MyD88

MyD88 is an adaptor molecule that links most Toll-like receptors (TLR) to the cell signaling machinery. We asked whether MyD88-coupled receptors play a role in tumor immunosurveillance. To that end, we subjected wild-type (wt) and mice deficient in TLR2, TLR4, TLR9, or MyD88, to a DMBA/TPA two-stage, RAS-dependent skin carcinogenesis protocol. TLR2-/-, TLR4-/-, and TLR9-/- mice developed papillomas with the same frequency and kinetics as in wt mice, virtually all of which developed skin papillomas by the end of the treatment period. Surprisingly, almost no MyD88-/- mice developed tumors in response to DMBA/TPA treatment. To address whether MyD88 acts in a cell-autonomous fashion, we generated mouse embryo fibroblasts (MEFs) from wt and MyD88-/- mice. MyD88-/- MEFs were completely resistant to cell transformation in vitro by DMBA/TPA or by transfection with an activated form of RAS, as assessed by the focus formation assay. However, MyD88-/- MEFs were transformed by SV40 T antigens, indicating that lack of MyD88 does not lead a general defect in cell transformation. Collectively, these data demonstrate a crucial role for MyD88 in RAS activation and transformation. The aim of the present study is to:  Elucidate the role of MyD88 in the RAS signalling cascade. We have shown that activation of the RAS/MAPK pathway is compromised in MyD88-deficient MEFs, as determined by ERK phosphorylation. Biochemical studies (co-immunoprecipitation, GST pulldown) will be carried out to pinpoint the interaction of MyD88 with members of the RAS/MAPK signalling cascade. Complementation experiments in MyD88-/- MEFs will be performed using activated members of the cascade such as RAS, c-RAF, B-RAF MEK, and ERK to establish the level of this signalling pathway at which the MyD88 protein functions.  Investigate the conseqences of MyD88 inactivation in vitro and in vivo. We have shown that knocking down MyD88 expression in human cancer cell lines using short interfering RNA (siRNA) leads to a block in proliferation at the G1/S transition of the cell cycle. We will investigate the molecular basis for this block by measuring the expression levels and functional status of different molecules implicated in cell cycle regulation such as c-Myc, cyclins, etc. Moreover, in order to study the consequences of MyD88 blockade in vivo, we will generate lentiviral vectors expressing recombinant MyD88-specific siRNAs which we will use to infect mice carrying RAS-dependent tumors. Tumor load, as well as the proliferative and apoptotic potential of infected tumor cells will be evaluated.  Analyse the status of the MyD88 gene and protein in human cancer samples. Based on prelimibanry results showing a variation in MyD88 protein levels in lung adenocarcinomas compared to peri-tumoral tissue, it is reasonable to postulate that alterations occur in this gene, leading to modifications in its expression levels, or in the stability of its product, in certain human cancers. We will first ask whether this observation applies to tumors of other histological origins. We will then sequence the MyD88 gene in a large panel of human tumor cell lines in normal or tumor tissue samples. We will also quantitate the levels of MyD88 mRNA in these tissues. Finally, immunohistochemical analysis will be performed to reveal the cell-type expression and sub-cellular localization of the MyD88 protein in neoplastic tissue. The above studies will provide important information on the mechanism of action of a novel mediator of cancer -MyD88- and on its potential as a therapeutic target in RAS-dependent tumors