The reverse Warburg Effect: Glycolysis inhibitors prevent the tumor promoting effects of caveolin-1 deficient cancer associated fibroblasts

We and others have previously identified a loss of stromal caveolin-1 (Cav-1) in cancer-associated fibroblasts (CAFs) as a powerful single independent predictor of breast cancer patient tumor recurrence, metastasis, tamoxifen-resistance, and poor clinical outcome. However, it remains unknown how loss of stromal Cav-1 mediates these effects clinically. To mechanistically address this issue, we have now generated a novel human tumor xenograft model. In this two-component system, nude mice are co-injected with i) human breast cancer cells (MDA-MB-231), and ii) stromal fibroblasts (wild-type (WT) versus Cav-1 (-/-) deficient). This allowed us to directly evaluate the effects of a Cav-1 deficiency solely in the tumor stromal compartment. Here, we show that Cav-1-deficient stromal fibroblasts are sufficient to promote both tumor growth and angiogenesis, and to recruit Cav-1 (+) micro-vascular cells. Proteomic analysis of Cav-1-deficient stromal fibroblasts indicates that these cells upregulate the expression of glycolytic enzymes, a hallmark of aerobic glycolysis (the Warburg effect). Thus, Cav-1-deficient stromal fibroblasts may contribute towards tumor growth and angiogenesis, by providing energy-rich metabolites in a paracrine fashion. We have previously termed this new idea the “Reverse Warburg Effect”. In direct support of this notion, treatment of this xenograft model with glycolysis inhibitors functionally blocks the positive effects of Cav-1-deficient stromal fibroblasts on breast cancer tumor growth. Thus, pharmacologically-induced metabolic restriction (via treatment with glycolysis inhibitors) may be a promising new therapeutic strategy for breast cancer patients that lack stromal Cav-1 expression. We also identify the stromal expression of PKM2 and LDH-B as new candidate biomarkers for the “Reverse Warburg Effect” or “Stromal-Epithelial Metabolic Coupling” in human breast cancers.     

Metabolic interplay between glycolysis and mitochondrial oxidation: The reverse Warburg effect and its therapeutic implication

Aerobic glycolysis, i.e., the Warburg effect, may contribute to the aggressive phenotype of hepatocellular carcinoma. However, increasing evidence highlights the limitations of the Warburg effect, such as high mitochondrial respiration and low glycolysis rates in cancer cells. To explain such contradictory phenomena with regard to the Warburg effect, a metabolic interplay between glycolytic and oxidative cells was proposed, i.e., the "reverse Warburg effect". Aerobic glycolysis may also occur in the stromal compartment that surrounds the tumor; thus, the stromal cells feed the cancer cells with lactate and this interaction prevents the creation of an acidic condition in the tumor microenvironment. This concept provides great heterogeneity in tumors, which makes the disease difficult to cure using a single agent. Understanding metabolic flexibility by lactate shuttles offers new perspectives to develop treatments that target the hypoxic tumor microenvironment and overcome the limitations of glycolytic inhibitors.     

Catabolic efficiency of aerobic glycolysis: The Warburg effect revisited

Background  

Cancer cells simultaneously exhibit glycolysis with lactate secretion and mitochondrial respiration even in the presence of oxygen, a phenomenon known as the Warburg effect. The maintenance of this mixed metabolic phenotype is seemingly counterintuitive given that aerobic glycolysis is far less efficient in terms of ATP yield per moles of glucose than mitochondrial respiration.     

Origin and function of tumor stroma fibroblasts

Tumor development is critically dependent on the formation of a supporting stroma consisting of neovasculature, inflammatory cells and activated fibroblasts. Activated fibroblasts present a heterogeneous cell population not only in regard to the expression of marker molecules but also to their origin and molecular signaling properties. The plasticity of this cell type is pointed out by the multiple transdifferentiation events that lead to the generation of activated fibroblasts which can arise from resting fibroblasts, epithelial and endothelial cells as well as from mesenchymal stem cells. Cellular in vitro and in vivo experiments have changed the perspective of fibroblasts from passive “bystanders” in the tumor microenvironment to that of important drivers of tumor progression. Here, we describe the multiple origins of fibroblast recruitment to the tumor tissue as well as the function of activated fibroblasts during tumor initiation, progression, metastasis and anti-VEGF resistance. The identification of markers present in activated fibroblasts as well as a better understanding how these cells influence other tumor compartments has led to the clinical development of anti-tumor therapies.     

Activation of fibroblasts in cancer stroma

Tumor microenvironment has emerged as an important target for cancer therapy. In particular, cancer-associated fibroblasts (CAF) seem to regulate many aspects of tumorigenesis. CAFs secrete a variety of soluble factors that act in a paracrine manner and thus affect not only cancer cells, but also other cell types present in the tumor stroma. Acting on cancer cells, CAFs promote tumor growth and invasion. They also enhance angiogenesis by secreting factors that activate endothelial cells and pericytes. Tumor immunity is mediated via cytokines secreted by immune cells and CAFs. Both immune cells and CAFs can exert tumor-suppressing and -promoting effects. CAFs, and the factors they produce, are attractive targets for cancer therapy, and they have proven to be useful as prognostic markers. In this review we focus mainly on carcinomas and discuss the recent findings regarding the role of activated fibroblasts in driving tumor progression.     

The Warburg effect and its cancer therapeutic implications

Increased aerobic glycolysis in cancer, a phenomenon known as the Warburg effect, has been observed in various tumor cells and represents a major biochemical alteration associated with malignant transformation. Although the exact molecular mechanisms underlying this metabolic change remain to be elucidated, the profound biochemical alteration in cancer cell energy metabolism provides exciting opportunities for the development of therapeutic strategies to preferentially kill cancer cells by targeting the glycolytic pathway. Several small molecules capable of inhibiting glycolysis in experimental systems have been shown to have promising anticancer activity in vitro and in vivo. This review article provides a brief summary of our current understanding of the Warburg effect, the underlying mechanisms, and its influence on the development of therapeutic strategies for cancer treatment.     

Hypoxia-inducible factor 1 activation by aerobic glycolysis implicates the Warburg effect in carcinogenesis

Cancer cells display high rates of aerobic glycolysis, a phenomenon known historically as the Warburg effect. Lactate and pyruvate, the end products of glycolysis, are highly produced by cancer cells even in the presence of oxygen. Hypoxia-induced gene expression in cancer cells has been linked to malignant transformation. Here we provide evidence that lactate and pyruvate regulate hypoxia-inducible gene expression independently of hypoxia by stimulating the accumulation of hypoxia-inducible Factor 1alpha (HIF-1alpha). In human gliomas and other cancer cell lines, the accumulation of HIF-1alpha protein under aerobic conditions requires the metabolism of glucose to pyruvate that prevents the aerobic degradation of HIF-1alpha protein, activates HIF-1 DNA binding activity, and enhances the expression of several HIF-1-activated genes including erythropoietin, vascular endothelial growth factor, glucose transporter 3, and aldolase A. Our findings support a novel role for pyruvate in metabolic signaling and suggest a mechanism by which high rates of aerobic glycolysis can promote the malignant transformation and survival of cancer cells.     

Aerobic Glycolysis in the Brain: Warburg and Crabtree Contra Pasteur

Neurochemical Research - Information processing is onerous. Curiously, active brain tissue does not fully oxidize glucose and instead generates a local surplus of lactate, a phenomenon termed...     

Hallmarks of cancer: Interactions with the tumor stroma

Ten years ago, Hanahan and Weinberg delineated six “Hallmarks of cancer” which summarize several decades of intense cancer research. However, tumor cells do not act in isolation, but rather subsist in a rich microenvironment provided by resident fibroblasts, endothelial cells, pericytes, leukocytes, and extra-cellular matrix. It is increasingly appreciated that the tumor stroma is an integral part of cancer initiation, growth and progression. The stromal elements of tumors hold prognostic, as well as response-predictive, information, and abundant targeting opportunities within the tumor microenvironment are continually identified. Herein we review the current understanding of tumor cell interactions with the tumor stroma with a particular focus on cancer-associated fibroblasts and pericytes. Moreover, we discuss emerging fields of research which need to be further explored in order to fulfil the promise of stroma-targeted therapies for cancer.     

Tumor microenvironment: the role of the tumor stroma in cancer

The tumor microenvironment, composed of non-cancer cells and their stroma, has become recognized as a major factor influencing the growth of cancer. The microenvironment has been implicated in the regulation of cell growth, determining metastatic potential and possibly determining location of metastatic disease, and impacting the outcome of therapy. While the stromal cells are not malignant per se, their role in supporting cancer growth is so vital to the survival of the tumor that they have become an attractive target for chemotherapeutic agents. In this review, we will discuss the various cellular and molecular components of the stromal environment, their effects on cancer cell dynamics, and the rationale and implications of targeting this environment for control of cancer. Additionally, we will emphasize the role of the bone marrow-derived cell in providing cells for the stroma.     

Autophagic tumor stroma: A biofuel for cancer growth

Comment on: Castello-Cros R, et al. Cell Cycle 2011; 10:2021-34.     

New aspects of the Warburg effect in cancer cell biology

Altered cellular metabolism is a defining feature of cancer [1]. The best studied metabolic phenotype of cancer is aerobic glycolysis--also known as the Warburg effect--characterized by increased metabolism of glucose to lactate in the presence of sufficient oxygen. Interest in the Warburg effect has escalated in recent years due to the proven utility of FDG-PET for imaging tumors in cancer patients and growing evidence that mutations in oncogenes and tumor suppressor genes directly impact metabolism. The goals of this review are to provide an organized snapshot of the current understanding of regulatory mechanisms important for Warburg effect and its role in tumor biology. Since several reviews have covered aspects of this topic in recent years, we focus on newest contributions to the field and reference other reviews where appropriate.     

Autophagy in cancer associated fibroblasts promotes tumor cell survival

Recently, using a co-culture system, we demonstrated that MCF7 epithelial cancer cells induce oxidative stress in adjacent cancer-associated fibroblasts, resulting in the autophagic/lysosomal degradation of stromal caveolin-1 (Cav-1). However, the detailed signaling mechanism(s) underlying this process remain largely unknown. Here, we show that hypoxia is sufficient to induce the autophagic degradation of Cav-1 in stromal fibroblasts, which is blocked by the lysosomal inhibitor chloroquine. Concomitant with the hypoxia-induced degradation of Cav-1, we see the upregulation of a number of well-established autophagy/mitophagy markers, namely LC3, ATG16L, BNIP3, BNIP3L, HIF-1α and NFκB. In addition, pharmacological activation of HIF-1α drives Cav-1 degradation, while pharmacological inactivation of HIF-1 prevents the downregulation of Cav-1. Similarly, pharmacological inactivation of NFκB-another inducer of autophagy-prevents Cav-1 degradation. Moreover, treatment with an inhibitor of glutathione synthase, namely BSO, which induces oxidative stress via depletion of the reduced glutathione pool, is sufficient to induce the autophagic degradation of Cav-1. Thus, it appears that oxidative stress mediated induction of HIF1- and NFκB-activation in fibroblasts drives the autophagic degradation of Cav-1. In direct support of this hypothesis, we show that MCF7 cancer cells activate HIF-1α- and NFκB-driven luciferase reporters in adjacent cancer-associated fibroblasts, via a paracrine mechanism. Consistent with these findings, acute knock-down of Cav-1 in stromal fibroblasts, using an siRNA approach, is indeed sufficient to induce autophagy, with the upregulation of both lysosomal and mitophagy markers. How does the loss of stromal Cav-1 and the induction of stromal autophagy affect cancer cell survival? Interestingly, we show that a loss of Cav-1 in stromal fibroblasts protects adjacent cancer cells against apoptotic cell death. Thus, autophagic cancer-associated fibroblasts, in addition to providing recycled nutrients for cancer cell metabolism, also play a protective role in preventing the death of adjacent epithelial cancer cells. We demonstrate that cancer-associated fibroblasts upregulate the expression of TIGAR in adjacent epithelial cancer cells, thereby conferring resistance to apoptosis and autophagy. Finally, the mammary fat pads derived from Cav-1 (-/-) null mice show a hypoxia-like response in vivo, with the upregulation of autophagy markers, such as LC3 and BNIP3L. Taken together, our results provide direct support for the "Autophagic Tumor Stroma Model of Cancer Metabolism," and explain the exceptional prognostic value of a loss of stromal Cav-1 in cancer patients. Thus, a loss of stromal fibroblast Cav-1 is a biomarker for chronic hypoxia, oxidative stress and autophagy in the tumor microenvironment, consistent with its ability to predict early tumor recurrence, lymph node metastasis and tamoxifen-resistance in human breast cancers. Our results imply that cancer patients lacking stromal Cav-1 should benefit from HIF-inhibitors, NFκB-inhibitors, anti-oxidant therapies, as well as autophagy/lysosomal inhibitors. These complementary targeted therapies could be administered either individually or in combination, to prevent the onset of autophagy in the tumor stromal compartment, which results in a "lethal" tumor microenvironment.     

Oxidative stress in cancer associated fibroblasts drives tumor-stroma co-evolution: A new paradigm for understanding tumor metabolism,the field effect and genomic instability in cancer cells

Loss of stromal fibroblast caveolin-1 (Cav-1) is a powerful single independent predictor of poor prognosis in human breast cancer patients, and is associated with early tumor recurrence, lymph node metastasis and tamoxifen-resistance. We developed a novel co-culture system to understand the mechanism(s) by which a loss of stromal fibroblast Cav-1 induces a “lethal tumor microenvironment.” Here, we propose a new paradigm to explain the powerful prognostic value of stromal Cav-1. In this model, cancer cells induce oxidative stress in cancer-associated fibroblasts, which then acts as a “metabolic” and “mutagenic” motor to drive tumor-stroma co-evolution, DNA damage and aneuploidy in cancer cells. More specifically, we show that an acute loss of Cav-1 expression leads to mitochondrial dysfunction, oxidative stress and aerobic glycolysis in cancer associated fibroblasts. Also, we propose that defective mitochondria are removed from cancer-associated fibroblasts by autophagy/mitophagy that is induced by oxidative stress. As a consequence, cancer associated fibroblasts provide nutrients (such as lactate) to stimulate mitochondrial biogenesis and oxidative metabolism in adjacent cancer cells (the “Reverse Warburg effect”). We provide evidence that oxidative stress in cancer-associated fibroblasts is sufficient to induce genomic instability in adjacent cancer cells, via a bystander effect, potentially increasing their aggressive behavior. Finally, we directly demonstrate that nitric oxide (NO) over-production, secondary to Cav-1 loss, is the root cause for mitochondrial dysfunction in cancer associated fibroblasts. In support of this notion, treatment with anti-oxidants (such as N-acetyl-cysteine, metformin and quercetin) or NO inhibitors (L-NAME) was sufficient to reverse many of the cancer-associated fibroblast phenotypes that we describe. Thus, cancer cells use “oxidative stress” in adjacent fibroblasts (1) as an “engine” to fuel their own survival via the stromal production of nutrients and (ii) to drive their own mutagenic evolution towards a more aggressive phenotype, by promoting genomic instability. We also present evidence that the “field effect” in cancer biology could also be related to the stromal production of ROS and NO species. eNOS-expressing fibroblasts have the ability to downregulate Cav-1 and induce mitochondrial dysfunction in adjacent fibroblasts that do not express eNOS. As such, the effects of stromal oxidative stress can be laterally propagated, amplified and are effectively “contagious”—spread from cell-to-cell like a virus—creating an “oncogenic/mutagenic” field promoting widespread DNA damage.Key words: caveolin-1, cancer associated fibroblasts, oxidative stress, reactive oxygen species (ROS), mitochondrial dysfunction, autophagy, nitric oxide (NO), DNA damage, aneuploidy, genomic instability, anti-oxidant cancer therapy, the “field effect” in cancer biology