Plakoglobin: Role in Tumorigenesis and Metastasis

Plakoglobin (γ-catenin) is a member of the Armadillo family of proteins and a homolog of β-catenin. As a component of both the adherens junctions and desmosomes, plakoglobin plays a pivotal role in the regulation of cell-cell adhesion. Furthermore, similar to β-catenin, plakoglobin is capable of participating in cell signaling. However, unlike β-catenin that has well-documented oncogenic potential through its involvement in the Wnt signaling pathway, plakoglobin generally acts as a tumor/metastasis suppressor. The exact roles that plakoglobin plays during tumorigenesis and metastasis are not clear; however, recent evidence suggests that it may regulate gene expression, cell proliferation, apoptosis, invasion, and migration. In this paper, we describe plakoglobin, its discovery and characterization, its role in regulating cell-cell adhesion, and its signaling capabilities in regulation of tumorigenesis and metastasis

A role for Dynlt3 in melanosome movement, distribution, acidity and transfer

Skin pigmentation is dependent on cellular processes including melanosome biogenesis, transport, maturation and transfer to keratinocytes. However, how the cells finely control these processes in space and time to ensure proper pigmentation remains unclear. Here, we show that a component of the cytoplasmic dynein complex, Dynlt3, is required for efficient melanosome transport, acidity and transfer. In Mus musculus melanocytes with decreased levels of Dynlt3, pigmented melanosomes undergo a more directional motion, leading to their peripheral location in the cell. Stage IV melanosomes are more acidic, but still heavily pigmented, resulting in a less efficient melanosome transfer. Finally, the level of Dynlt3 is dependent on beta -catenin activity, revealing a function of the Wnt/beta -catenin signalling pathway during melanocyte and skin pigmentation, by coupling the transport, positioning and acidity of melanosomes required for their transfer. Aktary et al. identify novel roles for the dynein light chain Dynlt3 in melanosome transport, maturation, and transfer to keratinocytes. They also find that the Wnt/beta catenin signalling pathway controls Dynlt3 levels and thus also contributes to the regulation of melanocyte transport and skin pigmentation

Caractérisation moléculaire de l’activité suppresseur de tumeur/métastases de plakoglobine

Plakoglobin (γ-catenin) is a member of the Armadillo family of proteins and a homolog of β-catenin with similar dual adhesive and signaling functions. The adhesive function of these proteins is mediated by their interactions with cadherins and their signaling function by association with various intracellular proteins, from signaling molecules to transcription factors. However, while β-catenin has well-documented oncogenic potential, plakoglobin signaling capabilities are typically associated with tumor/metastasis suppression through mechanisms that have remained unclear. The focus of this thesis was to elucidate the molecular mechanisms by which plakoglobin regulates tumorigenesis and metastasis. To this end, we expressed plakoglobin in plakoglobin-null human carcinoma cells and compared the mRNA and protein profiles of plakoglobin expressing cells with those of parental cells. We identified a number of oncogenes and tumor/metastasis suppressors whose mRNA/protein levels were decreased and increased, respectively, upon plakoglobin expression. Extensive characterization of the plakoglobin expressing cells showed that plakoglobin regulates tumorigenesis and metastasis by interacting with and altering the levels, localization and/or function of growth/metastasis regulating proteins and/or by associating with transcription factors that regulate the expression of genes involved in these processes. Plakoglobin interacted with and increased both the protein and mRNA levels of the metastasis suppressor Nm23-H1 while only increasing the protein levels of Nm23-H2. Furthermore, in plakoglobin expressing cells, Nm23-H1/H2 complex was redistributed from the cytoplasm to the adherens junction at the membrane.We also showed that plakoglobin interacted with p53 and together they regulated the expression of a number of p53-target genes, including tumor suppressors SFN and NME1 and the tumor promoter SATB1. Concurrent with these changes, there was a significant decrease in cell proliferation and in vitro migration and invasion of plakoglobin expressing cells.These results clearly demonstrate that plakoglobin plays an active role in suppressing tumorigenesis/metastasis through both the regulation of gene expression and by interacting with and altering the levels, localization and function of various intracellular proteins involved in these processes. The larger implication of this work is that plakoglobin may be a useful marker for diagnosis and prognosis as well as a therapeutic target for the treatment of various cancers.La plakoglobine (γ-caténine) est un membre de la famille des protéines Armadillo et un homologue de la β-caténine avec des fonctions adhésives et de signalisation doubles similaires. La fonction adhésive de ces protéines est médiée par leurs interactions avec les cadhérines et leur fonction de signalisation par association avec diverses protéines intracellulaires, des molécules de signalisation aux facteurs de transcription. Cependant, alors que la β-caténine a un potentiel oncogène bien documenté, les capacités de signalisation de la plakoglobine sont généralement associées à la suppression des tumeurs / métastases par des mécanismes qui sont restés peu clairs. L'objectif de cette thèse était d'élucider les mécanismes moléculaires par lesquels la plakoglobine régule la tumorigenèse et les métastases. À cette fin, nous avons exprimé la plakoglobine dans des cellules de carcinome humain nul en plakoglobine et comparé les profils d'ARNm et de protéines des cellules exprimant la plakoglobine avec ceux des cellules parentales. Nous avons identifié un certain nombre d'oncogènes et de suppresseurs de tumeurs / métastases dont les niveaux d'ARNm / protéines ont été diminués et augmentés, respectivement, lors de l'expression de la plakoglobine. Une caractérisation approfondie des cellules exprimant la plakoglobine a montré que la plakoglobine régule la tumorigenèse et les métastases en interagissant avec et en modifiant les niveaux, la localisation et / ou la fonction des protéines régulant la croissance / métastase et / ou en s'associant à des facteurs de transcription qui régulent l'expression des gènes impliqués dans ces protéines. processus. La plakoglobine a interagi avec et augmenté les niveaux de protéines et d'ARNm du suppresseur de métastases Nm23-H1 tout en augmentant seulement les niveaux de protéines de Nm23-H2. En outre, dans les cellules exprimant la plakoglobine, le complexe Nm23-H1 / H2 a été redistribué du cytoplasme à la jonction adhérente au niveau de la membrane.Nous avons également montré que la plakoglobine interagissait avec p53 et ensemble, ils régulaient l'expression d'un certain nombre de gènes cibles p53, y compris les suppresseurs de tumeur SFN et NME1 et le promoteur de tumeur SATB1. Parallèlement à ces changements, il y avait une diminution significative de la prolifération cellulaire et de la migration in vitro et de l'invasion des cellules exprimant la plakoglobine.Ces résultats démontrent clairement que la plakoglobine joue un rôle actif dans la suppression de la tumorigenèse / métastase à la fois par la régulation de l'expression génique et en interagissant avec et en modifiant les niveaux, la localisation et la fonction de diverses protéines intracellulaires impliquées dans ces processus. L'implication la plus large de ces travaux est que la plakoglobine peut être un marqueur utile pour le diagnostic et le pronostic ainsi qu'une cible thérapeutique pour le traitement de divers cancers

Plakoglobin represses SATB1 expression and decreases in vitro proliferation, migration and invasion.

Plakoglobin (γ-catenin) is a homolog of β-catenin with dual adhesive and signaling functions. Plakoglobin participates in cell-cell adhesion as a component of the adherens junction and desmosomes whereas its signaling function is mediated by its interactions with various intracellular protein partners. To determine the role of plakoglobin during tumorigenesis and metastasis, we expressed plakoglobin in the human tongue squamous cell carcinoma (SCC9) cells and compared the mRNA profiles of parental SCC9 cells and their plakoglobin-expressing transfectants (SCC9-PG). We observed that the mRNA levels of SATB1, the oncogenic chromatin remodeling factor, were decreased approximately 3-fold in SCC9-PG cells compared to parental SCC9 cells. Here, we showed that plakoglobin decreased levels of SATB1 mRNA and protein in SCC9-PG cells and that plakoglobin and p53 associated with the SATB1 promoter. Plakoglobin expression also resulted in decreased SATB1 promoter activity. These results were confirmed following plakoglobin expression in the very low plakoglobin expressing and invasive mammary carcinoma cell line MDA-MB-231 cells (MDA-231-PG). In addition, knockdown of endogenous plakoglobin in the non-invasive mammary carcinoma MCF-7 cells (MCF-7-shPG) resulted in increased SATB1 mRNA and protein. Plakoglobin expression also resulted in increased mRNA and protein levels of the metastasis suppressor Nm23-H1, a SATB1 target gene. Furthermore, the levels of various SATB1 target genes involved in tumorigenesis and metastasis were altered in MCF-7-shPG cells relative to parental MCF-7 cells. Finally, plakoglobin expression resulted in decreased in vitro proliferation, migration and invasion in different carcinoma cell lines. Together with the results of our previous studies, the data suggests that plakoglobin suppresses tumorigenesis and metastasis through the regulation of genes involved in these processes

Pasdar M. Plakoglobin: role in tumorigenesis and metastasis

Plakoglobin (γ-catenin) is a member of the Armadillo family of proteins and a homolog of β-catenin. As a component of both the adherens junctions and desmosomes, plakoglobin plays a pivotal role in the regulation of cell-cell adhesion. Furthermore, similar to β-catenin, plakoglobin is capable of participating in cell signaling. However, unlike β-catenin that has well-documented oncogenic potential through its involvement in the Wnt signaling pathway, plakoglobin generally acts as a tumor/metastasis suppressor. The exact roles that plakoglobin plays during tumorigenesis and metastasis are not clear; however, recent evidence suggests that it may regulate gene expression, cell proliferation, apoptosis, invasion, and migration. In this paper, we describe plakoglobin, its discovery and characterization, its role in regulating cell-cell adhesion, and its signaling capabilities in regulation of tumorigenesis and metastasis

Plakoglobin suppresses <i>SATB1</i> in mammary epithelial cell lines.

<p><b>A.</b> (Top) Total cellular RNA was isolated from MCF-7, MCF-7-shPG, MDA-231 and MDA-231-PG cells, reverse transcribed and processed for PCR using primers specific to SATB1 and GAPDH. (Bottom) Equal amounts of total cellular proteins from these cells were resolved by SDS-PAGE and processed for immunoblotting with antibodies to SATB1 and Actin.<b>B.</b> MCF-7, MDA-231 and MDA-231-PG cells were formaldehyde fixed and processed for chromatin immunoprecipitation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078388#pone-0078388-g001" target="_blank">Fig. 1B</a>. The purified DNA was then processed for PCR using SATB1 primers. As a positive control, total cellular DNA (Input) was amplified using the same primers. <b>C.</b> MCF-7, MCF-7-shPG, MDA-231 and MDA-231-PG cells were transfected with luciferase reporter constructs and processed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078388#pone-0078388-g001" target="_blank">Fig. 1C</a>. The <i>SATB1</i> promoter activity was normalized to the corresponding vector activity for each cell line and then normalized to MDA-231 or MCF-7, respectively (*p<0.01). PG, plakoglobin; RLU, Relative Light Units.</p

Plakoglobin associates with and activates <i>NME1</i>.

<p><b>A.</b> SCC9 and SCC9-PG cells were processed for chromatin immunoprecipitation using control IgG, plakoglobin and p53 antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078388#pone-0078388-g001" target="_blank">Fig. 1B</a>. The purified DNA was then processed for PCR using NME1 primers. As a positive control, total cellular DNA (Input) was amplified using the same primers. <b>B.</b> SCC9 and SCC9-PG cells were transfected with luciferase reporter constructs under the control of a 2 kb sequence of the <i>NME1</i> promoter. Luciferase activities were measured 48 hours post-transfection. The levels of luciferase activities from the vector and NME1 reporter constructs were determined from a minimum of three independent transfections and normalized for transfection efficiency by co-transfection with a β-galactosidase expression vector. The NME1 promoter activity was normalized to the corresponding vector activity for each cell line and then normalized to SCC9 (*p<0.01). PG, plakoglobin; RLU, Relative Light Units. <b>C.</b> (Top) Total cellular RNA was isolated from MCF-7, MCF-7-shPG, MDA-231 and MDA-231-PG cells, reverse transcribed and processed for PCR using primers specific to NME1, NME2 and GAPDH. (Bottom) Equal amounts of total cellular proteins from these cells were resolved by SDS-PAGE and processed for immunoblotting with antibodies to Nm23-H1, -H2 and Actin. <b>D.</b> MCF-7, MDA-231 and MDA-231-PG cells were processed for chromatin immunoprecipitation using control IgG, plakoglobin and p53 antibodies and the purified DNA processed for PCR using NME1 primers. As a positive control, total cellular DNA (Input) was amplified using the same primers. <b>E.</b> MCF-7, MCF-7-shPG, MDA-231 and MDA-231-PG cells were transfected with luciferase reporter constructs as described in Fig. 3B. The <i>NME1</i> promoter activity was normalized to the corresponding vector activity for each cell line and then normalized to MDA-231 or MCF-7, respectively (*p<0.01). PG, plakoglobin; RLU, Relative Light Units.</p

Plakoglobin knockdown changes the levels of SATB1 target genes.

<p><b>A.</b> Total cellular RNA was isolated from MCF-7 and MCF-7-shPG cells, reverse transcribed and processed for PCR using primers specific to SATB1 target genes c-Abl, MMP3, ErbB2, Snail, BRMS1, Kiss1 and Claudin-1. <b>B.</b> Equal amounts of total cellular proteins from these cells were resolved by SDS-PAGE and processed for immunoblotting with antibodies to c-Abl, MMP3, ErbB2, Snail, BRMS1, Kiss1 and Claudin-1. PG, plakoglobin.</p

Antibodies and their respective dilutions in specific assays.

<p>Antibodies and their respective dilutions in specific assays.</p