Uma Thanigai Arasu’s doctoral thesis delivers novel information about the role of hyaluronan (HA) synthesis on secretion of extracellular vesicles (EVs) and their content to stimulate oncogenic functions in recipient cells. The results suggest that mesenchymal stem cells and melanoma cells secrete HA-coated EVs with the ability to change the nature of their internalized target cells. Findings from this thesis work could be utilized as non-invasive prognostic and therapeutic tools in the future.
HA is an abundant polysaccharide found in the extracellular matrix; it is essential for the maintenance of normal tissues, but it also promotes cancer progression by creating a favorable microenvironment to allow the growth of tumour cells. HA is synthesized by specific plasma membrane-bound enzymes, hyaluronan synthases (HAS 1-3), producing a chain made up of repeating units of N-acetylglucosamine and glucuronic acid. EVs, including microvesicles, exosomes and apoptotic bodies, are membrane-derived extracellular particles that contain and transfer cytosolic components, proteins, RNA, ribosomes and selected plasma membrane proteins. The EVs that originate from cancer cells carry characteristics of their cellular origin and may serve as a surrogate for tumour biopsies, enabling real-time diagnosis and disease monitoring. It is surprising that even though all the body fluids known to contain an abundance of HA, such as synovial fluid, plasma and ascites of cancer patients, are rich in EVs, the data included in this thesis is the first time that EVs have been demonstrated to act as special carriers of HA.
This thesis aimed at achieving a more profound understanding of the functional relevance of HA synthesis and its effect on the secretion of EVs. Initially it was observed that bone marrow derived mesenchymal stem cells secreted high levels of endogenous HA, part of which was associated with the EVs. These HA coated MSC-EVs may be one of the factors mediating tissue regeneration and wound healing e.g. the interaction of these particles with other cells could be one mode of intercellular communication. Overexpression of GFP-HAS3 in MV3 metastatic melanoma cells promoted high levels of HA and EV secretion. Furthermore, enhancing the levels of UDP sugars not only increased the duration and proportion of GFP-HAS3 residing on the plasma membrane but it was also associated with released EVs, termed as HAS3-EVs. When the levels of UDP-sugars levels declined, an opposite effect was observed in the EV secretion and HAS3 plasma membrane residence duration and proportion. The results indicated that the presence of HAS3 on the plasma membrane was required for HA secretion and its release in the EVs.
The HAS3-EVs released by MV3 metastatic melanoma cells were able to induce tumorigenic properties in their target cells, HaCaT (keratinocytes) and WM115 (melanoma). It was observed that HAS3-EVs carry IHH (Indian Hedgehog) ligands which interacted with the target cells and were able to induce the hedgehog signaling (HH) pathway. The downstream target of the HH pathway, c-Myc, was upregulated with the subsequent expression of claspin. This signaling axis evoked an increase in proliferation, invasion and epithelial to mesenchymal transition in the target cells. In vivo staining of melanoma tissue sections revealed a correlation in the expression pattern between HA and claspin. The presence of IHH ligands in the EVs was associated with the HA synthesis rate of the donor melanoma cells. Inhibition of HH signaling also affected HA synthesis in melanoma cells. Inhibition of HA synthesis or HH signaling in MV3 melanoma cells resulted in decreased incorporation of IHH in HAS3-EVs. Conversely, increased HA synthesis increased the association of IHH with HAS3-EVs. The differential levels of IHH in HAS3-EVs were directly proportional to the level of HA synthesis and the proliferation rate in the target cells. The positive feedback mechanism displayed by HA and HH pathways in melanoma is a novel finding emerging from this study.
In summary, the results presented in this thesis reveal details of the molecular mechanisms involved in HAS3 trafficking and the related HA synthesis and its association with EV production. Moveover, the researchers unraveled the signaling axis activated by IHH which was mediated by HAS3-EVs in the target cells and we also observed that HAS3-EVs were capable of inducing tumorigenic properties in target cells. Understanding how HA-coated EVs from cancer cells induce oncogenic properties in target cells is important to address the development of diagnostic procedures and therapeutic interventions in HA rich cancer types.
The doctoral dissertation of Uma Thanigai Arasu, entitled Hyaluronan-coated extracellular vesicles – regulation of their secretion and interaction with their target cells will be examined at the Faculty of Health Sciences on 4 April 2020. The Opponent in the public examination will be Prof. Mattias Belting of the Department of Clinical Sciences, Lund University and the Custos will be Dr. Kirsi Rilla of the University of Eastern Finland. The public examination will be held in English.
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