HIF-1α Promotes the Metastasis of Esophageal Squamous Cell Carcinoma by Targeting SP1

Abstract

Background: In microenvironment of malignant tumors, Hypoxia-Inducible Factors (HIF), most importantly HIF-1α, play an important role in regulation of adaptive biological response to hypoxia, promoting angiogenesis and metastasis. However, the underlying mechanism that HIF-1α regulates metastasis needs to be further clarified. Methods: The expressions of HIF-1α and SP1 were detected in 182 samples of esophageal squamous cell carcinoma (ESCC) and adjacent normal tissues by immunohistochemistry (IHC), and the correlation between the expression levels of HIF-1α and SP1 was analyzed. The expression of HIF-1α in ESCC cell lines TE1 and KYSE30 was then detected using qRT-PCR and western blot. The potential binding sites of HIF-1α on the SP1 promoter were analyzed using UCSC and JASPAR databases, verified by chromosomal immunoprecipitation (ChIP) assay and qRT-PCR. The effects of HIF-1α and SP1 on ESCC cell migration and invasion were then tested with Transwell and Matrigel experiments. Results: The expression of HIF-1α in cancer tissues is higher than adjacent normal tissues, and is correlated with metastasis, recurrence and poor prognosis. Upon silencing HIF-1α by siRNA, the invasion and migration ability of ESCC cells were significantly inhibited, which could be restored by the overexpression of SP1. Hypoxic conditions significantly increased the expression of HIF-1α and SP1 at both protein and mRNA levels in ESCC cells. HIF-1α enhanced SP1 transcription through binding to the promoter region. The expression of protein and mRNA levels of SP1 was decreased by silencing HIF-1α in cells. In contrast, overexpression of HIF-1α significantly increased the mRNA and protein levels of SP1. The expression of SP1 in ESCC was positively correlated with the protein expression of HIF-1α and poor prognosis. Conclusion: The results of our study indicate that HIF-1α promotes metastasis of ESCC by targeting SP1 in a hypoxic microenvironment. Further study on this mechanism may elucidate the possibility of HIF-1α and SP1 as new targets for the treatment of ESCC.

Keywords: ESCC; HIF-1α; SP1; tumor metastasis.

Figure 1

The expression of HIF-1α in ESCC tissues and the functions of HIF-1α in ESCC cells. a The expression of HIF-1α in ESCC detected by IHC, and the immunoreactivity score of HIF-1α in ESCC tumor tissues and adjacent normal tissues(n=182); b the 3-year regional recurrence curve of patients with HIF-1α high/low expression; c the 5-year survival curve of patients with HIF-1α high/low expression; d silencing HIF-1α by siRNA significantly suppressed the migration and invasion of TE1 and KYSE30, (n=3). Data represent the mean ± S.D; e overexpression of HIF-1α promoted migration and invasion of TE1 and KYSE30, (n=3). Data represent the mean ± S.D. (** P<0.01, *** P<0.001).

Figure 2

Effects of SP1 overexpression on the migration and invasion of hypoxic ESCC cells. a, b migration and invasion of TE1 and KYSE30 cells can be blocked by HIF-1α silencing, and can be rescued by SP1 overexpression (n=3). Data represent the mean ± S.D. (*** P<0.001).

Figure 3

HIF-1α up-regulates the protein expression of SP1 and its transcriptional activity by directly binding to the SP1 promoter. a Schematic diagram depicting the positions of the primers used for the ChIP assay; b, c ChIP analysis was performed using a negative control immunoglobulin G (IgG) or anti- HIF-1α antibody in TE1 cells. Input positive control (anti- ARRDC3 antibodies); d HIF-1α and SP1 mRNA levels under both normoxic and hypoxic conditions were analyzed by real-time PCR. β-Actin was used as the internal control; e HIF-1α and SP1 protein levels under both normoxic and hypoxic conditions(48h) were analyzed by Western blotting. β-Actin was used as the internal control; f a knockdown efficiency of HIF-1α siRNA, and the mRNA levels of SP1 were detected by real-time PCR. β-Actin was used as the internal control (n=3); g a knockdown efficiency of HIF-1α siRNA, and the protein levels of SP1 were analyzed by Western blotting. β-Actin was used as the internal control (n=3); h the efficiency of over expression of HIF-1α, and the protein levels of SP1 were analyzed by Western blotting, the mRNA levels of SP1 were detected by real-time PCR. β-Actin was used as the internal control (n=3). (** P<0.01, *** P<0.001).

Figure 4

Co-expression of HIF-1α and SP1 in ESCC tissues. a The expression of SP1 in ESCC tissues detected by IHC, the immunoreactivity score of SP1 in ESCC tumor tissue and adjacent normal tissue (n=182); b the 3-year regional recurrence curve of patients with SP1 high/low expression; c the 5-year survival curve of patients with SP1 high/low expression; d the expression of HIF-1α and SP1 in ESCC detected by IHC, the immunoreactivity score of SP1 in HIF-1α high expression(n=102) and HIF-1α low expression(n=80) groups; e the correlation between the expression of HIF-1α and SP1 in esophageal cancer tissues; f, g the percent number of high/low expression of HIF-1α and SP1 in metastatic and non-metastatic groups; h the 5-year survival curve of patients with HIF-1α and SP1 positive/negative expression; i the 3-year regional recurrence curve of patients with HIF-1α and SP1 positive/negative expression. (** P<0.01, *** P<0.001).