Research Grants Awarded
Control of mammary epithelial hyperplasia and malignant progression by p21-activated kinase 1 (PAK1)
Tumor Cell Biology II
Background: Despite earlier diagnoses and improved treatment, breast carcinoma remains the second largest cause of female cancer deaths. Most breast malignancies are ductal carcinomas and thus represent derangements of the normal glandular structure of the breast. Since superfluous epithelial cells normally undergo a form of programmed-cell-death termed anoikis, loss of anoikis may be a primary factor in the initiating stages of breast cancer. Another critical element in breast cancer is increased and inappropriate expression and activation of specific proteases. Protease deregulation is important for metastasis, by facilitating invasion, but it is also vital for the development of breast cancer. The cysteine protease cathepsin B, for example, participates in the formation of premalignant lesions, in angiogenesis, and in the growth control of primary and metastatic tumors. Recent data have strongly implicated p21-activated kinase 1 (PAK1) in human breast cancer, where it is often over-expressed. Expression of a constitutively-activated PAK1, or over-expression of wild-type PAK1 in the presence of serum stimulation, is sufficient to suppress anoikis in breast epithelial cells. Objective, Hypothesis and Aims: The long-range goal of our work is to understand how critical events in early stage breast cancer and its malignant progression can be successfully exploited in improved strategies for prevention and treatment. The objective of this proposal is to determine the role of PAK1 in breast cancer. We will test our hypothesis that PAK1 is a key coordinator of both the suppression of anoikis and of the aberrant proteolysis that are characteristic of breast cancer progression through the following specific aims: 1. Determine the role of PAK1 activation in the proliferation and survival of MCF10A cells in 3-d culture. 2. Determine the role of PAK1 in the hyperplastic growth and survival of the MCF10A breast cancer progression model. 3. Define the role of PAK1 activation in the altered expression, subcellular localization, and activity of cathepsin B. Study Design: The hypothesis will be tested through experiments performed in 3-d cultures of the MCF10A-derived human breast cancer progression series. MCF10A is a spontaneously immortalized human breast epithelial cell line that retains many normal characteristics, including producing a luminal, acinar morphology in 3-d culture, and does not form tumors in mouse xenografts. The lines NeoT, AT-1, and DCIS represent a progression series from MCF10A cells that are defined by increasing tumor formation and malignancy. We will use retroviruses to manipulate PAK1 expression and activity. Our assays will be primarily based on functional imaging technology, supported by other techniques, such as western blotting, where appropriate. We will assay cell proliferation and survival, cell polarity and differentiation, and extracellular, cell surface and intracellular proteolysis by cathepsin B. Potential outcomes: This work will develop a useful model of human breast cancer that will be of further benefit to the research community in future studies. Protein kinases are being validated as pharmacological targets in many human diseases and there is intense effort to identify selective PAK1 inhibitors that are safe and effective. Successful completion of this project would be a step towards validating PAK1 as a therapeutic target for breast cancer.
We need to understand the very early events in breast cancer, as it is presumably at that stage that we will be most effective at curing, and potentially even preventing, the disease. We also need to understand the critical factors in the malignant progression of breast cancer, because it is the later stage, invasive disease that spreads to other tissues of the body (i.e. metastasis) that is the cause of most mortality. Cancer is a disease where one cell type proliferates and divides to such an extent that it imposes an unsupportable burden on the body. Recently we have come to realize that any extra cells that are produced by proliferation should automatically undergo a form of cellular suicide known as programmed-cell-death. We now think that this normal process of thinning out the population of cells in the glands of the breast is a fundamental mechanism that guards against cancer. Thus loss of this normal safeguard mechanism of cellular suicide is a primary factor in the initiating stages of breast cancer. Another critical element in breast cancer is increased and inappropriate destruction of proteins. It is impossible to study these events in women, and studies in animals may be limited by species differences. In order to characterize the factors that determine protein destruction and resistance of human breast cancer to cellular suicide we propose, therefore, to grow human mammary cells in an advanced three-dimensional matrix that models the physiological and pathological conditions of early breast cancer. Recent data have identified a protein kinase called p21-activated kinase 1 (PAK1) as able to supply a powerful and aberrant survival signal to breast cells. We propose that in addition to this cell survival function of PAK1, it is also a strong candidate to coordinate growth with inappropriate protein destruction, which is another hallmark of the malignant progression of breast cancer to metastatic disease. We will use genetically engineered retroviruses to manipulate PAK1 activity in three-dimensional cultures. We will use state-of-the-art functional probes to reveal cell proliferation and survival plus the course of protein destruction. The long-range goal of our work is to understand how critical events in early stage and malignant breast cancer can be successfully exploited in new strategies for effective prevention and therapy. The objective of this proposal is to determine the role of PAK1 in breast cancer through study of 3-d cultures of human mammary cells. We believe that these studies will develop a useful model of human breast cancer that will benefit the research community in future studies as well. The rationale for our proposed studies is that identification of these critical events will provide innovative pharmacological approaches to the prevention and treatment of breast cancer. Once a new therapeutic is identified then there is often a considerable delay before clinical trials can be started. In the case of PAK1, however, there is great current effort in both academic laboratories and in pharmaceutical companies to identify selective inhibitors that are safe and effective. Since such inhibitors have been found for many other protein kinases, it is reasonable to assume that if the hypothesis of this grant is validated, then initial clinical trails could be expected within five years.