We tested 25 ovarian cancer cell lines for expression of BRCA2 protein and found that BRCA2 protein was undetectable in PEO1 (). PEO1 was derived from the ascites of a patient with poorly differentiated ovarian serous adenocarcinoma that was still clinically responsive to cisplatin at the first relapse (14
). The clinical course of this patient is summarized in . Remarkably, in two ovarian cancer cell lines (PEO4 and PEO6) derived from the same patient obtained when her carcinoma had acquired clinical cisplatin resistance, BRCA2 protein was detectable (). PEO1 was sensitive to cisplatin and a PARP inhibitor (AG14361), while PEO4 was resistant ( and ), consistent with the BRCA2 protein expression status.
PEO1 is a BRCA2-deficient ovarian cancer cell line
Restoration of BRCA2 protein by secondary BRCA2 mutation in cisplatin-selected PEO1 clones
The patient turned out to be a heterozygous carrier of BRCA2.5193C>G (Y1655X) nonsense mutation (). Consistently, PEO1 had the hemizygous nonsense mutation (5193C>G). PEO1 is the first and only human BRCA2-deficient ovarian cancer cell line described, to the best of our knowledge.
Interestingly, PEO4 and PEO6 had a silent mutation (5193C>T, Y1655Y) instead (). In neoplastic cells in the ascites from which PEO4 and PEO6 were derived, at the relapse with clinical cisplatin resistance, 5193C>T was also detected. Thus, the secondary mutation (5193C>T) which canceled the inherited nonsense mutation (5193C>G) occurred in vivo.
Next, we selected PEO1 cells in cisplatin-containing medium (4μM) for four weeks in vitro and obtained 11 clones out of two million cells. Eight of them restored BRCA2 protein () and had a secondary BRCA2 mutation (5192A>T) (). This mutation is a single base pair substitution that changed the stop codon into an amino acid coding triplet leading to the restoration of the open reading frame (5192A>T; 5193C>G, Y1655L).
The eight BRCA2-restored PEO1 clones and PEO4 were cisplatin- and AG14361-resistant compared to parental PEO1 (), consistent with functionality of the restored BRCA2 proteins. Two of the three clones without BRCA2 restoration (C4-4, and C4-11) were cisplatin resistant, while the other clone (C4-13) was only slightly resistant. One of the cisplatin-resistant clones without restored BRCA2 (C4-4) was still sensitive to AG14361, consistent with the lack of BRCA2. Two cisplatin-resistant clones without restored BRCA2 (C4-11 and C4-13) showed intermediate sensitivity to AG14361, suggesting the existence of alternative mechanisms for the drug resistance.
Next, we analyzed the homologous recombination-based DNA double-strand-break repair function of the novel BRCA2 proteins using an I-SceI-dependent DR-GFP reporter assay in BRCA2-deficient V-C8 cells (16
) ( and Supplemental Fig S1
). In this assay, GFP expression correlates with the occurrence of homologous recombination. Transfection of a wild-type BRCA2
construct or constructs with the secondary BRCA2
mutations ((5192A>T; 5193C>G, Y1655L) or (5193C>T, Y1655Y)) resulted in seven to eight-fold more GFP-positive cells compared to control, whereas transfection of the 5193C>T (Y1655X) mutant construct resulted in impaired induction of GFP-positive cells. These results indicate that the secondary-mutated BRCA2 proteins efficiently promote homologous recombination.
The restored BRCA2 proteins with secondary BRCA2 mutations are functional
Functional BRCA2 is required for ionizing radiation-induced RAD51 foci formation (4
). Consistently, in parental PEO1 cells and the three BRCA2-non-restored PEO1 clones, ionizing radiation-induced RAD51 foci formation was severely impaired (). In contrast, it was restored in the eight BRCA2-restored PEO1 clones and in PEO4, again suggesting that the secondary-mutated BRCA2 proteins are functional.
Next, we depleted BRCA2 in two BRCA2-restored clones (C4-2 and C4-5) and PEO4 by siRNA transfection (). The BRCA2-depleted cells became sensitive to cisplatin/AG14361, indicating that the restored BRCA2 proteins are critical for the acquired cisplatin/AG14361 resistance. We confirmed this result using another BRCA2 siRNA (Supplemental Fig S2
). In contrast, BRCA2 siRNA had no effect on drug sensitivity of a BRCA2-non-restored clone C4-11 and parental PEO1 ().
BRCA2 restoration is critical for acquired drug resistance
We also selected PEO1 cells in AG14361-containing medium (4μM) for four weeks and obtained three clones out of one million cells. These clones restored BRCA2 protein, harbored the same secondary mutation (5192A>T), showed restored RAD51 foci formation, and were resistant to both cisplatin and AG14361 (Supplemental Fig S3A-D
). Depletion of BRCA2 sensitized these clones to cisplatin and AG14361 (Supplemental Fig S3E-F, S4
), indicating that restored BRCA2 was critical for the drug resistance.
These findings provide compelling evidence that restoration of functional BRCA2 protein by secondary BRCA2 mutation has a critical role in acquired platinum/PARP inhibitor resistance of BRCA2-mutated ovarian carcinomas. This concept has two important clinical implications. First, it suggests the importance of testing BRCA2 mutation status in recurrent ovarian carcinomas in BRCA2 mutation carriers to predict their response to platinum and PARP inhibitors. Second, it provides a theoretical basis for a strategy to overcome platinum/PARP inhibitor resistance. If the mechanism of resistance is restoration of BRCA2, inhibiting BRCA2 function is a logical way to re-sensitize the tumor to the drugs.
We may be able to apply the concept to other genes regulating DNA repair, such as BRCA1
. Indeed, secondary mutations of BRCA1
occur in BRCA1
-mutated ovarian cancer with platinum resistance (18
). Whether we can apply the concept to sporadic ovarian carcinomas with reduced BRCA1 or BRCA2 expression (19
) is also an important issue to be addressed in the future.