Journal of the National Cancer Institute Advance Access originally published online on June 12, 2007
JNCI Journal of the National Cancer Institute 2007 99(12):936-948; doi:10.1093/jnci/djm011
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© The Author 2007. Published by Oxford University Press.
ARTICLES |
Cell Cycle–Related Kinase: A Novel Candidate Oncogene in Human Glioblastoma
Affiliations of authors: Department of Chemistry, Open Laboratory of Chemical Biology, The University of Hong Kong, Pokfulam, Hong Kong, China (SSMN, YTC, XMA, ML, GHYL, WC, JS, MCL); The State Key Laboratory of Oncology in Southern China, Cancer Center, Sun Yat-Sen University, Guangzhou, China (YCC, DX, HFK); Stanley Ho Centre for Emerging Infectious Diseases, The Chinese University of Hong Kong, Shatin, Hong Kong, China (YCC, MLH, HFK); Institute of Molecular and Chemical Biology, East China Normal University, Shanghai, China (LL); Department of Neurology, The Second Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China (YP); Departments of Medicine (HHXX, BCYW) and Pathology (SYL), Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China
Correspondence to: Marie C. Lin, PhD, Department of Chemistry, Open Laboratory of Chemical Biology, The University of Hong Kong, Pokfulam, Hong Kong, China (e-mail: mcllin{at}hkusua.hku.hk) or Hsiang-Fu Kung, PhD, Centre for Emerging Infectious Diseases, The Chinese University of Hong Kong, Shatin, Hong Kong, China (e-mail: hkung{at}cuhk.edu.hk).
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ABSTRACT |
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Background: Median survival for patients with glioblastoma multiforme, the most aggressive glioma, is only 12–15 months, despite multimodal treatment that includes surgery, chemotherapy, and radiotherapy. Thus, identification of genes that control the progression of glioblastoma multiforme is crucial for devising new therapies. We investigated the involvement of cell cycle–related kinase (CCRK), a novel protein kinase that is homologous to cyclin-dependent kinase 7, in glioblastoma multiforme carcinogenesis.
Methods: We analyzed the expression levels of CCRK in 26 glioma patient samples (19 high-grade and seven low-grade) and normal brain by semiquantitative reverse transcription–polymerase chain reaction assays. CCRK expression was knocked down in human glioma U-373 MG and U-87 MG cells with small-interfering RNAs and short hairpin RNAs (siCCRK and shCCRK, respectively), and cell proliferation, cell cycle distribution, and cyclin-dependent kinase 2 (CDK2) phosphorylation were examined. A subcutaneous nude mouse xenograft model (n = 4 mice per group) was used to study the effect of CCRK knockdown and overexpression on tumorigenicity and growth of glioblastoma multiforme cells in vivo. All statistical tests were two-sided.
Results: CCRK mRNA was elevated at least 1.5-fold and as much as 3.7-fold in 14 (74%) of 19 high-grade glioblastoma multiforme patient samples and in four (80%) of five glioma cell lines examined compared with normal brain tissue. Suppression of CCRK by siCCRK inhibited the proliferation of U-373 MG and U-87 MG glioblastoma cells in a time- and dose-dependent manner. The growth-inhibiting effect of siCCRK was mediated via G1- to S-phase cell cycle arrest and reduced CDK2 phosphorylation. CCRK knockdown statistically significantly suppressed glioma cell growth in vivo as indicated by the mean tumor volumes at week 6 after tumor cell injection (U-373-control = 1352 mm3, U-373-shCCRK = 294 mm3, difference = 1058 mm3, 95% confidence interval [CI] = 677 to 1439 mm3, P<.001; U-87-control = 1910 mm3, U-87-shCCRK = 552 mm3, difference = 1358 mm3, 95% CI = 977 to 1739 mm3, P<.001).
Conclusions: CCRK is a candidate oncogene in glioblastoma multiforme tumorigenesis.
Prior knowledge Identification of genes that control the progression of glioblastoma multiforme, the most aggressive glioma, is needed to devise new therapies for patients with this cancer. Cell cycle–related kinase (CCRK), a novel protein kinase that is homologous to cyclin-dependent kinase 7, has been implicated in cancer cell proliferation, but its role in glioblastoma multiforme carcinogenesis is unknown. Study design Molecular study in human glioma cell lines, samples from glioma patients, normal brain tissue, and mouse xenograft models. Contribution Increased glioma cell proliferation and tumorigenicity were associated with the overexpression of CCRK, whereas suppression of CCRK expression was associated with the inhibition of glioma xenograft tumor growth. Implications CCRK is a candidate oncogene in glioblastoma multiforme tumorigenesis. Limitations The small number of patient samples precluded analysis of the association between CCRK expression and patient survival. The mechanistic evidence for how CCRK regulates cell cycle progression was indirect.
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Glioblastoma multiforme, which is classified by the World Health Organization [WHO; (1)] as a grade IV tumor that originates from poorly differentiated astrocytes, is the most severe and most common type of brain tumor. Glioblastoma multiforme is a highly aggressive and neurologically destructive tumor that frequently colonizes the cerebral hemispheres. Its ability to rapidly infiltrate the surrounding brain structures makes it one of the deadliest cancers. Despite multimodal treatment, which includes resection, chemotherapy, and radiotherapy, the prognosis for glioblastoma multiforme patients is poor, and their median survival is only 12–15 months (2,3).
Glioblastoma multiforme displays the largest number of genetic and epigenetic changes of all astrocytic neoplasms. These changes occur at several mutational hot spots that have been implicated in glioblastoma multiforme tumorigenesis, including the epidermal growth factor receptor gene (4), chromosomes 10 (5) and 19 (6), the murine double minute-2 gene (7), the genes encoding the cyclin-dependent kinase (CDK) inhibitors p15 and p16 (8), the TP53 gene (9), the platelet-derived growth factor receptor gene (10), and the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene (11). Although much effort has been made to understand the pathophysiology of glioblastoma multiforme, the molecular mechanisms of gliomagenesis have not been clearly defined. However, essential regulators of cell cycle progression, including CDKs, cyclins, and CDK inhibitors, have become the major focus of glioblastoma multiforme research. For example, hyperactivation of cyclin-dependent kinase 4 (12) and the D-type cyclins (13), and loss of p15 and p16 (8), have all been implicated in glioma development, suggesting the importance of cell cycle control in this cancer.
Cell cycle–related kinase (CCRK) (also called p42) is a 42-kD protein kinase that shares 43% sequence identity with cyclin-dependent kinase 7 [CDK7; (14)], a CDK-activating kinase that is important for both cell cycle and transcriptional regulation. CCRK has been reported to possess CDK-activating kinase activity (15) and phosphorylate male germ cell–associated kinase–related kinase at Thr-157 in mammalian cells (16). CCRK is essential for proliferation of cervical carcinoma HeLa cells, osteosarcoma U2OS cells, and colorectal carcinoma HCT116 cells (15,17), and its expression has been detected in various cancer cell lines (15). However, the function of CCRK in human carcinogenesis has not yet been assessed. In this study, we examined whether the CCRK gene is a candidate oncogene in human glioblastoma multiforme.
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Materials and Methods |
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Cell Lines, Cell Culture, and Human Tissue Samples
Human high-grade glioma cell lines (U-87 MG, U-118 MG, U-138 MG, and U-373 MG), human primitive neuroectodermal PFSK-1 cells, human low-grade glioma SW-1088 cells, and human embryonic kidney (HEK) 293T cells were purchased from American Type Culture Collection (Manassas, VA). U-87 MG, U-138 MG, and U-373 MG cells were cultured in minimal essential medium (MEM; Invitrogen, Carlsbad, CA). U-118 MG, PFSK-1, SW-1088, and HEK 293T cells were cultured in Dulbecco's modified Eagle medium (Invitrogen). Both media were supplemented with 10% fetal bovine serum (FBS; Invitrogen).
Archival frozen human tissue samples, including 19 high-grade gliomas (glioblastoma, WHO grade IV), seven low-grade gliomas (WHO grade II), and three samples of morphologically normal brain tissue in temporal lobectomy specimens from patients with epilepsy, were obtained from the Department of Pathology, University of Hong Kong. The use of these archival tissues in this study was approved by the Ethics Committee of the University of Hong Kong.
Reverse Transcription–Polymerase Chain Reaction
Total RNA was extracted from the five glioma cell lines and one primitive neuroectodermal cell line with the use of TRIzol reagent (Invitrogen) and from the 29 frozen human tissue samples by a standard guanidinium thiocyanate method. First-strand complementary DNA (cDNA) was reverse transcribed using the SuperScript First-Strand cDNA System (Invitrogen) and was used to amplify, by the polymerase chain reaction (PCR), the partial coding regions for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; GenBank accession number NM_002046) using the forward primer 5'-TGCCTCCTGCACCACCAACT-3' and the reverse primer 5'-CCCGTTCAGCTCAGGGCTGA-3' and for CCRK (GenBank accession number NM_178432) using the forward primer 5'-TCATCCTGGAGGGGTGAGAAGT-3' and the reverse primer 5'-CCACCTTAGCCATTTCCCTTGA-3'. The PCR conditions were 94 °C for 45 seconds, 59 °C for 45 seconds, and 72 °C for 1 minute for 27 cycles (for GAPDH) or for 35 cycles (for CCRK). The PCR products were resolved by electrophoresis on 1% ethidium bromide–stained agarose gels, and the band intensity was quantitated with the use of ImageQuant soft-ware (Molecular Dynamics, Sunnyvale, CA). The CCRK mRNA expression levels in the patient samples were considered to be elevated if they were more than 1.5-fold higher than those of three samples of normal tissues. This cut point was chosen according to previous gene expression–profiling studies in glioma patients (18,19).
Northern Blot Analysis
Northern blot analysis of CCRK mRNA expression was performed on a human multiple tissue northern blot that contained 10 µg total RNA per lane from the brain, heart, skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung, and peripheral blood leukocytes (Clontech, Palo Alto, CA). The membrane was hybridized with CCRK cDNA (PCR-amplified as described above) that was purified with the use of a GFX PCR DNA and Gel Band Purification kit (Amersham Biosciences, Piscataway, NJ), radioactively labeled with 50 µCi [
-32P]deoxycytidine triphosphate by using the Klenow fragment of DNA polymerase I, and purified on a Microspin G50 column (Amersham Biosciences). Hybridization was performed in ExpressHyb hybridization solution (Clontech) for 2 hours at 68 °C. The membrane was then washed sequentially with 2xstandard saline citrate (SSC)/0.05% sodium dodecyl sulfate (SDS) and 0.1x SSC/0.1% SDS (1x SSC = 0.15 M sodium chloride and 0.015 M sodium citrate) at 50 °C for 10 minutes and exposed to x-ray film for autoradiography.
Synthetic siRNA Oligonucleotides
Small-interfering RNA (siRNA) duplex oligonucleotides targeting human CCRK mRNA (siCCRK; 5'-GAAGGUGGCCCUAAGGC GG-3') or firefly (Photinus pyralis) luciferase mRNA (siLuc; 5'-CGUACGCGGAAUACUUCGA-3') were synthesized and purified by Proligo (Boulder, CO). The siRNAs were labeled at the 5' end with fluorescein isothiocyanate. An additional siRNA that targets a different region of CCRK mRNA, siCCRK2 (5'-GGCGGUUGGAGGACGGCUU-3'), was also synthesized according to a previously published sequence (15).
Transient Transfection of Glioma Cells With siRNAs
One day before transfection, U-373 MG or U-87 MG cells at 80% confluence were detached by treatment with 0.25% trypsin–EDTA (Invitrogen) and plated onto a 24-well plate at 3 x 104 cells per well. The cells were then transfected by incubation with siCCRK, siCCRK2, or siLuc at final concentrations of 40, 100, or 200 nM and Oligofectamine transfection reagent (Invitrogen) according to the manufacturer's instructions. Cells were incubated with MEM/10% FBS and transfection reagent as a negative control (mock transfection). Cell number and viability were determined by trypan blue dye exclusion at 24, 48, and 72 hours after transfection. Each transfection was done in triplicate, and the results were confirmed by at least three independent experiments.
Establishment of Glioma Cell Lines That Stably Express a Short Hairpin RNA Targeting CCRK
Short hairpin RNAs (shRNAs) that targeted the CCRK mRNA (shCCRK; 5'-GAAGGTGGCCCTAAGGCGGTTGGAAGACG-3') or the firefly luciferase mRNA (shLuc; 5'-GTGAACATCACGTACGCGGAATACTTCGA-3'; synthesized and purified by Proligo) were subcloned into the pGE-1 GeneEraser shRNA mammalian expression vector (Stratagene, La Jolla, CA) to generate the plasmids pGE-1-shCCRK or pGE-1-shLuc, respectively. U-373 MG or U-87 MG cells (2 x 106) were seeded onto 100-mm dishes 24 hours before transfection. The cells were transfected with 6 µg of pGE-1-shCCRK or pGE-1-shLuc with the use of GeneJuice Transfection Reagent (Novagen, San Diego, CA) according to the manufacturer's instructions and incubated for 48 hours at 37 °C. The cells were then passaged in medium that contained G418 (500 µg/mL; Calbiochem, San Diego, CA) to select for the neomycin resistance gene contained on the pGE-1 plasmids. G418-resistant colonies were isolated with the use of cloning cylinders (Sigma, St Louis, MO), and individual clones were expanded in the selection medium.
Cell Cycle Analysis
U-373 MG cells (7.5 x 105) were seeded onto a 100-mm dish and cultured for 24 hours. The cells were then transfected by incubation with siRNA duplexes (siCCRK, siCCRK2, or siLuc) at a final concentration of 200 nM and Oligofectamine transfection reagent and incubated for 48 hours at 37 °C. The transfected cells were then fixed in ice-cold 70% ethanol and stained with the use of a Coulter DNA-Prep Reagents kit (Beckman Coulter, Fullerton, CA). Cellular DNA content of 1 x 104 cells from each sample was determined with the use of an EPICS ALTRA flow cytometer (Beckman Coulter). Cell cycle phase distribution was analyzed with the use of ModFit LT 2.0 software (Verity Software House, Topsham, ME) using data obtained from two separate experiments in which each transfection was performed in triplicate.
CCRK Antibody Production and Immunoblot Analysis
A polyclonal antibody against human CCRK was raised by Boster Biological Technology (Wuhan, China) by immunizing rabbits with a synthetic 16–amino acid peptide (RIGEGAHGIVFKAKHV; Invitrogen) whose sequence is identical to amino acid residues 9–24 at the amino-terminus of human CCRK (NCBI Entrez protein accession number: AAN28684).
For immunoblotting, siLuc- or siCCRK-transfected U-373 MG or U-87 MG cells (1 x 106) were detached by treatment with 0.25% trypsin–EDTA (Invitrogen) at 72 hours after transfection. The cells were washed twice in phosphate-buffered saline (PBS) and lysed for 30 minutes in radioimmunoprecipitation assay lysis buffer (25 mM Tris–HCl, 150 mM sodium chloride, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) containing 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Sigma). The lysates were centrifuged at 15000 g for 15 minutes, the supernatants were recovered, and the protein concentrations of the supernatants were determined by using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Heat-denatured protein samples (20 µg per lane) were resolved by SDS–polyacrylamide gel electrophoresis (PAGE) and transferred to an Immobilon-P membrane (Millipore, Bedford, MA). The membrane was incubated for 30 minutes in PBS containing 0.1% Tween 20 and 3% skim milk to block nonspecific binding, followed by incubation for 1 hour at room temperature with a primary rabbit polyclonal antibody against human 
-actin, GAPDH, phosphorylated-CDK2 (1:500 dilution; all from Santa Cruz Biotechnology, Santa Cruz, CA), human CDK2 (1:500 dilution; Cell Signaling, Beverly, MA), or human CCRK (1:1000 dilution). The membrane was washed three times for 5 minutes in PBS with 0.1% Tween 20 and then incubated for 1 hour with a goat anti-rabbit horseradish peroxidase–conjugated secondary antibody (1:1000 dilution; Amersham Biosciences). The membrane was washed thoroughly in PBS containing 0.1% Tween 20, and bound antibody was detected with the use of enhanced chemiluminescence detection reagents (Amersham Biosciences) according to the manufacturer's instructions.
Coimmunoprecipitation Assay
Protein lysates (250 µg) prepared as described above from 1 x 106 U-373 MG cells or U-87 cells were incubated with 2 µg of human immunoglobulin G (IgG; Sigma) or human CCRK antibody (Boster Biological Technology) for 2 hours at 4 °C. Antibody–protein complexes were captured by binding to protein G agarose beads (Amersham Biosciences). The agarose beads were subsequently washed three times with ice-cold PBS, and the complexes were dissociated from the beads by boiling for 5 minutes. The beads were collected by centrifugation, and the supernatants containing the immunocomplexes were resolved by SDS–PAGE and transferred to a membrane for immunoblotting with a human CDK2 antibody (Santa Cruz Biotechnology). Bound CDK2 antibody was detected by a goat anti-rabbit horseradish peroxidase–conjugated antibody, which was visualized by enhanced chemiluminescence as described above.
Construction of a Kinase-Defective CCRK Expression Plasmid
A full-length cDNA encoding CCRK was amplified by PCR from a human brain cDNA library (Clontech). In humans, three CCRK transcript variants, which are thought to be generated from alternative splicing of the same precursor mRNA (16,17), have been identified: CCRK (which encodes a peptide of 346 amino acids; GenBank accession number NM_001039803), CCRK transcript variant 1 (which encodes a peptide of 338 amino acids; NM_178432), and CCRK transcript variant 2 (which encodes a peptide of 326 amino acids; NM_012119). We used the longest variant in our study because the shorter transcript variants lack an in-frame coding exon in the kinase domain and therefore may have less protein kinase activity than the longer variant (17). The full-length CCRK cDNA was inserted in the HindIII and XbaI sites of the pcDNA3.1 expression vector (Invitrogen) to generate pcDNA-CCRK. Using pcDNA-CCRK as a template, we generated a construct encoding a kinase-defective CCRK mutant, in which the T-loop threonine residue was replaced with an alanine (pcDNA-T161A), by PCR-based site-directed mutagenesis as described previously (15). The presence of the mutation was confirmed by DNA sequencing.
Colony Formation Assay
U-138 MG cells were transfected with pcDNA-CCRK, pcDNA-T161A, or empty expression vector pcDNA3.1 (Invitrogen) in the presence of Lipofectamine 2000 (Invitrogen) and incubated for 2 days in MEM supplemented with 10% FBS. The cells were then incubated for 2 weeks in medium containing G418 (500 µg/mL) to select for cells that were stably transfected with the pcDNA-based plasmids (i.e., U-138-CCRK, U-138-T161A, and U-138-pcDNA cells, respectively). The G418-resistant colonies were washed with PBS, stained for 10 minutes with 1% crystal violet in 10% ethanol, and then washed twice with PBS. Colonies were then counted while being viewed under a dissecting microscope.
Isolation of Nontumorigenic Glioma Cells That Stably Overexpress CCRK
U-138 MG cells were transfected with the pcDNA-CCRK plasmid or pcDNA3.1 expression vector by Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Two days after transfection, the cells were passaged at 1:10 ratio in selection medium (MEM containing 500 g/mL of G418 [Calbiochem]). Three weeks after G418 selection, resistant cell colonies that were stably transfected with pcDNA-CCRK (U-138-CCRK) or pcDNA3.1 (U-138-pcDNA) were isolated with the use of cloning cylinders (Sigma). The isolated cell clones were then expanded in the same selection medium.
Lentiviral Vector Preparation and Transduction
shCCRK and a cDNA encoding enhanced green fluorescent protein (EGFP) were cloned separately into the lentiviral transfer vector LUNIG (20) to produce the transfer vector constructs LUNIG-shCCRK and LUNIG-EGFP, respectively. The vesicular stomatitis virus glycoprotein (VSVG)–pseudotyped lentiviral vectors carrying shCCRK (lenti-shCCRK) or EGFP (lenti-EGFP) were produced by cotranfecting HEK 293T cells with the LUNIG-shCCRK or LUNIG-EGFP transfer vector constructs and three different packaging plasmids (pMDLg/p/RRE, pRSV-REV, and pCMV-VSVG) (21). The lentiviral vectors were purified using ultracentrifugation as described previously (20,21). Lenti-shCCRK was used to knock down CCRK expression in the subsequent cell proliferation assays and nude mouse xenograft studies. In these experiments, lenti-EGFP was used as a negative control.
Tumor Xenografts in Nude Mice
Female BALB/c athymic mice (nu/nu, 5–8 weeks old) were purchased from Charles River Laboratories (Wilmington, MA), housed under aseptic conditions, and cared for in accordance with the guidelines of the Laboratory Animal Unit of the University of Hong Kong. For the xenograft tumor growth assay, U-373 MG or U-87 MG cells stably transfected with pGE-1-shCCRK (U-373-shCCRK and U-87-shCCRK) or pGE-1-shLuc (U-373-shLuc and U-87-shLuc) (5 x 106 cells) were injected subcutaneously into the right flanks of the mice (n = 4 mice per group), and the experiment was conducted in triplicate. For the tumorigenicity assay, 10 clones each of U-138-pcDNA or U-138-CCRK G418-resistant cells were pooled, and the pooled U-138-pcDNA or U-138-CCRK cells (5 x 106) were injected subcutaneously into the right flanks of nude mice (n = 4 mice per group). Tumor size was measured once per week for 6 weeks with the use of a caliper, and tumor volume (V) was calculated according to the formula V = ab2/2, where a and b are major and minor axes of the tumor foci, respectively. Mice were killed by cervical dislocation when their tumors grew to a diameter of at least 20 mm or when the tumor burden exceeded 10% of their body weight. In another experiment to determine the effect of lenti-shCCRK transduction on the tumorigenicity of U-138-CCRK cells, U-138-CCRK cells were first transduced by either lenti-shCCRK or lenti-EGFP (multiplicity of infection [MOI] = 10) for 12 hours. Four days after transduction, the lenti-shCCRK– or lenti-EGFP–transduced U-138-CCRK cells (5 x 106) were injected subcutaneously at the right flanks of the nude mice (n = 4 mice per group). Tumor size was measured once per week for 6 weeks as described above. The animal studies and the experimental protocols were approved by the Department of Health of the Government of Hong Kong Special Administrative Region and by the Committee on the Use of Live Animals in Teaching and Research of the University of Hong Kong.
Cell Proliferation Assays
U-138 MG cells (5 x 104) were first transduced with either lenti-shCCRK or lenti-EGFP at different MOIs in the presence of 8 µg/mL Polybrene (Sigma) for 12 hours. Control cells were mock transduced with Polybrene alone. Viable cells were counted by trypan blue dye exclusion on days 2, 3, and 4 after transduction. The transduction efficiency of lenti-EGFP in U-138 MG cells was accessed by counting the number of transduced cells (i.e., those with green fluorescence) and nontransduced cells (i.e., without green fluorescence) in 10 randomly selected fields by using a fluorescence microscope at high-power magnification (x400).
Statistical Analysis
Experimental data are summarized as the mean values with 95% confidence intervals (CIs). All statistical analyses were performed using a two-tailed Student's t test (GraphPad Prism 3.0, GraphPad Software, San Diego, CA), and differences were considered to be statistically significant at a value of P less than .05.
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Expression of CCRK in Human Tissues, Glioma Samples, and Cell Lines
We first studied the tissue distribution of CCRK mRNA by northern blot analysis. The 2.2-kb CCRK transcript was expressed predominantly in the brain and kidney and to a lesser extent in the liver, heart, and placenta (Fig. 1, A). In all these tissues, another transcript with a larger size (4.2 kb) was also detected, suggesting that the CCRK gene is alternatively spliced or that the CCRK pre-mRNA is present along with the mature mRNA in the same tissue. To our knowledge, this is the first report of expression of the 4.2-kb transcript; its function is unclear.
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We next used semiquantitative reverse transcription (RT)–PCR to analyze CCRK mRNA expression in glioma samples from 26 patients and three normal samples; 19 samples were from patients with high-grade glioma and seven were from patients with low-grade glioma. We found that 14 (74%) of the high-grade glioma samples had CCRK mRNA expression levels that were more than 1.5-fold higher than those of three samples of normal brain tissue (Fig. 1, B). In some patient samples, the level of CCRK mRNA was elevated as much as 3.7-fold. This expression profile is concordant with results of a genome-wide expression study (22) showing that the expression level of CCRK was elevated by at least twofold in 18 (62%) of 29 glioblastoma multiforme tissue samples (accessible from the Stanford microarray database http://smd.stanford.edu/cgi-bin/exptsets/viewExptSets.pl?exptset_no=2677&del=no). By contrast, only two (29%) of the seven low-grade glioma samples had CCRK mRNA expression levels that were more than 1.5-fold higher than those of three samples of normal brain tissue (Fig. 1, C).
We next examined the expression of CCRK in four human glioma cell lines that were established from high-grade tumors (U-87 MG, U-118 MG, U-138 MG, and U-373 MG), one glioma cell line that was established from a low-grade tumor (SW-1088), and a cell line established from a malignant neuroectodermal tumor (PFSK-1). The CCRK mRNA level was elevated in four (80%) of the five glioma cell lines and in the neuroectodermal tumor cell line compared with normal brain tissue (Fig. 1, D); the five cell lines with elevated CCRK mRNA expression are tumorigenic in nude mice, according to data from American Type Culture Collection (http://www.atcc.org/). By contrast, the cell line that displayed the lowest level of CCRK mRNA (U-138 MG) was the only cell line studied that was not described by the ATCC as being tumorigenic in nude mice.
Effect of siRNA Suppression of CCRK Expression on the Growth of Glioma Cell Lines
We examined the effect of CCRK mRNA expression knockdown on glioma cell proliferation in vitro by transfecting U-373 MG cells and U-87 MG cells separately with siCCRK (which specifically targets the CCRK mRNA), siLuc (which targets an unrelated gene, the firefly luciferase mRNA), or transfection reagent alone (mock transfection). We first tested the effect of siRNA expression on CCRK mRNA levels (which were normalized to the GAPDH mRNA levels). The GAPDH-normalized CCRK mRNA levels in the mock-transfected cells was set as 100%. At 72 hours after transfection, U-373 MG cells and U-87 MG cells transfected with siCCRK had statistically significantly less CCRK mRNA than the respective cells transfected with siLuc (mean relative CCRK mRNA levels in U-373 cells transfected with siLuc versus siCCRK: 83.3% versus 33.2%, difference = 50.1%, 95% CI = 10.2% to 89.9%, P = .03; mean relative CCRK mRNA levels in U-87 cells transfected with siLuc versus siCCRK: 93.3% versus 38.7%, difference = 54.6%, 95% CI = 27.6% to 81.6%, P = .005) (Fig. 2, A). A corresponding depletion of endogenous CCRK protein was also observed in U-373 MG cells or U-87 MG cells transfected with siCCRK for 72 hours (Fig. 2, B).
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We next determined the number of viable cells at various times after siRNA transfection as a percentage of the number of viable cells in the mock transfection group. Transfection with siCCRK, but not with siLuc, statistically significantly inhibited the growth of both U-373 MG cells and U-87 MG cells in a time-dependent manner (mean number of siCCRK-transfected U-373 MG cells as percentage of the number of mock-transfected U-373 MG cells, 24 hours versus 72 hours after transfection = 96% versus 56%, difference = 40%, 95% CI = 6.4% to 73.6%, P = .024; mean number of siCCRK-transfected U-87 MG cells as percentage of the number of mock-transfected U-87 MG cells, 24 hours versus 72 hours after transfection = 81.5% versus 65.4%, difference = 16.1%, 95% CI = 0.2% to 32%, P = .048) (Fig. 2, C). The inhibiting effect of siCCRK on cell growth was also dose dependent (mean number of siCCRK-transfected U-373 MG cells as percentage of the number of mock-transfected U-373 MG cells, 40 nM versus 200 nM = 97% versus 58%, difference = 39%, 95% CI = 10.6% to 67.4%, P = .012; mean number of siCCRK-transfected U-87 MG cells as percentage of the mock-transfected U-87 MG cells, 40 nM versus 200 nM = 104.3% versus 69.7%, difference = 34.5%, 95% CI = 12.6% to 56.4%, P = .006) (Fig. 2, D). Parallel results were obtained by transfecting U-373 MG and U-87 MG cells with siCCRK2, an siRNA that targets a different region of the CCRK mRNA (data not shown).
We also examined the consequence of CCRK mRNA knockdown on cell growth in U-138 MG cells, which express the lowest level of CCRK mRNA compared with other glioma cells tested. Because U-138 MG cells are difficult to transfect using a liposome-mediated approach as in the previous experiment (i.e., they have a transfection efficiency of only approximately 10%, as determined by fluorescence microscopic monitoring of the fluorescein isothiocyanate label at the 5' end of the siRNAs in the transfected cells (data not shown), we used lentiviral transduction with lenti-shCCRK to produce U-138 MG cells expressing an shRNA that targets the CCRK mRNA. Control cells were transduced with lenti-EGFP or were mock transduced. Transduction efficiency was determined by dividing the number of transduced cells (i.e., with green fluorescence) by the total number of cells counted in 10 randomly selected fields. The transduction efficiency of lenti-EGFP in U-138 MG cells increased statistically significantly with increasing MOI (mean percentage of transduced cells at MOI of 10 versus MOI of 1: 86.0% versus 48.3%, difference = 37.7%, 95% CI = 29.6% to 45.7%, P<.001) (Fig. 3, A). U-138 MG cells transduced with lenti-shCCRK at an MOI of 10 had markedly lower CCRK mRNA and protein expression than that in mock and lenti-EGFP transduced cells, as determined by RT–PCR and immunoblotting, respectively (Fig. 3, B). Lenti-shCCRK inhibited the growth of U-138 MG cells in a time-dependent fashion (the mean percentage of cell number of the lenti-shCCRK–transduced cells as percentage of the number of mock-transduced cells, 48 hours versus 96 hours after transduction = 99.4% versus 76.0%, difference = 23.4%, 95% CI = 0.2% to 46.6%, P = .048). The inhibition of cell growth was also dose-dependent (the mean percentage of cell number of the lenti-shCCRK–transduced cells as percentage of the number of mock-transduced cells, MOI of 1 versus MOI of 10 = 99% versus 68%, difference = 31%, 95% CI = 7.7% to 54.3%, P = .014) (Fig. 3, C). Taken together, these data indicate that CCRK behaves as a candidate glioma cell oncogene because a decrease in its expression led to a marked reduction in tumor cell proliferation.
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Effect of siCCRK on Cell Cycle Progression in Glioma Cell Lines
To better understand the mechanism of the siCCRK-induced growth inhibition, we used flow cytometry to examine the cell cycle profiles of mock-transfected U-373 MG cells and U-373 MG cells transfected with siLuc or siCCRK (Fig. 4, Table 1). The siCCRK-transfected cells had a higher percentage of cells in G0/G1 phase than siLuc-transfected control cells (mean number of cells in G0/G1 phase as a percentage of the mean number of G0/G1-phase cells in the mock-transfected control, siCCRK versus siLuc: 136.5% versus 104.4%, difference = 32.1%, 95% CI = 26.1% to 38.1%, P<.001). Concomitantly, the number of cells in S phase and in G2/M phase were statistically significantly reduced, by approximately 68% and 18%, respectively, indicating that the growth-inhibiting effect of siCCRK occurs via arrest at the G1- to S-phase transition (mean number of cells in S phase as a percentage of the mean number of S-phase cells in the mock-transfected control, siCCRK versus siLuc: 28.5% versus 96.2%, difference = 67.7%, 95% CI = 55.2% to 80.2%, P<.001; mean percentage of cells in G2/M phase as compared with mock-transfected control, siCCRK versus siLuc: 74.0% versus 91.9%, difference = 17.9%, 95% CI = 1.5% to 34.3%, P = .04). We also observed a G1/S-phase arrest in U-373 MG cells transfected with siCCRK2 (data not shown).
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Effect of CCRK Expression on CDK2 Phosphorylation
To examine the mechanisms by which CCRK regulates cell cycle progression in glioma cells, we first examined the effect of CCRK expression on the phosphorylation of CDK2, an important regulator of the G1/S transition. Immunoblot analysis and densitometry of cell lysates showed that siCCRK-transfected U-373 MG cells had statistically significantly less CCRK protein and phosphorylated CDK2 than siLuc-transfected U-373 MG cells (mean relative CCRK protein level, siLuc versus siCCRK: 100.0% versus 19.3%, difference = 80.7%, 95% CI = 66.2% to 95.2%, P<.001; mean relative level of phosphorylated CDK2 protein, siLuc versus siCCRK: 100% versus 11%, difference = 89%, 95% CI = 80.1% to 97.9%, P<.001) (Fig. 5, A, upper panels). We also detected reductions in the levels of CCRK and phosphorylated CDK2 in U-87 cells transfected with siCCRK (mean relative CCRK protein level, siLuc versus siCCRK: 100.0% versus 31.3%, difference = 69.7%, 95% CI = 42.0% to 68.7%, P = .002; mean relative level of phosphorylated CDK2 protein, siLuc versus siCCRK: 100.0% versus 23.0%, difference = 77.0%, 95% CI = 49.0% to 105.4%, P = .002) (Fig. 5, A, lower panels).
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We next examined the association between CCRK protein levels and CDK2 phosphorylation in five glioma cell lines (U-373 MG, U-87 MG, U-118 MG, SW-1088, and U-138 MG). In general, the level of CCRK protein expression was positively associated with the level of CDK2 phosphorylation (Fig. 5, B and C). In addition, overexpression of CCRK in U-138 MG cells (which express low levels of CCRK protein) and U-87 MG and U-373 MG cells (which express high levels of CCRK protein) resulted in an increase in CDK2 phosphorylation even though the level of total CDK2 protein (i.e., phosphorylated and unphosphorylated) remained unchanged (Fig. 5, D). We also examined whether CCRK protein interacts with CDK2 in U-373 MG and U-87 MG cells by subjecting cell lysates to immunoprecipitation with either IgG or human CCRK antibody, followed by immunoblotting of the immunocomplexes with an antibody against CDK2 (Fig. 5, E). In both cell lines, CDK2 coimmunoprecipitated with an antibody that was specific for CCRK, suggesting that CCRK exerts its effects, at least in part, through direct or indirect binding to and phosphorylation of CDK2 in glioma cells.
Effect of CCRK Knockdown on Human Glioma Cell Tumorigenicity in a Mouse Xenograft Model
To examine the in vivo function of CCRK in glioblastoma multiforme carcinogenesis, nude mice were injected subcutaneously with U-373 MG or U-87 MG cells that had been stably transfected with pGE-1-shCCRK or pGE-1-shLuc (groups of four mice each) and measured tumor volume weekly for 6 weeks. The constructs expressed a small hairpin RNA that targets CCRK or luciferase, respectively. Stable transfection of U-373 MG and U-87 MG cells with pGE-1-shCCRK led to a marked reduction of CCRK expression at both the protein and mRNA levels (Fig. 6, A). By 4–6 weeks after cell injection, the mean tumor volumes of mice injected with the shCCRK-transfected cells (U-373-shCCRK and U-87-shCCRK) were statistically significantly smaller than those of mice injected with shLuc-transfected cells (U-373-shLuc and U-87-shLuc) (mean tumor volumes at week 6 after tumor cell injection, U373-shLuc versus U373-shCCRK: 1352 mm3 versus 294 mm3, difference = 1058 mm3, 95% CI = 677 to 1439 mm3, P<.001; U-87-shLuc versus U-87-shCCRK: 1910 mm3 versus 552 mm3, difference = 1358 mm3, 95% CI = 977 to 1739 mm3, P<.001) (Fig. 6, B).
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Effect of CCRK Overexpression on Tumorigenesis of Nontumorigenic Glioma Cells
We next performed reciprocal experiments to examine whether an elevated level of CCRK could induce malignant transformation in vitro and in vivo by using nontumorigenic U-138 MG cells that overexpressed CCRK (U-138-CCRK). In a colony formation assay, U-138-CCRK cells showed a marked increase in the formation of G418-resistant cell colonies compared with control cells transfected with the empty vector (U-138-pcDNA) (the mean number of cell colonies as percentage of the number of U-138-CCRK cell colonies, U-138-CCRK versus U-138-pcDNA = 100% versus 1.7%, difference = 98.3%, 95% CI = 61.9% to 134.7%, P = .002) (Fig. 7, A, upper and middle panels). By contrast, U-138 MG cells transfected with a construct that expressed a kinase-defective CCRK mutant protein produced statistically significantly fewer G418-resistant colonies (Fig. 7, A, lower panel). These data suggested that malignant transformation of U-138 MG cells was dependent on the kinase activity of CCRK (the mean number of cell colonies as percentage of the number of U-138-CCRK cell colonies, U-138-CCRK versus U-138-T161A = 100% versus 2.3%, difference = 97.7%, 95% CI = 58.8% to 136.6%, P = .002) (Fig. 7, A, right panel). In agreement with these results, we found that CCRK overexpression could induce the malignant conversion of the nontumorigenic U-138 MG cells in nude mouse xenograft in vivo. The growth of tumor xenografts could be detected in nude mice subcutaneously injected with U-138-CCRK cells or U-138-CCRK-lenti-EGFP cells. However, virtually no xenografts were formed in nude mice injected with U-138-pcDNA cells or U-138-CCRK-lenti-shCCRK cells (Fig. 7, B). The observation that lenti-shCCRK transduction, but not lenti-EGFP transduction, could reverse the tumorigenicity of the U-138-CCRK cells in nude mice suggested that the transformation of U-138-CCRK cells and U-138-CCRK-lenti-EGFP cells was attributable to an elevated level of CCRK (Fig. 7, B). The increase in the levels of CCRK protein in U-138-CCRK cells and U-138-CCRK-lenti-EGFP cells was also confirmed by immunoblotting. In these cell lines, an increase in CCRK protein level was accompanied by an elevation in the level of phosphorylated CDK2 protein (Fig. 7, C).
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Discussion |
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TopAbstractContext and CaveatsMaterials and MethodsResultsDiscussionReferencesNotes |
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In this study, we found that CCRK expression was elevated compared with normal brain tissue in a majority of glioblastoma multiforme patient samples and cell lines examined. We further showed that knockdown of CCRK expression statistically significantly inhibited the growth of two tumorigenic and high CCRK–expressing glioma cell lines (U-373 MG and U-87 MG) and one nontumorigenic and low CCRK–expressing glioma cell line (U-138 MG). We found that overexpression of CCRK induced malignant transformation of U-138 MG cells both in vitro and in vivo. U-138-CCRK cell tumorigenicity was conferred by CCRK overexpression because it was reversed by transduction of the cells with lenti-shCCRK. Furthermore, the tumorigenicity induced by CCRK overexpression appeared to be dependent on its kinase activity because expression of a kinase-defective mutant CCRK protein failed to transform U-138 MG cells. Taken together, our data indicate that increased glioma cell proliferation and tumorigenicity are associated with the overexpression of CCRK and suggest that CCRK is a candidate oncogene in glioblastoma multiforme.
It is unclear whether CCRK is a bona fide oncogene in other cancers. However, results from several studies have indicated that CCRK is expressed or overexpressed in various cancer types. For example, Boer et al. (23; http://www.ncbi.nlm.nih.gov/geo/gds/gds_browse.cgi?gds=8) showed that CCRK expression was increased by more than twofold in 81 renal cell carcinoma samples as compared with levels in corresponding paired normal kidney tissues. Expression of CCRK protein has also been detected in different cancer cell lines, including cervical adenocarcinoma (HeLa cells), osteogenic sarcoma (U2OS cells), breast adenocarcinoma (MCF-7 cells), and prostate cancer (PC3 cells) (15). We have also investigated the gene expression profiles of CCRK in paired normal and tumor tissues from colon cancer patients and colon cancer cell lines (An X, Ng SSM, Wang J, Sze J, Chen YC, Qiao L, et al.: unpublished observations). Our preliminary results indicated that CCRK mRNA and protein expression was higher in colon cancer tissues than their adjacent normal counterparts (data not shown). These observations suggest that CCRK may potentially act as an oncogene in various cancer types. Further expression-profiling studies are required to determine possible relationships between CCRK mRNA or protein levels and various clinicopathological and prognostic parameters in different grades of cancer patient samples.
The mechanism by which CCRK expression is dysregulated in glioma cells is not known. Possible mechanisms that might contribute to the increased expression of CCRK include gene amplification, regulation of promoter methylation and/or transcription, and CCRK gene mutations. We are currently examining each of these mechanisms in detail in glioma cell lines as well as in glioblastoma multiforme patient samples by using comparative genomic hybridization, methylation-specific PCR, and DNA sequencing. Elucidation of the mechanisms that regulate CCRK expression should provide important information regarding the molecular pathogenesis of glioblastoma multiforme.
The biologic role of CCRK in cell cycle progression has proven elusive. There has been controversy over whether CCRK functions as a second CDK-activating kinase (i.e., in addition to CDK7) (15,17). The function of a CDK-activating kinase is to phosphorylate CDKs that are important for cell cycle progression (24,25). Liu et al. (15) reported that, in HeLa cells, CCRK phosphorylates and activates CDK2 and that it is essential for cell growth. However, Wohlbold et al. (17) showed that although the depletion of CCRK from human colorectal carcinoma HCT116 cells and human osteosarcoma U2OS cells by RNA interference impaired cell proliferation, the intrinsic kinase activity of CCRK appeared to be rather weak and the low level of CDK-activating kinase activity of CCRK likely results from its apparent association with CDK7. Our observations, that siCCRK transfection reduced the level of phosphorylated CDK2 and that CCRK coimmunoprecipitated with CDK2, suggest that CCRK is a CDK-activating kinase.
The question of whether CDK7 is the sole CDK-activating kinase in mammals remains to be answered. If CCRK is another CDK-activating kinase, how is it different from CDK7 in function? One hypothesis is that instead of being a general CDK-activating kinase that activates a range of CDKs, CCRK may act as a CDK-specific CDK-activating kinase by preferentially binding to and modifying the activities of CDK2 and CDK7 (15,17). Another possibility is that CCRK has low kinase activity when it exists as a monomer but full CDK-activating kinase activity when bound to an unidentified coactivator in vivo. Further investigations, including yeast two-hybrid and DNA microarray studies, are needed to unravel the downstream targets and interacting partners of CCRK in cancer cells. These data are essential for elucidating whether CCRK is an oncogenic CDK-activating kinase in cell cycle control and carcinogenesis.
Our study has several limitations. First, because of the small number of glioblastoma multiforme patient samples, we could not test whether CCRK expression level was statistically significantly correlated with CDK2 phosphorylation and/or survival in glioblastoma multiforme patients. Second, our demonstration that siCCRK reduced CDK2 phosphorylation and that CCRK coimmunoprecipitated with CDK2 did not provide direct evidence that CCRK is a CDK-activating kinase or that CDK2 is a substrate of CCRK. It is possible, for example, that the reduction in CDK2 phosphorylation could be a secondary effect of the growth inhibition induced by CCRK knockdown. In addition, CCRK may bind and/or phosphorylate CDK7 (or other CDKs), which in turn bind to CDK2 and regulate its activity. Therefore, further studies such as in vitro kinase assays are needed to determine if CCRK interacts directly with CDK2.
The data presented here provide the first evidence, to our knowledge, that CCRK is an oncogene that encodes a key molecule in glioblastoma multiforme carcinogenesis. This conclusion is based on several lines of evidence: 1) CCRK expression was increased in the majority of the glioma patient samples and cell lines we examined; 2) siRNA-mediated CCRK knockdown inhibited the growth of the glioblastoma cells in vitro and in vivo; and 3) CCRK overexpression induced the malignant conversion of a nontumorigenic glioma cell line. Our results support the rationale for developing CCRK as a potential therapeutic and diagnostic target for glioblastoma multiforme and, possibly, other cancers. Further studies aimed at identifying the molecular mechanisms, signal transduction pathways, and additional substrates for CCRK-mediated oncogenesis, and how CCRK is dysregulated in various cancers may reveal novel mechanisms for cell cycle regulation.
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NOTES |
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TopAbstractContext and CaveatsMaterials and MethodsResultsDiscussionReferencesNotes |
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This work was supported by the Research Grants Council of the Hong Kong Special Administrative Region, China (HKU7243/02M to M. C. Lin), the Committee on Research and Conference Grants of the University of Hong Kong (200507176197 to S. S. M. Ng), Li Ka Shing Institute of Health Sciences (H.-F. Kung), AoE scheme of UGC, Shanghai Metropolitan Fund for Research and Development (04JC14096), and Foundation of Guangzhou Science and Technology Bureau (2005Z1-E0131). The study sponsors had no role in the design of the study; the collection, analysis, or interpretation of the data; the writing of the manuscript; or the decision to submit the manuscript for publication.
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Manuscript received November 8, 2006; revised April 20, 2007; accepted May 8, 2007.
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