To examine the anti-cancer properties of metformin, we first utilized an inducible transformation model consisting of non-transformed human mammary epithelial cells (MCF-10A) containing ER-Src, a fusion of the v-Src oncoprotein with the ligand-binding domain of estrogen receptor. When these cells are treated with tamoxifen, they become transformed within 24-36 hours. The transformed cell population contains 10% cancer stem cells, as defined by expression of the CD44 marker and the ability to form mammospheres, multicellular “micro-tumors” that are generated in non-adherent and non-differentiating conditions (18
). In addition, we analyzed three other mammary adenocarcinoma cell lines derived from genetically and phenotypically different tumors that are treated with different drugs: ER-positive MCF7 (13
); HER-positive SKBR3 (14
); triple-negative MDA-MB-468 (15
). These cell lines also contain a minority population of cancer stem cells capable of mammosphere formation. In all experiments, metformin was used at a concentration that does not affect the growth of non-transformed cells (0.1 or 0.3 mM; ). Previous experiments on cancer cell lines (7
) used much higher concentrations of metformin (typically 10-30 mM), conditions that are also toxic for non-transformed cells.
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 (more ...)
In the inducible MCF-10A model, metformin strongly inhibits morphological transformation (), invasive growth in wound-healing assays (), focus formation, formation of colonies in soft agar, and generation of mammospheres (). Furthermore, metformin treatment of mammospheres derived from all four breast cancer cell lines causes a dramatic reduction in the number of mammospheres within 48 hours () as a consequence of cell death. As mammospheres are composed primarily of cancer stem cells (18
), this latter observation suggests that metformin may kill cancer stem cells.
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.
Strikingly, metformin preferentially kills cancer stem cells (CD44high/CD24low) within a population of transformed MCF-10A or MCF-7 cells (). Similarly, when all four cancer cell lines are sorted, cancer stem cells are quite susceptible to metformin, whereas the standard cancer cell population remains essentially unaffected (). Furthermore, treatment of MCF-10A cancer stem cells with metformin for just 1 hour blocks the ability of these cells to form tumors in nude mice, even though the drug is not present for the month after injection (). The ability of metformin to selectively kill cancer stem cells is in marked contrast to doxorubicin, a chemotherapeutic agent that kills cancer cells, but not cancer stem cells. As expected from their distinct properties, metformin works together with doxorubicin to reduce both non-stem cancer cells and cancer stem cells in the mixed transformed population ().
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 (more ...)
In accord with the above results in cell lines, the synergy between metformin and doxorubicin is observed upon treatment of tumors that arise 10 days after injection of MCF-10A-ER-Src cells into nude mice. After 15 days of treatment (3 cycles every 5 days), this drug combination virtually eliminates tumors, whereas doxorubicin alone causes only a 2-fold decrease in tumor volume and metformin alone has little effect (). Doxorubin-treated mice show a further reduction in tumor volume after an additional 10 days (day 35). The minimal effect of metformin alone is in contrast to more significant effects seen in an independent report (8
), but there are many differences in experimental protocol between these studies.
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) (more ...)
To determine the basis for why the combination of metformin and doxorubicin is more effective than doxorubicin alone, we examined the population of cells recovered from tumors after 3 cycles of treatment (day 25). In accord with our results in cell lines, cancer stem cells are virtually absent from mice treated with the drug combination, whereas they are easily detected in tumors from mice treated with doxorubicin alone (). Thus, the therapeutic advantage of metformin in the context of conventional chemotherapy is linked to its ability to kill cancer stem cells.
The cancer stem cell hypothesis for the progression of human disease is based on the differential tumor-forming properties and responses to well-defined chemotherapy of cancer stem cells and non-stem cancer cells. A prediction of this model, heretofore untested, is that drugs that selectively inhibit cancer stem cells should function synergistically with chemotherapeutic drugs to delay relapse. Strikingly, mice treated with the combination of metformin and doxorubicin remain in remission for at least 60 days after treatment is ended (). In contrast, tumor growth resumes 20 days after mice are treated with doxorubicin alone, and the rate of tumor growth after relapse is comparable to that observed in the initial disease (i.e. in the absence of treatment). Thus, combinatorial therapy has a dramatic effect on prolonging remission, and indeed may even represent a cure of these xenograft-generated tumors. In addition to their potential medical significance, these observations provide independent and further support for the cancer stem cell hypothesis.
To our knowledge, the ability of metformin to selectively kill cancer stem cells and to function synergistically with doxorubicin to block both cancer stem cells and non-stem transformed cells is unique. In the case of breast cancer, herceptin and tamoxifen are useful drugs for cancer types that, respectively, express the HER2 and estrogen receptors, but some forms of breast cancer lack these receptors resist these treatments. For all of these types of breast cancer, metformin selectively inhibits cancer stem cell growth, and hence is likely to function synergistically with chemotherapeutic drugs. In addition, as metformin inhibits transformation of MCF10A-ER-Src cells, it might have a potential use in preventing the development of cancer, as opposed to treating cancer that has already occurred. Indeed, the ability of metformin to inhibit cellular transformation might underlie the epidemiological observation that diabetics treated with metformin have a lower incidence of cancer (5
). As a cancer preventative, metformin would be required on a long-term basis, and in this regard, the concentration of metformin needed for the anti-cancer effects observed here is considerably below that used for the treatment of diabetes. Lastly, the selectivity of metformin and doxorubicin for distinct types of cells in the tumor can explain the striking combinatorial effects on reducing tumor mass and prolonging remission in nude mice, and it provides the rationale for combining metformin with chemotherapy as a new treatment for breast (and possibly other) cancers.