Glycolytic cancer associated fibroblasts promote breast cancer tumor growth, without a measurable increase in angiogenesis: Evidence for stromal-epithelial metabolic coupling.

Abstract

Previously, we proposed a new model for understanding the Warburg effect in tumorigenesis and metastasis. In this model, the stromal fibroblasts would undergo aerobic glycolysis (a.k.a., the Warburg effect)-producing and secreting increased pyruvate/lactate that could then be used by adjacent epithelial cancer cells as "fuel" for the mitochondrial TCA cycle, oxidative phosphorylation, and ATP production. To test this model more directly, here we used a matched set of metabolically well-characterized immortalized fibroblasts that differ in a single gene. CL3 fibroblasts show a shift towards oxidative metabolism, and have an increased mitochondrial mass. In contrast, CL4 fibroblasts show a shift towards aerobic glycolysis, and have a reduced mitochondrial mass. We validated these differences in CL3 and CL4 fibroblasts by performing an unbiased proteomics analysis, showing the functional upregulation of 4 glycolytic enzymes, namely ENO1, ALDOA, LDHA and TP I1, in CL4 fibroblasts. Many of the proteins that were upregulated in CL4 fibroblasts, as seen by unbiased proteomics, were also transcriptionally upregulated in the stroma of human breast cancers, especially in the patients that were prone to metastasis. Importantly, when CL4 fibroblasts were co-injected with human breast cancer cells (MDA-MB-231) in a xenograft model, tumor growth was dramatically enhanced. CL4 fibroblasts induced a >4-fold increase in tumor mass, and a near 8-fold increase in tumor volume, without any measurable increases in tumor angiogenesis. In parallel, CL3 and CL4 fibroblasts both failed to form tumors when they were injected alone, without epithelial cancer cells. Mechanistically, under co-culture conditions, CL4 glycolytic fibroblasts increased mitochondrial activity in adjacent breast cancer cells (relative to CL3 cells), consistent with the "Reverse Warburg Effect". Notably, Western blot analysis of CL4 fibroblasts revealed a significant reduction in caveolin-1 (Cav-1) protein levels. In human breast cancer patients, a loss of stromal Cav-1 is associated with an increased risk of early tumor recurrence, metastasis, tamoxifen-resistance, and poor clinical outcome. Thus, loss of stromal Cav-1 may be an effective marker for predicting the "Reverse Warburg Effect" in the stroma of human breast cancer patients. As such, CL4 fibroblasts are a new attractive model for mimicking the "glycolytic phenotype" of cancer-associated fibroblasts. Nutrients derived from glycolytic cancer associated fibroblasts could provide an escape mechanism to confer drug-resistance during anti-angiogenic therapy, by effectively reducing the dependence of cancer cells on a vascular blood supply.