Primary Human Hepatocellular Carcinoma Samples Research Articles

Abstract 2106: Spatially resolved immune cell atlas of human liver cancer identifies the cellular interaction network underlying mucosal-associated invariant T (MAIT) cell dysfunction in hepatocellular carcinoma

Abstract Introduction: Hepatocellular Carcinoma (HCC) is considered a prototype of inflammation-derived cancer arising from chronic liver injury. The cellular composition of the HCC tumor immune microenvironment (TiME) has a major impact on cancer biology as the TiME can influence tumor initiation, progress, and response to therapy. Mucosal-associated invariant T (MAIT) cells can represent the most abundant T cell subtype in the human liver and have been found to be impaired in both number and function in liver cancer. These innate-like T cells are assigned crucial roles in regulating immunity and inflammation in the context of infection, albeit their role in HCC remains elusive. Methods: High-dimensional flow cytometry was used to analyze MAIT cell phenotypic changes in murine and human liver cancer. Highly multiplexed immunofluorescence microscopy was used to quantify immune cell infiltration in primary human HCC samples. We developed and validated a comprehensive 37-plex antibody panel for immunofluorescence imaging of human fresh frozen HCC samples. We applied co-detection by indexing (CODEX) technology to simultaneously profile in situ expression of 37 proteins at sub-cellular resolution in 15 HCC patient samples using whole slide scanning. Initial image analysis was performed using HALO quantitative image analysis software. Finally, we established an image analysis pipeline to quantify the MAIT cell interaction network at the HCC invasive front. Results: Profiling of human and murine HCC using flow cytometry and highly multiplexed CODEX imaging revealed substantial dysregulation/aberrant activation of MAITs in liver cancer. In situ phenotyping of 4,500,000 single cells (including 1,500,000 CD45+ immune cells) allowed for the quantification of 20 distinct immune cell phenotype clusters, differential analysis of activation markers and spatial features of each individual cell. CODEX imaging revealed detailed composition of the MAIT cell niche in human liver cancer tissue allowing for further distinct spatial analysis including infiltration and nearest-neighbor analysis. Importantly, flow cytometry data of paired samples correlated well with image-based immune phenotyping. Beyond that, whole slide imaging revealed spatial relationships and interactions within the MAIT cell hub localized in distinct tissue regions. Conclusion: Here, we demonstrate that spatially resolved, single-cell analysis of human liver cancer tissue allows for in-depth characterization of interacting immune cellular programs underlying MAIT cell dysfunction in HCC. Citation Format: Benjamin Ruf, Noemi Kedei, Matthias Bruhns, Sepideh Babaei, Bernd Heinrich, Varun Subramanyam, Chi Ma, Simon Wabitsch, Benjamin Green, Kylynda C. Bauer, Yuta Myojin, Jonathan Qi, Amran Nur, Justin McCallen, Layla Greten, William G. Telford, Merrill K. Stovroff, Kesha Oza, Jiman Kang, Alexander Kroemer, Manfred Claassen, Firouzeh Korangy, Tim F. Greten. Spatially resolved immune cell atlas of human liver cancer identifies the cellular interaction network underlying mucosal-associated invariant T (MAIT) cell dysfunction in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2106.

Silencing HOXD10 by promoter region hypermethylation activates ERK signaling in hepatocellular carcinoma

BackgroundHepatocellular carcinoma is the fifth most common malignancy and the third leading cause of cancer-related death worldwide. Dysregulation of HomeoboxD10 (HOXD10) was found to suppress or promote cancer progression in different cancer types. The function and regulation of HOXD10 remain unclear in human hepatocellular carcinoma (HCC).MethodsPrimary HCC samples (117), normal liver tissue samples (15), and 13 HCC cell lines (SNU182, SNU449, HBXF344, SMMC7721, Huh7, HepG2, LM3, PLC/PRF/5, BEL7402, SNU387, SNU475, QGY7703, and Huh1) were included in this study. Methylation-specific PCR, flow cytometry, western blot, transwell, siRNA, and chromatin immunoprecipitation assays were employed.ResultsHOXD10 was methylated in 76.9% (90/117) of human primary HCC samples. HOXD10 methylation was significantly associated with vessel cancerous embolus, tumor cell differentiation, and the 3-year overall survival rate (all P < 0.05). The expression of HOXD10 was regulated by promoter region methylation. HOXD10 suppressed colony formation, cell proliferation, cell invasion and migration, and induced G2/M phase arrest and apoptosis in HCC cells. HOXD10 suppressed HCC cell xenograft growth in mice. HOXD10 suppresses HCC growth by inhibiting ERK signaling.ConclusionHOXD10 is frequently methylated in human HCC, and the expression of HOXD10 is regulated by promoter region methylation. HOXD10 suppresses HCC cell growth both in vitro and in vivo. HOXD10 suppresses human HCC by inhibiting ERK signaling.

FOXP3 Is a HCC suppressor gene and Acts through regulating the TGF-\u03b2/Smad2/3 signaling pathway

BackgroundFOXP3 has been discovered to be expressed in tumor cells and participate in the regulation of tumor behavior. Herein, we investigated the clinical relevance and biological significance of FOXP3 expression in human hepatocellular carcinoma (HCC).MethodsExpression profile of FOXP3 was analyzed using real-time RT-PCR, western blotting and immunofluorescence on HCC cell lines, and immunostaing of a tissue microarray containing of 240 primary HCC samples. The potential regulatory roles of FOXP3 were dissected by an integrated approach, combining biochemical assays, analysis of patient survival, genetic manipulation of HCC cell lines, mouse xenograft tumor models and chromatin immunoprecipitation (ChIP) sequencing.ResultsFOXP3 was constitutively expressed in HCC cells with the existence of splice variants (especially exon 3 and 4 deleted, Δ3,4-FOXP3). High expression of FOXP3 significantly correlated with low serum α-fetoprotein (AFP) level, absence of vascular invasion and early TNM stage. Survival analyses revealed that increased FOXP3 expression was significantly associated with better survival and reduced recurrence, and served as an independent prognosticator for HCC patients. Furthermore, FOXP3 could potently suppress the proliferation and invasion of HCC cells in vitro and reduce tumor growth in vivo. However, Δ3,4-FOXP3 showed a significant reduction in the tumor-inhibiting effect. The inhibition of FOXP3 on HCC aggressiveness was acted probably by enhancing the TGF-β/Smad2/3 signaling pathway.ConclusionOur findings suggest that FOXP3 suppresses tumor progression in HCC via TGF-β/Smad2/3 signaling pathway, highlighting the role of FOXP3 as a prognostic factor and novel target for an optimal therapy against this fatal malignancy.

Serine protease inhibitor Kazal type 1 (SPINK1) downregulates E‐cadherin and induces EMT of hepatoma cells to promote hepatocellular carcinoma metastasis via the MEK/ERK signaling pathway

To investigate serine protease inhibitor Kazal type 1 (SPINK1) expression and its influence on the prognosis of human hepatocellular carcinoma (HCC) and to explore the underlying molecular mechanisms involved. Altogether 80 patients with HCC who underwent curative resection were followed up for a median of 58.6 months. SPINK1 expression was detected in the primary HCC samples by immunohistochemistry. Its role in tumor invasion and metastasis was evaluated in vitro by gene silencing using a small interfering RNA-mediated approach, recombinant SPINK1 and U0126 (an inhibitor of MEK/ERK). The proteins in the MEK/ERK signaling pathway were detected by Western blot. Patients with high SPINK1 expression showed poor overall survival (P = 0.0001) and recurrence-free survival (P = 0.001) compared with those with low SPINK1 expression. The suppression of SPINK1 resulted in reduced cell migration and invasion. SPINK1 overexpression was significantly associated with increased cell migration and invasion in vitro. Furthermore, SPINK1 promoted cancer cells motility and epithelial-mesenchymal transition (EMT) via the mitogen-activated protein kinase kinase (MAPK) and extracellular regulated kinase (ERK) pathway, resulting in increased vimentin expression and decreased E-cadherin expression. SPINK1 may be an oncogene that induces EMT via the MEK/ERK pathway and is a potential target for HCC therapy.

DNA methylation and expression of homeobox gene family as diagnostic and prognostic markers in human hepatocellular carcinoma

ABSTRACT Homeobox genes consist of a family of evolutionarily conserved genes that play important roles in morphogenesis, embryogenesis, and cell fate determination. Re-expression of embryogenic genes has been associated with carcinogenesis of human cancers. Aberrant expression of homeobox genes has been increasingly found to modulate diverse processes such as cell proliferation, cell death, metastasis, angiogenesis and DNA repair. We studied DNA methylation and expression of homeobox gene family in human hepatocellular carcinoma (HCC), the fifth most common cancer and the third leading cause of cancer mortality worldwide. We performed microarray for comprehensive DNA methylation and gene expression using primary HCC samples and healthy liver tissues. Confirmation using pyrosequencing and RT-PCR was then performed. Clustering both unsupervised and supervised methods using Qlucore software was then performed. Enrichment of homeobox genes both for DNA methylation and gene expression could differentiate HCC and the healthy liver tissues. Profile of homeobox gene methylation could further predict clinical outcome. Inverse correlation between DNA methylation and gene expression was shown ( HOXA9 , Spearman r=-0.49, p=0.002). Gain of DNA methylation in HOXA9 , HOXA13 , and MEOX1 correlated with shorter HCC survival (log-rank Mantel-Cox test p=0.02, with median survival 50 and 490 weeks, respectively). We demonstrated potential roles of DNA methylation and gene expression profiles of Homeobox gene family as diagnostic and prognostic marker in patients with HCC.

NUPR1, a new target in liver cancer: implication in controlling cell growth, migration, invasion and sorafenib resistance.

Sorafenib, an oral multikinase inhibitor, is the only approved agent for the treatment of advanced hepatocellular carcinoma (HCC). However, its benefits are modest, and as its mechanisms of action remain elusive, a better understanding of its anticancer effects is needed. Based on our previous study results, we investigated here the implication of the nuclear protein 1 (NUPR1) in HCC and its role in sorafenib treatment. NUPR1 is a stress-inducible protein that is overexpressed in various malignancies, but its role in HCC is not yet fully understood. We found that NUPR1 expression was significantly higher in primary human HCC samples than in the normal liver. Knockdown of NUPR1 significantly increased cell sensitivity to sorafenib and inhibited the cell growth, migration and invasion of HCC cells, both in vitro and in vivo. Moreover, NUPR1 silencing influenced the expression of RELB and IER3 genes. Unsurprisingly, RELB and IER3 knockdown also inhibited HCC cell viability, growth and migration. Using gene expression profiling of HCC cells following stable NUPR1 knockdown, we found that genes functionally involved in cell death and survival, cellular response to therapies, lipid metabolism, cell growth and proliferation, molecular transport and cellular movement were mostly suppressed. Network analysis of dynamic gene expression identified NF-κB and ERK as downregulated gene nodes, and several HCC-related oncogenes were also suppressed. We identified Runt-related transcription factor 2 (RUNX2) gene as a NUPR1-regulated gene and demonstrated that RUNX2 gene silencing inhibits HCC cell viability, growth, migration and increased cell sensitivity to sorafenib. We propose that the NUPR1/RELB/IER3/RUNX2 pathway has a pivotal role in hepatocarcinogenesis. The identification of the NUPR1/RELB/IER3/RUNX2 pathway as a potential therapeutic target may contribute to the development of new treatment strategies for HCC management.

Hepatocellular carcinoma: thyroid hormone promotes tumorigenicity through inducing cancer stem-like cell self-renewal.

Cancer stem-like cells (CSCs) play a key role in maintaining the aggressiveness of hepatocellular carcinoma (HCC), but the cell-biological regulation of CSCs is unclear. In the study, we report that thyroid hormone (TH) promotes cell self-renewal in HCC cells. TH also increases the percentage of CD90 + HCC cells and promotes drug resistance of HCC cells. By analyzing primary human HCC samples, we found that TRα transcript level is significantly elevated in primary liver cancer and portal vein metastatic tumor, compared to that of adjacent normal liver tissue. Knocking down TRα not only inhibits HCC self-renewal in vitro but also suppresses HCC tumor growth in vivo. Interestingly, treatment of TH leads to activation of NF-κB, which is required for the function of TH on inducing HCC cell self-renewal. We also found TRα and p65 cooperatively drive the expression of BMI1 by co-binding to the promoter region of BMI1 gene. In summary, our study uncovers a novel function of TH signaling in regulating the CSCs of HCC, and these findings might be useful for developing novel therapies by targeting TH function in HCC cells.

Adiponectin induces apoptosis in hepatocellular carcinoma through differential modulation of thioredoxin proteins

Adiponectin blocks hepatocellular carcinoma (HCC) progression by inducing cell apoptosis through the modulation of C-Jun N-terminal kinase and mammalian target of rapamycin. However, the precise upstream signaling pathways or molecules remain elusive. In the present study, we analyzed the role of antioxidant protein thioredoxin (Trx) in adiponectin-induced apoptosis in HCC. Adiponectin treatment decreased the viabilities of both HepG2 and Huh7 HCC cells accompanied by increased accumulation of intracellular reactive oxygen species, as evidenced by 2′,7′-dichlorodihydrofluorescein diacetate staining. Pretreatment of these cells with the deoxidant N-acetylcysteine blocked the inhibitory effect of adiponectin. Levels of Trx2 protein in both HCC cells were significantly decreased, and the level of Trx1 was significantly inhibited in Huh7 cells while unchanged in HepG2 cells. However, the redox state of Trx1 was altered from reduced to the oxidized form following adiponectin treatment in HepG2 cells. Overexpression of both Trx proteins rescued adiponectin-induced cell apoptosis, whereas mutated Trx proteins were less effective. Further analysis suggested that both ASK1 and JNK signaling are involved in this process. Trx1 and Trx2 proteins also manifested protective effects on HCC cells in response to adiponectin treatment in a xenograft tumor model. Furthermore, high levels of Trx proteins and low adiponectin expression levels were found in primary human HCC samples compared with paracancerous tissues. These results suggest that Trx proteins play important roles in mediating adiponectin-induced HCC cell apoptosis, thus providing new insights into the pathogenesis of HCC and identifying adiponectin and Trx proteins as potential combinational therapeutic targets for the treatment of HCC.

Identification of SLAMF3 (CD229) as an Inhibitor of Hepatocellular Carcinoma Cell Proliferation and Tumour Progression

Although hepatocellular carcinoma (HCC) is one of the most common malignancies and constitutes the third leading cause of cancer-related deaths, the underlying molecular mechanisms are not fully understood. In the present study, we demonstrate for the first time that hepatocytes express signalling lymphocytic activation molecule family member 3 (SLAMF3/CD229) but not other SLAMF members. We provide evidence to show that SLAMF3 is involved in the control of hepatocyte proliferation and in hepatocellular carcinogenesis. SLAMF3 expression is significantly lower in primary human HCC samples and HCC cell lines than in human healthy primary hepatocytes. In HCC cell lines, the restoration of high levels of SLAMF3 expression inhibited cell proliferation and migration and enhanced apoptosis. Furthermore, SLAMF3 expression was associated with inhibition of HCC xenograft progression in the nude mouse model. The restoration of SLAMF3 expression levels also decreased the phosphorylation of MAPK ERK1/2, JNK and mTOR. In samples from resected HCC patients, SLAMF3 expression levels were significantly lower in tumorous tissues than in peritumoral tissues. Our results identify SLAMF3 as a specific marker of normal hepatocytes and provide evidence for its potential role in the control of proliferation of HCC cells.

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Overexpression of p42.3 promotes cell growth and tumorigenicity in hepatocellular carcinoma

To investigate the association of p42.3 expression with clinicopathological characteristics and the biological function of p42.3 in human hepatocellular carcinoma (HCC). We used reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time RT-PCR and western blotting to detect p42.3 mRNA and protein expression in hepatic cell lines. We examined primary HCC samples and matched adjacent normal tissue by immunohistochemistry to investigate the correlation between p42.3 expression and clinicopathological features. HepG2 cells were transfected with a pIRES2-EGFP-p42.3 expression vector to examine the function of the p42.3 gene. Transfected cells were analyzed for their viability and malignant transformation abilities by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony formation assay, and tumorigenicity assay in nude mice. p42.3 is differentially expressed in primary HCC tumors and cell lines. Approximately 69.6% (96/138) of cells were p42.3-positive in hepatic tumor tissues, while 30.7% (35/114) were p42.3-positive in tumor-adjacent normal tissues. Clinicopathological characteristics of the HCC specimens revealed a significant correlation between p42.3 expression and tumor differentiation (P = 0.031). However, p42.3 positivity was not related to tumor tumor-node-metastasis classification, hepatitis B virus status, or hepatoma type. Regarding p42.3 overexpression in stably transfected HepG2 cells, we discovered significant enhancement of cancer cell growth and colony formation in vitro, and significantly enhanced tumorigenicity in nude mice. Western blot analysis of cell cycle proteins revealed that enhanced p42.3 levels promote upregulation of proliferating cell nuclear antigen, cyclin B1 and mitotic arrest deficient 2. p42.3 promotes tumorigenicity and tumor growth in HCC and may be a potential target for future clinical cancer therapeutics.

Concordant hypermethylation of intergenic microRNA genes in human hepatocellular carcinoma as new diagnostic and prognostic marker

Epigenetic inactivation by aberrant DNA methylation has been reported for many microRNA genes in various human malignancies. However, relatively little is known about microRNA gene methylation in hepatocellular carcinoma (HCC). Therefore, a systematic screen for identification of aberrantly hypermethylated microRNA genes in HCC was initiated. The methylation status of 39 intergenic CpG island associated microRNA genes was analyzed in HCC cell lines (n = 7), immortalized hepatocytes (n = 2) and normal liver samples (n = 5). Subsequently, 13 differentially methylated microRNA genes were analyzed in primary human HCC samples (n = 40), benign liver tumors (n = 15) and the adjacent liver tissues employing pyrosequencing. Expression of microRNA genes was measured using quantitative real-time polymerase chain reaction (RT-PCR). In addition, DNA methylation and expression of microRNA genes were measured after DNMT1 knockdown or DNMT inhibition. Aberrant hypermethylation and concomitant reduction in expression of intergenic microRNA genes is a frequent event in human HCC: hsa-mir-9-2 (23%), hsa-mir-9-3 (50 %), hsa-mir-124-1 (20%), hsa-mir-124-2 (13%), hsa-mir-124-3 (43%), hsa-mir-129-2 (58%), hsa-mir-596 (28%) and hsa-mir-1247 (38%). Altogether, it affects 90% of the HCC specimens under study. MicroRNA gene methylation is not found in hepatocellular adenoma (n = 10) and focal nodular hyperplasia (n = 5). DNMT1 knockdown or DNMT inhibition reduced microRNA gene methylation and stimulated expression. In primary human HCC specimens hypermethylation and expression of microRNA genes showed an inverse correlation. Concordant hypermethylation of three or more microRNA genes is a highly specific marker for the detection of HCC and for poor prognosis.

Comprehensive analysis of the independent effect of twist and snail in promoting metastasis of hepatocellular carcinoma

The epithelial-mesenchymal transition (EMT) is critical for induction of invasiveness and metastasis of human cancers. In this study we investigated the expression profiles of the EMT markers, the relationship between EMT markers and patient/tumor/viral factors, and the interplay between major EMT regulators in human hepatocellular carcinoma (HCC). Reduced E-cadherin and nonmembranous beta-catenin expression, the hallmarks of EMT, were shown in 60.2% and 51.5% of primary HCC samples, respectively. Overexpression of Snail, Twist, or Slug, the major regulators of EMT, was identified in 56.9%, 43.1%, and 51.4% of primary HCCs, respectively. Statistical analysis determined that Snail and Twist, but not Slug, are major EMT inducers in HCC: overexpression of Snail and/or Twist correlated with down-regulation of E-cadherin, nonmembranous expression of beta-catenin, and a worse prognosis. In contrast, there were no such significant differences in samples that overexpressed Slug. Coexpression of Snail and Twist correlated with the worst prognosis of HCC. Hepatitis C-associated HCC was significantly correlated with Twist overexpression. HCC cell lines with increased Snail and Twist expression (e.g., Mahlavu) exhibited a greater capacity for invasiveness/metastasis than cells with low endogenous Twist/Snail expression (e.g., Huh-7). Overexpression of Snail or/and Twist in Huh-7 induced EMT and invasiveness/metastasis, whereas knockdown of Twist or Snail in Mahlavu reversed EMT and inhibited invasiveness/metastasis. Twist and Snail were independently regulated, but exerted an additive inhibitory effect to suppress E-cadherin transcription. Our study provides a comprehensive profile of EMT markers in HCC, and the independent and collaborative effects of Snail and Twist on HCC metastasis were confirmed through different assays.

Epigenetic inactivation of SLIT2 in human hepatocellular carcinomas

Recent findings have shown that SLIT2 appears to function as a novel tumor suppressor gene. In addition, hypermethylation of its promoter region has been detected in various cancers, including breast and lung cancer, colorectal carcinoma, and gliomas. Here, we report for the first time that there is epigenetic silencing of SLIT2 in human hepatocellular carcinoma (HCC). Downregulation of SLIT2 was detected in 6 of 8 (75%) HCC cell lines by quantitative real-time RT-PCR (qRT-PCR), and the downregulation of SLIT2 was generally dependent on the degree of methylation at the promoter region. Furthermore, expression of SLIT2 was restored in relatively low-expressing cell lines after treatment with 5-aza-2-deoxycytidine (5-Aza-dC). Downregulation of SLIT2 expression was also detected in 45 of 54 primary HCC samples (83.3%), and the decrease in expression was significantly correlated with CpG island hypermethylation. This decrease of SLIT2 expression was also associated with lymph node metastasis in HCC. Moreover, overexpression of SLIT2 in SMMC-7721 cells induced by recombinant adenovirus suppressed cell growth, migration, and invasion, These results suggest that epigenetic inactivation of SLIT2 in HCC may be important in the development and progression of HCC. Thus, SLIT2 may be useful as a therapeutic target in the treatment of HCC.

Methylation silencing of SOCS-3 promotes cell growth and migration by enhancing JAK/STAT and FAK signalings in human hepatocellular carcinoma

We identified that suppressor of cytokine signaling-3 (SOCS-3) gene was aberrantly methylated in its CpG island in three of 10 human hepatocellular carcinoma (HCC) cell lines. SOCS-3 RNA was undetectable in five of the 10 HCC cell lines including the three methylated cell lines, and a demethylating agent, 5-aza-2'-deoxycytidine, reactivated SOCS-3 expression in three cell lines tested. The DNA region where we found aberrant DNA methylation includes a signal transducers and activators of transcription (STAT) binding consensus sequence. When the DNA region was used as a promoter, DNA methylation markedly reduced promoter activity. SOCS-3 was also aberrantly methylated in six of 18 primary HCC samples. SOCS-3 expression was reduced in three of the three methylated and one of the three unmethylated primary samples examined. Restoration of SOCS-3 in cells lacking SOCS-3 expression suppressed STAT3 phosphorylation and cell growth. We found that IL-6 acted as a growth factor in HCC cells. Inhibition of SOCS-3 expression in cells whose growth was induced by IL-6 enhanced STAT3 phosphorylation and cell growth. In addition, AG490, a chemical JAK2 inhibitor, suppressed cell growth and downregulated STAT3 phosphorylation, but not FAK phosphorylation. We also found that SOCS-3 physically interacted with phosphorylated FAK and Elongin B in HCC cells. Restoration of SOCS-3 decreased FAK phosphorylation as well as FAK protein level. Inhibition of SOCS-3 expression increased FAK phosphorylation, resulting in enhancement of cell migration. These data indicate that SOCS-3 negatively regulates cell growth and cell motility by inhibiting Janus kinase (JAK)/STAT and FAK signalings in HCC cells. Thus, loss of SOCS-3 by the associated DNA methylation confers cells advantage in growth and migration.