ALCAM in Melanoma Motility and Adhesion p.0.05; the average number of invasive MUM-2C cells per three 106 fields was 16; E). Growth and survival of MUM-2C and 2C-ALC is similar, as assayed and described in Fig. 3 (F). doi:10.1371/journal.pone.0039330.g004 (data not shown) or MUM-2C (Fig. 5A). Next, we quantified MMP-2 activity in the stable cell lines, sh5, sh6, and 2C-ALC (Fig. 5A, B). We found that MMP-2 activation was reduced in sh5 by nearly 80% compared to parental MUM-2B, control sh6 cells, and sh5rxd rescued cells (Fig. 5B). As expected from our previous results with the 2C-ALC cell line (Fig. 4) MMP-2 activity was not increased in 2C-ALC compared to parental MUM-2C (Fig. 5A, B); again, this suggests that ALCAM is necessary, but not sufficient, for an invasive cell phenotype in uveal melanoma. Pro-MMP-2 was detectable in MUM-2B, sh5, sh6, and sh5rxd cell lysates by western blot, indicating that even sh5 expressed this enzyme (Fig. 5C). Consistent with the decreased invasive capacity in sh5, the active form of MMP-2 was just barely detectable in sh5, yet was clearly present in MUM-2B, sh6, and sh5rxd (Fig. 5C). It is possible that sh5 ALCAM-silenced cells exhibit defects in both MMP2 secretion and MMP2 activation, based on our combined results. Cadherin-based Junctions are Disorganized in ALCAM- silenced Cells Another way in which ALCAM could influence tumor cell behavior is through the regulation of other adhesion molecules, particularly those that have been implicated in metastasis, such as classical cadherins (for reviews, see refs. [49–51]). Several previous lines of evidence support such a hypothesis. First, a report by Tomita and colleagues [52] described the coordinate recruitment of epithelial (E)-cadherin and ALCAM to cell contacts upon transfection of alpha-catenin into prostate cancer cell lines that have lost this protein. Second, ALCAM has been shown to colocalize with both alpha-catenin and filamentous actin in MV3- Tiam1 cells [53]. Finally, ALCAM could be co-immunoprecipi- tated with VE-cadherin and N-cadherin in PVMEC cells [54], and has been reported to be present in the same lipid microdomain compartments as cadherins [55]. We began by assessing the expression of cadherins in uveal melanoma cell lines. Both MUM-2B (Fig. 6G) and MUM-2C (Fig. 7E) expressed N-cadherin as well as ß-catenin; neither had detectable levels of E-cadherin (data not shown). To determine whether ALCAM silencing affects adherens junctions, we com- pared N-cadherin and ß-catenin staining in sh5 (ALCAM- silenced), sh6 (control), and sh5rxd (ALCAM-silenced+rescued) cells. In parental MUM-2B cells (data not shown) or in sh6 control cells, strong ß-catenin (Fig. 6B) and N-cadherin (Fig. 6E) staining was localized to cell-cell contacts that were also ALCAM-positive, and cells had a flattened, epithelioid shape. In contrast, sh5 silenced cells had disorganized ß-catenin (Fig. 6A) and N-cadherin (Fig. 6D) junctions, a more spindle-like shape, and often grew on top of each other. This phenotype was significantly rescued by re- expression of ALCAM in the sh5rxd cell line (Fig. 6C, F). Quantification (examples of ‘‘strong’’ and ‘‘weak’’ junctions are shown in Fig. 6H) showed that the percentage of cells with contiguous, strong ß-catenin-positive adherens junctions was reduced from nearly 75% in control lines to ,25% in sh5 (Fig. 6H). Neither silencing of ALCAM in sh5 nor its re-expression in sh5rxd appeared to affect levels of ß-catenin or N-cadherin expression (Fig. 6G). This phenomenon was not restricted to uveal melanoma cells. Transient transfection of HEK293 cells with an shRNA construct (sh2) confirmed to silence ALCAM also resulted in reduced ß- catenin localization to cell-cell contacts, and a lack of strong junctions (Fig. S1). In HEK293 cells transfected with a negative control, scrambled shRNA (sh0), ß-catenin localization to cell junctions was not perturbed (Fig. S1). ALCAM Expression Enhances Cadherin-mediated Cell-cell Contacts in the 2C-ALC Cell Line Finally, we addressed whether ALCAM expression would be sufficient to enhance the formation of cadherin-based adherens junctions in 2C-ALC cells. Parental MUM-2C cells exhibited weak, diffuse ß-catenin and N-cadherin staining, with few clear cell-cell junctions (Fig. 7A, C). Expression of ALCAM in the 2C- ALC cell line led to an increase in ß-catenin and N-cadherin staining along the length of cell junctions, which colocalized precisely with ALCAM (Fig. 7B, D). Despite this, cell morphology remained fairly similar to MUM-2C’s spindle shape, and no increase in total N-cadherin or ß-catenin levels was observed (Fig. 7E). In parental MUM-2C cells, only ,15% of cells had ß- catenin-positive junctions; in contrast, over 75% of 2C-ALC cells did (Fig. 7F). Together with our analysis of sh5 silenced cells, these data suggest that ALCAM expression is both necessary and sufficient to promote the recruitment of N-cadherin and ß-catenin to form adherens junctions in uveal melanoma cells. Discussion ALCAM has demonstrated functions in many critical devel- opmental processes such as hematopoiesis [6–8], neurite outgrowth [13–21], retinal ganglion cell targeting [23], and T- cell activation [10–12]. ALCAM has also been implicated in pathological states, such as cancer metastasis, but its role remains somewhat confusing. ALCAM has been identified as a marker of metastasis in many tumor cell types, and yet in other cases it has been associated with inhibition of metastasis. Reports from the literature thus present a paradox regarding ALCAM’s relation- ship to tumor cell motility and invasiveness. For example, initial studies described a positive correlation between ALCAM expression and metastatic capacity or progres- sion of cutaneous melanoma [25,32]. The role of ALCAM in cutaneous melanoma was first addressed directly by the laboratory of Guido Swart [56]. An amino terminal-truncated (dominant negative) form of ALCAM was transfected into cutaneous melanoma cells, and was found to diminish cell clustering and enhance both motility in vitro and the transition from primary tumor to tissue invasion in vivo. It appeared that the disruption of homophilic ALCAM contacts thus resulted in increased metastatic potential in cutaneous melanoma cell lines [56]; this was, however, in contrast to previous expression data that predicted ALCAM would promote invasion and metastasis. When other cancer types are considered, the picture becomes murkier – one study by Kristiansen and colleagues [35] found that ALCAM protein expression is high in low-grade prostate cancer, and is lost in higher-grade tumors. A study of colorectal cancer demonstrated overexpression of ALCAM neoplastic regions compared to normal surrounding tissue; membranous ALCAM staining correlated with reduced patient survival [31]. Studies of breast carcinoma also provide seemingly conflicting results: one study [28] found that low ALCAM expression correlated with high tumor grade and metastasis, while another [29] showed that ALCAM is associated with smaller tumor diameter and grade. PLoS ONE | www.plosone.org 9 June 2012 | Volume 7 | Issue 6 | e39330