3676

ITGA4

integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor);ITGA4;integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor)

269C wild type|CD49 antigen-like family member D|VLA-4 subunit alpha|antigen CD49D, alpha-4 subunit of VLA-4 receptor|integrin alpha 4|integrin alpha-4|integrin alpha-4 subunit|integrin alpha-IV|very late activation protein 4 receptor, alpha 4 subunit

2q31.3

protein-coding

PURPOSE: Although the altered expression of integrin alpha4 is known to be associated with transformation or metastasis in several human cancers, the information on cholangiocarcinoma (CC) is still poor. In this study, we investigated the promoter methylation status of integrin alpha4 gene in CC. METHODS: A total of 29 CC, 19 adjacent non-tumor-containing tissue and 15 normal liver specimens were used for identification of gene methylation status by methylation-specific polymerase chain reaction. RESULTS: The frequency of DNA methylation was 55.17% (16 of 29) in the CC specimens (P < 0.001). Also, transcripts of the integrin alpha4 gene were decreased in all CC tissues in which there was DNA methylation of the integrin alpha4 gene. In addition, the downregulated expression of integrin alpha4 in CC cells with hypermethylation of the integrin alpha4 gene was restored by treatment with 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor. Moreover, we found that DNA methylation of integrin alpha4 was detected in all CC tissues obtained from patients with LN metastasis (7/7). Furthermore, phosphorylation of paxillin, cell migration-related molecule, was regulated by silencing of integrin alpha4. CONCLUSIONS: Taken together, these results suggest that loss of the integrin alpha4 gene is caused by aberrant DNA methylation of the 5'-CpG island site of the gene, and methylation of the integrin alpha4 gene can be a useful marker of metastasis of CC.

The capacity of tumor cells to form metastatic foci correlates with their ability to interact with and migrate through endothelial cell layers. This process involves multiple adhesive interactions between tumor cells and the endothelium. Only little is known about the molecular nature of these interactions during extravasation of tumor cells. In human melanoma cells, the integrin alphavbeta3 is involved in transendothelial migration and its expression correlates with metastasis. However, many human melanoma cells do not express beta3 integrins. Therefore, it remained unclear how these cells undergo transendothelial migration. In this study we show that human melanoma cells with different metastatic potency, which do not express beta2 or beta3 integrins, express the VCAM-1 receptor alpha4beta1. VCAM-1 is up-regulated on activated endothelial cells and is known to promote transendothelial migration of leukocytes. Interestingly, despite comparable cell surface levels of alpha4beta1, only the highly metastatic melanoma cell lines MV3 and BLM, but not the low metastatic cell lines IF6 and 530, bind VCAM-1 with high affinity without further stimulation, and are therefore able to adhere to and migrate on isolated VCAM-1. Moreover, we demonstrate that function-blocking antibodies against the integrin alpha4beta1, as well as siRNA-mediated knock-down of the alpha4 subunit in these highly metastatic human melanoma cells reduce their transendothelial migration. These data imply that only high affinity interactions between the integrin alpha4beta1 on melanoma cells and VCAM-1 on activated endothelial cells may enhance the metastatic capacity of human beta2/beta3-negative melanoma cells.

Recent studies have shown that lymphangiogenesis or the growth of lymphatic vessels at the periphery of tumors promotes tumor metastasis to lymph nodes. We show here that the fibronectin-binding integrin alpha4beta1 and its ligand fibronectin are novel functional markers of proliferative lymphatic endothelium. tumors and lymphangiogenic growth factors, such as vascular endothelial growth factor-C (VEGF-C) and VEGF-A, induce lymphatic vessel expression of integrin alpha4beta1. Integrin alpha4beta1 then promotes growth factor and tumor-induced lymphangiogenesis, as genetic loss of integrin alpha4beta1 expression in Tie2Cre+ alpha4(loxp/loxp) mice or genetic loss of alpha4 signaling in alpha4Y991A knock-in mice blocks growth factor and tumor-induced lymphangiogenesis, as well as tumor metastasis to lymph nodes. In addition, antagonists of integrin alpha4beta1 suppress lymphangiogenesis and tumor metastasis. Our studies show that integrin alpha4beta1 and the signals it transduces regulate the adhesion, migration, invasion, and survival of proliferating lymphatic endothelial cells. As suppression of alpha4beta1 expression, signal transduction, or function in tumor lymphatic endothelium not only inhibits tumor lymphangiogenesis but also prevents metastatic disease, these results show that integrin alpha4beta1-mediated tumor lymphangiogenesis promotes metastasis and is a useful target for the suppression of metastatic disease.