Expression of Integrin Alpha5beta1 and MMPs Associated with Epithelioid Morphology and Malignancy of Uveal Melanoma

Purpose: Altered expression of the alpha5beta1 integrin and matrix metalloproteinases (MMPs) is recognized as a hallmark of invasive tumor cells. The purpose of the present study was to investigate the expression of integrin subunit alpha5, its corresponding ligand fibronectin (FN), and the expression pattern for MMPs in four highly proliferative human choroidal melanomas (TP17, TP31, SP8.0, and SP6.5) to evaluate whether any correlation can be established between these markers and cell tumorigenicity.

Methods: Cell tumorigenicity was evaluated by subcutaneous injection of uveal melanoma cell lines in immunodeficient nude mice. Anchorage dependency was evaluated by growth assays in soft agar. The invasive ability of each cell type was also determined using a modified Boyden chamber. Expression of both the alpha5 integrin subunit and FN was determined at the mRNA level by RT-PCR. The protein level (for alpha5) was determined by flow cytometry and inhibition of adhesion assays by using an antibody directed against the alpha5 subunit. Expression of MMPs was determined by standard gelatin zymography.

Results: Assays in nude mice provided evidence that the cell lines possess a range of tumorigenic ability of TP17>TP31>SP8.0>SP6.5. Antibody inhibition of cell adhesion and flow cytometry demonstrated that TP17 cells have no detectable membrane-bound alpha5beta1, whereas low levels are found in primary cultured melanocytes, as well as in SP6.5, SP8.0, and TP31 cells. RT-PCR analyses provided evidence that both FN and alpha5 expression may be regulated at the transcriptional level. Gelatin zymography revealed that all cell lines, as well as normal melanocytes, express MMP-2 at varying levels but that only the highly invasive TP17 cell line secretes a distinctive MMP with a high molecular weight of 117 kDa.

Conclusions: Among the four melanoma cell lines selected for the completion of this study, TP17 exhibited the most aggressive phenotype, which also correlated with the mostly epithelioid morphology of these cells. The cell morphology of the TP17 cell line could be related to the loss of alpha5beta1, whereas its invasive properties are more likely related to the expression of the 117-kDa MMP.