Loss of androgen receptor promotes HCC invasion and metastasis via activating circ-LNPEP/miR-532-3p/RAB9A signal under hypoxia.

Development of novel targeted therapies remains the priority in hepatocellular carcinoma (HCC) treatments. Early reports have demonstrated that androgen receptor (AR) plays a suppressive role in HCC progression. However, the underlying mechanisms by which AR attenuates HCC development are still elusive, especially under hypoxic conditions. Herein, we demonstrated that AR/circ-LNPEP/miR-532-3p/RAB9A signaling axis was tightly involved in hypoxia-induced cell invasion of HCC cells. AR worked as a transcription factor to reduce circ-LNPEP expression level, which released its sponge potential of miR-532-3p, leading to the downregulation of RAB9A and inhibiting cell invasion of HCC cells. In vitro and in vivo animal model also confirmed that overexpression of circ-LNPEP could reverse the suppressive effect of AR on HCC cell invasion or tumor metastasis. Overall, our study supplements a critical mechanism by which AR suppresses HCC invasion/metastasis under hypoxic conditions, providing compelling rationale to develop novel therapy for better treatments of HCC.

RNF2 inhibits E-Cadherin transcription to promote hepatocellular carcinoma metastasis via inducing histone mono-ubiquitination.

RNF2 is a RING domain-containing E3 ubiquitin ligase that mediate histone H2A mono-ubiquitination to repress gene transcription, but its expression patterns and molecular function in hepatocellular carcinoma (HCC) remain unclear. Herein, we extracted data from TGCA database and validated RNF2 expression in our own cohort, which revealed that RNF2 was highly expressed in HCC and was associated with malignant characteristics and poor prognosis of HCC. Moreover, RNF2 was demonstrated to promote HCC metastasis via enhancing epithelial-mesenchymal transition (EMT) both in vitro and in vivo. Mechanistically, RNF2 repressed E-Cadherin transcription by increasing the deposition of H2K119ub at the E-Cadherin promoter region. In addition, RNF2-regulated crosstalk between H2AK119ub, H3K27me3 and H3K4me3 synergistically reduced E-Cadherin transcription, which promoted EMT and HCC metastasis. These results indicate that RNF2 played an oncogenic role in HCC progression via inducing EMT, and RNF2 could be a potential therapeutic target for HCC.

A comprehensive analysis of FOX family in HCC and experimental evidence to support the oncogenic role of FOXH1.

Hepatocellular carcinoma (HCC) remains the second leading cause of cancer related deaths worldwide. Understanding about the molecular biology of HCC and development of targeted therapies are still the main focuses of this type of disease. Here, by connecting the expression levels of FOX proteins with their associated clinical characteristics using TCGA LIHC dataset, we found that 27/40 FOX proteins were highly expressed in HCC tumors compared to normal liver tissues and their expression levels were tightly associated with HCC tumor stage, tumor grade and overall survival. Our experimental results also confirmed that FOXH1 indeed played an oncogenic role in HCC development by promoting cell growth and cell migration/invasion. Mechanistic dissection demonstrated that FOXH1-induced cell growth and cell migration/invasion relied on mTOR signaling because inhibition of mTOR signaling by rapamycin could attenuate FOXH1-mediated phenotypic alterations of HCC cells. The results from orthotopic mouse model also validated that FOXH1 promoted HA22T tumor growth via triggering mTOR activation. Overall, this study not only comprehensively examines the clinical values of FOX proteins in HCC but also provides experimental evidence to support the role of FOXH1 in HCC development, building rationale to develop more effective therapies to treat HCC patients.

Mechanism of Liver Regeneration During ALPPS.

Liver cancer is one of the most lethal malignant tumors in the world, and surgical resection is the main treatment for liver cancer. Liver failure due to insufficient residual liver volume is a fatal complication after hepatectomy. How to effectively increase the residual liver volume after hepatectomy and improve the safety of hepatectomy has always been a problem to be solved in liver surgery. Associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) effectively reduces the occurrence of liver failure due to insufficient residual liver volume after hepatectomy, thereby increasing the probability of radical resection by inducing rapid proliferation of residual liver tissue. However, the molecular mechanism of residual liver tissue regeneration after primary ALPPS (combined liver partition and portal vein ligation) remains unclear. Here, we found that lots of circular RNAs (circRNAs) are upregulated after ALPPS in pig liver cells; then, we identified the orthologous circRNA in humans and pigs to detect their function in liver regeneration. The results showed that loss of circ-0067724 and circ-0016213 could suppress liver cell proliferation. Together, these findings suggest that circ-0067724 and circ-0016213 play an important role in liver cell proliferation, and this may help us to find new strategies to promote liver regeneration.

MAD2L1 Functions As a Novel Diagnostic and Predictive Biomarker in Cholangiocarcinoma.

Background: Most cholangiocarcinoma (CCA) patients are diagnosed at an advanced stage of disease, and the postoperational recurrence rates are high in those undergoing resection. The lack of satisfying biomarkers for early diagnoses and effective targeting of driver pathways is the leading reason for therapeutic failures. The goal of this study was to find a biomarker for making improved diagnoses with enhanced prognostic capabilities for CCA. Materials and Methods: Our study used bioinformatic analyses of microarray data from the Gene Expression Omnibus (GEO) database and investigated mitotic arrest deficient 2-like protein 1 (MAD2L1) expression in tumor and adjacent non-neoplastic biliary ducts through immunocytochemistry in 42 surgically removed primary CCAs from a single institute. In vitro and in vivo models were used to explore the function of MAD2L1. Results: In total, 297 high probability differentially expressed genes (DEGs) were obtained from overlapping the DEGs from the three individual data sets. Through enrichment assays and protein-protein interaction networks analyses, seven hub genes were identified. MAD2L1 was picked up as a novel biomarker based on hierarchical cluster analyses and Kaplan-Meier survival analyses. MAD2L1 was expressed in cancer tissues but not in the surrounding normal tissue, with 31 (73.81%) of 42 CCAs MAD2L1 positive by immunohistochemistry (IHC). MAD2L1 expression levels were significantly correlated with tumor size, pathological grade, and clinical stage. A Kaplan-Meier survival analysis demonstrated an inverse correlation with MAD2L1 expression. Real-time polymerase chain reaction and immunoblotting results further confirmed the results of IHC and bioinformatic analyses. In vitro and in vivo models demonstrated decreasing MAD2L1 could significantly suppress tumor growth, whereas increasing MAD2L1 could promote tumor growth. Conclusion: MAD2L1 could be used as a biomarker to predict prognosis and potential therapeutic target in CCA. Clinical Trial Registration Number: [2020]KY157-01.

R-2HG downregulates ERα to inhibit cholangiocarcinoma via the FTO/m6A-methylated ERα/miR16-5p/YAP1 signal pathway.

Isocitrate dehydrogenase (IDH) mutations increase (R)-2-hydroxyglutarate (R-2HG) production; however, functional mechanisms of R-2HG in regulating cholangiocarcinoma (CCA) development remain to be further investigated. We first applied the CRISPR-Cas9 gene-editing system to create IDH1R132H-mutated CCA cells. Interestingly, our data showed that R-2HG could function through downregulating estrogen receptor alpha (ERα) and Yes-associated protein 1 (YAP1) pathways to decrease CCA growth. Detailed mechanistic studies revealed that R-2HG could target and degrade the fat mass and obesity-associated protein (FTO), the first identified mRNA demethylase. This reduced FTO can increase the N 6-methyladenosine (m6A) to methylate the mRNA of ERα, and consequently decrease protein translation of the ERα. Further mechanistic studies revealed that ERα could transcriptionally suppress miR-16-5p expression, which could then increase YAP1 expression due to the reduced miR-16-5p binding to the 3' UTR of YAP1. Furthermore, data from the pre-clinical animal model with implantation of IDH1R132H QBC939 cells demonstrated that R-2HG generated by the IDH1 mutation could downregulate ERα and YAP1 to suppress CCA tumor growth. Taken together, our new findings suggested that IDH1 mutation-induced R-2HG could suppress CCA growth via regulating the FTO/m6A-methylated ERα/miR16-5p/YAP1 signaling pathway. Upregulating R-2HG or downregulating the ERα signal by short hairpin RNA ERα (shERα) or antiestrogen could be effective strategies to inhibit CCA.

Loss of ARID1A Promotes Hepatocellular Carcinoma Progression via Up-regulation of MYC Transcription.

BACKGROUND AND AIMS: AT-rich interactive domain-containing protein 1A (ARID1A) is frequently mutated or deficient in hepatocellular carcinoma (HCC). However, the role of ARID1A in HCC remains unclear. Therefore, the biological role of ARID1A in HCC was evaluated and a potential mechanism was investigated. METHODS: Arid1a was knocked out in the livers of mice using the CRISPR/Cas9 system delivered by hydrodynamic tail vein injection. The development of HCC was observed in different mouse models. The correlation of ARID1A and prognosis in patients with HCC was analyzed using cBioPortal. The effect of ARID1A on cell proliferation was assessed by MTT assay following the manipulation of candidate genes. RESULTS: ARID1A deficiency alone did not cause HCC in mice, but knockout of ARID1A accelerated liver tumorigenesis in response to diethylnitrosamine (DEN) or when a combination knockout of phosphatase and tensin homolog (Pten) plus tumor protein P53 (p53) was introduced. ARID1A mutations were associated with a poorer prognosis in HCC patients. The mRNA level of MYC was significantly higher in patients with an ARID1A mutation compared to those without a mutation. Ectopic expression of ARID1A inhibited HCC cell proliferation. ARID1A knockout increased HCC cell growth and resulted in disruptions to DNA damage repair and apoptosis following radiation stress. Furthermore, mechanistic studies revealed that ARID1A inhibited the proliferation of HCC cells via transcriptional down-regulation of MYC. CONCLUSIONS: These results describe ARID1A as a tumor suppressor in the liver. A deficiency in ARID1A predicts worse survival in HCC patients and promotes HCC progression via up-regulation of MYC transcription.

E2F3 promotes liver cancer progression under the regulation of circ-PRKAR1B.

Liver cancer is one of the most lethal malignant tumors in the world. The high recurrence and mortality rate make it urgent for scientists and clinicians to find new targets for better treatment of liver cancer. Here, we found that circ-PRKAR1B expression was increased in the paired intrahepatic metastasis sample through high-throughput sequencing. Further experiments also confirmed its high expression both in carcinoma and metastasis when compared to the paired para-carcinoma and the paired carcinoma, respectively. Mechanism study showed that circ-PRKAR1B could promote liver cancer progression through the miR-432-5p/E2F3 pathway, and microRNA-432-5p could directly target the 3' untranslated region (UTR) of E2F3 mRNA to suppress its translation, thereby influencing liver cancer cell invasion and migration capacities. Clinical data obtained by using online databases based on The Cancer Genome Atlas (TCGA) samples and the clinicopathological data of liver cancer patients who underwent surgery in our hospital in the past 2 years also confirmed the significance of circ-PRKAR1B/miR-432-5p/E2F3 signaling in liver cancer progression. Animal experiments also indicated that targeting this newly identified signaling by overexpressing microRNA-432-5p could suppress the progression of liver cancer. Together, our study suggests that circ-PRKAR1B plays an important role in the regulation of liver cancer progression, and targeting this new circ-PRKAR1B/miR-432-5p/E2F3 signaling may help us find new treatment strategies to better suppress liver cancer progression.

Androgen receptor (AR) decreases HCC cells migration and invasion via miR-325/ACP5 signaling.

Hepatocellular carcinoma (HCC) is the most 5th commonly diagnosed and 2nd most lethal tumor in the world. The obvious gender advantage of HCC indicates that androgen receptor (AR) may play an important role in the tumor occurrence, develop and metastasis of HCC. Here we found that decreased AR could alter miR-325 to increase ACP5 expression in HCC cells, to increase HCC cells migration and invasion capacities. Mechanism dissection revealed that AR could regulate miR-325 expression through transcriptional regulation and miR-325 might directly target the 3'UTR of ACP5-mRNA to suppress its translation. The in vivo orthotopic xenografts mouse model with oemiR-325 also validated in vitro data. Together, these findings suggest that AR may decrease HCC progression through miR-325/ACP5 signaling and targeting the AR/miR-325/ACP5 signaling may help in the development of the novel therapies to better suppress the HCC progression.

Testicular orphan receptor 4 (TR4) promotes papillary thyroid cancer invasion via activating circ-FNLA/miR-149-5p/MMP9 signaling.

The incidence and mortality of papillary thyroid cancer (PTC) have steadily increased. Although conventional therapies are very effective toward differentiated PTC patients, very limited therapeutic options are applicable to those patients with distant metastases. Therefore, better understanding of the molecular biology of metastatic PTC helps identify novel targets and facilitates the development of new therapies. In this study, we first found that testicular orphan receptor 4 (TR4) was significantly increased in PTC tumors spreading to lymph nodes compared to the paired primary tumors. Experimental evidence suggested that TR4 drove PTC progression via promoting its cell invasion and cell migration. Mechanistically, TR4 transcriptionally regulated the expression level of circ-filamin A (FLNA), which competed with matrix metalloproteinase 9 (MMP9) for microRNA (miR)-149-5p binding and led to an increased protein level of MMP9. Interruption assays with various gene manipulations verified that the TR4/circ-FLNA/miR-149-5p/MMP9 signaling axis played a central role in cell invasion and cell migration of PTC cells. Moreover, a xenografted mouse model also confirmed that the TR4/circ-FLNA signal promoted PTC tumor growth. Overall, our study pinpoints the oncogenic role of TR4 in PTC development, and the targeting of TR4/circ-FLNA/miR-149-5p/MMP9 signaling may be an alternative option for metastatic PTC patients.

Elevated CELSR3 expression is associated with hepatocarcinogenesis and poor prognosis.

Cadherin EGF LAG seven-pass G-type receptor 3 (CELSR3) has been reported to exhibit a cancer-specific pattern. The present study aimed to investigate the clinical value and functional role of CELSR3 in hepatocellular carcinoma (HCC), and determine the underlying molecular mechanism in patients with HCC. CELSR3 expression in tumor and paracancerous HCC tissues was obtained from The Cancer Genome Atlas. Differential expression analysis was performed using the edgeR package. Pearson correlation analysis was used to analyze the correlation between methylation and mRNA levels of CELSR3. Pathways affected by aberrant CELSR3 expression were identified through Gene Set Enrichment Analysis. The results demonstrated that CELSR3 was highly expressed in the early stage of cancer and was present throughout the entire cancer process, which suggested that CELSR3 may serve a key role in the carcinogenesis of HCC. In addition, upregulation of CELSR3 was associated with its methylation level; high CELSR3 expression indicated a shorter overall survival time. Multiple candidate genes were screened by integrating differentially expressed (DE) mRNAs and target genes of DE microRNAs (miRs). Subsequent pathway enrichment analysis demonstrated that the upregulated genes were predominantly enriched in the 'Neuroactive ligand-receptor interaction' and 'Cell cycle' pathways, whereas the downregulated genes were primarily enriched in 'Cytokine-cytokine receptor interaction' and 'Metabolic pathways'. CELSR3 and its connected nodes and edges were initially removed from the miRNA-mRNA regulatory network in order to prevent bias and compared with the network containing CELSR3 alone. The frequently dysregulated miRNAs were identified as miR-181 family members, and the results suggested that miR-181 and the Wnt/ß-catenin signaling pathway influenced CELSR3 expression.

SEMA3F Promotes Liver Hepatocellular Carcinoma Metastasis by Activating Focal Adhesion Pathway.

Previous studies have shown that semaphorin-3F (SEMA3F) functions as a tumor suppressor in several tumor types. However, the role of SEMA3F in the metastasis and prognosis of liver hepatocellular carcinoma (LIHC) remains unknown. In this study, by performing bioinformatics analysis on the transcriptome profiles from The Cancer Genome Atlas (TCGA), we demonstrated that SEMA3F was significantly upregulated in LIHC tissues, compared with normal controls. Moreover, the expression value of SEMA3F was positively correlated with patients' pathological stages and tumor metastasis, predicting a poor overall survival. Besides, SEMA3F expression level was negatively correlated with its methylation level, but positively correlated with its gene copy number. Differential expression analysis of LIHC samples with high or low SEMA3F expression values suggested that 983 genes were differentially expressed, among which 723 genes were upregulated and 260 genes were downregulated. Furthermore, enrichment analysis of differentially expressed genes revealed that SEMA3F was involved in the activation of focal adhesion pathway, which induced tumor metastasis. Taken together, our results suggested that the oncogenic function of SEMA3F promoted hepatocellular carcinoma metastasis by activating focal adhesion pathway.

IGF2BP3 and miR191-5p synergistically increase HCC cell invasiveness by altering ZO-1 expression.

Early studies have indicated that insulin-like growth factor II mRNA binding protein 3 (IGF2BP3/IMP3) may affect the progression of hepatocellular carcinoma (HCC); however, the detailed underlying mechanisms, particularly its linkage to tight junction protein-mediated cell invasion, remain unclear. The present study revealed that IGF2BP3 increased HCC cell invasiveness by suppressing zonula occludens-1 (ZO-1) expression, via direct binding to the 3' untranslated region (3'-UTR). Analysis of the molecular mechanisms demonstrated that IGF2BP3 binds to the overlapping targets of IGF2BP3-RNA cross-linkage and microRNA (miR)191-5p targeting sites, and promotes the formation of an miR191-5p-induced RNA-induced silencing complex. The knockdown of IGF2BP3 or the addition of a miR-191-5p inhibitor decreased cellular invasiveness and increased ZO-1 expression. Analysis of the human HCC database also confirmed the association between IGF2BP3 and HCC progression. Collectively, these preclinical findings suggest that IGF2BP3 increases HCC cell invasiveness by promoting the miR191-5p-induced suppression of ZO-1 signaling. This newly identified signaling effect on small molecule targeting may aid in the development of novel strategies with which to inhibit HCC progression more effectively.

M2 Macrophages Promote HCC Cells Invasion and Migration via miR-149-5p/MMP9 Signaling.

The roles of M2 macrophages on promoting tumor progression and chemotherapy resistance have been well studied in many cancers, such as pancreatic cancer, kidney cancer and so on, but its linkage to HCC cells still remains unclear. Here we found that M2 macrophages could alter miR-149-5p to increase MMP9 expression in HCC cells and mechanism dissection revealed that miR-149-5p might directly target the 3'UTR of MMP9-mRNA to suppress its translation. The in vivo orthotopic xenografts mouse model with oemiR-149-5p also validated in vitro data. Together, these findings suggest that M2 macrophages may through altering the miR-149-5p to promote HCC progression and targeting the M2 macrophages/miR149-5P/MMP9 signaling may help in the development of the novel therapies to better suppress the HCC progression.

The miR-92a-2-5p in exosomes from macrophages increases liver cancer cells invasion via altering the AR/PHLPP/p-AKT/ß-catenin signaling.

Early studies indicated that the androgen receptor (AR) might play important roles in the regulating of the initiation and progression of hepatocellular carcinoma (HCC), but its linkage to the surrounding macrophages and their impacts on the HCC progression remain unclear. Here we found that macrophages in liver cancer might function via altering the microRNA, miR-92a-2-5p, in exosomes to decrease liver cancer cells AR expression, which might then lead to increase the liver cancer cells invasion. Mechanism dissection revealed that miR-92a-2-5p from the exosomes could target the 3'UTR of AR mRNA to suppress AR translation, altering the PHLPP/p-AKT/ß-catenin signaling to increase liver cancer cells invasion. Preclinical studies demonstrated that targeting this newly identified signaling with miR-92a-2-5p inhibitors led to suppress liver cancer progression. Together, these findings suggest that macrophages in the liver cancer tumor microenvironment may function via exosomes to regulate liver cancer progression, and targeting this newly identified macrophages/exosomes-miR-92a-2-5p/AR/PHLPP/p-AKT/ß-catenin signaling may help in the development of novel treatment strategies to better suppress liver cancer progression.

Targeting the estrogen receptor alpha (ERα)-mediated circ-SMG1.72/miR-141-3p/Gelsolin signaling to better suppress the HCC cell invasion.

Early studies indicated that estrogen receptor α (ERα) might impact the progression of hepatocellular carcinoma (HCC). However, the detailed mechanisms, especially its linkage to the gelsolin (GSN)-mediated cell invasion, remain unclear. Here we found that ERα could decrease HCC cell invasion via suppressing the circular RNA-SMG1.72 (circRNA-SMG1.72) expression via transcriptional regulation through directly binding to the 5' promoter region of its host gene SMG1, We showed that ERα-suppressed circ-SMG1.72 could sponge and inhibit the expression of the microRNA (miRNA, miR), miR-141-3p, which could then result in increasing the GSN messenger RNA translation via reduced miR binding to its 3' untranslated region (3'UTR). The preclinical study using an in vivo mouse model with orthotopic xenografts of HCC cells confirmed the in vitro data, and the human HCC clinical sample survey and tissue staining also confirmed the linkage of ERα/miR-141-3p/GSN signaling to the HCC progression. Together, our findings suggest that ERα can suppress HCC cell invasion via altering the ERα/circRNA-SMG1.72/miR-141-3p/GSN signaling, and targeting this newly identified signaling with small molecules may help in the development of novel therapies to better suppress the HCC progression.