Syntenin Expression Research Articles

MDA-9/Syntenin and IGFBP-2 Promote Angiogenesis in Human Melanoma

Melanoma differentiation-associated gene-9 (mda-9/syntenin) encodes an adapter scaffold protein whose expression correlates with and mediates melanoma progression and metastasis. Tumor angiogenesis represents an integral component of cancer metastasis prompting us to investigate a possible role of mda-9/syntenin in inducing angiogenesis. Genetic (gain-of-function and loss-of-function) and pharmacologic approaches were used to modify mda-9/syntenin expression in normal immortal melanocytes, early radial growth phase melanoma, and metastatic melanoma cells. The consequence of modifying mda-9/syntenin expression on angiogenesis was evaluated using both in vitro and in vivo assays, including tube formation assays using human vascular endothelial cells, chorioallantoic membrane (CAM) assays and xenograft tumor animal models. Gain-of-function and loss-of-function experiments confirm that MDA-9/syntenin induces angiogenesis by augmenting expression of several proangiogenic factors/genes. Experimental evidence is provided for a model of angiogenesis induction by MDA-9/syntenin in which MDA-9/syntenin interacts with the extracellular matrix (ECM), activating Src and FAK resulting in activation by phosphorylation of Akt, which induces hypoxia inducible factor 1-α (HIF-1α). The HIF-1α activates transcription of insulin growth factor-binding protein-2 (IGFBP-2), which is secreted thereby promoting angiogenesis and further induces endothelial cells to produce and secrete VEGF-A augmenting tumor angiogenesis. Our studies delineate an unanticipated cell nonautonomous function of MDA-9/syntenin in the context of angiogenesis, which may directly contribute to its metastasis-promoting properties. As a result, targeting MDA-9/syntenin or its downstream-regulated molecules may provide a means of simultaneously impeding metastasis by both directly inhibiting tumor cell transformed properties (autonomous) and indirectly by blocking angiogenesis (nonautonomous).

Syntenin Is Expressed in Human Follicular Dendritic Cells and Involved in the Activation of Focal Adhesion Kinase

Syntenin is an adaptor molecule containing 2 PDZ domains which mediate molecular interactions with diverse integral or cytoplasmic proteins. Most of the results on the biological function of syntenin were obtained from studies with malignant cells, necessitating exploration into the role of syntenin in normal cells. To understand its role in normal cells, we investigated expression and function of syntenin in human lymphoid tissue and cells in situ and in vitro. Syntenin expression was denser in the germinal center than in the extrafollicular area. Inside the germinal center, syntenin expression was obvious in follicular dendritic cells (FDCs). Flow cytometric analysis with isolated cells confirmed a weak expression of syntenin in T and B cells and a strong expression in FDCs. In FDC-like cells, HK cells, most syntenin proteins were found in the cytoplasm compared to weak expression in the nucleus. To study the function of syntenin in FDC, we examined its role in the focal adhesion of HK cells by depleting syntenin by siRNA technology. Knockdown of syntenin markedly impaired focal adhesion kinase phosphorylation in HK cells. These results suggest that syntenin may play an important role in normal physiology as well as in cancer pathology.

Expression patterns of MDA-9/syntenin during development of the mouse embryo

Melanoma differentiation associated gene-9 (MDA-9)/syntenin is a PDZ domain-containing adaptor protein involved in multiple diverse cellular processes including organization of protein complexes in the plasma membrane, intracellular trafficking and cell surface targeting, synaptic transmission, and cancer metastasis. In the present study, we analyzed the expression pattern of MDA-9/syntenin during mouse development. MDA-9/syntenin was robustly expressed with tight regulation of its temporal and spatial expression during fetal development in the developing skin, spinal cord, heart, lung and liver, which are regulated by multiple signaling pathways in the process of organogenesis. Recent studies also indicate that MDA-9/syntenin is involved in the signaling pathways crucial during development such as Wnt, Notch and FGF. Taken together, these results suggest that MDA-9/syntenin may play a prominent role during normal mouse development in the context of cell proliferation as well as differentiation through modulating multiple signaling pathways as a crucial adaptor protein. Additionally, temporal regulation of MDA-9/syntenin expression may be required during specific stages and in specific tissues during development.

Mda-9/Syntenin Is Expressed in Uveal Melanoma and Correlates with Metastatic Progression

Uveal melanoma is an aggressive cancer that metastasizes to the liver in about half of the patients, with a high lethality rate. Identification of patients at high risk of metastases may provide indication for a frequent follow-up for early detection of metastases and treatment. The analysis of the gene expression profiles of primary human uveal melanomas showed high expression of SDCBP gene (encoding for syndecan-binding protein-1 or mda-9/syntenin), which appeared higher in patients with recurrence, whereas expression of syndecans was lower and unrelated to progression. Moreover, we found that high expression of SDCBP gene was related to metastatic progression in two additional independent datasets of uveal melanoma patients. More importantly, immunohistochemistry showed that high expression of mda-9/syntenin protein in primary tumors was significantly related to metastatic recurrence in our cohort of patients. Mda-9/syntenin expression was confirmed by RT-PCR, immunofluorescence and immunohistochemistry in cultured uveal melanoma cells or primary tumors. Interestingly, mda-9/syntenin showed both cytoplasmic and nuclear localization in cell lines and in a fraction of patients, suggesting its possible involvement in nuclear functions. A pseudo-metastatic model of uveal melanoma to the liver was developed in NOD/SCID/IL2Rγ null mice and the study of mda-9/syntenin expression in primary and metastatic lesions revealed higher mda-9/syntenin in metastases. The inhibition of SDCBP expression by siRNA impaired the ability of uveal melanoma cells to migrate in a wound–healing assay. Moreover, silencing of SDCBP in mda-9/syntenin-high uveal melanoma cells inhibited the hepatocyte growth factor (HGF)-triggered invasion of matrigel membranes and inhibited the activation of FAK, AKT and Src. Conversely syntenin overexpression in mda-9/syntenin-low uveal melanoma cells mediated opposite effects. These results suggest that mda-9/syntenin is involved in uveal melanoma progression and that it warrants further investigation as a candidate molecular marker of metastases and a potential therapeutic target.

Mda-9/Syntenin Protein Positively Regulates the Activation of Akt Protein by Facilitating Integrin-linked Kinase Adaptor Function during Adhesion to Type I Collagen

The integrin-linked kinase (ILK)-PINCH1-α-parvin (IPP) complex functions as a signaling platform for integrins that modulates various cellular processes. ILK functions as a central adaptor for the assembly of IPP complex. We report here that mda-9/syntenin, a positive regulator of cancer metastasis, regulates the activation of Akt (also known as protein kinase B) by facilitating ILK adaptor function during adhesion to type I collagen (COL-I) in human breast cancer cells. COL-I stimulation induced the phosphorylation and plasma membrane translocation of Akt. Inhibition of mda-9/syntenin or expression of mutant ILK (E359K) significantly blocked the translocation of both ILK and Akt to the plasma membrane. mda-9/syntenin associated with ILK, and this association was increased at the plasma membrane by COL-I stimulation. Knockdown of mda-9/syntenin impaired COL-I-induced association of ILK with Akt and plasma membrane targeting of ILK-Akt complex. These results demonstrated that mda-9/syntenin regulates the activation of Akt by controlling the plasma membrane targeting of Akt via a mechanism that facilitates the association of Akt with ILK at the plasma membrane during adhesion to COL-I. On a striking note, inhibition of mda-9/syntenin impaired COL-I-induced plasma membrane translocation of the IPP complex and assembly of integrin β1-IPP signaling complexes. Thus, our study defines the role of mda-9/syntenin in ILK adaptor function and describes a new mechanism of mda-9/syntenin for regulation of cell migration.

Abstract 1457: Raf kinase inhibitor protein (RKIP) negatively regulates melanoma differentiation associated gene-9/syntenin-mediated melanoma metastasis

Abstract Melanoma differentiation associated gene-9 (mda-9/syntenin) functions as a positive regulator of melanoma progression and metastasis by physically interacting with c-Src and promoting the formation of an active FAK/c-Src signaling complex leading to NFκB and matrix metalloproteinase (MMP) activation. In contrast, Raf Kinase Inhibitor Protein (RKIP), an inhibitor of RAF-mediated activation of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEKK), is strongly downregulated in metastatic melanoma cells suggesting a possible inverse relationship between expression of mda-9/syntenin and RKIP. We have now genetically modulated expression of mda-9/syntenin and RKIP in melanocytes and melanoma cells and evaluated biological consequences using invasion, anchorage-independent growth, Western blotting, immunoprecipitation and in vivo confocal imaging assays. These studies confirm an inverse correlation between mda-9/syntenin and RKIP expression. This reciprocal relationship was also documented in vivo using a chick embryo chorioallantoic membrane (CAM) model, which monitors spontaneous intravasation of cells from the upper to the lower layer by polymerase chain reaction (PCR) amplification of human Alu genomic DNA. Using tissue microarrays and a series of cell lines we confirmed a higher expression of MDA-9/syntenin and absence of RKIP in both malignant melanoma tumor sections and cell lines. We also established through gain-of-function and loss-of-function experiments that mda-9/syntenin transcriptionally downregulates RKIP, providing evidence for crosstalk between these two proteins. Moreover, transient overexpression of RKIP in metastatic melanoma cells overrides mda-9/syntenin-mediated signaling resulting in decreased cell invasion, anchorage-independent growth and spontaneous tumor cell dissemination in the CAM assay. RKIP through its RAF1 binding site (amino acids 77-108) physically interacts with MDA-9/syntenin and this interaction inversely correlates with downregulation of FAK and c-Src phosphorylation, crucial steps necessary for forming a stable FAK/c-Src complex and initiating a signaling cascade leading to an MDA-9/syntenin-mediated metastatic phenotype in melanoma cells. These provocative findings highlight a potential therapeutic role of RKIP in melanoma metastasis through its interaction with MDA-9/syntenin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1457. doi:10.1158/1538-7445.AM2011-1457

Abstract 1486: Mda-9/Syntenin regulates integrin signaling by facilitating membrane translocation of ILK/PINCH1/α-parvin complex

Abstract Integrin-linked kinase (ILK), PINCH1 and α-parvin form a ternary complex that plays crucial roles in integrins signaling. We report here that mda-9/syntenin is essential for the assembly of integrin β1-ILK-PINCH1-α-parvin signaling complexes during adhesion to collagen-I (COL-1). Modulation of mda-9/syntenin expression affects COL-1-induced activation of protein kinase B (PKB/Akt). mda-9/Syntenin associates with ILK and this association is increased at the plasma membrane in response to COL-1. mda-9/Syntenin regulates COL-1-induced association between ILK and PKB/Akt, and plasma membrane targeting of ILK-PKB/Akt, suggesting that mda-9/syntenin modulates the plasma membrane targeting of PKB/Akt via ILK. Strikingly, inhibition of mda-9/syntenin impairs COL-1-induced plasma membrane translocation of ILK-PINCH1-α-parvin complex and the assembly of integrin β1-ILK-PINCH1-α-parvin signaling complexes. Consistently, inhibition of mda-9/syntenin blocks the COL-1-induced reorganization of actin cytoskeleton and cell migration, as well as activation of Rac1 and ERK1/21/2. Thus, our study defines the role of mda-9/syntenin in COL-1-induced integrin signaling and describes new mechanism of mda-9/syntenin for the regulation of cell migration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1486. doi:10.1158/1538-7445.AM2011-1486

Abstract LB-372: Activation of focal adhesion kinase in cancer cells is regulated by crosstalk between PKCα and MDA-9/syntenin

Abstract mda-9/Syntenin, a tandem PDZ-domain containing adaptor protein, functions as a positive regulator of cancer cell progression in several human cancers. Protein kinase Cα (PKCα) is a key intermediate between integrins and focal adhesion kinase (FAK) signaling in response to fibronectin (FN), but the molecular mechanism by which PKCα regulates FN-induced FAK activation remains unclear. Here, we report that mda-9/syntenin links FN-induced activation of FAK and PKCα in human breast cancer and melanoma cells. FN induced mda-9/syntenin expression and PKCα activation in a similar manner prior to FAK activation. PKCα inhibition resulted in the suppression of the endogenous and FN-induced mda-9/syntenin expression, as well as mda-9/syntenin-induced cell migration and invasion toward FN. mda-9/Syntenin expression and PKCα activation was interdependently regulated; e.g., inhibition of mda-9/syntenin suppressed both FN-induced and endogenous phosphorylation of PKCα at Thr638/641, and inhibition of PKCα suppressed both FN-induced and endogenous expression of mda-9/syntenin. Moreover, inhibition of either mda-9/syntenin or PKCα suppressed the FN-induced association of integrin- 1/FAK/c-Src signaling complexes. Consistently, suppression of either mda-9/syntenin or PKCα significantly inhibited FN-induced phosphorylation of FAK at Tyr397 and c-Src at Tyr416, activation of downstream signaling molecules such p38 and ERK mitogen-activated protein kinases (MAPKs), Cdc42 GTPase, and nuclear factor-κB (NF-κB). These findings demonstrate that PKC -dependent mda-9/syntenin up regulation could play a critical role in FN-induced activation of FAK, and mda-9/syntenin may act as a molecular adaptor that links PKCα and FAK during adhesion to FN in human breast cancer and melanoma cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-372.

Src kinase activation is mandatory for MDA-9/syntenin-mediated activation of nuclear factor-κB

The scaffolding PDZ-domain containing protein MDA-9/syntenin is a tandem PDZ protein overexpressed in human melanoma, and breast and gastric cancer cells. MDA-9/syntenin affects cancer cell motility and invasion through distinct biochemical and signaling pathways, including focal adhesion kinase (FAK) and p38 mitogen-activated protein kinase (MAPK), resulting in activation of the NFκ B pathway. MDA9/syntenin also promotes melanoma metastasis by activating c-Src, but how c-Src regulates NFκ B activation is unclear. Using a human melanoma model, we document that MDA-9/syntenin/c-Src interactions are positive regulators of NFκ B activation. Inhibition of c-Src by PP2 treatment, by blocking c-Src or mda-9/syntenin expression with siRNA, or in c-Src (−/−) knockout cell lines, reduces NFκ B activation following overexpression of mda-9/syntenin or c-Src. Deletion or point mutations of the PDZ binding motif preventing MDA-9/syntenin association with c-Src reveals that both PDZ domains, with PDZ2 being the dominant module, are required for activating downstream signaling pathways, including p38 MAPK and NFκ B. We also document that MDA-9/syntenin/c-Src complexes functionally cooperate with NFκ B to promote anchorage-independent growth, motility and invasion of melanoma cells. These findings underscore PDZ domains of MDA-9/syntenin as promising potential therapeutic targets for intervening in a decisive component of cancer progression, namely metastatic tumor spread.

Regulation of myelopoiesis through syntenin-mediated modulation of IL-5 receptor output

AbstractThe granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin (IL)–3/IL-5 receptor family regulates the production and function of myeloid cells. These cytokines signal through receptor complexes that consist of unique ligand-binding α-chains and common signaling β-chains. IL-5 is distinct from IL-3 and GM-CSF in its capacity to induce eosinophil development, however, the molecular mechanisms that generate functional diversity within this receptor family are mostly unknown. Here, we characterized the selective IL-5Rα–binding adapter protein syntenin in IL-5R function. Syntenin and IL-5Rα colocalize at the plasma membrane and in early endosomal compartments. Manipulation of syntenin expression by ectopic expression or knockdown selectively modulated IL-5R but not GM-CSF receptor signaling, and severely affected IL-5–induced eosinophil differentiation from primary human CD34+ hematopoietic progenitor cells. We found syntenin up-regulated during eosinophilopoiesis but down-regulated during neutropoiesis. Syntenin forms complexes with multiple IL-5Rα chains, suggesting that syntenin-enhanced IL-5R output may result from stabilization of an IL-5–induced oligomeric receptor complex. These data demonstrate that cytokine-specific functions can be transduced by unique receptor α-chain–associating adapter proteins.

The tandem PDZ domains of syntenin promote cell invasion

Syntenin is a tandem PDZ protein that has recently been shown to be overexpressed in several cancer cells and tissues, and that might play an active role in tumor cell invasion and metastasis. Here we show that overexpression of the tandem PDZ domains of syntenin in non-invasive cells is necessary and sufficient to stimulate these cells to invade a collagen I matrix, and this effect can be regulated by ligand binding to the PDZ domains. Furthermore, we show that syntenin-induced invasion requires signaling through ras, rho and PI3K/MAPK signaling pathways and involves changes in cell–cell adhesion. Inversely, when we used RNA interference to inhibit syntenin expression in different invasive cancer cell lines, we observed a drastically decreased ability of these cells to migrate and invade into collagen type I or Matrigel ®. RNAi-treated cells also show increased cell aggregation, indicating that syntenin is important for cell–cell adhesion in epithelial cells. Together, these results suggest that downregulation of syntenin by RNA interference could provide a means of inhibiting tumor invasion and possibly metastasis in different cancers, and point to syntenin as a potential cancer biomarker and drug target.

RETRACTED: mda-9/Syntenin regulates the metastatic phenotype in human melanoma cells by activating nuclear factor-kappaB.

mda-9/Syntenin is a scaffolding PDZ domain-containing protein overexpressed in multiple human cancers that functions as a positive regulator of melanoma metastasis. Using a normal immortal human melanocyte cell line and weakly and highly metastatic human melanoma cell lines, we presently show that mda-9/syntenin initiates a signaling cascade that activates nuclear factor-kappaB (NF-kappaB) in human melanoma cells. As a consequence of elevated mda-9/syntenin expression, tumor cell growth and motility, fundamental components of tumor cell invasion and metastatic spread of melanoma cells, are enhanced through focal adhesion kinase (FAK)-induced and p38 mitogen-activated protein kinase (MAPK)-induced activation of NF-kappaB. Inhibiting mda-9/syntenin, using an adenovirus expressing antisense mda-9/syntenin, NF-kappaB, using an adenovirus expressing a mutant super-repressor of IkappaBalpha, or FAK, and using a dominant-negative mutant of FAK (FRNK), blocks melanoma cell migration, anchorage-independent growth, and invasion. Downstream signaling changes mediated by mda-9/syntenin, which include activation of FAK, p38 MAPK, and NF-kappaB, promote induction of membrane-type matrix metalloproteinase-1 that then activates pro-MMP-2-promoting migration and extracellular matrix invasion of melanoma cells. These results highlight the importance of mda-9/syntenin as a key component of melanoma metastasis providing a rational molecular target for potentially intervening in the metastatic process.

Cloning of eIF5A from shrimp Penaeus monodon, a highly expressed protein involved in the survival of WSSV-infected shrimp

Shrimp respond to viral infection with up- and down-regulation of many critical genes. In our previous work, we showed that white spot syndrome virus ( WSSV) infection of Penaeus monodon ( Pm) caused cellular syntenin levels to increase. In order to further explore the signal transduction pathway of syntenin, we constructed the cDNA library of WSSV-infected shrimp and performed a yeast two-hybrid screening of the library using syntenin as bait. Here we report that syntenin specifically binds eukaryotic translation initiation factor 5A (eIF5A), a 157 amino acid polypeptide that is implicated in cell proliferation and survival. GST-pull-down assays showed the presence of specific interaction between Pm-syntenin and eIF5A. Message analyses revealed that syntenin expression remained normal when shrimp were infected with WSSV but did not show any gross signs of disease. Syntenin expression, however, increased 1.4-fold when shrimp became dead. In contrast, eIF5A expression increased markedly during the stage of WSSV infection when there were no gross signs of disease, i.e., prior to the moribund stage. At the moribund stage, eIF5A expression was down-regulated. This data suggest that syntenin and eIF5A physically interact with each other in the setting of WSSV infection and that these two molecules exhibit dynamic changes in their expression patterns as infection progresses from grossly normal, infected shrimp to full-blown phase.

Mda-9/Syntenin: A Positive Regulator of Melanoma Metastasis

Metastasis is a significant event in cancer progression and continues to pose the greatest challenge for a cancer cure. Defining genes that control metastasis in vivo may provide new targets for intervening in this process with profound therapeutic implications. Melanoma differentiation associated gene-9 (mda-9) was initially identified by subtraction hybridization as a novel gene displaying biphasic expression during terminal differentiation in human melanoma cells. Mda-9, also known as syntenin, is a PDZ-domain protein overexpressed in many types of human cancers, where it is believed to function in tumor progression. However, a functional role of mda-9/syntenin in tumor growth and metastasis and the signaling pathways involved in mediating these biological activities remain to be defined. Evidence is now provided, using weakly and highly metastatic isogenic melanoma variants, that mda-9/syntenin regulates metastasis. Expression of mda-9/syntenin correlates with advanced stages of melanoma progression. Regulating mda-9/syntenin expression using a replication-incompetent adenovirus expressing either sense or antisense mda-9/syntenin modifies the transformed phenotype and alters metastatic ability in immortal human melanocytes and metastatic melanoma cells in vitro and in vivo in newborn rats. A direct relationship is observed between mda-9/syntenin expression and increased phosphorylation of focal adhesion kinase, c-Jun-NH2-kinase, and p38. This study provides the first direct link between mda-9/syntenin expression and tumor cell dissemination in vivo and indicates that mda-9/syntenin expression activates specific signal transduction pathways, which may regulate melanoma tumor progression. Based on its ability to directly alter metastasis, mda-9/syntenin provides a promising new focus for melanoma cancer research with potential therapeutic applications for metastatic diseases.