Intrahepatic Cholangiocarcinoma Cell Lines Research Articles

Proteomics analysis identified TPI1 as a novel biomarker for predicting recurrence of intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is the second most common tumor in primary liver cancer, but the prognostic factors associated with long-term outcomes after surgical resection remain poorly defined. This study aimed to develop a novel prognostic classifier for patients with ICC after surgery. Using a proteomics approach, we screened tumor markers that up-regulated in ICC tissues, and narrowed down by bioinformatics analysis, western blot and immunohistochemistry. Prognostic markers were identified using Cox regression analyses in primary training cohort and the predictive models for time to recurrence (TTR) were established. The predictive accuracy of predictive model was validated in external validation cohort and prospective validation cohort. MTT assay, clonal formation assay and trans-well assays were used to verify the effect on the proliferation and migration in ICC cell line. Triosephosphate isomerise (TPI1) was significantly up-regulated in ICC tissues and Kaplan-Meier analysis reveals that higher TPI1 expression was strongly correlated with higher recurrence rate of ICC patients. In the primary training cohort, mean TTR was significantly longer (p < 0.0001) than in the low-risk group (26.9months for TTR, 95% CI 22.4-31.5) than in the high-risk group (14.5months for TTR, 95% CI 10.6-18.4). Similar results were observed in two validation cohorts. In addition, a nomogram to predict recurrence was developed. Moreover, Knockdown of TPI1 by shRNA inhibited ICC cell growth, colony information, migration, invasion in vitro. Current prognostic models were accurate in predicting recurrence for ICC patients after surgical resection.

LncRNA MNX1-AS1 promotes progression of intrahepatic cholangiocarcinoma through the MNX1/Hippo axis

Long non-coding RNAs (lncRNAs) have extremely complex roles in the progression of intrahepatic cholangiocarcinoma (ICC) and remain to be elucidated. By cytological and animal model experiments, this study demonstrated that the expression of lncRNA MNX1-AS1 was remarkably elevated in ICC cell lines and tissues, and was highly and positively correlated with motor neuron and pancreas homeobox protein 1 (MNX1) expression. MNX1-AS1 significantly facilitated the proliferation, migration, invasion, and angiogenesis in ICC cells in vitro, and remarkably promoted tumor growth and metastasis in vivo. Further study revealed that MNX1-AS1 promoted the expression of MNX1 via recruiting transcription factors c-Myc and myc-associated zinc finger protein (MAZ). Furthermore, MNX1 upregulated the expression of Ajuba protein via binding to its promoter region, and subsequently, Ajuba protein suppressed the Hippo signaling pathway. Taken together, our results uncovered that MNX1-AS1 can facilitate ICC progression via MNX1-AS1/c-Myc and MAZ/MNX1/Ajuba/Hippo pathway, suggesting that MNX1-AS1 may be able to serve as a potential target for ICC treatment.

NRP1 is a Prognostic Factor and Promotes the Growth and Migration of Cells in Intrahepatic Cholangiocarcinoma.

BackgroundNeuropilin-1 (NRP-1) participates in cancer cell proliferation and metastasis as a multifunctional co-receptor by interacting with multiple signaling pathways. However, few studies have addressed the precise function and prognosis analysis of NRP1 in intrahepatic cholangiocarcinoma (ICC). We aimed to study the correlations between NRP1 and clinicopathological characteristics and NRP1 effect on ICC cell line functions.MethodsNRP1 mRNA and its protein levels in human ICC tissues and cell lines were detected by IHC, qRT-PCR, and WB method. Transwell, wound healing, and CCK-8 assays were performed to verify the effects of NRP1 knockdown and overexpression on cell migration and proliferation capability.ResultsNRP1 proteins and mRNA levels increased in ICC tissues compared to those in paired adjacent non-tumor tissues. High NRP1 expression of ICC tissues was related to poor prognosis. NRP1 expression level was expected to be an independent prognosticator for overall survival and cumulative tumor recurrence, and was closely related to tumor number (P=0.047). Knockdown of NRP1 inhibited cell proliferation and migration capability of RBE cells in vitro, and NRP1 overexpression in 9810 cells accelerated proliferation and migration. Additionally, NRP1 may promote cell proliferation and migration in ICC via the FAK/PI3-K/AKT pathway.ConclusionAs an oncogene, NRP1 may function as a candidate target and prognostic biomarker of value for ICC therapy.

Long non-coding RNA HAGLROS regulates lipid metabolism reprogramming in intrahepatic cholangiocarcinoma via the mTOR signaling pathway

Alternation of long non-coding RNA (lncRNA) is implicated in intrahepatic cholangiocarcinoma (ICC) development. HAGLROS is a lncRNA with a length of 699 bp, which is involved in the progression of various cancers. But the mechanism of HAGLROS in ICC remains unknown. In this study, the sh-HAGLROS-1 or sh-HAGLROS-2 was transfected into QBC939 cells, and overexpressing HAGLROS vector was transfected into KMCH cells. HAGLROS expression in ICC tissues and cell lines was detected, and its association with ICC prognosis was further analyzed. Lipid accumulation and lipid-related indicators (TG, LDL-C, TC and HDLC) in QBC939 and KMCH cells were measured. ICC cell viability, invasion and migration were measured. Western blot analysis was used to detect levels of the mTOR axis-related proteins and autophagy-related proteins (LC3I, LC3II, Beclin and P62). The levels of serum lipids and SREBP1 positive expression in transplanted tumors of nude mice were detected. HAGLROS was highly expressed in ICC and negatively correlated with prognosis. QBC939 cells with knocking down HAGLROS exhibited reduced lipid-related protein levels, blocked ICC cellular processes, inactivated mTOR axis, and increased autophagy. QBC939 cells with overexpressing HAGLROS showed opposite trends. The lipid-related protein levels in serum of nude mice and SREBP1 positive expression in transplanted tumors were diminished. Taken together, sh-HAGLROS inactivated the mTOR axis and promoted autophagy, thereby improving lipid metabolism reprogramming in ICC. This study may offer novel ICC treatments.

Integrative Analysis of Multi-omics Data Reveals AP-1 Is a Key Regulator in Intrahepatic Cholangiocarcinoma

Intrahepatic cholangiocarcinoma (ICC) is a malignant tumor with poor prognosis. Currently, its mechanism of pathogenesis remains elusive. In this study, we employed integrative -omics approaches (RNA-seq, ChIP-seq, and MNase-seq) to interrogate the epigenome in ICC. The results of ChIP-seq, differential gene expression, and MNase-seq analyses suggested that ICC cell lines exhibit less histone modification of cancer-related genes compared with a normal cell line. The region of different histone modification patterns is enrichment in sites of AP-1 motif. Subsequent analysis showed that ICC had different nucleosome occupancy in differentially expressed genes compared to a normal cell line. Furthermore, we found that AP-1 plays a key role in ICC and regulates ICC-related genes through its AP-1 binding site. This study is the first report showing the global features of histone modification, transcript, and nucleosome profiles in ICC; we also show that the transcription factor AP-1 might be a key target gene in ICC. Funding Statement: This work was supported by granted from the National Natural Science Foundation of China (No. 81071990, No. 81641110); Science and Technology Program of Guangzhou, China (No. 201707010305); Youth Science Foundation of Guangdong Second Provincial General Hospital (No. YQ2016-001). Declaration of Interests: The authors declare that they have no conflicts of interest.

Long non‑coding RNA ZEB1‑AS1 predicts a poor prognosis and promotes cancer progression through the miR‑200a/ZEB1 signaling pathway in intrahepatic cholangiocarcinoma.

Emerging evidence suggests that long non-coding RNAs (lncRNAs) play pivotal roles in cancer progression, including in intrahepatic cholangiocarcinoma (IHCC). The overexpression of lncRNA ZEB1 antisense 1 (ZEB1-AS1) has been discovered in several types of cancer; however, the clinical significance and functional role of ZEB1-AS1 in IHCC have not yet been determined. In the present study, ZEB1-AS1 was found to be upregulated in IHCC cell lines and tissues. A high ZEB1-AS1 expression was associated with clinical progression and a poor survival of patients with IHCC, and was identified as an independent risk factor for a poor prognosis. In addition, ZEB1-AS1 promoted the proliferation and metastasis of IHCC cells both in vitro and in vivo. ZEB1-AS1 was demonstrated to increase the expression of ZEB1 by sponging miR-200a and to thereby accelerate epithelial-mesenchymal transition (EMT). On the whole, the findings of the present study demonstrate that ZEB1-AS1 promotes proliferation and metastasis in IHCC, and induces EMT through the miR-200a/ZEB1 signaling pathway. ZEB1-AS1 may thus be a promising prognostic biomarker and essential therapeutic target for IHCC.

TGR5 promotes cholangiocarcinoma by interacting with mortalin

Takeda-G-protein-receptor-5 (TGR5) is a G-protein-coupled receptor (GPCR) activated by bile acids, and mortalin is a multipotent chaperone of the HSP70 family. In the present study, TGR5 was detected by immunohistochemistry (IHC) in extrahepatic cholangiocarcinoma (ECC) specimens, and TGR5 expression in ECC tissues and adjacent tissues was compared. In vitro TGR5 was overexpressed and knocked down in human intrahepatic cholangiocarcinoma (ICC) cell line RBE and human extrahepatic cholangiocarcinoma (ECC) cell line QBC-939 to observe its effects on the biological behavior of cholangiocarcinoma (CC) cells, including proliferation, apoptosis and migration. In vivo xenograft model was constructed to explore the role of TGR5 in CC growth. Proteins that interacted with TGR5 were screened using an immunoprecipitation spectrometry approach, and the identified protein was down-regulated to investigate its contribution to CC growth. The present study demonstrated that TGR5 is highly expressed in CC tissues, and strong TGR5 expression may indicate high malignancy in CC. Furthermore, TGR5 promotes CC cell proliferation, migration, and apoptosis resistance. TGR5 boosts CC growth in vivo. In addition, TGR5 combines with mortalin and regulates mortalin expression in the CC cell line. Mortalin participates in the TGR5-induced increase in CC cell proliferation. In conclusion, TGR5 is of clinical significance based on its implications for the degree of malignancy in patients with CC. Mortalin may be a downstream component regulated by TGR5, and TGR5 promotes cholangiocarcinoma at least partially by interacting with mortalin and upregulating its expression. Both TGR5 and mortalin are positive regulators, and may serve as potential therapeutic targets for CC.

Calcium-sensing receptor promotes tumor proliferation and migration in human intrahepatic cholangiocarcinoma by targeting ERK signaling pathway

The Calcium-sensing receptor (CaSR) is functionally expressed in the biliary epithelial cells and it has been verified to possess various regulatory functions in several different forms of human cancers. But its pathological role in human intrahepatic cholangiocarcinoma (ICC) development remains obscure. Here, we confirmed that CaSR expression was up-regulated in ICC tumor specimens and cell lines, which was positively correlated with number of tumors, lymph node metastasis and poor prognosis of ICC patients. CaSR activation induced by CaCl2 or Calindol (a selective CaSR agonist) markedly facilitated cell proliferation and migration in ICC cells, while knockdown of CaSR or NPS2143 treatment (a CaSR antagonist) dramatically suppressed the above effects. We also demonstrated that alteration of CaSR activity mediated tumorigenesis and growth of ICC in vivo. Mechanistically, CaSR activation could promote cell cycle progression and induce an upregulation of MMP-2 and MMP-9 expression partly via the simulation of ERK1/2 signaling pathway. And further inhibition of ERK pathway significantly suppressed ICC cell viability and migration capacity. Together, our findings shed novel light on the role of CaSR as an oncogene in ICC progression and indicated that modulation of CaSR might serve as a preventive or therapeutic strategy for ICC.

Therapeutic Targeting of CDK7 Suppresses Tumor Progression in Intrahepatic Cholangiocarcinoma

Intrahepatic cholangiocarcinoma (ICC) is a lethal malignancy with high mortality and lack of effective therapeutic targets. Here, we found that expression of cyclin-dependent kinase 7 (CDK7) was significantly associated with higher tumor grade and worse prognosis in 96 ICC specimens. Depletion of CDK7 significantly inhibited cell growth, induced a G2/M cell cycle arrest, and reduced the migratory and invasive potential in ICC cells. Subsequent experiments demonstrated that ICC cells were highly sensitive to the CDK7 inhibitor THZ1. A low concentration of THZ1 markedly inhibited cell growth, cell cycle, migration, and invasion in ICC cell lines. RNA-sequencing (RNA-seq) analysis revealed that THZ1 treatment decreased the levels of massive oncogene transcripts, particularly those associated with cell cycle and cell migration. Quantitative reverse transcriptase PCR (qRT-PCR) analysis confirmed that transcription of oncogenes involved in cell cycle regulation (AURKA, AURKB, CDC25B, CDK1, CCNA2, and MKI67) and the c-Met pathway (c-Met, AKT1, PTK2, CRK, PDPK1, and ARF6) was selectively repressed by THZ1. In addition, THZ1 exhibited significant anti-tumor activity in a patient-derived xenograft (PDX) model of ICC, without causing detectable side effects.

MicroRNA-129-2-3p directly targets Wip1 to suppress the proliferation and invasion of intrahepatic cholangiocarcinoma.

Accumulated studies showed that numerous microRNAs (miRNAs) were aberrantly expressed in human intrahepatic cholangiocarcinoma (ICC) and contributed to the tumorigenic processes. However, whether miR-129-2-3p is implicated in the ICC initiation and progression is still limited. Here, the results revealed that miR-129-2-3p expression was notably decreased in ICC tissues and cell lines, and that a low miR-129-2-3p expression was obviously associated with distant metastasis and clinical stage. Exogenous miR-129-2-3p expression evidently repressed the proliferative and invasive abilities of ICC cells. Mechanistic studies indicated that Wild-type p53-induced phosphatase 1 (Wip1) was a direct target gene for miR-129-2-3p in ICC cells. Furthermore, silencing Wip1 expression mimicked the suppressive effects of miR-129-2-3p upregulation on ICC cells. Interestingly, reintroduction of Wip1 expression partially abolished the miR-129-2-3p -reduced cell proliferation and invasion in ICC. Moreover, ectopic miR-129-2-3p expression hindered the ICC tumor growth in vivo. To the best of our knowledge, it is the first time to reveal that miR-129-2-3p plays a crucial role in tumor suppression in ICC pathogenesis through directly targeting Wip1. These results will aid in elucidating the roles of miR-129-2-3p in ICC, and suggest that this miRNA may provide a potential target for the treatment of ICC

Circulating osteopontin per tumor volume as a prognostic biomarker for resectable intrahepatic cholangiocarcinoma.

The role of osteopontin (OPN) in intrahepatic cholangiocarcinoma (ICC) remains controversial. This study aimed to explore the prognostic value of OPN in patients with ICC undergoing curative resection. Patients undergoing curative resection from 2005 to 2016 were identified for inclusion in this retrospective study. The expression level of OPN in tumors was measured in each of the 228 patients by immunohistochemistry. Circulating OPN in serum was tested in 124 patients by ELISA. Tumor volume was calculated according to preoperative imaging or operation record. Proliferation assay, wound healing assay, and invasion assay were performed to investigate the biological function. Low expression of OPN in tissue was associated with lymph node metastasis (P=0.009) and shorter overall survival (OS) (P=0.001). A low level of circulating OPN/volume was associated with multiple tumors (P<0.001), vascular invasion (P=0.027), visceral peritoneal perforation (P=0.001), and lymph node metastasis (P=0.002). It was also able to predict the invasive behavior, lymph node metastasis, and early recurrence with the area under the receiver operating curve (AUC) of being 0.719, 0.708 and 0.622 respectively. Patients with a low level of circulating OPN/volume had shorter OS (P=0.028) and disease-free survival (DFS) (P=0.004) and could benefit from adjuvant chemotherapy (P=0.011). Compared with negative controlled cells, ICC cell lines, which expressed more OPN, showed a decelerated proliferation rate, the weaker ability of migration and invasion, while the opposite was true for the cells expressed less OPN. MMP1, MMP10, and CXCR4 were negatively regulated by OPN. A low level of circulating OPN/volume could indicate aggressive characteristics, along with poor prognosis and efficacy of adjuvant chemotherapy in ICC patients. Over expression of OPN may inhibit phenotypes facilitating ICC metastasis by negatively regulating MMP1, MMP10, and CXCR4.

Notch1 Drives the Formation and Proliferation of Intrahepatic Cholangiocarcinoma.

The molecular mechanisms underlying the development of intrahepatic cholangiocarcinoma (ICC) are not clear yet. In this study, we investigated the involvement of Notch1 in the development of ICC. The cDNA microarray analysis showed that Notch1 expression was higher in ICC tissues than in normal biliary epithelial cells. Stable transfection of Notch1 receptor intracellular domain (NICD1) by hydrodynamic tail vein injection induced ICC formation in mice. Western blotting confirmed that Notch1 signaling was activated in human ICC cell lines and mouse ICC tissues. Silencing Notch1 with specific short interfering RNA (siRNA) inhibited the proliferation of ICC cells. Flow cytometry and Western blotting indicated that apoptosis was induced in Notch1-silenced ICC cells compared with controls. Additionally, Notch1 silencing was associated with the inhibition of hairy and enhancer of split-1 (Hes1) and activation of the phosphatase and tensin homolog (PTEN)/p53 pathway. Taken together, these data suggest that Notch1 drives ICC formation and proliferation; downregulation of Notch1 induces apoptosis in ICC cells; Notch1 signaling may serve as a novel therapeutic target for the treatment of ICC.

70P - New therapy for intrahepatic cholangiocarcinoma targeted to cancer associated fibroblasts

BackgroundThe microenvironment such as cancer associated fibroblasts (CAFs) was reported to be involved in progression of intrahepatic cholangiocarcinoma (ICC). Nintedanib (BIBF1120) is a tyrosine kinase inhibitor of PDGFR, FGFR and VEGFR which is approved for idiopathic pulmonary fibrosis. And nintedanib was reported to suppress liver fibrosis in mouse through the suppression of hepatic stellate cells activation (Ozturk Akcora B, et al. Sci Rep. 2017 Mar 14;7:44545). Therefore, we hypothesized that nintedanib can inhibit the activity of CAFs of ICC. The purpose of this study is to clarify the effect of CAFs on ICC and develop a new therapy for ICC targeted to CAFs. MethodsCAFs were established from surgically resected ICC tissues. The effect of nintedanib on proliferation and expression of alpha smooth muscle actin (αSMA) of CAFs was examined. The effect of CAF-CM (conditioned medium of CAFs) and nintedanib-CAF-CM (CM of CAFs treated with nintedanib) on proliferation and invasion of ICC cell lines (HuCCT1, RBE) was examined. The humoral factors of CAF-CM and nintedanib-CAF-CM were analyzed using cytokine array and ELISA. The effect of nintedanib and gemsitabine (GEM) treatment on xenograft model co-implanted with HuCCT1 and CAFs was examined in vivo. ResultsNintedanib(1µM) suppressed the proliferation of CAFs and expression of αSMA in CAFs. CAF-CM significantly promoted the proliferation and invasion of ICC cell lines. But in nintedanib-CAF-CM, all of those cancer promoting effects were cancelled. In cytokine array and ELISA of CAF-CMs, expression of IL-6, IL-8, VEGF, VCAM1, and Osteopontin were suppressed in nintedanib-CAF-CM compared with CAF-CM. HuCCT1 + CAFs xenograft was increased more than HuCCT1 alone in vivo. Combination treatment with nintedanib (30mg/kg) and GEM (50mg/kg) inhibited HuCCT1 + CAFs xenograft growth than nintedanib or GEM alone in vivo. The xenograft treated with nintedanib and GEM showed reduction of both αSMA-positive staining in stroma and proportion of Ki-67 positive ICC cells. ConclusionsNintedanib suppressed CAFs activity and the production of cancer-promoting cytokines produced by CAFs. Combination therapy with nintedanib and GEM may be a new promising therapy to overcome refractory ICC. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

From the Editor’s Desk…: October 2019

From the Editor’s Desk…: October 2019

INOS is associated with tumorigenicity as an independent prognosticator in human intrahepatic cholangiocarcinoma.

BackgroundInducible nitric oxide synthase (iNOS) has supposed to implicate in inflammation, infection, liver cirrhosis, and neoplastic diseases. This study was designed to explore the biological and clinical function of iNOS in intrahepatic cholangiocarcinoma (ICC).MethodsRT-PCR (Real-time quantitative PCR) and immunohistochemical staining were used to analyze the expression of iNOS in ICC and adjacent tissues. CCK8, transwell assays, flow cytometry were conducted to detect the proliferation, apoptosis, cell cycle. Western blotting was performed to detect the expression of target proteins. Multivariate analyses were conducted to analysis associates between clinicopathological values and survival.ResultsWe found that levels of iNOS mRNA and protein were dramatically increased in ICC samples and positively correlated with complicated bile duct stone, differentiation, pathology T, pathology M, Wip1, MMP-2, and MMP-9. iNOS expression was significantly correlated with the poor survival of ICC patients. Furthermore, iNOS was high expression in ICC cell lines (QBC-939, ICC-9810, SSP-25) compare with human normal biliary epithelium cell line (HIBEpic); both iNOS knockdown and iNOS inhibitor (1400 W) suppressed cell proliferation, invasion, and migration though nitric oxide production in ICC cells. Down-regulation of iNOS also induced G0/G1 cell cycle arrest and ICC cell apoptosis. Moreover, iNOS knockdown treatment significantly decreased Wip1, MMP-9, and MMP-2 gene expression.ConclusionLowly expressed iNOS-inhibited proliferation yet promoted apoptosis of ICC cells. Our data show targeted inhibition of iNOS in ICC may have therapeutic value.

PKM2 upregulation promotes malignancy and indicates poor prognosis for intrahepatic cholangiocarcinoma

Although pyruvate kinase M2 (PKM2) has been shown to be among the crucial enzymes that regulate aerobic glycolysis in multiple tumour cells, its role in the treatment and prognosis of intrahepatic cholangiocarcinoma (ICC) remains unclear. This study primarily aimed to determine whether the expression status of PKM2 is potentially associated with the clinical outcomes of ICC. PKM2 expression was evaluated in ICC cell lines and tissues via real-time quantitative reverse-transcription polymerase chain reaction, immunofluorescence assays, and Western blot, and its prognostic value was determined according to its impact on the overall survival of patients. We found that PKM2 is highly expressed in ICC, and this was correlated with patient survival. Moreover, we found that PKM2 knockdown could considerably inhibit ICC cell proliferation, invasion, and migration in vitro. PKM2 was overexpressed in ICC, and it may regulate proliferation, invasion, and migration and lead to poor prognosis. Thus, PKM2 might be a potential independent prognostic factor for ICC.

Long non-coding RNA UCA1 promotes proliferation and invasion of intrahepatic cholangiocarcinoma cells through targeting microRNA-122.

Long non-coding RNA urothelial carcinoma-associated 1 (UCA1) has a role in various common types of human malignancy, including cholangiocarcinoma; however, the expression and function of UCA1 in intrahepatic cholangiocarcinoma (ICC) has remained elusive. In the present study, it was observed that UCA1 expression was significantly upregulated in ICC tissues and cell lines compared with that in the adjacent non-tumour tissues and a human intrahepatic biliary epithelial cell line, respectively. The increased expression of UCA1 was significantly associated with lymph node metastasis and clinical T-stage in ICC. Furthermore, the ICC patients with high expression of UCA1 had a shorter survival time when compared with that of patients with low UCA1 expression. Knockdown of UCA1 caused a significant decrease in ICC cell proliferation and invasion, while ectopic overexpression of UCA1 significantly promoted the proliferation and invasion of ICC cells. Furthermore, it was revealed that UCA1 directly binds to microRNA (miR)-122 to negatively regulate its expression in ICC cells. In addition, miR-122 mimics abrogated the promoting effects of UCA1 on ICC cell proliferation and invasion. In addition, an inverse correlation between miR-122 and UCA1 expression in ICC tissues was observed. In conclusion, the present study demonstrates that the lncRNA UCA1 promotes ICC cell proliferation and invasion at least in part by targeting miR-122, suggesting that the UCA1/miR-122 interaction may become a potential therapeutic target for the treatment of ICC.

Downregulation of Fat Mass and Obesity Associated (FTO) Promotes the Progression of Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) ranks as the second most malignant type of primary liver cancer with a high degree of incidence and a very poor prognosis. Fat mass and obesity-associated protein (FTO) functions as an eraser of the RNA m6A modification, but its roles in ICC tumorigenesis and development remain unknown. We showed here that the protein level of FTO was downregulated in clinical ICC samples and cell lines and that FTO expression was inversely correlated with the expression of CA19-9 and micro-vessel density (MVD). A Kaplan-Meier survival analysis showed that a low expression of FTO predicted poor prognosis in ICC. in vitro, decreased endogenous expression of FTO obviously reduced apoptosis of ICC cells. Moreover, FTO suppressed the anchorage-independent growth and mobility of ICC cells. Through mining the database, FTO was found to regulate the integrin signaling pathway, inflammation signaling pathway, epidermal growth factor receptor (EGFR) signaling pathway, angiogenesis, and the pyrimidine metabolism pathway. RNA decay assay showed that oncogene TEAD2 mRNA stability was impaired by FTO. In addition, the overexpression of FTO suppressed tumor growth in vivo. In conclusion, our study demonstrated the critical roles of FTO in ICC.

Establishment and Characterization of a New Intrahepatic Cholangiocarcinoma Cell Line Resistant to Gemcitabine.

Intrahepatic cholangiocarcinoma (ICC) is one of the most lethal liver cancers. Late diagnosis and chemotherapy resistance contribute to the scarce outfit and poor survival. Resistance mechanisms are still poorly understood. Here, we established a Gemcitabine (GEM) resistant model, the MT-CHC01R1.5 cell line, obtained by a GEM gradual exposure (up to 1.5 µM) of the sensitive counterpart, MT-CHC01. GEM resistance was irreversible, even at high doses. The in vitro and in vivo growth was slower than MT-CHC01, and no differences were highlighted in terms of migration and invasion. Drug prediction analysis suggested that Paclitaxel and Doxycycline might overcome GEM resistance. Indeed, in vitro MT-CHC01R1.5 growth was reduced by Paclitaxel and Doxycycline. Importantly, Doxycycline pretreatment at very low doses restored GEM sensitivity. To assess molecular mechanisms underlying the acquisition of GEM resistance, a detailed analysis of the transcriptome in MT-CHC01R1.5 cells versus the corresponding parental counterpart was performed. Transcriptomic analysis showed that most up-regulated genes were involved in cell cycle regulation and in the DNA related process, while most down-regulated genes were involved in the response to stimuli, xenobiotic metabolism, and angiogenesis. Furthermore, additional panels of drug resistance and epithelial to mesenchymal transition genes (n = 168) were tested by qRT-PCR and the expression of 20 genes was affected. Next, based on a comparison between qRT-PCR and microarray data, a list of up-regulated genes in MT-CHC01R1.5 was selected and further confirmed in a primary cell culture obtained from an ICC patient resistant to GEM. In conclusion, we characterized a new GEM resistance ICC model that could be exploited either to study alternative mechanisms of resistance or to explore new therapies.

GNS561, a new lysosomotropic small molecule, for the treatment of intrahepatic cholangiocarcinoma.

Among the acquired modifications in cancer cells, changes in lysosomal phenotype and functions are well described, making lysosomes a potential target for novel therapies. Some weak base lipophilic drugs have a particular affinity towards lysosomes, taking benefits from lysosomal trapping to exert anticancer activity. Here, we have developed a new lysosomotropic small molecule, GNS561, and assessed its activity in multiple in vitro intrahepatic cholangiocarcinoma models (HuCCT1 and RBE cell lines and patient-derived cells) and in a chicken chorioallantoic membrane xenograft model. GNS561 significantly reduced cell viability in two intrahepatic cholangiocarcinoma cell lines (IC50 of 1.5 ± 0.2μM in HuCCT1 and IC50 of 1.7 ± 0.1μM in RBE cells) and induced apoptosis as measured by caspases activation. We confirmed that GNS561-mediated cell death was related to its lysosomotropic properties. GNS561 induced lysosomal dysregulation as proven by inhibition of late-stage autophagy and induction of a dose-dependent build-up of enlarged lysosomes. In patient-derived cells, GNS561 was more potent than cisplatin and gemcitabine in 2/5 and 1/5 of the patient-derived cells models, respectively. Moreover, in these models, GNS561 was potent in models with low sensitivity to gemcitabine. GNS561 was also efficient in vivo against a human intrahepatic cholangiocarcinoma cell line in a chicken chorioallantoic membrane xenograft model, with a good tolerance at doses high enough to induce an antitumor effect in this model. In summary, GNS561 is a new lysosomotropic agent, with an anticancer activity against intrahepatic cholangiocarcinoma. Further investigations are currently ongoing to fully elucidate its mechanism of action.