Mammography is an important screening modality for the early detection of DCIS and breast cancer lesions. More specifically, high mammographic density is associated with an increased risk of breast cancer. However, the biological processes underlying this phenomenon remain largely unknown. Here, we re-interrogated genome-wide transcriptional profiling data obtained from low-density (LD) mammary fibroblasts (n = 6 patients) and high-density (HD) mammary fibroblasts (n = 7 patients) derived from a series of 13 female patients. We used these raw data to generate a “breast density” gene signature consisting of >1250 transcripts that were significantly increased in HD fibroblasts, relative to LD fibroblasts. We then focused on the genes that were increased by ≥ 1.5-fold (P < 0.05) and performed gene set enrichment analysis (GSEA), using the molecular signatures database (MSigDB). Our results indicate that HD fibroblasts show the upregulation and/or hyper-activation of several key cellular processes, including the stress response, inflammation, stemness, and signal transduction. The transcriptional profiles of HD fibroblasts also showed striking similarities to human tumors, including head and neck, liver, thyroid, lung, and breast cancers. This may reflect functional similarities between cancer-associated fibroblasts (CAFs) and HD fibroblasts. This is consistent with the idea that the presence of HD fibroblasts may be a hallmark of a pre-cancerous phenotype. In these biological processes, GSEA predicts that several key signaling pathways may be involved, including JNK1, iNOS, Rho GTPase(s), FGF-R, EGF-R, and PDGF-R-mediated signal transduction, thereby creating a pro-inflammatory, pro-proliferative, cytokine, and chemokine-rich microenvironment. HD fibroblasts also showed significant overlap with gene profiles derived from smooth muscle cells under stress (JNK1) and activated/infected macrophages (iNOS). Thus, HD fibroblasts may behave like activated myofibroblasts and macrophages, to create and maintain a fibrotic and inflammatory microenvironment. Finally, comparisons between the HD fibroblast gene signature and breast cancer tumor stroma revealed that JNK1 stress signaling is the single most significant biological process that is shared between these 2 data sets (with P values between 5.40E-09 and 1.02E-14), and is specifically associated with tumor recurrence. These results implicate “stromal JNK1 signaling” in the pathogenesis of human breast cancers and the transition to malignancy. Augmented TGF-β signaling also emerged as a common feature linking high breast density with tumor stroma and breast cancer recurrence (P = 5.23E-05). Similarities between the HD fibroblast gene signature, wound healing, and the cancer-associated fibroblast phenotype were also noted. Thus, this unbiased informatics analysis of high breast density provides a novel framework for additional experimental exploration and new hypothesis-driven breast cancer research, with a focus on cancer prevention and personalized medicine.
EGF; FGF; JNK; PDGF; SAPK; TGF-beta; breast cancer; cancer-associated fibroblasts; fibrosis; gene signature; inflammation; mammographic density; mammography; microenvironment; stress signaling; tumor stroma; wound healing
Metabolic coupling, between mitochondria in cancer cells and catabolism in stromal fibroblasts, promotes tumor growth, recurrence, metastasis, and predicts anticancer drug resistance. Catabolic fibroblasts donate the necessary fuels (such as L-lactate, ketones, glutamine, other amino acids, and fatty acids) to anabolic cancer cells, to metabolize via their TCA cycle and oxidative phosphorylation (OXPHOS). This provides a simple mechanism by which metabolic energy and biomass are transferred from the host microenvironment to cancer cells. Recently, we showed that catabolic metabolism and “glycolytic reprogramming” in the tumor microenvironment are orchestrated by oncogene activation and inflammation, which originates in epithelial cancer cells. Oncogenes drive the onset of the cancer-associated fibroblast phenotype in adjacent normal fibroblasts via paracrine oxidative stress. This oncogene-induced transition to malignancy is “mirrored” by a loss of caveolin-1 (Cav-1) and an increase in MCT4 in adjacent stromal fibroblasts, functionally reflecting catabolic metabolism in the tumor microenvironment. Virtually identical findings were obtained using BRCA1-deficient breast and ovarian cancer cells. Thus, oncogene activation (RAS, NFkB, TGF-β) and/or tumor suppressor loss (BRCA1) have similar functional effects on adjacent stromal fibroblasts, initiating “metabolic symbiosis” and the cancer-associated fibroblast phenotype. New therapeutic strategies that metabolically uncouple oxidative cancer cells from their glycolytic stroma or modulate oxidative stress could be used to target this lethal subtype of cancers. Targeting “fibroblast addiction” in primary and metastatic tumor cells may expose a critical Achilles’ heel, leading to disease regression in both sporadic and familial cancers.
oncogene; tumor suppressor; RAS; NFkB; TGF-beta; BRCA1; oxidative stress; glycolysis; cancer-associated fibroblast; tumor microenvironment; stromal biomarkers; metabolic symbiosis
Caveolar domains act as platforms for the organization of molecular complexes involved in signal transduction. Caveolin proteins, the principal structural components of caveolae, have been involved in many cellular processes. Caveolin-1 (Cav-1) and caveolin-2 (Cav-2) are highly expressed in the lung. Cav-1-deficient mice (Cav-1−/−) and Cav-2-deficient mice (Cav-2−/−) exhibit severe lung dysfunction attributed to a lack of Cav-2 expression. Recently, Cav-1 has been shown to regulate lung fibrosis in different models. Here, we show that Cav-2 is also involved in modulation of the fibrotic response, but through distinct mechanisms. Treatment of wild-type mice with the pulmonary fibrosis-inducer bleomycin reduced the expression of Cav-2 and its phosphorylation at tyrosine 19. Importantly, Cav-2−/− mice, but not Cav-1−/− mice, were more sensitive to bleomycin-induced lung injury in comparison to wild-type mice. Bleomycin-induced lung injury was characterized by alveolar thickening, increase in cell density, and extracellular matrix deposition. The lung injury observed in bleomycin-treated Cav-2−/− mice was not associated with alterations in the TGF-β signaling pathway and/or in the ability to produce collagen. However, apoptosis and proliferation were more prominent in lungs of bleomycin-treated Cav-2−/− mice. Since Cav-1−/− mice also lack Cav-2 expression and show a different outcome after bleomycin treatment, we conclude that Cav-1 and Cav-2 have distinct roles in bleomycin induced-lung fibrosis, and that the balance of both proteins determines the development of the fibrotic process.
caveolin; caveolae; fibrosis; bleomycin; lung
The role of PPARγ in cancer therapy is controversial, with studies showing either pro-tumorigenic or antineoplastic effects. This debate is very clinically relevant, because PPARγ agonists are used as antidiabetic drugs. Here, we evaluated if the effects of PPARγ on tumorigenesis are determined by the cell type in which PPARγ is activated. Second, we examined if the metabolic changes induced by PPARγ, such as glycolysis and autophagy, play any role in the tumorigenic process. To this end, PPARγ was overexpressed in breast cancer cells or in stromal cells. PPARγ-overexpressing cells were examined with respect to (1) their tumorigenic potential, using xenograft models, and (2) regarding their metabolic features. In xenograft models, we show that when PPARγ is activated in cancer cells, tumor growth is inhibited by 40%. However, when PPARγ is activated in stromal cells, the growth of co-injected breast cancer cells is enhanced by 60%. Thus, the effect(s) of PPARγ on tumorigenesis are dependent on the cell compartment in which PPARγ is activated. Mechanistically, stromal cells with activated PPARγ display metabolic features of cancer-associated fibroblasts, with increased autophagy, glycolysis and senescence. Indeed, fibroblasts overexpressing PPARγ show increased expression of autophagic markers, increased numbers of acidic autophagic vacuoles, increased production of L-lactate, cell hypertrophy and mitochondrial dysfunction. In addition, PPARγ fibroblasts show increased expression of CDKs (p16/p21) and β-galactosidase, which are markers of cell cycle arrest and senescence. Finally, PPARγ induces the activation of the two major transcription factors that promote autophagy and glycolysis, i.e., HIF-1α and NFκB, in stromal cells. Thus, PPARγ activation in stromal cells results in the formation of a catabolic pro-inflammatory microenvironment that metabolically supports cancer growth. Interestingly, the tumor inhibition observed when PPARγ is expressed in epithelial cancer cells is also associated with increased autophagy, suggesting that activation of an autophagic program has both pro- or antitumorigenic effects depending on the cell compartment in which it occurs. Finally, when PPARγ is expressed in epithelial cancer cells, the suppression of tumor growth is associated with a modest inhibition of angiogenesis. In conclusion, these data support the “two-compartment tumor metabolism” model, which proposes that metabolic coupling exists between catabolic stromal cells and oxidative cancer cells. Cancer cells induce autophagy, glycolysis and senescence in stromal cells. In return, stromal cells generate onco-metabolites and mitochondrial fuels (L-lactate, ketones, glutamine/aminoacids and fatty acids) that are used by cancer cells to enhance their tumorigenic potential. Thus, as researchers design new therapies, they must be conscious that cancer is not a cell-autonomous disease, but rather a tumor is an ecosystem of many different cell types, which engage in metabolic symbiosis.
CDK inhibitors; PPARγ agonist therapy; Warburg effect; autophagy; cancer associated fibroblasts; catabolism; glycolysis; inflammatory microenvironment; lactate; mitophagy; oncometabolite; p16(INK4A); p21(WAF1/CIP1); premature aging; senescence
Cigarette smoke has been directly implicated in the disease pathogenesis of a plethora of different human cancer subtypes, including breast cancers. The prevailing view is that cigarette smoke acts as a mutagen and DNA damaging agent in normal epithelial cells, driving tumor initiation. However, its potential negative metabolic effects on the normal stromal microenvironment have been largely ignored. Here, we propose a new mechanism by which carcinogen-rich cigarette smoke may promote cancer growth, by metabolically “fertilizing” the host microenvironment. More specifically, we show that cigarette smoke exposure is indeed sufficient to drive the onset of the cancer-associated fibroblast phenotype via the induction of DNA damage, autophagy and mitophagy in the tumor stroma. In turn, cigarette smoke exposure induces premature aging and mitochondrial dysfunction in stromal fibroblasts, leading to the secretion of high-energy mitochondrial fuels, such as L-lactate and ketone bodies. Hence, cigarette smoke induces catabolism in the local microenvironment, directly fueling oxidative mitochondrial metabolism (OXPHOS) in neighboring epithelial cancer cells, actively promoting anabolic tumor growth. Remarkably, these autophagic-senescent fibroblasts increased breast cancer tumor growth in vivo by up to 4-fold. Importantly, we show that cigarette smoke-induced metabolic reprogramming of the fibroblastic stroma occurs independently of tumor neo-angiogenesis. We discuss the possible implications of our current findings for the prevention of aging-associated human diseases and, especially, common epithelial cancers, as we show that cigarette smoke can systemically accelerate aging in the host microenvironment. Finally, our current findings are consistent with the idea that cigarette smoke induces the “reverse Warburg effect,” thereby fueling “two-compartment tumor metabolism” and oxidative mitochondrial metabolism in epithelial cancer cells.
carcinogens; cigarette smoke; cancer prevention; autophagy; senescence; premature aging; mitochondrial dysfunction; lactate; ketone bodies; breast cancer; tumor growth; microenvironment
Here, we provide the necessary proof of concept, that it is possible to metabolically create a non-permissive or “hostile” stromal microenvironment, which actively prevents tumor engraftment in vivo. We developed a novel genetically engineered fibroblast cell line that completely prevents tumor formation in mice, with a 100% protection rate. No host side effects were apparent. This could represent a viable cellular strategy for preventing and treating a variety of human cancers. More specifically, we examined the autocrine and paracrine effects of the cellular delivery of TNFα on breast cancer tumor growth and cancer metabolism. For this purpose, we recombinantly overexpressed TNFα in human breast cancer cells (MDA-MB-231) or human immortalized fibroblasts (hTERT-BJ1). Our results directly show that TNFα functions as a potent tumor suppressor. Remarkably, TNFα-expressing breast cancer cells were viable, without any significant increases in their basal apoptotic rate. However, after 4 weeks post-implantation, TNFα-expressing breast cancer cells failed to form any tumors in xenografted mice (0 tumors/10 injections), ultimately conferring 100% protection against tumorigenesis. Similarly, TNFα-overexpressing fibroblasts were also viable, without any increases in apoptosis. Significantly, complete tumor suppression was obtained by co-injecting TNFα expressing stromal fibroblasts with human breast cancer cells, indicating that paracrine cell-mediated delivery of TNFα can also prevent tumor engraftment and growth (0 tumors/10 injections). Mechanistically, TNFα induced autophagy and mitochondrial dysfunction in both epithelial cancer cells and stromal fibroblasts, preventing energy transfer from the tumor microenvironment, likely “starving” the cancer cells to death. In addition, via qRT-PCR analysis of MDA-MB-231 cells, we observed that TNFα mediated the upregulation of gene transcripts associated with inflammation and senescence [IL-1-β, IL-6, IL-8, MCP-1, COX-2, p21(WAF1/CIP1)] and downregulated known tumor-promoting genes (collagen VI and MMP2). Recombinant overexpression of TNFα receptor(s) in MDA-MB-231 cells also significantly reduced tumor growth, but was not as effective as the TNFα ligand itself in preventing tumor growth. Thus, we propose that stromal cell-mediated delivery of TNFα to human tumors [using transfected fibroblasts or mesenchymal stem cells (hMSCs)] may be a novel and effective strategy for the prevention and treatment of human cancers.
tumor necrosis factor (TNF); cancer prevention; cellular therapy; fibroblast mediated delivery; mitochondrial dysfunction; breast cancer; tumor growth; tumor cell engraftment; autophagy; apoptosis
Improved clinical management of prostate cancer (PCa) has been impeded by an inadequate understanding of molecular genetic elements governing tumor progression. Gene signatures have provided improved prognostic indicators of human PCa. The TGFβ/BMP-SMAD4 signaling pathway, which induces epithelial mesenchymal transition (EMT), is known to constrain prostate cancer progression induced by Pten deletion. Herein, cyclin D1 inactivation reduced cellular proliferation in the murine prostate in vivo and in isogenic oncogene-transformed prostate cancer cell lines. The in vivo cyclin D1-mediated molecular signature predicted poor outcome of recurrence free survival for prostate cancer patients (K-means hazard ratio 3.75, P-value=0.02) and demonstrated that endogenous cyclin D1 restrains TGFβ, Snail, Twist and Goosecoid signaling. Endogenous cyclin D1 enhanced Wnt and ES cell gene expression and expanded a prostate stem cell population. In ChIP-Seq, cyclin D1 occupied genes governing stem cell expansion and induced their transcription. The coordination of EMT restraining and stem cell expanding gene expression by cyclin D1 in the prostate may contribute to its strong prognostic value for poor outcome in biochemical free recurrence in human prostate cancer.
Cyclin D1; gene network; prostate cancer; siRNA; shRNA
Mutations in the BRCA1 tumor suppressor gene are commonly found in hereditary breast cancer. Similarly, downregulation of BRCA1 protein expression is observed in the majority of basal-like breast cancers. Here, we set out to study the effects of BRCA1 mutations on oxidative stress in the tumor microenvironment. To mimic the breast tumor microenvironment, we utilized an in vitro co-culture model of human BRCA1-mutated HCC1937 breast cancer cells and hTERT-immortalized human fibroblasts. Notably, HCC1937 cells induce the generation of hydrogen peroxide in the fibroblast compartment during co-culture, which can be inhibited by genetic complementation with the wild-type BRCA1 gene. Importantly, treatment with powerful antioxidants, such as NAC and Tempol, induces apoptosis in HCC1937 cells, suggesting that microenvironmental oxidative stress supports cancer cell survival. In addition, Tempol treatment increases the apoptotic rates of MDA-MB-231 cells, which have wild-type BRCA1, but share a basal-like breast cancer phenotype with HCC1937 cells. MCT4 is the main exporter of L-lactate out of cells and is a marker for oxidative stress and glycolytic metabolism. Co-culture with HCC1937 cells dramatically induces MCT4 protein expression in fibroblasts, and this can be prevented by either BRCA1 overexpression or by pharmacological treatment with NAC. We next evaluated caveolin-1 (Cav-1) expression in stromal fibroblasts. Loss of Cav-1 is a marker of the cancer-associated fibroblast (CAF) phenotype, which is linked to high stromal glycolysis, and is associated with a poor prognosis in numerous types of human cancers, including breast cancers. Remarkably, HCC1937 cells induce a loss of Cav-1 in adjacent stromal cells during co-culture. Conversely, Cav-1 expression in fibroblasts can be rescued by administration of NAC or by overexpression of BRCA1 in HCC1937 cells. Notably, BRCA1-deficient human breast cancer samples (9 out of 10) also showed a glycolytic stromal phenotype, with intense mitochondrial staining specifically in BRCA1-deficient breast cancer cells. In summary, loss of BRCA1 function leads to hydrogen peroxide generation in both epithelial breast cancer cells and neighboring stromal fibroblasts, and promotes the onset of a reactive glycolytic stroma, with increased MCT4 and decreased Cav-1 expression. Importantly, these metabolic changes can be reversed by antioxidants, which potently induce cancer cell death. Thus, antioxidant therapy appears to be synthetically lethal with a BRCA1-deficiency in breast cancer cells and should be considered for future cancer prevention trials. In this regard, immunostaining with Cav-1 and MCT4 could be used as cost-effective biomarkers to monitor the response to antioxidant therapy.
hereditary breast cancer; tumor metabolism; BRCA1 mutations; hydrogen peroxide; oxidative stress; MCT4; caveolin-1 (Cav-1); triple-negative breast cancer; synthetic lethality
Here, we present new genetic and morphological evidence that human tumors consist of two distinct metabolic compartments. First, re-analysis of genome-wide transcriptional profiling data revealed that > 95 gene transcripts associated with mitochondrial biogenesis and/or mitochondrial translation were significantly elevated in human breast cancer cells, as compared with adjacent stromal tissue. Remarkably, nearly 40 of these upregulated gene transcripts were mitochondrial ribosomal proteins (MRPs), functionally associated with mitochondrial translation of protein components of the OXPHOS complex. Second, during validation by immunohistochemistry, we observed that antibodies directed against 15 markers of mitochondrial biogenesis and/or mitochondrial translation (AKAP1, GOLPH3, GOLPH3L, MCT1, MRPL40, MRPS7, MRPS15, MRPS22, NRF1, NRF2, PGC1-α, POLRMT, TFAM, TIMM9 and TOMM70A) selectively labeled epithelial breast cancer cells. These same mitochondrial markers were largely absent or excluded from adjacent tumor stromal cells. Finally, markers of mitochondrial lipid synthesis (GOLPH3) and mitochondrial translation (POLRMT) were associated with poor clinical outcome in human breast cancer patients. Thus, we conclude that human breast cancers contain two distinct metabolic compartments—a glycolytic tumor stroma, which surrounds oxidative epithelial cancer cells—that are mitochondria-rich. The co-existence of these two compartments is indicative of metabolic symbiosis between epithelial cancer cells and their surrounding stroma. As such, epithelial breast cancer cells should be viewed as predatory metabolic “parasites,” which undergo anabolic reprogramming to amplify their mitochondrial “power.” This notion is consistent with the observation that the anti-malarial agent chloroquine may be an effective anticancer agent. New anticancer therapies should be developed to target mitochondrial biogenesis and/or mitochondrial translation in human cancer cells.
two-compartment tumor metabolism; mitochondria; oxidative phosphorylation (OXPHOS); mitochondrial biogenesis; mitochondrial translation; cancer metabolism; metabolic reprogramming
Mutations in the BRCA1 tumor suppressor gene are commonly found in hereditary ovarian cancers. Here, we used a co-culture approach to study the metabolic effects of BRCA1-null ovarian cancer cells on adjacent tumor-associated stromal fibroblasts. Our results directly show that BRCA1-null ovarian cancer cells produce large amounts of hydrogen peroxide, which can be abolished either by administration of simple antioxidants (N-acetyl-cysteine; NAC) or by replacement of the BRCA1 gene. Thus, the BRCA1 gene normally suppresses tumor growth by functioning as an antioxidant. Importantly, hydrogen peroxide produced by BRCA1-null ovarian cancer cells induces oxidative stress and catabolic processes in adjacent stromal fibroblasts, such as autophagy, mitophagy and glycolysis, via stromal NFκB activation. Catabolism in stromal fibroblasts was also accompanied by the upregulation of MCT4 and a loss of Cav-1 expression, which are established markers of a lethal tumor microenvironment. In summary, loss of the BRCA1 tumor suppressor gene induces hydrogen peroxide production, which then leads to metabolic reprogramming of the tumor stroma, driving stromal-epithelial metabolic coupling. Our results suggest that new cancer prevention trials with antioxidants are clearly warranted in patients that harbor hereditary/familial BRCA1 mutations.
BRCA1 mutations; MCT4; NFκB activation; autophagy; caveolin-1 (Cav-1); hereditary ovarian cancer; hydrogen peroxide; mitophagy; oxidative stress; tumor metabolism
Our recent studies have mechanistically demonstrated that cancer-associated fibroblasts (CAFs) produce energy-rich metabolites that functionally support the growth of cancer cells. Also, several authors have demonstrated that DNA instability in the tumor stroma greatly contributes to carcinogenesis. To further test this hypothesis, we stably knocked-down BRCA1 expression in human hTERT-immortalized fibroblasts (shBRCA1) using an shRNA lentiviral approach. As expected, shBRCA1 fibroblasts displayed an elevated growth rate. Using immunofluorescence and immunoblot analysis, shBRCA1 fibroblasts demonstrated an increase in markers of autophagy and mitophagy. Most notably, shBRCA1 fibroblasts also displayed an elevation of HIF-1α expression. In accordance with these findings, shBRCA1 fibroblasts showed a 5.5-fold increase in ketone body production; ketone bodies function as high-energy mitochondrial fuels. This is consistent with the onset of mitochondrial dysfunction in BRCA1-deficient fibroblasts. Conversely, after 48 h of co-culturing shBRCA1 fibroblasts with a human breast cancer cell line (MDA-MB-231 cell), mitochondrial activity was enhanced in these epithelial cancer cells. Interestingly, our preclinical studies using xenografts demonstrated that shBRCA1 fibroblasts induced an ~2.2-fold increase in tumor growth when co-injected with MDA-MB-231 cells into nude mice. We conclude that a BRCA1 deficiency in the tumor stroma metabolically promotes cancer progression, via ketone production.
BRCA1; cancer metabolism; stromal fibroblasts; ketone bodies; HIF1; mitochondrial OXPHOS; autophagy; mitophagy
Here, we set out to test the novel hypothesis that increased mitochondrial biogenesis in epithelial cancer cells would “fuel” enhanced tumor growth. For this purpose, we generated MDA-MB-231 cells (a triple-negative human breast cancer cell line) overexpressing PGC-1α and MitoNEET, which are established molecules that drive mitochondrial biogenesis and increased mitochondrial oxidative phosphorylation (OXPHOS). Interestingly, both PGC-1α and MitoNEET increased the abundance of OXPHOS protein complexes, conferred autophagy resistance under conditions of starvation and increased tumor growth by up to ~3-fold. However, this increase in tumor growth was independent of neo-angiogenesis, as assessed by immunostaining and quantitation of vessel density using CD31 antibodies. Quantitatively similar increases in tumor growth were also observed by overexpression of PGC-1β and POLRMT in MDA-MB-231 cells, which are also responsible for mediating increased mitochondrial biogenesis. Thus, we propose that increased mitochondrial “power” in epithelial cancer cells oncogenically promotes tumor growth by conferring autophagy resistance. As such, PGC-1α, PGC-1β, mitoNEET and POLRMT should all be considered as tumor promoters or “metabolic oncogenes.” Our results are consistent with numerous previous clinical studies showing that metformin (a weak mitochondrial “poison”) prevents the onset of nearly all types of human cancers in diabetic patients. Therefore, metformin (a complex I inhibitor) and other mitochondrial inhibitors should be developed as novel anticancer therapies, targeting mitochondrial metabolism in cancer cells.
cancer metabolism; mitochondrial biogenesis; oxidative phosphorylation; OXPHOS; autophagy resistance; angiogenesis; two-compartment tumor metabolism
Soluble guanylyl cyclase (sGC) generates cyclic guanosine monophophate (cGMP) upon activation by nitric oxide (NO). Cardiac NO-sGC-cGMP signaling blunts cardiac stress responses, including pressure-overload induced hypertrophy. The latter itself depresses signaling via this pathway by reducing NO generation and enhancing cGMP hydrolysis.
We tested the hypothesis that the sGC response to NO also declines with pressure-overload stress, and assessed the role of heme-oxidation and altered intracellular compartmentation of sGC as potential mechanisms.
Methods and Results
C57BL/6 mice subjected to transverse aortic constriction developed cardiac hypertrophy and dysfunction. NO-stimulated sGC activity was markedly depressed, while NO- and heme-independent sGC activation by BAY 60-2770 was preserved. Total sGCα1 and β1 expression were unchanged by TAC, however sGCβ1 subunits shifted out of caveolin-enriched microdomains. NO-stimulated sGC activity was 2–3-fold greater in Cav3-containing lipid raft versus non-lipid raft domains in control, and 6-fold greater after TAC. In contrast, BAY 60-2770 responses were >10 fold higher in non-Cav3 domains with and without TAC, declining about 60% after TAC within each compartment. Mice genetically lacking Cav3 had reduced NO and BAY-stimulated sGC activity in microdomains containing Cav3 for controls, but no change within non-Cav3-enriched domains.
Pressure-overload depresses NO/heme-dependent sGC activation in the heart, consistent with enhanced oxidation. The data reveal a novel additional mechanism for reduced NO-coupled sGC activity related to dynamic shifts in membrane microdomain localization, with Cav3-microdomains protecting sGC from heme-oxidation and facilitating NO-responsiveness. Translocation of sGC out of this domain favors sGC oxidation and contributes to depressed NO-stimulated sGC activity.
hypertrophy; soluble guanylyl cyclase; caveolae; signaling; cardiomyocyte
We have previously suggested that ketone body metabolism is critical for tumor progression and metastasis. Here, using a co-culture system employing human breast cancer cells (MCF7) and hTERT-immortalized fibroblasts, we provide new evidence to directly support this hypothesis. More specifically, we show that the enzymes required for ketone body production are highly upregulated within cancer-associated fibroblasts. This appears to be mechanistically controlled by the stromal expression of caveolin-1 (Cav-1) and/or serum starvation. In addition, treatment with ketone bodies (such as 3-hydroxy-butyrate, and/or butanediol) is sufficient to drive mitochondrial biogenesis in human breast cancer cells. This observation was also validated by unbiased proteomic analysis. Interestingly, an MCT1 inhibitor was sufficient to block the onset of mitochondrial biogenesis in human breast cancer cells, suggesting a possible avenue for anticancer therapy. Finally, using human breast cancer tumor samples, we directly confirmed that the enzymes associated with ketone body production (HMGCS2, HMGCL and BDH1) were preferentially expressed in the tumor stroma. Conversely, enzymes associated with ketone re-utilization (ACAT1) and mitochondrial biogenesis (HSP60) were selectively associated with the epithelial tumor cell compartment. Our current findings are consistent with the “two-compartment tumor metabolism” model. Furthermore, they suggest that we should target ketone body metabolism as a new area for drug discovery, for the prevention and treatment of human cancers.
ketone body; 3-hydroxy-butyrate; cancer metabolism; BDH1; HMGCS2; ACAT isoforms; tumor growth; metastasis
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 MDA-MB-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.
caveolin-1; hypoxia-inducible factor; HIF-1alpha; HIF-2alpha; metabolic coupling; tumor stroma; cancer-associated fibroblasts; aerobic glycolysis; mitochondrial metabolism; OXPHOS
We have previously shown that a loss of stromal Cav-1 is a biomarker of poor prognosis in breast cancers. Mechanistically, a loss of Cav-1 induces the metabolic reprogramming of stromal cells, with increased autophagy/mitophagy, mitochondrial dysfunction and aerobic glycolysis. As a consequence, Cav-1-low CAFs generate nutrients (such as L-lactate) and chemical building blocks that fuel mitochondrial metabolism and the anabolic growth of adjacent breast cancer cells. It is also known that a loss of Cav-1 is associated with hyperactive TGF-β signaling. However, it remains unknown whether hyperactivation of the TGF-β signaling pathway contributes to the metabolic reprogramming of Cav-1-low CAFs. To address these issues, we overexpressed TGF-β ligands and the TGF-β receptor I (TGFβ-RI) in stromal fibroblasts and breast cancer cells. Here, we show that the role of TGF-β in tumorigenesis is compartment-specific, and that TGF-β promotes tumorigenesis by shifting cancer-associated fibroblasts toward catabolic metabolism. Importantly, the tumor-promoting effects of TGF-β are independent of the cell type generating TGF-β. Thus, stromal-derived TGF-β activates signaling in stromal cells in an autocrine fashion, leading to fibroblast activation, as judged by increased expression of myofibroblast markers, and metabolic reprogramming, with a shift toward catabolic metabolism and oxidative stress. We also show that TGF-β-activated fibroblasts promote the mitochondrial activity of adjacent cancer cells, and in a xenograft model, enhancing the growth of breast cancer cells, independently of angiogenesis. Conversely, activation of the TGF-β pathway in cancer cells does not influence tumor growth, but cancer cell-derived-TGF-β ligands affect stromal cells in a paracrine fashion, leading to fibroblast activation and enhanced tumor growth. In conclusion, ligand-dependent or cell-autonomous activation of the TGF-β pathway in stromal cells induces their metabolic reprogramming, with increased oxidative stress, autophagy/mitophagy and glycolysis, and downregulation of Cav-1. These metabolic alterations can spread among neighboring fibroblasts and greatly sustain the growth of breast cancer cells. Our data provide novel insights into the role of the TGF-β pathway in breast tumorigenesis, and establish a clear causative link between the tumor-promoting effects of TGF-β signaling and the metabolic reprogramming of the tumor microenvironment.
TGF beta; aerobic glycolysis; autocrine signaling; autophagy; cancer associated fibroblast; cancer metabolism; mitophagy; myofibroblast; oxidative stress; paracrine signaling; the field effect; tumor stroma; “Pied-Piper of Hamelin”
Recent studies in cancer metabolism directly implicate catabolic fibroblasts as a new rich source of i) energy and ii) biomass, for the growth and survival of anabolic cancer cells. Conversely, anabolic cancer cells upregulate oxidative mitochondrial metabolism, to take advantage of the abundant fibroblast fuel supply. This simple model of “metabolic-symbiosis” has now been independently validated in several different types of human cancers, including breast, ovarian, and prostate tumors. Biomarkers of metabolic-symbiosis are excellent predictors of tumor recurrence, metastasis, and drug resistance, as well as poor patient survival. New pre-clinical models of metabolic-symbiosis have been generated and they genetically validate that catabolic fibroblasts promote tumor growth and metastasis. Over 30 different stable lines of catabolic fibroblasts and >10 different lines of anabolic cancer cells have been created and are well-characterized. For example, catabolic fibroblasts harboring ATG16L1 increase tumor cell metastasis by >11.5-fold, despite the fact that genetically identical cancer cells were used. Taken together, these studies provide >40 novel validated targets, for new drug discovery and anti-cancer therapy. Since anabolic cancer cells amplify their capacity for oxidative mitochondrial metabolism, we should consider therapeutically targeting mitochondrial biogenesis and OXPHOS in epithelial cancer cells. As metabolic-symbiosis promotes drug-resistance and may represent the escape mechanism during anti-angiogenic therapy, new drugs targeting metabolic-symbiosis may also be effective in cancer patients with recurrent and advanced metastatic disease.
cancer metabolism; therapeutic targets; drug discovery; oncogenes; tumor suppressors; oxidative stress; glycolysis; cancer associated fibroblast; tumor microenvironment; metabolic symbiosis; anti-angiogenic therapy
Here, we developed a model system to evaluate the metabolic effects of oncogene(s) on the host microenvironment. A matched set of “normal” and oncogenically transformed epithelial cell lines were co-cultured with human fibroblasts, to determine the “bystander” effects of oncogenes on stromal cells. ROS production and glucose uptake were measured by FACS analysis. In addition, expression of a panel of metabolic protein biomarkers (Caveolin-1, MCT1, and MCT4) was analyzed in parallel. Interestingly, oncogene activation in cancer cells was sufficient to induce the metabolic reprogramming of cancer-associated fibroblasts toward glycolysis, via oxidative stress. Evidence for “metabolic symbiosis” between oxidative cancer cells and glycolytic fibroblasts was provided by MCT1/4 immunostaining. As such, oncogenes drive the establishment of a stromal-epithelial “lactate-shuttle”, to fuel the anabolic growth of cancer cells. Similar results were obtained with two divergent oncogenes (RAS and NFκB), indicating that ROS production and inflammation metabolically converge on the tumor stroma, driving glycolysis and upregulation of MCT4. These findings make stromal MCT4 an attractive target for new drug discovery, as MCT4 is a shared endpoint for the metabolic effects of many oncogenic stimuli. Thus, diverse oncogenes stimulate a common metabolic response in the tumor stroma. Conversely, we also show that fibroblasts protect cancer cells against oncogenic stress and senescence by reducing ROS production in tumor cells. Ras-transformed cells were also able to metabolically reprogram normal adjacent epithelia, indicating that cancer cells can use either fibroblasts or epithelial cells as “partners” for metabolic symbiosis. The antioxidant N-acetyl-cysteine (NAC) selectively halted mitochondrial biogenesis in Ras-transformed cells, but not in normal epithelia. NAC also blocked stromal induction of MCT4, indicating that NAC effectively functions as an “MCT4 inhibitor”. Taken together, our data provide new strategies for achieving more effective anticancer therapy. We conclude that oncogenes enable cancer cells to behave as selfish “metabolic parasites”, like foreign organisms (bacteria, fungi, viruses). Thus, we should consider treating cancer like an infectious disease, with new classes of metabolically targeted “antibiotics” to selectively starve cancer cells. Our results provide new support for the “seed and soil” hypothesis, which was first proposed in 1889 by the English surgeon, Stephen Paget.
oncogene; oxidative stress; glycolysis; reverse Warburg effect; RAS; inflammation; NFkB; cancer associated fibroblast; tumor microenvironment; HaCaT; MCT1; MCT4; caveolin-1; TOMM20; mitochondrial metabolism; wound healing; response to injury; field cancerization; metabolic parasite; autophagy; senescence; oncogenic stress; stromal biomarkers
Significance: Here, we review certain recent advances in oxidative stress and tumor metabolism, which are related to understanding the contributions of the microenvironment in promoting tumor growth and metastasis. In the early 1920s, Otto Warburg, a Nobel Laureate, formulated a hypothesis to explain the “fundamental basis” of cancer, based on his observations that tumors displayed a metabolic shift toward glycolysis. In 1963, Christian de Duve, another Nobel Laureate, first coined the phrase auto-phagy, derived from the Greek words “auto” and “phagy,” meaning “self” and “eating.” Recent Advances: Now, we see that these two ideas (autophagy and aerobic glycolysis) physically converge in the tumor stroma. First, cancer cells secrete hydrogen peroxide. Then, as a consequence, oxidative stress in cancer-associated fibroblasts drives autophagy, mitophagy, and aerobic glycolysis. Critical Issues: This “parasitic” metabolic coupling converts the stroma into a “factory” for the local production of recycled and high-energy nutrients (such as L-lactate)—to fuel oxidative mitochondrial metabolism in cancer cells. We believe that Warburg and de Duve would be pleased with this new two-compartment model for understanding tumor metabolism. It adds a novel stromal twist to two very well-established cancer paradigms: aerobic glycolysis and autophagy. Future Directions: Undoubtedly, these new metabolic models will foster the development of novel biomarkers, and corresponding therapies, to achieve the goal of personalized cancer medicine. Given the central role that oxidative stress plays in this process, new powerful antioxidants should be developed in the fight against cancer. Antioxid. Redox Signal. 16, 1264–1284.
The Nod-like receptor, Nlrp3, has been linked to inflammatory diseases and adjuvant-mediated immune responses. A wide array of structurally diverse agents does not interact directly with Nlrp3, but is thought to activate the Nlrp3 inflammasome by inducing a common upstream signal, such as lysosome rupture. To test the connection between lysosome integrity and Nlrp3 signaling, we analyzed inflammasome activation following stimulation of murine macrophages with lysosome-destabilizing agents and pyroptosis inducers. Here we provide evidence that lysosomal rupture and the corresponding release of lysosomal hydrolases is an early event in macrophages exposed to the lysosome-destabilizing adjuvants LLOMe and alum. Lysosome rupture preceded cell death induction mediated by these agents and was associated with the degradation of low-molecular weight proteins, including the inflammasome component caspase-1. Proteolysis of caspase-1 was controlled by specific cathepsins, but was independent of autocatalytic processes and Nlrp3 signaling. Consistent with these findings, lysosome-disrupting agents triggered only minimal caspase-1 activation and failed to cause caspase-1-dependent cell death (pyroptosis), generally associated with Nlrp3 signaling. In contrast, lysosome rupture was a late event in macrophages exposed to prototypical pyroptosis inducers. These agents triggered extensive Nlrp3 signaling prior to lysosome rupture with only minimal impact on the cellular proteome. Taken together, our findings suggest that lysosome impairment triggers a cascade of events culminating in cell death but is not crucial for Nlrp3 signaling. The significant differences observed between lysosome-disrupting agents and pyroptosis inducers might explain the distinct immunologic responses associated with these compounds.
Nlrp3 inflammasome; caspase-1; lysosome rupture; necrosis; pyroptosis
Both cell-autonomous and non-cell-autonomous factors contribute to tumor growth and metastasis of melanoma. The function of Caveolin-1 (Cav1), a multifunctional scaffold protein known to modulate several biological processes in both normal tissue and cancer, has been recently investigated in melanoma cancer cells, but its role in the melanoma microenvironment remains largely unexplored. Here, we show that orthotopic implantation of B16F10 melanoma cells in the skin of Cav1KO mice increases tumor growth, and co-injection of Cav1-deficient dermal fibroblasts with melanoma cells is sufficient to recapitulate the tumor phenotype observed in Cav1KO mice. Using indirect co-culture experiments with fibroblasts and melanoma cells combined with cytokine analysis, we found that Cav1-deficient fibroblasts promoted the growth of melanoma cells via enhanced paracrine cytokine signaling. Specifically, Cav1-deficient fibroblasts displayed increased ShhN expression, which heterotypically enhanced the Shh signaling pathway in melanoma cells. In contrast to primary tumor growth, the ability of B16F10 melanoma cells to form lung metastases was significantly reduced in Cav1KO mice. This phenotype was associated mechanistically with the inability of melanoma cells to adhere to and to transmigrate through a monolayer of endothelial cells lacking Cav1. Together, our findings demonstrate that Cav1 may regulate different mechanisms during primary melanoma tumor growth and metastatic dissemination.
Caveolin-1 (Cav-1) is a critical regulator of tumor progression in a variety of cancers where it has been shown to act as either a tumor suppressor or tumor promoter. In glioblastoma multiforme, it has been previously demonstrated to function as a putative tumor suppressor. Our studies here, using the human glioblastoma-derived cell line U-87MG, further support the role of Cav-1 as a negative regulator of tumor growth. Using a lentiviral transduction approach, we were able to stably overexpress Cav-1 in U-87MG cells. Gene expression microarray analyses demonstrated significant enrichment in gene signatures corresponding to downregulation of MAPK, PI3K/AKT and mTOR signaling, as well as activation of apoptotic pathways in Cav-1-overexpressing U-87MG cells. These same gene signatures were later confirmed at the protein level in vitro. To explore the ability of Cav-1 to regulate tumor growth in vivo, we further show that Cav-1-overexpressing U-87MG cells display reduced tumorigenicity in an ectopic xenograft mouse model, with marked hypoactivation of MAPK and PI3K/mTOR pathways. Finally, we demonstrate that Cav-1 overexpression confers sensitivity to the most commonly used chemotherapy for glioblastoma, temozolomide. In conclusion, Cav-1 negatively regulates key cell growth and survival pathways and may be an effective biomarker for predicting response to chemotherapy in glioblastoma.
Caveolin-1; glioma; brain cancer; tumor progression; tumor suppressor; microarray; mouse model; chemotherapy; temozolomide
Here, we interrogated head and neck cancer (HNSCC) specimens (n = 12) to examine if different metabolic compartments (oxidative vs. glycolytic) co-exist in human tumors. A large panel of well-established biomarkers was employed to determine the metabolic state of proliferative cancer cells. Interestingly, cell proliferation in cancer cells, as marked by Ki-67 immunostaining, was strictly correlated with oxidative mitochondrial metabolism (OXPHOS) and the uptake of mitochondrial fuels, as detected via MCT1 expression (p < 0.001). More specifically, three metabolic tumor compartments were delineated: (1) proliferative and mitochondrial-rich cancer cells (Ki-67+/TOMM20+/COX+/MCT1+); (2) non-proliferative and mitochondrial-poor cancer cells (Ki-67−/TOMM20−/COX−/MCT1−); and (3) non-proliferative and mitochondrial-poor stromal cells (Ki-67−/TOMM20−/COX−/MCT1−). In addition, high oxidative stress (MCT4+) was very specific for cancer tissues. Thus, we next evaluated the prognostic value of MCT4 in a second independent patient cohort (n = 40). Most importantly, oxidative stress (MCT4+) in non-proliferating epithelial cancer cells predicted poor clinical outcome (tumor recurrence; p < 0.0001; log-rank test), and was functionally associated with FDG-PET avidity (p < 0.04). Similarly, oxidative stress (MCT4+) in tumor stromal cells was specifically associated with higher tumor stage (p < 0.03), and was a highly specific marker for cancer-associated fibroblasts (p < 0.001). We propose that oxidative stress is a key hallmark of tumor tissues that drives high-energy metabolism in adjacent proliferating mitochondrial-rich cancer cells, via the paracrine transfer of mitochondrial fuels (such as L-lactate and ketone bodies). New antioxidants and MCT4 inhibitors should be developed to metabolically target “three-compartment tumor metabolism” in head and neck cancers. It is remarkable that two “non-proliferating” populations of cells (Ki-67−/MCT4+) within the tumor can actually determine clinical outcome, likely by providing high-energy mitochondrial “fuels” for proliferative cancer cells to burn. Finally, we also show that in normal mucosal tissue, the basal epithelial “stem cell” layer is hyper-proliferative (Ki-67+), mitochondrial-rich (TOMM20+/COX+) and is metabolically programmed to use mitochondrial fuels (MCT1+), such as ketone bodies and L-lactate. Thus, oxidative mitochondrial metabolism (OXPHOS) is a common feature of both (1) normal stem cells and (2) proliferating cancer cells. As such, we should consider metabolically treating cancer patients with mitochondrial inhibitors (such as Metformin), and/or with a combination of MCT1 and MCT4 inhibitors, to target “metabolic symbiosis.”
head and neck cancer; tumor recurrence; oxidative stress; stem cells; mitochondria; OXPHOS; glycolysis; monocarboxylate transporters (MCT); MCT1; MCT4; metabolic symbiosis; TOMM20; tumor stroma