Knockout of Vdac1 activates hypoxia-inducible factor through reactive oxygen species generation and induces tumor growth by promoting metabolic reprogramming and inflammation.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Local mitochondrial-endolysosomal microfusion cleaves voltage-dependent anion channel 1 to promote survival in hypoxia.

The oxygen-limiting (hypoxic) microenvironment of tumors induces metabolic reprogramming and cell survival, but the underlying mechanisms involving mitochondria remain poorly understood. We previously demonstrated that hypoxia-inducible factor 1 mediates the hyperfusion of mitochondria by inducing Bcl-2/adenovirus E1B 19-kDa interacting protein 3 and posttranslational truncation of the mitochondrial ATP transporter outer membrane voltage-dependent anion channel 1 in hypoxic cells. In addition, we showed that truncation is associated with increased resistance to drug-induced apoptosis and is indicative of increased patient chemoresistance. We now show that silencing of the tumor suppressor TP53 decreases truncation and increases drug-induced apoptosis. We also show that TP53 regulates truncation through induction of the mitochondrial protein Mieap. While we found that truncation was independent of mitophagy, we observed local microfusion between mitochondria and endolysosomes in hypoxic cells in culture and in patients' tumor tissues. Since we found that the endolysosomal asparagine endopeptidase was responsible for truncation, we propose that it is a readout of mitochondrial-endolysosomal microfusion in hypoxia. These novel findings provide the framework for a better understanding of hypoxic cell metabolism and cell survival through mitochondrial-endolysosomal microfusion regulated by hypoxia-inducible factor 1 and TP53.

Hypoxic VDAC1: a potential mitochondrial marker for cancer therapy.

Finding new therapeutic targets to fight cancer is an ongoing quest. Because of insufficiencies in tumor vasculature, cells often are exposed to a hostile microenvironment that is low in oxygen (hypoxic) and nutrients. Thus, tumor cells face the challenge of finding new sources of energy and defying apoptosis, which allow them to survive, grow, and colonize other tissues. Eradicating specifically these hypoxic cells is one of the many goals of anticancer therapies. The mitochondrial voltage-dependent anion channel (VDAC) is a protein at the crossroads of metabolic and survival pathways. As its name suggests, VDAC is involved in ion transport as well as adenosine triphosphate and NAD(+) transport. We recently reported the presence in tumor cells of a novel hypoxia-induced form of VDAC. This form, a C-terminal truncated protein (VDAC1-ΔC), was associated in some cancer cell lines with a high output of adenosine triphosphate and a strong resistance to chemotherapy-induced apoptosis. Furthermore, VDAC1-ΔC was detected in tissues of 50 % of 46 patients with lung cancer. This review examines the significance of this new form of VDAC1 for anticancer therapy.

Expression of a truncated active form of VDAC1 in lung cancer associates with hypoxic cell survival and correlates with progression to chemotherapy resistance.

Resistance to chemotherapy-induced apoptosis of tumor cells represents a major hurdle to efficient cancer therapy. Although resistance is a characteristic of tumor cells that evolve in a low oxygen environment (hypoxia), the mechanisms involved remain elusive. We observed that mitochondria of certain hypoxic cells take on an enlarged appearance with reorganized cristae. In these cells, we found that a major mitochondrial protein regulating metabolism and apoptosis, the voltage-dependent anion channel 1 (VDAC1), was linked to chemoresistance when in a truncated (VDAC1-ΔC) but active form. The formation of truncated VDAC1, which had a similar channel activity and voltage dependency as full-length, was hypoxia-inducible factor-1 (HIF-1)-dependent and could be inhibited in the presence of the tetracycline antibiotics doxycycline and minocycline, known inhibitors of metalloproteases. Its formation was also reversible upon cell reoxygenation and associated with cell survival through binding to the antiapoptotic protein hexokinase. Hypoxic cells containing VDAC1-ΔC were less sensitive to staurosporine- and etoposide-induced cell death, and silencing of VDAC1-ΔC or treatment with the tetracycline antibiotics restored sensitivity. Clinically, VDAC1-ΔC was detected in tumor tissues of patients with lung adenocarcinomas and was found more frequently in large and late-stage tumors. Together, our findings show that via induction of VDAC1-ΔC, HIF-1 confers selective protection from apoptosis that allows maintenance of ATP and cell survival in hypoxia. VDAC1-ΔC may also hold promise as a biomarker for tumor progression in chemotherapy-resistant patients.

Inactivation of androgen-induced regulator ARD1 inhibits androgen receptor acetylation and prostate tumorigenesis.

Androgen signaling through androgen receptor (AR) is critical for prostate tumorigenesis. Given that AR-mediated gene regulation is enhanced by AR coregulators, inactivation of those coregulators is emerging as a promising therapy for prostate cancer (PCa). Here, we show that the N-acetyltransferase arrest-defect 1 protein (ARD1) functions as a unique AR regulator in PCa cells. ARD1 is up-regulated in human PCa cell lines and primary tumor biopsies. The expression of ARD1 was augmented by treatment with synthetic androgen (R1881) unless AR is deficient or is inhibited by AR-specific siRNA or androgen inhibitor bicalutamide (Casodex). Depletion of ARD1 by shRNA suppressed PCa cell proliferation, anchorage-independent growth, and xenograft tumor formation in SCID mice, suggesting that AR-dependent ARD1 expression is biologically germane. Notably, ARD1 was critical for transcriptionally regulating a number of AR target genes that are involved in prostate tumorigenesis. Furthermore, ARD1 interacted physically with and acetylated the AR protein in vivo and in vitro. Because AR-ARD1 interaction facilitated the AR binding to its targeted promoters for gene transcription, we propose that ARD1 functions as a unique AR regulator and forms a positive feedback loop for AR-dependent prostate tumorigenesis. Disruption of AR-ARD1 interactions may be a potent intervention for androgen-dependent PCa therapy.

Hypoxia and energetic tumour metabolism.

The hypoxia-inducible factor (HIF-1), in addition to genetic and epigenetic changes, is largely responsible for alterations in cell metabolism in hypoxic tumour cells. This transcription factor not only favours cell proliferation through the metabolic shift from oxidative phosphorylation to glycolysis and lactic acid production but also stimulates nutrient supply by mediating adaptive survival mechanisms. These include epithelial-mesenchymal transition, angiogenesis, autophagy, and synthesis and storage of lipid and glycogen. HIF-1 also ensures survival by correcting tumour acidosis via increased expression of the carbonic anhydrase CA IX and the lactate/H+ symporter MCT4. The targeting of key HIF-1-mediated steps, responsible for exacerbated glycolysis and pHi-control, and of the 'guardian of cellular energy' AMP-kinase should offer novel therapeutic opportunities to fight cancer.

Hypoxic enlarged mitochondria protect cancer cells from apoptotic stimuli.

It is well established that cells exposed to the limiting oxygen microenvironment (hypoxia) of tumors acquire resistance to chemotherapy, through mechanisms not fully understood. We noted that a large number of cell lines showed protection from apoptotic stimuli, staurosporine, or etoposide, when exposed to long-term hypoxia (72 h). In addition, these cells had unusual enlarged mitochondria that were induced in a HIF-1-dependent manner. Enlarged mitochondria were functional as they conserved their transmembrane potential and ATP production. Here we reveal that mitochondria of hypoxia-induced chemotherapy-resistant cells undergo a HIF-1-dependent and mitofusin-1-mediated change in morphology from a tubular network to an enlarged phenotype. An imbalance in mitochondrial fusion/fission occurs since silencing of not only the mitochondrial fusion protein mitofusin 1 but also BNIP3 and BNIP3L, two mitochondrial HIF-targeted genes, reestablished a tubular morphology. Hypoxic cells were insensitive to staurosporine- and etoposide-induced cell death, but the silencing of mitofusin, BNIP3, and BNIP3L restored sensitivity. Our results demonstrate that some cancer cells have developed yet another way to evade apoptosis in hypoxia, by inducing mitochondrial fusion and targeting BNIP3 and BNIP3L to mitochondrial membranes, thereby giving these cells a selective growth advantage.

Tumour hypoxia induces a metabolic shift causing acidosis: a common feature in cancer.

Maintenance of cellular pH homeostasis is fundamental to life. A number of key intracellular pH (pHi) regulating systems including the Na(+)/H(+) exchangers, the proton pump, the monocarboxylate transporters, the HCO(3)(-) transporters and exchangers and the membrane-associated and cytosolic carbonic anhydrases cooperate in maintaining a pHi that is permissive for cell survival. A common feature of tumours is acidosis caused by hypoxia (low oxygen tension). In addition to oncogene activation and transformation, hypoxia is responsible for inducing acidosis through a shift in cellular metabolism that generates a high acid load in the tumour microenvironment. However, hypoxia and oncogene activation also allow cells to adapt to the potentially toxic effects of an excess in acidosis. Hypoxia does so by inducing the activity of a transcription factor the hypoxia-inducible factor (HIF), and particularly HIF-1, that in turn enhances the expression of a number of pHi-regulating systems that cope with acidosis. In this review, we will focus on the characterization and function of some of the hypoxia-inducible pH-regulating systems and their induction by hypoxic stress. It is essential to understand the fundamentals of pH regulation to meet the challenge consisting in targeting tumour metabolism and acidosis as an anti-tumour approach. We will summarize strategies that take advantage of intracellular and extracellular pH regulation to target the primary tumour and metastatic growth, and to turn around resistance to chemotherapy and radiotherapy.

Hypoxia-inducible carbonic anhydrase IX and XII promote tumor cell growth by counteracting acidosis through the regulation of the intracellular pH.

Acidosis of the tumor microenvironment is typical of a malignant phenotype, particularly in hypoxic tumors. All cells express multiple isoforms of carbonic anhydrase (CA), enzymes catalyzing the reversible hydration of carbon dioxide into bicarbonate and protons. Tumor cells express membrane-bound CAIX and CAXII that are controlled via the hypoxia-inducible factor (HIF). Despite the recognition that tumor expression of HIF-1alpha and CAIX correlates with poor patient survival, the role of CAIX and CAXII in tumor growth is not fully resolved. To understand the advantage that tumor cells derive from expression of both CAIX and CAXII, we set up experiments to either force or invalidate the expression of these enzymes. In hypoxic LS174Tr tumor cells expressing either one or both CA isoforms, we show that (a) in response to a "CO(2) load," both CAs contribute to extracellular acidification and (b) both contribute to maintain a more alkaline resting intracellular pH (pH(i)), an action that preserves ATP levels and cell survival in a range of acidic outside pH (6.0-6.8) and low bicarbonate medium. In vivo experiments show that ca9 silencing alone leads to a 40% reduction in xenograft tumor volume with up-regulation of ca12 mRNA levels, whereas invalidation of both CAIX and CAXII gives an impressive 85% reduction. Thus, hypoxia-induced CAIX and CAXII are major tumor prosurvival pH(i)-regulating enzymes, and their combined targeting shows that they hold potential as anticancer targets.

A dialogue between the hypoxia-inducible factor and the tumor microenvironment.

The hypoxia-inducible factor is the key protein responsible for the cellular adaptation to low oxygen tension. This transcription factor becomes activated as a result of a drop in the partial pressure of oxygen, to hypoxic levels below 5% oxygen, and targets a panel of genes involved in maintenance of oxygen homeostasis. Hypoxia is a common characteristic of the microenvironment of solid tumors and, through activation of the hypoxia-inducible factor, is at the center of the growth dynamics of tumor cells. Not only does the microenvironment impact on the hypoxia-inducible factor but this factor impacts on microenvironmental features, such as pH, nutrient availability, metabolism and the extracellular matrix. In this review we discuss the influence the tumor environment has on the hypoxia-inducible factor and outline the role of this factor as a modulator of the microenvironment and as a powerful actor in tumor remodeling. From a fundamental research point of view the hypoxia-inducible factor is at the center of a signaling pathway that must be deciphered to fully understand the dynamics of the tumor microenvironment. From a translational and pharmacological research point of view the hypoxia-inducible factor and its induced downstream gene products may provide information on patient prognosis and offer promising targets that open perspectives for novel "anti-microenvironment" directed therapies.

Hypoxia and cancer.

A major feature of solid tumours is hypoxia, decreased availability of oxygen, which increases patient treatment resistance and favours tumour progression. How hypoxic conditions are generated in tumour tissues and how cells respond to hypoxia are essential questions in understanding tumour progression and metastasis. Massive tumour-cell proliferation distances cells from the vasculature, leading to a deficiency in the local environment of blood carrying oxygen and nutrients. Such hypoxic conditions induce a molecular response, in both normal and neoplastic cells, that drives the activation of a key transcription factor; the hypoxia-inducible factor. This transcription factor regulates a large panel of genes that are exploited by tumour cells for survival, resistance to treatment and escape from a nutrient-deprived environment. Although now recognized as a major contributor to cancer progression and to treatment failure, the precise role of hypoxia signalling in cancer and in prognosis still needs to be further defined. It is hoped that a better understanding of the mechanisms implicated will lead to alternative and more efficient therapeutic approaches.

Hypoxia in cancer cell metabolism and pH regulation.

At a molecular level, hypoxia induces the stabilization and activation of the alpha-subunit of an alpha/beta heterodimeric transcription factor, appropriately termed HIF (hypoxia-inducible factor). Hypoxia is encountered, in particular, in tumour tissues, as a result of an insufficient and defective vasculature present in a highly proliferative tumour mass. In this context the active HIF heterodimer binds to and induces a panel of genes that lead to modification in a vast range of cellular functions that allow cancer cells to not only survive but to continue to proliferate and metastasize. Therefore HIF plays a key role in tumorigenesis, tumour development and metastasis, and its expression in solid tumours is associated with a poor patient outcome. Among the many genes induced by HIF are genes responsible for glucose transport and glucose metabolism. The products of these genes allow cells to adapt to cycles of hypoxic stress by maintaining a level of ATP sufficient for survival and proliferation. Whereas normal cells metabolize glucose through a cytoplasmic- and mitochondrial-dependent pathway, cancer cells preferentially use a cytoplasmic, glycolytic pathway that leads to an increased acid load due, in part, to the high level of production of lactic acid. This metabolic predilection of cancer cells is primarily dependent directly on the HIF activity but also indirectly through changes in the activity of tumour suppressors and oncogenes. A better understanding of HIF-dependent metabolism and pH regulation in cancer cells should lead to further development of diagnostic tools and novel therapeutics that will bring benefit to cancer patients.

SUMOylation of hypoxia-inducible factor-1alpha reduces its transcriptional activity.

The hypoxic response of mammalian cells is controlled through a transcriptional pathway that is mediated by the hypoxia-inducible factor (HIF). Here, we show that HIF-1alpha undergoes post-translational modification by the three isoforms of the small ubiquitin-related modifier (SUMO-1, -2 and -3) in vitro in proximity to and within the oxygen-dependent degradation domain (ODDD). SUMO conjugation is promoted in vitro by the E3 SUMO ligase RanBP2/Nup538 and SUMO modification in vivo does not change HIF-1alpha turnover rate. Using cotransfection of siRNA targeted to endogenous HIF-1alpha together with HIF-1alpha siRNA-resistant expression vectors carrying mutations for SUMO modification we demonstrate increased hypoxia-response element-dependent transcriptional activity for SUMO-deficient HIF-1alpha. These results indicate that when HIF-1alpha is conjugated to SUMO its transcriptional activity is decreased and that this is not mediated by a change in the protein's half-life.

Oxygen, a source of life and stress.

Oxygen is an essential element in the survival of complex organisms, however the level of oxygen, low or high, can be a source of stress depending on the biological context. Low levels of oxygen in tissues (hypoxia) can be the consequence of a number of pathophysiological conditions including ischemic disorders and cancer while relative, higher levels (hyperoxia) can lead to retinopathy of prematurity. The local oxygen environment and oxygen consumption dictate vascular homeostasis, vaso-proliferation and vaso-cessation, which is deregulated in these diseases through oxygen-dependent growth factors. In this review, we will introduce aspects of the physiology and biology of oxygen partial pressure and the molecular mechanisms implicated in oxygen sensing. We will outline the regulation and function of the key operator in cellular signalling of hypoxia, the transcription factor, hypoxia-inducible factor. In addition, we will focus on cancer cell hypoxia and on its role in driving cell metabolism, pH regulation and survival.

Hypoxia signalling controls metabolic demand.

It has been known for quite some time that cancer cells undergo far-reaching modifications in their metabolism, yet a full understanding of these changes and how they come about remains elusive. Even under conditions of plentiful oxygen, cancer cells choose to switch glucose metabolism from respiration to lactic acid formation. The mystery behind the molecular mechanisms of this phenomenon, known as the Warburg effect, is now being unravelled. The reduced respiration rate and increased glucose uptake associated with lactic acid production, and acidosis of the micro-environment, are primarily due to activation of the alpha/beta hypoxia-inducible transcription factor. This distinctive metabolic nature of cancer cells is already being exploited as a diagnostic tool but is yet to be harnessed as a therapeutic intervention.

Harnessing the hypoxia-inducible factor in cancer and ischemic disease.

The alpha/beta-heterodimeric transcription factor hypoxia-inducible factor (HIF) functions when the oxygen level in tissues is low, i.e. when the tissue microenvironment becomes hypoxic, and is non-functional when the level of oxygen is high. Certain pathophysiological conditions such as ischemic disorders and cancer encounter low levels of local tissue oxygenation due to a defective or insufficient vasculature. Highly proliferating tumour cells rapidly form into a mass that becomes located too far from the vasculature to be nourished and oxygenated. Under such conditions HIF activates or represses a vast array of genes that in particular, initiate the formation of new blood vessels and modify metabolism. In this way the tumour mass re-establishes conditions favourable for further proliferation. Interest is being expressed in the direct repression or stimulation of HIF activity, respectively, in the treatment of cancer and of ischemic disorders. The modulation of other HIF-target genes implicated, in particular, in tumour metabolism and intracellular pH control may also prove to be useful in cancer therapy. However, before going further a better understanding of the basics of the HIF signalling pathway is essential. This review will introduce the reader to the molecular mechanisms that regulate HIF and some of the biological consequences of its action, in particular in tumour metabolism, growth and invasion. Approaches to either enforce tumour regression or increase blood vessel formation through the targeting of HIF or its downstream effectors will also be discussed.

ARDent about acetylation and deacetylation in hypoxia signalling.

Given the key role that the alpha subunit of the alphabeta heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1) has in tumourigenesis, and in particular in angiogenesis, a full understanding of its regulation is crucial to the development of cancer therapeutics. Posttranslational acetylation and deacetylation of this subunit by an acetyltransferase called Arrest-defective-1 (ARD1) and by different histone deacetylases (HDACs), respectively, has been suggested as a mechanism. However, conflicting data bring into question the foundations of this mechanism and at present it is not clear what the precise role of these proteins is with respect to HIF. Nonetheless, the observation that small-molecule inhibitors of HDACs have anti-angiogenic activity suggests that acetylation and deacetylation of HIF or HIF modifiers represents a potential target in cancer therapy.