The Anticancer Potential of Peroxisome Proliferator

DOI: 10.1002/cmdc.201700703

Reviews

The Anticancer Potential of Peroxisome ProliferatorActivated Receptor Antagonists Laura De Lellis,[a, b] Annamaria Cimini,[c, d, e] Serena Veschi,[a, b] Elisabetta Benedetti,[c] Rosa Amoroso,*[a] Alessandro Cama,*[a, b] and Alessandra Ammazzalorso[a]

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T 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Reviews The effects on cancer-cell proliferation and differentiation mediated by peroxisome proliferator-activated receptors (PPARs) have been widely studied, and pleiotropic outcomes in different cancer models and under different experimental conditions have been obtained. Interestingly, few studies report and little preclinical evidence supports the potential antitumor

activity of PPAR antagonists. This review focuses on recent findings on the antitumor in vitro and in vivo effects observed for compounds able to inhibit the three PPAR subtypes in different tumor models, providing a rationale for the use of PPAR antagonists in the treatment of tumors expressing the corresponding receptors.

1. Introduction

eicosapentaenoic acids.[5, 10] PPARd is expressed ubiquitously, but it is particularly abundant in intestine, liver, kidney, and skeletal muscle, in which it participates in lipid metabolism. Activation of this receptor is also implicated in the regulation of glucose homeostasis, mainly in skeletal and cardiac muscles, but PPARd also regulates cholesterol levels and insulin sensitivity.[14–16] Among endogenous PPARd ligands, there are polyunsaturated fatty acids and eicosanoid metabolites, such as prostacyclin and 15-hydroxyeicosatetraenoic acid (15-HETE).[7, 17] Notably, different PPAR ligands appear to be implicated in the recruitment of coactivators or corepressors to the transcriptional complex, which may explain differences in the biological effects reported among the ligands.[17] Moreover, genome-wide binding profiles of PPARs obtained by using chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) approaches revealed a very complex PPAR binding profile that is largely cell or tissue specific. For example, PPAR binding sites that are peculiar for a cell type may be kept inaccessible by repressive chromatin mechanisms in a different cell, as reported for PPARg genomic binding sites in macrophages or adipocytes.[18] From a structural point of view, three PPAR subtypes share a common general organization, with distinct DNA binding domain (DBD) and ligand binding domain (LBD) (Figure 1 a).[19]

Peroxisome proliferator-activated receptors (PPARs), identified in the early 1990, are ligand-activated transcription factors belonging to the nuclear receptor family.[1, 2] The body of knowledge regarding these receptors has largely risen in recent years, and to date, their role in regulating lipid and glucose homeostasis, inflammation, proliferation, and differentiation is well known.[3, 4] Three PPAR subtypes, PPARa, PPARg, and PPARd, have been identified. They are encoded by distinct genes and show specific tissue distribution. They are activated by endogenous ligands, such as fatty acids, eicosanoids, and their metabolites, or by synthetic compounds, and they are able to regulate the transcription of genes involved in glucose and lipid metabolism.[5–7] Specifically, PPARa is expressed at high levels in several tissues, including liver, heart, kidney, and brown adipose tissue. It is a major transcriptional regulator of lipid metabolism in the regulation of energy homeostasis. Its activation is involved in the mobilization and catabolization of fatty acids, particularly in the liver during starvation.[5, 8, 9] Endogenous PPARa ligands include fatty acids and fatty-acid-derived metabolites, such as oleoylethanolamide and leukotriene B4.[5, 10] PPARg is mainly expressed in white and brown adipose tissues, the intestine, and the spleen, but its highest expression is in the adipocytes, in which PPARg is a major regulator of adipocyte biology. This receptor also participates in lipid biosynthesis, lipoprotein metabolism, and insulin sensitivity.[11–13] Natural PPARg ligands include polyunsaturated fatty acids, mainly docosahexaenoic and [a] Dr. L. De Lellis, Dr. S. Veschi, Prof. R. Amoroso, Prof. A. Cama, Dr. A. Ammazzalorso Department of Pharmacy, University of Chieti, Via dei Vestini 31, 66100 Chieti (Italy) E-mail: [email protected] [email protected] [b] Dr. L. De Lellis, Dr. S. Veschi, Prof. A. Cama Unit of General Pathology, CeSI-MeT, University of Chieti, Chieti (Italy) [c] Prof. A. Cimini, Dr. E. Benedetti Department of Life, Health and Environmental Sciences, University of L’Aquila, L’Aquila (Italy) [d] Prof. A. Cimini National Institute for Nuclear Physics (INFN), Gran Sasso National Laboratory (LNGS), Assergi (Aq) (Italy)

Figure 1. a) Functional domains of PPAR receptors. Amino-terminal domain (A/B) containing the transactivation activation function 1 (AF-1), domain C (DNA binding domain, DBD), domain D (Hinge), C-terminal domain (E/F or ligand binding domain, LBD) containing the transactivation activation function 2 (AF-2). b) Schematic representation of the PPAR mechanism of activation. In the absence of ligand, corepressors inhibit transcriptional activity; after agonist ligand binding, the heterodimer PPAR:RXR recruits coactivators to activate the transcription of specific target genes.

[e] Prof. A. Cimini Sbarro Institute for Cancer Research and Molecular Medicine and Center for Biotechnology, Temple University, 1900 N. 12th Street, Philadelphia, PA 19122 (USA) The ORCID identification number(s) for the author(s) of this article can be found under: https://doi.org/10.1002/cmdc.201700703.

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Reviews Dr. Laura De Lellis completed her BSc degree in Pharmaceutical Chemistry and Technology at the University of Chieti, Italy, in 2001. She obtained her PhD degree in Oncology and Molecular Pathology from the University of Chieti (2006), where she is currently an Assistant Professor in General Pathology in the Department of Pharmacy. She has broad experience in the genetics of cancer, and currently, her research interests focus on the biological evaluation of the antitumor potential of metabolic drugs.

Dr. Elisabetta Benedetti obtained her BSc degree in Biological Sciences in 2002 and her PhD in Cellular and Molecular Biology at the University of L’Aquila, Italy, in 2006. She is Assistant Professor at the Biotechnology faculty at the University of L’Aquila. Her research is based on the study of lipid metabolism on different models, such as glioblastoma and neuronal differentiation. Prof. Rosa Amoroso completed her BSc degree in Pharmaceutical Chemistry and Technology (1989) and her PhD in Organic Chemistry (1992) at University of Bologna, Italy. She is currently Associate Professor of Medicinal Chemistry in the Department of Pharmacy at the University of Chieti, Italy. Prof. Amoroso’s scientific interests are directed mainly toward medicinal chemistry, with research topics in cardiovascular agents and nitric oxide synthase inhibitors.

Prof. Annamaria Cimini completed her BSc degree in 1985 in Biological Sciences and is now a Full Professor at the University of L’Aquila, Italy. Moreover, she is Adjunct Full Professor at the Sbarro Institute for Cancer Research and Molecular Medicine and Center for Biotechnology at Temple University, USA. Her main scientific interests include studying the roles of peroxisome proliferator-activated receptors (PPARs) in neural cells and the development of potential therapy for neurodegenerative diseases.

Prof. Alessandro Cama received his MD degree (1981) and specialized in Endocrinology (1984) at the University of Rome “La Sapienza”, Italy. He trained in protein chemistry and molecular biology (1984–1991) at the Diabetes Branch of the National Institutes of Health, Bethesda, Maryland (USA) as a Fulbright Fellow, Fogarty International Visiting Fellow, and Fogarty International Visiting Associate. He is currently Full Professor of General Pathology at the Department of Pharmacy of the University of Chieti, Italy. He has a broad interest in genetics, metabolism, and cancer and has recently focused on evaluating the anticancer potential of novel compounds, metabolic and repurposed drugs.

Dr. Serena Veschi graduated in Biological Sciences at the University of L’Aquila, Italy (2000), and she holds a PhD in General Pathology (2008) and a specialization in Clinical Pathology (2010) at the University of Chieti. Her main research topics are related to cancer. Dr. Veschi is currently working in the Department of Pharmacy at the University of Chieti, and her research interests are focused on the potential antitumor activity of repurposed drugs and PPAR antagonists.

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Dr. Alessandra Ammazzalorso obtained her PhD in Pharmaceutical Sciences from the University of Chieti, Italy, in 2001. Since 2004 she has been an Assistant Professor of Medicinal Chemistry at the University of Chieti Department of Pharmacy. Her research interests include the design and synthesis of small-molecule drugs, mainly compounds active on PPARs.

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Reviews tion of cancer cells and interfere with the Warburg effect.[32, 33] Also, PPARg agonists may play an interesting role in chemoprevention. For example, overexpression of PPARg in breast cancer, found mainly in well-differentiated and ER-positive breast carcinomas, modulates estrogenic actions and has an inverse association with tumor size in humans.[34] Of note, recent studies have revealed that PPARg agonists exhibit antitumor activity and can induce apoptotic cell death in various malignant cell lineages, including malignant pleural mesothelioma, thyroid cancer, liposarcoma, breast adenocarcinoma, prostate carcinoma, colorectal carcinoma, non-small-cell lung carcinoma, pancreatic carcinoma, bladder cancer, and gastric carcinoma.[35–37] This potential antitumorigenic effect is contrasted by results in clinical studies, for which protumorigenic effects have been identified.[38] Notably, we cannot exclude that these antitumor effects may be associated with PPARg-independent mechanisms, as in the case of rosiglitazone, which has been shown to affect breast cancer-cell growth by inhibiting ACSL4.[39] Data related to PPARd and carcinogenesis are still controversial: the effects on differentiation and inflammation suggest anticancer potential for PPARd agonists, but antiapoptotic and growth stimulatory effects have also been reported.[40–44] In this regard, the PPARd antagonist GSK0660 only slightly decreases the viability of neuroblastoma cells if used alone.[45] On the other hand, Palkar et al. have shown that neither the PPARd agonist GW0742 nor the PPARd antagonist GSK3787 has any effect on cell proliferation in multiple human cancer cell lines, even if they are able to modulate in vitro and in vivo PPARd-dependent activity.[46] Paradoxically, despite this lack of activity on cell proliferation in multiple human cancer cell lines, in other studies the PPARd agonist GW0742 has been shown to be able to inhibit growth of in vitro and in vivo models of breast cancer, and it can inhibit chemically induced skin tumorigenesis in animal models.[47, 48] Further studies will be necessary to elucidate the basis of these conflicting results. PPARs also show relevant roles in angiogenesis, for which they influence the complex processes involved in the growth of tumors and the production of growth factors. PPARd ligands have been reported to increase or decrease key factors involved in angiogenesis, such as vascular endothelial growth factor (VEGF), so influencing the proliferation of endothelial cells.[49, 50] Similar effects have been reported for PPARg and PPARa agonists, described as both antiangiogenic and proangiogenic transcription factors.[51–53] To evaluate the anticancer properties of PPAR agonists, several clinical trials have been developed.[54] In this regard, the use of PPARg thiazolidinedione agonists, such as troglitazone and rosiglitazone, as antitumor agents has been evaluated in a wide panel of cancer types, including liposarcoma,[55, 56] prostatic,[57, 58] colorectal,[59, 60] and breast[61, 62] cancers. It should be noted that these trials show controversial data, with positive or negative impact on protection or progression of the different cancer types.[54] Moreover, some epidemiological studies indicate that PPARg agonists may be associated with an increased risk of bladder cancer,[63] although other studies report conflicting data on the possible association between bladder cancer and the assumption of these drugs.[64] On the other

In the absence of ligand, the heterodimer between the PPAR and the retinoid X receptor a (RXRa) is bound to chromatin, which produces basal repression of gene transcription. After binding an agonist, the dissociation of corepressors and the recruitment of coactivators takes place, thereby activating the transcriptional machinery (Figure 1 b).[17] Coactivators and corepressors are strongly involved in this process, and their different binding to the activated complex may favor and repress, respectively, target gene transcription.[20, 21] A key role in the activation process is played by a substructure of the LBD, helix 12 (H12), located in the C-terminus portion of PPARs. Specific studies have indicated that H12 folding is responsible for the global effect of a PPAR ligand. In particular, correct folding of H12 (holo-apo conformation) ensures receptor activation, whereas misfolding prevents biological effects, so resulting in antagonistic behavior.[22] This effect is clearly exemplified by the selective PPARa antagonist GW6471,[23] which induces repositioning of H12, and this supports the hypothesis of H12-mediated effects of PPAR ligands proposed by Hashimoto. As regards the mechanism of action, compounds able to modulate PPAR activity can be classified as agonists, antagonists, or inverse agonists. Whereas agonists allow the transcription of target genes, inducing structural modifications in the heterodimer PPAR:RXR, antagonists do not change receptor conformation and may or may not compete with other ligands.[17, 24] Inverse agonists produce a response similar to that of antagonists, and this leads to enhanced corepressor recruitment.[25] Weak agonists or inverse agonists are sometimes indicated as antagonists, because they produce receptor repression. Their actions may also depend on their concentrations in different tissues. From a therapeutic perspective, potent and subtype-selective PPAR synthetic agonists have been largely studied for their beneficial effects in metabolic disorders, such as atherosclerosis, type 2 diabetes, obesity, and metabolic syndrome.[26] Fibrates and thiazolidinediones, PPARa and PPARg agonists, respectively, represent important therapeutic options for treating dyslipidemias and diabetes.[27–30]

2. PPARs and Cancer Considering that metabolic syndrome, dyslipidemias, obesity, glucose intolerance, and chronic inflammation are associated with an increased risk of cancer, targeting PPARs could represent a new strategy for treating and preventing cancer.[17, 31] These receptors have a more direct role in malignancy, and they appear to modulate cancer-cell proliferation, differentiation, and survival.[17] However, given their multiple functions, PPARs have a pleiotropic role in cancer, and whether they function as tumor suppressors or inducers is context dependent. Indeed, such functions may relate to the cancer type and/or specific microenvironment of the tumor, supporting the potential of PPAR agonists or antagonists as antitumor molecules. As regards PPARa agonists, they represent molecules with potential in the prevention of different cancers, including colorectal cancer and leukemia, because they inhibit the proliferaChemMedChem 2018, 13, 209 – 219

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Reviews hand, although PPARa and PPARd agonists have been used in the clinic and in clinical trials for noncancerous diseases, their use as antitumor drugs has not yet been explored in clinical studies possibly as a result of the controversial roles of these receptor subtypes in tumor development, as reported in many preclinical evidences.[17] As far as PPAR antagonists, they have been studied in several noncancerous diseases.[65] In this review, we focus on the current findings about the antitumor effects displayed by different PPAR chemicals with antagonistic behavior in several preclinical cancer models, providing a rationale for the use of these molecules in the treatment of tumors expressing the corresponding PPARs.

A small number of PPARa synthetic antagonists have been identified to date, but little biological data has been reported for these compounds. Moreover, no selective PPARa antagonists have so far been tested in clinical trials. Figure 2 shows representative PPARa antagonists studied for their cytotoxic effects in cancer cells. MK886, originally discovered as a 5-lipoxygenase activating protein (FLAP) inhibitor, has also been described as a PPARa antagonist, and it is able to inhibit receptor activation at low micromolar concentrations. Operating through a noncompetitive mechanism, it prevents conformational changes that are responsible for receptor activation, as confirmed by binding curves obtained in the presence of arachidonic acid.[72] Different studies report that MK886 induces apoptosis and affects cancer-cell differentiation. For example, this compound potentiates TNFa-induced differentiation in human myeloid leukemia HL-60 cells, with induction of apoptosis and cell-cycle perturbation.[73] Further studies in leukemia cells demonstrate that MK886 inhibits HL-60 cell proliferation by affecting the activity of microsomal prostaglandin E synthase-1 (mPGES-1), a critical target for PGE2 production.[74] In addition, MK886 displays antitumor activity in cellular and animal models of chronic lymphocytic leukemia.[75] However, a recent study notes that MK886 is not selective for PPARa, as it inhibits a number of different nuclear receptors, and it has non-negligible cytotoxicity in normal cells, which hampers its therapeutic potential.[76] GW6471, discovered by GlaxoSmithKline, is a competitive PPARa antagonist at nanomolar concentrations. The ethylamide portion in the molecule is the structural motif involved in the antagonistic behavior: in fact, GW6471 adopts a U-shaped conformation, and the amide group prevents the formation of a H-bond with Tyr464, located in helix 3.[23] GW6471 is widely used as a pharmacological tool to block PPARa activation. Its antitumor effects have been studied in a kidney cancer cellular model, in which GW6471 has the ability to induce apoptosis and cell-cycle arrest at the G0/G1 phase, associated with a marked decrease in cyclin D1, CDK4, and c-Myc protein expression.[66] Notably, the specific silencing of PPARa by using a siRNA approach reduces the levels of different oncoproteins

3. PPAR Antagonists and Cancer Several studies propose antitumor potential for compounds able to antagonize PPARs. Reduced activation of PPARs has been related to beneficial effects on cell growth and survival in different cancer models, particularly in tumors overexpressing these receptors, which suggests new therapeutic options for PPAR antagonists. However, as for PPAR agonists, the anticancer effects reported for PPAR antagonists may be difficult to study, as they are strongly dependent from the specific tumor cell line or animal model used, the particular tumor microenvironment, the culture conditions employed, including relative serum levels, and the characteristics of the chemical compound tested. 3.1. PPARa antagonists Excessive PPARa expression has been found to lead to progression of tumor growth in several cancers, including renal cancer, hepatocellular carcinoma, breast cancer, and glioblastoma.[66–70] Notably, PPARa staining has been associated with worse overall survival in patients with liver metastases from colorectal cancer.[71] Thus, PPARa activation plays a critical role in tumor biology, and its inhibition by PPARa antagonists may be beneficial in tumors overexpressing this receptor.

Figure 2. Chemical structures of representative PPARa antagonists.

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Reviews (cyclin D1, CDK4, and c-Myc), induces apoptosis, and alters cell cycle consistently with the effects observed after GW6471 treatment, which thus supports the notion that the antitumor activities of GW6471 result from specific PPARa antagonism.[66] Indeed, although these alterations are certainly involved in the antitumor activity of PPARa antagonists, the exact mechanism underlying the observed effects remains unknown. Notably, the same research group has shown that treatment with GW6471 attenuates renal cell carcinoma (RCC) growth in a xenograft mouse model, with no adverse effects observed in the animals.[67] Recently, we have shown that in a unique model of head and neck paraganglioma (HNPGL), GW6471 reduces cell viability by interfering with the cell cycle and by inducing caspase-dependent apoptosis. In agreement with cell-cycle perturbation, GW6471 treatment of HNPGL cells is also associated with a sharp decrease in CDK4, cyclin D3, and cyclin B1 protein expression, along with an increase in the expression of the universal cell-cycle inhibitor p21. In addition, GW6471 treatment impacts HNPGL cell migration. The mechanisms affecting HNPGL cell viability involve repression of the PI3K/GSK3b/bcatenin pathway (Figure 3).[77] AA452 is a N-acylsulfonamide derivative able to revert PPARa activation promoted by the GW7647 agonist at low micromolar concentrations.[78] A time-resolved fluorescence resonance energy-transfer (TR-FRET) assay shows that AA452 is a competitive PPARa antagonist.[79] It has been tested in different tumor cell lines and has been shown to affect the viability of colorectal and pancreatic cancer cells.[80] In a recent study, this compound has been tested in human glioblastoma (GB) primary cells, inducing strong effects on cell viability, proliferation, and migration related to the mevalonate pathway. In addition, AA452 is able to increase the sensitivity of GB to gold standard radiation therapy, opening new possibilities to test this compound in preclinical studies.[81]

Very recent work reports the in vitro and in vivo pharmacology of NXT629, a selective competitive PPARa antagonist.[82] It inhibits agonist-mediated PPARa activation with an IC50 value of 78 nm, with great selectivity for the PPARa isoform over the PPARg and PPARd isoforms and also over a panel of other nuclear receptors. In other work, it has been shown that chronic lymphocytic leukemia (CLL) cells overexpress PPARa as compared to normal B cells, and that NXT629 can decrease CLL tumor burden in two different mouse models of CLL.[75, 76] In solid tumor settings, NXT629 inhibits experimental lung metastasis of syngeneic B16F10 melanoma cells that themselves overexpress PPARa and its target genes, probably by interfering in the adhesion and early proliferation of cancer cells; NXT629 also prevents the growth of subcutaneous B16F10 tumors if it was administrated preventatively. Interestingly, the chronic administration of the PPARa antagonist does not produce adverse side effects in animals.[76] Although NXT629 does not display a suitable pharmacokinetic profile for further clinical development, it represents a valuable tool that demonstrates in vivo proof of concept of PPARa antagonism.[82] 3.2. PPARg antagonists Several studies suggest that PPARg activation may enhance the growth of some tumors. For example, PPARg agonists increase polyp numbers in the APCmin model of familial adenomatous polyposis, although a specific PPARg-mediated effect of the thiazolidinediones used in this study has not been demonstrated.[83] Of note, one recent report notes a significant association between high levels of PPARg expression in pancreatic cancer cells and shorter overall survival.[84] In line with these observations, inhibition of PPARg might be beneficial in treating some neoplasms expressing this receptor, such as colon, breast, and hepatocellular carcinomas.[85–87] In Figure 4, the chemical structures of representative PPARg antagonists are shown. GW9662, a nitrobenzanilide discovered by GlaxoSmithKline, is a potent and irreversible PPARg antagonist that shows an IC50 value in the nanomolar range. Mass spectrometry analysis indicates the formation of a covalent interaction between GW9662 and Cys285 located in PPARg.[88] It has been tested for its anticancer potential in breast tumor cells with distinct phe-

Figure 3. Schematic representation of PPARa antagonist GW6471 antitumor activities. GW6471 affects cancer-cell viability and migration in tumors overexpressing PPARa by interfering with the expression or activity of key proteins involved in crucial biological processes (see text for details).[66, 67, 77]

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Figure 4. Chemical structures of representative PPARg antagonists.

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Reviews notypes and shows the inhibition of cell growth and survival.[89] Moreover, GW9662 treatment selectively decreases the cancer stem cell (CSC) population in ERBB2-positive human breast cancer cells by increasing intracellular reactive oxygen species (ROS) and inhibiting the lipogenesis pathway, a hallmark of cancer cells (Figure 5).[90] In fact, inhibition of PPARg reduces aldehyde dehydrogenase (ALDH)-positive cells, which identify the human breast cancer stem cell population, and suppresses the tumorsphere formation in ERBB2-positive cells; inhibition of PPARg also represses the expression of stem-cell related genes, such as KLF4 and ALDH. Furthermore, GW9662 treatment suppresses the expression of several lipogenic genes, including ACLY, FASN, and NR1D1. Also, GW9662 pretreatment effectively blocks tumor formation in a breast cancer cell derived animal model.[90] Its chemical analogue, T0070907, is a selective PPARg antagonist that is able to modify covalently a cysteine residue (Cys313) in helix 3 of human PPARg. In more detail, it blocks the recruitment of coactivators by affecting the conformation of helix 12 in the LBD of PPARg, which is responsible for receptor activation.[91] T0070907 has been tested in different tumor cell lines overexpressing PPARg. In two breast cancer cell lines (MB-231 and MCF-7), T0070907 treatment has been shown to decrease cell proliferation significantly, without affecting apoptosis.[92] Nakajima et al. have studied the effects of the inhibition of PPARg by T0070907 in pancreatic cancer cells Capan-1 and Panc-1: a strong decrease in cell motility is observed, associated with altered p120ctn localization and suppression of Rac1 and Cdc42 Rho GTPase activities, key regulators of cell motility and cell–cell or cell–extracellular matrix (ECM) adhesion (Figure 5).[93] Other studies support the role of T0070907 and the abovementioned GW9662 analogue in preventing tumor invasion and metastasis through inhibition of cell adhesion and induc-

tion of anoikis-mediated apoptosis in multiple epithelial cancer models. In particular, these PPARg antagonists have been shown to inhibit the focal adhesion kinase (FAK) pathway, which is functionally important in transducing intracellular messages associated with growth factor signaling and cell–extracellular matrix interactions, and to activate the mitogen-activated protein kinase (MAPK) cascade, including extracellular signal-regulated kinase (ERK). T0070907 and GW9662 also appear to decrease integrin a5 and CD151 expression, both implicated in cancer-cell migration and invasion (Figure 5).[87, 94, 95] Notably, the antitumor effects of the PPARg antagonist T0070907 appear to be PPARg mediated, as they are modified by knocking down the expression of the nuclear receptor by using a PPARg-specific siRNA.[87] The results of these investigations support PPARg inhibition as a potential therapeutic strategy for controlling cancer-cell dissemination and metastasis. The synthetic oleanane triterpenoid CDDO-Me (C-28 methyl ester of 2-cyano-3,12-dioxoolen-1,9-dien-28-oic acid) is considered a ligand for PPARg with antagonistic properties. Specifically, whereas CDDO is reported as a partial PPARg agonist, the C28 derivative shows an antagonistic profile, because of their different effects on the interaction of cofactors with PPARg. CDDO-Me is a weaker recruiter of the coactivator CBP, but it has no effect on the dissociation of corepressor NCoR, with a net effect of blocking PPARg activation.[96] It is worth noting that biological effects reported for CDDO-Me appear to be associated with multiple molecular targets.[97] As a multitarget and multifunctional drug, it has been exploited for the treatment of several diseases, including cancer.[97, 98] It has the potential to modulate distinct signaling pathways deregulated in malignant cells and shows inhibitory effects on cancer-cell growth and proliferation in a wide panel of in vitro and in vivo cancer models. However, it should be noted that growth sup-

Figure 5. Schematic representation of PPARg antagonists GW9662 and T0070907 antitumor activities. GW9662 and T0070907 affect cancer stem cell population, cell invasion, and cell migration in tumor cells by deregulating key molecules involved in crucial biological processes (see text for details).[89–95]

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Reviews pression by CDDO-Me appears to be largely independent of PPARg status.[97] In fact, modulation of both PPARg-dependent and PPARg-independent pathways in tumor cells has been described, and many biological responses have been reported for CDDO-Me. Specifically, CDDO-Me significantly induces the upregulation of the tumor suppressor gene caveolin-1 in colon cancer cells at concentrations that inhibit cell growth, and CDDO-Me-induced caveolin-1 expression is inhibited by the PPARg antagonist T0070907.[99] Conversely, higher doses of CDDO-Me Figure 6. Chemical structures of representative PPARd antagonists (SR13904) and inverse induce apoptosis and PARP cleavage, but these ef- agonists (ST247 and DG172). fects ae not inhibited by T0070907, which suggests the involvement of a PPARg-independent mechanism.[99] Moreover, Tabe et al. report that CDDO-Me enhances PPARd antagonists and inverse agonists displaying antitumor all-trans retinoic acid (ATRA)-induced differentiation and apopproperties have been identified to date (Figure 6). tosis in both ATRA-sensitive and ATRA-resistant acute promyeSR13904 is a competitive potent PPARd antagonist, also exlocytic leukemia (APL) cell lines. These effects are partially deerting weaker antagonistic activity on PPARg. This molecule pendent on PPARg, as inhibition of PPARg either by the specific has been shown to repress both PPARd agonist-induced transinhibitor T0070907 or by siRNA reduces CDDO-induced APL activation and cancer-cell proliferation, at least in part through differentiation.[100] In the same study, CDDO-Me markedly enthe inhibition of PPARd signaling.[107] SR13904 shows antitumor hances ATRA-induced maturation and extends the survival of a effects in several carcinoma cell lines, including lung, breast, mouse model of APL, providing a rationale for the combined and liver, with IC50 values ranging from 8 to > 30 mm dependtargeting of RAR and PPARg nuclear receptors in the therapy ing on the cancer cell line tested. In particular, this compound of APL. increases apoptosis and inhibits G1/S cell-cycle progression in In a yeast two-hybrid model, HL005, a 3-thiazolinone-modithe A549 lung cancer cell line. Consistently, SR13904 treatment fied benzoic acid derivative is reported to antagonize rosiglitaof A549 cells is associated with a marked decrease in the exzone-induced PPARg activation strongly by affecting coactivapression of some key cell cycle regulatory proteins, such as cytor recruitment. This PPARg antagonist inhibits proliferation of clins A and D and cyclin-dependent kinases CDK2 and CDK4 the MCF-7 breast cancer cell line in a dose-dependent fash(Figure 7). ion.[101] Specifically, HL005 induces apoptosis and inhibits the Recent studies report on the antitumor effects by PPARd incell growth of MCF-7 by arresting the cell cycle at the G2/M verse agonists ST247 and DG172. ST247 is a PPARd inverse agphase. onist that is able to enhance the interactions of the LBD of Overall, it is worth noting that the different PPARg antagoPPARd with corepressors. The long alkyl chain in the structure nists reviewed above show distinct antitumor effects. These of ST247 is probably the structural motif responsible for addidifferences might be related to the fact that some molecules tional hydrophobic interactions, leading to the stabilization of act through PPARg-dependent mechanisms, but they also PPARd with corepressors.[25] DG172 has been identified as a show PPARg-independent activities. The relative weight of the PPARg-dependent and PPARg-independent antitumor effects should be further investigated. 3.3. PPARd antagonists A number of studies report the activation of PPARd in several in vitro and in vivo tumor models, including colon, breast, prostatic, and hepatocellular carcinomas.[102–104] Remarkably, PPARd expression is associated with oncogenic processes in the APCmin + /@ mouse model for colorectal cancer, and PPARd knockout reduces the number and size of intestinal polyps.[105] More recently, a mouse model of intestinally targeted PPARd overexpression has been developed to simulate PPARd upregulation in human colon carcinogenesis.[106] In the animal model, PPARd overexpression modulates the gene expression profile of colonic epithelial cells in a pattern that activates pathways strongly associated with tumorigenesis.[106] This evidence suggests that the use of PPARd antagonists may be effective in tumors overexpressing the receptor, but only a few ChemMedChem 2018, 13, 209 – 219

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Figure 7. Schematic representation of PPARd antagonist SR13904 antitumor activities. SR13904 affects cancer-cell viability in tumors expressing PPARd by interfering with key proteins involved in crucial biological processes.[107]

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Reviews potent PPARd ligand, with high binding affinity and inverse agonistic properties. It represses agonist-induced PPARd activation and enhances corepressor recruitment.[108] These compounds are both able to inhibit cancer-cell invasion in a PPARd-mediated fashion by downregulating the ANGPTL4 PPARd target gene and other oncogenic signals in MDA-MB231 breast cancer cells.[109]

vasion and metastasis, and blocks tumor formation in several cellular and animal models.[90, 93–95] PPARd antagonists repress PPARd-mediated signaling related to cancer-cell proliferation and survival through modulation of crucial biological pathways,[107, 109] but the role of PPARd in cancer needs to be further elucidated owing to conflicting results obtained with agonists and antagonists.[40–43, 46–48, 113] Therefore, the use of PPAR antagonists as anticancer agents appears promising in preclinical studies. In this perspective, PPAR antagonists could have remarkable therapeutic value in the treatment of tumors expressing the corresponding receptors. This potential should be further investigated in preclinical models and eventually in clinical trials for the most promising molecules.

4. Summary and Perspectives Peroxisome proliferator-activated receptor (PPAR) antagonists have been shown to have potential anticancer effects. A better understanding of the anticancer mechanisms of these molecules may allow their rational use for the treatment of cancers or cancer-related diseases. Notably, some chemicals show both PPAR-dependent and PPAR-independent activities. In principle, it is possible that antitumor effects reported for some PPAR antagonists could be due to the effects of these molecules on different targets, especially for those that also show PPAR-independent activities. Further studies, for instance by including additional controls that demonstrate specificity for each PPAR by silencing the corresponding PPAR isoform, could help to dissect the relative weight of PPAR-dependent and PPAR-independent antitumor effects. In this regard, it should be considered that knockdown strategies frequently give misleading results, as partial depletion of transcription factors may not lead to their diminished binding to chromatin. Thus, knockout strategies, which are now becoming more widespread with the use of CRISPR-based methods, should be favored. Indeed, even if there is evidence that knocking out a PPAR isoform would modulate the activity of molecules involved in crucial biological processes, the specific mechanism linking the silencing of PPAR to the expression of critical oncoproteins and underlying the observed effects needs to be further investigated. Moreover, we cannot rule out the possibility that molecules that antagonize a nuclear receptor can also display agonist activities depending on the context of the cell or tissue, as it has been shown for selective estrogen receptor modulators (SERMs) that act as agonists or antagonists in a tissue-selective manner.[110] Although PPAR agonists are being tested in cancer clinical trials, there is a relative lack of clinical studies with PPAR antagonists.[54] In principle, given the central roles of PPARs in multiple essential biological functions, it cannot be excluded that interfering with the functions mediated by PPARs with an antagonist might cause negative effects. However, upon testing in animal models, these compounds appear to be well tolerated, without significant changes in weight and/or blood chemistry analysis, including organ function and glucose tolerance tests between controls and PPAR antagonist-treated animals.[46, 67, 76, 111] At present, only the CDDO-Me (C-28 methyl ester of 2-cyano-3,12-dioxoolen-1,9-dien-28-oic acid) PPARg antagonist is being tested in clinical trials against solid tumors and lymphoid malignancies on the basis of potent antitumor activity displayed in both cell and animal models.[97, 99, 112] In preclinical studies, antagonizing PPARa is associated with cancer-cell death and a decrease in tumor size.[66, 67, 70, 77] Inhibition of PPARg decreases cancer-cell growth, prevents tumor inChemMedChem 2018, 13, 209 – 219

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Abbreviations Peroxisome proliferator-activated receptors (PPARs), 15-hydroxyeicosatetraenoic acid (15-HETE), chromatin immunoprecipitation sequencing (ChIP-seq), DNA binding domain (DBD), ligand binding domain (LBD), retinoid X receptor (RXR), PPAR response elements (PPREs), helix 12 (H12), acyl-CoA synthetase long-chain family member 4 (ACSL4), vascular endothelial growth factor (VEGF), 5-lipoxygenase activating protein (FLAP), tumor necrosis factor alpha (TNFa) microsomal prostaglandin E synthase-1 (mPGES-1), cyclin-dependent kinase 4 (CDK4), renal cell carcinoma (RCC), head and neck paraganglioma (HNPGL), phosphoinositide 3-kinase (PI3K), glycogen synthase kinase 3 beta (GSK3b), time-resolved fluorescence resonance energy transfer (TR-FRET), glioblastoma (GB), chronic lymphocytic leukemia (CLL), adenomatous polyposis coli (APC), cancer stem cell (CSC), reactive oxygen species (ROS), aldehyde dehydrogenase (ALDH), Kruppel-like factor 4 (KLF4), ATP-citrate lyase (ACLY), fatty acid synthase (FASN), nuclear receptor subfamily 1 group D member 1 (NR1D1), Ras-related C3 botulinum toxin substrate 1 (Rac1), cell division control protein 42 homologue (Cdc42), extracellular matrix (ECM), focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK), extracellular signalregulated kinase (ERK), Creb binding protein (CBP), nuclear receptor co-repressor (NCoR), poly ADP (adenosine diphosphate)-ribose polymerase (PARP), all-trans retinoic acid (ATRA), acute promyelocytic leukemia (APL), short interfering RNA (siRNA), retinoic acid receptor (RAR), cyclin-dependent kinase 2 (CDK2), angiopoietin-like 4 (ANGPTL4), selective estrogen receptor modulators (SERMs).

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Manuscript received: November 11, 2017 Revised manuscript received: December 17, 2017 Accepted manuscript online: December 25, 2017 Version of record online: January 10, 2018

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