Tumorigenesis and Neoplastic Progression Pleiotropic Action of Renal Cell Carcinoma-Dysregulated miRNAs on Hypoxia-Related Signaling Pathways

The von Hippel-Lindau (VHL) gene is lost in 70% of all renal cell carcinomas (RCCs); however, increasing evidence supports the involvement of alternative mechanisms in the regulation of VHL expression, including suppression by microRNAs (miRNAs). miRNAs are small, noncoding RNA molecules that regulate gene expression through binding to target mRNAs. In this study, we found that miRNAs, which are dysregulated in cases of RCC, can target multiple members of RCCrelated signaling pathways. Importantly, both VHL and the hypoxia-inducible factor 1gene are experimentally validated and are likely direct targets of miR-17-5p and miR-224, as shown by both luciferase assay and Western blot analysis. We found a negative correlation between miR-17-5p and its two predicted targets, VEGF-A and EGLN3, and between miR-224 and its targets SMAD4 and SMAD5 in RCC specimens, suggesting that downstream signaling pathways are also modulated by clear cell RCC-dysregulated miRs. Results from our bioinformatics analysis show that a single miRNA molecule can target multiple components of the same pathway and that multiple miRNAs can target the same molecule. Our results also indicate that miRNAs represent a mechanism for the inactivation of VHL in cases of RCC and can elucidate a new dimension in cancer pathogenesis. As such, miRNAs exemplify new potential therapeutic targets with a significant effect on both tumor growth and metastatic potential. (Am J Pathol 2012, 180:1675–1687; DOI: 10.1016/j.ajpath.2011.12.030) Renal cell carcinoma (RCC) is the most common adult kidney neoplasm, with the clear cell RCC (ccRCC) subtype accounting for 70% of cases. At the molecular level, this subtype has been associated with loss of the von Hippel-Lindau (VHL) tumor suppressor protein. VHL acts as an E3 ubiquitin ligase. The best-understood VHL target, the transcription factor hypoxia-inducible factor 1(HIF1 ), is destabilized by VHL under normoxic conditions and targeted for ubiquitination and proteasomal degradation. During hypoxia, HIF1 translocates from the cytoplasm into the nucleus, where it initiates transcription of a number of growth and proangiogenic factors, including vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), as well as other downstream targets implicated in extracellular matrix assembly, epithelial-cell differentiation, and oxygen sensing. Loss of VHL function occurs in all hereditary forms and in 77% of sporadic cases of ccRCC. Other mechanisms of VHL down-regulation have been reported, including point mutations of the gene and hypermethylation of the VHL promoter. MicroRNAs (miRNAs) are a class of small, singlestranded, noncoding RNAs that regulate gene expression at the posttranscriptional level by binding to the 3= untranslated region (3=-UTR) of the target gene and causing suppression of translation and/or mRNA degradation. miRNAs play important roles in essential processes such as cell differentiation, growth, and cell death. It has been shown that miRNAs are aberrantly expressed or mutated in cancers, suggesting their involvement in cancer development, progression, and/or metastasis. Besides, the