RASSF1A independence and early galectin‐1 upregulation in PIK3CA‐induced hepatocarcinogenesis: new therapeutic venues

Abstract

Aberrant activation of the phosphoinositide 3-kinase (PI3K)/AKT/mTOR and Ras/mitogen-activated protein kinase (MAPK) pathways is a hallmark of hepatocarcinogenesis. In a subset of hepatocellular carcinomas (HCCs), PI3K/ AKT/mTOR signaling dysregulation depends on phosphatidylinositol-4,5bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutations, while RAS/MAPK activation is partly attributed to promoter methylation of the tumor suppressor Ras association domain-containing protein 1 (RASSF1A). To evaluate a possible cocarcinogenic effect of PIK3CA activation and RASSF1A knockout, plasmids expressing oncogenic forms of PIK3CA (E545K or H1047R mutants) were delivered to the liver of RASSF1A knockout and wildtype mice by hydrodynamic tail vein injection combined with sleeping beautymediated somatic integration. Transfection of either PIK3CA E545K or H1047R mutants sufficed to induce HCCs in mice irrespective of RASSF1A mutational background. The related tumors displayed a lipogenic phenotype with upregulation of fatty acid synthase and stearoyl-CoA desaturase-1 (SCD1). Galectin-1, which was commonly upregulated in preneoplastic lesions and tumors, emerged as a regulator of SCD1. Co-inhibitory treatment with PIK3CA inhibitors and the galectin-1 inhibitor OTX008 resulted in synergistic cytotoxicity in human HCC cell lines, suggesting novel therapeutic venues.

Identification of DUSP4/6 overexpression as a potential rheostat to NRAS-induced hepatocarcinogenesis

“…RASSF1A has been implied as a critical tumour suppressor in human hepatocarcinogenesis, where hypermethylation and inactivation of this gene frequently occur [18]. In accordance with our previous study, which did not show cooperativity with activating PIK3CA mutations [19], we could not find an increase in tumorigenesis resulting from NRAS G12V injections. Despite this, we were intrigued by the observation that the injection of NRAS G12V alone was sufficient to induce liver tumours (both in Rassf1a wildtype and knockout mice) after a latency as short as 3 months.…”

“…pT3-EF1α constituted the empty vector (EV) control. The control group's data have been published previously [19]. Purification of the plasmids was performed with the Endotoxin-free Maxi Prep Kit (Sigma-Aldrich, St.Louis, MO).…”

“…Note that the genetic background of Rassf1a WT and KO mice was C57BL/6 J x 129 Sv. As previously described [19], the control group of Rassf1a WT mice was generated by crossing Rassf1a KO mice with C57BL/6 J mice purchased from Charles River Laboratories (Sandhofer Weg, 97,633 Sulzfeld, Germany). Breeding conditions and PCR verification were identical to our report of Rassf1a KO with PIK3CA injections [19].…”

Chloroquine and cytosolic galectins affect endosomal escape of antisense oligonucleotides after Stabilin-mediated endocytosis

“… 42 Currently, OTX008 is being evaluated as a possible therapeutic against a variety of malignancies. 43 , 44 For our purposes, dosing our 190-HARE cells with 60 μM OTX008 significantly decreased Gal-1 expression while maintaining healthy cellular morphology ( Figure 8 A). Pretreatment of cells with 60 μM OTX008 for 3 days followed by 24 h treatment of ASO resulted in a further 50% reduction of malat-1 expression, suggesting that decreased Gal-1 expression results in more ASO escape into the cytoplasm ( Figure 8 B).…”

Smad3 Phospho-Isoform Signaling in Nonalcoholic Steatohepatitis

“…Recent experimental studies show increased lipogenesis in tumor cells based on an increase in two key up-regulators of lipogenesis, stearoyl-CoA desaturase and fatty acid synthase, as well as the appearance of a visibly lipid-rich phenotype among tumor cells, which were engorged with lipid droplets, as visualized by electron microscopy [ 130 ]. Such de novo lipogenesis is a hallmark of cancer.…”