Local and systemic effects of adipocyte-secreted factors in breast cancer

Abstract

Breast cancer is a complex disease that, once developed, progresses in response to multiple environmental factors, including local microenvironmental factors within the breast and systemic markers in circulation. Obesity affects one third of all New Zealand adults and is known to negatively impact breast cancer outcomes. Epidemiological studies have shown obese women with breast cancer have increased risk of recurrence and metastasis, poorer pathological response rates to chemotherapy, and worse overall survival. The biological mechanisms underlying these associations are complex and not yet completely understood. Cancer associated adipocytes (CAA) are fat cells located within close proximity to breast tumour cells. In vitro, CAA promote breast cancer cell migration, invasion, and resistance to therapy. Analysis of gene expression in breast cancer cells co-cultured with CAA has identified a number of genes which may be supporting disease progression. To further assess the influence of CAA on breast cancer cells, we identified and quantified changes in global protein abundance induced in breast cancer cells co-cultured with human breast adipocytes (CAA), and evaluated these changes by identifying key molecules and pathways that were significantly altered. Global differences in relative protein expression in MCF-7 (ER+, PR+, HER2-) and MDA-MB-231 (ER-, PR-, HER2-) breast cancer cells co-cultured with, or without, mature breast adipocytes in a transwell co-culture system, were measured using isobaric tags for relative and absolute quantification (iTRAQ) labelling and liquid chromatography tandem mass spectrometry (LC-MS/MS). In both control and co-cultured samples, a total of 1,126 proteins and 1,218 proteins were identified in MCF-7 and MDA-MB-231 breast cancer cells, respectively. Relative to controls, 85 proteins in MCF-7 cells (32 upregulated, 53 downregulated) and 63 proteins in MDA-MB-231 cells (51 upregulated, 12 downregulated) were differentially abundant by 1.5-fold or greater in co-cultured cells. Co-culture with CAA caused an enriched upregulation of tricarboxylic acid (TCA) cycle proteins in MCF-7 cells and glycolysis proteins in MDA-MB-231 cells. The glycolytic protein, phosphoglycerate kinase 1 (PGK1), was the only protein that was upregulated by more than 1.5-fold in both MCF-7 and MDA-MB-231 cells co-cultured with CAA. PGK1 is a kinase enzyme that plays an important role in the glycolytic pathway. In women with breast cancer, increased PGK1 expression in the tumour has been identified as a predictor of poor patient survival and marker of resistance to paclitaxel. As metabolic co-operation between adipocytes and breast cancer cells is a key mechanism promoting breast tumour progression, we investigated PGK1 overexpression in vitro. The transient transfection model for in vitro PGK1 overexpression utilised in this study induced differential effects in MCF-7 and MDA-MB-231 breast cancer cells. PGK1 overexpression increased sensitivity to chemotherapy in MCF-7 cells. Whereas, cell proliferation and viability were decreased, and conditioned media lactate concentrations were increased, in GFP and PGK1 expressing plasmid transfected MDA-MB-231 cells. In silico analysis showed PGK1 expression was higher in HER2 enriched compared to triple negative breast cancer cells, and was upregulated in HER2 overexpressing (HER2+) compared to HER2- breast tumours, suggesting that PGK1 expression may be particularly relevant to HER2+ breast cancers. Obesity is characterised by a state of low-grade chronic systemic inflammation. Breast cancer chemotherapies are predominantly metabolised in liver hepatocytes by cytochrome P450 (CYP) drug metabolising enzymes. Inflammatory cytokines have been shown to downregulate expression and activity of CYP enzymes in vitro. Additionally, CYP genotype-phenotype discordance has been observed in patients with advanced cancer. To investigate whether obesity-associated circulating inflammatory cytokines influence in vivo activity of CYP enzymes in women receiving chemotherapy for breast cancer, we carried out an exploratory patient study that recruited seven non-obese and five obese women receiving adriamycin-cyclophosphamide (AC) and paclitaxel chemotherapy for stage II or III breast cancer. During chemotherapy, serum levels of B-cell activating factor (BAFF), growth and differentiation factor 15 (GDF-15) and monocyte chemoattractant protein 1 (MCP-1) increased, whereas interleukin 10 (IL-10) levels decreased. Importantly, changes in the levels of circulating inflammatory cytokines during chemotherapy were not associated with differences in body morphometry or voluntary physical activity levels. Activity of the CYP enzymes (CYP2C9, CYP2C19, CYP2D6, and CYP3A4), measured using the ‘Inje’ probe drug cocktail, were largely unchanged over the course of chemotherapy, although varied between participants. However, increased serum MCP-1 levels correlated with decreased CYP3A4 activity during chemotherapy, and this finding provides preliminary evidence that circulating inflammatory cytokines may negatively influence CYP-mediated chemotherapy metabolism in women undergoing treatment for breast cancer. This study has provided, for the first time, an extensive list of breast cancer cell protein abundance alterations induced by co-culture with CAA, which can be used as a comprehensive platform for future investigations. Moreover, this study has validated, for the first time, the feasibility of using the ‘Inje’ cocktail to measure CYP activity in women receiving chemotherapy for breast cancer, and in doing so, has provided preliminary evidence to support the concept that changes in circulating inflammatory cytokines during chemotherapy treatment may impact CYP activity, and thus, chemotherapy metabolism in some patients.