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Abstract

It was shown previously that transiently activated tumour initiating cells (TICs) drove the progression and maintenance of the pancreatic ductal adenocarcinomas (PDACs). Previously, studies were performed to investigate the mechanisms and to identify potential regulatory genes of PDAC TIC activation. Three candidates for TIC activation were identified which named: guanine nucleotide-binding protein subunit beta 1 (GNB1), solute carrier family 35 member F5 (SLC35F5) and son of sevenless homolog 2 (SOS2). Preliminary validation experiments in immune-deficient mice were performed, and GNB1 was identified as a potential regulator of PDAC TIC activity.

This study aims at further 1) validating identified potential candidate of TIC regulators, 2) investigating the mechanisms of these candidate genes which induced TIC activation, and 3) identifing the binding partners and additional regulators of GNB1 in established PDAC cultures.

By using 10x Genomics Single Cell RNA sequencing technology, GNB1 is found heterogeneously expressed in the analyzed PDAC culture (n=1). β-arrestin signalling pathway was identified as the relevant signalling pathway correlating with high GNB1 expression. To investigate the mechanisms behind, the pathway alterations triggered by GNB1 were analyzed in normal pancreatic epithelial cells H6C7 with KRAS wild type and mutated background (n=2). The PI3K signalling pathway was identified as the most relevant signalling pathway. 3-phosphoinositide dependent protein kinase 1 (PDPK1), one of the critical molecules in the PI3K pathway showed a 6-fold increase of protein content in H6C7 KRAS wild type GNB1 overexpressed culture compared to non-overexpressed control, and 56-fold in mutated background. To investigate the binding partners and additional regulators of GNB1, GNB1 proteins were pulled down from primary PDAC cultures (n=3) and analyzed by mass-spectrometry. Guanine nucleotide-binding protein subunit gamma 12 (GNG12), guanine nucleotide- binding protein subunit alpha I1 (GNAI1), and potassium channel tetramerization domain containing 5 (KCTD5) were identified as the binding partners in primary PDACs and they were validated by co-immunoprecipitation.

To sum up, this study validated GNB1 as a PDAC TIC regulator. GNB1 is heterogeneously expressed in primary PDACs which strengthens the role as the tumour initiating cell regulator, rendering this as an interesting target for future studies. Moreover, the findings of GNB1 overexpressed H6C7 cells indicated that overexpressed GNB1 might induce PDAC TIC activation via the PI3K signalling pathway, and that PDPK1 was one critical downstream effector of GNB1. In addition, GNAI1, GNG12 and GNB1 as the combination of guanine nucleotide-binding protein subunits were characterized in primary PDAC cultures. Lastly, the findings in this study might provide a novel potential therapeutic target of pancreatic ductal adenocarcinoma in tumour initiating cell activation.