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Biochim Biophys Acta Mol Basis Dis. 2020 Jan 1;1866(1):165561. doi: 10.1016/j.bbadis.2019.165561. Epub 2019 Oct 19.

RAD6B is a major mediator of triple negative breast cancer cisplatin resistance: Regulation of translesion synthesis/Fanconi anemia crosstalk and BRCA1 independence.

Author information

1
Karmanos Cancer Institute, 421 E. Canfield Avenue, Detroit, MI 48201, USA; Department of Oncology, Wayne State University School of Medicine, 421 E. Canfield Avenue, Detroit, MI 48201, USA.
2
Karmanos Cancer Institute, 421 E. Canfield Avenue, Detroit, MI 48201, USA; Department of Oncology, Wayne State University School of Medicine, 421 E. Canfield Avenue, Detroit, MI 48201, USA; Department of Pathology, Wayne State University School of Medicine, 421 E. Canfield Avenue, Detroit, MI 48201, USA. Electronic address: shekharm@karmanos.org.

Abstract

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype with few therapy options besides chemotherapy. Although platinum-based drugs have shown initial activity in BRCA1-mutated TNBCs, chemoresistance remains a challenge. Here we show that RAD6B (UBE2B), a principal mediator of translesion synthesis (TLS), is overexpressed in BRCA1 wild-type and mutant TNBCs, and RAD6B overexpression correlates with poor survival. Pretreatment with a RAD6-selective inhibitor, SMI#9, enhanced cisplatin chemosensitivity of BRCA1 wild-type and mutant TNBCs. SMI#9 attenuated cisplatin-induced PCNA monoubiquitination (TLS marker), FANCD2 (Fanconi anemia (FA) activation marker), and TLS polymerase POL η. SMI#9-induced decreases in γH2AX levels were associated with concomitant inhibition of H2AX monoubiquitination, suggesting a key role for RAD6 in modulating cisplatin-induced γH2AX via H2AX monoubiquitination. Concordantly, SMI#9 inhibited γH2AX, POL η and FANCD2 foci formation. RAD51 foci formation was unaffected by SMI#9, however, its recruitment to double-strand breaks was inhibited. Using the DR-GFP-based assay, we showed that RAD6B silencing or SMI#9 treatment impairs homologous recombination (HR) in HR-proficient cells. DNA fiber assays confirmed that restart of cisplatin-stalled replicating forks is inhibited by SMI#9 in both BRCA1 wild-type and mutant TNBC cells. Consistent with the in vitro data, SMI#9 and cisplatin combination treatment inhibited BRCA1 wild-type and mutant TNBC growth as compared to controls. These RAD6B activities are unaffected by BRCA1 status of TNBCs suggesting that the RAD6B function in TLS/FA crosstalk could occur in HR-dependent and independent modes. Collectively, these data implicate RAD6 as an important therapeutic target for TNBCs irrespective of their BRCA1 status.

KEYWORDS:

BRCA1; Cisplatin; FANCD2; H2AX; Homologous recombination; POL η; Replication restart; Small molecule inhibitor; Triple negative breast cancer; Ubiquitination

PMID:
31639439
PMCID:
PMC6896319
[Available on 2021-01-01]
DOI:
10.1016/j.bbadis.2019.165561

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