Alternative Therapeutic Approaches in Blocking the Oncogenic Role of the Siglec-15/Sialyl-Tn Axis in Colorectal Cancer

Abstract

Colorectal cancer (CRC) malignancies are among the highest mortality and morbidity rates worldwide. Despite the emergence of numerous screening programs to reduce CRC incidence, the number of CRC and early-onset CRC diagnoses has risen exponentially, resulting in difficulties in curative surgical control and subsequent tumour related deaths. Thus, there is an unmet clinical need for developing targeted strategies in CRC treatment. As an emerging immune checkpoint protein, Siglec-15 is overexpressed in several malignancies and is correlated to the development of the tumour microenvironment through establishing immunosuppression upon binding with its putative ligand, Sialyl-Tn (STn). However, the role of Siglec-15 particularly relating to CRC remains elusive. The identification of regulatory mechanisms surrounding the Siglec-15/STn axis is poorly understood, and current therapeutic approaches have only shown a positive response in a small subset of cancer patients. Thus, underlining the clinical role of oncogenic targets such as Siglec-15 and the significant tumour heterogeneity that CRC tumours pose. As more recent approaches are focused on the development of monoclonal antibodies for Siglec-15 targeting, the development of small molecule therapeutic agents for Siglec-15 inhibition has not yet been elucidated. Hence, this study aimed to investigate the potential of key sialyltransferases as therapeutic targets in CRC by investigating the regulatory mechanisms that are associated with their expression profiles, the role they play in glycan biosynthesis, tumour progression, and the production of the STn antigen, respectively. Moreover, we aimed to experimentally demonstrate the characterisation of the cytotoxic profile of a b-amino carbonyl compound (SHG-8) and aleplasinin for Siglec- 15 targeting within in vitro cell models as feasible treatment alternatives. Initial datamining of the sialyltransferases ST6GALNAC1, ST6GALNAC2, ST3GAL4 and ST6GAL1 were performed using readily available transcriptomics tools including UALCAN and miRNA target prediction (TargetScan, MiRSystem, MiRDB and MirWalk) databases and binding site software for sialyltransferase expression profiles in colon adenocarcinoma tumours, pathologically the most frequently occurring CRC tumour type. Following this, tumour hallmarks associated with CRC progression were determined in relation to sialyltransferase expression through gene set enrichment analysis (GSEA). Similarly, their expression in association with the abundance of myeloid cell populations and prevalent immune checkpoint proteins were also demonstrated with integrated repositories readily available with the TIMER and TISIDB databases. Lastly, the expression of ST6GalNAc1 and ST6GalNAc2 at the protein level was determined via immunohistochemical analysis to underline their importance in the production of the STn antigen. Furthermore, molecular docking simulations were utilised to illustrate the potential of SHG-8 and aleplasinin as competitive small molecule inhibitors to the V-set binding domain of the Siglec-15 protein structure, rendering the STn antigen unable to directly bind to the R143 residue. Moreover, the characterisation of small molecule inhibitors on tumour viability, migration and colonisation via functional assays was determined in vitro on mammalian cell models SW480 and HCT116, both of which are representative of the large proportion of CRC diagnoses. In addition, apoptosis staining methods were performed to assess cellular arrest and apoptosis induction. Conversely, RNA sequencing methods were utilised to underline the expression profiles of miRNAs and gene targets following treatment exposure. Further exploration of these targets were also determined to elucidate potential CRC mechanisms of action. The sialyltransferases revealed downregulated expression profiles excluding ST6GAL1 in CRC adenocarcinoma tumours. Similarly, the identified interactive partners all exhibited varied expression profiles. Furthermore, common miRNAs involved in regulating sialyltransferases that play major roles in glycan biosynthesis were identified and exhibited high binding affinities. Enrichment analysis revealed several dysregulated tumorigenic hallmarks associated with CRC progression. Furthermore, the association of myeloid cells and prevalent immune checkpoint proteins underlined the multifaceted roles of the sialyltransferases in CRC. The immunohistochemistry analysis revealed that STn production is independent of ST6GalNAc1/2 activity in CRC. Moreover, aleplasinin and SHG-8 exhibited a high binding affinity to the V-set binding domain of the Siglec-15 protein structure. Both compounds exerted dose-dependent cytotoxicity on cellular viability, migration and colonisation. Apoptosis staining methods demonstrated late apoptosis induction and nuclear fragmentation at higher treatment concentrations. Sequencing analysis methods demonstrated that the miR-6715b- 3p/PTTG1IP and let-7a-5p/ACSL6 axes could be involved in CRC progression following treatment exposure. The evidence presented in this study suggested possible approaches for disrupting the Siglec- 15/Sia axis and underlined the roles of sialyltransferases as potential therapeutic targets in CRC tumours. Furthermore, we have expanded the therapeutic landscape of Siglec-15 and highlighted treatment alternatives in Siglec-15+ tumours in contrast to current conventional therapeutic agents. Therefore, this will greatly help in the transition of novel therapeutic agents towards a clinical setting to address the challenges that is facing Siglec-15 mediated CRC progression. Ultimately, a multifaceted treatment approach based on our findings would offer the novel development of further inhibitors in targeting Siglec-15 and significantly advance the field of oncology.