Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 7, 40446

Hypoxia Activates Wnt/β-catenin Signaling by Regulating the Expression of BCL9 in Human Hepatocellular Carcinoma

Affiliations

Hypoxia Activates Wnt/β-catenin Signaling by Regulating the Expression of BCL9 in Human Hepatocellular Carcinoma

Wei Xu et al. Sci Rep.

Abstract

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.

Figures

Figure 1
Figure 1. BCL9 expression in primary and metastastic HCC tissue.
Representative immunohistochemical stained imaging of BCL9 expression in different developing stages of HCC tissue containing 30 normal liver tissues, 360 primary HCC tissues and 72 bone metastatic HCC tissues. (A) No BCL9 staining in the normal mucosal epithelium; (B) low intensity expression in primary HCC tissue scored as negative (−); (C) moderate intensity expression in primary HCC tissue scored as weak (+); (D) high intensity expression in primary HCC tissue scored as strong (++); (E) BCL9 staining in bone metastatic HCC tissues. Scale bar: 40 μm for low magnitude image (10×); 10 μm for high magnitude images (40×).
Figure 2
Figure 2. BCL9 promotes cell proliferation, migration, and angiogenesis.
(A) Over-expression and knockdown BCL9 in HepG2 and smmc-7721 cell lines. (B) BCL9 over-expression significantly increases cell viability, while BCL9 knockdown reduces cell viability in HepG2 and smmc-7721 cell lines. (C) BCL9 over-expression significantly increases the cell migration ability, while BCL9 knockdown reduces the cell migration ability in HepG2 and smmc-7721 cell lines. (D) BCL9 over-expression significantly increases the angiogenesis activity, while BCL9 knockdown reduces the angiogenesis activity in HepG2 cell line. Data are presented as mean ± SD (n = 3). *: p < 0.01, Student’s t-test.
Figure 3
Figure 3. Hypoxia induces BCL9 expression in HCC cells.
Human HCC cell lines HepG2 and SMMC-7721 cells were cultured under the hypoxic condition for the indicated time periods. (A) The mRNA expression levels of BCL9 in these cells were determined by Taqman real-time PCR and normalized with actin. (B) The mRNA expression levels of VEGF in these cells were determined as a positive control. (C) The BCL9, HIF-1α and HIF-2α protein levels were determined by Western-blot assays. (D) Significant increase in TOPflash activity was observed under hypoxia treatment. Further, the TOPflash activity was significantly increased when BCL9 overexpressed while decreased when BCL9 knocked down. Data are presented as mean ± SD (n = 3).*p < 0.01, Student’s t-test.
Figure 4
Figure 4. Hypoxia transactivates hypoxia-responsive elements (HREs) in the BCL9 promoter which transcriptionally regulated by HIF-1α.
(A) The human BCL9 gene contains 3 putative HREs in its promoter region. (B) Hypoxia activates the luciferase activity of reporter vectors containing HRE-B or HRE-C sites in the BCL9 promoter. HepG2 and SMMC-7721 cells were transfected with the luciferase reporter vectors, and then subjected to hypoxia treatment for 36 h before measuring luciferase activities. Luciferase reporter vectors containing the HRE site in the VEGF promoter was included as a positive control. (C,D) HIF-1α but not HIF-2α binds to HRE-B and HRE-C sites in the BCL9 promoter under the hypoxic condition in HepG2 cells as determined by ChIP assays. Cells were cultured under the hypoxic or normoxic conditions for 36 h before assays. The HRE site in the VEGF promoter serves as a positive control. The amount of DNA fragments pulled- down was determined by real-time PCR (C) or conventional PCR (D). (E) Ectopic HIF-1α expression increases BCL9 protein levels in HepG2 cells as determined by Western-blot assays. (F,G) HIF-1α binds to HRE-B and HRE-C sites in the BCL9 promoter in HepG2 cells transfected with HIF-1α expression plasmids as determined by ChIP assays. The amount of DNA fragments pulled-down was determined by real-time PCR (F) or conventional PCR (F). The HRE site in the VEGF promoter serves as a positive control. Data are presented as mean ± SD (n = 3). *p < 0.01 (Student’s t-test).
Figure 5
Figure 5. Genetic engineering of HepG2 cells using TALENs.
(A) Schematic overview depicting the targeting strategy for BCL9 promoter. Primers are shown as red boxes; the blue arrow indicates the cut site by the TALENs. Donor plasmids: CMV promoter, human cytomegalovirus (CMV) immediate early promoter gene; eGFP, enhanced green fluorescent protein gene; Below, scheme of BCL9 TALENs and their recognition sequence. TALE repeat domains are colored to indicate the identity of the repeat variable diresidue (RVD); each RVD is related to the cognate targeted DNA base by the following code (N1 = A, HD = C, NN = G, NG = T). (B) Genomic PCR and restriction digestion characterization of BCL9-p-ko HepG2 cells. (C) Different TALEN pairs were designed. (D) 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. (E) The genomic sequences around the target site of the clones were detected. (F) BCL9-wt, BCL9-p-ko and BCL9-ko HepG2 cells were cultured under the hypoxic condition. The BCL9 and HIF-1α protein levels were determined by Western-blot assays. Data are presented as mean ± SD (n = 3). *p < 0.01, Student’s t-test.
Figure 6
Figure 6. BCL9 promotes the tumor growth in vivo.
(A) The HepG2 cells infected with BCL9-wt, BCL9-p-ko and BCL9-ko were injected subcutaneously into the nude mice. (B) Mice were sacrificed to remove the tumors, and images were taken with a Nikon camera. (C) Tumor weight was evaluate among BCL9-wt, BCL9-p-ko and BCL9-ko group. (D) HIF-1α (red) and BCL9 (green) detected by FISH and IF in tumors: HIF-1α normally expressed in BCL9-wt, BCL9-p-ko and BCL9-ko groups; BCL9 normally expressed in BCL9-wt, weakly expressed in BCL9-p-ko group and unexpressed in BCL9-ko group. (E,F) Angiogenesis was detected by IHC and RT-PCR: angiogenesis was significantly decreased in BCL9-p-ko and BCL9-ko group. (G) WNT/β-catenin target genes, including CD44, Axin-2 and Survivin, were detected by RT-PCR. CD44, Axin-2 and Survivin were significantly decreased in BCL9-p-ko and BCL9-ko group. Data are presented as mean ± SD (n = 3). *p < 0.01, Student’s t-test.
Figure 7
Figure 7. HIF-1α overexpression is associated with BCL9 overexpression in human HCC.
HIF-1α and BCL9 protein levels were detected by IHC staining in tissue microarrays containing 488 cases of human HCC specimens. (A) HIF-1α (red) and BCL9 (green) detected by FISH and IF in human HCC specimens. (B) HIF-1α overexpression is associated with BCL9 overexpression in human HCC (p < 0.000, Fisher exact test).

Similar articles

See all similar articles

Cited by 16 PubMed Central articles

See all "Cited by" articles

References

    1. El-Serag H. B. & Rudolph K. L. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 132(7), 2557–2576 (2007). - PubMed
    1. Miyoshi Y. et al. . Activation of the beta-catenin gene in primary hepatocellular carcinomas by somatic alterations involving exon 3. Cancer Res 58(12), 2524–2527 (1998). - PubMed
    1. Wands J. R. & Kim M. WNT/beta-catenin signaling and hepatocellular carcinoma. Hepatology 60(2), 452–454 (2014) - PubMed
    1. Clevers H. Wnt/beta-catenin signaling in development and disease. Cell 127(3), 469–480 (2006) - PubMed
    1. Klaus A. & Birchmeier W.: Wnt signalling and its impact on development and cancer. Nat Rev Cancer 8(5), 387–398 (2008). - PubMed

Publication types

MeSH terms

Substances

Feedback