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Objectives: Fatty acid synthase (FAS) is a key enzyme involved in fatty acid synthesis. It also plays an important role in tumorigenesis and is highly expressed in many cancers, including endometrial cancer. Orlistat is a weight loss medication that has been shown to be a potent inhibitor of FAS. The goal of this study was to evaluate the anti-tumorigenic potential of orlistat in endometrial cancer cell lines. Methods: The endometrial cancer cell lines ECC-1 and KLE were treated with varying doses of orlistat (0.01-500 μM). Cell proliferation was assessed using the MTT assay. Cell cycle progression was evaluated by Cellometer and apoptosis was assessed using the Annexin V assay. Reactive oxygen species (ROS) was measured using the DCFH-DA assay. Western immunoblotting was performed to determine changes in FAS, cellular stress, cell cycle progression and the AMPK/mTOR pathways. Results: Orlistat inhibited cell proliferation in a dose-dependent manner in both cell lines (IC50 = 81.7 μM in ECC-1; IC50 = 145.5 μM in KLE). Treatment with orlistat resulted in G1 arrest but did not affect apoptosis. Orlistat increased ROS at high doses (500 uM, p = 0.008 for ECC-1 cells and p = 0.012 for KLE cells) and induced expression of BIP and PERK. Western immunoblot analysis demonstrated that orlistat decreased expression of FAS, acetyl-CoA carboxylase (ACC), and carnitine palmitoyltransferase 1A (CPT1A), important proteins in fatty acid metabolism. Treatment with orlistat also increased expression of phosphorylated AMPK and decreased expression of the downstream S6 protein. Conclusions: Orlistat suppressed cell growth in endometrial cancer cell lines through inhibition of fatty acid metabolism, induction of cell cycle G1 arrest, and modulation of the AMPK/mTOR pathway. Given that patients with endometrial cancer have high rates of obesity, orlistat should be further investigated as a novel strategy for endometrial cancer treatment and prevention. Citation Format: Weiya Z. Wysham, Dario R. Roque, Jianjun Han, Hui Guo, Lu Zhang, Paola A. Gehrig, Chunxiao Zhou, Victoria L. Bae-Jump. Orlistat decreases endometrial cancer cell proliferation: implications for a novel treatment strategy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2994.

Objectives: Endometrial cancers (ECs) frequently harbor mutations in PI3K pathway genes. Our objective was to determine the mutation frequency of the PIK3 pathway in the tumors of EC patients prospectively enrolled on an IRB-approved institutional tumor sequencing initiative (UNCseq) and correlate these with clinical factors. Methods: Snap-frozen and FFPE tissue samples were collected from patients enrolled on UNCseq (NCT01457196). DNA was isolated using the Puregene DNA purification system, and Illumina libraries were prepared separately from tumor and a matched normal sample from each patient. Relevant targets, were enriched by a custom designed Agilent SureSelect hybrid capture enrichment library using standard protocols. Samples were sequenced on Illumina HiSeq machines in a variety of formats. Statistical analysis of clinical correlates was performed in R (v 3.1.1). Results: Data was collected from 109 EC patients. Thirty-six tumors (33.0%), 34 of which were endometrioid, had >2 mutations between PIK3CA and PIK3R1. 20 patients had multiple PIK3CA mutations (18.3%) and 11 had multiple PIK3R1 mutations (10.1%). In all but 5 cases, multiple mutations of PIK3R1 or PIK3CA were coincident with PTEN mutation (p = 0.018). There were strong correlations between PI3K pathway mutations and tumor grade and stage. High stage tumors (stage >2) were less likely (5/36) than stage 1 tumors (31/73) to have multiple PI3K mutations (p = 0.0056). Though not reaching significance, grade 3 tumors (8/37) were less likely than low-grade tumors (28/72) to demonstrate multiple PI3K mutations (p = 0.11). Of the 10 serous ECs, none exhibited multiple PI3K mutations. Conclusions: There is a strong relationship between mutated PIK3 pathway genes and clinically relevant tumor biology in ECs. Multiple mutations in the PIK3 pathway are common, suggesting that PI3K pathway inhibition in EC may need to be directed against PIK3CA and PIK3R1 simultaneously. These multiply mutated tumors are strongly linked to lower stage and grade tumors with concurrent PTEN mutations. We posit that initially PTEN mutant clones may give rise to subclonal populations with various PI3K mutations which may be relatively indolent until additional driver mutations are acquired such that one subpopulation predominates, resulting in higher grade and stage ECs. Citation Format: Dario R. Roque, Will R. Jeck, David N. Hayes, Arthur M. Dizon, Leslie Clark, Stuart Pierce, Weiya Z. Wysham, Tara Castellano, Paola A. Gehrig, Victoria Bae-Jump. Multiple PI3K pathway mutations predominate in low stage endometrial carcinomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1852.

Objective: To determine the effect of diet-induced obesity on the development of endometrial hyperplasia and cancer in genetically engineered mouse models. Methods: The PTEN heterozygous mouse model for endometrial hyperplasia and the LKB1/p53 endometrioid endometrial cancer mouse model were used. Mice were divided into two dietary groups and fed either a high-fat diet (HFD, 60% calories from fat, obese) or a low-fat diet (LFD, 10% calories from fat, lean) beginning at 3 weeks of age until sacrifice at 32 weeks (PTEN model) and 12 weeks (LKB1/p53 model). At the time of sacrifice, mouse weight and uterine/tumor size were documented. A dedicated mouse pathologist reviewed uterine pathology. For the PTEN heterozygous mouse model, uterine pathology was determined to be simple hyperplasia (SH) if increased number of simple tubular glands were seen without atypia. Complex hyperplasia (CH) was defined as branching, papillary lumenal infoldings, or back-to-back glandular crowding with or without nuclear atypic. Results: A total of 30 PTEN mice were examined, 14 in the obese group and 16 in the lean group. The HFD-fed mice were larger than the LFD-fed mice with a mean weight of 38.1 gm compared to 29.7 gm (p Conclusions: Diet-induced obesity led to increased mean body weight in both mouse models. In the PTEN heterozygous mouse model, a trend towards more complex endometrial hyperplasia was found in the obese group along with a higher rate of simple hyperplasia in the lean group. Increased endometrial tumor size was seen in the obese versus lean LKB1/p53 mice, suggesting that obesity may lead to increased aggressiveness in endometrial cancer. Citation Format: Leslie H. Clark, Chunxiao Zhou, Stephanie A. Montgomery, Paola A. Gehrig, Victoria Bae-Jump. Diet-induced obesity promotes tumor aggressiveness in genetically engineered mouse models of endometrial hyperplasia/cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 680.

e22259 Background: In a phase 0 clinical trial of metformin in endometrial cancer (EC) patients, we previously reported that metformin decreased proliferation in the tumors of the majority of patie...

e17090Background: We examined the effect of body mass index (BMI) on genetic mutations in ovarian cancer (OC) patients enrolled on UNCseq. Methods: Snap-frozen and FFPE tissue samples were collecte...

5593Background: We aimed to determine the most common mutations in endometrial cancers (EC) of patients enrolled in UNCSeq and to discern if there was any correlation between the detected mutations...

5592Background: We sought to evaluate the short-term response rate, toxicity and potential molecular changes following treatment with metformin for simple (SEH) and complex endometrial hyperplasia without atypia (CEH). Methods: Women with newly diagnosedsimple or complex endometrial hyperplasia without atypia by endometrial biopsy or dilation & curettage were enrolled onto a prospective multi-institutional single arm clinical trial, and treated with metformin for 3 months (850 mg daily x 4 weeks, then twice daily for 8 weeks), followed by repeat biopsy. Pathologic response and toxicity (CTCAE 4.1) were assessed. Expression of the estrogen (ER) and progesterone (PR) receptor and the metformin transporters (OCT1, OCT3, PMAT, MATE1, MATE2) were evaluated by immunohistochemistry in formalin-fixed, paraffin-embedded endometrial specimens pre- and post-treatment. Slides were scanned using the Aperio ScanScope, and digital images were analyzed using Aperio ImageScope software. We report proportions as %, along w...

BRCA1-associated breast and ovarian cancer risks can be modified by common genetic variants. To identify further cancer risk-modifying loci, we performed a multi-stage GWAS of 11,705 BRCA1 carriers (of whom 5,920 were diagnosed with breast and 1,839 were diagnosed with ovarian cancer), with a further replication in an additional sample of 2,646 BRCA1 carriers. We identified a novel breast cancer risk modifier locus at 1q32 for BRCA1 carriers (rs2290854, P = 2.7×10−8, HR = 1.14, 95% CI: 1.09–1.20). In addition, we identified two novel ovarian cancer risk modifier loci: 17q21.31 (rs17631303, P = 1.4×10−8, HR = 1.27, 95% CI: 1.17–1.38) and 4q32.3 (rs4691139, P = 3.4×10−8, HR = 1.20, 95% CI: 1.17–1.38). The 4q32.3 locus was not associated with ovarian cancer risk in the general population or BRCA2 carriers, suggesting a BRCA1-specific association. The 17q21.31 locus was also associated with ovarian cancer risk in 8,211 BRCA2 carriers (P = 2×10−4). These loci may lead to an improved understanding of the etiology of breast and ovarian tumors in BRCA1 carriers. Based on the joint distribution of the known BRCA1 breast cancer risk-modifying loci, we estimated that the breast cancer lifetime risks for the 5% of BRCA1 carriers at lowest risk are 28%–50% compared to 81%–100% for the 5% at highest risk. Similarly, based on the known ovarian cancer risk-modifying loci, the 5% of BRCA1 carriers at lowest risk have an estimated lifetime risk of developing ovarian cancer of 28% or lower, whereas the 5% at highest risk will have a risk of 63% or higher. Such differences in risk may have important implications for risk prediction and clinical management for BRCA1 carriers., Author Summary BRCA1 mutation carriers have increased and variable risks of breast and ovarian cancer. To identify modifiers of breast and ovarian cancer risk in this population, a multi-stage GWAS of 14,351 BRCA1 mutation carriers was performed. Loci 1q32 and TCF7L2 at 10q25.3 were associated with breast cancer risk, and two loci at 4q32.2 and 17q21.31 were associated with ovarian cancer risk. The 4q32.3 ovarian cancer locus was not associated with ovarian cancer risk in the general population or in BRCA2 carriers and is the first indication of a BRCA1-specific risk locus for either breast or ovarian cancer. Furthermore, modeling the influence of these modifiers on cumulative risk of breast and ovarian cancer in BRCA1 mutation carriers for the first time showed that a wide range of individual absolute risks of each cancer can be estimated. These differences suggest that genetic risk modifiers may be incorporated into the clinical management of BRCA1 mutation carriers.

Our goal was to evaluate the effect of rapamycin, an mTOR inhibitor, in type I and II human endometrial cancer tumor explants.Short-term tissue culture with fresh endometrial cancer tumor explants was performed. Cell proliferation was assessed by MTS assay after treatment with rapamycin. Akt and PTEN status were documented by Western blotting. The effect of rapamycin on phosphorylated-S6 and 4E-BP-1 was also assessed by Western blotting. Real-time RT-PCR was used to quantify hTERT mRNA expression. Telomere length was determined by terminal restriction fragment Southern blotting.Thirteen fresh endometrial cancer tumor explants (nine Type I, four Type II) were placed in short-term culture and treated with rapamycin. Nine of the endometrial cancer tumors responded to rapamycin, with a median IC₅₀ of 11.4 nM. Sensitivity to rapamycin was independent of PTEN and Akt status. Tumors (13/13) had a reduction in phosphorylated-S6 and 10/13 had a reduction in phosphorylated 4E-BP-1. Rapamycin decreased hTERT mRNA expression in all of the endometrial cancer tumors. Telomere length did not correspond with responsiveness to this drug.Rapamycin demonstrated activity in fresh endometrial tumor explants independent of PTEN and Akt status. Some tumors demonstrated a reduction in phosphorylated-S6 and 4E-BP-1 without a significant change in cellular proliferation, suggesting that additional pathways may modulate cellular proliferation. Thus, mTOR inhibitors may be a useful targeted therapy for both type I and type II endometrial cancers, but the search remains for a predictive biomarker of sensitivity to this therapy.

To discuss the novel agents which are being developed for the treatment of advanced and recurrent endometrial carcinoma and to review other molecular targets that may be interesting in the treatment of this disease. While the majority of women with endometrial cancer enjoy a relatively good prognosis, the options for those women who suffer from a disease recurrence are limited and there is a need to identify novel agents.A review of clinical trials of novel therapeutic agents and their molecular targets is provided. In addition, a review of the current literature on other potential molecular targets for endometrial cancer was performed.Several phase II trials of novel agents, both alone and in combination with traditional cytotoxic chemotherapy, have been completed or are nearing completion. It appears that the targeted agents may have the most efficacy in combination with cytotoxic chemotherapy or in a multi-targeted agent approach.Chemotherapy offers the opportunity for a meaningful response rate in women with endometrial cancer, but the responses are often short lived and cure is uncommon in the setting of recurrent disease. The recent increase in molecular targets has led to the availability of many novel therapies. Determining how these agents are to be used, alone or in combination with "standard" therapies, needs to be defined and translational studies are needed to develop rational combinations of these novel agents before we can move into clinical trials.

Mammalian target of rapamycin (mTOR) inhibitors modulate signaling pathways involved in cell cycle progression, and recent phase II trials demonstrate activity in patients with endometrial cancer. Our objective was to examine the effects of combination therapy with rapamycin and paclitaxel in endometrial cancer cell lines. Paclitaxel inhibited proliferation in a dose-dependent manner in both cell lines with IC(50) values of 0.1-0.5 nM and 1-5 nM for Ishikawa and ECC-1 cells, respectively. To assess synergy of paclitaxel and rapamycin, the combination index (CI) was calculated by the method of Chou and Talalay. Simultaneous exposure of cells to various doses of paclitaxel in combination with rapamycin (1 nM) resulted in a significant synergistic anti-proliferative effect (CI

Objective:To assess the attitudes regarding the use of colony-stimulating factor (CSF) and the maintenance of relative dose intensity (RDI) by gynecologic oncologists during the administration of chemotherapy to patients with epithelial ovarian cancer.Methods:A nationwide survey of 608 gynecologic oncologists was performed using a 19-point questionnaire. The questionnaire assessed the following domains: (1) demographic information, (2) patterns of CSF use during first-line and relapse chemotherapies for patients with epithelial ovarian cancer, and (3) use of CSFs to maintain RDI.Results:The response rate to the survey was 42% (n = 255). Eighty-six percent (220/255) of the respondents routinely administer chemotherapy. In the first-line setting, 67% of physicians who routinely administer chemotherapy preferred to use CSFs for secondary prophylaxis after a neutropenic complication, whereas only 2% would use CSFs for primary prophylaxis. In the recurrent disease setting, physicians were more likely to administer a regimen with minimal myelosuppression (74% reported "likely" or "very likely"), to dose delay or modify if neutropenic complications occur (78%), or to administer CSFs for secondary prophylaxis (85%) than to dose attenuate upon initiation of chemotherapy (49%) or to administer CSFs for primary prophylaxis (46%). Most physicians would administer CSFs to maintain RDI in both the first-line (75%) and palliative settings (62%), and 49% would strive to maintain a dose intensity of more than 85%.Conclusions:Most gynecologic oncologists use CSFs as secondary prophylaxis for neutropenic complications rather than as primary prophylaxis. Most gynecologic oncologists monitor RDI and use CSFs to maintain RDI in their patients with ovarian carcinoma.

Objective To determine factors related to recurrence and survival in women with stage IIIA endometrial cancer; to examine outcomes of women with IIIA1 disease. Methods Multi-institutional analysis of women with stage IIIA endometrial carcinoma undergoing hysterectomy, bilateral salpingo-oophorectomy, lymphadenectomy, and pelvic cytology between 1980 and 2008. Overall survival (OS) and recurrence-free disease specific survival (RFDSS) were compared using Kaplan–Meier method, univariate and multivariate analyses. Results 98 women underwent surgical staging for stage IIIA endometrial carcinoma. Pelvic washings were positive in 53%, serosa in 18%, and adnexae in 45%. Forty were IIIA1; 58 were IIIA2 (adnexal/serosal involvement). Median number of lymph nodes was 19 (range 1–73). Adjuvant treatment was given to 88%: radiotherapy — 21%, chemotherapy — 19%, chemotherapy and radiotherapy — 19%, hormonal therapy — 16%, and intraperitoneal P-32 — 11%. Five-year OS and RFDSS for IIIA1 were 77% and 76%, respectively; and for IIIA2 were 75% and 73%, respectively ( p =NS for both). Patients with IIIA1 disease were less likely to receive chemotherapy or radiotherapy than those with IIIA2 disease ( p =0.0035). Older age (Hazard ratio 1.24; 95% CI 1.00–1.54), non-Caucasian race (HR 5.35; 95% CI 1.96–14.5), and cervical metastases (HR 3.3; 95% CI 1.3–8.7) predicted lower RFDSS in multivariate analysis. Among 24 patients meeting NCCN's observation criteria (IIIA1, non-serous, and FIGO grade 1–2), 0/12 receiving adjuvant treatment recurred, while 1/12 not receiving adjuvant treatment recurred. Conclusions Surgically assessed stage IIIA endometrial adenocarcinoma recurs in approximately 20–25% of cases. A subset of stage IIIA1 with very low risk factors may be appropriate candidates for observation.

Given that prolonged exposure to unopposed estrogen has been implicated in endometrial carcinogenesis, our goal was to evaluate the effect of As(2)O(3) on regulation of estrogen receptor-alpha (ERa) expression in endometrial cancer cells. As(2)O(3) inhibited ER- mRNA and protein expression in a dose-dependent manner in both the Ishikawa and ECC-1 endometrial cancer cell lines. Treatment with As(2)O(3) resulted in rapid phosphorylation of the p42/p44 MAPK which could be abolished by addition of the MAPK inhibitor, U0126. Although treatment with U0126 alone resulted in up-regulation of ER- mRNA and protein, exposure to U0126 in combination with As(2)O(3) counteracted As(2)O(3)'s inhibitory effect on ER- expression. We provide evidence that As(2)O(3) inhibits ER- mRNA and protein expression in endometrial cancer cells, potentially through interaction with the MAPK pathway. Thus, As(2)O(3) may be valuable for its anti-estrogenic activity in combination with its anti-tumorigenic effects and be a novel therapeutic agent for endometrial cancer.

Epithelial ovarian carcinoma is the most lethal gynecological cancer due to its silent onset and recurrence with resistance to chemotherapy. Overexpression of oncogene c-Myc is one of the most frequently encountered events present in ovarian carcinoma. Disrupting the function of c-Myc and its downstream target genes is a promising strategy for cancer therapy. In this work, we aimed to evaluate the potential effects of small-molecule c-Myc inhibitor, 10058-F4, on ovarian cancer cells and the underlying mechanisms by which 10058-F4 exerts its actions. Using Cellometer image cytometry for cell cycle and Annexin V apoptosis assays, flow cytometry, MTT assay, and colony formation, we found that 10058-F4 significantly inhibited cell proliferation of both SKOV3 and Hey ovarian cancer cells in a dose dependent manner through induction of apoptosis and cell cycle G1 arrest. Treatment with 10058-F4 reduced cellular ATP production and ROS levels in SKOV3 and Hey cells. Consistently, primary cultures of ovarian cancer treated with 10058-F4 showed induction of caspase-3 activity and inhibition of cell proliferation in 15 of 18 cases. These novel findings suggest that targeting c-Myc-Max heterodimerization could be a potential therapeutic strategy for ovarian cancer. Note: This abstract was not presented at the meeting. Citation Format: Leo Li-Ying Chan, Jiandong Wang, Xiaoli Ma, Fang Song, Weiyuan Zhang, Hannah M. Jones, Victoria L. Bae-Jump, Chunxiao Zhou. Measuring antitumor effect of c-Myc-Max heterodimerization inhibitor 100258-F4 on ovarian cancer cells using cellometer image cytometry. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3469. doi:10.1158/1538-7445.AM2015-3469

Objectives: The anti-diabetic drug, metformin, is an AMPK activator with anti-tumorigenic effects. Indirectly, metformin improves insulin resistance and decreases insulin and glucose levels. Directly, metformin activates AMPK, leading to inhibition of the mTOR pathway. Everolimus is a traditional mTOR inhibitor, and its common toxicities include hyperglycemia and insulin resistance. Thus, we compared the efficacy of metformin versus everolimus in a genetically engineered mouse model of serous ovarian cancer (OC) under obese and non-obese conditions. Methods: We utilized the K18-gT121+/-; p53fl/fl;Brca1fl/fl (KpB) OC mouse model. KpB mice were subjected to 60% calories derived from fat in a high fat diet (HFD) to mimic diet-induced obesity versus 10% calories from fat in a low fat diet (LFD). Mice were treated with placebo, metformin orally (200 mg/kg/day) or everolimus intraperitoneally (3 mg/kg/day) following tumor onset for 4 weeks (N = 8-10 mice/group). Tumor size and volume were recorded weekly. Immunohistochemical analysis was performed on the ovarian tumors after treatment with placebo, metformin or everolimus to assess for effects on proliferation, apoptosis and downstream targets of the mTOR pathway. Individual slides were digitized using the Aperio ScanScope (Aperio Technologies, Vista, CA), and digital images were analyzed using Aperio ImageScope software. Results: Metformin inhibited tumor growth in the KpB mice fed a LFD (non-obese) and a HFD (obese), after one month of treatment. In the mice fed a HFD, metformin decreased tumor growth by 60% compared to control animals. Tumor growth was only decreased by 32% in the mice fed a LFD. A comparison of the anti-tumorigenic effects of metformin in mice fed a LFD versus a HFD demonstrated that metformin was more efficacious in those mice on the HFD (p = 0.003). Everolimus inhibited tumor growth by 39% and 38% in mice fed a HFD versus LFD, respectively. As compared to placebo-treated mice, metformin and everolimus decreased Ki-67, a marker of cell proliferation, and increased caspase-3, a marker of apoptosis, in the ovarian tumors from obese and non-obese KpB mice. In addition, metformin increased phosphorylation of AMPK, and both metformin and everolimus decreased phosphorylation of S6, a downstream target of the mTOR pathway. Conclusions: Metformin, an AMPK activator, was found to be more efficacious in the inhibition of ovarian tumor growth in the obese versus non-obese KpB mice, suggesting that obesity may be a biomarker for response to this agent. In contrast, everolimus, a mTOR inhibitor, was found to be equally efficacious in obese and non-obese mice. Further studies are underway to assess the potential differential effects of an AMPK activator versus a mTOR inhibitor on the interrelationship between the metabolic milieu and ovarian tumor growth. Citation Format: Weiya Z. Wysham, Yan Zhang, Hallum K. Dickens, Kimberly M. Malloy, Xiaoyun Han, Hui Guo, Paola A. Gehrig, Chunxiao Zhou, Victoria L. Bae-Jump. Differential efficacy of metformin versus everolimus in the setting of obesity in a mouse model of serous ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2305. doi:10.1158/1538-7445.AM2015-2305

Objectives: Obesity and diabetes are associated with increased risk and worse outcomes in endometrial cancer (EC). Anti-diabetic biguanide drugs such as metformin may have anti-tumorigenic effects by behaving as AMPK activators and mTOR inhibitors. Metformin requires organic cation transporters (OCTs) for entry into cells, and NT-1044 (NovaTarg Therapeutics) is an AMPK activator designed to have greater affinity for two of these transporters, OCT1 and 3. We sought to compare the effects of NT-1044 on cell proliferation and apoptosis in human EC cell lines. Methods: Cell proliferation was assessed in two EC cell lines, ECC-1 and Ishikawa, by MTT assay after exposure to NT-1044. Apoptosis was analyzed by Annexin V-FITC assay. Cell cycle progression was evaluated by flow cytometry. Phosphorylated (phos)-S6, phos-AMPK, Cyclin D-1 and CDK-6 were evaluated by Western immunoblotting. Results: NT-1044 significantly inhibited proliferation in a dose-dependent manner in both EC cell lines after 72 hours of exposure (IC50 218 uM for ECC-1; 87 uM for Ishikawa). Treatment with NT-1044 resulted in G1 cell cycle arrest and apoptosis in both cell lines. NT-1044 increased phosphorylation of AMPK and decreased phosphorylation of S6, a key downstream target of the mTOR pathway. Expression of Cyclin-D1 and CDK-6 also decreased in a dose dependent-fashion in both cell lines. Conclusions: NT-1044 suppressed EC cell growth through G1 cell cycle arrest, inducing apoptosis, and inhibition of the mTOR pathway. More work is needed to determine if this novel biguanide will be beneficial in the treatment of women with EC, a disease strongly impacted by obesity and diabetes. Citation Format: Dario R. Roque, Weiya W. Wysham, Chunxiao Zhou, Ken Batchelor, Wendy R. Brewster, Victoria L. Bae-Jump. The effects of NT-1044, a novel AMPK activator, on endometrial cancer cell proliferation and apoptosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4462. doi:10.1158/1538-7445.AM2015-4462

Objectives: The anti-diabetic drug, metformin, has been found to decrease proliferation, reduce estrogen receptor (ER) expression and inhibit downstream targets of the mTOR pathway in a pre-operative window study in endometrial cancer patients. Thus, we compared the endometrial cancers between patients with Type II diabetes taking metformin versus those not on metformin, in regards to expression of ER, progesterone receptor (PR) and PTEN. Methods: Patients with Type II diabetes who underwent surgical staging for endometrial cancer at our institution were identified. These patients were divided into two cohorts based on whether or not they were on metformin. The use of insulin and other anti-diabetic agents was recorded. Using triplicate cores from surgical specimens, a tissue microarray was constructed from formalin-fixed paraffin-embedded hysterectomy specimens. ER, PR and PTEN expression was measured by immunohistochemistry in both the epithelial and stromal components of the tumors. H scores were calculated, averaged among the triplicate cores, and compared between the two groups using Mann-Whitney U test and negative binomial regression analysis. Results: Triplicate cores were obtained from 162 endometrial cancers from patients with Type II diabetes. Of the 162 patients, 102 (63%) were taking metformin and 60 (37%) were not on metformin. Median BMI for metformin users and non-users was 40 and 37, respectively (p = 0.04). There was no significant difference in tumor stage, grade or histology between the two groups. There was no association between metformin use and PR or PTEN expression. There was a trend towards higher ER expression in the non-metformin users, which was statistically significant for stromal cells (p = 0.04) but not for epithelial cells (p = 0.13). However, when adjusting for age, BMI, race, tumor grade and stage, there was no association between metformin use and ER expression. The median H score for ER did differ between tumors from patients on metformin only (median 26) versus insulin only (median 91), but this was not statistically significant. Conclusions: Expression of ER, PR and PTEN in endometrial cancers did not differ amongst patients with Type II diabetes regardless of whether their diabetes treatment regimen involved metformin or insulin. Further studies are underway to compare the endometrial cancers of metformin users versus non-users in regards to other insulin and mTOR signaling targets. Citation Format: Dario R. Roque, Arthur M. Dizon, Brooke Rambally, Siobhan O'Connor, Paola A. Gehrig, Sheri A. Denslow, Victoria L. Bae-Jump. Expression of the estrogen receptor, progesterone receptor and PTEN in endometrial cancers from women with type II diabetes mellitus. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4996. doi:10.1158/1538-7445.AM2015-4996

5594 Background: Metformin, has been found to decrease proliferation and inhibit downstream targets of the mTOR pathway in a phase 0 trial in endometrial cancer (EC) patients. Given this, we aimed ...

e16513 Background: Combination chemotherapy and radiation is standard treatment for advanced stage endometrial cancer and can be given in a sequential or a sandwich fashion. Series have shown a sur...

e16522 Background: Endometrial cancers (ECs) frequently harbor mutations in PI3K pathway genes. Our objective was to determine the mutation frequency of the PIK3 pathway in the tumors of EC patients enrolled on UNCseq and correlate these findings with clinical outcomes. Methods: Snap-frozen and FFPE tissue samples were collected from EC patients enrolled on UNCseq (NCT01457196). DNA was isolated using the Puregene DNA purification system, and Illumina libraries were prepared separately from tumor and a matched normal sample from each patient. Relevant targets, were enriched by a custom designed Agilent SureSelect hybrid capture enrichment library using standard protocols. Samples were sequenced on Illumina HiSeq machines in a variety of formats. Statistical analysis of clinical correlates was performed in R (v 3.1.1). Results: Data was collected from 73 EC patients. Fifteen tumors (20.5%), 14 of which were endometrioid, had 2 or more mutations in PIK3CA or PIK3R1. Of 73 patients, 6 patients had 2 PIK3CA m...

Objectives To examine the effects of arsenic trioxide (As 2 O 3 ) on human endometrial carcinoma cell lines with respect to cytotoxicity and the induction of apoptosis and telomerase expression in vitro. Methods Four endometrial carcinoma cell lines (Ishikawa, ECC-1, RL-95-2 and Hec-1B) were treated with increasing concentrations of As 2 O 3 . Results As 2 O 3 inhibited proliferation of all cell lines in a concentration and time-dependent manner (IC50 range of 3–7 μM). Coincident with the inhibition of growth, As 2 O 3 also induced apoptosis in all cell lines as measured by the time-dependent increase in M30 antibody fluorescence (binds a caspase-cleaved epitope of cytokeratin 18) detected by flow cytometry, and reduced telomerase activity by decreasing the hTERT mRNA expression. Conclusion As 2 O 3 may exert anti-tumor effects through the induction of the apoptosis pathway and telomerase and hTERT may play an important role in the anti-apoptotic effects which are observed when endometrial cancer cells are treated in vitro with As 2 O 3 .

Rapamycin and its analogues have been shown to be promising as anti-neoplastic agents but have not been extensively studied in gynecologic malignancies. Our goal was to examine the ability of rapamycin to suppress growth and regulate telomerase activity in cervical and ovarian cancer cell lines.Cell proliferation was assessed after exposure to rapamycin. Cell cycle progression was determined by flow cytometry, and apoptosis was evaluated by DNA fragmentation. hTERT mRNA levels were quantified by real-time RT-PCR. Western blot analysis was performed to assess PTEN status, phosphorylated S6 and total S6 expression.Rapamycin inhibited growth of all the cervical cancer cell lines and 3 of the 4 ovarian cancer cell lines in a dose-dependent manner with IC50 values50 nM. Loss of PTEN protein expression was seen in only one of the cervical cancer cell lines. Rapamycin induced G1 arrest in those cell lines sensitive to its growth inhibitory effects. In all cell lines, rapamycin rapidly inhibited phosphorylation of S6 and resulted in decreased levels of total S6 protein. Treatment with rapamycin reduced hTERT mRNA expression in both rapamycin-sensitive and -resistant cell lines within 24 h. Thus, the effect of rapamycin on hTERT expression was not dependent on its ability to induce G1 cell cycle arrest.Our data suggest that rapamycin may potentially exert its anti-tumor effects through two independent pathways by G1 cell cycle arrest as well as suppression of telomerase activity by inhibition of hTERT mRNA transcription.

Objectives. Loss of PTEN expression is one of the most prevalent and earliest molecular abnormalities associated with endometrial carcinogenesis. Given that PTEN is often absent and telomerase is overexpressed by endometrial cancers, we hypothesize that PTEN signaling is important in telomerase regulation. Methods. PTEN expression was reconstituted in the PTEN-null Ishikawa endometrial cancer cells by adenovirus-mediated gene transduction. Cell proliferation was evaluated 12–96 h after infection. Western blot analysis was performed to assess PTEN status and phosphorylated Akt expression. Telomerase activity was determined by the telomeric repeat amplification protocol (TRAP) assay. hTERT mRNA levels were assessed by real-time RT-PCR. Ishikawa cells were also treated with LY294002, a PI3-kinase inhibitor. Results. Infection of Ishikawa cells by replication-defective recombinant adenovirus expressing wild-type PTEN, but not control adenovirus or adenovirus expressing lipid phosphatase defective PTEN GE mutant, inhibited constitutive Akt activation and suppressed proliferation of Ishikawa cells. Infection by wild-type PTEN adenovirus, but not control adenovirus, inhibited telomerase activity 24 h after infection. This inhibition of telomerase activity was parallel to decreased hTERT mRNA levels. LY294002 treatment resulted in dose-dependent inhibition of Akt activation and cellular proliferation. LY294002 suppressed telomerase activity and decreased hTERT transcript levels in a dose-dependent manner. Conclusions. Our data suggest that PTEN may regulate telomerase activity by a novel mechanism in which inhibition of Akt activation by PTEN leads to decreased hTERT mRNA levels. Thus, loss of PTEN may allow endometrial cells to continue to express high levels of telomerase activity, facilitating the neoplastic transformation of the endometrium.

See related article, page 531 Endometrial cancer, a postmenopausal disease often linked to obesity, is the most common gynecologic cancer in the United States. In 2010, 43,000 new cases were diagnosed and 7900 deaths occurred. The majority of endometrioid adenocarcinomas are confined to the uterus, and many patients do not require adjuvant treatment after surgery. However, nonendometrioid adenocarcinomas—papillary serous and clear cell adenocarcinomas—are associated with a worse prognosis and disease progression is unlike that of the more common endometrioid adenocarcinomas. In the United States, endometrial cancers are staged surgically; this is not the case in many European countries. Yet, the International Federation of Gynecology and Obstetrics (FIGO) issued updated recommendations in 2009 for the staging of endometrial malignancies (and other cancers), and these continue to incorporate prognostic factors found at surgery. Pelvic and periaortic lymph node assessment remain important aspects of staging. However, surgical staging can be associated with increased surgical morbidity, including injury to vessels, ureters, and nerves and lymphocyst formation. Additionally, because many women who develop endometrial cancer tend to be obese, the task of surgical staging can be technically challenging, although the robotic approach has remedied some of these technical difficulties. Nonetheless, it would be beneficial if recommendations for lymphadenectomy could be further refined to spare some patients the need for lymph node dissection.

Introduction: The anti-diabetic drugs, metformin and phenformin, are thought to have anti-tumorigenic benefits. Preclinical studies suggest that phenformin may be more potent for inhibiting tumor growth than metformin. Our objective was to assess the anti-tumorigenic effects of phenformin in endometrial cancer (EC) cell lines and primary cultures. Methods: The EC cell lines, ECC-1 and Ishikawa, were treated with phenformin under low glucose (2 mM), normal glucose (5.5 mM) and high glucose (25 mM) conditions. EC tumors were collected from consenting patients, at the time of surgical staging for primary culture. Cell proliferation was assessed by MTT assay. Apoptosis was analyzed by Annexin V-FITC assay. Cellometer evaluated cell cycle progression. Invasion was demonstrated by transwell invasion assay. Adhesion was assessed by ELISA. Western blotting was performed to determine expression of the downstream targets of phenformin. Results: Phenformin inhibited proliferation in a dose-dependent manner in both EC cell lines, within 48-72 hrs of exposure (IC50=1 mM, p=0.0004-0.009). Inhibition of proliferation was seen for all glucose concentrations, but greater potency was observed under low glucose conditions. Treatment with phenformin resulted in G1 arrest, induction of apoptosis (p=0.022-0.017) and inhibition of adhesion (p=0.0053-0.0144) and invasion (p=0.0063-0.05). Phenformin increased p21 and p27 and decreased cyclin D, cyclin E2, CDK4 and CDK 6, corresponding with G1 arrest. Phenformin significantly inhibited proliferation in 62% (8/13) of the EC primary cultures, within 48-72 hrs of exposure (IC50 0.1-5mM, p=0.00001-0.016). 3 additional EC samples (23%) were significantly inhibited by phenformin but did not reach an IC50 (p=0.0003-0.018), and 2/13 (15%) had no response to phenformin. Western blot analysis demonstrated that phenformin increased phosphorylation of AMPK and decreased phosphorylation of S6 coincident with inhibition of proliferation in the EC cell lines and primary cultures. Conclusion: Phenformin potently inhibited cell growth via G1 arrest and induced apoptosis in EC cell lines and the majority of primary cultures. Inhibition of adhesion/invasion was also seen with phenformin. Continued work is needed to explore whether phenformin has superior benefits over metformin. Citation Format: Amanda L. Jackson, Joshua E. Kilgore, Haifeng Qiu, Chunxiao Zhou, Paola A. Gehrig, Victoria L. Bae-Jump. Anti-tumorigenic effects of phenformin in human endometrial cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1313. doi:10.1158/1538-7445.AM2014-1313

Objectives: Epidemiologic and pre-clinical studies suggest that anti-diabetic biguanide drugs, such as metformin and buformin, may have anti-tumorigenic effects and behave as chemosensitizers. Thus, we evaluated the effects of buformin alone and in combination with paclitaxel on proliferation in human endometrial cancer (EC) cell lines as well as the impact of buformin on apoptosis and adhesion/invasion. Methods: The ECC-1 and Ishikawa EC cell lines were treated with varying doses of buformin alone and in combination with paclitaxel. Cell growth was determined by MTT assay. Cell cycle progression was assessed by Cellometer. Apoptosis was evaluated by Annexin V-FITC assay. Invasion was demonstrated by transwell invasion assay. Adhesion was assessed by ELISA. Effects on AMPK and S6 expression were documented by Western blotting. Results: Buformin inhibited proliferation in a dose-dependent manner in the EC cell lines (IC50 1000 μM for ECC-1, 10μM for Ishikawa, p=0.00001-0.0010). Treatment with buformin resulted in G1 arrest in the ECC-1 cell line and G2 arrest in the Ishikawa cell line. Buformin induced apoptosis in the Ishikawa cell line, but not in the ECC-1 cell line. Treatment with buformin resulted in inhibition of adhesion (p=0.42-0.0009) and invasion (p=0.00001-0.227) in both EC cell lines. Western blotting analysis demonstrated that buformin increased phosphorylation of AMPK and decreased phosphorylation of S6. Paclitaxel inhibited proliferation in a dose-dependent manner in both cell lines with IC50 values of 0.1-0.5 nM and 1-5 nM for Ishikawa and ECC-1 cells, respectively. To assess synergy of paclitaxel and buformin, the combination index (CI) was calculated by the method of Chou and Talalay. Simultaneous exposure of cells to various doses of paclitaxel in combination with buformin (0.1-1 μM) resulted in a significant synergistic anti-proliferative effect (CI Conclusions: Buformin inhibited proliferation in EC cell lines, via AMPK activation and mTOR pathway inhibition. Inhibition of adhesion/invasion was also seen with buformin. Buformin acted synergistically with paclitaxel to inhibit cell proliferation. More work is needed to determine if buformin and other biguanides alone or in combination with cytotoxic agents will be beneficial in the treatment of women with EC. Citation Format: Joshua Kilgore, Amanda Jackson, Haifeng Qiu, Chunxiao Zhou, Paola Gehrig, Victoria Bae-Jump. Buformin, an anti-diabetic biguanide, inhibits proliferation, invasion and adhesion, and acts synergistically with paclitaxel in endometrial cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1312. doi:10.1158/1538-7445.AM2014-1312

Objectives: Anti-diabetic biguanide drugs have been shown to have anti-tumorigenic effects by behaving as AMPK activators and mTOR inhibitors. Phenformin is a biguanide that is associated with a higher risk of lactic acidosis than is seen in metformin. In vitro and in vivo studies suggest that phenformin may be more potent for inhibiting tumor growth than metformin. We sought to examine the efficacy of phenformin in primary cultures of human ovarian cancers (OCs) as well as in a genetically engineered mouse model of serous OC. Methods: Serous OC tumors were collected from consenting patients, at the time of their surgical debulking for primary culture. Cell proliferation was assessed by MTT assay. Apoptosis was evaluated by Annexin V-FITC assay using Cellometer. Effects of phenformin on phosphorylated-AMPK and -S6 expression was documented by Western immunoblotting. For the in vivo studies, we utilized the KpB++ serous OC mouse model. This is a more aggressive derivative of the K18-gT121+/-; p53fl/fl;Brca1fl/fl (KpB) genetically engineered OC mouse model. The KpB++ mice were treated with placebo or phenformin (2 mg/kg body weight in drinking water) following tumor onset for one month. Immunohistochemical analysis was performed on the ovarian tumors after treatment with placebo or phenformin, in regards to Ki-67 expression, cleaved caspase 3 (a marker of apoptosis), phosphorylated AMPK and phosphorylated S6. Individual slides were digitized using the Aperio ScanScope (Aperio Technologies, Vista, CA), and digital images were analyzed using Aperio ImageScope software. Results: Phenformin significantly inhibited cell proliferation in a dose-dependent manner in 100% (7/7) of the primary cultures of ovarian tumors, within 48 to 72 hours of exposure (p=0.00001-0.015, IC50 range of 0.1-5 mM). Treatment with phenformin resulted in the induction of apoptosis in 71.4% (5/7) of the ovarian tumors. Western immunoblot analysis demonstrated that phenformin increased phosphorylation of AMPK and decreased phosphorylation of S6. Phenformin significantly inhibited tumor growth in the KpB++ mice (n=8-13 animals per group) by 68% as compared to placebo treated controls, after one month of treatment (p=0.016). As compared to the placebo treated control animals, phenformin was found to statistically decrease Ki67 staining, inhibit phosphorylated S6 expression and increase both cleaved caspase 3 and phosphorylated AMPK expression (p= 0.01-0.04). Conclusions: In primary culture of human serous OC tumors, phenformin potently inhibited cell growth and increased apoptotic cell death through AMPK activation and mTOR pathway inhibition. In vivo studies using the KpB++ mouse model found that phenformin was highly efficacious in inhibiting tumor growth. Although the risk/benefit ratio clearly favors metformin over phenformin for the treatment of diabetes, this may not hold true for the treatment of cancer. Citation Format: Amanda L. Jackson, Xiaoyun Han, Joshua E. Kilgore, Chunxiao Zhou, Liza Makowski, Victoria Bae-Jump. Phenformin has anti-tumorigenic effects in human ovarian cancer cells and in a genetically engineered mouse model of serous ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3113. doi:10.1158/1538-7445.AM2014-3113

Introduction: Endometrioid endometrial cancers (EEC) are commonly characterized by PTEN mutations and loss of function. JQ-1 is a selective small-molecule BET bromodomain inhibitor, and BET inhibition by JQ-1 leads to the downregulation of c-Myc transcription. Thus, JQ-1 functions as a novel c-Myc inhibitor and has been found to potently suppress tumor growth in many human cancers. Given the known interactions between c-Myc and the PI3K/Akt/mTOR pathway, we aim to assess the impact of JQ-1 on human EEC cell lines and further explore the mechanism underlying cellular resistance to JQ-1 treatment. Methods: Six EEC cancer cell lines were used in this study. Cell proliferation was assessed by MTT assay and colony formation assay after exposure to JQ-1. Cell cycle progression was evaluated by flow cytometry. Apoptosis was assessed by Annexin V-FITC assay. Expression of c-Myc, PTEN, phosphorylated-Akt, phosphorylated-S6, cyclins and cyclin-dependent kinases (CDKs) was detected by Western blotting analysis. Up- or down-regulation of PTEN was achieved by stable transfection with pcDNA3.1-PTEN or shRNA-PTEN, respectively. Results: JQ-1 significantly suppressed proliferation in the EEC cell lines that were PTEN positive (IC50=75 nM for HEC-1A, 550 nM for KLE and 1000 nM for ECC-1), but not in those that were PTEN negative (IC50>10000 nM for Ishikawa, AN3CA and RL 95-2). JQ-1 induced G1 cell cycle arrest in the PTEN positive HEC-1A and KLE cell lines, but had minimal effects on cell cycle progression in the PTEN negative Ishikawa and AN3CA cell lines. Western blotting analysis demonstrated that JQ-1 suppressed the expression of c-Myc, cyclins and CDKs. JQ-1 up-regulated PTEN expression and decreased phosphorylation of downstream targets of the PI3K/Akt/mTOR signaling pathway, including Akt and S6. Knock-down of PTEN led to cellular resistance to JQ-1 in the HEC-1A and KLE cells, while over-expression of PTEN sensitized Ishikawa and AN3CA cells to JQ-1 treatment. Conclusions: We find that JQ-1 significantly suppressed cellular proliferation through G1 cell cycle arrest in the EEC cell lines with abundant PTEN expression. Knock-down of PTEN caused cellular resistance while over-expression of PTEN sensitized EEC cells to JQ-1. This evidence suggests that JQ-1 may be a promising targeted therapy for EEC patients with PTEN positive tumors. Note: This abstract was not presented at the meeting. Citation Format: Haifeng Qiu, Amanda Jackson, Joshua Kilgore, Chunxiao Zhou, Victoria Lin Bae-Jump. Inhibition of BET bromodomain targets PTEN positive endometrioid endometrial cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4693. doi:10.1158/1538-7445.AM2014-4693