Nuevos mecanismos transformantes de MYC: inducción de SKP2 y degradación de p27= New MYC transforming mechanisms : induction of SKP2 and degradation of p27

  1. Bretones Sánchez, Gabriel
Supervised by:
  1. Javier León Serrano Director

Defence university: Universidad de Cantabria

Fecha de defensa: 27 March 2014

Committee:
  1. Oriol Bachs Valldeneu Chair
  2. María Dolores Delgado Villar Secretary
  3. David Santamaría Velilla Committee member

Type: Thesis

Teseo: 359974 DIALNET lock_openUCrea editor

Abstract

INTRODUCTION. MYC is one of the most frequently de-regulated oncogenes in human cancer. Through its role as a transcription factor, MYC regulates the expression of hundreds of target genes, involved in a wide range of cellular functions, such as cell cycle regulation, differentiation and metabolism. Expression of MYC at levels found in cancer cells is sufficient to drive primary quiescent cells into S phase, to accelerate rates of cell proliferation, and to prevent withdrawal from the cell cycle. p27 (KIP1) is a CDK inhibitor that restricts the transition of cells through the G1 and S phases of the cell cycle and arrests cell cycle progression. p27 levels are frequently down-regulated in human cancer and patients with tumors having low or undetectable levels of p27 protein have a very poor outcome. The levels of MYC and p27 inversely correlate in many human tumors, and it has been demonstrated in cell culture models that MYC overexpression antagonizes the antiproliferative effect of p27. However the mechanisms that mediated this antagonism are not understood at all. For this purpose, in the present study we have investigated the molecular mechanisms that underline the antagonism between MYC and p27 in different experimental contexts. RESULTS. Previously, using microarray technology we found that the activation of MYC up-regulates SKP2 mRNA expression in K562 cells (Acosta et al., 2008). SKP2 is an F-Box protein of the SCF ubiquitin-ligase complex, being p27 as one of its major substrates for proteolysis. SKP2 gene is frequently overexpressed in human cancers, inversely correlated with p27 and associated with tumor progression. We have used K562 human leukemia cell lines whit conditional MYC expression and we have found that MYC induces SKP2 expression at the mRNA (by RT-qPCR) and protein levels (by immunoblot). This SKP2 up-regulation was independent of MYC-induced cell proliferation. siRNA-mediated MYC silencing also resulted in SKP2 down-regulation in K562 cells. Moreover, MYC regulated SKP2 in a human lymphoid (P493.6) and mink epithelial (TM1) cell lines with conditional MYC expression and SKP2 was under-expressed in MYC null rat fibroblasts (HO.15.19 cells). MYC was able to up-regulate SKP2 mRNA expression in the presence of a protein synthesis inhibitor in K562 cells suggesting that MYC was a direct activator of SKP2. Luciferase assays showed that MYC activated human SKP2 promoter and chromatin immunoprecipitation experiments demonstrated that MYC bound to a 5´regulatory region of human SKP2 gene that includes two E-boxes, confirming that SKP2 was a new MYC target gene. Given that K562 cell line derives from human chronic myeloid leukemia (CML), we studied a cohort of 39 CML samples and we found a correlation between MYC and SKP2 mRNA levels. In addition, using Oncomine and GeneSapiens databases we found that MYC and SKP2 mRNA expression also correlates in two other hematological malignancies as acute myeloid leukemia and lymphoma. On the other hand, we observed that, in K562 cells, MYC promoted p27 phosphorylation at threonine 187 through the induction of cyclin A and the kinase activity associated to this cyclin. This phosphorylation is required for p27 recognition by the SCF SKP2 ubiquitin ligase complex. In addition, we found that, in mouse cells, MYC was able to promoting such phosphorylation independently of CDK2 or E-type cyclins. Importantly, the MYC-induced expression of SKP2 correlated with decreased p27 protein levels in K562-derived sublines. siRNA-mediated silencing of SKP2 resulted in increased p27 protein levels, led to a decrease of the proliferative rate of K562 cells and reduced the tumorigenic capacity of these cells in xenographs in nude mice. Interestingly, SKP2 silencing had no effect on MYC stability or activity. Finally, using gel filtration chromatography we found that p27 accumulates as free form when it was expressed at high levels in K562 cells and this was associated with a total proliferation block. The disappearance of p27 as free form correlates with MYC-mediated proliferative stimulus. CONCLUSION. Altogether, our data shows that, at least in human leukemia K562 cells, MYC orchestrates several mechanisms including up-regulation of the SKP2 oncogene and cyclin A, stimulation of CDK kinase activity associated to cyclin A and induction of p27 phosphorylation at Thr 187, to promote p27 down-regulation. Through these mechanisms, malignant cells overexpressing MYC acquire a proliferate advantage over normal non transformed cells.