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Supplementary Figure from Molecular Subgroups of Intrahepatic Cholangiocarcinoma Discovered by Single-Cell RNA Sequencing–Assisted Multiomics Analysis

Intrahepatic cholangiocarcinoma (ICC) is a relatively rare but highly aggressive tumor type that responds poorly to chemotherapy and immunotherapy. Comprehensive molecular characterization of ICC is essential for the development of novel therapeutics. Here, we constructed two independent cohorts from two clinic centers. A comprehensive multiomics analysis of ICC via proteomic, whole-exome sequencing (WES), and single-cell RNA sequencing (scRNA-seq) was performed. Novel ICC tumor subtypes were derived in the training cohort (n = 110) using proteomic signatures and their associated activated pathways, which were further validated in a validation cohort (n = 41). Three molecular subtypes, chromatin remodeling, metabolism, and chronic inflammation, with distinct prognoses in ICC were identified. The chronic inflammation subtype was associated with a poor prognosis. Our random forest algorithm revealed that mutation of lysine methyltransferase 2D (KMT2D) frequently occurred in the metabolism subtype and was associated with lower inflammatory activity. scRNA-seq further identified an APOE+C1QB+ macrophage subtype, which showed the capacity to reshape the chronic inflammation subtype and contribute to a poor prognosis in ICC. Altogether, with single-cell transcriptome-assisted multiomics analysis, we identified novel molecular subtypes of ICC and validated APOE+C1QB+ tumor-associated macrophages as potential immunotherapy targets against ICC.

Intrahepatic cholangiocarcinoma (ICC) is a relatively rare but highly aggressive tumor type that responds poorly to chemotherapy and immunotherapy. Comprehensive molecular characterization of ICC is essential for the development of novel therapeutics. Here, we constructed two independent cohorts from two clinic centers. A comprehensive multiomics analysis of ICC via proteomic, whole-exome sequencing (WES), and single-cell RNA sequencing (scRNA-seq) was performed. Novel ICC tumor subtypes were derived in the training cohort (n = 110) using proteomic signatures and their associated activated pathways, which were further validated in a validation cohort (n = 41). Three molecular subtypes, chromatin remodeling, metabolism, and chronic inflammation, with distinct prognoses in ICC were identified. The chronic inflammation subtype was associated with a poor prognosis. Our random forest algorithm revealed that mutation of lysine methyltransferase 2D (KMT2D) frequently occurred in the metabolism subtype and was associated with lower inflammatory activity. scRNA-seq further identified an APOE+C1QB+ macrophage subtype, which showed the capacity to reshape the chronic inflammation subtype and contribute to a poor prognosis in ICC. Altogether, with single-cell transcriptome-assisted multiomics analysis, we identified novel molecular subtypes of ICC and validated APOE+C1QB+ tumor-associated macrophages as potential immunotherapy targets against ICC.

Gastric cancer (GC) is one of the most common malignancies around the world, and the incidence of GC is increasing in the past decades. In addition to genetic modifications, epigenetic alterations catalyzed by DNA methyltransferases (DNMTs) are a well-characterized epigenetic hallmark in gastric cancer. Nowadays, DNA methylation landscape is essential for maintaining the silence of tumor suppressor genes (TSGs). As an important group of peptide, TFF family has been confirmed to function as a TSG in various kinds of cancers. However, whether TFFs could be modified by DNA methylation in gastric cancer remains unknown. In this study, we initially screened out two expression profiles about GC from Gene Expression Omnibus (GEO) database. The higher expressions of TFF1 and TFF2 were observed in GC tumor tissues than normal tissues. Additionally, we illustrated that the expressions of TFF1/TFF2 were associated to the overall survival (OS) and tumor free survival (TFS) of GC patients via through the Kaplan-Meier analysis. Subsequently, the integrative analysis was performed to estimate the DNA methylation level of each site in TFF1/TFF2 CpG islands. Importantly, our findings indicated that hyper-methylation of cg01886855 and cg26403416 were separately responsible for the downregulation of TFF1 and TFF2 in GC samples. Besides, utilizing the gain of function assay, we demonstrated that TFF1/TFF2 could suppress the proliferation of GC cells. Based on these results, We identified that TFF1 and TFF2 acted as the putative tumor suppressors in gastric cancer, which suggested that TFFs could be two candidate biomarkers for predicting tumor recurrence in gastric cancer patients. Furthermore, these findings highlight a potential therapeutic approach in targeting the TFFs for the treatment of gastric cancer.

As one of the most frequently occurring tumor types, the increasing incidence of gastric cancer (GC) has been observed in the past decades. The recent studies have illustrated that epigenetic modifications mediated by DNA methyltransferases (DNMTs) are the major epigenetic hallmark in GC progression. Nowadays, DNA methylation was considered to be necessary for inducing the silence of tumor suppressor genes (TSGs). As an important group of peptides, the TFF family has been confirmed to function as a TSG in various kinds of cancers. However, whether TFFs could be modified by DNA methylation in gastric cancer remains unknown. Here, we initially screened out two transcriptional sequencing profiles about GC from Gene Expression Omnibus (GEO) database. The lower expression levels of TFF1 and TFF2 were observed in GC tumor tissues as compared to those in normal tissues. Additionally, utilizing the Kaplan–Meier analysis, the expressions of TFF1 and TFF2 were identified to be associated with the prognosis of GC patients. Subsequently, the integrative analysis was performed to estimate the DNA methylation level of each site in TFF1/TFF2 CpG islands. Importantly, our findings indicated that hyper-methylation of cg01886855 and cg26403416 were separately responsible for the downregulation of TFF1 and TFF2 in GC samples. In addition, utilizing the experiments in vitro, we demonstrated that TFF1/TFF2 could suppress the proliferation of GC cells. Based on these results, we suspected that TFF1/TFF2 could potentially act as the putative tumor suppressor in GC, and these two TFFs were of great value for predicting the overall survival (OS) status in the gastric cancer cohort. Totally, our findings revealed a potential therapeutic method for targeting the TFFs for the treatment of GC.

As a highly conserved and ubiquitously expressed serine/threonine kinase, p21-activated kinase 2 (Pak2) participates in diverse biologic events. However, its roles in mouse oocyte meiotic maturation remain unclear. The present study revealed that mouse oocytes depleted of Pak2 were unable to completely progress through meiosis and that a majority were arrested at metaphase I. Pak2 depletion thus prompted MI arrest and induced meiotic apparatus assembly defects in mouse oocytes, in part due to a reduction in polo-like kinase (Plk1). We demonstrated that Pak2’s interaction with Plk1 protected it from degradation by APC/CCdh1, and that it promoted meiotic progression and bipolar spindle formation. Our data collectively display critical functions for Pak2 in meiotic progression and meiotic apparatus assembly in mouse oocytes and may mediate the effects of maternal aging on female reproduction.

At present, cerebral organoid culture technology is still complicated to operate and difficult to apply on a large scale. It is necessary to find a simple and practical solution. Therefore, a more feasible cerebral organoid protocol is proposed in the present study. To solve the unavoidable inconvenience in medium change and organoid transfer in the early stage, the current research optimizes the operation technology by applying the engineering principle. A soft light lamp was adopted to laterally illuminate the embryoid body (EB) samples, allowing the EBs to be seen by the naked eye through the enhanced diffuse reflection effect. Using the principle of secondary flow generated by rotation, the organoids gather toward the center of the well, which facilitates the operation of medium change or organoid transfer. Compared to the dispersed cell, the embryoid body settles faster in the pipette. Using this phenomenon, most of the free cells and dead cell fragments can be effectively removed in a simple way, preventing EBs from incurring damage from centrifugation. This study facilitates the operation of cerebral organoid culture and helps to promote the application of brain organoids.

The fetal-to-adult hemoglobin switch is regulated in a developmental stage-specific manner and reactivation of fetal hemoglobin (HbF) has therapeutic implications for treatment of β-thalassemia and sickle cell anemia, two major global health problems. Although significant progress has been made in our understanding of the molecular mechanism of the fetal-to-adult hemoglobin switch, the mechanism of epigenetic regulation of HbF silencing remains to be fully defined. Here, we performed whole-genome bisulfite sequencing and RNA sequencing analysis of the bone marrow-derived GYPA(+) erythroid cells from β-thalassemia-affected individuals with widely varying levels of HbF groups (HbF ≥ 95th percentile or HbF ≤ 5th percentile) to screen epigenetic modulators of HbF and phenotypic diversity of β-thalassemia. We identified an ETS2 repressor factor encoded by ERF, whose promoter hypermethylation and mRNA downregulation are associated with high HbF levels in β-thalassemia. We further observed that hypermethylation of the ERF promoter mediated by enrichment of DNMT3A leads to demethylation of γ-globin genes and attenuation of binding of ERF on the HBG promoter and eventually re-activation of HbF in β-thalassemia. We demonstrated that ERF depletion markedly increased HbF production in human CD34(+) erythroid progenitor cells, HUDEP-2 cell lines, and transplanted NCG-Kit-V831M mice. ERF represses γ-globin expression by directly binding to two consensus motifs regulating γ-globin gene expression. Importantly, ERF depletion did not affect maturation of erythroid cells. Identification of alterations in DNA methylation of ERF as a modulator of HbF synthesis opens up therapeutic targets for β-hemoglobinopathies.

BACKGROUND AND AIMS Methylation landscape is important for maintaining the silence of cannabinoid receptor-interacting protein 1 (CNRIP1) in some tumors. However, the role of CNRIP1 in intrahepatic cholangiocarcinoma (ICC) remains poorly defined. APPROACH AND RESULTS In our study, we showed that CNRIP1 was down-regulated in ICC tissues, and low expression of CNRIP1 was significantly associated with poor prognosis of patients with ICC in 3-year overall survival and tumor-free survival. Investigating the genomic DNA methylation profile, we disclosed a CpG island site named CNRIP1 MS-2 (CNRIP1 methylation site-2) that contributes to the down-regulation of CNRIP1. In addition, the methylation level of CNRIP1 MS-2 was correlated to the pathological grade, metastasis, and tumor-node-metastasis classification in ICC. Notably, we observed that CNRIP1 suppressed tumor cell migration, invasion, and proliferation by inhibiting the activity of pyruvate kinase M2 (PKM2). Sustained overexpression of CNRIP1 suppressed the in vivo tumor growth in a mouse xenograft model. It was also found that CNRIP1 overexpression activated Parkin (an E3 ubiquitin ligase), which resulted in the protein degradation of PKM2 in ICC cells. CONCLUSIONS We identified that CNRIP1 acted as a putative tumor suppressor in ICC, which suggested that CNRIP1 could be a candidate biomarker for predicting tumor recurrence in patients with ICC. Furthermore, these findings highlight a potential therapeutic approach in targeting the CNRIP1/Parkin/PKM2 pathway for the treatment of ICC.

Summary Background 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. Methods 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. Results 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. Conclusions 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.

Aim: To identify methylation-associated genes in the carcinogenesis of colorectal cancer (CRC). Materials & methods: Genome-wide patterns of DNA methylation and gene expression in CRC tissues and adjacent normal tissues were determined and further validated in The Cancer Genome Atlas data and Chinese CRC patients, respectively. Gene overexpression and knockdown cells were constructed to investigate their biological roles in CRC. Results: After validations, hypermethylation of eight genes were found to be correlated with their reduced transcription, and hypomethyaltion of three genes were associated with their upregulation. CADM3, CNRIP1, GRHL2, GRIA4, GSTM2 and NRXN1 were associated with the overall survival of CRC patients. CNRIP1 and GSTM2 were mainly responsible for the proliferation in CRC cells. Conclusion: A total of 11 genes may be promising biomarkers for CRC.

Intrahepatic cholangiocarcinoma (ICC) is a relatively rare but highly aggressive tumour type that responds poorly to chemotherapy and immunotherapy. Comprehensive molecular characterization of ICC is essential for the development of novel therapeutics. We performed a comprehensive multi-omics analysis of ICC via proteomic, whole-exon sequencing (WES) and single-cell RNA sequencing (scRNA-seq). We identified three molecular subtypes with deteriorating prognosis in ICC: chromatin remodelling, metabolism, and inflammation. The inflammation subtype was associated with a poor prognosis. Our random forest algorithm revealed that the mutation of KMT2D frequently occurred in the metabolism subtype and were associated with lower inflammatory activity. scRNA-seq further identified a novel APOE+C1QB+ macrophage subtype, which showed the capacity of promoting the inflammation subtype and contributing to a poor prognosis in ICC. Taking together, with a single-cell transcriptome-assisted multi-omics analysis, we identified novel molecular subtypes of ICC and valided APOE+C1QB+ TAMs as potential novel immunotherapy targets against ICC.

Background: Hepatocellular carcinomas (HCC) constantly rank among the malignancies with the highest death tolls on the global scale. Moreover, HCC are associated with a limited set of therapeutic options. This is particularly true in the case of advanced stage cancers, where long-term survival is uncommon. For the inoperable, advanced HCC patients, chemotherapy is the main modality of treatment. Due to the lack of known molecular targets, the efficacy of the chemotherapy is limited. Conclusion: These findings clearly indicate that DNA methylation plays a key role in regulating ACADS expression and that it can be a potential therapeutic target for treating HCC. Materials and methods: A thorough comparative analysis of 282 cancer samples with 47 normal samples from GEO datasets resulted in the observation that that the level of ACADS was significantly downregulated in HCC. Loss-of-function analyses were then conducted to understand the biological function of ACADS in HCC. It was noted that ACADS was involved in the proliferation and metastasis of HCC. Experiments involving the knockdown of DMNT expression led to the discovery that the expression of ACADS in the HCC cells was significantly increased. The TCGA database was then employed to identify tumor tissue samples which showed higher methylation levels at cg01535453, cg08618068, and cg10174836 (which are the target sites of the ACADS CpG islands) as compared with normal liver tissue samples. All these findings indicated that ACADS might be a novel methylation biomarker associated with HCC.

β‐thalassaemia is a prevalent hereditary haematological disease caused by mutations in the human haemoglobin β (HBB) gene. Among them, the HBB IVS2‐654 (C > T) mutation, which is in the intron, creates an aberrant splicing site. Bone marrow transplantation for curing β‐thalassaemia is limited due to the lack of matched donors. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9), as a widely used tool for gene editing, is able to target specific sequence and create double‐strand break (DSB), which can be combined with the single‐stranded oligodeoxynucleotide (ssODN) to correct mutations. In this study, according to two different strategies, the HBB IVS2‐654 mutation was seamlessly corrected in iPSCs by CRISPR/Cas9 system and ssODN. To reduce the occurrence of secondary cleavage, a more efficient strategy was adopted. The corrected iPSCs kept pluripotency and genome stability. Moreover, they could differentiate normally. Through CRISPR/Cas9 system and ssODN, our study provides improved strategies for gene correction of β‐Thalassaemia, and the expression of the HBB gene can be restored, which can be used for gene therapy in the future.

Background N6-methyladenosine (m6A) methylation, a well-known modification with new epigenetic functions, has been reported to participate in the tumorigenesis of hepatocellular carcinoma (HCC), providing novel insights into the molecular pathogenesis of this disease. However, as the key component of m6A methylation, Wilms tumor 1-associated protein (WTAP) has not been well studied in HCC. Here we investigated the biological role and underlying mechanism of WTAP in liver cancer. Methods We determined the expression of WTAP and its correlation with clinicopathological features using tissue microarrays and the Cancer Genome Atlas (TCGA) dataset. And we clarified the effects of WTAP on HCC cells using cell proliferation assay, colony formation, Edu assay and subcutaneous xenograft experiments. We then applied RNA sequencing combined with gene expression omnibus (GEO) data to screen candidate targets of WTAP. Finally, we investigated the regulatory mechanism of WTAP in HCC by m6A dot blot assay, methylated RNA immunoprecipitation (MeRIP) assay, dual luciferase reporter assay, RNA immunoprecipitation (RIP) assay and Chromatin immunoprecipitation (ChIP) assay. Results We demonstrated that WTAP was highly expressed in HCC which indicated the poor prognosis, and that WTAP expression served as an independent predictor of HCC survival. Functionally, WTAP promoted the proliferation capability and tumor growth of HCC cells in vitro and in vivo. Furthermore, ETS proto-oncogene 1 (ETS1) was identified as the downstream effector of WTAP. The m6A modification regulated by WTAP led to post-transcriptional suppression of ETS1, with the implication of Hu-Antigen R (HuR) as an RNA stabilizer. Then ETS1 was found to inhibit the progression of HCC and could rescue the phenotype induced by WTAP deficiency. Moreover, WTAP modulated the G2/M phase of HCC cells through a p21/p27-dependent pattern mediated by ETS1. Conclusion We have identified that WTAP is significantly up-regulated in HCC and promotes liver cancer development. WTAP-guided m6A modification contributes to the progression of HCC via the HuR-ETS1-p21/p27 axis. Our study is the first to report that WTAP-mediated m6A methylation has a crucial role in HCC oncogenesis, and highlights WTAP as a potential therapeutic target of HCC treatment. Electronic supplementary material The online version of this article (10.1186/s12943-019-1053-8) contains supplementary material, which is available to authorized users.

Homeobox B7 (HOXB7) protein is reported to be aberrantly expressed in a variety of cancers and to play an important role in multiple cellular processes. However, the specific mechanism by which HOXB7 promotes the malignant progression of intrahepatic cholangiocarcinoma (ICC) remains unclear. Therefore, we used quantitative real-time polymerase chain reaction (PCR) to detect the expression level of HOXB7 in 38 paired ICC tissue samples. Additionally, to assess correlation between HOXB7 and ICC prognosis, we performed immunohistochemistry (IHC) using 122 ICC tissues to detect HOXB7 expression. Cell Counting Kit-8 (CCK-8) and colony formation assays were employed to assess ICC cell proliferation, and Transwell assays were performed to estimate the invasion and migration abilities of ICC cells. The capillary tube formation assay was applied to explore the angiogenic effects of HOXB7. A xenograft tumor model was established in nude mice to assess the role of HOXB7 in tumor growth and lung metastasis. The results showed higher expression of HOXB7 in ICC tissues than in noncancerous tissues, and this increased expression was significantly associated with a poor prognosis. In addition, HOXB7 overexpression enhanced capillary tube formation, invasion and migration of ICC cells in vitro, whereas HOXB7 knockdown produced the opposite results in vitro. Moreover, the role of HOXB7 in promoting tumor growth and metastasis was verified in vivo. Further investigation revealed that the expression levels of MMP2, MMP9, VEGFa, and IL8 were elevated by HOXB7 and that the ERK pathway was activated. Our results demonstrate the prognostic value of HOXB7 and its role in metastasis and angiogenesis in ICC. HOXB7 upregulated MMP2, MMP9, VEGFa, and IL8 expression via the ERK pathway to accelerate the malignant progression of ICC.

Heterotopic pancreas (HP) is a developmental anomaly defined as aberrantly located pancreatic tissue with no anatomical, vascular, or neural connection to the pancreas. Bowel intussusception and gastrointestinal bleeding caused by isolated HP is extremely rare. This study explores the clinical, radiographical, pathologic, and treatment features of four patients with intussusception caused by incidental HP, with the aim of providing important insight into the diagnosis and management of these patients. In this paper, the charts of four patients who underwent emergency laparotomy between January 2013 and August 2015 at the First Affiliated Hospital of Zhejiang University, China were reviewed. Patient demographics, presenting symptoms, blood test results, pre-operative imaging, operative procedure, and histopathology were retrospectively reviewed. All four patients, including three males and one female, presented to the emergency room with severe abdominal pain and melena. Routine blood tests revealed hemoglobin levels ranging from 75 to 128 g/L. Contrast-enhanced computed tomography (CT) confirmed lesions involving the ileum with intussusception and bowel obstruction, and all cases immediately underwent emergency laparotomy. All cases were subsequently diagnosed with HP according to histopathology. HP should therefore be considered as a rare but possible differential diagnosis for gastrointestinal bleeding and bowel intussusception, and as a possible cause of these conditions.

β-thalassemia is a type of inherited hemolytic anemia caused by decreased globin production due to defect of the HBB gene. The pathogenesis of the disease is imbalance of α/β globin chains. The excess of α-globin chains will form hemichromes which can damage red blood cell membranes and lead to hemolysis, ineffective erythropoiesis, and secondary iron overload. Iron overload in turn can cause complications such as growth retardation, liver cirrhosis, cardiac insufficiency, and aggravate the disease phenotype. In recent decades, genes participating in iron metabolism have been discovered, and the mechanism of iron metabolism in the development of thalassemia has gradually been elucidated. Subsequently, by manipulating the expression of key genes in iron metabolism such as hepcidin and transferrin receptor, researchers have revealed that iron restriction can improve ineffective hematopoiesis and iron overload, which may provide a potential approach for the treatment of thalassemia. This article reviews the progress of research on iron metabolism-related genes and related pathways in β-thalassemia.

Additional file 1 Supplementary Table 1. Search strategy.

The artificial liver support system (ALSS) was first applied to treat acute liver failure in the 1970s with an attempt to replace the detoxification functions of the liver. In the past five decades, a variety of different types of ALSS have been developed to bridge patients with liver failure (LF) to liver transplantation or to support the failing liver temporarily, until it is able to regenerate. It has been demonstrated that some types of ALSS, such as plasma exchange (PE) and plasma bilirubin adsorption (PBA), are able to remove toxic substances, improve coagulopathy, and prevent bleeding. How to take the application of ALSS to bridge LF patients to liver transplantation has become a challenge? In this chapter, we summarized the role of ALSS played in perioperative period of liver transplantation patients.

Background Reactivation of fetal hemoglobin (HbF, α2γ2) holds a therapeutic target for β-thalassemia and sickle cell disease. Although many HbF regulators have been identified, the methylation patterns in β-globin cluster driving the fetal-to-adult hemoglobin switch remains to be determined. Results Here, we evaluated DNA methylation patterns of the β-globin cluster from peripheral bloods of 105 β0/β0 thalassemia patients and 44 normal controls. We also recruited 15 bone marrows and 4 cord blood samples for further evaluation. We identified that the CpG sites in the locus control region (LCR) DNase I hypersensitive site 4 and 3 (HS4-3) regions, and γ- and β-globin promoters displayed hypomethylation in β0/β0-thalassemia patients, especially for the patients with high HbF level, as compared with normal controls. Furthermore, hypomethylations in most of CpG sites of the HS4-3 core regions were also observed in bone marrows (BM) of β0/β0-patients compared with normal controls; and methylation level of γ-globin promoter -50 and + 17 CpG sites showed lower methylation level in patients with high HbF level compared with those with low HbF level and a negative correlation with HbF level among β0-thalassemia patients. Finally, γ-globin promoter + 17 and + 50 CpG sites also displayed significant hypomethylation in cord blood (CB) tissues compared with BM tissues from normal controls. Conclusions Our findings revealed methylation patterns in β-globin cluster associated with β0 thalassemia disease and γ-globin expression, contributed to understand the epigenetic modification in β0 thalassemia patients and provided candidate targets for the therapies of β-hemoglobinopathies.

Hepatocellular Carcinoma (HCC) is world fifth most common malignancy and third most common cause of cancer-associated...

Kruppel-like factors (KLFs) are a highly conserved family of transcription factors. We analysed expression profile data of KLFs and identified KLF6 as a new potential regulator of erythropoiesis. Knocking down the expression of KLF6 significantly raised γ-globin mRNA and protein levels in the erythroid cell line HUDEP-2 and haematopoietic progenitor (CD34+ ) cells. We found that overexpression of microRNA (miR)-2355-5p in HUDEP-2 and CD34+ cells correlated with increased γ-globin synthesis by suppressing expression of KLF6. Our discovery that the interaction between miR-2355-5p and KLF6 affects the expression of γ-globin may provide more information for the clinical management of β-thalassaemia patients.

Additional file 1: Phenotypic data of human subjects (Tables S1 and S2) and sequences of primers (Table S3) employed in this study.

The high activity of Histone deacetylases (HDACs) in hepatocellular carcinoma (HCC) usually positively correlates with poor prognosis of patients. Accordingly histone deacetylases inhibitors (HDACis) are considered to be potential agents treating patients with HCC. In our study, we evaluated effect of quisinostat alone and in combination with sorafenib in HCC cells via inducing G0/G1 phase arrest through PI3K/AKT/p21 pathway and apoptosis by JNK/c-Jun/caspase3 pathway in vitro and in vivo. The proliferation assay and flow cytometry were used to measure the viability, cell cycle and apoptosis. And Western blot assay was carried out to determine expression alternations of related proteins. Moreover HCCLM3 xenograft was further performed to detect antitumor effect of quisinostat in vivo. Here, we found that quisinostat impeded cell proliferation, and remarkably induced G0/G1 phase arrest and apoptosis in HCC cells in a dose-dependent manner. G0/G1 phase arrest was observed by alterations in PI3K/AKT/p21 proteins. Meanwhile the JNK, c-jun and caspase-3 were activated by quisinostat in a dose-dependent manner. Correspondingly quisinostat facilitated G0/G1 cycle arrest and apoptosis in HCC cells through PI3K/AKT/p21 pathways and JNK/c- jun/caspase3 pathways. Moreover, the potent tumor-suppressive effects facilitated by quisinostat, was significantly potentiated by combination with sorafenib in vitro and vivo. The combination treatment of quisinostat and sorafenib markedly suppressed cell proliferation and induced apoptosis in a synergistic manner. Moreover the therapy of quisinostat combined with sorafenib could apparently decrease tumor volume of a HCCLM3 xenograft model. Our study indicated that quisinostat, as a novel chemotherapy for HCC, exhibited excellent antitumor activity in vitro and vivo, which was even enhanced by the addition of sorafenib, implying combination of quisinostat with sorafenib a promising and alternative therapy for patients with advanced hepatocellular carcinoma.

Intrahepatic cholangiocarcinoma (ICC) is a fatal, malignant tumor of the liver; effective diagnostic biomarkers and therapeutic targets for ICC have not been identified yet. High expression of H2A histone family member Z (H2A.Z) is a high-risk factor for poor prognosis in patients with breast cancer and primary hepatocellular cancer. However, the significance of H2A.Z and its expression in ICC remains unknown. The present study demonstrated that H2A.Z is overexpressed in ICC and expression of H2A.Z correlated with poor prognosis in patients with ICC. H2A.Z regulated cell proliferation in vitro and in vivo via H2A.Z/S-phase kinase-associated protein 2/p27/p21 signaling. Inhibition of H2A.Z reduced cell proliferation and induced apoptosis in ICC. In addition, downregulation of H2AZ reduced tumor metastasis by repressing epithelial-mesenchymal transition and enhanced the antitumor effects of cisplatin in the treatment of ICC. Overall, H2A.Z promoted cell proliferation and epithelial-mesenchymal transition in ICC, suggesting that H2A.Z may be a novel biomarker and therapeutic target for ICC.