Terf2ip deficiency accelerates non-alcoholic steatohepatitis through regulating lipophagy and fatty acid oxidation via Sirt1/AMPK pathway.

BACKGROUND & AIMS: Our previous study has demonstrated that Telomeric repeat-binding factor 2-interacting protein 1(Terf2ip), played an important role in hepatic ischemia reperfusion injury. This study is aimed to explore the function and mechanism of Terf2ip in non-alcoholic steatohepatitis (NASH). METHODS: The expression of Terf2ip was detected in liver tissue samples obtained from patients diagnosed with NASH. Mice NASH models were constructed by fed with high-fat diet (HFD) or methionine/choline deficient diet (MCD) in Terf2ip knockout and wild type (WT) mice. To further investigate the role of Terf2ip in NASH, adeno-associated viruses (AAV)-Terf2ip was administrated to mice. RESULTS: We observed a significant down-regulation of Terf2ip levels in the livers of NASH patients and mice NASH models. Terf2ip deficiency was associated with an exacerbation of hepatic steatosis in mice under HFD or MCD. Additionally, Terf2ip deficiency impaired lipophagy and fatty acid oxidation (FAO) in NASH models. Mechanically, we discovered that Terf2ip bound to the promoter region of Sirt1 to regulate Sirt1/AMPK pathway activation. As a result, Terf2ip deficiency was shown to inhibit lipophagy through the AMPK pathway, while the activation of Sirt1 alleviated steatohepatitis in the livers of mice. Finally, re-expression of Terf2ip in hepatocyes alleviated liver steatosis, inflammation, and restored lipophagy. CONCLUSIONS: These results revealed that Terf2ip played a protective role in the progression of NASH through regulating lipophagy and FAO by binding to Sirt1 promoter. Our findings provided a potential therapeutic target for the treatment of NASH.

RBM10 C761Y mutation induced oncogenic ASPM isoforms and regulated ß-catenin signaling in cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma (CCA) comprises a heterogeneous group of biliary tract cancer. Our previous CCA mutation pattern study focused on genes in the post-transcription modification process, among which the alternative splicing factor RBM10 captured our attention. However, the roles of RBM10 wild type and mutations in CCA remain unclear. METHODS: RBM10 mutation spectrum in CCA was clarified using our initial data and other CCA genomic datasets from domestic and international sources. Real-time PCR and tissue microarray were used to detect RBM10 clinical association. Function assays were conducted to investigate the effects of RBM10 wild type and mutations on CCA. RNA sequencing was to investigate the changes in alternative splicing events in the mutation group compared to the wild-type group. Minigene splicing reporter and interaction assays were performed to elucidate the mechanism of mutation influence on alternative splicing events. RESULTS: RBM10 mutations were more common in Chinese CCA populations and exhibited more protein truncation variants. RBM10 exerted a tumor suppressive effect in CCA and correlated with favorable prognosis of CCA patients. The overexpression of wild-type RBM10 enhanced the ASPM exon18 exon skipping event interacting with SRSF2. The C761Y mutation in the C2H2-type zinc finger domain impaired its interaction with SRSF2, resulting in a loss-of-function mutation. Elevated ASPM203 stabilized DVL2 and enhanced ß-catenin signaling, which promoted CCA progression. CONCLUSIONS: Our results showed that RBM10C761Y-modulated ASPM203 promoted CCA progression in a Wnt/ß-catenin signaling-dependent manner. This study may enhance the understanding of the regulatory mechanisms that link mutation-altering splicing variants to CCA.

CircPCNXL2 promotes tumor growth and metastasis by interacting with STRAP to regulate ERK signaling in intrahepatic cholangiocarcinoma.

BACKGROUND: circular RNAs (circRNAs) have been reported to exert important effects in the progression of numerous cancers. However, the functions of circRNAs in intrahepatic cholangiocarcinoma (ICC) are still unclear. METHODS: circPCNXL2 (has_circ_0016956) were identified in paired ICC by circRNA microarray. Then, we assessed the biological functions of circPCNXL2 by CCK8, EdU, clone formation, transwell, wound healing assays, and xenograft models. RNA pull-down, mass spectrometry, and RNA immunoprecipitation (RIP) were applied to explore the interaction between cirrcPCNXL2 and serine-threonine kinase receptor-associated protein (STRAP). RNA pull-down, RIP and luciferase reporter assays were used to investigate the sponge functions of circPCNXL2. In the end, we explore the effects of circPCNXL2 and trametinib (a MEK1/2 inhibitor) in vivo. RESULTS: circPCNXL2 was upregulated in ICC tissues and cell lines, which promoted the proliferation and metastasis of ICC in vitro and in vivo. In terms of the mechanisms, circPCNXL2 could directly bind to STRAP and induce the interaction between STRAP and MEK1/2, resulting in the tumor promotion in ICC by activation of ERK/MAPK pathways. Besides, circPCNXL2 could regulate the expression of SRSF1 by sponging miR-766-3p and subsequently facilitated the growth of ICC. Finally, circPCNXL2 could partially inhibit the anti-tumor activity of trametinib in vivo. CONCLUSION: circPCNXL2 played a crucial role in the progression of ICC by interacting with STRAP to activate the ERK signaling pathway, as well as by modulating the miR-766-3p/SRSF1 axis. These findings suggest that circPCNXL2 may be a promising biomarker and therapeutic target for ICC.

USP21 deubiquitinates and stabilizes HSP90 and ENO1 to promote aerobic glycolysis and proliferation in cholangiocarcinoma.

Deubiquitylating enzymes (DUBs) play an essential role in targeted protein degradation and represent an emerging therapeutic paradigm in cancer. However, their therapeutic potential in cholangiocarcinoma (CCA) has not been explored. Herein, based on The Cancer Genome Atlas (TCGA) and The Gene Expression Omnibus (GEO) databases, we found that ubiquitin-specific protease 21 (USP21) was upregulated in CCA, high USP21 level was associated with poor prognosis. In vivo and in vitro, we identified USP21 as a master regulator of CCA growth and maintenance, which directly interacted with deubiquitinates and stabilized the heat shock protein 90 (HSP90) through K48-linked deubiquitination, and in turn, this stabilization increased HIF1A expression, thus upregulating key glycolytic enzyme genes ENO2, ENO3, ALDOC, ACSS2, and then promoted aerobic glycolysis, which provided energy for CCA cell proliferation. In addition, USP21 could directly stabilize alpha-Enolase 1 (ENO1) to promote aerobic glycolysis. Furthermore, increased USP21 level enhanced chemotherapy resistance to the gemcitabine-based regimen. Taken together, we identify a USP21-regulated aerobic glycolysis mechanism that involves the USP21/HSP90/HIF1A axis and USP21/ENO1 axis in CCA tumorigenesis, which could serve as a potential target for the treatment of CCA.

Exosome-derived miR-182-5p promoted cholangiocarcinoma progression and vasculogenesis by regulating ADK/SEMA5a/PI3K pathway.

BACKGROUND AND AIMS: Increasing evidence suggested that miRNAs regulated the expression of pivotal genes involved in oncogenesis and malignant phenotype. In this project, the purpose was to make an inquiry to the effect and mechanism of miR-182-5p in the progression of cholangiocarcinoma. METHODS: By analysing TCGA and GEO databases, combined with tissue expression levels, miR-182-5p was identified as one of the most valuable miRNAs for research. The function and relationships between miR-182-5p and downstream target genes were both verified by in vitro and in vivo experiments. Methylation-specific PCR and bisulphite sequencing were used to detect the methylation level changes of downstream gene promoter. RESULTS: We found that miR-182-5p could be taken up by exosomes secreted from cholangiocarcinoma. Moreover, exosomal derived miR-182-5p promoted vascular endothelial cell proliferation and migration and induced angiogenesis by targeting ADK/SEMA5a. Subsequently, the PI3K/AKT/mTOR signalling pathway was activated and ultimately caused resistance to gemcitabine and cisplatin. CONCLUSIONS: Our findings suggested that the miR-182-5p/ADK/SEMA5a axis might serve as a potential therapeutic target for cholangiocarcinoma.

Exosomal miR-30a-5p promoted intrahepatic cholangiocarcinoma progression by increasing angiogenesis and vascular permeability in PDCD10 dependent manner.

Tumor-associated angiogenesis positively associates with malignant metastasis of intrahepatic cholangiocarcinoma (ICCA). Cancer cell-derived exosomes carrying microRNAs involves in tumor microenvironment (TME) regulation. We aimed to evaluate exosomal miR-30a-5p in ICCA development. Our data showed that increased miR-30a-5p level was correlated with higher microvascular density (MVD) and worse prognosis. Augmented miR-30a-5p expression was induced by hypoxia induced factor 1α (HIF-1α) in ICCA cell. Further exploration revealed that ICCA-derived miR-30a-5p could be transferred to endothelial and increased endothelial cells recruitment and proliferation, induced angiogenesis and vascular permeability in exosome dependent manner. In addition, circulating exosomal miR-30a-5p was higher in ICCA patients, and correlated with ICCA tissues-expressing miR-30a-5p. Hypoxic stress enhanced the effects of exosomal miR-30a-5p on endothelial-associated phenotypes. Rescued experiments showed that exosomal miR-30a-5p modulated endothelial-associated phenotypes in a way relied on programmed cell death 10 (PDCD10). Moreover, we revealed that the packing of miR-30a-5p into ICCA cells-derived exosomes was mediated by eukaryotic translation initiation factor 4B (EIF4B). More importantly, the combined application of targeting miR-30a-5p and apatinib could synergistically improve antiangiogenic efficacy in ICCA. Combined, ICCA-derived exosomal miR-30a-5p could be an excellent therapeutic and monitoring indicator for ICCA patients.

Hypoxia-induced SKA3 promoted cholangiocarcinoma progression and chemoresistance by enhancing fatty acid synthesis via the regulation of PAR-dependent HIF-1a deubiquitylation.

BACKGROUND: Spindle and kinetochore-associated complex subunit 3 (SKA3) plays an important role in cell proliferation by regulating the separation of chromosomes and their division into daughter cells. Previous studies demonstrated that SKA3 was strongly implicated in tumor development and progression. However, the roles of SKA3 in cholangiocarcinoma (CCA) and the underlying mechanisms remain unclear. METHODS: Next-generation sequencing (NGS) was performed with paired CCA tissues and normal adjacent tissues (NATs). SKA3 was chose to be the target gene because of its remarkably upregulation and unknown function in cholangiocarcinoma in TCGA datasets, GSE107943 datasets and our sequencing results. RT-PCR and immunohistochemistry staining were used to detect the expression of SKA3 in paired CCA tissues and normal adjacent tissues. The SKA3 knockdown and overexpression cell line were constructed by small interfering RNA and lentivirus vector transfection. The effect of SKA3 on the proliferation of cholangiocarcinoma under hypoxic conditions was detected by experiments in vitro and in vivo. RNA-seq was used to find out the differentially expressed pathways in cholangiocarcinoma proliferation under hypoxia regulated by SKA3. IP/MS analysis and Western blot assays were used to explore the specific mechanism of SKA3 in regulating the expression of HIF-1a under hypoxia. RESULTS: SKA3 was up-regulated in NGS, TCGA and GSE107943 databases and was associated with poor prognosis. Functional experiments in vitro and in vivo showed that hypoxia-induced SKA3 promoted cholangiocarcinoma cell proliferation. RNA-sequencing was performed and verified that SKA3 enhanced fatty acid synthesis by up-regulating the expression of key fatty acid synthase, thus promoting cholangiocarcinoma cell proliferation under hypoxic conditions. Further studies indicated that under hypoxic conditions, SKA3 recruited PARP1 to bind to HIF-1a, thus enhancing the poly ADP-ribosylation (PARylation) of HIF-1a. This PARylation enhanced the binding between HIF-1a and USP7, which triggered the deubiquitylation of HIF-1a under hypoxic conditions. Additionally, PARP1 and HIF-1a were upregulated in CCA and promoted CCA cell proliferation. SKA3 promoted CCA cell proliferation and fatty acid synthesis via the PARP1/HIF-1a axis under hypoxic conditions. High SKA3 and HIF-1a expression levels were associated with poor prognosis after surgery. CONCLUSION: Hypoxia-induced SKA3 promoted CCA progression by enhancing fatty acid synthesis via the regulation of PARylation-dependent HIF-1a deubiquitylation. Furthermore, increased SKA3 level enhanced chemotherapy-resistance to gemcitabine-based regimen under hypoxic conditions. SKA3 and HIF-1a could be potential oncogenes and significant biomarkers for the analysis of CCA patient prognosis.

MAD2 activates IGF1R/PI3K/AKT pathway and promotes cholangiocarcinoma progression by interfering USP44/LIMA1 complex.

Spindle assembly checkpoint (SAC) plays an essential part in facilitating normal cell division. However, the clinicopathological and biological significance of mitotic arrest deficient 2 like 1 (MAD2/MAD2L1), a highly conserved member of SAC in cholangiocarcinoma (CCA) remain unclear. We aim to determine the role and mechanism of MAD2 in CCA progression. In the study, we found up-regulated MAD2 facilitated CCA progression and induced lymphatic metastasis dependent on USP44/LIMA1/PI3K/AKT pathway. MAD2 interfered the binding of USP44 to LIMA1 by sequestrating more USP44 in nuclei, causing impaired formation of USP44/LIMA1 complex and enhanced LIMA1 K48 (Lys48)-linked ubiquitination. In therapeutic perspective, the data combined eleven cases of CCA PDTX model showed that high-MAD2 inhibits tumor necrosis and diminishes the inhibition of cell viability after treated with gemcitabine-based regimens. Immunohistochemistry (IHC) analysis of tissue microarray (TMA) for CCA patients revealed that high-MAD2, low-USP44 or low-LIMA1 level are correlated with worse survival for patients. Together, MAD2 activates PI3K/AKT pathway, promotes cancer progression and induces gemcitabine chemo-resistance in CCA. These findings suggest that MAD2 might be an excellent indicator in prognosis analysis and chemotherapy guidance for CCA patients.

ACSL4 serves as a novel prognostic biomarker correlated with immune infiltration in Cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma (CHOL) is the second most common primary hepatic malignant tumor, following hepatocellular carcinoma (HCC). CHOL is highly aggressive and heterogeneous resulting in poor prognosis. The diagnosis and prognosis of CHOL has not improved in the past decade. Acyl-CoA synthetase long-chain family member 4 (ACSL4) is reported to be associated with tumors, however, its role in CHOL has not been revealed. This study is mainly for exploring the prognostic values and potential function of ACSL4 in CHOL. METHODS: We investigated the expression level and prognostic value of ACSL4 in CHOL based on The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. TIMER2.0, TISIDB and CIBERSORT databases were utilized to assess the associations between ACSL4 and immune infiltration cells in CHOL. Single-cell sequencing data from GSE138709 was analyzed to study the expression of ACSL4 in different types of cells. ACSL4 co-expressed genes were analyzed by Linkedomics. Additionally, Western Blot, qPCR, EdU assay, CCK8 assay, transwell assay and wound healing assay were performed to further confirm the roles of ACSL4 in the pathogenesis of CHOL. RESULTS: We found that the level of ACSL4 was higher in CHOL and it was correlated with the diagnosis and prognosis of CHOL patients. Then, we observed that the infiltration level of immune cells was related to the level of ACSL4 in CHOL. Moreover, ACSL4 and its co-expressed genes were mainly enriched in metabolism-related pathway and ACSL4 is also a key pro-ferroptosis gene in CHOL. Finally, knockdown of ACSL4 could reverse the tumor-promoting effect of ACSL4 in CHOL. CONCLUSIONS: The current findings demonstrated ACSL4 may as a novel biomarker for CHOL patients, which might regulate immune microenvironment and metabolism resulting in poor prognosis.

NUF2 Drives Cholangiocarcinoma Progression and Migration via Inhibiting Autophagic Degradation of TFR1.

Cholangiocarcinoma (CCA) is the second most common primary hepatic malignancy and associated with poor prognosis. Lack of therapeutic methods for CCA and insensitivity of targeted therapy and immunotherapy make its treatment challenging. NUF2, a component of Ndc80 kinetochore complex, is implicated in the initiation and development of multiple cancers. However, the role and mechanism of NUF2 in CCA is still unclear. In this research, we investigated the biological processes and underlying mechanisms of NUF2 in CCA. We discovered that the expression of NUF2 was upregulated in CCA and negatively correlated with prognosis. Changes in NUF2 levels had an impact on cell proliferation and migration. Moreover, NUF2 functioned as an oncogene to promote the progression of CCA through p38/MAPK signaling by inhibiting p62 binding of TFR1 and affecting its autophagic degradation. In addition, TFR1 promoted CCA progression and Kaplan-Meier analyses uncovered patients with high expression of TFR1 was associated with the poor survival. In conclusion, our study demonstrated that NUF2 promoted CCA progression by regulating TFR1 protein degradation, and the NUF2/TFR1/MAPK axis could be an excellent therapeutic target for CCA.

KIF14 promotes proliferation, lymphatic metastasis and chemoresistance through G3BP1/YBX1 mediated NF-κB pathway in cholangiocarcinoma.

Cholangiocarcinoma (CCA), a highly lethal and fetal cancer derived from the hepatobiliary system, is featured by aggressive growth and early lymphatic metastasis. Elucidating the underlying mechanism and identifying the effective therapy are critical for advanced CCA patients. In the study, we detected that KIF14 was upregulated in CCA samples, especially in patients with lymph node metastasis and vascular invasion. CCA patients with higher KIF14 were associated with worse overall survival and recurrence-free survival after surgery. Gain-of and loss-of function studies showed that KIF14 enhanced CCA cells proliferation, migration, invasion and lymphatic metastasis whereas its silencing abolished the effects in vivo and in vitro. Mechanistic investigation showed that KIF14 bound to the G3BP1/YBX1 complex and facilitated their interaction, causing increased activity of the NF-κB promoter and activation of NF-κB pathway. Furthermore, increased KIF14 level enhanced chemotherapy-resistance to gemcitabine-based regimen and induced immunosuppressive microenvironment. In addition, KIF14 was direct target of HNF4A and inversely regulated by HNF4A. Together, these findings suggested that KIF14 could be a potential oncogene and a good indicator in predicting prognosis and chemotherapy guidance for CCA patients.

Apolipoprotein A-1 protected hepatic ischaemia-reperfusion injury through suppressing macrophage pyroptosis via TLR4-NF-κB pathway.

BACKGROUND AND AIMS: Apolipoprotein A-1 (ApoA-1), the major apolipoprotein of high-density lipoprotein, plays anti-atherogenic role in cardiovascular diseases and exerts anti-inflammation effect in various inflammatory and infectious diseases. However, the role and mechanism of ApoA-1 in hepatic ischaemia-reperfusion (I/R) injury is unknown. METHODS: In this study, we measured ApoA-1 expression in human liver grafts after transplantation. Mice partial hepatic I/R injury model was made in ApoA-1 knockout mice, ApoA-1 mimetic peptide D-4F treatment mice and corresponding control mice to examine the effect of ApoA-1 on liver damage, inflammation response and cell death. Primary hepatocytes and macrophages were isolated for in vitro study. RESULTS: The results showed that ApoA-1 expression was down-regulated in human liver grafts after transplantation and mice livers subjected to hepatic I/R injury. ApoA-1 deficiency aggravated liver damage and inflammation response induced by hepatic I/R injury. Interestingly, we found that ApoA-1 deficiency increased pyroptosis instead of apoptosis during acute phase of hepatic I/R injury, which mainly occurred in macrophages rather than hepatocytes. The inhibition of pyroptosis compensated for the adverse impact of ApoA-1 deficiency. Furthermore, the up-regulated pyroptosis process was testified to be mediated by ApoA-1 through TLR4-NF-κB pathway and TLR4 inhibition significantly improved hepatic I/R injury. In addition, we confirmed that D-4F ameliorated hepatic I/R injury. CONCLUSIONS: Our study has identified the protective role of ApoA-1 in hepatic I/R injury through inhibiting pyroptosis in macrophages via TLR4-NF-κB pathway. The effect of ApoA-1 may provide a novel therapeutic approach for hepatic I/R injury.

The genomic landscape of cholangiocarcinoma reveals the disruption of post-transcriptional modifiers.

Molecular variation between geographical populations and subtypes indicate potential genomic heterogeneity and novel genomic features within CCA. Here, we analyze exome-sequencing data of 87 perihilar cholangiocarcinoma (pCCA) and 261 intrahepatic cholangiocarcinoma (iCCA) cases from 3 Asian centers (including 43 pCCAs and 24 iCCAs from our center). iCCA tumours demonstrate a higher tumor mutation burden and copy number alteration burden (CNAB) than pCCA tumours, and high CNAB indicates a poorer pCCA prognosis. We identify 12 significantly mutated genes and 5 focal CNA regions, and demonstrate common mutations in post-transcriptional modification-related potential driver genes METTL14 and RBM10 in pCCA tumours. Finally we demonstrate the tumour-suppressive role of METTL14, a major RNA N6-adenosine methyltransferase (m6A), and illustrate that its loss-of-function mutation R298H may act through m6A modification on potential driver gene MACF1. Our results may be valuable for better understanding of how post-transcriptional modification can affect CCA development, and highlight both similarities and differences between pCCA and iCCA.

Safety and Efficacy of Sintilimab and Anlotinib as First Line Treatment for Advanced Hepatocellular Carcinoma (KEEP-G04): A Single-Arm Phase 2 Study.

Purpose: Immune checkpoint inhibitors plus antiangiogenic tyrosine kinase inhibitors may offer a first-line treatment for advanced hepatocellular carcinoma (HCC). In this phase 2 trial [registered with clinicaltrials.gov (NCT04052152)], we investigated the safety and efficacy of first-line anti-PD-1 antibody sintilimab plus antiangiogenic TKI anlotinib for advanced HCC. Methods and Materials: Pathologically-proven advanced HCC patients received sintilimab (200 mg) on day 1 and anlotinib (12 mg) once daily on days 1 to 14 every 3 weeks, with a safety run-in for the first six participants to assess dose-limiting toxicities (DLTs). The primary endpoints were safety and objective response rate (ORR) per RECIST v1.1. Results: Twenty advanced HCC patients were enrolled. No DLTs occurred in the safety run-in. All patients had treatment-related adverse events (TRAEs). Grade 3 TRAEs occurred in 8 (40.0%) patients, the most common being decreased platelet count (10.0%) and increased γ-glutamyl transferase (10.0%). No grade 4/5 TRAEs occurred. Five (25%) patients developed immune-related AEs. The ORR was 35.0% (95%CI 15.4%-59.2%) per RECIST v1.1 and 55.0% (95%CI 31.5%-76.9%) per modified RECIST. At data cutoff (March 31, 2021), the median progression-free survival was 12.2 months (95%CI, 3.8 to not reached). The median PFS was significantly longer in patients with lower LDH levels (not reached [NR], 95% CI, 8.7 to NR vs. higher LDH levels 5.2 months, 95% CI 3.4 to NR; P=0.020) and a CONUT score ≤2 (NR, 95% CI 5.1 to NR vs. CONUT score >2 6.2 months, 95% CI 1.8 to NR; P=0.020). Furthermore, patients showing tumor response had a significantly higher median proportion of CD16+CD56+ NK cells than patients who had stable or progressive disease (21.6% vs. 14.6%; P=0.026). Conclusion: Sintilimab plus anlotinib showed promising clinical activities with manageable toxicity as first-line treatment of advanced HCC.

ROBO1 p.E280* Loses the Inhibitory Effects on the Proliferation and Angiogenesis of Wild-Type ROBO1 in Cholangiocarcinoma by Interrupting SLIT2 Signal.

Background: Cholangiocarcinoma (CCA) remains one of the most lethal malignancies with an increasing incidence globally. Through whole-exome sequencing of 67 CCA tissues, we identified new mutated genes in CCA, including MACF1, METTL14, ROBO1, and so on. The study was designed to explore the effects and mechanism of ROBO1 wild type (ROBO1WT) and ROBO1E280* mutation on the progression of CCA. Methods: Whole-exome sequencing was performed to identify novel mutations in CCAs. In vitro and in vivo experiments were used to examine the function and mechanism of ROBO1WT and ROBO1E280* in cholangiocarcinoma. A tissue microarray including 190 CCA patients and subsequent analyses were performed to indicate the clinical significance of ROBO1. Results: Through whole-exome sequencing, we identified a novel CCA-related mutation, ROBO1E280*. ROBO1 was downregulated in CCA tissues, and the downregulation of ROBO1 was significantly correlated with poor prognosis. ROBO1WT suppressed the proliferation and angiogenesis of CCA in vitro and in vivo, while ROBO1E280* lost the inhibitory effects. Mechanically, ROBO1E280* translocated from the cytomembrane to the cytoplasm and interrupted the interaction between SLIT2 and ROBO1. We identified OLFML3 as a potential target of ROBO1 by conducting RNA-Seq assays. OLFML3 expression was downregulated by ROBO1WT and recovered by ROBO1E280*. Functionally, the silence of OLFML3 inhibited CCA proliferation and angiogenesis and was sufficient to repress the loss-of-function role of ROBO1E280*. Conclusions: These results suggest that ROBO1 may act as a tumor suppressor and potential prognostic marker for CCA. ROBO1E280* mutation is a loss-of-function mutation, and it might serve as a candidate therapeutic target for CCA patients.

Outcomes and recurrence patterns following curative hepatectomy for hepatocellular carcinoma patients with different China liver cancer staging.

This study aimed to investigate outcomes and recurrence patterns after hepatectomy for hepatocellular carcinoma (HCC) patients with different China Liver Cancer staging (CNLC), and then analyze the risk factors of different recurrence patterns. A total of 731 HCC patients undergoing curative resection were reviewed from 6 independent institutions. Data on preoperative and clinicopathological parameters, operation and tumor recurrence information, recurrence management and long-term outcomes were analyzed. Our results showed that 1-, 3-, and 5-year OS rate for Ia was 96.6%, 88.5%, and 77.4%, while 1-, 3-, and 5-year of Ib was 84.2%, 65.5%, and 51.3%, respectively. Compared to Ia, the patients in IIa and IIb staging had poorer 1-, 3-, and 5-year OS and DFS. Furthermore, the 1-, 3-, and 5-year OS rate in IIIa was 59.3%, 37.3%, and 27.7%, while the 1-, 3-, and 5-year OS of IIIb was 25.6%, 12.8%, and 0%, respectively. The mostly site of recurrence after liver surgery was intrahepatic recurrence (CNLC Ia: 89.4%; Ib: 65.9%; IIa: 68.9%; IIb: 91.7%; IIIa: 63.8%). However, the CNLC IIIb patients have higher percentage of extrahepatic recurrence (56.5%). The main recurrence pattern of time course was late recurrence in CNLC Ia patients (61.1%). However, the rate of early recurrence in Ib, IIa, IIb, IIIa, IIIb patients was 69.0%, 62.2%, 62.5%, 78.3% and 95.7% respectively. In conclusion, the outcomes and recurrence patterns of HCC patients after resection vary with different CNLC staging, which defined the prognosis of patients with HCC after resection. The HCC patients with CNLC IIIa can also benefit from liver resection. The CNLC staging could be considered in forming management strategies, treatment choice and surveillance for HCC patients.

Single-cell profiling of human CD127+ innate lymphoid cells reveals diverse immune phenotypes in hepatocellular carcinoma.

BACKGROUND AND AIMS: Innate lymphoid cells (ILCs) are tissue-resident lymphocytes that play critical roles in cytokine-mediated regulation of homeostasis and inflammation. However, relationships between their immune phenotypic characteristics and HCC remain largely unexplored. APPROACH AND RESULTS: We performed single-cell RNA sequencing analysis on sorted hepatic ILC cells from human patients with HCC and validated using flow cytometry, multiplex immunofluorescence staining, and functional experiments. Moreover, we applied selection strategies to enrich ILC populations in HCC samples to investigate the effects of B cells on the immune reaction of inducible T cell costimulator (ICOS)+ ILC2 cells. Dysregulation of ILCs was manifested by the changes in cell numbers or subset proportions in HCC. Seven subsets of 3433 ILCs were identified with unique properties, of which ICOS+ ILC2a were preferentially enriched in HCC and correlated with poor prognosis. Mechanistically, we report that B cells, particularly resting naïve B cells, have a previously unrecognized function that is involved in inflammatory differentiation of ILC2 cells. B cell-derived ICOSL signaling was responsible for exacerbating inflammation through the increased production of IL-13 in ICOS+ ILC2a cells. Heat shock protein 70 (HSP70) genes Heat Shock Protein Family A Member 1A (HSPA1A) and Heat Shock Protein Family A Member 1B (HSPA1B) were highly expressed in ILC2s in late-stage HCC, and targeting to ICOS and its downstream effector HSP70 in ILC2s suppressed tumor growth and remodeled the immunosuppressive tumor microenvironment. CONCLUSIONS: This in-depth understanding sheds light on B cell-driven innate type 2 inflammation and provides a potential strategy for HCC immunotherapy.

Identification of a ferroptosis-related gene signature for predicting the prognosis of cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma (CCA) is a highly intractable malignancy with poor prognosis. Ferroptosis, a newly explored type of programmed cell death, plays a critical role in the initiation and progression of a tumor. Herein, we aimed to identify a ferroptosis-related risk model to evaluate the prognosis of CCA. METHODS: Differentially expressed genes (DEGs) were retrieved from three GEO cohorts. Univariate and LASSO analysis were employed to build a ferroptosis-related gene signature. Next, the predictive value was assessed in a training and a validation cohort. Metascape Online analysis, ESTIMATE and CIBERSORT algorithms, and ssGSEA were employed to perform the functional analysis between different risk groups. Finally, the expression of prognostic genes was validated with RT-qPCR. RESULTS: We identified 51 differentially expressed ferroptosis genes and established the prognostic signature containing five ferroptosis-related genes. The K-M curves and the ROC curves revealed a favorable predictive efficacy of the prognostic signature. Functional enrichment analysis indicated that immune-related responses were greatly enriched between different risk groups. Five prognostic genes were also differentially expressed in CCA cell lines. CONCLUSIONS: We developed a novel ferroptosis-related gene signature for CCA with high predictive accuracy. The analysis of the immune infiltration status may provide a potential therapeutic alternative to CCA.

Hepatic stellate cell activation and senescence induced by intrahepatic microbiota disturbances drive progression of liver cirrhosis toward hepatocellular carcinoma.

BACKGROUND: The significance of the relationship between the microbiota and diseases is increasingly being recognized. However, the characterization of tumor microbiome and their precise molecular mechanisms through which microbiota promotes hepatocellular carcinoma (HCC) development are still unclear. METHODS: The intrahepatic microbiota was investigated from tumor, normal adjacent tissues in 46 patients with HCC and normal hepatic tissues in 33 patients with hemangioma by 16S rRNA gene sequencing. Taxonomic composition differences in patients were evaluated using Linear discriminant analysis Effect Size (LefSe) and Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) to predict microbial functional pathways. Associations between the most relevant taxa and clinical characteristics of HCC patients were analyzed by Spearman rank correlations. The effects of microbe on hepatic stellate cells (HSCs) activation and HCC progression were examined. RESULTS: We observed intrahepatic microbiota disturbances by reduced microbial diversity in HCC. The tumor microbiota of the HCC patients with cirrhosis showed higher abundance of Stenotrophomonas maltophilia (S. maltophilia). S. maltophilia provoked senescence-associated secretory phenotype (SASP) in HSCs by activating TLR-4-mediated NF-κB signaling pathway, which in turn induced NLRP3 inflammasome complex formation and secreted various inflammatory factors in the liver, thus facilitating HCC progression in mice. Moreover, signs of SASP were also observed in the HSCs in the area of HCC with higher S. maltophilia enrichment arising in patients with cirrhosis. CONCLUSIONS: Our analysis of the hepatic microbiota revealed for the first time that patients with HCC exhibited a dysbiotic microbial community with higher S. maltophilia abundance, which induced the expression SASP factors of HSCs and cirrhosis in the liver, concurring in the process of hepatocarcinogenesis.

Protein profiling reveals potential isomiR-associated cross-talks among RNAs in cholangiocarcinoma.

Cholangiocarcinomas (CCAs) are tumors that arise from the cholangiocytes. Although some genes have been shown with important roles in pathological process, interactions or cross-talks among different RNAs are important to understand the detailed molecular mechanisms in cancer development, especially discussing cross-talks among isomiRs and other RNAs. Herein, to characterize crucial genes in CCA, the protein expression profile was performed to survey potential crucial mRNAs and related non-coding RNAs (ncRNAs) in mRNA-ncRNA network, mainly including miRNAs/isomiRs and lncRNAs. Deregulated mRNAs were firstly obtained if consistent expression patterns were found at protein and mRNA levels, and related miRNAs/isomiRs were screened according to regulatory relationships. Diverse isomiRs from a given miRNA locus also contributed to interactions between the small RNAs and target mRNAs, and miRNAs were further used to survey related lncRNAs to expand the interactions. Thus, several groups of RNAs were constructed as candidate competitive endogenous RNA (ceRNA) networks. Finally, we found that RAB11FIP1:miR-101-3p:MIR3142HG may be a potential ceRNA network, and the interactions among them may be more complex due to variety of isomiRs. Simultaneously, RAB11FIP1 and miR-194-5p were also detected other related lncRNAs (FBXL19-AS1, SNHG1 and PVT1) that may be crucial in coding-non-coding RNA regulatory network. Our results show that diverse isomiRs with sequence and expression heterogeneities contribute to ceRNA regulatory network that may have crucial roles in CCA, which will expand our understanding of interactions among diverse RNAs and their contributions in cancer development.