CCL2/CCR2 chemokine signaling modulates ductal breast cancer progression by enhancing glycolysis and c-MET activation

Abstract

Despite therapeutic advancement, ductal carcinoma in situ (DCIS) remains the most common form of pre- invasive breast cancer diagnosed among women with 60,000 cases every year. Treatment for DCIS involves a combination of lumpectomy and radiotherapy in which 10-35% of patients experience disease recurrence that is often accompanied by invasive ductal carcinoma. By developing a molecular approach to evaluate the prognosis of DCIS we can identify biomarkers for DCIS patients capable of predicting which cases will become invasive and potentially spare women from non-necessary surgeries. The CCL2/CCR2 chemokine signaling is overexpressed in breast cancer biopsies and malignant breast cell lines. Here we demonstrated additional molecular factors by which CCL2/CCR2 might drive DCIS progression, by RPPA analysis on DCIS.com breast cancer cells we identified that CCL2 induction phosphorylated c-MET tyrosine kinase. By PLA we confirmed that CCR2 interacts with c-MET and by CO-IP we demonstrated that this interaction is potentially mediated by SRC dependent mechanisms. Additionally, global metabolomic analysis revealed that CCL2 alters glycolysis. Specifically, CCL2/CCR2 regulate glycolysis by enhancing glucose consumption, intracellular lactate, and upregulation of Hexokinase 2. In vitro studies targeting c-MET induced by CCL2 with the c-MET pharmacological Merestinib, resulted in blocked cellular migration, proliferation, survival, growth, and glycolysis in basal like breast cancer cells. By injecting SUM225 CCR2 overexpressing cells via the MIND Mammary Intraductal Injection model and dosing the animals orally with Merestinib, we demonstrated that c-MET inhibition mediated by CCL2/CCR2 resulted in decreased lesion mass, proliferation, and expression of glycolytic enzymes associated with formation of fewer invasive lesions compared to vehicle control. To investigate the effects of inhibiting c-MET pathway on metabolites involved in the glycolytic pathway in vivo, MIND model lesions were analyzed by IC-MS and showed that Merestinib blocked consumption of glycolytic intermediates. Lastly, we identified the physiological and clinical relevance of the CCR2 and c-MET receptors in normal, DCIS and IDC (invasive ductal carcinoma). By datamining analysis of TCGA data sets we found positive and significant mRNA correlations between CCR2 and c-MET. We further analyzed the protein expression in TMAs from DCIS and DCIS patient samples and identified that there was a positive and significant correlation between CCR2 and c-MET in DCIS and IDC tissues. Co-staining of CCR2 and c-MET demonstrated that co-expression of these receptors is higher in IDC tissues compared to DCIS and normal. These data indicate that CCR2 and c-MET mRNA, and protein expression have an important role in DCIS transition to IDC and that can potentially be identified as biomarkers use to predict DCIS progression. Implications: This project identifies a unique association between CCL2/CCR2 expression, c-MET activity, and enhancement of glycolysis with important implications into prognosis and potential management of DCIS progression. These associations can be considered as novel molecular targets for alternative therapeutic for DCIS patients expressing high levels of CCR2 and c-MET.