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 4

MLH1 resolves R-loops in BRCA2-deficient cells.

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MLH1 resolves R-loops in BRCA2-deficient cells. (A) Quantitation of PLA ...
(A) Quantitation of PLA foci formed between S9.6-PCNA in Mlh1-silenced Brca2KO/KO-r or Brca2cKO/KO-mi clones. (B) DRIP assay showing the enrichment of R-loops in Brca2KO/KO-r or Brca2cKO/KO-mi clones by RT-qPCR. IgG was used as a negative control, and RNaseH treatment was used to confirm R-loops. Values are expressed as percentages of input. Actb genes analyzed are depicted (insert). (C) TCGA analysis showing copy number alteration frequency of RNASEH1 in BRCA2-low breast carcinoma samples under MLH1-unaltered versus MLH1-low (MLH1 Exp: <-1) versus MLH1-high (MLH1 Exp: >1) conditions. Data are from ref. 51 (left) and ref. 52(right). (D) ChIP-immunoblot showing levels of MLH1 on approximately 500 bp fragmented genomic DNA from KB2P1.21 pulled down with IgG or S9.6. Input of 1% was used as a control. (E) ChIP-immunoblot showing levels of MLH1 on in vitro–developed R-loop structure (DDR) pulled down with IgG or S9.6. MLH1 pure protein was used for this assay. (F) In vitro nuclease assay showing degradation of RNA strand in P32-labeled R-loop substrate by MLH1 (0, 250 ng, and 500 ng) and FEN1 protein (0, 25 ng, or 50 ng). RNA strand with 5′ overhangs is labeled in red. (G) DRIP assay showing enrichment of R-loops in Actb gene in Mlh1-silenced Brca2KO/KO-r or Brca2cKO/KO-mi clones by RT-qPCR. (H) Representative images of PLA foci formed between S9.6-γH2AX in Mlh1- or Mlh3-silenced or combination of Mlh1/3-silenced Brca2KO/KO-r or Brca2cKO/KO-mi clones (left panel). Quantitation of PLA foci (right panel). Lower panel shows immunoblot confirmation of Mlh1 or Mlh3 silencing. Original magnification, ×63. (I) Model showing replication-transcription conflict in MLH1-high/low conditions in Brca2KO/KO-r clones. Data are represented as mean ± SEM for A, B, G, and H (n = 3 biological replicate). Data were analyzed using unpaired, 2-tailed Student’s t test (A and H) and unpaired, 2-tailed Student’s t test with Holm-Šidák multiple-comparison test (B and G). *P < 0.05; **P < 0.01; ***P < 0.001.