CCA Cell Research Articles

Abstract 4325: CX-4945 (Silmitasertib) induces cell death through impairment of lysosomal utilization in KRAS mutant cholangiocarcinoma cell lines

Abstract Purpose: Macropinocytosis is a non-selective endocytosis that uptakes extracellular substances such as nutrients and macromolecules into the cells. In KRAS-driven cancer such as pancreatic ductal adenocarcinoma, the macropinocytosis and following lysosomal utilization are enhanced to overcome metabolic stress. In this study, we investigated the role of CK2 in micropinocytosis and the following metabolic process in the KRAS mutant CCA cell line. Materials and Methods: The BSA uptake indicating micropinocytosis was performed by flow cytometry using the HuCCT1, cholangiocarcinoma cell line harboring KRAS mutation. To validate macropinosome, the Rab7 and LAMP2 were labeled and analyzed via immunocytochemistry and Western blot. The CX-4945, CK2 inhibitor, was used to investigate the role of CK2 in micropinocytosis and following lysosomal metabolism. Results: The HuCCT1, a KRAS mutant CCA cell line, showed micropinocytosis. Although CX-4945, a CK2 inhibitor, induced morphological changes accompanied by accumulation of intracellular vacuoles and cell death rate, the level of micropinocytosis was not changed. The vacuoles accumulated in the cell were identified as late macropinosomes representing Rab7 before fusion with lysosomes. In addition, the CX-4945 suppressed LAMP2 expression following inhibition of the Akt-mTOR signaling pathway which interrupts mature macropinosome and lysosomal metabolic utilization. Conclusion: These results support the utilization of micropinocytosis as an energy source even in CCA cell line harboring KRAS mutation. The inhibition of CK2, the regulator of macropinosome maturation, by CX-4945, alters lysosome dependent metabolism and leads to cell death in KRAS mutant CCA cell lines. Citation Format: Chorong Kim, Da Sol Lee, Minwoo Han, Seonmin Lee, Kyu-pyo Kim, Changhoon Yoo. CX-4945 (Silmitasertib) induces cell death through impairment of lysosomal utilization in KRAS mutant cholangiocarcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4325.

Abstract 3600: Reversal of iNKT cell dysfunction in cholangiocarcinoma

Abstract Invariant natural killer T (iNKT) cells are CD1d-restricted unconventional T cells possessing innate and adaptive immunity with high anti-tumor activities. Yet, the effectiveness of iNKT cell therapy in clinical trials is limited due to iNKT cell dysfunction after exposure to the tumor microenvironment. Aggressive cholangiocarcinoma (CCA) cell lines were used to study iNKT cell reactivity. These CCA cell lines lacked CD1d molecules and responded poorly to iNKT cells. The iNKT cells became dysfunctional after the exposure to CCA by 48 h. In this study, we overcame iNKT cell dysfunction by searching for a small molecule to prevent or reverse the gene sets regulating iNKT cell dysfunction, thus prolonging anti-tumor activity. It was found that P53 and estrogen early response pathway gene sets were enriched in dysfunctional iNKT cells which were reversed by vorinostat, an HDAC inhibitor. Anti-CCA activity of iNKT cells was regained with a subtherapeutic dose of vorinostat as protection. Interestingly, vorinostat could not totally recover the anti-tumor effect of iNKT cells that have already developed into the dysfunctional stage. Significant tumor size reduction was seen in the humanized mouse model after iNKT cell adoptive therapy was given in combination with vorinostat compared to monotherapy with either iNKT cell or vorinostat alone. This study demonstrated that inhibiting the gene sets regulating iNKT cell dysfunction by vorinostat could prevent iNKT cell dysfunction, and prolong its anti-tumor activities. This combination could be a novel treatment option for CD1d-negative CCA patients resistant to standard chemotherapy. Citation Format: Khin Su Su Htwe, Kitipong Soontrapa, Siwanon Jirawatnotai, Sunisa Prasopporn, Pimkanya More-Krong, Somponnat Sampattavanich, Adisak Wongkajornsilp. Reversal of iNKT cell dysfunction in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3600.

Abstract 3160: PufA promotes proliferation, migration and tumorigenesis through NF-kB pathway in cholangiocarcinoma

Abstract Cholangiocarcinoma (CCA) exhibits a poor prognosis despite the recent advances in targeted therapy and immunotherapy. Therefore, exploring the novel mechanism of CCA tumorigenesis and identifying the effective targeted therapy is critical for advanced CCA to improve future treatment. A novel biogenesis factor of the 90S pre-ribosome named PufA, is higher expressed in tumors than normal tissue based on the TCGA databank implying the role of CCA tumorigenesis. Our results showed that PufA expression was significantly associated with larger tumor size, more lymph node metastasis, more tumor relapse, death, and unfavorable survivals in 205 patients with resectable CCA in Chang Gung Memorial Hospital. We demonstrated the inhibited effect of cell survival, migration ability, and the sphere-formation potential by silencing of PufA in CCA cell lines. Tumor growth is also suppressed in PufA knockdown xenograft mice. Mechanistic investigation showed that PufA was expression associated with nuclear factor kappa B p65 (RELA) activity and facilitated activity of the NF-κB transactivation. RELA, a transcription factor, is the principal regulator of the inflammatory response and is crucial for the homeostasis of the immune system and the expression of the immune checkpoint. Furthermore, decreased PufA levels also downregulated PD-L1 expression in CCA. Together, these findings suggested that the PufA-RELA axis could be a potential target for further application of targeted therapy in combination with immunotherapy. Citation Format: Chiao-Ping Chen, Chun-Nan Yeh, Yi-Ru Pan, Yu-Tien Hsiao, Chih-Hong Lo, Chia-Hsien Lin, Chiao-En Wu. PufA promotes proliferation, migration and tumorigenesis through NF-kB pathway in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3160.

Circ_0007534 promotes cholangiocarcinoma stemness and resistance to anoikis through DDX3X-mediated positive feedback regulation of parental gene DDX42

Cholangiocarcinoma (CCA) is a malignancy with an extremely poor prognosis, and much remains unknown about its pathogenesis and treatment modalities. Circular RNA (circRNA) has been proven to play regulatory roles in various tumorigenesis, yet its potential function and mechanism in cholangiocarcinoma require further investigation. This study is the first to identify the aberrant expression and functional role of a novel circRNA, circ_0007534, derived from the DDX42 gene, in cholangiocarcinoma. Compared to the normal control group, the expression of circ_0007534 was significantly elevated in the tissues and cells with CCA and that high expression correlated with lymph node invasion and poor prognosis. Functional experiments indicated that downregulating circ_0007534 markedly inhibited the proliferation, migration, invasion, stemness, and anti-anoikis ability of CCA cells, as well as the tumor growth and liver and lung metastasis in nude mice. Mechanistic studies revealed that DDX42, as the parent gene of circ_0007534, can mutually regulate each other's expression. Predominantly located in the cytoplasm, circ_0007534 can form a complex with the RNA-binding protein DDX3X, which enhances the stability of DDX42 mRNA, thereby upregulating the expression of DDX42. This creates a positive feedback loop among the three, collectively promoting the progression of cholangiocarcinoma. In conclusion, this study sheds light on the pivotal role and molecular mechanism of circ_0007534 in the development of CCA, offering potential new targets for early diagnosis and treatment.

FAXC interacts with ANXA2 and SRC in mitochondria and promotes tumorigenesis in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is one of the most difficult malignancies to treat as the therapeutic options are limited. Although several driver genes have been identified, most remain unknown. In this study, we identified a failed axon connection homolog (FAXC), whose function is unknown in mammals, by analyzing serially passaged CCA xenograft models. Knockdown of FAXC reduced subcutaneous tumorigenicity in mice. FAXC was bound to annexin A2 (ANXA2) and c-SRC, which are tumor-promoting genes. The FAXC/ANXA2/c-SRC complex forms in the mitochondria. FAXC enhances SRC-dependent ANXA2 phosphorylation at tyrosine-24, and the C-terminal amino acid residues (351-375) of FAXC are required for ANXA2 phosphorylation. Transcriptome data from a xenografted CCA cell line revealed that FAXC correlated with epithelial-mesenchymal transition, hypoxia, and KRAS signaling genes. Collectively, these findings advance our understanding of CCA tumorigenesis and provide candidate therapeutic targets.

The importance of preclinical models in cholangiocarcinoma

Cholangiocarcinoma (CCA) is an adenocarcinoma of the hepatobiliary system with a grim prognosis. Incidence is rising globally and surgery is currently the only curative treatment, but is only available for patients who are fit and diagnosed in an early-stage of disease progression. Great importance has been placed on developing preclinical models to help further our understanding of CCA and potential treatments to improve therapeutic outcomes. Preclinical models of varying complexity and cost have been established, ranging from more simplistic in vitro 2D CCA cell lines in culture, to more complex in vivo genetically engineered mouse models. Currently there is no single model that faithfully recaptures the complexities of human CCA and the in vivo tumour microenvironment. Instead a multi-model approach should be used when designing preclinical trials to study CCA and potential therapies.

Trilobolide-6-O-isobutyrate exerts anti-tumor effects on cholangiocarcinoma cells through inhibiting JAK/STAT3 signaling pathway

Trilobolide-6-O-isobutyrate exhibits significant antitumor effects on cholangiocarcinoma (CCA) cells by effectively inhibiting the JAK/STAT3 signaling pathway. This study aims to investigate the mechanisms underlying the antitumor properties of trilobolide-6-O-isobutyrate, and to explore its potential as a therapeutic agent for CCA. This study illustrates that trilobolide-6-O-isobutyrate efficiently suppresses CCA cell proliferation in a dose- and time-dependent manner. Furthermore, trilobolide-6-O-isobutyrate stimulates the production of reactive oxygen species, leading to oxidative stress and initiation of apoptosis via the activation of the mitochondrial pathway. Data from xenograft tumor assays in nude mice confirms that TBB inhibits tumor growth, and that there are no obvious toxic effects or side effects in vivo. Mechanistically, trilobolide-6-O-isobutyrate exerts antitumor effects by inhibiting STAT3 transcriptional activation, reducing PCNA and Bcl-2 expression, and increasing P21 expression. These findings emphasizes the potential of trilobolide-6-O-isobutyrate as a promising therapeutic candidate for the treatment of CCA.

RPL35A promotes the progression of cholangiocarcinoma by mediating HSPA8 ubiquitination

BackgroundCholangiocarcinoma (CCA) is a biliary epithelial malignant tumor with an increasing incidence worldwide. Therefore, further understanding of the molecular mechanisms of CCA progression is required to identify new therapeutic targets.MethodsThe expression of RPL35A in CCA and para-carcinoma tissues was detected by immunohistochemical staining. IP-MS combined with Co-IP identified downstream proteins regulated by RPL35A. Western blot and Co-IP of CHX or MG-132 treated CCA cells were used to verify the regulation of HSPA8 protein by RPL35A. Cell experiments and subcutaneous tumorigenesis experiments in nude mice were performed to evaluate the effects of RPL35A and HSPA8 on the proliferation, apoptosis, cell cycle, migration of CCA cells and tumor growth in vivo.ResultsRPL35A was significantly upregulated in CCA tissues and cells. RPL35A knockdown inhibited the proliferation and migration of HCCC-9810 and HUCCT1 cells, induced apoptosis, and arrested the cell cycle in G1 phase. HSPA8 was a downstream protein of RPL35A and overexpressed in CCA. RPL35A knockdown impaired HSPA8 protein stability and increased HSPA8 protein ubiquitination levels. RPL35A overexpression promoted CCA cell proliferation and migration. HSPA8 knockdown inhibited CCA cell proliferation and migration, and reversed the promoting effect of RPL35A. Furthermore, RPL35A promoted tumor growth in vivo. In contrast, HSPA8 knockdown suppressed tumor growth, while was able to restore the effects of RPL35A overexpression.ConclusionRPL35A was upregulated in CCA tissues and promoted the progression of CCA by mediating HSPA8 ubiquitination.

Role of Wisteria floribunda agglutinin binding glycans in carcinogenesis and metastasis of cholangiocarcinoma.

Aberrant glycosylation is an important factor in facilitating tumor progression and therapeutic resistance. In this study, using Wisteria floribunda agglutinin (WFA), we examined the expression of WFA-binding glycans (WFAG) in cholangiocarcinoma (CCA). The results showed that WFAG was highly detected in precancerous and cancerous lesions of human CCA tissues, although it was rarely detected in normal bile ducts. The positive signal of WFAG in the cancerous lesion accounted for 96.2% (50/52) of the cases. Overexpression of WFAG was significantly associated with lymph node and distant metastasis (P < 0.05). The study using the CCA hamster model showed that WFAG is elevated in preneoplastic and neoplastic bile ducts as early as 1month after being infected with liver fluke and exposed to N-nitrosodimethylamine. Functional analysis was performed to reveal the role of WFAG in CCA. The CCA cell lines KKU-213A and KKU-213B were treated with WFA, followed by migration assay. Our data suggested that WFAG facilitates the migration of CCA cells via the activation of the Akt and ERK signaling pathways. In conclusion, we have demonstrated the association of WFAG with carcinogenesis and metastasis of CCA, suggesting its potential as a target for the treatment of the disease.

GOLPH3 promotes tumor malignancy via inhibition of ferroptosis by upregulating SLC7A11 in cholangiocarcinoma.

Golgi phosphoprotein 3 (GOLPH3) has been reported as an oncogene in various tumors; however, the role and function of GOLPH3 and its relevant molecular mechanism in cholangiocarcinoma (CCA) are unclear. Herein, GOLPH3 expression in CCA tissues was observed to be significantly higher than that in paired adjacent noncancerous tissues. Clinicopathological analysis showed that GOLPH3 expression correlated positively with the tumor-node-metastasis stage. In addition, GOLPH3 expression correlated inversely with the overall survival of patients with CCA. Multivariate analysis showed that GOLPH3 was an independent prognostic factor for patients with CCA. Transcriptome analysis (RNA sequencing) of GOLPH3 knockdown cells showed that the expression levels of nine ferroptosis-related genes were significantly changed, indicating the important biological function of GOLPH3 in ferroptosis in CCA cells. Furthermore, GOLPH3 knockdown could significantly promote Erastin-induced ferroptosis in vitro and suppress tumor growth in vivo. Overexpression of GOLPH3 had the opposite effect on this phenotype. Further studies revealed that GOLPH3 knockdown was significantly associated with a decrease in cysteine content, an accumulation of the lipid peroxidation product malondialdehyde, an increase in reactive oxygen species, and sensitized CCA cells to Erastin-induced ferroptosis. Moreover, changes in GOLPH3 expression were found to be consistent with the expression of light chain subunit solute carrier family 7 member 11 (SLC7A11). Thus, our study suggested that GOLPH3 functions as an oncoprotein in CCA and may suppress ferroptosis by facilitating SLC7A11 expression, suggesting that GOLPH3 could serve as a therapeutic target for CCA treatment.

PIN1 promotes the metastasis of cholangiocarcinoma cells by RACK1-mediated phosphorylation of ANXA2.

Cholangiocarcinoma (CCA), a primary hepatobiliary malignancy, is characterized by a poor prognosis and a lack of effective treatments. Therefore, the need to explore novel therapeutic approaches is urgent. While the role of Peptidylprolyl Cis/Trans Isomerase, NIMA-Interacting 1 (PIN1) has been extensively studied in various tumor types, its involvement in CCA remains poorly understood. In this study, we employed tissue microarray (TMA), reverse transcription-polymerase chain reaction (RT-PCR), and The Cancer Genome Atlas (TCGA) database to assess the expression of PIN1. Through in vitro and in vivo functional experiments, we investigated the impact of PIN1 on the adhesion and metastasis of CCA. Additionally, we explored downstream molecular pathways using RNA-seq, western blotting, co-immunoprecipitation, immunofluorescence, and mass spectrometry techniques. Our findings revealed a negative correlation between PIN1 overexpression and prognosis in CCA tissues. Furthermore, high PIN1 expression promoted CCA cell proliferation and migration. Mechanistically, PIN1 functioned as an oncogene by regulating ANXA2 phosphorylation, thereby promoting CCA adhesion. Notably, the interaction between PIN1 and ANXA2 was facilitated by RACK1. Importantly, pharmacological inhibition of PIN1 using the FDA-approved drug all-trans retinoic acid (ATRA) effectively suppressed the metastatic potential of CCA cells in a nude mouse lung metastasis model. Overall, our study emphasizes the critical role of the PIN1/RACK1/ANXA2 complex in CCA growth and functionality, highlighting the potential of targeting PIN1 as a promising therapeutic strategy for CCA.

Cholangiocarcinoma Malignant Traits Are Promoted by Schwann Cells through TGFβ Signaling in a Model of Perineural Invasion.

The term cholangiocarcinoma (CCA) defines a class of epithelial malignancies originating from bile ducts. Although it has been demonstrated that CCA patients with perineural invasion (PNI) have a worse prognosis, the biological features of this phenomenon are yet unclear. Our data show that in human intrahepatic CCA specimens with documented PNI, nerve-infiltrating CCA cells display positivity of the epithelial marker cytokeratin 7, lower with respect to the rest of the tumor mass. In an in vitro 3D model, CCA cells move towards a peripheral nerve explant allowing contact with Schwann cells (SCs) emerging from the nerve. Here, we show that SCs produce soluble factors that favor the migration, invasion, survival and proliferation of CCA cells in vitro. This effect is accompanied by a cadherin switch, suggestive of an epithelial-mesenchymal transition. The influence of SCs in promoting the ability of CCA cells to migrate and invade the extracellular matrix is hampered by a specific TGFβ receptor 1 (TGFBR1) antagonist. Differential proteomic data indicate that the exposure of CCA cells to SC secreted factors induces the upregulation of key oncogenes and the concomitant downregulation of some tumor suppressors. Taken together, these data concur in identifying SCs as possible promoters of a more aggressive CCA phenotype, ascribing a central role to TGFβ signaling in regulating this process.

Molecular mechanism of Spatholobi Caulis treatment for cholangiocarcinoma based on network pharmacology, molecular docking, and molecular dynamics simulation.

Cholangiocarcinoma (CCA) is a type of malignant tumor originating from the intrahepatic, periportal, or distal biliary system. The treatment means for CCA is limited, and its prognosis is poor. Spatholobi Caulis (SC) is reported to have effects on anti-inflammatory and anti-tumor, but its role in CCA is unclear. First, the potential molecular mechanism of SC for CCA treatment was explored based on network pharmacology, and the core targets were verified by molecular docking and molecular dynamics simulation. Then, we explored the inhibitory effect of SC on the malignant biological behavior of CCA in vitro and in vivo and also explored the related signaling pathways. The effect of combination therapy of SC and cisplatin (DDP) in CCA was also explored. Finally, we conducted a network pharmacological study and simple experimental verification on luteolin, one of the main components of SC. Network pharmacology analysis showed that the core targets of SC on CCA were AKT1, CASP3, MYC, TP53, and VEGFA. Molecular docking and molecular dynamics simulation indicated a good combination between the core target protein and the corresponding active ingredients. In vitro, SC inhibited proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of CCA cells. In vivo experiments, the results were consistent with in vitro experiments, and there was no significant hepatorenal toxicity of SC at our dosage. Based on KEGG enrichment analysis, we found PI3K/AKT signaling pathway might be the main signaling pathway of SC action on CCA by using AKT agonist SC79. To explore whether SC was related to the chemotherapy sensitivity of CCA, we found that SC combined with DDP could more effectively inhibit the progression of cholangiocarcinoma. Finally, we found luteolin may inhibit the proliferation and invasion of CCA cells. Our study demonstrates for the first time that SC inhibits the progression of CCA by suppressing EMT through the PI3K-AKT signaling pathway, and SC could enhance the effectiveness of cisplatin therapy for CCA.

Therapeutic Implications of Ceritinib in Cholangiocarcinoma beyond ALK Expression and Mutation.

Cholangiocarcinoma (CCA) is a difficult-to-treat cancer, with limited therapeutic options and surgery being the only curative treatment. Standard chemotherapy involves gemcitabine-based therapies combined with cisplatin, oxaliplatin, capecitabine, or 5-FU with a dismal prognosis for most patients. Receptor tyrosine kinases (RTKs) are aberrantly expressed in CCAs encompassing potential therapeutic opportunity. Hence, 112 RTK inhibitors were screened in KKU-M213 cells, and ceritinib, an approved targeted therapy for ALK-fusion gene driven cancers, was the most potent candidate. Ceritinib's cytotoxicity in CCA was assessed using MTT and clonogenic assays, along with immunofluorescence, western blot, and qRT-PCR techniques to analyze gene expression and signaling changes. Furthermore, the drug interaction relationship between ceritinib and cisplatin was determined using a ZIP synergy score. Additionally, spheroid and xenograft models were employed to investigate the efficacy of ceritinib in vivo. Our study revealed that ceritinib effectively killed CCA cells at clinically relevant plasma concentrations, irrespective of ALK expression or mutation status. Ceritinib modulated multiple signaling pathways leading to the inhibition of the PI3K/Akt/mTOR pathway and activated both apoptosis and autophagy. Additionally, ceritinib and cisplatin synergistically reduced CCA cell viability. Our data show ceritinib as an effective treatment of CCA, which could be potentially explored in the other cancer types without ALK mutations.

Exosomal circ_0037104 derived from Hu-MSCs inhibits cholangiocarcinoma progression by sponging miR-620 and targeting AFAP1.

Exosomes are membrane-enclosed nanovesicles that shuttle active cargoes, such as circular RNAs (circRNAs) and microRNAs (miRNAs), between different cells. Human umbilical cord-derived mesenchymal stem cells (Hu-MSCs) can migrate to tumor sites and exert complex functions throughout tumor progression. In this study, we successfully isolated Hu-MSCs from human umbilical cords based on their surface marker expression. Hu-MSC-derived exosomes significantly reduced the invasion, migration, and proliferation of cholangiocarcinoma (CCA) cells. Furthermore, circ_0037104 was downregulated in CCAand inhibited the proliferation and metastasis of CCAcells. Then, we investigated the effect of Hu-MSC-derived exosomal circ_0037104 on CCA. Circ_0037104 mainly regulates miR-620 and enhances APAF1 expression, inhibiting CCA cell proliferation and metastasis. Overall, Hu-MSC exosomal circ_0037104 contributes to the progression and stemness of CCAcells via miR-620/APAF1. In conclusion, Hu-MSC-derived exosomal circ_0037104 sponges miR-620 directly and negatively targets APAF1 to suppress CCA.

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.

Aurora B facilitates cholangiocarcinoma progression by stabilizing c-Myc.

Cholangiocarcinoma (CCA), a malignancy that arises from biliary epithelial cells, has a dismal prognosis, and few targeted therapies are available. Aurora B, a key mitotic regulator, has been reported to be involved in the progression of various tumors, yet its role in CCA is still unclarified. Human CCA tissues and murine spontaneous CCA models were used to assess Aurora B expression in CCA. A loss-of-function model was constructed in CCA cells to determine the role of Aurora B in CCA progression. Subcutaneous and liver orthotopic xenograft models were used to assess the therapeutic potential of Aurora B inhibitors in CCA. In murine spontaneous CCA models, Aurora B was significantly upregulated. Elevated Aurora B expression was also observed in 62.3% of human specimens in our validation cohort (143 CCA specimens), and high Aurora B expression was positively correlated with pathological parameters of tumors and poor survival. Knockdown of Aurora B by siRNA and heteroduplex oligonucleotide (HDO) or an Aurora B kinase inhibitor (AZD1152) significantly suppressed CCA progression via G2/M arrest induction. An interaction between Aurora B and c-Myc was found in CCA cells. Targeting Aurora B significantly reduced this interaction and accelerated the proteasomal degradation of c-Myc, suggesting that Aurora B promoted the malignant properties of CCA by stabilizing c-Myc. Furthermore, sequential application of AZD1152 or Aurora B HDO drastically improved the efficacy of gemcitabine in CCA. Aurora B plays an essential role in CCA progression by modulating c-Myc stability and represents a new target for treatment and chemosensitization in CCA.

Expanding immunotherapy options in cholangiocarcinoma: Role of CD27 agonist in combination with PD-L1 and MEK inhibition on antitumor effect and CD8+ T cells.

518 Background: Despite addition of PD-L1 therapy to unresectable cholangiocarcinoma (CCA) standard of care, there remains an unmet need for novel therapies. Our recent phase II trial of dual PD-L1 inhibition (PD-L1i) and MEK inhibition (MEKi) significantly improved progression-free survival (PFS). While MEKi enhances tumor antigen presentation and T cell tumor infiltration, it also impairs T cell priming and activation with notable CD27 upregulation. Our work reveals that CD27 agonists (CD27Ag) foster T cell stem-like and resident memory phenotypes, negating inhibitory effect of MEKi on CD8+ T cells. We hypothesized that addition of a CD27Ag to PD-L1i/MEKi combination therapy would rescue T cell activation and improve antitumor efficacy. Methods: Immune cells from pmel-1 mice were isolated, activated with gp100, and cultured in vitro with 100 IU/mL IL-2 and single, dual, or triple therapy with CD27Ag, cobimetinib (MEKi) and/or αPD-L1. Day 3 flow cytometry characterized CD8+ T cell phenotype and cytokine expression. Additionally, syngeneic murine CCA cell line URCCA4.3 (KrasG21D, tp53-/-) was injected into C57BL/6 mice, subcutaneously or intrahepatic, followed by ~3 weeks of treatment with mono, dual, or triple therapy. For CD8 depletion studies, CD8-depleting antibody was given on the day prior to and following surgery and every 3-4 days afterwards. Tumor and tumor-draining lymph nodes were harvested at study endpoint. Lymphocytes were characterized by flow cytometry. Results: MEKi, alone or with αPD-L1, reduced frequency of activated T cells in vitro (dose-dependent effect on CD69+). CD27Ag combined with MEKi maintained prominent T cell activation. Triple therapy PD-L1i/MEKi/CD27Ag significantly reduced tumor growth rate in mice versus mono or dual therapies (p&lt;0.05). Tumor-infiltrating lymphocytes comprised a stem-like (Tcf-1+), effector memory (CD44+CD62L-), and tissue resident memory (CD103+CD69+) phenotype in triple therapy versus single or dual therapy (p&lt;0.01, p&lt;0.0001, p&lt;0.0001 respectively). All groups had similar numbers of CD8+ stem-like cells (Tcf-1+Tim-3-) in draining lymph nodes, but these cells were only present in tumor from the triple therapy arm (p&lt;0.05). Cell depletion studies showed that CD8+ T cells were required for efficacy of triple therapy in vivo. Finally, an aggressive orthotopic liver tumor model showed improved median survival in triple therapy mice versus control (37.5 days versus 29.5 days, p&lt;0.01). Conclusions: CD27 agonism prevents the impairment of T cell activation that was mediated by MEKi. Moreover, this CD27Ag/MEKi/PDL1i triple therapy bolstered trafficking of T cells with stem-like, effector, or resident memory properties, in turn enhancing the antitumor response. This therapy is being evaluated in metastatic CCA patients in an ongoing Phase II clinical trial.

Degradation of AZGP1 suppresses apoptosis and facilitates cholangiocarcinoma tumorigenesis via TRIM25.

Alpha-2-Glycoprotein 1, Zinc-binding (AZGP1, ZAG) is a secreted protein that is synthesized by adipocytes and epithelial cells; it is downregulated in several malignancies such as breast, prostate, liver and lung cancers. However, its function remains unclear in cholangiocarcinoma (CCA). Here, we evaluated the impact AZGP1 in CCA using Gene Expression Omnibus (GEO) and GEPIA. In addition, we analysed AZGP1 expression using quantitative reverse transcription PCR and western blotting. Expression of AZGP1 was nearly deficient in CCA patients and cell lines and was associated with poor prognosis. AZGP1 overexpression upregulated apoptosis markers. Co-immunoprecipitation experiments showed that AZGP1 interacts with tripartite motif-containing protein 25 (TRIM25), and tissue microarray and bioinformatic analysis showed that AZGP1 is negatively correlated with TRIM25 expression in CCA. Thereafter, TRIM25 knockdown led to AZGP1 upregulation and induced cancer cell apoptosis. TRIM25 targets AZGP1 for degradation by catalysing its ubiquitination. AZGP1 overexpression significantly suppressed tumour growth in a xenograft mouse model. This study findings suggest that AZGP1 is a potential therapeutic target or a diagnostic biomarker for treating patients with CCA.

Metformin Increases the Response of Cholangiocarcinoma Cells to Gemcitabine by Suppressing Pyruvate Kinase M2 to Activate Mitochondrial Apoptosis.

Cholangiocarcinoma (CCA) is a malignant tumor with a high mortality rate. Resistance to chemotherapy remains a major challenge related to cancer treatment, and increasing the sensitivity of cancer cells to therapeutic drugs is a major focus of cancer treatment. We purposed to explore the role of Metformin in CCA involved in chemotherapeutic sensitivity and Pyruvate kinase M2 (PKM2) through regulating mitochondrial apoptosis in the present study. CCA cell lines of HCC9810 and RBE were treated with Metformin companied with antagonists or agonists of PKM2, cells sensitivity to Gemcitabine, cell migration and invasion along with apoptosis, which is mediated by JC-1 and LDH were assayed. Our results indicated that Metformin and Gemcitabine exhibit synergistic effect on inhibition of cholangiocarcinoma cell viability, cell migration and invasion as well as promotion apoptosis of cholangiocarcinoma cells. In vivo, Metformin combined with Gemcitabine has cooperation in inhibiting the growth of cholangiocarcinoma cell-derived tumors. Moreover, Metformin and Gemcitabine inhibited expression of PKM2 and PDHB in HCC9810 and RBE. Our study suggested that Metformin may increase the response of cholangiocarcinoma cells to Gemcitabine by suppressing PKM2 to activate mitochondrial apoptosis.