p63 controls cell migration and invasion by transcriptional regulation of MTSS1.

Metastasis is a multistep cell-biological process, which is orchestrated by many factors, including metastasis activators and suppressors. Metastasis Suppressor 1 (MTSS1) was originally identified as a metastasis suppressor protein whose expression is lost in metastatic bladder and prostate carcinomas. However, recent findings indicate that MTSS1 acts as oncogene and pro-migratory factor in melanoma tumors. Here, we identify and characterized a molecular mechanism controlling MTSS1 expression, which impinges on a pro-tumorigenic role of MTSS1 in breast tumors. We found that in normal and in cancer cell lines ΔNp63 is able to drive the expression of MTSS1 by binding to a p63-binding responsive element localized in the MTSS1 locus. We reported that ΔNp63 is able to drive the migration of breast tumor cells by inducing the expression of MTSS1. Notably, in three human breast tumors data sets the MTSS1/p63 co-expression is a negative prognostic factor on patient survival, suggesting that the MTSS1/p63 axis might be functionally important to regulate breast tumor progression.

miR-143 regulates hexokinase 2 expression in cancer cells.

Tumor cells activate pathways that facilitate and stimulate glycolysis even in the presence of adequate levels of oxygen in order to satisfy their continuous need of molecules, such as nucleotides, ATP and fatty acids, necessary to support their rapid proliferation. Accordingly, a variety of human tumors are characterized by elevated expression levels of the hexokinase 2 isoform (HK2). Although different molecular mechanisms, including genetic and epigenetic mechanisms, have been suggested to account for the altered expression of HK2 in tumors, the potential role of microRNAs (miRNAs) in the regulation of HK2 expression has not been evaluated. Here, we report that miR-143 inhibits HK2 expression via a conserved miR-143 recognition motif located in the 3'-untranslated region (3'UTR) of HK2 mRNA. We demonstrate that miR143 inhibits HK2 expression both in primary keratinocytes and in head and neck squamous cell carcinoma (HNSCC)-derived cell lines. Importantly, we found that miR-143 inversely correlates with HK2 expression in HNSCC-derived cell lines and in primary tumors. We also report that the miRNA-dependent regulation of hexokinase expression is not limited to HK2 as miR-138 targets HK1 via a specific recognition motif located in its 3'UTR. All these data unveil a new miRNA-dependent mechanism of regulation of hexokinase expression potentially important in the regulation of glucose metabolism of cancer cells.

FLASH is essential during early embryogenesis and cooperates with p73 to regulate histone gene transcription.

Replication-dependent histone gene expression is a fundamental process occurring in S-phase under the control of the cyclin-E/CDK2 complex. This process is regulated by a number of proteins, including Flice-Associated Huge Protein (FLASH) (CASP8AP2), concentrated in specific nuclear organelles known as HLBs. FLASH regulates both histone gene transcription and mRNA maturation, and its downregulation in vitro results in the depletion of the histone pull and cell-cycle arrest in S-phase. Here we show that the transcription factor p73 binds to FLASH and is part of the complex that regulates histone gene transcription. Moreover, we created a novel gene trap to disrupt FLASH in mice, and we show that homozygous deletion of FLASH results in early embryonic lethality, owing to arrest of FLASH(-/-) embryos at the morula stage. These results indicate that FLASH is an essential, non-redundant regulator of histone transcription and cell cycle during embryogenesis.

FLASH degradation in response to UV-C results in histone locus bodies disruption and cell-cycle arrest.

Eucaryotic cell nuclei contain a number of different organelles that are highly dynamic structures and respond to a variety of stimuli. Here we investigated the effect of UV irradiation on a recently identified group of organelles, Histone Locus Bodies. Histone Locus Bodies contain at least two main proteins, FLASH and NPAT, and have been shown to be involved in replication-dependent histone gene transcription. We show that these organelles are disrupted after sublethal irradiation and both FLASH and NPAT are degraded, which in turn results in cell-cycle arrest at the S/G2 transition. The effect on the cell cycle is due to reduced transcription of histone genes and restoring normal histone protein levels by stabilizing histone mRNA allows cells to progress through the cell cycle. This provides a novel mechanism of S-phase arrest in response to DNA damage that potentially allows DNA repair before cells continue into mitosis, and thus prevents transmission of genomic alterations.

FLASH is required for histone transcription and S-phase progression.

Cajal bodies are nuclear subdomains that are involved in maturation of small ribonucleoproteins and frequently associate with small nuclear RNA and histone gene clusters in interphase cells. We have recently identified FADD-like IL-1beta-converting enzyme (FLICE) associated huge protein (FLASH) as an essential component of Cajal bodies. Here we show that FLASH associates with nuclear protein, ataxia-telangiectasia, a component of the cell-cycle-dependent histone gene transcription machinery. Reduction of FLASH expression by RNA interference results in disruption of the normal Cajal body architecture and relocalization of nuclear protein, ataxia-telangiectasia. Furthermore, FLASH down-regulation results in a clear reduction of histone transcription and a dramatic S-phase arrest of the cell cycle. Chromatin immunoprecipitation reveals that FLASH interacts with histone gene promoter sequences. These results identify FLASH as an important component of the machinery required for histone precursor mRNA expression and cell-cycle progression.

FLASH is an essential component of Cajal bodies.

Cajal bodies are small nuclear organelles with a number of nuclear functions. Here we show that FLICE-associated huge protein (FLASH), originally described as a component of the apoptosis signaling pathway, is mainly localized in Cajal bodies and is essential for their structure. Reduction in FLASH expression by short hairpin RNA results in disruption of the normal architecture of the Cajal body and relocalization of its components. Because the function of FLASH in the apoptosis receptor signaling pathway has been strongly questioned, we have now identified a clear function for this protein.

A tricistronic retroviral vector expressing natural antiangiogenic factors inhibits angiogenesis in vitro, but is not able to block tumor progression in vivo.

Angiogenesis, the formation of new blood vessels out of pre-existing capillaries, is essential for tumor progression. Many factors have been identified that are able to inhibit angiogenesis. Here, we report the construction of a tricistronic retroviral vector encoding two inhibitors of angiogenesis expressed in mammals: the N-terminal fragment of rat prolactin (16KrPRL) and a secreted form of human platelet factor 4 (sPF4). When transduced by this retroviral vector, a rat glioblastoma cell line loses its ability of promoting endothelial cell locomotion, the initial step of angiogenesis, and the formation of an endothelial cell tube network. In spite of this encouraging in vitro result, however, the anti-angiogenic vector cannot block glioblastoma progression in animal models. These results suggest that therapeutic strategies aiming to block tumor progression through the inhibition of tumor-associated angiogenesis, should not only provide large numbers of angiogenesis inhibitors, but also target the angiogenic factors produced by tumor cells. Moreover, the data described herein may confirm recent findings from other groups which indicate that in order to successfully counteract tumor progression, drugs inhibiting new blood vessel formation should be employed in combination with traditional anti-tumor strategies, such as chemotherapy or radiotherapy.

Specificity of the binding of synapsin I to Src homology 3 domains.

Synapsins are synaptic vesicle-associated phosphoproteins involved in synapse formation and regulation of neurotransmitter release. Recently, synapsin I has been found to bind the Src homology 3 (SH3) domains of Grb2 and c-Src. In this work we have analyzed the interactions between synapsins and an array of SH3 domains belonging to proteins involved in signal transduction, cytoskeleton assembly, or endocytosis. The binding of synapsin I was specific for a subset of SH3 domains. The highest binding was observed with SH3 domains of c-Src, phospholipase C-gamma, p85 subunit of phosphatidylinositol 3-kinase, full-length and NH(2)-terminal Grb2, whereas binding was moderate with the SH3 domains of amphiphysins I/II, Crk, alpha-spectrin, and NADPH oxidase factor p47(phox) and negligible with the SH3 domains of p21(ras) GTPase-activating protein and COOH-terminal Grb2. Distinct sites in the proline-rich COOH-terminal region of synapsin I were found to be involved in binding to the various SH3 domains. Synapsin II also interacted with SH3 domains with a partly distinct binding pattern. Phosphorylation of synapsin I in the COOH-terminal region by Ca(2+)/calmodulin-dependent protein kinase II or mitogen-activated protein kinase modulated the binding to the SH3 domains of amphiphysins I/II, Crk, and alpha-spectrin without affecting the high affinity interactions. The SH3-mediated interaction of synapsin I with amphiphysins affected the ability of synapsin I to interact with actin and synaptic vesicles, and pools of synapsin I and amphiphysin I were shown to associate in isolated nerve terminals. The ability to bind multiple SH3 domains further implicates the synapsins in signal transduction and protein-protein interactions at the nerve terminal level.