The E2F family of transcription factors is essential in the regulation of the cell cycle and apoptosis. While the activity of E2F1-3 is tightly controlled by the retinoblastoma family of proteins, the expression of these factors is also regulated at the level of transcription, post-translational modifications and protein stability. Recently, a new level of regulation of E2Fs has been identified, where micro-RNAs (miRNAs) from the mir-17-92 cluster influence the translation of the E2F1 mRNA. We now report that miR-20a, a member of the mir-17-92 cluster, modulates the translation of the E2F2 and E2F3 mRNAs via binding sites in their 3-untranslated region. We also found that the endogenous E2F1, E2F2, and E2F3 directly bind the promoter of the mir-17-92 cluster activating its transcription, suggesting an autoregulatory feedback loop between E2F factors and miRNAs from the mir-17-92 cluster. Our data also point toward an antiapoptotic role for miR-20a, since overexpression of this miRNA decreased apoptosis in a prostate cancer cell line, while inhibition of miR-20a by an antisense oligonucleotide resulted in increased cell death after doxorubicin treatment. This anti-apoptotic role of miR-20a may explain some of the oncogenic capacities of the mir-17-92 cluster. Altogether, these results suggest that the autoregulation between E2F1-3 and miR-20a is important for preventing an abnormal accumulation of E2F1-3 and may play a role in the regulation of cellular proliferation and apoptosis.

Interferons are cytokines with potent antiviral and antiproliferative activities. We report that although a transient exposure to ␤-interferon induces a reversible cell cycle arrest, a sustained treatment triggers a p53-dependent senescence program. ␤-Interferon switched on p53 in two steps. First, it induced the acetylation of p53 at lysine 320 and its dephosphorylation at serine 392 but not p53 activity. Later on, it triggered a DNA signaling pathway, the phosphorylation of p53 at serine 15 and its transcriptional activity. In agreement, ␤-interferon-treated cells accumulated ␥-H2AX foci and phosphorylated forms of ATM and CHK2. The DNA damage signaling pathway was activated by an increase in reactive oxygen species (ROS) induced by interferon and was inhibited by the antioxidant N-acetyl cysteine. More important, RNA interference against ATM inhibited p53 phosphorylation at serine 15, p53 activity and senescence in response to ␤-interferon. ␤-Interferon-induced senescence was more efficient in cells expressing either, p53, or constitutive allele of ERK2 or RasV12. Hence, ␤-interferon-induced senescence targets preferentially cells with premalignant changes. INTRODUCTIONMany cell types are able to enter a stable and viable postmitotic state in response to oncogenic stresses such as DNA damage, short telomeres, and certain oncogenes. This condition, known as cellular senescence, is regulated by tumor suppressors such as p53, RB, p16INK4a , p19 ARF , and PML and engages a specific gene expression program (Ferbeyre, 2002;Lowe et al., 2004;Shay and Roninson, 2004). By preventing the expansion of potentially malignant cells, senescence may act as a barrier to tumor formation. Several signaling pathways must connect different stressors to the senescence program. For example, short telomeres or DNA damage activate the senescence program through the checkpoint proteins ATM, ATR, CHK1, and CHK2 (d'Adda di Fagagna et al., 2003;Herbig et al., 2004;Zglinicki et al., 2005). On the other hand, oncogenic stresses do not regulate senescence through telomere shortening and they may signal to senescence regulators through the production of reactive oxygen species (Lee et al., 1999;Wu et al., 2004;Catalano et al., 2005). In addition, it is established that some senescence regulators (p53, PML, and IFI16) are also targets of interferon-stimulated transcription factors (Lavau et al., 1995;Stadler et al., 1995;Takaoka et al., 2003;Xin et al., 2003Xin et al., , 2004de Stanchina et al., 2004). Accordingly, senescence prevented ras-transformation in wild-type fibroblasts, but not in fibroblasts from mice lacking the interferon regulated transcription factor IRF1 (Tanaka et al., 1994). Together, these studies suggest that signaling through the interferon pathway may play a role in senescence.Interferons comprise a family of cytokines with antiviral and antiproliferative activity. They include the type I interferon family (mainly ␣ and ␤-interferon) and type II or ␥-interferon (Taniguchi and Takaoka, 2002). Type-I interferon, usually pr...

The tumor-suppressive role of p53 at the level of tumor initiation is well documented. It has also been shown previously that p53 acts against tumor progression/metastasis. However, its role in modulating cell migration and invasion leading to metastasis is poorly understood. In this study, using vascular smooth muscle cells and NIH 3T3 fibroblast cells, we have shown that p53 potently suppresses Src-induced podosome/rosette formation, extracellular matrix digestion, cell migration, and invasion. The overexpression of exogenous wild-type p53 or the activation of the endogenous p53 function suppresses, while the short hairpin RNA-mediated knockdown of p53 expression or the blocking of its function exacerbates, Src-induced migratory and invasive phenotypes. We have also found that p53 expression and function are downregulated in cells stably transformed with constitutively active Src that exhibit aggressive invasive properties. Lastly, p53 upregulates the expression of caldesmon, an actin-binding protein that has been shown to be an inhibitor of podosome/invadopodium formation. The ability of p53 to suppress Src phenotypes in transformed cells was largely abolished by knocking down caldesmon. This study reports a novel molecular mechanism (caldesmon), as well as a structural basis (podosomes/rosettes), to show how p53 can act as an anti-motility/invasion/metastasis agent.

SummaryInvadopodia are actin-rich membrane protrusions that promote extracellular matrix degradation and invasiveness of tumor cells. Src protein-tyrosine kinase is a potent inducer of invadopodia and tumor metastases. Cdc42-interacting protein 4 (CIP4) adaptor protein interacts with actin regulatory proteins and regulates endocytosis. Here, we show that CIP4 is a Src substrate that localizes to invadopodia in MDA-MB-231 breast tumor cells expressing activated Src (MDA-SrcYF). To probe the function of CIP4 in invadopodia, we established stable CIP4 knockdown in MDA-SrcYF cell lines by RNA interference. Compared with control cells, CIP4 knockdown cells degrade more extracellular matrix (ECM), have increased numbers of mature invadopodia and are more invasive through matrigel. Similar results are observed with knockdown of CIP4 in EGF-treated MDA-MB-231 cells. This inhibitory role of CIP4 is explained by our finding that CIP4 limits surface expression of transmembrane type I matrix metalloprotease (MT1-MMP), by promoting MT1-MMP internalization. Ectopic expression of CIP4 reduces ECM digestion by MDA-SrcYF cells, and this activity is enhanced by mutation of the major Src phosphorylation site in CIP4 (Y471). Overall, our results identify CIP4 as a suppressor of Srcinduced invadopodia and invasion in breast tumor cells by promoting endocytosis of MT1-MMP.

We have recently shown that Src induces the formation of podosomes and cell invasion by suppressing endogenous p53, while enhanced p53 strongly represses the Src-induced invasive phenotype. However, the mechanism by which Src and p53 play antagonistic roles in cell invasion is unknown. Here we show that the Stat3 oncogene is a required downstream effector of Src in inducing podosome structures and related invasive phenotypes. Stat3 promotes Src phenotypes through the suppression of p53 and the p53-inducible protein caldesmon, a known podosome antagonist. In contrast, enhanced p53 attenuates Stat3 function and Srcinduced podosome formation by upregulating the tumor suppressor PTEN. PTEN, through the inactivation of Src/Stat3 function, also stabilizes the podosome-antagonizing p53/caldesmon axis, thereby further enhancing the anti-invasive potential of the cell. Furthermore, the protein phosphatase activity of PTEN plays a major role in the negative regulation of the Src/Stat3 pathway and represses podosome formation. Our data suggest that cellular invasiveness is dependent on the balance between two opposing forces: the proinvasive oncogenes Src-Stat3 and the anti-invasive tumor suppressors p53-PTEN.p53 is a potent tumor suppressor that plays a critical role in the regulation of cell cycle progression, DNA repair, apoptosis, and senescence (3,10,32,57). Approximately half of all human tumors have compromised p53 function (25, 62). Loss of p53 function has also been implicated in the evolution of aggressive and metastatic cancers (28,33,42,43), suggesting an antiinvasive and migration role of p53. Recent studies have increasingly unveiled this relatively less known aspect of p53 function in the regulation of cell migration and invasion (19,20,45,63,66). We have recently shown that p53, acting downstream of Src, strongly suppresses the formation of podosomes (also called invadopodia in cancer cells) and extracellular matrix (ECM) digestion by upregulating the expression of caldesmon, a known antagonist of podosomes (44).Src, a proto-oncogenic nonreceptor tyrosine kinase, induces migratory and invasive phenotypes in various cell types by initiating extensive cytoskeletal rearrangements (38,51,67). Activated Src induces the formation of podosomes and rosettes of podosomes, which are dynamic, actin-rich membrane protrusions (9,24,40), specialized in the degradation of the ECM by the recruitment and secretion of matrix metalloproteinases (MMPs) (8,38,60,64