The role of aromatic hydrocarbons in the genesis of breast cancer.

The incidence of breast cancer in women has increased dramatically over the last decade. Epidemiological markers of this increased incidence include: endocrine related phenomena (early menarche, age of first parity and age of menopause); exposure of the breast to X-radiation; and a group of seemingly disparate factors--urban residence, dietary selection and alcohol consumption. Although experimental breast cancer may be induced by estrogenic hormones, X-radiation and aromatic hydrocarbons, only aromatic hydrocarbons have not been previously implicated in human mammary carcinogenesis. The seemingly unrelated human factors can best be understood by examining the role of breast tissue in aromatic hydrocarbon metabolism. Aromatic hydrocarbons are important environmental chemicals produced by the incomplete combustion of hydrocarbons for use in energy production. Benzene, benz(a)pyrene, dibenz(ah)anthracene and 1-nitropyrene, known experimental breast carcinogens, are produced in this way. Human exposure to aromatic hydrocarbon metabolites induces and promotes altered DNA by mechanisms described as increased intracellular pro-oxidant production as well as direct adduction to DNA. The breast is anatomically embedded in a major fat depot which stores and concentrates aromatic hydrocarbons and can metabolize these hydrocarbons to carcinogenic metabolites. Ductal cells concentrate these metabolites and themselves become target cells for carcinogenesis. Some lifestyle factors increase the amount of carcinogens produced or enhance their activity. A unitary model for mammary carcinogenesis in humans as well as in experimental carcinogenesis is hypothesized. If correct, the hypothesis would account for some of the increase in breast cancer incidence in industrial countries--and would suggest environmental and dietary modifications that would inhibit hydrocarbon induced mammary carcinogenesis.     

Increased erbB3 promotes erbB2/neu-driven mammary tumor proliferation and co-targeting of erbB2/erbB3 receptors exhibits potent inhibitory effects on breast cancer cells

The kinase deficient erbB3 receptor frequently co-expresses and interacts with erbB2 in human breast cancer to activate the oncogenic signaling pathways, and thus promote breast cancer cell survival/proliferation. In the current study, we discovered that the expression of endogenous mouse erbB3 was increased in the mammary tumors-derived from wild type (wt) rat erbB2/neu-transgenic mice, and the co-expression of erbB2 and erbB3 significantly promoted mammary tumor proliferation in vivo. Co-immunoprecipitation assays detected a heterodimeric complex consisting of the transgene encoded protein rat erbB2 and the endogenous mouse erbB3 in the mammary tumors. Specific knockdown of mouse erbB3 dramatically inhibited proliferation of the mammary tumor cell lines-derived from the transgenic mice. Elevated expression of erbB3 protein, but not mRNA, was abserved in human breast cancer cells upon ectopic expression of erbB2. Additional studies revealed that overexpression of erbB2 downregulated three erbB3-targeting miRNAs, miR-125a, miR-125b, and miR-205, whereas the erbB2 kinase inhibitor (lapatinib) significantly enhanced expression of the three miRNAs in breast cancer cells, suggesting that erbB2 might regulate erbB3 expression through a miRNA-dependent mechanism. Furthermore, an anti-erbB3 monoclonal IgG1 antibody (Ab) in combination with Herceptin mainly inactivated Akt and significantly inhibited proliferation of erbB2-overexpressing breast cancer cells. Collectively, our data indicate that increased expression of erbB3 plays a pivotal role in activating downstream PI-3K/Akt pathway and promoting erbB2-driven mammary/breast tumorigenesis. Simultaneous targeting of erbB2 and erbB3 with two IgG1 Abs may be an effective strategy to treat breast cancer patients whose tumors overexpress both erbB2 and erbB3.     

DMBA-induced mammary pathologies are angiogenic in vivo and in vitro

We have previously shown that human pre-invasive diseases of the breast are angiogenic. In addition, normal epithelium from women with coincident or subsequent invasive breast cancer is more vascular than normal epithelium from women with no breast cancer. To develop a model in which to study the regulation of angiogenesis in pre-invasive mammary pathologies, we examined 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tissues for the presence of neovascularization in pre-invasive histopathologies. These studies included morphometric analysis of tissue vascularity in pre-invasive lesions. In addition, we isolated fresh tumors and histologically normal epithelium (organoids) from DMBA or vehicle-treated control rats to test their ability to induce endothelial cell tubule formation in vitro. Finally, we examined tumors for their ability to produce vascular endothelial cell growth factor. The morphometric studies documented that with epithelial progression, the ability of individual cells to elicit angiogenesis increases. The in vitro studies showed that isolated tumors from these animals stimulate angiogenesis. Furthermore, normal epithelium from DMBA-treated rats is more angiogenic than epithelium from control animals. Finally, DMBA-induced tumors produce vascular endothelial growth factor (VEGF) mRNA, therefore, DMBA-induced mammary tumorigenesis is one model in which to test the dependency of progression on angiogenesis.     

Genetic mapping in mice identifies DMBT1 as a candidate modifier of mammary tumors and breast cancer risk

Low-penetrance breast cancer susceptibility alleles seem to play a significant role in breast cancer risk but are difficult to identify in human cohorts. A genetic screen of 176 N2 backcross progeny of two Trp53(+/-) strains, BALB/c and C57BL/6, which differ in their susceptibility to mammary tumors, identified a modifier of mammary tumor susceptibility in an approximately 25-Mb interval on mouse chromosome 7 (designated SuprMam1). Relative to heterozygotes, homozygosity for BALB/c alleles of SuprMam1 significantly decreased mammary tumor latency from 70.7 to 61.1 weeks and increased risk twofold (P = 0.002). Dmbt1 (deleted in malignant brain tumors 1) was identified as a candidate modifier gene within the SuprMam1 interval because it was differentially expressed in mammary tissues from BALB/c-Trp53(+/-) and C57BL/6-Trp53(+/-) mice. Dmbt1 mRNA and protein was reduced in mammary glands of the susceptible BALB/c mice. Immunohistochemical staining demonstrated that DMBT1 protein expression was also significantly reduced in normal breast tissue from women with breast cancer (staining score, 1.8; n = 46) compared with cancer-free controls (staining score, 3.9; n = 53; P < 0.0001). These experiments demonstrate the use of Trp53(+/-) mice as a sensitized background to screen for low-penetrance modifiers of cancer. The results identify a novel mammary tumor susceptibility locus in mice and support a role for DMBT1 in suppression of mammary tumors in both mice and women.     

Development of spontaneous mammary tumors in BALB/c p53 heterozygous mice. A model for Li-Fraumeni syndrome

Breast cancer is the most frequent tumor type among women in the United States and in individuals with Li-Fraumeni syndrome. The p53 tumor suppressor gene is altered in a large proportion of both spontaneous breast malignancies and Li-Fraumeni breast cancers. This suggests that loss of p53 can accelerate breast tumorigenesis, yet p53-deficient mice rarely develop mammary tumors. To evaluate the effect of p53 loss on mammary tumor formation, the p53(null) allele was back-crossed onto the BALB/c genetic background. Median survival was 15.4 weeks for BALB/c-p53(-/-) mice compared to 54 weeks for BALB/c-p53(+/-) mice. Sarcomas and lymphomas were the most frequent tumor types in BALB/c-p53(-/-) mice, whereas 55% of the female BALB/c-p53(+/-) mice developed mammary carcinomas. The mammary tumors were highly aneuploid, frequently lost the remaining wild-type p53 allele, but rarely lost BRCA1. Although mammary tumors were rarely detected in BALB/c-p53(-/-) female mice, when glands from BALB/c-p53(-/-) mice were transplanted into wild-type BALB/c hosts, 75% developed mammary tumors. The high rate of mammary tumor development in the BALB/c background, but not C57Bl/6 or 129/Sv, suggests a genetic predisposition toward mammary tumorigenesis. Therefore, the BALB/c-p53(+/-) mice provide a unique model for the study of breast cancer in Li-Fraumeni syndrome. These results demonstrate the critical role that the p53 tumor suppressor gene plays in preventing tumorigenesis in the mammary gland.     

Mutagens in human breast lipid and milk: the search for environmental agents that initiate breast cancer

Epidemiological studies indicate the involvement of environmental factors in the etiology of breast cancer, but have not provided clear indications of the nature of the agents responsible. Several environmental carcinogens are known to induce mammary tumors in rodents, and the abundance of adipose tissue in the human breast suggests that the epithelial cells, from which breast tumors commonly arise, could be exposed to lipid-soluble carcinogens sequestered by the adipose tissue. In this report we review our studies in which we have examined human mammary lipid, obtained from elective reduction mammoplasties from healthy donors, and human milk from healthy mothers, for the presence of components with genotoxic activity in several in vitro assays. A significant proportion of lipid extracts induced mutations in bacteria and micronuclei in mammalian cells. They also caused DNA damage, detected as single-strand breaks in the alkaline single-cell gel electrophoresis (comet) assay, in both the MCL-5 cell line and in primary cultures of human mammary epithelial cells. Genotoxic activity was also found in a significant proportion of extracts of human breast milk. Viable cells recovered from milk samples showed evidence of DNA damage and were susceptible to comet formation by genotoxic agents in vitro. Genotoxic activity was found to be less prevalent in milk samples from countries of lower breast cancer incidence (the Far East) compared with that in samples from the UK. The agents responsible for the activity in milk appear to be moderately polar lipophilic compounds and of low molecular weight. Identification of these agents and their sources may hold clues to the origins of breast cancer.     

Tobacco smoke carcinogens and breast cancer

Cigarette smoking is an established cause of a variety of cancer types, but its role in breast cancer etiology is not clear. In this report, the potential role of cigarette smoke carcinogens as causes of human breast cancer is evaluated. Of over 60 known carcinogens in tobacco smoke, several are known to induce mammary tumors in laboratory animals: benzo[a]pyrene (B[a]P), dibenzo[a,l]pyrene (DB[a,l]P), 2-toluidine, 4-aminobiphenyl, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 1,3-butadiene, isoprene, nitromethane, ethylene oxide, and benzene. Studies in humans demonstrate that tobacco constituents can reach breast tissue. The uptake and metabolic activation of mammary carcinogens such as polycyclic aromatic hydrocarbons (PAHs) and 4-aminobiphenyl are frequently higher in smokers than in nonsmokers. Although it is likely that specific mammary carcinogens in tobacco smoke can reach breast tissue, evidence is lacking at the present time. Some PAHs present in cigarette smoke can be metabolized to sterically hindered diol epoxides, which are potent mammary carcinogens. Thus, compounds such as benzo[c]phenanthrene (B[c]P), not classically considered to be a strong carcinogen in rodents, could nevertheless be metabolized in humans to diol epoxides carcinogenic to the breast. Collectively, the link between smoking and breast cancer is plausible but has been difficult to establish, probably because of the low carcinogen dose.     

PPAR gamma signaling exacerbates mammary gland tumor development

Breast cancer cell lines that express the nuclear peroxisome proliferator-activated receptor gamma (PPAR gamma) can be prompted to undergo growth arrest and differentiation when treated with synthetic PPAR gamma ligands. To evaluate the therapeutic potential of increased PPAR gamma signaling in vivo, we generated transgenic mice that express a constitutively active form of PPAR gamma in mammary gland. These mice are indistinguishable from their wild-type littermates. However, when bred to a transgenic strain prone to mammary gland cancer, bigenic animals develop tumors with greatly accelerated kinetics. Surprisingly, in spite of their more malignant nature, bigenic tumors are more secretory and differentiated. The molecular basis of this tumor-promoting effect may be an increase in Wnt signaling, as ligand activation of PPAR gamma potentiates Wnt function in an in vivo model of this pathway. These results suggest that once an initiating event has taken place, increased PPAR gamma signaling serves as a tumor promoter in the mammary gland.     

Induction of cell proliferation, micronuclei and hyperdiploidy/polyploidy in the mammary cells of DDT- and DMBA-treated pubertal rats

The environmental estrogen, dichlorodiphenyltrichloroethane (DDT), and its metabolites have been implicated in the development of breast cancer through mechanisms that remain to be elucidated. It has been hypothesized that exposure to DDT and its metabolites, during critical periods of development, can contribute to an elevated risk for breast cancer in adults. In the present study, we have investigated the effect of o,p'-DDT on mammary gland cell proliferation and chromosomal alterations, in a rat mammary cancer model (commonly used to study human cancer), to gain insights into its potential role in the development of breast cancer. Twenty-one-day-old female Sprague-Dawley (SD) rats were administered o,p'-DDT, 7,12-dimethylbenz[a]anthracene (DMBA), genistein, DDT+DMBA, or DDT+DMBA+genistein, over a 14-day period. To determine changes in chromosome number and structure, we used the micronucleus assay as well as multicolor fluorescence in situ hybridization (FISH) region-specific DNA probes for rat chromosomes 4 and 19. Cell proliferation was evaluated using 5-bromo-2'-deoxyuridine (BrdU). Significant increases in BrdU-incorporated cells were seen in the rats treated with DDT+DMBA. Although micronucleus frequencies were somewhat elevated in several of the treatment groups, significant increases were not seen in any of them. Significant increases in numerical chromosomal aberrations were detected in all of the DDT- and DMBA-treated groups. Genistein significantly reduced BrdU incorporation and polyploidy in the DDT+DMBA-treated rats. These initial studies indicate that DDT and DMBA can induce cellular and chromosomal alterations in the rat mammary gland, which is consistent with the hypothesis that these agents can induce early events in mammary carcinogenesis.     

Caveolin-1 and accelerated host aging in the breast tumor microenvironment: chemoprevention with rapamycin, an mTOR inhibitor and anti-aging drug

Increasing chronological age is the most significant risk factor for human cancer development. To examine the effects of host aging on mammary tumor growth, we used caveolin (Cav)-1 knockout mice as a bona fide model of accelerated host aging. Mammary tumor cells were orthotopically implanted into these distinct microenvironments (Cav-1(+/+) versus Cav-1(-/-) age-matched young female mice). Mammary tumors grown in a Cav-1-deficient tumor microenvironment have an increased stromal content, with vimentin-positive myofibroblasts (a marker associated with oxidative stress) that are also positive for S6-kinase activation (a marker associated with aging). Mammary tumors grown in a Cav-1-deficient tumor microenvironment were more than fivefold larger than tumors grown in a wild-type microenvironment. Thus, a Cav-1-deficient tumor microenvironment provides a fertile soil for breast cancer tumor growth. Interestingly, the mammary tumor-promoting effects of a Cav-1-deficient microenvironment were estrogen and progesterone independent. In this context, chemoprevention was achieved by using the mammalian target of rapamycin (mTOR) inhibitor and anti-aging drug, rapamycin. Systemic rapamycin treatment of mammary tumors grown in a Cav-1-deficient microenvironment significantly inhibited their tumor growth, decreased their stromal content, and reduced the levels of both vimentin and phospho-S6 in Cav-1-deficient cancer-associated fibroblasts. Since stromal loss of Cav-1 is a marker of a lethal tumor microenvironment in breast tumors, these high-risk patients might benefit from treatment with mTOR inhibitors, such as rapamycin or other rapamycin-related compounds (rapalogues).     

Mitogen activated protein kinase pathway is involved in RhoC GTPase induced motility,invasion and angiogenesis in inflammatory breast cancer

Inflammatory breast cancer (IBC) is the most lethal form of locally advanced breast cancer known. IBC carries a guarded prognosis primarily due to rapid onset of disease, typically within six months, and the propensity of tumor emboli to invade the dermal lymphatics and spread systemically. Although the clinical manifestations of IBC have been well documented, until recently little was known about the genetic mechanisms underlying the disease. In a comprehensive study aimed at identifying the molecular mechanisms responsible for the unique IBC phenotype, our laboratory identified overexpression of RhoC GTPase in over 90% of IBC tumors in contrast to 36% of stage-matched non-IBC tumors. We also demonstrated that overexpression of RhoC GTPase in human mammary epithelial (HME) cells nearly recapitulated the IBC phenotype with regards to invasion, motility and angiogenesis. In the current study we sought to delineate which signaling pathways were responsible for each aspect of the IBC phenotype. Using well-established inhibitors to the mitogen activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) pathways. We found that activation of the MAPK pathway was responsible for motility, invasion and production of angiogenic factors. In contrast, growth under anchorage independent conditions was dependent on the PI3K pathway. This revised version was published online in July 2006 with corrections to the Cover Date.     

Gene expression analysis of immune-mediated arrest of tumorigenesis in a transgenic mouse model of HER-2/neu-positive basal-like mammary carcinoma

We previously showed that a vaccine combining interleukin 12 and allogeneic p185(neu)-positive mammary carcinoma cells completely prevented multifocal mammary carcinogenesis in HER-2/neu transgenic mice. To identify the molecular events responsible for effective tumor prevention and to define the tumor gene expression signature, we used microarrays to analyze the expression profile of mammary tissue of untreated transgenic mice and of vaccine-treated, tumor-free mice at different time points. Mammary tissue from vaccinated mice displayed a gene expression profile different from that of untreated, tumor-bearing mice but similar to that of normal/hyperplastic mammary gland. Comparison of treated and untreated mice at 15 weeks of age revealed up-regulation of genes encoding antibodies, chemokines, gamma-interferon-induced genes and inflammatory molecules, and down-regulation of early genes induced by tumor development. The gene expression signature of HER-2/neu-transformed tumor cells showed modulation of genes promoting proliferation, angiogenesis, migration, invasion, and metastasis and inhibiting apoptosis and immune response. Meta-analysis of microarray data on human breast cancer showed that the signature of tumors arising in murine HER-2/neu transgenic model correctly classified human HER-2/neu-expressing tumors and normal breast tissue. Moreover murine and human HER-2/neu-positive tumors share the signature of basal-like breast cancers. This gene expression analysis reveals the immune events associated with prevention of tumor development and shows that HER-2/neu transgenic mice represent a good model of a poor-prognosis group of human breast tumors.     

Mammary tumor induction and premature ovarian failure in ApcMin mice are not enhanced by Brca2 deficiency

Inherited BRCA2 mutations predispose individuals to breast cancer and increase risk at other sites. Recent studies have suggested a role for the APC I1307K allele as a low-penetrance breast cancer susceptibility gene that enhances the phenotypic effects of BRCA1 and BRCA2 mutations. To model the consequences of inheriting mutant alleles of the BRCA2 and APC tumor suppressor genes, we examined tumor outcome in C57BL/6 mice with mutations in the Brca2 and Apc genes. We hypothesized that if the Brca2 and Apc genes were interacting to influence mammary tumor susceptibility, then mammary tumor incidence and/or multiplicity would be altered in mice that had inherited mutations in both genes. Female and male offspring treated with a single IP injection of 50 mg/kg N-ethyl-N-nitrosourea (ENU) at 35 days of age developed mammary adenoacanthomas by 100 days of age. The female Apc-mutant and Brca2/Apc double-mutant progeny had mean mammary tumor multiplicities of 6.7+/-2.8 and 7.2+/-2.7, respectively, compared to wild-type and Brca2-mutant females, which had mean mammary tumor multiplicities of 0.1+/-0.4 and 0.3+/-0.5, respectively. Female ENU-treated Apc-mutant and Brca2/Apc double heterozygotes were also susceptible to premature ovarian failure. Thus, the inheritance of an Apc mutation predisposes ENU-treated female and male mice to mammary tumors and, in the case of female mice, to ovarian failure. These results indicate that mammary tumor development in Apc-mutant mice can progress independently of ovarian hormones. The Apc mutation-driven phenotypes were not modified by mutation of Brca2, perhaps because Brca2 acts in a hormonally dependent pathway of mammary carcinogenesis.     

Targeting monocyte chemotactic protein-1 synthesis with bindarit induces tumor regression in prostate and breast cancer animal models

Prostate and breast cancer are major causes of death worldwide, mainly due to patient relapse upon disease recurrence through formation of metastases. Chemokines are small proteins with crucial roles in the immune system, and their regulation is finely tuned in early inflammatory responses. They are key molecules during inflammatory processes, and many studies are focusing on their regulatory functions in tumor growth and angiogenesis during metastatic cell seeding and spreading. Bindarit is an anti-inflammatory indazolic derivative that can inhibit the synthesis of MCP-1/CCL2, with a potential inhibitory function in tumor progression and metastasis formation. We show here that in vitro, bindarit can modulate cancer-cell proliferation and migration, mainly through negative regulation of TGF-β and AKT signaling, and it can impair the NF-κB signaling pathway through enhancing the expression of the NF-κB inhibitor IkB-α. In vivo administration of bindarit results in impaired metastatic disease in prostate cancer xenograft mice (PC-3M-Luc2 cells injected intra-cardially) and impairment of local tumorigenesis in syngeneic Balb/c mice injected under the mammary gland with murine breast cancer cells (4T1-Luc cells). In addition, bindarit treatment significantly decreases the infiltration of tumor-associated macrophages and myeloid-derived suppressor cells in 4T1-Luc primary tumors. Overall, our data indicate that bindarit is a good candidate for new therapies against prostate and breast tumorigenesis, with an action through impairment of inflammatory cell responses during formation of the tumor-stroma niche microenvironment.     

Leptin receptor signaling supports cancer cell metabolism through suppression of mitochondrial respiration in vivo

Obesity represents a risk factor for certain types of cancer. Leptin, a hormone predominantly produced by adipocytes, is elevated in the obese state. In the context of breast cancer, leptin derived from local adipocytes is present at high concentrations within the mammary gland. A direct physiological role of peripheral leptin action in the tumor microenvironment in vivo has not yet been examined. Here, we report that mice deficient in the peripheral leptin receptor, while harboring an intact central leptin signaling pathway, develop a fully mature ductal epithelium, a phenomenon not observed in db/db mice to date. In the context of the MMTV-PyMT mammary tumor model, the lack of peripheral leptin receptors attenuated tumor progression and metastasis through a reduction of the ERK1/2 and Jak2/STAT3 pathways. These are tumor cell-autonomous properties, independent of the metabolic state of the host. In the absence of leptin receptor signaling, the metabolic phenotype is less reliant on aerobic glycolysis and displays an enhanced capacity for β-oxidation, in contrast to nontransformed cells. Leptin receptor-free tumor cells display reduced STAT3 tyrosine phosphorylation on residue Y705 but have increased serine phosphorylation on residue S727, consistent with preserved mitochondrial function in the absence of the leptin receptor. Therefore, local leptin action within the mammary gland is a critical mediator, linking obesity and dysfunctional adipose tissue with aggressive tumor growth.     

Evidence that an early pregnancy causes a persistent decrease in the number of functional mammary epithelial stem cells--implications for pregnancy-induced protection against breast cancer

A completed pregnancy at a young age reduces a woman's lifetime risk of breast cancer by up to 50%. A similar protective effect of an early pregnancy has been observed in rodent models using chemical carcinogens. However, the mechanisms responsible for this protective effect remain unclear. Stem cells have been proposed to be the cells of origin for breast cancer. We hypothesized that an early pregnancy reduces adult levels of either mammary stem cells or mammary multipotent progenitor cells. Unsorted mammary cells from adult mice that had undergone an early parity had the same mammosphere formation efficiency as cells from age-matched virgin mice. However, when we transplanted adult mammary cells in limiting dilutions into cleared fat pads of syngeneic mice, we found a significant reduction in the outgrowth potential of the cells from early parous mice compared with age-matched virgin mice. The extent of fat pad filling in successful outgrowths did not change, suggesting that although mammary stem cells in parous mice retained their functional competence, the number of mammary stem cells was reduced. Our results provide the first direct evidence that an early pregnancy has an effect on mammary stem cells.     

Inhibition of VEGFR2 prevents DMBA-induced mammary tumor formation

Preinvasive mammary pathologies in humans and rat chemical carcinogenesis model systems have an increased microvascular density relative to normal tissue. This suggests the possibility of preventing invasive breast cancer by inhibiting angiogenesis. Vascular endothelial cell growth factor (VEGF) is a potent angiogenic growth factor, commonly involved in tumor-induced angiogenesis. Here, we show that both VEGF and VEGFR2 expression increase with histological progression to invasive disease in the rat 7,12-dimethylbenz[a]anthracene (DMBA) model. Other VEGF receptors, VEGFR1, neuropilin 1 and neuropilin 2, are constitutively expressed throughout progression. To examine whether VEGF signaling is functionally relevant to tumor-induced endothelial tubule formation in vitro and for tumor formation in vivo, we utilized the VEGFR2 inhibitor, ZD6474. In vitro endothelial cell tubulogenesis induced by isolated mammary organoids or carcinoma in situ from DMBA-treated rats is inhibited by ZD6474, in a dose-dependent fashion. The administration of ZD6474 to DMBA-treated rats inhibits the formation of atypical ductal hyperplasia and carcinoma in situ by greater than 95% (P < 0.05), when administered 1 week or 6 weeks post-DMBA initiation. Invasive disease was absent in all ZD6474 cohorts. These data support the hypothesis that progression of DMBA-induced preinvasive mammary pathologies to palpable disease requires angiogenesis via a VEGF-dependent mechanism.     

Chemically induced mammary gland adenomyoepitheliomas and myoepithelial carcinomas of mice. Immunohistochemical and ultrastructural features.

Myoepithelial cell tumors of the mammary gland have been observed in several mammalian species and are composed of a single cell type (myoepithelium) or, more often, present as a biphasic process including neoplastic ductal epithelial cells. In dogs, these are common tumors, but in humans they are rare neoplasms of the breast, and little is yet known of their pathogenesis, particularly with respect to myoepithelial origin. The present report describes bicellular mammary gland tumors arising from the duct epithelium that were induced in (C57BL/6NCr X DBA/2NCr)F1 (B6D2F1) mice by four weekly oral applications of 1 mg 7,12-dimethylbenz[a]anthracene (DMBA) starting at 8 weeks of age. Mammary tumors developed 7 to 8 months later in 14 of 57 mice, and most showed great morphologic resemblance to human adenomyoepitheliomas and myoepithelial carcinomas. Ultrastructurally, the induced tumors were composed of cuboidal epithelium with a microvillous border originating from the lining duct epithelium and plump oval or highly elongated cells that were identified as myoepithelial in origin. These spindle cells contained abundant microfilaments in parallel orientation, some with focal densities and intermediate filaments that frequently formed loose bundles or compact tonofibrils. The myoepithelial cells possessed well-developed desmosomes and plasma membrane caveolae and were regularly bordered by single or reduplicated basement membranes. By immunohistochemistry, strong immunoreactivity was observed for actin in the myoepithelial tumor component only, whereas cytokeratin was variably present in both duct epithelium and myoepithelium. Neoplastic myoepithelial cells stained purple with phosphotungstic acid hematoxylin (PTAH) and brilliant red with Masson's trichrome. It is suggested that DMBA-induced mouse mammary gland adenomyoepitheliomas and myoepithelial carcinomas may serve as very useful animal models to study myoepithelial tumorigenesis.