TIP30 overcomes gefitinib resistance by regulating cytoplasmic and nuclear EGFR signaling in non-small-cell lung cancer

Abstract

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) (eg, gefitinib) exert potent therapeutic efficacy in non-small-cell lung cancer (NSCLC) harboring EGFR-activating mutations. However, the resistance to EGFR TKIs limits their clinical therapeutic efficacy. TIP30, a newly identified tumor suppressor, appears to be involved in the regulation of cytoplasmic and nuclear EGFR signaling in NSCLC. Our previous study demonstrated that TIP30 regulated EGF-dependent cyclin D1 transcription in human lung adenocarcinoma and suppressed tumorigenesis. In the present study, the involvement of TIP30 in combating gefitinib resistance in NSCLC was determined for the first time in vitro and in vivo. Gain and loss of function studies showed that overexpression of TIP30 effectively sensitized cells to gefitinib in vitro, whereas TIP30 inhibition promoted gefitinib cell resistance. Moreover, TIP30 negatively regulated the activation of the p-AKT and p-MEK signaling pathways in PC9/GR. Importantly, PC9/GR harbored high levels of nuclear EGFR, and overexpression of TIP30 restored irregular EGFR trafficking and degradation from early endosomes to the late endosomes, decreasing the nuclear accumulation of EGFR, which may partly or totally inhibit EGFR-mediated induction of c-Myc transcription. Xenographic tumors induced by overexpression of TIP30 by PC9/GR cells in nude mice were suppressed compared with their original counterparts. Overall, it was revealed that TIP30 overexpression restored gefitinib sensitivity in NSCLC cells and attenuated the cytoplasmic and nuclear EGFR signaling pathways and may be a promising biomarker in gefitinib resistance in NSCLC.

Keywords: EGFR; TIP30; gefitinib resistance; non-small-cell lung cancer (NSCLC).

FIGURE 1

Overexpression/downregulation of TIP30 increases/decreases the sensitivity of PC9/GR and PC9 cells to gefitinib. A, The growth inhibitory effects of gefitinib on PC9 and two different gefitinib‐resistant clones of PC9/GR cells were determined by MTT assays. B, C, The mRNA and protein levels of TIP30 expressed in PC9, PC9/GR, PC9/GR2 + TIP30, Mock, and negative control cell lines. D, Western blotting and RT‐qPCR analysis of the TIP30 levels in PC9 + shTIP30 and shRNA‐Con cells. E, Gefitinib sensitivities of PC9, PC9/GR2 + Mock, PC9/GR2 + TIP30, PC9 + TIP30‐SH2, and PC9 + shRNA‐Con cells were evaluated using the MTT assay. F, Transfected PC9 cells or PC9/GR2 cells were treated with 0.1 μM gefitinib or 5 μM gefitinib for 24 h, respectively, followed by flow cytometry to evaluate the apoptotic rate. The same concentration of DMSO was used as the control. G, Colony formation assay for transfected PC9/GR2 and transfected PC9 cells under the stimulation of 5 μM or 0.1 μM gefitinib, respectively.^* P < .05; ^** P < .01; ^*** P < .001

FIGURE 2

Cytotoxic effects of gefitinib on PC9, PC9/GR, and PC9/GR + TIP30 cells. A, PC9 and PC9/GR cells were pretreated with different concentrations of gefitinib (0‐1 μM) for 24 h. Expression of p‐AKT, total AKT, p‐EGFR, total EGFR, p‐ERK, and total ERK in the cell lysates was evaluated by western blotting. B, PC9/GR + TIP30 and PC9/GR + Mock cells were pretreated with different concentrations of gefitinib (0‐1 μM) for 24 h. Expression of p‐AKT, total AKT, p‐EGFR, total EGFR, p‐ERK, and total ERK was investigated by western blotting. ^* P < .05; ^*** P < .001

FIGURE 3

TIP30 modulates lysosome‐mediated EGFR degradation and nuclear localization upon EGF treatment in gefitinib‐resistant NSCLC cells. A, Confocal microscopy analysis of EGFR localization was carried out in PC9/GR + Mock and PC9/GR + TIP30 cells. B, Western blot analysis of EGFR expression in the nuclear fraction and total fraction of PC9, PC9/GR + Mock, and PC9/GR + TIP30 cells following EGF treatment for 30 min. C, D, Fluorescence microscopy analysis of the EGFR localization in PC9/GR + Mock and PC9/GR+TIP30 cells pre‐incubated with or without 5 μM gefitinib at 37°C, following stimulation of EGF for 0 min, 30 min, or 60 min. The quantitative analysis of EGFR and LAMP1 co‐lozalizations was shown in Figure S1. ^* P < .05; ^** P < .01;^*** P < .001. (Scale bars, 10 μm)

FIGURE 4

TIP30 negatively regulates c‐Myc transcription in PC9/GR cells. A, B, The c‐Myc mRNA and protein expressions were analyzed after 96 h of transduction of Mock or TIP30 in PC9/GR cells. C, Schematic of the STAT3‐binding sites in the c‐Myc promoter. D, ChIP analysis of STAT3 binding to the c‐Myc promoter in PC9/GR + Mock and PC9/GR + TIP30 cells. The lysates were analyzed using the anti‐histone 3 antibody as the positive control, and anti‐IgG was used as the negative control. ^** P < .01, ^*** P < .001

FIGURE 5

Overexpression of TIP30 attenuates growth of PC9/GR xenograft tumors in nude mice cells. A, Subcutaneous tumors were generated in nude mice injected with PC9 and PC9/GR cells that overexpressed TIP30 or carried a Mock vector. Representative images of the xenograft tumors dissected after treatment with gefitinib or vehicle for 12 d. B, Representative tumor burdens in the 4 investigated groups. In vivo tumor growth curves for the 4 groups were plotted and compared using Student t test. C, Representative immunohistochemical staining of c‐Myc, nuclear EGFR, and TIP30 in xenograft tumors. ^** P < .01; n = 5 for each group

FIGURE 6

Schematic diagram of this study. A, EGFR signaling network in gefitinib resistance in NSCLC cells. B, TIP30 overexpression in gefitinib resistance NSCLC cells