BACKGROUND: Lung cancer is emerging rapidly as the leading death cause in Chinese cancer patients. The causal factors for Chinese lung cancer 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 line BEAS-2B. METHODS/RESULTS: Soft agar assays were conducted for screening BEAS-2B cells infected with the retroviral shRNA library with the acquired feature of anchorage-independent growth, large (>0.5mm in diameter) and well-separated 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 BEAS-2B via activation of Akt-mTOR-p70S6K signaling, whereas ectopic expression of Sens2 re-suppressed the malignant 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.