Abstract 4836: Ly6-neurotoxin1 is a potential target in pancreatic ductal adenocarcinoma cells
Authors: Ali Doaa M. , Nedyalkova Nevena , Zepp Michael , Berger Martin R. 
Published: August 2019

Ly6/neurotoxin1 (Lynx1) is a cholinergic transmission modulator, which was described as a tumor suppressor in lung cancer. In addition, the expression of Lynx1 was downregulated in rat PDAC cells colonizing rat liver. As cholinergic signaling via the vagus nerve may slow pancreatic tumor progression, we investigated the role of Lynx1 in human PDAC cell lines.

Transient gene knockdown (KD) by siRNA was used to modulate the expression of Lynx1 in BXPC3 and Miapaca cells and KD was confirmed by qRT-PCR and western blot (WB). Resulting effects were determined by respective assays for proliferation, migration and colony formation. Induction of apoptosis was assessed by Hoechst and Annexin V-FITC staining. Induction of autophagy was determined by acridine orange and immunofluorescence (IF) staining for LC3b. Finally, we performed microarrays for gene expression in cells with Lynx1 KD and analyzed by WB cancer pathways of related signaling molecules.

Following successful KD of Lynx1, the response of Miapaca and BXPC3 cells was altered regarding proliferation (-60% and -10%), migration (+200% and -10%) at 72 h after transfection and colony formation (-50% and -40%) at 9 d after transfection. Hoechst staining revealed increased rates of apoptosis in both cell lines. This was confirmed by Annexin V assay in Miapaca cells and these changes were associated with significantly reduced BCL2 levels. Autophagy was increased in both cell lines as were LC3b levels detected by IF. Alterations in mRNA expression, as assessed by microarray, were analyzed by IPA software using a 1.5 fold cutoff. Miapaca cells showed significant activation of 43 pathways including phospholipase C signaling, cholecystokinin/gastrin mediated signaling, and cell cycle regulation. In addition, five pathways showed uniform downregulation, i.e estrogen mediated S phase entry, sirtuin signaling, small cell lung cancer signaling, cyclins and cell cycle regulation, and ataxia-telangiectasia-mutated (ATM) signaling. In contrast to the more sensitive Miapaca cells, BXPC3 cells showed modulation of few pathways only: activation of G protein beta gamma signaling and osteoarthritis pathways, as well as downregulation of the sirtuin signaling pathway. At protein level, the mTOR pathway was downregulated in both cell lines (including phosphorylated forms of mTOR, Rictor, Raptor, PRAS40), as were the upstream regulators PI3K and p-AKT.

In conclusion, KD of Lynx1 caused changes in gene product levels, which are related to cholinergic signal transmission as well as to DNA damage and repair systems. The observed decreased colony formation in Lynx1 KD cells is also indicative of defective cholinergic signaling. Reduced mTOR pathway signaling caused induction of autophagy. These findings suggest that Lynx1 is beneficial for regular cellular functions and its lack will contribute to increased apoptosis, DNA damage and autophagy.

Citation Format: Doaa M. Ali, Nevena Nedyalkova, Michael Zepp, Martin R. Berger. Ly6-neurotoxin1 is a potential target in pancreatic ductal adenocarcinoma 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 4836.