title: Screening for tumor suppressors: Loss of ephrin receptor A2 cooperates with oncogenic KRas in promoting lung adenocarcinoma.
abstract: Lung adenocarcinoma, a major form of non-small cell lung cancer, is the leading cause of cancer deaths. The Cancer Genome Atlas analysis of lung adenocarcinoma has identified a large number of previously unknown copy number alterations and mutations, requiring experimental validation before use in therapeutics. Here, we describe an shRNA-mediated high-throughput approach to test a set of genes for their ability to function as tumor suppressors in the background of mutant KRas and WT Tp53. We identified several candidate genes from tumors originated from lentiviral delivery of shRNAs along with Cre recombinase into lungs of Loxp-stop-Loxp-KRas mice. Ephrin receptorA2 (EphA2) is among the top candidate genes and was reconfirmed by two distinct shRNAs. By generating knockdown, inducible knockdown and knockout cell lines for loss of EphA2, we showed that negating its expression activates a transcriptional program for cell proliferation. Loss of EPHA2 releases feedback inhibition of KRAS, resulting in activation of ERK1/2 MAP kinase signaling, leading to enhanced cell proliferation. Intriguingly, loss of EPHA2 induces activation of GLI1 transcription factor and hedgehog signaling that further contributes to cell proliferation. Small molecules targeting MEK1/2 and Smoothened hamper proliferation in EphA2-deficient cells. Additionally, in EphA2 WT cells, activation of EPHA2 by its ligand, EFNA1, affects KRAS-RAF interaction, leading to inhibition of the RAS-RAF-MEK-ERK pathway and cell proliferation. Together, our studies have identified that (i) EphA2 acts as a KRas cooperative tumor suppressor by in vivo screen and (ii) reactivation of the EphA2 signal may serve as a potential therapeutic for KRas-induced human lung cancers.
authors.affiliation: Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA 92037;
authors.forename: Narayana
authors.initials: N
authors.lastname: Yeddula
authors.affiliation: Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA 92037;
authors.forename: Yifeng
authors.initials: Y
authors.lastname: Xia
authors.affiliation: Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA 92037;
authors.forename: Eugene
authors.initials: E
authors.lastname: Ke
authors.affiliation: Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA 92037; Hubrecht Institute, Utrecht, The Netherlands.
authors.forename: Joep
authors.initials: J
authors.lastname: Beumer
authors.affiliation: Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA 92037; verma@salk.edu.
authors.forename: Inder M
authors.initials: IM
authors.lastname: Verma
mesh_terms: D000230:Adenocarcinoma / Q000378:metabolism*; D000077192:Adenocarcinoma of Lung; D000818:Animals; D001483:Base Sequence; D063646:Carcinogenesis / Q000473:pathology; D049109:Cell Proliferation; D004789:Enzyme Activation; D055785:Gene Knockdown Techniques; D015894:Genome, Human; D053823:Hedgehog Proteins / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006801:Humans; D008024:Ligands; D008175:Lung Neoplasms / Q000378:metabolism*; D020935:MAP Kinase Signaling System; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D036104:Receptor, EphA2 / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D023041:Xenograft Model Antitumor Assays
chemical_list: D053823:Hedgehog Proteins; D008024:Ligands; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; D036104:Receptor, EphA2; C495971:Hras protein, mouse; D016283:Proto-Oncogene Proteins p21(ras)
keywords: EphA2; Hedgehog signaling; KrasG12D; in vivo shRNA screen; lung adenocarcinoma