In this study, we demonstrate that L1CAM protein is expressed in NSCLC and is correlated with vessel infiltration, metastasis, and a poor prognosis. In-vivo data suggests that L1CAM is involved in EMT of lung cancer. Further, we provide evidence that L1CAM is involved in NSCLC cell invasion.
L1CAM expression was found in 25% of NSCLC by immunohistochemistry, using our previously described monoclonal anti-L1CAM antibody (clone 14.10), which is directed to the ectodomain of L1CAM (CD171).
Three hundred fifty-three tumors with two L1CAM negative cores were defined as negative. It cannot be ruled out that some of these cases have L1CAM expression due to intratumoral heterogeneity. However, we found L1CAM expression heterogeneity of 13.5%. In these cases, different staining scores were observed in the two cores of the tumor. Of these, 34 tumors had the score "0 and 1" or "1 and 0". These are less than 10% compared to the 353 cases that are L1CAM negative for both cores. In 10 randomly chosen L1CAM negative cases identified by TMA we did not observe focal L1CAM positivity on whole sections. Our finding of L1CAM expression in non-small cell lung cancer is in contrast to a previous protein expression profile study across several human tissues, which was not able to identify L1CAM expression in NSCLC . This discrepancy is most probably due to the low case number (10 SCC/5 ADCA) in that recent expression analysis . Importantly, other studies identified L1CAM expression in small cell lung carcinoma, in pulmonary carcinoids and in large cell lung carcinomas [10, 25]. Interestingly, a relationship to prognosis was also demonstrated in large cell lung carcinomas , corroborating our findings in NSCLC. Only L1CAM was of uni- as well as multivariate prognostic significance for OS and PFS among all biomarkers tested. These data are in line with published prognostic data on L1CAM expression in extrahepatic cholangiocarcinoma, gastric, breast, colorectal, ovarian, endometrial, and pancreatic duct carcinoma [6, 21, 26–30].
We observed a negative correlation of L1CAM with E-cadherin and a positive correlation with slug/vimentin/beta-catenin, indicating a potential role of L1CAM in EMT of NSCLC. Our results are in concordance with findings in colorectal, breast and pancreatic carcinoma where L1CAM expression was clearly involved in the EMT program [9, 16, 23]. It is unclear how L1CAM up-regulation exactly occurs in NSCLC. Several mechanisms have been described so far: In colorectal carcinoma, aberrant L1CAM expression was attributed to a hyperactive beta-catenin/TCF pathway  or to DNA hypomethylation at CpG islands of the L1CAM promoter . In a pancreatic cancer cell line L1CAM expression was regulated via binding of the transcription factor slug to the L1CAM promoter, induced by TGF-beta1 and mediated by c-jun NH2-terminal kinase .
In A549 cells, L1CAM expression was inducible by TGF-beta1 which also induced beta-catenin and vimentin and downregulated E-cadherin. We found the EMT transcription factors slug and snail induced by TGF-beta1 on the mRNA level. Our results would favor a model in which aberrant L1CAM expression in NSCLC is mediated by slug and an activated beta-catenin pathway. A knockdown of slug or snail was not performed so that we cannot prove this hypothesis. L1CAM knockdown in the cell lines SK-LU-1 and SK-LC-LL reduced matrigel invasion but we did not observe further up-regulation of L1CAM expression by TGF-beta1 in the latter cell lines.
Recently, EMT has been linked to the cancer stem cell (CSC) phenotype , and L1CAM was shown to be co-expressed with the CSC marker CD133 in glioma cells . Molecular targeting of L1CAM in CD133+ glioma cells reduced tumor growth and increased survival in-vivo . In a widely accepted hypothesis CSC's are believed to be endowed with increased drug resistance . The existence of bronchoalveolar stem cells (BASC's) and their key role in KRAS-induced lung cancer was proven although it is not assured that BASC's can induce a histophenocopy of the initial tumor in secondary or tertiary hosts (for review see ). It remains to be seen if the dismal prognosis that we observe in the L1CAM positive NSCLC subgroup is related to the presence of CSC's.
We observed a correlation of L1CAM protein up-regulation with blood vessel invasion and metastasis in NSCLC. Further, L1CAM was accentuated at the tumor-stroma interface but decreased in central parts, suggesting an L1CAM involvement in tumor cell invasion. SiRNA knockdown of L1CAM conferred a less invasive phenotype in the two cell lines tested, supporting our in-vivo observation. On whole sections, we found strong L1CAM expression of tumor cells close to and inside intratumoral blood vessels. Up-regulation of L1CAM at the invasive front was also found in colorectal cancer . NSCLC has a highly desmoplastic stroma similar to pancreatic duct carcinoma, often with formation of a central scar. It is unclear whether this prominent fibrotic reaction is inhibiting or rather promoting tumor cell migration. Therefore the question arises whether up-regulation of L1CAM is a prerequisite for infiltrating tumor cells to reach and invade neo-angiogenetic blood vessels. This hypothesis requires further systematic investigation by in-vitro and in-vivo vessel invasion assays. Two recent studies showed that a soluble form of L1CAM acts pro-angiogenic and L1CAM expression in the tumor endothelium mediates selective tumor cell transmigration in pancreatic adenocarcinoma [35, 36].
Anti-L1CAM therapy altered L1CAM gene expression in-vitro as well as reduced tumor growth in a mouse model of intraperitoneally transplanted ovarian cancer cells [37–40]. Thus, anti-L1CAM agents may be used in the setting of intracavitary chemotherapy of malignant effusions. Since protein expression in the carcinoma cells was of similar intensity compared with adjacent peripheral nerves and proximal tubules of the kidney, systemic therapy with mAb to L1CAM could potentially lead to neurological or renal complications.