Mitochondrial metabolism in cancer metastasis: Visualizing tumor cell mitochondria and the “reverse Warburg effect” in positive lymph node tissue

We have recently proposed a new two-compartment model for understanding the Warburg effect in tumor metabolism. In this model, glycolytic stromal cells produce mitochondrial fuels (L-lactate and ketone bodies) that are then transferred to oxidative epithelial cancer cells, driving OXPHOS and mitochondrial metabolism. Thus, stromal catabolism fuels anabolic tumor growth via energy transfer. We have termed this new cancer paradigm the “reverse Warburg effect,” because stromal cells undergo aerobic glycolysis, rather than tumor cells. To assess whether this mechanism also applies during cancer cell metastasis, we analyzed the bioenergetic status of breast cancer lymph node metastases, by employing a series of metabolic protein markers. For this purpose, we used MCT4 to identify glycolytic cells. Similarly, we used TOMM20 and COX staining as markers of mitochondrial mass and OXPHOS activity, respectively. Consistent with the “reverse Warburg effect,” our results indicate that metastatic breast cancer cells amplify oxidative mitochondrial metabolism (OXPHOS) and that adjacent stromal cells are glycolytic and lack detectable mitochondria. Glycolytic stromal cells included cancer-associated fibroblasts, adipocytes and inflammatory cells. Double labeling experiments with glycolytic (MCT4) and oxidative (TOMM20 or COX) markers directly shows that at least two different metabolic compartments co-exist, side-by-side, within primary tumors and their metastases. Since cancer-associated immune cells appeared glycolytic, this observation may also explain how inflammation literally “fuels” tumor progression and metastatic dissemination, by “feeding” mitochondrial metabolism in cancer cells. Finally, MCT4(+) and TOMM20(-) “glycolytic” cancer cells were rarely observed, indicating that the conventional “Warburg effect” does not frequently occur in cancer-positive lymph node metastases.Key words: caveolin-1, oxidative stress, MCT4, metabolic coupling, tumor stroma, SLC16A3, monocarboxylic acid transporter, two-compartment tumor metabolism, metastasis, TOMM20, complex IV, OXPHOS, mitochondria, inflammation     

Could cancer and infection be adverse effects of mesenchymal stromal cell therapy?

Multipotent mesenchymal stromal cells [also referred to as mesenchymal stem cells(MSCs)] are a heterogeneous subset of stromal cells. They can be isolated from bone marrow and many other types of tissue. MSCs are currently being tested for therapeutic purposes(i.e., improving hematopoietic stem cell engraftment, managing inflammatory diseases and regenerating damaged organs). Their tropism for tumors and inflamed sites and their context-dependent potential for producing trophic and immunomodulatory factors raises the question as to whether MSCs promote cancer and/or infection. Thisarticle reviews the effect of MSCs on tumor establishment, growth and metastasis and also susceptibility to infection and its progression. Data published to date shows a paradoxical effect regarding MSCs, which seems to depend on isolation and expansion, cells source and dose and the route and timing of administration. Cancer and infection may thus be adverse or therapeutic effects arising form MSC administration.     

ROS,autophagy, mitochondria and cancer: Ras,the hidden master?

Recent advances have highlighted the complex web of biological mechanisms and pathways involved in oncogenic transformation and maintenance of the cancer phenotype. To that end, a number of key factors have been identified and thoroughly investigated over the past couple of decades, such as redox regulation of cell fate decisions, cellular metabolism and bioenergetics, autophagy induction as a survival signal, and how these pathways interplay with oncogene-induced transformation. This has been particularly well documented for oncoprotein Ras-induced carcinogenesis, and recent reports provide ample evidence to indicate a well-coordinated crosstalk between these diverse cellular pathways in the process of cancer initiation and progression. Here we provide a brief summary of the recent advances in the field to illustrate the dual role of autophagy as a tumor suppressor and as a survival mechanism required for cancer maintenance as well as its implication in the complex relationship between Ras-mediated carcinogenesis, mitochondrial metabolism, cellular redox status and bioenergetics.     

Downregulation of stromal BRCA1 drives breast cancer tumor growth via upregulation of HIF-1α, autophagy and ketone body production

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.     

PRMT2β suppresses autophagy and glycolysis pathway in human breast cancer MCF-7 cell lines

Autophagy as a novel therapeutic target can inhibit or increase treatment efficacy in various types of breast cancer in a cell-type-dependent manner [1,2].Several studies have revealed that the coordination between Akt and the glycolytic pathway plays an indispensable role in mediating autophagy and caspase-dependent apoptosis,suggesting that a new regulatory mechanism for the process [3,4].Protein arginine N-methyltransferases(PRMTs)are eukaryotic enzymes that catalyze the transfer of methyl groups from S-adenosylmethionine to arginine residues of numerous PRMT substrates [5,6].PRMT2(also known as HRMT1L1)belongs to the arginine methyltransferase family [7].PRMT2β is a novel PRMT2 splice variant isolated from breast cancer cell [8].It occurs at the 3′ end of the PRMT2,resulting in loss of exons 7–9 and downstream frame-shifting [9].PRMT2β possesses 83 new amino acids at the C-terminus and its size is 301 amino acids.Our previous study reported that PRMT2β has potential antitumor effect by suppressing cyclin D1 expression [10].However,little is known about whether PRMT2β could regulate autophagy and glycolysis of MCF-7 cells.     

Biotin attenuation of oxidative stress,mitochondrial dysfunction,lipid metabolism alteration and 7β-hydroxycholesterol-induced cell death in 158N murine oligodendrocytes

Mitochondrial dysfunction and oxidative stress are involved in neurodegenerative diseases associated with an enhancement of lipid peroxidation products such as 7β-hydroxycholesterol (7β-OHC). It is, therefore, important to study the ability of 7β-OHC to trigger mitochondrial defects, oxidative stress, metabolic dysfunctions and cell death, which are hallmarks of neurodegeneration, and to identify cytoprotective molecules. The effects of biotin were evaluated on 158N murine oligodendrocytes, which are myelin synthesizing cells, exposed to 7β-OHC (50?µM) with or without biotin (10 and 100?nM) or α-tocopherol (positive control of cytoprotection). The effects of biotin on 7β-OHC activities were determined using different criteria: cell adhesion; plasma membrane integrity; redox status. The impact on mitochondria was characterized by the measurement of transmembrane mitochondrial potential (ΔΨm), reactive oxygen species (ROS) overproduction, mitochondrial mass, quantification of cardiolipins and organic acids. Sterols and fatty acids were also quantified. Cell death (apoptosis, autophagy) was characterized by the enumeration of apoptotic cells, caspase-3 activation, identification of autophagic vesicles, and activation of LC3-I into LC3-II. Biotin attenuates 7β-OHC-induced cytotoxicity: loss of cell adhesion was reduced; antioxidant activities were normalized. ROS overproduction, protein and lipid oxidation products were decreased. Biotin partially restores mitochondrial functions: attenuation of the loss of ΔΨm; reduced levels of mitochondrial O₂^?? overproduction; normalization of cardiolipins and organic acid levels. Biotin also normalizes cholesterol and fatty acid synthesis, and prevents apoptosis and autophagy (oxiapoptophagy). Our data support that biotin, which prevents oligodendrocytes damages, could be useful in the treatment of neurodegeneration and demyelination.     

Nigericin inhibits epithelial ovarian cancer metastasis by suppressing the cell cycle and epithelial−mesenchymal transition

Epithelial ovarian cancer (EOC) has the highest mortality among various types of gynecological malignancies. Most patients die of metastasis and recurrence due to cisplatin resistance. Thus, it is urgent to develop novel therapies to cure this disease. CCK-8 assay showed that nigericin exhibited strong cytotoxicity on A2780 and SKOV3 cell lines. Flow cytometry indicated that nigericin could induce cell cycle arrest at G0/G1 phase and promote cell apoptosis. Boyden chamber assay revealed that nigericin could inhibit migration and invasion in a dose-dependent manner by suppressing epithelial–mesenchymal transition (EMT) in EOC cells. These effects were mediated, at least partly, by the Wnt/β-catenin signaling pathway. Our results demonstrated that nigericin could inhibit EMT during cell invasion and metastasis through the canonical Wnt/β-catenin signaling pathway. Nigericin may prove to be a novel therapeutic strategy that is effective in patients with metastatic EOC.     

Localization of thymosin β10 in breast cancer cells: relationship to actin cytoskeletal remodeling and cell motility

Beta-thymosins are polypeptides involved in the regulation of actin polymerization and thymosin β10 and β4 have been implicated in sequestration of monomeric (G-) actin. Additionally, experimental overexpression of thymosin β10 has been found to result in increases in F-actin bundles as well as in cell motility and spreading. We have studied the distribution of endogenously expressed thymosin β10 in cultured human breast cancer cell lines. Both unperturbed monolayer cultures and wound-healing models were examined using double-staining for thymosin β10 and polymerized (F-) actin. Our findings show that thymosin β10 is expressed in all three-cancer cell lines (SK-BR-3, MCF-7 and MDA-MB-231) studied. No or little staining was detected in confluent cells, whereas strong staining occurred in semiconfluent cells and in cells populating monolayer wounds. Importantly, the distribution of staining for thymosin β10 was inverse of staining for F-actin. These data support a physiological role for thymosin β10 in sequestration of G-actin as well as in cancer cell motility.     

Loss of Ssq1 leads to mitochondrial dysfunction,activation of autophagy and cell cycle arrest due to iron overload triggered by mitochondrial iron–sulfur cluster assembly defects in Candida albicans

Iron–sulfur clusters perform essential functions in enzymatic catalysis and homeostatic regulation. Here we for the first time identified Ssq1 as an essential component for iron–sulfur cluster assembly in Candida albicans. Ssq1 played an important role in cell growth. Shutting off SSQ1 led to accumulation of intracellular iron, especially in mitochondria, and disorder of intracellular iron regulation. In tetO-SSQ1, iron overloading triggered the oxidative damage of mitochondrial function. Surprisingly, disruption of SSQ1 activated autophagic pathway. The mitochondrial dysfunction was further aggravated when CCZ1 (which is essential for autophagy) and SSQ1 was simultaneously deleted, suggesting that autophagy played a critical role in maintenance of mitochondrial function in tetO-SSQ1. In addition, double deletion of SSQ1 and CCZ1 further elevated cellular iron levels in comparison with tetO-SSQ1, indicating that autophagy participated in maintenance of iron homeostasis. Furthermore, we found that loss of SSQ1 led to increasing protein expression of Rnr1 and redistribution of Rnr2 from the nucleus to cytoplasm, and further resulted in cell cycle arrest. The results implied that cell cycle arrest was caused by activating the checkpoint pathway because of impairing the iron–sulfur cluster assembly in tetO-SSQ1. Shutting off SSQ1 led to a significant defect in filamentous development. Interestingly, the tetO-SSQ1ccz1Δ/Δ growth was inhibited on hyphae-inducing solid media. Both tetO-SSQ1 and tetO-SSQ1ccz1Δ/Δ exhibited extremely attenuated virulence, indicating that Ssq1 might provide a promising target for antifungal drugs development. In summary, our findings provide new insights into the understanding of iron–sulfur cluster assembly-related gene in C. albicans.     

miR-125a regulates HAS1 and inhibits the proliferation,invasion and metastasis by targeting STAT3 in non–small cell lung cancer cells

MicroRNA-125a (miR-125a) is related to the occurrence, development, and prognosis of various cancers according to relevant reports. However, its function role and mechanism in non–small cell lung cancer (NSCLC) is yet to be explored. Herein, we investigated the role and preliminary mechanism of miR-125a in NSCLC. First, miR-125a was noticeably downregulated in NSCLC tissues in contrast to adjacent normal tissues through the real-time quantitative polymerase chain reaction (RT-qPCR) assay. The inverted result was observed on the STAT3 and HAS1 expressions. Moreover, miR-125a was expressed at highest level in A549 among four human NSCLC cell lines. Second, functional studies indicated miR-125a restrained proliferation, invasion, migration, metastasis, and advocated apoptosis of NSCLC cells, but had no obvious effect on cell cycle. Next, results indicated that a target of miR-125a was STAT3 on the basis of prediction and confirmation by the dual-luciferase reporter assay. RT-qPCR and Western blot assays displayed that miR-125a overexpression conspicuously constrained STAT3 expression at messenger RNA and protein levels. Finally, the binding between HAS1 promoter region and STAT3 was predicted by PROMO database analysis and verified by chromatin immunoprecipitation assay, suggesting that STAT3 was bound with the HAS1 promoter regions. STAT3 overexpression exerted positive effects on HAS1 expression at protein and mRNA levels. Additionally, HAS1-related functional studies illustrated HAS1 pronouncedly suppressed the proliferative, invasive, and migratory potential of NSCLC cells in vitro. Collectively, our findings demonstrated that miR-125a prohibited the proliferation, invasion, and migration of NSCLC cells by HAS1 expression reduction as a result of inhibiting STAT3 expression in NSCLC. This study indicated that miR-125a might be of potential or value for NSCLC treatment.