ALCAM in Melanoma Motility and Adhesion Figure 6. Silencing of ALCAM disrupts N-cadherin and ß-catenin junctions. (A-F) Immunostaining of sh5 (A, D), sh6 (B,E), and sh5rxd (C,F) cells for nuclei (DAPI; blue), ß-catenin or N-Cadherin (green), and ALCAM (red) reveal that adherens junctions are disorganized when ALCAM is absent (A, D). Control sh6 cells and sh5rxd rescue cells, in contrast, have strong localization of ß-catenin and N-Cadherin at cell-cell contacts (B,E; C, F), where ALCAM colocalizes. Overall levels of adhesion molecules were assayed in MUM-2B, sh5, sh5rxd, and sh6 by western blot (G). The levels of expression detected by pan-cadherin, N-cadherin, and ß-catenin antibodies were similar across all cell lines. The proportion of cells that exhibited strong ß- catenin-labeled cell-cell junctions was quantified and is shown in (H), as are representative examples of cell-cell junctions classified as strong or weak. The number of sh5 cells forming strong ß-catenin junctions is significantly reduced in sh5 compared to sh5rxd (t-test, p,0.0001). Error bars are mean 6 S.E.M. Scale bars are 10 microns. doi:10.1371/journal.pone.0039330.g006 Our results have implicated ALCAM as a regulator of cadherin- based adherens junctions in uveal melanoma cells. The disruption of N-cadherin/ß-catenin-positive junctions we observe in AL- CAM-silenced sh5 cells is striking. Typically, N-cadherin and ß- catenin localize strongly to cell-cell contacts, colocalizing with ALCAM. In ALCAM-silenced cells, however, both N-cadherin and ß-catenin localization at cell contacts is markedly reduced – it appears as if adherens junctions ‘‘fall apart’’ in the absence of ALCAM. This is consistent with earlier findings by Ofori-Acquah and colleagues [54], in which ALCAM co-immunoprecipitated in multiple adherens junction complexes. We were not, however, able to co-immunoprecipitate ALCAM with N-cadherin in uveal melanoma cells, suggesting that any interaction may not be direct or may be sensitive to our lysis conditions. The cadherins have long been implicated in invasion and metastasis, with N-cadherin/E-cadherin expression often dictating invasive potential in cancer cells. In addition to mediating intercellular and cell-matrix adhesive interactions, cell adhesion molecules also modulate signaling pathways. Thus, changes in the expression and localization of cell adhesion molecules can influence tumor progression by both modulating the adhesion status of a cell and by altering cell signaling. In many human cancer types, including melanoma, the loss of E-cadherin function is concomitant with expression of mesenchymal cadherins, including N-cadherin [57,58]. N-cadherin has been shown to promote cell motility and migration – in stark contrast to the anti-migratory properties of E-cadherin [59,60]. N-cadherin is capable of overcoming E- cadherin-mediated cell adhesion, resulting in induction of an invasive phenotype [59,61]. This so-called ‘cadherin switch’ not only occurs during the transition of cancer cells to an invasive phenotype, but is also a hallmark of the epithelial-to-mesenchy- mal transition that occurs during embryonic development. Given that ALCAM expression can modulate N-cadherin localization at cell-cell junctions, we can envision the following possibilities as to how ALCAM status might influence the migratory and invasive properties of uveal melanoma cell lines. ALCAM-induced N-cadherin junction formation might en- hance the ability of tumor cells to move into different surround- ings. Stromal cells, fibroblasts, and blood vessel endothelial cells express N-cadherin [62]. Positive regulation of N-cadherin junctions by ALCAM could allow cancerous cells to successfully move through adjacent N-cadherin-positive tissues by promoting interactions with these cells, thereby enhancing the probability of metastasis. This is an attractive hypothesis, given that the choroid of the eye is rich in blood vessels, and that endothelial cells express N-cadherin and vascular endothelial (VE)-cadherin [62]. When accompanied by a loss of E-cadherin, the tumor cells may lose their ability to interact with adjacent epithelial cell types. Therefore, expression of ALCAM could promote a state of dynamic adhesion, whereby cells dissociate from their primary site and subsequently interact with adjacent stromal cells and endothelial cells. In cases where adjacent cells are devoid of N- cadherin expression, we speculate that ALCAM expression might not serve to promote metastasis, as it would not increase the interaction between the two cell types. Since our cells were devoid of detectable E-cadherin expression, we were not able to observe whether ALCAM has a similar effect on this or other cadherin family members, though the literature suggests it does [52,54]. In addition to modulating adhesion specificity, ALCAM- induced N-cadherin junction formation might also provide cells with a pro-migratory signal. N-cadherin is capable of inducing motility and invasion independent of E-cadherin status [61]. Breast cancer cells transfected with N-cadherin show increased metastatic potential when injected into nude mice [59]. What N- cadherin signaling pathway might lead to increased motility and invasion? Fibroblast growth factor receptors (FGFRs) have been shown to physically interact with N-cadherin, likely through interactions between the fourth extracellular domain of N- cadherin and the first two Ig-like domains of FGFRs [63,64]. It is hypothesized that N-cadherin interaction with FGFRs facilitates binding of fibroblast growth factor-2 (FGF2) to its receptor but helps prevent internalization of FGFR. This, in turn, leads to increased expression of FGFRs at the cell surface, which contributes to sustained MAPK signaling. The end result is increased motility, invasiveness, and secretion of matrix metallo- proteinases, including MMP-9 [64]. The cutaneous-derived BLM cell line, being devoid of cadherin expression, might not be subject to the same changes in signaling induced by decreasing ALCAM-ALCAM interactions as would our uveal melanoma cell lines. The reduction of homophilic ALCAM interactions in a cell line lacking cadherins might free the cells from interacting with each other, allowing migration of individual cells into surrounding tissue. The reduction in invasiveness we observe appears at odds with the finding that amino-truncated ALCAM expression served to disrupt ALCAM junctions and to reduce MMP-2 activation, but actually increased the invasive capacity of BLM cutaneous melanoma cells [56] [40]. An attractive hypothesis that could account for the increased invasiveness caused by a dominant-negative ALCAM [56] versus our own results in which silencing ALCAM results in decreased invasiveness, centers around the cadherin status of the cell lines used in each study. BLM cells are devoid of N-, E-, and P-cadherin expression [65], while both cell lines used in our study strongly express N-cadherin (but not E-cadherin; P-cadherin was not assayed). Overall, our work confirms a previously suggested link between ALCAM and cadherins [52,54], and provides a new example of the regulation of cadherins by IgSF members. Nectins are IgSF molecules that localize to adherens junctions in epithelial cells [66,67], and influence E-cadherin-mediated adhesion [68,69] appears to increase the overall strength of adhesion between cells. All nectins associate with an intracellular binding partner, afadin, which directly links nectins to the actin cytoskeleton [70]. Afadin also associates with alpha-catenin [71,72]. As ALCAM’s intracel- lular interaction partners are completely unknown, a key component of our work going forward will be focused on identifying such partners, and the signaling pathways associated with them. It will also be important to determine the specificity of the interaction between ALCAM and cadherins: can silencing of PLoS ONE | www.plosone.org 12 June 2012 | Volume 7 | Issue 6 | e39330