Peroxisome Proliferator-Activated Receptor γ and PGC-1α in Cancer: Dual Actions as Tumor Promoter and Suppressor

Peroxisome proliferator-activated receptor γ (PPARγ) is part of a nuclear receptor superfamily that regulates gene expression involved in cell differentiation, proliferation, immune/inflammation response, and lipid metabolism. PPARγ coactivator-1α (PGC-1α), initially identified as a PPARγ-interacting protein, is an important regulator of diverse metabolic pathways, such as oxidative metabolism and energy homeostasis. The role of PGC-1α in diabetes, neurodegeneration, and cardiovascular disease is particularly well known. PGC-1α is also now known to play important roles in cancer, independent of the role of PPARγ in cancer. Though many researchers have studied the expression and clinical implications of PPARγ and PGC-1α in cancer, there are still many controversies about the role of PPARγ and PGC-1α in cancer. This review examines and summarizes some recent data on the role and action mechanisms of PPARγ and PGC-1α in cancer, respectively, particularly the recent progress in understanding the role of PPARγ in several cancers since our review was published in 2012.


Introduction
Peroxisome proliferator-activated receptor (PPAR ) belongs to a nuclear hormone receptor superfamily that regulates the expression of genes involved in cell differentiation, proliferation, the immune/inflammation response, and lipid metabolism [1]. Ligand binding and activation of PPAR result in heterodimer formation with the retinoid X receptor (RXR) and binding to a PPAR response element (PPRE) to regulate the transcription of numerous target genes [2,3]. PPAR consists of a ligand-independent transcriptional activation domain, DNA binding domain (DBD), hinge region for cofactor docking, and ligand binding domain (LBD) (Figure 1(a)). Two PPAR isoforms are known, PPAR 1 and PPAR 2 [4,5]. PPAR 2, which is generated by alternative splicing, contains an additional 28 amino acids in mice and 30 amino acids in humans, at the N-terminus compared to PPAR 1. PPAR 2 is expressed selectively in adipose tissue and plays an important role in adipocyte differentiation, lipid storage in white adipose tissue, and energy dissipation in brown adipose tissue [4,6]. PPAR 1 is expressed in the colon, immune system, and hematopoietic cells and plays an important role in the control of inflammation, macrophage maturation, and embryo implantation. PPAR 1 is a molecular target of antidiabetic thiazolidinediones [7,8]. Our previous review summarized the role and action mechanisms of PPAR in colorectal cancer [8], but the role of PPAR in cancer is still debated. Thus, this review updates the progress in understanding the role and molecular mechanisms of PPAR in cancer.
The PPAR coactivator-1 (PGC-1) family is composed of PGC-1 , PGC-1 , and PGC-1-related coactivator (PRC). PGC-1 was initially identified as a transcriptional coactivator involved in mitochondrial function and thermogenesis in brown fat [9]. PGC-1 and PRC were discovered in sequence homology searches [10][11][12][13]. The PGC-1 family members have similar activity to increase mitochondrial function when overexpressed and have a related modular structure (Figure 1(b)). The most common functional domains are shared between PGC-1 and PGC-1 . The N-terminal activation domain interacts with several transcriptional coactivators, including p300 and steroid receptor coactivator-1 (SRC-1). A domain involved in inhibition of PGC-1 activity is located adjacent to the N-terminal region. Through several LXXLL motifs, the N-terminal half of PGC-1 interacts with many transcription factors, whereas the C-terminal end of PGC-1 interacts with the TRAP/DRIP/Mediator complex. PGC-1 has a Ser/Arg-rich domain and RNA binding motif that plays an important role in mRNA splicing [14,15]. Because PGC-1 was described initially as a PPAR interacting protein, some investigators recently studied the expression and clinical significance of PGC-1 in cancer [16,17]. However, the expression and the roles of PGC-1 in cancer were not significantly related to the expression of PPAR . In addition, controversies still exist whether PGC-1 acts as a tumor promoter or a tumor suppressor in cancer. This review focuses on the expression and actions of PGC-1 in order to understand the clinical significance of PGC-1 expression in cancer.

The Role and Action Mechanisms of PPAR in Cancer
PPAR is expressed in various malignant tissues, including bladder, colon, prostate, and breast cancer [18][19][20][21][22]. Natural ligands that activate PPAR include long-chain polyunsaturated fatty acids, eicosanoids, components of oxidized low density lipoproteins (oxLDLs), and 15-deoxy-Δ 12,14prostaglandin J 2 (15d-PGJ 2 ) [23]. Synthetic ligands include the antidiabetic thiazolidinedione (TZD) class of drugs [23].  Figure 2). Understanding the role of PPAR in cancer was improved by developing new synthetic and natural ligands of PPAR and performing overexpression and knockdown experiments. PPAR agonist troglitazone inhibits colon cancer cell growth through the inactivation of NF-B by suppressing GSK-3 activity [24]. Emerging data suggest that PPAR acts as a tumor suppressor by inactivating NF-B through different mechanisms. For example, Lee et al. demonstrated that 4-O-methylhonokiol (MH), a PPAR agonist, has antitumor activity in prostate cancer through increased PPAR activity and p21-mediated suppression of NF-B activity as observed by the loss of MH-induced growth inhibition and NF-B inhibition in a p21 siRNA knockdown experiment [25]. In addition, overexpression of PPAR was shown to inhibit cell proliferation and tumor growth via degradation of NF-B by acting as an E3 ligase [26]. Hou et al. demonstrated that PPAR inhibits mucin 1-(MUC1-) C-mediated cell proliferation via MUC1-C ubiquitination and degradation [27]. MUC1-C is known as an oncoprotein and interacts with I B kinase, NF-B/p65, and signal transducer and activator of transcription factor 3 (Stat3), p53, or BAX in order to activate the downstream pathway associated with tumor growth [47][48][49][50][51][52]. Efatutazone, a third-generation PPAR agonist, has been reported to inhibit esophageal squamous cell Inhibition of cell proliferation and tumor growth; increased p21 protein levels by inactivation of Akt [28] Pioglitazone and 6-OH-11-O-hydroxy phenanthrene (PPAR and RXR agonist treatment) Breast cancer MCF-7 cells, breast cancer associated fibroblast Inhibition of cancer stem cell survival; inhibition of IL-6 promoter and reduced MMP-2, MMP-9 expression and activity [29] Pioglitazone treatment (PPAR ligand)

Chronic myeloid leukemia cells, leukemia stem cell (LSC)
Inhibition of cancer stem cell survival; decreased expression of STAT5 and HIF-1 [30] PPAR overexpression by PPAR plasmid carcinoma (ESCC) cell proliferation in vitro and in vivo through increased p21Cip protein levels via inactivation of Akt [28].
The Wnt/ -catenin signaling pathway plays an important role in the occurrence and development of cancer [56,57]. Guo et al. reported that PPAR overexpression inhibits the proliferation and migration of gastric cancer cells through downregulation of telomerase reverse transcriptase (TERT) and enabled homolog (ENAH) via inhibition of -catenin [31]. Mammalian enabled (Mena), encoded by ENAH, is an actin-regulatory protein involved in controlling cell motility and cell-cell adhesion, which are important for the development of metastatic potential [58]. TERT and ENAH are new targets of the Wnt/ -catenin signaling pathway [59,60]. Recently, activation of canonical Wnt signaling was reported to directly act on aerobic glycolysis and increase vessel formation in colon cancer through the Wnt target gene pyruvate dehydrogenase kinase 1 (PDK1) [61]. Via PDK1 activation, pyruvate is converted into acetyl-CoA, which enters the TCA cycle and is converted into citrate, which stimulates protein synthesis. Accumulation of metabolic intermediates (such as aspartate, glycine, serine, and ribose) in cells promotes de novo nucleotide synthesis, contributing to growth and proliferation [62]. In addition, blocking the Wnt pathway decreases PDK1 expression via transcriptional regulation and inhibits in vivo tumor growth [61].
Pseftogas et al. reported that PPAR activation has a tumor suppressive effect by upregulating the expression of tumor suppressor Cyld, as the Cyld promoter has PPAR binding sites [32]. Cyld was identified as a tumor suppressor gene that is causally associated with the development of inherited cylindromas [63]. The gene encodes a protein (CYLD) possessing a carboxyl-terminal ubiquitin-specific protease domain that selectively hydrolyzes K63-and M1linked polyubiquitin chains [64]. A number of studies have suggested a role for CYLD in the growth suppression of different types of cancer cells, such as colon, hepatocellular, lung, melanoma, and breast cancer (reviewed in [65]). CYLD can inhibit several growth and antiapoptotic signaling pathways, including the NF-B, JNK, p38, Wnt, Akt, and Notch pathways [65].
Rovito et al. demonstrated that PPAR activation downregulates CXCR4 gene expression through recruitment of the silencing mediator of retinoid and thyroid hormone receptor (SMRT) corepressor to PPRE within the CXCR4 promoter and then inhibits breast cancer cell migration and invasion [33]. CXCR4, a seven-transmembrane G-proteincoupled receptor for stromal-cell derived factor-1 (SDF-1 ), has been shown to be expressed in human breast cancer cells, and activation of the SDF-1 /CXCR4 axis is important in breast cancer migration and metastasis [66,67].

PPAR as a Tumor Promoter in Cancer.
Our previous review described that PPAR has tumor-promoting activity through the upregulation of -catenin and c-Myc expression, upregulation of COX-2, upregulation of the expression of vascular endothelial growth factor (VEGF) and VEGF receptors, and upregulation of MMP-1 [8]. This review briefly introduces the action mechanisms of PPAR as a tumor promoter ( Figure 3). Recently, increasing evidence has indicated that PPAR acts as a tumor promoter [68][69][70][71][72][73][74]. Downregulation of PPAR by siRNA knockdown or treatment with PPAR antagonist GW9662 has been shown to inhibit the growth of cancer cells, suggesting a tumor-promoting effect for PPAR in these cells [68][69][70]. PPAR was shown to protect ErbB2-positive breast cancer cells from palmitate-induced toxicity [75]. In addition, PPAR was demonstrated to play a crucial role in the maintenance of stemness in ErbB2-positive breast cancer cells; PPAR antagonist GW9662 induces apoptosis and inhibits tumorsphere formation and tumor formation through the inhibition of lipogenic genes (ACLY, MIG12, FASN, and NR1D1) and stem cell-related genes (KLF4 and ALDH) [71]. CSCs have been identified as subpopulations of cells within tumors that promote tumor growth and recurrence [76][77][78].
Kesanakurti et al. demonstrated that PPAR is involved in radiation-induced epithelial-to-mesenchymal transition (EMT) in glioma by interacting with p21-activated kinase 4 (PAK4), resulting in increased Nox1 expression and reactive oxygen species (ROS) [72]. EMT is a developmental transdifferentiation program facilitating the formation of highly motile cells with stem cell characteristics [79,80]. EMT is also involved in increased metastatic potential and treatment resistance in cancer [81,82]. The PAKs are a family of serine/threonine kinases involved in embryonic development, cytoskeletal remodeling, cell motility, and cell proliferation [83,84], and aberrant expression of PAK4 has been shown to promote cancer cell proliferation and invasion [85][86][87].
A recent study using PPAR siRNA showed that PPAR suppression inhibits cell proliferation, colony formation, and tumorigenicity in vivo [73]. In addition, PPAR upregulated VEGF expression through the binding of PPAR in the promoter region of VEGF in prostate cancer cells [73]. Patitucci et al. demonstrated that PPAR activation is involved in steatosis-associated liver cancer and provided evidence supporting the pharmacological modulation of hepatic PPAR activity as a therapeutically relevant strategy in hepatic malignancy associated with activated Akt2 and PPAR signaling [74].

The Role and Action Mechanisms of PGC-1 in Cancer
Many studies have examined the role of PGC-1 in cancer by observing its expression in several cancers and performing PGC-1 overexpression and siRNA knockdown experiments. PGC-1 expression has been shown in some studies to be 6 PPAR Research decreased in some types of cancer, including colon [88], breast [89], and ovarian cancer [41], whereas other studies have shown that PGC-1 expression is increased in cancer [17,90]. Even though many studies have been published, the role of PGC-1 in cancer is still controversial. Therefore, this review describes the role and action mechanisms of PGC-1 in cancer (Table 2).  [17]. In addition, PGC-1 was increased in tumor samples from arsenic-induced skin cancer patients and may be associated with increased cell proliferation and enhanced mitochondrial biogenesis [34]. Bhalla et al. showed that PGC-1 promotes carcinogenesis and tumor growth through the induction of lipogenic enzymes (acetyl-CoA carboxylase and fatty acid synthase) using genetically modified PGC-1 mice [35].

Tumor-Promoting
That study demonstrated that PGC-1 knockout mice had decreased chemically induced liver and colon carcinogenesis, suggesting that PGC-1 may stimulate carcinogenesis [35]. Similarly, Shin et al. first demonstrated that overexpression of PGC-1 enhances cell proliferation and tumorigenesis via the upregulation of Sp1 and acyl-CoA binding protein [36]. It was also reported that PGC-1 overexpression leads to increased antioxidant enzymes (catalase, superoxide dismutase) and decreased ROS-induced apoptosis [36]. Similarly, PGC-1 knockdown significantly decreased cell number and induced apoptosis in PGC-1 positive melanoma cell lines, suggesting that PGC-1 is crucial in the survival of PGC-1 positive melanoma cells [37]. In addition, superoxide dismutase 2 protein levels were decreased in PGC-1 depleted melanoma cells. Moreover, ectopic expression of PGC-1 in melanoma cells increased the expression of ROS detoxifying genes. These data support the hypothesis that PGC-1 plays an important role in activating the ROS detoxification gene program to maintain melanoma cell survival [37]. Vazquez et al. also demonstrated that there was a significant reduction in tumor size in PGC-1 depleted cells, implying PGC-1 may be important in tumor progression [37]. De novo lipogenesis is a distinctive anabolic feature of malignant cells [94]. Carbons from glucose and glutamine supply cytoplasmic citrate for fatty acid synthesis with the help of lipogenic enzymes [94]. Glutamine can serve as an anaplerotic mitochondrial fuel and seems to be important for tumor survival [95]. In ErbB2-positive breast cancer cells, the PGC-1 /ERR complex directly regulates the expression of glutamine metabolism enzymes, leading to the provision of glutamine carbons to de novo fatty acid synthesis [38]. PGC-1 overexpression, or ERR activation, confers growth advantages of breast cancer cells even under limited nutrients, supporting the correlative clinical data that high expression of PGC-1 is associated with poor prognosis, possibly related to the activation of its downstream glutamine pathway target genes [38]. It was reported that PGC-1 expression is affected by various transcriptional pathways. One example is that melanocyte-lineage transcription master regulator and oncogene MITF activated PGC-1 expression in melanoma [37,91]. The decrease in mitochondrial membrane potential and increased ROS production with a decrease in glutathione, cystathionine, and 5-adenosylhomocysteine were observed in PGC-1 -depleted melanoma cell lines, suggesting that intrinsic apoptotic pathway is activated in PGC-1 -depleted melanoma cells [37]. Another example is that the androgen receptor-AMP-activated protein kinase (AMPK) signaling axis increased expression of PGC-1 to drive growth advantages in prostate cancers [39]. It was also shown that PGC-1 expression was significantly higher in lung adenocarcinomas with wild type p53 than in tumors with mutant p53 [40]. Cell proliferation was inhibited by PGC-1 siRNA knockdown experiments in H1944 lung adenocarcinoma cells [40]. In metabolic stress conditions, PGC-1 was shown, in complex with p53, to coactivate the transcription of cell cycle inhibitors, while it was also shown to promote the expression of genes related to mitochondrial biogenesis. These two functions are cooperative processes that promote cell survival. Moreover, oxidative stress in PGC-1 knockdown cells resulted in p53-induced apoptosis [96]. In turn, it was also shown that increased expression of PGC-1 might prevent p53-induced cell death by maintaining an adequate balance between oxidative phosphorylation and glycolysis [97]. Some studies have examined the effect of PGC-1 on angiogenesis. PGC-1 has been reported to activate the production of VEGF through the estrogen-related receptor -(ERR -) dependent pathway [98]. PGC-1 was shown to regulate HIF-1 activity. Increased PGC-1 expression enhances oxygen consumption, resulting in decreased local oxygen tension and increased HIF-1 stability [99]. In addition, HIF-2 is a transcriptional target of PGC-1 , even though the involved transcriptional mechanism is not clear [100]. ERR is overexpressed in many cancers and its inhibition reduces cell proliferation. Recent studies suggest an important role for the interaction between PGC-1 and ERR in cancer (reviewed in [15]). Kinase suppressor of ras 1 (KSR1), a molecular scaffold of the Raf/MEK/extracellular signal-regulated kinase (ERK) cascade, has been demonstrated to promote oncogenic Ras-dependent anchorage-independent growth through the activation of PGC-1 and ERR [92]. Interestingly, recent study shows that PGC-1 plays an important role in the metastatic switch. LeBleu et al. demonstrated that circulating mammary epithelial cancer cells exhibit increased PGC-1 expression, enhanced mitochondrial biogenesis, and oxidative phosphorylation, which may contribute to distant metastasis and poor patient outcome [93]. In addition, PGC-1 knockdown decreased ATP production, reduced actin cytoskeleton remodeling, lowered anchorage-independent survival, and decreased intra-/extravasation, which are all checkpoints that prevent metastasis in MDA-MB-231 breast cancer and B16F10 melanoma cells [93]. LeBleu et al. also showed that PGC-1 expression in invasive cancer cells was significantly associated with the formation of distant metastases in a clinical analysis of human invasive breast cancers [93].

Anticancer Functions of PGC-1 .
As opposed to the tumor-promoting role of PGC-1 described above, several studies have shown that PGC-1 has anticancer effects. As described above, PGC-1 is decreased in colon [88], breast [89], and ovarian cancer cells [41], and PGC-1 overexpression in human ovarian cancer cell line Ho-8910 has been shown to induce apoptosis via downregulation of Bcl-1 and upregulation of Bax, suggesting that PGC-1 may be a contributor to the inhibition of tumor growth [41]. Lee [101]. HIF-1 is known to induce the expression of transcriptional repressor Dec1, which leads to the suppression of PGC-1 expression and the inhibition of mitochondrial respiration [102]. However, the enforced PGC-1 expression in VHL-deficient cells, despite the restoration of mitochondrial function, did not block the inhibition of cell growth and enhanced sensitivity to cytotoxic therapies in oxidative stress conditions [102]. This is in line with clinical clear cell carcinoma data that showed the correlation of higher mitochondrial mass with reduced tumor aggressiveness [103], and the association of lower PGC-1 levels with worse patient outcome [102]. It was shown that PGC-1 attenuates stress responses necessary for cancer cell survival, by interacting with heat-shock factor 1 [104]. Wang and Moraes revealed that increased PGC-1 expression due to treatment with PPAR panagonist (bezafibrate) increased mitochondrial biogenesis, resulting in an inhibition of cancer cell proliferation under glycolytic conditions and inhibition of invasion [44]. In addition, PGC-1 downregulation by miRNA-217 led to the promotion of cancer cell proliferation in breast cancer cells, suggesting a role of PGC-1 as a tumor suppressor [105]. Recently, Torrano et al. showed that PGC-1 suppresses metastasis of prostate carcinoma through an ERR -dependent transcriptional program [45]. Highly metastatic melanoma cells expressed lower levels of PGC-1 [46,106]. In turn, these PGC-1 -low cells expressed higher levels of integrin, TGF , and Wnt signaling components involved in metastasis. It was shown that genetic depletion of PGC-1 increased metastasis in poorly invasive melanoma cells [46]. In contrast, PGC-1 overexpression in melanoma cells by ectopic expression or exposure to BRAF V600E inhibitor vemurafenib suppressed metastasis through the direct regulation of inhibitor of DNA binding protein 2 (ID2) and inhibition of TCF-mediated gene transcription [46].
As described above, there have been many studies of the role of PGC-1 in tumor progression. However, it is still not sure if PGC-1 acts as a tumor promoter or tumor suppressor, and to date it is thought that its effect on tumor varies depending on the tissue context and tumor type (reviewed in [107]).

Conclusions
PPAR and PGC-1 are emerging proteins involved in tumorigenesis and attractive topics to study for further understanding of cancer biology. Originally, PGC-1 was identified as a PPAR interacting protein. However, most of the reported actions of PGC-1 in cancer were not related to the expression of PPAR . Despite the fact that PPAR and PGC-1 can each act as both tumor promoter and tumor suppressor, there is no clearly defined mechanism that can explain the contradictory dual effects. However, their dual actions can be explained, in part, by their cell type-specific effects and variable interacting proteins. Therefore, each of the molecular interactions of PPAR and PGC-1 with other transcriptional partners needs to be further investigated to understand the role of PPAR and PGC-1 in cancer.

Conflicts of Interest
The authors declare that they have no conflicts of interest.