MicroRNAs in clear cell renal cell carcinoma: biological functions and applications

MicroRNAs (miRs) are small noncoding RNAs that govern many biological processes. They frequently acquire a gain or a loss of function in cancer and hence play a causative role in the development and progression of neoplasms. They could be used as biomarkers to improve our knowledge on diagnosis, prognosis and drug resistance, and to attempt therapeutic approaches in several types of cancer including clear cell renal cell carcinoma (ccRCC). ccRCC is the most predominant subtype of RCC that accounts for about 90% of all renal cancers. Since ccRCC is generally asymptomatic until very late, it is difficult to diagnose early. Moreover, in the absence of preventive treatments for metastatic ccRCC after surgical resection of the primary cancer, predictive prognostic biomarkers are needed in order to achieve appropriate therapies. Herein the role of miRs in the biology of ccRCC and the potential applications of these molecules are discussed. Moreover, future applications in the diagnostic and prognostic field, as well as their impact on drug response and therapeutic targets are also explored. Their use in clinical practice as molecular biomarkers alone, or in combination with other biological markers could accelerate progress, help design personalized therapies, limit side effects, and improve quality of life of ccRCC patients

Preferential expression of the transcription coactivator HTIF1alpha gene in acute myeloid leukemia and MDS-related AML

HTIF1α, a transcription coactivator which is able to mediate RARα activity and functionally interact with PML, is encoded by a gene on chromosome 7q32–34, which is a critical region in acute myeloid leukemias (AML). With the assumption that this gene may be related to AML, we investigated the HTIF1α DNA structure and RNA expression in leukemic cells from 36 M1–M5 AML patients (28 ‘de novo’ and eight ‘secondary’ to myelodysplastic syndrome (MDS)). Abnormal HTIF1α DNA fragments were never found, whereas loss of HTIF1α DNA was observed in the patients with chromosome 7q32 deletion and translocation, and in one case without detectable chromosome 7 abnormality. HTIF1α RNA was found in acute myelocytic leukemic blasts, and was almost undetectable in normal mononuclear cells. The expression varied among the patients: higher in M1 to M3 subtypes, with the highest values in M1; low levels were constantly observed in M4 and M5 AML. In addition, HTIF1α was significantly overexpressed in MDS-related AML (MDR-AML), but not in MDS. We also found that HTIF1α expression was high in myeloid cell lines. In myeloblastic HL60 and promyelocytic NB4 cells, induced to differentiate along the monocytic–macrophage pathway by TPA or vitamin D3, HTIF1α expression decreased, whereas it was maintained at high levels on induction to granulocytic differentiation by RA or DMSO. In K562 cells, HTIF1α RNA levels did not change after hemin-induced erythroid differentiation. These results suggest that HTIF1α could play a role in myeloid differentiation, being distinctly regulated in hematopoietic lineages

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

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

New Therapeutic Interventions for Kidney Carcinoma: Looking to the Future

Patients suffering from metastatic renal cell carcinoma (mRCC) show an overall survival rate of lower than 10% after 5 years from diagnosis. Currently, the first-line treatment for mRCC patients is based on antiangiogenic drugs that are able to inhibit tyrosine kinase receptors (TKI) in combination with immuno-oncology (IO) therapy or IO-IO treatments. Second-line therapy involves the use of other TKIs, immunotherapeutic drugs, and mTOR inhibitors. Nevertheless, many patients treated with mTOR and TK inhibitors acquire drug resistance, making the therapy ineffective. Therefore, the research of new therapeutic targets is crucial for improving the overall survival and quality of life of mRCC patients. The investigation of the molecular basis of RCC, especially in clear cell renal cell carcinoma (ccRCC), has led to the identification of different signaling pathways that are involved in renal carcinogenesis. Most of ccRCCs are associated with mutation in VHL gene, which mediates the degradation of hypoxia-inducible factors (HIFs), that, in turn, regulate the pathways related to tumorigenesis, including angiogenesis and invasion. Renal tumorigenesis is also associated with the activation of tyrosine kinases that modulate the PI3K-Akt-mTOR pathway, promoting cell proliferation and survival. In ccRCC, the abnormal activity of mTOR activates the MDM2 protein, which leads to the degradation of tumor suppressor p53 via proteasome machinery. In addition, p53 may be degraded by autophagy in a mechanism involving the enzyme transglutaminase 2 (TG2). Suppression of wild-type p53 promotes cell growth, invasion, and drug resistance. Finally, the activation of ferroptosis appears to inhibit cancer progression in RCC. In conclusion, these pathways might represent new therapeutic targets for mRCC

Inhibition of the lncRNA Coded within Transglutaminase 2 Gene Impacts Several Relevant Networks in MCF-7 Breast Cancer Cells

Long non-coding RNAs are nucleotide molecules that regulate transcription in numerous cellular processes and are related to the occurrence of many diseases, including cancer. In this regard, we recently discovered a polyadenylated long non-coding RNA (named TG2-lncRNA) encoded within the first intron of the Transglutaminase type 2 gene (TGM2), which is related to tumour proliferation in human cancer cell lines. To better characterize this new biological player, we investigated the effects of its suppression in MCF-7 breast cancer cells, using siRNA treatment and RNA-sequencing. In this way, we found modifications in several networks associated to biological functions relevant for tumorigenesis (apoptosis, chronic inflammation, angiogenesis, immunomodulation, cell mobility, and epithelial-mesenchymal transition) that were originally attributed only to Transglutaminase type 2 protein but that could be regulated also by TG2-lncRNA. Moreover, our experiments strongly suggest the ability of TG2-lncRNA to directly interact with important transcription factors, such as RXRα and TP53, paving the way for several regulatory loops that can potentially influence the phenotypic behaviour of MCF-7 cells. These considerations imply the need to further investigate the relative relevance of the TG2 protein itself and/or other gene products as key regulators in the organization of breast cancer program

MicroRNA501-5p activating the mTOR independent autophagy induces p53 degradation in kidney cancer cells

Aim of the work. In advanced kidney carcinoma, autophagy could enhances tumor survival increasing energy metabolism and promoting cell migration and metastasis. We have observed that miR501-5p overexpression is associated with poor prognosis in ccRCC patients [1]. Moreover, the upregulation of this miR causes p53 degradation activating the mTOR pathway that is a regulator of autophagy. Therefore, we have investigated the role of autophagy in kidney carcinoma cells overexpressing miR501-5p. Materials and methods. The analysis of autophagy was performed in KJ29 and Caki-2 kidney carcinoma cells transfected with a plasmid (PL501) expressing miR501-5p sequences. Autophagy was analysed by western blot using antibodies that recognize the autophagic markers LC3II and p62. Autophagy was also studied detecting cellular autophagosomes by florescence microscopy in cells transfected with a plasmid expressing LC3II protein linked with GFP. The phosphorylation state of mTOR and AMP kinases as well as p53 and ATG7 protein expression were analysed by immunoblotting. The inhibition of autophagy was performed transducing KJ29 and Caki-2 ccRCC cells with lentiviruses containing a recombinant vector (394) expressing specific shRNAs for the silencing of ATG7 gene. The function of p53 was tested by luciferase assay using a plasmid containing the p53 response-element of p21 gene promoter. Statistical analysis was carried out by ANOVA test. Results. The upregulation of miR501-5p increases the expression of LC3II and reduces the levels of p62, protein markers of autophagy. Moreover, the co-transfection with PL501 and LC3II-GFP plasmids promotes the formation of autophagosomes in KJ29 ccRCC cells, suggesting that miR501-5p stimulates the activation of autophagy. Nevertheless, the inhibition of mTOR does not affect autophagy indicating that the autophagic flux driven by the upregulation of miR501-5p is mTOR-independent. In fact, the overexpression of this miR strongly activates the AMP kinase that regulates non-canonical autophagy. Furthermore, the analysis of p53 localization shows that the most of p53 protein is trapped in structures that co-localizes with autophagosomes. Thus, the autophagic system may be used by kidney cancer cell to remove p53. Consistently, the inhibition of autophagy by ATG7 gene silencing restores p53 protein levels and increases p21-luciferase activity, confirming the re-activation of p53 function. Conclusion. The inhibition of autophagy is able to reactivate p53 expression and function in different kidney carcinoma cell lines, therefore, the molecular/pharmacological targeting of this biological process could represent a new therapeutic approach to treat metastatic renal carcinom

MicroRNA501-5p induces p53 proteasome degradation through the activation of the mTOR/MDM2 pathway in ADPKD cells.

Cell proliferation and apoptosis are typical hallmarks of autosomal dominant polycystic kidney disease (ADPKD) and cause the development of kidney cysts that lead to end-stage renal disease (ESRD). Many factors, impaired by polycystin complex loss of function, may promote these biological processes, including cAMP, mTOR and EGFR signalling pathways. In addition, microRNAs (miRs) may also regulate the ADPKD related signalling network and their dysregulation contributes to disease progression. However, the role of miRs in ADPKD pathogenesis has not been fully understood, but also the function of p53 is quite obscure, especially its regulatory contribution on cell proliferation and apoptosis. Here, we describe for the first time that miR501-5p, upregulated in ADPKD cells and tissues, induces the activation of mTOR kinase by PTEN and TSC1 gene repression. The increased activity of mTOR kinase enhances the expression of E3 ubiquitin ligase MDM2 that in turn promotes p53 ubiquitination, leading to its degradation by proteasome machinery in a network involving p70S6K. Moreover, the overexpression of miR501-5p stimulates cell proliferation in kidney cells by the inhibition of p53 function in a mechanism driven by mTOR signalling. In fact, the downregulation of this miR as well as the pharmacological treatment with proteasome and mTOR inhibitors in ADPKD cells reduces cell growth by the activation of apoptosis. Consequently, the stimulation of cell death in ADPKD cells may occur through the inhibition of mTOR/MDM2 signalling and the restoring of p53 function. The data presented here confirm that the impaired mTOR signalling plays an important role in ADPKD

Mitochondria: Insights into Crucial Features to Overcome Cancer Chemoresistance

Mitochondria are key regulators of cell survival and are involved in a plethora of mechanisms, such as metabolism, Ca2+ signaling, reactive oxygen species (ROS) production, mitophagy and mitochondrial transfer, fusion, and fission (known as mitochondrial dynamics). The tuning of these processes in pathophysiological conditions is fundamental to the balance between cell death and survival. Indeed, ROS overproduction and mitochondrial Ca2+ overload are linked to the induction of apoptosis, while the impairment of mitochondrial dynamics and metabolism can have a double-faceted role in the decision between cell survival and death. Tumorigenesis involves an intricate series of cellular impairments not yet completely clarified, and a further level of complexity is added by the onset of apoptosis resistance mechanisms in cancer cells. In the majority of cases, cancer relapse or lack of responsiveness is related to the emergence of chemoresistance, which may be due to the cooperation of several cellular protection mechanisms, often mitochondria-related. With this review, we aim to critically report the current evidence on the relationship between mitochondria and cancer chemoresistance with a particular focus on the involvement of mitochondrial dynamics, mitochondrial Ca2+ signaling, oxidative stress, and metabolism to possibly identify new approaches or targets for overcoming cancer resistance

The inhibition of MDM2 slows cell proliferation and activates apoptosis in ADPKD cell lines

Introduction Autosomal dominant polycystic kidney disease (ADPKD) is characterised by progressive cysts formation and renal enlargement that in most of cases leads to end stage of renal disease (ESRD). This pathology is caused by mutations of either PKD1 or PKD2 genes that encode for polycystin-1 (PC1) and polycystin-2 (PC2), respectively. These proteins function as receptor-channel complex able to regulate calcium homeostasis. PKD1/2 loss of function impairs different signalling pathways including cAMP and mTOR that are considered therapeutic targets for this disease. In fact, Tolvaptan, a vasopressin-2 antagonist that reduces cAMP levels, is the only drug approved for ADPKD treatment. Nevertheless, some ADPKD patients developed side effects in response to Tolvaptan including liver damage. Conversely, mTOR inhibitors that induced disease regression in ADPKD animal models failed the clinical trials. Results Here, we show that the inhibition of mTOR causes the activation of autophagy in ADPKD cells that could reduce therapy effectiveness by drug degradation through the autophagic vesicles. Consistently, the combined treatment with rapamycin and chloroquine, an autophagy inhibitor, potentiates the decrease of cell proliferation induced by rapamycin. To overcome the dangerous activation of autophagy by mTOR inhibition, we targeted MDM2 (a downstream effector of mTOR signalling) that is involved in TP53 degradation by using RG7112, a small-molecule MDM2 inhibitor used for the treatment of haematologic malignancies. The inhibition of MDM2 by RG7112 prevents TP53 degradation and increases p21 expression leading to the decrease of cell proliferation and the activation of apoptosis. Conclusion The targeting of MDM2 by RG7112 might represent a new therapeutic option for the treatment of ADPKD