endothelin-1 and Carcinoma--Renal-Cell

This review highlights new developments in miRNA as diagnostic and surveillance tools in diseases damaging the renal proximal tubule mediated by endothelin in the field of renal carcinoma, proteinuric kidney disease and tubulotoxicity. A new mechanism in the miRNA regulation of proteins leads to the binding of the miRNA directly to the DNA with premature transcriptional termination and hence the formation of truncated protein isoforms (Mxi2, Vim3). These isoforms are mediated through miRNA15a or miRNA 498, respectively. ET-1 can activate a cytoplasmic complex consisting of NF-κB p65, MAPK p38α, and PKCα. Consequently, PKCα does not transmigrate into the nucleus, which leads to the loss of suppression of a primiRNA15a, maturation of this miRNA in the cytoplasm, tubular secretion and detectability in the urine. This mechanism has been shown in renal cell carcinoma and in proteinuric disease as a biomarker for the activation of the signalling pathway. Similarly, ET-1 induced miRNA 498 transmigrates into the nucleus to form the truncated protein Vim3, which is a biomarker for the benign renal cell tumour, oncocytoma. In tubulotoxicity, ET-1 induced miRNa133a down-regulating multiple-drug-resistant related protein-2, relevant for proteinuric and cisplatin/cyclosporine A toxicity. Current advantages and limitations of miRNAs as urinary biomarkers are discussed.

Topics: Animals; Biomarkers; Carcinoma, Renal Cell; Endothelin-1; Gene Expression Profiling; Humans; Kidney Neoplasms; Kidney Tubules, Proximal; Mice; MicroRNAs; Proteinuria; Rats; Signal Transduction

Angiogenesis inhibition with sunitinib, a multitarget tyrosine kinase inhibitor of the vascular endothelial growth factor receptor, is associated with hypertension and cardiac toxicity, of which the underlying pathophysiological mechanism is unknown. We investigated the effects of sunitinib on blood pressure (BP), its circadian rhythm, and potential mechanisms involved, including the endothelin-1 system, in 15 patients with metastatic renal cell carcinoma or gastrointestinal stromal tumors. In addition, we investigated in rats the effect of sunitinib on BP, serum endothelin-1 levels, coronary microvascular function, cardiac structure, and cardiac mitochondrial function. In patients, BP increased by ≈15 mm Hg, whereas heart rate decreased after 4 weeks of treatment. Furthermore, the nocturnal dipping of BP diminished. Plasma endothelin-1 concentration increased 2-fold (P<0.05) and plasma renin decreased (P<0.05), whereas plasma catecholamines and renal function remained unchanged. In rats, 8 days of sunitinib administration induced an ≈30-mm Hg rise in BP, an attenuation of the circadian BP rhythm, and a 3-fold rise in serum endothelin-1 and creatinine, of which all but the rise in creatinine reversed after sunitinib withdrawal. Coronary microvascular function studies after 8 days of sunitinib administration showed decreased responses to bradykinin, angiotensin II, and sodium nitroprusside, all normalizing after sunitinib withdrawal. Cardiac structure and cardiac mitochondrial function did not change. In conclusion, sunitinib induces a reversible rise in BP in patients and in rats associated with activation of the endothelin-1 system, suppression of the renin-angiotensin system, and generalized microvascular dysfunction.

Topics: Aged; Angiotensin II; Animals; Blood Pressure; Bradykinin; Carcinoma, Renal Cell; Cells, Cultured; Coronary Circulation; Endothelial Cells; Endothelin-1; Female; Gastrointestinal Stromal Tumors; Heart; Heart Rate; Humans; Hypertension; In Vitro Techniques; Indoles; Kidney Neoplasms; Male; Middle Aged; Pyrroles; Rats; Rats, Inbred WKY; Receptor Protein-Tyrosine Kinases; Renin; Sunitinib; Vascular Endothelial Growth Factor A

The DNA-damaging agent camptothecin (CPT) and its analogs demonstrate clinical utility for the treatment of advanced solid tumors, and CPT-based nanopharmaceuticals are currently in clinical trials for advanced kidney cancer; however, little is known regarding the effects of CPT on hypoxia-inducible factor-2α (HIF-2α) accumulation and activity in clear cell renal cell carcinoma (ccRCC). Here we assessed the effects of CPT on the HIF/p53 pathway. CPT demonstrated striking inhibition of both HIF-1α and HIF-2α accumulation in von Hippel-Lindau (VHL)-defective ccRCC cells, but surprisingly failed to inhibit protein levels of HIF-2α-dependent target genes (VEGF, PAI-1, ET-1, cyclin D1). Instead, CPT induced DNA damage-dependent apoptosis that was augmented in the presence of pVHL. Further analysis revealed CPT regulated endothelin-1 (ET-1) in a p53-dependent manner: CPT increased ET-1 mRNA abundance in VHL-defective ccRCC cell lines that was significantly augmented in their VHL-expressing counterparts that displayed increased phosphorylation and accumulation of p53; p53 siRNA suppressed CPT-induced increase in ET-1 mRNA, as did an inhibitor of ataxia telangiectasia mutated (ATM) signaling, suggesting a role for ATM-dependent phosphorylation of p53 in the induction of ET-1. Finally, we demonstrate that p53 phosphorylation and accumulation is partially dependent on mTOR activity in ccRCC. Consistent with this result, pharmacological inhibition of mTORC1/2 kinase inhibited CPT-mediated ET-1 upregulation, and p53-dependent responses in ccRCC. Collectively, these data provide mechanistic insight into the action of CPT in ccRCC, identify ET-1 as a p53-regulated gene and demonstrate a requirement of mTOR for p53-mediated responses in this tumor type.

Topics: Apigenin; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Camptothecin; Carcinoma, Renal Cell; Cell Hypoxia; Cell Line, Tumor; DNA Damage; Endothelin-1; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney Neoplasms; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Multiprotein Complexes; Protein Biosynthesis; RNA, Messenger; Signal Transduction; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53

Cellular senescence, leading to cell death through prevention of regular cell renewal, is associated with the upregulation of the tumor suppressor gene p16(INK4a). While this mechanism has been described as leading to progressive nephron loss, p16(INK4a) upregulation in renal cell carcinoma has been linked to a disease-specific improved patient survival rate. While in both conditions endothelin-1 is also upregulated, the signaling pathway connecting ET-1 to p16(INK4a) has not been characterized until this study.. Cell culture, qRT-PCR, Western Blot, immunoprecipitation (IP), proximity ligation assay (PLA), and non-radioactive electrophoretic mobility shift assay (EMSA).. In malignant renal proximal tumor cells (Caki-1), an activation of p16(INK4a) and p21(waf1/cip1) was observed. An increased expression of E-26 transformation-specific (ETS) transcription factors was detectable. Using specific antibodies, a complex formation between ETS1 and extracellular signal-regulated kinase-2 (ERK2) was shown. A further complex partner was Mxi2. EMSA with supershift analysis for ETS1 and Mxi2 indicated the involvement of both factors in the protein-DNA interaction. After specifically blocking the endothelin receptors, ETS1 expression was significantly downregulated. However, the endothelin B receptor dependent downregulation was stronger than that of the A receptor. In contrast, primary proximal tubule cells showed a nuclear decrease after ET-1 stimulation. This indicates that other ETS members may be involved in the observed p16(INK4a) upregulation (as described in the literature).. ETS1, ERK2 and Mxi2 are important complex partners initiating increased p16(INK4a) and p21w(af1/cip1) activation in renal tumor cells.

Topics: Carcinoma, Renal Cell; Cell Line; Cell Line, Tumor; Cellular Senescence; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Endothelin-1; Humans; Kidney Neoplasms; Kidney Tubules, Proximal; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 14; Proto-Oncogene Protein c-ets-1; Proto-Oncogene Proteins c-ets; Up-Regulation

Topics: Blood Pressure; Carcinoma, Renal Cell; Endothelin-1; Gastrointestinal Stromal Tumors; Heart Rate; Humans; Hypertension; Indoles; Kidney Neoplasms; Models, Biological; Proteinuria; Pyrroles; Receptor Protein-Tyrosine Kinases; Renin; Sunitinib; Vascular Endothelial Growth Factor A

Endothelin-1 (ET-1) is a potent vasoconstrictor that has been shown to significantly impact many benign and malignant tissues by signaling through its two cognate receptors: ET(A) and ET(B). As ET-1 has a role in both normal and diseased kidney, we initiated studies to investigate endothelin axis expression and function in renal cell carcinoma (RCC). In this study, relatively high levels of ET-1 were detected in all six human RCC cell lines investigated. RT-PCR and Southern analyses revealed that all six RCC cell lines expressed ET(A) receptor mRNA, while 3/6 cell lines also expressed ET(B) mRNA. High affinity ET-1 binding occurred in all but one RCC cell line and quantitative RT-PCR demonstrated ET(A) mRNA expression in all six cell lines. Methylation of the ET(B) promoter (EDNRB) in 4/6 RCC cell lines was observed, suggesting a mechanism for repressed ET(B) expression. Moreover, methylation occurred in 32/48 of renal tumors and in 27/55 of histologically normal adjacent tissue samples studied, while no methylation was evident in any normal tissue isolated from nephrectomy or at autopsy. Functionally, ET-1 significantly inhibited paclitaxel-induced apoptosis in RCC cells through binding ET(A) with the ET-1 signaling mediated via the PI3-kinase/Akt pathway. Collectively, these data support the therapeutic targeting of the ET(A) receptor as a novel treatment strategy for RCC.

Topics: Androstadienes; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Survival; Chromones; DNA Methylation; Endothelin A Receptor Antagonists; Endothelin-1; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Kidney Neoplasms; Morpholines; Paclitaxel; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Promoter Regions, Genetic; Protein Binding; Proto-Oncogene Proteins c-akt; Receptor, Endothelin A; Receptor, Endothelin B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Wortmannin

Endothelin-1 and its receptors ETAR and ETBR, commonly referred to as the Endothelin-axis, are emerging to play a role in cancer. The Endothelin-axis has been shown to be involved in proliferation, angiogenesis and metastasis in various human tumours. To assess the role of the Endothelin-axis in renal cell carcinoma, we analysed its expression in archival tumour tissue of 183 patients. Representative tumour blocks were selected for constructing a tissue microarray. Paraffin sections were assessed immunohistochemically using monoclonal and polyclonal antibodies for Endothelin-1, ETAR and ETBR. Staining intensities were analysed semiquantitatively and the results were correlated with various histopathologic factors. Overexpression of Endothelin-1, ETAR and ETBR was identified in 12.8%, 84.1% and 93.3% of cases, respectively. No association with pathological tumour stage and histologic grading was found. Papillary renal cell carcinomas expressed highly significantly more Endothelin-1 than clear cell renal cell carcinomas (34.5% vs. 6.7%, p<0.001), while there was no difference between ETAR- and ETBR-expression in these histologic subtypes. However, ETAR tended to be overexpressed in the subgroup of G3-tumours (p=0.044). Studies are underway assessing the role of the Endothelin-axis and its potential use as a molecular target in renal cell carcinoma.

Topics: Antibodies, Monoclonal; Apoptosis; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Endothelin-1; Endothelins; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Kidney Neoplasms; Neovascularization, Pathologic; Oligonucleotide Array Sequence Analysis; Receptor, Endothelin A; Receptor, Endothelin B

The endothelin axis has been implicated in cancer growth, angiogenesis, and metastasis, but to the authors' knowledge the expression of endothelin genes has not been defined in renal cell carcinoma (RCC).. Tissue specimens were harvested from both normal and tumor-affected regions at the time of radical nephrectomy from 35 patients with RCC (22 with clear cell RCC [ccRCC] and 13 with papillary RCC [PRCC]). Real-time reverse transcriptase-polymerase chain reaction analysis determined the expression profile of the preproendothelins (PPET-1, PPET-2, and PPET-3), the endothelin receptors (ET(A) and ET(B)), and the endothelin-converting enzymes (ECE-1 and ECE-2).. PPET-1 was found to be up-regulated in ccRCC tumor specimens and down-regulated in PRCC tumor specimens. ET(A) was significantly down-regulated in PRCC tumor specimens. ECE-1 was expressed in all tissue specimens at comparable levels, with moderate but significant elevation in normal tissue specimens associated with PRCC. Of the other genes, PPET-2 and ET(B) were expressed in all tissue specimens and no differences were observed between tumor subtypes or tumor-affected and normal tissue specimens, whereas PPET-3 and ECE-2 were present in all tissue specimens but were barely detectable.. The endothelin axis was expressed differently in the two main subtypes of RCC and appeared to match macroscopic features commonly observed in these tumors (i.e., high expression of PPET-1 in hypervascular ccRCC contrasted against low PPET-1 and ET(A) expression in hypovascular PRCC). The presence of ECE-1 mRNA in these tissue specimens suggested that active endothelin ligands were present, indicating endothelin axis activity was elevated in ccRCC compared with normal kidney, but impaired in PRCC. The current study provided further evidence that it is not appropriate to consider ccRCC and PRCC indiscriminately in regard to treatment.

Topics: Aspartic Acid Endopeptidases; Biomarkers, Tumor; Carcinoma, Papillary; Carcinoma, Renal Cell; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Metalloendopeptidases; Nephrectomy; Protein Precursors; Receptor, Endothelin A; Receptor, Endothelin B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic

The vasodilator hydralazine, used clinically in cardiovascular therapy, relaxes arterial smooth muscle by inhibiting accumulation of intracellular free Ca2+ via an unidentified primary target. Collagen prolyl hydroxylase is a known target of hydralazine. We therefore investigated whether inhibition of other members of this enzyme family, namely the hypoxia-inducible factor (HIF)-regulating O2-dependent prolyl hydroxylase domain (PHD) enzymes, could represent a novel mechanism of action. Hydralazine induced rapid and transient expression of HIF-1alpha and downstream targets of HIF (endothelin-1, adrenomedullin, haem oxygenase 1, and vascular endothelial growth factor [VEGF]) in endothelial and smooth muscle cells and induced endothelial cell-specific proliferation. Hydralazine dose-dependently inhibited PHD activity and induced nonhydroxylated HIF-1alpha, evidence for HIF stabilization specifically by inhibition of PHD enzyme activity. In vivo, hydralazine induced HIF-1alpha and VEGF protein in tissue extracts and elevated plasma VEGF levels. In sponge angiogenesis assays, hydralazine increased stromal cell infiltration and blood vessel density versus control animals. Thus, hydralazine activates the HIF pathway through inhibition of PHD activity and initiates a pro-angiogenic phenotype. This represents a novel mechanism of action for hydralazine and presents HIF as a potential target for treatment of ischemic disease.

Topics: Adrenomedullin; Angiogenesis Inducing Agents; Animals; Breast Neoplasms; Carcinoma; Carcinoma, Renal Cell; Cell Hypoxia; Cell Line, Tumor; Cells, Cultured; DNA-Binding Proteins; Dose-Response Relationship, Drug; Endothelial Cells; Endothelin-1; Enzyme Inhibitors; Gene Expression Regulation; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Humans; Hydralazine; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Implants, Experimental; Kidney Neoplasms; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Nude; Myocytes, Smooth Muscle; Neovascularization, Physiologic; Nuclear Proteins; Peptides; Procollagen-Proline Dioxygenase; Transcription Factors; Vascular Endothelial Growth Factor A; Vasodilator Agents