Recent insights into the pathogeneses and therapeutic targets of liver diseases: Summary of the 4th Chinese American liver Society/Society of Chinese Bioscientists in America Hepatology Division Symposium in 2021

Abstract

The 4th Chinese American Liver Society (CALS)/Society of Chinese Bioscientists in America (SCBA) Hepatology Division Annual Symposium was held virtually on October 29–30, 2021. The goal of the CALS Symposium was to present and discuss the recent research data on the pathogenesis and therapeutic targets of liver diseases among the CALS members, trainees and invited speakers. Here we briefly introduce the history of the CALS/SCBA Hepatology Division and highlight the presentations that focus on the current progresses on basic and translational research in liver metabolism, bile acid biology, alcohol-related liver disease, drug-induced liver injury, cholestatic liver injury, non-alcoholic fatty liver disease/non-alcoholic steatohepatitis and liver cancer.

1. Introduction

The liver is a vital metabolic organ for the regulation of plasma protein synthesis, gluconeogenesis and glycogen storage, cholesterol metabolism, and bile acid synthesis, as well as drug metabolism and detoxification. Liver diseases, particularly chronic liver diseases, are a growing cause of morbidity and mortality worldwide, which accounts for approximately 2 million deaths per year worldwide, with half due to complications of cirrhosis and another half, viral hepatitis and hepatocellular carcinoma (HCC).^1,2 Owning to the prevalence of overnutrition and obesity, non-alcoholic fatty liver disease (NAFLD) has become one of the most prominent causes of liver diseases worldwide, affecting one quarter of the world population, and will probably emerge as the leading cause of end-stage liver disease affecting both adults and children.³ The high prevalence of the metabolic liver disease and the lack of effective treatment ask for more collaborations among scientists and clinicians from different disciplines through exchange of ideas, working together and learning from each other. Scientific societies and scientific meetings provide some of the most effective platforms to facilitate such interactions and collaborations.

One of the largest meetings is the Experimental Biology (EB), which is held annually and participated by more than 20,000 researchers from all over the world. It is organized by multiple scientific societies in the United States (US), including the American Society of Investigative Pathology (ASIP), American Society for Pharmacology and Experimental Therapeutics (ASPET) and American Society of Biochemistry and Molecular Biology (ASBMB). This large interdisciplinary meeting offers great opportunities for researchers affiliated with different societies but with common interests to share their findings and to learn from each other. Several Chinese American researchers working in the liver field from different universities met regularly during this meeting. As many of these interactions occurred after the meeting and at the dinner table, a “Dinner Club” was organized by Dr. Xiao-Ming Yin (then Indiana University, now at Tulane University), and Dr. Wen-Xing Ding (University of Kansas Medical Center). Early participants include Drs. Min You (University of South Florida), Li Wang (University of Connecticut), Bin Gao (National Institutes of Health), Huiping Zhou (Virginia Commonwealth University), Cynthia Ju (then University of Colorado, now University of Health Science Center at Houston), Mengwei Zang (then Boston University, now University of Texas Health San Antonio) and Grace L. Guo (Rutgers University). The “Dinner Club” grew quickly from a few people to dozens. However, these activities often diminished when the EB meetings ended. A critical change occurred when several of these investigators met at another large annual international meeting in early 2010's, which is organized by the American Association for the Study of Liver Diseases (AASLD), also known as The Liver Meeting, where more than ten thousands of participants from the world meet. WeChat, a popular messaging platform launched in January 2011, was caught the attention by the member of the Dinner Club as the way to maintain the interaction even after the meeting. Dr. Xiao-Ming Yin started the WeChat Group “Tan Tian Shuo Gan” around 2013–2014 during a gathering at the AASLD meeting, which quickly grew from the initial dozens of participants to more than 400 people from the US, China and other countries and regions.

The WeChat group became the ideal platform to connect liver researchers from different places together. Many active members expressed the desire to formalize the scientific activities in the form of a scientific society. Following the discussions in 2015 AASLD meeting and 2016 EB meeting, a growing consensus was reached to establish a society for Chinese American liver investigators and hepatologists in North America with a goal to exchange and discuss the latest scientific progresses from basic liver biology to clinical management of liver diseases, and furthermore, to foster collaborations to advance liver-related research and clinical translation. Subsequently, a planning committee was formed. Several names for this society were proposed and discussed in the WeChat Group, which eventually evolved into the name “Chinese American Liver Society (CALS)”. Soon a call for the society logo was sent to the member of the group and several interesting designs emerged. The one designed by Dr. Xiao-Ming Yin was adopted, which is based on the 4 letters of CALS that form the shape of the liver (Fig. 1). Both the name and the logo have been in use since 2016.

Fig. 1.

The logo of Chinese American Liver Society (CALS). Courtesy of Dr. Xiao-Ming Yin.

The WeChat group provides the platform for many of the discussions and for the organization of some activities during the EB meetings and AASLD meetings. In particular a meeting was held during 2017 AASLD annual meeting to discuss how CALS could develop its scientific activities beyond the virtual social platform through international collaborations. However, there were many challenging logistic issues for establishing a professional society such as the membership service, registration as a non-profit organization with the Internal Revenue Service (IRS), and operation of website that promotes the society. With their experiences in working with the well-established Society of Chinese Bioscientists in America (SCBA), Drs. Bin Gao and Xin Wei Wang (National Institutes of Health, NIH) proposed and facilitated the association of CALS in early 2018 with SCBA, to become the affiliated Hepatology Division (https://scbasociety.org/hepatology-division). SCBA is a non-profit organization founded in 1984 and currently has approximately 2000 members from various universities, academies, medical institutions, and biotechnology and pharmaceutical companies. A supplemental bylaw is crafted for the Division of Hepatology/CALS with Dr. Xiao-Ming Yin providing the first draft and finalized and approved by the members in February 2019. The mission of CALS is specified to promote collaborations and interactions among basic scientists and hepatologists in basic research, pre-clinical and clinical studies, and to help development of novel therapies related to liver diseases. CALS also aims to help the training and professional development opportunities for the next generation of scientists and hepatologists.

With the support of SCBA, CALS formally launched its activity by organizing Annual Symposiums that open to all who are interested in. On November 9, 2018, CALS successfully held the first CALS Annual Symposium at the Doubletree Hotel of San Francisco, California with approximately 100 attendants including senior and junior investigators, postdoctoral fellows and graduate students (Fig. 2). The symposium was organized by the inaugural president Dr. Bin Gao with the assistance of Dr. Li Wang (secretary) and Dr. Wen-Xing Ding (treasurer). Dr. Jake Liang from NIH/NIDDK, who is also a past president of AASLD, delivered a keynote talk entitled “An unexpected journey: Insights from hepatitis C virus infection to general human biology”. On November 7, 2019, the second CALS Annual Symposium was successfully held at the Embassy Suite in Boston, Massachusetts, which was organized by the president Dr. Xiao-Ming Yin with the assistance of Dr. Mengwei Zang (secretary), and Dr. Wen-Xing Ding (treasurer) (Fig. 3). Over sixty of registrants and invited guests attended the meeting in Boston. Many liver researchers and clinicians as well as postdoctoral fellows and graduate students from the US and China were invited to the Boston meeting. Dr. John Y.L. Chiang from Northeast Ohio Medical University delivered a keynote lecture on bile acid receptors in liver metabolism and diseases.

Fig. 2.

First Chinese AmericanLiverSociety (CALS)AnnualSymposium on November 9, 2018 at theDoubletreeHotel of San Francisco, California. Courtesy of Dr. Yuan Li.

Fig. 3.

Second Chinese AmericanLiverSociety (CALS)AnnualSymposium on November 7, 2019 at theEmbassy Suite in Boston, Massachusetts. Courtesy of Dr. Yuan Li.

Due to the pandemic of coronavirus disease in 2019, the 3rd CALS Annual Symposium was successfully held online on October 30–31, 2020. The meeting was organized by Dr. Huiping Zhou (president) and assisted by Dr. Yanqiao Zhang (secretary, Northeast Ohio Medical University) and Dr. Wen-Xing Ding (treasurer) with the participation of other executive council members. The zoom platform provided the convenience to allow more people to attend with a very successful campaign by the organization committee. Over 200 registrants and invited guests from North America and China attended this event. The participants included basic, translational, and clinical experts, junior faculty, and post-doctoral/doctoral trainees. The keynote speaker was Dr. Shelly Lu from Cedars-Sinai Medical Center at Los Angeles, who presented her works on the role of methionine adenosyltransferase in HCC. For the first time, the CALS Organizing Committees reviewed a total of 79 submitted abstracts and selected 27 abstracts for oral presentations and 34 for poster presentations in this symposium. Thirty-three investigators and trainees received the Junior Investigator Research Award, Postdoctoral Fellow Research Award, and Graduate Student Research Award. In addition, 9 trainees received the Best Poster Presentation Award.

The 4th CALS Annual Symposium was successfully held virtually on October 29–30, 2021. Over 190 registrants and invited speakers from North America and China attended this exciting event. The participants were composed of faculty, postdoctoral fellows and graduate students from both basic and clinical science disciplines. The meeting was organized by Dr. Xin Wei Wang (president) and assisted by Dr. Yanqiao Zhang (secretary), Dr. Wen-Xing Ding (treasurer) and Dr. Lisa Zhang (co-treasurer, University of Kansas Medical Center) with the participation of other executive council members. The organizers received a total of 73 abstracts. Twenty-nine trainees were presented either Postdoctoral Fellow Research Award or Graduate Student Research Award. In addition, 5 trainees were selected by Session Chairs to receive the Best Poster Presentation Award. There were two keynote speakers in this symposium. Dr. Anna Lok is the assistant dean for clinical research at University of Michigan and a past president of AASLD. In her lecture, Dr. Lok elegantly summarized the current global status of hepatitis B and hepatitis C, and their impact on the development of liver cancer. The second keynote speaker, Dr. Hao Zhu, a well-respected pediatric hepatologist at the University of Texas Southwestern Medical Center, discussed a fascinating topic on the heterogeneity and “mosaic” hepatocyte populations in the liver in response to liver injury, regeneration, and liver cancer.

In addition to the two keynote lectures, the 4th CALS Annual Symposium had several major sessions that covered basic liver physiology and basic and translational research on liver diseases, including lipid metabolism, liver injury, inflammation, and liver cancer. The followings are the summaries of six sessions that addressed different aspects of liver biology and liver diseases.

2. Alcohol-related liver disease (ALD)

ALD is a major cause of global liver related morbidity and mortality, which accounts for 1% all deaths worldwide with 23.6 million of alcohol-associated compensated cirrhosis and 2.5 million decompensated cirrhosis.⁴ Decades of research has enriched our understanding on the pathogeneses of ALD that include hepatic steatosis, inflammation and fibrosis, which further progress to cirrhosis and liver cancer.5, 6, 7 Despite of extensive ALD research, no effective pharmacological treatments are currently available to treat ALD patients. In the 4th CALS Annual Symposium, many exciting talks related to ALD were presented, which revealed some potential therapeutic targets on liver inflammation, lipid homeostasis, organelle homeostasis, autophagy signaling, noncoding RNA (microRNA, miR), and gut-liver axis to improve the pathogenesis of ALD.

The role of gut-liver crosstalk in ALD has been well appreciated.⁸ Toll-like receptors (TLRs) play critical roles in innate immunity, and several of TLRs including TLR2, TLR4, and TLR9 have been implicated in the development of ALD. A study from Dr. Ekihiro Seki's group demonstrated that 1Z1, a synthetic ligand of TLR7, protected against liver injury in a mouse model of alcoholic hepatitis (AH).⁹ Oral administration of 1Z1 prevented alcohol-mediated gut leakage and bacterial translocation without noticeable systemic side effects. Mechanistically, 1Z1 increased interleukin-22 (IL-22) production from the intestinal immune cells. These findings suggest that 1Z1 is a promising drug for treating ALD patients. Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that is critical for xenobiotics, dietary, microbial, and metabolic cues. A study from Dr. Lirui Wang's group showed that intestinal epithelial cell (IEC)-specific Ahr knockout mice were more susceptible to alcohol-induced liver injury.¹⁰ Lack of AHR in IEC altered the intestinal metabolite composition and promoted the overgrowth of Helicobacter hepaticus and Helicobacter ganmani in the gut as well as their translocation to mesenteric lymph nodes and liver, resulting in more liver injury. More importantly, administration of AHR agonists, 6-formylindolo[3,2-b]carbazole and indole-3-carbinol, protected against ALD in mice by specifically activating intestinal Ahr without affecting liver Ahr function. Probiotics have been shown to improve alcohol-induced gut leakiness and endotoxemia and liver injury in animal models.¹¹ In an unpublished study from Dr. Wenke Feng's group, they showed that probiotic Lactobacillus rhamnosus GG-derived exosome-like nanoparticles (LDNPs) protected against ALD by modulation of intestinal miR-194-farnesoid X receptor (FXR)-fibroblast growth factor (FGF)15-mediated bile acid synthesis pathway.

Mitochondria damage and adaption have also been implicated in the pathogenesis of ALD in both experimental ALD models and human ALD samples.^12,13 Alcohol consumption affects hepatic mitochondrial dynamics resulting in the accumulation of megamitochondria.^12,14 However, the role of hepatic megamitochondria in mitochondrial homeostasis and alcohol-induced liver injury is not clear. Moreover, how alcohol causes accumulation of megamitochondria is also not known. One simple hypothesis is that large mitochondria may be difficult to be removed by mitophagy, a selective autophagy process that helps eliminate damaged mitochondria and protects against ALD.^15,16 In an unpublished study from Dr. Wen-Xing Ding's group, they reported that hepatic expression of Drp1, the key GTPase protein that regulates mitochondrial fission, was downregulated in both alcohol-fed mice and human alcoholic hepatitis. Liver-specific deletion of Drp1 in mice led to accumulation of megamitochondria and exacerbated alcohol-induced liver injury by inhibiting mitophagy and promoting mtDNA-mediated cGAS-STING-IRF3/7 activation.

There were several other presentations in the symposium that reported additional interesting mechanisms underlying alcoholic liver injury including an important role of stress-responsive gene FK506-binding protein 5 (FKBP5)-YAP-TEAD1-CXCL1 axis, patatin-like phospholipase domain containing 3 (PNPLA3) variant, secreting receptors, cell senescence, serine-arginine protein kinase 2-mediated lipogenesis in hepatocytes, sphinogosine kinase 2 deficiency and TFEB in the pathogenesis of ALD and alcohol-associated liver cancer.¹⁷ In summary, these findings presented in the 4th CALS Annual Symposium provided novel molecular mechanisms underlying the pathogeneses of ALD and potential therapeutic targets for the treatment of ALD.

3. Drug-induced liver injury (DILI)

Liver is the major site for the metabolism of drugs and xenobiotics. Drugs can be harmful to the liver when patients are exposed to toxic doses that lead to DILI.¹⁸ Among DILIs, acetaminophen (APAP, also called paracetamol) overdose is one of the most common DILIs, which leads to more than 78,000 emergency visits and 33,000 hospitalizations in the US.¹⁹ At the therapeutic dose, APAP is safe but overdose of APAP can lead to hepatocyte necrosis due to the mitochondria damage triggered by the APAP metabolites and protein adducts in the early injury phase.²⁰ Following the early injury phase, several adaptive events occur in the liver including autophagy, senescence, the innate immune response, and blood coagulation events, which may either improve or exacerbate the recovery phase of the APAP-induced liver injury (AILI).21, 22, 23, 24 It has been well documented that depletion of hepatic glutathione (GSH) and formation of mitochondrial APAP-adducts are early events that trigger mitochondrial damage and necrosis after the APAP overdose. In a study conducted by Dr. Lisa Zhang's group, they found that liver-specific deletion of small heterodimer partner (SHP) protected against AILI. Mechanistically, liver-specific SHP knockout mice had increased nuclear factor erythroid 2-related factor 2 (Nrf2) activation and faster recovery of hepatic GSH contents resulting in the protection against AILI.

Infiltrated macrophages can trigger neutrophil apoptosis and help remove necrotic cells to promote the resolution of the injury.²⁵ In this symposium, Dr. Cynthia Ju's group reported that eosinophils accumulated in patients with APAP-induced liver failure. In mice treated with APAP, carbon tetrachloride (CCl₄) or concanavalin A, eosinophils were recruited into the liver and played a profound protective role. Mice with depletion of macrophages or IL-33 exhibited impaired hepatic eosinophil recruitment during acute liver injury. These results demonstrated that hepatic recruitment of and protection by eosinophils occur commonly in various acute liver injury conditions including AILI. The same group also identified α-chitinase 3-like-1 (α-Chi3l1) that plays a critical role in hepatic platelet recruitment during AILI.²⁶ Increased Chi3l1 and platelets in the liver were observed in both AILI patients and mice. Chil1^−/− mice had reduced hepatic platelet accumulation and AILI. Mechanistically, they found that Chi3l1 signaled through CD44 on macrophages to induce podoplanin expression, which mediated platelet recruitment through C-type lectin-like receptor 2. Importantly, anti-Chi3l1 monoclonal antibodies effectively inhibited hepatic platelet accumulation and AILI. These studies suggest that platelets could be detrimental for the later recovery phase of AILI. Autophagy is known to protect against AILI by selective removal of APAP-adducts and damaged mitochondria.27, 28, 29 In a separate study conducted by Dr. Wen-Xing Ding's group to investigate the autophagy receptor protein p62/SQSTM1 in AILI, they found that p62/SQSTM1 plays a dual role in AILI. In the early injury phase, p62/SQMSTM1 was protective by promoting selective autophagy for APAP-adducts and damaged mitochondria. However, in the later recovery phase, p62/SQSTM1 was detrimental by promoting the accumulation of platelet-adhesive protein von Willebrand factor (VWF) and platelets in the liver that inhibited liver regeneration in the late recovery phase of AILI. While it remains unclear how p62/SQSTM1 regulates VWF and platelet recruitment, these findings are generally in agreement with the findings from Dr. Cynthia Ju's group on the detrimental role of platelet in AILI. Taken together, these studies may offer a novel therapeutic avenue by targeting Chi3l1 and platelet for treating AILI.

4. New insights on bile acids (BAs) metabolism and diseases related to BA dysregulation

BAs are amphiphilic molecules synthesized from cholesterol in the liver and secreted into the bile and small intestine, where they facilitate the intestinal digestion and absorption of dietary lipids and fat-soluble vitamins. Most of the BAs are reabsorbed in the terminal ileum and returned to the liver through portal circulation to complete enterohepatic circulation. BAs are not only important for the intestinal dietary lipid absorption but also signaling molecules that regulate hepatic metabolism and maintain intestinal microbiota homeostasis.³⁰ Disruption of BA homeostasis may result in intrahepatic accumulation of cytotoxic BAs, leading to cholestatic liver disease. In the 4th CALS Annual Symposium, many exciting abstracts related to BAs were presented, which provided new insights to advance our understanding of BA metabolism and diseases mediated by BA dysregulation.

Primary sclerosing cholangitis (PSC) is a chronic and progressive cholestatic liver disease characterized by increased hepatic BA levels, ductular reaction, and liver fibrosis with infiltration and activation of immune cells including mast cells.31, 32, 33 In a study evaluating the regulation of enterohepatic BA circulation and gut-liver histamine signaling by mast cells in PSC mice, Dr. Heather Francis's group at the Indiana University knocked out histidine decarboxylase (Hdc), a key enzyme involved in histamine synthesis, in Mdr2^−/− mice. After treating Mdr2^−/− and Hdc^−/-/Mdr2^−/− mice with exogenous histamine, the group found infiltration of mast cells into the intestine and liver during cholestatic liver disease, resulting in increases in total BAs, FGF15 secretion, enterohepatic histamine circulation, and biliary BA cholehepatic shunting. Their studies highlight the importance of mast cell activation in the pathogenesis of PSC and inhibition of mast cell activation may provide therapeutic intervention for patients with PSC. The upregulation of hepatic long noncoding RNA H19 (lncRNAH19) is associated with liver fibrosis in the Mdr2^−/− mouse, a PSC model, and PSC patients.34, 35, 36 To understand the impact of lncRNAH19 on hepatic miRNA expression and cholestatic liver injury, Dr. Huiping Zhou's group at the Virginia Commonwealth University examined miRNA expression profiles in Mdr2^−/− and Mdr2^−/−/H19^−/− mice and showed that lncRNAH19-induced dysregulation of hepatic miRNAs represents a major molecular mechanism underlying cholestatic liver injury in Mdr2^−/− mice. Their study suggests that identification of specific targets of dysregulated miRNAs may provide important information for developing novel diagnostic and therapeutic strategies for PSC. Dysregulation of sphingosine-1-phosphate (S1P)-mediated signaling pathways is associated with various diseases, including cholestatic liver disease and neurological disorders.37, 38, 39 The effects of hepatic cholestatic liver injury on sphingolipid metabolism and brain function remain unclear. Another study conducted by Dr. Huiping Zhou's group demonstrated that cholestatic liver injury in Mdr2^−/− mice was associated with brain dysfunction, especially memory loss. Mechanistically, the aberrant upregulation of semipermeable membrane devices (Smpds) in Mdr2^−/− mice resulted in the loss of sphingomyelin and the imbalance of S1P and ceramide, the two major sphingolipids involved in regulating both liver and brain functions. Their study suggests that further investigation is warranted to explore the role of sphingolipids in the liver-brain axis. The intrahepatic accumulation of cytotoxic BAs often acts as a carcinogenic factor in the initiation and progression of liver cancer.40, 41, 42 The hippo kinase Mst1/2 has been shown to regulate BA homeostasis in liver injury and carcinogenesis.⁴³ The study from Dr. Yingzi Yang's group at the Harvard School of Dental Medicine demonstrated that Yap, but not Mst1/2, is a critical regulator of BA homeostasis in the liver by controlling extracellular regulated protein kinase (ERK) activation, a key pathway in BA metabolism. Their study identified a novel mechanism by which Yap promotes liver carcinogenesis via regulating BAs metabolism.

Intestinal canonical FXR and FGF15/19 signaling plays a critical role in BA homeostasis. In a study searching for post-transcriptional regulators of FGF15 in neonatal piglet model of parenteral nutrition-associated liver disease (PNALD), Dr. Junkai Yan's group at the Shanghai Jiaotong University identified insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) acting as an m6A reader to stabilize FGF15 mRNA transcript and promote translation. Their study highlights the IGF2BP1-FGF15/FGF19 axis as a potential therapeutic target for PNALD. BAs homeostasis is well known to be regulated by circadian rhythm.^44,45 The study from Dr. Helmut Zarbl's group at the Rutgers University reported that disruption of circadian rhythm by long-term night shift work affected the circadian pattern of plasma FGF19 levels, which may contribute to the development of NAFLD in night shift workers. Their findings suggest that FGF15/19 may serve as a biomarker for circadian desynchronization with liver lipid metabolism in night shift workers and FGF15/19 may also serve as a therapeutic target for NAFLD associated with circadian disruption. Taken together, these studies have significantly improved our understanding of BAs metabolism and diseases mediated by BA dysregulation. These findings could also facilitate the development of potential diagnostic biomarkers and therapeutic targets for the treatment of BA-related diseases.

5. Non-alcoholic steatohepatitis (NASH): Emerging mechanisms and therapeutic targets

NASH is characterized by hepatic accumulation of fat (lipid droplets), accompanied by inflammation, degeneration of hepatocytes, and fibrosis.^46,47 NASH is a serious chronic liver disease that often carries no symptoms at its early stage, but if untreated, NASH can progress to cirrhosis, HCC, and the need for liver transplantation.⁴⁸ Fibrosis is the major determinant of clinical outcomes in patients with NASH and has an increased risk of cirrhosis and HCC. Due to the rapid increase of the obesity epidemic, NASH is the most common cause of chronic liver disease.^47,49 NASH is associated with increased risks for cardiovascular disease as well as for the progression to cirrhosis and HCC.⁴⁶ Currently NASH has no the US Food and Drug Administration (FDA)-approved therapies, and liver fibrosis shows poor response to existing pharmacotherapy. Thus, identification of pharmaceutical targets is a great unmet need for an increasingly obese population and demand a comprehensive understanding of pathological mechanisms of NASH.

Hepatocyte glutathione S-transferase Mu 2 (GSTM2) is a phase II detoxification enzyme,⁵⁰ but it is less understood whether hepatocyte GSTM2 plays a role in the control of NASH and its potential mechanisms. Studies from Dr. Tian Lan at Guangdong Pharmaceutical University have shared new insight into the function of GSTM2 in NASH. They found that hepatic GSTM2 was significantly downregulated during NASH progression. Hepatocyte GSTM2 overexpression attenuated insulin resistance, hepatic steatosis, inflammation and fibrosis induced by a high-fat diet (HFD) and a high-fat/high-cholesterol diet. Mechanistic studies indicated that GSTM2 directly bound to the amino-terminal region of apoptosis signal-regulating kinase 1 (ASK1) and subsequently inhibited the formation of ASK1 dimerization and ASK1 activity under metabolic stress. Dr. Lan's study provides evidence that GSTM2 acts as an endogenous inhibitor of ASK1 during NASH and targeting GSTM2 could be a potential therapeutic strategy to treat NASH.

Liver and adipose crosstalk is another important factor of lipid metabolism and NAFLD development. Dr. Rongya Tao at Harvard Medical School has shared a new research concept that hepatokine Follistatin (Fst) promotes adipose tissue insulin resistance and thereby leads to fat accumulation in the liver of mice fed with a NASH diet.⁵¹ Obesity and diabetes are risk factors for the progression of NAFLD to life-threatening NASH.⁴⁹ Dr. Tao's studies showed that hepatic insulin resistance caused by inactivation of Irs1 and Irs2 in hepatocytes in liver double knockout mice exacerbated the progression of NAFLD/NASH during consumption of the Amylin liver NASH (AMLN) diet containing high fat and high fructose. This effect likely acted through Forkhead box protein O1-dependent Fst secreted from the liver. Because recent studies indicate that circulating Fst is liver-derived and is elevated in patients with Type 2 diabetes (T2D) compared with lean and obese nondiabetic individuals,⁵² inhibition of hepatic Fst might provide a new therapeutic target for fatty liver disease associated with insulin resistance and T2D.

In addition to liver-adipose axis, the brain-liver axis is also important for regulating lipid metabolism and NAFLD. Studies from Dr. Ling Yang's group at University of Iowa Carver College of Medicine has shared new insight into the role of the disruption of pituitary-liver unfolded protein response (UPR) interaction on NAFLD. Although abnormal pituitary hormone levels are positively correlated with the incidence of NAFLD and NASH in rodents and humans,⁵³ the molecular mechanisms underlying the cell autonomous defect of pituitary hormone production are incompletely understood. As a result of chronic substrate overload, some steatotic hepatocytes undergo cell death due to sustained lipotoxicity and endoplasmic reticulum (ER) stress.⁵⁴ Using the bulk and single-cell RNA-sequencing analyses, chemical or genetic approaches for gain and loss function of UPR regulators, and in vivo mouse metabolic profiling to determine inter-organ communication during NASH, they demonstrated that obesity, the major risk factor for NAFLD, impaired pituitary ER homeostasis by inhibiting the inositol-requiring enzyme α (IRE1α)-mediated adaptive UPR. They further showed that overexpression of hepatic sXBP1 improved the metabolic defects in IRE1α pituitary-deletion mice. Therefore, these studies suggest that dysregulation of pituitary hormone-mediated inter-organ UPR communication drives NAFLD pathologies associated with obesity.

In addition to the above studies, other studies presented in the CALS Annual Symposium have identified several dysregulation signaling pathways in the pathogenesis of NAFLD and NASH, including miR-34, SIRT6, retinoic acid receptor α, vacuole membrane protein 1, gut microbiota, Takeda G protein-coupled receptor 5 and the substance P/neurokinin receptor signaling. These pathways become maladaptive and ultimately a culprit in the progression of NASH associated with obesity and T2D. Studies from Dr. Lirui Wang at China Pharmaceutical University also revealed new insights into the relationship between gut microbiota and NASH. Taken together, these studies have advanced our understanding on the complex multiplayers in NASH pathogenesis, and hold promise to identify novel therapeutic targets for NASH.

6. New mechanisms of fibrosis

Liver fibrosis occurs in most types of chronic liver diseases, which is featured with excessive accumulation of extracellular matrix proteins such as collagen. Advanced liver fibrosis can lead to cirrhosis, portal hypertension and even liver failure, which often requires liver transplantation. Activation of hepatic stellate cells (HSCs) is a key pathogenic event in liver fibrosis. Studies from Dr. Wen Xie's group at University of Pittsburgh showed that hepatic cytochrome 1B1 (CYP1B1) expression was induced in fibrotic human and mouse livers via the transcription factor Wilms' tumor 1 (WT1). Cyp1b1 ablation or pharmacological inhibition of CYP1B1 by 2,4,3′5′-tetramethoxystilbene (TMS) attenuated HSC activation and liver fibrosis. In a separate study, Dr. Wen Xie's group showed that fibrotic mouse and human livers expressed less glutaredoxin-1 (GLRX), also known as thioltransferase-1 (TTase-1), which functions to remove protein S-glutathionylation (PSSG) of cysteine residues by releasing glutathione. Decreased GLRX led to increased PSSG. Glrx depletion exacerbated liver fibrosis whereas overexpression of Glrx inhibited PSSG and liver fibrosis. The antifibrotic drug pirfenidone (PFD) inhibited HSC activation and liver fibrosis in a GLRX-dependent manner. Mechanistically, GLRX deglutathionylated Smad3, leading to inhibition of Smad3 phosphorylation and fibrogenic gene expression.

PSC is a rare, chronic cholestatic disease characterized by inflammation and fibrosis of bile ducts, followed by liver damage.⁵⁵ Dr. Gianfranco Alpini's group at University of Indiana School of Medicine showed that the melatonin receptor 1 (MT1) was increased in PSC patients, Mdr2^−/− mice and bile duct-ligated (BDL) mice. Treatment of Mdr2^−/− mice or BDL mice with MT1 Vivo-Morpholinos reduced liver inflammation and fibrosis by inhibiting G protein-coupled receptor 50 receptor-promoted constitutive transforming growth factor (TGF) β1 receptor signaling. Another study from Guangfu Li's group at University of Missouri showed that TGFβ-induced protein (TGFB1) was induced in mice with choline low-HFD-induced NASH, and CCl₄-induced liver fibrosis. Overexpression of TGFB1 promoted the activation of HSCs and NASH progression. These findings have significant enriched our understanding on liver fibrosis.

7. New perspectives on liver cancer

HCC, a main type of liver cancer, is the third leading cause of cancer deaths worldwide in 2020, which claims for more than 830,000 deaths annually.⁵⁶ It is well known that there is large inter-tumor and intratumor heterogeneity at the phenotypic and molecular levels in HCC.⁵⁷ To better understand the complexity of liver cancer heterogeneity including its tumor cell evolution in response to therapy, the implementation of new strategies including single-cell analysis has been carried out in recent years.⁵⁸

It is known that PTPN11/Shp2 is a non-receptor tyrosine phosphatase that contains two Src-homology 2 (SH2) domains, and hyperactivation of Shp2 has been found in many cancers including HCC.⁵⁹ Dr. Gen-Sheng Feng's lab presented interesting single-cell transcriptomics results demonstrating opposing roles of Shp2 in Myc-driven liver tumor cells and microenvironment. They showed that hepatocyte-specific Ptpn11/Shp2 deletion aggravated Myc-driven HCC in mice. Intriguingly, Myc-induced tumors developed selectively from the rare Shp2-positive hepatocytes in liver-specific Shp2 knockout mouse livers, and Myc-driven oncogenesis requires an intact Ras-Erk signaling promoted by Shp2 to sustain Myc stability.⁶⁰ Dr. Lichun Ma and Dr. Man Hsin Hung from Dr. Xin Wei Wang's lab have successfully utilized single cell technologies to dissect liver tumor cell evolution in response to therapy and to exploit tumor methionine metabolism as liver cancer vulnerability. They used transcriptomic clusters as a surrogate for functional clonality and found an increase in tumor cell state heterogeneity that is tightly linked to HCC prognosis and response to immunotherapy. More importantly, they found that enhanced functional clonality coincident with a polarized immune cell landscape including an increase in pre-exhausted T cells.⁶¹

Cancer metabolism has attracted much attention in the study of HCC. Recent evidence indicates that β-catenin activation in zone 3 hepatocytes leads to high mechanistic target of rapamycin complex 1 (mTORC1) activity downstream of elevated glutamine synthetase expression and intracellular glutamine, which promotes HCC.⁶² Dr. Wei-Xing Zong of Rutgers University kicked off the basic session of liver cancer by presenting his work on glutamine anabolism in pancreatic and liver cancers, and his work raised a novel concept that the homoeostasis rather than the increased or decreased levels of glutamine is critical for liver tumor formation. Dr. Bangyan Stiles of University of Southern California presented her exciting work on heterogenous liver cancer development promoted by PTEN-regulated liver steatosis, providing evidence to link steatosis for promoting HCC. There are many other outstanding presentations that further cover the role of alcohol consumption, autophagy, mTOR, and miRNA for liver tumorigenesis, programmed death ligand-1 (PDL-1) and immune-based therapies for HCC as well as vessels encapsulating tumor clusters (VETC)-dependent and invasion-dependent metastasis of HCC.^17,63,64 Taken together, the conference brought a timely discussion on recent advances in the understanding of liver cancer development as well as clinical efforts to target this dreadful disease.

8. Summary

CALS Annual Symposium has provided an excellent platform for liver investigators and their trainees to exchange state-of-the-art research progress and novel ideas for broad topics related to liver biology and diseases. The 2021 CALS Annual Symposium has provided great opportunities for establishing productive collaborations among different groups and disciplines. More importantly, it has provided an excellent opportunity for trainees to present their work and receive constructive feedback from the senior investigators as well as their own peer trainees. The studies presented in the symposium clearly enhanced our understanding on the pathogeneses of ALD, DILI, cholestasis, NAFLD/NASH and HCC.

Authors’ contributions

W.-X. Ding, H. Wang, Y. Zhang, and the Symposium committee members conceived the idea and wrote the manuscript.

Declaration of competing interest

The authors declare that they have no conflicts of interest.