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Responses of cancer cells with wild-type or tyrosine kinase domain-mutated epidermal growth factor receptor (EGFR) to EGFR-targeted therapy are linked to downregulation of hypoxia-inducible factor-1alpha.

Lu Y, Liang K, Li X, Fan Z - Mol. Cancer (2007)

Bottom Line: Searching for novel molecular markers that dependably predict or indicate responses of human cancer cells to epidermal growth factor receptor (EGFR)-targeted therapy is strongly warranted.The purpose of the current study was to evaluate hypoxia-inducible factor-1alpha (HIF-1alpha) as a novel response marker compared with previously explored markers following treatment with an EGFR-blocking monoclonal antibody (cetuximab) and a small-molecule EGFR tyrosine kinase inhibitor (gefitinib) in a group of cancer cell lines containing wild-type or tyrosine kinase domain-mutated EGFR.To demonstrate a critical role of HIF-1alpha downregulation by EGFR-targeted treatment, we introduced a constitutively expressed HIF-1alpha mutant (HIF-1alpha/DeltaODD) that is resistant to cetuximab-induced downregulation in a cetuximab-responsive cell line (A431); we found that the HIF-1alpha/DeltaODD-transfected cells remained sensitive to cetuximab-induced inhibition of Akt and ERK phosphorylation but were remarkably less responsive to cetuximab-induced growth inhibition compared with corresponding control cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Experimental Therapeutics, The University of Texas M, D, Anderson Cancer Center, Houston, Texas 77030, USA. ylu@mdanderson.org

ABSTRACT

Background: Searching for novel molecular markers that dependably predict or indicate responses of human cancer cells to epidermal growth factor receptor (EGFR)-targeted therapy is strongly warranted. The purpose of the current study was to evaluate hypoxia-inducible factor-1alpha (HIF-1alpha) as a novel response marker compared with previously explored markers following treatment with an EGFR-blocking monoclonal antibody (cetuximab) and a small-molecule EGFR tyrosine kinase inhibitor (gefitinib) in a group of cancer cell lines containing wild-type or tyrosine kinase domain-mutated EGFR.

Results: We found that, compared with previously studied response markers, including EGFR per se and three EGFR downstream signal molecules (ERK, Akt, and STAT3), which showed variable post-treatment changes in levels of phosphorylation and no consistent link of the changes to therapeutic responses, HIF-1alpha showed a selective decrease in protein levels only in responsive cell lines. To demonstrate a critical role of HIF-1alpha downregulation by EGFR-targeted treatment, we introduced a constitutively expressed HIF-1alpha mutant (HIF-1alpha/DeltaODD) that is resistant to cetuximab-induced downregulation in a cetuximab-responsive cell line (A431); we found that the HIF-1alpha/DeltaODD-transfected cells remained sensitive to cetuximab-induced inhibition of Akt and ERK phosphorylation but were remarkably less responsive to cetuximab-induced growth inhibition compared with corresponding control cells.

Conclusion: Our data indicates that downregulation of HIF-1alpha is associated with positive therapeutic responses of cancer cells to EGFR-targeted therapy and suggest further investigation using HIF-1alpha as an indicator of tumor response to EGFR-targeted therapy in preclinical studies and in the clinical setting.

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Expression of HIF-1α/ΔODD mutant leads to cellular resistance to cetuximab without affecting cellular sensitivity to cetuximab-induced inhibition of EGFR signaling. (a) A431neo and A431/HIF-1α/ΔODD cells were treated as indicated for 16 hours (overnight). Cell lysates were prepared and subjected to Western blot analysis with the indicated antibodies. (b) Cells were left untreated or treated with the indicated concentrations of cetuximab in culture with 0.5% FBS for 5 days. Relative cell numbers were measured by the MTT assay and are presented as a percentage of the untreated control. (c) A431neo and A431/HIF-1α/ΔODD cells (300 cells/dish) were cultured, with or without cetuximab (2 nM), for 9 days. After treatment, cells were fixed and the colonies were counted, as described in the Methods section.
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Figure 6: Expression of HIF-1α/ΔODD mutant leads to cellular resistance to cetuximab without affecting cellular sensitivity to cetuximab-induced inhibition of EGFR signaling. (a) A431neo and A431/HIF-1α/ΔODD cells were treated as indicated for 16 hours (overnight). Cell lysates were prepared and subjected to Western blot analysis with the indicated antibodies. (b) Cells were left untreated or treated with the indicated concentrations of cetuximab in culture with 0.5% FBS for 5 days. Relative cell numbers were measured by the MTT assay and are presented as a percentage of the untreated control. (c) A431neo and A431/HIF-1α/ΔODD cells (300 cells/dish) were cultured, with or without cetuximab (2 nM), for 9 days. After treatment, cells were fixed and the colonies were counted, as described in the Methods section.

Mentions: To provide experimental evidence supporting a critical role of HIF-1α downregulation in mediating cellular responses to EGFR-targeted therapy, we introduced a HIF-1α mutant (HIF-1α/ΔODD) in A431 cells. In the HIF-1α/ΔODD mutant, the oxygen-dependent degradation (ODD) domain of HIF-1α was removed and therefore the mutant became insensitive to VHL ubiquitin ligase-mediated proteasomal degradation, rendering the expressed truncated protein stable in normoxia [29]. After neomycin selection, pooled A431 transfectant cells were obtained and their response to cetuximab was compared with that of control-vector transfected cells. Figure 6a shows that the level of HIF-1α/ΔODD was minimally affected by cetuximab, whereas the level of wild-type HIF-1α was decreased in A431neo and, to a lesser degree, in A431/HIF-1α/ΔODD cells. Importantly, A431/HIF-1α/ΔODD cells remained as sensitive to cetuximab-induced inhibition of ERK and Akt as A431neo cells, as shown by decreased levels of activation-specific phosphorylation in the two molecules. However, the A431/HIF-1α/ΔODD cells were considerably more resistant to cetuximab-induced growth inhibition, as measured by an MTT assay (Fig. 6b). Clonogenic survival assays showed that A431/HIF-1α/ΔODD had markedly more surviving colonies when cultured in the presence of cetuximab than did untreated A431neo cells, indicating that constitutive expression of HIF-1α can indeed render cells resistant to cetuximab treatment (Fig. 6c).


Responses of cancer cells with wild-type or tyrosine kinase domain-mutated epidermal growth factor receptor (EGFR) to EGFR-targeted therapy are linked to downregulation of hypoxia-inducible factor-1alpha.

Lu Y, Liang K, Li X, Fan Z - Mol. Cancer (2007)

Expression of HIF-1α/ΔODD mutant leads to cellular resistance to cetuximab without affecting cellular sensitivity to cetuximab-induced inhibition of EGFR signaling. (a) A431neo and A431/HIF-1α/ΔODD cells were treated as indicated for 16 hours (overnight). Cell lysates were prepared and subjected to Western blot analysis with the indicated antibodies. (b) Cells were left untreated or treated with the indicated concentrations of cetuximab in culture with 0.5% FBS for 5 days. Relative cell numbers were measured by the MTT assay and are presented as a percentage of the untreated control. (c) A431neo and A431/HIF-1α/ΔODD cells (300 cells/dish) were cultured, with or without cetuximab (2 nM), for 9 days. After treatment, cells were fixed and the colonies were counted, as described in the Methods section.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2117021&req=5

Figure 6: Expression of HIF-1α/ΔODD mutant leads to cellular resistance to cetuximab without affecting cellular sensitivity to cetuximab-induced inhibition of EGFR signaling. (a) A431neo and A431/HIF-1α/ΔODD cells were treated as indicated for 16 hours (overnight). Cell lysates were prepared and subjected to Western blot analysis with the indicated antibodies. (b) Cells were left untreated or treated with the indicated concentrations of cetuximab in culture with 0.5% FBS for 5 days. Relative cell numbers were measured by the MTT assay and are presented as a percentage of the untreated control. (c) A431neo and A431/HIF-1α/ΔODD cells (300 cells/dish) were cultured, with or without cetuximab (2 nM), for 9 days. After treatment, cells were fixed and the colonies were counted, as described in the Methods section.
Mentions: To provide experimental evidence supporting a critical role of HIF-1α downregulation in mediating cellular responses to EGFR-targeted therapy, we introduced a HIF-1α mutant (HIF-1α/ΔODD) in A431 cells. In the HIF-1α/ΔODD mutant, the oxygen-dependent degradation (ODD) domain of HIF-1α was removed and therefore the mutant became insensitive to VHL ubiquitin ligase-mediated proteasomal degradation, rendering the expressed truncated protein stable in normoxia [29]. After neomycin selection, pooled A431 transfectant cells were obtained and their response to cetuximab was compared with that of control-vector transfected cells. Figure 6a shows that the level of HIF-1α/ΔODD was minimally affected by cetuximab, whereas the level of wild-type HIF-1α was decreased in A431neo and, to a lesser degree, in A431/HIF-1α/ΔODD cells. Importantly, A431/HIF-1α/ΔODD cells remained as sensitive to cetuximab-induced inhibition of ERK and Akt as A431neo cells, as shown by decreased levels of activation-specific phosphorylation in the two molecules. However, the A431/HIF-1α/ΔODD cells were considerably more resistant to cetuximab-induced growth inhibition, as measured by an MTT assay (Fig. 6b). Clonogenic survival assays showed that A431/HIF-1α/ΔODD had markedly more surviving colonies when cultured in the presence of cetuximab than did untreated A431neo cells, indicating that constitutive expression of HIF-1α can indeed render cells resistant to cetuximab treatment (Fig. 6c).

Bottom Line: Searching for novel molecular markers that dependably predict or indicate responses of human cancer cells to epidermal growth factor receptor (EGFR)-targeted therapy is strongly warranted.The purpose of the current study was to evaluate hypoxia-inducible factor-1alpha (HIF-1alpha) as a novel response marker compared with previously explored markers following treatment with an EGFR-blocking monoclonal antibody (cetuximab) and a small-molecule EGFR tyrosine kinase inhibitor (gefitinib) in a group of cancer cell lines containing wild-type or tyrosine kinase domain-mutated EGFR.To demonstrate a critical role of HIF-1alpha downregulation by EGFR-targeted treatment, we introduced a constitutively expressed HIF-1alpha mutant (HIF-1alpha/DeltaODD) that is resistant to cetuximab-induced downregulation in a cetuximab-responsive cell line (A431); we found that the HIF-1alpha/DeltaODD-transfected cells remained sensitive to cetuximab-induced inhibition of Akt and ERK phosphorylation but were remarkably less responsive to cetuximab-induced growth inhibition compared with corresponding control cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Experimental Therapeutics, The University of Texas M, D, Anderson Cancer Center, Houston, Texas 77030, USA. ylu@mdanderson.org

ABSTRACT

Background: Searching for novel molecular markers that dependably predict or indicate responses of human cancer cells to epidermal growth factor receptor (EGFR)-targeted therapy is strongly warranted. The purpose of the current study was to evaluate hypoxia-inducible factor-1alpha (HIF-1alpha) as a novel response marker compared with previously explored markers following treatment with an EGFR-blocking monoclonal antibody (cetuximab) and a small-molecule EGFR tyrosine kinase inhibitor (gefitinib) in a group of cancer cell lines containing wild-type or tyrosine kinase domain-mutated EGFR.

Results: We found that, compared with previously studied response markers, including EGFR per se and three EGFR downstream signal molecules (ERK, Akt, and STAT3), which showed variable post-treatment changes in levels of phosphorylation and no consistent link of the changes to therapeutic responses, HIF-1alpha showed a selective decrease in protein levels only in responsive cell lines. To demonstrate a critical role of HIF-1alpha downregulation by EGFR-targeted treatment, we introduced a constitutively expressed HIF-1alpha mutant (HIF-1alpha/DeltaODD) that is resistant to cetuximab-induced downregulation in a cetuximab-responsive cell line (A431); we found that the HIF-1alpha/DeltaODD-transfected cells remained sensitive to cetuximab-induced inhibition of Akt and ERK phosphorylation but were remarkably less responsive to cetuximab-induced growth inhibition compared with corresponding control cells.

Conclusion: Our data indicates that downregulation of HIF-1alpha is associated with positive therapeutic responses of cancer cells to EGFR-targeted therapy and suggest further investigation using HIF-1alpha as an indicator of tumor response to EGFR-targeted therapy in preclinical studies and in the clinical setting.

Show MeSH
Related in: MedlinePlus