Metformin-induced preferential killing of breast cancer initiating CD44+CD24-/low cells is sufficient to overcome primary resistance to trastuzumab in HER2+ human breast cancer xenografts

Abstract

Trastuzumab-refractory breast cancer stem cells (CSCs) could also explain the high rate of primary resistance to single-agent trastuzumab in HER2 gene-amplified breast cancer patients. The identification of agents with strong selective toxicity for trastuzumab-resistant breast CSCs may have tremendous relevance for how HER2+ breast cancer patients should be treated. Using the human breast cancer cell line JIMT-1, which was established from the pleural metastasis of a patient who was clinically resistant to trastuzumab ab initio, we examined whether preferential killing of the putative CD44+CD24-/low breast CSC population might be sufficient to overcome primary resistance to trastuzumab in vivo. Because recent studies have shown that the anti-diabetic biguanide metformin can exert antitumor effects by targeted killing of CSC-like cells, we explored whether metformin's ability to preferentially kill breast cancer initiating CD44+CD24-/low cells may have the potential to sensitize JIMT-1 xenograft mouse models to trastuzumab. Upon isolation for breast cancer initiating CD44+CD24-/low cells by employing magnetic activated cell sorting, we observed the kinetics of metformin-induced killing drastically varied among CSC and non-CSC subpopulations. Metformin's cell killing effect increased dramatically by more than 10-fold in CD44+CD24-/low breast CSC cells compared to non-CD44+CD24-/low immunophenotypes. While seven-weeks treatment length with trastuzumab likewise failed to reduce tumor growth of JIMT-1 xenografts, systemic treatment with metformin as single agent resulted in a significant two-fold reduction in tumor volume. When trastuzumab was combined with concurrent metformin, tumor volume decreased sharply by more than four-fold. Given that metformin-induced preferential killing of breast cancer initiating CD44+CD24-/low subpopulations is sufficient to overcome in vivo primary resistance to trastuzumab, the incorporation of metformin into trastuzumab-based regimens may provide a valuable strategy for treatment of HER2+ breast cancer patients.

Figure 1

A. Top. CD44 and CD24 expression patterns in CD44⁺CD24^−/low and CD44⁺CD24⁺ subpopulations of JIMT-1 sorted by sequential sorting (depletion followed by positive selection) using MACS MicroBeads (MACS^® Technology). Enrichment of target cells by magnetic MicroBeads was carried out according to the manufacturer’s (Milteny Biotec, Bergisch Gladbach, Germany) protocol. CD44⁺CD24^−/low were isolated from the parental JIMT-1 cell line by firstly depleting CD24⁺ cells using the CD24 MicroBead Kit and then positively selected for CD44 using the CD44 MicroBeads. Cells were fluorescently stained with combinations of fluorochrome-conjugated monoclonal antibodies obtained from BD Biosciences (San Diego, CA, USA) against human CD44 (FITC; cat.#555478) and CD24 (PE; cat.#555428) or their respective isotype controls. Representative expression (n=5) in pre-sorted and post-sorted JIMT-1 cells is shown. Bottom. Parental JIMT-1 cells and cells sorted for BCSC⁺CD44⁺CD24^−/low and non-BCSC⁻CD44⁺CD24⁺ markers were treated simultaneously with increasing concentrations of metformin for 5 days. Cell viability was determined using a standard colorimetric MTT (3-4, 5-dimethylthiazol-2-yl-2, 5-diphenyl-tetrazolium bromide) reduction assay. For each treatment, cell viability was evaluated as a percentage using the following equation: (OD₅₇₀ of treated sample/OD₅₇₀ of untreated sample) × 100. Cell sensitivity to metformin was expressed in terms of the concentration of drug required to decrease by 50% cell viability (IC₅₀ value). Since the percentage of control absorbance was considered to be the surviving fraction of cells, the IC₅₀ values were defined as the concentration of agents that produced 50% reduction in control absorbance. B. Tumor xenografts were established by subcutaneous injection of 5 × 10⁶ JIMT-1 cells suspended in 100 μl of culture medium into the flank of female athymic nude mice (four to five weeks old, 23 to 25 g; Harlan Laboratories —France-). Animals were randomized into four groups with five mice in each group: control (vehicles), trastuzumab, metformin, and trastuzumab + Metformin. Trastuzumab (5 mg/kg) was given intraperitoneally (i.p.) once per week. Both the “metformin group” and the “trastuzumab + metformin group” received daily a single i.p. injection of metformin (250 mg/kg). Mice were weighed once per week after dosing, tumors were measured daily with electronic callipers, and tumor volumes were calculated using the following formula: volume (mm³) = length x width² x 0.5. Figure shows mean tumor volumes (±SD) of JIMT-1 xenograft-bearing nude mice following injection with trastuzumab, metformin, and trastuzumab + metformin until seven weeks.