In this study, we found the increased expression of HIF-1α in HCC samples obtained from surgical resection. Ectopic expression profile of HIF-1α is correlated with poor prognosis and enhanced HCC invasion and metastasis. Further analysis showed that increased HIF-1α level was associated with loss of E-cadherin and overexpression of SNAI1, N-cadherin and Vimentin. Our data suggest that hypoxia may induce EMT of cancer cells in HCC.
To test this hypothesis, we treated HCC cells under hypoxic condition. We found that hypoxia could induce EMT in HCC cells and enhance cell migration and invasion. Furthermore, we found that induction of EMT by hypoxia was reversible when cells were returned to normoxic condition. In addition, we confirmed that hypoxia led to G0/G1 arrest of HCC cells, which is coincident with previous reports [37–41]. CoCl2-induced HIF-1α stabilization also promoted EMT in HCC cells. And shRNA-mediated HIF-1α suppression was able to prevent EMT. All these data confirm that HIF-1α is an important stimulatory factor of EMT process in HCC cells.
The downstream target genes regulated by HIF-1α are involved in angiogenesis, hypoxic metabolism, cancer cell survival and invasion [10, 42–46]. HIF-1α is also documented to be an upstream regulatory factor of many EMT modulators, such as SNAI1, twist, Zeb1, SIP1 and LOX . Recent studies revealed that HIF-1α-induced LOX overexpression promoted the metastasis of breast cancers in a mouse model and was correlated with poor prognosis of ER negative patients . Response to hypoxia was also utilized in tumor therapy in the field of gene therapy. Oncolytic adenoviruses were shown to selectively and effectively proliferate in cancer cells, when its E1B gene expression was driven by HRE-modulated promoters . It is well demonstrated that SNAI1 is an inducer of EMT and it plays an important role in induction of EMT in HCC cells [50, 51]. Thus, we investigated the potential effect of HIF-1α on SNAI1 expression.
Bioinformatics analysis on SNAI1 promoter identified two putative HREs, providing the possibility that HIF-1α can directly bind these sites and promoter SNAI1 transcription. Using luciferase report systems, we determined that vectors containing either of these two HREs had high luciferase activity in CoCl2-treated HCC cells. The vector containing -651 bp HRE apparently had higher luciferase expression than that harboring -541 bp HRE. Previous study has shown that hypoxia could induce Snail expression during EMT . Recently, Luo et al. demonstrated that HIF could directly regulated mouse Snail expression . Furthermore, it was reported that hypoxia induced EMT in melanoma via regulation of Snail by HIF-2α . So we confirmed that HIF-1α promoted the transcription of one of central EMT-inducer, SNAI1, in hypoxia-simulating HCC model.
Collectively, we present our hypothesis of hypoxia participating in EMT of HCC cells (Figure 5C). In hypoxic conditions of the primary solid tumor, the oxygen required for proline hydroxylase activity is absent. HIF-1α in turn escapes proteolysis, allowing for its entry into the nucleus. Then, it can dimerize with HIF-1β to form the active transcription-stimulating complex, which binds HRE in SNAI1 promoter to promote SNAI1 expression. The tumor cells acquire mesenchymal phenotype, disseminate from the primary tumors, penetrate extracellular matrix (ECM) and enter blood or lymphatic vessels. As soon as some of these tumors cells penetrate ECM and enter the parenchyma of targeting tissues or organs on the condition of reoxygenation, HIF-1α is rapidly oxidized at either or both of two proline residues by a proline hydroxylase enzyme. This hydroxylation permits the binding of the von hippel-landau protein (pVHL) to HIF-1α. Once bound, HIF-1α is polyubiquitinated and subsequently degraded in the proteasome. Subsequently, the mesenchymal tumor cells undergo MET. HIF-1α may play a central role in EMT induced by hypoxia. HIF-1α-SNAI1-EMT may be one of the key signal pathways.