Levels of caveolin-1 (Cav-1) in tumour epithelial cells increase during prostate cancer progression. Conversely, Cav-1 expression in the stroma can decline in advanced and metastatic prostate cancer. In a large cohort of 724 prostate cancers, we observed significantly decreased levels of stromal Cav-1 in concordance with increased Gleason score (p = 0.012). Importantly, reduced expression of Cav-1 in the stroma correlated with reduced relapse-free survival (p = 0.009), suggesting a role for stromal Cav-1 in inhibiting advanced disease. Silencing of Cav-1 by shRNA in WPMY-1 prostate fibroblasts resulted in up-regulation of Akt phosphorylation, and significantly altered expression of genes involved in angiogenesis, invasion, and metastasis, including a > 2.5-fold increase in TGF-β1 and γ-synuclein (SNCG) gene expression. Moreover, silencing of Cav-1 induced migration of prostate cancer cells when stromal cells were used as attractants. Pharmacological inhibition of Akt caused down-regulation of TGF-β1 and SNCG, suggesting that loss of Cav-1 in the stroma can influence Akt-mediated signalling in the tumour microenvironment. Cav-1-depleted stromal cells exhibited increased levels of intracellular cholesterol, a precursor for androgen biosynthesis, steroidogenic enzymes, and testosterone. These findings suggest that loss of Cav-1 in the tumour microenvironment contributes to the metastatic behaviour of tumour cells by a mechanism that involves up-regulation of TGF-β1 and SNCG through Akt activation. They also suggest that intracrine production of androgens, a process relevant to castration resistance, may occur in the stroma.
caveolin-1; stroma; prostate cancer; prognosis
Trypanosoma cruzi infection in humans and experimental animals causes Chagas disease which is often accompanied by myocarditis, cardiomyopathy and vasculopathy. T. cruzi-derived thromboxane A2 (TXA2) modulates vasculopathy and other pathophysiological features of Chagasic cardiomyopathy. Here, we provide evidence that epimastigotes, trypomastigotes and amastigotes of T. cruzi (Brazil and Tulahuen strains) express a biologically active thromboxane prostanoid (TP) receptor that is responsive to TXA2 mimetics, e.g. IBOP. This putative receptor, TcTP, is mainly localized in the flagellar membrane of the parasites and shows a similar glycosylation pattern to that of TPs obtained from human platelets. Furthermore, TXA2-TP signal transduction activates T. cruzi specific MAPK pathways. While mammalian TP is a G-protein coupled receptor (GPCR); T. cruzi genome sequencing has not demonstrated any GPCRs in these parasites. Based on this genome sequencing it is likely that TcTP is unique in these protists with no counterpart in mammals. TXA2 is a potent vasoconstrictor which contributes to the pathogenesis of Chagasic cardiovascular disease. It may, however, also control parasite differentiation and proliferation in the infected host allowing the infection to progress to a chronic state.
Thromboxane; thromboxane receptor; MAPK; Trypanosoma cruzi; Chagas disease; Prostanoid receptor
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
Herein, murine prostate cancer cell lines, generated via selective transduction with a single oncogene (c-Myc, Ha-Ras, and v-Src), demonstrated oncogene-specific prostate cancer molecular signatures that were recapitulated in human prostate cancer, and developed lung metastasis in immune competent mice. Interrogation of two independent retrospective cohorts of patient samples using the oncogene signature demonstrated an ability to distinguish tumor from normal prostate with a predictive value for prostate cancer of 98 – 99%. In a blinded study, the signature algorithm demonstrated independent substratification of reduced recurrence free survival by Kaplan-Meier analysis. The generation of new oncogene-specific prostate cancer cell lines that recapitulate human prostate cancer gene expression, that metastasize in immune-competent mice, are a valuable new resource for testing targeted therapy while the molecular signatures identified herein provides further value over current gene signature markers of prediction and outcome.
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
The laboratory and clinical evidence that exists for calorie restriction is reviewed, showing compelling evidence through the molecular pathways calorie restriction induces about how it may be used as a treatment in tandem with radiation therapy to improve rates of disease control.
CME Learning Objectives
Identify molecular pathways that are potential targets of calorie restriction combined with radiation therapy.Identify cancer patients for whom calorie restriction would be contraindicated.
Calorie restriction (CR), or a diet modification aiming to reduce the total intake of calories by 20%–40%, has been shown to increase longevity across multiple species. Recently, there has been growing interest in investigating the potential role of CR as a treatment intervention for age-related diseases, such as cancer, because an increasing body of literature has demonstrated a metabolic component to both carcinogenesis and tumor progression. In fact, many of the molecular pathways that are altered with CR are also known to be altered in cancer. Therefore, manipulation of these pathways using CR can render cancer cells, and most notably breast cancer cells, more susceptible to standard cytotoxic treatment with radiation and chemotherapy. In this review article we demonstrate the laboratory and clinical evidence that exists for CR and show compelling evidence through the molecular pathways CR induces about how it may be used as a treatment in tandem with radiation therapy to improve our rates of disease control.
Calorie restriction; Intermittent fasting; Radiotherapy; Cancer
Cancer Stem Cells (CSCs) are a small subpopulation of cells within tumors with capabilities of self-renewal, differentiation, and tumorigenicity when transplanted into an animal host. A number of cell surface markers such as CD44, CD24, and CD133 are often used to identify and enrich CSCs. A regulatory network consisting of microRNAs and Wnt/β-catenin, Notch, and Hedgehog signaling pathways controls the CSC properties. The clinical relevance of CSCs has been strengthened by emerging evidence, demonstrating that CSCs are resistant to conventional chemotherapy and radiation treatment and that CSCs are very likely to be the origin of cancer metastasis. CSCs are believed to be an important target for novel anti-cancer drug discovery. Herein we summarize the current understanding of CSCs, with a focus on the role of miRNA and epithelial mesenchymal transition (EMT), and discuss the clinical application of targeting CSCs for cancer treatment.
Cancer stem cells; tumorigenesis; relapse; metastasis; miRNA
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
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
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
Previous studies have identified cholesterol as an important regulator of breast cancer development. High-density lipoprotein (HDL) and its cellular receptor, the scavenger receptor class B type I (SR-BI) have both been implicated in the regulation of cellular cholesterol homeostasis, but their functions in cancer remain to be established.
In the present study, we have examined the role of HDL and SR-BI in the regulation of cellular signaling pathways in breast cancer cell lines and in the development of tumor in a mouse xenograft model.
Our data show that HDL is capable of stimulating migration and can activate signal transduction pathways in the two human breast cancer cell lines, MDA-MB-231 and MCF7. Furthermore, we also show that knockdown of the HDL receptor, SR-BI, attenuates HDL-induced activation of the phosphatidylinositol 3-kinase (PI3K)/protein Kinase B (Akt) pathway in both cell lines. Additional investigations show that inhibition of the PI3K pathway, but not that of the mitogen-activated protein kinase (MAPK) pathway, could lead to a reduction in cellular proliferation in the absence of SR-BI. Importantly, whereas the knockdown of SR-BI led to decreased proliferation and migration in vitro, it also led to a significant reduction in tumor growth in vivo. Most important, we also show that pharmacological inhibition of SR-BI can attenuate signaling and lead to decreased cellular proliferation in vitro. Taken together, our data indicate that both cholesteryl ester entry via HDL-SR-BI and Akt signaling play an essential role in the regulation of cellular proliferation and migration, and, eventually, tumor growth.
These results identify SR-BI as a potential target for the treatment of breast cancer.
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
Reactive oxygen species can induce premature senescence. Caveolin-1 promotes oxidative stress–induced activation of the p53/p21Waf1/Cip1 pathway and development of premature senescence by acting as an endogenous inhibitor of the transcription factor Nrf2.
Reactive oxygen species (ROS) can induce premature cellular senescence, which is believed to contribute to aging and age-related diseases. The nuclear erythroid 2 p45–related factor-2 (Nrf2) is a transcription factor that mediates cytoprotective responses against stress. We demonstrate that caveolin-1 is a direct binding partner of Nrf2, as shown by the binding of the scaffolding domain of caveolin-1 (amino acids 82–101) to the caveolin-binding domain of Nrf2 (amino acids 281–289). Biochemical studies show that Nrf2 is concentrated into caveolar membranes in human and mouse fibroblasts, where it colocalizes with caveolin-1, under resting conditions. After oxidative stress, caveolin-1 limits the movement of Nrf2 from caveolar membranes to the nucleus. In contrast, Nrf2 is constitutively localized to the nucleus before and after oxidative stress in caveolin-1–null mouse embryonic fibroblasts (MEFs), which do not express caveolin-1. Functional studies demonstrate that caveolin-1 acts as an endogenous inhibitor of Nrf2, as shown by the enhanced up-regulation of NQO1, an Nrf2 target gene, in caveolin-1–null MEFs and the activation or inhibition of a luciferase construct carrying an antioxidant responsive element (ARE) after down-regulation of caveolin-1 by small interfering RNA or overexpression of caveolin-1, respectively. Expression of a mutant form of Nrf2 that cannot bind to caveolin-1 (Φ→A-Nrf2) hyperactivates ARE and inhibits oxidative stress–induced activation of the p53/p21Waf1/Cip1 pathway and induction of premature senescence in fibroblasts. Finally, we show that overexpression of caveolin-1 in colon cancer cells inhibits oxidant-induced activation of Nrf2-dependent signaling, promotes premature senescence, and inhibits their transformed phenotype. Thus, by inhibiting Nrf2-mediated signaling, caveolin-1 links free radicals to the activation of the p53/senescence pathway.
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”
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
Breast cancer is a leading form of cancer in the world. The Drosophila Dac gene was cloned as an inhibitor of the hyperactive epidermal growth factor (EGFR), ellipse. Herein, endogenous DACH1 co-localized with p53 in a nuclear, extranucleolar compartment and bound to p53 in human breast cancer cell lines, p53 and DACH1 bound common genes in Chip-Seq. Full inhibition of breast cancer contact-independent growth by DACH1 required p53. The p53 breast cancer mutants R248Q and R273H, evaded DACH1 binding. DACH1 phosphorylation at serine residue (S439) inhibited p53 binding and phosphorylation at p53 amino-terminal sites (S15, S20) enhanced DACH1 binding. DACH1 binding to p53 was inhibited by NAD-dependent deacetylation via DACH1 K628. DACH1 repressed p21CIP1 and induced RAD51, an association found in basal breast cancer. DACH1 inhibits breast cancer cellular growth in an NAD and p53-dependent manner through direct protein-protein association.
p53; breast cancer; cell fate; stem cells; dach
Dietary modification such as caloric restriction (CR) has been shown to decrease tumor initiation and progression. We sought to determine if nutrient restriction could be used as a novel therapeutic intervention to enhance cytotoxic therapies such as radiation (IR) and alter the molecular profile of triple-negative breast cancer (TNBC), which displays a poor prognosis. In two murine models of TNBC, significant tumor regression is noted with IR or diet modification, and a greater regression is observed combining diet modification with IR. Two methods of diet modification were compared, and it was found that a daily 30% reduction in total calories provided more significant tumor regression than alternate day feeding. At the molecular level, tumors treated with CR and IR showed less proliferation and more apoptosis. cDNA array analysis demonstrated the IGF-1R pathway plays a key role in achieving this physiologic response, and multiple members of the IGF-1R pathway including IGF-1R, IRS, PIK3ca and mTOR were found to be downregulated. The innovative use of CR as a novel therapeutic option has the potential to change the biology of tumors and enhance the opportunity for clinical benefit in the treatment of patients with TNBC.
caloric restriction; breast cancer; radiation; IGF; tumor regression; cytotoxic therapy