Hypoxia activates Wnt/β-catenin signaling by regulating the expression of BCL9 in human hepatocellular carcinoma

The Wnt/β-catenin signaling is abnormally activated in the progression of hepatocellular carcinoma (HCC). BCL9 is an essential co-activator in the Wnt/β-catenin signaling. Importantly, BCL9 is absent from tumors originating from normal cellular counterparts and overexpressed in many cancers including HCC. But the mechanism for BCL9 overexpression remains unknown. Ample evidence indicates that hypoxia inducible factors (HIFs) play a role in the development of HCC. It was found in our study that BCL9 was overexpressed in both primary HCC and bone metastasis specimens; loss of BCL9 inhibited the proliferation, migration and angiogenesis of HCC; and that that hypoxia mechanically induced the expression of BCL9. BCL9 induction under the hypoxic condition was predominantly mediated by HIF-1α but not HIF2α. In vitro evidence from xenograft models indicated that BCL9 promoter/gene knockout inhibited HCC tumor growth and angiogenesis. Notably, we found that BCL9 and HIF-1α were coordinately regulated in human HCC specimen. The above findings suggest that hypoxia may promote the expression of BCL9 and associate with the development of HCC. Specific regulation of BCL9 expression by HIF-1α may prove to be an underlying crosstalk between Wnt/β-catenin signaling and hypoxia signaling pathways.

Scientific RepoRts | 7:40446 | DOI: 10.1038/srep40446 complex 8 . Efficient β -catenin-mediated transcription is required in mammalian cells 11 . In addition, BCL9 enhances β -catenin-mediated transcription activity regardless of the mutational status of the Wnt signaling components and increases cell proliferation, invasion and migration. Importantly, BCL9 is absent from the normal cellular counterparts from which tumors originate 12 . BCL9 is frequently overexpressed in a variety of solid tumors including colorectal cancer, multiple myeloma and HCC. Overexpression of BCL9 is associated with poor prognosis of HCC patients 13 . There is evidence that BCL9 is a bona fide oncogene 12,14,15 . However, the mechanism of BCL9 overexpression in tumors remains unclear.
Hypoxia is a common feature of all solid tumors and plays an essential role in tumor occurrence and development 16 . The hypoxia microenvironment could be found in HCC because of imbalance between oxygen supply and consumption in proliferating tumors 17 . Ample evidence indicates that hypoxia-inducible factors (HIFs) play an important role in the pathogenesis and pathophysiology of HCC 18 . Thus, HIF inhibitors have been considered as promising drug targets to be exploited in oncology 19,20 . Human HIFs are heterodimeric transcription factors consisting of a constitutively expressed subunit (ARNT) and an oxygen-regulated subunit, mainly HIF1α and HIF2α . They promote adaptation of tumor cells to hypoxic stress by regulating the expression of genes involved in metabolism, angiogenesis, cell proliferation and apoptosis [21][22][23][24] . Both in vitro and in vivo experiments have demonstrated the existence of a crosstalk between the Wnt/β -catenin and HIF pathways 25 . An interaction was found between β -catenin and HIF-1α , implying an underlying competition for β -catenin between HIF-1α and T-cell factor-4 26 . Given the complex mechanism underlying this crosstalk, further efforts should be made to investigate the network of Wnt/β -catenin and hypoxia signaling pathways [24][25][26][27] .
In this study, we studied the effect of hypoxia on BCL9 expression in HCC cells. We firsty found that BCL9 was transcriptionally induced by hypoxia in HCC cell lines in a HIF1α -dependent manner. HIF1α and BCL9 were coordinately regulated in HCC. We demonstrated that the mechanism of regulation on BCL9 was through the hypoxia signaling pathway, thus providing convincing evidence that BCL9 functions as a crucial molecule in hypoxia modulation of Wnt/β -catenin and plays a vital role in carcinogenesis of human HCC.

Results
Overexpression of BCL9 protein in human HCC specimens. Immunohistochemical studies on primary HCC have demonstrated that BCL9 overexpression is associated with poor prognosis of HCC patients 13 . To further confirm and evaluate the prognostic significance of BCL9 in HCC, we detected the protein expression of BCL9 in the normal liver, primary HCC and bone metastatic specimens by immunohistochemical staining. The sections were scored as negative (− ), weak (+ ) and strong (+ + ) in cases examined. Different distributions of stained tumor cells are shown in Fig. 1. The specimens comprised 30 normal liver tissues, 360 primary HCC tissues and 72 bone metastatic tissues (Table 1). It was found that the expression of BCL9 was significantly increased in HCC specimens, especially in bone metastatic specimens, as compared with that in the normal liver specimens (34.4% and 90.3% vs. 0.00%, P = 0.00). The correlations between BCL9 expression and clinicopathological parameters in primary HCC are shown in Supplementary Table 1. The expression of BCL9 was correlated with Edmondson invasion (P = 0.00), Microvascular invasion (P = 0.01) and T-stage (P = 0.02). No significant correlation was observed between BCL9 expression and age (P = 0.09), gender (P = 0.10), tumor size (P = 0.92), lymph node metastasis (P = 0.15) and distance metastasis (P = 0.26). These data together indicate that BCL9 was closely associated with the prognosis of human HCC.

BCL9 promotes cell proliferation, migration and angiogenesis.
To evaluate the role of BCL9 in human HCC, we used gain-of-function and loss-of-function approaches to evaluate the function of BCL9 in human liver cancer cell lines. Two distinct BCL9 siRNAs were used to avoid the off-target RNA interference effect. (Fig. 2A) Our results suggest that transient BCL9 over-expression significantly increased the cell viability, cell migration and angiogenesis of HepG2 and smmc-7721 cell lines, while BCL9 knockdown reduced the cell viability, migration and angiogenesis of these cell lines. (Figure 2B,C and D) These results suggest that BCL9 was an essential protein for the proliferation, migration and angiogenesis of human HCC cells.

BCL9 functions as a crucial molecule in hypoxia activation of Wnt/β-catenin pathway.
The previous results showed that BCL9 was overexpressed in primary HCC and bone metastatic specimens, but the underlying mechanism was unclear. Knowing that hypoxia is a common characteristic of the microenvironment of solid tumors including HCC 18,28 , we subjected HepG2 and smmc-7721 cells to hypoxia treatment (0.1% O 2 ) to see whether hypoxia could modulate the expression of BCL9 in human HCC. The effective induction of hypoxia in cells was confirmed by the increased expression of vascular endothelial growth factor (VEGF), a hypoxia-responsive gene, at mRNA levels ( Fig. 3B) and the increased protein levels of HIF-1α and HIF-2α , two major HIFs (Fig. 3C). Interestingly, we found that BCL9 expression was induced by hypoxia ( Fig. 3A and C). The results showed that BCL9 mRNA and protein levels were significantly increased in both HepG2 and smmc-7721 cell lines cultured under the hypoxic condition, indicating that the expression of BCL9 was modulated by hypoxia. To test whether hypoxia activated the WNT/β -catenin pathway, a TOPflash luciferase assay was performed. A significant increase in TOPflash activity was observed under hypoxia treatment. ( Figure 3D) In addition, the TOPflash activity was significantly increased when BCL9 was overexpressed, and decreased when BCL9 was knocked down. ( Figure 3D) These results suggest that BCL9 may function as a crucial molecule in hypoxia activation of the Wnt/β -catenin pathway.
HIF1α rather than HIF2α binds to HRE-B and HRE-C in BCL9 promoter-induced BCL9 expression under the hypoxia condition. The transcriptional response to hypoxia in cells is largely mediated by HIFs. Although HIF1a and HIF2a have shared targets such as VEGF, they also regulate unique gene targets 29,30 . To study whether the transcriptional induction of BCL9 by hypoxia was mediated by HIFs, we searched for the hypoxia-responsive element (HRE) consensus sequence in the promoter region of the BCL9 gene from 3 kb upstream of transcriptional site to exon 1. Three putative HRE sites (HRE-A, HRE-B and HRE-C) were identified in the promoter region of the BCL9 gene. ( Figure 4A) To study whether these putative HREs were responsible for the hypoxia-mediated induction of BCL9, the DNA fragments containing one copy of these putative HRE sites were inserted into a pGL3 luciferase reporter plasmid. Knowing that VEGF is a hypoxia-inducible gene, we inserted the DNA fragment containing the HRE in VEGF promoter into a pGL3 luciferase reporter plasmid to serve as a positive control. HepG2 and smmc-7721 cells were transiently transfected with these reporter constructs, and then exposed to hypoxia for 36 h. TK100 plasmids were co-transfected as an internal standard to normalize transfection efficiency. The results showed that hypoxia significantly increased the luciferase activity of VEGF promoter, the HRE-B and HRE-C from the BCL9 promoter, while HRE-A from the BCL9 promoter did not respond to hypoxia in both HepG2 and smmc-7721 cells. ( Figure 4B) These results demonstrate that hypoxia transactivated the BCL9 promoter containing functional HRE, including HRE-B and HRE-C.
To further investigate which HIF-α (HIF1α or HIF2α ) mediated the hypoxia-induced BCL9 expression, chromatin immunoprecipitation (ChIP) assays were applied to determine whether HIF1α and HIF2α physically bound to HREs, especially HRE-B and HRE-C in the BCL9 promoter. HepG2 cells were cultured under the normoxic or hypoxic condition for 36 h, and ChIP assays were undertaken by using antibodies against HIF1α or HIF2α . It was found that HIF1a, but not HIF2a, bound to the HRE-B and HRE-C region in the BCL9 promoter, using VEGF as the positive control. ( Figure 4C) The specific pull-down of chromatin fragment containing HRE-B and HRE-C but not HRE-A by the HIF1α antibody under the hypoxic condition was further confirmed by conventional PCR followed by agarose gel electrophoresis (Fig. 4D). It was found that HIF1α rather than HIF2α bound to HRE-B and HRE-C in the BCL9 promoter under the hypoxic condition, which further mediated the hypoxia-induced BCL9 expression. To directly confirm that it was HIF1α that transcriptionally induced the BCL9 expression, HepG2 cells were transfected with the plasmids expressing HIF1α (pcDNA3-Flag-HIF1α ). The ectopic expression of HIF1α clearly increased the expression of BCL9 at protein levels as determined by Western-blot assays. ( Figure 4E) Furthermore, HIF1a could also bind to the HRE-B and HRE-C region of the BCL9 promoter. ( Figure 4F) These results further confirmed that HIF1α interacted with HRE-B and HRE-C in the BCL9 promoter, which is consistent with the ChIP result obtained in cells under the hypoxic condition. ( Figure 4G) Collectively, these results suggest that HIF1α rather than HIF2α interacted with HRE-B and HRE-C in the BCL9 promoter to directly induce BCL9 expression under the hypoxic condition.

Construction of stable knockout cell lines using TALENs.
To gain a more comprehensive understanding of the regulatory role of BCL9 in the development of HCC, we established stable BCL9 promoter knockout (BCL9-p-ko) and BCL9 gene knockout (BCL9-ko) HepG2 cell lines by using TALENs. The schematic overview of the targeting strategy for BCL9 promoter is depicted in Fig. 5A. HepG2 cells were co-transfected with the TALEN pairs targeting the BCL9 promoter region along with Donor plasmids. After G418 treatment, cell clones were expanded and identified by genome PCR and sequencing. ( Figure 5B) The plasmids of TALENs for BCL9 gene were designed and constructed using Fast TALE TM TALEN Assembly kit from SiDanSai biotechnology (SiDanSai, China). Three left TALEN plasmids and three right TALEN plasmids were constructed and the target sites are shown in Fig. 5C. The activities of each two TALEN constructs were examined by SSA assay, and construct L1-R1 showed the highest activity among the constructs in the assay (Fig. 5D). Then, construct L1-R1 was introduced into HepG2 by lipofection. After 14-day culture, 50 clones were obtained and the genomic sequences around the target site of the clones were detected. The BCL9-p-ko, BCL9-ko and wild type HepG2 cell lines were treated by hypoxia (0.1% O 2 ). The induction of BCL9 expression by hypoxia was confirmed at the protein levels by using Western-blot assay (Fig. 5E). The TOPflash experiments were performed by using WT, BCL-9-ko and BCL9-ko cells. The results were similar to those obtained by siRNA. (Figure 5F).

BCL9 promotes tumor growth in vivo.
To further evaluate the role of BCL9 in the progression of human HCC, we established a xenograft tumor model. Eighteen male nude mice aged 6 weeks were equally randomized to 3 groups. 2 × 10 6 HepG2 cells transfected with BCL9-wt, BCL9-p-ko and BCL9-ko were injected subcutaneously into the right forearm. Tumor formation was monitored weekly after initiation of tumor cell implantation (Fig. 6A). Mice were euthanized and tumors were removed for further analysis 6 weeks after implantation.   The tumor volume and weight were obviously decreased in BCL9-p-ko and BCL9-ko groups. (Figure 6B and C) HIF1α (red) and BCL9 (green) were detected by immunofluorescence. It was found that HIF1α was normally expressed in BCL9-wt, BCL9-p-ko and BCL9-ko groups, and that BCL9 was normally expressed in BCL9-wt, weakly expressed in BCL9-p-ko group, and unexpressed in BCL9-ko group (Fig. 6D). Angiogenesis was detected by immunohistochemistry and RT-PCR. It was found that angiogenesis was significantly decreased in BCL9-p-ko and BCL9-ko groups (P < 0.01) (Fig. 6E and F). WNT/β -catenin target genes, including CD44, Axin-2 and Survivin, were detected by RT-PCR. It was found that CD44, Axin-2 and Survivin were significantly decreased in BCL9-p-ko and BCL9-ko groups (P < 0.01). ( Figure 6G).

HIF-1α overexpression is associated with BCL9 overexpression in human HCC specimens.
Knowing that hypoxia-induced BCL9 plays an essential role in the progression of HCC in vitro and vivo, we examined whether the expression of HIF1α and BCL9 was correlated with the human HCC specimen using double immunofluorescence co-localization. Altogether 488 human HCC specimens were examined. The typical immunofluorescence images of BCL9 and HIF1α are shown in Fig. 7A. 54.5% human HCC specimens showed positive BCL9 staining, and 67.2% showed positive HIF1α staining. Notably, HIF-1α overexpression was significantly correlated with BCL9 over-expression (Fig. 7B)  Immunohistochemical and immunofluorescence staining. Immunnohistochemical staining for BCL9 was performed by using the standard histological procedure described in the manual for Histostain-Plus IHC Kit, DAB (Phygene). BCL9 staining was scored according to two investigators with previous protocols as negative (− ), weak positive (+ ), and strong (+ + ). The results were analyzed by standard light microscopy. Scoring was assessed blindly with respect to the histologic grade of HCC specimens. Immunofluorescence staining was performed with primary and secondary antibodies diluted in 10% BSA, and the nucleus was stained by DAPI (Sigma). The antibody against HIF-1α (sc-13515, Santa Cruz Biotechnology, 1:500), antibody against HIF-2α (sc-13596, Santa Cruz Biotechnology, 1:500), antibody against BCL9 (ab37305, Abcam, 1:500 dilution) and antibody against β -actin (A5441, Sigma) were used in this study. The antibodies are anti-rabbit in this study. All fluorescent secondary antibodies were used at a dilution of 1:200 for 30 min (invitrogen). Quantification of immunofluorescence was performed using NIH Image J.
Cell lines and culture conditions. Human HCC HepG2 and smmc-7721 cells were obtained from the American Type Culture Collection (ATCC), and maintained in DMEM supplemented with 10% fetal bovine serum (Gibico) in the cell incubator (37 °C, 5% CO2). For hypoxic treatment, cells at 50-60% confluence were incubated in a hypoxia chamber (0.1% O 2 , STEMCELL).

Cell proliferation, migration and angiogenesis. BCL9 expression plasmids (pcDNA3-Flag-BCL9) and
HIF1α (pcDNA3-Flag-HIF1α ) were purchased from GENEwiz TM . Two different siRNA oligos against BCL9 were obtained from Ribobio TM for siRNA knockdown. SiRNA targeting BCL9: siRNA-1: 5-CAG ACU UUA UGU UCA UAG UUC UUC CUC-3; siRNA-2: 5-CUU CCA CAA CUA CAU AGG GUA UUG UUU-3. Expression plasmids and siRNA oligos were transfected into cells using X-tremeGENE HP (Roche). MTS assay was used for cell viability analysis according to the manufacturer's instructions (Promega, Fitchburg, WI, USA). The results were expressed as viability indices representing relative percentages compared to the controls. For migration assay, HepG2 and smmc-7721 cells were seeded in the upper chamber of Transwell plates (BD Bioscience) without or with Matrigel, under serum-free conditions. Medium supplemented with 10% fetal calf serum (FCS) and 50% ug/mL fibronectin (BD Bioscience) was used as a chemoattractant in the lower chamber. After 10-h incubation, cells remaining in the upper chamber were removed with a cotton swab, while cells adhering to the lower membrane were stained with 0.1% crystal violet and photographed with an inverted microscope (Zeiss). The area of positive staining was measured using image analysis software (Image-Pro Plus 6.0, Media Cybernetics). Migration was calculated as the positive area percentages. For angiogenesis analysis, 50 μ l chilled Matrigel (BD Bioscience, Franklin Lakes, NJ, USA) was added to a 96-well plate and incubated at 37 °C for 30 min. HUVECs (1 × 104 cells) were suspended in 100 μ l EBM-2 and conditioning medium from each treated cells, and added to the solidified Matrigel. After 18-h incubation, angiogenesis was assessed on the basis of capillary-like structure formation. Tubes in randomly chosen five microscopic fields were photographed and counted. At least three independent experiments were performed for each condition.
RT-PCR. Total RNA was isolated by using RNAiso Plus (TAKARA) following the manufacturer's instruction. RNA was reverse transcribed into cDNA by using the Taqman Reverse Transcription Reagents kit (Applied Biosystems) with random hexamers. Human BCL9, VEGF, HIF1α , HIF2α , actin, survivin, CD44 and axin 2 mRNA levels were determined in 7900HT Fast Real-time PCR system (Life Technology Corporation, USA). All primers were purchased from Applied Biosystems. RT-PCR was done in triplicate with TaqMan PCR mixture (Applied Biosystems). The expression of genes was normalized to the actin gene. Luciferase reporter assay. To test whether hypoxia activated the WNT/β -catenin pathway, HepG2 and smmc-7721 cells were transfected with DNA plasmids of β -catenin-LEF/TCF-sensitive (TOP-flash) or β -catenin-LEF/TCF insensitive (FOP-flash) reporter vectors (Addgene, Cambridge, MA). The following amounts of plasmids were used per well of a 24-well plate: 1 μ g and 0.5 μ g of TOPflash as well as 1 μ g and 0.5 μ g of FOPflash for HepG2 and smmc-7721 cells, respectively. To test whether hypoxia transactivated the pGL3-reporter plasmids described above, HepG2 and smmc-7721 cells were transfected with the pGL3-reporter plasmids containing one copy of each potential HRE site. To further test the transactivation activity of HIF1α on pGL3-reporter plasmids, cells were co-transfected with pGL3-reporter plasmids together with HIF1α expression plasmids. The X-tremeGENE HP (Roche) transfection agent was used according to the manufacturer's instructions. phRL-TK plasmid (Promega, Madison, WI) was co-transfected as internal controls. Forty-eight hours after transfection, cells were then treated by hypoxia for 48 h. The luciferase activity was measured using the Dual Luciferase assay kit (Promega) and normalized with the internal standard. At least three independent experiments were carried out for each condition.
ChIP assay. ChIP assay was performed by using a ChIP assay kit (Millipore) in accordance with the manufacturer's instructions. HepG2 and smmc-7721 cells treated with hypoxic or normoxic conditions or transfected with HIF1α expression plasmids were lysed in 150ul SDS buffer. Chromatins in these cells were then cross-linked with 1% formaldehyde for 10 min at room temperature, and then sonicated to generate 200 to 500 bp DNA fragments in lysis buffer. The DNA fragments were subjected to ChIP assays with anti-HIF-1α or anti-HIF2α antibodies. Normal IgG was used as a control for nonspecific binding of genomic DNA. Also, we employed transcription factor KLF4 to regulate WNT5A gene as control, knowing that it is not affected by hypoxia 32 . The enrichment of the eluted DNA was assessed by RT-PCR and conventional PCR by using the PCR primer sets described above with the omission of restriction enzyme recognition sequence.
Construction of stable knockout cell lines using TALENs. The schematic overview of the targeting strategy for BCL9 promoter is depicted in Fig. 5A. HepG2 cells were co-transfected with the TALEN pairs targeting the BCL9 promoter region along with Donor plasmids. After G418 treatment, cell clones were expanded and identified by genome PCR and sequencing. The plasmids of TALENs for BCL9 gene were designed and constructed using Fast TALETM TALEN Assembly kit from SiDanSai biotechnology (SiDanSai, China). The constructed TALEN constructs were transfected into 293 T cells. To validate the activities of synthesized endonucleases, an equal amount of expression vectors for the left and right TALENs or sgRNA were transferred into HEK293 cells, and T7E1 mismatch-specific nuclease assay was performed. The TALENs for BCL9 gene were transfected into HepG2. After selection with puromycin, resistant colonies were picked up and examined by genomic PCR and gene sequencing. Clones with successful knockout of BCL9 promotor and BCL9 gene were selected for the xenografts.
Animals. Animal use was according to the National Institutes of Health (NIH) guidelines, and the care and treatment of all animals were in strict accordance with protocols approved by the Institutional Animal Experiment Committees of the Second Military Medical University. Eighteen male nude mice aged 6 weeks were equally randomized to 3 groups. To detect the effect of BCL9 on the tumorigenicity of HCC cells, 2 × 10 6 HepG2 cells transfected with BCL9-p-ko, BCL9-ko and BCL9-wt were injected subcutaneously into the right forearm of the animal. Tumor formation was monitored weekly after initiation of implantation. Mice were euthanized and tumors were removed for further analysis 6 weeks after implantation. The tumor volume and weight were estimated. BCL9 and HIF-1α level and angiogenesis were detected. The β -catenin pathway targeted genes, including CD44, Actin-2 and Survivin expression were also measured 33-35 . Statistical analysis. The data were expressed as mean ± SD. The association between HIF-1α expression levels and BCL9 expression levels was analyzed by Fisher exact test. All other p values were obtained by using two-tailed Student's t-test. Values of p < 0.05 were considered to be statistically significant.

Discussion
The canonical Wnt/ β -catenin signaling pathway is known to be involved in the pathogenesis of a wide variety of cancers. Nuclear β -catenin plays a key role in this pathway by functioning as a transcription factor to activate the expression of cell proliferation, migration, and survival genes such as c-MYC and CyclinD1 6,7 . BCL9 is an essential co-activator in the Wnt/β -catenin signaling pathway by mediating the recruitment of pygopus to the nuclear β -catenin-TCF complex 8 . Despite the mutational status of the Wnt signaling components, BCL9 could also enhance β -catenin-mediated transcription activity and increase cell proliferation, invasion and migration 12 . BCL9 is absent in normal cells and frequently over-expressed in a variety of solid tumors including colorectal cancers, multiple myeloma and HCC 12 . Importantly, the over-expression of BCL9 in tumors is associated with poor prognosis of HCC patients 13 . There is evidence that BCL9 is a bona fide oncogene 12,14,15 . However, the mechanism underlying the effect of BCL9 over-expression in tumors remains unclear.
It was found in this study that BCL9 was overexpressed in HCC specimens, especially in bone metastasis specimens. BCL9 was reported to be an important factor in determining the proliferation, migration, invasion and the metastatic potential of multiple myeloma and colon carcinoma cells 12 . In this study, we also found that BCL9 promoted cell proliferation, migration and angiogenesis by overexpressing or knocking down BCL9 in HepG2 and smmc-7721 cell lines. These data together indicate that BCL9 plays a role in the progression and metastasis of human HCC.
Previous studies 15,36 showed reduced BCL9 mRNA and protein expressions upon ectopic expression of miR-30s, suggesting that the expression of BCL9 was regulated by miR-30s post-tanscriptionally. However, the mechanism for BCL9 transcription in tumors has not been reported. Here we provided evidence for the first time that hypoxia, which is very common in solid tumors, increased the expression of BCL9 in human HCC cell lines. Cellular responses to hypoxia are mediated by HIFs. In humans, HIFs are heterodimeric transcription factors consisting of HIF-α and HIF-β that are expressed constitutively at the transcriptional and translational levels.
Once activated under hypoxia, HIFs bind to DNA containing a HRE (5′ -G/ACGTG-3′ ) 37 . HIF-1α and HIF-2α are the two best-studied members of the HIF-α family 38 . HIF-1α and HIF-2α proteins can form dimers with HIF-β and bind to HRE to effectively regulate the expression of their target genes 39 . Although HIF-1α and HIF-2α induce a number of common genes, and at the same time they have their respective unique target genes 40,41 . In addition, the difference in HIF1α and HIF2α target gene selection can be attributed to their interactions with different transcription factors and/or chromatin context 42,43 . Therefore, experiments elaborating the precise roles of each subunit are needed 19,44 . In the current study, we found that BCL9 was a target gene of HIF-1α that interacted with HRE-B and HRE-C but not HRE-A in the BCL9 promoter under the hypoxic condition. Of relevance the expression of BCL9 and HIF1α transcripts was significantly correlated, because both transcripts were highly expressed in HCC specimens. To the best of our knowledge, it is the first time to demonstrate that HIF1α rather than HIF2α interacted with HRE-B and HRE-C in the BCL9 promoter to directly induce BCL9 expression under the hypoxic condition.
The hypoxia signaling pathway is known to regulate multiple steps of tumorigenesis and related to poor clinical outcomes 45 . Due to restricted blood flow, solid tumor cells may experience hypoxia, which reduces cell proliferation or death, or even energy metabolism from the oxidative phosphorylation pathway to the glycolysis pathway as a response to hypoxia, resulting in modifications of hypoxia-inducible genes expression and treatment outcome in cancer patients 46 . HCC was also reported to display hypoxia, which could be a potential therapeutic target 47 . It was reported that the Wnt/β -catenin signaling pathway was activated at least 95% HCC patients 48 . Several studies 26,49 . reported the crosstalk between Wnt/β -catenin and hypoxia signaling pathway, but the mechanism has not well documented due to the complexity of the network between Wnt/β -catenin and hypoxia signaling pathway. In this study, we for the first time provided the evidence of crosstalk between canonical Wnt/β -catenin and hypoxia signaling in that hypoxia led to the upregulation of the key β -catenin coactivator BCL9 in a HIF1α -dependent manner, suggesting that the hypoxia pathway might promote oncogenic Wnt/β -catenin signaling by recruiting HIF-α /BCL9 interaction. These findings may enlarge our views on the interaction of Wnt/β -catenin and hypoxia signaling pathways.
In conclusion, this study demonstrated that BCL9 promoted cell proliferation, migration and angiogenesis in human HCC cell lines. BCL9 expression could be stimulated by hypoxia. BCL9 induction under the hypoxic condition was predominantly mediated by HIF1α . Due to the critical role of BCL9 in promoting tumor development in the Wnt/β -catenin signaling pathway, the specific regulation of BCL9 expression by HIF1α is considered to be an underlying crosstalk between Wnt/β -catenin signaling and hypoxia signaling pathways. These results could serve as a foundation for study of new targets for development of potential therapeutic strategies by blocking HIF-1α /BCL9 signaling related overexpression in human HCC.