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Overexpression of a Novel Activator of PAK4, the CDK5 Kinase–Associated Protein CDK5RAP3, Promotes Hepatocellular Carcinoma Metastasis

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Overexpression of a Novel Activator of PAK4, the CDK5 Kinase–Associated Protein CDK5RAP3, Promotes Hepatocellular Carcinoma Metastasis Published OnlineFirst March 8, 2011; DOI: 10.1158/0008-5472.CAN-10-4046 Cancer Molecular and Cellular Pathobiology Research Overexpression of a Novel Activator of PAK4, the CDK5 Kinase–Associated Protein CDK5RAP3, Promotes Hepatocellular Carcinoma Metastasis Grace Wing-Yan Mak1, Mandy Man-Lok Chan1, Veronica Yee-Law Leong1, Joyce Man-Fong Lee2,3, Tai-On Yau2, Irene Oi-Lin Ng2,3, and Yick-Pang Ching1,3 Abstract The CDK5 kinase regulatory subunit-associated protein 3 (CDK5RAP3 or C53/LZAP) regulates apoptosis induced by genotoxic stress. Although CDK5RAP3 has been implicated in cancer progression, its exact role in carcinogenesis is not well established. In this article, we report that CDK5RAP3 has an important prometastatic function in hepatocarcinogenesis. An examination of human hepatocellular carcinoma (HCC) samples revealed at least twofold overexpression of CDK5RAP3 transcripts in 58% (39/67) of HCC specimens when compared with corresponding nontumorous livers. CDK5RAP3 overexpression was associated with more aggressive biological behavior. In HCC cell lines, stable overexpression of CDK5RAP3 promoted, and small interfering RNA–mediated knockdown inhibited, tumorigenic activity and metastatic potential. We found that over- expression of CDK5RAP3 and p21-activated protein kinase 4 (PAK4) correlated in human HCCs, and that CDK5RAP3 was a novel binding partner of PAK4, and this binding enhanced PAK4 activity. siRNA-mediated knockdown of PAK4 in CDK5RAP3-expressing HCC cells reversed the enhanced cell invasiveness mediated by CDK5RAP3 overexpression, implying that PAK4 is essential for CDK5RAP3 function. Taken together, our findings reveal that CDK5RAP3 is widely overexpressed in HCC and that overexpression of CDK5RAP3 promotes HCC metastasis through PAK4 activation. Cancer Res; 71(8); 2949–58. Ó2011 AACR. Introduction suggests that CDK5RAP3 may function as a tumor suppressor. On the contrary, stable overexpression of the CDK5RAP3 The CDK5 kinase regulatory subunit-associated protein 3 isoform has been shown to promote hepatocellular carcinoma (CDK5RAP3, also called C53/LZAP) was first identified as a (HCC) and cardiac cell proliferation (6, 7), which indicates, binding partner of cyclin-dependent kinase 5 activator, furthermore, that CDK5RAP3 may enhance cell growth. p35nck5a, in yeast 2-hybrid screening (1). Northern analysis CDK5RAP3 is located at chromosome region 17q21.32, indicated that CDK5RAP3 is widely expressed in human which has been reported to be amplified in HCC; however, tissues, and the expression level is relatively constant in the the role of CDK5RAP3 in HCC has not been explored so far (8). heart, brain, skeletal muscle, placenta, lung, liver, kidney, and In this study, we found that the expression of CDK5RAP3 was pancreas (2). Overexpression of CDK5RAP3 has been shown to frequently upregulated in human HCCs at both transcript and sensitize cells to apoptosis induced by genotoxic stress (3). protein levels. More importantly, we detected a remarkable CDK5RAP3 can interact with a well-known tumor suppressor, enhancement of CDK5RAP3 expression in metastatic HCC. namely, the alternate reading frame (ARF; p14ARF), by which it Although little information is available on how CDK5RAP3 stabilizes and promotes the transcription activity of p53 (4). regulates cancer metastasis, we found that CDK5RAP3 is a More recently, CDK5RAP3 has been found to be underex- novel activator of p21-activated protein kinase 4 (PAK4) and pressed in head and neck cancers, and forced expression of activation of PAK4 can promote HCC cell migration. There- CDK5RAP3 can negatively regulate NF-kB activity (5), which fore, we provided, in this study, a novel mechanism by which CDK5RAP3 contributes to the metastasis of HCC by activation Authors' Affiliations: Departments of 1Anatomy and 2Pathology, Li Ka of PAK4. Shing Faculty of Medicine, and 3State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong, China Materials and Methods Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Cell culture Corresponding Author: Yick-Pang Ching, Department of Anatomy, The University of Hong Kong, Room L1-43, Laboratory Block, Faculty of Human hepatoma cell lines PLC/PRF/5 and HepG2 were Medicine Building, Hong Kong. Phone: 852-28199434; Fax: 852- purchased from the American Type Culture Collection. The 28170857; E-mail: [email protected] authentication of these cell lines was ensured by the provider doi: 10.1158/0008-5472.CAN-10-4046 through cytogenetic analysis. No additional test was conducted Ó2011 American Association for Cancer Research. specifically for this study. The human HCC cell line www.aacrjournals.org 2949 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2011 American Association for Cancer Research. Published OnlineFirst March 8, 2011; DOI: 10.1158/0008-5472.CAN-10-4046 Mak et al. SMMC-7721 was a gift from the Shanghai Institute of Biochem- Purification of protein istry and Cell Biology, Chinese Academy of Sciences. Cells were GST fusion proteins were purified using reduced glu- maintained in Dulbecco's modified Eagle medium (DMEM)- tathione (GSH)-sepharose beads by a procedure described high glucose (Life Technologies) supplemented with 1 mmol/L previously (1). His-tagged proteins were purified using Nickel- sodium pyruvate and 10% heat-inactivated FBS (JRH Bios- NTA agarose beads (Qiagen). Bacterial cells were lysed in a ciences). Cells were transfected with DNA constructs, using lysis buffer containing 20 mmol/L Tris (pH 7.5), 500 mmol/L Lipofectamine 2000 (Invitrogen) according to the man- NaCl, 5 mmol/L imidazole, 1 mmol/L dithiothreitol (DTT), ufacturer's protocol. For constructing the stable clones, cells 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L leupep- were transfected with expression constructs (Myc-CDK5RAP3 tin, and 10 mg/mL lysozyme. The protein was eluted with or shCDK5RAP3) and were selected with the correspond- 100 mmol/L imidazole in Tris buffer. ing antibiotic puromycin at 0.4 mg/mL (Sigma) or G418 at 0.8 mg/mL (Calbiochem), 48 hours after transfection. PAK4 kinase assay An in vitro gel kinase assay was conducted as described CDK5RAP3 antibody previously (12). GST-CDK5RAP3 and its mutants were incu- Rabbit anti-CDK5RAP3 polyclonal antibody was generated bated with His-PAK4 and GST-PAK4 peptide substrate [amino using purified glutathione S-transferase (GST)-CDK5RAP3 acid (aa) sequence AARRLSVASAK, named PAK4tide, designed fusion protein as antigen (Genscript Company). Then, the on the basis of the published result (13)] in PAK4 kinase buffer antibody was affinity purified by (CNBr)-GST-CDK5RAP3 and [50 mmol/L HEPES (pH 7.5), 5 mmol/L MgCl2, 100 mmol/L GST column. NaCl, and 1 mmol/L DTT] containing 10 mCi [g-32P]ATP. For peptide-kinase assay, GST and GST-CDK5RAP3 were incu- Immunohistochemistry bated with His-PAK4 in the presence of 1 mmol/L PAK4tide Immunohistochemical staining for CDK5RAP3 and phos- and 10 mCi [g-32P]ATP at 30C for 10 minutes (14). Peptides pho-PAK4 (p-PAK4) was done as described previously (9). with single mutation (AARRLAVASAK) were included as a Purified anti-CDK5RAP3 antibody and anti-p-PAK4 (Ser474) negative control. antibody (Cell Signaling Technology) were used at 1:100 and 1:50 dilutions, respectively. Scoring of the protein expression GST affinity pull-down assay was assessed by an experienced pathologist. GST and GST-CDK5RAP3 immobilized by GSH-sepharose beads (GE Healthcare) were incubated with His-PAK4 for 2 Real-time quantitative reverse transcriptase PCR hours at 4C, followed by washing 3 times with NETN buffer Quantitative PCR (qPCR) was done as described previously [50 mmol/L NaCl, 5 mmol/L EDTA, 50 mmol/L Tris (pH 8.0), (9). The sequence of TaqMan probes (Applied Biosystems) for 1% NP40]. The bound proteins were then visualized by CDK5RAP3 and PAK4 are 50-AGGAAAGATGGAGGACCAT- Western blotting. CAGCAC-30 and 50-GCGGCGCCGAGCCGATGAGTAACCC-30, respectively; cellular 18S rRNA was used as an internal control. Coimmunoprecipitation HEK293T cells, which were ectopically expressed with GFP- Colony formation assay PAK4 and Myc-CDK5RAP3, were lysed with NETN buffer sup- Cells were transiently transfected with plasmids expressing plemented with freshly prepared protease inhibitors (1 mmol/L CDK5RAP3 and shCDK5RAP3 and were selected with corre- phenylmethylsulfonyl fluoride, 1 mg/mL leupeptin, 2 mg/mL sponding antibiotics for 2 weeks. The experiments were done aprotinin, and 1 mmol/L DTT). Antibodies used for immuno- 3 times independently. precipitation include anti-Myc and rabbit anti-PAK4, and anti- bodies used for immunoblotting are rabbit anti-Myc, rabbit anti- Cell proliferation, soft agar growth, cell migration, and GFP, and rabbit anti-CDK5RAP3. invasion assays The procedure described by Leung and colleagues (10) and Confocal microscopy Wong and colleagues (11) was adopted. For soft agar assay, Cells were fixed with 4% paraformaldehyde and permeabi- colonies with diameter greater than 50 mmin10fieldswere lized with 0.2% Triton X-100 (9). Images were captured by a counted and data were shown as average number of colonies per confocal laser scanning microscope LSM510 (Carl Zeiss). field. The experiments were carried out 3 times independently. Statistical analysis Nude mouse xenograft assay The Student t test and the Mann–Whitney test were used CDK5RAP3 stable knockdown PLC/PRF/5 cells and control for statistical analysis of data. Tests were considered signifi- cells (5  106) were administered by s.c. injection into the right cant with P < 0.05. and left flanks of 6-week-old male nude mice, respectively. The larger (a) and smaller (b) diameters of a tumor were measured Results weekly. Tumor volume was estimated according to the formula: volume ¼ 1/2  a  b2; and it was measured from week 2 to CDK5RAP3 was overexpressed in human HCCs week 5. After 5 weeks, the mice were sacrificed in accordance To elucidate the role of CDK5RAP3 in human HCCs, we with institutional regulations for animal experiments. examined CDK5RAP3 transcripts in human HCCs by using 2950 Cancer Res; 71(8) April 15, 2011 Cancer Research Downloaded from cancerres.aacrjournals.org on September 25, 2021.
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