Abstract 2485: Secretome signature associated with TITF1/NKX2-1-negative non-small cell lung cancer
Authors: Taguchi Ayumu , Celiktas Muge , Dilsher Dhillon , Zhang Qing , Wong Chee-Hong , Chin Alice , Gazdar Adi , Hanash Samir 
Published: May 2015

Abstract
Background: TITF1/NKX2-1 is a master transcription factor of peripheral airway cells and a known lineage-specific oncogene in lung cancer cells, whereas it also suppresses tumor progression and is associated with favorable prognosis of lung adenocarcinoma. In our previous study, a protein signature for TITF1 was found in plasmas of mouse models of lung adenocarcinoma, leading to the identification of concordant circulating protein signatures in human non-small cell lung cancer (NSCLC). Following study demonstrated one of TITF1 signature proteins, pro-SFTPB, as a predictive biomarker for NSCLC. To identify additional biomarkers which would complement the performance of pro-SFTPB, we studied secretome from forty NSCLC cell lines in terms of TITF1 expression.
Materials and Methods: Forty NSCLC cell lines were cultured according to the standard SILAC protocol. The same batch of cells was used for analysis of whole cell extract and conditioned media. Samples were fractionated and subjected to mass spectrometric analysis. Gene expression data for the same set of NSCLC cell lines were obtained using the Illumina Human WG-6 v3.0 Expression BeadChips.
Results: NSCLC cell lines were divided into two groups by median of TITF1 gene expression (TITF1-positive and negative). In secretome analysis, overall 10,399 proteins were identified and semi-quantified with using total number of spectral counts. Interestingly, top abundant secretome proteins in TITF1-negative NSCLC cell lines contain many cytokine/chemokines (IL6, CXCL1, CXCL2 and CXCL5) and growth factors (CTGF, PDGFA, PDGFC, and VEGFC). Among top abundant secretome proteins, gene expression of SRGN was most significantly and inversely correlated with TITF1 gene expression. SRGN is a proteoglycan and physiologically associated with some proteases and cytokine secretion in hematopoietic cells. In addition recent studies have indicated that SRGN is also associated with aggressive phenotype in some cancers, thus considered as a promising biomarker candidate for TITF1-negative NSCLC. Then we determined whether SRGN would be directly regulated by TITF1. Suppression of TITF1 by siRNA or TNF alpha treatment increased SRGN expression, suggesting potential transcriptional regulation of SRGN by TITF1. Finally we studied functional roles of SRGN in TITF1-negative cell lines. Knockdown of SRGN by siRNA did not affect cell proliferation. However two chemokines, CXCL1 and CXCL5, which are abundant in secretome of TITF1-negative cell lines, were decreased by SRGN knockdown. In addition, macrophage migration was also decreased in the conditioned media of SRGN knockdown cells compared with control cells.
Conclusion: Our findings suggest functional relevance of SRGN in tumor microenvironment and tumor aggressiveness in TITF1/NKX2-1-negative NSCLC. Validation study for plasma levels of CXCL1 and CXCL5 in NSCLC subjects is now on-going.
Citation Format: Ayumu Taguchi, Muge Celiktas, Dhillon Dilsher, Qing Zhang, Chee-Hong Wong, Alice Chin, Adi Gazdar, Samir Hanash. Secretome signature associated with TITF1/NKX2-1-negative non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2485. doi:10.1158/1538-7445.AM2014-2485