ALCAM Regulates Motility, Invasiveness, and Adherens Junction Formation in Uveal Melanoma Cells Karry M. Jannie, Christopher S. Stipp, Joshua A. Weiner* Department of Biology, The University of Iowa, Iowa City, Iowa, United States of America Abstract ALCAM, a member of the immunoglobulin superfamily, has been implicated in numerous developmental events and has been repeatedly identified as a marker for cancer metastasis. Previous studies addressing ALCAM’s role in cancer have, however, yielded conflicting results. Depending on the tumor cell type, ALCAM expression has been reported to be both positively and negatively correlated with cancer progression and metastasis in the literature. To better understand how ALCAM might regulate cancer cell behavior, we utilized a panel of defined uveal melanoma cell lines with high or low ALCAM levels, and directly tested the effects of manipulating these levels on cell motility, invasiveness, and adhesion using multiple assays. ALCAM expression was stably silenced by shRNA knockdown in a high-ALCAM cell line (MUM-2B); the resulting cells displayed reduced motility in gap-closure assays and a reduction in invasiveness as measured by a transwell migration assay. Immunostaining revealed that the silenced cells were defective in the formation of adherens junctions, at which ALCAM colocalizes with N-cadherin and ß-catenin in native cells. Additionally, we stably overexpressed ALCAM in a low-ALCAM cell line (MUM-2C); intriguingly, these cells did not exhibit any increase in motility or invasiveness, indicating that ALCAM is necessary but not sufficient to promote metastasis-associated cell behaviors. In these ALCAM-overexpressing cells, however, recruitment of ß-catenin and N-cadherin to adherens junctions was enhanced. These data confirm a previously suggested role for ALCAM in the regulation of adherens junctions, and also suggest a mechanism by which ALCAM might differentially enhance or decrease invasiveness, depending on the type of cadherin adhesion complexes present in tissues surrounding the primary tumor, and on the cadherin status of the tumor cells themselves. Citation: Jannie KM, Stipp CS, Weiner JA (2012) ALCAM Regulates Motility, Invasiveness, and Adherens Junction Formation in Uveal Melanoma Cells. PLoS ONE 7(6): e39330. doi:10.1371/journal.pone.0039330 Editor: Mohammed Akaaboune, University of Michigan, United States of America Received February 17, 2012; Accepted May 23, 2012; Published June 26, 2012 Copyright: 2012 Jannie et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by a grant from the E. Matilda Ziegler Foundation for the Blind (http://emzfoundation.com/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: joshua-weiner@uiowa.edu Introduction The immunoglobulin superfamily (IgSF), a class of proteins with 765 putative members in humans [1] represents one of the most ancient and diverse families of cell adhesion proteins. Not surprisingly, IgSF members are key players in numerous developmental and pathological processes [2–5]. Activated Leu- kocyte Cell Adhesion Molecule (ALCAM; also reported as CD166, DM-GRASP, neurolin, and BEN), an IgSF member, has been implicated in the regulation of many developmental events, including hematopoiesis [6–8], osteogenesis [9], T cell activation [10–12], and neurite outgrowth, fasciculation, and targeting [13–21]. Some of these studies suggested roles for ALCAM based on its expression pattern alone, while others utilized a variety of in vitro assays to identify ALCAM functions. To assess in vivo roles, we generated the first mutation of ALCAM in any model organism by targeting the Alcam locus in mice [22]. ALCAM-null mice are viable and fertile, suggesting that ALCAM’s functions in vivo may not be as broad as assumed from these earlier studies, although a high degree of functional redundancy among IgSF members is also likely. Nevertheless, we found that ALCAM-null mice do display several nervous system defects predicted by previous studies, including disrupted fasciculation of both motor and retinal ganglion cell axons [22], and mistargeting of retinal ganglion cell axons within the superior colliculus [23]. In addition, ALCAM- mice on a mixed C57BL/6-129 background exhibit retinal dysplasias, including disrupted organization of the outer nuclear layer photoreceptor neurons and invagination of the adjacent retinal pigment epithelium (RPE) and choroid (choriocapillaris) [22]; these dysplasias are greatly reduced on a congenic C57BL/6 background, however (data not shown). This last phenotype was initially puzzling, since expression of ALCAM in the retina is restricted to retinal ganglion cells and a subset of inner nuclear layer amacrine cells, with no expression detectable in photorecep- tor neurons or the RPE. We found, however, high levels of ALCAM expression in melanocytes and stromal cells of the choroid [22], a pigmented tissue that nourishes the RPE and photoreceptor cells and provides much of the blood supply to the eye (for review see ref. [24]). This previously undocumented expression, as well as the fact that choroidal melanocytes were found within ectopic retinal folds [22] suggests that in the absence of ALCAM, the structure and/or function of melanocytes in the uvea, which includes the choroid, iris, and ciliary body, might be disrupted. We found this phenotype to be particularly interesting in light of dozens of reports identifying ALCAM as a potential regulator of tumor cell behavior. Indeed, ALCAM has been implicated in the progression and metastasis of cutaneous melanoma [25], prostate carcinoma [26,27], breast cancer [28–30], colorectal carcinoma PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e39330