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1.  Utility of MLH1 Methylation Analysis in the Clinical Evaluation of Lynch Syndrome in Women with Endometrial Cancer 
Current pharmaceutical design  2014;20(11):1655-1663.
Clinical screening criteria, such as young age of endometrial cancer diagnosis and family history of signature cancers, have traditionally been used to identify women with Lynch Syndrome, which is caused by mutation of a DNA mismatch repair gene. Immunohistochemistry and microsatellite instability analysis have evolved as important screening tools to evaluate endometrial cancer patients for Lynch Syndrome. A complicating factor is that 15-20% of sporadic endometrial cancers have immunohistochemical loss of the DNA mismatch repair protein MLH1 and high levels of microsatellite instability due to methylation of MLH1. The PCR-based MLH1 methylation assay potentially resolves this issue, yet many clinical laboratories do not perform this assay. The objective of this study was to determine if clinical and pathologic features help to distinguish sporadic endometrial carcinomas with MLH1 loss secondary to MLH1 methylation from Lynch Syndrome-associated endometrial carcinomas with MLH1 loss and absence of MLH1 methylation. Of 337 endometrial carcinomas examined, 54 had immunohistochemical loss of MLH1. 40/54 had MLH1 methylation and were designated as sporadic, while 14/54 lacked MLH1 methylation and were designated as Lynch Syndrome. Diabetes and deep myometrial invasion were associated with Lynch Syndrome; no other clinical or pathological variable distinguished the 2 groups. Combining Society of Gynecologic Oncology screening criteria with these 2 features accurately captured all Lynch Syndrome cases, but with low specificity. In summary, no single clinical/pathologic feature or screening criteria tool accurately identified all Lynch Syndrome-associated endometrial carcinomas, highlighting the importance of the MLH1 methylation assay in the clinical evaluation of these patients.
PMCID: PMC3895501  PMID: 23888949
Lynch Syndrome; molecular diagnostics; MLH1 methylation; immunohistochemistry; endometrial cancer
2.  Sex hormone regulation of survivin gene expression 
The Journal of endocrinology  2010;207(2):237-243.
Survivin (BIRC5) is a cell survival gene that is overexpressed in endometrial cancer and has been implicated to have a physiological role in normal endometrial function. To determine whether survivin gene expression is regulated by reproductive steroid hormones in the human endometrium, RNA was prepared from normal cycling women in the proliferative and secretory phases of the menstrual cycle. RNA was also isolated from 21 endometrial biopsies from premenopausal women at baseline and following 3 months of treatment with depot medroxyprogesterone acetate. Finally, RNA was isolated from endometrial biopsies from ten healthy postmenopausal women participating in a clinical trial of estrogen replacement therapy at baseline and following 6 months of treatment with conjugated equine estrogen. Quantitative RT-PCR analysis was used to determine survivin, insulin-like growth factor binding protein 1 (IGFBP1), Ki67, and IGF1 gene expression levels. Survivin gene expression was highest in the proliferative phase of the menstrual cycle and showed a statistically significant 4-fold increase in expression following chronic treatment with estrogens; this was strongly correlated with increased Ki67, a marker of proliferation. Survivin gene expression decreased 4·6-fold following chronic progestin treatment in the human endometrium. These data suggest that survivin transcript is regulated by estrogens and progestins in the disease-free human endometrium. The data also suggest that survivin transcript may be used as a biomarker of estrogen and progestin treatment efficacy, but validation studies must be conducted to support this conclusion.
doi:10.1677/JOE-10-0128
PMCID: PMC4270120  PMID: 20798131
3.  Clinicodemographic factors influencing outcomes in patients with low-grade serous ovarian carcinoma 
Cancer  2011;117(16):3741-3749.
Background
Low-grade serous carcinoma (LGSC) of the ovary is a rare tumor that is distinct from its high-grade counterpart. Our aim was to estimate if patient demographic factors and clinical treatment histories affected survival in a population of women with this disease.
Methods
A review of patients with pathologically-confirmed LGSC of the ovary diagnosed between 1977- 2009 was performed. Abstracted data included medical and social histories, anthropometric measurements, and details about diagnosis, treatment, and follow-up. Statistical analyses included Fisher's exact test, Cox proportional hazards models, and the Kaplan-Meier method.
Results
The study sample included 194 patients with a median follow-up of 60.9 months (range 1-383). By multivariable analyses, smoking was negatively associated with both overall survival (OS) (HR 1.73; 95% CI [1.03-2.92], p=0.04) and progression-free survival (PFS) (HR 1.72; 95% CI [1.00-2.96], p=0.05). Median OS was shorter in current smokers than former/never smokers (48.0 vs. 79.9 months; p=.002). PFS was also predicted by year of diagnosis >1994 (HR1.74, p=0.01). Although not statistically significant, hormone consolidation appeared to be associated with better OS (HR 0.15, p=.06) and better PFS (HR 0.44; p=.07). A smaller proportion of patients treated with hormone consolidation experienced recurrence compared to those who did not receive hormone consolidation (66.7% vs. 87.6%, p=0.07).
Conclusion
Smoking is negatively associated with survival outcomes in women with LGSC of the ovary, while consolidation treatment with hormone antagonists demonstrated a protective associative trend with survival. Both lifestyle modification and innovative treatment plans should be considered in this group of patients.
doi:10.1002/cncr.25929
PMCID: PMC4254832  PMID: 21319148
4.  Another Surprise from Metformin: Novel Mechanism of Action via K-Ras Influences Endometrial Cancer Response to Therapy 
Molecular cancer therapeutics  2013;12(12):10.1158/1535-7163.MCT-13-0439.
Metformin is an oral biguanide commonly used for the treatment of type II diabetes and has recently been demonstrated to possess anti-proliferative properties that can be exploited for the prevention and treatment of a variety of cancers. The mechanisms underlying this effect have not been fully elucidated. Using both in vitro and in vivo models, we examined the effects of metformin on endometrial tumors with defined aberrations in the PI3K/PTEN/mTOR and MAPK signaling pathways to understand metformin mechanism of action and identify clinically useful predictors of response to this agent. In vitro assays of proliferation, cytotoxicity, and apoptosis were used to quantify the effects of metformin on endometrial cancer cell lines with mutations in the PI3K/PTEN/mTOR and MAPK signaling pathways. The in vivo effects of oral metformin on tumor progression were further examined using xenograft mouse models of endometrial cancer. K-Ras localization was analyzed by confocal microscopy using GFP-labeled oncogenic K-Ras and by immunoblot following subcellular fractionation. Metformin inhibited cell proliferation, induced apoptosis, and decreased tumor growth in preclinical endometrial cancer models, with the greatest response observed in cells harboring activating mutations in K-Ras. Furthermore, metformin displaces constitutively active K-Ras from the cell membrane, causing uncoupling of the MAPK signaling pathway. These studies provide a rationale for clinical trials using metformin in combination with PI3K targeted agents for tumors harboring activating K-Ras mutations, and reveal a novel mechanism of action for metformin.
doi:10.1158/1535-7163.MCT-13-0439
PMCID: PMC3883498  PMID: 24077915
metformin; endometrial cancer; K-Ras; PTEN
5.  Primary Peritoneal Cancer After Bilateral Salpingo-oophorectomy in Two Patients With Lynch Syndrome 
Obstetrics and gynecology  2010;115(2 0 2):432-434.
Background
Women with Lynch syndrome or hereditary nonpolyposis colorectal carcinoma (HNPCC) have a 40-60% lifetime risk of endometrial cancer and a 7-12% lifetime risk of ovarian cancer. Risk-reducing surgery, including hysterectomy and bilateral salpingooophorectomy (BSO), is currently recommended once childbearing is complete. We describe two cases of primary peritoneal cancer following BSO in women with Lynch syndrome or HNPCC.
Cases
The first patient was a 44 year-old woman who underwent hysterectomy with BSO for benign disease. She presented 12 years later with a pelvic mass and was diagnosed with a high-grade serous primary peritoneal cancer. Genetic testing showed a mutation in the MSH2 DNA mismatch repair gene. The second case wasa 58 year-old woman who had a hysterectomy and BSO for endometrial cancer. She developed a high-grade serous primary peritoneal cancer 8 years later and was found to have a mutation in the PMS2 DNA mismatch repair gene.
Conclusion
Women with Lynch syndrome or HNPCC should be counseled that they may be at risk for developing primary peritoneal cancer despite undergoing gynecologic cancer risk-reducing surgery. The magnitude of this risk remains to be determined.
doi:10.1097/AOG.0b013e3181b6f4f9
PMCID: PMC4250042  PMID: 20093870
6.  Pathological Scoring of PTEN Immunohistochemistry in Endometrial Carcinoma is Highly Reproducible 
Endometrial carcinomas show frequent PTEN-PI3K pathway abnormalities, and there are currently multiple trials focused on PI3K pathway inhibitors in endometrial carcinoma patients. PTEN immunohistochemistry may help select patients with potential for response to targeted therapy making it important to develop and validate this stain in formalin-fixed, paraffin embedded tissue. Immunohistochemistry for PTEN was performed and scored independently on 118 cases of endometrial carcinomas from 2 cancer centers using monoclonal DAKO 6H2.1antibody. Cases were scored as positive, negative or heterogeneous; reproducibility of PTEN staining and interpretation was assessed.
Overall interobserver agreement was good (weighted κ = 0.80), with 82% concordance, similar for non endometrioid (81%) and endometrioid carcinomas (85%). 21 of 118 cases showed discrepant results (17%); that resulted from differences in interpretation and not staining.
Our study shows that evaluation of PTEN loss by immunohistochemistry is highly reproducible with the application of standard immunohistochemical techniques and simple scoring criteria.
doi:10.1097/PGP.0b013e3182230d00
PMCID: PMC4244710  PMID: 22123723
Endometrial carcinoma; PI3K pathway; PTEN immunohistochemistry
7.  OvMark: a user-friendly system for the identification of prognostic biomarkers in publically available ovarian cancer gene expression datasets 
Molecular Cancer  2014;13(1):241.
Background
Ovarian cancer has the lowest survival rate of all gynaecologic cancers and is characterised by a lack of early symptoms and frequent late stage diagnosis. There is a paucity of robust molecular markers that are independent of and complementary to clinical parameters such as disease stage and tumour grade.
Methods
We have developed a user-friendly, web-based system to evaluate the association of genes/miRNAs with outcome in ovarian cancer. The OvMark algorithm combines data from multiple microarray platforms (including probesets targeting miRNAs) and correlates them with clinical parameters (e.g. tumour grade, stage) and outcomes (disease free survival (DFS), overall survival). In total, OvMark combines 14 datasets from 7 different array platforms measuring the expression of ~17,000 genes and 341 miRNAs across 2,129 ovarian cancer samples.
Results
To demonstrate the utility of the system we confirmed the prognostic ability of 14 genes and 2 miRNAs known to play a role in ovarian cancer. Of these genes, CXCL12 was the most significant predictor of DFS (HR = 1.42, p-value = 2.42x10−6). Surprisingly, those genes found to have the greatest correlation with outcome have not been heavily studied in ovarian cancer, or in some cases in any cancer. For instance, the three genes with the greatest association with survival are SNAI3, VWA3A and DNAH12.
Conclusions/Impact
OvMark is a powerful tool for examining putative gene/miRNA prognostic biomarkers in ovarian cancer (available at http://glados.ucd.ie/OvMark/index.html). The impact of this tool will be in the preliminary assessment of putative biomarkers in ovarian cancer, particularly for research groups with limited bioinformatics facilities.
Electronic supplementary material
The online version of this article (doi:10.1186/1476-4598-13-241) contains supplementary material, which is available to authorized users.
doi:10.1186/1476-4598-13-241
PMCID: PMC4219121  PMID: 25344116
8.  Critical tumor suppressor function mediated by epithelial Mig-6 in endometrial cancer 
Cancer research  2013;73(16):5090-5099.
Endometrial cancer is preceded by endometrial hyperplasia, unopposed estrogen exposure and genetic alterations, but the precise causes of endometrial cancer remain uncertain. Mig-6, mainly known as a negative regulator of the EGF receptor, is an important mediator of progesterone signaling in the uterus, where it mediates tumor suppression by modulating endometrial stromal-epithelial communications. In this study, we investigated the function of Mig-6 in the uterine epithelium using a tissue-specific gene knockout strategy, in which floxed Mig-6 (Mig-6f/f) mice were crossed to Wnt7a-Cre mice (Wnt7acre+ Mig-6f/f). Wnt7acre+ Mig-6f/f mice developed endometrial hyperplasia and estrogen-dependent endometrial cancer, exhibiting increased proliferation in epithelial cells as well as apoptosis in sub-epithelial stromal cells. We documented increased expression of NOTCH1 and BIRC3 in epithelial cells of Wnt7acre+ Mig-6f/f mice and decreased expression of the progesterone receptor (PR) in stromal cells. Progesterone therapy controls endometrial growth and prevents endometrial cancer, but the effectiveness of progesterone as a treatment for women with endometrial cancer is less clear. We noted that the hyperplasic phenotype of Wnt7acre+ Mig-6f/f mice was prevented by progesterone treatment, whereas this treatment had no effect in PRcre/+ Mig-6f/f mice where Mig-6 was deleted in both the epithelial and stromal compartments of the uterus. In contrast, activation of progesterone signaling in the stroma regulated proliferation and apoptosis in the epithelium via suppression of ERα signaling there. In summary, our results establish that epithelial Mig-6 functions as a critical tumor suppressor that mediates the ability of progesterone to prevent the development of endometrial cancer.
doi:10.1158/0008-5472.CAN-13-0241
PMCID: PMC3745525  PMID: 23811943
Mig-6; Endometrial cancer; Tumor suppressor; Progesterone; Uterus
9.  Molecular Pathogenesis of Endometrial Cancers in Lynch Syndrome 
Cancer  2013;119(16):3027-3033.
Objective
We hypothesized that Lynch Syndrome (LS)-associated endometrial cancer (EC) develops from morphologically normal endometrium that accumulates molecular changes to progress through a continuum of hyperplasia to carcinoma, similar to sporadic EC. The primary objective of this study was to determine if LS-associated EC involves progression through a pre-invasive lesion. The secondary objective was to identify molecular changes contributing to endometrial carcinogenesis in LS.
Methods
Women with a confirmed mismatch repair gene mutation for LS undergoing a prophylactic or therapeutic hysterectomy were eligible. Case controls were matched for EC and hyperplasia based preferentially on age and histology. Mutation status of PIK3CA, KRAS, AKT, LKB1, CTNNB1, and PTEN was assessed.
Results
Concurrent complex atypical hyperplasia (CAH) was found in EC in 11 (39.3%) and 21 (46.6%) of LS and sporadic cases, respectively. Loss of PTEN expression was common in both sporadic (69%) and LS EC (86.2%). There was no significant difference in frequency of KRAS mutation in sporadic EC (10.3%) compared to LS EC (3.4%). AKT and LKB1 mutations were rarely observed. Mutations in PIK3CA and CTNNB1 occurred more frequently in sporadic EC compared to LS EC.
Conclusions
Hyperplasia, particularly CAH, is part of the pre-invasive spectrum of disease in LS-associated EC as indicated by the presence of complex hyperplasia and CAH in LS cases. While PTEN loss is common in both LS and sporadic EC cases, there was a lack of additional mutations in LS associated EC cases. This suggests that in the context of the mismatch repair defects in LS, fewer additional molecular changes are required to progress from pre-invasive lesions to carcinoma.
doi:10.1002/cncr.28152
PMCID: PMC4120439  PMID: 23760948
10.  Prospective, Multi-center Randomized Intermediate Biomarker Study of Oral Contraceptive vs. Depo-Provera for Prevention of Endometrial Cancer in Women with Lynch Syndrome 
Women with Lynch syndrome have a 40–60% 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 demonstrated that progestin-containing oral contraceptives (OCP) reduce the risk of endometrial cancer by 50% in women at general population risk. It is unknown if they are effective in women with Lynch syndrome. Asymptomatic women age 25–50 with Lynch syndrome were randomized to receive the progestin compounds depo-Provera (depoMPA) or OCP for three months. An endometrial biopsy and transvaginal ultrasound were performed 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 depoMPA 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 demonstrate that women with Lynch syndrome do show an endometrial response to short term exogenous progestins, suggesting that OCP and depoMPA may be reasonable chemopreventive agents in this high-risk patient population.
doi:10.1158/1940-6207.CAPR-13-0020
PMCID: PMC3737517  PMID: 23639481
endometrial cancer; chemoprevention; Lynch syndrome; progestin
11.  β-Catenin activation contributes to the pathogenesis of adenomyosis through epithelial–mesenchymal transition 
The Journal of pathology  2013;231(2):210-222.
Adenomyosis is defined by the presence of endometrial glands and stroma within the myometrium. Despite its frequent occurrence, the precise aetiology and physiopathology of adenomyosis is still unknown. WNT/β-catenin signalling molecules are important and should be tightly regulated for uterine function. To investigate the role of β-catenin signalling in adenomyosis, the expression of β-catenin was examined. Nuclear and cytoplasmic β-catenin expression was significantly higher in epithelial cells of human adenomyosis compared to control endometrium. To determine whether constitutive activation of β-catenin in the murine uterus leads to development of adenomyosis, mice that expressed a dominant stabilized β-catenin in the uterus were used by crossing PR-Cre mice with Ctnnb1f(ex3)/+ mice. Uteri of PRcre/+ Ctnnb1f(ex3)/+ mice displayed an abnormal irregular structure and highly active proliferation in the myometrium, and subsequently developed adenomyosis. Interestingly, the expression of E-cadherin was repressed in epithelial cells of PRcre/+ Ctnnb1f(ex3)/+ mice compared to control mice. Repression of E-cadherin is one of the hallmarks of epithelial–mesenchymal transition (EMT). The expression of SNAIL and ZEB1 was observed in some epithelial cells of the uterus in PRcre/+ Ctnnb1f(ex3)/+ mice but not in control mice. Vimentin and COUP-TFII, mesenchymal cell markers, were expressed in some epithelial cells of PRcre/+ Ctnnb1f(ex3)/+ mice. In human adenomyosis, the expression of E-cadherin was decreased in epithelial cells compared to control endometrium, while CD10, an endometrial stromal marker, was expressed in some epithelial cells of human adenomyosis. These results suggest that abnormal activation of β-catenin contributes to adenomyosis development through the induction of EMT.
doi:10.1002/path.4224
PMCID: PMC4105844  PMID: 23784889
β-catenin; adenomyosis; epithelial–mesenchymal transition; uterus
12.  Loss of Inhibitory IRS-1 Phosphorylation is an Early Event in mTOR-Dependent Endometrial Hyperplasia and Carcinoma 
IGF-I receptor signaling contributes to the development of endometrial hyperplasia, the precursor to endometrioid-type endometrial carcinoma, in humans and in rodent models. This pathway is under both positive and negative regulation, including S6K phosphorylation of IRS-1 at serine (636/639), which occurs downstream of mTOR activation to inhibit this adapter protein. We observed activation of mTOR with a high frequency in human endometrial hyperplasia and carcinoma, but an absence of IRS-1 phosphorylation, despite high levels of activated S6K. To explore when during disease progression mTOR activation and loss of negative feedback to IRS-1 occurred, we utilized the Eker rat (Tsc2Ek/+) model, where endometrial hyperplasia develops as a result of loss of Tsc2, a “gatekeeper” for mTOR. We observed mTOR activation early in progression in hyperplasias and in some histologically normal epithelial cells, suggesting that event(s) in addition to loss of Tsc2 were required for progression to hyperplasia. In contrast, while IRS-1 S636/639 phosphorylation was observed in normal epithelium, it was absent from all hyperplasias, indicating loss of IRS-1 inhibition by S6K occurred during progression to hyperplasia. Treatment with an mTOR inhibitor (WAY-129327) significantly decreased hyperplasia incidence and proliferative indices. Since progression from normal epithelium to carcinoma proceeds via endometrial hyperplasia, these data suggest a progression sequence where activation of mTOR is followed by loss of negative feedback to IRS-1 during the initial stages of development of this disease.
doi:10.1158/1940-6207.CAPR-09-0199
PMCID: PMC4103009  PMID: 20179297
Endometrial Hyperplasia; Endometrial Cancer; IRS-1; mTOR; WAY-129327; rat
13.  Chemopreventive effects of metformin on obesity-associated endometrial proliferation 
American journal of obstetrics and gynecology  2013;209(1):10.1016/j.ajog.2013.03.008.
Objective
Obesity is a significant contributing factor to endometrial cancer risk. We previously demonstrated that estrogen-induced endometrial proliferation is enhanced in the context of hyperinsulinemia and insulin resistance. In this study we investigate whether pharmacologic agents that modulate insulin sensitivity or normalize insulin levels will diminish the proliferative response to estrogen.
Study Design
Zucker fa/fa obese rats and lean controls were used as models of hyperinsulinemia and insulin resistance. Insulin levels were depleted in ovariectomized rats following treatment with Streptozotocin (STZ), or modulated by metformin treatment. The number of BrdU incorporated cells, estrogen dependent proliferative and anti-proliferative gene expression, and activation of mTOR and Erk1/2 MAPK signaling were studied. A rat normal endometrial cell line RENE1 was used to evaluate the direct effects of metformin on endometrial cell proliferation and gene expression in vitro.
Results
STZ lowered circulating insulin levels in obese rats and decreased the number of BrdU labeled endometrial cells even in the presence of exogenous estrogen. Treatment with the insulin-sensitizing drug metformin attenuated estrogen-dependent proliferative expression of c-myc and c-fos in the obese rat endometrium compared to untreated controls and was accompanied by inhibition of phosphorylation of the insulin and IGF1 receptors (IRβ/IGF1R) and ERK1/2. In vitro studies indicated metformin inhibited RENE1 proliferation in a dose dependent manner.
Conclusion
These findings suggest that drugs that modulate insulin sensitivity, such as metformin, hinder estrogen-mediated endometrial proliferation. Therefore, these drugs may be clinically useful for the prevention of endometrial cancer in obese women.
doi:10.1016/j.ajog.2013.03.008
PMCID: PMC3819145  PMID: 23500454
Obesity; estrogen; insulin resistance
14.  CGRRF1 as a novel biomarker of tissue response to metformin in the context of obesity 
Gynecologic oncology  2014;133(1):83-89.
Objective
Obesity-associated hyperestrogenism and hyperinsulinemia contribute significantly to the pathogenesis of endometrial cancer. We recently demonstrated that metformin, a drug long used for treatment of type 2 diabetes, attenuates both insulin- and estrogen-mediated proliferative signaling in the obese rat endometrium. In this study, we sought to identify tissue biomarkers that may prove clinically useful to predict tissue response for both prevention and therapeutic studies. We identified CGRRF1 (Cell growth regulator with ring finger domain 1) as a novel metformin-responsive gene and characterized its possible role in endometrial cancer prevention.
Methods
CGRRF1 mRNA expression was evaluated by RT-qPCR in the endometrium of obese and lean rats, and also in normal and malignant human endometrium. CGRRF1 levels were genetically manipulated in endometrial cancer cells, and its effects on proliferation and apoptosis were evaluated by MTT and western blot.
Results
CGRRF1 is significantly induced by metformin treatment in the obese rat endometrium. In vitro studies demonstrate that overexpression of CGRRF1 inhibits endometrial cancer cell proliferation. Analysis of human endometrial tumors reveal that CGRRF1 expression is significantly lower in hyperplasia, Grade 1, Grade 2, Grade 3, MMMT, and UPSC endometrial tumors compared to normal human endometrium (p<0.05), suggesting that loss of CGRRF1 is associated with the presence of disease.
Conclusion
CGRRF1 represents a novel, reproducible tissue marker of metformin response in the obese endometrium. Furthermore, our preliminary data suggests that up-regulation of CGRRF1 expression may prove clinically useful in the prevention or treatment of endometrial cancer.
doi:10.1016/j.ygyno.2013.12.006
PMCID: PMC4077340  PMID: 24680596
Obesity; metformin; estrogen; insulin resistance
15.  The synergistic effect of Mig-6 and Pten ablation on endometrial cancer development and progression 
Oncogene  2010;29(26):3770-3780.
Ablation of Mig-6 in the murine uterus leads to the development of endometrial hyperplasia and estrogen-induced endometrial cancer. An additional endometrial cancer mouse model is generated by ablation of Pten (either as heterozygotes or by conditional uterine ablation). To determine the interplay between Mig-6 and the PTEN/PI3K signaling pathway during endometrial tumorigenesis, we have generated mice with Mig-6 and Pten conditionally ablated in progesterone receptor positive cells (PRcre/+Mig-6f/fPtenf/f ; Mig-6d/dPtend/d). The ablation of both Mig-6 and Pten dramatically accelerated the development of endometrial cancer compared to single ablation of either gene. The epithelium of Mig-6d/dPtend/d mice showed a significant decrease in the number of apoptotic cells compared to Ptend/d mice. The expression of the estrogen-induced apoptotic inhibitors Birc1 was significantly increased in the Mig-6d/dPtend/d mice. We identified ERK2 as a MIG-6 interacting protein by co-immunoprecipitation and demonstrated that the level of ERK2 phosphorylation was increased upon Mig-6 ablation either singly or in combination with Pten ablation. These results suggest that Mig-6 exerts a tumor suppressor function in endometrial cancer by promoting epithelial cell apoptosis through the down-regulation of the estrogen-induced apoptosis inhibitors Birc1 and the inhibition of ERK2 phosphorylation.
doi:10.1038/onc.2010.126
PMCID: PMC4013686  PMID: 20418913
Mig-6; Pten; Uterus; Endometrial cancer
16.  Relationship between PTEN, DNA Mismatch Repair, and Tumor Histotype in Endometrial Carcinoma: Retained Positive Expression of PTEN Preferentially Identifies Sporadic Non-Endometrioid Carcinomas 
Loss of PTEN (phosphatase and tensin homolog) expression and microsatellite instability are two of the more common molecular alterations in endometrial carcinoma. From the published literature, it is controversial as to whether there is a relationship between these different molecular mechanisms. Therefore, a cohort of 187 pure endometrioid and non-endometrioid endometrial carcinomas, carefully characterized as to clinical and pathological features, was examined for PTEN sequence abnormalities and the immunohistochemical expression of PTEN and the DNA mismatch repair proteins MLH1, MSH2, MSH6 and PMS2. MLH1 methylation analysis was performed when tumors had loss of MLH1 protein. Mismatch repair protein loss was more frequent in endometrioid carcinomas compared to non-endometrioid carcinomas, a difference primarily attributable to the presence of MLH1 methylation in a greater proportion of endometrioid tumors. Among the non-endometrioid group, mixed endometrioid/non-endometrioid carcinomas were the histotype that most commonly had loss of a mismatch repair protein. In endometrioid tumors, the frequency of PTEN loss measured by immunohistochemistry and mutation did not differ significantly between the mismatch repair protein intact or mismatch repair protein loss groups, suggesting that PTEN loss is independent of mismatch protein repair status in this group. However, in non-endometrioid carcinomas, both intact positive PTEN immunohistochemical expression and PTEN wild type were highly associated with retained positive expression of mismatch repair proteins in the tumor. Relevant to screening endometrial cancers for Lynch Syndrome, an initial PTEN immunohistochemistry determination may be able to replace the use of four mismatch repair immunohistochemical markers in 63% of patients with non-endometrioid endometrial carcinoma. Therefore, PTEN immunohistochemistry, in combination with tumor histotype, is a useful adjunct in the clinical evaluation of endometrial carcinomas for Lynch Syndrome.
doi:10.1038/modpathol.2013.67
PMCID: PMC3720775  PMID: 23599155
17.  Integrated analyses identify a master microRNA regulatory network for the mesenchymal subtype in serous ovarian cancer 
Cancer cell  2013;23(2):186-199.
Summary
Integrated genomic analyses revealed a miRNA-regulatory network, which further defined a robust integrated mesenchymal subtype associated with poor overall survival in 459 cases of serous ovarian cancer (OvCa) from The Cancer Genome Atlas and 560 cases from independent cohorts. Eight key miRNAs, including miR-506, miR-141 and miR-200a, were predicted to regulate 89% of the targets in this network. Follow-up functional experiments illustrate that miR-506 augmented E-cadherin expression, inhibited cell migration and invasion, and prevented TGFβ-induced epithelial-mesenchymal transition (EMT) by targeting SNAI2, a transcriptional repressor of E-cadherin. In human OvCa, miR-506 expression was correlated with decreased SNAI2 and VIM, elevated E-cadherin, and beneficial prognosis. Nanoparticle delivery of miR-506 in orthotopic OvCa mouse models led to E-cadherin induction and reduced tumor growth.
doi:10.1016/j.ccr.2012.12.020
PMCID: PMC3603369  PMID: 23410973
18.  Human Omental-Derived Adipose Stem Cells Increase Ovarian Cancer Proliferation, Migration, and Chemoresistance 
PLoS ONE  2013;8(12):e81859.
Objectives
Adipose tissue contains a population of multipotent adipose stem cells (ASCs) that form tumor stroma and can promote tumor progression. Given the high rate of ovarian cancer metastasis to the omental adipose, we hypothesized that omental-derived ASC may contribute to ovarian cancer growth and dissemination.
Materials and Methods
We isolated ASCs from the omentum of three patients with ovarian cancer, with (O-ASC4, O-ASC5) and without (O-ASC1) omental metastasis. BM-MSCs, SQ-ASCs, O-ASCs were characterized with gene expression arrays and metabolic analysis. Stromal cells effects on ovarian cancer cells proliferation, chemoresistance and radiation resistance was evaluated using co-culture assays with luciferase-labeled human ovarian cancer cell lines. Transwell migration assays were performed with conditioned media from O-ASCs and control cell lines. SKOV3 cells were intraperitionally injected with or without O-ASC1 to track in-vivo engraftment.
Results
O-ASCs significantly promoted in vitro proliferation, migration chemotherapy and radiation response of ovarian cancer cell lines. O-ASC4 had more marked effects on migration and chemotherapy response on OVCA 429 and OVCA 433 cells than O-ASC1. Analysis of microarray data revealed that O-ASC4 and O-ASC5 have similar gene expression profiles, in contrast to O-ASC1, which was more similar to BM-MSCs and subcutaneous ASCs in hierarchical clustering. Human O-ASCs were detected in the stroma of human ovarian cancer murine xenografts but not uninvolved ovaries.
Conclusions
ASCs derived from the human omentum can promote ovarian cancer proliferation, migration, chemoresistance and radiation resistance in-vitro. Furthermore, clinical O-ASCs isolates demonstrate heterogenous effects on ovarian cancer in-vitro.
doi:10.1371/journal.pone.0081859
PMCID: PMC3847080  PMID: 24312594
19.  Restoration of miR-200c to ovarian cancer reduces tumor burden and increases sensitivity to paclitaxel 
Molecular cancer therapeutics  2012;11(12):2556-2565.
A therapeutic intervention that could decrease tumor burden and increase sensitivity to chemotherapy would have a significant impact on the high morbidity rate associated with ovarian cancer. MicroRNAs (miRNAs) have emerged as potential therapeutic candidates due to their ability to down regulate multiple targets involved in tumor progression and chemoresistance. MiR-200c is down regulated in ovarian cancer cell lines and stage III ovarian tumors, and low miR-200c correlates with poor prognosis. MiR-200c increases sensitivity to taxanes in vitro, by targeting TUBB3, a tubulin known to mediate chemoresistance. Indeed, we find that patients with tumors with low TUBB3 had significantly prolonged survival (average survival 52.73 ± 4.08 months) compared to those with high TUBB3 (average survival 42.56 ±3.19 months). MiR-200c also targets TrkB, a mediator of resistance to anoikis. We demonstrate that restoration of miR-200c to ovarian cancer cells results in increased anoikis sensitivity and reduced adherence to biological substrates in vitro. Since both chemo- and anoikis-resistance are critical steps in the progression of ovarian cancer, we sought to determine how restoration of miR-200c affects tumor burden and chemosensitivity in an in vivo preclinical model of ovarian cancer. Restoration of miR-200c in an intraperitoneal xenograft model of human ovarian cancer, results in decreased tumor formation and tumor burden. Furthermore, even in established tumors, restoration of miR-200c, alone or in combination with paclitaxel, results in significantly decreased tumor burden. Our study suggests that restoration of miR-200c immediately prior to cytotoxic chemotherapy may allow for a better response or lower effective dose.
doi:10.1158/1535-7163.MCT-12-0463
PMCID: PMC3519949  PMID: 23074172
miR-200; Hey cells; chemotherapy; IVIS; paclitaxel
20.  Application of Immunohistochemistry and Molecular Diagnostics to Clinically Relevant Problems in Endometrial Cancer Bojana Djordjevic, Shannon Westin, Russell R. Broaddus 
Surgical pathology clinics  2012;5(4):859-878.
Synopsis
A number of different clinical scenarios are presented in which lab-based analyses beyond the usual diagnosis based on light microscopic examination of H&E stained slides – immunohistochemistry and PCR-based assays such as sequencing, mutation testing, microsatellite instability analysis, and determination of MLH1 methylation - are most helpful for guiding diagnosis and treatment of endometrial cancer.
The central goal of this information is to provide a practical guide of key current and emerging issues in diagnostic endometrial cancer pathology that require the use of ancillary laboratory techniques, such as immunohistochemistry and molecular testing. The authors present the common diagnostic problems in endometrial carcinoma pathology, types of endometrial carcinoma, description of tissue testing and markers, pathological features, and targeted therapy.
doi:10.1016/j.path.2012.08.004
PMCID: PMC3653323  PMID: 23687522
endometrial cancer; targeted therapy; Lynch Syndrome
21.  HE4 Transcription- and Splice Variants-Specific Expression in Endometrial Cancer and Correlation with Patient Survival 
We investigated the HE4 variant-specific expression patterns in various normal tissues as well as in normal and malignant endometrial tissues. The relationships between mRNA variants and age, body weight, or survival are analyzed. ICAT-labeled normal and endometrial cancer (EC) tissues were analyzed with multidimensional liquid chromatography followed by tandem mass spectrometry. Levels of HE4 mRNA variants were measured by real-time PCR. Mean mRNA levels were compared among 16 normal endometrial samples, 14 grade 1 and 14 grade 3 endometrioid EC, 15 papillary serous EC, and 14 normal human tissue samples. The relationship between levels of HE4 variants and EC patient characteristics was analyzed with the use of Pearson correlation test. We found that, although all five HE4 mRNA variants are detectable in normal tissue samples, their expression is highly tissue-specific, with epididymis, trachea, breast and endometrium containing the highest levels. HE4-V0, -V1, and -V3 are the most abundant variants in both normal and malignant tissues. All variants are significantly increased in both endometrioid and papillary serous EC, with higher levels observed in grade 3 endometrioid EC. In the EC group, HE4-V1, -V3, and -V4 levels inversely correlate with EC patient survival, whereas HE4-V0 levels positively correlate with age. HE4 variants exhibit tissue-specific expression, suggesting that each variant may exert distinct functions in normal and malignant cells. HE4 levels appear to correlate with EC patient survival in a variant-specific manner. When using HE4 as a biomarker for EC management, the effects of age should be considered.
doi:10.3390/ijms141122655
PMCID: PMC3856083  PMID: 24252907
HE4; transcription; splice variants; gene expression; endometrial cancer
22.  Somatic Deletions of the PolyA Tract in the 3′ Untranslated Region of Epidermal Growth Factor Receptor Are Common in Microsatellite Instability–High Endometrial and Colorectal Carcinomas 
Context
Epidermal growth factor receptor (EGFR) is overexpressed in up to 80% of colorectal and endometrial carcinomas. Deletions of the polyA tract in the 3′ untranslated region (3′ UTR) have been reported in microsatellite instability–high (MSI-H) colonic carcinomas, but their impacts on EGFR expression and downstream pathways are unclear. This phenomenon has not been reported in other MSI-H tumors.
Objective
To assess the 3′ UTR polyA tract of EGFR in both endometrial and colorectal carcinomas and the mutational status of EGFR downstream pathways.
Design
Ninety-eight colorectal carcinomas and 47 endometrial carcinomas were included. EGFR 3′ UTR polyA status was detected by capillary electrophoresis and Sanger sequencing. EGFR gene expression, EGFR copy numbers, and KRAS and BRAF mutation status were analyzed accordingly.
Results
The 3′ UTR polyA tract was deleted in 18 of 23 (78%) MSI-H versus 0 of 24 microsatellite-stable endometrial carcinomas (P < .001). Similar observations were seen in colorectal carcinomas, in which 29 of 36 (81%) MSI-H, 1 of 62 (1.6%) microsatellite instability–low, and none of the microsatellite-stable tumors harbored the deletion (P < .001). A moderate increase in EGFR mRNA level was observed in endometrial carcinomas with 3′ UTR polyA deletions versus those with wild-type polyA tract. Amplification of the EGFR gene was not observed. Deletions in polyA tract do not seem to affect the frequency of KRAS and BRAF mutations.
Conclusions
Deletions of EGFR 3′ UTR polyA are frequent in endometrial and colorectal carcinomas, are confined almost exclusively to MSI-H tumors, and do not affect KRAS and BRAF mutations.
doi:10.5858/arpa.2010-0638-OA
PMCID: PMC3807134  PMID: 22540299
23.  Endometrial Cancer and Lynch Syndrome: Clinical and Pathologic Considerations 
Background
Approximately 2% to 5% of endometrial cancers may be due to an inherited susceptibility. Lynch syndrome, also known as hereditary nonpolyposis colorectal cancer (HNPCC) syndrome, an autosomal-dominant inherited cancer susceptibility syndrome caused by a germline mutation in one of the DNA mismatch repair genes, accounts for the majority of inherited cases. Lynch syndrome is associated with early onset of cancer and the development of multiple cancer types, particularly colon and endometrial cancer.
Methods
The current status of knowledge regarding Lynch syndrome-associated endometrial cancer and methods for diagnosis, screening and prevention of cancers are reviewed.
Results
The lifetime cumulative risk of endometrial cancer for women with Lynch syndrome is 40% to 60%, which equals or exceeds their risk of colorectal cancer. No current evidence suggests either a survival advantage or disadvantage to endometrial cancer that is associated with Lynch syndrome when these cases are compared with sporadic cases. A combination of family and personal medical history and tumor testing provides an efficient combination for diagnosing Lynch syndrome in women with endometrial cancer. Current gynecologic cancer screening guidelines for women with Lynch syndrome include annual endometrial sampling and transvaginal ultrasonography beginning at age 30 to 35 years.
Conclusions
Diagnosing endometrial cancer patients with Lynch syndrome has important clinical implications for the individual and family members. Screening and prevention practices can decrease the likelihood of developing additional cancers.
PMCID: PMC3693757  PMID: 19078925
24.  Effects of Gut-Targeted 15-LOX-1 Transgene Expression on Colonic Tumorigenesis in Mice 
Expression of 15-lipoxygenase-1 (15-LOX-1) is decreased in many human cancers; however, the mechanistic significance of its decreased expression has been difficult to determine because its mouse homolog 12/15-LOX has opposing functions. We generated a mouse model in which expression of a human 15-LOX-1 transgene was targeted to the intestinal epithelium via the villin promoter. Targeted expression was confirmed by real-time reverse transcription–polymerase chain reaction and immunoblotting. When the 15-LOX-1 transgene was expressed in colonic epithelial cells of two independent mouse lines (B6 and FVB), azoxymethane-inducible colonic tumorigenesis was suppressed (mean number of tumors: wild type [WT] = 8.2, 15-LOX-1+/− = 4.91, 15-LOX-1+/+ = 3.57; WT vs 15-LOX-1+/− two-sided P = .003, WT vs 15-LOX-1+/+ two-sided P < .001; n = 10–14 mice per group). 15-LOX-1 transgene expression was always decreased in the tumors that did develop. In the presence of expression of the 15-LOX-1 transgene, expression of tumor necrosis factor alpha and its target inducible nitric oxide synthase were decreased and activation of nuclear factor-kappa B in colonic epithelial cells was inhibited.
doi:10.1093/jnci/djs187
PMCID: PMC3341308  PMID: 22472308
25.  Uterine Serous Carcinoma: Increased Familial Risk for Lynch-Associated Malignancies 
Purpose
Serous uterine cancer is not a feature of any known hereditary cancer syndrome. This study evaluated familial risk of cancers for serous uterine carcinoma patients, focusing on Lynch syndrome malignancies.
Experimental design
Fifty serous or mixed serous endometrial carcinoma cases were prospectively enrolled. Pedigrees were developed for 29 probands and tumors were assessed for DNA mismatch repair abnormalities. Standardized incidence ratios for cancers in relatives were estimated. A second stage analysis was undertaken using data from GOG-210. Incidence data for cancers reported in relatives of 348 serous and mixed epithelial and 624 endometrioid carcinoma patients were compared.
Results
Nineteen of 29 (65.5%) patients in the single institution series reported a Lynch-related cancer in relatives. Endometrial and ovarian cancers were significantly over-represented and a high number of probands (6/29, 20.7%) reported pancreatic cancers. None of the probands’ tumors had DNA mismatch repair abnormalities. There was no difference in endometrial or ovarian cancer incidence in relatives of serous and endometrioid cancer probands in the case-control study. Pancreatic cancers were, however, significantly more common in relatives of serous cancer patients (OR 2.39, 95% CI 1.06–5.38).
Conclusions
We identified an excess of endometrial, ovarian, and pancreatic cancers in relatives of serous cancer patients in a single institution study. Follow-up studies suggest only pancreatic cancers are over-represented in relatives. DNA mismatch repair defects in familial clustering of pancreatic and other Lynch-associated malignancies are unlikely. The excess of pancreatic cancers in relatives may reflect an as yet unidentified hereditary syndrome that includes uterine serous cancers.
doi:10.1158/1940-6207.CAPR-11-0499
PMCID: PMC3294192  PMID: 22246618
Endometrial cancer; Uterine serous carcinoma; Lynch syndrome; Mismatch repair; Familial clustering of cancers

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