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 RCC-related signaling pathways. Importantly, both VHL and the hypoxia-inducible factor 1-α gene 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.

The von Hippel-Lindau tumor suppressor protein and Egl-9-Type proline hydroxylases regulate the large subunit of RNA polymerase II in response to oxidative stress.

Human renal clear cell carcinoma (RCC) is frequently associated with loss of the von Hippel-Lindau (VHL) tumor suppressor (pVHL), which inhibits ubiquitylation and degradation of the alpha subunits of hypoxia-inducible transcription factor. pVHL also ubiquitylates the large subunit of RNA polymerase II, Rpb1, phosphorylated on serine 5 (Ser5) within the C-terminal domain (CTD). A hydroxylated proline 1465 within an LXXLAP motif located N-terminal to the CTD allows the interaction of Rpb1 with pVHL. Here we report that in RCC cells, pVHL regulates expression of Rpb1 and is necessary for low-grade oxidative-stress-induced recruitment of Rpb1 to the DNA-engaged fraction and for its P1465 hydroxylation, phosphorylation, and nondegradative ubiquitylation. Egln-9-type prolyl hydroxylases, PHD1 and PHD2, coimmunoprecipitated with Rpb1 in the chromatin fraction of VHL(+) RCC cells in response to oxidative stress, and PHD1 was necessary for P1465 hydroxylation while PHD2 had an inhibitory effect. P1465 hydroxylation was required for oxidative-stress-induced Ser5 phosphorylation of Rpb1. Importantly, overexpression of wild-type Rpb1 stimulated formation of kidney tumors by VHL(+) cells, and this effect was abolished by P1465A mutation of Rpb1. These data indicate that through this novel pathway involving P1465 hydroxylation and Ser5 phosphorylation of Rbp1, pVHL may regulate tumor growth.

Regulation of tyrosine hydroxylase promoter activity by the von Hippel-Lindau tumor suppressor protein and hypoxia-inducible transcription factors.

Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is induced by hypoxia in oxygen-sensitive cells of the carotid body and pheochromocytoma-derived PC12 cells. TH is also regulated by the von Hippel-Lindau tumor suppressor protein (pVHL). Here, we report that induction of TH gene expression involves activation of the hypoxia-inducible transcription factors (HIFs) that interact with a specific hypoxia-responsive element (HRE) in the proximal region of the TH promoter. We also show that some of the effects of pVHL on activity of the TH promoter are mediated through HIFs. Low levels of pVHL are associated with decreased HIFalpha ubiquitination, increased accumulation of HIFalpha proteins, increased binding of HIFs to the HRE within the TH promoter, and increased activity of a TH promoter-reporter construct. In contrast, high levels of pVHL repress HIF accumulation and inhibit its activity in hypoxic cells. These results indicate that HIFs may play an important role in regulation of TH gene expression in oxygen-sensitive cells and also in the development of hypercatecholaminemia in pheochromocytoma tumors.

von Hippel-Lindau protein binds hyperphosphorylated large subunit of RNA polymerase II through a proline hydroxylation motif and targets it for ubiquitination.

The transition from transcription initiation to elongation involves phosphorylation of the large subunit (Rpb1) of RNA polymerase II on the repetitive carboxyl-terminal domain. The elongating hyperphosphorylated Rpb1 is subject to ubiquitination, particularly in response to UV radiation and DNA-damaging agents. By using computer modeling, we identified regions of Rpb1 and the adjacent subunit 6 of RNA polymerase II (Rpb6) that share sequence and structural similarity with the domain of hypoxia-inducible transcription factor 1 alpha (HIF-1 alpha) that binds von Hippel-Lindau tumor suppressor protein (pVHL). pVHL confers substrate specificity to the E3 ligase complex, which ubiquitinates HIF-alpha and targets it for proteasomal degradation. In agreement with the computational model, we show biochemical evidence that pVHL specifically binds the hyperphosphorylated Rpb1 in a proline-hydroxylation-dependent manner, targeting it for ubiquitination. This interaction is regulated by UV radiation.

Transition C2718T in the AR gene, resulting in generation of a termination codon and truncated form of the androgen receptor, causes complete androgen insensitivity syndrome.

The action of testosterone and 5 alpha-dihydrotestosterone are essential to the development of the male phenotype. Patients with karyotype 46,XY, resistant to these hormones, exhibit a wide spectrum of phenotypes: from phenotypic female, through a range of incomplete masculinization, to under-virilized, infertile man. These disturbances are caused by mutations in the androgen receptor gene (AR). We studied a 46,XY fenotypic female with typical symptoms of Complete Androgen Insensitivity Syndrome (CAIS). Multiple temperature single-stranded conformation polymorphism (MSSCP) and sequence analysis of exon 6 of the AR gene in a patient revealed a C2718T transition causing R786X mutation in the loop between helices VII and VIII of the LBD of the androgen receptor. The R786X mutation has been described in a patient with CAIS only once and no such mutations have been described in Eastern Europe.