Research Grants Awarded
The Role Of Pmca2 In Mammary Tumorigenesis
Career Catalyst Research
The acquisition of resistance to apoptosis is an important milestone in the progression of many tumors, including breast cancers. It is generally recognized that intracellular calcium (Ca) dynamics are integrally related to programmed cell death. Cellular Ca homeostasis is controlled by Ca entry, Ca sequestration in intracellular organelles, buffering by Ca-binding proteins, and the removal of Ca from the cell. The plasma membrane Ca-ATPases (PMCA?s) are thought to be the primary pumps in the plasma membrane responsible for maintaining the basal resting cytosolic Ca concentration by extruding excess Ca into the extracellular space. Dysfunction of PMCA?s can alter intracellular Ca responses and cause apoptosis. In the mammary gland, PMCA2 expression increases dramatically with the onset of lactation, and this PMCA isoform is responsible for bulk transport of Ca across the apical plasma membrane into milk. PMCA2 levels drop back to baseline immediately upon weaning, concomitant with the massive wave of apoptosis characteristic of post-lactational involution. In mice lacking functional PMCA2, a large proportion of mammary epithelial cells (MECs) aberrantly undergo apoptosis at the end of pregnancy. This suggests that loss of PMCA2 expression may contribute to involution by disrupting Ca balance and causing mammary epithelial cell apoptosis due to cytosolic Ca overload. As a corollary, increased PMCA2 expression might protect breast cancer cells against apoptosis by preventing Ca overload. In support of this possibility, W.J. Lee and colleagues have reported that breast cancer cells express higher levels of PMCA2 than non-malignant breast cells. In preliminary studies, we have found that MDA-MB-231 breast cancer cells, which express 400-fold higher levels of PMCA2 mRNA than T47D breast cancer cells, show less apoptosis at baseline and after taxol treatment as compared to T47D cells. Finally, in clinical studies of patients with breast cancer, tumor PMCA2 expression correlated positively with tumor grade, the presence of metastases, docetaxol resistance, and 5-year mortality. Therefore, the objective of this study is to determine whether PMCA2 overexpression protects breast cancer cells from apoptosis, thereby promoting the development or progression of the disease.
Our first Aim will examine the relationship between PMCA2 expression, cytosolic Ca, and apoptosis in human breast cancer cell lines. We will knockdown PMCA2 expression in MDA-MB-231 cells, and overexpress PMCA2 in T47D cells using tetracycline-regulated expression systems. We will utilize confocal ratiometric imaging of Fluo-4/Fura-Red Ca indicator dyes to evaluate the cytosolic Ca responses to taxol in these breast cancer cell lines. We will then measure taxol-induced apoptosis in the cells with altered PMCA2 expression using a cell death detection ELISA, morphological criteria, and cleaved caspase-3 staining. If our hypothesis is correct, PMCA2 knockdown in MDA-MB-231 cells should increase the cytosolic Ca response and the rate of apoptosis in response to taxol. Conversely, overexpression of PMCA2 in T47D cells should reduce the Ca response and desensitize the cells to apoptosis.
The second and third Aims will determine whether the absence of PMCA2, or overexpression of PMCA2 will alter tumor incidence, tumor growth, or taxol-resistance in a mouse model of breast cancer. We will mate ?deafwaddler? (dfw-2J) mice, lacking PMCA2, with MMTV-neu mice to generate a PMCA2-deficient transgenic mouse breast cancer model. To create a PMCA2 overexpressing model, we will generate mice carrying a tetracycline-responsive PMCA2 transgene (TetO-PMCA2). We will then mate these mice to MMTV-rtTA mice and MMTV-neu mice to obtain MMTV-neu/MMTV-rtTA/TetO-PMCA2 transgenic mice, which will express PMCA2 in MECs only when induced with doxycycline. We will measure tumor latency, size, number, and the occurrence of metastases in the resulting mice. We will also examine the mammary gland/mammary tumor histology by standard methods and perform TUNEL staining and cleaved caspase-3 staining to detect apoptotic cells. In addition, we will treat tumor-bearing mice with taxol to determine if over- or under-expression of PMCA2 modulates sensitivity to the drug. If our hypothesis is correct, then tumors should emerge more slowly and/or be smaller in MMTV-neu/dfw-2J mice (no PMCA2) due to increased apoptosis of developing tumor cells. In addition, tumors in MMTV-neu/dfw-2J mice should be more susceptible to taxol-induced apoptosis. Conversely, we expect PMCA2 overexpression to accelerate tumor progression and promote taxol-resistance by enabling tumor cells to escape apoptosis in the MMTV-neu/MMTV-rtTA/TetO-PMCA2 transgenic mice on doxycycline.
Understanding how breast cancer cells escape apoptosis is a key question. Our hope is that a better understanding of this process will lead to novel treatment strategies, especially for late-stage and drug-resistant breast cancers. If our hypotheses are correct, this research will identify PMCA2 as a potential therapeutic target for new drug development. More generally, this research may also highlight the machinery regulating calcium handling by breast cells as an area to examine for other viable drug targets in breast cancer.
Early in the development of breast cancer, tumors usually grow slowly because many of the newly formed tumor cells undergo programmed cell death (apoptosis). But in later stages of breast cancer, the tumor cells escape death, contributing to more rapid growth, invasion of surrounding tissue, metastasis, and the resistance to chemotherapeutic drugs. Understanding how breast cancer cells escape cell death is a key question, because then we could devise ways to prevent the escape of cell death and treat late-stage and drug-resistant breast cancers more effectively. Taxol (paclitaxel, docetaxol, etc.) is one of the most commonly employed chemotherapy drugs for treatment of breast cancer. One of the ways taxol works is by causing breast cancer cells to die by programmed cell death (apoptosis). When cells are treated with taxol, the calcium levels inside the cells increase, and we think that this is necessary for taxol to induce apoptosis in breast cancer cells. PMCA2 is a protein that is often found on the surface of breast cancer cells in higher amounts than normal cells. Our preliminary studies show that breast cancer cells with more PMCA2 are relatively resistant to taxol (less apoptosis, or cell death, goes along with more PMCA2 expression).
One objective of this study is to determine whether the amount of PMCA2 in breast cancer cell lines changes the increase in calcium inside the cells, and the subsequent cell death, when treated with taxol. We will genetically engineer a breast cancer cell line with low PMCA2 levels to overexpress PMCA2, and we will reduce PMCA2 expression in a breast cancer cell line with high expression. If our hypothesis is correct, increasing the amount of PMCA2 should decrease cell calcium and apoptosis, while decreasing PMCA2 levels should increase cell calcium and apoptosis in response to taxol treatment. Another objective is to determine, in living animals, whether the development, progression, or chemotherapy resistance of breast cancer is changed by manipulating the amount of PMCA2. We will use a mouse model of breast cancer in which mammary tumors develop that resemble human breast cancer tissue. Through mating genetically engineered mice, we will be able to generate two complementary mouse models: a) mice that develop mammary tumors and have no PMCA2 whatsoever, and b) mice that develop tumors that express high levels of PMCA2. These mice will allow us to determine, in the setting of live animals, whether the amount of PMCA2 in tumor cells changes how quickly the tumors develop, how large the tumors grow, how invasive the tumors become, and how extensively the tumors metastasize. In addition, by treating the tumor-bearing mice with a taxol chemotherapy regimen, we can measure how the level of PMCA2 in the tumors affects their response to the drug.
If breast cancers become refractory to the initiation of cell death because they increase their amount of PMCA2, then, in the future, targeting PMCA2 with drugs (or other approaches) could significantly improve the current therapies for breast cancer. If the effect of PMCA2 on the level of calcium inside the cell is the key to how PMCA2 de-sensitizes breast cancer cells to cell death, this could lead to the development of a whole class of therapies designed to alter cellular calcium levels. These therapies would increase the tumor cell calcium levels, so that taxol, or other existing therapies, would be more effective at initiating cell death. Because the potential therapies resulting from targeting PMCA2 would be especially useful in chemotherapy-resistant breast cancers, this research has the potential to greatly improve morbidity and mortality resulting from the disease.