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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Liver cirrhosis is a predominant risk factor for hepatocellular carcinoma (HCC). However, the mechanism underlying the progression from cirrhosis to HCC remains unclear. Herein we report the concurrent increase of liver progenitor cells (LPCs) and transforming growth factor-β (TGF-β) in diethylnitrosamine (DEN)-induced rat hepatocarcinogenesis and cirrhotic livers of HCC patients. Using several experimental approaches, including 2-acetylaminofluorene/partial hepatectomy (2-AAF/PHx) and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-elicited murine liver regeneration, we found that activation of LPCs in the absence of TGF-β induction was insufficient to trigger hepatocarcinogenesis. Moreover, a small fraction of LPCs was detected to coexpress tumor initiating cell (T-IC) markers during rat hepatocarcinogenesis and in human HCCs, and TGF-β levels were positively correlated with T-IC marker expression, which indicates a role of TGF-β in T-IC generation. Rat pluripotent LPC-like WB-F344 cells were exposed to low doses of TGF-β for 18 weeks imitating the enhanced TGF-β expression in cirrhotic liver. Interestingly, long-term treatment of TGF-β on WB-F344 cells impaired their LPC potential but granted them T-IC properties including expression of T-IC markers, increased self-renewal capacity, stronger chemoresistance, and tumorigenicity in NOD-SCID mice. Hyperactivation of Akt but not Notch, signal transducer and activator of transcription 3 (STAT3), or mammalian target of rapamycin (mTOR) was detected in TGF-β-treated WB-F344 cells. Introduction of the dominant-negative mutant of Akt significantly attenuated T-IC properties of those transformed WB-F344 cells, indicating Akt was required in TGF-β-mediated-generation of hepatic T-ICs. We further demonstrate that TGF-β-induced Akt activation and LPC transformation was mediated by microRNA-216a-modulated phosphatase and tensin homolog deleted on chromosome 10 (PTEN) suppression. Conclusion: Hepatoma-initiating cells may derive from hepatic progenitor cells exposed to chronic and constant TGF-β stimulation in cirrhotic liver, and pharmaceutical inhibition of microRNA-216a/PTEN/Akt signaling could be a novel strategy for HCC prevention and therapy targeting hepatic T-ICs. (HEPATOLOGY 2012;56:2255–2267)

Liver cancer is the fifth most common cancer globally and the second leading cause of cancer death in men, among which hepatocellular carcinoma (HCC) accounts for 70% to 85% of total cancer burden.1 Despite the current advance in the diagnosis of HCC, the majority of patients are not eligible for surgical treatment due to late diagnosis.2 The high heterogeneity of HCC makes it difficult to eliminate the cancer cells with chemotherapy alone. Recurrence and metastasis result in a poor prognosis of HCC and the 5-year survival rate of patients undergoing surgical resection is disappointingly low.3 It is thereby urgent to elucidate the molecular pathogenesis of HCC so that a novel strategy for HCC prevention and treatment can be developed. Chronic infection of hepatitis B virus (HBV) or hepatitis C virus (HCV) is considered the major cause of cirrhosis and liver cancer.4 Epidemiological studies have revealed that cirrhosis with hepatitis virus infection is the most predominant risk factor for HCC development, and only 10% to 20% of HCCs occur in patients without cirrhosis.5 Therefore, prevention of HCC in the high-risk population, particularly in those with established cirrhosis, would be highly desirable. Unfortunately, the molecular mechanism of hepatocarcinogenesis in those patients with cirrhosis remains elusive and effective approaches for HCC prevention and therapy are scarce to date.

Liver regeneration normally counts on the proliferation of hepatocytes and cholangiocytes. In cirrhotic liver, however, the ability of those parenchymal cells to divide and repopulate damaged tissue is apparently compromised. Therefore, bipotential liver progenitor cells (LPCs), which reside quiescently within the canals of Hering in adults, are activated for compensative proliferation and differentiation into both hepatic and biliary lineages.6, 7 Recently, the concept that HCC originates from liver cancer stem cells (tumor-initiating cells) has captured much attention. It is believed that cancer stem cells mostly derive from normal stem/progenitor cells in certain pathological microenvironments.8 There is also evidence demonstrating that dysregulated LPCs possess tumor-initiating ability in vivo, which suggests that LPCs may participate in hepatocarcinogenesis.9, 10 Our most current study showed that LPCs in HBx knockin mice could be transformed and develop bilineage liver cancer in the presence of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC).11 Therefore, whether malignant transformation of LPCs in the persistent cirrhotic microenvironment could initiate HCC deserves exploration.

Transforming growth factor β (TGF-β) is the most potent hepatic profibrogenic cytokine predominantly produced by activated mesenchymal cells upon chronic liver damage.12 Moreover, TGF-β has also been reported as a multifunctional cytokine that exerts its biological effects on tissue and organ development, cellular proliferation, differentiation, survival, and apoptosis.13 In the liver, TGF-β is hypothesized to serve as the important link among chronic injury, cirrhosis, and HCC.14 Accumulating evidence has demonstrated that TGF-β modulates the expression of numerous genes relevant to tumor development.15 It has been assigned a central role in the epithelial-mesenchymal transition, which is a critical cellular event during tumor metastasis.16 It has been well established that HCCs usually occur in those cirrhotic livers where TGF-β is highly expressed compared with healthy controls, which suggests a possible pro-oncogenic role of TGF-β in HCC initiation.17 Although the mechanism remains to be defined, TGF-β seems to be very important in HCC occurrence in patients with cirrhosis. In this study we investigated the influence of hepatic TGF-β on the transition of LPCs to T-ICs and the underlying molecular mechanism. The results provide new insight into hepatic T-ICs-targeted HCC prevention and therapy.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Experimental Animal Models.

Thirty male Wistar rats and 20 male C57BL/6 mice were purchased from Shanghai Experimental Center of Chinese Academy of Science and maintained under pathogen-free conditions. The hepatocarcinogenesis model in rats was induced by intraperitoneal injections of diethylinitrosamine (DEN; Sigma-Aldrich, St. Louis, MO) once a week at 70 mg/kg for 10 weeks. Two rats were sacrificed biweekly thereafter to monitor HCC development. All of the remaining rats were sacrificed 22 weeks after the first DEN administration. The activation of liver progenitor cells in rats and mice was induced by the 2-acetylaminofluorene (2-AAF; Sigma)/partial hepatectomy approach and the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC; Sigma) treatment as described.18 These studies were approved by the Ethical Committee of the Second Military Medical University.

Cell Culture and Adenovirus.

The rat pluripotent LPC-like cell line WB-F34419, 20 was purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences and cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 0.5% fetal bovine serum (FBS; Gibco, Invitrogen). WB-F344 cells treated with TGF-β (Miltenyi Biotec, Auburn, CA) at 0.25 ng/mL or saline for 18 weeks were termed WB-TβLT or WB-CON cells, respectively. The mouse liver progenitor cell line LEPC was cultured in DMEM supplemented with 10% FBS.21 Adenovirus encoding dominant-negative mutant of Akt (AdDN-Akt) and green fluorescent protein (AdGFP) were generated using AdMax Adenovirus Vector (Microbix, Ontario, Canada).

Patients and Liver Samples.

All human liver tissues were obtained from surgical resections of patients without preoperative treatment at the Eastern Hepatobiliary Surgery Hospital (Shanghai, China). (See Supporting Table 1 for detailed clinicopathologic information.) The procedure for human sample collection was approved by the Ethics Committee of Eastern Hepatobiliary Surgery Hospital.

Immunohistochemistry and Immunofluorescence Staining.

Formaldehyde-fixed, paraffin-embedded sections of liver tissue were subjected to hematoxylin and eosin (H&E) staining and immunohistochemistry following routine protocols as described.22 The antibody information is provided in Supporting Table 2. Frozen sections of fresh human or rat liver tissue were incubated with rabbit anti-CD133 (Abcam, Cambridge, MA) and mouse anti-OV-6 (R&D Systems, Minneapolis, MN), followed by fluorescent staining with Alexa Fluor 488-conjugated antimouse IgG and Alexa Fluor 555-conjugated antirabbit IgG (Invitrogen). Mice samples were stained by rabbit anti-A6 and FITC-conjugated antirabbit IgG (Invitrogen). Nuclear staining was performed by Hoechst 33342 in tissue samples. Rabbit anti-forkhead family of transcriptional regulators subfamily O, 3a (FOXO3a; Epitomics, Burlingame, CA) and Alexa Fluor 555-conjugated antirabbit IgG were used to detect the cellular localization of FOXO3a in WB-CON and WB-TβLT cells, and 4′,6-diamidino-2-phenylindole (DAPI) was applied to show the nucleus. Representative images were captured with an Olympus IX70.

Real-Time Polymerase Chain Reaction (PCR) Analysis.

Quantitative PCR was performed using SYBR Green PCR Kit (Applied Biosystems, Foster City, CA) and ABI 7900HT Fast Real-Time PCR System (Applied Biosystems). The messenger RNA (mRNA) level of specific genes was normalized against β-actin. Primers used are listed in Supporting Table 3.

Quantification and Silencing of MicroRNAs.

Total RNAs were isolated using TriZol (Invitrogen). The level of microRNA (miRNA) was determined using specific primers (RiboBio Biotechnology, Guangzhou, China) and normalized against U6. The delivery of antagomirs against miRNAs (antagomir-216a, antagomir-217; RiboBio) was mediated by Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions and antagomir-negative (antagomir-NC; RiboBio) was used as control.

Flow Cytometry Analysis.

The flow cytometry analysis was carried out using a Moflo XDP from Beckman Coulter. WB-CON and WB-TβLT cells were incubated with Alexa Fluor 488-conjugated antirat CD90 (BioLegend, San Diego, CA) or rabbit anti-CD133 (Abcam) with FITC-conjugated antirabbit IgG (Invitrogen) as secondary antibody.

Spheroid Assay.

WB-CON or WB-TβLT cells were plated in 6-well ultra-low attachment culture dishes at 1 × 106 cell per well and cultured in DMEM/F12 (Gibco, Invitrogen) supplemented with 10% FBS for 7 days. The number of spheroids was counted and representative views are shown.

In Vitro Limiting Dilution Assay.

WB-CON or WB-TβLT cells were seeded into 96-well ultra-low attachment culture dishes at cell doses described in Tables 1, 2, and Supporting Table 4 (8 wells per dose) and incubated under spheroid condition for 7 days. Colony formation was assessed by visual inspection. Based on the frequency of wells without colony, the proportion of stem cells was determined using Poisson distribution statistics and the L-Calc v, 1.1 software (Stem Cell Technologies, Vancouver, Canada).

Table 1. Proportion of Stem Cells in WB-TβLT Cells Infected With AdDN-Akt
 Number of Cells Seeded Each WellStem Cell FrequencyP Value
Frequency of colony formation1,5001,200900600300100EstimateUpper and Lower LimitsRatio of Prop.'sRatio of Prop.'s=1
WB-CON + AdGFP8/86/86/85/83/81/81:633(1:423-1:948)  
WB-CON + AdDN-Akt7/87/85/85/83/81/81:696(1:463-1:1,044)1.10.37
WB-TβLT + AdGFP8/88/87/87/85/82/81:319(1:206-1:4,94)  
WB-TβLT + AdDN-Akt6/85/85/83/80/80/81:1290(1:817-1:2,036)4.040.0001
Table 2. Proportion of Stem Cells in WB-TβLT Cells Transfected With Antagomirs
 Number of Cells Seeded Each WellStem Cell FrequencyP Value
Frequency of colony formation900600300100EstimateUpper and Lower LimitsRatio of Prop.'sRatio of Prop.'s=1
WB-CON + Anta-NC5/84/83/81/81:821(1:468-1:1,441)  
WB-CON + Anta-216a5/84/82/81/81:907(1:508-1:1,619)1.10.4
WB-CON + Anta-2175/84/82/81/81:907(1:508-1:1,619)1.10.4
WB-TβLT + Anta-NC7/86/84/83/81:393(1:240-1:645)  
WB-TβLT + Anta-216a5/84/82/81/81:907(1:508-1:1,619)2.30.016
WB-TβLT + Anta-2177/85/84/82/81:474(1:273-1:783)1.20.3

Anchor-Independent Growth Assay.

WB-CON or WB-TβLT cells were diluted to 1.2 × 104 cells/mL in DMEM, mixed with Matrigel Basement Membrane Matrix (BD Bioscience, Bedford, MA) at a ratio of 2:1 to a final volume of 150 μL and then cultured in 96-well plates for 7 days. Colonies formed within the gel were counted and representative pictures were taken.

Cord Formation Assay.

A 96-well plate was coated with a mixture of DMEM and Matrigel at a ratio of 2:1 to a final volume of 100 μL for 2 hours. Then 6,000 cells were seeded on the top of a gelled mixture and cultured for 12 hours. Cord angles were counted on a view basis and representative pictures were taken.

Xenograft Formation.

WB-CON or WB-TβLT cells were mixed with Matrigel at a ratio of 1:1 and then injected subcutaneously into eight NOD-SCID mice at 2 × 106 cells per mouse. Mice were sacrificed 3 months postinoculation and tumors were measured and collected.

Data Analysis.

Statistical analysis in this study was calculated with SPSS 14.0 (Chicago, IL). Data are expressed as mean value ± standard error of the mean (SEM). The significance of mean values between two groups was analyzed by Student's t test. Pearson correlation analysis was performed to determine the correlation statistics between two variables. All differences except for limiting dilution assay were two-sided. P < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Subset of LPCs Exhibits T-IC Characteristics During DEN-Induced HCC.

To explore the role of LPCs in hepatocarcinogenesis, we examined the LPCs status in the livers of Wistar rats administrated with DEN. As shown in Fig. 1A and Supporting Fig. 1A, H&E staining and immunohistochemistry indicated the fibrosis, cirrhosis, and tumorigenesis after DEN treatment. Development of both HCC and cholangiocarcinoma (CC) in rat liver suggested that liver progenitor cells could be involved in DEN-elicited carcinogenesis (Supporting Fig. 1B). To our interest, the increasing level of TGF-β was in concomitance with the up-regulation of OV-6-positive LPCs (Supporting Fig. 1C), and proliferation of LPCs was also detected (Supporting Fig. 1D). Additionally, sustained activation of Smad2 in LPCs and time-dependent up-regulation of TGF-β target genes during hepatocarcinogenesis were noted (Supporting Fig. 1E,F). Intriguingly, although LPCs were remarkably activated, neither the rats undergoing 2-acetylaminofluorene/partial hepatectomy (2-AAF/PHx) approach (Supporting Fig. 2) nor the mice subjected to DDC treatment (Supporting Fig. 3) developed HCC without the up-regulation of TGF-β in liver, which implies the essential role of TGF-β in HCC occurrence. Notably, expression of hepatoma stem cell markers which include EpCAM, CD133, and CD90 was also increased during rat hepatocarcinogenesis and closely correlated with the up-regulation of TGF-β (Fig. 1B,C). More important, a small portion of LPCs in DEN-treated rat cirrhotic livers were found to coexpress CD133, indicating that LPCs may acquire tumor initiating cell features during hepatocarcinogenesis (Fig. 1D). These data suggest the importance of TGF-β in the generation of hepatic T-ICs during hepatocarcinogenesis.

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Figure 1. A subset of LPCs exhibits T-ICs characteristics during DEN-induced HCC. (A) Liver sections of DEN-treated rats were subjected to H&E staining and immunohistochemical analysis of α-SMA, OV-6, and TGF-β at the indicated time intervals; scale bar = 100 μm. (B) The mRNA levels of TGF-β, CD90, CD133, and EpCAM in the liver tissue obtained from rats administrated with DEN at the indicated time intervals. (C) The bivariate relation between the transcription levels of TGF-β and that of CD90, CD133, or EpCAM was assessed by Pearson's correlation test; R, Pearson correlation coefficient; P, P-value; linear curve estimation shown as indicated. (D) Frozen liver sections of rats treated with DEN for 22 weeks and that of rats in the control group were stained with CD133 (red), OV-6 (green), and Hoechst 33342 (blue); white arrow indicates CD133+OV-6+ cells; scale bar = 100 μm.

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TGF-β Levels Correlate with T-ICs Marker Expression in Human Cirrhotic Liver.

To explore the influence of TGF-β on the transition of human LPCs to hepatoma-initiating cells, 32 samples of human cirrhotic liver and 24 samples of normal liver were collected. As expected, OV-6-positive LPCs preferentially existed in cirrhotic livers and only a few LPCs were detected in the portal area of normal livers (Fig. 2A). Interestingly, CD133, CD90, and EpCAM were scarcely expressed in normal livers but highly expressed in cirrhotic livers in parallel with the up-regulated TGF-β (Fig. 2B). Furthermore, TGF-β levels were positively correlated with CD90 and CD133 expression in cirrhotic livers, implying the effect of TGF-β on the transformation of LPCs into T-ICs (Fig. 2C). As illustrated in Fig. 2D, a small portion of LPCs were found to coexpress T-IC marker CD133 in the cirrhotic liver of an HCC patient. Considering the consistent results of DEN-induced rat hepatocarcinogenesis, we speculated that the activated liver progenitor cells may undergo malignant transformation towards hepatic T-ICs in cirrhotic liver, where the unique TGF-β exposure may play an important role.

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Figure 2. TGF-β levels correlate with T-ICs marker expression in human cirrhotic liver. (A) Formalin-fixed, paraffin-embedded cirrhotic liver tissues (lower row) or para-tumorous normal tissues from patients with liver hemangioma (upper row) were stained with OV-6; scale bar = 100 μm. (B) The para-cancerous tissues of 32 HCC patients with cirrhosis and 24 HCC patients without cirrhosis were subjected to real-time PCR analysis of TGF-β, CD90, CD133, and EpCAM. (C) The Pearson correlation between the mRNA level of TGF-β and that of CD90, CD133 was calculated in the 32 samples of cirrhotic liver; R, Pearson correlation coefficient. (D) Frozen sections of liver tissues from patients with cirrhosis were stained with OV-6 (green) and CD133 (red). Red frame indicates OV-6+CD133+ cells, whereas blue frame refers to OV-6+CD133- cells; scale bar = 100 μm.

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TGF-β Exposure Promotes Malignant Transformation of LPC Cells.

To test whether long-term TGF-β exposure could mediate the transformation of LPCs into hepatic T-ICs, WB-F344 cells were treated with a low dose of TGF-β or saline control for 18 weeks, and the cells were then termed WB-TβLT or WB-CON cells, respectively. Consistent with previous studies, TGF-β-treated cells exhibited enhanced phosphorylation of Smad2 and Smad3, a distinct expression profile of TGF-β response genes,23 reduced proliferation, and marginally increased apoptosis (Supporting Fig. 4A-D). Interestingly, the discrepancy in morphology between WB-TβLT and WB-CON cells implied a transformative change mediated by TGF-β (Fig. 3A). WB-TβLT cells exhibited a distinct gene expression pattern of transformed cell with down-regulation of E-cadherin, ZO-1, p53, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), Rb, and cyclin D1, and up-regulation of fibronectin, vimentin, c-Jun, c-myc, alpha fetal protein (AFP), and ABCG2 (Fig. 3B). WB-TβLT cells exhibited mesenchymal characteristics and enhanced migration capacity (Supporting Fig. 4E-G). Cord formation assay revealed that WB-CON cells were able to assemble bile duct-like structures while the differentiation potential of WB-TβLT cells was evidently impaired (Fig. 3C). Moreover, suspension cultured WB-TβLT cells formed much more spheroids (Fig. 3D) and exhibited a higher proportion of stem cells in limiting dilution assay compared with WB-CON cells (Fig. 3E; Supporting Table 4), indicating that TGF-β exposure enhanced the self-renewal capacity of LPCs.

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Figure 3. TGF-β exposure promotes malignant transformation of LPC cells. (A) WB-F344 cells were exposed to TGF-β at 0.25 ng/mL (WB-TβLT cells) or saline (WB-CON cells) for 18 weeks. Representative images of their morphology are shown; scale bar = 100 μm. (B) The difference of indicated genes between WB-TβLT and WB-CON cells are shown as relative mRNA expression. (C) WB-CON and WB-TβLT cells were subjected to tube formation assay and representative images were taken; red arrow indicates tube-like structures; scale bar = 100 μm. Cord angles were counted six fields per well and the average number is shown. (D) Spheroid formation assay was carried out at cell numbers indicated and the numbers of spheroids were counted. Representative images are shown; scale bar = 100 μm. (E) WB-CON and WB-TβLT cells were subjected to limiting dilution assay and the results were shown as natural logarithm of the proportion of stem cells.

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TGF-β Treatment Confers T-IC Characteristics to LPCs.

To test if WB-TβLT cells possess hepatic T-IC characteristics, expression of putative hepatic T-IC markers was examined. As shown in Fig. 4A, expression of CD90 and CD133 were much higher in WB-TβLT cells than in WB-CON cells, which was further confirmed by fluorescence-activated cell sorting (FACS) assay (Fig. 4B). The spheroids derived from WB-TβLT cells presented higher levels of CD90 and CD133 compared with that from WB-CON cells as well (Supporting Fig. 5). Resistance to chemotherapy is one of the key hallmarks of T-ICs. Our data illustrated that WB-TβLT cells exhibit robust proliferation ability and reduced apoptosis upon 5′-fluorouracil (5-FU) and etoposide (ETO) treatment (Fig. 4C; Supporting Fig. 6). More important, WB-TβLT cells presented potent anchor-independent growth capacity in colony formation assay, whereas WB-CON cells did not (Fig. 4D). To explore the tumorigenicity of WB-TβLT cells, NOD/SCID mice were subcutaneously inoculated with WB-TβLT and WB-CON cells. As shown in Fig. 4E, six out of eight mice in the WB-TβLT group exhibited xenograft tumors, whereas none of the mice in the WB-CON group developed tumor. Histological analysis revealed the disrupted histological structure of the xenograft tumor and the aberrant expression of AFP and CD133 as well (Fig. 4F). These results imply that LPCs could achieve T-IC characteristics after long-term TGF-β treatment.

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Figure 4. TGF-β treatment confers T-IC characteristics to LPCs. (A) WB-CON and WB-TβLT cells were subjected to real-time PCR analysis of CD90 and CD133. (B) The protein levels of CD90 and CD133 were determined by FACS analysis. The percentage of cells positively stained by isotype controls was deducted from the value shown. (C) WB-CON and WB-TβLT cells were treated with 100 μM 5-FU for 48hrs and then subjected to FACS analysis. The percentage of apoptotic cells is shown. (D) WB-CON and WB-TβLT cells were subjected to anchor-independent growth assay and representative images are shown; scale bar = 20 μm. The clone numbers were measured in three independent experiments. (E) 2 × 106 WB-CON or WB-TβLT cells were subcutaneously inoculated into NOD/SCID mice. Xenografted tumors were excised 11 weeks postinoculation. Pictures of tumors were taken and tumor volumes and weights are shown. (F) Representative images of H&E staining, immunohistochemical staining of AFP, and immunofluorescence against CD133 of xenografted tumors are shown; scale bar = 100 μm.

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Activation of Akt Is Required in LPCs Transformation upon TGF-β Exposure.

TGF-β signaling has been reported to interact with NOTCH, JAK/STAT3 (signal transducer and activator of transcription 3), and the Akt/PKB signaling pathway, which facilitates the survival and self-renewal of T-ICs.24-26 As shown in Fig. 5A, there was no significant difference of cleaved Notch intracellular domain (ICD) and STAT3 phosphorylation between WB-CON and WB-TβLT cells, whereas phosphorylation of Akt was evidently enhanced in WB-TβLT cells compared with WB-CON control. We further tested if mammalian target of rapamycin (mTOR) and FOXO3a, two major Akt downstream functional mediators of CSCs maintenance,27, 28 were involved in the T-ICs generation in WB-TβLT cells. The results in Fig. 5B demonstrate that no distinct difference in the phosphorylation of mTORSer2448, which usually indicates mTOR activation,29 was detected between WB-CON and WB-TβLT cells. But the expression of FOXO3a was significantly reduced in WB-TβLT cells in comparison with WB-CON cells. Immunofluorescence analysis illustrated that nucleus FOXO3a was dramatically decreased in WB-TβLT cells compared with WB-CON cells (Fig. 5C), and it could be restored by overexpression of the dominant-negative mutant of Akt (Fig. 5D), which implies that Akt-mediated exportation and subsequent degradation of FOXO3a might be, at least partially, involved in LPCs transformation upon TGF-β treatment. More important, overexpression of DN-Akt diminished the proportion of T-ICs (Fig. 5E, Table 1) in WB-TβLT cells and attenuated their self-renewal capacity (Fig. 5F).

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Figure 5. Activation of Akt is required in LPCs transformation upon TGF-β exposure. The whole cell lysates of WB-CON and WB-TβLT cells were subjected to immunoblotting against intracellular domain of Notch1 and Notch3, phosphorylated and total STAT3, phosphorylated and total Akt (A), phosphorylated and total mTOR, FOXO3a, and GAPDH (B). Immunofluorescent staining of FOXO3a was performed to visualize the cellular localization of FOXO3a in WB-CON and WB-TβLT cells (C). Nuclear staining was performed by DAPI. WB-TβLT cells were infected with adenovirus overexpressing dominant-negative mutant of Akt (AdDN-Akt) or green fluorescent protein (AdGFP) for 48 hours and then subjected to immunofluorescence analysis of FOXO3a (D). WB-CON and WB-TβLT cells were infected with AdDN-Akt or AdGFP for 48 hours followed by limiting dilution assay (E) and spheroid formation assay (F).

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PTEN Suppression by MiR-216a Accounts for TGF-β-Mediated Akt Hyperactivation.

To clarify how TGF-β regulates the activation of Akt, we determined the PI3K activity in WB-TβLT cells. As shown in Fig. 6A, there was no significant difference of PI3K activity between WB-TβLT and the control cells. Interestingly, WB-TβLT cells with elevated levels of phosphorylated Akt displayed dramatically reduced PTEN expression (Fig. 6B), suggesting PTEN was involved in the activation of Akt. Among the three miRNAs previously reported to suppress PTEN expression,30, 31 the level of miR-216a was obviously up-regulated in WB-TβLT cells compared with WB-CON, whereas miR-217 was only slightly increased and miR-21 remained invariable (Fig. 6C). Mouse-derived liver progenitor cell line (LEPCs) was subjected to long-term treatment with TGF-β and consistent results were achieved (Supporting Fig. 7). Specific antagomir against miR-216a notably rescued PTEN expression and attenuated Akt phosphorylation, whereas down-regulation of miR-217 had a marginal effect (Fig. 6D). Moreover, antagomir-216a evidently reduced the proportion of stem cells in WB-TβLT cells (Fig. 6E, Table 2) and suppressed their self-renewal capacity (Fig. 6F). Suppression of Smad3 by its specific inhibitor or repression of Smad2 by small interference RNA not only attenuated the up-regulation of miR-216a and down-regulation of PTEN, but also impaired the T-IC characteristics of WB-TβLT cells (Supporting Fig. 8). Therefore, constant activation of Akt elicited by miR-216a-mediated PTEN suppression is involved in the T-ICs generation from LPCs exposed to TGF-β.

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Figure 6. PTEN suppression by miR-216a accounts for TGF-β-mediated Akt hyperactivation. (A) PI3k activity was determined by the amount of PIP3 catalyzed in the lysis of WB-CON and WB-TβLT cells. (B) WB-CON and WB-TβLT cells were lysated and subjected to western blot assay as indicated. (C) The RNA levels of miR-21, miR-216a, and miR-217 in WB-CON and WB-TβLT cells were measured by real-time PCR analysis. WB-CON and WB-TβLT cells were transfected with specific antagomirs against miR-216a or miR-217 and then subjected to immunoblotting assay (D), limiting dilution assay (E), and spheroid assay (F) as indicted; antagomir-negative control (miR-NC) was used as negative control.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

TGF-β has been well accepted to be critical in the process of liver fibrosis and cirrhosis. However, the role of TGF-β in HCC occurrence remains elusive.4, 32 With this report we first proposed the association of TGF-β with hepatic T-ICs generation during hepatocarcinogenesis. Our data revealed that TGF-β exposure could induce the transformation of LPCs and give rise to hepatic T-ICs. We also demonstrated that hyperactivation of Akt was required in TGF-β-induced malignant transformation of LPCs. Suppression of PTEN by miR-216a was responsible for Akt hyperactivation in LPCs upon TGF-β exposure. We thereby clarified a novel signaling cascade involved in LPCs transformation and hepatic T-ICs generation, which not only led to new insight on the molecular mechanism of hepatocarcinogenesis, but also provided a novel strategy for HCC prevention and therapy.

It is traditionally believed that accumulation of genetic and epigenetic mutations in regenerating mature hepatocytes during chronic liver injury leads to HCC occurrence.33 However, more and more evidence favors the hypothesis of cancer stem cells/T-ICs, which occupy a rare subpopulation within tumor and are responsible for tumor initiation and chemoresistance.8, 34 Besides rare adult hepatocytes undergoing dedifferentiation in certain pathological conditions, the neoplastic mutation of proliferating LPCs is considered the principle origin of hepatic cancer stem cells.9, 10 Both liver-specific and nonspecific risk factors contribute to genetic disruptions in LPCs, which include integration of HBV DNA, mutation of p53 and RB1 (retinoblastoma 1), or aberrant activation of β-catenin, etc.35, 36 Recent advances in molecular pathogenesis revealed a substantial heterogeneity and hierarchical organizations within hepatoma, which also supports that hepatic T-ICs could be the origins of HCCs.37, 38 Therefore, neoplastic transformation of LPCs should be a critical molecular event during hepatocarcinogenesis.

We previously reported the expression of LPC marker OV-6 in some human HCC samples, and the sorted OV-6-positive hepatoma cells exhibited greater tumorigenicity and chemoresistance than OV-6-negative cells, implying that HCC may originate from the transformed LPCs.18 Interestingly, You et al.39 unveiled that TGF-β was capable of epigenetically modulating CD133 expression by way of inhibition of DNA methyltransferases in Huh7 cells, implying a novel role of TGF-β in the regulation of liver cancer stem cells. In this study, we identified a minor portion of OV-6+ LPCs coexpressing T-IC marker CD133 in the liver of patients with cirrhosis and DEN-administrated rats. The expression of T-IC markers was closely associated with the TGF-β levels in cirrhotic livers, suggesting the important role of TGF-β in T-ICs generation and hepatocarcinogenesis. In addition, it took about 3-4 months before TGF-β-treated LPCs progressively acquired T-IC characteristics, which was consistent with the clinical observation that HCCs usually arise from those cirrhotic livers where TGF-β has been at comparatively high levels for a long time.14, 40

TGF-β is most well known for its antiproliferative effect and it has been demonstrated to reversibly suppress the proliferative response of hepatocytes following partial hepatectomy. Hepatocytes of TβR-II+/− mice exhibited enhanced proliferation and increased vulnerability to DEN.41, 42 Recent studies also indicated that ablation of TGF-β signaling promoted expansion of Oct3/4-positive cells and facilitated spontaneous HCC occurrence.43, 44 Moreover, it was reported that LPCs exhibited impaired sensitivity to the growth inhibitory effect of TGF-β treatment compared with hepatocytes due to the deficiency of Smad6.45 Nevertheless, the role of aberrant TGF-β induction in HPC-associated hepatocarcinogenesis remains elusive. In the present study we found that LPCs survived long-term TGF-β treatment and underwent neoplastic transformation and exhibited T-IC characteristics. It has been well established that TGF-β levels are notably increased in cirrhotic liver and compensatory proliferation of LPCs during cirrhosis preceding HCC is secondary to sustained liver injury. Our results presented here suggest the chronic and progressively enhanced transforming effect of TGF-β on LPCs in the context of sustained liver damage.

Maintenance and proliferation of stem/progenitor cells are tightly regulated by comprehensive signaling network involving JAK/STAT3, NOTCH, PTEN, Akt/FOXO3a, etc.46 Dysregulation of these pathways may lead to aberrant proliferation or neoplastic transition of stem/progenitor cells. With this report, we unveiled that long-term TGF-β exposure down-regulated PTEN expression and up-regulated Akt phosphorylation in LPCs, which subsequently led to the nuclear exportation of FOXO3a and neoplastic transformation of LPCs. FOXO transcription factors participate in a variety of cellular events including the maintenance of cell differentiation.47 The FOXO family consists of four members: FOXO1, FOXO3a, FOXO4, and FOXO6, and functions in the nucleus of the cell. FOXO3a has been considered the key mediator for the maintenance of hematopoietic stem cells and the nuclear exportation of FOXO3a by phosphorylated Akt was proved to be a critical event during the transformation of stem/progenitor cells.28, 48 In the current study, our data indicate that Akt is responsible for FOXO3a inactivation and T-ICs generation in LPCs exposed to TGF-β.

Akt phosphorylation is usually enhanced as a consequence of PI3-K activation or PTEN suppression.49 In the present study, PTEN suppression but not PI3-K activation was observed in LPCs upon long-term TGF-β treatment. Dysfunction of PTEN has been widely detected in various cancers and accumulating studies have implicated the pivotal role of PTEN in the maintenance of stem cells.50, 51 Impaired PTEN function could induce the transformation of stem cells into cancer stem cells, sequentially initiating tumorigenesis. Fu et al.52 reported that PTEN deficiency in mice and zebrafish induced myelodysplasia with aberrant infiltration of myeloid progenitor cells. Enhancement of PTEN signaling not only depleted leukemia-initiating cells but also restored normal HSC function, which indicates the regulatory mechanistic difference between normal stem cells and cancer stem cells, and suggests that PTEN might be pharmaceutically targeted to deplete cancer stem cells without damaging normal stem cells.53 Herein, our data also indicate that PTEN is an indispensable moderator for LPC maintenance and is significantly reduced in hepatic T-ICs. Therefore, molecular therapy targeting PTEN might be a promising approach for HCC therapy.

MiRNAs are small (∼22 nt) noncoding RNAs regulating gene expression by way of inhibiting the translation of mRNAs or accelerating their degradation.54 It has been demonstrated that various cellular events including self-renewal and tumorigenicity of cancer stem cells could be regulated by miRNAs.55 Previous studies have elucidated that TGF-β signaling promotes the transaction of primary miRNAs to precursor miRNAs and facilitates miRNA maturation by way of Smad/Drosha-dependent machinery.56 Of note, we showed that miR-216a instead of miR-21, which was reported to regulate PTEN expression in human HCC,30 was involved in PTEN suppression and hepatic T-ICs generation in LPCs exposed to TGF-β. These data indicate the discrepant expression patterns of miRNA between hepatic stem cells and hepatoma-initiating cells, and suggests the potential therapeutic significance of miRNA in HCC targeted therapy.

To summarize, our results suggest that TGF-β in cirrhotic liver promotes the neoplastic transformation of LPCs to hepatic T-ICs and facilitates hepatocarcinogenesis by way of an miR216a/PTEN/Akt-dependent pathway. These findings not only provide important insight into the molecular mechanism of hepatocarcinogenesis, but also shed new light on the targeting strategy for HCC prevention and therapy.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_26007_sm_SuppFig1to1.tif6029KSupporting Information Figure 1-1. (A) DEN-treated rats were sacrificed at indicated time intervals and the gross appearance of their livers were shown. (B) Tumor nodules of rats in the 22nd week were assessed by H&E staining. Representative images of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) like regions were shown as indicated. (C) Liver sections of rats treated with DEN were subjected to immnuohistochemistry against OV-6 and proliferation of oval cells was determined based on the percentage of positively stained cells. (D) Liver sections of DEN-treated rats were stained with OV-6 (green), Ki67 (red) and Hochest 33342 (blue); white arrow indicated Ki67+OV-6+ cells; scale bar, 25 μm. (E) Liver sections of DEN-treated rats were stained with OV-6 (green), p-Smad2 (red) and Hochest 33342 (blue); white arrow indicated p-Smad2+OV-6+ cells; scale bar, 25 μm. (F) The mRNA expression of PAI-1, CDKN1A, CDKN2B and TGFB1I1 in the liver tissue of DEN-treated rats.
HEP_26007_sm_SuppFig1to2.tif2323KSupporting Information Figure Part 1-2.
HEP_26007_sm_SuppFig2.tif2892KSupporting Information Figure 2. Rats following 2-acetylaminofluorene/partial hepatectomy (2-AAF/PHx) approach were sacrificed seven days after the partial hepatectomy and their liver sections were subjected to H&&amp;E staining, immnuohistochemistry against α-SMA, OV-6 and TGF-β. Rats treated with the partial hepatectomy only were used as control; scale bar, 100 μm.
HEP_26007_sm_SuppFig3.tif3393KSupporting Information Figure 3. Liver sections of mice fed with diethyldithiocarbamate (DDC) or saline for 4 months were subjected to H&E staining, immnuohistochemistry of TGF-β and immnuofluorescence of A6 (green) and DAPI (blue); scale bar, 100 μm.
HEP_26007_sm_SuppFig4to1.tif1787KSupporting Information Figure 4-1. (A) WB-F344 cells treated with saline or TGF-β for indicated time intervals were subjected to immnuoblotting assay. (B) Relative expression analysis of TGF-β response genes in WB-CON and WB-TβLT cells. Data are presented in a heatmap format in which rows and columns represent genes and samples at indicated time intervals, respectively. Proliferation (C) and apoptosis (D) of TGF-β-treated WB-F344 and control cells were determined by Cell Counting Kit 8 and FACS assay. (E) WB-CON and WB-TβLT cells were subjected to immunofluorescent staining of E-cadherin and Vimentin (red). DAPI were used to show the location of nucleus (blue); scale bar, 50 μm. (F) WB-CON and WB-TβLT cells were seeded into 12-well plates at 70% confluence and then subjected to wound healing assay. Representative images were taken at indicated time point and the relative migration rate was shown. (G) 1×105 of WB-CON or WB-TβLT cells were seeded into the upper chamber of Corning 3422 transwell plates with serum-free DMEM. The lower chamber was filled with DMEM with 10% FBS. The cells were fixed and stained with crystal violet.
HEP_26007_sm_SuppFig4to2.tif3073KSupporting Information Figure 4-2.
HEP_26007_sm_SuppFig5.tif331KSupporting Information Figure 5. WB-CON and WB-TβLT cells were cultured in spheroid condition for seven days and the spheroids were collected. The mRNA level of CD90 and CD133 were measured using Realtime PCR analysis.
HEP_26007_sm_SuppFig6.tif316KSupporting Information Figure 6. WB-CON and WB-TβLT cells were treated with 5'-fluorouracil (5-FU) at 200 ?μM and etoposide (ETO) at 50 μM for 24hrs and their relative proliferation rates were analyzed by CCK-8.
HEP_26007_sm_SuppFig7.tif854KSupporting Information Figure 7. (A) LEPCs were treated with saline or TGF-β at 0.25 ng/mL for 6 weeks and subjected to spheroid assay at 10000 cells per well. Spheroids were counted 7 days later and representative images were shown; scale bar, 100 μm. (B) TGF-β-treated LEPCs and control cells were subjected to limiting dilution assay at doses of 25, 100, 200 and 400 cells per well. The results were shown as natural logarithm of the proportion of stem cells. (C) TGF-β-treated LEPCs and control cells were subjected to colony formation assay at 3000 cells per well and colonies formed were counted 7 days later. (D) TGF-β-treated LEPCs and control cells were subjected to immnuoblotting analysis as indicated. (E) The microRNA levels of TGF-β-treated LEPCs and control cells were determined by Realtime-PCR assay. *, p<0.05.
HEP_26007_sm_SuppFig8.tif840KSupporting Information Figure 8. WB-CON and WB-TβLT cells were treated with SIS3 (Merck KGaA, Germany) at 10 μM and then subjected to spheroid assay (A) and limiting dilution assay (B), DMSO was used as solvent control. WB-CON and WB-TβLT cells were transfected with small interference RNA against Smad2 (RiboBio Biotechnology, Guangzhou, China) followed by spheroid assay (C) and limiting dilution assay (D). (E) Realtime-PCR analysis of microRNA-216a expression in siSmad2-transfected WB-CON and WB-TβLT cells. Scramble siRNA was used as negative control. WB-CON and WB-TβLT cells were treated with SIS3 at indicated concentration and then subjected to immnuoblotting analysis of PTEN (F) and phosphorylation of Akt (G), GAPDH was used as loading control.
HEP_26007_sm_SuppInfo.doc94KSupporting Information

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