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RASEF Is a Novel Diagnostic Biomarker and a Therapeutic Target for Lung Cancer

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RASEF Is a Novel Diagnostic Biomarker and a Therapeutic Target for Lung Cancer Published OnlineFirst May 16, 2013; DOI: 10.1158/1541-7786.MCR-12-0685-T Molecular Cancer Oncogenes and Tumor Suppressors Research RASEF is a Novel Diagnostic Biomarker and a Therapeutic Target for Lung Cancer Hideto Oshita1,3, Ryohei Nishino1, Atsushi Takano1,2, Takashi Fujitomo1, Masato Aragaki1, Tatsuya Kato1,6, Hirohiko Akiyama4, Eiju Tsuchiya5, Nobuoki Kohno3, Yusuke Nakamura1, and Yataro Daigo1,2 Abstract Genome-wide gene expression profiling revealed that the Ras and EF-hand domain containing (RASEF) transcript was significantly transactivated in the majority of lung cancers. Using lung cancer cells, transient expression of RASEF promoted cell growth, whereas RASEF knockdown not only reduced its expression but resulted in growth suppression of the cancer cells. Immunohistochemical staining using tumor tissue microarrays consisting of 341 archived non–small cell lung cancers (NSCLC) revealed the association of strong RASEF positivity with poor prognosis (P ¼ 0.0034 by multivariate analysis). Mechanistically, RASEF interacted with extracellular signal-regulated kinase (ERK) 1/2 and enhanced ERK1/2 signaling. Importantly, inhibiting the interaction between RASEF and ERK1/2 using a cell-permeable peptide that corresponded to the ERK1/2-interacting site of RASEF, suppressed growth of lung cancer cells. This study demonstrates that elevated RASEF promoted cell growth via enhanced ERK signaling and is associated with poor prognosis of NSCLC. Implications: RASEF may play an important role in lung carcinogenesis and could serve as a vaiable prognostic biomarker and target for the development of new molecular therapies. Mol Cancer Res; 11(8); 937–51. Ó2013 AACR. Introduction non–small cell lung cancer (NSCLC) who had undergone Lung cancer is the most common cause of cancer-related curative surgery suffered recurrent diseases (2), it is impor- death in the world and its incidence has been increasing (1). tant to develop more precise prognostic biomarkers for In spite of the use of advanced surgical treatments combined selecting patients who should be treated with adjuvant with radiotherapy and chemotherapy, the overall 5-year therapies and intensively followed after surgical treatment. survival rate of patients with lung cancer still remains at Systematic analysis of expression levels of thousands of 20% (2). Development of molecular-targeted drugs such as genes using a cDNA microarray technology is an effective fi approach for identifying molecules involved in pathways of ge tinib and bevacizumab have improved treatment modal- fi ities of lung cancer, but fatal adverse events such as interstitial carcinogenesis or those associated with ef cacy/resistance to pneumonia by gefitinib or severe hemorrhage by bevacizu- anticancer therapy; some of such genes or their gene products mab were reported (3–4). Therefore, further development of may be good target molecules for the development of novel new agents targeting cancer-specific molecules with no or therapies and/or cancer biomarkers (5). To identify such molecules, particularly oncoantigens, we had conducted minimum risk of adverse effect is urgently awaited. To date, fi clinical and pathologic staging have been the most reliable genome-wide expression pro le analysis of 120 clinical lung information for physicians in the choice of therapy. How- cancer tissue samples, coupled with enrichment of tumor cells ever, considering that about 30% of patients with stage I by laser microdissection, and then compared the expression profile data with those in 31 normal human tissues (27 adult and 4 fetal organs; refs. 6–10). To verify the clinicopathologic Authors' Affiliations: 1Laboratory of Molecular Medicine, Human Genome fi Center, Institute of Medical Science, The University of Tokyo, Tokyo; signi cance of the respective gene products, we have estab- 2Department of Medical Oncology and Cancer Center, Shiga University lished a screening system by a combination of the tumor- of Medical Science, Otsu; 3Department of Molecular and Internal Medicine, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima; tissue microarray analysis of clinical lung cancer materials and 4Department of Thoracic Surgery, Saitama Cancer Center, Saitama; 5Kana- RNA interference technique (11–41). This systematic gawa Cancer Center Research Institute, Yokohama; and 6Department of approach revealed that RAS and EF-hand domain-containing Surgical Oncology, Hokkaido University Graduate School of Medicine, Sapporo, Japan protein (RASEF) is likely to be a novel molecule that was overexpressed commonly in primary lung cancers and was Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). essential for cell growth and/or survival of cancer cells. RASEF contains a Rab GTPase domain in the C-terminal Corresponding Author: Yataro Daigo, Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of region and is considered as a member of Rab GTPase protein Tokyo, Tokyo 108-8639. Phone: 81-3-5449-5457; Fax: 81-3-5449-5406; family. Unlike other Rab proteins, RASEF contains 2 EF- E-mail: [email protected] hand domains which are generally known to be important doi: 10.1158/1541-7786.MCR-12-0685-T for binding to calcium ions in the N-terminus and a coiled- Ó2013 American Association for Cancer Research. coil motif in an internal region (42). The functional www.aacrjournals.org 937 Downloaded from mcr.aacrjournals.org on September 25, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst May 16, 2013; DOI: 10.1158/1541-7786.MCR-12-0685-T Oshita et al. relevance of RASEF activation to carcinogenesis as well as Semiquantitative reverse transcription-PCR its detailed biologic function has not yet been defined. Here A total of 3 mg aliquot of mRNA from each sample was we report the first evidence that RASEF plays a significant reversely transcribed to single-stranded cDNAs using ran- role in lung cancer cell growth possibly through its interac- dom primer (Roche Diagnostics) and SuperScript II (Invi- tion with extracellular signal-regulated kinase (ERK) 1/2, trogen). Semiquantitative reverse transcription-PCR (RT- and suggest that RASEF could be a promising prognostic PCR) experiments were carried out with the following sets of biomarker and therapeutic target for lung cancer. synthesized gene-specific primers or with b-actin (ACTB)- specific primers as an internal control: RASEF,50-GGC- Materials and Methods TGACATTCGTGACACTG-30 and 50-GGAATTGGTC- Cell lines and tissue samples CCGGTTAGAT-30; cyclin D1 (CCND1), 50-CCTCGG- Lung cancer cell lines and human bronchial epithelial cells TGTCCTACTTCAAA-30 and 50-CCAGGTTCCACTT- (BEAS-2B) used in this study are listed in Supplementary GAGCTTGT-30; cyclin B1 (CCNB1), 50-TTGGTGTCA- Table S1. A427, A549, NCI-H1373, NCI-H1781, NCI- CTGCCATGTTT-30 and 50-GATGCTCTCCGAA- H358, NCI-H226, NCI-H520, NCI-H2170, NCI- GGAAGTG-30; cyclin-dependent kinase inhibitor 1A H1703, DMS114, DMS273, NCI-H196, NCI-H446, and (CDKN1A), 50-TTAGCAGCGGAACAAGGAGT-30 and BEAS-2B cells were from American Type Culture Collec- 50-ATTCAGCATTGTGGGAGGAG-30; and ACTB,50- tion in 2003, 2010, and 2011, and tested and authenticated GAGGTGATAGCATTGCTTTCG-30 and 50-CAAGTC- by DNA profiling for polymorphic short-tandem repeat AGTGTACAGGTAAGC-30. PCRs were optimized for the (STR) markers. PC-3, SBC-3, and SBC-5 cells were from number of cycles to ensure product intensity to be within the Japanese Collection of Research Bioresources (JCRB) in linear phase of amplification. 2001 and 2010, and tested and authenticated by DNA profiling for polymorphic STR markers. PC-14, EBC-1, and Western blot analysis RERF-LC-AI cells were from RIKEN BioResource Center Cell lysates from lung cancer cell line or normal airway in 2001 and 2010, and tested and authenticated by DNA epithelial cells were subjected to Western blotting. In brief, profiling for polymorphic STR markers. LC319 cells were cells were incubated in 1 mL lysis buffer (0.5% NP-40, 50 from Aichi Cancer Center in 2003, and tested and authen- mmol/L Tris-HCl, 150 mmol/L NaCl) in the presence of ticated by DNA profiling for single-nucleotide polymor- protease inhibitor (Protease Inhibitor Cocktail Set III; Cal- phism, mutation, and deletion analysis. PC-9 and LX1 cells biochem). Western blotting were done using an ECL West- were from Tokyo Medical University and Central Institute ern-blotting analysis system (GE Healthcare Bio-sciences) as for Experimental Animals and European Collection of described previously (11–15). A commercially available Animal Cell Cultures in 2002, and tested and authenticated rabbit polyclonal antibody to human RASEF (Catalog No. by DNA profiling for single-nucleotide polymorphism, 11569-1-AP, Proteintech Group, Inc.) was confirmed to be mutation, and deletion analysis. All cells were grown in specific to endogenous RASEF protein by Western-blot monolayer in appropriate medium supplemented with 10% analysis using lysates of lung cancer cell lines as well as fetal calf serum (FCS) and maintained at 37C in humidified normal airway epithelial cells (negative control). air with 5% CO2. Primary lung cancer samples had been obtained earlier as previously described (6, 10). All tumors Immunocytochemical analysis were staged on the basis of the pTNM pathologic classifi- Cells were plated onto glass coverslips (Becton Dickinson cation of the UICC (International Union Against Cancer; Discovery Labware), fixed with 4% paraformaldehyde, and ref.
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