Joshua Kilgore

Recurrence-free and 5-year survival following robotic-assisted surgical staging for endometrial carcinoma.

OBJECTIVE The aim of this study is to report recurrence-free and overall survival for women with endometrial adenocarcinoma who were surgically staged using robotic-assisted laparoscopy. METHODS A retrospective chart review was performed for all consecutive endometrial adenocarcinoma patients surgically staged with robotic-assisted laparoscopy at the University of North Carolina Hospital from 2005 to 2010. Demographic data, 5-year survival, and recurrence-free intervals were analyzed. Statistical analysis using Chi-square, t-test, and Kaplan-Meier curves were performed with SAS software. Study results were compared to endometrial cancer statistics from the Surveillance Epidemiology and End Results database from the National Cancer Institute. RESULTS A total of 499 patients were identified and included in the study. Recurrence-free intervals after robotic-assisted surgical staging were 85.2% for stage IA, 80.2% for stage IB, 69.8% for stage II, and 69% for stage III. Projected 5-year survival was 88.7% for all patients included in the study. Nearly 82% of cases were endometrioid adenocarcinoma, with papillary serous, clear cell or mixed histology comprising 17.4% of cases. Median follow up time was 23 months, with a range of 0 to 80 months. Among stage IA, IB, II, and III patients, projected overall survival was 94.2%, 85.9%, 77.4%, and 68.6%, respectively. CONCLUSIONS The results from this study demonstrate that robotic-assisted surgical staging for endometrial cancer does not adversely affect rates of recurrence or survival. These findings provide further evidence that robotic-assisted laparoscopic surgical staging is not associated with inferior results when compared to laparotomy or traditional laparoscopy.

JQ1 suppresses tumor growth via PTEN/PI3K/AKT pathway in endometrial cancer

Overexpression of c-Myc is associated with worse outcomes in endometrial cancer, indicating that c-Myc may be a promising target for endometrial cancer therapy. A novel small molecule, JQ1, has been shown to block BRD4 resulting in inhibition of c-Myc expression and tumor growth. Thus, we investigated whether JQ1 can inhibit endometrial cancer growth in cell culture and xenograft models. In PTEN-positive endometrial cancer cells, JQ1 significantly suppressed cell proliferation via induction of G1 phase arrest and apoptosis in a dose-dependent manner, accompanied by a sharp decline in cyclin D1 and CDK4 protein expression. However, PTEN-negative endometrial cancer cells exhibited intrinsic resistance to JQ1, despite significant c-Myc inhibition. Moreover, we found that PTEN and its downstream PI3K/AKT signaling targets were modulated by JQ1, as evidenced by microarray analysis. Silencing of PTEN in PTEN-positive endometrial cancer cells resulted in resistance to JQ1, while upregulation of PTEN in PTEN-negative endometrial cancer cells increased sensitivity to JQ1. In xenografts models of PTEN-positive and PTEN-knock-in endometrial cancer, JQ1 significantly upregulated the expression of PTEN, blocked the PI3K/AKT signaling pathway and suppressed tumor growth. These effects were attenuated in PTEN-negative and PTEN-knockdown xenograft models. Thus, JQ1 resistance appears to be highly associated with the status of PTEN expression in endometrial cancer. Our findings suggest that targeting BRD4 using JQ1 might serve as a novel therapeutic strategy in PTEN-positive endometrial cancers.

Evaluation of the anti-tumor effects of lactate dehydrogenase inhibitor galloflavin in endometrial cancer cells.

High rates of aerobic glycolysis represent a key mechanism by which endometrial cancer cells consume glucose as its primary energy source. The up-regulated glycolytic pathway is a common therapeutic target whose inhibition has implications for anti-tumor activity in cancer cells. The present study was aimed at evaluating the potential of a novel lactate dehydrogenase (LDH) inhibitor, Galloflavin, as a therapeutic agent for endometrial cancer. Our results revealed that Galloflavin effectively inhibited cell growth in endometrial cancer cell lines and primary cultures of human endometrial cancer through its involvement in multiple signaling pathways that regulate metabolism, cell cycle, apoptosis, cell stress and metastasis.

NT1014, a novel biguanide, inhibits ovarian cancer growth in vitro and in vivo

BackgroundNT1014 is a novel biguanide and AMPK activator with a high affinity for the organic cation-specific transporters, OCT1 and OCT3. We sought to determine the anti-tumorigenic effects of NT1014 in human ovarian cancer cell lines as well as in a genetically engineered mouse model of high-grade serous ovarian cancer.MethodsThe effects of NT1014 and metformin on cell proliferation were assessed by MTT assay using the human ovarian cancer cell lines, SKOV3 and IGROV1, as well as in primary cultures. In addition, the impact of NT1014 on cell cycle progression, apoptosis, cellular stress, adhesion, invasion, glycolysis, and AMPK activation/mTOR pathway inhibition was also explored. The effects of NT1014 treatment in vivo was evaluated using the K18 − gT121+/−; p53fl/fl; Brca1fl/fl (KpB) mouse model of high-grade serous ovarian cancer.ResultsNT1014 significantly inhibited cell proliferation in both ovarian cancer cell lines as well as in primary cultures. In addition, NT1014 activated AMPK, inhibited downstream targets of the mTOR pathway, induced G1 cell cycle arrest/apoptosis/cellular stress, altered glycolysis, and reduced invasion/adhesion. Similar to its anti-tumorigenic effects in vitro, NT1014 decreased ovarian cancer growth in the KpB mouse model of ovarian cancer. NT1014 appeared to be more potent than metformin in both our in vitro and in vivo studies.ConclusionsNT1014 inhibited ovarian cancer cell growth in vitro and in vivo, with greater efficacy than the traditional biguanide, metformin. These results support further development of NT1014 as a useful therapeutic approach for the treatment of ovarian cancer.

Obesity is associated with worse overall survival in women with low-grade papillary serous epithelial ovarian cancer.

Objective The objective of this study was to evaluate prognostic risk factors for survival in women with low-grade serous epithelial ovarian cancer (LGSC). Methods A multicenter retrospective analysis of patients with LGSC was conducted. Potential epidemiologic risk factors evaluated included obesity, age, parity, race, smoking, oral contraceptive pill and/or hormonal replacement therapy use, and previous hysterectomy or surgery on fallopian tubes and/or ovaries. Additional factors included stage, extent of debulking, residual disease, and disease status. Results Eighty-one patients were identified, and pathologic diagnosis was independently confirmed. Median age at diagnosis was 56 years (range, 21–86 years). Thirty-four percent were obese, and 80% had optimally debulked disease. Forty-six percent were alive, 14% with disease, whereas 25% were dead of disease, 2% died of intercurrent disease, and 27% had an unknown status. In a univariate analysis, optimal surgical debulking was associated with improved progression-free survival (P = 0.01), disease-specific survival (P = 0.03), and overall survival (OS) (P < 0.001) and body mass index with worse OS (P = 0.05). On multivariate analysis, obesity (hazard ratio, 2.8; 95% confidence interval, 1.05–7.3; P = 0.04) and optimal tumor debulking (hazard ratio, 0.05; 95% confidence interval, 0.008–0.29; P = 0.001) were a significant predictor of OS. Conclusions In a multivariate analysis, obesity and optimal tumor cytoreduction were significant predictors of OS. However, obesity was not associated with worse disease-specific survival, suggesting that mortality of obese patients with LGSC may result from other comorbidities. Interventions addressing obesity may improve survival for women diagnosed with LGSC, and further study is warranted to address the role of obesity in LGSC.

Abstract 1312: Buformin, an anti-diabetic biguanide, inhibits proliferation, invasion and adhesion, and acts synergistically with paclitaxel in endometrial cancer cell lines

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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 <1, range 0.009-0.85). 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

Abstract 4693: Inhibition of BET bromodomain targets PTEN positive endometrioid endometrial cancers

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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

Abstract 3113: Phenformin has anti-tumorigenic effects in human ovarian cancer cells and in a genetically engineered mouse model of serous ovarian cancer

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

Abstract 1313: Anti-tumorigenic effects of phenformin in human endometrial cancer cells

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

Abstract 2442: Antitumorigenic effects of phenformin in human ovarian cancer cell lines.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: Anti-diabetic biguanide drugs, such as metformin, have been shown to have anti-tumorigenic effects by behaving as AMPK activators and mTOR inhibitors. Phenformin is another biguanide with anti-diabetic activity that was withdrawn from the market due to a risk of lactic acidosis that was higher than that seen with metformin. In vitro and in vivo studies suggest that phenformin may be more potent for inhibiting tumor growth than metformin. Thus, our objective was to assess the effect of phenformin on proliferation and apoptosis in ovarian cancer cell lines. Methods: Three ovarian cell lines, SKOV3, IGROV1, and HEY, were used. Cell proliferation was assessed by MTT assay after exposure to phenformin and after exposure to phenformin under varying glucose levels. Apoptosis was analyzed by Annexin V-FITC assay. Cell cycle progression was evaluated by Cellometer. Western immunoblotting was performed to determine expression of the downstream targets of phenformin, including AMPK, ribosomal protein S6, cyclin D and E2, cyclin dependent kinase (CDK) 4 and 6, p21 and p27. Inhibition of adhesion by phenformin was assessed by in vitro adhesion assay. Results: Phenformin potently inhibited proliferation in a dose-dependent manner in all three ovarian cancer cell lines (IC50 = 2.5mM for SKOV3 and HEY, 1mM for IGROV1 at 48 hours) (p = 0.0035 – 0.00001). Inhibition of proliferation was found at normal, low and high glucose concentrations, but greater potency was observed under low glucose concentrations. Treatment with phenformin resulted in G1 cell cycle arrest and induction of apoptosis. Cellular adhesion was decreased by 14-32% in the ovarian cancer cell lines at a phenformin dose of 2.5mM (p = 0.009 – 0.0001). Western immunoblot analysis demonstrated that phenformin induced phosphorylation of AMPK, its immediate downstream mediator, within 18 hours of exposure. In parallel, treatment with phenformin decreased phosphorylation of the S6 protein, a key target of the mTOR pathway. Phenformin was also found to increase the cell cycle inhibitors, p21 and p27, and decrease cyclin D, cyclin E2, CDK4 and CDK 6, corresponding with G1 arrest in these cell lines. Conclusion: Phenformin potently inhibited ovarian cancer cell growth via G1 arrest and increased apoptosis. 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 if phenformin were found to have superior anti-tumorigenic activity. Continued work is needed to explore the benefits of both metformin and phenformin for the treatment of ovarian cancer and the optimal therapeutic index. Citation Format: Amanda L. Jackson, Joshua E. Kilgore, Haifeng Qiu, Chunxiao Zhou, Paola A. Gehrig, Victoria L. Bae-Jump. Antitumorigenic effects of phenformin in human ovarian cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2442. doi:10.1158/1538-7445.AM2013-2442