The Role of PPARs in Cancer

1. INTRODUCTION

Peroxisomeproliferator-activated receptors (PPARs) are ligand-activated transcriptionfactors that belong to the nuclear hormone receptor superfamily [1]. PPARs bindto a direct repeat of two hexanucleotides, spaced by one or two nucleotides(the DR1 or DR2 motif) as heterodimers with the retinoid X receptor (RXR), andactivate several target genes [2–4]. Theseperoxisome proliferator responsive elements (PPREs) are found in various genesthat are involved in lipid metabolism and energy homeostasis,including lipidstorage or catabolism, and fatty acid transport, uptake, and intracellularbinding [5]. Three subtypes, PPARα, PPARδ (also known as PPARβ), and PPARγ, have been identified and thesesubtypes with a high degree of sequence conservation of each subtype acrossvarious species have been characterized. The DNA-binding domains of the three subtypes are 80% identical, while their ligand-binding domains exhibit a lowerdegree (approximately 65%) of identity (Figure 1). Consistent with thisrelatively high divergence among the subtype-specific ligand-binding domains,differential activation of PPARs by endogenous and exogenous compounds mayaccount for the specific biological activity of the three PPAR subtypes [6, 7].

PPARα is expressed in the liver, kidney, smallintestine, heart, and muscle, where it activates fatty acid catabolism and isinvolved in the control of lipoprotein assembly [8]. PPARα is activated by several molecules, suchas long chain unsaturated fatty acids, eicosanoids, and hypolipidemic drugs(e.g., fenofibrate) [9–12]. PPARα activators have been used to treatdyslipidemia, causing a reduction in plasma triglyceride and elevation of high-densitylipoprotein (HDL) cholesterol [13, 14]. PPARδ is expressed ubiquitously and isimplicated in fatty acid oxidation, keratinocyte differentiation, wound healing,and the response of macrophages for very low-density lipoprotein [15–19]. PPARδ activators have been proposed for thetreatment of metabolic disease and are under clinical trial [20, 21]. There aretwo PPARγ isoforms: PPARγ1 and γ2 [22, 23]. PPARγ2, which is generated by alternativesplicing, contains an additional 28 amino acids at the N-terminal compared toPPARγ1. PPARγ3 is a splicing variant of PPARγ1 and gives rise to the same protein[24]. PPARγ2 is expressed exclusively in adiposetissue and plays a pivotal role in adipocyte differentiation, lipid storage inthe white adipose tissue, and energy dissipation in the brown adipose tissue [22, 25]. On the other hand, PPARγ1 is expressed in the colon, the immunesystem (e.g., monocytes and macrophages), and others. Except for the functionof PPARγ2 in adipose tissue, PPARγ also participates in inflammation, cellcycle regulation, and other functions [26]. PPARγ is involved in glucose metabolism throughthe improvement of insulin sensitivity and represents a potential therapeutictarget of type 2 diabetes [26]. Indeed, insulin-sensitizing thiazolidinedione(TZD) drugs are PPARγ ligands [27]. Thus PPARs are moleculartargets for the development of drugs to treat type 2 diabetes and metabolicsyndrome. On the other hand, PPARs also play a role in the regulation of cancercell growth.

2. PPARα AND CANCER

Fibrates,which are relatively weak PPARα ligands, are useful for the treatmentof dyslipidemia [7, 9–11]. Fibrateslower serum triglyceride levels and increase HDL levels through the activationof PPARα [5]. PPARα induces lipoprotein lipase (LPL)expression, reduces the expression levels of apolipoprotein C-III (ApoC-III), anatural LPL inhibitor, and stimulates the uptake of cellular fatty acids andthe conversion of fatty acids to acyl-CoA derivatives [5, 28, 29]. Thesecatabolism functions are mediated by upregulating the expression of a series ofgenes-related carbohydrate and lipid metabolism [5, 30]. In addition, PPARα increases the expressions of ApoA-I andApoA-II, resulting in raising HDL cholesterol levels in humans [31, 32]. ThusPPARα plays a central role in the control offatty acid and lipoprotein metabolism, and improves plasma lipid profiles.Although peroxisome proliferators have carcinogenic consequences in the liverof rodents, epidemiological studies suggest that similar effects are unlikelyto occur in humans [10, 33–36].

Several mechanisms have beenproposed to explain the carcinogenesis of peroxisome proliferators in rodents.Peters et al. reported thatwild-type mice treated with the Wy-14,643 showed increase of replicative DNAsynthesis in hepatic cells and developing liver tumors with 100% incidence,whereas PPARα-null mice were refractory to thiseffect [37]. Peroxisome proliferators increase the peroxisome volume and numberand result in an increase in hydrogen peroxide (H₂O₂)levels [38–40]. Theseeffects may be mediated in part by the increased expression of peroxisomalenzymes that produce H₂O₂, such as acyl CoA oxidase (ACO) [39–41]. PPARα upregulates the expression levels of ACO via PPRE in the promoter region [42, 43]. A stably transfected Africangreen monkey kidney cells (CV-1) overexpressing rat ACO increased H₂O₂ production, formed transformed foci, and grew efficiently in soft agar when thecells were treated with linoleic acid [44]. Furthermore, when these cells weretransplanted into nude mice, these cells formed solid tumors [44]. An increaseof intracellular levels of H₂O₂ could lead to DNA damagevia a variety of mechanisms [45]. Any reduced iron present can catalyze thecleavage of H₂O₂, via the Fenton reaction, to producehydroxyl radicals (HO•) [46]. The HO• attacks guanineresidues, producing residues of 8-oxo-7,8-dihydroguanine (8-oxoguanine). WhenDNA synthesis occurs before the 8-oxoguanine is repaired, this damaged basewill have a chance to pair with adenine nucleotide, resulting in a mutation inthe daughter cells [47]. In addition, antioxidants inhibit ciprofibrate-inducedhepatic tumorigenesis by scavenging active oxygen [48]. Thus oxidative stressby peroxisome proliferators acts as a driving force to malignancy. Theactivation of PPARα also leads to increased hepatocellularproliferation and inhibition of apoptosis. Chronic administration of nafenopin,PPARα agonist, to mice causes significantincrease in the liver weight, hepatic DNA synthesis, and the development ofhepatocellular carcinomas [49]. Nafenopin treatment of primary cultures ofadultrat hepatocytes also stimulated DNA synthesis [50]. Indeed, Peters et al. showed that mRNAs encoding cyclin-dependent kinase (CDK) 1, CDK4,cyclin D1, and c-myc and theirproteins, which induce cell proliferation, increased in wild-type mice fed byWy-14,643 but not in PPARα-null mice [51]. Increase of the averageliver weight and the levels of mRNAs encoding cell cycle regulation, such asCDK4, proliferating cell nuclear antigen (PCNA) and cyclin B1, were also foundin wild-type mice fed by bezafibrate, the less specific PPARα agonist, and these effects were notfound in PPARα-null mice [52]. Moreover, the treatmentof the primary culture of rat hepatocytes and the rat hepatoma cell line, FaO,with nafenopin suppressed apoptosis [53, 54]. Thus the activation of PPARα leads to the increase of oxidativestress, induction of cell proliferation and inhibition of apoptosis, indicatingthat PPARα increases hepatocarcinogenesis in mice.

A number of experimentalobservations suggest that there is a species difference between rodents andhumans in the response to PPARα agonists, although the functionaldifferences of PPARα derived from species are not clear(Table 1). One possible explanation for the difference is the expression levelsof PPARα in the liver. The expression levels ofPPARα in human liver are approximately oneorder less than that observed in mouse liver [55]. Small expression levels ofPPARα could allow PPREs to be occupied by othermembers of the nuclear receptor superfamily,such as RXR, the chicken ovalbuminupstream promoter transcription factor I (COUP-TFI), COUP-TFII, hepatocytenuclear factor-4 (HNF4), retinoic acid receptor (RAR), and thyroid hormonereceptor (TR), and affect responsiveness to peroxisome proliferators [56–62]. We andothers have shown that elevated expression of PPARα in HepG2 cells dramatically increasedthe expression of several target genes, such as 3-hydroxy-3-methylglutaryl-CoAsynthase 2 (mitochondrial) (HMGCS2), carnitine palmitoyltransferase 1A (CPT1A),and long chain fatty acyl-CoA synthetase (ACS) [30, 63, 64]. In this way, thelower expression levels of PPARα in human liver might contribute toholding down peroxisome proliferation and subsequent pathologic effects. Anotherexplanation is that several PPARα variants, which lack the entire exon 6or contain mutations, are detected in human cells and these variants act as adominant negative regulator of PPAR-mediated gene transcription [55, 65, 66]. Butthis has not been found in rodents yet. One PPARα variant containing the mutationprevents the suppression of hepatocyte apoptosis by nafenopin [55, 65, 66].Thus the expression levels of PPARα variants might affect the response toperoxisome proliferators. Next, there appears to be sequence differences in thePPRE found in the promoter region of ACO. Osumi et al. identified ACO to be a direct PPARα target gene and a functional PPRElocated in the proximal promoter of the rat ACO gene [42]. In contrast to therodent ACO gene promoter, the human ACO gene promoter differs at three baseswithin the PPRE from the rat ACO promoter and appears refractory to PPARα [42, 67, 68]. This human PPRE wasunable to drive peroxisome proliferators-induced gene transcription in cell-basedassays [67–69]. Indeed,human liver cell lines and primary hepatocytes did not induce ACO mRNA bytreatment with fibrates or other PPARα agonists [63, 64]. A similar pattern,such differences between human and other species, was observed in theexpression of ApoA-I gene [31]. Fibrates influence the ApoA-I gene expression,raising it in humans, and lowering it in rodents. These differences are due toa combination of two distinct mechanisms implicating the nuclear receptors PPARα and Rev-erbα, a negative regulator of genetranscription [31]. The species-distinct regulation is due to sequencedifferences in cis-acting elements intheir respective promoters leading to repression by Rev-erbα of rat ApoA-I and activation by PPARα of human ApoA-I. There is a positivePPRE in the human ApoA-I promoter but not in rats. The expression of Rev-erbα is induced by fibrates [3, 31]. In thecase of rat, this induction leads to the repression of the ApoA-I geneexpression through an Rev-erbα response element. On the other hand,there is no Rev-erbα response element in the human ApoA-Igene [31]. Thus the sequence differences in cis-actingelements cause the species-distinct regulation of target genes expression byperoxisome proliferators. However, the mechanism of the species differences isnot known in detail.

To determine the mechanism of species difference in response to peroxisome proliferators, Gonzalez et al.generated a liver-specific PPARα humanized mouse line (hPPARα^TetOff mice) in which thehuman PPARα was expressed in the liver in a PPARα-null background under the control ofthe tetracycline (Tet) responsive regulatory system [70–72]. Theexpression of several target genes encoding peroxisomal and mitochondrial fattyacid metabolizing enzymes were elevated in hPPARα^TetOff mice fed Wy-14,643 orfenofibrate, resulting in the decrease of serum triglycerides [70, 73]. However, the expressions of various genes involved in cell cycle regulation(PCNA, c-myc, CDK1, CDK4, andcyclins) in the liver were unaffected by Wy-14,643. In addition, hPPARα^TetOff mice were resistant toWy-14,643-induced hepatocarcinogenesis [70, 73]. Recently, Shah et al. showed that Wy-14,643regulated mice hepatic MicroRNA (miRNA) expression via a PPARα-dependent pathway [74]. miRNAs are aclass of nonprotein-coding, endogenous,small RNAs, and regulate geneexpression by translational repression and mRNA cleavage [75]. Some miRNAsregulate cell proliferation and apoptosis processes that are important incancer formation [76]. The activation of PPARα with Wy-14,643 inhibits the expressionof miRNA let-7C, which functions as a tumor suppressor gene [74]. let-7Cdegrades c-myc mRNA by binding to 3'untranslated region (UTR) of the c-myc gene. Treatment of mice with Wy-14,643 showed that let-7C expression wasdecreased and a subsequent increase in c-myc was observed. Following an increase in c-myc,the levels of the oncogenic mir-17 miRNA cluster were increased [74]. In thisway, inhibition of the let-7C signaling cascade may lead to increasedhepatocellular proliferation and tumorigenesis. In contrast, hPPARα^TetOff mice do not exhibitdownregulation of let-7C and induced c-myc and mir-17 expression [74]. Furthermore, Yang et al. generated another type of PPARα humanized mice, hPPARα^PAC mice, that has thecomplete human PPARα gene sequence including 5' and 3' flankingsequences on a P1 phage artificial chromosome (PAC) genomic clone, introducedonto the mouse PPARα-null background [71]. Upon treatmentwith the peroxisome proliferators (Wy-14,643 or fenofibrate), hPPARα^PAC mice exhibited peroxisomeproliferation, lowering of serum triglycerides, and induction of PPARα target genes encoding enzymes involvedin fatty acid metabolism. However, let-7C expression was not decreased and theexpression levels of c-myc, cyclin D1and CDK4 were not increased [71]. Thus these observations suggest that thespecies differences in response to peroxisome proliferators could be due inpart to a differential ability of the mouse and human PPARα to suppress let-7C gene expression[74]. However, the mechanism involved in PPARα-dependent repression of let-7C isunclear. The differences between the wild-type mice and PPARα humanized mice could be caused by thestructural differences between human and mouse PPARα and differential coactivatorrecruitment. However, additional investigation is required to better understandand clarify the mechanism of action of PPARα in causing hepatocarcinogenesis.

3. PPARδ AND CANCER

The role of PPARδ in oncogenesis is controversial,especially in colon cancer. Some reports show that PPARδ promotes tumorigenesis by increasingcell proliferation. Indeed, the levels of PPARδ mRNA are increased in both human androdent colorectal carcinomas [77, 78]. PPARδ is a potential downstream target geneof the adenomatous polyposis coli (APC)/β-catenin/T cell factor-4 (TCF-4) pathway[77]. APC is a tumor suppressor gene and is mutated in familial adenomatouspolyposis (FAP) and most sporadic colorectal tumors [79–83]. β-catenin, which binds to APC and axin ina large protein complex, can be phosphorylated by glycogen synthase kinase-3β (GSK3β) and is followed by ubiquitination anddegradation. Mutation of APC results in the accumulation of β-catenin, which in turn translocates tothe nucleus and associates with the transcription factor TCF-4 [84]. The β-catenin-TCF-4 transcription complexincreases the transcription of growth-promoting genes, such as c-myc and cyclin D1 [85, 86]. The β-catenin-TCF-4 transcription complexalso activates the human PPARδ promoter activity via TCF-4 bindingsites, namely, APC suppresses the PPARδ expression through the degradation of β-catenin [77]. K-Ras mutation is foundin colorectal cancer [80, 87]. Activation mutations in Ras result in theactivation of the mitogen-activated protein kinase (MAPK) pathway and inducetumor growth and progression [88]. The expression levels and activity of PPARδ were increased by the induction ofmutated K-Ras in conditionally K-Ras-transformed rat intestinal epithelialcells [89]. Thus PPARδ is also a downstream target gene of Ras/Raf/MAPK andextracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathway [89]. Inthis way, PPARδ may play a role in colon cancer.

Epidemiological studies haveshown that nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, indomethacin, and sulindac, reduce the overall number and size of adenomas inpatients with FAP. Healthy individuals using NSAIDs regularly can lead to a 40–50% reduction inthe relative risk of developing colon cancer [90]. NSAIDs inhibit cyclooxygenase(COX) activity and thereby reduce prostaglandin synthesis [91]. COX is a keyenzyme in arachidonic acid metabolism and prostaglandin production. COX catalyzes a two-step reaction that converts arachidonic acid to prostaglandin H₂ (PGH₂), which in turn serves as the precursor for the synthesis ofall biologically active prostaglandins, including PGD₂, PGE₂,PGF₂α, prostacyclin (PGI₂), andthromboxane A₂ (TXA₂) [92]. COX exists in two isoformsthat are encoded by two separate genes. COX-1 is constitutively expressed inmost tissues, on the other hand, the expression of COX-2 is normally low orabsent in most tissues but is rapidly upregulated by proinflammatory cytokines[93]. Expression of COX-2 is also elevated in colorectal cancer and in a subsetof adenomas [94]. Moreover, since both the introduction of the knockoutmutation of the COX-2 gene into Apc^Δ716 mice, a model of humanFAP, and treating Apc^Δ716 mice with NSAIDs reducethe development of intestinal tumors, COX-2 inhibitors have been considered astherapeutic agents for colorectal polyposis and cancer [95]. He et al. reported that NSAIDs inhibitedthe transcriptional activity of PPARδ by disruption of the DNA bindingability of PPARδ/RXR heterodimers, and ectopicexpression of PPARδ in the human colorectal cancer cellline, HCT116, protected the cells from sulindac-induced apoptosis [77]. PPARδ and COX-2 mRNA are expressed in similarregions in human colon cancer, and the stable PGI₂ analog,carbaprostacyclin (cPGI), acts as a PPARδ ligand [11, 78]. Indeed, ectopicexpression of COX-2 and PGI synthase (PGIS) in the human osteosarcoma cellline, U2OS, produced high levels of endogenous PGI₂ andtransactivation of PPARδ [78]. PGE₂ levels are alsoelevated in human colorectal cancers and adenomas, and PGE₂ increases the growth and motility of colorectal carcinoma cells [96, 97]. D. Wang et al. showed that PGE₂ promoted resistance to serum starvation-induced apoptosis of cultured humancolon carcinoma cells, LS-174T, through indirectly upregulation PPARδ transcriptional activity via a phosphotidylinositol-3-kinase(PI3K)-Akt pathway [98]. Furthermore, PGE₂ accelerates intestinaladenoma growth of Apc^min mice, a model of human FAP that harbors a mutation in the apc gene, via PPARδ [98]. Xu et al. showed that PGE₂ activated cytosolicphospholipase A₂α (cPLA₂α) through PI3K or MAPK pathway, andsubsequently cPLA₂α enhanced PPARδ activity in the humancholangiocarcinoma cells [99]. They also showed that PPARδ enhanced COX2 expression and PGE₂ production. This positive feedback loop may play an important role incholangiocarcinoma cell growth, although it is not known whether this kind ofpositive feedback loop exists in the colorectal cancer cells [99]. Thus PPARδ induces the cell proliferation throughthe inhibition of apoptosis. However, sulindac sulfide induces apoptosis notonly in wild-type HCT116, but also in HCT116 PPARδ-null cell lines [100]. On the basis ofthese observations, although NSAIDs may reduce tumorigenesis through theinhibition of PPARδ activity, PPARδ is not a major mediator ofsulindac-mediated apoptosis.

Recent evidence supports thehypothesis that PPARδ promotes tumor progression. HCT116 PPARδ-null cell lines grew slightly moreslowly than wild-type HCT116 cells, and exhibited a decreased ability to formtumors compared with wild-type mice when inoculated as xenografts in nude mice[100]. Gupta et al. showed thatexposure of Apc^min mice to10 mg/kg of GW501516, a high-affinity PPARδ-selective agonist, led to a two-foldincrease in polyp number in the small intestine [101]. The most prominent effect was on polyp size, mice treated with the PPARδ activator had a five-fold increase inthe number of polyps larger than 2 mm, suggesting that PPARδ activation primarily affected the rateof polyp growth rather than initiating polyp formation. Pretreatment ofwild-type HCT116 cells with GW501516 significantly suppressed serumstarvation-induced apoptosis in a dose-dependent manner, but not HCT116 PPARδ-null cells [101]. Furthermore, D. Wang et al. showed that PPARd^−/−/Apc^min mice decreased intestinal adenoma growth andinhibited the tumor-promoting effect of GW501516 [102]. They also showed that PPARδ activation with GW501516 upregulated vascularendothelial growth factor (VEGF) transcription, expression, and peptide releasein intestinal epithelial tumor cells, and subsequently activated PI3K-Aktsignaling [102]. Similar results were obtained in the human endothelial cells[103, 104]. Piqueras et al. showed that GW501516 induced VEGF mRNA and peptide release, and thus PPARδ induced endothelial cell proliferationand angiogenesis [103]. Stephen et al.showed that the activation of PPARδ resulted in increased expression ofVEGF and its receptor fms-related tyrosine kinase 1 (FLT-1), and they suggestedthat PPARδ might initiate an autocrine loop forcellular proliferation and possibly angiogenesis [104]. These resultsdemonstrate that VEGF mediates the antiapoptotic effects of PPARδ in intestinal epithelial tumor cells byactivating the PI3K-Akt cell survival pathway, and the VEGF autocrine loopplays an important role in cell survival. Diminished apoptosis is also linkedto downregulated 15-lipoxygenase-1 (15-LOX-1) expression in colorectal cancercells. 13-S-hydroxyoctadecadienoic acid (13-S-HODE), which is the primaryproduct of 15-LOX-1 metabolism of linoleic acid, inhibits cell proliferationand induces cell cycle arrest and apoptosis in transformed colonic epithelialcells [105]. 15-LOX-1 protein expression and 13-S-HODE intracellular levels aredecreased in human colonic tumors [105]. Shureiqi et al. showed that 13-S-HODE bound to PPARδ and then downregulated PPARδ expression and activation in colorectalcancer cells, DLD-1 and RKO, and that the loss of PPARδ expression in HCT116 markedlysuppressed 13-S-HODE-mediated apoptosis [106]. 15-LOX-1 expression alsodownregulated PPARδ expression and transcriptional activityin these colorectal cancer cells [106]. Furthermore, NSAIDs increase 15-LOX-1protein expression and its product 13-S-HODE levels and downregulate PPARδ expression in association withsubsequent growth inhibition and apoptosis [106, 107]. Thus it is consideredpossible that PPARδ promotes the growth of colon cancers.

On the contrary, other reportssuggest that ligand activation of PPARδ promotes the induction of terminaldifferentiation and inhibition of cell growth. PPARδ was found in intestinal epithelialcells in both the normal intestine and adenomas of Apc^min mice [101]. Reed et al. reported that targeted deletion of the APC alleles inmouse intestines decreased the expression levels of PPARδ mRNA and protein, although β-catenin and c-myc were increased [108]. Marin et al. showed that PPARδ expression was reduced in both the Apc^min mouse colon polyps andazoxymethane (AOM)-treated wild-type mouse polyps, though the expression levelsof PPARδ mRNA in colonic epithelium were not different between Apc^min mice and wild-type mice with or without AOM-treatment[109]. Several reportsidentified that the transcription factor binding sites for AP-1,CCAAT/enhancer-binding proteins, vitamin D receptor, and others were found inhuman or mouse PPARδ promoter, and these transcriptionfactors regulated PPARδ expression [16, 110, 111]. However,further investigation is required to certify the regulation of PPARδ expression in cancer.

Hollingshead et al. reported that GW501516 andGW0742, highly specific PPARδ ligands, did not increase the growth ofhuman colon cancer cell lines (HT-29, HCT116, and LS-174T) and liver cancercell lines (HepG2 and HuH7) cultured in the presence or absence of serum [112].In addition, treatment of these cell lines with either GW501516 or GW0742 did not change the phosphorylation of Akt, and no increase in the expression levelsof COX2 or VEGF were detected [112]. Similar results were observed in the colon or liver of Apc^min mice treated withGW501516 or GW0742 [109, 112]. Barak etal. showed that the average number of intestinal polyps was notsignificantly different between PPARd^+/+/Apc^min, PPARd^+/−/Apc^min, and PPARd^−/−/Apc^min mice, although thisstudy was limited to a small number [113]. On the other hand, several studiesshowed that colon polyp formation was enhanced in the absence of PPARδ expression in both PPARd^−/−/Apc^min and AOM-treated PPARd^−/− mice [108, 109, 114]. Moreover, Marin etal. showed that the administration of GW0742 had no effect on colon orsmall intestinal tumorigenesis in either PPARd^−/−/Apc^min or PPARd^+/+/Apc^min mice as compared with controls [109]. Inaddition, decrease of colon polyp multiplicity was observed in PPARd^+/+ AOM-treated miceadministrated with GW0742 compared with control wild-type mice. This effect waslikely due in part to PPARδ-dependent induction of colonocytedifferentiation and enhancement of apoptosis [109]. Indeed, PPARδ induces keratinocyte terminaldifferentiation, which normally opposes cell proliferation [115, 116]. Hatae et al. also showed that intracellularPGI₂, an endogenous PPARδ ligand, formed by expressing PGIS inhuman embryonic kidney 293 (HEK293) cells, promoted apoptosis by activatingPPARδ [117]. In this way, PPARδ inhibits tumor growth by inducing apoptosis or differentiation.

Thus the conflicting reports inthe literature suggest that PPARδ either potentiates or attenuates coloncancer. Similar discrepancies were observed in other tissues. Di-Poï et al. showed that the activation ofPPARδ inhibited apoptosis in keratinocyte[118]. The activation of PPARδ by L-165041, one type of PPARδ ligand, upregulates3-phosphoinositide-dependent kinase-1 (PDK1) and integrin-linked kinase (ILK)gene expression via PPRE and downregulates phosphatase and tensin homolog(PTEN) protein expression, and subsequently leads to the activation of Akt1 ina PI3K- dependent manner in mouse primary keratinocytes and human keratinocyteHaCaT cells [118]. Yin et al.showed that PPARδ ligand GW501516 accelerated progestin-and carcinogen-induced mouse mammary carcinogenesis [119]. Stephen et al. reported that PPARδ selective agonists stimulated theproliferation of human breast and prostate cancer cell lines and primary endothelial cells [104]. On theother hand, Burdick et al.reported that ligand activation of PPARδ with GW0742 inhibited the cell growthof either human keratinocyte cell line N/TERT1 or mouse primary keratinocytes[120]. In these cells, ligand activation of PPARδ by GW0742 did not alter expressionand/or modulation of the PTEN/PDK1/ILK1/Akt pathway [120]. Girroir et al. reported that both GW0742 andGW501516 inhibited the growth of the human breast cancer cell line, MCF7, andhuman melanoma cell line, UACC903 [121].

To date, however, the reason forthe contradiction in these observations is unclear. One explanation for these conflictingresults may be the ability of PPARδ to repress the transcription of targetgenes. We and others observed that unliganded PPARδ repressed target gene expression,though ligand-activated PPARδ induced these genes [30, 122–124]. It has beenreported that unliganded PPARδ bound to PPRE and recruitedcorepressors, such as B-cell lymphoma 6 (BCL-6), silencing mediator forretinoid and thyroid hormone receptor (SMRT), nuclear receptor corepressor(NCoR), and others. On the other hand, liganded PPARδ is thought to release the corepressorand form a complex with coactivators [122–124].Furthermore, binding of ligand to the PPARδ or deletion of PPARδ expression may lead to the release ofBCL-6. Subsequently, BCL-6 represses the transcription of a number ofinflammatory cytokine genes [124]. Thus the PPARδ activity may be influenced by thecellular environment, such as the existence of PPARδ ligands, cofactors, and others. Fromthis viewpoint, the conflicting results may be due to differences in thecondition of cell cultures or the genetic background of animal models. Secondly, prostaglandins, some of which act as PPAR ligands, have a variety of biologicalactivities. Prostaglandins, synthesized via the COX pathway from arachidonicacid, are released outside the cells and lead to changes in the cellular levelsof cyclic AMP and Ca²⁺ through binding to G-protein-coupledreceptors on the plasma membrane [90]. Indeed, Hatae et al. suggested that cAMP produced by the PGI₂-PGIreceptor (IP)-cAMP pathway might protect vascular endothelial cells fromintracellular PGI₂-PPARδ-mediated apoptosis[117]. On the otherhand, Fauti et al. showed thatthe ectopic expression of COX-2 and PGIS in HEK293 cells results in a dramaticinduction of PGI₂ synthesis, but no increase in PPARδ transcriptional activity is observed[125]. Thus they suggest that PGI₂ lacks agonistic activity for PPARδ. Since PGI₂ is unstable andrapidly hydrolyzed to 6-keto-PGF_1α within minutes and increases theproduction of intracellular cAMP via stimulation of adenylyl cyclase throughthe cell surface IP receptor, further investigation is necessary to certify themechanism of the effect of the PGI₂ on PPARδ activity in detail. Therefore, additionalanalyses are necessary to define the PPARδ functions in cancer (Figure 2).

4. PPARγ AND CANCER

Cancer cellsrepresent dysregulaton of the cell cycle and lead to cell proliferation. Inthis viewpoint, modulators of the cell cycle and/or apoptosis are useful aschemotherapeutic agents for cancer [126, 127]. A number of investigators haveshown that PPARγ was expressed in a variety of tumorcells, and the activation of PPARγ by ligands led to either inhibition ofcell proliferation or induction of apoptosis (Table 2) [128, 129]. PPARγ is expressed in colonic tumors, normalcolonic mucosa, and colon cancer cell lines [130–135]. Kitamura et al. showed that TZDs, such astroglitazone and rosiglitazone, inhibited the cell growth and induced G1 cellcycle arrest of rat intestinal epithelial cells [136]. The cell growthinhibition by TZDs was caused by the decrease of the expression of cyclin D1,critical for entering the S phase of the cell cycle. TZDs suppressed the cyclinD1 promoter activity through inhibition of the transcriptional activities of AP-1and Ets [136]. Shao et al.demonstrated that treatment with rosiglitazone inhibited the K-Ras-inducedelevation of the expression levels of cyclin D1 by inhibition of the K-Ras-induced phosphorylation of Akt, resulting in the G1 cell cycle arrest[89]. Furthermore, J.-A. Kim et al.showed that treatment of the human colorectal cell line, HCT15, withtroglitazone induced the expression of p21^Cip1/Waf1, that is, a CDKinhibitor (CKI) and negatively regulates the cell cycle progression, throughthe ERK pathway, and inhibited HCT15 cell growth [155]. PPARγ ligands also induce apoptosis in humancolon cancer cells [156]. Chen et al.showed that PPARγ ligands, 15-Deoxy-Δ^12,14-prostaglandinJ₂⁢ (15dPGJ₂), or ciglitazone, induced apoptosis in HT-29by inhibiting nuclear factor kappa B (NF-κB) activity, which upregulates variousantiapoptotic genes, and suppressing the expression of BCL-2, which protectscells against apoptosis [133]. Furthermore, using the in vivo mouse model, the administration of TZD to mice reduced AOMand/or dextran sodium sulfate-induced formation of aberrant crypts foci andcolon carcinogenesis [131, 157]. In addition, PPARγ ligands also inhibit the cell growth ofseveral breast cancer cell lines and mammary gland tumor development [137, 158–162]. Elstner et al. showed that PPARγ ligands, troglitazone, 15dPGJ₂, and indomethacin, caused inhibitionof proliferation in several human breast cancer cell lines, such as MCF7,MDA-MB-231, BT474, and T47D [162]. Troglitazone also inhibited MCF7 tumorgrowth in triple-immunodeficient BNX nude mice [162]. Clay et al. reported that 15dPGJ₂ and troglitzaone attenuated cellular proliferation of MDA-MB-231 by blockingcell cycle progression and inducing apoptosis [160]. Pretreatment of MDA-MB-231cells with 15dPGJ₂ attenuated the capacity of these cells to inducetumors in nude mice [160]. Yin et al. showed that treatment of MCF7with troglitazone also decreased the expression of several regulators of pRbphosphorylation, such as cyclin D1, CDK4, CDK6, and CDK2 [158]. pRB is aretinoblastoma tumor suppressor gene product, and phosphorylated pRB inducescell cycle progression [163]. Troglitazone induced the G1 cell cycle arrest byattenuation of pRb phosphorylation, resulting in inhibition of cellproliferation [158]. Suh et al.showed that GW7845, synthetic PPARγ ligand, prevented mammarycarcinogenesis in the rat model that used nitrosomethylurea as the carcinogen[159]. Mehta et al. alsoreported that troglitazone prevented the induction of preneoplastic lesions by7, 12-dimethylbenz[a]anthracene in amouse mammary gland organ culture model [161]. Moreover, PPARγ ligands inhibit the cell proliferationin other types of cancer. PPARγ ligands inhibited the growth ofesophageal squamous carcinoma cell lines by inducing G1 arrest associated withan increased level of several CKIs, such as p27^Kip1, p21^Cip1/Waf1, and p18^Ink4c [138]. PPARγ ligands also induced apoptosis and G1cell cycle arrest in human gastric cancer cells, and that inhibited cellproliferation [139, 164]. In human pancreatic cancer cells, PPARγ ligands induced apoptosis and growthinhibition associated with G1 cell cycle arrest through increasing p27^Kip1 protein expression [140, 165–167]. In humanhepatocellular carcinoma cell lines, PPARγ ligands induced cell cycle arrestthrough increased expression of p21^Cip1/Waf1, p27^Kip1, andp18^Ink4c protein levels [141, 168]. Troglitazone also induced theactivation of the cell death protease, caspase 3, and that induced apoptosis ofhuman liver cancer cell lines [169]. PPARγ is abundantly expressed in humanadrenal tumors including adrenocortical carcinomas and normal adrenal tissues.PPARγ agonists suppress adrenocortical tumorcell proliferation, increase apoptosis, and induce adrenal differentiation[142, 170]. Moreover, PPARγ ligand showed antitumor effect againsthuman prostate cancer cells and human lung cancer cells [143, 144, 171–173]. Thus PPARγ ligands could suppress thetumorigenesis. Therefore, PPARγ ligands could be used as antineoplasticdrugs.

In contrast, both troglitazoneand rosiglitazone treatment increased the frequency and size of colon tumors in Apc^min mice [174, 175].Treatment with rosiglitazone also increased the expression levels of β-catenin, a protein involved in Wntsignaling and correlating with enhanced cell proliferation, in the colon of Apc^min mice and HT-29 cells[174]. To investigate the basis for this contradiction, Girnun et al. used mice heterozygous for PPARγ with both chemical and genetic modelsof human colon cancer [176]. Heterozygous loss of PPARγ caused a greater incidence of coloncancer when these mice were treated with AOM. Although there was no differencein β-catenin expression levels in colorectaltumors between AOM-treated PPARg^+/−and wild-type mice, β-catenin expression levels in the colonicepithelium of untreated PPARg^+/−mice were greater than that of untreated wild-type mice. When crossing to Apc^1638Nmice, the mousemodel for FAP, there were also no difference in β-catenin levels between PPARg^+/−/Apc^1638N and PPARg^+/+/Apc^1638N mice before polypformation. Survival and the number of tumors formed in the colon also showed nodifference in both mice. Thus although PPARγ has the potential to suppress β-catenin levels and coloncarcinogenesis, PPARγ has no effect on β-catenin levels or tumorigenesis in thepresence of APC signaling dysfunction [176]. Furthermore, PPARγ mutations, some of which show the lossof the transactivation ability, are found in colon cancers in humans, and thatPPARγ may be considered as a tumor suppressorgene [134]. On the other hand, to evaluate the contribution of PPARγ to breast cancer, Saez et al. generated transgenic mice,MMTV-VpPPARγ mice, that express a constitutivelyactive form of PPARγ in mammary gland [177]. MMTV-VpPPARγ mice showed normal development ofmammary gland and no increased tendency to develop tumors. To assess theinfluence of increased PPARγ signaling on mammary gland neoplasia,MMTV-VpPPARγ mice were crossed to mice that expressa polyoma virus middle T antigen (PyV-MT) in mammary tissue, MMTV-PyV mice,which rapidly develop tumors. These mice that expressed both activated PPARγ and PyV-MT showed accelerateddevelopment of mammary tumors. Therefore, although increased PPARγ activation does not initiate tumorformation in normal mammary tissue, once a tumor-initiating event occurs, PPARγ signaling serves as a tumor promoter inthe mammary gland. Furthermore, there is no difference in tumor developmentbetween MMTV-PyV mice and the mice, generated by crossing PPARg^+/−mice to MMTV-PyV mice [177]. Thus in thismodel, PPARγ does not act as a tumor suppressorgene.

Furthermore, PPARγ ligands exert their biological effectsthrough a PPARγ-independent pathway. Palakurthi et al. reported that troglitazone andciglitazone induced G1 arrest by inhibiting translation initiation in both PPARg^−/−and PPARg^+/+mouse embryonic stemcells. Thus TZDs inhibit cell proliferation and tumor growth in a PPARγ-independent manner[178]. Therefore, althoughPPARγ ligands are used as insulinsensitizers, further investigation is needed to clarify whether PPARγ ligands are effective chemotherapeuticagents for cancer in humans.

5. SUMMARY

PPARs arelinked to metabolic disorders and are interesting pharmaceutical targets. Amongthe synthetic ligands, fibrates are hypolipidemic compounds that activate PPARα, and TZDs, which selectively activatePPARγ, are hypoglycaemic molecules that areused to treat type 2 diabetes. PPARδ agonists might form effective drugs forobesity, diabetes, and cardiovascular disease. Moreover, recent evidencesuggests that PPAR modulators may have beneficial effects as chemopreventiveagents [179]. However, as mentioned above, it remains unclear whether PPARs actas oncogenes or as tumor suppressors. From this viewpoint, current strategiesare aimed at reducing the side effects and improving the efficacy and safetyprofile of PPAR agonists, termed selective PPAR modulators (SPPARMs) [180, 181]. This model proposes that SPPARMs bind in distinct manners to the ligandbinding pocket of PPAR and induce distinct conformational changes of thereceptor, resulting in differential interactions with cofactors according tothe combination of their expression levels in different organs. Thus eachSPPARM leads to differential gene expression and biological response. However,what kinds of cofactors are recruited to PPAR by each SPPARM is still unknown.Thus it is important to identify the cofactor complex for PPAR with each SPPARMand the expression patterns of cofactors in various tissues. Furthermore,recent evidence suggests that the ligand binding protein in the cytosol thattransports ligands into the nucleus is important to modulate the action ofnuclear receptors. Long-chain fatty acids, endogenous PPAR ligands, are highlyhydrophobic and fatty acids are bound to fatty acid binding proteins (FABPs) inthe aqueous intracellular compartment [182]. FABPs also bind to PPAR ligandsand transport them from the cytosol into the nucleus [183–191]. In thenucleus, FABPs interact directly with PPARs and deliver ligands to PPARs, andthe activity of PPARs is modulated [186, 187, 190–192]. Recently,Schug et al. showed that whenthe cellular retinoic acid binding protein-II (CRABP-II) expression levels werehigher than FABP5 in the cells, retinoic acid (RA) bound to CRABP-II.Subsequently, CRABP-II relocated to the nucleus and delivered RA to RAR,resulting in inhibition of cell proliferation and induction of apoptosis [187, 193]. On the contrary, when the FABP5 to CRABP-II ratio is high, RA serves as aphysiological ligand for PPARδ, which induces cell survival andproliferation [187, 194]. Therefore, it is important to identify the cytosolicligand binding proteins and the expression levels of the proteins for definingthe physiological effects of ligands. Furthermore, several ligands exert theirbiological effects through a PPAR-independent pathway [195]. Thus furtherstudies are required to elucidate the role of PPARs for developing newefficiently and safety chemotherapeutic agents for cancer.