Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

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

Metformin-induced preferential killing of breast cancer initiating CD44+CD24−/low cells is... www.impactjournals.com/oncotarget/ Oncotarget, April, Vol.3, No 4 Metformin-induced preferential killing of breast cancer initiating + -/low CD44 CD24 cells is sufficient to overcome primary resistance to trastuzumab in HER2+ human breast cancer xenografts 1,2 1,2 1,2 Sílvia Cufí , Bruna Corominas-Faja , Alejandro Vazquez-Martin , Cristina 1,2 2,3 2,3 Oliveras-Ferraros , Joan Dorca , Joaquim Bosch-Barrera , Begoña 2,4 1,2 Martin-Castillo , Javier A. Menendez Translational Research Laboratory, Catalan Institute of Oncology (ICO), Girona, Spain Girona Biomedical Research Institute (IDIBGi), Girona, Spain Medical Oncology, Catalan Institute of Oncology (ICO), Girona, Spain Clinical Research Unit, Catalan Institute of Oncology (ICO), Girona, Spain Correspondence to: Javier A. Menendez, email: [email protected]; [email protected] Keywords: Metformin, trastuzumab, HER2, cancer stem cells, breast cancer Received: April 27, 2012, Accepted: April 28, 2012, Published: May 4, 2012 Copyright: © Cufi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which per - mits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT: Trastuzumab-refractory breast cancer stem cells (CSCs) could 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 + -/low CD44 CD24 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 + -/low initiating CD44 CD24 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 + -/low + -/low CD44 CD24 breast CSC cells compared to non-CD44 CD24 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 + -/low CD44 CD24 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. tumors overexpress the human epidermal growth factor 2 INTRODUCTION + -/ (HER2) [1]. Evidence is mounting that the CD44 CD24 low cell subpopulation, which is enriched with potential De novo (i. e. primary) resistance to the monoclonal breast cancer stem cells (CSCs), could explain clinical antibody trastuzumab (Herceptin) remains a prevalent resistance to HER2-targeted therapies [1-3]. Therefore, challenge in the treatment of breast cancer patients whose www.impactjournals.com/oncotarget 395 Oncotarget 2012; 3: 395-398 future clinical trials should involve the integration of novel estimated IC value of 1±0.2 mmol/L metformin whereas + -/low anti-breast cancer stem cells approaches to prevent and that in the corresponding non-CD44 CD24 population overcome the inherent unresponsiveness to trastuzumab was 11±2 mmol/L metformin (Fig. 1A, bottom right). across clinically important subgroups of HER2-positive The IC of metformin in the unsorted JIMT-1 parental breast cancer patients. Although in vitro studies have population was 8±2 mmol/L (Fig. 1A, bottom right). recently suggested that the anti-diabetic drug metformin These findings, altogether, confirmed that the breast cancer can efficiently eliminate treatment-resistant stem/ initiating subpopulation within a heterogeneous population progenitor cell populations in heterogeneous breast cancer of trastuzumab-refractory, HER2-positive breast cancer populations [4-6], it remained to be evaluated whether cells notably exhibit greater sensitivity to the growth- systemic metformin might overcome primary resistance inhibitory effects of metformin. Indeed, the growth of + -/low to trastuzumab in HER2-gene amplified human breast the breast cancer initiating CD44 CD24 population cancer xenografts. Here we explored for the first time the was more significantly affected by metformin at all tested in vivo sensitizing efficacy of metformin on trastuzumab concentrations. Because it has been shown that gradual therapy by using xenografts of the human breast cancer loss of stem cell markers takes place in cells growing cell line JIMT-1, which was established from the pleural as a monolayer, these data may further underestimate metastasis of a patient who was clinically resistant to hypersensitivity of the breast cancer initiating + -/low trastuzumab ab initio [7]. The JIMT-1 model is unique CD44 CD24 cells to metformin. Indeed, JIMT-1 + -/low because of displaying at the same time several co-existing cells sorted for the CD44 CD24 immunophenotype mechanisms of resistance to trastuzumab present at repopulated all the parental cell fractions after few days in variable levels in other breast cancer cell lines, including monolayer culture (data not shown). moderate expression levels of HER2 (despite HER2 gene amplification), low expression of PTEN (phosphatase and Systemic metformin inhibits tumor growth in tensin homolog), an activating mutation of the PIK3CA trastuzumab-refractory breast cancer xenografts gene, high expression of NRG1 (neuregulin-1), and enhanced expression of mesenchymal markers, including a The effects of metformin and trastuzumab alone naturally enriched subpopulation of breast cancer initiating + -/low and in combination on tumor growth were next studied CD44 CD24 CSC-like cells [2, 8-10]. in vivo using a JIMT-1 xenograft animal model (Fig. 1B). Compared to the control group after seven weeks RESULTS AND DISCUSSION of treatment (940±170 mm ), the trastuzumab-treated group likewise failed to exhibit significant reductions in mean tumor size (891±135 mm ). Compared to the Metformin preferentially kills breast cancer mean xenograft tumor size in both the control and the + - initiating CD44 CD24 /low cells trastuzumab group, the mean tumor size in the metformin group was significantly smaller (390±64 mm ), which confirmed that the inhibitory effect of metformin at the We first examined whether breast cancer + -/low + -/low tested concentration (250 mg/kg/day) was notably stronger initiating CD44 CD24 and non-CD44 CD24 cell than that of trastuzumab (5 mg/kg/week) on JIMT-1 tumor subpopulations from the trastuzumab-refractory JIMT-1 growth. When the two drugs were combined, the xenograft cell line exhibited differential sensitivities to the growth tumor size (213±75 mm ) was smaller than those of the inhibitory effects of metformin. We employed MTT-based groups treated with trastuzumab or metformin alone, cell viability assays to compare the degree of sensitivity indicating that the combination of the drugs was much to metformin of parental JIMT-1 cells and that of JIMT- more effective at reducing tumor volume. Consequently, 1 cell subpopulations isolated and purified for breast + -/low + -/low the days required for four-fold increase in tumor volume cancer initiating CD44 CD24 and non-CD44 CD24 was 27±5 after treatment with trastuzumab whereas more immunophenotypes by employing magnetic activated cell than 50 days were required after the combined treatment sorting (MACS; Fig. 1A, top panels). Cells were treated with trastuzumab and metformin (Fig. 1B). No significant side-by-side on the following sorting day with graded difference in body weight was observed in xenograft concentrations of metformin for five days. While unsorted + -/low + + tumor-bearing mice between the treatment groups (data JIMT-1, CD44 CD24 , and CD44 CD24 populations not shown). all showed an inhibitory response to metformin that Our current findings reveal that: a.) Metformin augmented with the increase in metformin concentration as single agent is active against JIMT-1-derived tumor (Fig. 1A, bottom left), each separate cell population xenografts with primary resistance to trastuzumab; b.) differed in their extend of cytotoxic response to metformin. + -/low metformin’s ability to distinctively kill breast cancer The breast cancer initiating CD44 CD24 subpopulation + -/low initiating CD44 CD24 mesenchymal subpopulations was significantly more sensitive than was the parental or + + + -/low is sufficient to overcome in vivo primary resistance to CD44 CD24 populations. CD44 CD24 cells had an www.impactjournals.com/oncotarget 396 Oncotarget 2012; 3: 395-398 + -/low + + CD44 CD24 -enriched A Unsorted CD44 CD24 -enriched 40% 3% 77% 60% 97% 23% CD44 CD44 CD44 Unsorted + + JIMT-1 CD44 CD24 110 + -/low CD44 CD24 0 0.5 1 5 10 JIMT-1 M ETFORMIN (mmol/L) Control Trastuzumab Metformin 200 M etformin + Trastuzumab 0 7 14 21 28 35 42 49 D ays post-inoculation + -/low + + Figure 1: A. Top. CD44 and CD24 expression patterns in CD44 CD24 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. + -/low + CD44 CD24 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 + + -/low - + + is shown. Bottom. Parental JIMT-1 cells and cells sorted for BCSC CD44 CD24 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: (OD of treated sample/OD of untreated sample) × 100. Cell sensitivity to metformin was 570 570 expressed in terms of the concentration of drug required to decrease by 50% cell viability (IC value). Since the percentage of control absorbance was considered to be the surviving fraction of cells, the IC 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 x 10 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 (mm ) = length x width 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. www.impactjournals.com/oncotarget 397 Oncotarget 2012; 3: 395-398 CD24 M T T up take (% of untreated control) Tumor vol ume (mm ) IC v alue ( mmol/L) Unsorted + + CD44 CD24 + -/low CD44 CD24 6. Iliopoulos D, Hirsch HA, Struhl K. Metformin decreases trastuzumab in HER2-gene amplified breast tumors. A the dose of chemotherapy for prolonging tumor remission combination of trastuzumab and metformin may provide in mouse xenografts involving multiple cancer cell types. a valuable strategy for treatment of HER2-overexpressing Cancer Res. 2011; 71:3196-3201. breast cancer, and a phase II trial recently has opened in Spain to evaluate the efficacy of trastuzumab plus 7. Tanner M, Kapanen AI, Junttila T, Raheem O, Grenman S, metformin as neo-adjuvant therapy for patients with Elo J, Elenius K, Isola J. Characterization of a novel cell HER2-positive breast cancer [11]. line established from a patient with Herceptin-resistant breast cancer. Mol Cancer Ther. 2004; 3:1585-1592. ACKNOWLEDGMENTS 8. Rennstam K, Jönsson G, Tanner M, Bendahl PO, Staaf J, Kapanen AI, Karhu R, Baldetorp B, Borg A, Isola J. This work was financially supported through funding Cytogenetic characterization and gene expression profiling from the Instituto de Salud Carlos III (Ministerio de of the trastuzumab-resistant breast cancer cell line JIMT-1. Sanidad y Consumo, Fondo de Investigación Sanitaria Cancer Genet Cytogenet. 2007; 172:95-106. (FIS), Spain, Grants CP05-00090 and PI06-0778 and 9. Köninki K, Barok M, Tanner M, Staff S, Pitkänen J, RD06-0020-0028), the Fundación Científica de la Hemmilä P, Ilvesaro J, Isola J. Multiple molecular Asociación Española Contra el Cáncer (AECC, Spain), mechanisms underlying trastuzumab and lapatinib and the Ministerio de Ciencia e Innovación (SAF2009- resistance in JIMT-1 breast cancer cells. Cancer Lett. 2010; 11579, Plan Nacional de I+D+ I, MICINN, Spain). 294:211-219. Alejandro Vazquez-Martin received the Sara Borrell post- 10. Oliveras-Ferraros C, Vazquez-Martin A, Martin-Castilló doctoral contract (CD08/00283, Ministerio de Sanidad y B, Pérez-Martínez MC, Cufí S, Del Barco S, Bernado L, Consumo, Fondo de Investigación Sanitaria –FIS-, Spain). Brunet J, López-Bonet E, Menendez JA. Pathway-focused Sílvia Cufí received a Research Fellowship (Formación de proteomic signatures in HER2-overexpressing breast cancer Personal Investigador, FPI) from the Ministerio de Ciencia with a basal-like phenotype: new insights into de novo e Innovación (MICINN, Spain). resistance to trastuzumab (Herceptin). Int J Oncol. 2010; 37:669-678. CONFLICT OF INTEREST 11. Martin-Castillo B, Dorca J, Vazquez-Martin A, Oliveras- Ferraros C, Lopez-Bonet E, Garcia M, Del Barco S, The authors declare no conflict of interest. Menendez JA. Incorporating the antidiabetic drug metformin in HER2-positive breast cancer treated with REFERENCE neo-adjuvant chemotherapy and trastuzumab: an ongoing clinical-translational research experience at the Catalan 1. Bedard PL, Cardoso F, Piccart-Gebhart MJ. Stemming Institute of Oncology. Ann Oncol. 2010; 21:187-189. resistance to HER-2 targeted therapy. J Mammary Gland Biol. 2009; 14:55-66. 2. Oliveras-Ferraros C, Vazquez-Martin A, Martin-Castillo B, Cufí S, Del Barco S, Lopez-Bonet E, Brunet J, Menendez JA. Dynamic emergence of the mesenchymal CD44(pos) CD24(neg/low) phenotype in HER2-gene amplified breast cancer cells with de novo resistance to trastuzumab (Herceptin). Biochem Biophys Res Commun. 2010; 397:27- 3. Fumagalli D, Michiels S, Sotiriou C. CD44+ CD24-/low phenotype and resistance to trastuzumab in HER2-positive breast cancer cell lines. Pharmacogenomics. 2011; 12:12- 4. Hirsch HA, Iliopoulos D, Tsichlis PN, Struhl K. Metformin selectively targets cancer stem cells, and acts together with chemotherapy to block tumor growth and prolong remission. Cancer Res. 2009; 69:7507-7511. 5. Vazquez-Martin A, Oliveras-Ferraros C, Del Barco S, Martin-Castillo B, Menendez JA. The anti-diabetic drug metformin suppresses self-renewal and proliferation of trastuzumab-resistant tumor-initiating breast cancer stem cells. Breast Cancer Res Treat. 2011; 126:355-364. www.impactjournals.com/oncotarget 398 Oncotarget 2012; 3: 395-398 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Oncotarget Unpaywall

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

Download PDF
4 pages

Loading next page...
 
/lp/unpaywall/metformin-induced-preferential-killing-of-breast-cancer-initiating-dXuTeMdlBJ

References

References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.

Publisher
Unpaywall
ISSN
1949-2553
DOI
10.18632/oncotarget.488
Publisher site
See Article on Publisher Site

Abstract

www.impactjournals.com/oncotarget/ Oncotarget, April, Vol.3, No 4 Metformin-induced preferential killing of breast cancer initiating + -/low CD44 CD24 cells is sufficient to overcome primary resistance to trastuzumab in HER2+ human breast cancer xenografts 1,2 1,2 1,2 Sílvia Cufí , Bruna Corominas-Faja , Alejandro Vazquez-Martin , Cristina 1,2 2,3 2,3 Oliveras-Ferraros , Joan Dorca , Joaquim Bosch-Barrera , Begoña 2,4 1,2 Martin-Castillo , Javier A. Menendez Translational Research Laboratory, Catalan Institute of Oncology (ICO), Girona, Spain Girona Biomedical Research Institute (IDIBGi), Girona, Spain Medical Oncology, Catalan Institute of Oncology (ICO), Girona, Spain Clinical Research Unit, Catalan Institute of Oncology (ICO), Girona, Spain Correspondence to: Javier A. Menendez, email: [email protected]; [email protected] Keywords: Metformin, trastuzumab, HER2, cancer stem cells, breast cancer Received: April 27, 2012, Accepted: April 28, 2012, Published: May 4, 2012 Copyright: © Cufi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which per - mits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT: Trastuzumab-refractory breast cancer stem cells (CSCs) could 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 + -/low CD44 CD24 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 + -/low initiating CD44 CD24 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 + -/low + -/low CD44 CD24 breast CSC cells compared to non-CD44 CD24 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 + -/low CD44 CD24 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. tumors overexpress the human epidermal growth factor 2 INTRODUCTION + -/ (HER2) [1]. Evidence is mounting that the CD44 CD24 low cell subpopulation, which is enriched with potential De novo (i. e. primary) resistance to the monoclonal breast cancer stem cells (CSCs), could explain clinical antibody trastuzumab (Herceptin) remains a prevalent resistance to HER2-targeted therapies [1-3]. Therefore, challenge in the treatment of breast cancer patients whose www.impactjournals.com/oncotarget 395 Oncotarget 2012; 3: 395-398 future clinical trials should involve the integration of novel estimated IC value of 1±0.2 mmol/L metformin whereas + -/low anti-breast cancer stem cells approaches to prevent and that in the corresponding non-CD44 CD24 population overcome the inherent unresponsiveness to trastuzumab was 11±2 mmol/L metformin (Fig. 1A, bottom right). across clinically important subgroups of HER2-positive The IC of metformin in the unsorted JIMT-1 parental breast cancer patients. Although in vitro studies have population was 8±2 mmol/L (Fig. 1A, bottom right). recently suggested that the anti-diabetic drug metformin These findings, altogether, confirmed that the breast cancer can efficiently eliminate treatment-resistant stem/ initiating subpopulation within a heterogeneous population progenitor cell populations in heterogeneous breast cancer of trastuzumab-refractory, HER2-positive breast cancer populations [4-6], it remained to be evaluated whether cells notably exhibit greater sensitivity to the growth- systemic metformin might overcome primary resistance inhibitory effects of metformin. Indeed, the growth of + -/low to trastuzumab in HER2-gene amplified human breast the breast cancer initiating CD44 CD24 population cancer xenografts. Here we explored for the first time the was more significantly affected by metformin at all tested in vivo sensitizing efficacy of metformin on trastuzumab concentrations. Because it has been shown that gradual therapy by using xenografts of the human breast cancer loss of stem cell markers takes place in cells growing cell line JIMT-1, which was established from the pleural as a monolayer, these data may further underestimate metastasis of a patient who was clinically resistant to hypersensitivity of the breast cancer initiating + -/low trastuzumab ab initio [7]. The JIMT-1 model is unique CD44 CD24 cells to metformin. Indeed, JIMT-1 + -/low because of displaying at the same time several co-existing cells sorted for the CD44 CD24 immunophenotype mechanisms of resistance to trastuzumab present at repopulated all the parental cell fractions after few days in variable levels in other breast cancer cell lines, including monolayer culture (data not shown). moderate expression levels of HER2 (despite HER2 gene amplification), low expression of PTEN (phosphatase and Systemic metformin inhibits tumor growth in tensin homolog), an activating mutation of the PIK3CA trastuzumab-refractory breast cancer xenografts gene, high expression of NRG1 (neuregulin-1), and enhanced expression of mesenchymal markers, including a The effects of metformin and trastuzumab alone naturally enriched subpopulation of breast cancer initiating + -/low and in combination on tumor growth were next studied CD44 CD24 CSC-like cells [2, 8-10]. in vivo using a JIMT-1 xenograft animal model (Fig. 1B). Compared to the control group after seven weeks RESULTS AND DISCUSSION of treatment (940±170 mm ), the trastuzumab-treated group likewise failed to exhibit significant reductions in mean tumor size (891±135 mm ). Compared to the Metformin preferentially kills breast cancer mean xenograft tumor size in both the control and the + - initiating CD44 CD24 /low cells trastuzumab group, the mean tumor size in the metformin group was significantly smaller (390±64 mm ), which confirmed that the inhibitory effect of metformin at the We first examined whether breast cancer + -/low + -/low tested concentration (250 mg/kg/day) was notably stronger initiating CD44 CD24 and non-CD44 CD24 cell than that of trastuzumab (5 mg/kg/week) on JIMT-1 tumor subpopulations from the trastuzumab-refractory JIMT-1 growth. When the two drugs were combined, the xenograft cell line exhibited differential sensitivities to the growth tumor size (213±75 mm ) was smaller than those of the inhibitory effects of metformin. We employed MTT-based groups treated with trastuzumab or metformin alone, cell viability assays to compare the degree of sensitivity indicating that the combination of the drugs was much to metformin of parental JIMT-1 cells and that of JIMT- more effective at reducing tumor volume. Consequently, 1 cell subpopulations isolated and purified for breast + -/low + -/low the days required for four-fold increase in tumor volume cancer initiating CD44 CD24 and non-CD44 CD24 was 27±5 after treatment with trastuzumab whereas more immunophenotypes by employing magnetic activated cell than 50 days were required after the combined treatment sorting (MACS; Fig. 1A, top panels). Cells were treated with trastuzumab and metformin (Fig. 1B). No significant side-by-side on the following sorting day with graded difference in body weight was observed in xenograft concentrations of metformin for five days. While unsorted + -/low + + tumor-bearing mice between the treatment groups (data JIMT-1, CD44 CD24 , and CD44 CD24 populations not shown). all showed an inhibitory response to metformin that Our current findings reveal that: a.) Metformin augmented with the increase in metformin concentration as single agent is active against JIMT-1-derived tumor (Fig. 1A, bottom left), each separate cell population xenografts with primary resistance to trastuzumab; b.) differed in their extend of cytotoxic response to metformin. + -/low metformin’s ability to distinctively kill breast cancer The breast cancer initiating CD44 CD24 subpopulation + -/low initiating CD44 CD24 mesenchymal subpopulations was significantly more sensitive than was the parental or + + + -/low is sufficient to overcome in vivo primary resistance to CD44 CD24 populations. CD44 CD24 cells had an www.impactjournals.com/oncotarget 396 Oncotarget 2012; 3: 395-398 + -/low + + CD44 CD24 -enriched A Unsorted CD44 CD24 -enriched 40% 3% 77% 60% 97% 23% CD44 CD44 CD44 Unsorted + + JIMT-1 CD44 CD24 110 + -/low CD44 CD24 0 0.5 1 5 10 JIMT-1 M ETFORMIN (mmol/L) Control Trastuzumab Metformin 200 M etformin + Trastuzumab 0 7 14 21 28 35 42 49 D ays post-inoculation + -/low + + Figure 1: A. Top. CD44 and CD24 expression patterns in CD44 CD24 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. + -/low + CD44 CD24 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 + + -/low - + + is shown. Bottom. Parental JIMT-1 cells and cells sorted for BCSC CD44 CD24 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: (OD of treated sample/OD of untreated sample) × 100. Cell sensitivity to metformin was 570 570 expressed in terms of the concentration of drug required to decrease by 50% cell viability (IC value). Since the percentage of control absorbance was considered to be the surviving fraction of cells, the IC 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 x 10 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 (mm ) = length x width 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. www.impactjournals.com/oncotarget 397 Oncotarget 2012; 3: 395-398 CD24 M T T up take (% of untreated control) Tumor vol ume (mm ) IC v alue ( mmol/L) Unsorted + + CD44 CD24 + -/low CD44 CD24 6. Iliopoulos D, Hirsch HA, Struhl K. Metformin decreases trastuzumab in HER2-gene amplified breast tumors. A the dose of chemotherapy for prolonging tumor remission combination of trastuzumab and metformin may provide in mouse xenografts involving multiple cancer cell types. a valuable strategy for treatment of HER2-overexpressing Cancer Res. 2011; 71:3196-3201. breast cancer, and a phase II trial recently has opened in Spain to evaluate the efficacy of trastuzumab plus 7. Tanner M, Kapanen AI, Junttila T, Raheem O, Grenman S, metformin as neo-adjuvant therapy for patients with Elo J, Elenius K, Isola J. Characterization of a novel cell HER2-positive breast cancer [11]. line established from a patient with Herceptin-resistant breast cancer. Mol Cancer Ther. 2004; 3:1585-1592. ACKNOWLEDGMENTS 8. Rennstam K, Jönsson G, Tanner M, Bendahl PO, Staaf J, Kapanen AI, Karhu R, Baldetorp B, Borg A, Isola J. This work was financially supported through funding Cytogenetic characterization and gene expression profiling from the Instituto de Salud Carlos III (Ministerio de of the trastuzumab-resistant breast cancer cell line JIMT-1. Sanidad y Consumo, Fondo de Investigación Sanitaria Cancer Genet Cytogenet. 2007; 172:95-106. (FIS), Spain, Grants CP05-00090 and PI06-0778 and 9. Köninki K, Barok M, Tanner M, Staff S, Pitkänen J, RD06-0020-0028), the Fundación Científica de la Hemmilä P, Ilvesaro J, Isola J. Multiple molecular Asociación Española Contra el Cáncer (AECC, Spain), mechanisms underlying trastuzumab and lapatinib and the Ministerio de Ciencia e Innovación (SAF2009- resistance in JIMT-1 breast cancer cells. Cancer Lett. 2010; 11579, Plan Nacional de I+D+ I, MICINN, Spain). 294:211-219. Alejandro Vazquez-Martin received the Sara Borrell post- 10. Oliveras-Ferraros C, Vazquez-Martin A, Martin-Castilló doctoral contract (CD08/00283, Ministerio de Sanidad y B, Pérez-Martínez MC, Cufí S, Del Barco S, Bernado L, Consumo, Fondo de Investigación Sanitaria –FIS-, Spain). Brunet J, López-Bonet E, Menendez JA. Pathway-focused Sílvia Cufí received a Research Fellowship (Formación de proteomic signatures in HER2-overexpressing breast cancer Personal Investigador, FPI) from the Ministerio de Ciencia with a basal-like phenotype: new insights into de novo e Innovación (MICINN, Spain). resistance to trastuzumab (Herceptin). Int J Oncol. 2010; 37:669-678. CONFLICT OF INTEREST 11. Martin-Castillo B, Dorca J, Vazquez-Martin A, Oliveras- Ferraros C, Lopez-Bonet E, Garcia M, Del Barco S, The authors declare no conflict of interest. Menendez JA. Incorporating the antidiabetic drug metformin in HER2-positive breast cancer treated with REFERENCE neo-adjuvant chemotherapy and trastuzumab: an ongoing clinical-translational research experience at the Catalan 1. Bedard PL, Cardoso F, Piccart-Gebhart MJ. Stemming Institute of Oncology. Ann Oncol. 2010; 21:187-189. resistance to HER-2 targeted therapy. J Mammary Gland Biol. 2009; 14:55-66. 2. Oliveras-Ferraros C, Vazquez-Martin A, Martin-Castillo B, Cufí S, Del Barco S, Lopez-Bonet E, Brunet J, Menendez JA. Dynamic emergence of the mesenchymal CD44(pos) CD24(neg/low) phenotype in HER2-gene amplified breast cancer cells with de novo resistance to trastuzumab (Herceptin). Biochem Biophys Res Commun. 2010; 397:27- 3. Fumagalli D, Michiels S, Sotiriou C. CD44+ CD24-/low phenotype and resistance to trastuzumab in HER2-positive breast cancer cell lines. Pharmacogenomics. 2011; 12:12- 4. Hirsch HA, Iliopoulos D, Tsichlis PN, Struhl K. Metformin selectively targets cancer stem cells, and acts together with chemotherapy to block tumor growth and prolong remission. Cancer Res. 2009; 69:7507-7511. 5. Vazquez-Martin A, Oliveras-Ferraros C, Del Barco S, Martin-Castillo B, Menendez JA. The anti-diabetic drug metformin suppresses self-renewal and proliferation of trastuzumab-resistant tumor-initiating breast cancer stem cells. Breast Cancer Res Treat. 2011; 126:355-364. www.impactjournals.com/oncotarget 398 Oncotarget 2012; 3: 395-398

Journal

Oncotarget – Unpaywall

Published: Apr 30, 2012

There are no references for this article.