Abstract 3381: Investigation of C/EBPG transcription factor role in regulation of ERCC4 and ERCC5 in human lung cancer cells
Authors: Zhang Xiaolu , Yeo Jiyoun , Crawford Erin L. , Willey James C. 
Published: May 2015

Abstract
Background: ERCC4 and ERCC5 are key nucleotide excision DNA repair genes and are expressed abundantly in normal bronchial epithelial cells (NBEC). DNA sequence variation in ERCC4 or ERCC5 is associated with risk for lung cancer in multiple independent studies. C/EBPG expression is correlated with that of ERCC4, ERCC5 and many other key genes in BEC, suggesting a regulatory role, supported by experimental observation that up-regulation of CCAAT/enhancer-binding protein gamma (C/EBPG) transcription factor up-regulates ERCC5 expression in H23 lung cancer cell line. The purpose of this study was to test the hypothesis that knockdown of C/EBPG in lung cancer cells would reduce transcription of ERCC5 and ERCC4. Methods: We knocked-down C/EBPG transcript level by C/EBPG siRNA transfection in three non-small cell lung cancer cell lines: H23, H520 and H1703. Following transfection, RNA was extracted after 24 and 48 hours. Reduction of C/EBPG transcript abundance measured by competitive multiplex RT-PCR in H23, H520, and H1703 was 56%, 84%, and 92% at 24 hours, and 82%, 89%, and 76% at 48 hours. After confirming C/EBPG knockdown, we measured allele-specific expression (ASE) and total expression of C/EBPG, ERCC4 ERCC5 through competitive multiplex PCR-based amplicon sequencing library preparation followed by Illumina HiSeq next generation sequencing (NGS) (Blomquist et al, PLOS one, 2013). This NGS method a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. ASE was measured at ERCC4 SNPs rs2276466, rs3743538 and ERCC5 SNPs rs1047768, rs17655, rs4150316. Results: In H23 C/EBPG knock-down was associated with no change in expression at any of the SNPs for ERCC4 or ERCC5, while in H520 one ERCC5 rs1047768 allele decreased (3-fold) and the other allele had no change, and in H1703 both ERCC5 rs1047768 alleles increased (10-fold, and 2-fold). In H520, ERCC4 measured at two SNPs were different with increase at one allele but not the other at rs3743538 and decrease for both alleles at rs2276466. In H1703 each allele at ERCC4 rs3743538 increased, 2-fold and 45-fold respectively and each allele at ERCC4 rs2276466 increased, 5-fold and 2.7-fold respectively. Conclusions: These results support prior evidence that C/EBPG regulates ERCC5 transcription, and provides evidence that it also contributes to regulation of ERCC4. The inter-allelic and inter-cell line variation in response to C/EBPG knockdown data supports that hypothesis that cis-regulatory DNA variants interact with C/EBPG in regulation of these genes. The lack of response in H23 may be in part due to the low constitutive level of ERCC4 and ERCC5 expression in this cell line.
Citation Format: Xiaolu Zhang, Jiyoun Yeo, Erin L. Crawford, James C. Willey. Investigation of C/EBPG transcription factor role in regulation of ERCC4 and ERCC5 in human lung cancer cells. [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 3381. doi:10.1158/1538-7445.AM2014-3381