Tumor-derived exosomes induce immunosuppressive macrophages to foster intrahepatic cholangiocarcinoma progression.

BACKGROUND AND AIMS: Macrophages are prominent components of solid tumors and exhibit distinct functions in different tumor microenvironments. Exosomes are emerging as necessary mediators of the cross-talk between tumor cells and the microenvironment. However, the underlying mechanisms of exosomes involving into crosstalk between tumor cells and macrophages during disease progression of intrahepatic cholangiocarcinoma (ICC) have not been yet fully realized. APPROACH AND RESULTS: We found that the macrophages of ICC tumor tissues up-regulated the expression levels of immunosuppressive molecule programmed death-ligand 1 (PD-L1). Increased PD-L1+ macrophages in tumor tissues effectively suppressed T-cell immunity and correlated with poor survival rates in patients with ICC. High-throughput RNA-sequencing analysis that was performed to identify differential levels of microRNAs (miRNAs) between exosomes derived from ICC cells and primary human intrahepatic biliary epithelial cells revealed that miR-183-5p was increased in ICC cell-derived exosomes. Exosomal miR-183-5p inhibited phosphatase and tensin homolog (PTEN) expression, to subsequently affect the elevations on both phosphorylated AKT and PD-L1 expression in macrophages. Furthermore, macrophages that treated with ICC cell-derived exosomes significantly suppressed T-cell immunity in vitro and contributed to the growth and progression of ICC in vivo, which were reversible through blockages on PD-L1 of these macrophages. Finally, clinical data showed that up-regulated levels of plasma exosomal miR-183-5p correlated with poor prognosis of patients with ICC after curative resection. CONCLUSIONS: Tumor-derived exosomal miR-183-5p up-regulates PD-L1-expressing macrophages to foster immune suppression and disease progression in ICC through the miR-183-5p/PTEN/AKT/PD-L1 pathway. Exosomal miR-183-5p is a potential predictive biomarker for ICC progression and a potential target for development of therapeutic strategies against immune tolerance feature of ICC.

Genomic evolution and the impact of SLIT2 mutation in relapsed intrahepatic cholangiocarcinoma.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (ICC) is aggressive and has high rates of relapse, conferring poor long-term survival after curative resection. Little is known about the genomic evolution that occurs during ICC relapse. APPROACH AND RESULTS: We conducted whole-exome sequencing of 30 paired primary and relapsed tumors from 10 patients with ICC who received curative resection. We sought to identify frequently altered genes, infer tumor subclonal architectures, and track genomic evolution from primary to relapsed tumors. We examined functional effects and the mechanism of action of SLIT2, a gene specifically mutated in relapsed tumors, on tumor growth and metastasis and the tumor microenvironment. Our results indicated that relapsed ICCs were genetically derived from intrahepatic dissemination of primary tumors. However, they acquired additional mutations while maintaining most drivers, such as TP53 and IDH1. Multiregion sequencing suggested polyclonal seeding of ICC dissemination. Four of 10 relapsed ICCs acquired SLIT2 mutations that were not present in the corresponding primary tumors. Validation in an expanded sample revealed SLIT2 mutations in 2.3% (1/44) of primary ICCs and 29.5% (13/44) of relapsed ICCs. Biofunctional investigations revealed that inactivating mutation of SLIT2 resulted in activation of PI3K-Akt signaling in ICC cells, directly enhanced neutrophil chemotaxis, mediated tumor-associated neutrophil infiltration, and contributed to ICC growth and metastasis. CONCLUSIONS: We characterized genomic evolution during ICC relapse and identified SLIT2 as a driver of tumor dissemination and tumor-associated neutrophil infiltration.

Long noncoding RNA lnc-H2AFV-1 promotes cell growth by regulating aberrant m6A RNA modification in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the most common malignant tumor in the oral and maxillofacial regions, and long noncoding RNAs (lncRNAs) play crucial roles in the occurrence and progression of HNSCC. The lncRNA lnc-H2AFV-1 was found to be upregulated in HNSCC tissues; however, the function of lnc-H2AFV-1 in regulating HNSCC proliferation and the potential molecular mechanism is unclear. The present study evaluated the expression of lnc-H2AFV-1 in HNSCC tissues using quantitative real-time PCR (qPCR) and associated abundant lnc-H2AFV-1 expression with tumor size. Functionally, lnc-H2AFV-1 significantly promoted the proliferation of HNSCC cells in vitro and in vivo. Quantified N6-methyladenosine (m6A) RNA methylation and dot blot assays revealed that total m6A methylation in HNSCC cells was accompanied by lnc-H2AFV-1 expression. Western blotting showed that the expression of methyltransferase-like (METTL) 3 and METTL14 was consistent with that of lnc-H2AFV-1, whereas the expression of demethylase fat mass and obesity-associated (FTO) was contrary to that of lnc-H2AFV-1. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and MeRIP-qPCR revealed that lnc-H2AFV-1 overexpression led to the elevated expression and maximal m6A methylation of intraflagellar transport (IFT) 80 in HNSCC. In addition, METTL3/14 knockdown decreased IFT80 expression. Thus, our findings suggested that lnc-H2AFV-1 might be a biomarker that alters m6A modification by regulating the m6A methylases METTL3/14 and FTO and then mediating the downstream target IFT80 to promote HNSCC progression.

WDR5 facilitates EMT and metastasis of CCA by increasing HIF-1α accumulation in Myc-dependent and independent pathways.

Cholangiocarcinoma (CCA) is a highly aggressive malignancy with extremely poor prognoses. The oncogenic role and prognostic value of c-Myc in CCA is not well elucidated. WD repeat domain 5 (WDR5) is a critical regulatory factor directly interacting with c-Myc to regulate c-Myc recruitment at chromosomal locations, but the interaction of WDR5 and c-Myc in CCA was uncovered. In our study, we detected WDR5 and c-Myc expression in all CCA types, including intrahepatic (iCCA), perihilar (pCCA), and distal (dCCA) CCA, and evaluated their prognostic significance. Consequently, we demonstrated that WDR5 was significantly correlated with poor prognosis of CCA and that WDR5 and c-Myc co-expression was a more sensitive prognostic factor. With in vitro and in vivo experiments and bioinformatics, we showed that WDR5 interacted with the Myc box IIIb (MBIIIb) motif of c-Myc and facilitated Myc-induced HIF1A transcription, thereby promoting the epithelial-mesenchymal transition (EMT), invasion, and metastasis of CCA. Moreover, WDR5 enhanced hypoxia-inducible factor 1 subunit α (HIF-1α) accumulation by binding with histone deacetylase 2 (HDAC2) and increasing histone 3 lysine 4 acetylation (H3K4ac) deacetylation of the prolyl hydroxylase domain protein 2 (PHD2) promoter, resulting in the attenuation of chromatin opening and PHD2 expression, and eventually leading to HIF-1α stabilization and accumulation. In conclusion, WDR5 facilitated EMT and metastasis of CCA by increasing HIF-1α accumulation in a Myc-dependent pathway to promote HIF-1α transcription and a Myc-independent pathway to stabilize HIF-1α.

Characterization of tumor microbiome and associations with prognosis in intrahepatic cholangiocarcinoma.

BACKGROUND: The tumor microbiome has been characterized in several malignancies; however, no previous studies have investigated its role in intrahepatic cholangiocarcinoma (ICC). Hence, we explored the tumor microbiome and its association with prognosis in ICC. METHODS: One hundred and twenty-one ICC tumor samples and 89 adjacent normal tissues were profiled by 16S rRNA sequencing. Microbial differences between tumor and adjacent nontumoral liver tissues were assessed. Tumor microbial composition was then evaluated to detect its association with prognosis. Finally, a risk score calculated by the tumor microbiota was accessed by the least absolute shrinkage and selector operator method (Lasso) to predict prognosis of ICC. RESULTS: The tumor microbiome displayed a greater diversity than that in adjacent nontumoral liver tissues. Tumor samples were characterized by a higher abundance of Firmicutes, Actinobacteria, Bacteroidetes, and Acidobacteriota. Higher tumor microbial α diversity was associated with lymph node metastasis and predicted shortened overall survival (OS) and recurrence-free survival (RFS). A total of 11 bacteria were selected to generate the risk score by Lasso. This score showed potential in predicting OS, and was an independent risk factor for OS. CONCLUSION: In conclusion, our study characterized the tumor microbiome and revealed its role in predicting prognosis in ICC.

Development and Validation of a Stromal-Immune Signature to Predict Prognosis in Intrahepatic Cholangiocarcinoma.

Background: Intrahepatic cholangiocarcinoma (ICC) is a highly desmoplastic tumor with poor prognosis even after curative resection. We investigated the associations between the composition of the ICC stroma and immune cell infiltration and aimed to develop a stromal-immune signature to predict prognosis in surgically treated ICC. Patients and methods: We recruited 359 ICC patients and performed immunohistochemistry to detect α-smooth muscle actin (α-SMA), CD3, CD4, CD8, Foxp3, CD68, and CD66b. Aniline was used to stain collagen deposition. Survival analyses were performed to detect prognostic values of these markers. Recursive partitioning for a discrete-time survival tree was applied to define a stromal-immune signature with distinct prognostic value. We delineated an integrated stromal-immune signature based on immune cell subpopulations and stromal composition to distinguish subgroups with different recurrence-free survival (RFS) and overall survival (OS) time. Results: We defined four major patterns of ICC stroma composition according to the distributions of α-SMA and collagen: dormant (α-SMAlow/collagenhigh), fibrogenic (α-SMAhigh/collagenhigh), inert (α-SMAlow/collagenlow), and fibrolytic (α-SMAhigh/collagenlow). The stroma types were characterized by distinct patterns of infiltration by immune cells. We divided patients into six classes. Class I, characterized by high CD8 expression and dormant stroma, displayed the longest RFS and OS, whereas Class VI, characterized by low CD8 expression and high CD66b expression, displayed the shortest RFS and OS. The integrated stromal-immune signature was consolidated in a validation cohort. Conclusion: We developed and validated a stromal-immune signature to predict prognosis in surgically treated ICC. These findings provide new insights into the stromal-immune response to ICC.

Transcriptome Mapping of the Internal N7-Methylguanosine Methylome in Messenger RNAs in Human Oral Squamous Cell Carcinoma.

BACKGROUND: Internal N7-methylguanosine (m7G) methylation in mammalian messenger RNAs (mRNAs) is essential in disease development. However, the status of internally m7G-modified mRNAs in oral squamous cell carcinoma (OSCC) remains poorly understood. METHODS: Methylated RNA immunoprecipitation sequencing (MeRIP-seq) was used to identify the m7G modification level of mRNAs and the expression of mRNAs between OSCC and normal tissues. These differentially methylated and expressed genes were subjected to Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and construction of protein-protein interaction (PPI) networks. Quantitative real-time PCR (qPCR) assay was performed to detect the expression of Baculoviral IAP Repeat Containing 3 (BIRC3) in vitro. The biological function of BIRC3 in OSCC was clarified using CCK-8, Transwell migration and Western blot assays. RESULTS: The m7G-mRNA profile showed 9514 unique m7G peaks within 7455 genes in OSCC tissues. In addition, the most conserved m7G motif within mRNAs in OSCC was GGARG (R = G/A). The identified m7G peaks were mainly distributed in the coding sequence region within mRNAs in OSCC. GO enrichment and KEGG pathway analyses showed that m7G-modified genes were closely related to cancer progression. m7G-modified hub genes were screened from the constructed PPI networks. Furthermore, BIRC3 with high m7G methylation showed high expression in OSCC cell lines, as confirmed by qPCR assay. Functionally, the knockdown of BIRC3 significantly inhibited the proliferation and migration ability of CAL-27 cells in vitro functional assays. In addition, the relative expression of E-cadherin expression was elevated, while Vimentin and N-cadherin protein expression was decreased in CAL-27 cells transfected with si-BIRC3. This study suggests that BIRC3 could promote OSCC proliferation and migration, which may be associated with involvement in epithelial-mesenchymal transition (EMT) progression. CONCLUSIONS: This paper constructed a transcriptome map of internal m7G in mRNAs, which provides potential research value to study the role of m7G methylation in OSCC.

Association of BRAF Variants With Disease Characteristics, Prognosis, and Targeted Therapy Response in Intrahepatic Cholangiocarcinoma.

Importance: BRAF variants are associated with tumor progression; however, the prevalence of BRAF variant subtypes and their association with disease characteristics, prognosis, and targeted therapy response in patients with intrahepatic cholangiocarcinoma (ICC) are largely unknown. Objective: To explore the association of BRAF variant subtypes with disease characteristics, prognosis, and targeted therapy response in patients with ICC. Design, Setting, and Participants: In this cohort study, 1175 patients who underwent curative resection for ICC from January 1, 2009, through December 31, 2017, were evaluated at a single hospital in China. Whole-exome sequencing, targeted sequencing, and Sanger sequencing were performed to identify BRAF variants. The Kaplan-Meier method and log-rank test were used to compare overall survival (OS) and disease-free survival (DFS). Univariate and multivariate analyses were performed using Cox proportional hazards regression. Associations between BRAF variants and targeted therapy response were tested in 6 BRAF-variant, patient-derived organoid lines and in 3 of the patient donors of those lines. Data were analyzed from June 1, 2021, to March 15, 2022. Interventions: Hepatectomy in patients with ICC. Main Outcomes and Measures: The association of BRAF variant subtypes with OS and DFS. Results: Of 1175 patients with ICC, the mean (SD) age was 59.4 (10.4) years and 701 (59.7%) were men. A total of 20 different subtypes of BRAF somatic variance affecting 49 patients (4.2%) were identified; V600E was the most frequent allele in this cohort, accounting for 27% of the identified BRAF variants, followed by K601E (14%), D594G (12%), and N581S (6%). Compared with patients with non-V600E BRAF variants, patients with BRAF V600E variants were more likely to have large tumor size (10 of 13 [77%] vs 12 of 36 [33%]; P = .007), multiple tumors (7 of 13 [54%] vs 8 of 36 [22%]; P = .04), and more vascular/bile duct invasion (7 of 13 [54%] vs 8 of 36 [22%]; P = .04). Multivariate analysis revealed that BRAF V600E variants, but not overall BRAF variants or non-V600E BRAF variants, were associated with poor OS (hazard ratio [HR], 1.87; 95% CI, 1.05-3.33; P = .03) and DFS (HR, 1.66; 95% CI, 1.03-2.97; P = .04). There were also broad differences among organoids with different BRAF variant subtypes in sensitivity to BRAF or MEK inhibitors. Conclusions and Relevance: The findings of this cohort study suggest that there are broad differences among organoids with different BRAF variant subtypes in sensitivity to BRAF or MEK inhibitors. Identifying and classifying BRAF variants may be able to help guide precise treatment for patients with ICC.

Association of KRAS Variant Subtypes With Survival and Recurrence in Patients With Surgically Treated Intrahepatic Cholangiocarcinoma.

Importance: KRAS variants are associated with tumor progression; however, the prevalence of KRAS variant subtypes and their association with survival and recurrence in patients with intrahepatic cholangiocarcinoma (ICC) after curative resection are largely unknown. Objective: To explore the prognostic association of KRAS variant subtypes with survival and recurrence in patients with ICC. Design, Setting, and Participants: In this cohort study, patients who underwent curative resection for ICC from January 2009 through December 2016 at a single hospital in China were recruited, and whole-exome sequencing, targeted sequencing, and Sanger sequencing were performed to identify KRAS variants. Kaplan-Meier and log-rank tests were used to compare overall survival (OS) and disease-free survival (DFS). Univariate and multivariate analyses were performed using the Cox proportional hazards regression model. Data were analyzed from April 2020 to January 2021. Interventions: Hepatectomy in patients with ICC. Main Outcomes and Measures: The association of KRAS variant subtypes with OS and DFS. Results: Of 1024 included patients with ICC, 621 (60.6%) were male, and the mean (SD) age was 59.2 (10.2) years. A total of 14 different subtypes of KRAS somatic variants affecting 127 patients (12.4%) were identified. G12D was the most frequent allele in this cohort, accounting for 55 of 127 identified KRAS variants (43.3%), followed by G12V (25 [19.7%]), G12C (9 [7.1%]), and G13D (8 [6.3%]). Compared with patients with wild-type KRAS, patients with variant KRAS were more likely to have high levels of carbohydrate antigen 19-9 (92 of 127 [72.4%] vs 546 of 897 [60.9%]; P = .01) and γ-glutamyltransferase (72 of 127 [56.7%] vs 420 of 897 [46.8%]; P = .04). Multivariable analysis revealed that G12 KRAS variants but not non-G12 KRAS variants were independently associated with worse OS (hazard ratio [HR], 1.69; 95% CI, 1.31-2.18; P < .001) and DFS (HR, 1.47; 95% CI, 1.16-1.88; P = .002). Among the patients with G12 KRAS variants, the G12V KRAS variant was the strongest prognostic determinant for the worst OS (HR, 3.05; 95% CI, 1.94-4.79; P < .001) and DFS (HR, 1.79; 95% CI, 1.13-2.85; P = .01). Conclusions and Relevance: In this cohort study, the distribution of KRAS variant subtypes was characterized in a large cohort of patients with ICC from China. The presence of G12 KRAS variants but not non-G12 KRAS variants was associated with worse survival and increased risk of recurrence. Patients with the G12V variant exhibited the worst outcomes in the whole cohort.

HMGA1-TRIP13 axis promotes stemness and epithelial mesenchymal transition of perihilar cholangiocarcinoma in a positive feedback loop dependent on c-Myc.

BACKGROUND: Cholangiocarcinoma is a highly malignant cancer with very dismal prognosis. Perihilar cholangiocarcinoma(pCCA) accounts for more than 50% of all cholangiocarcinoma and is well-characterized for its low rate of radical resection. Effects of radiotherapy and chemotherapy of pCCA are very limited. METHODS: Here we screened potential biomarkers of pCCA with transcriptome sequencing and evaluated the prognostic significance of HMGA1 in a large cohort pCCA consisting of 106 patients. With bioinformatics and in vitro/vivo experiments, we showed that HMGA1 induced tumor cell stemness and epithelial-mesenchymal-transition (EMT), and thus facilitated proliferation, migration and invasion by promoting TRIP13 transcription. Moreover, TRIP13 was also an unfavorable prognostic biomarker of pCCA, and double high expression of HMGA1/TRIP13 could predict prognosis more sensitively. TRIP13 promoted pCCA progression by suppressing FBXW7 transcription and stabilizing c-Myc. c-Myc in turn induced the transcription and expression of both HMGA1 and TRIP13, indicating that HMGA-TRIP13 axis facilitated pCCA stemness and EMT in a positive feedback pathway. CONCLUSIONS: HMGA1 and TRIP13 were unfavorable prognostic biomarkers of pCCA. HMGA1 enhanced pCCA proliferation, migration, invasion, stemness and EMT, by inducing TRIP13 expression, suppressing FBXW7 expression and stabilizing c-Myc. Moreover, c-Myc can induce the transcription of HMGA1 and TRIP13, suggesting that HMGA-TRIP13 axis promoted EMT and stemness in a positive feedback pathway dependent on c-Myc.

Tumor-associated neutrophils and macrophages interaction contributes to intrahepatic cholangiocarcinoma progression by activating STAT3.

BACKGROUND: Tumor-associated neutrophils (TANs) and macrophages (TAMs) can each influence cancer growth and metastasis, but their combined effects in intrahepatic cholangiocarcinoma (ICC) remain unclear. METHODS: We explored the distributions of TANs and TAMs in patient-derived ICC samples by multiplex immunofluorescent staining and tested their separate and combined effects on ICC in vitro and in vivo. We then investigated the mechanistic basis of the effects using PCR array, western blot analysis and ELISA experiments. Finally, we validated our results in a tissue microarray composed of primary tumor tissues from 359 patients with ICC. RESULTS: The spatial distributions of TANs and TAMs were correlated with each other in patient-derived ICC samples. Interaction between TANs and TAMs enhanced the proliferation and invasion abilities of ICC cells in vitro and tumor progression in a mouse xenograft model of ICC. TANs and TAMs produced higher levels of oncostatin M and interleukin-11, respectively, in co-culture than in monoculture. Both of those cytokines activated STAT3 signaling in ICC cells. Knockdown of STAT3 abolished the protumor effect of TANs and TAMs on ICC. In tumor samples from patients with ICC, increased TAN and TAM levels were correlated with elevated p-STAT3 expression. All three of those factors were independent predictors of patient outcomes. CONCLUSIONS: TANs and TAMs interact to promote ICC progression by activating STAT3.

Characterization of immune infiltration in sarcomatoid hepatocellular carcinoma.

Sarcomatoid hepatocellular carcinoma (sHCC) is a rare type of liver malignancy. Currently, the tumor immune features of sHCC are poorly understood. We recruited 31 patients with resected sHCC for whom tissue samples and complete clinicopathologic and follow-up data were available. To understand the immune infiltration of sHCC, immunohistochemical staining was performed on the resected sHCC samples to compare the expressions of programmed death-1 (PD-1), programmed death-ligand 1 (PD-L1), B7-H3, indoleamine 2,3-dioxygenase (IDO), lymphocyte-activation gene 3 (LAG-3), CD8, FOXP3, and CD68 in tumor and peritumoral tissues. Kaplan-Meier and Cox regression analyses were used to assess the predictive value of immune markers. Sarcomatoid components were characterized with significantly higher expression of PD-L1 and B7-H3 in tumor cells than in conventional HCC components, as well as in peritumoral tissue. Additionally, sarcomatoid components had a higher density of FOXP3+ and LAG-3+ cells and a lower density of CD8+ cells than conventional HCC components or peritumoral tissue. Higher expression of PD-L1 in tumor cells significantly correlated with higher densities of CD8+, PD-1+, and LAG-3+ cells. Increased tumor PD-L1 expression and decreased CD8+ T-cell density were associated with poor overall survival (OS) and disease-free survival (DFS) in patients of sHCC. These findings suggest further characterization on relative mechanism of sHCC immune infiltration may identify therapeutic targets for immunotherapy.

LINC01133 promotes hepatocellular carcinoma progression by sponging miR-199a-5p and activating annexin A2.

BACKGROUND: Long noncoding RNAs (lncRNAs) are functionally associated with cancer development and progression. Although gene copy number variation (CNV) is common in hepatocellular carcinoma (HCC), it is not known how CNV in lncRNAs affects HCC progression and recurrence. We aimed to identify a CNV-related lncRNA involved in HCC progression and recurrence and illustrate its underlying mechanisms and prognostic value. METHODS: We analyzed the whole genome sequencing (WGS) data of matched cancerous and noncancerous liver samples from 49 patients with HCC to identify lncRNAs with CNV. The results were validated in another cohort of 238 paired HCC and nontumor samples by TaqMan copy number assay. We preformed Kaplan-Meier analysis and log-rank test to identify lncRNA CNV with prognostic value. We conducted loss- and gain-of-function studies to explore the biological functions of LINC01133 in vitro and in vivo. The competing endogenous RNAs (ceRNAs) mechanism was clarified by microRNA sequencing (miR-seq), quantitative real-time PCR (qRT-PCR), western blot, and dual-luciferase reporter assays. We confirmed the binding mechanism between lncRNA and protein by RNA pull-down, RNA immunoprecipitation, qRT-PCR, and western blot analyses. RESULTS: Genomic copy numbers of LINC01133 were increased in HCC, which were positively related with the elevated expression of LINC01133. Increased copy number of LINC01133 predicted the poor prognosis in HCC patients. LINC01133 overexpression in HCC cells promoted proliferation and aggressive phenotypes in vitro, and facilitated tumor growth and lung metastasis in vivo, whereas LINC01133 knockdown had the opposite effects. LINC01133 sponged miR-199a-5p, resulting in enhanced expression of SNAI1, which induced epithelial-to-mesenchymal transition (EMT) in HCC cells. In addition, LINC01133 interacted with Annexin A2 (ANXA2) to activate the ANXA2/STAT3 signaling pathway. CONCLUSIONS: LINC01133 promotes HCC progression by sponging miR-199a-5p and interacting with ANXA2. LINC01133 CNV gain is predictive of poor prognosis in patients with HCC.

Decreased expression of serum semaphorin 3B is associated with poor prognosis of patients with hepatocellular carcinoma.

Semaphorin 3B (SEMA-3B), which belongs to the semaphorin family, has an important role in cell apoptosis and inhibition of angiogenesis. A previous study by our group revealed that SEMA-3B was downregulated in tumor tissues of patients with hepatocellular carcinoma (HCC) and exerts anti-motility and anti-invasion effects on tumor cells. However, the serum levels of SEMA-3B and their clinical significance have remained elusive; therefore, the aim of the present study was to monitor its expression in HCC and investigate its clinical significance. ELISA was used to determine the serum levels of SEMA-3B in 132 patients with HCC and 57 healthy individuals. The association between SEMA-3B and clinicopathological parameters was investigated. Serum SEMA-3B was indicated to be significantly decreased in patients with HCC as compared with that in the controls (P<0.05) and it was negatively associated with tumor size (P=0.039), encapsulation (P=0.002) and TNM stage (P=0.034). The prognosis of patients with low expression of SEMA-3B was poor. In conclusion, the results of the present study revealed that serum SEMA-3B is decreased in HCC and is negatively associated with prognosis; therefore, it may be used as a prognostic marker in HCC.

PTPN3 suppresses the proliferation and correlates with favorable prognosis of perihilar cholangiocarcinoma by inhibiting AKT phosphorylation.

BACKGROUND: Perihilar cholangiocarcinoma (PHCCA) is the most common type of human cholangiocarcinoma with a very dismal prognosis. Tumor markers and target drugs of PHCCA are desperately needed. Protein phosphatase N3 (PTPN3) has dual roles in the progression of human cancers, but its expression and functions in PHCCA have not been elucidated. MATERIALS AND METHODS: The expression of PTPN3 in PHCCA was detected with western blotting, qRT-PCR and immunohistochemistry. The clinical significance of PTPN3 was identified by analyzing the correlations between its expression and the clinicopathological variables, and the prognostic value was evaluated by univariate and multivariate analyses. The functions of PTPN3 in the progression of PHCCA were estimated with both in vitro and in vivo experiments. RESULTS: PTPN3 expression was down-regulated in PHCCA compared with normal bile duct. Low PTPN3 expression was markedly associated with large tumor size and unfavorable prognosis. After knocking down PTPN3, the percentages of G2/S phase of PHCCA cells were elevated, and the proliferation increased significantly. Moreover, we demonstrated that the phosphorylation of AKT was elevated by PTPN3 knockdown, and it was required in PTPN3-involved proliferation of PHCCA. Within vivo experiments, PTPN3 and AKT inhibitor MK-2206 were demonstrated to suppress tumor size of PHCCA. CONCLUSION: PTPN3 was a favorable prognostic biomarker of PHCCA. PTPN3 suppressed the proliferation of PHCCA by inhibiting AKT phosphorylation and arresting cell cycle. Our results suggested thatpost-operative detection of PTPN3 would be a helpful approach to stratify the PHCCA patients with high-risk.

Transcription factor 7 promotes the progression of perihilar cholangiocarcinoma by inducing the transcription of c-Myc and FOS-like antigen 1.

BACKGROUND: Perihilar cholangiocarcinoma (PHCC) is the most common type of cholangiocarcinoma with the worst prognosis. Radical resection of PHCC is difficult; thus, few effective biomarkers or useful molecular profiles for PHCC have been reported in recent years. Therefore, in this study, we aimed to assess biomarkers for PHCC. METHODS: We screened potential biomarkers for PHCC using exome and transcriptome sequencing with PHCC tissues and paired normal tissues. Transcription factor 7 (TCF7) expression was evaluated using quantitative reverse transcription polymerase chain reaction, western blotting, and immunohistochemistry. The correlations between TCF7 and clinicopathological factors were analyzed with Chi-square test, and the prognostic significance of TCF7 was evaluated with univariate and multivariate analyses. The functions of TCF7 and its main effectors in PHCC cells were investigated in vitro and in vivo. FINDINGS: TCF7 expression was upregulated in PHCC and was an unfavorable prognostic biomarker. c-Myc was a main effector of TCF7 in PHCC cells and modulated TCF7-induced proliferation, invasion, and migration. FOS-like antigen 1 (FOSL1) was identified as a downstream target of TCF7 and was required in TCF7-induced PHCC proliferation. Triple-positive expression of TCF7, c-Myc, and FOSL1 predicted a much worse prognosis in patients with PHCC than TCF7 expression alone. INTERPRETATION: Postoperative detection of TCF7, c-Myc, and FOSL1 may be useful for stratifying patients with a high risk of unfavorable prognosis, and suppressing TCF7 or its downstream effectors may be a promising strategy for the treatment of PHCC.

Annexin10 promotes extrahepatic cholangiocarcinoma metastasis by facilitating EMT via PLA2G4A/PGE2/STAT3 pathway.

BACKGROUND: Cholangiocarcinoma (CCA), consisting of intrahepatic (IHCCA), perihilar (PHCCA), and distal (DCCA) CCA, is a type of highly aggressive malignancy with a very dismal prognosis. Potential biomarkers and drug targets of CCA are urgently needed. As a new member of the Annexin (ANXA) family, the role of ANXA10 in the progression and prognosis of CCA is unknown. METHODS: Potential PHCCA biomarkers were screened by transcriptome sequencing of 5 pairs of PHCCA and adjacent tissues. The clinical significance of ANXA10 was evaluated by analyzing its correlation with clinicopathological variables, and the prognostic value of ANXA10 was evaluated with univariate and multivariate analyses. The function of ANXA10 in the epithelial-mesenchymal transition (EMT), proliferation, invasion and metastasis was detected with in vitro and in vivo experiments. Moreover, we screened the key molecule in ANXA10-induced CCA progression by mRNA sequencing and evaluated the correlation between PLA2G4A and ANXA10. The effect of PLA2G4A downstream signaling, including Cyclooxygenase 2, Prostaglandin E2(PGE2) and Signal transducer and activator of transcription 3(STAT3), on EMT and metastasis was further detected with in vitro and in vivo experiments. FINDINGS: ANXA10 expression was upregulated in PHCCA and DCCA but not in IHCCA. High ANXA10 expression was significantly associated with poor tumor differentiation and prognosis. ANXA10 promoted the proliferation, migration and invasion of the PHCCA cells. PLA2G4A expression was regulated by ANXA10 and high PLA2G4A predicted poor prognosis in PHCCA and DCCA. ANXA10 facilitated EMT and promoted metastasis by upregulating PLA2G4A expression, thus increasing PGE2 levels and activating STAT3. INTERPRETATION: ANXA10 was an independent prognostic biomarker of PHCCA and DCCA but not IHCCA. ANXA10 promoted the progression of PHCCA and facilitated metastasis by promoting the EMT process via the PLA2G4A/PGE2/STAT3 pathway. ANXA10, PLA2G4A and their downstream molecules, such as COX2 and PGE2, may be promising drug targets of PHCCA and DCCA.

Sprouty4 correlates with favorable prognosis in perihilar cholangiocarcinoma by blocking the FGFR-ERK signaling pathway and arresting the cell cycle.

BACKGROUND: Perihilar cholangiocarcinoma (PHCC) is the most common subtype of cholangiocarcinoma(CCA). We previously investigated the expression pattern of Sprouty(SPRY) in intrahepatic cholangiocarcinoma(ICC), but the expression and clinical significance of SPRY family members in PHCC are still unknown. METHODS: The expression of SPRY family members(SPRY1-4) was detected in different subtypes of CCA and corresponding adjacent tissues. SPRY4 expression in 142 cases of PHCC was detected by immunohistochemistry, and its clinical significance was evaluated using univariate and multivariate analyses. The functions of SPRY4 in the FGFR-induced proliferation and migration of PHCC cells were investigated through in vitro and in vivo experiments. We further investigated the effects and mechanisms of SPRY4 on FGFR-induced ERK phosphorylation and cell cycle distribution in the presence of FGFR and ERK inhibitors. FINDINGS: SPRY4 was the only SPRY family member associated with PHCC prognosis, and it was identified as an independent factor predicting favorable prognosis. In PHCC, SPRY4 expression was extensively associated with FGFR2, and its expression can be induced by ectopic FGFR2 activation. Through in vitro and in vivo experiments, we demonstrated that SPRY4 suppressed FGFR-induced proliferation and migration by inhibiting ERK phosphorylation. Moreover, SPRY4 knockdown was shown to decrease the percentage of cells in the G1 phase and promote the percentage of cells in the S and G2/M phases by increasing cyclin D1 expression, which also required FGFR-induced ERK phosphorylation. INTERPRETATION: High expression of SPRY4 was an independent biomarker of favorable prognosis in PHCC. SPRY4 expression can be induced by ectopic FGFR2 activation in PHCC. SPRY4 arrested the cell cycle at G1 phase and suppressed FGFR-induced proliferation and migration by inhibiting ERK phosphorylation, indicating that SPRY4 may be a potential therapeutic target in PHCC.

Note: A data acquisition system for a high phase contrast medical computed tomography prototype.

A Data Acquisition (DAQ) system is designed for a high phase contrast medical computed tomography prototype, which is based on the three-grating interferometry technology. The DAQ system contains 43 detector boards including 16 512 channels, 3 data collection boards, and one data transmission board (DTB). All digitized data from 43 detector board are collected and transmitted to the DTB through a slip ring. The communication between the DTB and Personal Computer is based on gigabit Ethernet. The efficient resolution of readout electronics is up to 14.6 bit. For all channels, the integral nonlinearity is no more than 0.1%. An air scan algorithm is employed to calibrate the channel gain non-uniformity.