Article Info

Author Affiliations: Department of Hepatobiliary and Pancreatic Surgery, Shulan Hospital, Hangzhou 310000, China (Zhang W); Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health, Zhejiang University School of Medicine, Hangzhou 310003, China (Zhang W, Xie HY, Zhou L and Zheng SS); Department of Hepatobiliary and Pancreatic Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China (Zhang W, Ding SM, Xing CY, Chen A, Lai MC and Zheng SS)

Corresponding Author: Prof. Shu-Sen Zheng, MD, PhD, FACS, Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health, and Department of Hepatobiliary and Pancreatic Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China (Tel: +86-571-87236570; Fax: +86-571-87236628; Email: shusenzheng@zju.edu.cn)

Contributors: ZW, XHY, ZL and ZSS contributed to the conception and design of the study. DSM and LMC performed the experiments. XCY performed the statistical analysis. ZW and XHY contributed to the interpretation of data. ZW and CA drafted and revised the manuscript. ZW and XHY contributed equally to this work. All authors read and approved the final manuscript. ZSS is the guarantor.

Funding: This study was supported by grants from the National S&T Major Project (2012ZX10002-017); National Natural Science Foundation of China (81201944); Health Science and Technology Program of Zhejiang Province (2013KYB083); and Education Department of Zhejiang Province Scientific Research Project (Y201430751).

Ethical approval: This study was approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine (2013-0022). All mouse experiments were approved by the Institutional Animal Care and Use Committee of Zhejiang University.

BACKGROUND: Increasing evidence indicates that downregulation of cell adhesion molecule 1 (CADM1) contributes to tumorigenesis in various cancers. The present study was undertaken to investigate the CADM1 expression pattern in human hepatocellular carcinoma (HCC), and to elucidate the mechanism underlying CADM1-mediated tumor suppression.

METHODS: CADM1 expression in HCC cell lines was measured by quantitative real-time PCR. The function of CADM1 in the context of tumor suppression in HCC cells was determined using proliferation assays, cell cycle analysis, EdU incorporation assays, in vitro colony formation analysis, and in vivo tumorigenicity assays. The mechanism by which CADM1 acts as a tumor suppressor gene in HCC was investigated using Western blotting analysis.

RESULTS: Downregulation of CADM1 expression is frequently detected in both HCC cells and clinical samples. Restoration of CADM1 expression in HCC cell lines significantly inhibits cell growth and negatively regulates the G1/S transition. CADM1 overexpression can inhibit the tumorigenicity of HCC cells both in vitro and in vivo. Western blotting analysis revealed that ectopic expression of CADM1 in HCC cells is associated with increased expression of Retinoblastoma (Rb) protein.

CONCLUSIONS: Our results showed that suppression of tumorigenesis by CADM1 may be mediated by the Rb-E2F pathway, involving upregulation of Rb protein levels. This pathway could therefore represent an attractive target for HCC therapy.

Hepatocellular carcinoma (HCC) has a notably poor prognosis, with an annual death exceeding 800 000 worldwide, making it the second most common cancer-related cause of death.^[1] Resection or liver transplantation (LT) is the best option for a potential cure for patients with early HCC; however, the overall 5-year survival rate of HCC remains low.^[2] The development of HCC is a complex process; multiple genetic and epigenetic alterations that occur during the pathogenesis of this malignancy have been described.^{[3, 4]} A number of tumor suppressor genes are frequently inactivated in human HCC, including ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), scavenger receptor class A, member 5 (SCARA5), deleted in liver cancer 1 (DLC1), XAF1 and Tip30.^[5-9] However, the molecular pathogenesis of HCC remains largely unknown; the evidence accumulated to date does not fully account for the initiation and progression of HCC.

Cell adhesion molecule 1 (CADM1), also known as TSLC1, IGSF4, Necl-2, SynCAM1 and SgIGSF, maps to chromosome 11q23.2, and was identified as a tumor suppressor gene based on its influence on tumor formation in BALB/c nu/nu mice.^[10] Silencing of CADM1 is frequently observed in various cancers, including lung, prostate, gastric, breast, pancreatic, nasopharyngeal and cervical cancer, and particularly in the invasive forms of these cancers.^[11] Overexpression of CADM1 results in a significant reduction of proliferation and a coincidental induction of apoptosis in neuroblastoma cells and non-small-cell lung cancer cells (NSCLC).^{[12, 13]} Recently, two teams reported that miR-1246 and miR-10b, by down-regulation of CADM1, enhance migration and invasion in HCC cells.^{[14, 15]} However, the molecular mechanism of CADM1 involvement in HCC progression has not yet been thoroughly investigated.

Aiming to address this in the present study, we investigated the influence of CADM1 on cell growth, cell cycle control and tumorigenesis. Furthermore we attempted to elucidate a potential mechanism for the anticancer function of CADM1 in HCC.

Ten HCC cell lines, HepG2, Hep3B, SK-HEP-1, PLC/PRF/5, Bel-7402, SMMC-7721, Huh7 (Cell Bank of the Chinese Academy of Sciences, Shanghai, China), MHCC-97L, MHCC-97H and MHCC-LM3 (Liver Cancer Institute, Fudan University, Shanghai, China), and one immortalized normal liver cell line, L02 (Cell Bank of the Chinese Academy of Sciences), were cultured according to the manufacturer’s instructions. According to the eligibility criteria used in our previous study,^[16] 10 patients (8 males and 2 females; mean age 48.7 years, range 26-67) who underwent surgery in our center were enrolled in this study. Primary tumor samples and corresponding non-tumorous tissues were stored in liquid nitrogen. The diagnosis of HCC was confirmed by pathological examination. Written informed consent was obtained from each patient, and the study was approved by the local Ethics Committee and performed according to the Declaration of Helsinki.

Total RNA was extracted using TRIzol reagent (Invitrogen). First-strand cDNA was synthesized with the SuperScript III First-Strand Synthesis System (Invitrogen). Real-time reverse transcription-PCR (RT-PCR) was performed using an ABI Prism 7500™ PCR System (Applied Biosystems). The CADM1-specific primers were as follows: sense, 5’-CTG TGA TTC AGC TAC TGA ATC-3’; antisense, 5’-TAG AAA AAT TCA GCA ACT GAA AC-3’(reverse). GAPDH was used as an endogenous control. The relative expression was calculated using the comparative C_t method.

The full-length CADM1 ORF [130-1458 nt; (GeneBank: NM_014333.3)] was amplified by PCR with a human cDNA panel (Clontech) as a template. Primers were designed with XhoI and KpnI restriction sites as linkers. The primers were as follows: sense, 5’-GTG CTC GAG TAT GGC GAG TGT AGT GCT G-3’; antisense, 5’-GCG GGT ACC GGC TGA TCT AGA TGA AGT ACT CT-3’. After digestion with XhoI and KpnI, the PCR product was ligated into the pcDNA3.1 (-) vector (Clontech). The recombinant plasmid was sequence-verified. The siRNA sequence (Genepharma) targeting CADM1 was from position +1255 to +1274, relative to the first adenine of the start codon at position 1. The corresponding DNA sequence of a control siRNA did not match any known mammalian genomic sequence.

To evaluate cell proliferation, HepG2 and MHCC-97L cells were transfected with the pcDNA3.1-CADM1 or pcDNA3.1-vector using Lipofectamine 2000 (Invitrogen). At 24 hours post-transfection, transfected cells were seeded at 2×10³ cells per well in triplicate in 96-well plates. After culturing for 1, 3, 5 and 7 days, cell viability was measured using the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories), according to the manufacturer’s instructions. The absorbance value of each well was determined at 450 nm using a microplate reader. Each experiment was performed in triplicate and repeated at least three times.

For 5-ethynyl-2’-deoxyuridine (EdU; Ruibo Biotech) incorporation experiments, transiently transfected cells were seeded at a density of 2×10³ cells per well into 96-well plates. At 3 days after seeding, cell proliferation was evaluated by assessing the incorporation of EdU, according to the manufacturer’s instructions. Briefly, cells were incubated with 50 µmol EdU for 2 hours and fixed with 100 µL of 4% paraformaldehyde for 30 minutes. Cell nuclei were stained with Hoechst 33342 for 30 minutes after permeabilization. The number of Hoechst 33342-positive nucleated cells incorporating EdU was evaluated by fluorescence microscopy.

Cell cycle analysis was performed using a DNA Prep Reagent Kit, according to the manufacturer’s instructions (Beckman Coulter, Fullerton, CA, USA). At 48 hours after transient transfection, cells were harvested and resuspended at a density of 1×10⁶ cells/mL in cold PBS/2% FBS. Then, 100 µL of single-cell-suspension was added to 100 µL of lysing and permeabilizing reagent, and incubated with 2 mL of DNA Prep Stain for 30 minutes. The cells were analyzed using a Beckman Coulter FC500 Flow Cytometry System (Beckman Coulter) within 1 hour. Cell cycle distribution was determined using the Multicycle Software for Windows. Each experiment was performed three times independently.

To assay the effect of CADM1 on colony formation, the recombinant vector pcDNA3.1-CADM1 was transfected into HepG2 and MHCC-97L cells (with the empty vector as a control), as described previously. At 24 hours after transfection, cells were trypsinized, counted, and seeded into six-well plates. Then, G418 (Sigma-Aldrich) was added to the medium to a final concentration of 400 µg/mL. After 3 weeks of selection, the cells were stained with 0.1% crystal violet and cell colonies with >50 cells/colony were scored. All experiments were performed independently at least three times.

The recombinant vector pcDNA3.1-CADM1 and the empty vector were transfected into MHCC-97L cells using Lipofectamine 2000. A pool of transfectants was selected using G418 at a final concentration of 400 µg/mL for 1 month. Ectopic expression of CADM1 was confirmed by Western blotting analysis. After G418 selection, 1×10⁷ cells were injected subcutaneously into the flank of 6 to 8-week-old BALB/c nu/nu mice. Primary tumor growth was analyzed by measuring tumor length (L) and width (W), and tumor volume was calculated according to V=π/6×L×W², as described previously.^[6] All mouse experiments were approved by the Institutional Animal Care and Use Committee at Zhejiang University School of Medicine. Experimental procedures were performed according to the institutional ethical guidelines for animal experiments.

Western blotting analysis was performed as described previously.^[17] Protein lysates were obtained from transiently transfected HCC cells and frozen samples of HCC tissue. Mouse anti-p21 mAb, mouse anti-cyclin D1 mAb, mouse anti-cyclin D3 mAb, mouse anti-cyclin-dependent kinase 4 (CDK4) mAb, mouse anti-cyclin-dependent kinase 6 (CDK6) mAb, mouse anti-phospho-Retinoblastoma (Rb) mAb, mouse anti-Rb mAb, rabbit anti-p15 polyclonal antibody, rabbit anti-p16 polyclonal antibody, rabbit anti-p27 polyclonal antibody, rabbit anti-E2F1 polyclonal antibody, rabbit anti-CADM1 polyclonal antibody, and rabbit anti-β-actin mAb were used according to the manufacturers’ instructions. All antibodies were purchased from Cell Signaling Technology, with the exception of the CADM1 antibody, which was purchased from Abcam.

To establish the gene expression pattern of CADM1 in HCC cells, we tested all 10 currently available human HCC cell lines as mentioned above and the normal liver cell L02 was used as a control. Expression of CADM1 mRNA was reduced in nine HCC cell lines, but enhanced in Bel-7402 cells, compared to the normal liver cell line L02 (Fig. 1A). These results suggested that decreased CADM1 expression is a frequent event in human HCC and may be involved in HCC oncogenesis.

The frequent silencing of CADM1 in HCC cells suggests that CADM1 is likely a tumor suppressor. To investigate this hypothesis, we evaluated the effect of CADM1 expression on cell proliferation. A mammalian expression vector containing CADM1 was transiently transfected into HepG2 and MHCC-97L cells with silenced endogenous CADM1. CCK-8 analysis revealed that treatment of HCC cells with recombinant CADM1 protein significantly suppressed cell proliferation (Fig. 1B, C). To further confirm the tumor suppressor characteristic of CADM1, an EdU incorporation assay was performed. EdU is an ideal alternative to BrdU, and can be used to sensitively visualize newly synthesized DNA.^[18] Our data showed a decreased proportion of EdU-positive cells in CADM1-transfected cultures, compared to vector-transfected cultures (Fig. 2A), confirming the inhibitory effect of CADM1 on cell proliferation in HCC cells.

To explore the molecular mechanism by which CADM1 suppresses HCC cell growth, we investigated the effects of CADM1 on cell cycle distribution. Flow cytometric analysis revealed that in CADM1-transfected cells, compared with vector-transfected cells, the proportion of cells in the G1 phase of the cell cycle increased coincidentally with a decrease in the proportion of cells in the S phase (HepG2, 68.6%±5.5% vs 53.6%±5.4%, P<0.05; MHCC-97L, 70.5%±6.8% vs 54.8%±4.7%, P<0.05; Fig. 2B).

To investigate the potential role of CADM1 in tumorigenicity, in vitro colony formation and in vivo tumor growth assays were performed. HepG2 cells and MHCC-97L cells were transfected with the pcDNA3.1-CADM1 vector or the empty vector, and then allowed to grow at a very low density. Compared with the vector control, CADM1-transfected cells showed markedly fewer and smaller colonies after 3 weeks of selection using G418. As shown in Fig. 3A and B, colony numbers of HepG2 cells and MHCC-97L cells transfected with the pcDNA3.1-CADM1 vector were 85±35 and 79±20, respectively. In both cases, the numbers were significantly lower than those in the control group (350±50 and 165±33, respectively, P<0.05).

To validate the growth inhibitory function of CADM1 in an in vivo context, an xenograft model of tumorigenicity was established in nude mice. Empty vector and CADM1-transfected MHCC-97L cells were injected separately into two groups of nude mice (n=8). One month after injection, we observed a significant reduction in the frequency of tumor formation in the mice injected with CADM1-transfected MHCC-97L cells (empty-vector versus CADM1-transfectants, 8/8 vs 4/8). The tumors formed by CADM1-transfected cells were significantly smaller than those formed by cells transfected with the empty vector (Fig. 3C). Taken together, both the in vitro and the in vivo studies suggest that CADM1 can suppress tumor formation in HCC.

To explore the mechanism by which CADM1 suppresses tumor formation by inhibiting the G1/S transition in HCC cells, we assessed the expression of positive regulators of the G1/S transition using Western blotting analysis. We selected P15, P16, P21, P27, Cyclin D1, Cyclin D3, CDK4, CDK6, phopho-Rb, Rb and E2F1 as attractive candidates because they are crucial regulators involved in cell cycle progression. Interestingly, ectopic expression of CADM1 induced an obvious increase in Rb protein expression in both HepG2 and MHCC-97L cells, while expression of all other proteins examined was unchanged (Fig. 4A).

The Rb protein is a tumor suppressor that plays a pivotal role in the negative control of cell cycle progression.^[19] We therefore examined the correlation between CADM1 and Rb expression in vivo. As shown in Fig. 4B, increased CADM1 expression correlated strongly with enhanced expression of Rb protein in human HCC tissue samples. The expression of E2F1 was also detected in the same set of specimens, but no correlation with CADM1 or Rb expression was observed (Fig. 4B). To further confirm the relationship between CADM1 and Rb, we knocked down the expression of CADM1 in Bel-7402 cells using small interfering RNA (siRNA). Interestingly, the silencing of CADM1 was accompanied by decreased expression of Rb (Fig. 4C). The CADM1-Rb-E2F pathway could represent a novel mechanism involved in HCC tumorigenesis (Fig. 4D). In summary, these data suggested that there is a strong correlation between the expression of CADM1 and Rb, indicating that CADM1 may inhibit the G1/S transition by inducing Rb expression.

CADM1 was identified as a novel tumor suppressor in human non-small cell lung cancer (NSCLC) through a series of functional complementation analyses.^[10] CADM1 encodes a transmembrane protein whose extracellular domain shows close homology to members of the immunoglobulin superfamily, and whose roles involve Ca²⁺-independent cell-cell adhesion and cell signal transduction.^{[21, 22]} Silencing of CADM1 is frequently observed in various cancers, including NSCLC, nasal NK/T-cell lymphoma, colorectal carcinoma and cervical cancer.^{[10, 23-26]} Recently, many reports have demonstrated that CADM1 may act as a tumor suppressor gene via induction of tumor cell apoptosis and inhibition of growth.^{[12, 13, 26]} However, to date, less progress has been achieved in terms of elucidating the molecular mechanisms of CADM1-mediated tumor suppression.

The current study showed that inactivation of CADM1 occurs frequently in HCC cell lines and in clinical samples. Based on the downregulation of CADM1 in HepG2 and MHCC-97L cells, we introduced full-length CADM1 into these cells in order to obtain functional evidence of the involvement of this gene in HCC pathogenesis. Results of the in vitro proliferation assay and the cell cycle analysis showed that ectopic expression of CADM1 can inhibit cell growth and negatively regulate the G1/S transition. The in vitro and in vivo experimental data also showed that CADM1 overexpression can inhibit tumorigenicity. Taken together, these data suggested that CADM1 may serve as a tumor suppressor during hepatocarcinogenesis.

In an effort to explore the molecular mechanisms of CADM1 in tumor suppression, we investigated several key regulators of the cell cycle. Most interestingly, overexpression of CADM1 is associated with upregulation of Rb protein, but not phospho-Rb protein. It is well known that Rb protein plays a central role in governing cell cycle progression and DNA replication by controlling the expression of cell cycle E2F-dependent genes.^[19] Loss of the Rb tumor suppressor pathway represents a relatively common event in HCC, suggesting that it plays an important role in the pathogenesis of HCC.^{[27, 28]} Our data also demonstrated that there is a strong correlation in the expression patterns of CADM1 and Rb protein in vivo, based on the analysis of HCC clinical samples. Furthermore, knockdown of CADM1 using siRNA is accompanied by decreased expression of Rb protein. Therefore, the CADM1-Rb-E2F pathway could represent a novel mechanism involved in HCC tumorigenesis (Fig. 4D). To our knowledge, this is the first description of an association between CADM1 and Rb.

Surprisingly, our immunohistochemistry results showed that CADM1 appeared to be located in both cytoplasm and cell membrane (data not shown). The same phenomenon has been reported in cutaneous melanoma, colorectal carcinoma, lung adenocarcinoma, and human umbilical vein endothelial cells.^{[24, 29-31]} Interestingly, Overmeer has detected the presence of full-length CADM1 protein in the nucleus of cervical cancer cells.^[17] It appears that the location of CADM1 is dependent upon the epithelial origin of the cells. Similar to the findings in CADM1, several transmembrane glycoproteins such as EGFR/ErbB1, ErbB2, ErbB3 and ErbB4 have also been detected in the nucleus of many tissues, where they may act as transcriptional regulators.^{[32, 33]} Elucidating the exact roles of nuclear CADM1 requires further investigation.

In conclusion, our data demonstrated that downregulation of CADM1 expression negatively regulates the G1/S transition and suppresses the development of HCC. Given its association with the Rb-E2F pathway, which has been shown to be critical in HCC, CADM1 may serve as a valuable target for HCC therapy.

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