Screening for tumor suppressors: Loss of ephrin receptor A2 cooperates with oncogenic KRas in promoting lung adenocarcinoma
- aLaboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA 92037;
- bHubrecht Institute, Utrecht, The Netherlands
-
Contributed by Inder M. Verma, October 12, 2015 (sent for review July 28, 2015; reviewed by Anton Berns, Tyler Jacks, and Frank McCormick)
Significance
A large number of genetic alterations have been found in the cancer genome of lung adenocarcinoma. However, they need experimental validation to determine their tumorigenic potential, as well as the therapeutic utility of individual alterations. Here, we provide an shRNA-mediated screen to validate a large set of genes for their tumor suppressor efficacy in vivo in a mouse lung adenocarcinoma model. We identified several tumor suppressors, including ephrin receptor A2 (EphA2) loss of which promotes adenocarcinoma in the context of oncogenic mutant KRas mutation. EphA2 loss promotes cell proliferation by activating ERK MAP kinase signaling and hedgehog signaling pathways, leading to tumorigenesis. Identification of these pathways provides important therapeutic targets for 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.
Footnotes
- ↵1To whom correspondence should be addressed. Email: verma{at}salk.edu.
-
Author contributions: N.Y., Y.X., and I.M.V. designed research; N.Y., Y.X., J.B., and I.M.V. performed research; N.Y. and E.K. analyzed data; N.Y. and I.M.V. wrote the paper; and I.M.V. supervised the project.
-
Reviewers: A.B., Netherlands Cancer Institute; T.J., Massachusetts Institute of Technology; and F.M., University of California, San Francisco.
-
The authors declare no conflict of interest.
-
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1520110112/-/DCSupplemental.
