Women with ovarian cancer generally have a high initial response to platinum-based chemotherapy, but over time the majority of ovarian carcinomas become refractory, and most patients die with progressive chemoresistant disease 2
. The molecular basis of initial platinum sensitivity and acquired resistance remains largely unknown. Individuals with germline mutations in BRCA1
) have an increased risk of developing cancer in the breast and ovary 5
as well as in some other organs including the pancreas and prostate 6
is identical to the Fanconi anemia (FA) gene FANCD1 7
. The BRCA2 protein directly binds to and regulates RAD51, an essential protein for DNA repair through homologous recombination (HR) 8
. Tumors from BRCA1/2
mutation carriers usually have deletion of the wild-type allele at the BRCA1/2
locus and are BRCA1/2-deficient 9–11
. BRCA1/2-deficient cancer cells are hypersensitive to DNA-crosslinking agents including cisplatin 1, 12
. Therefore, cisplatin or its derivative, carboplatin, is a logical choice for the treatment of BRCA1/2
-mutated tumors 13
and women with BRCA1/2
-mutated ovarian carcinoma have a better prognosis than those without BRCA1/2
mutation if they receive platinum-based therapy 14, 15
. However, even BRCA1/2
-mutated tumors eventually develop platinum resistance. In several genetic disorders such as FA, spontaneous genetic alterations compensating for inherited disease-causing mutations have been described 16
. These alterations in FA include secondary genetic changes of one of the mutated FA alleles, such as back-mutations to wild-type, compensatory mutations in cis
, intragenic crossovers, and gene conversion 16–18
. We speculated that secondary mutations in BRCA2
-mutated alleles might also occur in cancer cells during chemotherapy.
To study the mechanism of acquired cisplatin resistance of BRCA2
-mutated cancer, first we screened 12 human breast cancer cell lines for alterations in BRCA2 protein expression (). A pancreatic cancer cell line, Capan-1 3, 4
, with a truncated BRCA2 protein, was used as a control.
HCC1428 is a cisplatin-resistant breast cancer cell line with a secondary BRCA2 mutation
From breast cancer cell line, HCC1428 19
, BRCA2 protein was undetectable by western blotting using anti-BRCA2 (Ab-1), which recognizes the middle part of BRCA2 () but was detectable with anti-BRCA2 (Ab-2) that recognizes the BRCA2 C-terminus. The small size of HCC1428 BRCA2 identified by Ab-2 suggested that the protein might have an internal in-frame deletion. Constitutional DNA from a lymphoblast cell line (HCC1428BL) from the same patient had one wild-type BRCA2
allele and one mutant allele with a frameshift mutation, 6174delT, which is common in the Ashkenazi Jewish population ( and S1
Sequence of genomic DNA from HCC1428 cancer cells revealed no wild-type allele and a mutant allele with a 2135-basepair deletion surrounding the original 6174delT mutation and the exon 11/intron 11 junction ( and S1
). Sequence of cDNA indicated that this deletion activated two cryptic splice donor sites in exon 11, resulting in expression of two transcripts with in-frame deletions (, and S1
). HCC1428 transcript 1 had a 2640-basepair deletion and encoded a protein lacking amino acids 1401 to 2281. Transcript 2 had a 2187-basepair deletion and encoded a protein lacking amino acids 1552 to 2281 ( and S1
). Both HCC1428 BRCA2 proteins retain the single-strand DNA (ssDNA) binding domain and the C-terminus nuclear localization signals (NLS), whereas these domains are lost in BRCA2.6174delT
(). A recent report that only 1 BRC repeat plus ssDNA binding domain and NLS are sufficient for BRCA2 function 21
suggests that the novel BRCA2 proteins in HCC1428 might be functional. Indeed, HCC1428 cells were resistant to cisplatin (). Furthermore, depletion of these novel BRCA2 proteins with siRNA restored sensitivity of HCC1428 cells to cisplatin ().
HCC1428 was derived after chemotherapy from the pleural effusion of a 49 year-old woman with stage IV breast carcinoma who died 6 months later 19
. We speculate that the patient’s chemotherapy selected in vivo
for secondary mutations in BRCA2
-mutated breast tumor cells.
To test this hypothesis, we selected in vitro
for cisplatin-resistant clones from the cisplatin-sensitive BRCA2
-mutated pancreatic cancer cell line Capan-1. Capan-1 has the mutant allele BRCA2.6174delT
, but no wild-type allele (Fig. S5a
) 3, 4
. Fluorescence in situ hybridization (FISH) revealed that Capan-1 harbors at least two copies of the BRCA2
gene indicating duplication of the chromosome 13 with the mutant BRCA2
gene (Fig. S2
After selection in cisplatin, we obtained 14 cisplatin-resistant Capan-1 clones out of 12 million cells (Fig. S3 and Table S1
). Importantly, in 7 of these 14 clones, BRCA2 expression, at close to the length of the wild-type protein, was now detectable (). The other 7 cisplatin-resistant clones still lacked BRCA2 protein expression.
Secondary genetic changes in mutated BRCA2 in cisplatin-resistant clones of a pancreatic cancer cell line, Capan-1
In all 7 cisplatin-resistant clones with restored, nearly full-length BRCA2 protein expression, we identified additional BRCA2
mutations that corrected the frameshift caused by the 6174delT mutation (, S4, and S5, and Table S1
). These secondary genetic changes included a small deletion, insertion, and deletion/insertion at sites close to the original mutation, and in-frame deletions surrounding the original mutation site. Interestingly, in each clone we observed both the original mutant BRCA2.6174delT
sequence and the 6174delT sequence with additional mutations (Figs. S4 and S5
) indicating that the secondary mutations occurred on only one of the duplicated mutant BRCA2
copies (Fig. S5g
). None of the 7 cisplatin-resistant clones lacking BRCA2 protein expression harbored additional mutations in BRCA2.
Next, we assessed the function of the restored BRCA2 proteins in the 14 cisplatin-resistant Capan-1 clones by evaluating RAD51 foci formation after exposure to ionizing radiation (IR) (). IR-induced RAD51 foci formation was impaired in parental Capan-1 1
. In contrast, in 6 of the 7 cisplatin-resistant Capan-1 clones with restored BRCA2 expression, IR-induced RAD51 foci formation was significantly improved, suggesting that these novel BRCA2 isoforms are functional.
Functional analyses of the restored BRCA2 proteins
In most of the cisplatin-resistant Capan-1 clones without secondary mutations, IR-induced RAD51 foci formation remained impaired (), suggesting that these clones acquired cisplatin resistance through mechanisms other than the restoration of the BRCA2-RAD51 pathway.
Next, we analyzed the homologous recombination (HR)-based DNA double-strand-break (DSB) repair function of some of the novel BRCA2 proteins using an I-SceI-dependent DR-GFP reporter assay in BRCA2-deficient VC-8 Chinese hamster cells 22
. The proportions of GFP-positive cells arising through the repair of I-SceI-induced DSB by HR after transfection of various mutant BRCA2
constructs were compared ( and S6, Table S2
). Transfection of a wild-type BRCA2
construct resulted in about 5-fold more GFP-positive cells compared to transfection of vector control or the BRCA2.6174delT
mutant. Transfection of constructs with any of the secondary BRCA2
mutations resulted in GFP-positive frequencies equal to or greater than that of the construct with wild-type BRCA2
. These results indicate that the novel BRCA2 proteins efficiently promote HR.
Poly(ADP-ribose) polymerase (PARP) inhibitors selectively kill BRCA1/2-deficient tumor cells 23, 24
and are expected to become a therapeutic option for patients with BRCA1/2
-mutated cancers 13
. However, it remains unclear whether BRCA1/2
-mutated tumors with acquired cisplatin resistance are sensitive to PARP inhibitors. We tested the sensitivity of cisplatin-resistant Capan-1 clones to a PARP inhibitor (AG14361) (). Both parental Capan-1 and cisplatin resistant clones without secondary mutations were sensitive to the PARP inhibitor. In contrast, Capan-1 clones with secondary BRCA2
mutations were resistant, consistent with the restoration of functional BRCA2 in these clones, although the differences of the sensitivity between parental Capan-1 and these clones were relatively small.
Finally, we analyzed tissues of 5 patients with BRCA2
mutations and recurrent ovarian carcinomas previously treated with platinum. Three recurrent tumors were clinically refractory to platinum and two were sensitive (Table S3
). Platinum-refractory tumor UW3548 revealed genetic reversion of BRCA2.6174delT
(). Evidence for genetic reversion is as follows. Constitutional DNA of this patient was heterozygous for BRCA2.6174delT
and at two single nucleotide polymorphisms (SNPs). As expected, the microdissected specimen of the recurrent tumor was hemizygous at each SNP, reflecting loss of heterozygosity of BRCA2
in the tumor. However, in the same tumor sample, both sequences of BRCA2.6174delT
and wild-type BRCA2
were detected, suggesting that the recurrent tumor had acquired wild-type BRCA2
by genetic reversion, or back mutation to wild-type. We speculate that the appearance of both mutant and wild-type sequences resulted from duplication of the mutant BRCA2
followed by genetic reversion of one of the duplicates, similar to the situation occurring in Capan-1 clones with secondary mutations (Figs. S4 and S5
). A larger clinical study is warranted to determine the prevalence and clinical significance of in vivo
secondary mutations in BRCA2
in human tumors.
Genetic reversion of BRCA2 in platinum-resistant recurrent BRCA2-mutated ovarian cancer
The second platinum-refractory relapsed tumor (CS2) presented a complex profile. This patient carried germline mutations in both BRCA1
). The primary ovarian tumor was sensitive to platinum and showed loss of the wild-type BRCA1
allele and heterozygosity of BRCA
2, indicating that carcinogenesis was primarily driven by the BRCA1
mutation. Importantly, the recurrent platinum-refractory tumor had lost the mutant BRCA2
allele. We speculate that loss of the mutant BRCA2
allele was the result of selection by chemotherapy and contributed to platinum resistance.
The tumor of a third patient (UW174) recurred 5 months after her primary chemotherapy, so her tumor is defined as clinically refractory to platinum. No secondary mutation in BRCA2 was detectable in the tumor specimen of this patient. Recurrent tumors of two other patients (CS9 and CS15) were sensitive to cisplatin. No secondary genetic alterations in BRCA2 were identified in either of these tumors.
Taken together, our data demonstrate that secondary mutations that correct the frameshift caused by mutated BRCA2
alleles are a mechanism of acquired resistance to cisplatin (Fig. S8
). Testing for secondary mutations in platinum-treated BRCA2
-mutated cancers may be clinically important, because tumors with secondary mutations are likely to be resistant to both cisplatin and PARP inhibitors. Theoretically, it might be possible to re-sensitize these tumors to cisplatin and to PARP inhibitors by treatment with drugs such as proteasome inhibitors that inhibit RAD51 recruitment to sites of DNA repair 25
. In contrast, platinum-resistant BRCA2
-mutated tumors without secondary BRCA2
mutations may remain sensitive to PARP inhibitors.
Treatment with cisplatin could facilitate secondary mutations by increasing the mutation rate via DNA damaging effects. Alternatively, since BRCA2 is involved in error-free DNA repair, HR, it is possible that BRCA2 deficiency itself promotes secondary mutations through compensatory utilization of more error-prone DNA repair processes 26
Secondary mutations are relevant as a mechanism of resistance only in those tumors that contain frameshift mutations in BRCA2
, which occurs in only 5% of ovarian carcinomas. However, BRCA2
mRNA expression is undetectable in 13% of ovarian carcinoma 27
. We speculate that other mechanisms yet to be identified could restore BRCA2 in acquired cisplatin resistance in these sporadic cases.
Our findings may be applicable to cancers other than ovary. Secondary mutations in BRCA2
occurred in a mitomycin C-resistant human acute myeloid leukemia cell line with biallelic BRCA2
mutations derived from an FA (D1 subtype) patient 28
-mutated Chinese hamster fibroblast lines 29
, suggesting that secondary mutations in BRCA2
may have a general role in resistance to DNA-crosslinking agents in cells derived from various organs.
Secondary mutation of mutated BRCA2
is reminiscent of mechanisms of acquired resistance of Philadelphia-chromosome-positive leukemia to imatinib 30
, such as BCR-ABL point mutations that prevent imatinib binding. In both cases, the disease-causing mutations increase tumor sensitivity to specific agents, but development of further mutations in the disease-causing genes during targeted drug treatment leads to drug resistance. It will be important to explore mechanisms for restoring drug sensitivity.