Apoptosis In CCA Cells Research Articles

Lenvatinib inhibits cholangiocarcinoma progression by targeting the FGF19/PI3K/AKT signaling pathway.

Cholangiocarcinoma (CCA) is known for its high aggressiveness and dismal prognosis, whose effectiveness of systemic therapy remains limited. As a multi-target drug, lenvatinib has exhibited promising effects in many solid tumors. However, the therapeutic role of lenvatinib in CCA is rarely investigated. Here, the in vitro assays including EdU, colony formation, transwell, wound healing, and apoptosis analyses demonstrated that lenvatinib significantly inhibited the proliferation, migration, and invasion, while simultaneously inducing apoptosis of CCA cells. Mechanistically, lenvatinib downregulated the expression of FGF19 and inactivated the PI3K/AKT signaling pathway. Depletion of FGF19 enhanced the anti-tumor effects of lenvatinib, which was attributed to the inhibition of p-PI3K and p-AKT expression in CCA cells. In contrast, overexpression of FGF19 activated the PI3K/AKT signaling pathway, thereby impairing the inhibitory effects of lenvatinib against CCA. In addition, the AKT inhibitor, MK-2206, reinforced the lenvatinib-induced CCA inhibition. Notably, the in vivo experiment confirmed that the subcutaneous tumorigenicity of CCA cells in nude mice was weakened by lenvatinib. Lenvatinib markedly downregulated the expression of FGF19, p-AKT, Ki-67, vimentin, and VEGF in the xenograft tumor tissues. Collectively, these findings demonstrated that lenvatinib inhibits CCA progression by targeting the FGF19/PI3K/AKT signaling pathway. The present study provides novel experimental evidence for the potential clinical application of lenvatinib in CCA, which also highlights the promising role of targeting FGF19 in combined therapeutic approaches for CCA.

Curcumin synergistically enhances the efficacy of gemcitabine against gemcitabine-resistant cholangiocarcinoma via the targeting LAT2/glutamine pathway

Cholangiocarcinoma (CCA) is often diagnosed late, leading to incomplete tumor removal, drug resistance and reduced chemotherapy efficacy. Curcumin has the potential for anti-cancer activity through various therapeutic properties and can improve the efficacy of chemotherapy. We aimed to investigate the synergistic effect of a combination of curcumin and gemcitabine against CCA, targeting the LAT2/glutamine pathway. This combination synergistically suppressed proliferation in gemcitabine-resistant CCA cells (KKU-213BGemR). It also resulted in a remarkable degree of CCA cell apoptosis and cell cycle arrest, characterized by a high proportion of cells in the S and G2/M phases. Knockdown of SLC7A8 decreased the expressions of glutaminase and glutamine synthetase, resulting in inhibited cell proliferation and sensitized CCA cells to gemcitabine treatment. Moreover, in vivo experiments showed that a combination curcumin and gemcitabine significantly reduced tumor size, tumor growth rate and LAT2 expression in a gemcitabine-resistant CCA xenograft mouse model. Suppression of tumor progression in an orthotopic CCA hamster model provided strong support for clinical application. In conclusion, curcumin synergistically enhances gemcitabine efficacy against gemcitabine-resistant CCA by induction of apoptosis, partly via inhibiting LAT2/glutamine pathway. This approach may be an alternative strategy for the treatment of gemcitabine-resistant in CCA patients.

Iodine-125 seed inhibits proliferation and promotes apoptosis of cholangiocarcinoma cells by inducing the ROS/p53 axis

With advances in radioactive particle implantation in clinical practice, Iodine-125 (125I) seed brachytherapy has emerged as a promising treatment for cholangiocarcinoma (CCA), showing good prognosis; however, the underlying molecular mechanism of the therapeutic effect of 125I seed is unclear. To study the effects of 125I seed on the proliferation and apoptosis of CCA cells. CCA cell lines, RBE and HCCC-9810, were treated with reactive oxygen species (ROS) scavenger acetylcysteine (NAC) or the p53 functional inhibitor, pifithrin-α hydrobromide (PFTα). Cell counting kit-8 (CCK-8) assay, 5-bromo-2-deoxy-uridine (BrdU) staining, and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and flow cytometry assay were performed to test the radiation-sensitivity of 125I seed toward CCA cells at different radiation doses (0.4 mCi and 0.8 mCi). 2,7-dichlorofluorescein diacetate (DCF-DA) assay, real-time quantitative polymerase chain reaction (RT-qPCR), and western blot analysis were performed to assess the effect of 125I seed on the ROS/p53 axis. A dose-dependent inhibitory effect of 125I seeds on the proliferation of CCA cells was observed. The 125I seed promoted apoptosis of CCA cells and induced the activation of the ROS/p53 pathway in a dose-dependent manner. NAC or PFTα treatment effectively reversed the stimulatory effect of 125I seed on the proliferation of CCA cells. NAC or PFTα suppressed apoptosis and p53 protein expression induced by the 125I seed. 125I seed can inhibit cell growth mainly through the apoptotic pathway. The mechanism may involve the activation of p53 and its downstream apoptotic pathway by up-regulating the level of ROS in cells.

LOXL1-AS1 inhibits JAK2 ubiquitination and promotes cholangiocarcinoma progression through JAK2/STAT3 signaling.

This study thoroughly investigated the role of the long non-coding RNA LOXL1-AS1 in the pathogenesis of cholangiocarcinoma (CCA). Through bioinformatics analysis and tissue samples validation, the study found that LOXL1-AS1 was significantly elevated in CCA, with its high expression closely tied to clinical pathological features and prognosis. In vitro and in vivo experiments revealed that LOXL1-AS1 was crucial in regulating CCA cell apoptosis, proliferation, migration, and invasion. Further investigations using FISH, subcellular localization experiments, RNA pull down, and RIP uncovered that LOXL1-AS1 primarily resided in the cytoplasm and influenced CCA progression by modulating the JAK2/STAT3 signaling pathway. Notably, LOXL1-AS1 might regulate the activity of JAK2 through modulating its ubiquitination and degradation. YY1 had also been found to act as an upstream transcription factor of LOXL1-AS1 to impact CCA cell malignancy. These findings shed light on the pivotal role of LOXL1-AS1 in CCA and offered potential directions for novel therapeutic strategies, providing a fresh perspective on tumor pathogenesis.

The Human Placental Amniotic Membrane Mesenchymal-Stromal-Cell-Derived Conditioned Medium Inhibits Growth and Promotes Apoptosis of Human Cholangiocarcinoma Cells In Vitro and In Vivo by Suppressing IL-6/JAK2/STAT3 Signaling.

Mesenchymal stromal cells (MSCs) have recently been shown to play an important role in the growth and progression of many solid tumors, including cholangiocarcinoma (CCA). The human placental amniotic membrane (hPAM) is one of the most favorable sources of MSCs due to its availability and non-invasive harvesting procedure. However, the role of human placental amniotic membrane mesenchymal stromal cells (hPAMSCs) in the growth and progression of human CCA has not yet been determined. This study investigates the effects of conditioned medium derived from hPAMSCs (PA-CM) on the properties of three human CCA cell lines and explores possible mechanisms of action. Varying concentrations of PA-CM were used to treat CCA cells to determine their effects on the proliferation and apoptosis of CCA cells. The results showed that PA-CM inhibited the proliferation and colony-forming capacity of KKU100, KKU213A, and KKU213B cells. PA-CM also promoted the apoptosis of these CCA cells by causing the loss of mitochondrial membrane potential. Western Blotting confirmed that PA-CM induced CCA cell apoptosis by increasing the levels of the Bax/Bcl-2 ratio, cleaved caspase 3, and cleaved PARP, possibly by inhibiting the IL-6/JAK2/STAT3 signaling pathway. Moreover, our in vivo study also confirmed the suppressive effect of hPAMSCs on CCA cells by showing that PA-CM reduced tumor volume in nude mice transplanted with human CCA cells. Taken together, our results demonstrate that PA-CM has potent tumor-suppressive effects on human CCA cells and could potentially be used in combination with chemotherapy to develop a more effective treatment for CCA patients.

Mitochondrial transplantation inhibits cholangiocarcinoma cells growth by balancing oxidative stress tolerance through PTEN/PI3K/AKT signaling pathway

BackgroundCholangiocarcinoma (CCA) is a serious threat to human health, and tumor development is associated with abnormal mitochondrial function. It is believed that the introduction of healthy mitochondria into tumor cells can induce the oxidative stress in tumor cells to return to normal levels, thus exerting an inhibitory effect on tumor growth. MethodsMitochondria isolated from 143BρW cells were co-cultured with HuCCT1 cells, and the mitochondria were stained with MitoTracker dye as a tracking label. Changes in apoptosis, proliferation, oxidative stress, and PTEN/PI3K/AKT signaling pathway were assessed. In addition, a CCA nude mouse transplantation tumor model was constructed to analyze the effects of mitochondrial transplantation on the above factors in nude mice. Furthermore, the expression of PTEN was interfered to observe the effect and mechanism of mitochondrial transplantation on the proliferation and apoptosis of CCA cells. ResultsMitochondrial transplantation promoted apoptosis and inhibited cell proliferation in CCA cell line. SOD, GSH, and CAT activities were significantly increased, the expression of PTEN was activated, and the expression of p-PI3K and p-AKT were inhibited after mitochondrial transplantation. After mitochondrial transplantation + si-PTEN treatment, cell apoptosis, SOD, GSH, CAT activity, and the expression of PTEN were decreased, while the expression of p-PI3K and p-AKT were significantly enhanced. ConclusionThis study reveals the anti-tumor potential of mitochondrial transplantation through PTEN/PI3K/AKT signaling pathway to regulate cellular oxidative stress in CCA.

The m6A reader YTHDC1-mediated lncRNA CTBP1-AS2 m6A modification accelerates cholangiocarcinoma progression

BackgroundCholangiocarcinoma (CCA) is a serious malignancy originating from the bile ducts and the second most common primary liver cancer. Long non-coding RNA (lncRNA) is a functional lncRNA that plays an important role in human cancers. However, the role and underlying mechanisms of CTBP1-AS2 in CCA remain unknown. PurposeIn this study, we investigated the functional role and mechanism of long-stranded non-coding RNA (lncRNA) C-terminal binding protein 1 antisense RNA 2 (CTBP1-AS2) in CCA progression. ResultIn the present study, the bioinformatics analysis revealed that YTHDC1 and CTBP1-AS2 were significantly upregulated, and it was confirmed in cholangiocarcinoma tissues from CCA patients. Meanwhile, we demonstrated that knockdown of YTHDC1 or lncRNA CTBP1-AS2 inhibited CCA cell proliferation, migration and invasion, blocked the cell cycle in G2/M phase and promoted apoptosis of CCA cells. In addition, lncRNA CTBP1-AS2-mediated N6-methyladenosine (m6A) methylation levels were significantly elevated in cholangiocarcinoma tissues, whereas knockdown of YTHDC1 resulted in a significant down-regulation of m6A methylation levels by lncRNA CTBP1-AS2. ConclusionOur results suggest that YTHDC1 affects cholangiocarcinoma progression by modifying the lncRNA CTBP1-AS2 m6A, and CTBP1-AS2 may be a promising therapeutic target for CCA.

Cucurbitacin C as an effective anti-cancer agent: unveiling its potential role against cholangiocarcinoma and mechanistic insights.

Cholangiocarcinoma (CCA) is a malignant epithelial tumor characterized by a dismal prognosis. Given the lack of therapeutic strategies and durable treatment options currently available, identifying innovative treatments for CCA is an urgent unmet clinical need. Cucurbitacin C (CuC) is a distinct variant of the cucurbitacin family, displaying promising anti-cancer activity against various tumor types. The primary objective of our research is to elucidate the promising effects of CuC on CCA. The impact of CuC on CCA cell lines was assessed by cell count kit-8 assay, EdU staining assay, colony formation assay, wound-healing assay, and Transwell assay. Flow cytometric analysis was conducted to explore the function of CuC treatments on cell-cycle distribution and apoptosis in CCA cells. Computational biology and network pharmacology approaches were utilized to predict potential targets of CuC. Furthermore, a tumor xenograft mouse model was established using CCA cells to explore the anti-cancer effects of CuC in vivo. Our research findings revealed that CuC exerted a suppressive effect on CCA cell progression. Cell viability assays, EdU staining assays, and colony formation assays demonstrated that CuC effectively suppressed viability and proliferation of CCA cells. Wound-healing assays and Transwell assays indicated that CuC effectively inhibits the migratory and invasive capabilities of CCA cells. Flow cytometry analysis elucidated that CuC played its anti-proliferative role in CCA cells by arresting G0/G1 phase and increasing apoptosis. Through bioinformatics and network pharmacology analysis, in conjunction with western blot analysis, we demonstrated CuC mediated the inhibition of CCA cell progression through modulation of JAK2/STAT3 pathway. Additionally, the CCA xenograft tumor model was established, and the results supported the inhibition of CuC treatment against CCA progression in vivo. Our study demonstrates that CuC possesses notable capabilities to suppress cell proliferation, migration, and invasion in CCA. Importantly, the inhibitory effects of CuC on CCA progression are attributed to its modulation of the JAK2/STAT3 signaling pathway. Altogether, our study demonstrated that CuC holds promise as a prospective therapeutic agent for treating CCA.

Identification of SPRYD4 as a tumour suppressor predicts prognosis and correlates with immune infiltration in cholangiocarcinoma

Cholangiocarcinoma (CCA) is an aggressive solid tumour with a 5-year survival rate ranging from 7% to 20%. It is, therefore, urgent to identify novel biomarkers and therapeutic targets to improve the outcomes of patients with CCA. SPRY-domain containing protein 4 (SPRYD4) contains SPRY domains that modulate protein–protein interaction in various biological processes; however, its role in cancer development is insufficiently explored. This study is the first to identify that SPRYD4 is downregulated in CCA tissues using multiple public datasets and a CCA cohort. Furthermore, the low expression of SPRYD4 was significantly associated with unfavourable clinicopathological characteristics and poor prognosis in patients with CCA, indicating that SPRYD4 could be a prognosis indicator of CCA. In vitro experiments revealed that SPRYD4 overexpression inhibited CCA cells proliferation and migration, whereas the proliferative and migratory capacity of CCA cells was enhanced after SPRYD4 deletion. Moreover, flow cytometry showed that SPRYD4 overexpression triggered the S/G2 cell phase arrest and promoted apoptosis in CCA cells. Furthermore, the tumour-inhibitory effect of SPRYD4 was validated in vivo using xenograft mouse models. SPRYD4 also showed a close association with tumour-infiltrating lymphocytes and important immune checkpoints including PD1, PD-L1 and CTLA4 in CCA. In conclusion, this study elucidated the role of SPRYD4 during CCA development and highlighted SPRYD4 as a novel biomarker and tumour suppressor in CCA.

Plumbagin Suppresses Growth, Induces Apoptosis, and Inhibits Migration in Cholangiocarcinoma via Reactive Oxygen Species Generation and Mitochondrial Function

Objectives: Plumbagin has been displayed on growth inhibition in several cancer cells; however, the anticancer effects of plumbagin on cholangiocarcinoma (CCA) cell are still less information. This article will explore the potential anticancer effects and underlying mechanisms of plumbagin on CCA cells. Materials and Methods: Two types of CCA cell, including KKU-100 with slow growth and migration and KKU-M452 with rapid growth and migration, were treated with plumbagin and measured the growth inhibition and migratory suppression by sulforhodamine B, colony formation, acridine orange/ethidium bromide staining, wound healing, and Matrigel migration. Apoptosis induction, reactive oxygen species (ROS) formation, and mitochondrial function were analyzed by the flow cytometric method. Results: To compare the plumbagin effects on two types of CCA cells, the data indicated that plumbagin strongly reduced cell proliferation in a concentration- and time-dependent manner. At 72 h, IC50 values were 1.11 ± 0.12 µM for KKU-100 and 2.52 ± 0.40 µM for KKU-M452 cells, with inhibition of the cell cycle in the G0/G1 phase for KKU-100 and the G2/M phase for KKU-M452, respectively. Moreover, plumbagin decreased cell migration in both CCA cells in a dose-dependent manner. Furthermore, late apoptosis was significantly induced after treatment with plumbagin. Mechanistically, plumbagin increased ROS along with decreased mitochondrial function in CCA cells. Finally, the position of 5-hydroxy on the naphthoquinone ring of plumbagin was in a good position to form an H-bond with the amino group for all caspase 3, cytochrome C, and p21 binding sites, and these data indicated that plumbagin suppressed CCA cells via modulation of these proteins. Conclusion: Plumbagin strongly suppresses cell viability and stimulates apoptosis in CCA cells via induction of ROS and inhibition of mitochondrial function.

Inhibition of neuropilin-1 enhances the therapeutic effects of lenvatinib in suppressing cholangiocarcinoma cells via the c-Met pathway

Neuropilin-1 (NRP-1) participates in the progression of cholangiocarcinoma (CCA) and lenvatinib is an approved tyrosine kinase inhibitor treating several other cancers. Our exploratory study reveals that the inhibitory activities of lenvatinib largely rely on the expression levels of NRP-1 in CCA cells, leading to the present study that aims to investigate whether inhibition of NRP-1 could enhance the effects of lenvatinib in suppressing CCA. By using stable transfected CCA cells depleted of NRP-1 and EG00229, a specific NRP-1 inhibitor, we examined cell proliferation, cell cycle distribution and apoptosis, and detected the expression of key molecules and the involvement of the c-Met pathway. Xenograft mouse models were employed to verify the in vitro results. NRP-1 depletion and EG00229 strengthened lenvatinib in inhibiting the proliferation and promoting the apoptosis of CCA cells, and their additive or synergistic effects were confirmed in animal models. Mechanistically, lenvatinib induced the activation of the c-Met pathway, while either NRP-1 depletion or EG00229 inhibited this activation, which could be stimulated by its ligand hepatocyte growth factor. NRP-1 inhibition resulted in a significant alteration in the expression/activation of downstream pathways and molecules, which are key factors regulating cell proliferation and apoptosis. In conclusion, the present results indicate that the inhibition of NRP-1 enhances the efficacy of lenvatinib via the c-Met pathway, and warrant further studies on the pharmacological utility of EG00229, particularly, in the combination of lenvatinib as a promising adjunct therapeutic strategy for combating CCA.

Abstract PO032: Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-TRAIL-R Mediates Cholangiocarcinoma Tumor Immune Evasion by Enhancing Myeloid-Derived Suppressive Cell Population

Abstract Background and Aims: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of TNF superfamily, is predominantly expressed on immune cells. Engagement of TRAIL with its cognate receptor, TRAIL-R (TR), leads to cancer cells apoptosis. Although the anti-tumor role of TRAIL-TR has been extensively studied, TRAIL agonists have had very limited anti-cancer activity in human clinical trials. TRAIL signaling can facilitate the myeloid response. However, this potential immunosuppressive function of TRAIL has not been examined extensively in cancer biology. Cholangiocarcinoma (CCA), a highly lethal biliary tract cancer, has a dense immunosuppressive microenvironment. Accordingly, CCA provides a model to examine the potential immune regulatory function of TRAIL in cancer biology. Methods: Using syngeneic, orthotopic murine models of CCA, (PMID: 29464042), murine CCA cells (SB cells) that express both Trail and Trail receptor (Tr) were implanted into livers of WT Tr− / − and LyzcreTrf/f mice, the latter have a specific deletion of TRAIL-R on myeloid cells. Hence, in this model the host immune cells express Trail but not the receptor; therefore, they would be capable of inducing TRAIL-mediated apoptosis in CCA cells but would be resistant to TRAIL-mediated immunosuppression. After 4 weeks of tumor growth, mice were sacrificed, and tumors were characterized using flow cytometry. Results: We observed that Tr− / − mice had a significant reduction in tumor burden compared to WT mice. Myeloid-derived suppressor cells (MDSCs) were significantly decreased in Tr− / − tumors compared to WT tumors. Implantation of SB cells into LyzcreTrf/f mice resulted in a marked reduction in tumor burden and attenuation of MDSC infiltration compared to Lyzcre mice. In vitro functional studies employing MDSCs from mice deficient in Tr (MDSC-Tr −/−) were carried out. Compared to WT MDSCs, coculture of MDSC-Tr− /- with SB cells resulted in a significant reduction in the proliferation and immunosuppressive function of MDSCs. Moreover, following cocultured with SB cells, immunofluorescence analysis demonstrated that MDSC-Tr− / − had a reduction in the nuclear translocation of the NFkB subunit P65 compared to WT MDSCs. These data suggest that TRAIL-TR augments MDSC proliferation via NFkB. In conclusion, we have demonstrated that Tr− / − mice have a significant reduction in CCA tumor burden and MDSC infiltration. These results indicate that TRAIL-TR facilitates tumor immune escape and progression by fostering MDSC immunosuppressive function and infiltration, in an NFkB-dependent manner. Direct targeting of TRAIL on CCA cells is a potential anti-tumor strategy. Citation Format: Emilien Loeuillard, Jingchun Yang, Dong Haidong, Gregory Gores, Sumera Ilyas. Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-TRAIL-R Mediates Cholangiocarcinoma Tumor Immune Evasion by Enhancing Myeloid-Derived Suppressive Cell Population [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO032.

Albendazole-induced autophagy blockade contributes to elevated apoptosis in cholangiocarcinoma cells through AMPK/mTOR activation

Albendazole (ABZ) is a broad-spectrum anti-parasitic drug that exhibits antitumor effects against several carcinomas. The effects of ABZ on cholangiocarcinoma (CCA) and its underlying mechanisms are still unclear. Our study aims to investigate the role of ABZ in inducing autophagy-mediated apoptosis of cholangiocarcinoma cells. The antitumor effects of ABZ were evaluated against CCA cells and HIBEC intrahepatic biliary epithelial cells. Furthermore, the apoptosis rates, and autophagy flux in RBE and FRH-0201 cells treated with ABZ were investigated. ABZ inhibited proliferation, induced cell death and apoptosis in CCA cells in vitro. In vivo, tumors from ABZ- treated BALB/c nude mice were significantly smaller than untreated mice. ABZ also induced the initiation of autophagy via AMPK/mTOR pathways, resulting in the formation of autophagosome. In addition, ABZ blocked autophagic flux by inhibiting the fusion of autophagosome-lysosome, which increased the apoptotic death of CCA cells. However, the apoptotic death of CCA cells induced by ABZ was reversed by 3-methyladenine (3-MA), an autophagosome formation inhibitor, but increased by chloroquine (CQ), an autophagosome-lysosome fusion inhibitor.Our work provides novel mechanisms for anti-tumor effects of ABZ on CCA, suggesting that ABZ may be used as a potent autophagy inhibitor in the treatment of CCA.

Human chorion-derived mesenchymal stem cells suppress JAK2/STAT3 signaling and induce apoptosis of cholangiocarcinoma cell lines

Cholangiocarcinoma (CCA) is an aggressive malignancy arising from the damaged epithelial cells of the biliary tract. Previous studies have reported that the multi-potent mesenchymal stem cells (MSCs) activate a series of tumor signaling pathways by releasing several cytokines to influence tumor cell development. However, the roles and mechanisms of human chorion-derived MSCs (CH-MSCs) in cholangiocarcinoma progression have not been fully addressed. This present study aims to examine the effects of conditioned media derived from CH-MSCs (CH-CM) on CCA cell lines and investigate the respective underlying mechanism of action. For this purpose, MSCs were isolated from chorion tissue, and three cholangiocarcinoma cell lines, namely KKU100, KKU213A, and KKU213B, were used. MTT assay, annexin V/PI analysis, and JC-1 staining were used to assess the effects of CH-CM on proliferation and apoptosis of CCA cells, respectively. Moreover, the effect of CH-CM on caspase-dependent apoptotic pathways was also evaluated. The western blotting assay was also used for measuring the expression of JAK2/STAT3 signaling pathway-associated proteins. The results showed that CH-CM suppressed proliferation and promoted apoptosis of CCA cell lines. CH-CM treatment-induced loss of mitochondrial membrane potential (∆Ψm) in CCA cell lines. The factors presented in the CH-CM also inhibited JAK2/STAT3 signaling, reduced the expression of BCL-2, and increased BAX expression in CCA cells. In conclusion, our study suggests that the CH-CM has a potent anti-cancer effect on cholangiocarcinoma cells and thus provides opportunities for use in alternative cell therapy or in combination with a conventional chemotherapeutic drug to increase the efficiency of CCA treatment.

Abstract 1315: Tumor necrosis factor related apoptosis inducing ligand fosters myeloid derived suppressor cell mediated tumor immune evasion in cholangiocarcinoma

Abstract Background and Aims: Tumor necrosis factor related apoptosis inducing ligand (TRAIL) is predominantly expressed on immune cells. Although TRAIL biology has garnered considerable attention as a potential anti-cancer strategy, TRAIL agonists have had very limited anti-cancer activity in humans. TRAIL signaling can attenuate the T lymphocyte response. However, this potential immunosuppressive function of TRAIL has not been examined in cancer biology. Cholangiocarcinoma (CCA), a highly lethal biliary tract cancer, has a dense immunosuppressive microenvironment. Accordingly, CCA provides a model to examine the potential immune regulatory function of TRAIL in cancer biology. Methods: Using a syngeneic, orthotopic murine model of CCA (PMID: 29464042), murine CCA cells (SB cells) that express both Trail and Trail receptor (Tr) were implanted into livers of WT and Tr-/- mice. Hence, in this model the host immune cells express Trail but not the receptor; therefore, they would be capable of inducing TRAIL-mediated apoptosis in CCA cells but would be resistant to TRAIL-mediated immunosuppression. After 4 weeks of tumor growth, mice were sacrificed, and tumors were characterized using flow cytometry. Results: We observed that Tr-/- mice had a significant reduction in tumor burden compared to WT mice. Moreover, tumor bearing Tr-/- mice had a significant increase in cytotoxic T lymphocytes (CTLs) and enhanced CTL effector function. Myeloid-derived suppressor cells (MDSCs) were significantly decreased in Tr-/- tumors compared to WT tumors. Furthermore, implantation of SB cells devoid of Trail (SB-Trail-/-) into WT mice resulted in a significant reduction in tumor burden and MDSC infiltration. In vitro functional studies employing SB cells deficient in Tr (SB-Tr-/-) and MDSCs were carried out. There was a significant reduction in proliferation and immunosuppressive function of MDSCs when cocultured with SB-Tr-/- compared to SB cells. These results indicate that TRAIL-TR fosters MDSC growth and immunosuppressive function. In conclusion, we have demonstrated that Tr-/- mice have a significant reduction in CCA tumor burden and MDSC infiltration. Consequently, Tr-/- tumors have enhanced CTL infiltration and function. These data suggest that the TRAIL-TR system mediates tumors immune evasion via MDSCs, and direct targeting of TRAIL on CCA cells is a potential anti-tumor strategy. Citation Format: Emilien Loeuillard, Jingchun Yang, Haidong Dong, Gregory J. Gores, Sumera I. Ilyas. Tumor necrosis factor related apoptosis inducing ligand fosters myeloid derived suppressor cell mediated tumor immune evasion in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1315.

CircSETD3 (hsa_circ_0000567) inhibits proliferation and induces apoptosis in cholangiocarcinoma cells via downregulation of microRNA-421 expression

ABSTRACT Cholangiocarcinoma (CCA) is a fatal tumor associated with chronic inflammation. Circular RNAs (circRNAs) have been evidenced to be involved in tumorigenesis and tumor progression. This study aimed to explore the effects and potential molecular mechanism of circSETD3 in CCA progression. Levels of CircSETD3 and microRNA (miR)-421 in CCA tissue and cell lines were measured using quantitative real-time polymerase-chain reaction (qRT-PCR). A direct target of miR-421 was predicted using TargetScan and further confirmed by a dual-luciferase reporter assay. Cell proliferation and apoptosis were measured using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and flow cytometry, respectively. The activity of caspase-3 was also examined using caspase-3 activity detection kits. Moreover, the levels of B-cell lymphoma-2 modifying factor (BMF), B-cell lymphoma 2 (BCL2), and Bcl-2-associated X protein (BAX) in TFK1 cells were assessed using qRT-PCR and western blot analysis. We found that circSETD3 was downregulated, while miR-421 was upregulated in CCA tissues and cell lines. CircSETD3 negatively regulated miR-421 levels in TFK1 cells. Functional assays revealed that circSETD3-plasmid inhibited cell proliferation, induced apoptosis, promoted caspase-3 activity, enhanced Bax and cleaved-Caspase 3 expression, and reduced Bcl-2 levels, and these effects were reversed by miR-421 mimic. Meanwhile, similar results were observed in miR-421 inhibitor-transfected TFK1 cells, and these results were abolished by BMF-siRNA. BMF, a direct target of miR-421, was downregulated in CCA tissues and cell lines. These findings demonstrate that circSETD3 inhibits proliferation and induces apoptosis in CCA cells by regulating the miR-421/BMF axis, indicating its potential as a promising candidate for CCA therapy.

MCM2 promotes the proliferation, migration and invasion of cholangiocarcinoma cells by reducing the p53 signaling pathway.

The abnormal expressions of minichromosome maintenance protein 2 (MCM2) are closely related to the development of various kinds of cancers. We aimed to explore the functions and potential molecular mechanisms of MCM2 gene in cholangiocarcinoma (CCA) cell lines (Huh28 and RBE). First, the cell counting kit-8 (CCK-8), plate clone formation, transwell and invasion assays showed that MCM2 promotes the proliferation, migration and invasion of CCA cells. Flow cytometry assays showed that MCM2 significantly promotes the cell cycle, and inhibits the apoptosis of CCA cells. Further, by analyzing the RNA sequencing data of cholangiocarcinoma, we found that MCM2 gene is significantly negatively correlated with p53 signaling pathway. Quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting (WB) assays confirmed that MCM2 in CCA cells significantly down-regulated the mRNA and protein expression levels of p53 and BAX, and up-regulated the mRNA and protein expression levels of BCL2 and CCND1. Flow cytometry, qRT-PCR and WB assays confirmed that MCM2 promotes CCA through p53 pathway. Finally, we found that MCM2 is up-regulated in CCA tissues compared to the matched non-tumor cholangiocarcinoma tissues, and the high expressions of MCM2 are significantly associated with the poor clinical outcomes of CCA patients. In conclusion, this study revealed that MCM2 promotes the development of CCA by reducing the p53 pathway, and its high expression levels predict poor prognosis in CCA patients. These results provide a theoretical basis for the development of new clinical diagnosis and treatment of cholangiocarcinoma in the future.

Abstract P072: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) foster myeloid-derived suppressor cell-mediated tumor immune evasion in cholangiocarcinoma

Abstract Background and Aims: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is predominantly expressed on immune cells. Although TRAIL biology has garnered considerable interest as a potential anti-cancer strategy, TRAIL agonists have had very limited anti-cancer activity in humans. TRAIL signaling in T cells may also potentially provide an immune checkpoint function as it can inhibit T cell activation and proliferation by interfering with T cell receptor signaling. However, this potential immune checkpoint function of TRAIL has not been examined in cancer biology. Cholangiocarcinoma (CCA), a highly lethal biliary tract cancer, provides a model to examine the potential immune checkpoint function of TRAIL. Methods: Using a syngeneic, orthotopic murine model of CCA (PMID: 29464042), murine CCA cells (SB cells) that express both TRAIL and TRAIL receptor (TR) were implanted into livers of WT and Tr−/− mice. Hence, in this model the host immune cells express TRAIL but not the receptor; therefore, they would be capable of inducing TRAIL-mediated apoptosis in CCA cells but would be resistant to TRAIL-mediated immunosuppression. After 4 weeks of tumor growth, mice were sacrificed, and tumors were characterized using flow cytometry. Results: We observed that Tr−/− mice had a significant reduction in tumor burden compared to WT mice. Moreover, tumor bearing Tr−/− mice had a significant increase in cytotoxic T lymphocytes (CTLs) and enhanced CTL effector function. However, coculture of T cells with SB cells or SB cells deficient in Tr (SB-Tr−/−) did not result in a significant difference in T cell apoptosis or function, implying that TRAIL-TR is not a direct T cell checkpoint. Myeloid derived suppressor cells (MDSCs) were significantly decreased in Tr−/− tumors compared to WT tumors. Furthermore, implantation of SB cells devoid of Trail (SB-Trail−/−) into WT mice resulted in a significant reduction in tumor burden and MDSC infiltration. Coculture of SB cells with MDSCs from Tr−/− mice attenuated MDSC proliferation and immunosuppression compared to WT MDSCs. Moreover, treatment of MDSCs from Tr−/− mice with TRAIL recombinant protein resulted in a reduction in their proliferation and immunosuppressive function compare to MDSCs from WT mice implying that TRAIL-TR fosters MDSC growth and immunosuppressive function. In conclusion, we have demonstrated that Tr−/− mice have a significant reduction in CCA tumor burden and MDSC infiltration. Consequently, Tr−/− mice bearing tumors have enhanced CTL infiltration and function. These data suggest that the TRAIL-TR system mediates tumor immune evasion via MDSCs. Herein, we suggest that TRAIL-TR appears to function as an indirect T cell checkpoint by augmenting the immunosuppressive effects of MDSCs. Citation Format: Emilien Loeuillard, Juan Wang, Jingchun Yang, Haidong Dong, Gregory Gores, Sumera Ilyas. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) foster myeloid-derived suppressor cell-mediated tumor immune evasion in cholangiocarcinoma [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P072.

125I seeds inhibit proliferation and promote apoptosis in cholangiocarcinoma cells by regulating the AGR2-mediated p38 MAPK pathway.

125I seeds can effectively inhibit the growth of a variety of cancer cells. It has been used in the treatment of a variety of cancers, and has achieved certain curative effect. However, to the best of our knowledge, no report has described the effects of 125I seeds on the biological functions of cholangiocarcinoma (CCA) and the mechanisms underlying the effects of the seeds on this cancer. In this study, we demonstrated that 125I seeds could inhibit the proliferation, migration and invasion of CCA cells, as well as promoting apoptosis and blocking the cell cycle in these cells. Moreover, 125I seeds inhibited the growth of CCA xenografts and promoted the apoptosis of CCA cells in vivo. Furthermore, transcriptome sequencing showed that 125I seeds could inhibit the growth of CCA by inhibiting the expression of AGR2 and regulating p38 MAPK pathway. Finally, this finding indicated that 125I seeds can inhibit proliferation and promote apoptosis in CCA cells by inhibiting the expression of AGR2 and DUSP1 and increasing the expression of p-p38 MAPK and p-p53. This study provides a new research direction for studies investigating the mechanisms underlying the effects of 125I seeds on CCA.

Abstract 2738: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediates tumor immune evasion in cholangiocarcinoma

Abstract Background and Aims: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor superfamily, is predominantly expressed on immune cells. Although TRAIL biology has garnered considerable interest as a potential anti-cancer strategy, TR agonists have had very limited anti-cancer activity in humans. TRAIL signaling in T cells may also potentially provide an immune checkpoint function as it can inhibit T cell activation and proliferation by interfering with T cell receptor signaling. However, this potential immune checkpoint function of TRAIL has not been examined in cancer biology. Cholangiocarcinoma (CCA), a malignancy of the bile ducts, provides a model to examine the potential immune checkpoint function of TRAIL. Methods: Using a syngeneic, orthotopic murine model of CCA (PMID: 29464042), murine CCA cells (SB cells) that express both TRAIL and TRAIL receptor (TR) were implanted into livers of WT C57BL/6J and Tr−/− mice. Hence, in this model the host immune cells express TRAIL but not the receptor; they would be capable of inducing TRAIL-mediated apoptosis in CCA cells but would be resistant to potential TRAIL-mediated immunosuppression. After 4 weeks of tumor growth, mice were sacrificed and tumor and immune characterization (via flow cytometry) was conducted. Results: Implantation of SB cells into Tr−/− mice result in a significant reduction in tumor volumes compared to WT mice. Tumor-bearing Tr−/− mice had a significant infiltration of cytotoxic T lymphocytes (CTLs) (CD45+CD3+CD8+CD11a+) and enhanced CTL effector function. Moreover, Tr−/− mice tumors had a significant decrease in granulocytic and monocytic myeloid-derived suppressor cell (MDSCs) as well as tumor-associated macrophages (TAMs) (CD45+F4/80+CD11b+CD206+) compared to WT mice tumors. Conclusion: Using a unique syngeneic orthotopic implantation model of murine CCA, we demonstrate that CCA cells expressing TRAIL in a mouse genetically deficient for TR exhibit reduced tumor volumes enhanced CTL infiltration and function, and reduced MDSC and TAM infiltration into the tumors. These data suggest that TRAIL has a potential immune checkpoint function, and targeting TRAIL signaling with consequent augmentation of CTL function maybe a promising therapeutic approach in human cancers. Citation Format: Emilien Loeuillard, Jingchun Yang, Haidong Dong, Gregory J. Gores, Sumera Ilyas. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediates tumor immune evasion in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2738.