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Identification of the angiogenic gene signature induced by EGF and hypoxia in colorectal cancer.

Khong TL, Thairu N, Larsen H, Dawson PM, Kiriakidis S, Paleolog EM - BMC Cancer (2013)

Bottom Line: Stimulation with EGF resulted in EGFR tyrosine autophosphorylation, activation of p42/p44 MAP kinases and stabilisation of HIF-1α and HIF-2α proteins.These findings suggest that although EGFR phosphorylation activates the MAP kinase signalling and promotes HIF stabilisation in CRC, this alone is not sufficient to induce angiogenic gene expression.Finally, HIF activation synergises with EGF-mediated signalling to additionally induce a unique sub-group of candidate angiogenic genes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Kennedy Institute of Rheumatology, Faculty of Medicine, Imperial College, London, UK. ewa.paleolog@kennedy.ox.ac.uk.

ABSTRACT

Background: Colorectal cancer (CRC) is characterised by hypoxia, which activates gene transcription through hypoxia-inducible factors (HIF), as well as by expression of epidermal growth factor (EGF) and EGF receptors, targeting of which has been demonstrated to provide therapeutic benefit in CRC. Although EGF has been demonstrated to induce expression of angiogenic mediators, potential interactions in CRC between EGF-mediated signalling and the hypoxia/HIF pathway remain uncharacterised.

Methods: PCR-based profiling was applied to identify angiogenic genes in Caco-2 CRC cells regulated by hypoxia, the hypoxia mimetic dimethyloxallylglycine (DMOG) and/or EGF. Western blotting was used to determine the role of HIF-1alpha, HIF-2alpha and MAPK cell signalling in mediating the angiogenic responses.

Results: We identified a total of 9 angiogenic genes, including angiopoietin-like (ANGPTL) 4, ephrin (EFNA) 3, transforming growth factor (TGF) β1 and vascular endothelial growth factor (VEGF), to be upregulated in a HIF dependent manner in Caco-2 CRC cells in response to both hypoxia and the hypoxia mimetic dimethyloxallylglycine (DMOG). Stimulation with EGF resulted in EGFR tyrosine autophosphorylation, activation of p42/p44 MAP kinases and stabilisation of HIF-1α and HIF-2α proteins. However, expression of 84 angiogenic genes remained unchanged in response to EGF alone. Crucially, addition of DMOG in combination with EGF significantly increased expression of a further 11 genes (in addition to the 9 genes upregulated in response to either DMOG alone or hypoxia alone). These additional genes included chemokines (CCL-11/eotaxin-1 and interleukin-8), collagen type IV α3 chain, integrin β3 chain, TGFα and VEGF receptor KDR.

Conclusion: These findings suggest that although EGFR phosphorylation activates the MAP kinase signalling and promotes HIF stabilisation in CRC, this alone is not sufficient to induce angiogenic gene expression. In contrast, HIF activation downstream of hypoxia/DMOG drives expression of genes such as ANGPTL4, EFNA3, TGFβ1 and VEGF. Finally, HIF activation synergises with EGF-mediated signalling to additionally induce a unique sub-group of candidate angiogenic genes. Our data highlight the complex interrelationship between tumour hypoxia, EGF and angiogenesis in the pathogenesis of CRC.

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Angiogenic gene expression in Caco-2 cells exposed to EGF and/or DMOG. Caco-2 cells were stimulated with 20 ng/mL EGF and/or 1 mM DMOG for 24 hours. Changes in (a) ANGPTL4, (b) EFNA3, (c) TGFβ1 and (d) VEGF mRNA levels were determined by Q-PCR using the 2-ΔΔCt method and are expressed relative to HKG 18S. Data are mean ± SEM from 3 representative experiments, and were analysed by 1-way ANOVA of ΔCt values versus normoxia (unless otherwise indicated): ns = not significant, *** p < 0.001. Dashed line shows response of unstimulated cells.
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Figure 6: Angiogenic gene expression in Caco-2 cells exposed to EGF and/or DMOG. Caco-2 cells were stimulated with 20 ng/mL EGF and/or 1 mM DMOG for 24 hours. Changes in (a) ANGPTL4, (b) EFNA3, (c) TGFβ1 and (d) VEGF mRNA levels were determined by Q-PCR using the 2-ΔΔCt method and are expressed relative to HKG 18S. Data are mean ± SEM from 3 representative experiments, and were analysed by 1-way ANOVA of ΔCt values versus normoxia (unless otherwise indicated): ns = not significant, *** p < 0.001. Dashed line shows response of unstimulated cells.

Mentions: The above cell signalling studies clearly demonstrate that EGF is capable of activating downstream signalling in Caco-2 cells, inducing rapid phosphorylation of tyrosine residues in EGFR, activation of ERK1/2 and stabilisation of HIF proteins. However, in spite of the observed changes, and in particular despite stabilisation of HIF-1α, expression of the 4 angiogenic HIF-1 target genes, namely ANGPTL4 (Figure 6a), EFNA3 (Figure 6b), TGFβ1 (Figure 6c) and VEGF (Figure 6d), was unaffected by addition of EGF alone. Furthermore, responses induced by DMOG alone were not further altered by addition of EGF (p > 0.05 versus DMOG alone) specifically for these 4 angiogenic genes.


Identification of the angiogenic gene signature induced by EGF and hypoxia in colorectal cancer.

Khong TL, Thairu N, Larsen H, Dawson PM, Kiriakidis S, Paleolog EM - BMC Cancer (2013)

Angiogenic gene expression in Caco-2 cells exposed to EGF and/or DMOG. Caco-2 cells were stimulated with 20 ng/mL EGF and/or 1 mM DMOG for 24 hours. Changes in (a) ANGPTL4, (b) EFNA3, (c) TGFβ1 and (d) VEGF mRNA levels were determined by Q-PCR using the 2-ΔΔCt method and are expressed relative to HKG 18S. Data are mean ± SEM from 3 representative experiments, and were analysed by 1-way ANOVA of ΔCt values versus normoxia (unless otherwise indicated): ns = not significant, *** p < 0.001. Dashed line shows response of unstimulated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4228238&req=5

Figure 6: Angiogenic gene expression in Caco-2 cells exposed to EGF and/or DMOG. Caco-2 cells were stimulated with 20 ng/mL EGF and/or 1 mM DMOG for 24 hours. Changes in (a) ANGPTL4, (b) EFNA3, (c) TGFβ1 and (d) VEGF mRNA levels were determined by Q-PCR using the 2-ΔΔCt method and are expressed relative to HKG 18S. Data are mean ± SEM from 3 representative experiments, and were analysed by 1-way ANOVA of ΔCt values versus normoxia (unless otherwise indicated): ns = not significant, *** p < 0.001. Dashed line shows response of unstimulated cells.
Mentions: The above cell signalling studies clearly demonstrate that EGF is capable of activating downstream signalling in Caco-2 cells, inducing rapid phosphorylation of tyrosine residues in EGFR, activation of ERK1/2 and stabilisation of HIF proteins. However, in spite of the observed changes, and in particular despite stabilisation of HIF-1α, expression of the 4 angiogenic HIF-1 target genes, namely ANGPTL4 (Figure 6a), EFNA3 (Figure 6b), TGFβ1 (Figure 6c) and VEGF (Figure 6d), was unaffected by addition of EGF alone. Furthermore, responses induced by DMOG alone were not further altered by addition of EGF (p > 0.05 versus DMOG alone) specifically for these 4 angiogenic genes.

Bottom Line: Stimulation with EGF resulted in EGFR tyrosine autophosphorylation, activation of p42/p44 MAP kinases and stabilisation of HIF-1α and HIF-2α proteins.These findings suggest that although EGFR phosphorylation activates the MAP kinase signalling and promotes HIF stabilisation in CRC, this alone is not sufficient to induce angiogenic gene expression.Finally, HIF activation synergises with EGF-mediated signalling to additionally induce a unique sub-group of candidate angiogenic genes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Kennedy Institute of Rheumatology, Faculty of Medicine, Imperial College, London, UK. ewa.paleolog@kennedy.ox.ac.uk.

ABSTRACT

Background: Colorectal cancer (CRC) is characterised by hypoxia, which activates gene transcription through hypoxia-inducible factors (HIF), as well as by expression of epidermal growth factor (EGF) and EGF receptors, targeting of which has been demonstrated to provide therapeutic benefit in CRC. Although EGF has been demonstrated to induce expression of angiogenic mediators, potential interactions in CRC between EGF-mediated signalling and the hypoxia/HIF pathway remain uncharacterised.

Methods: PCR-based profiling was applied to identify angiogenic genes in Caco-2 CRC cells regulated by hypoxia, the hypoxia mimetic dimethyloxallylglycine (DMOG) and/or EGF. Western blotting was used to determine the role of HIF-1alpha, HIF-2alpha and MAPK cell signalling in mediating the angiogenic responses.

Results: We identified a total of 9 angiogenic genes, including angiopoietin-like (ANGPTL) 4, ephrin (EFNA) 3, transforming growth factor (TGF) β1 and vascular endothelial growth factor (VEGF), to be upregulated in a HIF dependent manner in Caco-2 CRC cells in response to both hypoxia and the hypoxia mimetic dimethyloxallylglycine (DMOG). Stimulation with EGF resulted in EGFR tyrosine autophosphorylation, activation of p42/p44 MAP kinases and stabilisation of HIF-1α and HIF-2α proteins. However, expression of 84 angiogenic genes remained unchanged in response to EGF alone. Crucially, addition of DMOG in combination with EGF significantly increased expression of a further 11 genes (in addition to the 9 genes upregulated in response to either DMOG alone or hypoxia alone). These additional genes included chemokines (CCL-11/eotaxin-1 and interleukin-8), collagen type IV α3 chain, integrin β3 chain, TGFα and VEGF receptor KDR.

Conclusion: These findings suggest that although EGFR phosphorylation activates the MAP kinase signalling and promotes HIF stabilisation in CRC, this alone is not sufficient to induce angiogenic gene expression. In contrast, HIF activation downstream of hypoxia/DMOG drives expression of genes such as ANGPTL4, EFNA3, TGFβ1 and VEGF. Finally, HIF activation synergises with EGF-mediated signalling to additionally induce a unique sub-group of candidate angiogenic genes. Our data highlight the complex interrelationship between tumour hypoxia, EGF and angiogenesis in the pathogenesis of CRC.

Show MeSH
Related in: MedlinePlus