Lian-Yue Yang

MicroRNA‐140‐5p suppresses tumor growth and metastasis by targeting transforming growth factor β receptor 1 and fibroblast growth factor 9 in hepatocellular carcinoma

By comparing the expression profiles of microRNAs (miRNAs) in different hepatocellular carcinoma (HCC) subtypes, we identified miR‐140‐5p as an HCC‐related miRNA. We found that miR‐140‐5p was significantly decreased in HCC tissues and all of six liver cancer cell lines examined and its expression levels were correlated with multiple nodules, vein invasion, capsular formation, and differentiation, as well as overall and disease‐free survival of HCC. We also found that miR‐140‐5p suppressed HCC cell proliferation and HCC metastasis. Multipathway reporter arrays suggested that miR‐140‐5p inhibited transforming growth factor β (TGF‐β) and mitogen‐activated protein kinase / extracellular signal‐regulated kinase (MAPK/ERK) signaling. TGFB receptor 1 (TGFBR1) and fibroblast growth factor 9 (FGF9) were then characterized as the direct targets for miR‐140‐5p after it was found that ectopic miR‐140‐5p expression suppressed TGFBR1 and FGF9 expression. Silencing TGFBR1 and FGF9 by small interfering RNA (siRNA) resembled the phenotype resulting from ectopic miR‐140‐5p expression, while overexpression of TGFBR1 and FGF9 attenuated the effect of miR‐140‐5p on HCC growth and metastasis. Conclusion: These data elucidated a tumor suppressor role for miR‐140‐5p in HCC development and progression with therapeutic potential. Our correlation studies in clinical HCC samples further suggest that miR‐140‐5p could be a valuable biomarker for HCC prognosis. (Hepatology 2013 )

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 Coxs 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.

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

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. (Hepatology 2014;60:1251–1263)

Increased Expression of Wiskott-Aldrich Syndrome Protein Family Verprolin-Homologous Protein 2 Correlated with Poor Prognosis of Hepatocellular Carcinoma

Purpose: Because of its role in cell migration, the Wiskott-Aldrich syndrome protein family verprolin-homologous protein (WAVE) 2 has been implicated in cancer metastasis. Evidence to support such a role of WAVE2 in human cancer, however, is lacking. We thus examined the expression of WAVE2 in hepatocellular carcinoma (HCC) tissues to test whether the levels of WAVE2 expression correlated to the progression of HCC. Experimental Design: Samples of 112 HCC patients were determined immunohistochemically for WAVE2 expression and the correlation of WAVE2 levels with prognosis was analyzed. Among the 112 cases, 31 paired HCC and paracarcinomatous liver tissue specimens were analyzed for WAVE2 levels by reverse transcription-PCR and Western blotting, respectively. Results: Among 112 cases of HCCs, the immunohistochemistry data indicated significant increase of WAVE2 expression levels in 71 cases. Importantly, the increased WAVE2 expression correlated with the multiple tumor nodules (P = 0.008), the absence of capsular formation (P = 0.035), Edmondson-Steiner grade (P = 0.009), vein invasion (P = 0.023), and a shortened median survival time (326 versus 512 days; P = 0.003). Multivariable Cox regression analysis revealed the WAVE2 expression level was an independent factor for prognosis. The immunohistochemistry data were further confirmed by results of reverse transcription-PCR and Western analysis of 31 HCC cases, in which the WAVE2 mRNA and protein in HCC tissues were significantly elevated when compared with paracarcinomatous liver tissue (P < 0.001). Conclusions: WAVE2 expression is elevated in HCC tissues, which correlates with a poor prognosis, suggesting WAVE2 as a candidate prognostic marker of 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.

Inhibition of Invasion and Metastasis of Hepatocellular Carcinoma Cells via Targeting RhoC In vitro and In vivo

Purpose: Hepatocellular carcinoma (HCC) is one of the most deadly human cancers because of its high incidence of metastasis. Our previous work identified a strong correlation between increased expression of RhoC and HCC metastasis. Here, we investigate to define the role of RhoC in HCC metastasis. Furthermore, we sought to determine whether inhibition of the expression of RhoC might block the metastasis of HCC in vivo. Experimental Design: A stable retroviral small interfering RNA approach was employed to selectively knockdown the expression of RhoC in vitro and in vivo. Invasion and migration assay, MTT and fluorescence-activated cell sorting analysis, Rho activity assay, and immunofluorescence staining were carried out to characterize RhoC in vitro. An anti-RhoC retroviral gene delivery BALB/c nude mice model was established to investigate whether knockdown of the expression of RhoC might inhibit the metastasis of HCC in vivo. Results: We confirmed the correlation of RhoC expression and metastatic potentials of HCC cell lines. We also showed that suppression of RhoC expression resulted in inhibition of invasion and migration without an apparent effect on cell survival and proliferation in HCCLM3 cells. Furthermore, a similar effect of RhoC on autotaxin-induced invasion of HCCLM3 cells was also observed. Significantly, we successfully adopted an HCC metastatic mouse model that allowed us to show that knockdown of the RhoC expression resulted in inhibition of metastasis of HCC in vivo for the first time. Conclusions: Our results show a critical role of RhoC in metastasis of HCC, implicating RhoC as a potential therapeutic target to block HCC metastasis.

Determination of High Mobility Group A1 (HMGA1) Expression in Hepatocellular Carcinoma: A Potential Prognostic Marker

Our objective was to investigate the expression of HMGA1 mRNA and protein in hepatocellular carcinoma (HCC) and the correlation between its expression and clinical pathological characteristics and prognosis. HMGA1 expression was determined at both the mRNA level and the protein level in 30 HCC tissues and their corresponding paracancer liver tissues (PCLTs) and 2 normal liver tissues by RT-PCR and IHC. Follow-up study was done on the 30 patients involved in this research. HMGA1 mRNA was detected in nine cases of HCC tissues and two PCLTs, for a positivity rate of 30% and 6.7%, respectively (P < 0.05), whereas no HMGA1 mRNA expression was found in normal liver tisssues. Clinicopathological analysis revealed that HMGA1 mRNA expression was significantly correlated with Edmondsons grade (P < 0.05). HMGA1 protein was detected in four HCC tissues by IHC and located mainly in the nuclei; no positive staining was found in PCLTs. Follow-up study showed that HMGA1 mRNA-positive patients had a higher risk of recurrence/metastasis and a shorter survival than negative cases (P < 0.05). Our findings indicate that HMGA1 may be involved in the carcinogenesis and invasiveness of HCC and the determination of HMGA1 can be of great value in predicting the prognosis of patients with HCC.

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

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. (Hepatology 2016;63:1256–1271)

RhoC is essential for angiogenesis induced by hepatocellular carcinoma cells via regulation of endothelial cell organization

The angiogenesis induced by tumor cells is essential for metastasis of hepatocellular carcinoma. Available information suggests that RhoC participates in angiogenesis through regulation of vascular endothelial growth factor expression in tumor cells. For its broad functions in cell migration and cytoskeletal organization, we hypothesized that RhoC regulating angiogenesis does not exclusively depend on regulation of vascular endothelial growth factor expression. To address this question, in the present study, we used a retroviral small interfering RNA approach to selectively knockdown the expression of RhoC in a neovascularization model in vivo and in vitro. Our present results indicate that RhoC is the downstream regulator of vascular endothelial growth factor in endothelial cells and is essential for angiogenesis induced by vascular endothelial growth factor, notwithstanding that RhoC regulates the expression of vascular endothelial growth factor in tumor cells. Furthermore, we show that knockdown of RhoC is associated with the inhibition of invasion and migration but not apoptosis of endothelial cells. Knockdown of RhoC results in inhibition of endothelial cell organization through restraining the reorganization of F‐actin filaments, which represses endothelial cell network and sprout formation. In conclusion, our results demonstrate that knockdown of RhoC inhibits angiogenesis induced by tumor cells not only through effecting the release of vascular endothelial growth factor, but also through inhibiting endothelial cell migration and organization, which implies that it blocks tumor metastasis by specifically inhibiting RhoC in endothelial cells. (Cancer Sci 2008; 99: 2012–2018)

The expression of Egfl7 in human normal tissues and epithelial tumors.

Background and aims To investigate the expression of Egfl7 in normal adult human tissues and human epithelial tumors. Methods RT-PCR and Western blot were employed to detect Egfl7 expression in normal adult human tissues and 10 human epithelial tumors including hepatocellular carcinoma (HCC), lung cancer, breast cancer, prostate cancer, colorectal cancer, gastric cancer, esophageal cancer, malignant glioma, ovarian cancer and renal cancer. Immunohistochemistry and cytoimmunofluorescence were subsequently used to determine the localization of Egfl7 in human epithelial tumor tissues and cell lines. ELISA was also carried out to examine the serum Egfl7 levels in cancer patients. In addition, correlations between Egfl7 expression and clinicopathological features as well as prognosis of HCC and breast cancer were also analyzed on the basis of immunohistochemistry results. Results Egfl7 was differentially expressed in 19 adult human normal tissues and was overexpressed in all 10 human epithelial tumor tissues. The serum Egfl7 level was also significantly elevated in cancer patients. The increased Egfl7 expression in HCC correlated with vein invasion, absence of capsule formation, multiple tumor nodes and poor prognosis. Similarly, upregulation of Egfl7 in breast cancer correlated strongly with TNM stage, lymphatic metastasis, estrogen receptor positivity, Her2 positivity and poor prognosis. Conclusions Egfl7 is significantly upregulated in human epithelial tumor tissues, suggesting Egfl7 to be a potential biomarker for human epithelial tumors, especially HCC and breast cancer.