Elizabeth Alli

Synergistic Chemosensitivity of Triple-Negative Breast Cancer Cell Lines to Poly(ADP-Ribose) Polymerase Inhibition, Gemcitabine, and Cisplatin

The basal-like subtype of breast cancer is characterized by a triple-negative (TN) phenotype (estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2/neu negative). TN breast cancers share similar gene expression profiles and DNA repair deficiencies with BRCA1-associated breast cancers. BRCA1-mutant cells exhibit sensitivity to gemcitabine, cisplatin, and poly(ADP-ribose) polymerase (PARP) inhibition; therefore, we hypothesized that TN cancer cells may also exhibit sensitivity to these drugs. In this study, we report that TN breast cancer cells are more sensitive to these drugs compared with non-TN breast cancer cells. Moreover, combination treatments indicated that PARP inhibition by the small-molecule inhibitor PJ34 or siRNA knockdown synergized with gemcitabine and cisplatin in TN cells but not in luminal cancer cells. TN cells exhibited reduced repair of UV-induced cyclobutane pyrimidine dimers after PARP inhibition, suggesting that the synergistic effect of PJ34 and gemcitabine or cisplatin reflected inefficient nucleotide excision repair. Mechanistic investigations revealed that in TN cells, PJ34 reduced the levels of ΔNp63α with a concurrent increase in p73 and its downstream target p21. Thus, the sensitivity to combination treatment seemed to be mediated by sustained DNA damage and inefficient DNA repair triggering p63/p73-mediated apoptosis. Our results suggest a novel therapeutic strategy to treat women with TN breast cancer, an aggressive disease that presently lacks effective treatment options.

Defective Repair of Oxidative DNA Damage in Triple-Negative Breast Cancer Confers Sensitivity to Inhibition of Poly(ADP-Ribose) Polymerase

Subtypes of breast cancer that represent the two major types of epithelial cells in the breast (luminal and basal) carry distinct histopathologic profiles. Breast cancers of the basal-like subtype, which include the majority of hereditary breast cancers due to mutations in the breast cancer susceptibility gene 1 (BRCA1), frequently assume triple-negative status, i.e., they lack expression of estrogen receptor-alpha and progesterone receptor, and lack overexpression or amplification of the HER2/NEU oncogene. Defects in DNA damage response pathways result in genome instability and lead to carcinogenesis, but may also be exploited for therapeutic purposes. We analyzed repair of oxidative DNA damage by the base-excision repair (BER) pathway, which when aberrant leads to genomic instability and breast carcinogenesis, in cell lines that represent the different subtypes of breast cancer and in the presence of BRCA1 deficiency. We found that basal-like and BRCA1-mutated breast cancer cells were defective in BER of oxidative DNA damage, and that this defect conferred sensitivity to inhibition of poly(ADP-ribose) polymerase, a DNA repair enzyme. The defect may be attributed, at least in part, to a novel role for BRCA1 in the BER pathway. Overall, these data offer preventive, prognostic, and therapeutic usefulness.

Reversal of stathmin-mediated resistance to paclitaxel and vinblastine in human breast carcinoma cells.

Antimicrotubule agents are commonly used chemotherapy drugs for the treatment of breast and other cancers. However, these agents have variable activity partly because of microtubule regulatory proteins. Stathmin, an 18-kDa phosphoprotein that promotes microtubule depolymerization, was found to be frequently overexpressed in breast cancer. We previously identified stathmin-mediated mechanisms of resistance to antimicrotubule agents, including altered drug binding and delayed transit from G2 into M phase, where these agents are effective in disrupting microtubule dynamics. We hypothesized that by reversing stathmin-mediated depolymerization of microtubules or by promoting entry into mitosis, this could increase sensitivity to antimicrotubule agents in human breast cancer cells overexpressing stathmin. We found that targeting stathmin or wee-1 expression with RNA interference can induce microtubule polymerization and promote G2/M progression, respectively, and sensitize stathmin-overexpressing breast cancer cells to paclitaxel and vinblastine. Furthermore, targeting wee-1 led to the phosphorylation of stathmin, which is known to attenuate its activity. Therefore, these data suggest a novel approach to improving the efficacy of certain antimicrotubule agents against breast cancer by regulating the function of stathmin.

Silencing of stathmin induces tumor-suppressor function in breast cancer cell lines harboring mutant p53

Cancers harboring dominant-negative p53 mutations are often aggressive and difficult to treat. Direct attempts to restore wild-type p53 function have produced little clinical benefit. We investigated whether targeting a p53-target gene could induce certain tumor-suppressor characteristics. We found that inhibition of stathmin, a microtubule regulator that can be transcriptionally repressed by wild-type p53, restored certain wild-type functions to cancer cells with mutant p53. Silencing of stathmin by small interfering RNA (siRNA) in mutant p53 cell lines lowered expression to that observed following activation of wild-type p53 by DNA damage in wild-type p53 cell lines. siRNA-induced repression of stathmin decreased cell proliferation, viability and clonogenicity in mutant p53 cell lines. Furthermore, knockdown of stathmin partially restored cell-cycle regulation and activation of apoptosis. Therefore, targeting stathmin, a gene product that is overexpressed in the presence of mutant p53, may represent a novel approach to treating cancers with aberrant p53 function.

BRCA1: Beyond double-strand break repair.

Since its discovery, the BRCA1 tumor suppressor has been shown to play a role in multiple DNA damage response pathways. Here, we will review the involvement of BRCA1 in base-excision DNA repair and highlight its clinical implications.

Therapeutic targeting of BRCA1-mutated breast cancers with agents that activate DNA repair

Cancers due to germline mutations in the BRCA1 gene tend to lack targets for approved chemoprevention agents. This study aimed at a targeted chemoprevention strategy for BRCA1-associated malignancies. Mutant BRCA1 limits the base-excision DNA repair activity that addresses oxidative DNA damage, the accumulation of which heightens ones risk for cancer. Therefore, we conducted a high-throughput chemical screen to identify drug candidates that could attenuate the inhibitory effects of mutant BRCA1 on this repair activity, thereby describing a new class of DNA repair-activating chemopreventive agents. In the screen design, such drugs functioned by enhancing base-excision DNA repair of oxidative DNA damage in the presence of mutant BRCA1, with minimal cytotoxicity. We identified at least one new agent that decreased malignant properties associated with tumorigenesis, including anchorage-independent growth and tumor progression. This work offers a preclinical proof-of-concept for a wholly new approach to chemoprevention in carriers of BRCA1 mutations as a strategy to reduce the prevalence of BRCA1-associated malignancy.

BRCA1: a movement toward cancer prevention.

Breast cancer susceptibility gene 1 (BRCA1) was first identified in 1994 and has since been shown to encode a tumor suppressor protein that maintains genetic stability through DNA damage response pathways. Carriers of mutations in BRCA1 are predisposed to breast and ovarian cancer; however, their cancers lack the targets for existing anticancer drugs. We describe a novel chemoprevention approach that uses DNA repair-activating agents to enhance the repair of oxidative DNA damage and, in turn, prevent tumorigenesis in the presence of mutant BRCA1.

Abstract 2966: Targeting defective DNA repair as a novel chemoprevention strategy for BRCA1-mutated breast cancer

Carriers of germline mutations in the Breast Cancer Susceptibility Gene 1 (BRCA1) have an increased risk for developing breast cancer. Unfortunately, BRCA1-mutated cancers are not amenable to current chemoprevention options, often associate with an aggressive clinical course, and thus, are in need of an effective prevention strategy. We previously found that BRCA1 plays a role in DNA base-excision repair (BER) of oxidative DNA damage, and that BRCA1-mutated breast cancers exhibit a compromised ability for BER of oxidative DNA damage. Given that excessive oxidative DNA damage leads to tumorigenesis, we hypothesized that small molecules may be used to enhance the repair of oxidative DNA damage, and in turn, prevent tumorigenesis of BRCA1-mutated breast cancer cells. First, a high-throughput chemical screen identified small molecules that enhance BER of oxidative DNA damage in the presence of mutant BRCA1. These molecules have been termed DNA repair-activating agents. At least two DNA repair-activating agents significantly enhanced BER in mutant BRCA1 but not wild-type BRCA1 cell lines. These molecules also decreased basal levels of oxidative DNA damage as determined by flow cytometry using a FITC-conjugated 8oxoG-binding protein and decreased H 2 O 2 -induced oxidative DNA damage as determined by the alkaline comet assay modified for detection of oxidized lesions. Both DNA repair-activating agents directly activated BER, rather than indirectly as a result of induction of DNA damage, as evidenced by the alkaline comet assay for DNA strand breaks. Both agents also showed no cytotoxicity at concentrations that enhanced BER of oxidative DNA damage, which is ideal for chemoprevention. Finally, at least one of the DNA repair-activating agents decreased BRCA1-associated tumorigenesis in vitro and in vivo. The DNA repair-activating agent decreased anchorage-independent growth of BRCA1-mutant/deficient cells without a significant effect on cell viability, as well as delayed tumor formation and decreased tumor burden in a dose-response manner in a xenograft mouse model. Taken together, these data suggest that enhancing DNA base-excision repair of oxidative DNA damage may be a novel strategy for the targeted chemoprevention of BRCA1-associated breast cancers. Citation Format: Elizabeth Alli, David Solow-Cordero, Stephanie C. Casey, James M. Ford. Targeting defective DNA repair as a novel chemoprevention strategy for BRCA1-mutated breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2966. doi:10.1158/1538-7445.AM2014-2966

Abstract A043: Targeting defective DNA repair for the prevention of BRCA1-mutated breast cancer

Carriers of germline mutations in the Breast Cancer Susceptibility Gene 1 (BRCA1) have an increased risk for developing breast cancer. Unfortunately, BRCA1-mutated cancers are not amenable to current chemoprevention options, often associate with an aggressive clinical course, and thus, are in need of an effective prevention strategy. We previously found that BRCA1 plays a role in DNA base-excision repair (BER) of oxidative DNA damage (ODD), and that BRCA1-mutated breast cancers exhibit a compromised ability for BER of ODD. Given that excessive ODD leads to tumorigenesis, we hypothesized that repair of ODD may be enhanced to prevent tumorigenesis of BRCA1-mutated breast cancer. To test this hypothesis, we first identified drugs that enhance BER of ODD in the presence of mutant BRCA1. To do so, we conducted a high-throughput chemical screen using small molecules with known pharmacological activity. Two of these compounds, which have been approved for other clinical indications, enhanced BER in mutant BRCA1 but not wild-type BRCA1 cell lines. These molecules also decreased basal levels of ODD as determined by flow cytometry using a FITC-conjugated 8oxoG-binding protein and decreased H2O2-induced ODD as determined by the alkaline comet assay modified for detection of oxidized lesions. Second, we analyzed certain effects of these drugs that would negatively affect their ability to function as chemoprevention agents. Both drugs showed no cytotoxicity at concentrations that enhanced BER of ODD as seen by the MTT assay and Hoechst-staining for live cells. Both drugs directly activated BER, rather than indirectly as a result of induction of DNA damage, as evidenced by the alkaline comet assay for DNA strand breaks. Finally, at least one of these drugs decreased in vitro tumorigenesis of BRCA1-mutant and isogenic BRCA1-deficient cells using the soft agar colony formation assay and simultaneously had no significant effect on cell viability as determined by the trypan blue exclusion assay. Taken together, these data suggest a novel strategy for the targeted chemoprevention of BRCA1-associated breast cancers. Citation Format: Elizabeth Alli, Dave Solow-Cordero. Targeting defective DNA repair for the prevention of BRCA1-mutated breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A043.

Abstract 1761: Breast cancers with compromised DNA repair exhibit selective sensitivity to elesclomol-induced oxidative DNA damage

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Hereditary breast cancers due to germline mutations in BRCA1 and sporadic breast cancers of the basal-like subtype are often triple-negative, i.e. lack expression of estrogen and progesterone receptors and lack overexpression/amplification of the HER2/neu oncogene. Given that the triple-negative status of these cancers renders them relatively insensitive to existing “targeted” therapy, improved treatment options are needed. We tested a panel of cell lines that represent the different molecular subtypes of breast cancer for sensitivity to elesclomol, an experimental therapeutic that produces elevated levels of oxidative DNA damage, using the MTT assay. We found that both BRCA1-mutated and basal-like breast cancer cell lines with defective base-excision DNA repair (BER) were markedly more sensitive to elesclomol than cell lines that represent the normal breast or other subtypes of breast cancer with proficient BER. We also assessed sensitivity to elesclomol in breast cancer cell lines that were stably transduced with shRNA to OGG1, a BER glycosylase required for the repair of the most common type of oxidative DNA damage. Two different cell lines with decreased levels of OGG1 (shOGG1A, 50% knock-down; shOGG1B, 20% knock-down) were more sensitive to elesclomol (IC50 = 0.04nM and 0.3nM, respectively) compared to two different isogenic control cell lines (IC50 = 2nM and 3nM, respectively). Taken together, these data suggest that compromised repair of oxidative DNA damage by BER represents a functional target for elesclomol. Overall, BRCA1-mutated and/or basal-like breast cancers may benefit from treatment regimens that include elesclomol. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1761. doi:1538-7445.AM2012-1761