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ARTICLE IN PRESS Pathology – Research and Practice xxx (2016) xxx–xxx

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Article Series of Liver Tumors from PR China

Downregulated DYRK2 expression is associated with poor prognosis and Oxaliplatin resistance in hepatocellular carcinoma Xiubing Zhang a,1 , Pan Xu b,1 , Wenkai Ni c , Hui Fan a , Jian Xu a , Yongmei Chen a , Wei Huang d , Shumin Lu d , Li Liang d , Jinxia Liu c , Buyou Chen b,∗ , Weidong Shi a,∗ a Department of Medical Oncology, the Second Peoples Hospital of Nan Tong, 43 Tangzha Xinglong Road, Nantong 226002, Jiangsu Province, People’s Republic of China b Department of Radiotherapy, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong 226001, Jiangsu Province, People’s Republic of China c Department of Gastroenterology, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong 226001, Jiangsu Province, People’s Republic of China d Department of Pathogen Biology, Jiangsu Province Key Laboratory for Information and Molecular Drug Target, Nantong University, 9 Qiangyuan Road, Nantong 226019, Jiangsu Province, People’s Republic of China

a r t i c l e

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Article history: Received 11 July 2015 Received in revised form 5 October 2015 Accepted 5 January 2016 Keywords: Hepatocellular carcinoma DYRK2 Poor prognosis Oxaliplatin resistance

a b s t r a c t We aimed to investigate the molecular mechanisms of DYRK2 and the HCC sensitivity to Oxaliplatin in DYRK2-depleted HCC cells. HCC tissue specimens were obtained from 86 HCC patients during hepatectomy. We used immunohistochemistry and western blot to analyze DYRK2 expression in HCC tissues and cell lines, and used siRNA transfection to decrease DYRK2 expression in HCC cells. Flow cytometry and CCK-8 assay were detected in cell cycle progression, cell proliferation and the efficacy of Oxaliplatin, DYRK2 was down-regulated in HCC tissues, compared with adjacent nontumor ones. The significant correlation between DYRK2 expression and clinicopathologic factors was apparently shown in the immunohistochemical and statistical analyses. The expression of DYRK2 was significantly associated with histological grade of HCC patients. Univariate and multivariate survival analyses revealed that DYRK2 was a significant predictor for overall survival of HCC patients. The depletion of DYRK2 promoted HCC cell proliferation, and increased resistance to Oxaliplatin. These data showed that the downregulated expression of DYRK2 in HCC tumor tissues could promote the proliferation of HCC cells. In addition, reducing DYRK2 expression was associated with poor prognosis and Oxaliplatin resistance in HCC. © 2016 Elsevier GmbH. All rights reserved.

1. Introduction Hepatocellular carcinoma (HCC) is the fifth most frequent tumor worldwide, HCC patients often have a poor prognosis [1,2]. Owing to increased incidence of tumor recurrence and intrahepatic metastasis after surgical resection. Therefore, most patients will receive some forms of chemotherapy in hope of prolonging life. However, HCC cells resistance to chemotherapy drugs is one of the most important factors that restrict the curative effect and survival. For example, epirubicin, cisplatin, 5-fluorouracil, etoposide and their combinations have demonstrated lower efficacy for the treatment of HCC [3,4]. Nowadays Oxaliplatin (Oxa) is considered to be one of the most potential advantage of the few cytotoxic drugs

∗ Corresponding authors. E-mail addresses: [email protected] (B. Chen), [email protected] (W. Shi). 1 These authors contributed equally to this work.

in the treatment of HCC, but the latest study found Oxaliplatin could increase the autophagy role of HCC cells, which eventually result in a tumor resistance response in HCC [5,6]. However, the molecular mechanism of Oxaliplatin-resistance in HCC remains undetermined. DYRK2, a dual-specificity tyrosine-(Y)-phosphorylationregulated kinase gene, is a member of an evolutionarily conserved family of DYRKs [7–9]. DYRK2 associates with an E3 ligase complex containing EDD, DDB1 and VPRBP proteins (EDVP complex). The formation of the EDD–DDB1–VPRBP complex were disrupted by small-interfering-RNA-mediated depletion of DYRK2. These observations indicate protein kinase DYRK2 is a scaffold that facilitates assembly of an E3 ligase [10]. Recent studies have also linked DYRK2 into the tumorigenesis of various cancer types. For example, DYRK2 was identified as the most frequently amplified and overexpressed gene in esophageal carcinoma and lung adenocarcinomas [11]. However, (DYRK2) can be a favorable prognostic marker in pulmonary adenocarcinoma, using immunohistochemical analysis and real-time reverse-transcriptase polymerase chain

http://dx.doi.org/10.1016/j.prp.2016.01.002 0344-0338/© 2016 Elsevier GmbH. All rights reserved.

Please cite this article in press as: X. Zhang, et al., Downregulated DYRK2 expression is associated with poor prognosis and Oxaliplatin resistance in hepatocellular carcinoma, Pathol. – Res. Pract (2016), http://dx.doi.org/10.1016/j.prp.2016.01.002

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reaction to determine the expression of DYRK2 and compared this with the clinicopathologic factors and survival [12]. Moreover, DYRK2 could control the epithelial–mesenchymal transition in breast cancer and ovarian cancer by degrading snail [13]. Another study further demonstrated that DYRK2 may function as a tumor suppressor. The study reported DYRK2 regulated p53 to induce apoptosis in response to DNA damage via phosphorylation of Ser 46, meanwhile DYRK2 translocated from the cytoplasm to the nucleus [14]. Dysregulation of the G1/S transition in the cell cycle contributed to tumor development. Degradation of c-Jun/c-Myc was a critical process for the G1/S transition. Recent report displayed that DYRK2 functioned as a kinase could prime the phosphorylation of c-Jun and c-Myc. Knockdown of DYRK2 in human cancer cells shortened the G1 phase and accelerated cell proliferation. Unphosphorylated c-Jun and c-Myc escape from the ubiquitin-mediated proteasomal degradation to induce their target genes, such as cyclin E [15]. However, the underlying mechanism of DYRK2 regulation of tumorigenesis in HCC is little understood. Furthermore, DYRK2 expression may be a predictive marker for chemotherapy in non-small cell lung cancer. Patients with high DYRK2 expression could be good candidates for chemotherapy [16]. It is of our interest to investigate whether downregulated DYRK2 expression contributed to Oxaliplatin Resistance in HCC. We speculated DYRK2 may be a tumor suppressor in HCC. The objective of this study is to understand that downregulated DYRK2 expression is associated with poor prognosis and Oxaliplatin resistance in HCC. Firstly, We intended to conduct a comprehensive analysis of DYRK2 as a prognostic marker in HCC and further investigate the associations between DYRK2 expression and clinicopathological factors. Moreover, we showed for the first time that DYRK2 was significantly downregulated in human HCC tissues and HCC cell lines. After knockdowning DYRK2 expression in HCC cells, we further observed the effects of DYRK2-deleted expression on cell proliferation and Oxaliplatin sensitivity of HCC cells. These findings

may provide a novel insight into the mechanism underlying HCC progression. 2. Materials and methods 2.1. Patients 86 samples from surgically treated HCC patients without preoperative systemic chemotherapy were obtained at the Affiliated Hospital of Nantong University between 2005 and 2008. All HCC tissues were collected using protocols approved by the Ethics Committee of Affiliated Hospital of Nantong University and written informed consent was obtained from every patient. The main clinical and pathologic variables of the patients are shown in Table 1. The 86 HCC cases comprised 69 males and 17 females. Their ages ranged from 21 to 69 (mean, 47.93 years). 69 patients were positive for HBsAg, 68 were positive for cirrhosis. According to the Edmondson grading system, histological grades were classified to well differentiated (grade I; n = 18), moderately differentiated (grade II; n = 36), and poorly differentiated (grade III; n = 32). The follow-up time was 5 years with a range of 1–82 months (median, 35.83 months). None of the patients received postoperative adjuvant therapy. The clinical data (patient history, diagnosis, staging, and survival) were obtained from the National Cancer Institute “Regina Elena” databases. Survival data were integrated by periodic interviews with their relatives. Tissue specimens were immediately processed after surgical removal. For histological examination, all tumorous and surrounding non-tumorous tissue portions were processed into 10% formalin-fixed and embedded in paraffin. The diagnosis was confirmed histologically in all cases, based mainly on examination of sections stained with hematoxylin and eosin (H&E). All the sections were viewed and diagnosed by three pathologists. DYRK2 expression were analyzed in 8 paired HCC and adjacent nontumorous samples by Western blot (anti-DYRK2 antibody, 1:200, Santa Cruz Biotechnology).

Table 1 DYRK2 Ki67 expression and clinicopathological features in 86 HCC specimens. Clinicopathological features

Total

DYRK2

Ki67

Low score < 5 n = 52

High score ≥ 5 n = 34

P value

2 value

Low score < 5 n = 45

High score ≥ 5 n = 41

P value

2 value

Age (years)

Downregulated DYRK2 expression is associated with poor prognosis and Oxaliplatin resistance in hepatocellular carcinoma.

We aimed to investigate the molecular mechanisms of DYRK2 and the HCC sensitivity to Oxaliplatin in DYRK2-depleted HCC cells. HCC tissue specimens wer...
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