Metformin selectively targets cancer stem cells, and acts together with chemotherapy to block tumor growth and prolong remission

Abstract

The cancer stem cell hypothesis suggests that, unlike most cancer cells within a tumor, cancer stem cells resist chemotherapeutic drugs and can regenerate the various cell types in the tumor, thereby causing relapse of the disease. Thus, drugs that selectively target cancer stem cells offer great promise for cancer treatment, particularly in combination with chemotherapy. Here, we show that low doses of metformin, a standard drug for diabetes, inhibits cellular transformation and selectively kills cancer stem cells in four genetically different types of breast cancer. The combination of metformin and a well-defined chemotherapeutic agent, doxorubicin, kills both cancer stem cells and non-stem cancer cells in culture. Furthermore, this combinatorial therapy reduces tumor mass and prevents relapse much more effectively than either drug alone in a xenograft mouse model. Mice seem to remain tumor-free for at least 2 months after combinatorial therapy with metformin and doxorubicin is ended. These results provide further evidence supporting the cancer stem cell hypothesis, and they provide a rationale and experimental basis for using the combination of metformin and chemotherapeutic drugs to improve treatment of patients with breast (and possibly other) cancers.

Figures

Figure 1

Metformin prevents transformation of MCF10A-ER-Src cells. A, Number of cells grown in the presence or absence of 1 μM 4-hydroxy tamoxifen (TAM) with the indicated concentrations of metformin for 24 hours. B, Phase-contrast images of cells grown in the presence or absence of 0.1 mM metformin and/or TAM for 36 hours. C, Wound-healing/invasion response assay of cells grown in the presence or absence of 0.1 mM metformin and/or TAM. D, Relative number of foci, colonies in soft agar, and mammospheres in untreated or TAM-treated cells in the presence of the indicated concentration of metformin.

Figure 2

Metformin inhibits growth of mammospheres. 6-day old mammospheres from the indicated cell lines were or were not treated with 0.1 mM metformin for 48 hr, and the number of mammospheres counted.

Figure 3

Metformin selectively kills cancer stem cells and functions synergistically with doxorubicin. A, Number of cancer stem cells (CD44high/CD24low; black) and cancer cells (CD44low/CD24high; grey) in the transformed (36 h TAM treatment) MCF-10A population that was treated with doxorubicin, 0.1 mM metformin, or both (n = 3). B, Cancer stem cells (SC) and non-stem cancer cells (NSC) obtained by sorting were treated with 0.1 mM metformin for 0, 24, and 48 hours. C, Tumor volume in nude mice at the indicated number of days after injection of MCF10A-ER-Src cancer stem cells that were or were not treated with 0.1 mM metformin for 1 hr prior to injection.

Figure 4

Metformin and doxorubicin act in combination to reduce tumor mass and prolong remission in nude mice. A, Tumor volume (mean values and 95% confidence intervals) of mice injected with transformed MCF10A-ER-Src cells (time 0 indicates the time of injection) that were untreated, or treated by intraperitoneal injections every 5 days (3 cycles; arrows indicate the day or injections) with 4 mg/kg doxorubicin (Dox), 100 μg/ml metformin (Met), or both. B, Number of cancer stem cells (CD44high/CD24low) in cells obtained from tumors treated with Dox or the combination of Dox + Met after 3 cycles of treatment (day 25).