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Clinical implications of hypoxia inducible factor in renal cell carcinoma
Urologic Oncology: Seminars and Original Investigations 27 (2009) 238 –245
Translational studies in urologic oncology
Clinical implications of hypoxia inducible factor in renal cell carcinoma Marc C. Smaldone, M.D., Jodi K. Maranchie, M.D., FACS* Department of Urology, University of Pittsburgh Medical Center and University of Pittsburgh Cancer Institute, Pittsburgh, PA 15232, USA
Abstract Management of renal cell carcinoma (RCC) has made considerable strides in the past decade, due in large part to identification of the von Hippel Lindau (VHL) tumor suppressor as a negative regulator of hypoxia inducible factor ␣ (HIF-␣) protein expression. Stabilization of HIF-␣ appears to be critical for renal tumorigenesis, and is observed even in VHL-independent RCC. Thus, an understanding of the pathways that regulate expression and activation of the different HIF-␣ isoforms is key to delineating the mechanism of renal transformation and for the development of novel therapeutics. A number of agents targeting HIF-␣ or its transcriptionally-regulated genes have shown promise in treatment of RCC. However, more effective treatment strategies are still needed. This report provides a directed review of recent discoveries defining the role of HIF in renal tumorigenesis and their relevance to the clinical advances in targeted therapy for advanced RCC. © 2009 Elsevier Inc. All rights reserved. Keywords: Renal cell carcinoma; HIF; VHL; VEGF; Therapy
Introduction The incidence of renal cell carcinoma (RCC) has increased steadily over the past several decades. An estimated 38,000 new cases of RCC were diagnosed in 2006, with greater than 12,000 expected deaths from the disease [1]. This rise has been attributed to the widespread use of noninvasive abdominal imaging procedures for improved detection [2]. However, despite a higher proportion of patients with localized disease at diagnosis, mortality has also risen steadily over the same time period [3], suggesting a fundamental shift in cancer biology. Although tumors localized to the kidney are potentially cured by surgical resection, onethird of patients present with advanced disease, and half of those remaining will ultimately relapse. These lesions are both radio- and chemo-resistant, and standard immunotherapies lead to complete response in fewer 15% of patients [4]. Clear cell RCC is well known for its intense vascularity and high expression of angiogenic factors. Insight into this biology came with the 2000 discovery that the von Hippel Lindau tumor suppressor gene (VHL), lost in greater than 75% of clear cell RCCs, functions as a negative regulator of hypoxia inducible factor-␣ (HIF-␣). However, HIF-␣ in-
* Corresponding author. Tel.: ⫹1-412-605-3019; fax: ⫹1-412-6053030. E-mail address: [email protected] (J.K. Maranchie). 1078-1439/09/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.urolonc.2007.12.001
duction is not limited to the subset of clear cell RCC with VHL loss, suggesting that it plays a fundamental role in renal transformation. Novel therapeutic agents targeting HIF-␣ or its transcription targets have demonstrated promising antitumor activity in clinical trials.
Hypoxia inducible factor 1 The heterodimer transcription factor HIF-1 was first identified in 1991 as a regulator of renal production of erythropoietin (Epo), the glycoprotein hormone that controls RBC production and maintains physiologic oxygen homeostasis. Deletion analysis of the 3= flanking region of Epo revealed the minimal essential sequence of 5=CTACGTGCT-3= [5] required for oxygen-dependent regulation. HIF-1 was subsequently purified from this hypoxiaresponse element (HRE), yielding two subunits, HIF-1␣ and HIF-1 [6]. The latter proved to be the aryl hydrocarbon receptor nuclear translocator (ARNT), which is constitutively expressed in all cell types [7]. In contrast, HIF-1␣ is tightly regulated at the protein level by oxygen-dependent ubiquitination followed by proteasomal degradation, now known to be mediated by VHL. Under physiologic oxygen conditions, HIF-1␣ protein is virtually undetectable. Hypoxia leads to abundant protein levels, nuclear translocation, and transactivation of target genes harboring the HRE sequence [8,9]. More than 100 HIF transcription targets have
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changes required for CBP/p300 binding [12,13]. FIH-1 is inhibited by low oxygen levels, iron sequestration, or heavy metals (cobalt), conditions known to promote HIF activation. Binding of CBP/p300 was also recently shown to be regulated by the mammalian target of rapamycin (mTOR) via binding of an mTOR-signaling motif (TOS) located in the N-terminus of HIF-1␣ [14].
VHL regulation of HIF-1␣
Fig. 1. Pathways and representative genes transcriptionally regulated by hypoxia inducible factors HIF1 and HIF2. Although significant overlap exists, array studies indicate differential induction by the two isoforms with apoptosis and gluconeogenesis preferentially induced by HIF-1 and angiogenesis preferentially induced by HIF-2. Single asterisks indicate genes specifically regulated by HIF-2 whereas double asterisks indicate those specifically regulated by HIF-1.
been described to date, involving a myriad of diverse pathways required for response to hypoxia or environmental stress, including erythropoiesis, angiogenesis, proliferation, apoptosis [10], and anaerobic metabolism (Fig. 1). The N-terminus of HIF-1␣ contains the sequences required for dimerization and DNA binding. The C-terminus harbors two distinct activation domains: the oxygen dependent degradation domain (ODD), and the carboxy-terminal activation domain (CAD) (Fig. 2). As its name implies, the ODD contains the residues required for oxygen-mediated degradation and can confer this property to other proteins when expressed as a fusion [11]. Stabilized protein, however, is not transcriptionally active until a second oxygendependent event occurs at the CAD to permit binding of co-factor CBP/p300, which promotes dimerization and nuclear translocation. Under normal oxygen conditions, an asparagine within the CAD is hydroxylated by factor inhibiting HIF (FIH-1), a 2-oxoglutarate-dependent oxygenase that requires oxygen, iron (Fe(II)) and 2-oxoglutarate as substrates. Hydroxylation prevents tertiary structural
Greater than 75% of clear cell RCCs harbor biallelic loss of the von Hippel Lindau (VHL) tumor suppressor gene [15]. These tumors are uniquely vascular and characterized by elevated expression of VEGF and glucose transporter-1 (Glut-1). By 1999, the multi-subunit von Hippel Lindau tumor suppressor complex, comprised of elongin B, elongin C, Cul2, and RBx1, had been characterized. Due to striking homology to the SCF (Skp1-Cdc53/Cul-1-F-box protein) complex in yeast, it was identified as an E3 ubiquitin ligase with VHL as the substrate recognition component [16]. The discovery of HIF-1␣ and HIF-2␣ as the first two confirmed targets of VHL-mediated degradation greatly advanced our understanding of renal tumorigenesis, and opened the door to novel molecularly targeted therapies [17]. VHL binds to the ODD under normal oxygen conditions, leading to polyubiquitination and subsequent degradation of HIF-␣. VHL binding requires hydroxylation of one of two proline residues within the ODD by a family of prolyl hydroxylases (PHD 1–3) [18]. Analogous to FIH-1, PHDs are dioxygenases, dependent upon molecular oxygen, 2-oxoglutarate, and iron and inhibited by hypoxia, iron chelation, or cobalt [19,20]. When oxygen levels are low, hydroxylation does not occur and VHL cannot bind HIF-␣, leading to stabilization and protein accumulation.
Alternate HIF-␣ isoforms A homologous HIF-2␣ subunit (the product of the EPAS-1 gene) with 48% sequence identity with HIF-1␣ was isolated from endothelial cells in 1997 [21]. Like HIF-1␣, HIF-2␣ is stabilized and activated by hypoxia and dimerizes
Fig. 2. Schematic of HIF-1␣ and HIF-2␣ indicating conserved activation and binding domains. Shaded boxes represent critical hydroxylation sites. ODD: Oxygen dependent degradation domain. CAD: Carboxy-terminal activation domain.
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with ARNT to form HIF-2. Further, HIF-2 activates transcription of target genes by binding the same HRE as HIF-1␣. However, HIF-1␣ and HIF-2␣ are not functionally redundant. Whereas HIF-1␣ homozygous inactivation is embryonic lethal due to lack of vascular formation and global hypoxia [22], embryos of HIF-2␣ double knockout mice demonstrate normal systemic vasculature but impaired lung maturation and catecholamine production [23]. Further, HIF-2␣ expression is more tightly controlled and undetected in most cells. It promotes an undifferentiated phenotype in pluripotent stem cells emphasizing the need for persistent silencing in many tissues after development [24]. HIF-2␣ is the predominant isoform, however, in developing kidney, small vessels of the CNS, adrenal medulla, lung and intestine. Interestingly, although mature normal kidney and early cystic lesions predominantly express the HIF-1␣ isoform [25] clear cell RCC demonstrates a shift toward expression of HIF-2␣ [26,27]. Conversely, expression of HIF3␣, described in 1998, is down-regulated with progression to cancer [28]. This isoform is highly abundant in heart, placenta, and skeletal muscle. Six different splice variants have been described, three of which are targeted by VHLmediated degradation [29]. HIF-3␣ resembles HIF-1␣ and -2␣ in the ODD, but lacks the CAD transactivation domain. It appears to function as a dominant negative regulator of hypoxia-inducible gene expression in the human kidney [30]. HIF-␣ as a renal oncogene Recent studies have shown that HIF-␣, particularly HIF2␣, plays an important role in the development in VHLdeficient RCC and is not solely a marker for disruption of the VHL pathway. Immunohistochemical examination of early kidney lesions from VHL patients show a coordinated loss of VHL and increase in HIF-1␣. Intriguingly, there is an apparent switch from HIF-1␣ accumulation to HIF-2␣ accumulation in such lesions coincident with increasing dysplasia [26]. While the relative contributions of HIF-1␣ and HIF-2␣ to the pathogenesis of the VHL phenotype have yet to be defined, these findings suggest that HIF-2␣ is more oncogenic than HIF-1␣ in the setting of VHL-defective RCC. The evidence supporting this theory was nicely summarized by Kim et al. in a recent review [31]. Briefly, human RCC lines express either both HIF-1␣ and HIF-2␣ or HIF-2␣ alone, suggesting that there may be a selection pressure to maintain HIF-2␣ expression or lose HIF-1␣ expression [17]. Second, HIF-2␣ variants lacking prolyl hydroxylation sites prevent tumor inhibition by VHL in animal models, whereas analogous HIF-1␣ mutants do not [32,33]. Third, down-regulation of HIF-2␣ with viral vector hairpin RNAs in human VHL–/– RCC cells is sufficient to prevent tumor formation in nude mice [34,35]. Finally, pathologic changes observed in mice engineered to lack VHL can be prevented by simultaneous deletion of HIF-1
[36]. Bias toward HIF-2␣ in renal transformation is perhaps in part due to the fact that HIF-1␣ preferentially induces pro-apoptotic pathways not targeted by HIF-2␣ [10]. Despite the fact that both target the same HRE, array studies demonstrate that expression of genes involved in the glycolytic pathway are driven preferentially by HIF-1␣ [37], whereas HIF-2␣ preferentially promotes growth and angiogenesis [38]. Consistent with this, although both HIFs equally activate exogenous reporter constructs containing the Epo HRE, HIF-2 preferentially binds the endogenous Epo promoter [39] suggesting the involvement of isoformspecific nuclear co-factors.
Nonhypoxic regulation of HIF There is a growing body of evidence that HIF-␣ subunits are alternatively activated by reactive oxygen species (ROS) under normal oxygen conditions [40]. Transcriptional induction and activation of HIF-␣ are seen in response to a wide array of inflammatory cytokines and growth factor stimuli, including TNF-␣ [41,42], angiotensin II [43], IL-1 [44], and thrombin [45]. We showed that in VHL-deficient RCC cells, where HIF-2␣ protein levels are abundant, normoxic transcriptional activity is critically dependent upon expression of the Nox4 NADPH oxidase, an endogenous generator of ROS found in greatest abundance in the distal renal tubules [46,47]. A role in tumorigenesis was confirmed by Nox inhibition in xenografts, demonstrating that NADPH oxidases promote tumor growth [48]. These studies suggest a role for Nox4 as a future target for treatment of RCC. HIF-␣ protein is also stabilized by binding to heat shock protein 90 (hsp90) via a mechanism that is independent of both oxygen and VHL. The specific hsp90 inhibitor geldanamycin leads to HIF-␣ degradation [49]. ARNT competes with hsp90 for binding of HIF-␣ and protects the protein from geldanamycin-mediated degradation [50]. This pathway is thought to play a role in adaptation to hypoxia by attenuating HIF-␣ activation.
HIF in non-clear cell RCC Although the HIF pathway was originally linked to VHL-deficient clear cell RCC, over-expression of HIF is documented in all renal histologic subtypes, including nascent renal tumors expected to have limited artifact from tumor ischemia. Increased expression of HIF-1␣ or HIF-2␣ was seen in 50% and 100% of chromophobe tumors and in 25% and 50% of hereditary type I papillary tumors (HPRC), respectively [27]. Loss of fumarate hydrogenase in hereditary leiomyomatosis and papillary RCC (HLPRC) leads to elevated fumarate, which directly stabilizes HIF-␣ by competitive inhibition of HIF prolyl hydroxylases [51]. HIF is also elevated in clear cell RCC of tuberous sclerosis where
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the VHL pathway is intact [52]. Although the impact of HIF in non-VHL tumorigenesis is less well defined, these studies hint at a fundamental role in renal transformation.
Direct targeting of HIF Over 40 inhibitors of HIF-1 activity have been identified, including inhibitors of mRNA expression, protein expression, DNA-binding activity, or HIF-1 mediated gene transcription [53], with varying degrees of overlap with HIF-2. HIF-␣ mRNA translation requires expression of mammalian target of rapamycin (mTOR) via a PI3 kinase-dependent pathway [54]. Inhibitors of mTOR (rapamycin, temsirolimus, everolimus) bind FK506 binding protein 12 (FKBP12). The resulting FKBP12/drug complex then binds mTOR, inducing G1 growth arrest. This prevents translational initiation of proteins including HIF-␣ [55]. Preclinical studies show that VHL loss sensitizes cells to mTOR inhibition in a HIF-dependent manner [56]. A Phase II study of single-agent temsirolimus (CCI-779) in cytokine refractory advanced RCC yielded a 7% objective response rate, and a 51% stable disease rate at 24 weeks [57]. In combination with interferon-␣, temsirolimus resulted in 11% partial response with median time to progression of 9 months [58]. A large (n ⫽ 626) Phase III, randomized study of first-line temsirolimus vs. IFN vs. temsirolimus plus IFN in poor-risk RCC revealed a survival benefit for temsirolimus alone (median survival 10.9 months) compared with IFN (7.3 months) or the combination (8.4 months) [59]. The survival benefit in this trial was greatest in patients with poorest risk features and with non-clear cell histology. Everolimus (RAD-001) is an orally administered mTOR inhibitor that has demonstrated antitumor activity as well as prolonged time to progression in a Phase II trial of heavily pretreated patients [60]. A randomized Phase III trial of everolimus vs. placebo is ongoing.
HIF-responsive growth factors and angiogenesis Many of the HIF-responsive genes described to date encode growth factor receptors or their ligands, including vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), and transforming growth factor-␣ (TGF-␣) [61]. Disruption of these pathways with neutralizing antibodies or small molecule inhibitors has shown promise in clinical trials. VEGF stimulates endothelial cell proliferation and survival and suppresses the antitumor immune response [62,63]. Furthermore, the VEGF tyrosine kinase receptor is expressed by RCC cells, suggesting the possibility of an autocrine feedback loop in addition to paracrine effects of VEGF on endothelial cells [64]. Multiple VEGF isoforms have been described. VEGF-A is involved in angiogenesis, while VEGF-C and VEGF-D have been linked to lym-
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phangiogenesis [65]. Bevacizumab (Avastin) is a humanized monoclonal antibody that binds and neutralizes all the major isoforms of VEGF. In a randomized Phase II trial, bevacizumab led to a significant delay in time-to-progression relative to placebo (4.8 vs. 2.5 months) in patients with advanced RCC who had failed prior high dose IL-2 [66]. Two randomized, Phase III trials comparing interferon vs. interferon plus bevacizumab have been completed [67] and will help determine the role of bevacizumab as a primary treatment modality. Early analysis of one study shows a response rate of 31% for the combination relative to 13% for interferon alone, with a corresponding benefit in progression free survival of 10.2 vs. 5.4 months [68]. Neutralization of serum VEGF with a fusion of the VEGF receptor and human immunoglobulin (the VEGF-trap) showed minimal response in an early Phase I trial of 15 patients with advanced solid malignancies. However, one subject with metastatic RCC maintained stable disease for greater than 6 months [69], and Phase II trials for advanced RCC are planned. Tyrosine kinase inhibitors also demonstrate single-agent activity in advanced RCC. Sunitinib (SU11248) inhibits the receptor tyrosine kinases VEGFR-2, PDGFR, FMS-like tyrosine kinase 3 (FLT-3), and c-KIT. Initial trials demonstrated sunitinib’s utility as a second-line agent for patients with metastatic RCC who had failed initial cytokine therapy [70,71]. A recent randomized multicenter Phase III clinical trial of sunitinib vs. interferon as first-line treatment for metastatic RCC revealed a response rate of 37% vs. 9% with progression free survival of 11 vs. 5 months and improved quality of life [72]. Initially developed as an RAF-1 inhibitor, sorafenib (BAY43-9006), also inhibits VEGFR-2, VEGFR-3, FLT-3, c-KIT, and PDGFR, and has shown promising activity and a good safety profile in RCC [73]. A Phase II “randomized discontinuation” trial of sorafenib vs. placebo showed a progression free survival of 24 weeks vs. 6 weeks [74]. In a subsequent Phase III trial, sorafenib produced definitive responses in 10% of patients, and stabilized disease in an additional 74%. There was a significant improvement in progression-free survival vs. placebo (median 5.5 vs. 2.8 months) [75]. These encouraging early results and acceptable side effect profiles formed the basis for recent FDA approval of both Sunitinib and sorafenib. Other VEGFR inhibitors are currently under investigation. Axitinib (AG013736) inhibits VEGFR-1, VEGFR-2, PDGFR, and c-KIT. Early results have shown promising activity and a good tolerance [76]. Pazopanib (GW786034) targets VEGFR-1, VEGFR-2, VEGFR-3, PDGFR␣, PDGFR, and c-kit [77]. It recently completed Phase I testing and is currently being evaluated in both European and U.S. trials. Platelet-derived growth factor (PDGF)-B promotes vascular pericytes and maintenance of established vasculature [78]. Preclinical data suggest that involution of blood vessels may require dual inhibition of VEGF and PDGF [79,80]. Many of the TKIs described above also demonstrate activity against the PDGF receptor (PDGFR), re-
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flecting their structural similarities. Imatinib mesylate (Gleevec), an inhibitor of c-Abl and c-Kit used to treat chronic myeloid leukemia and gastrointestinal stromal tumors, is also a potent inhibitor of PDGFR. Although initial studies reported minimal single-agent activity in advanced RCC, combination trials of imatinib with VEGFR inhibitors are underway. TGF-␣ is another HIF-responsive growth factor, which promotes proliferation by activating epidermal growth factor receptor (EGFR) expressed by RCC [81]. EGFR inhibition abrogates RCC tumor growth in xenografts [82,83]. Small molecule inhibitors (erlotinib and gefitinib), and monoclonal antibodies (cetuximab) targeting EGFR are now FDA-approved for other indications. Unfortunately, they have thus far displayed limited single-agent activity against advanced RCC [84,85]. Lapatinib, an EGFR/Erb1 tyrosine kinase inhibitor, showed no overall survival benefit as second-line therapy following cytokine failure, but did benefit a subset of patients with tumor overexpression of EGFR [86].
Combination therapy Combining agents that target different points in the VHL-HIF pathway may enhance therapeutic efficacy. A Phase II trial combining bevacizumab and erlotinib in patients with metastatic RCC reported a 25% overall response rate in 59 patients who had failed prior cytokine therapy [87]. This led to a randomized Phase II trial comparing bevacizumab plus erlotinib vs. bevacizumab plus placebo as first line therapy for metastatic RCC that unfortunately demonstrated no discernible difference in response rate or progression free survival [88]. Studies to evaluate the safety of combination of small molecule inhibitors with monoclonal antibodies or inhibitors targeting different pathways of angiogenesis or proliferation are underway. Until clinical trials demonstrate conclusive evidence that combination therapy is both safe and effective, combinations should be avoided.
Future targets Many other HIF target genes are currently under investigation as potential therapy targets. Cyclin D1 and its catalytic partner cyclin-dependent kinase 4 (cdk4) promote cell proliferation via phosphorylation of RB1 [38]. Flavopiridol, a cdk inhibitor, showed an overall response rate of 12% and stable disease rate of 41% in a Phase II study of 34 patients with advanced RCC. Unfortunately, this was associated with significant drug-related toxicity [89]. Carbonic anhydrase IX (CAIX) is another HIF transcription target upregulated in RCC [90]. In conjunction with low dose IL-2, direct targeting of CAIX with G250, a chimeric anti-CAIX monoclonal antibody, has shown promising results in early
clinical trials [91]. There is evidence that the tyrosine kinase receptor c-Met and its ligand, hepatocyte growth factor, are induced by HIF [92,93]. Activating mutations of the c-MET proto-oncogene, characteristic of HPRC, have also been reported in a subset of sporadic clear cell tumors [94,95]. Inhibitors of c-MET are in development for both clear cell and papillary RCC. The small molecule, chemotin, disrupts binding of p300 to the CAD of HIF-␣. It has been shown to abrogate tumor growth in RCC xenografts [96]. Inhibitors of HSP90 including geldanamycin reduce HIF-␣ protein expression and activity [97]. As noted, suppression of the Nox4 NADPH oxidase decreases HIF-␣ mRNA expression and activity and abrogates tumor growth in xenografts. These and other targets that disrupt HIF␣/HIF or HIFcoactivator interactions will provide new therapeutic targets in the treatment of RCC.
Conclusions Recent advances in the understanding of the role of HIF in RCC biology have had a dramatic impact on the development of novel targeted therapies in patients with advanced disease. Small molecule inhibitors (temsirolimus, sunitinib, and sorafenib), and monoclonal antibodies (bevacizumab) have demonstrated promising results in Phase II and Phase III clinical trials as both adjunct and primary therapies. The tyrosine kinase inhibitors sunitinib and sorafenib have been approved by the FDA and are currently being implemented in clinical practice. Combination therapy with and without adjuvant cytokine therapy is still under early investigation. Further definition of the VHLHIF pathway will continue to provide insight into RCC tumorigenesis and novel therapeutic targets for this complex patient population.
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Translational studies in urologic oncology
Clinical implications of hypoxia inducible factor in renal cell carcinoma Marc C. Smaldone, M.D., Jodi K. Maranchie, M.D., FACS* Department of Urology, University of Pittsburgh Medical Center and University of Pittsburgh Cancer Institute, Pittsburgh, PA 15232, USA
Abstract Management of renal cell carcinoma (RCC) has made considerable strides in the past decade, due in large part to identification of the von Hippel Lindau (VHL) tumor suppressor as a negative regulator of hypoxia inducible factor ␣ (HIF-␣) protein expression. Stabilization of HIF-␣ appears to be critical for renal tumorigenesis, and is observed even in VHL-independent RCC. Thus, an understanding of the pathways that regulate expression and activation of the different HIF-␣ isoforms is key to delineating the mechanism of renal transformation and for the development of novel therapeutics. A number of agents targeting HIF-␣ or its transcriptionally-regulated genes have shown promise in treatment of RCC. However, more effective treatment strategies are still needed. This report provides a directed review of recent discoveries defining the role of HIF in renal tumorigenesis and their relevance to the clinical advances in targeted therapy for advanced RCC. © 2009 Elsevier Inc. All rights reserved. Keywords: Renal cell carcinoma; HIF; VHL; VEGF; Therapy
Introduction The incidence of renal cell carcinoma (RCC) has increased steadily over the past several decades. An estimated 38,000 new cases of RCC were diagnosed in 2006, with greater than 12,000 expected deaths from the disease [1]. This rise has been attributed to the widespread use of noninvasive abdominal imaging procedures for improved detection [2]. However, despite a higher proportion of patients with localized disease at diagnosis, mortality has also risen steadily over the same time period [3], suggesting a fundamental shift in cancer biology. Although tumors localized to the kidney are potentially cured by surgical resection, onethird of patients present with advanced disease, and half of those remaining will ultimately relapse. These lesions are both radio- and chemo-resistant, and standard immunotherapies lead to complete response in fewer 15% of patients [4]. Clear cell RCC is well known for its intense vascularity and high expression of angiogenic factors. Insight into this biology came with the 2000 discovery that the von Hippel Lindau tumor suppressor gene (VHL), lost in greater than 75% of clear cell RCCs, functions as a negative regulator of hypoxia inducible factor-␣ (HIF-␣). However, HIF-␣ in-
* Corresponding author. Tel.: ⫹1-412-605-3019; fax: ⫹1-412-6053030. E-mail address: [email protected] (J.K. Maranchie). 1078-1439/09/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.urolonc.2007.12.001
duction is not limited to the subset of clear cell RCC with VHL loss, suggesting that it plays a fundamental role in renal transformation. Novel therapeutic agents targeting HIF-␣ or its transcription targets have demonstrated promising antitumor activity in clinical trials.
Hypoxia inducible factor 1 The heterodimer transcription factor HIF-1 was first identified in 1991 as a regulator of renal production of erythropoietin (Epo), the glycoprotein hormone that controls RBC production and maintains physiologic oxygen homeostasis. Deletion analysis of the 3= flanking region of Epo revealed the minimal essential sequence of 5=CTACGTGCT-3= [5] required for oxygen-dependent regulation. HIF-1 was subsequently purified from this hypoxiaresponse element (HRE), yielding two subunits, HIF-1␣ and HIF-1 [6]. The latter proved to be the aryl hydrocarbon receptor nuclear translocator (ARNT), which is constitutively expressed in all cell types [7]. In contrast, HIF-1␣ is tightly regulated at the protein level by oxygen-dependent ubiquitination followed by proteasomal degradation, now known to be mediated by VHL. Under physiologic oxygen conditions, HIF-1␣ protein is virtually undetectable. Hypoxia leads to abundant protein levels, nuclear translocation, and transactivation of target genes harboring the HRE sequence [8,9]. More than 100 HIF transcription targets have
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changes required for CBP/p300 binding [12,13]. FIH-1 is inhibited by low oxygen levels, iron sequestration, or heavy metals (cobalt), conditions known to promote HIF activation. Binding of CBP/p300 was also recently shown to be regulated by the mammalian target of rapamycin (mTOR) via binding of an mTOR-signaling motif (TOS) located in the N-terminus of HIF-1␣ [14].
VHL regulation of HIF-1␣
Fig. 1. Pathways and representative genes transcriptionally regulated by hypoxia inducible factors HIF1 and HIF2. Although significant overlap exists, array studies indicate differential induction by the two isoforms with apoptosis and gluconeogenesis preferentially induced by HIF-1 and angiogenesis preferentially induced by HIF-2. Single asterisks indicate genes specifically regulated by HIF-2 whereas double asterisks indicate those specifically regulated by HIF-1.
been described to date, involving a myriad of diverse pathways required for response to hypoxia or environmental stress, including erythropoiesis, angiogenesis, proliferation, apoptosis [10], and anaerobic metabolism (Fig. 1). The N-terminus of HIF-1␣ contains the sequences required for dimerization and DNA binding. The C-terminus harbors two distinct activation domains: the oxygen dependent degradation domain (ODD), and the carboxy-terminal activation domain (CAD) (Fig. 2). As its name implies, the ODD contains the residues required for oxygen-mediated degradation and can confer this property to other proteins when expressed as a fusion [11]. Stabilized protein, however, is not transcriptionally active until a second oxygendependent event occurs at the CAD to permit binding of co-factor CBP/p300, which promotes dimerization and nuclear translocation. Under normal oxygen conditions, an asparagine within the CAD is hydroxylated by factor inhibiting HIF (FIH-1), a 2-oxoglutarate-dependent oxygenase that requires oxygen, iron (Fe(II)) and 2-oxoglutarate as substrates. Hydroxylation prevents tertiary structural
Greater than 75% of clear cell RCCs harbor biallelic loss of the von Hippel Lindau (VHL) tumor suppressor gene [15]. These tumors are uniquely vascular and characterized by elevated expression of VEGF and glucose transporter-1 (Glut-1). By 1999, the multi-subunit von Hippel Lindau tumor suppressor complex, comprised of elongin B, elongin C, Cul2, and RBx1, had been characterized. Due to striking homology to the SCF (Skp1-Cdc53/Cul-1-F-box protein) complex in yeast, it was identified as an E3 ubiquitin ligase with VHL as the substrate recognition component [16]. The discovery of HIF-1␣ and HIF-2␣ as the first two confirmed targets of VHL-mediated degradation greatly advanced our understanding of renal tumorigenesis, and opened the door to novel molecularly targeted therapies [17]. VHL binds to the ODD under normal oxygen conditions, leading to polyubiquitination and subsequent degradation of HIF-␣. VHL binding requires hydroxylation of one of two proline residues within the ODD by a family of prolyl hydroxylases (PHD 1–3) [18]. Analogous to FIH-1, PHDs are dioxygenases, dependent upon molecular oxygen, 2-oxoglutarate, and iron and inhibited by hypoxia, iron chelation, or cobalt [19,20]. When oxygen levels are low, hydroxylation does not occur and VHL cannot bind HIF-␣, leading to stabilization and protein accumulation.
Alternate HIF-␣ isoforms A homologous HIF-2␣ subunit (the product of the EPAS-1 gene) with 48% sequence identity with HIF-1␣ was isolated from endothelial cells in 1997 [21]. Like HIF-1␣, HIF-2␣ is stabilized and activated by hypoxia and dimerizes
Fig. 2. Schematic of HIF-1␣ and HIF-2␣ indicating conserved activation and binding domains. Shaded boxes represent critical hydroxylation sites. ODD: Oxygen dependent degradation domain. CAD: Carboxy-terminal activation domain.
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with ARNT to form HIF-2. Further, HIF-2 activates transcription of target genes by binding the same HRE as HIF-1␣. However, HIF-1␣ and HIF-2␣ are not functionally redundant. Whereas HIF-1␣ homozygous inactivation is embryonic lethal due to lack of vascular formation and global hypoxia [22], embryos of HIF-2␣ double knockout mice demonstrate normal systemic vasculature but impaired lung maturation and catecholamine production [23]. Further, HIF-2␣ expression is more tightly controlled and undetected in most cells. It promotes an undifferentiated phenotype in pluripotent stem cells emphasizing the need for persistent silencing in many tissues after development [24]. HIF-2␣ is the predominant isoform, however, in developing kidney, small vessels of the CNS, adrenal medulla, lung and intestine. Interestingly, although mature normal kidney and early cystic lesions predominantly express the HIF-1␣ isoform [25] clear cell RCC demonstrates a shift toward expression of HIF-2␣ [26,27]. Conversely, expression of HIF3␣, described in 1998, is down-regulated with progression to cancer [28]. This isoform is highly abundant in heart, placenta, and skeletal muscle. Six different splice variants have been described, three of which are targeted by VHLmediated degradation [29]. HIF-3␣ resembles HIF-1␣ and -2␣ in the ODD, but lacks the CAD transactivation domain. It appears to function as a dominant negative regulator of hypoxia-inducible gene expression in the human kidney [30]. HIF-␣ as a renal oncogene Recent studies have shown that HIF-␣, particularly HIF2␣, plays an important role in the development in VHLdeficient RCC and is not solely a marker for disruption of the VHL pathway. Immunohistochemical examination of early kidney lesions from VHL patients show a coordinated loss of VHL and increase in HIF-1␣. Intriguingly, there is an apparent switch from HIF-1␣ accumulation to HIF-2␣ accumulation in such lesions coincident with increasing dysplasia [26]. While the relative contributions of HIF-1␣ and HIF-2␣ to the pathogenesis of the VHL phenotype have yet to be defined, these findings suggest that HIF-2␣ is more oncogenic than HIF-1␣ in the setting of VHL-defective RCC. The evidence supporting this theory was nicely summarized by Kim et al. in a recent review [31]. Briefly, human RCC lines express either both HIF-1␣ and HIF-2␣ or HIF-2␣ alone, suggesting that there may be a selection pressure to maintain HIF-2␣ expression or lose HIF-1␣ expression [17]. Second, HIF-2␣ variants lacking prolyl hydroxylation sites prevent tumor inhibition by VHL in animal models, whereas analogous HIF-1␣ mutants do not [32,33]. Third, down-regulation of HIF-2␣ with viral vector hairpin RNAs in human VHL–/– RCC cells is sufficient to prevent tumor formation in nude mice [34,35]. Finally, pathologic changes observed in mice engineered to lack VHL can be prevented by simultaneous deletion of HIF-1
[36]. Bias toward HIF-2␣ in renal transformation is perhaps in part due to the fact that HIF-1␣ preferentially induces pro-apoptotic pathways not targeted by HIF-2␣ [10]. Despite the fact that both target the same HRE, array studies demonstrate that expression of genes involved in the glycolytic pathway are driven preferentially by HIF-1␣ [37], whereas HIF-2␣ preferentially promotes growth and angiogenesis [38]. Consistent with this, although both HIFs equally activate exogenous reporter constructs containing the Epo HRE, HIF-2 preferentially binds the endogenous Epo promoter [39] suggesting the involvement of isoformspecific nuclear co-factors.
Nonhypoxic regulation of HIF There is a growing body of evidence that HIF-␣ subunits are alternatively activated by reactive oxygen species (ROS) under normal oxygen conditions [40]. Transcriptional induction and activation of HIF-␣ are seen in response to a wide array of inflammatory cytokines and growth factor stimuli, including TNF-␣ [41,42], angiotensin II [43], IL-1 [44], and thrombin [45]. We showed that in VHL-deficient RCC cells, where HIF-2␣ protein levels are abundant, normoxic transcriptional activity is critically dependent upon expression of the Nox4 NADPH oxidase, an endogenous generator of ROS found in greatest abundance in the distal renal tubules [46,47]. A role in tumorigenesis was confirmed by Nox inhibition in xenografts, demonstrating that NADPH oxidases promote tumor growth [48]. These studies suggest a role for Nox4 as a future target for treatment of RCC. HIF-␣ protein is also stabilized by binding to heat shock protein 90 (hsp90) via a mechanism that is independent of both oxygen and VHL. The specific hsp90 inhibitor geldanamycin leads to HIF-␣ degradation [49]. ARNT competes with hsp90 for binding of HIF-␣ and protects the protein from geldanamycin-mediated degradation [50]. This pathway is thought to play a role in adaptation to hypoxia by attenuating HIF-␣ activation.
HIF in non-clear cell RCC Although the HIF pathway was originally linked to VHL-deficient clear cell RCC, over-expression of HIF is documented in all renal histologic subtypes, including nascent renal tumors expected to have limited artifact from tumor ischemia. Increased expression of HIF-1␣ or HIF-2␣ was seen in 50% and 100% of chromophobe tumors and in 25% and 50% of hereditary type I papillary tumors (HPRC), respectively [27]. Loss of fumarate hydrogenase in hereditary leiomyomatosis and papillary RCC (HLPRC) leads to elevated fumarate, which directly stabilizes HIF-␣ by competitive inhibition of HIF prolyl hydroxylases [51]. HIF is also elevated in clear cell RCC of tuberous sclerosis where
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the VHL pathway is intact [52]. Although the impact of HIF in non-VHL tumorigenesis is less well defined, these studies hint at a fundamental role in renal transformation.
Direct targeting of HIF Over 40 inhibitors of HIF-1 activity have been identified, including inhibitors of mRNA expression, protein expression, DNA-binding activity, or HIF-1 mediated gene transcription [53], with varying degrees of overlap with HIF-2. HIF-␣ mRNA translation requires expression of mammalian target of rapamycin (mTOR) via a PI3 kinase-dependent pathway [54]. Inhibitors of mTOR (rapamycin, temsirolimus, everolimus) bind FK506 binding protein 12 (FKBP12). The resulting FKBP12/drug complex then binds mTOR, inducing G1 growth arrest. This prevents translational initiation of proteins including HIF-␣ [55]. Preclinical studies show that VHL loss sensitizes cells to mTOR inhibition in a HIF-dependent manner [56]. A Phase II study of single-agent temsirolimus (CCI-779) in cytokine refractory advanced RCC yielded a 7% objective response rate, and a 51% stable disease rate at 24 weeks [57]. In combination with interferon-␣, temsirolimus resulted in 11% partial response with median time to progression of 9 months [58]. A large (n ⫽ 626) Phase III, randomized study of first-line temsirolimus vs. IFN vs. temsirolimus plus IFN in poor-risk RCC revealed a survival benefit for temsirolimus alone (median survival 10.9 months) compared with IFN (7.3 months) or the combination (8.4 months) [59]. The survival benefit in this trial was greatest in patients with poorest risk features and with non-clear cell histology. Everolimus (RAD-001) is an orally administered mTOR inhibitor that has demonstrated antitumor activity as well as prolonged time to progression in a Phase II trial of heavily pretreated patients [60]. A randomized Phase III trial of everolimus vs. placebo is ongoing.
HIF-responsive growth factors and angiogenesis Many of the HIF-responsive genes described to date encode growth factor receptors or their ligands, including vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), and transforming growth factor-␣ (TGF-␣) [61]. Disruption of these pathways with neutralizing antibodies or small molecule inhibitors has shown promise in clinical trials. VEGF stimulates endothelial cell proliferation and survival and suppresses the antitumor immune response [62,63]. Furthermore, the VEGF tyrosine kinase receptor is expressed by RCC cells, suggesting the possibility of an autocrine feedback loop in addition to paracrine effects of VEGF on endothelial cells [64]. Multiple VEGF isoforms have been described. VEGF-A is involved in angiogenesis, while VEGF-C and VEGF-D have been linked to lym-
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phangiogenesis [65]. Bevacizumab (Avastin) is a humanized monoclonal antibody that binds and neutralizes all the major isoforms of VEGF. In a randomized Phase II trial, bevacizumab led to a significant delay in time-to-progression relative to placebo (4.8 vs. 2.5 months) in patients with advanced RCC who had failed prior high dose IL-2 [66]. Two randomized, Phase III trials comparing interferon vs. interferon plus bevacizumab have been completed [67] and will help determine the role of bevacizumab as a primary treatment modality. Early analysis of one study shows a response rate of 31% for the combination relative to 13% for interferon alone, with a corresponding benefit in progression free survival of 10.2 vs. 5.4 months [68]. Neutralization of serum VEGF with a fusion of the VEGF receptor and human immunoglobulin (the VEGF-trap) showed minimal response in an early Phase I trial of 15 patients with advanced solid malignancies. However, one subject with metastatic RCC maintained stable disease for greater than 6 months [69], and Phase II trials for advanced RCC are planned. Tyrosine kinase inhibitors also demonstrate single-agent activity in advanced RCC. Sunitinib (SU11248) inhibits the receptor tyrosine kinases VEGFR-2, PDGFR, FMS-like tyrosine kinase 3 (FLT-3), and c-KIT. Initial trials demonstrated sunitinib’s utility as a second-line agent for patients with metastatic RCC who had failed initial cytokine therapy [70,71]. A recent randomized multicenter Phase III clinical trial of sunitinib vs. interferon as first-line treatment for metastatic RCC revealed a response rate of 37% vs. 9% with progression free survival of 11 vs. 5 months and improved quality of life [72]. Initially developed as an RAF-1 inhibitor, sorafenib (BAY43-9006), also inhibits VEGFR-2, VEGFR-3, FLT-3, c-KIT, and PDGFR, and has shown promising activity and a good safety profile in RCC [73]. A Phase II “randomized discontinuation” trial of sorafenib vs. placebo showed a progression free survival of 24 weeks vs. 6 weeks [74]. In a subsequent Phase III trial, sorafenib produced definitive responses in 10% of patients, and stabilized disease in an additional 74%. There was a significant improvement in progression-free survival vs. placebo (median 5.5 vs. 2.8 months) [75]. These encouraging early results and acceptable side effect profiles formed the basis for recent FDA approval of both Sunitinib and sorafenib. Other VEGFR inhibitors are currently under investigation. Axitinib (AG013736) inhibits VEGFR-1, VEGFR-2, PDGFR, and c-KIT. Early results have shown promising activity and a good tolerance [76]. Pazopanib (GW786034) targets VEGFR-1, VEGFR-2, VEGFR-3, PDGFR␣, PDGFR, and c-kit [77]. It recently completed Phase I testing and is currently being evaluated in both European and U.S. trials. Platelet-derived growth factor (PDGF)-B promotes vascular pericytes and maintenance of established vasculature [78]. Preclinical data suggest that involution of blood vessels may require dual inhibition of VEGF and PDGF [79,80]. Many of the TKIs described above also demonstrate activity against the PDGF receptor (PDGFR), re-
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flecting their structural similarities. Imatinib mesylate (Gleevec), an inhibitor of c-Abl and c-Kit used to treat chronic myeloid leukemia and gastrointestinal stromal tumors, is also a potent inhibitor of PDGFR. Although initial studies reported minimal single-agent activity in advanced RCC, combination trials of imatinib with VEGFR inhibitors are underway. TGF-␣ is another HIF-responsive growth factor, which promotes proliferation by activating epidermal growth factor receptor (EGFR) expressed by RCC [81]. EGFR inhibition abrogates RCC tumor growth in xenografts [82,83]. Small molecule inhibitors (erlotinib and gefitinib), and monoclonal antibodies (cetuximab) targeting EGFR are now FDA-approved for other indications. Unfortunately, they have thus far displayed limited single-agent activity against advanced RCC [84,85]. Lapatinib, an EGFR/Erb1 tyrosine kinase inhibitor, showed no overall survival benefit as second-line therapy following cytokine failure, but did benefit a subset of patients with tumor overexpression of EGFR [86].
Combination therapy Combining agents that target different points in the VHL-HIF pathway may enhance therapeutic efficacy. A Phase II trial combining bevacizumab and erlotinib in patients with metastatic RCC reported a 25% overall response rate in 59 patients who had failed prior cytokine therapy [87]. This led to a randomized Phase II trial comparing bevacizumab plus erlotinib vs. bevacizumab plus placebo as first line therapy for metastatic RCC that unfortunately demonstrated no discernible difference in response rate or progression free survival [88]. Studies to evaluate the safety of combination of small molecule inhibitors with monoclonal antibodies or inhibitors targeting different pathways of angiogenesis or proliferation are underway. Until clinical trials demonstrate conclusive evidence that combination therapy is both safe and effective, combinations should be avoided.
Future targets Many other HIF target genes are currently under investigation as potential therapy targets. Cyclin D1 and its catalytic partner cyclin-dependent kinase 4 (cdk4) promote cell proliferation via phosphorylation of RB1 [38]. Flavopiridol, a cdk inhibitor, showed an overall response rate of 12% and stable disease rate of 41% in a Phase II study of 34 patients with advanced RCC. Unfortunately, this was associated with significant drug-related toxicity [89]. Carbonic anhydrase IX (CAIX) is another HIF transcription target upregulated in RCC [90]. In conjunction with low dose IL-2, direct targeting of CAIX with G250, a chimeric anti-CAIX monoclonal antibody, has shown promising results in early
clinical trials [91]. There is evidence that the tyrosine kinase receptor c-Met and its ligand, hepatocyte growth factor, are induced by HIF [92,93]. Activating mutations of the c-MET proto-oncogene, characteristic of HPRC, have also been reported in a subset of sporadic clear cell tumors [94,95]. Inhibitors of c-MET are in development for both clear cell and papillary RCC. The small molecule, chemotin, disrupts binding of p300 to the CAD of HIF-␣. It has been shown to abrogate tumor growth in RCC xenografts [96]. Inhibitors of HSP90 including geldanamycin reduce HIF-␣ protein expression and activity [97]. As noted, suppression of the Nox4 NADPH oxidase decreases HIF-␣ mRNA expression and activity and abrogates tumor growth in xenografts. These and other targets that disrupt HIF␣/HIF or HIFcoactivator interactions will provide new therapeutic targets in the treatment of RCC.
Conclusions Recent advances in the understanding of the role of HIF in RCC biology have had a dramatic impact on the development of novel targeted therapies in patients with advanced disease. Small molecule inhibitors (temsirolimus, sunitinib, and sorafenib), and monoclonal antibodies (bevacizumab) have demonstrated promising results in Phase II and Phase III clinical trials as both adjunct and primary therapies. The tyrosine kinase inhibitors sunitinib and sorafenib have been approved by the FDA and are currently being implemented in clinical practice. Combination therapy with and without adjuvant cytokine therapy is still under early investigation. Further definition of the VHLHIF pathway will continue to provide insight into RCC tumorigenesis and novel therapeutic targets for this complex patient population.
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