Victoria L. Bae-Jump

Metformin is a potent inhibitor of endometrial cancer cell proliferation—implications for a novel treatment strategy

OBJECTIVES Obesity and diabetes are strong risk factors that drive the development of type I endometrial cancers. Recent epidemiological evidence suggests that metformin may lower cancer risk and reduce rates of cancer deaths among diabetic patients. In order to better understand metformins anti-tumorigenic potential, our goal was to assess the effect of metformin on proliferation and expression of key targets of metformin cell signaling in endometrial cancer cell lines. METHODS The endometrial cancer cell lines, ECC-1 and Ishikawa, were used. Cell proliferation was assessed after exposure to metformin. Cell cycle progression was evaluated by flow cytometry. Apoptosis was assessed by ELISA for caspase-3 activity. hTERT expression was determined by real-time RT-PCR. Western immunoblotting was performed to determine the expression of the downstream targets of metformin. RESULTS Metformin potently inhibited growth in a dose-dependent manner in both cell lines (IC(50) of 1 mM). Treatment with metformin resulted in G1 arrest, induction of apoptosis and decreased hTERT expression. Western immunoblot analysis demonstrated that metformin induced phosphorylation of AMPK, its immediate downstream mediator, within 24 h of exposure. In parallel, treatment with metformin decreased phosphorylation of S6 protein, a key target of the mTOR pathway. CONCLUSIONS We find that metformin is a potent inhibitor of cell proliferation in endometrial cancer cell lines. This effect is partially mediated through AMPK activation and subsequent inhibition of the mTOR pathway. This work should provide the scientific foundation for future investigation of metformin as a strategy for endometrial cancer prevention and treatment.

Metformin is associated with improved survival in endometrial cancer.

OBJECTIVE Preclinical evidence suggests that metformin exhibits anti-tumorigenic effects in endometrial cancer. We sought to investigate the association of metformin on endometrial cancer outcomes. METHODS A multi-institutional IRB-approved retrospective cohort analysis was conducted comparing endometrial cancer patients with diabetes mellitus who used metformin (based on medication review at the time of diagnosis) to those who did not use metformin from 2005 to 2010. Metformin use on treatment related outcomes (TTR: time to recurrence; RFS: recurrence free survival; OS: overall survival) were evaluated using univariate and multivariate modeling. RESULTS 24% (363/1495) endometrial cancer patients were diabetic, of whom 54% used metformin. Metformin users were younger and heavier than non-users, though nearly all were postmenopausal and obese. 75% of both groups had endometrioid histology. Stage, grade, and adjuvant therapy distributions were similar. Metformin users had improved RFS and OS. Non-metformin users had 1.8 times worse RFS (95% CI: 1.1-2.9, p = 0.02) and 2.3 times worse OS (95% CI: 1.3-4.2, p = 0.005) after adjusting for age, stage, grade, histology and adjuvant treatment. Metformin use was not associated with TTR. CONCLUSION Metformin use was associated with improved RFS and OS but not TTR, most likely due to improving all-cause mortality. Its role in modifying cancer recurrence remains unclear. Prospective studies that capture metformin exposure prior to, during and post endometrial cancer treatment may help define the role of metformin upon cancer specific and overall health outcomes.

Metformin potentiates the effects of paclitaxel in endometrial cancer cells through inhibition of cell proliferation and modulation of the mTOR pathway

OBJECTIVES To examine the effects of combination therapy with metformin and paclitaxel in endometrial cancer cell lines. METHODS ECC-1 and Ishikawa endometrial cancer cell lines were used. Cell proliferation was assessed after exposure to paclitaxel and metformin. Cell cycle progression was assessed by flow cytometry. hTERT expression was determined by real-time RT-PCR. Western immunoblotting was performed to determine the effect of metformin/paclitaxel on the mTOR pathway. RESULTS Paclitaxel inhibited proliferation in a dose-dependent manner in both cell lines with IC(50) values of 1-5nM and 5-10nM for Ishikawa and ECC-1 cells, respectively. Simultaneous exposure of cells to various doses of paclitaxel in combination with metformin (0.5mM) resulted in a significant synergistic anti-proliferative effect in both cell lines (Combination Index<1). Metformin induced G1 arrest in both cell lines. Paclitaxel alone or in combination with metformin resulted in predominantly G2 arrest. Metformin decreased hTERT mRNA expression while paclitaxel alone had no effect on telomerase activity. Metformin stimulated AMPK phosphorylation and decreased phosphorylation of the S6 protein. In contrast, paclitaxel inhibited AMPK phosphorylation in the ECC-1 cell line and induced phosphorylation of S6 in both cell lines. Treatment with metformin and paclitaxel resulted in decreased phosphorylation of S6 in both cell lines but only had an additive effect on AMPK phosphorylation in the ECC-1 cell line. CONCLUSIONS Metformin potentiates the effects of paclitaxel in endometrial cancer cells through inhibition of cell proliferation and modulation of the mTOR pathway. This combination may be a promising targeted therapy for endometrial cancer.

A multicenter evaluation of sequential multimodality therapy and clinical outcome for the treatment of advanced endometrial cancer.

OBJECTIVES The appropriate sequencing of chemotherapy and radiation for the treatment of advanced endometrial cancer has not yet been determined. We sought to evaluate the outcome and adverse effects in patients with advanced stage endometrial cancer treated with postoperative chemotherapy and radiation to determine whether there was an advantage to a particular sequencing modality. METHODS A multicenter retrospective analysis of patients with surgical stages III and IV endometrial cancer from 1993 to 2007 was conducted. Inclusion criteria were comprehensive staging procedure including hysterectomy, bilateral salpingo-oophorectomy, +/- selective pelvic/aortic lymphadenectomy, and treatment with adjuvant chemotherapy and radiation. Differences in frequencies of adverse events were tested with Pearsons chi-square test for comparing proportions. OS and PFS rates were calculated using Kaplan-Meier estimates. Hazard Ratios (HR) were estimated from multivariate Cox proportional hazards models. RESULTS One hundred and nine patients with advanced stage endometrial cancer were identified who received postoperative adjuvant therapies; 41% (n=45) chemotherapy followed by radiation and then further chemotherapy (CRC), 17% (n=18) radiation followed by chemotherapy (RC), and 42% (n=46) chemotherapy followed by radiation (CR). The median age was 62 years (range: 35-83); 48% had endometrioid tumors; and 90% underwent optimal cytoreduction. There was no difference in the frequency of adverse effects due to either chemotherapy (p=0.35) or radiotherapy (p=0.14); dose modifications (p=0.055); or delays (p=0.80) between the various sequencing modalities. There was a significant difference between adjuvant treatment groups for both OS (log rank p=0.011) and PFS (log rank p=0.025), with those receiving CRC having a superior 3-year OS (88%) and PFS (69%) compared to RC (54% and 47%) or CR (57% and 52%). After adjusting for stage, age, grade, race, histology and cytoreduction status the OS HR for therapy was 5.74 (95% CI, 1.96 to 16.77) for RC and 2.60 (95% CI, 1.01 to 6.71) for CR, compared to CRC, p=0.003. When the analysis was restricted to optimally cytoreduced patients, those who were treated with RC were at higher risk for disease progression [HR=3.53 (95% CI, 1.29 to 9.71)], p=0.024, and death [HR=7.24 (95% CI, 2.25 to 23.37)], p=0.001, than patients who received sequential CRC. CONCLUSIONS Sequential CRC was associated with improved survival in women with advanced stage disease compared to other sequencing modalities with a similar adverse effect profile. Future clinical trials are needed to prospectively evaluate appropriate sequencing and types of adjuvant chemotherapy and radiotherapy for the treatment of advanced stage endometrial cancer.

Antiproliferative and metabolic effects of metformin in a preoperative window clinical trial for endometrial cancer

We conducted a preoperative window study of metformin in endometrial cancer (EC) patients and evaluated its antiproliferative, molecular and metabolic effects. Twenty obese women with endometrioid EC were treated with metformin (850 mg) daily for up to 4 weeks prior to surgical staging. Expression of the proliferation marker Ki‐67, estrogen receptor (ER), progesterone receptor (PR), adenosine monophosphate‐activated protein kinase (AMPK), and downstream targets of the mammalian target of rapamycin (mTOR) pathway were measured by immunohistochemistry. Global, untargeted metabolomics analysis of serum pre‐ and postmetformin treatment, and matched tumor, was performed. Metformin reduced proliferation by 11.75% (P = 0.008) based on the comparison of pre‐ and posttreatment endometrial tumors. A total of 65% of patients responded to metformin as defined by a decrease in Ki‐67 staining in their endometrial tumors post‐treatment. Metformin decreased expression of phosphorylated (p)‐AMPK (P = 0.00001), p‐Akt (P = 0.0002), p‐S6 (51.2%, P = 0.0002), p‐4E‐BP‐1 (P = 0.001), and ER (P = 0.0002) but not PR expression. Metabolomic profiling of serum indicated that responders versus nonresponders to treatment were more sensitive to metformins effects on induction of lipolysis, which correlated with increased fatty acid oxidation and glycogen metabolism in matched tumors. In conclusion, metformin reduced tumor proliferation in a pre‐operative window study in obese EC patients, with dramatic effects on inhibition of the mTOR pathway. Metformin induced a shift in lipid and glycogen metabolism that was more pronounced in the serum and tumors of responders versus nonresponders to treatment.This study provides support for therapeutic clinical trials of metformin in obese patients with EC.

Rapamycin potentiates the effects of paclitaxel in endometrial cancer cells through inhibition of cell proliferation and induction of apoptosis

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 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 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 <1, range 0.131–0.920). Rapamycin alone did not induce apoptosis, but combined treatment with paclitaxel increased apoptosis over that of paclitaxel alone. Treatment with rapamycin and paclitaxel resulted in decreased phosphorylation of S6 and 4E‐BP1, two critical downstream targets of the mTOR pathway. Rapamycin decreased hTERT mRNA expression by real‐time RT‐PCR while paclitaxel alone had no effect on telomerase activity. Paclitaxel increased polymerization and acetylation of tubulin, and rapamycin appeared to enhance this effect. Thus, in conclusion, we demonstrate that rapamycin potentiates the effects of paclitaxel in endometrial cancer cells through inhibition of cell proliferation, induction of apoptosis and potentially increased polymerization and acetylation of tubulin. This suggests that the combination of rapamycin and paclitaxel may be a promising effective targeted therapy for endometrial cancer.

Translational Studies of Phenotypic Probes for the Mononuclear Phagocyte System and Liposomal Pharmacology

As nanoparticles (NPs) are cleared via phagocytes of the mononuclear phagocyte system (MPS), we hypothesized that the function of circulating monocytes and dendritic cells (MO/DC) in blood can predict NP clearance (CL). We measured MO/DC phagocytosis and reactive oxygen species (ROS) production in mice, rats, dogs, and patients with refractory solid tumors. Pharmacokinetic studies of polyethylene glycol (PEG)-encapsulated liposomal doxorubicin (PEGylated liposomal doxirubicin [PLD]), CKD-602 (S-CKD602), and cisplatin (SPI-077) were performed at the maximum tolerated dose. MO/DC function was also evaluated in patients with recurrent epithelial ovarian cancer (EOC) administered PLD. Across species, a positive association was observed between cell function and CL of PEGylated liposomes. In patients with EOC, associations were observed between PLD CL and phagocytosis (R2 = 0.43, P = 0.04) and ROS production (R2 = 0.61, P = 0.008) in blood MO/DC. These findings suggest that probes of MPS function may help predict PEGylated liposome CL across species and PLD CL in patients with EOC.

The complex triad of obesity, diabetes and race in Type I and II endometrial cancers: Prevalence and prognostic significance

BACKGROUND We examined the distribution of obesity, diabetes, and race in Type I and Type II endometrial cancers (EC) and their associations with clinical outcomes. METHODS A multi-institutional retrospective analysis of Type I and II EC cases from January 2005 to December 2010 was conducted. Type I (endometrioid), Type II (serous and clear cell), low grade (LG) (grade 1 and 2 endometrioid), and high grade (HG) (grade 3 endometrioid, serous, clear cell) cohorts were compared. Univariate and multivariate analyses were used to determine time-to-recurrence (TTR), recurrence-free survival (RFS), and overall survival (OS). RESULTS Type I EC patients were more frequently obese than Type II (66% versus 51%, p<0.0001) and had similar rates of diabetes (25% versus 23%, p=0.69). African-Americans (AA) had higher median BMI than Caucasians in both Type I (p<0.001) and II (p<0.001) ECs, and were twice as likely to have diabetes (p<0.001). In Type I EC, DM was associated with worse RFS and OS in unadjusted and adjusted models (RFS HR 1.38, 95%CI 1.01-1.89; OS HR 1.86, 95%CI 1.30-2.67), but not with TTR. BMI was associated with improved TTR in the adjusted analysis for Type I EC (HR 0.98, 95%CI 0.95-1.0), but not with RFS or OS. There was no association between DM or BMI and outcomes in Type II or HG EC. AA race was not associated with RFS or OS on adjusted analyses in any group. CONCLUSIONS Obesity and diabetes are highly prevalent in Type I and II ECs, especially in AA. DM was associated with worse RFS and OS in Type I EC. Neither DM nor BMI was associated with outcomes in Type II or HG EC.

Metformin and the risk of endometrial cancer: A population-based cohort study

OBJECTIVE While some observational studies have suggested a protective effect of metformin on incident cancer, concerns about potential bias remain. We compared the incidence of endometrial cancer in metformin versus sulfonylurea initiators. Research design and methods We conducted a retrospective cohort analysis using US healthcare claims (MarketScan®), 2000-2011. We identified new users of metformin versus sulfonylureas with no prior cancer diagnoses and followed them until a diagnosis of endometrial cancer, hysterectomy, treatment change, or disenrollment. We estimated hazard ratios (HR) and 95% confidence intervals (CI) using Cox proportional hazards, using an as-treated analytic approach. Stabilized inverse probability of treatment weights were used to adjust for potential confounding at baseline. RESULTS Of 541,128 eligible women, 456,838 (84%) initiated metformin and 84,290 (16%) initiated sulfonylurea. The treatment groups differed at baseline in terms of age and recent diagnosis codes for diabetes, polycystic ovarian syndrome, and endometrial hyperplasia. Over a median follow-up of 1.2 (IQR 0.4-2.3) years and a total of 2,030,914 person-years, 729 women developed endometrial cancer. Metformin initiation was associated with a lower risk of endometrial cancer in the unadjusted analysis (HR 0.81, 95% CI 0.67-0.97). However, after balancing baseline covariates across groups, metformin was not associated with a reduced risk of endometrial cancer (HR 1.09, 95% CI 0.88-1.35). This finding was consistent across multiple sensitivity analyses and subgroup analyses in diabetic patients and relevant age groups. CONCLUSIONS In this population-based cohort of >500,000 women, initiating metformin compared with sulfonylureas was not associated with a reduced risk of developing endometrial cancer.

Glucose promotes cell proliferation, glucose uptake and invasion in endometrial cancer cells via AMPK/mTOR/S6 and MAPK signaling

OBJECTIVES Obesity and diabetes are well-known risk factors for the development of endometrial cancer. A high rate of aerobic glycolysis represents 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. This study aimed to investigate the effect of various concentrations of glucose on cell proliferation in endometrial cancer. METHODS ECC-1 and Ishikawa cells were treated with low glucose (1mM), normal glucose (5mM) and high glucose (25mM), and cytotoxicity, apoptosis, cell cycle, adhesion/invasion, and changes of AMPK/mTOR/S6 and MAPK pathways were evaluated. RESULTS Our results revealed that high glucose increased cell growth and clonogenicity in two endometrial cancer cell lines in a dose dependent manner. Low glucose induced the activity of cleaved caspase 3 and caused cell cycle G1 arrest. High glucose increased the ability of adhesion and invasion by decreasing E-cadherin and increasing Snail expression. In addition, high glucose increased glucose uptake and glycolytic activity through modulating the AMPK/mTOR/S6 and MAPK pathways. CONCLUSIONS Our findings suggest that glucose stimulated cell proliferation through multiple complex signaling pathways. Targeting glucose metabolism may be a promising therapeutic strategy in the treatment of endometrial cancer.