Abstract 2616: Stemness factors nanog and oct4 contribute to epithelial-to-mesenchymal transition and are predictive for outcome in esophageal adenocarcinoma
Authors: van der Zalm Amber Perenna , Dings Mark P. , Janssen Reimer , Bailey Peter , Koster Jan , Zwijnenburg Danny , Volckmann Richard , Waasdorp Cynthia , Blokhuis Jeroen , Oyarce César , Hooijer Gerrit , Meijer Sybren L. , Medema Jan Paul , van Laarhoven Hanneke W. , Bijlsma Maarten F. 
Published: June 2022

Abstract
Background and Aims: The incidence of esophageal adenocarcinoma (EAC) has increased six-fold in Western countries over the last decades, and 5-year survival rates remain low at 5-20%. While multimodality treatment strategies for curative treatment of esophageal cancer, including the CROSS regimen (chemoradiotherapy followed by surgery) have increased median overall survival, the majority of patients develop recurrences after several months. Epithelial to mesenchymal transition (EMT) has been recently shown by our group to be one of the major underlying mechanisms of resistance to therapy. Paradoxically, therapeutic pressure of effective therapies such as the CROSS regimen are found to instruct a mesenchymal, resistant phenotype in models for EAC. In this study, the aim was to delineate the heterogeneity for the propensity to undergo EMT after chemoradiation and which mechanisms underpin this propensity.
Methods: A panel of 8 EAC cell lines (5 primary and 3 ATCC cell lines) were treated with chemoradiotherapy and ranked by their propensity to undergo EMT, based on morphology when EMT occurred and protein marker expression. Next, the cell line panel as well as 44 pre-treated esophageal biopsies were RNA-sequenced. Expression data of the cell line panel were linked to their ranked in vitro EMT response. By means of Leave-one-out cross validation with Ridge Regression, EMT score prediction in pre-treated biopsies was validated. Gene expression profiles were related to clinical outcome data to identify markers that associated with propensity for EMT in patients.
Results: In the panel of in vitro EAC models, a strong heterogeneity was observed for the propensity to EMT after chemoradiation. For each marker, Ridge regression analysis identified the top 50 highly correlating genes. Combining all positively correlating genes of days to EMT, NCAD and ZEB1, known key transcription factors of pluripotency including NANOG and OCT4 emerged. Expression of NANOG and OCT4 in pre-treatment biopsies was highly predictive for response to neoadjuvant chemoradiation, occurrence of recurrences, and survival in patients. Genetic perturbation by knockout and inhibition of NANOG and OCT4 reduced the onset of EMT and sensitized cells for chemoradiation.
Conclusions: In conclusion, we were able to identify patients who are disproportionally prone to develop EMT in response to chemoradiation. Moreover, stemness factors NANOG and OCT4 are crucial regulators in plasticity of EAC and are promising predictive markers in pre-treatment biopsies of patients. By targeting NANOG and OCT4 in vitro, cells were sensitized to chemoradiation, holding promise for stemness inhibition to prevent therapy resistance in EAC.
Citation Format: Amber Perenna van der Zalm, Mark P. Dings, Reimer Janssen, Peter Bailey, Jan Koster, Danny Zwijnenburg, Richard Volckmann, Cynthia Waasdorp, Jeroen Blokhuis, César Oyarce, Gerrit Hooijer, Sybren L. Meijer, Jan Paul Medema, Hanneke W. van Laarhoven, Maarten F. Bijlsma. Stemness factors nanog and oct4 contribute to epithelial-to-mesenchymal transition and are predictive for outcome in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2616.