Children with Hirschsprung disease exhibited alterations in host-microbial co-metabolism after pull-through operation.

PURPOSE: This study aims to compare the fecal metabolome in post pull-through HD with and without HAEC patients and healthy young children using nuclear magnetic resonance (NMR) spectroscopy. METHODS: Fresh fecal samples were collected from children under 5 years of age in both post-pull-through HD patients and healthy Thai children. A total of 20 fecal samples were then analyzed using NMR spectroscopy. RESULTS: Thirty-four metabolites identified among HD and healthy children younger than 5 years were compared. HD samples demonstrated a significant decrease in acetoin, phenylacetylglutamine, and N-acetylornithine (corrected p value = 0.01, 0.04, and 0.004, respectively). Succinate and xylose significantly decreased in HD with HAEC group compared to HD without HAEC group (corrected p value = 0.04 and 0.02, respectively). Moreover, glutamine and glutamate metabolism, and alanine, aspartate, and glutamate metabolism were the significant pathways involved, with pathway impact 0.42 and 0.50, respectively (corrected p value = 0.02 and 0.04, respectively). CONCLUSION: Differences in class, quantity, and metabolism of protein and other metabolites in young children with HD after pull-through operation were identified. Most of the associated metabolic pathways were correlated with the amino acids metabolism, which is required to maintain intestinal integrity and function.

Integrative multiomics enhancer activity profiling identifies therapeutic vulnerabilities in cholangiocarcinoma of different etiologies.

OBJECTIVES: Cholangiocarcinoma (CCA) is a heterogeneous malignancy with high mortality and dismal prognosis, and an urgent clinical need for new therapies. Knowledge of the CCA epigenome is largely limited to aberrant DNA methylation. Dysregulation of enhancer activities has been identified to affect carcinogenesis and leveraged for new therapies but is uninvestigated in CCA. Our aim is to identify potential therapeutic targets in different subtypes of CCA through enhancer profiling. DESIGN: Integrative multiomics enhancer activity profiling of diverse CCA was performed. A panel of diverse CCA cell lines, patient-derived and cell line-derived xenografts were used to study identified enriched pathways and vulnerabilities. NanoString, multiplex immunohistochemistry staining and single-cell spatial transcriptomics were used to explore the immunogenicity of diverse CCA. RESULTS: We identified three distinct groups, associated with different etiologies and unique pathways. Drug inhibitors of identified pathways reduced tumour growth in in vitro and in vivo models. The first group (ESTRO), with mostly fluke-positive CCAs, displayed activation in estrogen signalling and were sensitive to MTOR inhibitors. Another group (OXPHO), with mostly BAP1 and IDH-mutant CCAs, displayed activated oxidative phosphorylation pathways, and were sensitive to oxidative phosphorylation inhibitors. Immune-related pathways were activated in the final group (IMMUN), made up of an immunogenic CCA subtype and CCA with aristolochic acid (AA) mutational signatures. Intratumour differences in AA mutation load were correlated to intratumour variation of different immune cell populations. CONCLUSION: Our study elucidates the mechanisms underlying enhancer dysregulation and deepens understanding of different tumourigenesis processes in distinct CCA subtypes, with potential significant therapeutics and clinical benefits.

The Hallmarks of Liver Fluke Related Cholangiocarcinoma: Insight into Drug Target Possibility.

Cholangiocarcinoma (CCA) is a malignant tumor of the biliary tree that is classified into three groups based on its anatomic location: intrahepatic (iCCA), perihilar (pCCA), and distal (dCCA). Perihilar CCA is the most common type and accounts for 50-60% of CCA cases. It is followed by distal CCA and then intrahepatic CCA that account for 20-30% and 10-20% of cases, respectively. This chapter discusses the hallmarks of liver fluke related CCA and explores insights into drug target possibilities.

Overexpression of a panel of cancer stem cell markers enhances the predictive capability of the progression and recurrence in the early stage cholangiocarcinoma.

BACKGROUND: Cancer recurrence is the important problem of cholangiocarcinoma (CCA) patients, lead to a very high mortality rate. Therefore, the identification of candidate markers to predict CCA recurrence is needed in order to effectively manage the disease. This study aims to examine the predictive value of cancer stem cell (CSC) markers on the progression and recurrence of CCA patients. METHODS: The expression of 6 putative CSC markers, cluster of differentiation 44 (CD44), CD44 variant 6 (CD44v6), CD44 variants 8-10 (CD44v8-10), cluster of differentiation 133 (CD133), epithelial cell adhesion molecule (EpCAM), and aldehyde dehydrogenase 1A1 (ALDH1A1), was investigated in 178 CCA tissue samples using immunohistochemistry (IHC) and analyzed with respect to clinicopathological data and patient outcome including recurrence-free survival (RFS) and overall survival (OS). The candidate CSC markers were also investigated in serum from CCA patients, and explored for their predictive ability on CCA recurrence. RESULTS: Elevated protein level of CD44 and positive expression of CD44v6 and CD44v8-10 were significantly associated with short RFS and OS, while high levels of ALDH1A1 were correlated with a favorable prognosis patient. The elevated CD44v6 level was also correlated with higher tumor staging, whereas a decreasing level of ALDH1A1 was correlated with lower tumor staging. The levels of CD44, CD44v6 and CD44v8-10 were also correlated and were associated with a poor outcome. Furthermore, soluble CD44, CD44v6, CD44v8-10 and EpCAM were significantly increased in the recurrence group for early stage CCA; they also correlated with high levels of the tumor marker CA19-9. Elevated levels of CD44, CD44v6, CD44v8-10 or EpCAM alone or in combination has the potential to predict CCA recurrence. CONCLUSIONS: The overexpression of CD44, CD44v6, CD44v8-10 and EpCAM increases predictability of post-operative CCA recurrence. Moreover, the overexpression of the panel of CSC markers combined with CA19-9 could improve our predictive ability for tumor recurrence in early stage CCA patients. This result may be beneficial for the patients in order to predict the outcome after treatment and may be useful for clinical intervention in order to improve patient survival.

A panel of protein kinase high expression is associated with postoperative recurrence in cholangiocarcinoma.

BACKGROUND: Cancer recurrence is one of the most concerning clinical problems of cholangiocarcinoma (CCA) patients after treatment. However, an identification of predictive factor on Opisthorchis viverrini (OV)-associated CCA recurrence is not well elucidated. In the present study, we aimed to investigate the correlation of twelve targeted protein kinases with CCA recurrence. METHODS: Twelve protein kinases, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2, 3, 4 (HER2, HER3, HER4), vascular endothelial growth factor receptor 3 (VEGFR3), vascular endothelial growth factor-C (VEGF-C), erythropoietin-producing hepatocellular carcinoma receptor type-A3 (EphA3), EphrinA1, phosphor-serine/threonine kinase 1 (p-Akt1), serine/threonine kinase 1 (Akt1), beta-catenin and protein Wnt5a (Wnt5a) were examined using immunohistochemistry. Pre-operative serum tumor markers, CA19-9 and CEA were also investigated. RESULTS: Among twelve protein kinases, EGFR, HER4, and EphA3 were associated with tumor recurrence status, recurrence-free survival (RFS) and overall survival (OS). Multivariate cox regression demonstrated that EGFR, HER4, EphA3 or the panel of high expression of these proteins was an independent prognostic factor for tumor recurrence. The combination of high expression of these proteins with a high level of CA19-9 could improve the predictive ability on tumor recurrence. Moreover, the patients were stratified more accurately when analyzed using the combination of high expression of these proteins with primary tumor (T) or lymph node metastasis (N) status. CONCLUSION: EGFR, HER4, EphA3 or the panel of high expression of these proteins is an independent prognostic factor for post-operative CCA recurrence.

Opposing Roles of FoxA1 and FoxA3 in Intrahepatic Cholangiocarcinoma Progression.

Cholangiocarcinoma (CCA), a malignancy of biliary epithelium, is related to liver stem cell deregulation. FoxAs are a group of transcription factors that play critical roles in liver stem cell differentiation. In this study, the expression levels of FoxAs (i.e., FoxA1, FoxA2 and FoxA3) were detected in intrahepatic CCA tissues and the functions of FoxAs were studied in CCA cell lines. FoxA1 and FoxA2 were mainly localized in the nuclei of normal bile duct (NBD) cells and some of the cancer cells. Low expression of FoxA1 in CCA tissues (72%) was significantly correlated with poor prognosis. FoxA3 expression of CCA cells was localized in the nucleus and cytoplasm, whereas it was slightly detected in NBDs. High expression of FoxA3 in cancer tissues (61%) was significantly related to high metastasis status. These findings suggest the opposing roles of FoxA1 and FoxA3 in CCA. Moreover, the FoxA1-over-expressing CCA cell line exhibited a significant reduction in proliferative and invasive activities compared to control cells. Knockdown of FoxA3 in CCA cells resulted in a significant decrease in proliferative and invasive activities compared with control cells. Taken together, in CCA, FoxA1 is down-regulated and has tumor suppressive roles, whereas FoxA3 is up-regulated and has oncogenic roles.

Cholangiocarcinoma: Recent Advances in Molecular Pathobiology and Therapeutic Approaches.

Cholangiocarcinomas (CCA) pose a complex challenge in oncology due to diverse etiologies, necessitating tailored therapeutic approaches. This review discusses the risk factors, molecular pathology, and current therapeutic options for CCA and explores the emerging strategies encompassing targeted therapies, immunotherapy, novel compounds from natural sources, and modulation of gut microbiota. CCA are driven by an intricate landscape of genetic mutations, epigenetic dysregulation, and post-transcriptional modification, which differs based on geography (e.g., for liver fluke versus non-liver fluke-driven CCA) and exposure to environmental carcinogens (e.g., exposure to aristolochic acid). Liquid biopsy, including circulating cell-free DNA, is a potential diagnostic tool for CCA, which warrants further investigations. Currently, surgical resection is the primary curative treatment for CCA despite the technical challenges. Adjuvant chemotherapy, including cisplatin and gemcitabine, is standard for advanced, unresectable, or recurrent CCA. Second-line therapy options, such as FOLFOX (oxaliplatin and 5-FU), and the significance of radiation therapy in adjuvant, neoadjuvant, and palliative settings are also discussed. This review underscores the need for personalized therapies and demonstrates the shift towards precision medicine in CCA treatment. The development of targeted therapies, including FDA-approved drugs inhibiting FGFR2 gene fusions and IDH1 mutations, is of major research focus. Investigations into immune checkpoint inhibitors have also revealed potential clinical benefits, although improvements in survival remain elusive, especially across patient demographics. Novel compounds from natural sources exhibit anti-CCA activity, while microbiota dysbiosis emerges as a potential contributor to CCA progression, necessitating further exploration of their direct impact and mechanisms through in-depth research and clinical studies. In the future, extensive translational research efforts are imperative to bridge existing gaps and optimize therapeutic strategies to improve therapeutic outcomes for this complex malignancy.

Metabolomic analyses uncover an inhibitory effect of niclosamide on mitochondrial membrane potential in cholangiocarcinoma cells.

Background: Niclosamide is an oral anthelminthic drug that has been used for treating tapeworm infections. Its mechanism involves the disturbance of mitochondrial membrane potential that in turn inhibits oxidative phosphorylation leading to ATP depletion. To date, niclosamide has been validated as the potent anti-cancer agent against several cancers. However, the molecular mechanisms underlying the effects of niclosamide on the liver fluke Opisthorchis viverrini (Ov)-associated cholangiocarcinoma (CCA) cell functions remain to be elucidated. The aims of this study were to investigate the effects of niclosamide on CCA cell proliferation and on metabolic phenoconversion through the alteration of metabolites associated with mitochondrial function in CCA cell lines. Materials and Methods: The inhibitory effect of niclosamide on CCA cells was determined using SRB assay. A mitochondrial membrane potential using tetramethylrhodamine, ethyl ester-mitochondrial membrane potential (TMRE-MMP) assay was conducted. Liquid chromatography-mass spectrometry-based metabolomics was employed to investigate the global metabolic changes upon niclosamide treatment. ATP levels were measured using CellTiter-Glo® luminescent cell viability assay. NAD metabolism was examined by the NAD+/NADH ratio. Results: Niclosamide strongly inhibited CCA cell growth and reduced the MMP of CCA cells. An orthogonal partial-least square regression analysis revealed that the effects of niclosamide on suppressing cell viability and MMP of CCA cells were significantly associated with an increase in niacinamide, a precursor in NAD synthesis that may disrupt the electron transport system leading to suppression of NAD+/NADH ratio and ATP depletion. Conclusion: Our findings unravel the mode of action of niclosamide in the energy depletion that could potentially serve as the promising therapeutic strategy for CCA treatment.

Toward novel treatment against filariasis: Insight into genome-wide co-evolutionary analysis of filarial nematodes and Wolbachia.

Infectious diseases caused by filarial nematodes are major health problems for humans and animals globally. Current treatment using anti-helminthic drugs requires a long treatment period and is only effective against the microfilarial stage. Most species of filarial nematodes harbor a specific strain of Wolbachia bacteria, which are essential for the survival, development, and reproduction of the nematodes. This parasite-bacteria obligate symbiosis offers a new angle for the cure of filariasis. In this study, we utilized publicly available genome data and putative protein sequences from seven filarial nematode species and their symbiotic Wolbachia to screen for protein-protein interactions that could be a novel target against multiple filarial nematode species. Genome-wide in silico screening was performed to predict molecular interactions based on co-evolutionary signals. We identified over 8,000 pairs of gene families that show evidence of co-evolution based on high correlation score and low false discovery rate (FDR) between gene families and obtained a candidate list that may be keys in filarial nematode-Wolbachia interactions. Functional analysis was conducted on these top-scoring pairs, revealing biological processes related to various signaling processes, adult lifespan, developmental control, lipid and nucleotide metabolism, and RNA modification. Furthermore, network analysis of the top-scoring genes with multiple co-evolving pairs suggests candidate genes in both Wolbachia and the nematode that may play crucial roles at the center of multi-gene networks. A number of the top-scoring genes matched well to known drug targets, suggesting a promising drug-repurposing strategy that could be applicable against multiple filarial nematode species.

Mismatch Repair Deficiency Is a Prognostic Factor Predicting Good Survival of Opisthorchis viverrini-Associated Cholangiocarcinoma at Early Cancer Stage.

BACKGROUND: The mismatch repair (MMR) system prevents DNA mutation; therefore, deficient MMR protein (dMMR) expression causes genetic alterations and microsatellite instability (MSI). dMMR is correlated with a good outcome and treatment response in various cancers; however, the situation remains ambiguous in cholangiocarcinoma (CCA). This study aims to evaluate the prevalence of dMMR and investigate the correlation with clinicopathological features and the survival of CCA patients after resection. MATERIALS AND METHODS: Serum and tissues were collected from CCA patients who underwent resection from January 2005 to December 2017. Serum OV IgG was examined using ELISA. The expression of MMR proteins MLH1, MSH2, MSH6 and PMS2 was investigated by immunohistochemistry; subsequently, MMR assessment was evaluated as either proficient or as deficient by pathologists. The clinicopathological features and MMR status were compared using the Chi-square test. Univariate and multivariate analyses were conducted to identify prognostic factors. RESULTS: Among the 102 CCA patients, dMMR was detected in 22.5%. Survival analysis revealed that dMMR patients had better survival than pMMR (HR = 0.50, p = 0.008). In multivariate analysis, dMMR was an independent factor for a good prognosis in CCA patients (HR = 0.58, p = 0.041), especially at an early stage (HR = 0.18, p = 0.027). Moreover, subgroup analysis showed dMMR patients who received adjuvant chemotherapy had better survival than surgery alone (HR = 0.28, p = 0.012). CONCLUSION: This study showed a high prevalence of dMMR in cholangiocarcinoma with dMMR being the independent prognostic factor for good survival, especially in early-stage CCA and for patients who received adjuvant chemotherapy. dMMR should be the marker for selecting patients to receive a specific adjuvant treatment after resection for CCA.