Abstract 5212: GM-CSF and MMP9, targets of metformin, are crucial mediators of the tumor-promoting role of adipose tissue cells in breast cancer

Abstract

Abstract The white adipose tissue (WAT) promotes breast cancer (BC) progression with mechanisms that are only partially understood. We have found that WAT cells with progenitor-like phenotype (CD45-CD34+) are able to promote angiogenesis, motility, invasiveness, local and metastatic neoplastic progression in orthotopic murine models of triple negative BC (Martin-Padura et al., 2012; Orecchioni et al., 2013). Here we report the results of our screening for molecular signals involved in human WAT and BC interplay. Subpopulations of human WAT CD34+CD45- cells were cultured with several triple negative BC cell lines. An up-regulation of more than 10 proteins was detected in WAT- cells, suggesting a strong impact on their secretoma. In cellular supernatants two molecules were significantly up-regulated: GM-CSF and MMP9. Results were further confirmed by ELISA and western blotting using WAT cells from 10 different BC patients. GM-CSF was found to be 2 to 47 fold up-regulated both in transwell and in direct co-culture systems, thus suggesting a soluble factor implication. MMP9 showed up to 12 fold up-regulation in direct co-culture and a weak up-regulation in transwell system, suggesting a prevalent cell-to-cell mechanism. Quantitative RT-PCR revealed that WAT-progenitors were responsible for the observed up-regulation for both GM-CSF and MMP9, whereas BC cells did not change their basal expression levels of the two factors. Up-regulation was conserved among species both at RNA and protein levels. As we reported (Orecchioni et al., 2013) WAT CD45-CD34+ cells include two sub-populations of progenitors formally identified as mesenchymal (ASCs) and endothelial (EPCs). Both populations were able to contribute to GM-CSF and MMP9 up-release in a synergistic manner. In vivo studies in NODSCIDIL2Rgnull (NSG) mice orthotopic BC models confirmed the up-regulation of both factors: a significant increase of circulating human GM-CSF was detected in the plasma of mice injected with human WAT progenitors and BC compared to mice injected with BC alone. This up-regulation was statistically significant (p<0.001) in the early phases of tumor growth. BC investigation revealed an increased expression of MMP9 in mice injected with WAT progenitors. Accordingly, the qRT-PCR on tumor tissues did not detect an up-regulation of the two factors, confirming that the observed up-regulation were due to WAT cells. As we recently published (Orecchioni et al, 2014), metformin can target in vitro and in vivo WAT progenitors. In this context, we found in vitro that metformin targeted GM-CSF and MMP9, significantly reducing mRNA in WAT progenitors and protein levels in co-culture systems. Moreover, in our NSG orthotopic BC models metformin significantly reduced BC-specific circulating human GM-CSF. We are currently investigating the effect of GM-CSF and MMP9 inhibition in vivo, alone or in combination with metformin. Citation Format: Francesca Reggiani, Patrizia Mancuso, Cristina Rabascio, Stefania Orecchioni, Giovanna Talarico, Cinzia Massaro, Valentina Labanca, Angelica Calleri, Francesco Bertolini. GM-CSF and MMP9, targets of metformin, are crucial mediators of the tumor-promoting role of adipose tissue cells in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5212. doi:10.1158/1538-7445.AM2015-5212