Abstract P5-05-02: Whole Genome In Vivo RNA Interference Screening Identifies the Leukemia Inhibitory Factor Receptor as a Novel Breast Tumor Suppressor

Abstract

Abstract Background: Cancer is caused by mutations in oncogenes and tumor suppressor genes resulting in the deregulation of processes fundamental to the normal behavior of cells. The identification and characterization of oncogenes and tumor suppressors has led to new treatment strategies that have significantly improved cancer outcome. The advent of next generation sequencing has allowed the elucidation of the fine structure of cancer genomes, however, the identification of pathogenic changes is complicated by the inherent genomic instability of cancer cells. Therefore, functional approaches for the identification of novel genes involved in the initiation and development of tumors are critical. Methods: In order to identify functionally important tumor suppressor genes we have conducted the first human whole genome in vivo RNA interference (RNAi) screen. Partially transformed human mammary epithelial cells (HMLEs), which do not form tumors in immunodeficient mice, were infected with the Expression Arrest™ GIPZ lentiviral shRNA library consisting of 62,000 shRNAs targeting the whole human genome, and injected into the mammary fat pad of immunodeficient mice. shRNAs that silenced tumor suppressor genes fully transformed the mammary epithelial cells resulting in tumor formation. Candidate tumor suppressor genes were identified by PCR amplification and sequencing of tumor integrated shRNAs. For validation, candidate tumor suppressor genes were silenced in HMLEs and ectopically expressed in fully transformed breast cancer cells. The effect of modifying gene expression on the transformed phenotype was assessed using soft agar colony formation assays. Clinical significance was determined by comparing expression in normal and cancerous human breast tissue using Oncomine Research. Results and Discussion: Using our novel approach, we identify previously validated tumor suppressor genes including TP53 and MNT, as well as several novel candidate tumor suppressor genes including leukemia inhibitory factor receptor (LIFR). Silencing LIFR expression with multiple shRNA constructs fully transformed human mammary epithelial cells resulting in enhanced colony formation in soft agar (P<0.05). Furthermore, overexpression of LIFR significantly inhibited colony formation in soft agar of fully transformed MDA231 and MCF7 breast cancer cells (P<0.01). In addition, our analysis of clinical data revealed that LIFR expression is significantly decreased in a large percentage of human cancers including breast (P<0.0001), lung (P<0.0001), hepatocellular (P<0.0001) and gastrointestinal tumors (P<0.0001). These results validate LIFR as a previously unidentified highly significant tumor suppressor, and also demonstrate the power of whole genome in vivo RNAi screens as a method for identifying novel genes regulating tumorigenesis. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-05-02.