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. 2009 Dec;11(12):1371-82.
doi: 10.1593/neo.91326.

Regulation of membrane-type 4 matrix metalloproteinase by SLUG contributes to hypoxia-mediated metastasis (VSports手机版)

Chi-Hung Huang  1 , Wen-Hao Yang, Shyue-Yih Chang, Shyh-Kuan Tai, Cheng-Hwei Tzeng, Jung-Yie Kao, Kou-Juey Wu, Muh-Hwa Yang
Affiliations

Regulation of membrane-type 4 matrix metalloproteinase by SLUG contributes to hypoxia-mediated metastasis

Chi-Hung Huang (V体育官网入口) et al. Neoplasia. 2009 Dec.

Abstract

The hypoxic tumor environment has been shown to be critical to cancer metastasis through the promotion of angiogenesis, induction of epithelial-mesenchymal transition (EMT), and acquisition of invasive potential VSports手机版. However, the impact of hypoxia on the expression profile of the proteolytic enzymes involved in invasiveness is relatively unknown. Membrane-type 4 matrix metalloproteinase (MT4-MMP) is a glycosyl-phosphatidyl inositol-anchored protease that has been shown to be overexpressed in human cancers. However, detailed mechanisms regarding the regulation and function of MT4-MMP expression in tumor cells remain unknown. Here, we demonstrate that hypoxia or overexpression of hypoxia-inducible factor-1alpha (HIF-1alpha) induced MT4-MMP expression in human cancer cells. Activation of SLUG, a transcriptional factor regulating the EMT process of human cancers, by HIF-1alpha was critical for the induction of MT4-MMP under hypoxia. SLUG regulated the transcription of MT4-MMP through direct binding to the E-box located in its proximal promoter. Short-interference RNA-mediated knockdown of MT4-MMP attenuated in vitro invasiveness and in vivo pulmonary colonization of tumor cells without affecting cell migratory ability. MT4-MMP promoted invasiveness and pulmonary colonization through modulation of the expression profile of MMPs and angiogenic factors. Finally, coexpression of HIF-1alpha and MT4-MMP in human head and neck cancer was predictive of a worse clinical outcome. These findings establish a novel signaling pathway for hypoxia-mediated metastasis and elucidate the underlying regulatory mechanism and functional significance of MT4-MMP in cancer metastasis. .

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Figures

Figure 1
Figure 1
Hypoxia or constitutive expression of HIF-1α(ΔODD) upregulates MT4-MMP expression. (A) Upper: Fold change of mRNA levels of HIF-1α and MT4-MMP by real-time RT-PCR analysis in FADU, SAS, and OECM-1 cells under normoxia versus hypoxia. Lower: Western blot analysis of HIF-1α and MT4-MMP expression in FADU, SAS, and OECM-1 cells under normoxia versus hypoxia. (B) Upper: relative mRNA expression levels of HIF-1α and MT4-MMP in FADU-HIF1α(ΔODD) versus FADU-cDNA3 (left) and SAS-HIF1α(ΔODD) versus SAS-cDNA3. Lower: HIF-1α and MT4-MMP protein levels in FADU-HIF1α(ΔODD) versus FADU-cDNA3 (left) and SAS-HIF1α(ΔODD) versus SAS-cDNA3. (C) siRNA-mediated repression of endogenous HIF-1α abolishes the induction of MT4-MMP (mRNA and protein levels) in SAS cells under hypoxia. Transfection of the vector containing a scrambled sequence against human transcriptome (si-scr) was used as a control for siRNA experiments. The Western blot of MT4-MMP in all panels revealed two bands (upper indicates pro form; lower, active form) indicated by black arrows. GAPDH was used as a loading control for Western blot analysis. N indicates normoxia; H, hypoxia. The asterisk (*) indicated statistical significance (P < .05) between experimental and control clones.
Figure 2
Figure 2
Mapping of the major regulatory region in MT4-MMP promoter responsible for HIF-1α-induced transcriptional activation, and hypoxia/HIF-1α upregulates the expression levels of SNAIL, TWIST, and SLUG. (A) Schematic representation of the promoter region of MT4-MMP and reporter constructs containing different lengths of MT4-MMP promoter. The E-boxes and hypoxia response element (HRE) are indicated. (B) Activation of MT4-Luc 862, MT4-Luc606, MT4-Luc433, MT4-Luc290, or MT4-Luc57 by hypoxia/HIF-1α overexpression. The luciferase activity/β-galactosidase of 293T cells cotransfected with MT4-Luc862/pcDNA3.1 was applied as the baseline control of other experiments (mean ± SD, n = 3; *P < .05 between experimental and control transfections). (C) Western blot analysis of TWIST, SNAIL, and SLUG expression in FADU and SAS cells under normoxia (N) versus hypoxia (H). (D) Western blot analysis of TWIST, SNAIL, and SLUG expression in FADU-HIF1α(ΔODD) versus FADU-cDNA3 (left) and SAS-HIF1α(ΔODD) versus SAS-cDNA3 (right).
Figure 3
Figure 3
SLUG activates MT4-MMP expression and is critical for HIF-1α-mediated MT4-MMP induction. (A) Schematic representation of the reporter constructs containing proximal promoter of MT4-MMP (MT4-Luc862) or E-cadherin (Ecad-Luc486). (B) Left: Activation of MT4-Luc862 by overexpression of TWIST, SNAIL, or SLUG. Right: Repression of Ecad-Luc486 by overexpression of TWIST, SNAIL, or SLUG. The luciferase activity/β-galactosidase of 293T cells cotransfected with MT4-Luc862/pcDNA3.1/pFLAG-CMV (left) or Ecad-Luc486/pcDNA3.1/pFLAG-CMV (right) was applied as the baseline control of experiments (mean ± SD, n = 3; *P < .05 between experimental and control transfections). (C) Upper: Fold change of mRNA levels of MT4-MMP by real-time RT-PCR analysis in SAS cells overexpressing TWIST, SNAIL, or SLUG. Lower: Western blot analysis of MT4-MMP expression in SAS cells overexpressing TWIST, SNAIL, or SLUG. (D) Upper: Relative mRNA expression levels of MT4-MMP in SAS-SLUG versus SAS-cDNA3. Lower: Western blot analysis of SLUG and MT4-MMP expression in SAS-SLUG versus SAS-cDNA3. (E) siRNA-mediated repression of SLUG abolishes the HIF-1α-mediated induction of MT4-MMP expression in SAS cells. Transfection of the vector containing a scrambled sequence against human transcriptome (si-scr) was used as a control for siRNA experiments. The Western blot of MT4-MMP in (C), (D), and (E) revealed two bands (upper indicates pro form; lower, active form) indicated by black arrows. GAPDH was used as a loading control for Western blot analysis. *Statistical significance (P < .05) between experimental and control clones.
Figure 4
Figure 4
Direct regulation of MT4-MMP by SLUG. (A) Schematic representation of the genomic organization of the promoter region of MT4-MMP and reporter constructs used in transient transfection assays. The constructs were wild-type (MT4-Luc862), E-Box-deleted (MT4-Luc433), or E-box-mutated (MT4-Luc862Mut). (B) Transcriptional activation of MT4-Luc862, but not MT4-Luc433 and MT4-Luc862Mut by SLUG, hypoxia or SLUG + hypoxia. The luciferase activity/β-galactosidase of 293T cells cotransfected with MT4-Luc862/pcDNA3.1 was applied as the baseline control of other experiments (mean ± SD, n = 3; *P < .05 between experimental and control transfections). (C) ChIP analysis of SAS-SLUG, SAS-HIF1α(ΔODD) versus SAS-cDNA3. Chromatin was incubated without antibody, with an IgG, or with an anti-SLUG antibody. The 196-bp fragment contains the Slug binding sequence, whereas the 146-bp fragment does not contain any Slug binding sequence. Schematic representation of the design of ChIP and control primers was shown in the upper panel. Input: 2% of total input lysate.
Figure 5
Figure 5
MT4-MMP is critical in HIF-1α or SLUG mediated invasion and pulmonary colonization of tumor cells. (A) Fold change of migratory ability of SAS-HIF1α(ΔODD) receiving siRNA against SLUG or MT4-MMP, and SAS-SLUG receiving siRNA-mediated MT4-MMP repression. (B) Fold change of invasiveness of SAS-HIF1α(ΔODD) receiving siRNA against SLUG or MT4-MMP, and SAS-SLUG receiving siRNA-mediated MT4-MMP repression. (C) Representative pictures of metastatic pulmonary nodules (indicated by black arrows) in mice receiving SAS-cDNA3, SAS-HIF1α(ΔODD)-si-scr, SAS-HIF1α(ΔODD)-si-SLUG, SAS-HIF1α(ΔODD)-si-MT4-MMP, SAS-SLUG-si-scr, and SAS-SLUG-si-MT4-MMP injections. (D) Fold change of pulmonary tumor nodules of SAS-HIF1α(ΔODD) receiving siRNA against SLUG or MT4-MMP, and SAS-SLUG receiving siRNA-mediated MT4-MMP repression. The migration/invasion/metastasis of SAS-cDNA3 clone was used as the baseline control of all experiments, whereas transfection of the vector containing a scrambled sequence (si-scr) was used as a control of siRNA experiments. *Statistical significance (P < .05) between the baseline control clone (SAS-cDNA3) and SAS-HIF1α(ΔODD)-si-scr/SAS-SLUG-si-scr; **statistical significance (P < .05) between siRNA experimental clones (si-SLUG or si-MT4-MMP) and si-scr clones.
Figure 6
Figure 6
MT4-MMP contributes to invasiveness and pulmonary colonization of tumor cells through an EMT-independent mechanism. (A) Western blot analysis MT4-MMP in SAS-MT4-MMP versus SAS-cDNA3. GAPDH was used as a loading control for Western blot analysis. The Western blot of MT4-MMP revealed two bands (upper indicates pro form; lower, active form) indicated by black arrows. (B) Fold change of migratory ability (left) and invasiveness (right) of SAS-MT4-MMP versus SAS-cDNA3. (C) Western blot analysis the epithelial (E-cadherin) and mesenchymal (N-cadherin) markers in SAS-MT4-MMP versus SAS-cDNA3. (D) Left: Representative pictures of metastatic pulmonary nodules (indicated by black arrows) in mice receiving SAS-MT4-MMP versus SAS-cDNA3 injections. Right: Number of metastatic nodules counted in mice receiving SAS-MT4-MMP versus SAS-cDNA3 injections. *Statistical significance (P < .05) between experimental clones and baseline control clone (SAS-cDNA3).
Figure 7
Figure 7
Coexpression of HIF-1α and MT4-MMP in HNSCC cases indicates a worse survival, and a proposed model of hypoxia induced metastasis through activation of MT4-MMP. (A) IHC staining of HIF-1α and MT4-MMP in two representative HNSCC cases with coexpression of HIF-1α/MT4-MMP (upper panel, cases A) and negative for both markers (lower panel, case B). N indicates normal epithelium; T, tumor tissues. The red arrows indicate the nucleus expression of HIF-1α, whereas the blue arrows indicate the membranocytoplasmic expression of MT4-MMP. Scale bars, 200 µm. (B) Left: Comparison of the overall survival period of patients categorized by HIF-1α/MT4-MMP IHC result. Right: Survival difference in HNSCC cases with or without HIF-1α/MT4-MMP coexpression. (C) A proposed model of hypoxia induced metastasis through MT4-MMP.
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