Yuan-Yuan Sheng

MicroRNA-26a suppresses tumor growth and metastasis of human hepatocellular carcinoma by targeting interleukin-6-Stat3 pathway†‡

Down‐regulation of microRNA‐26a (miR‐26a) is associated with poor prognosis of hepatocellular carcinoma (HCC), but its functional mechanism in HCC remains unclear. In this study, we investigated the roles of miR‐26a in tumor growth and metastasis of HCC and found that miR‐26a was frequently down‐regulated in HCC tissues. Down‐regulation of miR‐26a correlated with HCC recurrence and metastasis. Through gain‐ and loss‐of‐function studies, miR‐26a was demonstrated to significantly inhibit in vitro cell proliferation, migration, and invasion. In addition, miR‐26a induced G1 arrest and promoted apoptosis of HCC cells. Importantly, miR‐26a suppressed in vivo tumor growth and metastasis in nude mice models bearing human HCC. Interleukin‐6 (IL‐6) was identified as a target of miR‐26a. Knockdown of IL‐6 induced effects on HCC cells similar to those induced by miR‐26a. In contrast, IL‐6 treatment abrogated the effects induced by miR‐26a up‐regulation. Moreover, miR‐26a dramatically suppressed expression of signal transducer and activator of transcription 3 (Stat3) target genes, including Bcl‐2, Mcl‐1, cyclin D1, and MMP2. IL‐6 treatment antagonized this effect, while knockdown of IL‐6 by IL‐6 short hairpin RNA (shIL‐6) induced inhibitory effects on the expression of p‐Stat3 and its main target genes, similar to miR‐26a. The messenger RNA and protein levels of IL‐6 inversely correlated with miR‐26a in HCCs. Patients with high miR‐26a or low IL‐6 in HCC tissues had a better prognosis with longer overall survival (OS) and time to recurrence (TTR). In multivariate analysis, miR‐26a, IL‐6, and their combination were demonstrated to be independent prognostic indicators for OS and TTR of HCC patients. Conclusion: miR‐26a could suppress tumor growth and metastasis of HCC through IL‐6‐Stat3 signaling and is a novel prognostic marker and therapeutic target for HCC. (HEPATOLOGY 2013)

Osteopontin promotes epithelial-mesenchymal transition of hepatocellular carcinoma through regulating vimentin.

Our previous studies have found that osteopontin (OPN) is a promoter for hepatocellular carcinoma (HCC) progression. However, the molecular mechanism by which OPN enhances HCC metastasis remains elusive. Epithelial-mesenchymal transition (EMT) of cancer cells plays a pivotal role in promoting metastatic process. In this study, we demonstrated that OPN promotes HCC metastasis by inducing an EMT-like, more aggressive cellular phenotype in vitro and in vivo. Furthermore, OPN was identified to interact with vimentin by reciprocal OPN and vimentin immunoprecipitation as well as co-immunofluorescence examination. By using deletion mutants, we found that the residues between 246 and 406 in vimentin are required for binding to OPN. Importantly, OPN significantly increased vimentin stability through inhibition of its protein degradation. Knockdown of vimentin neutralized the EMT induced by OPN both in vitro and in vivo. Moreover, a significant correlation between OPN and vimentin levels was found in clinical HCC specimens and their combination had a worse prognosis with shorter overall survival (OS) and time to recurrence (TTR). In multivariate analysis, OPN and their combination were demonstrated to be independent prognostic indicators for OS and TTR of HCC patients. Collectively, this study indicates that OPN can induce EMT of HCC cells through increasing vimentin stability, which provides more in-depth understanding about the molecular mechanisms of OPN in promoting HCC metastasis and opens tantalizing therapeutic possibilities in HCC.

FOXQ1/NDRG1 axis exacerbates hepatocellular carcinoma initiation via enhancing crosstalk between fibroblasts and tumor cells

Cancer associated fibroblast (CAF) is a well-known microenvironment contributor for the development of hepatocellular carcinoma (HCC), while forkhead box (FOX) proteins are also critical to exacerbate HCC malignancy. However, whether FOX proteins are involved in the crosstalk between CAFs and HCC cells remains unclear. In the present study, we reveal that CAFs induce forkhead box Q1 (FOXQ1) expression, and N-myc downstream-regulated gene 1 (NDRG1) is therefore trans-activated to enhance HCC initiation. Intriguingly, pSTAT6/C-C motif chemokine ligand 26 (CCL26) signaling is induced by FOXQ1/NDRG1 axis, thus recruiting hepatic stellate cells (HSCs), the main cellular source of CAFs, to the tumor microenvironment. Thereby, tumor initiating properties are enhanced at least partly through a positive feedback loop between CAFs and HCC cells. Importantly, leflunomide, a pSTAT6 inhibitor that has been approved for the treatment of rheumatoid arthritis, significantly blocks the loop and HCC progression. High expression of CAF marker, ACTA2, and induced FOXQ1/NDRG1 axis in HCC tissues predict unfavorable prognosis. Collectively, our findings uncover a positive feedback loop between CAFs and FOXQ1/NDRG1 axis in neoplastic cells to drive HCC initiation, thus providing new potential therapeutic targets for HCC.

Whole-exome mutational and transcriptional landscapes of combined hepatocellular cholangiocarcinoma and intrahepatic cholangiocarcinoma reveal molecular diversity

BACKGROUND Primary liver cancer (PLC) is the third largest contributor to cancer mortality in the world. PLC is a heterogeneous disease that encompasses several biologically distinct subtypes including hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC) and combined hepatocellular-cholangiocarcinoma (CHC). CHC is a distinct, albeit rare, subtype of PLC and is comprised of cells with histopathological features of both HCC and ICC. Several studies have focused on the mutation and expression landscapes of HCC and ICC. However, studies of CHC were rare. OBJECTIVE The aim of the current study was to identify genetic and gene expression alterations in the carcinogenesis and development of CHC and ICC in the Chinese population. Unraveling both similar and differing patterns among these subtypes may help to identify personalized medicine approaches that could improve patient survival. METHODS Whole genome sequencing (WGS), whole exome sequencing (WES) and RNA-seq were performed on 10 ICC and 10 CHC samples, matched with adjacent non-tumor liver tissue specimens. Comparative analysis was performed using HCC datasets from The Cancer Genome Atlas (TCGA). RESULTS Mutational and transcriptional landscapes of CHC and ICC were clearly delineated. TP53 and CTNNB1 were identified as exhibiting mutations in CHC. ARID1A, PBRM1, and IDH1 were frequently mutated in ICC. RYR3, FBN2, and KCNN3 are associated with cell migration and metastasis and might be driver genes in CHC. KCNN3 was identified as also exhibiting mutations in ICC. The ECM-receptor interaction pathway associated fibrogenic hepatic progenitor cell differentiation and liver fibrosis may play an important role in carcinogenesis of PLC. Chromatin remodeling and chromosome organization are key processes in carcinogenesis and development in PLC. P53 related pathways showed alterations in CHC and HCC. Inflammation may be a key factor involved in ICC carcinogenesis. CONCLUSION CHC and ICC are different subtypes of PLC. This study discusses predominantly the molecular genetic details of PLC subtypes and highlights the need for an accurate diagnosis and treatment of specific PLC subtypes to optimize patient management.

Osteopontin promotes hepatocellular carcinoma progression via the PI3K/AKT/Twist signaling pathway

The epithelial-mesenchymal transition (EMT) serves critical roles in the migration, invasion and metastasis of human cancer cells. This process is initiated by regulation of E-cadherin expression by the major inducers of EMT. Previous studies reported that osteopontin (OPN) is essential for hepatocellular carcinoma (HCC) metastasis as it facilitates the EMT in HCC. However, the role and clinical significance of OPN as an EMT regulator in HCC remains unknown. The present study revealed that OPN regulated the expression of Twist by activating RAC serine/threonine-protein kinase (Akt), a critical EMT regulator. Interfering with the phosphoinositide 3-kinase (PI3K)/Akt pathway may suppress the expression of Twist enhanced by OPN. Increased Twist levels in HCC were associated with poor survival and tumor recurrence in patients with HCC following surgery. A significant association was observed between OPN expression and Twist levels in HCC, and a combination of these two parameters was revealed to be a more powerful predictor of poor patient prognosis. The findings of the present study indicate that Twist serves an notable role in OPN-mediated metastasis of HCC through activation of the PI3K/Akt pathway. Twist may be a potential therapeutic target for the prevention of HCC metastasis in patients exhibiting high OPN expression.

The dual blockade of MET and VEGFR2 signaling demonstrates pronounced inhibition on tumor growth and metastasis of hepatocellular carcinoma

BackgroundThe application of VEGF signaling inhibitors have been associated with more invasive or metastatic behavior of cancers including hepatocellular carcinoma (HCC). We explored the contribution of MET pathway to the enhanced HCC invasion and metastasis by VEGF signaling inhibition, and investigated the antitumor effects of NZ001, a novel dual inhibitor of MET and VEGFR2, in HCC.MethodsImmunocompetent orthotopic mice model of hepal-6 was established to investigate the effects of either VEGF antibody alone or in combination with the selective MET inhibitor on tumor aggressiveness. The antitumor effects of NZ001 were examined in cultured HCC cells as well as in vivo models. MET gene amplification was determined by SNP 6.0 assay. MET/P-MET expression was detected by IHC.ResultsSelective VEGF signaling inhibition by VEGF antibody significantly reduced in vivo tumor growth of the orthotopic mice models, simultaneously also enhanced tumor invasion and metastasis, but inhibiting MET signaling attenuated this side-effect. Further study revealed that hypoxia caused by VEGF signaling inhibition induced HIF-1α nuclear accumulation, subsequently leading to elevated total-MET expression, and synergized with HGF in inducing invasion. NZ001, a novel dual inhibitor of MET and VEGFR2, markedly inhibited both tumor growth and metastasis of HCC, which showed obvious advantages over sorafenib in not inducing more invasive and metastatic behaviors. This effect is more pronounced in HCC with MET amplification and overexpression.ConclusionsThe activation of MET is responsible for the metastasis-promoting effects induced by VEGF inhibition. MET and VEGFR2 dual blockade, NZ001, has advantages over sorafenib in not inducing more invasive and metastatic behaviors; MET amplification and overexpression can be used to identify the subgroup of patients most likely to get the optimal benefit from NZ001 treatment.

Osteopontin alters DNA methylation through up-regulating DNMT1 and sensitizes CD133+/CD44+ cancer stem cells to 5 azacytidine in hepatocellular carcinoma

BackgroundIn hepatocellular carcinoma (HCC), CD133+/CD44+ cells are one subgroup with high stemness and responsible for metastatic relapse and resistance to treatment. Our previous studies have demonstrated that osteopontin (OPN) plays critical roles in HCC metastasis. We further investigated the molecular mechanism underlying the role of OPN in regulating the stemness of HCC epigenetically and explored possible targeting strategy.MethodsCD133+/CD44+ subgroup sorting from HCC cell lines and HCC tissues was used to investigate the effects of OPN knockdown on stemness. iTRAQ and MedIP-sequencing were applied to detect the protein profile and epigenetic modification of CD133+/CD44+ subgroup with or without OPN knockdown. The antitumor effects of 5 Azacytidine were examined in cultured HCC cells and patient derived xenograft (PDX) models.ResultsOPN was accumulated in CD133+/CD44+ subgroup of HCC cells. Knocking down OPN significantly inhibited the sphere formation and stemness-related genes expression, and delayed tumor initiation of CD133+/CD44+ subgroup of HCC cells. Employing MedIP-sequencing, dot blot and iTRAQ analyses of CD133+/CD44+ SCR and CD133+/CD44+ shOPN cells, we found that OPN knockdown leaded to reduction in DNA methylation with particular enrichment in CGI. Meanwhile, DNA (cytosine-5)-methyltransferase 1 (DNMT1), the main methylation maintainer, was downregulated via proteomics analysis, which mediated OPN altering DNA methylation. Furthermore, DNMT1 upregulation could partially rescue the properties of CD133+/CD44+ shOPN cells. Both in vitro and in vivo assays showed that CD133+/CD44+ cells with high OPN levels were more sensitive to DNA methylation inhibitor, 5 Azacytidine (5 Aza). The above findings were validated in HCC primary cells, a more clinically relevant model.ConclusionsOPN induces methylome reprogramming to enhance the stemness of CD133+/CD44+ subgroup and provides the therapeutic benefits to DNMT1 targeting treatment in HCC.

Osteopontin promotes metastasis of intrahepatic cholangiocarcinoma through recruiting MAPK1 and mediating Ser675 phosphorylation of β-Catenin

The incidence and mortality of intrahepatic cholangiocarcinoma (ICC) are increasing worldwide in recent decades. Osteopontin (OPN) plays an important role in cancer metastasis, but its functional mechanism in ICC is not clear yet. In this study, we found that OPN level was elevated both in plasma and tumor tissues of ICC patients, which was closely related to a shorter overall survival (OS) and high probability of tumor relapse after curative resection. The gain- and loss-of-function studies determined that OPN could promote ICC growth and metastasis. OPN selectively interacted with β-Catenin and knockdown of β-Catenin abrogated the effects induced by OPN. OPN recruited MAPK1 and activated MEK–MAPK1 pathway to mediate the S675 phosphorylation of β-Catenin and nucleus accumulation, which induced the activation of Wnt signaling. Moreover, a significant correlation between OPN and β-Catenin was found in ICC tissues. OPN, β-Catenin, and their combination were independent prognostic indicator for ICC patients. In conclusion, OPN promotes ICC progression through recruiting MAPK1 and activating the Wnt/β-Catenin pathway and can serve as a novel prognostic marker and therapeutic target for ICC.

C-C chemokine receptor type 1 mediates osteopontin-promoted metastasis in hepatocellular carcinoma

In the hepatocellular carcinoma (HCC) microenvironment, chemokine receptors play a critical role in tumorigenesis and metastasis. Our previous studies have found that osteopontin (OPN) is a promoter for HCC metastasis. However, the role of chemokine receptors in OPN‐induced HCC metastasis remains unclear. In this study, we demonstrate that OPN is dramatically elevated in HCC tissues with metastasis and that high expression of OPN correlates with poorer overall survival and higher recurrence rate. OPN upregulates chemokine receptor expression, migration, invasion and pulmonary metastasis in HCC. We find that C‐C chemokine receptor type 1 (CCR1) and C‐X‐C chemokine receptor type 6 (CXCR6) are the most upregulated chemokine receptors induced by OPN. CCR1 knockdown results in reduction of migration, invasion and pulmonary metastasis induced by OPN in vitro and in vivo, whereas CXCR6 knockdown does not reverse OPN‐promoted migration and invasion. Moreover, OPN upregulates the expression of CCR1 through activating phosphoinositide 3‐kinase (PI3K)/AKT and hypoxia‐inducible factor 1α (HIF‐1α) in HCC cells. Furthermore, blockade of OPN‐CCR1 axis with CCR1 antagonist significantly restrains the promoting effects of OPN on HCC progression and metastasis. In human HCC tissues, OPN expression shows significantly positive correlation with CCR1 expression, and the patients with high levels of both OPN and CCR1 have the most dismal prognosis. Collectively, our results indicate that the OPN‐CCR1 axis in HCC is important for accelerating tumor metastasis and that CCR1 is a potential therapeutic target for controlling metastasis in HCC patients with high OPN.

MicroRNA-219-5p Promotes Tumor Growth and Metastasis of Hepatocellular Carcinoma by Regulating Cadherin 1

MicroRNAs play significant roles in the development of cancer and may serve as promising therapeutic targets. In our previous work, miR-219-5p was identified as one of the important metastasis-related microRNAs in HCC. Here we demonstrated that miR-219-5p expression was elevated in HCC tissues and was associated with vascular invasion and dismal prognosis. In multivariate analysis, miR-219-5p was identified as an independent prognostic indicator for HCC patients. Functional mechanism analyses showed that miR-219-5p promoted HCC cell proliferation and invasion in in vitro, as well as in vivo, tumor growth and metastasis in nude mice models bearing human HCC tumors. In addition, cadherin 1 (CDH1) was revealed to be a downstream target of miR-219-5p in HCC cells. In conclusion, miR-219-5p promotes tumor growth and metastasis of HCC by regulating CDH1 and can serve as a prognostic marker for HCC patients.