Abstract 4456: Novel oncogenic function of Notch4 in Hispanic lung cancer
Authors: Gordian Edna , Gimbrone Nicholas , Pannuti Antonio , Miele Lucio , Cress W. Douglas , Muñoz-Antonia Teresita 
Published: July 2017

Abstract
Background: Molecular drivers of ~40% of lung adenocarcinomas are still unknown. We reasoned that since the frequencies of driver mutations have been shown to be different between ethnic groups, comparison of targeted exome sequencing data would identify genes with significant alteration frequencies between White Non-Hispanics (WNH) and other racial/ethnic groups. Notch signaling in cancer is context- and tissue-specific and may be both oncogenic and tumor suppressive. The mechanisms of action and genetic alterations involved in Notch oncogenesis have been studied for Notch1 and Notch3; specifically in Non-Small Cell Carcinoma (NSCLC), translocations in Notch3, and gain of function Notch1 mutations have been found in 1 in 10 patients. Notch4 is a mammary oncogene, but little is known about its role in NSCLC tumorigenesis. Truncations in Notch4 have been reported in lung cancer cell lines, and in public databases the frequency of Notch4 alterations in WNH adenocarcinoma is ~5.5%. However, little is known about the mechanisms by which Notch4 acts in lung oncogenesis.
Methods: Using data from the TCCTM protocol, we analyzed targeted sequencing of 1,321 genes of interest to determine the mutations driving lung cancer in Hispanic (H/L) patients. The functional relevance of the Notch4 mutations was tested by cloning the intracellular domain (ICD) of Notch4 containing one of the mutations identified in the ANK domain (P1663Q) into pcDNA3.1. Plasmids expressing either the WT or a mutated Notch 4 (P1663Q) were transfected into A549 cells and the effect of this mutation tested on the ability to induce the expression of Notch4 expression genes.
Results: In our H/L cohort we see Notch4 altered in about 20% of our samples, and 7/12 of the Notch4 single amino acid substitutions are in the Negative Regulatory Region (NRR). PROVEAN protein analysis of the Notch4 mutations shows that more than half of the mutations would disrupt the NRR. Introduction of the P1663Q mutation in the ANK repeats of the Notch4 ICD, which mediates binding to CSL and is essential for Notch transcriptional activity, resulted in a shift in the amount of transcript of known Notch4 target genes, such as Hes1 and Snai1. This effect is specific, as it is not observed with other genes, previously reported to be targets for other Notch paralogs such as Smad3 and Zeb1. A similar effect of the P1663Q Notch4 mutation on Hes1 transcript levels was observed in breast cancer cells, suggesting that this is not lung cancer specific. Transcriptional activity of the Notch-responsive luciferase construct (pHESLuc) was increased in the presence of Wt Notch4, and reduced in the presence of the P1663Q Notch4 Mu.
Conclusion: The nature and high number of genetic alterations in Notch4 in H/L lung cancer patients, suggests that Notch4 may play a critical role in NSCLC tumorigenesis. Our data suggests that mutations in the ANK domain of Notch4 can interfere with its transcriptional activity and function, and should be further characterized.
Citation Format: Edna Gordian, Nicholas Gimbrone, Antonio Pannuti, Lucio Miele, W. Douglas Cress, Teresita Muñoz-Antonia. Novel oncogenic function of Notch4 in Hispanic lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4456. doi:10.1158/1538-7445.AM2017-4456