Rosalia C. M. Simmen

Repression of Mammosphere Formation of Human Breast Cancer Cells by Soy Isoflavone Genistein and Blueberry Polyphenolic Acids Suggests Diet-Mediated Targeting of Cancer Stem-Like/Progenitor Cells

Mammary stem cells are undifferentiated epithelial cells, which initiate mammary tumors and render them resistant to anticancer therapies, when deregulated. Diets rich in fruits and vegetables are implicated in breast cancer risk reduction, yet underlying mechanisms are poorly understood. Here, we addressed whether dietary factors selectively target mammary epithelial cells that display stem-like/progenitor subpopulations with previously recognized tumor-initiating potential. Using estrogen receptor-positive MCF-7 and estrogen receptor-negative MDA-MB-231 human breast cancer cell lines and freshly isolated epithelial cells from MMTV-Wnt-1 transgenic mouse mammary tumors, we demonstrate that sera of adult mice consuming soy isoflavone genistein (GEN) or blueberry (BB) polyphenol-containing diets alter the population of stem-like/progenitor cells, as measured by their functional ability to self-renew and form anchorage-independent spheroid cultures in vitro at low frequency (1-2%). Serum effects on mammosphere formation were dose-dependently replicated by GEN (40 nM >2 μM) and targeted the basal stem-like CD44+/CD24-/ESA+ and the luminal progenitor CD24+ subpopulations in MDA-MB-231 and MCF-7 cells. GEN inhibition of mammosphere formation was mimicked by the Akt inhibitor perifosine and was associated with enhanced tumor suppressor phosphatase and tensin homologue deleted on chromosome ten (PTEN) expression. In contrast, a selected mixture of BB phenolic acids was only active in MDA-MD-231 cells and its CD44+/CD24-/ESA+ subpopulation, and this activity was independent of induction of PTEN expression. These findings delineate a novel and selective function of distinct dietary factors in targeting stem/progenitor cell populations in estrogen receptor-dependent and -independent breast cancers.

Soy isoflavone genistein upregulates epithelial adhesion molecule E-cadherin expression and attenuates β-catenin signaling in mammary epithelial cells

Breast cancer risk is highly modifiable by diet; however, mechanisms underlying dietary protection against mammary tumorigenesis remain poorly understood. A proportion of breast carcinomas is associated with deregulation of beta-catenin stability and amplification of c-Myc expression. We recently showed that dietary exposure to the soy isoflavone genistein (Gen) inhibited Wnt transduction in rat mammary epithelial cells in vivo. Here, we explored the role of Gen on cell adhesion protein, E-cadherin, expression to downregulate beta-catenin proto-oncogene function. In mammary glands of female rats exposed to dietary Gen, E-cadherin and beta-catenin protein levels were increased, concurrent with higher beta-casein gene expression. In HC11 mouse mammary epithelial cells, Gen diminished basal and Wnt-1-induced cell proliferation and attenuated Wnt-1 targets c-Myc and Cyclin D1 expression. Whereas, Gen had no effect on E-cadherin transcript levels, the abundance of membrane E-cadherin protein and of E-cadherin-beta-catenin adhesion complex was increased by Gen, attendant with downregulation of Wnt-1-induced free beta-catenin accumulation in cytosol. Gen inhibition of Wnt-induced c-Myc expression was mimicked by an estrogen receptor (ER)-beta-specific but not ER-alpha-specific agonist and was attenuated with loss of ER-beta expression, concordant with decreased E-cadherin expression. E-cadherin small-interfering RNA targeting eliminated Gen inhibition of Wnt-stimulated c-Myc expression and promoted Gen induction of basal c-Myc transcript levels and subsequent proliferation. Our studies identify E-cadherin as a Gen cellular target and demonstrate that the dichotomy in mammary epithelial response to Gen may be a function of cellular E-cadherin expression.

Soy Protein Isolate and Protection Against Cancer

Objective: Results from epidemiological and animal studies suggest that consuming soy-containing diets reduces the incidence of certain cancers. The purpose of this presentation was to evaluate the potential of soy protein to prevent occurrence of prostate, breast and colon cancer. Methods: Meta-analyses of published epidemiologic studies associating cancer risk with soy intake were performed. The incidence of chemically-induced mammary or colon tumors was determined for rats fed AIN-93G diets made with either casein or soy protein isolate (SPI). Western and Northern blot and microarray analyses were performed on rat mammary and colon tissues to study mechanisms underlying the effects of soy. Results: Meta-analyses revealed reductions in the mean overall risk estimate for mammary (0.78, p < 0.001), colon (0.70, p < 0.001) and prostate (0.66, p < 0.001) cancer for soy consumers. The incidence of AOM-induced colon tumors and DMBA-induced mammary tumors was reduced (p < 0.05) in rats fed SPI-containing diets. Lower incidence of mammary tumors in SPI-fed rats was associated with: 1) reduced terminal end bud numbers (p < 0.05), 2) lower expression of the phase I enzyme CYP1B1 (p < 0.05) and 3) reduced expression of the Ah Receptor and ARNT (p < 0.05). Conclusions: SPI may protect against cancer via multiple mechanisms, including: 1) increased mammary gland differentiation, 2) decreased activation of procarcinogens to carcinogens and 3) regulation of genes in signal transduction pathways underlying tumor initiation, promotion and/or progression.

Secretory leukocyte protease inhibitor mediates proliferation of human endometrial epithelial cells by positive and negative regulation of growth-associated genes.

Secretory leukocyte protease inhibitor (SLPI) inhibits chymotrypsin, trypsin, elastase, and cathepsin G. This protein also exhibits proliferative effects, although little is known about the molecular mechanisms underlying this activity. We have generated SLPI-ablated epithelial sublines by stably transfecting the Ishikawa human endometrial cell line with an antisense human SLPI RNA expression vector. We demonstrate a positive correlation between cellular SLPI production and proliferation. We further show that Ishikawa sublines expressing low to undetectable SLPI have correspondingly increased and decreased expression, respectively, of transforming growth factor-β1 and cyclin D1 genes, relative to parental cells. SLPI selectively increased cyclin D1 gene expression, with the effect occurring in part at the level of promoter activity. Cellular SLPI levels negatively influenced the anti-proliferative and pro-apoptotic insulin-like growth factor-binding protein-3 expression. We also identified lysyl oxidase, a phenotypic inhibitor of the ras oncogenic pathway and a tumor suppressor, as SLPI-repressed gene, whose expression is up-regulated by transforming growth factor-β1. Our results suggest that SLPI acts at the node(s) of at least three major interacting growth inhibitory pathways. Because expression of SLPI is generally high in epithelial cells exhibiting abnormal proliferation such as in carcinomas, SLPI may define a novel pathway by which cellular growth is modulated.

Krüppel-Like Factor 9 and Progesterone Receptor Coregulation of Decidualizing Endometrial Stromal Cells: Implications for the Pathogenesis of Endometriosis

CONTEXT Endometriosis is characterized by progesterone resistance and associated with infertility. Krüppel-like factor 9 (KLF9) is a progesterone receptor (PGR)-interacting protein, and mice null for Klf9 are subfertile. Whether loss of KLF9 expression contributes to progesterone resistance of eutopic endometrium of women with endometriosis is unknown. OBJECTIVE The aims were to investigate 1) KLF9 expression in eutopic endometrium of women with and without endometriosis, 2) effects of attenuated KLF9 expression on WNT-signaling component expression and on WNT inhibitor Dickkopf-1 promoter activity in human endometrial stromal cells (HESC), and 3) PGR and KLF9 coregulation of the stromal transcriptome network. METHODS Transcript levels of KLF9, PGR, and WNT signaling components were measured in eutopic endometrium of women with and without endometriosis. Transcript and protein levels of WNT signaling components in HESC transfected with KLF9 and/or PGR small interfering RNA were analyzed by quantitative RT-PCR and Western blot. KLF9 and PGR coregulation of Dickkopf-1 promoter activity was evaluated using human Dickkopf-1-luciferase promoter/reporter constructs and by chromatin immunoprecipitation. KLF9 and PGR signaling networks were analyzed by gene expression array profiling. RESULTS Eutopic endometrium from women with endometriosis had reduced expression of KLF9 mRNA together with those of PGR-B, WNT4, WNT2, and DKK1. KLF9 and PGR were recruited to the DKK1 promoter and modified each others transactivity. In HESC, KLF9 and PGR coregulated components of the WNT, cytokine, and IGF gene networks that are implicated in endometriosis and infertility. CONCLUSION Loss of KLF9 coregulation of endometrial stromal PGR-responsive gene networks may underlie progesterone resistance in endometriosis.

Molecular Cloning of Porcine Estrogen Receptor-β Complementary DNAs and Developmental Expression in Periimplantation Embryos

Abstract In the pig, estrogens transiently produced by embryos and progestins of maternal origin target the uterine endometrium, causing alterations in gene expression and secretory activity, both of which are important for the initiation of embryo attachment. The potential direct embryotrophic roles of estrogens and progestins are, however, unknown. Here we report the cloning of porcine embryonic estrogen receptor-beta (ER-β) mRNA by reverse transcription-polymerase chain reaction (RT-PCR) using specific primer sets designed initially within conserved regions of human and bovine ER-β mRNAs, and subsequently within regions of identified porcine ER-β cDNA sequences. The ER-β mRNA has an open reading frame of 1578 nucleotides and encodes a 526 amino acid polypeptide that displays greater than 90% identity with other mammalian ER-β proteins. Northern and Western blot analyses using porcine filamentous embryos from Day 12 of pregnancy demonstrated the presence of multiple ER-β mRNA transcripts of approximately 9.5, 4.9, and 3.5 kilobases, and a ∼64-kDa protein corresponding in size to human ovarian granulosa cell ER-β, respectively. In Day 12 filamentous embryos, ER-β expression was immunolocalized to trophoblastic cell nuclei, coincident with that of proliferative cell nuclear antigen (PCNA). The developmental ontogeny of ER-β mRNA was evaluated in embryos of different morphologies (spherical, tubular, and filamentous) by semiquantitative RT-PCR, along with those for other steroid hormone receptors (ER-α and progesterone receptor) and known embryonic genes associated with cell differentiation (cytochrome P450 aromatase type III) and growth (cyclin D1). ER-β mRNA levels varied with embryo morphology (filamentous maximum at Day 12), coincident with that of cyclin D1. Progesterone receptor mRNA levels were maximal in tubular embryos, similar to that of P450 aromatase, whereas the expression of the ER-α gene was barely detectable and appeared constitutive for all developmental stages examined. Estradiol-17β treatment of Day 12 filamentous embryos in culture up-regulated ER-β and P450 aromatase (type III) mRNA levels, respectively, but decreased those of PCNA, and had no effect on cyclin D1 mRNA levels. These studies taken together suggest that embryonic ER-β likely mediates the autocrine functions of estrogens in the dynamic regulation of embryonic growth and development at periimplantation.

Insulin-like growth factors and blastocyst development

Abstract The interactions between the maternal uterus and the developing conceptus that underlie normal blastocyst development and successful progression of pregnancy are complex. Recent studies implicate peptide growth factors in the initiation, establishment and maintenance of pregnancy and in maternal-embryonic communication. The insulin-like family of growth factors appear to function as key mediators of the coordinate development of the uterus and conceptus during early pregnancy by virtue of their ability to influence directly or indirectly, the synthesis and secretion of uterine and conceptus secretory proteins. The autocrine and paracrine modes of action of the insulin-like growth factors (IGFs) are modulated within the uterine microenvironment by Type I IGF receptors and IGF binding proteins (IGFBPs), which are themselves subject to local control within the uterus and conceptus. An understanding of the mechanisms by which IGFs regulate and are regulated by, the expression of conceptus signals for maternal recognition of pregnancy may provide novel insights and practical applications towards increasing reproductive efficiency in large animals.

Direct Interaction of the Krüppel-like Family (KLF) Member, BTEB1, and PR Mediates Progesterone-Responsive Gene Expression in Endometrial Epithelial Cells

The present study was undertaken to evaluate the underlying mechanism(s) by which PR and a Kruppel-like family member, basic transcription element binding protein (BTEB1), mediate endometrial epithelial expression of pregnancy-associated genes. Human endometrial carcinoma cell lines (Hec-1-A) expressing high and low levels of BTEB1 were transiently transfected with a human PR isoform (PR-B) expression construct and a luciferase reporter gene driven by the uteroferrin gene promoter that is responsive to both BTEB1 and the PR ligand progesterone. Unliganded PR inhibited luciferase activity in low and high BTEB backgrounds, and this effect was reversed by the synthetic progestin R5020 in both lines. Transactivation by PR of uteroferrin promoter activity (∼4-fold) was maximal at lower R5020 concentrations (10 nm) in endometrial cells with higher BTEB1 expression, suggesting that nuclear BTEB1content influenced target gene promoter sensitivity to progesterone. BTEB1 and PR-B were found to physically interact i...

The emerging role of Krüppel-like factors in endocrine-responsive cancers of female reproductive tissues

Krüppel-like factors (KLFs), of which there are currently 17 known protein members, belong to the specificity protein (Sp) family of transcription factors and are characterized by the presence of Cys(2)/His(2) zinc finger motifs in their carboxy-terminal domains that confer preferential binding to GC/GT-rich sequences in gene promoter and enhancer regions. While previously regarded to simply function as silencers of Sp1 transactivity, many KLFs are now shown to be relevant to human cancers by their newly identified abilities to mediate crosstalk with signaling pathways involved in the control of cell proliferation, apoptosis, migration, and differentiation. Several KLFs act as tumor suppressors and/or oncogenes under distinct cellular contexts, underscoring their prognostic potential for cancer survival and outcome. Recent studies suggest that a number of KLFs can influence steroid hormone signaling through transcriptional networks involving steroid hormone receptors and members of the nuclear receptor family of transcription factors. Since inappropriate sensitivity or resistance to steroid hormone actions underlies endocrine-related malignancies, we consider the intriguing possibility that dysregulation of expression and/or activity of KLF members is linked to the pathogenesis of endometrial and breast cancers. In this review, we focus on recently described mechanisms of actions of several KLFs (KLF4, KLF5, KLF6, and KLF9) in cancers of the mammary gland and uterus. We suggest that understanding the mode of actions of KLFs and their functional networks may lead to the development of novel therapeutics to improve current prospects for cancer prevention and cure.

Insulin-Like Growth Factor-I and Insulin-Like Growth Factor Binding Protein-2 and -5 in Equine Seminal Plasma: Association with Sperm Characteristics and Fertility

Abstract The objectives of this study were 1) to determine whether insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding proteins (IGFBPs) were present in seminal plasma of stallions; 2) to compare semen parameters (IGF proteins, sperm numbers, morphology, and motility) from stallions at sexual rest (SR) and when sexually active (SA); 3) to compare semen parameters between stallions with high and low seminal plasma IGF-I concentrations; and 4) to examine the relationship between seminal plasma IGF-I concentrations and fertility parameters of stallions. Ejaculates were collected from stallions at SR (n = 51) and SA (n = 46). Concentrations of IGF-I and IGFBP-2 in seminal plasma samples were determined by radioimmunoassay. Presence of IGFBPs in equine seminal plasma was verified using immunoprecipitation and Western ligand blot procedures. IGF-I, IGFBP-2, and IGFBP-5 were present in equine seminal plasma. Concentrations of IGF-I, IGF-I/protein, total IGF-I, IGFBP-2, IGFBP-2/protein, and total IGFBP-2 were not significantly different (P ≥ 0.13) in seminal plasma between stallions at either SR or SA. At SR, stallions with higher seminal plasma IGF-I had more total IGFBP-2 per ejaculate (P < 0.01), more morphologically normal sperm (P = 0.05), and higher first-cycle pregnancy rates (P = 0.02). At SA, stallions with higher seminal plasma IGF-I had fewer cycles per pregnancy (P = 0.02). An association of seminal plasma IGF-I concentration with sperm motility, sperm morphology, and pregnancy rates in bred mares suggests that IGF-I may play a role in sperm function.