Mitochondria "fuel" breast cancer metabolism: fifteen markers of mitochondrial biogenesis label epithelial cancer cells, but are excluded from adjacent stromal cells

Abstract

Here, we present new genetic and morphological evidence that human tumors consist of two distinct metabolic compartments. First, re-analysis of genome-wide transcriptional profiling data revealed that > 95 gene transcripts associated with mitochondrial biogenesis and/or mitochondrial translation were significantly elevated in human breast cancer cells, as compared with adjacent stromal tissue. Remarkably, nearly 40 of these upregulated gene transcripts were mitochondrial ribosomal proteins (MRPs), functionally associated with mitochondrial translation of protein components of the OXPHOS complex. Second, during validation by immunohistochemistry, we observed that antibodies directed against 15 markers of mitochondrial biogenesis and/or mitochondrial translation (AKAP1, GOLPH3, GOLPH3L, MCT1, MRPL40, MRPS7, MRPS15, MRPS22, NRF1, NRF2, PGC1-α, POLRMT, TFAM, TIMM9 and TOMM70A) selectively labeled epithelial breast cancer cells. These same mitochondrial markers were largely absent or excluded from adjacent tumor stromal cells. Finally, markers of mitochondrial lipid synthesis (GOLPH3) and mitochondrial translation (POLRMT) were associated with poor clinical outcome in human breast cancer patients. Thus, we conclude that human breast cancers contain two distinct metabolic compartments-a glycolytic tumor stroma, which surrounds oxidative epithelial cancer cells-that are mitochondria-rich. The co-existence of these two compartments is indicative of metabolic symbiosis between epithelial cancer cells and their surrounding stroma. As such, epithelial breast cancer cells should be viewed as predatory metabolic "parasites," which undergo anabolic reprogramming to amplify their mitochondrial "power." This notion is consistent with the observation that the anti-malarial agent chloroquine may be an effective anticancer agent. New anticancer therapies should be developed to target mitochondrial biogenesis and/or mitochondrial translation in human cancer cells.

Figure 1. AKAP1, a mitochondrial marker, is predominantly confined to epithelial cancer cells, and largely absent from adjacent stromal cells, in human breast cancer tissues. Paraffin-embedded sections of human breast cancer tumor tissue were immunostained with antibodies directed against AKAP1. Slides were then counter-stained with hematoxylin. Note that AKAP1 is highly expressed in the epithelial compartment (brown color). Two representative images are shown. Original magnification is 60x, as indicated.

Figure 7. MCT1, a metabolic marker for the uptake of high-energy mitochondrial fuels, is predominantly localized to epithelial cancer cells, and absent from adjacent tumor stromal cells, in human breast cancers. Paraffin-embedded sections of human breast cancer tumor tissue were immunostained with antibodies directed against MCT1. Slides were then counter-stained with hematoxylin. Note that MCT1 immunostaining is largely absent from the stromal compartment and confined to the epithelial compartment (brown color). Original magnification is 60x, as indicated.

Figure 2. GOLPH3 and GOLPH3L, markers of mitochondrial lipid biosynthesis, are localized mainly to epithelial cancer cells in human breast cancer tissues. Paraffin-embedded sections of human breast cancer tumor tissue were immunostained with antibodies directed against GOLPH3 and GOLPH3L. Slides were then counter-stained with hematoxylin. Note that both GOLPH3 family members are largely absent from the stromal compartment and confined to the epithelial compartment (brown color). Original magnification is 40x, as indicated.

Figure 3. Mitochondrial ribosomal proteins (MRPL40, MRPS7, MRPS15, and MRPS22) are localized to epithelial cancer cells, but absent from adjacent tumor stroma, in human breast cancers. Paraffin-embedded sections of human breast cancer primary tumors were immunostained with antibodies directed against MRPL40, MRPS7, MRPS15, and MRPS22 (all mitochondrial ribosomal proteins). Note that immunostaining (brown color) is largely confined to the epithelial cancer cells. Original magnification, 40x.

Figure 4. PGC1-α, a key mitochondrial transcription factor, is largely confined to epithelial cancer cells, and absent from stromal cells, in human breast cancers. Paraffin-embedded sections of human breast cancer primary tumors were immunostained with antibodies directed against PGC1-α. Note that PGC1-α immunostaining is largely confined to the epithelial cancer cells. A red arrow points at an area that is further magnified below and is shown as an inset. Original magnification, 60x.

Figure 5. NRF1 and NRF2 family members preferentially label epithelial cancer cells in human breast cancers, but not adjacent stromal cells. Paraffin-embedded sections of human breast cancer primary tumors were immunostained with antibodies directed against either NRF1 (panel A) or NRF2 (panel B). Note that NRF1/2 immunostaining is largely confined to the epithelial cancer cells. Original magnification is as indicated.

Figure 6. Markers of mitochondrial biogenesis (TFAM, POLRMT, TOMM70A, and TIMM9) are all predominantly confined to epithelial cancer cells in human breast cancer tumor tissues, but are largely absent from adjacent stromal cells. Paraffin-embedded sections of human breast cancer tumor tissue were immunostained with antibodies directed against TFAM, POLRMT, TOMM70A and TIMM9. Slides were then counter-stained with hematoxylin. Note that TFAM, POLRMT, TOMM70A and TIMM9 are all largely absent from the stromal compartment and confined to the epithelial compartment (brown color). Original magnifications, 40x and 60x, are as indicated.

Figure 8. GOLPH3 (a marker of mitochondrial lipid synthesis) and POLRMT (a marker of mitochondrial translation) both predict poor clinical outcome in human breast cancer patients. Note that the expression levels of the gene transcripts for GOLPH3 (A) and POLRMT (B) predict poor overall survival, especially in ER-positive (A) patients. Numbers of cases with annotation are shown. P values are as indicated. X-Tile software was employed to identify subpopulation cut-points to observe maximum survival differences between the high expression and low expression subpopulations. The Log-rank test was used to evaluate the significance of differences in survival curves for high vs. low expressing populations.

Figure 9. Two-compartment tumor metabolism (2CTM) reflects metabolic symbiosis. We suggest that aggressive breast cancers consist of two distinct metabolic compartments. In the tumor microenvironment, stromal fibroblasts (and other cell types) show signs of mitochondrial dysfunction, are mitochondrial-deficient, and metabolically shift toward aerobic glycolysis (the “reverse Warburg effect”). This results in the stromal production of high-energy mitochondrial fuels, such as L-lactate, ketone bodies, glutamine and free fatty acids. These recycled nutrients are then available to “feed” neighboring cancer cells. In response to this energy-rich microenvironment, epithelial cancer cells undergo mitochondrial biogenesis, amplifying their capacity for oxidative mitochondrial metabolism (OXPHOS). Thus, the tumor stroma and epithelial breast cancer cells are metabolically linked in a “symbiotic/parasitic” relationship, related to energy transfer or an energy imbalance.