HBx enhances CPAP expression via interacting with CREB to promote hepatocarcinogenesis in HBV-associated HCC

Hepatitis B virus (HBV) encoded non-structure protein X (HBx) can promote cell proliferation, migration, and anti-apoptosis via activating several transcription factors and increasing their downstream gene expression in HBV-infected liver cells. Our previous report suggested that centrosomal P4.1-associated protein (CPAP) is required for HBx-mediated NF-κB activation. Here, we found that, upon HBV infection, overexpressed HBx can transcriptionally up-regulate CPAP via interacting with CREB. CPAP can directly interact with HBx to promote HBx-mediated cell proliferation and migration; and SUMO modification of CPAP is involved in interacting with HBx. Interestingly, CPAP can increase the HBx protein stability in an NF-κB-dependent manner; and overexpressed CPAP and HBx is positively correlated with the activation status of NF-κB in HCC. Increased expression of CREB and CPAP mRNAs exists in the high-risk group with a lower survival rate in hepatocellular carcinoma (HCC). These results suggest that the reciprocal regulation between CPAP and HBx may provide a microenvironment to facilitate HCC development via enhancing NF-κB activation, inflammatory cytokine production, and cancer maligancies. The findings of this study not only shed light on the role of CPAP in HBV-associated HCC, but also provide CPAP as a potential target for HBV-related HCC therapy. Author Summary In this study, we address a novel molecular mechanism for the collaboration between overexpressed HBx and CPAP in promoting hepatocarcinogenesis in HBV-associated HCC. Upon HBV infection, HBx is overexpressed and interacts with CREB to transcriptionally activate CPAP; the HBx/CPAP interaction promotes hepatocarcinogenesis. Clinical analysis found that co-overexpressed CPAP and CREB exist in the high-risk group with a lower survival rate in HCC. Additionally, overexpressed CPAP contributes to HBx protein stability in a NF-κB-dependent pathway. Our study provides a potential translational application in targeting CREB-CPAP axis in HBV-associated HCC.


Introduction
HBV infection is a major etiological factor in acute and chronic hepatitis and enhances the development of liver diseases such as cirrhosis and HCC. Among the different HBV proteins encoded by HBV genome, the X protein of HBV (HBx) plays a critical role in HBV-associated HCC development possibly through triggering specific oncogenic pathways and causing an accumulation of genetic and epigenetic alterations in regulatory genes (1)(2)(3)(4).
HBx is a multifunctional protein that modulates the expression of various cellular and viral genes involving in cell survival, cell cycle progression, DNA repair, invasion, protein degradation and regulates several signaling pathways such as Ras/Raf/MAPK, PI3K/Akt, NF-κB or JNK (5)(6)(7)(8)(9)(10). Additionally, functions of HBx on cytoplasmic signal transduction cascades and transcriptional activation imply that HBx is both a cytoplasmic and nuclear protein (11). It is known that HBx serves as a powerful transcriptional activator that up-regulates several transcription factors including NF-κB, AP-1, and even the HBV genome (12,13). However, HBx does not bind DNA directly. HBx affects transcription by interacting with several transcription factors, including DNA-binding factors and complexes of transcriptional machinery(14). For example, HBx interacts with TATA box-binding protein (TBP), RNA polymerase II subunit 5 (RPB5), and transcription factor IIB (TFIIB) to regulate RNA polymerase transcription (15)(16)(17). In the nucleus, HBx associates with C-terminal binding protein (CBP/p300) and binds to the CREB-targeting site of the promoters of IL-8 and proliferating cell nuclear antigen (PCNA) (18,19). Recently, an HBx transgenic mouse model showed a high incidence of liver tumor formation without fibrosis in 90% of cases and has been widely used as an animal model for studying the detailed mechanisms of chronic HBV infection in HCC development (20,21). Although the role of HBx in the pathogenesis of HCC is well understood, the mechanism by which HBx regulates the gene expression network is not fully clear.
Previously, we showed that the expression of CPAP in HBV-associated HCC correlates with a poor prognosis (22). CPAP has been reported to be part of the γ-tubulin complex, which is associated with γ-tubulin in both the centrosomal and cytosolic fractions throughout the cell cycle, and plays an essential role in microtubule nucleation and procentriole elongation (23)(24)(25). Interestingly, CPAP also regulates cell apoptosis and the growth of neural precursor cells (26,27). There are three nuclear localization signals and two nuclear export signals within the CPAP polypeptide(28), indicating CPAP can shuttle between the nucleus and cytoplasm. Furthermore, CPAP has been shown to act as a transcriptional coactivator of STAT5 and NF-κB(28, 29). TNF-α-induced SUMO modification of CPAP is required for IKK-mediated NF-κB activation in HCC cell lines and promotes the growth of HCC cells, suggesting that CPAP is critical for the association between NF-κB and inflammation-related diseases, such as HCC (22). In addition, the cooperation of CPAP and HBx in regulating the transcriptional activity of NF-κB, provides evidence that CPAP plays an important role in HBx-mediated HCC development (22). However, the relationship between CPAP and HBx and the physiological role of CPAP in HBV-associated HCC are still unclear.
In this study, we investigated the interaction between CPAP and HBx and determined the functional role of the CPAP-HBx interaction in HBx-mediated hepatocarcinogenesis. HBx transcriptionally increased the expression of CPAP via interacting with CREB, and overexpressed CPAP increased the protein stability of HBx in an NF-B-dependent manner, both of which resulted in an increased activity and target gene expression of NF-κB. The reciprocal regulatory loop between CPAP and HBx at the transcriptional and post-transcriptional levels presents a complex relationship during early and late hepatocarcinogenesis in HBV-related HCC. HCC patients with co-overexpressed CREB and CPAP mRNAs have a poor prognostic value. Taken together, our results provide strong evidence that CPAP is crucial for HBx-induced HCC development and may offer opportunities to develop mechanism-based therapies.

Overexpression of CPAP in HBV-associated HCC
Our previous studies have showed that CPAP expression positively correlates with a poor prognosis in HBV-HCC (22). To further assess the clinical significance of CPAP expression in HBV-HCC, we evaluated the association between CPAP and the major clinicopathological features of 132 HBV-HCCs (Supplementary Table 2). The results showed a significant correlation between high CPAP expression levels with the disease-free survival rate, AST, ALT, differentiation, tumor size, and AJCC stage (Table 1).

HBx transcriptionally up-regulates CPAP by interacting with CREB
By Western blot analysis, we found that HBx can increase CPAP expression in HCC cells ( Figure 1A, left panel). HBx stable expression cell lines Hep3BX or HepG2X also exhibited a higher CPAP mRNA expression than Hep3B or HepG2   Figure 3).
Given that HBx has been shown to interact with CREB-binding protein/p300 to mediate CREB-dependent transcription(3, 31), we assumed that HBx might activate the CPAP promoter through the interaction with CREB. As anticipated, overexpression of CREB or HBx increased CPAP promoter activity, which was further enhanced by co-expression of HBx and CREB ( Figure 1C and Supplementary We further investigated whether the transcriptional up-regulation of CPAP by HBx was due to HBx interacting with CREB and binding to the CPAP promoter. ChIP assay confirmed that overexpressed CREB can enhance the association of HBx with the CPAP promoter, re-Chip assay further demonstrated that CREB can form a complex with HBx to bind to the CPAP promoter ( Figure 1E). By contrast, CREB and HBx did not associate with the CPAP/M1 promoter ( Figure 1F). These results indicated that the CPAP promoter -86 to -79 bp is a cis-regulatory element for HBx-mediated transcriptional activation; HBx enhances CPAP expression by binding onto the CPAP promoter through the association with CREB.

CPAP and HBx cooperatively enhance NF-B activation
It has been well recognized that NF-κB is a transcription factor regulated by HBx,  Figure 2B).
To further investigate how CPAP and HBx cooperate to enhance NF-κB activity, we first checked the association between CPAP and HBx in the NF-κB pathway.  Figure 2E).

Co-immunoprecipitation analysis and in situ
Furthermore, the interaction between CPAP and HBx was increased upon TNF-α stimulation (Supplementary Figure 7B). These findings suggest that SUMO modification is essential for CPAP to associate with HBx and cooperatively enhance NF-κB activity.

SUMO modification is required for CPAP to promote HCC proliferation and tumorigenicity
Next, we examined the role of CPAP in HCC development. To further validate the effect of CPAP in these observations, CPAP was knocked down in Hep3BX cells, and the results showed an impairment of HBx-induced cell growth and migration ( Figure 3D). These results support that CPAP is crucial for HBx-induced tumorigenesis in HCC.

CPAP is required for maintaining TNF--mediated HBx protein stability through enhancing the NF-κB activity
Interestingly, we found that the protein expression of GFP-HBx was increased in HA-CPAP-overexpressing cells ( Figure 4A), no changes in GFP-HBx mRNA expression were observed ( Figure 4A), indicating that the prolonged protein stability of GFP-HBx may under the regulation of HA-CPAP. Previous study demonstrated that TNF-α can induce a notable accumulation of HBx by increasing protein stability due to reduced proteasomal degradation through NF-κB signaling(33). Our previous report showed that CPAP is a co-activator of IKK-mediated NF-κB activation in response to TNF-α treatment (22). Therefore, we further clarified whether CPAP can SUMO modification is also required for the interaction of CPAP and HBx and for the enhanced effect of CPAP on TNF-α-induced HBx stabilization (22). It remains to be elucidated whether the complex of SUMOylated CPAP and NF-κB/p65 can prevent the association of HBx with proteasome subunits to increase HBx protein stability.
Here, we demonstrate a novel regulatory mechanism between CPAP and HBx in inflammation-related HCC development. The HBx/inflammatory cytokines/CPAP regulatory loop resulted in marked NF-κB activation in HBV-associated HCC, which provides a microenvironment for tumor development. Additionally, overexpressed CREB/CPAP indicated a poor prognostic value in HBV-associated HCC. Taken together, our findings provide an alternative therapeutic target in the NF-κB pathway to reduce the immunodeficiency caused by NF-κB inhibition, which may lead to a novel therapeutic strategy for HBV-association HCC and other chronic inflammatory diseases.

Xenograft tumorigenicity assay
Hep3B cells (2×10 6 ) stably expressed GFP, GFP-CPAP/WT, GFP-CPAP/MT were injected subcutaneously into the right flank region of 5-week-old male NOD-SCID mice. The tumor volumes were measured using calipers every 3 days. Tumor size was measured using the formula: length X width 2 X 0.5. At 28 days after injection, all mice were sacrificed and tumors were weighed and photographed.

Co-immunoprecipitation assay and Western blot analysis
Co-Immunoprecipitation assay and Western blots were performed as described (22).
Lysates were analyzed by immunoblott analysis using the specific antibodies as indicated in the text. Specific bands were detected with a horseradish peroxidase-conjugated antibody and revealed by an enhanced chemiluminescence (ECL) Western blot system (PerkinElmer).

Chromatin immunoprecipitation (ChIP) and re-chromatin immunoprecipitation analysis
The ChIP assay was performed as described previously(36

Inducible expression of HBV and HBV infection
HepAD38/Tet-off cells were grown in DMEM with 0.3 μg/ml doxycycline, and the production of HBV was induced by removing doxycycline. HepG2-hNTCP-C4 cells were seeded at 5×10 5 cells/well in a 6 cm collagen-coated plate. HBV derived from

RNA extraction and quantitative real-time PCR
Total RNA was extracted using TRIsure reagent (Bioline, London, UK

Colony formation assay
For clonogenicity analysis, 3~5×10 3 cells/well were seeded in 6-well plate and cultured in complete medium for 10~15 days. Colonies were fixed with formaldehyde and stained with 0.1% crystal violet.

Cell proliferation assay
Cells (3×10 3 cells/well) were seeded in 96-well plate and maintained in complete medium overnight. Cell proliferation was performed using the CCK-8 and BrdU incorporation at indicated times according to the manufacturer's instructions.

In vitro trans-well migration assay
Cells were resuspended in serum-free medium and 400 μl of cell suspension (1.

The Cancer Genome Atlas (TCGA) data set
Data from the TCGA data set was used to analyze the overall survival curves using SurvExpress biomarker validation tool(34).

Ethics Statement
The use of clinical HCC specimens was in accordance with the Declaration of

List of supplementary material online Supplementary Figures
Supplementary Figure Legends Supplementary Tables