NGAL Mediates Anaplastic Thyroid Carcinoma Cells Survival Through FAS/CD95 Inhibition.

Neutrophil gelatinase-associated lipocalin (NGAL), a siderophore-mediated iron binding protein, is highly expressed in human anaplastic thyroid carcinomas (ATCs) where it plays pleiotropic protumorigenic roles including that of a prosurvival protein. Here we show that NGAL inhibits FAS/CD95 death receptor to control ATC cell survival. FAS/CD95 expression in human specimens from patients with ATC and in ATC-derived cell lines negatively correlate with NGAL expression. Silencing of NGAL in ATC cells leads to FAS/CD95 upregulation, whereas NGAL overexpression determines the opposite effect. As a result, an agonist anti-FAS/CD95 antibody induces cell death in NGAL-silenced cells while it is ineffective on NGAL-overexpressing cells. Interestingly, the inhibitory activity of NGAL on FAS/CD95 is due to its iron carrier property given that perturbing iron homeostasis of NGAL-proficient and -deficient ATC cells directly influences FAS/CD95 expression. Accordingly, conditioned media containing a mutant form of NGAL unable to bind siderophores cannot rescue cells from FAS/CD95-dependent death, whereas NGAL wild type-containing conditioned media abolish the effects of the agonist antibody. We also find that downregulation of FAS/CD95 expression is mediated by iron-dependent NGAL suppression of p53 transcriptional activity. Our results indicate that NGAL contributes to ATC cell survival by iron-mediated inhibition of p53-dependent FAS/CD95 expression and suggest that restoring FAS/CD95 by NGAL suppression could be a helpful strategy to kill ATC cells.

Trabectedin suppresses escape from therapy-induced senescence in tumor cells by interfering with glutamine metabolism.

Conventional and targeted cancer therapies may induce a cellular senescence program termed therapy-induced senescence. However, unlike normal cells, cancer cells are able to evade the senescence cell cycle arrest and to resume proliferation, driving tumor recurrence after treatments. Cells that escape from therapy-induced senescence are characterized by a plastic, cancer stem cell-like phenotype, and recent studies are beginning to define their unique metabolic features, such as glutamine dependence. Here, we show that the antineoplastic drug trabectedin suppresses escape from therapy-induced senescence in all cell lines studied, and reduces breast cancer stem-like cells, at concentrations that do not affect the viability of senescent tumor cells. We demonstrate that trabectedin downregulates both the glutamine transporter SLC1A5 and glutamine synthetase, thereby interfering with glutamine metabolism. On the whole, our results indicate that trabectedin targets a glutamine-dependent cancer stem-like cell population involved in evasion from therapy-induced senescence and suggest a therapeutic potential for trabectedin combined with pro-senescence chemotherapy in tumor treatment.

Glutamine promotes escape from therapy-induced senescence in tumor cells.

Therapy-induced senescence (TIS) is a major cellular response to anticancer therapies. While induction of a persistent growth arrest would be a desirable outcome in cancer therapy, it has been shown that, unlike normal cells, cancer cells are able to evade the senescence cell cycle arrest and to resume proliferation, likely contributing to tumor relapse. Notably, cells that escape from TIS acquire a plastic, stem cell-like phenotype. The metabolic dependencies of cells that evade senescence have not been thoroughly studied. In this study, we show that glutamine depletion inhibits escape from TIS in all cell lines studied, and reduces the stem cell subpopulation. In line with a metabolic reliance on glutamine, escaped clones overexpress the glutamine transporter SLC1A5. We also demonstrate a central role of glutamine synthetase that mediates resistance to glutamine deprivation, conferring independence from exogenous glutamine. Finally, rescue experiments demonstrate that glutamine provides nitrogen for nucleotides biosynthesis in cells that escape from TIS, but also suggest a critical involvement of glutamine in other metabolic and non-metabolic pathways. On the whole, these results reveal a metabolic vulnerability of cancer stem cells that recover proliferation after exposure to anticancer therapies, which could be exploited to prevent tumor recurrence.

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.

The chemokine scavenging receptor D6/ACKR2 is a target of miR-146a in thyroid cancer.

We have previously shown that miR-146a, a NF-κB-regulated microRNA, is strongly expressed in human specimens and cell lines derived from anaplastic thyroid carcinomas (ATC) where it mediates some of the NF-κB pro-tumorigenic functions. By using a bioinformatic analysis, we identified the chemokine scavenger receptor D6/ ACKR2 as a target of miR146a in human ATC. We found that the expression of D6/ ACKR2 was up-regulated in miR-146a-null ATC cell lines and that the 3' UTR of D6/ ACKR2 mRNA was able to inhibit its expression in parental, but not in miR-146a-null ATC cells. Since human specimens from primary ATC showed a low expression of D6/ ACKR2 compared to normal thyroid tissues, we analyzed the effects of D6/ACKR2 over-expression in ATC cells. Different chemokines added to the conditioned medium of D6/ACKR2 over-expressing ATC cells partially failed to drive in vitro monocyte migration, and tumors derived from the injection of the same cells in nude mice showed a decreased number of infiltrating macrophages. Taken together, these results indicate that ATC cells down-regulate D6/ACKR2 expression through miR-146a activity to sustain leukocyte trafficking inside tumor microenvironment and shed light on a novel mechanism by which NF-κB indirectly inhibits the expression and the function of anti-tumorigenic gene in thyroid cancer.

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.

Cytokine expression and ultrastructural alterations in fresh-frozen, freeze-dried and γ-irradiated human amniotic membranes.

The aim of this work was to compare the effects on human amniotic membrane of freeze-drying and γ-irradiation at doses of 10, 20 and 30 kGy, with freezing. For this purpose, nine cytokines (interleukin 10, platelet-derived growth factor-AA, platelet-derived growth factor-BB, basic fibroblast growth factor, epidermal growth factor, transforming growth factor beta 1, and tissue inhibitors of metalloproteinase-1, -2, and -4) were titrated in 5 different preparations for each of 3 amniotic membranes included in the study. In addition, the extracellular matrix structure of each sample was assessed by transmission electron microscopy. While freeze-drying did not seem to affect the biological structure or cytokine content of the different amniotic membrane samples, γ-irradiation led to a significant decrease in the tissue inhibitors of metalloproteinase-4, basic fibroblast growth factor and epidermal growth factor, and induced structural damage to the epithelium, basement membrane and lamina densa. The higher the irradiation dose the more severe the damage to the amniotic membrane structure. In conclusion, the Authors recommend processing amniotic membrane under sterile conditions to guarantee safety at every step rather than final sterilization with γ-irradiation, thereby avoiding alteration to the biological characteristics of the amniotic membrane.

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.

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 Ser(15) and to inhibit DNA damage response. However, we also uncover a regulatory pathway whereby suppression of p53 Ser(15) phosphorylation is associated with enhanced phosphorylation at Ser(46), 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.

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.

Nuclear factor-{kappa}B 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.

ABIN-1 binds to NEMO/IKKgamma and co-operates with A20 in inhibiting NF-kappaB.

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

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-kappaB 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-kappaB in protecting cells from ER stress-induced apoptosis.

Oncogenic and anti-apoptotic activity of NF-kappa B in human thyroid carcinomas.

Thyroid cancer includes three types of carcinomas classified as differentiated thyroid carcinomas (DTC), medullary thyroid carcinomas, and undifferentiated carcinomas (UTC). DTC and medullary thyroid carcinomas generally have a good prognosis, but UTC are usually fatal. Consequently, there is a need for new effective therapeutic modalities to improve the survival of UTC patients. Here we show that NF-kappa B is activated in human thyroid neoplasms, particularly in undifferentiated carcinomas. Thyroid cell lines, reproducing in vitro the different thyroid neoplasias, also show basal NF-kappa B activity and resistance to drug-induced apoptosis, which correlates with the level of NF-kappa B activation. Activation of NF-kappa B in the DTC cell line NPA renders these cells resistant to drug-induced apoptosis. Stable expression of a super-repressor form of I kappa B alpha (I kappa B alpha M) in the UTC cell line FRO results in enhanced sensitivity to drug-induced apoptosis, to the loss of the ability of these cells to form colonies in soft agar, and to induce tumor growth in nude mice. In addition, we show that FRO cells display a very low JNK activity that is restored in FRO-I kappa B alpha M clones. Moreover, inhibition of JNK activity renders FRO-I kappa B alpha M clones resistant to apoptosis induced by chemotherapeutic agents. Our results indicate that NF-kappa B plays a pivotal role in thyroid carcinogenesis, being required for tumor growth and for resistance to drug-induced apoptosis, the latter function very likely through the inhibition of JNK activity. Furthermore, the strong constitutive NF-kappa B activity in human anaplastic thyroid carcinomas, besides representing a novel diagnostic tool, makes NF-kappa B a target for the development of novel therapeutic strategies.

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.

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.

Promoter identification of CIKS, a novel NF-kappaB activating gene, and regulation of its expression.

We have recently identified a novel gene, named CIKS (Connection to IKK-complex and SAPK), able to activate the transcription factor NF-kappaB, after interaction with the regulatory subunit NEMO/IKKgamma of IKK complex, and the stress-activated protein kinase (SAPK)/JNK. CIKS mRNA is ubiquitously expressed, although its levels differ greatly among different tissues. The aim of this study is to identify and characterize the promoter region of CIKS gene and to analyse the regulation of its expression by different cytokines. The transcription start site of CIKS mRNA was mapped both by primer extension and by a polymerase chain reaction (PCR)-based strategy. The proximal 5'-flanking region of CIKS gene was 'TATA-less', but contained other consensus promoter elements including an initiator (Inr), 'GC' and 'CAAT' boxes. Transfection of luciferase reporter plasmids containing 1.8 kb of the 5'-flanking region increased luciferase activity in epithelial MDCK cells, but not in endothelial HUVEC cells. Deletion analysis identified a sequence from -464 to -220 bp of the 5'-flanking region of CIKS gene essential for basal promoter activity in MDCK cells. Competitive reverse transcriptase-PCR, Northern and Western blot assays showed that different cytokines, such as tumor necrosis factor (TNF)-alpha, Interleukin (IL)-1beta and transforming growth factor (TGF)-beta, dramatically increased CIKS mRNA expression in HeLa cells. We conclude that the proximal 5'-flanking region of CIKS gene contains a functional promoter and binding sites for nuclear proteins leading to its basal transcription. Moreover, we demonstrate that the expression of CIKS is up-regulated by different cytokines.