Involvement of miR-106b in tumorigenic actions of both prolactin and estradiol.

Prolactin promotes a variety of cancers by an array of different mechanisms. Here, we have investigated prolactin's inhibitory effect on expression of the cell cycle-regulating protein, p21. Using a miRNA array, we identified a number of miRNAs upregulated by prolactin treatment, but one in particular that was strongly induced by prolactin and predicted to bind to the 3'UTR of p21 mRNA, miR-106b. By creating a p21 mRNA 3'UTR-luciferase mRNA construct, we demonstrated degradation of the construct in response to prolactin in human breast, prostate and ovarian cancer cell lines. Increased expression of miR-106b replicated, and anti-miR-106b counteracted, the effects of prolactin on degradation of the 3'UTR construct, p21 mRNA levels, and cell proliferation in breast (T47D) and prostate (PC3) cancer cells. Increased expression of miR-106b also stimulated migration of the very epithelioid T47D cell line. By contrast, anti-miR-106b dramatically decreased expression of the mesenchymal markers, SNAIL-2, TWIST-2, VIMENTIN, and FIBRONECTIN. Using signaling pathway inhibitors and the 3'UTR construct, induction of miR-106b by prolactin was determined to be mediated through the MAPK/ERK and PI3K/Akt pathways and not through Jak2/Stat5 in both T47D and PC3 cells. Prolactin activation of MAPK/ERK and PI3K/Akt also activates ERα in the absence of an ERα ligand. 17β-estradiol promoted degradation of the construct in both cell lines and pre-incubation in the estrogen antagonist, Fulvestrant, blocked the ability of both prolactin and 17β-estradiol to induce the construct-degrading activity. Together, these data support a convergence of the prolactin and 17β-estradiol miR-106b-elevating signaling pathways at ERα

An N-terminal splice variant of human Stat5a that interacts with different transcription factors is the dominant form expressed in invasive ductal carcinoma

AbstractWe have identified a new variant of human Stat5a, found at higher ratios to full-length Stat5a in invasive ductal carcinoma versus contiguous normal tissue. The variant, missing exon 5, inhibits p21 and Bax production and increases cell number. After prolactin stimulation, only full-length Stat5a interacts with the vitamin D and retinoid X receptors, whereas only Δ5 Stat5a interacts with activating protein 1–2 and specificity protein 1. Prolactin also oppositely regulates interaction of the two Stat5a forms with β-catenin. We propose that a change in splicing leading to upregulation of this new isoform is a pathogenic aspect of invasive ductal carcinoma

Trans-Epithelial Immune Cell Transfer during Suckling Modulates Delayed-Type Hypersensitivity in Recipients as a Function of Gender

INTRODUCTION: Breast feeding has long term effects on the developing immune system which outlive passive immunization of the neonate. We have investigated the transfer of milk immune cells and examined the result of transfer once the recipients were adult. METHODS: Non-transgenic mouse pups were foster-nursed by green fluorescent protein (GFP) transgenic dams for 3 weeks and the fate of GFP+ cells was followed by FACS analysis, immunohistochemistry and RT-PCR for GFP and appropriate immune cell markers. Pups suckled by non-transgenic dams served as controls. RESULTS: Despite a preponderance of B cells and macrophages in the stomach contents of the pups, most cells undergoing trans-epithelial migration derived from the 3-4% of milk cells positive for T lymphocyte markers. These cells homed to the spleen and thymus, with maximal accumulation at 3-4 weeks. By sensitizing dams with an antigen which elicits a T cell-mediated delayed-type-hypersensitivity (DTH) response, we determined that nursing by a sensitized dam (compared to a non-sensitized dam) amplified a subsequent DTH response in females and yet suppressed one in males. DISCUSSION: These results suggest that clinical evaluation weighing the pros and cons of nursing male versus female children by mothers with genetically-linked hypersensitivity diseases, such as celiac disease and eczema, or those in regions of the world with endemic DTH-eliciting diseases, such as tuberculosis, may be warranted

Lactation-Based Maternal Educational Immunity Crosses MHC Class I Barriers and Can Impart Th1 Immunity to Th2-Biased Recipients

We have previously demonstrated lactational transfer of T cell-based immunity from dam to foster pup. In the short term, a significant part of transferred immunity is passive cellular immunity. However, as time progresses, this is replaced by what we have described as maternal educational immunity such that by young adulthood, all immune cells responding to a foster dam immunogen are the product of the foster pup's thymus. To reduce confounding factors, this original demonstration used congenic/syngeneic dam and foster pup pairs. In this study, we investigated lactational transfer of immunity to Mycobacterium tuberculosis in MHC class I-mismatched animals, as well as from Th1-biased dams to Th2-biased foster pups. Using immunized C57BL/6J dams, lactational transfer to nonimmunized BALB/cJ foster pups resulted in much greater immunity than direct immunization in 5-wk-old pups (ex vivo assay of pup splenocytes). At this age, 82% of immunogen-responding cells in the pup spleen were produced through maternal educational immunity. FVB/NJ nonimmunized foster recipients had a greater number of maternal cells in the spleen and thymus but a much larger percentage was Foxp3+, resulting in equivalent immunity to direct immunization. Depletion of maternal Foxp3+ cells from pup splenocytes illustrated a substantial role for lactationally transferred dam regulatory T cells in suppression of the ex vivo response in FVB/NJ, but not BALB/cJ, recipients. We conclude that lactational transfer of immunity can cross MHC class I barriers and that Th1 immunity can be imparted to Th2-biased offspring; in some instances, it can be greater than that achieved by direct immunization

Involvement of miR-106b in tumorigenic actions of both prolactin and estradiol.

Prolactin promotes a variety of cancers by an array of different mechanisms. Here, we have investigated prolactin's inhibitory effect on expression of the cell cycle-regulating protein, p21. Using a miRNA array, we identified a number of miRNAs upregulated by prolactin treatment, but one in particular that was strongly induced by prolactin and predicted to bind to the 3'UTR of p21 mRNA, miR-106b. By creating a p21 mRNA 3'UTR-luciferase mRNA construct, we demonstrated degradation of the construct in response to prolactin in human breast, prostate and ovarian cancer cell lines. Increased expression of miR-106b replicated, and anti-miR-106b counteracted, the effects of prolactin on degradation of the 3'UTR construct, p21 mRNA levels, and cell proliferation in breast (T47D) and prostate (PC3) cancer cells. Increased expression of miR-106b also stimulated migration of the very epithelioid T47D cell line. By contrast, anti-miR-106b dramatically decreased expression of the mesenchymal markers, SNAIL-2, TWIST-2, VIMENTIN, and FIBRONECTIN. Using signaling pathway inhibitors and the 3'UTR construct, induction of miR-106b by prolactin was determined to be mediated through the MAPK/ERK and PI3K/Akt pathways and not through Jak2/Stat5 in both T47D and PC3 cells. Prolactin activation of MAPK/ERK and PI3K/Akt also activates ERα in the absence of an ERα ligand. 17β-estradiol promoted degradation of the construct in both cell lines and pre-incubation in the estrogen antagonist, Fulvestrant, blocked the ability of both prolactin and 17β-estradiol to induce the construct-degrading activity. Together, these data support a convergence of the prolactin and 17β-estradiol miR-106b-elevating signaling pathways at ERα

Enzyme-linked oligonucleotide hybridization assay for direct oligo measurement in blood.

Small oligonucleotides (oligos) are increasingly being utilized as diagnostics or treatments for disease. An impediment to broader use is the ability to readily measure oligos in biological fluids. Here, we describe a very straightforward assay with detection in the sub-picomole range that does not require extraction from serum/plasma or polymerization chain reaction amplification. As a result, there are no losses or errors due to sample handling, and the assay can be used to measure oligos modified in a variety of ways that increase therapeutic efficacy. The enzyme-linked oligonucleotide hybridization assay (ELOHA) is based on competition with a detection oligo for hybridization to a capture oligo covalently linked to a solid substrate. The versatility of ELOHAs is demonstrated by application to the measurement of three oligos, including two morpholino-oligos with 3'-octaguanidine derivatization for efficient cell uptake. The third oligo is unmodified and has a DNA sequence equivalent to miR93. The assays have sensitivity as low as 0.28 pmol/sample reaction at 50% hybridization. Adding to clinical utility is the need for only a simple 96-well absorbance plate reader and the finding that neither EDTA nor heparin interferes with detection