Lack of PPARβ/δ-Inactivated SGK-1 Is Implicated in Liver Carcinogenesis

Objective The present study examined the role of PPARβ/δ in hepatocellular carcinoma (HCC). Methods The effect of PPARβ/δ on HCC development was analyzed using PPARβ/δ-overexpressed liver cancer cells and PPARβ/δ-knockout mouse models. Results PPARβ/δ(-/-) mice were susceptible to diethylnitrosamine- (DEN-) induced HCC (87.5% vs. 37.5%, p < 0.05). In addition, PPARβ/δ-overexpressed HepG2 cells had reduced proliferation, migration, and invasion capabilities accompanied by increased apoptosis and cell cycle arrest at the G0/G1 phase. Moreover, differential gene expression profiling uncovered that the levels of serine/threonine-protein kinase (SGK-1) mRNA and its encoded protein were reduced in PPARβ/δ-overexpressed HepG2 cells. Consistently, elevated SGK-1 levels were found in PPARβ/δ(-/-) mouse livers as well as PPARβ/δ-knockdown human SMMC-7721 HCC cells. Chromatin immunoprecipitation (ChIP) assays followed by real-time quantitative polymerase chain reaction (qPCR) assays further revealed the binding of PPARβ/δ to the SGK-1 regulatory region in HepG2 cells. Conclusions Due to the known tumor-promoting effect of SGK1, the present data suggest that PPARβ/δ-deactivated SGK1 is a novel pathway for inhibiting liver carcinogenesis.

Hepatocellular carcinoma (HCC) is one of the deadliest forms of cancer, and very limited data are available on the role of PPARβ/δ in HCC development. Studies have indicated that PPARβ/δ is a feasible target for chemoprevention in the last 10 years [14], although the functional outcomes of PPARβ/δ activation in some cancers are contradictory [15,16]. However, the Human Protein Atlas database indicates that PPARβ/δ is undetectable in 80% of HCCs [14]. Nevertheless, it has been shown that PPARβ/δ activation promotes the proliferation and growth of human hepatic cancer cell lines through the upregulation of cyclooxygenase-2 (COX-2) and prostaglandin E 2 production [17]. In contrast, another study has demonstrated that the COX-2 expression was not affected when human HCC cell lines were treated with PPARβ/δ ligands [18]. Therefore, the role of PPARβ/δ in hepatocarcinogenesis warrants further investigation. The aim of this study was to investigate the functional significance of PPARβ/δ in liver cancer cells and mouse models. Our data revealed the anti-HCC effect of PPARβ/δ and that PPARβ/δ-regulated serine/threonine-protein kinase (SGK-1) is implicated in the anti-HCC effect. In summary, PPARβ/δdeactivated SGK-1 is a novel pathway for inhibiting tumor growth and linking metabolism and liver carcinogenesis together.

Experimental Animals and Study
Design. PPARβ/δ-null mice in the C57BL/6 background were provided by Dr. Frank J. Gonzalez at the National Cancer Institute, National Institutes of Health, Bethesda, MD [19]. Genotyping was confirmed using the polymerase chain reaction (PCR), and animals were housed under controlled temperature (21 ± 1°C) conditions with a 12 h light-dark cycle and were allowed free access to food and water. Wild-type or PPARβ/δ-null mice (male, 15 days old; 8 per group) were given a single intraperitoneal injection of diethylnitrosamine (DEN) (5 mg/kg body weight; Sigma Chemical Co., St. Louis, MO) [19]. The mice were anesthetized by chloroform and were sacrificed without fasting at the indicated time points. Blood was collected by cardiac puncture, and the livers were excised and weighed. The presence and dimensions of the surface nodules were evaluated and recorded. Each liver was cut into strips of 2-3 mm in thickness to examine the presence of macroscopically visible lesions. HCC was diagnosed by an experienced pathologist based on gross or histological examination. All of the animal experiments were conducted in accordance with the guidelines provided by the Animal Experimentation Ethics Committee of Guangzhou Medical University. 2.4. RNA Interference and Transfection. The SMMC-7721-NC and SMMC-7721-shPPARD cells were generated using lentiviral transduction of LV008-shPPARβ/δ (shPPARD) or control LV008 vectors (NC) (Forevergen. China) into SMMC-7721 cells, respectively, followed by selection of stable cell lines in puromycin (2 μg/mL). The sequence of shPPARD was 5 ′ -AACT CAGTGATATCATTGAGCCTAATTCAA GAGATTAGGCTCAATGATATCACGTTTTTTC-3 ′ .

Colony Formation Assay.
HepG2 cells were transfected with GV230-PPARβ/δ or an empty vector to the preseeded cells in 6-well plates at a density of 50, 100, or 200 cells per well. After 14 days of stationary culture, the cells were fixed with 70% ethanol and stained with crystal violet (Sigma, St. Louis, MO). Colonies with more than 50 cells/colony were counted under a microscope to calculate the rate of colony formation. All of the data were obtained from three independent experiments.

Migration and Invasion
Assays. The wound-healing assay was performed in vitro for cell migration analysis. Briefly, HepG2_PPARβ/δ and HepG2_mock cells (5 × 10 5 cells/well) were cultured in 6-well plates until they reached 90% confluency [20]. Sterile tips were used to scratch the cell layers. Images of the wound closure areas were taken at 0, 24, and 48 h. Matrigel migration and invasion assays were performed on HepG2_PPARβ/δ and HepG2_mock stably transfected liver cancer cells using 24-well Matrigel-biocoated migration and invasion chambers (Becton Dickinson, Waltham, MA), as previously described [21].
2.12. Microarray Analysis. The gene expression profiles of PPARβ/δ-overexpressed and empty vector-treated cells were obtained by oligonucleotide microarray analysis using an Illumina kit, according to the manufacturer's instructions. Data were collected using the Illumina Genome Studio software. Functional annotation was carried out using gene lists submitted to a variety of online software tools, including the Database for Annotation, Visualization and Integrated Discovery (DAVID) [22] and Gene Set Enrichment Analysis (GSEA) [23].
2.13. Chromatin Immunoprecipitation (ChIP) Assay. ChIP assays were performed on HepG2 cells transfected with pEGFP-PPARβ/δ or pEGFP vectors (used as a control) using an EZ-Magna ChIP A kit (Millipore, Billerica, MA). The cells were cross-linked with 1% formaldehyde (Sigma-Aldrich) for 10 min and quenched by glycine. The cross-linked cells were collected in cold phosphate-buffered saline and sonicated to reduce the total DNA size to 200-1000 bp. The chromatin DNA fragments were precipitated overnight with 10 μg of PPARβ/δ antibody (Santa Cruz Biotechnology) or normal rabbit IgG at 4°C. The magnetic bead-antibody-chromatin complexes were washed, eluted, and incubated at 62°C for 2 h. The immunoprecipitated and input DNA was subjected to qPCR analysis using primers. The sequences of the SGK-1 promoter 1 were F 5 ′ -CAAATAGAGGTTCAAGGGAT-3′ and R 5′-TTAGGAGGCTTAGGTGGA-3′.

PPARβ/δ Deficiency Accelerates Hepatocarcinogenesis.
The mice developed HCC induced by DEN at 8 months. DEN induced HCC in 37.5% (3/8) of the wild-type mice, while the prevalence of HCC was much higher in the PPARβ/δ (-/-) mice (87.5%, 7/8, p < 0:05). Moreover, the average number of tumors per animal was 2.8-fold higher in the PPARβ/δ (-/-) mice compared with the wild-type mice (p < 0:05). Thus, PPARβ/δ deficiency increased the susceptibility of mice to DEN-induced hepatocarcinogenesis. No marked differences in the macroscopic or histological features of the HCCs were observed between the wild-type and PPARβ/δ-deficient mice, as evaluated by a pathologist (Figure 1).

Overexpression of PPARβ/δ Reduces Cell Proliferation and Induces Cell Cycle Arrest As Well As Apoptosis in HepG2
Cells. An elevated PPARβ/δ protein level was observed in human HCC SMMC7721 cells, while HepG2 and MHCC97H cells did not express PPARβ/δ protein (Figure 2(a)). Therefore, HepG2 cells were used for PPARβ/δ overexpression, and overexpression was confirmed by qRT-PCR and western blotting in HepG2 cells transfected with pEGFP-PPARβ/δ (Figures 2(b) and 2(c)).
The effect of PPARβ/δ overexpression on the cell viability of HepG2 cells was analyzed by the CCK-8 assay. The enhanced PPARβ/δ expression suppressed the cell viability in a time-dependent fashion (Figure 2(d)). The suppressive effect on cancer cell growth was further confirmed by the colony formation assay in stably transfected cells. The colony numbers of pEGFP-PPARβ/δ-transfected cells were reduced to 38% of that of the control cells (p < 0:01; Figure 2(e)). To further characterize the influence of PPARβ/δ on cell growth, flow cytometry was used to analyze the cell cycle distribution in HepG2 cells transfected with pEGFP-PPARβ/δ or control pEGFP vectors. We found that the overexpression of PPARβ/δ in HepG2 cells resulted in significant inhibition of cell cycle progression and the accumulation of G0-G1 phase cells (61:7 ± 1:72% vs. 49:1 ± 3:2%, p < 0:05 Figure 2(f)). Cell apoptosis was determined by annexin V-FITC/propidium iodide fluorescence-activated cell sorting (FACS) analysis. The results showed an increase in the number of early apoptotic cells (25:67 ± 0:531% vs. 13:71 ± 0:364%, p < 0:05) in HepG2 cells transfected with pEGFP-PPARβ/δ, as compared to the vector-transfected cells (Figure 2(g)).

Overexpression of PPARβ/δ Suppresses HepG2
Migration and Invasion. Wound-healing assays were conducted to evaluate migration in PPARβ/δ-overexpressed HepG2 cells. As shown in Figure 3

PPARβ/δ Modulates the Expression Profiles of Cancer-Related Genes in HepG2
Cells. To elucidate the molecular mechanisms underlying the inhibitory effect of PPARβ/δ on HCC growth, the gene expression profiles in pEGFP-PPARβ/δ-transfected HepG2 cells were analyzed using whole-genome expression arrays from Illumina (humanHT-12_v4 beadchips). Principal component analysis utilizing the entire gene expression dataset showed the relatively tight clustering of the two groups and the clear separation of the experimental group from the control group. Compared with mock transfection, 222 upregulated and 382 downregulated genes were found in HepG2_PPARβ/δ cells. GSEA of the PPARβ/δ target genes revealed a significant drop in the average expression of genes related to metastasis and cell migration, cell adhesion, proliferation, angiogenesis, epithelial-tomesenchymal transition, nuclear factor-κB, and transforming growth factor β signaling pathways, while upregulation in the average gene expression of cell cycle regulators (Figure 4(f)).

PPARβ/δ Transcriptionally Downregulates SGK-1
Expression. Expression array analysis indicated a 7.79-fold decrease in the abundance of SGK-1 expression in PPARβ/δ-overexpressed HepG2 cells. SGK-1 was one of the most downregulated genes. The downregulation of the SGK-1 expression by PPARβ/δ was confirmed by western blot (Figure 4(a)). The mRNA level of SGK-1 was noticeably  BioMed Research International increased when the PPARβ/δ activity was suppressed in SMMC-7721 cells infected with LV008-shPPARD (Figures 4(b) and 4(c)). A higher expression of SGK-1 protein was also detected in the livers of the PPARβ/δ (-/-) mice compared to that of the wild-type mice by immunohistochemistry (Figure 4(d)). These results indicated that PPARβ/δ might play a catalytic role through binding to the SGK-1 gene promoter. ChIP assays were performed on pEGFP-PPARβ/δ-or control vector-transfected HepG2 cells. Primarily, the transcription factor binding sites in the SGK-1 regulatory regions were evaluated using the JASPAR database (http://jaspar.genereg.net/cgi-bin/jaspar_db.pl), and the PPARβ/δ recognition site (CCAGGCTAAAGTGC A) was found in the 5′-regulatory region of the SGK-1 gene, which points to the role of the transcription factor PPARβ/δ in the expression of SGK-1. The immunoprecipitation was performed using an anti-PPARβ/δ antibody in chromatin DNA fragments, and a 163 bp fragment of the SGK-1 sequence was amplified from the immunoprecipitated DNA, indicating the direct binding of PPARβ/δ to SGK-1 (Figure 4(e)).

Discussion
Over the past decade, many studies have revealed the health benefits of PPARβ/δ in combating inflammation, 8 BioMed Research International lipogenesis, and insulin resistance. Activation of PPARβ/δ has been shown to have anticarcinogenic effects in skin cancer [24], pancreatic cancer [19], and prostate cancer [18], albeit not without controversy [15]. The role of PPARβ/δ in liver tumorigenesis has been established as well. Using a DEN-induced murine model of HCC, we demonstrated that a lack of PPARβ/δ increased the susceptibility to HCC formation. Our results were consistent with other studies using PPARβ/δ-knockout mice that showed an increased incidence of skin cancer [21], larger intestinal tumors [25], and chemically induced liver toxicity [23]. In addition, it has been reported that PPARβ/δ has an antiproliferative influence on prostate cancer cells, keratinocytes, and melanoma cells [24,26,27]. In order to investigate the effect of endogenous transactivation of PPARβ/δ in liver carcinogenesis, we examined its functional consequences by overexpressing PPARβ/δ in human HepG2 liver cancer cells. We found that the overexpression of PPARβ/δ resulted in inhibition of HepG2 cell proliferation in a time-dependent manner. The subsequent Hoechst staining and flow cytometry assays revealed that PPARβ/δ could induce apoptotic cell death and cell cycle arrest. Consistently, Coleman et al. have demonstrated that PPARβ/δ activation prevents the invasion and migration abilities of pancreatic cancer cells by activating the B cell lymphoma 6 pathway [19,28]. Moreover, the current study revealed that overexpression of PPARβ/δ inhibited the liver cancer cell migration and invasion abilities. It is well established that PPARβ/δ plays an important role in lipid and glucose metabolism and that it could be a potential molecule that links metabolism and carcinogenesis.
The current study demonstrated by microarray analysis that SGK1, a member of the protein kinase A, G, and C families, is downregulated by PPARβ/δ. The immunohistochemistry results also supported this observation as the SGK-1 level was higher in PPARβ/δ -/mice. Previous data have shown that PPARγ agonists induce the SGK-1 gene expression by direct binding [29]. The current study is the first to show that PPARβ/δ also regulates the SGK-1 gene expression but in a negative way. SGK-1 transcription is stimulated by excessive glucose levels and diabetes, oxidative stress, DNA damage, ischemia, neuronal injury, and a high-fat diet [30][31][32][33]. In addition, active SGK-1 induces insulin release, adipocyte differentiation, and adipogenesis [31,34]. The Human Protein Atlas database also shows elevated SGK-1 levels in liver cancer, colon cancer, myeloma, medulloblastoma, prostate cancer, ovarian tumors, and non-small-cell lung cancer [35]. Moreover, SGK-1-knockout mice are resistant to chemically induced colon carcinogenesis [31]. Recent findings also have shown that SGK-1 regulates cell survival, proliferation, and differentiation in several types of cancer cells such as kidney [31], breast [36], and liver cancer [37]. Additionally, SGK-1 may promote the survival of cholangiocarcinoma cells by mediating the IL-6-related pathway [38]. Furthermore, angiotensin II protects fibrosarcoma-derived cells from apoptosis by increasing SGK-1 phosphorylation [39]. Meanwhile, activated PPARβ/δ prevents IL-6-induced insulin resistance by inhibiting the signal transducer and activator of transcription 3 pathway in adipocytes, which was enhanced in PPARβ/δ-null mice [10]. Another study has suggested that PPARβ/δ protects against lipid accumulation  9 BioMed Research International and oxidative stress by reducing angiotensin II-induced activation of the Wnt signaling pathway [40]. Thus, through different signaling pathways, PPARβ/δ is implicated in metabolism and growth.

Conclusions
In conclusion, our data suggest that PPARβ/δ is a tumor suppressor in HCC and that downregulation of SGK-1 may be implicated in its tumor-suppressive effect.

Data Availability
The datasets generated and analyzed during the present study are available from the corresponding author on reasonable request.

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