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 6

MLH1 loss is lethal during BRCA2 deficiency.

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MLH1 loss is lethal during BRCA2 deficiency. (A) DRIP assay showing the ...
(A) DRIP assay showing the enrichment of R-loops in Actb genes in MEFs from Brca2L2431P/L2431P (under genotypes of Mlh1KO/KO;Mlh1KO/+;Mlh1+/+) (n = 3 biological replicates for Mlh1KO/KO; n = 2 biological duplicates from 2 independent MEFs for Mlh1KO/+; n = 2 biological replicates from 2 independent MEFs for Mlh1+/+) and Brca2L2431P/KO (under genotypes of Mlh1KO/+;Mlh1+/+) (n = 3 biological replicates) by RT-qPCR. (B) Immunoblot confirmation of doxycycline-induced knockdown of Mlh1 using Mlh1 shRNA in KB2P1.21 cells. (C) Quantitation of colony formation assay showing the normalized cell viability of doxycycline-induced knockdown of Mlh1 using Mlh1 shRNA in KB2P1.21 cells. Control shRNA and no doxycycline treatment was used as a control (n = 3 biological replicates). (D) Allograft model showing the tumor volume of short hairpin RNA control (shControl) or shMlh1 KB2P1.21 cells under doxycycline induction (n = 10 animals). Tumor volume was measured every 2 days using digital caliper. (E) Allograft model showing the tumor volume of shControl or shMlh1 KB2P1.21 under no-doxycycline induction (n = 8 animals). Tumor volume was measured every 2–3 days using digital calipers. (F) TCGA data set analysis (TCGA, Firehose Legacy) showing the percentage of genomes altered in BRCA2-low; MLH1-median versus BRCA2-low; MLH1-low breast cancer patient samples. (G) TCGA data set analysis (TCGA, Firehose Legacy) showing the phosphorylation status of CHEK1 and CHEK2 in BRCA2-low; MLH1-median versus BRCA2-low; MLH1-low versus BRCA2-low; MLH1-high breast cancer patient samples. The levels of total CHEK1 and CHEK2 act as a control. Note: TCGA breast data set (TCGA, Cell and Firehose Legacy) has some overlapping samples. Data were analyzed using unpaired, 2-tailed Student’s t test with Holm-Šidák multiple-comparison test (A), unpaired, 2-tailed Student’s t test and Wilcoxon’s rank-sum test (C), unpaired, 2-tailed Student’s t test with Holm multiple-comparison test (D and E), Wilcoxon’s rank-sum test (F), and unpaired, 2-tailed Student’s t test (G). *P < 0.05; **P < 0.01.