Endocytosis and Trafficking of Heparan Sulfate Proteoglycans in Triple-Negative Breast Cancer Cells Unraveled with a Polycationic Peptide.

The process of heparan sulfate proteoglycan (HSPG) internalization has been described as following different pathways. The tumor-specific branched NT4 peptide has been demonstrated to bind HSPGs on the plasma membrane and to be internalized in tumor cell lines. The polycationic peptide has been also shown to impair migration of different cancer cell lines in 2D and 3D models. Our hypothesis was that HSPG endocytosis could affect two important phenomena of cancer development: cell migration and nourishment. Using NT4 as an experimental tool mimicking heparin-binding ligands, we studied endocytosis and trafficking of HSPGs in a triple-negative human breast cancer cell line, MDA-MB-231. The peptide entered cells employing caveolin- or clathrin-dependent endocytosis and macropinocytosis, in line with what is already known about HSPGs. NT4 then localized in early and late endosomes in a time-dependent manner. The peptide had a negative effect on CDC42-activation triggered by EGF. The effect can be explained if we consider NT4 a competitive inhibitor of EGF on HS that impairs the co-receptor activity of the proteoglycan, reducing EGFR activation. Reduction of the invasive migratory phenotype of MDA-MB-231 induced by NT4 can be ascribed to this effect. RhoA activation was damped by EGF in MDA-MB-231. Indeed, EGF reduced RhoA-GTP and NT4 did not interfere with this receptor-mediated signaling. On the other hand, the peptide alone determined a small but solid reduction in active RhoA in breast cancer cells. This result supports the observation of few other studies, showing direct activation of the GTPase through HSPG, not mediated by EGF/EGFR.

Heparan Sulfate Proteoglycans Can Promote Opposite Effects on Adhesion and Directional Migration of Different Cancer Cells.

Heparan sulfate proteoglycans take part in crucial events of cancer progression, such as epithelial-mesenchymal transition, cell migration, and cell invasion. Through sulfated groups on their glycosaminoglycan chains, heparan sulfate proteoglycans interact with growth factors, morphogens, chemokines, and extracellular matrix (ECM) proteins. The amount and position of sulfated groups are highly variable, thus allowing differentiated ligand binding and activity of heparan sulfate proteoglycans. This variability and the lack of specific ligands have delayed comprehension of the molecular basis of heparan sulfate proteoglycan functions. Exploiting a tumor-targeting peptide tool that specifically recognizes sulfated glycosaminoglycans, we analyzed the role of membrane heparan sulfate proteoglycans in the adhesion and migration of cancer cell lines. Starting from the observation that the sulfated glycosaminoglycan-specific peptide exerts a different effect on adhesion, migration, and invasiveness of different cancer cell lines, we identified and characterized three cell migration phenotypes, where different syndecans are associated with alternative signaling for directional cell migration.

Unraveling Heparan Sulfate Proteoglycan Binding Motif for Cancer Cell Selectivity.

Membrane heparan sulfate proteoglycans (HSPG) regulate cell proliferation, migration, and differentiation and are therefore considered key players in cancer cell development processes. Here, we used the NT4 peptide to investigate how the sulfation pattern of HSPG on cells drives binding specificity. NT4 is a branched peptide that binds the glycosaminoglycan (GAG) chains of HSPG. It has already been shown to inhibit growth factor-induced migration and invasiveness of cancer cells, implying antagonist binding of HSPG. The binding affinity of NT4 with recombinant HSPG showed that NT4 bound glypican-3 and -4 and, with lower affinity, syndecan-4. NT4 binding to the cancer cell membrane was inversely correlated with sulfatase expression. NT4 binding was higher in cell lines with lower expression of SULF-1 and SULF-2, which confirms the determinant role of sulfate groups for recognition by NT4. Using 8-mer and 9-mer heparan sulfate (HS) oligosaccharides with analog disaccharide composition and different sulfation sites, a possible recognition motif was identified that includes repeated 6-O-sulfates alternating with N- and/or 2-O-sulfates. Molecular modeling provided a fully descriptive picture of binding architecture, showing that sulfate groups on opposite sides of the oligosaccharide can interact with positive residues on two peptide sequences of the branched structure, thus favoring multivalent binding and explaining the high affinity and selectivity of NT4 for highly sulfated GAGs. NT4 and possibly newly selected branched peptides will be essential probes for reconstructing and unraveling binding sites for cancer-involved ligands on GAGs and will pave the way for new cancer detection and treatment options.

The GAG-specific branched peptide NT4 reduces angiogenesis and invasiveness of tumor cells.

Heparan sulfate proteoglycans, HSPGs, modulate major transformations of cancer cells, leading to tumor growth, invasion and metastasis. HSPGs also regulate neo-angiogenesis which prompts cancer progression and metastatic spread. A different aspect of heparin and analogs is their prominent role in the coagulation of blood. The interplay between coagulation and metastasis is being actively studied: anticoagulants such as heparin-derivatives have anticancer activity and procoagulants, such as thrombin, positively modulate proliferation, migration and invasion. The branched peptide NT4 binds to HSPGs and targets selectively cancer cells and tissues. For this, it had been extensively investigated in the last years and it proved to be efficient as chemotherapeutic and tumor tracer in in vivo models of cancer. We investigated the effects of the branched peptide in terms of modulation of angiogenesis and invasiveness of cancer cells. NT4 proved to have a major impact on endothelial cell proliferation, migration and tube formation, particularly when induced by FGF2 and thrombin. In addition, NT4 had important effects on aggressive tumor cells migration and invasion and it also had an anticoagulant profile.The peptide showed very interesting evidence of interference with tumor invasion pathways, offering a cue for its development as a tumor-targeting drug, and also for its use in the study of links between coagulation and tumor progression involving HSPGs.

Coupling to a cancer-selective heparan-sulfate-targeted branched peptide can by-pass breast cancer cell resistance to methotrexate.

Cancer-selective tetra-branched peptides, named NT4, can be coupled to different functional units for cancer cell imaging or therapy. NT4 peptides specifically bind to lipoprotein receptor-related proteins (LRP) receptors and to heparan sulfate chains on membrane proteoglycans and can be efficiently internalized by cancer cells expressing these membrane targets. Since binding and internalization of NT4 peptides is mediated by specific NT4 receptors on cancer cell membranes and this may allow drug resistance produced by drug membrane transporters to be by-passed, we tested the ability of drug-armed NT4 to by-pass drug resistance in cancer cell lines. We found that MTX-conjugated NT4 allows drug resistance to be by-passed in MTX-resistant human breast cancer cells lacking expression of folate reduced carrier. NT4 peptides appear to be extremely promising cancer-selective targeting agents that can be exploited as theranostics in personalized oncological applications.

Insights into the role of sulfated glycans in cancer cell adhesion and migration through use of branched peptide probe.

The tetra-branched peptide NT4 selectively binds to different human cancer cells and tissues. NT4 specifically binds to sulfated glycosaminoglycans on cancer cell membranes. Since sulfated glycosaminoglycans are involved in cancer cell interaction with the extracellular matrix, we evaluated the effect of NT4 on cancer cell adhesion and migration. We demonstrated here that the branched peptide NT4 binds sulfated glycosaminoglycans with high affinity and with preferential binding to heparan sulfate. NT4 inhibits cancer cell adhesion and migration on different proteins, without modifying cancer cell morphology or their ability to produce protrusions, but dramatically affecting the directionality and polarity of cell movement. Results obtained by taking advantage of the selective targeting of glycosaminoglycans chains by NT4, provide insights into the role of heparan sulfate proteoglycans in cancer cell adhesion and migration and suggest a determinant role of sulfated glycosaminoglycans in the control of cancer cell directional migration.