Acquired resistance to EGFR tyrosine kinase inhibitors in cancer cells is mediated by loss of IGF-binding proteins
Marta Guix, … , Carlos L. Arteaga, Jeffrey A. Engelman
Marta Guix, … , Carlos L. Arteaga, Jeffrey A. Engelman
Published June 20, 2008
Citation Information: J Clin Invest. 2008;118(7):2609-2619. https://doi.org/10.1172/JCI34588.
View: Text | PDF
Research Article

Acquired resistance to EGFR tyrosine kinase inhibitors in cancer cells is mediated by loss of IGF-binding proteins

Abstract

Although some cancers are initially sensitive to EGFR tyrosine kinase inhibitors (TKIs), resistance invariably develops. We investigated mechanisms of acquired resistance to the EGFR TKI gefitinib by generating gefitinib-resistant (GR) A431 squamous cancer cells. In GR cells, gefitinib reduced phosphorylation of EGFR, ErbB-3, and Erk but not Akt. These cells also showed hyperphosphorylation of the IGFI receptor (IGFIR) and constitutive association of IRS-1 with PI3K. Inhibition of IGFIR signaling disrupted the association of IRS-1 with PI3K and restored the ability of gefitinib to downregulate PI3K/Akt signaling and to inhibit GR cell growth. Gene expression analyses revealed that GR cells exhibited markedly reduced IGF-binding protein 3 (IGFBP-3) and IGFBP-4 RNA. Addition of recombinant IGFBP-3 restored the ability of gefitinib to downregulate PI3K/Akt signaling and to inhibit cell growth. Finally, gefitinib treatment of mice with A431 xenografts in combination with an IGFIR-specific monoclonal antibody prevented tumor recurrence, whereas each drug given alone was unable to do so. These data suggest that loss of expression of IGFBPs in tumor cells treated with EGFR TKIs derepresses IGFIR signaling, which in turn mediates resistance to EGFR antagonists. Moreover, combined therapeutic inhibition of EGFR and IGFIR may abrogate this acquired mechanism of drug resistance and is thus worthy of prospective clinical investigation.

Authors

Marta Guix, Anthony C. Faber, Shizhen Emily Wang, Maria Graciela Olivares, Youngchul Song, Sherman Qu, Cammie Rinehart, Brenda Seidel, Douglas Yee, Carlos L. Arteaga, Jeffrey A. Engelman

×

Figure 3

Blockade of IGFIR in combination with gefitinib inhibits PI3K/Akt signaling and cell growth.

Options: View larger image (or click on image) Download as PowerPoint
Blockade of IGFIR in combination with gefitinib inhibits PI3K/Akt signal...
(A) A431 GR cells were treated with control, gefitinib (1 μM), AEW541 (1 μM), or gefitinib and AEW541 at the indicated concentrations in either full serum (10% FBS) or low serum (0.5% FBS) for 6 hours. The cells were lysed and Western blots were probed with the indicated antibodies. (B) The A431 GR cells were treated with single-agent gefitinib, AEW541, Mk-0646, or combinations of gefitinib and AEW541 or gefitinib and Mk-0646 at the indicated concentrations for 6 hours. Cells were lysed as in Figure 2A, and the extracts were immunoprecipitated with an anti-p85 antibody. IPs were probed with the indicated antibodies. Extracts from the same lysates were probed with antibodies against p-Akt (Ser473) and total Akt. (C) A schematic depicting the 2 pathways leading to PI3K/Akt signaling in A431 GR cells: the EGFR/ErbB-3 and the IGFIR/IRS-1 pathways. (D) A431 GR cells were grown in Matrigel with or without gefitinib (1 μM), AEW541 (1 μM), Mk-0646 (10 μg/ml), or combinations of these drugs as specified. Photographs of the colonies were taken after 10 days. Original magnification, ×10. Right panel shows cell numbers from Matrigel experiments. Cells were harvested by trypsinization and then counted. Cell numbers are represented as percentages of untreated cells. Bars represent the mean ± SD of 3 wells. (E) A431 GR cells were grown in 12-well plates in 0.5% FBS–containing medium for 72 hours with or without drugs (same concentrations as in D) and harvested by trypsinization. Cell numbers were determined with a Coulter Counter. Bars represent the mean ± SD of 3 wells. Student’s t test was used for statistical comparisons.