L1 cell adhesion molecule overexpression in hepatocellular carcinoma associates with advanced tumor progression and poor patient survival

Hepatocellular carcinoma (HCC) is one of the most frequent human malignancy worldwide. Especially in China, it has become a major cause of cancer-related death [1]. There are many risk factors associated with HCC disease etiology, such as hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, genetic makeup and environmental exposure [2]. The main features of HCC are fast infiltrating growth, early metastasis, high-grade malignancy, and poor therapeutic efficacy. Although surgical resection is the best method to ensure long-term survival for HCC patients, frequent recurrence and a high incidence of metastasis often make the overall prognosis unsatisfactory. The progression of HCC is associated with cumulative genomic alterations, including oncogene upregulation and tumor suppressor downregulation. Therefore, it is necessary to clarify the molecular pathogenesis of HCC.

The L1 cell adhesion molecule (L1CAM, also named as CD171), as a member of the immunoglobulin superfamily, is a 220 kDa type I transmembrane glycoprotein [3]. It is comprised of six IgG-like domains and five fibronectin-type III repeats, followed by a transmembrane region and a highly conserved cytoplasmic tail [4]. L1CAM was first described in neural cell migration [5]. Then, several studies demonstrated that this protein plays pivotal roles in mediating the correct formation of neuronal connections during embryo neurogenesis and performs important functions in neuron-neuron adhesion, neurite fasciculation, synaptogenesis, neurite outgrowth on Schwann cells, and neuronal cell migration [6‐8]. In other normal cells such as hematopoietic, endothelial, and intestinal crypt cells, L1CAM is also detected, however, its function is unclear [9, 10]. Recent studies have found the association of L1CAM expression with various human malignancies by analysis using established cell lines, and fresh frozen or fixed paraffin-embedded tissues. As the results, L1CAM expression was observed in lung cancer cell lines, gallbladder carcinomas, colon cancer, pancreatic cancer, renal cell cancer, gliomas, ovarian carcinomas, endometrial carcinomas, and melanomas [11‐15]. However, no data of L1CAM are available for HCC. The aim of this study was to investigate the expression of L1CAM in HCC and determine its correlation with tumor progression and prognosis.

In this study, we first dmonstrate that L1CAM protein and mRNA expression in human HCC tissue was significantly associated with tumor progression and clinicopathologic features. Immunohistochemical analysis of a large set of HCCs revealed that 63.08% of HCC were high expression for L1CAM. Notably, L1CAM immunoreactivity was distinctly increased in a substantial proportion of HCC cases compared with their adjacent nonneoplastic liver tissue, which was further confirmed by Western bloting analysis and Q-PCR analysis. Then, the expression of L1CAM in HCC tissues with advanced tumor stage and grade was significantly higher than that in early tumor stage and low tumor grade HCC, suggesting that L1CAM expression might be of clinical relevance in the aggressiveness of HCC. The impact of L1CAM expression on clinical outcome was assessed by Kaplan-Meier analyses. High L1CAM expression was associated with a significant trend toward both poorer disease-free and overall survival. Univariate and multivariate analyses clearly demonstrated that L1CAM expression was an independent risk factor predicting overall survival and disease-free survival of patients with HCC. The statistically significant impact of L1CAM expression for overall survival (P = 0.008) was more significant than the tumor stage (P = 0.009) that is widely used at present, suggesting that L1CAM expression could be a useful marker to predict patient survival.

It is necessary to identify biological markers associated with the advancement of tumor progression for early diagnosis of patients with aggressive tumors and poor prognosis, and for the development of new therapeutic strategies and the selection of the appropriate treatment. The cell dhesion molecule families, such as integrins, cadherins, immunoglobulin-like CAMs and selectins, are often aberrantly regulated in human malignancies, leading to the tumor progression [19]. L1CAM, a member of immunoglobulin-like CAMs, was first reported to be involved in human cancers by investigating its expression in B16 melanoma cells [20]. After that, L1CAM overexpression has been found in various other tumors. Especially in degestive system, the study of Issa et al. [21] observed that L1CAM expression was selectively enhanced on endothelium associated with pancreatic adenocarcinoma in situ and on cultured pancreatic tumor-derived endothelial cells in vitro; Kodera et al. [22] detected the expression of L1CAM in gastric cancer specimens, more often among the intestinal-type cancer, and further demonstrated the prognositc value of L1CAM expression in pT3-stage gastric cancer; Choi et al. [23] also indicated that L1CAM was not expressed in the normal epithelium of the gallbladder but in 63.8% of gallbladder carcinomas, remarkably at the invasive front of the tumors; regarding the clinical significance, they demonstrated that L1CAM expression was significantly associated with the aggressiveness and poor prognosis of gallbladder carcinomas. With the similar results of these previous studies, our data also shown the association of L1CAM overexpression with the advancement and short survival of HCC patients.

The function of L1CAM determines its contribution to the tumorigensis. At first, L1CAM is a target gene of β-catenin-TCF signaling, which is an important cancer-related pathway. Many β-catenin target genes including metalloproteases, cell-extracellular matrix components, transcription factors, and cell adhesion molecules have been demonstrated to be involved in later stages of tumorigenesis that can confer invasive and metastatic capacities [24]. In 2006, Huszar et al. [25] identified L1CAM as a novel target gene of β-catenin-TCF signaling which is implicated in human colon cancer development. LEF/TCF binding sites were detected in the L1CAM promoter and an inducible dominant negative TCF, or an siRNA to β-catenin, suppressed the expression of L1CAM in colon cancer cells [26]. In addition, L1CAM induces ERK activation and ERK-regulated genes, including various integrin genes associated with cell motility and invasion. It was linked to activation of ERK and focal adhesion kinase to apoptosis protection in ovarian carcinoma [27]. Thirdly, L1CAM does not only mediate homophilic binding between cells, but also forms heterophilic interactions with various ECM proteins and their receptors. The L1CAM-induced cell motility was shown to involve a direct interaction of the shed L1CAM ectodomain, or the full-length L1CAM, with integrins, implying that L1CAM may play a role in cancer promotion and metastasis by also mediating cell-ECM interactions [28]. Because of its involvement in a wide variety of human cancers, L1CAM has been considered as a target molecule for cancer therapeutics. For example, the study of Bao et al. [29] found that L1CAM is required for maintaining the growth and survival of CD133+ glioma cells both in vitro and in vivo, and L1CAM may represent a cancer stem cell specific therapeutic target for improving the treatment of malignant gliomas and other brain tumors; Hung et al. [30] reported that targeting L1CAM using lentivirus-mediated shRNA may be a useful molecular pharmaceutical approach for the treatment of advanced oral squamous cell carcinoma; These previous studies suggested that using L1CAM as a drug target might improve the cancer patients’ outcome. The value of L1CAM in HCC therapy also needs further evaluation.

In conclusion, our data suggest that L1CAM is overexpressed in HCC tissues compared with their benign counterparts. To the best our knowledge, this is the first study evaluating the expression levels of L1CAM mRNA and protein in HCC tissues and its association with clinicopathologic parameters. Especially, the most important finding of this study is that L1CAM also is a novel and potential factor for predicting the poorer prognosis of HCC patients after surgery.