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 3

RNF168 depletion suppresses the in vitro and in vivo growth of BRCA1- and BRCA2-deficient breast and ovarian tumor cells, respectively.

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RNF168 depletion suppresses the in vitro and in vivo growth of BRCA1- an...
(A) Representative images of the indicated BRCA1-deficient cells. Scale bar: 500 μm. The arrows indicate large, flat senescent cells. (B) Long-term proliferation of indicated MDA-MB-436 cells stained with crystal violet; 100,000 cells were seeded per 6 cm plate; cells were fixed after 21 days (n = 3). (C) Cumulative growth curve in indicated cells (n = 3). (D) In vivo growth of indicated xenografts (n = 4). p/s, photons/second. Representative images are shown (scale bar: 1 cm). (E) Representative images of staining in tumors from D (n = 4) and quantification. Scale bar: 100 μm. (F) Quantification of tumor mass from the experiments in D and E. (G) Relative growth of the indicated TNBC PDXs (n = 3). RLU, relative luciferase unit. (H) In vivo growth of BRCA2-mutant PEO1 control and RNF168-depleted xenografts. Representative images are shown (n = 3). Scale bar: 1 cm. (I) Quantification of Ki67 immunofluorescence in indicated cells. (J) Quantification of tumor mass from H. Data were analyzed by Welch’s t test (B), 2-way ANOVA with Tukey’s multiple-comparison test (C), 2-way ANOVA with Sidak’s multiple-comparison test (D), 2-tailed, unpaired Student’s t test (E, F, I, and J), 2-tailed, paired Student’s t test (G), and 2-tailed Wilcoxon matched-pairs signed-rank test (G). Data are presented as the mean ± SEM.