Inactivation of kindlin-3 increases human melanoma aggressiveness through the collagen-activated tyrosine kinase receptor DDR1.

The role of the focal adhesion protein kindlin-3 as a tumor suppressor and its interaction mechanisms with extracellular matrix constitute a major field of investigation to better decipher tumor progression. Besides the well-described role of kindlin-3 in integrin activation, evidence regarding modulatory functions between melanoma cells and tumor microenvironment are lacking and data are needed to understand mechanisms driven by kindlin-3 inactivation. Here, we show that kindlin-3 inactivation through knockdown or somatic mutations increases BRAFV600mut melanoma cells oncogenic properties via collagen-related signaling by decreasing cell adhesion and enhancing proliferation and migration in vitro, and by promoting tumor growth in mice. Mechanistic analysis reveals that kindlin-3 interacts with the collagen-activated tyrosine kinase receptor DDR1 (Discoidin domain receptor 1) modulating its expression and its interaction with ß1-integrin. Kindlin-3 knockdown or mutational inactivation disrupt DDR1/ß1-integrin complex in vitro and in vivo and its loss improves the anti-proliferative effect of DDR1 inhibition. In agreement, kindlin-3 downregulation is associated with DDR1 over-expression in situ and linked to worse melanoma prognosis. Our study reveals a unique mechanism of action of kindlin-3 in the regulation of tumorigenesis mediated by the collagen-activated tyrosine kinase receptor DDR1 thus paving the way for innovative therapeutic targeting approaches in melanoma.

Novel treatment strategy for NRAS-mutated melanoma through a selective inhibitor of CD147/VEGFR-2 interaction.

More than 70% of human NRASmut melanomas are resistant to MEK inhibitors highlighting the crucial need for efficient therapeutic strategies for these tumors. CD147, a membrane receptor, is overexpressed in most cancers including melanoma and is associated with poor prognosis. We show here that CD147i, a specific inhibitor of CD147/VEGFR-2 interaction represents a potential therapeutic strategy for NRASmut melanoma cells. It significantly inhibited the malignant properties of NRASmut melanomas ex vivo and in vivo. Importantly, NRASmut patient's-derived xenografts, which were resistant to MEKi, became sensitive when combined with CD147i leading to decreased proliferation ex vivo and tumor regression in vivo. Mechanistic studies revealed that CD147i effects were mediated through STAT3 pathway. These data bring a proof of concept on the impact of the inhibition of CD147/VEGFR-2 interaction on melanoma progression and represents a new therapeutic opportunity for NRASmut melanoma when combined with MEKi.

CD147 Promotes Tumor Lymphangiogenesis in Melanoma via PROX-1.

Malignant melanoma is one of the most aggressive skin cancers and is characterized by early lymph node metastasis and the capacity to develop resistance to therapies. Hence, understanding the regulation of lymphangiogenesis through mechanisms contributing to lymphatic vessel formation represents a treatment strategy for metastatic cancer. We have previously shown that CD147, a transmembrane glycoprotein overexpressed in melanoma, regulates the angiogenic process in endothelial cells. In this study, we show a correlation between high CD147 expression levels and the number of lymphatic vessels expressing LYVE-1, Podoplanin, and VEGFR-3 in human melanoma lymph nodes. CD147 upregulates in vitro lymphangiogenesis and its related mediators through the PROX-1 transcription factor. In vivo studies in a melanoma model confirmed that CD147 is involved in metastasis through a similar mechanism as in vitro. This study, demonstrating the paracrine role of CD147 in the lymphangiogenesis process, suggests that CD147 could be a promising target for the inhibition of melanoma-associated lymphangiogenesis.

CD147 Is a Promising Target of Tumor Progression and a Prognostic Biomarker.

Microenvironment plays a crucial role in tumor development and progression. Cancer cells modulate the tumor microenvironment, which also contribute to resistance to therapy. Identifying biomarkers involved in tumorigenesis and cancer progression represents a great challenge for cancer diagnosis and therapeutic strategy development. CD147 is a glycoprotein involved in the regulation of the tumor microenvironment and cancer progression by several mechanisms-in particular, by the control of glycolysis and also by its well-known ability to induce proteinases leading to matrix degradation, tumor cell invasion, metastasis and angiogenesis. Accumulating evidence has demonstrated the role of CD147 expression in tumor progression and prognosis, suggesting it as a relevant tumor biomarker for cancer diagnosis and prognosis, as well as validating its potential as a promising therapeutic target in cancers.

Discoidin domain receptors: A promising target in melanoma.

The discoidin domain receptor 1 (DDR1) is a member of the receptor tyrosine kinase family that signals in response to collagen and that has been implicated in cancer progression. In the present study, we investigated the expression and role of DDR1 in human melanoma progression. Immunohistochemical staining of human melanoma specimens (n = 52) shows high DDR1 expression in melanoma lesions that correlates with poor prognosis. DDR1 expression was associated with the clinical characteristics of Clark level and ulceration and with BRAF mutations. Downregulation of DDR1 by small interfering RNA (siRNA) in vitro inhibited melanoma cells malignant properties, migration, invasion, and survival in several human melanoma cell lines. A DDR tyrosine kinase inhibitor (DDR1-IN-1) significantly inhibited melanoma cell proliferation in vitro, and ex vivo and in tumor xenografts, underlining the promising potential of DDR1 inhibition in melanoma.

Endogenous Enzymes in Root Caries.

Similar to coronal caries, root caries results from a disequilibrium of the de-remineralization balance in favor of the demineralization process. It mainly involves a bacterial shift in favor of an increase in the proportion of acidogenic and aciduric bacteria. This process permanently damages the dental mineralized tissues, namely the dental cementum and dentin. In addition to the demineralization and exposure of the dentin or the cementum organic matrix, acid production by cariogenic bacteria induces the activation of endogenous (host-derived) enzymes within the dentin and saliva. These enzymes include matrix metalloproteinases and cathepsins. Once activated, these potent proteolytic enzymes collectively have the capacity to degrade all the components of the exposed organic dentin and cementum matrices. In this chapter, the description of the healthy cement and dentin organic matrices and their endogenous proteases will be followed by the role of these proteases in the root caries process.

EMMPRIN/CD147 is an independent prognostic biomarker in cutaneous melanoma.

CD147 has been implicated in melanoma invasion and metastasis mainly through increasing metalloproteinase synthesis and regulating VEGF/VEGFR signalling. In this study, the prognostic value of CD147 expression was investigated in a cohort of 196 cutaneous melanomas including 136 consecutive primary malignant melanomas, 30 lymph nodes, 16 in-transit and 14 visceral metastases. A series of 10 normal skin, 10 blue nevi and 10 dermal nevi was used as control. CD147 expression was assessed by immunohistochemistry, and the association of its expression with the clinicopathological characteristics of patients and survival was evaluated using univariate and multivariate statistical analyses. Univariate analysis showed that high CD147 expression was significantly associated with metastatic potential and with a reduced overall survival (P < 0.05 for both) in primary melanoma patients. CD147 expression level was correlated with histological factors which were associated with prognosis: Clark level, ulceration status and more particularly with Breslow index (r = 0.7, P < 10(-8) ). Multivariate analysis retained CD147 expression level and ulceration status as predicting factors for metastasis and overall survival (P < 0.05 for both). CD147 emerges as an important factor in the aggressive behaviour of melanoma and deserves further evaluation as an independent prognostic biomarker.

Claudin-16 Deficiency Impairs Tight Junction Function in Ameloblasts, Leading to Abnormal Enamel Formation.

Claudin-16 protein (CLDN16) is a component of tight junctions (TJ) with a restrictive distribution so far demonstrated mainly in the kidney. Here, we demonstrate the expression of CLDN16 also in the tooth germ and show that claudin-16 gene (CLDN16) mutations result in amelogenesis imperfecta (AI) in the 5 studied patients with familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC). To investigate the role of CLDN16 in tooth formation, we studied a murine model of FHHNC and showed that CLDN16 deficiency led to altered secretory ameloblast TJ structure, lowering of extracellular pH in the forming enamel matrix, and abnormal enamel matrix protein processing, resulting in an enamel phenotype closely resembling human AI. This study unravels an association of FHHNC owing to CLDN16 mutations with AI, which is directly related to the loss of function of CLDN16 during amelogenesis. Overall, this study indicates for the first time the importance of a TJ protein in tooth formation and underlines the need to establish a specific dental follow-up for these patients.

EMMPRIN/CD147 is a novel coreceptor of VEGFR-2 mediating its activation by VEGF.

EMMPRIN/CD147 is mainly known for its protease inducing function but a role in promoting tumor angiogenesis has also been demonstrated. This study provides evidence that EMMPRIN is a new coreceptor for the VEGFR-2 tyrosine kinase receptor in both endothelial and tumor cells, as it directly interacts with it and regulates its activation by its VEGF ligand, signalling and functional consequences both in vitro and in vivo. Computational docking analyses and mutagenesis studies identified a molecular binding site in the extracellular domain of EMMPRIN located close to the cell membrane and containing the amino acids 195/199. EMMPRIN is overexpressed in cancer and hence is able to further potentiate VEGFR-2 activation, suggesting that a combinatory therapy of an antiangiogenic drug together with an inhibitor of EMMPRIN/VEGFR-2 interaction may have a greater impact on inhibiting angiogenesis and malignancy.

EMMPRIN regulates ß1 integrin-mediated adhesion through Kindlin-3 in human melanoma cells.

EMMPRIN is known to promote tumor invasion through extracellular matrix (ECM) degradation. Here we report that EMMPRIN can regulate melanoma cell adhesion to the ECM through an interaction with ß1 integrin involving kindlin-3. In this study, EMMPRIN knockdown in the human melanoma cell line M10 using siRNA decreased cell invasion and significantly increased cell adhesion and spreading. A morphological change from a round to a spread shape was observed associated with enhanced phalloidin-labelled actin staining. In situ proximity ligation assay and co-immunoprecipitation revealed that EMMPRIN silencing increased the interaction of ß1 integrin with kindlin-3, a focal adhesion protein. This was associated with an increase in ß1 integrin activation and a decrease in the phosphorylation of the downstream integrin kinase FAK. Moreover, the expression at both the transcript and protein level of kindlin-3 and of ß1 integrin was inversely regulated by EMMPRIN. EMMPRIN did not regulate either talin expression or its interaction with ß1 integrin. These results are consistent with our in vivo demonstration that EMMPRIN inhibition increased ß1 integrin activation and its interaction with kindlin-3. To conclude, these findings reveal a new role of EMMPRIN in tumor cell migration through ß1 integrin/kindlin-3-mediated adhesion pathway.

Increased extracellular matrix metalloproteinase inducer (EMMPRIN) expression in the conjunctival epithelium exposed to antiglaucoma treatments.

OBJECTIVE: To analyze the effect of preserved antiglaucoma eye drops on the expression of extracellular matrix (ECM) metalloproteinase inducer (EMMPRIN) in conjunctival epithelial cells. METHODS: A total of 18 patients treated for primary open-angle glaucoma with benzalkonium chloride (BAK) preserved eye drops and eight age-matched controls were included in this study. Glaucoma patients were divided into two groups according to their daily exposure to BAK: high-exposure (HE) group and low-exposure (LE) group. HLA-DR and EMMPRIN were quantified on conjunctival impression cytology specimens using flow cytometry. In parallel, IOBA-NHC conjunctival epithelial cells were exposed to different BAK concentrations, in the presence or absence of cyclosporine A (CsA), and their total and surface expressions of EMMPRIN were assessed by flow cytometry and results are given in relative fluorescence intensities (RFIs). RESULTS: Compared to the control group (1.71 ± 0.39 RFI), EMMPRIN was significantly increased in the HE (4.19 ± 1.50 RFI, p < 0.001) and LE groups (2.55 ± 0.40 RFI, p = 0.029). Similar increase was observed in HLA-DR expression in the HE (4.58 ± 1.38 RFI, p < 0.001) and LE groups (2.52 ± 0.47 RFI, p = 0.046) as compared to control subjects (1.75 ± 0.27 RFI). Across all subjects enrolled in the study, there was a significant correlation between HLA-DR and EMMPRIN (R(2) = 0.875, p < 0.0001). IOBA-NHC cells exposed to BAK presented a significant increase in EMMPRIN, which was proportional to the concentration of BAK. The surface expression of EMMPRIN was inhibited by CsA. CONCLUSIONS: The increased expression of EMMPRIN in patients topically treated with multiple antiglaucoma BAK-preserved eye drops suggests a matrix metalloproteinase-related modification of conjunctival ECM remodeling. In vitro results suggest that CsA has the potential to limit BAK effects on EMMPRIN.

Grape seed extracts inhibit dentin matrix degradation by MMP-3.

UNLABELLED: Since Matrix metalloproteinases (MMPs) have been suggested to contribute to dentin caries progression, the hypothesis that MMP inhibition would affect the progression of dentin caries is clinically relevant. Grape seed extracts (GSE) have been previously reported to be natural inhibitors of MMPs. OBJECTIVE: To evaluate the capacity of a GSE mouthrinse to prevent the degradation of demineralized dentin matrix by MMP-3 (stromelysin-1). MATERIALS AND METHODS: Standardized blocks of dentin obtained from sound permanent teeth extracted for orthodontic reasons were demineralized with Ethylenediaminetetraacetic acid (EDTA) and pretreated either with (A) GSE (0.2% w/v), (B) amine fluoride (AmF) (20% w/v), (C) a mouthrinse which contains both, (D) placebo, (E) sodium fluoride (0.15 mg.ml(-1)), (F) PBS, (G) Chlorhexidine digluconate (CHX), or (H) zinc chloride (ZnCl2). The dentin blocks were then incubated with activated recombinant MMP-3. The supernatants were analyzed by Western Blot for several dentin matrix proteins known to be MMP-3 substrate. In parallel, scanning electron microscopy (SEM) was performed on resin replica of the dentin blocks. RESULTS: Western blot analysis of the supernatants revealed that MMP-3 released from the dentin matrix small proteoglycans (decorin and biglycan) and dentin sialoprotein (DSP) in the AmF, sodium fluoride, PBS and placebo pretreated groups, but not in the GSE and mouthrinse pretreated groups. SEM examination of resin replica showed that the mouthrinse and its active components not only had an anti-MMP action but also modified the dentin surface accessibility. CONCLUSION: This study shows that GSE either alone or combined with AmF as in the evaluated mouthrinse limits dentin matrix degradation. This association may be promising to prevent the progression of caries within dentin. However, the procedure should be adapted to clinically relevant durations.

A novel tumor suppressor function of Kindlin-3 in solid cancer.

Kindlin-3 (FERMT-3) is known to be central in hemostasis and thrombosis control and its deficiency disrupts platelet aggregation and causes Leukocyte Adhesion Deficiency disease. Here we report that Kindlin-3 has a tumor suppressive role in solid cancer. Our present genetic and functional data show that Kindlin-3 is downregulated in several solid tumors by a mechanism involving gene hypermethylation and deletions. In vivo experiments demonstrated that Kindlin-3 knockdown in 2 tumor cell models (breast cancer and melanoma) markedly increases metastasis formation, in accord with the in vitro increase of tumor cell malignant properties. The metastatic phenotype was supported by a mechanism involving alteration in ß3-integrin activation including decreased phosphorylation, interaction with talin and the internalization of its active form leading to less cell attachment and more migration/invasion. These data uncover a novel and unexpected tumor suppressor role of Kindlin-3 which can influence integrins targeted therapies development.

EMMPRIN/CD147 deficiency disturbs ameloblast-odontoblast cross-talk and delays enamel mineralization.

Tooth development is regulated by a series of reciprocal inductive signaling between the dental epithelium and mesenchyme, which culminates with the formation of dentin and enamel. EMMPRIN/CD147 is an Extracellular Matrix MetalloPRoteinase (MMP) INducer that mediates epithelial-mesenchymal interactions in cancer and other pathological processes and is expressed in developing teeth. Here we used EMMPRIN knockout (KO) mice to determine the functional role of EMMPRIN on dental tissue formation. We report a delay in enamel deposition and formation that is clearly distinguishable in the growing incisor and associated with a significant reduction of MMP-3 and MMP-20 expression in tooth germs of KO mice. Insufficient basement membrane degradation is evidenced by a persistent laminin immunostaining, resulting in a delay of both odontoblast and ameloblast differentiation. Consequently, enamel volume and thickness are decreased in adult mutant teeth but enamel maturation and tooth morphology are normal, as shown by micro-computed tomographic (micro-CT), nanoindentation, and scanning electron microscope analyses. In addition, the dentino-enamel junction appears as a rough calcified layer of approximately 10±5µm thick (mean±SD) in both molars and growing incisors of KO adult mice. These results indicate that EMMPRIN is involved in the epithelial-mesenchymal cross-talk during tooth development by regulating the expression of MMPs. The mild tooth phenotype observed in EMMPRIN KO mice suggests that the direct effect of EMMPRIN may be limited to a short time window, comprised between basement membrane degradation allowing direct cell contact and calcified matrix deposition.

EMMPRIN/CD147-encriched membrane vesicles released from malignant human testicular germ cells increase MMP production through tumor-stroma interaction.

BACKGROUND: Elevated levels of EMMPRIN/CD147 in cancer tissues have been correlated with tumor progression but the regulation of its expression is not yet understood. Here, the regulation of EMMPRIN expression was investigated in testicular germ cell tumor (TGCTs) cell lines. METHODS: EMMPRIN expression in seminoma JKT-1 and embryonal carcinoma NT2/D1 cell lines was determined by Western blot, immunofluorescence and qRT-PCR. Membrane vesicles (MVs) secreted from these cells, treated or not with EMMPRIN siRNA, were isolated by differential centrifugations of their conditioned medium. MMP-2 was analyzed by zymography and qRT-PCR. RESULTS: The more aggressive embryonic carcinoma NT2/D1 cells expressed more EMMPRIN mRNA than the seminoma JKT-1 cells, but surprisingly contained less EMMPRIN protein, as determined by immunoblotting and immunostaining. The protein/mRNA discrepancy was not due to accelerated protein degradation in NT2/D1 cells, but by the secretion of EMMPRIN within MVs, as the vesicles released from NT2/D1 contained considerably more EMMPRIN than those released from JKT-1. EMMPRIN-containing MVs obtained from NT2/D1, but not from EMMPRIN-siRNA treated NT2/D1, increased MMP-2 production in fibroblasts to a greater extent than those from JKT-1 cells. CONCLUSION AND GENERAL SIGNIFICANCE: The data presented show that the more aggressive embryonic carcinoma cells synthesize more EMMPRIN than seminoma cells, but which they preferentially target to secreted MVs, unlike seminoma cells which retain EMMPRIN within the cell membrane. This cellular event points to a mechanism by which EMMPRIN expressed by malignant testicular cells can exert its MMP inducing effect on distant cells within the tumor microenvironment to promote tumor invasion. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

Dentin matrix degradation by host matrix metalloproteinases: inhibition and clinical perspectives toward regeneration.

Bacterial enzymes have long been considered solely accountable for the degradation of the dentin matrix during the carious process. However, the emerging literature suggests that host-derived enzymes, and in particular the matrix metalloproteinases (MMPs) contained in dentin and saliva can play a major role in this process by their ability to degrade the dentin matrix from within. These findings are important since they open new therapeutic options for caries prevention and treatment. The possibility of using MMP inhibitors to interfere with dentin caries progression is discussed. Furthermore, the potential release of bioactive peptides by the enzymatic cleavage of dentin matrix proteins by MMPs during the carious process is discussed. These peptides, once identified, may constitute promising therapeutical tools for tooth and bone regeneration.

Cell membrane vesicles as a tool for the study of direct epithelial-stromal interaction: lessons from CD147.

Communication between the epithelial and stromal tissue layers, separated by basement membrane, is known to provide the information necessary for development, differentiation, and homeostasis. These interactions are altered in benign or malignant diseases, in particular when the basement membrane barrier is disrupted allowing a greater proximity between the two cell layers that triggers tissue remodeling. Epithelial-stromal interactions (ESI) have been examined in vitro by various approaches that can be broadly divided into interactions arising from secreted diffusible factors and interactions through direct cell-cell contact. Here we describe a method for the study of direct ESI through CD147, an adhesion molecule present on the epithelial cell surface and which is known to interact with stromal cells, such as fibroblasts and endothelial cells, and signal them to increase production of matrix metalloproteinases. This method can be extended to other adhesion molecules involved in ESI.

Early events of overused supraspinatus tendons involve matrix metalloproteinases and EMMPRIN/CD147 in the absence of inflammation.

BACKGROUND: The principal feature of tendon degeneration is structural change of the extracellular matrix (ECM) including collagens. In painful tendons, alterations of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) have been described; however, the initial molecular mechanism at the origin of these alterations is still poorly understood. A rat model of supraspinatus tendon overuse has been developed, which may be predictive of pathological tendon alterations. PURPOSE: To determine which MMPs are involved in early ECM remodeling during overuse and their relationship with the inflammatory context. STUDY DESIGN: Controlled laboratory study. METHODS: Analyses were performed on rat supraspinatus tendons at 2 and 4 weeks of overuse on a downhill treadmill. Transcript levels of MMPs and TIMPs were assessed by semiquantitative reverse transcription polymerase chain reaction. Western blotting and/or immunolabeling were used for MMP-2, MMP-3, MMP-13, and extracellular MMP inducer (EMMPRIN, also called cluster of differentiation [CD] 147) detection. In situ and/or sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gelatin zymography was performed for MMP-2 and MMP-9. TIMP activity was revealed by reverse zymography. Inflammation was assessed by cytokine antibody array and/or immunolabeling. RESULTS: Compared with a control, overused supraspinatus tendons showed a significantly higher gelatinolytic activity at 2 weeks, which slightly decreased at 4 weeks. MMP-9 and MMP-13 were undetectable; MMP-3 was downregulated in overused tendons. Only MMP-2, particularly its active form, and the MMP-2 activator MMP-14 were upregulated at 2 weeks of overuse when an increase in TIMP-2 transcripts was observed. MMP-2 upregulation occurred in the absence of inflammation but was associated with an increase of EMMPRIN/CD147. CONCLUSION: EMMPRIN/CD147-regulated MMP-2 and MMP-14, associated with low MMP-3, appear as the main characteristics of ECM remodeling in early overused tendons. Whether alterations in the pattern of these MMPs are an adaptive response or a repair response that may degenerate into tendinosis, is still uncertain. Moreover, there seems to be no indication for an inflammatory response to overuse, suggesting that the increased metalloproteinase activity is rather a response to a mechanical stress than an inflammatory one. CLINICAL RELEVANCE: Any strategy aimed at preventing full-thickness tears resulting from initial tendon matrix alterations should consider these changes in MMP-3, MMP-2, and MMP-14, or further upstream, EMMPRIN.

Loss of receptor protein tyrosine phosphatase ß/ζ (RPTPß/ζ) promotes prostate cancer metastasis.

BACKGROUND: The role of pleiotrophin and its receptors RPTPß/ζ and Syndecan-3 during tumor metastasis remains unknown. RESULTS: RPTPß/ζ knockdown initiates EMT, promotes pleiotrophin-mediated migration and attachment through Syndecan-3 and induces in vivo metastasis. CONCLUSION: RPTPß/ζ plays a suppressor-like role in prostate cancer metastasis. SIGNIFICANCE: Boosting RPTPß/ζ or attenuating Syndecan-3 signaling pathways may lead to more effective therapeutic strategies in treating prostate cancer metastasis. Pleiotrophin is a growth factor that induces carcinogenesis. Despite the fact that many published reports focused on the role of pleiotrophin and its receptors, receptor protein tyrosine phosphatase (RPTPß/ζ), and syndecan-3 during tumor development, no information is available regarding their function in tumor metastasis. To investigate the mechanism through which pleiotrophin regulates tumor metastasis, we used two different prostate carcinoma cell lines, DU145 and PC3, in which the expression of RPTPß/ζ or syndecan-3 was down-regulated by the RNAi technology. The loss of RPTPß/ζ expression initiated epithelial-to-mesenchymal transition (EMT) and increased the ability of the cells to migrate and invade. Importantly, the loss of RPTPß/ζ expression increased metastasis in nude mice in an experimental metastasis assay. We also demonstrate that RPTPß/ζ counterbalanced the pleiotrophin-mediated syndecan-3 pathway. While the inhibition of syndecan-3 expression inhibited the pleiotrophin-mediated cell migration and attachment through the Src and Fak pathway, the inhibition of RPTPß/ζ expression increased pleiotrophin-mediated migration and attachment through an interaction with Src and the subsequent activation of a signal transduction pathway involving Fak, Pten, and Erk1/2. Taken together, these results suggest that the loss of RPTPß/ζ may contribute to the metastasis of prostate cancer cells by inducing EMT and promoting pleiotrophin activity through the syndecan-3 pathway.

Multivalent pseudopeptides targeting cell surface nucleoproteins inhibit cancer cell invasion through tissue inhibitor of metalloproteinases 3 (TIMP-3) release.

Blockage of the metastasis process remains a significant clinical challenge, requiring innovative therapeutic approaches. For this purpose, molecules that inhibit matrix metalloproteinases activity or induce the expression of their natural inhibitor, the tissue inhibitor of metalloproteinases (TIMPs), are potentially interesting. In a previous study, we have shown that synthetic ligands binding to cell surface nucleolin/nucleophosmin and known as HB 19 for the lead compound and NucAnt 6L (N6L) for the most potent analog, inhibit both tumor growth and angiogenesis. Furthermore, they prevent metastasis in a RET transgenic mice model which develops melanoma. Here, we investigated the effect of N6L on the invasion capacity of MDA-MB-435 melanoma cells. Our results show that the multivalent pseudopeptide N6L inhibited Matrigel invasion of MDA-MB-435 cells in a modified Boyden chamber model. This was associated with an increase in TIMP-3 in the cell culture medium without a change in TIMP-3 mRNA expression suggesting its release from cell surface and/or extracellular matrix. This may be explained by our demonstrated N6L interaction with sulfated glycosaminoglycans and consequently the controlled bioavailability of glycosaminoglycan-bound TIMP-3. The implication of TIMP-3 in N6L-induced inhibition of cell invasion was evidenced by siRNA silencing experiments showing that the loss of TIMP-3 expression abrogated the effect of N6L. The inhibition of tumor cell invasion by N6L demonstrated in this study, in addition to its previously established inhibitory effect on tumor growth and angiogenesis, suggests that N6L represents a promising anticancer drug candidate warranting further investigation.