Metastasis Of Gallbladder Carcinoma Research Articles

TGF-β1 facilitates gallbladder carcinoma metastasis by regulating FOXA1 translation efficiency through m6A modification

TGF-β1 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-β1 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-β1 expression in GBC. Overexpression of FOXA1 inhibited TGF-β1-induced migration and epithelial-mesenchymal transition (EMT) in GBC cells. Mechanistically, we confirmed that TGF-β1 suppressed the translation efficiency of FOXA1 mRNA through polysome profiling analysis. Importantly, both in vivo and in vitro experiments showed that TGF-β1 promoted m6A modification on the coding sequence (CDS) region of FOXA1 mRNA, which was responsible for the inhibition of FOXA1 mRNA translation by TGF-β1. 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-β1 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-β1 in regulating TGF-β1/FOXA1-induced GBC EMT and metastasis by inhibiting FOXA1 translation efficiency through m6A modification.

Prognosis and Treatment Outcomes of Bone Metastasis in Gallbladder Adenocarcinoma: A SEER-Based Study.

Gallbladder carcinoma (GBC) is a rare, aggressive malignancy comprising 0.5% of gastrointestinal cancers. It has poor survival outcomes due to its insidious onset, lack of standardized screening, and limited therapies. Advanced-stage diagnosis with liver, lymph node, and peritoneal metastasis is common, while bone metastasis is rare. The knowledge on bone metastasis in GBC is limited to case reports and small series, and its clinical significance is largely unexplored. The study extracted the demographic and clinical variables of patients with metastatic (M1) gallbladder adenocarcinoma from the Surveillance, Epidemiology, and End Results (SEER) database between 2011 and 2020. Descriptive statistics were used to analyze the demographic characteristics. The multivariate Cox regression analysis was used to calculate the hazard ratio. The overall survival (OS) was assessed using the Kaplan-Meier method, and the log-rank test was utilized to compare the survival between the groups. A total of 2724 patients were included in the study. A total of 69% of the patients were female, and the median age was 68 (range 24-90+). A total of 7.4% of the patients had bone metastasis on diagnosis. The multivariate Cox analysis identified bone metastasis as an independent mortality risk factor in metastatic GBC (HR 1.50, p < 0.001). The patients were divided into two age groups: a younger age group (18-74 years) and an older age group (75+ years). In the younger group, the median OS with and without bone metastasis was 3 and 5 months, respectively (p < 0.0001). In the older age group, there was no significant difference in the OS between the patients with and without bone metastasis (p = 0.35). In the younger group who were treated with chemotherapy, the patients with bone metastasis had a significantly worse OS (median OS 5 months vs. 8 months, p < 0.0001). In the untreated group, the patients with bone metastasis in the younger age group had a significantly worse OS (median OS 1 month vs. 2 months, p = 0.014). In the patients with bone metastasis, those who did not receive chemotherapy had a significantly worse OS than those who were treated with chemotherapy in both age groups (younger age group: median OS 1 month vs. 5 months, p < 0.0001 and older age group: median OS 1 month vs. 5 months, p = 0.041). Our findings suggest that the presence of bone metastasis in gallbladder adenocarcinoma is an independent prognostic factor associated with unfavorable survival outcomes in the younger age group (18-74 years). However, in the older age group (75+ years), the presence of bone metastasis did not impact the survival. Treatment with chemotherapy was associated with extended survival in all patients. Thus, early detection and aggressive management of bone metastasis, including the consideration of chemotherapy, may be crucial in improving the OS and quality of life for individuals with gallbladder adenocarcinoma.

EMP3 as a key downstream target of miR-663a regulation interferes with MAPK/ERK signaling pathway to inhibit gallbladder cancer progression

Gallbladder carcinoma (GBC) is the most common malignancy of the biliary tract, and its molecular pathogenesis remains unclear. Here we explore the functional roles of epithelial membrane protein 3 (EMP3) in GBC progression, which is aberrantly expressed in various types of cancers. The results showed that the expression level of EMP3 was reduced in human GBC tissues compared with non-malignant tissues. Further, the low expression of EMP3 was associated with the poor prognosis of GBC patients by Kaplan-Meier analysis. The ectopic expression of EMP3 inhibited GBC cell proliferation, migration and invasion in vitro and in vivo. Conversely, the depletion of EMP3 promoted GBC cell growth and metastasis. In addition, we found that EMP3 was a target gene of miR-663a, and the downregulation of EMP3 in GBC was attributed to the overexpression of miR-663a. MiR-663a was also shown to be a tumor-promoting factor mediating GBC development. In this study, we demonstrate that downregulation of EMP3 activates MAPK/ERK signaling, which regulates GBC progression. These data reveal the mechanism by which EMP3 inhibits the progression of GBC, suggesting that the miR-663a/EMP3/MAPK/ERK axis may be a new therapeutic target for GBC treatment.

MiR-4733-5p promotes gallbladder carcinoma progression via directly targeting kruppel like factor 7

ABSTRACT Gallbladder carcinoma (GBC) is highly aggressive with poor prognosis. Accumulating reports show that miRNAs play critical roles in tumor progression. Previous studies have identified several miRNAs that promoted or inhibited GBC cell proliferation and/or metastasis. Here, we used the Gene Expression Omnibus (GEO) dataset to identify dysregulated miRNAs in GBC, followed by validating the upregulation of the miR-4733-5p and downregulation of kruppel-like factor 7 (KLF7) in GBC biopsies by quantitative real-time PCR (RT-qPCR), in situ hybridization (ISH) staining, and immunohistochemistry (IHC) assays. GBC cell proliferation and invasion capacities mediated by miR-4733-5p were evaluated by a series of function assays in vitro, including CCK-8, colony formation assay, wound healing assay and transwell assay. Xenograft tumor model found that miR-4733-5p promoted GBC tumor growth in vivo. This study clarified that miR-4733-5p was upregulated in GBC and promoted GBC cell proliferation via directly binding to 3’ untranslated region (UTR) of KLF, which was downregulated and prohibited the proliferation and migration of GBC cells.

MiR-4461 inhibits the progression of Gallbladder carcinoma via regulating EGFR/AKT signaling

ABSTRACT Increasing evidence has demonstrated that microRNAs (miRNAs) participated in the tumorigenesis, progression and recurrence of various malignancies including Gallbladder carcinoma (GBC). miR-4461 was reported to work as a tumor suppressor gene in renal cell carcinoma. However, the role of miR-4461 in GBC remains unknown. Herein, we show that miR-4461 is downregulated in gallbladder cancer stem cells (CSCs). Forced miR-4461 expression attenuates the self-renewal, tumorigenicity of gallbladder CSCs, and inhibits proliferation and metastasis of GBC cells. Conversely, miR-4461 knockdown promotes the self-renewal of gallbladder CSCs, and facilities proliferation and metastasis of GBC cells. Mechanistically, miR-4461 inhibits GBC progression via downregulating EGFR/AKT pathway. Special EGFR siRNA or AKT overexpression virus abolishes the discrepancy of self-renewal, tumorigenesis, growth, and metastasis between miR-4461 overexpression GBC cells and their control cells. In conclusion, miR-4461 suppresses GBC cells self-renewal, tumorigenicity, proliferation, and metastasis by inactivating EGFR/AKT signaling, and may therefore prove to be a potential therapeutic target for GBC patients.

MiR-365 inhibits the progression of gallbladder carcinoma and predicts the prognosis of Gallbladder carcinoma patients

ABSTRACT Gallbladder carcinoma (GBC) is one of the most common fatal biliary tract tumors in the world. Its 3-year survival rate is 30% and the recurrence rate remains very high. miR-365 was downregulated in numerous tumors and worked as tumor suppressor gene. However, the role of miR-365 in GBC was unclear. In this study, our results found that the expression of miR-365 in GBC tissues was reduced rather than that in non-cancerous tissues. miR-365 overexpression inhibited the proliferation, metastasis and expansion of GBC CSCs. Mechanically, bioinformatic and luciferase reporter analysis identified Ras-related C3 botulinum toxin substrate 1 (RAC1) as a direct target of miR-365. Overexpression of miR-365 in GBC cells reduced the RAC1 mRNA and protein expression. The special RAC1 inhibitor EHop-106 abolished the discrepancy of growth, metastasis and self-renewal ability between miR-365-overexpression GBC cells and their control cells, which further demonstrated that RAC1 was involved in miR-365-disrupted GBC cells growth, metastasis and self-renewal. More importantly, reduced expression of miR-365 was a predictor of poor prognosis of GBC patients. In conclusion, miR-365 inhibited GBC cell growth, metastasis and self-renewal capacity by directly targeting RAC1, and may therefore prove to be a novel prognosis biomarker for GBC patients.

Phosphorylation at Ser10 triggered p27 degradation and promoted gallbladder carcinoma cell migration and invasion by regulating stathmin1 under glucose deficiency

Gallbladder carcinoma (GBC) is a considerable challenge because of its high metastatic potential. The tumor microenvironment is characterized by nutrient starvation, which promotes tumor metastasis. Stathmin1, an important microtubuleregulating protein, is overexpressed and promotes metastasis in GBC. It remains unclear how the harsh tumor microenvironment regulates stathmin1 expression to affect GBC metastasis. We employed glucose deficiency to mimic nutrient starvation and found that glucose deficiency upregulated stathmin1 transcription. However, glucose deficiency also promoted p27 degradation. There was a significant negative correlation between stathmin1 and p27 protein levels under glucose deficiency. Further study revealed that, under glucose deficiency, human kinase interacting with stathmin (hKIS) induced phosphorylation at Ser10 of p27 and its translocation to the cytoplasm for degradation, which upregulated the transcription factor E2F1 to promote stathmin1 transcription. hKIS knockdown significantly inhibited p27 cytoplasmic translocation and its consequent degradation. Stathmin1 knockdown significantly inhibited GBC cell migration and invasion in vitro. Our study revealed the role of the hKIS/p27/E2F1 axis in upregulating stathmin1 transcription to promote GBC cell migration and invasion under glucose deficiency conditions

Cyclin D1 and Its Expression in Neoplastic Gall Bladder Lesions

Carcinoma of Gall bladder is an infrequent tumour of the gastrointestinal tract and has long been recognized as a highly lethal disease. Tumour markers are glycoprotein that can be determined in blood, saliva, urine and tissues by certain immunological methods. Cyclin D1 is such a marker which is seen to be expressed in various neoplasms. Its role has also been confirmed in gall bladder neoplastic conditions. The onset and progression of gallbladder carcinoma are accompanied with multiple genetic changes that result in qualitative and quantitative alterations in individual gene expression. Immuno-histochemical methods can be used to analyze cyclin D1 levels in gallbladder carcinomas, adenomas and cholecystitis to evaluate their relationships with the pathogenesis, development and metastasis of gallbladder carcinoma.

Intramuscular Metastasis of Gallbladder Carcinoma

Introduction: Gallbladder cancer is a highly fatal malignancy. Even though it is a rare malignancy, it is the most common biliary tract malignancy in USA. Majority of the patients are asymptomatic and among symptomatic patients, the most common complains include pain, anorexia, nausea or vomiting. Patients with GBC may also present with obstructive jaundice, either from direct invasion of the biliary tree or from metastatic liver diseases. Gall bladder carcinoma mainly metastasizes to liver, intra-abdominal LNs and biliary tract. Intramuscular and subcutaneous manifestations are very rare from gallbladder carcinoma. In this article we present a rare case of Intramuscular metastasis of gallbladder carcinoma in an African American lady with no other history of gallbladder disease. Case narration: 55 year old African American lady with history of poly-substance abuse disorder presented to Boston Medical Centre with 2 months of progressive Right Upper Quadrant abdominal pain. She had CT scan showing 6.3 cm mass in gallbladder invading into the liver which on biopsy was consistent with adenocarcinoma of gallbladder. She presented again in the Emergency Department with a new 3 x 3 cm lump in her left arm and 5 x 5 cm lump in her left thigh. A CT Scan of the thigh mass showed an extensive lesion in her left thigh muscles again which on USG guided biopsy confirmed intramuscular metastasis with IHC positive for CK7, AE1:3 and CAM5.2 consistent with metastatic gallbladder adenocarcinoma. Conclusion: Although rare, intramuscular lesions should raise suspicion of metastatic disease in the gall bladder carcinoma patients. In our case, patient reported two new rapidly growing lesions consistent with rapid progression of disease. Palliative radiation therapy should be considered in patients with severe pain associated with intramuscular metastasis. Intramuscular metastasis is usually associated with poorer prognosis

The Role of 18F-FDG PET/CT in staging of gallbladder carcinomas.

Gallbladder Carcinoma (GBC) is the most common and aggressive tumor of the biliary tract. Patients are typically diagnosed during advanced stages, and the mean overall survival is short. In our study, we aimed to demonstrate the uptake patterns of 18F-FDG PET/CT in GBC, as well as its association with survival and diagnostic value during the initial stage. Overall, 17 patients with GBC were retrospectively included in the study. 18F-FDG PET/CT study was performed for pretreatment staging. Two different standardized uptake values (SUVmax and SUVmean), metabolic tumor volume 40% (MTV40), and tumor lesion glycolysis (TLG) of the primary tumors were compared between the clinical and histopathological groups. Of the 17 patients, 11 were women (64.7%), and 6 (35.3%) were men. The mean age of the patients was 69.7±8.8 years. 18F-FDG uptake was detected in all lesions. Mean SUVmax was calculated to be 15.4±13.7 (median=10.6, range=3.4-46.8). All distant metastases (52.9%) were detected in the liver. Semiquantitative metabolic parameters (SUVmax and SUVmean, MTV40, and TLG) obtained from patients with distant metastasis were not significantly higher than those without distant metastasis. Similar results were obtained in patients with and without nodal metastasis. No statistically significant intergroup difference was observed regarding metabolic parameters. However, a statistically significant negative correlation was observed between the patient's age and the SUVmax of the primary lesion and metastatic lymph nodes (r=-0.564, p=0.018). During 10.7±10.4 months of mean follow-up, the mean survival of patients with distant metastases (6.1±11.0 months) was significantly shorter than that of patients with no organ metastases (15.8±7.1 months). In our study, distant metastases and age were observed to be crucial prognostic factors in patients with gallbladder carcinoma (GBC). In addition, we believe that 18F-FDG PET/CT imaging will help to stage the GBC, detect nodal and distant metastasis, and evaluate the metabolic state of gallbladder lesions.

Metastasis of Gall Bladder carcinoma - A case Report

Introduction: Metastasis of Gall Bladder Carcinoma (GBC) is very uncommon, aggressive and fatal cancer. World wide incidence rate reported as in women than men, older age more affected and coexisting Gall stonesare seen in majority cases. Because of vague symptoms at early stage tumour will not be encountered, most of the patients present in advanced stage, soprognosis is poor, hence Metastasis of GBC forms a diagnostic and therapeutic challenge. Case Report: A 53 years old male, known diabetic past 10 years presented with intermittent vague symptoms of vomiting, nausea, fever, fatigue, right upper quadrant pain since 3 months. Family history of Gallstones and cancer was present. On examination 2-3 bilateral cervical swellings present, abdominal examination reveals liver was palpable 4 cms below the right costal margin. Relevant investigations done. Complete blood count & Liver function tests were abnormal. Ultrasonography (USG) of abdomen and pelvis, USG guided FNAC of liver, CT of abdomen and pelvis, PET scan of abdomen, Histopathological and Immunohistochemistry report of left supraclavicularlymphnode biopsy were suggestive of metastatic adenocarcinoma of GB origin. After appropriate staging adjuvant chemotherapy started, after third chemotherapy cycle he died of due to metastasis of GBC, two months from his initial diagnosis. Conclusion: In the present study patient was diagnosed in advanced stage, early diagnosis could be missed due to vague symptoms and as GBC is uncommon entity, So proper evaluation of the patient in early stage may prolong the survival. Keywords: Gall Bladder, Carcinoma.

TASP1 Promotes Gallbladder Cancer Cell Proliferation and Metastasis by Up-regulating FAM49B via PI3K/AKT Pathway.

The highly conserved protease TASP1 not only takes part in critical site-specific proteolysis, but also plays an important role in numerous liquid and solid malignancies. However, the TASP1 expression and its biological regulation function in malignant gallbladder carcinoma (GBC) remain fully unknown. Here we observed that TASP1 levels were substantially overexpressed in GBC samples compared with non-tumor tissues. High TASP1 level was closely associated with T stage and metastasis, and was also correlated with poor prognosis in GBC patients. The depletion of TASP1 inhibited GBC cell proliferation and metastasis in vitro and in vivo. Furthermore, we first revealed that FAM49B had biological function and was positively regulated by TASP1 activating PI3K/AKT signaling pathway in GBC. At the same time, FAM49B also promoted GBC cell proliferation and migration. Inhibition of PI3K/AKT with LY294002 or FAM49B expression abrogated Myc-TASP1/Lv-shTASP1-induced GBC cell proliferation and motility. In conclusion, these findings demonstrate that TASP1 is critical for GBC progression via TASP1-PI3K/AKT-FAM49B axis and it may be a novel prognostic factor. The therapeutic targeting TASP1 may be a potential treatment approach for GBC patients.

MiR-324-5p inhibits gallbladder carcinoma cell metastatic behaviours by downregulation of transforming growth factor beta 2 expression

Increasing studies have demonstrated that microRNAs (miRNAs) are associated with the metastasis of gallbladder carcinoma (GBC). Recently, miR-324-5p has been reported to be a tumour-suppressive miRNA in many types of malignant cancer. However, the biological function and molecular mechanism of miR-324-5p in GBC still remain largely unknown. Here, we found that miR-324-5p expression was notably down-regulated in both GBC tissues and cells compared with that in normal controls. Downregulated miR-324-5p expression was negatively associated with the status of local invasion and lymph node metastasis and predicted a poor prognosis in GBC patients. Further functional assays revealed that restoration of miR-324-5p significantly suppressed GBC cell migration, invasion and epithelial-mesenchymal transition (EMT) in vitro and impeded the metastasis of GBC cells in vivo. Moreover, RNA immunoprecipitation (RIP) and dual-luciferase reporter assay confirmed that the transforming growth factor beta 2 (TGFB2) was a direct target gene of miR-324-5p in GBC cells. Mechanically, small interfering RNA (siRNA)-mediated knockdown of TGFB2 partially phenocopied the inhibitory effects of miR-324-5p overexpression on GBC cell metastatic phenotypes. In summary, our findings demonstrated that miR-324-5p targets TGFB2 expression to inhibit GBC cell metastatic behaviors, and implying miR-324-5p as a potential biomarker for diagnostic and therapeutic strategies in GBC.

LncRNA-HGBC stabilized by HuR promotes gallbladder cancer progression by regulating miR-502-3p/SET/AKT axis

BackgroundsLong non-coding RNAs (lncRNAs) are essential factors that regulate tumor development and metastasis via diverse molecular mechanisms in a broad type of cancers. However, the pathological roles of lncRNAs in gallbladder carcinoma (GBC) remain largely unknown. Here we discovered a novel lncRNA termed lncRNA Highly expressed in GBC (lncRNA-HGBC) which was upregulated in GBC tissue and aimed to investigate its role and regulatory mechanism in the development and progression of GBC.MethodsThe expression level of lncRNA-HGBC in GBC tissue and different cell lines was determined by quantitative real-time PCR. The full length of lncRNA-HGBC was obtained by 5′ and 3′ rapid amplification of the cDNA ends (RACE). Cellular localization of lncRNA-HGBC was detected by fluorescence in situ hybridization (FISH) assays and subcellular fractionation assay. In vitro and in vivo assays were preformed to explore the biological effects of lncRNA-HGBC in GBC cells. RNA pull-down assay, mass spectrometry, and RNA immunoprecipitation (RIP) assay were used to identify lncRNA-HGBC-interacting proteins. Dual luciferase reporter assays, AGO2-RIP, and MS2-RIP assays were performed to verify the interaction between lncRNA-HGBC and miR-502-3p.ResultsWe found that lncRNA-HGBC was upregulated in GBC and its upregulation could predict poor survival. Overexpression or knockdown of lncRNA-HGBC in GBC cell lines resulted in increased or decreased, respectively, cell proliferation and invasion in vitro and in xenografted tumors. LncRNA-HGBC specifically bound to RNA binding protein Hu Antigen R (HuR) that in turn stabilized lncRNA-HGBC. LncRNA-HGBC functioned as a competitive endogenous RNA to bind to miR-502-3p that inhibits target gene SET. Overexpression, knockdown or mutation of lncRNA-HGBC altered the inhibitory effects of miR-502-3p on SET expression and downstream activation of AKT. Clinically, lncRNA-HGBC expression was negatively correlated with miR-502-3p, but positively correlated with SET and HuR in GBC tissue.ConclusionsOur study demonstrates that lncRNA-HGBC promotes GBC metastasis via activation of the miR-502-3p-SET-AKT cascade, pointing to lncRNA-HGBC as a new prognostic predictor and a therapeutic target.

High-throughput screening identified miR-7-2-3p and miR-29c-3p as metastasis suppressors in gallbladder carcinoma.

Gallbladder carcinoma (GBC) is one of the most aggressive and lethal tumors, with extremely high metastatic activity and poor prognosis. Previously we have studied miRNAs that promote metastasis and progression of GBC, the aim of present study was to systematically elucidate the metastasis suppressor miRNAs in GBC. A novel designed high-throughput screening method that combined high content screening (HCS) and miRNA microarray analysis was conducted to filter out anti-metastatic miRNAs of GBC. Frozen samples were analyzed for the expression of goal miRNAs by real-time PCR. The biological functions of miRNAs were studied by transwell, immunoblot. Liver metastasis model via spleen injection was further examined in nude mice. Kaplan-Meier and Cox regression analyses were used to analyze the effect of goal miRNAs on overall survival. The target genes and interaction network of goal miRNAs were determined by whole transcriptome genome sequencing. Out of the miRNAs library, a series of prominent metastatic suppressor miRNA candidates were filtered out. Among them, miR-7-2-3p and miR-29c-3p were discovered downregulated in GBC, and upregulation of them could reverse epithelial-mesenchymal transition and decrease the metastasis ability of GBC cells in vitro and in vivo, which was dominated by the miRNA-mRNA-lncRNA co-expression network. And DCLK1 and SLC36A1 are the direct target genes of miR-7-2-3p and miR-29c-3p. Moreover, the deficiency of miR-7-2-3p and miR-29c-3p was closely associated with poor prognosis of GBC patients. Our findings indicate that miR-7-2-3p and miR-29c-3p play crucial roles in the pathogenesis and worse prognosis of GBCs, which may serve as prognosis biomarkers and promise potential therapeutic targets in the future.

Axillary Lymph Node Metastasis in Gallbladder Carcinoma with Incidentally Detected Coexistence of Aberrant Right Subclavian Artery with Left-Sided Superior Vena Cava.

The sequential development of port site recurrence, followed by recurrence in the axillary lymph node in gallbladder carcinoma is very infrequently reported in the literature. The representing 18F-fluorodeoxyglucose positron emission tomography-computed tomography image shows a metastatic right axillary lymph node in a case of gallbladder cancer developed following surgical removal of port site recurrence and six cycles of chemotherapy. The image also shows coexistence of two incidentally detected vascular anomalies, i.e., aberrant right subclavian artery and left-sided superior vena cava. Coexistence of both the vascular anomalies is rare among the general population and have their own clinical implications as described.

Tumor suppressor LKB1 inhibits the progression of gallbladder carcinoma and predicts the prognosis of patients with this malignancy.

Gallbladder carcinoma(GBC) represents the most common fatal tumors of the biliary tract. The 3-year or 5-year survival rate for patients with this disease are 30 and 5%, respectively. Liver kinase B1 (LKB1), a primary upstream kinase of adenosine monophosphate-activated protein kinase (AMPK) necessary for maintaining cell metabolism and energy homeostasis, has been found to be an important tumor suppressor gene in recent years, and its inactivation has also found to be closely associated with tumor growth, metastasis and cancer stem cell (CSC) proliferation. Nevertheless, the function of LKB1 in GBC remains unclear. In this study, we found that the expression of LKB1 in GBC tissues was decreased compared with that in non-cancerous tissues. LKB1 overexpression suppressed the proliferation, metastasis and expansion of GBC CSCs. Mechanically, LKB1 suppressed GBC cell progression via the JAK/signal transducer and activator of transcription 3 (STAT3) pathway. The use of the JAK2 inhibitor, AZD‑1480, attenuated the suppressive effects of LKB1 overexpression on the growth, metastasis and self-renewal ability of the GBC cells, which further demonstrated that JAK/STAT3 was involved in the LKB1-induced suppression of GBC cell growth, metastasis and self-renewal ability. More importantly, the decreased expression of LKB1 was a predictor of a poor prognosis of patients with GBC. On the whole, our data indicate that LKB1 inhibits GBC cell growth, metastasis and self-renewal ability by disrupting JAK/STAT3 signaling, and may thus prove to be a novel prognostic biomarker for patients with GBC.

P-028 - Lipoxin A4 inhibits the paracrine of Nodal in CAFs by suppressing FPRL1/ROS/NF-κB signaling to attenuate invasion and metastasis of gallbladder carcinoma

P-028 - Lipoxin A4 inhibits the paracrine of Nodal in CAFs by suppressing FPRL1/ROS/NF-κB signaling to attenuate invasion and metastasis of gallbladder carcinoma

RIP1 regulates TNF-α-mediated lymphangiogenesis and lymphatic metastasis in gallbladder cancer by modulating the NF-κB-VEGF-C pathway.

BackgroundTumor necrosis factor alpha (TNF-α) enhances lymphangiogenesis in gallbladder carcinoma (GBC) via activation of nuclear factor (NF-κB)-dependent vascular endothelial growth factor-C (VEGF-C). Receptor-interacting protein 1 (RIP1) is a multifunctional protein in the TNF-α signaling pathway and is highly expressed in GBC. However, whether RIP1 participates in the signaling pathway of TNF-α-mediated VEGF-C expression that enhances lymphangiogenesis in GBC remains unclear.MethodsThe RIP1 protein levels in the GBC-SD and NOZ cells upon stimulation with increasing concentrations of TNF-α as indicated was examined using Western blot. Lentiviral RIP1 shRNA and siIκBα were constructed and transduced respectively them into NOZ and GBC-SD cells, and then PcDNA3.1-RIP1 vectors was transduced into siRIP1 cell lines to reverse RIP1 expression. The protein expression of RIP1, inhibitor of NF-κB alpha (IκBα), p-IκBα, TAK1, NF-κB essential modulator were examined through immunoblotting or immunoprecipitation. Moreover, VEGF-C mRNA levels were measured by quantitative real-time polymerase chain reaction, VEGF-C protein levels were measured by immunoblotting and enzyme-linked immunosorbent assay, and VEGF-C promoter and NF-κB activities were quantified using a dual luciferase reporter assay. The association of NF-κB with the VEGF-C promoter was analysed by chromatin immunoprecipitation assay. A three-dimensional coculture method and orthotopic transplantation nude mice model were used to evaluate lymphatic tube-forming and metastasis ability in GBC cells. The expression of RIP1 protein, TNF-α protein and lymphatic vessels in human GBC tissues was examined by immunohistochemistry, and the dependence between RIP1 protein with TNF-α protein and lymphatic vessel density was analysed.ResultsTNF-α dose- and time-dependently increased RIP1 protein expression in the GBC-SD and NOZ cells of GBC, and the strongest effect was observed with a concentration of 50 ng/ml. RIP1 is fundamental for TNF-α-mediated NF-κB activation in GBC cells and can regulate TNF-α-mediated VEGF-C expression at the protein and transcriptional levels through the NF-κB pathway. RIP1 can regulate TNF-α-mediated lymphatic tube formation and metastasis in GBC cells both in vitro and vivo. The average optical density of RIP1 was linearly related to that of TNF-α protein and the lymphatic vessel density in GBC tissues.ConclusionWe conclude that RIP1 regulates TNF-α-mediated lymphangiogenesis and lymph node metastasis in GBC by modulating the NF-κB-VEGF-C pathway.

Therapeutic effects of long-circulating miR-135a-containing cationic immunoliposomes against gallbladder carcinoma

Gallbladder carcinoma (GBC) is the most common malignant tumour in the biliary tract, but effective therapeutics are lacking. Based on our previous studies, miR-135a is a potential tool to inhibit GBC proliferation. In this study, we constructed miR-135a-loaded DSPE-PEG2000 liposomes modified with Anti-EGFR antibodies (Anti-EGFR-CIL-miR-135a). The results of an analysis of their physicochemical properties indicated the particle size of it was 222.0 ± 2.1 nm in diameter with an uptake efficiency of 86.5%. Next, the post-treatment biological behaviours of GBC, specifically, invasion, metastasis and apoptosis, were evaluated. miR-135a inhibited GBC invasion and metastasis and promoted apoptosis compared to controls. Additionally, miR-135a targeted and regulated the expression of ROCK1, HOXA10 and BCL-2. Due to the targeted effects of Anti-EGFR-CIL-miR-135a, the GBC tumour growth rate was 60% lower in an in vivo xenograft-bearing mouse model compared to controls. Thus, Anti-EGFR-CIL-miR-135a is a promising therapeutic strategy to combat GBC.