Ruth A. Lininger

Elevated Endometrial Androgen Receptor Expression in Women with Polycystic Ovarian Syndrome

Abstract Androgen receptors (AR) have been identified in human endometrium; however, their role in endometrial cyclic development and function remains poorly understood. The objective of the present study was to investigate the profile of endometrial AR in normal menstrual cycles and in the endometrium of women with polycystic ovarian syndrome (PCOS). This syndrome is characterized by chronic hyperandrogenism and oligo-ovulation, and it is often associated with poor reproductive performance. Using immunohistochemistry and reverse transcription-polymerase chain reaction, we found that women with PCOS exhibited elevated endometrial AR expression compared to normal, fertile controls. This increase was most apparent in glandular and luminal epithelium. Furthermore, when compared to endometrium from fertile women, PCOS endometrium showed other abnormalities in endometrial development, including delay or absence of the αvβ3 integrin, a well-characterized biomarker of uterine receptivity described previously (Lessey et al., JCI 1992; 90:188–195). To better understand and to gain insights regarding these findings, we used in vitro cell-culture models to study the regulation of AR in primary endometrial stromal and the well-differentiated epithelial cell line (Ishikawa). Based on Western blot analysis, epithelial AR is up-regulated by estrogens and androgens and is inhibited by progestins and epidermal growth factor (EGF). On the other hand, EGF significantly induced the expression of αvβ3, whereas estrogen and androgen treatment inhibited its expression. Collectively, these results suggest that the poor reproductive performance observed in women with PCOS may be due, in part, to the concomitant increase in both serum androgens and elevations in endometrial AR. This combination may reduce endometrial receptivity as judged by the down-regulation of αvβ3 integrin.

Adult-Derived Stem Cells from the Liver Become Myocytes in the Heart in Vivo

Recent evidence suggests that adult-derived stem cells, like their embryonic counterparts, are pluripotent. These simple, undifferentiated and uncommitted cells are able to respond to signals from their host tissue microenvironment and differentiate, producing progeny that display a phenotype characteristic of the mature cells of that tissue. We used a clonal stem cell line (termed WB-F344) that was derived from an adult male rat liver to investigate the possibility that uncommitted stem cells from a nonmyogenic tissue source would respond to the tissue microenvironment of the heart in vivo and differentiate into cardiac myocytes. Male WB-F344 cells that carry the Escherichia coli beta-galactosidase gene were identified in the left ventricular myocardium of adult female nude mice 6 weeks after transplantation. We confirmed the presence of a rat Y-chromosome-specific repetitive DNA sequence exclusively in the beta-galactosidase-positive myocytes by polymerase chain reaction and fluorescence in situ hybridization. Immunohistochemistry, using a cardiac troponin T-specific monoclonal antibody, and ultrastructural analysis confirmed a cardiac myocyte phenotype of the stem cell-derived myocytes. The beta-galactosidase-positive myocytes ranged from < 20 microm to 110 microm in length. The longer of these cells contained well-organized sarcomeres and myofibrils, and formed intercalated disks and gap junctions with endogenous (host-derived) myocytes, suggesting that WB-F344-derived myocytes participate in the function of the cardiac syncytium. These results demonstrate that adult liver-derived stem cells respond to the tissue microenvironment of the adult heart in vivo and differentiate into mature cardiac myocytes.

Estrogen receptor-alpha (ER-alpha) and defects in uterine receptivity in women

Endometriosis is a disorder that affects 5% of the normal population but is present in up to 40% of women with pelvic pain and/or infertility. Recent evidence suggests that the endometrium of women with endometriosis exhibits progesterone insensitivity. One endometrial protein that fluctuates in response to progesterone is the estrogen receptor-alpha (ER alpha), being down-regulated at the time of peak progesterone secretion during the window of implantation. Here we demonstrate that the biomarker of uterine receptivity, beta 3 integrin subunit, is reduced or absent in some women with endometriosis and that such defects are accompanied by inappropriate over-expression of ER alpha during the mid-secretory phase. Using a well-differentiated endometrial cell line we showed that the beta 3 integrin protein is negatively regulated by estrogen and positively regulated by epidermal growth factor (EGF). By competing against estrogen with various selective estrogen receptor modulators (SERMs) and estrogen receptor agonists and antagonists, inhibition of expression of the beta 3 integrin by estrogen can be mitigated. In conclusion, we hypothesize that certain types of uterine receptivity defects may be caused by the loss of appropriate ER alpha down-regulation in the mid-secretory phase, leading to defects in uterine receptivity. Such changes might be effectively treated by timely administration of the appropriate anti-estrogens to artificially block ER alpha and restore normal patterns of gene expression. Such treatments will require further clinical studies.

Invasive carcinomas of the male breast: a morphologic study of the distribution of histologic subtypes and metastatic patterns in 778 cases

The current investigation was conducted to evaluate the proportional distribution of the various histologic subtypes (including newly recognized variants) of male breast carcinomas, to determine whether any histologic subtypes occur with a frequency that is markedly discordant with the expected frequencies from published data on parallel female breast tumors. We also aimed to document the distribution of malignancies metastatic to the breast. Seven hundred fifty-nine archived cases of primary invasive carcinoma involving the male breast were retrieved and subcategorized into histologic subtypes according to contemporary criteria. Six hundred forty-three (84.7%) tumors were pure infiltrating ductal carcinoma (IDC) not otherwise specified. The most common of the remainder included papillary carcinoma with invasion in the form of IDC (n = 34), mixed IDC and mucinous carcinoma (n = 26), and pure mucinous carcinoma (n = 21). In 19 cases, metastases from other sites involved the breast, most commonly (58%) cutaneous melanoma. Invasive carcinoma of the male breast appears to display a morphologic spectrum and distribution of histologic subtypes that is comparable to those of the female breast, with some expected variation. Compared with published experience on their female counterparts, there is a two-fold increase in the frequency of invasive papillary carcinoma in the male breast. Finally, the most common tumor metastatic to the male breast in this series was cutaneous melanoma.

Focal adhesion kinase overexpression in endometrial neoplasia.

Focal adhesion kinase (FAK) is a protein tyrosine kinase that is a critical mediator of signaling events between cells and their extracellular matrix. Elevations in FAK mRNA and protein overexpression have been linked to tumor cell capacity for invasion and metastasis. FAK expression has been shown to be elevated in a variety of solid tumors. The purpose of this study was to evaluate for FAK upregulation in endometrial neoplasia. Tissue microarray blocks were made from formalin-fixed, paraffin-embedded archival tissue, including 115 carcinoma (100 endometrioid, 10 serous, and 5 clear cell), 28 hyperplasia, and 38 normal specimens using 1-mm punches. The tissue was immunostained with monoclonal antibody for FAK and p53. Immunoreactivity was scored by intensity (0–4+ scale) and percent positive staining. FAK overexpression was categorized as 4+ cytoplasmic intensity in more than 90% of neoplastic cells. Positive p53 was categorized at least 2+ nuclear intensity in more than 10% of neoplastic cells. Higher rates of FAK upregulation were identified in endometrial hyperplasia (P = 0.025) and carcinoma (P < 0.001) versus normal endometrium. FAK overexpression in carcinoma correlated with higher FIGO grade (P = 0.025) and p53 overexpression (P < 0.001). FAK was consistently overexpressed in high-grade tumors regardless of subtype, including 8 of 10 serous tumors, 4 of 5 clear cell tumors, and 16 of 23 grade 3 endometrioid tumors. In conclusion, upregulation of FAK is seen in both endometrial hyperplasia and carcinoma, implying that FAK may play an important role in endometrial carcinogenesis. FAK overexpression in endometrial carcinoma correlates with higher FIGO grade and p53 overexpression.

Endometrial Development and Function in Experimentally Induced Luteal Phase Deficiency

CONTEXT It is generally assumed that delayed endometrial development observed in luteal phase deficiency (LPD) is the result of abnormally low progesterone (P) levels. This hypothesis has never been tested by direct experiment. OBJECTIVE Our objective was to evaluate the effects of P concentrations on human endometrium. DESIGN AND SETTING A randomized trial was conducted at an academic medical center. SUBJECTS Twenty-nine healthy, ovulatory 18- to 35-yr-old women participated. INTERVENTION Endometrial samples were obtained from women in natural cycles and two groups of experimentally modeled cycles. Women undergoing modeled cycles were treated with GnRH agonist and a fixed physiological dose of transdermal estradiol, followed by randomization to 10 or 40 mg daily im P administration to achieve either normal circulating luteal P or 4-fold lower P concentrations, the latter representing an experimental model of LPD. MAIN OUTCOME MEASURES Tissue specimens, obtained after 10 days of P exposure, were analyzed by histological dating, immunohistochemistry, immunoblot, and real-time quantitative RT-PCR (qRT-PCR). RESULTS Histological dating of endometrium, immunohistochemistry for endometrial integrins, and qRT-PCR analysis for nine putative functional markers showed no differences between the three groups. Preliminary data from Western analysis suggest that some proteins may be affected by low serum P concentrations. CONCLUSIONS Histological endometrial dating does not reflect circulating P concentrations and cannot serve as a reliable bioassay of the quality of luteal function. Assessment of selected functional markers by either immunohistochemistry or qRT-PCR is similarly insensitive to decreased circulating P. Preliminary evidence suggests that abnormally low luteal phase serum P concentrations may have important functional consequences not otherwise detected.

Endometrial expression of Cyr61: a marker of estrogenic activity in normal and abnormal endometrium.

OBJECTIVE: To compare the expression of Cyr61 in normal cycling endometrium with endometrium from women with polycystic ovarian syndrome (PCOS) and endometrial hyperplasia and adenocarcinoma. METHODS: This is a retrospective study of 59 samples of normal and abnormal endometrium. Endometrial biopsies were obtained from normal fertile controls throughout the menstrual cycle and compared with endometrium from ovulatory and anovulatory women with PCOS and complex endometrial hyperplasia and endometrioid adenocarcinoma. Cyr61 expression was evaluated by using immunohistochemistry and reverse transcription PCR for Cyr61, estrogen receptor (ER)-α, a marker of cell proliferation (Ki67), and another marker of early estrogen action, cFos. Regulation of Cyr61 protein was studied in a steroid-responsive endometrial carcinoma cell line, ECC1. RESULTS: Cyr61 protein was regulated by estrogen. In normal endometrium, Cyr61 was highest in the proliferative phase and lowest in the normal midsecretory phase. In contrast, elevated levels of Cyr61, ER-α, Ki67, and cFos were all found in the midsecretory endometrium of ovulatory PCOS patients, endometrial cancer patients, and hyperplasia patients. CONCLUSION: Cyr61 is overexpressed in PCOS endometrium, reflecting a heightened responsiveness to estrogen. As a unique marker of estrogen action, Cyr61 may be an early biomarker for the development of hyperplasia or adenocarcinoma in this group of women. LEVEL OF EVIDENCE: II

G Protein Coupled Receptor Kinase 3 Regulates Breast Cancer Migration, Invasion, and Metastasis

Triple negative breast cancer (TNBC) is a heterogeneous disease that has a poor prognosis and limited treatment options. Chemokine receptor interactions are important modulators of breast cancer metastasis; however, it is now recognized that quantitative surface expression of one important chemokine receptor, CXCR4, may not directly correlate with metastasis and that its functional activity in breast cancer may better inform tumor pathogenicity. G protein coupled receptor kinase 3 (GRK3) is a negative regulator of CXCR4 activity, and we show that GRK expression correlates with tumorigenicity, molecular subtype, and metastatic potential in human tumor microarray analysis. Using established human breast cancer cell lines and an immunocompetent in vivo mouse model, we further demonstrate that alterations in GRK3 expression levels in tumor cells directly affect migration and invasion in vitro and the establishment of distant metastasis in vivo. The effects of GRK3 modulation appear to be specific to chemokine-mediated migration behaviors without influencing tumor cell proliferation or survival. These data demonstrate that GRK3 dysregulation may play an important part in TNBC metastasis.

Cyclic Regulation of T-Bet and GATA-3 in Human Endometrium

Endometrial cytokine expression is poorly understood. T-Bet and GATA-3 regulate cytokine expression in T-lymphocytes. Previous work has demonstrated expression of T-Bet in human endometrium. Changes in human endometrial T-Bet and GATA-3 mRNA and protein expression during the normal menstrual cycle were characterized. Human endometrium from each phase of the menstrual cycle underwent real-time reverse-transcriptase polymerase chain reaction and immunohistochemistry to examine expression and localization. T-Bet and GATA-3 mRNA were increased in the late secretory phase. Progesterone receptor (PR) mRNA was increased during the proliferative and early secretory phases. T-Bet and GATA-3 proteins localized cytoplasmically in the late secretory phase. PR protein displayed nuclear localization and maximal immunostaining during the early secretory phase. T-Bet and GATA-3 are expressed in endometrial epithelium cyclically during the menstrual cycle. T-Bet and GATA-3 are both upregulated during the late secretory phase and in the same cell types. The expression patterns of T-Bet and GATA-3 oppose PR, suggesting antagonistic function and/or regulation between PR and T-Bet/GATA-3.

Effects of variations in serum estradiol concentrations on secretory endometrial development and function in experimentally induced cycles in normal women

Eighteen normal women underwent pituitary down-regulation with leuprolide, followed by a 10-day treatment with 0.2 mg/d transdermal estradiol (E(2)) with subsequent allocation to one of two 10-day estradiol regimens plus 40 mg daily intramuscular P: supraphysiologic (0.2 mg/d transdermal E(2) mg/d vaginal micronized E(2)) or subphysiologic (no exogenous E(2) treatment). Average E(2) and P in the supraphysiologic, physiologic, and subphysiologic groups were 1,175.9 pg/mL and 17.5 ng/mL, 136.9 pg/mL and 21.2 pg/mL, and 23.8 ng/mL and 22.0 ng/mL, respectively, and there were no differences between groups in endometrial histology or expression of biomarkers of receptivity.