TIGIT is the central player in T-cell suppression associated with CAR T-cell relapse in mantle cell lymphoma.

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy using brexucabtagene autoleucel (BA) induces remission in many patients with mantle cell lymphoma (MCL), and BA is the only CAR T-cell therapy approved by the FDA for MCL. However, development of relapses to BA is recognized with poor patient outcomes. Multiple CAR T-cell therapies have been approved for other lymphomas and the resistance mechanisms have been investigated. However, the mechanisms underlying BA relapse in MCL have not been investigated and whether any previously reported resistance mechanisms apply to BA-relapsed patients with MCL is unknown. METHODS: To interrogate BA resistance mechanisms in MCL, we performed single-cell RNA sequencing on 39 longitudinally collected samples from 15 BA-treated patients, and multiplex cytokine profiling on 80 serial samples from 20 patients. RESULTS: We demonstrate that after BA relapse, the proportion of T cells, especially cytotoxic T cells (CTLs), decreased among non-tumor cells, while the proportion of myeloid cells correspondingly increased. TIGIT, LAG3, and CD96 were the predominant checkpoint molecules expressed on exhausted T cells and CTLs; only TIGIT was significantly increased after relapse. CTLs expanded during remission, and then contracted during relapse with upregulated TIGIT expression. Tumor cells also acquired TIGIT expression after relapse, leading to the enhanced interaction of tumor cell TIGIT with monocyte CD155/PVR. In myeloid cells, post-relapse HLA-II expression was reduced relative to pretreatment and during remission. Myeloid-derived suppressor cells (MDSCs) were enriched after relapse with elevated expression of activation markers, including CLU (clusterin) and VCAN (versican). Extracellular chemokines (CCL4, CXCL9, CXCL13), soluble checkpoint inhibitors (sPD-L1, sTIM3, s4-1BB), and soluble receptors (sIL-2R, sTNFRII) were decreased during remission but elevated after relapse. CONCLUSIONS: Our data demonstrate that multiple tumor-intrinsic and -extrinsic factors are associated with T-cell suppression and BA relapse. Among these, TIGIT appears to be the central player given its elevated expression after BA relapse in not only CTLs but also MCL cells. The acquisition of TIGIT expression on tumor cells is MCL-specific and has not been reported in other CAR T-treated diseases. Together, our data suggest that co-targeting TIGIT may prevent CAR T relapses and thus promote long-term progression-free survival in MCL patients.

Electroacupuncture ameliorates inflammatory response induced by retinal ischemia-reperfusion injury and protects the retina through the DOR-BDNF/Trkb pathway.

Objectives: Retinal ischemia-reperfusion injury (RIRI) is the common pathological basis of many ophthalmic diseases in the later stages, and inflammation is the primary damage mechanism of RIRI. Our study aimed to assess whether electroacupuncture (EA) has a protective effect against RIRI and to elucidate its related mechanisms. Methods: A high-intraocular pressure (HIOP) model was used to simulate RIRI in Wistar rats. EA was applied to the EA1 group [Jingming (BL1) + Shuigou (GV26)] and the EA2 group [Jingming (BL1) + Hegu (LI4)] respectively for 30 min starting immediately after the onset of reperfusion and repeated (30 min/time) at 12 h and then every 24 h until days 7 after reperfusion. The pathological changes in the retina were observed by H and E staining after HIOP. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was utilized to observe retinal cell apoptosis. The mRNA expression of IL1-ß, TNF-α, IL-4, IL-10, δ-opioid receptor (DOR), brain-derived neurotrophic factor (BDNF), and tropomyosin-related kinase B (TrkB) in the retina was measured by quantitative real-time PCR. Results: HIOP caused structural disorders of the retina, decreased RGCs, and increased retinal cell apoptosis. At 1 and 3 days of RIRI, retinal apoptotic cells in the EA group were significantly reduced, while there was no distinct difference in the EA group compared with the HIOP group at 7 days of RIRI. Compared with that in the HIOP group, the expression of anti-inflammatory factors, DOR and TrkB was increased, and the expression of pro-inflammatory factors was decreased in the EA group. In contrast, HIOP had no appreciable effect on BDNF expression. Conclusion: EA at Jingming (BL1) and Shuigou (GV26) or at Jingming (BL1) and Hegu (LI4) may inhibit RIRI induced inflammation through activating the DOR-BDNF/TrkB pathway to protect the retina, especially the pair of Jingming (BL1) and Shuigou (GV26) has better inhibitory effects on inflammation.

Applying Lean Six Sigma to Reduce the Incidence of Unplanned Surgery Cancellation at a Large Comprehensive Tertiary Hospital in China.

Unplanned surgery cancellation (USC) was an important quality management issue in the course of medical care for surgical patients, which caused inappropriate use of hospital resources and had negative impacts on quality and safety. This study used Lean Six Sigma to reduce the incidence of USC. Following the Lean Six Sigma DMAIC (Define, Measure, Analyze, Improve, and Control) process, the main factors influencing the USC were identified, such as the time of informing patient admission, the time of submitting operation notice, and the management of test report follow-up. A series of measures were implemented including improving the health education content of virtual bed patients, standardizing the way of communication between the Admission Management Center and the patients, improving the timing of anesthesia evaluation, optimizing the process of operation notice with an information system, and implementing the regulations of virtual bed management. The incidence of USC reduced from 10.21% in Jan. 2016 to 3.8% in Dec. 2016, and the Z-score increased from 1.25 to 1.68, which improved patient safety and demonstrated that Lean Six Sigma was an effective method to solve cross-department issues in hospital.

NKT Cells Coexpressing a GD2-Specific Chimeric Antigen Receptor and IL15 Show Enhanced In Vivo Persistence and Antitumor Activity against Neuroblastoma.

PURPOSE: Vα24-invariant natural killer T cells (NKT) are attractive carriers for chimeric antigen receptors (CAR) due to their inherent antitumor properties and preferential localization to tumor sites. However, limited persistence of CAR-NKTs in tumor-bearing mice is associated with tumor recurrence. Here, we evaluated whether coexpression of the NKT homeostatic cytokine IL15 with a CAR enhances the in vivo persistence and therapeutic efficacy of CAR-NKTs. EXPERIMENTAL DESIGN: Human primary NKTs were ex vivo expanded and transduced with CAR constructs containing an optimized GD2-specific single-chain variable fragment and either the CD28 or 4-1BB costimulatory endodomain, each with or without IL15 (GD2.CAR or GD2.CAR.15). Constructs that mediated robust CAR-NKT cell expansion were selected for further functional evaluation in vitro and in xenogeneic mouse models of neuroblastoma. RESULTS: Coexpression of IL15 with either costimulatory domain increased CAR-NKT absolute numbers. However, constructs containing 4-1BB induced excessive activation-induced cell death and reduced numeric expansion of NKTs compared with respective CD28-based constructs. Further evaluation of CD28-based GD2.CAR and GD2.CAR.15 showed that coexpression of IL15 led to reduced expression levels of exhaustion markers in NKTs and increased multiround in vitro tumor cell killing. Following transfer into mice bearing neuroblastoma xenografts, GD2.CAR.15 NKTs demonstrated enhanced in vivo persistence, increased localization to tumor sites, and improved tumor control compared with GD2.CAR NKTs. Importantly, GD2.CAR.15 NKTs did not produce significant toxicity as determined by histopathologic analysis. CONCLUSIONS: Our results informed selection of the CD28-based GD2.CAR.15 construct for clinical testing and led to initiation of a first-in-human CAR-NKT cell clinical trial (NCT03294954).

Small molecule inhibitor agerafenib effectively suppresses neuroblastoma tumor growth in mouse models via inhibiting ERK MAPK signaling.

Neuroblastoma (NB) is the most common extracranial solid tumor in early childhood. Despite intensive multimodal therapy, nearly half of children with high-risk disease will relapse with therapy-resistant tumors. Dysregulation of MAPK pathway has been implicated in the pathogenesis of relapsed and refractory NB patients, which underscores the possibility of targeting MAPK signaling cascade as a novel therapeutic strategy. In this study, we found that high expressions of RAF family kinases correlated with advanced tumor stage, high-risk disease, tumor progression, and poor overall survival. Targeted inhibition of RAF family kinases with the novel small molecule inhibitor agerafenib abrogated the activation of ERK MAPK pathway in NB cells. Agerafenib significantly inhibited the cell proliferation and colony formation ability of NB cells in vitro, and its combination with traditional chemotherapy showed a synergistic pro-apoptotic effect. More importantly, agerafenib exhibited a favorable toxicity profile, potently suppressed tumor growth, and prolonged survival in NB mouse models. In conclusion, our preclinical data suggest that agerafenib might be an effective therapeutic agent for NB treatment, both as a single-agent and in combination with chemotherapy.

Targeting LRH­1 in hepatoblastoma cell lines causes decreased proliferation.

Hepatoblastoma is the most common malignant liver tumor in children. Since it is often unresectable and exhibits drug resistance, the treatment of advanced hepatoblastoma is challenging. The orphan nuclear receptor liver receptor homolog­1 (LRH­1) serves prominent roles in malignancy; however, to the best of our knowledge, the role of LRH­1 in hepatoblastoma remains unknown. In the present study, human hepatoblastoma cell lines were analyzed; the mRNA and protein expression levels of LRH­1 were significantly higher in HepG2 and HuH6 cells compared with those in HepT1 cells and control THLE­2 cells. Knockdown of LRH­1 resulted in decreased HepG2 and HuH6 cell proliferation via downregulation of cyclin D1 (CCND1) and c­Myc. Furthermore, treatment with an LRH­1 antagonist (LRA) inhibited the proliferation and colony formation of cell lines in a dose­dependent manner, and induced cell cycle arrest at G1 phase through inhibition of CCND1 expression. Finally, LRA treatment enhanced the cytotoxic effects of doxorubicin on hepatoblastoma cells. Collectively, these findings suggested that LRH­1 may have an important role in the progression of hepatoblastoma and implicated LRA as a novel, potential therapeutic agent for the treatment of hepatoblastoma.

IL-21 Selectively Protects CD62L+ NKT Cells and Enhances Their Effector Functions for Adoptive Immunotherapy.

T cells expressing CD19-specific chimeric Ag receptors (CARs) produce high remission rates in B cell lymphoma, but frequent disease recurrence and challenges in generating sufficient numbers of autologous CAR T cells necessitate the development of alternative therapeutic effectors. Vα24-invariant NKTs have intrinsic antitumor properties and are not alloreactive, allowing for off-the-shelf use of CAR-NKTs from healthy donors. We recently reported that CD62L+ NKTs persist longer and have more potent antilymphoma activity than CD62L- cells. However, the conditions governing preservation of CD62L+ cells during NKT cell expansion remain largely unknown. In this study, we demonstrate that IL-21 preserves this crucial central memory-like NKT subset and enhances its antitumor effector functionality. We found that following antigenic stimulation with α-galactosylceramide, CD62L+ NKTs both expressed IL-21R and secreted IL-21, each at significantly higher levels than CD62L- cells. Although IL-21 alone failed to expand stimulated NKTs, combined IL-2/IL-21 treatment produced more NKTs and increased the frequency of CD62L+ cells versus IL-2 alone. Gene expression analysis comparing CD62L+ and CD62L- cells treated with IL-2 alone or IL-2/IL-21 revealed that the latter condition downregulated the proapoptotic protein BIM selectively in CD62L+ NKTs, protecting them from activation-induced cell death. Moreover, IL-2/IL-21-expanded NKTs upregulated granzyme B expression and produced more TH1 cytokines, leading to enhanced in vitro cytotoxicity of nontransduced and anti-CD19-CAR-transduced NKTs against CD1d+ and CD19+ lymphoma cells, respectively. Further, IL-2/IL-21-expanded CAR-NKTs dramatically increased the survival of lymphoma-bearing NSG mice compared with IL-2-expanded CAR-NKTs. These findings have immediate translational implications for the development of NKT cell-based immunotherapies targeting lymphoma and other malignancies.

Methylation of PCDH19 predicts poor prognosis of hepatocellular carcinoma.

OBJECTIVE: Several members of protocadherins (PCDHs) have been identified as tumor suppressor genes in human carcinogenesis, but little is known about PCDH19. The aim of the present study was to assess the expression and methylation of PCDH19 in hepatocellular carcinoma (HCC). METHODS: The RNA-seq data from The Cancer Genome Atlas Database were downloaded and used for analyzing PCDH19 expression in HCC patients and normal liver tissues. We collected 63 paired tumor and nontumor liver tissues from hepatitis B virus-related HCC patients. The expression of PCDH19 was detected by real-time quantitative RT-PCR assay. The methylation of PCDH19 gene was analyzed by DNA methylation-sensitive endonuclease digestion and the sequential quantitative PCR. The prognostic value of PCDH19 gene methylation was evaluated by Kaplan-Meier analyses. RESULTS: PCDH19 expression was downregulated in HCC tissues and seven HCC cell lines compared to nontumor tissues. PCHD19 promoter was frequently hypermethylated in three (SMMC7721, Hep3B and SNU387) of seven HCC cell lines and 5-aza-dC treatment could significantly increased the PCDH19 expression in these methylated cells. In addition, HCC tumor tissues exhibited significantly increased PCDH19 hypermethylation both in frequency (30.15% vs 9.52%, P = 0.003) and in intensity (P = 0.002) compared to that in nontumor tissues. Kaplan-Meier survival analysis revealed that PCDH19 hypermethylation was correlated with the poor overall survival of HCC patients. CONCLUSION: PCDH19 expression was downregulated in HCC, which was mediated at least in part by promoter hypermethylation. PCDH19 hypermethylation might present a potential prognostic marker in HCC patients.

A Novel Cell Line Based Orthotopic Xenograft Mouse Model That Recapitulates Human Hepatoblastoma.

Currently, preclinical testing of therapies for hepatoblastoma (HB) is limited to subcutaneous and intrasplenic xenograft models that do not recapitulate the hepatic tumors seen in patients. We hypothesized that injection of HB cell lines into the livers of mice would result in liver tumors that resemble their clinical counterparts. HepG2 and Huh-6 HB cell lines were injected, and tumor growth was monitored with bioluminescence imaging (BLI) and magnetic resonance imaging (MRI). Levels of human α-fetoprotein (AFP) were monitored in the serum of animals. Immunohistochemical and gene expression analyses were also completed on xenograft tumor samples. BLI signal indicative of tumor growth was seen in 55% of HepG2- and Huh-6-injected animals after a period of four to seven weeks. Increased AFP levels correlated with tumor growth. MRI showed large intrahepatic tumors with active neovascularization. HepG2 and Huh-6 xenografts showed expression of ß-catenin, AFP, and Glypican-3 (GPC3). HepG2 samples displayed a consistent gene expression profile most similar to human HB tumors. Intrahepatic injection of HB cell lines leads to liver tumors in mice with growth patterns and biologic, histologic, and genetic features similar to human HB tumors. This orthotopic xenograft mouse model will enable clinically relevant testing of novel agents for HB.

EWS-FLI1 and RNA helicase A interaction inhibitor YK-4-279 inhibits growth of neuroblastoma.

Treatment failure in high risk neuroblastoma (NB) is largely due to the development of chemotherapy resistance. We analyzed the gene expression changes associated with exposure to chemotherapy in six high risk NB tumors with the aid of the Connectivity Map bioinformatics platform. Ten therapeutic agents were predicted to have a high probability of reversing the transcriptome changes associated with neoadjuvant chemotherapy treatment. Among these agents, initial screening showed the EWS-FLI1 and RNA helicase A interaction inhibitor YK-4-279, had obvious cytotoxic effects on NB cell lines. Using a panel of NB cell lines, including MYCN nonamplified (SK-N-AS, SH-SY5Y, and CHLA-255), and MYCN amplified (NB-19, NGP, and IMR-32) cell lines, we found that YK-4-279 had cytotoxic effects on all lines tested. In addition, YK-4-279 also inhibited cell proliferation and anchorage-independent growth and induced cell apoptosis of these cells. YK-4-279 enhanced the cytotoxic effect of doxorubicin (Dox). Moreover, YK-4-279 was able to overcome the established chemoresistance of LA-N-6 NB cells. In an orthotopic xenograft NB mouse model, YK-4-279 inhibited NB tumor growth and induced apoptosis in tumor cells through PARP and Caspase 3 cleavage in vivo. While EWS-FLI1 fusion protein is not frequently found in NB, using the R2 public database of neuroblastoma outcome and gene expression, we found that high expression of EWSR1 was associated with poor patient outcome. Knockdown of EWSR1 inhibited the oncogenic potential of neuroblastoma cell lines. Taken together, our results indicate that YK-4-279 might be a promising agent for treatment of NB that merits further exploration.

The second-generation ALK inhibitor alectinib effectively induces apoptosis in human neuroblastoma cells and inhibits tumor growth in a TH-MYCN transgenic neuroblastoma mouse model.

Activating germline mutations of anaplastic lymphoma kinase (ALK) occur in most cases of hereditary neuroblastoma (NB) and the constitutively active kinase activity of ALK promotes cell proliferation and survival in NB. Therefore, ALK kinase is a potential therapeutic target for NB. In this study, we show that the novel ALK inhibitor alectinib effectively suppressed cell proliferation and induces apoptosis in NB cell lines with either wild-type ALK or mutated ALK (F1174L and D1091N) by blocking ALK-mediated PI3K/Akt/mTOR signaling. In addition, alectinib enhanced doxorubicin-induced cytotoxicity and apoptosis in NB cells. Furthermore, alectinib induced apoptosis in an orthotopic xenograft NB mouse model. Also, in the TH-MYCN transgenic mouse model, alectinib resulted in decreased tumor growth and prolonged survival time. These results indicate that alectinib may be a promising therapeutic agent for the treatment of NB.

Activation of CDK4 Triggers Development of Non-alcoholic Fatty Liver Disease.

The development of non-alcoholic fatty liver disease (NAFLD) is a multiple step process. Here, we show that activation of cdk4 triggers the development of NAFLD. We found that cdk4 protein levels are elevated in mouse models of NAFLD and in patients with fatty livers. This increase leads to C/EBPα phosphorylation on Ser193 and formation of C/EBPα-p300 complexes, resulting in hepatic steatosis, fibrosis, and hepatocellular carcinoma (HCC). The disruption of this pathway in cdk4-resistant C/EBPα-S193A mice dramatically reduces development of high-fat diet (HFD)-mediated NAFLD. In addition, inhibition of cdk4 by flavopiridol or PD-0332991 significantly reduces development of hepatic steatosis, the first step of NAFLD. Thus, this study reveals that activation of cdk4 triggers NAFLD and that inhibitors of cdk4 may be used for the prevention/treatment of NAFLD.

Cooperation of C/EBP family proteins and chromatin remodeling proteins is essential for termination of liver regeneration.

UNLABELLED: Liver cancer is the fifth most common cancer. A highly invasive surgical resection of the liver tumor is the main approach used to eliminate the tumor. Mechanisms that terminate liver regeneration when the liver reaches the original size are not known. The aims of this work were to generate an animal model that fails to stop liver regeneration after surgical resections and elucidate mechanisms that are involved in termination of liver regeneration. Because epigenetic control of liver function has been previously implicated in the regulation of liver proliferation, we generated C/EBPα-S193A knockin mice, which have alterations in formation of complexes of C/EBP family proteins with chromatin remodeling proteins. The C/EBPα-S193A mice have altered liver morphology and altered liver function leading to changes of glucose metabolism and blood parameters. Examination of the proliferative capacity of C/EBPα-S193A livers showed that livers of S193A mice have a higher rate of proliferation after birth, but stop proliferation at the age of 2 months. These animals have increased liver proliferation in response to liver surgery as well as carbon tetrachloride (CCl4 )-mediated injury. Importantly, livers of C/EBPα-S193A mice fail to stop liver regeneration after surgery when livers reach the original, preresection, size. The failure of S193A livers to stop regeneration correlates with the epigenetic repression of key regulators of liver proliferation C/EBPα, p53, FXR, SIRT1, PGC1α, and TERT by C/EBPß-HDAC1 complexes. The C/EBPß-HDAC1 complexes also repress promoters of enzymes of glucose synthesis PEPCK and G6Pase. CONCLUSION: Proper cooperation of C/EBP and chromatin remodeling proteins is essential for the termination of liver regeneration after surgery and for maintenance of liver functions.

Age-associated change of C/EBP family proteins causes severe liver injury and acceleration of liver proliferation after CCl4 treatments.

The aged liver is more sensitive to the drug treatments and has a high probability of developing liver disorders such as fibrosis, cirrhosis, and cancer. Here we present mechanisms underlying age-associated severe liver injury and acceleration of liver proliferation after CCl4 treatments. We have examined liver response to CCl4 treatments using old WT mice and young C/EBPα-S193D knockin mice, which express an aged-like isoform of C/EBPα. Both animal models have altered chromatin structure as well as increased liver injury and proliferation after acute CCl4 treatments. We found that these age-related changes are associated with the repression of key regulators of liver biology: C/EBPα, Farnesoid X Receptor (FXR) and telomere reverse transcriptase (TERT). In quiescent livers of old WT and young S193D mice, the inhibition of TERT is mediated by HDAC1-C/EBPα complexes. After CCl4 treatments, TERT, C/EBPα and FXR are repressed by different mechanisms. These mechanisms include the increase of a dominant negative isoform, C/EBPß-LIP, and subsequent repression of C/EBPα, FXR, and TERT promoters. C/EBPß-LIP also disrupts Rb-E2F1 complexes in C/EBPα-S193D mice after CCl4 treatments. To examine if these alterations are involved in drug-mediated liver diseases, we performed chronic treatments of mice with CCl4. We found that C/EBPα-S193D mice developed fibrosis much more rapidly than WT mice. Thus, our data show that the age-associated alterations of C/EBP proteins create favorable conditions for the increased liver proliferation after CCl4 treatments and for development of drug-mediated liver diseases.

Farnesoid X receptor directly regulates xenobiotic detoxification genes in the long-lived Little mice.

Activation of xenobiotic metabolism pathways has been linked to lifespan extension in different models of aging. However, the mechanisms underlying activation of xenobiotic genes remain largely unknown. Here we showed that although farnesoid X receptor (FXR, Nr1h4) mRNA levels do not change significantly, FXR protein levels are elevated in the livers of the long-lived Little mice, leading to increased DNA binding activity of FXR. Hepatic FXR expression is sex-dependent in wild-type mice but not in Little mice, implying that up-regulation of FXR might be dependent on the reduction of growth hormone in Little mice. Growth hormone treatment decreased hepatic expression of FXR and xenobiotic genes Abcb1a, Fmo3 and Gsta2 in both wild-type and Little mice, suggesting an association between FXR and xenobiotic gene expression. We found that Abcb1a is transactivated by FXR via direct binding of FXR/retinoid X receptor α (RXRα) heterodimer to a response element at the proximal promoter. FXR also positively controls Fmo3 and Gsta2 expression through direct interaction with the response elements in these genes. Our study demonstrates that xenobiotic genes are direct transcriptional targets of FXR and suggests that FXR signaling may play a critical role in the lifespan extension observed in Little mice.

Transcriptional and translational regulation of C/EBPß-HDAC1 protein complexes controls different levels of p53, SIRT1, and PGC1α proteins at the early and late stages of liver cancer.

Cancer changes biological processes in the liver by altering gene expression at the levels of transcription, translation, and protein modification. The RNA binding protein CUGBP1 is a key regulator of translation of CCAAT enhancer binding protein ß and histone deacetylase 1 (HDAC1). These proteins form complexes that are involved in the regulation of liver biology. Here we show a critical role of the translational activation of CCAAT/enhancer binding protein ß-HDAC1 complexes in the development of liver cancer mediated by diethylnitrosamine. We found that diethylnitrosamine increases the levels of CUGBP1 and activates CUGBP1 by phosphorylation, leading to the formation of the CUGBP1-eIF2 complex, which is an activator of translation of CUGBP1-dependent mRNAs. The elevation of the CUGBP1-eIF2 complex increases translation of C/EBPß and HDAC1, resulting in an increase of C/EBPß-HDAC1 complexes at later stages of liver cancer. We found that C/EBPß-HDAC1 complexes repress promoters of three key regulators of liver functions: p53, SIRT1, and PGC1α. As the result of this suppression, the p53-, SIRT1-, and PGC1α-dependent downstream pathways are reduced, leading to increased liver proliferation. We also found that the proper regulation of C/EBPß-HDAC1 complexes is required for the maintenance of biological levels of p53, SIRT1, and PGC1α in quiescent livers and at early stages of liver cancer. Taken together, these studies showed that the development of liver cancer includes a tight regulation of levels of C/EBPß-HDAC1 complexes on the levels of transcription, translation, and posttranslational modifications.

Increased expression of enzymes of triglyceride synthesis is essential for the development of hepatic steatosis.

Molecular mechanisms underpinning nonalcoholic fatty liver disease (NAFLD) are not well understood. The earliest step of NAFLD is hepatic steatosis, which is one of the main characteristics of aging liver. Here, we present a molecular scenario of age-related liver steatosis. We show that C/EBPα-S193D knockin mice have age-associated epigenetic changes and develop hepatic steatosis at 2 months of age. The underlying mechanism of the hepatic steatosis in old wild-type (WT) mice and in young S193D mice includes increased amounts of tripartite p300-C/EBPα/ß complexes that activate promoters of five genes that drive triglyceride synthesis. Knockdown of p300 in old WT mice inhibits hepatic steatosis. Indeed, transgenic mice expressing dominant-negative p300 have fewer C/EBPα/ß-p300 complexes and do not develop age-dependent hepatic steatosis. Notably, the p300-C/EBPα/ß pathway is activated in the livers of patients with NAFLD. Thus, our results show that p300 and C/EBP proteins are essential participants in hepatic steatosis.

Farnesoid X receptor inhibits gankyrin in mouse livers and prevents development of liver cancer.

UNLABELLED: One of the early events in the development of liver cancer is a neutralization of tumor suppressor proteins Rb, p53, hepatocyte nuclear factor 4α (HNF4α), and CCAAT/enhancer binding protein (C/EBP) α. The elimination of these proteins is mediated by a small subunit of proteasome, gankyrin, which is activated by cancer. The aim of this study was to determine the mechanisms that repress gankyrin in quiescent livers and mechanisms of activation of gankyrin in liver cancer. We found that farnesoid X receptor (FXR) inhibits expression of gankyrin in quiescent livers by silencing the gankyrin promoter through HDAC1-C/EBPß complexes. C/EBPß is a key transcription factor that delivers HDAC1 to gankyrin promoter and causes epigenetic silencing of the promoter. We show that down-regulation of C/EBPß in mouse hepatoma cells and in mouse livers reduces C/EBPß-HDAC1 complexes and activates the gankyrin promoter. Deletion of FXR signaling in mice leads to de-repression of the gankyrin promoter and to spontaneous development of liver cancer at 12 months of age. Diethylnitrosoamine (DEN)-mediated liver cancer in wild-type mice also involves the reduction of FXR and activation of gankyrin. Examination of liver cancer in old mice and liver cancer in human patients revealed that FXR is reduced, while gankyrin is elevated during spontaneous development of liver cancer. Searching for animal models with altered levels of FXR, we found that long-lived Little mice have high levels of FXR and do not develop liver cancer with age and after DEN injections due to failure to activate gankyrin and eliminate Rb, p53, HNF4α and C/EBPα proteins. CONCLUSION: FXR prevents liver cancer by inhibiting the gankyrin promoter via C/EBPß-HDAC1 complexes, leading to subsequent protection of tumor suppressor proteins from degradation.