Abstract 2644: Histone deacetylase 6 plays a critical role in sulfiredoxin-mediated activation of the epidermal growth factor receptor signaling in human colorectal cancer

Mammalian cells develop various transmembrane receptors including the family of receptor tyrosine kinases (RTKs) to respond to extracellular stimuli. Epidermal growth factor receptor (EGFR) is the first identified member of the RTKs and it plays a pivotal role in colorectal cancer by stimulating cell proliferation and survival. Binding of extracellular ligands, mainly EGF, leads to the dimerization of EGFR that triggers the activation of downstream mitogen activated protein kinases. In addition to the amount of ligand binding, the strength and duration of EGFR signaling are also affected by the posttranslational modifications of the receptor itself, such as phosphorylation, ubiquitination, acetylation, etc. In the previous study, we demonstrated that Sulfiredoxin (Srx) enhances the EGFR signaling in colorectal cancer cells through the inhibition of receptor acetylation at lysine residue 1061. However, the molecular mechanism by which Srx regulates the level of EGFR acetylation is still unclear. In this study, we investigated (1) the mechanism of EGFR acetylation and its regulation by Srx; (2) the functional consequence of EGFR acetylation on EGF-induced receptor dimerization, recycling, trafficking and degradation. Human colon cancer cell lines were cultured and maintained in standard conditions. Western blotting, immunoprecipitation and Mass spectrometry were used to identify cysteine oxidation of HDAC6 and characterize enzymes that are involved in the acetylation of EGFR. Stable cells that overexpress or loss of target genes, such as Srx, HDAC6, EGFR and/or mutants, were established using the 3rd generation lentiviral ShRNA infection or the CRISPR-Cas9 system. The activity of histone deacetylases was determined using commercial kit in the presence/absence of pan- or isoform-specific chemical inhibitors of HDACs. Upon EGF stimulation, the dimerization, recycling, trafficking and degradation of EGFR were demonstrated through a combination of immunoblotting, flow-cytometry and immunofluorescent imaging. We demonstrated that HDAC6 plays a central role in the regulation of EGFR acetylation through its interaction with EGFR. Depletion of Srx in human colon cancer cells increases the levels of intracellular reactive oxygen species, leading to inhibition of HDAC6 activity through regulating the redox state of cysteines 407/417. Depletion of HDAC6 not only leads to increased level of EGFR acetylation but also suppresses the binding between EGFR and its adaptor proteins. EGF-induced receptor dimerization and recycling are altered in HDAC6 knockout cells, result in inhibition of EGFR downstream signaling. Taken together, this study indicates that Srx enhances the strength and duration of EGF signaling through the ROS-HDAC6-EGFR axis in human colon cancer cells. Citation Format: Qi Ying, Hong Jiang, B. Mark Evers, Qiou Wei. Histone deacetylase 6 plays a critical role in sulfiredoxin-mediated activation of the epidermal growth factor receptor signaling in human colorectal cancer [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 2644.