Hyperactivated JNK Is a Therapeutic Target in Pvhl-Deficient Renal Cell Carcinoma

Published OnlineFirst February 7, 2013; DOI: 10.1158/0008-5472.CAN-12-2362

Cancer Therapeutics, Targets, and Chemical Biology Research

Hyperactivated JNK Is a Therapeutic Target in pVHL-Deﬁcient Renal Cell Carcinoma

Jiabin An1,3, Huiren Liu1,3, Clara E. Magyar4, Yanchuan Guo7, Mysore S. Veena2,5, Eri S. Srivatsan2,5, Jiaoti Huang4, and Matthew B. Rettig1,3,6

Abstract Clear cell renal cell carcinomas (RCC), the major histologic subtype of RCC accounting for more than 80% of cases, are typiﬁed by biallelic inactivation of the von Hippel–Lindau (VHL) tumor suppressor gene. Although accumulation of hypoxia-inducible factor alpha (HIF-a) is the most well-studied effect of VHL inactivation, direct inhibition of HIFa or restoration of wild-type pVHL protein expression has not proved readily feasible, given the limitations associated with pharmacologic targeting of transcription factors (i.e., HIF-a) and gene replacement therapy of tumor suppressor genes (i.e., VHL). Here, we have established that phosphorylated c-Jun, a substrate of

the c-Jun-NH2-kinase (JNK), is selectively activated in clear cell RCC patient specimens. Using multiple isogenic cell lines, we show that HIF-a–independent JNK hyperactivation is unique to the pVHL-deficient state. Importantly, pVHL-deficient RCCs are dependent upon JNK activity for in vitro and in vivo growth. A multistep signaling pathway that links pVHL loss to JNK activation involves the formation of a CARD9/BCL10/TRAF6 complex as a proximal signal to sequentially stimulate TAK1 (MAPKKK), MKK4 (MAPKK), and JNK (MAPK). JNK stimulates c-Jun phosphorylation, activation, and dimerization with c-Fos to form a transcriptionally competent AP1 complex that drives transcription of the Twist gene and induces epithelial–mesenchymal transition. Thus, JNK represents a novel molecular target that is selectively activated in and drives the growth of pVHL-deficient clear cell RCCs. These findings can serve as the preclinical foundation for directed efforts to characterize potent pharmacologic inhibitors of the JNK pathway for clinical translation. Cancer Res; 73(4); 1–12. 2012 AACR.

Introduction devoid of VHL gene alterations and express wild-type pVHL (2). The protein product of the VHL gene, pVHL, serves many Reexpression of pVHL suppresses tumor formation in pVHL- fi a functions, the most well-characterized of which relate to its de cient murine models (3), and HIF- expression is required fi role in the E3 ligase complex that polyubiquitinates the tran- for tumorigenesis in the context of pVHL de ciency (4, 5). scription factor, hypoxia-inducible factor alpha (HIF-a), there- Thus, there is a preclinical rationale for drug development – a by marking it for proteasome-mediated degradation (1). In this aimed at inhibition of the pVHL HIF interaction. Unfortu- fi VHL capacity, pVHL plays a central role in mammalian cellular nately, ef cient and selective restoration of the gene in responses to ambient oxygen tension. Biallelic inactivation of tumor cells of actual patients is not achievable with current the VHL gene characterizes both hereditary and sporadic forms gene therapy technologies, and transcription factors such as a of clear cell renal cell carcinoma (RCC). Approximately 90% of HIF- are not readily amenable to drug development. There- – a clear cell RCCs manifest biallelic VHL inactivation through fore, despite the etiologic role of the pVHL HIF relationship genetic and epigenetic mechanisms, whereas nonclear cell in renal carcinogenesis, alternative strategies aimed at target- RCC variants such as papillary and chromophobe RCCs are ing more traditional "druggable" targets are required. In the case of clear cell RCC, proto-oncoproteins that are negatively regulated by pVHL may recapitulate the state of nononcogene addiction under the conditions of biallelic VHL fi 1 Authors' Af liations: Department of Medicine, Division of Hematology- inactivation. That is, the identification of biochemical signals, Oncology and 2Department of Surgery, VA Greater Los Angeles Healthcare System; Departments of 3Urology, 4Pathology and Laboratory Medicine, especially "druggable" kinases that are activated in response to 5Surgery, and 6Medicine, Division of Hematology-Oncology, David Geffen VHL loss in a HIF-a–independent fashion could provide alter- School of Medicine at UCLA, Los Angeles, California; and 7Technical Institute of Physics and Chemistry, Chinese Academy of Science, Beijing, native opportunities for therapeutic development. Indeed, China there is evidence for the existence of HIF-a–independent Note: Supplementary data for this article are available at Cancer Research effects of pVHL. For example, pVHL regulates an inhibitory Online (http://cancerres.aacrjournals.org/). phosphorylation of caspase recruitment domain 9 protein Corresponding Author: Matthew B. Rettig, UCLA VAGLAHS, 11301 (CARD9) by CK2 and downregulates activated PKC activity Wilshire Blvd, Bldg. 500, Room 4237, Los Angeles, CA, 90073. Phone: (6, 7). Important genotype–phenotype correlations in the 310-794-3565; Fax: 310-268-4508; E-mail: [email protected] subtypes of von Hippel–Lindau syndrome provide further doi: 10.1158/0008-5472.CAN-12-2362 evidence for HIF-a–independent effects of pVHL. In type 2C 2012 American Association for Cancer Research. von Hippel–Lindau syndrome, the associated VHL mutations

www.aacrjournals.org OF1

An et al.

do not lead to HIF-a dysregulation, yet patients develop stain. The accuracy of thresholding was verified by a licensed pheochromocytomas (8). Thus, biochemical and clinical cor- pathologist before analysis. For assessing nuclear staining, relative evidence points to the existence of HIF-a–independent positive 3,30-diaminobenzidine staining was calculated by effects of pVHL that are germane to oncogenesis. applying 2 color thresholds with one recognizing blue back- The c-Jun-NH2-kinase (JNK) is a mitogen-activated protein ground (hematoxylin stained) cells and another recognizing kinase (MAPK) required for Ras-induced transformation (9). It brown-positive cells and blue, nonpositive cells (total cell is protumorigenic in many tumor model systems. Its effects are number). Individual cells were discriminated by incorporating principally mediated through phosphorylation of members of the shape and size thresholds, providing, together with the the AP1 family of transcription factors, such as c-Jun (10). JNK color thresholds, actual cell counts. Percent of positivity was shares common upstream activators with components of the determined by dividing the cell number detected by the brown classical NF-kB pathway, which is constitutively activated in threshold by the total cell number, detected by the sum of the VHL-inactivated clear cell RCC in an HIF-a–independent brown and blue thresholds. Total tissue area analyzed was also manner. Accordingly, we investigated the potential for JNK to included in the final analysis. All investigations involving function in a nononcogene addiction fashion in the context of human specimens were carried out in accordance with the pVHL deficiency. principles of the Declaration of Helsinki.

Materials and Methods Cell growth assay Reagents The MTT assay was conducted as described (17). The isogenic pairs of ACHN, SN12C, 786-0, UOK121, and UMRC6 cell lines have been described (11, 12). The ACHN and Statistical analyses SN12C isogenic pairs were obtained from George Thomas Data are presented as mean SD. Significance was deter- (Oregon Health Sciences University, Portland, OR) and were mine with a 2-tailed Student t test. tested for pVHL and HIF-a expression by Western blotting (Supplementary Fig. S1). The 786-0 cell lines were obtained Results from W. Kaelin (Dana Farber Cancer Institute, Boston, MA); Activation of c-Jun in human clear cell RCC specimens UOK121 cells were from J. Gnarra (Louisiana State University, We first assessed the state of JNK/AP1 activation in the Baton Rouge, LA); and UMRC6 cells were from B. Zbar context of pVHL deficiency in human tissue by examining the (National Cancer Institute, Bethesda, MD). The 786-0, UOK121, extent of c-Jun phosphorylation by immunohistochemical and UMRC6 isogenic pairs of cell lines were originally tested for staining in clear cell versus nonclear cell RCC radical nephrec- pVHL and HIF-a expression by Western blotting as described tomy specimens. Here, we used a phospho-specific antibody earlier and are retested quarterly (12). The TRAF6-DN was used that detects c-Jun when phosphorylated at serine 73, a residue as described (13). Lentiviral plasmids designed to express that is a substrate for JNK phosphorylation and thus is marker target-specific or non-silencing (NS) short hairpin RNA (shRNA) for JNK activity (18–20). Quantitative assessment by image were obtained from Open Biosystems, as was the Twist lentiviral analysis confirmed that the percentage of cells exhibiting nu- plasmid. Transduction of a pVHL-resistant HIF-1a mutant clear p-c-Jun staining was statistically significantly greater (P ¼ (P402A;P564A) or HIF-2a mutant (P405A;P531A) expressed in 7.2 10 23) in clear cell RCCs than in the nonclear cell þ the pBabe retrovirus was used to express HIF-a in VHL cells counterparts (Fig. 1A and B and Supplementary Table S1). (14, 15). The pRL-SV40 Renilla luciferase reporter for normal- The marked difference in p-c-Jun staining was preserved when ization of transfection efficiency and the fireflyluciferase results for clear cell RCCs were compared with individual reporter (pGL4.24) for cloning segments of the Twist promoter histologic subtypes of nonclear cell renal neoplasms (Fig. 1B). were from Promega. The JNK inhibitor, SP600125, was from EMD Biosciences, and the TAK1 inhibitor, (5Z)-7-oxozeaenol, VHL Inactivation results in JNK/AP1 activation was from Tocris Bioscience. Wild-type and phospho-deficient We compared the activation state of the JNK/AP1 signaling myc-tagged CARD9 plasmids were from Addgene, deposited axis in pVHL-replete versus pVHL-deficient RCC cell models. by W. Kaelin, and have been described earlier (6). The HA- We employed 2 groups of isogenic pairs of RCC cell lines. In one ubiquitin, HA-ubiquitin-K63, and HA-ubiquitin K48 plasmids group of RCC cell lines (786-0, UOK121, and UMRC6), the were also from Addgene and deposited by Ted Dawson, Johns parental cell line manifests biallelic VHL gene inactivation Hopkins University, Baltimore, MD. (VHL ), and pVHL expression is restored in the isogenic þ counterparts (VHL ) by stable transduction of a retrovirus Immunohistochemistry and image analysis expressing the wild-type VHL gene. These isogenic pairs of RCC P-c-Jun immunohistochemistry was conducted as described cell lines, and their baseline expression levels of pVHL and HIF- (16). To quantitate p-c-Jun staining in human nephrectomy a have been widely reported (12, 15). In the second set (ACHN specimens, slides were analyzed using the Ariol SL-50 auto- and SN12C), the parental cell line expresses endogenous wild- þ mated slide scanner (Applied Imaging) for each area of interest. type VHL (VHL ). Isogenic partners that are pVHL-deficient Thresholds for each image were applied using the Ariol ana- (VHLlow) have been created by stable transduction of VHL- lytical software based on multiple parameters: RGB algorithm, specific shRNA, as described (Supplementary Fig. S1; ref. 14). þ shape, and size. All analyses were conducted with the Multi- Compared with VHL cells, all pVHL-deficient cell lines (5/5) Stain script. Thresholded classifiers were customized for each manifested heightened constitutive JNK/AP1 activity by

OF2 Cancer Res; 73(4) February 15, 2013 Cancer Research

JNK/AP1 Activation in pVHL-Deﬁcient RCCs

Figure 1. Activation of JNK/AP1 in pVHL-deficient RCC cells. A, increased AP1 activity as represented by nuclear phosphorylated c-Jun expression observed in clear cell RCCs. Representative hematoxylin and eosin (H&E) and phosphorylated c-Jun (p-c-Jun) stains of renal neoplasms at top and bottom, respectively. Arrowheads point to cells with nuclear p-c-Jun staining. B, box plot of percentage of cells exhibiting nuclear p-c-Jun in renal neoplasms. P values are comparisons to clear cell RCC subgroup. Whiskers are ranges; boxes are 25th to 75th percentiles; horizontal lines in boxes are medians. C, in vitro kinase assays of JNK activity. D, Western blots for indicated proteins. E, AP1-driven reporter gene activity. a, ACHN and SN12C cells. , P ¼ 0.00010; , P ¼ 0.019. b, 786-0, UOK121, and UMRC6 cells. , P < 0.005. Results are means of triplicates SD. F, EMSAs and EMSSAs. a, AP1 EMSA for ACHN and SN12C cells, with EMSSA shown in lanes 5 to 7 at the top. Oct-1 EMSA serves as a specificity control. wt, m ¼ excess cold wild-type, mutant probes, respectively. b, AP1 and Oct-1 EMSAs for 786-0 (lanes 1–2), UOK121 (lanes 3–4), and UMRC6 (lanes 5–9) cells. RLU, relative luminescence units. multiple complementary assays: JNK in vitro kinase assays, assays (EMSSA) established that heterodimers of c-Jun and expression of phosphorylated (Ser73) c-Jun (p-c-Jun), phospho- c-Fos, another AP1 family member, represent the components JNK expression, AP1-driven reporter gene activity, and binding of the AP1 complex that form in the setting of pVHL deficiency of nuclear extracts to a consensus AP1 response element (i.e., (Fig. 1F, a, lanes 5–7). an AP1 DNA binding site) in electrophoretic mobility shift We next investigated our isogenic cell models for potential assays (EMSA; Fig. 1C–F). Electrophoretic mobility supershift interactions between HIF-a and JNK/AP1 activity. Expression

www.aacrjournals.org Cancer Res; 73(4) February 15, 2013 OF3

An et al.

of functionally active pVHL-resistant mutants of HIF-2a in In vitro and in vivo growth of pVHL-deficient cells is þ þ 786-0-VHL and HIF-1a in ACHN-VHL cells did not induce dependent upon constitutive JNK activity JNK activation (Fig. 2A; refs. 14, 15). Moreover, HIF-1a or HIF- We next explored the biologic significance of JNK hyper- 2a suppression with HIF-a–specific siRNA did not reduce p-c- activation on in vitro and in vivo growth. The JNKi inhibited the þ Jun (Ser73) levels in pVHL-deficient cells (Fig. 2B). Conversely, growth of pVHL-deficient but not VHL cells in vitro in a dose- RNAi of c-Jun did not influence HIF-1a or HIF-2a expression dependent manner (Fig. 3A). ACHN-VHLlow xenografts, which (Fig. 2C). Pharmacologic inhibition of JNK with SP600125 exhibited heightened expression of phosphorylated c-Jun (Fig. þ (hereafter termed JNKi), did not affect endogenous HIF-1a or 3B, top), grew more rapidly than the VHL counterparts in HIF-2a expression, in pVHL-deficient UMRC6 or UOK121 and nude mice (Fig. 3B, bottom). Inhibition of JNK expression by 786-0 cells, respectively, but did reduce JNK activity and p-c-Jun transduction of lentivirus expressing JNK-specific shRNA levels (Fig. 2D and Supplementary Fig. S2A). These findings largely abrogated the heightened tumorigenesis observed in implicate pVHL-dependent, HIF-a–independent biochemical pVHL-deficient ACHN cells. In a similar fashion, the JNKi signaling events in the JNK/AP1 activation that occurs in the prevented the growth of pVHL-deficient ACHN cells (Fig. 3D). context of pVHL deficiency. Next, we confirmed the role of JNK in the tumorigenesis of In addition to the effects of the JNKi on p-c-Jun levels UOK-121 cells, which manifest endogenous VHL inactivation described above (Supplementary Fig. S2A), RNA interference and exhibit intrinsically more delayed and slower tumorigen- (RNAi) of JNK by transduction of a lentivirus expressing JNK- esis (3–5). Approximately 7 to 8 weeks after tumor cell inoc- specific shRNA also markedly reduced c-Jun Ser73 phosphor- ulation to allow for the formation of established tumors, mice ylation (Supplementary Fig. S2B), and the JNKi reduced AP1 harboring subcutaneous UOK121-VHL xenografts were trea- reporter activity in a dose-dependent fashion (Supplementary ted with the JNKi or vehicle control. Pharmacologic JNK Fig. S2C). These results confirm that JNK is the operative MAPK inhibition prevented the further growth and actually induced that regulates c-Jun and AP1 activity in our pVHL-deficient regression of established pVHL-deficient UOK121 xenografts models. (Fig. 3E).

Identification of MKK4 and TAK1 as the MAPKK and MAPKKK, respectively, that drive JNK activity in pVHL- deficient RCC cells JNK activation is regulated by the MAPKKs, MKK4, and/or MKK7 (21, 22). MKK7 activation status, as determined by phosphorylation of MKK7, was similar between pVHL-deficient and pVHL-expressing cell lines (Fig. 4A). In contrast, phospho-MKK4 expression was increased in pVHL-deficient cell lines (Fig. 4A). Inhibition of endogenous MKK4 activity by ectopic expression of a dominant negative MKK4 construct suppressed AP1 activity (Fig. 4B), a finding that implicates MKK4 as the essential MAPKK in this pathway. A major branch point in the upstream activation of the JNK signaling axis occurs at the level of the MAPKKKs, including MEKK1, ASK1, MLK, and others (10). We focused on TGF-b activating kinase-1 (TAK1), a MAPKKK that not only can function in the JNK pathway but also operates in the NF-kB pathway, which has been previously linked to VHL inactivation (6, 12, 15, 23). Indeed, elevated constitutive TAK1 activation, as measured by TAK1 in vitro kinase assays and phosphorylated TAK1 levels, was observed in all (5/5) pVHL-deficient cell lines (Fig. 4C and D). Exposure of pVHL-deficient cells to a pharmacologic TAK1 inhibitor (TAK1i), (5Z)-7-oxozeaenol, reduced pMKK4 levels (Fig. 4E), inhibited JNK activity (Fig. 4F), and reduced AP1 reporter activity (Fig. 4H). In addition, TAK1- specific shRNA inhibited JNK activity, (Fig. 4G). Taken togeth- ! ! ! a in vitro er, these results identify TAK1 MKK4 JNK c-Jun as a Figure 2. JNK/AP1 activation is HIF- independent. A, kinase VHL assays showing that expression of a pVHL-resistant HIF-1a or HIF-2a in MAPK signaling cascade that is activated in response to VHLþ cells does not induce JNK activity. B, siRNA-mediated silencing of inactivation. HIF-1a or HIF-2a in pVHL-deficient cells does not affect p-c-Jun expression. C, siRNA-mediated silencing of c-Jun in pVHL-deficient cells pVHL and CARD9-Dependent TRAF6 lysine 63 does not affect HIF-a expression. D, pharmacologic inhibition of JNK ! (4-hour drug exposure) inhibits phosphorylation of c-Jun in a dose- polyubiquitination drives TAK1 JNK activity dependent fashion but does not influence expression of HIF-1a or HIF-2a pVHL facilitates an inhibitory phosphorylation of the in pVHL-deficient cells. CARD9 by casein kinase 2 (CK2) in a HIFa-independent

OF4 Cancer Res; 73(4) February 15, 2013 Cancer Research

JNK/AP1 Activation in pVHL-Deﬁcient RCCs

Figure 3. JNK blockade inhibits the growth of pVHL-deficient RCCs. A, in vitro growth of pVHL-deficient but not pVHL-replete cells is inhibited by pharmacologic JNK blockade. Cells were exposed to the JNKi for 120 hours before assessing overall cell viability by MTT assay. Results were normalized to that of vehicle-treated controls and are means of 8 experiments SD. B, pVHL deficiency leads to rapid tumorigenesis that is associated with AP1 activation. After SN12C cells were inoculated subcutaneously into the flanks of nude mice (n ¼ 6/group), tumor volume was measured weekly. Results are averages SD. , P ¼ 0012. Top, Western blotting on protein extracts obtained from frozen xenografts harvested at the time of animal sacrifice. C, tumorigenesis of ACHN-VHLlow cells transduced with JNK-specific or NS shRNA (n ¼ 6/group). , P ¼ 0.0068; , P ¼ 1.8 10 5. Inset, JNK Western blotting on protein extracts obtained 48 hours after lentiviral transduction. Untransduced cells serve as a control for JNK expression. D, pharmacologic inhibition of JNK prevents tumor progression of ACHN-VHLlow xenografts. Once subcutaneous tumors reached a volume of approximately 50 mm3, animals (n ¼ 6/group) were treated with 25 mg/kg of the JNKi by i.p. injection every other day, and tumor volume was monitored. , P ¼ 0.0011. E, tumorigenesis of UOK121-VHL cells is markedly delayed by JNKi administration. When subcutaneous xenografts (n ¼ 6/group) became palpable, mice were treated with JNKi (12.5 mg/kg i.p. twice per week). P < 0.001. Results are means of tumor volumes SD. fashion, and, consequently, in the context of VHL loss, there ciated factors), that subsequently oligomerize and transauto- is constitutive activation of CARD9, an upstream trigger for NF- ubiquitinate. The latter process results in lysine 63 (K63)-linked kB activation (6). Upon activation, CARD9 typically forms a polyubiquitination, which, unlike K48 polyubiquitin linkages multiprotein complex with other proteins including BCL10 and that classically result in proteasome-dependent degradation, Malt1 (24), which in turn recruit TRAFs (TNF receptor–asso- is a proteasome-independent, activating event. Importantly,

www.aacrjournals.org Cancer Res; 73(4) February 15, 2013 OF5

An et al.

Figure 4. VHL inactivation results in activation of TAK1 and MKK4. A, Western blotting for P-MKK4, MKK4, P-MKK7, and MKK7. B, transient transfection of an MKK4- DN inhibits AP1 reporter activity in a dose-dependent fashion. Results are means of triplicates SD. C, TAK1 in vitro kinase assay shows the increased constitutive TAK1 activity in pVHL-deficient RCC cells. D, phosphorylated TAK1 (P-TAK1) is greater in pVHL-deficient cells. Total TAK1 was immunoprecipitated followed by immunoblotting for the indicated proteins. E, pharmacologic inhibition of TAK1 by the TAK1i (250 nmol/L for 4 hours) reduces the expression of P-MKK4. F, TAK1i (250 nmol/L for 4 hours) inhibits JNK activity measured in an in vitro kinase assay. G, TAK1-specific shRNA reduces JNK activity in pVHL-deficient cells. Top, Western blots for TAK1 in the presence of indicated shRNA. Bottom, JNK in vitro kinase assay. H, TAK1i (24- hour exposure) reduces AP1 reporter activity in a dose- dependent fashion.

þ activated TRAFs recruit and activate TAK1 (in complex with with VHL cells (Fig. 5D, top), yet BCL10 expression did not the adaptor proteins TAB1/2). Thus, we postulated that VHL vary in a pVHL-dependent fashion (Fig. 5D, bottom). inactivation is linked to TAK1 through disinhibition of CARD9 These biochemical findings suggested to us that mitigation followed by a CARD9- and TRAF-dependent activation and of the inhibitory phosphorylation of CARD9 by CK2 in the recruitment of TAK1. context of VHL inactivation, allows for the formation of a We initially evaluated the ubiquitination state of TRAF6, complex that includes CARD9, BCL10, and TRAF6, which which is known to function upstream of TAK1 (25). pVHL- serves as a proximal signal for TAK1 and ultimately JNK/AP1 deficient cells showed appreciably more constitutive TRAF6 activation. To directly test this notion, we transfected cells that ubiquitination (Fig. 5A); the TRAF6 polyubiquitination was endogenously express wild-type pVHL (ACHN and SN12C) mediated through K63 linkages (Fig. 5B). Transient transfec- with a wild-type myc-tagged CARD9 construct or a phos- tion of a TRAF6-dominant negative resulted in a dose-depen- pho-deficient mutant of the CARD9 construct in which all dent reduction in AP1-driven reporter gene expression in CK2 phosphorylation sites were mutated to alanine, and is thus pVHL-deficient cells, a finding that mechanistically links resistant to the CK2-dependent inhibitory phosphorylation. As TRAF6 to the JNK/AP1 pathway (Fig. 5C). predicted, the phospho-deficient CARD9 mutant more readily Next, we postulated that CARD9–TRAF6 interactions form coimmunoprecipitated with TRAF6 (Fig. 5E), indicating that more robustly in pVHL-deficient cells. Indeed, appreciably the phosphorylation of CARD9 by CK2 functions to inhibit the higher amounts of CARD9 coimmunoprecipitated with TRAF6 formation of a CARD9–TRAF6 complex. þ in all pVHL-deficient cells compared with their VHL counter- To determine whether the CARD9–TRAF6 interaction drives þ parts (Fig. 5D, top) despite the fact that VHL cells constitu- the activation of the JNK signaling axis in a pVHL-deficient tively express higher levels of CARD9 (Fig. 5D, bottom). TRAFs context, we conducted RNA interference of CARD9 and exam- deliver their downstream signals in the context of protein ined the downstream effects on TRAF6 K63 polyubiquitination complexes that contain not only CARD9 but also other mod- and TAK1 activation. CARD9 silencing led to a sharp reduction ulatory proteins, such as BCL10. In fact, in pVHL-deficient cells, in TRAF6 K63 polyubiquitination in pVHL-deficient cells (Fig. more BCL10 coimmunoprecipitated with TRAF6 compared 5F and G) and inhibited AP1 reporter activity as well as TAK1

OF6 Cancer Res; 73(4) February 15, 2013 Cancer Research

JNK/AP1 Activation in pVHL-Deﬁcient RCCs

Figure 5. VHL inactivation promotes CARD9/TRAF6 complex formation and TRAF6 K63 ubiquitination. A, increased TRAF6 ubiquitination in pVHL-deficient cells. B, increased TRAF6 ubiquitination occurs through K63 in pVHL-deficient cells. C, TRAF6-DN inhibits AP1 reporter activity. Results are means of triplicates SD. D, increased TRAF6 immunoprecipitation with CARD9 and BCL10 in pVHL-deficient cells. Bottom, Western blotting. Top, coimmunoprecipitation studies. E, mutagenesis of the CK2 phosphorylation sites within CARD9 promotes the interaction between CARD9 and TRAF6. Bottom, Western blotting shows equivalent expression of the wild-type and mutant CARD9 proteins. Top, coimmunoprecipitation studies. F, CARD9 siRNA reduces TRAF6 K63 ubiquitination. 786-0-VHL cells were transiently transfected with one of 2 CARD9 siRNAs. Top, Western blotting. Bottom, CARD9 siRNA reduces TRAF6 K63 polyubiquitination. G, Same as F but assay also conducted on additional cell lines with CARD9 siRNA (C9-1). H, CARD9 siRNA inhibits AP1 reporter activity. Results are means of triplicates SD. , P ¼ 0.00032; , P ¼ 0.0016; #, P ¼ 0.00025; ##, P ¼ 2.3 105; þ, P ¼ 1.7 105; þþ, P ¼ 3.7 105. I, schematic of biochemical pathway whereby pVHL deficiency leads to JNK/AP1 activation. www.aacrjournals.org Cancer Res; 73(4) February 15, 2013 OF7

An et al.

þ Figure 6. An EMT occurring in the context of VHL inactivation is dependent upon JNK/AP1 activation of Twist expression. A, morphology of VHL and VHLlow cells. Scale bar, 100 mmol/L. B, Western blotting shows a mesenchymal pattern of cadherin expression and increased Twist and Slug expression in pVHL-deficient cells. C, heightened invasiveness of pVHL-deficient cells in a Matrigel chamber invasion assay. Results are means of cells counts in 3 100 fields SD. Scale bar ¼ 100 mmol/L. , P ¼ 0.00024; , P ¼ 0.00058. Comparisons are VHLþ versus VHLlow cells. Importantly, cell proliferation of pVHL-deficient and VHLþ cells does not differ over the time course of the Matrigel invasion assay. D, Western blotting showing effects of c-Jun siRNA on expression of cadherins and indicated transcription factors in pVHL-deficient cells. E, same as D but JNK shRNA in lieu of c-Jun siRNA. F, dose-dependent effects of JNKi on expression of indicated proteins (Western blots). G, c-Jun siRNA reduces invasiveness of pVHL-deficient RCCs in Matrigel chamber assay. , P ¼ 0.00041; , P ¼ 0.00012. H, JNKi reduces invasiveness of pVHL-deficient cells.

kinase activity (Fig. 5H and Supplementary Fig. S3). In sum- JNK/AP1 Induces an EMT phenotype mediated by Twist mary, the results of our biochemical studies along with pre- We previously reported differences in cellular morphology þ viously published work indicate that pVHL deﬁciency results in between VHL and VHLlow ACHN and SN12C cells (14). þ the disinhibition of CARD9 (6), formation of a protein complex Whereas VHL cells grew in cellular clusters with individual amongst CARD9, BCL10, and TRAF6, and activation of TRAF6 cells taking on a polygonal shape, VHLlow cells often grew as through K63 polyubiquitination, which triggers the sequential single cells that manifested an elongated, ﬁbroblastic mor- activation of TAK1, MKK4, and JNK (Fig. 5I). phology consistent with an epithelial–mesenchymal transition

OF8 Cancer Res; 73(4) February 15, 2013 Cancer Research

JNK/AP1 Activation in pVHL-Deﬁcient RCCs

Figure 7. Twist expression induces JNK-dependent EMT. A, Twist siRNA inhibits invasiveness of pVHL-deficient ACHN and SN12C cells. a, Western blotting shows effective silencing of Twist. b, effect of Twist silencing on invasiveness. , P ¼ 0.0068; , P ¼ 0.0031; #, P ¼ 0.0011; ##, P ¼ 0.0012. c, light micrographs of cells that have invaded into Matrigel. Scale bar, 100 mmol/L. B, same as A but in 786-0-VHL cells. , P ¼ 0.00070; , P ¼ 0.00039. C, ectopic Twist (eTwist) expression restores invasiveness of JNKi-treated VHLlow ACHN and SN12C cells. Cells were transduced with lentiviral particles expressing eTwist or red fluorescent protein, and, 72 hours later, were analyzed in a Matrigel invasion assay with or without the JNKi (10 mmol/L for 24 hours). n.s., not statistically significant for comparisons of red fluorescent protein transduced þ vehicle treated to Twist transduced þ JNKi treated; P ¼ 0.37 and P ¼ 0.99 for ACHN and SN12C, respectively. D, Western blotting for indicated proteins show that eTwist expression prevents the induction of E-cadherin by JNKi and that eTwist-expressing cells exposed to the JNKi (10 mmol/L) have similar total Twist expression as red fluorescent protein-expressing, vehicle-treated cells. E, schematic figure of the Twist gene and promoter (modified from NCBI database). Approximately 2.5 kb upstream of the transcription start site was analyzed for putative AP1 binding sites with the aid of the M-Match search engine, and 5 PCR primers sets spanning predicted AP1 sites were generated for ChIP analysis (see Supplementary Table S2). F, ChIP analysis of Twist promoter. PCR products (Supplementary Table S2) from sets 1, 2, and 3 are shown.

www.aacrjournals.org Cancer Res; 73(4) February 15, 2013 OF9

An et al.

(EMT; Fig. 6A). As reported (14), VHLlow cells also manifested a indicate that the mesenchymal phenotype of pVHL-deficient "cadherin switch," whereby the characteristic pattern of cells is dependent upon JNK-induced Twist expression, increased E-cadherin and reduced N-cadherin expression typ- although JNK-independent pathways mediated by HIF-a have þ ified by epithelial cells and observed in VHL cells transitioned also been described and may be operative (26–28). to a mesenchymal pattern of heightened N-cadherin and suppressed E-cadherin expression (Fig. 6B and Supplementary c-Jun/c-Fos Heterodimers transactivate Twist through þ Fig. S4A). Moreover, compared with VHL cells, pVHL-defi- an upstream AP1 cis-acting element cient cells showed markedly heightened invasiveness in a We next investigated the transcriptional regulation of Twist Matrigel chamber, a finding that further supports the notion expression by AP1. A virtual analysis of the 2.5 kb region of EMT induced by pVHL deficiency (Fig. 6C and Supplemen- upstream of the Twist transcription start site identified numer- tary Fig. S4B) that has been described (26–28). Although these ous potential AP1 binding sites with varying degrees of homol- prior studies have implicated HIF-a as a pivotal factor in the ogy to the consensus AP1 sequence (Fig. 7E and Supplementary EMT observed in pVHL-deficient RCCs, we investigated the Table S2). Using chromatin immunoprecipitation (ChIP) potential for JNK/AP1 to function in a parallel pathway to experiments, we identified a 350 bp region (2,589–2,238 mediate EMT in this context. relative to the transcription start site), hereafter termed the RNAi with c-Jun–specific siRNA reversed the mesenchy- set 1 segment, that contains 4 predicted AP1 binding sites to malcadherinexpressionpatterninVHLlow cells (Fig. 6D). which c-Jun was recruited (Fig. 7F). On the basis of the EMSSAs Similar effects on cadherin expression were observed when shown in Fig. 1F, we predicted that c-Jun forms heterodimers JNK-specific shRNA was introduced (Fig. 6E). In pVHL- with c-Fos on the AP1 DNA binding sites of the Twist promoter. deficient cells, the JNKi induced a dose- and time-dependent Indeed, ChIP experiments confirmed that c-Fos was recruited þ reversion of the "cadherin switch" back to that of VHL cells to the set 1 region of the Twist promoter in a similar manner as (Fig. 6F and Supplementary Fig. S5). Similarly, the height- c-Jun (Fig. 7F). ened invasiveness of pVHL-deficient cells was reversed by To determine whether the set 1 region manifests functional theJNKiaswellasc-Jun–specific siRNA (Fig. 6G and H and activity, we cloned the set 1 region into a firefly luciferase Supplementary Fig. S6); the JNKi did not influence the reporter construct. Increased reporter gene activity driven by þ invasiveness of VHL cells (not shown). JNK inhibition did the set 1 segment of the Twist promoter was observed in þ not result in a reversion of the morphology of pVHL-defi- VHLlow compared with VHL cells (Fig. 7G). In contrast, a cient cells from a mesenchymal to epithelial phenotype, a similarly sized segment of the Twist promoter, which did finding that implicates JNK-independent effects on cellular not show AP1 binding in ChIP assays (set 3, Fig. 7E), had þ morphology. shown similar reporter gene activity in VHLlow and VHL cells We screened for differential expression of transcription (Fig. 7G). Differential reporter gene activity attributable to factors, including Twist, Slug, Snail, Zeb1, and Zeb2, which the set 1 segment was inhibited by exposure of VHLlow cells are known to regulate EMT and E-cadherin expression (29–31). to the JNKi (Fig. 7H). Taken together, our results indicate Twist and Slug expression was augmented in pVHL-deficient that an AP1 complex, composed of c-Jun/c-Fos heterodimers, þ cells compared with their VHL counterparts (Fig. 6B and binds to AP1 response elements in the regulatory region Supplementary Fig. S4), whereas the expression of Zeb1, Zeb2, upstream of the Twist promoter, thereby driving Twist protein and Snail was unaffected by VHL status (Fig. 6B and Supple- expression. mentary Fig. S4). RNAi of c-Jun sharply decreased the aug- mentation of Twist expression in VHLlow cells but had no effect Discussion on Slug or Snail expression (Fig. 6D). In the same manner, the We have provided several lines of evidence that pVHL JNKi reduced Twist, but not Slug or Snail expression, in both a deficiency causally results in JNK activation, whereby the dose- and time-dependent fashion (Fig. 6F and Supplementary inhibitory phosphorylation of CARD9 by CK2 is hindered by Fig. S5), as did lentiviral-mediated introduction of JNK-specific the inactivation of VHL (6), CARD9 forms a complex with shRNA (Fig. 6E). TRAF6 and BCL10, resulting in TRAF 6 activation followed in Suppression of Twist expression by Twist siRNA reduced the sequence by TAK1, MKK4, and JNK activation. Importantly, invasiveness of pVHL-deficient cells (Fig. 7A and B). Ectopic tumorigenesis of pVHL-deficient RCCs is dependent upon JNK, Twist expression driven by the AP1-independent CMV pro- which may represent a suitable biochemical target for drug moter in pVHL-deficient cells was sufficient to overcome the discovery efforts. The finding of frequent expression of nuclear inhibitory effects of the JNKi on invasion and E-cadherin p-c-Jun, a substrate of JNK, in clear cell RCCs further validates expression (Fig. 7C and D). Taken together, these findings JNK as a target for clinical translation.

PCR amplification of DNA not subjected to immunoprecipitation served as a positive control, and amplification of water was a negative control. G, increased reporter gene activity attributable to the set 1 segment in VHLlow compared with VHLþ cells. The regions from 2589 to 2238 and 782 to 442 representing the ChIP primer set 1 and set 3 amplification products were cloned into a basal promoter (i.e., TATA box) luciferase reporter, pGL4.24, for transient transfection studies. , P ¼ 7.6 105; , P ¼ 7.5 105;¶,P ¼ 0.23; and x, P ¼ 0.088 for comparisons of VHLþ to VHLlow cells. H, JNK-dependent transcriptional activity of the set 1 segment. The set 1 segment reporter was transiently transfected into VHLlow cells and exposed to the indicated concentrations of the JNKi for 24 hours starting the day after transfection. ¶, P ¼ 0.0032; ¶¶, P ¼ 0.0037; x, P ¼ 0.050; xx, P ¼ 0.0093 for comparisons to vehicle-treated cells. Results of C and D are averages SD.

OF10 Cancer Res; 73(4) February 15, 2013 Cancer Research

JNK/AP1 Activation in pVHL-Deﬁcient RCCs

We and others have observed that VHL inactivation can only been partially identified and elucidated but include muta- induce a mesenchymal phenotype (14, 26–28). In the process of tions in genes that regulate chromatin remodeling and struc- elucidating the molecular underpinnings that drive EMT in ture, accumulate and are required to initiate RCC (35, 36). pVHL-deficient RCCs, we found that JNK/AP1 induces Twist In summary, we have presented several lines of evidence that transcription. Specifically, AP1 complexes composed of c-Jun JNK/AP1 is constitutively activated in the pVHL-deficient and c-Fos heterodimers transcriptionally upregulate Twist state. pVHL defi ciency leads to dependence on the activity of expression by directly binding to and transactivating AP1 nonmutated JNK for proliferation and tumorigenesis in a state cis-acting elements upstream of the transcription start site of of nononcogene addiction. This dependence of clear cell RCCs the Twist gene. To our knowledge, JNK/AP1 has not been on the kinase activity of JNK can potentially be exploited for previously reported to regulate expression of Twist.Asa clinical translation. Thus, JNK/AP1 hyperactivation may rep- consequence of increased Twist expression, pVHL-deficient resent a nononcogene addiction target, and further optimiza- cells manifest reduced E-cadherin and increased N-cadherin tion of commercially available JNK inhibitors may constitute a expression as well as other characteristics of EMT, including viable endeavor for clinical application to the treatment of increased invasiveness and a fibroblastic morphology. clear cell RCC. Previous reports have found that EMT and reduced E- cadherin expression in pVHL-deficient cells occurs in an Disclosure of Potential Conflicts of Interest HIF-a–dependent manner (26–28). For example, one group No potential conflicts of interest were disclosed. found that HIF-1a induces Twist protein expression by binding to hypoxia response elements in the Twist promoter (32). Some Authors' Contributions fi a Conception and design: J. An, M. Veena, E. Srivatsan, M.B. Rettig reports speci cally indicted HIF-1 as the principal mediator Development of methodology: J. An, H. Liu, J. Huang, M.B. Rettig of EMT (28), others point to HIF-2a (27), whereas HIF-1a and Acquisition of data (provided animals, acquired and managed patients, a provided facilities, etc.): J. An, H. Liu, J. Huang, M.B. Rettig HIF-2 were found to function in this regard by yet others (26). Analysis and interpretation of data (e.g., statistical analysis, biostatistics, No clear explanation for these discrepancies can be readily computational analysis): J. An, H. Liu, C.E. Magyar, M. Veena, E. Srivatsan, J. discerned from the methodologies in these studies, although Huang, M.B. Rettig Administrative, technical, or material support (i.e., reporting or orga- some of the differential results may be partially accounted for nizing data, constructing databases): J. An, Y. Guo, M.B. Rettig by the use of different cell lines. Nonetheless, our results that Study supervision: J. An, J. Huang, M.B. Rettig EMT regulatory transcription factors and EMT itself are reg- Writing, review, and/or revision of the manuscript: C.E. Magyar, M.B. Rettig ulated in a JNK/AP1-dependent fashion are not mutually exclusive with HIF-a–dependent regulation of these same Acknowledgments The authors thank G. Thomas (Institute of Cancer Research, United King- processes. In point of fact, it seems that HIF-a as well as JNK dom) for the isogenic pairs of ACHN and SN12C cell lines, W. Kaelin (Dana Farber can both transcriptionally upregulate the expression of tran- Cancer Institute) for the 786-0 lines and the CARD9 plasmids, and B. Zbar (National Cancer Institute) for the UMRC6 and UOK121 pairs. The Vector Core scription factors that mediate EMT, which supports the notion (NIH grant 2P30DK041301) at the David Geffen School of Medicine prepared and that JNK and HIF-a function in parallel to coordinately drive titered lentiviral particles. RCC growth and tumorigenesis. On the basis of the finding of somatic VHL allele inactivation Grant Support observed in the premalignant renal cysts of patients with von This work was supported by a Merit Review grant (M.B. Rettig) from the – VHL Department of Veterans Affairs. Hippel Lindau syndrome, the biallelic inactivation of - is The costs of publication of this article were defrayed in part by the thought to represent an early mutational event in renal car- payment of page charges. This article must therefore be hereby marked advertisement cinogenesis (33, 34). Accordingly, we postulate that not only in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. constitutive HIF-a expression but also JNK activation repre- VHL sents an early molecular event that occurs in response to Received June 18, 2012; revised November 2, 2012; accepted November 15, 2012; inactivation. Additional acquired genetic lesions, which have published OnlineFirst February 7, 2013.

References 1. Kondo K, Kaelin WG Jr. The von Hippel–Lindau tumor suppressor ylation of the NF-kappaB agonist Card9 by CK2. Mol Cell 2007; gene. Exp Cell Res 2001;264:117–25. 28:15–27. 2. Nickerson ML, Jaeger E, Shi Y, Durocher JA, Mahurkar S, Zaridze D, 7. Razorenova OV, Finger EC, Colavitti R, Chernikova SB, Boiko AD, et al. Improved identiﬁcation of von Hippel–Lindau gene alterations in Chan CK, et al. VHL loss in renal cell carcinoma leads to upregulation of clear cell renal tumors. Clin Cancer Res 2008;14:4726–34. CUB domain-containing protein 1 to stimulate PKC{delta}-driven 3. Iliopoulos O, Kibel A, Gray S, Kaelin WG Jr. Tumour suppression by the migration. Proc Natl Acad Sci U S A 2011;108:1931–6. human von Hippel–Lindau gene product. Nat Med 1995;1:822–6. 8. Hoffman MA, Ohh M, Yang H, Klco JM, Ivan M, Kaelin WG Jr. von 4. Kondo K, Kim WY, Lechpammer M, Kaelin WG Jr. Inhibition of Hippel–Lindau protein mutants linked to type 2C VHL disease HIF2alpha is sufﬁcient to suppress pVHL-defective tumor growth. preserve the ability to downregulate HIF. Hum Mol Genet 2001; PLoS Biol 2003;1:E83. 10:1019–27. 5. Kondo K, Klco J, Nakamura E, Lechpammer M, Kaelin WG Jr. Inhibition 9. Kennedy NJ, Davis RJ. Role of JNK in tumor development. Cell Cycle of HIF is necessary for tumor suppression by the von Hippel–Lindau 2003;2:199–201. protein. Cancer Cell 2002;1:237–46. 10. Dunn C, Wiltshire C, MacLaren A, Gillespie DA. Molecular mechanism 6. YangH,MinamishimaYA,YanQ,SchlisioS,EbertBL,ZhangX, and biological functions of c-Jun N-terminal kinase signalling via the c- et al. pVHL acts as an adaptor to promote the inhibitory phosphor- Jun transcription factor. Cell Signal 2002;14:585–93.

www.aacrjournals.org Cancer Res; 73(4) February 15, 2013 OF11

An et al.

11. Thomas GV, Tran C, Mellinghoff IK, Welsbie DS, Chan E, Fueger B, cytotoxicity by suppressing the nuclear factor-kappaB-dependent et al. Hypoxia-inducible factor determines sensitivity to inhibitors of antiapoptotic pathway. Cancer Res 2003;63:7076–80. mTOR in kidney cancer. Nat Med 2006;12:122–7. 24. Hara H, Saito T. CARD9 versus CARMA1 in innate and adaptive 12. An J, Fisher M, Rettig MB. VHL expression in renal cell carcinoma immunity. Trends Immunol 2009;30:234–42. sensitizes to bortezomib (PS-341) through an NF-kappaB-dependent 25. Adhikari A, Xu M, Chen ZJ. Ubiquitin-mediated activation of TAK1 and mechanism. Oncogene 2005;24:1563–70. IKK. Oncogene 2007;26:3214–26. 13. An J, Mo D, Liu H, Veena MS, Srivatsan ES, Massoumi R, et al. 26. Esteban MA, Tran MG, Harten SK, Hill P, Castellanos MC, Chandra A, Inactivation of the CYLD deubiquitinase by HPV E6 mediates hypox- et al. Regulation of E-cadherin expression by VHL and hypoxia-induc- ia-induced NF-kappaB activation. Cancer Cell 2008;14:394–407. ible factor. Cancer Res 2006;66:3567–75. 14. Pantuck AJ, An J, Liu H, Rettig MB. NF-kappaB-dependent plasticity 27. EvansAJ,RussellRC,RocheO,BurryTN,FishJE,ChowVW,etal. of the epithelial to mesenchymal transition induced by Von Hippel- VHL promotes E2 box-dependent E-cadherin transcription by HIF- Lindau inactivation in renal cell carcinomas. Cancer Res 2010;70: mediated regulation of SIP1 and snail. Mol Cell Biol 2007;27: 752–61. 157–69. 15. An J, Rettig MB. Mechanism of von hippel-lindau protein-mediated 28. Krishnamachary B, Zagzag D, Nagasawa H, Rainey K, Okuyama H, suppression of nuclear factor kappa B (NF-kB) activity. Mol Cell Biol Baek JH, et al. Hypoxia-inducible factor-1-dependent repression of E- 2005;25:7546–56. cadherin in von Hippel–Lindau tumor suppressor-null renal cell carci- 16. Vivanco I, Palaskas N, Tran C, Finn SP, Getz G, Kennedy NJ, et al. noma mediated by TCF3, ZFHX1A, and ZFHX1B. Cancer Res Identification of the JNK signaling pathway as a functional target of the 2006;66:2725–31. tumor suppressor PTEN. Cancer Cell 2007;11:555–69. 29. Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal 17. An J, Sun Y, Fisher M, Rettig MB. Maximal apoptosis of renal cell transitions in development and disease. Cell 2009;139:871–90. carcinoma by the proteasome inhibitor bortezomib is nuclear factor- 30. Berx G, Raspe E, Christofori G, Thiery JP, Sleeman JP. Pre-EMTing kappaB dependent. Mol Cancer Ther 2004;3:727–36. metastasis? Recapitulation of morphogenetic processes in cancer. 18. Smeal T, Binetruy B, Mercola DA, Birrer M, Karin M. Oncogenic and Clin Exp Metastasis 2007;24:587–97. transcriptional cooperation with Ha-Ras requires phosphorylation of c- 31. Weinberg RA. Twisted epithelial-mesenchymal transition blocks Jun on serines 63 and 73. Nature 1991;354:494–6. senescence. Nat Cell Biol 2008;10:1021–3. 19. Smeal T, Binetruy B, Mercola D, Grover-Bardwick A, Heidecker G, 32. Yang MH, Wu MZ, Chiou SH, Chen PM, Chang SY, Liu CJ, et al. Direct Rapp UR, et al. Oncoprotein-mediated signalling cascade stimulates regulation of TWIST by HIF-1alpha promotes metastasis. Nat Cell Biol c-Jun activity by phosphorylation of serines 63 and 73. Mol Cell Biol 2008;10:295–305. 1992;12:3507–13. 33. Lubensky IA, Gnarra JR, Bertheau P, Walther MM, Linehan WM, 20. Pulverer BJ, Kyriakis JM, Avruch J, Nikolakaki E, Woodgett JR. Zhuang Z. Allelic deletions of the VHL gene detected in multiple Phosphorylation of c-Jun mediated by MAP kinases. Nature 1991; microscopic clear cell renal lesions in von Hippel–Lindau disease 353:670–4. patients. Am J Pathol 1996;149:2089–94. 21. Kishimoto H, Nakagawa K, Watanabe T, Kitagawa D, Momose H, Seo 34. Mandriota SJ, Turner KJ, Davies DR, Murray PG, Morgan NV, Sowter J, et al. Different properties of SEK1 and MKK7 in dual phosphorylation HM, et al. HIF activation identifies early lesions in VHL kidneys: of stress-induced activated protein kinase SAPK/JNK in embryonic evidence for site-specific tumor suppressor function in the nephron. stem cells. J Biol Chem 2003;278:16595–601. Cancer Cell 2002;1:459–68. 22. Wada T, Nakagawa K, Watanabe T, Nishitai G, Seo J, Kishimoto H, 35. Dalgliesh GL, Furge K, Greenman C, Chen L, Bignell G, Butler A, et al. et al. Impaired synergistic activation of stress-activated protein kinase Systematic sequencing of renal carcinoma reveals inactivation of SAPK/JNK in mouse embryonic stem cells lacking SEK1/MKK4: histone modifying genes. Nature 2010;463:360–3. different contribution of SEK2/MKK7 isoforms to the synergistic acti- 36. Varela I, Tarpey P, Raine K, Huang D, Ong CK, Stephens P, et al. vation. J Biol Chem 2001;276:30892–7. Exome sequencing identifies frequent mutation of the SWI/SNF 23. Qi H, Ohh M. The von Hippel–Lindau tumor suppressor protein sensi- complex gene PBRM1 in renal carcinoma. Nature 2011;469: tizes renal cell carcinoma cells to tumor necrosis factor-induced 539–42.

OF12 Cancer Res; 73(4) February 15, 2013 Cancer Research

Hyperactivated JNK Is a Therapeutic Target in pVHL-Deficient Renal Cell Carcinoma

Jiabin An, Huiren Liu, Clara E. Magyar, et al.

Cancer Res Published OnlineFirst February 7, 2013.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-12-2362

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2012/12/31/0008-5472.CAN-12-2362.DC1

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/early/2013/02/06/0008-5472.CAN-12-2362. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.