Secretory NPC2 Protein-Mediated Free Cholesterol Levels Were Correlated with the Sorafenib Response in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the most common primary malignant tumor in the world. Sorafenib is the first-line drug for patients with advanced HCC. However, long-term treatment with sorafenib often results in reduced sensitivity of tumor cells to the drug, leading to acquired resistance. Identifying biomarkers which can predict the response to sorafenib treatment may represent a clinical challenge in the personalized treatment era. Niemann-Pick type C2 (NPC2), a secretory glycoprotein, plays an important role in regulating intracellular free cholesterol homeostasis. In HCC patients, downregulation of hepatic NPC2 is correlated with poor clinical pathological features through regulating mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) activation. This study aimed to investigate the roles of secretory NPC2-mediated free cholesterol levels as biomarkers when undergoing sorafenib treatment and evaluate its impact on acquired sorafenib resistance in HCC cells. Herein, we showed that NPC2 downregulation and free cholesterol accumulation weakened sorafenib’s efficacy through enhancing MAPK/AKT signaling in HCC cells. Meanwhile, NPC2 overexpression slightly enhanced the sorafenib-induced cytotoxic effect. Compared to normal diet feeding, mice fed a high-cholesterol diet had much higher tumor growth rates, whereas treatment with the free cholesterol-lowering agent, hydroxypropyl-β-cyclodextrin, enhanced sorafenib’s tumor-inhibiting ability. In addition, sorafenib treatment induced higher NPC2 secretion, which was mediated by inhibition of the Ras/Raf/MAPK kinase (MEK)/ERK signaling pathway in HCC cells. In both acquired sorafenib-resistant cell and xenograft models, NPC2 and free cholesterol secretion were increased in culture supernatant and serum samples. In conclusion, NPC2-mediated free cholesterol secretion may represent a candidate biomarker for the likelihood of HCC cells developing resistance to sorafenib.

Obesity and Gallstones

<b><i>Background:</i></b> The prevalence of obesity has been increasing globally and represents the main risk factor for the development of gallstone disease (GD). <b><i>Summary:</i></b> Excess body weight represents the main cause for the development of GD; nevertheless, there have been described multiple risk factors for its development, among them modifiable risk factors as diet, lifestyle, physical inactivity, and non-modifiable risk factors as ethnicity, female sex, advanced age, parity, and genetic mutations. Body mass index, abdominal perimeter, and waist-hip index have been used to determine the degree of adiposity of a person. Hence, central abdominal fat has been mostly associated with insulin resistance with the consequent increase in the hepatic cholesterol secretion; contributing as one of the multiple mechanisms associated with the development of gallstones. This disease has a low mortality; however, it has been associated with multiple diseases such as cardiovascular diseases, carotid atherosclerosis, metabolic associated fatty liver disease, and gallbladder cancer, probably because they share many of the risk factors. <b><i>Key Messages:</i></b> GD continues to be considered a disease with a high medical burden, in which it is sought to intervene in modifiable risk factors to reduce its development.

Molecular Regulation of Canalicular ABC Transporters

The ATP-binding cassette (ABC) transporters expressed at the canalicular membrane of hepatocytes mediate the secretion of several compounds into the bile canaliculi and therefore play a key role in bile secretion. Among these transporters, ABCB11 secretes bile acids, ABCB4 translocates phosphatidylcholine and ABCG5/G8 is responsible for cholesterol secretion, while ABCB1 and ABCC2 transport a variety of drugs and other compounds. The dysfunction of these transporters leads to severe, rare, evolutionary biliary diseases. The development of new therapies for patients with these diseases requires a deep understanding of the biology of these transporters. In this review, we report the current knowledge regarding the regulation of canalicular ABC transporters’ folding, trafficking, membrane stability and function, and we highlight the role of molecular partners in these regulating mechanisms.

MiRNA-223 Inhibits MouseGallstone Formation by Targeting Key Transporters in Hepatobiliary Cholesterol Secretion Pathway

BackgroudMiRNA-223 has previously been reported to play an essential role in hepatic cholesterol homeostasis by suppressing cholesterol synthesis, attenuating cholesterol uptake by hepatocyte from and promoting cholesterol efflux into the blood. However, its role in regulation of biliary cholesterol secretion and gallstone formation remains unknown.MethodsMice with conventional knockout (KO), hepatocyte-specific knockout (ΔHepa) / knockdown (KD) or gain expression of miRNA-223 were included in the study and were subjected to lithogenic diet (LD) for various weeks. The gallbladders were harvested and subjected to cholesterol crystal imaging and gallstone mass measurement. Liver tissues were collected for western blotting, RT-qPCR, and immunohistochemistry staining. Levels of cholesterol, bile salt, phospholipids, and triglyceride were determined in serum, liver tissues, and bile by enzyme color reactive assays. 3’ UTR reporter gene assays were used to verify the direct target genes for miRNA-223.ResultsLD-induced gallstone formation was remarkably accelerated in miRNA-223 KO, ΔHepa, and KD mice with concurrent enhancement in total cholesterol levels in liver tissue and bile. Key biliary cholesterol transporters ABCG5 and ABCG8 were identified as direct targets of miRNA-223. Reversely, AAV-mediated hepatocyte-specific miRNA-223 overexpression prevented gallstone progression with reduced targets protein expression.ConclusionThe present study demonstrates a novel role of miRNA-223 in the regulation of hepatic bile cholesterol secretion pathway and gallstone formation by targeting ABCG5 and ABCG8 expression. Therefore, elevating miRNA-223 would be a potentially novel approach to overcome the sternness of cholesterol gallstone disease.Graphical Abstract

ABCG5/G8: a structural view to pathophysiology of the hepatobiliary cholesterol secretion

The ABCG5/G8 heterodimer is the primary neutral sterol transporter in hepatobiliary and transintestinal cholesterol excretion. Inactivating mutations on either the ABCG5 or ABCG8 subunit cause Sitosterolemia, a rare genetic disorder. In 2016, a crystal structure of human ABCG5/G8 in an apo state showed the first structural information on ATP-binding cassette (ABC) sterol transporters and revealed several structural features that were observed for the first time. Over the past decade, several missense variants of ABCG5/G8 have been associated with non-Sitosterolemia lipid phenotypes. In this review, we summarize recent pathophysiological and structural findings of ABCG5/G8, interpret the structure-function relationship in disease-causing variants and describe the available evidence that allows us to build a mechanistic view of ABCG5/G8-mediated sterol transport.

Simultaneous Determination of Biliary and Intestinal Cholesterol Secretion Reveals That CETP (Cholesteryl Ester Transfer Protein) Alters Elimination Route in Mice

Objective: Determine the impact of CETP (cholesteryl ester transfer protein) on the route of cholesterol elimination in mice. Approach and Results: We adapted our protocol for biliary cholesterol secretion with published methods for measuring transintestinal cholesterol elimination. Bile was diverted and biliary lipid secretion maintained by infusion of bile acid. The proximal small bowel was perfused with bile acid micelles. In high-fat, high-cholesterol–fed mice, the presence of a CETP transgene increased biliary cholesterol secretion at the expense of transintestinal cholesterol elimination. The increase in biliary cholesterol secretion was not associated with increases in hepatic SR-BI (scavenger receptor BI) or ABCG5 (ATP-binding cassette G5) ABCG8. The decline in intestinal cholesterol secretion was associated with an increase in intestinal Niemann-Pick disease, type C1, gene-like 1 mRNA. Finally, we followed the delivery of HDL (high-density lipoprotein) or LDL (low-density lipoprotein) cholesteryl esters (CE) from plasma to bile and intestinal perfusates. HDL-CE favored the biliary pathway. Following high-fat feeding, the presence of CETP directed HDL-CE away from the bile and towards the intestine. The presence of CETP increased LDL-CE delivery to bile, whereas the appearance of LDL-CE in intestinal perfusate was near the lower limit of detection. Conclusions: Biliary and intestinal cholesterol secretion can be simultaneously measured in mice and used as a model to examine factors that alter cholesterol elimination. Plasma factors, such as CETP, alter the route of cholesterol elimination from the body. Intestinal and biliary cholesterol secretion rates are independent of transhepatic or transintestinal delivery of HDL-CE, whereas LDL-CE was eliminated almost exclusively in the hepatobiliary pathway.

Hdac3, Setdb1, and Kap1 mark H3K9me3/H3K14ac bivalent regions in young and aged liver

SummaryPost-translational modifications of histone tails play a crucial role in gene regulation. Here, we performed chromatin profiling by quantitative targeted mass spectrometry to assess all possible modifications of the core histones. We discovered a novel bivalent combination, a dually-marked H3K9me3/H3K14ac modification in the liver, that is significantly decreased in old hepatocytes. Subsequent genome-wide location analysis (ChIP-Seq) identified 1032 and 668 bivalent regions in young and old livers, respectively, with 280 in common. Histone H3K9 deacetylase Hdac3, as well as H3K9 methyltransferase Setdb1, found in complex Kap1, occupied bivalent regions in both young and old livers, correlating to presence of H3K9me3. Expression of genes associated with bivalent regions in young liver, including those regulating cholesterol secretion and triglyceride synthesis, is upregulated in old liver once the bivalency is lost. Hence, H3K9me3/H3K14ac dually-marked regions define a poised inactive state that is resolved with loss of one or both of the chromatin marks, which subsequently leads to change in gene expression.