Secondary BRCA1 and BRCA2 alterations and acquired chemoresistance

Abstract

Tumor suppressor BRCA1 and BRCA2 are frequently mutated in familial breast and ovarian cancer. More than ten percent of women with breast or ovarian cancer carry BRCA1 or BRCA2 (BRCA1/2) mutations. Cancers that arise in mutation carriers have often lost the wild-type allele through somatic alterations during tumor progression. BRCA1/2 play important roles in homologous recombination repair of DNA double-strand breaks. Because of this, BRCA1/2-deficient cancers often have a better response to DNA cross-linking agents such as platinum analogues and to poly(ADP-ribose) polymerase (PARP) inhibitors. However, over time, the majority of these BRCA1/2-deficient cancers become resistant and patients die from refractory diseases. Three recent studies demonstrated that acquired resistance to platinum analogues or PARP inhibitors in tumors carrying frame-shift BRCA1/2 mutations came from restored BRCA1/2 expression and HR function due to secondary intragenic mutations that corrected the open reading frames of mutated BRCA1/2.

Tumor suppressor BRCA1 and BRCA2 are frequently mutated in familial breast and ovarian cancer. More than ten percent of women with breast or ovarian cancer carry BRCA1 or BRCA2 (BRCA1/2) mutations. Cancers that arise in mutation carriers have often lost the wild-type allele through somatic alterations during tumor progression [1, 2]. BRCA1/2 play important roles in homologous recombination (HR) repair of DNA double-strand breaks [1, 2]. Because of this, BRCA1/2-deficient cancers often have a better response to DNA cross-linking agents such as platinum analogues and to poly(ADP-ribose) polymerase (PARP) inhibitors [3-5]. However, over time, the majority of these BRCA1/2-deficient cancers become resistant and patients die from refractory diseases.

Three recent studies demonstrated that acquired resistance to platinum or PARP inhibitors in tumors carrying frame-shift BRCA1/2 mutations came from restored BRCA1/2 expression and HR function due to secondary intragenic mutations that corrected the open reading frames (ORFs) of mutated BRCA1/2 [6-8]. Pancreatic cancer cell line CAPAN1 lacks a wild-type BRCA2 but carries a BRCA2 with a 6174delT frame-shift mutation, a founder mutation often detected in the Ashkenazi Jewish population [9, 10]. This 6174delT mutation results in expression of a truncated BRCA2 protein that lacks two BRC repeats, the DNA-binding/DSS1 (DBD) interaction domain, the second RAD51-binding domain (TR2) and the nuclear localization sequences (NLS) [6, 7, 10]. Ashworth's and Taniguchi's groups isolated PARP inhibitor-resistant [6] and cisplatin-resistant [7] clones from BRCA2-deficient CAPAN1 cells. These clones were resistant to both PARP inhibitors and cisplatin, but not to the microtubule-stabilizing agent, docetaxel [6, 7]. Most resistant clones possessed multiple copies of the BRCA2 gene. All resistant clones still carried the original 6174delT allele and the allele with secondary mutations [6, 7]. Ashworth's group found that the PARP inhibitor-resistant clones expressed BRCA2 alleles missing the region containing 6174delT mutation due to deletions ranging from 458 bp to 58 kb, and most of these deletions occurred in regions with small tracts of homology, possibly due to error-prone repair caused by BRCA2 deficiency. As a result, BRCA2 ORF was restored that included five BRC repeats, the C-terminal NLS and the TR2 RAD51 interaction domain [6].

Taniguchi's group found that, in cisplatin-resistant clones with restored BRCA2 expression, the BRCA2 ORF was restored by deletion, insertion, or deletion/insertion at sites close to the original 6174delT mutation site, or by in-frame deletions flanking the original mutation site [7]. They also found a 2,135 bp in-frame deletion flanking the 6174delT mutation in one cisplatin-resistant breast cancer cell line, HCC1428, that resulted in alternative splicing and expression of truncated BRCA2 proteins and restoration of BRCA2 function [7]. However, not all the cisplatin-resistant clones were found to carry the secondary BRCA2 mutations. In a half of resistant clones, there were no secondary mutations in BRCA2 and no restored BRCA2 expression and HR function were observed. These clones were also not resistant to PARP inhibition. This suggested that these clones acquired cisplatin resistance through mechanisms other than the restoration of HR function [7].

The HR function was restored in resistant clones expressing BRCA2 isoforms [6, 7]. By performing RNA interference experiments knocking down the expression of variant BRCA2 in resistant clones, and by reconstituting variant BRCA2 expression in BRCA2-deficient CAPAN1 and VC8 cells, they demonstrated that the restoration of HR function and acquired chemoresistance did indeed result from the expression of these variant BRCA2 proteins [6, 7].

Sequencing of DNAs from recurrent platinum-resistant ovarian tumors indicated that BRCA2 ORF was restored by deletions downstream of 6174delT mutation [6], or by reversion of 6174delT mutation back to wild-type [7].

In another study, Taniguchi's group showed that the acquired cisplatin resistance in recurrent BRCA1-deficient ovarian cancers resulted from the restoration of BRCA1 expression [8]. They analyzed cisplatin-resistant ovarian cancer tissues with a frame-shift 185delAG mutation in BRCA1, a founder mutation commonly found in the Ashkenazi Jewish population [11], and observed that recurrent tumors regained wild-type BRCA1 by genetic reversion of 185delAG to wild-type. In another case, the primary tumor was detected carrying a wild-type BRCA1 resulting from genetic reversion of a frame-shift 2594delC mutation in BRCA1, making this primary tumor resistant to cisplatin. In a recurrent tumor derived from this patient, the genetically reverted wild-type allele was lost, but a secondary deletion (2606-2628del23) was found in sequence with the inherited mutation (2594delC), resulting in the restoration of BRCA1 expression [8]. As expected, in recurrent cisplatin-sensitive tumors, no secondary genetic changes in BRCA1 were observed [8].

These studies provided insights on mechanisms of acquired chemoresistance to platinum analogues and PARP inhibitors in tumors carrying frame-shift BRCA1/2 mutations. It remains to be seen how resistance might develop in tumors carrying other types of BRCA1/2 mutations such as large segments of deletions or truncations. It is also unclear what roles other DNA repair mechanisms might play in acquired chemoresistance in BRCA1/2-defective cancers since increased nucleotide excision repair and loss of mismatch repair proteins have been associated with cisplatin-resistant ovarian cancers [12-16].