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Silencing of SNX1 by Sirna Stimulates the Ligand-Induced Endocytosis of EGFR and Increases EGFR Phosphorylation in Gefitinib-Resistant Human Lung Cancer Cell Lines

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Silencing of SNX1 by Sirna Stimulates the Ligand-Induced Endocytosis of EGFR and Increases EGFR Phosphorylation in Gefitinib-Resistant Human Lung Cancer Cell Lines 1520 INTERNATIONAL JOURNAL OF ONCOLOGY 41: 1520-1530, 2012 Silencing of SNX1 by siRNA stimulates the ligand-induced endocytosis of EGFR and increases EGFR phosphorylation in gefitinib-resistant human lung cancer cell lines YUKIO NISHIMURA1, SOICHI TAKIGUCHI2, KIYOKO YOSHIOKA3, YUSAKU NAKABEPPU4 and KAZUYUKI ITOH3 1Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582; 2Institute for Clinical Research, National Kyushu Cancer Center, Fukuoka 811-1395; 3Department of Biology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka 537-8511; 4Division of Neurofunctional Genomics, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan Received May 8, 2012; Accepted July 6, 2012 DOI: 10.3892/ijo.2012.1578 Abstract. Gefitinib is known to suppress the activation of delivery of pEGFR from early endosomes to late endosomes. EGFR signaling, which is required for cell survival and Further, western blot analysis revealed that silencing of SNX1 proliferation in non-small cell lung cancer (NSCLC) cell expression by siRNA in the gefitinib-resistant cells leads to lines. We previously demonstrated that the gefitinib-sensitive an accelerated degradation of EGFR along with a dramatic NSCLC cell line PC9 shows efficient ligand-induced endo- increase in the amounts of pEGFR after EGF stimulation. cytosis of phosphorylated EGFR (pEGFR). In contrast, the Based on these findings, we suggest that SNX1 is involved in gefitinib-resistant NSCLC cell lines QG56 and A549 showed the negative regulation of ligand-induced EGFR phosphory- internalized pEGFR accumulation in the aggregated early lation and mediates EGFR/pEGFR trafficking out of early endosomes, and this was associated with SNX1, a protein that endosomes for targeting to late endosomes/lysosomes via the interacts with and enhances the degradation of EGFR upon early/late endocytic pathway in human lung cancer cells. EGF stimulation. In the present study, to investigate the role of SNX1 on EGF-stimulated EGFR/pEGFR endocytosis via Introduction the endocytic pathway, we examined the effect of depletion of SNX1 expression by siRNA in human NSCLC cell lines. The endocytosis of epidermal growth factor receptor (EGFR) Using immunofluorescence, we demonstrated that transfection serves as a model for studying ligand-induced receptor- of SNX1 siRNA into gefitinib-resistant NSCLC cells resulted mediated endocytosis. Upon EGF stimulation, the dimerizd in the disappearance of a large amounts of SNX1 staining. EGF-EGFR complexes are internalized and transported via In addition, upon 15 min of EGF stimulation, we observed clathrin-coated vesicles to early endosomes. EGFR then an efficient EGFR phosphorylation and a rapid endocytic recruits and phosphorylates signaling molecules, leading to the activation of the MAPK-signal transduction cascade, an important mechanism for regulating cell growth (1-4). To turn off EGF signaling, the EGF-EGFR complexes are delivered to the lysosomes for degradation by a process known as receptor Correspondence to: Dr Yukio Nishimura, Division of Pharma- downregulation. Therefore, endocytosis of EGF-EGFR ceutical Cell Biology, Graduate School of Pharmaceutical Sciences, complexes is closely related to attenuation of intracellular Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, EGFR signaling. Furthermore, EGFR mediates an important Japan role in the pathogenesis of different tumors, and therapies E-mail: [email protected] directed at inhibiting EGFR function have potential as anti- Abbreviations: EGFR, epidermal growth factor receptor; pEGFR, cancer treatments (5,6). phosphorylated epidermal growth factor receptor; SNX1, sorting Gefitinib, a selective EGFR tyrosine kinase inhibitor, has nexin 1; NSCLC, non-small cell lung cancer; LIMPII, lysosomal been shown to block the signal transduction pathways impli- integral membrane protein II cated in the proliferation and survival of cancer cells (7-10). It was reported previously that of the 9 non-small cell lung Key words: sorting nexin 1, phosphorylated EGF receptor, endo- cancer (NSCLC) cell lines examined, the PC9 cell line was cytosis, endosomes/lysosomes, gefitinib, non-small cell lung cancer most sensitive to the effect of gefitinib with respect to EGFR cell lines phosphorylation and activation of EGFR downstream effec- tors such as AKT and those in the ERK1/2 pathway, which are required for EGFR-stimulated proliferation (11). In contrast, NISHIMURA et al: ROLE OF SNX1 IN THE REGULATION OF EGFR ENDOCYTOSIS IN HUMAN LUNG CANCER CELLS 1521 the other NSCLC lines such as QG56 and A549 cells showed Materials and methods greater resistance to gefitinib (11). Consequently, we hypoth- esize that the mechanism responsible for determining the Materials. Texas red-labeled human transferrin, Texas red- sensitivity of the EGFR endocytic pathway could be useful in labeled EGF, and SlowFade anti-fade reagent were purchased predicting the potential effectiveness of gefitinib in NSCLC from Molecular Probes (Eugene, OR, USA). DAPI, recombinant patients. We have previously investigated the endocytosis of human EGF was purchased from PeproTech (London, UK). Texas red-labeled EGF in the absence or presence of gefitinib Bafilomycin A1, and cycloheximide (CHX) were obtained from in three NSCLC cell lines, and then assessed the amounts of Sigma (St. Louis, MO, USA). Other chemicals were of reagent internalized Texas red-EGF or phosphorylated EGFR (pEGFR) grade and were obtained from commercial sources. by using confocal immunofluorescence microscopy (12-14). We showed that an aberration in certain steps of EGF-EGFR/ Cell culture. Cell lines PC9, QG56 and A549 (National Kyushu pEGFR trafficking from the early endosomes to the late endo- Cancer Center, Fukuoka, Japan) were cultured in RPMI somes/lysosomes does occur in the gefitinib-resistant human supplemented with 10% fetal bovine serum (FBS). Cells were lung cancer cell line QG56 and A549, whereas endocytosis maintained under standard cell culture conditions at 37˚C and of EGFR/pEGFR is normal in gefitinib-sensitive PC9 cells 5% CO2 in a humid environment. (12-14). Accordingly, we suggested that impairment of certain steps of EGF-EGFR/pEGFR trafficking from early endosomes Small interfering RNA. siRNA targeting SNX1 was purchased to late endosomes/lysosomes might confer gefitinib-resistance from Dharmacon (Boulder, CO, USA). The target sequence of in NSCLC cell lines. Furthermore, we made a novel observation the siRNA was as follows: 5'-AAGAACAAGACCAAGAGC that large amounts of sorting nexin 1 (SNX1) are localized in CAC-3'. Scramble sequence was used as a control. The 3 NSCLC the aggregated vesicular structures of early endosomes where cell lines were transfected with Lipofectamine 2000 (Life the internalized pEGFR is also accumulated (13,14). Therefore, Technologies, Gaithersburg, MD, USA) in the presence of we postulate that impairment of protein function, such as the 40 nM siRNA targeting SNX1 according to the manufacturer's SNX1 regulation of EGFR/pEGFR trafficking in the early protocol. Knockdown efficiency was determined by qRT-PCR, endocytic pathway, might perturb EGFR/pEGFR endocytosis, and confocal immunofluorescence microscopy analysis. which subsequently leads to gefitinib-resistance in NSCLC cell lines. qRT-PCR analysis. The 3 NSCLC cell lines PC9, QG56 or SNX1 was previously demonstrated to be a protein that A549 cells transfected with siRNA-control or siRNA-SNX1 interacts with EGFR (15) and is localized to early endo- were stimulated with EGF (100 ng/ml) at 37˚C for 5, 15, or somes through its phospholipid-binding motif termed the 30 min, and total RNA was extracted from each cell line using phox homology (PX) domain (16). SNX1 is homologous to an RNeasy RNA isolation kit (Qiagen, Hilden, Germany) Vps5p, a yeast protein that is required for endosome-to-Golgi according to the manufacturer's instructions. Transcription trafficking (17-19). Previous studies also revealed that over- into cDNA was done in a 20-µl volume using ThermoScript expression of SNX1 causes enhanced EGFR degradation and RT-PCR System with random hexamer (Invitrogen, Carlsbad, that deletion mutant of SNX1 blocked EGFR degradation but CA, USA) according to the manufacturer's instructions. All failed to inhibit receptor endocytosis (15,20). Therefore, it was PCR reactions were carried out in a final volume of 25 µl and suggested that SNX1 interacts with EGFR and enhances the were performed in the ABI PRISM 7000 Sequence Detection degradation of the receptor upon EGF stimulation, thereby System (Applied Biosystems, Foster City, CA, USA) according implying that SNX1 plays a role in endosome-lysosome traf- to the manufacturer's protocol. Sequence-specific primers ficking. However, recent evidence has failed to support the were quoted from an official website ‘PrimerBank’ (http:// intracellular colocalization of SNX1 with EGFR and its direct pga.mgh.harvard.edu/primerbank/) for the indicated genes role in EGFR degradation, raising the possibility that alterna- (Table I). The reaction mix consisted of SYBR Premix Ex Taq tive mechanisms are involved in the function of SNX1 (21,22). (2x) (Takara Bio., Shiga, Japan) 12.5 µl, ROX Reference Dye Consequently, the molecular mechanism underlying EGFR (x50) (Takara Bio.) 0.5 µl, 0.2 µM of each specific forward membrane trafficking remains to be elucidated. and reverse primer, and 9 µl of diluted cDNA (equivalent to
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