouveaux biomarqueurs et des cibles thérapeutiques pour améliorer la prise en charge des patients. Les microARNs, des petits ARN non codants de 22 nucléotides, qui ont été précédemment montrés comme favorisant l'initiation et la progression tumoral, semblent être de bons candidats. Nous avons focalisé notre étude sur (i) miR-21 qui est le principal oncomiR surexprimé dans le cRCC et (ii) le récepteur nucléaire PPARα (Peroxisome Proliferator Activated Receptor), l'une des cibles de miR-21.D'une part, sur une cohorte de 99 échantillons de cRCC primaires, nous avons montré que l'expression de miR-21 était plus élevée dans les tissus cancéreux que dans les tissus non tumoraux adjacents. In vitro, miR-21 est également surexprimé dans les lignées cellulaires de carcinomes rénaux comparées à la lignée cellulaire épithéliale HK-2 provenant de tubes proximaux humains. De plus, nous avons également montré que la surexpression de miR-21 augmente les propriétés de migration et d'invasion des cellules cancéreuses rénales ainsi que les voies de signalisation prolifératives et anti-apoptotiques, alors que des résultats opposés ont été observés en utilisant une stratégie d'inhibition anti-miR-21. Enfin, nous avons évalué le rôle du miR-21 dans la chimiorésistance du cRCC et montré, en outre, que l'inhibition de miR-21 augmentait significativement la chimiosensibilité au paclitaxel, au 5-fluorouracile, à l'oxaliplatine et au dovitinib, diminuait l'expression des transporteurs à efflux MRP1-6/ABCC1-6 et augmentait l'expression des transporteurs à influx SLC22A1/OCT1, SLC22A2/OCT2 et SLC31A1/CTR1. Ces résultats ont permis la publication d'un article dans Tumor Biology se trouvant en annexe.D'autre part, dans les tissus de patients atteints de cRCC, nous avons montré pour la première fois que la surexpression de miR-21 est en corrélation avec une perte d'expression de PPARα. In vitro, nous avons montré que miR-21 cible le 3'-UTR de PPARα et diminue son expression protéique et que la surexpression de miR-21 diminue l'activité transcriptionnelle de PPARα. En outre, la surexpression et l'activation de PPARα diminuent l'expression de miR-21. En effet, PPARα interagit avec les facteurs de transcription AP-1 et NF-κB et empêche ainsi leur liaison au promoteur de miR-21 diminuant ainsi sa transcription.En conclusion, nous avons montré que (i) miR-21 est un acteur clé de la progression du cancer du rein et joue un rôle important dans la résistance aux chimiothérapies et (ii) qu'il existe une boucle de régulation négative entre miR-21 et PPARα dans le cRCC.Renal clear cell carcinoma (cRCC) is the major histological type of renal carcinoma and one of the most chemo- and radio-resistant tumors. The absence of biomarkers for early detection and for monitoring patients is responsible of a poor prognosis. It is necessary to identify new biomarkers and therapeutic targets to improve patient care. MicroRNAs, small noncoding RNAs of 22 nucleotides, which have been previously shown to promote malignant initiation and progression, appear to be good candidates.We focused our study on (i) miR-21 which is the main overexpressed oncomirs in cRCC and (ii) the nuclear receptor PPARα (Peroxisome Proliferator Activated Receptor), one of miR-21 targets.In one hand, by using a cohort of 99 primary cRCC samples, we showed that miR-21 expression in cancer tissues was higher than in adjacent non-tumor tissues. In vitro, miR-21 was also overexpressed in renal carcinoma cell lines compared to HK-2 human proximal tubule epithelial cell line. Moreover, we also showed that miR-21 overexpression increased migratory, invasive, proliferative, and anti-apoptotic signaling pathways whereas opposite results were observed using an anti-miR-21-based silencing strategy. Finally, we assessed the role of miR-21 in mediating cRCC chemoresistance and further showed that miR-21 silencing significantly increased chemosensitivity of paclitaxel, 5-fluorouracil, oxaliplatin and dovitinib, decreased expression of multi-drug resistance genes and increased SLC22A1/OCT1, SLC22A2/OCT2 and SLC31A1/CTR1 platinum influx transporter expression. These results led to the publication of an article in Tumor Biology in annex.In other hand, in cRCC tissue patients, we showed for the first time that miR-21 overexpression correlates with a loss of expression of PPARα. In vitro, we showed that miR-21 targets PPARα 3'-UTR and decreases its protein expression and miR-21 overexpression decreases the transcriptional activity of PPARα. Furthermore, PPARα overexpression and activation decrease miR-21 expression. In fact, PPARα interacts with AP-1 and NF-kappaB transcription factors and thus prevents their binding to the miR-21 promoter thus decreasing its transcription.In conclusion, we have shown that (i) miR-21 is a key actor of renal cancer progression and plays an important role in the resistance to chemotherapeutic drugs and (ii) there is a negative regulatory loop between miR-21 and PPARα in cRCC

Rôle de miR-21 dans la progression tumorale et la chimiorésistance des carcinomes rénaux à cellules claires : étude de la boucle de régulation entre miR-21 et PPARα

Renal clear cell carcinoma (cRCC) is the major histological type of renal carcinoma and one of the most chemo- and radio-resistant tumors. The absence of biomarkers for early detection and for monitoring patients is responsible of a poor prognosis. It is necessary to identify new biomarkers and therapeutic targets to improve patient care. MicroRNAs, small noncoding RNAs of 22 nucleotides, which have been previously shown to promote malignant initiation and progression, appear to be good candidates.We focused our study on (i) miR-21 which is the main overexpressed oncomirs in cRCC and (ii) the nuclear receptor PPARα (Peroxisome Proliferator Activated Receptor), one of miR-21 targets.In one hand, by using a cohort of 99 primary cRCC samples, we showed that miR-21 expression in cancer tissues was higher than in adjacent non-tumor tissues. In vitro, miR-21 was also overexpressed in renal carcinoma cell lines compared to HK-2 human proximal tubule epithelial cell line. Moreover, we also showed that miR-21 overexpression increased migratory, invasive, proliferative, and anti-apoptotic signaling pathways whereas opposite results were observed using an anti-miR-21-based silencing strategy. Finally, we assessed the role of miR-21 in mediating cRCC chemoresistance and further showed that miR-21 silencing significantly increased chemosensitivity of paclitaxel, 5-fluorouracil, oxaliplatin and dovitinib, decreased expression of multi-drug resistance genes and increased SLC22A1/OCT1, SLC22A2/OCT2 and SLC31A1/CTR1 platinum influx transporter expression. These results led to the publication of an article in Tumor Biology in annex.In other hand, in cRCC tissue patients, we showed for the first time that miR-21 overexpression correlates with a loss of expression of PPARα. In vitro, we showed that miR-21 targets PPARα 3'-UTR and decreases its protein expression and miR-21 overexpression decreases the transcriptional activity of PPARα. Furthermore, PPARα overexpression and activation decrease miR-21 expression. In fact, PPARα interacts with AP-1 and NF-kappaB transcription factors and thus prevents their binding to the miR-21 promoter thus decreasing its transcription.In conclusion, we have shown that (i) miR-21 is a key actor of renal cancer progression and plays an important role in the resistance to chemotherapeutic drugs and (ii) there is a negative regulatory loop between miR-21 and PPARα in cRCC.Le carcinome rénal à cellules claires (cRCC) est le principal type histologique de carcinome rénal et l'une des tumeurs les plus résistantes à la chimio et à la radiothérapie. L'absence de biomarqueurs pour la détection précoce et pour le suivi des patients est responsable d'un mauvais pronostic. Il est nécessaire d'identifier de nouveaux biomarqueurs et des cibles thérapeutiques pour améliorer la prise en charge des patients. Les microARNs, des petits ARN non codants de 22 nucléotides, qui ont été précédemment montrés comme favorisant l'initiation et la progression tumoral, semblent être de bons candidats. Nous avons focalisé notre étude sur (i) miR-21 qui est le principal oncomiR surexprimé dans le cRCC et (ii) le récepteur nucléaire PPARα (Peroxisome Proliferator Activated Receptor), l'une des cibles de miR-21.D'une part, sur une cohorte de 99 échantillons de cRCC primaires, nous avons montré que l'expression de miR-21 était plus élevée dans les tissus cancéreux que dans les tissus non tumoraux adjacents. In vitro, miR-21 est également surexprimé dans les lignées cellulaires de carcinomes rénaux comparées à la lignée cellulaire épithéliale HK-2 provenant de tubes proximaux humains. De plus, nous avons également montré que la surexpression de miR-21 augmente les propriétés de migration et d'invasion des cellules cancéreuses rénales ainsi que les voies de signalisation prolifératives et anti-apoptotiques, alors que des résultats opposés ont été observés en utilisant une stratégie d'inhibition anti-miR-21. Enfin, nous avons évalué le rôle du miR-21 dans la chimiorésistance du cRCC et montré, en outre, que l'inhibition de miR-21 augmentait significativement la chimiosensibilité au paclitaxel, au 5-fluorouracile, à l'oxaliplatine et au dovitinib, diminuait l'expression des transporteurs à efflux MRP1-6/ABCC1-6 et augmentait l'expression des transporteurs à influx SLC22A1/OCT1, SLC22A2/OCT2 et SLC31A1/CTR1. Ces résultats ont permis la publication d'un article dans Tumor Biology se trouvant en annexe.D'autre part, dans les tissus de patients atteints de cRCC, nous avons montré pour la première fois que la surexpression de miR-21 est en corrélation avec une perte d'expression de PPARα. In vitro, nous avons montré que miR-21 cible le 3'-UTR de PPARα et diminue son expression protéique et que la surexpression de miR-21 diminue l'activité transcriptionnelle de PPARα. En outre, la surexpression et l'activation de PPARα diminuent l'expression de miR-21. En effet, PPARα interagit avec les facteurs de transcription AP-1 et NF-κB et empêche ainsi leur liaison au promoteur de miR-21 diminuant ainsi sa transcription.En conclusion, nous avons montré que (i) miR-21 est un acteur clé de la progression du cancer du rein et joue un rôle important dans la résistance aux chimiothérapies et (ii) qu'il existe une boucle de régulation négative entre miR-21 et PPARα dans le cRCC

Targeting miR-21 decreases expression of multi-drug resistant genes and promotes chemosensitivity of renal carcinoma

International audienceRenal cell carcinoma, the most common neoplasm of adult kidney, accounts for about 3% of adult malignancies and is usually highly resistant to conventional therapy. MicroRNAs are a class of small non-coding RNAs, which have been previously shown to promote malignant initiation and progression. In this study, we focused our attention on miR-21, a well described oncomiR commonly upregulated in cancer. Using a cohort of 99 primary renal cell carcinoma samples, we showed that miR-21 expression in cancer tissues was higher than in adjacent non-tumor tissues whereas no significant difference was observed with stages, grades, and metastatic outcome. In vitro, miR-21 was also overexpressed in renal carcinoma cell lines compared to HK-2 human proximal tubule epithelial cell line. Moreover, using Boyden chambers and western blot techniques, we also showed that miR-21 overexpression increased migratory, invasive, proliferative, and anti-apoptotic signaling pathways whereas opposite results were observed using an anti-miR-21-based silencing strategy. Finally, we assessed the role of miR-21 in mediating renal cell carcinoma chemoresistance and further showed that miR-21 silencing significantly (1) increased chemosensitivity of paclitaxel, 5-fluorouracil, oxaliplatin, and dovitinib; (2) decreased expression of multi-drug resistance genes; and (4) increased SLC22A1/OCT1, SLC22A2/OCT2, and SLC31A1/CTR1 platinum influx transporter expression. In conclusion, our results showed that miR-21 is a key actor of renal cancer progression and plays an important role in the resistance to chemotherapeutic drugs. In renal cell carcinoma, targeting miR-21 is a potential new therapeutic strategy to improve chemotherapy efficacy and consequently patient outcome

A Double-Negative Feedback Interaction between miR-21 and PPAR-α in Clear Renal Cell Carcinoma

International audienceClear cell renal cell carcinoma (ccRCC) is the main histotype of kidney cancer, which is typically highly resistant to conventional therapies and known for abnormal lipid accumulation. In this context, we focused our attention on miR-21, an oncogenic miRNA overexpressed in ccRCC, and peroxysome proliferator-activated receptor-α (PPAR- α), one master regulator of lipid metabolism targeted by miR-21. First, in a cohort of 52 primary ccRCC samples, using RT-qPCR and immunohistochemistry, we showed that miR-21 overexpression was correlated with PPAR-α downregulation. Then, in ACHN and 786-O cells, using RT-qPCR, the luciferase reporter gene, chromatin immunoprecipitation, and Western blotting, we showed that PPAR-α overexpression (i) decreased miR-21 expression, AP-1 and NF-κB transcriptional activity, and the binding of AP-1 and NF-κB to the miR-21 promoter and (ii) increased PTEN and PDCD4 expressions. In contrast, using pre-miR-21 transfection, miR-21 overexpression decreased PPAR-α expression and transcriptional activity mediated by PPAR-α, whereas the anti-miR-21 (LNA-21) strategy increased PPAR-α expression, but also the expression of its targets involved in fatty acid oxidation. In this study, we showed a double-negative feedback interaction between miR-21 and PPAR-α. In ccRCC, miR-21 silencing could be therapeutically exploited to restore PPAR-α expression and consequently inhibit the oncogenic events mediated by the aberrant lipid metabolism of ccRCC

Dual role of MUC1 mucin in kidney ischemia-reperfusion injury: Nephroprotector in early phase, but pro-fibrotic in late phase

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MUC1-C nuclear localization drives invasiveness of renal cancer cells through a sheddase/gamma secretase dependent pathway

International audienceMUC1 is a membrane-anchored mucin and its cytoplasmic tail (CT) can interact with many signaling pathways and act as a co-transcription factor to activate genes involved in tumor progression and metastasis. MUC1 is overexpressed in renal cell carcinoma with correlation to prognosis and has been implicated in the hypoxic pathway, the main renal carcinogenetic pathway. In this context, we assessed the effects of MUC1 overexpression on renal cancer cells properties. Using shRNA strategy and/or different MUC1 constructs, we found that MUC1-extracellular domain and MUC1-CT are involved in increase of migration, cell viability, resistance to anoikis and in decrease of cell aggregation in cancer cells. Invasiveness depends only on MUC1-CT. Then, by using siRNA strategy and/or pharmacological inhibitors or peptides, we showed that sheddases ADAM10, ADAM17 and gamma-secretase are necessary for MUC1 C-terminal subunit (MUC1-C) nuclear location and in increase of invasion property. Finally, MUC1 overexpression increases ADAM10/17 protein expression suggesting a positive regulatory loop. In conclusion, we report that MUC1 acts in renal cancer progression and MUC1-C nuclear localization drives invasiveness of cancer cells through a sheddase/gamma secretase dependent pathway. MUC1 appears as a therapeutic target by blocking MUC1 cleavage or nuclear translocation by using pharmacological approach and peptide strategies