Stefano Mellone

ABIN-1 Binds to NEMO/IKKγ and Co-operates with A20 in Inhibiting NF-κB

Nuclear factor κB (NF-κB) plays a pivotal role in inflammation, immunity, stress responses, and protection from apoptosis. Canonical activation of NF-κB is dependent on the phosphorylation of the inhibitory subunit IκBα that is mediated by a multimeric, high molecular weight complex, called IκB kinase (IKK) complex. This is composed of two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, NEMO/IKKγ. The latter protein is essential for the activation of IKKs and NF-κB, but its mechanism of action is not well understood. Here we identified ABIN-1 (A20 binding inhibitor of NF-κB) as a NEMO/IKKγ-interacting protein. ABIN-1 has been previously identified as an A20-binding protein and it has been proposed to mediate the NF-κB inhibiting effects of A20. We find that both ABIN-1 and A20 inhibit NF-κB at the level of the IKK complex and that A20 inhibits activation of NF-κB by de-ubiquitination of NEMO/IKKγ. Importantly, small interfering RNA targeting ABIN-1 abrogates A20-dependent de-ubiquitination of NEMO/IKKγ and RNA interference of A20 impairs the ability of ABIN-1 to inhibit NF-κB activation. Altogether our data indicate that ABIN-1 physically links A20 to NEMO/IKKγ and facilitates A20-mediated de-ubiquitination of NEMO/IKKγ, thus resulting in inhibition of NF-κB.

Nuclear Factor-κB Contributes to Anaplastic Thyroid Carcinomas through Up-Regulation of miR-146a

CONTEXT Micro-RNAs (miRNAs) have been recently involved in the modulation of several biological activities including cancer. Many human tumors show deregulated expression of miRNAs targeting oncogenes and/or tumor suppressors, thus identifying miRNAs as new molecular targets for cancer therapy. OBJECTIVES Nuclear factor (NF)-kappaB is strongly activated in human anaplastic thyroid carcinomas (ATCs). Because the regulation of miRNA expression is under control of RNA polymerase II-dependent transcription factors, we stably inactivated NF-kappaB in the ATC-derived FRO cell line and analyzed its miRNA profile in comparison with the parental counterpart by using a miRNA chip microarray. RESULTS The analysis revealed that a number of miRNAs were differentially expressed in the two cell lines. Among others, the miR-146a showed a strong down-regulation that was confirmed by quantitative real time RT-PCR. The expression of miR-146a was almost undetectable in mouse embryonic fibroblasts isolated from the RelA knockout mice and was restored after reexpression of RelA, thus indicating that miR-146a transcription was controlled by NF-kappaB. The inhibition of miR-146a expression in FRO cells decreased their oncogenic potential and increased the susceptibility to chemotherapeutic drug-induced apoptosis. No difference was found in the growth rate between untransfected and miR-146a-null FRO cells. Importantly, the miR-146a resulted in overexpression of human ATC specimens compared with the normal thyroid tissue. CONCLUSIONS Our results show that NF-kappaB contributes to anaplastic thyroid cancer up-regulating the expression of miR-146a.

Central Role of the Scaffold Protein Tumor Necrosis Factor Receptor-associated Factor 2 in Regulating Endoplasmic Reticulum Stress-induced Apoptosis

The endoplasmic reticulum represents the quality control site of the cell for folding and assembly of cargo proteins. A variety of conditions can alter the ability of the endoplasmic reticulum (ER) to properly fold proteins, thus resulting in ER stress. Cells respond to ER stress by activating different signal transduction pathways leading to increased transcription of chaperone genes, decreased protein synthesis, and eventually to apoptosis. In the present paper we analyzed the role that the adaptor protein tumor necrosis factor-receptor associated factor 2 (TRAF2) plays in regulating cellular responses to apoptotic stimuli from the endoplasmic reticulum. Mouse embryonic fibroblasts derived from TRAF2-/- mice were more susceptible to apoptosis induced by ER stress than the wild type counterpart. This increased susceptibility to ER stress-induced apoptosis was because of an increased accumulation of reactive oxygen species following ER stress, and was abolished by the use of antioxidant. In addition, we demonstrated that the NF-κB pathway protects cells from ER stress-induced apoptosis, controlling ROS accumulation. Our results underscore the involvement of TRAF2 in regulating ER stress responses and the role of NF-κB in protecting cells from ER stress-induced apoptosis.

Role of the adaptor protein CIKS in the activation of the IKK complex

Nuclear factor kappaB (NF-kappaB) plays a pivotal role in numerous cellular processes, including stress response, inflammation, and protection from apoptosis. Therefore, the activity of NF-kappaB needs to be tightly regulated. We have previously identified a novel gene, named CIKS (connection to IkappaB-kinase and SAPK), able to bind the regulatory sub-unit NEMO/IKKgamma and to activate NF-kappaB. Here, we demonstrate that CIKS forms homo-oligomers, interacts with NEMO/IKKgamma, and is recruited to the IKK-complex upon cell stimulation. In addition, we identified the regions of CIKS responsible for these functions. We found that the ability of CIKS to oligomerize, and to be recruited to the IKK-complex is not sufficient to activate the NF-kappaB. In fact, a deletion mutant of CIKS able to oligomerize, to interact with NEMO/IKKgamma, and to be recruited to the IKK-complex does not activate NF-kappaB, suggesting that CIKS needs a second level of regulation to efficiently activate NF-kappaB.

NGAL Controls the Metastatic Potential of Anaplastic Thyroid Carcinoma Cells

CONTEXT We have previously identified neutrophil gelatinase-associated lipocalin (NGAL) as one of the genes mediating the oncogenic activity of nuclear factor-κB in human anaplastic thyroid carcinomas (ATCs). OBJECTIVES To further investigate the role of NGAL in thyroid cancer, we established NGAL knocked-down and NGAL overexpressing ATC cell lines. RESULTS We found that the ability of NGAL knocked-down cells to degrade Matrigel in a transwell invasion assay and to form lung metastasis in nude mice was decreased. Because NGAL binds matrix metalloproteinase-9 (MMP-9), to form a macromolecular complex involved in the regulation of metastatic spread of cancer cells and given the strong expression of both genes in tissue specimens from human ATCs, we analyzed the MMP-9 enzymatic activity in NGAL-null ATC cells. Enzymatic immunoassays show that MMP-9 activity is reduced in NGAL-null ATC cells, even if its expression is not affected by NGAL inhibition. Ectopic expression of NGAL in an ATC cell line not expressing NGAL determines an increase of its metastatic property. The use of a mutated form of NGAL, unable to bind MMP-9, has no positive effect on the invasive potential of ATC cells and does not improve the MMP-9 enzymatic activity. CONCLUSIONS Our results indicate NGAL as a novel target of nuclear factor-κB prometastatic activity in thyroid cancer through enhancement of MMP-9 enzymatic activity.

Down-regulation of Wild-type p53-induced Phosphatase 1 (Wip1) Plays a Critical Role in Regulating Several p53-dependent Functions in Premature Senescent Tumor Cells

Background: Wip1 is a phosphatase involved in DNA-damage response. Results: Wip1 expression is down-regulated in premature senescent cancer cells. Failure to down-regulate Wip1 expression results in cell death and polyploidy. Conclusion: Wip1 down-regulation is important for maintenance of permanent cell cycle arrest in premature senescent tumor cells. Significance: These findings improve our understanding of the mechanism by which Wip1 promotes tumor progression. Premature or drug-induced senescence is a major cellular response to chemotherapy in solid tumors. The senescent phenotype develops slowly and is associated with chronic DNA damage response. We found that expression of wild-type p53-induced phosphatase 1 (Wip1) is markedly down-regulated during persistent DNA damage and after drug release during the acquisition of the senescent phenotype in carcinoma cells. We demonstrate that down-regulation of Wip1 is required for maintenance of permanent G2 arrest. In fact, we show that forced expression of Wip1 in premature senescent tumor cells induces inappropriate re-initiation of mitosis, uncontrolled polyploid progression, and cell death by mitotic failure. Most of the effects of Wip1 may be attributed to its ability to dephosphorylate p53 at Ser15 and to inhibit DNA damage response. However, we also uncover a regulatory pathway whereby suppression of p53 Ser15 phosphorylation is associated with enhanced phosphorylation at Ser46, increased p53 protein levels, and induction of Noxa expression. On the whole, our data indicate that down-regulation of Wip1 expression during premature senescence plays a pivotal role in regulating several p53-dependent aspects of the senescent phenotype.

The RHL-1 subunit of the asialoglycoprotein receptor of thyroid cells: cellular localization and its role in thyroglobulin endocytosis

The rat hepatic lectin (RHL)-1 is the major component of the rat liver asialoglycoprotein receptor (ASGPr), a membrane receptor highly expressed on the basolateral side of hepatocytes, which mediates endocytosis of serum desialated glycoproteins. We have recently shown that RHL-1 is expressed in rat thyroid tissue and thyroid differentiated cell lines. Both in vitro and in vivo assays show that thyrotropin up-regulates thyroid RHL-1 expression, while neoplastic transformation of thyroid cells exerts a down-regulation of receptor expression. Moreover, RHL-1 expressed on the surface of differentiated thyroid cells is able to bind thyroglobulin (Tg), the macromolecular site of synthesis and storage of thyroid hormones. In the present work, we demonstrate, by immunohistochemistry analysis, that RHL-1 is localized on the apical surface of thyrocytes, at a variance with its basolateral localization on hepatocytes. Moreover, albeit its expression in thyroid is less abundant than in liver, the receptor is able to bind asialorosomucoid (ASOR), the best-known ligand of hepatic ASGPr, and to mediate endocytosis of a significative amount of Tg on the surface of differentiated PC Cl3 thyroid cells. Taken together, the data suggest that RHL-1, even if expressed in thyroid at lower levels than in liver, could serve as a receptor for endocytosis of colloidal Tg and, likely, for its delivery to lysosomes.

Diatom-derived oxylipins induce cell death in sea urchin embryos activating caspase-8 and caspase 3/7.

Diatoms are an important class of unicellular algae that produce bioactive secondary metabolites with cytotoxic activity collectively termed oxylipins, including polyunsaturated aldehydes (PUAs), hydroxyacids (HEPEs), oxo-acids and epoxyalcohols. Previous results showed that at higher concentrations, the PUA decadienal induced apoptosis on copepods and sea urchin embryos via caspase-3 activation; at lower concentrations decadienal affected the expression levels of the caspase-8 gene in embryos of the sea urchin Paracentrotus lividus. In the present work, we studied the effects of other common oxylipins produced by diatoms: two PUAs (heptadienal and octadienal) and four hydroxyacids (5-, 9- 11- and 15-HEPE) on P. lividus cell death and caspase activities. Our results showed that (i) at higher concentrations PUAs and HEPEs induced apoptosis in sea urchin embryos, detected by microscopic observation and through the activation of caspase-3/7 and caspase-8 measured by luminescent assays; (ii) at low concentrations, PUAs and HEPEs affected the expression levels of caspase-8 and caspase-3/7 (isolated for the first time here in P. lividus) genes, detected by Real Time qPCR. These findings have interesting implications from the ecological point of view, given the importance of diatom blooms in nutrient-rich aquatic environments.

NGAL promotes recruitment of tumor infiltrating leukocytes

We have previously shown that Neutrophil Gelatinase-Associated Lipocalin (NGAL) is strongly expressed in thyroid carcinomas, especially of anaplastic type, where it protects neoplastic cells from serum deprivation-induced apoptosis and enhances tumor invasivity by regulating MMP-9 activity. Here we demonstrate that NGAL-containing conditioned medium from human anaplastic thyroid carcinoma (ATC) cells is able to induce monocyte migration via up-regulation of a number of different chemokines. The enhanced chemokines transcription is due to the NGAL-mediated intracellular iron uptake. Very importantly, mice tumor allografts raised from subcutaneous injection of syngeneic colon carcinoma cell lines, expressing high levels of NGAL, show a dense leukocyte infiltrate which strongly decreases in tumor allografts from NGAL-depleted cell injected mice. Our results indicate that the NGAL promotes leukocytes recruitment in tumor microenvironment through iron-mediated chemokines production.