J Mol Neurosci DOI 10.1007/s12031-015-0600-z

Knockdown of PFTK1 Inhibits the Migration of Glioma Cells Shaochen Fan 1,2 & Chengjin Zhao 3 & Li Zhang 4 & Shirong Dai 3 & Jianbing Ren 3 & Xiubing Zhang 3 & Na Ban 4 & Xiaojuan He 4 & Lixiang Yang 5 & Zhen Bao 5 & Wenjuan Chen 4 & Jie Sun 4 & Yilu Gao 1,2 & Tao Tao 4

Received: 27 April 2015 / Accepted: 3 June 2015 # Springer Science+Business Media New York 2015

Abstract The prognosis of glioma patients is generally poor, so it is urgent to find out the underlying molecular mechanisms. PFTK1 is a member of cyclin-dependent kinases (Cdks) family and has been reported to contribute to tumor migration and invasion. In this study, we aimed to explore the expression and function in human glioma. Western blot and immunohistochemistry were used to evaluate the expression of PFTK1. PFTK1 expression was higher in glioma tissues compared with normal brain tissues, and its level was associated with the WHO grade in Western blot analysis. The suppression of PFTK1 expression by RNA interference was

shown to inhibit the migration of glioma cells. Knockdown of PFTK1 increases E-cadherin expression and decreases vimentin expression. These data show that PFTK1 may participate in the pathogenic process of glioma, suggesting that PFTK1 can become a potential therapeutic strategy for gastric cancer. Keywords Glioma . PFTK1 . Migration . Knockdown . E-cadherin

Introduction Shaochen Fan and Chengjin Zhao have contributed equally to this work. Tao Tao and Yilu Gao have contributed equally to this work. * Yilu Gao [email protected] * Tao Tao [email protected] 1

Department of Neurosurgery, The Affiliated Hospital of Nantong University, Xisi Road No. 20, Nantong 226001, People’s Republic of China

2

Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong 226001, People’s Republic of China

3

Department of Neurosurgery, Nantong Second People Affiliated Hospital of Nantong University, 43 Xinglong Road, Nantong 226001, Jiangsu Province, People’s Republic of China

4

Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Medical College of Nantong University, Nantong, Jiangsu Province 226001, People’s Republic of China

5

Department of Neurosurgery, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu Province 215006, People’s Republic of China

Glioma, with poor prognosis for patients, is the most frequent type of primary central nervous system tumor (Assem et al. 2012; Deorah et al. 2006; Reardon et al. 2012). Despite that the operation and adjuvant therapy have been improving, gliomas still stay highly resistant to conventional treatments (Clarke et al. 2010; Wen and Kesari 2008; Stupp et al. 2005) and less than 3 % of diagnosed patients hold a 5-year survival (Ohgaki and Kleihues 2005). This phenomenon is due to the highly invasive nature of glioma cells (Manji et al. 2003). Therefore, it is urgent to find out the underlying molecular mechanisms leading to glioma to offer proposals for glioma therapies. Altered regulation of the cell cycle is closely related to the proliferation of cancer and carcinogenesis (Sherr 1996), and research has reported that the family of cyclin-dependent kinases (Cdks) could operate the progression of the cell cycle (Chang et al. 2000). PFTK1, also named as PFTAIRE1 or cyclin-dependent kinase (CDK) 14, belongs to the CDK family, which includes mammalian PFTAIRE, PCTAIRE, PITSLRE, PISSLRE, etc. (Yang and Chen 2001; Lazzaro and Julien 1997; Besset et al. 1998; Meyerson et al. 1992; Li et al. 2002; Charrasse et al. 1999; Malumbres and Barbacid

J Mol Neurosci

2005) and has different functions, for instance, CDKs regulate cell cycle progression (CDK1, CDK2, CDK3, CDK4, and CDK6), transcription (CDK7, CDK8, CDK9, and CDK10), differentiation (CDK5), and other processes. In some cancers, CDKs may be related with the prognosis of patients (Marone et al. 1998; Mihara et al. 2001; Kim et al. 2008). PFTK1, a serine/threonine-protein kinase protein, is highly expressed in the brain, pancreas, kidney, heart, testis, and ovary (Yang and Chen 2001). PFTK1 can promote the cell cycle as classical CDKs (Marone et al. 1998) and control the hepatocellular carcinoma (HCC) cell migration as a regulatory factor (Mihara et al. 2001). Miyagaki also proved that PFTK1 is useful as a prognostic marker (Miyagaki et al. 2012). However, the role of PFTK1 in gliomas has not yet been reported. In this study, we aimed to investigate the expression and function of PFTK1 in gliomagenesis, using immunohistochemistry and Western blot. We first showed that PFTK1 expression was upregulated in glioma specimens and glioma cells and had a negative correlation with E-cadherin. Besides, we showed that targeted silencing of PFTK1 gene could inhibit glioma cell migration. Explaining novel functions of PFTK1 would not only contribute to understand the molecular mechanisms leading to glioma, but also provide a potential drug target for glioma therapy.

Immunohistochemistry Sections were dewaxed in xylene and rehydrated in graded ethanols. Endogenous peroxidase activity was blocked by immersion in 3 % methanolic peroxide for 10 min. Then, the sections were processed in 0.1 M citrate buffer (pH 6.0) and heated to 105 ° C in an autoclave for 10 min to retrieve the antigen. Tissue sections were incubated with anti-PFTK1 antibody and anti-E-cadherin antibody for 2 h at room temperature. After washing in phosphate-buffered saline (PBS), tissues were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse Ig polymer as a second antibody (Envision kit, Dako) for 30 min at room temperature, according to the manufacturer’s instructions. Finally, sections were counterstained with hematoxylin, dehydrated, and mounted in resin mount. Stained sections were observed under a microscope. The degree of immunostaining was viewed and scored separately by two independent pathologists. Tumor cell proportion was scored as follows: 0, <1 %; 1, 1–25 %; 2, 26– 50 %; 3, 51–75 %; and 4, >75 %. Staining intensity was graded according to the following criteria: 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining). Scores from the two scales were combined, and each section was classified as low/no PFTK1/E-cadherin expression (0–2) or high PFTK1/E-cadherin expression. Tissue Samples and Cell Culture

Materials and Methods Pathological Samples Seventy-five glioma sections and six glioma tissue samples were obtained from 2008 to 2010 at the Affiliated Hospital of Nantong University. All fresh frozen human glioma tissue samples were obtained and in accordance with an institutional review board (IRB) protocol approved by the Partners Human Research Committee. All tumors were from patients with newly diagnosed glioma who had received no chemotherapy or radiation therapy before sample collection. All the tissues for immunoblot analysis were frozen immediately after surgery. Formalin-fixed, paraffin-embedded sections were prepared for all tissues.

Tissue samples were immediately processed after surgical removal. Normal brain specimens were acquired from eight patients undergoing surgery for epilepsy and were reviewed to verify the absence of tumor. Protein was analyzed in nine snap-frozen tumors that were stored at −80 °C. Informed consent was obtained from all patients. The human glioma cell lines U87MG, H4, U251MG, and A172 were purchased from the cell library of the Chinese Academy of Sciences and were cultured in DMEM (GibCo BRL, Grand Island, NY, USA) supplemented with 10 % fetal bovine serum 2 mM L-glutamine, 100 U/mL penicillin-streptomycin mixture (GibCo BRL) at 37 °C, and 5 % CO2. The medium was changed every 2–3 days, and cultures were split using 0.25 % trypsin. Western Blot

Antibodies The antibodies used in this study included the following: antiPFTK1 antibody (1:500; Santa Cruz Biotechnology), Ecadherin (1:500; Santa Cruz Biotechnology), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000; Santa Cruz Biotechnology), vimentin (1:1000; Santa Cruz Biotechnology), Snail (1:1000; Santa Cruz Biotechnology).

Cells were washed with ice-cold PBS, resuspended in 2 lysis buffer (50 mM Tris-HCl, 120 mM NaCl, 100 mM NaF, 0.5 % Nonidet P-40, 200 mM Na3VO4, and protease inhibitor mixture), and incubated for 20 min at 4 °C while rocking. Lysates were cleared by centrifugation (10 min×12,000 rpm, 4 °C), and 60 mg total protein was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane (Immobilon; Millipore). The membranes were first blocked

J Mol Neurosci

and then incubated with the primary antibodies for 2 h at room temperature. The secondary antibodies were visualized using LumiGLO Reagent and Peroxide (Cell Signaling). The optical density on the film was measured with a computer imaging system (Imaging Technology, Ontario, Canada).

Expression Plasmid and Transient Transfection Oligonucleotides containing the short hairpin RNA (shRNA) target sequences were designed and synthesized by Shanghai Genechem (China). Four shRNAs targeting PFTK1 (gene ID NM_012395) gene were designed and synthesized, and the most effective shRNA identified by Western blot was applied for the further experiments. The sequence of shRNA is as follows: 5′-GTTCATTCTTTACCACATT-3′, 5′-AGGTTG CATCTTTGTTGAA-3′, 5′-AAAGAGTCACCTAAAG TTA-3′, and 5′-ACCCATACAGGAAATCCAA-3′. Transient transfections were performed using Lipofectamine 2000,

Fig. 1 The correlation between PFTK1 and glioma grade, E-cadherin. a Western blot analysis of PFTK1 expression in two normal brain tissue samples and ones of glioma tissues (grades II–IV). b The bar chart shows the ratio of PFTK1 protein to GAPDH for the above by densitometry. The

following the manufacturer’s recommendation (Life Technologies). Monolayer Wound-Healing Assays U87 cells were seeded to nearly complete confluence in a monolayer in 6-well plates. After transfected 36 h, cells were serum starved for 12 h. Then scratching the monolayer with a 10-μL micropipette tip, cells were washed with PBS for three times, cultured in 5 % fetal bovine serum-Dulbecco's modified eagle medium (FBS-DMEM) at 5 % CO2 and 37 °C, and photographs were taken by an inverted Leica phase-contrast microscope (Leica DFC 300 FX) at 0-, 24-, and 48-h time points. Transwell Migration Assay U87 cells which transfected with PFTK1 shRNAs were starved overnight in DMEM media with 0.1 % FBS, then were

data are mean±SD of three independent experiment, *P<0.01. c Paraffinembedded glioma tissue sections (grades II–IV) were stained with antibodies against PFTK1 and E-cadherin and counterstained with hematoxylin (SP×400). d The relationship between PFTK1 and E-cadherin

J Mol Neurosci

trypsinized and resuspended into DMEM containing 0.1 % bovine serum albumin. Cells (1×105) were added to the top chambers of 24-well transwell plates (Corning, 8-μm pore size), and DMEM with 10 % FBS was added to the bottom chambers. After overnight incubation, top (nonmigrated) cells were removed, and bottom (migrated) cells were fixed and stained with crystal violet to visualize nuclei. The number of migrating cells in five fields was counted under ×200 magnification, and the means for each chamber were determined. All experiments were conducted in triplicate and repeated twice.

Table 1 PFTK1 expression and clinicopathologic characteristics on 75 glioma specimens Characteristics

Total

PFTK1 expression

P*

Low

High

14

6

8

61

26

35

29 46

13 19

16 27

0.764

Frontal Parietal

28 9

11 2

17 7

0.215

Occipital Temporal

6 13

1 7

5 6

Unknown Surgery Biopsy

19

11

8

15

4

11

25 35

9 19

16 16

17 58

9 23

8 35

0.330

50 25

23 9

27 16

0.409

21 29 25

14 13 5

7 16 20

0.006*

Age <45 >45 Gender Female Male

0.987

Tumor location

Statistical Analysis Statistical analysis was performed using SPSS 13.0 statistical software (SPSS, Inc. Chicago, IL). The statistical significance of the correlations between PFTK1 and E-cadherin expression and the clinicopathologic features were analyzed by the χ2test. Expression of PFTK1 and E-cadherin was studied by using the Spearman rank correlation test because the data were not normally distributed. Survival analysis was undertaken using the Kaplan–Meier method, and curves were compared using the log-rank test. Data were presented as (mean±SD), and P<0.05 was considered significant.

Results PFTK1 Overexpression in Glioma Tissues To reveal whether or not PFTK1 associated with glioma, we first evaluated the expression of PFTK1 in two normal brain samples and six glioma samples by Western blot. As shown in Fig. 1a and b, the expression level of PFTK1 was higher in tumor tissues than the normal brain. Furthermore, its level was raised as the grade was increasing from grade II to grade IV. Next, we confirmed the association of PFTK1 with glioma progression by immunohistochemistry for 75 glioma samples. As expected, PFTK1 expressed higher in poorly differentiated samples than well-differentiated ones (Fig. 1c). However, its expression was negatively correlated with E-cadherin (Fig. 1d), which and vimentin are the markers of epithelial cells and mesenchymal cells, respectively. To further investigate the pathophysiological significance of PFTK1 with glioma characteristics, the immunohistochemical results of 75 glioma specimens were summarized in Table 1. Interestingly, we found that the expression of PFTK1 was significantly related with glioma grade (P=0.006). However, there was no correlation with patients’ gender, age, tumor location, type of surgery, vessel density, or necrosis in 75 glioma cases.

Partial resection Gross total resection Tumor diameter <4 cm >4 cm Necrosis Absence Presence WHO grade II III IV

0.138

Statistical analyses were performed by the Pearson χ2 -test *P<0.05 was considered significant

PFTK1 Expression and Patients’ Survival To analyze the association between PFTK1 expression and 75 patients’ survival, Kaplan–Meier survival curves were made. As shown in Fig. 2, we found that high expression of PFTK1 significantly associated with poor survival. The Expression of PFTK1 in Glioma Cell Lines Next, we examined the expression of PFTK1 in glioma cell lines by using Western blot. The results indicated that PFTK1 was highly expressed in glioma cells especially in U87 cells (Fig. 3a, b). Previous study found that PFTK1 could promote the migration of HCC, and E-cadherin and vimentin, which were the markers of epithelial-mesenchymal transition (EMT), contribute to cancer metastasis. Therefore, to investigate whether E-cadherin and vimentin are related with the cell migration

J Mol Neurosci

Fig. 2 Kaplan–Meier postoperative survival curve for patterns of patients with glioma and PFTK1 expression in 75 patients with glioma. Patients were divided into high PFTK1 expressers and low PFTK1 expressers according to the mean PFTK1 percentages. Patients in the high-expression PFTK1 group had a significantly shorter overall survival (P<0.01). The log-rank test was used to calculate P values

and invasion induced by the PFTK1, we analyzed the expression of E-cadherin and vimentin in U87 cells and U251 cells by using Western blot; as shown in Fig. 3c, E-cadherin expression was increased in U87 cells and decreased in U251 cells. However, the vimentin expression was opposite to E-cadherin.

PFTK1 expression was evaluated by using Western blot. The results showed that PFTK1 expression was decreased in U87 cells transfected with PFTK1-shRNAs especially PFTK1-shRNA#3 compared with the ones transfected with control-shRNA (Fig. 3d). Meanwhile, we examined the expression of E-cadherin and vimentin and found that knockdown of PFTK1 resulted in an upregulation of E-cadherin and downregulation of vimentin (Fig. 4a, b). Previous study has found that E-cadherin could be suppressed by a series of factors, including Snail and Slug. Then, we test the expression level of Snail; as shown in Fig. 4a, its expression was decreased in PFTK1-shRNA/U87 cells. Next, we performed wound healing and transwell assays to determine the potential for PFTK1 to induce cell motility. For wound-healing assay, we conducted three groups: control, vector, and PFTK1shRNA#3 cells. The representative photo-micrographs were taken at 0, 24, and 48 h after the cell surfaces were scratched. A decrease in wound-healing cell migration was clearly seen in cells transfected with PFTK1-shRNA#3 after 24 h compared with cells transfected with control and vector cells. Particularly, at 48 h, U87 cells transfected with PFTK1-shRNA#3 exhibited a significant decrease compared with control and vector cells, and the relative migrating distance of cells was significantly shorter (Fig. 5a, b). Meanwhile, knockdown of PFTK1 inhibited cell migration to the bottom chambers compared to control and vector cells (Fig. 5c, d). Taken together, these above data indicated that downregulation of PFTK1 did suppress glioma cell migration.

Discussion Downregulation of PFTK1 Inhibited Glioma Cell Migration To further study the role of PFTK1 in glioma cell migration, U87 cells were transfected with control-shRNA, PFTK1shRNA#1, PFTK1-shRNA#2, and PFTK1-shRNA#3, and Fig. 3 The expression of PFTK1, E-cadherin, and vimentin in glioma cells. a PFTK1 and GAPDH protein in the different glioma cell lines analyzed by Western blot analysis. b The relative differences of the expression among cell lines were normalized with GAPDH, *P<0.01. c PFTK1, E-cadherin, vimentin, and GAPDH protein in U87 and U251 cell lines analyzed by Western blot analysis. d Western blot analysis of PFTK1 in control, vector, sh1, sh2, and sh3

Glioma, with a high rate of mortality, is the most common primary malignant brain tumor. Although with a combination of surgery, chemotherapy, and radiotherapy, most of the patients die to the disease within 2 years of diagnosis (Jansen et al. 2010; Rock et al. 2012). To date, the underlying

J Mol Neurosci Fig. 4 Knockdown of PFTK1 downregulates E-cadherin expression in U87 cells. a, b Western blot analysis of Ecadherin, vimentin, and its repressors in control, vector, and sh3 cells. The bar chart shows the ratio of E-cadherin, vimentin, and Snail to GAPDH by densitometry. The data are mean±SD

molecular mechanisms that determine the biologic behavior of glioma remain poorly understood. Therefore, it is urgent to identify the genes or proteins that effectively regulate tumor growth, migration, and invasion (Eyler et al. 2011). In the present study, we demonstrated the functional role and clinical significance of PFTK1 expression in glioma. PFTK1 is a member of Cdc2-related kinase family, which contains 21 genes encoding CDKs (Malumbres et al. 2009).

Many CDKs play important roles in cell cycle progression, transcription, and differentiation. Although it has been long considered that cell cycle proteins would not influence the migration of cells, many studies showed that the overexpression of Cdc2 could increase cell migration and associated with the motile phenotype of cancer cells (Manes et al. 2003; Juliano 2003). Recently, studies have demonstrated that PFTK1 may be involved in the progress of cancers (Pang

Fig. 5 Knockdown of PFTK1 inhibits glioma cell migration ability. a, b Wound healing assays with control, vector, and sh3 cells. Migration of the cells to the wound was visualized at 0, 24, and 48 h with an inverted Leica phase-contrast microscope (×200 magnification). *P < 0.05. c, d

Knockdown of PFTK1 expression inhibit the migration ability by transwell assays. Each time point is derived from three independent experiments

J Mol Neurosci

et al. 2007; Miyagaki et al. 2012). In this study, we explored the potential role of PFTK1 in glioma development. PFTK1 was found to show a significantly high expression in human glioma samples using Western blot analysis. Immunohistochemistry (IHC) analysis also revealed that the expression of PFTK1 was positively correlated with glioma grade. E-cadherin is a calcium-dependent cell adhesion protein that mediates intracellular adhesion in normal epithelial cells (van Roy and Berx 2008). Knockdown of E-cadherin is associated with invasion and metastasis of tumors including HCC and breast cancer (Wang et al. 2012). Previous study has reported that E-cadherin was downregulated in the majority of glioma tissues. In addition, PFTK1 has been shown to promote the migration of HCC cells. Thus, we sought to explore the effects of PFTK1 on E-cadherin expression. The data showed that E-cadherin expression was negatively correlated with PFTK1 protein level in glioma. Western blot analysis showed that knockdown of PFTK1 could increase the expression of E-cadherin but decrease the expression of vimentin and Snail in glioma cells, suggesting that downregulation of PFTK1 might be an effective approach for blocking EMT. In addition, our results indicated a significantly worse overall survival for patients whose tumors had high expression of PFTK1, suggesting that high PFTK1 protein level is a marker of poor prognosis for patients with glioma. In summary, our study first demonstrated that the expression of PFTK1 significantly was increased in the human glioma tissues and was significantly correlated with WHO grade. Downregulation of PFTK1 by RNA interference could inhibit the migration of glioma cell U87 with a possible involvement of E-cadherin and vimentin. From these data, we can expect that PFTK1 might function as a therapy target for glioma. However, the further precise mechanism between PFTK1 and the proliferation and invasion in glioma need further research.

Acknowledgments This work was supported by the National Basic Research Program of China (973 Program, No. 2012CB822104), the National Natural Science Foundation of China (No. 81202368), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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