RB1 deletion in RB-pathway disrupted cells results in DNA damage and cancer progression

Proliferative control in cancer cells is frequently disrupted by mutations in the RB-pathway. Paradoxically, RB1 mutations can arise late in tumorigenesis in cancer cells whose RB-pathway is already compromised by another mutation. In this study, we present evidence for increased DNA damage in CDKN2A deleted cancer cells when RB1 mutations are induced. We created isogenic RB1 wild type, heterozygous, and null genotypes. Cells with even one mutant copy of RB1 have increased basal levels of DNA damage and increased mitotic errors. Elevated levels of reactive oxygen species as well as impaired homologous recombination repair underlie this DNA damage. When xenografted into immune compromised mice RB1 mutant cells exhibit an elevated propensity to seed new tumors in recipient lungs. This study offers evidence that late arising RB1 mutations can facilitate genome instability and cancer progression that are beyond the pre-existing proliferative control deficit. γH2AX localization was studied in three different genotypes of U2OS osteosarcoma cells: RB1+/+, RB1+/- and RB1-/-. As a control for possible histone density differences, histone 4 (H4) ChIP-Seq was also performed.