Integrative analyses of bulk and single-cell transcriptomics reveals the infiltration and crosstalk of cancer-associated fibroblasts as a novel predictor for prognosis and microenvironment remodeling in intrahepatic cholangiocarcinoma.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is a highly malignant neoplasm and characterized by desmoplastic matrix. The heterogeneity and crosstalk of tumor microenvironment remain incompletely understood. METHODS: To address this gap, we performed Weighted Gene Co-expression Network Analysis (WGCNA) to identify and construct a cancer associated fibroblasts (CAFs) infiltration biomarker. We also depicted the intercellular communication network and important receptor-ligand complexes using the single-cell transcriptomics analysis of tumor and Adjacent normal tissue. RESULTS: Through the intersection of TCGA DEGs and WGCNA module genes, 784 differential genes related to CAFs infiltration were obtained. After a series of regression analyses, the CAFs score was generated by integrating the expressions of EVA1A, APBA2, LRRTM4, GOLGA8M, BPIFB2, and their corresponding coefficients. In the TCGA-CHOL, GSE89748, and 107,943 cohorts, the high CAFs score group showed unfavorable survival prognosis (p < 0.001, p = 0.0074, p = 0.028, respectively). Additionally, a series of drugs have been predicted to be more sensitive to the high-risk group (p < 0.05). Subsequent to dimension reduction and clustering, thirteen clusters were identified to construct the single-cell atlas. Cell-cell interaction analysis unveiled significant enhancement of signal transduction in tumor tissues, particularly from fibroblasts to malignant cells via diverse pathways. Moreover, SCENIC analysis indicated that HOXA5, WT1, and LHX2 are fibroblast specific motifs. CONCLUSIONS: This study reveals the key role of fibroblasts - oncocytes interaction in the remodeling of the immunosuppressive microenvironment in intrahepatic cholangiocarcinoma. Subsequently, it may trigger cascade activation of downstream signaling pathways such as PI3K-AKT and Notch in tumor, thus initiating tumorigenesis. Targeted drugs aimed at disrupting fibroblasts-tumor cell interaction, along with associated enrichment pathways, show potential in mitigating the immunosuppressive microenvironment that facilitates tumor progression.

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.

Bortezomib in previously treated phosphatase and tension homology-deficient patients with advanced intrahepatic cholangiocarcinoma: An open-label, prospective and single-centre phase II trial.

INTRODUCTION: Intrahepatic cholangiocarcinoma (ICC) is characterized by a dismal prognosis with limited therapeutic alternatives. To explore phosphatase and tension homolog (PTEN) as a biomarker for proteasome inhibition in ICC, we conducted a phase II trial to assess the second-line efficacy of bortezomib in PTEN-deficient advanced ICC patients. METHODS: A total of 130 patients with advanced ICC in our centre were screened by PTEN immunohistochemical staining between 1 July 2017, and 31 December 2021, and 16 patients were ultimately enrolled and treated with single-agent bortezomib 1.3 mg/m2 on days 1, 4, 8 and 11 of a 21-day cycle. The primary endpoint was the objective response rate (ORR) according to Response Evaluation Criteria in Solid Tumors v1.1. RESULTS: The median follow-up was 6.55 months (95% confidence interval [CI]: 0.7-19.9 months). Among the 16 enrolled patients, the ORR was 18.75% (3/16) and the disease control rate was 43.75% (7/16). The median progress-free survival was 2.95 months (95% CI: 2.1-5.1 months) and the median overall survival (mOS) was 7.2 months (95% CI: 0.7-21.6 months) in the intent-to-treat-patients. Treatment-related adverse events of any grade were reported in 16 patients, with thrombopenia being the most common toxicity. Patients with PTEN staining scores of 0 were more likely to benefit from bortezomib than those with staining scores > 0. CONCLUSIONS: Bortezomib yielded an encouraging objective response and a favourable OS as a second-line agent in PTEN-deficient ICC patients. Our findings suggest bortezomib as a promising therapeutic option for patients with PTEN-deficient ICC. HIGHLIGHTS: There is a limited strategy for the second-line option of intrahepatic cholangiocarcinoma (ICC). This investigator-initiated phase 2 study evaluated bortezomib in ICC patients with phosphatase and tension homology deficiency. The overall response rate was 18.75% and the overall survival was 7.2 months in the intent-to-treat cohort. These results justify further developing bortezomib in ICC patients with PTEN deficiency.

Cholangiocarcinoma combined with biliary obstruction: an exosomal circRNA signature for diagnosis and early recurrence monitoring.

Cholangiocarcinoma (CCA) is a highly malignant biliary tract cancer with currently suboptimal diagnostic and prognostic approaches. We present a novel system to monitor CCA using exosomal circular RNA (circRNA) via serum and biliary liquid biopsies. A pilot cohort consisting of patients with CCA-induced biliary obstruction (CCA-BO, n = 5) and benign biliary obstruction (BBO, n = 5) was used to identify CCA-derived exosomal circRNAs through microarray analysis. This was followed by a discovery cohort (n = 20) to further reveal a CCA-specific circRNA complex (hsa-circ-0000367, hsa-circ-0021647, and hsa-circ-0000288) in both bile and serum exosomes. In vitro and in vivo studies revealed the three circRNAs as promoters of CCA invasiveness. Diagnostic and prognostic models were established and verified by two independent cohorts (training cohort, n = 184; validation cohort, n = 105). An interpreter-free diagnostic model disclosed the diagnostic power of biliary exosomal circRNA signature (Bile-DS, AUROC = 0.947, RR = 6.05) and serum exosomal circRNA signature (Serum-DS, AUROC = 0.861, RR = 4.04) compared with conventional CA19-9 (AUROC = 0.759, RR = 2.08). A prognostic model of CCA undergoing curative-intent surgery was established by calculating early recurrence score, verified with bile samples (Bile-ERS, C-index=0.783) and serum samples (Serum-ERS, C-index = 0.782). These models, combined with other prognostic factors revealed by COX-PH model, enabled the establishment of nomograms for recurrence monitoring of CCA. Our study demonstrates that the exosomal triple-circRNA panel identified in both bile and serum samples serves as a novel diagnostic and prognostic tool for the clinical management of CCA.

Current considerations on intraductal papillary neoplasms of the bile duct and pancreatic duct.

Pancreatobiliary intraductal papillary neoplasms (IPNs) represent precursors of pancreatic cancer or bile duct cholangiocarcinoma that can be detected and treated. Despite advances in diagnostic methods, identifying these premalignant lesions is still challenging for treatment providers. Modern imaging, biomarkers and molecular tests for genomic alterations can be used for diagnosis and follow-up. Surgical intervention in combination with new chemotherapeutic agents is considered the optimal treatment for malignant cases. The balance between the risk of malignancy and any risk of resection guides management policy; therefore, treatment should be individualized based on a meticulous preoperative assessment of high-risk stigmata. IPN of the bile duct is more aggressive; thus, early diagnosis and surgery are crucial. The conservative management of low-risk pancreatic branch-duct lesions is safe and effective.

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

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

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.

Identification and validation of a lactate metabolism-related six-gene prognostic signature in intrahepatic cholangiocarcinoma.

PURPOSE: Intrahepatic cholangiocarcinoma (iCCA) is a highly malignant and fatal liver tumor with increasing incidence worldwide. Lactate metabolism has been recently reported as a crucial contributor to tumor progression and immune regulation in the tumor microenvironment. However, it remains poorly identified about the biological functions of lactate metabolism in iCCA, which hinders the development of prognostic tools and therapeutic interventions. METHODS: The univariate Cox regression analysis and Boruta algorithm were utilized to identify key lactate metabolism-related genes (LMRGs), and a prognostic signature was constructed based on LMRG scores. Genomic variations and immune cell infiltration were evaluated in the high and low LMRG score groups. Finally, the biological functions of key LMRGs were verified with in vitro and in vivo experiments. RESULTS: Patients in the high LMRG score group exhibit a poor prognosis compared to those in the low LMRG score group, with a high frequency of TP53 and KRAS mutations. Moreover, the infiltration and function of NK cells were compromised in the high LMRG score group, consistent with the results from two independent single-cell RNA sequencing datasets and immunohistochemistry of tissue microarrays. Experimental data revealed that lactate dehydrogenase A (LDHA) knockdown inhibited proliferation and migration in iCCA cell lines and tumor growth in immunocompetent mice. CONCLUSION: Our study revealed the biological roles of LDHA in iCCA and developed a reliable lactate metabolism-related prognostic signature for iCCA, offering promising therapeutic targets for iCCA in the clinic.

Identification of Novel Cuproptosis-Related Genes Mediating the Prognosis and Immune Microenvironment in Cholangiocarcinoma.

BACKGROUND: Cuproptosis is a novel type of mediated cell death strongly associated with the progression of several cancers and has been implicated as a potential therapeutic target. However, the role of cuproptosis in cholangiocarcinoma for prognostic prediction, subgroup classification, and therapeutic strategies remains largely unknown. METHODS: A systematic analysis was conducted among 146 cuproptosis-related genes and clinical information based on independent mRNA and protein datasets to elucidate the potential mechanisms and prognostic prediction value of cuproptosis-related genes. A 10-cuproptosis-related gene prediction model was constructed, and its effects on cholangiocarcinoma prognosis were significantly connected to poor patient survival. Additionally, the expression patterns of our model included genes that were validated with several cholangiocarcinoma cancer cell lines and a normal biliary epithelial cell line. RESULTS: First, a 10-cuproptosis-related gene signature (ADAM9, ADAM17, ALB, AQP1, CDK1, MT2A, PAM, SOD3, STEAP3, and TMPRSS6) displayed excellent predictive performance for the overall survival of cholangiocarcinoma. The low-cuproptosis group had a significantly better prognosis than the high-cuproptosis group with transcriptome and protein cohorts. Second, compared with the high-risk and low-risk groups, the 2 groups displayed distinct tumor microenvironments, reduced proportions of endothelial cells, and increased levels of cancer-associated fibroblasts based on CIBERSORTx and EPIC analyses. Third, patients' sensitivities to chemotherapeutic drugs and immune checkpoints revealed distinctive differences between the 2 groups. Finally, in replicating the expression patterns of the 10 genes, these results were validated with quantitative real-time polymerase chain reaction results validating the abnormal expression pattern of the target genes in cholangiocarcinoma. CONCLUSIONS: Collectively, we established and verified an effective prognostic model that could separate cholangiocarcinoma patients into 2 heterogeneous cuproptosis subtypes based on the molecular or protein characteristics of 10 cuproptosis-related genes. These findings may provide potential benefits for unveiling molecular characteristics and defining subgroups could improve the early diagnosis and individualized treatment of cholangiocarcinoma patients.

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.

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.

Generation of Metastatic Cholangiocarcinoma Patient-Derived Xenograft Models.

Cholangiocarcinoma (CCA) poses a substantial clinical hurdle as it is often detected at advanced metastatic stages with limited therapeutic options. To enhance our understanding of advanced CCA, it is imperative to establish preclinical models that faithfully recapitulate the disease's characteristics. Patient-derived xenograft (PDX) models have emerged as a valuable approach in cancer research, offering an avenue to reproduce and study the genomic, histologic, and molecular features of the original human tumors. By faithfully preserving the heterogeneity, microenvironmental interactions, and drug responses observed in human tumors, PDX models serve as highly relevant and predictive preclinical tools. Here, we present a comprehensive protocol that outlines the step-by-step process of generating and maintaining PDX models using biopsy samples from patients with advanced metastatic CCA. The protocol encompasses crucial aspects such as tissue processing, xenograft transplantation, and subsequent monitoring of the PDX models. By employing this protocol, we aim to establish a robust collection of PDX models that accurately reflect the genomic landscape, histologic diversity, and therapeutic responses observed in advanced CCA, thereby enabling improved translational research, drug development, and personalized treatment strategies for patients facing this challenging disease.

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.

The histone lysine acetyltransferase KAT2B inhibits cholangiocarcinoma growth: evidence for interaction with SP1 to regulate NF2-YAP signaling.

BACKGROUND: Cholangiocarcinoma (CCA) is a highly malignant cancer of the biliary tract with poor prognosis. Further mechanistic insights into the molecular mechanisms of CCA are needed to develop more effective target therapy. METHODS: The expression of the histone lysine acetyltransferase KAT2B in human CCA was analyzed in human CCA tissues. CCA xenograft was developed by inoculation of human CCA cells with or without KAT2B overexpression into SCID mice. Western blotting, ChIP-qPCR, qRT-PCR, protein immunoprecipitation, GST pull-down and RNA-seq were performed to delineate KAT2B mechanisms of action in CCA. RESULTS: We identified KAT2B as a frequently downregulated histone acetyltransferase in human CCA. Downregulation of KAT2B was significantly associated with CCA disease progression and poor prognosis of CCA patients. The reduction of KAT2B expression in human CCA was attributed to gene copy number loss. In experimental systems, we demonstrated that overexpression of KAT2B suppressed CCA cell proliferation and colony formation in vitro and inhibits CCA growth in mice. Mechanistically, forced overexpression of KAT2B enhanced the expression of the tumor suppressor gene NF2, which is independent of its histone acetyltransferase activity. We showed that KAT2B was recruited to the promoter region of the NF2 gene via interaction with the transcription factor SP1, which led to enhanced transcription of the NF2 gene. KAT2B-induced NF2 resulted in subsequent inhibition of YAP activity, as reflected by reduced nuclear accumulation of oncogenic YAP and inhibition of YAP downstream genes. Depletion of NF2 was able to reverse KAT2B-induced reduction of nuclear YAP and subvert KAT2B-induced inhibition of CCA cell growth. CONCLUSIONS: This study provides the first evidence for an important tumor inhibitory effect of KAT2B in CCA through regulation of NF2-YAP signaling and suggests that this signaling cascade may be therapeutically targeted for CCA treatment.

Bioinformatics and system biology approaches to determine the connection of SARS-CoV-2 infection and intrahepatic cholangiocarcinoma.

INTRODUCTION: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease 2019 (COVID-19), has infected millions of individuals worldwide, which poses a severe threat to human health. COVID-19 is a systemic ailment affecting various tissues and organs, including the lungs and liver. Intrahepatic cholangiocarcinoma (ICC) is one of the most common liver cancer, and cancer patients are particularly at high risk of SARS-CoV-2 infection. Nonetheless, few studies have investigated the impact of COVID-19 on ICC patients. METHODS: With the methods of systems biology and bioinformatics, this study explored the link between COVID-19 and ICC, and searched for potential therapeutic drugs. RESULTS: This study identified a total of 70 common differentially expressed genes (DEGs) shared by both diseases, shedding light on their shared functionalities. Enrichment analysis pinpointed metabolism and immunity as the primary areas influenced by these common genes. Subsequently, through protein-protein interaction (PPI) network analysis, we identified SCD, ACSL5, ACAT2, HSD17B4, ALDOA, ACSS1, ACADSB, CYP51A1, PSAT1, and HKDC1 as hub genes. Additionally, 44 transcription factors (TFs) and 112 microRNAs (miRNAs) were forecasted to regulate the hub genes. Most importantly, several drug candidates (Periodate-oxidized adenosine, Desipramine, Quercetin, Perfluoroheptanoic acid, Tetrandrine, Pentadecafluorooctanoic acid, Benzo[a]pyrene, SARIN, Dorzolamide, 8-Bromo-cAMP) may prove effective in treating ICC and COVID-19. CONCLUSION: This study is expected to provide valuable references and potential drugs for future research and treatment of COVID-19 and ICC.

Cisplatin Disrupts Proteasome Bounce-Back Effect through Suppressing ZEB1/Nfe2l1 in Cholangiocarcinoma.

BACKGROUND: Bortezomib (BTZ) is a powerful proteasome inhibitor that has been approved for the treatment of haematologic malignancies. Its effectiveness has been assessed against different types of solid tumours. BTZ is ineffective in most solid tumours because of drug resistance, including cholangiocarcinoma, which is associated with a proteasome bounce-back effect. However, the mechanism through which proteasome inhibitors induce the proteasome bounce-back effect remains largely unknown. METHODS: Cholangiocarcinoma cells were treated with BTZ, cisplatin, or a combination of both. The mRNA levels of Nfe2l1 and proteasome subunit genes (PSMA1, PSMB7, PSMD1, PSMD11, PSMD14, and PSME4) were determined using quantitative real time polymerase chain reaction (qPCR). The protein levels of nuclear factor-erythroid 2-related factor 1 (Nfe2l1) and proteasome enzyme activity were evaluated using western blotting and proteasome activity assays, respectively. Transcriptome sequencing was performed to screen for potential transcription factors that regulate Nfe2l1 expression. The effect of zinc finger E-box-binding homeobox 1 (ZEB1) on the expression of Nfe2l1 and proteasome subunit genes, as well as proteasome enzyme activity, was evaluated after the knockdown of ZEB1 expression with siRNA before treatment with BTZ. The transcriptional activity of ZEB1 on the Nfe2l1 promoter was detected using dual-luciferase reporter gene and chromatin immunoprecipitation assays. Cell viability was measured using the cell counting kit-8 (CCK-8) assay and cell apoptosis was assessed using western blotting and flow cytometry. RESULTS: Cisplatin treatment of BTZ-treated human cholangiocarcinoma cell line (RBE) suppressed proteasome subunit gene expression (proteasome bounce-back) and proteasomal enzyme activity. This effect was achieved by reducing the levels of Nfe2l1 mRNA and protein. Our study utilised transcriptome sequencing to identify ZEB1 as an upstream transcription factor of Nfe2l1, which was confirmed using dual-luciferase reporter gene and chromatin immunoprecipitation assays. Notably, ZEB1 knockdown using siRNA (si-ZEB1) hindered the expression of proteasome subunit genes under both basal and BTZ-induced conditions, leading to the inhibition of proteasomal enzyme activity. Furthermore, the combination treatment with BTZ, cisplatin, and si-ZEB1 significantly reduced the viability of RBE cells. CONCLUSIONS: Our study uncovered a novel mechanism through which cisplatin disrupts the BTZ-induced proteasome bounce-back effect by suppressing the ZEB1/Nfe2l1 axis in cholangiocarcinoma. This finding provides a theoretical basis for developing proteasome inhibitor-based strategies for the clinical treatment of cholangiocarcinoma and other tumours.

Role of long non-coding RNAs in cholangiocarcinoma: A systematic review and meta-analysis.

BACKGROUND: Cholangiocarcinoma (CCA), as a rare malignancy of the biliary tree, has a poor prognosis most of the time. CCA is highly epigenetically regulated and several long non-coding RNAs (lncRNA) have been investigated to have a diagnostic and prognostic role in CCA. The current study aimed to assess the studies finding relevant lncRNAs in CCA systematically. METHODS: International databases, including PubMed, Cochrane Library, and Embase, were comprehensively searched in order to identify studies investigating any lncRNA in CCA. After screening by title/abstract and full-text, necessary data were extracted. Random-effect meta-analysis was performed for pooling the areas under the curve (AUCs), specificity, and sensitivity of lncRNAs for the diagnosis of CCA. RESULTS: A total of 33 studies were chosen to be included in the final analysis, comprised of 2677 patients. Meta-analysis of AUCs for evaluation of CCA resulted in pooled AUC of 0.79 (95% CI: 0.75-0.82; I2 = 69.11, p < .01). Additionally, overall sensitivity of 0.80 (95% CI 0.75-0.84) and specificity of 0.77 (95% CI: 0.68-0.84) were observed. Measurement of lncRANs in the assessment of CCA also improved overall survival significantly (effect size 1.61, 95% CI: 1.39-1.82). A similar result was found for progression-free survival (effect size 1.57, 95% CI: 1.20-1.93). CONCLUSION: Based on our findings, lncRNAs showed promising results as biomarkers in the diagnosis of CCA since they had acceptable sensitivity and specificity, in addition to the fact that improved survival in this poor prognosis cancer. Further studies might be needed to address this issue and find the best clinically useful lncRNA.

Harnessing the supremacy of MEG3 LncRNA to defeat gastrointestinal malignancies.

Evidence suggests that long non-coding RNAs (lncRNAs) play a pivotal role in the carcinogenesis and progression of various human malignancies including gastrointestinal malignancies. This comprehensive review reports the functions and mechanisms of the lncRNA maternally expressed gene 3 (MEG3) involved in gastrointestinal malignancies. It summarizes its roles in mediating the regulation of cellular proliferation, apoptosis, migration, invasiveness, epithelial-to-mesenchymal transition, and drug resistance in several gastrointestinal cancers such as colorectal cancer, gall bladder cancer, pancreatic cancer, gastric cancer, esophageal cancer, cholangiocarcinoma, gastrointestinal stromal tumors and most importantly, hepatocellular carcinoma. In addition, the authors briefly highlight its implicated mechanistic role and interactions with different non-coding RNAs and oncogenic signaling cascades. This review presents the rationale for developing non coding RNA-based anticancer therapy via harnessing the power of MEG3 in gastrointestinal malignancies.