Research Grants Awarded
Genetic Determinants Of Chemosensitivity In Triple Negative Breast Cancer Cells
Sporadic breast cancers of the basal-like subtype are similar to breast tumors with BRCA1 mutations in their lack of expression of the estrogen, progesterone and HER2 receptors (triple negative). These triple negative sporadic basal-like breast cancers are refractory to commonly used chemotherapeutic agents and are often associated with a poor prognosis. Previously we demonstrated that compared to wild-type BRCA1 cells, BRCA1-deficient breast cancer cells are more sensitive to the chemotherapeutic drug gemcitabine. Our preliminary studies reveal that triple negative breast cancer cell lines show dramatically increased sensitivity to gemcitabine compared to luminal derived breast carcinoma cells. p63 and p73 are members of the p53 family, and the dNp63a isoform of p63 is upregulated in many cancers. Gemcitabine treatment decreased expression of dNp63a protein in triple-negative cells. Together, these data suggest that dNp63a protein may contribute to the chemosensitivity of the basal-like breast cancer cells to gemcitabine treatment. Human equilibrative nucleoside transporter (hENT1) mediates gemcitabine uptake. Once gemcitabine is inside mammalian cells, it undergoes a series of phosphorylations by deoxycytidine kinase (dCK), converting it to gemcitabine di- and triphosphate which act as the active drug metabolites. Gemcitabine cannot inhibit cell growth if it is not transported via hENT1 or phosphorylated by dCK, making them potential targets for therapeutics. Interestingly, in certain tumors, despite being readily converted to di- and triphosphates, gemcitabine is not very effective, suggesting that the response of tumors to this agent is also likely to depend on its ability to activate effectors of apoptosis. We therefore hypothesize that the drug transportation and metabolism pathway along with the p63/p73 signaling cascade may play an important role in chemosensitizing basal-like breast cancer cells. Our initial study also established sensitivity of triple negative breast cancer cells to PARP inhibition. Gemcitabine is frequently used in combination with other drugs like taxanes, vinorelbine, platinum salts for treatment of metastatic breast cancers, we hypothesize that combination therapy of gemcitabine and PARP inhibitor will be more effective than individual treatment. Moreover, combination treatment will help in reducing drug toxicity and delay or prevent drug resistance. The goal of this project is to study the genetic determinants of chemosensitivity of triple negative cells with the following specific aims: (i) To study the role of the p63/p73 signaling pathway in sensitizing basal-like cells to gemcitabine (ii) To investigate the role of human equilibrative nucleoside transporter-1 (hENT1) and deoxycytidine kinase (dCK) in chemosensitizing triple negative cells to gemcitabine (iii) study the combination drug regimen of gemcitabine and PARP inhibitor (KU58948) for treatment of basal-like breast cancer cells. Thus, studies proposed in this project will result in better understanding of the molecular mechanisms of these highly aggressive tumors and provide us with new biomarkers, therapeutic targets and novel treatment options.
Basal-like breast carcinomas are a newly defined subset of breast cancer with an aggressive phenotype that account for 15-30% of all breast cancers. Recently Carolina breast cancer study found the highest prevalence of basal-like tumors among premenopausal African American breast cancer patients. This study also showed that basal-like breast cancers exhibited aggressive features, including high proliferative capacity, high histologic grade, high nuclear grade, and frequent TP53 mutations. Sporadic breast cancers of the basal-like subtype are similar to breast tumors with BRCA1 mutations in their lack of expression of the estrogen (ER), progesterone (PR) and HER2 receptors (triple negative). Basal-like tumors are particularly enigmatic because the genes that are responsible for their aggressive phenotype are not well understood, a major barrier to developing targeted therapies. The urgent need for new therapies is underscored by the fact that basal-like breast carcinomas do not express ER or HER2 and thus are typically refractory to endocrine therapy and to trastuzumab, a humanized monoclonal antibody that targets HER2. Thus there is an urgent need of understanding the molecular mechanisms behind this aggressive phenotype and for developing targeted therapeutic. This proposal will study genetic determinants of chemosensitivity of triple negative breast cancer cells. Our preliminary study suggested that basal-like breast cancer cells are very sensitivity to gemcitabine, and gemcitabine is more potent than cisplatin, a chemotherapeutic drug found to be specifically effective in BRCA1 deficient breast cancers. Our study also found that basal-like breast cancer cells are sensitive to PARP inhibition, which is being widely tested for targeted therapeutics, particularly in breast cancers with mutations in BRCA1 and BRCA2. p63 and p73 belongs to a family of p53-related nuclear transcription factors. p63 is an essential regulatory factor of mammary epithelial development. In adult breast tissue, p63 expression is restricted to the basal myoepithelial cell layer, which is known to contribute to proliferation, differentiation, and polarity of mammary epithelia. Studies have shown that the dNp63a isoform of p63 is upregulated in many cancers and promotes tumor cell survival through repression of p73-dependent cell death. In basal-like breast carcinomas, dNp63a is upregulated anywhere up to 30%. Therefore targeting dNp63a for therapeutics seems to be critical due to (i) importance in mammary development, (ii) as a marker of stem cells , and (iii) its likely expression in basal-like breast cancers. Our preliminary data suggests that gemcitabine, decreases the protein expression of dNp63a in basal-like breast cancer cells. We therefore hypothesize that p63/p73 pathway may sensitize basal-like breast cancer cells to gemcitabine treatment. We will address this by studying the protein interaction of dNp63a and p73, and by knocking down the expression of dNp63a to investigate if deletion of this gene increases the sensitivity of basal-like breast cancer cells to gemcitabine. Many chemotherapeutic drugs including gemcitabine cannot inhibit cell growth unless transported inside the cell or converted to its active metabolite. Thus genes involved in transportation and metabolism of drugs make them potential targets for therapeutics. We will study the expression of these genes in basal-like breast cancer cells by quantitative PCR. Analyzing the levels of transportation and metabolism genes will allow us to prescreen patients that maybe sensitive/resistant to therapy. Combination drug regimens for the treatment of cancer often achieve therapeutic efficacy greater than that achieved with monotherapy. Other benefits may include decreased toxicity and delay or prevention of drug resistance development. We will test if combination treatment of gemcitabine and PARP inhibition has synergistic, additive, or antagonistic effects in basal-like breast cancer cells. We anticipate that studies proposed in this project will result in better understanding of the molecular mechanisms of these highly aggressive tumors and provide us with new biomarkers, therapeutic targets and novel treatment options.