Phase II clinical trial of metformin as a cancer stem cell-targeting agent in ovarian cancer

Abstract

BACKGROUNDEpidemiologic studies suggest that metformin has antitumor effects. Laboratory studies indicate metformin impacts cancer stem-like cells (CSCs). As part of a phase II trial, we evaluated the impact of metformin on CSC number and on carcinoma-associated mesenchymal stem cells (CA-MSCs) and clinical outcomes in nondiabetic patients with advanced-stage epithelial ovarian cancer (EOC).METHODSThirty-eight patients with stage IIC (n = 1)/III (n = 25)/IV (n = 12) EOC were treated with either (a) neoadjuvant metformin, debulking surgery, and adjuvant chemotherapy plus metformin or (b) neoadjuvant chemotherapy and metformin, interval debulking surgery, and adjuvant chemotherapy plus metformin. Metformin-treated tumors, compared with historical controls, were evaluated for CSC number and chemotherapy response. Primary endpoints were (a) a 2-fold or greater reduction in aldehyde dehydrogenase-positive (ALDH+) CD133+ CSCs and (b) a relapse-free survival at 18 months of more than 50%.RESULTSMetformin was well tolerated. Median progression-free survival was 18.0 months (95% CI 14.0-21.6) with relapse-free survival at 18 months of 59.3% (95% CI 38.6-70.5). Median overall survival was 57.9 months (95% CI 28.0-not estimable). Tumors treated with metformin had a 2.4-fold decrease in ALDH+CD133+ CSCs and increased sensitivity to cisplatin ex vivo. Furthermore, metformin altered the methylation signature in CA-MSCs, which prevented CA-MSC-driven chemoresistance in vitro.CONCLUSIONTranslational studies confirm an impact of metformin on EOC CSCs and suggest epigenetic change in the tumor stroma may drive the platinum sensitivity ex vivo. Consistent with this, metformin therapy was associated with better-than-expected overall survival, supporting the use of metformin in phase III studies.TRIAL REGISTRATIONClinicalTrials.gov NCT01579812.

Keywords: Human stem cells; Oncology.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. Clinical study trial design.

Patients were treated with metformin before debulking surgery either for 2 weeks or along with 3 cycles of neoadjuvant chemotherapy. Surgical specimens were used for later CSC studies. Metformin was continued along with adjuvant chemotherapy for a total of 6 cycles.

Figure 2. Enrollment and exclusion in the clinical trial.

Of 91 patients enrolled, 38 were evaluable. Excluded patients included those who withdrew before trial initiation, were ineligible due to benign or lower stage pathology, or withdrew after treatment was initiated but before receiving chemotherapy due to intolerance of metformin, noncompliance, or other reasons, such as moving or choosing to receive adjuvant treatment at another institution.

Figure 3. Kaplan-Meier estimates of progression-free and overall survival.

(A and B) Kaplan-Meier estimates of PFS and OS for the entire population. Median PFS was 18.0 months (14.0–21.6). Median OS was 57.9 months (28.0–not estimable). (C and D) Kaplan-Meier estimates of PFS and OS by stage.

Figure 4. Tumors treated with metformin have decreased cancer stemness.

(A) Summary of FACS analysis of ALDH+CD133+ CSCs in metformin-treated (n = 22) and matched control ovarian cancers (n = 22) demonstrating a 2.4-fold decrease in CSCs in metformin-treated tumors. (B) Cell viability of tumor cells from metformin-treated patients (n = 6) or control patients (n = 7) grown in suspension, passaged weekly, and treated with cisplatin (5 replicates each). Tumor cells from metformin-treated patients maintain platinum sensitivity with serial passage, while control tumor cells increase platinum resistance over time. (C) Evaluation of ALDH and CD133 expression in metformin-treated and control tumor cells grown in suspension and after serial passages. Metformin-treated samples start at a lower baseline and increase less over time relative to controls. Lines in boxes represent averages. The whiskers depict the minimum and maximum values, and the length of the box represents the interquartile range. Statistical significance between passages was assessed with 2-sided Student’s t tests and comparisons made between metformin and nonmetformin samples at each passage, using 1-/2-way ANOVA and Tukey’s post hoc analysis to determine specific significant differences (P < 0.05). All data are expressed as mean ± SEM.

Figure 5. Metformin does not affect DNA methylation of bulk cancer cells.

(A) Primary 2-dimensional UMAP analysis showing intermixed metformin-treated tumor cells (mtTumor, red, n = 10) and nontreated control tumor cells (ctrlTumor, blue, n = 6). (B) Dendrogram of hierarchical clustering using the top 5% most variable CpGs in tumor cells from control and metformin-treated patients.

Figure 6. Metformin impacts DNA methylation of host CA-MSCs and prevents CA-MSC–induced chemoresistance.

(A) Primary 2-dimensional UMAP analysis for the MSC groups, including metformin-CA-MSCs (mtCAMSC, red, n = 11), control-CA-MSCs (CA-MSC, blue, n = 9), and normal adipose MSCs (MSC, green, n = 6). (B) Dendrogram from hierarchical clustering using the top 5% most variable CpGs for the MSCs. Metformin-CA-MSC samples were split into 2 groups as indicated. (C) DNA methylation heatmap of 14,791 probes differentially methylated between group I and group II. MIR200C promoter DNA methylation level (unmethylated in epithelial cells and methylated in mesenchymal cells) is included as a measure of CA-MSC purity. Group I metformin-CA-MSCs segregate with normal MSCs, distinct from control-CA-MSCs. (D) Viable cancer cell number following coculture with the indicated MSC types and the indicated doses of cisplatin. A Student’s 2-tailed t test was used for comparison.