Extracellular modulators of epithelial mesenchymal transition: functional insights into tumour angiogenesis

Thesis with publications.

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Biochemistry and Genetics, School of Molecular Science, College of Science, Health and Engineering, La Trobe University, Victoria.

Epithelial-mesenchymal transition (EMT), a morphogenic process defined by the loss of epithelial characteristics and acquisition of mesenchymal phenotype, is associated with increased metastatic potential in carcinoma cells. Using Madin-Darby canine kidney (MDCK) cells, and MDCK cells transformed with oncogenic H-Ras (21D1 cells) as a model, previous studies from our lab identified significantly elevated levels of the master transcriptional regulator YBX1 and the metalloproteinase MMP-1 in the extracellular culture medium (secretome). Following on from these findings, I surmised that YBX1 and MMP1 might facilitate tumorigenesis and angiogenesis. In this thesis, I first examined the overexpression of YBX1 in MDCK cells (MDCKYBX1) and revealed that elevated YBX1 levels led to induction of a partial EMT phenotype (Chapter 2). MDCKYBX1 cells displayed cytosolic relocalisation of E-cadherin, increased cell scattering and anchorageindependent growth. Interestingly, subcutaneous injection of MDCKYBX1 cells into NOD/SCID mice formed tumours, in contrast to MDCK cells that could not establish a tumour xenograft. Further, mass-spectrometry-based sequencing of the MDCKYBX1 cell secretome identified secretion of factors known to enhance angiogenesis (VGF, ADAM9 and ADAM17). These findings define YBX1 as an important oncogenic enhancer that can promote angiogenesis (Gopal et al., 2016, Oncotarget, PMID: 25980435). Next, I investigated the functional utility of exosomes (30-150 nm extracellular vesicles) derived from MDCKYBX1 cells (Chapter 3). Unlike parental MDCK-derived exosomes, MDCKYBX1 exosomes significantly enhanced the motility and tube formation (length and branching) of recipient 2F-2B endothelial cells. Significantly, injection of 2F-2B cells treated with MDCKYBX1 exosomes into mice led to in vivo systemic perfusion of the MatrigelTM plug. Proteomic analysis of MDCKYBX1 exosomes revealed elevated levels of activated Rac1/PAK2 which promotes angiogenesis in recipient 2F-2B cells (Gopal et al., 2016, Oncotarget, PMID: 26919098). Finally, in Chapter 4 I investigated the contribution of MMP1 during EMT using 21D1 cells with attenuated MMP1 expression (RNAi). Characterisation of these cells revealed decreased cell migration, invasion, anchorageindependent growth in vitro and reduced xenograft tumour volume in vivo. Interestingly, I identified LAMA5 as a novel biological substrate of MMP1 which produces a proteolytic fragment that can bind to integrin αvβ3 on endothelial cell membranes and induce angiogenesis (Gopal et al., 2016, Scientific Reports, PMID: 2732484). Collectively, my studies reveal novel molecular mechanisms by which EMT cells can promote tumour angiogenesis.