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.

Figure 1. A431 GR cells maintain PI3K/Akt signaling in the presence of gefitinib and remain sensitive to PI3K inhibitors.

(A) Parental and A431 GR cells were grown for 72 hours in 0.5% FBS containing medium with or without gefitinib at the indicated μM concentrations. Cell number was determined in a Coulter Counter. Each data point represents the mean ± SD of 3 wells. (B) Parental and A431 GR cells were grown in Matrigel in the absence or presence of gefitinib (1 μM). Pictures were taken after 10 days. Original magnification, ×10. (C) Parental and A431 GR cells were treated for 6 hours in growth medium containing 1 μM gefitinib. Whole-cell lysates were prepared and probed in immunoblots with the indicated antibodies. (D) Parental and GR cells were treated with control, gefitinib (3 μM), or erlotinib (3 μM), and the extracts were probed with the indicated antibodies. (E) Cell growth assays were performed as in A. Cells were treated with the indicated drugs and concentrations. (Gef, gefitinib; Erlot, erlotinib; LY, LY294002). Student’s t test was used for statistical comparisons. Error bars represent SD.

Figure 2. IRS-1 associates with PI3K in GR but not sensitive A431 cells.

(A) Parental and A431 GR cells were treated with or without 1 μM gefitinib for 6 hours. Cell lysates were subjected to immunoprecipitation with an anti-p85 antibody followed by Western blot analysis with an anti–p-Tyr antibody. The blots were then stripped and reprobed with antibodies against IRS-1 and p85. (B) The parental and A431 GR cells were treated as in A, and the extracts were probed with antibodies against the indicated proteins. Note that IGFIR phosphorylation is substantially greater in the A431 GR cells.

Figure 3. Blockade of IGFIR in combination with gefitinib inhibits PI3K/Akt signaling and cell growth.

(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.

Figure 4. IGFBPs are downregulated in GR cells.

(A) Parental and A431 GR cells (10⁶ cells) were incubated for 24 hours in serum-free medium. The conditioned medium (C.M.) was concentrated by ultrafiltration, and IGF-I and IGF-II levels were determined by immunoassay as indicated in Methods. Bars represent the mean ± SD of 3 experiments. (B) Left panel: conditioned medium from 2 × 10⁶ parental and A431 GR cells was collected after 24 hours incubation and concentrated 20-fold by ultrafiltration. Medium was subjected to electrophoresis under nonreducing conditions, transferred to a nitrocellulose membrane, and incubated with ¹²⁵I–IGF-I overnight at 4°C. Signal was captured with a phosphorimager. Human serum and MCF-7 cells were used as positive controls for ¹²⁵I–IGF-I binding and IGFBP-4. Right panel: conditioned medium or 50 μg total protein from whole-cell lysates was subjected to immunoblot analysis with IGFBP-3, IGFBP-4, or Akt antibodies. (C) A431 GR cells were grown in 12-well plates in 0.5% FBS-containing medium for 72 hours with or without gefitinib (1 μM) and/or IGFBP-3 (1 μg/ml) and harvested by trypsinization. Cell numbers were determined with a Coulter Counter. Error bars represent the mean ± SD of 3 wells. Student’s t test was used for statistical comparisons. (D) A431 GR cells were treated with vehicle or gefitinib (1 μM) ± IGFBP-3 at the indicated concentrations for 6 hours. Cell lysates were prepared and analyzed with Western blots using the indicated antibodies.

Figure 5. IGF-I activates PI3K/Akt and promotes gefitinib resistance in A431 cells but not in EGFR mutant HCC827 cells.

(A) A431 and HCC827 cells were exposed to IGF-I (10 nM), gefitinib (1 μM), AEW541 (1 μM), or a combination as indicated for 6 hours. Cells were lysed and probed with the indicated antibodies. (B) A431 and HCC827 cells were subjected to a 72-hour MTS survival assay (see Methods) in increasing doses of gefitinib and in the presence or absence of 10 nM IGF-I. Results are presented as percentage of survival compared with cells grown in the absence of gefitinib. Experiments with each concentration of gefitinib were performed 6 times and the mean ± SD are shown.

Figure 6. An independent cell-line model of acquired resistance, HN11 GR, is overcome by combined EGFR and IGFIR blockade.

(A) Parental HN11 and HN11 GR cells (see Methods) were subjected to an MTS survival assay in increasing concentrations of gefitinib as in Figure 5. (B) Parental HN11 and HN11 GR cells were treated with the indicated inhibitors for 6 hours and lysed. Lysates were examined by Western blot analyses with the indicated antibodies. (C) Parental HN11 and HN11 GR cells were subjected to an MTS survival assay in increasing concentrations of gefitinib or AEW541 or a combination. When the combination was used, both drugs were used at the identical concentrations during the dose escalation. Error bars show ± SD.

Figure 7. Inhibition of IGFIR abrogates the emergence of resistance to gefitinib.

(A) Parental A431 cells were cultured in regular growth medium with the indicated drugs for 28 days in T-25 flasks. Photographs show the crystal violet staining of the surviving cells after 4 weeks of treatment. Original magnification, ×4. (B) Female athymic mice were injected with parental A431 cells as indicated in Methods. Once tumors reached a volume of approximately 40 mm³, 7 mice per group were randomized to no therapy or treatment with gefitinib (100 mg/kg daily via orogastric gavage), Mk-0646 (500 μg loading dose followed by 250 μg i.p. twice a week), or a combination of gefitinib and Mk-0646. Treatment was administered for 30 days. Tumors were measured twice a week with calipers. Data in the figure represent mean tumor volume ± SD.