Dissecting tumor metabolic heterogeneity: Telomerase and large cell size metabolically define a sub-population of stem-like, mitochondrial-rich, cancer cells

// Rebecca Lamb 1,2 , Bela Ozsvari 1,2 , Gloria Bonuccelli 1,2 , Duncan L. Smith 3 , Richard G. Pestell 4 , Ubaldo E. Martinez-Outschoorn 4 , Robert B. Clarke 1 , Federica Sotgia 1,2 and Michael P. Lisanti 1,2 1 The Breast Cancer Now Research Unit, Institute of Cancer Sciences, University of Manchester, Manchester, UK 2 The Manchester Centre for Cellular Metabolism (MCCM), Institute of Cancer Sciences, University of Manchester, Manchester, UK 3 The Cancer Research UK Manchester Institute, University of Manchester, Manchester, UK 4 The Sidney Kimmel Cancer Center, Philadelphia, PA, USA Correspondence to: Rebecca Lamb, email: // Federica Sotgia, email: // Michael P. Lisanti, email: // Keywords : hTERT, telomerase, cell size, mitochondrial biogenesis, cancer stem cells, proteomic analysis, tumor metabolism Abbreviations : CSCs, cancer stem-like cells, TICs, tumor-initiating cells Received : June 16, 2015 Accepted : July 13, 2015 Published : August 27, 2015 Abstract Tumor cell metabolic heterogeneity is thought to contribute to tumor recurrence, distant metastasis and chemo-resistance in cancer patients, driving poor clinical outcome. To better understand tumor metabolic heterogeneity, here we used the MCF7 breast cancer line as a model system to metabolically fractionate a cancer cell population. First, MCF7 cells were stably transfected with an hTERT-promoter construct driving GFP expression, as a surrogate marker of telomerase transcriptional activity. To enrich for immortal stem-like cancer cells, MCF7 cells expressing the highest levels of GFP (top 5%) were then isolated by FACS analysis. Notably, hTERT-GFP(+) MCF7 cells were significantly more efficient at forming mammospheres (i.e., stem cell activity) and showed increased mitochondrial mass and mitochondrial functional activity, all relative to hTERT-GFP(-) cells. Unbiased proteomics analysis of hTERT-GFP(+) MCF7 cells directly demonstrated the over-expression of 33 key mitochondrial proteins, 17 glycolytic enzymes, 34 ribosome-related proteins and 17 EMT markers, consistent with an anabolic cancer stem-like phenotype. Interestingly, MT-CO2 (cytochrome c oxidase subunit 2; Complex IV) expression was increased by >20-fold. As MT-CO2 is encoded by mt-DNA, this finding is indicative of increased mitochondrial biogenesis in hTERT-GFP(+) MCF7 cells. Importantly, most of these candidate biomarkers were transcriptionally over-expressed in human breast cancer epithelial cells in vivo . Similar results were obtained using cell size (forward/side scatter) to fractionate MCF7 cells. Larger stem-like cells also showed increased hTERT-GFP levels, as well as increased mitochondrial mass and function. Thus, this simple and rapid approach for the enrichment of immortal anabolic stem-like cancer cells will allow us and others to develop new prognostic biomarkers and novel anti-cancer therapies, by specifically and selectively targeting this metabolic sub-population of aggressive cancer cells. Based on our proteomics and functional analysis, FDA-approved inhibitors of protein synthesis and/or mitochondrial biogenesis, may represent novel treatment options for targeting these anabolic stem-like cancer cells.