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Objective: Protein convertase subtilisin/Kexin type 9 (PCSK9) has been found to be closely associated with the occurrence and development of numerous tumors. However, the precise role of PCSK9 and its relationship to the development of hepatocellular carcinoma (HCC) remain largely unknown. This study aimed to clarify these issues. Methods: The expression levels of PCSK9 in HCC tissues and HCC cell lines were determined by the quantitative reverse transcription polymerase chain reaction, Western blot, and immunohistochemical analyses, and the effects of PCSK9 expression on HCC cell biological traits were investigated by overexpressing and downregulating PCSK9 expression in vivo and in vitro. Additionally, the mechanism by which PCSK9 mediated dissociation of glutathione S-transferase Pi 1 (GSTP1) dimers and phosphorylation of the Jun N-terminal kinase (JNK) pathway components were investigated. Results: PCSK9 expression levels were significantly lower in HCC tissues than in adjacent non-tumor samples. In vivo and in vitro experiments suggested that PCSK9 inhibited HCC cell proliferation and metastasis. Further analysis showed that PCSK9 interacted with GSTP1 and promoted GSTP1 dimer dissociation and JNK signaling pathway inactivation in HCC cells. Moreover, the relationships between PCSK9 protein expressions and clinical outcomes were investigated. The PCSK9-lo group displayed a significantly shorter overall survival (OS; median OS: 64.2 months vs. 83.2 months; log-rank statistic: 4.237; P = 0.04) and recurrence-free survival (RFS; median RFS: 26.5 months vs. 46.6 months; log-rank statistic: 10.498; P = 0.001) time than the PCSK9-hi group. Conclusions: PCSK9 inhibited HCC cell proliferation, cell cycle progression, and apoptosis by interacting with GSTP1 and suppressing JNK signaling, suggesting that PCSK9 might act as a tumor suppressor and be a therapeutic target in HCC patients.

Summary The discovery that mutations in the EGFR gene are detected in up to 50% of lung adenocarcinoma patients, along with the development of highly efficacious epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), has revolutionized the treatment of this frequently occurring lung malignancy. Indeed, the clinical success of these TKIs constitutes a critical milestone in targeted cancer therapy. Three generations of EGFR-TKIs are currently approved for the treatment of EGFR mutation-positive non-small cell lung cancer (NSCLC). The first-generation TKIs include erlotinib, gefitinib, lapatinib, and icotinib; the second-generation ErbB family blockers include afatinib, neratinib, and dacomitinib; whereas osimertinib, approved by the FDA on 2015, is a third-generation TKI targeting EGFR harboring specific mutations. Compared with the first- and second-generation TKIs, third-generation EGFR inhibitors display a significant advantage in terms of patient survival. For example, the median overall survival in NSCLC patients receiving osimertinib reached 38.6 months. Unfortunately, however, like other targeted therapies, new EGFR mutations, as well as additional drug-resistance mechanisms emerge rapidly after treatment, posing formidable obstacles to cancer therapeutics aimed at surmounting this chemoresistance. In this review, we summarize the molecular mechanisms underlying resistance to third-generation EGFR inhibitors and the ongoing efforts to address and overcome this chemoresistance. We also discuss the current status of fourth-generation EGFR inhibitors, which are of great value in overcoming resistance to EGFR inhibitors that appear to have greater therapeutic benefits in the clinic., Graphical abstract, Public summary • EGFR gene mutations are detected in about 50% of non-small cell lung cancer (NSCLC) patients worldwide • The three generations of EGFR tyrosine kinase inhibitors (TKIs) are critical milestones for NSCLC patients • Like other targeted therapies, new EGFR mutations and coupled drug resistances emerge rapidly after TKI treatment, posing formidable obstacles to cancer management • The investigational fourth-generation EGFR inhibitors are of great promise, through a number of novel mechanisms, in overcoming these resistances after third-generation TKI treatment, and will bring more benefits to NSCLC patients

Background Fibrinogen-like protein 1 (FGL1)—Lymphocyte activating gene 3 (LAG-3) pathway is a promising immunotherapeutic target and has synergistic effect with programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1). However, the prognostic significance of FGL1-LAG-3 pathway and the correlation with PD-L1 in hepatocellular carcinoma (HCC) remain unknown. Methods The levels of LAG-3, FGL1, PD-L1 and cytotoxic T (CD8+T) cells in 143 HCC patients were assessed by multiplex immunofluorescence. Associations between the marker’s expression and clinical significances were studied. Results We found FGL1 and LAG-3 densities were elevated while PD-L1 and CD8 were decreased in HCC tissues compared to adjacent normal liver tissues. High levels of FGL1 were strongly associated with high densities of LAG-3+cells but not PD-L1. CD8+ T cells densities had positive correlation with PD-L1 levels and negative association with FGL1 expression. Elevated densities of LAG-3+cells and low levels of CD8+ T cells were correlated with poor disease outcome. Moreover, LAG-3+cells deteriorated patient stratification based on the abundance of CD8+ T cells. Patients with positive PD-L1 expression on tumor cells (PD-L1 TC+) tended to have an improved survival than that with negative PD-L1 expression on tumor cells (PD-L1 TC−). Furthermore, PD-L1 TC− in combination with high densities of LAG-3+cells showed the worst prognosis, and PD-L1 TC+ patients with low densities of LAG-3+cells had the best prognosis. Conclusions LAG-3, FGL1, PD-L1 and CD8 have distinct tissue distribution and relationships with each other. High levels of LAG-3+cells and CD8+ T cells represent unfavorable and favorable prognostic biomarkers for HCC respectively.

Identifying novel prognostic biomarkers for hepatocellular carcinoma (HCC) and then, develop an effective individualized treatment strategy remain extremely warranted. The prognostic role of sulfiredoxin‐1(SRXN1), an antioxidant enzyme, remains unknown in HCC. This study aimed to explore the prognostic implications of SRXN1 in HCC patients after partial hepatectomy. The expression of SRXN1 in HCC and normal tissue were analyzed using the patients from the public databases and Zhongshan Hospital. The Cox regression, Kaplan‐Meier survival analysis, and time‐dependent receiver operating characteristic curves were performed to identify the predictive role of SRXN1 expression on HCC patients. A prognostic nomogram based on SRXN1 expression was constructed and validated to further confirm the predictive power of SRXN1 as a prognostic biomarker. Finally, functional enrichment analysis and protein‐protein interaction network analysis of SRXN1 and its associated genes were conducted. The results showed that SRXN1 was upregulated in HCC samples compared with the normal liver tissues. Patients with SRXN1 upregulation had shorter survival time. SRXN1 overexpression was significantly correlated with advanced clinicopathological parameters. The prognostic nomogram based on SRXN1 expression was proved to be more accurate than routine staging systems for the prediction of overall survival. Protein‐protein interaction network analysis demonstrated the first neighbor genes of SRXN1 mainly participated in response to oxidative stress. In brief, SRXN1 could be a prognostic biomarker for the management of HCC., In this work, we evaluated the prognostic power of SRXN1 for hepatocellular carcinoma using clinical specimens from both the public databases and Zhongshan Hospital. We also established a prognostic nomogram based on SRXN1 expression, which was proved to be more accurate than the routine staging systems for the prediction of overall survival. Finally, functional and pathway enrichment analysis and PPI network analysis of SRXN1 and its related genes were conducted, respectively, which may shed light on the mechanism of SRXN1 as an oncogene.

Background/Aims: Intrahepatic cholangiocarcinoma (ICC) is a complicated condition, with difficult diagnosis and poor prognosis. The expression and clinical significance of the farnesoid X receptor (FXR), an endogenous receptor of bile acids, in ICC is not well understood. Methods: Western blotting and immunochemical analyses were used to determine the levels of FXR in 4 cholangiocarcinoma cell lines, a human intrahepatic biliary epithelial cell line (HIBEpic) and 322 ICC specimens, respectively, while quantitative reverse transcription polymerase chain reaction was used to detect the mRNA levels of FXR in cholangiocarcinoma cell lines. We evaluated the prognostic value of FXR expression and its association with clinical parameters. We determined the biological significance of FXR in ICC cell lines by agonist-mediated activation and lentivirus-mediated silence. IL-6 expression was tested by an enzyme-linked immunosorbent assay and flow cytometry. In vitro, cell proliferation was examined by Cell Counting Kit-8, migration and invasion were examined by wound healing and transwell assays; in vivo, tumor migration and invasion were explored in NOD-SCID mice. Results: FXR was downregulated in ICC cell lines and clinical ICC specimens. Loss of FXR was markedly correlated with aggressive tumor phenotypes and poor prognosis in patients with ICC. Moreover, FXR expression also had significant prognostic value in carbohydrate antigen 19-9 (CA19-9) negative patients. The expression of FXR was negatively correlated with IL-6 levels in clinical ICC tissues. FXR inhibited the proliferation, migration, invasion and epithelial mesenchymal transition (EMT) of ICC cells via suppression of IL-6 in vitro. Obeticholic acid, an agonist of FXR, inhibited IL-6 production, tumor growth and lung metastasis of ICC in vivo. Conclusions: FXR could be a promising ICC prognostic biomarker, especially in CA19-9 negative patients with ICC. FXR inhibits the tumor growth and metastasis of ICC via IL-6 suppression.

Developing predictive biomarkers that can detect the tipping point before metastasis of hepatocellular carcinoma (HCC), is critical to prevent further irreversible deterioration. To discover such early-warning signals or biomarkers of pulmonary metastasis in HCC, we analyse time-series gene expression data in spontaneous pulmonary metastasis mice HCCLM3-RFP model with our dynamic network biomarker (DNB) method, and identify CALML3 as a core DNB member. All experimental results of gain-of-function and loss-of-function studies show that CALML3 could indicate metastasis initiation and act as a suppressor of metastasis. We also reveal the biological role of CALML3 in metastasis initiation at a network level, including proximal regulation and cascading influences in dysfunctional pathways. Our further experiments and clinical samples show that DNB with CALML3 reduced pulmonary metastasis in liver cancer. Actually, loss of CALML3 predicts shorter overall and relapse-free survival in postoperative HCC patients, thus providing a prognostic biomarker and therapy target in HCC.

Three carbapenem-resistant clinical isolates of the Enterobacter cloacae complex (ECC) were recovered from different patients in a hospital. All 3 isolates carried 2 carbapenemase genes blaKPC-2 and blaNDM-1. A study was performed to characterize their relatedness and to investigate possible links among the patients. Whole genome sequencing revealed that the isolates were Enterobacter hormaechei and belonged to ST177 of the ECC. There were 19-142 single nucleotide polymorphisms (SNPs) between the isolates, suggesting that the isolates were likely from a central reservoir, which might have existed for some time. blaKPC-2 and blaNDM-1 were carried on 2 different IncF-type plasmids in the isolates. The 3 blaNDM-1-carrying plasmids were almost identical and were self-transmissible, while the blaKPC-2-carrying plasmids were only transmissible in the presence of the blaNDM-1-carrying plasmid. The source of and direct links among them were not identified, suggesting a hospital transmission of a common multidrug resistant strain.

Cytokines play an important role in the development of tumors. The purpose of the present study was to evaluate the mechanisms and cytokine level changes after transarterial chemoembolization (TACE) in patients with hepatocellular carcinoma (HCC). The Short Time-series Expression Miner (STEM) program was utilized to cluster cytokine expression profiles from the day before TACE to day 21 post-TACE. Based on the identified significant signatures, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed. Cytokines were serially monitored in 60 evaluable patients to identify the results of the STEM program. Examination of the significant signatures identified 6 significant time-varied expression patterns for 507 cytokines (profiles 16, 18, 28, 41, 42 and 43). GO analysis was enriched in ‘cytokine receptor-binding’ and ‘cytokine receptor activity’, and the identified signaling pathways included ‘cytokine-cytokine receptor interaction’ and the ‘JAK-STAT signaling pathway’. Ciliary neurotrophic factor (CNTF) level was increased early after TACE, reaching a peak on day 7 before finally decreasing from day 14 onwards, and was significantly positively correlated with aminotransferase level. Serum levels of pre-TACE IL-10 predicted the local tumor response and overall survival (OS) of the patients, while serum levels of post-TACE IL-1β only indicated the local tumor response of the patient. Overall, the present study identified cytokine time-series expression profiles of patients with HCC undergoing TACE. Early phase increases in CNTF after TACE were associated with post-treatment hepatic injury. IL-1β may reflect an objective response after TACE, while IL-10 may represent a biomarker for OS and the objective response pre-TACE, which may help patients with HCC to benefit from TACE.

Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide that responds poorly to existing therapies. The Casein kinase 1 (CK1) isoforms CK1δ and CK1e are reported to be highly expressed in several tumor types, and both genetic and pharmacological inhibition of CK1δ/e activity has deleterious effects on tumor cell growth. IC261, an CK1δ/e selectively inhibitor, shows anti-tumor effect against pancreatic tumor and glioblastoma, but its role in HCC remains poorly characterized. In our research, IC261 displayed time- and dose-dependent inhibition of HCC cell proliferation, and induced G2/M arrest and cell apoptosis in vitro. However, the anti-tumor effects of IC261 was independent of CK1δ/e. Additionally, IC261 was verified to induce centrosome fragmentation during mitosis independent of CK1δ status, and intraperitoneal injection of IC261 to HCCLM3 xenograft models inhibited tumor growth. Taken together, our data indicated that IC261 has therapeutic potential for HCC.

Ethnopharmacological relevance Many studies have confirmed that traditional Chinese herbs exert potential anti-tumor effects. Actinidia Chinensis Planch root has been used as a traditional Chinese medicine (TCM) for thousands of years. However, the mechanism of anti-tumor effects of Actinidia Chinensis Planch root has not been clearly clarified. Aim of the study To explore the molecular biological mechanisms underlying the inhibitory effect of Actinidia Chinensis Planch root extract (acRoots) on hepatocellular carcinoma (HCC). Materials and methods In our previous study, we used mRNA chip analyses to identify genes regulated by acRoots. Further analyses of altered genes led to the identification of a key regulator of genes that responds to acRoots. We explored the effects of acRoots on the proliferation and invasion of HCC cells via cell counting as well as transwell assays, and further explored the molecular mechanisms underlying the effects of acRoots on HCC cells using qRT-PCR, western blot, and Chip-PCR. Results Increasing the concentration of acRoots as well as prolonging its action time enhanced the inhibitory activity of acRoots as well as its cytotoxicity against HCC cells. High TARBP2 expression in HCC cells, which is associated with advanced-stage HCC and poor prognoses in HCC patients, was downregulated by treatment with acRoots. Furthermore, acRoots inhibited proliferation, invasion, and epithelial-to-mesenchymal transition by downregulating TARBP2 expression. HCC cells with higher TARBP2 expression were more sensitive to acRoots. The expression of TARBP2 and DLX2 in HCC patients and HCC cell lines was significantly positively correlated, and DLX2 as a transcription factor may promote TARBP2 expression, thereby further activating the JNK/AKT signaling pathway leading to the inhibition of HCC. Conclusions acRoots inhibited the malignant behavior of HCC cells by inhibiting TARBP2 expression, which is affected by the transcription factor DLX2, leading to a reduction in JNK/AKT signaling pathway activation.

TIPE1 (tumor necrosis factor-α-induced protein 8-like 1) contributes to cell death in diverse cancers. However, the expression and biological functions of TIPE1 in colon cancer remain unclear. In the present study, we report that TIPE1 was downregulated in colon cancer tissues and positively correlates with prognosis of colon cancer patients. TIPE1 overexpression significantly inhibits colon cancer cell growth both in vitro and in vivo through impairing stemness, accompanied with downregulation of the stemness-related markers, ALDH, CD133, CD44 and SOX-9. Mechanically, TIPE1 directly targets β-catenin and promotes β-catenin degradation in a protease-dependent manner, and Wnt/β-catenin signaling plays a crucial role during TIPE1-mediated stemness inhibition in colon cancer. These findings reveal that TIPE1 exerts anti-tumor effects in colon cancer and suggest that TIPE1 would be a therapeutic target for cancers.

Objectives LncRNA nuclear‐enriched abundant transcript 1 (NEAT1) participates in the development and progression of multiple malignancies. However, the molecular mechanism by which NEAT1 contributes to colorectal cancer (CRC) remains unclear. Methods The association between lncRNA NEAT1 expression and clinicopathological characteristics and prognosis in patients with CRC was analysed by TCGA RNA‐sequencing data. MTT, colony formation, flow cytometry, transwell assays and a xenograft tumour model were used to assess the functions of NEAT1. Bioinformatics and spearman correlation analysis were used to identify the NEAT1‐specific binding with miRNAs, and luciferase gene report and RIP assays were performed to confirm the interaction between miR‐193a‐3p (miR‐193a) and NEAT1 in CRC cells. Results Upregulation of NEAT1 expression was significantly correlated with TNM stage, poor survival and tumour recurrence in patients with CRC, and acted as an independent prognostic factor for tumour recurrence. Knockdown of NEAT1 suppressed cell proliferation, colony formation abilities and invasive potential and induced cell apoptosis, but overexpression of NEAT1 reversed these effects. Furthermore, NEAT1 was confirmed to act as a sponge of miR‐193a, and knockdown of NEAT1 attenuated miR‐193a inhibitor‐induced tumour promoting effects and L17RD expression in CRC cells. miR‐193a harboured negative correlation with NEAT1 and IL17RD expression in CRC specimens. In vivo experiment further validated the inhibitory effects of NEAT1 knockdown on xenograft tumour growth. Conclusion Our findings demonstrate that lncRNA NEAT1 acts as an oncogenic role in CRC cells by sponging miR‐193a and may represent a potential marker for CRC patients.

Aim Phospholipase C-γ1 (PLCG1) was previously found to be involved in a variety of oncogenic behaviors such as cell motility, cell proliferation, cell migration, and invasion. However, its function in hepatocellular carcinoma (HCC) was unknown. Here, we explored the expression pattern and function of PLCG1 in HCC progression. Methods Expression of PLCG1 was examined by western blotting in hepatoma cells and human tumor tissues. Expression was also detected by immunohistochemistry in 150 HCC clinical samples, and its clinical significance was analyzed. The influence of PLCG1 on HCC carcinogenesis were determined in vitro and in vivo. The underlying mechanisms were explored by detecting the expression of critical molecules of signaling pathways. Results The results showed that PLCG1 was overexpressed in hepatoma cell lines and clinical HCC tissues. Increased PLCG1 expression in tumor tissues was remarkably correlated with poor clinical features of HCC. Patients with positive PLCG1 expression in tumor tissues had shorter overall survival and relapse-free survival. Phospholipase C gamma 1 could substantially promote cell proliferation, anchor growth, and cell invasion in vitro. The in vivo study showed that inhibition of PLCG1 in hepatoma cells significantly repressed tumor growth in nude mice. Furthermore, we showed that PLCG1 might exert its function by activating the mitogen-activated protein kinase and nuclear factor-κB signaling pathways. Conclusion Our data indicated that PLCG1 could act as an oncogene in HCC carcinogenesis and could serve as a valuable prognostic marker and potential therapeutic target for HCC.

Background Hepatocellular carcinoma (HCC) develops in a complex microenvironment characterized by chronic inflammation. In recent years, cholesterol metabolic abnormalities have been implicated the importance in cancer cell physiology. This study was designed to investigate the relationship between inflammation and cholesterol accumulation in HCC cells. Methods Human HCC cells HepG2 and Huh7 were cultured and stimulated with lipopolysaccharide (LPS) for 24 h. The changes of HCC cells related to cholesterol metabolism including intracellular cholesterol concentrations, cholesterol uptake, and the expression of cholesterol-related genes 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), LDL receptor (LDLR), sterol regulatory element-binding transcription factor 2 (SREBF2), and proprotein convertase subtilisin/kexin 9 (PCSK9) were comparatively analyzed. Simultaneously, the effects of nuclear factor-kappa B (NF-κB) signaling pathway on cholesterol metabolism were clarified by knocking-down of nuclear factor kappa-B kinase subunit alpha (IKKα) and TGF-beta-activated kinase 1 and MAP3K7-binding protein 3 (TAB3) via RNAi and microRNA (miR)-195. Subsequently, the roles of cholesterol accumulation in LPS induced pro-inflammatory effects were further investigated. Results Pro-inflammatory factor LPS significantly increased intracellular cholesterol accumulation by upregulating the expression of HMGCR, LDLR, and SREBF2, while downregulating the expression of PCSK9. These effects were revealed to depend on NF-κB signaling pathway by knocking-down and overexpression of IKKα and TAB3. Additionally, miR-195, a regulator directly targeting IKKα and TAB3, blocked the effects of cholesterol accumulation, further supporting the critical role of pro-inflammation NF-κB signaling in regulating cholesterol accumulation. Intriguingly, the accumulation of cholesterol conversely exerted an augmented pro-inflammation effects by further activating NF-κB signaling pathway. Conclusions These results indicated that pro-inflammation effects of NF-κB signaling could be augmented by a positive feedback via enhancing the cholesterol accumulation in liver cancer cells.

The development of hepatocellular carcinoma (HCC) is strongly associated with chronic inflammation. p66Shc is an oxidase previously shown to promote androgen-independent cell growth through generation of reactive oxygen species. However, the importance and biologic functions of p66Shc in HCC are unclear. The clinical significance of p66Shc was assessed in a large cohort of patients with HCC. High Shc1 expression was closely correlated with poor clinical outcomes and early recurrence of HCC. p66Shc expression was also determined in HCC samples and cell lines and found to be increased. Moreover, knockdown of p66Shc significantly inhibited cell proliferation, motility in vitro and tumor growth in vivo and could attenuate the proliferation, and motility of cells stimulated by activated macrophage conditioned media. Mechanically, p66Shc knockdown inhibited phosphorylation of STAT3 on serine 727 in vitro and in vivo. Our results show that high p66Shc expression in HCC predicts a worse prognosis for survival. Furthermore, p66Shc may serve as a novel candidate target for HCC therapy.