Autotaxin (ATX/ENPP2) is a secreted phospholipase D enzyme that converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA). LPA is the agonist for at least six G protein coupled receptors whose activation stimulates cell proliferation, survival and migration. Overexpression of ATX and increased levels of LPA have been linked to fibrotic and inflammatory diseases and to several cancers. ATX is a therapeutic target and the ATX inhibitor IOA-289 is currently in Phase 1B clinical trials for the treatment of patients with metastatic pancreatic cancer, in combination with gemcitabine.

ENPP2 is overexpressed in both pancreatic adenocarcinoma (PDAC) and hepatocellular carcinoma (HCC). In HCC, ATX is produced by both the tumour microenvironment (TME) and tumour cells and our data show that ATX signalling can drive tumour cell growth in an autocrine manner. In contrast, in PDAC the TME primarily expresses ATX. The cell lines, PANC-1 and MIA PaCa-2 and the cancer associated fibroblast (CAF) derived cell line, 0082T, were used as a model to explore ATX signalling in PDAC. Consistent with publicly available mRNA expression data, 0082T cells, but not the PDAC cancer cells, secrete ATX and its substrate LPC. Conditioned media from 0082T cells increased the growth of both PANC-1 and MIA PaCa-2 cells. Treatment of 0082T CAF cells with IOA-289 during media conditioning reduced PDAC cell growth, without impacting CAF cell numbers.

Lipidomic analysis showed IOA-289 and PF8380, another ATX inhibitor, reduce the generation of LPA in 0082T CAF conditioned media. 22:6 LPA was shown to be generated by an ATX independent pathway, and production of 22:6 LPA was upregulated upon ATX inhibition. Additionally, 22:6 LPA accumulates in conditioned media when 0082T, PANC-1 and MIA PaCa-2 cells are present, but species of LPA generated by ATX are specifically degraded (16:0,16:1 18:0,18:1). Cell surface lipid phosphate phosphatases (LPPs) dephosphorylate LPA to produce monoacylglycerol. This presents the first evidence for LPA species substrate specificity of LPPs and suggests LPPs specifically regulate ATX generated species of LPA. RNA-seq analysis of ATX inhibitor and DMSO treated CAF cells revealed significantly altered expression of genes encoding extracellular proteins, such as CTGF. A combination of modulating the mitogenic CAF secretome and a reduction in LPA is proposed as the mechanism of ATX inhibition in regulating CAF-driven PDAC growth.

ATX has non-catalytic functions that could be mediated by specific protein-protein interactions. An HRP-based proximity labelling method was optimised for investigating the extracellular protein interactors of ATX. Several potential interactors are identified, including fatty acid binding protein 5 (FABP5). Modelling with AlphaFold-Multimer indicates FABP5 may interact with ATX at the opening to ATX’s lipid binding active site. Further investigation will be required to validate this idea.