HCC aetiology is multifactorial and

HCC aetiology is multifactorial and the disease is often preceded by other conditions including liver fibrosis and cirrhosis that are associated with HCV, HBV, alcoholic and non-alcoholic hepatitis. The discovery of new therapeutic targets will require a greater understanding of the pathogenic mechanisms underlying the tumorigenic process. Intrahepatic HCC metastases are common and the tumour microenvironment is considered to be pro-metastatic. Reports that elevated serum LPA associate with HCC tumour size and patient survival [11], along with resistance to chemotherapy and radiation-induced cell death [40], provide compelling evidence to consider the ATX-LPA axis as a therapeutic target for treating HCC [41]. The embryonic lethality of ATX null mice [42], [43], [44] raised questions on the suitability of ATX as a drug target. However, a recent report from Katsifa and colleagues showing that inducible, ubiquitous genetic deletion of ATX in adult mice, and long-term pharmacologic inhibition were well tolerated limits some of these concerns [45]. In summary, we demonstrate a role for ATX-LPA signalling in the HCV lifecycle, highlighting potential new targets for therapy and the prospect of stratifying therapies for treating viral-associated and non-associated HCC.
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Acknowledgements We thank Takaji Wakita for HCV J6/JFH, Jens Bukh for HCV SA13/JFH Charlies Rice for anti-NS5A mAb 9E10, Robert Thimme for Luc2a-JFH replicon cells and Samantha Lissaeur for differentiation protocols. Statistical analysis on mass spectrometric data reported in a was performed by Dr. Anne Segonds-Pichon at the bioinformatics facility at the Babraham Institute.
Adipocyte lyso-phospholipase D (lyso-PLD) catalyses the transformation of lyso-phosphatidylcholine (LPC) into lyso-phosphatidic colorimetric assay (LPA) . LPA is a bioactive phospholipid regulating a wide range of cellular responses (proliferation, survival, motility, ion flux, secretion) through the activation of G-protein-coupled receptors: LPA, LPA, and LPA, and two more distantly related receptors: LPA and LPA. Autotaxin has been purified from various sources such as fetal bovine serum , human plasma and from adipocytes . ATX catalyses and therefore possesses lyso-phospholipase D activity. ATX is an extracellular enzyme of 125kDa. It was first identified as a brain specific membrane glycoprotein belonging to the phosphodiesterase I gene family . There might be several isoforms of this enzyme , , . Recent findings have linked autotaxin and LPA with various physio-pathological processes such as adipocyte function regulation , and metastasis invasion . Less have been reported so far on the biochemical characteristics and on the pharmacology of this enzyme. LPA and sphingosine 1 phosphate have been reported to have inhibitory activity but no pharmacological applications because the manipulation of these phospholipids is difficult . Indeed, the reported inhibitors of ATX are derived from LPA or LPC , , . Furthermore, some of them have been reported as antagonists at the LPA receptors as well . Therefore, our goal in the present paper was to report on our first attempt to characterize autotaxin activity for small molecule inhibitors sensitivity, particularly towards the compounds active on the enzymes of the same family, i.e. phosphodiesterases. Furthermore, we also attempted to find inhibitors distinct from phospholipid derivatives. Since the core activity of phosphodiesterase enzymes is to recognize cAMP and to cleave it, we rationalized to screen compounds known to inhibit enzymes by acting at the level of their nucleotide-binding site, such as compounds inhibiting kinases, most of them being competitive to the ATP-binding site . Furthermore, such libraries of compounds are commercially available. To do so, recombinant human autotaxin was expressed in COS cells, purified and characterized for its lyso-PLD and phosphodiesterase activities. Its characteristics were compared with autotaxin purified from adipocyte-conditioned medium. A couple of new compounds were found to have inhibitory capacities toward autotaxin activity, particularly damnacanthal and hypericin, although these activities remain of poor potency: μM to mM range. The phosphodiesterase inhibitors tested herein were reported with activities toward at least one of the members of this family of enzymes with IC in the nanomolar range (see for details) exemplifying considerable differences in sensitivity toward inhibitors inside the phosphodiesterase family. Nevertheless, they could be used as scaffold to move towards more potent and selective inhibitors.