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

BLG-e1 – A novel regulatory element in the distal region of the β-lactoglobulin gene promoter

Abstractβ-Lactoglobulin (BLG) is a major ruminant milk protein. A regulatory element, termed BLG-e1, was defined in the distal region of the ovine BLG gene promoter. This 299-bp element lacks the established cis-regulatory sequences that affect milk-protein gene expression. Nevertheless, it alters the binding of downstream BLG sequences to histone H4 and the sensitivity of the histone–DNA complexes to trichostatin A treatment. In mammary cells cultured under favorable lactogenic conditions, BLG-e1 acts as a potent, position-independent silencer of BLG/luciferase expression, and similarly affects the promoter activity of the mouse whey acidic protein gene. Intragenic sequences upstream of BLG exon 2 reverse the silencing effect of BLG-e1 in vitro and in transgenic mice

Cultured bMEC populations do not differ in their CD49f/CD24 expression, but maintain their distinct parental characteristics.

<p>A: Schematic representation of the experimental procedure. B: FACS histograms depicting the levels of CD24 and CD49f in freshly isolated bMECs compared with their cultured counterparts. C. FACS dot-plots depicting the subpopulations sorted from the cultured cells. D: Immunofluorescence staining of the lineage markers CK14 and CK18 in organized and non-organized colonies. E: Regardless their different cell-surface marker expression, organized colonies were significantly more frequent in sorted cultured cells originated from the puStm and Basal populations. Bar = 50 µm.</p

Mammospheres are formed by freshly dissociated bMECs, whereas sorting procedures induce non-spherical floating colonies (NSFCs).

<p>A: Representative mammosphere, formed by freshly dissociated Lin⁻ bMECs. Nuclei are stained with DAPI. B: Supplementation of conditioned mammary medium enhances mammosphere formation. *,**Significantly different at <i>P</i>≤0.05 and <i>P</i>≤0.01, respectively. C: The mammosphere is comprised of cells expressing CK14 and CK18. D: Antibody labeling and the sorting process prevent mammosphere formation. E: Representative NSFC formed by sorted bMECs. F: NSFC is comprised mainly of live cells. Trypan blue-stained dead cell (blue) is marked by an arrow. G: The NFSC is comprised of cells expressing CK14 and CK18. Columns represent average±SEM of three wells analyzed for each group. Bar = 50 µm.</p

Multipotency, high propagation potential and clone-formation capability characterize the puStm population.

<p>A: Percentage of colony-forming cells out of the total sorted cell population. B: Immunofluorescence staining of adherent clones, demonstrating two types: luminal clones expressing CK18 (left) and basal clones expressing CK14 (right). C: Composition of clone types formed by each of the sorted populations. Numbers of defined clones are relative to their total number. D: Differences in propagation were observed among the sorted populations during the first 7 days in culture. Columns represent mean±SEM of three analyses. Different letters above the columns indicate statistically significant (<i>P</i><0.05) differences. Bar = 50 µm.</p

Luminal and basal/myoepithelial layers in the bovine mammary gland can be distinguished by immunofluorescence analysis.

<p>A: H&E staining of sections from a heifer's mammary gland reveals ductal structures penetrating the fibrous stroma. B–F: Immunofluorescence detection of mammary lineage markers. Inset: 2× magnification. L = lumen. Bar = 50 µm.</p

List of antibodies used in the study.

<p>List of antibodies used in the study.</p