HBP1 regulates AFP expression
We applied survival analysis to the data on 71 hepatoma patients who received conservative surgery and then were monitored for up to 10 years. We observed a significant inverse correlation between HBP1 and AFP protein levels in hepatoma tissues by immunohistochemical staining (Fig. 1A). As the degree of malignancy increased, the expression of HBP1 gradually decreased from phase Ⅰ to phase Ⅳ under the TNM staging system. In contrast, AFP gradually increased from phase Ⅰ to phase Ⅳ. We defined HBP1 or AFP expression greater than mean as HBP1 or AFP high expression group, and vice versa low expression group. Survival analysis demonstrated that HBP1 high expression, or AFP low expression was associated with higher survival rate (Fig.1B, top panel). The expression level of HBP1 decreased with the increase of tumor TNM stage, and the survival rate after surgery also decreased (Fig. 1B, middle and bottom panels). The data suggest that the inverse expressions of HBP1 and AFP correlate with relapse and survival in hepatoma patients.
To determine whether HBP1 represses AFP expression, we overexpressed HBP1 in HepG2 and PLC5 cells (Fig. 1C). AFP protein (left panel) and mRNA (right panel) levels were reduced by HBP1 overexpression in both HepG2 and PLC5 cells. To confirm the endogenous regulation of HBP1 on AFP expression, we knocked down the expression of HBP1 using short hairpin RNAs (shRNAs). As shown in Fig. 1D, HBP1 knockdown resulted in increased levels of AFP protein (left panel) and mRNA (right panel) in the two cell lines. These results suggest that HBP1 inhibits AFP expression at the transcriptional level.
HBP1 represses the AFP gene through binding an affinity site in the AFP promoter
We next investigated whether HBP1 regulates the promoter activity of AFP in a DNA-binding-dependent manner. We found that the AFP promoter contains an affinity site (TGAATGGG) for HBP1 at position -1512 to -1505 bp from the transcriptional start site. We co-transfected 293T cells with plasmids expressing HBP1 and different deletion mutants of AFP promoter reporter genes (Fig. 2A). HBP1 inhibited the reporter genes containing the HBP1 affinity site (-1871 to +134 and -1545 to +134), whereas HBP1 had no effect on the reporter genes lacking the HBP1 affinity site (-1500 to +134, -1004 to +134, -448 to +134 and -215 to +134). HBP1 also inhibited AFP promoter activity in a dose-dependent manner (Fig. 2B).
To further verify the DNA binding requirement for the effects of HBP1, we constructed a mutant reporter for the AFP promoter with point mutations in the binding element at -1510 (changing AA to CC). HBP1 inhibited the activity of the wild-type AFP promoter, but had no effect on the mutant (Fig. 2C). To further investigate whether the transcriptional repression of HBP1 depends on DNA binding, we used two mutants of HBP1: pmHMG, which has three amino acid mutations in the HMG domain and lacks DNA binding ability, and DelEx7, which was isolated from breast cancer tissue and lacks the DNA binding domain and part of the repression domain [17, 20]. As shown in Fig. 2D and 2E, wild-type HBP1 decreased the activity of AFP promoter and protein level, but overexpression of pmHMG and DelEx7 had no effect. Since HBP1 inhibited the activity of the AFP promoter, we tested whether HBP1 directly binds to the AFP promoter. Electrophoretic mobility shift assay (EMSA) (Fig. 2F) and chromatin immunoprecipitation (ChIP) assay (Fig. 2G) demonstrated that HBP1 bound directly to the specific affinity site in the AFP promoter in vivo and in vitro. In contrast, pmHMG and DelEx7 did not bind the AFP promoter.
HBP1 acetylation at K419 is required for HBP1 suppression of AFP transcription
Our previous studies showed that HBP1 is acetylated by p300/CBP at five lysine sites (K292, K305, and K307 in the Repression domain, and K171 and K419 in the P domain). Acetylation of HBP1 is required for its transcriptional activity . To investigate whether acetylation of HBP1 affects its transcriptional repression of AFP, we used various HBP1 mutants: K171R, K419R, 2KR (K171/419R), 3KR (K297/305/307R), and 5KR (K171/419/297/305/307R) (Fig. 3A). We co-transfected the AFP luciferase reporter with either wild-type HBP1 or the HBP1 acetylation mutants into 293T cells. Wild-type HBP1 repressed AFP promoter activity. The 3KR acetylation-deficient mutant in the Repression domain showed similar results as wild-type HBP1, whereas mutant 2KR and 5KR showed no repression of the AFP promoter-luciferase construct (Fig. 3B, left panel). Consistent with these results, wild-type HBP1 and mutant 3KR decreased AFP mRNA and protein levels, however, overexpressing mutant 2KR or 5KR had no effect on AFP expression compared with control vector (Fig. 3C and 3D, left panel). We further found that HBP1 acetylation at K419 was required for its suppression of AFP transcription (Fig. 3B, right panel). Likewise, the regulation of AFP mRNA and protein levels (Fig. 3C and 3D, right panel) depends on acetylation of HBP1 at K419. These data indicate that HBP1 acetylation at K419 is crucial for its repression of AFP transcription.
HBx attenuates the suppression of HBP1 on AFP through inhibiting binding of HBP1 to the AFP promoter
The development of hepatoma is closely related to hepatitis B virus (HBV) infection . HBx is a HBV regulatory protein that up-regulates AFP expression and promotes the development of HBV-associated hepatoma [30,31]. To determine whether HBx promotes the development of hepatoma through inhibiting the downregulation of AFP by HBP1, we overexpressed HBP1 with or without HBx in HepG2 cells. As shown in Fig. 4A–4C, HBx rescued the HBP1-mediated decrease of AFP protein level, mRNA level and promoter activity. We next examined whether HBx affects the binding of HBP1 to the AFP promoter. ChIP results confirmed that HBx reduced the binding of HBP1 to the AFP promoter (Fig. 4D). We also performed co-IP assays and found that exogenous HBx can interact with HBP1 (Fig. 4E). Therefore, we speculated that HBx may inhibit the binding of HBP1 to the AFP promoter by interacting with HBP1.
Icaritin promotes the suppression of HBP1 on AFP through enhancing HBP1 binding to AFP promoter
Icaritin is an active ingredient of the Chinese herb epimedium that has been proven to have a wide range of biological and pharmacological functions, such as enhancing immunity and antioxidative and anticancer functions . Previous studies reported that Icaritin inhibits the growth of liver cancer cells through promoting the apoptosis of hepatoma cells by activating the caspase pathway and inhibiting the IL-6/Jak2/Stat3 signaling pathway [33,34]. Zhang et al. demonstrated that Icaritin reduces the expression of AFP by promoting miRNA-mediated degradation of AFP mRNA . We thus next examined whether Icaritin interferes with the suppression of HBP1 on AFP. We overexpressed HBP1 with or without Icaritin treatment in HepG2 cells and examined the protein level, mRNA level and promoter activity of AFP. As shown in Fig. 5A–5C, HBP1 more significantly inhibited AFP expression in the presence of Icaritin compared with HBP1 alone. ChIP assay showed that Icaritin enhanced the binding of HBP1 to the AFP promoter (Fig. 5D). To further investigate how Icaritin enhances the transcriptional repression of HBP1, we evaluated the protein and mRNA levels of HBP1 in HepG2 cells after Icaritin treatment. As shown in Fig. 5E, Icaritin increased HBP1 protein expression but had no effect on mRNA level. Furthermore, Icaritin inhibited HBP1 ubiquitination-mediated proteasome degradation (Fig. 5F). Thus, we concluded that Icaritin increases HBP1 expression by inhibiting the ubiquitination of HBP1, thereby enhances HBP1 binding to AFP promoter.
The repression of AFP by HBP1 attenuates AFP effect on PTEN, MMP9 and caspase-3 protein levels in hepatoma cells
Previous studies reported that AFP exerts its tumor promotion through interacting with PTEN, MMP9, and caspase-3 and blocking their functions on the PI3K/AKT, metastasis and caspase signaling [8,9]. We then tested whether the repression of AFP by HBP1 attenuates AFP effect on PTEN, MMP9 and caspase-3 in HepG2 cells. As shown in Fig.6A, HBP1 overexpression increased PTEN protein expression and decreased phosphorylated AKT level. Meanwhile, HBP1 enhanced pro-caspase-3 processing to cleaved-caspase-3 and decreased the protein level of MMP9, whereas co-expressing AFP rescued the HBP1-mediated changes in PTEN, caspase-3, and MMP9. Furthermore, knockdown of HBP1 increased AFP expression, thereby decreased PTEN and cleaved-caspase-3 levels, and increased phosphorylated AKT and MMP9 levels, but there was no effect when AFP was also knocked down (Fig. 6B). We also examined the K419R mutant and found that it had no effect on expression of the proteins (Fig. 6E). These results indicated that HBP1 increases protein levels of PTEN and cleaved-caspase-3 and decreases protein level of MMP9 through suppressing AFP, and HBP1 exerts the role depending on its acetylation at K419.
We also tested the role of HBx or Icaritin in regulating HBP1-mediated expression of these proteins. As shown in Fig. 6C and 6D, HBx rescued HBP1-mediated expression of these proteins, while Icaritin enhanced HBP1-mediated expression of these proteins, suggesting that HBx or Icaritin influences PTEN/AKT, caspase-3, and MMP9 signals through regulating HBP1-AFP axis.
HBP1 inhibits malignancy through suppressing AFP in hepatoma cells
Since HBP1 upregulates AKT and caspase-3 signals and decreases metastasis-related protein of MMP9 through suppressing AFP, we next investigated the role of HBP1 suppression on AFP expression in cell proliferation, migration and apoptosis in HepG2 cells. Consistent with previous studies, HBP1 decreased cell proliferation and cell migration as shown by growth curve (Fig.7A, top panel) and Transwell assay (Fig.7A, middle panel), also increased cell apoptosis response to H2O2 induction, as demonstrated by FACS (Fig.7A, bottom panel). AFP rescued the HBP1-induced decrease of cell proliferation and migration, and also rescued the increase of cell apoptosis (Fig. 7A). HBP1-knockdown cells showed higher growth rate and cell migration ability, but lower apoptosis rate response to H2O2 induction, while AFP knockdown by shRNA in HBP1-knockdown cells rescued the elevated proliferation and migration, and the decreased apoptosis induced by knockdown of HBP1 (Fig. 7B).
To further determine whether HBx or Icaritin treatment alters the degree of malignancy in hepatoma cells, HepG2 cells were stably transfected with HBP1 with or without lentivirus expressing HBx or Icaritin treatment. As shown in Fig. 7C and 7D, HBx rescued HBP1-mediated decrease of cell proliferation and migration, and the increase of cell apoptosis, while Icaritin enhanced HBP1-mediated decrease of cell proliferation and migration, and the and the increase of cell apoptosis. These results indicate that HBP1 inhibits malignancy in hepatoma cells through suppressing AFP, and HBx or Icaritin influences hepatoma cell malignancy through regulating HBP1-AFP axis. We also examined the K419R mutant and confirmed that the effect of HBP1 on cell proliferation, migration and apoptosis is dependent on its acetylation of K419 (Fig. 7E).
HBP1 deletion aggravates DEN/CCl4-induced hepatoma
To further investigate the function of HBP1 in the development of hepatoma, we constructed HBP1 knockout mice using the CRISPR/Cas9 system. The removal of 194 base pairs in the third exon of the HBP1 gene prevented the HBP1 gene transcription is prematurely terminated, resulting in the deletion of HBP1 protein. After genetic identification of newborn mice, we selected two groups of wild-type and HBP1-deficient mice and randomly assigned six wild-type and six HBP1-deficient mice per group. One group was treated with DEN/CCl4 to construct a mouse hepatic fibrosis and hepatoma models, and the other group was treated with saline/olive oil as a negative control. About 12 weeks later, the mouse hepatic fibrosis model was firstly established. We sacrificed the mice and excised the livers for analyses. As shown in Fig. S1A, more inflammatory cell infiltrations and necrotic foci were formed in the liver of HBP1-deficient mice treated with DEN/CCl4 compared with that of HBP1 wild-type mice. Masson staining and Sirius Red staining showed that hepatic fibrosis only occurred in the mouse group treated with DEN/CCl4, and more severe hepatic fibrosis occurred in HBP1-deficient mice (Fig. S1B). There was no liver damage and fibrosis in wild-type or HBP1-deficient mice in the groups not treated with DEN/CCl4 (Fig. S2A, S2B). We then detected the protein and mRNA levels of several inflammatory factors associated with hepatic fibrosis in the liver of mice. As shown in Fig. S1C and S1D, the protein and mRNA levels of TypeⅠcollagen, Type Ⅲ collagen and TNF-α were increased in the liver of HBP1-deficient mice, while IL-1β expression was unchanged compared with levels in wild-type HBP1 mice. We also tested ALT and AST levels in mice serum. ALT and AST levels increased after DEN/CCl4 treatment and both levels increased more significantly in HBP1-deficient mice compared with wild-type mice (Fig. S1E), indicating that HBP1-deficient mice had more severe liver function damage after DEN/CCl4 treatment, whereas ALT and AST levels did not increase in the group without DEN/CCl4 treatment (Fig. S2C).
In separate experiments, mice were treated with DEN/CCl4 or saline/olive oil (as a negative control) for longer (about 20 weeks) to induce hepatoma. HBP1-deficient mice had more liver tumor tissue blocks, and the tumor tissue volume was larger compared with wild-type mice (Fig. 8A). In addition, multiple nodules were clearly visible on the HBP1-deficient mouse liver surface, while the wild-type mouse liver was relatively smooth. As shown in Fig. 8B and 8C, the protein level and mRNA level of AFP were increased in the liver of HBP1-deficient mice. Since AFP is a serum marker of primary liver cancer, we evaluated the AFP content in mouse serum using ELISA and found that serum AFP level of HBP1-deficient mice was higher than that of wild-type mice (Fig. 8D). IHC staining of paraffin sections showed that Ki67 expression was higher in HBP1-deficient mice compared with wild-type mice (Fig. 8E), indicating that HBP1-deficient mouse hepatoma is more malignant in the DEN/CCl4-induced hepatoma model. Together, the data suggest that HBP1 deletion aggravates DEN/CCl4-induced liver damage, hepatic fibrosis and hepatoma in mice. Our results are thus consistent with a model in which HBP1 inhibits hepatoma by repressing the expression of AFP (see model in Fig. 8F).