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• W2039442869 abstract "Kinases execute pivotal cellular functions and are therefore widely investigated as potential targets in anticancer treatment. Here we analyze the kinase gene expression profiles of various tumor types and reveal the wee1 kinase to be overexpressed in glioblastomas. We demonstrate that WEE1 is a major regulator of the G2 checkpoint in glioblastoma cells. Inhibition of WEE1 by siRNA or small molecular compound in cells exposed to DNA damaging agents results in abrogation of the G2 arrest, premature termination of DNA repair, and cell death. Importantly, we show that the small-molecule inhibitor of WEE1 sensitizes glioblastoma to ionizing radiation in vivo. Our results suggest that inhibition of WEE1 kinase holds potential as a therapeutic approach in treatment of glioblastoma. Kinases execute pivotal cellular functions and are therefore widely investigated as potential targets in anticancer treatment. Here we analyze the kinase gene expression profiles of various tumor types and reveal the wee1 kinase to be overexpressed in glioblastomas. We demonstrate that WEE1 is a major regulator of the G2 checkpoint in glioblastoma cells. Inhibition of WEE1 by siRNA or small molecular compound in cells exposed to DNA damaging agents results in abrogation of the G2 arrest, premature termination of DNA repair, and cell death. Importantly, we show that the small-molecule inhibitor of WEE1 sensitizes glioblastoma to ionizing radiation in vivo. Our results suggest that inhibition of WEE1 kinase holds potential as a therapeutic approach in treatment of glioblastoma. In silico analysis reveals WEE1 kinase to be overexpressed in glioblastomas WEE1 kinase is a major regulator of the G2 checkpoint in glioblastoma cells Inhibition of WEE1 pushes DNA-damaged glioblastoma cells into mitotic catastrophe WEE1 inhibitors may prove therapeutically attractive for glioblastoma treatment The resistance of glioblastoma cells to irradiation and chemotherapy is partly due to their proficient ability to repair treatment-induced DNA damage during the G2 cell-cycle arrest. Many kinase inhibitors intend to prolong cell-cycle arrest to halt cancer cell division. Here, we inhibit the WEE1 kinase, a gatekeeper of the DNA damage-induced G2 arrest, “pushing” glioblastoma cells through the G2 arrest phase and thereby inducing mitotic catastrophe and cell death. This strategy results in extensive cytotoxicity in vitro and, more importantly, eradication of irradiated brain tumors in mice, without showing adverse side effects. Therefore, manipulation of WEE1 activity may prove therapeutically attractive as a sensitizing approach for glioblastoma treatment. Glioblastoma (GBM) is one of the most aggressive human cancers and the most common primary brain tumor. The median survival of GBM patients is <15 months because this tumor is inherently resistant to conventional therapy (Stupp et al., 2009Stupp R. Hegi M.E. Mason W.P. van den Bent M.J. Taphoorn M.J. Janzer R.C. Ludwin S.K. Allgeier A. Fisher B. Belanger K. et al.Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial.Lancet Oncol. 2009; 10: 459-466Abstract Full Text Full Text PDF PubMed Scopus (4777) Google Scholar). Although the conventional treatment with surgery, irradiation (IR), and temozolomide (TMZ) postpones tumor progression and extends patients survival to some extent, these tumors universally recur and unrelentingly result in patient death. Despite recent advances in understanding the underlying molecular mechanisms, there has been little improvement in clinical outcome (Wen and Kesari, 2008Wen P.Y. Kesari S. Malignant gliomas in adults.N. Engl. J. Med. 2008; 359: 492-507Crossref PubMed Scopus (2969) Google Scholar). Manipulation of oncogenic kinase activity has become a therapeutic concept in human cancer because kinases regulate crucial cellular functions such as proliferation, apoptosis, cell metabolism, migration, DNA damage repair, and responses to the microenvironment (Manning et al., 2002Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanam S. The protein kinase complement of the human genome.Science. 2002; 298: 1912-1934Crossref PubMed Scopus (5791) Google Scholar). Several human cancers are considered to be kinase-driven (Weinstein, 2002Weinstein I.B. Cancer. Addiction to oncogenes—the Achilles heal of cancer.Science. 2002; 297: 63-64Crossref PubMed Scopus (1403) Google Scholar, Krause and Van Etten, 2005Krause D.S. Van Etten R.A. Tyrosine kinases as targets for cancer therapy.N. Engl. J. Med. 2005; 353: 172-187Crossref PubMed Scopus (1105) Google Scholar), and inhibitors of several cancer-driving kinases are under evaluation as potential therapeutic agents (Cohen, 2002Cohen P. Protein kinases—the major drug targets of the twenty-first century?.Nat. Rev. Drug Discov. 2002; 1: 309-315Crossref PubMed Scopus (1734) Google Scholar, Vieth et al., 2005Vieth M. Sutherland J.J. Robertson D.H. Campbell R.M. Kinomics: characterizing the therapeutically validated kinase space.Drug Discov. Today. 2005; 10: 839-846Crossref PubMed Scopus (153) Google Scholar). Most of these inhibitors aim at stagnation of tumor growth by interrupting the replicative cycle of cancer cells. Examples include inhibitors of epidermal growth factor receptor (EGFR), polo-like kinase 1 (PLK1), v-akt murine thymoma viral oncogene homologue (AKT), mitogen-activated protein kinase (MAPK), protein kinase C (PKC), vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR) (reviewed by Zhang et al., 2009Zhang J. Yang P.L. Gray N.S. Targeting cancer with small molecule kinase inhibitors.Nat. Rev. Cancer. 2009; 9: 28-39Crossref PubMed Scopus (1901) Google Scholar). These therapeutic kinase targets are mainly deregulated by either mutation, protein fusion or gene overexpression (Krause and Van Etten, 2005Krause D.S. Van Etten R.A. Tyrosine kinases as targets for cancer therapy.N. Engl. J. Med. 2005; 353: 172-187Crossref PubMed Scopus (1105) Google Scholar). Thus far, results from clinical trials testing the efficacy of kinase inhibitors in patients with GBM have been disappointing (Omuro et al., 2007Omuro A.M.P. Faivre S. Raymond E. Lessons learned in the development of targeted therapy for malignant gliomas.Mol. Cancer Ther. 2007; 6: 1909-1919Crossref PubMed Scopus (170) Google Scholar, De Witt Hamer, 2010De Witt Hamer P.C. Small molecule kinase inhibitors in glioblastoma: a systematic review of clinical studies.Neuro-oncol. 2010; 12: 304-316Crossref PubMed Scopus (113) Google Scholar). In this study we focused on the kinase gene expression profile of GBM using publicly available gene expression data sets to identify additional putative therapeutic targets. To identify kinase targets, we used a cancer-wide approach and selected 34 cancer-versus-normal data sets (Table S1 available online), including two GBM data sets obtained by different microarray platforms in distinct laboratories (Bredel et al., 2005Bredel M. Bredel C. Juric D. Harsh G.R. Vogel H. Recht L.D. Sikic B.I. Functional network analysis reveals extended gliomagenesis pathway maps and three novel MYC-interacting genes in human gliomas.Cancer Res. 2005; 65: 8679-8689Crossref PubMed Scopus (246) Google Scholar, Kotliarov et al., 2006Kotliarov Y. Steed M.E. Christopher N. Walling J. Su Q. Center A. Heiss J. Rosenblum M. Mikkelsen T. Zenklusen J.C. et al.High-resolution global genomic survey of 178 gliomas reveals novel regions of copy number alteration and allelic imbalances.Cancer Res. 2006; 66: 9428-9436Crossref PubMed Scopus (97) Google Scholar). Two parameters of gene expression were determined, details of which are described in Supplemental Experimental Procedures. First, to compare the differential kinase gene expression of cancer-versus-normal samples among data sets, the fold change was determined for all genes within a data set. Then, the percentile of fold change was determined for each kinase within a data set to relate its expression value to all genes in that data set. This allowed comparison of expression levels of each kinase between data sets. Second, to compare the frequency at which overexpression occurs in cancer samples between data sets, the frequency of overexpression was determined for each kinase within each cancer-versus-normal data set. To recover the human kinome from the genes present in the data sets, the Entrez GeneIDs of the human protein kinase family (518 kinases) and the human lipid kinase family (33 kinases) were retrieved (Figures 1A , Figure S1, and Table S2) (Manning et al., 2002Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanam S. The protein kinase complement of the human genome.Science. 2002; 298: 1912-1934Crossref PubMed Scopus (5791) Google Scholar, Peri et al., 2003Peri S. Navarro J.D. Amanchy R. Kristiansen T.Z. Jonnalagadda C.K. Surendranath V. Niranjan V. Muthusamy B. Gandhi T.K.B. Gronborg M. et al.Development of human protein reference database as an initial platform for approaching systems biology in humans.Genome Res. 2003; 13: 2363-2371Crossref PubMed Scopus (810) Google Scholar). The vast majority of kinase genes were represented on the microarray platforms (Table S1 and Table S2). We found distinct kinase expression profiles in various cancer types (Figure 1A). Thus, potential therapeutic targets overexpressed in a specific type of cancer may not necessarily be valid in other cancer types. To validate our approach, we selected kinases previously demonstrated to be overexpressed in specific cancers and determined their expression across our kinase profiles (Figure 1B). Our analysis revealed v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (kit) to be selectively overexpressed in seminomas and gastrointestinal stromal tumors, which show clinical response to anti-KIT treatment (Pedersini et al., 2007Pedersini R. Vattemi E. Mazzoleni G. Graiff C. Complete response after treatment with imatinib in pretreated disseminated testicular seminoma with overexpression of c-KIT.Lancet Oncol. 2007; 8: 1039-1040Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, Demetri et al., 2002Demetri G.D. von Mehren M. Blanke C.D. Van den Abbeele A.D. Eisenberg B. Roberts P.J. Heinrich M.C. Tuveson D.A. Singer S. Janicek M. et al.Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors.N. Engl. J. Med. 2002; 347: 472-480Crossref PubMed Scopus (3547) Google Scholar). Moreover, egfr overexpression was confirmed in head and neck cancers, nonsmall cell lung cancers, and renal cell cancers, for which anti-EGFR treatment can prolong overall survival (Lynch et al., 2004Lynch T.J. Bell D.W. Sordella R. Gurubhagavatula S. Okimoto R.A. Brannigan B.W. Harris P.L. Haserlat S.M. Supko J.G. Haluska F.G. et al.Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib.N. Engl. J. Med. 2004; 350: 2129-2139Crossref PubMed Scopus (9495) Google Scholar, Agulnik et al., 2007Agulnik M. da Cunha Santos G. Hedley D. Nicklee T. Dos Reis P.P. Ho J. Pond G.R. Chen H. Chen S. Shyr Y. et al.Predictive and pharmacodynamic biomarker studies in tumor and skin tissue samples of patients with recurrent or metastatic squamous cell carcinoma of the head and neck treated with erlotinib.J. Clin. Oncol. 2007; 25: 2184-2190Crossref PubMed Scopus (76) Google Scholar, Ravaud et al., 2008Ravaud A. Hawkins R. Gardner J.P. von der Maase H. Zantl N. Harper P. Rolland F. Audhuy B. Machiels J.P. Petavy F. et al.Lapatinib versus hormone therapy in patients with advanced renal cell carcinoma: a randomized phase III clinical trial.J. Clin. Oncol. 2008; 26: 2285-2291Crossref PubMed Scopus (64) Google Scholar). Although we retrieved the kinase expression profiles of several tumor types, our prime focus was to identify potential therapeutic targets in glioblastoma. We considered overexpression at a level surpassing 95% of all genes in a data set as substantial. Additionally, we considered overexpression in >20% of the patient population as frequent. A listing of nine kinases was obtained that were both substantially (Figure 2A ) and frequently (Figure 2B) overexpressed in the two available GBM data sets. In previous investigations four of these nine selected kinases, aurora kinase A (aurka) (Klein et al., 2004Klein A. Reichardt W. Jung V. Zang K.D. Meese E. Urbschat S. Overexpression and amplification of STK15 in human gliomas.Int. J. Oncol. 2004; 25: 1789-1794PubMed Google Scholar), egfr (Nicholas et al., 2006Nicholas M.K. Lukas R.V. Jafri N.F. Faoro L. Salgia R. Epidermal growth factor receptor - mediated signal transduction in the development and therapy of gliomas.Clin. Cancer Res. 2006; 12: 7261-7270Crossref PubMed Scopus (162) Google Scholar), maternal embryonic leucine zipper kinase (melk) (Nakano et al., 2008Nakano I. Masterman-Smith M. Saigusa K. Paucar A.A. Horvath S. Shoemaker L. Watanabe M. Negro A. Bajpai R. Howes A. et al.Maternal embryonic leucine zipper kinase is a key regulator of the proliferation of malignant brain tumors, including brain tumor stem cells.J. Neurosci. Res. 2008; 86: 48-60Crossref PubMed Scopus (125) Google Scholar), and cyclin-dependent kinase 1 (cdc2) (Hodgson et al., 2009Hodgson J.G. Yeh R.F. Ray A. Wang N.J. Smirnov I. Yu M. Hariono S. Silber J. Feiler H.S. Gray J.W. et al.Comparative analyses of gene copy number and mRNA expression in GBM tumors and GBM xenografts.Neuro. Oncol. 2009; 11: 477-487Crossref PubMed Scopus (95) Google Scholar), were described as relevantly overexpressed in human GBM samples, emphasizing the biological plausibility of our results. The other five kinases were not previously associated with human GBM. pdgfr, kinase insert domain receptor (kdr), fms-related tyrosine kinase 4 (flt4), transforming growth factor, beta receptor 1 (tgfbr1), and transforming growth factor, beta receptor 2 (tgfbr2) had been described to be overexpressed in GBM (Rich and Bigner, 2004Rich J.N. Bigner D.D. Development of novel targeted therapies in the treatment of malignant glioma.Nat. Rev. Drug Discov. 2004; 3: 430-446Crossref PubMed Scopus (197) Google Scholar) but they were not included in our list. Despite marked overexpression in the two GBM data sets (Table S3 and Table S4), these kinases did not meet our selection criteria. Given the noise levels in microarray data, we preferred high specificity over sensitivity. The individual data for the nine overexpressed kinases retrieved from the GBM studies is presented in Figures 3A and 3B and Figure S2, according to glioma grade and histological subtype. To validate the results obtained in silico, we determined mRNA expression of these kinases in GBM cell lines and primary samples of GBM and normal brain using quantitative reverse transcription polymerase chain reaction (RT-PCR) (Figure 3C, Table S5, and Table S6). Because WEE1 kinase plays an important role in controlling the cell-cycle progression (Russell and Nurse, 1987Russell P. Nurse P. Negative regulation of mitosis by wee1+, a gene encoding a protein kinase homolog.Cell. 1987; 49: 559-567Abstract Full Text PDF PubMed Scopus (700) Google Scholar) and because it topped the ranking of overexpressed kinases (Figure 2, Figure 3, and Figure S2), we further studied its role in GBM. We first determined the levels of WEE1 protein expression in normal brain and GBM tissue sections using immunohistochemistry. Consistent with the gene expression data, GBM cell nuclei contained high levels of WEE1 protein, as compared to non-neoplastic brain regions (Figure 3D). We then investigated the correlation between wee1 mRNA level and patient survival using published data from 267 GBM patients (Lee et al., 2008Lee Y. Scheck A.C. Cloughesy T.F. Lai A. Dong J. Farooqi H.K. Liau L.M. Horvath S. Mischel P.S. Nelson S.F. Gene expression analysis of glioblastomas identifies the major molecular basis for the prognostic benefit of younger age.BMC Med. Genomics. 2008; 1: 52Crossref PubMed Google Scholar). Expression of wee1 was classified as high or low, based on whether the values were above or below the median wee1 expression levels, respectively. This showed high tumor wee1 expression to be significantly correlated with worse patient survival (log rank p < 0.0059), patients with high wee1-expressing GBM had a median survival time of 308 days and a 2-year survival rate of 14% versus a median survival time of 402 days and a 2-year survival rate of 26% for patients with low wee1-expressing GBM (Figure 3E). wee1 expression level was significantly associated with patient survival after correction for confounding by age, gender, tumor recurrence, and prior treatment modalities, in a multivariate Cox proportional-hazards regression model (hazard ratio: 0.69, 95% confidence interval: 0.53–0.89, p = 0.0047 and Table S7). After DNA damage induction, GBM cells mainly arrest in the G2 phase, due to an impaired G1 checkpoint (Hirose et al., 2001Hirose Y. Berger M.S. Pieper R.O. p53 effects both the duration of G2/M arrest and the fate of temozolomide-treated human glioblastoma cells.Cancer Res. 2001; 61: 1957-1963PubMed Google Scholar). Indeed, U251MG GBM cells arrested in the G2 phase after IR and TMZ treatment (Figures 4A and 4C ). We then focused on the role of WEE1 in the G2 checkpoint activation in response to DNA damage. To specifically inhibit WEE1 function, we transfected GBM cells with siRNA directed against the wee1 mRNA. As determined by quantitative RT-PCR, the wee1 siRNA efficiently knocked down wee1 mRNA levels, as compared to nontargeting control siRNA (Figure S3A). After 48 hr the transfected cells were either exposed to 6 Gray (Gy) of IR or treated with 100 μM TMZ and 16 hr later cell-cycle distribution was monitored by fluorescence activated cell sorting. DNA content was monitored by propidium iodide staining and phospho-histone-H3 staining was used as an indicator of mitotic entry. Knock down of wee1 resulted in significant abrogation of the IR- and TMZ-induced G2 arrest and decrease in the amount of mitotic cells (Figures 4A and 4C and Figures S3B and S3C). Similar abrogation of the DNA damage-induced arrest in U251MG cells was achieved by using the WEE1 inhibitor PD0166285 (Wang et al., 2001Wang Y. Li J. Booher R.N. Kraker A. Lawrence T. Leopold W.R. Sun Y. Radiosensitization of p53 mutant cells by PD0166285, a novel G(2) checkpoint abrogator.Cancer Res. 2001; 61: 8211-8217PubMed Google Scholar) (Figures 4A and4C, Figure S3D, and Figure S4E). Comparative cell-cycle results were obtained using additional established GBM cell lines (U118MG, U87MG, U373MG) and primary GBM cells (VU147, VU148, E98) (Figures S3D and S3E). These results indicate that WEE1 is a major determinant of the DNA damage-induced G2 arrest in GBM cells. Importantly, no adverse effects of WEE1 inhibition on cell cycle were observed in primary human fibroblasts and astrocytes, showing no loss of G1 arrest after exposure to IR or TMZ in the presence or absence of WEE1 inhibitor or siRNA against wee1 (Figure 4B and Figures S3D and S3E). Furthermore, no effects of PD0166285 on fibroblasts and astrocytes were observed by WST-1 cell viability measurements and microscopical monitoring of cellular morphology (data not shown). Inhibition of WEE1 also reduces phosphorylation of its downstream substrate CDC2 (Figure 4D and Figure S3F). This suggests that the inhibitory CDC2 phosphorylation is reduced by inhibition of WEE1 and thereby the CDC2-mediated G2 arrest is abolished in GBM cells. We then investigated the effects of WEE1 inhibition on the viability of cells treated with IR, TMZ, or both. wee1 silencing by siRNA or WEE1 inhibition by PD0166285 had a significant sensitizing effect in combination with 6 Gy of IR, 100 μM of TMZ, or both (Figures 4E and 4F and Figure S3G). At concentrations up to 1 μm, PD0166285 had no effects on the viability of GBM cells in the absence of IR and TMZ (Figure 4E). In addition, radiosensitizing effects of PD0166285 in GBM cells could be measured by colony formation assay (Figure 4G), which correlated with wee1 gene expression levels in the GBM cells. U373MG cells with a fold change log2wee1 expression (FC) compared to six non-neoplastic brain samples of 13.0 (see Figure 3C) showed the largest sensitization, with a sensitizing enhancement ratio (SER) of 1.95, U251MG cells (FC: 9.1, SER: 1.73) and U118MG cells (FC: 9.2, SER: 1.45) showed intermediate sensitization, whereas U87MG cells (FC 5.2 that is within the upper threshold for normal expression of FC 6.4 and SER of 1.19) showed the least sensitization (Figure 3C and Figure 4G). In contrast, no correlation was observed between p53 mutation status and WEE1 inhibitor sensitivity in the GBM cells analyzed here. However, U87 cells, which express wee1 mRNA levels within the normal range (Figure 3C and Figure 4G) and express the wild-type p53 (Figure S3H), showed the least PD0166285-mediated radiosensitization (Figure 4G). There was no significant effect of PD0166285 on colony formation in non-irradiated GBM cells. A subpopulation of GBM cells, the so-called cancer stem-like cells, has been described to be particularly resistant to chemo- and radiotherapy (Bao et al., 2006Bao S. Wu Q. McLendon R.E. Hao Y. Shi Q. Hjelmeland A.B. Dewhirst M.W. Bigner D.D. Rich J.N. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response.Nature. 2006; 444: 756-760Crossref PubMed Scopus (4442) Google Scholar, Liu et al., 2006Liu G. Yuan X. Zeng Z. Tunici P. Ng H. Abdulkadir I.R. Lu L. Irvin D. Black K.L. Yu J.S. Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma.Mol. Cancer. 2006; 5: 67Crossref PubMed Scopus (1384) Google Scholar). In silico analysis using the ONCOMINE database (Rhodes et al., 2004Rhodes D.R. Yu J. Shanker K. Deshpande N. Varambally R. Ghosh D. Barrette T. Pandey A. Chinnaiyan A.M. ONCOMINE: a cancer microarray database and integrated data-mining platform.Neoplasia. 2004; 6: 1-6PubMed Google Scholar) showed that wee1 was overexpressed to an even higher extent in a subpopulation of GBM cells cultured in neural basal medium (Lee et al., 2006Lee J. Kotliarova S. Kotliarov Y. Li A. Su Q. Donin N.M. Pastorino S. Purow B.W. Christopher N. Zhang W. et al.Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines.Cancer Cell. 2006; 9: 391-403Abstract Full Text Full Text PDF PubMed Scopus (1658) Google Scholar) (Figure S3I). GBM neurospheres formed by such cells expressed CD133, Nestin, and SOX2 (Figure S3J), putative GBM stem-like cell markers (Lee et al., 2006Lee J. Kotliarova S. Kotliarov Y. Li A. Su Q. Donin N.M. Pastorino S. Purow B.W. Christopher N. Zhang W. et al.Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines.Cancer Cell. 2006; 9: 391-403Abstract Full Text Full Text PDF PubMed Scopus (1658) Google Scholar). Next, the radiosensitizing effect of WEE1 inhibitor PD0166285 on primary GBM neurospheres was determined. GBM neurospheres failed to respond to exposure to IR alone, confirming their radioresistance (Bao et al., 2006Bao S. Wu Q. McLendon R.E. Hao Y. Shi Q. Hjelmeland A.B. Dewhirst M.W. Bigner D.D. Rich J.N. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response.Nature. 2006; 444: 756-760Crossref PubMed Scopus (4442) Google Scholar). Importantly, dramatic cell death was measured when primary GBM neurospheres were exposed to IR in the presence of PD0166285. Although GBM stem-like cell markers are still subject of continuous debate (Chen et al., 2010Chen R. Nishimura M.C. Bumbaca S.M. Kharbanda S. Forrest W.F. Kasman I.M. Greve J.M. Soriano R.H. Gilmour L.L. Rivers C.S. et al.A hierarchy of self-renewing tumor-initiating cell types in glioblastoma.Cancer Cell. 2010; 17: 362-375Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar), we isolated primary CD133 positive and negative GBM cells with high wee1 expression and intermediate wee1 expression, respectively, as determined by qRT-PCR (Figure S3I). Subsequently we treated the sorted cells with 0.5 μM of PD0166285 and 6 Gy of IR. After 4 days the radiation-resistant CD133 positive GBM cells were efficiently killed as determined by WST-1 cell viability assay (Figure 4H). These results suggest that radiation resistance of GBM stem-like cell populations can be overcome by WEE1 inhibition in vitro. After induction of DNA damage, WEE1 activates and sustains the G2 cell-cycle arrest until DNA damage is sufficiently repaired (Rowley et al., 1992Rowley R. Hudson J. Young P.G. The wee1 protein kinase is required for radiation-induced mitotic delay.Nature. 1992; 356: 353-355Crossref PubMed Scopus (102) Google Scholar). Therefore, inhibition of WEE1 should abrogate G2 arrest activation and result in increased amounts of unrepaired DNA damage in cells prematurely entering mitosis. To determine the effect of WEE1 inhibition on the rejoining of double-strand breaks (DSBs) and on DSB-induced cell-cycle arrest, we exposed GBM cells to IR in the presence or absence of the WEE1 inhibitor. IR resulted in induction of DSBs as evidenced by formation of ionizing radiation-induced foci (IRIF) by a DSB marker γ-H2AX (Rogakou et al., 1999Rogakou E.P. Boon C. Redon C. Bonner W.M. Megabase chromatin domains involved in DNA double-strand breaks in vivo.J. Cell Biol. 1999; 146: 905-916Crossref PubMed Scopus (1853) Google Scholar), and by repair factors ATMp1981, MDC1, and MRE11 (Goldberg et al., 2003Goldberg M. Stucki M. Falck J. D'Amours D. Rahman D. Pappin D. Bartek J. Jackson S.P. MDC1 is required for the intra-S-phase DNA damage checkpoint.Nature. 2003; 421: 952-956Crossref PubMed Scopus (416) Google Scholar, Lavin, 2007Lavin M.F. ATM and the Mre11 complex combine to recognize and signal DNA double-strand breaks.Oncogene. 2007; 26: 7749-7758Crossref PubMed Scopus (211) Google Scholar) (Figures 5A and 5B ). Phase-contrast time-lapse imaging of the U251MG cells at 10-min intervals showed that IR induced a considerable delay in the onset of mitosis (Figures 5C and 5D). In contrast, cells irradiated in the presence of PD0166285 failed to delay their mitotic entry (Figures 5C and 5D), in agreement with the role of WEE1 in activating and maintaining the G2 arrest. In the absence of a functional G2 arrest, irradiated GBM cells may not be capable of repairing the DNA damage before entering mitosis, leading to mitotic catastrophe. We irradiated GBM cells and then time-lapse imaged the cells in the presence or absence of WEE1 inhibitor (Movie S1). Cells were fixed and stained for γ-H2AX and MDC1 and analyzed for completion of mitosis by fluorescence microscopy. Exposure to IR alone resulted in only a few residual DSBs in the daughter cell nuclei, indicating near-complete DSB repair before the cells entered mitosis (Figures 5E and 5G). In contrast, cells irradiated in the presence of WEE1 inhibitor presented their daughter cells with considerable amount of unrepaired DSBs (Figures 5F and 5G). Additionally, the presence of PD0166285 resulted in largely fragmented nuclei of the daughter cells (Figure 5F and Movie S1), indicative of mitotic catastrophe due to DNA fragments devoid of centromeres, which can not be properly segregated during mitosis (Loffler et al., 2006Loffler H. Lukas J. Bartek J. Kramer A. Structure meets function–centrosomes, genome maintenance and the DNA damage response.Exp. Cell Res. 2006; 312: 2633-2640Crossref PubMed Scopus (98) Google Scholar). Together, these results support the concept that G2 abrogation by WEE1 inhibition results in mitotic catastrophe due to unrepaired DSBs that explains the radiosensitizing properties of the WEE1 inhibitor. To compare the antitumor efficacy of short hairpin-mediated wee1 knock down alone or in combination with IR in vivo, we first used a bioluminescent orthotopic model of human U251-Fluc-mCherry cells (U251-FM) (Candolfi et al., 2007Candolfi M. Curtin J.F. Nichols W.S. Muhammad A.G. King G.D. Pluhar G.E. McNiel E.A. Ohlfest J.R. Freese A.B. Moore P.F. et al.Intracranial glioblastoma models in preclinical neuro-oncology: neuropathological characterization and tumor progression.J. Neurooncol. 2007; 85: 133-148Crossref PubMed Scopus (234) Google Scholar). U251-FM cells were transduced with lentivectors encoding a control shRNA or a shRNA directed against wee1 (shWEE11702). First, the efficiency of wee1 knock down and reduction of cell viability in response to wee1 knock down after IR-mediated DNA damage was determined in these cells (Figure S4A). Second, control or shWEE1 transduced U251-FM cells were implanted in the brain of nude mice. The growth of tumors was monitored weekly by bioluminescence imaging. Two weeks after intracranial injection of the transduced cells, 50% of the mice in each group were exposed to a single dose of 6 Gy. In mice injected with U251-FM cells expressing shControl strong tumor progression was observed in both irradiated and non-irradiated groups at 6 weeks after injection of the cells (Figures 6A and 6B ). Similarly, the nonirradiated U251-FM-shWEE1 cells showed strong increase in bioluminescence signal after 6 weeks, whereas mice injected with U251-FM-shWEE1 cells showed significant tumor regression after IR at 6 weeks after injection (Figures 6A and 6B). Tumor burden was markedly reduced in irradiated mice carrying shWEE1 transduced cells (Figure 6C). Survival analysis showed a significant (p = 0.001) advantage for combining irradiation with wee1 knock down (Figure 6D). These results indicate that wee1 knockdown sensitizes U251-FM GBMs to IR in vivo." @default.
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• W2039442869 date "2010-09-01" @default.
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• W2039442869 title "In Silico Analysis of Kinase Expression Identifies WEE1 as a Gatekeeper against Mitotic Catastrophe in Glioblastoma" @default.
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