Knockout of Vdac1 activates hypoxia-inducible factor through reactive oxygen species generation and induces tumor growth by promoting metabolic reprogramming and inflammation
- M. Christiane Brahimi-Horn†1,
- Sandy Giuliano†1,
- Estelle Saland2,
- Sandra Lacas-Gervais3,
- Tatiana Sheiko4,
- Joffrey Pelletier1,
- Isabelle Bourget5,
- Frédéric Bost6,
- Chloé Féral5,
- Etienne Boulter5,
- Michel Tauc7,
- Mircea Ivan8,
- Barbara Garmy-Susini9,
- Alexandra Popa10,
- Bernard Mari10,
- Jean-Emmanuel Sarry2,
- William J. Craigen4,
- Jacques Pouysségur1, 11 and
- Nathalie M. Mazure1Email author
© Brahimi-Horn et al. 2015
Received: 9 January 2015
Accepted: 20 May 2015
Published: 26 August 2015
Mitochondria are more than just the powerhouse of cells; they dictate if a cell dies or survives. Mitochondria are dynamic organelles that constantly undergo fusion and fission in response to environmental conditions. We showed previously that mitochondria of cells in a low oxygen environment (hypoxia) hyperfuse to form enlarged or highly interconnected networks with enhanced metabolic efficacy and resistance to apoptosis. Modifications to the appearance and metabolic capacity of mitochondria have been reported in cancer. However, the precise mechanisms regulating mitochondrial dynamics and metabolism in cancer are unknown. Since hypoxia plays a role in the generation of these abnormal mitochondria, we questioned if it modulates mitochondrial function. The mitochondrial outer-membrane voltage-dependent anion channel 1 (VDAC1) is at center stage in regulating metabolism and apoptosis. We demonstrated previously that VDAC1 was post-translationally C-terminal cleaved not only in various hypoxic cancer cells but also in tumor tissues of patients with lung adenocarcinomas. Cells with enlarged mitochondria and cleaved VDAC1 were also more resistant to chemotherapy-stimulated cell death than normoxic cancer cells.
Transcriptome analysis of mouse embryonic fibroblasts (MEF) knocked out for Vdac1 highlighted alterations in not only cancer and inflammatory pathways but also in the activation of the hypoxia-inducible factor-1 (HIF-1) signaling pathway in normoxia. HIF-1α was stable in normoxia due to accumulation of reactive oxygen species (ROS), which decreased respiration and glycolysis and maintained basal apoptosis. However, in hypoxia, activation of extracellular signal-regulated kinase (ERK) in combination with maintenance of respiration and increased glycolysis counterbalanced the deleterious effects of enhanced ROS, thereby allowing Vdac1 −/− MEF to proliferate better than wild-type MEF in hypoxia. Allografts of RAS-transformed Vdac1 −/− MEF exhibited stabilization of both HIF-1α and HIF-2α, blood vessel destabilization, and a strong inflammatory response. Moreover, expression of Cdkn2a, a HIF-1-target and tumor suppressor gene, was markedly decreased. Consequently, RAS-transformed Vdac1 −/− MEF tumors grew faster than wild-type MEF tumors.
Metabolic reprogramming in cancer cells may be regulated by VDAC1 through vascular destabilization and inflammation. These findings provide new perspectives into the understanding of VDAC1 in the function of mitochondria not only in cancer but also in inflammatory diseases.
While the Warburg effect, or aerobic glycolysis, is considered to be primarily responsible for the metabolic reprogramming of cancer cells , mitochondrial respiration remains functional. However, it is not clear how mitochondria impact on proliferation or transformation of cancer cells, but as the «powerhouse» of cells, any change in metabolism can strongly influence the survival of the cancerous cell. Mitochondria are not only crucial in metabolic reprogramming; they also play an important role in delivering the message of cell death i.e., apoptosis. When the mitochondrial membrane potential (Δψm) is lost, mitochondria lose the integrity of their outer membrane, ATP synthesis is stopped, and proteins such as cytochrome C activate a cascade of caspases, ensuring certain death of the cell [2, 3].
The voltage-dependent anion channel (VDAC) is a major protein of the mitochondrial outer membrane that functions at the intersection of metabolism and apoptosis. The mammalian mitochondrial porin family includes three isoforms: VDAC1, VDAC2, and VDAC3 . However, their expression levels differ according to the type of tissue, as do their physiological function. Mice lacking Vdac1 or Vdac3 are viable, whereas mice lacking Vdac2 are not. While heterozygous Vdac1 +/− mice appear to have no obvious phenotype, when mated, they showed a reduced number of homozygous deficient mice when compared to the expected Mendelian ratio . Only 40 % of the expected number of Vdac1 −/− mice survived, suggesting partial lethality, the reason for which has not yet been elucidated. VDAC1 is a pore that regulates the passage of molecules, including NADH, ATP/ADP, citrate, succinate, glutamate, pyruvate, and even glucose, as well as Mg2+, Ca2+, Cl−, K+, and Na+ ions . It regulates the mitochondrial phenotype, apoptosis, and glycolysis through binding to hexokinase I/II (HKI/II), the first enzyme of the glycolytic pathway. Binding of HK to VDAC is a crucial event that coordinates mitochondrial ATP generation, cytoplasmic glycolytic flux, and possibly cytochrome C release. Moreover, HKI/II are upregulated by hypoxia-inducible factors (HIFs), the key transcription factors that direct the cell response to hypoxia, a condition in which tumors are inadequately oxygenated . It is well known that the hypoxic nature of tumors is associated with poor clinical outcome, as hypoxia confers resistance to radio- and chemotherapy.
The role of VDAC1 in cancer has not been extensively investigated [7, 8, 4]. We recently reported the presence in tumor cells of a novel hypoxia-induced form of VDAC1 lacking the C-terminus (VDAC1-ΔC) [7, 9]. This new form was produced after long-term exposure to hypoxia. Cells expressing VDAC1-ΔC produced more ATP and were more resistant to stimulated apoptosis than cells expressing only full length VDAC1. In hypoxia, the level of full length VDAC1 decreased by about at least 50 % while the level of VDAC1-ΔC conversly increased by about 50 %. We hypothesized that knockout of VDAC1 in hypoxia would have a substantial impact on cell proliferation, oxidative and glycolytic metabolism, as well as apoptosis in hypoxic cells.
Herein, we characterized the cellular and molecular phenotype of Vdac1-deficient mouse embryonic fibroblasts (MEF) in normoxia or hypoxia. We report that VDAC1 participates in apoptosis and oxidative phosphorylation via reactive oxygen species (ROS) production, which modulates proliferation. We report connection to the Raf/MEK/ERK signaling pathway that counterbalances the deleterious effects of ROS in hypoxia. Finally, we show that knockout of Vdac1 in MEF expressing oncogenic RAS potentiates tumor development in mice by promoting metabolic reprogramming, accelerating vascular destabilization and inflammation.
Cell culture, transfection, and animals
MEF cells were grown in Dulbecco’s modified eagle’s medium (DMEM) (Gibco-BRL) supplemented with 10 % fetal bovine serum with penicillin G (50 U/ml) and streptomycin sulfate (50 μg/ml). An INVIVO2 200 anaerobic workstation (Ruskinn Technology Biotrace International Plc) set at 1 % oxygen, 94 % nitrogen, and 5 % carbon dioxide was used for hypoxic conditions. MEF were transformed with the pBabe-RASV12 vector, and puromycin-resistant cells were collected. Animal procedures were approved by the Animal Care and Use Committee of the Unité Mixte de Service 006 of Toulouse (approval number 13-U1037-JES-08).
Cells were fixed in situ with 1.6 % glutaraldehyde in 0.1 M phosphate buffer at room temperature (RT) and stored overnight at 4 °C. Samples were rinsed in the same buffer and then postfixed with 1 % osmium tetroxide and 1 % potassium ferrocyanide in 0.1 M cacodylate buffer for 1 h at RT to enhance the staining of cytoplasmic membranes. Cells were rinsed with distilled water, embedded in epoxy resin, sectioned, and examined with a Philips CM12 transmission electron microscope equipped with an Olympus SIS CCD camera. The area of the mitochondria of cells was calculated as an ellipse (0.785ab; n.2, 50 mitochondria per experiment; mean ± SD).
MEF (Wt and Vdac1 −/−) and RAS MEF (Wt and Vdac1 −/−) were incubated in hypoxia for 72 h and then lysed. Quantification of ATP was done using a luciferin/luciferase-based assay (Cell Titer Glo kit, Promega) according to the manufacturer’s instructions, and results are expressed as relative luminescence units (RLU). Each condition was tested eight times, and the entire experiment was done twice.
The lactate concentration in the supernatant of cells incubated either in normoxia or hypoxia for 48 h was determined by an enzyme-based assay using 900 μM β-NAD (BioChemika) and 175 μg/ml l-lactate dehydrogenase (BioChemika), and 100 μg/ml glutamate-pyruvate transaminase (Roche) were diluted in a sodium carbonate (620 mM)-l-gultamate (79 mM) buffer adjusted to pH 10. Lithium lactate was used as a standard. Measurement was done with a microplate reader after incubation for 30 min at 37 °C. For each condition, the protein concentration was determined to express the lactate concentration as mmole/μg protein.
Respirometry and extracellular acidification
The cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were obtained using a Seahorse XF96 extracellular flux analyzer from Seahorse Bioscience (North Billerica, MA, USA). The final concentrations of the agents are given in the legends. Experiments were performed according to the manufacturer’s instructions. Protein standardization was performed after each experiment, with no noticeable differences in protein concentration and cell phenotype.
Determination of glutamate production
Glutamate concentrations were measured in media supernatants using a Ysi 2300 STAT Plus analyzer (YSI Life Sciences). Measurements were done in triplicate, and the experiment was repeated two times.
Quantification of the caspase 3/7 activity was done using a luciferin/luciferase-based assay (Caspase-Glo 3/7 kit, Promega) according to the manufacturer’s instructions. Each condition was performed eight times, and the entire experiment was done three times. Significant differences are based on the Student’s t test (p < 0.005). STS was added 4 h prior to assay for caspase 3/7 activity.
Flow cytometry and ROS measurement
For measuring intracellular cytosolic ROS levels, cells were cultured in hypoxia or normoxia for 48 h. Cells were then treated with oxidation sensitive DCF-DA (a final concentration of 10 μM) in culture medium for 30 min at 37 °C. After trypsination, the fluorescence of DCF in cells was measured using a fluorescence-activated cell sorter (BD FACSCalibur, analyzer). Mitochondrial H2O2 production was determined using Amplex UltraRed as described  but with a Xenius XC Safas spectrofluorimeter (Monaco) at λexcitation = 560 nm, λemision = 590 nm. Whole digitonin permeabilized cells were used instead of isolated mitochondria, as described . The assay was performed with 0.6 mg protein/ml of each cell type suspended in KHEP buffer without BSA (120 mM KCl, 2.5 mM MgCl2, 1 mM EGTA, 5 mM HEPES pH 7.2 at 37 °C) together with 5 U/ml horseradish peroxidase, 25 U/ml superoxide dismutase, and 50 μM Amplex UltraRed. The reaction was monitored and digitonin (final 10 μg/ml), then potassium succinate (final 5 mM), and then antimycin A (final 2 mM) were added successively. The results are expressed as the mean slope ratio of antimycin A/succinate.
Cells were lysed in 1.5x SDS buffer and the protein concentration determined using the BCA assay. 40 μg of protein of whole cell extracts was resolved by SDS-PAGE and transferred onto a PVDF membrane (Millipore). Membranes were blocked in 5 % non-fat milk in TN buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl) and incubated in the presence of the primary and then secondary antibodies in 5 % non-fat milk in TN buffer.
The Bak antibody was purchased from Abcam, Bid and tBid from R&D Systems, Bax and Bcl-XL from Santa Cruz, Bcl-2 from Novus, and Mcl-1 from Sigma. Rat anti-mouse CD31 (MEC 13.3) was from BD Bioscience (San Diego, CA). VDAC1 antibody (ab15895) was purchased from Abcam. Rabbit polyclonal anti-HIF-1α antibody (antiserum 2087) was produced and characterized in our laboratory . ECL signals were normalized to either β-tubulin or ARD1 . After washing in TN buffer containing 1 % Triton-X100 and then in TN buffer, immunoreactive bands were visualized with the ECL system (Amersham Biosciences).
MEF were incubated in normoxia or hypoxia for 72 h (1 % O2) and then lysed prior to RNA isolation. RNA was assessed for integrity by using an Agilent BioAnalyser 2100 (Agilent Technologies) (RIN above 9). RNA samples were then labeled with Cy3 dye using the low RNA input QuickAmp kit (Agilent) as recommended by the supplier. Labeled cRNA probe (825 ng) was hybridized on 8x60K high density SurePrint G3 gene expression mouse microarrays. The experimental data are deposited on the NCBI Gene Expression Omnibus (GEO) under the series record number GSE63247 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE63247). Normalization of microarray data was performed using the Limma package available from Bioconductor (http://www.bioconductor.org). Inter-slide normalization was performed using quantile methods. Means of ratios from all comparisons were calculated and a B test analysis using paired analysis was performed. Differentially expressed genes were selected based on an adjusted p value below 0.01 and a log2 (fold change) >1. Data were analyzed for enrichment in biological themes (diseases and functions, canonical pathways, upstream analysis) using Ingenuity Pathway Analysis software (http://www.ingenuity.com/).
All values are the means ± SEM. Statistical analysis were performed using the Student’s t test as provided by Microsoft Excel. The p values are indicated. All categorical data used numbers and percentages. Quantitative data were presented using the median and range or mean. Differences between groups were evaluated using the chi-square test for categorical variables and the Student’s t test for continuous variables. Analyses were performed using SPSS 16.0 statistical software (SPSS Inc., Chicago, Ill). All statistical tests were two-sided, and p values <0.05 indicated statistical significance, whereas p values between 0.05 and 0.10 indicated a statistical tendency (Additional file 1).
The online version of the article contains a data supplement
Additional file 2: Table S1, Additional file 3: Table S2, Additional file 4: Table S3, Additional file 5: Table S4, Additional file 6: Figure S1 and Additional file 7: Figure S2 show detailed data related to the microarray analysis. Additional file 8: Figure S3 shows expression of COX4-2. Additional file 9: Figure S4 shows the ROS status. Additional file 10: Figure S5 shows expression of GPX7 and the effect of ebselen. Additional file 11: Figure S6 shows changes in metabolic pathways. Additional file 12: Figure S7 shows data on glucose and glutamine catabolism. Additional file 13: Figure S8 shows the level of apoptosis. Additional file 14: Figure S9 shows electron micrograph and immunoblotting data related to autophagy. Additional file 15: Figure S11 shows data on vascularization of the RAS-MEF tumor tissue. Additional file 16: Figure S12 shows the immunohistochemistry to VEGF of tumor sections. Additional file 17: Figure S13 shows an in silico analysis of the Vdac1 gene in cancer.
High-throughput gene expression profiling of wild-type and Vdac1 −/− mouse embryonic fibroblasts
Vdac1 −/− MEF proliferated faster than Wt MEF in hypoxia through activation of the ERK1/2 pathway
Vdac1 −/− MEF showed alterations in mitochondrial respiration
Respiratory control of Wt and Vdac1 −/− MEF
Vdac1 −/− MEF
Vdac1 −/− MEF
(a) Basal respiration
20.52 ± 2.5
16.19 ± 1.8
4.5 ± 0.52
10.4 ± 2.41
(b) Oligomycin-insensitve respiration (leak)
5.96 ± 0.9
5.04 ± 0.9
2.4 ± 0.27
7.1 ± 2.73
(c) Oligomycin-sensitive (ATP turnover) respiration
14.56 ± 1.6
11.16 ± 0.8
1.8 ± 0.70
3.2 ± 0.95
(d) Maximal respiration in the presence of FCCP
29.21 ± 3.2
19.29 ± 1.7
5.4 ± 1.19
6.0 ± 0.29
(c/a) Coupling efficiency
0.71 ± 0.02
0.71 ± 0.01
0.4 ± 0.12
0.3 ± 0.13
(d/b) Respiratory control ratio
4.91 ± 0.2
3.95 ± 0.4
2.3 ± 0.71
0.8 ± 0.44
(d-a) Spare respiratory capacity
8.19 ± 1.2
3.10 ± 0.1
1.3 ± 0.50
−5.4 ± 2.83
Blockade of complex I with rotenone in Wt MEF markedly decreased basal respiration, suggesting a normal complex I activity in normoxia (Fig. 3c). Inhibition with rotenone did not affect complex I activity in hypoxia as it is known that its activity is already reduced in hypoxia . However, rotenone did not modify the OCR of Vdac1 −/− MEF, which suggests that the complex I activity was already diminished. We then quantified the levels of ROS (Fig. 3d). We found a strong correlation between the inactivation of complex I and ROS production. In addition, using a cuvette-based Amplex UltraRed assay for mitochondrial production of H2O2, Vdac1 −/− MEF mitochondria produced more H2O2 than did the Wt MEF (Additional file 9: Figure S4A). N-acetyl-l-cysteine restored proliferation of Vdac1 −/− MEF in normoxia to a similar level to that of Wt MEF (Fig. 3e), decreased P-ERK in hypoxia (Fig. 3f), and decreased HIF-1α in normoxia (Fig. 3g). Addition of hydrogen peroxide to Wt MEF decreased proliferation to a level similar to that of Vdac1 −/− MEF (Fig. 3h). The expression of enzymes involved in upstream or downstream transformation of H2O2 including superoxide dismutase 3 (Sod3) and glutathione peroxidase 7 (Gpx7) were tightly regulated in Vdac1 −/− MEF (Additional file 5: Table S4). However, SOD1, SOD2, and SOD3 showed no difference in expression in normoxia (Additional file 10: Figure S5B) or hypoxia (data not shown) in Wt MEF compared to Vdac1 −/− MEF. GPX7, which detoxifies hydroperoxide substrates, was absent in Vdac1 −/− MEF in both normoxia and hypoxia (Fig. 3i, Additional file 10: Figure S5A). Moreover, ebselen, a mimetic of GPX , did not modify HIF-1α stability (Additional file 10: Figure S5B). It decreased slightly the proliferation of Wt MEF, but not Vdac1 −/− MEF (Additional file 10: Figure S5C) and increased slightly the viability of Vdac1 −/− MEF (Additional file 10: Figure S5D), suggesting a putative protection against ROS in these cells.
These results showed that the accumulation of ROS in Vdac1 −/− MEF, probably due to down-regulation of GPX7 and the stabilization of HIF-1α, participate in the blockade of proliferation in normoxia. However, in hypoxia, activation of P-ERK and induced expression of COX4-2 and COX7A1 compensated for the reduced mitochondrial complex I activity in Vdac1 −/− MEF.
Vdac1 −/− MEF were more glycolytic in hypoxia
Vdac1 −/− MEF showed a higher level of apoptosis than Wt MEF
Vdac1 −/− RASV12-transformed MEF form inflammatory tumors that grow faster than Wt tumors
Herein, we characterized the gene expression profile and cellular phenotype of Vdac1-deficient MEF in normoxia or hypoxia. Our results revealed important functions of VDAC1, in cultured cells and in in vivo allograft tumors, which were primarily driven by ROS and HIF-1.
The gene expression profiles of Wt vs Vdac1 −/− MEF in normoxia and hypoxia were compared keeping in mind that the other isoforms VDAC2 and VDAC3 may compensate . However, we did not detect the VDAC2 protein, and the expression of VDAC3 protein remained constant. When comparing Vdac1 −/− to Wt MEF, the largest difference in expression was noted in normoxia, as HIF-1α and HIF-2α were already stabilized by ROS in Vdac1 −/− MEF. Therefore, the hypoxic effect on gene expression was diminished in Vdac1 −/− MEF. The mRNAs affected by the loss of VDAC1 are involved in cancer and metastasis, but also in fibrosis, neuromuscular disease and finally in the inflammatory response. The role of VDAC1 in cancer has only just started to be investigated [7, 8, 4]. The results of the present study reveal that the role of VDAC1 in cancer is connected to modulation of energy and cell death, which are closely interconnected. We noted a decrease in glycolysis in Vdac1 −/− MEF possibly due to the decrease in HKII expression. HKII has been shown to protect cancer cells from entering apoptosis by blocking the interaction of the pro-apoptotic protein Bax with VDAC1 . Two critical anti-apoptotic factors, Bcl-XL and Bcl-2, were expressed in Vdac1 −/− MEF. Bcl-XL has been shown to critically regulate the opening of VDAC1 and to thus influence apoptosis, possibly through the permeability transition pore complex V [26-29]. Interestingly, in Vdac1 −/− MEF, neither Bcl-XL nor Bcl-2, two critical anti-apoptotic factors, were expressed. As Bcl-XL may influence metabolism , it is tempting to hypothesize that the absence of Bcl-XL, like HKII, may play a role in the reduced capacity to produce energy of Vdac1 −/− MEF in normoxia. A decrease in expression of HKII and Bcl-XL would certainly affect apoptosis. Indeed, mitochondria-associated HKII has been shown to protect cancer cells from entering apoptosis by blocking the interaction of the pro-apoptotic protein Bax with VDAC1 . Moreover, a decrease in the pool of anti-apoptotic proteins such as Bcl-XL or Bcl-2 will inevitably destabilize the balance of anti-apoptotic/pro-apoptotic factors. We observed a basal level of apoptosis (around 10 % cell death), suggesting that VDAC1 is indispensable for mitochondrial cell death. This contrasts with data of a previous study using the same MEF .
In hypoxia in Vdac1 −/− MEF, we observed both enlarged mitochondria with abundant cristae and mitochondria similar to those in normoxia. The former may reflect the improved proliferation, thus better bioenergetics of the cells, and the later of mitochondria that had not or could not adapt. However, one question remains: how do these cells survive in normoxia with a low level of glycolysis and respiration? Fatty acid alpha oxidation by Vdac1 −/− MEF could be a possible explanation. The deficiency in Vdac1 in both MEF and RAS-transformed MEF resulted in a reduction in proliferation in normoxia. Examination of the analytic technique for assessment of RNAi by similarity (ATARiS) , a computational method to assess the effects of gene suppression on cell survival/proliferation, in the Project Achilles database  supported this result. We specifically focused on cell lines with K-Ras mutations. The ATARiS gene-level score of dependency showed that knockout of Vdac1 decreased proliferation, even in human cancer cells driven by oncogenic K-Ras (Additional file 17: Figure S13A). Based on these results, could Vdac1 be a putative cancer driver gene in human cancer? The Cancer Genome Atlas (TCGA) data sets from 89 cancer studies were analyzed for gene mutation, deletion, gain, and amplification in the Vdac1 gene with cBioPortal (http://www.cbioportal.org) [34, 35]. The alteration frequency varied from 80 to 1 % depending on the type of cancer. The Vdac1 gene was mainly heterologously lost (light blue) and/or gained (pink). Homologous loss (dark blue) and amplification (red) were also observed, which suggested a putative role in development of human cancers (Additional file 17: Figure S13B). However, Vdac1 −/− MEF grew better than Wt MEF in hypoxia, and ERK activation was required. Chan et al. showed that ROS can participate in the degradation of DUSP6 . Since ROS accumulated in Vdac1 −/− MEF, due in part to a decrease in GPX7 expression, DUSP6 was degraded in normoxia and hypoxia.
The data from Vdac1 −/− RAS MEF tumors in mice emphasized two major events: i) destructuration of blood vessels and ii) inflammation. The increase in endostatin, an inhibitor of angiogenesis that inhibits endothelial cell proliferation, migration/invasion, and tube formation, may explain the decrease in the number of blood vessels in these tumors. Moreover, the low level of VEGFA expression in the Vdac1 −/− RAS MEF tumors reinforced the implication of VDAC1 in vascular development. This newly identified role of VDAC1 in modulating the structure of blood vessels may explain why the expected Mendelian ratio of 1:2:1 was not observed for heterozygous mice (Vdac1 +/−) . Only 40 % of the expected number of Vdac1 −/− mice survived and, using timed matings, the fetal loss was determined to occur between embryonic day 10.5 and 11.5, a time during which blood vessel formation occurs. We speculate that 60 % of the Vdac1 −/− mice may develop dysfunctional blood vessels during embryogenesis. Why the other 40 % survive and show no obvious outward signs of deficiency remains obscure. The Vdac1 −/− RAS MEF tumors showed less blood vessels than Wt tumors and exhibited an inflammatory response that may result from infiltration of red blood cells (RBCs) through permeabilization of the remaining blood vessels. RBCs could serve as a potential source of ROS since they contain a large pool of O2 that is autoxidized in a hypoxic microenvironment, thereby promoting inflammation . One of the early responses to tissue damage due to ROS is production of IL-8, a pro-inflammatory cytokine often associated with advanced stage cancer and with poor prognosis. RBCs can also bind inflammatory mediators such as IL-8. The mRNA level of Il-8 and Cxcl5 were markedly elevated in Vdac1 −/− RAS MEF, which supports the hypothesis of an enhanced inflammatory response. Finally, it has been shown that RBCs may induce the secretion of matrix metalloproteinases such as mmp-3 by fibroblasts , which impacts on tissue remodeling. Thrombomodulin, a protein that maintains vascular homeostasis via its anti-inflammatory properties , was not expressed in the Vdac1 −/− RAS MEF, so the pro-inflammatory activity is likely not repressed. The cascade of events leading to tumor development was accompanied by a notable decrease in the mRNA level of Cdkn2A, a tumor suppressor gene.
While we observed more rapid tumor growth of Vdac1 −/− RAS MEF compared to Wt RAS MEF, a study using A549 cells (human lung adenocarcinoma epithelial cells) reported that the silencing of Vdac1 expression with siRNA inhibited cancer cell proliferation and tumor growth in vivo . However, we used a mouse/mouse system (tumor/host) while Arif et al. used a human/mouse system (tumor/host). In addition, we used MEF rather than lung cancer cells. Finally and most importantly, our system was a total knockout of Vdac1 and not on partial and temporary silencing as for siRNA. Is angiogenesis induced in the same way in these tumors? We suggest that the Vdac1 −/− RAS MEF adapted and offset an imbalance in some metabolic, angiogenic, or inflammatory processes to proliferate.
- 2-Deoxy-d-glucose; αSMA:
alpha-smooth muscle actin
arrest defective 1
analytic technique for assessment of RNAi by similarity
chemokine (C-X-C motif) ligand 5
cyclin-dependent kinase inhibitor 2A
dual specificity phosphatase
extracellular acidification rate
extracellular signal-regulated kinase
- IPATM :
ingenuity pathway analysisTM
matrix metallopeptidase 3
mouse embryonic fibroblasts
oxygen consumption rate
reactive oxygen species
The Cancer Genome Atlas
voltage-dependent anion channel
This research was supported by grants from the Fondation ARC, Fondation de France, the ANR, the INCA, la Ligue Nationale Contre le Cancer (équipe labellisée LNCC), METOXIA and Canceropôle PACA. The laboratory is funded by the Centre A. Lacassagne, CNRS, and INSERM. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. We acknowledge the excellent support of the Nice-Sophia Antipolis Functional Genomics Platform and the PICMI microscopy facility of IRCAN. The facility was supported financially by the Conseil Général 06, FEDER, Ministère de lEnseignement Supérieur and INSERM. We thank T Rudel of the University of Wuerzburg for the Flag-VDAC1 plasmid and G Rios for microarray hybridization Mircea Ivan's research is supported by NCI R01 CA155332.
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