Avasimibe Dampens Cholangiocarcinoma Progression by Inhibiting FoxM1-AKR1C1 Signaling

Avasimibe is a bioavailable acetyl-CoA acetyltransferase (ACAT) inhibitor and shows a good antitumor effect in various human solid tumors, but its therapeutic value in cholangiocarcinoma (CCA) and underlying mechanisms are largely unknown. In the study, we proved that avasimibe retard cell proliferation and tumor growth of CCAs and identified FoxM1/AKR1C1 axis as the potential novel targets of avasimibe. Aldo-keto reductase 1 family member C1 (AKR1C1) is gradually increased along with the disease progression and highly expressed in human CCAs. From survival analysis, AKR1C1 could be a vital predictor of tumor recurrence and prognostic factor. Enforced Forkhead box protein M1 (FoxM1) expression results in the upregulation of AKR1C1, whereas silencing FoxM1 do the opposite. FoxM1 directly binds to promoter of AKR1C1 and triggers its transcription, while FoxM1-binding site mutation decreases AKR1C1 promoter activity. Moreover, over-expressing exogenous FoxM1 reverses the growth retardation of CCA cells induced by avasimibe administration, while silencing AKR1C1 in FoxM1-overexpressing again retard cell growth. Furthermore, FoxM1 expression significantly correlates with the AKR1C1 expression in human CCA specimens. Our study demonstrates a novel positive regulatory between FoxM1 and AKR1C1 contributing cell growth and tumor progression of CCA and avasimibe may be an alternative therapeutic option for CCA by targeting this FoxM1/AKR1C1 signaling pathway.

Hepatitis C virus infection mediates cholesteryl ester synthesis to facilitate infectious particle production

Cholesterol is a critical component of the hepatitis C virus (HCV) life cycle, as demonstrated by its accumulation within infected hepatocytes and lipoviral particles. To cope with excess cholesterol, hepatic enzymes ACAT1 and ACAT2 produce cholesteryl esters (CEs), which are destined for storage in lipid droplets or for secretion as apolipoproteins. Here we demonstrate in vitro that cholesterol accumulation following HCV infection induces upregulation of the ACAT genes and increases CE synthesis. Analysis of human liver biopsy tissue showed increased ACAT2 mRNA expression in liver infected with HCV genotype 3, compared with genotype 1. Inhibiting cholesterol esterification using the potent ACAT inhibitor TMP-153 significantly reduced production of infectious virus, but did not inhibit virus RNA replication. Density gradient analysis showed that TMP-153 treatment caused a significant increase in lipoviral particle density, suggesting reduced lipidation. These data suggest that cholesterol accumulation following HCV infection stimulates the production of CE, a major component of lipoviral particles. Inhibition of CE synthesis reduces HCV particle density and infectivity, suggesting that CEs are required for optimal infection of hepatocytes.

Abstract 66: SR-BI Mediates Cholesteryl Ester (CE) Sorting in Tetraspanin Microdomains with CD81 Facilitating CE Nano-endocytosis and CE Transport to Lipid Droplets

SR-BI functions as a neutral lipid transporter facilitating cholesteryl ester (CE) sorting, internalization and accumulation in LDs. The mechanism of CE transport remains obscure. We hypothesized that HDL lipid transport requires CD81 as a co-receptor and tetraspanin enriched domains (TED) for CE sorting, similar to hepatitis C uptake. Utilizing BODIPY-CE labeled recombinant HDL (rHDL/BP-CE) based on either human apoA-I or an apoA-I mimetic peptide, L-37pA, and confocal and TIRF microscopy, we found that after initially binding to SR-BI at the cellular filopodia and lamellipodia, HDL CE was laterally transported to the bases of filopodia, accumulating in 1-2 μm plasma membrane microdomains. While CE was rapidly internalized, neither apoA-I, L37pA nor SR-BI was, suggesting that these microdomains are an important part of the CE sorting mechanism. Internalized CE was transported in 50-100 nm CE-containing intracellular nano-vesicles. In a process of multiple collisions potentially involving transient nano-vesicle/LD fusion, BODIPY-CE was transferred to LDs. To identify CE-sorting microdomains, SR-BI expressing HeLa cells were transfected with GFP-CD81 or GFP-CD82. After a 1 h incubation with rHDL/BP-CE, CE was co-localized with both tetraspanin proteins demonstrating that these are TED (the observation was confirmed by anti-CD81 and -CD-82 antibody immunostaining). Following a 2 h chase, only GFP-CD81, not CD82, was found internalized and co-localized with CE transporting nano-vesicles. CE transport was not affected by inhibitors of de novo neutral lipid synthesis such as the ACSL-3 inhibitor, triacsin c, ACAT inhibitor, Sandoz 58-035 or ATGL inhibitor, A926500, indicating that CE was directly transported through a novel nano-endocytosis mechanism involving CD81 and not requiring CE hydrolysis and de novo synthesis. We conclude that TED function as CE sorting sites where CD81 helps to form nano-vesicles delivering CE to LDs. Further understanding of their function will help develop novel anti-atherogenic compounds accelerating reverse cholesterol transport.

Abstract 284: Cholesterol Handling in Bone Marrow--Derived Macrophages from Apoe-Deficient AKR and DBA/2 Mice

It has been demonstrated in humans that about 50% of the susceptibility to atherosclerosis is heritable. Therefore, it is not surprising that mice from distinct genetic backgrounds present different susceptibility to atherosclerosis. For example, apoE -/- DBA/2 mice have aortic root atherosclerosis lesion areas >10-fold larger than apoE -/- AKR mice. In order to better understand the mechanisms underlying this difference, we studied cholesterol metabolism in bone marrow-derived macrophages from these two strains. In the unloaded state, macrophages from both strains had equivalent amounts of total, esterified, and free cholesterol (TC, CE, and FC, respectively). Cells were loaded with acetylated low density lipoproteins (AcLDL) for 48h and DBA/2 macrophages had 36% higher TC (p<0.0001) vs. AKR macrophages, mainly due to 72% higher CE accumulation (p<0.0001). In contrast the loaded DBA/2 macrophages had 6% lower FC than the AKR macrophages (p<0.05). No difference was seen in AcLDL uptake or acetyl-coenzyme A acyltransferase (ACAT) activity between the two strains. When cells were loaded with AcLDL, the DBA/2 cells released cholesterol less efficiently than the AKR cells to apoAI (18% lower, p=0.01) or HDL (25% lower, p=0.001). However when the loading was performed in presence of ACAT inhibitor, blocking the formation of CE, the efflux from the two strains was equivalent suggesting a blockage of CE hydrolysis in DBA/2 cells. In loaded cells, when a 24h chase period was done in presence of ACAT inhibitor to prevent cholesterol re-esterification, the CE was reduce by 69% in AKR cells (t 1/2 = 47h) compared to chase without inhibitor while it was only reduced by 42% in DBA/2 cells (t 1/2 = 101h). Furthermore, when lalistat, an inhibitor of lysosomal acid lipase (LAL), and ACAT inhibitor were added during the chase, CE levels were equivalent to the one observed in chase without any inhibitors, suggesting that CE hydrolysis occurs primarily in the lysosome. In conclusion, our data support the hypothesis that DBA/2 cells accumulate more CE than AKR cells due to a defect in CE hydrolysis by LAL. Autophagy has been recently described as the pathway in macrophages by which lipid droplets can be delivered to the lysosome, and we are investigating the role of this pathway in our cells.