These authors contributed equally to this work.
Targeting cadherin-17 inactivates Wnt signaling and inhibits tumor growth in liver carcinoma†
Article first published online: 24 JUN 2009
Copyright © 2009 American Association for the Study of Liver Diseases
Volume 50, Issue 5, pages 1453–1463, November 2009
How to Cite
Liu, L. X., Lee, N. P., Chan, V. W., Xue, W., Zender, L., Zhang, C., Mao, M., Dai, H., Wang, X. L., Xu, M. Z., Lee, T. K., Ng, I. O., Chen, Y., Kung, H.-f., Lowe, S. W., Poon, R. T.P., Wang, J. H. and Luk, J. M. (2009), Targeting cadherin-17 inactivates Wnt signaling and inhibits tumor growth in liver carcinoma. Hepatology, 50: 1453–1463. doi: 10.1002/hep.23143
Potential conflict of interest: Nothing to report.
- Issue published online: 29 OCT 2009
- Article first published online: 24 JUN 2009
- Accepted manuscript online: 24 JUN 2009 12:00AM EST
- Manuscript Accepted: 18 JUN 2009
- Manuscript Received: 21 MAY 2009
- General Research Fund from the Research Grants Council of Hong Kong. Grant Number: HKU771607M
- Collaborative Research Fund. Grant Number: HKU1/06C
- National Cancer Institute. Grant Number: CA13106
Hepatocellular carcinoma (HCC) is a lethal malignancy for which there are no effective therapies. To develop rational therapeutic approaches for treating this disease, we are performing proof-of-principle studies targeting molecules crucial for the development of HCC. Here, we show that cadherin-17 (CDH17) adhesion molecule is up-regulated in human liver cancers and can transform premalignant liver progenitor cells to produce liver carcinomas in mice. RNA interference–mediated knockdown of CDH17 inhibited proliferation of both primary and highly metastatic HCC cell lines in vitro and in vivo. The antitumor mechanisms underlying CDH17 inhibition involve inactivation of Wnt signaling, because growth inhibition and cell death were accompanied by relocalization of β-catenin to the cytoplasm and a concomitant reduction in cyclin D1 and an increase in retinoblastoma. Conclusion: Our results identify CDH17 as a novel oncogene in HCC and suggest that CDH17 is a biomarker and attractive therapeutic target for this aggressive malignancy. (HEPATOLOGY 2009.)
Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal malignancies. It is the fifth most common cancer and ranks as the third leading cause of cancer-related deaths worldwide.1 The number of incident cases is over 600,000 per year, which is almost the same as the number of deaths, owing to the difficulty in early detection and high postsurgical recurrence rate. The prognosis of HCC is extremely poor, and only ≈5% of patients survive more than 5 years.2 Patients afflicted with HCC are often asymptomatic, and the lack of sensitive and reliable biomarkers for early detection of HCC and cancer surveillance in at-risk populations (hepatitis B or C virus carriers and those with cirrhosis) means that diagnosis normally occurs late, when surgical intervention is not an option.3 Today, there are no effective drugs for curing liver cancer.
Using integrative genomic and proteomic approaches in mouse models,4–6 we have begun to identify novel oncogenes and tumor suppressors in liver cancer. Our previous studies using clinical cohorts identified a cell surface adhesion molecule, cadherin-17 (CDH17), also known as liver-intestine cadherin, as a potential disease marker for HCC.7 It consists of seven cadherin-like ectodomains and a short cytoplasmic tail of 24 amino acid residues.8 Expression of CDH17 is restricted to the colon, intestine, and pancreas in humans; it is not found in the healthy adult liver and stomach.7, 9–12 It plays an important role during embryonic gastrointestinal development and also functions as a peptide transporter.13, 14 Most, if not all, cadherin molecules interact with the cytosolic β-catenin network and thereby regulate the Wnt signaling pathway, but there are no published reports that CDH17 does so.
Increased CDH17 expression has been reported in liver and stomach cancer.7, 9, 15, 16 In our earlier studies, we identified an isoform that lacks exon 7 and correlates with poor prognostic outcomes in HCC patients.11 Furthermore, this specific CDH17 haplotype is associated with increased risk of HCC in Chinese subjects.17 Despite these significant clinical findings, the molecular pathogenesis of CDH17 remains unknown, and its tumorigenic role in HCC has not yet been confirmed. Here, we aimed to dissect the oncogenic signaling mechanisms of CDH17 in the HCC context and evaluated the feasibility of targeting CDH17 using RNA interference (RNAi) as a potential therapeutic approach for HCC.
Materials and Methods
HCC Cell Lines and CDH17 Monoclonal Antibodies.
Hepatoma cell lines PLC/PRF/5, HuH-7, Hep3B, and HepG2 were obtained from the American Type Culture Collection (Manassas, VA) or Japanese Cancer Research Resources Bank (Tokyo, Japan). Primary and metastatic HCC cell lines H2-P, H2-M,18 MHCC97H, and MHCC97L19 and the immortalized human hepatocyte cell line MIHA20 were obtained and used as described. A mouse monoclonal anti-CDH17 antibody (Lic-3, IgG2a) was established using the recombinant amino-terminal domain 1-2 (amino acid residues: 30–244) of human CDH17 as an antigen according to previously reported procedures.21
MicroRNA-Based CDH17 Short Hairpin RNA (CDH17 shRNAmir).
The procedure for suppressing the expression of CDH17 using a lentiviral-mediated approach was performed as described.22 Several target regions spanning exon 3 (nt 244–262), 4 (nt 414–432), 12 (nt 1558–1576), and 18 (nt 2506–2524) of human CDH17 (NM_004063.2) were chosen. These target sequences were cloned into an Expression Arrest pSHAG-MAGIC2 (pSM2) vector (Open Biosystems, Huntsville, AL). Testing showed that the construct targeting exon 3 yielded the best suppression efficiency (data not shown); therefore, this construct was chosen. The construct was cloned into a LUNIG vector,22 which was then transfected into 293T/17 (American Type Culture Collection). The lentivirus was used to transduce MHCC97H cells, and the stable transfectants were selected using G418 sulfate for 2 weeks. Two controls were included, MHCC97H cells that received either no treatment (97H) or a nontargeted RNAi vector (Mock). Construction of the CDH17 short hairpin RNA (shRNA) vector is described in the Supporting Methods.
Luciferase Reporter Assay.
Tumor cells were seeded onto 24-well plates and cultured for 24 hours. TOPFlash (T cell factor reporter plasmid) luciferase construct (Upstate-Millipore, Billerica, MA) and pRL-TK-Luc vector (Promega) for normalization were used to cotransfect cells using FuGENE-6 reagent (Roche). After 48 hours, cells were lysed and assayed for luciferase activities using the Dual-Luciferase Reporter Assay System (Promega).
Cell Cycle and Apoptotic Assays.
Cells were fixed in 1% paraformaldehyde and stained with 0.5 mL PI/RNase Staining Buffer (BD Biosciences, San Jose, CA) for 15 minutes at room temperature. Stained cells were then analyzed using flow cytometry (Beckman Coulter Cytomics FC500). Apoptotic cells were detected using the ApopTag Red In Situ Apoptosis Detection Kit (Chemicon-Millipore, Billerica, MA) as described.23
In Vitro Assays to Assess Tumor Phenotypes.
The experimental procedures for cell proliferation, colony formation, cell adhesion, wound healing, cell invasion, and cell migration are described in the Supporting Methods.
In Vivo Mouse Models of HCC.
Tumor xenograft and genetically defined mouse models for HCC are described in the Supporting Methods. For the liver tumor–bearing mouse model, subcutaneous tumors were induced in nude mice using MHCC97H cells as mentioned. One week later, mice with inoculated tumors were subjected to different experimental treatments: recombinant adeno-associated virus expressing TP53 (rAAV-TP53), lentivirus-mediated suppression using CDH17 shRNAmir (CDH17 shRNAmir), or a combined treatment of both rAAV-TP53 and CDH17 shRNAmir. Five mice were used in each group. For each group, mice received 100 μL therapeutic agents by way of intratumoral injections. For the treatment groups, 1 × 109 copies of the corresponding virus for each gene therapy were injected twice weekly for 2 weeks. For the 97H control and Mock groups, intratumoral injections of 1 × Tris-buffered saline and 1 × 109 copies of lentivirus with the nontargeted RNAi vector were performed. Tumor formation and growth were monitored daily. To monitor virus infection in each group, the expression levels of genes associated with virus infection and neutrophilic granulocyte-related inflammation, such as 2'-5'-oligoadenylate synthetase 1, interleukin-8, and protein-kinase, interferon-inducible double-stranded RNA dependent inhibitor, repressor of (P58 repressor) (PRKRIR), in the subcutaneous tumors were assessed by way of quantitative polymerase chain reaction.
Statistical analyses were performed using PRISM version 4.0 software (GraphPad, San Diego, CA). A Student t test and χ2 test were used for calculating the significance between different groups. Values are expressed as the mean ± standard error of the mean. A P value <0.05 was considered significant. Copy number variation (CNV) was measured using the Ilumina platform that contains 650,000 single-nucleotide polymorphisms, and the results are expressed as the logR ratio (that is, the intensity ratio of the studied sample to a number of reference samples). The genomic locations of CDH17 single-nucleotide polymorphism probes were identified. CNV of CDH17 was obtained by averaging the logR ratio from those single-nucleotide polymorphism probes. The same procedure was used to study the CNV of all ≈40,000 transcripts. In addition to the continuous CNV data, the hidden Markov model (HMM) was applied to determine if there was copy number gain or loss.24
Tumorigenic and Metastatic Properties of CDH17 in HCC.
We used a genetically defined mouse model in which the CDH17 gene was subcloned into the murine stem cell virus SV40–green fluorescent protein recipient vector, then constitutively induced in immortalized premalignant liver progenitor cells.4, 6, 25 The CDH17-overexpressing cells gave rise to subcutaneous tumors by 18 days after implantation in mice, whereas the vector-carrying cells did not (Fig. 1A). This is the first direct demonstration of tumorigenesis by CDH17 in the HCC context.
Next, we measured the CDH17 messenger RNA level in a panel of human HCC cell lines with different metastatic potential by way of quantitative polymerase chain reaction analysis. The control cell line MIHA is an immortalized normal human hepatocyte that expressed very little or undetectable CDH17. Primary HCC cell lines expressed some CDH17 transcript, whereas strong expression was seen in their metastatic counterparts (e.g. MHCC97H, MHCC97L, and H2-M) (Fig. 1B).
These cell line data were supported by results from 46 pairs of tumor and adjacent liver tissues from HCC patients who received curative surgery. Overexpression of CDH17 was strongly associated with advanced tumor stages (pathological tumor–node–metastasis III and IV) (P = 0.022) and tumor venous invasion (P = 0.022). No significant correlation was found for other clinico-pathologic parameters (Table 1). Most strikingly, copy number variation analysis revealed genomic amplification of the CDH17 gene in the tumor compared with adjacent nontumor tissues in 49% of HCC cases that were analyzed (n = 231) (Fig. 1C).
|Variables||Frequency (%)||CDH17 Overexpression||P Value|
|Tumor size, cm||0.786|
|Alpha fetoprotein, ng/mL||0.226|
|Hepatitis B surface antigen||0.126|
|Pathological tumor–node–metastasis stage||0.022†|
|Early (I, II)||18 (39.1)||12||6|
|Late (III, IV)||28 (60.9)||9||19|
Taken together, these results suggest that CDH17 is a candidate target for intervening in the initiation and metastasis of HCC. To test this principle, we used primary and metastatic HCC cell lines HuH-7, PLC, and MHCC97H, which express CDH17 most strongly and have high metastatic properties, to examine the clinical potential of reducing the expression of CDH17 RNA.
Targeting CDH17 Expression Alleviated Malignant Phenotypes in HCC Cells.
shRNA in pcDNA and lentiviral vectors was employed to test the knockdown efficiency at different sites in the CDH17 open-reading frame. The target sequences in exon 3 and exon 5 yielded >50% reduction in both messenger RNA and protein levels (Supporting Fig. 1 and 2A). We then assessed the tumorigenic and metastatic properties (cell proliferation, colony formation, adhesion, invasion, and apoptosis) of the HCC cells either transduced with CDH17 shRNAmir (CDH17 shRNAmir cells) or transfected with CDH17 shRNA (CDH17 shRNA cells) compared with the control cells—the parental line transfected with vector (vector) or scrambled shRNA (Mock). CDH17 shRNAmir MHCC97H cells in culture showed a reduction in cell proliferation (by 49.6% at 6 days), substrate-adhering ability (by 62.7% at 45 minutes), cell migration ability (by 90.2% at 12 hours), colony-forming ability (by 93.1% at 14 days), and cell invasion ability (by 88.3% at 36 hours) (Fig. 2A-E). We also observed an increase in the number of apoptotic cells and in the amount of cleaved caspase-3 fragment (Fig. 2F). More CDH17 shRNAmir cells were arrested in G0/G1 phase, with fewer cells in S phase, whereas there was no significant alteration in cell numbers in G2/M-phase (Fig. 2G). A similar reduction in tumorigenicity was observed when we employed the shRNA knockdown method (Supporting Fig. 2B-E). The antitumor effect of CDH17 shRNA was also seen in two other primary HCC cell lines: PLC and HuH-7. Knockdown of CDH17 expression remarkably impaired their migration activities (Supporting Fig. 3). These results provide strong evidence that in vitro delivery of CDH17 shRNA into hepatic carcinoma cells could reduce the tumorigenicity of HCC.
CDH17 as an In Vivo Target for Liver Cancer Therapy.
We further examined the effect of CDH17 shRNA in liver tumors in vivo using a xenograft mouse model. Our initial observation showed that the tumorigenic potential of the primary HCC line PLC was significantly hampered when the CDH17 gene was silenced. We then moved to test the CDH17 knockdown effect on MHCC97H cells, which expresses the highest transcript level of CDH17. At 8 weeks after tumor cell inoculation, large tumors were seen in the control groups, but the tumor volume was still minimal in those mice transplanted with the CDH17 shRNAmir (Fig. 3A) or CDH17 shRNA (Fig. 3B) MHCC97H cells. Of great interest, all control animals (Mock group) developed metastasis in the lungs as shown by the presence of green fluorescent protein–positive tumor cells at 8 weeks regardless of whether the MHCC97H tumor cells were transplanted subcutaneously or injected systemically. By contrast, no lung metastasis was observed in animals from the CDH17 shRNAmir treatment group (Fig. 4). Thus, knockdown of CDH17 not only reduced tumor growth but also diminished the metastatic potential of hepatic carcinoma. This has a great clinical implication, because most of the HCC patients die from tumor recurrence due to intrahepatic or extrahepatic metastasis.
We then investigated whether in vivo delivery of CDH17 shRNAmir could impede the growth of an established tumor xenograft derived from parental MHCC97H cells in nude mice. At 1 week after tumor inoculation, when the tumor had reached approximately 0.5 cm in diameter, the animals were injected at the tumor site with placebo (Tris-buffered saline), mock reagents, rAAV-TP53, CDH17 shRNAmir, or combined rAAV-TP53 and CDH17 shRNAmir regimen. Mice in the control groups (97H/placebo and mock) grew large, solid, vascularized tumors, whereas the groups treated with rAAV-TP53 or CDH17 shRNAmir developed much smaller subcutaneous tumors. A further reduction in tumor size was observed when CDH17 shRNAmir and rAAV-TP53 were used in combination (Fig. 5; Supporting Fig. 4). No extensive coverage of blood vessels was observed in the tumors. This shows that knockdown of CDH17 is an effective means of shrinking tumor growth of HCC in a tumor-bearing mouse model. On the other hand, no distortion or rupture of tissue architecture was noticed in other organs, including the brain, heart, kidney, liver, lungs, and stomach of the animals treated as above (Supporting Fig. 5A). There was no significant difference in the expression levels of three genes (2'-5'-oligoadenylate synthetase 1, interleukin-8, protein-kinase, interferon-inducible double-stranded RNA–dependent inhibitor, repressor of [P58 repressor]) associated with virus infection and neutrophilic granulocyte-related inflammation due to the use of lentiviral vector for delivery in the subcutaneous tumors among the five experimental groups (Supporting Fig. 5B).
Knockdown of CDH17 Inactivated Wnt/Catenin Signaling Pathway.
Our preliminary observation indicates CDH17-mediated oncogenic signaling associated with the Wnt/catenin pathway. In a TOPFlash reporter luciferase assay (Fig. 6A), knockdown of CDH17 by shRNA in MHCC97H cells significantly decreased the strength of TCF/LEF signals compared with the vector and mock controls. Furthermore, as shown by way of immunoblot analysis of CDH17 shRNAmir cells, we detected a significant reduction of both the total and nuclear β-catenin protein, but no change in the p53 level, when compared with the 97H and Mock controls (Fig. 6B). Then we evaluated the involvement of other Wnt pathway signaling components. Knockdown of CDH17 resulted in down-regulation of the phosphorylated glycogen synthase kinase (GSK)-3β, β-catenin, and cyclin D1 proteins, but the tumor suppressor retinoblastoma (Rb) level was increased compared with the rAAV-TP53, 97H, and mock controls (Fig. 6C). In addition, rAAV-TP53 enhanced Bax expression but suppressed Bcl-XL expression, whereas CDH17 shRNAmir did not. Nevertheless, both rAAV-TP53 and CDH17 shRNAmir enhanced caspase-3 cleavage, and there was an additive effect on the pro-apoptotic mechanism (caspase 3, Bax, and Bcl-XL levels) in the combined shRNAmir & rAAV-TP53 regimen.
To confirm the effect of CDH17 knockdown on the Wnt pathway in vivo, we dissected MHCC97H xenograft tumors from the animals and conducted immuno-histochemical analyses on the tissues. Consistent with the above findings, we observed relocalization of β-catenin to the cytoplasm, with concomitant reduction in cyclin D1 and increase in Rb in HCC tumors treated with CDH17 shRNA (Fig. 6D).
The CDH17 oncogene is an attractive therapeutic target for HCC, because it is highly expressed in tumor tissues but not in the normal liver. Over 80% of HCCs are CDH17-positive, and half of these patients reveal genomic copy gain of this gene. Our previous work first demonstrated the overexpression and prognostic significance of CDH17 associated with poor overall survival and disease-free survival times in HCC patients.7, 11 A high CDH17 level has also been reported in a stem-like progenitor HCC subpopulation driven by c-Met, which exhibited high metastasis of liver tumors.26 In the present study, we provide new evidence correlating high expression of CDH17 and HCC tumorigenesis, showing the oncogenic properties of CDH17 in a genetically defined mouse model. The expression of CDH17 in HCC cell lines is positively correlated with metastatic potential, and in HCC patients a high level of CDH17 clinically correlates with tumor venous invasion, a strong risk factor for cancer metastasis. Furthermore, HCC patients at advanced stages usually have high expression of CDH17 in the tumors, which is often associated with aggressive and malignant phenotypes of the cancer.
Today, there is accumulating evidence that CDH17 is a disease marker for other gastrointestinal malignancies.27, 28 For instance, high expression of CDH17 is associated with high metastatic potential, positive lymph node metastasis and short overall survival in gastric cancer patients.16, 29 However, down-regulation of CDH17 is found in human colorectal cancers and its reduced expression indicates the presence of lymph node metastasis.12 The difference in the expression pattern of CDH17 between HCC and colon cancer are perhaps context-dependent. For instance, CDH17 may support normal physiology and/or epithelial integrity in colon tissue, while aberrant expression of CDH17 may drive oncogenesis in liver and gastric cancers. Furthermore, we identify different isoforms of CDH17 in HCC, but there is no information on whether the wild-type or splice variants are present in the colon.
The present study demonstrates the use of shRNA vectors to knock down CDH17 expression in a highly tumorigenic and metastatic HCC cell line, MHCC97H, rendering the cells less tumorigenic in vitro in terms of cell proliferation, cell adhesion, cell migration, cell invasion, and anchorage-independent colony formation. This was confirmed in vivo when constitutive knockdown of CDH17 expression in MHCC97H cells markedly impaired growth of a tumor xenograft. In tumor-bearing mice, local delivery of lentiviral-based CDH17 shRNAmir vector inhibited tumor growth, and further reduction was seen after combination with rAAV-TP53 treatment, showing an additive antitumor effect on HCC.
Although we focus on targeting CDH17 as a novel therapeutic approach in HCC, deficiency or loss of tumor suppressor p53 is common in various cancers, including HCC, for which restoration of the TP53 gene may induce tumor apoptosis.30, 31 Furthermore, a 249(ser) TP53 mutation has been found in ≈50% of Asian patients associated with poor clinical outcomes.32 This mutation is also found in the metastatic MHCC97H cell line used in this study (data not shown). Our data revealed no direct link between CDH17 and TP53, because there was no obvious change in the level or localization of p53 when CDH17 expression was suppressed, or vice versa. They may be involved in distinct pathways during the development and progression of HCC, although the loss of p53 and deregulation of the Wnt pathway (as reflected by β-catenin nuclear localization) frequently occur in HCC. Therefore, targeting these two molecules could be used to develop a personalized treatment strategy for those patients with high CDH17 and deficient p53 in their tumors, particularly at the late advanced stages for which there are no effective treatments.
Altered expression levels of phosphorylated GSK, cyclin D1, and Rb were found in tumor xenografts with reduced CDH17 expression. GSK is a protein kinase associated with the destruction complex of β-catenin, consisting of at least adenomatous polyposis coli and axin, whereas cyclin D1 is a Wnt-responsive target gene. Loss or down-regulation of Rb expression in HCC was shown to be associated with poor cell differentiation and metastasis.33 Herein, CDH17 knockdown in HCC decreased GSK-3β phosphorylation, accompanied by a concomitant reduction of cyclin D1 and induction of Rb. Moreover, CDH17 knockdown in MHCC97H cells led to cytoplasmic sequestration (or nuclear extravasation) and potentially degradation of β-catenin, which subsequently reduced TCF/LEF transactivation activity. In the normal hepatocyte cell line MIHA, our studies revealed that overexpression of CDH17 induced epithelial-to-mesenchymal transition and enhanced cyclooxygenase 2 activities (unpublished data). In agreement with our observations, prostaglandin 2 was recently shown to regulate the Wnt signaling in vivo.34 Furthermore, a recent report showed a trans-interaction between E-cadherin and CDH17 in enterocytes during development of the intestinal epithelium,35, 36 suggesting that CDH17 might intersect with the Wnt pathway through its coordination with E-cadherin and/or associated partners. Together, these observations point to a potential oncogenic role for CDH17 in HCC, eventually leading to stabilization, nuclear shuffling, and localization of β-catenin (Fig. 7). In the proposed model, we hypothesize that CDH17 may (1) destabilize the cadherin–catenin network through interaction with E-cadherin, unleashing the β-catenin molecule from cytoplasmic membrane, (2) inactivate the GSK-3β activities and prevent cytosolic β-catenin from degradation, and (3) up-regulate the cyclooxygenase 2 pathway, thereby activating Wnt signaling.
In conclusion, the present study presents a novel therapeutic approach to liver cancer therapy by targeting CDH17 alone or in combination with p53, and suggests beneficial effects of targeting multiple pathways. This study also provides the first direct evidence of CDH17 as a bona fide oncogene, with tumorigenic properties giving rise to aggressive phenotypes of HCC.
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Additional Supporting Information may be found in the online version of this article.
|HEP_23143_sm_suppfig1.tif||749K||Supplemental Figure 1.|
|HEP_23143_sm_suppfig2.tif||181K||Supplemental Figure 2.|
|HEP_23143_sm_suppfig3.tif||620K||Supplemental Figure 3.|
|HEP_23143_sm_suppfig4.tif||788K||Supplemental Figure 4.|
|HEP_23143_sm_suppfig5.tif||4261K||Supplemental Figure 5.|
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.