Endometrial cancer is the most common gynecologic malignancy diagnosed among women in developed countries. One recent biomarker strongly associated with disease progression and survival is epithelial membrane protein-2 (EMP2), a tetraspan protein known to associate with and modify surface expression of certain integrin isoforms. In this study, we show using a xenograft model system that EMP2 expression is necessary for efficient endometrial tumor formation, and we have started to characterize the mechanism by which EMP2 contributes to this malignant phenotype. In endometrial cancer cells, the focal adhesion kinase (FAK)/Src pathway appears to regulate migration as measured through wound healing assays. Manipulation of EMP2 levels in endometrial cancer cells regulates the phosphorylation of FAK and Src, and promotes their distribution into lipid raft domains. Notably, cells with low levels of EMP2 fail to migrate and poorly form tumors in vivo. These findings reveal the pivotal role of EMP2 in endometrial cancer carcinogenesis, and suggest that the association of elevated EMP2 levels with endometrial cancer prognosis may be causally linked to its effect on integrin-mediated signaling.

Epithelial membrane protein-2 (EMP2) is a tetraspan protein predicted to regulate placental development. Highly expressed in secretory endometrium and trophectoderm cells, previous studies suggest that it may regulate implantation by orchestrating the surface expression of integrins and other membrane proteins. In order to test the role of EMP2 in pregnancy, mice lacking EMP2 (Emp2−/−) were generated. Emp2−/− females are fertile but have reduced litter sizes when carrying Emp2−/− but not Emp2+/− fetuses. Placentas of Emp2−/− fetuses exhibit dysregulation in pathways related to neoangiogenesis, coagulation, and oxidative stress, and have increased fibrin deposition and altered vasculature. Given that these findings often occur due to placental insufficiency resulting in an oxygen-poor environment, the expression of hypoxia-inducible factor-1 alpha (HIF-1α) was examined. Placentas from Emp2−/− fetuses had increased total HIF-1α expression in large part through an increase in uterine NK (uNK) cells, demonstrating a unique interplay between uNK cells and trophoblasts modulated through EMP2. To determine if these results translated to human pregnancy, placentas from normal, term deliveries or those complicated by placental insufficiency resulting in intrauterine growth restriction (IUGR) were stained for EMP2. EMP2 was significantly reduced in both villous and extravillous trophoblast populations in IUGR placentas. Experiments in vitro using human trophoblast cells lines indicate that EMP2 modulates angiogenesis by altering HIF-1α expression. Our results reveal a novel role for EMP2 in regulating trophoblast function and vascular development in mice and humans and suggest it may be a new biomarker for placental insufficiency.

BackgroundPMP22, a member of the GAS3 family of tetraspan proteins, is associated with a variety of neurological diseases. Previous studies have shown that PMP22 is expressed in proliferative endometrium, but its function within this tissue is poorly understood. In this study, we first characterized the expression of PMP22 in the human menstrual cycle and began to characterize its function in the endometrium.MethodsUsing a combination of immunohistochemistry and quantitative PCR, we characterized the expression of PMP22 in both proliferative and secretory endometrium. Differences in PMP22 expression between proliferative and secretory endometrium were determined using a Mann-Whitney U test. In order to investigate the influence of PMP22 on α6 integrin expression, cells were created that ectopically overexpressed PMP22 or expressed a siRNA to inhibit its expression. These cells were analyzed for changes in integrins and binding to extracellular matrices.ResultsIn this study, we show that PMP22 expression is higher in proliferative phase than secretory phase. Functionally, we have begun to characterize the functional significance of this expression. Previous studies have suggested a link between PMP22 and α6 integrin, and therefore we asked whether PMP22 could associate or potentially modulate the expression of α6 integrin. Expression of both PMP22 and α6 integrin were detectable in endometrial epithelial and stromal cells, and we show that both proteins can associate and colocalize with each other. To understand if PMP22 directly altered the expression of a6 integrin, we examined cell lines with modulated levels of the protein. Overexpression of PMP22 was sufficient to increase α6 integrin surface expression with a concominant increase in binding to the extracellular matrix laminin, while a reduction in PMP22 suppressed α6 integrin surface expression.ConclusionThese findings suggest a physiologic role for PMP22 on the expression of α6 integrin. We predict that this may be important for the maintainence of endometrial integrity and to the disease biology associated with altered levels of α6 integrin expression in the endometrium.

Objective: Progestin therapy is a fertility-sparing treatment option for women with complex atypical hyperplasia (CAH) or low grade endometrial adenocarcinoma (Grade 1 EA). However, no biological markers have yet been identified that reliably predict response to treatment. Expression of progesterone receptor and activation of the PI3K/Akt/mTOR pathway have been hypothesized to impact the effectiveness of progestin treatment. We investigated whether the expression of progesterone receptor isoforms PR-A and PR-B, and of pAkt and p4EBP1 as indicators of PI3K/Akt/mTOR pathway signaling in pretreatment biopsy specimens predict the response of patients with CAH and Grade 1 EA to progestin treatment. Methods: Premenopausal women with CAH or Grade 1 EA who underwent progestin therapy for a minimum of 8 weeks were identified. Pre-treatment endometrial biopsy specimens and matched second specimens obtained while on progestin treatment were stained by immunohistochemistry for expression of PR-A, PR-B, pAkt and p4EBP1. The H-score (staining intensity × % of cells) was determined for tumor cells and stroma. T tests were used to compare mean H-scores. Standardized resolution ratios (SRR) were calculated to assess relationship with resolution. Results: 38 subjects were identified with a median age of 36 years (range 23-48). On initial sampling, pathology showed G1EAC in 35% and CAH in 65% of subjects. 50% of subjects had documented resolution to normal histopathology with a median time to resolution of 7 months. Staining for PR-A, PR-B, pAkt and p4EBP1 was found to be positive in 84%, 87%, 84%, and 24% of all patients. The H-score was significantly higher in tumor tissue compared to stromal tissue (p<.0003) for all four markers. PR-A and PR-B expression was significantly decreased in the second specimen under progestin treatment for subjects both with and without resolution (p<.00001). In contrast, pAkt expression decreased significantly after treatment only in subjects with resolution (p = .02). Expression of p4EBP1 did not change significantly with treatment (p = .24). Strong positive PR-A or PR-B staining in tumor tissue of pretreatment biopsies showed a positive correlation with resolution (SRR=1.95, CI 1.15-3.18; SRR = 2.37, CI 1.25-4.13, respectively). In addition, p4EBP1 positivity was associated with resolution (SRR = 2.3, CI 1.48-3.52). Expression of pAkt did not correlate significantly with resolution (SRR = 1.5, CI 0.66-3.29). Conclusions: Our data suggest that the response of premenopausal women with CAH or Grade 1 EA to progestin treatment is significantly correlated with expression of PR-A, PR-B or p4EBP1 in pre-treatment tumor tissue. We propose that the expression of PR-A, PR-B and p4EBP1 may serve as valuable molecular predictors of response to progestin therapy in CAH and Grade 1 EA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3157. doi:10.1158/1538-7445.AM2011-3157