Mechanisms of IRES driven Laminin B1 translation in tumor metastasis

Laminin B1 (LamB1) is an extracellular matrix protein regulating cancer cell migration and invasion. Metastasis of carcinoma cells is frequently associated with epithelial to mesenchymal transition (EMT) which allows tumor cells to gain an invasive phenotype and to dissociate from epithelial cell clusters of the tumor. We recently showed that LamB1 is translationally upregulated in malignant hepatocytes that have undergone EMT by the collaboration of oncogenic H-Ras and transforming growth factor (TGF)-ß signaling. Increased LamB1 translation in metastatic hepatocytes proved to be mediated by an internal ribosome entry site (IRES) located in the 5`-untranslated region (UTR) of the LamB1 transcript. We identified La as an IRES trans-acting factor (ITAF) that translocates into the cytoplasm and binds the minimal LamB1 IRES motif to enhance its activity in EMT-transformed hepatocytes. The translocation of La is triggered by platelet derived growth factor (PDGF) signaling that is downstream of TGF-ß. The aim of this investigation is to determine the significance of IRES-mediated translation in EMT and carcinoma progression. First, we will reconstitute LamB1 expression in epithelial and EMT-transformed hepatocytes with LamB1 knock-down by expressing LamB1 transcripts containing the entire 5-UTR or lacking the IRES motif. Second, we will examine the impact of IRES-driven LamB1 expression on angiogenesis and metastatic colonization in xeno- and allograft mouse models. IRES activity of LamB1 in experimental primary tumors and metastases will be monitored with a bicistronic reporter containing the LamB1 IRES. Third, we aim to identify novel ITAFs that regulate LamB1 IRES activity on its own or in cooperation with La by RNA affinity chromatography and mass spectrometry. RNA immunoprecipitation for La or novel identified ITAFs and bicistronic reporter assays in (La/ITAF) knock-down cells will determine their regulatory potential on LamB1 IRES activity and putative interactions of La with other ITAFs. Fourth, the subcellular localization and regulation of ITAFs will be studied during EMT. In particular, the dependence of La translocation on phosphorylation or cleavage of the nuclear localisation signal will be analyzed by mutation of regulatory sites. This approach will be extended to the analysis of ITAFs in patient samples of hepatocellular carcinoma by subcellular fractionation of frozen specimen and correlation with LamB1 levels, tumor grading, vessel invasion and patient survival. This study will provide novel insights into the relevance of IRESmediated LamB1 translation during cancer progression and assess the therapeutic potential of IRES inhibition in cancer treatment.

Tumor metastasis is the main reason for a bad prognosis in cancer disease. During metastasis cancer cells disconnect from the tumor and form new nodes in the body. The basic cause of metastasis is the activation of a cell program called EMT (Epithelial to Mesenchymal Transition) which leads to the loss of cell contacts and allows the detachment of cells from the tumor. Tumor cells that undergo the EMT transition elevate the production of laminin b1 (LamB1), a protein that regulates cell movement.The project aimed to investigate the role of LamB1during metastasis of liver cancer. New functional links and mechanisms that highlight the influence of LamB1on metastasis were identified and could provide targets of future therapies.Analysis of samples from liver cancer patients showed high levels of laminin b1 at the tumor border in tumors that produce keratin 19 (K19). The study of liver cancer cell lines revealed a novel mechanistic connection showing that the production of K19 is controlled by LamB1. K19 in liver cancer is associated with a very poor prognosis.The regulation of LamB1in K19 positive tumors was investigated. The unusual mechanism is based on a structural regulatory element located in the RNA of LamB1. The activity of the IRES element is elevated during metastasis and could be a potential target of future cancer therapies.To find out whether LamB1influences the EMT processes it was deactivated in liver cancer cells. The cells that lost the ability to produce LamB1 were unable to disintegrate the cell connection protein E-cadherin and thus failed to disconnect from each other. Cells that had already undergone EMT started to form new connections between the cells after loss of LamB1. The presence of LamB1 could therefore be important for the metastasis process.