Understand the Role of Glycans in the Oncogenesis of Pediatric Sarcomas

Abstract

Sarcoma, a cancer originating from mesenchymal cells, is more prevalent in children and adolescents (<18) than in adults. The most common pediatric sarcoma (PS) subtypes are rhabdomyosarcoma (RMS), osteosarcomas (OS) and Ewing sarcoma (ES). Diagnosing and treating these cancers remains a clinical challenge, highlighting the need for new biomarkers to improve diagnosis and develop more effective therapies. Aberrant glycans expression is a feature in cancer, with alterations in cellular glycosylation having a role in tumor development and progression. This thesis aimed to characterize aberrant glycan expression in the most common PS subtypes and explore their biological roles in PS oncogenesis. We started by a retrospective epidemiological analysis of PS cases in the North of Portugal. Further, we assessed, by immunofluorescence and flow cytometry analysis, the expression of described cancer-associated glycans, in commercially available cell lines from the three most prevalent PS subtypes. To unveil the biological roles of the identified glycans, the cell lines were glycoengineered, by CRISPR/Cas9 technology, and parameters such as proliferation, motility, invasion and activation of receptor tyrosine kinase (RTK) were assessed. Promising identified glycans were further validated in tissues samples from our retrospective patient cohort. Our epidemiological study revealed that along the past 23 years, 15 different PS subtypes were diagnosed, with ES, RMS and OS being the most common. A differential expression of cancer-associated glycans was observed across the different cell lines in study. Terminal α2,6 sialylation was highly expressed in ES and RMS cell lines, while heparan sulfate (HS) was prominent in the OS cell line. To investigate their functional roles, we knocked-out (KO) the enzymes responsible for their biosynthesis, resulting in the loss of terminal α2,6 sialylation in ES and RMS KO clones and a substantial reduction of HS in OS KO clones. Additionally, KO of HS led to a marked decrease in metabolic activity of OS cells, as well as reduction in the activation of few RTK such as PDGFRα. Ultimately, HS expression was validated in tissues samples from pediatric OS patients from our cohort, with positivity observed in seven out of ten cases.