David S. Loose

Secreted Frizzled-Related Protein 4 Regulates Two Wnt7a Signaling Pathways and Inhibits Proliferation in Endometrial Cancer Cells

In the endometrium, hormonal effects on epithelial cells are often elicited through stromal hormone receptors via unknown paracrine mechanisms. Several lines of evidence support the hypothesis that Wnts participate in stromal-epithelial cell communication. Wnt7a is expressed in the luminal epithelium, whereas the extracellular modulator of Wnt signaling, secreted frizzled-related protein 4 (SFRP4), is localized to the stroma. Studies have reported that SFRP4 expression is significantly decreased in endometrial carcinoma and that both SFRP4 and Wnt7a genes are differentially regulated in response to estrogenic stimuli. Aberrant Wnt7a signaling irrevocably causes organ defects and infertility and contributes to the onset of disease. However, specific frizzled receptors (Fzd) that bind Wnt7a and the particular signal transduction pathway each Wnt7a-Fzd pair activates have not been identified. Additionally, the function of SFRP4 in the endometrium has not been addressed. We show here that Wnt7a coimmunoprecipitates with Fzd5, Fzd10, and SFRP4 in Ishikawa cells. Wnt7a binding to Fzd5 was shown to activate β-catenin/canonical Wnt signaling and increase cellular proliferation. Conversely, Wnt7a signaling mediated by Fzd10 induced a noncanonical c-Jun NH2-terminal kinase–responsive pathway. SFRP4 suppresses activation of Wnt7a signaling in both an autocrine and paracrine manner. Stable overexpression of SFRP4 and treatment with recombinant SFRP4 protein inhibited endometrial cancer cell growth in vitro. These findings support a mechanism by which the nature of the Wnt7a signal in the endometrium is dependent on the Fzd repertoire of the cell and can be regulated by SFRP4. (Mol Cancer Res 2008;6(6):1017–28)

Overexpression of the insulin-like growth factor I receptor and activation of the AKT pathway in hyperplastic endometrium.

Purpose: Although there is considerable information on the molecular aberrations associated with endometrial cancer, very little is known of the changes in gene expression associated with endometrial hyperplasia. Experimental Design: To address this, we have compared the level of expression of estrogen-regulated genes and components of the insulin-like growth factor I (IGF-I) signaling pathway in endometrial biopsies from subjects with normal endometrium, complex atypical endometrial hyperplasia, and endometrial adenocarcinoma (type I). Results: There was a significant increase in the expression of the IGF-I receptor (IGF-IR) in biopsies from hyperplastic endometrium and endometrial carcinoma compared with the proliferative endometrium. The receptor was also activated, as judged by increased tyrosine phosphorylation. In addition, in endometrial hyperplasia and carcinoma, the downstream components of the IGF-IR pathway are activated, as reflected in increased Akt phosphorylation. Loss of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) expression in endometrial hyperplasia did not correlate with increased activation of IGF-IR. However, the simultaneous loss of PTEN expression and increased IGF-IR activation in hyperplasia was associated with an increased incidence of endometrial carcinoma. Conclusions: These results suggest that up-regulation of IGF-IR and loss of PTEN may be independent events that give rise to complementary activation of the IGF-I pathway and increase the probability of the development of cancer. These studies suggest that increased expression of IGF-IR may be an important contributor to the risk of endometrial hyperplasia and cancer.

DNA methylation inhibits p53-mediated survivin repression

The molecular progression of endometrial cancer is poorly understood, and both genetic and epigenetic factors play a role. Survivin is a member of the inhibitor of apoptosis (IAP) gene family and contains a canonical CpG island that has been described as epigenetically regulated. As survivin is overexpressed in endometrial tumors, we hypothesized that hypomethylation could explain this expression pattern. Surprisingly, methylation-specific PCR and pyrosequencing showed that survivin was hypermethylated in endometrial tumors and correlated with increased survivin expression. We speculated that methylation could inhibit the binding of p53, a repressor of survivin expression. Our data indicates that demethylation of the survivin promoter by decitabine results in p53-dependent survivin repression and that p53 binding can be inhibited by DNA methylation. We are the first to report survivin de-repression by DNA methylation. We also present microarray data, which suggest that de-repression by methylation is a general mechanism of p53 regulation. Demethylation induced by decitabine is traditionally thought to be active in tumors by allowing the re-expression of tumor suppressor genes. However, our results indicate that an additional important mechanism is to decrease the expression of oncogenes.

Comparative gene expression profiling in three primary human cell lines after treatment with a novel inhibitor of Rho kinase or atorvastatin.

Inhibitors of Rho kinase (ROCK) are a relatively new class of drugs with potential benefits in oncology, neurology, and fibrotic and cardiovascular diseases. ROCK inhibitors modulate many cellular functions, some of which are similar to the pleiotropic effects of statins, suggesting additive or synergistic properties. Studies to date have used compounds that inhibit both isoforms of ROCK, ROCK1 and ROCK2. This study was designed to compare gene expression profiles of atorvastatin with the newly developed ROCK2 inhibitor SLx-2119 in primary cultures of normal human endothelial cells, smooth muscle cells, and fibroblasts. Cells were treated with each compound for 24 h, after which total RNA was isolated and genome-wide gene-expression profiles were obtained with Illumina arrays. Because of the known effect of statins on the actin cytoskeleton and on connective tissue growth factor, a prominent growth factor involved in tissue fibrosis, the effects of SLx-2119 and atorvastatin on the actin cytoskeleton and connective tissue growth factor mRNA were also examined in cultures of smooth muscle cells with a fibrotic phenotype, isolated from biopsies of human intestine with radiation-induced fibrosis. Although SLx-2119 and atorvastatin affected expression of genes belonging to the same biological processes, individual genes were mostly different, consistent with synergistic or additive properties. Both SLx-2119 and atorvastatin reduced connective tissue growth factor mRNA and remodeled the actin cytoskeleton in fibrosis-derived smooth muscle cells, suggesting that both compounds have antifibrotic properties. These results form the basis for further studies to assess the possible therapeutic benefit of combined treatments.

Hypomethylation-induced expression of S100A4 in endometrial carcinoma

Expression of various S100 genes has been associated with clinically aggressive subtypes in a variety of different cancers. We hypothesized that S100A4 would be overexpressed in endometrial carcinoma compared to benign endometrium. Quantitative real-time RT-PCR (qRT-PCR) was used to quantify the mRNA level of S100A4 in benign endometrium (n=19), endometrioid adenocarcinoma (n=87), and non-endometrioid tumors (n=21). Immunohistochemistry was used to verify the results of qRT-PCR and to assess protein localization. Possible mechanisms of S100A4 gene regulation were also examined. S100A4 was overexpressed in the grade 3 endometrioid tumors, uterine papillary serous carcinoma, and uterine malignant mixed müllerian tumor. Expression in grade 1 and grade 2 endometrioid tumors was comparable to that of normal endometrium, which was quite low. Expression was significantly higher in stage III and IV tumors compared with stage I. By immunohistochemistry, S100A4 was expressed in the tumor cell cytoplasm of poorly differentiated tumors, but was not detected in normal endometrial glandular epithelium. In benign endometrium, S100A4 expression was confined to stromal cells. S100A4 was not regulated by estrogen or progesterone, and its expression in tumors was not significantly correlated to estrogen receptor or progesterone receptor content. However, methylation of the S100A4 gene was detected in benign endometrium and grade 1 tumors with low S100A4 expression. In contrast, grade 3 endometrioid tumors with high S100A4 mRNA and protein expression showed no methylation of the gene. These methylation results were verified in endometrial cancer cell lines with differential baseline levels of S100A4 protein. These results suggest that hypomethylation is an important mechanism of regulating the expression of the S100A4 gene. These results support the emerging concept that hypomethylation may play a role in the upregulation of genes during later stages of tumorigenesis.

Prospective Multicenter Randomized Intermediate Biomarker Study of Oral Contraceptive versus Depo-Provera for Prevention of Endometrial Cancer in Women with Lynch Syndrome

Women with Lynch syndrome have a 40% to 60% lifetime risk for developing endometrial cancer, a cancer associated with estrogen imbalance. The molecular basis for endometrial-specific tumorigenesis is unclear. Progestins inhibit estrogen-driven proliferation, and epidemiologic studies have shown that progestin-containing oral contraceptives (OCP) reduce the risk of endometrial cancer by 50% in women at general population risk. It is unknown whether they are effective in women with Lynch syndrome. Asymptomatic women ages 25 to 50 with Lynch syndrome were randomized to receive the progestin compounds Depo-Provera (depo-MPA) or OCP for three months. An endometrial biopsy and transvaginal ultrasound were conducted before and after treatment. Endometrial proliferation was evaluated as the primary endpoint. Histology and a panel of surrogate endpoint biomarkers were evaluated for each endometrial biopsy as secondary endpoints. A total of 51 women were enrolled, and 46 completed treatment. Two of the 51 women had complex hyperplasia with atypia at the baseline endometrial biopsy and were excluded from the study. Overall, both depo-MPA and OCP induced a dramatic decrease in endometrial epithelial proliferation and microscopic changes in the endometrium characteristic of progestin action. Transvaginal ultrasound measurement of endometrial stripe was not a useful measure of endometrial response or baseline hyperplasia. These results show that women with Lynch syndrome do show an endometrial response to short-term exogenous progestins, suggesting that OCP and depo-MPA may be reasonable chemopreventive agents in this high-risk patient population. Cancer Prev Res; 6(8); 774–81. ©2013 AACR.

Clinical implications of DNA methylation in hepatocellular carcinoma

BACKGROUND Epigenetics is a rapidly evolving field of genetic study applicable to nearly every aspect of genome-related research. The importance of epigenetics has been recognised in human hepatocellular carcinoma (HCC). Changes in DNA methylation patterns, including global hypomethylation and promoter hypermethylation, are thought to be early events in hepatocarcinogenesis. OBJECTIVES This review aimed to summarise the role of epigenetics in HCC, to describe the mechanisms of epigenetic changes in HCC and to examine the clinical relevance of epigenetics in HCC. METHODS This review examines the role of CpG-rich regions and DNA methylation, and describes an epigenetic model of cancer, tumour type-specific methylation, the relationships among methylation, cirrhosis and hepatocarcinogenesis, and the role of DNA methylation in HCC. The clinical implications of epigenetics in HCC are discussed. RESULTS A multivariate predictor model based on traditional clinical factors and DNA methylation profile may have important applications in the early detection of neoplastic transformation in populations at high risk for HCC. CpG methylation may be valuable in HCC prognostics. DNA methylation profiles may enable clinical prediction in pre-therapy patient biopsies, paraffin-embedded samples or plasma DNA. CONCLUSIONS Epigenetic changes and profiles may correlate to the biological behaviour of tumours and clinical outcome of HCC patients. The use of DNA methylation profiles as a surrogate biomarker remains an active area of clinical cancer research.

Development of a bioassay for detection of Wnt-binding affinities for individual frizzled receptors.

Wnts are secreted lipid-modified glycoproteins that carry out various signaling functions during development and in adult tissue. Wnt signaling is mediated by frizzled receptors (Fzds) at the cell surface and can be modulated by the secreted frizzled-related proteins (SFRPs) and other molecular antagonists. Abnormal Wnt signaling has been implicated in several diseases. However, due to the complexity of the Wnt signal and the lack of knowledge pertaining to the binding properties of different Wnt ligands, no therapeutic agents that target this pathway exist. Using a novel enzyme-linked immunosorbent assay (ELISA)-based technique, we were able to determine the first measurements of binding affinity for specific Wnt interactions. This study shows that purified Wnt3a, Wnt7a, and Wnt5a have different binding specificities for Fzds and SFRPs.

Identification of a Novel Estrogen-Regulated Gene, EIG121, Induced by Hormone Replacement Therapy and Differentially Expressed in Type I and Type II Endometrial Cancer

Purpose: The identification of genes and pathways that are affected by estrogenization may shed light on the mechanisms of estrogen action. Here, we describe the expression pattern of a novel estrogen-induced gene, EIG121, in distinct types of endometrial cancer. Experimental Design: EIG121 was identified by cDNA microarray analysis of endometrial RNA from women receiving either placebo or estrogen replacement therapy. The expression level of EIG121 was then measured by real-time quantitative reverse transcription-PCR in benign, hyperplastic, and malignant endometrial samples. A polyclonal antibody was used to detect EIG121 protein by immunohistochemistry. Results: In postmenopausal endometrium, estrogen replacement therapy with Premarin and synthetic estrogen sulfate conjugates induced the expression of EIG121 2- and 3-fold, respectively. In premenopausal endometrium, the expression of EIG121 was higher in the estrogen-dominated proliferative phase than the secretory phase. In endometrial complex, hyperplasia, and endometrioid adenocarcinoma, neoplastic proliferations associated with estrogen excess, the expression of EIG121 was significantly elevated (on average 3.8-fold in hyperplasias and 21-fold in grade 1 tumors). Although the level of EIG121 mRNA in grade 3 endometrioid carcinoma was still 3.5-fold of that in benign endometrium, EIG121 expression tended to decline with increasing tumor grade and disease stage. Immunohistochemistry showed faint staining of normal endometrial epithelium, but intense staining of endometrioid tumors. In sharp contrast, EIG121 expression was significantly suppressed in both uterine papillary serous carcinoma and uterine malignant mixed mullerian tumor, two tumors not associated with estrogen exposure, to <5% of the level in benign endometrium. Conclusions: Our results suggest that EIG121 is a good endometrial biomarker associated with a hyperestrogenic state and estrogen-related type I endometrial adenocarcinoma.

Influence of Sublethal Total-Body Irradiation on Immune Cell Populations in the Intestinal Mucosa

Abstract The intestinal immune system is the largest in the body. This study analyzed changes in intestinal immune cell populations, cytokine protein levels, and transcript profiles after total-body irradiation (TBI) in CD2F1 mice. A single dose of 8.0 Gy γ radiation caused negligible 30-day lethality but induced significant histological damage in jejunal mucosa that was maximal at 3.5 days and that had seemingly recovered by day 21 after irradiation. These changes were accompanied by decreased numbers of mucosal macrophages, neutrophils, and B and T lymphocytes, mostly coinciding with similar reductions in peripheral blood cell counts. Recovery of mucosal macrophages occurred within 1 week, whereas mucosal granulocytes and lymphocytes remained low until 3 weeks after TBI. Maximal suppression of T-helper cell (TH)-related transcripts occurred at 3.5 days, but there was no obvious TH1 or TH2 bias. Genome-wide transcriptional profiling revealed a preponderance of differentially regulated genes involved in cell cycle control, cell death and DNA repair between 4 h and 3.5 days after irradiation. Genes involved in tissue recovery predominated from day 7 onward. We conclude that the intestinal immune system undergoes profound changes after sublethal TBI and that these changes likely contribute to postirradiation pathophysiological manifestations.