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VHL substrate transcription factor ZHX2 as an oncogenic driver in clear cell renal cell carcinoma

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Science  20 Jul 2018:
Vol. 361, Issue 6399, pp. 290-295
DOI: 10.1126/science.aap8411

Mechanistic insights into kidney cancer

Many clear cell renal cell carcinomas (ccRCCs) have alterations to the gene encoding the von Hippel-Lindau protein (VHL). VHL is a ubiquitin ligase that degrades target proteins when they are prolyl-hydroxylated. Zhang et al. performed a genome-wide search for VHL target (see the Perspective by Sanchez and Simon). They identified ZHX2, a protein with structural motifs that indicate DNA binding. ZHX2 has been implicated in tumor suppression. Loss of ZHX2 inhibited signaling through the transcription factor NF-κB, and ZHX2 bound to many NF-κB target genes. Depletion of ZHX2 slowed growth of ccRCC cells in vitro and in a mouse model.

Science, this issue p. 290; see also p. 226

Abstract

Inactivation of the von Hippel-Lindau (VHL) E3 ubiquitin ligase protein is a hallmark of clear cell renal cell carcinoma (ccRCC). Identifying how pathways affected by VHL loss contribute to ccRCC remains challenging. We used a genome-wide in vitro expression strategy to identify proteins that bind VHL when hydroxylated. Zinc fingers and homeoboxes 2 (ZHX2) was found as a VHL target, and its hydroxylation allowed VHL to regulate its protein stability. Tumor cells from ccRCC patients with VHL loss-of-function mutations usually had increased abundance and nuclear localization of ZHX2. Functionally, depletion of ZHX2 inhibited VHL-deficient ccRCC cell growth in vitro and in vivo. Mechanistically, integrated chromatin immunoprecipitation sequencing and microarray analysis showed that ZHX2 promoted nuclear factor κB activation. These studies reveal ZHX2 as a potential therapeutic target for ccRCC.

Clear cell renal cell carcinoma (ccRCC) makes up ~70% of all renal malignancies, and up to 92% of these cancers have inactivated the von Hippel-Lindau (VHL) gene (1, 2). Therapies that indirectly target the canonical VHL substrate hypoxia-inducible factor (HIF), such as vascular endothelial growth factor (VEGF) inhibitors, are the standard of care for ccRCC, but drug resistance occurs in most patients (3). Therefore, identification of additional VHL substrates could improve therapeutic options for ccRCC.

Prolyl hydroxylation of HIFα paralogs by Egl nine homolog (EglN) family proteins promotes their binding with the VHL complex (VBC, including VHL and elongin B and C), which leads to their ubiquitination and degradation (47). Other potential VHL targets might undergo similar prolyl hydroxylation. Therefore, hydroxylated (p-OH) but not nonhydroxylated HIF1α peptide should compete with potential VHL targets for binding with VBC. We validated this by incubating 35S-labeled HIF2α protein with glutathione S-transferase VBC (GST-VBC) in the presence of p-OH HIF1α peptide in a competition assay (fig. S1A). Next, a genome-wide human cDNA library was divided into approximately 700 pools with 24 cDNAs/pool (8), which were translated in vitro followed by binding assays with the GST-VBC in the presence of either unmodified or p-OH HIF1α peptide. Pools containing a potential binding partner were further analyzed so as to identify individual proteins (Fig. 1A). We mixed the HIF2α cDNA with a cDNA pool and found that even in the ratio of 33:1 (cDNA pool:HIF2α), HIF2α can be retrieved as a positive hit (fig. S1B). We discovered a pool that contained a protein whose binding to VBC was displaced by the p-OH HIF1α peptide and identified Zinc fingers and homeoboxes 2 (ZHX2) as the relevant protein in the pool (Fig. 1, B and C). Similar to HIF2α, the prolyl hydroxylase inhibitors dimethyloxalylglycine (DMOG), deferoxamine (DFO), or CoCl2 inhibited binding of ZHX2 to GST-VBC (Fig. 1D).

Fig. 1 ZHX2 is a VHL target, and its stability is regulated through prolyl hydroxylation.

(A) Schematic representation of VHL substrate screen. (B and C) Binding assays of 35S-methionine–labeled in vitro–translated (B) cDNA poolsor (C) ZHX2 and GST-VBC in the presence of wild-type (WT) or prolyl hydroxylated (p-OH) HIF peptide. (D) ZHX2/HIF2α binding to GST-VBC in the presence of prolyl hydroxylase inhibitors. (E to H) Immunoblots (IB) of whole-cell extracts (WCE) and immunoprecipitations (IP) of lysates from 786-O cells infected with lentivirus encoding either control vector (Ctrl) or hemagglutinin (HA)–tagged VHL and treated as indicated for 8 hours. (I) IB of lysates from UMRC2, UMRC6, or RCC4 cells transfected with indicated plasmids. (J and K) IB of WCE and IP of RCC4 cells transfected with indicated plasmids followed by (J) densitometry analysis of Flag-VHL or (K) ubiquitination assays in UMRC2 cells transfected with indicated plasmids.

ZHX2 was reported to be a tumor suppressor in hepatocellular carcinoma (HCC) and lymphoma (9, 10). Recently, mRNA levels of its related family members ZHX1 and ZHX3 were reported to associate with the pathological stage of ccRCC (11). The amount of ZHX2 protein, but not ZHX1 or -3, in VHL-deficient ccRCC cells decreased if VHL was reintroduced (fig. S1, C and D), and inhibition of prolyl hydroxylation or proteasomal degradation increased ZHX2 protein levels (Fig. 1, E and F, and fig. S1E). ZHX2 was predominantly localized in the nucleus (fig. S1F). Prolyl hydroxylation inhibition led to decreased binding of ZHX2 to VHL (Fig. 1G and fig. S1G). DMOG, DFO, proteasomal inhibitor MG132, and CRISPR/Cas9–mediated elimination of VHL increased the abundance of ZHX2 protein in VHL-proficient human kidney cells (fig. S1, H to J). Conversely, reintroduction of VHL into VHL-deficient ccRCC cells increased the ubiquitination and degradation of endogenous ZHX2 (Fig. 1H). Similar effects were observed with exogenous ZHX2 (Fig. 1I and fig. S1, K to M). Thus, ZHX2 is regulated by VHL through prolyl hydroxylation and proteasomal degradation. Next, we performed mass spectrometry and identified three ZHX2 prolyl hydroxylation sites: proline 427, 440, and 464 (fig. S2, A to E). We generated single proline-to-alanine mutants (P427A, P440A, and P464A) and a triple mutant that harbors three mutations (P3A). The single mutants, and especially the P3A mutant, exhibited decreased VHL binding, ubiquitination, and a concomitant increase of ZHX2 (Fig. 1, J and K, and fig. S2, F and G). The sensitivity of the single mutants to VHL was variable in different ccRCC cell lines (fig. S2, F and G). The importance of this is unclear but may reflect cell line–dependent differences on hydroxylating the remaining prolyl hydroxylation sites due to variable expression of relevant hydroxylase(s).

We obtained seven tumors from ccRCC patients and confirmed, by means of sequencing, VHL loss-of-function mutations important for HIFα regulation in all seven (table S1) (2, 1215), most of which contained greater amounts of ZHX2, HIF1α, and HIF2α than those of the paired normal tissues (Fig. 2A). For two tumors with VHL missense mutations (332 and 778), we did not observe distinctive up-regulation of ZHX2 compared with that of normals, possibly because such mutations are less critical for ZHX2 regulation. Normal kidney tissues contained variable amounts of ZHX2, HIF1α, and HIF2α, which could be due to tissue heterogeneity or some degree of tumor contamination. In some cases, protein levels of ZHX2 and HIFα did not correlate with one another, possibily because of distinct VHL-independent regulatory pathways. ZHX2, HIF1α, and HIF2α up-regulation were also found for another two pairs of ccRCC tumor tissues harboring VHL frameshift mutations (table S1), but not ccRCC tumors with intact VHL (Fig. 2B). Despite the lack of ZHX2 protein detected with Western blot, ZHX2 displayed cytoplasmic and apical membrane immunohistochemical staining patterns in normals, similar to HIF2α. This discrepancy remains to be resolved. On the other hand, ZHX2 was exclusively in the nucleus of tumors that harbor VHL frameshift mutations (Fig. 2C and fig. S3, A to C). These findings were corroborated by using ccRCC tissue microarray (Fig. 2, D and E, and table S2). Thus, VHL loss usually increases the abundance and nuclear levels of ZHX2 in ccRCC tumors.

Fig. 2 ZHX2 accumulation in ccRCC patients.

(A and B) IB of lysates from paired ccRCC patient nontumor (N) and tumor (T) tissues. (C) Representative ZHX2 immunohistochemistry staining for ccRCC patient tissues. (D and E) Representative hematoxylin and eosin (H&E), ZHX2 immunohistochemistry staining of tumor (T) and nontumor (N) tissues (D) and quantification of ZHX2 nuclear/cytoplasmic staining ratio (E) from ccRCC tissue microarray (TMA) slides. Error bars represent SEM (unpaired Student’s t test).

Depletion of ZHX2 in multiple VHL-deficient ccRCC cells with several independent short-hairpin RNAs (shRNAs) or single-guide RNAs (sgRNAs) decreased cell proliferation and growth in soft agar (Fig. 3, A to F, and figs. S4, A to G, and S5, A to H). These phenotypic defects were rescued by exogenously expressing shRNA-resistant or sgRNA-resistant ZHX2 cDNAs, respectively (Fig. 3, G to I, and figs. S4, H and I, and S5, I to M). These rescues were incomplete, however, possibly because the exogenous ZHX2 was incompletely localized to nuclei compared with endogenous ZHX2 (fig. S4J). In addition, ZHX2 depletion decreased orthotopic tumor growth (Fig. 3, J and K, and fig. S6A). To ask whether ZHX2 was required for established tumors, we introduced two doxycycline-inducible ZHX2 shRNAs into 786-O cells. Depletion of ZHX2 in the presence of doxycycline correlated with decreased cell proliferation in vitro (fig. S6, B and C). Next, 786-O cells expressing either ZHX2 shRNAs (45) cells or the control were injected into the renal capsules of immuno-deficient mice. Upon tumor formation, mice were fed doxycycline. Whereas cells expressing control shRNA grew readily after 6 weeks, cells expressing ZHX2 shRNA failed to proliferate, as determined with serial in vivo live tumor imaging and tumor-bearing kidney weights at necropsy (Fig. 3, L and M, and fig. S6, D and E).

Fig. 3 Requirement of ZHX2 for ccRCC cell proliferation, anchorage-independent growth, and tumorigenesis.

(A to F) IB of [(A) and (C)] cell lysates, [(B) and (D)] cell proliferation, and [(E) and (F)] soft agar growth of 786-O and UMRC2 cells infected with lentivirus encoding control (Ctrl) or ZHX2 shRNAs (43 and 45) (n = 3 replicates per group). Soft agar quantitation results are available in fig. S4, A and B. (G to I) IB of (G) cell lysates and (H) representative soft agar growth assays and (I) their quantification of UMRC2 cells transfected with ZHX2 sh45-resistant HA-ZHX2 or control (Ctrl) vector, followed by ZHX2 sh45 or control (Ctrl) shRNA infection (n = 3 replicates per group). (J to M) Representative bioluminescence imagings of (J) 1 and 7 weeks after implantation and (K) quantification of bioluminescence imaging from 786-O luciferase stable cells infected with either ZHX2 sh45 or control (Ctrl) shRNA, or (L) imagings of 0 week and 6 weeks post-doxycycline treatment and (M) quantification of imaging from 786-O luciferase stable cells infected with lentivirus encoding either Teton-ZHX2 sh45 or Teton-control (Teton-Ctrl) shRNA injected orthotopically into the renal subcapsule of nonobese diabetic (NOD) severe combined immunodeficient (SCID) γ (NSG) mice as indicated. The Mann-Whitney test was used to calculate the P values. Error bars represent SEM, ***P < 0.001 (unpaired Student’s t test) in (B), (D), and (I).

Next, we performed gene expression profiling of 786-O cells after ZHX2 knockdown followed by gene set enrichment analysis (GSEA) adjusted for gene function associated with oncogenic pathways. ZHX2 depletion caused decreased expression of multiple genes linked with anti-apoptosis, cell proliferation, invasion/metastasis, and metabolism (fig. S7, A to F). GSEA analyses also demonstrated that nuclear factor κB (NF-κB) activity was suppressed by means of ZHX2 depletion (fig. S8, A and B). Real-time polymerase chain reaction (RT-PCR) analysis confirmed that ZHX2 depletion decreased the expression of canonical NF-κB target genes, including c-c motif chemokine ligand 2 (CCL2), interleukin-8 (IL8), and IL6 (Fig. 4A). Generally, the more effective ZHX2 shRNA (sh45) suppressed the NF-κB–responsive mRNAs better. The CCL2 and IL8 mRNAs were, however, profoundly suppressed by both ZHX2 shRNAs, possibly because both shRNAs suppressed NF-κB below a threshold required for these two mRNAs (Fig. 4A).

Fig. 4 ZHX2 regulates NF-κB activation.

(A and B) Quantitative RT-PCR quantification of (A) mRNA of NF-κB target genes (n = 3 replicates per group) or (B) IB of cell fractions from 786-O cells infected with ZHX2 shRNAs (43 and 45) or Ctrl. (C) IB of WCE and IP of 786-O cells infected with either Ctrl or HA-VHL. (D) Venn diagram showing ChIP-seq binding peak overlap between ZHX2 and NF-κB–p65. ZHX2 ChIP-seq experiments were performed in duplicate and intersected. (E) ChIP-seq signal intensity in the 3 kb surrounding the midpoint of specific ZHX2 (green), specific NF-κB–p65 (yellow), and common (purple) sites. (F) Heatmap for genes down-regulated because of ZHX2 and p65 silencing (adjusted P < 0.05) are shown. (G) Heatmap for activated genes that were strongly bound by both ZHX2 and NF-κB–p65 and were significantly associated with ccRCC prognosis (adjusted P < 0.01). The log2 Cox Hazard Ratio was colored red (higher expression associated with poorer prognosis). (H) ChIP–quantitative PCR of NF-κB–p65 binding at IL6 and IKBKE promoters after silencing of indicated genes (n = 3 replicates per group). Error bars represent SEM, ***P < 0.001 (unpaired Student’s t test).

Loss of VHL constitutively activates the NF-κB pathway (1618). NF-κB activation is characterized by degradation of inhibitor of NF-κB (IκBα) and phosphorylation of RelA/p65, which then accumulates in the nucleus (1921). Depletion of ZHX2 had no major effect on IκB degradation or RelA/p65 phosphorylation but inhibited translocation of RelA/p65 into the nucleus (Fig. 4B and fig. S8, C and D). We detected binding of ZHX2 to RelA/p65 with endogenous and exogenous proteins (Fig. 4C and fig. S8, E and F). By contrast, we have thus far not detected binding of ZHX2 to other NF-κB subunits (fig. S8F). Inhibiting NF-κB with RelA/p65 shRNAs or with a specific IκB kinase (IKK) inhibitor compound A (CMPDA) suppressed VHL-deficient ccRCC cell proliferation and growth in soft agar (fig. S9, A to L) (22). We performed chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) to determine genome-wide chromatin occupancy of ZHX2 and RelA/p65, which revealed that 75% of p65 binding sites overlapped with those of ZHX2 (Fig. 4D and fig. S10, A and B). ChIP–quantitative PCR confirmed the binding by ZHX2 and p65 at the promoters of several genes (fig. S10, C and D). DNA sequences bound by both NF-κB–p65 and ZHX2 were enriched for the NF-κB consensus motif (fig. S10E). ZHX2 and RelA/p65 overlapping sites also displayed a strong enrichment for H3K4me3 and H3K27ac but not H3K4me1 (Fig. 4E) (23), indicating that ZHX2 and RelA/p65 bound to active gene promoters. ZHX2 and HIF2α positively regulated genes showed minimal overlap (fig. S11A and table S3), and Gene Ontology (GO) analysis showed that ZHX2 regulated distinct pathways, including NF-κB (fig. S11B). Integrated analyses of ZHX2 and NF-κB–p65 localization and gene expression showed 390 genes regulated by both ZHX2 and RelA/p65 positively (Fig. 4F and table S4), among which higher expression of 32 genes was associated with a worse prognosis for ccRCC patients (Fig. 4G and table S5). These 32 genes were further analyzed by means of hierarchical clustering analysis of The Cancer Genome Atalas (TCGA) RCC cases, which showed that 18 had high correlations with each other (fig. S12A). A metagene representing the median expression of these 18 was a very strong predictor of a worse prognosis (fig. S12B). ZHX2 depletion impaired RelA/p65 occupancy on IL6 and inhibitor of NF-κB kinase subunit epsilon (IKBKE) promoters (Fig. 4H). VHL binding–defective ZHX2 promoted ccRCC cell growth on soft agar, with this effect ameliorated by treatment with CMPDA (fig. S13, A and B). Thus, our results suggest that ZHX2 promotes NF-κB activation and ccRCC carcinogenesis.

HIF2α and its downstream targets [such as VEGF, glucose transporter member 1 (GLUT1), perilipin (PLIN2), and c-Myc] contribute to ccRCC (3, 2426). Ηowever, the HIF2α inhibitor PT2399 is effective in only a subset of ccRCC (27, 28). We found that ZHX2 depletion or IKK inhibition inhibited soft agar growth of UMRC2 and UMRC6 cells (Fig. 3 and figs. S4, S5, and S9), whereas inhibition or depletion of HIF2α did not (27). ZHX2 has been reported to be an HCC tumor suppressor and to repress cyclin A, cyclin E, α fetoprotein (AFP), and multidrug resistance 1 (MDR1) expression (10, 29, 30). We did not detect suppression of these mRNAs in ccRCC cells (fig. S14). ZHX2 targets may be context dependent, allowing it to act as an oncoprotein in ccRCC. The oncogenic role of ZHX2, via control of NF-κB activation, might provide additional therapeutic avenues for ccRCC.

Supplementary Materials

www.sciencemag.org/content/361/6399/290/suppl/DC1

Materials and Methods

Figs. S1 to S14

Tables S1 to S5

References (3152)

References and Notes

Acknowledgments: The initial screen was performed in the Kaelin laboratory when Q.Z. was a postdoctoral fellow. We thank all members of the Zhang, Kaelin, and Baldwin laboratories for helpful discussions and suggestions; G.Wang for help with ChIP-seq; W.Yu for providing VHL sgRNAs; UNC Tissue Procurement Facility; and UNC Translational Pathology Laboratory. Funding: This work was supported in part by Department of Defense (DOD) Career Development Award (Q.Z.) W81XWH-15-1-0599), University Cancer Research Fund innovator award (J.Z.) NINDS-P30NS045892 (J.S), and the National Cancer Institute (Q.Z., R01CA211732 and R21CA223675, and A.B., R35CA197684). Q.Z. is a V Scholar, Kimmel Scholar, Susan G. Komen Career Catalyst awardee and Mary Kay Foundation awardee. J.Z. is supported by a DOD Fellowship Award (W81XWH-17-1-0016). W.G.K. is a HHMI investigator and is supported by R35 CA210068 from NCI. X.Y. is supported by the National Medical Research Council (OFYIRG17May057) and Biomedical Research Council (BRMC YIG grant 1510851024) from Singapore. Author contributions: Q.Z., J.Z., and T.W. conceived the project. J.Z., T.W., M.T., R.S., X.-D.L., L.X., X.Z., C.La., X.L., L.H., J.W., C.Li., K.H., G.Z., and Q.Z. performed experiments. C.F. performed the patient data analysis. J.F. and J.G. performed TMA immunohistochemistry staining quantification. J.S.P. and J.T.A. performed tumor sample sequencing and analysis. E.J., X.C., C.M.P., W.K.R., W.Y.K., M.W.K., W.G.K., and A.S.B. helped to supervise the study and provide critical advice and reagents for the paper. X.Y., B.T.T., and P.T. provided the unpublished histone ChIP-seq data set. J.S. provided all of bioinformatics analysis. M.L. provided help on obtaining patient samples in kidney cancer. J.Z., W.G.K., A.S.B., and Q.Z. wrote the manuscript with critical comments from all authors. Competing interests: The authors declare no conflicts of interest. Data and materials availability: Additional data are available in the supplementary materials. The ChIP-seq data and microarray data are available at the Gene Expression Omnibus under accession nos. GSE109953 and GSE110094.
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