Hepatitis B virus infection disrupts homologous recombination in hepatocellular carcinoma by stabilizing resection inhibitor ADRM1
Ming Zeng, … , Antony M. Carr, Cong Liu
Ming Zeng, … , Antony M. Carr, Cong Liu
Published October 10, 2023
Citation Information: J Clin Invest. 2023;133(23):e171533. https://doi.org/10.1172/JCI171533.
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Research Article Hepatology Virology

Hepatitis B virus infection disrupts homologous recombination in hepatocellular carcinoma by stabilizing resection inhibitor ADRM1

Abstract

Many cancers harbor homologous recombination defects (HRDs). A HRD is a therapeutic target that is being successfully utilized in treatment of breast/ovarian cancer via synthetic lethality. However, canonical HRD caused by BRCAness mutations do not prevail in liver cancer. Here we report a subtype of HRD caused by the perturbation of a proteasome variant (CDW19S) in hepatitis B virus–bearing (HBV-bearing) cells. This amalgamate protein complex contained the 19S proteasome decorated with CRL4WDR70 ubiquitin ligase, and assembled at broken chromatin in a PSMD4Rpn10- and ATM-MDC1-RNF8–dependent manner. CDW19S promoted DNA end processing via segregated modules that promote nuclease activities of MRE11 and EXO1. Contrarily, a proteasomal component, ADRM1Rpn13, inhibited resection and was removed by CRL4WDR70-catalyzed ubiquitination upon commitment of extensive resection. HBx interfered with ADRM1Rpn13 degradation, leading to the imposition of ADRM1Rpn13-dependent resection barrier and consequent viral HRD subtype distinguishable from that caused by BRCA1 defect. Finally, we demonstrated that viral HRD in HBV-associated hepatocellular carcinoma can be exploited to restrict tumor progression. Our work clarifies the underlying mechanism of a virus-induced HRD subtype.

Authors

Ming Zeng, Zizhi Tang, Laifeng Ren, Haibin Wang, Xiaojun Wang, Wenyuan Zhu, Xiaobing Mao, Zeyang Li, Xianming Mo, Jun Chen, Junhong Han, Daochun Kong, Jianguo Ji, Antony M. Carr, Cong Liu

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Figure 2

CDW19S engages DSB-proximal chromatin.

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CDW19S engages DSB-proximal chromatin. (A) TAP-affinity-purified spWdr70...
(A) TAP-affinity-purified spWdr70-interacting proteins separated by gradient SDS-PAGE. Proteins identified by MALDI-TOF mass spectrometry are shown on the right. See peptide coverage in Supplemental Table 1. Subdomains of interface (Int), PCI, MPN, and ATPase (ATP) are indicated for RP subunits using human and yeast nomenclatures. IgH, heavy chain of rabbit IgG; TEV, tobacco etch virus endopeptidase. (B) Immunoblotting for p-RPA32 and H2B monoubiquitination (uH2B) in L02 cells with indicated siRNA and CPT treatment. (C) Left: Illustration for XbaI-based resection assay at the selected AsiSI-dependent DSB site (chromosome 1: 89,458,595–89,458,603). Right: Example of monitoring of ssDNA production by semiquantitative PCR. (D) Quantification for XbaI-based resection assay showing DSB processing in DIvA cells depleted for the indicated CDW19S subunits. Inset: Excessive p-RPA32 immunostaining implies hypersection in siADRM1Rpn13 RPE1 cells. Data normalized to control (siScr) with 4-OHT induction. Original magnification (inset): ×400. (E) ChIP assay 2.5 kb distal to an AsiSI-induced DSB showing break association of FLAG-tagged CDW19S subunits upon PSMD4Rpn10 ablation relative to control transfection. DIvA cells all treated with 4-OHT. (F) Left: ChIP assay for FLAG-tagged WDR70/DDB1 at indicated distances from AsiSI-induced DSB ends. Right: Representative PCR products. (G) ChIP assay of FLAG-PSMD4Rpn10 2.5 kb from an AsiSI-induced DSB upon PSMD4WT or PSMD4dUIM expression. Anti-FLAG immunoblotting is shown in the right panel. (H) Equivalent ChIP assay for indicated CDW19S subunits in the presence of PSMD4WT or PSMD4dUIM expression. (I) Top: Representative images of p-RPA32 and BRCA1 IRIF in the presence of PSMD4Rpn10 or PSMD4dUIM (4 hours after IR). Nuclei counterstained with DAPI. Scale bars: 10 μm. Bottom: Quantification of fluorescent intensity or foci numbers. In H and I, PSMD4 plasmids are FLAG-less and siRNA resistant, and cells were cotransfected with siPSMD4Rpn10. (J and K) PSMD4Rpn10 enrichment upon 4-OHT induction at 0.5 kb from an AsiSI-induced DSB after treatment with the indicated siRNA or inhibitors.