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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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Induction of apoptosis in cancer cells with wild-type EGFR or tyrosine kinase domain-mutated EGFR by cetuximab and gefitinib. Cells from each line were left untreated or were treated with vehicle (DMSO), 5 nM cetuximab, or 0.5 μM gefitinib in a medium containing 0.5% FBS. After 16 hours of treatment, the cells were harvested and lysed for quantitative apoptosis measurement by (a) an enzyme-linked immunosorbent assay, as described in the Methods section, and (b) Western blot analysis with anti-PARP antibodies. *P < 0.05, **P < 0.01 compared with corresponding controls.
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Figure 3: Induction of apoptosis in cancer cells with wild-type EGFR or tyrosine kinase domain-mutated EGFR by cetuximab and gefitinib. Cells from each line were left untreated or were treated with vehicle (DMSO), 5 nM cetuximab, or 0.5 μM gefitinib in a medium containing 0.5% FBS. After 16 hours of treatment, the cells were harvested and lysed for quantitative apoptosis measurement by (a) an enzyme-linked immunosorbent assay, as described in the Methods section, and (b) Western blot analysis with anti-PARP antibodies. *P < 0.05, **P < 0.01 compared with corresponding controls.

Mentions: To determine whether cetuximab or gefitinib induced apoptosis in the cell lines, we used two independent apoptosis assays: an enzyme-linked immunosorbent assay to measure the cytoplasmic levels of histone-associated DNA fragments characteristic of apoptotic cells and a Western blot analysis to detect the proteolytic cleavage of poly(adenosine diphosphate-ribose) polymerase (PARP) (Fig. 3a and 3b). Clear evidence of apoptosis was found in three cell lines: DiFi, HCC827, and H3255. At the doses tested, gefitinib seemed to induce a higher cytoplasmic level of histone-associated DNA fragments than did cetuximab (Fig. 3a), but both agents had similar effects on PARP cleavage (Fig. 3b). DiFi cells contained a high basal level of cleaved PARP when cultured in low-serum medium, but the cleaved PARP fragment level clearly increased after treatment. Although cell proliferation was strongly inhibited, no clear sign of apoptosis was detected in A431 cells after treatment with cetuximab or gefitinib under the treatment condition consisting of 0.5% FBS in culture medium. As expected, HCC2279 and H1975 cells showed modest or poor anti-proliferative responses to the treatments, with no apoptosis detected in these cell lines with either treatment.


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)

Induction of apoptosis in cancer cells with wild-type EGFR or tyrosine kinase domain-mutated EGFR by cetuximab and gefitinib. Cells from each line were left untreated or were treated with vehicle (DMSO), 5 nM cetuximab, or 0.5 μM gefitinib in a medium containing 0.5% FBS. After 16 hours of treatment, the cells were harvested and lysed for quantitative apoptosis measurement by (a) an enzyme-linked immunosorbent assay, as described in the Methods section, and (b) Western blot analysis with anti-PARP antibodies. *P < 0.05, **P < 0.01 compared with corresponding controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Induction of apoptosis in cancer cells with wild-type EGFR or tyrosine kinase domain-mutated EGFR by cetuximab and gefitinib. Cells from each line were left untreated or were treated with vehicle (DMSO), 5 nM cetuximab, or 0.5 μM gefitinib in a medium containing 0.5% FBS. After 16 hours of treatment, the cells were harvested and lysed for quantitative apoptosis measurement by (a) an enzyme-linked immunosorbent assay, as described in the Methods section, and (b) Western blot analysis with anti-PARP antibodies. *P < 0.05, **P < 0.01 compared with corresponding controls.
Mentions: To determine whether cetuximab or gefitinib induced apoptosis in the cell lines, we used two independent apoptosis assays: an enzyme-linked immunosorbent assay to measure the cytoplasmic levels of histone-associated DNA fragments characteristic of apoptotic cells and a Western blot analysis to detect the proteolytic cleavage of poly(adenosine diphosphate-ribose) polymerase (PARP) (Fig. 3a and 3b). Clear evidence of apoptosis was found in three cell lines: DiFi, HCC827, and H3255. At the doses tested, gefitinib seemed to induce a higher cytoplasmic level of histone-associated DNA fragments than did cetuximab (Fig. 3a), but both agents had similar effects on PARP cleavage (Fig. 3b). DiFi cells contained a high basal level of cleaved PARP when cultured in low-serum medium, but the cleaved PARP fragment level clearly increased after treatment. Although cell proliferation was strongly inhibited, no clear sign of apoptosis was detected in A431 cells after treatment with cetuximab or gefitinib under the treatment condition consisting of 0.5% FBS in culture medium. As expected, HCC2279 and H1975 cells showed modest or poor anti-proliferative responses to the treatments, with no apoptosis detected in these cell lines with either treatment.

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