• Cancer cells metabolically "fertilize" the tumor microenvironment with hydrogen peroxide, driving the Warburg effect: implications for PET imaging of human tumors.

      Martinez-Outschoorn, U E; Lin, Z; Trimmer, C; Flomenberg, N; Wang, C; Pavlides, S; Pestell, R G; Howell, Anthony; Sotgia, F; Lisanti, M P; et al. (2011-08-01)
      Previously, we proposed that cancer cells behave as metabolic parasites, as they use targeted oxidative stress as a "weapon" to extract recycled nutrients from adjacent stromal cells. Oxidative stress in cancer-associated fibroblasts triggers autophagy and  mitophagy, resulting in compartmentalized cellular catabolism, loss of mitochondrial function, and the onset of aerobic glycolysis, in the tumor stroma. As such, cancer-associated fibroblasts produce high-energy nutrients (such as lactate and ketones) that fuel mitochondrial biogenesis, and oxidative metabolism in cancer cells. We have termed this new energy-transfer mechanism the "reverse Warburg effect." To further test the validity of this hypothesis, here we used an in vitro MCF7-fibroblast co-culture system, and quantitatively measured a variety of metabolic parameters by FACS analysis (analogous to laser-capture micro-dissection).  Mitochondrial activity, glucose uptake, and ROS production were measured with highly-sensitive fluorescent probes (MitoTracker, NBD-2-deoxy-glucose, and DCF-DA). Interestingly, using this approach, we directly show that cancer cells initially secrete hydrogen peroxide that then triggers oxidative stress in neighboring fibroblasts. Thus, oxidative stress is contagious (spreads like a virus) and is propagated laterally and vectorially from cancer cells to adjacent fibroblasts. Experimentally, we show that oxidative stress in cancer-associated fibroblasts quantitatively reduces mitochondrial activity, and increases glucose uptake, as the fibroblasts become more dependent on aerobic glycolysis.  Conversely, co-cultured cancer cells show significant increases in mitochondrial activity, and corresponding reductions in both glucose uptake and GLUT1 expression. Pre-treatment of co-cultures with extracellular catalase (an anti-oxidant enzyme that detoxifies hydrogen peroxide) blocks the onset of oxidative stress, and potently induces the death of cancer cells, likely via starvation.  Given that cancer-associated fibroblasts show the largest increases in glucose uptake, we suggest that PET imaging of human tumors, with Fluoro-2-deoxy-D-glucose (F-2-DG), may be specifically detecting the tumor stroma, rather than epithelial cancer cells.
    • Caveolin-1 and mitochondrial SOD2 (MnSOD) function as tumor suppressors in the stromal microenvironment: a new genetically tractable model for human cancer associated fibroblasts.

      Trimmer, C; Sotgia, F; Whitaker-Menezes, D; Balliet, Renee M; Eaton, Gregory; Martinez-Outschoorn, Ubaldo E; Pavlides, Stephanos; Howell, Anthony; Iozzo, Renato V; Pestell, Richard G; et al. (2011-02-15)
      We have recently proposed a new model for understanding tumor metabolism, termed: "The Autophagic Tumor Stroma Model of Cancer Metabolism". In this new paradigm, catabolism (autophagy) in the tumor stroma fuels the anabolic growth of aggressive cancer cells. Mechanistically, tumor cells induce autophagy in adjacent cancer-associated fibroblasts via the loss of caveolin-1 (Cav-1), which is sufficient to promote oxidative stress in stromal fibroblasts. To further test this hypothesis, here we created human Cav-1 deficient immortalized fibroblasts using a targeted sh-RNA knock-down approach. Relative to control fibroblasts, Cav-1 deficient fibroblasts dramatically promoted tumor growth in xenograft assays employing an aggressive human breast cancer cell line, namely MDA-MB-231 cells. Co-injection of Cav-1 deficient fibroblasts, with MDA-MB-231 cells, increased both tumor mass and tumor volume by ~4-fold. Immuno-staining with CD31 indicated that this paracrine tumor promoting effect was clearly independent of angiogenesis. Mechanistically, proteomic analysis of these human Cav-1 deficient fibroblasts identified > 40 protein biomarkers that were upregulated, most of which were associated with i) myofibroblast differentiation, or ii) oxidative stress/hypoxia. In direct support of these findings, the tumor promoting effects of Cav-1 deficient fibroblasts could be functionally suppressed (nearly 2-fold) by the recombinant over-expression of SOD2 (superoxide dismutase 2), a known mitochondrial enzyme that de-activates superoxide, thereby reducing mitochondrial oxidative stress. In contrast, cytoplasmic soluble SOD1 had no effect, further highlighting a specific role for mitochondrial oxidative stress in this process. In summary, here we provide new evidence directly supporting a key role for a loss of stromal Cav-1 expression and oxidative stress in cancer-associated fibroblasts, in promoting tumor growth, which is consistent with "The Autophagic Tumor Stroma Model of Cancer". The human Cav-1 deficient fibroblasts that we have generated are a new genetically tractable model system for identifying other suppressors of the cancer-associated fibroblast phenotype, via a genetic "complementation" approach. This has important implications for understanding the pathogenesis of triple negative and basal breasts cancers, as well as tamoxifen-resistance in ER+ breast cancers, which are all associated with a Cav-1 deficient "lethal" tumor micro-environment, driving poor clinical outcome.
    • Cytokine production and inflammation drive autophagy in the tumor microenvironment: role of stromal caveolin-1 as a key regulator.

      Martinez-Outschoorn, U E; Whitaker-Menezes, D; Lin, Z; Flomenberg, N; Howell, Anthony; Pestell, R G; Lisanti, M P; Sotgia, F; Thomas Jefferson University, Philadelphia, PA, USA. (2011-06-01)
      Recently, we proposed a new paradigm for understanding the role of the tumor microenvironment in breast cancer onset and progression. In this model, cancer cells induce oxidative stress in adjacent fibroblasts. This, in turn, results in the onset of stromal autophagy, which produces recycled nutrients to "feed" anabolic cancer cells. However, it remains unknown how autophagy in the tumor microenvironment relates to inflammation, another key driver of tumorigenesis. To address this issue, here we employed a well-characterized co-culture system in which cancer cells induce autophagy in adjacent fibroblasts via oxidative stress and NFκB-activation. We show, using this co-culture system, that the same experimental conditions that result in an autophagic microenvironment, also drive in the production of numerous inflammatory mediators (including IL-6, IL-8, IL-10, MIP1a, IFNg, RANTES (CCL5) and GMCSF). Furthermore, we demonstrate that most of these inflammatory mediators are individually sufficient to directly induce the onset of autophagy in fibroblasts. To further validate the in vivo relevance of these findings, we assessed the inflammatory status of Cav-1 (-/-) null mammary fat pads, which are a model of a bonafide autophagic microenvironment. Notably, we show that Cav-1 (-/-) mammary fat pads undergo infiltration with numerous inflammatory cell types, including lymphocytes, T-cells, macrophages and mast cells. Taken together, our results suggest that cytokine production and inflammation are key drivers of autophagy in the tumor microenvironment. These results may explain why a loss of stromal Cav-1 is a powerful predictor of poor clinical outcome in breast cancer patients, as it is a marker of both (1) autophagy and (2) inflammation in the tumor microenvironment. Lastly, hypoxia in fibroblasts was not sufficient to induce the full-blown inflammatory response that we observed during the co-culture of fibroblasts with cancer cells, indicating that key reciprocal interactions between cancer cells and fibroblasts may be required.
    • Evidence for a stromal-epithelial "lactate shuttle" in human tumors: MCT4 is a marker of oxidative stress in cancer-associated fibroblasts.

      Whitaker-Menezes, D; Martinez-Outschoorn, U E; Lin, Z; Ertel, A; Flomenberg, N; Witkiewicz, A K; Birbe, R C; Howell, Anthony; Pavlides, S; Gandara, R; et al. (2011-06-01)
      Recently, we proposed a new mechanism for understanding the Warburg effect in cancer metabolism. In this new paradigm, cancer-associated fibroblasts undergo aerobic glycolysis, and extrude lactate to "feed" adjacent cancer cells, which then drives mitochondrial biogenesis and oxidative mitochondrial metabolism in cancer cells. Thus, there is vectorial transport of energy-rich substrates from the fibroblastic tumor stroma to anabolic cancer cells. A prediction of this hypothesis is that cancer-associated fibroblasts should express MCT4, a mono-carboxylate transporter that has been implicated in lactate efflux from glycolytic muscle fibers and astrocytes in the brain. To address this issue, we co-cultured MCF7 breast cancer cells with normal fibroblasts. Interestingly, our results directly show that breast cancer cells specifically induce the expression of MCT4 in cancer-associated fibroblasts; MCF7 cells alone and fibroblasts alone, both failed to express MCT4. We also show that the expression of MCT4 in cancer-associated fibroblasts is due to oxidative stress, and can be prevented by pre-treatment with the anti-oxidant N-acetyl-cysteine. In contrast to our results with MCT4, we see that MCT1, a transporter involved in lactate uptake, is specifically upregulated in MCF7 breast cancer cells when co-cultured with fibroblasts. Virtually identical results were also obtained with primary human breast cancer samples. In human breast cancers, MCT4 selectively labels the tumor stroma, e.g., the cancer-associated fibroblast compartment. Conversely, MCT1 was selectively expressed in the epithelial cancer cells within the same tumors. Functionally, we show that overexpression of MCT4 in fibroblasts protects both MCF7 cancer cells and fibroblasts against cell death, under co-culture conditions. Thus, we provide the first evidence for the existence of a stromal-epithelial lactate shuttle in human tumors, analogous to the lactate shuttles that are essential for the normal physiological function of muscle tissue and brain. These data are consistent with the "reverse Warburg effect," which states that cancer-associated fibroblasts undergo aerobic glycolysis, thereby producing lactate, which is utilized as a metabolic substrate by adjacent cancer cells. In this model, "energy transfer" or "metabolic-coupling" between the tumor stroma and epithelial cancer cells "fuels" tumor growth and metastasis, via oxidative mitochondrial metabolism in anabolic cancer cells. Most importantly, our current findings provide a new rationale and novel strategy for anti-cancer therapies, by employing MCT inhibitors.
    • Hydrogen peroxide fuels aging, inflammation, cancer metabolism and metastasis: the seed and soil also needs "fertilizer".

      Lisanti, Michael P; Martinez-Outschoorn, U E; Lin, Z; Pavlides, S; Whitaker-Menezes, D; Pestell, R G; Howell, Anthony; Sotgia, F; The Jefferson Stem Cell Biology and Regenerative Medicine Center, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA. (2011-08-01)
      In 1889, Dr. Stephen Paget proposed the "seed and soil" hypothesis, which states that cancer cells (the seeds) need the proper microenvironment (the soil) for them to grow, spread and metastasize systemically. In this hypothesis, Dr. Paget rightfully recognized that the tumor microenvironment has an important role to play in cancer progression and metastasis. In this regard, a series of recent studies have elegantly shown that the production of hydrogen peroxide, by both cancer cells and cancer-associated fibroblasts, may provide the necessary "fertilizer," by driving accelerated aging, DNA damage, inflammation and cancer metabolism, in the tumor microenvironment. By secreting hydrogen peroxide, cancer cells and fibroblasts are mimicking the behavior of immune cells (macrophages/neutrophils), driving local and systemic inflammation, via the innate immune response (NFκB). Thus, we should consider using various therapeutic strategies (such as catalase and/or other anti-oxidants) to neutralize the production of cancer-associated hydrogen peroxide, thereby preventing tumor-stroma co-evolution and metastasis. The implications of these findings for overcoming chemo-resistance in cancer cells are also discussed in the context of hydrogen peroxide production and cancer metabolism.
    • Ketones and lactate increase cancer cell "stemness," driving recurrence, metastasis and poor clinical outcome in breast cancer: achieving personalized medicine via Metabolo-Genomics.

      Martinez-Outschoorn, U E; Prisco, M; Ertel, A; Tsirigos, A; Lin, Z; Pavlides, S; Wang, C; Flomenberg, N; Knudsen, E S; Howell, Anthony; et al. (2011-04-15)
      Previously, we showed that high-energy metabolites (lactate and ketones) "fuel" tumor growth and experimental metastasis in an in vivo xenograft model, most likely by driving oxidative mitochondrial metabolism in breast cancer cells. To mechanistically understand how these metabolites affect tumor cell behavior, here we used genome-wide transcriptional profiling. Briefly, human breast cancer cells (MCF7) were cultured with lactate or ketones, and then subjected to transcriptional analysis (exon-array). Interestingly, our results show that treatment with these high-energy metabolites increases the transcriptional expression of gene profiles normally associated with "stemness," including genes upregulated in embryonic stem (ES) cells. Similarly, we observe that lactate and ketones promote the growth of bonafide ES cells, providing functional validation. The lactate- and ketone-induced "gene signatures" were able to predict poor clinical outcome (including recurrence and metastasis) in a cohort of human breast cancer patients. Taken together, our results are consistent with the idea that lactate and ketone utilization in cancer cells promotes the "cancer stem cell" phenotype, resulting in significant decreases in patient survival. One possible mechanism by which these high-energy metabolites might induce stemness is by increasing the pool of Acetyl-CoA, leading to increased histone acetylation, and elevated gene expression. Thus, our results mechanistically imply that clinical outcome in breast cancer could simply be determined by epigenetics and energy metabolism, rather than by the accumulation of specific "classical" gene mutations. We also suggest that high-risk cancer patients (identified by the lactate/ketone gene signatures) could be treated with new therapeutics that target oxidative mitochondrial metabolism, such as the anti-oxidant and "mitochondrial poison" metformin. Finally, we propose that this new approach to personalized cancer medicine be termed "Metabolo-Genomics," which incorporates features of both 1) cell metabolism and 2) gene transcriptional profiling. Importantly, this powerful new approach directly links cancer cell metabolism with clinical outcome, and new therapeutic strategies for inhibiting the TCA cycle and mitochondrial oxidative phosphorylation in cancer cells.
    • Understanding the metabolic basis of drug resistance: therapeutic induction of the Warburg effect kills cancer cells.

      Martinez-Outschoorn, U E; Lin, Z; Ko, Y H; Goldberg, A F; Flomenberg, N; Wang, C; Pavlides, S; Pestell, R G; Howell, Anthony; Sotgia, F; et al. (2011-08-01)
      Previously, we identified a form of epithelial-stromal metabolic coupling, in which cancer cells induce aerobic glycolysis in adjacent stromal fibroblasts, via oxidative stress, driving autophagy and mitophagy. In turn, these cancer-associated fibroblasts provide recycled nutrients to epithelial cancer cells, "fueling" oxidative mitochondrial metabolism and anabolic growth. An additional consequence is that these glycolytic fibroblasts protect cancer cells against apoptosis, by providing a steady nutrient stream of to mitochondria in cancer cells. Here, we investigated whether these interactions might be the basis of tamoxifen-resistance in ER(+) breast cancer cells. We show that MCF7 cells alone are Tamoxifen-sensitive, but become resistant when co-cultured with hTERT-immortalized human fibroblasts. Next, we searched for a drug combination (Tamoxifen + Dasatinib) that could over-come fibroblast-induced Tamoxifen-resistance. Importantly, we show that this drug combination acutely induces the Warburg effect (aerobic glycolysis) in MCF7 cancer cells, abruptly cutting off their ability to use their fuel supply, effectively killing these cancer cells. Thus, we believe that the Warburg effect in tumor cells is not the "root cause" of cancer, but rather it may provide the necessary clues to preventing chemo-resistance in cancer cells. Finally, we observed that this drug combination (Tamoxifen + Dasatinib) also had a generalized anti-oxidant effect, on both co-cultured fibroblasts and cancer cells alike, potentially reducing tumor-stroma co-evolution. Our results are consistent with the idea that chemo-resistance may be both a metabolic and stromal phenomenon that can be overcome by targeting mitochondrial function in epithelial cancer cells. Thus, simultaneously targeting both (1) the tumor stroma and (2) the epithelial cancer cells, with combination therapies, may be the most successful approach to anti-cancer therapy. This general strategy of combination therapy for overcoming drug resistance could be applicable to many different types of cancer.