RNF168 regulates R-loop resolution and genomic stability in BRCA1/2-deficient tumors
Parasvi S. Patel, … , Anne Hakem, Razqallah Hakem
Parasvi S. Patel, … , Anne Hakem, Razqallah Hakem
Published February 1, 2021
Citation Information: J Clin Invest. 2021;131(3):e140105. https://doi.org/10.1172/JCI140105.
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Research Article Cell biology Oncology

RNF168 regulates R-loop resolution and genomic stability in BRCA1/2-deficient tumors

Abstract

Germline mutations in BRCA1 and BRCA2 (BRCA1/2) genes considerably increase breast and ovarian cancer risk. Given that tumors with these mutations have elevated genomic instability, they exhibit relative vulnerability to certain chemotherapies and targeted treatments based on poly (ADP-ribose) polymerase (PARP) inhibition. However, the molecular mechanisms that influence cancer risk and therapeutic benefit or resistance remain only partially understood. BRCA1 and BRCA2 have also been implicated in the suppression of R-loops, triple-stranded nucleic acid structures composed of a DNA:RNA hybrid and a displaced ssDNA strand. Here, we report that loss of RNF168, an E3 ubiquitin ligase and DNA double-strand break (DSB) responder, remarkably protected Brca1-mutant mice against mammary tumorigenesis. We demonstrate that RNF168 deficiency resulted in accumulation of R-loops in BRCA1/2-mutant breast and ovarian cancer cells, leading to DSBs, senescence, and subsequent cell death. Using interactome assays, we identified RNF168 interaction with DHX9, a helicase involved in the resolution and removal of R-loops. Mechanistically, RNF168 directly ubiquitylated DHX9 to facilitate its recruitment to R-loop–prone genomic loci. Consequently, loss of RNF168 impaired DHX9 recruitment to R-loops, thereby abrogating its ability to resolve R-loops. The data presented in this study highlight a dependence of BRCA1/2-defective tumors on factors that suppress R-loops and reveal a fundamental RNF168-mediated molecular mechanism that governs cancer development and vulnerability.

Authors

Parasvi S. Patel, Karan Joshua Abraham, Kiran Kumar Naidu Guturi, Marie-Jo Halaby, Zahra Khan, Luis Palomero, Brandon Ho, Shili Duan, Jonathan St-Germain, Arash Algouneh, Francesca Mateo, Samah El Ghamrasni, Haithem Barbour, Daniel R. Barnes, Jonathan Beesley, Otto Sanchez, Hal K. Berman, Grant W. Brown, El Bachir Affar, Georgia Chenevix-Trench, Antonis C. Antoniou, Cheryl H. Arrowsmith, Brian Raught, Miquel Angel Pujana, Karim Mekhail, Anne Hakem, Razqallah Hakem

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Figure 2

Loss of RNF168 increases genomic instability, impairs DSB repair, and causes senescence in BRCA1-deficient cells.

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Loss of RNF168 increases genomic instability, impairs DSB repair, and ca...
(A) Quantification of primary MECs with the indicated genotypes exhibiting 10 or more spontaneous γH2AX foci (n = 3). (B) Representative images of γH2AX staining in indicated cells under untreated or irradiated conditions. Scale bar: 50 μm. (C) Quantification of cells with 10 or more γH2AX foci from B (n = 3–6). (D) Quantification of indicated cells exhibiting 10 or more spontaneous γH2AX foci. Western blot for shRNF168 knockdown efficiency is shown in Supplemental Figure 5A (n = 3–4). (E) Efficiency of DSB repair pathways (homologous recombination, NHEJ, and a-EJ) in mediating the repair of I-SceI–induced DSBs in indicated cells (n = 3). shRNF168 knockdown efficiency and cell counts are shown in Supplemental Figure 5, I and J. (F) Quantification of chromosomal bridges and micronuclei in the indicated cells. Representative images of RNF168-depleted MDA-MB-436 cells showing chromosomal bridges (arrowhead) and micronuclei (arrow) are shown (n = 4). Scale bar: 25 μm. (G) Cell cycle distribution analysis using propidium iodide staining of the indicated cells (n = 3). (H) Representative images of SA-β-gal staining of the indicated MECs and quantification (n = 3–6). Scale bar: 25 μm. For all experiments, a minimum of 3 independent experiments and 100 cells per condition and genotype were scored from at least 5 fields of view. A 1-way ANOVA followed by Tukey’s multiple-comparison test (A, C, and H), Welch’s t test (E), unpaired Student’s t test (G), or 1-way ANOVA followed by Dunnett’s multiple-comparison (D and F) test were performed. Data are presented as the mean ± SEM.