This article was downloaded by: [183.250.91.47] On: 26 August 2015, At: 23:42 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: 5 Howick Place, London, SW1P 1WG
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Metabolic reprogramming and two-compartment tumor metabolism ab
Barbara Chiavarina , Ubaldo E. Martinez-Outschoorn d
d
Howell , Herbert B. Tanowitz , Richard G. Pestell
abc
abc
ab
, Diana Whitaker-Menezes , Anthony abd
, Federica Sotgia
& Michael P.
abcd
Lisanti a
The Jefferson Stem Cell Biology and Regenerative Medicine Center; Kimmel Cancer Center; Thomas Jefferson University; Philadelphia, PA USA b
Departments of Stem Cell Biology & Regenerative Medicine and Cancer Biology; Kimmel Cancer Center; Thomas Jefferson University; Philadelphia, PA USA c
Department of Medical Oncology; Kimmel Cancer Center; Thomas Jefferson University; Philadelphia, PA USA d
Manchester Breast Centre & Breakthrough Breast Cancer Research Unit; Paterson Institute for Cancer Research; School of Cancer, Enabling Sciences and Technology; Manchester Academic Health Science Centre; University of Manchester; Manchester, UK Published online: 16 Aug 2012.
To cite this article: Barbara Chiavarina, Ubaldo E. Martinez-Outschoorn, Diana Whitaker-Menezes, Anthony Howell, Herbert B. Tanowitz, Richard G. Pestell, Federica Sotgia & Michael P. Lisanti (2012) Metabolic reprogramming and two-compartment tumor metabolism, Cell Cycle, 11:17, 3280-3289, DOI: 10.4161/cc.21643 To link to this article: http://dx.doi.org/10.4161/cc.21643
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Metabolic reprogramming and two-compartment tumor metabolism Opposing role(s) of HIF1α and HIF2α in tumor-associated fibroblasts and human breast cancer cells
1 The Jefferson Stem Cell Biology and Regenerative Medicine Center; Kimmel Cancer Center; Thomas Jefferson University; Philadelphia, PA USA; 2Departments of Stem Cell Biology & Regenerative Medicine and Cancer Biology; Kimmel Cancer Center; Thomas Jefferson University; Philadelphia, PA USA; 3Department of Medical Oncology; Kimmel Cancer Center; Thomas Jefferson University; Philadelphia, PA USA; 4Manchester Breast Centre & Breakthrough Breast Cancer Research Unit; Paterson Institute for Cancer Research; School of Cancer, Enabling Sciences and Technology; Manchester Academic Health Science Centre; University of Manchester; Manchester, UK
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Keywords: caveolin-1, hypoxia-inducible factor, HIF-1a, HIF-2a, metabolic coupling, tumor stroma, cancer-associated fibroblasts, aerobic glycolysis, mitochondrial metabolism, OXPHOS Abbreviations: Cav-1, caveolin-1; COX, cytochrome C oxidase; HIF, hypoxia-inducible factor; MCT, monocarboxylate transporter; VHL, von Hippel-Lindau
Hypoxia-inducible factor (HIF) 1α and 2α are transcription factors responsible for the cellular response to hypoxia. The functional roles of HIF1α and HIF2α in cancer are distinct and vary among different tumor types. The aim of this study was to evaluate the compartment-specific role(s) of HIF1α and HIF2α in breast cancer. To this end, immortalized human fibroblasts and MDA-MB-231 breast cancer cells carrying constitutively active HIF1α or HIF2α mutants were analyzed with respect to their metabolic function(s) and ability to promote tumor growth in an in vivo setting. We observed that activation of HIF1α, but not HIF2α, in stromal cells promotes a shift toward aerobic glycolysis, with increased L-lactate production and a loss of mitochondrial activity. In a xenograft model, HIF1α-activated fibroblasts promoted the tumor growth of co-injected MDA-MB-231 cells without an increase in angiogenesis. Conversely, HIF2α-activated stromal cells did not favor tumor growth and behaved as the empty vector controls. Similarly, activation of HIF1α, but not HIF2α, in MDA-MB-231 cells promoted a shift toward aerobic glycolysis, with increased glucose uptake and L-lactate production. In contrast, HIF2α activation in cancer cells increased the expression of EGFR, Ras and cyclin D1, which are known markers of tumor growth and cell cycle progression. In a xenograft model, HIF1α activation in MDA-MB-231 cells acted as a tumor suppressor, resulting in an almost 2-fold reduction in tumor mass and volume. Interestingly, HIF2α activation in MDAMB-231 cells induced a significant ~2-fold-increase in tumor mass and volume. Analysis of mitochondrial activity in these tumor xenografts using COX (cytochrome C oxidase) staining demonstrated elevated mitochondrial oxidative metabolism (OXPHOS) in HIF2α-tumors. We conclude that the role(s) of HIF1α and HIF2α in tumorigenesis are compartment-specific. HIF1α acts as a tumor promoter in stromal cells but as a tumor suppressor in cancer cells. Conversely, HIF2α is a tumor promoter in cancer cells. Mechanistically, HIF1α-driven aerobic glycolysis in stromal cells supports cancer cell growth via the paracrine production of nutrients (such as L-lactate) that can “feed” cancer cells. However, HIF1α-driven aerobic glycolysis in cancer cells inhibits tumor growth. Finally, HIF2α activation in cancer cells induces the expression of known pro-oncogenic molecules and promotes the mitochondrial activity of cancer cells.
Introduction Most solid tumors contain poorly oxygenated regions, as compared with normal tissues. Tumor hypoxia is typically associated with changes in metabolism, neo-vascularization, invasion, metastasis, drug resistance and, ultimately, poor clinical outcome. The transcription factor primarily responsible for the cellular responses to hypoxia is the hypoxia-inducible factor (HIF). HIF
is a hetero-dimer formed by the oxygen-regulated and growthfactor-sensitive α subunit and the constitutively expressed β subunit. Under normoxic conditions, the α subunit forms a complex with the von Hippel-Lindau (VHL) protein, which mediates its ubiquitination and continuous degradation by the proteasome. However, under hypoxic conditions, the α subunit is stabilized and translocates to the nucleus, where it dimerizes with the β subunit and activates the transcription of highly specific target genes.
*Correspondence to: Federica Sotgia and Michael P. Lisanti; Email:
[email protected] and
[email protected] Submitted: 07/17/12; Accepted: 07/26/12 http://dx.doi.org/10.4161/cc.21643 3280
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Barbara Chiavarina,1,2 Ubaldo E. Martinez-Outschoorn,1,2,3 Diana Whitaker-Menezes,1,2 Anthony Howell,4 Herbert B. Tanowitz,4 Richard G. Pestell,1,2,3 Federica Sotgia1,2,4,* and Michael P. Lisanti1,2,3,4,*
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There are three isoforms of the α subunit, HIF1α, HIF2α or HIF3α, and one β subunit, HIF1β. HIF1α is ubiquitously expressed, whereas HIF2α expression is more restricted to particular cell types, including endothelial cells and subsets of cells in the kidney, brain, heart, lung, liver, pancreas and small intestine. Compared with HIF1α and HIF2α, relatively little data are available regarding the biological function(s) and localization of HIF3α. While it is believed that HIF1α and HIF2α share several functions, it is also now clear that HIF1α and HIF2α can regulate distinct processes. For example, HIF1α appears to preferentially induce the expression of glycolytic enzymes,1 and to repress mitochondrial function and electron transport chain activity.2,3 In contrast, HIF2α preferentially activates angiogenesis, inducing the expression of VEGF and its receptor Flt-1,4,5 and other pro-oncogenic molecules, including EGFR, cyclin D1, Oct-4 and c-Myc.6,7 Recent studies have shown that HIF2α, but not HIF1α, promotes tumor growth in xenograft models. In animal models, it has been shown that HIF2α activation in cancer cells8 or HIF1α replacement by HIF2α9,10 favors aggressive tumor growth and invasion, whereas overexpression of stable HIF1α inhibits tumor growth.8 However, the role of HIF1α and HIF2α in the tumor stroma is still largely unexplored. Recently, we and others have shown that a loss of caveolin-1 (Cav-1) in the tumor stroma is a predictor of early tumor recurrence, lymph-node metastasis, tamoxifen resistance and poor clinical outcome in breast cancer patients. Importantly, the predictive value of stromal Cav-1 is independent of epithelial marker status and is valid in all the different subtypes of invasive ductal carcinoma (IDC).11,12 In a coculture system of breast cancer cells (MCF7) and human-immortalized fibroblasts, we observed that tumor cells promote the conversion of fibroblasts to cancer-associated fibroblasts, by inducing oxidative stress, resulting in mitochondrial dysfunction and increased aerobic glycolysis in the stromal compartment.13,14 Oxidative stress in fibroblasts induces the autophagy-mediated downregulation of Cav-1 and the activation of HIF1α and NFkB.15 We have also shown that acute loss of Cav-1 in fibroblasts is sufficient to increase the expression of HIF1α, resulting in a switch toward aerobic glycolysis.15 To evaluate whether HIF1α activation in cancer-associated fibroblasts is sufficient to promote tumor growth, we stably expressed wild-type (WT) and an activated mutant of HIF1α in human-immortalized fibroblasts.16 We observed that, relative to control cells, HIF1α activation in fibroblasts induced autophagy/mitophagy, with a consequent loss of mitochondrial activity and a shift toward aerobic glycolysis, driving increased L-lactate production. During coculture, HIF1α-activated fibroblasts promoted the mitochondrial activity of adjacent tumor cells (MDA-MB-231 breast cancer cells). We hypothesized that HIF1α activation in stromal cells triggers stromal-epithelial metabolic coupling, providing the energy necessary to fuel tumor growth. In direct support of this hypothesis, in a xenograft model, HIF1αactivated fibroblasts promoted the tumor growth of co-injected MDA-MB-231 cells, without an increase in angiogenesis.16
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We also evaluated the effects of HIF1α activation directly in MDA-MB-231 cells. In vivo, HIF1α activation in breast cancer cells resulted in a significant reduction in tumor mass and volume.16 Based on these studies, we proposed that the downstream effects of HIF1α activation on tumorigenesis are compartmentspecific. In cancer-associated fibroblasts, HIF1α activation functions as a tumor promoter, via the paracrine secretion of recycled nutrients and L-lactate, fueling mitochondrial activity and growth in cancer cells. In contrast, HIF1α activation in cancer cells acts as a tumor suppressor, via their autophagy-mediated self-digestion. Recently, using cytochrome C oxidase (COX, mitochondrial complex IV) activity staining in human samples derived from breast cancer patients, we have demonstrated that the mitochondrial activity is selectively enhanced in epithelial cancer cells and severely decreased in the surrounding tumor stroma.17 Thus, two distinct metabolic “comparments” co-exist, side-by-side, in human tumors, resulting in glycolyic-oxidative metabolic coupling between the two compartments. The aim of the current study was to define the compartmentspecific role of HIF1α and HIF2α activation in cancer-associated fibroblasts and breast cancer cells. To this end, human-immortalized fibroblasts and MDA-MB-231 cells were stably transfected with well-established mutants of HIF1α or HIF2α, which cannot be degraded, and are, thus, constitutively active. Here, we show that HIF1α activation in cancer-associated fibroblasts promotes tumor growth by an angiogenesis-independent mechanism; conversely, HIF2α activation in stromal cells did not exert any effects, relative to the empty vector control. Interestingly, HIF1α activation in tumor cells inhibited tumor growth, whereas HIF2α activation in cancer cells acted as a tumor promoter. Thus, we propose that the tumor-promoting and tumor-suppressing effects of HIF1α and HIF2α are compartment- and cell type- specific, reflecting the existence of “two-compartment tumor metabolism.” Results HIF1α-activated fibroblasts show downregulation of Cav-1 expression. We previously demonstrated that HIF1α activation in human fibroblasts is sufficient to induce the downregulation of Cav-1 expression levels. We also reported that in xenograft models, HIF1α activation in stromal fibroblasts functions as a tumor promoter, but HIF1α activation in breast cancer cells functions as a tumor suppressor. However, certain tumors are known to express HIF2α. The compartment-specific role(s) of HIF1α and HIF2α activation in modulating tumor growth remains to be elucidated. To study the effects of HIF1α and HIF2α activation in the tumor stroma, we stably overexpressed a mutant form of HIF1α or HIF2α in human-immortalized fibroblasts (hTERT-BJ1 cells) (Fig. 1A and B). These mutants are constitutively stabilized under normoxia because of proline to alanine substitutions at the VHL binding site, which are critical for their effective ubiquitination and degradation. First, we examined the expression levels of Cav-1, because a loss of Cav-1 in fibroblasts is a marker of an activated tumor stroma.
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Figure 1. Activation of HIF1α, but not HIF2α, decreases Cav-1 expression in fibroblasts. (A and B) hTERT-BJ1 fibroblasts were stably transfected with plasmids carrying the cDNA for HIF1α or HIF2α mutants (in pBabe vector). These mutants stabilize HIF1α and HIF2α proteins, leading to their constitutive expression and activation. After selection with puromycin, cells were subjected to immunoblot analysis to confirm HIF1α (A) and HIF2α (B) constitutive expression, relative to pBabe empty vector controls. Immunoblotting with β-actin served as equal loading control. (C) Fibroblasts harboring activated HIF1α showed a dramatic downregulation of Cav-1 levels. In contrast, HIF2α-activated fibroblasts have the same Cav-1 protein levels as the empty vector control. Two different antibodies against Cav-1 were employed. Equal loading was assessed by immunoblotting with β-actin.
decrease in angiogenesis in HIF1α-tumors (Fig. 3B), indicating that the tumor-promoting effects of HIF1α are not dependent on angiogenesis. Activation of HIF1α, but not HIF2α, in human breast cancer cells promotes a shift toward aerobic glycolysis, with increased glucose uptake and L-lactate release. To study the compartment-specific effects of HIF1α and HIF2α activation on breast cancer cell growth, we directly expressed the same stable mutants of HIF1α or HIF2α in MDA-MB-231 breast cancer cells (Fig. 4A and B). First, we assessed the metabolic status of HIF1α- and HIF2αactivated MDA-MB-231 cells. Our in vitro results show that HIF1α activation in MDA-MB-231 cells drives a shift toward aerobic glycolysis, with an increase of glucose uptake (Fig. 4C) and lactate production (Fig. 4D). Conversely, HIF2α activation in breast cancer cells is not associated with aerobic glycolysis and does not promote glucose uptake (Fig. 4C) or lactate production (Fig. 4D). Thus, activation of HIF1α, but not HIF2α, in breast cancer cells induces the classical “Warburg effect.” HIF2α activation in breast cancer cells increases the expression of proteins involved in proliferation and cell cycle progression. We next asked if HIF1α or HIF2α activation in MDA-MB-231 cells induces the expression of markers of proliferation and cell cycle progression.
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Figure 1C shows that only HIF1α, but not HIF2α, activation induces the downregulation of Cav-1 levels in human fibroblasts. Activation of HIF1α, but not HIF2α, in fibroblasts induces a loss of mitochondrial activity, with a shift toward aerobic glycolysis. To examine the metabolic effects of HIF1α and HIF2α activation in stromal cells, we first assessed the mitochondrial activity of HIF1α- and HIF2α-activated fibroblasts, by staining with a specific mitochondrial dye, namely MitoTracker. MitoTracker only labels functional mitochondria with an active membrane potential and is, thus, a measure of mitochondrial activity. Figure 2A shows that only HIF1α activation in fibroblasts induces a significant decrease in mitochondrial activity, consistent with a shift toward aerobic glycolysis. HIF2α-activated fibroblasts showed similar mitochondrial activity as control cells. Next, we measured L-lactate release into the cell culture media from control, HIF1α- and HIF2α-activated fibroblasts. Figure 2B shows that HIF1α-activated fibroblasts display a 2-fold increase in L-lactate generation. Conversely, HIF2α-activated fibroblasts display a slight, but significant, decrease in L-lactate levels relative to control cells. These data indicate that only HIF1α-activated fibroblasts show a shift toward aerobic glycolysis, with increased lactate production. We also evaluated the expression levels of known lactate transporters, such as monocarboxylate transporter MCT1 and MCT4, by immunoblotting with specific antibody probes. MCT4 mediates lactate efflux from cells that rely on glycolysis for ATP production, whereas MCT1 is the transporter responsible for lactate uptake. Interestingly, HIF1α-activated fibroblasts show the upregulation of MCT4 and the downregulation of MCT1, indicating that activation of HIF1α induces L-lactate efflux out of the fibroblasts (Fig. 2C). Conversely, HIF2α-activated fibroblasts robustly express both MCT4 and MCT1 (Fig. 2C), indicating that the lactate flux is not unidirectional in HIF2α-activated fibroblasts, and that lactate can be extruded, but can also re-enter the cells, to be used as substrate for mitochondrial respiration in HIF2α-activated fibroblasts. Activation of HIF1α, but not HIF2α, in fibroblasts promotes tumor growth in vivo. To determine the in vivo role of HIF1α and HIF2α activation in the tumor stroma, we employed a humanized xenograft model consisting of fibroblasts (control, HIF1α- or HIF2α-activated) co-injected with MDA-MB-231 breast cancer cells into the flanks of nude mice. Tumor weight and volume were assessed at 4 wk post-injection. Figure 3A shows that only HIF1α-activated fibroblasts promote tumor growth, resulting in a 2.4-fold increase in tumor mass and volume. This is consistent with the idea that HIF1α-driven aerobic glycolysis in the tumor stromal compartment promotes tumor growth, by producing nutrients (such as L-lactate) that can feed cancer cells. However, as expected from our in vitro results, HIF2αactivated fibroblasts do not promote tumor growth (Fig. 3A). Thus, HIF2α activation in cancer-associated fibroblasts exerts a completely different effect than HIF1α on tumorigenesis in vivo. To evaluate if the tumor-promoting effects of HIF1αactivated fibroblasts are due to increased tumor angiogenesis, frozen tumor sections were immunostained with anti-antibodies directed against CD31, an endothelial cell marker. Surprisingly, vessel density quantification shows a slight but significant
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Discussion In this study, we have used a compartment-specific approach to evaluate the role of HIF1α and HIF2α activation in a model of breast cancer. Here, we show that HIF1α and HIF2α have
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Figure 2. HIF1α, but not HIF2α, activation induces aerobic glycolysis and decreases mitochondrial activity in fibroblasts. (A) MitoTracker staining (red) was used to visualize mitochondrial activity. Nuclei were stained with Dapi (blue). MitoTracker accumulates in functional mitochondria with an active membrane potential, thus correlating with mitochondrial activity. Note that fibroblasts harboring activated HIF1α show reduced MitoTracker staining, consistent with a shift toward aerobic glycolysis. Conversely, fibroblasts harboring activated HIF2α show similar MitoTracker staining as the empty vector control cells. (B) Empty vector control, HIF1α- or HIF2α-activated fibroblasts were grown for 48 h in media with 2% FBS. Then, the media was collected and subjected to a biochemical analysis to determine the concentration of lactate. The graph shows that HIF1α-activated fibroblasts secrete 2-fold more lactate than empty vector control cells. Conversely, HIF2α overexpressing fibroblasts show a 1.5-fold reduction in lactate production, as compared with control cells. p values, Student t-test. (C) Immunoblotting was performed on control, HIF1α- and HIF2α-fibroblasts with antibodies against MCT4 (the transporter for lactate efflux) and MCT1 (the transporter for lactate uptake). HIF1α-activated fibroblasts show MCT4 upregulation, and MCT1 downregulation, relative to control cells, consistent with increased lactate production. Conversely, HIF2α-activated fibroblasts show robust expression of both MCT1 and MCT4, suggesting that the lactate flux is not unidirectional in HIF2α-activated cells. Equal loading was assessed by immunoblotting with β-actin.
Figure 5 shows that HIF2α activation in cancer cells increases EGFR and Ras expression, which are known to be critical for tumor growth, and also promotes the expression of cyclin D1, a positive regulator of cell cycle progression. These results are consistent with previous studies showing a critical role for HIF2α in promoting cellular proliferation, by regulating the expression of genes involved in cell cycle control and progression. Conversely, HIF1α activation in MDA-MB-231 cancer cells does not induce the expression of EGFR and Ras, but it suppresses the expression of PI3K (Fig. 5), indicating that HIF1α activation in cancer cells may inhibit cell survival. Role of HIF1α and HIF2α activation in breast cancer cells in vivo: HIF1α inhibits tumor growth, while HIF2α promotes tumor growth. To study the in vivo effects of HIF1α and HIF2α activation in human breast cancer cells, MDA-MB-231 cells harboring activated HIF1α or HIF2α were injected into the flanks of nude mice. Mice were sacrificed at 2 wk post-injection. Figure 6A shows that the activation of HIF1α in breast cancer cells inhibits tumor growth, resulting in a 1.8-fold decrease in tumor weight and 1.9-fold decrease in tumor volume. Conversely, HIF2α activation in cancer cells drives a significant increase in both tumor weight (1.6-fold) and volume (1.9-fold). CD31 immunostaining on frozen sections derived from these xenograft tumors shows that activation of both HIF1α and HIF2α in breast cancer cells promotes a slight, but significant, increase in angiogenesis (23% and 15%, respectively, for HIF1α and HIF2α). Thus, we conclude that the tumor promoting effects of HIF2α activation in cancer cells is likely independent of angiogenesis (Fig. 6B). HIF2α activation in cancer cells promotes oxidative metabolism in cancer cells. To evaluate the impact of HIF1α and HIF2α activation on mitochondrial respiration, we performed COX (mitochondrial complex IV) activity staining on frozen tumor sections. COX staining detects the in situ activity of mitochondrial oxidative phosphorylation. Figure 7 shows that COX activity is sustained in tumor xenografts derived from control pBabe-MDA-MB-231 cells. However, in tumors derived from HIF1α-activated MDA-MB-231 cells, COX activity is selectively found in the tumor stromal compartment, but is low in cancer cells. Conversely, in tumor sections derived from HIF2αactivated MDA-MB-231 cells, COX activity is highly elevated within the cancer cells. These findings suggest that HIF2α activation promotes oxidative metabolism in cancer cells, and that may drive aggressive tumor growth. Thus, we conclude that HIF1α-expression in cancer cells induces the Warburg effect in vivo, by driving mitochondrial dysfunction, resulting in decreased tumor growth. However, HIF2α-expression in cancer cells has just the opposite effect; instead, HIF2α-expression increases mitochondrial OXPHOS, leading to enhanced tumor growth.
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distinct function(s) in the pathogenesis of breast cancer, and their effects are compartmentspecific (Fig. 8). In fact, we show that HIF1α acts as a tumor promoter when activated in the tumor stroma, but acts as a tumor suppressor in breast cancer cells. Conversely, HIF2α activation in cancer-associated fibroblasts does not affect tumor growth, while HIF2α activation in epithelial tumor cells functions as a tumor promoter. Role of HIF1α and HIF2α in the tumor microenvironment. We demonstrate that HIF1α activation in stromal cells induces stromal aerobic glycolysis (the “reverse Warburg effect”). Fibroblasts expressing stable HIF1α display increased L-lactate secretion and increased MCT4 protein levels. MCT4 is a HIF1α target gene that mediates the efflux of L-lactate from cells that largely rely on aerobic glycolysis to produce energy and lack functional mitochondria.18 We propose that energy-rich metabolites, like lactate and pyruvate, are secreted by glycolytic fibroblasts and utilized by adjacent cancer cells to support their oxidative metabolism. This stromal-epithelial metabolic coupling provides the energy necessary to support tumor growth.19,20 Consistent with this hypothesis, we have recently demonstrated that enhancement of stromal glycolysis via the overexpression of the glycolytic enzymes pyruvate kinase M1 and M2 (PKM1 and PKM2) in stromal cells, is sufficient to promote tumor growth.21 In addition, silencing Figure 3. Fibroblasts harboring activated HIF1α, but not HIF2α, promote breast cancer of stromal TFAM, a key transcription factor of tumor growth, without increases of angiogenesis. (A) pBabe-control, HIF1α- or HIF2αmitochondrial genes, impairs mitochondrial oxifibroblasts were co-injected with MDA-MB-231 cells in the flanks of nude mice. After 4 wk, the tumors were harvested. Note that HIF1α-activated fibroblasts induce a 2.4-fold dative phosphorylation (OXPHOS), increases increase of tumor mass and volume, relative to control fibroblasts. However, activation of L-lactate generation and sustains the growth HIF2α in fibroblasts does not promote tumor growth. The indicated p values are relative to 22 of breast cancer cells in a xenograft model. the pBabe control group. (B) Quantification of tumor angiogenesis. Frozen sections from Finally, elevated stromal expression of MCT4 xenograft tumors were immuno-stained with anti-CD31 antibodies, and vessel density is a powerful biomarker that predicts poor sur(vessels per field) was quantitated. The graph shows that HIF1α-tumors have a 20% decrease in vessel density, suggesting that the tumor promoting effects of HIF1α-activated vival in human triple-negative breast cancers, fibroblasts are independent of angiogenesis. * p = 0.007. whereas expression of MCT4 in epithelial cancer 23 cells does not have any prognostic value. These results indicate that stromal aerobic glycolysis is a driver of aggressive tumor growth, whereas aerobic glycolysis Interestingly, the activation of HIF2α in cancer-associated in epithelial cancer cells does not exert any significant effects on fibroblasts did not induce significant changes relative to the tumorigenesis. empty vector control, both in terms of glycolysis induction in Energy transfer from glycolytic stromal cells to epithelial can- vitro and tumor growth in vivo. Our findings are consistent with cer cells closely resembles physiological processes of metabolic a recent study showing that expression of HIF1α, but not HIF2α, cooperativity, such as in “neuron-glia metabolic coupling” in the in the tumor microenvironment is required for tumor growth brain, and the “lactate shuttle” in the skeletal muscle. Activation and metastasis. Implantation of B16F10 melanoma cells in mice of glycolysis in astrocytes and MCT-mediated transfer of lactate with a monocyte/macrophage-selective deletion of HIF1α or to neurons supports neuron mitochondrial oxidative phosphory- HIF2α shows that tumor growth and metastatic dissemination lation and energy demands.24 Similarly, lactate generation in to the lung is greatly inhibited in HIF1α (-/-) null background. the MCT4-positive-fast-twitch muscle fibers (glycolytic fibers) However, no effects on tumor growth and metastasis formation sustains mitochondrial respiration in the MCT1-positive-slow- were observed in HIF2α (-/-) null background, relative to control twitch muscle fibers (oxidative fibers).25 animals.26
Very few studies have evaluated the prognostic value of HIF1α and HIF2α overexpression in the tumor stroma. In one study in hepatocellular carcinoma, patients with elevated HIF1α in the adjacent non-malignant liver tissue had a significantly poorer
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Figure 4. Activation of HIF1α, but not HIF2α, induces glycolysis in human breast cancer cells. (A and B) To evaluate the possible compartment-specific effects of HIF1α or HIF2α activation on breast cancer tumor growth, we stably expressed a mutationally activated form of HIF1α or HIF2α in MDA-MB-231 cells. Cells were analyzed by western blot to confirm the overexpression of HIF1α (A) or HIF2α (B). Equal loading was assessed by immunoblotting with β-actin. (C-D) MDA-pBabe, MDA-HIF1α or MDA-HIF2α cells were serum-starved for 2 h, and then treated with 1 mM metformin or vehicle alone for 16 h. Then, cells were analyzed for glucose uptake (C) or lactate release (D). (C) Glucose uptake was performed using radiolabeled 3H-glucose. The graph shows that HIF1α-activated breast cancer cells have 2.5-fold increase in glucose uptake, as compared with vector alone and HIF2α-cells. (D) Lactate assay. Cell culture media was subjected to a biochemical analysis to determine the lactate concentration. Note that HIF1α-activated MDA-MB-231 cells display a 1.7-fold increase in lactate production, as compared with vector alone and HIF2α-cells, indicating a shift toward aerobic glycolysis. For both panels 4C and 4D, results are expressed as ratio between metformin-treated vs. vehicle-treated cells. Significance was evaluated using the Mann-Whitney test.
prognosis, relative to patients with low HIF1α.27 In conclusion, it appears that HIF1α, but not HIF2α, activation plays an important pro-tumorogenic role in the tumor microenvironment. Role of HIF1α and HIF2α in the epithelial tumor compartment. Our data show that HIF1α activation in epithelial tumor cells induces a shift toward aerobic glycolysis, with increased glucose uptake and L-lactate production (the classical Warburg effect), with weak mitochondrial activity in cancer cells, as judged by COX activity staining on HIF1α-tumor xenografts. In vivo, HIF1α activation strongly inhibits tumor growth, indicating that the classical Warburg effect does not support tumor growth. Conversely, the overexpression of stable HIF2α in MDA-MB-231 cells induces a significant increase in tumor growth in vivo. Mechanistically, the activation of HIF2α in cancer cells increases the expression of EGFR and Ras, which are known to be critical for tumor growth, and of cyclin D1, an important positive regulator of cell cycle progression. Moreover, assessment of mitochondrial activity in situ in HIF2α-tumor xenograft samples shows highly elevated levels of COX staining in cancer cells, but not in stromal compartment. This suggests that HIF2α activation in cancer cells promotes mitochondrial oxidative metabolism. Similarly, we have shown that hyper-activation of oxidative mitochondrial metabolism (OXPHOS) in epithelial cancer cells is a common feature of human invasive breast cancers.17 Our findings are consistent with recent studies showing that HIF2α is required for proper mitochondrial function. Analysis of HIF2α (-/-) null animals has shown multiple-organ pathology, with mitochondrial abnormalities and degeneration, increased lactate and ketone levels, altered Krebs cycle function and dysregulated fatty acid β-oxidation.28 Notably, HIF2α is a PGC1α target gene, the key transcription factor for positively regulating mitochondrial gene expression, and is required for the switch from glycolytic muscle fibers (fast-twitch) to oxidative muscle fibers (slow-twitch). Consistent with this idea, HIF2α (-/-) null skeletal muscles are mainly constituted by glycolytic muscle fibers (fast-twitch).29 These results indicate that HIF2α activation promotes mitochondrial function and cellular respiration. Similarly to our results, in a xenograft model of clear-cell renal carcinoma, HIF2α significantly increased, and HIF1α significantly decreased tumor growth rates.30 Interestingly, HIF2αxenografts demonstrate elevated expression of key mitochondrial proteins, such as pyruvate dehydrogenase (PDH, catalyzing the first step of the Krebs cycle) and TOMM20, the decreased expression of the Krebs cycle inhibitor pyruvate dehydrogenase kinase (PDK1), and decreased intra-tumoral lactate levels, indicating that HIF2α-tumors rely less on glycolysis and more on oxidative metabolism.30 Conversely, HIF1α-xenografts demonstrated elevated expression of PDK1 and the mitophagy marker BNIP3, suggesting that HIF1α in renal cancer cells functions as a tumor suppressor and promotes autophagic cell death.30 HIF2α in epithelial cancer cells plays an important role in the pathogenesis and prognosis of some type of cancers. Several clinical studies have indicated that HIF2α expression in epithelial cancer cells is an independent prognostic factor associated with
poor clinical outcome in invasive breast cancer,31 non-small cell lung cancer,32 colorectal cancer,33 hepatocellular carcinoma 34 and is correlated with radiotherapy failure in head and neck cancer.35 In the same studies, when evaluated, HIF1α expression did not predict clinical outcome.31-33
Materials. The following antibodies were used: anti-HIF1α (Novus Biologicals, NB100–123); anti-HIF2α (Novus Biologicals, NB100–122); anti-caveolin-1 (N20) (Santa Cruz, cat#sc-894); anti-caveolin-1 (BD Biosciences, clone 2297); antiSLC16A1 (MCT1) (Sigma, cat#AV43841); anti-MCT4 (generous gift of Dr. Nancy Philips); anti-EGFR (1005) (Santa Cruz, cat#sc-03); anti-Ras (BD Biosciences, cat#610001); anti-cyclin D1 (clone DCS6) (Thermo Scientific, cat#MS-210-P1); antiPI3 Kinase p110 α (Cell Signaling, cat#4255) and anti-β-actin (Sigma-Aldrich, cat#A5441). Cell cultures. Human skin-immortalized fibroblasts (hTERTBJ1) and human GFP-positive breast cancer cells (MDA-MB231-GFP) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a 37°C, 5% CO2 incubator. Retroviruses. Retroviral plasmids pBabe-puro (empty vector), HA-HIF1α-P402A/P564A-pBabe-puro and HA-HIF2αP405A/P531A-pBabe-puro (all from Addgene) were transfected into Phoenix Amphotropic packaging cells using FuGene 6 reagent (Roche Molecular Biochemicals), as described by the manufacturer. After 48 h post-transfection, retrovirus-containing culture medium was collected, passed through a 0.45 μm filter and added to hTERT-BJ1 fibroblasts or MDA-MB-231-GFP cancer cells in the presence of 5 μg/ml Polybrene. Infected cells were selected with puromycin (1.5 μg/ml for hTERT-BJ1 cells and 2 μg/ml for MDA-MB-231 cells). Immuno-blot analysis. Cells were harvested in lysis buffer (10 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100 and 60 mM n-octyl-glucoside), containing protease (Roche Applied Science) and phosphatase inhibitors (Sigma). For HIF1α and HIF2α detection, cells were lysed in EBC lysis buffer (5 mM Tris pH 8.0, 120 mM NaCl, 0.5% NP-40, with protease and phosphatase inhibitors). After rotation at 4°C for 40 min, cell lysates were centrifuged at 10,000 × g for 15 min at 4°C to remove insoluble debris. Protein concentrations were determined using the BCA reagent (Pierce). Cell lysates were then separated by SDS-PAGE (10% to 15% acrylamide) and transferred to nitrocellulose. After blocking for 1 h in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween 20) supplemented with 5% non-fat dry milk, membranes were incubated with primary antibodies diluted in TBST with 1% bovine serum albumin. Horseradish peroxidaseconjugated secondary antibodies [anti-mouse, 1:6,000 dilution (Pierce) or anti-rabbit 1:5,000 (BD PharMingen)] were used to visualize bound primary antibodies, with the Supersignal chemiluminescence substrate (Pierce). MitoTracker. To measure mitochondrial activity, cells were stained with the rosamine-based MitoTracker Orange CMTMRos (cat#M7510, Invitrogen), whose accumulation
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Figure 5. Activation of HIF2α in human breast cancer cells induces the upregulation of proteins involved in cell cycle progression and proliferation. MDA-pBabe, MDA-HIF1α or MDA-HIF2α cancer cells were analyzed by immunoblotting using markers of proliferation and cell cycle progression. HIF2α activation induces the upregulation of EGFR, Ras and cyclin D1. Conversely, HIF1α activation suppresses the expression of PI3K, indicating that HIF1α may inhibit cell survival. Equal loading was assessed by β-actin immunoblotting.
in mitochondria is dependent upon membrane potential. Lyophilized MitoTracker product was dissolved in DMSO to 1 mM stock solution. Then, the stock solution was diluted in serum-free DMEM to a final concentration of 25 nM. Cells were incubated with the pre-warmed MitoTracker staining solution for 12 min at 37°C. Then, cells were washed in PBS three times and fixed with 2% PFA. L-lactate assay. The pBabe (vector alone), HIF1α- and HIF2α-fibroblasts were seeded (1.2 × 105 per well) in 12-well plates in 300 μl of complete media. The day after, the media was changed to DMEM containing 2% FBS. After 48 h, the media was collected for measuring the lactate concentration using the EnzyChromTM L-Lactate Assay Kit (BioAssay Systems, cat#ECLC-100). The L-lactate concentration was normalized for protein concentration. The assay was performed also on MDApBabe, MDA-HIF1α and MDA-HIF2α tumor cells (8 × 104 per well). Cells were serum-starved for 2 h, then treated for 16 h with 1 mM metformin (Aldrich, cat#D150959) or vehicle alone, in DMEM containing 2% FBS. After 16 h, the media was collected for measuring lactate concentration. Results were expressed as ratio between metformin-treated vs. vehicle-treated control. 3H-deoxy-glucose uptake assay. For glucose uptake experiments, MDA-pBabe, MDA-HIF1α and MDA-HIF2α cells were plated in 12-well plates at a density of 1.2 × 105 cells in complete medium. The day after, cells were washed twice in PBS and incubated in serum-free medium for 2 h, then treated for 16 h with 1 mM metformin (Aldrich, cat#D150959) or vehicle alone, in DMEM containing 1% FBS. After one wash in PBS, cells were incubated in PBS containing 1 μCi/ml of Deoxy-D-Glucose, 2-[1,2–3H (N)]- (PerkinElmer) for 5 min, washed three times in ice-cold PBS and solubilized in 0.4 ml of 1% SDS. To measure incorporated radioactivity, 0.3 ml of each sample were counted
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Materials and Methods
in a liquid scintillation β-counter. Twenty microliters of each sample were used for protein determination. Radioactivity values in counts per minute (cpm) were normalized for the protein concentration. Results were expressed as a ratio between metformintreated vs. vehicle-treated controls. Tumor xenografts. All animals were housed and maintained in a barrier facility at the Kimmel Cancer Center at Thomas Jefferson University under National Institutes of Health (NIH) guidelines. Mice were kept on a 12-h light/dark cycle with ad libitum access to chow and water. Animal protocols used for this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). Briefly, MDA-MB-231GFP cancer cells (1 × 106 cells) were co-injected with pBabe-, HIF1α- or HIF2α-fibroblasts (3 × 105 cells) in 100 μl of sterile PBS into the flanks of athymic NCr nude mice (NCRNU; Taconic Farms; 6–8 wk of age). Mice were then sacrificed at 4 wk post-injection; tumors were dissected out to determine weight and size. Tumor size was measured bi-directionally using calipers, and the tumor volume was calculated using the formula X 2Y/2, where X and Y are, respectively, the short and long tumor dimensions. MDA-pBabe, MDA-HIF1α and MDA-HIF2α cancer cells (1 × 106 cells) were injected into the flanks of athymic NCr nude mice. Then, mice were sacrificed at 2 wk, post-injection. Quantification of tumor angiogenesis. Immunohistochemical staining for CD31 antibody was performed on 6-micron frozen sections using a three-step biotin-streptavidin-horseradish peroxidase method. Frozen tumor sections were fixed in 2% paraformaldehyde in PBS for 10 min and washed with PBS. After fixation, the sections were blocked with 10% rabbit serum and incubated overnight at 4°C with rat monoclonal CD31 antibody (#550274, BD Biosciences). The sections were then incubated with biotinylated rabbit anti-rat IgG (Vector Labs) and streptavidin-HRP (Dako). Immuno-reactivity was revealed with 3, 3' diaminobenzidine.
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Complex IV (COX) staining. Seven- or 10-micron thick cryostat sections were prepared from frozen tumor samples (n = 4 per group) and stored at -80°C until use. For the Cytochrome C Oxidase (COX) mitochondrial activity assay, frozen sections were brought to room temperature, washed for 5 min with 25 mM sodium phosphate buffer pH 7.4, and then incubated for 30 min at 37°C with the COX incubation mixture. The COX solution consisted of 10 mg Cytochrome C (Sigma-Aldrich, #C7752), 10 mg 3, 3' diaminobenzidine tetrahydrochloride hydrate (Sigma, #D5637) and 2 mg catalase (Sigma, #C1345) in 10 ml 25 mM sodium phosphate buffer. The solution was filtered after preparation and the pH adjusted to 7.2–7.4 with 1 N NaOH. After incubation, the slides were rinsed in 25 mM phosphate buffer, fixed in 10% formalin for 5 min and rinsed in tap water. The slides were counterstained with hematoxylin, dehydrated and coverslipped with permount mounting medium. Concluding Remarks In conclusion, we show here a novel, compartment-specific role for HIF1α and HIF2α in breast cancers. HIF1α acts as a tumor promoter in stromal cells, but as a tumor suppressor in cancer cells. Conversely, HIF2α is a tumor promoter in cancer cells, but does not play a role in stromal cells. Mechanistically, HIF1α-driven aerobic glycolysis in stromal cells supports cancer cell growth via the paracrine production of nutrients (such as L-lactate), which can directly “feed” cancer cells. However, HIF1α-driven aerobic glycolysis in cancer cells inhibits tumor growth. Conversely, expression of stable HIF2α activation in cancer cells induces the expression of known oncogenic molecules and promotes mitochondrial activity in cancer cells. Notably, the tumor-promoting effects of HIF1α and HIF2α are independent of angiogenesis, indicating that HIF1α-driven aerobic glycolysis in stromal cells, and HIF2α-driven mitochondrial oxidative
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Figure 6. HIF1α activation in human breast cancer cells suppresses tumor growth, while HIF2α activation promotes tumor growth. (A) MDA-pBabe, MDA-HIF1α or MDA-HIF2α breast cancer cells were injected into the flanks of nude mice. Two weeks, post-injections, tumor were harvested. Activation of HIF1α in MDA-MB-231 cells inhibits tumor growth, resulting in 1.8-fold reduction in tumor mass and a 1.9-fold reduction in tumor volume. Conversely, HIF2α activation in MDA-MB-231 cancer cells determines an increase of tumor growth (1.6-fold) and tumor volume (1.9-fold). * p < 0.05. (B) Quantification of tumor angiogenesis. Frozen sections from xenograft tumors were immuno-stained with CD31 antibodies and vessel density was quantitated. Note that HIF1α and HIF2α activation in breast cancer cells determines a slight but significant increase in angiogenesis, suggesting that the tumor promoting effects of HIF2α activation in breast cancer cells are independent of angiogenesis.
Figure 7. HIF2α activation promotes oxidative metabolism in tumor cells in vivo. To evaluate the mitochondrial activity in situ, COX activity staining was performed on frozen sections derived from tumor xenografts. In MDA-pBabe tumors (top panels), COX activity is found in MDA-MB-231 cells. However, in MDA-HIF1α tumor sections (middle panels), COX activity is low in cancer cells (see arrows in enlarged area), but strongly positive in tumor-associated fibroblasts. This is consistent with our in vitro results showing that HIF1α-activated MDA-MB-231 cells display a glycolytic metabolism. Conversely, in MDA-HIF2α tumor sections (bottom panels), strong COX activity is evident in cancer cells (arrows in enlarged area), indicating that HIF2α-activated tumor cells show an increased oxidative metabolism. Original magnification, 60X.
metabolism (OXPHOS) in cancer cells, are sufficient to support tumor growth. Acknowledgments
F.S. and her laboratory were supported by grants from the Breast Cancer Alliance (BCA) and the American Cancer Society (ACS). References Hu CJ, Sataur A, Wang L, Chen H, Simon MC. The N-terminal transactivation domain confers target gene specificity of hypoxia-inducible factors HIF-1alpha and HIF-2alpha. Mol Biol Cell 2007; 18:4528-42; PMID:17804822; http://dx.doi.org/10.1091/mbc. E06-05-0419 2. Kim JW, Tchernyshyov I, Semenza GL, Dang CV. HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia. Cell Metab 2006; 3:17785; PMID:16517405; http://dx.doi.org/10.1016/j. cmet.2006.02.002 3. Papandreou I, Cairns RA, Fontana L, Lim AL, Denko NC. HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption. Cell Metab 2006; 3:187-97; PMID:16517406; http:// dx.doi.org/10.1016/j.cmet.2006.01.012 4. Scortegagna M, Morris MA, Oktay Y, Bennett M, Garcia JA. The HIF family member EPAS1/HIF2alpha is required for normal hematopoiesis in mice. Blood 2003; 102:1634-40; PMID:12750163; http:// dx.doi.org/10.1182/blood-2003-02-0448
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U.E.M. was supported by a Young Investigator Award from the Margaret Q. Landenberger Research Foundation. M.P.L. was supported by grants from the NIH/NCI (R01-CA-080250; R01-CA-098779; R01-CA-120876; R01-AR-055660) and the Susan G. Komen Breast Cancer Foundation. R.G.P. was supported by grants from the NIH/NCI (R01-CA-70896, R01-CA-75503, R01-CA-86072 and R01-CA-107382) and the Dr. Ralph and Marian C. Falk Medical Research Trust. The Kimmel Cancer Center was supported by the NIH/ NCI Cancer Center Core grant P30-CA-56036 (to R.G.P.). Funds were also contributed by the Margaret Q. Landenberger Research Foundation (to M.P.L.). This project is funded, in part, under a grant with the Pennsylvania Department of Health (to M.P.L. and F.S.). The Department specifically disclaims responsibility for any analyses, interpretations or conclusions. This work was also supported, in part, by a Centre grant in Manchester from Breakthrough Breast Cancer in the UK (to A.H.) and an Advanced ERC Grant from the European Research Council.
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9. Covello KL, Simon MC, Keith B. Targeted replacement of hypoxia-inducible factor-1alpha by a hypoxia-inducible factor-2alpha knock-in allele promotes tumor growth. Cancer Res 2005; 65:2277-86; PMID:15781641; http://dx.doi.org/10.1158/00085472.CAN-04-3246 10. Koh MY, Lemos R Jr., Liu X, Powis G. The hypoxiaassociated factor switches cells from HIF-1α- to HIF2α-dependent signaling promoting stem cell characteristics, aggressive tumor growth and invasion. Cancer Res 2011; 71:4015-27; PMID:21512133; http:// dx.doi.org/10.1158/0008-5472.CAN-10-4142 11. Witkiewicz AK, Dasgupta A, Sotgia F, Mercier I, Pestell RG, Sabel M, et al. An absence of stromal caveolin-1 expression predicts early tumor recurrence and poor clinical outcome in human breast cancers. Am J Pathol 2009; 174:2023-34; PMID:19411448; http://dx.doi. org/10.2353/ajpath.2009.080873 12. Sloan EK, Ciocca DR, Pouliot N, Natoli A, Restall C, Henderson MA, et al. Stromal cell expression of caveolin-1 predicts outcome in breast cancer. Am J Pathol 2009; 174:2035-43; PMID:19411449; http://dx.doi. org/10.2353/ajpath.2009.080924
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Figure 8. Compartment-specific role of HIF1α and HIF2α in breast cancer. Our data indicate that the effects of HIF1α and HIF2α in breast cancer are distinct and compartment-specific. In fact, we show that HIF1α acts as a tumor promoter when activated in the tumor stroma, but acts as a tumor suppressor in breast cancer cells. Conversely, HIF2α activation in cancer-associated fibroblasts does not affect tumor growth, while HIF2α activation in epithelial tumor cells support tumor growth.
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21. Chiavarina B, Whitaker-Menezes D, MartinezOutschoorn UE, Witkiewicz AK, Birbe RC, Howell A, et al. Pyruvate kinase expression (PKM1 and PKM2) in cancer-associated fibroblasts drives stromal nutrient production and tumor growth. Cancer Biol Ther 2011; 12:12; PMID:22236875 22. Balliet RM, Capparelli C, Guido C, Pestell TG, Martinez-Outschoorn UE, Lin Z, et al. Mitochondrial oxidative stress in cancer-associated fibroblasts drives lactate production, promoting breast cancer tumor growth: understanding the aging and cancer connection. Cell Cycle 2011; 10:4065-73; PMID:22129993; http://dx.doi.org/10.4161/cc.10.23.18254 23. Witkiewicz AK, Whitaker-Menezes D, Dasgupta A, Philp NJ, Lin Z, Gandara R, et al. Using the “reverse Warburg effect” to identify high-risk breast cancer patients: stromal MCT4 predicts poor clinical outcome in triple-negative breast cancers. Cell Cycle 2012; 11:1108-17; PMID:22313602; http://dx.doi. org/10.4161/cc.11.6.19530 24. Magistretti PJ. Neuron-glia metabolic coupling and plasticity. J Exp Biol 2006; 209:230411; PMID:16731806; http://dx.doi.org/10.1242/ jeb.02208 25. Brooks GA. Lactate shuttles in nature. Biochem Soc Trans 2002; 30:258-64; PMID:12023861; http:// dx.doi.org/10.1042/BST0300258 26. Roda JM, Sumner LA, Evans R, Phillips GS, Marsh CB, Eubank TD. Hypoxia-inducible factor-2α regulates GM-CSF-derived soluble vascular endothelial growth factor receptor 1 production from macrophages and inhibits tumor growth and angiogenesis. J Immunol 2011; 187:1970-6; PMID:21765015; http:// dx.doi.org/10.4049/jimmunol.1100841 27. Simon F, Bockhorn M, Praha C, Baba HA, Broelsch CE, Frilling A, et al. Deregulation of HIF1-alpha and hypoxia-regulated pathways in hepatocellular carcinoma and corresponding non-malignant liver tissue-influence of a modulated host stroma on the prognosis of HCC. Langenbecks Arch Surg 2010; 395:395-405; PMID:20165955; http://dx.doi.org/10.1007/s00423009-0590-9 28. Scortegagna M, Ding K, Oktay Y, Gaur A, Thurmond F, Yan LJ, et al. Multiple organ pathology, metabolic abnormalities and impaired homeostasis of reactive oxygen species in Epas1-/- mice. Nat Genet 2003; 35:331-40; PMID:14608355; http://dx.doi. org/10.1038/ng1266
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29. Rasbach KA, Gupta RK, Ruas JL, Wu J, Naseri E, Estall JL, et al. PGC-1alpha regulates a HIF2alpha-dependent switch in skeletal muscle fiber types. Proc Natl Acad Sci USA 2010; 107:21866-71; PMID:21106753; http:// dx.doi.org/10.1073/pnas.1016089107 30. Biswas S, Troy H, Leek R, Chung YL, Li JL, Raval RR, et al. Effects of HIF-1alpha and HIF2alpha on Growth and Metabolism of Clear-Cell Renal Cell Carcinoma 786-0 Xenografts. J Oncol 2010; 2010:757908; PMID:20652061; http://dx.doi. org/10.1155/2010/757908. 31. Helczynska K, Larsson AM, Holmquist Mengelbier L, Bridges E, Fredlund E, Borgquist S, et al. Hypoxiainducible factor-2alpha correlates to distant recurrence and poor outcome in invasive breast cancer. Cancer Res 2008; 68:9212-20; PMID:19010893; http://dx.doi. org/10.1158/0008-5472.CAN-08-1135 32. Wu XH, Qian C, Yuan K. Correlations of hypoxia-inducible factor-1α/hypoxia-inducible factor-2α expression with angiogenesis factors expression and prognosis in non-small cell lung cancer. Chin Med J (Engl) 2011; 124:11-8; PMID:21362301 33. Yoshimura H, Dhar DK, Kohno H, Kubota H, Fujii T, Ueda S, et al. Prognostic impact of hypoxia-inducible factors 1alpha and 2alpha in colorectal cancer patients: correlation with tumor angiogenesis and cyclooxygenase-2 expression. Clin Cancer Res 2004; 10:8554-60; PMID:15623639; http://dx.doi.org/10.1158/10780432.CCR-0946-03 34. Bangoura G, Liu ZS, Qian Q, Jiang CQ, Yang GF, Jing S. Prognostic significance of HIF-2alpha/ EPAS1 expression in hepatocellular carcinoma. World J Gastroenterol 2007; 13:3176-82; PMID:17589895 35. Koukourakis MI, Bentzen SM, Giatromanolaki A, Wilson GD, Daley FM, Saunders MI, et al. Endogenous markers of two separate hypoxia response pathways (hypoxia inducible factor 2 alpha and carbonic anhydrase 9) are associated with radiotherapy failure in head and neck cancer patients recruited in the CHART randomized trial. J Clin Oncol 2006; 24:72735; PMID:16418497; http://dx.doi.org/10.1200/ JCO.2005.02.7474
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13. Martinez-Outschoorn UE, Pavlides S, WhitakerMenezes D, Daumer KM, Milliman JN, Chiavarina B, et al. Tumor cells induce the cancer associated fibroblast phenotype via caveolin-1 degradation: implications for breast cancer and DCIS therapy with autophagy inhibitors. Cell Cycle 2010; 9:2423-33; PMID:20562526; http://dx.doi.org/10.4161/cc.9.12.12048 14. Martinez-Outschoorn UE, Balliet RM, Rivadeneira DB, Chiavarina B, Pavlides S, Wang C, et al. Oxidative stress in cancer associated fibroblasts drives tumorstroma co-evolution: A new paradigm for understanding tumor metabolism, the field effect and genomic instability in cancer cells. Cell Cycle 2010; 9:325676; PMID:20814239; http://dx.doi.org/10.4161/ cc.9.16.12553 15. Martinez-Outschoorn UE, Trimmer C, Lin Z, Whitaker-Menezes D, Chiavarina B, Zhou J, et al. Autophagy in cancer associated fibroblasts promotes tumor cell survival: Role of hypoxia, HIF1 induction and NFκB activation in the tumor stromal microenvironment. Cell Cycle 2010; 9:351533; PMID:20855962; http://dx.doi.org/10.4161/ cc.9.17.12928 16. Chiavarina B, Whitaker-Menezes D, Migneco G, Martinez-Outschoorn UE, Pavlides S, Howell A, et al. HIF1-alpha functions as a tumor promoter in cancer associated fibroblasts, and as a tumor suppressor in breast cancer cells: Autophagy drives compartment-specific oncogenesis. Cell Cycle 2010; 9:353451; PMID:20864819; http://dx.doi.org/10.4161/ cc.9.17.12908 17. Whitaker-Menezes D, Martinez-Outschoorn UE, Flomenberg N, Birbe RC, Witkiewicz AK, Howell A, et al. Hyperactivation of oxidative mitochondrial metabolism in epithelial cancer cells in situ: visualizing the therapeutic effects of metformin in tumor tissue. Cell Cycle 2011; 10:4047-64; PMID:22134189; http://dx.doi.org/10.4161/cc.10.23.18151 18. Ullah MS, Davies AJ, Halestrap AP. The plasma membrane lactate transporter MCT4, but not MCT1, is up-regulated by hypoxia through a HIF-1alphadependent mechanism. J Biol Chem 2006; 281:90307; PMID:16452478; http://dx.doi.org/10.1074/jbc. M511397200 19. Sotgia F, Martinez-Outschoorn UE, Howell A, Pestell RG, Pavlides S, Lisanti MP. Caveolin-1 and cancer metabolism in the tumor microenvironment: markers, models, and mechanisms. Annu Rev Pathol 2012; 7:423-67; PMID:22077552; http://dx.doi. org/10.1146/annurev-pathol-011811-120856 20. Sotgia F, Martinez-Outschoorn UE, Pavlides S, Howell A, Pestell RG, Lisanti MP. Understanding the Warburg effect and the prognostic value of stromal caveolin-1 as a marker of a lethal tumor microenvironment. Breast Cancer Res 2011; 13:213; PMID:21867571; http:// dx.doi.org/10.1186/bcr2892
