Mismatch repair protein MLH1 suppresses replicative stress in BRCA2-deficient breast tumors
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
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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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 5

MLH1 suppresses genomic instability in BRCA2-deficient cells.

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MLH1 suppresses genomic instability in BRCA2-deficient cells. (A) Repres...
(A) Representative images of chromosomal aberrations in BRCA2Y3308X, Brca2KO/KO-r (n = 2 clones), and Brca2cKO/KO-mi clones (n = 1 clone) as assessed by metaphase spreads. (B) Quantitation of chromosomal aberrations in BRCA2Y3308X, Brca2KO/KO-r (n = 2 clones), and Brca2cKO/KO-mi clones (n = 2 clones) from A. Aberrations per nuclei are plotted in a superplot (30 nuclei; n = 3 biological replicate; 10 nuclei per replicate). (C) Quantitation of chromosomal aberrations in Brca2KO/KO-r (n = 2 clones) and Brca2cKO/KO-mi clones (n = 1 clone) treated with and/or without olaparib (100 nM). Aberrations per nuclei are plotted in a superplot (25–30 nuclei; n = 3 biological replicate; 5–10 nuclei per replicate). (D) WGS analysis showing the SVs in BRCA2Y3308X, Brca2KO/KO-r (n = 2 clones), and Brca2cKO/KO-mi clones (n = 19 clones). SVs such as translocations (BND), deletion (DEL), and insertion (INS) are expressed as unique SVs. (E) Quantitation of chromosomal aberrations in Mlh1-silenced Brca2KO/KO-r and Brca2cKO/KO-mi clones. Aberrations per nuclei are plotted in a superplot (30 nuclei; n = 3 biological replicate; 10 nuclei per replicate). (F) Circos plot depicting levels of SVs in Brca2KO/KO-r and Brca2cKO/KO-mi clones upon silencing Mlh1. Levels of SVs were normalized to Brca2cKO/KO-mi (control siRNA) clone. (G) Quantitation of γH2AX foci in control or Mlh1-silenced Brca2KO/KO-r and Brca2cKO/KO-mi clones using immunofluorescence analysis. ImageJ was used to count γH2AX foci manually. (H) Quantitation of chromosomal aberrations in control (pLVX_control) or Mlh1-overexpressing (pLVX_Mlh1) BRCA2Y3308X clone. Aberrations per nuclei are plotted in a superplot (25 nuclei; n = 3 biological replicate; 5–10 nuclei per replicate). (I) Circos plot depicting the levels of SVs in BRCA2Y3308X (normalized to Brca2cKO/KO-mi clone) and upon Mlh1 overexpression in BRCA2Y3308X clones (n = 2; BRCA2Y3308X; pLVX_Mlh1 #1 and BRCA2Y3308X; pLVX_Mlh1 #2) (normalized to BRCA2Y3308X; pLVX_control). Data were analyzed using unpaired, 2-tailed Student’s t test (B, C, E, and H) and unpaired, 2-tailed Student’s t test with Holm-Šidák multiple-comparison test (G). *P < 0.05; **P < 0.01; ***P < 0.001.