Pasquale Verde

Neoplastic transformation of rat thyroid cells requires the junB and fra-1 gene induction which is dependent on the HMGI-C gene product.

The expression of the high mobility group I (HMGI)‐C chromatin component was shown previously to be essential for the establishment of the neoplastic phenotype in retrovirally transformed thyroid cell lines. To identify possible targets of the HMGI‐C gene product, we have analyzed the AP‐1 complex in normal, fully transformed and antisense HMGI‐C‐expressing rat thyroid cells. We show that neoplastic transformation is associated with a drastic increase in AP‐1 activity, which reflects multiple compositional changes. The strongest effect is represented by the dramatic junB and fra‐1 gene induction, which is prevented in cell lines expressing the antisense HMGI‐C. These results indicate that the HMGI‐C gene product is essential for the junB and fra‐1 transcriptional induction associated with neoplastic transformation. The inhibition of Fra‐1 protein synthesis by stable transfection with a fra‐1 antisense RNA vector significantly reduces the malignant phenotype of the transformed thyroid cells, indicating a pivotal role for the fra‐1 gene product in the process of cellular transformation.

A novel complex between the p65 subunit of NF-kappa B and c-Rel binds to a DNA element involved in the phorbol ester induction of the human urokinase gene.

The NF‐kappa B subunits, p50 and p65, have extensive sequence homology with the c‐rel proto‐oncogene and the Drosophila morphogen dorsal. It has recently been shown that in vitro translated c‐Rel can bind to DNA and form a complex with p50. However, the conditions for DNA binding of c‐Rel in vivo and its DNA sequence specificity have not been established. Here we report the identification a novel heterodimeric complex that binds to a kappa B‐like, phorbol ester (TPA) responsive DNA sequence, 5′‐GGGAAAGTAC‐3′, in the 5′ flanking region of the human urokinase (uPA) gene. This sequence was shown to bind two protein complexes, LC and UC. LC was indistinguishable from NF‐kappa B as it reacted with antibodies recognizing the p50 subunit of NF‐kappa B, and was shown by UV crosslinking to contain the p50 and p65 subunits of NF‐kappa B. UC, on the other hand, strongly reacted with anti‐v‐Rel, but not with the anti‐p50 antibodies, and was shown by crosslinking to contain 75 kDa and 85 kDa protein‐DNA adducts. The 75 kDa and the 85 kDa adducts could be immunoprecipitated only by anti‐p65 and anti‐c‐Rel antibodies, respectively, showing that c‐Rel formed a heterodimer with p65. Both protein complexes were present in inactive forms in HeLa cell cytosol, and their nuclear translocation was induced by TPA. DNA binding of UC and LC could, furthermore, be inhibited by I kappa B‐alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

A regulatory element that mediates co-operation between a PEA3-AP-1 element and an AP-1 site is required for phorbol ester induction of urokinase enhancer activity in HepG2 hepatoma cells.

We have characterized a transcriptional enhancer of the human urokinase‐type plasminogen activator (uPA) gene and found a regulatory element required for co‐operation between a PEA3‐‐AP‐1 element and an AP‐1 site in the enhancer. We designated this regulatory element co‐operation mediator (COM). Both the PEA3‐‐AP‐1 element, the AP‐1 site and the COM are required for efficient phorbol ester induction of transcription from the uPA promoter in the HepG2 hepatoma cell line. We show that the COM is also required for co‐operation between the PEA3‐‐AP‐1 element and a glucocorticoid response element, both in the presence or absence of TPA, indicating that the COM is generally capable of mediating synergism between inducible enhancer elements. The COM contains multiple overlapping binding sites for nuclear proteins, designated uPA enhancer factors 1–4 (UEF‐1‐4). We have identified putative binding sites for UEF‐1, −2 and −3. The UEF‐1 and −3 sites in the uPA enhancer are highly conserved between species. We demonstrate the binding of UEF‐3 to the NIP element, a previously characterized regulatory element in the human interleukin‐3 and stromelysin promoters, suggesting that this factor plays a role in regulation of a variety of genes.

Identification of microRNA-regulated gene networks by expression analysis of target genes.

MicroRNAs (miRNAs) and transcription factors control eukaryotic cell proliferation, differentiation, and metabolism through their specific gene regulatory networks. However, differently from transcription factors, our understanding of the processes regulated by miRNAs is currently limited. Here, we introduce gene network analysis as a new means for gaining insight into miRNA biology. A systematic analysis of all human miRNAs based on Co-expression Meta-analysis of miRNA Targets (CoMeTa) assigns high-resolution biological functions to miRNAs and provides a comprehensive, genome-scale analysis of human miRNA regulatory networks. Moreover, gene cotargeting analyses show that miRNAs synergistically regulate cohorts of genes that participate in similar processes. We experimentally validate the CoMeTa procedure through focusing on three poorly characterized miRNAs, miR-519d/190/340, which CoMeTa predicts to be associated with the TGFβ pathway. Using lung adenocarcinoma A549 cells as a model system, we show that miR-519d and miR-190 inhibit, while miR-340 enhances TGFβ signaling and its effects on cell proliferation, morphology, and scattering. Based on these findings, we formalize and propose co-expression analysis as a general paradigm for second-generation procedures to recognize bona fide targets and infer biological roles and network communities of miRNAs.

Deciphering AP-1 Function in Tumorigenesis : Fra-Ternizing on Target Promoters

Multi-gene families of transcription factors pose a formidable challenge to molecular and functional analysis. Dissecting distinct functions for individual family members requires a combination of approaches in different cellular and animal models. The AP-1 transcription factor complex serves as a paradigm for understanding the dynamics of transcriptional regulation. Knockout, knockdown and transgenic strategies, inducible alleles, mutational analysis, chemical genetics, etc.; researchers have applied all the tricks of the trade to understand how AP-1 works. AP-1 refers to a mixture of dimers formed between members of the Jun, Fos and ATF families. The complexity of the AP-1 biological functions reflects the wide combinatorial diversity of its components.1 AP-1 has been linked to cancer and neoplastic transformation ever since the first jun and fos genes were cloned as cellular homologues of viral oncogenes twenty years ago. Because of the oncogenic or tumor suppressive activity exhibited by distinct Jun and Fos nuclear proteins depending on the cell context and the genetic background of the tumor, the AP-1 complex has been called a “double-edged sword” in tumorigenesis.2 The cumulating results over the last decade are finally leading to the identification of specific functions for individual AP-1 components and their contribution to neoplastic disease. Here, we focus on the Fra-1 protein in tumorigenesis, which offers an illustrative example of this helter-skelter voyage.

miR-340 inhibits tumor cell proliferation and induces apoptosis by targeting multiple negative regulators of p27 in non-small cell lung cancer.

MicroRNAs (miRNAs) control cell cycle progression by targeting the transcripts encoding for cyclins, CDKs and CDK inhibitors, such as p27KIP1 (p27). p27 expression is controlled by multiple transcriptional and posttranscriptional mechanisms, including translational inhibition by miR-221/222 and posttranslational regulation by the SCFSKP2 complex. The oncosuppressor activity of miR-340 has been recently characterized in breast, colorectal and osteosarcoma tumor cells. However, the mechanisms underlying miR-340-induced cell growth arrest have not been elucidated. Here, we describe miR-340 as a novel tumor suppressor in non-small cell lung cancer (NSCLC). Starting from the observation that the growth-inhibitory and proapoptotic effects of miR-340 correlate with the accumulation of p27 in lung adenocarcinoma and glioblastoma cells, we have analyzed the functional relationship between miR-340 and p27 expression. miR-340 targets three key negative regulators of p27. The miR-340-mediated inhibition of both Pumilio family RNA-binding proteins (PUM1 and PUM2), required for the miR-221/222 interaction with the p27 3′-UTR, antagonizes the miRNA-dependent downregulation of p27. At the same time, miR-340 induces the stabilization of p27 by targeting SKP2, the key posttranslational regulator of p27. Therefore, miR-340 controls p27 at both translational and posttranslational levels. Accordingly, the inhibition of either PUM1 or SKP2 partially recapitulates the miR-340 effect on cell proliferation and apoptosis. In addition to the effect on tumor cell proliferation, miR-340 also inhibits intercellular adhesion and motility in lung cancer cells. These changes correlate with the miR-340-mediated inhibition of previously validated (MET and ROCK1) and potentially novel (RHOA and CDH1) miR-340 target transcripts. Finally, we show that in a small cohort of NSCLC patients (n=23), representative of all four stages of lung cancer, miR-340 expression inversely correlates with clinical staging, thus suggesting that miR-340 downregulation contributes to the disease progression.

Accumulation of Fra-1 in ras-transformed cells depends on both transcriptional autoregulation and MEK-dependent posttranslational stabilization.

ABSTRACT The AP-1 transcription factor plays an essential role in cell proliferation and tumorigenesis. It was previously shown that the fra-1 gene product is upregulated by various oncogenes and is involved in the in vitro and in vivo transformation of thyroid cells. Here we show that the ras oncogene-dependent accumulation of Fra-1 is mediated by a positive feedback mechanism which requires both transcriptional autoregulation and posttranslational stabilization of the protein. The oncogene-dependent transcriptional activation involves the cooperation between both Raf-dependent and Raf-independent pathways and is mediated by an AP-1 site within the fra-1 first intron, which becomes stably occupied by a transcriptionally active Fra-1-containing complex in ras-transformed cells. The posttranslational stabilization results in a drastic increase in the Fra-1 half-life in ras-transformed cells and is totally dependent on the activity of the MEK/ERK phosphorylation pathway. The analysis of the Fra-1 transactivation potential shows that the protein is able to stimulate a heterologous promoter in a ras-dependent manner, but the transactivating activity requires the recruitment of a heterodimeric partner. These data show that the alteration of multiple regulatory mechanisms is required for the constitutive activation of Fra-1 as a nuclear target of ras transformation.

Increase in Ap-1 activity is a general event in thyroid cell transformation in vitro and in vivo

We have recently reported that neoplastic transformation of two rat thyroid epithelial cell lines by retroviruses carrying the v-mos and v-ras Ki oncogenes is associated with a drastic increase of AP-1 activity. The most important effects were represented by the dramatic junB and fra-1 gene induction, which was abolished by the block of the transformation-induced HMGI-C protein synthesis. Here, we have further characterized the transformation-dependent AP-1 activity, by analysing the expression of different jun- and fos-related components, in rat thyroid cell lines transformed by several oncogenes, in human thyroid carcinoma cell lines, and in naturally occurring human thyroid tumours. A significant increase of Fra-1 and JunB protein levels was detected in all oncogene transformed rat thyroid cell lines. Fra-1 gene induction was demonstrated to occur also in human thyroid carcinoma cell lines and tissues. Conversely, c-Jun and JunD proteins, rather than JunB, accumulated in human thyroid carcinoma cell lines. An induction of AP-1 target genes was also detected both in rat and human thyroid transformed cell lines. Therefore, in vivo and in vitro thyroid cell transformation is associated with important compositional changes in the AP-1 complex and an increased transcriptional activity.

Urokinase plasminogen activator receptor affects bone homeostasis by regulating osteoblast and osteoclast function.

The uPAR and its ligand uPA are expressed by both osteoblasts and osteoclasts. Their function in bone remodeling is unknown. We report that uPAR‐lacking mice display increased BMD, increased osteogenic potential of osteoblasts, decreased osteoclasts formation, and altered cytoskeletal reorganization in mature osteoclasts.

Role of Distinct Mitogen-Activated Protein Kinase Pathways and Cooperation between Ets-2, ATF-2, and Jun Family Members in Human Urokinase-Type Plasminogen Activator Gene Induction by Interleukin-1 and Tetradecanoyl Phorbol Acetate

ABSTRACT We have investigated the in vivo and in vitro regulation of the human urokinase-type plasminogen activator (uPA) gene by interleukin-1 (IL-1) and analyzed the transcription factors and signalling pathways involved in the response of the −2.0-kb uPA enhancer to IL-1 induction and to tetradecanoyl phorbol acetate (TPA) induction. Mutational analysis showed the cooperative activity of the Ets-binding site (EBS) and the two AP-1 elements of the enhancer. The results reveal that the EBS is required for the response to both inducers mediated by Ets-2, which is regulated at a level subsequent to DNA binding, by an IL-1- and phorbol ester-inducible transactivation domain. Both the IL-1 and the TPA-mediated induction result in a drastic increase of AP-1 binding to the downstream site of the enhancer (uPA 3′ TPA-responsive element), while a mostly qualitative change, resulting from the interplay between ATF-2 homodimers and c-Jun–ATF-2 heterodimers, takes place at the upstream AP-1 element. The analysis of two distinct mitogen-activated protein kinase pathways shows that stress-activated protein kinase–Jun N-terminal kinase activation, resulting in the phosphorylation of ATF-2, c-Jun, and JunD, is required not only for the IL-1- but also for the TPA-dependent induction, while the extracellular signal-related kinase 1 (ERK-1) and ERK-2 activation is involved in the TPA- but not in the IL-1-dependent stimulation of the uPA enhancer.