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APPL1 Promotes the Migration of Gastric Cancer Cells by Regulating Akt2 Phosphorylation

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APPL1 Promotes the Migration of Gastric Cancer Cells by Regulating Akt2 Phosphorylation INTERNATIONAL JOURNAL OF ONCOLOGY 51: 1343-1351, 2017 APPL1 promotes the migration of gastric cancer cells by regulating Akt2 phosphorylation YINGXUN LIU1*, CHUNLI ZHANG1*, LINGYU ZHAO1, NING DU3, NI HOU1,2, TUSHENG SONG1 and CHEN HUANG1,2 1Department of Cell Biology and Genetics, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi 710061; 2Key Laboratory of Environment and Genes Related to Diseases (Xi'an Jiaotong University), Ministry of Education of China, Xi'an, Shaanxi 710061; 3Department of Oncology Surgery, The First Affiliated Hospital of Medical College, Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China Received May 22, 2017; Accepted August 25, 2017 DOI: 10.3892/ijo.2017.4121 Abstract. As a multifunctional adaptor protein, APPL1 results demonstrate an important new function of APPL1 in (adaptor protein containing pleckstrin homology domain, regulating cell migration through a mechanism that depends phosphotyrosine binding domain and a leucine zipper motif 1) on Akt2 phosphorylation. is overexpressed in many cancers, and has been implicated in tumorigenesis and tumor progression. The present study Introduction investigated the expression of APPL1 in gastric carcinoma and the function in regulating cell migration. We investigated Gastric cancer (GC) is one of the most common human the expression of APPL1 in gastric carcinoma based upon malignant diseases and the second leading cause of cancer The Cancer Genome Atlas (TCGA) database. The expression mortality worldwide (1). The reason for the high fatality of APPL1 in collected gastric carcinoma tissues and cultured rate associated with this disease is that most cases of GC cells was measured by qRT-PCR and western blot analysis. are clinically detected only at an advanced stage with distant Transwell assay and wound healing assay were used to analyze metastasis. Metastasis is the spread of cancer cells from their the effects of APPL1 on tumor cell migration. The statistical primary location to other parts of the body. Unfortunately, results based upon TCGA database showed significantly once cancer becomes metastatic it cannot be treated higher expression of APPL1 in gastric carcinoma compared effectively by surgical and radiation therapies (2). Tumor to adjacent normal tissues, and we confirmed these findings by metastasis is a complex process that requires the integration measuring APPL1 expression in collected gastric carcinoma of signaling events that occur in distinct locations within the tissues and cultured cells. The results of Transwell assay and cell. However, little is known about molecular mechanisms wound healing assay showed that when APPL1 was silenced underlying tumor metastasis (3,4). Therefore, a highly critical by siRNA, cell migration was inhibited and overexpression of issue is to explore the molecular mechanisms and to identify APPL1 promoted migration. Western blot results demonstrated ‘key’ molecular markers related to metastasis in gastric that changes in several mesenchymal markers were consistent cancer, which will provide new targets for intervention in the with the observed reduction or enhancement of cell migration. metastatic recurrence of gastric cancer (5). Importantly, the expression of APPL1 significantly affected The recent discovery of altered adaptor proteins in cancer the phosphorylation of Akt2. In addition, MMP2 and MMP9, has identified a fundamental change involved in cell migration downstream effectors of Akt2 changed accordingly, which is and invasion (6-8). Adaptor proteins are composed exclusively a critical requirement for Akt2-mediated cell migration. The of domains and motifs that mediate molecular interactions, and can thereby link signaling proteins such as activated cell- surface receptors to downstream effectors. Adaptor proteins are emerging as important regulators of key signaling events Correspondence to: Professor Chen Huang, Department of Cell that control cellular behavior underlying many biological and Biology and Genetics, School of Basic Medical Sciences, Xi'an pathological processes (6). Adaptor proteins accomplish this Jiaotong University Health Science Center, Xi'an, Shaanxi 710061, through their multiple functional domains by bringing together P. R. Ch i na and targeting protein-binding partners to specific locations E-mail: [email protected] within cells. For example, APPL1 is a 709 amino acid, endo- * somal protein, containing a pleckstrin-homology (PH) domain, Contributed equally phosphotyrosine-binding (PTB) domain and leucine zipper motif. APPL1 interacts with 14 proteins, including follicle- Key words: APPL1, gastric carcinoma, cell migration, p-Akt2, miR-145, The Cancer Genome Atlas stimulating hormone receptor, deleted in colorectal carcinoma (DCC), Rab5a and Akt2 (9). These membrane receptors and signaling molecules take part in various signaling pathways to 1344 Liu et al: APPL1 PROMOTES MIGRATION OF GC CELLS BY REGULATING AKT2 PHOSPHORYLATION mediate apoptosis, development (10), cell proliferation, chro- Human gastric carcinoma tissue collection and cell culture. matin remodeling (11), DNA repair (12) and cell survival (13). Human gastric carcinoma and adjacent normal tissues were Recently, emerging data suggest that APPL1 also plays a key consecutively collected between April 2013 and November role in the regulation of cell migration (14). However, the 2015 at the Pathology Department of the First Affiliated molecular mechanism is not well understood. Hospital of Medical College, Xi'an Jiaotong University. No APPL1 was originally identified as an Akt2-binding local or systemic treatment was conducted prior to operation. protein in a yeast two-hybrid screening system and Akt2 Clinicopathological data such as age and gender, as well as is a known regulator of cell migration (15). Akt is a serine/ lymph node metastasis status, lymphatic and venous inva- threonine kinase that is activated downstream of phosphati- sion status, tumor stage, and pTNM stage were obtained by dylinositol 3-kinase. Subsequently, active Akt phosphorylates reviewing their pathology records. Tumor stage was deter- its downstream effectors to regulate several cellular processes, mined according to the American Joint Committee on Cancer including cell growth, survival and proliferation (16). (AJCC) staging criteria. A total of 32 paired samples were Moreover, there has recently been growing interest in the func- performed to examine APPL1 expression, including 18 (56.3%) tion of Akt in the regulation of cell migration. Akt has been men and 14 (43.7%) women. The age distribution of the patients shown to stimulate the migration of epithelial cells, fibroblasts examined ranged from 42 to 78 years of age. Informed consent and fibrosarcomas (17,18). was obtained from each patient and the study was approved by Recent studies have indicated that APPL1 gene amplifica- the Institute Research Ethics Committee at the Cancer Center tion is common in breast (20), prostate cancer (7) and several of Xi'an Jiaotong University. cell lines, including pancreatic carcinoma cells (12), HCT116 Stomach adenocarcinoma cell lines (including AGS, and SW480 colorectal cancer cell lines (9). Importantly, BGC-823, SGC-7901 and MKN-45) and a GES-1 cell line were the expression of APPL1 protein and mRNA were highly obtained from the Shanghai Genechem Co., Ltd. (Shanghai, upregulated in gastric cancer. It was also reported that the China). The cells were grown at 37˚C in a 5% CO2 incubator, expression of APPL1 in GC was statistically associated in RPMI-1640 culture media supplemented with fetal bovine with depth of infiltration and lymph node metastasis (19). serum (FBS). The observation suggested that expression of APPL1 had a role in tumor infiltration and metastasis. However, little is RNA extraction and quantitative real-time PCR. Total RNA known about the molecular mechanism of APPL1 in tumor was isolated from prepared gastric issues and cell lines using metastasis in gastric cancer. In the present study, we inves- Trizol reagent (Invitrogen, Carlsbad, CA, USA), measured tigated the expression of APPL1 protein in gastric cancer spectrophotometrically using NanoDrop (Thermo Fisher and its direct effect on cell migration. We also showed that Scientific, Inc., Wilmington, DE, USA). cDNA was synthesized APPL1 promoted cell migration via the Akt2 pathway using by using PrimeScript® RT reagent kit (DRR037A; Takara, loss of function assays. Our results suggest that APPL1 Dalian, China), according to the manufacturer's protocol. promotes invasion and metastasis of gastric cancer cells and Quantitative real-time PCR (qRT-PCR) was performed using the underlying molecular mechanism may facilitate Akt2 the SYBR-Green PCR kit (Takara), and was conducted in phosphorylation and activation of downstream effectors. the IQ5 Optical System real-time PCR machine. The relative expression of APPL1 genes was calculated with the 2-ΔΔCt Materials and methods method. The primers used are listed in Table I. Retrieval of TCGA public data. Based on The Cancer Genome Western blot analysis. Protein expression levels were Atlas (TCGA) (http://cancergenome.nih.gov/) public datasets, assessed using western blot analysis. In brief, total cell we evaluated and analyzed APPL1 expression in gastric carci- lysates from different experiments were obtained by lysing noma. Clinical information and gene expression profile data the cells in RIPA buffer. Following protein concentra- were downloaded at the website of the UCSC cancer
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