to be distinct from the other NFAT isoforms (Lopez-2 Department of Adult Oncology Rodriguez et al., 1999; Miyakawa et al., 1999; Trama et The Dana-Farber Cancer Institute al., 2000). Harvard Medical School It has been extensively demonstrated that NFATc1, Boston, Massachusetts 02115 c2, and c3 require the continued activation of calcineurin 3 AMC Cancer Research Center and (Loh et al., 1996) and AP-1 elements (Macian et al., Donald Monk Cancer Research Foundation 2001) in order to regulate the production of numerous Denver, Colorado 80214 lymphokine elements. Calcineurin functions to dephos-4 University of Colorado Cancer Center phorylate serine/threonine residues within the amino Denver, Colorado 80262 terminus of NFAT, allowing NFAT to translocate to the 5 Skirball Institute of Biomolecular Medicine nucleus and bind to DNA. Inhibition of the phosphatase New York University School of Medicine activity of calcineurin by FK506 or cyclosporin A (CsA) New York, New York 10016 results in the relocalization of NFAT to the cytosol and loss of the ability to bind to DNA (Kiani et al., 2000). Targeted disruption of NFATc2 (Xanthoudakis et al., Summary 1996), NFATc1 (Ranger et al., 1998), and NFATc3 (Oukka et al., 1998) has confirmed the roles played by NFAT The calcineurin-regulated transcription factor, nuclear family members in lymphocyte development and proliffactor of activated T cells (NFAT), controls many aseration. However, targeted disruption of calcineurin A␣ pects of T cell function. Here, we demonstrate that did not reveal dramatic changes in overall T cell function the calcineurin/NFAT pathway negatively regulates and showed only slight impairment in antigen-depenthe expression of cyclin-dependent kinase 4 (CDK4). dent responses (Zhang et al., 1996). This was unex-A canonical NFAT binding site was identified and found pected given that calcineurin A␣ accounts for 70%-80% to be sensitive to calcium signals, FK506/CsA, and of the overall phosphatase activity of T cells in the pehistone deacetylase activity and to not require AP-1. riphery (Jiang et al., 1997). The ␤ form of calcineurin Ectopic expression of NFATc2 inhibited the basal acaccounts for the remaining 20%-30% of calcineurin activity of the human CDK4 promoter. Additionally, both tivity in the periphery and is thought to be more imporcalcineurin A␣ Ϫ/Ϫ and NFATc2 Ϫ/Ϫ mice had elevated tant in influencing thymocyte activation and negative protein levels of CDK4, confirming a negative regulaselection (Hollander et al., 1994; Jiang et al., 1997). The tory role for the calcineurin/NFAT pathway. This pathremaining isoform of calcineurin, ␥, is testis specific and way may thus regulate the expression of CDK4 at the does not contribute to lymphocyte function (11). transcriptional level and control how cells re-enter a CDK4 functions as an important G0/G1 restriction resting, nonproliferative state. point kinase to allow cells to leave the resting state and commit to entrance into the cell cycle (Ladha et al.,

Autophagy is a protein and organelle degradation pathway that is involved in diverse diseases including cancer. Recent evidence suggests that autophagy is a cell survival mechanism in tumor cells and that its inhibition especially in combination with other therapy could be beneficial but it remains unclear if all cancer cells behave the same way when autophagy is inhibited. We inhibited autophagy in a panel of breast cancer cell lines and found that some of them are dependent on autophagy for survival even in nutrient rich conditions without any additional stress while others need autophagy only when stressed. Survival under unstressed conditions is due to cell type specific autophagy regulation of STAT3 activity and this phenotype is enriched in triple negative cell lines. This autophagy-dependency affects response to therapy because autophagy inhibition reduced tumor growth in vivo in autophagy-dependent but not in autophagy-independent breast tumors while combination treatment with autophagy inhibitors and other agent was preferentially synergistic in autophagy-dependent cells. These results imply that autophagy-dependence represents a tumor cell specific characteristic where autophagy inhibition will be more effective. Moreover, our results suggest that autophagy inhibition might be a potential therapeutic strategy for triple negative breast cancers, which currently lack an effective targeted treatment.

Hemangiosarcoma (HSA) is a common untreatable cancer of dogs that resembles human angiosarcoma. Detailed studies of these diseases have been historically hindered by the paucity of suitable reagents. Here, we show that expression of CD117 (c-Kit) can distinguish primitive (malignant) from mature (benign) proliferative endothelial lesions, and we describe eight independent cell lines derived from canine HSA explants. Endothelial origin was confirmed by sustained expression of surface CD105 (endoglin), CD146 (MUC18), and CD51/CD61 (a v b 3 integrin). The cell lines showed anchorage-independent growth and were motile and invasive when cultured on a basement membrane matrix. They required endothelial growth factors for growth and survival, and they could be induced to form tubular structures resembling blood vessels when cultured under low calcium conditions. The formation of vessel-like structures was blocked by nicotine, and restored by FK506, suggesting that 'nuclear factor of activated T cells' activity prevents differentiation of these cells. In summary, these cell lines represent a unique and novel resource to improve our understanding of endothelial cell biology in general and canine HSA in particular.