Survival signals generated by estrogen and phospholipase D in MCF-7 breast cancer cells are dependent on Myc.

A role for estrogens in the development of breast cancer has been indicated for over a century. It was demonstrated in 1896 that breast cancer remission could be induced by removal of the ovaries in a subset of breast cancer patients (3). Although not originally understood, the observed effects were the result of eliminating the primary source of estrogen in premenopausal woman. The effectiveness of antiestrogen therapies such as tamoxifen and aromatase inhibitors in the prevention of breast cancer (22, 23) also supports a critical role for estrogens in the promotion of breast cancer. The very low incidence of breast cancer in men is also consistent with a role for estrogens. The role that estrogens, predominantly 17-β-estradiol (E2), play in the development of breast cancer is largely that of a tumor promoter, i.e., a substance that does not directly lead to the mutations that convert a normal cell to a cancerous one. Tumor promoters typically stimulate the proliferation of cells that have acquired an activating mutation that facilitates the amplification of these “initiated” cells (18). One component of tumor promotion is the suppression of default apoptotic programs that are activated in cells with inappropriate growth signals or damaged genomes (17). In this regard, it is of significance that E2 has been shown to suppress apoptosis in the estrogen receptor (ER)-positive breast cancer cell line MCF-7 (1, 12, 20, 35). Breast epithelial cells typically express the ER in 15 to 25% of the cells of normal resting breast epithelia, whereas most (70%) breast cancers are ER positive (2). These data indicate that in the development of breast cancer there is a selection for breast epithelial cells that express the ER. The apparent selection of ER-positive breast epithelial cells in breast cancer further implicates estrogen in the development of breast cancer. Interestingly, most breast cancer cell lines are ER negative, indicating that the selection of breast cancer cell lines in culture involves the loss of estrogen responsiveness, a hallmark of poor prognosis in breast cancer (34). Thus, while breast cancers are still dependent upon estrogen, the tumors remain less aggressive. Gaining estrogen independence apparently provides a means for a tumor to become more aggressive. The ability to gain independence from estrogen likely involves the activation of signals that lead to the suppression of apoptosis that was provided by estrogen. A candidate for replacing the estrogen-dependent suppression of apoptosis is phospholipase D (PLD), which is commonly elevated in breast cancer tissues (28, 36) and in breast cancer cell lines (6, 40). PLD was also shown to provide a survival signal in the ER-negative breast cancer cell line MDA-MB-231 (7, 40). PLD cooperates with tyrosine kinases to transform rat fibroblasts (19, 24, 27), suppresses apoptosis induced by high-intensity Raf signaling (25), and prevents upregulation of the tumor suppressor p53 (21). Thus, PLD is a good candidate oncogene in cancers where elevated tyrosine kinase signaling is common, such as breast cancer. Elevated expression of tyrosine kinases such as the epidermal growth factor (EGF) receptor, Her2/Neu, and c-Src are commonly observed in breast cancer (4). These studies suggest that elevated PLD activity in breast cancer could provide survival signals that could allow an emerging breast cancer to progress to estrogen independence. Consistent with the hypothesis that PLD could generate survival signals, phosphatidic acid, the metabolite of PLD activity, has been shown to target mTOR (6, 10, 11), which has long been implicated as a signal that suppresses apoptosis and promotes survival (14, 32, 37). In this study, we have investigated the ability of E2 to suppress apoptosis in the ER-positive MCF-7 human breast cancer cell line. We provide evidence that an mTOR- and PLD-dependent activation of Myc is necessary for the suppression of apoptosis by E2 and that elevated PLD activity in these cells provides an estrogen-independent survival signal.