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    Molecular Targets of Breast Cancer Prevention by n-3 PUFAs

    Scientific Abstract:
    Identification of Molecular Targets of n-3 PUFAs in Breast Cancer The incidence of breast cancer in the world is highest among American women and lowest among Japanese and Chinese women living in their native countries. However, the incidence of breast cancer among second and third generation American women of Chinese or Japanese ancestry approaches that of the general American population suggesting that some dietary or environmental factor(s) either promote breast cancer in the U.S. or interfere with breast cancer in Asian countries. One critical difference between the diets of Asian and American woman is that Asian diets are richer in n-3 polyunsaturated fatty acids (n-3 PUFAs). Epidemiological observations suggest that n-3 PUFAs such as eicosapentaenoic (EPA) in the diet influence the incidence of breast cancer. Studies of breast cancer cells and animal models of human breast cancer indicate that EPA inhibits proliferation of breast cancer cells in vitro and mammary tumor growth in vivo. However, the molecular basis of anti-cancer activity of EPA and its molecular effectors are not known. The n-3 PUFA EPA causes depletion of internal Ca++ stores and thereby phosphorylation of the alpha subunit of eIF2 (eIF2a) in human cancer cells. eIF2 is a guanine nucleotide binding translation initiation factor that is active only in the GTP bound form. Phosphorylation of eIF2a interferes with the GDP-GTP exchange and thus causes inhibition of translation initiation that preferentially abrogates translation of mRNAs coding for oncogenes while inducing the expression of tumor suppressors such BRCA1. Based on data published by others and us and on our preliminary data we hypothesize that the anti-cancer activity of EPA is mediated by phosphorylation of eIF2a and inhibition of translation initiation. The goal of the proposed research is to generate a translation-initiation specific animal model of breast cancer to determine if the anti-cancer activity of EPA is mediated by inhibition of translation initiation (Specific Aim 1), and to elucidate the direct molecular target and the cellular effectors of EPA (Specific Aim 2). To accomplish our first Specific Aim, we will transfect human breast cancer cells with an expression vector that codes either for a non-phosphorylatable mutant of eIF2a (eIF2a-51A) and yellow fluorescent protein (YFP) or wild type eIF2a and cyan fluorescent protein (CFP). The selected colonies of both types will be infected with a retroviral expression vector that codes for a small interfering RNA that will suppress expression of endogenous but not transfected (mutant or wild type) eIF2a. A mixture of these cells will be injected into the mammary gland of nude mice to form mammary tumors. The tumor-bearing mice will be treated with either EPA or vehicle and evaluated for the relative contribution of wild type (CFP) or mutant eIF2a expressing breast cancer cells (YFP) to tumor in EPA or vehicle treated mice will be determined. The tumors will also be stained with antibodies specific for phosphorylated eIF2a, total eIF2a, cyclin D1, and BRCA1. For Specific Aim 2, we will construct breast cancer cells expressing red fluorescent protein (RFP) under the control of the CHOP promoter, which is induced only when eIF2a is phosphorylated. We will infect these cells with a retroviral gene-trapping vector that also codes for promoter-less green fluorescent protein (GFP) and treat the cells with EPA (which will induce RFP expression). We will sort the cells that express GFP (indicating that retrovirus has interrupted an active gene) but fail to express RFP (suggesting that interrupted gene plays a role in induction of CHOP promoter by EPA). These cells will be subjected to two more rounds of screening and those that express GFP but not RFP will be cloned and the cellular gene interrupted by the retroviral vector will be identified. If these studies are successful they will identify translation initiation as a bona fide target for prevention/treatment of breast cancer, and provide critical preliminary information for the design of clinical trials aimed at defining effective methods to increase the consumption of n-3 PUFAs in amounts adequate to reduce the incidence of breast cancer. Furthermore they will identify novel molecular targets (i.e., effectors of EPA and similar agents) for the development of more potent anti-cancer agents for breast cancer prevention and/or therapy.

    Lay Abstract:
    Identification of Molecular Targets of Omega-3 Fatty Acids in Breast Cancer The incidence of breast cancer in the world is highest among American women and lowest among Japanese and Chinese women living in their native countries. First generation Japanese and Chinese woman in the U.S. similarly have a low incidence of breast cancer. However, their daughters and granddaughters suffer from breast cancer at the rate of Caucasian American women suggesting that some dietary or environmental factor(s) either promote breast cancer in the U.S. or interfere with breast cancer in Asian countries. One critical difference between the diets of Asian and American woman is that Asian diets contain less animal fat and are richer in omega-3 fatty acids found in fish, other marine animals and flaxseed. Asian diets are also high in soy. However, plant estrogens found in soy may promote breast cancer rather than prevent it. Some observational studies suggest that the dietary content of omega-3 polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), influences the incidence of breast cancer. Studies of breast cancer cells and animal models of human breast cancer indicate that EPA prevents multiplication of breast cancer cells in the laboratory and growth of mammary tumors in mice. The molecular basis of the anti-cancer activity of omega-3 fatty acids is, however, a matter of controversy. Omega-3 fatty acids such as EPA cause inactivation of an enzyme called eIF2. This enzyme is needed for making proteins. When the activity of eIF2 is reduced, fewer cancer-promoting proteins (called oncogenes) are made, while the amount of proteins needed for routine cellular functions remains about the same. Without these oncogenes the cancer cells cannot multiply. Furthermore a group of genes are activated when eIF2 activity is low. One of these genes called BRCA1 has been proven to prevent breast cancer. This gene is mutated in most familial breast cancers but in fewer cases of sporadic breast cancer. In most sporadic breast cancers there is no mutation in the BRCA1 gene but it isn’t able to generate as much BRCA1 protein compared to normal breast cells. We believe that EPA will cause cells to make fewer oncogenes and more BRCA1 proteins. Our hypothesis is that by simultaneously decreasing the amount of oncogenes that promote breast cancer while increasing the amount of BRCA1 protein that prevents breast cancer, EPA prevents multiplication of breast cancer cells and growth of mammary tumors. We plan to generate an animal model to test this hypothesis and propose a new strategy to identify the genes that play a role in the anti-cancer effects of EPA. These genes are currently unknown but most probably are novel targets for the development of effective agents for the prevention and/or therapy of breast cancer. If we are successful in our endeavor, eIF2 will emerge as a bona fide target for prevention and/or treatment of breast cancer. Furthermore, our findings will form a basis for the design of clinical trials aimed at defining effective methods to increase the consumption of omega-3 fatty acids in amounts adequate to reduce the incidence of breast cancer. Finally our studies will identify novel molecular targets for the development of more effective agents for breast cancer prevention and/or therapy.