Anomalies moléculaires de la voie MAPK et cancer papillaire de la thyroïde : étude de deux phosphatases spécifiques de ERK, DUSP5 et DUSP6

Papillary thyroid cancer (PTC) is the most common endocrine malignancy. Mutually exclusive and activating alterations of the MAPK pathway (Mitogen-Activated Protein Kinases) are identified in 70% of cases. Common mutations found in PTCs are point mutation of the B-RAF (50%) and RAS genes (10%) as well as RET/PTC chromosomal rearrangements (10%). The hot spot B-RAFV600E mutation is the most frequently alteration identified and is connected with agressive clinical characteristics (high stage at diagnosis, high recurrence risk and death). These molecular events lead to constitutive activation of the MAPK pathway, resulting in MEK (Mitogen-activated Extracellular signal-Regulated Kinase) and ERK (Extracellular signal-Regulated Kinase) phosphorylation. ERK is negatively regulated by phosphatases and among them, Dual Specificity Phosphatases (DUSPs), ubiquitary expressed, in particular two ERK-specific phosphatases DUSP5 (nuclear) and DUSP6 (cytosolic). We hypothesized that these phosphatases could have tumor supressor properties (i.e. their loss would be associated with an increase in MAPK pathway activation) or may serve as a surrogate marker of MAPK pathway activation in the context of a negative feedback loop. We analysed regulation and expression of both phosphatases in 3 models: three PCCL3 cell lines (rat thyroid cells) expressing one of the most common oncogene identified in PTCs (RET/PTC3 or H-RASV12 or B-RAFV600E) under the control of a doxycycline-inducible promoter, human PTC-derived cell lines and human PTC. We demonstrated that MAPK pathway activation was correlated with induction of DUSP5 and DUSP6. These phosphatases are involved in a negative feedback loop that contributes to a tight regulation of phospho-ERK levels. DUSP5 and DUSP6 mRNA are overexpressed in human PTCs, especially in B-RAF mutated tumors suggesting a higher MAPK signaling output in these agressive PTCs. Silencing of DUSP5 and/or DUSP6 by small interfering RNA does not affect proliferation of human B-RAFV600E thyroid carcinoma-derived cell lines, suggesting the lack of tumor suppressor gene role. Compensatory changes in expression of DUSPs when a specific one is inactivated may explain this lack of effect. On the opposite, a DUSP6 pharmacological inhibitor induced a concentration dependent decrease in proliferation of human B-RAFV600E cells, suggesting « off-target » effect of this inhibitor. In a second part, we analysed the regulation of DUSP5 expression, which is a target of the MAPK pathway activation. We demonstrated, using pharmacological inhibitors, that DUSP5 is an early response gene, regulated mostly by the MAPK pathway, at the transcriptional level. Two contiguous CArG boxes that bind serum response factor (SRF) were found in a 1Kb promoter region, as well as several E twenty-six transcription factor family binding sites (EBS). These sites potentially bind Elk-1, a transcription factor activated by ERK1/2. Using wild type or mutated DUSP5 promoter reporters, we demonstrated that SRF plays a crucial role in serum induction of DUSP5 promoter activity, the proximal CArG box being important for SRF binding in vitro and in living cells. Moreover Elk-1 was bound in vitro to a promoter region containing the proximal CArG box and a putative EBS. Its specific binding to SRF was necessary to elicit promoter response to dominant positive Elk-VP16 and to enhance the response to serum stimulation. Altogether our results suggest that the MAPK pathway is more active in B-RAFV600E PTC than in PTC with other genetic alteration and could explain their clinical agressivity. DUSP5 and DUSP6, as well as phosphorylated MEK, are markers of activation of the MAPK pathway. Neither phosphatase has tumor suppressor properties in our thyroid cancer cell models. Our results suggest redundancy and functional compensation among DUSPs. (...)Le cancer papillaire de la thyroïde (CPT) est la tumeur endocrine la plus fréquente. Des anomalies moléculaires activant la voie des MAPK (Mitogen-Activated Protein Kinases) sont identifiées, de façon mutuellement exclusive, dans environ 70% des cas. Il s’agit de réarrangements chromosomiques, le plus souvent de type RET/PTC (10%), de mutations ponctuelles activatrices des trois isoformes de l’oncogène RAS (H, N et K-RAS) (10%), ou de l’oncogène B-RAF (50%). La mutation « hot spot » B-RAFV600E est la plus fréquemment identifiée, elle est associée à une plus grande agressivité clinique (diagnostic à un stade tardif, risque de récidives et de décès accru). Ces évènements moléculaires ont pour conséquence commune l’activation de la voie des MAPK, se traduisant en aval par la phosphorylation de MEK (Mitogen-activated Extracellular signal-Regulated Kinase) puis de ERK (Extracellular signal-Regulated Kinase). Cette dernière est régulée négativement par des phosphatases, appartenant à la famille des Dual Specificity Phosphatases (DUSPs), d’expression ubiquitaire, et en particulier de deux phosphatases spécifiques de ERK, l’une cytoplasmique (DUSP6) et l’autre nucléaire (DUSP5). Nous avons fait l’hypothèse que ces phosphatases pouvaient être soit des gènes suppresseurs de tumeurs (leur perte d’expression conduisant à une augmentation de phosphorylation de ERK et une prolifération accrue), soit des marqueurs du degré d’activation de la voie MAPK dans le cadre d’une boucle de rétrocontrôle négatif. Ceci nous a conduits à analyser la régulation et l’expression de ces phosphatases dans trois modèles : la lignée cellulaire PCCL3 (thyroïde de rat), exprimant l’un des trois principaux oncogènes mutés dans les CPT (RET/PTC3 ou H-RASV12 ou B-RAFV600E) sous le contrôle d’un promoteur inductible par la doxycycline, des lignées cellulaires humaines dérivant de CPT et des CPT humains. (...