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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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Dose- and time-dependent responses of wild-type EGFR and tyrosine kinase domain-mutated EGFR cells to cetuximab and gefitinib treatment. (a) Absolute cell numbers in each treatment group (control [DMSO]), 5 nM cetuximab, and 0.5 μM gefitinib, all in 0.5% FBS medium) were plotted against the duration of treatment. (b) The inhibition of cell proliferation after treatment with cetuximab was measured by an MTT assay and is shown as a percentage of the optical density value of control cells (untreated) for each concentration tested. (c) The inhibition of cell proliferation after treatment with gefitinib was measured as in (b) and is shown as a percentage of the optical density value of vehicle-treated cells (DMSO) for each concentration tested. Results are shown as the mean of five independent measurements, plus or minus the standard deviation (SD). The magnitude of some SDs was smaller than the symbol size; thus some bars do not appear in the figure.
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Figure 2: Dose- and time-dependent responses of wild-type EGFR and tyrosine kinase domain-mutated EGFR cells to cetuximab and gefitinib treatment. (a) Absolute cell numbers in each treatment group (control [DMSO]), 5 nM cetuximab, and 0.5 μM gefitinib, all in 0.5% FBS medium) were plotted against the duration of treatment. (b) The inhibition of cell proliferation after treatment with cetuximab was measured by an MTT assay and is shown as a percentage of the optical density value of control cells (untreated) for each concentration tested. (c) The inhibition of cell proliferation after treatment with gefitinib was measured as in (b) and is shown as a percentage of the optical density value of vehicle-treated cells (DMSO) for each concentration tested. Results are shown as the mean of five independent measurements, plus or minus the standard deviation (SD). The magnitude of some SDs was smaller than the symbol size; thus some bars do not appear in the figure.

Mentions: Figure 2a shows the time-dependent responses of these individual cell lines to treatment with 10 nM cetuximab or 0.5 μM gefitinib for 4 days. Four cell lines (DiFi, HCC827, H3255, and A431) showed marked growth inhibition responses after cetuximab or gefitinib treatment, whereas HCC2279 and H1975 cells showed only moderate or poor growth inhibition. The degrees of growth inhibition of DiFi cells after cetuximab (10 nM) and gefitinib (0.5 μM) treatments were comparable, but in A431 and HCC827 cells more growth inhibition was induced by cetuximab than by gefitinib. In contrast, H3255 cells responded more strongly to gefitinib than to cetuximab: massive cell death was microscopically visible just a few hours after exposure to gefitinib, whereas the response of H3255 cells to cetuximab was slower and not evident until those cells had been exposed to the treatment overnight (data not shown). HCC2279 and H1975 cell lines demonstrated much less growth inhibition than did other cell lines in response to treatment with either agent. The growth rate of HCC2279 cells was much slower than that in the other cell lines, which may partly explain the moderate inhibitory effects of the agents on HCC2279 cell proliferation. H1975 cells, which also contain a second mutation (T790M) linked to gefitinib resistance [25], responded poorly to either agent.


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)

Dose- and time-dependent responses of wild-type EGFR and tyrosine kinase domain-mutated EGFR cells to cetuximab and gefitinib treatment. (a) Absolute cell numbers in each treatment group (control [DMSO]), 5 nM cetuximab, and 0.5 μM gefitinib, all in 0.5% FBS medium) were plotted against the duration of treatment. (b) The inhibition of cell proliferation after treatment with cetuximab was measured by an MTT assay and is shown as a percentage of the optical density value of control cells (untreated) for each concentration tested. (c) The inhibition of cell proliferation after treatment with gefitinib was measured as in (b) and is shown as a percentage of the optical density value of vehicle-treated cells (DMSO) for each concentration tested. Results are shown as the mean of five independent measurements, plus or minus the standard deviation (SD). The magnitude of some SDs was smaller than the symbol size; thus some bars do not appear in the figure.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Dose- and time-dependent responses of wild-type EGFR and tyrosine kinase domain-mutated EGFR cells to cetuximab and gefitinib treatment. (a) Absolute cell numbers in each treatment group (control [DMSO]), 5 nM cetuximab, and 0.5 μM gefitinib, all in 0.5% FBS medium) were plotted against the duration of treatment. (b) The inhibition of cell proliferation after treatment with cetuximab was measured by an MTT assay and is shown as a percentage of the optical density value of control cells (untreated) for each concentration tested. (c) The inhibition of cell proliferation after treatment with gefitinib was measured as in (b) and is shown as a percentage of the optical density value of vehicle-treated cells (DMSO) for each concentration tested. Results are shown as the mean of five independent measurements, plus or minus the standard deviation (SD). The magnitude of some SDs was smaller than the symbol size; thus some bars do not appear in the figure.
Mentions: Figure 2a shows the time-dependent responses of these individual cell lines to treatment with 10 nM cetuximab or 0.5 μM gefitinib for 4 days. Four cell lines (DiFi, HCC827, H3255, and A431) showed marked growth inhibition responses after cetuximab or gefitinib treatment, whereas HCC2279 and H1975 cells showed only moderate or poor growth inhibition. The degrees of growth inhibition of DiFi cells after cetuximab (10 nM) and gefitinib (0.5 μM) treatments were comparable, but in A431 and HCC827 cells more growth inhibition was induced by cetuximab than by gefitinib. In contrast, H3255 cells responded more strongly to gefitinib than to cetuximab: massive cell death was microscopically visible just a few hours after exposure to gefitinib, whereas the response of H3255 cells to cetuximab was slower and not evident until those cells had been exposed to the treatment overnight (data not shown). HCC2279 and H1975 cell lines demonstrated much less growth inhibition than did other cell lines in response to treatment with either agent. The growth rate of HCC2279 cells was much slower than that in the other cell lines, which may partly explain the moderate inhibitory effects of the agents on HCC2279 cell proliferation. H1975 cells, which also contain a second mutation (T790M) linked to gefitinib resistance [25], responded poorly to either agent.

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