Distinct gene-expression profiles characterize mammary tumors developed in transgenic mice expressing constitutively active and C-terminally truncated variants of STAT5

<p>Abstract</p> <p>Background</p> <p>Stat5 is a latent transcription factor that regulates essential growth and survival functions in normal cells. Constitutive activity of Stat5 and the involvement of its C-terminally truncated variant have been implicated in blood cell malignancies and mammary or breast cancer. To distinguish the individual contributions of the Stat5 variants to mammary tumorigenesis, global gene-expression profiling was performed on transgenic STAT5-induced tumors.</p> <p>Results</p> <p>We identified 364 genes exhibiting differential expression in mammary tumors developed in transgenic mice expressing constitutively active STAT5 (STAT5ca) vs. its C-terminally truncated variant (STAT5Δ750). These genes mediate established Stat5 effects on cellular processes such as proliferation and cell death, as well as yet-unrelated homeostatic features, e.g. carbohydrate metabolism. A set of 14 genes linked STAT5Δ750 expression to the poorly differentiated carcinoma phenotype and STAT5ca to the highly differentiated papillary adenocarcinoma.</p> <p>Specifically affected genes exhibited differential expression in an individual tumor set vs. its counterpart and the intact mammary gland: 50 genes were specifically affected by STAT5ca, and 94% of these were downregulated, the latter involved in suppression of tumor suppressors and proliferation antagonistics. This substantial downregulation distinguishes the STAT5ca-induced tumorigenic consequences from the relatively equal effect of the STAT5Δ750 on gene expression, which included significant elevation in the expression of oncogenes and growth mediators.</p> <p>STAT5Δ750 mRNA expression was below detection levels in the tumors and the amount of STAT5ca transcript was not correlated with the expression of its specifically affected genes. Interestingly, we identified several groups of three to eight genes affected by a particular STAT5 variant with significant correlated expression at distinct locations in the clustergram.</p> <p>Conclusion</p> <p>The different gene-expression profiles in mammary tumors caused by the STAT5Δ750 and STAT5ca variants, corroborated by the absence of a direct link to transgenic STAT5 expression, imply distinct metabolic consequences for their oncogenic role which probably initiate early in tumor development. Tumorigenesis may involve induction of growth factor and oncogenes by STAT5Δ750 or suppression of tumor suppressors and growth antagonists by STAT5ca. The list of genes specifically affected by the STAT5 variants may provide a basis for the development of a marker set for their distinct oncogenic role.</p

Cell Hierarchy and Lineage Commitment in the Bovine Mammary Gland

The bovine mammary gland is a favorable organ for studying mammary cell hierarchy due to its robust milk-production capabilities that reflect the adaptation of its cell populations to extensive expansion and differentiation. It also shares basic characteristics with the human breast, and identification of its cell composition may broaden our understanding of the diversity in cell hierarchy among mammals. Here, Lin− epithelial cells were sorted according to expression of CD24 and CD49f into four populations: CD24medCD49fpos (putative stem cells, puStm), CD24negCD49fpos (Basal), CD24highCD49fneg (putative progenitors, puPgt) and CD24medCD49fneg (luminal, Lum). These populations maintained differential gene expression of lineage markers and markers of stem cells and luminal progenitors. Of note was the high expression of Stat5a in the puPgt cells, and of Notch1, Delta1, Jagged1 and Hey1 in the puStm and Basal populations. Cultured puStm and Basal cells formed lineage-restricted basal or luminal clones and after re-sorting, colonies that preserved a duct-like alignment of epithelial layers. In contrast, puPgt and Lum cells generated only luminal clones and unorganized colonies. Under non-adherent culture conditions, the puPgt and puStm populations generated significantly more floating colonies. The increase in cell number during culture provides a measure of propagation potential, which was highest for the puStm cells. Taken together, these analyses position puStm cells at the top of the cell hierarchy and denote the presence of both bi-potent and luminally restricted progenitors. In addition, a population of differentiated luminal cells was marked. Finally, combining ALDH activity with cell-surface marker analyses defined a small subpopulation that is potentially stem cell- enriched

Translational Mechanisms Governing Milk Protein Levels and Composition

Original objectives: The long-term goal of the research is to achieve higher protein content in the milk of ruminants by modulating the translational apparatus of the mammary gland genetically, nutritionally, or pharmacologically. The short-term objectives are to obtain a better understanding of 1) the role of amino acids (AA) as regulators of translation in bovine and mouse mammary epithelial cells and 2) the mechanism responsible for the synergistic enhancement of milk-protein mRNA polyadenylation by insulin and prolactin. Background of the topic: In many cell types and tissues, individual AA affect a signaling pathway which parallels the insulin pathway to modulate rates and levels of protein synthesis. Diverse nutritional and hormonal conditions are funneled to mTOR, a multidomain serine/threonine kinase that regulates a number of components in the initiation and elongation stages of translation. The mechanism by which AA signal mTOR is largely unknown. During the current grant period, we have studied the effect of essential AA on mechanisms involved in protein synthesis in differentiated mammary epithelial cells cultured under lactogenic conditions. We also studied lactogenic hormone regulation of milk protein synthesis in differentiated mammary epithelial cells. In the first BARD grant (2000-03), we discovered a novel mechanism for mRNA-specific hormone-regulated translation, namely, that the combination of insulin plus prolactin causes cytoplasmic polyadenylation of milk protein mRNAs, which leads to their efficient translation. In the current BARD grant, we have pursued the signaling pathways of this novel hormone action. Major conclusions/solutions/achievements: The positive and negative signaling from AA to the mTOR pathway, combined with modulation of insulin sensitization, mediates the synthesis rates of total and specific milk proteins in mammary epithelial cells. The current in vitro study revealed cryptic negative effects of Lys, His, and Thr on cellular mechanisms regulating translation initiation and protein synthesis in mammary epithelial cells that could not be detected by conventional in vivo analyses. We also showed that a signaling pathway involving Jak2 and Stat5, previously shown to lead from the prolactin receptor to transcription of milk protein genes, is also used for cytoplasmic polyadenylation of milk protein mRNAs, thereby stabilizing these mRNAs and activating them for translation. Implications: In vivo, plasma AA levels are affected by nutritional and hormonal effects as well as by conditions of exercise and stress. The amplitude in plasma AA levels resembles that applied in the current in vitro study. Thus, by changing plasma AA levels in the epithelial cell microenvironment or by sensitizing the mTOR pathway to their presence, it should be possible to modulate the rate of milk protein synthesis. Furthermore, knowledge that phosphorylation of Stat5 is required for enhanced milk protein synthesis in response to lactogenic opens the possibility for pharmacologic approaches to increase the phosphorylation of Stat5 and, thereby, milk protein production.</p

Abstract 1229: The oncogenic aspects of deregulated Stat5 activity in the mammary gland

Abstract Stat5 is a latent transcription factor, regulating growth and survival functions in normal cells. Deregulated Stat5 activity has been implicated in blood cell malignancies and mammary / breast cancer. We studied Stat5 oncogenic role in transgenic mice overexpressing its native, constitutively active (STAT5ca) and C-terminally truncated (STAT5Δ750) forms in the mammary gland and in culture. Overexpression and forced activation of the oStat5 (homologous to the mStat5a) cause parity-dependent formation of differentiated papillary or micropapillary tumors in the mammary gland of postestrapusal transgenic mice. In contrast, expression of its truncated form, mainly initiates the poorly differentiated carcinomas. Array analyses distinct tumors caused by STAT5ca and the STAT5Δ750. 50 genes were specifically affected by STAT5ca, and 94% of these were downregulated, the latter involved in suppression of tumor suppressors and proliferation antagonistics. This substantial downregulation distinguishes the STAT5ca-induced tumorigenic consequences from the relatively equal effect of the STAT5Δ750 on gene expression, which included significant elevation in the expression of oncogenes and growth mediators. The different gene-expression profiles in mammary tumors caused by the STAT5Δ750 and STAT5ca variants, corroborated by the absence of a direct link to transgenic STAT5 expression, imply distinct metabolic consequences for their oncogenic role. Gene expression profile was also compared between the resulting papillary adenocarcinomas and the poorly differentiated carcinomas. At least six metabolic pathways support the morphological and functional differences between these tumors. In the carcinoma, stronger expression of genes coding for specific integrins, cytoskeletal proteins and calcium-binding proteins highlight cell-adhesion and motility features of the tumor cells. This is supported by the higher expression of genes involved in O-glycan synthesis, TGF-β pathway, as well as the Notch ligands and members of the γ-secretase complex that enable Notch nuclear localization. The Wnt pathway was also a target for differential gene expression which resulted in membranal accumulation of β-catenin in the papillary tumors, in contrast to its nuclear translocation in the carcinoma. Genes involved in cell-cycle arrest at G1 and response to DNA damage were more highly expressed in the papillary adenocarcinomas, as opposed to favored G2/M regulation in the carcinoma tumors. In culture, transfected CID-9 cells with STAT5ca, accumulated DNA damage within 3 days after the addition of lactogenic hormones. Significant induction in the expression of Chk2, H2A.X. Brca1, p53 and c-Myc was noted. Surprisingly, no sign for cell senescence was observed. We propose that mutation accumulated in damaged cells surviving the repeated reproduction cycles may participate in the oncogenic effect of Stat5 that is observed in aged females. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1229.</jats:p