Obesity is a known risk factor for breast cancer. Since obesity rates are constantly rising worldwide, understanding the molecular details of the interaction between adipose tissue and breast tumors becomes an urgent task. To investigate potential molecular changes in breast cancer cells induced by co-existing adipocytes, we used a co-culture system of different breast cancer cell lines (MCF-7 and T47D: ER+/PR+/HER2− and MDA-MB-231: ER−/PR−/HER2−) and murine 3T3-L1 adipocytes. Here, we report that co-culture with adipocytes revealed distinct changes in global gene expression pattern in the different breast cancer cell lines. Our microarray data revealed that in both ER+ cell lines, top upregulated genes showed significant enrichment for hormone receptor target genes. In triple-negative MDA-MB-231 cells, co-culture with adipocytes led to the induction of pro-inflammatory genes, mainly involving genes of the Nf-κB signaling pathway. Moreover, co-cultured MDA-MB-231 cells showed increased secretion of the pro-inflammatory interleukins IL-6 and IL-8. Using a specific NF-κB inhibitor, these effects were significantly decreased. Finally, migratory capacities were significantly increased in triple-negative breast cancer cells upon co-culture with adipocytes, indicating an enhanced aggressive cell phenotype. Together, our studies illustrate that factors secreted by adipocytes have a significant impact on the molecular biology of breast cancer cells.

◥Recent studies indicate that adipose tissue in obesity promotes breast cancer progression by secreting protumorigenic chemokines, growth factors, and fatty acids. However, the detailed mechanisms by which hypertrophic adipose tissue influences breast cancer cells are still not well understood. Here we show that co-culture with adipose tissue from high-fat diet induced obese C57BL/6 mice alters transcriptome profiles in triple-negative breast cancer (TNBC) cells, leading to upregulation of genes involved in inflammation and lipid metabolism, such as IL1B, PLIN2, and ANGPTL4. Similar results were obtained by treating TNBC cells with adipose tissue conditioned media (ACM) generated from fat tissue of obese female patients. Many of the upregulated genes were activated by PPAR nuclear receptors, as shown by pathway analyses and gene expression experiments using PPAR agonists and antagonists. Metabolic analysis revealed that TNBC cells cultivated with ACM had signif-icantly higher levels of b-oxidation. Furthermore, ACM-treated TNBC cells displayed a pronounced aggressive cell phenotype, with enhanced wound healing, proliferation, and invasion capabilities. ACM-induced invasion was dependent on the PPAR-target ANGPTL4 and activated FAK signaling, as shown by ANGPTL4 depletion and FAK inhibition. Together, our data suggest that factors released by adipose tissue change PPAR-regulated gene expression and lipid metabolism and induce a more aggressive TNBC cell phenotype. These effects are, at least in parts, mediated by fatty acids provided by the adipose tissue.Implications: Adipose tissue provides factors for increased progression of TNBC cells, identifying PPAR-and FAK-signaling as potential novel targets for treatment of TNBC, especially in obese women.

<p>Table S1: Primersequences used for human and murine cell lines. Table S2: Upstream regulators of genes with increased expression in MDA-MB-231 cells co-cultured with murine adipose tissue (HFD). Table S3: Upstream regulators of genes with decreased expression in MDA-MB-231 cells co-cultured with murine adipose tissue (HFD). Table S4: Characterization of the free fatty acid profile of ACMs<30 and ACMs>40. Table S5: Enriched GO terms (Biological Process) in genes with {greater than or equal to}1.5-fold downregulation in MDAMB-231 cells co-cultured with adipose tissue of HFD mice vs control. Table S6: Enriched GO terms (Biological Process) in genes with {greater than or equal to}1.5-fold upregulation in MDA-MB231 cells co-cultured with adipose tissue of HFD mice vs control. Fig. S1: Pathway Analysis and gene expression data of co-cultured MDA-MB-231 cells. Fig. S2: ACM increases PPAR target gene expression in murine E0771 cells. Fig. S3: ANGPTL4 mRNA is increased in human triple negative breast cancer and associated with reduced survival. Fig. S4: PPAR target gene expression is dependent on PPARα. Fig. S5: Free fatty acids activate PPAR signaling in TNBC cells. Fig. S6: Incubation of TNBC cells with ACM or BSA-OA decreases de novo FA synthesis. Fig. S7: Cultivation of TNBC cells with ACM or BSA-OA induces the expression of β-oxidation genes. Fig. S8: Treatment with oleic acid increases intracellular lipid droplet formation in TNBC cells. Fig S9: ACM promotes proliferation and migration of E0771 cells. Fig. S10: ANGPTL4 knockdown in MDA-MB-231 cells does not affect cell proliferation. Fig. S11: MMP2 is upregulated in MDA-MB-231 cells upon ACM cultivation.</p>

<p>Table S1: Primersequences used for human and murine cell lines. Table S2: Upstream regulators of genes with increased expression in MDA-MB-231 cells co-cultured with murine adipose tissue (HFD). Table S3: Upstream regulators of genes with decreased expression in MDA-MB-231 cells co-cultured with murine adipose tissue (HFD). Table S4: Characterization of the free fatty acid profile of ACMs<30 and ACMs>40. Table S5: Enriched GO terms (Biological Process) in genes with {greater than or equal to}1.5-fold downregulation in MDAMB-231 cells co-cultured with adipose tissue of HFD mice vs control. Table S6: Enriched GO terms (Biological Process) in genes with {greater than or equal to}1.5-fold upregulation in MDA-MB231 cells co-cultured with adipose tissue of HFD mice vs control. Fig. S1: Pathway Analysis and gene expression data of co-cultured MDA-MB-231 cells. Fig. S2: ACM increases PPAR target gene expression in murine E0771 cells. Fig. S3: ANGPTL4 mRNA is increased in human triple negative breast cancer and associated with reduced survival. Fig. S4: PPAR target gene expression is dependent on PPARα. Fig. S5: Free fatty acids activate PPAR signaling in TNBC cells. Fig. S6: Incubation of TNBC cells with ACM or BSA-OA decreases de novo FA synthesis. Fig. S7: Cultivation of TNBC cells with ACM or BSA-OA induces the expression of β-oxidation genes. Fig. S8: Treatment with oleic acid increases intracellular lipid droplet formation in TNBC cells. Fig S9: ACM promotes proliferation and migration of E0771 cells. Fig. S10: ANGPTL4 knockdown in MDA-MB-231 cells does not affect cell proliferation. Fig. S11: MMP2 is upregulated in MDA-MB-231 cells upon ACM cultivation.</p>