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Expression of immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling proteins is normally restricted to hematopoietic tissues. The basal activity of ITAM-containing proteins is mediated through negative regulation by coreceptors restricted to hematopoietic tissues. We have identified an ITAM signaling domain encoded within the env gene of murine mammary tumor virus (MMTV). Three-dimensional structures derived in vitro from murine cells stably transfected with MMTV env display a depolarized morphology in comparison with control mammary epithelial cells. This effect is abolished by Y>F substitution within the Env ITAM, as well as inhibitors of Syk and Src protein tyrosine kinases. Env-expressing cells bear hallmarks of cell transformation such as sensitivity to apoptosis induced by tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) or TNFα, as well as down-regulation of E-cadherin and Keratin-18. Human normal mammary epithelial cells expressing MMTV Env also develop transformed phenotype, as typified by growth in soft agar and Matrigel invasion. These disruptions are abrogated by Y>F substitutions. We conclude that ITAM-dependent signals are generated through MMTV Env and trigger early hallmarks of transformation of mouse and human mammary epithelial cells. Therefore, these data suggest a heretofore unappreciated potential mechanism for the initiation of breast cancer and identify MMTV Env and ITAM-containing proteins in human breast tumors as probable oncoproteins.

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Sporadic breast cancer, the most common cancer diagnosed in American and Northern European women, is gradually increasing in incidence in most Western countries. Prevention would be the most efficient way of eradicating this disease. This goal, however, cannot be accomplished until the specific agent(s) or mechanisms that initiate the neoplastic process are identified. Experimental studies have demonstrated that mammary cancer is a hormone-dependent multistep process that can be induced by a variety of compounds and mechanisms, that is, hormones, chemicals, radiation, and viruses, in addition to or in combination with genetic factors. Although estrogens have been shown to play a central role in breast cancer development, their carcinogenicity on human breast epithelial cells (HBECs) has not yet been clearly demonstrated. Breast cancer initiates in the undifferentiated lobules type 1, which are composed of three cell types: highly proliferating cells that are estrogen-receptor negative (ERϪ), nonproliferating cells that are ER positive (ERϩ), and very few (Ͻ1%) ERϩ cells that proliferate. Interestingly, endogenous 17␤-estradiol (E 2 ) is metabolized by the cytochrome P450 enzyme isoforms CYP1A1 and CYP1B1, which also activate benzo[a]pyrene (B[a]P), a carcinogen contained in cigarette smoke. We postulate that if estrogens are carcinogenic in HBECs, they should induce the same transformation phenotypes induced by chemical carcinogens and ultimately genomic changes observed in spontaneously developing primary breast cancers. To test this hypothesis we compared the transforming potential of E 2 on the HBEC MCF-10F with that of B[a]P. Both E 2 and B[a]P induced anchorage-independent growth, colony formation in agar methocel, and loss of ductulogenic capacity in collagen gel, all parameters indicative of cell transformation. In addition, the DNA of E 2 -transformed cells expressed LOH in chromosome 11 at 11q23.3, 11q24.2-q25, and LOH at 13q12-q13. B[a]P-induced cell transformation was also associated with LOH at 13q12-q13 and at 17p13.2. The relevance of these findings is highlighted by the observation that E 2 -and B[a]P-induced genomic alterations in the same loci found in ductal hyperplasia, ductal carcinoma in situ, and invasive ductal carcinoma of the breast. Environ. Mol. Mutagen. 39:254-263, 2002.