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.

Growth arrest and DNA damage-inducible-beta (Gadd45 beta) is a stress-response protein involved in a number of processes, including cell cycle control, DNA repair, survival and death control, and stress signaling, depending on its interactions. Gadd45 beta expression is dysregulated in numerous types of cancer, functioning as either a tumor promoter or a tumor suppressor. However, the functions of Gadd45 beta in cholangiocarcinoma (CCA), particularly in metastasis, has not been studied. The immunohistochemical analysis of Gadd45 beta expression revealed that 75% of histological specimens from patients with CCA expressed high levels of Gadd45 beta, and that high Gadd45 beta expression was associated with metastasis. The role of Gadd45 beta in CCA was examined using siRNA-mediated gene knockdown in HuCCA-1, a human CCA cell line established from a Thai patient. The effects of Gadd45 beta downregulation upon cell viability and death, invasion, migration, matrix metalloproteinase (MMP) activity and epithelial-mesenchymal transition (EMT) marker expression were investigated. Gadd45 beta knockdown impaired cell viability, which was associated with the induction of apoptosis. In addition, there was a marked reduction in invasion and migration, although MMP activity was unaffected. Impairment of these metastatic properties was accompanied by the decreased expression of EMT markers, including Slug, vimentin, claudin-1 and zona occludens protein 1, whereas E-cadherin expression was increased. The present study suggests that Gadd45 beta is involved in regulating the viability and the metastatic potential of CCA cells, which may be mediated by the modulation of the EMT pathway.

Cholangiocarcinoma (CCA) is a heterogenous group of malignancies in the bile duct, which proliferates aggressively. CCA is highly prevalent in Northeastern Thailand wherein it is associated with liver fluke infection, or Opisthorchis viverrini (OV). Most patients are diagnosed in advanced stages, when the cancer has metastasized or severely progressed, thereby limiting treatment options. Several studies investigate the effect of traditional Thai medicinal plants that may be potential therapeutic options in combating CCA. Galangin is one such herbal flavonoid that has medicinal properties and exhibits anti-tumor properties in various cancers. In this study, we investigate the role of Galangin in inhibiting cell proliferation, invasion, and migration in OV-infected CCA cell lines. We discovered that Galangin reduced cell viability and colony formation by inducing apoptosis in CCA cell lines in a dose-dependent manner. Further, Galangin also effectively inhibited invasion and migration in OV-infected CCA cells by reduction of MMP2 and MMP9 enzymatic activity. Additionally, using proteomics, we identified proteins affected post-treatment with Galangin. Enrichment analysis revealed that several kinase pathways were affected by Galangin, and the signature corroborated with that of small molecule kinase inhibitors. Hence, we identified putative targets of Galangin using an in silico approach which highlighted c-Met as candidate target. Galangin effectively inhibited c-Met phosphorylation and subsequent signaling in in vitro CCA cells. In addition, Galangin was able to inhibit HGF, a mediator of c-Met signaling, by suppressing HGF-stimulated invasion, as well as migration and MMP9 activity. This shows that Galangin can be a useful anti-metastatic therapeutic strategy in a subtype of CCA patients.

Background/Objectives: Cholangiocarcinoma (CCA) is an aggressive malignancy with a poor prognosis, creating an urgent need for novel biomarkers to improve risk stratification. The prognostic significance of the transmembrane glycoprotein CD44 and its isoforms (CD44s, v5, v6) in CCA remains controversial. This preliminary study aimed to investigate whether the combined loss of these isoforms could serve as a distinct prognostic indicator. Methods: We evaluated the expression of CD44s, CD44v5, and CD44v6 via immunohistochemistry on a retrospective cohort of 61 paraffin-embedded CCA patient tissue blocks from Ramathibodi Hospital, Bangkok, Thailand. Expression levels were correlated with clinicopathological parameters. Survival analyses, including Kaplan–Meier and Cox proportional hazards models, were used to determine the prognostic value of individual isoforms and the complete absence of all three. Results: Expression of CD44s, CD44v5, and CD44v6 was found in 52.5%, 47.5%, and 82.0% of tumors, respectively. In univariate and multivariate analyses, the expression of any single isoform was not a significant predictor of overall survival. However, a subgroup of 8 patients (13.1%) was identified whose tumors were negative for all three isoforms, a phenotype we termed “CD44s-v5-v6 Null”. This status was significantly associated with advanced TNM stages (p = 0.022). Patients with these Null tumors also showed a clinically relevant, though not statistically significant, trend towards poorer survival (median 7.0 vs. 12.0 months, p = 0.336). Conclusions: Individual CD44 isoforms did not serve as reliable independent prognostic markers in this cohort. Instead, the complete loss of the CD44 expression program characterizes a potential “CD44s-v5-v6 Null” phenotype associated with advanced-stage disease.

Background: Cancer immune evasion is a multifaceted process that synchronizes pro-tumoral immune infiltration, immunosuppressive inflammation, and inhibitory immune checkpoint expression (IC). Current immunotherapies combat this issue by reinstating immunosurveillance of tumors; however, it benefits a limited patient population. Thus, a more effective immunotherapeutic strategy is warranted to cater to specific patient populations. This investigation introduces a novel immunotherapeutic strategy via inhibition of master regulators of immune evasion (MR-IE). Methods: Samples of the TCGA Pan-Cancer Atlas transcriptomic data were subset and stratified based on IC and estimated immune cell infiltration. Transcriptomic analysis was conducted to unravel pathways associated with the immune evasion process. Transcription factor enrichment and survival analyses were conducted to identify and rank candidate MR-IEs per cancer type. Results: Inhibition of the top-ranking MR-IE candidate of cholangiocarcinoma (CCA), MYC, modulated the gene and protein expression of PD-L1. Moreover, pro-tumoral inflammatory markers, IFNA21 and CX3CL1, were downregulated, and anti-tumoral cytokines, IL-18 and IL-16, were upregulated. Lastly, MYC inhibition potentiated fourth-generation anti-folate receptor alpha (FRα) CAR-T cell therapy against CCA cells. Conclusions: Cumulatively, this study highlights the promise of MR-IE inhibition as a novel potent immunotherapeutic strategy for the treatment of CCA and offers a candidate list of MR-IEs per cancer type for further validation.