Mismatch repair protein MLH1 suppresses replicative stress in BRCA2-deficient breast tumors
Satheesh K. Sengodan, … , Subhajyoti De, Shyam K. Sharan
Satheesh K. Sengodan, … , Subhajyoti De, Shyam K. Sharan
Published January 25, 2024
Citation Information: J Clin Invest. 2024;134(7):e173718. https://doi.org/10.1172/JCI173718.
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Research Article Genetics

Mismatch repair protein MLH1 suppresses replicative stress in BRCA2-deficient breast tumors

Abstract

Loss of BRCA2 (breast cancer 2) is lethal for normal cells. Yet it remains poorly understood how, in BRCA2 mutation carriers, cells undergoing loss of heterozygosity overcome the lethality and undergo tissue-specific neoplastic transformation. Here, we identified mismatch repair gene mutL homolog 1 (MLH1) as a genetic interactor of BRCA2 whose overexpression supports the viability of Brca2-null cells. Mechanistically, we showed that MLH1 interacts with Flap endonuclease 1 (FEN1) and competes to process the RNA flaps of Okazaki fragments. Together, they restrained the DNA2 nuclease activity on the reversed forks of lagging strands, leading to replication fork (RF) stability in BRCA2-deficient cells. In these cells, MLH1 also attenuated R-loops, allowing the progression of stable RFs, which suppressed genomic instability and supported cell viability. We demonstrated the significance of their genetic interaction by the lethality of Brca2-mutant mice and inhibition of Brca2-deficient tumor growth in mice by Mlh1 loss. Furthermore, we described estrogen as inducing MLH1 expression through estrogen receptor α (ERα), which might explain why the majority of BRCA2 mutation carriers develop ER-positive breast cancer. Taken together, our findings reveal a role of MLH1 in relieving replicative stress and show how it may contribute to the establishment of BRCA2-deficient breast tumors.

Authors

Satheesh K. Sengodan, Xiaoju Hu, Vaishnavi Peddibhotla, Kuppusamy Balamurugan, Alexander Y. Mitrophanov, Lois McKennett, Suhas S. Kharat, Rahul Sanawar, Vinod Kumar Singh, Mary E. Albaugh, Sandra S. Burkett, Yongmei Zhao, Bao Tran, Tyler Malys, Esta Sterneck, Subhajyoti De, Shyam K. Sharan

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Figure 3

MLH1 protects reversed RFs from DNA2 nuclease.

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MLH1 protects reversed RFs from DNA2 nuclease. (A) Quantitation of IdU/C...
(A) Quantitation of IdU/CldU ratios of control, Mlh1-, or Dna2-silenced or a combination in Brca2KO/KO-r (n = 2), Brca2cKO/KO-mi (n = 1), or Brca2cKO/KO (n = 1) mESCs. (B) mRNA level expression status of panel of nucleases in Brca2KO/KO-r(n = 22) and Brca2cKO/KO-mi(n = 2) clones. Each dot in the graph represents an individual Brca2KO/KO-r or Brca2cKO/KO-mi clone. Red arrows are used to indicate Brca2cKO/KO-mi clones. (C) Immunoblot showing confirmation of MLH1 and/or DNA2 overexpression in KB2P1.21 cells. (D) Quantitation of IdU/CldU ratios of pcDNA_Mlh1- or pcDNA_Dna2-overexpressing KB2P1.21 cells. (E) Quantitation of IdU/CldU ratios of Dna2-overexpressing or Mlh1-overexpressing KB2P1.21 cells upon control or Fen1 or Mlh1 silencing. (F) In vitro nuclease assay of RNA flap substrate using pure MLH1 and FEN1 proteins. Processed products are marked by arrows. (G) Quantitation of IdU/CldU ratios of pcDNA_Mlh1-overexpressing KB2P1.21 cells upon Mlh1, Fen1, or Smarcal1 silencing under C5-treated or untreated conditions. (H) Quantitation of IdU/CldU ratios of pcDNA_Mlh1-overexpressing KB2P1.21 cells upon Mlh1, Fen1, or Dna2 silencing under C5- or mirin-treated or untreated conditions. (I) Quantitation of IdU/CldU ratios of pcDNA_Dna2-overexpressing KB2P1.21 cells upon treating with DNA2 inhibitor C5 or MRE11 inhibitor mirin. (J) Schematic representation of model to show RF protection and/or degradation in MLH1 or FEN1-high/low cells under DNA2-high conditions. MLH1/FEN1 binds and processes flaps of Okazaki fragments. MLH1/FEN1 knockdown generates longer Okazaki fragment flaps, which are then bound by DNA2, leading to RF degradation upon fork stalling (with HU). Each dot represents an individual fiber, and at least 150 fibers pooled from 3 biological replicates (>50 fibers per replicate) were used to generate the superplots in A, D, E, G, H, and I. Each replicate is color coded. Data are represented as means ± SEM. Data were analyzed by unpaired, 2-tailed Student’s t test (A, D, E, G, H, and I) using the mean from 3 biological replicates. *P < 0.05; **P < 0.01; ***P < 0.001.