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

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 ?. SMI#9-induced decreases in ?H2AX levels were associated with concomitant inhibition of H2AX monoubiquitination, suggesting a key role for RAD6 in modulating cisplatin-induced ?H2AX via H2AX monoubiquitination. Concordantly, SMI#9 inhibited ?H2AX, POL ? 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.

SUBMITTER: Haynes B

PROVIDER: S-EPMC6896319 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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

Haynes Brittany B Gajan Ambikai A Nangia-Makker Pratima P Shekhar Malathy P MP

Biochimica et biophysica acta. Molecular basis of disease 20191019 1

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 ...[more]

PMID: 31639439

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Project description:BackgroundPlatinum compounds such as cisplatin and carboplatin are DNA crosslinking agents widely used for cancer chemotherapy. However, the effectiveness of platinum compounds is often tempered by the acquisition of cellular drug resistance. Until now, no pharmacological approach has successfully overcome cisplatin resistance in cancer treatment. Since the Fanconi anemia (FA) pathway is a DNA damage response pathway required for cellular resistance to DNA interstrand crosslinking agents, identification of small molecules that inhibit the FA pathway may reveal classes of chemicals that sensitize cancer cells to cisplatin.ResultsThrough a cell-based screening assay of over 16,000 chemicals, we identified 26 small molecules that inhibit ionizing radiation and cisplatin-induced FANCD2 foci formation, a marker of FA pathway activity, in multiple human cell lines. Most of these small molecules also compromised ionizing radiation-induced RAD51 foci formation and homologous recombination repair, indicating that they are not selective toward the regulation of FANCD2. These compounds include known inhibitors of the proteasome, cathepsin B, lysosome, CHK1, HSP90, CDK and PKC, and several uncharacterized chemicals including a novel proteasome inhibitor (Chembridge compound 5929407).Isobologram analyses demonstrated that half of the identified molecules sensitized ovarian cancer cells to cisplatin. Among them, 9 demonstrated increased efficiency toward FA pathway-proficient, cisplatin-resistant ovarian cancer cells. Six small molecules, including bortezomib (proteasome inhibitor), CA-074-Me (cathepsin B inhibitor) and 17-AAG (HSP90 inhibitor), synergized with cisplatin specifically in FA-proficient ovarian cancer cells (2008 + FANCF), but not in FA-deficient isogenic cells (2008). In addition, geldanamycin (HSP90 inhibitor) and two CHK1 inhibitors (UCN-01 and SB218078) exhibited a significantly stronger synergism with cisplatin in FA-proficient cells when compared to FA-deficient cells, suggesting a contribution of their FA pathway inhibitory activity to cisplatin sensitization.ConclusionOur findings suggest that, despite their lack of specificity, pharmaceutical inhibition of the FA pathway by bortezomib, CA-074-Me, CHK1 inhibitors or HSP90 inhibitors may be a promising strategy to sensitize cisplatin-resistant, FA pathway-proficient tumor cells to cisplatin. In addition, we identified four new small molecules which synergize with cisplatin. Further development of their analogs and evaluation of their combination with cisplatin may lead to the development of efficient cancer treatments.

Dataset Information

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

Publications

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

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