Inflammatory microenvironment of fibrotic liver promotes hepatocellular carcinoma growth,metastasis and sorafenib resistance through STAT3 activation

The pro-inflammatory and pro-fibrotic liver microenvironment facilitates hepatocarcinogenesis. However, the effects and mechanisms by which the hepatic fibroinflammatory microenvironment modulates intrahepatic hepatocellular carcinoma (HCC) progression and its response to systematic therapy remain largely unexplored. We established a syngeneic orthotopic HCC mouse model with a series of persistent liver injury induced by CCl₄ gavage, which mimic the dynamic effect of hepatic pathology microenvironment on intrahepatic HCC growth and metastasis. Non-invasive bioluminescence imaging was applied to follow tumour progression over time. The effect of the liver microenvironment modulated by hepatic injury on sorafenib resistance was investigated in vivo and in vitro. We found that the persistent liver injury facilitated HCC growth and metastasis, which was positively correlated with the degree of liver inflammation rather than the extent of liver fibrosis. The inflammatory cytokines in liver tissue were clearly increased after liver injury. The two indicated cytokines, tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6), both promoted intrahepatic HCC progression via STAT3 activation. In addition, the hepatic inflammatory microenvironment contributed to sorafenib resistance through the anti-apoptotic protein mediated by STAT3, and STAT3 inhibitor S3I-201 significantly improved sorafenib efficacy impaired by liver inflammation. Clinically, the increased inflammation of liver tissues was accompanied with the up-regulated STAT3 activation in HCC. Above all, we concluded that the hepatic inflammatory microenvironment promotes intrahepatic HCC growth, metastasis and sorafenib resistance through activation of STAT3.     

EYA4 serves as a prognostic biomarker in hepatocellular carcinoma and suppresses tumour angiogenesis and metastasis

Eye absent homolog 4 (EYA4) has been demonstrated to be down‐regulated in hepatocellular carcinoma (HCC), but its biological function and the mechanism in HCC angiogenesis and metastasis remain largely unknown. Herein, we showed that EYA4 expression was frequently low in HCC tissue samples compared with matched adjacent non‐tumourous tissues. In the analysis of 302 HCC specimens, we revealed that decreased expression of EYA4 correlated with tumour differentiation status. Univariate and multivariate analyses identified EYA4 as an independent risk factor for recurrence‐free survival (RFS) and overall survival (OS) among the 302 patients. Functional assays showed that forced expression of EYA4 suppressed HCC cell migration, invasion and capillary tube formation of endothelial cells in vitro, as well as in vivo tumour angiogenesis and metastasis in a mouse model. Furthermore, mechanism study exhibited that EYA4 could inhibit HCC angiogenesis and metastasis by inhibiting c‐JUN/VEGFA pathway. Together, we provide proof that EYA4 is a novel tumour suppressor in HCC and a new prognostic biomarker and therapeutic target in HCC.     

CCAT2 enhances autophagy-related invasion and metastasis via regulating miR-4496 and ELAVL1 in hepatocellular carcinoma

Autophagy is thought to contribute to the pathogenesis of many diseases, including cancer. Long non-coding RNA (lncRNA) CCAT2 functions as an oncogene in a variety of tumours. However, it is still unknown whether CCAT2 is involved in autophagy and metastasis of hepatocellular carcinoma (HCC). In our study, we found that lncRNA CCAT2 expression was significantly increased in HCC tissue and was correlated with advanced stage and venous invasion. Further experiments revealed that CCAT2 induced autophagy and promoted migration and invasion in vitro and in vivo. Mechanistic investigations found that CCAT2 involved in HCC by regulating miR-4496/Atg5 in cytoplasm. In nucleus, CCAT2 bound with ELAVL1/HuR to facilitate HCC progression. Our findings suggest that CCAT2 is an oncogenic factor in the progression of HCC with different regulatory mechanisms and may serve as a target for HCC therapy.     

Betulinic acid induces apoptosis and suppresses metastasis in hepatocellular carcinoma cell lines in vitro and in vivo

Hepatocellular carcinoma (HCC) is a high incidence and mortality malignant tumour globally. Betulinic acid (BA) is a pentacyclic triterpenoid with potential pro‐apoptotic activities which widely found in many plants. In this study, we determined the effects of BA on proliferation, apoptosis, invasion, and metastasis in HCC cell lines and on tumour growth and pulmonary metastasis in mice. The results suggested that BA could inhibit cell viability and proliferation of HCC cell lines including HepG2, LM3, and MHCC97H. In addition, BA induced apoptosis of HepG2 cells characterised condensed nuclei and nuclear fragmentation. Moreover, western blot analysis showed that BA‐induced apoptosis associated with increasing of pro‐apoptotic protein Bax and cleaved caspase‐3 and decreasing of anti‐apoptotic protein Bcl‐2. Meanwhile, BA also reduced the reactive oxygen species (ROS) level. Furthermore, BA also significantly inhibited HCC growth in vivo and blocked pulmonary metastasis of HCC by regulating the metastasis‐related proteins including MMP‐2, MMP‐9, and TIMP2 without obvious toxicity. In all, the present study suggested that BA might be a promising anti‐HCC drug candidate by inhibiting proliferation, inducing apoptosis, and blocking metastasis.     

Overexpression of phosphatase regenerating liver 3 in oesophageal squamous cell carcinoma associated with metastasis and its comparison with phosphatase regenerating liver 1

Expression of PRL3 (phosphatase of regenerating liver 3) protein was examined with immunohistochemistry in 60 cases of ESCC (oesophageal squamous cell carcinoma) with matched lymph node metastasis (n = 40) and 6 cases of oesophageal adenocarcinoma. Its associations with PRL1 and clinicopathological parameters were analysed. The results showed the frequency of PRL3 protein expression was significantly higher in ESCC (39/60, 65%) than in normal oesophageal mucosa (0/20, P < 0.001); higher in ESCC with lymph node metastasis (30/40, 75%) than in ESCC without lymph node metastasis (9/20, P = 0.022), as well as higher in metastatic ESCC in lymph node (38/40, 95%) than in the primary ESCC (39/60, 65%, P < 0.001). PRL3 was expressed in 1 out of 6 oesophageal adenocarcinomas, but showed no nuclear staining of PRL1. Expression of PRL3 protein was positively associated with the grade and partially with the stage of ESCC. These results suggest that expression of PRL3 protein may be involved in the metastasis of ESCC and serve as a biomarker for prediction of ESCC metastasis.     

Prediction of lymph node metastasis in oral squamous cell carcinoma based on protein profile

Objective: Lymph node metastasis leads to high mortality rates of oral squamous cell carcinoma (OSCC). However, it is still controversial to define clinically negative neck (cN0) and positive neck (cN1-3).

Methods: We retrieved candidate biomarkers identified by proteomic analysis in OSCC from published works of literature. In training stage, immunohistochemistry (IHC) analysis was used to determine the expression of proteins and logistic regression models with stepwise variable selection were used to identify potential factors that might affect lymph node metastasis and life status. Furthermore, the prediction model was validated in validating stage.

Results: We screened eight highly expressed proteins related to lymph node metastasis in OSCC and found that the expression levels of SOD2, BST2, CAD, ITGB6, and PRDX4 were significantly elevated in patients with lymph node metastasis compared to the patients without lymph node metastasis. Furthermore, in training and validating stages, the prediction model base on the combination of CAD, SOD2 expression levels, and histopathologic grade was developed and validated in patients with OSCC.

Conclusions: Our findings showed that the developed model well predicts the lymph node metastasis and life status in patients with OSCC, independent of TNM stage.     

Promotion of hepatocellular carcinoma metastasis through matrix metalloproteinase activation by epithelial‐mesenchymal transition regulator Twist1

E‐cadherin loss is a key biological mechanism in tumour invasion. As a main regulator of epithelial‐mesenchymal transition (EMT) mechanism‐mediated invasion and metastasis, Twist1 plays an important role through its regulation of E‐cadherin expression. However, whether or not Twist2 has the same function in tumour metastasis remains unclear. The purpose of this study is to investigate the expressions and different roles of Twist1 and Twist2 in human hepatocellular carcinoma (HCC). The expressions of Twist1 and Twist2 in HCC tissue were evaluated by immunohistochemical staining. The role of Twist1 and Twist2 in invasiveness was also evaluated in vitro by using HCC cell lines. Twist1 nuclear overexpression is found to be correlated with HCC metastasis, and its expression is negatively correlated with E‐cadherin expression in human tissue. Twist2, a Twist1 homology protein, only expresses in the cytoplasm and shows no significant correlation with HCC metastasis. By ectopic transfection of Twist1 and Twist2 into the HCC cells, HepG2 and PLC, Twist1 is able to down‐regulate E‐cadherin expression and promote matrix metalloproteinase (MMP) activation, specifically in MMP2 and MMP9. In functional assays, Twist1 is found to promote invasion in HepG2 and PLC cells, but the invasion ability of the groups is not affected Twist2. Our findings indicate that Twist1 induces HCC invasion via increased activity in MMPs, leading to poor clinical prognoses. The results of this study also demonstrate a novel cogitation in Twist2, which has no effect on HCC invasion and metastasis. Twist1 may contribute to HCC invasion and metastasis and may be used as a novel therapeutic target for the inhibition of HCC metastasis.     

Polycomb chromobox 4 enhances migration and pulmonary metastasis of hepatocellular carcinoma cell line MHCC97L

We recently report that the expression of polycomb chromobox 4(Cbx4)is significantly correlated with the overall survival of a great cohort of hepatocellular carcinoma(HCC)patients and it enhances hypoxia-induced vascular endothelial growth factor(VEGF)expression and angiogenesis in HCC cells through enhancing sumoylation of hypoxia inducible factor-1alpha(HIF-1α).Here we continue to investigate the potential effects of Cbx4 on the migration and metastasis of the metastatic HCC cell line MHCC97L.Our results show that Cbx4 overexpression in the cell line increases the in vitro vessel formation of vascular endothelial cells in its SUMO interaction motifs-dependent manner,and promotes the in vitro migration of the cancer cell,which can be effectively abrogated by anti-VEGF antibody.Although Cbx4 expression does not impact the in vitro growth of MHCC97L cells,it still promotes the progression and metastasis of orthotopically transplanted tumors in nude mice.These results further support the role of Cbx4 as a SUMO E3 ligase in the progression and metastasis of HCC.     

SRXN1 stimulates hepatocellular carcinoma tumorigenesis and metastasis through modulating ROS/p65/BTG2 signalling

Sulfiredoxin 1 (SRXN1) is a pivotal regulator of the antioxidant response in eukaryotic cells. However, the role of SRXN1 in hepatocellular carcinoma (HCC) is far from clear. The present study aims to elucidate whether SRXN1 participates in tumorigenesis and metastasis of HCC and to determine the molecular mechanisms. We found that SRXN1 expression was up‐regulated in HCC tissue samples and correlated with poor prognosis in HCC patients. We also observed that SRXN1 knockdown by transient siRNA transfection inhibited HCC cell proliferation, migration and invasion. Overexpression of SRXN1 increased HCC cell migration and invasion. B‐cell translocation gene 2 (BTG2) was identified as a downstream target of SRXN1. Mechanistic studies revealed that SRXN1‐depleted reactive oxygen species (ROS) modulated migration and invasion of HCC cells. In addition, the ROS/p65/BTG2 signalling hub was found to regulate the epithelial‐mesenchymal transition (EMT), which mediates the pro‐metastasis role of SRXN1 in HCC cells. In vivo experiments showed SRXN1 promotes HCC tumour growth and metastasis in mouse subcutaneous xenograft and metastasis models. Collectively, our results revealed a novel pro‐tumorigenic and pro‐metastatic function of SRXN1 in HCC. These findings demonstrate a rationale to exploit SRXN1 as a therapeutic target effectively preventing metastasis of HCC.     

Astrocyte elevated gene 1 (AEG-1) promotes anoikis resistance and metastasis by inducing autophagy in hepatocellular carcinoma

Astrocyte elevated gene 1 (AEG-1) is overexpressed in hepatocellular carcinoma (HCC) and is strongly associated with tumor metastasis. Anoikis resistance and autophagy may play an important role in the survival of circulating tumor cells. However, the relationship among AEG-1, anoikis resistance, autophagy, and metastasis in HCC is still not clear. The results of this study indicate that AEG-1 expression is increased in HCC cell lines grown in suspension culture. AEG-1 could enhance anoikis resistance to promote the survival of detached HCC cells. Moreover, the anoikis resistance appears to be partly dependent on autophagy. Regulating AEG-1 expression changed the autophagy levels to modulate anoikis resistance, likely acting via the protein kinase RNA-like ER kinase (PERK)-eIF2α-ATF4-CHOP signaling axis. Finally, inhibiting autophagy by RNA interference prevented the AEG-1-promoted metastasis of HCC xenografts to the liver and lungs of nude mice. Taken together, AEG-1 is a key contributor to anoikis resistance and metastasis by inducing autophagy in vitro and in vivo, and it may be a potential target for therapeutic intervention in HCC.     

Hepatoma‐derived growth factor promotes growth and metastasis of hepatocellular carcinoma cells

We aimed to elucidate the effects of hepatoma‐derived growth factor (HDGF) on growth and metastasis of hepatocellular carcinoma (HCC) cells. Tissue microarrays with 236 HCC specimens and 18 extrahepatic metastases were utilized to detect the HDGF expression by immunohistochemistry. Meanwhile, HDGF expressions in HCC cell lines with different metastatic potentials were examined using immunofluorescence staining, real‐time PCR and western blotting. After HDGF silencing, the growth and metastatic potentials of HCC cells were evaluated by soft agar assay, invasion assay, together with tumorigenicity assay in nude mice. The gelatin zymography was performed by detecting MMP‐2 and MMP‐9 levels. Additionally, western blotting was conducted to determine the levels of total and phosphorylated ERK1/2, JNK, p38 and Akt. The results showed that HDGF was overexpressed in HCC metastasis tumour, and the expression increased with the differentiation degree of tumours (Grade I 44.0%, Grade II 48.4% and Grade III 65.6%). Consistently, HDGF levels were positively associated with the metastatic capability of HCC cells (MHCC97L < MHCC97H < HCCLM3). The growth and metastasis were suppressed by HDGF‐siRNA. Gelatinolytic activities were enhanced in the three metastatic HCC cell lines, but had no significant difference among them. The tumourigenicity and metastatic capability of HCCLM3 cells in nude mice were inhibited after silencing HDGF. Meanwhile, HDGF‐siRNA specifically suppressed the total and phosphorylated protein levels of ERK1/2, while not JNK, p38 and Akt. In conclusion, HDGF was overexpressed in HCC patients and cells, and HDGF might be closely correlated with HCC metastasis via regulating ERK signalling pathway. Copyright © 2016 John Wiley & Sons, Ltd.     

High CCNB2 expression correlates with poor prognosis in hepatocellular carcinoma

Cyclin B2 (CCNB2), a member of the cyclin protein family, has a key role in the progression of G2/M transition. However, the clinical value of CCNB2 in hepatocellular carcinoma (HCC) is still unknown. The aim of our study is to identify the role of CCNB2 in HCC patients. Immunohistochemical analysis using tissue microarray (TMA) was employed to evaluate the expression of CCNB2 in HCC and the correlation between CCNB2 expression and clinicopathological features in HCC patients. The relationship between CCNB2 expression and the prognosis of HCC patients was analyzed using Oncomine and Kaplan-Meier Plotter online resources. High CCNB2 cytoplasmic expression was observed in 77.22% of patients with HCC, which was related to differentiation (P<0.001), tumor diameter (P=0.025), and hepatitis B virus infection (P=0.008). High CCNB2 nuclear expression was seen among 43.43% of cases, which was associated with differentiation (P=0.001). CCNB2 levels were inversely proportional to patient prognosis. The study suggests that CCNB2 expression could be an effective prognostic biomarker for HCC.     

Immunohistochemical method identifies lymphovascular invasion in a majority of oral squamous cell carcinomas and discriminates between blood and lymphatic vessel invasion.

Tumor invasion into blood and/or lymphatic channels is an important component of cancer staging and prognosis. Standard pathological methods do not provide sufficient contrast to discriminate between invasion into each type of vessel and are complicated by tissue retraction artifacts. We evaluated the ability of a triple-stain immunohistochemical method, combining cytokeratin, CD34, and podoplanin stains in a single section, to distinguish blood from lymphatic vascular invasion in oral squamous cell carcinoma and confirmed its results using multispectral analysis. The triple-stain method was significantly more sensitive in detecting invasive events than the standard hematoxylin and eosin staining method and easily discriminated between blood and lymphatic vessel invasion. Invasive events were present in blood and/or lymphatic vessels in the majority of patients with and without presentation of lymph node metastasis, indicating that vessel invasion in this cancer model is common and is not a rate-limiting step for lymph node metastasis.     

RAB25 expression is epigenetically downregulated in oral and oropharyngeal squamous cell carcinoma with lymph node metastasis

Oral and oropharyngeal squamous cell carcinoma (OOSCC) have a low survival rate, mainly due to metastasis to the regional lymph nodes. For optimal treatment of these metastases, a neck dissection is required; however, inaccurate detection methods results in under- and over-treatment. New DNA prognostic methylation biomarkers might improve lymph node metastases detection. To identify epigenetically regulated genes associated with lymph node metastases, genome-wide methylation analysis was performed on 6 OOSCC with (pN+) and 6 OOSCC without (pN0) lymph node metastases and combined with a gene expression signature predictive for pN+ status in OOSCC. Selected genes were validated using an independent OOSCC cohort by immunohistochemistry and pyrosequencing, and on data retrieved from The Cancer Genome Atlas. A two-step statistical selection of differentially methylated sequences revealed 14 genes with increased methylation status and mRNA downregulation in pN+ OOSCC. RAB25, a known tumor suppressor gene, was the highest-ranking gene in the discovery set. In the validation sets, both RAB25 mRNA (P = 0.015) and protein levels (P = 0.012) were lower in pN+ OOSCC. RAB25 mRNA levels were negatively correlated with RAB25 methylation levels (P < 0.001) but RAB25 protein expression was not. Our data revealed that promoter methylation is a mechanism resulting in downregulation of RAB25 expression in pN+ OOSCC and decreased expression is associated with lymph node metastasis. Detection of RAB25 methylation might contribute to lymph node metastasis diagnosis and serve as a potential new therapeutic target in OOSCC.     

Pleiotrophin,a target of miR‐384, promotes proliferation,metastasis and lipogenesis in HBV‐related hepatocellular carcinoma

Hepatitis B virus (HBV) infection plays a crucial role and is a major cause of hepatocellular carcinoma (HCC) in China. microRNAs (miRNAs) have emerged as key players in hepatic steatosis and carcinogenesis. We found that down‐regulation of miR‐384 expression was a common event in HCC, especially HBV‐related HCC. However, the possible function of miR‐384 in HBV‐related HCC remains unclear. The oncogene pleiotrophin (PTN) was a target of miR‐384. HBx inhibited miR‐384, increasing PTN expression. The PTN receptor N‐syndecan was highly expressed in HCC. PTN induced by HBx acted as a growth factor via N‐syndecan on hepatocytes and further promoted cell proliferation, metastasis and lipogenesis. PTN up‐regulated sterol regulatory element‐binding protein 1c (SREBP‐1c) through the N‐syndecan/PI3K/Akt/mTORC1 pathway and the expression of lipogenic genes, including fatty acid synthesis (FAS). PTN‐mediated de novo lipid synthesis played an important role in HCC proliferation and metastasis. PI3K/AKT and an mTORC1 inhibitor diminished PTN‐induced proliferation, metastasis and lipogenesis. Taken together, these data strongly suggest that the dysregulation of miR‐384 could play a crucial role in HBV related to HCC, and the target gene of miR‐384, PTN, represents a new potential therapeutic target for the prevention of hepatic steatosis and further progression to HCC after chronic HBV infection.     

Transmembrane protein 106C promotes the development of hepatocellular carcinoma

Several protein-coding genes have been identified to play essential roles in cancer biology, and they are dysregulated in many tumors. Transmembrane protein 106C (TMEM106C) is differentially expressed in several human and porcine diseases; however, the expression and biological functions of TMEM106C in hepatocellular carcinoma (HCC) are not clear. In our study, we obtained paired tissue samples from patients undergoing resection for HCC and public databases, which were analyzed for TMEM106C expression using quantitative real-time polymerase chain reaction (qRT-PCR). We further conducted in vitro and in vivo experiments in HCC cell lines and nude mice, respectively, in which TMEM106C was overexpressed or knocked down. Cell-Counting Kit-8 and colony formation experiments were used to determine the influence of TMEM106C on cell proliferation, flow cytometric assays were used to detect the influence on cell cycle distribution and apoptosis, and transwell assays were used for detecting changes in cell migration and invasion. TMEM106C levels were significantly elevated in HCC tissues and cell lines from public databases and our collected specimens from patients. Moreover, higher TMEM106C expression levels predicted a poor prognosis in HCC patients in survival analysis. Overexpression of TMEM106C in HCC cells accelerated cell growth, migration, and invasion, but it inhibited cell apoptosis by targeting forkhead box O-1 (FOXO1) and FOXO3. Conversely, TMEM106C knockdown impeded cell proliferation and metastasis, whereas it enhanced the rate of apoptosis. More important, knockdown of the expression of TMEM106C in HCC cells inhibited the growth of xenograft tumors in vivo. Collectively, these results suggest that TMEM106C acts as an oncogene and can serve as a potential therapeutic target for HCC in the future.     

Derepression of c‐Fos caused by MicroRNA‐139 down‐regulation contributes to the metastasis of human hepatocellular carcinoma

This study investigates whether the anti‐metastasis effect of microRNA‐139 (miR‐139) on hepatocellular carcinoma (HCC) is mediated through regulating c‐fos expression. The expression levels of miR‐139 and c‐fos in human HCC cell sublines with high (MHCC97H) and low (MHCC97L) spontaneous metastatic potentials were quantified using QPCR or Western blot. miR‐139 mimics was transfected into MHCC97H cells to overexpress miR‐139, and miR‐139 inhibitor was transfected into MHCC97L cells to down‐express miR‐139. The effect of overexpression or down‐expression of miR‐139 on c‐fos expression of MHCC97H and MHCC97L cells was evaluated using QPCR and Western blot. The 3′ untranslated region segments of FOS containing the miR‐139 binding sites were amplified by PCR, and the luciferase activity in the transfected cells was assayed. In comparison with the expression level of miR‐139 in MHCC97L cells, the expression level in MHCC97H cells was significantly decreased, whereas c‐Fos was significantly up‐regulated in MHCC97H. The overexpression of miR‐139 significantly inhibited the expression of c‐fos in MHCC97H cells, and the down‐expression of miR‐139 significantly promoted the expression of c‐fos in MHCC97L cells. miR‐139 suppressed the luciferase activity of the pGL‐FOS by approximately 40% compared with the negative control. In vitro cell migration analysis demonstrated that depletion of c‐fos or overexpression of miR‐139 in MHCC97H cells reduced cell migration, whereas overexpression of c‐fos or depletion of miR‐139 in MHCC97L cells increased cell migration. Thus, we got the conclusion that miR‐139 expression is down‐regulated in human HCC cell sublines with high spontaneous metastatic potentials (MHCC97H). Derepression of c‐Fos caused by miR‐139 down‐regulation contributes to the metastasis of HCC. Copyright © 2012 John Wiley & Sons, Ltd.     

二甲双胍与肝细胞癌

二甲双胍作为一种口服降糖药,已被用于2型糖尿病的一线治疗,有报道指出其可以预防和控制多种癌症的发展。肝细胞癌(肝癌)是原发性肝癌的最常见形式,也是最常见的恶性肿瘤之一。目前,流行病学显示二甲双胍可以降低肝细胞癌的发生风险或改善肝癌患者的预后,实验室研究也证实二甲双胍可抑制肝癌细胞生长。因此文章将探讨二甲双胍抑制肝癌生长的相关机制。