Study of role of sulphated proteoglycans in cancer

Pancreatic cancer is projected to become the second leading cause of death by 2030, due to an extremely immune-suppressive tumour environment and lack of early-stage markers. Over the last decade, new insights have emerged regarding the mechanism and biological significance of the interactions between heparan sulphate proteoglycans (HSPGs) and their ligands. HSPGs are found in cell surface and extracellular matrix (ECM) and contribute to the binding of growth factors and chemokines to their receptors, thus HSPGs can modulate cell-growth, proliferation and play an important role in cell migration. HSPGs overexpression in some tumours has enabled the concrete hypothesis of their use as possible new markers and target treatment. In this study, we investigated the tumour specificity and the bioactivity of different tetra-branched peptides that could target HSPGs and be effective as new cancer drugs. Particularly, we focused on peptide’s specificity on pancreatic cancer cell lines. The use of the tetra-branched structure overcomes the sensitivity to proteolytic degradation of the linear monomeric structure, increasing their half-life and maintaining their target specificity. BOPs proved to be selective in binding and killing tumour cells, particularly pancreatic cancer cells, PANC-1 and Mia Paca-2, compared to cells with no tumoral origin RAW 264.7 and PgsA-745. The promising cancer selectivity exhibited by the two peptides (BOP7 and BOP9) is likely attributed to their repeated cationic sequences, enabling multivalent binding to heparan sulphate proteoglycans (HSPGs) bearing anionic sulfation patterns in cancer cells, that typically exhibit increased surface negativity. This interaction of BOPs with HSPGs not only fosters an anti-metastatic effect in vitro, as evidenced by reduced adhesion and migration of PANC-1 cancer cells, but also demonstrated promising specific tumour cytotoxicity and low haemolytic activity. A preliminary in vivo assay in nude mice, showed an encouraging ability of BOP9 to inhibit tumour grafting and growth by 20% in a pancreatic cancer model. In the perspective of increasing the chances of early diagnosis of pancreatic cancer, tumour conditioned medium of pancreatic cell cultures was analysed for whole RNA from exosomes. Lastly, to understand the regulation of proteoglycans intracellular pathways on pancreatic cancer cells, we investigate Syndecan-1 (Sdc-1) and Glypican-1 (Gpc-1) role in mTOR signalling. mTOR is involved in migration, proliferation, autophagy, survival and catabolism processes and its inhibition led to proliferative arrest in KRAS/PTEN deficient tumours, but not in KRAS/p53 mutated. On the other hand, Sdc-1 and Gpc-1 seem to be related to AKT and ERK pathway in several tumours. So, we first treated all the primary pancreatic cell lines with AZD2014 (mTORC2 inhibitor) and Rapamycin (mTORC1 inhibitor) to verify the effect of the drug on proteoglycans cell surface expression. Then, we overexpressed Sdc-1 to investigate its role in AKT/ERK cascade in PA14C cells. In fact, the ability of syndecan-1 to enhance TGFβ binding on cell surface receptors let us suppose its involvement in the AKT/ERK pathway. Furthermore, AKT overactivation is present in 33% ductal carcinoma in situ lesions, while phosphorylation of ERK1/2 is known to be necessary for TGFβ-induced epithelial–mesenchymal transition (EMT) in pancreatic cancer cells, and their inhibition reduce CDK2 levels and prevent EMT due to the downstream regulation of c-MYC, with effects on cancer cell growth suppression and cellular senescence. To summarize, peptides demonstrated a great specificity for cancer cells and the ability to reduce their migration and proliferation properties. BOPs target, HSPGs, could play a role in mTOR signalling opening new perspectives in cancer treatment.