The mammary gland microenvironment during postlactational involution shares similarities with inflammation, including high matrix metalloproteinase activity, fibrillar collagen deposition, and release of bioactive fragments of fibronectin and laminin. Because inflammation can promote tumorigenesis, we evaluated whether the tissue microenvironment of the involuting gland is also promotional. Extracellular matrix was isolated from mammary glands of nulliparous rats or rats with mammary glands undergoing weaning-induced involution. Using these matrices as substratum, nulliparous matrix was found to promote ductal organization of normal mammary epithelial MCF-12A cells in three-dimensional culture and to suppress invasion of mammary tumor MDA-MB-231 cells in transwell filter assays. Conversely, involution matrix failed to support ductal development in normal cells and promoted invasiveness in tumor cells. To evaluate the effects of these matrices on metastasis in vivo, MDA-MB-231 cells, premixed with Matrigel, nulliparous matrix, or involution matrix, were injected into mammary fat pads of nude mice. Metastases to lung, liver, and kidney were increased in the involution matrix group, and correlated with a twofold increase in tumor vascular endothelial growth factor expression and increased angiogenesis. These data suggest that the mammary gland microenvironment becomes promotional for tumor cell dissemination during involution, thus providing a plausible mechanism to explain the high rate of metastases that occur with pregnancy-associated breast cancer.

To address whether reproductive state alters mammary gland extracellular matrix (ECM) composition and function, ECM was isolated from nulliparous, pregnant, lactating, involuting, and regressed rat mammary glands. The ECM composition of fibronectin, tenascin, laminin, clusterin, and MMPs was found to vary dramatically with reproductive state. In 3-dimensional (3-D) culture, we identified novel effects of these endogenous mammary matrices on mammary epithelial cells. Specifically we found that (1) matrix isolated from nulliparous animals promoted the formation of epithelial ducts with bifurcation, (2) matrix isolated from mid-involuting mammary glands induced cell death, (3) matrix isolated from late-stage involuting glands restored glandular development, while (4) matrix isolated from parous animals restricted glandular morphogenesis. Our data were consistent with mammary gland ECM facilitating epithelial cell proliferation, differentiation, death, and glandular reorganization that occur during the pregnancy and involution cycle. Further, we show that the parous gland has persistent changes in ECM function. Cumulatively, our data demonstrated that the microenvironment of the normal adult mammary gland is highly plastic, which has important implications for mammary tumor cell progression and dormancy. These data also raised the possibility of targeting mammary matrix production with preventive or therapeutic interventions.

Activation of protein kinase C (PKC)-A decreases normal and neoplastic cell proliferation by inhibiting epidermal growth factor receptor (EGFR)-related signaling. The molecular interactions upstream to PKC-A that influence its suppression of EGFR, however, are poorly understood. We have found that caveolin-1, tetraspanin CD82, and ganglioside GM3 enable the association of EGFR with PKC-A, ultimately leading to inhibition of EGFR signaling. GM3-and CD82-induced inhibition of EGFR signaling requires PKC-A translocation and serine/threonine phosphorylation, which eventually triggers EGFR Thr 654 phosphorylation and receptor internalization. Within this ordered complex of signaling molecules, the ability of CD82 to associate with PKC-A requires the presence of caveolin-1, whereas the interaction of caveolin-1 or PKC-A with EGFR requires the presence of CD82 and ganglioside GM3. Disruption of the membrane with methyl-B-cyclodextrin dissociates the EGFR/GM3/caveolin-1/CD82/PKC-A complex and prevents the inhibitory effect of PKC-A on EGFR phosphorylation, suggesting that caveolin-1, CD82, and ganglioside interact with EGFR and PKC-A within intact cholesterolenriched membrane microdomains. Given the role of these membrane molecules in suppressing EGFR signaling, upregulation of GM3, caveolin-1, and CD82 function may be an effective adjunctive therapy for treating epithelial cell malignancies. [Cancer Res 2007;67(20):9986-95]

ESX is an epithelial-restricted member of a large family of transcription factors known as the Ets family. ESX expression has been shown to be correlated with Her2/neu proto-oncogene amplification in highly aggressive breast cancers and induced by Her2/neu in breast cell lines, but its role in tumorigenesis is unknown. Previously, we have shown that ESX enhances breast cell survival in colonyformation assays. In order to determine whether ESX can act as a transforming gene, we stably transfected MCF-12A human mammary epithelial cells with the ESX expression vector, pCGN2-HA-ESX. The MCF-12A cell line is immortalized, but nontransformed, and importantly, these cells fail to express endogenous ESX protein.We used pCGN2-HA-Ets-2 and pSVRas expression vectors as positive controls for transformation. Like HA-Ets-2 and V12-Ras, stable expression of ESX induced EGF-independent proliferation, serum-independent MAPK phosphorylation and growth in soft agar. Additionally, stable ESX expression conferred increased cell adhesion, motility and invasion in two-dimensional and transwell filter assays, and an epithelial to mesenchymal morphological transition. In three-dimensional cultures, parental and vector control (pCGN2) cells formed highly organized duct-like structures with evidence of cell polarity, ECM adhesion-dependent proliferation and cell survival, and lack of cellular invasion into surrounding matrix. Remarkably, the ESX stable cells formed solid, disorganized structures, with lack of cell polarity, loss of adhesion junctions and cytokeratin staining and loss of dependence on ECM adhesion for cell proliferation and survival. In addition, ESX cells invaded the surrounding matrix, indicative of a transformed and metastatic phenotype. Taken together, these data show that ESX expression alone confers a transformed and in vitro metastatic phenotype to otherwise normal MCF-12A cells.

Introduction The functional unit of the mammary gland has been defined as the epithelial cell plus its microenvironment, a hypothesis that predicts changes in epithelial cell function will be accompanied by concurrent changes in mammary stroma. To test this hypothesis, the question was addressed of whether mammary stroma is functionally altered by the anti-oestrogen drug tamoxifen.

Obesity increases the risk for postmenopausal breast cancer. We have modeled this metabolic context using female Wistar rats that differ in their polygenic predisposition for obesity under conditions of high-fat feeding and limited physical activity. At 52 days of age, rats were injected with 1-methyl-1-nitrosourea (MNU, 50 mg/kg) and placed in an obesogenic environment. At 19 weeks of age, the rats were separated into lean, mid-weight, and obese rats, based upon their weight gained during this time. The rats were ovariectomized (OVX) at ~24 weeks of age and the change in tumor multiplicity and burden, weight gain, energy intake, tumor estrogen receptor (ER) status, and humoral metabolite and cytokine profiles were examined. The survival and growth of tumors increased in obese rats in response to OVX. OVX induced a high rate of weight gain during post-OVX weeks 1–3, compared to SHAM-operated controls. During this time, feed efficiency (mg gain/kcal intake) was lower in obese rats, and this reduced storage efficiency of ingested fuels predicted the OVX-induced changes in tumor multiplicity (r = −0.64, P < 0.001) and burden (r = −0.57, P < 0.001). Tumors from obese rats contained more cells that expressed ERα, and post-OVX plasma from rats with the lowest feed efficiency had lower interleukin (IL)-2 and IL-4 levels. Our observations suggest a novel link between obesity and mammary tumor promotion that involves impaired fuel metabolism during OVX-induced weight gain. The metabolically inflexible state of obesity and its inability to appropriately respond to the OVX-induced energy imbalance provides a plausible explanation for this relationship and the emergence of obesity’s impact on breast cancer risk after menopause.

Previous work using an adolescent rat model for breast cancer showed increased tumor occurrence in rats fed a chemopreventive dose of vitamin A. Preclinical models for nutrient-cancer interactions utilizing defined diets do not replicate the complexity of the human diet and may be inadequate to investigate food patterns associated with reduced cancer risk in humans. To evaluate this concept, the effects of vitamin A on sexual maturation, mammary gland development, and sensitivity to carcinogenesis were determined in the context of a human food-based diet (whole food diet). At 20 d of age (p20), female rats received either a whole-food diet with adequate levels of vitamin A, a diet with a 5.5-fold increase in vitamin A from fruits and vegetables (S diet), or a diet with a 6.2-fold increase in vitamin A provided as retinyl palmitate (RP diet). To determine the effect of dietary intervention on pubertal mammary gland development, the dietary intervention period was restricted to postnatal d 21-63. Rats were injected with 50 mg 1-methyl-1-nitrosourea/kg body weight at d 66. Compared with adolescent rats that consumed the Ad diet, consumption of S and RP diets reduced mammary cancer multiplicity (relative risk approximately 0.7, P < or = 0.002), which was associated with a reduction in alveolar gland development. The S diet suppressed the onset of sexual maturation (P < 0.001) and inhibited markers of mammary alveologenesis more than the RP diet. These data demonstrate that the amount and source of vitamin A consumed by adolescent female rats can influence the onset of puberty, mammary gland alveolar development, and breast cancer risk and highlight the relevance of utilizing whole-food diets to evaluate the role of dietary factors in cancer prevention.

Supplementary Figure Legends 1-4 from Suppression of Epidermal Growth Factor Receptor Signaling by Protein Kinase C-α Activation Requires CD82, Caveolin-1, and Ganglioside