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, 12 (9), 1371-84

Cancer Metabolism, Stemness and Tumor Recurrence: MCT1 and MCT4 Are Functional Biomarkers of Metabolic Symbiosis in Head and Neck Cancer

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Cancer Metabolism, Stemness and Tumor Recurrence: MCT1 and MCT4 Are Functional Biomarkers of Metabolic Symbiosis in Head and Neck Cancer

Joseph M Curry et al. Cell Cycle.

Abstract

Here, we interrogated head and neck cancer (HNSCC) specimens (n = 12) to examine if different metabolic compartments (oxidative vs. glycolytic) co-exist in human tumors. A large panel of well-established biomarkers was employed to determine the metabolic state of proliferative cancer cells. Interestingly, cell proliferation in cancer cells, as marked by Ki-67 immunostaining, was strictly correlated with oxidative mitochondrial metabolism (OXPHOS) and the uptake of mitochondrial fuels, as detected via MCT1 expression (p < 0.001). More specifically, three metabolic tumor compartments were delineated: (1) proliferative and mitochondrial-rich cancer cells (Ki-67+/TOMM20+/COX+/MCT1+); (2) non-proliferative and mitochondrial-poor cancer cells (Ki-67-/TOMM20-/COX-/MCT1-); and (3) non-proliferative and mitochondrial-poor stromal cells (Ki-67-/TOMM20-/COX-/MCT1-). In addition, high oxidative stress (MCT4+) was very specific for cancer tissues. Thus, we next evaluated the prognostic value of MCT4 in a second independent patient cohort (n = 40). Most importantly, oxidative stress (MCT4+) in non-proliferating epithelial cancer cells predicted poor clinical outcome (tumor recurrence; p < 0.0001; log-rank test), and was functionally associated with FDG-PET avidity (p < 0.04). Similarly, oxidative stress (MCT4+) in tumor stromal cells was specifically associated with higher tumor stage (p < 0.03), and was a highly specific marker for cancer-associated fibroblasts (p < 0.001). We propose that oxidative stress is a key hallmark of tumor tissues that drives high-energy metabolism in adjacent proliferating mitochondrial-rich cancer cells, via the paracrine transfer of mitochondrial fuels (such as L-lactate and ketone bodies). New antioxidants and MCT4 inhibitors should be developed to metabolically target "three-compartment tumor metabolism" in head and neck cancers. It is remarkable that two "non-proliferating" populations of cells (Ki-67-/MCT4+) within the tumor can actually determine clinical outcome, likely by providing high-energy mitochondrial "fuels" for proliferative cancer cells to burn. Finally, we also show that in normal mucosal tissue, the basal epithelial "stem cell" layer is hyper-proliferative (Ki-67+), mitochondrial-rich (TOMM20+/COX+) and is metabolically programmed to use mitochondrial fuels (MCT1+), such as ketone bodies and L-lactate. Thus, oxidative mitochondrial metabolism (OXPHOS) is a common feature of both (1) normal stem cells and (2) proliferating cancer cells. As such, we should consider metabolically treating cancer patients with mitochondrial inhibitors (such as Metformin), and/or with a combination of MCT1 and MCT4 inhibitors, to target "metabolic symbiosis."

Keywords: MCT1; MCT4; OXPHOS; TOMM20; glycolysis; head and neck cancer; metabolic symbiosis; mitochondria; monocarboxylate transporters (MCT); oxidative stress; stem cells; tumor recurrence; tumor stroma.

Figures

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Figure 1. Distribution of proliferative and mitochondrial biomarkers in normal mucosa. Normal mucosal tissue was subjected to staining (brown color) with a variety of metabolic markers. Note that the basal “stem cell” layer is highly enriched in markers of proliferation (Ki-67), oxidative mitochondrial metabolism (TOMM20 and COX) and L-lactate/ketone body utilization (MCT1). Based on these studies, MCT1 is a highly selective marker of the basal stem cell layer. S, underlying connective tissue layer; B, basal stem cell layer; E, differentiating squamous epithelial layer. Original magnification: 20× (Ki-67 and COX); 40x (TOMM20 and MCT1).
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Figure 2. MCT1 is a specific marker for basal stem cells, and co-distributes with markers of proliferation (Ki-67) and mitochondrial metabolism (TOMM20 and COX). As in Figure 1, except that selected areas are shown at a higher magnification to better appreciate the staining of the basal stem cell layer. An arrow points at MCT1 staining, which is a specific marker for basal stem cells.
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Figure 3. Morphology and proliferation in well-differentiated HNSCC tumor tissue: H&E and Ki-67 immunostaining. Note that carcinoma cells are more differentiated in the center of clusters or nests, with occasional keratin pearls. The carcinoma cells in the periphery of the nests stain strongly for Ki-67, which is absent in the center of the nests. Original magnification: 20× and 40×, as indicated.
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Figure 4. Morphology and proliferation in poorly differentiated HNSCC tumor tissue: H&E and Ki-67 immunostaining. Note that carcinoma cells form small clusters or nests without differentiation or keratin pearls. The carcinoma cells in the nests diffusely stain strongly for Ki-67. Note also that the tumor stroma is negative for Ki-67 staining. Original magnification: 20× and 40×, as indicated.
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Figure 5. TOMM20 and MCT1 immunostaining in well-differentiated and poorly differentiated HNSCC specimens. In well-differentiated, note that carcinoma cells in the periphery of nests have high TOMM20 staining (brown), while well differentiated cells in the center of nests have low TOMM20 staining. In poorly differentiated, note that carcinoma cells in the nests strongly stain for TOMM20 (brown). Virtually identical results were obtained for MCT1, as well. Note also that the tumor stroma is negative for both TOMM20 and MCT1-staining. Original magnification: 20×, as indicated.
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Figure 6. COX mitochondrial activity staining in well-differentiated and poorly differentiated HNSCC specimens. In well-differentiated, note that carcinoma cells in the periphery of nests have high COX activity (brown), while as differentiated cells in the center of nests have low COX activity. In poorly differentiated, note that carcinoma cells in the nests diffusely stain strongly for COX. Note also that the tumor stroma is negative COX staining. Original magnification: 40×, as indicated.
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Figure 7. Higher power views of MCT1 immunostaining in HNSCC specimens. As in Figure 5, except higher power view are shown to better appreciate the plasma membrane staining of MCT1+ tumor cells. Also, note that MCT1 staining is absent from the the tumor stroma and cancer-associated fibroblasts. Original magnification: 40×, as indicated.
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Figure 8. MCT4 epithelial immunostaining in HNSCC tumor tissue. Three different tumor regions or metabolic compartments are as indicated: (1) is the tumor stroma; (2) represents the proliferating cancer cell compartment and (3) is the non-proliferating cancer cell compartment. Note that MCT4 staining is primarily localized to the tumor stroma (red arrows) and the non-proliferating epithelial cancer cell compartment (black arrow). Note also that the non-proliferating MCT4+ cancer cells appear enlarged or hypertrophic, which is consistent with a senescent phenotype. Original magnification: 40×, as indicated.
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Figure 9. MCT4 stromal immunostaining in HNSCC tumor tissue. Note that the stromal cells separating nests of proliferating carcinoma cells have the highest MCT4 expression (brown). Original magnification: 40×, as indicated.
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Figure 10. LDHB stromal immunostaining in HNSCC tumor tissue. Note that the stromal cells separating nests of proliferating carcinoma cells have the highest LDHB expression of all the cells in the sample (brown). Original magnification: 40×, as indicated.
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Figure 11. MCT4 immunostaining distinguishes tumor tissue from normal adjacent tissue. Note that MCT4 preferentially stains cancer-associated fibroblasts (CAFs) and not normal fibroblasts (NFs), allowing one to distinguish tumor tissue from normal tissue, even at low-magnification. Original magnification: 2×, as indicated. N, normal tissue; T, tumor tissue.
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Figure 12. Epithelial MCT4 is a negative prognostic marker in HNSCC: Kaplan-Meier analysis and disease-free survival (DFS). Kaplan-Meier survival curves are shown for the 40 subjects analyzed. Note that higher levels of epithelial MCT4 expression are significantly associated with lower DFS (higher tumor recurrence; p = 0.0001; log-rank test).
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Figure 13. Three-compartment metabolism in normal mucosa. Note that in normal mucosa, three morphological and metabolic compartments can be distinguished. The basal stem cell layer is hyper-proliferative (Ki-67+), mitochondrial-rich (TOMM20+/COX+) and uses mitochondrial fuels (MCT1+). In contrast, the underlying connective tissue and the differentiated epithelial cells are non-proliferative and mitochondrial-poor. Importantly, all three compartments are MCT4-negative.
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Figure 14. Three-compartment metabolism in HNSCC tumors. Note that in tumor tissue, three morphological and metabolic compartments could be identified. The poorly differentiated cancer cells are hyper-proliferative (Ki-67+), mitochondrial-rich (TOMM20+/COX+) and use mitochondrial fuels (MCT1+). In contrast, the tumor stroma and well-differentiated cancer cells are non-proliferative and mitochondrial-poor. However, in a subset of patients, both of these non-proliferating compartments are MCT4-positive, resulting in a higher disease stage or lower disease-free survival (DFS). MCT4 is a marker of oxidative stress and mitochondrial dysfunction, resulting in increased L-lactate and ketone body production, as well as export into the microenvironment. Thus, MCT1/MCT4 expression drives metabolic symbiosis in head and neck cancers.

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