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.
View: Text | PDF
Research Article Genetics Article has an altmetric score of 6

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

×

Figure 1

MLH1 rescues and promotes survival of BRCA2-deficient cells.

Options: View larger image (or click on image) Download as PowerPoint
MLH1 rescues and promotes survival of BRCA2-deficient cells. (A) Schemat...
(A) Schematic representation of experimental approach to generating Brca2KO/KO-r mESC clones from (Brca2cKO/KO) mESCs after mirin (10 μM for 3 hours) pretreatment. Conditional allele was deleted in DMSO or mirin-treated Brca2cKO/KO cells by Cre expression followed by selection in HAT media. (B) Heatmap showing mRNA expression of upregulated and downregulated genes in Brca2KO/KO-r (n = 22) and Brca2cKO/KO-mi clones (n = 2). (C) RT-qPCR analysis of Mlh1, Msh2, and Mlh3 in Brca2KO/KO-r and Brca2cKO/KO-mi clones (n = 3 biological replicates). Independent clones are marked by number symbols. (D) Representative images showing colony formation upon Mlh1 and Msh2 silencing in Brca2KO/KO-r (n = 3 clones) and Brca2cKO/KO-mi clones (n = 1 clone). (E) Quantitation of D, showing the normalized cell viability in Brca2KO/KO-r and Brca2cKO/KO-mi clones (n = 3 biological replicates). (F) Immunoblot showing overexpression of MLH1 in Brca2cKO/KO mESCs. (G) Southern blot showing rescue of Brca2KO/KO clones in control vector or MLH1-overexpressing Brca2cKO/KO mESCs (left panel). Asterisks indicate Brca2KO/KO clones. Quantitation of percentage rescue in vector or Mlh1-overexpressing Brca2cKO/KO mESCs (n = 3 biological replicates) (right panel). (H) Kaplan-Meier analysis showing survival status of patients from MLH1-high (n = 111) versus MLH1-low (n = 80) breast cancer patient samples obtained from the TCGA Nature breast cancer data set. (I) Kaplan-Meier analysis showing the survival status of patients from BRCA2-low; MLH1-high (n = 100) versus BRCA2-low; MLH1 low (n = 134) breast cancer patient samples pooled from TCGA breast cancer data sets (TCGA Nature, Cell, Firehose Legacy). (J) Kaplan-Meier analysis showing survival status of patients from MLH1-high (n = 68) versus MLH1-low (n = 74) colorectal cancer patient samples obtained from TCGA Pancancer data set. Data were analyzed using unpaired, 2-tailed Student’s t test (G) with Holm-Šidák multiple-comparison test (C), Holm multiple-comparison test (E), and log-rank (Mantel-Cox) test (H–J). *P < 0.05; ***P < 0.001.