Diacylglycerol kinase alpha is a proliferation marker of intrahepatic cholangiocarcinoma associated with the prognosis.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) has a high recurrence rate and a poor prognosis. Thus, the development of effective treatment and prognostic biomarkers is required. High expression of diacylglycerol kinase alpha (DGKα) is a prognostic factor for the recurrence of hepatocellular carcinoma. However, the relationship between DGKα expression and prognosis in ICC has not been reported. METHODS: Immunohistochemistry (IHC) with anti-DGKα antibody was performed on surgical specimens of ICC (n = 69). First, DGKα expression in cancer cells was qualitatively classified into four groups (-, 1+, 2+, 3+) and divided into two groups (DGKα- and DGKα+1 + to 3+). The relationship between clinical features and DGKα expression was analyzed. Second, Ki-67 expression was evaluated as a cell proliferation marker. The number of Ki-67-positive cells was counted, and the relationship with DGKα expression was examined. RESULTS: DGKα IHC divided the patients into a DGKα+ group (1+: n = 15; 2+: n = 5; 3+: n = 5) and a DGKα- group (-: n = 44). In the DGKα+ group, patients were older and had advanced disease. Both overall survival and recurrence-free survival (RFS) were significantly worse in the DGKα+ patients. DGKα+ was identified as an independent prognostic factor for RFS by multivariate analysis. Furthermore, the number of Ki-67-positive cells increased in association with the staining levels of DGKα. CONCLUSION: Pathological DGKα expression in ICC was a cancer proliferation marker associated with recurrence. This suggests that DGKα may be a potential therapeutic target for ICC.

The deubiquitinase USP5 promotes cholangiocarcinoma progression by stabilizing YBX1.

AIMS: Ubiquitin specific peptidase 5 (USP5), a member of deubiquitinating enzymes, has garnered significant attention for its crucial role in cancer progression. This study aims to explore the role of USP5 and its potential molecular mechanisms in cholangiocarcinoma (CCA). MAIN METHODS: To explore the effect of USP5 on CCA, gain-of-function and loss-of-function assays were conducted in human CCA cell lines RBE and HCCC9810. The CCK8, colony-forming assay, EDU, flow cytometry, transwell assay and xenografts were used to assess cell proliferation, migration and tumorigenesis. Western blot and immunohistochemistry were performed to measure the expression of related proteins. Immunoprecipitation and immunofluorescence were applied to identify the interaction between USP5 and Y box-binding protein 1 (YBX1). Ubiquitination assays and cycloheximide chase assays were carried out to confirm the effect of USP5 on YBX1. KEY FINDINGS: We found USP5 is highly expressed in CCA tissues, and upregulated USP5 is required for the cancer progression. Knockdown of USP5 inhibited cell proliferation, migration and epithelial-mesenchymal transition (EMT) in vitro, along with suppressed xenograft tumor growth and metastasis in vivo. Mechanistically, USP5 could interact with YBX1 and stabilize YBX1 by deubiquitination in CCA cells. Additionally, silencing of USP5 hindered the phosphorylation of YBX1 at serine 102 and its subsequent translocation to the nucleus. Notably, the effect induced by USP5 overexpression in CCA cells was reversed by YBX1 silencing. SIGNIFICANCE: Our findings reveal that USP5 is required for cell proliferation, migration and EMT in CCA by stabilizing YBX1, suggesting USP5-YBX1 axis as a promising therapeutic target for CCA.

Expression of Fascin-1 and its diagnostic value in liver cancer.

Although some studies have reported on the expression and clinical significance of Fascin-1 (FSCN1) in liver cancer, the clinical application and differential diagnosis value of FSCN1 in liver cancer are still unclear. The aim of this study was to analyze the expression level of FSCN1 protein in liver cancer tissues and explore its diagnostic and application value in differentiating between hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). The immunehistochemical analysis was used to detect the expression of FSCN1 in 108 cases of HCC, 26 cases of ICC, 23 cases of liver cirrhosis, and 11 cases of normal liver tissues. The differences in the positive expression rate and strong positive expression rate of FSCN1 among different groups were analyzed. The positive rate of FSCN1 in normal liver tissues, liver cirrhosis, HCC, and ICC tissues was 0.0% (0/11), 0.0% (0/23), 13.9% (15/108), and 92.3% (24/26), respectively, while the strong positive rate was 0.0% (0/11), 0.0% (0/23), 0.9% (1/108), and 69.2% (18/26), respectively. Both the positive rate and strong positive rate of FSCN1 in ICC tissues were significantly higher than those in HCC, liver cirrhosis, and normal liver tissues. Additionally, the positive rate of FSCN1 in moderately to poorly differentiated HCC tissues was 18.8% (15/80), significantly higher than in well-differentiated HCC (0.0%, 0/28) (P = 0.031). In liver cancer, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of FSCN1 positive prediction for ICC were 92.3%, 86.1%, 61.5%, and 97.9%, respectively, whereas the sensitivity, specificity, PPV, and NPV of FSCN1 strong positive prediction for ICC were 69.2%, 99.1%, 94.7%, and 93.0%, respectively. These results suggest that FSCN1 may play an important role in the occurrence and progression of liver cancer, and it can be used as a novel diagnostic marker for ICC.

FGFR inhibition blocks NF-ĸB-dependent glucose metabolism and confers metabolic vulnerabilities in cholangiocarcinoma.

Genomic alterations that activate Fibroblast Growth Factor Receptor 2 (FGFR2) are common in intrahepatic cholangiocarcinoma (ICC) and confer sensitivity to FGFR inhibition. However, the depth and duration of response is often limited. Here, we conduct integrative transcriptomics, metabolomics, and phosphoproteomics analysis of patient-derived models to define pathways downstream of oncogenic FGFR2 signaling that fuel ICC growth and to uncover compensatory mechanisms associated with pathway inhibition. We find that FGFR2-mediated activation of Nuclear factor-κB (NF-κB) maintains a highly glycolytic phenotype. Conversely, FGFR inhibition blocks glucose uptake and glycolysis while inciting adaptive changes, including switching fuel source utilization favoring fatty acid oxidation and increasing mitochondrial fusion and autophagy. Accordingly, FGFR inhibitor efficacy is potentiated by combined mitochondrial targeting, an effect enhanced in xenograft models by intermittent fasting. Thus, we show that oncogenic FGFR2 signaling drives NF-κB-dependent glycolysis in ICC and that metabolic reprogramming in response to FGFR inhibition confers new targetable vulnerabilities.

SRC inhibition enables formation of a growth suppressive MAGI1-PP2A complex in isocitrate dehydrogenase-mutant cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive bile duct malignancy that frequently exhibits isocitrate dehydrogenase (IDH1/IDH2) mutations. Mutant IDH (IDHm) ICC is dependent on SRC kinase for growth and survival and is hypersensitive to inhibition by dasatinib, but the molecular mechanism underlying this sensitivity is unclear. We found that dasatinib reduced p70 S6 kinase (S6K) and ribosomal protein S6 (S6), leading to substantial reductions in cell size and de novo protein synthesis. Using an unbiased phosphoproteomic screen, we identified membrane-associated guanylate kinase, WW, and PDZ domain containing 1 (MAGI1) as an SRC substrate in IDHm ICC. Biochemical and functional assays further showed that SRC inhibits a latent tumor-suppressing function of the MAGI1-protein phosphatase 2A (PP2A) complex to activate S6K/S6 signaling in IDHm ICC. Inhibiting SRC led to activation and increased access of PP2A to dephosphorylate S6K, resulting in cell death. Evidence from patient tissue and cell line models revealed that both intrinsic and extrinsic resistance to dasatinib is due to increased phospho-S6 (pS6). To block pS6, we paired dasatinib with the S6K/AKT inhibitor M2698, which led to a marked reduction in pS6 in IDHm ICC cell lines and patient-derived organoids in vitro and substantial growth inhibition in ICC patient-derived xenografts in vivo. Together, these results elucidated the mechanism of action of dasatinib in IDHm ICC, revealed a signaling complex regulating S6K phosphorylation independent of mTOR, suggested markers for dasatinib sensitivity, and described a combination therapy for IDHm ICC that may be actionable in the clinic.

EIF3B stabilizes PCNA by counteracting SYVN1-mediated ubiquitination to serve as a promotor in cholangiocarcinoma.

Cholangiocarcinoma, a prevalent hepatic malignancy, exhibits a progressively rising incidence. While Eukaryotic translation initiation factor 3 subunit B (EIF3B) has been implicated in the occurrence and development of various cancers, its specific roles in cholangiocarcinoma remain unexplored. Immunohistochemical (IHC) analysis was employed to detect EIF3B/PCNA expression in cholangiocarcinoma. Cells were manipulated using short hairpin RNA (shRNA)-mediated lentiviruses or overexpression plasmids. Statistical significance was assessed using the Student's t-test and one-way ANOVA, with P < 0.05 considered statistically significant. EIF3B exhibited robust expression in cholangiocarcinoma, demonstrating a significant correlation with the pathological grade of cholangiocarcinoma patients. Furthermore, modulation of EIF3B expression, either depletion or elevation, demonstrated the ability to inhibit or enhance cholangiocarcinoma cell survival and migration in vitro. Mechanistically, we identified Proliferating Cell Nuclear Antigen (PCNA) as a downstream gene of EIF3B, driving cholangiocarcinoma. EIF3B stabilized PCNA by inhibiting PCNA ubiquitination, a process mediated by E3 ligase SYVN1. Similar to EIF3B, PCNA levels were also abundant in cholangiocarcinoma, and knocking down PCNA impeded cholangiocarcinoma development. Intriguingly, silencing PCNA attenuated the promotion induced by EIF3B overexpression. Furthermore, the elevated P21 protein level in shEIF3B RBE cells was partially attenuated after UC2288 (P21 signaling pathway inhibitor) treatment. Our findings underscored the potential of EIF3B as a therapeutic target for cholangiocarcinoma. Unraveling its functions holds promise for the development of more specific and effective targeted therapy strategies.

CX-4945 (Silmitasertib) Induces Cell Death by Impairing Lysosomal Utilization in KRAS Mutant Cholangiocarcinoma Cell Lines.

BACKGROUND/AIM: Macropinocytosis is a non-selective form of endocytosis that facilitates the uptake of extracellular substances, such as nutrients and macromolecules, into the cells. In KRAS-driven cancers, including pancreatic ductal adenocarcinoma, macropinocytosis and subsequent lysosomal utilization are known to be enhanced to overcome metabolic stress. In this study, we investigated the role of Casein Kinase 2 (CK2) inhibition in macropinocytosis and subsequent metabolic processes in KRAS mutant cholangiocarcinoma (CCA) cell lines. MATERIALS AND METHODS: The bovine serum albumin (BSA) uptake indicating macropinocytosis was performed by flow cytometry using the HuCCT1 KRAS mutant CCA cell line. To validate macropinosome, the Rab7 and LAMP2 were labeled and analyzed via immunocytochemistry and western blot. The CX-4945 (Silmitasertib), CK2 inhibitor, was used to investigate the role of CK2 in macropinocytosis and subsequent lysosomal metabolism. RESULTS: The TFK-1, a KRAS wild-type CCA cell line, showed only apoptotic morphological changes. However, the HuCCT1 cell line showed macropinocytosis. Although CX-4945 induced morphological changes accompanied by the accumulation of intracellular vacuoles and cell death, the level of macropinocytosis did not change. These intracellular vacuoles were identified as late macropinosomes, representing Rab7+ vesicles before fusion with lysosomes. In addition, CX-4945 suppressed LAMP2 expression following the inhibition of the Akt-mTOR signaling pathway, which interrupts mature macropinosome and lysosomal metabolic utilization. CONCLUSION: Macropinocytosis is used as an energy source in the KRAS mutant CCA cell line HuCCT1. The inhibition of CK2 by CX-4945 leads to cell death in HuCCT1 cells through alteration of the lysosome-dependent metabolism.

Gut microbiota regulates the ALK5/NOX1 axis by altering glutamine metabolism to inhibit ferroptosis of intrahepatic cholangiocarcinoma cells.

Intrahepatic cholangiocarcinoma (ICC) is a kind of hepatobiliary tumor that is increasing in incidence and mortality. The gut microbiota plays a role in the onset and progression of cancer, however, the specific mechanism by which the gut microbiota acts on ICC remains unclear. In this study, feces and plasma from healthy controls and ICC patients were collected for 16S rRNA sequencing or metabolomics analysis. Gut microbiota analysis showed that gut microbiota abundance and biodiversity were altered in ICC patients compared with controls. Plasma metabolism analysis showed that the metabolite glutamine content of the ICC patient was significantly higher than that of the controls. KEGG pathway analysis showed that glutamine plays a vital role in ICC. In addition, the use of antibiotics in ICC animals further confirmed that changes in gut microbiota affect changes in glutamine. Further experiments showed that supplementation with glutamine inhibited ferroptosis and downregulated ALK5 and NOX1 expression in HuCCT1 cells. ALK5 overexpression or NOX1 overexpression increased NOX1, p53, PTGS2, ACSL4, LPCAT3, ROS, MDA and Fe2+ and decreased FTH1, SLC7A11 and GSH. Knockdown of NOX1 suppressed FIN56-induced ferroptosis. In vivo, supplementation with glutamine promoted tumor growth. Overexpression of ALK5 repressed tumor growth and induced ferroptosis in nude mice, which could be reversed by the addition of glutamine. Our results suggested that the gut microbiota altered glutamine metabolism to inhibit ferroptosis in ICC by regulating the ALK5/NOX1 axis.

Circulating tumor cell-derived exosome-transmitted long non-coding RNA TTN-AS1 can promote the proliferation and migration of cholangiocarcinoma cells.

BACKGROUND: Exosomes assume a pivotal role as essential mediators of intercellular communication within tumor microenvironments. Within this context, long noncoding RNAs (LncRNAs) have been observed to be preferentially sorted into exosomes, thus exerting regulatory control over the initiation and progression of cancer through diverse mechanisms. RESULTS: Exosomes were successfully isolated from cholangiocarcinoma (CCA) CTCs organoid and healthy human serum. Notably, the LncRNA titin-antisense RNA1 (TTN-AS1) exhibited a conspicuous up-regulation within CCA CTCs organoid derived exosomes. Furthermore, a significant elevation of TTN-AS1 expression was observed in tumor tissues, as well as in blood and serum exosomes from patients afflicted with CCA. Importantly, this hightened TTN-AS1 expression in serum exosomes of CCA patients manifested a strong correlation with both lymph node metastasis and TNM staging. Remarkably, both CCA CTCs organoid-derived exosomes and CCA cells-derived exosomes featuring pronounced TTN-AS1 expression demonstrated the capability to the proliferation and migratory potential of CCA cells. Validation of these outcomes was conducted in vivo experiments. CONCLUSIONS: In conclusion, our study elucidating that CCA CTCs-derived exosomes possess the capacity to bolster the metastasis tendencies of CCA cells by transporting TTN-AS1. These observations underscore the potential of TTN-AS1 within CTCs-derived exosomes to serve as a promising biomarker for the diagnosis and therapeutic management of CCA.

N6-methyladenosine-modified circSLCO1B3 promotes intrahepatic cholangiocarcinoma progression via regulating HOXC8 and PD-L1.

BACKGROUND: Refractoriness to surgical resection and chemotherapy makes intrahepatic cholangiocarcinoma (ICC) a fatal cancer of the digestive system with high mortality and poor prognosis. Important function invests circRNAs with tremendous potential in biomarkers and therapeutic targets. Nevertheless, it is still unknown how circRNAs contribute to the evolution of ICC. METHODS: CircRNAs in paired ICC and adjacent tissues were screened by circRNAs sequencing. To explore the impact of circRNAs on ICC development, experiments involving gain and loss of function were conducted. Various experimental techniques, including quantitative real-time PCR (qPCR), western blotting, RNA immunoprecipitation (RIP), luciferase reporter assays, RNA pull-down, chromatin immunoprecipitation (ChIP), ubiquitination assays and so on were employed to identify the molecular regulatory role of circRNAs. RESULTS: Herein, we reported a new circRNA, which originates from exon 9 to exon 15 of the SLCO1B3 gene (named circSLCO1B3), orchestrated ICC progression by promoting tumor proliferation, metastasis and immune evasion. We found that the circSLCO1B3 gene was highly overexpressed in ICC tissues and related to lymphatic metastasis, tumor sizes, and tumor differentiation. Mechanically, circSLCO1B3 not only promoted ICC proliferation and metastasis via miR-502-5p/HOXC8/SMAD3 axis, but also eradicated anti-tumor immunity via suppressing ubiquitin-proteasome-dependent degradation of PD-L1 by E3 ubiquitin ligase SPOP. We further found that methyltransferase like 3 (METTL3) mediated the m6A methylation of circSLCO1B3 and stabilizes its expression. Our findings indicate that circSLCO1B3 is a potential prognostic marker and therapeutic target in ICC patients. CONCLUSIONS: Taken together, m6A-modified circSLCO1B3 was correlated with poor prognosis in ICC and promoted ICC progression not only by enhancing proliferation and metastasis via potentiating HOXC8 expression, but also by inducing immune evasion via antagonizing PD-L1 degradation. These results suggest that circSLCO1B3 is a potential prognostic marker and therapeutic target for ICC.

YBX1 promotes stemness and cisplatin insensitivity in intrahepatic cholangiocarcinoma via the AKT/ß-catenin axis.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive malignancy characterized by a poor prognosis and closely linked to tumor stemness. However, the key molecules that regulate ICC stemness remain elusive. Although Y-box binding protein 1 (YBX1) negatively affects prognosis in various cancers by enhancing stemness and chemoresistance, its effect on stemness and cisplatin sensitivity in ICC remains unclear. METHODS: Three bulk and single-cell RNA-seq datasets were analyzed to investigate YBX1 expression in ICC and its association with stemness. Clinical samples and colony/sphere formation assays validated the role of YBX1 in stemness and sensitivity to cisplatin. AZD5363 and KYA1979K explored the interaction of YBX1 with the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT) and WNT/ß-catenin pathways. RESULTS: YBX1 was significantly upregulated in ICC, correlated with worse overall survival and shorter postoperative recurrence time, and was higher in chemotherapy-non-responsive ICC tissues. The YBX1-high group exhibited significantly elevated stemness scores, and genes linked to YBX1 upregulation were enriched in multiple stemness-related pathways. Moreover, YBX1 expression is significantly correlated with several stemness-related genes (SOX9, OCT4, CD133, CD44 and EPCAM). Additionally, YBX1 overexpression significantly enhanced the colony- and spheroid-forming abilities of ICC cells, accelerated tumor growth in vivo and reduced their sensitivity to cisplatin. Conversely, the downregulation of YBX1 exerted the opposite effect. The transcriptomic analysis highlighted the link between YBX1 and the PI3K/AKT and WNT/ß-catenin pathways. Further, AZD5363 and KYA1979K were used to clarify that YBX1 promoted ICC stemness through the regulation of the AKT/ß-catenin axis. CONCLUSIONS: YBX1 is upregulated in ICC and promotes stemness and cisplatin insensitivity via the AKT/ß-catenin axis. Our study describes a novel potential therapeutic target for improving ICC prognosis.

Circ_0007534 promotes cholangiocarcinoma stemness and resistance to anoikis through DDX3X-mediated positive feedback regulation of parental gene DDX42.

Cholangiocarcinoma (CCA) is a malignancy with an extremely poor prognosis, and much remains unknown about its pathogenesis and treatment modalities. Circular RNA (circRNA) has been proven to play regulatory roles in various tumorigenesis, yet its potential function and mechanism in cholangiocarcinoma require further investigation. This study is the first to identify the aberrant expression and functional role of a novel circRNA, circ_0007534, derived from the DDX42 gene, in cholangiocarcinoma. Compared to the normal control group, the expression of circ_0007534 was significantly elevated in the tissues and cells with CCA and that high expression correlated with lymph node invasion and poor prognosis. Functional experiments indicated that downregulating circ_0007534 markedly inhibited the proliferation, migration, invasion, stemness, and anti-anoikis ability of CCA cells, as well as the tumor growth and liver and lung metastasis in nude mice. Mechanistic studies revealed that DDX42, as the parent gene of circ_0007534, can mutually regulate each other's expression. Predominantly located in the cytoplasm, circ_0007534 can form a complex with the RNA-binding protein DDX3X, which enhances the stability of DDX42 mRNA, thereby upregulating the expression of DDX42. This creates a positive feedback loop among the three, collectively promoting the progression of cholangiocarcinoma. In conclusion, this study sheds light on the pivotal role and molecular mechanism of circ_0007534 in the development of CCA, offering potential new targets for early diagnosis and treatment.

FUT4 promotes the progression of Cholangiocarcinoma by modulating epithelial-mesenchymal transition.

Cholangiocarcinoma (CCA) is a common gastrointestinal malignancy characterized by a poor prognosis. Considering its prevalence, exploring its underlying molecular biological mechanisms is of paramount clinical importance. In this study, bioinformatics techniques were utilized to analyze CCA sample data obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. The analysis revealed a notable upregulation in FUT4 expression in CCA samples. To further investigate the functional implications of FUT4, in vivo and in vitro experiments were conducted, which demonstrated that FUT4 overexpression significantly enhances the proliferative and migratory capabilities of tumor cells. Subsequent sequencing analysis unveiled a correlation between FUT4 and epithelial-mesenchymal transition (EMT). Indeed, the pioneering discovery of elevated FUT4 expression in CCA was highlighted in this study. Further investigations into the function of FUT4 in CCA provided initial insights into its role in driving cancer progression via EMT. These findings present promising avenues for the diagnosis and treatment of CCA.[Figure: see text].

Comprehensive analysis of the clinical and biological significances for chemokine CXCL3 in cholangiocarcinoma.

Cholangiocarcinoma (CHOL) is a race malignant cancer arising from bile duct epithelial cells in clinical practice. C-X-C motif chemokine ligand 3 (CXCL3) is a member of chemokines family, which participates in the pathogenesis of various tumors. However, the association between CXCL3 and CHOL is unclear. This present study was to assess the role of CXCL3 expression in the progress of CHOL. TIMER, GEPIA, UALCAN, GSCA, LinkedOmics, Metascape and STRING databases were performed to evaluate the clinical and biological significances for CXCL3 with CHOL patients including expression, clinicopathological factors, immune cell infiltration, GO enrichment and KEGG pathway analyses, as well as PPI network analysis. The immunohistochemistry analysis of tissue microarray was conducted to detect the protein expression level, subcellular localization, clinicopathological factors and prognosis of CXCL3 in CHOL. The mRNA and protein expression levels of CXCL3 were markedly increased in CHOL tissues. The overexpression of CXCL3 was strongly associated with maximum tumor diameter of patients with CHOL. Additionally, there were negative correlations between the expression of CXCL3 and monocyte as well as Th17. Low infiltration of neutrophil indicated significantly shorter cumulative survival in CHOL patients. And CXCL3 was significantly associated with arm-level deletion of CD8+ T cell. Furthermore, functional network analysis suggested that CXCL3 and its associated genes were mainly enriched for chemotaxis, secretory granule membrane, cytokine activity and IL-17 signaling pathway. CXCL3 might potentially participate in the carcinogenesis of CHOL, which provided a direction for future research on the mechanism of CXCL3 in CHOL.

Escherichia coli-Induced cGLIS3-Mediated Stress Granules Activate the NF-κB Pathway to Promote Intrahepatic Cholangiocarcinoma Progression.

Patients with concurrent intrahepatic cholangiocarcinoma (ICC) and hepatolithiasis generally have poor prognoses. Hepatolithiasis is once considered the primary cause of ICC, although recent insights indicate that bacteria in the occurrence of hepatolithiasis can promote the progression of ICC. By constructing in vitro and in vivo ICC models and patient-derived organoids (PDOs), it is shown that Escherichia coli induces the production of a novel RNA, circGLIS3 (cGLIS3), which promotes tumor growth. cGLIS3 binds to hnRNPA1 and G3BP1, resulting in the assembly of stress granules (SGs) and suppression of hnRNPA1 and G3BP1 ubiquitination. Consequently, the IKKα mRNA is blocked in SGs, decreasing the production of IKKα and activating the NF-κB pathway, which finally results in chemoresistance and produces metastatic phenotypes of ICC. This study shows that a combination of Icaritin (ICA) and gemcitabine plus cisplatin (GP) chemotherapy can be a promising treatment strategy for ICC.

GOLPH3 promotes tumor malignancy via inhibition of ferroptosis by upregulating SLC7A11 in cholangiocarcinoma.

Golgi phosphoprotein 3 (GOLPH3) has been reported as an oncogene in various tumors; however, the role and function of GOLPH3 and its relevant molecular mechanism in cholangiocarcinoma (CCA) are unclear. Herein, GOLPH3 expression in CCA tissues was observed to be significantly higher than that in paired adjacent noncancerous tissues. Clinicopathological analysis showed that GOLPH3 expression correlated positively with the tumor-node-metastasis stage. In addition, GOLPH3 expression correlated inversely with the overall survival of patients with CCA. Multivariate analysis showed that GOLPH3 was an independent prognostic factor for patients with CCA. Transcriptome analysis (RNA sequencing) of GOLPH3 knockdown cells showed that the expression levels of nine ferroptosis-related genes were significantly changed, indicating the important biological function of GOLPH3 in ferroptosis in CCA cells. Furthermore, GOLPH3 knockdown could significantly promote Erastin-induced ferroptosis in vitro and suppress tumor growth in vivo. Overexpression of GOLPH3 had the opposite effect on this phenotype. Further studies revealed that GOLPH3 knockdown was significantly associated with a decrease in cysteine content, an accumulation of the lipid peroxidation product malondialdehyde, an increase in reactive oxygen species, and sensitized CCA cells to Erastin-induced ferroptosis. Moreover, changes in GOLPH3 expression were found to be consistent with the expression of light chain subunit solute carrier family 7 member 11 (SLC7A11). Thus, our study suggested that GOLPH3 functions as an oncoprotein in CCA and may suppress ferroptosis by facilitating SLC7A11 expression, suggesting that GOLPH3 could serve as a therapeutic target for CCA treatment.

Role of Wisteria floribunda agglutinin binding glycans in carcinogenesis and metastasis of cholangiocarcinoma.

Aberrant glycosylation is an important factor in facilitating tumor progression and therapeutic resistance. In this study, using Wisteria floribunda agglutinin (WFA), we examined the expression of WFA-binding glycans (WFAG) in cholangiocarcinoma (CCA). The results showed that WFAG was highly detected in precancerous and cancerous lesions of human CCA tissues, although it was rarely detected in normal bile ducts. The positive signal of WFAG in the cancerous lesion accounted for 96.2% (50/52) of the cases. Overexpression of WFAG was significantly associated with lymph node and distant metastasis (P < 0.05). The study using the CCA hamster model showed that WFAG is elevated in preneoplastic and neoplastic bile ducts as early as 1 month after being infected with liver fluke and exposed to N-nitrosodimethylamine. Functional analysis was performed to reveal the role of WFAG in CCA. The CCA cell lines KKU-213A and KKU-213B were treated with WFA, followed by migration assay. Our data suggested that WFAG facilitates the migration of CCA cells via the activation of the Akt and ERK signaling pathways. In conclusion, we have demonstrated the association of WFAG with carcinogenesis and metastasis of CCA, suggesting its potential as a target for the treatment of the disease.

RPL35A promotes the progression of cholangiocarcinoma by mediating HSPA8 ubiquitination.

BACKGROUND: Cholangiocarcinoma (CCA) is a biliary epithelial malignant tumor with an increasing incidence worldwide. Therefore, further understanding of the molecular mechanisms of CCA progression is required to identify new therapeutic targets. METHODS: The expression of RPL35A in CCA and para-carcinoma tissues was detected by immunohistochemical staining. IP-MS combined with Co-IP identified downstream proteins regulated by RPL35A. Western blot and Co-IP of CHX or MG-132 treated CCA cells were used to verify the regulation of HSPA8 protein by RPL35A. Cell experiments and subcutaneous tumorigenesis experiments in nude mice were performed to evaluate the effects of RPL35A and HSPA8 on the proliferation, apoptosis, cell cycle, migration of CCA cells and tumor growth in vivo. RESULTS: RPL35A was significantly upregulated in CCA tissues and cells. RPL35A knockdown inhibited the proliferation and migration of HCCC-9810 and HUCCT1 cells, induced apoptosis, and arrested the cell cycle in G1 phase. HSPA8 was a downstream protein of RPL35A and overexpressed in CCA. RPL35A knockdown impaired HSPA8 protein stability and increased HSPA8 protein ubiquitination levels. RPL35A overexpression promoted CCA cell proliferation and migration. HSPA8 knockdown inhibited CCA cell proliferation and migration, and reversed the promoting effect of RPL35A. Furthermore, RPL35A promoted tumor growth in vivo. In contrast, HSPA8 knockdown suppressed tumor growth, while was able to restore the effects of RPL35A overexpression. CONCLUSION: RPL35A was upregulated in CCA tissues and promoted the progression of CCA by mediating HSPA8 ubiquitination.