Promotion of Cancer Stem-Like Cell Properties in Hepatitis C Virus-Infected Hepatocytes

ABSTRACT

We have previously reported that hepatitis C virus (HCV) infection of primary human hepatocytes (PHH) induces the epithelial mesenchymal transition (EMT) state and extends hepatocyte life span (S. K. Bose, K. Meyer, A. M. Di Bisceglie, R. B. Ray, and R. Ray, J Virol 86:13621–13628, 2012, http://dx.doi.org/10.1128/JVI.02016-12). These hepatocytes displayed sphere formation on ultralow binding plates and survived for more than 12 weeks. The sphere-forming hepatocytes expressed a number of cancer stem-like cell (CSC) markers, including high levels of the stem cell factor receptor c-Kit. The c-Kit receptor is regarded as one of the CSC markers in hepatocellular carcinoma (HCC). Analysis of c-Kit mRNA displayed a significant increase in the liver biopsy specimens of chronically HCV-infected patients. We also found c-Kit is highly expressed in transformed human hepatocytes (THH) infected in vitro with cell culture-grown HCV genotype 2a. Further studies suggested that HCV core protein significantly upregulates c-Kit expression at the transcriptional level. HCV infection of THH led to a significant increase in the number of spheres displayed on ultralow binding plates and in enhanced EMT and CSC markers and tumor growth in immunodeficient mice. The use of imatinib or dasatinib as a c-Kit inhibitor reduced the level of sphere-forming cells in culture. The sphere-forming cells were sensitive to treatment with sorafenib, a multikinase inhibitor, that is used for HCC treatment. Further, stattic, an inhibitor of the Stat3 molecule, induced sphere-forming cell death. A combination of sorafenib and stattic had a significantly stronger effect, leading to cell death. These results suggested that HCV infection potentiates CSC generation, and selected drugs can be targeted to efficiently inhibit cell growth.

IMPORTANCE HCV infection may develop into HCC as an end-stage liver disease. We focused on understanding the mechanism for the risk of HCC from chronic HCV infection and identified targets for treatment. HCV-infected primary and transformed human hepatocytes (PHH or THH) generated CSC. HCV-induced spheres were highly sensitive to cell death from sorafenib and stattic treatment. Thus, our study is highly significant for HCV-associated HCC, with the potential for developing a target-specific strategy for improved therapies.

INTRODUCTION

Over 180 million people are estimated to be infected with hepatitis C virus (HCV) worldwide. HCV infection often causes liver fibrosis/cirrhosis and is an increasingly important factor in the etiology of hepatocellular carcinoma (HCC) (1–3). Sustained virologic response (SVR) correlated with disease severity at the point of treatment (4). The eradication of HCV by recently emerged direct-acting antivirals (DAAs) does not completely eliminate the risk of HCC (5, 6). A strong link exists between chronic HCV infection and HCC, although the mechanism for disease promotion remains poorly understood. Significant challenges remain in deploying modern antivirals for patients with asymptomatic HCV infection.

The HCV genome does not integrate into its host genome, and it has a predominantly cytoplasmic life cycle. Somatic cells have the ability to become pluripotent cells when transiently exposed to strong stimuli that they would not normally experience in their living environments (7, 8). This reprogramming does not require nuclear transfer or genetic manipulation. Therefore, HCV-mediated end-stage liver disease progression appears to involve indirect mechanisms related to the persistent infection of hepatocytes. HCC remains largely incurable because of late presentation and tumor recurrence. Studying the underlying mechanisms of HCV-mediated end-stage liver disease progression is challenging due to the lack of a naturally susceptible small-animal model.

We have previously reported that primary human hepatocytes, a principal host cell type for HCV, when infected in vitro with cell culture-grown virus induces EMT and cell growth promotion (9). EMT is a major mechanism of tumor progression, local invasion, metastasis, and therapeutic resistance. EMT also may be linked to the development of stem-like properties of cancer cells (10–14). The use of a human mammary epithelial cell model showed that the acquisition of a mesenchymal trait links to the expression of stem cell markers (10). Further, the transformed human mammary epithelial cells undergoing EMT form spheres on soft agar and tumors more efficiently.

Here, we examined CSC generation in HCV-infected primary human hepatocytes (PHH) and transformed human hepatocytes (THH) and analyzed the underlying molecular changes. Our results suggested that HCV-infected hepatocytes display sphere formation on ultralow binding plates with characteristic activation of CSC signaling mechanism and tumorigenicity in NOD-SCID IL2Rgamma^null (NSG) mice. Treatment with sorafenib and stattic revealed an increased effect leading to HCV-associated CSC death.

MATERIALS AND METHODS

Patient materials.

Chronically HCV-infected liver biopsy specimens from 11 patients and non-HCV liver biopsy specimens from 4 patients were used. Archived liver samples were used. Specimens were collected from subjects with their written consent, and the human studies protocol (number 10592) was approved by the Saint Louis University Internal Review. RNA was prepared from liver specimens by using TRIzol (Invitrogen) as previously described (15). Commercially available control liver RNAs (Clontech, CA, and Lonza, NJ) were used for comparison.

Hepatocytes and HCV.

Commercially available PHH (Lonza, MD) were maintained in SAGM (Lonza, MD) as previously described (9). Cells were infected with cell culture-grown HCV genotype 2a, clone JFH1 (multiplicity of infection [MOI] of 0.2). HCV inoculum was incubated with hepatocytes for 8 h, washed, and incubated with fresh medium. Infected PHH were examined for phenotypic and molecular changes. Immortalized human hepatocytes (IHH) were generated previously (16) by stable transfection of the HCV core (genotype 1a) genomic region into primary human hepatocytes. IHH exhibited a weak level of HCV core protein expression and were differentiated (16). Multiple passages of IHH generated a transformed and tumorigenic phenotype that was named THH.

Maintenance of hepatocytes on ultralow binding plate.

PHH or THH infected with HCV were plated (10,000 cells/well) on ultralow attachment plastic plates (Corning) in SAGM medium without serum. Hepatocytes were incubated for 3 weeks and examined microscopically. Cell spheres (>50-μm diameter) were counted and compared to total numbers of cells plated. To propagate, spheres were passaged to 100-mm ultralow binding plates (Corning) using Accutase (Gibco, Life Technologies).

Cancer stem cell PCR array.

RNA was extracted from PHH and sphere-forming cells using TRIzol (Invitrogen, Carlsbad, CA). A cancer stem cell pathway-specific PCR array (SA Biosciences) was performed by following the manufacturer's instructions.

Immunoblotting.

Cells were lysed in sample-reducing buffer and subjected to SDS-PAGE using specific antibodies. Separated proteins were transferred onto nitrocellulose (Bio-Rad Laboratories). The blot was blocked with 5% skim milk and incubated with a primary antibody, followed by a secondary antibody conjugated with horseradish peroxidase (HRP) (Bio-Rad Laboratories). The protein bands were detected with SuperSignal West Pico ECL reagents (Pierce). Primary antibody to CD133 (PA1217) (Boster Biochemical Technology); Nanog (D73G4), Oct-4A (C30A3), LIN28A (D84C11), Vimentin (R28), c-Myc (D84C12), Notch1 (D1E11), c-Kit (D13A2), and cleaved poly(ADP-ribose)polymerase (PARP; Cell Signaling Technology); Snail (H-130), Twist (H-81), and actin HRP-conjugated antibody (I-19) (Santa Cruz Biotechnology); and caspase 8 (BD Pharmingen) were used.

Real-time PCR analysis.

HCV RNA and c-Kit mRNA were analyzed by real-time PCR (Applied Biosystems, Foster City, CA) using specific primers as previously described (9). RNA from experimental cells was isolated by treatment with TRIzol, and cDNA was transcribed using a Superscript III first-strand synthesis system (Invitrogen). HCV RNA was analyzed with a TaqMan gene expression kit (Applied Biosystems). A Power SYBR green assay kit (Applied Biosystems) was used for c-Kit mRNA quantification by following the manufacturer's protocol. The mRNA status of c-Kit was determined by using specific forward (5′-GAGTTGGCCCTAGACTTAGAAG-3′) and reverse (5′-TCTTTGTGATCCGACCATGAG-3′) primers and normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific forward (5′-CATCATCCCTGCCTCTACTG-3′) and reverse (5′-GCCTGCTTCACCTTCTT-3′) primers. All reactions were performed in triplicate and analyzed with an ABI Prism 7500 analyzer.

Luciferase reporter assay.

The human c-Kit response promoter tagged with a luciferase reporter plasmid in pGL3 was kindly provided by Menashe Bar-Eli (University of Texas MD Anderson Cancer Center, Houston, TX). THH were transfected with the c-Kit promoter-driven luciferase reporter construct (100 ng/well) alone or together with HCV core, NS2, NS3, NS5A, or a full-length infectious cDNA (JFH1-FL) construct (400 ng/well) in a 24-well plate. Cells were lysed with reporter lysis buffer (Promega) 48 h after transfection, and the clarified lysates were subjected to the luciferase reporter assay (Glomax; Promega).

Drug treatment and cell viability assay.

A sorafenib derivative (SC-1; Sigma), sorafenib, imatinib, dasatinib, stattic, PI-103, and Notch inhibitor (DAPT) (Santa Cruz) were purchased for use as CSC signaling inhibitors. All of these drugs were suspended in dimethyl sulfoxide (DMSO) except imatinib, which was dissolved in water, at a stock concentration by following the manufacturer's instructions and stored in aliquots at −70°C until used. Drugs were diluted into the culture medium at the time of addition to cells and incubated for 1 to 7 days. Cells were monitored for viability using Trypan Blue dye exclusion and/or CellTiter 96 AQueous One cell viability assay (Promega).

Tumor xenograft in NSG mouse model.

Tumor xenografts were established in 12-week-old female NSG mice (Jackson Laboratory) by direct injection of cells into the flanks. All animal experiments were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee of Saint Louis University. A total of 10³, 10⁴, or 10⁵ parental THH after trypsinization (as a positive control) or THH-HCV sphere-forming cells after Accutase treatment were resuspended in Opti-MEM and mixed with 40% Matrigel for subcutaneous injections. Tumor growth was examined weekly until the end of each experiment. Tumor size was measured with a slide caliper and the volume calculated using the formula (L × W²) × 0.5, where L is length and W is width. Mice were sacrificed with large tumors (after 6 weeks), and cells were harvested, grown in vitro, and stored frozen for future cellular and biochemical work.

Statistical analysis.

Results were expressed as the means ± standard deviations (SD) from at least three independent experiments, and statistical analyses were performed using a two-tailed unpaired Student t test. A P value of <0.05 was considered significant.

RESULTS

Characterization of sphere-forming HCV-infected primary human hepatocytes.

Tumors can arise from stem or progenitor cells, and EMT can endow CSC characteristics (10, 17–19). In our previous study, we observed the generation of EMT in HCV-infected PHH (9). To test whether HCV-infected hepatocytes displaying EMT generate CSC, we performed a sphere-forming assay to assess the capacity of self-renewal under ultralow binding culture conditions. After 2 to ∼3 weeks, some of these cells (∼1% of the total cell population) displayed sphere formation of different sizes and slow proliferation (Fig. 1A). These sphere-forming hepatocytes survived for more than 8 weeks in culture. We examined HCV RNA on days 3 and 42 postinfection by real-time PCR (Fig. 1B). HCV RNA was almost undetectable at day 42 compared to the level at day 3 postinfection. The altered morphology of hepatocytes compared to that of sphere-forming cells prompted us to examine the induction of CSC signaling molecules. A CSC pathway-specific PCR array was performed using total RNA from sphere-forming cells and normal PHH as a control for comparison. A differential gene expression of CSC markers was observed (Table 1). The majority of the cancer stem cell markers analyzed, including c-Kit, ENG, PTPRC, and CD133 (PROM1), were upregulated in sphere-forming cells compared to levels in mock-infected control cells. These data suggested that HCV infection induces and maintains CSC despite a decrease in HCV RNA over time to an almost undetectable level in infected hepatocytes.

FIG 1.

Generation, identification, and characterization of sphere-forming cells from HCV-infected primary human hepatocytes and THH. (A) EMT-induced cells from HCV genotype 2a (clone JFH1)-infected PHH were incubated with serum-free medium on ultralow attachment plates for 2 to ∼3 weeks and displayed sphere formation. (B) HCV RNA level in HCV-infected PHH at 3 and 42 days postinfection (DPI) is shown. (C) Immunoblot for enhanced c-Kit protein expression in HCV-infected PHH after 4 weeks. Tubulin is shown as a loading control. (D) Mock-infected THH and HCV-infected THH were incubated in serum-free SAGM medium on ultralow attachment plates for 3 weeks to facilitate sphere formation. The numbers of spheres (diameter, >50 μm) were counted in 10 randomly chosen microscopic fields. The means and standard deviations from the 10 counts are shown. (E) HCV RNA levels were determined in virus-infected THH at different days (4, 9, 18, or 27) postinfection by real-time PCR. (F) THH infected with HCV were lysed at different days (9, 18, and 27) postinfection and immunoblotted against c-Kit or CD133 antibody.

TABLE 1.

Cancer stem cell PCR array using HCV (genotype 2a)-infected primary human hepatocytes displaying sphere formation versus mock-treated control

Marker(s) and gene name	Fold change in expression (upregulation)
Cancer stem cell
ENG (CD105)	250
ITGA6	3
KIT (CD117)	3,000
PTCH1	10
PTPRC (CD45)	227
PROM1 (CD133)	87
Proliferation
LIN28B	197
NOS2	71
Pluripotency
SOX2	154
LIN28A	4
NANOG	19
Migration and metastasis
KLF17	52
SNAIL1	24
TWIST1	42
TWIST2	17
Transcription factors
NOTCH1	5
WNT1	19

We also examined HCV-mediated sphere formation using THH. For this, THH were infected with HCV genotype 2a (MOI, 0.2), and infected parental THH were grown under ultralow binding conditions for 3 weeks. The number of sphere-forming cells was significantly increased in HCV-infected THH (>10-fold) compared to that in parental THH (Fig. 1D). The HCV RNA level following infection peaked at day 4 and decreased to an almost undetectable level at days 18 and 27 postinfection (Fig. 1E).

Sphere-forming cells from THH display CSC and EMT marker proteins.

c-Kit is a stem cell factor (SCF) receptor. SCF is a major cytokine for the self-renewal, proliferation, and differentiation of numerous embryonic, adult hematopoietic, neural, and primordial stem cells (20). The uncontrolled activity of c-Kit contributes to the formation of human tumors. We have observed significant upregulation of c-Kit in our array. We next observed c-Kit upregulation in HCV-infected PHH (4 weeks postinfection) at the protein level compared to that of mock-treated normal PHH (Fig. 1C). We found c-Kit mRNA (data not shown) and protein expression in HCV-infected THH to be increased (Fig. 1F). We also examined CD133, a membrane protein known as a stem and progenitor cell marker, in HCV-infected THH. CD133 expression increased upon HCV infection in THH (Fig. 1F). CD133 was detected throughout the study period. CD133 is significantly upregulated in our array data in spheres generated from HCV-infected primary human hepatocytes. Taken together, our results suggested that HCV infection induced the CSC markers c-Kit and CD133.

We performed immunoblot analysis of the sphere-forming cell lysates from THH following HCV infection to determine the expression of selected stem cell/pluripotency marker proteins and compared their levels to those in the control cells (Fig. 2). NANOG, a member of the homeobox family of DNA binding transcription factors, was identified in a screen for pluripotency-promoting genes. NANOG is a master transcription factor essential for maintaining cell stemness, and it is specifically expressed in embryonic pluripotent stem cells. NANOG acts together with Oct4 and Sox2 to maintain the proliferation and self-renewal of embryonic stem cells (21). We found that NANOG was upregulated in HCV-infected THH. CD133 was upregulated in spheres from HCV-infected THH compared to the level in the mock-infected control (Fig. 2A). Other transcription factors (Oct4A, Lin28A, and c-Myc) also were upregulated in THH/HCV-infected sphere-forming cells (Fig. 2B). Notch signaling is activated in several solid tumors, including in human HCCs (22), and plays an important role in the EMT/CSC program (23). Notch1 was significantly enhanced in THH/HCV-infected sphere-forming cells (Fig. 2C). These data suggest that HCV infection induces the generation of CSCs that have proliferation and self-renewal ability in a hepatocyte-derived model.

FIG 2.

CSC and EMT marker genes in sphere-forming cells from HCV-infected THH. Immunoblot analysis shows that cancer stem cell marker proteins are differentially regulated in sphere-forming cells from HCV-infected THH compared to mock-infected control cells. Protein expression status of surface marker CD133 and transcription factor Nanog (A), Lin28A and Oct4A (B), and Notch1 and c-Myc (C) are shown. Protein expression statuses of Snail, Twist, Vimentin (D), and Slug (E) were similarly analyzed by immunoblotting using specific antibodies. The expression level of actin is shown as a loading control for comparison.

EMT relating to the emergence of a CSC phenotype may be a prerequisite for cancer metastasis (10). In order to know the relationship between EMT and sphere-forming CSCs, we examined the Snail, Slug, and Twist transcription factors and their downstream effector, Vimentin. Snail, Twist, and Vimentin were increased in THH-HCV-infected sphere-forming cells compared to levels for the control (Fig. 2D and E). The expression of Slug also was increased in THH-HCV-infected sphere-forming cells. We also observed HCV-induced EMT (9), and our results suggest that HCV-induced CSCs are linked to EMT.

Enhanced c-Kit mRNA expression in liver biopsy specimens of chronically HCV-infected patients.

We determined the expression status of c-Kit mRNA by quantitative PCR analysis in liver biopsy specimens from a randomly chosen cohort (15, 24) of chronically HCV-infected patients (Fig. 3A and B). c-Kit mRNA expression was enhanced in all patients and was significantly higher (>50-fold) in 8 of 11 chronically HCV-infected liver biopsy specimens than in two healthy liver RNA samples or 4 non-HCV liver samples. These data suggested that c-Kit is upregulated in HCV-infected human liver specimens and suggested clinical relevance.

FIG 3.

c-Kit expression in HCV-associated liver biopsy samples. c-Kit mRNA expression in liver specimens from healthy control, non-HCV-, and chronically HCV genotype 1a-infected patients is shown as a box plot (A) and stratified results (B). The results were compared to those for non-HCV-infected and healthy liver tissues (C1 and C2) and normalized with GAPDH RNA. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

HCV core protein upregulates c-Kit promoter.

c-Kit mRNA expression was upregulated in HCV-infected THH (Fig. 4A). HCV core and NS5A are reported to induce EMT (25, 26). For this, luciferase-tagged c-Kit promoter reporter plasmid (−1215 to +1) was cotransfected with HCV protein expressing plasmid (Core, NS2, NS3, or NS5A) as well as the full-length HCV genome to THH. Cells were lysed 48 h after transfection, and promoter activity was measured by luciferase assay. c-Kit promoter activity was increased by HCV core or full-length HCV plasmid (Fig. 4A). In comparison, HCV NS2, NS3, or NS5A did not have a significant role in c-Kit promoter regulation (Fig. 4B and C). c-Kit promoter activity is known to be upregulated by Sp1 (27). In previous studies, we and others have reported that HCV core enhances Sp1 promoter activity (28, 29), which may be a mechanism by which c-Kit expression is enhanced in the presence of HCV.

FIG 4.

Activation of c-Kit promoter by HCV core protein. (A) c-Kit mRNA expression status in PHH, THH, and THH-HCV are shown for comparison. (B) A change in c-Kit promoter activity by pcDNA3 vector control, HCV core, NS2, NS3, NS5A, and HCV full-length genome (HCV FL) at 0.4 μg/well dose in a luciferase reporter assay is shown. (C) Response of c-Kit promoter at two doses (0.2 and 0.4 μg) of core protein is shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Sphere-forming cells are tumorigenic in NSG mice.

We examined the ability of THH/HCV-infected sphere-forming cells for tumor formation in NSG mice. Three groups of NSG mice were injected with three different doses of cells (10³, 10⁴, or 10⁵) per injection site. Cells were subcutaneously injected into two flanks of 4 mice in each group. Even the lowest number of THH/HCV-infected sphere-forming cells (10³) displayed tumorigenicity in mice. Tumor formation was observed at ∼16 days postinjection in mice which received the highest number of injected cells (10⁵), and tumor volume increased with time (Fig. 5A). On the other hand, similar numbers of implanted THH did not form tumors in the same time frame. THH/HCV-infected spheres formed multinodular tumors (without interstitial fluid) (Fig. 5B). Mice were sacrificed beginning at week 5 postimplantation. EMT markers (Snail, Twist, or Vimentin) were observed in xenografted tumors when examined by real-time PCR or Western blot analysis (data not shown). Thus, sphere-forming cells showing characteristic CSC properties generated from THH displayed an aggressive tumorigenic phenotype.

FIG 5.

Tumorigenicity of sphere-forming cells in NSG mice. (A) Larger tumor volume with increasing number of injected sphere-forming cells at different time points is shown. (B) Representative mouse showing tumor formation by sphere-forming cells after subcutaneous injection of 10³ cells. (Inset) Representative multinodular tumor formation is shown by arrows after sacrificing and skin removal.

Effect of inhibitors on HCV-induced sphere-forming cells.

In order to examine the effect of c-Kit on CSCs, we used the c-Kit inhibitors imatinib (0.5 μM) and dasatinib (0.5 μM) and examined their effect on sphere formation and cell viability. Incubation of drugs for 7 days significantly reduced sphere numbers (∼50%), implying cell growth arrest (Fig. 6A and B). These data suggested that c-Kit expression is necessary for HCV-mediated CSC generation, although the reduced sphere-forming cells did not show altered viability (Fig. 6C). We used two other cell growth inhibitors, DAPT (20 μM) as a Notch inhibitor and PI-103 (2 μM) as a phosphatidylinositol 3-kinase (PI3K) inhibitor, at known inhibitory concentrations. DAPT did not affect CSC viability; however, PI-103 displayed a 30% inhibition of CSC viability by Trypan blue staining. On the other hand, the sorafenib derivative SC-1 reduced cell viability by ∼80% much earlier (24 to 72 h). The combination treatment of c-Kit inhibitors and SC-1 did not have an additive or synergistic effect on CSC growth inhibition (Fig. 6A, B, and C). Since SC-1 alone significantly reduced the number of spheres (Fig. 6A and B), a combination of c-Kit inhibitor and SC-1 failed to provide further detectable decreases in sphere numbers. To clarify limitations with sphere formation assay, we further analyzed cell growth after incubation with one drug or a combined-drug treatment described below. Thus, our results from this set of experiments showed that c-Kit is important for generating CSC/sphere formation, but blocking c-Kit did not affect hepatocyte viability. Similar observations also were noted with lung cancer cells upon treatment with these inhibitors (30).

FIG 6.

Effect of signaling pathway inhibitors on sphere-forming cells. Single-cell suspensions, after Accutase treatment of THH/HCV sphere-forming cells, were plated and cultured on ultralow attachment plates overnight. Imatinib (Imat; 0.5 μM), dasatinib (Dasa; 0.5 μM), and/or SC-1 (5 μM) were added alone or in combination. (A) Microscopic view of cells before and after treatment of spheres with the inhibitors are shown. (B) The sphere numbers were counted microscopically. (C) Cell proliferation was analyzed by MTS assay after treatment of the inhibitors. (D) Sphere-forming cells were separately treated with sorafinib (Sora) and/or stattic (Statt) at the indicated concentrations, and cell proliferation was measured by MTS assay. Parental THH were treated similarly with the inhibitors and analyzed for comparison. (E) Apoptosis assay results analyzing endpoint signaling molecules (cleaved caspase 8 and PARP) are shown. The actin level in each lane is shown for comparison of protein load.

Sorafenib and stattic increased inhibition of sphere-forming cell growth.

Sorafenib is used for HCC treatment, although it is effective for only a short time. SC-1 inhibits Stat3 phosphorylation and induces apoptosis in a breast cancer cell line (31). We treated cells with sorafenib (multikinase inhibitor, including c-Kit) and/or stattic (Stat3 inhibitor). Sphere-forming cells were treated with different concentrations of sorafenib and/or stattic (1.25 to 10 μM), and cell viability was examined at 24-h intervals using a Trypan blue or MTS assay. Sorafenib or stattic exerted a concentration-dependent inhibitory effect on CSCs within 24 h, with a 50% inhibitory concentration (IC₅₀) of 0.625 μM for sorafenib and 2.5 μM for stattic. We next treated sphere-forming cells with a combination of sorafenib (1.25 or 2.5 μM) and stattic (2.5 or 5 μM) and observed an increased inhibitory effect on CSC growth (Fig. 6D). In contrast, sorafenib (1.25 to 2.5 μM) had a similar effect on parental THH, but stattic (2.5 to 5.0 μM) did not alter cell growth, and the stronger effect seen in combination treatment of CSC was not observed with parental THH. However, the blocking of downstream receptor tyrosine kinase (RTK) pathways related to cell proliferation could be affected by sorafenib and/or stattic. Similar results were obtained for viability by Trypan blue staining (data not shown).

We next investigated whether the apoptotic signaling pathway is activated by these two inhibitors as a mechanism of sphere-forming cell growth inhibition. Treatment of sphere-forming cells with sorafenib or stattic indicated that cell death occurs via a caspase-dependent pathway. We analyzed caspase activation and PARP cleavage in inhibitor-treated sphere-forming cells by immunoblot analysis. Apoptosis of sphere-forming cells did not involve procaspase 9 activation, indicating that the intrinsic pathway is not involved in the process. An increase in cleaved caspase 8 inhibitor-treated cells compared to the level for the mock-treated control suggested an activation of caspase-8 (Fig. 6E). The DNA-repairing enzyme PARP plays a key role in the execution phase of apoptosis. An ∼116-kDa polypeptide of the DNA repair enzyme PARP was cleaved to its ∼86-kDa signature peptide in inhibitor-treated cells (Fig. 6E). Stat3 inhibition is reported to induce apoptosis by reducing Bcl2 expression (32). However, our Bax and Bcl2 analyses did not suggest a significant change upon treatment of sphere-forming cells with stattic. These results indicated that both sorafenib and stattic initiate apoptosis of sphere-forming cells utilizing an extrinsic caspase-dependent signaling pathway.

DISCUSSION

Our observations suggest that HCV infection of PHH alters cell morphology and important cellular gene expression. These hepatocytes displayed sphere formation under ultralow binding conditions and survived for more than 8 weeks in culture, although HCV RNA significantly decreased. A CSC pathway-specific PCR array from these sphere-forming cells revealed a significant increase in stem cell markers (c-Kit, CD133, ENG, and PTPRC), pluripotency (Lin28a), migration/metastasis (Snail and Twist1), and transcription factors (Notch1 and Nanog) compared to levels for mock-treated hepatocytes. c-Kit is regarded as one of the CSC markers in HCC. A significantly higher level of c-Kit mRNA in liver biopsy specimens of chronically HCV-infected patients was observed. c-Kit also was increased in cell culture-grown HCV-infected hepatocytes, and viral core protein significantly upregulated c-Kit at the transcriptional level. This indicates that c-Kit is more abundant and active in HCV-mediated liver disease progression to HCC.

Binding of SCF to c-Kit can trigger pathways involved in the maintenance of progenitor cells besides functioning as a transcriptional factor in the regulation of developmental processes (33). Cancer patients with overexpression or mutation of c-Kit in their tumors have a poor prognosis (34). c-Kit is expressed in most of the hepatocytes of the corresponding peritumoral noncirrhotic and, even more often, those of the peritumoral cirrhotic liver parenchyma (35). Here, we have shown that EMT markers are induced in c-Kit-activated cells and potentiate sphere-forming ability. The use of the c-Kit-specific inhibitor imatinib or dasatinib impaired sphere formation. These data implicate a novel role of c-Kit in inducing EMT and link with CSC.

c-Kit stimulation activates downstream effector proteins, including PI3K/Akt, STAT, and RAS/MAPK pathways (36). Imatinib treatment downregulates the PI3K-Akt pathway (37, 38). Blocking the SCF/c-Kit signaling axis may lead to interference with the proliferation or survival of c-Kit-positive CSCs. Imatinib was used as monotherapy in clinical trials against different solid tumors, including breast, ovarian, and small cell lung cancer; however, no clinical responses were detected (39–41). Interestingly, we also did not observe a significant effect of imatinib or dasatinib treatment upon hepatocyte growth. Antitumor therapy of patients with advanced liver cancer might be significantly improved by combining conventional chemotherapy with inhibitors such as imatinib or dasatinib that would target the SCF/c-Kit axis. We also observed that the treatment of sorafenib or stattic exerted an inhibitory effect on CSCs. Sorafenib, a multikinase inhibitor, is currently in use for HCC treatment. Stattic selectively inhibits activation, dimerization, and nuclear translocation of Stat3 and increases the apoptotic rate of Stat3-dependent cancer cells (42). We observed that a combination of sorafenib and stattic enhanced the inhibition of HCV-associated CSC growth.

Tumor stem cells display plasticity to transition between alternative states, including a relatively quiescent, invasive, mesenchymal-like state and a more proliferative epithelial-like state (43). The results from our study highlight that HCV induces signaling molecules promoting primary hepatocyte growth and EMT/CSC generation. HCV infection transcriptionally regulates a number of cellular genes and may contribute to HCC. HCV-specific drug treatment most likely will inhibit viral replication; however, an advanced or established disease state may continue in the absence of strong host clearance mechanisms. Targeting critical convergences of several EMT signaling pathways may provide a realistic strategy to effectively control EMT and the progression of human epithelial cancers (44, 45).

In summary, we have shown: (i) HCV infection generates CSC in PHH/THH, (ii) cellular changes in HCV-infected hepatocytes occur and are maintained even when viral RNA is almost undetectable, (iii) HCV core upregulates c-Kit expression, (iv) sphere-forming cells are tumorigenic in immunodeficient mice, and (v) targeting multiple growth promotion pathways of CSCs effectively prevents cell growth. Our observations further suggested that a spontaneous decrease or eradication of HCV RNA occurs in sphere-forming hepatocytes and THH. This interesting observation does not rule out the possibility that HCC could arise even after the apparent clearance of HCV infection. Future studies using multiple targets in a preclinical trial from orthotopic transplantation of CSCs will suggest prevention or delay of a metastatic property for HCV-associated CSCs. Thus, the results from our study are highly significant for HCV-associated HCC, with the potential for developing target-specific strategies for improved therapies.