Targeting PDAC aggressiveness through AHR mediated epidermal-mesenchymal transition

Introduction: Aryl hydrocarbon receptor (AHR) is a transcription fac -tor that’s commonly upregulated in pancreatic ductal adenocarcinoma(PDAC). There are evidence that upregulated AHR increases cancer aggressiveness, invasiveness, migration and decreases survival rates. This can be linked to epithelial-mesenchymal transition, when cells acquire a migratory mesenchymal phenotype. AHR expression levels have been shown to influence epithelial or mesenchymal phenotype, however the exact mechanism is not yet know. EMT transcription factors (EMT-TF)(ZEB1, ZEB2, SNAI1, SNAI2, TWIST) can regulate EMT, but their relation-ship with AHR or the possibility to decrease PDAC migration and invasion by targeting AHR has not been widely studied. Aims & Methods: The aim of this study was to investigate correlations of AHR and EMT-TF RNA expression in PDAC tissue, patient outcomes and the effects of AHR modulation on PDAC cell migration and invasion. Pancreatic carcinoma tissue and next-to-cancer tissue samples were obtained from 86 patients who underwent partial duodenopancreatectomy (Whip-pleresection) for pancreatic carcinoma. The presence of cancerous tissue in the samples was confirmed histologically. Cancerous and next-to-cancer tissues were taken from the same patients. Next-to-cancer tissues were used as reference controls. Total RNA was extracted after which it was converted to cDNA and RNA expression was analyzed using real time polymerase chain reaction using AHR, GAPDH, ZEB1, ZEB2, SNAI1, SNAI2and TWIST primers. Expression changes were calculated using 2(-Delta Delta CT) method, correlations were calculated using Spearman method, Survival analysis was done using Log-Rank method. PDAC cells (BxPC-3;Su.86.86) were grown in RPMI medium with additives and treated for 24hours with AHR inhibitor (BAY) after which they were seeded for cell migration analysis using scratch assay and 3D spheroid assays and invasive-ness analysis using 3D spheroid method utilizing a basement membranematrix. Results: The results showed that AHR expression change in cancer com-pared to next-to-cancer tissue positively correlated with ZEB1 (r=0.801),SNAI1 (r=0.687) and SNAI2 (r=0.802) expression changes but did not correlate with ZEB2 or TWIST expression changes. Grouping the patient into AHR low (1/2 lowest AHR expression patients) and AHR high (1/2 highest AHR expression patients) showed similar results, with most EMT-TF relative expression being decreased compared to next-to-cancer tissue (AHR0.19-fold, ZEB1 0.15-fold, ZEB2 0.62-fold, SNAI1 0.12-fold, SNAI2 0.21-fold,TWIST – no change). In AHR high group most of EMT-TF relative expression was increased (AHR 11.7-fold, ZEB1 6.6-fold, ZEB2 – no change, SNAI112.4-fold, SNAI2, 67.2-fold, TWIST – no change). Survival analysis of AHR low and AHR high groups revealed a significantly higher survival rates in AHR low group (54 months median) compared to AHR high group (19months median). The results of PDAC cell line migration showed a statistically significant decrease (by 30-49 % depending on cell line and method)as well as decreased invasiveness (20-45 % depending on cell line).Conclusion: AHR plays a role in PDAC patient survival possibly through its involvement with a number of EMT-TF. AHR inhibition could prove a viable strategy in reducing PDAC aggressiveness through EMT process modulation.