AUY922 effectively overcomes MET- and AXL-mediated resistance to EGFR-TKI in lung cancer cells

Abstract

The activation of bypass signals, such as MET and AXL, has been identified as a possible mechanism of EGFR-TKI resistance. Because various oncoproteins depend on HSP90 for maturation and stability, we investigated the effects of AUY922, a newly developed non-geldanamycin class HSP90 inhibitor, in lung cancer cells with MET- and AXL-mediated resistance. We established resistant cell lines with HCC827 cells harboring an exon 19-deletion mutation in of the EGFR gene via long-term exposure to increasing concentrations of gefitinib and erlotinib (HCC827/GR and HCC827/ER, respectively). HCC827/GR resistance was mediated by MET activation, whereas AXL activation caused resistance in HCC827/ER cells. AUY922 treatment effectively suppressed proliferation and induced cell death in both resistant cell lines. Accordingly, the downregulation of EGFR, MET, and AXL led to decreased Akt activation. The inhibitory effects of AUY922 on each receptor were confirmed in gene-transfected LK2 cells. AUY922 also effectively controlled tumor growth in xenograft mouse models containing HCC827/GR and HCC827/ER cells. In addition, AUY922 reduced invasion and migration by both types of resistant cells. Our study findings thus show that AUY922 is a promising therapeutic option for MET- and AXL-mediated resistance to EGFR-TKI in lung cancer.

Fig 1. EGFR-TKI and AUY922 cytotoxicity in parental HCC827 and resistant cell lines.

Cells were treated with the indicated doses of gefitinib, erlotinib, afatinib, or AUY922 for 72 hours in medium containing 1% FBS. Cell viability was determined using the MTT assay.

Fig 2. Suppression of MET and AXL by AUY922 in resistant cell lines.

A, Cells were treated with the indicated doses of AUY922 for 72 hours in medium containing 1% FBS. Attached cells were stained with trypan blue solution (top). Cell viability based on cell counting is also shown (bottom). Bars represent the mean ±SD of three wells. B, Cells treated with AUY922, similar to panel A. After 48 hours, cells were harvested and EGFR-related signaling molecules were evaluated using western blotting. C, LK2 cells were treated with vector-containing wild-type EGFR, del E746-E750, MET, or AXL and the indicated doses of AUY922 for 12 hours.

Fig 3. Induction of cell death in response to AUY922.

A and B, Cells were treated with 0.1 μmol/L AUY922 for 24–72 hours. To analyze the cell cycle distribution, the harvested cells were analyzed by flow cytometry. C, Cell lysates were analyzed using western blotting. The indicated antibodies were used to evaluate apoptotic cell death.

Fig 4. Efficacy of AUY922 in HCC827/GR and HCC827/ER tumor xenograft models.

A, SCID mice with established HCC827/GR and HCC827/ER tumor cell xenografts were treated with AUY922. Tumor size was measured on the indicated days, and tumor volume was calculated. Arrows mean last day of drug treatment (red arrow, HCC827/GR; purple arrow, HCC827/ER). Bars represent the mean tumor volume ± SD. B and C, Immunohistochemical staining for Ki-67 and TUNEL followed by quantitative analysis of proliferation and apoptosis in (B) Ki-67⁺ cells and (C) TUNEL⁺ cells. *p< 0.01 and **p< 0.001 in comparison with the control.

Fig 5. Reduced migratory and invasive capabilities of cancer cells following AUY922 treatment.

Cells were seeded onto either collagen or matrigel-coated polycarbonate filters to determine their migratory and invasive potentials, respectively. Cells were incubated in modified Boyden chambers with or without the indicated doses of AUY922 for 24 hours, and the cells that penetrated the filter were stained and counted using a light microscope. Bars represent the mean ± SD of three wells. *p< 0.01 and **p< 0.001 in comparison with HCC827 cells.