Abstract 1851: E6-associated protein-dependent estrogen receptor modulation of protein kinase A regulatory subunit R2A expression in neuroblastoma

Abstract

Abstract Purpose/Objective(s): Steroid hormone receptors, upon hormonal activation, act to modulate gene expression in receptive cells through interactions with coregulator proteins. Protein Kinase A (PKA) is a particularly potent downstream effector, and is conservatively regulated in a number of biochemical pathways. The current project aims to elucidate the mechanisms of action of E6AP, a known critical player in endocrine cancer on the expression/activity of PKA in neuroblastoma. Materials/Methods: A microarray genetic analysis yielded a decreased PKA-R2A expression dependent upon decreased levels of estradiol (E2) or E6AP. This correlation was validated via a PCR determined downregulation of PKA-R2A mRNA with E6AP knockdown in Neuro2a cells. Bioinformatics provided four potential Estrogen Response Element (ERE) sequences in the PKA-R2A promoter region. These sequences were cloned to a Luciferase reporter gene vector. HeLa cells harboring these constructs were incubated with and without E2. Transcriptional activity was surrogated by bioluminescence via a Luciferase-Luciferin reaction, normalized to Renilla luminal output. Electrophoretic mobility shift assay (EMSA) was performed to detect direct ER binding to biotin-labeled ERE in the presence and absence of E2. ER-specific antibodies generated a super-shift. Unlabeled and consensus ERE allowed for competitive binding. Chromatin immunoprecipitation (ChIP) investigated recruitment of the complete transcriptional complex E6AP-ER onto the promoter region of the PKA-R2A gene and its association with active expression of the gene in Neuro2a cells. Results: A Luciferase luminal output to unit Renilla luminal output ratio was obtained in the absence and presence of E2. The scrambled control yielded ratios of 2.77 and 1.97, respectively. The consensus control yielded 9.55 and 85.37. ERE-1 yielded significant ratios of 8.57 and 55.7. EMSA evaluation of ERE-1 demonstrated a biotin-labeled ERE band shift in the presence of ER incubated with E2 relative to that without E2, and a super-shift with addition of an Anti-ER antibody. Competitive saturation with both the unlabeled and consensus ERE sequences negated these observed shifts. ChIP results show, upon E2 treatment in Neuro2a cells, increased recruitment of the E6AP-ER complex to the same ERE-1 associated with increased H3K14 acetylation and p300 recruitment at the same site which was coupled with the recruitment of pRNAPolII to the transcriptional start site of the PKA-R2A gene indicating active transcription and expression of the gene. Conclusion: ERE sequences exist and appear to function in response to E2 within the PKA-R2A promoter region, modulating the transcriptional status of a ubiquitous and crucial protein. Novel therapies targeting this hormonal foundation may potentially play a role in ameliorating the symptomatic manifestations associated with neuroblastoma. Citation Format: Jean-Pierre Obeid, Nawal Zafar, Jimmy El Hokayem. E6-associated protein-dependent estrogen receptor modulation of protein kinase A regulatory subunit R2A expression in neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1851.