Understanding the Warburg effect and the prognostic value of stromal caveolin-1 as a marker of a lethal tumor microenvironment

Abstract

Cancer cells show a broad spectrum of bioenergetic states, with some cells using aerobic glycolysis while others rely on oxidative phosphorylation as their main source of energy. In addition, there is mounting evidence that metabolic coupling occurs in aggressive tumors, between epithelial cancer cells and the stromal compartment, and between well-oxygenated and hypoxic compartments. We recently showed that oxidative stress in the tumor stroma, due to aerobic glycolysis and mitochondrial dysfunction, is important for cancer cell mutagenesis and tumor progression. More specifically , increased autophagy/mitophagy in the tumor stroma drives a form of parasitic epithelial-stromal metabolic coupling. These findings explain why it is effective to treat tumors with either inducers or inhibitors of autophagy, as both would disrupt this energetic coupling. We also discuss evidence that glutamine addiction in cancer cells produces ammonia via oxidative mitochondrial metabolism. Ammonia production in cancer cells, in turn, could then help maintain autophagy in the tumor stromal compartment. In this vicious cycle, the initial glutamine provided to cancer cells would be produced by autophagy in the tumor stroma. Thus, we believe that parasitic epithelial-stromal metabolic coupling has important implications for cancer diagnosis and therapy, for example, in designing novel metabolic imaging techniques and establishing new targeted therapies. In direct support of this notion, we identified a loss of stromal caveolin-1 as a marker of oxidative stress, hypoxia, and autophagy in the tumor microenvironment, explaining its powerful predictive value. Loss of stromal caveolin-1 in breast cancers is associated with early tumor recurrence, metastasis, and drug resistance, leading to poor clinical outcome.

Figure 1

The autophagic tumor stroma model of cancer metabolism. Cancer cells induce oxidative stress in adjacent cancer-associated fibroblasts (CAFs). This activates reactive oxygen species (ROS) production and autophagy. ROS production in CAFs, via the bystander effect, serves to induce random mutagenesis in epithelial cancer cells, leading to double-strand DNA breaks and aneuploidy. Cancer cells mount an anti-oxidant defense and up-regulate molecules that protect them against ROS and autophagy, preventing them from undergoing apoptosis. So, stromal fibroblasts conveniently feed and mutagenize cancer cells, while protecting them against death. See the text for more details. A+, autophagy positive; A-, autophagy negative; A^R, autophagy resistant.

Figure 2

The reverse Warburg effect. (a) Via oxidative stress, cancer cells activate two major transcription factors in adjacent stromal fibroblasts (hypoxia-inducible factor (HIF)1α and NFκB). This leads to the onset of both autophagy and mitophagy, as well as aerobic glycolysis, which then produces recycled nutrients (such as lactate, ketones, and glutamine). These high-energy chemical building blocks can then be transferred and used as fuel in the tricarboxylic acid cycle (TCA) in adjacent cancer cells. The outcome is high ATP production in cancer cells, and protection against cell death. ROS, reactive oxygen species. (b) Homotypic cultures (upper panels) of MCF7 cells (right) and hTERT-fibroblasts (left) were immunostained with a mitochondrial membrane antibody (red). Note that mitochondrial mass is lower in mono-cultures of MCF7 cells compared to fibroblasts. However, co-culture of MCF7 cells with fibroblasts (lower panel) induces a dramatic increase in mitochondrial mass in the 'central MCF7 cell colony', outlined by the dotted white oval. In contrast, mitochondrial mass is decreased in co-cultured fibroblasts. Panel (b) was modified and reproduced with permission from [41,78].

Figure 3

Evidence supporting a 'lactate shuttle' in human tumors: compartmentalized distribution of monocarboxylate transporter (MCT)1/4. (a) MCT4 is expressed in the fibroblastic stromal compartment of human breast cancers. Note that MCT4 staining is absent from the tumor epithelial cells, but is present in the surrounding stroma. MCT4 staining outlines the cancer-associated fibroblasts that surround nests of epithelial cancer cells. (b) MCT1 is expressed in the epithelial compartment of human breast cancers. Note that MCT1 staining is present in the tumor epithelial cells, but is absent in the surrounding stroma. (c) The lactate shuttle: an energy transfer mechanism in normal tissue and human cancers. MCT4 functions primarily as a transporter that extrudes lactate from cells that are undergoing aerobic glycolysis and lack functional mitochondria. After lactate is extruded by MCT4 in cancer-associated fibroblasts (CAFs), lactate is then taken up by MCT1 in adjacent cancer cells. Similarly, ketones are transported by the same MCTs that handle lactate. Our studies suggest that metabolic coupling occurs between CAFs and adjacent tumor cells. Modified and reproduced with permission from [67].

Figure 4

Molecular profiling of a Cav-1 deficient tumor micro-environment in breast cancer patients. (a) The transcriptional profiles of caveolin-1 (Cav-1)-positive (+) tumor stroma (N = 4) versus Cav-1-negative (-) tumor stroma (N = 7) were compared, via laser-capture microdissection. We identified 238 gene transcripts that were up-regulated and 232 gene transcripts that were down-regulated in the stroma of tumors showing a loss of Cav-1 expression. Note that the two patient populations are transcriptionally distinct. (b) The Cav-1-deficient stromal gene signature is associated with poor survival in estrogen receptor-positive and luminal A breast cancer patients. Note that the Cav-1-deficient stromal signature is clearly associated with decreased overall survival. (c) Heat maps of the gene transcripts associated with the response to hypoxia, glycolysis, and autophagy. Note that Cav-1-deficient stroma shows the up-regulation of hypoxia target genes (65 transcripts), glycolysis/pyruvate metabolism (15 transcripts), and autophagy (22 transcripts). Modified and reproduced with permission from [98].

Figure 5

Glutamine utilization in cancer cells and the tumor stroma. Oxidative mitochondrial metabolism of glutamine in cancer cells produces ammonia. Ammonia production is sufficient to induce autophagy. Thus, autophagy in cancer-associated fibroblasts provides cancer cells with an abundant source of glutamine. In turn, the ammonia produced maintains the autophagic phenotype of the adjacent stromal fibroblasts. See text for details. TCA, tricarboxylic acid.

Figure 6

Lactate treatment of estrogen receptor-positive breast cancer cells drives mitochondrial biogenesis, leading to poor survival in a subset of luminal A breast cancer patients. (a) Lactate-treated (10 mM) MCF7 cells show a significant increase in mitochondrial mass, as visualized by staining with antibodies against mitochondrial marker proteins. (b) Lactate-treated (10 mM) MCF7 cells were subjected to transcriptional profiling (exon-array) to generate a gene signature. Interestingly, this lactate-induced gene signature predicts poor clinical outcome in estrogen receptor-positive/luminal A breast cancer patients, which represent nearly 60 to 70% of cases, and is the most common type of breast cancer. Modified and reproduced with permission from [124].