BTB/POZ domain‐containing protein 7/hypoxia‐inducible factor 1 alpha signalling axis modulates hepatocellular carcinoma metastasis

Dear Editor, This study is the first to demonstrate that HIF-1α could activate BTBD7 under hypoxia, thus promotingHCC metastasis through tumor cell adhesion andEMT.Hypoxia in the tumour microenvironment influences the entire stages of hepatocellular carcinoma (HCC) metastasis with unarticulated mechanisms.1 Activation of hypoxiainducible factor (HIF) signalling promotes the invasion of tumour cells and infiltrating immune cells and naturally selects the tumour cells that survived under hypoxia.2 Previously, we have confirmed that BTBD7 in HCC microenvironment canpromote epithelial-mesenchymal transition (EMT) and stimulate angiogenesis in vitro and in vivo.3 However, BTBD7 does not be concerned about hypoxiaassociated particular mechanisms. We observed increased BTBD7 mRNA and protein expression in HCC cells (MHCC97L and HCCML3) under hypoxia comparedwith normoxia condition (Figure 1A, B). The qPCR results of 78 paired HCC tissues and adjacent non-tumorous tissues demonstrated that HIF-1α mRNA was significantly overexpressed in HCC (Figure S1), suggesting that HIF-1α was probably involved in hypoxiamediated BTBD7 upregulation, which was preliminarily confirmed by using theHIF-1α activators (CoCl2 andDFO) or inhibitors (17-AAG andDeguelin) (Figures 1C and S2) in HCC cells. The correlation between HIF-1α and BTBD7 expression in HCCwas further investigated in a training cohort of 104 HCC patients using a western blot, showing higher expressions of HIF-1α and BTBD7 in HCC tissues characterizedwith tumour haemorrhage plus necrosis (THPN) (Figure 1D, E). THPN subtype in HCC reflected much severer hypoxia level, higher incidence of intrahepatic metastasis and vessel invasion (Figure S3). Immunohistochemistry of HIF-1α and BTBD7 also gave consistent results (Figure 1F). HIF-1α protein positively correlates with BTBD7 protein


BTB/POZ domain-containing protein 7/hypoxia-inducible factor 1 alpha signalling axis modulates hepatocellular carcinoma metastasis
Dear Editor, This study is the first to demonstrate that HIF-1α could activate BTBD7 under hypoxia, thus promoting HCC metastasis through tumor cell adhesion and EMT. Hypoxia in the tumour microenvironment influences the entire stages of hepatocellular carcinoma (HCC) metastasis with unarticulated mechanisms. 1 Activation of hypoxiainducible factor (HIF) signalling promotes the invasion of tumour cells and infiltrating immune cells and naturally selects the tumour cells that survived under hypoxia. 2 Previously, we have confirmed that BTBD7 in HCC microenvironment can promote epithelial-mesenchymal transition (EMT) and stimulate angiogenesis in vitro and in vivo. 3 However, BTBD7 does not be concerned about hypoxiaassociated particular mechanisms.
We observed increased BTBD7 mRNA and protein expression in HCC cells (MHCC97L and HCCML3) under hypoxia compared with normoxia condition ( Figure 1A, B). The qPCR results of 78 paired HCC tissues and adjacent non-tumorous tissues demonstrated that HIF-1α mRNA was significantly overexpressed in HCC ( Figure S1), suggesting that HIF-1α was probably involved in hypoxiamediated BTBD7 upregulation, which was preliminarily confirmed by using the HIF-1α activators (CoCl 2 and DFO) or inhibitors (17-AAG and Deguelin) ( Figures 1C and S2) in HCC cells.
The correlation between HIF-1α and BTBD7 expression in HCC was further investigated in a training cohort of 104 HCC patients using a western blot, showing higher expressions of HIF-1α and BTBD7 in HCC tissues characterized with tumour haemorrhage plus necrosis (THPN) (Figure 1D, E). THPN subtype in HCC reflected much severer hypoxia level, higher incidence of intrahepatic metastasis and vessel invasion ( Figure S3). Immunohistochemistry of HIF-1α and BTBD7 also gave consistent results ( Figure 1F Figure 1G). These data indicated that HIF-1α might upregulate BTBD7, thus enhancing HCC metastasis.
Next, we performed invasion assays to explore the roles of BTBD7 and HIF-1α in HCC metastasis under hypoxia. The invasion capability of HCC cells was critically disrupted after the co-knockdown of BTBD7 and HIF-1α, and was moderately decreased when si-BTBD7 or si-HIF-1α was transfected separately ( Figure S4). Besides, the overexpression of BTBD7 or HIF-1α enhanced the invasion activity of HepG2 cells and exhibited a combined effect when BTBD7 and HIF-1α were co-overexpressed ( Figure 2E). To recapitulate this in vivo, we established HepG2 cells with stable over-expressed BTBD7 or HIF-1α or both, then built xenograft tumour-bearing nude mice. The results showed that the most increased intrahepatic and pulmonary metastatic nodules and microvascular density were found in the co-overexpressed group compared with other groups (Figures 2F-H).
Cell adhesion was measured after the overexpression of BTBD7 or HIF-1α with the consideration of the crosstalk between HIF-1α and the α5β1-integrin signalling pathway in cell-matrix adhesion. 4 The cell adhesion to collagen, fibronectin and laminin increased 1.5-to 2.5-fold (Figure 3A, B). Then, western blot was performed to identify the adhesion-associated molecules regulated by BTBD7. We found elevated expression of α5β1-integrin, fibronectin, p-FAK, p-Smad2, p-STAT3, Ikk-β and MMP-9 in HepG2 cells that overexpressed BTBD7 or HIF-1α or both (Figure 3C). The antibodies against α5-integrin and β1-integrin showed a synergistic effect with si-BTBD7 in decreasing the HCC cell-matrix adhesion ( Figure 3D). Besides, the enhanced adhesion to collagen and laminin was blocked by an anti-fibronectin antibody ( Figure S5). These results indicated that BTBD7 and HIF-1α could positively modulate HCC cell adhesion.
Both cell adhesion and EMT are induced by hypoxia to promote HCC metastasis. 5,6 Therefore, we explored whether BTBD7 played a significant role in hypoxiainduced EMT through α5β1-integrin. Either knockdown of BTBD7 or anti-α5β1-integrin significantly reduced invasion ability in MHCC97L cells induced by hypoxia (Figure 3E), inhibited the formation of typical pipe-like structure within the Matrigel and decreased the in vitro F-actin stress fibre formation induced by hypoxia in MHCC97L cells ( Figure 3F). Western blot and qRT-PCR assays showed the upregulation of E-cadherin expression and abolishment of fibronectin and vimentin upregulation in MHCC97L cells under si-BTBD7 or anti-α5β1-integrin (Figures 3G and S6). These results supported that activation of BTBD7 by HIF-1α was required for hypoxia-induced HCC cells EMT via the α5β1-integrin pathway.
Given the central role of the HIF-1α/BTBD7 axis in HCC adhesion and EMT, we chose Fasudil, the HIF-1α inhibitor, 7 as a potential therapeutic agent in BTBD7-positive HCC. Fasudil treatment induced a dosedependent decrease expression of HIF-1α and BTBD7 in the MHCC97L cells under hypoxia ( Figure S7). Administration of Fasudil in HepG2-LV-BTBD7 xenograft tumourbearing nude mice obviously inhibited tumour growth ( Figure 4A) and metastasis ( Figure 4B, C). Meanwhile, the administration of Fasudil significantly downregulated the levels of BTBD7, HIF-1α, TGF-β1 and α5β1-integrin and upregulated the level of E-cadherin ( Figure 4D).
Univariate and multivariate analyses revealed that BTBD7 and HIF-1α expression were independent predictors of cumulative overall survival (OS) and time to recurrence in the training cohort (Table S1). BTBD7 and HIF-1α could act as a combined biomarker on the prognosis of HCC because three subgroups stratified according to BTBD7 and HIF-1α showed a significant difference in OS and recurrence rates in both training and validation cohorts ( Figure 4E, F). High expression of both BTBD7 and HIF-1α indicated a poor prognosis.
In summary, this study is the first to elucidate that HIF-1α and BTBD7 induced by hypoxic tumour microenvironment can promote HCC tumour cell adhesion and EMT with clinical prognostic and therapeutic significance ( Figure 4G). TNPH may serve as a visualized and convenient phenotype to indicate high expression of HIF-1α and BTBD7 for preliminary clinical screening.

Funding information
This study was supported by the National Natural Science