Vazquez-Martin A, Oliveras-Ferraros C, Del Barco S, et al. The anti-diabetic drug metformin suppresses self-renewal and proliferation of trastuzumab-resistant tumor-initiating breast cancer stem cells

Catalan Institute of Oncology, Girona (ICO-Girona), Dr. Josep Trueta University Hospital, Ctra. França s/n, 17007, Girona, Catalonia, Spain.
Breast Cancer Research and Treatment (Impact Factor: 3.94). 05/2010; 126(2):355-64. DOI: 10.1007/s10549-010-0924-x
Source: PubMed


We here demonstrate that the anti-diabetic drug metformin interacts synergistically with the anti-HER2 monoclonal antibody trastuzumab (Tzb; Herceptin™) to eliminate stem/progenitor cell populations in HER2-gene-amplified breast carcinoma cells. When using the mammosphere culture technique, graded concentrations of single-agent metformin (range 50-1,000 μmol/l) were found to dose-dependently reduce the number of mammospheres formed by SKBR3 (a Tzb-naïve model), SKBR3 TzbR (a model of acquired auto-resistance to Tzb) and JIMT-1 (a model of refractoriness to Tzb and other HER2-targeted therapies ab initio) HER2-overexpressing breast cancer cells. Single-agent Tzb likewise reduced mammosphere-forming efficiency (MSFE) in Tzb-naïve SKBR3 cells, but it failed to significantly decrease MSFE in Tzb-resistant SKBR3 TzbR and JIMT-1 cells. Of note, CD44-overexpressing Tzb-refractory SKBR3 TzbR and JIMT-1 cells retained an exquisite sensitivity to single-agent metformin. Concurrent combination of metformin with Tzb synergistically reduced MSFE as well as the size of mammospheres in Tzb-refractory SKBR3 TzbR and JIMT-1 cells. Flow cytometry analyses confirmed that metformin and Tzb functioned synergistically to down-regulate the percentage of Tzb-refractory JIMT-1 cells displaying the CD44(pos)/CD24(neg/low) stem/progenitor immunophenotype. Given that MSFE and mammosphere size are indicators of stem self-renewal and progenitor cell proliferation, respectively, our current findings reveal for the first time that: (a) Tzb refractoriness in HER2 overexpressors can be explained in terms of Tzb-resistant/CD44-overexpressing/tumor-initiating stem cells; (b) metformin synergistically interacts with Tzb to suppress self-renewal and proliferation of cancer stem/progenitor cells in HER2-positive carcinomas.

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Available from: Alejandro Vazquez-Martin,
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    • "various cancer stem cells (Janzer et al. 2014; Honjo et al. 2014; Lonardo et al. 2013; Shank et al. 2012; Song et al. 2012; Vazquez-Martin et al. 2011; Hirsch et al. 2009; Viale et al. 2014). Indeed, a number of clinical trials of metformin are ongoing as therapy for a variety of cancers (Pollak 2013). "
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    • "Cells were next incubated for 20 min with FITC-conjugated anti-CD44 antibody (BD Biosciences, Franklin Lakes, NJ) followed by incubation with phycoerythrine-conjugated anti-CD24 antibody (BioLegend, San Diego, CA) for 20 min in the dark at 4°C. After washing with PBS containing 1% (v/v) FCS, CD44high/CD24low cells were identified with flow cytometry [6],[15],[27]–[29],[32], and the % of CD44high/CD24low cells was estimated. The same method was applied to identify CD44high/CD24high cells, which are CSCs of MIA PaCa-2 cells [25]. "
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    ABSTRACT: Metformin, the most widely prescribed drug for treatment of type 2 diabetes, has been shown to exert significant anticancer effects. Hyperthermia has been known to kill cancer cells and enhance the efficacy of various anti-cancer drugs and radiotherapy. We investigated the combined effects of metformin and hyperthermia against MCF-7 and MDA-MB-231 human breast cancer cell, and MIA PaCa-2 human pancreatic cancer cells. Incubation of breast cancer cells with 0.5-10 mM metformin for 48 h caused significant clonogenic cell death. Culturing breast cancer cells with 30 µM metformin, clinically relevant plasma concentration of metformin, significantly reduced the survival of cancer cells. Importantly, metformin was preferentially cytotoxic to CD44(high)/CD24(low) cells of MCF-7 cells and, CD44(high)/CD24(high) cells of MIA PaCa-2 cells, which are known to be cancer stem cells (CSCs) of MCF-7 cells and MIA PaCa-2 cells, respectively. Heating at 42°C for 1 h was slightly toxic to both cancer cells and CSCs, and it markedly enhanced the efficacy of metformin to kill cancer cells and CSCs. Metformin has been reported to activate AMPK, thereby suppressing mTOR, which plays an important role for protein synthesis, cell cycle progression, and cell survival. For the first time, we show that hyperthermia activates AMPK and inactivates mTOR and its downstream effector S6K. Furthermore, hyperthermia potentiated the effect of metformin to activate AMPK and inactivate mTOR and S6K. Cell proliferation was markedly suppressed by metformin or combination of metformin and hyperthermia, which could be attributed to activation of AMPK leading to inactivation of mTOR. It is conclude that the effects of metformin against cancer cells including CSCs can be markedly enhanced by hyperthermia.
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    • "Afterwards, several papers have reported a selective sensitivity to metformin by CSCs in several cancer models. Vazquez-Martin et al. showed that metformin acts synergistically with trastuzumab both in vivo and in vitro to repress proliferation and survival of CSC in HER2-positive CD44+/CD24− breast cancer cell lines [97]. Since metformin can overcome in vivo primary resistance to trastuzumab, the authors proposed metformin as promising strategy for treatment of HER2+ breast cancer patients [98]. "
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