Protein tyrosine phosphatase 4A3 (PTP4A3/PRL-3) promotes the aggressiveness of human uveal melanoma through dephosphorylation of CRMP2.

Uveal melanoma (UM) is an aggressive tumor in which approximately 50% of patients develop metastasis. Expression of the PTP4A3 gene, encoding a phosphatase, is predictive of poor patient survival. PTP4A3 expression in UM cells increases their migration in vitro and invasiveness in vivo. Here, we show that CRMP2 is mostly dephosphorylated on T514 in PTP4A3 expressing cells. We also demonstrate that inhibition of CRMP2 expression in UM cells expressing PTP4A3 increases their migration in vitro and invasiveness in vivo. This phenotype is accompanied by modifications of the actin microfilament network, with shortened filaments, whereas cells with a inactive mutant of the phosphatase do not show the same behavior. In addition, we showed that the cell cytoplasm becomes stiffer when CRMP2 is downregulated or PTP4A3 is expressed. Our results suggest that PTP4A3 acts upstream of CRMP2 in UM cells to enhance their migration and invasiveness and that a low level of CRMP2 in tumors is predictive of poor patient survival.

PRL-3/PTP4A3 phosphatase regulates integrin ß1 in adhesion structures during migration of human ocular melanoma cells.

In a previous transcriptomic analysis of 63 ocular melanomas of the uvea, we found that expression of the PRL-3/PTP4A3 gene, encoding a phosphatase that is anchored to the plasma membrane, was associated with the risk of metastasis, and a poor prognosis. We also showed that PRL-3 overexpression in OCM-1 ocular melanoma cells significantly increased cell migration in vitro and invasiveness in vivo, suggesting a direct role for PRL-3 in the metastatic spreading of uveal melanoma. Here, we aimed to identify PRL-3 substrates at the plasma membrane involved in adhesion to the extracellular matrix. We focused on integrin ß1, which is the most highly expressed integrin in our cohort of uveal melanomas. We show that preventing PRL-3 anchorage to the plasma membrane i) abolishes PRL-3-induced migration in OCM-1 cells, ii) specifically enhances the spreading of OCM-1 cells overexpressing PRL-3, and iii) favors the maturation of large focal adhesions (FAs) containing integrin ß1 on collagen I. Knockdown experiments confirmed integrin ß1 involvement in PRL3-induced migration. We identified interactions between PRL-3 and integrin ß1, as well as with FAK P-Y397, an auto-activated form of Focal Adhesion Kinase found in FAs. We also show that integrin ß1 may be dephosphorylated by PRL-3 in its intracytoplasmic S/T region, an important motif for integrin-mediated cell adhesion. Finally, we observed that PRL-3 regulated the clustering of integrin ß1 in FAs on collagen I but not on fibronectin. This work identifies PRL-3 as a new regulator of cell adhesion structures to the extracellular matrix, and further supports PRL-3 as a key actor of metastasis in uveal melanoma, of which molecular mechanisms are still poorly understood.

Protein Tyrosine Phosphatase 4A3 (PTP4A3) Promotes Human Uveal Melanoma Aggressiveness Through Membrane Accumulation of Matrix Metalloproteinase 14 (MMP14).

PURPOSE: To study PTP4A3 phosphatase and MMP14 metalloprotease synergy in uveal melanoma aggressiveness. METHODS: Cell membrane localization of matrix metalloprotease 14 (MMP14) in uveal melanoma cells expressing protein tyrosine phosphatase A3 (PTP4A3) was assessed by flow cytometry or immunohistochemistry. The vesicular trafficking of MMP14 in the presence of PTP4A3 was evaluated in OCM-1 cells expressing either the wild-type or mutated phosphatase. Finally, MMP14 localization at the cell membrane of OCM-1 cells was impaired using RNA interference, and the PTP4A3-related migration in vitro and invasiveness in vivo of the treated cells were evaluated. RESULTS: We found that the membrane-anchored MMP14 is enriched at the cell surface of OCM-1 cells, patient-derived xenograft cells, and human primary uveal melanoma tumors expressing PTP4A3. Moreover, we show that PTP4A3 and MMP14 colocalize and that the vesicular trafficking of MMP14 is faster in the presence of active PTP4A3. Finally, we demonstrate that inhibition of MMP14 expression in uveal melanoma cells expressing PTP4A3 impairs their migration in vitro and invasiveness in vivo. CONCLUSIONS: Our observations indicate that PTP4A3 increases cell membrane accumulation of MMP14 as a result of increased cellular trafficking of the metalloprotease. We also show that downregulation of MMP14 expression reduced PTP4A3-induced cell migration and invasiveness. Taken together, our findings suggest that PTP4A3-related subcellular localization of MMP14 is an important event in metastasis induction.

Protein tyrosine phosphatase 4A3 (PTP4A3) is required for Xenopus laevis cranial neural crest migration in vivo.

Uveal melanoma is the most common intraocular malignancy in adults, representing between about 4% and 5% of all melanomas. High expression levels of Protein Tyrosine Phosphatase 4A3, a dual phosphatase, is highly predictive of metastasis development and PTP4A3 overexpression in uveal melanoma cells increases their in vitro migration and in vivo invasiveness. Melanocytes, including uveal melanocytes, are derived from the neural crest during embryonic development. We therefore suggested that PTP4A3 function in uveal melanoma metastasis may be related to an embryonic role during neural crest cell migration. We show that PTP4A3 plays a role in cephalic neural crest development in Xenopus laevis. PTP4A3 loss of function resulted in a reduction of neural crest territory, whilst gain of function experiments increased neural crest territory. Isochronic graft experiments demonstrated that PTP4A3-depleted neural crest explants are unable to migrate in host embryos. Pharmacological inhibition of PTP4A3 on dissected neural crest cells significantly reduced their migration velocity in vitro. Our results demonstrate that PTP4A3 is required for cephalic neural crest migration in vivo during embryonic development.

High PTP4A3 phosphatase expression correlates with metastatic risk in uveal melanoma patients.

A high percentage of uveal melanoma patients develop metastatic tumors predominantly in the liver. We studied the molecular profiles derived from gene expression microarrays and comparative genomic hybridization microarrays, to identify genes associated with metastasis in this aggressive cancer. We compared 28 uveal melanomas from patients who developed liver metastases within three years of enucleation with 35 tumors from patients without metastases or who developed metastases more than 3 years after enucleation. Protein tyrosine phosphatase type IV A member 3 (PTP4A3/PRL3), was identified as a strong predictor of metastasis occurrence. We demonstrated that the differential expression of this gene, which maps to 8q24.3, was not merely a consequence of 8q chromosome overrepresentation. PTP4A3 overexpression in uveal melanoma cell lines significantly increased cell migration and invasiveness in vivo, suggesting a direct role for this protein in metastasis. Our findings suggest that PTP4A3 or its cellular substrates could constitute attractive therapeutic targets to treat metastatic uveal melanomas.

Matricellular protein CCN3 (NOV) regulates actin cytoskeleton reorganization.

CCN3 (NOV), a putative ligand for integrin receptors, is tightly associated with the extracellular matrix and mediates diverse cellular functions, including cell adhesion and proliferation. CCN3 has been shown to negatively regulate growth although it promotes migration in a cell type-specific manner. In this study, overexpression of CCN3 reduces growth and increases intercellular adhesion of breast cancer cells. Interestingly, CCN3 overexpression also led to the formation of multiple pseudopodia that are enriched in actin, CCN3, and vinculin. Breast cancer cells preincubated with exogenous CCN3 protein also induced the same phenotype, indicating that secreted CCN3 is sufficient to induce changes in cell morphology. Surprisingly, extracellular CCN3 is internalized to the early endosomes but not to the membrane protrusions, suggesting pseudopodia-enriched CCN3 may derive from a different source. The presence of an intracellular variant of CCN3 will be consistent with our finding that the cytoplasmic tail of the gap junction protein connexin43 (Cx43) associates with CCN3. Cx43 is a channel protein permitting intercellular communication to occur. However, neither the channel properties nor the protein levels of Cx43 are affected by the CCN3 protein. In contrast, CCN3 proteins are down-regulated in the absence of Cx43. Finally, we showed that overexpression of CCN3 increases the activity of the small GTPase Rac1, thereby revealing a pathway that links Cx43 directly to actin reorganization.

Domain-specific CCN3 antibodies as unique tools for structural and functional studies.

CCN3 is a member of the CCN family of cell growth and differentiation regulators that play key roles during embryonic development, and are associated with severe human pathologies. The level of CCN genes' expression is of prognostic value in several types of tumors. In the present manuscript, we report the isolation and characterization of a new set of antibodies targeted against each individual module of the human CCN3 protein. The need for module-specific antibodies stemmed from recent reports indicating that the expression of truncated CCN variant proteins was associated with development of cancers. Each of the four CCN3 modules were expressed as GST fusion proteins and used for rabbits immunization. Polyclonal IgGs purified by two rounds of affinity-chromatography specifically detected both the individual CCN3 domains and the full length CCN3 protein expressed in mammalian cell lines and tissues, as well as recombinant full length and truncated CCN3 proteins. The purified module-specific antibodies were successfully used for Western blotting, immunoprecipitation, immunofluorescence and immunocytochemistry. These antibodies permitted the detection of CCN3 proteins under native and denaturing conditions, and confirmed the sublocalisation of CCN3 proteins in the extracellular compartment, at the cell membrane, in the cytoplasm and in the nucleus of positive cells. Immunocytochemistry and Western blotting studies performed with the module-specific antibodies identified truncated CCN3 proteins in kidney tumor samples. The detection of these rearranged variants provides clues for their involvement in tumorigenesis. Therefore, these antibodies constitute unique tools for the identification of truncated CCN3 proteins in human tissues and may be of great interest in molecular medicine.

CCN3 controls 3D spatial localization of melanocytes in the human skin through DDR1.

Melanocytes reside within the basal layer of the human epidermis, where they attach to the basement membrane and replicate at a rate proportionate to that of keratinocytes, maintaining a lifelong stable ratio. In this study, we report that coculturing melanocytes with keratinocytes up-regulated CCN3, a matricellular protein that we subsequently found to be critical for the spatial localization of melanocytes to the basement membrane. CCN3 knockdown cells were dissociated either upward to the suprabasal layers of the epidermis or downward into the dermis. The overexpression of CCN3 increased adhesion to collagen type IV, the major component of the basement membrane. As the receptor responsible for CCN3-mediated melanocyte localization, we identified discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase that acts as a collagen IV adhesion receptor. DDR1 knockdown decreased melanocyte adhesion to collagen IV and shifted melanocyte localization in a manner similar to CCN3 knockdown. These results demonstrate an intricate and necessary communication between keratinocytes and melanocytes in maintaining normal epidermal homeostasis.

Nuclear trafficking of secreted factors and cell-surface receptors: new pathways to regulate cell proliferation and differentiation, and involvement in cancers.

Secreted factors and cell surface receptors can be internalized by endocytosis and translocated to the cytoplasm. Instead of being recycled or proteolysed, they sometimes translocate to the nucleus. Nuclear import generally involves a nuclear localization signal contained either in the secreted factor or its transmembrane receptor, that is recognized by the importins machinery. In the nucleus, these molecules regulate transcription of specific target genes by direct binding to transcription factors or general coregulators. In addition to the transcription regulation, nuclear secreted proteins and receptors seem to be involved in other important processes for cell life and cellular integrity such as DNA replication, DNA repair and RNA metabolism. Nuclear secreted proteins and transmembrane receptors now appear to induce new signaling pathways to regulate cell proliferation and differentiation. Their nuclear localization is often transient, appearing only during certain phases of the cell cycle. Nuclear secreted and transmembrane molecules regulate the proliferation and differentiation of a large panel of cell types during embryogenesis and adulthood and are also potentially involved in wound healing. Secreted factors such as CCN proteins, EGF, FGFs and their receptors are often detected in the nucleus of cancer cells. Nuclear localization of these molecules has been correlated with tumor progression and poor prognosis for patient survival. Nuclear growth factors and receptors may be responsible for resistance to radiotherapy.

A novel mechanism for BCR-ABL action: stimulated secretion of CCN3 is involved in growth and differentiation regulation.

Chronic myeloid leukemia (CML) is characterized by the presence of the constitutively active BCR-ABL protein tyrosine kinase. Using a multipotent hemopoietic cell line, FDCP-Mix, expressing BCR-ABL tyrosine kinase, we investigated the initial effects of this kinase in primitive hematopoietic stem cells. We identified down-regulation of a novel gene, CCN3, as a direct consequence of BCR-ABL kinase activity. CCN3 has been reported to function as a tumor suppressor gene in solid tumors. Northern and Western blotting plus immunocytochemical analysis confirmed CCN3 expression is decreased and is tyrosine-phosphorylated in BCR-ABL kinase active FDCP-Mix cells. Decreased cellular CCN3 correlated with increased CCN3 secretion in BCR-ABL kinase active cells. In vitro treatment of human CML cell lines with imatinib or siRNA directed against BCR-ABL significantly reduced BCR-ABL while increasing CCN3 expression. Cells from patients responding to imatinib showed a similar decrease in BCR-ABL and increase in CCN3. CML CD34+ cells treated with imatinib in vitro demonstrated increased CCN3 protein. Transfecting CCN3 into BCR-ABL+ cells inhibited proliferation and decreased clonogenic potential. CCN3 plays an important role in internal and external cell-signaling pathways. Thus, BCR-ABL can regulate protein levels by governing secretion, a novel mechanism for this tyrosine kinase.

Nuclear addressing provides a clue for the transforming activity of amino-truncated CCN3 proteins.

CCN3 is a founding member of the CCN (Cyr61, Ctgf, Nov) family of cell growth and differentiation regulators. These secreted proteins are key regulators in embryonic development, and are associated with severe pathologies including fibrotic diseases and cancers. CCN3 was discovered as a MAV integration site in an avian nephroblastoma. Previous work established that the amino-truncated protein expressed in this tumor was inducing morphological transformation of chicken embryo fibroblasts, whereas the full-length secreted CCN3 protein was inhibiting cell growth. Amino-truncated variants were identified in cancer cell lines. Since the lack of signal peptide was expected to alter the fate of the truncated proteins, we hypothesized that modifications of CCN3 subcellular addressing could be responsible for the oncogenic activities of CCN3. The CCN proteins are composed of four structural modules (IGFBP, TSP1, VWC, and CT). We report that amino-truncated variants of CCN3 are addressed to the nucleus and that the carboxyterminal (CT) module of CCN3 is responsible for the nuclear addressing. Furthermore, our data identify nuclear CCN3 variants as potential transcriptional regulators. In this context, the CT module confers on nuclear CCN3 proteins a negative regulatory effect on transcription. We propose that the nuclear localization of amino-truncated CCN3 proteins be correlated to oncogenicity.

CCN proteins and cancer: two to tango.

The CCN genes encode secreted proteins, associated to the extracellular matrix. They are involved in diverse biological processes such as regulation of cell- adhesion, migration, proliferation, differentiation and survival. They play important roles in pregnancy, development, angiogenesis, wound repair and inflammation. Several lines of evidence support a role for CCN genes in fibrotic disorders and tumorigenesis. We will focus our attention in this review on two CCN proteins: CCN1 and CCN3, that appear to exert distinct and opposite effects. Recent data suggest that CCN1 acts as a tumor-promoting factor and a key regulator in cancer progression, while CCN3 exhibits suppressive capabilities. The possible opposite functions of CCN1 and CCN3 in tumorigenesis, and the relevance of the distinct expression profiles of these two genes observed in many cancers are discussed below.

Microphthalmia transcription factor induces both retinal pigmented epithelium and neural crest melanocytes from neuroretina cells.

Mitf encodes a basic helix-loop-helix transcription factor that plays an essential role in the differentiation of the retinal pigmented epithelium (RPE) and neural crest-derived melanocytes. As cells containing melanogenic enzymes (TRP2) are found in Mitf mouse mutants, it is not clear whether Mitf is a downstream factor or a master regulator of melanocyte differentiation. To further study the role of Mitf in committing cells to the melanocyte lineage, we express Mitf in the cultured quail neuroretina cells. This leads to the induction of two types of pigmented cells: neural crest-derived melanocytes, according to their dendritic morphology, physiology, and gene expression pattern are observed together with pigmented epithelial RPE-like cells. The expression of Mitf is lower in pigmented epithelial RPE-like cells than in neural crest-derived melanocytes. Accordingly, overexpression of Mitf in cultured quail RPE causes cells to develop into neural crest-like pigmented cells. Thus, Mitf is sufficient for the proper differentiation of crest-like pigmented cells from retinal cells and its expression level may determine the type of pigment cell induced.

CCN3 and calcium signaling.

The CCN family of genes consists presently of six members in human (CCN1-6) also known as Cyr61 (Cystein rich 61), CTGF (Connective Tissue Growth Factor), NOV (Nephroblastoma Overexpressed gene), WISP-1, 2 and 3 (Wnt-1 Induced Secreted Proteins). Results obtained over the past decade have indicated that CCN proteins are matricellular proteins, which are involved in the regulation of various cellular functions, such as proliferation, differentiation, survival, adhesion and migration. The CCN proteins have recently emerged as regulatory factors involved in both internal and external cell signaling. CCN3 was reported to physically interact with fibulin-1C, integrins, Notch and S100A4. Considering that, the conformation and biological activity of these proteins are dependent upon calcium binding, we hypothesized that CCN3 might be involved in signaling pathways mediated by calcium ions.In this article, we review the data showing that CCN3 regulates the levels of intracellular calcium and discuss potential models that may account for the biological effects of CCN3.

A structural approach to the role of CCN (CYR61/CTGF/NOV) proteins in tumourigenesis.

The CCN (CYR61 [Cystein-rich61]/CTGF [connective tissue growth factor]/NOV [Nephroblastoma overexpressed]) proteins constitute a family of regulatory factors involved in many aspects of cell proliferation and differentiation. An increasing body of evidence indicates that abnormal expression of the CCN proteins is associated to tumourgenesis. The multimodular architecture of the CCN proteins, and the production of truncated isoforms in tumours, raise interesting questions regarding the participation of each individual module to the various biological properties of these proteins. In this article, we review the current data regarding the involvement of CCN proteins in tumourigenesis. We also attempt to provide structural basis for the stimulatory and inhibitory functions of the full length and truncated CCN proteins that are expressed in various tumour tissues.

Microphthalmia transcription factor analysis in posterior uveal melanomas.

PURPOSE: The protein encoded by the Microphthalmia gene (MITF) is a transcription factor essential for the development and survival of melanocytes. It serves as a master regulator in modulating extracellular signals. Because of its central role in melanocytes survival and to assess its potential use as a histopathological marker for melanoma, MITF expression was examined in human choroidal melanomas. METHODS: Fifty-seven paraffin-embedded sections of choroidal melanoma specimens and 1 choroidal melanoma cell line were analyzed using immunochemistry and RT-PCR. Normal choroids and normal choroidal melanocyte cells were used as control. RESULTS: Sixty-five percent of the tumoral specimens stained positively for MITF with a predominant nuclear pattern of reactivity. MITF-M and MITF-A isoforms were detected by RT-PCR in all specimens examined. Using a chimeric protein resulting from the fusion of each Mitf protein with the GFP, Mitf-M exhibited an exclusive nuclear staining whereas Mitf-A exhibited a mixed nuclear and cytoplasmic staining. No correlation between MITF-positivity and parameters such as cell type, largest tumor diameter, sclera invasion, mitotic figures was observed. In contrast, a significant negative association was found between MITF staining and the pigmentation (p=0.02) and a positive correlation between MITF staining and the proliferative marker Ki67 was found (p=0.02). CONCLUSION: MITF may be implicated in choroidal melanoma pigmentation and proliferation. Further analysis should provide new insights into the mechanisms underlying the molecular and cellular changes of choroidal melanomas.

Activated MAPK/ERK kinase (MEK-1) induces transdifferentiation of pigmented epithelium into neural retina.

During vertebrate eye development, the optic vesicle originating from the neuroectoderm is partitioned into a domain that will give rise to the neural retina (NR) and another that will give rise to the retinal pigmented epithelium (RPE). Previous studies have shown that ectopic expression of FGFs in the RPE induces RPE-to-NR transdifferentiation. Similarly, a naturally occurring mutation of the transcription factor Mitf in mouse resulted in the formation of a second neural retina in place of the dorsal RPE, but the putative signaling pathway linking FGF to Mitf regulation is presently unknown. In cultures of neural crest-derived melanocytes, the MAPK pathway was recently shown to target the Mitf transcription factor for ubiquitin-dependent proteolysis, resulting in a rapid degradation and downregulation. In the present study, we show that ectopic expression of a constitutively activated allele of MEK-1, the immediate upstream activator of the MAPK ERK, in chicken embryonic retina in ovo, induces transdifferentiation of the RPE into a neural-like epithelium that is correlated with a downregulation of Mitf expression in the presumptive RPE.

O-glycosylation of the nuclear forms of Pax-6 products in quail neuroretina cells.

Many transcription factors are demonstrated as being glycosylated with O-N-acetylglucosamine (GlcNAc) residue in transfected insect cell lines, but rarely in the original cells. For the first time, we demonstrate the O-GlcNAc modification of the p48/p46 Pax-6 gene (a developmental control gene involved in the eye morphogenesis) products in the quail neuroretina (QNR). In conjunction with a systematic PNGase F treatment, we used wheat germ agglutinin (WGA) binding, in vitro labeling with bovine galactosyltransferase, and labeling of cultured QNR with [14C]GlcNH2. Glycosylated forms of Pax-6 proteins were found in the nucleus of the neuroretina cells. WGA-selected Pax-6 proteins produced in the reticulocyte lysate were able to bind a DNA target, as well as to the unglycosylated form. The O-GlcNAc may, however, modulate protein interactions, mainly with other factors involved in the transcription process. Characterization of products released after reductive alkaline treatment of the proteins clearly demonstrates that N-acetylglucosamine is directly linked to serine or threonine residues. Examination of Pax-6 primary sequence allowed us to determine potential O-GlcNAc attachment sites. Most of these expected glycosylation sites appear to be located on the two DNA binding domains and on the carboxyterminal transactivation domain, while experimental evidence taken from WGA-selected proteins experiment points in favor of a main localization on the paired-box domain.