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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: 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.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.

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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Cancer cell lines with wild-type or tyrosine kinase domain-mutated EGFR. (a) PCR fragments from the indicated cell lines were compared with the wild-type sequence of EGFR. The black arrows indicate the codons E746 to A750, which are present in the EGFR in DiFi cells but have been deleted in HCC827 and HCC2279 cells. Codon L858R substitution in H1975 and H3255 cells is indicated by arrows. (b) Lysates from the indicated cell lines maintained in regular culture medium were prepared for Western blot analysis using antibodies directed against EGFR, HER2, and HER3, and antibodies directed against total and activation-specific phosphorylated downstream signaling molecules (ERK, Akt, and STAT3). The level of β-actin was used as a reference of lysate protein loading control of each cell line.
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Figure 1: Cancer cell lines with wild-type or tyrosine kinase domain-mutated EGFR. (a) PCR fragments from the indicated cell lines were compared with the wild-type sequence of EGFR. The black arrows indicate the codons E746 to A750, which are present in the EGFR in DiFi cells but have been deleted in HCC827 and HCC2279 cells. Codon L858R substitution in H1975 and H3255 cells is indicated by arrows. (b) Lysates from the indicated cell lines maintained in regular culture medium were prepared for Western blot analysis using antibodies directed against EGFR, HER2, and HER3, and antibodies directed against total and activation-specific phosphorylated downstream signaling molecules (ERK, Akt, and STAT3). The level of β-actin was used as a reference of lysate protein loading control of each cell line.

Mentions: Figure 1 shows the genetic and biochemical characteristics of the cell lines used in our study. Compared with the EGFR coding sequences in the GenBank, which originated from A431 cells, no mutations were found in the tyrosine kinase domain of EGFR in DiFi colorectal carcinoma cells (Fig. 1a). In contrast, HCC827 and HCC2279 NSCLC cells had a ΔE746-A750 deletion mutation, and H3255 and H1975 cells had an L858R point mutation. Figure 1b shows the levels of protein expression in the EGFR family, including EGFR (HER1), HER2, and HER3, and the levels of activation-specific phosphorylation of the three most common EGFR substrates, ERK, Akt, and STAT3. At baseline, HCC827, HCC2279, and H3255 cells expressed intermediate levels of EGFR, whereas A431 and DiFi cells expressed high levels. In contrast, H1975 cells expressed the lowest level of EGFR, which was barely detectable unless the film was overexposed. HER2 and HER3 were readily detectable in the cell lines except in H3255 cells (low in HER3) and HCC2279 cells (low in both HER2 and HER3). The basal levels of EGFR downstream signaling, shown by the levels of activation-specific phosphorylation of Akt, ERK, and STAT3, were not consistently associated with the HER family expression levels or EGFR sequence-coding status in a positive or negative manner among the cell lines.


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)

Cancer cell lines with wild-type or tyrosine kinase domain-mutated EGFR. (a) PCR fragments from the indicated cell lines were compared with the wild-type sequence of EGFR. The black arrows indicate the codons E746 to A750, which are present in the EGFR in DiFi cells but have been deleted in HCC827 and HCC2279 cells. Codon L858R substitution in H1975 and H3255 cells is indicated by arrows. (b) Lysates from the indicated cell lines maintained in regular culture medium were prepared for Western blot analysis using antibodies directed against EGFR, HER2, and HER3, and antibodies directed against total and activation-specific phosphorylated downstream signaling molecules (ERK, Akt, and STAT3). The level of β-actin was used as a reference of lysate protein loading control of each cell line.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Cancer cell lines with wild-type or tyrosine kinase domain-mutated EGFR. (a) PCR fragments from the indicated cell lines were compared with the wild-type sequence of EGFR. The black arrows indicate the codons E746 to A750, which are present in the EGFR in DiFi cells but have been deleted in HCC827 and HCC2279 cells. Codon L858R substitution in H1975 and H3255 cells is indicated by arrows. (b) Lysates from the indicated cell lines maintained in regular culture medium were prepared for Western blot analysis using antibodies directed against EGFR, HER2, and HER3, and antibodies directed against total and activation-specific phosphorylated downstream signaling molecules (ERK, Akt, and STAT3). The level of β-actin was used as a reference of lysate protein loading control of each cell line.
Mentions: Figure 1 shows the genetic and biochemical characteristics of the cell lines used in our study. Compared with the EGFR coding sequences in the GenBank, which originated from A431 cells, no mutations were found in the tyrosine kinase domain of EGFR in DiFi colorectal carcinoma cells (Fig. 1a). In contrast, HCC827 and HCC2279 NSCLC cells had a ΔE746-A750 deletion mutation, and H3255 and H1975 cells had an L858R point mutation. Figure 1b shows the levels of protein expression in the EGFR family, including EGFR (HER1), HER2, and HER3, and the levels of activation-specific phosphorylation of the three most common EGFR substrates, ERK, Akt, and STAT3. At baseline, HCC827, HCC2279, and H3255 cells expressed intermediate levels of EGFR, whereas A431 and DiFi cells expressed high levels. In contrast, H1975 cells expressed the lowest level of EGFR, which was barely detectable unless the film was overexposed. HER2 and HER3 were readily detectable in the cell lines except in H3255 cells (low in HER3) and HCC2279 cells (low in both HER2 and HER3). The basal levels of EGFR downstream signaling, shown by the levels of activation-specific phosphorylation of Akt, ERK, and STAT3, were not consistently associated with the HER family expression levels or EGFR sequence-coding status in a positive or negative manner among the cell lines.

Bottom Line: 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.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.

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