The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the pRB tumor suppressor protein. Cyclin D1 is overexpressed in 20 -30% of human breast tumors and is induced both by oncogenes including those for Ras, Neu, and Src, and by the ␤-catenin/lymphoid enhancer factor (LEF)/T cell factor (TCF) pathway. The ankyrin repeat containing serine-threonine protein kinase, integrinlinked kinase (ILK), binds to the cytoplasmic domain of ␤ 1 and ␤ 3 integrin subunits and promotes anchorageindependent growth. We show here that ILK overexpression elevates cyclin D1 protein levels and directly induces the cyclin D1 gene in mammary epithelial cells. ILK activation of the cyclin D1 promoter was abolished by point mutation of a cAMP-responsive element-binding protein (CREB)/ATF-2 binding site at nucleotide ؊54 in the cyclin D1 promoter, and by overexpression of either glycogen synthase kinase-3␤ (GSK-3␤) or dominant negative mutants of CREB or ATF-2. Inhibition of the PI 3-kinase and AKT/protein kinase B, but not of the p38, ERK, or JNK signaling pathways, reduced ILK induction of cyclin D1 expression. ILK induced CREB transactivation and CREB binding to the cyclin D1 promoter CRE. Wnt-1 overexpression in mammary epithelial cells induced cyclin D1 mRNA and targeted overexpression of Wnt-1 in the mammary gland of transgenic mice increased both ILK activity and cyclin D1 levels. We conclude that the cyclin D1 gene is regulated by the Wnt-1 and ILK signaling pathways and that ILK induction of cyclin D1 involves the CREB signaling pathway in mammary epithelial cells.The cyclin D1 gene encodes a regulatory subunit of a serinethreonine kinase that phosphorylates and inactivates the tumor suppressor pRB (1). The abundance of cyclin D1 was shown to be rate-limiting in cellular proliferation induced by diverse signaling pathways in fibroblasts and breast epithelial cells, including MCF7 cells (2, 3). Homozygous deletion of the cyclin D1 gene in mice results in defects in mammary gland development (4, 5) and in serum-induced cellular proliferation (6). The abundance of cyclin D1 is increased in more than 30% of human breast tumors, and overexpression of cyclin D1 under control of the MMTV 1 promoter in transgenic mice induces mammary adenocarcinoma (7). The majority of breast cancer cell lines and mammary tumors induced by transgenic overexpression of either pp60 v-src or ErbB-2 oncogenes overexpress cyclin D1, suggesting that the induction of cyclin D1 may play an important role in mammary tumorigenesis (8). The cyclin D1 gene is activated by mitogenic stimuli induced by G-protein-

Telomerase, an enzyme that maintains telomere length, plays major roles in cellular immortalization and cancer progression. We found that an exogenous BRCA1 gene strongly inhibited telomerase enzymatic activity in human prostate and breast cancer cell lines and caused telomere shortening in cell lines expressing wild-type BRCA1 (wtBRCA1) but not a tumor-associated mutant BRCA1 (T300G). wtBRCA1 inhibited the expression of the catalytic subunit (telomerase reverse transcriptase [TERT]) but had no effect on the expression of a subset of other components of the telomerase holoenzyme or on the expression of c-Myc, a transcriptional activator of TERT. However, endogenous BRCA1 associated and partially colocalized with c-Myc; exogenous wtBRCA1 strongly suppressed TERT promoter activity in various cell lines. The TERT inhibition was due, in part, to suppression of c-Myc E-box-mediated transcriptional activity. Suppression of TERT promoter and c-Myc activity required the amino terminus of BRCA1 but not the carboxyl terminus. Finally, endogenous BRCA1 and c-Myc were detected on transfected mouse and human TERT promoter segments in vivo. We postulate that inhibition of telomerase may contribute to the BRCA1 tumor suppressor activity.

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Bone morphogenetic protein (BMP) signaling is an important regulator of hematovascular development. However, the progenitor population that responds to BMP signaling is undefined, and the relative role of downstream mediators including Smad1 is unclear. We find that Smad1 shows a distinctive expression profile as embryonic stem (ES) cells undergo differentiation in the embryoid body (EB) system, with peak levels in cell populations enriched for the hemangioblast. To test the functional relevance of this observation, we generated an ES cell line that allows temporal control of ectopic Smad1 expression. Continuous expression of Smad1 from day 2 of EB culture does not disturb hematopoiesis, according to colony assays. In contrast, a pulse of Smad1 expression exclusively between day 2 and day 2.25 expands the population of progenitors for primitive erythroblasts and other hematopoietic lineages. This effect correlates with increased levels of transcripts encoding markers for the hemangioblast, including Runx1, Scl, and Gata2. Indeed, the pulse of Smad1 induction also expands the blast colonyforming cell (BL-CFC) population at a level that is fully sufficient to explain subsequent increases in hematopoiesis. Our data demonstrate that Smad1 expression is sufficient to expand the number of cells that commit to hemangioblast fate. IntroductionThe progenitors that generate the early hematovascular system are derived from ventral mesoderm, which in mammals contributes to the yolk sac primitive blood lineage and associated vasculature. 1 The bone morphogenetic protein (BMP) signaling pathway is an essential regulator of the process by which mesoderm acquires ventral character. 2 Thus, ectopic activation of BMP signaling expands ventral mesoderm derivatives including increased numbers of embryonic hematopoietic cells, whereas inhibition of the pathway causes a corresponding loss in hematopoiesis. Genetic loss-of-function experiments in mice and fish have confirmed the conserved requirement for BMPs and downstream signaling components for the generation of blood cells. [3][4][5] However, because the pathway is essential first for the formation of normal ventral mesoderm, it has been more difficult to define regulatory functions that act on the subsequent commitment steps of mesoderm toward a hematopoietic fate. BMPs can influence hematopoietic progenitors in vitro, 6,7 but whether this reflects the normal process of stem cell commitment during embryogenesis is less clear.The Smads comprise a family of transcriptional cofactors that specifically transduce activated TGF-␤/BMP signals. 8 Smads 1, 5, and 8 constitute the receptor-activated Smads (R-Smads) that on phosphorylation by a type I BMP receptor bind the co-Smad4, common to all TGF-␤/BMP pathways, to form heteromeric protein complexes. These R-Smad/co-Smad complexes translocate to the nucleus and cooperate with other transcription factors to modulate target gene expression. Mice deficient for Smad1 or Smad5 display varying degrees of defects in hematopoietic and vascula...

Breast cancer is the most commonly diagnosed cancer in American women. The underlying mechanisms that cause aberrant cell proliferation and tumor growth involve conserved pathways, which include components of the cell cycle machinery. Proto-oncogenes, growth factors, and steroids have been implicated in the pathogenesis of breast cancer. Surgery, local irradiation, and chemotherapy have been the mainstay of treatment for early and advanced stage disease. Potential targets for selective breast cancer therapy are herein reviewed. Improved understanding of the biology of breast cancer has led to more specific "targeted therapies" directed at biological processes that are selectively deregulated in the cancerous cells. Examples include tamoxifen for estrogen receptor positive tumors and imunoneutralizing antibodies such as trastuzumab for Her2/neu overexpressing tumors. Other novel anticancer agents such as paclitaxel, a microtubule binding molecule, and flavopiridol, a cyclin dependent kinase inhibitor, exert their anticancer effects by inhibiting cell cycle progression.