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.
Highlights
Telomerase plays a central role both in the biology of normal aging, as well as in the development of human cancers [1, 2]
MCF7-hTERTeGFP cells were subjected to FACS analysis to visualize the broad distribution of eGFP expression, which serves as a surrogate marker of telomerase activity
To determine the clinical relevance of our findings, we assessed whether the hTERT proteomic targets that we identified in GFP-high cells were transcriptionally over-expressed, in human breast cancer cells in vivo
Summary
Telomerase plays a central role both in the biology of normal aging, as well as in the development of human cancers [1, 2]. Clarke and colleagues have taken advantage of the properties of human telomerase (hTERT), to enrich for a population of osteosarcoma cells with stemlike properties [7] For this purpose, they fused a 1.5-kB fragment of the hTERT promoter to GFP, in order to select a sub-population of osteosarcoma cells with hightelomerase transcriptional activity by FACS analysis. They fused a 1.5-kB fragment of the hTERT promoter to GFP, in order to select a sub-population of osteosarcoma cells with hightelomerase transcriptional activity by FACS analysis They demonstrated that these hTERThigh-activity osteosarcoma cells showed increased stemlike activity, anchorage-independent growth, invasiveness and metastatic activity, as well as chemo-therapy resistance [7]. This hTERT-based approach for the purification of cancer stem-like cells (CSCs) has already been functionally validated
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