Cell Cycle–Related Kinase: A Novel Candidate Oncogene in Human Glioblastoma

Samuel S. M. Ng Yuen-Ting Cheung Xiao-Meng An Yang Chao Chen Ming Li Gloria Hoi-Yee Li William Cheung Johnny Sze Lihui Lai Ying Peng Harry H. X. Xia Benjamin C. Y. Wong Suet-Yi Leung Dan Xie Ming-Liang He Hsiang-Fu Kung Marie C. Lin 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. We analyzed the expression levels of CCRK in 26 glioma patient samples (19 high-grade and seven lowgrade) 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. 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). Methods Results Conclusions CCRK is a candidate oncogene in glioblastoma multiforme tumorigenesis. 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 1215 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 plateletderived 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 cyclerelated kinase (CCRK) (also called p42) is a 42-kD protein kinase that shares 43% sequence identity with cyclindependent 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 cellassociated 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. C O N T E X T A N D C A V E A T S 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. Reverse TranscriptionPolymerase 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 Northern Blot Analysis Northern blot analysis of CCRK mRNA expression was performed on a human multiple tissue northern blot that contained 10 g 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 (...truncated)