Ly6-Neurotoxin1 Knockout In Pdac Cells Inhibits Their Growth In Vitro And In Vivo

Ly6/neurotoxin1 (Lynx1) functions as a brake for nicotinic receptors and was defined as a tumor suppressor in lung cancer. As pancreatic cancer development may be slowed down by cholinergic signaling, we investigated the role of Lynx1 in pancreatic ductal adenocarcinoma (PDAC) cell lines, both in vitro and in vivo. Lynx1 knockout cell clones were generated by transfecting the CRISPRCas9 plasmid - (pSpCas9 (BB)-2A-Puro) into Miapacaluci and BXPC3luci PDAC cells using jet Pei and jet Prime as transfecting agents, respectively. The annealed gRNA was directed towards exon 3 of the Lynx1 gene. Control clones were transfected with the plasmid alone. Clones from both cell lines were tested by qRT-PCR and Western blot for knockout efficiency as well as by proliferation assay. In addition, the expression levels of p-MEK, p-MAPK, p-mTOR and p-Rictor were verified in two BXPC3 cell clones in relation to Lynx1 expression. Furthermore, 4x106 cells of four BXPC3 cell clones (one control, 3 knockout clones) were injected, respectively, into the portal vein of nude rats to control for a possibly reduced tumor growth in the liver. The knockdown of Lynx1 was incomplete at mRNA level and ranged from 20 to 60% in BXPC3 clones and from 0 to 30% in Miapaca clones. At protein level, the respective values ranged from 0 to 90% in BXPC3 and from 22 to 50% in Miapaca clones. However, Lynx1 protein levels increased at later time points. All BXPC3 clones proliferated less quickly than the respective control when tested by MTT assay. Miapaca clones, however, didn9t show a significant difference from the respective control, although their growth was clearly disturbed shortly after transfection. Concomitantly with reduced Lynx1 protein levels, there was reduction of p-mTOR (90%), p-Rictor (30%) and p-MEK (40%) in BXPC3 cells. In vivo, the BXPC3 clones showed a lag period of 1 to 2 weeks till the appearance of a first bioluminescence signal indicating tumor growth. Rats injected with cells from the control clone showed a steady increase in the bioluminescence signal (n=7 of 10) as compared to the most sensitive knockdown clone, which didn9t show any signal in any of 4 injected rats (p=0.05). Cells of 2 other BXPC3 clones showed a reduced growth rate at best in 2 of 2 rats used for each clone, respectively. In conclusion, knockout of Lynx1 was incomplete at both mRNA and protein levels. Nevertheless, the respective BXPC3 clones exhibited reduced proliferation in vitro, which was associated with diminished p-mTOR, p-Rictor and p-MEK levels. In addition, they failed to establish a tumor in vivo or showed a reduced tumor growth rate. These findings suggest that Lynx1 is a vital gene and may play an important role in the growth and establishment of PDAC cells. Citation Format: Doaa Ali, Michael Zepp, Matthias Bozza, Maria Nikolova, Richard Harbottle, Martin R. Berger. Ly6-neurotoxin1 knockout in PDAC cells inhibits their growth in vitro and in vivo [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4717.