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
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
Here, we identified the milk protein α-casein as a novel suppressor of tumor growth and metastasis. Briefly, Met-1 mammary tumor cells expressing α-casein showed a ~5-fold reduction in tumor growth and a near 10-fold decrease in experimental metastasis. To identify the molecular mechanism(s), we performed genome-wide transcriptional profiling. Interestingly, our results show that α-casein upregulates gene transcripts associated with interferon/STAT1 signaling and downregulates genes associated with “stemness.” These findings were validated by immunoblot and FACS analysis, which showed the upregulation and hyperactivation of STAT1 and a decrease in the number of CD44(+) “cancer stem cells.” These gene signatures were also able to predict clinical outcome in human breast cancer patients. Thus, we conclude that a lactation-based therapeutic strategy using recombinant α-casein would provide a more natural and non-toxic approach to the development of novel anticancer therapies.
breast cancer; lactation; milk proteins; casein; STAT1; interferon signaling; cancer stem cells; 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
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.
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
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.
Cav1; caveolin; skin cancer; skin; two stage carcinogenesis
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.
aging; mitochondria; cancer metabolism; autophagy; mitophagy; aerobic glycolysis; oxidative phosphorylation; Metformin; drug resistance; chemoresistance; Warburg effect; metabolic compartments; parasite; PGC1a; PGC1b; NRF1; two-compartment tumor metabolism
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.
MCT4; alcohol consumption; breast cancer prevention; cancer metabolism; caveolin-1; ethanol; ketone bodies; mitochondrial biogenesis; myofibroblast; oxidative phosphorylation (OXPHOS); oxidative stress; reactive oxygen species (ROS)
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.
chemoprevention; Metformin; mitochondrial dysfunction; breast cancer; tumor growth; UCP; mitochondrial uncoupling proteins; autophagy; ketone body production; fatty acid beta-oxidation; ATP-rich vesicles
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.
drug resistance; tamoxifen; metformin; tumor stroma; microenvironment; Warburg Effect; aerobic glycolysis; mitochondrial oxidative phosphorylation; TIGAR; glucose uptake; oxidative stress; reactive oxygen species (ROS); cancer associated fibroblasts
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.
caveolae; caveolin-1; epithelial-mesenchymal transition; E-cadherin; pancreatic cancer; cell differentiation; chemoresistance
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.
ketone body; 3-hydroxy-butyrate; cancer metabolism; BDH1; HMGCS2; OXCT isoforms; ACAT isoforms; tumor growth; metastasis
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.
CDK inhibitors; PD0332991; aging; autophagy; cancer metabolism; cancer-associated fibroblast; cell cycle arrest; glycolysis; mitophagy; senescence; tumor initiation; tumor stroma
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.
TGF-beta signaling; aerobic glycolysis; cancer-associated fibroblasts; metabolic coupling; migration stimulating factor (MSF); myofibroblast; tumor stroma
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.
hydrogen peroxide; tumor stroma; microenvironment; aging; DNA damage; inflammation; NFκB; cancer metabolism; HIF1; autophagy; hypoxia; oxidative stress; seed and soil hypothesis