Functional analysis of Laminin B1 IRES in cancer progression

Translational control represents a crucial event in gene expression and occurs in a wide range of cellular processes. Translation initiation by cellular internal ribosome entry sites (IRESs) is a mechanism which allows specific messenger (m)RNA translation due to unique RNA sequences and secondary structures in their untranslated regions (UTRs). IRES motives are of particular significance for tumorigenesis as the production of regulatory (onco)proteins such as c-myc are directed by IRES elements. In a recent investigation, we have been able to identify the IRES-mediated translation control of Laminin B1 (LamB1) during cancer progression. These data provide strong evidence that the upregulated biosynthesis of the extracellular matrix protein LamB1 depends on a bona fide IRES element in the 5`-UTR of LamB1 mRNA. In this project we will determine the sequence motif of the LamB1 IRES by employing genetic analyses. Knowledge of the cis-acting regulatory IRES element will allow to identify the trans-acting factors which physically interact with LamB1 IRES. In a further step we will address the question what signaling cascades affect the function of the LamB1 IRES upon tumor progression, and thus are responsible for the regulation of LamB1 translation. The novel insights into the molecular mechanism of the IRES-dependent LamB1 translation and its aberrations are considered as an important step in the understanding of tumor progression which might be relevant for efficient anti-cancer concepts.

Translational control is an important mechanism in the regulation of gene expression and occurs mainly at the level of translation initiation. Internal ribosome entry sites (IRESs) are regulatory motifs in the 5-untranslated regions (5-UTRs) of individual messenger (m)RNAs that allow the selective control of de novo protein synthesis in a cap-independent manner. The major goal of this project was to functionally analyze the IRES in the 5-UTR of the extracellular matrix component Laminin B1 (LamB1) during the epithelial to mesenchymal transition (EMT) of malignant hepatocytes, a process relevant for hepatocellular carcinoma (HCC) progression and metastasis. We demonstrated the IRES activity of LamB1 by independent bicistronic reporter assays. Strong evidences exclude an impact of cryptic promoter or splice sites on IRES-driven translation of LamB1, suggesting a bona fide IRES element in LamB1. Mapping of the LamB1 5-UTR revealed the minimal LamB1 IRES motif between -293 to -1 upstream of the start codon. RNA affinity purification showed that the La protein interacts with the LamB1 IRES. This interaction is upregulated during EMT of malignant hepatocytes and positively modulates LamB1 IRES translation. Platelet-derived growth factor (PDGF) enhances the IRES activity of LamB1 by the increasing cytoplasmic localization of La during the EMT of hepatocytes. Accordingly, cells expressing dominant negative PDGF receptor display reduced cytoplasmic accumulation of La and show no elevation of IRES activity or endogenous LamB1 levels after stimulation with PDGF. Furthermore, La mediated regulation of LamB1 IRES activity predominantly depends on MAPK/ERK signaling downstream of PDGF. Notably, LamB1 expression is not significantly downregulated by the impairment of the translation initiation factor eIF4E. In vivo, knock-down of La decreased LamB1 expression and reduced tumor growth. Together, these data suggest that PDGF/MAPK/ERK signaling is required for the cytoplasmic accumulation of La that triggers IRES-dependent translation of LamB1 during EMT and HCC progression.Pharmacological intervention with the PDGF/LamB1 axis could therefore be of paramount importance for combating HCC development.