The von Hippel-Lindau Tumor Suppressor Protein and Clear Cell Renal Carcinoma William G. Kaelin, Jr.

Abstract Germ line VHL tumor suppressor gene loss-of-function mutations cause von Hippel-Lindau disease, which is associated with an increased risk of central nervous system hemangioblas- tomas, clear cell renal carcinomas, and pheochromocytomas. Somatic VHL mutations are also common in sporadic clear cell renal carcinomas.TheVHL gene product, pVHL, is part of a ubiquitin ligase complex that targets the a-subunits of the heterodimeric transcription factor hypoxia- inducible factor (HIF) for polyubiquitylation, and hence, proteasomal degradation, when oxygen is available. pVHL-defective clear cell renal carcinomas overproduce a variety of mRNAs that are under the control of HIF, including the mRNAs that encode vascular endothelial growth factor, platelet-derived growth factor B, and transforming growth factor a. In preclinical models, down- regulation of HIF-a, especially HIF-2a, is both necessary and sufficient for renal tumor suppres- sion by pVHL. These observations are probably relevant to the demonstrated clinical activity of vascular endothelial growth factor antagonists in clear cell renal carcinoma and form a foundation for the testing of additional agents that inhibit HIF, or HIF-responsive gene products, in this disease.

Inactivation of the von Hippel-Lindau tumor suppressor gene site for pVHL. pVHL is the substrate recognition component of (VHL), which is located on chromosome 3p25, plays an a multisubunit ubiquitin ligase complex that also contains important role in hereditary (VHL disease) and sporadic clear elongin B, elongin C, Cul2, and Rbx1 (also called Roc1). cell renal carcinoma, which is the most common form of pVHL directs the polyubiquitylation of HIF-a, which in turn, kidney cancer (1). The VHL gene product, pVHL, has multiple earmarks HIF-a for proteasomal degradation (13–16). In cells functions, but the one that has been most extensively studied that lackfunctional pVHL or that are exposed to low oxygen relates to the regulation of the transcription factor called (hypoxia), HIF-a subunits accumulate and bind to a HIF-h hypoxia-inducible factor (HIF). HIF is a heterodimer that partner protein (17). This HIF heterodimer binds to specific consists of an unstable a-subunit (such as HIF-1a) and a DNA sequences and transcriptionally activates genes involved stable h-subunit (such as HIF-1h, also called ARNT1). There in acute or chronic adaptation to hypoxia, such as vascular are three HIF-a genes in the human genome, although only endothelial growth factor (VEGF) and erythropoietin. Over- HIF1-a and HIF2-a have been well documented to activate production of such hypoxia-inducible mRNAs is a hallmarkof transcription in mammalian cells under physiologic condi- pVHL-defective tumors (18). tions. In fact, some HIF3-a splice variants probably act as Humans carrying one wild-type VHL allele and one mutated dominant-negative inhibitors of HIF activity (2–4). In the VHL allele in their germ line develop VHL disease, which is presence of oxygen, HIF-a (‘‘HIF-a’’ will be used in the associated with an increased riskof a variety of tumors, remainder of this review when referring to the three HIFa including central nervous system (especially cerebellum and family members generically) subunits become hydroxylated on spinal cord) hemangioblastomas and pheochromocytomas, in one (or both) of two prolyl residues (5–9). Hydroxylation of addition to kidney cancer (19). The risk of these different either site, which is carried out by members of the EglN family tumors is influenced by the nature of the VHL mutation. In (also called PHDs or HPHs; refs. 10–12), generates a binding other words, there are strong genotype-phenotype correlations in VHL disease. VHL families have been subdivided into those with a low riskof pheochromocytoma (type 1 VHL disease) and those with a high riskof pheochromocytoma (type 2 VHL Author’s Affiliation: Howard Hughes Medical Institute, Dana-Farber Cancer disease). Type 2 families almost invariably have missense VHL Institute and Brigham and Women’s Hospital, Harvard Medical School, Boston, mutations, whereas many different types of mutations, inclu- Massachusetts ding nonsense and deletion VHL mutations, have been linked Received 7/27/06; revised 11/7/06; accepted 11/10/06. Presented at the Second Cambridge Conference on Innovations and Challenges in to type 1 VHL disease. Type 2 VHL disease has been subdivided Renal Cancer, March 24-25, 2006, Cambridge, Massachusetts. into type 2A (low riskof renal carcinoma), type 2B (high riskof Requests for reprints: William G. Kaelin, Jr., Howard Hughes Medical Institute, renal carcinoma), and type 2C (familial pheochromocytoma Dana-Farber Cancer Institute and Brigham and Women’s Hospital, Harvard Medical without hemangioblastoma and clear cell renal carcinoma). School, Boston, MA 02115. Phone: 617-632-3975; Fax: 617-632-4760; E-mail: VHL [email protected]. alleles linked to type 1, type 2A, and type 2B VHL disease F 2007 American Association for Cancer Research. encode proteins that are at least partially defective with respect doi:10.1158/1078-0432.CCR-06-1865 to HIF-a regulation, whereas the products of type 2C VHL

Clin Cancer Res 2007;13(2 Suppl) January 15, 2007 680s www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2007 American Association for Cancer Research. VHL Protein and Renal Carcinoma alleles are grossly normal with respect to HIF (20, 21). The products of type 2C VHL alleles are defective, however, with respect to another pVHL function, i.e., down-regulation of atypical protein kinase C activity (22–25). Increased atypical protein kinase C activity, and consequent up-regulation of JunB, seems to promote the survival of pheochromocytoma cells when growth factors such as nerve growth factor become limiting (22). In VHL disease, stochastic loss of the remaining wild-type VHL allele in the kidney causes the development of renal cysts (26). The precise cell of origin in these lesions is debated but might be a distal renal tubular epithelial cell (26). Regardless of the cell of origin, it is presumed that mutations at other loci are required to convert these cysts to renal cell carcinomas. A total of 40% to 80% of sporadic clear cell renal carcinomas are linked to biallelic VHL inactivation, and in some VHL+/+ clear cell renal carcinomas, little or no VHL mRNA is produced as a result of promoter hypermethylation (1). VHLÀ À Restoration of wild-type pVHL function in / renal Fig.1. Potential cancer drug targets linked to pVHL and HIF. pVHL inhibits the carcinoma cells suppresses their ability to form tumors in nude heterodimeric transcription factor HIF by targeting it for proteasomal degradation. mice (27, 28). Although pVHL does not grossly alter cell HIF protein levels are also sensitive to changes in histone deacetylase (HDAC), heat shock protein 90 (HSP90), and mTOR activities. HIF transcriptionally activates >10 0 proliferation under standard cell culture conditions, it induces genes, including many suspected or known to play roles in tumorigenesis (solid a number of phenotypes in vitro that are likely to correlate with arrows). Sorafinib and sunitinib inhibit signaling by theVEGF receptor KDR and tumor suppression in vivo. These include enhanced cell cycle platelet-derived growth factor receptor (PDGFR). See text for details. exit under low serum conditions and enhanced cell differen- tiation in monolayer and spheroid growth assays (29–34). In particular, restoration of pVHL function in VHLÀ/À renal Fourth, tumor suppression by pVHL can be overridden by carcinoma cells is able to induce a mesenchymal to epithelial HIF-2a but not by HIF-1a (41, 43–45). Finally, type 2B pVHL transition. mutants, which are associated with a high riskof renal VHL+/À mice develop liver hemangiomas but do not deve- carcinoma, are more defective with respect to HIF-a regulation 1 lop the lesions typical of human VHL disease (35). Similar than type 2A mutants (46). Collectively, these results hepatic lesions develop after Cre recombinase–mediated implicate dysregulation of HIF target genes as playing a causal inactivation of VHL in the livers of VHL flox/flox mice role in the pathogenesis of pVHL-defective clear cell renal (35, 36). These lesions are characterized by elevated levels of carcinomas. HIF-a and HIF-responsive gene products such as VEGF. Why pVHL and HIF, both of which are ubiquitously Importantly, these lesions do not develop in mice that lack expressed, play such an important role in human clear cell HIF-1h, also called aryl hydrocarbon receptor nuclear trans- renal carcinoma is not clear, but several observations might be locator (ARNT1), suggesting that HIF-a function is necessary relevant. First, renal epithelia seem to be particularly sensitive for these pathologic changes (37). In a complementary set of to the mitogenic effects of the HIF-responsive growth factor experiments, we found that hepatic expression of a stabilized transforming growth factor a among various epithelia tested version of HIF-2a in genetically engineered mice is sufficient to (47). Second, hypoxia and HIF cause the up-regulation of cyclin induce many of the pathologic changes attributed to pVHL D1 (Fig. 1), which with its catalytic partners, cdk4 and cdk6, loss (38). stimulates cell proliferation by directing the phosphorylation of VHLÀ/À embryos are not viable, and conditional, systemic, the pRB tumor suppressor protein, in renal epithelia but not in inactivation of VHL in adult mice is lethal (36, 39). Haase and other cells types (45, 48–50). Finally, portions of the kidney in coworkers recently reported that inactivation of VHL in the mammals (especially the medulla) are hypoxic at rest (51). mouse kidney, using Cre recombinase under the control of Epigenetic differences between the kidney and other organs that the phosphoenolpyruvate carboxykinase promoter, caused the allow renal cells to proliferate in a hypoxic environment might development of polycythemia and renal cysts (40). These also make them more susceptible to the oncogenic effects of results, however, are complicated by the fact that the Cre pVHL loss/HIF activation. transgene was also expressed in the liver. Renal pathology was Kidney cancers are notoriously resistant to standard chemo- not observed in a mouse in which VHL was inactivated in a therapy and radiotherapy. It has been suggested that this systemic mosaic pattern (36). resistance might be due, at least in part, to increased levels of HIF-a, especially HIF-2a, seems to play a special role with the transcription factor nuclear factor nB (52, 53). Loss of VHL respect to VHLÀ/À renal carcinogenesis. First, VHLÀ/À renal leads to increased nuclear factor nB activity and resistance to carcinomas seem to produce both HIF-1a and HIF-2a or apoptosis (54, 55). One study suggested that increased levels of HIF-2a alone (17). Second, the appearance of HIF-2a in HIF-a were necessary and sufficient for the activation of nuclear VHLÀ/À preneoplastic lesions arising from VHL+/À kidneys is factor nB, although this workneeds to be independently associated with increased dysplasia (26). Third, the elimina- tion of HIF-2a, like the restoration of pVHL function, is sufficient to suppress VHLÀ/À tumor growth in vivo (41, 42). 1Lianjie Li and Kaelin W.G., unpublished data.

www.aacrjournals.org 681s Clin Cancer Res 2007;13(2 Suppl) January 15, 2007 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2007 American Association for Cancer Research. corroborated (56). We have obtained evidence of a HIF- robust clinical activity from VEGF pathway inhibitors in the independent linkbetween pVHL and nuclear factor nB.2 form of complete responses? These considerations suggest that drugs which target HIF, Dr. Kaelin: I am a believer of the preclinical workper- or HIF-responsive gene products, should be effective in the formed by Ellie Keshet in Israel and Doug Hannahan at the treatment of renal carcinomas. Unfortunately, DNA-binding University of California, San Francisco, which strongly suggests transcription factors, with the exception of the steroid hor- that newly sprouting blood vessels, before they are properly mone receptors, have proven difficult to inhibit with drug-like invested with pericytes and surrounding stroma, are exquisitely small organic molecules. However, a number of drugs have sensitive to, and will regress upon, VEGF withdrawal. However, been identified that indirectly down-regulate HIF-a, including once you have a mature vessel that is properly enveloped with drugs that inhibit mTOR (57–61), HSP90 (62, 63), and pericytes and stroma, VEGF inhibitors no longer cause regres- histone deacetylases (64). These drugs clearly warrant inves- sion; they cause stasis. tigation in pVHL-defective renal carcinomas. In one phase 3 Dr. Kwon: Is there any thinking that HIF may be a relatively trial, patients with poor prognosis renal carcinoma treated late player in tumor progression? with the mTOR inhibitor, CCI-779, fared better than patients Dr. Kaelin: I cannot prove that VHL loss is an early event treated with IFN with respect to time to progression and versus a late event in sporadic clear cell carcinoma. At least in overall survival (65). VHL disease, it appears that VHL loss is an early event and HIF-responsive gene products that are suspected or known sufficient to cause renal cysts. Therefore, your question really is to play a role in renal tumorigenesis include VEGF, platelet- why would VHL loss give rise to renal cysts? VHL loss leads to derived growth factor B, c-Met (66–68), transforming growth up-regulation of cyclin D1 and gives rise to transforming growth factor a (47, 69, 70), transforming growth factor h (71), factor a. We know that one of the features of renal cystic diseases CXCR4 (and its ligand SDF1; refs. 72, 73), and certain matrix is increased proliferation of renal epithelial cells, which might metalloproteinase (refs. 74, 75; Fig. 1). It is worth noting be due to cyclin D1 or transforming growth factor-a. Another that overproduction of VEGF probably occurs very early feature is alterations in epithelial stromal interactions, and there during the development of VHLÀ/À renal cell carcinomas is a role for VHL in the regulation of the extracellular matrix. and therefore reduces the selection pressure to activate Again, it is partly related to HIF. Several groups have shown that ‘‘collateral’’ angiogenic pathways (26, 76, 77). This might when you inactivate VHL in a renal epithelial cell, the cell explain why renal cell carcinomas are the only solid tumors undergoes an epithelial to mesenchymal transition. You can where agents that inhibit VEGF, or its receptor KDR, have reverse that by putting VHL backin. There are a lot of effects of significant activity as single agents. Indeed, two such agents VHL loss that might translate into renal cyst formation. (sorafenib and sunitinib) were recently approved by the Food Although you might have expected HIF deregulation to be a and Drug Administration for this indication. These drugs late event, at least in renal cancer, it can be an early event. both inhibit KDR in addition to some other receptor tyrosine Dr. Kwon: Is there a possibility that the inadequacy of kinases, including the platelet-derived growth factor receptor. angiogenesis inhibition may be in part related to exacerbation This might be fortuitous because dual inhibition of VEGF of hypoxia of the tumor and subsequent induction of more and platelet-derived growth factor signaling is more effective HIF? at inducing the regression of established tumor blood vessels Dr. Kaelin: As the tumor expands and hypoxia occurs in the than is VEGF blockade alone in preclinical models (78, 79). surrounding normal tissues that are being compressed, the It will be important to determine whether the VHL genotype normal host becomes a source of angiogenic growth factors. influences the responsiveness to VEGF inhibitors, as well as Therefore, as you treat these tumors with an angiogenesis the molecular basis for acquired or de novo resistance. VEGF inhibitor, it is likely that you exacerbate hypoxia. inhibitors can now serve as a platform for building rational Dr. Kwon: What are the driving molecules that push the combinations that target additional HIF-responsive growth inception of the tumor? factors and/or incorporate drugs that down-regulate HIF Dr. Kaelin: I have satisfied myself as best I can that HIF is itself. the driver. Dr. Sosman: Some recent data from Neal Rosen and his colleagues has shown that if you blockmTOR, you induce a Open Discussion feedbackthat enhances AKT activity. Is that pathway important in renal cancer outside mTOR-regulating HIF? Dr. Atkins: Is something other than the hypoxia-inducible Dr. Kaelin: mTOR inhibitors down-regulate HIF, at least in factor driving the von Hippel-Lindau wild-type clear cell renal cell culture. I do not know whether indirect effects on AKT cancer? signaling might offset any benefits of down-regulating HIF. Dr. Kaelin: In most VHL wild-type renal carcinoma cell Dr. Sukhatme: At least two reports have shown that if lines, it looks like HIF is regulated appropriately, meaning at you introduce a nondegradable HIF1 into a tumorigenic cell least it is responsive to hypoxia. I don’t know what is driving line, you get a decreased growth rate, but if you introduce VHL wild-type clear cell renal cancer. a dominant-negative you get even faster growth. Are you Dr. Figlin: You said that with VHL loss, angiogenesis concerned about HIF inhibitors in this context or is there through the vascular endothelial growth factor pathway something peculiar about that data with HIF1-a versus dominates. If that were true, wouldn’t we have seen more HIF2-a? Dr. Kaelin: Emerging data suggest that whether HIF1 can promote or inhibit tumor growth depends on what cell type are 2 Haifeng Yang and Kaelin W.G., unpublished data. you looking at and in what context that cell is growing.

Clin Cancer Res 2007;13(2 Suppl) January 15, 2007 682s www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2007 American Association for Cancer Research. VHL Protein and Renal Carcinoma

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Clin Cancer Res 2007;13(2 Suppl) January 15, 2007 684s www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2007 American Association for Cancer Research. The von Hippel-Lindau Tumor Suppressor Protein and Clear Cell Renal Carcinoma

William G. Kaelin, Jr.

Clin Cancer Res 2007;13:680s-684s.

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