Pharmacological inhibition of MDA-9/Syntenin blocks breast cancer metastasis through suppression of IL-1ß.

Melanoma differentiation associated gene-9 (MDA-9), Syntenin-1, or syndecan binding protein is a differentially regulated prometastatic gene with elevated expression in advanced stages of melanoma. MDA-9/Syntenin expression positively associates with advanced disease stage in multiple histologically distinct cancers and negatively correlates with patient survival and response to chemotherapy. MDA-9/Syntenin is a highly conserved PDZ-domain scaffold protein, robustly expressed in a spectrum of diverse cancer cell lines and clinical samples. PDZ domains interact with a number of proteins, many of which are critical regulators of signaling cascades in cancer. Knockdown of MDA-9/Syntenin decreases cancer cell metastasis, sensitizing these cells to radiation. Genetic silencing of MDA-9/Syntenin or treatment with a pharmacological inhibitor of the PDZ1 domain, PDZ1i, also activates the immune system to kill cancer cells. Additionally, suppression of MDA-9/Syntenin deregulates myeloid-derived suppressor cell differentiation via the STAT3/interleukin (IL)-1ß pathway, which concomitantly promotes activation of cytotoxic T lymphocytes. Biologically, PDZ1i treatment decreases metastatic nodule formation in the lungs, resulting in significantly fewer invasive cancer cells. In summary, our observations indicate that MDA-9/Syntenin provides a direct therapeutic target for mitigating aggressive breast cancer and a small-molecule inhibitor, PDZ1i, provides a promising reagent for inhibiting advanced breast cancer pathogenesis.

Pharmacological inhibition of syntenin PDZ2 domain impairs breast cancer cell activities and exosome loading with syndecan and EpCAM cargo.

Exosomes support cell-to-cell communication in physiology and disease, including cancer. We currently lack tools, such as small chemicals, capable of modifying exosome composition and activity in a specific manner. Building on our previous understanding of how syntenin, and its PDZ partner syndecan (SDC), impact on exosome composition we optimized a small chemical compound targeting the PDZ2 domain of syntenin. In vitro , in tests on MCF-7 breast carcinoma cells, this compound is non-toxic and impairs cell proliferation, migration and primary sphere formation. It does not affect the size or the number of secreted particles, yet it decreases the amounts of exosomal syntenin, ALIX and SDC4 while leaving other exosomal markers unaffected. Interestingly, it also blocks the sorting of EpCAM, a bona fide target used for carcinoma exosome immunocapture. Our study highlights the first characterization of a small pharmacological inhibitor of the syntenin-exosomal pathway, of potential interest for exosome research and oncology.

Syntenin regulates melanogenesis via the p38 MAPK pathway.

Melanogenesis is the synthesis of the skin pigment melanin, which serves a critical role in the study of pigmentary skin diseases. Syntenin has been identified as a melanosome protein, but its role in melanogenesis is not completely understood. The present study aimed to investigate the effects and mechanisms underlying syntenin on melanogenesis in immortalized human melanocytes. Depletion of syntenin expression increased both tyrosinase (Tyr) activity and melanin content. Syntenin silencing also increased the protein expression levels of Tyr, pre­melanosomal protein and microphthalmia­associated transcription factor. In addition, the results indicated that syntenin regulated melanogenesis by upregulating the phosphorylation of p38 mitogen­activated protein kinase (p38 MAPK). Taken together, these findings suggested that the regulation of melanogenesis by syntenin may be mediated by the activation of p38 MAPK and that syntenin might provide new insights into the pathogenesis of pigmented diseases.

MDA-9/Syntenin: An emerging global molecular target regulating cancer invasion and metastasis.

With few exceptions, metastasis is the terminal stage of cancer with limited therapeutic options. Metastasis consists of numerous phenotypic and genotypic alterations of cells that are directly and indirectly induced by multiple intrinsic (cellular) and extrinsic (micro-environmental) factors. To metastasize, a cancer cell often transitions from an epithelial to mesenchymal morphology (EMT), modifies the extracellular matrix, forms emboli and survives in the circulation, escapes immune surveillance, adheres to sites distant from the initial tumor and finally develops a blood supply (angiogenesis) and colonizes in a secondary niche (a micrometastasis). Scientific advances have greatly enhanced our understanding of the precise molecular and genetic changes, operating independently or collectively, that lead to metastasis. This review focuses on a unique gene, melanoma differentiation associated gene-9 (also known as Syntenin-1; Syndecan Binding Protein (sdcbp); mda-9/syntenin), initially cloned and characterized from metastatic human melanoma and shown to be a pro-metastatic gene. In the last two decades, our comprehension of the diversity of actions of MDA-9/Syntenin on cellular phenotype has emerged. MDA-9/Sytenin plays pivotal regulatory roles in multiple signaling cascades and orchestrates both metastatic and non-metastatic events. Considering the relevance of this gene in controlling cancer invasion and metastasis, approaches have been developed to uniquely and selectively target this gene. We also provide recent updates on strategies that have been successfully employed in targeting MDA-9/Syntenin resulting in profound pre-clinical anti-cancer activity.

Rethinking Glioblastoma Therapy: MDA-9/Syntenin Targeted Small Molecule.

Effective therapies for glioblastoma multiforme (GBM) are not currently available. A small molecule has been identified using fragment-based drug-discovery guided by NMR that targets important protein-protein interactions controlling GBM invasion and pathogenicity. This first generation drug displays excellent pharmacokinetic properties, passes through the blood-brain barrier and is effective in preclinical animal models of GBM, particularly when combined with radiation therapy.

Syntenin-targeted peptide blocker inhibits progression of cancer cells.

The multidomain adaptor protein syntenin is known to mediate cancer cell metastasis and invasion through its tandem PDZ1 and PDZ2 domains, leading to the postulation that the PDZ tandem may serve as a potential drug target for cancer treatment. Here we report the development of high-affinity peptide blockers to target the syntenin tandem PDZ domain, and elucidate that blocking syntenin correlates with the inhibition of cell migration and spreading. Two strategies are employed to derive high-affinity blockers from the low-affinity natural binding peptides: first, dimerization of the C termini of natural syntenin-binding peptides confers dimer peptides with much higher affinity than the monomers; second, unnatural amino acid substitution at P-1 and P-2 positions of the PDZ-binding sequence increases the binding affinity. Through several rounds of optimization, we discovered a dimeric peptide that binds tightly to syntenin tandem PDZ domain, with a dissociation constant of 0.21 µM based on fluorescence polarization measurement. The peptide dimer inhibits the migration and invasion of syntenin high-expression human cancer cells through attenuating the ERK phosphorylation of the MAPK kinase pathway. This work showcases an effective strategy to derive high-affinity blocker of multidomain adaptor proteins, which resulted in a syntenin-targeted antagonist with potential pharmaceutical values for the treatment of syntenin over-expressing cancers.

Novel function of MDA-9/Syntenin (SDCBP) as a regulator of survival and stemness in glioma stem cells.

Glioblastoma multiforme (GBM) is an aggressive cancer with current therapies only marginally impacting on patient survival. Glioma stem cells (GSCs), a subpopulation of highly tumorigenic cells, are considered major contributors to glioma progression and play seminal roles in therapy resistance, immune evasion and increased invasion. Despite clinical relevance, effective/selective therapeutic targeting strategies for GSCs do not exist, potentially due to the lack of a definitive understanding of key regulators of GSCs. Consequently, there is a pressing need to identify therapeutic targets and novel options to effectively target this therapy-resistant cell population. The precise roles of GSCs in governing GBM development, progression and prognosis are under intense scrutiny, but key upstream regulatory genes remain speculative. MDA-9/Syntenin (SDCBP), a scaffold protein, regulates tumor pathogenesis in multiple cancers. Highly aggressive cancers like GBM express elevated levels of MDA-9 and contain increased populations of GSCs. We now uncover a unique function of MDA-9 as a facilitator and determinant of glioma stemness and survival. Mechanistically, MDA-9 regulates multiple stemness genes (Nanog, Oct4 and Sox2) through activation of STAT3. MDA-9 controls survival of GSCs by activating the NOTCH1 pathway through phospho-Src and DLL1. Once activated, cleaved NOTCH1 regulates C-Myc expression through RBPJK, thereby facilitating GSC growth and proliferation. Knockdown of MDA-9 affects the NOTCH1/C-Myc and p-STAT3/Nanog pathways causing a loss of stemness and initiation of apoptosis in GSCs. Our data uncover a previously unidentified relationship between MDA-9 and GSCs, reinforcing relevance of this gene as a potential therapeutic target in GBM.

Processing of heparanase is mediated by syndecan-1 cytoplasmic domain and involves syntenin and α-actinin.

Heparanase activity plays a decisive role in cell dissemination associated with cancer metastasis. Cellular uptake of heparanase is considered a pre-requisite for the delivery of latent 65-kDa heparanase to lysosomes and its subsequent proteolytic processing and activation into 8- and 50-kDa protein subunits by cathepsin L. Heparan sulfate proteoglycans, and particularly syndecan, are instrumental for heparanase uptake and activation, through a process that has been shown to occur independent of rafts. Nevertheless, the molecular mechanism underlying syndecan-mediated internalization outside of rafts is unclear. Here, we examined the role of syndecan-1 cytoplasmic domain in heparanase processing, utilizing deletion constructs lacking the entire cytoplasmic domain (Delta), the conserved (C1 or C2), or variable (V) regions. Heparanase processing was markedly increased following syndecan-1 over-expression; in contrast, heparanase was retained at the cell membrane and its processing was impaired in cells over-expressing syndecan-1 deleted for the entire cytoplasmic tail. We have next revealed that conserved domain 2 (C2) and variable (V) regions of syndecan-1 cytoplasmic tail mediate heparanase processing. Furthermore, we found that syntenin, known to interact with syndecan C2 domain, and α actinin are essential for heparanase processing.

Syntenin-a promotes spinal cord regeneration following injury in adult zebrafish.

In contrast to mammals, adult zebrafish recover locomotor function after spinal cord injury, in part due to the capacity of the central nervous system to repair severed connections. To identify molecular cues that underlie regeneration, we conducted mRNA expression profiling and found that syntenin-a expression is upregulated in the adult zebrafish spinal cord caudal to the lesion site after injury. Syntenin is a scaffolding protein involved in mammalian cell adhesion and movement, axonal outgrowth, establishment of cell polarity, and protein trafficking. It could thus be expected to be involved in supporting regeneration in fish. Syntenin-a mRNA and protein are expressed in neurons, glia and newly generated neural cells, and upregulated caudal to the lesion site on days 6 and 11 following spinal cord injury. Treatment of spinal cord-injured fish with two different antisense morpholinos to knock down syntenin-a expression resulted in significant inhibition of locomotor recovery at 5 and 6 weeks after injury, when compared to control morpholino-treated fish. Knock-down of syntenin-a reduced regrowth of descending axons from brainstem neurons into the spinal cord caudal to the lesion site. These observations indicate that syntenin-a is involved in regeneration after traumatic insult to the central nervous system of adult zebrafish, potentially leading to novel insights into the cellular and molecular mechanisms that require activation in the regeneration-deficient mammalian central nervous system.

Raf kinase inhibitor RKIP inhibits MDA-9/syntenin-mediated metastasis in melanoma.

Melanoma differentiation associated gene-9 (MDA-9), also known as syntenin, functions as a positive regulator of melanoma progression and metastasis. In contrast, the Raf kinase inhibitor, RKIP, a negative modulator of RAF-stimulated MEKK activation, is strongly downregulated in metastatic melanoma cells. In this study, we explored a hypothesized inverse relationship between MDA-9 and RKIP in melanoma. Tumor array and cell line analyses confirmed an inverse relationship between expression of MDA-9 and RKIP during melanoma progression. We found that MDA-9 transcriptionally downregulated RKIP in support of a suggested cross-talk between these two proteins. Furthermore, MDA-9 and RKIP physically interacted in a manner that correlated with a suppression of FAK and c-Src phosphorylation, crucial steps necessary for MDA-9 to promote FAK/c-Src complex formation and initiate signaling cascades that drive the MDA-9-mediated metastatic phenotype. Finally, ectopic RKIP expression in melanoma cells overrode MDA-9-mediated signaling, inhibiting cell invasion, anchorage-independent growth, and in vivo dissemination of tumor cells. Taken together, these findings establish RKIP as an inhibitor of MDA-9-dependent melanoma metastasis, with potential implications for targeting this process therapeutically.

Activation of the integrin effector kinase focal adhesion kinase in cancer cells is regulated by crosstalk between protein kinase Calpha and the PDZ adapter protein mda-9/Syntenin.

Aberrant adhesion signaling pathways in cancer cells underlie their deadly invasive capabilities. The adhesion-related PDZ adapter protein mda-9/syntenin is a positive regulator of cancer cell progression in breast cancer, melanoma, and other human cancers. In this study, we report that mda-9/syntenin mediates adhesion-mediated activation of protein kinase Calpha (PKCalpha) and focal adhesion kinase (FAK) by fibronectin (FN) in human breast cancer and melanoma cells. FN rapidly stimulated the expression of mda-9/syntenin and the activation of PKCalpha prior to activation of FAK. Inhibiting PKCalpha suppressed basal or FN-induced expression of mda-9/syntenin, as well as cell migration and invasion toward FN stimulated by mda-9/syntenin. Several lines of evidence suggested that activation of PKCalpha and expression of mda-9/syntenin were interdependent. First, mda-9/syntenin inhibition suppressed basal or FN-induced phosphorylation of PKCalpha at Thr(638/641), whereas PKCalpha inhibition suppressed basal or FN-induced expression of mda-9/syntenin. Second, inhibiting either mda-9/syntenin or PKCalpha suppressed FN-induced formation of integrin-beta(1)/FAK/c-Src signaling complexes. Third, inhibiting either mda-9/syntenin or PKCalpha suppressed FN-induced phosphorylation of FAK Tyr(397) and c-Src Tyr(416) and the induction of downstream effector signals to p38 and mitogen-activated protein kinase, Cdc42, and NF-kappaB. In summary, our findings offer evidence that mda-9/syntenin acts as a molecular adaptor linking PKCalpha and FAK activation in a pathway of FN adhesion by human breast cancer and melanoma cells.

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.