Cancer Gene Therapy (2011) 18, 399–406 r 2011 Nature America, Inc. All rights reserved 0929-1903/11 www.nature.com/cgt

ORIGINAL ARTICLE The tumor suppressor function of LECT2 in human makes it a potential therapeutic target HT Ong1, PK Tan1, SM Wang1, DT Hian Low1, LL PJ Ooi2 and KM Hui1 1Bek Chai Heah Laboratory of Cancer Genomics, Division of Cellular and Molecular Research, Humphrey Oei Institute of Cancer Research, Singapore, Singapore and 2Division of Surgical Oncology, National Cancer Centre, Singapore, Singapore

Vascular invasion is one of the clinicopathologic features that are associated with early recurrence of human hepatocellular carcinoma (HCC). In this study, we have employed high-density Affymetrix oligonucleotide GeneChips (Affymetrix, Santa Clara, CA) to compare the expression profiles of HCC with and without vascular invasion. Data mining of the database established revealed that leukocyte cell-derived chemotaxin-2 (LECT2) transcripts were downregulated in HCC patients with vascular invasion. Expression of LECT2 in human HCC biopsies was significantly reduced (Po0.0001, fold change ¼À7.2) when compared with non-tumorous adjacent liver tissues. The reduction of LECT2 expression was significantly correlated with the early recurrent and poor prognosis of the patient (P ¼ 0.024). To validate the ability of LECT2 to repress the growth of HCC, an adenoviral vector encoding the secreted human LECT2 (AdLECT2) was introduced into the human HCC cell lines Hep3B and PLC/PRF/5, which do not express endogenous LECT2. Over-expression of LECT2 resulted in the significant inhibition of in vitro migration and invasion of the AdLECT2-transfected HCC cells. Additionally, over-expression of AdLECT2 in subcutaneous Hep3B tumor xenografts in athymic nude mice resulted in significant inhibition of tumor growth (Po0.05). In summary, our data not only demonstrated that LECT2 is a candidate prognostic marker of human HCC, but also that therapeutic strategies targeting LECT2 expression is a promising therapy for human HCC. Cancer Gene Therapy (2011) 18, 399–406; doi:10.1038/cgt.2011.5; published online 11 March 2011 Keywords: adenovirus-mediated gene therapy; human hepatocellular carcinoma (HCC); leukocyte cell-derived chemotaxin-2 (LECT2); tumor invasion and metastasis

6–10 Introduction survival. Data-mining studies of a gene expression database that we have established earlier for Hepatitis B 9 Hepatocellular carcinoma (HCC) is the most common virus þ HCC patients revealed that the transcripts of primary malignancy of liver and exhibits a broad the leukocyte cell-derived chemotaxin-2 (LECT2) gene spectrum of clinical behaviors, with considerable mole- were specifically downregulated in the subgroup of HCC cular heterogeneity in tumor development and progres- patients presented with vascular invasion at diagnosis. sion.1 HCC is a difficult disease to manage clinically, LECT2 is a multi-functional that involves in and surgical resection and liver transplantation provide , cell proliferation, , immuno- 11–17 the best options for cure in selected patients.2,3 However, modulation and carcinogenesis. It was originally the long-term clinical outcome for HCC following tumor identified as a novel -chemotactic protein by 17 resection and liver transplantation remains unsatisfactory Yamagoe et al. in 1996. LECT2 is a 16-kDa protein that because of the high tumor-recurrence rate.4,5 Although consists of 133 amino acids and three intra-molecular 18,19 various clinicopathologic variables have been described disulfide bonds. The human LECT2 gene consists of 19 as predictive of post-transplant recurrence, the exact four and three . It has been mapped to molecular mechanism(s) underlying HCC recurrence are 5q31.1-q32, a cluster harboring several genes still poorly understood. Vascular invasion is one of encoding immuno-regulatory cytokines. These include the clinicopathologic features reported to be strongly (IL)-3, IL-4, granulocyte macrophage-colony 19 associated with the duration of tumor recurrence and stimulating factor and IL-5. Subsequently, LECT2 has been shown to be synthesized by human hepatocytes20,21 and most hepatocytes show diffuse immune-staining for Correspondence: Dr KM Hui, Division of Cellular and Molecular LECT2 16,20 Research, National Cancer Centre Singapore, 11 Hospital Drive, within the cytoplasm. Singapore, 169610, Singapore. LECT2 is involved in many pathologic conditions. E-mail: [email protected] Recently, LECT2 has been identified as one of the 22,23 Received 18 May 2010; revised 4 October 2010; accepted 26 associated with human systemic . November 2010; published online 11 March 2011 LECT2 was discovered in a patient who had LECT2 inhibits growth of human HCC cells HT Ong et al 400 nephritic syndrome and slowly progressive renal failure (Affymetrix, Santa Clara, CA) as per the manufacturer’s over a number of years.22 However, clinical and instructions. The HCC profiling data sets can be obtained pathological features of earlier phases of this type of using E-MEXP-84 and E-TABM-292 at ArrayExpress, amyloidosis have yet to be determined. Kameoka et al.11 European Bioinformatics Institutes of the European reported a polymorphism of the human LECT2 gene in Molecular Biology Laboratory.9 the Japanese population associated with a severe form of that leads to progressive joint Real-time PCR destruction. Using the concanavalin A-induced hepatic HCC specimens with and without vascular invasion were injury model, Segawa et al.15 reported that LECT2 expres- selected randomly and subjected to real-time PCR sion in the mouse liver was inversely regulated by TNF-a amplification. Each sample was amplified in duplicates and/or IFN-g. Saito et al.14 observed severe liver injury in using Rotor-Gene 2000 Real Time Cycler (Corbett LECT2À/À knockout mice compared with wild-type mice, Research, Mortlake, VIC, Australia). Gene expression following treatment with concanavalin A and correlated was normalized against the corresponding 18S mRNA with the significant increase of natural killer T cells in the expression level. liver of the LECT2À/À knockout mice. These authors concluded that the increase of hepatic natural killer T cells Adenoviral vector expressing recombinant could result from the excessive expression of IL-4 and Fas human LECT2 ligand in these mice, suggesting that LECT2 might All adenovirus vectors used in this study were replication regulate the homeostasis of natural killer T cells in the deficient (E1/E3 deleted) and based on the serotype-5 liver.14 Using immunohistochemistry, Uchida et al.16 adenovirus genome.26 The cDNA of human LECT2 or demonstrated the reduction in LECT2 expression with firefly luciferase were cloned into a transfer plasmid, with the progression of hepatocarcinogenesis and its expres- expression driven by an human cytomegalovirus immediate sion was abrogated in malignant HCC cells. Despite these early gene region promoter/enhancer, and mRNA stability observations, the direct function of LECT2 in hepato- and polyadenylation directed by a rabbit b-globin / carcinogenesis has not been well studied and its role in polyadenylation signal. The expression cassettes were intro- vascular invasion of human HCC has not been reported. duced into a bacterial plasmid-borne adenovirus genome, In this study, we showed that re-expression of LECT2 using a recombination in bacteria.27,28 The resulting adeno- significantly reduced the migration and invasion of virus genomes (AdLECT2 and AdLuc) were linearized with human HCC cells in vitro and significantly reduced their SpeI enzyme, transfected into E1-complementing human growth in vivo. embryonic kidney 293 cells and were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitro- gen, Grand Island, NY) until a cytophathic effect was apparent 5–7d post-transfection. Cells were collected and Materials and methods subjected to four freeze/thaw cycles. The supernatant was Patients and tissue specimens stored at À80 1C as crude viral lysate. An inoculum was Human HCC tissues and some of the corresponding distal added to 293 cells and the cycle was repeated three times noncancerous liver tissues were obtained from patients to amplify the virus. The resulting recombinant Ad who had undergone partial hepatectomy at the National vectors were propagated in 293 cells and purified using Cancer Centre, Singapore, with informed consent and AdenoPACK (Sartorius Stedium Biotech, Epsom, UK). conducted in accordance with the policies of the Institu- Vector stocks were frozen at À80 1C until use. Quantifica- tional Review Board. This study consisted of specimens tion of viruses was based on protein content,29 using the from vascular invasive (N ¼ 29) and non-invasive (N ¼ 51) conversion 1 mg of viral protein ¼ 3.4 Â 1012 viral parti- HCC. As a control, histologically normal liver tissues cles (vp).30 Typical vector stock titers were 1012vp/ml with (NN) from colorectal patients with liver metastases a 1:100 infectious plaque forming unit to vector particle (N ¼ 10) and distal histologically normal surrounding ratio. tissues (ST) of HCC patients (N ¼ 27) were included in the analyses. Immediately after surgical resection, tissues were Cell culture and virus transduction snap-frozen and stored in liquid nitrogen. Frozen sections Human HCC cell lines Hep3B and PLC/PRF/5 were of all tumor samples studied were evaluated by qualified cultured in DMEM, supplemented with 10% heat-inacti- pathologists to ascertain that the tissues studied were vated fetal bovine serum (HyClone Labs, Logan, UT) and either cancerous (480% cancerous tissues) or normal. non-essential amino acids (Invitrogen) at 37 1Cin5% CO2-humidified incubator. For transduction experiments, Gene expression analysis: differential gene expression cells were incubated with virus at a multiplicity of infection analysis of human HCC samples with or without of 100vp in serum-free medium for 2 h at 37 1C, after which vascular invasion the virus inoculum was removed and cells were further Experiments were carried out as described previously.24,25 maintained in culture medium for 2 days. In brief, 5 mg of total RNA was extracted from HCC specimens, reversibly transcribed and biotin labeled. Immunoblotting The final cRNA obtained was hybridized to human At 48 h post-viral transduction, cell lysates were har- HG-U133A and HG-U133B oligonucleotide probe arrays vested. Protein concentrations were determined by the

Cancer Gene Therapy LECT2 inhibits growth of human HCC cells HT Ong et al 401 Bradford method. Cell lysates were mixed with loading used to wipe off non-invading cells on the upper surface buffer, denatured for 5 min at 95 1C, fractionated on a of the membrane. The invading cells on the lower surface continuous SDS-polyacrylamide gel and blotted onto of the membrane were fixed and stained with Hoescht nitrocellulose membrane (Invitrogen). The membrane was 333548 (Molecular Probes, Eugene, OR). Using  200 immunoblotted with anti-LECT2 (1:6000 dilution; Med- magnification, cells invading the membranes were ical & Biological Labs, Nagoya, Japan), anti-FLAG counted in three microscopic fields. For each group, (1:1000 dilution; Stratagene, Cedar Creek, TX) and anti- three duplicate wells were studied. The invasion assay was Ad5 (1:1000 dilution; Abcam, Cambridge, UK) for 1 h at done in a similar manner but with growth factor-reduced room temperature after which the membrane was washed Matrigel coating on the polycarbonate membranes. and incubated with horseradish peroxidase conju- gated goat anti-mouse or horseradish peroxidase-goat Tumor model anti-rabbit immunoglobulin (1:2500 dilution; Pierce, All procedures involving animals were reviewed and Rockford, IL). Protein expression was normalized using approved by SingHealth Institutional Animal Care and an antibody against actin (Neomarker, Fremont, CA). Use Committee. To establish subcutaneous HCC tumors, Detection of positive signal was achieved using Super- a 6-week-old athymic nu/nu female mice (Animal Signal West Pico Substrate (Pierce). Resource Centre, Canning Vale, WA, Australia) were injected with 5  106 Hep3B cells. When tumors reached Reverse transcription-PCR 0.3–0.4 cm in diameter, mice were injected intra-tumoral All human tissue samples were obtained from the with AdLECT2 (N ¼ 13) or AdLuc (N ¼ 12), at 1  1010vp National Cancer Centre Singapore Tissue Repository. per dose, 3 doses per week for 3 weeks (total Hep3B and PLC/PRF/5 cells were transduced with dose ¼ 9  1010vp). Equal volumes of saline were given AdLECT2 or AdLuc at a multiplicity of infection of to each of the three mice as controls for the effect of vp 100vp for 48 h. All samples were lyzed and total RNA was administration on tumor growth. Animals were killed at extracted with RNeasy mini kit (Qiagen, Valencia, CA). day 28 after injection of the first therapeutic dose. Tumor Reverse transcription-PCR was performed using the dimensions were measured using a digital caliper and the SuperScript III First-Strand Synthesis System (Invitro- tumor volume (V) was calculated using the formula: gen) according to manufacturer’s instruction, using V ¼ p/6  length  (width)2. Growth curves were plotted primer pairs as described previously.13 The products were using relative tumor volume for each experimental group separated by electrophoresis on 1.5% agarose gel and at the set time points. stained with ethidium bromide. Statistical analysis In vitro experiments on migration and invasion: Analysis of variance (ANOVA) and the Student’s t-test scratch wound assay were used for comparison among groups and between Adenoviral vectors transduced cells were seeded in 6-well matched data, respectively. We used GraphPad Prism 5.0 plate and incubated until a monolayer of cells was (GraphPad Software, San Diego, CA) for the statistical formed. A scratch was made in the middle of the well calculations. A P value of less than 0.05 was considered to with a sterile paper clip. The debris was washed away and be statistically significant. conditioned media was added to the wells. Cells were imaged at  100 microscopy magnification, and the initial area of the scratch (area devoid of cells) for the field of view was measured with Image-Pro Plus software Results (Media Cybernetics, Acton, MA). The plate was incu- Expression of LECT2 in human HCC biopsy samples bated at 37 1C and the same field of view was imaged from patients with vascular invasion is significantly at 24, 48 and 72 h. The percentage of wound closure by reduced migrating cells over time was derived from the final area Vascular invasion is an adverse prognostic factor in of the scratch wound divided by the initial area. human HCC associated with early recurrence. Data- mining studies of a gene expression database that we have In vitro experiments on migration and invasion: established for Hepatitis B virus þ HCC patients revealed Boyden chamber assay that expression of LECT2 was reduced in HCC biopsy The effect of LECT2 on the migration of HCC cells was samples (N ¼ 80) compared with adjacent histologically evaluated using a transwell chamber with 8 mm pore size normal liver tissues (N ¼ 37). In Figure 1, we showed that polycarbonate membranes (Becton Dickson, Bedford, MA). LECT2 expression in HCC without vascular invasion Cells were transduced with AdLECT2 at multiplicity of (NI, N ¼ 51) is significantly differed from matched- infection of 100vp, cultured for 48 h before seeding at a normal surrounding tissues (ST) (Po0.0001, fold density of 2  104 cells in 250 ml of 1% bovine serum change ¼À2.35). In comparison, LECT2 expression in albumin supplemented DMEM in the upper transwell HCC with vascular invasion (VI, N ¼ 29) is even more chamber. In the lower chamber, 750 ml DMEM contain- significantly differed from ST (Po0.0001, fold ing 10% fetal bovine serum was added. Cells were allowed change ¼À7.29) (Figure 1a). Expression of LECT2 to migrate across the polycarbonate membranes and between HCC with and without vascular invasion is analyzed at 72 h after cell seeding. A cotton swab was also significantly differed with P ¼ 0.0006 and fold

Cancer Gene Therapy LECT2 inhibits growth of human HCC cells HT Ong et al 402

Figure 1 Consistent downregulation of LECT2 expression in HCC surgical biopsy samples with vascular invasion at diagnosis. (a) Gene expression analysis. Signal intensity of LECT2 obtained after hybridization with Affymetrix HG-U133A and HG-U133B oligonucleotide probe arrays for liver biopsy samples with vascular invasion (N ¼ 29), non-vascular invasion (N ¼ 51), ST (N ¼ 27) and NN (N ¼ 10). (b) Real-time PCR analysis. Relative signal intensity determined after real-time PCR for liver biopsy samples with vascular invasion (N ¼ 19), non-vascular invasion (N ¼ 28), ST (N ¼ 12) and NN (N ¼ 12). Results were normalized against 18S RNA of the same sample to adjust for variations in sample loading. Differences in LECT2 expression between tissue sample categories were analyzed by t-test. N, number of patients; NN, histological normal liver tissues from colorectal cancer patients with liver metastases; ST, histological normal surrounding liver tissues of HCC patients; NI, HCC with no vascular invasion at diagnosis; VI, HCC samples with vascular invasion at diagnosis; NS, not significant; FC, fold change, negative values indicate downregulation.

(P ¼ 0.029) (Table 1). Nearly 40% of HCC patients with their LECT2 expression downregulated recurred in less than 6 months post-resection compared with an observed recurrent rate of less than 20% for HCC patients with comparatively higher LECT2 expression (P ¼ 0.024, Figure 3). These data corroborated well with the suggestion that LECT2 has a significant role in vascular invasion, and the recurrence of human HCC and HCC cells with downregulated LECT2 expression could have Figure 2 Tissue type-specific gene expression of LECT2. Total enhanced invasion/metastasis potentials. RNA of various human tissues were extracted and subjected to reverse transcription PCR. Only fetal liver, adult liver and testis tissues were positive for the LECT2 transcript. 18S was used as Construction of adenoviral vector carrying the LECT2 a loading control. gene Adenoviral vector encoding secreted human LECT2 was constructed and was employed to deliver the LECT2 into change ¼À3.1 (Figure 1a). To validate the results the human HCC cell line Hep3B. LECT2 gene could be obtained from the microarray data on the downregulation detected in AdLECT2-transduced cells but not in the of LECT2 in HCC biopsy samples with VI, the mRNA parental or AdLuc-transduced control cells (Figure 4a). level of LECT2 in randomly selected VI–HCC biopsy This was further confirmed at the protein level using samples (N ¼ 19) and NI–HCC biopsy samples (N ¼ 28) western blot. The presence of adenoviral vectors in the used in genome-wide gene expression analysis were transduced cells was confirmed with anti-Ad5 antibody at determined by real-time PCR. Consistent with the results 24 and 48 h post-infection (Figure 4b), but at 96 h post- obtained from genome-wide gene expression analysis, infection, viral fiber protein could no longer be detected, expression of LECT2 was significantly reduced in VI– whereas LECT2 protein was still present (data not HCC samples compared with NI samples (Po0.05, fold shown). This observation is likely due to the leaky change ¼À3.98) (Figure 1b). No significant difference expression of the early and late genes of the replication was detected between normal liver tissues (NN) and the defective Ads immediately following infection. adjacent histologically normal tissues of HCC specimens (ST). Furthermore, using reverse transcription-PCR, Over-expression of LECT2 in Hep3B and PLC/PRF/5 it was determined that LECT2 was only expressed in human HCC cells suppressed the migration and fetal liver, adult liver and testis normal tissues (Figure 2). invasion ability of these cells in vitro To elucidate the functional role of LECT2 in HCC, we First, we employed the scratch wound-healing assay to correlated the level of expression of LECT2 with the evaluate the effect of LECT2 on the migration ability of various major clinicopathologic variables available at HCC cells in vitro. As shown in Figure 5a, re-expression diagnosis. Univariate analysis showed that the down- of LECT2 in Hep3B cells following adenoviral delivery regulation of LECT2 was statistically significantly significantly reduced the ability of these cells to migrate correlated with VI (P ¼ 0.001) and tumor recurrence across a scratch wound. Semi-quantitative analysis also

Cancer Gene Therapy LECT2 inhibits growth of human HCC cells HT Ong et al 403 Table 1 Univariate analyses to examine the association of clinicopathological factors with LECT2 expression Univariate analysis

RR (95% Cl) P value

Gender 0.773 Male (n ¼ 61) vs female (n ¼ 15) 0.85 (0.27–2.63) Age 0.818 460 year (n ¼ 41) vs p60 year 0.899 (0.36–2.22) (n ¼ 35) Hepatitis 0.845 HBV (n ¼ 59) vs non-B/C 2.0 (0.15–27.45) 0.604 (n ¼ 14) HCV (n ¼ 3) vs non-B/C (n ¼ 14) 2.7 (0.18–24.07) 0.561 Encapsulation 0.174 Figure 3 Comparatively low level of LECT2 expression correlated Yes (n ¼ 17) vs no (n ¼ 59) 2.17 (0.71–6.65) with short duration of HCC recurrence. On the basis of signal Tumor size 0.818 intensity obtained for LECT2 after hybridization with Affymetrix HG- 45cm(n ¼ 35) vs p5cm 1.11 (0.45–2.74) U133A and HG-U133B oligonucleotide probe arrays, the HCC (n ¼ 41) samples analyzed (N ¼ 76) were classified into high LECT2 expres- sion (signal intensity X16421, N ¼ 38) and low LECT2 expression AFP level 0.795 (o16421, N ¼ 38). Statistical significance was calculated using log 20–200 ng/ml (n ¼ 22) vs 0.83 (0.28–2.46) 0.741 rank test. o20 ng/ml (n ¼ 33) 4200 ng/ml (n ¼ 20) vs 0.68 (0.22–2.08) 0.50 o20 ng/ml (n ¼ 33) Lesion 0.186 Multiple (n ¼ 10) vs single 2.63 (0.63–11.08) (n ¼ 66) Differentiation 0.693 G2 (n ¼ 42) vs G1 (n ¼ 10) 0.67 (0.16–2.71) 0.571 G3 (n ¼ 18) vs G1 (n ¼ 10) 0.42 (0.09–2.06) 0.288 G4 (n ¼ 5) vs G1 (n ¼ 10) 1.0 (0.11–8.95) 1 Cirrhosis 0.818 Figure 4 Adenovirus-mediated gene delivery of LECT2. Human Yes (n ¼ 41) vs no (n ¼ 35) 0.90 (0.36–3.22) HCC Hep3B was transduced with AdLECT2 for 48 h. Reverse Vascular invasion 0.001** transcription PCR and western blotting analyses were then performed Yes (n ¼ 29) vs no (n ¼ 47) 6.09 (2.15–17.28) on the transduced cells. (a) RNA extracted was analyzed by reverse transcription PCR. A PCR product of 333 bp, corresponding to LECT2 Recurrence 0.029* gene, could be detected in AdLECT2-transduced cells. 18S was used Rp6(n ¼ 21) vs NR (n ¼ 41) 0.28 (0.09–0.88) as a control. (b) Western blots were performed with cell lysates Abbreviations: AFP, alpha-fetoprotein; HBV, Hepatitis B virus; harvested from Hep3B cells 48 h post-viral transduction with anti-Ad5, HCV, Hepatitis C virus; NR, non-recurrent; R, recurrent; RR, relative anti-Flag and anti-LECT2 antibodies. Anti-b-actin antibody was used risk. as loading control. Lane 1: parental cells; lane 2: AdLuc-transduced **Po0.001, *Po0.05. cells; lane 3: AdLECT2-transduced cells. indicated a significant decrease (450%) in wound closure Po0.01) (Figure 6b). Similar trends of suppression of in Hep3B cells expressing LECT2 in comparison with the invasion could also be observed with PLC/PRF/5, control cells (Po0.001) (Figure 5b). following transduction with AdLECT2. However, the Using Boyden chambers, the ability of LECT2 to P-value obtained (P ¼ 0.052) was not statistically sig- inhibit the migration and invasion of HCC cells was also nificant (Figure 6b). These findings indicate that over- studied. Hep3B and PLC/PRF/5 cells were transduced expression of LECT2 could have a significant inhibitory with AdLECT2. Following transduction with AdLECT2, effect on the migration and invasion of human HCC cells. the migratory ability of Hep3B and PLC/PRF/5 cells through the polycarbonate membranes was significantly Over-expression of LECT2 significantly reduced the reduced (Po0.01 and Po0.001, respectively) (Figure 6a). tumorigenicity of HCC cells in vivo The number of cells invaded through the matrigel-coated The therapeutic effect of LECT2 in vivo was evaluated membrane in the AdLECT2-transduced Hep3B cells was using a Hep3B subcutaneous xenograft model. Hep3B reduced by more than 80% when compared with AdLuc- tumor-bearing mice were given AdLECT2 (N ¼ 13) or transduced control cells (216±7 versus 1151±187, AdLuc (N ¼ 12) at a dose of 1 Â 1010vp in 100 ml saline by

Cancer Gene Therapy LECT2 inhibits growth of human HCC cells HT Ong et al 404

Figure 5 Over-expression of LECT2 inhibited migration of Hep3B cells in vitro.(a) Scratch wound assay. Migration of Hep3B cells following AdLuc or AdLECT2 transduction was compared in a scratch wound assay. Images were captured at 0, 24, 48 and 72 h, following incubation at 37 1C with a phase-contrast microscope at  100 magnification. (b) The rate of migration was determined by quantifying the total area that the cells had moved from the edge of the scratch towards the center of the scratch (to estimate the total area devoid of cells). The results were presented as percentage of inhibition by comparing the total area devoid of cells obtained following transduction of AdLECT2 and AdLuc at the same time point.

Figure 6 Over-expression of LECT2 in human Hep3B and PLC/PRF/5 cells inhibited their ability to migrate and invade in vitro. Boyden chamber assays were performed to test the migratory (a) and invasion (b) ability of Hep3B and PLC/PRF/5 cells following transduction with AdLECT2. Adenoviral vector transduced-cells were seeded on polycarbonate membrane with or without coating with matrigel. Cells on the lower surface of the membranes were fixed, stained and counted under microscope at 72 h post-seeding. Data shown are averages of triplicates ± s.e. (***Po0.001, **Po0.01).

Figure 7 Intra-tumoral injection of AdLECT2 suppressed growth of Hep3B-derived human HCC xenografts in vivo. Athymic nude mice bearing subcutaneous Hep3B tumors were treated with intra-tumoral injections of either saline (N ¼ 3), AdLuc (N ¼ 12) or AdLECT2 (N ¼ 13). A total of 9 Â 1010 vp each for AdLuc and AdLECT2 were given in nine injections over a period of 3 weeks. Student’s t-test was used to determine the statistical significance of the data. The short lines within the data points in the left panel represent the median distribution of the relative tumor burden. The right panel summarized the results of the groups treated with AdLuc and AdLECT2.

direct injection into the subcutaneous tumor nodule of relative tumor volume in the recipient tumor-bearing mice tumor-bearing nude mice. Saline only was given to tumor- compared with mice similarly treated with AdLuc or bearing nude mice controls (N ¼ 3). A total of nine saline (Figure 7, Po0.05). No statistically significant injections were given over the duration of 3 weeks. It was difference was observed between saline-treated controls demonstrated that AdLECT2 significantly reduced the and AdLuc-treated mice (Figure 7). These data provide

Cancer Gene Therapy LECT2 inhibits growth of human HCC cells HT Ong et al 405 direct evidence that LECT2 can be a potential therapeutic is an inhibitor of the Wnt signaling pathway. The Wnt strategy for human HCC. signaling pathway has been shown to have critical roles in cell development, proliferation and differentiation.38,39 Therefore, alterations of this pathway within cells and between cells could disrupt the balance between cell-to- Discussion cell interactions.40,41 Loss of this delicate cell-to-cell contact resulting from the reduction of LECT2 during Recurrent disease remains one of the major challenges for hepatocarcinogenesis could lead to the increase migration the management of HCC patients following resection observed in tumor cells, resulting in tumor invasion and or liver transplantation, accounting for 50–90% of post- metastasis.40,41 This hypothesis supports our observation operative death.4,8,31 Vascular invasion is an adverse that reduced expression of LECT2 in HCC cells enhanced prognostic factor in human HCC associated with early their migration and invasion properties. In accordance, recurrence. Tumor invasion and metastasis are complicated increased LECT2 expression in HCC cells following processes involving many different classes of proteins, such Ad-mediated LECT2 gene expression inhibit tumor as cell-adhesion molecules, extracellular proteases, angio- growth in vivo and tumor migration and invasion in vitro. genetic factors, cytokines and growth factors. In this study, Hence, besides being a candidate-prognostic marker of we have shown that reduction in LECT2 is significantly human HCC, the loss of LECT2 expression in human associated with HCC vascular invasion. HCC is of functional importance and relevant to the A 16-kDa secreted protein, LECT2 was first isolated development and progression of the disease. Therefore, as a possible chemotactic factor for .17 This LECT2 could potentially be a key player in the protein is synthesized by hepatocytes and has been development of HCC, and manipulating the expression associated with repair and cell growth after da- of LECT2 in HCC cells is a promising therapeutic mage.16,32–34 Accumulating evidence suggests that LECT2 strategy for human HCC therapy. is a pleiotropic protein, as are many cytokines. LECT2 has an important role in neuronogenesis, and its mutation 35 is the major cause of primary microencephaly. Despite Conflict of interest these observations, the significance of LECT2 in human cancer has not been fully investigated. Here, we showed The authors declare no conflict of interest. that the majority of samples from HCC patients that had vascular invasion at diagnosis had downregulated LECT2 expression compared with most of the non-recurrent Acknowledgements HCC samples that did not have vascular invasion at We thank the National Cancer Centre Tissue Repository diagnosis. The expression of LECT2 was three-fold lower for providing human tissue specimens for this study. in HCC samples with vascular invasion at diagnosis This research was supported in part by grants from compared with HCC with no vascular invasion, and the Singapore Biomedical Research Council and the approximately eight-fold reduced compared with histolo- Singapore Millennium Foundation. gically normal adjacent tissues (Figure 1). Uchida et al.16 had reported that the expression of LECT2 was almost abrogated in full-blown HCC cells compared with low- grade malignant HCC (LGM-HCC) and pre-malignant References atypical hyperplasia (AH). The decreased expression of LECT2 in HCC is also in accordance with other reports.13,36 1 Hui KM. Human hepatocellular carcinoma: expression To analyze the relationship of the expression level profiles-based molecular interpretations and clinical applica- of the LECT2 gene with the clinicopathologic features of tions. Cancer Lett 2009; 286: 96–102. HCC patients made available at diagnosis, univariate 2 Llovet JM, Bru C, Bruix J. Prognosis of hepatocellular carcinoma: the BCLC staging classification. Semin Liver Dis analysis was performed and demonstrated that only 1999; 19: 329–338. vascular invasion and recurrent status statistically 3 Mann CD, Neal CP, Garcea G, Manson MM, Dennison AR, significantly correlated with the expression of LECT2. Berry DP. Prognostic molecular markers in hepatocellular carci- This was further supported by the significant difference in noma: a systematic review. 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Cancer Gene Therapy