Primary Hepatocellular Carcinoma Cells Research Articles

Application of IL-36 receptor antagonist weakens CCL20 expression and impairs recovery in the late phase of murine acetaminophen-induced liver injury.

Overdosing of the analgesic acetaminophen (APAP, paracetamol) is a major cause of acute liver injury. Whereas toxicity is initiated by hepatocyte necrosis, course of disease is regulated by mechanisms of innate immunity having the potential to serve in complex manner pathogenic or pro-regenerative functions. Interleukin (IL)-36γ has been identified as novel IL-1-like cytokine produced by and targeting epithelial (-like) tissues. Herein, we investigated IL-36γ in acute liver disease focusing on murine APAP-induced hepatotoxicity. Enhanced expression of hepatic IL-36γ and its prime downstream chemokine target CCL20 was detected upon liver injury. CCL20 expression coincided with the later regeneration phase of intoxication. Primary murine hepatocytes and human Huh7 hepatocellular carcinoma cells indeed displayed enhanced IL-36γ expression when exposed to inflammatory cytokines. Administration of IL-36 receptor antagonist (IL-36Ra) decreased hepatic CCL20 in APAP-treated mice. Unexpectedly, IL-36Ra likewise increased late phase hepatic injury as detected by augmented serum alanine aminotransferase activity and histological necrosis which suggests disturbed tissue recovery upon IL-36 blockage. Finally, we demonstrate induction of IL-36γ in inflamed livers of endotoxemic mice. Observations presented introduce IL-36γ as novel parameter in acute liver injury which may contribute to the decision between unleashed tissue damage and initiation of liver regeneration during late APAP toxicity.

Establishment of a novel system for the culture and expansion of hepatic stem-like cancer cells

Hepatocellular carcinoma (HCC) is a major primary liver malignancy in adults. Despite the progress made, the outcome of the treatment to this disease is less than satisfactory as the post therapy tumor recurrence is almost inevitable. Accumulating pieces of evidence have suggested that the recurrence is due to the existence of a subpopulation of the HCC cells that possess the properties of stem cells and are resistant to radiation and chemotherapy. It is therefore important to understand the characteristics of this subpopulation of HCC cells, and which requires the establishment of an in vitro system to study these stem-like cancer cells. However, despite extensive efforts, the progress in establishing such an in vitro system has been slow largely due to the lack of definitive biomarkers in the isolation and expansion of these cells. In order to successfully maintain and expand HCC CSCs, we first optimized the culture system. We establish a novel medium system that allows the culture and enrichment of these hepatic stem-like cancer cells from both hepatoma cells and human primary HCC cells. These cells exhibited typical stem cell properties, such as enhanced stem cell markers, gain of EMT properties and drug resistance, and more importantly, stronger tumor-initiating capabilities. The medium may help to establish an in vitro model for hepatic cancer stem cell (HCSC) studies, which may contribute to the development of novel cell therapies and new drugs for the treatment of HCC.

Inactivation of Cyclin E1 acts pro-apoptotic and anti-proliferative in primary hepatoma cells and protects from hepatocarcinogenesis in mice

Background & Aims: E-type cyclins (CcnE1, CcnE2) are regulatory subunits of Cyclin-dependent kinase 2 (Cdk2). There is evidence that CcnE/Cdk2 complexes control the transition of quiescent cells into the cell cycle. Surprisingly, genetic deletion of CcnE1, CcnE2 or Cdk2 does not inhibit cell proliferation during embryonic development or liver regeneration. The aim of the present study was to evaluate if E-type cyclins or Cdk2 may have a specific role for hepatocarcinogenesis.

Establishment and characterization of a novel primary hepatocellular carcinoma cell line with metastatic ability in vivo

BackgroundHepatocellular carcinoma (HCC) is a highly aggressive and heterogeneous disease. HCC cell lines established from different patients would be useful in elucidating the molecular pathogenesis. However, success of HCC primary culture establishment remains at low rate. We aim to establish and characterize HCC primary culture and the derived cell line.MethodsFresh tumor tissues were collected from 30 HCC patients. Culture conditions were optimized for the attachment and growth of the isolated hepatocytes. Granulin-epithelin precursor (GEP), a growth factor reported to associate with cancer stem cell properties, was examined by flow cytometry to elucidate its role on primary culture establishment. The primary cell line was characterized in detail.ResultsCells isolated from 16 out of 30 HCC cases (53%) had viability more than 70% and were subject to subsequent in vitro culture. 7 out of 16 cases (44%) could give rise to cells that were able to attach and grow in culture. GEP expression levels significantly correlated with the viability of isolated hepatocytes and success rate of subsequent primary culture establishment. Cells from HCC patient 21 grew and expanded rapidly in vitro and was selected to be further characterized. The line, designated HCC21, derived from a Hong Kong Chinese female patient with HCC at Stage II. The cells exhibited typical epithelial morphology and expressed albumin, AFP and HBV antigens. The cell line was authenticated by short tandem repeat analysis. Comparative genome hybridization analysis revealed chromosomal loss at 1p35-p36, 1q44, 2q11.2-q24.3, 2q37, 4q12-q13.3, 4q21.21-q35.2, 8p12-p23, 15q11.2-q14, 15q24-q26, 16p12.1-p13.3, 16q, 17p, 22q and gain at 1q21-q43 in both HCC21 cells and the original clinical tumor specimen. Sequence analysis revealed p53 gene mutation. Subcutaneous injection of HCC21 cells into immunodeficient mice showed that the cells were able to form tumors at the primary injection sites and metastatic tumors in the peritoneal cavity.ConclusionsThe newly established cell line could serve as useful in vitro and in vivo models for studying primary HCC that possess metastasis ability.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-014-0103-y) contains supplementary material, which is available to authorized users.

Tumor-Associated Macrophages Produce Interleukin 6 and Signal via STAT3 to Promote Expansion of Human Hepatocellular Carcinoma Stem Cells

Cancer stem cells (CSCs) can contribute to hepatocellular carcinoma (HCC) progression and recurrence after therapy. The presence of tumor-associated macrophages (TAMs) in patients with HCC is associated with poor outcomes. It is not clear whether TAMs interact with CSCs during HCC development. We investigated whether TAMs affect the activities of CSCs in the microenvironment of human HCCs. HCCs were collected from 17 patients during surgical resection and single-cell suspensions were analyzed by flow cytometry. CD14(+) TAMs were isolated from the HCC cell suspensions and placed into co-culture with HepG2 or Hep3B cells, and CSC functions were measured. The interleukin 6 (IL6) receptor was blocked with a monoclonal antibody (tocilizumab), and signal transducer and activator of transcription 3 was knocked down with small hairpin RNAs in HepG2 cells. Xenograft tumors were grown in NOD-SCID/Il2Rg(null) mice from human primary HCC cells or HepG2 cells. CD44(+) cells from human HCCs and cell lines formed more spheres in culture and more xenograft tumors in mice than CD44(-) cells, indicating that CD44(+) cells are CSCs. Incubation of the CD44(+) cells with TAMs promoted expansion of CD44(+) cells, and increased their sphere formation in culture and formation of xenograft tumors in mice. In human HCC samples, the numbers of TAMs correlated with the numbers of CD44(+) cells. Of all cytokines expressed by TAMs, IL6 was increased at the highest level in human HCC co-cultures, compared with TAMs not undergoing co-culture. IL6 was detected in the microenvironment of HCC samples and induced expansion of CD44(+) cells in culture. Levels of IL6 correlated with stages of human HCCs and detection of CSC markers. Incubation of HCC cell lines with tocilizumab or knockdown of signal transducer and activator of transcription 3 in HCC cells reduced the ability of TAMs to promote sphere formation by CD44+ cells in culture and growth of xenograft tumors in mice. CD44(+) cells isolated from human HCC tissues and cell lines have CSC activities in vitro and form a larger number of xenograft tumors in mice than CD44(-) cells. TAMs produce IL6, which promotes expansion of these CSCs and tumorigenesis. Levels of IL6 in human HCC samples correlate with tumor stage and markers of CSCs. Blockade of IL6 signaling with tocilizumab, a drug approved by the Food and Drug Administration for treatment of rheumatoid arthritis, inhibits TAM-stimulated activity of CD44(+) cells. This drug might be used to treat patients with HCC.

Heat shock protein 70 serum levels differ significantly in patients with chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma.

Members of the heat shock protein 70 (HSP70) family play an important role in assisting protein folding, preventing protein aggregation and transport of proteins across membranes under physiological conditions. Following environmental (i.e., irradiation, chemotherapy), physiological (i.e., cell growth, differentiation), and pathophysiological (i.e., inflammation, tumorigenesis) stress, the synthesis of heat shock proteins (HSPs) is highly up-regulated, whereas protein synthesis in general is reduced. In contrast to normal cells, many tumor entities including hepatocellular carcinoma (HCC) overexpress HSP70, the major-stress-inducible member of the HSP70 family, present it on their cell surface and secrete it into the extracellular milieu. Herein, the prognostic relevance of serum HSP70 levels in patients with chronic hepatitis (CH; n = 50), liver cirrhosis (LC; n = 46), and HCC (n = 47) was analyzed. Similar to other tumor entities, HSP70 is also present on the surface of primary HCC cells. The staining intensity of intracellular HSP70 in HCC tissue is stronger compared to control and cirrhotic liver sections. HSP70 serum levels in all HCC patients were significantly higher compared to a control group without liver disease (n = 40). No significant age- and gender-related differences in HSP70 serum levels were observed in male and female healthy human volunteers (n = 86). Patients with CH (n = 50) revealed significantly higher HSP70 serum levels compared to the control group, however, these values were significantly lower than those of HCC patients (n = 47). Furthermore, a subgroup of patients with LC who subsequently developed HCC (LC-HCC, n = 13) revealed higher HSP70 serum levels than patients with LC (n = 46, p = 0.05). These data indicate that serum HSP70 levels are consecutively increased in patients with CH, LC and liver carcinomas and thus might have a prognostic value.

In vitro genotoxic and mutagenic evaluation of the aqueous extract of Codiaeum variegatum and its amoebicidal sub-fraction

Ethnopharmacological relevanceCodiaeum variegatum, grown in many varieties, has been widely used as a houseplant based on its brightly decorative foliage. In addition, a variety of this plant has been used for a long time against bloody diarrhea by the local population in Cameroon. Aim of the studyIn our previous study, the aqueous extract of this plant and an isolated sub-fraction exhibited significant anti-amoebic activity on axenic culture of Entamoeba histolytica. Due to the medicinal value of these extracts, we promptly initiated to investigate their genotoxic and mutagenic potential in order to assure their safe and rationale usage in traditional healthcare system. Material and methodsBoth extracts were incubated with L5178Y mouse lymphoma cells, primary hepatic cells and HepG2 human hepatocellular carcinoma cells and their genotoxicity and mutagenicity were evaluated by quantifying DNA damage and chromosomal aberrations through comet assay, micronucleus assay and mouse lymphoma mutation assay. ResultsThe aqueous extract of Codiaeum variegatum is not cytotoxic up to 2000µg/mL while the amoebicidal fraction is significantly cytotoxic (≤ 40–55%) on L5178Y mouse lymphoma and HepG2 cells at concentrations higher than 500µg/mL. Besides, no significant DNA damage and induction of micronucleus formation were identified at concentrations up to 2000µg/mL. Moreover, the mutagenic potential of these extracts after short (4h) and long term (24h) treatment, revealed no significant gene mutation induction. ConclusionThe aqueous extract of Codiaeum variegatum and the amoebicidal fraction SF9B are neither genotoxic on non-competent or metabolic competent cell lines, nor mutagenic in mouse lymphoma mutation assay and therefore they could be safely used at lower doses for medicinal purpose.

Androgen receptor enhances cell adhesion and decreases cell migration via modulating β1-integrin-AKT signaling in hepatocellular carcinoma cells

The androgen receptor (AR) has been shown to promote the initiation and development of hepatocellular carcinoma (HCC) during the early stage of the disease process and to suppress HCC cell invasion during the later stages of the disease. The mechanisms governing these dual yet opposite roles have yet to be elucidated. Using carcinogen-induced HCC in vivo mouse models and the in vitro human HCC cell line SKhep1, we found that knockout of AR in primary HCC cells led to a decrease in HCC cell focal adhesion capacity compared to cells from wildtype mice. Similar results were obtained after adding functional AR into human HCC SKhep1 cells. Further analysis revealed that the role AR plays in adhesion of HCC cells is governed, at least in part, by its ability to up-regulate β1-integrin and activate the PI3K/AKT pathway. We also found that AR-β1-integrin-mediated cell adhesion suppresses cell migration. Those findings indicate that the AR-β1-integrin-PI3K/AKT signaling pathway might play a role in the bimodal function of AR on cell adhesion and migration at the cellular level.

Dihydromyricetin promotes hepatocellular carcinoma regression via a p53 activation-dependent mechanism

The development of antitumor chemotherapy drugs remains a key goal for oncologists, and natural products provide a vast resource for anti-cancer drug discovery. In the current study, we found that the flavonoid dihydromyricetin (DHM) exhibited antitumor activity against liver cancer cells, including primary cells obtained from hepatocellular carcinoma (HCC) patients. In contrast, DHM was not cytotoxic to immortalized normal liver cells. Furthermore, DHM treatment resulted in the growth inhibition and remission of xenotransplanted tumors in nude mice. Our results further demonstrated that this antitumor activity was caused by the activation of the p53-dependent apoptosis pathway via p53 phosphorylation at serine (15Ser). Moreover, our results showed that DHM plays a dual role in the induction of cell death when administered in combination with cisplatin, a common clinical drug that kills primary hepatoma cells but not normal liver cells.

Overexpression of the Transcription Factor MEF2D in Hepatocellular Carcinoma Sustains Malignant Character by Suppressing G2–M Transition Genes

The underlying molecular pathogenesis in hepatocellular carcinoma remains poorly understood. The transcription factor MEF2D promotes survival in various cell types and it seems to function as an oncogene in leukemia. However, its potential contributions to solid cancers have not been explored. In this study, we investigated MEF2D expression and function in hepatocellular carcinoma, finding that MEF2D elevation in hepatocellular carcinoma clinical specimens was associated with poor prognosis. MEF2D-positive primary hepatocellular carcinoma cells displayed a faster proliferation rate compared with MEF2D-negative cells, and silencing or promoting MEF2D expression in these settings limited or accelerated cell proliferation, respectively. Notably, MEF2D-silencing abolished hepatocellular carcinoma tumorigenicity in mouse xenograft models. Mechanistic investigations revealed that MEF2D-silencing triggered G2-M arrest in a manner associated with direct downregulation of the cell-cycle regulatory genes RPRM, GADD45A, GADD45B, and CDKN1A. Furthermore, we identified MEF2D as an authentic target of miR-122, the reduced expression of which in hepatocellular carcinoma may be responsible for MEF2D upregulation. Together, our results identify MEF2D as a candidate oncogene in hepatocellular carcinoma and a potential target for hepatocellular carcinoma therapy.

BVES Inhibition Triggers Epithelial-Mesenchymal Transition in Human Hepatocellular Carcinoma

Metastasis contributes to the poor prognosis of hepatocellular carcinoma (HCC). However, the mechanism through which a primary HCC cell develops into a metastatic phenotype is not well understood. In this study, we set out to elucidate how blood vessel epicardial substance (BVES), a novel adhesion molecule regulating tight junction formation, mediates invasion and metastasis in human HCC cells. qRT-PCR, western blot and IHC were used to detect the expression of BVES in HCC samples and HCC cell lines. Small interfering RNAs (siRNAs) against human BVES were synthesized and used to transfect Huh7 cells. Then, the interference efficiency and the expression of mesenchymal marker vimentin and epithelial marker E-cadherin were measured by qRT-PCR and western blot. F-actin cytoskeleton was detected using TRITC-conjugated phalloidin. After inhibition of BVES, wound healing experiment and transwellassay were used to analyze the migratory and invasive ability of Huh7 cells. BVES was down-regulated in human HCC tissues and HCC cell lines with high metastatic potential. After BVES inhibition, Huh7 cells exhibited some morphological changes including cytoskeleton rearrangement and junctional disruption. Cell migration and invasion were increased concomitant with increased expression of vimentin, IL-6, MMP2, MMP9 and decreased expression of E-cadherin. Finally, we found the expression of epithelial-mesenchymal transition (EMT) transcription factors Snail1 and Twist1 was significantly increased in BVES knockdown cells. Our results suggest that down-regulation of BVES in HCC induces EMT, thus promoting invasion and metastasis in HCC cells.

Disulfiram Eradicates Tumor-Initiating Hepatocellular Carcinoma Cells in ROS-p38 MAPK Pathway-Dependent and -Independent Manners

Tumor-initiating cells (TICs) play a central role in tumor development, metastasis, and recurrence. In the present study, we investigated the effect of disulfiram (DSF), an inhibitor of aldehyde dehydrogenase, toward tumor-initiating hepatocellular carcinoma (HCC) cells. DSF treatment suppressed the anchorage-independent sphere formation of both HCC cells. Flow cytometric analyses showed that DSF but not 5-fluorouracil (5-FU) drastically reduces the number of tumor-initiating HCC cells. The sphere formation assays of epithelial cell adhesion molecule (EpCAM)+ HCC cells co-treated with p38-specific inhibitor revealed that DSF suppresses self-renewal capability mainly through the activation of reactive oxygen species (ROS)-p38 MAPK pathway. Microarray experiments also revealed the enrichment of the gene set involved in p38 MAPK signaling in EpCAM+ cells treated with DSF but not 5-FU. In addition, DSF appeared to downregulate Glypican 3 (GPC3) in a manner independent of ROS-p38 MAPK pathway. GPC3 was co-expressed with EpCAM in HCC cell lines and primary HCC cells and GPC3-knockdown reduced the number of EpCAM+ cells by compromising their self-renewal capability and inducing the apoptosis. These results indicate that DSF impaired the tumorigenicity of tumor-initiating HCC cells through activation of ROS-p38 pathway and in part through the downregulation of GPC3. DSF might be a promising therapeutic agent for the eradication of tumor-initiating HCC cells.

Studies on the Identification of Constituents in Ethanol Extract of Radix Glycyrrhizae and Their Anti-Primary Hepatoma Cell Susceptibility

The objective of this paper is to study the chemical constituents of Radix Glycyrrhizae and to apply the resulting natural products in the study of drug susceptibility of hepatoma cells so as to provide a scientific basis for quality standards and clinical application of medicinal Radix Glycyrrhizae. Chromatographic materials were used for isolation and purification; structural identification was performed based on physicochemical properties and spectral data. MTT colorimetry was used to detect the proliferation inhibition rate against primary hepatoma cells by natural products, and flow cytometry was used to detect the changes in cell cycle progression. Five compounds were isolated and identified, namely, liquiritigenin (1), liquiritin (2), isoliquiritigenin (3), betulinic acid (4), and oleanolic acid (5). In the study, 5-FU (5-fluorouracil) is used as a positive control to the hepatoma cells. Primary hepatoma cells were highly susceptible to 5-FU and liquiritigenin, both of which markedly inhibited the proliferation of hepatoma cells; flow cytometry results showed an increase in G0/G1 phase cells, a decrease in S phase cells, and a relative increase in G2/M phase cells. Primary hepatoma cells are highly susceptible to liquiritigenin, a natural product; the testing of tumor cell susceptibility is of important significance to the improvement of therapeutic effect of cancer.

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.

Evaluation of miR-122-regulated suicide gene therapy for hepatocellular carcinoma in an orthotopic mouse model.

Intratumoral administration of adenoviral vector encoding herpes simplex virus (HSV) thymidine kinase (TK) gene (Ad-TK) followed by systemic ganciclovir (GCV) is an effective approach in treating experimental hepatocellular carcinoma (HCC). However, hepatotoxicity due to unwanted vector spread and suicide gene expression limited the application of this therapy. miR-122 is an abundant, liver-specific microRNA whose expression is decreased in human primary HCC and HCC-derived cell lines. These different expression profiles provide an opportunity to induce tumor-specific gene expression by miR-122 regulation. By inserting miR-122 target sequences (miR-122T) in the 3' untranslated region (UTR) of TK gene, we constructed adenovirus (Ad) vectors expressing miR-122-regulated TK (Ad-TK-122T) and report genes. After intratumoral administration of Ad vectors into an orthotopic miR-122-deficient HCC mouse model, we observed the miR-122-regulated transgene expression and assessed the antitumor activity and safety of Ad-TK-122T. Insertion of miR-122T specifically down-regulated transgene expression in vitro and selectively protected the miR-122-positive cells from killing by TK/GCV treatment. Insertion of miR-122T led to significant reduction of tansgene expression in the liver without inhibition of its expression in tumors in vivo, resulting in an 11-fold improvement of tumor-specific transgene expression. Intratumoral injection of Ad vectors mediated TK/GCV system led to a vector dosage-dependent regression of tumor. The insertion of miR-122T does not influence the antitumor effects of suicide gene therapy. Whereas mice administrated with Ad-TK showed severe lethal hepatotoxicity at the effective therapeutic dose, no liver damage was found in Ad-TK-122T group. miR-122-regulated TK expression achieved effective anti-tumor effects and increased the safety of intratumoral delivery of adenovirus-mediated TK/GCV gene therapy for miR-122-deficient HCC.

Exchange of cytosolic content between T cells and tumor cells activates CD4 T cells and impedes cancer growth.

BackgroundT cells are known to participate in the response to tumor cells and react with cytotoxicity and cytokine release. At the same time tumors established versatile mechanisms for silencing the immune responses. The interplay is far from being completely understood. In this study we show contacts between tumor cells and lymphocytes revealing novel characteristics in the interaction of T cells and cancer cells in a way not previously described.Methods/ FindingsExperiments are based on the usage of a hydrophilic fluorescent dye that occurs free in the cytosol and thus transfer of fluorescent cytosol from one cell to the other can be observed using flow cytometry. Tumor cells from cell lines of different origin or primary hepatocellular carcinoma (HCC) cells were incubated with lymphocytes from human and mice. This exposure provoked a contact dependent uptake of tumor derived cytosol by lymphocytes – even in CD4+ T cells and murine B cells – which could not be detected after incubation of lymphocytes with healthy cells. The interaction was a direct one, not requiring the presence of accessory cells, but independent of cytotoxicity and TCR engagement.Electron microscopy disclosed 100-200nm large gaps in the cell membranes of connected cells which separated viable and revealed astonishing outcome. While the lymphocytes were induced to proliferate in a long term fashion, the tumor cells underwent a temporary break in cell division. The in vitro results were confirmed in vivo using a murine acute lymphoblastic leukemia (ALL) model. The arrest of tumor proliferation resulted in a significant prolonged survival of challenged mice. ConclusionsThe reported cell-cell contacts reveal new characteristics i.e. the enabling of cytosol flow between the cells including biological active proteins that influence the cell cycle and biological behaviour of the recipient cells. This adds a completely new aspect in tumor induced immunology.

Epithelial–mesenchymal transition markers expressed in circulating tumor cells in hepatocellular carcinoma patients with different stages of disease

The presence of circulating tumor cells (CTCs) in peripheral blood is associated with metastasis and prognosis in hepatocellular carcinoma (HCC) patients. The epithelial–mesenchymal transition (EMT) has a pivotal role in tumor invasion and dissemination. To identify more sensitive biomarkers for evaluating metastasis and prognosis, we investigated the expression of EMT markers, including vimentin, twist, ZEB1, ZEB2, snail, slug and E-cadherin in CTCs, primary HCC tumors and adjacent non-tumoral liver tissues. After isolating viable CTCs from the peripheral blood of HCC patients using asialoglycoprotein receptors (ASGPRs), the CTCs were identified with immunofluorescence staining. CTCs were detected in the peripheral blood obtained from 46 of 60 (76.7%) HCC patients. Triple-immunofluorescence staining showed that twist and vimentin expression could be detected in CTCs obtained from 39 (84.8%) and 37 (80.4%) of the 46 patients, respectively. The expression of both twist and vimentin in CTCs was significantly correlated with portal vein tumor thrombus. Coexpression of twist and vimentin in CTCs could be detected in 32 (69.6%) of the 46 patients and was highly correlated with portal vein tumor thrombus, TNM classification and tumor size. Quantitative fluorescence western blot analysis revealed that the expression levels of E-cadherin, vimentin and twist in HCC tumors were significantly associated with the positivity of isolated CTCs (P=0.013, P=0.012, P=0.009, respectively). However, there was no significant difference in ZEB1, ZEB2, snail and slug expression levels in CTCs, primary HCC tumors and adjacent non-tumoral liver tissues across samples with regard to the clinicopathological parameters. Our results demonstrate that the EMT has a role in promoting the blood-borne dissemination of primary HCC cells, and the twist and vimentin expression levels in CTCs could serve as promising biomarkers for evaluating metastasis and prognosis in HCC patients.

Cyclin E facilitates dysplastic hepatocytes to bypass G1/S checkpoint in hepatocarcinogenesis

By array-comparative genomic hybridization, we demonstrated cyclin E as one of seven genes associated with hepatocellular carcinoma (HCC) development in Ku70 DNA repair-deficient mice. We therefore explored the hypothesis that during hepatocarcinogenesis, cyclin E kinase can overcome the inhibitory effects of p53 and establish whether abnormal miRNA(mi-R)-34, a co-regulator of cyclin E and p53, can account for their interactions as "drivers" of HCC. Dysplastic hepatocytes (DNs) and HCCs were generated from diethylnitrosamine (DEN)-injected C57BL/6 male mice at 3-12 months. Cyclin E/cdk2 was barely expressed in normal liver, but was readily detected in dysplastic hepatocytes, localizing to glutathione-S transferase pi-form positive cells dissected by laser-dissection. Cyclin E kinase activity preceded cyclin D1, proliferating cell nuclear antigen expression in DNs and HCCs despite maximal p53 and p21 expression. We confirmed that cyclin E, rather than cyclin D1, is the proliferative driver in hepatocarcinogenesis by immunoprecipitation experiments demonstrating preferential binding of p21 to cyclin D1, allowing cyclin E-mediated "escape" from G1/S checkpoint. We then showed cyclin E was responsible for regulating wild-type p53 by knockdown experiments in primary HCC cells; cyclin E-knockdown increased p53 and p21, diminished anti-apoptotic Bcl-XL and reduced cell viability. Conversely, blocking p53 augmented cyclin E, Bcl-XL expression and increased proliferation. Physiological interactions between cyclin E/p53/p21 were confirmed in primary hepatocytes. miR-34a,c were upregulated in dysplastic murine, human liver and HCCs compared with normal liver, and appeared to be linked to cyclin E/p53. Upregulation of functionally active cyclin E via miR34 with loss of p53 function is associated with cell-cycle checkpoint failure increasing proliferative drive that favors hepatocarcinogenesis.

Abstract 272: Identification of MicroRNAs unique to cancer stem cells by small RNA deep sequencing.

Abstract Cancer stem cells (CSCs) are undifferentiated cancer initiating cells with normal stem cell traits and they are responsible for cancer progression, tumor relapse and resistance to chemotherapy. While therapies that target CSCs hold promise in eliminating cancer burden, normal stem cells are likely to be targeted due to their similarities with CSCs. Recently we have demonstrated that EpCAM+ AFP+ hepatocellular carcinoma (HCC) cells are CSCs. EpCAM and AFP are two cellular proteins that are highly expressed in fetal livers and are silenced in adult livers but they are reactivated in a subset of HCC. Since microRNAs play a key role in the maintenance of normal stem cells and CSCs, we hypothesized that abnormal microRNA expression could be a unique feature specific to EpCAM+ AFP+ CSCs but not to normal liver stem cells. In this study, we used the SOLiD-4 system to perform genome-wide measurement of small RNA transcriptome. We constructed 16 small RNA libraries derived from EpCAM+ AFP+ CSCs and corresponding differentiated EpCAM- AFP- tumor cells from primary HCC clinical specimens and HCC cell lines, human embryonic stem cells, primary normal liver stem cells and their lineage restricted hepatoblasts, and differentiated hepatocytes. We generated a total of 290.9 million reads. Using the RNA2MAP (v0.5) computational program, the 35 nucleotide-filtered reads were aligned to the reference human miRBase (v17.0), then to the human genome (hg19). The alignment yielded an average of 10.3 million high-quality mapped reads, of which 59.3% were aligned to miRBase. Further, we developed an in-house bioinformatics analysis pipeline to process mapped reads and identified 83.2% of known microRNA hairpins (n=1426) and 71.0% of known mature microRNAs within the human miRBase (n=1733). Using a stringent comparison criterion between undifferentiated and differentiated cells as well as between normal and cancer cells, we identified 10 microRNAs unique to HCC CSC but not to normal stem cells and 15 microRNAs unique to normal liver stem cells. We also identified several microRNAs that contain candidate somatic mutations. These microRNAs may possess unique functions to HCC CSCs and thus they are strong candidates as therapeutic targets for HCC. Further investigation would allow us to develop novel therapies to specifically eliminate CSC without sacrificing normal stem cells to avoid unwanted side effect. Citation Format: Junfang Ji, Marshonna Forgues, Taro Yamashita, Eliane Wauthier, Lola M. Reid, Xinyu Wen, Young Song, Jun Wei, Javed Khan, Snorri S. Thorgeirsson, Xin W. Wang. Identification of MicroRNAs unique to cancer stem cells by small RNA deep sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 272. doi:10.1158/1538-7445.AM2013-272

Abstract LB-313: Development of a novel anti-MYC therapeutic with efficacy against liver and kidney cancers.

Abstract One of the most attractive, yet elusive, targets for cancer therapy is the MYC oncogene. MYC is overexpressed in the majority of human cancers, and we and others have shown that suppression of this oncogene is sufficient to induce sustained tumor regression, a phenomenon known as oncogene addiction. However, no viable therapy has been developed as yet that targets this crucial regulator of the tumor phenotype. Here, we report the development of a novel antisense therapeutic that targets the MYC oncogene. This inhibitor can potently suppress MYC expression in primary tumors in vivo, as demonstrated by immunohistochemistry and real-time PCR. Importantly, we show that treatment with the anti-MYC drug significant reduces tumor onset and progression in transgenic mouse models of primary hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC) cancer. We imaged tumor growth via MRI and utilized quantitative image analysis to demonstrate the inhibition of HCC and RCC progression, confirming our results on total tumor burden histologically after four weeks of drug treatment. Additionally, this antisense therapy against MYC can also attenuate the growth of human HCC cells in vivo, irrespective of the inducing oncogene. In contrast with other reported MYC inhibitors, our approach results in no discernible toxicity due to MYC suppression on any organs examined in the treated animals. Thus, we have established a novel therapy that directly inhibits the MYC oncogene, representing a potentially exciting advance in the field of targeted cancer therapeutics. Citation Format: David I. Bellovin, Aleksey Yevtodiyenko, Stacey J. Adam, Hanan Fernando, Julia Arzeno, Meital Gabay, Dean W. Felsher. Development of a novel anti-MYC therapeutic with efficacy against liver and kidney cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-313. doi:10.1158/1538-7445.AM2013-LB-313 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.