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p53 mutations are rarely detected in clear-cell renal cell carcinoma (CCRCC), but paradoxically these tumors remain highly resistant to chemotherapy and death receptor-induced death. Here, we show that the accumulation of HIF2α, a critical oncogenic event in CCRCC upon the loss of von Hippel-Lindau (VHL) tumor suppressor protein, leads to Hdm2-mediated suppression of p53. Primary CCRCC specimens exhibiting strong hypoxic signatures show increased levels of activated nuclear phospho-Hdm2(Ser166) that is concomitant with low p53 expression. The abrogation of Hdm2/p53 interaction using a small molecule Hdm2 inhibitor nutlin-3 or the downregulation of HIF2α via HIF2α-specific shRNA or wild-type VHL reconstitution restores p53 function and reverses the resistance of CCRCC cells to Fas-mediated and chemotherapy-induced cell death. These findings unveil a mechanistic link between HIF2α and p53 and provide a rationale for combining Hdm2 antagonists with chemotherapy for the treatment of CCRCC.
Renal cell carcinoma (RCC) accounts for approximately 3% of all malignancies and patients with metastatic RCC have a median survival of 13 months. The most common form (75%) of kidney cancer is of the clear-cell histology (CCRCC), which is highly aggressive and unresponsive to radiation or chemotherapy 1. Surgery by radical or partial nephrectomy is the most effective treatment option for localized disease. However, in one-third of patients tumors recur post-operatively as distant metastases, and only 4-6% of these tumors respond to chemotherapy. The standard non-surgical treatment for advanced CCRCC has been the administration of Interleukin-2 (IL-2). However, high-dose IL-2 regiment has a response rate of only 21% and causes significant toxicities 1. Recently, clinical trials of receptor tyrosine kinase (RTK) inhibitors, such as the VEGF receptor 2 (VEGFR2) and PDGF receptor β (PDGFRβ) inhibitors sorafenib (Nexavar) and sunitinib (Sutent) have yielded promising results in clinical trials by prolonging progression-free survival in approximately 70% of patients with metastatic CCRCC. However, neither drug has had a significant effect on overall patient survival 1, 2.
The efficacy of most chemotherapies is dependent on a successful execution of p53-mediated apoptosis to override the survival signals acquired by cancer cells 3, 4. Consequently, tumors harboring p53 mutations are associated with chemoresistance and in general, predict a considerably worse patient prognosis in comparison to malignancies with wild-type p53 5. Intriguingly, p53 mutations are infrequently detected in CCRCC 6, 7, but nevertheless these tumors are very resistant to chemotherapy. While there is no general consensus, several models have been proposed to explain the resistance of CCRCC to apoptosis, which may contribute to chemoresistance. For example, CCRCC cells devoid of VHL are resistant to death receptor TNFR-mediated cell death due, at least in part, to the increased activity of NFκB and downstream NFκB-mediated expression of anti-apoptotic proteins 8. Yang et al. showed that VHL acts as an adaptor molecule that binds and promotes the inhibitory phosphorylation of the NFκB agonist Card9 by casein kinase 2 in a hypoxia-inducible factor (HIF)-independent manner. Downregulation of Card9 in VHL−/− CCRCC normalized NFκB activity and sensitivity to cytokine-induced cell death, and attenuated the tumorigenic potential of CCRCC cells 9. The impact of the other major death receptor Fas-mediated signaling in CCRCC is unknown. There are limited and conflicting reports regarding the significance of p53 in CCRCC. In particular, Gurova et al. suggests that p53 is inactive via unknown dominant-negative mechanisms independent of Hdm2 7, 10, while Warburton et al. showed that p53 in several CCRCC cell lines can respond to ultraviolet radiation and is negatively regulated by Hdm2 10, 11. Furthermore, Hdm2 positivity was found significantly more frequently in CCRCC tumors of higher grade 12. The presence of a specific single nucleotide polymorphism in the Hdm2 promoter (SNP309), which results in elevated Hdm2 transcription and expression 13, has also been identified to be predictive of poor prognosis and survival in RCC 14. These findings suggest a possible oncogenic involvement of Hdm2 in CCRCC.
Approximately 80% of sporadic CCRCC arise due to the biallelic inactivation of the von Hippel-Lindau (VHL) tumor suppressor protein. In addition, individuals who inherit one faulty copy of VHL develop a rare multisystemic VHL cancer syndrome characterized by the development of retinal and cerebellar hemangioblastoma and pheochromocytoma, as well as CCRCC upon the loss of the remaining wild-type VHL allele in a susceptible cell. VHL is the substrate-specifying component of the multiprotein E3 ubiquitin ligase ECV (Elongins B and C/Cullin 2/VHL) that catalyzes the polyubiquitylation of prolyl-hydroxylated HIFα for subsequent destruction via the 26S proteasome. HIFα is hydroxylated on conserved proline residues by prolyl hydroxylase domain-containing enzymes (PHDs) in an oxygen-dependent manner. Under hypoxia, the unhydroxylated HIFα escapes recognition by VHL and thereby escapes ECV-mediated degradation. Stabilized HIFα associates with the constitutively stable partner HIFβ to form an active heterodimeric HIF transcription factor, which binds to hypoxia-responsive elements (HREs) located in the promoter/enhancer regions of numerous hypoxia-inducible genes to initiate the various adaptive responses to hypoxia, such as anaerobic metabolism, erythropoiesis and angiogenesis 15, 16.
Several lines of evidence have strengthened the notion that HIF2α stabilization is critical for CCRCC progression. For example, the inhibition of HIF2α, but not HIF1α, in CCRCC cells was sufficient to abolish the tumorigenic potential of CCRCC cells in a mouse xenograft assay 17, 18. Conversely, the stable expression of non-degradable HIF2α in VHL-reconstituted CCRCC cells overcame the tumor suppressive role of VHL 17. Moreover, a subset of CCRCC is caused by an inactivation of TSC1/2 tumor suppressor complex. The loss of function mutations in TSC1/2 result in increased translation of HIFα via mTOR-dependent and independent mechanisms 19. In the Eker rat renal tumor model, HIF2α was shown to be upregulated in RCC with a loss of TSC2 20. Recently, VHL-null CCRCC exclusively expressing HIF2α showed elevated c-Myc activity associated with enhanced proliferation and resistance to replication stress in comparison to CCRCC overexpressing both HIF1α and HIF2α 21. These observations suggest that while HIF1α antagonizes c-Myc, HIF2α promotes c-Myc activity associated with increased disease aggressiveness 21. Although these findings further support a critical role of HIF2α in the progression of CCRCC, the role of HIF2α in chemoresistance is unknown.
Here, we show that HIF2α suppresses p53 expression and function via Hdm2. We show in patient CCRCC samples, an increased accumulation of nuclear phospho-Hdm2(Ser166) and correspondingly negligible levels of p53, and provide evidence that HIF2α-dependent Hdm2-mediated suppression of p53 contributes to the resistance of CCRCC cells to Fas or chemotherapy-induced cell death. Importantly, CCRCC cells can be rendered highly sensitive to apoptotic stimuli by restoring p53 function via pharmacologic Hdm2 inhibitors. These findings provide the first mechanistic link between HIF2α and p53-dependent resistance to apoptosis in CCRCC, and support the notion that CCRCC can be successfully sensitized to conventional chemotherapy if combined with modalities designed to reactivate p53.
HEK293A embryonic kidney cells, MCF7 breast carcinoma cells and A498 (VHL−/−; HIF1α−/−) and 786-O (VHL−/−; HIF1α−/−) CCRCC cell lines were obtained from the American Type Culture Collection (Rockville, MD) and maintained in Dulbecco’s modified Eagle’s medium (DMEM)supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich, Oakville, ON)at 37°C in a humidified 5% CO2 atmosphere. 786-VHL(WT), 786-VHL(C162F) and 786-VHL(L188V) were previously described 22, 23. 786-VHL(WT) isogenic subclone harboring mutant p53(R248W) was described previously 24. RCC4-VHL(WT) and RCC4-MOCK were previously described 25. 786-O subclones stably expressingpRetroSUPER-empty (786-RetroEMPTY) or pRetroSUPER-HIF2α shRNA (786-RetroshHIF2α) were previouslydescribed 26 and generously provided by Dr. William G. Kaelin.
Monoclonal anti-HA antibody (12CA5) was obtained from Roche Molecular Biochemicals (Laval, PQ). Polyclonal anti-HA (Y-11) and anti-Hdm2 (SMP14) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-cleaved PARP, anti-PUMA, anti-phospho-Akt(Ser473), and anti-phospho-Hdm2(Ser166) antibodies were obtained from Cell Signaling Technology (Beverly, MA). Anti-HIF2α and anti-carbonic anhydrase IX (CAIX) antibodies were obtained from Novus Biologicals (Littleton, CO). Anti-HIF1α antibody was obtained from BD Biosciences (Mississauga, ON). Anti-Hdm2(Ab-1), anti-p53 (DO-1), anti-T7, and anti-Noxa antibodies were obtained from Calbiochem (San Diego, CA). Anti-Vinculin and anti-Fas (CH-11) antibodies were obtained from Upstate (Lake Placid, NY). Anti-β-Actin and anti-α-Tubulin antibodies were obtained from Sigma-Aldrich (Oakville, ON). Polyclonal anti-p53, anti-GLUT-1, anti-cleaved Caspase-3, and anti-hnRNP C1+C2 antibodies were obtained from Abcam (Cambridge, MA). Anti-Cul2 antibody was obtained from Zymed (San Francisco, CA).
LY294002 and nutlin-3 were obtained from Upstate (Lake Placid, NY) and Cedarlane Laboratories (Hornby, ON), respectively. Doxorubicin, etoposide, cisplatin, taxol, 5-fluorouracil, desferrioxamine (DFO) and neocarzinostatin (NCS) were obtained from Sigma-Aldrich (Oakville, ON).
Immunoprecipitation and Western blotting were performed as describedpreviously 27.
siRNA duplex targeted to p53 (5′-GUGAGCGCUUCGAGAUGUUUU-3′) (Dharmacon, Austin, TX) and a non-targeting scrambled siRNA duplex (5′-CCAUUCCGAUCCUGAUCCG-3′) (Dharmacon), as well as ON-TARGETplus SMARTpool siRNA targeted to HIF2α (Dharmacon) and siGENOME RISC-Free Control siRNA (Dharmacon) were transfected into indicated cell lines according to manufacturer’s instructions.
Subcellular fractionation was performed as previously described 28.
In vitro binding assay was performed as previously described 29
VHL was amplified from patient samples as previously described 30.
Formalin-fixed paraffin-embedded sections from 10 nephrectomy CCRCC specimens and matched normal tissue and TMA consisting of quadruplicate representative 1.0-mm needle-biopsy cores from 56 CCRCC were obtained from the files of The Department of Pathologyand Laboratory Medicine at The University Health Network (Toronto, ON). These samples were used and processed in accordancewith a University Health Network Research Ethics Board-approvedprotocol concerning gene expression in RCC, and analyzed as previously described 31.
The acquisition of mechanisms promoting resistance to apoptosis is a common feature of all transformed cells. The significance and deregulation of the Fas-mediated apoptotic pathway has been observed in several cancer types including ovarian 32 and prostate 33 cancers, as well as T cell leukemia 34. However, the status of Fas-mediated apoptosis in CCRCC is unknown.
786-O (VHL−/−; HIF1α−/−) CCRCC cells ectopically expressing empty plasmid (786-MOCK) or HA-VHL(WT) (786-VHL) were treated with the agonistic anti-Fas receptor antibody CH-11. 786-VHL(WT) cells were sensitive to CH-11 and showed high levels of cleaved Caspase-3 and 86kDa PARP cleavage product, as measures of apoptosis (Fig. 1a and Supplemental Fig. S1). In contrast, 786-MOCK cells exhibited very low levels of cleaved PARP and Caspase-3 (Fig. 1a and Supplemental Fig. S1). As expected, PARP cleavage inversely correlated with cell viability as measured by Trypan Blue exclusion assay (Fig. 1a, graph). Similar results were obtained using another CCRCC cell line system (RCC4-MOCK and RCC4-VHL) (Supplemental Fig. S2)
VHL promotes the destruction of HIFα via the ubiquitin-proteasome pathway. Thus, we asked whether the resistance to Fas-mediated apoptosis in CCRCC is HIF-dependent. 786-O cells stably reconstituted with wild-type VHL or disease-causing VHL mutants possessing varying abilities to degrade HIF2α were treated with CH-11 and monitored for cleaved PARP. Similar to 786-MOCK, 786-VHL(C162F) (high HIF2α) showed negligible PARP cleavage upon CH-11 treatment (Fig. 1b). However, in response to CH-11 treatment, 786-VHL(L188V) (low HIF2α) showed significant levels of cleaved PARP, comparable to levels observed in 786-VHL(WT) cells (Fig. 1b). In addition, CH-11 treatment of 786-O cells with stable integration of retrovirus encoding HIF2α-specific shRNA (786-RetroshHIF2α) showed significantly higher levels of cleaved PARP in otherwise CH-11-resistant parental 786-O or 786-RetroEMPTY cells (Fig. 1c and data not shown). Similarly, siRNA-mediated knockdown of HIF2α markedly increased the sensitivity of CCRCC cell lines A498 (VHL−/−; HIF1α−/−) and RCC4-MOCK (VHL−/−), the latter of which overexpresses both HIF1α and HIF2α, to CH-11-induced apoptosis (Fig. 1d). These results suggest that the stabilization of HIF2α contributes to the resistance of CCRCC to Fas-mediated apoptosis irrespective of HIF1α status. Consistent with this notion, ectopic expression of HIF2α likewise increased the resistance of MCF7 breast carcinoma cells to Fas-mediated apoptosis (Supplemental Fig. S3).
p53 influences Fas-mediated apoptosis 35, 36. We asked whether HIF2α-dependent sensitivity of CCRCC cells to Fas-mediated apoptosis is dependent on p53 by first determining the level of p53 and p53-target gene products in 786-MOCK and 786-VHL cells. 786-MOCK cells possess significantly reduced levels of p53 and p53-regulated gene products PUMA and Noxa in comparison to 786-O cells reconstituted with VHL (Fig. 2a), suggesting that the loss of VHL leads to the suppression of p53 expression and activity. Importantly, siRNA-mediated knockdown of endogenous wild-type p53 in 786-VHL(WT) cells resulted in diminution of PARP cleavage upon CH-11 treatment (Fig. 2b). In addition, 786-VHL isogenic subclone harboring a sporadic inactivating p53(R248W) mutation 24 exhibited considerable resistance to CH-11-induced apoptosis similar to 786-MOCK cells (Fig. 2c). These results suggest that VHL-mediated restoration of p53 activity is required for Fas-induced apoptosis.
Akt-mediated phosphorylation of the E3 ubiquitin ligase Hdm2 on Serine 166 or 186 leads to nuclear localization of Hdm2 and enhanced degradation of p53 37, 38. The activation of Akt is triggered upon Serine 473 phosphorylation, which represents a well-established survival signaling event downstream of growth factor receptors, many of which, including EGFR and PDGFR, have been shown to be hyperstimulated in response to HIF2α-mediated production of secreted growth factors 39–41. Thus, the overexpression of HIF2α upon the loss or inactivation of VHL could lead to the downregulation of p53 activity via Akt/Hdm2 pathway (see Fig. 3a, left panel). Consistent with this model, 786-MOCK cells that overexpress HIF2α showed elevated levels of phospho-Akt(Ser473) and phospho-Hdm2(Ser166) accompanied by a significant reduction in p53 level in comparison to 786-VHL(WT) cells (Fig. 3a, right panel). In keeping with the notion that Serine 166 phosphorylation promotes Hdm2 nuclear localization, 786-MOCK cells showed greater levels of nuclear Hdm2 than 786-VHL cells (Supplemental Fig. S4). To directly assess the contribution of Hdm2 in regulating p53 expression and function, 786-MOCK cells were treated with nutlin-3, a small molecule inhibitor of Hdm2-p53 interaction. Nutlin-3 treatment resulted in a marked increase in p53 levels and p53 transcriptional activity as detected by upregulation of the p53 target gene product PUMA (Fig. 3b). Nutlin-3 treatment likewise increased p53 expression in A498 cells (Supplemental Fig. S5). Furthermore, treatment of 786-MOCK cells with LY294002, a small molecule negative regulator of Akt 42, decreased the level of phospho-Akt(Ser473), as well as concomitant attenuation of phospho-Hdm2(Ser166) level, predictably resulting in increased p53 and PUMA levels (Fig. 3c, left panel). Notably, total Hdm2 levels also decreased in the presence of LY294002 (Fig. 3c), which is consistent with the notion that Akt-mediated phosphorylation of Hdm2 leads to increased Hdm2 stability 43. To determine whether hypoxia or HIF2α-dependent production of growth factors was responsible, at least in part, for the activation of Akt as evidenced by increased levels of phospho-Akt(Ser473) in 786-MOCK cells (see Fig. 3a, right panel), we maintained 786-VHL(WT) cells in various conditioned media and subsequently measured phospho-Akt(Ser473) levels (Fig. 3c, right panel). The treatment of 786-VHL(WT) cells with 786-MOCK (i.e., high HIF2α)-conditioned media as well as with conditioned media from 786-VHL(WT) cells grown under hypoxia (i.e., high HIF2α) resulted in increased levels of phospho-Akt(Ser473) (Fig. 3c, right panel). In contrast, treatment with conditioned media from 786-VHL(WT) cells grown under normoxia (i.e., low HIF2α) had comparably negligible effect on phospho-Akt(Ser473) level (Fig. 3c, right panel). These results support the notion that the activation of Akt in CCRCC cells devoid of VHL is driven, at least in part, by receptor-mediated signaling initiated by HIF2α-dependent secretion of growth factors. In addition, 786-RetroshHIF2α cells infected with retrovirus driving shRNA specifically targeting HIF2α showed significant reduction in nuclear Hdm2 levels, indicative of reduced phospho-Hdm2(Ser166) levels (Supplemental Fig. S6), and restoration of p53 expression in comparison to 786-RetroEMPTY cells (Fig. 3d). Similarly, siRNA-mediated HIF2α knockdown in A498 cells increased p53 levels (Supplemental Fig. S6). These results support a novel link between HIF2α and p53, in which the stabilization of HIF2α in CCRCC leads to the suppression of p53 activity via Akt/Hdm2 pathway.
High levels of intense membranous and positive cytoplasmic staining of a well-established HIF-target carbonic anhydrase (CA) IX was observed in CCRCC tumor cells from all 10 nephrectomy specimens, while normal renal cortex showed an absence of CAIX staining in the glomerulus and a moderate cytoplasmic staining in the adjacent proximal convoluted tubules (Fig. 4a). We next assessed phospho-Hdm2(Ser166) levels and showed that the normal renal cortex displayed negative nuclear staining of phospho-Hdm2(Ser166) in proximal convoluted tubular epithelium along with positive nuclear staining in scattered glomerular epithelial cells and mesangial cells (Fig. 4b, left panel). However, tumor cells in 9 out of 10 CCRCC nephrectomy samples exhibited intense nuclear phospho-Hdm2(Ser166) staining pattern (Fig. 4b, right panel), demonstrating for the first time an increased nuclear staining of the activated phospho-Hdm2(Ser166) in CCRCC. Normal renal cortex showed weak nuclear p53 staining in glomerular cells and tubular epithelium (Fig. 4c, left panel). Tumor cells in all 10 CCRCC nephrectomy samples showed negative nuclear p53 staining (Fig. 4c, top right panel and data not shown). Breast carcinoma specimen served as a positive control for anti-p53 (DO-1) antibody (Fig. 4c, bottom right panel).
Previous studies have shown that VHL status invariably correlates with HIF2α expression 21, 44. For example, Turner et al. showed that 94% of VHL-negative tumors overexpress HIF2α 21, 44, while Gordan et al. showed that 100% of VHL-negative tumors overexpress HIF2α 21, 44. Thus, VHL negativity implies HIF2α overexpression. Thus, we examined phospho-Hdm2 and p53 status in the context of VHL status on tissue microarray (TMA) consisting of 56 quadruplicate representatives of needle-core CCRCC biopsies (Fig. 4d). Consistent with the above nephrectomy specimens, the majority (82%) of CCRCC exhibited negative p53 staining and the majority (73%) of p53-negative tumors with known VHL status, as determined by IHC and direct DNA sequencing since subtle mutations can give ‘false-positive’ VHL immunostaining, was determined to be VHL-negative (Fig. 4d). Furthermore, the majority of phospho-Hdm2(Ser166)-positive samples showed negative VHL (69%) and p53 (63%) profile (Fig. 4d). These results collectively support the notion that p53 expression is influenced by VHL/HIF2α via Hdm2.
We asked whether the restoration of p53 expression or activity could sensitize 786-MOCK cells to CH-11-induced apoptosis. 786-MOCK cells, which are resistant to CH-11-induced apoptosis (Fig. 5a, lane 2; see Fig. 1 and and2),2), were sensitive to CH-11 treatment in combination with nutlin-3 as evidenced by a dramatic induction of cleaved PARP (Fig. 5a, lane 4). Notably, nutlin-3 treatment alone was not sufficient to cause death in CCRCC cells, as the apoptotic stimulus provided by CH-11 was required to execute the apoptotic process in the presence of restored p53 expression (Fig. 5a, compare lanes 3 and 4). These results demonstrate that the restoration of p53 activity can sensitize CCRCC devoid of VHL to apoptotic signals.
We therefore asked whether the HIF2α-dependent suppression of p53 contributes to the chemoresistance of CCRCC since the efficacy of most chemotherapies is highly dependent on p53-mediated apoptosis to override the survival signals acquired by cancer cells 3, 4. CCRCC is one of most resistant tumors to conventional chemotherapy 1. Consistent with this view, 786-MOCK cells compared to their wild-type VHL-reconstituted counterparts displayed considerable resistance to doxorubicin (Fig. 5b, left panel), a DNA-damaging agent currently used in the clinic for the treatment of various tumors such as breast and ovarian cancers 45, 46. If the chemoresistance of CCRCC is indeed attributed to the impairment of p53 function via HIF2α overexpression, a prediction is that the suppression of HIF2α or restoring the activity of p53 activity via nutlin-3 treatment would reverse the chemoresistance of 786-MOCK cells to doxorubicin-induced death. Suppression of HIF2α via HIF2α-specific shRNA caused de-repression of p53 expression via the inactivation of Hdm2 (see Fig. 3d) and as predicted, 786-RetroshHIF2α cells displayed increased sensitivity to doxorubicin-induced cell death (Fig. 5b, right panel). In accord, HIF2α knockdown enhanced p53 expression in A498 cells (Supplemental Fig. S7) and led to enhanced sensitivity to etoposide-induced apoptosis (Supplemental Fig. S8). 786-MOCK and A498 cells treated with nutlin-3 increased the levels of p53 and were dramatically sensitized to doxorubicin, as well as etoposide, induced cell death (Fig. 5c). Notably, such treatment strategy has been adopted in preclinical trials against a variety of cancers including leukemias, lung cancer, and neuroblastoma 47–49. These results demonstrate that a death-inducing stimulus (i.e., DNA-damaging agent or death receptor activation) is required in addition to the restoration of p53 activity, via nutlin-3 or HIF2α suppression, to promote the death of CCRCC cells.
Neocarzinostatin (NCS) is a DNA-damaging radiomimetic drug known to induce sequence-specific single and double strand breaks, as well as PI3K-dependent Hdm2 autoubiquitylation that results in Hdm2 destabilization and consequential accumulation of p53 50, 51. A prediction is that, unlike conventional DNA-damaging chemotherapeutic agents including cisplatin, taxol and 5-fluorouracil that are unable to destabilize Hdm2 or induce apoptosis in 786-MOCK cells (Supplemental Fig. S9), NCS treatment alone would induce 786-MOCK cell death since NCS induces both DNA damage and p53 expression via destabilizing Hdm2. Notably, similar low doses of cisplatin, taxol and 5-fluorouracil sufficiently killed several other tumor cell lines, including H1299 lung carcinoma, SW480 colorectal adenocarcinoma, HCT-116 colorectal, and SCC-9 head and neck squamous cell carcinoma cell lines (data not shown). As expected, 786-MOCK cells treated with NCS rapidly decreased Hdm2 levels as early as 1h post-treatment and sustained this effect up to 24h (Fig. 5d, left panel and Supplemental Fig. S10). Importantly, NCS treatment significantly increased p53 protein levels and PARP cleavage (Fig. 5d, right panel, lane 3), while CH-11 treatment alone resulted in insignificant induction of PARP cleavage (Fig. 5d, right panel, lane 2; see also Fig. 1, ,22 and and5a).5a). These results support the notion that CCRCC cells can be rendered highly sensitive to chemotherapy-induced cell death by first restoring p53 function.
Amongst urologic malignancies, CCRCC is the most lethal with greater than one third of the patients succumbing to this disease. Approximately one quarter of the patients present with metastatic disease and one third of patients who have been treated by surgery for clinically localized CCRCC eventually develop metastases. Patients with metastatic CCRCC have a median survival of 13 months with a chemotherapy response rate of only 4–6% 1.
Majority of human cancers have compromised p53-induced apoptotic pathway due to mutations in p53, which is tightly correlated with poor treatment response 5. CCRCC is arguably one of the most chemoresistant tumors despite rarely harboring p53 gene mutations 6, 7. This paradox suggests that p53 may be inactivated via non-mutational mechanisms. In support of this notion, Gurova et al. showed that despite normal p53 status, p53-dependent transactivation in CCRCC cells is repressed via unknown mechanisms 10. Galban et al. showed that the reintroduction of VHL in CCRCC cells results in p53 accumulation, in part by enhanced binding of RNA-stabilizing protein HuR to the 3′-untranslated region of p53 mRNA 52. Recently, Roe et al. reported that VHL stabilizes p53 via direct protein-protein interaction 53. However, p53 expression level and activity in VHL−/− CCRCC cells could be restored upon shRNA-mediated attenuation of HIF2α (see Fig. 3d), which argues that VHL-dependent upregulation of p53 is indirect via HIF2α. Moreover, we did not observe any appreciable binding between VHL and p53 (Supplementary Fig. S11).
Accumulation of HIF2α is regarded as a critical oncogenic event in CCRCC 17. Here, we show that HIF2α induces Akt-mediated phosphorylation of Hdm2(Ser166), which activates and promotes the nuclear localization of Hdm2 resulting in the downregulation of p53. In support, multiple patient-derived CCRCC tumor samples almost invariably exhibited both strong nuclear phospho-Hdm2(Ser166) and membranous expression of CAIX, a classical marker of hypoxia and a reliable indicator of HIF activity. Consistent with increased nuclear phospho-Hdm2(Ser166) expression, the level of nuclear p53 was negligible in virtually all CCRCC tumor samples tested, providing in vivo support for the link between aberrant HIF2 activity and inactivation of p53 in kidney cancer. These results provide a mechanistic explanation of why CCRCC cells are resistant to apoptosis triggered via Fas death receptor or DNA damage, and support a rationale for combining conventional chemotherapy drugs, such as doxorubicin or etoposide, with modalities that disarm Hdm2 such as nutlin-3 for the treatment of CCRCC.
We thank the Ohh lab for helpful discussions and T. Nadine Burry for her technical assistance. A.M.R. is a recipient of the Canadian Cancer Society Harold E. Johns Studentship Award. I.R.W. is a recipient of the Canadian Institutes of Health Research (CIHR) Canada Graduate Scholarship. M.S.I. and M.O. are Canada Research Chairs.
Financial support: This work was supported by funds from the Canadian Cancer Society (18460 to M.O. and 18054 to M.S.I.).