Cholesteryl Esters Modulate Lipid Droplet Rigidity and Monolayer Organization during Liver Cancer Progression

Macrophage foam cells are a defining pathologic feature of atherosclerotic lesions. Recent studies have demonstrated that at high concentrations associated with hypercholesterolemia, native LDL induces macrophage lipid accumulation. LDL particles are taken up by macrophages as part of bulk fluid pinocytosis. However, the uptake and metabolism of cholesterol from native LDL during foam cell formation has not been clearly defined. Previous reports have suggested that selective cholesteryl ester (CE) uptake might contribute to cholesterol uptake from LDL independently of particle endocytosis. In this study we demonstrate that the majority of macrophage LDL-derived cholesterol is acquired by selective CE uptake in excess of LDL pinocytosis and degradation. Macrophage selective CE uptake does not saturate at high LDL concentrations and is not down-regulated during cholesterol accumulation. In contrast to CE uptake, macrophages exhibit little selective uptake of free cholesterol (FC) from LDL. Following selective uptake from LDL, CE is rapidly hydrolyzed by a novel chloroquine-sensitive pathway. FC released from LDL-derived CE hydrolysis is largely effluxed from cells but also is subject to ACAT-mediated reesterification. These results indicate that selective CE uptake plays a major role in macrophage metabolism of LDL.

This study examined the molecular mechanisms underlying the roles of the microRNAs miR-18a and miR-25 in the progression of human liver cancer. Liver cancer biopsies obtained from early-stage liver cancer patients were examined by qRT-PCR and Northern blotting to examine the expression of miR-18a and miR-25. Both microRNAs were overexpressed in mouse primary hepatocytes following transfection of the cells with vectors encoding the microRNAs. An analysis of biopsy samples from liver cancer patients indicated that both miR-18a and miR-25 were overexpressed during the early stages of liver cancer. Further, qRT-PCR and Northern blotting confirmed that both of these microRNAs play crucial roles in the progression of liver cancer. Our findings clearly indicate that miR-18a and miR-25 can be used as prognostic biomarkers for early-stage liver cancer. Hence, miR-18a and miR-25 may have value as prognostic indicators and may facilitate the development of novel therapeutics for liver cancer.

The Wnt pathway, which controls crucial steps of the development and differentiation programs, has been proposed to influence lipid storage and homeostasis. In this paper, using an unbiased strategy based on high-content genome-wide RNAi screens that monitored lipid distribution and amounts, we find that Wnt3a regulates cellular cholesterol. We show that Wnt3a stimulates the production of lipid droplets and that this stimulation strictly depends on endocytosed, LDL-derived cholesterol and on functional early and late endosomes. We also show that Wnt signaling itself controls cholesterol endocytosis and flux along the endosomal pathway, which in turn modulates cellular lipid homeostasis. These results underscore the importance of endosome functions for LD formation and reveal a previously unknown regulatory mechanism of the cellular programs controlling lipid storage and endosome transport under the control of Wnt signaling.

Cellular cholesterol can be metabolized to its fatty acid esters, cholesteryl esters (CEs), to be stored in lipid droplets (LDs). With triacylglycerols (TGs), CEs represent the main neutral lipids in LDs. However, while TG melts at ~4 °C, CE melts at ~44 °C, raising the question of how CE-rich LDs form in cells. Here, we show that CE forms supercooled droplets when the CE concentration in LDs is above 20% to TG and, in particular, liquid-crystalline phases when the fraction of CEs is above 90% at 37 °C. In model bilayers, CEs condense and nucleate droplets when the CE/phospholipid ratio reaches over 10-15%. This concentration is reduced by TG pre-clusters in the membrane that thereby facilitate CE nucleation. Accordingly, blocking TG synthesis in cells is sufficient to strongly dampen CE LD nucleation. Finally, CE LDs emerged at seipins, which cluster and nucleate TG LDs in the ER. However, when TG synthesis is inhibited, similar numbers of LDs are generated in the presence and absence of seipin, suggesting that seipin controls CE LD formation via its TG clustering capacity. Our data point to a unique model whereby TG pre-clusters, favorable at seipins, catalyze the nucleation of CE LDs.

Lipid droplets (LDs) are ubiquitous and specialized organelles in eukaryotic cells. Consisting of a triacylglycerol core surrounded by a monolayer of membrane lipids, LDs are decorated with proteins and have myriad functions, from carbon/energy storage to membrane lipid remodeling and signal transduction. The biogenesis and turnover of LDs are therefore tightly coordinated with cellular metabolic needs in a fluctuating environment. Lipid droplet turnover requires remodeling of the protein coat, lipolysis, autophagy and fatty acid β-oxidation. Several key components of these processes have been identified in Chlamydomonas (Chlamydomonas reinhardtii), including the major lipid droplet protein, a CXC-domain containing regulatory protein, the phosphatidylethanolamine-binding DTH1 (DELAYED IN TAG HYDROLYSIS1), two lipases and two enzymes involved in fatty acid β-oxidation. Here, we review LD turnover and discuss its physiological significance in Chlamydomonas, a major model green microalga in research on algal oil.

To study macrophage lipid droplet composition and the effects of TG on cholesteryl ester (CE) physical state, hydrolysis, and cholesterol efflux, a technique was developed to remove the majority of accumulated TG with minimal effect on CE content. THP-1 macrophages were incubated with acetylated LDL, and the accumulated TG was depleted by incubation with the acyl-CoA synthetase inhibitor triacsin D in the presence of albumin. Before TG removal, all cellular lipid droplets were isotropic as determined by polarizing light microscopy. When the TG concentration was reduced, anisotropic lipid droplets were visible, indicating a change in physical state, and suggesting that TG and CE originally accumulated in mixed lipid droplets. This change in physical state of lipid droplets was associated with slower rates of CE hydrolysis and cholesterol efflux. Although lipid droplets within the same cell had a similar physical state after TG depletion, there was considerable variability among cells in the physical state of their lipid droplets.▪In conclusion, THP-1 macrophages store accumulated CE and TG in mixed droplets, and the proportion of CE to TG varies among cells. Reducing accumulated TG altered CE physical state, which in turn affected hydrolysis of CE and cholesterol efflux.—Lada, A. T., M. C. Willingham, and R. W. St. Clair. Triglyceride depletion in THP-1 cells alters cholesteryl ester physical state and cholesterol efflux. J. Lipid Res. 2002. 43: 618–628.

Lecithin:cholesterol acyltransferase (LCAT) is the major determinant of the cholesteryl ester (CE) content of high density lipoprotein (HDL) in plasma. The selective uptake of HDL-CE is postulated to participate in delivery of tissue-derived cholesterol both to the liver and steroidogenic tissues. Recent studies comparing mice with similarly low levels of HDL, due to the absence of either of the two major HDL-associated apolipoproteins apoA-I and apoA-II, suggest that apoA-I is crucial in modulating this process, possibly through interaction with scavenger receptor class B type I (SR-BI). Because of the central role of LCAT in determining the size, lipid composition, and plasma concentration of HDL, we have created LCAT-deficient mice by gene targeting to examine the effect of LCAT deficiency on HDL structure and composition and adrenal cholesterol delivery. The HDL in the LCAT-deficient mice was reduced in its plasma concentration (92%) and CE content (96%). The HDL particles were heterogeneous in size and morphology and included numerous discoidal particles, mimicking those observed in LCAT-deficient humans. The adrenals of the male Lcat (-/-) mice were severely depleted of lipid stores, which was associated with a 2-fold up-regulation of the adrenal SR-BI mRNA. These studies demonstrate that LCAT deficiency, similar to apoA-I deficiency, is associated with a marked decrease in adrenal cholesterol delivery and supports the hypothesis that adrenal SR-BI expression is regulated by the adrenal cholesterol.

Lipid droplets (LD) are organelles present in all cell types, consisting of a hydrophobic core of triacylglycerols and cholesteryl esters, surrounded by a monolayer of phospholipids and cholesterol. This work shows that LD biogenesis induced by serum, by long-chain fatty acids, or the combination of both in CHO-K1 cells was prevented by phospholipase A(2) inhibitors with a pharmacological profile consistent with the implication of group IVA cytosolic phospholipase A(2) (cPLA(2)alpha). Knocking down cPLA(2)alpha expression with short interfering RNA was similar to pharmacological inhibition in terms of enzyme activity and LD biogenesis. A Chinese hamster ovary cell clone stably expressing an enhanced green fluorescent protein-cPLA(2)alpha fusion protein (EGFP-cPLA(2)) displayed higher LD occurrence under basal conditions and upon LD induction. Induction of LD took place with concurrent phosphorylation of cPLA(2)alpha at Ser(505). Transfection of a S505A mutant cPLA(2)alpha showed that phosphorylation at Ser(505) is key for enzyme activity and LD formation. cPLA(2)alpha contribution to LD biogenesis was not because of the generation of arachidonic acid, nor was it related to neutral lipid synthesis. cPLA(2)alpha inhibition in cells induced to form LD resulted in the appearance of tubulo-vesicular profiles of the smooth endoplasmic reticulum, compatible with a role of cPLA(2)alpha in the formation of nascent LD from the endoplasmic reticulum.

Tumour are characterised by a high content of cholesteryl esters (CEs) stored in lipid droplets purported to be due to a high rate of intracellular esterification of cholesterol. To verify whether and which pathways involved in CE accumulation are essential in tumour proliferation, the effect of CE deprivation, from both exogenous and endogenous sources, on CEM-CCRF cells was investigated. Cholesterol synthesis, esterification and content, low-density lipoprotein (LDL) binding and high-density lipoprotein (HDL)-CE uptake were evaluated in cultured in both conventional and delipidated bovine serum with or without oleic or linoleic acids, cholesteryl oleate, LDL and HDL. High content of CEs in lipid droplets in this cell line was due to esterification of both newly synthesised cholesterol and that obtained from hydrolysis of LDL; moreover, a significant amount of CE was derived from HDL-CE uptake. Cell proliferation was slightly affected by either acute or chronic treatment up to 400 μM with Sz-58035, an acyl-cholesteryl cholesterol esterification inhibitor (ACAT); although when the enzyme activity was continuously inhibited, CE content in lipid droplets was significantly higher than those in control cells. In these cells, analysis of intracellular and medium CEs revealed a profile reflecting the characteristics of bovine serum, suggesting a plasma origin of CE molecules. Cell proliferation arrest in delipidated medium was almost completely prevented in the first 72 h by LDL or HDL, although in subsequent cultures with LDL, it manifested an increasing mortality rate. This study suggests that high content of CEs in CEM-CCRF is mainly derived from plasma lipoproteins and that part of CEs stored in lipid droplets are obtained after being taken up from HDL. This route appears to be up-regulated according to cell requirements and involved in low levels of c-HDL during cancer. Moreover, the dependence of tumour cells on a source of lipoprotein provides a novel impetus in developing therapeutic strategies for use in the treatment of some tumours.

Liver cancer is a malignant tumour with high morbidity and fatality rates that is common worldwide. At present, the clinical approaches to treating primary liver cancer include partial hepatectomy, systemic or local chemotherapy, radiotherapy, radiofrequency ablative surgery and liver transplantation. However, all of these approaches have shortcomings, including poor prognosis and numerous side-effects. A large number of studies have proven that many effective ingredients in traditional Chinese medicine, particularly the flavonoid compounds extracted from plants, have achieved breakthroughs in terms of enhancing the effects and reducing the toxicity of chemotherapy and radiotherapy, preventing tumour metastasis and relapse after surgery, alleviating the clinical symptoms of advanced tumours, improving the quality of life of the patient with tumours and extending patient long‑term survival. The purpose of the present study was to investigate the impact of isoquercitrin, the flavonoid from Bidens bipinnata L. extract, on the progression of liver cancer and to achieve a deeper understanding of the biological characteristics of isoquercitrin's involvement in the progression of liver cancer. In the in vitro experiments, isoquercitrin was found to strongly inhibit the proliferation of human liver cancer cells, promote the apoptosis of human liver cancer cells, and block the cell cycle in the G1 phase. Isoquercitrin activated caspase-3, -8 and -9, inhibited the expression level of ERK and p38MAPK protein phosphorylation, and promoted the phosphorylation of JNK. Additionally, isoquercitrin reduced the expression level of PKC in human liver cancer cells. In the in vivo experiments, isoquercitrin was also found to significantly inhibit the growth of transplanted tumours in nude mice. The present study confirmed that isoquercitrin could inhibit the progression of human liver cancer in vivo and in vitro, and the molecular mechanism of isoquercitrin may be closely associated with the MAPK and PKC signalling pathways.

Background: Metabolic disorders and chronic liver disease (CLD) play crucial roles in the development and progression of liver cancer (LC). Since the ethnic minority population increasingly suffers from CLD and LC, it is vital to understand the biosocial factors contributing to CLD and LC. The ’All of Us’ database, with significant participation from minority populations, provides a valuable tool for studies in different racial/ethnic groups. Using different databases, including the ‘All of Us’ and ‘The Cancer Genome Atlas’, this study aimed to understand the biosocial factors contributing to CLD and LC. Methods: Using ‘All of Us’ data, confounding factors like the lack of immunization, comorbidities, and socioeconomic status (SES) barriers were analyzed in a cohort of 33767 CLD [non-alcoholic fatty liver disease, alcoholic liver disease, and Hepatitis B and C] patients. Among the 556 LC patients in the ‘All of Us’ database, 92% had CLD. Since hypoxanthine is known to be increased in the urine of LC patients, purine metabolic pathway genes were analyzed using different databases and validated using publicly available RNASeq data. Results: We identified several confounding factors associated with CLD in Hispanic (HA) and African American (AA) populations compared to the non-Hispanic White (NHW) populations. HA and AA CLD patients suffer from high SES barriers. While most of the genes related to the purine metabolic pathway were upregulated in LC, xanthine dehydrogenase (XDH), which converts hypoxanthine to uric acid, showed a downregulation in the tumor compared to the normal tissues. The TCGA data among different racial/ethnic groups showed that only in Asian (AN) LC tumors the XDH expression was significantly lower compared to the NHW. The decreased XDH mRNA expression in AN LC compared to benign tissues was further validated using publicly available RNAseq datasets. Survival analysis confirmed poor overall survival among the AN LC patients with lower XDH expression in their tumors. Conclusions: Our study identified several confounding factors contributing to the minority CLD population. This study also identified decreased XDH expression as a critical metabolic alteration that has clinical significance in AN LC patients.

Liver cancer, the advanced stage of various chronic liver diseases, has garnered attention due to its high incidence and insidious progression. Lipid droplets (LDs), unique lipid storage organelles in hepatocytes, play a pivotal role in lipid metabolism. Lipophagy, a selective autophagy process initially identified in hepatocytes, regulates lipid homeostasis by selectively degrading LDs. This process offers a novel therapeutic avenue for addressing lipid metabolism disorders in liver cancer. This review highlights the regulatory role of lipophagy in liver cancer progression and its therapeutic potential. It elaborates on the molecular mechanisms underlying lipophagy-mediated LDs degradation and discusses the dual regulatory role of lipophagy in liver cancer. While lipophagy can suppress liver cancer development, under specific conditions, it may promote cancer cell proliferation, inhibit apoptosis, facilitate invasion and metastasis, and contribute to treatment resistance. Consequently, strategies targeting lipophagy for liver cancer prevention and therapy hold significant promise. These include interventions through traditional Chinese and Western medicine, as well as lifestyle modifications. This review evaluates current research, hotspots, and controversies in the field, aiming to provide innovative therapeutic strategies for liver cancer associated with abnormal lipid metabolism.

There is a growing belief that depression was positively associated with the progression of liver cancer. However, the driving molecular events behind the depression in liver cancer are poorly understood and need to be elucidated. Since hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis during depression leads to the excessive release of glucocorticoids (GCs), which suppress the activity of NK cells, we hypothesized that high levels of GCs during depression may inhibit function of tumor infiltrating NK cells during the progress of the liver cancer. Using chronic unpredictable mild stress (CUMS) induced depressed mice model, we showed that the progression of liver cancer was significantly accelerated in the depressed mice. The high levels of GCs were observed in both depressed mice and depressed liver cancer patients. Importantly, the expression of PD-1 on NK cells was specifically increased in the tumor microenvironment rather than that in blood or spleen. Co-culture studies demonstrated that the expression of PD-1 was significantly increased and cytotoxicity of NK92 cells was remarkably decreased by the dexamethasone treatment through PD-L1-dependent pathway. To our best knowledge we firstly found that PD-1/PD-L1 mediated exhaustion of infiltrated NK cells promoted hepatocellular carcinoma progression under depression and provided a novel strategy for GC-mediated anti-depressant therapy in liver cancer patients.

Simple SummaryNeuropilin-1 (NRP1) is a transmembrane protein which has had recently increased interest from cancer researchers. Liver cancer and colorectal cancer (CRC) are two of the most frequent and deadly tumors worldwide. Here, we assessed the prognostic, diagnostic and clinicopathological value of NRP1 in liver cancer and CRC patients by systematic searches in PubMed, Scopus, Web of Science, Embase and Cochrane Library and a meta-analysis. Results obtained showed that NRP1 overexpression was significantly correlated with lower survival in liver cancer patients and with tumor development in hepatocarcinoma patients, and high levels of NRP1 were strongly correlated with an increased risk of vascular invasion in liver cancer and metastasis in CRC and liver tumors. Therefore, these findings could establish novel interest of NRP1 as a useful biomarker for patient prognosis as well as for invasive-related characteristics in patients with liver cancer or CRC.Neuropilin-1 (NRP1) is a transmembrane protein involved in numerous cellular functions which has had increasing interest from cancer researchers. Liver cancer and colorectal cancer (CRC) are two of the most frequent and deadly tumors with a complex pharmacological framework. Here, we assessed the prognostic, diagnostic and clinicopathological value of NRP1 in liver cancer and CRC patients. We searched PubMed, Scopus, Web of Science, Embase and Cochrane Library databases for articles evaluating the NRP1 correlation with survival parameters, tumor development or clinicopathological features. Hazard ratios and odds ratios with 95% confidence intervals were extracted or estimated by Parmar method and pooled to evaluate the overall effect size with STATA 16 software. Heterogeneity was analyzed by chi-square-based Q test and I2 statistic, along with meta-regression and subgroup analysis, and publication bias was assessed by funnel plot asymmetry and Egger’s test. The study protocol was registered in PROSPERO (CRD42022307062). NRP1 overexpression was significantly correlated with lower survival in liver cancer patients and with tumor development in hepatocarcinoma patients, and was strongly correlated with an increased risk of vascular invasion in liver cancer and metastasis in CRC and liver tumors. These results support the role of NRP1 as a potential and useful biomarker in both types of cancer.

The expression of apolipoprotein A-V (apoA-V) in hepatoma cells results in homing of this protein to intracellular lipid droplets. When hepatoma cells transfected with a full-length apoA-V-green fluorescent protein fusion protein were cultured in medium that was not supplemented with oleic acid (OA), intracellular lipid droplet size and number were reduced compared with those of cells supplemented with OA. Confocal microscopy studies revealed that apoA-V associates with lipid droplets under both conditions. To define the structural requirements for apoA-V lipid droplet association, hepatoma cells were transfected with a series of C-terminal truncated apoA-V variants. Confocal microscopy analysis revealed that, in a manner similar to mature full-length apoA-V (343 amino acids), truncation variants apoA-V(1-292), apoA-V(1-237), and apoA-V(1-191) associated with lipid droplets, while apoA-V(1-146) did not. Western blot analysis of the relative abundance of apoA-V in cell lysates versus conditioned medium indicated that apoA-V variants associated with lipid droplets were poorly secreted while apoA-V(1-146) was efficiently secreted. Ultracentrifugation of conditioned medium revealed that, unlike full-length apoA-V, which associates with lipoproteins, apoA-V(1-146) was present solely in the lipoprotein-deficient fraction. Deletion of the N-terminal signal peptide from apoA-V resulted in an inability of the protein to be secreted into the medium, although it associated with lipid droplets. Taken together, these data suggest that the C terminus of apoA-V is essential for lipid droplet association in transfected hepatoma cells and lipoprotein association in conditioned medium while the signal peptide is required for extracellular trafficking of this protein.