Invasion Of Liver Cancer Cells Research Articles

Hepatitis B virus-regulated growth of liver cancer cells occurs through the microRNA-340-5p-activating transcription factor 7-heat shock protein A member 1B axis.

Hepatocellular carcinoma (HCC) is a common cancer with poor prognosis. Hepatitis B virus (HBV) is one of the leading causes of HCC, but the precise mechanisms by which this infection promotes cancer development are not fully understood. Recently, miR‐340‐5p, a microRNA (miRNA) that has been identified as a cancer suppressor gene, was found to inhibit the migration and invasion of liver cancer cells. However, the effect of miR‐340‐5p on cell proliferation and apoptosis in HBV‐associated HCC remains unknown. In our study, we show that miR‐340‐5p plays an important role during HBV infection and hepatocellular carcinoma development. Specifically, this miRNA directly binds to the mRNA encoding activating transcription factor 7 (ATF7), a protein that both promotes cell proliferation and suppresses apoptosis through its interaction with heat shock protein A member 1B (HSPA1B). We further found that miR‐340‐5p is downregulated by HBV, which enhances ATF7 expression, leading to enhanced cell proliferation and inhibition of apoptosis. Notably, ATF7 is upregulated in HCC tissue, suggesting that HBV may target miR‐340‐5p in vivo to promote ATF7/HSPA1B‐mediated proliferation and apoptosis and regulate liver cancer progression. This work helps to elucidate the complex interactions between HBV and host miRNAs and further suggests that miR‐340‐5p may represent a promising candidate for the development of improved therapeutic strategies for HCC.

ZEB1 causes the production of hsa-microRNA-99b/let-7e/microRNA-125a cluster and promotes invasion of liver cancer cells.

To explore the role of hsa-microRNA-99b/let-7e/microRNA-125a cluster in the progression of liver cancer and its possible regulatory mechanisms. Ten liver cancer tissues were randomly selected and matched with normal liver tissue samples. The mRNA expression levels of miR-99b, let-7e and miR-125a were detected by Real Time-quantitative Polymerase Chain Reaction (RT-qPCR). The three miRNA mimics were transfected alone or together into hepatoma cells SMMC-7721; at the same time, the knockdown of the three miRNAs was also performed. Then the cell invasive and migratory abilities were examined through transwell assay. Bioinformatics analysis was used to detect the potential transcription factors that bind to the hsa-microRNA-99b/let-7e/microRNA-125a promoter sequence, and the binding of the two was verified by the Luciferase reporting assay. The level of hsa-microRNA-99b/let-7e/microRNA-125a mature mRNA was detected after ZEB1 was inhibited by ZEB1 siRNA. Meanwhile, after interfering with Drosha and ZEB1, the expression level of hsa-microRNA-99b/let-7e/microRNA-125a of the primary transcript was examined. Rescue experiments were carried out to assess the role of hsa-microRNA-99b/let-7e/microRNA-125a in the ZEB1 regulation of invasive cell capacity. The mRNA levels of microRNA-99b, let-7e, and microRNA-125a in 10 selected hepatocarcinoma tissues were significantly higher than those in the matched paracancerous tissues. After overexpressing the three miRNA mimics either alone or together, cell invasive and migratory abilities were extensively enhanced, and vice versa. It was found that there is a binding site in the upstream sequence of the promoter region of the hsa-microRNA-99b/let-7e/microRNA-125a cluster for ZEB1. The Luciferase reporter gene results showed an increase in the Luciferase activity of the cells transfected with E-box element wild-type sequence, while mutant E-box element group did not change. After knocking out ZEB1, the levels of mature microRNA-99b, let-7e and microRNA-125a were reduced. However, when DROSHA was knocked out, the levels of immature microRNA-99b, let-7e and microRNA-125a were increased, while simultaneous knocking out ZEB1 reversed this effect. Besides, the invasive ability of SMMC-7721 cells decreased after KEB1 was knocked down, while the opposite result was observed after transfection of hsa-microRNA-99b/let-7e/microRNA-125a alone or together. Hsa-microRNA-99b/let-7e/microRNA-125a cluster is highly-expressed in hepatocarcinoma, and its expression can be regulated by ZEB1. In addition, the overexpression of this cluster can promote the invasion of liver cancer cells and advance liver cancer progression.

WDR76 is a RAS binding protein that functions as a tumor suppressor via RAS degradation

Stability regulation of RAS that can affect its activity, in addition to the oncogenic mutations, occurs in human cancer. However, the mechanisms for stability regulation of RAS involved in their activity and its roles in tumorigenesis are poorly explored. Here, we identify WD40-repeat protein 76 (WDR76) as one of the HRAS binding proteins using proteomic analyses of hepatocellular carcinomas (HCC) tissue. WDR76 plays a role as an E3 linker protein and mediates the polyubiquitination-dependent degradation of RAS. WDR76-mediated RAS destabilization results in the inhibition of proliferation, transformation, and invasion of liver cancer cells. WDR76−/− mice are more susceptible to diethylnitrosamine-induced liver carcinogenesis. Liver-specific WDR76 induction destabilizes Ras and markedly reduces tumorigenesis in HRasG12V mouse livers. The clinical relevance of RAS regulation by WDR76 is indicated by the inverse correlation of their expressions in HCC tissues. Our study demonstrates that WDR76 functions as a tumor suppressor via RAS degradation.

The Down-Regulation of SUZ12 Accelerates the Migration and Invasion of Liver Cancer Cells via Activating ERK1/2 Pathway.

The suppressor of zest 12 (SUZ12), an essential subunit of the transcription polycomb repressive complex 2 (PRC2), has been found to be involved in HBV X-induced oncogenic transformation in hepatocellular carcinoma (HCC). However, the specific function of SUZ12 has not yet been determined in the pathogenesis of migration and invasion of HBV-associated HCC. Here, our results showed that SUZ12 was significantly down-regulated in HBV-related HCC tissues compared with adjacent non-tumor tissues by immunohistochemical and Western blot assays. The 5-years survival rate was worse in patients with low expression level of SUZ12. SUZ12 silencing increased the migration and invasion of HCC cells, and its overexpression impaired HCC cells migration and invasion. Knockdown of SUZ12 activated ERK1/2 pathway and increased MMP9 (matrix metallopeptidase 9) and MMP2 (matrix metallopeptidase 2) expression, whereas SUZ12 overexpression had opposite effects. Specific ERK1/2 inhibitor (SCH772984) significantly decreased HCC cells migration and invasion caused by SUZ12 shRNA. Thus, the liver cancer-down-regulated SUZ12 accelerated the invasion and metastasis of HCC cells. These effects might be associated with deregulation of SUZ12 activating ERK1/2, MMP2 and MMP9 in HCC cells.

Ginsenoside Rg3 inhibits the migration and invasion of liver cancer cells by increasing the protein expression of ARHGAP9.

Ginsenoside Rg3, a naturally occurring phytochemical, serves an important role in the prevention and treatment of cancer. In the present study, with the aim to reveal the molecular mechanism of Rg3 in liver cancer cell metastasis, the anti-migration and anti-invasion effects of Rg3 on liver cancer cells were investigated. It was demonstrated that Rg3 caused marked inhibition of cell migration and invasion of human liver cancer cells, HepG2 and MHCC-97L, in vitro, and the growth of HepG2 and MHCC-97L tumors in BABL/c nude mice. The protein expression of Rho GTPase activating protein 9 (ARHGAP9) was increased both in HepG2 and MHCC-97L cells. Following ARHGAP9 knockdown, the results of Transwell and tumorigenesis assays revealed that the anti-migration, anti-invasion and anti-tumor growth effects of Rg3 were impaired significantly. The increased expression of ARHGAP9 protein induced by Rg3 was remarkably suppressed. All results suggested that ARHGAP9 protein may be a vital regulator in the anti-metastatic role of Rg3. To the best of our knowledge, the present study is the first to report that Rg3 effectively suppressed the migration and invasion of liver cancer cells by upregulating the protein expression of ARHGAP9, indicating a novel natural therapeutic agent and a therapeutic target for the treatment of liver cancer.

Ginkgolic acid suppresses the invasion of HepG2 cells via downregulation of HGF/c‑Met signaling.

Liver cancer is one of the most devastating types of cancer worldwide. Despite years of improvements in treatment, the prognosis of patients with this type of malignancy remains poor due to frequent recurrence and metastasis after surgical resection. Ginkgolic acid (GA) is a botanical drug extracted from the seed coat of Ginkgobiloba L. that possesses a wide range of bioactive properties. However, to the best of our knowledge, whether GA can inhibit the invasion of liver cancer cells and the underlying mechanisms remains unknown. The aim of the present study was to investigate the effects of GA on the migration and invasion abilities of liver cancer cells and the underlying molecular mechanism. The results revealed that GA suppressed the migration and invasion abilities of HepG2 cells. In addition, GA treatment inhibited the expression of invasion‑related molecules (MMP‑2 and MMP‑9) and prevented the epithelial‑mesenchymal transition (EMT) of HepG2 cells. Further experiments revealed that GA‑reduced hepatocyte growth factor (HGF) production and suppressed c‑Met phosphorylation may be the underlying mechanisms. Exogenous recombinant HGF supplementation improved the cell invasion ability impaired by GA. Moreover, the invivo experiment revealed that GA inhibited the tumor growth of liver cancer and prevented EMT. Collectively, these data indicated that GA effectively suppressed the invasion and EMT of HepG2 cells via downregulation of HGF/c‑Met signaling, thus GA may serve as a novel chemotherapeutic agent for the treatment of HCC.

Corrigendum to "NANOG promotes liver cancer cell invasion by inducing epithelial-mesenchymal transition through NODAL/SMAD3 signaling pathway" [Int. J. Biochem. Cell Biol. (2013) 1099-1108

Corrigendum to "NANOG promotes liver cancer cell invasion by inducing epithelial-mesenchymal transition through NODAL/SMAD3 signaling pathway" [Int. J. Biochem. Cell Biol. (2013) 1099-1108

EMT induced by loss of LKB1 promotes migration and invasion of liver cancer cells through ZEB1-induced YAP signaling.

Liver cancer cells often exhibit mesenchymal phenotypes, a critical phenotypic alteration of cancer cells termed the epithelial-mesenchymal transition (EMT). To examine whether liver kinase B1 (LKB1) serves a potential role in EMT in liver carcinogenesis, in the present study, it was determined that the expression of LKB1 decreased in the hepatocellular carcinoma (HCC) cell line, compared with a normal liver cell line. LKB1 overexpression decreased cell motility and invasiveness. Furthermore, the loss of LKB1 induced the expression of several EMT marker proteins, including that of Zinc Finger E-Box Binding Homeobox 1 (ZEB1). Notably, the expression of Yes-associated protein (YAP) was positively associated with that of ZEB1 in LKB1-knockdown cells with a mesenchymal phenotype. Here, we describe the direct regulation of the Hippo pathway effector YAP by ZEB1. The findings of the present study demonstrate that ZEB1 regulates the expression of YAP and regulates the expression of downstream target genes to promote malignant progression.

Effect on the liver cancer cell invasion ability by studying the associations between autophagy and TRAP1 expression.

Liver cancer is one of the leading causes of cancer associated mortality, particularly in eastern Asia. Autophagy serves an important role in carcinogenesis. Previous studies have reported that TRAP1 is a novel and efficient therapeutic target in various tumors. However, the associations between autophagy and TRAP1 is not clear. In the present study, autophagy activity and TRAP1 expression were examined in 4 different liver cancer cell lines (HepG2, Hep3B2.1–7, Sk-hep1 and HepG2.2.15) with or without rapamycin induction. The cell autophagy level was validated by monodansylcadaverine fluorescent staining, and the expression levels of Beclin1 and light chain (LC)-3-II/LC3-I. The mRNA and protein expression levels of tumor necrosis factor receptor-associated protein-1 (TRAP-1), Beclin1 and LC3-II/LC3-I were measured by reverse transcription-quantitative polymerase chain reaction, Protein Simple Western and western blot analysis. HepG2 cells, with medium invasive ability, exerted the highest basal level of autophagy and TRAP1 expression. In addition, hepatitis B (HBV) infection in HepG2 cells inhibited autophagy activity and TRAP1 expression. Rapamycin treatment also significantly enhanced autophagy in the 4 liver cancer cell lines and increased TRAP1 expression in HepG2, Hep3B2.1–7 and Sk-hep1 cells. Thus, the cell invasive ability, HBV infection and autophagy induction had different effects on TRAP1 expression, and TRAP1 may be associated with autophagy in liver cancer.

Anesthetic effects of isoflurane and the molecular mechanism underlying isoflurane‑inhibited aggressiveness of hepatic carcinoma.

Anesthesia is produced by drugs or other methods, and refers to the attenuation of pain via reversible suppression of neuronal transmission in the central and peripheral nervous systems, during surgery. Clinical investigations have indicated that the anesthetic action of isoflurane is efficient to alleviate pain during tumor resection clinical trials. In addition, it has been reported that isoflurane can induce caspase-3 activation and is associated with apoptosis of tumor cells. The present study investigated the anesthetic effects and molecular mechanisms underlying isoflurane-induced apoptosis in patients with hepatic carcinoma. Furthermore, the pain of patients with hepatic carcinoma was evaluated during the perioperative period according to the pain index. The apoptotic rate of hepatic carcinoma cells was analyzed in tumor tissues using TUNEL assay. The expression levels of apoptosis-associated proteins were detected in liver cancer cells following anesthesia in patients. Phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and nuclear factor (NF)-κB signaling pathways were also analyzed in liver cancer cells following treatment with isoflurane. The results demonstrated that isoflurane inhibited growth and decreased viability of liver cancer cells in vitro and in vivo. In addition, the apoptotic rate was increased in cells obtained from isoflurane-treated patients. The results also demonstrated that isoflurane upregulated the expression levels of proapoptotic genes and downregulated anti-apoptotic mRNA expression. In addition, a molecular mechanism analysis indicated that isoflurane inhibited PI3K and AKT expression in liver cancer cells. Isoflurane also induced caspase-3 activation in liver cancer cells. Furthermore, isoflurane treatment attenuated NF-κB activity and inhibited migration and invasion of liver cancer cells. In conclusion, these findings indicated that isoflurane treatment efficiently attenuated surgical pain and inhibited tumor aggressiveness via regulation of NF-κB activity and the PI3K/AKT signaling pathway, thus suggesting that isoflurane is an efficient anesthetic drug that induces pain remission and promotes apoptosis of liver cancer cells.

Circular RNA circCDK13 suppresses cell proliferation, migration and invasion by modulating the JAK/STAT and PI3K/AKT pathways in liver cancer.

Circular RNAs have recently been disclosed as potential biomarkers for human cancers. This study aimed to characterize the expression and function of a novel circular RNA, circCDK13, in liver cancer progression, as well as to elucide the underlying mechanisms. For this purpose, circCDK13 expression was quantitatively analyzed by RT-PCR in various liver cancer cell lines and human cancerous tissues. The migration, cell cycle progression, proliferation and invasion of liver cancer cells with an enhanced circCDK13 expression were evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, flow cytometry and the Transwell culture system. Microarray and western blot analyses were performed to explore the underlying signaling mechanisms. The role of circCDK13 in liver cancer was finally examined by tumorigenicity assay using nude mice. The results revealed that circCDK13 expression was suppressed in various liver cancer lines and tissue samples from patients with liver cancer (hepatocellular carcinoma). The induced overexpression of circCDK13 in the liver cancer cells markedly inhibited their migration rates, altered cell cycle progression, and suppressed the cell migratory and invasive capacities. Microarray analysis also identified numerous downstream genes regulated by circCDK13, particularly those in the Janus tyrosine kinase(JAK)/signal transducer and activator of transcription(STAT) and phosphoinositide3-kinase(PI3K)/AKT signaling pathways. The results of the tumorigenicity assay revealed that circCDK13 overexpression significantly inhibited liver cancer progression in nude mice. On the whole, the findings of this study indicate that circCDK13 is a novel circular RNA that suppresses liver cancer progression, and that these suppressive effects are possibly mediated via the JAK/STAT and PI3K/AKT signaling pathways.

Benzo(a)pyrene promotes Hep-G2 cell migration and invasion by upregulating phosphorylated extracellular signal-regulated kinase expression.

Benzo(a)pyrene (BaP), a carcinogenic component of cigarette smoke, has been reported to activate extracellular signal-regulated kinase (ERK) in cancer cells. Furthermore, activated ERK is associated with liver cancer cell invasion and metastasis. Therefore, the aim of the present study was to investigate the potential role of phosphorylated (p)-ERK in BaP-induced Hep-G2 cell migration and invasion. An MTT assay was used to determine the effects of BaP treatment on Hep-G2 cell proliferation. Wound-healing and Transwell invasion assays were employed to assess the migration and invasion abilities of Hep-G2 cells. Western blot analysis was applied to detect the expression of proteins. The results of the present study demonstrated that BaP treatment was able to increase the level of p-ERK protein expression in Hep-G2 cells. BaP treatment promoted Hep-G2 cell migration and invasion. The ERK inhibitor, U0126, was able to block the migration and invasion abilities of Hep-G2 cells induced by BaP. The results of the present study demonstrated that BaP treatment promoted the migration and invasion of Hep-G2 cells by upregulating p-ERK expression.

Copper/MYC/CTR1 interplay: a dangerous relationship in hepatocellular carcinoma.

Free serum copper correlates with tumor incidence and progression of human cancers, including hepatocellular carcinoma (HCC). Copper extracellular uptake is provided by the transporter CTR1, whose expression is regulated to avoid excessive intracellular copper entry. Inadequate copper serum concentration is involved in the pathogenesis of Non Alcoholic Fatty Liver Disease (NAFLD), which is becoming a major cause of liver damage progression and HCC incidence. Finally, MYC is over-expressed in most of HCCs and is a critical regulator of cellular growth, tumor invasion and metastasis.The purpose of our study was to understand if higher serum copper concentrations might be involved in the progression of NAFLD-cirrhosis toward-HCC. We investigated whether high exogenous copper levels sensitize liver cells to transformation and if it exists an interplay between copper-related proteins and MYC oncogene.NAFLD-cirrhotic patients were characterized by a statistical significant enhancement of serum copper levels, even more evident in HCC patients. We demonstrated that high extracellular copper concentrations increase cell growth, migration, and invasion of liver cancer cells by modulating MYC/CTR1 axis. We highlighted that MYC binds a specific region of the CTR1 promoter, regulating its transcription. Accordingly, CTR1 and MYC proteins expression were progressively up-regulated in liver tissues from NAFLD-cirrhotic to HCC patients.This work provides novel insights on the molecular mechanisms by which copper may favor the progression from cirrhosis to cancer. The Cu/MYC/CTR1 interplay opens a window to refine HCC diagnosis and design new combined therapies.

MicroRNA-493-5p promotes apoptosis and suppresses proliferation and invasion in liver cancer cells by targeting VAMP2.

The aim of the present study was to explore the role of miR‑493-5p in liver cancer tissues and cell lines, and its effect on cell behavioral characteristics. The expression of miR-493-5p was detected by reverse transcription-quantitative polymerase chain reaction(RT-qPCR) in liver cancer tissues and cell lines (hepatic cell line HL-7702 and the liver cancer cell lines HCCC-9810, HuH-7 and HepG2). In addition, the mechanism by which miR-493-5p mediates its effects was analyzed via the transfection of miR-493-5p mimic and negative control miRNA into HepG2cells. The viability, proliferation, apoptosis and invasion of the cells were analyzed using MTT assay, flow cytometry and Transwell chamber experiments. Furthermore, the effect of miR-493-5p on the expression of vesicle associated membrane protein2(VAMP2) was assayed using a dual-luciferase reporter system, and VAMP2 protein levels were determined by western blot analysis. In addition, following the cotransfection of HepG2 cells with pcDNA3.1‑VAMP2 plasmid and miR‑493-5p mimic, the role of miR-493-5p as a regulator of VAMP2 was evaluated using MTT assay, flow cytometry and Transwell chamber experiments. RT-qPCR analysis indicated that the expression of miR-493-5p in liver cancer tissues and cell lines was decreased significantly compared with that in adjacent normal liver tissues and normal liver cell lines, respectively. Compared with the control group, the cells transfected with miR-493-5p mimic (the miR-493-5p overexpression group) exhibited reduced cell viability, a reduced percentage of cells in the Sphase and an increased percentage of apoptotic cells. In addition, fewer cells passed through the Transwell membrane in the miR-493-5p overexpression group compared with the control group. In the dual-luciferase reporter assay, luciferase activity in the miR‑493-5p overexpression group was attenuated compared with that in the control group. Inaddition, western blot analysis indicated that the VAMP2 protein levels in the miR‑493-5p overexpression group were lower than those in the control group. Furthermore, in cells overexpressing miR-493-5p and VAMP2 simultaneously, the biological behavior of the cells, including cell viability, cell cycle and cell invasiveness, was significantly rescued compared with that of the control group transfected with miR‑493-5p alone. In conclusion, miR-493-5p is indicated to be a tumor suppressor gene, and is downregulated in human liver cancer. miR-493-5p overexpression promotes cell apoptosis and inhibits the proliferation and migration of liver cancer cells by negatively regulating the expression of VAMP. These observations suggest the potential of treating liver cancer by the overexpression of microRNA-493-5p.

Artemisia capillaris extract AC68 induces apoptosis of hepatocellular carcinoma by blocking the PI3K/AKT pathway

Artemisia capillaris Thunberg (AC) has been widely used to treat various diseases including hepatitis and is known to affect many cellular events such as cell proliferation and apoptosis. Herein a potent ethyl acetate fraction (AC68) was newly extracted from AC, and was assessed for its anti-cancer efficacy in progression and growth of hepatocellular carcinoma (HCC). AC68 dose-dependently inhibited the growth and proliferation of two HCC cell lines. The AC68-induced apoptosis was observed by increased levels of cleaved caspase-3 and decreased survivin, XIAP, and MCL-1 expression via mitochondria membrane potential change, as well as elevated numbers of TUNEL-positive apoptotic cells. AC68 was also found to suppress invasion and migration of HCC cells. Moreover, it inhibited PI3K/AKT signaling pathway in vitro and in vivo. In vivo study showed that AC68 significantly inhibited tumor growth in HCC mouse xenograft model, and induced apoptosis by increasing the expression of cleaved caspase-3. The expression of PCNA was decreased by the treatment of AC68. Taken together, our data demonstrated that AC68 not only induced apoptosis but also inhibited cell growth, migration, and invasion of liver cancer cells by blocking the PI3K/AKT pathway. We suggest that AC68 may be a potent chemotherapeutic candidate for the treatment of HCC.

STAT3 aggravates TGF-β1-induced hepatic epithelial-to-mesenchymal transition and migration

Signal transducer and activator of transcription 3 (STAT3) has been shown to affect epithelial-to-mesenchymal transition (EMT) in cancers. We investigated the underlying molecular mechanisms of STAT3 crosstalk with Snail-Smad3/transforming growth factor (TGF)-β1 signaling pathways during the EMT in hepatocellular carcinoma (HCC). STAT3 and TGF-β1 expressions are examined in liver tissues of HCC patients and rats. The effect of IL-6/ STAT3 crosstalk with Snail-Smad3/TGF-β1 on EMT, carcinogenesis, migration and invasion are tested in vitro and in vivo. Phosphorylation of STAT3 and TGF-β1 proteins are universally high and positively co-expressed in HCC tissues from human and rats. Hepatic lower p-STAT3 proteins are related to earlier tumor stages in HCC patients. AG490 (a JAK2-specific inhibitor) treatment could reduce tumor numbers and sizes depending on suppression of STAT3 signaling in HCC rats. TGF-β1 could induce EMT along with an E-cadherin decrease, while vimentin, Snail, p-Smad2/3, and p-STAT3/STAT3 increase in HepG2. SIS3 (a specific inhibitor of Smad3) could markedly inhibit Snail, Vim and p-STAT3 along with blocking phosphorylation of Smad3, but E-cadherin could be activated in HepG2. IL-6 activates STAT3 signaling and then has cascading consequences for activating Snail-Smad3/TGF-β1 and vimentin as well as migration and invasion in liver cancer cells. In contrast, AG490 has an effect that inhibits phosphorylation of STAT3, lowers Snail-p-Smad3 protein levels, decreases TGF-β1-related PAI-1 promoter activation and then reduces migration or invasion of liver cancer cells. STAT3 functions as a positive regulator to activate TGF-β1-induced EMT and metastasis of HCC. STAT3 and the Snail-Smad3/TGF-β1 signaling pathways synergistically augment EMT and migration in HCC.

S-adenosyl-methionine (SAM) alters the transcriptome and methylome and specifically blocks growth and invasiveness of liver cancer cells.

S-adenosyl methionine (SAM) is a ubiquitous methyl donor that was reported to have chemo- protective activity against liver cancer, however the molecular footprint of SAM is unknown. We show here that SAM selectively inhibits growth, transformation and invasiveness of hepatocellular carcinoma cell lines but not normal primary liver cells. Analysis of the transcriptome of SAM treated and untreated liver cancer cell lines HepG2 and SKhep1 and primary liver cells reveals pathways involved in cancer and metastasis that are upregulated in cancer cells and are downregulated by SAM. Analysis of the methylome using bisulfite mapping of captured promoters and enhancers reveals that SAM hyper-methylates and downregulates genes in pathways of growth and metastasis that are upregulated in liver cancer cells. Depletion of two SAM downregulated genes STMN1 and TAF15 reduces cellular transformation and invasiveness, providing evidence that SAM targets are genes important for cancer growth and invasiveness. Taken together these data provide a molecular rationale for SAM as an anticancer agent.

Transforming growth factor β1 promotes migration and invasion in HepG2 cells: Epithelial‑to‑mesenchymal transition via JAK/STAT3 signaling.

Transforming growth factor β1 (TGFβ1) is a cytokine with multiple functions. TGFβ1 significantly induces migration and invasion of liver cancer cells. However, the molecular mechanisms underlying this effect remain unclear. Epithelial-to-mesenchymal transition (EMT) is crucial for the development of invasion and metastasis in human cancers. The aim of the present study was to determine whether TGFβ1-induced EMT promoted migration and invasion in HepG2 cells. The underlying mechanism and the effect of EMT on HepG2 cells were also investigated. The results demonstrated that TGFβ1 may induce EMT to promote migration and invasion of HepG2 cells, and this effect depends on activation of the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) signaling pathway. JAK/STAT3 signaling is involved in human malignancies, including lung cancer, and is implicated in cell transformation, tumorigenicity, EMT and metastasis. In the present study, TGFβ1 also activated JAK/STAT3 signaling in HepG2 cells and promoted Twist expression, but these events were abolished by treatment with the STAT3 inhibitor AG490. Additionally, Twist siRNA blocked TGFβ1-induced EMT. Thus, TGFβ1 was shown to induce EMT, thereby promoting the migration and invasion of HepG2 cells via JAK/STAT3/Twist signaling.

MiRNA-346 promotes proliferation, migration and invasion in liver cancer.

Liver cancer primarily accounts for the majority of malignancies of the liver. MicroRNAs (miRNAs) are endogenous non-coding RNAs, which are important in tumorigenesis. Abnormal expression of microRNA-346 (miR-346) has been demonstrated in various types of human cancer, however, its expression and potential molecular mechanism in liver cancer remains to be elucidated. Expression levels of miR-346 in liver cancer cell lines were determined by quantitative polymerase chain reaction. The effect of miR-346 on proliferation was evaluated by an MTT assay; cell migration and invasion were evaluated by Transwell migration and invasion assays and target protein expression was determined by western blotting. The present study observed that miR-346 was upregulated in liver cancer cell lines. miR-346 overexpression promoted cell proliferation, migration and invasion in liver cancer cells and conversely, inhibition of miR-346 resulted in the opposite effects. Furthermore, F-Box and leucine rich repeat protein (FBXL)2 was identified as a direct target of miR-346. miR-346 promoted proliferation, migration and invasion of liver cancer via FBXL2. Overall, these findings demonstrated that miR-346 may act as a potential prognostic marker and therapeutic target against liver cancer in the future.

Abstract 4472: A subunit of splicing factor 3b, SF3B4, functions as driver of liver cancer via aberrant splicing activity on tumor suppressor KLF4

Abstract Alternative splicing is one of the crucial mechanism that contribute to proteome diversity and, as reported, up to 90% of genes are alternatively spliced in human. In the last few years, a large number of mRNA isoforms revealed to contribute many cellular development and disease, including cancer. SF3B4 is one of six subunits of the splicing factor 3b (SF3b) complex which is an important protein complex in U2 snRNP super complex. In this study, we show that SF3B4 expression was overexpressed in a large cohort of human hepatocellular carcinoma (HCC) patients. SF3B4 knockdown caused G1/S cell cycle arrest by recovering p27 expression and simultaneously suppressing cyclins, and CDKs in liver cancer cell. Consistently, Spliceostatin A, a SF3b complex inhibitor, also suppressed liver cancer cell growth with similar effect on cell cycle regulation. In addition, sustained suppression of SF3B4 reduced the in vivo tumor growth rate in mouse xenograft models. Furthermore, SF3B4 knockdown repressed in vitro tumor cell motility and invasion of liver cancer cells. Notably, we observed that SF3B4 knockdown increased alternative transcript splicing event of a large number of genes in liver cancer cells. From this, we found that SF3B4 knockdown caused induction of wildtype KLF4 transcript expression, and thereby exerted anti-tumor growth effect on liver cancer cells. Indeed, ectopic expression of KLF4 mimicked SF3B4 knockdown effect on same cells. Our results suggest that aberrant regulation of SF3B4 contribute to malignant transformation and growth of liver cancer cells by repression of tumor suppressor via unmodulated alternative splicing activity during liver tumorigenesis. Citation Format: Qingyu Shen, Jung Woo Eun, Hyung Seok Kim, Hee Doo Yang, Sang Yean Kim, Suk Woo Nam. A subunit of splicing factor 3b, SF3B4, functions as driver of liver cancer via aberrant splicing activity on tumor suppressor KLF4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4472. doi:10.1158/1538-7445.AM2017-4472