Proteomics

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Codon Specific Readthrough as a Mechanism of BRCA2 Restoration in Acquired PARP Inhibitor and Chemotherapy Resistance

ABSTRACT: Alteration of the gene encoding BRCA2, a protein involved in homologous recombination (HR), by missense or nonsense mutations results in sensitivity of a subset of breast and ovarian cancers to platinating agents and poly(ADP-ribose) polymerase (PARP) inhibitors. Conversely, resistance to these agents is often accompanied by secondary BRCA2 mutations that restore a functional protein. Here we characterized clones derived from the BRCA2-mutant ovarian cancer line PEO1 by selection with the PARP inhibitor veliparib. Colony forming assays demonstrated cross-resistance of the resulting clones to multiple PARP inhibitors and cisplatin. Despite the continued presence of a 4965C>A (p.Y1655X) BRCA2 nonsense mutation, HR was restored in the resistant cells. Immunoprecipitation followed by immunoblotting or mass spectrometry demonstrated expression of low levels of BRCA2 protein, including peptides distal to the premature termination codon. Reporter assays demonstrated readthrough selective for the UAG stop codon in the veliparib-resistant clones but not parental PEO1 cells; and mass spectrometry revealed insertion of tyrosine rather than termination in the readthrough product. Mass spectrometry and immunoblotting provided evidence for readthrough of UAGs in additional proteins in the resistant clones. BRCA2 gene interruption 3’ to the stop codon restored sensitivity of the resistant clones to PARP inhibitors and cisplatin, implicating the low level BRCA2 expression in resistance. Accordingly, the present results identify low level readthrough of nonsense codons as a potential mechanism of drug resistance that might need to be considered when assessing the impact of mutations on the biology of cancers.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

DISEASE(S): Malignant Neoplasm Of Ovary

SUBMITTER: Xinyan Wu  

LAB HEAD: Xinayn Wu

PROVIDER: PXD037989 | Pride | 2025-10-27

REPOSITORIES: Pride

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Publications

BRCA2 mutations contribute to the pathogenesis and treatment sensitivity of a subset of ovarian, breast, prostate, and pancreatic cancers. When these cancers become therapy resistant, secondary mutations that restore the BRCA2 open reading frame are found in half the cases, but other causes of resistance remain incompletely understood. Here, we identified translational readthrough of a premature termination codon (PTC) as a cause of resistance to poly(ADP-ribose) polymerase inhibitors (PARPis) a  ...[more]

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