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Abstract Single-cell technology depicts integrated tumor profiles including both tumor cells and tumor microenvironments, which theoretically enables more robust diagnosis than traditional diagnostic standards based on only pathology. However, the inherent challenges of single-cell RNA sequencing (scRNA-seq) data, such as high dimensionality, low signal-to-noise ratio (SNR), sparse and non-Euclidean nature, pose significant obstacles for traditional diagnostic approaches. The diagnostic value of single-cell technology has been largely unexplored despite the potential advantages. Here, we present a graph neural network-based framework tailored for molecular diagnosis of primary liver tumors using scRNA-seq data. Our approach capitalizes on the biological plausibility inherent in the intercellular communication networks within tumor samples. By integrating pathway activation features within cell clusters and modeling unidirectional inter-cellular communication, we achieve robust discrimination between malignant tumors (including hepatocellular carcinoma, HCC, and intrahepatic cholangiocarcinoma, iCCA) and benign tumors (focal nodular hyperplasia, FNH) by scRNA data of all tissue cells and immunocytes only. The efficacy to distinguish iCCA from HCC was further validated on public datasets. Through extending the application of high-throughput scRNA-seq data into diagnosis approaches focusing on integrated tumor microenvironment profiles rather than a few tumor markers, this framework also sheds light on minimal-invasive diagnostic methods based on migrating/circulating immunocytes.

Abstract Intratumoral immune status influences tumor therapeutic response, but it remains largely unclear how the status determines therapies for patients with intrahepatic cholangiocarcinoma. Here, we examine the single-cell transcriptional and TCR profiles of 18 tumor tissues pre- and post- therapy of gemcitabine plus oxaliplatin, in combination with lenvatinib and anti-PD1 antibody for intrahepatic cholangiocarcinoma. We find that high CD8 GZMB+ and CD8 proliferating proportions and a low Macro CD5L+ proportion predict good response to the therapy. In patients with a poor response, the CD8 GZMB+ and CD8 proliferating proportions are increased, but the CD8 GZMK+ proportion is decreased after the therapy. Transition of CD8 proliferating and CD8 GZMB+ to CD8 GZMK+ facilitates good response to the therapy, while Macro CD5L+–CD8 GZMB+ crosstalk impairs the response by increasing CTLA4 in CD8 GZMB+. Anti-CTLA4 antibody reverses resistance of the therapy in intrahepatic cholangiocarcinoma. Our data provide a resource for predicting response of the combination therapy and highlight the importance of CD8+T-cell status conversion and exhaustion induced by Macro CD5L+ in influencing the response, suggesting future avenues for cancer treatment optimization.

Abstract Advanced intrahepatic cholangiocarcinoma (ICC) has a dismal prognosis. Here, we report the efficacy and safety of combining toripalimab, lenvatinib, and gemcitabine plus oxaliplatin (GEMOX) as first-line therapy for advanced ICC. Thirty patients with pathologically confirmed advanced ICC received intravenous gemcitabine (1 g/m2) on Days 1 and 8 and oxaliplatin (85 mg/m2) Q3W for six cycles along with intravenous toripalimab (240 mg) Q3W and oral lenvatinib (8 mg) once daily for one year. The expression of programmed death-ligand 1 (PD-L1) and genetic status was investigated in paraffin-embedded tissues using immunohistochemistry and whole-exome sequencing (WES) analysis. The primary endpoint was the objective response rate (ORR). Secondary outcomes included safety, overall survival (OS), progression-free survival (PFS), disease control rate (DCR) and duration of response (DoR). As of July 1, 2022, the median follow-up time was 23.5 months, and the ORR was 80%. Twenty-three patients achieved partial response, and one achieved complete response. Patients (21/30) with DNA damage response (DDR)-related gene mutations showed a higher ORR, while patients (14/30) with tumor area positivity ≥1 (PD-L1 staining) showed a trend of high ORR, but without significant difference. The median OS, PFS, and DoR were 22.5, 10.2, and 11.0 months, respectively. The DCR was 93.3%. Further, 56.7% of patients experienced manageable grade ≥3 adverse events (AEs), commonly neutropenia (40.0%) and leukocytopenia (23.3%). In conclusion, toripalimab plus lenvatinib and GEMOX are promising first-line regimens for the treatment of advanced ICC. A phase-III, multicenter, double-blinded, randomized study to validate our findings was approved by the National Medical Products Administration (NMPA, No. 2021LP01825). Trial registration Clinical trials: NCT03951597.

Abstract Objective This study evaluated the antitumor activity and safety of pemigatinib in previously treated Chinese patients with advanced cholangiocarcinoma and fibroblast growth factor receptor 2 (FGFR2) fusions or rearrangements. Background Pemigatinib provided clinical benefits for previously treated patients with cholangiocarcinoma carrying FGFR2 fusions or rearrangements and was approved for this indication in multiple countries. Methods In this ongoing, multicenter, single‐arm, phase II study, adult patients with locally advanced or metastatic cholangiocarcinoma carrying centrally confirmed FGFR2 fusions or rearrangements who had progressed on ≥1 systemic therapy received 13.5 mg oral pemigatinib once daily (3‐week cycle; 2 weeks on, 1 week off) until disease progression, unacceptable toxicity, or consent withdrawal. The primary endpoint was objective response rate (ORR) assessed by an independent radiology review committee. Results As of January 29, 2021, 31 patients were enrolled. The median follow‐up was 5.1 months (range, 1.5–9.3). Among 30 patients with FGFR2 fusions or rearrangements evaluated for efficacy, 15 patients achieved partial response (ORR, 50.0%; 95% confidence interval [CI], 31.3–68.7); 15 achieved stable disease, contributing to a disease control rate of 100% (95% CI, 88.4–100). The median time to response was 1.4 months (95% CI, 1.3–1.4), the median duration of response was not reached, and the median progression‐free survival was 6.3 months (95% CI, 4.9–not estimable [NE]). Eight (25.8%) of 31 patients had ≥grade 3 treatment‐emergent adverse events. Hyperphosphatemia, hypophosphatasemia, nail toxicities, and ocular disorders were mostly

Abstract Multidrug resistance is a major challenge in treating advanced hepatocellular carcinoma (HCC). Although recent studies have reported that the multidrug resistance phenotype is associated with abnormal DNA methylation in cancer cells, the epigenetic mechanism underlying multidrug resistance remains unknown. Here, we reported that the level of 5-hydroxymethylcytosine (5-hmC) in human HCC tissues was significantly lower than that in adjacent liver tissues, and reduced 5-hmC significantly correlated with malignant phenotypes, including poor differentiation and microvascular invasion; additionally, loss of 5-hmC was related to chemotherapy resistance in post-transplantation HCC patients. Further, the 5-hmC level was regulated by ten-eleven translocation 2 (TET2), and the reduction of TET2 in HCC contributes to chemotherapy resistance through histone acetyltransferase P300/CBP-associated factor (PCAF) inhibition and AKT signaling hyperactivation. In conclusion, loss of 5-hmC induces chemotherapy resistance through PCAF/AKT axis and is a promising chemosensitivity prediction biomarker and therapeutic target for HCC patients.

Hepatocellular carcinoma (HCC) has a complex and changeable tumor microenvironment. Despite emerging evidence focusing on autophagy process within immune cells, the function and regulatory mechanism of macrophage autophagy in tumor progression remains unclear. Our results of multiplex-immunohistochemistry and RNA-sequencing identified the reduced levels of autophagy in tumor macrophages in the HCC microenvironment, associated with a poor prognosis and increased microvascular metastasis in HCC patients. Specifically, HCC suppressed the macrophage autophagy initiation through the up-regulation of mTOR and ULK1 phosphorylation at Ser757. Knockdown of autophagy-related proteins to further inhibit autophagy significantly boosted the metastatic potential of HCC. Mechanistically, the accumulation of NLRP3 inflammasome mediated by autophagy inhibition promoted the cleavage, maturation, and release of IL-1β, which facilitated the HCC progression, eventually accelerating HCC metastasis via the epithelial-mesenchymal transition. Autophagy inhibition provoked macrophage self-recruitment through the CCL20-CCR6 signaling was also a crucial account of HCC progression. Recruited macrophages mediated the cascade amplification of IL-1β and CCL20 to form a novel pro-metastatic positive feedback loop through promoting HCC metastasis and increased macrophage recruitment, respectively. Notably, targeting IL-1β/IL-1 receptor signaling impaired lung metastasis induced by macrophage autophagy inhibition in a mice HCC lung metastasis model. In summary, this study highlighted that inhibition of tumor macrophage autophagy facilitated HCC progression by increasing IL-1β secretion via NLRP3 inflammasome accumulation and by macrophage self-recruitment through the CCL20 signaling pathway. Interruption of this metastasis-promoting loop by IL-1β blockade may provide a promising therapeutic strategy for HCC patients.

Abstract Background Kinase suppressor of Ras 2 (KSR2) is a regulator of MAPK signaling that is overactivated in most hepatocellular carcinoma (HCC). We sought to determine the role of KSR2 in HCC pathogenesis. Methods We tested the level of KSR2 in HCC tissues and cell lines by tissue microarray, qPCR, and western blotting. Functionally, we determined the effects of KSR2 on the proliferation, migration, and invasion of HCC cells through colony formation assays, scratch assays, transwell migration assays, and xenograft tumor models. Co-immunoprecipitation (co-IP) experiments were used to assess the interaction of phospho-serine binding protein 14–3-3ζ and KSR2, and the effects of this interaction on growth and proliferation of human HCC cells were tested by co-overexpression and knockdown experiments. Additionally, we used flow cytometry to examine whether the KSR2 and 14–3-3ζ interaction conveys HCC resistance to sorafenib. Results KSR2 was significantly upregulated in HCC tissues and cell lines, and high KSR2 expression associated with poor prognosis in HCC patients. KSR2 knockdown significantly suppressed HCC cell proliferation, migration, and invasion in vitro and in vivo. Mechanistically, co-IP experiments identified that 14–3-3ζ complexed with KSR2, and elevated 14–3-3ζ increased KSR2 protein levels in HCC cells. Importantly, Kaplan–Meier survival analysis showed that patients with both high KSR2 and high 14–3-3ζ expression levels had the shortest survival times and poorest prognoses. Interestingly, HCC cells overexpressing both KSR2 and 14–3-3ζ, rather than either protein alone, showed hyperactivated MAPK signaling and resistance to sorafenib. Conclusions Our results provide new insights into the pro-tumorigenic role of KSR2 and its regulation of the MAPK pathway in HCC. The KSR2–14–3-3ζ interaction may be a therapeutic target to enhance the sorafenib sensitivity of HCC.

Abstract Background While the correlation between PD-L1 expression and KRAS mutation has been previously reported in other solid tumors such as non-small cell lung cancer (NSCLC), whether PD-L1 can be modulated by ERK signaling downstream of KRAS in intrahepatic cholangiocarcinoma (iCCA) and the underlying molecular regulatory mechanism remain unclear. Methods The expression of ERK, p-ERK, PD-L1 and autophagy markers following KRAS knockdown or Ras/Raf/MEK/ERK signaling inhibitors treatment was examined in two human iCCA cell lines (HuCCT1 and RBE) using western blotting and immunofluorescence. Both pharmacological autophagy inhibitors and short-interfering RNA against ATG7 were applied to inhibit autophagy. The apoptosis rates of iCCA cell lines were detected by flow cytometry and CCK-8 after co-culturing with CD3/CD28-activated human CD8+ T lymphocytes. Immunohistochemistry was applied to detect the correlation of ERK, p-ERK and PD-L1 in 92 iCCA tissues. Results The present study demonstrated that the PD-L1 expression level was distinctly reduced in KRAS-mutated iCCA cell lines when ERK signaling was inhibited and ERK phosphorylation levels were lowered. The positive association between p-ERK and PD-L1 was also verified in 92 iCCA tissue samples. Moreover, ERK inhibition induced autophagy activation. Both inhibiting autophagy via autophagy inhibitors and genetically silencing the ATG7 expression partially reversed the reduced PD-L1 expression caused by ERK inhibition. In addition, ERK-mediated down-regulation of PD-L1 via autophagy pathways induced the apoptosis of iCCA cells when co-cultured with CD3/CD28-activated human CD8+ T lymphocytes in vitro. Conclusions Our results suggest that ERK signaling inhibition contributes to the reduction of PD-L1 expression through the autophagy pathway in iCCA. As a supplement to anti-PD-1/PD-L1 immunotherapy, ERK-targeted therapy may serve as a potentially novel treatment strategy for human KRAS-mutated iCCA.

Abstract Background Immune checkpoint blockade resistance narrows the efficacy of cancer immunotherapies, but the underlying mechanism remains elusive. Delineating the inherent mechanisms of anti-PD1 resistance is important to improve outcome of patients with advanced HCC. Method The level of cricTMEM181 was measured in HCC patients with anti-PD1 therapy by RNA sequencing and then confirmed by qPCR and Sanger sequencing. Immune status in tumor microenvironment of HCC patients or mice models was evaluated by flow cytometry and IHC. Exosomes from HCC cell lines were isolated by ultracentrifugation, and their internalization by macrophage was confirmed by immunofluorescence. The underlying mechanism of HCC-derived exosomal circTMEM181 to macrophage was confirmed by SILAC, RNA FISH and RNA immunoprecipitation. The ATP–ADO pathway amplified by HCC–macrophage interaction was evaluated through ATP, AMP and ADO measurement and macrophage-specific CD39 knockout mice. The role of circTMEM181 in anti-PD1 therapy and its clinical significance were also determined in our retrospective HCC cohorts. Results Here, we found that circTMEM181 was elevated in hepatocellular carcinoma (HCC) patients responding poorly to anti-PD1 therapy and in HCC patients with a poor prognosis after operation. Moreover, we also found that high exosomal circTMEM181 favored the immunosuppressive microenvironment and endowed anti-PD1 resistance in HCC. Mechanistically, exosomal circTMEM181 sponged miR-488-3p and upregulated CD39 expression in macrophages. Using macrophage-specific CD39 knockout mice and pharmacologic approaches, we revealed a novel mode of anti-PD1 resistance in HCC. We discovered that cell-specific CD39 expression in macrophages and CD73 expression in HCC cells synergistically activated the eATP–adenosine pathway and produced more adenosine, thereby impairing CD8+ T cell function and driving anti-PD1 resistance. Conclusion In summary, HCC-derived exosomal circTMEM181 contributes to immunosuppression and anti-PD1 resistance by elevating CD39 expression, and inhibiting the ATP–adenosine pathway by targeting CD39 on macrophages can rescue anti-PD1 therapy resistance in HCC. Graphical Abstract

Abstract Background Cirrhosis is a recognized risk factor for developing hepatocellular carcinoma (HCC). Few studies have reported the expression profile of circRNAs in HCC samples compared to paratumour dysplastic nodule (DN) samples. Methods The Arraystar Human circRNA Array combined with laser capture microdissection (LCM) was used to analyse the expression profile of circRNAs in HCC samples compared to paratumour DN samples. Then, both in vitro and in vivo HCC models were used to determine the role and mechanism of key circRNA in HCC progression and treatment sensitivity. Results We found that circMEMO1 was significantly downregulated in HCC samples and that the level of circMEMO1 was closely related to the OS and disease-free survival (DFS) of HCC patients. Mechanistic analysis revealed that circMEMO1 can modulate the promoter methylation and gene expression of TCF21 to regulate HCC progression by acting as a sponge for miR-106b-5p, which targets the TET family of genes and increases the 5hmC level. More importantly, circMEMO1 can increase the sensitivity of HCC cells to sorafenib treatment. Conclusion Our study determined that circMEMO1 can promote the demethylation and expression of TCF21 and can be considered a crucial epigenetic modifier in HCC progression.

Abstract Objective Natural killer (NK) cells play a critical role in the innate antitumor immune response. Recently, NK cell dysfunction has been verified in various malignant tumors, including hepatocellular carcinoma (HCC). However, the molecular biological mechanisms of NK cell dysfunction in human HCC are still obscure. Methods The expression of circular ubiquitin-like with PHD and ring finger domain 1 RNA (circUHRF1) in HCC tissues, exosomes, and cell lines was detected by qRT-PCR. Exosomes were isolated from the culture medium of HCC cells and plasma of HCC patients using an ultracentrifugation method and the ExoQuick Exosome Precipitation Solution kit and then characterized by transmission electronic microscopy, NanoSight and western blotting. The role of circUHRF1 in NK cell dysfunction was assessed by ELISA. In vivo circRNA precipitation, RNA immunoprecipitation, and luciferase reporter assays were performed to explore the molecular mechanisms of circUHRF1 in NK cells. In a retrospective study, the clinical characteristics and prognostic significance of circUHRF1 were determined in HCC tissues. Results Here, we report that the expression of circUHRF1 is higher in human HCC tissues than in matched adjacent nontumor tissues. Increased levels of circUHRF1 indicate poor clinical prognosis and NK cell dysfunction in patients with HCC. In HCC patient plasma, circUHRF1 is predominantly secreted by HCC cells in an exosomal manner, and circUHRF1 inhibits NK cell-derived IFN-γ and TNF-α secretion. A high level of plasma exosomal circUHRF1 is associated with a decreased NK cell proportion and decreased NK cell tumor infiltration. Moreover, circUHRF1 inhibits NK cell function by upregulating the expression of TIM-3 via degradation of miR-449c-5p. Finally, we show that circUHRF1 may drive resistance to anti-PD1 immunotherapy in HCC patients. Conclusions Exosomal circUHRF1 is predominantly secreted by HCC cells and contributes to immunosuppression by inducing NK cell dysfunction in HCC. CircUHRF1 may drive resistance to anti-PD1 immunotherapy, providing a potential therapeutic strategy for patients with HCC.

Abstract Background Amplification of chromosome 7q21-7q31 is associated with tumor recurrence and multidrug resistance, and several genes in this region are powerful drivers of hepatocellular carcinoma (HCC). We aimed to investigate the key circular RNAs (circRNAs) in this region that regulate the initiation and development of HCC. Methods We used qRT-PCR to assess the expression of 43 putative circRNAs in this chromosomal region in human HCC and matched nontumor tissues. In addition, we used cultured HCC cells to modify circRNA expression and assessed the effects in several cell-based assays as well as gene expression analyses via RNA-seq. Modified cells were implanted into immunocompetent mice to assess the effects on tumor development. We performed additional experiments to determine the mechanism of action of these effects. Results circMET (hsa_circ_0082002) was overexpressed in HCC tumors, and circMET expression was associated with survival and recurrence in HCC patients. By modifying the expression of circMET in HCC cells in vitro, we found that circMET overexpression promoted HCC development by inducing an epithelial to mesenchymal transition and enhancing the immunosuppressive tumor microenvironment. Mechanistically, circMET induced this microenvironment through the miR-30-5p/Snail/ dipeptidyl peptidase 4(DPP4)/CXCL10 axis. In addition, the combination of the DPP4 inhibitor sitagliptin and anti-PD1 antibody improved antitumor immunity in immunocompetent mice. Clinically, HCC tissues from diabetic patients receiving sitagliptin showed higher CD8+ T cell infiltration than those from HCC patients with diabetes without sitagliptin treatment. Conclusions circMET is an onco-circRNA that induces HCC development and immune tolerance via the Snail/DPP4/CXCL10 axis. Furthermore, sitagliptin may enhance the efficacy of anti-PD1 therapy in a subgroup of patients with HCC.

Intrahepatic cholangiocarcinoma (ICC) is highly invasive and carries high mortality due to limited therapeutic strategies. In other solid tumors, immune checkpoint inhibitors (ICIs) target cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD1), and the PD1 ligand PD-L1 has revolutionized treatment and improved outcomes. However, the relationship and clinical significance of CTLA-4 and PD-L1 expression in ICC remains to be addressed. Deciphering CTLA-4 and PD-L1 interactions in ICC enable targeted therapy for this disease. In this study, immunohistochemistry (IHC) was used to detect and quantify CTLA-4, forkhead box protein P3 (FOXP3), and PD-L1 in samples from 290 patients with ICC. The prognostic capabilities of CTLA-4, FOXP3, and PD-L1 expression in ICC were investigated with the Kaplan–Meier method. Independent risk factors related to ICC survival and recurrence were assessed by the Cox proportional hazards models. Here, we identified that CTLA-4+ lymphocyte density was elevated in ICC tumors compared with peritumoral hepatic tissues (P

Abstract Background Recently, the dysregulation of circular RNA (circRNA) have been shown to have important regulatory roles in cancer development and progression, including hepatocellular carcinoma (HCC). However, the roles of most circRNAs in HCC are still unknown. Methods The expression of circular tripartite motif containing 33–12 (circTRIM33–12) in HCC tissues and cell lines was detected by qRT-PCR. The role of circTRIM33–12 in HCC progression was assessed by western blotting, CCK-8, flow cytometry, transwell and a subcutaneous tumor mouse assays both in vitro and in vivo. In vivo circRNA precipitation, RNA immunoprecipitation, luciferase reporter assays were performed to evaluate the interaction between circTRIM33–12 and miR-191. Results Here, we found that circTRIM33–12, is downregulated in HCC tissues and cell lines. The downregulation of circTRIM33–12 in HCC was significantly correlated with malignant characteristics and served as an independent risk factor for the overall survival (OS) and recurrence-free survival (RFS) of patients with HCC after surgery. The reduced expression of circTRIM33–12 in HCC cells increases tumor proliferation, migration, invasion and immune evasion. Mechanistically, we demonstrated that circTRIM33–12 upregulated TET1 expression by sponging miR-191, resulting in significantly reduced 5-hydroxymethylcytosine (5hmC) levels in HCC cells. Conclusions These results reveal the important role of circTRIM33–12 in the proliferation, migration, invasion and immune evasion abilities of HCC cells and provide a new perspective on circRNAs in HCC progression.

Abstract Background Oxaliplatin-based chemotherapy is widely used to treat hepatocellular carcinoma (HCC). Recent studies suggested that therapeutic resistance of tumors was affected by tumor microenvironment (TME). As a major component of TME, the role of tumor-associated macrophages (TAMs) on drug resistance in HCC is largely unknown. Methods 26 HCC samples were obtained from patients who had underwent transarterial chemoembolization (TACE) within 3 months before receiving curative resections. Immunohistochemistry was applied to detect the density of TAMs in these tissues. SMMC-7721 and Huh-7 cell lines were used to co-culture with THP-1 derived macrophages. Under oxaliplatin treatment, cell death was measured using MTT and annexin V/propidium iodide assays. Autophagy activation was evaluated by GFP-LC3 redistribution and LC3 conversion in SMMC-7721 and Huh-7. Short-interfering RNA against ATG5 gene was applied to inhibit autophagy. In vivo validation was conducted in Huh-7 with or without macrophages using an HCC xenograft model in nude mice after oxaliplatin administration. Results We found that the density of TAMs in HCC samples was associated with the efficacy of TACE. Macrophages inhibited cell death induced by oxaliplatin in HCC cells. Autophagy was functionally activated in HCC cells after co-culturing with macrophages. Suppression of autophagy using RNA interference of ATG5 in HCC cells promoted the oxaliplatin cytotoxicity in the co-culture system. Critically, co-implantation with macrophages in HCC xenografts weakens cytotoxic effect of oxaliplatin through inducing autophagy to avoid apoptosis. Conclusions Our results suggest that TAMs induce autophagy in HCC cells which might contribute to oxaliplatin resistance. Targeting TAMs is a promising therapeutic strategy to enhance the effects of chemotherapy oxaliplatin in HCC patients.

Abstract Background RING finger protein 38 (RNF38), a member of the RNF protein family, has just emerged as a vital driver of cancer progression. However, the oncogenic mechanisms of RNF38 remain unexplored. Methods Using frozen tumor tissue and tissue microarray from hepatocellular carcinoma (HCC) patients, we tried to probe the expression of RNF38 in HCC and its clinical value. Then the biological functions of RNF38 were analyzed in vivo and vitro. Stable isotope labeling with amino acids (SILAC) in cell culture and co-immunoprecipitation proteomic analyses were combined to reveal the potential mechanism of RNF38 in HCC progression. Results We report that RNF38 expression was markedly higher in HCC tissues than in peritumor tissues. Correspondingly, RNF38 overexpression promoted the HCC cell migration and invasion and inhibited apoptosis both in vitro and in vivo. And elevated RNF38 expression induced HCC cell epithelial-mesenchymal transition by facilitating transforming growth factor-β (TGF-β) signaling via ubiquitinating and degrading neuroblast differentiation-associated protein (AHNAK), a well-established inhibitor of TGF-β signaling. Furthermore, AHNAK interference restored the HCC cell invasion and metastasis deprived by RNF38 downregulation. Clinically, elevated RNF38 and transforming growth factor beta receptor 1 (TGFBR1) expression was related to short overall survival (OS) and high cumulative recurrence rates in HCC patients. Conclusions High levels of RNF38 promote HCC by facilitating TGF-β signaling and are a novel marker for predicting the prognosis of HCC patients and a potential therapeutic target in HCC.

Abstract Background This study was performed to investigate the role of nucleotide-binding oligomerization domain (NOD)-like receptor X1 (NLRX1) in regulating hepatocellular carcinoma (HCC) progression. Methods Expression levels of NLRX1 in clinical specimens and cell lines were determined by reverse transcription-polymerase chain reaction (RT-PCR) and western blot (WB). Transwell assays were conducted to evaluate the effect of NLRX1 on cell invasion, and flow cytometry was used to assess apoptosis. Expression patterns of key molecules in the phosphoinositide 3-kinase (PI3K)-AKT pathways were determined via WB. The effect of NLRX1 on cell senescence was evaluated with β-galactosidase assays. Kaplan-Meier analyses and Cox regression models were used for prognostic evaluation. Results NLRX1 was downregulated in tumor tissue compared with adjacent normal liver tissue. Low tumor NLRX1 expression was identified as an independent indicator for HCC prognosis (recurrence: hazard ratio [HR] 1.87, 95% confidence interval [CI] 1.26–2.76, overall survival [OS] 2.26, 95% CI 1.44–3.56). NLRX1 over-expression (OE) significantly inhibited invasiveness ability and induced apoptosis in HCC cells. In vivo experiments showed that NLRX1 knock-down (KD) significantly promoted HCC growth. Mechanistically, NLRX1 exhibited a suppressor function by decreasing phosphorylation of AKT and thus downregulating Snail1 expression, which inhibited epithelial-mesenchymal-transition (EMT) in HCC cells. Moreover, NLRX1 OE could induce cell senescence via an AKT-P21-dependent manner. Conclusions NLRX1 acted as a tumor suppressor in HCC by inducing apoptosis, promoting senescence, and decreasing invasiveness by repressing PI3K-AKT signaling pathway. Future investigations will focus on restoring expression of NLRX1 to provide new insights into HCC treatment.

Abstract Background The conception that serological hepatitis markers determined surgical prognosis of hepatocellular carcinoma (HCC) associated with hepatitis B (HBV) or hepatitis C (HCV) has been well defined. However, little is known about the relationship between surgical outcomes and serological hepatitis markers in patients with dual HBV and HCV related HCC. Methods A retrospective analysis of the clinical data of 39 HCC patients with HBV-HCV coinfection who underwent curative hepatectomy between 2001 and 2011 was performed. HBV DNA quantification, expression of HBV antigens, anti-HCV signal-to-cutoff ratio (S/CO) and some clinicopathological characteristics were investigated to show the potential relationship among them and the surgical prognosis. Results The Cox proportional hazards model identified that HBV DNA quantification of 1,000 IU/mL or higher, HBeAg seropositivity, tumor size of greater than 5 cm, multiple tumors, and vascular invasion were risk factors for HCC prognosis. Thus, HBV DNA quantification, HBsAg level, HBeAg status and HCV-Ab level which may reveal the hepatitis status were further analyzed. The overall survival time in the group with high (≥1,000 IU/mL) HBV DNA quantification was significantly lower than the group with low (

The aim of this study is to investigate the expression profile of multiple epithelial mesenchymal transition (EMT)-related molecules in intrahepatic cholangiocarcinoma (ICC) and the related prognostic significance.Immunohistochemistry was performed to determine the expression of E-cadherin, Vimentin, Snail, slug and β-catenin in a tissue microarray consisting of tumor tissues of 140 ICC patients undergoing curative resection. The correlation between the expression of these molecules and the clinicopathological characteristics of ICC patients was analyzed, and their prognostic implication was evaluated.Reduced E-cadherin and increased Vimentin expression, the characteristic changes of EMT, identified in 55.0% and 55.7% of primary ICCs, respectively, were correlated with lymphatic metastasis and poorer overall survival (OS) and disease-free survival (DFS) of ICCs. The overexpression of snail and nonmembranous β-catenin, which are the major regulators of the EMT, were identified in 49.2% and 45.7% of primary ICCs, while little slug expression was detected in ICCs. Cytoplasmic/nuclear β-catenin did not significantly predict worse DFS and was not related with E-cadherin loss. The overexpression of snail predicted worse OS and DFS. Snail overexpression correlated with the down-regulation of E-cadherin and the up-regulation of Vimentin. Inhibition of snail in an ICC cell line decreased the expression of E-cadherin, enhanced the expression of Vimentin and impaired the invasion and migration ability of ICC cells.These data support the hypothesis that EMT plays vital roles in ICC progression and suggest that snail but not slug and β-catenin plays a crucial role in the EMT induction of ICC.

Frequent deletions of the kinesin-like protein gene 1B (KIF1B) have been reported in neural tumors. Recently, a genome-wide association study revealed an association between polymorphisms in the KIF1B gene and the risk of hepatocellular carcinoma (HCC), and several case-control studies have further investigated this relationship. However, these studies have yielded controversial results. We therefore performed a meta-analysis to derive a more precise estimation of the association between the KIF1B gene polymorphisms and HCC risk.PubMed, EMBASE, the ISI Web of Science and the CNKI databases were systematically searched to identify relevant studies. A total of 5 studies containing 13 cohorts with 5,773 cases and 6,404 controls were included. Odds ratios (ORs) with corresponding 95% confidence intervals (CIs) were used to assess the strength of the associations. Subgroup analyses were conducted based on ethnicities, sample sizes and quality scores. Overall, the G allele at rs17401966 of the KIF1B gene was associated with a significantly decreased risk for HCC (OR = 0.81, 95%CI: 0.70-0.93; P = 0.003). Furthermore, subgroup analyses showed that the G allele at rs17401966 of the KIF1B gene significantly reduced the risk for HCC in Chinese cohorts (OR = 0.76, 95%CI: 0.64-0.90; P = 0.002), large-sample-size cohorts (OR = 0.80, 95%CI: 0.73-0.88, P

BACKGROUND: Calpain small subunit 1 (Capn4) has been shown to correlate with the metastasis/invasion of hepatocellular carcinoma. This study aimed to investigate the role of Capn4 in intrahepatic cholangiocarcinoma (ICC). METHODS: Capn4 expression was measured in 33 ICC tissues by quantitative real-time polymerase chain reaction and western blot. The role of Capn4 in the migration, invasion and proliferation of ICC cells and matrix metalloproteinase 2 (MMP2) expression were assessed after Capn4 depletion by specific small interfering RNA. Capn4 expression was further examined by immunohistochemistry in a tissue microarray consisting of 140 ICC patients and 13 normal liver tissues, and the prognostic role of Capn4 in ICC was evaluated by Kaplan-Meier and Cox regression analyses. RESULTS: Capn4 expression was significantly higher in the ICC tissues compared to the peritumor tissues. Capn4 down-regulation impaired the migration/invasion ability of HCCC-9810 and QBC939 cells in vitro and decreased MMP2 expression. Capn4 overexpression significantly correlated with the presence of lymphatic metastasis of ICC (p = 0.026) and the tumor-node-metastasis (TNM) stage (p = 0.009). The postoperative 2- and 5-year overall survivals in patients with Capn4(low) were higher than those in the Capn4(high) group. The cumulative recurrence rate in patients with Capn4(low) was much lower than in the Capn4(high) group. Multivariate analysis showed that Capn4 overexpression was an independent prognostic marker in ICC. CONCLUSIONS: Capn4 overexpression was implicated in ICC metastasis/invasion, and Capn4 overexpression may be used as a molecular therapeutic target for ICC.

BACKGROUND: Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional receptor involved in receptor-mediated endocytosis and cell signaling. The aim of this study was to elucidate the expression and mechanism of LRP1 in hepatocellular carcinoma (HCC). METHODS: LRP1 expression in 4 HCC cell lines and 40 HCC samples was detected. After interruption of LRP1 expression in a HCC cell line either with specific lentiviral-mediated shRNA LRP1 or in the presence of the LRP1-specific chaperone, receptor-associated protein (RAP), the role of LRP1 in the migration and invasion of HCC cells was assessed in vivo and in vitro, and the expression of matrix metalloproteinase (MMP) 9 in cells and the bioactivity of MMP9 in the supernatant were assayed. The expression and prognostic value of LRP1 were investigated in 327 HCC specimens. RESULTS: Low LRP1 expression was associated with poor HCC prognosis, with low expression independently related to shortened overall survival and increased tumor recurrence rate. Expression of LRP1 in non-recurrent HCC samples was significantly higher than that in early recurrent samples. LRP1 expression in HCC cell lines was inversely correlated with their metastatic potential. After inhibition of LRP1, low-metastatic SMCC-7721 cells showed enhanced migration and invasion and increased expression and bioactivity of MMP9. Correlation analysis showed a negative correlation between LRP1 and MMP9 expression in HCC patients. The prognostic value of LRP1 expression was validated in the independent data set. CONCLUSIONS: LRP1 modulated the level of MMP9 and low level of LRP1 expression was associated with aggressiveness and invasiveness in HCCs. LRP1 offered a possible strategy for tumor molecular therapy.

BACKGROUND:Recurrence prediction of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) patients undergoing liver transplantation (LT) present a great challenge because of a lack of biomarkers. Genetic variations play an important role in tumor development and metastasis. METHODS:Oligonucleotide microarrays were used to evaluate the genetic characteristics of tumor DNA in 30 HBV-related HCC patients who were underwent LT. Recurrence-related single-nucleotide polymorphism were selected, and their prognostic value was assessed and validated in two independent cohorts of HCC patients (N = 102 and N = 77), using pretransplant plasma circulating DNA. Prognostic significance was assessed by Kaplan-Meier survival estimates and log-rank tests. Multivariate analyses were performed to evaluate prognosis-related factors. RESULTS:rs894151 and rs12438080 were significantly associated with recurrence (P = .003 and P = .004, respectively). Multivariate analyses demonstrated that the co-index of the 2 SNPs was an independent prognostic factor for recurrence (P = .040). Similar results were obtained in the third cohort (N = 77). Furthermore, for HCC patients (all the 3 cohorts) exceeding Milan criteria, the co-index was a prognostic factor for recurrence and survival (P

Tetraspanin CD151 has been implicated in metastasis through forming complexes with different molecular partners. In this study, we mapped tetraspanin web proteins centered on CD151, in order to explore the role of CD151 complexes in the progression of hepatocellular carcinoma (HCC). Immunoprecipitation was used to isolate tetraspanin complexes from HCCLM3 cells using a CD151 antibody, and associated proteins were identified by mass spectrometry. The interaction of CD151 and its molecular partners, and their roles in invasiveness and metastasis of HCC cells were assayed through disruption of the CD151 network. Finally, the clinical implication of CD151 complexes in HCC patients was also examined. In this study, we identified 58 proteins, characterized the tetraspanin CD151 web, and chose integrin β1 as a main partner to further investigate. When the CD151/integrin β1 complex in HCC cells was disrupted, migration, invasiveness, secretion of matrix metalloproteinase 9, and metastasis were markedly influenced. However, both CD151 and integrin β1 expression were untouched. HCC patients with high expression of CD151/integrin β1 complex had the poorest prognosis of the whole cohort of patients. Together, our data show that CD151 acts as an important player in the progression of HCC in an integrin β1-dependent manner.