Junction plakoglobin regulates and destabilizes HIF2α to inhibit tumorigenesis of renal cell carcinoma

Abstract

Background: Increased hypoxia-inducible factor 2α (HIF2α) activation is a common event in clear cell renal cell carcinoma (ccRCC) progression. However, the function and underlying mechanism of HIF2α in ccRCC remains uninvestigated. We conducted this study to access the potential link between junction plakoglobin (JUP) and HIF2α in ccRCC.

Methods: Affinity purification and mass spectrometry (AP-MS) screening, glutathione-s-transferase (GST) pull-down and co-immunoprecipitation (Co-IP) assays were performed to detect the interacting proteins of HIF2α. Quantitative PCR (qPCR) and Western blotting were used to detect the expression of JUP in human ccRCC samples. Luciferase reporter assays, chromatin immunoprecipitation (ChIP), cycloheximide chase assays, and ubiquitination assays were conducted to explore the regulation of JUP on the activity of HIF2α. Cell Counting Kit-8 (CCK-8) assays, colony formation assays, transwell assays, and xenograft tumor assays were performed to investigate the effect of JUP knockdown or overexpression on the tumorigenicity of renal cancer cells.

Results: We identified JUP as a novel HIF2α-binding partner and revealed an important role of JUP in recruiting von Hippel-Lindau (VHL) and histone deacetylases 1/2 (HDAC1/2) to HIF2α to regulate its stability and transactivation. JUP knockdown promoted and overexpression suppressed the tumorigenicity of renal cell carcinoma in vitro and in vivo. Importantly, the low expression of JUP was found in clinical ccRCC samples and correlated with enhanced hypoxia scores and poor treatment outcomes.

Conclusion: Taken together, these data support a role of JUP in modulating HIF2α signaling during ccRCC progression and identify JUP as a potential therapeutic target.

Keywords: hypoxia-inducible factor 2α; junction plakoglobin; renal cell carcinoma; transcriptional activity; ubiquitination.

FIGURE 1

JUP interaction with HIF2α. A and B, HEK293T cells were transfected with Flag‐HIF2α and HA‐JUP. The cell lysates were immunoprecipitated with anti‐Flag (A) or anti‐HA antibody (B), and co‐immunoprecipitated proteins were detected by Western blotting analysis with anti‐HA or anti‐Flag antibody, respectively. C, Purified GST or GST‐JUP was incubated with Flag‐HIF2α‐expressing 786‐O lysates and precipitated with glutathione‐agarose beads. Pulled‐down Flag‐HIF2α proteins by GST‐JUP were detected by immunoblotting with the HIF2α antibody. Purified GST or GST‐JUP was visualized by Western blotting analysis using the GST antibody. D and E, 786‐O cells were harvested and subjected to endogenous Co‐IP analysis with JUP antibody. Rabbit IgG was used as a control. F, Schematic diagram of the various domains and relevant mutant fusion expression constructs of HIF2α (upper). HA‐JUP and Flag‐HIF2α full‐length and deletion mutants were ectopically expressed in HEK293T cells, followed by Flag‐immunoprecipitation and immunoblotting with HA antibody. Inputs are shown in the bottom panels. G, GST pull‐down assay detecting the regions of JUP that bind to HIF2α. Schematic diagram of the various domains and relevant mutant fusion expression constructs of JUP (upper). Flag‐HIF2α and GST‐JUP deletion mutants were ectopically expressed in HEK293T cells, followed by Western blotting analysis of Flag‐HIF2α pulled down by GST or GST‐JUP fusion proteins. Inputs are shown in the bottom panels. Abbreviations: HIF, hypoxia‐inducible factor; JUP, junction plakoglobin; bHLH, basic helix‐loop‐helix; PAS, Per‐Arnt‐Sim; PAC, PAS‐associated C‐terminal; ODD, oxygen‐dependent degradation; N‐TAD, N‐terminal transactivation domain; C‐TAD, C‐terminal transactivation domain; IH, inhibitory domain; ARM, armadillo; NTD, N‐terminal domain; CTD, C‐terminal domain; GST, glutathione‐S‐transferase; Co‐IP, co‐immunoprecipitation

FIGURE 2

JUP expression in ccRCC tissues. A, qPCR analysis of JUP expression in 12 specimens of ccRCC and adjacent normal tissues. B, JUP protein levels in the paired tumor (T) and adjacent normal (N) tissues. C, Quantification of JUP protein levels in a tissue array containing 15 specimens of ccRCC and adjacent normal tissues. JUP levels were classified into three grades (negative [‐], weak positive [+], and strong positive [++]) according to results from immunofluorescence staining. D, JUP expression was significantly downregulated in ccRCC tumors compared with normal tissues. E, There was low expression of JUP in stage III and IV ccRCCs. F, Kaplan–Meier analysis identified that patients with low JUP expression exhibited shorter survival compared to those with high JUP expression. Abbreviations: RSEM, RNA‐Seq by Expectation‐Maximization; OS, overall survival; DSS, disease‐specific survival; PFS, progression‐free survival; JUP, junction plakoglobin; ccRCC, clear cell renal cell carcinoma

FIGURE 3

JUP suppresses renal cancer proliferation and metastasis. A, JUP inhibited the proliferation of ACHN and 786‐O cells. The cells were stably transfected with a vector control or JUP and analyzed by colony formation assays. Images of the whole plate are shown in the upper panels, and the number of colonies was quantified in the lower panels. Data are plotted as the mean ± SD of three independent experiments. B, Wound healing assay of ACHN and 786‐O cells expressing a vector control or JUP. Data are plotted as the mean ± SD of 3 independent experiments. C, Migration and invasion assays for RCC cells. Migrated and invaded cells from each treatment group were counted in five random images. Three experiments were conducted, and a mean ± SD of relative cell numbers was plotted. D–F, ACHN cells stably expressing sh‐JUP‐1# and sh‐LacZ (control) were subcutaneously injected into the left and right flanks of nude mice as indicated. The tumor volumes were measured every week after transplantation (D), and the macroscopic appearances (E) and weight (F) of tumors at week 7 post‐transplantation are shown. G, Representative hematoxylin and eosin‐stained xenograft tumors corresponding to week 7 after injection. H, Metastatic colonization was assessed by fluorescence small‐animal imaging system. I, Kaplan‐Meier survival analysis for mice injected by the tail vein with sh‐LacZ (n = 9) or sh‐JUP ACHN cells (n = 9). *P < 0.05, ** P < 0.01, and ***P < 0.001. Abbreviations: JUP, junction plakoglobin; RCC, renal cell carcinoma

FIGURE 4

JUP negatively regulates the activity of HIF2α in RCC cells. A, The relative luciferase activity and Western blotting analysis of HEK293T cells transfected with Flag‐HIF2α (100 ng) with the indicated doses of HA‐JUP (0, 200, or 600 ng). B, JUP inhibits 6×HRE‐driven luciferase activity in VHL‐null 786‐O cells (expressing HIF2α, but not HIF1α). C, The N‐terminal of JUP is required for the transcriptional inhibition of HIF2α. 786‐O cells were transfected with 6×HRE‐driven luciferase reporter and pGL4.73 along with serial deletion mutants of GST­tagged JUP or GST (control). D and E, qPCR showed that JUP‐KD enhanced VEGFA, TGFA, and CCND1 transcription in 786‐O cells (D) and OSRC‐2 cells (E) expressing sh‐JUP‐1#, sh‐JUP‐2#, or sh–LacZ (control). F, Biological pathway analysis of JUP repressed genes showed enrichment of the HIF pathway and VEGF signaling in 786‐O cells. G Venn diagram showing the intersection of HIF2α‐activated and JUP‐repressed transcriptomes revealed 86 co‐regulated genes. H, Validation of RNA‐seq results by qPCR of six genes was performed. The experiments were repeated at least three times, and the results are shown as the mean ± SD and were analyzed by the unpaired two‐tailed Student's t‐test (n = 3). I, Scatterplot of hypoxic signature score against gene expression levels of JUP in ccRCC samples from a TCGA cohort. J, JUP was highly expressed in ccRCC patients with a low hypoxic score. *P < 0.05, ** P < 0.01, and ***P < 0.001. Abbreviations: TBP, TATA box binding protein; JUP, junction plakoglobin; RCC, renal cell carcinoma; qPCR, quantitative polymerase chain reaction; JUP‐KD, JUP knockdown; n.s. not significant

FIGURE 5

JUP promotes HIF2α ubiquitination and degradation by facilitating HIF2α‐VHL interaction. A, JUP affected HIF2α protein levels in ACHN cells. ACHN cells were transfected with HA‐JUP (0, 0.5, or 2 μg) plasmids. Cell lysates were subjected to SDS‐PAGE followed by immunoblotting with anti‐HIF2α, anti‐HA, or anti‐GAPDH antibody. B, Downregulation of HIF2α level by JUP was reversed by MG132. Cells transfected with HA‐JUP were left untreated or were treated with MG132, and HIF2α level was examined by Western blotting. C and D, ACHN and SN12‐PM6 cells were stably transfected with JUP shRNAs, and the HIF2α level was examined. E, ACHN cells stably expressing HIF2α/LacZ shRNA or HIF2α/JUP shRNA were treated with 100 μmol/L cycloheximide and harvested at the indicated time points to examine HIF2α levels. F, Quantification of HIF2α protein expression level in panel E shows the effect of JUP on HIF2α stability. G, ACHN cells stably expressing HIF2α/HA‐JUP or HIF2α were treated with 100 μmol/L cycloheximide and harvested at the indicated time points to examine HIF2α levels. H, Quantification of HIF2α protein expression level shown in panel G. I, JUP enhanced HIF2α ubiquitination in ACHN cells. Cells were stably transfected with the indicated plasmids and then treated with MG132 (10 μg/mL) for 6 h. Ubiquitinated proteins were pulled down by Ni‐NTA agarose under denaturing conditions, and then analyzed by Western blotting with Myc, HIF2α, and HA antibodies. J, GST pull‐down assay detecting JUP‐VHL interaction. GST or GST‐VHL purified from E. coli was incubated with 786‐O cell lysates and precipitated with glutathione‐agarose beads, followed by Western blotting analysis of anti‐JUP. K, Co‐IP of endogenous JUP from extracts of 786‐O cells expressing Flag‐VHL. L, Co‐IP of HA‐JUP with VHL in 786‐O cells. The 786‐O cells transfected with HA‐JUP and Flag‐VHL were subjected to Co‐IP with anti‐HA antibody. M, GST pull‐down assay detecting the regions of JUP that bind to VHL. Flag‐VHL and GST‐JUP deletion mutants were ectopically expressed in HEK293T cells, followed by Western blotting analysis of Flag‐VHL pulled down by GST‐JUP fusion deletion mutants. Inputs are shown in the bottom panels. N, JUP promoted the HIF2α‐VHL interaction. 786‐O cells were co‐transfected with Flag‐VHL (0.6 μg) and HA‐JUP (0 or 1 μg) plasmids. Endogenous HIF2α co‐immunoprecipitated with Flag antibody. Abbreviations: CHX, cycloheximide; JUP, junction plakoglobin

FIGURE 6

Recruitment of HDAC1/2 to HIF2α by JUP decreases H3K27ac levels at HIF2α‐binding sites in RCC cells. A, Endogenous HIF2α and JUP co‐immunoprecipitated with Flag‐HDAC1/2 in 786‐O cells. 786‐O cells stably expressing Flag‐HDAC1 or Flag‐HDAC2 were lysed and immunoprecipitated with anti‐Flag antibody. B, GST pull‐down assay detecting the regions of JUP that bind to HDAC1. Flag‐HDAC1 and GST‐JUP deletion mutants were ectopically expressed in HEK293T cells, followed by Western blotting analysis of Flag‐HDAC1 pulled down by GST or GST‐JUP deletion mutants. Inputs are shown in the bottom panels. C, JUP promoted both HDAC1 and HDAC2 binding to HIF2α. 786‐O cells were co‐transfected with Flag‐HDAC1 or Flag‐HDAC2 (0.6 μg) and HA‐JUP (0 or 1 μg) plasmids. Endogenous HIF2α co‐immunoprecipitated with Flag antibody. D, GST pull‐down assay detecting JUP‐HDAC1/HDAC2 interaction. E, Schematic diagram of regulatory regions in VEGFA promoter and ZNF395 enhancer shows the HIF DNA‐binding sites. Numbers indicate nucleotides with the transcription start site indicated by +1. F and G, ChIP analysis with anti‐JUP or anti‐HDAC1 antibody in WT‐ or HIF2α‐specific depleted 786‐O cells. Precipitated DNAs were quantified by qPCR of fragments around the HIF‐binding site at the VEGFA promoter (F) and ZNF395 enhancer (G). H and I, ChIP analysis with anti‐HIF2α antibody in WT‐, JUP‐, or HDAC1‐specific depleted 786‐O cells. Precipitated DNAs were quantified by qPCR of fragments around the HIF‐binding site at the VEGFA promoter (H) and ZNF395 enhancer (I). J and K, ChIP‐qPCR validated the recruitment of HDAC1 by JUP at the VEGFA promoter (J) and ZNF395 enhancer (K) in 786‐O cells. L and M, ChIP‐qPCR of p300 binding and H3K27ac enrichment at the VEGFA promoter (L) and ZNF395 enhancer (M) in WT‐ or JUP‐specific depleted 786‐O cells. *P < 0.05, ** P < 0.01, and ***P < 0.001. Abbreviations: ZNF395, zinc finger protein 395; H3K27ac, histone H3K27 acetylation; ChIP, chromatin immunoprecipitation; JUP, junction plakoglobin; RCC, renal cell carcinoma

FIGURE 7

JUP suppresses RCC cell migration and invasion in part through HIF2α. A, 786‐O cells were infected with different combinations of lentivirus as indicated. At 72 h after infection, Western blotting analysis was performed to determine the protein levels of JUP and HIF2α. GAPDH is an internal control. B and C, 786‐O cells stably expressing sh‐JUP, sh‐HIF2α, or sh‐LacZ as in panel A were analyzed by transwell assay (B) and colony formation assay (C). D, ACHN and 786‐O cells were infected with lentivirus with control PCDH (Control), PCDH‐HIF2α, or/and sh‐JUP as indicated. Western blotting analysis was performed to evaluate the expression of JUP and HIF2α. GAPDH is an internal control. E, After infection as in panel D, cells were used for transwell assays. The number of migrated and invaded cells from each group was quantified in 4 random images. F, 786‐O cells were transfected with PCDH‐HIF2α along with HA‐JUP or HA‐JUP△N/C, as indicated. Western blotting analysis was performed to determine the protein levels of JUP and HIF2α. GAPDH is an internal control. G, After infection as in panel D, cells were used for transwell assays. The number of migrated and invaded cells from each group was quantified in 4 random images. *P < 0.05, ** P < 0.01, and ***P < 0.001. Abbreviations: JUP, junction plakoglobin; RCC, renal cell carcinoma; JUP△N/C, both N‐ and C‐terminus deletion of JUP