HBXIP activates the PPARδ/NF-κB feedback loop resulting in cell proliferation

Hepatitis B X-interacting protein (HBXIP, also termed as LAMTOR5) plays a crucial role in regulation of cancer progression, while the mechanism is still unclear. Here we found that HBXIP increased the expression of PPARδ (peroxisome proliferator-activated receptor-δ) in gene and protein levels of SW480 or HT-29 colonic cancer cells. Chromatin immunoprecipitation and luciferase reporter assays showed that HBXIP occupied the core promoter (−1079/−239 nt) regions of PPARδ and that HBXIP activated the transcription activity of PPARδ in an NF-κB (p65)-dependent manner. Moreover, Co-immunoprecipitation and immunofluorescence analysis showed that HBXIP bound to NF-κB/p65 in the cells. Interestingly, we found that PPARδ could conversely increase the expression of NF-κB/p65 through activating its transcription activity. In addition, the clinical observations showed that both HBXIP and PPARδ were highly expressed in colonic carcinoma, and HBXIP expression was positively associated with that of PPARδ in the clinical specimen. Importantly, HBXIP expression levels were positively correlated with the clinical pathological parameters including lymph node metastasis and advanced TNM stage. These findings suggest that HBXIP served as a co-activator to activate the positive feedback regulations of NF-κB/PPARδ, which promoted the fast proliferation of the colonic cancer cells. Therapeutically, HBXIP may serve as a potential drug target of colonic cancer cells.


INTRODUCTION
Colonic cancer is the third death-related cancer in the worldwide, accounting for more than 1,300,000 new cases annually and its incidence has sharply increased over the past two decades [1]. Hepatitis B X-interacting protein (HBXIP), a new oncoprotein also known as LAMTOR5 [2], is a conserved ～18 KDa protein originally identified by its interaction with the hepatitis B virus X protein [3]. Previous studies have reported that HBXIP functions as a coactivator of multiple oncogenic transcription factors, such as TF-IID, SP1, STAT3 on the promotion of proliferation and metastasis of breast cancer cells [4][5][6]. However, the mechanism by which HBXIP enhances the growth of colonic cancer remains poorly documented.
Peroxisome proliferator-activated receptors (PPARs) are a nuclear receptor family of ligand-inducible transcription factors, which have three isoforms: PPARα, δ and γ, which are expressed in all cell types of the brain [7,8]. Many researchers have investigated the neuroprotective properties of PPAR agonists [9][10][11]. The efficiency of PPARδ agonists in neurodegenerative disease animal models have been reviewed [12,13]. To date, studies demonstrate that PPARδ also play crucial role in the regulation of cell growth and metabolism of glucose and lipids [14][15][16]. PPARδ is highly expressed in colonic cells and implicated in colonic tumorigenesis, and its Research Paper expression is elevated in human colorectal carcinoma specimen [17]. PPARδ also enhances the expression of VEGF, an angiogenic factor, in colonic carcinoma cells [18]. Moreover, the activation of PPARδ is associated with oncogenic pathways such as the K-Ras and APC/β-catenin/Tcf pathway [19]. PPARδ deficiency disrupts hypoxia-mediated tumorigenic potential of colonic cancer cells [20]. Additionally, PPARδ is required for chronic colonic inflammation and colitisassociated carcinogenesis [21].
In the present study, we investigated the mechanism by which HBXIP promoted the proliferation of colonic cancer cells. Our data indicate that the oncoprotein HBXIP, functioned as a co-activator of NF-κB (p65) or PPARδ, transactivated the promoter activity of PPARδ or NF-κB, and enhanced the expression of PPARδ and NF-κB in mRNA and protein levels in a positive feedback loop manner, leading to the fast growth of colonic cancer cells. These findings contribute new insight into the mechanism by which HBXIP enhances the proliferation of colonic cancer cells, and also provide a potential target for cancer treatment.

HBXIP up-regulates PPARδ expression in colonic cancer cells
Studies had demonstrated that HBXIP (LAMTOR5) expression was up-regulated in many types of cancers [4,22,23]. And PPARδ is highly expressed in colonic cells and implicated in colonic tumorigenesis [17]. Then we wondered whether the expression of HBXIP was associated with that of PPARδ in the colonic cancer cells. Intriguingly, we observed that the mRNA and protein levels of PPARδ were significantly elevated by transiently transfecting HBXIP expression plasmids (pCMV-HBXIP) in colonic cancer SW480 and HT-29 cells ( Figure 1A, 1B and Supplementary Figure 1B). Conversely, the expression of PPARδ was remarkably reduced when HBXIP was knockdown by transiently transfecting HBXIP siRNA (si-HBXIP) in colonic cancer SW480 or HT-29 cells respectively ( Figure 1C and 1D). Therefore, we conclude that the oncoprotein HBXIP can up-regulate the expression of PPARδ in colonic cancer cells.

HBXIP activates PPARδ promoter in an NF-κBdependent manner
We next explored the -1079/-239 nt promoter regions for a possible transcription factor binding sites by using GPMiner (http://gpminer.mbc.nctu.edu.tw/). The promoter region −1079/−239 contains various different promoter elements, such as Hsf1 and NF-κB/p65. It has been reported that NF-κB/p65 signaling was involved in carcinogenesis [24]. Thus, we hypothesized that HBXIP might activate the PPARδ promoter via NF-κB/p65. Interestingly, luciferase reporter gene assays indicated that PPARδ promoter activity was remarkably increased in a dose-dependent manner in SW480 cells treated with p65 expression plasmid (pCMV-p65) ( Figure 3A), suggesting that NF-κB can activate the transcription activity of PPARδ. To further validate whether NF-κB is an important transcription factor of PPARδ promoter, we reconstructed the pGL3-841 mutant, which deleted the NF-κB binding sequence (PPARδ promoter mutant), and performed the luciferase reporter gene assays. Data showed that the activity of pGL3-841 mutant sharply decreased compared to that of the normal promoter. Moreover, HBXIP failed to activate the pGL3-841 mutant ( Figure 3B), indicating that HBXIP activates the PPARδ promoter activity in an NF-κB-dependent manner. To further verify that HBXIP activates PPARδ transcription activity via NF-κB, the quantitative real-time PCR was performed and the results showed that the mRNA levels of PPARδ of SW480 cells co-transfected with pCMV-p65/ pCMV-HBXIP markedly increased compare to that of the cells transfected with pCMV-p65. The mRNA levels of PPARδ of cells co-transfected with pCMV-p65/ si-HBXIP decreased compared to that of cells transfected with pCMV-p65 ( Figure 3C and Supplementary Figure 2A), indicating that HBXIP up-regulates PPARδ expression via NF-κB. Moreover, the mRNA levels of PPARδ of SW480 cells transfected with si-p65 decreased relative to that of cells transfected with si-Control, and HBXIP failed to enhance the PPARδ mRNA levels in the cells treated with transfecting si-p65 ( Figure 3D), indicating that HBXIP up-regulated PPARδ expression in a NF-κBdependent manner. We then detected the protein levels of PPARδ by Western blotting. The results demonstrated that over-expression of HBXIP increased PPARδ expressions in SW480 or HT-29 cells. However, the knockdown of p65 abolished the increase of PPARδ proteins-induced by HBXIP ( Figure 3E, 3F and Supplementary Figure 2B), indicating that HBXIP boosts the expression of PPARδ in a NF-κB-dependent manner in cancer cells.
To further elucidate the relationship between HBXIP and NF-κB, co-immunoprecipitation (Co-IP) assays were performed and the results displayed that HBXIP and NF-κB could bind to each other in HT-29 cells ( Figure 4A). Moreover, immunofluorescence (IF) images showed that the proteins of HBXIP and p65 were mainly located in the cytoplasm and a small amount of HBXIP and p65 proteins co-localized in the nucleus ( Figure 4B), the similar results were also confirmed by confocal laser scanning microscope ( Figure 4C     HBXIP could elevate the expression of NF-κB/p65 both in the nucleus and cytoplasm (Supplementary Figure 2E). Taken together, we conclude that HBXIP, as a c o-activator of NF-κB, promotes the transcription activity of PPARδ, and enhances its expression in mRNA and protein levels.

PPARδ regulates the expression of NF-κB in colonic cancer cells
Next, we wondered whether PPARδ could enhance the expression of NF-κB in colonic cancer cells. Interestingly, we observed that PPARδ expression could positively regulate the expression of NF-κB in the cells. Western blot assays were performed in SW480 and HT-29 colonic cancer cells and HEK293T cells. The results indicated that the expression of NF-κB was decreased in SW480 and HT-29 cells transfected with PPARδ siRNA (si-PPARδ) ( Figure 5A). The similar results were also confirmed in the HEK293T cells (Supplementary Figure 2D), suggesting that PPARδ could up-regulate the expression of NF-κB in the colonic cancer cells. Then, quantitative real-time PCR (qRT-PCR) were performed and the data showed that the mRNA levels of NF-κB could be remarkably decreased in the cells transfected with si-PPARδ ( Figure 5B and Supplementary Figure 2C), suggesting that PPARδ up-regulated the transcription activity of NF-κB. We detected the promoter activity of NF-κB by luciferase reporter gene assays, data showed that the promoter activity of NF-κB could be decreased in the cells treated with si-PPARδ ( Figure  5C and Supplementary Figure 4C), suggesting that PPARδ could activate the transcription activity of NF-κB. Taken together, we conclude that PPARδ up-regulated the expression of NF-κB via activating its transcription activity in colonic cancer cells.

PPARδ is required for HBXIP-induced proliferation of colonic cancer cells
Based on the hypothesis that PPARδ is involved in the HBXIP-induced cell proliferation, we performed Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assays in SW480 or HT-29 cells by transfecting with pCMV (0.5 μg), pCMV-HBXIP (0.5 μg), pCMV-HBXIP (0.5 μg)/si-Control (100 nM) or pCMV-HBXIP (0.5 μg)/ si-PPARδ (100 nM) respectively. Consistent with the hypothesis, the data clearly indicated that the proliferation  Figure 4A), the similar results were also confirmed by colony formation assays (Supplementary Figure 4B), suggesting that HBXIP promoted the proliferation of colonic cancer cells through activating PPARδ. Moreover, ethynyldeoxyuridine (EdU) assays were performed and the data showed the similar results of MTT assays, that is HBXIP expression enhanced the proliferation of SW480 or HT-29 cells, and the increase was abolished by the addition of si-PPARδ (100 nM) ( Figure 6C, 6D and Supplementary Figure 4D). Taken together, these data strongly suggest that HBXIP promoted the proliferation of colonic cancer cells via activating PPARδ.

Association between HBXIP and clinicopathological characteristics
We have found that HBXIP could promote the proliferation of colonic cancer cells. Then we wondered whether the expression levels of HBXIP were associated with specific clinicopathological parameters in colonic cancer patients. 148 patients (Supplementary Table 1) were separated equally into two groups according to the expression levels of HBXIP measured by quantitative realtime PCR (qRT-PCR). We found that HBXIP expression levels were strongly correlated with the clinical stage including lymph node metastasis and advanced TNM stage (Table 1). However, there were no statistically significant associations between HBXIP expression and other clinicopathological data, including Age, Gender, Tumor size and Differentiation. The clinicopathological parameters showed that HBXIP expression was positively associated with the development and progression of colonic carcinoma, which suggests that HBXIP may play important roles in the colonic carcinoma and function as a potential factor for predicting tumor malignancy.

HBXIP has a positive correlation with PPARδ in clinical colonic carcimoma tissues
Literatures reported that PPARδ, a ligand-inducible transcription factor, is highly expressed in colonic cells and involved in colonic tumorigenesis. Thus, we wondered whether the expression of HBXIP was associated with that of PPARδ in the clinical colonic cancer patients. We then tested the expression levels of HBXIP ( Figure 7A) and PPARδ ( Figure 7B) of 83 cases of colonic paraffinembedded specimen and 12 cases of normal colonic tissues by immunohistochemical assays (Supplementary Table 3), the results demonstrated that Both HBXIP and PPARδ were highly expressed in the colonic tumor tissues and showed a positive correlation between HBXIP and PPARδ expression in the carcinoma tissues, which suggests that HBXIP had played important roles in the progression of colonic carcinoma cells. Moreover, quantitative realtime PCR were performed to test the expression levels of HBXIP and PPARδ of the colonic carcinoma tissues from 148 colonic carcinoma paraffin-embedded specimen. The data demonstrated that the expression levels of PPARδ had a significant positive correlation with those of HBXIP in colonic carcinoma tissues (p < 0.001 Wilcoxon signedrank, Figure 7C). Taken together, we conclude that the expression levels of HBXIP are positively associated with those of PPARδ in colonic carcinoma tissues, and the HBXIP/ PPARδ signaling may have important roles in the colonic tumorigenesis.

DISCUSSION
HBXIP (LAMTOR5) plays an important role in the promotion of proliferation of breast cancer cells [4][5][6]. Research reported that HBXIP showed a high expression in the colonic cancer cells (https://www. proteinatlas.org/ENSG00000134248-LAMTOR5/tissue) and we also found the different expression levels of HBXIP in the colonic cancer cells by RT-PCR assays (Supplementary Figure 1A). Previous report indicated that HBXIP functioned in the cytoplasm [25], which is consistent with our immunofluorescent results indicated that HBXIP mainly localized in the cytoplasm, while a small amount of HBXIP proteins located in the nuclues ( Figure 4H), suggesting HBXIP may have some functions in the nucleus, which is consistent with our previous studies [4,6]. However, the mechanism by which HBXIP enhances the proliferation of colonic cancer cells is still unclear. Many studies demonstrated that PPARδ, a ligand-inducible transcription factor, plays a critical role in regulating cancer progression, and PPARδ-related tumorigenesis was first identified in colorectal cancer [26]. PPARδ is also highly expressed in colonic epithelial cells and closely linked to colonic carcinogenesis [20]. Thus, we are interested in whether PPARδ is involved in HBXIP-enhanced proliferation of colonic cancer cells.
In our study, we investigate how HBXIP functions with PPARδ in promotion of colonic cancer cells.
Many studies indicated that HBXIP promotes the proliferation of breast cancer cells by different signal pathways [27,28], and HBXIP expression occurs in nearly all tissues [3]. PPARδ is also found high expression in colonic epithelial cells and involved in colonic carcinogenesis. Thus, we first tested whether HBXIP is associated with PPARδ in colonic cancer cells, we found that HBXIP could up-regulate the expression of PPARδ in mRNA and protein levels in SW480 and HT-29 colonic cancer cells, respectively. In order to explore the underlying mechanism by which HBXIP regulates PPARδ expression, and according to our previous research results that is HBXIP functioned as a co-activator of transcription factors to activate the transcription activity of target genes [4,6,27]. ChIP assay results indicated that HBXIP could occupy on the PPARδ promoter, and luciferase report gene data showed that HBXIP could activate the transcription activity of PPARδ. We next reconstructed various length of PPARδ promoter regions and performed the luciferase reporter gene assays, we found the core region of PPARδ promoter, which showed the maximum activity among these different PPARδ promoter regions. Our previous study demonstrated that HBXIP could not bind to DNA sequence directly [4]. Next, we predicted the possible transcription factors of PPARδ promoter using the GPMiner (http://gpminer.mbc. nctu.edu.tw/). NF-κB transcription factor was screened out and previous literature reported that NF-κB signaling was involved in carcinogenesis [24]. Luciferase reporter analysis results validate that NF-κB plays critical roles in regulating the transcription activity of PPARδ. Co-IP, immunofluorescence and confocal laser scanning microscope assays displayed that HBXIP and NF-κB could bind to each other in the cells, indicating that HBXIP up-regulates the expression of PPARδ via NF-κB. Thus, HBXIP functioned as a co-activator of NF-κB activates PPARδ transcription. These findings are consistent with our previous report that HBXIP may function as a co-activator [4,29]. Previous studies indicated that PPARδ negatively regulated the transcription of NF-κB, such as in inflammation [30] or in diabetic nephropathy [31]. Interestingly, we observed that PPARδ up-regulated the expression of NF-κB through activating its transcription activity. One possible explanation is that the mechanisms by which PPARδ regulates NF-κB in inflammation might be different from that in colonic cancer. Thus, HBXIP accelerated the expression of PPARδ/NF-κB feedback loop in the cells. In function, MTT, EdU and colony formation data demonstrated that HBXIP enhanced the proliferation of colonic cancer cells in PPARδ-dependent manner. In clinical colonic cancer samples, we detected the relationship between HBXIP and clinicopathologic parameters in colonic cancer patients. Interestingly, we found that HBXIP is positively correlated with the lymph node metastasis and advanced TNM stage, suggesting HBXIP expression is positively correlated with the development and progression of colonic carcinoma.
In summary, we show a model that the oncoprotein HBXIP promotes the proliferation of colonic cancer cells via activating PPARδ, in which HBXIP, functioned as a co-activator of NF-κB, up-regulated the expression of PPARδ, and the PPARδ conversely up-regulated the expression of NF-κB through activating its transcription activity (Supplementary Figure 5). The HBXIP/PPARδ/ NF-κB signaling may be a critical key driver in the development and progression of colonic carcinogenesis. Our findings provide new insights into the mechanism by which HBXIP promoted the proliferation of colonic cancer cells. Additionally, HBXIP may serve as a potential biomarker or drug target for diagnosis and treatment for colonic cancer patients.

Clinical specimen collection
A total of 148 colonic carcinoma paraffin-embedded specimen were obtained from patients who underwent surgical resection at the Changzhou Wujin People's Hospital. This study was approved by the Human Ethics Committee board of Changzhou Wujin People's Hospital and informed consent was obtained from patients prior to the research commencing with no patients receiving preoperative treatments. The information of patients with colonic cancer is presented in Supplementary Table 1, which was tested by quantitative real-time PCR, and 83 cases from the 148 colonic carcinoma paraffin-embedded specimens were detected by immunohistochemistry assays. The documented clinicopathological characteristics were: Age, Gender, Tumor size, Differentiation, Lymph node metastasis and TNM stage.

RT-PCR, qRT-PCR and Western blotting assays
The total RNA of paraffin-embedded specimens was extracted by using the Recover All Total Nucleic Acid Isolation Optimized for FFPE Sample Kit (Ambion Inc., Texas, USA) following the manufacturer's protocol [32]. And the total RNA from the cells was extracted using Trizol (Invitrogen) according to the manufacturer's protocol. For HBXIP, PPARD and P65 mRNA level detection, the total RNA from colonic cancer SW480, HT-29 cells or from the paraffin-embedded carcinoma specimens were directly reverse transcribed. The qRT-PCR was performed as described in the method of SYBR Premix Ex Tag Kit (TaKaRa, Japan). All primers used in this study are listed in Supplementary Table 2. Western blotting was performed as previously described [33]. The primary antibodies used in this study were rabbit polyclonal anti-HBXIP, anti-PPARδ, anti-p65 (BBI, Sangon Biotech) and anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA).

Immunohistochemistry
Immunohistochemistry assay was performed as described previously [33]. The slides provided by Pathology of Changzhou Wujin People's Hospital were incubated with rabbit anti-HBXIP (or rabbit anti-PPARδ) antibody at 4°C for overnight. After incubation at room temperature for 30 min with biotinylated secondary antibody, the slides were stained with the 3-amino-9ethylcarbazole according to the protocol of AEC substrate staining Kit (Solarbio).

Co-immunoprecipitation (Co-IP) assay
The co-immunoprecipitation protocol was described in detail on our previously published paper [4]. SW480 cells (2 × 10 6 ) were harvested and lysed in a lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.3% Triton X-100, 1 mM protease inhibitor PMSF). The lysates were incubated with antibodies at 4°C overnight, next added the pre-washed protein A-conjugated agarose beads at 4°C for 2 h. The precipitates were washed eight times with ice-cold lysis buffer, resuspended in the PBS and resolved by SDS-PAGE followed by western blotting.

Luciferase reporter gene assay
Adherent cells (SW480 and HT-29 cells) were seeded into 24-well plates and, respectively, transfected with the constructs containing different length fragments of PPARδ promoter or pGL3-Basic as a negative control, with the pRL-TK plasmid (Promega, Madison, WI) which was used as internal normalization. Cell extracts were harvested after 36 hours and lysed using lysis buffer (Promega). Luciferase reporter gene assay was measured using the GLOMA MULTI DETECTION SYSTEM (Promega) as previously described [4]. All experiments were performed at least three times.

Chromatin immunoprecipitation (ChIP) assays
The chromatin immunoprecipitation assays were performed by the published methods [34], and the detailed procedures were carried out according to the protocol of the EZ-Magna ChIP™ A-Chromatin Immunoprecipitation Kit (Merck Millipore). Protein-DNA complexes were immunoprecipitated with HBXIP antibodies and with mouse IgG as a negative control antibody. DNA collected by these antibodies was subjected to PCR analysis, followed by sequencing. Amplification of soluble chromatin prior to immunoprecipitation was used as an input control.

Analysis of cell proliferation
SW480 and HT-29 cells were seeded respectively onto 96 well plates (800 cells/well) for 24 h before transfection and the 3-(4,5-dimethylthiazol2-yl) 22, 5-diphenyltetrazolium bromide (MTT) (Sigma) assays [4] were used to assess cell proliferation every day from the 24 h until the 96 h after transfection. In addition, 5-ethynyl-2 0 -deoxyuridine (EdU) incorporation assay was carried out to assess cell proliferation using the Cell-Light TM EdU imaging detecting kit according to the manufacturer's instructions (RiboBio, China) as well. For the colony formation assays, approximately 1000 SW480 transfected cells or 3000 HT29 transfected cells were placed in six-well plates after 36 h transfection and maintained in complete medium for 10 days, respectively. www.impactjournals.com/oncotarget The colonies were fixed by methanol for 20 min then stained with methylene blue for 30 min, washed with water and take pictures as previous described [4].

Statistical analysis
Each experiment was repeated at least three times. Statistical significance was assessed by comparing mean values (±SD) using a Student's t-test for independent groups and was assumed for p < 0.05 ( * ), p < 0.01 ( ** ) and p < 0.001 ( *** ). Correlation between expression levels of HBXIP and PPARδ in tumor tissues was explored using Pearson's correlation coefficient by the Statistical Analysis System (GraphPad Prism 6, GraphPad Software Inc, CA, USA)