Abstract 3037: Adipokines modulate human mammary stem cell self-renewal through mTOR

Abstract Rationale: Excess visceral adiposity is associated with an increased incidence of a variety of human ailments, including cancer. We tested the hypothesis that adipose-derived factors drive normal mammary stem cell self-renewal. Methods: Visceral adipose tissue explants from obese F344 rats and cultured mouse 3T3-L1 adipocytes were grown with serum-free media for 48 hours. Primary human mammary epithelial cells, MCF10A, SUM149 and MCF7 cells were exposed to rhleptin (500 ng/ml), or adipocyte conditioned media and expanded in a non-adherent sphere forming assay for 10 days. Spheres were counted and size was quantified in Image J. Adipokine concentrations were assayed with the Proteome Profiler Adipokine Array. Other cultures were pre-treated with mTOR inhibitors rapamycin (100 nM) or everolimus (100 nM) for 2 hours prior to exposure to rhleptin or adipocyte conditioned media for 2 hours. S6 protein phosphorylation was quantified from Western blots. Results: Data shown in Table 1 suggest that conditioned media from adipose tissue and from 3T3-L1 adipocytes increase human mammary stem cell self-renewal by 103% and 189%, respectively, whereas rhleptin increases stem cell self-renewal by 37%. Blockade of leptin activity with neutralizing antibodies or chimeric soluble receptors reduced the adipocyte conditioned media's stimulation of mammary epithelial cell self-renewal by 10-15%. Media conditioned by the mouse 3T3-L1 adipocytes or primary rat visceral adipose explants increased pS6 protein by 170% in MCF10A cells. mTOR inhibitors reduced rhleptin and adipocyte conditioned media stimulation of self-renewal in MCF10A and normal human mammary stem cells by 78-85%. Conclusion: Adipocyte secreted factors and rhleptin promote increased mammary epithelial cell self-renewal through a process involving the activation of mTOR. Leptin contributes to a small fraction of the activity in adipocyte conditioned media, and is sufficient but not required for mammary stem cell self-renewal. Change in stem cell self-renewalrhLeptin3T3-L1 Adipocyte Conditioned MediaRat Adipose Tissue Conditioned MediaBaseline # Spheres% Change (± SEM)P Value% Change (± SEM)P Value% Change (± SEM)P ValuePrimary human organoids19737 ± 7.005104 ± 20.006190 ± 26.001MCF-10A cells28834 ± 16.043320 ± 32.002389 ± 27.002MCF-7 cells57638 ± 9.007142 ± 15.001262 ± 40.006SUM149 cells95435 ± 6.09174 ± 20.012167 ± 21.003 Citation Format: Ray Esper, Dame Michael, Zora Djuric, William Smith, Max Wicha, Dean Brenner. Adipokines modulate human mammary stem cell self-renewal through mTOR. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3037. doi:10.1158/1538-7445.AM2014-3037