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DUSP6/MKP-3 is a cytoplasmic dual-specificity phosphatase specific for the MAP kinases ERK1/2. Previous data have shown that the MEK/ERK axis exerts a retro-control on its own signaling through transcriptional and post-translational regulation of DUSP6. We first confirm the key role of MEK/ERK in maintaining the levels of dusp6 mRNA, while PI3K/mTOR, p38 MAPK, and JNK signaling pathways had no significant effects. We further show that regulation of dusp6 mRNA stability plays a critical role in ERK-dependent regulation of dusp6 expression. Luciferase reporter constructs indicated that MEK/ERK signaling increased the half-life of dusp6 mRNA in a 3'untranslated region (3'UTR)-dependent manner. In addition, hypoxia, a hallmark of tumor growth, was found to increase both endogenous levels of dusp6 mRNA and the stability of the luciferase reporter constructs containing its 3'UTR, in a HIF-1-dependent manner. Nevertheless, a basal ERK activity was required for the response to hypoxia. Finally, Tristetraprolin (TTP), a member of the TIS11 CCCH zinc finger protein family, and PUM2, an homolog of drosophila pumilio, two proteins regulating mRNA stability reduced the levels of endogenous dusp6 mRNA and the activity of the dusp6/3'UTR luciferase reporter constructs. This study shows that post-transcriptional regulation is a key process in the control of DUSP6 expression.

Post-transcriptional regulation plays a central role in cell differentiation and proliferation. Among the regulatory factors involved in this mechanism, Tristetraprolin (ZFP36 or TTP) is the prototype of a family of RNA-binding proteins that bind to adenylate and uridylate (AU)-rich sequences in the 3'UTR of mRNAs, which promotes their physiological decay. Here, we investigated whether TTP correlates with tumor aggressiveness in breast cancer and is a novel prognostic factor for this neoplasia. By immunoblot analysis, we determined the amount of TTP protein in different breast cancer cell lines and found an inverse correlation between aggressiveness and metastatic potential. TTP mRNA levels were very variable among cells lines and did not correlate with protein levels. Interestingly, by sequencing the entire TTP coding region in Hs578T cells that do not express the TTP protein, we identified a synonymous polymorphism (rs3746083) that showed a statistically significant association with a lack of response to Herceptin/Trastuzumab in HER2-positive-breast cancer patients. Even though this genetic change did not modify the corresponding amino acid, we performed functional studies and showed an effect on protein translation associated with the variant allele with respect to the wild-type. These data underline the importance of synonymous variants on gene expression and the potential role of TTP genetic polymorphisms as a prognostic marker for breast cancer.

Increased angiogenesis in bone marrow (BM) is one of the characteristics of chronic myeloid leukemia (CML), a clonal myeloproliferative disorder that expresses a chimeric Bcr/Abl protein. Recently, the therapeutic strategy in CML has been totally modified with the development of a new drug: imatinib mesylate (STI571), a specific inhibitor of Bcr/Abl tyrosine kinase activity. The aim of our study was to determine, in patients with CML, the capacity of imatinib mesylate to modulate one of the most potent regulators of angiogenesis, the vascular endothelial growth factor (VEGF). In newly diagnosed CML, we observed significantly increased VEGF secretion by CML BM cells and significantly increased VEGF plasma concentrations. We showed that low plasma VEGF concentrations could be one of the characteristics of complete cytogenetic remission. To understand the molecular mechanisms leading to the inhibition of VEGF production by imatinib, we focused our experiments on the human cell line K562, which is Bcr/Abl positive. We demonstrated that imatinib inhibits VEGF gene transcription by targeting the Sp1 and Sp3 transcription factors. Taken IntroductionVascular endothelial growth factor (VEGF) principally targets endothelial cells and regulates several of their functions, including mitogenesis, permeability, and migration. VEGF is one of the most potent and specific regulators of angiogenesis 1 and, as a consequence, is required for viability and growth of solid tumors. Recently, an increase in the microvessel density was reported in the bone marrow of patients with chronic myeloid leukemia (CML), 2,3 suggesting a role for angiogenesis in the pathophysiology of hematologic malignancies. CML is a clonal myeloproliferative disorder of pluripotent hematopoietic stem cells with a specific cytogenetic abnormality, a balanced translocation between chromosomes 9 and 22. This translocation results in a Bcr/Abl chimeric gene that expresses an abnormal fusion protein showing constitutive tyrosine kinase activity. In the murine cell line Ba/F3, overexpression of Bcr/Abl induces VEGF expression. 4 Previously, VEGF secretion was shown to be increased in myeloid cells derived from day-6 granulocyte-macrophage colony-forming unit (GM-CFU) colonies isolated from patients with CML. 5 These data are supported by solid-phase radioimmunoassay (RIA) analysis of the VEGF protein in the bone marrow of patients with CML. 6 Furthermore, VEGF plasma concentrations are significantly increased in CML. 2 Recently, the therapeutic strategy in CML has been totally modified with the development of an effective new drug: imatinib mesylate (STI571), a specific inhibitor of Bcr/Abl protein tyrosine kinase activity. The aim of our study was to monitor the effect of imatinib on VEGF plasma concentrations in patients with CML. We also analyzed the effects of imatinib on the molecular mechanisms leading to VEGF expression in the human Bcr/Abl-positive cell line K562. Patients, materials, and methods PatientsAll samples were obtained from patients, healthy dono...