• © 2005 by American Society of Clinical Oncology

Targeted for Destruction: The Molecular Basis for Development of Novel Therapeutic Strategies in Renal Cell Cancer

Renal cell carcinoma (RCC) accounts for 3% of all adult malignancies, with approximately 31,000 new cases diagnosed in the United States every year.¹ For reasons that are unclear, the incidence of kidney cancer has shown a steady increase of 2% to 4% per year since the 1970s.² Although progress has been made in the development of immunologic therapy for RCC, most patients with advanced RCC do not respond to such therapy. Cytokine therapy with interleukin-2 (IL-2) remains the standard for patients with metastatic disease, with overall response rates of 15% to 22%; most patients with advanced RCC succumb to their disease. These statistics clearly highlight the need for the development of novel approaches aimed at improving outcome in RCC.

Current cancer research priorities include deciphering the molecular mechanisms underlying oncogenesis and applying this knowledge to the development of treatment strategies directed against malignant disorders. The successful development of the tyrosine kinase inhibitor STI-571 as a treatment option in patients with chronic myelogenous leukemia⁴ and gastrointestinal stromal tumors⁵ has galvanized efforts to develop similar strategies in other tumors. Several groups, including our own, have attempted to elucidate the molecular pathogenesis of renal cancer, leading to the identification of several genes that play critical roles in the development and progression of these tumors. This leaves us favorably poised, in this era of targeted molecular therapy, to address the development of rational therapeutic strategies directed against RCC.

RCC is a heterogenous group of diseases, each characterized by unique pathologic features and distinct molecular signatures. Our understanding of the common sporadic renal cancers has been greatly aided by the recognition that these tumors often have familial counterparts. At least four distinct familial renal cancer syndromes have been identified to date. The most extensively studied of these is von Hippel Lindau (VHL). Affected individuals in VHL families are at risk for the development of multiple foci of clear-cell renal cancer, as well as CNS hemangioblastomas, pancreatic tumors, pheochromocytomas, retinal angiomas, and endolymphatic sac tumors.^3,6 Loss of function of the tumor suppressor VHL gene was identified as the underlying abnormality in these families.⁷ The VHL gene product, as part of a multiunit complex that includes proteins such as elongin C/B and Cul2, targets the α subunits of the hypoxia inducible factors HIF-1 and HIF-2, for ubiquitination and subsequent degradation. The hypoxia-inducible factors are important components of the hypoxia response and are upregulated in normal cells in response to hypoxic stress. However, their intracellular levels are tightly regulated by the VHL gene product under normoxia. In the absence of functional VHL, there is unhindered accumulation of HIFα despite normal tissue oxygenation, and consequent overexpression of its downstream transcriptional targets such as vascular endothelial growth factor (VEGF), transforming growth factor-alpha (TGF-α), platelet derived growth factor (PDGF), GLUT-1, and erythropoietin. Several of the aforementioned proteins favor the development and sustenance of renal tumors by providing unopposed growth stimuli or angiogenic support. Although originally described in the familial VHL syndrome, VHL inactivation by mutation or promoter hypermethylation has also been identified in 60% to 80% of patients with sporadic clear-cell RCC.

Other well-defined familial renal cancer syndromes include: (1) hereditary papillary renal carcinoma (HPRC), in which activating tyrosine kinase mutations in the c-met gene lead to the development of bilateral multifocal type I papillary renal carcinoma. Mutations in c-met are also described in some patients with sporadic type I papillary renal carcinoma.^8,9 (2) Another is hereditary leiomyomatosis renal cell carcinoma (HLRCC), a syndrome characterized by the presence of cutaneous and uterine leiomyomata and type 2 papillary renal carcinoma. Affected individuals have a germline mutation in the Krebs’ cycle enzyme fumarate hydratase (FH), and the mechanisms by which inactivation of this enzyme leads to the development of type 2 papillary RCC are under investigation.¹⁰ (3) Birt-Hogg-Dube syndrome, is a syndrome in which affected individuals are at risk for the development of pulmonary cysts, fibrofolliculoma of hair follicles, and multifocal renal tumors (chromophobe, oncocytoma, hybrid chromophobe/oncocytic tumors or rarely, clear-cell variants). The BHD gene has been recently identified, and its function is currently under investigation.¹¹

The partial unraveling of the molecular code of kidney cancer has already enabled us to reap rich dividends. Several targeted small molecule protein kinase inhibitors and antibodies have shown activity in phase II trials of metastatic RCC and are currently under evaluation in phase III trials.^12-15 These therapeutic agents seem to exert their antitumor activity by inactivating one or more elements of growth factor or angiogenic pathways. For instance, bevacizumab is a monoclonal antibody that binds to and inhibits the activity of VEGF, while BAY 43-9006 (sorafenib) owes its activity to antagonism of both angiogenesis (inhibition of VEGF receptor) and the TGF-α/EGFR and PDGF growth signaling pathways. We are still in the early stages of development of these drugs, and it is too early to determine what percentage of patients is likely to derive clinical benefit in the form of disease response or prolonged disease stabilization from these agents. Hence, continued efforts at identifying new therapeutic targets are underway.

In this issue of the Journal of Clinical Oncology, Mizutani et al.¹⁶ report their evaluation of second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO) expression in renal cancer. Smac/DIABLO is a mitochondrial protein that is activated and released into the cytosol in response to apoptotic stimuli. Once in the cytosol, Smac/DIABLO displaces caspase-9 from regulatory cytosolic proteins called inhibitor of apoptotic proteins (IAPs).¹⁷ This interaction removes the inhibition of the caspase cascade imposed by the IAPs and allows apoptosis to proceed unfettered. Mizutani et al make several important observations in their article. (1) The expression of Smac/DIABLO is downregulated in RCC, with only 50% of stage III/IV tumors expressing the protein as determined by Western blot analysis. This is in contrast to normal renal tissue and early stage RCC, where 100% and 96%, respectively, of evaluated samples expressed Smac/DIABLO. (2) Second, although the data is not presented, the authors point out in their discussion that Smac/DIABLO expression in stage III/IV patients correlates with longer survival, suggesting its potential use as a prognostic marker. (3) Lastly, and perhaps most interesting from a therapeutic standpoint, the introduction of Smac/DIABLO into RCC cells lacking the protein enhances the ability of chemotherapy and proapoptotic agents like tumor necrosis factor–related apoptosis-inducing ligand to induce apoptotic death in these cells.

Might these findings suggest new and meaningful strategies for therapeutic intervention in RCC? Manipulating the apoptotic pathway in tumors by activating pro-apoptotic caspases is an appealing concept that deserves further exploration. Enthusiasm for strategies that would result in the activation of caspases is, however, rightly tempered by the consideration that caspase activators might be unable to discriminate between normal and tumor tissue, resulting in an unacceptably narrow therapeutic window. Recent evidence, however, suggests that unlike in normal cells, tumor cells already possess active caspases such as caspase-3. What seems to hold these caspases in check are the regulatory IAPs, which, by directly binding to several components of the caspase pathway, prevent cellular annihilation.¹⁸ Indeed, X-linked inhibitor of apoptosis protein (XIAP), the best studied IAP, seems to be upregulated in several cancers, including clear-cell RCC, presumably conferring resistance to both intrinsic and extrinsic activators of apoptosis.¹⁹ Schimmer et al have reported the identification of several compounds from a screen of small molecule combinatorial libraries that can release caspase 3 from XIAP and promote apoptosis.²⁰ These agents have shown activity against tumor cells both in vitro and in xenograft models. Furthermore, their activity seems to be selective for tumors, with sparing of normal tissues. The identification of Smac/DIABLO as an endogenous inhibitor of IAPs has also led to the investigation of the antitumor properties of short synthetic peptides comprising the XIAP-interacting domain of Smac. These peptides seem to enhance chemotherapy induced apoptosis in tumor cell lines and are the subject of active investigation in preclinical models.²¹ The issues raised by Mizutani et al, and available preclinical data, should provide the impetus for the enthusiastic investigation of IAP-antagonists, either as single agents or as chemosensitizers in RCC, a malignancy that is notoriously chemoresistant.