Tumor marker nucleoporin 88 kDa regulates nucleocytoplasmic transport of NF-kappaB.

Nucleoporin 88 kDa (Nup88) is a tumor marker, overexpressed in various types of cancer. In Drosophila Nup88 (mbo) was reported to selectively mediate the nucleocytoplasmic transport of NF-kappaB, an ubiquitous transcription factor involved in immune responses, apoptosis, and cancer. We addressed the function of Nup88 in mammalian cells. Selective depletion of Nup88 by small interfering RNA (siRNA) inhibited NF-kappaB-dependent reporter gene activation and the nuclear translocation of NF-kappaB without affecting the upstream activation pathway in NIH3T3 cells. In contrast, nuclear translocation of glucocorticoid receptor was not reduced by the depletion of Nup88. In metastatic melanoma cells overexpressing Nup88, constitutive activation of NF-kappaB was found both in nucleus and cytoplasm. Nup88 depletion in these cells reduced TNF-induced nuclear accumulation of NF-kappaB subunits. We conclude that Nup88 regulates the activity of NF-kappaB at the level of nucleocytoplasmic transport. Overexpression of Nup88 in tumor cells may, thus be involved in the constitutive NF-kappaB activation.

Activated leukocyte cell adhesion molecule (ALCAM/CD166/MEMD), a novel actor in invasive growth, controls matrix metalloproteinase activity.

Activated leukocyte cell adhesion molecule (ALCAM/CD166/MEMD) could function as a cell surface sensor for cell density, controlling the transition between local cell proliferation and tissue invasion in melanoma progression. We have tested the hypothesis that progressive cell clustering controls the proteolytic cascade for activation of gelatinase A/matrix metalloproteinase-2 (MMP-2), which involves formation of an intermediate ternary complex of membrane type 1 MMP (MT1-MMP/MMP-14), tissue inhibitor of metalloproteinase-2 (TIMP-2), and pro-MMP-2 at the cell surface. Surprisingly, truncation of ALCAM severely impaired MMP-2 activation in a nude mouse xenograft model, in which we previously observed diminished primary tumor growth and enhanced melanoma metastasis. Comparative studies of two-dimensional monolayer and three-dimensional collagen-gel cultures revealed that extensive cell-to-cell contacts, wild-type ALCAM, and cell-to-matrix interactions were all indispensable for efficient conversion of pro-MMP-2 to its active form in metastatic melanoma cells. Truncated, dominant-negative ALCAM diminished MMP-2 activation via reduced transcript levels and decreased processing of MT1-MMP. Failure of the proteolytic cascade after selective ALCAM depletion by RNA interference was mainly due to incomplete MT1-MMP processing, which was otherwise promoted by extensive cell-to-cell contacts. These data attribute a novel signaling role to ALCAM in regulation of proteolysis and support its previously postulated sensor function in invasive growth.

Activated leukocyte cell adhesion molecule (CD166/ALCAM): developmental and mechanistic aspects of cell clustering and cell migration.

Activated leukocyte cell adhesion molecule (ALCAM/CD166) is a member of the immunoglobulin superfamily and belongs to a recent subgroup with five extracellular immunoglobulin-like domains (VVC2C2C2). ALCAM mediates both heterophilic (ALCAM-CD6) and homophilic (ALCAM-ALCAM) cell-cell interactions. While expressed in a wide variety of tissues, ALCAM is usually restricted to subsets of cells involved in dynamic growth and/or migration, including neural development, branching organ development, hematopoiesis, immune response and tumor progression. Recent structure-function analyses of ALCAM hint at how its cytoskeletal anchoring and the integrity of the extracellular immunoglobulin-like domains may regulate complex cellular properties in regard to cell adhesion, growth and migration. Accumulating evidence suggests that ALCAM expression may reflect the onset of a cellular program for homeostatic control of growth saturation, which induces either growth arrest or cell migration when the upper limits are exceeded.

Truncation of activated leukocyte cell adhesion molecule: a gateway to melanoma metastasis.

Progression of human cutaneous primary melanoma is, among others, accompanied by de novo expression of activated leukocyte cell adhesion molecule (ALCAM/CD166) and enhanced activity of proteolytic cascades in the invasive, vertical growth phase (VGP) of lesions. The homophilic cell adhesion function of wild-type ALCAM mediates homotypic clustering of melanoma cells and would, thus, antagonize cell release from the primary tumor, an early prerequisite for metastasis. Stable transfection of a transmembrane, amino-terminally truncated ALCAM (DeltaN-ALCAM) into metastatic cells diminished cell clustering mediated by wild-type ALCAM. We have addressed the biological effects of DeltaN-ALCAM on tumorigenicity and found that the relief of cell clustering constraints promoted motility in vitro and the transition from expansive tumor growth to tissue invasion in reconstructed skin in culture. In a transplant tumor model, the changes were reflected in reduced subcutaneous tumor growth and in accelerated, spontaneous lung metastasis. These data indicate that the intact cell adhesion function of ALCAM may both favor primary tumor growth and represent a rate-limiting step for tissue invasion from VGP melanoma. ALCAM induction could, thus, provide an attractive target for proteolysis as a part of a more complex cellular program that couples growth and migration and facilitates dissemination.

Modulation of activated leukocyte cell adhesion molecule-mediated invasion triggers an innate immune gene response in melanoma.

Activated leukocyte cell adhesion molecule (ALCAM/CD166) is a progression marker of a variety of cancers, including melanoma, and is a marker for mesenchymal stem cells. ALCAM expression triggers matrix metalloproteinase activity and correlates with the transition between superficial melanoma growth and deep dermal invasion in vivo. We previously showed that manipulating ALCAM functionality could both decrease and increase melanoma invasion, depending on the manner by which ALCAM function was altered. How ALCAM exerts these opposing invasive phenotypes remained elusive. In the present study, we analyzed differences in melanoma cell gene expression in two- and three-dimensional cultures as function of ALCAM-mediated adhesion. We identified a cluster of genes highly responsive to ALCAM functionality and relevant for melanoma invasion. This cluster is characterized by known invasion-related genes similar to L1 neuronal cell adhesion molecule and showed a remarkable induction of several innate immune genes. Unexpectedly, we identified major variations in the expression of genes related to an immunological response when modulating ALCAM function, including complement factors C1r and C1s. The expression and function of these proteinases were confirmed in protein assays and in vivo. Together, our results demonstrate a link between ALCAM functionality and the immune transcriptome, and support the assumption that ALCAM-ALCAM interactions could function as a cell signaling complex to promote melanoma tumor invasion.

ALCAM/CD166: cancer-related issues.

Activated leucocyte adhesion molecule (ALCAM) was originally identified as a transmembrane receptor which is involved in T-cell activation and has other still unresolved functions in hematopoiesis, development, inflammation and transendothelial migration of neutrophils. ALCAM is a member of a subfamily of immunoglobulin receptors with five immunoglobulin-like domains (VVC2C2C2) in the extracellular domain and is expressed in many types of tumors. The tumor-type-dependent impact of its expression level with respect to prognosis points to a possible context-dependent function. Most functional investigations have focused on malignant melanoma, in which high ALCAM expression at the protein level correlates with a poor prognosis. ALCAM mediates low-affinity homophilic interactions and much stronger interactions with CD6. Modulation of ALCAM function with agents such as transfected dominant negative ALCAM and ligand-binding secreted ALCAM both lead to inhibition of matrix metalloproteinase-2 activation, but their impact with respect to invasion in vitro and metastasis in vivo are different. Single-chain Fv fragments directed against ALCAM are efficiently internalized, paving the way for exploration of immunoconjugates as therapeutic agents. Validation experiments of the target with modulatory agents for possible therapeutic application in oncology are discussed.

Keratinocytes drive melanoma invasion in a reconstructed skin model.

Melanoma progression is a multistep progression from a common melanocytic nevus through the radial growth phase, the invasive vertical growth phase finally leading to metastatic spread into distant organs. Migration and invasion of tumor cells requires secretion of proteases to facilitate remodeling of the extracellular matrix including basement membranes. Here we used a reconstructed skin model to investigate melanoma growth and invasion in vitro. Using this model we show that the dermoepidermal basement membrane prevents the invasion of metastatic melanoma BLM and MV3 cells in the absence of a stratified epidermis. In the reconstructed skin model, matrix metalloproteinase-9, a protease activated early in melanoma development, is secreted by the keratinocytes and subsequently activated by an unknown soluble factor secreted by the melanoma cells. The dynamic interplay between keratinocytes and melanoma cells is further shown by an altered growth pattern of melanoma cells and the finding that a reconstructed epidermis induces invasion. Overall, our findings show that the invasive behavior of melanoma cells is determined by the melanoma cells themselves, but that the interplay between surrounding keratinocytes and the melanoma cells plays an important role in melanoma invasion.

Soluble adhesion molecules in human cancers: sources and fates.

Adhesion molecules endow tumor cells with the necessary cell-cell contacts and cell-matrix interactions. As such, adhesion molecules are involved in cell signalling, proliferation and tumor growth. Rearrangements in the adhesion repertoire allow tumor cells to migrate, invade and form metastases. Besides these membrane-bound adhesion molecules several soluble adhesion molecules are detected in the supernatant of tumor cell lines and patient body fluids. Truncated soluble adhesion molecules can be generated by several conventional mechanisms, including alternative splicing of mRNA transcripts, chromosomal translocation, and extracellular proteolytic ectodomain shedding. Secretion of vesicles (ectosomes and exosomes) is an alternative mechanism mediating the release of full-length adhesion molecules. Soluble adhesion molecules function as modulators of cell adhesion, induce proteolytic activity and facilitate cell signalling. Additionally, adhesion molecules present on secreted vesicles might be involved in the vesicle-target cell interaction. Based on currently available data, released soluble adhesion molecules contribute to cancer progression and therefore should not be regarded as unrelated and non-functional side products of tumor progression.

Antigenic profiles of individual-matched pairs of primary and melanoma metastases.

A unique collection of individual-matched pairs of primary and melanoma metastases were studied immunohistochemically with a panel of 6 monoclonal antibodies directed to gp-100, pigmentation-associated antigen, tyrosinase-related protein, human leukocyte antigen DR, MAA-1, and MAA-2 (high molecular weight melanoma-associated antigens). The antigenic profile of immunoreactive pigment cells was compared with the stage of tumor progression. Our data show consistent antigenic profiles of primary melanomas and their metastases within the same patient. Expression of tyrosinase-related protein and pigmentation-associated antigen was observed in the radial growth phase of primary melanomas but showed diminished or complete loss of expression in the vertical growth phase and in metastatic melanomas. HLA-DR was negative in the most primary lesions, but melanoma cells and a larger proportion of immunoreactive cells were observed at the metastatic site. The melanoma-associated antigens MAA-1 and MAA-2 were expressed throughout tumor progression. Although no clear distinction could be made between primary and secondary melanoma lesions for both melanoma-associated antigens, there was a profound variability in the topographical antigen distribution when compared with HLA-DR. The loss of expression of pigmentation-associated antigen and tyrosinase-related protein in the vertical growth phase of the primary lesions and metastatic melanomas did not reach statistical significance but still may be related to tumor progression. This indicates that primary melanomas can be distinguished from their metastases by evaluation of the antigenic profile and in this respect facilitate the recognition of tumor progression stages.

Attenuation of melanoma invasion by a secreted variant of activated leukocyte cell adhesion molecule.

Activated leukocyte cell adhesion molecule (ALCAM/CD166/MEMD), a marker of various cancers and mesenchymal stem cells, is involved in melanoma metastasis. We have exploited a secreted NH(2)-terminal fragment, sALCAM, to test the hypothesis that ALCAM coordinates tissue growth and cell migration. Overexpression of sALCAM in metastatic melanoma cells disturbed clustering of endogenous ALCAM and inhibited activation of matrix metalloproteinase-2 (MMP-2). Exposure of HT1080 fibrosarcoma cells to sALCAM similarly inhibited MMP-2, suggesting a broader effect on ALCAM-positive tumor cells. In contrast to the previously reported, promotive effects of an NH(2)-terminally truncated, transmembrane variant (DeltaN-ALCAM), sALCAM impaired the migratory capacity of transfected cells in vitro, reduced basement membrane penetration in reconstituted human skin equivalents, and diminished metastatic capacity in nude mice. Remarkably, L1 neuronal cell adhesion molecule (L1CAM/CD171), another progression marker of several cancers including melanoma, was suppressed upon sALCAM overexpression but was up-regulated by DeltaN-ALCAM. The partially overlapping and opposite effects induced by alternative strategies targeting ALCAM functions collectively attribute an integrative role to ALCAM in orchestrating cell adhesion, growth, invasion, and proteolysis in the tumor tissue microenvironment and disclose a therapeutic potential for sALCAM.

Activated leukocyte cell adhesion molecule (ALCAM/CD166): signaling at the divide of melanoma cell clustering and cell migration?

Orchestrated modulation of cell adhesion is essential for development and homeostasis in multicellular organisms. It optimizes embedding of the cell in its dynamic environment and facilitates appropriate cell responses and intercellular communication. Chronic disturbance of this delicate equilibrium causes defects in tissue architecture and sometimes cancer. In tumor cell biology, dynamic control of adhesion molecules is important to proceed through the metastatic cascade and to allow cell release from the primary tumor, invasion of the surrounding matrix, intravasation and adhesion to vascular endothelial cells to facilitate extravasation. Intertwined and multiple adhesive interactions rather than individual interactions presumably play critical roles in neoplastic development. Yet, knowledge of the contribution of each individual adhesion molecule is essential to unravel this network of interactions. This review will focus on activated leukocyte cell adhesion molecule (ALCAM/CD166) and its role in human melanoma progression. It is hypothesized that ALCAM may function as a cell surface sensor to register local growth saturation and to regulate cellular signaling and dynamic responses.

Gene expression patterns in melanocytic cells: candidate markers for early stage and malignant transformation.

Different stages of differentiation of human melanocytic cells, such as normal melanocytes, naevus and melanoma cells, reflect distinct gene expression patterns. A PCR-based subtractive hybridization and display method was applied to identify genes that are differentially expressed in melanocytic cells in relation to early stage and malignant transformation. This resulted in the identification of a number of candidate cDNAs differentially expressed among melanocytes, naevus cells, and (non)-metastatic melanoma cells. Out of this collection of cDNAs, 16 clones were screened that comprised 12 novel genes, one previously identified expressed sequence tag related to vesicular trafficking (Ras-related protein Rab5b). The other three were also known genes that were either related to cell motility (beta-tubulin), pre-mRNA splicing (small nuclear protein U1A), or of unknown function (the human TI227-H gene). The differential expression patterns of Rab5b and two novel gene fragments (pCMa1, pCMn2) were further assessed in melanocytic cells. pCMa1 was expressed more in metastatic melanoma than in primary melanoma cells. In contrast, pCMn2 was expressed in both non-metastatic and metastatic melanoma cells, but was not detectable in either normal melanocytes or naevus cells. The Ras-related protein Rab5b showed lower levels of expression in highly metastatic than in other melanoma cells. These three cDNAs may therefore be involved in the early stage and malignant transformation of melanocytes.

Multiple complementary transcripts of pCMa1, a novel gene located at chromosome 11p15.1-2, and melanocytic cell transformation.

The cDNA clone pCMa1 (0.45 kb) is one of the 12 novel cDNAs, previously identified when comparing RNA expression profiles of melanocytes, naevus cells, and non-metastatic melanoma cells. This clone did not reveal a unique long open reading frame. The pCMa1 gene localized to the distal, telomere proximal region on the short arm of chromosome 11.p15.1-2. Northern blot analyses with single-stranded cRNA probes revealed the presence of various complementary pCMa1 transcripts of different lengths, which are not enriched in the poly(A)(+) RNA fraction. The arbitrarily defined plus strand (used as a probe) mainly hybridized to 0.45 kb and 4.0 kb minus transcripts in total RNA samples, and the minus strand (used as a probe) hybridized to a major plus transcript of 4.0 kb. By RNA in situ hybridization, the highest levels of the plus transcripts were observed in melanocytic naevi (12/12), particularly in congenital naevi, whereas normal skin melanocytes (12/12) were negative. pCMa1 plus transcripts were detected in naevus cell nests (100%) near the dermo-epidermal junction. Expression, however, diminished to some extent in the deeper parts of the melanocytic naevi. Although most of the cutaneous primary melanoma lesions (11/15) showed detectable, but variable levels of plus transcripts of pCMa1 in the papillary to reticular dermis, not more than 10% of the melanoma cells were positive. The majority of melanoma metastases (6/7) were negative, while the positive lesion originated from a patient with a positive primary melanoma. Furthermore, plus transcripts were present in the nuclei of non-metastatic melanoma cells in culture, whereas metastatic cells showed elevated expression both in the nucleus and in the cytoplasm. Briefly, the data show transient up-regulation of pCMa1 in neoplastic progression of melanocytic cells, with peak levels occurring during naevus stages, and suggest that pCMa1 is a molecular marker in melanocytes for the early changes from the proliferating phenotype to malignant transformation.