BRCA-mutated Cancer Research Articles

Abstract 2509: CDDO-Im induces DNA damage, G2/M arrest and apoptosis in BRCA1-mutated breast cancer cells

Abstract The breast cancer-associated gene 1 (BRCA1) is the most frequently mutated tumor suppressor gene in familial breast cancers. BRCA1 mutations also predispose to other types of cancers, pointing to a fundamental role of this pathway in tumor suppression. BRCA1 plays important roles in DNA damage and repair, homologous recombination, cell-cycle regulation, and apoptosis. The synthetic triterpenoid 1-[2-cyano-3,12-dioxooleana-1, 9(11)-dien-28-oyl]imidazole (CDDO-Im) is a promising anticancer agent with potent anti-proliferative and apoptotic activities against a wide variety of cancer types. However the mechanisms responsible for the selective apoptotic effects of CDDO-Im in cancer cells remain elusive. In the present work, treatment of BRCA1-mutated mammary tumor cells with CCDO-Im (0.3-1 μM) led to apoptosis as characterized by decreased thymidine incorporation, increased annexin V-binding, and cleavage of PARP and caspases-3. In addition, 0.3 μM CDDO-Im treatment depleted the G1 population of cells, which was accompanied by an accumulation of cells at G2 phase. CDDO-Im also activated the DNA damage response by inducing the phosphorylation of Chk1/2 and the up-regulation of p21, prior to the induction of apoptosis in BRCA1-mutated cancer cells. The inhibition of Chk1 by UCN-01, a Chk1 inhibitor abrogated G2/M arrest induced by CDDO-Im. To investigate how CDDO-Im activates the DNA damage response in BRCA1-mutated breast cancer cells, we performed a comet assay and Western Blot for γH2AX, a marker of DNA damage. CDDO-Im induced DNA damage and the phosphorylation of H2AX in BRCA1-mutated mammary tumor cells. Pretreatment with 0.3 μM CDDO-Im sensitized BRCA1-mutated cancer cells to carboplatin, a DNA-damaging agent, suggesting that CDDO-Im potentiates the effect of this drug. Moreover, CDDO-Im also induced reactive oxygen species (ROS) generation in these tumor cells, which is associated with the induction of DNA damage. The inhibition of ROS generation by uric acid prevented CDDO-Im-induced DNA damage. Furthermore, treatment with CDDO-Im was not cytotoxic to normal mouse 3T3 fibroblasts, highlighting a selective therapeutic potential of CDDO-Im for BRCA1-associated breast cancer. Taken together, our results demonstrate that CDDO-Im induces ROS and subsequent DNA damage, thereby facilitating the activation of the DNA damage checkpoint, G2/M arrest and finally apoptosis in Brca1-mutated cancer cells. These findings are being explored in vivo to determine whether CDDO-Im could be used therapeutically for BRCA1-associated breast cancer. This study was supported by Breast Cancer Research Foundation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2509.

A Genomic-Profile Derived Summary Measure of Chromosomal Breakpoints Predicts Response to Treatment with the DNA-Damaging Agent Cisplatin.

Abstract Cisplatin-based chemotherapy promotes DNA inter- and intra-strand cross-links and is effective treatment in multiple cancer types, yet it is rarely used for first-line therapy of breast cancer. Cisplatin is an effective cytotoxic in BRCA1 mutant cancer cell lines and in breast and ovarian cancers with BRCA1 mutations, prompting speculation that defective DNA double-strand break repair is associated with cisplatin sensitivity. Furthermore, tumors with BRCA1 mutation are presumed to be defective in various aspects of DNA repair and also display increased levels of chromosomal instability. Thus, we hypothesized that the total number of chromosomal breakpoints in a specific tumor may reflect defective DNA repair and/or chromosomal instability, and may therefore be used as a predictor of cisplatin sensitivity. Supporting this relationship, we identified a correlation (r = 0.8, P = 0.08) between the total number of chromosomal breakpoints, estimated by the number of genomic regions showing allelic imbalance, and cisplatin sensitivity in five triple negative breast cancer cell lines. To validate this relationship in a clinical setting we compared the total number of chromosomal breakpoints to therapy response in a cisplatin treated breast cancer cohort. A total of 28 women with stage II or III ER-/PR-/HER2- breast cancer were treated with cisplatin in the neo-adjuvant setting followed by surgery and standard adjuvant chemotherapy. A core biopsy was obtained before treatment commenced, tumor cells were enriched by needle microdissection, and DNA was extracted for genotyping. As only limited amounts of DNA were available, we used a Molecular Inversion Probe assay (in collaboration with Affymetrix), which allowed us to genotype 42,000 SNPs with only 40 ng of starting DNA. We then estimated the total number of putative genomic breakpoints in 21 samples containing at least 75% tumor cells. We compared this summary genomic measure to the response rate as quantitated by the Miller-Payne score determined on the pathological specimen obtained at surgery. This genomic measure showed a remarkably accurate separation of patients by degree of response, correctly classifying 20 out of 21 patients (P < 0.001).These results suggest that the total number of DNA breakpoints may be an accurate biomarker of breast cancers which may be sensitive to cisplatin treatment. The molecular explanations for these findings await further work. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 111.

The Expression of Phosphorylated mTOR in Breast Cancer from BRCA1-Mutation Carriers.

Abstract Background: The mammalian target of rapamycin (mTOR) is a serine-threonine kinase coupling growth stimuli and metabolic signals to cell cycle progression and protein synthesis. mTOR represents also a novel therapeutic target in various types of cancer. The role of mTOR in development and progression of breast cancer is not fully understood. There are studies suggesting that mTOR is responsible for the resistance of breast cancer to tamoxifen, trastuzumab and various chemotherapeutic agents. Since there are no studies evaluating the activation (phosphorylation) of mTOR in breast cancer cells carrying a germinal mutation of BRCA1, we have decided to assess activation of this kinase in tumor specimens in BRCA1-mutation carriers.Materials and methods: Formalin fixed paraffin-embedded tumor samples were obtained from 19 patients carrying BRCA1 germinal mutation and 24 patients with intact BRCA1 gene. All patients have been treated in the Great Poland Cancer Center between 2000-2008. The blocks were retrieved from the archives of the Department of Pathology and immunostained for phospho mTOR (Phospho-mTOR (Ser 2448), rabbit antibody, Cell Signaling).Results: Phosphorylation of mTOR was not detected in any of 19 patients carrying the mutated BRCA1 gene. Most of the BRCA1-mutated cancers were ER and HER2 negative (18 (95%) and 16 (94%), respectively). In the tumors with a wildtype BRCA1 gene, mTOR activation was detected in 12 samples (50%). Lack of mTOR phosphorylation was again observed mostly in ER and HER2 negative tissue samples (10 (83%) and 11 (92%), respectively).Discussion: Our findings have demonstrated differential activation of mTOR between tumors carrying a wild-type and mutated BRCA1 gene. We have also observed a negative correlation between ER and HER2 status and phosphorylation of mTOR. Although our study included a relatively low number of cases, the results indicate that activation of mTOR and probably also of its upstream targets do not play a crucial role in biology of tumors with a BRCA1 mutation or with the triple-negative phenotype. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3144.

Altered Proliferation and Differentiation Properties of Primary Mammary Epithelial Cells from BRCA1 Mutation Carriers

Female BRCA1 mutation carriers have a nearly 80% probability of developing breast cancer during their life-time. We hypothesized that the breast epithelium at risk in BRCA1 mutation carriers harbors mammary epithelial cells (MEC) with altered proliferation and differentiation properties. Using a three-dimensional culture technique to grow MECs ex vivo, we found that the ability to form colonies, an indication of clonality, was restricted to the aldehyde dehydrogenase 1-positive fraction in MECs but not in HCC1937 BRCA1-mutant cancer cells. Primary MECs from BRCA1 mutation carriers (n = 9) had a 28% greater ability for clonal growth compared with normal controls (n = 6; P = 0.006), and their colonies were significantly larger. Colonies in controls and BRCA1 mutation carriers stained positive for BRCA1 by immunohistochemistry, and 79% of the examined single colonies from BRCA1 carriers retained heterozygosity for BRCA1 (ROH). Colonies from BRCA1 mutation carriers frequently showed high epidermal growth factor receptor (EGFR) expression (71% EGFR positive versus 44% in controls) and were negative for estrogen receptor (ERalpha; 32% ER negative, 44% mixed, 24% ER positive versus 90% ER positive in controls). Expression of CK14 and p63 were not significantly different. Microarray studies revealed that colonies from BRCA1-mutant PMECs anticipate expression profiles found in BRCA1-related tumors, and that the EGFR pathway is up-regulated. We conclude that BRCA1 haploinsufficiency leads to an increased ability for clonal growth and proliferation in the PMECs of BRCA1 mutation carriers, possibly as a result of EGFR pathway activation. These altered growth and differentiation properties may render BRCA1-mutant PMECs vulnerable to transformation and predispose to the development of ER-negative, EGFR-positive breast cancers.

Basal-like subtype and BRCA1 dysfunction in breast cancers

Basal-like breast cancers are characterized by their unique expression profile, with the frequent loss of BRCA1, caused by such mechanisms as promoter methylation and the overexpression of high-mobility group proteins of the A type 1 or inhibitor of differentiation 4. Clinicopathologically, basal-like cancers are estrogen receptor-, progesterone receptor-, and human epidermal growth factor receptor type 2 (HER2)-negative; they are of high grade and have a poor prognosis. The fundamental similarity between BRCA1-mutated and basal-like cancers indicates that disruption of BRCA1 may be an essential common initial pathogenic event. Furthermore, p53 mutation and EGFR overexpression occur similarly in BRCA1-mutated and basal-like cancers; these shared alterations provide very important information for understanding not only the genetic and epigenetic carcinogenic pathways in these tumors but also therapeutic strategies. Despite the limited available clinical data about response to chemotherapy, anthracycline-based chemotherapy seems to be effective in a distinct subset of basal-like cancers. Both disrupted BRCA1 and overexpressed topoisomerase II-alpha possibly found in basal-like cancers are speculated to be associated with their increased sensitivity to anthracyclines. If these tumors respond to this chemotherapy, a favorable prognosis might be expected; however, in patients who do not respond, the prognosis is poor. Currently, the sensitivity of basal-like cancers to taxanes is not clear, but considering that these tumors have disrupted mitotic checkpoint function, a poor response may be suggested. On the basis of in vitro studies, BRCA1-disrupted basal-like cancers may be sensitive to DNA-damaging agents including platinum-based compounds, topoisomerase I and II inhibitors, and alkylating agents. In future, new therapeutic approaches for patients with basal-like cancers that are unlikely to respond to chemotherapy should focus on molecules that are involved in the pathogenic pathways of this disease.

Interplay among BRCA1, SIRT1, and Survivin during BRCA1-Associated Tumorigenesis

Germline mutations of BRCA1 predispose women to breast and ovarian cancers. However, the downstream mediators of BRCA1 function in tumor suppression remain elusive. We found that human BRCA1-associated breast cancers have lower levels of SIRT1 than their normal controls. We further demonstrated that mammary tumors from Brca1 mutant mice have low levels of Sirt1 and high levels of Survivin, which is reversed by induced expression of Brca1. BRCA1 binds to the SIRT1 promoter and increases SIRT1 expression, which in turn inhibits Survivin by changing the epigenetic modification of histone H3. Absence of SIRT1 blocks the regulation of Survivin by BRCA1. Furthermore, we demonstrated that activation of Sirt1 and inhibition of Survivin expression by resveratrol elicit a more profound inhibitory effect on Brca1 mutant cancer cells than on Brca1-wild-type cancer cells both in vitro and in vivo. These findings suggest that resveratrol treatment serves as an excellent strategy for targeted therapy for BRCA1-associated breast cancer.

Secondary BRCA1 Mutations in BRCA1-Mutated Ovarian Carcinomas with Platinum Resistance

Although ovarian carcinomas with mutated BRCA1 or BRCA2 are sensitive to platinum compounds, such carcinomas eventually develop platinum resistance. Previously, we showed that acquired resistance to cisplatin in BRCA2-mutated tumors can be mediated by secondary intragenic mutations in BRCA2 that restore the wild-type BRCA2 reading frame. Here, we show that secondary mutations of BRCA1 also occur in BRCA1-mutated ovarian cancer with platinum resistance. We evaluated nine recurrent BRCA1-mutated ovarian cancers previously treated with platinum compounds, including five with acquired platinum resistance, one with primary platinum resistance, and three with platinum sensitivity. Four of the six recurrent platinum-resistant tumors had developed secondary genetic changes in BRCA1 that restored the reading frame of the BRCA1 protein, whereas none of the three platinum-sensitive recurrent tumors developed BRCA1 sequence alterations. We immunohistochemically confirmed restored expression of BRCA1 protein in two cases with secondary mutations. Intriguingly, the case with primary platinum resistance showed back mutation of BRCA1 in the primary tumor and showed another secondary mutation in the recurrent tumor. Our results suggest that secondary mutations in BRCA1 can mediate resistance to platinum in BRCA1-mutated ovarian tumors.

Secondary mutations as a mechanism of cisplatin resistance in BRCA2-mutated cancers

Ovarian carcinomas with mutations in the tumour suppressor BRCA2 are particularly sensitive to platinum compounds. However, such carcinomas ultimately develop cisplatin resistance. The mechanism of that resistance is largely unknown. Here we show that acquired resistance to cisplatin can be mediated by secondary intragenic mutations in BRCA2 that restore the wild-type BRCA2 reading frame. First, in a cisplatin-resistant BRCA2-mutated breast-cancer cell line, HCC1428, a secondary genetic change in BRCA2 rescued BRCA2 function. Second, cisplatin selection of a BRCA2-mutated pancreatic cancer cell line, Capan-1 (refs 3, 4), led to five different secondary mutations that restored the wild-type BRCA2 reading frame. All clones with secondary mutations were resistant both to cisplatin and to a poly(ADP-ribose) polymerase (PARP) inhibitor (AG14361). Finally, we evaluated recurrent cancers from patients whose primary BRCA2-mutated ovarian carcinomas were treated with cisplatin. The recurrent tumour that acquired cisplatin resistance had undergone reversion of its BRCA2 mutation. Our results suggest that secondary mutations that restore the wild-type BRCA2 reading frame may be a major clinical mediator of acquired resistance to platinum-based chemotherapy.

Identification and characterization of cancer initiating cells from BRCA1 related mammary tumors using markers for normal mammary stem cells.

It is hypothesized that cancer stem cells arise either from normal stem cells or from progenitor cells that have gained the ability to self-renew. Here we determine whether mammary cancer stem cells can be isolated by using antibodies that have been used for the isolation of normal mammary stem cells. We show that BRCA1 mutant cancer cell lines contained a subpopulation of CD24+CD29+ or CD24+CD49f+ cells that exhibited increased proliferation and colony forming ability in vitro, and enhanced tumor-forming ability in vivo. The purified CD24+CD29+ cells could differentiate and reconstitute the heterogeneity found in parental cells when plated as a monolayer. Under low-attachment conditions, we detected "tumorspheres" only in the presence of double positive cells, which maintained their ability to self-renew. Furthermore, CD24+CD29+ cells could form tubular structures reminiscent of the mammary ductal tree when grown in three-dimensional cultures, implying that these cancer cells maintain some of the characteristics of the normal stem cells. Nevertheless, they could still drive tumor formation since as low as 500 double positive cells immediately after sorting from BRCA1 mutant primary tumors were able to form tumors with the same heterogeneity found in the original tumors. These data provide evidence that breast cancer stem cells originate from normal stem cells and advance our understanding of BRCA1-associated tumorigenesis with possible implications for future cancer treatment.

Double-strand break repair deficiency and radiation sensitivity in BRCA2 mutant cancer cells.

The protein product of the BRCA2 gene mediates repair of double-strand breaks in DNA. Because a number of cancer therapies exert cytotoxic effects via the initiation of double-strand breaks, cancers comprised of cells carrying BRCA2 gene mutations may be more amenable to treatment with agents that cause such breaks. We identified a human pancreatic adenocarcinoma cell line lacking one copy of the BRCA2 gene and containing a mutation (6174delT) in the remaining copy. In vitro and in vivo experiments were conducted with this cell line and with other carcinoma cell lines matched for similar genetic mutations, similar differentiation status, and/or similar carcinoma type to examine double-strand break repair, sensitivity to drugs that induce double-strand breaks, and radiation sensitivity. BRCA2-defective cells were unable to repair the double-strand DNA breaks induced by ionizing radiation. These cells were also markedly sensitive to mitoxantrone, amsacrine, and etoposide (drugs that induce double-strand breaks) (two-sided P = .002) and to ionizing radiation (two-sided P = .001). Introduction of antisense BRCA2 deoxyribonucleotides into cells possessing normal BRCA2 function led to increased sensitivity to mitoxantrone (two-sided P = .008). Tumors formed by injection of BRCA2-defective cells into nude mice were highly sensitive (>90% tumor size reduction, two-sided P = .002) to both ionizing radiation and mitoxantrone when compared with tumors exhibiting normal BRCA2 function. Histologic analysis of irradiated BRCA2-defective tumors showed a large degree of necrosis compared with that observed for control tumors possessing normal BRCA2 function. BRCA2-defective cancer cells are highly sensitive to agents that cause double-strand breaks in DNA.