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. 2010 Aug 10;5(8):e12038.
doi: 10.1371/journal.pone.0012038.

Hypoxia-inducible factor 1alpha determines gastric cancer chemosensitivity via modulation of p53 and NF-kappaB

Nadine Rohwer  1 , Christof Dame, Anja Haugstetter, Bertram Wiedenmann, Katharina Detjen, Clemens A Schmitt, Thorsten Cramer
Affiliations

Hypoxia-inducible factor 1alpha determines gastric cancer chemosensitivity via modulation of p53 and NF-kappaB

Nadine Rohwer et al. PLoS One. .

Abstract

Background: Reduced chemosensitivity of solid cancer cells represents a pivotal obstacle in clinical oncology. Hence, the molecular characterization of pathways regulating chemosensitivity is a central prerequisite to improve cancer therapy. The hypoxia-inducible factor HIF-1alpha has been linked to chemosensitivity while the underlying molecular mechanisms remain largely elusive. Therefore, we comprehensively analysed HIF-1alpha's role in determining chemosensitivity focussing on responsible molecular pathways.

Methodology and principal findings: RNA interference was applied to inactivate HIF-1alpha or p53 in the human gastric cancer cell lines AGS and MKN28. The chemotherapeutic agents 5-fluorouracil and cisplatin were used and chemosensitivity was assessed by cell proliferation assays as well as determination of cell cycle distribution and apoptosis. Expression of p53 and p53 target proteins was analyzed by western blot. NF-kappaB activity was characterized by means of electrophoretic mobility shift assay. Inactivation of HIF-1alpha in gastric cancer cells resulted in robust elevation of chemosensitivity. Accordingly, HIF-1alpha-competent cells displayed a significant reduction of chemotherapy-induced senescence and apoptosis. Remarkably, this phenotype was completely absent in p53 mutant cells while inactivation of p53 per se did not affect chemosensitivity. HIF-1alpha markedly suppressed chemotherapy-induced activation of p53 and p21 as well as the retinoblastoma protein, eventually resulting in cell cycle arrest. Reduced formation of reactive oxygen species in HIF-1alpha-competent cells was identified as the molecular mechanism of HIF-1alpha-mediated inhibition of p53. Furthermore, loss of HIF-1alpha abrogated, in a p53-dependent manner, chemotherapy-induced DNA-binding of NF-kappaB and expression of anti-apoptotic NF-kappaB target genes. Accordingly, reconstitution of the NF-kappaB subunit p65 reversed the increased chemosensitivity of HIF-1alpha-deficient cells.

Conclusion and significance: In summary, we identified HIF-1alpha as a potent regulator of p53 and NF-kappaB activity under conditions of genotoxic stress. We conclude that p53 mutations in human tumors hold the potential to confound the efficacy of HIF-1-inhibitors in cancer therapy.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. HIF-1α mediates resistance towards the chemotherapeutic agent 5-FU in AGS cells.
(A) Proliferation of AGS KD and SCR cells 24 h after treatment with increasing concentrations of 5-FU under normoxic conditions. Cell numbers are shown as percent of untreated cells (*, P<0.05; **, P<0.01). (B) AGS wild-type cells were transfected with HIF-1α expression vector (pcDNA HIF-1α) or empty vector (pcDNA 3.1) and treated with 5-FU 24 h post transfection. Cell numbers were determined 24 h after treatment with 5-FU, and are shown as percent of untreated control cells (**, P<0.01). (C) AGS wild-type cells were co-transfected with either pcDNA HIF-1α or pcDNA 3.1 plus HRE-Luciferase reporter (pHRE-Luc) and Renilla reporter (phRL-null) as internal control. Cells were harvested 48 h post transfection. HRE luciferase activity, normalised for Renilla luciferase activity, was expressed relative to that of control transfected cells (***, P<0.001).
Figure 2
Figure 2. HIF-1α mediates resistance towards 5-FU by blocking p53-dependent G1 arrest and apoptosis.
(A) AGS KD and SCR cells were treated for 24 h with 10 µg/ml 5-FU, and cell cycle distribution was determined by FACS analysis (**, P<0.01). (B) Chemotherapy-induced senescence was quantified in AGS KD and SCR cells 4 days after treatment with 5-FU by measurement of SA-β-Gal activity (**, P<0.01). (C) AGS KD and SCR cells were treated for 48 h with 10 µg/ml 5-FU and apoptosis was quantitated based on detection of active caspase-3 using flow cytometry (**, P<0.01). (D) Representative immunoblot analysis of p53, p21, CDK2, cyclin A and pRb protein levels in AGS KD and SCR cells treated with 10 µg/ml 5-FU for 6 and 24 h. Actin served as loading control. ppRb, phosphorylated pRb.
Figure 3
Figure 3. siRNA silencing of p53 reverses the chemosensitization of AGS KD cells.
AGS cells were transfected with control siRNA (si scr) or siRNA against p53 (si p53), and 10 µg/ml 5-FU was added 24 h post transfection. (A) Immunoblot analysis for p53, p21 and MDM2 in whole cell extracts from AGS KD cells after 5-FU treatment. Actin served as loading control. (B and C) Cell numbers of si scr and si p53 transfected AGS cells was determined 24 h after treatment with 5-FU. Results are shown as percent of untreated control cells (**, P<0.01). Cells in the G1 phase (D) and thesub-G1 population (E) were evaluated from DNA histograms of AGS KD cells transfected with si p53 or si scr and treated for 24 h with 5-FU (*, P<0.05; **, P<0.01).
Figure 4
Figure 4. Effects of 5-FU on MKN28 cells with mutant p53.
(A) Cell numbers of MKN28 KD and SCR cells 24 h after treatment with 5-FU under normoxic conditions. Data are shown as percent of untreated cells. Cells in G1 phase (B) and apoptotic cells (C) were quantitated from DNA histograms of MKN28 KD and SCR cells treated for 48 h with 10 µg/ml 5-FU. (D) Immunoblot analysis of p53, p21 and pRb protein levels in MKN28 KD and SCR cells treated with 10 µg/ml 5-FU for 6 and 24 h. Actin served as loading control.
Figure 5
Figure 5. HIF-1α-mediated activation of NF-κB limits the toxicity of 5-FU.
(A) Nuclear extracts of AGS KD and SCR cells were prepared at the indicated time points after treatment with 10 µg/ml 5-FU or TNFα as a positive control, and DNA-binding activity for NF-κB was examined by EMSA. For supershift experiments, nuclear extracts were incubated with an anti-p65 antibody. (B) Expression of NF-κB target genes cIAP1 and A20 mRNA in total RNA extracts from AGS KD and AGS SCR cells 48 h after treatment with 10 µg/ml 5-FU. Data were expressed relative to mRNA levels in untreated AGS SCR cells, set at 1.0 (*, P<0.05; **, P<0.01). (C) AGS KD cells were co-transfected with either pcDNA p65 or pcDNA 3.1 and treated with 5-FU 24 h post transfection. Cell numbers were after another 24 h and are presented as percent of untreated control cells (***, P<0.001). (D) DNA binding activity for NF-κB was examined by EMSA using nuclear extracts of MKN28 KD and SCR cells treated with 10 µg/ml 5-FU or TNFα for the indicated times. Antibody inhibition was performed with an anti-p65 antibody.
Figure 6
Figure 6. ROS as molecular mediator of the HIF-1α effect on chemosensitivity in AGS KD cells.
(A–D) AGS KD cells were pretreated for 16 h with different concentrations of the NADPH oxidase inhibitors diphenyleneiodonium (DPI) or apocynin. Proliferation of DPI-pretreated (A) or apocynin-pretreated (B) AGS KD cells 24 h after treatment with 10 µg/ml 5-FU under normoxic conditions (***, P<0.001). Cell numbers are shown as percent of untreated cells. (C and D) The effect of DPI (C) and apocynin (D) on p53 and p21 protein levels was determined by immunoblot analysis using whole cell extracts of AGS KD cells treated for 24 h with 10 µg/ml 5-FU. (E) Proposed model for HIF-1-dependent regulation of chemosensitivity by ROS-induced modulation of p53. (left panel) Chemotherapy-induced response in HIF-1-competent cells. HIF-1 counteracts generation of ROS at the mitochondrial level. Decreased ROS levels in turn abate activation of p53 and allow for cell cycle progression despite chemotherapy. Hence, HIF-1-competent cells display a more chemoresistant phenotype. Ub, ubiquitin; P, phosphate. (right panel) Chemotherapy-induced response in HIF-1-deficient cells. Inactivation of HIF-1 leads to accelerated mitochondrial ROS generation. ROS are potent inducers of p53 and thus boost activation of p53 by chemotherapeutic agents. P53 in turn transactivates -among others - the cyclin-dependent kinase (CDK) inhibitor p21 and inhibits NF-κB activity. The combined activation of p53 and inhibition of NF-κB, result in apoptosis and/or senescence of HIF-1-deficient cells, hence a more chemosensitive phenotype.
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