NSCLC that accounts for more than 80% of all lung malignancy cases can be further divided into adenocarcinoma (~48%), squamous cell carcinoma (~28%) and large cell carcinoma (~24%) [1,3]. relation to Sesn2 protein expression levels. (DOC) pone.0124033.s004.doc (35K) GUID:?9AC38E73-60C3-4F4B-9D51-568849FF1C51 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Background Lung malignancy is usually emerging rapidly as the leading death cause in Chinese malignancy patients. The causal factors for Chinese lung malignancy development remain largely unclear. Here we employed an shRNA library-based loss-of-function screen in a genome-wide and unbiased manner to interrogate potential tumor suppressor candidates in the immortalized human lung epithelial cell collection BEAS-2B. Methods/Results Soft agar assays were conducted for screening BEAS-2B cells Rabbit polyclonal to cyclinA infected with the retroviral shRNA library with the acquired feature of anchorage-independent growth, large (>0.5mm in diameter) and wellseparated colonies were isolated for proliferation. PCRs were performed to amplify the integrated shRNA fragment from individual genomic DNA extracted from each colony, and each PCR product is submitted for DNA sequencing to reveal the integrated shRNA and its target gene. A total of 6 candidate transformation suppressors including INPP4B, Sesn2, TIAR, ACRC, Nup210, LMTK3 were identified. We validated Sesn2 as the candidate of lung cancer tumor suppressor. Knockdown of Sesn2 by an shRNA targeting 3 UTR of Sesn2 transcript potently stimulated the proliferation and malignant transformation of lung bronchial epithelial cell GSK726701A BEAS-2B via activation of Akt-mTOR-p70S6K signaling, whereas ectopic expression of Sens2 re-suppressed the malignant GSK726701A transformation elicited by the Sesn2 shRNA. Moreover, knockdown of Sesn2 in BEAS-2B cells promoted the BEAS-2B cell-transplanted xenograft tumor growth in nude mice. Lastly, DNA sequencing indicated mutations of Sesn2 gene are rare, the protein levels of Sesn2 of 77 Chinese lung cancer patients varies greatly compared to their adjacent normal tissues, and the low expression level of Sesn2 associates with the poor survival in these examined patients by Kaplan Meier analysis. Conclusions Our shRNA-based screen has demonstrated Sesn2 is a potential tumor suppressor in lung epithelial cells. The expression level of Sesn2 may serve as a prognostic marker for Chinese lung cancer patients in the clinic. Introduction Lung cancer is emerging as the most common and deadly malignancy in China as well as in the world [1,2]. Based on pathological features, lung cancer can be divided into two major subtypes, non-small-cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC). NSCLC that accounts for more than 80% of all lung cancer cases can be further divided into adenocarcinoma (~48%), squamous cell carcinoma (~28%) and large cell carcinoma (~24%) [1,3]. Despite the great advances achieved in the diagnostics, surgical operation, radiotherapy and targeted therapies, lung cancer still holds a quite poor prognosis and its 5 year survival rate remains as low as 10%-15% in the past 30 years [3]. The mechanisms driving lung cancer development are complex, genetic alterations, smoking and various environmental pollutions are common causal factors attributed to lung cancer occurrence. Tumor suppressors with loss-of-function mutations, deletions, and/or epigenetic silencing often play a crucial role in lung tumorigenesis [4]. For example, the mutation rate of p53 gene in non-small cell lung cancer (NSCLC) can reach to 60%, even goes up to 80% in small cell lung cancer (SCLC) [5]. Other tumor suppressors such as PTEN with much lower mutation rate also involve in lung adenocarcinoma GSK726701A [6]. In addition to better understanding the molecular alterations occurred during lung cell malignant transformation, discovery of lung cancer related tumor suppressor genes also provides more effective and personalized therapies for lung cancer treatment [7]. To this end, to GSK726701A identify novel tumor suppressors in a genome-wide and unbiased manner is one of the central tasks for lung cancer research. However, identifying the new tumor suppressor genes is rather difficult due to their recessive expression nature. Cancer whole genomic analysis indicates that there are many low ratio mutations in the tumor cells, and the mutations vary between different origins of tissues [8]. An shRNA library-based loss-of-function screen targeting human transcriptome to interrogate potential tumor suppressor candidates systematically in immortalized human cells has been proven to be a powerful approach for.