Hereditary ovarian cancer and two-compartment tumor metabolism: Epithelial loss of BRCA1 induces hydrogen peroxide production,driving oxidative stress and NFκB activation in the tumor stroma

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 drive oxidative stress and glycolysis in the tumor microenvironment

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.     

Oncogenes induce the cancer-associated fibroblast phenotype: Metabolic symbiosis and “fibroblast addiction” are new therapeutic targets for drug discovery

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.     

Cancer cells metabolically “fertilize” the tumor microenvironment with hydrogen peroxide,driving the Warburg effect: Implications for PET imaging of human tumors

Previously, we proposed that cancer cells behave as metabolic parasites, as they use targeted oxidative stress as a “weapon” to extract recycled nutrients from adjacent stromal cells. Oxidative stress in cancer-associated fibroblasts triggers autophagy and mitophagy, resulting in compartmentalized cellular catabolism, loss of mitochondrial function, and the onset of aerobic glycolysis, in the tumor stroma. As such, cancer-associated fibroblasts produce high-energy nutrients (such as lactate and ketones) that fuel mitochondrial biogenesis and oxidative metabolism in cancer cells. We have termed this new energy-transfer mechanism the “reverse Warburg effect.” To further test the validity of this hypothesis, here we used an in vitro MCF7-fibroblast co-culture system and quantitatively measured a variety of metabolic parameters by FACS analysis (analogous to laser-capture micro-dissection). Mitochondrial activity, glucose uptake and ROS production were measured with highly-sensitive fluorescent probes (MitoTracker, NBD-2-deoxy-glucose and DCF-DA). Interestingly, using this approach, we directly show that cancer cells initially secrete hydrogen peroxide that then triggers oxidative stress in neighboring fibroblasts. Thus, oxidative stress is contagious (spreads like a virus) and is propagated laterally and vectorially from cancer cells to adjacent fibroblasts. Experimentally, we show that oxidative stress in cancer-associated fibroblasts quantitatively reduces mitochondrial activity and increases glucose uptake, as the fibroblasts become more dependent on aerobic glycolysis. Conversely, co-cultured cancer cells show significant increases in mitochondrial activity and corresponding reductions in both glucose uptake and GLUT1 expression. Pre-treatment of co-cultures with extracellular catalase (an anti-oxidant enzyme that detoxifies hydrogen peroxide) blocks the onset of oxidative stress and potently induces the death of cancer cells, likely via starvation. Given that cancer-associated fibroblasts show the largest increases in glucose uptake, we suggest that PET imaging of human tumors, with Fluoro-2-deoxy-D-glucose (F-2-DG), may be specifically detecting the tumor stroma, rather than epithelial cancer cells.Key words: tumor stroma, microenvironment, hydrogen peroxide, aerobic glycolysis, mitochondrial oxidative phosphorylation, glucose uptake, oxidative stress, reactive oxygen species (ROS), cancer associated fibroblasts, PET imaging, the field effect, caveolin-1     

Cancer cells metabolically "fertilize" the tumor microenvironment with hydrogen peroxide,driving the Warburg effect

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

Metabolic reprogramming of cancer-associated fibroblasts by TGF-β drives tumor growth: Connecting TGF-β signaling with “Warburg-like” cancer metabolism and L-lactate production

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.     

Loss of heterozygosity studies in tumors from families with breast-ovarian cancer syndrome

A gene (BRCA1) predisposing for familial breast and ovarian cancer has been mapped to chromosome band 17q12-21. Based on the observation that ovarian tumors from families with breast and ovarian cancer lose the wild-type allele in the region for the BRCA1 locus, it has been suggested that the gene functions as a tumor suppressor gene. We have studied chromosomal deletions in the BRCA1 region in seven breast tumors, three ovarian tumors, one bladder cancer, and one colon cancer from patients in six families with breast-ovarian cancer, in order to test the hypothesis of the tumor suppressor mechanism at this locus. We have found a low frequency of loss of heterozygosity at this region, and our results do not support the idea that BRCA1 is a tumor suppressor gene. Alternatively, the disease segregating in these families is linked to one or more different loci.     

Metabolic reprogramming of cancer-associated fibroblasts by TGF-β drives tumor growth: Connecting TGF-β signaling with “Warburg-like” cancer metabolism and L-lactate production

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.     

Immunolocalization of BRCA1 protein in tumor breast tissue: prescreening of BRCA1 mutation in Tunisian patients with hereditary breast cancer?

BRCA1 is a tumor suppressor gene which is inactivated by mutation in familial breast and ovarian cancers. Over 300 different disease causing germ-line mutations have been described; 60% are unique to an individual family. This diversity and the large size of the gene lead us to search for a prescreening method for BRCA1 mutations. Since BRCA1 is a nuclear protein in normal cells, but reported by some authors to be cytoplasmic in breast tumor cells of patients with BRCA1 mutation, we evaluated immunohistochemistry as a prescreening technique to identify BRCA1 mutations in patients with familial presentation of breast cancer. Using a monoclonal antibody against the carboxy-terminal region of BRCA1, we performed immunohistochemistry on 18 tumor samples from patients with hereditary breast cancer. Cytoplasmic staining of BRCA1 was observed in 10 cases. Of the 18 tumors, 12 (66%) showed either BRCA mutation or BRCA1 accumulation or both, indicating that BRCA1 function might be lost in breast tumor cells not only through mutation, but also via abnormal cytoplasmic location. The immunohistochemical test used in this study would not be efficient as a pre-screening method of deleterious mutations, but it appeared useful to investigate tumor physiology.     

Brca1 and differentiation

Breast cancer is one of the most frequent malignancies affecting women. The human breast cancer gene 1 (BRCA1) gene is mutated in a distinct proportion of hereditary breast and ovarian cancers. Tumourigenesis in individuals with germline BRCA1 mutations requires somatic inactivation of the remaining wild-type allelle. Although, this evidence supports a role for BRCA1 as a tumour suppressor, the mechanisms through which its loss leads to tumourigenesis remain to be determined. Neither the expression pattern nor the described functions of human BRCA1 and murine breast cancer gene 1 (Brca1) can explain the specific association of mutations in this gene with the development of breast and ovarian cancer. Investigation of the role of Brca1 in normal cell differentiation processes might provide the basis to understand the tissue-restricted properties.     

Oxidative stress in cancer associated fibroblasts drives tumor-stroma co-evolution

Loss of stromal fibroblast caveolin-1 (Cav-1) is a powerful single independent predictor of poor prognosis in human breast cancer patients, and is associated with early tumor recurrence, lymph node metastasis, and tamoxifen-resistance. We developed a novel co-culture system to understand the mechanism(s) by which a loss of stromal fibroblast Cav-1 induces a "lethal tumor micro-environment". Here, we propose a new paradigm to explain the powerful prognostic value of stromal Cav-1. In this model, cancer cells induce oxidative stress in cancer associated fibroblasts, which then acts as a "metabolic" and "mutagenic" motor to drive tumor-stroma co-evolution, DNA damage, and aneuploidy in cancer cells. More specifically, we show that an acute loss of Cav-1 expression leads to mitochondrial dysfunction, oxidative stress, and aerobic glycolysis in cancer associated fibroblasts. Also, we propose that defective mitochondria are removed from cancer-associated fibroblasts by autophagy/mitophagy that is induced by oxidative stress. As a consequence, cancer associated fibroblasts provide nutrients (such as lactate) to stimulate mitochondrial biogenesis and oxidative metabolism in adjacent cancer cells (the "Reverse Warburg Effect"). We provide evidence that oxidative stress in cancer associated fibroblasts is sufficient to induce genomic instability in adjacent cancer cells, via a bystander effect, potentially increasing their aggressive behavior. Finally, we directly demonstrate that nitric oxide (NO) over-production, secondary to Cav-1 loss, is the root cause for mitochondrial dysfunction in cancer associated fibroblasts. In support of this notion, treatment with anti-oxidants (such as N-acetyl-cysteine, metformin, and quercetin), or NO inhibitors (L-NAME) was sufficient to reverse many of the cancer-associated fibroblast phenotypes that we describe. Thus, cancer cells use "oxidative stress" in adjacent fibroblasts i) as an "engine" to fuel their own survival via the stromal production of nutrients, and ii) to drive their own mutagenic evolution towards a more aggressive phenotype, by promoting genomic instability. We also present evidence that the "field effect" in cancer biology could also be related to the stromal production of ROS and NO species. eNOS-expressing fibroblasts have the ability to down-regulate Cav-1 and induce mitochondrial dysfunction in adjacent fibroblasts that do not express eNOS. As such, the effects of stromal oxidative stress can be laterally propagated, amplified, and are effectively "contagious"-spread from cell-to-cell like a virus-creating an "oncogenic/mutagenic" field promoting widespread DNA damage.     

Functional characterization of a novel BRCA1-null ovarian cancer cell line in response to ionizing radiation

The breast and ovarian cancer susceptibility gene BRCA1 plays a major role in the DNA damage response pathway. The lack of well-characterized human BRCA1-null cell lines has limited the investigation of BRCA1 function, particularly with regard to its role in ovarian cancer. We propagated a novel BRCA1-null human ovarian cancer cell line UWB1.289 from a tumor of papillary serous histology, the most common form of ovarian carcinoma. UWB1.289 carries a germline BRCA1 mutation within exon 11 and has a deletion of the wild-type allele. UWB1.289 is estrogen and progesterone receptor negative and has an acquired somatic mutation in p53, similar to the commonly used BRCA1-null breast cancer cell line HCC1937. We used ionizing radiation to induce DNA damage in both UWB1.289 and in a stable UWB1.289 line in which wild-type BRCA1 was restored. We examined several responses to DNA damage in these cell lines, including sensitivity to radiation, cell cycle checkpoint function, and changes in gene expression using microarray analysis. We observed that UWB1.289 is sensitive to ionizing radiation and lacks cell cycle checkpoint functions that are a normal part of the DNA damage response. Restoration of wild-type BRCA1 function in these cells partially restores DNA damage responses. Expression array analysis not only supports this partial functional correction but also reveals interesting new information regarding BRCA1-positive regulation of the expression of claudin 6 and other metastasis-associated genes and negative regulation of multiple IFN-inducible genes.     

A paradox and three egnimas about the role of BRCA1 in breast and ovarian cancers

More than 50% of the hereditary forms are associated with germ line mutation in either BRCA1 or BRCA2 genes (BReast CAncer 1/2). The BRCA1 protein is expressed ubiquitously and is likely to play a role in several fundamental processes, including the maintenance of genomic integrity. Paradoxically, BRCA1 appears as a gene essential for proliferation of embryonic cells that simultaneously carries tumor suppressor activity. The nature of the role of BRCA1 in DNA repair and maintenance of genome integrity remains enigmatic. BRCA1 may indeed be a sensor of "abnormal" DNA structures that undergo heterochromatinisation. This model finds some support in the recent report that BRCA1 participates in the maintenance of X-chromosome inactivation, a paradigm for facultative heterochromatinisation. Why are epithelial cells from mammary glands and ovaries the privileged targets for tumorigenesis in women carrying germline mutations in BRCA1? The inheritance of a single defective copy of BRCA1 by women confers a status of susceptibility for developing breast and/or ovarian cancer. The loss of the wild-type allele inherited from the unaffected parent (LOH), commonly observed in the primary breast and ovarian tumors in these susceptible women, represents the event that initiates the tumorigenesis process. This classical two hit model, which assumes that heterozygote cells are "normal" until the LOH occurs stochastically, remains enigmatic.     

BRCA1 and BRCA2 protect against oxidative DNA damage converted into double-strand breaks during DNA replication

BRCA1 and BRCA2 mutation carriers are predisposed to develop breast and ovarian cancers, but the reasons for this tissue specificity are unknown. Breast epithelial cells are known to contain elevated levels of oxidative DNA damage, triggered by hormonally driven growth and its effect on cell metabolism. BRCA1- or BRCA2-deficient cells were found to be more sensitive to oxidative stress, modeled by treatment with patho-physiologic concentrations of hydrogen peroxide. Hydrogen peroxide exposure leads to oxidative DNA damage induced DNA double strand breaks (DSB) in BRCA-deficient cells causing them to accumulate in S-phase. In addition, after hydrogen peroxide treatment, BRCA deficient cells showed impaired Rad51 foci which are dependent on an intact BRCA1–BRCA2 pathway. These DSB resulted in an increase in chromatid-type aberrations, which are characteristic for BRCA1 and BRCA2-deficient cells. The most common result of oxidative DNA damage induced processing of S-phase DSB is an interstitial chromatid deletion, but insertions and exchanges were also seen in BRCA deficient cells. Thus, BRCA1 and BRCA2 are essential for the repair of oxidative DNA damage repair intermediates that persist into S-phase and produce DSB. The implication is that oxidative stress plays a role in the etiology of hereditary breast cancer.