Regulation of translation during cancer progression

Laminin B1 (LamB1) ist ein Bestandteil der extrazellulären Matrix und auf entscheidende Weise in der Regulation der Tumorzellmigration und –invasion während der Karzinogenese involviert. Ein essentieller Aspekt der Tumorprogression ist die Umwandlung epithelialer Tumorzellen zu einem mesenchymalen Phänotyp (EMT), der mit metastatischen Eigenschaften korreliert. Ein Expressionsprofil polysom-assozierter mRNA maligner Hepatozyten zeigte die Aktivierung der Translation von LamB1 in EMT-transformierten Zellen. Es stellte sich heraus, das die erhöhte Translation von LamB1 in metastasierenden Hepatozyten von einer internen Ribosomen-Bindungsstelle (IRES) reguliert wird, welche in der 5‘-untranslatierten Region (UTR) des LamB1 Transkripts lokalisiert ist. Zwei unabhängige bicistronische Reportersysteme wurden für die Bestimmung der IRES-Aktivität von LamB1 verwendet. Zur Verifikation der IRES Aktivität wurde die Präsenz kryptischer Promotoren und Spleißstellen im 5’-UTR von LamB1 ausgeschlossen. Die minimale cis-agierende IRES Sequenz, welche für die „Cap“-unabhängige Translation notwendig ist, wurde mit einer Länge von 293 Nukleotiden direkt oberhalb des Startcodons lokalisiert. Mithilfe einer RNA Affinitätschromatography wurde der IRES trans-aktivierende Faktor (ITAF) La als Bindungspartner identifiziert, welcher mit der LamB1 5’-UTR in vitro interagiert. Diese Interaktion wurde durch eine RNA-Immunopräzipitation in vivo verifiziert. Weiters konnte die Bindung einer erhöhten Menge von La Protein an das minimale LamB1 IRES Motiv nachgewiesen werden. In Übereinstimmung mit dieser Beobachtung konnten erhöhte zytoplasmatische Mengen von La in EMT-transformierten Zellen detektiert werden. Überdies führte die Gegenwart von La zu einer gesteigerten IRES-meditierten Translation von LamB1 in vitro. Platelet derived growth factor (PDGF) wurde als auslösender Signaltransduktionsweg für die zytoplasmatische Translokation von La identifiziert. Zusammenfassend konnte damit nachgewiesen werden, dass der PDGF Signalweg für die zytoplasmatische Akkumulation von La und damit der Interaktion mit dem LamB1 IRES verantwortlich ist, wodurch die cap-unabhängige Translation von LamB1 in malignen Hepatozyten nach EMT aktiviert wird.Laminin B1 (LamB1) is a main component of the extracellular matrix and is involved in the regulation of tumor cell migration and invasion of during carcinogenesis. Metastasis of carcinoma cells is crucially linked to the process of epithelial to mesenchymal transition (EMT), which allows tumor cells to acquire a more motile phenotype and to dissociate from the epithelial cell cluster of the tumor. Expression profiling of polysome-associated mRNA revealed LamB1 to be translationally upregulated upon EMT of malignant hepatocytes. The enhanced translation of LamB1 in metastatic hepatocytes proved to be regulated by an internal ribosome entry site (IRES) located within the 5’-untranslated region (UTR) of the LamB1 transcript. IRES activity was detected by employing two independent reporter systems and verified by stringent assays for the presence of cryptic promoter or splice sites. The minimal cis-acting IRES sequence of 293 nucleotides that is required for cap-independent translation was localized directly upstream of the start codon. Notably, the IRES trans-acting factor (ITAF) La was identified by RNA affinity purification as regulatory factor that interacts with LamB1 5’-UTR. This interaction was verified by RNA-immunoprecipitation in vivo, which revealed enhanced binding of La to the minimal IRES motif of LamB1 after EMT. Consistently, cytoplasmic levels of La were elevated in EMT-transformed cells and correlated with increased LamB1 protein expression. Furthermore, IRES-driven translation of LamB1 was elevated in the presence of La in vitro. Importantly, the EMT-induced cytoplasmic translocation of La was found to be triggered by platelet derived growth factor (PDGF) that is downstream of transforming growth factor (TGF)-β signaling. Together, these data demonstrate that La interacts with the LamB1 IRES in the cytoplasm, resulting in enhanced cap-independent translation of LamB1 in malignant hepatocytes that have undergone EMT

The leader region of Laminin B1 mRNA confers cap-independent translation

Translation initiation of eukaryotic mRNAs generally occurs by cap-dependent ribosome scanning. However, certain mRNAs contain internal ribosome entry sites (IRES) allowing cap-independent translation. Several of these IRES-competent transcripts and their corresponding proteins are involved in tumourigenesis. This study focused on IRES-driven translation control during the epithelial to mesenchymal transition (EMT) of hepatocytes that reflects crucial aspects of carcinoma progression. Expression profiling of EMT revealed Laminin B1 (LamB1) to be translationally upregulated. The 5′-untranslated region (UTR) of LamB1 was potent to direct IRES-dependent mRNA utilization of a bicistronic reporter construct. Stringent assays for cryptic promoter and splice sites showed no aberrantly expressed transcripts, suggesting that the reporter activity provided by the leader region of LamB1 mRNA exclusively depends on IRES. In accordance, LamB1 expression increased upon negative interference with cap-dependent translation by expression of human rhinovirus 2A protease or heat shock of cells. Finally, the enhanced expression of LamB1 during EMT correlated with an elevated IRES activity. Together, these data provide first evidence that the 5′-UTR of LamB1 contains a bona fide IRES that directs translational upregulation of LamB1 during stress conditions and neoplastic progression of hepatocytes

La enhances IRES-mediated translation of laminin B1 during malignant epithelial to mesenchymal transition

The majority of transcripts that harbor an internal ribosome entry site (IRES) are involved in cancer development via corresponding proteins. A crucial event in tumor progression referred to as epithelial to mesenchymal transition (EMT) allows carcinoma cells to acquire invasive properties. The translational activation of the extracellular matrix component laminin B1 (LamB1) during EMT has been recently reported suggesting an IRES-mediated mechanism. In this study, the IRES activity of LamB1 was determined by independent bicistronic reporter assays. Strong evidences exclude an impact of cryptic promoter or splice sites on IRES-driven translation of LamB1. Furthermore, no other LamB1 mRNA species arising from alternative transcription start sites or polyadenylation signals were detected that account for its translational control. Mapping of the LamB1 5′-untranslated region (UTR) revealed the minimal LamB1 IRES motif between −293 and −1 upstream of the start codon. Notably, RNA affinity purification showed that the La protein interacts with the LamB1 IRES. This interaction and its regulation during EMT were confirmed by ribonucleoprotein immunoprecipitation. In addition, La was able to positively modulate LamB1 IRES translation. In summary, these data indicate that the LamB1 IRES is activated by binding to La which leads to translational upregulation during hepatocellular EMT

A Letter to Émile Durkheim

Émile Durkheim, a classical social theorist, is considered to be one of the founders of modern social science, and a father of sociology. Despite his historically controversial views of society, he is now recognised as a scientist of the social order. This paper examines a number of Durkheim’s theories and applies them to the struggles of a modern day entrepreneur. This paper is non-traditional essay formatted into two separate letters. The first letter is written through the eyes of a struggling young entrepreneur. She is writing to Émile Durkheim to seek advice on her business endeavours. More specifically, she feels restless and anxious about her future and is under constant pressure to succeed. The second letter is written as Émile Durkheim offering his advice and expertise to this struggling entrepreneur. As a sociologist, he provides a unique diagnosis that is non-psychological or therapeutic, but rather structural in nature. Émile assures her that what she is experiencing is a structural problem of modern society. Finally he outlines the social changes that must occur in order to greatly reduce the struggles experienced by entrepreneurs

PDGF enhances IRES-mediated translation of Laminin B1 by cytoplasmic accumulation of La during epithelial to mesenchymal transition

The extracellular matrix protein Laminin B1 (LamB1) regulates tumor cell migration and invasion. Carcinoma cells acquire invasive properties by epithelial to mesenchymal transition (EMT), which is a fundamental step in dissemination of metastatic cells from the primary tumor. Recently, we showed that enhanced translation of LamB1 upon EMT of malignant hepatocytes is mediated by an internal ribosome entry site (IRES). We demonstrated that the IRES transacting factor La binds the minimal IRES motif and positively modulates IRES activity of LamB1. Here, we show that platelet-derived growth factor (PDGF) enhances IRES activity of LamB1 by the increasing cytoplasmic localization of La during EMT. 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 , knockdown of La associated with decreased LamB1 expression and reduced tumor growth. Together, these data suggest that PDGF is required for the cytoplasmic accumulation of La that triggers IRES-dependent translation of LamB1 during EMT.(VLID)458954

LamB1 translation after heat shock or intervention with ribosome scanning through 2A protease-dependent cleavage of eIF4G

<p><b>Copyright information:</b></p><p>Taken from "The leader region of Laminin B1 mRNA confers cap-independent translation"</p><p></p><p>Nucleic Acids Research 2007;35(8):2473-2482.</p><p>Published online 29 Mar 2007</p><p>PMCID:PMC1885646.</p><p>© 2007 The Author(s)</p> () LamB1 expression in MIM-R cells 4, 6 and 8 h after heat shock as detected by western blotting. () Firefly luciferase assay of MIM-R hepatocytes transfected either with pR-EMCV-F or pR-Lam-F bicistronic plasmids. Cells were exposed to heat shock 12 h post-transfection. 48 hours after transfection, Firefly luciferase activity was determined and normalized to the RNA level after reverse transcription and quantitation of cDNA. () Western blot analysis of MIM-R cells showing the cleavage of eIF4GI/II. MIM-R cells were transfected with wild-type 2A protease expressing plasmid (p2Awt) and lysed at the indicated times. () Firefly luciferase assay of MIM-R hepatocytes co-transfected with pR-F and either p2Amut or p2Awt. () Firefly luciferase assay of MIM-R cells co-transfected either with pR-EMCV-F or pR-Lam-F and p2Amut or p2Awt, respectively. Cells were lysed 48 h post-transfection and the Firefly luciferase activity was normalized to the RNA level after reverse transcription and quantitation of cDNA