N6-methyladenosine modified TGFB2 triggers lipid metabolism reprogramming to confer pancreatic ductal adenocarcinoma gemcitabine resistance.

Gemcitabine resistance is a major obstacle to the effectiveness of chemotherapy in pancreatic ductal adenocarcinoma (PDAC). Therefore, new strategies are needed to sensitize cancer cells to gemcitabine. Here, we constructed gemcitabine-resistant PDAC cells and analyzed them with RNA-sequence. Employing an integrated approach involving bioinformatic analyses from multiple databases, TGFB2 is identified as a crucial gene in gemcitabine-resistant PDAC and is significantly associated with poor gemcitabine therapeutic response. The patient-derived xenograft (PDX) model further substantiates the gradual upregulation of TGFB2 expression during gemcitabine-induced resistance. Silencing TGFB2 expression can enhance the chemosensitivity of gemcitabine against PDAC. Mechanistically, TGFB2, post-transcriptionally stabilized by METTL14-mediated m6A modification, can promote lipid accumulation and the enhanced triglyceride accumulation drives gemcitabine resistance by lipidomic profiling. TGFB2 upregulates the lipogenesis regulator sterol regulatory element binding factor 1 (SREBF1) and its downstream lipogenic enzymes via PI3K-AKT signaling. Moreover, SREBF1 is responsible for TGFB2-mediated lipogenesis to promote gemcitabine resistance in PDAC. Importantly, TGFB2 inhibitor imperatorin combined with gemcitabine shows synergistic effects in gemcitabine-resistant PDAC PDX model. This study sheds new light on an avenue to mitigate PDAC gemcitabine resistance by targeting TGFB2 and lipid metabolism and develops the potential of imperatorin as a promising chemosensitizer in clinical translation.

ADAR1 promotes cisplatin resistance in intrahepatic cholangiocarcinoma by regulating BRCA2 expression through A-to-I editing manner.

Aberrant A-to-I RNA editing, mediated by ADAR1 has been found to be associated with increased tumourigenesis and the development of chemotherapy resistance in various types of cancer. Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive malignancy with a poor prognosis, and overcoming chemotherapy resistance poses a significant clinical challenge. This study aimed to clarify the roles of ADAR1 in tumour resistance to cisplatin in iCCA. We discovered that ADAR1 expression is elevated in iCCA patients, particularly in those resistant to cisplatin, and associated with poor clinical outcomes. Downregulation of ADAR1 can increase the sensitivity of iCCA cells to cisplatin treatment, whereas its overexpression has the inverse effect. By integrating RNA sequencing and Sanger sequencing, we identified BRCA2, a critical DNA damage repair gene, as a downstream target of ADAR1 in iCCA. ADAR1 mediates the A-to-I editing in BRCA2 3'UTR, inhibiting miR-3157-5p binding, consequently increasing BRCA2 mRNA and protein levels. Furthermore, ADAR1 enhances cellular DNA damage repair ability and facilitates cisplatin resistance in iCCA cells. Combining ADAR1 targeting with cisplatin treatment markedly enhances the anticancer efficacy of cisplatin. In conclusion, ADAR1 promotes tumour progression and cisplatin resistance of iCCA. ADAR1 targeting could inform the development of innovative combination therapies for iCCA.

High Bile Titer and High Bile to Serum Ratio of CYFRA 21 - 1 Reliably Discriminate Malignant Biliary Obstruction Caused by Cholangiocarcinoma.

INTRODUCTION: Previously we demonstrated that elevated serum CYFRA 21 - 1 is a reliable diagnostic and prognostic biomarker for biliary tract cancers. This study aims to explore the diagnostic performance of bile CYFRA 21 - 1 (bCYFRA 21 - 1) in discriminating malignant biliary obstruction (MBO) caused by cholangiocarcinoma (CCA). METHODS: 77 CCA patients ((17 intrahepatic CCA (iCCA), 49 perihilar CCA (pCCA) and 11 distal CCA (dCCA)) and 43 benign patients with biliary obstruction were enrolled. Serum and bile levels of CYFRA 21 - 1, carcinoembryonic antigen (CEA) and carbohydrate antigen 19 - 9 (CA19-9) were quantified. Diagnostic performances of these biomarkers were estimated by receiver operator characteristic curves. Subgroups analysis of these tumor markers among CCA subtypes was performed. RESULTS: High bCYFRA 21 - 1 (cut-off value of 59.25 ng/mL with sensitivity of 0.889 and specificity of 0.750) and high bile to serum ratio of CYFRA 21 - 1 (b/sCYFRA 21 - 1, cut-off value of 31.55 with sensitivity of 0.741 and specificity of 0.778) achieved better diagnostic performance than any other biomarker in discriminating MBO. Subgroup analysis revealed that bCYFRA 21 - 1 was significantly elevated in all CCA subtypes; moreover b/sCYFRA 21 - 1 was upregulated in pCCA and dCCA (the mean b/sCYFRA 21 - 1 of pCCA was highest among CCA subtypes: 57.90, IQR 29.82-112.27). CONCLUSIONS: Both high biliary CYFRA 21 - 1 and high bile to serum ratio of CYFRA 21 - 1 were reliable diagnostic biomarkers for MBO caused by CCA.

CSNK2A1 confers gemcitabine resistance to pancreatic ductal adenocarcinoma via inducing autophagy.

Gemcitabine, a pivotal chemotherapeutic agent for pancreatic ductal adenocarcinoma (PDAC), frequently encounters drug resistance, posing a significant clinical challenge with implications for PDAC patient prognosis. In this study, employing an integrated approach involving bioinformatic analyses from multiple databases, we unveil CSNK2A1 as a key regulatory factor. The patient-derived xenograft (PDX) model further substantiates the critical role of CSNK2A1 in gemcitabine resistance within the context of PDAC. Additionally, targeted silencing of CSNK2A1 expression significantly enhances sensitivity of PDAC cells to gemcitabine treatment. Mechanistically, CSNK2A1's transcriptional regulation is mediated by H3K27 acetylation in PDAC. Moreover, we identify CSNK2A1 as a pivotal activator of autophagy, and enhanced autophagy drives gemcitabine resistance. Silmitasertib, an established CSNK2A1 inhibitor, can effectively inhibit autophagy. Notably, the combinatorial treatment of Silmitasertib with gemcitabine demonstrates remarkable efficacy in treating PDAC. In summary, our study reveals CSNK2A1 as a potent predictive factor for gemcitabine resistance in PDAC. Moreover, targeted CSNK2A1 inhibition by Silmitasertib represents a promising therapeutic strategy to restore gemcitabine sensitivity in PDAC, offering hope for improved clinical outcomes.

Development and Verification of a novel cuproptosis- and immune-associated based prognostic genetic signature for pancreatic ductal adenocarcinoma.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is a malignancy with a dismal prognosis. Cuproptosis, a novel mechanism mediated by protein lipoylation, results in acute proteotoxic stress and ultimately cell death. However, the clinical impacts of cuproptosis-associated genes and their relationship with immune status in PDAC have not been documented. In this study, we aimed at constructing a cuproptosis- and immune-associated prognostic signature to stratify and predict the prognosis for PDAC patients. METHODS: The gene expression profiles of 176 PDAC patients from The Cancer Genome Atlas and 167 normal pancreas tissues from the Genotype-Tissue Expression Project were analyzed for differentially expressed genes (DEGs) between PDAC and normal tissues. Pearson correlation analyses were performed to screen out cuproptosis- and immune-associated DEGs. The risk signature of DEGs was constructed using the least absolute shrinkage and selection operator (LASSO) Cox regression analysis, which was validated in the Gene Expression Omnibus (GEO) cohort (n = 114). The immune characteristics in the two risk groups were evaluated using single-sample gene set enrichment analysis and ESTIMATE algorithms. RESULTS: A total of 91 cuproptosis- and immune-associated DEGs were screened out, and eight prognostic-related genes were identified using LASSO Cox regression. The prognostic-related genes were then used to construct a risk scoring model, which stratified patients into low- and high-risk groups and were further verified in the external GEO database. The patients in the high-risk group had significantly shorter overall survival than those in the low-risk group. A nomogram based on the risk signature was then constructed. Immune infiltration evaluation suggested that immune status was more activated in the low-risk group. The mutation spectrum also differed between high- and low-risk groups. CONCLUSIONS: Our cuproptosis- and immune-associated genetic risk signature could be a prognostic biomarker for PDAC. Cuproptosis might be a promising therapeutic target for PDAC.

Imatinib facilitates gemcitabine sensitivity by targeting epigenetically activated PDGFC signaling in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a malignant tumor with poor prognosis. Gemcitabine-based chemotherapy has become one of the main modalities of its management. However, gemcitabine resistance frequently occurs, leading to failure of PDAC therapy. Platelet-derived growth factors (PDGFs) and their receptors play important roles in cancer progression and chemoresistance. We aimed to investigate the biological function and therapeutic significance of platelet-derived growth factor C (PDGFC) in drug-resistant PDAC. Our study showed that PDGFC was abnormally highly expressed in gemcitabine-resistant PDAC. Silencing PDGFC expression can enhance the therapeutic effect of gemcitabine on PDAC. Mechanistically, the transcription of PDGFC is mediated by H3K27 acetylation, and PDGFC promotes gemcitabine resistance by activating the PDGFR-PI3K-AKT signaling pathway. The PDGFR inhibitor imatinib inhibits the PDGFR pathway. Imatinib and gemcitabine have a synergistic effect on the treatment of PDAC, and imatinib can significantly enhance the anti-tumor effect of gemcitabine in a drug-resistant PDAC patient-derived xenograft model. In conclusion, PDGFC is a potential predictor of gemcitabine-resistant PDAC. Imatinib inhibits PDGFR activation to promote gemcitabine sensitivity in PDAC. Combined modality regimen of imatinib and gemcitabine is likely to translate into clinical trial for the treatment of PDGFC-associated gemcitabine-resistant patients.

Values of a novel pyroptosis-related genetic signature in predicting outcome and immune status of pancreatic ductal adenocarcinoma.

Background: Pyroptosis is an emerging form of programmed cell death associated with progression in malignancies. Yet, there are few studies reporting on the association between pancreatic ductal adenocarcinoma (PDAC) and pyroptosis. Therefore, we aimed to construct a pyroptosis-related genetic signature to predict the clinical outcome and immune status in PDAC patients. Methods: RNA-seq data of 176 PDAC patients from The Cancer Genome Atlas (TCGA) and 167 PDAC patients from the Genotype-Tissue Expression Project were analysed for pyroptosis-related differentially expressed genes (DEGs) between PDAC and normal pancreas. The risk signature of DEGs was analysed using the least absolute shrinkage and selection operator (LASSO) Cox regression analysis and its accuracy was validated in the Gene Expression Omnibus (GEO) cohort (n = 190). Functional enrichment analyses were performed to explore the mechanisms of the DEGs. The immune characteristics were evaluated using single-sample gene set enrichment analysis and ESTIMATE algorithms for each group. Results: A nine-gene risk signature was generated from LASSO Cox regression analysis and classified PDAC patients into either a high- or low-risk group according to the median risk score. The high-risk group had significantly shorter overall survival than the low-risk group and it was verified in the external GEO database. A nomogram based on the risk signature was constructed and showed an ideal prediction performance. Functional enrichment analyses revealed that pyroptosis might regulate the tumor immune microenvironment in PDAC. Immune infiltration evaluation suggested that immune status was more activated in the low-risk group than in the high-risk group. Conclusion: The risk signature encompassing nine pyroptosis-related genes may be a prognostic marker for PDAC. Pyroptosis might affect the prognosis of PDAC patients via regulating the tumor immune microenvironment.

YTHDF2 promotes intrahepatic cholangiocarcinoma progression and desensitises cisplatin treatment by increasing CDKN1B mRNA degradation.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is an aggressive cancer with exceedingly poor prognosis, and chemoresistance is a huge challenge for treatment. N6-methyladenosine (m6 A) modification plays an important role in the progression and chemoresistance of cancers. We aimed to investigate the oncogenic function and therapeutic significance of the m6 A binding protein, YTH domain family 2 (YTHDF2), in ICC progression and cisplatin-based chemotherapy. METHODS: Several independent data sets were used to assess the expression of YTHDF2 in ICC, particularly in chemoresistant ICC. Knockdown and overexpression were used to evaluate the effects of YTHDF2 on tumourigenesis and cisplatin response in ICC. Multi-omics sequencing was performed to identify target genes. RIP, dual luciferase reporter, RNA stability experiment and loss-of-function assays were conducted to study the mechanisms underlying the oncogenic function of YTHDF2. Furthermore, patient-derived xenograft (PDX) model was established to determine the effect of combination treatment of YTHDF2 siRNA and cisplatin in ICC. RESULTS: Our study showed that YTHDF2 was upregulated in ICC tissues, particularly in chemoresistant ICC tissues, and correlated with poor prognosis. Furthermore, silencing YTHDF2 led to inhibited proliferation, promoted apoptosis and G0/G1 cell cycle arrest. Its downregulation also enhanced DNA damage and sensitised ICC cells to cisplatin. YTHDF2 overexpression exerted the opposite results. Integration analysis using RNA-seq, MeRIP-seq and anti-YTHDF2 RIP-seq elucidated the role of YTHDF2 in tumourigenesis and cisplatin-desensitising function by promoting the degradation of cyclin-dependent kinase inhibitor 1B (CDKN1B) mRNA in an m6 A-dependent manner. Downregulation of CDKN1B increased the YTHDF2 silencing-induced influence on tumourigenesis and cisplatin response to ICC. In addition, the combination treatment of YTHDF2 siRNA and cisplatin significantly enhanced the anti-tumour effect of cisplatin in a chemoresistant ICC PDX model. CONCLUSIONS: YTHDF2 exhibits tumour oncogenic and cisplatin-desensitising properties, which may offer insight into the development of novel combination therapeutic strategies for ICC.

METTL3 promotes intrahepatic cholangiocarcinoma progression by regulating IFIT2 expression in an m6A-YTHDF2-dependent manner.

N6-methyladenosine (m6A) RNA methylation has recently been found involving in regulatory mechanism of the tumor progression. Our aim was to explore the biological function and clinical significance of the m6A methyltransferase METTL3 in intrahepatic cholangiocarcinoma (ICC). In this study, we revealed that METTL3 was upregulated and predicted poor prognosis of patients with ICC. Multivariate regression analysis demonstrated that METTL3 expression was an independent predictor for overall survival in patients with ICC. Moreover, METTL3 knockdown inhibited ICC progression, while METTL3 overexpression showed the opposite effect. METTL3 inhibitor STM2457 also showed anti-tumor effect in ICC. Mechanistically, METTL3 transcription was driven by H3K4me3 activation. Upregulation of METTL3 mediated m6A modification of IFIT2 mRNA and accelerated IFIT2 mRNA decay in a YTHDF2-dependent manner, which promoted the development of ICC and lead to poorer prognosis. In summary, our findings revealed that H3K4me3 activation-driven METTL3 transcription promotes ICC progression by YTHDF2-mediated IFIT2 mRNA degradation, suggesting that METTL3 may serve as a potential target for human ICC therapy.

Nanomaterial-Facilitated Cyclin-Dependent Kinase 7 Inhibition Suppresses Gallbladder Cancer Progression via Targeting Transcriptional Addiction.

Gallbladder cancer (GBC) is the most aggressive malignancy of the biliary tract cancer, and there is a lack of effective treatment. Here, we developed a nanoparticle platform (8P4 NP) that can deliver THZ1, a cyclin-dependent kinase 7 (CDK7) inhibitor, to treat GBC. Analysis of datasets demonstrated that CDK7 was positively correlated with poor prognosis. CDK7 inhibition suppressed cell proliferation, induced apoptosis, and caused cell cycle block in GBC cells. THZ1 downregulated CDK7-mediated phosphorylation of RNA polymerase II (RNAPII), resulting in a significant downregulation of transcriptional programs, with a preferential repression of oncogenic transcription factors. To improve the tumor targeting efficiency of THZ1, 8P4 NPs were prepared and assembled with THZ1 to form THZ1@8P4 NPs. Compared with free THZ1, THZ1@8P4 NPs showed more advantages in prolonging blood circulation, escaping from lysosomes and increasing cellular uptake. Importantly, THZ1@8P4 NPs demonstrated a more significant inhibition effect on GBC cells than free THZ1 in vitro. In addition, THZ1@8P4 NPs could efficiently deliver THZ1 to tumor sites in a patient-derived xenograft model of early recurrence, leading to tumor regression and transcriptional inhibition with minimal toxicity. In summary, we conclude that THZ1@8P4 NPs provide a potent therapeutic strategy that targets CDK7-mediated transcriptional addiction in GBC.

HNRNPC impedes m6A-dependent anti-metastatic alternative splicing events in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a malignancy with poor prognosis due to early metastasis. The aberrant N6-methyladenosine (m6A) RNA modification has emerged as an important mechanism in cancer progression and metastasis, but its role in PDAC remained largely unknown. Here, we demonstrated that an m6A regulator, heterogeneous nuclear ribonucleoprotein C (HNRNPC), modulated alternative splicing events to promote PDAC metastasis. In clinical PDAC tissues, high expression of HNRNPC was correlated with metastasis, resulting in poor prognosis in PDAC patients. Knockdown of HNRNPC significantly reduced PDAC cell invasion in vitro and metastasis in vivo. In contrast, overexpression of HNRNPC provoked malignant phenotypes of PDAC cells. Mechanistically, HNRNPC antagonized the anti-metastatic isoform of TAF8 (TAF8L) but increased the pro-metastatic alternative splicing isoform of TAF8 (TAF8S). Mutation of the m6A-site of TAF8 attenuated the interaction between HNRNPC and TAF8 transcript, leading to the decrease of TAF8S. Furthermore, experimental manipulation of the anti-metastasis splicing isoform TAF8L revealed that splice isoform switching of TAF8 is crucial for PDAC metastasis. In conclusion, our findings demonstrate the essentiality of HNRNPC-mediated alternative splicing events that impinges on metastatic PDAC.

Use of Nodal Staging Score in Evaluating the Accuracy of Pathologic Nodal Status in Node-Negative Ampullary Carcinoma.

BACKGROUND: The minimum number of lymph nodes (LNs) that should be resected for accurate nodal staging in patients with ampullary carcinoma (AC) remains controversial. This study aimed to establish a nodal staging score (NSS) to evaluate whether a pathological node-negative AC patient is indeed free of a nodal disease. METHODS: A total of 2539 AC patients with stages I-III were retrieved from the Surveillance, Epidemiology and End Result database (design cohort [DC], n = 2382) and First Affiliated Hospital of Sun Yat-sen University (validation cohort [VC], n = 157). NSS was developed to represent the probability that a node-negative patient was correctly staged as a function of the number of examined LNs (ELNs) and pathologic T stage with a beta-binomial model. Its prognostic value in node-negative patients was assessed by survival analysis. RESULTS: The probability of missing a metastatic LN decreased as the number of the ELNs increased. NSS was escalated as the number of ELNs increased. For patients with early-stage (T1-T2) and late-stage (T3-T4) tumors, examining 7 and 33 lymph nodes could ensure an NSS of 80.0%, respectively. Multivariate analysis showed that higher NSS was an independent favorable prognostic factor for overall survival in node-negative patients with AC (DC, p < 0.001; VC, p = 0.001). CONCLUSIONS: NSS model could be used to evaluate the accuracy of nodal staging and predict the prognosis of node-negative AC patients. It could assist in making clinical strategies in node-negative AC patients.

Histone methyltransferase G9a inhibitor-loaded redox-responsive nanoparticles for pancreatic ductal adenocarcinoma therapy.

Survival data have shown little therapeutic improvement in pancreatic ductal adenocarcinoma (PDAC) over the past several decades, mostly due to aggressive growth and resistance to therapy. Glutathione (GSH) depletion in PDAC may serve as a strategy to suppress tumour malignancy and sensitize tumour cells to therapy. Herein, novel l-cysteine-based poly(disulfide amide) polymers were fabricated to deliver a histone methyltransferase G9a inhibitor (UNC0638) that can simultaneously block GSH biosynthesis and clear cellular GSH levels in PDAC. The optimal UNC0638 nanodrug (NPUNC0638) had the desired particle size, reasonable drug loading capacity, and GSH-controlled drug release. Moreover, compared to UNC0638 alone, NPUNC0638 showed better efficacy in inhibiting cell viability, arresting the cell cycle, inducing apoptosis, and suppressing the invasion and self-renewal capacity of PDAC cells. Furthermore, NPUNC0638 was found to be tumour-specific and well tolerated with no apparent toxicity to vital organs and haematopoietic stem and progenitor cells. Additionally, treatment with NPUNC0638 provided favourable outcomes in the PDAC xenograft model. Therefore, this work presents a potent drug delivery platform to overcome the GSH-induced malignant potential of PDAC.

Targeting Super-Enhancers via Nanoparticle-Facilitated BRD4 and CDK7 Inhibitors Synergistically Suppresses Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignant cancer with complex genomic variations, and no targetable genomic lesions have been found yet. Super-enhancers (SEs) have been found to contribute to the continuous and robust oncogenic transcription. Here, histone H3 lysine 27 acetylation (H3K27ac) is profiled in PDAC cell lines to establish SE landscapes. Concurrently, it is also shown that PDAC is vulnerable to the perturbation of the SE complex using bromodomain-containing protein 4 (BRD4) inhibitor, JQ1, synergized with cyclin-dependent kinase 7 (CDK7) inhibitor, THZ1. Formulations of hydrophobic l-phenylalanine-poly (ester amide) nanoparticles (NPs) with high drug loading of JQ1 and THZ1 (J/T@8P4s) are further designed and developed. J/T@8P4s is assessed for size, encapsulation efficiency, morphology, drug release profiles, and drug uptake in vitro. Compared to conventional free drug formulation, the nanodelivery system dramatically reduces the hepatotoxicity while significantly enhancing the tumor inhibition effects and the bioavailability of incorporated JQ1 and THZ1 at equal doses in a Gemcitabine-resistant PDAC patient-derived xenograft (PDX) model. Overall, the present study demonstrates that the J/T@8P4s can be a promising therapeutic treatment against the PDAC via suppression of SE-associated oncogenic transcription, and provides a strategy utilizing NPs to assist the drug delivery targeting SEs.

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.

MFAP5 facilitates the aggressiveness of intrahepatic Cholangiocarcinoma by activating the Notch1 signaling pathway.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer. The dismal outcome of ICC patients is due to lack of early diagnosis, the aggressive biological behavior of ICC and the lack of effective therapeutic options. Early diagnosis and prognosis of ICC by non-invasive methods would be helpful in providing valuable information and developing effective treatment strategies. METHODS: Expression of microfibrillar-associated protein 5 (MFAP5) in the serum of ICC patients was detected by ELISA. Human ICC specimens were immunostained by MFAP5 antibodies. The growth rate of human ICC cell lines treated with MFAP5 or MFAP5 shRNAs was examined by CCK8 and colony formation assays. Cell cycle analysis was performed with PI staining. The effect of MFAP5 inhibition was assessed by xenograft models in nude mice. RNA-seq and ATAC-seq analyses were used to dissect the molecular mechanism by which MFAP5 promoted ICC aggressiveness. RESULTS: We identified MFAP5 as a biomarker for the diagnosis and prognosis of ICC. Upregulated MFAP5 is a common feature in aggressive ICC patients' tissues. Importantly, MFAP5 level in the serum of ICC patients and healthy individuals showed significant differential expression profiles. Furthermore, we showed that MFAP5 promoted ICC cell growth and G1 to S-phase transition. Using RNA-seq expression and ATAC-seq chromatin accessibility profiling of ICC cells with suppressed MFAP5 secretion, we showed that MFAP5 regulated the expression of genes involved in the Notch1 signaling pathway. Furthermore, FLI-06, a Notch signaling inhibitor, completely abolished the MFAP5-dependent transcriptional programs. CONCLUSIONS: Raised MFAP5 serum level is useful for differentiating ICC patients from healthy individuals, and could be helpful in ICC diagnosis, prognosis and therapies.

N6-Methyladenosine Modulates Nonsense-Mediated mRNA Decay in Human Glioblastoma.

The N6-methyladenosine (m6A) modification influences various mRNA metabolic events and tumorigenesis, however, its functions in nonsense-mediated mRNA decay (NMD) and whether NMD detects induced carcinogenesis pathways remain undefined. Here, we showed that the m6A methyltransferase METTL3 sustained its oncogenic role by modulating NMD of splicing factors and alternative splicing isoform switches in glioblastoma (GBM). Methylated RNA immunoprecipitation-seq (MeRIP-seq) analyses showed that m6A modification peaks were enriched at metabolic pathway-related transcripts in glioma stem cells (GSC) compared with neural progenitor cells. In addition, the clinical aggressiveness of malignant gliomas was associated with elevated expression of METTL3. Furthermore, silencing METTL3 or overexpressing dominant-negative mutant METTL3 suppressed the growth and self-renewal of GSCs. Integrated transcriptome and MeRIP-seq analyses revealed that downregulating the expression of METTL3 decreased m6A modification levels of serine- and arginine-rich splicing factors (SRSF), which led to YTHDC1-dependent NMD of SRSF transcripts and decreased SRSF protein expression. Reduced expression of SRSFs led to larger changes in alternative splicing isoform switches. Importantly, the phenotypes mediated by METTL3 deficiency could be rescued by downregulating BCL-X or NCOR2 isoforms. Overall, these results establish a novel function of m6A in modulating NMD and uncover the mechanism by which METTL3 promotes GBM tumor growth and progression. SIGNIFICANCE: These findings establish the oncogenic role of m6A writer METTL3 in glioblastoma stem cells.

EYA4 inhibits hepatocellular carcinoma by repressing MYCBP by dephosphorylating ß-catenin at Ser552.

Hepatocellular carcinoma (HCC) is one of the most common malignancies and the fourth leading cause of cancer-related death worldwide. Our previous study showed that EYA4 functioned by suppressing growth of HCC tumor cells, but its molecular mechanism is still not elucidated. Based on the results of gene microassay, EYA4 was inversely correlated with MYCBP and was verified in human HCC tissues by immunohistochemistry and western blot. Overexpressed and KO EYA4 in human HCC cell lines confirmed the negative correlation between EYA4 and MYCBP by qRT-PCR and western blot. Transfected siRNA of MYCBP in EYA4 overexpressed cells and overexpressed MYCBP in EYA4 KO cells could efficiently rescue the proliferation and G2/M arrest effects of EYA4 on HCC cells. Mechanistically, armed with serine/threonine-specific protein phosphatase activity, EYA4 reduced nuclear translocation of ß-catenin by dephosphorylating ß-catenin at Ser552, thereby suppressing the transcription of MYCBP which was induced by ß-catenin/LEF1 binding to the promoter of MYCBP. Clinically, HCC patients with highly expressed EYA4 and poorly expressed MYCBP had significantly longer disease-free survival and overall survival than HCC patients with poorly expressed EYA4 and highly expressed MYCBP. In conclusion, EYA4 suppressed HCC tumor cell growth by repressing MYCBP by dephosphorylating ß-catenin S552. EYA4 combined with MYCBP could be potential prognostic biomarkers in HCC.

LncRNA FLVCR1-AS1 acts as miR-513c sponge to modulate cancer cell proliferation, migration, and invasion in hepatocellular carcinoma.

In the present study, we aimed to search for dysregulated lnRNAs in Hepatocellular carcinoma (HCC) tissues, and analyze the relationship of its expression level with the clinicopathological feature and patient prognosis. The biological function of FLVCR1-AS1, the identified lncRNA, in the process of HCC development, and progression was investigated in vitro and in vivo. The underlying molecular mechanism was further explored. We determined FLVCR1-AS1 expression in HCC tissues and peri-tumor tissues by bioinformatic analysis, qRT-PCR, Northern blot and in situ hybridization. The relationship between FLVCR1-AS1 expression level and prognosis was determined by analyzing clinical samples. The effects of FLVCR1-AS1 knockdown on HCC cell proliferation, apoptosis, migration, and invasion were investigated by CCK8, FACS, and tanswell assay, respectively. Tumor xenograft model was used to determine the influence of down-regulated FLVCR1-AS1 on tumor growth and metastasis. lncRNA FLVCR1-AS1 was extremely up-regulated in HCC tissues and cell lines. FLVCR1-AS1 expression level was positively correlated with tumor severity. FLVCR1-AS1 knockdown remarkably inhibited HCC cell proliferation, migration, and invasion in vitro and in vivo while induced cell apoptosis. In mechanism, FLVCR1-AS1 acted as a competitive endogenous RNAs to sponge miR-513c which targeted the mRNA of MET for degradation. By directly sponging miR-513c, FLVCR1-AS1 increased MET expression in HCC, and then promoted HCC progression. It was demonstrated that FLVCR1-AS1 played a positive role in HCC development and progression according to the study in its mechanism, function and clinical manifestation, so that it could be expected to become a new target in HCC prevention and treatment.