Overexpression of PDZ-binding kinase confers malignant phenotype in prostate cancer via the regulation of E2F1by Jia-Hong Chen, Yu-Xiang Liang, Hui-Chan He, Jin-Yan Chen, Jian-Ming Lu, Guo Chen, Zhuo-Yuan Lin, Xin Fu, Xiao-Hui Ling, Zhao-Dong Han, Fu-Neng Jiang, Wei-De Zhong

International Journal of Biological Macromolecules


Biochemistry / Molecular Biology / Structural Biology


International Journal of Biological Macromolecules 81 (2015) 615–623

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules j ourna l h o mepa ge: www.elsev ier .com/ locate / i jb iomac

Overexpression of PDZ-binding kinase confers m in prostate cancer via the regulation of E2F1

Jia-Hong Yan

Guo Che -Do

Wei-De Z a Department o Guang

Guangzhou Me b Department o China c Reproductive 16001 d Guangdong P gzhou e Department of Urology, Huadu District People’s Hospital, Southern Medical University, Guangzhou, 510800, China f Urology Key Laboratory of Guangdong Province, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University,

Guangzhou, 510230, China a r t i c l

Article history:

Received 11 Fe

Received in re

Accepted 21 A

Available onlin


Prostate cance

PDZ-binding k

Biochemical re 1. Introdu

Prostate cer and the men in Un indolent tu sive metast often succe ever, diseas ∗ Correspon pital, Guangzh

Tel.: +86 20 81

E-mail add 1 These auth http://dx.doi.o 0141-8130/© e i n f o bruary 2015 vised form 16 August 2015 ugust 2015 e 24 August 2015 r inase currence-free survival a b s t r a c t

Roles and mechanisms of cell cycle-specific transcription factor E2F1 on prostate cancer (PCa) have not been fully elucidated. To address this problem, we here identified PDZ-binding kinase (PBK) as a direct target for E2F1 through bioinformatics binding site prediction, combined with chromatin immunoprecipitation-PCR (ChIP-PCR), quantitative (Q)-PCR and Western blot analysis. Then, we observed that the knockdown of both E2F1 and PBK could suppress cell proliferation, invasion and migration of PCa cell lines in vitro. Based on Taylor dataset, we found that PBK upregulation occurred more frequently in PCa patients with the older age of patients (P = 0.044), the higher Gleason score (P < 0.001), the advanced clinical pathological stage (P = 0.019), the presence of metastasis (P = 0.008), the overall survival (P < 0.001) and PSA failure (P = 0.004). More interestingly, the survival analysis identified PBK as an independent factor for predicting the biochemical recurrence-free survival of PCa patients (P = 0.041).

Taken together, these findings offer the convincing evidence for the first time that the overexpression of

PBK may lead to high malignant phenotype in PCa cells via the regulation of E2F1. PBK may function as a biomarker that can differentiate patients with biochemical recurrent and non-biochemical recurrent disease following radical prostatectomy, highlighting its potential as a therapeutic target. © 2015 Published by Elsevier B.V. ction cancer (PCa) represents the most commonly solid cansecond leading cause of cancer-related deaths among ited States [1]. Clinical behavior of PCa ranges from mors with no or little clinical significance to aggresatic and lethal diseases [2]. Locally defined disease is ssfully treated with surgery and/or radiotherapy; howe recurs in an estimated 15 ∼ 30% of patients [3]. In ding author at: Department of Urology, Guangzhou First People’s Hosou Medical University, Guangzhou 510180, China. 048312; fax: +86 20 83373322. ress: wdezhong@21cn.com (W.-D. Zhong). ors contributed equally to this article. spite of several clinical parameters, such as serum prostate specific antigen (PSA) levels, age and underlying health of men, the extent of tumor spread, appearance under the microscope, and the response to initial treatment, which may provide some prognostic utility in the treatment settings, there are currently no definitive clinical methods that can reliably predict the responses to clinical therapies for PCa [4]. As the molecular etiology of PCa becomes better understood, it is clear that PCa tumor progression is influenced by a multistep process, involving both genetic insults to epithelial cells and changes in epithelial–stromal interactions [5]. Therefore, it is extremely necessary to clarify the molecular mechanism underlying the progressive process of PCa in order to identify novel and effective biomarkers, to strengthen the efficiency of early diagnosis and to help create the efficient therapeutic strategies of this cancer.

Cell cycle-specific transcription factor E2F1 was originally identified as a member of the E2F family of transcription factors, rg/10.1016/j.ijbiomac.2015.08.048 2015 Published by Elsevier B.V. Chena,b,1, Yu-Xiang Lianga,1, Hui-Chan Hea,1, Jinna, Zhuo-Yuan Lina, Xin Fua, Xiao-Hui Lingc, Zhao honga,d,e,f,∗ f Urology, Guangdong Key Laboratory of Clinical Molecular Medicine and Diagnostics, dical University, Guangzhou 510180, China f Urology, Huizhou Municipal Central People’s Hospital, Huizhou, Guangdong, 516001,

Medicine Centre, Huizhou Municipal Central People’s Hospital, Huizhou, Guangdong, 5 rovincial Institute of Nephrology, Nanfang Hospital, Southern Medical University, Guanalignant phenotype

Chena, Jian-Ming Lua, ng Hana, Fu-Neng Jianga, zhou First People’s Hospital, , China , 510515, China 616 J.-H. Chen et al. / International Journal of Biological Macromolecules 81 (2015) 615–623

Fig. 1. E2F1 re including oPO (HI-E2F1) and (HI-E2F1) and

E2F1 (HI-E2F1

E2F1 (HI-E2F1 negative contr which cons (E2F1–E2F3 to either ac tionally, E2 regulating t thesis and c role in the

S-phase tra show that role in carc cycle, overr primary cel in various h gene in eryt protein in b lines [11,12 cancer and sion associa

Especially, ment of E2F that E2F1 e that E2F1 kgulated PBK expression in PCa cell lines in vitro. (A) Putative E2F1-PBK binding region (seq

SSUM, Consite and PROMO; (B) relative expression levels of E2F1 mRNA in DU145 and L knock-down of PBK (si-PBK); (C) relative expression levels of PBK mRNA in DU145 and L knock-down of PBK (si-PBK); (D) relative expression levels of E2F1 protein in DU145 a ) and knock-down of PBK (si-PBK); (E) relative expression levels of PBK protein in DU145 ) and knock-down of PBK (si-PBK); (F) ChIP-PCR analysis. Compared to the negative con ol; si-E2F1, siRNA for E2F1; si-PBK, siRNA for PBK; Hi-E2F1: reexpression of E2F1. Data w ists of eight proteins commonly classified as activators a) or repressors (E2F3b–E2F8), based on their ability tivate or repress target gene transcription [6]. FuncFs control the progression through the cell cycle by he transcription of genes that are essential for DNA synell cycle progression [7]. Especially, E2F1 has a critical initiation and direction of genes required for the G1 to nsition as well as in apoptosis [8]. Growing evidence several E2Fs, including E2F1, may play an important inogenesis by inducing quiescent cells to enter the cell iding various growth-arrest signals, and transforming ls [9]. Abnormal expression of E2F1 has been observed uman cancer types, including amplification of the E2F1 hroleukemia cell lines [10], elevated expression of E2F1 reast cancer cell lines and head and neck carcinoma cell ], and overexpression of E2F1 in invasive ductal breast non-small cell lung cancer, where high E2F1 exprestes with advanced disease and poor prognosis [13,14]. accumulating studies also demonstrated the involve1 in human PCa. For example, Ren et al. [15] observed xpression was elevated in advanced PCa and found nockdown could inhibit prostate tumor growth in vitro and in vivo anti-immun of E2F1 as elevated E2 receptor (A of hormone could induc of EGR1 tra