Abstract 3024: Transcriptional and epigenetic regulation of resistance markers in cetuximab sensitive HNSCC cells
Authors: Kagohara Luciane T. , Zamuner Fernando , Considine Michael , Stein-O'Brien Genevieve , Sherman Thomas , Gaykalova Daria A. , Fertig Elana J. 
Published: August 2019

Abstract
Acquired resistance is pervasive in cancers treated with targeted therapies. The understanding of resistance mechanisms and the timing of the driver molecular changes are crucial for alternative interventions while tumors are sensitive. In this study, we focus on cetuximab, the only targeted therapeutic approved to treat head and neck cancers (HNSCC). We and others have shown that signatures of resistance arise in early sensitive states of treatment, including notably EMT and TFAP2 activation. Because TFAP2 regulates both EMT genes and compensatory growth factor receptors and resistance is associated with epigenetic alterations, we hypothesized that epigenetic regulation of TFAP2 is a master regulator of future mechanisms of resistance.To test our central hypothesis, we treated three HNSCC cell lines (SCC1, SCC6 and SCC25) with CTX for 5 days, a time frame that cells are known to be sensitive. We performed RNA-seq and ATAC-seq to understand the global molecular changes induced by CTX. To verify the role of TFAP2A in cell growth and EMT control in response to CTX, we used siRNA for gene silencing and measured cell proliferation and migration ability (scratch assay). In order to identify a potential combination therapy, we also treated the cells with JQ1, a bromodomain inhibitor with a major impact in delaying acquired CTX resistance.RNA-seq analysis shows that CTX sensitive cells up-regulate growth factor receptors and EMT genes. Chromatin changes were also evident in the same cells. Enrichment of accessible chromatin areas for genes from the TFAP2A and EMT pathways suggests epigenetic regulation of CTX resistance associated genes as an early response to therapy. In both, RNA-seq and ATAC-seq analysis, we observed alterations in different sets of genes for each of the cell lines, indicating that resistance signatures vary in the cell models as observed in patients. TFAP2A plays a pivotal role in cell growth. Lack of TFAP2A results in lower proliferation rates in untreated cells and a synergistic effect is observed with CTX, JQ1 and CTX + JQ1 therapies. TFAP2A loss reflected in increased migration ability in SCC1 and SCC25, while in SCC6 we observed less cell motility. However, in all cell lines EMT markers were up-regulated and suggests EMT development, due to down-regulation of TFAP2A, is a cell type and time dependent mechanism with specific cell types requiring more time to translate the transcriptional changes.Overall, we demonstrate that resistance-associated genes in HNSCC sensitive cells are epigenetically regulated. These mechanisms are also cell type and time dependent reflecting observations from patients’ cohorts. We show that TFAP2A regulates growth factor receptors and EMT genes in CTX sensitive cells and could be a potential target for drug development. We also show that the CTX + JQ1 combined therapy is a potential alternative and more effective approach while patients are sensitive to therapy.
Citation Format: Luciane T. Kagohara, Fernando Zamuner, Michael Considine, Genevieve Stein-O'Brien, Thomas Sherman, Daria A. Gaykalova, Elana J. Fertig. Transcriptional and epigenetic regulation of resistance markers in cetuximab sensitive HNSCC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3024.