Hepatocellular carcinoma cells remodel the pro-metastatic tumour microenvironment through recruitment and activation of fibroblasts via paracrine Egfl7 signaling.

BACKGROUND: The tumour microenvironment consists of a complex and dynamic milieu of cancer cells, including tumour-associated stromal cells (leukocytes, fibroblasts, vascular cells, etc.) and their extracellular products. During invasion and metastasis, cancer cells actively remodel the tumour microenvironment and alterations of microenvironment, particularly cancer-associated fibroblasts (CAFs), can promote tumour progression. However, the underlying mechanisms of the CAF formation and their metastasis-promoting functions remain unclear. METHODS: Primary liver fibroblasts and CAFs were isolated and characterized. CAFs in clinical samples were identified by immunohistochemical staining and the clinical significance of CAFs was also analysed in two independent cohorts. A transwell coculture system was used to confirm the role of HCC cells in CAF recruitment and activation. qRT-PCR, western blotting and ELISA were used to screen paracrine cytokines. The role and mechanism of Egfl7 in CAFs were explored via an in vitro coculture system and an in vivo mouse orthotopic transplantation model. RESULTS: We showed that CAFs in hepatocellular carcinoma (HCC) are characterized by the expression of α-SMA and that HCC cells can recruit liver fibroblasts (LFs) and activate them to promote their transformation into CAFs. High α-SMA expression, indicating high CAF infiltration, was correlated with malignant characteristics. It was also an independent risk factor for HCC survival and could predict a poor prognosis in HCC patients. Then, we demonstrated that EGF-like domain multiple 7 (Egfl7) was preferentially secreted by HCC cells, and exhibited high potential to recruit and activate LFs into the CAF phenotype. The ability of Egfl7 to modulate LFs relies upon increased phosphorylation of FAK and AKT via the receptor ανß3 integrin. Strikingly, CAFs activated by paracrine Egfl7 could further remodel the tumour microenvironment by depositing fibrils and collagen and in turn facilitate HCC cell proliferation, invasion and metastasis. CONCLUSION: Our data highlighted a novel role of Egfl7 in remodelling the tumour microenvironment: it recruits LFs and activates them to promote their transformation into CAFs via the ανß3 integrin signaling pathway, which further promotes HCC progression and contributes to poor clinical outcomes in HCC patients. Video Abstract.

Piezo1 promoted hepatocellular carcinoma progression and EMT through activating TGF-ß signaling by recruiting Rab5c.

BACKGROUND: Piezo1 has been revealed to play a regulatory role in vascular development and progression of variety tumors. However, whether and how the progression of hepatocellular carcinoma (HCC) regulated by Piezo1 remains elusive. This study aimed to elucidate the effect and mechanisms of Piezo1 in HCC. METHODS: The mRNA and protein expression level of Piezo1 in HCC samples and cell lines was determined by qRT-PCR, western blot and immunohistochemistry analyses. Two independent study cohorts containing 280 patients were analyzed to reveal the association between Piezo1 expression and clinicopathological characteristics. Series of in vitro and in vivo experiments were used to validate the function of Piezo1 in HCC. Gene set enrichment analysis (GSEA) was performed to explore the signaling pathway of Piezo1. Immunoprecipitation, immunofluorescence and in vitro and in vivo experiments were used to explore the molecular mechanism of Piezo1 in HCC progression. RESULTS: Our results demonstrated the Piezo1 expression was significantly upregulated in HCC tissues and cell lines, and upregulation of Piezo1 closely correlated with aggressive clinicopathological features and poor prognosis. Knockdown of Piezo1 in HCCLM3 and Hep3B cells significantly restrained proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of HCC cells in vitro, and tumor growth, metastasis, EMT in vivo. TGF-ß signaling pathway was most significant enriched pathway in GSEA. Finally, tumor promotion effect of Piezo1 was found to exerted through recruiting and combining Rab5c to activating TGF-ß signaling pathway. CONCLUSIONS: Piezo1 significantly related to poor prognosis and promotes progression of hepatocellular carcinoma via activating TGF-ß signaling, which suggesting that Piezo1 may serve as a novel prognostic predictor and the potential therapeutic target for HCC patients.

GNA14's interaction with RACK1 inhibits hepatocellular carcinoma progression through reducing MAPK/JNK and PI3K/AKT signaling pathway.

Gαq subfamily proteins play critical roles in many biological functions including cardiovascular development, angiogenesis, and tumorigenesis of melanoma. However, the understanding of G Protein Subunit Alpha 14 (GNA14) in diseases, especially in cancers is limited. Here, we revealed that GNA14 was significantly low expression in Human hepatocellular carcinoma (HCC) samples. Low GNA14 expression was correlated with aggressive clinicopathological features. Moreover, the overall survival (OS) and disease-free survival (DFS) of high GNA14 expression HCC patients were much better than low GNA14 expression group. Lentivirus-mediated GNA14 knockdown significantly promoted the growth of liver cancer in vitro and in vivo. However, opposing results were observed when GNA14 is upregulated. Mechanistically, We identified receptor for activated C kinase 1 (RACK1) as a binding partner of GNA14 by co-immunoprecipitation and mass spectrometry (MS). Glutathione-S-transferase (GST) pull-down assay further verified the direct interaction between GNA14 and RACK1. RNA-Seq and loss- and gain-of-function assays also confirmed that GNA14 reduced the activity of both MAPK/JNK and PI3K/AKT signaling pathways through RACK1. GNA14 synergized with U73122 (PLC inhibitor) to enhance this effect. Further studies suggested that GNA14 potentially competed with protein kinase C (PKC) to bind with RACK1, consequently reducing the stability of PKC. Moreover, we also showed that GNA14'supression of p-AKT protein level depended on sufficient RACK1 expression. In conclusion, we indicated a different role of GNA14, which acted as a suppressor inhibiting liver cancer progression through MAPK/JNK and PI3K/AKT signaling pathways. Due to this, GNA14 served as a potentially valuable prognostic biomarker for liver cancer.

NAD(P)HX epimerase downregulation promotes tumor progression through ROS/HIF-1α signaling in hepatocellular carcinoma.

Reactive oxygen species (ROS) derived from aberrant tumor metabolism could contribute to tumor invasion and metastasis. NAD(P)HX Epimerase (NAXE), an epimerase that allows the repair of damaged forms of antioxidant NADPH, is a potential cellular ROS scavenger and its role in tumor development is still elusive. Here, we found that NAXE is significantly downregulated in hepatocellular carcinoma (HCC) tissues and cell lines. NAXE downregulation is associated with poor clinicopathological characteristics and is an independent risk factor for overall and disease-free survival of HCC patients after liver resection. In addition, low NAXE expression could identify worse prognosis of HCC patients before vascular invasion or in early stages of disease. In particularly, low NAXE expression in HCC is markedly associated with microvascular invasion (MVI) and its combination with MVI predicts poorer prognosis of HCC patients after liver resection. Furthermore, in vitro and in vivo experiments both showed that knockdown of NAXE expression in HCC cells promoted migration, invasion, and metastasis by inducing epithelial-mesenchymal transition (EMT), whereas NAXE overexpression causes the opposite effects. Mechanistically, low NAXE expression reduced NADPH levels and further caused ROS level increase and hypoxia-inducible factor-1α (HIF-1α) activation, thereby promoting invasion and metastasis of HCC by facilitating EMT. What is more, the tumor-promoting effect of NAXE knockdown in HCC xenograft can be abolished by giving mice N-acetyl-l-cysteine (NAC) in drinking water. Taken together, our findings uncovered a tumor suppressor role for NAXE in HCC by scavenging excessive ROS and inhibiting tumor-promoting signaling pathways, suggesting a new strategy for HCC therapy by targeting redox signaling.

Serum epidermal growth factor-like domain 7 serves as a novel diagnostic marker for early hepatocellular carcinoma.

BACKGROUND: Epidermal growth factor-like domain 7 (Egfl7), a recently identified secreted protein, was significantly increased in patients with HCC by our previous studies. However, its efficacy in the diagnosis of early HCC remains unknown. In this study, we therefore evaluate the efficacy of serum Egfl7 for early HCC diagnosis and compare it with alpha-fetoprotein (AFP). METHODS: Serum Egfl7 levels in testing cohort (1081 participants) and validation cohort (476 participants) were measured by a sandwich enzyme-linked immunoassay (ELISA). The cut-off value of Egfl7 was determined by Youden's index and the efficacies of Egfl7 and AFP in diagnosing early HCC were estimated by receiver operating characteristic (ROC). RESULTS: Serum Egfl7 was significantly elevated in patients with early HCC than all non-HCC controls in whatever Testing Cohort or Validation Cohort. In the Testing Cohort, ROC curves showed the optimum cut-off value of Egfl7 was 2610 ng/mL and Egfl7 showed a significantly higher sensitivity than AFP in discriminating early HCC from healthy individuals (77.4% vs. 65.3%, P = 0.0013) but the area under ROC (AUROC) and accuracy of Egfl7 and AFP were similar (0.860 vs. 0.868, P = 0.704; 80.2% vs. 83.8%, P = 0.184). In distinguishing patients with early HCC from patients with chronic liver disease (CLD), the AUROC, sensitivity, specificity and accuracy of Egfl7 were 0.800, 75.2, 71.7 and 73.5%, which were all significantly higher than AFP (0.675, 61.8, 62.0 and 61.9% in order). Egfl7 also showed a significant higher sensitivity and accuracy than AFP (76.6% vs. 64.0%, P = 0.0031; 79.9% vs. 66.1%, P < 0.0001) in differentiating early HCC patients from non-HCC individuals. Additionally, 70.8% of early HCC patients with negative AFP could be diagnosed by Egfl7 and the combined use of Egfl7 and AFP increased the sensitivity to 91.0%. These results were confirmed by a validation cohort. CONCLUSION: Egfl7 is a valuable serum marker in the diagnosis of early HCC and could complement the efficacy of AFP, especially in distinguishing early HCC from CLD and identifying patients with AFP-negative early HCC.

EB2 promotes hepatocellular carcinoma proliferation and metastasis via MAPK/ERK pathway by modulating microtubule dynamics.

Metastasis is the main cause of poor postoperative survival of hepatocellular carcinoma (HCC) patients. Cytoskeleton rearrangement is a key event in cancer metastasis. However, the significance of microtubule (MT), one of the core components of cytoskeleton, in this process is only beginning to be revealed. Here, we find that the MT dynamics regulator end-binding protein 2 (EB2) is highly expressed in HCC and predicts poor prognosis of HCC patients. Functional studies show that EB2 overexpression promotes HCC proliferation, invasion and metastasis in vitro and in vivo, while EB2 knockdown has opposite results. Mechanistically, EB2 mediates MTs destabilization, increases Src (Src proto-oncogene non-receptor tyrosine kinase) activity, and thus facilitates extracellular signal-regulated kinase (ERK) signaling activation, which could in turn promote EB2 expression in HCC, eventually resulting in enhanced HCC proliferation, invasion and metastasis. Furthermore, U0126, a specific ERK inhibitor, could effectively inhibit EB2-mediated HCC proliferation and metastasis in vitro and in vivo. In conclusion, EB2 coordinates MT cytoskeleton and intracellular signal transduction, forming an EB2-MT-ERK positive feedback loop, to facilitate HCC proliferation, invasion and metastasis. EB2 could serve as a promising prognostic biomarker and potential therapeutic target for HCC; HCC patients with high EB2 expression may benefit from treatment with ERK inhibitors.

HEG1 indicates poor prognosis and promotes hepatocellular carcinoma invasion, metastasis, and EMT by activating Wnt/ß-catenin signaling.

Heart development protein with EGF-like domains 1 (HEG1) plays critical roles in embryo development and angiogenesis, which are closely related to tumor progression. However, the role of HEG1 in hepatocellular carcinoma (HCC) remains unknown. In the present study, we explored the clinical significance, biological function and regulatory mechanisms of HEG1 in HCC and found that HEG1 is significantly up-regulated in HCC cell lines and primary tumor samples. Additionally, high HEG1 expression is correlated with aggressive clinicopathological features. Patients with high HEG1 expression had shorter overall survival (OS) and disease-free survival (DFS) than those with low HEG1 expression, which indicated that HEG1 is an independent factor for poor prognosis. Lentivirus-mediated HEG1 overexpression significantly promotes HCC cell migration, invasion and epithelial-mesenchymal transition (EMT) in vitro and promotes intrahepatic metastasis, lung metastasis and EMT in vivo Opposing results are observed when HEG1 is silenced. Mechanistically, HEG1 promotes ß-catenin expression and maintains its stability, leading to intracellular ß-catenin accumulation, ß-catenin nuclear translocation and Wnt signaling activation. Loss- and gain-of-function assays further confirmed that ß-catenin is essential for HEG1-mediated promotion of HCC invasion, metastasis and EMT. In conclusion, HEG1 indicates poor prognosis; plays important roles in HCC invasion, metastasis and EMT by activating Wnt/ß-catenin signaling; and can serve as a potentially valuable prognostic biomarker and therapeutic target for HCC.

Actin-like 6A predicts poor prognosis of hepatocellular carcinoma and promotes metastasis and epithelial-mesenchymal transition.

UNLABELLED: Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide because of metastasis. Epithelial-mesenchymal transition (EMT) is widely considered to be crucial to the invasion-metastasis cascade during cancer progression. Actin-like 6A (ACTL6A) is initially verified important for cell proliferation, differentiation, and migration. In this study, we find that ACTL6A plays an essential role in metastasis and EMT of HCC. ACTL6A expression is up-regulated in HCC cells and tissues. A high level of ACTL6A in HCCs is correlated with aggressive clinicopathological features and is an independent poor prognostic factor for overall and disease-free survival of HCC patients. Ectopic expression of ACTL6A markedly promotes HCC cells migration, invasion, as well as EMT in vitro and promotes tumor growth and metastasis in the mouse xenograft model. Opposite results are observed when ACTL6A is knocked down. Mechanistically, ACTL6A promotes metastasis and EMT through activating Notch signaling. ACTL6A knockdown has the equal blockage effect as the Notch signaling inhibitor, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butylester, in HCC cells. Further studies indicate that ACTL6A might manipulate SRY (sex determining region Y)-box 2 (SOX2) expression and then activate Notch1 signaling. CONCLUSIONS: ACTL6A promotes metastasis and EMT by SOX2/Notch1 signaling, indicating a prognostic biomarker candidate and a potential therapeutic target for HCC.

MicroRNA-130b promotes proliferation and EMT-induced metastasis via PTEN/p-AKT/HIF-1α signaling.

Hepatocellular carcinoma (HCC) is a major cause of cancer-related deaths owing to its high rate of postoperative recurrence and metastasis. New research is continuously identifying novel metastasis-associated oncogenes and tumor suppressor genes. miRNAs are noncoding RNAs that regulate protein synthesis post-translationally. miR-130b is one of several miRNAs involved in tumor metastasis. However, the role of miR-130b in HCC remains controversial. Here, we demonstrate that miR-130b is highly expressed in HCC and that it correlates with tumor number, vascular invasion, and TNM stage-important predictors of postoperative recurrence and metastases. Moreover, high levels of miR-130b predicted poor overall and disease-free survival of HCC patients, and in vitro and in vivo research revealed that knockdown or overexpression of miR-130b inhibited and promoted proliferation and metastasis of HCC cells, respectively. We identified PTEN as a direct functional target of miR-130b using miRNA databases and a dual luciferase report assay. Next, using a gain and loss assay and epithelial-mesenchymal transition (EMT) relative assays, we show that miR-130b may promote proliferation and EMT-induced metastasis via PTEN/p-AKT/HIF-1α signaling. Collectively, our data suggests that miR-130b may have prognostic value in HCC. Additionally, the miR-130b/PTEN/p-AKT/HIF-1α axis identified in this study provides novel insight into the mechanisms of HCC metastasis, which may facilitate the development of new therapeutics against HCC.

MicroRNA-188-5p suppresses tumor cell proliferation and metastasis by directly targeting FGF5 in hepatocellular carcinoma.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. However, the detailed molecular mechanisms underlying HCC progression are still not completely clear. Given the crucial role of microRNAs (miRNAs) in cancer metastasis, we aimed to analyze the expression and function of a metastasis-associated miRNA named miR-188-5p in HCC. METHODS: miRNA array analysis was performed to search for metastasis-associated miRNAs in HCC. miR-188-5p expressions in tumor tissues and adjacent non-tumorous liver tissues of HCC patients and cell lines were evaluated by real-time PCR. The protein expression levels were analyzed by Western blot and immunohistochemistry. Luciferase reporter assays was used to validate the target of miR-188-5p. The effect of miR-188-5p on HCC progression was studied in vitro and in vivo. RESULTS: miR-188-5p was significantly decreased in HCC and its expression levels were highly correlated with multiple nodules, microvascular invasion, overall and disease-free survival of HCC. Ectopic expression of miR-188-5p suppressed HCC cell proliferation and metastasis in vitro and in vivo. Fibroblast growth factor 5 (FGF5) was identified as a major target of miR-188-5p. Enforced expression of miR-188-5p inhibited the expression of FGF5 significantly and the restoration of FGF5 expression reversed the inhibitory effects of miR-188-5p on HCC cell proliferation and metastasis. CONCLUSIONS: These findings collectively demonstrate a tumor suppressor role of miR-188-5p in HCC progression via targeting FGF5, suggesting that miR-188-5p could serve as a potential prognostic biomarker and therapeutic target for HCC.

MicroRNA-331-3p promotes proliferation and metastasis of hepatocellular carcinoma by targeting PH domain and leucine-rich repeat protein phosphatase.

UNLABELLED: Hepatocellular carcinoma (HCC) is a highly invasive tumor with frequent intrahepatic or pulmonary metastasis, which is the main reason for high recurrence and poor survival of HCC after liver resection. However, the mechanisms for metastasis remain incompletely clear. Given that microRNAs (miRNAs) are implicated in HCC progression, we explored a potential role of miRNAs in metastasis by performing miRNA expression profiling in three subtypes of HCC with different metastatic potentials. We discovered miR-331-3p as one of most significantly overexpressed miRNAs and highly associated with metastasis of HCC. Increased expression of miR-331-3p was correlated with poor long-term survival of HCC. We provided both in vivo and in vitro evidence demonstrating that miR-331-3p promoted proliferation and metastasis of HCC cells. Using an integrated approach, we uncovered that PH domain and leucine-rich repeat protein phosphatase (PHLPP) was a novel target of miR-331-3p. Indeed, the miR-331-3p-mediated effects were antagonized by reexpression of PHLPP or mimicked by silencing of PHLPP. We further showed that miR-331-3p-mediated inhibition of PHLPP resulted in stimulation of protein kinase B (AKT) and subsequent epithelial mesenchymal transition (EMT). Finally, inhibition of miR-331-3p through a jetPEI-mediated delivery of anti-miR-331-3p vector resulted in marked inhibition of proliferation and metastasis of HCC in xenograft mice. CONCLUSION: miR-331-3p promotes proliferation and EMT-mediated metastasis of HCC through suppression of PHLPP-mediated dephosphorylation of AKT. Our work implicates miR-331-3p as a potential prognostic biomarker and a novel therapeutic target.

Novel roles of Vmp1: inhibition metastasis and proliferation of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most deadly human cancers because of its high incidence of metastasis. Despite extensive efforts, therapies against metastasis of HCC remain underdeveloped. Vacuole membrane protein 1 (Vmp1) was recently identified to be involved in cancer-relevant processes; however, its expression, clinical significance and biological function in HCC progression are still unknown. Therefore, we evaluated the expression of Vmp1 in human HCC specimens. To functionally characterize Vmp1 in HCC, we upregulated its expression in HCCLM3 cells using a plasmid transfection approach, following which both in vitro and in vivo models were used to elucidate its role. A significant downregulation of Vmp1 was found in human HCC tissues and closely correlated with multiple tumor nodes, absence of capsular formation, vein invasion and poor prognosis of HCC. Such expression was verified with HCC cell lines including HepG2, MHCC97-L and HCCLM3, and the Vmp1 expression levels negatively correlated with metastatic potential. Interestingly, upregulation of Vmp1 significantly affects proliferation, migration, invasion and adhesion of HCCLM3 cells. Using a mouse model, we demonstrated that upregulation of Vmp1 was associated with suppression of growth and pulmonary metastases of HCC. Therefore, our data suggest Vmp1 is a novel prognostic marker and potential therapeutic target for metastasis of HCC.

ATE1 Inhibits Liver Cancer Progression through RGS5-Mediated Suppression of Wnt/ß-Catenin Signaling.

Arginyltransferase (ATE1) plays critical roles in many biological functions including cardiovascular development, angiogenesis, adipogenesis, muscle contraction, and metastasis of cancer. However, the role of ATE1 in hepatocellular carcinoma (HCC) remains unknown. In this study, we find that ATE1 plays an essential role in growth and malignancy of liver cancer. ATE1 expression is significantly reduced in human HCC samples compared with normal liver tissue. In addition, low ATE1 expression is correlated with aggressive clinicopathologic features and is an independent poor prognostic factor for overall survival and disease-free survival of patients with HCC. Lentivirus-mediated ATE1 knockdown significantly promoted liver cancer growth, migration, and disease progression in vitro and in vivo. Opposing results were observed when ATE1 was upregulated. Mechanistically, ATE1 accelerated the degradation of ß-catenin and inhibited Wnt signaling by regulating turnover of Regulator of G Protein Signaling 5 (RGS5). Loss- and gain-of-function assays confirmed that RGS5 was a key effector of ATE1-mediated regulation of Wnt signaling. Further studies indicated that RGS5 might be involved in regulating the activity of GSK3-ß, a crucial component of the cytoplasmic destruction complex. Treatment with a GSK inhibitor (CHIR99021) cooperated with ablation of ATE1 or RGS5 overexpression to promote Wnt/ß-catenin signaling, but overexpression of ATE1 or RGS5 knockdown did not reverse the effect of GSK inhibitor. IMPLICATIONS: ATE1 inhibits liver cancer progression by suppressing Wnt/ß-catenin signaling and can serve as a potentially valuable prognostic biomarker for HCC.

STMN2 mediates nuclear translocation of Smad2/3 and enhances TGFß signaling by destabilizing microtubules to promote epithelial-mesenchymal transition in hepatocellular carcinoma.

Metastasis remains the major obstacle of improving the survival of patients with hepatocellular carcinoma (HCC). Epithelial-mesenchymal transition (EMT) is critical to cancer metastasis. Successful induction of EMT requires dramatic cytoskeleton rearrangement. However, the significance of microtubule (MT), one of the core components of cell cytoskeleton, in this process remains largely unknown. Here we revealed that STMN2, an important MT dynamics regulator, is barely expressed in normal live tissues but markedly up-regulated in HCCs, especially in those with early recurrence. High STMN2 expression correlates with aggressive clinicopathological features and predicts poor prognosis of HCC patients. STMN2 overexpression in HCC cells promotes EMT, invasion and metastasis in vitro and in vivo, whereas STMN2 knockdown has opposite results. Mechanistically, STMN2 modulates MTs disassembly, disrupts MT-Smad complex, and facilitates release from MT network, phosphorylation and nuclear translocation of Smad2/3 even independent of TGFß stimulation, thereby enhancing TGFß signaling. Collectively, STMN2 orchestrates MT disassembly to facilitate EMT via TGF-ß signaling, providing a novel insight into the mechanisms underlying cancer metastasis. STMN2 is a promising prognostic biomarker and potential therapeutic target for HCC.

Programmed cell death 10 promotes metastasis and epithelial-mesenchymal transition of hepatocellular carcinoma via PP2Ac-mediated YAP activation.

Tumour metastasis is the main cause of postoperative tumour recurrence and mortality in patients with hepatocellular carcinoma (HCC), but the underlying mechanism remains unclear. Accumulating evidence has demonstrated that programmed cell death 10 (PDCD10) plays an important role in many biological processes. However, the role of PDCD10 in HCC progression is still elusive. In this study, we aimed to explore the clinical significance and molecular function of PDCD10 in HCC. PDCD10 is significantly upregulated in HCC, which also correlates with aggressive clinicopathological characteristics and predicts poor prognosis of HCC patients after liver resection. High PDCD10 expression promotes HCC cell proliferation, migration, and invasion in vitro and tumour growth, metastasis in vivo. In addition, PDCD10 could facilitate epithelial-to-mesenchymal transition (EMT) of HCC cells. In terms of the mechanism, PDCD10 directly binds to the catalytic subunit of protein phosphatase 2A (PP2Ac) and increases its enzymatic activity, leading to the interaction of YAP and dephosphorylation of the YAP protein. This interaction contributes to YAP nuclear translocation and transcriptional activation. PP2Ac is necessary for PDCD10-mediated HCC progression. Knocking down PP2Ac abolished the tumour-promoting role of PDCD10 in the migration, invasion and EMT of HCC. Moreover, a PP2Ac inhibitor (LB100) could restrict tumour growth and metastasis of HCC with high PDCD10 expression. Collectively, PDCD10 promotes EMT and the progression of HCC by interacting with PP2Ac to promote YAP activation, which provides new insight into the mechanism of cancer metastasis. PDCD10 may be a potential prognostic biomarker and therapeutic target for HCC.

Novel role for epidermal growth factor-like domain 7 in metastasis of human hepatocellular carcinoma.

UNLABELLED: Epidermal growth factor-like domain 7 (Egfl7) is a recently identified secreted protein that is believed to be primarily expressed in endothelial cells (ECs). Although its expression was reported elevated during tumorigenesis, whether and how Egfl7 contributes to human malignancies remains unknown. In the present study overexpression of Egfl7 was found predominantly in hepatocellular carcinoma (HCC) cells in HCC tissues and closely correlated with poor prognosis of HCC. The expression of Egfl7 in cancer cells was further verified with HCC cell lines including HepG2, MHCC97-L, and HCCLM3, and the Egfl7 expression levels positively correlated with metastatic potential of HCC cell lines was tested. To functionally characterize Egfl7 in HCC, we depleted its expression in HCCLM3 cells by using small interfering RNA. Interestingly, reduction of Egfl7 expression resulted in significant inhibition of migration but not growth of HCCLM3 cells. Biochemical analysis indicated that Egfl7 could facilitate the phosphorylation of focal adhesion kinase (FAK) and therefore promote the migration of HCCLM3 cells. In addition, this effect was almost completely blocked by inhibition of epidermal growth factor receptor (EGFR), indicating that the activation of FAK by Egfl7 is mediated through EGFR. Finally, we used a mouse model to demonstrate that down-regulation of Egfl7 was associated with suppression of intrahepatic and pulmonary metastases of HCC. Collectively, our study shows for the first time that overexpression of Egfl7 in HCC and Egfl7 promotes metastasis of HCC by enhancing cell motility through EGFR-dependent FAK phosphorylation. CONCLUSION: Our study suggests Egfl7 as a novel prognostic marker for metastasis of HCC and a potential therapeutic target.

Decreased expression of inhibitor of growth 4 correlated with poor prognosis of hepatocellular carcinoma.

OBJECTIVE: Inhibitor of growth 4 (ING4) is a candidate tumor suppressor that plays an important role in tumor growth and angiogenesis. Here, we examined the expression of ING4 in hepatocellular carcinoma (HCC) tissues and analyzed its correlation with the progression of HCC. METHODS: Specimens from 136 HCC patients were determined immunohistochemically for ING4 expression. The correlation of ING4 levels with clinicopathologic variables, prognosis, and metastatic potential was analyzed. Among the 136 cases, 36 paired HCC and paracarcinomatous liver tissue specimens were analyzed for ING4 expression levels by real-time quantitative reverse transcription-PCR and Western blotting. MVD was determined by CD34 immunostaining to test whether it correlated with ING4 protein expression level. RESULTS: The ING4 mRNA and protein levels were significantly lower in HCC than paracarcinomatous liver tissue from both real-time quantitative reverse transcription-PCR and Western blotting (P = 0.039 and 0.012, respectively). Importantly, the ING4 protein level correlated with the Edmondson-Steiner grade (P = 0.035), vein invasion (P = 0.015), and microvessel density (P = 0.005). Survival and metastasis analysis indicated that HCC patients with lower ING4 expression had poorer overall survival and disease-free survival than those with high expression (P = 0.0001 and 0.0065; respectively). Multivariable Cox regression analysis revealed that the ING4 expression level was an independent factor for prognosis (hazard risk, 9.63; P = 0.001). CONCLUSIONS: ING4 expression is down-regulated in HCC tissues. ING4 expression level correlates with prognosis and metastatic potential, which suggests that ING4 is a candidate prognostic marker of HCC.

Solitary large hepatocellular carcinoma: a specific subtype of hepatocellular carcinoma with good outcome after hepatic resection.

OBJECTIVE: To evaluate and compare the clinical and pathologic characteristics and outcomes after hepatic resection of large hepatocellular carcinoma (SLHCC), small HCC (SHCC), and nodular HCC (NHCC). SUMMARY BACKGROUND DATA: Traditional viewpoint insists that the classification and prognosis of HCC are determined by the size of HCC. As a result, large HCC is often considered as advanced and unresectable. However, we have observed a unique type of HCC-SLHCC, which is large in size but exhibits good clinical, pathologic, and molecular biologic characteristics as well as prognosis. METHODS: From January 1992 to December 2002, a total of 481 consecutive patients were diagnosed with HCC and received hepatic resection. In this series of patients, the clinical and pathologic data, surgical outcome, and long-term survival of patients with SLHCC (group A, n = 260) were analyzed retrospectively and compared with patients who had SHCC (group B, n = 135) or NHCC (group C, n = 86). Postresection prognostic factors of HCC were also evaluated by univariate and multivariate analysis using Cox's proportional hazards model. RESULTS: The clinical and pathologic characteristics of SLHCC and SHCC were similar except for tumor necrosis and tumor size. Patients of SLHCC had significantly longer operative time, higher intraoperative blood loss, higher intraoperative blood transfusion, and higher postoperative morbidity than SHCC. However, the 2 groups were similar in duration of hospital stay and overall morbidity. Overall survival and disease-free survival in group A and group B were similar and significantly better than those in group C. Multivariate analysis revealed that large amount of intraoperative blood transfusion and vein invasion were independently significant factors for overall survival of patients with HCC. CONCLUSION: The clinical and pathologic characteristics and the outcome after hepatic resection of SLHCC are similar to that of SHCC, but significantly better than NHCC. We propose SLHCC as a specific subtype of HCC and hepatic resection as its choice of standard treatment.

Identification of gene expression profiling in hepatocellular carcinoma using cDNA microarrays.

The aim of this study was systematic investigation of the differentially expressed genes during carcinogenesis in hepatocellular carcinoma (HCC) using cDNA microarray technology. The differentially expressed genes between 22 fresh HCC tissues and para-cancerous liver tissues (PCLT) were displayed using cDNA microarray technology. The result was verified by the reverse transcriptase polymerase chain reaction. Among the 8,464 human genes, 507 (5.99%) genes were expressed differentially at the mRNA levels between HCC and PCLT. Two hundred (2.36%) genes were down-regulated, whereas 307 (3.63%) genes were up-regulated. The mRNA expression levels of RhoC and protocadherin LKC detected by reverse transcription polymerase chain reaction (RT-PCR) were consistent with the microarray experiment. This study describes a gene expression profiling of HCC, which provides an extensive list of potential molecular markers for early diagnosis and molecular targets for the development of drugs to treat patients with primary HCC.

[Down-regulated expression of UNC5b related to hepatocellular carcinoma angiogenesis].

OBJECTIVE: To investigate the relationship between UNC5b gene expression and angiogenesis of hepatocellular carcinoma (HCC). METHODS: In situ hybridization was performed to detect the expression of UNC5b mRNA in HCC samples, paracarcinomatous liver tissues samples and normal liver samples. The relationship between UNC5b mRNA expression and the HCC clinicopathological features were also analyzed. Human umbilical artery endothelial cells were isolated and stimulated with HCC tissues homogenate, vascular endothelial growth factor and basic fibroblast growth factor. Then RT-PCR was employed to detect the expression of UNC5b mRNA in normal HUAEC as well as activated HUAEC. RESULTS: In situ hybridization results showed that UNC5b mRNA expression was detected majorly in endothelial cells of all normal liver tissues, and partial PCLTs but was weak or even undetectable in endothelial cells of the corresponding HCC tissues. The expression levels of UNC5b gene in PCLTs were significantly correlated with capsular formation of HCC. Furthermore, RT-PCR results showed that the expression levels of UNC5b mRNA in activated HUAEC were significantly higher than those in normal HUAEC. CONCLUSIONS: Down-regulation of UNC5b gene expression is related to angiogenesis of HCC, which may be associated with the progression of HCC.