In silico analysis unveiling potential biomarkers in gallbladder carcinogenesis.

Gallbladder cancer (GBC) is a rare but very aggressive most common digestive tract cancer with a high mortality rate due to delayed diagnosis at the advanced stage. Moreover, GBC progression shows asymptomatic characteristics making it impossible to detect at an early stage. In these circumstances, conventional therapy like surgery, chemotherapy, and radiotherapy becomes refractive. However, few studies reported some molecular markers like KRAS (Kirsten Rat Sarcoma) mutation, upregulation of HER2/neu, EGFR (Epidermal Growth Factor Receptor), and microRNAs in GBC. However, the absence of some specific early diagnostic and prognostic markers is the biggest hurdle for the therapy of GBC to date. The present study has been designed to identify some specific molecular markers for precise diagnosis, and prognosis, for successful treatment of the GBC. By In Silico a network-centric analysis of two microarray datasets; (GSE202479) and (GSE13222) from the Gene Expression Omnibus (GEO) database, shows 50 differentially expressed genes (DEGs) associated with GBC. Further network analysis revealed that 12 genes are highly interconnected based on the highest MCODE (Molecular Complex Detection) value, among all three genes; TRIP13 (Thyroid Receptor Interacting Protein), NEK2 (Never in Mitosis gene-A related Kinase 2), and TPX2 (Targeting Protein for Xklp2) having highest network interaction with transcription factors and miRNA suggesting critically associated with GBC. Further survival analysis data corroborate the association of these genes; TRIP13, NEK2, and TPX2 with GBC. Thus, TRIP13, NEK2, and TPX2 genes are significantly correlated with a greater risk of mortality, transforming them from mere biomarkers of the GBC for early detections and may emerge as prognostic markers for treatment.

TGF-ß1 facilitates gallbladder carcinoma metastasis by regulating FOXA1 translation efficiency through m6A modification.

TGF-ß1 plays a pivotal role in the metastatic cascade of malignant neoplasms. N6-methyladenosine (m6A) stands as one of the most abundant modifications on the mRNA transcriptome. However, in the metastasis of gallbladder carcinoma (GBC), the effect of TGF-ß1 with mRNA m6A modification, especially the effect of mRNA translation efficiency associated with m6A modification, remains poorly elucidated. Here we demonstrated a negative correlation between FOXA1 and TGF-ß1 expression in GBC. Overexpression of FOXA1 inhibited TGF-ß1-induced migration and epithelial-mesenchymal transition (EMT) in GBC cells. Mechanistically, we confirmed that TGF-ß1 suppressed the translation efficiency of FOXA1 mRNA through polysome profiling analysis. Importantly, both in vivo and in vitro experiments showed that TGF-ß1 promoted m6A modification on the coding sequence (CDS) region of FOXA1 mRNA, which was responsible for the inhibition of FOXA1 mRNA translation by TGF-ß1. We demonstrated through MeRIP and RIP assays, dual-luciferase reporter assays and site-directed mutagenesis that ALKBH5 promoted FOXA1 protein expression by inhibiting m6A modification on the CDS region of FOXA1 mRNA. Moreover, TGF-ß1 inhibited the binding capacity of ALKBH5 to the FOXA1 CDS region. Lastly, our study confirmed that overexpression of FOXA1 suppressed lung metastasis and EMT in a nude mice lung metastasis model. In summary, our research findings underscore the role of TGF-ß1 in regulating TGF-ß1/FOXA1-induced GBC EMT and metastasis by inhibiting FOXA1 translation efficiency through m6A modification.

LINC00662 Promotes Aggressive Traits by Modulating OCT4 Expression through miR-335-5p in Gallbladder Cancer Cells.

Long non-coding RNAs (lncRNAs) are nucleotide sequences that participate in different biological processes and are associated with different pathologies, including cancer. Long intergenic non-protein-coding RNA 662 (LINC00662) has been reported to be involved in different cancers, including colorectal, prostate, and breast cancer. However, its role in gallbladder cancer has not yet been described. In this article, we hypothesize that LINC00662 has an important role in the acquisition of aggressiveness traits such as a stem-like phenotype, invasion, and chemoresistance in gallbladder cancer. Here, we show that LINC00662 is associated with larger tumor size and lymph node metastasis in patients with gallbladder cancer. Furthermore, we show that the overexpression of LINC00662 promotes an increase in CD133+/CD44+ cell populations and the expression of stemness-associated genes. LINC00662 promotes greater invasive capacity and the expression of genes associated with epithelial-mesenchymal transition. In addition, the expression of LINC00662 promotes resistance to cisplatin and 5-fluorouracil, associated with increased expression of chemoresistance-related ATP-binding cassette (ABC) transporters in gallbladder cancer (GBC) cell lines. Finally, we show that the mechanism by which LINC00662 exerts its function is through a decrease in microRNA 335-5p (miR-335-5p) and an increase in octamer-binding transcription factor 4 (OCT4) in GBC cells. Thus, our data allow us to propose LINC00662 as a biomarker of poor prognosis and a potential therapeutic target for patients with GBC.

TRIM21-mediated ubiquitylation of TAT suppresses liver metastasis in gallbladder cancer.

Liver metastasis is common in patients with gallbladder cancer (GBC), imposing a significant challenge in clinical management and serving as a poor prognostic indicator. However, the mechanisms underlying liver metastasis remain largely unknown. Here, we report a crucial role of tyrosine aminotransferase (TAT) in liver metastasis of GBC. TAT is frequently up-regulated in GBC tissues. Increased TAT expression is associated with frequent liver metastasis and poor prognosis of GBC patients. Overexpression of TAT promotes GBC cell migration and invasion in vitro, as well as liver metastasis in vivo. TAT knockdown has the opposite effects. Intriguingly, TAT promotes liver metastasis of GBC by potentiating cardiolipin-dependent mitophagy. Mechanistically, TAT directly binds to cardiolipin and leads to cardiolipin externalization and subsequent mitophagy. Moreover, TRIM21 (Tripartite Motif Containing 21), an E3 ubiquitin ligase, interacts with TAT. The histine residues 336 and 338 at TRIM21 are essential for this binding. TRIM21 preferentially adds the lysine 63 (K63)-linked ubiquitin chains on TAT principally at K136. TRIM21-mediated TAT ubiquitination impairs its dimerization and mitochondrial location, subsequently inhibiting tumor invasion and migration of GBC cells. Therefore, our study identifies TAT as a novel driver of GBC liver metastasis, emphasizing its potential as a therapeutic target.

SOAT1 in gallbladder cancer: Clinicopathological significance and avasimibe therapeutics.

The aim of this investigation was to evaluate the differential expression of the sterol O-acyltransferase 1 (SOAT1) protein in gallbladder cancer tissues and cells, investigate the impact of Avastin on the proliferation, migration, invasion capabilities of gallbladder cancer cells, and its potential to induce cell apoptosis. Immunohistochemical analysis of samples from 145 gallbladder cancer patients was conducted, along with analysis of SOAT1 protein, mRNA expression levels, and cholesterol content in gallbladder cancer cell lines SGC-996, NOZ, and gallbladder cancer (GBC)-SD using Western blot and q-PCR techniques. Furthermore, the effects of Avastin on the proliferation, migration, and invasion capabilities of these gallbladder cancer cell lines were studied, and its ability to induce cell apoptosis was evaluated using flow cytometry, Western blot, and immunohistochemical methods. Additionally, gene expression and pathway analysis were performed, and the synergistic therapeutic effects of Avastin combined with gemcitabine were tested in a gallbladder cancer xenograft model. The study found that SOAT1 expression was significantly upregulated in GBC tissues and positively correlated with lymph node metastasis and TNM staging. In vitro experiments demonstrated that Avastin significantly inhibited the proliferation, migration, and invasion capabilities of SGC-996 and GBC-SD cell lines and induced apoptosis. RNA sequencing analysis revealed multiple differentially expressed genes in cells treated with Avastin, primarily enriched in biological pathways such as signaling transduction, malignant tumors, and the immune system. In vivo, experiments confirmed that Avastin could effectively suppress tumor growth in a gallbladder cancer xenograft model and enhanced the treatment efficacy when used in combination with gemcitabine. Overall, these findings provide new insights and strategies for targeted therapy in gallbladder cancer.

Fatty Acid Binding Protein 1 is an Independent Prognostic Biomarker for Gallbladder Cancer with Direct Hepatic Invasion.

Background: Direct liver invasion (DI) is a predominant pathway of gallbladder cancer (GBC) metastasis, but the molecular alterations associated with DI remain addressed. This study identified specific genes correlated with DI, which may offer a potential biomarker for the diagnosis and prognosis of advanced GBC. Methods: RNA samples from 3 patients with DI of GBC were used for RNA-seq analysis. Differentially expressed genes and metabolic pathways between primary tumor (T) and DI tissue was used to analyze aberrant gene expressions. Immunohistochemistry (IHC) of fatty acid binding protein 1 (FABP1) in 62 patients with DI was engaged to evaluate its association with clinicopathological characteristics and prognosis. IHC of CD3+ and CD8+ T cells was analyzed for their correlation with FABP1 expression, clinicopathological features and prognosis. Univariate and multivariate Cox hazards regression analyses were performed to identify independent prognostic factors for disease-free survival (DFS) and overall survival (OS). Results: FABP1 mRNA levels were significantly upregulated in DI region compared to T tissue. IHC results showed identical results with elevated FABP1 (p < 0.0001). Expression of FABP1 in DI region was significantly associated with lymph node metastasis (P = 0.028), reduced DFS (P = 0.013) and OS (P = 0.022); in contrast, its expression in T region was not associated with clinicopathological characteristics and prognosis (P > 0.05). The density of CD8+ T cells in DI region with higher FABP1 expression was significantly lower than that with lower FABP1 expression (p = 0.0084). Multivariate analysis unveiled those hepatic metastatic nodules (HR = 3.35, 95%CI: 1.37-8.15, P = 0.008) and FABP1 expression in DI region (HR = 2.01, 95%CI: 1.05-3.88, P = 0.036) were high risk factors for OS, and FABP1(HR = 2.05, 95%CI: 1.04-4.06, P = 0.039) was also a high risk factor for DFS. Conclusions: Elevated expression of FABP1 in DI region serves as a potential prognostic biomarker for advanced GBC with DI.

YTHDF1 promotes gallbladder cancer progression via post-transcriptional regulation of the m6A/UHRF1 axis.

Gallbladder cancer is a rare but fatal malignancy. However, the mechanisms underlying gallbladder carcinogenesis and its progression are poorly understood. The function of m6A modification and its regulators was still unclear for gallbladder cancer. The current study seeks to investigate the function of YTH m6A RNA-binding protein 1 (YTHDF1) in gallbladder cancer. Transcriptomic analysis and immunochemical staining of YTHDF1 in gallbladder cancer tissues revealed its upregulation compared to paracancerous tissues. Moreover, YTHDF1 promotes the proliferation assays, Transwell migration assays, and Transwell invasion assays of gallbladder cancer cells in vitro. And it also increased tumour growth in xenograft mouse model and metastases in tail vein injection model in vivo. In vitro, UHRF1 knockdown partly reversed the effects of YTHDF1 overexpression. Mechanistically, dual-luciferase assays proved that YTHDF1 promotes UHRF1 expression via direct binding to the mRNA 3'-UTR in a m6A-dependent manner. Overexpression of YTHDF1 enhanced UHRF1 mRNA stability, as demonstrated by mRNA stability assays, and Co-IP studies confirmed a direct interaction between YTHDF1 and PABPC1. Collectively, these findings provide new insights into the progression of gallbladder cancer as well as a novel post-transcriptional mechanism of YTHDF1 via stabilizing target mRNA.

SIRT7: A potential prognostic marker and therapeutic target in gallbladder cancer.

Gallbladder cancer (GBC) is a highly aggressive malignancy with limited treatment options and poor prognosis. In this study, we aimed to investigate the role of SIRT7, a member of the sirtuin family, in GBC and its potential as a prognostic marker and therapeutic target. Through immunohistochemistry analysis of GBC tissue samples, we observed elevated levels of SIRT7, which were correlated with worse clinicopathological parameters and shorter overall survival in GBC patients. Additionally, through cellular and animal experiments, we have discovered that interfering with SIRT7 can effectively suppress the proliferation, migration, and invasive capabilities of GBC cells. Conversely, overexpressing SIRT7 yields the opposite outcome. Furthermore, interference with SIRT7 triggers cell cycle arrest and enhances apoptosis in GBC cells. Mechanistically, we found that SIRT7 inhibition led to reduced activation of the NF-κB signaling pathway, suggesting its involvement in modulating GBC cell behavior. Our findings shed light on the oncogenic role of SIRT7 in GBC and highlight its potential as a promising prognostic marker and therapeutic target. Further research is warranted to explore the therapeutic implications of targeting SIRT7 in GBC treatment.

Exosomal long non-coding RNA TRPM2-AS promotes angiogenesis in gallbladder cancer through interacting with PABPC1 to activate NOTCH1 signaling pathway.

BACKGROUND: Abnormal angiogenesis is crucial for gallbladder cancer (GBC) tumor growth and invasion, highlighting the importance of elucidating the mechanisms underlying this process. LncRNA (long non-coding RNA) is widely involved in the malignancy of GBC. However, conclusive evidence confirming the correlation between lncRNAs and angiogenesis in GBC is lacking. METHODS: LncRNA sequencing was performed to identify the differentially expressed lncRNAs. RT-qPCR, western blot, FISH, and immunofluorescence were used to measure TRPM2-AS and NOTCH1 signaling pathway expression in vitro. Mouse xenograft and lung metastasis models were used to evaluate the biological function of TRPM2-AS during angiogenesis in vivo. EDU, transwell, and tube formation assays were used to detect the angiogenic ability of HUVECs. RIP, RAP, RNA pull-down, dual-luciferase reporter system, and mass spectrometry were used to confirm the interaction between TRPM2-AS, IGF2BP2, NUMB, and PABPC1. RESULTS: TRPM2-AS was upregulated in GBC tissues and was closely related to angiogenesis and poor prognosis in patients with GBC. The high expression level and stability of TRPM2-AS benefited from m6A modification, which is recognized by IGF2BP2. In terms of exerting pro-angiogenic effects, TRPM2-AS loaded with exosomes transported from GBC cells to HUVECs enhanced PABPC1-mediated NUMB expression inhibition, ultimately promoting the activation of the NOTCH1 signaling pathway. PABPC1 inhibited NUMB mRNA expression through interacting with AGO2 and promoted miR-31-5p and miR-146a-5p-mediated the degradation of NUMB mRNA. The NOTCH signaling pathway inhibitor DAPT inhibited GBC tumor angiogenesis, and TRPM2-AS knockdown enhanced this effect. CONCLUSIONS: TRPM2-AS is a novel and promising biomarker for GBC angiogenesis that promotes angiogenesis by facilitating the activation of the NOTCH1 signaling pathway. Targeting TRPM2-AS opens further opportunities for future GBC treatments.

Decoding the role of long non-coding RNAs in gallbladder cancer pathogenesis: A review focus on signaling pathways interplay.

Gallbladder cancer (GBC) is one of the most aggressive types of biliary tree cancers and the commonest despite its rarity. It is infrequently diagnosed at an early stage, further contributing to its poor prognosis and low survival rate. The lethal nature of the disease has underlined a crucial need to discern the underlying mechanisms of GBC carcinogenesis which are still largely unknown. However, with the continual evolution in the research of cancer biology and molecular genetics, studies have found that non-coding RNAs (ncRNAs) play an active role in the molecular pathophysiology of GBC development. Dysregulated long non-coding RNAs (lncRNAs) and their interaction with intracellular signaling pathways contribute to malignancy and disease development. LncRNAs, a subclass of ncRNAs with over 200 nucleotides, regulate gene expression at transcriptional, translational, and post-translational levels and especially as epigenetic modulators. Thus, their expression abnormalities have been linked to malignancy and therapeutic resistance. lnsRNAs have also been found in GBC patients' serum and tumor tissue biopsies, highlighting their potential as novel biomarkers and for targeted therapy. This review will examine the growing involvement of lncRNAs in GBC pathophysiology, including related signaling pathways and their wider clinical use.

Acylcarnitines promote gallbladder cancer metastasis through lncBCL2L11-THOC5-JNK axis.

BACKGROUND: The progression of gallbladder cancer (GBC) is accompanied by abnormal fatty acid ß-oxidation (FAO) metabolism. Different types of lipids perform various biological functions. This study aimed to determine the role of acyl carnitines in the molecular mechanisms of GBC progression. METHODS: Distribution of lipids in GBC was described by LC-MS-based lipidomics. Cellular localization, expression level and full-length of lncBCL2L11 were detected using fluorescence in situ hybridization (FISH) assays, subcellular fractionation assay and 5' and 3' rapid amplification of the cDNA ends (RACE), respectively. In vitro and in vivo experiments were used to verify the biological function of lncBCL2L11 in GBC cells. Methylated RNA Immunoprecipitation (MeRIP) was performed to detect the methylation levels of lncBCL2L11. RNA pull-down assay and RNA immunoprecipitation (RIP) assay were used to identify lncBCL2L11 interacting proteins. Co-Immunoprecipitation (Co-IP) and Western blot assay were performed to validate the regulatory mechanism of lncBCL2L11 and THO complex. RESULTS: Acylcarnitines were significantly up-regulated in GBC tissues. High serum triglycerides correlated to decreased survival in GBC patients and promoted tumor migration. LncBCL2L11 was identified in the joint analysis of highly metastatic cells and RNA sequencing data. LncBCl2L11 prevented the binding of THOC6 and THOC5 and causes the degradation of THOC5, thus promoting the accumulation of acylcarnitines in GBC cells, leading to the malignant progression of cancer cells. In addition, highly expressed acylcarnitines stabilized the expression of lncBCL2L11 through N6-methyladenosine methylation (m6A), forming a positive feedback regulation in tumor dissemination. CONCLUSIONS: LncBCL2L11 is involved in gallbladder cancer metastasis through FAO metabolism. High lipid intake is associated with poor prognosis of GBC. Therefore, targeting lncBCL2L11 and its pathway-related proteins or reducing lipid intake may be significant for the treatment of GBC patients.

Adavosertib-encapsulated metal-organic frameworks for p53-mutated gallbladder cancer treatment via synthetic lethality.

Adavosertib (ADA) is a WEE1 inhibitor that exhibits a synthetic lethal effect on p53-mutated gallbladder cancer (GBC). However, drug resistance due to DNA damage response compensation pathways and high toxicity limits further applications. Herein, estrone-targeted ADA-encapsulated metal-organic frameworks (ADA@MOF-EPL) for GBC synthetic lethal treatment by inducing conditional factors are developed. The high expression of estrogen receptors in GBC enables ADA@MOF-EPL to quickly enter and accumulate near the cell nucleus through estrone-mediated endocytosis and release ADA to inhibit WEE1 upon entering the acidic tumor microenvironment. Ultrasound irradiation induces ADA@MOF-EPL to generate reactive oxygen species (ROS), which leads to a further increase in DNA damage, resulting in a higher sensitivity of p53-mutated cancer cells to WEE1 inhibitor and promoting cell death via conditional synthetic lethality. The conditional factor induced by ADA@MOF-EPL further enhances the antitumor efficacy while significantly reducing systemic toxicity. Moreover, ADA@MOF-EPL demonstrates similar antitumor abilities in other p53-mutated solid tumors, revealing its potential as a broad-spectrum antitumor drug.

Deciphering the role of Enterococcus faecium cytidine deaminase in gemcitabine resistance of gallbladder cancer.

Gemcitabine-based chemotherapy is a cornerstone of standard care for gallbladder cancer (GBC) treatment. Still, drug resistance remains a significant challenge, influenced by factors such as tumor-associated microbiota impacting drug concentrations within tumors. Enterococcus faecium, a member of tumor-associated microbiota, was notably enriched in the GBC patient cluster. In this study, we investigated the biochemical characteristics, catalytic activity, and kinetics of the cytidine deaminase of E. faecium (EfCDA). EfCDA showed the ability to convert gemcitabine to its metabolite 2',2'-difluorodeoxyuridine. Both EfCDA and E. faecium can induce gemcitabine resistance in GBC cells. Moreover, we determined the crystal structure of EfCDA, in its apo form and in complex with 2', 2'-difluorodeoxyuridine at high resolution. Mutation of key residues abolished the catalytic activity of EfCDA and reduced the gemcitabine resistance in GBC cells. Our findings provide structural insights into the molecular basis for recognizing gemcitabine metabolite by a bacteria CDA protein and may provide potential strategies to combat cancer drug resistance and improve the efficacy of gemcitabine-based chemotherapy in GBC treatment.

Metabolomics and miRNA profiling reveals feature of gallbladder cancer-derived biliary extracellular vesicles.

BACKGROUND: Although there are several studies in the development of various human cancers, the role of exosomes is poorly understood in the progression of gallbladder cancer. This study aims to characterize the metabolic changes occurring in exosomes obtained from patients with gallbladder cancer compared with those from other gallbladder disease groups. METHODS: Biliary exosomes were isolated from healthy donors (n = 3) and from patients with gallbladder cancer (n = 3), gallbladder polyps (n = 4), or cholecystitis (n = 3) using a validated exosome isolation kit. Afterward, we performed miRNA profiling and untargeted metabolomic analysis of the exosomes. The results were validated by integrating the results of the miRNA and metabolomic analyses. RESULTS: The gallbladder cancer group exhibited a significant reduction in the levels of multiple unsaturated phosphatidylethanolamines and phosphatidylcholines compared to the normal group, which resulted in the loss of exosome membrane integrity. Additionally, the gallbladder cancer group demonstrated significant overexpression of miR-181c and palmitic acid, and decreased levels of conjugated deoxycholic acid, all of which are strongly associated with the activation of the PI3K/AKT pathway. CONCLUSIONS: Our findings demonstrate that the contents of exosomes are disease-specific, particularly in gallbladder cancer, and that altered metabolites convey critical information regarding their phenotype. We believe that our metabolomic and miRNA profiling results may provide important insights into the development of gallbladder cancer.

Survival analysis in association with GST gene polymorphism and Treatment outcomes of Gemcitabine and Cisplatin/Carboplatin-based chemotherapy among patients with Gallbladder Carcinoma.

PURPOSE: Majority of the gallbladder cancer (GBC) cases are diagnosed at an advanced stage where chemotherapy alone (or in combination with other treatment methods) is mainly opted as therapeutic approach. However, success or failure of this approach largely depends on the interindividual genetic differences. Careful consideration on the genetic association could assist in the evaluation of patient's treatment response and survival rate. Hence, the present study aims to investigate the survival of patients with GBC and their treatment response to gemcitabine and cisplatin/carboplatin-based chemotherapy in association with Glutathione S-transferase (GSTs) gene polymorphism. MATERIAL AND METHODS: A total of 216 histologically confirmed cases of gallbladder cancer were recruited. A total of 180 patients were treated with gemcitabine and cisplatin/carboplatin-based chemotherapy. GSTM1, GSTT1, and GSTP1 genotypes were determined by multiplex PCR and by PCR restriction fragment length polymorphism (PCR-RFLP), respectively. The influence of genetic polymorphism on overall survival was analyzed by Kaplan-Meier method, survival rate difference was analyzed by log-rank test, and hazard ratio for mortality outcomes was estimated using Cox regression method. RESULTS: GBC patients having genotype GSTP1 (AG + GG) showed poor 3-year survival rate of 0.8% compared to 10.9% of GSTP1 (AA) genotype (χ2 = 6.456, P = 0.011). The multivariate Cox regression results showed that the death risk was significantly higher in GSTP1 (AG + GG) genotype (HR = 3.858, P = 0.050). We found no association of GSTM1 and GSTT1 gene polymorphism with the survival; however, the combined genotypes of GSM1/GSTP1, GSTT1/GSTP1, and GSTM1/GSTT1/GSTP1 were associated with survival (P = 0.053, 0.006, and 0.058, respectively). Increased death hazard was noted by the genotype combinations of GSTM1+/GSTP1AG + GG (HR = 3.484, P = 0.024), GSTM1-/GSTP1AG + GG (HR = 2.721, P = 0.014), GSTT1+/GSTP1AG + GG (HR = 20.690, P = 0.001), and GSTT1-/GSTP1AA (HR = 26.111, P < 0.0001). Our findings indicate that chemotherapy treatment response of GSTP1 (AG + GG) has 1.62-fold increased risk for progression compared to GSTP1 (AA) genotype (p = 0.018); however, none of the genotypes showed association with overall survival and death risk after chemotherapeutic treatment. CONCLUSION: We found that the presence of GSTP1 (AG + GG) genotype showed survival disadvantage and poor treatment outcomes in response to gemcitabine and cisplatin/carboplatin-based chemotherapy. This could serve as biomarker, and future research in pharmacogenomics will definitely pave the way for the development of better treatment approach for GBC.

Clinicopathological and prognostic significance of VEGF, PDGF-B, and HER2/neu expression in gallbladder cancer.

AIM: Gallbladder cancer (GBC) is usually diagnosed in advanced stages with poor survival. The molecular mechanisms of GBC still remain unexplored. Several angiogenesis factors play a pivotal role in tumor progression. We aimed to study the expression of VEGF, PDGF-B, and human epidermal growth factor receptor 2 (HER2/neu) and its association with clinicopathological features and survival in GBC. MATERIALS AND METHODS: VEGF, PDGF-B, and HER2/neu expression was studied by immunohistochemistry (IHC) after histological evaluation in 91 GBC cases. The relationship between these markers and clinicopathological features and survival was explained through the Cox regression model and Kaplan-Meier method. RESULTS: VEGF, PDGF-B, and HER2/neu overexpressed in 45, 79, and 68% GBC cases, respectively. VEGF was significantly overexpressed in GBC without gall stones (GS) (p = 0.007) and with moderately and poorly differentiated tumors (p = 0.012). HER2/neu was significantly overexpressed in GBC with GS (p = 0.022). Median overall survival (OS) was 39 months (95% CI: 23-55). In univariate analysis, histological type (adenocarcinoma and papillary) vs. others (signet ring/mucinous/adenosquamous) (p = 0.004), depth of tumor infiltration (p = 0.017), distant metastasis (p = 0.012), and adjuvant therapies (chemotherapy/radiotherapy) (p = 0.083) were associated with poor prognosis. Multivariate survival analysis showed histological type (p = 0.004) and distant metastasis (p = 0.032) to be independent prognostic factors for OS. Histological type (p = 0.002), distant metastasis (p = 0.003), and depth of tumor infiltration (T3-T4) (p = 0.012) showed poor median survival. Poor survival was seen in VEGF and HER2/neu positive cases. CONCLUSION: Overexpression of VEGF, PDGF-B, and HER2/neu might be possible prognostic biomarkers in GBC. Poor survival of VEGF and HER2/neu positive cases indicates the possibilities of using their blockers as therapeutic agents.

Functional relevance of MMP2 promoter variants in gallbladder cancer: A case-control study in an Eastern Indian Population.

Gallbladder cancer (GBC) is a prevalent and deadly form of bile duct cancer, associated with poor prognosis. This study aimed to investigate the genetic factors contributing to the high incidence of GBC in certain geographical regions, particularly in the Northern and Eastern parts of India. The present case-control study focused on MMP2, a gene involved in tumor progression and metastasis, as a potential candidate in GBC pathogenesis. We scanned MMP2 promoter for twelve SNPs using Sanger's sequencing and carried out a case-control study in 300 cases and 300 control samples. We found five rare variants (rs1961998763, rs1961996235, rs1391392808, rs1488656253, and rs17859816) and one nonpolymorphic SNP (rs17859817). Our results revealed a significant association between GBC and MMP2 promoter SNPs, rs243865 (Allelic-Padjusted = 0.0353) and g.55477735G > A (Allelic-Padjusted = 9.22E-05). Moreover, the haplotype "C-C-A-C-C" exhibited a significant association with GBC (P = 4.23E-05). Genotype-phenotype correlation for variant rs243865, in the GBC patient tissue samples, established that 'T' risk allele carriers had higher expression levels of MMP2. Additionally, luciferase reporter assay in HEK293T cells revealed the probable regulatory role of rs243865 variant allele 'T' in MMP2 expression. Our study uncovers the association of MMP2 promoter SNPs with GBC and their role in regulating its expression.

PP4R1 promotes glycolysis and gallbladder cancer progression through facilitating ERK1/2 mediated PKM2 nuclear translocation.

Gallbladder cancer (GBC) is a common solid tumor of the biliary tract with a high mortality rate and limited curative benefits from surgical resection. Here, we aimed to elucidate the pathogenesis of GBC from the perspective of molecular mechanisms and determined that protein phosphatase 4 regulator subunit 1 (PP4R1) is overexpressed in GBC tissues and contributes to poor prognosis. Through a series of in vitro and in vivo experiments, we demonstrated that PP4R1 overexpression improved tumorigenesis in GBC cells. Further mechanistic exploration revealed that PP4R1 directly interacts with pyruvate kinase-M2 (PKM2), a key regulator of glycolysis. PP4R1 promotes the extracellular signal-related kinase 1 and 2 (ERK1/2)-mediated PKM2 nuclear translocation, thereby participating in the regulation of tumor glycolysis. Interestingly, we determined that PP4R1 strengthens the interaction between ERK1/2 and PKM2. Furthermore, PP4R1 enhanced the suppressive effects of the ERK inhibitor SCH772984 on GBC. In conclusion, our data showed that PP4R1 is a promising biomarker associated with GBC and confirmed that PP4R1 regulates PKM2-mediated tumor glycolysis, which provides a metabolic growth advantage to GBC cells, thereby promoting GBC tumor growth and metastasis1.

Downregulated circRNA_CDKN1A promotes gallbladder cancer progression through activation of the NF-κB pathway.

This study uncovered the potential clinical value and molecular driving mechanisms of circular RNAs (circRNAs) in gallbladder cancer (GBC). Differentially expressed circRNAs in GBC cells were screened by high-throughput sequencing. CircRNA_CDKN1A (circBase ID: hsa_circ_0076194) was knocked out in BGC-SD cells through transfection with sh-circRNA_CDKN1A. Then, proliferation was investigated via CCK8 and EdU assays, apoptosis via flow cytometry, migration via wound healing assays, and invasion via Transwell assays. Bioinformatics analysis of circRNA_CDKN1A-related signaling pathways was performed using MetScape and g:Profiler. Results showed that the knockdown of circRNA_CDKN1A enhanced the proliferation, migration, and invasion of GBC cells and inhibited apoptosis. In addition, knocking out circRNA_CDKN1A promoted GBC cell proliferation and enhanced the dry indices of the OCT4 protein and CD34 expression levels. The knockdown of circRNA_CDKN1A activated the epithelial-mesenchymal transition pathway. Bioinformatics analysis revealed that the biological role of circRNA_CDKN1A in GBC cells involved the NF-κB pathway. LY2409881, which is an NF-κB inhibitor, reversed the effects induced by the knockdown of circRNA_CDKN1A in GBC-SD cells. In summary, the knockdown of circRNA_CDKN1A promoted the progression of GBC by activating the NF-κB signaling pathway. For the first time, this study revealed the mechanism of circRNA_CDKN1A-mediated regulatory action in GBC and identified the newly discovered circRNA_CDKN1A-NF-κB signaling axis as a potentially important candidate for clinical therapy and prognostic diagnosis of GBC.

Study of morphology with assessment of expression of proliferation marker Ki67 antigen and P53 protein in lesions of gall bladder.

OBJECTIVE: To study the spectrum and distribution of histopathological changes and evaluate immunohistochemistry markers p53 protein and Ki67 antigen in various lesions of gall bladder. MATERIALS AND METHODS: A total of 804 consecutive gall bladder specimens were evaluated. Forty cases were selected for immunohistochemical analysis to evaluate expression of p53 and ki67 proliferation index, including 20 carcinoma gall bladder cases and 20 cases of inflammatory pathology associated with metaplasia, atypia, hyperplasia, dysplasia, and adenoma. p53 immunostaining was categorized as wild type and mutant type. ki67 of >20% was considered high expression. RESULTS: The majority of the gall bladder lesions were inflammatory in origin, most common being chronic cholecystitis. In the group of 20 gall bladder carcinoma cases, 65% were p53 mutant and the remaining 35% cases had a p53 wild-type immunophenotype. 55% cases showed high expression for ki67 labeling. However, significant correlation ( P < 0.05) was seen with lympho-vascular invasion. Among non-malignant lesions, normal/wild-type p53 expression was seen with increasing intensity and positivity in lesions with atypia and intra-epithelial neoplasms. Ki67 index also showed the same trend in all cases. CONCLUSIONS: p53 and ki-67 expression increases in inflammation, and further increment occurs in premalignant and malignant lesions of the gall bladder epithelium and can be used as a marker of aggression of histopathological lesions. The results emphasize the potential of Ki-67 and p53 as biomarkers of carcinogenesis in gall bladder carcinoma.