The Eph family of receptor tyrosine kinases and their ligands, known as ephrins, play a crucial role in vascular development during embryogenesis. The function of these molecules in adult angiogenesis has not been well characterized. Here, we report that blocking Eph A class receptor activation inhibits angiogenesis in two independent tumor types, the RIP-Tag transgenic model of angiogenesis-dependent pancreatic islet cell carcinoma and the 4T1 model of metastatic mammary adenocarcinoma. Ephrin-A1 ligand was expressed in both tumor and endothelial cells, and EphA2 receptor was localized primarily in tumor-associated vascular endothelial cells. Soluble EphA2-Fc or EphA3-Fc receptors inhibited tumor angiogenesis in cutaneous window assays, and tumor growth in vivo. EphA2-Fc or EphA3-Fc treatment resulted in decreased tumor vascular density, tumor volume, and cell proliferation, but increased cell apoptosis. However, EphA2-Fc had no direct effect on tumor cell growth or apoptosis in culture, yet inhibited migration of endothelial cells in response to tumor cells, suggesting that the soluble receptor inhibited blood vessel recruitment by the tumor. These data provide the first functional evidence for Eph A class receptor regulation of pathogenic angiogenesis induced by tumors and support the function of A class Eph receptors in tumor progression.

Stromal fibroblasts regulate epithelial cell behavior through direct and indirect cell-cell interactions. To clarify the role of TGF-beta signaling in stromal fibroblasts during mammary development and tumorigenesis, we conditionally knocked out the TGF-beta type II receptor gene in mouse mammary fibroblasts (Tgfbr2(fspKO)). Tgfbr2(fspKO) mice exhibit defective mammary ductal development, characterized in part by increased ductal epithelial cell turnover associated with an increase in stromal fibroblast abundance. Tgfbr2(fspKO) mammary fibroblasts transplanted with mammary carcinoma cells promote growth and invasion, which is associated with increased activating phosphorylation of the receptors: erbB1, erbB2, RON, and c-Met. Furthermore, the increased receptor phosphorylation correlates with increased secretion of the cognate ligands by Tgfbr2(fspKO) fibroblasts. Treatment of tumor cells with fibroblast-conditioned medium leads to increased tumor cell proliferation and motility, which are blocked by addition of pharmacologic inhibitors of TGF-alpha signaling or neutralizing antibodies to macrophage-stimulating protein (MSP), HGF, or c-Met. These studies characterize a significant role for stromal TGF-beta signaling in mammary tissue homeostasis and mammary tumor progression via regulation of TGF-alpha, MSP, and HGF signaling pathways.

MethodsFc:TβRII and transgenic mice. Fc:TβRII has been described previously (11). FVB MMTV-Polyomavirus middle T antigen (MMTV-PyV mT) mice (13) (The Jackson Laboratories, Bar Harbor, Maine, USA) were housed in the Animal Care Facility at Vanderbilt University following The American Association for the Accrediation of Laboratory Animal Care guidelines. Three-week-old transgenic mice were treated twice weekly with Fc:TβRII in PBS (5 mg/kg) by intraperitoneal injection. At 110 days, tissues were harvested and fixed in formalin or were snap-frozen. Serum levels of Fc:TβRII were measured by TGF-βs are potent inhibitors of epithelial cell proliferation. However, in established carcinomas, autocrine/paracrine TGF-β interactions can enhance tumor cell viability and progression. Thus, we studied the effect of a soluble Fc:TGF-β type II receptor fusion protein (Fc:TβRII) on transgenic and transplantable models of breast cancer metastases. Systemic administration of Fc:TβRII did not alter primary mammary tumor latency in MMTV-Polyomavirus middle T antigen transgenic mice. However, Fc:TβRII increased apoptosis in primary tumors, while reducing tumor cell motility, intravasation, and lung metastases. These effects correlated with inhibition of Akt activity and FKHRL1 phosphorylation. Fc:TβRII also inhibited metastases from transplanted 4T1 and EMT-6 mammary tumors in syngeneic BALB/c mice. Tumor microvessel density in a mouse dorsal skin window chamber was unaffected by Fc:TβRII. Therefore, blockade of TGF-β signaling may reduce tumor cell viability and migratory potential and represents a testable therapeutic approach against metastatic carcinomas.

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TGF-betas are potent inhibitors of epithelial cell proliferation. However, in established carcinomas, autocrine/paracrine TGF-beta interactions can enhance tumor cell viability and progression. Thus, we studied the effect of a soluble Fc:TGF-beta type II receptor fusion protein (Fc:TbetaRII) on transgenic and transplantable models of breast cancer metastases. Systemic administration of Fc:TbetaRII did not alter primary mammary tumor latency in MMTV-Polyomavirus middle T antigen transgenic mice. However, Fc:TbetaRII increased apoptosis in primary tumors, while reducing tumor cell motility, intravasation, and lung metastases. These effects correlated with inhibition of Akt activity and FKHRL1 phosphorylation. Fc:TbetaRII also inhibited metastases from transplanted 4T1 and EMT-6 mammary tumors in syngeneic BALB/c mice. Tumor microvessel density in a mouse dorsal skin window chamber was unaffected by Fc:TbetaRII. Therefore, blockade of TGF-beta signaling may reduce tumor cell viability and migratory potential and represents a testable therapeutic approach against metastatic carcinomas.

The nuclear factor-B (NF-B) family of transcription factors has been shown to regulate proliferation in several cell types. Although recent studies have demonstrated aberrant expression or activity of NF-B in human breast cancer cell lines and tumors, little is known regarding the precise role of NF-B in normal proliferation and development of the mammary epithelium. We investigated the function of NF-B during murine early postnatal mammary gland development by observing the consequences of increased NF-B activity in mouse mammary epithelium lacking the gene encoding IB␣, a major inhibitor of NF-B. Mammary tissue containing epithelium from inhibitor B␣ (IB␣)-deficient female donors was transplanted into the gland-free mammary stroma of wild-type mice, resulting in an increase in lateral ductal branching and pervasive intraductal hyperplasia. A two-to threefold increase in epithelial cell number was observed in IB␣-deficient epithelium compared with controls. Epithelial cell proliferation was strikingly increased in IB␣-deficient epithelium, and no alteration in apoptosis was detected. The extracellular matrix adjacent to IB␣-deficient epithelium was reduced. Consistent with in vivo data, a fourfold increase in epithelial branching was also observed in purified IB␣-deficient primary epithelial cells in three-dimensional culture. These data demonstrate that NF-B positively regulates mammary epithelial proliferation, branching, and functions in maintenance of normal epithelial architecture during early postnatal development. INTRODUCTIONThe mammary gland is an organ designed to deliver nourishment and passive immunity to infant mammals. It consists of an epithelium that synthesizes and secretes lipid and milk proteins, as well as a fatty stroma that provides support and local growth regulatory cues to the epithelium (reviewed by Medina, 1996). Although the mammary gland rudiment is established during embryogenesis, the majority of mammary gland development occurs postnatally. During puberty, the epithelium proliferates and branches in response to hormonal signals, eventually extending throughout the entire stroma. More extensive growth and differentiation of the epithelium occurs during each round of pregnancy. The distal tips of each epithelial branch proliferate and differentiate into lobuloalveoli, which synthesize and secrete milk during lactation. Upon cessation of nursing, the majority of the epithelium undergoes apoptosis in a process called involution (reviewed by Furth, 1999). After involution, the epithelium remains relatively quiescent until the next pregnancy, when the morphogenetic cycle is repeated.The nuclear factor-B (NF-B) family of transcription factors regulates growth, differentiation, and apoptosis in several tissues, including lymphocytes, embryonic limb, lung and liver, skin, and bone (Beg et al., 1995;Klement et al., 1996;Boothby et al., 1997;Franzoso et al., 1997;Bushdid et al., 1998;Kanegae et al., 1998;Seitz et al., 1998;Bendall et al., 1999;Hu et al., 1999; Li et al., 1999a,b,c;Takeda et...

ErbB2/Neu destabilizes the cyclin-dependent kinase (Cdk) inhibitor p27 and increases expression of cyclin D1. Therefore, we studied the roles of p27 and cyclin D1 in ErbB2-mediated mammary epithelial cell transformation. Overexpression of ErbB2 or cyclin D1 in p27 ؉/؊ primary murine mammary epithelial cells resulted in increased proliferation, cyclin D1 nuclear localization, and colony formation in soft agar compared to those in p27 ؉/؉ cells. In contrast, ErbB2-or cyclin D1-overexpressing p27 ؊/؊ cells displayed reduced proliferation, anchorage-independent growth, Cdk4 activity, cyclin D1 expression, and cyclin D1 nuclear localization compared to wild-type cells. A cyclin D1 mutation in its nuclear export sequence (T286A) partially rescued nuclear localization of cyclin D1 in p27 ؊/؊ cells but did not increase proliferation or Cdk4 kinase activity. Overexpression of E2F1, however, increased proliferation to the same degree in p27 ؉/؉ , p27 ؉/؊ , and p27 ؊/؊ cells. Mammary glands from MMTV (mouse mammary tumor virus)-neu/p27؉/؊ mice exhibited alveolar hyperplasia, enhanced proliferation, decreased apoptosis, and accelerated tumor formation compared to MMTV-neu/p27 glands. However, MMTV-neu/p27؊/؊ glands showed decreased proliferation, cyclin D1 expression, and Cdk4 activity, as well as markedly prolonged tumor latency, compared to MMTV-neu/p27 ؉/؉ glands. These results suggest that p27 ؉/؊ mammary epithelium may be more susceptible to oncogene-induced tumorigenesis, whereas p27-null glands, due to severely impaired cyclin D1/Cdk4 function, are more resistant to transformation.

We have studied the role of the cyclin-dependent kinase (Cdk) inhibitor p27Kip1 in postnatal mammary gland morphogenesis. Based on its ability to negatively regulate cyclin/Cdk function, loss of p27 may result in unrestrained cellular proliferation. However, recent evidence about the stabilizing effect of p27 on cyclin D1–Cdk4 complexes suggests that p27 deficiency might recapitulate the hypoplastic mammary phenotype of cyclin D1–deficient animals. These hypotheses were investigated in postnatal p27-deficient (p27 −/−), hemizygous (p27 +/−), or wild-type (p27 +/+) mammary glands. Mammary glands from p27 +/− mice displayed increased ductal branching and proliferation with delayed postlactational involution. In contrast, p27 −/− mammary glands or wild-type mammary fat pads reconstituted with p27 −/− epithelium produced the opposite phenotype: hypoplasia, low proliferation, decreased ductal branching, impaired lobuloalveolar differentiation, and inability to lactate. The association of cyclin D1 with Cdk4, the kinase activity of Cdk4 against pRb in vitro, the nuclear localization of cyclin D1, and the stability of cyclin D1 were all severely impaired in p27 −/− mammary epithelial cells compared with p27 +/+ and p27 +/− mammary epithelial cells. Therefore, p27 is required for mammary gland development in a dose-dependent fashion and positively regulates cyclin D–Cdk4 function in the mammary gland.