Conditional overexpression of active transforming growth factor beta1 in vivo accelerates metastases of transgenic mammary tumors.

To address the role of transforming growth factor (TGF) beta in the progression of established tumors while avoiding the confounding inhibitory effects of TGF-beta on early transformation, we generated doxycycline (DOX)-inducible triple transgenic mice in which active TGF-beta1 expression could be conditionally regulated in mouse mammary tumor cells transformed by the polyomavirus middle T antigen. DOX-mediated induction of TGF-beta1 for as little as 2 weeks increased lung metastases >10-fold without a detectable effect on primary tumor cell proliferation or tumor size. DOX-induced active TGF-beta1 protein and nuclear Smad2 were restricted to cancer cells, suggesting a causal association between autocrine TGF-beta and increased metastases. Antisense-mediated inhibition of TGF-beta1 in polyomavirus middle T antigen-expressing tumor cells also reduced basal cell motility, survival, anchorage-independent growth, tumorigenicity, and metastases. Therefore, induction and/or activation of TGF-beta in hosts with established TGF-beta-responsive cancers can rapidly accelerate metastatic progression.

Increased malignancy of Neu-induced mammary tumors overexpressing active transforming growth factor beta1.

To determine if Neu is dominant over transforming growth factor beta (TGF-beta), we crossed mouse mammary tumor virus (MMTV)-Neu mice with MMTV-TGF-beta1(S223/225) mice expressing active TGF-beta1 in the mammary gland. Bigenic (NT) and Neu-induced mammary tumors developed with a similar latency. The bigenic tumors and their metastases were less proliferative than those occurring in MMTV-Neu mice. However, NT tumors exhibited less apoptosis and were more locally invasive and of higher histological grade. NT mice exhibited more circulating tumor cells and lung metastases than Neu mice, while NT tumors contained higher levels of phosphorylated (active) Smad2, Akt, mitogen-activated protein kinase (MAPK), and p38, as well as vimentin content and Rac1 activity in situ than tumors expressing Neu alone. Ex vivo, NT cells exhibited higher levels of P-Akt and P-MAPK than Neu cells. These were inhibited by the TGF-beta inhibitor-soluble TGF-beta type II receptor (TbetaRII:Fc), suggesting they were activated by autocrine TGF-beta. TGF-beta stimulated migration of Neu cells into surrounding matrix, while the soluble TGF-beta inhibitor abrogated motility and invasiveness of NT cells. These data suggest that (i) the antimitogenic and prometastatic effects of TGF-beta can exist simultaneously and (ii) Neu does not abrogate TGF-beta-mediated antiproliferative action but can synergize with TGF-beta in accelerating metastatic tumor progression.

Blockade of TGF-beta inhibits mammary tumor cell viability, migration, and metastases.

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.

ErbB2/Neu-induced, cyclin D1-dependent transformation is accelerated in p27-haploinsufficient mammary epithelial cells but impaired in p27-null cells.

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.

Cyclin-dependent kinase inhibitor p27(Kip1) is required for mouse mammary gland morphogenesis and function.

We have studied the role of the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) 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.

State health care reforms: how they affect children and adolescents with emotional disorders and their families.

This article reports on the Health Care Reform Tracking Project, a national study designed to describe and analyze state health care reforms and their impact on children and adolescents with emotional disorders and their families. It summarizes the results of the baseline survey of states conducted in 1995, exploring the nature and extent of the reforms in which states are engaged, most of which involve applying managed care technologies to their Medicaid programs. Trends across states are identified with respect to mental health service delivery, particularly with respect to children and adolescents. The article concludes with a discussion of issues and concerns related not only to mental health service delivery for children and adolescents with emotional disorders and their families but also to the systems of care that have been developing over the past decade to serve them. Some of these concerns include the lack of pilots or demonstrations, limited mental health coverage in some reforms, the lack of integration between mental health and substance abuse systems, the lack of special provisions for children, the need for more reliable bases for deriving capitation rates, the limited incorporation of systems of care, the need to incorporate interagency treatment planning and service delivery approaches, the lack of outcome measures specific to and appropriate for children, and the need for greater family involvement in the planning and implementation of these reforms.