Effect of TNFα and RELA inhibition on gene expression in human PDAC cells

The canonical NF-κB transcription factor RELA is a master regulator of immune and stress responses and is upregulated in PDAC tumours. Here, we characterised previously unknown endogenous RELA-GFP dynamics in PDAC cell lines by live single cell imaging, which revealed rapid, sustained and non-oscillatory nuclear RELA following TNFα stimulation. Using Bayesian analysis of single cell datasets with variation in nuclear RELA, we predicted that RELA heterogeneity in PDAC cell lines is dependent on F-actin dynamics. Using RNA-seq, we identified the actin regulators NUAK2 and ARHGAP31 as transcriptionally regulated by RELA. In turn, NUAK2 or ARHGAP31 siRNA depletion downregulates TNFα-stimulated RELA nuclear localisation in PDAC cells, establishing a novel negative feedback loop regulating RELA activation by TNFα. We identify an additional actin-independent feedback loop involving RELB, which suppresses TNFα-mediated RELA nuclear localisation following RELA mediated upregulation of RELB. Taken together, we provide computational and experimental support for interdependence between the F-actin network and RELA translocation dynamics in PDAC. To study the role of TNFα-activated RELA on gene expression in PDAC cells, we introduced doxycycline-inducible lentiviral IκB-SR (RELA inhibitor) into the human PDAC cell lines MIA PaCa2 and PANC1. We performed RNA-seq analysis on cells treated with TNFα at 0, 0.01, 0.1 or 10 ng/ml in the presence or absence of IκB-SR induction.