Salman M. Hyder - US grants

VEGF is a central regulator of angiogenesis, and cancer is dependent upon this process since tumors cannot grow past a microscope size without the generation of new blood vessels to provide a sufficient nutrient supply. The VEGF system is thus an attractive therapeutic target for disease such as breast and uterine cancer, which require angiogenesis for growth and metastasis. Little is known about the regulation of VEGF expression in hormone related cancers such as those of the mammary gland and endometrium, and it is unclear how one might prevent VEGF expression or actions in these diseases. We have been involved in studying the hormonal regulation of VEGF expression in these tissues, and recently made the novel discovery that progesterone regulates expression of this key angiogenic factor in some human breast cancer cells, specifically in the well characterized T47-D cell line. The aim of this proposal is to determine the molecular mechanisms involved in the regulation of VEGF by progesterone in human breast cancer cells, in normal rodent mammary cells in vivo, in several in vivo mammary tumor models and in human xenografts grown in nude mice. The hypothesis we will test is that progestins (P) regulate transcription of VEGF directly via classical progesterone (PR) mediated signaling pathways. To test this hypothesis we propose 4 specific aims: (1) To determine the mechanism by which progestins regulate expression of VEGF in human breast cancer cells, (2) To determine which form(s) of the PR regulates VEGF expression, the receptor domains necessary for regulation, and the regulatory elements in the VEGF gene that mediate PR induction, (3) To define VEGF regulation by progestins and anti- progestins in vivo in the normal rodent mammary gland, in rodent mammary tumor models and in human tumor xenografts grown in nude mice and (4) to determine if PO induced VEGF in breast cancer cells participates in the process of angiogenesis and tumor growth. Elucidating the mechanisms that control VEGF production in normal and neoplastic mammary cells is essential to understand the regulation of angiogenesis in breast cancer and to design new therapeutic approaches to this disease that modulate VEGF-induced angiogenesis.

Wild-type p53 tumor suppressor protein (wtp53) promotes cell cycle arrest and apoptosis and inhibits VEGF-dependent angiogenesis, which is required to support rapid tumor growth. In contrast, mutant p53 (mtp53) fails to inhibit VEGF-dependent angiogenesis and thereby permits rapid tumor growth. However, apoptosis can be restored in tumor cells by co-expression of wtp53 and mtp53 or by treatment with PRIMA-1 (p53 reactivation and induction of massive apoptosis), a small molecule activator of mtp53. We recently observed that in many tumor cells, the effect of mtp53 on VEGF is mediated by progestins and the progesterone receptor. Importantly, PRIMA-1 also blocks this reaction. These data suggest that mtp53, which is often expressed at a high level in tumor cells, might be a useful molecular target for anti-tumor drugs, if its wild-type function could be efficiently re-activated in vivo. Furthermore, such an approach might reduce breast cancer risk in women exposed to progestins (for example, during hormone replacement therapy). This proposal will explore the potential of this idea. The proposed research will test the following hypothesis: PRIMA-1 re-activates mtp53 and restores wtp53 function, thereby inhibiting cell proliferation, expression of VEGF, angiogenesis and metastasis, and stimulating apoptosis in p53-deficient breast tumor cells. The specific aims of the proposed research are: (1) Determine the mechanism by which PRIMA-1 induces cell-cycle arrest and/or apoptosis in p53-defective human breast tumor cells. (2) Determine the mechanism by which PRIMA-1 regulates expression of VEGF in p53-defective human breast tumor cells. (3) Characterize the effects of PRIMA-1 on incidence and progression of human breast tumor xenografts in nude mice. (4) Characterize the effects of PRIMA-1 on metastasis of human breast cancer cells in nude mice. The methods to be used include FACS, ELISA, Western blotting, immunohistochemistry, real-time RT-PCR, gelshift, chromatin immunoprecipitation and in vivo xenograft tumor mouse models. It is expected that the proposed research will yield valuable insights into the role of wtp53 and mtp53 in carcinogenesis. These studies should lead to the development of a novel approach for treating progestin-dependent and progestinindependent breast cancer in humans. Furthermore, we contend that the research will provide critical data relevant to minimizing breast cancer risk and metastasis in post-menopausal women and other women exposed to exogenous progestins.