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    Awarded Grants
    Molecular Mechanisms and Therapeutic Implications of a Novel Bi-Directional Cross-Talk Between the Lipogenic Enzyme Fatty Acid Synthase (FAS) and the HER-2/neu Oncogene in Breast Cancer

    Scientific Abstract:
    BRCTR0403141: Molecular mechanisms and therapeutic implications of a novel bi-directional cross-talk between the lipogenic enzyme Fatty Acid Synthase (FAS) and the HER-2/neu oncogene in breast cancer BACKGROUND: Activity and expression of Fatty Acid Synthase (FAS), a critical enzyme in the de novo biosynthesis of fatty acids in mammals, are exquisitely sensitive to nutritional regulation of lipogenesis in liver or adipose tissue. Thus, most human tissues express very low levels of FAS, since the requirement for fatty acids is sufficiently met by dietary intake under normal conditions. Surprisingly, a number of studies have demonstrated hyperactivity and overexpression of FAS in virulent human breast cancer, suggesting that FAS-dependent neoplastic lipogenesis is unresponsive to nutritional regulation. Indeed, FAS-overexpressing patients demonstrate shortened disease-free intervals or overall survivals, even in node negative disease. FAS up-regulation has also been identified in intraductal and lobular in situ breast carcinoma, lesions associated with increased risk for the development of infiltrating breast cancer. Notably, the relative absence of FAS in normal tissue has led to the notion that its inhibition should provide a highly selective basis for a novel breast cancer therapy. Unfortunately, very little is known about the molecular mechanisms that lead to FAS dysregulation in human breast cancer. RATIONALE: We have recently identified a novel link between FAS and HER-2/neu, one of the most well characterized oncogenes in human breast cancer. HER-2/neu overexpression occurs in approximately 30% of breast carcinomas, and has been correlated with a particularly aggressive course and poor clinical outcome. HER-2/neu overexpression is linked to elevated tumorigenicity, metastatic potential, and resistance to chemotherapy. In HER-2/neu breast cancer overexpressors, we demonstrated that inhibition of FAS activity by the natural mycotoxin cerulenin or by the related synthetic FAS inhibitor C75, did negatively regulate HER-2/neu mRNA, protein, and tyrosine-kynase activity. FAS blockade induced up-regulation of PEA3 (a transcriptional repressor of HER-2/neu) and promoted a re-localization of HER-2/neu from the plasma membrane to the cytoplasmic/nuclear compartments. Co-exposure to FAS inhibitors and trastuzumab (a humanized monoclonal antibody directed against the extracellular domain of HER-2/neu) synergistically down-regulated HER-2/neu, and was synergistically cytotoxic by promoting apoptosis. Interestingly, the degree of HER-2/neu oncogene expression but not FAS status predicted hypersensitivity to cellular toxicity after FAS inhibition. Remarkably, the activation of HER-2/neu signaling by both HER-2/neu overexpression and Heregulin (HRG)-induced transactivation of HER-2/neu, induced up-regulation of FAS expression, a phenotype that was prevented by trastuzumab, strongly suggesting that a novel bi-directional molecular cross-talk between HER-2/neu and FAS may take place in human breast cancer. OBJECTIVE/HYPOTHESIS: The purpose of this project will be to characterize the HER-2/neu oncogene as a novel molecular sensor for the maintenance of an elevated FAS-dependent endogenous fatty acid synthesis in human breast cancer. We will evaluate how FAS activity is contributing to HER-2/neu-promoted tumorigenicity, metastasis, chemoresistance, and angiogenesis. This project will also evaluate whether FAS repression can reverse HER-2/neu-induced transformation and breast cancer progression, and whether FAS repression can sensitize breast cancer to both conventional chemotherapy and HER-2/neu-targeted therapies. STUDY DESIGN: We have formulated the following aims: 1) to evaluate whether FAS repression modulates HER-2/neu-induced tumorigenicity. Using RNA silencing interference techniques, we will assess whether FAS signaling modulates HER-2/neu expression and function. The ability of FAS blockade to inhibit HER-2/neu-induced transformation in vivo will be tested in breast cancer xenografts; 2) to assess whether FAS blockade prevents HER-2/neu-induced chemoresistance. The chemosensitivity of HER-2/neu-overexpressors to taxanes, vinca alkaloids, anthracyclines, alkylators, and anti-metabolites will be examined before and after FAS gene silencing. By using the median effect-combination index methodology, we will establish the nature of the interaction (antagonism, addition, or synergism) between pharmacological inhibitors of FAS activity and chemotherapy in HER-2/neu-overexpressing breast cancer models. To investigate clinically relevant transcriptional events after FAS blockade, gene expression profiling of pro-apoptotic and apoptosis inhibitors-related genes will be performed by microarray analysis; 3) to evaluate the in vivo tumor growth of HER-2/neu-overexpressing breast cancer xenografts co-treated with FAS inhibitors and anti-HER-2/neu therapies. We will investigate whether down-regulation of HER-2/neu, better induction of apoptosis, and reduced Vascular Endothelial Growth Factor (VEGF)-promoted angiogenesis are the underlying mechanisms for the expected tumor growth inhibition after FAS inhibition; 4) to investigate whether CYR61, an angiogenic factor overexpressed in HRG-positive breast cancer cells, is sufficient to bypass the HER-2/neu requirement for HRG-induced activation of FAS. RELEVANCE: Our study proposes that HER-2/neu oncogene is a novel molecular sensor that plays a key role in the cellular response to a metabolic stress after perturbation of abnormal breast cancer associated-FAS hyperactivity. In terms of a clinical perspective, a sensitization of tumor cells to both conventional chemotherapy and HER-2/neu-based therapies by FAS blockade raises the possibility that a therapeutic targeting of FAS may be clinically useful in the treatment of HER-2/neu-overexpressing breast carcinomas.

    Lay Abstract:
    BRCTR0403141: Molecular mechanisms and therapeutic implications of a novel bi-directional cross-talk between the lipogenic enzyme Fatty Acid Synthase (FAS) and the HER-2/neu oncogene in breast cancer Dietary or exogenously derived fatty acids have emerged as an intense focus of research and controversy to breast cancer genesis, prevention and/or therapy. Thus, the high level of fat in the Western diet has been implicated in the development of many human malignancies including breast cancer. However, despite this link between dietary fatty acids and the development of breast cancer, relatively little information exists on the role of “endogenously” synthesized fatty acids. Endogenous fatty acid metabolism is an important part of normal human physiology. Thus, fatty acids or their derivatives comprise biological membranes, serve as hormones and intracellular “messengers”, and act as fuel molecules to provide energy. In well-nourished individuals, the role of Fatty Acid Synthase (FAS), a very complex protein working as a “factory” of endogenous fatty acids, is of minor importance due to high levels of dietary fat. In other words, most normal cells and tissues under use endogenous fatty acids, and FAS concentration in lipogenic tissues (liver and adipose tissue) remains at very low levels because the mechanism that regulates FAS levels in normal tissues is exquisitely sensitive to nutritional and hormonal status. Unexpectedly, although possibly related, a number of experimental and clinical studies have reported that human breast cancer cells exhibit gigantic amounts of FAS. Remarkably, patients expressing high levels of FAS demonstrate a poorer prognosis, and hence, shortened overall survivals. Enormous amounts of FAS have been observed in lesions associated with increased risk for breast cancer development. Moreover, elevated levels of FAS have also been identified in the blood of breast cancer patients compared with healthy subjects. Notably, the relative absence of FAS in normal tissues has led to the notion that its inhibition should provide a highly selective basis for a novel anticancer therapy. In light of these evidences, it is understandable that FAS-dependent endogenous production of fatty acids, an “energy-storage” pathway largely considered of minor importance in human, may be ultimately be used for diagnosis, prognosis, early intervention, and treatment of a very aggressive subset of human breast tumors. Unfortunately, very little is known about the mechanisms controlling this abnormal role of FAS in malignant cells. We have recently identified a novel connection between FAS and HER-2/neu oncogene, one of the most well-characterized malignant proteins in human breast cancer. High levels of this pro-malignant protein occur in approximately 20-30% of human breast cancers, and its presence promotes a particularly aggressive course and poor prognosis of breast cancer disease. Accordingly, high levels of HER-2/neu enhance the growth of tumor cells, promotes the ability of breast cancer cells to migrate to distal sites (metastasis), and also increase the resistance of breast tumor cells against the injury induced by chemotherapy. Our experimental observations demonstrated that inhibition of FAS activity by a natural fungal compound (called cerulenin) or a related synthetic molecule (C75, a novel anti-obesity drug) dramatically diminished HER-2/neu levels in breast cancer cells. Moreover, the exposure of breast cancer cells expressing high levels of HER-2/neu to FAS inhibitors and an antibody directed against HER-2/neu (called trastuzumab or HerceptinÒ), decreased HER-2/neu levels in a synergistic fashion. Interestingly, this novel “connection” appeared to have therapeutic relevance because simultaneous inhibition of FAS and HER-2/neu was extraordinarily toxic by promoting a “programmed cell death” or “suicide” of breast cancer cells. Interestingly, the presence of high levels of HER-2/neu in breast cancer cells predicted a higher sensitivity to cellular toxicity induced by FAS inhibition. Remarkably, the activation of HER-2/neu signaling leaded to FAS accumulation, and this increase of FAS was prevented by the anti-HER-2/neu antibody trastuzumab, suggesting that this “molecular connection” between FAS and HER-2/neu oncogene in human breast cancer was bi-directional. The purpose of this project will be to characterize a novel role of HER-2/neu in the cellular response to a “metabolic stress” such as the perturbation of FAS-dependent synthesis of endogenous fatty acids. In addition, this project will evaluate whether blockade of the lipogenic enzyme FAS can reverse the malignant behavior promoted by HER-2/neu in breast tumors. In terms of a clinical perspective, this project will evaluate whether repression of FAS can enhance the efficacy of conventional chemotherapeutic drugs and novel therapies directed against HER-2/neu, raising the possibility that FAS inhibition may be clinically useful against breast carcinomas carrying high levels of HER-2/neu. Therefore, the results derived from this study should offer a molecular rationale for a novel therapeutic approach by targeting endogenous fatty acid metabolism (an “energy-storage” cellular route largely considered of minor importance in humans) in breast cancer tumors expressing high levels of the pro-malignant HER-2/neu protein.