Brown adipose tissue (BAT) and white adipose tissue (WAT) and adipocytes are targets of Trypanosoma cruzi infection. Adipose tissue obtained from CD-1 mice 15 days after infection, an early stage of infection revealed a high parasite load. There was a significant increase in macrophages in infected adipose tissue and a reduction in lipid accumulation, adipocyte size, and fat mass and increased expression of lipolytic enzymes. Infection increased levels of Toll-like receptor (TLR) 4 and TLR9 and in the expression of components of the mitogen-activated protein kinase pathway. Protein and messenger RNA (mRNA) levels of peroxisome proliferator-activated receptor γ were increased in WAT, whereas protein and mRNA levels of adiponectin were significantly reduced in BAT and WAT. The mRNA levels of cytokines, chemokines, and their receptors were increased. Nuclear Factor Kappa B levels were increased in BAT, whereas Iκκ-γ levels increased in WAT. Adipose tissue is an early target of T. cruzi infection.

The c-jun gene regulates cellular proliferation and apoptosis via direct regulation of cellular gene expression. Alternative splicing of pre-mRNA increases the diversity of protein functions and alternate splicing events occur in tumors. Here, by targeting the excision of the endogenous c-jun gene within the mouse mammary epithelium, we have identified its selective role as an inhibitor of RNA splicing. Microarray-based assessment of gene expression, on laser capture micro-dissected c-jun−/− mammary epithelium, demonstrated that endogenous c-jun regulates the expression of approximately 50 genes governing RNA splicing. In addition, genome-wide splicing arrays demonstrated that endogenous c-jun regulated the alternate exon of approximately 147 genes, and 18% of these were either alternatively spliced in human tumors or involved in apoptosis. Endogenous c-jun also was shown to reduce splicing activity, which required the c-jun dimerization domain. Together, our findings suggest that c-jun directly attenuates RNA splicing efficiency, which may be of broad biological importance as alternative splicing plays an important role in both cancer development and therapy resistance.

The role of caveolin and caveolae in the pathogenesis of infection has only recently been appreciated. In this chapter, we have highlighted some important new data on the role of caveolin in infections due to bacteria, viruses and fungi but with particular emphasis on the protozoan parasites Leishmania spp., Trypanosoma cruzi and Toxoplasma gondii. This is a continuing area of research and the final chapter has not been written on this topic.

Diet and obesity, and their associated metabolic alterations, are some of the fastest-growing causes of disease and death in America. Findings from epidemiological studies correlating obesity, the sources of dietary fat and prostate cancer (PCa) are conflicting. We have previously shown that 15% of PB-ErbB-2 x pten+/− mice developed PCa and exhibited increased phosphorylated 4E-BP1, but not the key PI3-kinase intermediary phospho-protein, mTOR, when maintained on unrefined mouse chow.

We report herein that 100% of animals fed refined, westernized AIN-93-based diets containing corn oil developed PCa by 12 months of age. Increases in visceral fat and mTOR activation in the tumors were also observed. Furthermore, nuclear cyclin E levels were significantly induced by the AIN-93-corn oil-based diets versus chow. Replacing 50% of the corn oil with menhaden oil, with 21% of its triglycerides being n-3 PUFA’s, had no effect on tumorigenesis, fat deposition, cyclin E or mTOR. Phosphorylated BAD levels were similar in the tumors of mice in all three diets.

Our data demonstrated that in the context of our preclinical model, components of crude chow, but not dietary n-3 PUFAs, protect against PCa progression. In addition, these data establish phosphorylated mTOR, nuclear cyclin E and visceral fat deposits as possible biomarkers of increased dietary risk for PCa.

Caveolin-1 (Cav1) is the main protein component of the membrane lipid rafts caveolae. Cav1 serves as a scaffolding protein that compartmentalizes a multitude of signaling molecules and sequesters them in their inactive state. Due to its function in the negative regulation of signal transduction, loss of Cav1 has been implicated in the pathogenesis of many cancers, but its role in cutaneous squamous cell carcinoma (cSCC) is largely unexplored. cSCC is a multi-stage disease characterized by the development of benign, premalignant lesions and their progression into malignant cancer. Here, we use a two-stage carcinogenesis protocol to elucidate the function of Cav1 in the different stages of benign papilloma development: initiation and promotion. First, we demonstrate that Cav1 knock-out (KO) mice are more susceptible to benign papilloma development after being subjected to a DMBA/TPA initiation/promotion protocol. Treatment of wild-type (WT) and Cav1 KO mice with DMBA alone shows that both groups have similar rates of apoptosis. In contrast, treatment of these groups with TPA alone indicates that Cav1 KO mice are more susceptible to promoter treatment as evidenced by increased epidermal proliferation. Furthermore, primary keratinocytes isolated from Cav1 KO mice have a proliferative advantage over WT keratinocytes in both low- and high-calcium medium, conditions that promote proliferation and induce differentiation, respectively. Collectively, these data indicate that Cav1 functions to suppress proliferation in the epidermis, and loss of this function promotes the development of benign skin tumors.

Aging drives large systemic reductions in oxidative mitochondrial function, shifting the entire body metabolically toward aerobic glycolysis, a.k.a, the Warburg effect. Aging is also one of the most significant risk factors for the development of human cancers, including breast tumors. How are these two findings connected? One simplistic idea is that cancer cells rebel against the aging process by increasing their capacity for oxidative mitochondrial metabolism (OXPHOS). Then, local and systemic aerobic glycolysis in the aging host would provide energy-rich mitochondrial fuels (such as L-lactate and ketones) to directly “fuel” tumor cell growth and metastasis. This would establish a type of parasite-host relationship or “two-compartment tumor metabolism,” with glycolytic/oxidative metabolic coupling. The cancer cells (“the seeds”) would flourish in this nutrient-rich microenvironment (“the soil”), which has been fertilized by host aging. In this scenario, cancer cells are only trying to save themselves from the consequences of aging by engineering a metabolic mutiny, through the amplification of mitochondrial metabolism. We discuss the recent findings of Drs. Ron DePinho (MD Anderson) and Craig Thomspson (Sloan-Kettering) that are also consistent with this new hypothesis, linking cancer progression with metabolic aging. Using data mining and bioinformatics approaches, we also provide key evidence of a role for PGC1a/NRF1 signaling in the pathogenesis of (1) two-compartment tumor metabolism and (2) mitochondrial biogenesis in human breast cancer cells.

Little is known about how alcohol consumption promotes the onset of human breast cancer(s). One hypothesis is that ethanol induces metabolic changes in the tumor microenvironment, which then enhances epithelial tumor growth. To experimentally test this hypothesis, we used a co-culture system consisting of human breast cancer cells (MCF7) and hTERT-immortalized fibroblasts. Here, we show that ethanol treatment (100 mM) promotes ROS production and oxidative stress in cancer-associated fibroblasts, which is sufficient to induce myofibroblastic differentiation. Oxidative stress in stromal fibroblasts also results in the onset of autophagy/mitophagy, driving the induction of ketone body production in the tumor microenvironment. Interestingly, ethanol has just the opposite effect in epithelial cancer cells, where it confers autophagy resistance, elevates mitochondrial biogenesis and induces key enzymes associated with ketone re-utilization (ACAT1/OXCT1). During co-culture, ethanol treatment also converts MCF7 cells from an ER(+) to an ER(-) status, which is thought to be associated with “stemness,” more aggressive behavior and a worse prognosis. Thus, ethanol treatment induces ketone production in cancer-associated fibroblasts and ketone re-utilization in epithelial cancer cells, fueling tumor cell growth via oxidative mitochondrial metabolism (OXPHOS). This “two-compartment” metabolic model is consistent with previous historical observations that ethanol is first converted to acetaldehyde (which induces oxidative stress) and then ultimately to acetyl-CoA (a high-energy mitochondrial fuel), or can be used to synthesize ketone bodies. As such, our results provide a novel mechanism by which alcohol consumption could metabolically convert “low-risk” breast cancer patients to “high-risk” status, explaining tumor recurrence or disease progression. Hence, our findings have clear implications for both breast cancer prevention and therapy. Remarkably, our results also show that antioxidants [such as N-acetyl cysteine (NAC)] can effectively reverse or prevent ethanol-induced oxidative stress in cancer-associated fibroblasts, suggesting a novel strategy for cancer prevention. We also show that caveolin-1 and MCt4 protein expression can be effectively used as new biomarkers to monitor oxidative stress induced by ethanol.

Metformin is a well-established diabetes drug that prevents the onset of most types of human cancers in diabetic patients, especially by targeting cancer stem cells. Metformin exerts its protective effects by functioning as a weak “mitochondrial poison,” as it acts as a complex I inhibitor and prevents oxidative mitochondrial metabolism (OXPHOS). Thus, mitochondrial metabolism must play an essential role in promoting tumor growth. To determine the functional role of “mitochondrial health” in breast cancer pathogenesis, here we used mitochondrial uncoupling proteins (UCPs) to genetically induce mitochondrial dysfunction in either human breast cancer cells (MDA-MB-231) or cancer-associated fibroblasts (hTERT-BJ1 cells). Our results directly show that all three UCP family members (UCP-1/2/3) induce autophagy and mitochondrial dysfunction in human breast cancer cells, which results in significant reductions in tumor growth. Conversely, induction of mitochondrial dysfunction in cancer-associated fibroblasts has just the opposite effect. More specifically, overexpression of UCP-1 in stromal fibroblasts increases β-oxidation, ketone body production and the release of ATP-rich vesicles, which “fuels” tumor growth by providing high-energy nutrients in a paracrine fashion to epithelial cancer cells. Hence, the effects of mitochondrial dysfunction are truly compartment-specific. Thus, we conclude that the beneficial anticancer effects of mitochondrial inhibitors (such as metformin) may be attributed to the induction of mitochondrial dysfunction in the epithelial cancer cell compartment. Our studies identify cancer cell mitochondria as a clear target for drug discovery and for novel therapeutic interventions.

Recent evidence has identified substantial overlap between metabolic and oncogenic biochemical pathways, suggesting novel approaches to cancer intervention. For example, cholesterol lowering statins and the antidiabetes medication metformin both act as chemopreventive agents in prostate and other cancers. The natural compound resveratrol has similar properties: increasing insulin sensitivity, suppressing adipogenesis, and inducing apoptotic death of cancer cells in vitro. However, in vivo tumor xenografts acquire resistance to resveratrol by an unknown mechanism, while mouse models of metabolic disorders respond more consistently to the compound. Here we demonstrate that castration-resistant human prostate cancer C4-2 cells are more sensitive to resveratrol-induced apoptosis than isogenic androgen-dependent LNCaP cells. The MEK inhibitor U0126 antagonized resveratrol-induced apoptosis in C4-2 cells, but this effect was not seen with other MEK inhibitors. U0126 was found to inhibit mitochondrial function and shift cells to aerobic glycolysis independently of MEK. Mitochondrial activity of U0126 arose through decomposition, producing both mitochondrial fluorescence and cyanide, a known inhibitor of complex IV. Applying U0126 mitochondrial inhibition to C4-2 cell apoptosis, we tested the possibility that glutamine supplementation of citric acid cycle intermediate α-ketoglutarate may be involved. Suppression of the conversion of glutamate to α-ketoglutarate antagonized resveratrol-induced death in C4-2 cells. A similar effect was also seen by reducing extracellular glutamine concentration in the culture medium, suggesting that resveratrol-induced death is dependent on glutamine metabolism, a process frequently dysregulated in cancer. Further work on resveratrol and metabolism in cancer is warranted to ascertain if the glutamine dependence has clinical implications.

Colorectal cancer is a heterogeneous disease resulting from a combination of genetic and environmental factors. The C57BL/6J (B6) ApcMin/+ mouse develops polyps throughout the gastrointestinal tract and has been a valuable model for understanding the genetic basis of intestinal tumorigenesis. ApcMin/+ mice have been used to study known oncogenes and tumor suppressor genes on a controlled genetic background. These studies often utilize congenic knockout alleles, which can carry an unknown amount of residual donor DNA. The ApcMin model has also been used to identify modifer loci, known as Modifier of Min (Mom) loci, which alter ApcMin-mediated intestinal tumorigenesis. B6 mice carrying a knockout allele generated in WW6 embryonic stem cells were crossed to B6 ApcMin/+ mice to determine the effect on polyp multiplicity. The newly generated colony developed significantly more intestinal polyps than ApcMin/+ controls. Polyp multiplicity did not correlate with inheritance of the knockout allele, suggesting the presence of one or more modifier loci segregating in the colony. Genotyping of simple sequence length polymorphism (SSLP) markers revealed residual 129X1/SvJ genomic DNA within the congenic region of the parental knockout line. An analysis of polyp multiplicity data and SSLP genotyping indicated the presence of two Mom loci in the colony: (1) Mom12, a dominant modifier linked to the congenic region on chromosome 6 and (2) Mom13, which is unlinked to the congenic region and whose effect is masked by Mom12. The identification of Mom12 and Mom13 demonstrates the potential problems resulting from residual heterozygosity present in congenic lines.

Here, we show that tamoxifen resistance is induced by cancer-associated fibroblasts (CAFs). Coculture of estrogen receptor positive (ER+) MCF7 cells with fibroblasts induces tamoxifen and fulvestrant resistance with 4.4 and 2.5-fold reductions, respectively, in apoptosis compared with homotypic MCF7 cell cultures. Treatment of MCF7 cells cultured alone with high-energy mitochondrial “fuels” (L-lactate or ketone bodies) is sufficient to confer tamoxifen resistance, mimicking the effects of coculture with fibroblasts. To further demonstrate that epithelial cancer cell mitochondrial activity is the origin of tamoxifen resistance, we employed complementary pharmacological and genetic approaches. First, we studied the effects of two mitochondrial “poisons,” namely metformin and arsenic trioxide (ATO), on fibroblast-induced tamoxifen resistance. We show here that treatment with metformin or ATO overcomes fibroblast-induced tamoxifen resistance in MCF7 cells. Treatment with the combination of tamoxifen plus metformin or ATO leads to increases in glucose uptake in MCF7 cells, reflecting metabolic uncoupling between epithelial cancer cells and fibroblasts. In coculture, tamoxifen induces the upregulation of TIGAR (TP53-induced glycolysis and apoptosis regulator), a p53 regulated gene that simultaneously inhibits glycolysis, autophagy and apoptosis and reduces ROS generation, thereby promoting oxidative mitochondrial metabolism. To genetically mimic the effects of coculture, we next recombinantly overexpressed TIGAR in MCF7 cells. Remarkably, TIGAR overexpression protects epithelial cancer cells from tamoxifen-induced apoptosis, providing genetic evidence that increased mitochondrial function confers tamoxifen resistance. Finally, CAFs also protect MCF7 cells against apoptosis induced by other anticancer agents, such as the topoisomerase inhibitor doxorubicin (adriamycin) and the PARP-1 inhibitor ABT-888. These results suggest that the tumor microenvironment may be a general mechanism for conferring drug resistance. In summary, we have discovered that mitochondrial activity in epithelial cancer cells drives tamoxifen resistance in breast cancer and that mitochondrial “poisons” are able to re-sensitize these cancer cells to tamoxifen. In this context, TIGAR may be a key “druggable” target for preventing drug resistance in cancer cells, as it protects cancer cells against the onset of stress-induced mitochondrial dys-function and aerobic glycolysis.

Pancreatic cancer is one of the deadliest cancers due to early rapid metastasis and chemoresistance. Recently, epithelial to mesenchymal transition (EMT) was shown to play a key role in the pathogenesis of pancreatic cancer. To understand the role of caveolin-1 (Cav-1) in EMT, we overexpressed Cav-1 in a pancreatic cancer cell line, Panc 10.05, that does not normally express Cav-1. Here, we show that Cav-1 expression in pancreatic cancer cells induces an epithelial phenotype and promotes cell-cell contact, with increased expression of plasma membrane bound E-cadherin and β-catenin. Mechanistically, Cav-1 induces Snail downregulation and decreased activation of AKT, MAPK and TGFβ-Smad signaling pathways. In vitro, Cav-1 expression reduces cell migration and invasion, and attenuates doxorubicin-chemoresistance of pancreatic cancer cells. Importantly, in vivo studies revealed that Cav-1 expression greatly suppresses tumor formation in a xenograft model. Most interestingly, Panc/Cav-1 tumors displayed organized nests of differentiated cells that were totally absent in control tumors. Confirming our in vitro results, these nests of differentiated cells showed reexpression of E-cadherin and β-catenin at the cell membrane. Thus, we provide evidence that Cav-1 functions as a crucial modulator of EMT and cell differentiation in pancreatic cancer.

Despite decades of research, cerebral malaria remains one of the most serious complications of Plasmodium infection and is a significant burden in Sub-Saharan Africa, where, despite effective antiparasitic treatment, survivors develop long-term neurological sequelae. Although much remains to be discovered about the pathogenesis of cerebral malaria, The American Journal of Pathology has been seminal in presenting original research from both human and experimental models. These studies have afforded significant insight into the mechanism of cerebral damage in this devastating disease. The present review highlights information gleaned from these studies, especially in terms of their contributions to the understanding of cerebral malaria.

We have previously proposed that catabolic fibroblasts generate mitochondrial fuels (such as ketone bodies) to promote the anabolic growth of human cancer cells and their metastasic dissemination. We have termed this new paradigm “two-compartment tumor metabolism.” Here, we further tested this hypothesis by using a genetic approach. For this purpose, we generated hTERT-immortalized fibroblasts overexpressing the rate-limiting enzymes that promote ketone body production, namely BDH1 and HMGCS2. Similarly, we generated MDA-MB-231 human breast cancer cells overexpressing the key enzyme(s) that allow ketone body re-utilization, OXCT1/2 and ACAT1/2. Interestingly, our results directly show that ketogenic fibroblasts are catabolic and undergo autophagy, with a loss of caveolin-1 (Cav-1) protein expression. Moreover, ketogenic fibroblasts increase the mitochondrial mass and growth of adjacent breast cancer cells. However, most importantly, ketogenic fibroblasts also effectively promote tumor growth, without a significant increase in tumor angiogenesis. Finally, MDA-MB-231 cells overexpressing the enzyme(s) required for ketone re-utilization show dramatic increases in tumor growth and metastatic capacity. Our data provide the necessary genetic evidence that ketone body production and re-utilization drive tumor progression and metastasis. As such, ketone inhibitors should be designed as novel therapeutics to effectively treat advanced cancer patients, with tumor recurrence and metastatic disease. In summary, ketone bodies behave as onco-metabolites, and we directly show that the enzymes HMGCS2, ACAT1/2 and OXCT1/2 are bona fide metabolic oncogenes.

Mitogen-activated protein kinase (MAPK) pathways are major signal transduction systems by which eukaryotic cells convert environmental cues to intracellular events, such as cell proliferation and differentiation. Toxoplasma gondii is an obligate intracellular protozoan that is both a human and animal pathogen. This Apicomplexan causes significant morbidity and mortality in immune-competent and immune-compromised hosts. In humans, the most common manifestations of T. gondii infections are chorioretinitis in congenital infection and encephalitis in immune-compromised patients, such as patients with advanced AIDS. We have identified a T. gondii homolog of the MAPK family that we have called TgMAPK2. Sequence analyses demonstrated that TgMAPK2 has homology with lower eukaryotic ERK2 but has significant differences from mammalian ERK2. TgMAPK2 has an open reading frame of 2,037 bp, 678 amino acids, and its molecular weight is 73.1 kDa. It contains the typical 12 subdomains of a MAPK and has a TDY motif in the dual phosphorylation and activation subdomains. This suggests that TgMAPK2 may play an important role in stress response. Recombinant TgMAPK2 was catalytically active and was not inhibited by a human ERK2 inhibitor, FR180204. A partial TgMAPK2 lacking the ATP-binding motifs GxGxxGxV was successfully regulated by a ligand-controlled destabilization domain (ddFKBP) expression vector system in T. gondii. Since TgMAPK2 is significantly different from its mammalian counterpart, it may be useful as a drug target. This work establishes a foundation for further study for this unique kinase.

The development of new small molecule-based therapeutic drugs requires accurate quantification of drug bioavailability, biological activity and treatment efficacy. Rapidly measuring these endpoints is often hampered by the lack of efficient assay platforms with high sensitivity and specificity. Using an in vivo model system, we report a simple and sensitive liquid chromatography-tandem mass spectrometry assay to quantify the bioavailability of a recently developed novel cyclin-dependent kinase inhibitor VMY-1-103, a purvalanol B-based analog whose biological activity is enhanced via dansylation. We developed a rapid organic phase extraction technique and validated wide and functional VMY-1-103 distribution in various mouse tissues, consistent with its enhanced potency previously observed in a variety of human cancer cell lines. More importantly, in vivo MRI and single voxel proton MR-Spectroscopy further established that VMY-1-103 inhibited disease progression and affected key metabolites in a mouse model of hedgehog-driven medulloblastoma.

Here, we investigated the compartment-specific role of cell cycle arrest and senescence in breast cancer tumor growth. For this purpose, we generated a number of hTERT-immortalized senescent fibroblast cell lines overexpressing CDK inhibitors, such as p16(INK4A), p19(ARF) or p21(WAF1/CIP1). Interestingly, all these senescent fibroblast cell lines showed evidence of increased susceptibility toward the induction of autophagy (either at baseline or after starvation), as well as significant mitochondrial dysfunction. Most importantly, these senescent fibroblasts also dramatically promoted tumor growth (up to ~2-fold), without any comparable increases in tumor angiogenesis. Conversely, we generated human breast cancer cells (MDA-MB-231 cells) overexpressing CDK inhibitors, namely p16(INK4A) or p21(WAF1/CIP1). Senescent MDA-MB-231 cells also showed increased expression of markers of cell cycle arrest and autophagy, including β-galactosidase, as predicted. Senescent MDA-MB-231 cells had retarded tumor growth, with up to a near 2-fold reduction in tumor volume. Thus, the effects of CDK inhibitors are compartment-specific and are related to their metabolic effects, which results in the induction of autophagy and mitochondrial dysfunction. Finally, induction of cell cycle arrest with specific inhibitors (PD0332991) or cellular stressors [hydrogen peroxide (H₂O₂) or starvation] indicated that the onset of autophagy and senescence are inextricably linked biological processes. The compartment-specific induction of senescence (and hence autophagy) may be a new therapeutic target that could be exploited for the successful treatment of human breast cancer patients.

Migration stimulating factor (MSF) is a genetically truncated N-terminal isoform of fibronectin that is highly expressed during mammalian development in fetal fibroblasts, and during tumor formation in human cancer-associated myofibroblasts. However, its potential functional role in regulating tumor metabolism remains unexplored. Here, we generated an immortalized fibroblast cell line that recombinantly overexpresses MSF and studied their properties relative to vector-alone control fibroblasts. Our results indicate that overexpression of MSF is sufficient to confer myofibroblastic differentiation, likely via increased TGF-b signaling. In addition, MSF activates the inflammation-associated transcription factor NFκB, resulting in the onset of autophagy/mitophagy, thereby driving glycolytic metabolism (L-lactate production) in the tumor microenvironment. Consistent with the idea that glycolytic fibroblasts fuel tumor growth (via L-lactate, a high-energy mitochondrial fuel), MSF fibroblasts significantly increased tumor growth, by up to 4-fold. Mechanistic dissection of the MSF signaling pathway indicated that Cdc42 lies downstream of MSF and fibroblast activation. In accordance with this notion, Cdc42 overexpression in immortalized fibroblasts was sufficient to drive myofibroblast differentiation, to provoke a shift towards glycolytic metabolism and to promote tumor growth by up to 2-fold. In conclusion, the MSF/Cdc42/NFκB signaling cascade may be a critical druggable target in preventing “Warburg-like” cancer metabolism in tumor-associated fibroblasts. Thus, MSF functions in the metabolic remodeling of the tumor microenvironment by metabolically reprogramming cancer-associated fibroblasts toward glycolytic metabolism.

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 antioxidants) 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.

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.

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 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 chemoresistance 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.

Recent studies have suggested that cancer cells behave as metabolic parasites, by inducing oxidative stress in adjacent normal fibroblasts. More specifically, oncogenic mutations in cancer cells lead to ROS production and the “secretion” of hydrogen peroxide species. Oxidative stress in stromal fibroblasts then induces their metabolic conversion into cancer-associated fibroblasts. Such oxidative stress drives the onset of autophagy, mitophagy, and aerobic glycolysis in fibroblasts, resulting in the local production of high-energy mitochondrial fuels (such as L-lactate, ketone bodies, and glutamine). These recycled nutrients are then transferred to cancer cells, where they are efficiently burned via oxidative mitochondrial metabolism (OXPHOS). We have termed this new energy-transfer mechanism “Two-Compartment Tumor Metabolism”, to reflect that the production and consumption of nutrients (L-lactate and other catabolites) is highly compartmentalized. Thus, high-energy onco-catabolites are produced by the tumor stroma.

Here, we used a genetic approach to stringently test this energy-transfer hypothesis. First, we generated hTERT-immortalized fibroblasts which were genetically re-programmed towards catabolic metabolism. Metabolic re-programming towards glycolytic metabolism was achieved by the recombinant over-expression of MFF (mitochondrial fission factor). MFF over-expression results in extensive mitochondrial fragmentation, driving mitochondrial dysfunction. Our results directly show that MFF-fibroblasts undergo oxidative stress, with increased ROS production, and the onset of autophagy and mitophagy, both catabolic processes. Mechanistically, oxidative stress induces autophagy via NF-kB activation, also providing a link with inflammation. As a consequence MFF-fibroblasts showed intracellular ATP depletion and the extracellular secretion of L-lactate, a critical onco-catabolite. MFF-fibroblasts also showed signs of myofibroblast differentiation, with the expression of SMA and calponin.

Importantly, MFF-fibroblasts signficantly promoted early tumor growth (up to 6.5-fold), despite a 20% overall reduction in angiogenesis. Thus, catabolic metabolism in cancer-associated fibroblasts may be a critical event during tumor intiation, allowing accelerated tumor growth, especially prior to the onset of neo-angiogenesis.

Cancer cells don’t exist as pure homogeneous populations in vivo. Instead they are embedded in “cancer cell nests” that are surrounded by stromal cells, especially cancer associated fibroblasts. Thus, it is not unreasonable to suspect that stromal fibroblasts could influence the metabolism of adjacent cancer cells, and visa versa. In accordance with this idea, we have recently proposed that the Warburg effect in cancer cells may be due to culturing cancer cells by themselves, out of their normal stromal context or tumor microenvironment. In fact, when cancer cells are co-cultured with fibroblasts, then cancer cells increase their mitochondrial mass, while fibroblasts lose their mitochondria. An in depth analysis of this phenomenon reveals that aggressive cancer cells are “parasites” that use oxidative stress as a “weapon” to extract nutrients from surrounding stromal cells. Oxidative stress in fibroblasts induces the autophagic destruction of mitochondria, by mitophagy. Then, stromal cells are forced to undergo aerobic glycolysis, and produce energy-rich nutrients (such as lactate and ketones) to “feed” cancer cells. This mechanism would allow cancer cells to seed anywhere, without blood vessels as a food source, as they could simply induce oxidative stress wherever they go, explaining how cancer cells survive during metastasis. We suggest that stromal catabolism, via autophagy and mitophagy, fuels the anabolic growth of tumor cells, promoting tumor progression and metastasis. We have previously termed this new paradigm “The Autophagic Tumor Stroma Model of Cancer Metabolism”, or the “Reverse Warburg Effect”. We also discuss how glutamine addiction (glutaminolysis) in cancer cells fits well with this new model, by promoting oxidative mitochondrial metabolism in aggressive cancer cells.

Cancer is thought to be a disease associated with aging. Interestingly, normal aging is driven by the production of ROS and mitochondrial oxidative stress, resulting in the cumulative accumulation of DNA damage. Here, we discuss how ROS signaling, NFκB- and HIF1-activation in the tumor micro-environment induces a form of “accelerated aging,” which leads to stromal inflammation and changes in cancer cell metabolism. Thus, we present a unified model where aging (ROS), inflammation (NFκB) and cancer metabolism (HIF1), act as co-conspirators to drive autophagy (“self-eating”) in the tumor stroma. Then, autophagy in the tumor stroma provides high-energy “fuel” and the necessary chemical building blocks, for accelerated tumor growth and metastasis. Stromal ROS production acts as a “mutagenic motor” and allows cancer cells to buffer—at a distance—exactly how much of a mutagenic stimulus they receive, further driving tumor cell selection and evolution. Surviving cancer cells would be selected for the ability to induce ROS more effectively in stromal fibroblasts, so they could extract more nutrients from the stroma via autophagy. If lethal cancer is a disease of “accelerated host aging” in the tumor stroma, then cancer patients may benefit from therapy with powerful antioxidants. Antioxidant therapy should block the resulting DNA damage, and halt autophagy in the tumor stroma, effectively “cutting off the fuel supply” for cancer cells. These findings have important new implications for personalized cancer medicine, as they link aging, inflammation and cancer metabolism with novel strategies for more effective cancer diagnostics and therapeutics.

Caveolin-1 (Cav-1), the principal structural component of caveolae, participates in the pathogenesis of several fibrotic diseases, including systemic sclerosis (SSc). Interestingly, affected skin and lung samples from patients with SSc show reduced levels of Cav-1, as compared to normal skin. In addition, restoration of Cav-1 function in skin fibroblasts from SSc patients reversed their pro-fibrotic phenotype. Here, we further investigated whether Cav-1 mice are a useful preclinical model for studying the pathogenesis of SSc. For this purpose, we performed quantitative transmission electron microscopy, as well as biochemical, biomechanical, and immuno-histochemical analysis, of the skin from Cav-1-/- null mice. Using these complementary approaches, we now show that skin from Cav-1 null mice exhibits many of the same characteristics as SSc skin from patients. These changes include a decrease in collagen fiber diameter, increased maximum stress (a measure tensile strength) and modulus (a measure of stiffness), as well as mononuclear cell infiltration. Furthermore, an increase in autophagy/mitophagy was observed in the stromal cells of the dermis from Cav-1-/- mice. These findings suggest that changes in cellular energy metabolism (e.g., a shift towards aerobic glycolysis) in these stromal cells may provide a survival mechanism in this “hostile” or pro-inflammatory microenvironment. Taken together, our results demonstrate that Cav-1-/- mice are a valuable new pre-clinical model for studying scleroderma. Most importantly, our results suggest that inhibition of autophagy and/or aerobic glycolysis may represent a new promising therapeutic strategy for halting fibrosis in SSc patients. Finally, Cav-1-/- mice are also a pre-clinical model for a “lethal” tumor microenvironment, possibly explaining the link between fibrosis, tumor progression and cancer metastasis.