Gene expression signature of atypical breast hyperplasia and regulation by SFRP1.

BACKGROUND: Atypical breast hyperplasias (AH) have a 10-year risk of progression to invasive cancer estimated at 4-7%, with the overall risk of developing breast cancer increased by ~ 4-fold. AH lesions are estrogen receptor alpha positive (ERα+) and represent risk indicators and/or precursor lesions to low grade ERα+ tumors. Therefore, molecular profiles of AH lesions offer insights into the earliest changes in the breast epithelium, rendering it susceptible to oncogenic transformation. METHODS: In this study, women were selected who were diagnosed with ductal or lobular AH, but no breast cancer prior to or within the 2-year follow-up. Paired AH and histologically normal benign (HNB) tissues from patients were microdissected. RNA was isolated, amplified linearly, labeled, and hybridized to whole transcriptome microarrays to determine gene expression profiles. Genes that were differentially expressed between AH and HNB were identified using a paired analysis. Gene expression signatures distinguishing AH and HNB were defined using AGNES and PAM methods. Regulation of gene networks was investigated using breast epithelial cell lines, explant cultures of normal breast tissue and mouse tissues. RESULTS: A 99-gene signature discriminated the histologically normal and AH tissues in 81% of the cases. Network analysis identified coordinated alterations in signaling through ERα, epidermal growth factor receptors, and androgen receptor which were associated with the development of both lobular and ductal AH. Decreased expression of SFRP1 was also consistently lower in AH. Knockdown of SFRP1 in 76N-Tert cells resulted altered expression of 13 genes similarly to that observed in AH. An SFRP1-regulated network was also observed in tissues from mice lacking Sfrp1. Re-expression of SFRP1 in MCF7 cells provided further support for the SFRP1-regulated network. Treatment of breast explant cultures with rSFRP1 dampened estrogen-induced progesterone receptor levels. CONCLUSIONS: The alterations in gene expression were observed in both ductal and lobular AH suggesting shared underlying mechanisms predisposing to AH. Loss of SFRP1 expression is a significant regulator of AH transcriptional profiles driving previously unidentified changes affecting responses to estrogen and possibly other pathways. The gene signature and pathways provide insights into alterations contributing to AH breast lesions.

Oncogenic transformation of mammary epithelial cells by transforming growth factor beta independent of mammary stem cell regulation.

BACKGROUND: Transforming growth factor beta (TGFß) is transiently increased in the mammary gland during involution and by radiation. While TGFß normally has a tumour suppressor role, prolonged exposure to TGFß can induce an oncogenic epithelial to mesenchymal transition (EMT) program in permissive cells and initiate the generation of cancer stem cells. Our objective is to mimic the transient exposure to TGFß during involution to determine the persistent effects on premalignant mammary epithelium. METHOD: CDßGeo cells, a transplantable mouse mammary epithelial cell line, were treated in vitro for 14 days with TGFß (5 ng/ml). The cells were passaged for an additional 14 days in media without TGFß and then assessed for markers of EMT and transformation. RESULTS: The 14-day exposure to TGFß induced EMT and transdifferentiation in vitro that persists after withdrawal of TGFß. TGFß-treated cells are highly tumorigenic in vivo, producing invasive solid de-differentiated tumours (100%; latency 6.7 weeks) compared to control (43%; latency 32.7 weeks). Although the TGFß-treated cells have initiated a persistent EMT program, the stem cell population was unchanged relative to the controls. The gene expression profiles of TGFß-treated cells demonstrate de-differentiation with decreases in the expression of genes that define luminal, basal and stem cells. Additionally, the gene expression profiles demonstrate increases in markers of EMT, growth factor signalling, TGFß2 and changes in extra cellular matrix. CONCLUSION: This model demonstrates full oncogenic EMT without an increase in stem cells, serving to separate EMT markers from stem cell markers.

The role of activin in mammary gland development and oncogenesis.

TGFß contributes to mammary gland development and has paradoxical roles in breast cancer because it has both tumor suppressor and tumor promoter activity. Another member of the TGFß superfamily, activin, also has roles in the developing mammary gland, but these functions, and the role of activin in breast cancer, are not well characterized. TGFß and activin share the same intracellular signaling pathways, but divergence in their signaling pathways are suggested. The purpose of this review is to compare the spatial and temporal expression of TGFß and activin during mammary gland development, with consideration given to their functions during each developmental period. We also review the contributions of TGFß and activin to breast cancer resistance and susceptibility. Finally, we consider the systemic contributions of activin in regulating obesity and diabetes; and the impact this regulation has on breast cancer. Elevated levels of activin in serum during pregnancy and its influence on pregnancy associated breast cancer are also considered. We conclude that evidence demonstrates that activin has tumor suppressing potential, without definitive indication of tumor promoting activity in the mammary gland, making it a good target for development of therapeutics.

Estrogens, regulation of p53 and breast cancer risk: a balancing act.

The paradoxical effects of ovarian hormones in both the promotion and prevention of breast cancer have been debated for over 30 years. Genetic studies have demonstrated that ovarian hormones act through NF-kappaB to stimulate proliferation and ductal elongation, whereas the p53 tumor suppressor protein plays a central role in rendering the mammary epithelium resistant to tumorigenesis. Transcriptional profiles now suggest that ovarian hormones stimulate a constellation of genes that interact with NF-kappaB and p53 to arbitrate the competing demands for proliferation and surveillance. Genes that participate in chromatin remodeling are among the acute transcriptional responses to estrogens and progestins. These genes are proposed to initiate epigenetic programs that influence the balance between proliferation and surveillance, and render the breast epithelium resistant to tumors.

Exposure to Propylparaben During Pregnancy and Lactation Induces Long-Term Alterations to the Mammary Gland in Mice.

The mammary gland is a hormone sensitive organ that is susceptible to endocrine-disrupting chemicals (EDCs) during the vulnerable periods of parous reorganization (ie, pregnancy, lactation, and involution). Pregnancy is believed to have long-term protective effects against breast cancer development; however, it is unknown if EDCs can alter this effect. We examined the long-term effects of propylparaben, a common preservative used in personal care products and foods, with estrogenic properties, on the parous mouse mammary gland. Pregnant BALB/c mice were treated with 0, 20, 100, or 10 000 µg/kg/day propylparaben throughout pregnancy and lactation. Unexposed nulliparous females were also evaluated. Five weeks post-involution, mammary glands were collected and assessed for changes in histomorphology, hormone receptor expression, immune cell number, and gene expression. For several parameters of mammary gland morphology, propylparaben reduced the effects of parity. Propylparaben also increased proliferation, but not stem cell number, and induced modest alterations to expression of ERα-mediated genes. Finally, propylparaben altered the effect of parity on the number of several immune cell types in the mammary gland. These results suggest that propylparaben, at levels relevant to human exposure, can interfere with the effects of parity on the mouse mammary gland and induce long-term alterations to mammary gland structure. Future studies should address if propylparaben exposures negate the protective effects of pregnancy on mammary cancer development.

Stress tolerance and environmental fitness of Pseudomonas fluorescens A506, which has a mutation in RpoS.

Establishment of suppressive populations of bacterial biological control agents on aerial plant surfaces is a critical phase in biologically based management of floral diseases. Periodically, biocontrol agents encounter inhospitable conditions for growth on plants; consequently, tolerance of environmental stresses may contribute to their fitness. In many gram-negative bacteria, including strains of Pseudomonas spp., the capacity to survive environmental stresses is influenced by the stationary phase sigma factor RpoS. This study focused on the role of RpoS in stress response and epiphytic fitness of Pseudomonas fluorescens A506, a well-studied bacterial biological control agent. We detected a frameshift mutation in the rpoS of A506 and demonstrated that the mutation resulted in a truncated, nonfunctional RpoS. Using site-directed mutagenesis, we deleted a nucleotide from rpoS, which then encoded a full-length, functional RpoS. We compared the stress response and epiphytic fitness of A506 with derivative strains having the functional full-length RpoS or a disrupted, nonfunctional RpoS. RpoS had little effect on stress response of A506 and no consistent influence on epiphytic population size of A506 on pear or apple leaves or flowers. Although the capacity of strain A506 to withstand exposure to environmental stresses was similar to that of other fluorescent pseudomonads, this capacity was largely independent of rpoS.

Transcriptional responses to estrogen and progesterone in mammary gland identify networks regulating p53 activity.

Estrogen and progestins are essential for mammary growth and differentiation but also enhance the activity of the p53 tumor suppressor protein in the mammary epithelium. However, the pathways by which these hormones regulate p53 activity are unknown. Microarrays were used to profile the transcriptional changes within the mammary gland after administration of either vehicle, 17beta-estradiol (E), or progesterone (P) individually and combined (EP). Treatment with EP yielded 1182 unique genes that were differentially expressed compared to the vehicle-treated group. Although 30% of genes were responsive to either E or P individually, combined treatment with both EP had a synergistic effect accounting for 60% of the differentially regulated genes. Analysis of protein-protein interactions identified p53, RelA, Snw1, and Igfals as common targets of genes regulated by EP. RelA and p53 form hubs within a network connected by genes that are regulated by EP and that may coordinate the competing functions of RelA and p53 in proliferation and survival of cells. Induction of early growth response 1 (Egr1) and Stratifin (Sfn) (also known as 14-3-3sigma) by EP was confirmed by reverse transcription-quantitative PCR and shown to be p53 independent. In luciferase reporter assays, Egr1 was shown to enhance transcriptional activation by p53 and inhibit nuclear factor kappaB activity. These results identify a gene expression network that provides redundant activation of RelA to support proliferation as well as sensitize p53 to ensure proper surveillance and integration of their competing functions through factors such as Egr1, which both enhance p53 and inhibit RelA.