Two-compartment tumor metabolism: Autophagy in the tumor microenvironment and oxidative mitochondrial metabolism (OXPHOS) in cancer cells

Previously, we proposed a new paradigm to explain the compartment-specific role of autophagy in tumor metabolism. In this model, autophagy and mitochondrial dysfunction in the tumor stroma promotes cellular catabolism, which results in the production of recycled nutrients. These chemical building blocks and high-energy “fuels” would then drive the anabolic growth of tumors, via autophagy resistance and oxidative mitochondrial metabolism in cancer cells. We have termed this new form of stromal-epithelial metabolic coupling: “two-compartment tumor metabolism.” Here, we stringently tested this energy-transfer hypothesis, by genetically creating (1) constitutively autophagic fibroblasts, with mitochondrial dysfunction or (2) autophagy-resistant cancer cells, with increased mitochondrial function. Autophagic fibroblasts were generated by stably overexpressing key target genes that lead to AMP-kinase activation, such as DRAM and LKB1. Autophagy-resistant cancer cells were derived by overexpressing GOLPH3, which functionally promotes mitochondrial biogenesis. As predicted, DRAM and LKB1 overexpressing fibroblasts were constitutively autophagic and effectively promoted tumor growth. We validated that autophagic fibroblasts showed mitochondrial dysfunction, with increased production of mitochondrial fuels (L-lactate and ketone body accumulation). Conversely, GOLPH3 overexpressing breast cancer cells were autophagy-resistant, and showed signs of increased mitochondrial biogenesis and function, which resulted in increased tumor growth. Thus, autophagy in the tumor stroma and oxidative mitochondrial metabolism (OXPHOS) in cancer cells can both dramatically promote tumor growth, independently of tumor angiogenesis. For the first time, our current studies also link the DNA damage response in the tumor microenvironment with “Warburg-like” cancer metabolism, as DRAM is a DNA damage/repair target gene.     

Two-compartment tumor metabolism: Autophagy in the tumor microenvironment and oxidative mitochondrial metabolism (OXPHOS) in cancer cells

Previously, we proposed a new paradigm to explain the compartment-specific role of autophagy in tumor metabolism. In this model, autophagy and mitochondrial dysfunction in the tumor stroma promotes cellular catabolism, which results in the production of recycled nutrients. These chemical building blocks and high-energy “fuels” would then drive the anabolic growth of tumors, via autophagy resistance and oxidative mitochondrial metabolism in cancer cells. We have termed this new form of stromal-epithelial metabolic coupling: “two-compartment tumor metabolism.” Here, we stringently tested this energy-transfer hypothesis, by genetically creating (1) constitutively autophagic fibroblasts, with mitochondrial dysfunction or (2) autophagy-resistant cancer cells, with increased mitochondrial function. Autophagic fibroblasts were generated by stably overexpressing key target genes that lead to AMP-kinase activation, such as DRAM and LKB1. Autophagy-resistant cancer cells were derived by overexpressing GOLPH3, which functionally promotes mitochondrial biogenesis. As predicted, DRAM and LKB1 overexpressing fibroblasts were constitutively autophagic and effectively promoted tumor growth. We validated that autophagic fibroblasts showed mitochondrial dysfunction, with increased production of mitochondrial fuels (L-lactate and ketone body accumulation). Conversely, GOLPH3 overexpressing breast cancer cells were autophagy-resistant, and showed signs of increased mitochondrial biogenesis and function, which resulted in increased tumor growth. Thus, autophagy in the tumor stroma and oxidative mitochondrial metabolism (OXPHOS) in cancer cells can both dramatically promote tumor growth, independently of tumor angiogenesis. For the first time, our current studies also link the DNA damage response in the tumor microenvironment with “Warburg-like” cancer metabolism, as DRAM is a DNA damage/repair target gene.     

The tumor suppressor WW domain-containing oxidoreductase modulates cell metabolism

The WW domain-containing oxidoreductase (WWOX) encodes a tumor suppressor that is frequently altered in cancer. WWOX binds several proteins and thus is postulated to be involved in a variety of cellular processes. Interestingly, Wwox-knockout mice develop normally in utero but succumb to hypoglycemia and other metabolic defects early in life resulting in their death by 3–4 weeks of age. Cumulative evidence has linked WWOX with cellular metabolism including steroid metabolism, high-density lipoprotein cholesterol (HDL-C) metabolism, bone metabolism and, more recently, glucose metabolism. In this review, we discuss these evolving functions for WWOX and how its deletion affects cellular metabolism and neoplastic progression.     

Hereditary ovarian cancer and two-compartment tumor metabolism

Mutations in the BRCA1 tumor suppressor gene are commonly found in hereditary ovarian cancers. Here, we used a co-culture approach to study the metabolic effects of BRCA1-null ovarian cancer cells on adjacent tumor-associated stromal fibroblasts. Our results directly show that BRCA1-null ovarian cancer cells produce large amounts of hydrogen peroxide, which can be abolished either by administration of simple antioxidants (N-acetyl-cysteine; NAC) or by replacement of the BRCA1 gene. Thus, the BRCA1 gene normally suppresses tumor growth by functioning as an antioxidant. Importantly, hydrogen peroxide produced by BRCA1-null ovarian cancer cells induces oxidative stress and catabolic processes in adjacent stromal fibroblasts, such as autophagy, mitophagy and glycolysis, via stromal NFκB activation. Catabolism in stromal fibroblasts was also accompanied by the upregulation of MCT4 and a loss of Cav-1 expression, which are established markers of a lethal tumor microenvironment. In summary, loss of the BRCA1 tumor suppressor gene induces hydrogen peroxide production, which then leads to metabolic reprogramming of the tumor stroma, driving stromal-epithelial metabolic coupling. Our results suggest that new cancer prevention trials with antioxidants are clearly warranted in patients that harbor hereditary/familial BRCA1 mutations.     

Cancer metabolism,stemness and tumor recurrence

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.”     

Mitochondrial metabolism in cancer metastasis

We have recently proposed a new two-compartment model for understanding the Warburg effect in tumor metabolism. In this model, glycolytic stromal cells produce mitochondrial fuels (L-lactate and ketone bodies) that are then transferred to oxidative epithelial cancer cells, driving OXPHOS and mitochondrial metabolism. Thus, stromal catabolism fuels anabolic tumor growth via energy transfer. We have termed this new cancer paradigm the “reverse Warburg effect,” because stromal cells undergo aerobic glycolysis, rather than tumor cells. To assess whether this mechanism also applies during cancer cell metastasis, we analyzed the bioenergetic status of breast cancer lymph node metastases, by employing a series of metabolic protein markers. For this purpose, we used MCT4 to identify glycolytic cells. Similarly, we used TO MM20 and COX staining as markers of mitochondrial mass and OXPHOS activity, respectively. Consistent with the “reverse Warburg effect,” our results indicate that metastatic breast cancer cells amplify oxidative mitochondrial metabolism (OXPHOS) and that adjacent stromal cells are glycolytic and lack detectable mitochondria. Glycolytic stromal cells included cancer-associated fibroblasts, adipocytes and inflammatory cells. Double labeling experiments with glycolytic (MCT4) and oxidative (TO MM20 or COX) markers directly shows that at least two different metabolic compartments co-exist, side-by-side, within primary tumors and their metastases. Since cancer-associated immune cells appeared glycolytic, this observation may also explain how inflammation literally “fuels” tumor progression and metastatic dissemination, by “feeding” mitochondrial metabolism in cancer cells. Finally, MCT4(+) and TO MM20(-) “glycolytic” cancer cells were rarely observed, indicating that the conventional “Warburg effect” does not frequently occur in cancer-positive lymph node metastases.     

Matrix remodeling stimulates stromal autophagy, “fueling” cancer cell mitochondrial metabolism and metastasis

We have previously demonstrated that loss of stromal caveolin-1 (Cav-1) in cancer-associated fibroblasts is a strong and independent predictor of poor clinical outcome in human breast cancer patients. However, the signaling mechanism(s) by which Cav-1 downregulation leads to this tumor-promoting microenvironment are not well understood. To address this issue, we performed an unbiased comparative proteomic analysis of wild-type (WT) and Cav-1^-/- null mammary stromal fibroblasts (MSFs). Our results show that plasminogen activator inhibitor type 1 and type 2 (PAI-1 and PAI-2) expression is significantly increased in Cav-1^-/- MSFs. To establish a direct cause-effect relationship, we next generated immortalized human fibroblast lines stably overexpressing either PAI-1 or PAI-2. Importantly, PAI-1/2(+) fibroblasts promote the growth of MDA-MB-231 tumors (a human breast cancer cell line) in a murine xenograft model, without any increases in angiogenesis. Similarly, PAI-1/2(+) fibroblasts stimulate experimental metastasis of MDA-MB-231 cells using an in vivo lung colonization assay. Further mechanistic studies revealed that fibroblasts overexpressing PAI-1 or PAI-2 display increased autophagy (“self-eating”) and are sufficient to induce mitochondrial biogenesis/activity in adjacent cancer cells, in co-culture experiments. In xenografts, PAI-1/2(+) fibroblasts significantly reduce the apoptosis of MDA-MB-231 tumor cells. The current study provides further support for the “Autophagic Tumor Stroma Model of Cancer” and identifies a novel “extracellular matrix”-based signaling mechanism, by which a loss of stromal Cav-1 generates a metastatic phenotype. Thus, the secretion and remodeling of extracellular matrix components (such as PAI-1/2) can directly regulate both (1) autophagy in stromal fibroblasts and (2) epithelial tumor cell mitochondrial metabolism.     

Ketone bodies and two-compartment tumor metabolism: Stromal ketone production fuels mitochondrial biogenesis in epithelial cancer cells

We have previously suggested that ketone body metabolism is critical for tumor progression and metastasis. Here, using a co-culture system employing human breast cancer cells (MCF7) and hTERT-immortalized fibroblasts, we provide new evidence to directly support this hypothesis. More specifically, we show that the enzymes required for ketone body production are highly upregulated within cancer-associated fibroblasts. This appears to be mechanistically controlled by the stromal expression of caveolin-1 (Cav-1) and/or serum starvation. In addition, treatment with ketone bodies (such as 3-hydroxy-butyrate, and/or butanediol) is sufficient to drive mitochondrial biogenesis in human breast cancer cells. This observation was also validated by unbiased proteomic analysis. Interestingly, an MCT1 inhibitor was sufficient to block the onset of mitochondrial biogenesis in human breast cancer cells, suggesting a possible avenue for anticancer therapy. Finally, using human breast cancer tumor samples, we directly confirmed that the enzymes associated with ketone body production (HMGCS2, HMGCL and BDH1) were preferentially expressed in the tumor stroma. Conversely, enzymes associated with ketone re-utilization (ACAT1) and mitochondrial biogenesis (HSP60) were selectively associated with the epithelial tumor cell compartment. Our current findings are consistent with the “two-compartment tumor metabolism” model. Furthermore, they suggest that we should target ketone body metabolism as a new area for drug discovery, for the prevention and treatment of human cancers.     

Ketone bodies and two-compartment tumor metabolism: Stromal ketone production fuels mitochondrial biogenesis in epithelial cancer cells

We have previously suggested that ketone body metabolism is critical for tumor progression and metastasis. Here, using a co-culture system employing human breast cancer cells (MCF7) and hTERT-immortalized fibroblasts, we provide new evidence to directly support this hypothesis. More specifically, we show that the enzymes required for ketone body production are highly upregulated within cancer-associated fibroblasts. This appears to be mechanistically controlled by the stromal expression of caveolin-1 (Cav-1) and/or serum starvation. In addition, treatment with ketone bodies (such as 3-hydroxy-butyrate, and/or butanediol) is sufficient to drive mitochondrial biogenesis in human breast cancer cells. This observation was also validated by unbiased proteomic analysis. Interestingly, an MCT1 inhibitor was sufficient to block the onset of mitochondrial biogenesis in human breast cancer cells, suggesting a possible avenue for anticancer therapy. Finally, using human breast cancer tumor samples, we directly confirmed that the enzymes associated with ketone body production (HMGCS2, HMGCL and BDH1) were preferentially expressed in the tumor stroma. Conversely, enzymes associated with ketone re-utilization (ACAT1) and mitochondrial biogenesis (HSP60) were selectively associated with the epithelial tumor cell compartment. Our current findings are consistent with the “two-compartment tumor metabolism” model. Furthermore, they suggest that we should target ketone body metabolism as a new area for drug discovery, for the prevention and treatment of human cancers.     

Metabolic coupling in urothelial bladder cancer compartments and its correlation to tumor aggressiveness

Monocarboxylate transporters (MCTs) are vital for intracellular pH homeostasis by extruding lactate from highly glycolytic cells. These molecules are key players of the metabolic reprogramming of cancer cells, and evidence indicates a potential contribution in urothelial bladder cancer (UBC) aggressiveness and chemoresistance. However, the specific role of MCTs in the metabolic compartmentalization within bladder tumors, namely their preponderance on the tumor stroma, remains to be elucidated. Thus, we evaluated the immunoexpression of MCTs in the different compartments of UBC tissue samples (n = 111), assessing the correlations among them and with the clinical and prognostic parameters. A significant decrease in positivity for MCT1 and MCT4 occurred from normoxic toward hypoxic regions. Significant associations were found between the expression of MCT4 in hypoxic tumor cells and in the tumor stroma. MCT1 staining in normoxic tumor areas, and MCT4 staining in hypoxic regions, in the tumor stroma and in the blood vessels were significantly associated with UBC aggressiveness. MCT4 concomitant positivity in hypoxic tumor cells and in the tumor stroma, as well as positivity in each of these regions concomitant with MCT1 positivity in normoxic tumor cells, was significantly associated with an unfavourable clinicopathological profile, and predicted lower overall survival rates among patients receiving platinum-based chemotherapy. Our results point to the existence of a multi-compartment metabolic model in UBC, providing evidence of a metabolic coupling between catabolic stromal and cancer cells’ compartments, and the anabolic cancer cells. It is urgent to further explore the involvement of this metabolic coupling in UBC progression and chemoresistance.     

Oncogenes and inflammation rewire host energy metabolism in the tumor microenvironment

Here, we developed a model system to evaluate the metabolic effects of oncogene(s) on the host microenvironment. A matched set of “normal” and oncogenically transformed epithelial cell lines were co-cultured with human fibroblasts, to determine the “bystander” effects of oncogenes on stromal cells. ROS production and glucose uptake were measured by FACS analysis. In addition, expression of a panel of metabolic protein biomarkers (Caveolin-1, MCT1, and MCT4) was analyzed in parallel. Interestingly, oncogene activation in cancer cells was sufficient to induce the metabolic reprogramming of cancer-associated fibroblasts toward glycolysis, via oxidative stress. Evidence for “metabolic symbiosis” between oxidative cancer cells and glycolytic fibroblasts was provided by MCT1/4 immunostaining. As such, oncogenes drive the establishment of a stromal-epithelial “lactate-shuttle”, to fuel the anabolic growth of cancer cells. Similar results were obtained with two divergent oncogenes (RAS and NFκB), indicating that ROS production and inflammation metabolically converge on the tumor stroma, driving glycolysis and upregulation of MCT4. These findings make stromal MCT4 an attractive target for new drug discovery, as MCT4 is a shared endpoint for the metabolic effects of many oncogenic stimuli. Thus, diverse oncogenes stimulate a common metabolic response in the tumor stroma. Conversely, we also show that fibroblasts protect cancer cells against oncogenic stress and senescence by reducing ROS production in tumor cells. Ras-transformed cells were also able to metabolically reprogram normal adjacent epithelia, indicating that cancer cells can use either fibroblasts or epithelial cells as “partners” for metabolic symbiosis. The antioxidant N-acetyl-cysteine (NAC) selectively halted mitochondrial biogenesis in Ras-transformed cells, but not in normal epithelia. NAC also blocked stromal induction of MCT4, indicating that NAC effectively functions as an “MCT4 inhibitor”. Taken together, our data provide new strategies for achieving more effective anticancer therapy. We conclude that oncogenes enable cancer cells to behave as selfish “metabolic parasites”, like foreign organisms (bacteria, fungi, viruses). Thus, we should consider treating cancer like an infectious disease, with new classes of metabolically targeted “antibiotics” to selectively starve cancer cells. Our results provide new support for the “seed and soil” hypothesis, which was first proposed in 1889 by the English surgeon, Stephen Paget.     

Ethanol exposure induces the cancer-associated fibroblast phenotype and lethal tumor metabolism

Little is known about how alcohol consumption promotes the onset of human breast cancer(s). One hypothesis is that ethanol induces metabolic changes in the tumor microenvironment, which then enhances epithelial tumor growth. To experimentally test this hypothesis, we used a co-culture system consisting of human breast cancer cells (MCF7) and hTERT-immortalized fibroblasts. Here, we show that ethanol treatment (100 mM) promotes ROS production and oxidative stress in cancer-associated fibroblasts, which is sufficient to induce myofibroblastic differentiation. Oxidative stress in stromal fibroblasts also results in the onset of autophagy/mitophagy, driving the induction of ketone body production in the tumor microenvironment. Interestingly, ethanol has just the opposite effect in epithelial cancer cells, where it confers autophagy resistance, elevates mitochondrial biogenesis and induces key enzymes associated with ketone re-utilization (ACAT1/OXCT1). During co-culture, ethanol treatment also converts MCF7 cells from an ER(+) to an ER(-) status, which is thought to be associated with “stemness,” more aggressive behavior and a worse prognosis. Thus, ethanol treatment induces ketone production in cancer-associated fibroblasts and ketone re-utilization in epithelial cancer cells, fueling tumor cell growth via oxidative mitochondrial metabolism (OXPHOS). This “two-compartment” metabolic model is consistent with previous historical observations that ethanol is first converted to acetaldehyde (which induces oxidative stress) and then ultimately to acetyl-CoA (a high-energy mitochondrial fuel), or can be used to synthesize ketone bodies. As such, our results provide a novel mechanism by which alcohol consumption could metabolically convert “low-risk” breast cancer patients to “high-risk” status, explaining tumor recurrence or disease progression. Hence, our findings have clear implications for both breast cancer prevention and therapy. Remarkably, our results also show that antioxidants [such as N-acetyl cysteine (NAC)] can effectively reverse or prevent ethanol-induced oxidative stress in cancer-associated fibroblasts, suggesting a novel strategy for cancer prevention. We also show that caveolin-1 and MCT4 protein expression can be effectively used as new biomarkers to monitor oxidative stress induced by ethanol.     

Metabolic strategies of melanoma cells: Mechanisms,interactions with the tumor microenvironment,and therapeutic implications

Melanomas are metabolically heterogeneous, and they are able to adapt in order to utilize a variety of fuels that facilitate tumor progression and metastasis. The significance of metabolism in melanoma is supported by growing evidence of impact on the efficacy of contemporary therapies for this disease. There are also data to support that the metabolic phenotypes of melanoma cells depend upon contributions from both intrinsic oncogenic pathways and extrinsic factors in the tumor microenvironment. This review summarizes current understanding of the metabolic processes that promote cutaneous melanoma tumorigenesis and progression, the regulation of cancer cell metabolism by the tumor microenvironment, and the impact of metabolic pathways on targeted and immune therapies.