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Triple-negative breast cancers (TNBC) are typically resistant to treatment, and strategies that build upon frontline therapy are needed. Targeting the murine double minute 2 (Mdm2) protein is an attractive approach, as Mdm2 levels are elevated in many therapy-refractive breast cancers. The Mdm2 protein–protein interaction inhibitor Nutlin-3a blocks the binding of Mdm2 to key signaling molecules such as p53 and p73α and can result in activation of cell death signaling pathways. In the present study, the therapeutic potential of carboplatin and Nutlin-3a to treat TNBC was investigated, as carboplatin is under evaluation in clinical trials for TNBC. In mutant p53 TMD231 TNBC cells, carboplatin and Nutlin-3a led to increased Mdm2 and was strongly synergistic in promoting cell death in vitro. Furthermore, sensitivity of TNBC cells to combination treatment was dependent on p73α. Following combination treatment, γH2AX increased and Mdm2 localized to a larger degree to chromatin compared with single-agent treatment, consistent with previous observations that Mdm2 binds to the Mre11/Rad50/Nbs1 complex associated with DNA and inhibits the DNA damage response. In vivo efficacy studies were conducted in the TMD231 orthotopic mammary fat pad model in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Using an intermittent dosing schedule of combined carboplatin and Nutlin-3a, there was a significant reduction in primary tumor growth and lung metastases compared with vehicle and single-agent treatments. In addition, there was minimal toxicity to the bone marrow and normal tissues. These studies demonstrate that Mdm2 holds promise as a therapeutic target in combination with conventional therapy and may lead to new clinical therapies for TNBC. Mol Cancer Ther; 14(12); 2850–63. ©2015 AACR.

Treatment of drug-resistant cancer cells remains a difficult problem in cancer therapy because most resistant cells can pump out drugs or upregulate other survival pathways to bypass a targeted therapy. To study cancers that are resistant to the common cancer drug, paclitaxel, a novel paclitaxel-resistant cell line was generated from the breast cancer cell line MDA-MB-231. A “spiking” method of paclitaxel treatment was used to select for a population of cells that are resistant to the drug. This method mimics the development of resistance in recurrent tumors in patients. The IC50 of paclitaxel in these cells was 75 μM compared to the 0.037 μM IC50 of the parent cell line, a 2000-fold increase in resistance. In this very heterogeneous population, the mechanism of resistance was not due to increased protein levels of the efflux protein, p-glycoprotein, as quantitated by western blot analysis. To better study these cells, the paclitaxel-resistant cell line was cloned using a limiting dilution method to provide more homogeneous populations of resistant cells. The 29 clones obtained exhibited a paclitaxel IC50 range of 8 μM to 78 μM which was equivalent to a 200- to 2000-fold increase in resistance compared to the parent line. It has been suggested that the polyphenol natural compound, resveratrol, which has been shown to inhibit cell growth of multiple cancer types, may be useful as a combination anti-cancer treatment or novel therapeutic for drug-resistant cancer cells. There was no significant difference among the 72 hour IC50 of resveratrol in the parent line, the heterogeneous resistant line, the least paclitaxel-resistant clone or the most paclitaxel-resistant clone. We observed that treatment with 10-100 μM concentrations of resveratrol in all cell lines showed a reduction in cell proliferation and increased apoptosis within 72 hours (p Citation Format: Alyssa A. Sprouse, Brittney-Shea Herbert. Molecular mechanisms of paclitaxel-resistance and resveratrol sensitivity in MDA-MB-231 breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 876. doi:10.1158/1538-7445.AM2013-876

Treatment of drug-resistant cancer cells remains a difficult problem in cancer therapy because most resistant cells can pump out drugs or upregulate other survival pathways to bypass a targeted therapy. The polyphenol natural compound, resveratrol, has been shown to inhibit cell growth of multiple cancer types, but it is not cytotoxic to normal cells. However, the effects of resveratrol in triple negative breast cancer cells (estrogen receptor-, progesterone receptor-, and HER2-negative) as well as cancers that are resistant to the common cancer drug, paclitaxel, are not well understood. In this study, the effects of resveratrol were investigated in the triple negative breast cancer cell line MDA-MB-231 as well as a novel MDA-MB-231 derived paclitaxel-resistant line generated in our laboratory. Assays for cell proliferation, apoptosis, and protein expression changes related to apoptosis and survival were utilized in order to determine the differential effects of resveratrol on the parental and resistant cell lines. At lower (< 10 μM) concentrations, resveratrol induced cell proliferation in both cell lines, consistent with other studies demonstrating lifespan extension of normal cells with resveratrol treatment. On the other hand, after treatment with 10-100 μM concentrations of resveratrol, both cell lines exhibited a reduction in cell proliferation, with the paclitaxel-resistant cells to a greater extent. In addition, resveratrol decreased the ability of both cell lines to form colonies when plated at low density (an indication of reduced cell survival capacity). Furthermore, resveratrol treatment increased the amount of DNA fragmentation associated with apoptosis compared to untreated controls in both cell lines, but the paclitaxel resistant cells were more sensitive to resveratrol treatment than the parental cells. By protein expression analyses, we observed that in both the parental and paclitaxel-resistant cell lines, resveratrol may be acting through NAD-dependent deacetylase sirtuin (SIRT1) activity by decreasing the expression of the inhibitor-of-apoptosis protein, surviving and increasing the activator-of-apoptosis, caspase 3. Based on these data, we propose that resveratrol can inhibit proliferation and induce apoptosis in triple negative breast cancer cells, including paclitaxel-resistant cells. These results provide rationale for the use of resveratrol as an important starting point for the development of a novel anti-cancer agent for drug resistant, aggressive cancers as well as in combination with other anti-cancer drugs without significant toxicity to normal cells. 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 5676. doi:1538-7445.AM2012-5676