ZNF452 Facilitates Tumor Proliferation and Invasion Via Activating AKT-Gsk3β Signaling Pathway and Predicts Poor Prognosis of Non-Small Cell Lung Cancer Patients
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www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 24), pp: 38863-38875 Research Paper ZNF452 facilitates tumor proliferation and invasion via activating AKT-GSK3β signaling pathway and predicts poor prognosis of non-small cell lung cancer patients Xiupeng Zhang1, Haijing Zhou1, Yong Zhang2, Lin Cai1, Guiyang Jiang1, Ailin Li3, Yuan Miao1, Qingchang Li1, Xueshan Qiu1 and Enhua Wang1 1Department of Pathology, College of Basic Medicine Science and First Affiliated Hospital of China Medical University, Shenyang, China 2Department of Pathology, Cancer Hospital of China Medical University, Shenyang, China 3Department of Radiotherapy, First Affiliated Hospital of China Medical University, Shenyang, China Correspondence to: Yuan Miao, email: [email protected] Keywords: ZNF452, NSCLC, AKT signaling, proliferation, invasion and metastasis Received: January 17, 2017 Accepted: February 24, 2017 Published: March 21, 2017 Copyright: Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT ZNF452 is a zinc-finger protein family member which contains an isolated SCAN (SRE-ZBP, CTfin51, AW-1 and Number 18 cDNA) zinc-finger domain. Despite the SCAN N-terminus domain is known to play a role in transcriptional regulation of genes involved in cell survival and differentiation, there are no precise cellular functions that have been assigned to ZNF452. In the present study, we found that either endogenous or exogenous ZNF452 was overexpressed in the cytoplasm of NSCLC cells and positive ratio of ZNF452 in NSCLC samples (50.8%, 93/183) was significantly higher than that in normal lung tissues (22.4%, 13/58, P<0.001). ZNF452 overexpression was correlated with advanced TNM stage (P=0.033), positive lymph node metastasis (P=0.002) and predicted poor overall survival of NSCLC patients (P<0.001). ZNF452 facilitated tumor growth, colony formation, G1-S phase arrest, migration and invasion through upregulating the levels of CyclinD1, CyclinE1, p-Rb, or Snail, and downregulating the expression of Zo-1. In nude mice xenografts, overexpressing ZNF452 also promoted tumor proliferation and metastasis. Subsequently, we found that the effect of ZNF452 on facilitating tumor proliferation and invasion was through activating its downstream AKT-GSK3β signaling pathway. Treatment of AKT inhibitor markedly prevented the phosphorylation of AKT and GSK3β which subsequently counteracted increasing expression of CyclinD1, CyclinE1 or Snail and restored the decreasing expression of Zo-1, as well as the upregulation of tumor proliferation and invasion, caused by ZNF452 overexpression. Taken together, the present study indicated that ZNF452 may be an upstream regulator of AKT-GSK3β signaling pathway and facilitates proliferation and invasion of NSCLC. INTRODUCTION domain in diverse transcription factors, and they are often seen as functional domains in the C2H2-type ZNF family ZNF452 (also known as SCAND3) contains a [2, 3]. Recent studies showed that C2H2 ZNF proteins SCAN (SRE-ZBP, CTfin51, AW-1 and Number 18 cDNA) played important roles in cancer progression through zinc-finger (ZNF) domain at the N-terminus, an INT core regulating transcription of downstream genes, which at the flank of the N-terminus and a TPase-derived hATd were involved in proliferation, apoptosis, migration and dimerization module [1]. SCAN zinc-finger domain invasion [4]. Interestingly, existing literatures showed that often co-exists with the KRAB (Krüppel-associated box) ZNFs played different roles in different cancer types, some www.impactjournals.com/oncotarget 38863 Oncotarget functioned as oncogenes and some functioned as tumor normalized protein level of ZNF452 in lung cancer suppressors [5–12]. However, except SCAN domains, (0.84±0.096) was obviously higher than that in ZNF452 does not contain C2H2 ZNF domains or KRAB noncancerous tissues (0.60±0.083, P=0.031, Figure 1I- domains. Despite the SCAN N-terminus domain is 1J). known to play a role in transcriptional regulation of genes involved in cell survival and differentiation, there are no ZNF452 expression and localization in precise cellular functions have been assigned to ZNF452 NSCLC cell lines [2-3, 13]. In this study, we explored the protein level and We evaluated ZNF452 expression and subcellular subcellular distribution of ZNF452 in both lung cancer localization in NSCLC cell lines. ZNF452 was expressed tissues and cell lines, as well as their clinicopathological in all the 8 cell lines and 6 of 7 NSCLC cell lines which relevances. We also investigated the effects of ZNF452 showed higher ZNF452 expression than normal bronchial on tumor proliferation and invasiveness after transfected epithelial cells (HBE, Figure 2A). In A549, H460 and with ZNF452 plasmid or ZNF452-siRNA. In conclusion, H1299 cells, endogenous ZNF452 localized in the we identified that ZNF452 enhanced proliferation and cytoplasm (Figure 2B). After overexpressing ZNF452 invasion of NSCLC cells through facilitating the activation in A549 cells, we found that exogenous ZNF452 also of AKT-GSK3β signaling pathway. localized in the cytoplasm (Figure 2C). RESULTS ZNF452 promoted NSCLC cells proliferation and accelerated cell cycle progression The expression of ZNF452 in NSCLC specimens After overexpressing ZNF452 in A549 and Initially, we evaluated ZNF452 expression in depleting ZNF452 in H1299 cells, MTT and colony 183 cases NSCLC samples and 58 cases corresponding formation assay results suggested that upregulating noncancerous tissues using IHC staining. We found that ZNF452 expression enhanced tumor growth and colony ZNF452 was dimly expressed in normal lung tissues formation abilities in A549 cells, whereas downregulating (Figure 1A-1B) but showed strong expression in the ZNF452 expression depressed tumor growth and colony cytoplasm of NSCLC specimens (Figure 1C-1D). The formation abilities in H1299 cells (Figure 3A-3B). Flow positive ratio of ZNF452 expression in NSCLC (50.8%, cytometry analysis was employed to characterize the 93/183) was significantly higher than that in normal cell cycle status in A549 cells with ZNF452 plasmid lung tissues (22.4%, 13/58, P<0.001, Figure1E-1G). transfecting or in H1299 cells with ZNF452-siRNA Statistical analysis indicated that ZNF452 overexpression transfection. We found that G1 phase was decreased and S was obviously correlated with advanced TNM stage phase were increased after transfecting ZNF452 plasmids (P=0.033) and positive lymph node metastasis (P=0.002). in A549 cells; however, G1 phase was enhanced and S There was no significant association between ZNF452 phase was depressed followed by depletion of ZNF452 overexpression and sex, age, histological type or with siRNA in H1299 cells (Figure 3C). The G2/M phase histological differentiation (Table 1). As the prognostic showed no visible changes after either upregulating or biological meaning or definition of tumor differentiation is downregulating the protein levels of ZNF452 (Figure different between lung adenocarcinoma and squamous cell 3C). Subsequently, the protein levels of cell cycle related carcinoma, we also investigated the associations between molecules were also examined by western blot followed ZNF452 expression and histological differentiation by overexpressing ZNF452 in A549 cells or inhibiting in lung adenocarcinoma or squamous cell carcinoma ZNF452 in H1299 cells. The protein levels of CyclinD1 separately. No significant correlation was seen either and CyclinE1, as well as p-Rb, were upregulated after in lung adenocarcinoma or squamous cell carcinoma ZNF452 overexpression, and they were correspondingly (Supplementary Table 1). Kaplan–Meier analysis decreased by ZNF452 knock-down (Figure 3D). The result showed that the overall survival of patients with other cyclin proteins showed no obvious changes either positive ZNF452 expression (42.73 ±2.87 months) was after upregulating or downregulating the protein levels of significantly shorter than those with negative ZNF452 ZNF452 (Supplementary Figure 1). expression (62.67 ±1.91 months, P<0.001, Figure 1H). Subsequent univariate analysis (UA) and multivariate ZNF452 increased NSCLC cells migration analysis (MA) revealed that, along with positive lymph and invasion node metastasis (P<0.001 for UA and P=0.023 for MA), overexpression of ZNF452 (P<0.001 for UA and P<0.001 Tumor migration (Figure 4A) and invasion (Figure for MA, Table 2) could be considered as independent 4B) were also enhanced by transfecting ZNF452 plasmid prognostic factors in NSCLC patients. in A549 cells or depressed by transfecting ZNF452 Then, we performed WB to test ZNF452 protein siRNA in H1299 cells. Western blotting results revealed levels in 20 cases paired fresh NSCLC samples. The that Snail was increased and Zo-1 was decreased after www.impactjournals.com/oncotarget 38864 Oncotarget overexpressing ZNF452 in A549 cells (Figure 4C). ZNF452 accelerated NSCLC malignant Accordingly, Snail was upregulated and Zo-1 was phenotype in vivo downregulated followed by depleting ZNF452 in H1299 cells (Figure 4C). Other proteins such as Slug, Occludin, In order to evaluate the effect of ZNF452 on tumor E-cadherin, α-catenin, N-cadherin and Vimentin proliferation in vivo, we subcutaneously injected ZNF452- presented no visible alterations (Supplementary transfected A549 cells (A549-ZNF452 (+)) or control cells Figure 2). into