TLR2 promotes tumour proliferation in KRAS-dependant PDAC in the absence of ARF6

Metastasis is responsible for nearly 90% of all cancer-related deaths. Despite global efforts to prevent aggressive tumours, cancers such as pancreatic ductal adenocarcinoma (PDAC) are poorly diagnosed in the primary stage, resulting in lethal metastatic disease. RAS mutations are known to promote tumour spread, with mutant KRAS present in almost 90% of cases. Until recently, mutant KRAS remained untargeted and, despite the recent development of inhibitors, results show that tumour cells develop resistance. Another strategy for targeting mutant KRAS-dependent PDAC metastasis may come from targeting the downstream effectors of KRAS. One such axis, which controls tumour proliferation, invasiveness and immune evasion, is represented by ARF6-ASAP1. Here we show that targeting ARF6 results in adaptive rewiring that can restore proliferation and invasion potential over time. Using time-series RNA and ATAC sequencing approaches, we identified TLR-dependent NF?B, TNFa and hypoxia signalling as key drivers of adaptation in ARF6-depleted KRAS-dependent PDAC. Using in vitro and in vivo assays, we show that knocking down TLR2 with ARF6 significantly reduces proliferation, migration and invasion. Taken together, our data shed light on a novel co-targeting strategy with the therapeutic potential to counteract PDAC proliferation and metastasis. Overall design: To understand the adaptive landscape of PDAC when KRAS-downstream signalling nodes at different hierarchical levels are perturbed, we treated MIA PaCa-2 cells with shRNAs targeting either ARF6 or ASAP1, and monitored them for a prolonged period of up to 21 days. We performed time-resolved RNA sequencing (RNA-seq) for cells treated with shNT, shARF6 or shASAP1 at time points Day 3, 7, 14 and 21