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. 2015 Jun 1;75(11):2363-74.
doi: 10.1158/0008-5472.CAN-14-2928. Epub 2015 Apr 8.

PLK1 and HOTAIR Accelerate Proteasomal Degradation of SUZ12 and ZNF198 during Hepatitis B Virus-Induced Liver Carcinogenesis

Hao Zhang  1 , Ahmed Diab  1 , Huitao Fan  1 , Saravana Kumar Kailasam Mani  1 , Ronald Hullinger  1 , Philippe Merle  2 , Ourania Andrisani  3
Affiliations

PLK1 and HOTAIR Accelerate Proteasomal Degradation of SUZ12 and ZNF198 during Hepatitis B Virus-Induced Liver Carcinogenesis

Hao Zhang et al. Cancer Res. .

Abstract

Elucidating mechanisms of hepatitis B virus (HBV)-mediated hepatocarcinogenesis is needed to gain insights into the etiology and treatment of liver cancer. Cells where HBV is replicating exhibit increased expression of Plk1 kinase and reduced levels of two transcription repression factors, SUZ12 and ZNF198. SUZ12 is an essential subunit of the transcription repressive complex PRC2. ZNF198 stabilizes the transcription repressive complex composed of LSD1, Co-REST, and HDAC1. These two transcription repressive complexes are held together by binding the long noncoding RNA HOTAIR. In this study, we linked these regulatory events mechanistically by showing that Plk1 induces proteasomal degradation of SUZ12 and ZNF198 by site-specific phosphorylation. Plk1-dependent ubiquitination of SUZ12 and ZNF198 was enhanced by expression of HOTAIR, significantly reducing SUZ12 and ZNF198 stability. In cells expressing the HBV X protein (HBx), downregulation of SUZ12 and ZNF198 mediated global changes in histone modifications. In turn, HBx-expressing cells propagated an altered chromatin landscape after cell division, as exemplified by changes in histone modifications of the EpCAM promoter, a target of PRC2 and LSD1/Co-REST/HDAC1 complexes. Notably, liver tumors from X/c-myc bitransgenic mice exhibited downregulation of SUZ12 and ZNF198 along with elevated expression of Plk1, HOTAIR, and EpCAM. Clinically, similar effects were documented in a set of HBV-related liver tumors consistent with the likelihood that downregulation of SUZ12 and ZNF198 leads to epigenetic reprogramming of infected hepatocytes. Because both Plk1 and HOTAIR are elevated in many human cancers, we propose that their combined effects are involved in epigenetic reprogramming associated broadly with oncogenic transformation.

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Figures

Figure 1
Figure 1. Plk1 down-regulates SUZ12 and ZNF198 proteins
A. (left panel) 4pX-1 cells, transiently transfected with SUZ12-HA and ZNF198-GFP encoding plasmids, were grown with (+) or without (−) HBx expression by tetracycline removal for 48h; whole cell extracts (WCE) were immunoblotted with GFP or HA antibodies. (Right panel) WCE from 4pX-1 cells treated with nocodazole (100 nM) for 12h and grown with (+) or without (−) HBx expression for 16h, were immunoblotted with ZNF198 or SUZ12 antibodies; B. HEK293T cells transiently transfected with SUZ12-HA and ZNF198-GFP encoding plasmids, were grown with (+) or without (−) BI2536 (250 nM) for 12h or MG132 (2.5 μM). (Left panel) DMSO serves as vehicle control. (Right panel) Transfected cells were synchronized in G2/M by addition of nocodazole (100 nM) for 12 hr. Lysates were immunoblotted with HA or GFP antibodies C. Immunoblots of indicated proteins using WCE from HepAD38 cells grown with (+) or without (−) HBV replication by tetracycline removal for 5 and 10 days. D. Immunofluorescence microscopy of indicated proteins, +/- HBV replication in HepAD38 cells.
Figure 2
Figure 2. In vitro Plk1 kinase assays of SUZ12 and ZNF198 and site-directed mutants
A. Affinity purified SUZ12-HA and ZNF198-GFP isolated from transfected HEK293T cells and Plk1 (35, 36) were used in in vitro Plk1 kinase assays. Reactions were performed with (+) or without (−) Plk1, in the presence of γ-32P-ATP and analyzed by SDS PAGE and autoradiography (35, 36). B. Functional regions of SUZ12 (40) and ZNF198 (23) and putative Plk1 phosphorylation sites are shown. Ser to Ala substitutions constructed at highlighted residues. C. and D., in vitro kinase assays of wildtype (WT) SUZ12-HA (C.) and ZNF198-GFP (D.) and site-directed mutants, as indicated. Commassie blue staining shows the same gel used for autoradiography. Relative intensity quantified by ImageJ software is ratio of signal from in vitro kinase reaction vs. corresponding commassie blue stained band, expressed relative to WT. (n=3)
Figure 3
Figure 3. Plk1-mediated phosphorylations of SUZ12 and ZNF198 disrupt interaction with respective chromatin modifying complexes
A. Transient co-expression of SUZ12-HA and Plk1CA or Plk1KD in HEK293T cells. WCE of transfected cells immunoprecipitated with IgG or HA antibodies; immunoprecipitates (IP) were immunoblotted (WB) with EZH2 or HA antibodies. Relative intensity quantified vs. Input. B. Transient co-expression of ZNF198-GFP and Plk1CA or Plk1KD in HEK293T cells. WCE of transfected cells immunoprecipitated with IgG or GFP antibodies; IPs were immunoblotted for GFP, HDAC1 or LSD1. C. and D., transient co-expression of indicated site-directed mutants of SUZ12 (C.) and ZNF198 (D.) in HEK293T cells. IPs immunoblotted with indicated antibodies.( n=3)
Figure 4
Figure 4. Plk1 dependent ubiquitination of SUZ12 and ZNF198 facilitated by lncRNA HOTAIR
Ubiquitination assays of SUZ12-HA (A.) and ZNF198-GFP (B.) in HEK293T cells with addition (+) of BI2536 (500 nM for 20h) or (−) addition of DMSO. HEK293T cells co-transfected with 4.0μg each of indicated plasmids (+) or pCDNA3 empty vector (−). In A., WCE (0.5mg) immunoprecipitated with HA antibody and immunoblotted with FLAG antibody. In B., WCE (1.0 mg) immunoprecipitated with ZNF198 antibody and immnunobloted with FLAG. C. and D., ubiquitination assays of WT SUZ12-HA (C.) and WT ZNF198-GFP (D.), and the respective Ser to Ala mutants, in HEK293T cells transfected with 4.0μg each of plasmid DNA encoding Plk1CA and HOTAIR (+) or pcDNA3 empty vector (−). WCE (0.5mg) utilized for immunoprecipitations. (n=3) E. Quantification of transfected protein (SUZ12 and ZNF198) remaining, in a time course (0-6h) after cyclohexamide (CHX) addition. The indicated WT and Ser to Ala mutants of SUZ12-HA (A.) and ZNF198-GFP (F.) were co-transfected with Plk1CA and HOTAIR expression vectors; Plk1KD and pcDNA3 served as negative controls, respectively. WCE were harvested at 0-6h following addition of 20μg/ml of CHX to HEK293T cells; CHX was added 24h after transfection. Results represent the average from two independent experiments. Error bars are standard deviation. G. HepAD38 cells grown (−) HBV replication by addition of 5 μg/ml tetracycline or with (+) HBV replication by tetracycline removal. HOTAIR and Plk1 CA expression vectors were transfected on day-1 after tetracycline removal and cells were harvested 48h later. WCE were innumoblotted with indicated antibodies. (n=3)
Figure 5
Figure 5. HBx mediates global histone changes during cell cycle progression
A. Immunoblots of endogenous proteins with indicated antibodies, employing WCE isolated at 0-10h after release from dTB. Tetracycline regulated HBx-expressing 4pX-1 cells, grown without (−) or with (+) HBx, were synchronized in G1/S as described (27). BI2536 (500nM) was added 2h prior to cell-harvesting. ( n=3) B. Chromatin immunoprecipitation assays (ChIP) with indicated antibodies employing 4pX-1 cells arrested in G2/M by nocodazole (100nM) addition for 12h, and released from nocodazole block for 4h. Expression of HBx was by tetracycline removal for 16h. C. Immunoblots of SUZ12 and ZNF198 employing WCE from 4pX-1 cells isolated at 4h after release from nocodazole block, without (−) or with (+) HBx expression for 16hr, without (−) or with (+) BI2536 addition for 4h, without (−) or with (+) knockdown of HOTAIR by siRNA (siHOTAIR) transfection. ( n=3) D. Immunoblots of SUZ12 and ZNF198 employing WCE from 4pX-1 cells isolated in a time course (0-6 hr) after release from nocodazole and addition of CHX (20μg/ml), with (+) HBx expression, with (+) or without (−) BI2536.
Figure 6
Figure 6. Plk1 and HOTAIR overexpression in liver tumors from X/c-myc mice and HBV-infected patients
PCR quantification of Plk1 and HOTAIR RNAs using total RNA isolated from A. liver tumors of X/c-myc bitransgenic mice compared to control (Ctrl) RNA (normal mouse liver or peritumoral tissue), shown as box plots or histograms in liver tumors from individual mice. B. PCR quantification of Plk1 and HOTAIR RNAs using RNA isolated from HBV-related HCCs compared to peritumoral tissue. Quantitative PCR reactions were performed in identical triplicates (Plk1 and HOTAIR) using GAPDH as internal control. C. Quantification of EpCAM mRNA in liver tumors from X/c-myc mice (left panel) and HBV-mediated HCCs (right panel), expressed relative to Ctrl RNA. PCR quantification of RNAs from HBV-related HCC samples were performed in duplicates employing PCR arrays, and expressed relative to normal liver (average value from eight patients). p values are shown. D. Model illustrates the mechanism by which HBx-activated Plk1 by phosphorylation signals ubiquitination and proteasomal degradation of SUZ12 and ZNF198. HOTAIR accelerates ubiquitination and proteasomal degradation of SUZ12 and ZNF198, likely acting as scaffold for recruitment of RNA binding E3 ligases, as described by Yoon et al (41).
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