The control of lymphatic vascular remodelling in cancer by microRNAs

Abstract

Metastasis is the lethal aspect of cancer for most patients. Remodelling of lymphatic vessels associated with a tumour is a key initial step in metastasis because it facilitates the entry of cancer cells into the lymphatic vasculature and their spread to lymph nodes and distant organs. Although it is clear that vascular endothelial growth factors (VEGFs), such as VEGFC and VEGFD, are key drivers of lymphatic remodelling in cancer, the means by which many signalling pathways in endothelial cells are co-ordinately regulated to drive growth and remodelling of lymphatics in cancer is not understood. In this thesis, I seek to understand the broader molecular mechanisms that control cancer metastasis through the analysis of microRNAs which act to co-ordinately regulate signalling pathways involved in complex biological responses, such as lymphatic remodelling, in health and disease. Here, using high-throughput small RNA sequencing, I found that a specific microRNA, miR-132, is up-regulated in expression in lymphatic endothelial cells (LECs) in response to stimulation with VEGFC and VEGFD. Interestingly, inhibiting the effects of miR-132 in LECs in vitro blocked proliferation and tube formation of these cells induced by VEGFC and VEGFD - LEC proliferation and tube formation are key steps in lymphatic remodelling. Moreover, I demonstrated that miR-132 is expressed in the lymphatic vessels of a subset of human breast tumours which were previously found to express high levels of VEGFD. In order to dissect the complexity of molecular regulation by miR-132 in lymphatic biology, my collaborators and I identified miR-132 target mRNAs in LECs, using high-throughput sequencing after RNA-protein cross-linking and immunoprecipitation of the Argonaute protein (Argonaute HITS-CLIP), which led us to define the miR-132-mRNA interactome in LECs. We found that this microRNA in LECs is involved in the control of many different molecular pathways mainly involved in cell proliferation and regulation of the extracellular matrix and cell-cell junctions. It is logical that miR-132 regulates such pathways given they are involved in the processes of LEC proliferation and tube formation, which I showed are dependent on miR-132 in my in vitro studies. Finally, I demonstrated that inhibiting the effects of miR-132 in a mouse ear model of lymphangiogenesis, using an antagomiR inhibitor of miR-132 coupled to cholesterol, blocked the complex remodelling of lymphatic vessels stimulated by VEGFC, in vivo. It was noteworthy that all aspects of lymphatic remodelling induced by VEGFC were restricted by inhibition of miR-132, including the enlargement, branching and sprouting of lymphatic vessels. Thus the inhibitory effect of targeting this microRNA on lymphangiogenesis and lymphatic remodelling can be considered comprehensive. The research described in this thesis identified miR-132 as a critical regulator of lymphangiogenesis and lymphatic remodelling, and delineated molecular mechanisms by which this microRNA influences these important biological processes. This work also identified new molecular pathways which are involved in modifying the lymphatic vasculature in response to key lymphangiogenic growth factors. In-so-doing, these studies identified potential therapeutic targets for drugs designed to block the growth and remodelling of tumour lymphatics, and thereby restrict the metastatic spread of cancer.