Cancer Biology and Signal Transduction Genome-wide siRNA Screen Identifies the Radiosensitizing Effect of Downregulation of MASTL and FOXM1 in NSCLC Remco Nagel 1 , Marijke Stigter-van Walsum 1 , Marijke Buijze 1 , Jaap van den Berg 2 , Ida H. van der Meulen 3,4 , Jasmina Hodzic 4 , Sander R. Piersma 5 , Thang V. Pham 5 , Connie R. Jim enez 5 , Victor W. van Beusechem 3,4 , and Ruud H. Brakenhoff 1 Abstract Lung cancer is the most common cancer worldwide and on top of that has a very poor prognosis, which is reflected by a 5-year survival rate of 5% to 15%. Radiotherapy is an integral part of most treatment regimens for this type of tumor, often combined with radiosensitizing cytotoxic drugs. In this study, we identified many genes that could potentially be exploited for targeted radiosensitization using a genome-wide siRNA screen in non–small cell lung cancer (NSCLC) cells. The screen identified 433 siRNAs that potentially sensitize lung cancer cells to radiation. Validation experiments showed that knockdown of expression of Forkhead box M1 (FOXM1) or microtubule- associated serine/threonine kinase-like (MASTL) indeed causes radiosensitization in a panel of NSCLC cells. Strikingly, this effect was not observed in primary human fibroblasts, suggest- ing that the observed radiosensitization is specific for cancer cells. Phosphoproteomics analyses with and without irradia- tion showed that a number of cell-cycle–related proteins were significantly less phosphorylated after MASTL knockdown in comparison to the control, while there were no changes in the levels of phosphorylation of DNA damage response proteins. Subsequent analyses showed that MASTL knockdown cells respond differently to radiation, with a significantly shortened G 2 –M phase arrest and defects in cytokinesis, which are fol- lowed by a cell-cycle arrest. In summary, we have identified many potential therapeutic targets that could be used for radiosensitization of NSCLC cells, with MASTL being a very promising and druggable target to combine with radiotherapy. Mol Cancer Ther; 14(6); 1434–44. Ó2015 AACR. Introduction Lung cancer is the most frequently diagnosed cancer, repre- senting 12% of all cancers worldwide (1). It is a heterogeneous disease and is divided into multiple classes. The major division is made between small cell lung cancer (SCLC) and non–small cell lung cancer (NSCLC), which make up 20% and 80% of the lung cancer cases, respectively (2). It has been shown that the different subtypes of lung cancer exhibit large variations in sensitivity to cytotoxic compounds and radiation, where SCLC was shown to be the most sensitive (3). Clinical treatment regimens for lung cancer consist of surgery, radiotherapy, or chemotherapy, but often a combination of these modalities is applied. Radiotherapy is an integral part in almost all treatment schedules for lung cancer (4). Despite an initial remission after the start of treatment, a relapse is frequently observed after which the tumor can no longer be treated effectively (5). The lack of treatment efficacy has become a major obstacle in the curative treatment of lung cancer, which is reflected in the current 5-year survival rate for patients with lung cancer of only 5% to 15% (6). It has become clear that the tumor subtype and the presence of specific genetic aberrations in the tumor cell determine the intrinsic capacity to respond to therapy. Several studies have shown that specific mutations, for example, in components of the EGFR pathway, are associated with a more radioresistant phenotype in NSCLC (7, 8). The identification of genes involved in radiation response in lung cancer could therefore provide leads for novel druggable targets that could be exploited to enhance radiotherapy-containing treatment modalities. Here, we report the results of a genome-wide loss-of-function screen and the identification of multiple novel targets that may cause radio- sensitization in NSCLC cells when inhibited. Materials and Methods Cell culture All cell lines used were cultured in DMEM supplemented with 5% FCS, 2 mmol/L L-glutamine, and 1% penicillin/streptomycin in a humidified atmosphere of 5% CO 2 at 37  C. NSCLC cell lines SW1573, A549, H322, and H1299 were obtained from the 1 Department of Otolaryngology–Head and Neck Surgery, VU Univer- sity Medical Center, Amsterdam, the Netherlands. 2 Department of Radiation Oncology, VU University Medical Center, Amsterdam, the Netherlands. 3 RNA Interference Functional Oncogenomics Laborato- ry, VU University Medical Center, Amsterdam, the Netherlands. 4 Department of Medical Oncology, VU University Medical Center, Amsterdam, the Netherlands. 5 OncoProteomics Laboratory, Depart- ment of Medical Oncology,VU University Medical Center, Amsterdam, the Netherlands. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Corresponding Author: Ruud H. Brakenhoff, Tumor Biology Section, Depart- ment of Otolaryngology–Head and Neck Surgery, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, the Netherlands. Phone: 31-20-444-0953; Fax: 31-20-444-3688; E-mail: rh.brakenhoff@vumc.nl doi: 10.1158/1535-7163.MCT-14-0846 Ó2015 American Association for Cancer Research. Molecular Cancer Therapeutics Mol Cancer Ther; 14(6) June 2015 1434 Downloaded from http://aacrjournals.org/mct/article-pdf/14/6/1434/1932797/1434.pdf by guest on 23 August 2023