Summary The von Hippel-Lindau tumor-suppressor gene (VHL) is lost in most clear cell renal cell carcinomas (ccRCC). Here, using human ccRCC specimens, VHL-deficient cells, and xenograft models, we show that miR-204 is a VHL-regulated tumor suppressor acting by inhibiting macroautophagy, with MAP1LC3B (LC3B) as a direct and functional target. Importantly, higher tumor grade of human ccRCC was correlated with a concomitant decrease in miR-204 and increase in LC3B levels, indicating that LC3B-mediated macroautophagy is necessary for RCC progression. VHL, in addition to inducing endogenous miR-204, triggered the expression of LC3C, an HIF-regulated LC3B paralog, that suppressed tumor growth. These data reveal a function of VHL as a tumor suppressing regulator of autophagic programs.

Summary Autophagy promotes tumor growth by generating nutrients from the degradation of intracellular structures. Here we establish, using shRNAs, a dominant negative mutant, and a pharmacologic inhibitor, mefenamic acid (MFA), that the Transient Receptor Potential Melastatin 3 (TRPM3) channel promotes growth of clear cell renal cell carcinoma (ccRCC) and stimulates MAP1LC3A (LC3A) and MAP1LC3B (LC3B) autophagy. Increased expression of TRPM3 in RCC leads to Ca2+ influx, activation of CAMKK2, AMPK, and ULK1, and phagophore formation. In addition, TRPM3 Ca2+ and Zn2+ fluxes inhibit miR-214 which directly targets LC3A and LC3B. The von Hippel-Lindau tumor suppressor (VHL) represses TRPM3 through miR-204 directly and indirectly through another miR-204 target, Caveolin 1 (CAV1).

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 hypoxiainducible 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 oxidativestress-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.pVHL is the main tumor suppressor for which loss of activity is causatively linked to renal clear cell carcinoma (RCC), the most malignant and common form of kidney cancer. The VHL gene is mutated or hypermethylated in about 40 to 70% of sporadic RCC. Hereditary loss of pVHL function in von Hippel-Lindau (VHL) disease also results in highly vascularized RCC and capillary tumors of other organs, such as hemangioblastoma of the central nervous system and retinal angioma (19,20). A body of experimental evidence, based on a subcutaneous xenograft model system, supports the idea that accumulation of the alpha subunit of the hypoxia-inducible transcription factor (HIF) HIF-2␣ and induction of HIF target gene products, resulting from the loss of pVHL-mediated ubiquitylation, are necessary and sufficient to promote growth of RCC tumors (22,23). HIF activation has also been demonstrated as an early tumorigenesis event in kidneys from VHL patients (32). Biochemically, pVHL is the substrate-recognizing component of a multiprotein E3 ubiquitin ligase complex containing elongins C and B, Cullin 2, and the RING-H2 finger protein Rbx-1 (for a review, see reference 19). pVHL-dependent ubiquitylation of HIF-␣s is preceded by hydroxylation of conserved proline residues located within LXXLAP motifs (16,17) by the O 2 -, Fe(II)-, and oxyglutarate-regulated Egl-9-type proline hydroxylases (PHDs) (7). Thus, an important aspect of pVHL's tumor suppressing activity is the prevention of HIF-␣ accumulation, which in turn suppresses induction of the HIF target genes. Clearly, however, pVHL activity is not limited to regulation of HIFs. Other targets of pVHL-associated E...

Von Hippel-Lindau tumor suppressor (VHL) is lost in the majority of clear cell renal cell carcinomas (ccRCC). Folliculin (FLCN) is a tumor suppressor whose function is lost in Birt-Hogg-Dubé syndrome (BHD), a disorder characterized by renal cancer of multiple histological types including clear cell carcinoma, cutaneous fibrofolliculoma, and pneumothorax. Here we explored whether there is connection between VHL and FLCN in clear cell renal carcinoma cell lines and tumors. We demonstrate that VHL regulates expression of FLCN at the mRNA and protein levels in RCC cell lines, and that FLCN protein expression is decreased in human ccRCC tumors with VHL loss, as compared with matched normal kidney tissue. Knockdown of FLCN results in increased formation of tumors by RCC cells with wild-type VHL in orthotopic xenografts in nude mice, an indication that FLCN plays a role in the tumor-suppressing activity of VHL. Interestingly, FLCN, similarly to VHL, is necessary for the activity of LC3C-mediated autophagic program that we have previously characterized as contributing to the tumor suppressing activity of VHL. The results show the existence of functional crosstalk between two major tumor suppressors in renal cancer, VHL and FLCN, converging on regulation of autophagy.

Purpose: We have previously shown that von Hippel-Lindau (VHL) regulates ubiquitylation and proline 1465 hydroxylation of the large subunit of RNA polymerase II, Rpb1, in human renal clear cell carcinoma (RCC) cell lines. Here, our goal was to determine the effect of this VHL function and the status of P1465 hydroxylation in human RCC tumors.Experimental Design: Primary human tumors and matched normal kidney samples were probed for expression levels of the large subunit of RNA polymerase II (Rpb1), Rpb1 hydroxylated on P1465 [Rpb1 (OH)], Rpb1 phosphorylated on Ser5 [Rpb1(S5P)], and proline hydroxylases PHD1, PHD2, and PHD3. Results from RCC tumors were categorized according to the status of VHL gene. Mechanistic analysis was performed in orthotopic xenograft model using 786-O RCC cells with wild-type (WT) VHL and knockdown of PHD2, characterized by high levels of Rpb1(OH) and PHD1.Results: Levels of Rpb1(OH), PHD1, and PHD2 were significantly higher in RCC tumors compared with normal kidneys. RCC tumors with WT VHL had higher levels of Rpb1(OH) and PHD1 and lower levels of PHD2 than tumors with VHL gene alterations. Levels of Rpb1(OH) significantly correlated with levels of PHD1 in tumors and normal kidneys. Knockdown of PHD2 in 786-O VHL(+) cells resulted in a more malignant phenotype in orthotopic xenografts and higher expression of specific cell cycle regulators (CDC25A, cyclin-dependent kinase 2, CCNA2) compared with VHL(−) RCC cells.Conclusions: Elevated PHD1 concomitant with decreased PHD2 are causatively related to Rpb1 hydroxylation and oncogenesis in human RCC tumors with WT VHL gene. Thus, P1465-hydroxylated Rpb1 and PHD1 represent attractive drug targets for new RCC treatments. Clin Cancer Res; 16(21); 5142-52. ©2010 AACR.Renal clear cell carcinoma (RCC) is the most prevalent and malignant histologic type of kidney cancer. The majority of RCC (60-80%) is associated with intragenic mutations or hypermethylation of the von Hippel-Lindau (VHL) tumor suppressor gene and loss of heterozygosity at the VHL locus (3p26). In VHL-deficient tumors, activation of metabolic, angiogenic, and survival pathways resulting from VHL loss is an early and critical event in RCC tumorigenesis. In cases of RCC with the wild-type (WT) VHL gene, oncogenesis is not well understood. That is, it is not clear which activities of VHL are retained in those tumors.VHL is a substrate recognition molecule of an E3 ubiquitin ligase complex, recognizing hydroxylation of proline within the LxxLAP motif, which leads to ubiquitylation of the α subunits of hypoxia-inducible transcription factor (HIF; ref. 1). This hydroxylation is mediated by two of the O 2 -, Fe(II)-, and oxyglutarate-regulated Egln9-type proline hydroxylases, PHD2 and PHD3 (2). The function of the third enzyme from this group, PHD1, is less well understood, but it is known to hydroxylate conserved LQYLAP motifs of IKKβ and, by doing so, inhibit activation of the NF-κB transcription factor (3).Importantly, we have discovered that VHL mediates ubiquitylation of the l...

Summary The large subunit of RNA Polymerase II, Rpb1, undergoes hydroxylation on proline 1465, which in turn triggers Ser5 hydroxylation. While Egln2 prolyl hydroxylase appears to mediate P1465 hydroxylation, Egln1 has an inhibitory activity and its knockdown stimulates constitutive hydroxylation and Ser5 phosphorylation of Rpb1, but only in cells that are VHL(+). In this study we have analyzed protein factors affected by the knockdown of Egln1 in VHL(+) and VHL(−) cells. We found that, in VHL(+) cells, several proteins were inhibited but none were induced by Egln2 knockdown. The function of several of those proteins was related to calcium metabolism and the cytoskeleton. In contrast, in VHL(−) cells Egln1 knockdown caused upregulation of several mitochondrial proteins including subunits of ATP synthase. Several of the proteins repressed in VHL(−) cells by Egln1 knockdown were involved in the function of RNA polymerase II during transcription from chromatin templates. These data suggest that the effects of Egln1 knockdown depend on the status of pVHL and can be correlated with effects on Rpb1.