Supplementary Data from Activation of YAP1 by N6-Methyladenosine–Modified circCPSF6 Drives Malignancy in Hepatocellular Carcinoma

Supplementary Data from Activation of YAP1 by N6-Methyladenosine–Modified circCPSF6 Drives Malignancy in Hepatocellular Carcinoma

Abstract Circular RNAs (circRNA) and N6-methyladenosine (m6A) modification are extensively involved in the progression of diverse tumors, including hepatocellular carcinoma (HCC). However, the cross-talk between circRNAs and m6A remains elusive in the pathogenesis of HCC. Here we investigated m6A-mediated regulation of circRNAs in HCC. m6A-related circRNAs were identified by integrating information from two published studies, revealing circular cleavage and polyadenylation specific factor 6 (circCPSF6) as a novel m6A-modified circRNA. circCPSF6 was dominated by ALKBH5-mediated demethylation, followed by the recognization and destabilization by YTHDF2. Meanwhile, circCPSF6 was upregulated in HCC specimens, and elevated circCPSF6 expression served as an independent prognostic factor for worse survival of patients with HCC. Loss-of-function assays demonstrated that circCPSF6 maintained cell proliferation and tumorigenicity and reinforced cell motility and tumor metastasis. circCPSF6 triggered expression of YAP1, further activating its downstream cascade. Mechanistically, circCPSF6 competitively bound PCBP2, blunting its binding to YAP1 mRNA, thereby sustaining the stability of YAP1. Functionally, removal of YAP1 reversed the effects of circCPSF6 in vitro and in vivo. Aberrant activation of the circCPSF6-YAP1 axis promoted HCC malignancy. These findings offer novel insights into the regulation of circRNAs by m6A modifications and the role of this epigenetic reprogramming in HCC. Significance: This study advances the understanding of the interplay between m6A methylation and circRNAs in hepatocellular carcinoma, highlighting the potential of circCPSF6 as a therapeutic target.

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.

Background: Acute cellular rejection (ACR) frequently occurs after liver transplantation (LT) and can result in permanent damage of the liver allograft. Specific and sensitive biomarkers for predicting and monitoring ACR are vital for guiding post-transplantation care. In the present study, we aimed to investigate the function of high-mobility group box 1 (HMGB1) in predicting ACR and prognosis after LT. Material/Methods: A total of 113 LT recipients were enrolled in the study, including 62 patients in an ACR group and 51 patients in a non-rejection group. Using tissues from the 113 patients, HMGB1 expression was examined by immunohistochemistry, and the total score for HMGB1 expression was calculated by multiplying the percentage of immunoreactive cells score and the staining intensity score. We then analyzed the association between HMGB1 expression and clinical features. Finally, the function of HMGB1 in predicting the prognosis of LT was determined using Kaplan-Meier (K-M) survival and Cox multivariate analyses. Results: Immunohistochemical staining results demonstrated that the expression of HMGB1 was significantly increased in the ACR group, compared with that in the non-rejection group (P<0.05). Clinical characteristic analysis revealed that high HMGB1 levels were related to ACR (P<0.05). Moreover, K-M survival analysis showed that patients with high HMGB1 expression displayed poorer prognosis (P<0.05). Cox multivariate analysis demonstrated that HMGB1 was an independent prognostic predictor for post-LT survival (odds ratio, 3.283; P=0.008). Conclusions: LT recipients' HMGB1 levels may be a useful and noninvasive biomarker for the prediction of ACR and prognosis after LT.

BACKGROUND:Our previous study demonstrated that RBBP4 was upregulated in colon cancer and correlated with poor prognosis of colon cancer and hepatic metastasis. However, the potential biological function of RBBP4 in colon cancer is still unknown. AIM:To investigate the biological role and the potential mechanisms of RBBP4 in colon cancer progression. METHODS:Real-time polymerase chain reaction and western blot analysis were used to detect the expression of RBBP4 in colon cancer cell lines. The cell proliferation and viability of SW620 and HCT116 cells with RBBP4 knockdown was detected by Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine staining. The transwell assay was used to detect the invasion and migration capabilities of colon cancer cells with RBBP4 knockdown. Flow cytometry apoptosis assay was used to detect the apoptosis of colon cancer cells. Western blotting analysis was used to detect the expression of epithelial-mesenchymal transition and apoptosis related markers in colon cancer. The nuclear translocation of β-catenin was examined by Western blotting analysis in colon cancer cells with RBBP4 knockdown. The TOPFlash luciferase assay was used to detect the effect of RBBP4 on Wnt/β-catenin activation. The rescue experiments were performed in colon cancer cells treated with Wnt/β-catenin activator LiCl and RBBP4 knockdown. RESULTS:We found that RBBP4 was highly expressed in colon cancer cell lines. The 5-ethynyl-2'-deoxyuridine assay showed that knockdown of RBBP4 significantly inhibited cell proliferation. RBBP4 inhibition reduced cell invasion and migration via regulating proteins related to epithelial-mesenchymal transition. Knockdown of RBBP4 significantly inhibited survivin-mediated apoptosis. Mechanistically, the TOPFlash assay showed that RBBP4 knockdown increased activity of the Wnt/β-catenin pathway. Meanwhile, RBBP4 knockdown suppressed nuclear translocation of β-catenin. With Wnt/β-catenin activator, rescue experiments suggested that the role of RBBP4 in colon cancer progression was dependent on Wnt/β-catenin pathway. CONCLUSION:RBBP4 promotes colon cancer development via increasing activity of the Wnt/β-catenin pathway. RBBP4 may serve as a novel therapeutic target in colon cancer.

Exosomes play an important role in intercellular communication and metastatic progression of hepatocellular carcinoma (HCC). However, cellular communication between heterogeneous HCC cells with different metastatic potentials and the resultant cancer progression are not fully understood in HCC. Here, HCC cells with high-metastatic capacity (97hm and Huhm) were constructed by continually exerting selective pressure on primary HCC cells (MHCC-97H and Huh7). Through performing exosomal miRNA sequencing in HCC cells with different metastatic potentials (MHCC-97H and 97hm), many significantly different miRNA candidates were found. Among these miRNAs, miR-92a-3p was the most abundant miRNA in the exosomes of highly metastatic HCC cells. Exosomal miR92a-3p was also found enriched in the plasma of HCC patient-derived xenograft mice (PDX) model with high-metastatic potential. Exosomal miR-92a-3p promotes epithelial-mesenchymal transition (EMT) in recipient cancer cells via targeting PTEN and regulating its downstream Akt/Snail signaling. Furthermore, through mRNA sequencing in HCC cells with different metastatic potentials and predicting potential transcription factors of miR92a-3p, upregulated transcript factors E2F1 and c-Myc were found in high-metastatic HCC cells promote the expression of cellular and exosomal miR-92a-3p in HCC by directly binding the promoter of its host gene,miR17HG. Clinical data showed that a high plasma exosomal miR92a-3p level was correlated with shortened overall survival and disease-free survival, indicating poor prognosis in HCC patients. In conclusion, hepatoma-derived exosomal miR92a-3p plays a critical role in the EMT progression and promoting metastasis by inhibiting PTEN and activating Akt/Snail signaling. Exosomal miR92a-3p is a potential predictive biomarker for HCC metastasis, and this may provoke the development of novel therapeutic and preventing strategies against metastasis of HCC.

BackgroundMyb-binding protein 1A (Mybbp1a) is a nucleolar protein that can regulate rRNA metabolism, the stress response and carcinogenesis. However, the function of Mybbp1a in the progression of hepatocellular carcinoma (HCC) is unclear.We aimed to determine the role of Mybbp1a in HCC and the underlying mechanism.MethodsWe investigated the function of Mybbp1a in HCC cell models and the xenograft mouse model. The relationship between Mybbp1a and IGFBP5 was found through expression profile chip. The molecular mechanism of Mybbp1a regulating IGFBP5 was proved through CO-IP, CHIP, Bisulfite Sequencing and Pyrosequencing.FindingsIn this study, we observed that Mybbp1a was overexpressed in HCC tissues and associated with the poor prognosis of HCC patients. Suppression of Mybbp1a led to a reduction in the proliferation and migration ability of HCC cells through inhibiting the IGF1/AKT signaling pathway. Further study found that Mybbp1a could form a complex with DNMT1 and induce aberrant hyper-methylation of CpG islands of IGFBP5, which inhibits secretion of IGFBP5 and then activates IGF1/AKT signaling pathway.InterpretationThese findings extend our understanding of the function of Mybbp1a in the progression of HCC. The newly identified Mybbp1a may provide a novel biomarker for developing potential therapeutic targets of HCC.FundScience Technology Department of Zhejiang Province (No. 2015C03034), National Health and Family Planning Commission of China (No. 2016138643), Innovative Research Groups of National Natural Science Foundation of China (No. 81721091), Major program of National Natural Science Foundation of China (No. 91542205).

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.

Liver cancer is a kind of lethal and aggressive malignant neoplasm with a high rate of relapse and metastasis after therapy. An important cause for the relapse and metastasis is the existence of liver cancer stem cells (CSCs), which have high resistance to chemotherapy and high tumorigenic potential. Therefore, it is crucial to develop new methods to eradicate CSCs in tumors. Herein, we develop a photodynamic therapy (PDT) that features bimodal metallacage-loaded nanoparticles (MNPs) for integrated chemotherapy. This platform achieves chemo-photodynamic combinational therapy. Organoplatinum(ii) metallacage-loaded nanoparticles show excellent ability to kill liver CSCs, decreasing their mobility and sphenoid formation ability under near-infrared laser irradiation. Importantly, MNPs can successfully penetrate into 3D tumor spheroids, which display higher drug resistance compared to traditional 2D cultured cells. This destroys CSCs and prevents subsequent tumor formation in vivo. With the excellent combinational therapeutic results in hand, the working mechanisms of MNPs were then studied. MNPs under NIR light irradiation can generate reactive oxygen species (ROS), resulting in damage of mitochondrial membrane and subsequent cell apoptosis with chemotherapeutic platinum. This study proves the great potential of MNPs for combinational cancer therapy, providing a new insight for the next generation of nanomedicines.

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. (C) 2019 Elsevier Masson SAS. All rights reserved.

Background: Retinoblastoma binding protein 4 (RBBP4) plays an essential role in the development of multiple cancers. However, its relationship with prognosis in colon cancer and colon cancer hepatic metastasis has not been elucidated. The aim of this study was to explore the relationship between RBBP4 expression and prognosis of colon cancer patients and to evaluate RBBP4 as a new prognostic marker in these patients. Methods: Eighty colon cancer patients underwent surgical resection of the colon were enrolled. Among them, forty colon cancer patients suffered with hepatic metastasis. The colon cancer tissues, para-colon cancer tissues, and hepatic metastatic cancer tissues were collected from the pathological department for further analysis. The expression of RBBP4 proteins was examined by immunohistochemistry and correlated with clinicopathological parameters. The Cancer Genome Atlas (TCGA) database was used to validate the expression and explore its relationship with clinical characteristics. Results: RBBP4 was up-regulated in the colon cancer tissues compared with the para-colon cancer tissues. The analysis of TCGA database verified the upregulation of RBBP4 in the colon cancer tissues and RBBP4 overexpression was correlated with nerve invasion and poor outcomes of chemotherapy. Moreover, the positive rate of RBBP4 expression in 40 colon cancer patients with hepatic metastasis was higher in the hepatic metastatic cancer tissues (39/40, 97.5%) than in the colon cancer tissues (26/40, 65.0%). Our clinicopathological analysis showed that RBBP4 expression was significantly correlated with vascular invasion, hepatic metastasis, and lymph node involvement (all P < 0.05). Additionally, the survival analysis demonstrated that RBBP4 over-expression was correlated with poor prognosis. Conclusions: RBBP4 was upregulated in the colon cancer. RBBP4 may be a novel predictor for poor prognosis of colon cancer and colon cancer hepatic metastasis. (C) 2019 First Affiliated Hospital, Zhejiang University School of Medicine in China. Published by Elsevier B.V. All rights reserved.

Rationale: Lymphangiomas are benign lymphatic malformations that mostly occur in the neck and axillary regions. Abdominal lymphangioma is a rare type of this tumor, and pancreatic lymphangioma accounts for less than 1% of all lymphangiomas. In this report, we firstly reveal the application of ultrasound-guided puncture drainage combined with cell morphological examination for the diagnosis of pancreatic lymphangioma. Patient concerns: A 35-year-old male patient was admitted to our hospital with recurrent abdominal pain and general weakness for 1 week. From abdominal ultrasound (US) showed that a large cystic lesion occupied the abdomen, about 30.0cm×25.0cm, leading to suspicion of lymphatic cyst. Computed tomography (CT) was performed for further diagnosis and staging. Diagnoses: According to pathological findings in combination with immunohistochemical features, pancreatic lymphangioma was made. Interventions: To relieve symptoms of discomfort in the patient, distal pancreatectomy and splenectomy was carried out 1 week after the CT scan. Outcomes: The patient recovered to normal status within 19 days after surgery. Lessons: The abdominal cystic lymphangiomas are difficult to be differential diagnosed from other cystic lesions. And the origin of the tumor is also hard to be detected before operation. We should combine image and pathological examination to clarify a diagnosis. Although lymphangiomas are benign tumours, they can encroach on adjacent organs and grow to an enormous size and that, resection of these invaded organs may be required for a complete excision.

e24283 Background: PI3K/mTOR signaling pathway plays a central role in cancer biology. Previously studies had shown that mTOR inhibitors might be a promising therapeutic approach in patients (pts) with PIK3CA/PTEN mutations. Pan-cancer study about characteristics of PIK3CA/PTEN variations in Chinese population had not been fully evaluated. Methods: FFPE tumor and matched blood samples of 515 Chinese pts including 317 males and 198 females with a median age of 58 years were collected for NGS based 450-gene panel assay. The whole cohort included 314 lung adenocarcinomas (ADC), 128 colorectal carcinomas (CRC) and 73 hepatocellular carcinomas (HCC). Genomic alterations including single base substitutions, short and long insertions/deletions, copy number variations, gene rearrangements in selected genes were assessed. Results: PIK3CA variations were detected in 8.7% of Chinese cancer pts, in which the hotspot mutations included E542, E545 and H1047 accounted for 71.1%. PTEN variations were detected in 4.5% of the pts, whereas 6 pts had both PIK3CA and PTEN mutations. The rates of PIK3CA/PTEN variations in CRC, ADC and HCC were 23.4%, 8.6% and 6.7%, respectively. Compared with TCGA dataset, more PIK3CA/PTEN alterations were ovserved in Chinese CRC patients than Western ones (23.4% vs 12.1%), but similar frequency in ADC (8.6% vs 6.1%) and HCC (6.7% vs 7.1%). In CRC patients with PIK3CA/PTEN mutations, 63.3% and 53.3% of them were found possessing mutations in KRAS and APC respectively. 14 pts with PIK3CA or PTEN alterations including 8 NSCLC, 3 cholangiocarcinoma, 1 lung neuroendocrine carcinoma, 1 soft tissue sarcoma and 1 osteosarcoma had been treated with Everolimus. Two pts (both NSCLC) achieved partial response (PR), and 9 pts showed stable disease (SD), resulting in a disease control rate (DCR) of 78%. Conclusions: Our study revealed the pan-cancer genomic profile differed in different cancer types, more PIK3CA/PTEN mutated in Chinese CRC pts than western population. The retrospective analysis of efficacy of everolimus monotherapy in a small cohort of pts with PIK3CA/PTEN alterations across various cancers showed DCR of 78%, which indicated the regimen was effective in PI3K/mTOR pathway activated solid tumors.

BACKGROUND:Accumulating evidence demonstrates that microRNAs (miRNAs) play essential roles in tumorigenesis and cancer progression of hepatocellular carcinoma (HCC). Average targets of a miRNA were more than 100. And one miRNA may act in tumor via regulating several targets. The present study aimed to explore more potential targets of miR-449a by proteomics technology and further uncover the role of miR-449a in HCC tumorigenesis.METHODS:Technologies such as iTRAQ-based quantitative proteomic were used to investigate the effect of miR-449a on HCC. The expression of c-Met and miR-449a was detected by qRT-PCR in HCC samples. Gain- and loss-of-function experiments were performed to identify the function and potential target of miR-449a in HCC cells.RESULTS:In HCC, miR-449a was significantly downregulated, while c-Met was upregulated concurrently. Quantitative proteomics and luciferase reporter assay identified c-Met as a direct target of miR-449a. Moreover, miR-449a inhibited HCC growth not only by targeting CDK6 but also by suppressing c-Met/Ras/Raf/ERK signaling pathway. Furthermore, the inhibition of c-Met expression with a specific siRNA significantly inhibited cells growth and deregulated the ERK pathway in HCC.CONCLUSION:The tumor suppressor miR-449a suppresses HCC tumorigenesis by repressing the c-Met/ERK pathway.

This study aims to investigate how microRNA-375 (miR-375) improves immune function by regulating liver macrophages (Kupffer cells) in mice with liver failure. Forty mice were divided into ConA-1h, ConA-3h, ConA-6h, and control groups, with 10 mice in each group. Mice models of liver failure were established by injecting concanavalin A (ConA) solution via the tail veins of mice, and then primary Kupffer cells were isolated and cultured. Reverse transcription quantitative polymerase chain reaction, Western blot analysis, and enzyme-linked immunosorbent assay were conducted to examine the expressions of miR-375, astrocyte elevated gene-1 (AEG-1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and IL-1β in Kupffer cells of mice with liver failure as well as after silencing of miR-375. Flow cytometry was used to determine cell apoptosis. During the liver failure process, miR-375, IL-6, TNF-α, and IL-1β expressions were increased over time, while AEG-1 expression decreased over time in the control, ConA-1h, ConA-3h, and ConA-6h groups. Opposite alternations were observed after silencing of miR-375. Dual-luciferase reporter gene assay showed that AEG-1 was a target gene of miR-375. Flow cytometry determination showed that the ratio of apoptotic Kupffer cells decreased after silencing of miR-375. Overexpression of AEG-1 could rescue the suppression of IL-6, TNF-α, and IL-1β expressions in Kupffer cells after the short-term induction of ConA and further inhibit cell apoptosis. Our study provides evidence that miR-375 could regulate Kupffer cells to improve immune function in mice with liver failure.

Background/Aims: Pancreatic cancer is an aggressive malignancy as a result of highly metastatic potential. The current study was carried out to alter the expression of LINC01121 in pancreatic cancer, with the aim of elucidating its effects on the biological processes of cell proliferation, migration, invasion, and apoptosis. We hypothesized that both the GLP1R gene and cAMP/PKA signaling pathway participate in the aforementioned process. Methods: Microarray data (GSE14245, GSE27890 and GSE16515) and annotating probe files linked to pancreatic cancer were downloaded through the GEO database. The Multi Experiment Matrix (MEM) site was used to predict the target gene of lncRNA. Both pancreatic cancer tissues (n = 56) and paracancerous tissues (n = 45) were collected from patients diagnosed with pancreatic cancer. Immunohistochemistry was applied to identify the positive expression rate of GLP1R protein. Isolated pancreatic cancer cells and PANC-1 cells were independently classified into the blank, negative control (NC), LINC01121 vector, siRNA-LINC01121, siRNA-GLP1R and siRNA-LINC01121 + siRNA-GLP1R groups. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis were applied to detect the expressions of LINC01121, GLP1R, cAMP, PKA, CREB, Bcl-2, Bad and PCNA. Cell proliferation, migration, invasion, cycle progression, and apoptosis were examined by MTT assay, scratch test, Transwell assay and flow cytometry analyses of Annexin V-FITC/PI staining. Results: Observations were made indicating that LINC01121 was highly expressed, while low expressions of GLP1R in pancreatic cancer were detected based on microarray data, which was largely in consistent with the data collected of LINC01121 and GLP1R within the tissues. The target prediction program and luciferase activity analysis was testament to the notion suggesting that GLP1R was indeed a target of LINC01121. In contrast to the blank and NC groups, the LINC01121 vector group exhibited increased expressions of LINC01121; decreased mRNA and protein levels of GLP1R, Bad, cAMP, and PKA; increased protein levels of CREB, Bcl-2, PCNA, p-PKA and p-CREB; increased cell proliferation, migration and invasion; and decreased cell apoptosis. There was no significant difference detected among the blank, NC, and siRNA-LINC01121 + siRNA-GLP1R groups, except that decreased LINC01121 expression was determined in the siRNA-LINC01121 + siRNA-GLP1R group. Parallel data were observed in the pancreatic cancer cells and PANC-1 cells. Conclusion: The current study presents evidence indicating that LINC01121 might inhibit apoptosis while acting to promote proliferation, migration, and invasion of pancreatic cancer cells, supplementing the stance held that LINC01121 functions as a tumor promoter by means of its involvement in the process of translational repression of the GLP1R and inhibition of the cAMP/PKA signaling pathway.

The aim of the study is to demonstrate the effect of Romidepsin in hepatocellular carcinoma (HCC) by inducing G2/M phase arrest via Erk/cdc25C/cdc2/cyclinB pathway and apoptosis through JNK/c-Jun/caspase3 pathway in vitro and in vivo. Human HCC cell lines were cultured with Romidepsin and DMSO (negative control) and 5-fluorouracil (positive control). Then the cells' viability and apoptosis were determined by cell proliferation assay and flow cytometry. Protein concentrations and expression changes were measured by Western blot. Subsequently, Huh7 cells were subcutaneously inoculated into the nude mice, which were employed to further probe the tumor-suppressive effect of Romidepsin in vivo. Romidepsin treatment led to a time- and dose-dependent induction of cell cycle arrest in the G2/M phase and apoptosis. G2/M phase arrest inhibited the proliferation of HCC cells by alterations in p21/cdc25C/cdc2/cyclinB proteins. Increased concentrations of Erk and JNK phosphorylations were observed in a dose-dependent manner in the Romidepsin group, but p38 phosphorylation was not affected. G2/M phase arrest and the apoptosis of HCC cells induced by Romidepsin were mediated by the activation of Erk/MAPK pathways and JNK/MAPK pathways. The tumor size was significantly larger in the negative control group compared to Romidepsin group and no significant loss in body weight was observed in the Romidepsin group. Our findings offer proof-of-concept for use of Romidepsin as a novel class of chemotherapy in the treatment of HCC.

A lack of effective prognostic biomarkers and molecular targets is a serious problem in hepatocellular carcinoma. KCTD11, reported as a tumor suppressor, are still not well understood. In this study, KCTD11 was found low-expressed in HCC tissues and cell lines. The HCC patients with low expression of KCTD11 suggested shorter overall survival. We found KCTD11 inhibiting cell proliferation in vitro and tumor growth in vivo, by activating p21 and repressing cycle related proteins. KCTD11 also inhibited cell adhesion by decreasing CTGF and CLDN1. We found CTGF binding COL3A1 in HCCLM3, which might lead to reduction of COL3A1 expression. KCTD11 also inhibited cell migration and invasion in HCC, by repressing MMPs and EMT. We found the tumor suppression function of KCTD11 was at least partly through activating Hippo pathway in HCC. Base on the enhanced Hippo pathway, KCTD11 could activate p21 by stabilizing p53 or promoting the MST1/ GSK3β/p21 signaling in HCC. Overall, these results suggest that KCTD11 works as a tumor suppressor and owns prognostic and therapeutic potentials in HCC.