Potential conflict of interest: Nothing to report.
MicroRNAs (miRNAs) are endogenous small noncoding RNAs that regulate gene expression with functional links to tumorigenesis. Hepatocellular carcinoma (HCC) is the most common type of liver cancer, and it is heterogeneous in clinical outcomes and biological activities. Recently, we have identified a subset of highly invasive epithelial cell adhesion molecule (EpCAM)+ HCC cells from alpha-fetoprotein (AFP)+ tumors with cancer stem/progenitor cell features, that is, the abilities to self-renew, differentiate, and initiate aggressive tumors in vivo. Here, using a global microarray-based miRNA profiling approach followed by validation with quantitative reverse transcription polymerase chain reaction, we have demonstrated that conserved miR-181 family members were up-regulated in EpCAM+AFP+ HCCs and in EpCAM+ HCC cells isolated from AFP+ tumors. Moreover, miR-181 family members were highly expressed in embryonic livers and in isolated hepatic stem cells. Importantly, inhibition of miR-181 led to a reduction in EpCAM+ HCC cell quantity and tumor initiating ability, whereas exogenous miR-181 expression in HCC cells resulted in an enrichment of EpCAM+ HCC cells. We have found that miR-181 could directly target hepatic transcriptional regulators of differentiation (for example, caudal type homeobox transcription factor 2 [CDX2] and GATA binding protein 6 [GATA6]) and an inhibitor of Wnt/β-catenin signaling (nemo-like kinase [NLK]). Taken together, our results define a novel regulatory link between miR-181s and human EpCAM+ liver cancer stem/progenitor cells and imply that molecular targeting of miR-181 may eradicate HCC. (HEPATOLOGY 2009.)
Human epithelial cell–derived cancers are highly heterogeneous, often composed of a hierarchy of mixed tumor cells with different biological properties. Tumor-initiating cells (TICs) with self-renewal and differentiation capabilities constitute a small proportion of this hierarchy and are thought to give rise to tumor heterogeneity.1 Some TICs may be cancer stem cells (CSCs) because of their potential derivation from adult stem cells. TICs or CSCs have been associated with aggressive and metastatic cancers of the breast, brain, colon, and liver.2–8 TIC eradication may be critical to achieve stable remission, and even a cure, of aggressive malignancies.9 Normal stem cells and CSCs share many common cellular properties and signaling pathways (e.g. Wnt/β-catenin, transforming growth factor beta, and Notch).9 MicroRNAs (miRNAs, miRs), a novel class of small, noncoding RNAs that post-transcriptionally regulate gene expression through complementary base pairing to messenger RNAs (mRNAs),10 are functionally linked to stem cells.11 The miRNA pathway affects stem cell division, and the loss of dicer1, a critical endonuclease for generating mature miRNAs, can reduce stem cell populations and induce embryonic lethality.12, 13 Moreover, a distinct miRNA subset is specifically expressed in pluripotent embryonic stem cells but not in adult tissues.14 The role of miRNAs in CSCs, however, remains undetermined.15
Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related mortality worldwide, with observable heterogeneity and a cellular origin that has yet to be identified.16 Recent studies indicate that epithelial cell adhesion molecule (EpCAM), a membrane-associated glycoprotein encoded by tumor-associated calcium signal transducer 1 (TACSTD1), could serve as a hepatic stem/progenitor cell-specific marker.17–19 Similarly, alpha-fetoprotein (AFP) is one of the earliest markers detected in the liver bud.20 Using HCC EpCAM and AFP status together with HCC transcriptome analyses, we recently identified two distinct prognostic HCC subtypes, EpCAM+AFP+ HCC (referred to as HpSC-HCC; hepatic stem cell–like HCC) with venous metastases and poor survival and EpCAM−AFP− HCC (referred to as MH-HCC; mature hepatocyte-like HCC) with relatively good outcome, which differ significantly in their molecular profiles.21 Furthermore, EpCAM+ HCC cells isolated with an EpCAM-specific antibody by fluorescence-activated cell sorting (FACS) from AFP+ HCC cell lines or AFP+ HCC clinical specimens are hepatic TICs with stem/progenitor cell features.7 Here, we identify a conserved gene family, miR-181, that is functionally critical in the maintenance of EpCAM+AFP+ HCC cells, possibly by inhibiting hepatic cell differentiation and promoting HCC stemness through targeting the transcriptional regulators caudal type homeobox transcription factor 2 (CDX2), GATA binding protein 6 (GATA6), and the Wnt signaling inhibitor nemo-like kinase (NLK). Our results suggest that miR-181 may serve as a novel biomarker and a molecular target for hepatic TICs.
HCC samples were obtained with informed consent from patients who underwent radical resection at the Liver Cancer Institute of Fudan University. The study was approved by the Institutional Review Board of the respective institutes. Human fetal liver RNAs, obtained from Clontech Inc. (Mountain View, CA), were isolated from a pool of 63 fetal livers from spontaneously aborted male/female fetuses between 22 and 40 weeks old. Normal liver tissue samples were obtained from eight disease-free liver donors as previously described.22 Purified human hepatic stem cells, hHpSCs, from fetal liver were isolated as previously described.28 The miRNA microarray profiling data, previously described,22 are publicly available (GEO accession number: GSE6857). Other materials and methodologies are described in detail in the Supporting Text.
A Unique miRNA Signature in HpSC-HCC.
We searched for miRNAs unique to EpCAM+AFP+ HCC by interrogating miRNA expression profiles of 53 HpSC-HCC and 95 MH-HCC clinical specimens using the array dataset recently described.22 We performed multivariate nearest neighbor class prediction and found 20 unique miRNAs that could significantly predict HpSC-HCC and MH-HCC cases with 78% overall accuracy (multivariate P < 0.01) (Fig. 1A). Noticeably, multiple miR-181 transcripts, that is, miR-181a-1, miR-181a-2, miR-181b-1, miR-181b-2, miR-181c, along with several miRNAs in the miR-17-92 cluster, that is, 17, 20a, 25, 92, 93, and 106b, were up-regulated in HpSC-HCC, but down-regulated in MH-HCC (Fig. 1A). The expression of miR-181s was inversely correlated with mature hepatocyte-specific genes in the same clinical specimens (Supporting Fig. 1).
Examination of the miRBase database (http://microrna.sanger.ac.uk/) showed that six human miR-181s—miR-181a-1, miR-181a-2, miR-181b-1, miR-181b-2, miR-181c, and miR-181d—were previously identified. Strikingly, five of these were up-regulated in HpSC-HCC by our microarray study. Genome structure analyses indicated that these six miR-181s were encoded in three independent transcripts located on three separate chromosomes (Fig. 1B) (Supporting Fig. 2A). The corresponding precursors appeared to yield four sets of mature miR-181s, that is, miR-181a, miR-181b, miR-181c, and miR-181d, based on their sequence homology, where miR-181a-1 and miR-181a-2, as well as miR-181b-1 and miR-181b-2, were identical in their mature forms (Fig. 1C, Supporting Fig. 2A). Their genomic organization appeared to be evolutionarily conserved from bony fishes to mammals, suggesting an important role in development (Supporting Fig. 2B). It appeared that miR-181b and miR-181d were closely linked, whereas miR-181a and miR-181c were closely linked (Supporting Fig. 2C,D). Although sequence variations were evident among the four mature miR-181s, each miR-181 was highly conserved among species (Supporting Fig. 2E-H). Each transcript contained two miR-181 paralogs in most species, whereas the same 5′ “seed” region (nucleotides 2-8) and 3′ “variable region” were found in all four miR-181s (Fig. 1B, Supporting Fig. 2E-H). Thus, the four miR-181 family members are evolutionarily conserved among the vertebrate lineage with high homology implicating their functional redundancy.
We performed quantitative reverse transcription polymerase chain reaction (RT-PCR) to validate the microarray-based miR-181 expression, in 20 HCC cases (10 HpSC-HCC and 10 MH-HCC). We found that all four mature miR-181s were significantly elevated in HpSC-HCC than MH-HCC (Fig. 2A with a significant correlation between the RT-PCR and microarray data (Supporting Fig. 3). An examination of miR-181 expression levels in several HCC cell lines with different EpCAM and AFP status23 showed that miR-181 expression was much higher in Hep3B cells (all cells expressing EpCAM and AFP) than HuH1 or HuH7 cells (heterogeneous in EpCAM and AFP expression) (Fig. 2B,C). Furthermore, miR-181 levels were much higher in human fetal livers and isolated HpSCs from fetal liver than in adult livers or freshly isolated mature hepatocytes (Fig. 2D,E). On examination of different mouse fetal liver stages, we found that miR-181 levels were highest at E10.5, at which time the liver bud is rich in liver stem/progenitor cells, with a gradual decline in their expression between E11.5 and 15.5 to reach that of adult livers (15 weeks) (Fig. 2E). Taken together, the miR-181s are highly expressed in embryonic liver tissues, pluripotent hepatic stem/progenitor cells in human livers, and EpCAM+AFP+ HCC samples.
Elevated Levels of miR-181s in EpCAM+ HCC Cells.
FACS-isolated EpCAM+ from HuH1 or HuH7 cell populations had significantly higher miR-181 expression than EpCAM− cells (P < 0.01) (Fig. 3A-C). Consistently,7 the EpCAM+ fraction decreased in sorted EpCAM+ cells after 7 days of culture to a level similar to unsorted cells (Fig. 3A,B). In parallel, mature miR-181 levels decreased with time following incubation with sorted EpCAM+ HuH1 or HuH7 cells (Fig. 3D). We also compared the effects of an “enrichment of stem/progenitor cell culture” (knockout medium referred to as enrichment of stem/progenitor cell culture [EnSC]) that was optimized to maintain undifferentiated embryonic stem cells while hindering the proliferation of differentiated cells, to a regular culture (RC) in an attempt to enrich hepatic CSCs after prolonged culture.24 After 8 days in EnSC, the EpCAM+ fraction of HuH7 or HuH1 cells increased, but decreased when further cultured in RC medium (Fig. 3E,F). Consistently, EpCAM+ cell enrichment in HuH1 or HuH7 cells resulted in elevated miR-181 levels (Fig. 3G), which decreased after further culture in RC medium (Fig. 3H). Therefore, miR-181 expression patterns appear to correlate with an enriched EpCAM+ HCC cell population.
Augmentation of EpCAM+ HCC Cells by miR-181s.
Our findings that four miR-181 family members, encoded by different transcripts, were highly elevated in HpSC-HCCs and EpCAM+ HCC cells suggested that the miR-181s may be critical in maintaining a stem cell phenotype. To test this hypothesis, we examined the effect of miR-181 on EpCAM+ cell distribution by expressing each miR-181 in unsorted HuH1 or HuH7 cells. Individual expression of miR-181s resulted in an enrichment of EpCAM+ HuH7 cells compared with a control (Fig 4A,B). A similar and reproducible observation was made in HuH1 cells. However, the smaller induction could be attributable to low basal miR-181 levels and poor transfection efficiency (Supporting Fig. 4). The ability to specifically induce miR-181 expression by each miRVec-181 construct was verified (Supporting Fig. 5A).
Similar to mammary, hepatic, and neural stem cells as well as melanoma CSCs,25–28 EpCAM+ HuH1 cells could efficiently form spheroids, whereas EpCAM− cells failed to do so.7 Inhibition of endogenous miR-181s by various anti-miRNA miR-181 inhibitors resulted in a significant reduction of spheroid formation in HuH1 cells (Fig. 4D). Meanwhile, forced expression of miR-181s led to a modest induction of spheroid formation (Fig. 4D). The specificity of each miR-181 inhibitor was also validated (Supporting Fig. 5B). We also found that the miR-181a and miR-181b inhibitors could inhibit the tumorigenicity of EpCAM+ HuH1 cells (Fig. 4E and data not shown).
The “stemness” of EpCAM+ HCC cells was further supported by their association with activated Wnt/β-catenin signaling, measured by target gene induction (CCND1 and TACSTD1), as well as the reduction of genes specific for differentiated hepatocytes (uridine diphosphate-glucuronosyltransferase-2B7 [UGT2B7] and cytochrome P450 3A4 [CYP3A4]).7, 21, 23 If miR-181 was critical for maintaining an undifferentiated hepatic CSC phenotype, then forced miR-181expression would lead to Wnt target up-regulation, whereas inactivation of endogenous miR-181 would lead to up-regulation of differentiation markers. Consistently, the mRNA level of UGT2B7 and CYP3A4 was down-regulated whereas CCND1 and TACSTD1 were up-regulated after ectopic miR-181 expression (Fig. 4F). In contrast, inhibition of endogenous miR-181s resulted in UGT2B7 and CYP3A4 induction with CCND1 and TACSTD1 reduction (Fig. 4G). CDX2, a positive regulator of hepatocyte differentiation, had a similar expression trend as UGT2B7 and CYP3A4 (Fig. 4). Wnt/β-catenin signaling activation by miR-181expression was verified by the TOP-FLASH/FOP-FLASH reporter system (a luciferase reporter system containing wild-type or mutant beta-catenin responsive elements) (Supporting Fig. 6). Furthermore, we used the EnSC-RC strategy to alter the differentiation status of HCC cells. HuH7/HuH1 cells were grown in either RC (8D RC) or EnSC (8D EnSC) for 8 days followed by another 8 days in RC (EnSC-RC). Consistently, we found that miR-181 levels were negatively correlated with the expression of hepatocyte differentiation markers but positively correlated with β-catenin–associated genes (Supporting Fig. 7).
CDX2, GATA6, and NLK Are miR-181 Target Genes.
In-silico screening with TargetScan29 and PicTar30 programs showed potential miR-181 target genes. Among them, CDX2 and GATA6 were previously implicated as regulators of hepatocyte gene expression31, 32 and NLK as a negative regulator of Wnt/β-catenin signaling.33 Sequence analyses showed that the 3′ untranslated region (3′-UTR) of CDX2, GATA6, and NLK mRNA contain putative sites that are partially complementary to miR-181s (Fig. 5A) and evolutionarily conserved among human, mouse, and rat (Supporting Fig. 8A-C). To experimentally validate these targets, we measured their levels upon alteration of miR-181s (Supporting Fig. 9). Western blotting analysis showed that blockage of endogenous miR-181s in Hep3B cells, which highly express miR-181s, resulted in an induction of CDX2, GATA6, and NLK (Fig. 5B). In contrast, forced expression of miR-181s in HCC cells resulted in a reduction of CDX2, GATA6, and NLK (Fig 5C). To further determine whether CDX2, GATA6, and NLK were bona fide targets of miR-181–mediated small interfering RNA (siRNA) silencing, the miR-181 binding sites in the 3′-UTR of these three genes were cloned into a luciferase reporter. We found that forced expression of miR-181s resulted in decreased luciferase activity when the wild-type sequences were present (Fig. 5D). This effect was significantly reduced when the corresponding miR-181 binding sites (five nucleotides within the complementary seed sequences) were mutated (Fig. 5D). Thus, miR-181s directly target CDX2, GATA6, and NLK, and a single site within each gene is sufficient for miR-181–mediated gene silencing.
To further test whether miR-181s may maintain HCC stemness by inhibiting CDX2, GATA6, or NLK, we used RNA interference technology and successfully knocked-down GATA6 and NLK expression (Fig. 5E,F). We were unable to identify a functional siRNA specific to CDX2, and thus its functional role in HCC could not be assessed at this time. Consistently, GATA6 or NLK knock-down in HCC cells resulted in a reduction of both adult hepatocyte-specific genes UGT2B7 and CYP3A4 (Fig. 5E,F). Moreover, similar to miR-181 overexpression experiments, silencing of GATA6 and NLK resulted in a modest induction of the EpCAM+ cell fraction (Supporting Fig. 10), suggesting that GATA6 and NLK may be directly involved in hepatocyte differentiation.
Similar to observations by Lee et al.,34 we recently used global mRNA profiling of HCC clinical specimens to identify an EpCAM+ AFP+ HCC subtype resembling hepatic stem/progenitor cells.21 Furthermore, we demonstrated that EpCAM+ HCC cells from AFP+ tumors are a subpopulation of undifferentiated hepatic TICs with normal HpSC-like phenotypes.7 Using miRNA expression profiling, we recently identified unique miRNAs associated with HCC metastases and patient survival.22 With this technology, we have now identified a highly conserved miR-181 family that may contribute to the maintenance of EpCAM+ hepatic TIC and EpCAM+ normal HpSC activities. We presented the following evidence: First, all conserved mature miR-181 members were highly expressed in HpSC-HCCs and isolated EpCAM+ HCC cells. Second, human fetal livers and early stage mouse embryonic livers, rich in HpSCs, had a high miR-181 level compared with adult livers. Consistently, isolated HpSCs expressed high miR-181 levels. Third, miR-181 levels correlated with HCC cell differentiation. Fourth, forced miR-181 expression enriched EpCAM+ HCC cells with stem cell properties, whereas miR-181 blockage reduced EpCAM+ HCC cells and induction of hepatic differentiation. Fifth, miR-181s could directly target CDX2, GATA6, and NLK, known regulators of hepatic cell differentiation. Collectively, our results suggest that miR-181s are important components of human EpCAM+ hepatic CSCs and may maintain HCC “stemness” by inactivating critical cellular transcriptional regulators that induce hepatocyte differentiation.
In addition to EpCAM, CD133 (PROM1) and CD90 (THY1) have also been proposed as hepatic CSC markers.6, 8 We compared EpCAM+, CD133+, or CD90+ HCC cells and found that although EpCAM and CD133 could be detected in HuH1 and HuH7 cells, these markers appeared to overlap in HuH7 but not in HuH1 cells7 (data not shown). Noticeably, EpCAM+ HuH1 cells showed marked tumor-initiating capacity compared with CD133+ HuH1 cells.7 Our current results indicate that miR-181 is highly expressed in EpCAM+ or CD133+ cells when compared with EpCAM− or CD133− cells isolated from HuH1 and HuH7 cells (Supporting Fig. 11). Noticeably, miR-181 expression is much higher in EpCAM+CD133+ cells than double-negative or single-positive cells. However, CD90 is undetectable in these cells. Taken together, our results indicate that EpCAM is a better marker than CD133 to define HCC CSCs, and miR-181 is more closely associated with EpCAM+ cells than CD133+ cells, further emphasizing the role of miR-181 in HCC stemness.
The roles of miR-181 in cellular differentiation have recently been explored, with evidence as both positive and negative regulators of this phenotype, but their mechanisms remain unclear. In murine muscle, miR-181 is up-regulated during fiber regeneration, returning to basal levels at the end of the regeneration, and is poorly expressed in terminal differentiated muscle.35 The authors suggest that miR-181 may be involved in establishing the differentiated phenotype but probably not its maintenance.35 However, during hematopoiesis, it appears that miR-181 has opposing roles. In B-lymphocytes, miR-181a is up-regulated in differentiated cells when compared with undifferentiated progenitor cells and acts to positively regulate B-cell differentiation.36 However, during megakaryocytic differentiation, miR-181b and miR-181c are down-regulated.37 In addition, increased miR-181a and miR-181b levels are associated with erythroid differentiation,38, 39 whereas miR-181 expression gradually decreases after erythroid terminal differentiation.39 In our study, we found that all members of miR-181 are up-regulated in hepatic stem cell populations and HCC cells with progenitor cell features, implying that miR-181 functions in maintaining an undifferentiated state of hepatic progenitor cells. The apparent opposing roles of miR-181 in cellular differentiation may be attributable to repression of different target genes with tissue/cell type specificity. Of note, skeletal muscle and blood cells are derived from mesoderm, whereas liver is derived from endoderm during embryonic development.
Several signaling pathways are involved in regulating stem cell functions (for example, Wnt, transforming growth factor-beta, Notch). MicroRNAs are also involved in this process, possibly by regulating cell cycle progression.12 Our results suggest that miR-181s may be activators of hepatic progenitor cells and HCCs through alterations in at least two cellular signaling pathways (Supporting Fig. 12). One pathway is the blockage of HCC cell differentiation through inhibition of GATA6 or CDX2, two transcriptional activators regulating hepatocyte differentiation.31, 32 The second may be to activate the Wnt/β-catenin pathway by down-regulating NLK, a Wnt/β-catenin signaling inhibitor. Our results indicated that miR-181, EpCAM+ cell populations, and Wnt/β-catein signaling were elevated in HuH1 or HuH7 cells after a prolonged EnSC culture, whereas a down-regulation of differentiated genes was observed. Thus, miR-181s may bolster progenitor cell functions by affecting both pathways to ensure hepatic “stemness” (Supporting Fig. 12).
Our results indicate that all four independently encoded miR-181 family members are elevated in hepatic stem cells and HCC cells with features of hepatic CSCs and are similarly activated to maintain “stemness.” This is intriguing and implies that a common cellular signaling pathway may converge to activate miR-181s. One candidate “master key” activator is the Wnt signaling pathway, because it is involved in virtually every aspect of embryonic development and also controls homeostatic self-renewal of adult stem cells.40 Consistently, conditioned media derived from WNT10B-overexpressing cells could enhance the levels of all four mature miR-181s in HCC cells (data not shown). Examination of the genome database revealed several classical TCF4 binding sites located within the three putative promoter regions of miR-181s on chromosomes 1, 9, and 19 (data not shown). Thus, it is plausible that Wnt/β-catenin may induce miR-181s to regulate hepatic stem cells during early-stage embryogenesis and to ensure a positive feedback mechanism to maintain stemness. These could be achieved by inactivating Wnt/β-catenin inhibitors such as NLK and by blocking genes such as GATA6 or CDX2 to prevent hepatic cell differentiation. Whether miR-181s are transcriptional targets of Wnt/β-catenin signaling is currently being investigated in our laboratory using the same strategy previously described.23 A future challenge is to explore miR-181 as a molecular target for eradicating hepatic TICs.
The authors thank Prof. Reuven Agami for the miRVec-control and 181s plasmids, and Barbara Taylor for expertise in cell sorting.