Cancer cells differ

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Cancer cells differ from their normal counterparts in several aspects including energy production pathways, which in most cancer cells are executed through glycolysis even in the presence of ample oxygen. Initially it was thought that aerobic glycolysis, or Warburg effect was a result of irreversibly impaired mitochondrial respiration, but recent studies have provided evidence that this switch to alternative metabolic pathways is a consequence of oncogenic activity and mutations occurring in genes involved in energy metabolism. The important role of transcription factors such as p53 and Hypoxia Inducible Factor-1a (HIF-1a) in the regulation of the expression of numerous enzymes involved in energy production has been well documented in recent studies. p53 regulates energy metabolism through its target genes Tp53-Induced Glycolysis and Apoptosis Regulator (TIGAR) and Synthesis of Cytochrome C Oxidase 2 (SCO2). On the other hand HIF-1a modulates the expression of glucose transporters (GLUT-1) and glycolytic enzymes including lactate dehydrogenase-A (LDH-A). In addition, the identification of Hypoxia Responsive Elements (HREs) in the promoters of TIGAR and SCO2 indicate that those two genes are putative HIF-1a transcription targets. The aim of our study is to investigate the role of p53 and HIF-1a in the acquisition of specific metabolic phenotypes by tumour cells by differentially regulating TIGAR and/or SCO2 in response to diverse stress signals. At this stage, we have found out few HREs in the promoters of TIGAR and SCO2. Also, we have shown that p53 is not the only transcription factor that regulates TIGAR under hypoxia. We have shown the importance of HAT (Histone Acetyl Transferase) activity in the apoptosis/survival pattern of p53+/+ and p53-/- cells. In future, these findings need to be correlated to the expressions of TIGAR and/or SCO2 under hypoxia.


1.1 Warburg effect and its possible causes

In normal cells, metabolism of glucose starts with glycolysis, which occurs in the cytoplasm. This oxygen independent pathway produces pyruvate as end product. Pyruvate then enters the mitochondria and is metabolized through tricarboxylic acid cycle (TCA cycle) and electron transport chain (ETC) to carbon-dioxide (CO2), water and energy in the form of Adenosine 5'-triphosphate (ATP). Each molecule of glucose metabolized this way, produces thirty six (36) molecules of ATP. When the tissue oxygen supply is inadequate (during hypoxia), cells tend to adapt some changes in glucose metabolism. During hypoxia, the pyruvate produced by glycolysis is converted to lactate instead of entering mitochondrial respiration. The net energy produced in this pathway is only 2 ATP molecules per one molecule of glucose (Murray, 1996).

Marathon runners and other athletes benefit from this shift (Garber, 2004) when prolonged physical exercise reduces the oxygen supply to the muscles. This cellular response to hypoxia becomes a basic metabolic phenomenon in tumour cells. In 1920s German Biochemist Otto Warburg discovered that tumour cells prefer to produce energy through glycolysis even in the presence of adequate oxygen (Warburg, 1956). This phenomenon is called Warburg effect or aerobic glycolysis. Figure 1 schematically explains the Warburg hypothesis. Warburg hypothesized that the irreversible damage of mitochondrial respiration (oxidative phosphorylation) is the cause of increased glycolysis in cancer cells and that is the cause of cancer (Warburg, 1956; Weinhouse, 1976). The lack of experimental evidence however for impaired mitochondrial respiration led his hypothesis to controversies (Racker, 1983; Weinhouse, 1976). The Warburg effect has attracted the interest of several research groups and is currently studied as the potential eighth hallmark of cancer. Extensive research in this field, has led to the identification of several pathways through which tumour cells increase their glycolytic conduit. These include oncogenes, mutations in mtDNA, mutations in tumour suppressor genes and certain genes which encode the enzymes of the glycolysis and TCA cycle (Kim and Dang, 2006). Elstrom RL et al., show that oncogene Akt stimulates glucose intake in transformed cells without affecting the rate of oxidative phosphorylation (Elstrom et al., 2004). Kim et al., postulate that oncogene MYC transcriptionally activates all the genes encoding glycolytic enzyme and binds to various glycolytic enzymes including hexokinase-2 (HK2), enolase, and LDH-A hence enhances aerobic glycolysis (Kim and Dang, 2005). Powers JT et al show that elevated or sustained MYC activity stimulates the generation of reactive oxygen species (ROS) which in turn mutates mitochondrial DNA which leads to the impairment of mitochondrial respiration (Powers et al., 2004). Matoba et al suggest that Tp53, a tumour suppressor gene which is frequently mutated in human cancer cells transcriptionally activates SCO2. SCO2 is essential for the assembly of COX which facilitates mitochondrial respiration. Loss of p53 activity or disruption of SCO2 gene in turn disturbs mitochondrial respiration (Matoba et al., 2006). The above mentioned findings are schematically explained in figure 2.

Interestingly two recent reports relate HIF-1a to the inhibition of mitochondrial functions. Papandreou et al report that pyruvate dehydrogenase kinase 1 (PDK1), a HIF-1a target gene inhibits pyruvate dehydrogenase (PDH) from using pyruvate to fuel mitochondrial TCA cycle which in turn reduces oxidative phosphorylation (Mason et al., 2007). Kim et al suggest that HIF-1a directly stimulates PDK-1 and inhibits PDH activity thereby reduces oxidative phosphorylation (Kim et al., 2006). Figure 2 (modified from Kim and Dang, 2006) shows the connection between all the above mentioned findings. More recent reports link two TCA cycle enzymes succinate dehydrogenase (SDH) and fumarate hydratase (FH) to mitochondrial dysfunction and Warburg effect (King et al.)

1.2 Adaptive and oncogenic activation of metabolism

Several studies report that many human tumours are severely hypoxic because of inefficient and insufficient vascular system (Helmlinger et al., 1997). The adaptive response to hypoxia results in the stabilization of HIF-1 and/or HIF-2 which in turn transcriptionally activate genes encoding glucose transporters, glycolytic enzymes and angiogenic factors (Semenza, 2000). At the same time, hypoxia is not the only signal that stabilizes HIF-1. Recent studies provide evidence that HIF-1 can be stabilized by factors other than hypoxia, such as inactivation of pVHL, stimulation of insulin-like growth factor and activation of v-Src or c-Src and ras (Janssen et al., 2002). Furthermore, Kim et al indicate that Akt has HIF-1 dependent and HIF-1 independent effects on glycolysis. This shows oncogenic activation of several pathways could stabilize HIF-1 under normoxic conditions (Kim et al., 2005) and these findings support adaptive as well as oncogenic activation of glycolysis (Elstrom et al., 2004; Kim et al., 2005).

1.3 Possible advantages of increased glycolysis in cancer cells

It is important to know why tumour cells prefer less energy efficient glycolysis (produces only 2 ATPs) instead of high energy yielding oxidative phosphorylation (produces 36 ATPs) when there is a need for enormous ATP production to facilitate rapid tumour growth. The reports following suggest that tumour cells benefit from high glycolytic shift in several ways. Pedersen et al explain that glycolysis is a rich source of precursors which are essential for the biosynthesis of nucleic acids, phospholipids, fatty acids, porphyrins and cholesterol. This suggests high glycolytic rates in cancer cells facilitate not only survival but also a high growth rate. Furthermore lactic acid, the end product of aerobic glycolysis with its low pH, create acidic environment which helps the tumour cells to protect themselves from body's immune system. Cancer cells can survive with glycolysis even when there is no oxygen, because the enzymes involved in glycolysis are oxygen independent and can be stimulated by hypoxia (Pedersen, 2007). The increased glycolytic rate is deduced to surpass reactive oxygen species (ROS) produced by oxidative phosphorylation which has been implicated in cellular senescence and apoptosis. The immortalization potential of glycolytic enzymes explains the possibility that this phenotype (aerobic glycolysis) could be a step toward tumorigenesis through cellular immortalization (Kim and Dang, 2005).

1.4 Targeting glycolysis for cancer therapy

Since elevated glycolysis is very common in most of the human cancer cells, altering glycolytic pathway at various steps has become one of the major targets for cancer treatment and prevention (Semenza, 2003). In advanced metastatic cancers, understanding the molecular and physiological causes and consequences of upregulated glycolysis may lead to targeted therapies (Gatenby and Gillies, 2007). As discussed at the beginning, the increase in glycolytic rate is connected with significant malfunction of mitochondrial respiration due to various factors and that lead the tumour cells depend more on glycolysis. On the other hand, normal cells with competent mitochondrial respiration may produce energy through alternative energy sources such as fatty acids and amino acids when glycolysis is inhibited. This has led to the development of a therapeutic concept to inhibit glycolysis as a strategy to preferentially kill cancer cells (Pelicano et al., 2006).

1.5 Known treatment strategies

Several reports confirm that inhibition of glycolysis effectively kills cancer cells with defective mitochondria (Xu et al., 2005). One of the effective ways of inhibiting glycolysis is to inhibit the glycolytic enzymes. Among the glycolytic enzymes hexokinase (HXK), phospho fructo kinase (PFK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH) can be targeted by known chemical inhibitors (Chen et al., 2007). Refer Figure 3 for the glycolytic pathway and the sites of action of these enzymes.

1.5.1 Hexokinase

HXK plays a role in the first rate-limiting step of glycolysis i.e. the formation of glucose-6-phosphate from glucose. In human cells there are four isoforms of hexokinase (I-IV) identified (Sebastian et al., 1999). Pedersen et al report that HIF-1a and mutant p53 may stimulate HXK II expression (Pedersen et al., 2002) and most immortalized and malignant cells display increased expression of HXK II, which might contribute to elevated glycolysis. Arora et al reported that the percentage of HXK binding to mitochondria is also significantly increased in certain cancer cells (Arora and Pedersen, 1988). Apart from enzymatic activity, mitochondria associated HXK II seems to regulate apoptosis (Robey et al., 2005). Additionally, inhibition of HXK will affect both glycolysis and it's off shoot pentose phosphate pathway (PPP) because glucose-6-phosphate is a common intermediate for both pathways. These findings confirm that inhibition of this enzyme is likely to have profound effects on cellular energy metabolism and survival. Thus, hexokinase is an attractive target for anticancer agents. The available inhibitors of HXK include 2-deoxyglucose, 3-bromo pyruvate, 5-thioglucose and mannoheptulose (Chen et al., 2007).

1.5.2 Phosphofructokinase

PFK facilitates phosphorylation of fructose-6-phosphate and converts it to fructose-1, 6-bisphosphate. In this energy consuming step ATP acts as negative regulator and fructose-1, 6-bisphosphate act as positive regulator. Phosphofructokinase/fructose-2,6-bisphosphatase 3 (PFK/FBPase 3) isozyme is overexpressed in leukaemia, solid tumours and ras-transformed cells and is activated by hypoxia. Targeting this regulatory mechanism by suppressing of PFK/FBPase 3 isozyme has been proposed as strategy to preferentially kill Ras-transformed cells (Chesney, 2006). Recently Bensaad et al reported that a new p53 target gene TIGAR inhibits the formation of fructose-1, 6-bisphosphate and inhibits glycolysis (Bensaad et al., 2006). TIGAR as one of the genes that will be studied further in this thesis will be discussed in the following chapters more extensively.

1.5.3 Glyceraldehyde-3-phosphate dehydrogenase

GAPDH converts glyceraldehyde-3-phosphate to 1, 3-bisphosphoglycerate. GAPDH is unique among the glycolytic enzymes because of its ability to bind NAD+ or NADH, and also to DNA and RNA. Apart from enzymatic activity, GAPDH also affects multiple cellular functions such as endocytosis, membrane fusion, vesicular secretory, nuclear tRNA transport, and DNA replication and repair (Michael, 2005). Known inhibitors of GAPDH include a-chlorohydrin, ornidazole and iodoacetate.

1.5.4 Lactate dehydrogenase

LDH is a tetramer of A and B subunits, encoded by two separate genes. It catalyzes the conversion of pyruvate to lactate coupled with an oxidation of NADH to NAD+, which is essential for the glycolytic pathway. Since NAD+ is required by GAPDH, regeneration of NAD+ by LDH catalyzed reaction is very important to maintain glycolysis, especially when mitochondrial respiration is compromised. LDH-A gene is controlled by HIF-1a. Interestingly, Fantin et al report that siRNA knockdown of LDH-A in tumour cells stimulate mitochondrial respiration, decreases cell proliferation in hypoxia and suppress tumorigenicity (Fantin et al., 2006). Oxamate is a known metabolic inhibitor of LDH.

1.6 Hypoxia inducible factor (HIF-1a)

The important role of HIF-1a and the p53 transcription factors in the regulation of the expression of numerous enzymes involved in energy production has been reported in several recent studies (Mason et al., 2007; Matoba et al., 2006).

Cellular hypoxia is largely controlled by HIF-1. HIF-1 belongs to the basic helix-loop-helix (bHLH) superfamily of eukaryotic transcription factors that contain PAS domain, an additional active dimerization site. The name indicates the first three proteins this motif was found PER-ARNT-SIM (Frede et al., 2007). The basic domain and carboxy-terminus of PAS are important for DNA binding of HIF-1 and HLH and the amino-terminus of PAS is required for dimerization. HIF is a heterodimer composed of one of the three a subunits and a constitutively expressed ß subunit. The stability of HIF-1a is dependent on ODD domain where as its transcriptional activity is dependent on N or C - terminal transcription activation domains. These TADs are important for the interactions with transcriptional coactivators like CBP/p300. The TADs are important for post translational modifications such as phosphorylation and acetylation (Frede et al., 2007). Figure 4 schematically shows the domain structure of HIF-1a and HIF-1ß.

In hypoxia, HIF-1a regulates the transcription of several genes which are involved in erythropoiesis, angiogenesis, iron homeostasis and energy metabolism, which help in cellular adaptation of hypoxia. HIF-1a is over expressed in many different types of tumours (Goonewardene et al., 2002). Any hypoxia targeted therapy such as limiting the accumulation of HIF-1a, blocking the binding sites of HIF-1a (HREs), reducing the tissue hypoxia, using hypoxia-activated prodrugs might shut off tumour cells from their supply of oxygen and nutrients.

1.7 p53 regulation and energy metabolism

p53, a tumour suppressor protein is mutated in most of the tumours. Like HIF, p53 is also accumulated in hypoxia. P53 controls cellular homeostasis by affecting cell cycle progression and apoptosis. These essential tumour suppressor functions are lost when it is mutated. In cells under normoxic conditions the amount of p53 is very low and it has very short half life. But under stress like hypoxia, DNA damage or oncogene activation p53 is stabilized by post translational modifications and acts as a transcription factor.

There are several genes activated by p53, in which some are involved in the cell cycle arrest such as p21WAF1/CIP1, and some are involved in apoptosis such as bax and some others are involved in energy metabolism such as TIGAR and SCO2. The degradation of p53 occurs in an autoregulatory feedback loop mechanism. The oncoprotein Mdm2 binds to the transcriptional activation domain of p53 and blocks its ability to regulate target genes and p53 activates the expression of Mdm2 (Steven M. Picksley, 1993). The interval between p53 activation and Mdm2 accumulation is the time where p53 exhibits its effects. Mdm2 promoted degradation of p53 opens new ways to terminate p53 signals (Haupt et al., 1997).

As shown in Figure 5. (Modified from Bensaad et al., 2006) p53 targets different genes under different stress conditions. In mild and reversible conditions it targets genes involved in cell cycle arrest, DNA repair and anti oxidant genes to facilitate cell survival. Under extended and irreversible stress, the targets are different such as pro apoptotic genes to facilitate senescence or apoptosis (Bensaad et al., 2006). p53 can regulate the balance between glycolysis and oxidative phosphorylation. The high glycolytic rate in cancer cells could be because of many reasons such as, mutations in p53, loss of enzymatic activity of several enzymes involved in oxidative phosphorylation and activation of several oncogenes such as Myc, Ras, Src and Akt (Bensaad et al., 2006; Pelicano et al.).

1.8 p53 target genes involved in glycolysis and OXPHOS

p53 regulates genes involved in glycolysis directly and indirectly. p53 directly upregulates type II hexo kinase, and downregulates phospho glycerate mutase (Corcoran et al., 2006). Bensaad et al identified a p53 target gene called TIGAR (Bensaad et al., 2006) and showed that it alters the pathway in which cell uses glucose. TIGAR belongs to phospho glycerate mutase (PGM) family and its expression lowers the fructose-1, 6-bisphosphate levels in cells, which leads to inhibition of glycolysis and overall reduction in intracellular ROS levels.

TIGAR shows functional sequence similarities with the bisphosphatase domain of the bifunctional enzyme 6-phosphofructokinase-2/fructose-2, 6-bisphosphatase (PFK-2/FBPase-2) which degrades fructose-2, 6-bisphosphate. Fructose-2, 6-bisphosphate stimulates 6-phospho-1-kinase to convert fructose-6-phosphate to fructose-1,6-bisphosphate. When the formation of fructose-2,6-bisphosphate decreases the accumulation of fructose-6-phosphate is favoured. Similarly TIGAR decreases the fructose-2,6-bisphosphate level and blocks glycolysis at the third step (Green and Chipuk, 2006). Figure 6 schematically explains this. Furthermore Fructose-2,6-bisphosphate has also been found to both enhance expression of glucokinase (GK), the hepatic isoform of HXK, and concomitantly inhibits the expression of glucose-6-phosphatase (G6Pase), which reverses the phosphorylation of glucose performed by GK (Discher et al., 1998; Ruiz-Lozano et al., 1999). Several reports suggest that p53 controls intracellular ROS levels through its target genes (Polyak et al., 1997).

p53 can increase ROS levels to enhance ROS induced apoptosis (Yoon et al., 2004) or decrease intracellular ROS levels to allow the cells to undergo repair mechanism in case of mild stress conditions (Sablina et al., 2005). Bensaad et al suggest that TIGAR facilitates the accumulation of fructose-6-phosphate which stops glycolysis and enters pentose phosphate pathway. The increased flow through PPP lowers apoptosis due to an increased generation of reduced glutathione and removal of ROS in cells (Bensaad et al., 2006). Interestingly ROS can positively regulate p53 transcriptional activity thus creating a positive feedback loop between p53 and ROS generated by p53 target genes (Jennifer L. Martindale, 2002).

Matoba et al identified a p53 target gene SCO2 which is required for the assembly of COX that is directly responsible for the reduction of oxygen during aerobic respiration (Matoba et al., 2006). Disruption of the SCO2 gene in human cancer cells with wild-type p53 shows similarity to the metabolic switch toward glycolysis that is exhibited by p53-deficient cells (Matoba et al., 2006). Figure 7 explains the influence of p53 on glycolysis and oxidative phosphorylation in relationship with TIGAR and SCO2. Interestingly the products of glycolysis and oxidative phosphorylation regulate p53. Pyruvate, a major metabolic product of glycolysis induces p53 (Xu and Finkel, 2002). Furthermore ATP and NAD+ have been shown to induce the dissociation of p53 from p53-DNA complex that act as negative feedback loop for p53 activity (McLure et al., 2004; Okorokov and Milner, 1999).

1.9 TIGAR and SCO2 regulation by p53 and HIF-1a

The studies by Bensaad and Matoba show that p53 regulates TIGAR and SCO2 (Bensaad et al., 2006; Matoba et al., 2006). Gene TIGAR is located on chromosome 12p13-3 and contains six potential coding exons and two possible p53 binding sites, one upstream of the first exon (BS1) and one within the first intron (BS2) (Bensaad et al., 2006). The unpublished observation in our laboratory shows that TIGAR contains six HREs (Rajendran R, 2007; Xenaki et al., 2008). Interestingly SCO2 also contains the binding sites for p53 and HIF. Identification of binding sites for both p53 and HIF-1a within TIGAR and SCO2 raises the question whether these are differentially regulated by p53 and HIF-1a in hypoxic conditions and whether post translational modifications of the two transcription factors play any role in the modulation of the expression of these two genes.

1.10 Post translational modifications of p53 and HIF-1a

HIF and p53 are reported to interact directly or in complex with the coactivator proteins p300 and PCAF (Blagosklonny et al., 1998; Liu and Simon, 2004; Xenaki et al., 2008). Loss of p53 activity increases HIF-1a and increases its transcriptional activation of several genes (Semenza et al., 2001). Reports contradict in defining the ability of HIF-1a to stabilize p53 levels (Blagosklonny et al., 1998; Wenger et al., 1998). Recent reports suggest that hypoxia alone cannot stabilize or activate transcriptional activity of p53 but also requires other signals like DNA damage or extended stress (Kaluzova et al., 2004; Pan et al., 2004). Studies reveal that only under severe hypoxic conditions, considerable amount of p53 is accumulated and destabilizes HIF-1a. Hansson et al report that in hypoxia, where prolyl residues of HIF-1a are not hydroxylated which is required for the degradation through ubiquitination, p53 degrades HIF-1a through different route (Semenza et al., 2001). p53 in the p53-HIF-1a complex may recruit Mdm2 which in turn ubiquitinates and degrades HIF-1a (Hansson et al., 2002). The post translational modifications of both these genes play major roles in their stabilization, accumulation and activation (Arnesen et al., 2005; Bilton et al., 2005; Liu and Simon, 2004). HIF undergoes post translational modifications of Hydroxylation, phosphorylation and acetylation of various amino acids at different sites (Cho et al., 2007). P53 undergoes post translational modifications of phosphorylation, acetylation, and sumoylation (Bode and Dong, 2004). Such post translational modifications of transcriptional factors HIF-1a or p53 may lead to differential activation of genes involved in energy metabolism such as TIGAR and SCO2.

Functional crosstalk between the HIF-1a and p53 pathways occurs at several levels, including p53 protein stability (Semenza et al., 2001), attenuated HIF-1 transcriptional activity by p53 (Blagosklonny et al., 1998) and vice versa (Chen et al., 2003). Interference of each one of these transcription factors with the transcriptional activity of the other has been attributed to the availability of the transcriptional co-activator p300 (Arany et al., 1996; Frede et al., 2007) since both p53 and HIF-1a share a common binding site on these cofactors (Freedman et al., 2002). Post translational modifications of both HIF-1a and p53 play major roles in their stabilization, accumulation activation as well as selective induction of their target genes (Bilton et al., 2005; Liu and Simon, 2004). For example, differential acetylation of p53 by p300 and other Histone Acetyl Transferases (HATs) mediates differential responses to diverse signals and adjustment to the cellular requirements depending on the environmental conditions (Liu and Simon, 2004). Acetylation of HIF-1a mediated by Arrest-Defective-1 (ARD1), has also been reported (Jeong et al., 2002), although contradictory results have been presented with regard to the effect of acetylation on HIF-1a transcriptional activity and protein stability (Bilton et al., 2005). Other post translational modifications regulating HIF-1a and p53 function by the same enzymatic cascades include phosphorylation, nitrosylation and SUMOylation (Brahimi-Horn et al., 2005).

Recent studies in our lab (Xenaki et al., 2008; Xenaki G, 2007) have provided evidence for an additional molecular mechanism linking the function of p53 and HIF-1a by the cofactor PCAF, which was shown to regulate the fine-tuning of the transcriptional activity of both p53 and HIF-1a in hypoxic conditions as well as the protein stability of both transcription factors (Jin et al., 2002; Linares et al., 2007). In addition, the HAT activity of PCAF regulates the HIF-1a transcriptional selectivity targeting the transcription factor to a selective subset of its target genes (Xenaki et al., 2008) (Figure 8). In particular, PCAF was found to regulate the expression of the known HIF-1a transcription targets Carbonic Anhydrase IX (CA-IX) (Wykoff et al., 2000), VEGF (Forsythe et al., 1996) PGK-1a and LDH-A (Semenza et al., 1996). Luciferase reporter assays shown in Figure 8 point out that PCAF functions as HIF-1a co-activator in the case of the CA-IX-Luc and VEGF-Luc reporters (Figure 8A and 8B), but did not have any effect in the expression of PGK-Luc and LDH-A-Luc reporters (Figure 8C and 8D).

In addition, PCAF HAT played an important role in the co-activation of CA-IX-Luc reporter (Figure 8A) but did not appear to have significant effect on the VEGF-Luc and PGK-Luc reporters (Figure 8B and 8C) and exerted a negative effect on the expression of LDH-A-Luc reporter (Figure 8D). The reason for the presence of binding sites for both HIF-1a and p53 transcription factors in the regulatory regions of the promoters of TIGAR and SCO2, two genes involved in two alternative energy production pathways, might be necessary for the cell to be able to selectively express one of the two genes depending on its energy needs. This selective expression of either TIGAR or SCO2 might be determined by post-translational modifications regulating the transcriptional activity of HIF-1a and p53, hypothesis which will be investigated as part of this thesis.

1.11 Aims

This project aims to study the gene regulation of TIGAR and SCO2 in hypoxia and investigate whether TIGAR and SCO2 are targets of HIF-1a. Part of this study will explore whether PCAF mediated acetylation of p53 and HIF-1a selectively targets anyone or both of these transcription factors to either TIGAR or SCO2 promoter. This project also aims to investigate the role of p53 and HIF-1a in the acquisition of specific metabolic phenotypes by tumour cells by differentially regulating TIGAR and/or SCO2 in response to diverse stress signals.

2. Materials and Methods

2.1 Cell lines, Culture conditions and Transfection

Human osteosarcoma U2OS (p53+/+) and SAOS2 (p53-/-) cell lines were cultured in Dulbecco's Modified Essential Medium (DMEM, Lonza) supplemented with 10% Fetal Bovine Serum (FBS, Lonza) and 1% 10U/ml penicillin and streptomycin and maintained at 37°C and 5% CO2. Media was changed once in two days and four hours before splitting. Cells were collected 16 h after 250 mM Desferrioxamine (DSFX) treatment (Sigma) and 16 h after incubation with 10 mM Etoposide (Sigma) unless otherwise indicated.

The pCiFlag-PCAF (wt) and pCiFlag-PCAF (?HAT) from Dr. Talianidis (University of Crete) and pcDNA3 (Invitrogen) were used. The cells were transfected the day after slitting using Calcium Phosphate method (Demonacos et al 2001) or Polyfect® Transfection reagent (Qiagen).

2.2 Western Blotting

For Immunoblotting (IB), when 80% confluence is achieved, U2OS and SAOS2 cells were splitted into 100cm plates. The cells were transfected after 24 hours and the media was changed after 16 hours. DSFX or Etoposide was treated for 16 hours and then washed twice with ice-cold Phosphate Buffered Saline (137mM NaCl; 10mM Phosphate; 2.7mM Kcl; pH 7.4) and harvested using 120mM TNN buffer (50mM Tris-Hcl, pH 7.4; 120mM NaCl; 5mM EDTA; 5% Nonidet P-40) added with 1mM DTT, 1mM PMSF, 1µg/ml Protease Inhibitor cocktail (PI Consisting of Pepstatin, Aprotinin and Leupeptin), 20mM ß-glycerol phosphate, 5mM Sodium pyrophosphate and 2mM Sodium orthovandate, placed on a rotator for 20 minutes at 4°C and centrifuged at 13,000 rpm and 4°C for 20 minutes. Protein extracts were normalized using Bradford assay (Bio-Rad) and equal amount of protein is added with SDS loading buffer. The proteins were incubated at 95°C for 3 minutes and then resolved by SDS-PAGE using s PageRulerTM protein ladder (Fermentas) as a molecular weight marker. Proteins were transferred to ImmobilonTM P Membrane (Millipore) by western transfer. Membranes were blocked with 5% milk for 1 hour and then incubated overnight at 4°C in 2.5% milk/PBS/0.1% Tween (Sigma) with diluted primary antibody (See section 2.6). Membranes were washed 3 times with PBS/0.1% Tween and then incubated for 90 minutes in 2.5% milk/PBS/0.1% Tween containing diluted (1:3000) relevant anti-rabbit or anti-mouse IgG horseradish peroxidise conjugated antibody (Amersham Biosciences). Proteins then were visualized using ECL (Pierce) according to the manufacturer's instructions.

2.3 Quantitative Real Time PCR

After reaching 80% confluence, U2OS and SAOS2 cells were splitted into 30mm plates. After transfecting with the desired plasmid (either pcDNA3 or PCAF or PCAF?HAT) the cells were treated with DSFX or Etoposide for 16 hours. The RNA was extracted using the RNeasy Plus Kit (Qiagen). RNA concentrations were measured and 1mg/ml of RNA was reverse-transcribed using BioscriptTM Reverse Transcriptase (Bioline). The cDNA was then diluted 6-fold, and then used for quantitative PCR using SYBR Green Tag Man Kit (Sigma) on qRT-PCR plate (Bio-Rad). For the primer sequence please refer section 2.6. The results were analyzed using Opticon monitor Software 2.1.

2.4 Molecular Cloning

Primers were used to amplify a particular region of TIGAR or SCO2 gene which contains the binding sites of p53 and HREs. This amplified fragment was then inserted into the pCR® -Blunt II-TOPO vector (Invitrogen) using the manufacturer's instructions. Restriction enzyme Kpn1 (Roche) was used to digest the vector and the insert was recovered.

2.5 Flow Cytometry

Cells were transfected with the indicated expression vectors, treated with 250µMDSFX or 20µMEtoposide and harvested 48 hours after transfection and fixed in 50% ethanol. Propidiumiodide and RNase A were added to final concentrations of 50 and100 mg/ml, respectively and transfected cells were stained with a CD-20-FITC antibody for CD20 expression. Cell-cycle profile was determined by FACS can flow cytometry and analysed by CellQuest software (Becton Dickinson) as previously described (Demonacos et al., 2001). Cell count represents the cells exhibiting the CD20 antigen used as a transfection efficiency control. The average Transfection efficiency was 30% for U2OS and 12% for SAOS2 cells. Only the CD20-transfected population of cells (sorted by CD20- FITC-conjugated antibody) has been taken into account for the FACS analysis. The average of two independent FACS, experiments is shown in Figures 12a and 12b.

2.6 Antibodies and Primers

3. Results

3.1 Identification of HREs in the promoters of TIGAR and SCO2

The studies by Matoba et al have shown that SCO2 has p53 binding sites and is directly regulated by p53(Matoba et al., 2006). Recently Bensaad et al have provided evidence for the presence of p53 binding sites in the promoter region of TIGAR(Bensaad et al., 2006). Since these two genes are involved in the energy production of cancer cells, we asked whether hypoxia induced HIF-1a has any effect on these two genes. To find out the binding sites of HIF-1a in the promoters of TIGAR and SCO2, we carried out bio-informatics searches in the gene bank. Interestingly, both these genes have HREs in their promoter region. There are 6 HREs identified in the promoter of TIGAR and the following table shows the positions of HREs in the promoter region of TIGAR.

In SCO2 promoter region we have identified 2 HREs. The positions of HREs are shown in the following table.

To investigate, whether these HREs present in the promoters of TIGAR and SCO2 genes are functionally active, we have decided to carry out Luciferase assays. The figures 9a and 9b schematically explain the construction of the Luciferase vector with the HREs present in TIGAR and SCO2 promoters.

3.2. PCAF is a cofactor that regulates the expression of TIGAR protein

Cofactor PCAF has the ability to acetylate the lysine residues present in the promoters of p53 and HIF-1a. Since TIGAR is a known p53 target (Bensaad et al., 2006), expression of TIGAR protein in p53 knocked out cells would explain the involvement of any other transcription factors in the regulation of TIGAR. To investigate the same, we transfected pcDNA3, Flag-PCAF and Flag-PCAF?HAT into U2OS cells which express endogenous p53 (p53+/+ cells) and SAOS2 cells which do not express p53 (p53-/- cells). To explore the behaviour of TIGAR in hypoxia mimicking (HIF-1a stabilized) conditions we used 250µM DSFX. After 16 hours of treatment with DSFX, the protein levels of HIF-1a, Flag, Actin and TIGAR were measured using specific antibodies.

In both cell lines, HIF-1a expression was observed in hypoxia mimicking conditions. This confirmed that HIF-1a is stabilized in hypoxia mimicking condition. However, the expression of HIF-1a was higher in the cells which are over expressing PCAF. This showed that PCAF upregulated the expression of HIF-1a.

Expression of Flag shows that equal levels of PCAF and PCAF?HAT were transfected in both the cell lines. For the transfection efficiencies of these two cell lines please refer section 3.4.

TIGAR expression in U2OS (p53+/+) cells was observed in all the conditions. However, the expression of TIGAR was more in hypoxia mimicking conditions. Since both p53 and HIF-1a are stabilized in hypoxia mimicking conditions the expression of TIGAR can be related to any of these two transcription factors. In cells transfected with PCAF the expression of TIGAR was more compared to the cells transfected with PCAF or PCAF?HAT. This shows that the hyper acetylated form of any of these to transcriptional factors upregulate the expression of TIGAR.

In Figure 10 the expression of TIGAR in p53-/- cells where there is no p53 expressed indicated that TIGAR was regulated by some other factors apart from p53. Since the expressions were seen only in hypoxia mimicking conditions, we can say that the other transcription factor regulates TIGAR in hypoxia dependant manner. However, the enhanced expressions of TIGAR in PCAF transfected cells indicated that the hyper acetylated form of that transfection factor upregulates TIGAR in hypoxia dependant manner. This could be probably because of HIF-1a or p57.

3.3. The HAT activity of PCAF is involved in the expression of TIGAR Protein

To find out the effects of HAT activity of PCAF in the mRNA expression of TIGAR, in HIF-1a and p53 stabilized conditions, total RNA was extracted from cultures of U2OS (p53 +/+) and Saos2 (p53 -/-) cells transfected with either PCDNA3, PCAFwt or PCAF?HAT. Each of these were exposed to DSFX, etoposide (a topoisomerase II inhibitor causing DNA double stand breaks and activation of p53), or left untreated. Reverse transcription of mRNA then gave cDNA which was analyzed by quantitative real time polymerize chain reaction (qRT-PCR). Two samples were analyzed in duplicates. Average and standard deviation for each group were obtained and the amounts were neutralized compared to untreated cells so that data shows relative increase in mRNA levels by DSFX or etoposide treatment.

In figure 11 p53+/+ cells subjected to both DSFX treatment and treatment with etoposide showed significant increase in TIGAR mRNA expression. All p53 +/+ cell samples treated with etoposide causing activation of p53 resulted in increased TIGAR mRNA expression irrespective of acetylation. However cells overexpressing PCAF?HAT showed a two-fold increase in TIGAR mRNA expression compared to cells containing only endogenous PCAF (and the empty vector, PCDNA3). Compared to cells overexpressing the wild type PCAF, cells transfected with PCAF?HAT showed three-fold increase in mRNA expression suggesting p53+/+ cells hindered from acetylation by PCAF PCAF?HAT favoured the induction of TIGAR expression. Induction of TIGAR mRNA expression was greater on treatment with etoposide, causing activation of p53, than DSFX which can potentially stabilize both HIF-1a and p53 in p53 +/+ cells. Under these conditions p53 +/+ cells overexpressing PCAF?HAT showed slightly more TIGAR mRNA expression than cells with the capability to acetylate both endogenously and via overexpression of PCAF-wt which showed similar levels of TIGAR transcript.

Similarly in p53-/- cell lines, TIGAR mRNA expression was increased following treatment with both etoposide and DSFX. Treatment with DSFX, stabilizing HIF-1a, produced an increase in TIGAR mRNA expression independent of p53 expression. This increase in expression was particularly pronounced in cells with an overexpression of PCAF?HAT suggesting greater induction of TIGAR in hypoxia in the absence of acetylation by PCAF. p53 -/- cells were influenced more by absence of acetylation in DSFX compared to p53 +/+ cells showing a 2-fold increase in TIGAR mRNA expression in cells transfected with PCAF?HAT whilst p53 -/- cells only showed a moderate change. The double stand DNA damage caused by etoposide treatment caused considerable increase in TIGAR mRNA even in the absence of p53. This increase in expression observed, once again, was greater than that induced by conditions mimicking hypoxia. Post-treatment with etoposide, p53 -/- cells overexpressing wild type PCAF showed a repression of TIGAR expression of TIGAR mRNA compared to the mutant form and cells with endogenous acetylation only, which both displayed similar levels of mRNA expression.

Quantitative analysis of SCO2 mRNA expression in p53 +/+ cells showed the largest increase in the presence of etoposide particularly in cells overexpressing the PCAF mutant, PCAF?HAT. Cells transfected with PCDNA3 showed an increase in mRNA significantly greater than cells overexpressing PCAFwt which only showed only a negligible increase. Hence cells expressing p53 which were hyperacetylated by PCAF showed a massive decrease in ability to induce expression of SCO2 post-treatment with etoposide. A small but significant increase in SCO2 mRNA expression resulted following DSFX treatment in p53+/+ cells suggesting hypoxic conditions could cause a mild induction of SCO2. The largest amount of SCO2 transcript obtained from p53 +/+ cells was in those with endogenous acetylation suggesting that, in hypoxic conditions, expression of SCO2 is neither favoured by hypoacetylation by PCAF nor hyperacetylation by PCAF. As the standard deviation for the results obtained for SCO2 expression in p53-/- cells was too large the results from mRNA expression analyses were inconclusive. This could have occurred for a number of reasons but is most likely to be due to impurities introduced to the sample at some point in the process.

3.4. The apoptotic program executed by PCAF in p53+/+ and p53-/- cells under hypoxia require its HAT activity

The role of the HAT activity of PCAF on cell cycle progression and apoptosis was investigated by FACS analysis performed in DSFX treated or untreated p53+/+ and p53-/- cells transfected with either Flag-PCAFwt or Flag-PCAF-?HAT expression vectors. In hypoxia mimicking conditions, PCAF reduced the apoptosis in p53+/+ cells and increased apoptosis in p53-/- cells (Compare PCAF and PCAF?HAT bars in 12a and 12b). To investigate the role of acetylated HIF-1a in apoptosis, we compared the apoptosis by PCAF in p53+/+ and p53-/-. In the absence of p53, acetylated HIF-1a increased the apoptosis (Compare blue bars in Fig 12b) and in the presence of p53, PCAF reduced the apoptosis (Compare Blue bars in Fig 12a). To find out the role of HIF-1a, we transfected both the cell lines with RNAi-HIF1a. PCAF transfected p53+/+ cells have shown considerable reduction compared to PCAF?HAT transfected cells (Compare black bars in Fig 12a). Interestingly PCAF transfected p53-/- cells have shown more than twofold increase compared to PCAF?HAT transfected cells. (Compare black bars in Fig 12b). Figure 12 show that PCAF HAT activity is required for the apoptotic program executed by p53+/+ and p53-/- cells under hypoxia mimicking conditions.

4. Discussion

The studies by Matoba et al (Matoba et al., 2006) and Bensaad et al (Bensaad et al., 2006) showed that SCO2 and TIGAR are p53 target genes. Recent study in our lab (Xenaki et al., 2008) suggested that acetylated p53 regulates the transcriptional activity of HIF-1a under hypoxia mimicking conditions. Since TIGAR and SCO2 are involved in tumour metabolism and HIF-1a is an important oncogene that is regulated by hypoxia, it is important to know whether SCO2 and TIGAR are regulated by HIF-1a. The presence of HREs in the promoters of SCO2 and TIGAR regulatory regions shows that there are possibilities for these two genes to be regulated by HIF-1a. To investigate the HREs present in these genes are functionally active, we have amplified the regulatory region that contain HREs and cloned them into pGL3 promoter vector. This work will further continued with firefly luciferase assay.

The studies by Xenaki et al showed that PCAF is a cofactor for both p53 and HIF-1a and the acetylated p53 regulates the transcriptional ability of HIF-1a (Xenaki et al., 2008). To investigate whether TIGAR and SCO2 proteins are regulated by PCAF mediated acetylation of p53 and HIF-1a, the protein expression of TIGAR was measured in p53+/+ and p53-/- cells transfected with either pcDNA3 or PCAF or PCAF?HAT. Under hypoxia mimicking conditions, HIF-1a was expressed in both the cell lines. The cells expressing ectopic PCAF increased the expression of HIF-1a compared to pcDNA3 and PCAF?HAT transfected cells (Compare lanes 2, 4 and 6 in Figure 10a for p53+/+ cells and 8, 10 and 12 in Figure 10b). This upregulation of HI-1a by PCAF comply with the published results.

The expression of TIGAR was increased in a PCAF dependent manner under hypoxia mimicking conditions. In p53+/+ cells, TIGAR is expressed in all pcDNA3 and PCAF and PCAF?HAT cells. This shows that p53 regulates TIGAR and the increase in TIGAR protein expression in PCAF transfected cells shows that acetylated p53 at K320 targets TIGAR which is a pro-survival gene. This result is matching with the published reports, which say acetylation of p53 by PCAF targets a set of pro-survival genes such as p21 (Ma et al., 2007). The expression of TIGAR in p53-/- cells in hypoxia mimicking conditions, confirms that, apart from p53, there are other factors involved in the regulation of TIGAR. Under hypoxia, the increased expression of TIGAR in PCAF transfected cells (compared to pcDNA3 and PCAF?HAT transfected cells) shows that TIGAR is regulated in PCAF and hypoxia dependent manner.

In p53+/+ cells, the mRNA levels were elevated in etoposide treated cells compared to DSFX treated cells. Since etoposide treatment stabilizes p53, the elevated mRNA levels in etoposide treated cells confirmed that TIGAR is a p53 target gene. In p53+/+ cells, under DNA damage conditions, PCAF HAT reduced the mRNA expression. It is possible that hypoacetylated p53 at K320 is recruited to the promoters of pro-survival genes such as p21. TIGAR is also a pro-survival gene as it inhibits the p53 dependent ROS induced apoptosis. (Please refer section 1.8). In p53-/- cells, where there is no endogenous p53 expressed, under DNA damage conditions, mRNA of TIGAR is reduced to a lesser extent compared to the p53+/+ cells where there is endogenous p53 expression. However the reduction of mRNA levels in DSFX treated p53-/- cells could be related to any transcription factor which is regulated by hypoxia e.g. HIF-1a.

To know the effect of PCAF HAT in programmed cell death in p53+/+ and p53-/- cells, we performed FACS analysis (Figure 12a and 12b). Under hypoxia mimicking conditions PCAF HAT reduced apoptosis in p53+/+ cells and increased apoptosis in p53-/- cells. It is possible that acetylated p53 at K320 is recruited by its pro-survival target genes and reduced the p53 dependent programmed cell death. On the other hand, in p53-/- cells which do not express p53, under hypoxia mimicking conditions, the apoptosis is increased possibly by HIF-1a which is regulated by hypoxia. However, when the HIF-1a is silenced in p53+/+ cells the PCAF HAT reduces the apoptosis. This explains that there is a competition for PCAF between p53 and HIF-1a and the hypoacetylated p53 or hypoacetylated HIF-1a was recruited by any of its pro-survival genes and reduces the apoptosis. This relationship between the apoptosis executed in p53+/+, p53-/- cells and TIGAR, SCO2 needs to be clarified by future experiments.

5. Future Experiments:

Investigate the HREs present in the promoters of TIGAR and SCO2 are functional. Luciferase Firefly Assay and Chromatin Immunoprecipitation (ChIP) methods will be used to determine the functionality of HREs.

Verify the role PCAF HAT activity in the p53 and HIF-1a mediated expression of TIGAR and SCO2

Investigate the role of TIGAR and SCO2 in the p53 and HIF-1a mediated apoptosis/survival events

Investigate whether PCAF HAT can modulate these survival/apoptosis events executed by p52 and HIF-1a.

Investigate the interaction between HIF-1a and TIGAR and SCO2 at protein level (Immunoprecipitation assay)

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