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. 2009 Mar 10;100(5):772-81.
doi: 10.1038/sj.bjc.6604919. Epub 2009 Feb 17.

HIF-1alpha determines the metastatic potential of gastric cancer cells

N Rohwer  1 , S Lobitz, K Daskalow, T Jöns, M Vieth, P M Schlag, W Kemmner, B Wiedenmann, T Cramer, M Höcker
Affiliations

HIF-1alpha determines the metastatic potential of gastric cancer cells

N Rohwer et al. Br J Cancer. .

Abstract

Gastric adenocarcinoma is characterised by rapid emergence of systemic metastases, resulting in poor prognosis due to vanished curative treatment options. Better understanding of the molecular basis of gastric cancer spread is needed to design innovative treatments. The transcription factor HIF-1alpha (hypoxia-inducible factor 1alpha) is frequently overexpressed in human gastric cancer, and inhibition of HIF-1alpha has proven antitumour efficacy in rodent models, whereas the relevance of HIF-1alpha for the metastatic phenotype of gastric adenocarcinoma remains elusive. Therefore, we have conducted a comprehensive analysis of the role of HIF-1alpha for pivotal metastasis-associated processes of human gastric cancer. Immunhistochemistry for HIF-1alpha showed specific staining at the invading tumour edge in 90% of human gastric cancer samples, whereas normal gastric tissue was negative and only a minority of early gastric cancers (T1 tumours) showed specific staining. Hypoxia-inducible factor 1alpha-deficient cells showed a significant reduction of migratory, invasive and adhesive properties in vitro. Furthermore, the HIF-1alpha-inhibitor 2-methoxy-estradiol significantly reduced metastatic properties of gastric cancer cells. The accentuated expression at the invading edge together with the in vitro requirement of HIF-1alpha for migration, invasion and adherence argues for a pivotal role of HIF-1alpha in local invasion and, ultimately, systemic tumour spread. These results warrant the exploration of HIF-1alpha-inhibiting substances in clinical treatment studies of advanced gastric cancer.

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Figures

Figure 1
Figure 1
Expression pattern of HIF-1α in human gastric cancer tissues. Paraffin sections were pretreated as described in Materials and Methods, and HIF-1α was visualised by means of immunohistochemistry. (A) Negative control staining. (B–E) Expression of HIF-1α in established human gastric cancers, showing that the vast majority of tumour cells were positively stained for HIF-1α over nuclei. Magnification × 100 (A and B) and × 200 (C–E).
Figure 2
Figure 2
Expression and inhibition of HIF-1α in the human gastric cancer cell line AGS. Cells were cultured under normoxia (N) or hypoxia (H) or treated with 100 μM DFO. (A) HIF-1α protein levels were analysed in nuclear extracts by western blot analysis, with YY1 serving as nuclear loading control. Induction of HIF-1α protein was quantified by densitometry. Hypoxia-inducible factor 1 protein was found to be expressed in AGS cells under both hypoxia and DFO treatment. (B) Transcription of the HIF-1 target genes 3-phosphoglycerate kinase (PGK) and carbonic anhydrase IX (CA IX) was analysed by quantitative real-time PCR. Hypoxic culture and treatment with DFO resulted in induction of PGK (**P⩽0.0086) and CA IX (**P⩽0.0060) mRNAs. Values were normalised to that of β-actin, and relative expression was compared with the same cell line in hypoxia or DFO treatment vs normoxia. Values are means±s.e.m. (C) Western blot analysis of nuclear extracts from knockdown (KD) and control (SCR) AGS cells under normoxia (N) or hypoxia (H) for 16 h. AGS KD cells were unable to express HIF-1α protein under hypoxic culture. Differences in hypoxia-induced nuclear HIF-1α protein levels were quantified by densitometry. (D) Confirmation of loss of HIF-1α function by HRE-luc reporter assay. AGS KD and SCR cells were co-transfected with an HRE-luc reporter and phRL-null Renilla as an internal control and incubated under either normoxia or hypoxia for 24 h. Inhibition of HIF-1α resulted in a significant decrease of HRE-luc reporter activity under hypoxic conditions (***P<0.0001). Luciferase activity, normalised to Renilla luciferase activity, was expressed relative to that of transfected control cells (SCR) under normoxia, set at 1.0. Results shown are representative of three independent experiments, and values represent the mean±s.e.m. of triplicate determinations. (E) Expression of HIF-1 target genes PGK and CA IX was measured relative to β-actin by quantitative real-time PCR. Inhibition of HIF-1α protein by RNAi resulted in decreased transcription of HIF-1 target genes PGK (*P=0.0125) and CA IX (*P=0.0421) in AGS KD cells. Transcription levels were expressed relative to that of control cells (SCR) under normoxia, set at 1.0. Values represent the means±s.e.m.
Figure 3
Figure 3
Effects of HIF-1α inhibition on growth of AGS and MKN28 cells in vitro. Anchorage-dependent proliferation of AGS (A) and MKN28 (B) knockdown (KD) and control (SCR) cells under normoxia (left panels) or hypoxia (right panels). Cells were counted every other day from day 2 to 6 using a hemacytometer. Gastric cancer KD cells grew at approximately the same rate as SCR cells under both normoxia and hypoxia. Results shown are representative of three independent experiments and values represent the mean±s.e.m. of duplicate determinations.
Figure 4
Figure 4
Effects of HIF-1α inactivation on migration, invasion and adhesion of AGS and MKN28 cells. Migration of AGS (A) and MKN28 (B) knockdown (KD) and control (SCR) cells was evaluated in 24-well Transwell chambers (8 μm pore size) under normoxia or hypoxia for 24 h. Directed migration of KD cells was significantly impaired compared with SCR cells under both normoxia and hypoxia (**P⩽0.0056; *P⩽0.0281). The number of migrated cells on the bottom side of the filter was determined and normalised to the number of migrated SCR cells under normoxic conditions. Data represent mean±s.e.m. of a representative out of three experiments, each performed in duplicate. Photos show the bottom of representative migration filters. (C) For invasion assay, AGS KD and SCR cells were seeded into Matrigel-coated transwell inserts (8 μm pore size) and incubated under normoxia or hypoxia for 24 h. Hypoxia decreased the invasion of both AGS KD and SCR cells. Inactivation of HIF-1α reduced directed invasion of AGS cells significantly under normoxia and hypoxia (*P⩽0.0266; **P=0.0048). The number of invading cells was normalised to the number of invading SCR cells under normoxic conditions. Bars show means±s.e.m. of a representative experiment out of two experiments, each performed in duplicate. Photos show the bottom of representative invasion filters. (D) For adhesion assay, BCECF/AM labelled AGS KD and SCR cells were added to an HUVEC monolayer and allowed to adhere for 30 min under normoxia or hypoxia. Adhesion of AGS cells to HUVEC endothelial cells was reduced under normoxic and hypoxic conditions by loss of HIF-1α (*P=0.0327; **P⩽0.0025). Adhesion was expressed relative to that of control cells (SCR) under normoxic conditions. Shown are mean±s.e.m. of three independent experiments, each performed in triplicate.
Figure 5
Figure 5
Effects of 2ME2 on HIF-1α protein and transcriptional activity and on metastatic properties of AGS cells. (A) AGS cells were treated for 24 h with vehicle or increasing concentrations of 2ME2, and inhibition of HIF-1α was analysed by western blot analysis. Treatment with 2ME2 led to a dose-dependent reduction of HIF-1α protein. (B) HRE-luc reporter assay. AGS cells transfected with an HRE-luc reporter, and phRL-null Renilla were treated with vehicle or increasing concentrations of 2ME2 under either normoxia or hypoxia for 24 h. Exposure to 2ME2 resulted in a significant decrease of HRE-luc reporter activity under hypoxic conditions (***P=0.0004; **P⩽0.0089). Luciferase activity, normalised to Renilla luciferase activity, was expressed relative to that of transfected control cells under normoxia, set at 1.0. Results shown are representative of three independent experiments, and values represent the mean±s.e.m. of triplicate determinations. (C) Migration of AGS cells was evaluated in 24-well transwell chambers (8 μm pore size) after treatment with either vehicle or increasing concentrations of 2ME2 under normoxia for 24 h. Directed migration of 2ME2-treated cells was significantly impaired compared with vehicle-treated cells (*P=0.0246; **P=0.002; ***P=0.001). The number of migrated cells on the bottom side of the filter was determined and normalised to the number of vehicle-treated cells. Results shown are representative of three independent experiments, and values represent the mean±s.e.m. of duplicate determinations. Photos show the bottom of representative migration filters. (D) For invasion assay, AGS cells were seeded into Matrigel-coated transwell inserts (8 μm pore size) after pretreatment with either vehicle or 10 μM 2ME2 for 24 h. Invasion of 2ME2-treated cells was significantly reduced compared with vehicle-treated cells (***P<0.0001). Bars show means±s.e.m. of a representative experiment out of two experiments, each performed in duplicate. Photos show the bottom of representative invasion filters. (E) For adhesion assay, AGS cells were pretreated with either vehicle or 10 μM 2ME2 for 24 h, labelled with BCECF/AM and allowed to adhere to HUVEC endothelial cells for 30 min. Treatment with 2ME2 significantly reduced adhesion of AGS cells to an HUVEC monolayer (***P=0.0002). Shown are means±s.e.m. of two independent experiments, each performed in triplicate.
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