Low glucose microenvironment of normal kidney cells stabilizes a subset of messengers involved in angiogenesis.

As glucose is a mandatory nutrient for cell proliferation and renewal, it is suspected that glucose microenvironment is sensed by all cell types to regulate angiogenesis. Several glucose-sensing components have been partially described to respond to high glucose levels. However, little is known about the response to low glucose. Here, we used well-differentiated isolated normal rat renal tubules under normal oxygenation conditions to assess the angiogenic response to low glucose. In apparent paradox, but confirming observations made separately in other models, high glucose but also low glucose increased mRNA level of vascular endothelial growth factor A (VEGFA). A subset of mRNAs including hypoxia-inducible factor 1A (HIF1A), angiopoietin receptor (TIE-2), and VEGF receptor 2 (FLK1) were similarly glucose-sensitive and responded to low glucose by increased stability independently of HIF1A and HIF2A proteins. These results contribute to gain some insights as to how normal cells response to low glucose may play a role in the tumor microenvironment.

TRIM8 anti-proliferative action against chemo-resistant renal cell carcinoma.

In some tumours, despite a wild-type p53 gene, the p53 pathway is inactivated by alterations in its regulators or by unknown mechanisms, leading to resistance to cytotoxic therapies. Understanding the mechanisms of functional inactivation of wild-type p53 in these tumours may help to define prospective targets for treating cancer by restoring p53 activity. Recently, we identified TRIM8 as a new p53 modulator, which stabilizes p53 impairing its association with MDM2 and inducing the reduction of cell proliferation. In this paper we demonstrated that TRIM8 deficit dramatically impairs p53-mediated cellular responses to chemotherapeutic drugs and that TRIM8 is down regulated in patients affected by clear cell Renal Cell Carcinoma (ccRCC), an aggressive drug-resistant cancer showing wild-type p53. These results suggest that down regulation of TRIM8 might be an alternative way to suppress p53 activity in RCC. Interestingly, we show that TRIM8 expression recovery in RCC cell lines renders these cells sensitive to chemotherapeutic treatments following p53 pathway re-activation. These findings provide the first mechanistic link between TRIM8 and the drug resistance of ccRCC and suggest more generally that TRIM8 could be used as enhancer of the chemotherapy efficacy in cancers where p53 is wild-type and its pathway is defective.

Endoplasmic reticulum calcium release through ITPR2 channels leads to mitochondrial calcium accumulation and senescence.

Senescence is involved in various pathophysiological conditions. Besides loss of retinoblastoma and p53 pathways, little is known about other pathways involved in senescence. Here we identify two calcium channels; inositol 1,4,5-trisphosphate receptor, type 2 (ITPR2) (also known as inositol 1,4,5-triphosphate receptor 2 (IP3R2)) and mitochondrial calcium uniporter (MCU) as new senescence regulators in a loss-of-function genetic screen. We show that loss of ITPR2, known to mediate endoplasmic reticulum (ER) calcium release, as well as loss of MCU, necessary for mitochondrial calcium uptake, enable escape from oncogene-induced senescence (OIS). During OIS, ITPR2 triggers calcium release from the ER, followed by mitochondrial calcium accumulation through MCU channels. Mitochondrial calcium accumulation leads to a subsequent decrease in mitochondrial membrane potential, reactive oxygen species accumulation and senescence. This ER-mitochondria calcium transport is not restricted to OIS, but is also involved in replicative senescence. Our results show a functional role of calcium release by the ITPR2 channel and its subsequent accumulation in the mitochondria.

Conventional techniques to monitor mitochondrial oxygen consumption.

Following several key discoveries on hypoxia-inducible factors, we have observed an explosion of studies investigating how the hypoxic microenvironment provokes bioenergetic alterations. This is particularly relevant for cancer cells, as they are often exposed to hypoxic conditions in the course of tumor progression. Thus, interest in the measurement of oxygen consumption at the tissue, cell, or mitochondrion level has been revived. Here, we describe the basic principles of cellular respiration and survey some of the conventional methods for measuring O2 consumption in intact or permeabilized cells.

Repression of PLA2R1 by c-MYC and HIF-2alpha promotes cancer growth.

Loss of secreted phospholipase A2 receptor (PLA2R1) has recently been found to render human primary cells more resistant to senescence whereas increased PLA2R1 expression is able to induce cell cycle arrest, cancer cell death or blockage of cancer cell transformation in vitro, suggesting that PLA2R1 displays tumor suppressive activities. Here we report that PLA2R1 expression strongly decreases in samples of human renal cell carcinoma (RCC). Knockdown of PLA2R1 increases renal cancer cell tumorigenicity supporting a role of PLA2R1 loss to promote in vivo RCC growth. Most RCC result from Von Hippel-Lindau (VHL) tumor suppressor loss-of-function and subsequent gain-of-function of the oncogenic HIF-2alpha/c-MYC pathway. Here, by genetically manipulating VHL, HIF-2alpha and c-MYC, we demonstrate that loss of VHL, stabilization of HIF-2alpha and subsequent increased c-MYC activity, binding and transcriptional repression, through induction of PLA2R1 DNA methylation closed to PLA2R1 transcriptional start site, results in decreased PLA2R1 transcription. Our results describe for the first time an oncogenic pathway leading to PLA2R1 transcriptional repression and the importance of this repression for tumor growth.

The CXCL7/CXCR1/2 axis is a key driver in the growth of clear cell renal cell carcinoma.

Mutations in the von Hippel-Lindau gene upregulate expression of the central angiogenic factor VEGF, which drives abnormal angiogenesis in clear cell renal cell carcinomas (ccRCC). However, the overexpression of VEGF in these tumors was not found to correlate with overall survival. Here, we show that the proangiogenic, proinflammatory cytokine CXCL7 is an independent prognostic factor for overall survival in this setting. CXCL7 antibodies strongly reduced the growth of ccRCC tumors in nude mice. Conversely, conditional overexpression of CXCL7 accelerated ccRCC development. CXCL7 promoted cell proliferation in vivo and in vitro, in which expression of CXCL7 was induced by the central proinflammatory cytokine interleukin (IL)-1ß. ccRCC cells normally secrete low amounts of CXCL7; it was more highly expressed in tumors due to high levels of IL-1ß there. We found that a pharmacological inhibitor of the CXCL7 receptors CXCR1 and CXCR2 (SB225002) was sufficient to inhibit endothelial cell proliferation and ccRCC growth. Because CXCR1 and CXCR2 are present on both endothelial and ccRCC cells, their inhibition affected both the tumor vasculature and the proliferation of tumor cells. Our results highlight the CXCL7/CXCR1/CXCR2 axis as a pertinent target for the treatment of ccRCC.

PLA2R1 kills cancer cells by inducing mitochondrial stress.

Little is known about the biological functions of the phospholipase A2 receptor (PLA2R1) except that it has the ability to bind a few secreted phospholipases A2 (sPLA2's). We have previously shown that PLA2R1 regulates senescence in normal human cells. In this study, we investigated the ability of PLA2R1 to control cancer cell growth. Analysis of expression in cancer cells indicates a marked PLA2R1 decrease in breast cancer cell lines compared to normal or nontransformed human mammary epithelial cells. Accordingly, PLA2R1 ectopic expression in PLA2R1-negative breast cancer cell lines led to apoptosis, whereas a prosenescence response was predominantly triggered in normal cells. PLA2R1 structure-function studies and the use of chemical inhibitors of sPLA2-related signaling pathways suggest that the effect of PLA2R1 is sPLA2-independent. Functional experiments demonstrate that PLA2R1 regulation of cell death is driven by a reactive oxygen species (ROS)-dependent mechanism. While screening for ROS-producing complexes involved in PLA2R1 biological responses, we identified a critical role for the mitochondrial electron transport chain in PLA2R1-induced ROS production and cell death. Taken together, this set of data provides evidence for an important role of PLA2R1 in controlling cancer cell death by influencing mitochondrial biology.

PLA2R1 mediates tumor suppression by activating JAK2.

Little is known about the physiological role of the phospholipase A2 receptor (PLA2R1). PLA2R1 has been described as regulating the replicative senescence, a telomerase-dependent proliferation arrest. The downstream PLA2R1 signaling and its role in cancer are currently unknown. Senescence induction in response to activated oncogenes is a failsafe program of tumor suppression that must be bypassed for tumorigenesis. We now present evidence that PLA2R1 functions in vitro as a tumor suppressor, the depletion of which is sufficient to escape oncogene-induced senescence (OIS), thereby facilitating oncogenic cell transformation. Furthermore, mice that are genetically deficient in PLA2R1 display increased sensitivity to RAS-induced tumorigenesis by facilitating OIS escape, highlighting its physiological role as a tumor suppressor. Unexpectedly, PLA2R1 activated JAK2 and its effector signaling, with PLA2R1-mediated inhibition of cell transformation largely reverted in JAK2-depleted cells. This finding was unexpected as the JAK2 pathway has been associated mainly with protumoral functions and several inhibitors are currently in clinical trials. Taken together, our findings uncover an unanticipated tumor suppressive role for PLA2R1 that is mediated by targeting downstream JAK2 effector signaling.

HIF and reactive oxygen species regulate oxidative phosphorylation in cancer.

A decrease in oxidative phosphorylation (OXPHOS) is characteristic of many cancer types and, in particular, of clear cell renal carcinoma (CCRC) deficient in von Hippel-Lindau (vhl) gene. In the absence of functional pVHL, hypoxia-inducible factor (HIF) 1-alpha and HIF2-alpha subunits are stabilized, which induces the transcription of many genes including those involved in glycolysis and reactive oxygen species (ROS) metabolism. Transfection of these cells with vhl is known to restore HIF-alpha subunit degradation and to reduce glycolytic genes transcription. We show that such transfection with vhl of 786-0 CCRC (which are devoid of HIF1-alpha) also increased the content of respiratory chain subunits. However, the levels of most transcripts encoding OXPHOS subunits were not modified. Inhibition of HIF2-alpha synthesis by RNA interference in pVHL-deficient 786-0 CCRC also restored respiratory chain subunit content and clearly demonstrated a key role of HIF in OXPHOS regulation. In agreement with these observations, stabilization of HIF-alpha subunit by CoCl(2) decreased respiratory chain subunit levels in CCRC cells expressing pVHL. In addition, HIF stimulated ROS production and mitochondrial manganese superoxide dismutase content. OXPHOS subunit content was also decreased by added H(2)O(2.) Interestingly, desferrioxamine (DFO) that also stabilized HIF did not decrease respiratory chain subunit level. While CoCl(2) significantly stimulates ROS production, DFO is known to prevent hydroxyl radical production by inhibiting Fenton reactions. This indicates that the HIF-induced decrease in OXPHOS is at least in part mediated by hydroxyl radical production.

Clonal expansion of mutated mitochondrial DNA is associated with tumor formation and complex I deficiency in the benign renal oncocytoma.

Mutations in mitochondrial DNA (mtDNA) are frequent in cancers but it is not yet clearly established whether they are modifier events involved in cancer progression or whether they are a consequence of tumorigenesis. Here we show a benign tumor type in which mtDNA mutations that lead to complex I (CI) enzyme deficiency are found in all tumors and are the only genetic alteration detected. Actually renal oncocytomas are homogeneous tumors characterized by dense accumulation of mitochondria and we had found that they are deficient in electron transport chain complex I (CI, NADH-ubiquinone oxidoreductase). In this work total sequencing of mtDNA showed that 9/9 tumors harbored point mutations in mtDNA, seven in CI genes, one in complex III, and one in the control region. 7/8 mutations were somatic. All tumors were somatically deficient for CI. The clonal amplification of mutated mtDNA in 8/9 tumors demonstrates that these alterations are selected and therefore favor or trigger growth. No nuclear DNA rearrangement was detected beside mtDNA defects. We hypothesize that functional deficiency of the oxidative phosphorylation CI could create a loop of amplification of mitochondria during cell division, impair substrates oxidation and increase intermediary metabolites availability.

Actuality of Warburg's views in our understanding of renal cancer metabolism.

More than 50 years ago, Warburg proposed that the shift in glucose metabolism from oxidative phosphorylation (OXPHOS) to glycolysis occurring in spite of an adequate oxygen supply was at the root of cancer. This hypothesis often disregarded over the following years has recently stirred up much interest due to progress made in cancer genetics and proteomics. Studies related to renal cancers have been particularly informative to understand how abnormal use of glucose and decrease in OXPHOS are linked to cell proliferation in tumors. Indeed, in aggressive tumors such as clear cell renal carcinoma, the von Hippel-Lindau factor invalidation stabilizes the hypoxia-inducible factor (HIF) in the presence of oxygen. HIF stimulating glycolytic gene expression increases the glycolytic flux. Deficiencies in genes involved in oxidative phosphorylation that can explain the down-regulation of OXPHOS components also begin to be identified. These findings are important in the search for novel therapeutic approaches to cancer treatment.

Mitochondria and reactive oxygen species in renal cancer.

In most cancer cells, the ATP necessary for survival and proliferation is derived from glycolysis rather than from oxidative phosphorylations (OXPHOS) even when oxygen supply would be adequate to sustain them. This phenomenon, named "aerobic glycolysis" by Warburg many years ago, can now be explained by a mechanism up-regulating the expression of genes involved in glucose transport, glucose metabolism, lactate formation and exit from the cell. In clear cell renal carcinoma, this mechanism is due to the stabilization of the hypoxia-inducible transcription factor HIF occurring when the tumor suppressor gene vhl is invalidated. HIF increases the transcription of genes involved in glycolysis and lactate metabolism. Although respiratory chain complex activities and subunit amounts are severely diminished, the transcription of genes involved in the structure and biogenesis of these complexes does not seem to be significantly decreased in these cancers but reactive oxygen species (ROS) production is increased. In this review, we discuss the roles that ROS may play in the decrease of OXPHOS in cancer and in the regulation of the mitochondria-induced initiation of apoptosis.

Physiological oxygenation status is required for fully differentiated phenotype in kidney cortex proximal tubules.

Hypoxia has been suspected to trigger transdifferentiation of renal tubular cells into myofibroblasts in an epithelial-to-mesenchymal transition (EMT) process. To determine the functional networks potentially altered by hypoxia, rat renal tubule suspensions were incubated under three conditions of oxygenation ranging from normoxia (lactate uptake) to severe hypoxia (lactate production). Transcriptome changes after 4 h were analyzed on a high scale by restriction fragment differential display. Among 1,533 transcripts found, 42% were maximally expressed under severe hypoxia and 8% under mild hypoxia (Po(2) = 48 mmHg), suggesting two different levels of oxygen sensing. Normoxia was required for full expression of the proximal tubule-specific transcripts 25-hydroxyvitamin D 1-hydroxylase (Cyp27b1) and l-pyruvate kinase (Pklr), transcripts involved in tissue cohesion such as fibronectin (Fn1) and N-cadherin (Cdh2), and non-muscle-type myosin transcripts. Mild hypoxia increased myogenin transcript level. Conversely, severe hypoxia increased transcripts involved in extracellular matrix remodeling, those of muscle-type myosins, and others involved in creatine phosphate synthesis and lactate transport (Slc16a7). Accordingly, microscopy showed loss of tubule aggregation under hypoxia, without tubular disruption. Hypoxia also increased the levels of kidney-specific transcripts normally restricted to the less oxygenated medullary zone and others specific for the distal part of the nephron. We conclude that extensive oxygen supply to the kidney tubule favors expression of its differentiated functions specifically in the proximal tubule, whose embryonic origin is mesenchymal. The phenotype changes could potentially permit transient adaptation to hypoxia but also favor pathological processes such as tissue invasion.

Inhibition of cytochrome c oxidase subunit 4 precursor processing by the hypoxia mimic cobalt chloride.

Cobalt is often used as a hypoxia mimic in cell culture, because it stabilizes the alpha subunits of the transcription factor, HIF (hypoxia-inducible factor). We have previously shown that HIF stabilization due to a deficiency of the von Hippel Lindau protein (pVHL) in clear cell renal carcinoma (CRCC) was correlated to a down-regulation of oxidative phosphorylation. To better understand this mechanism, we have used CoCl2 in CRCC expressing stably transfected vhl. We show that, in addition to its effect on HIF-alpha subunits, CoCl2 prevented the normal processing of the precursor of cytochrome c oxidase (COX) subunit 4 and induced COX degradation very likely by inhibiting the mitochondrial intermediate peptidase (MIP) that cleaves the COX4 precursor protein. This cobalt-induced MIP inhibition was however not observed in other human mitochondrial precursor sequences as previously predicted from comparison between human and yeast mitochondrial precursor sequences.

A new role for the von Hippel-Lindau tumor suppressor protein: stimulation of mitochondrial oxidative phosphorylation complex biogenesis.

Although mitochondrial deficiency in cancer has been described by Warburg, many years ago, the mechanisms underlying this impairment remain essentially unknown. Many types of cancer cells are concerned and, in particular, clear cell renal carcinoma (CCRC). In this cancer, the tumor suppressor gene, VHL (von Hippel-Lindau factor) is invalidated. Previous studies have shown that the transfection of the VHL gene in VHL-deficient cells originating from CCRCs could suppress their ability to form tumors when they were injected into nude mice. However, various additional genetic alterations are observed in such cancer cells. In order to investigate whether VHL invalidation was related to the mitochondrial impairment, we have studied the effects of wild-type VHL transfection into VHL-deficient 786-0 or RCC10 cells on their oxidative phosphorylation (OXPHOS) subunit contents and functions. We show that the presence of wild-type VHL protein (pVHL) increased mitochondrial DNA and respiratory chain protein contents and permitted the cells to rely on their mitochondrial ATP production to grow in the absence of glucose. In parallel to mtDNA increase, the presence of pVHL up regulated the mitochondrial transcription factor A, as shown by western blot analysis. In conclusion, in CCRCs, pVHL deficiency is one of the factors responsible for down-regulation of the biogenesis of OXPHOS complexes.

Natural antisense transcripts of HIF-1alpha are conserved in rodents.

A natural antisense transcript (aHIF), which sequence is strictly complementary to the 3' untranslated region (3'UTR) of HIF-1alpha mRNA, has been identified in human and shown to be overexpressed in renal carcinomas. We searched for aHIF in different rodent tissues. Two candidate expressed sequence tag (EST) were identified in silico and their PCR products (1.1 and 1.0 kb) were cloned and sequenced in mouse and rat, respectively. These transcripts were rigorously complementary to the 3'UTR of rodent HIF-1alpha mRNA and were broadly expressed in all mouse and rat tissues we tested. The conservation of aHIF in rodents underlined its potential importance in cell regulations. Therefore the responses of aHIF and HIF-1alpha transcripts were investigated in various types of hypoxic conditions. In freshly isolated rat renal tubules, aHIF RNA level was increased by acute hypoxia and low in normal supply of oxygen. In a rat strain raised in chronic hypobaric altitude hypoxia, aHIF transcript was greatly induced in the oxidative-type soleus and heart muscles of 3 month-old animals. By contrast, in the glycolytic-type extensor digitorum longus muscle aHIF transcript amount was lowered by hypoxia whereas HIF-1alpha transcript was highly expressed. In brain, where oxidative glycolysis takes place, HIF-1alpha mRNA and its antisense transcript levels were high and not significantly changed by altitude. Tumour cell lines cultured for 6 h in conditions mimicking hypoxia expressed lower amounts of HIF-1alpha mRNA. In two rat cell lines, aHIF transcript levels were greatly augmented after a 6-h incubation in these conditions, whereas in a mouse cell line, aHIF level was significantly reduced.

Mitochondrial complex I is deficient in renal oncocytomas.

Renal oncocytomas are benign tumors characterized by dense accumulation of mitochondria the cause of which remains unknown so far. Consistently, mitochondrial DNA content and the amounts and catalytic activities of several oxidative phosphorylation (OXPHOS) complexes were known to be increased in these tumors, but it was not ascertained that the OXPHOS system was functional. Here we investigated mitochondrial complex I and found that its NADH dehydrogenase activity and protein content were specifically decreased in oncocytomas, in stark contrast with the parallel decrease of all respiratory chain complexes in other, malignant, renal tumors. We conclude that deficiency of complex I in oncocytomas might be the early event causing the increased mitochondrial biogenesis, attempting to compensate for the loss of OXPHOS function. Since other tumors were found to be linked to mitochondrial deficiencies like genetic alterations of fumarate hydratase or succinate dehydrogenase, oncocytoma could be the third type of benign tumor associated with impairment of mitochondrial ATP production in an oxidative, quiescent tissue. Besides, complex I enzyme activity was moderately decreased in the vicinity of oncocytomas, when compared with normal tissue adjacent to other renal tumors. This suggested that oncocytomas are the result of at least two serial modifications altering the mitochondrial respiratory chain.

Low mitochondrial respiratory chain content correlates with tumor aggressiveness in renal cell carcinoma.

A mechanism decreasing oxidative metabolism during normal cell division and growth is expected to direct substrates toward biosyntheses rather than toward complete oxidation to CO(2). Hence, any event decreasing oxidative phosphorylations (OXPHOS) could provide a proliferating advantage to a transformed or tumor cell in an oxidative tissue. To test this hypothesis, we studied mitochondrial enzymes, DNA and OXPHOS protein content in three types of renal tumors from 25 patients. Renal cell carcinomas (RCCs) of clear cell type (CCRCCs) originate from the proximal tubule and are most aggressive. Chromophilic RCCs, from similar proximal origin, are less aggressive. The benign renal oncocytomas originate from collecting duct cells. Mitochondrial enzyme and DNA contents in all tumor types or grades differed significantly from normal tissue. Mitochondrial impairment increased from the less aggressive to the most aggressive RCCs, and correlated with a considerably decreased content of OXPHOS complexes (complexes II, III, and IV of the respiratory chain, and ATPase/ATP synthase) rather than to the mitochondrial content (citrate synthase and mitochondrial (mt)DNA). In benign oncocytoma, some mitochondrial parameters (mtDNA, citrate synthase, and complex IV) were increased 4- to 7-fold, and some were slightly increased by a factor of 2 (complex V) or close to normal (complexes II and III). A low content of complex V protein was found in all CCRCC and chromophilic tumors studied. However F(1)-ATPase activity was not consistently decreased and its impairment was associated with increased aggressiveness in CCRCCs. Immunodetection of free F(1)-sector of complex V demonstrated a disturbed assembly/stability of complex V in several CCRCC and chromophilic tumors. All results are in agreement with the hypothesis that a decreased OXPHOS capacity favors faster growth or increased invasiveness.