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The signaling network between cancer and stromal cells plays a crucial role in tumor microenvironment. The fate of tumor progression mainly depends on the huge amount of information that these cell populations exchange from the onset of neoplastic transformation. Interfering with such signaling has been producing exciting results in cancer therapy: just think of anti-PD-1/anti-PD-L1/anti-CTLA-4 antibodies that, acting as immune checkpoint inhibitors, interrupt the inhibitory signaling exerted by cancer cells on immune cells or the CAR-T technology that fosters the reactivation of anti-tumoral immunity in a restricted group of leukemias and lymphomas. Nevertheless, many types of cancers, in particular solid tumors, are still refractory to these treatments, so the identification of novel molecular targets in tumor secretome would benefit from implementation of current anti-cancer therapeutical strategies. Neutrophil Gelatinase-Associated Lipocalin (NGAL) is a secreted protein abundantly expressed in the secretome of various human tumors. It represents a promising target for the multiple roles that are played inside cancer and stromal cells, and also overall in their cross-talk. The review focuses on the different roles of NGAL in tumor microenvironment and in cancer senescence-associated secretory phenotype (SASP), highlighting the most crucial functions that could be eventually targetable in cancer therapy.

Context: We have previously identified neutrophil gelatinase-associated lipocalin (NGAL) as one of the genes mediating the oncogenic activity of nuclear factor-?B 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-?B prometastatic activity in thyroid cancer through enhancement of MMP-9 enzymatic activity.

OBJECTIVE: We recently reported that the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2b is the SERCA form preferentially expressed in rat thyroid. Moreover, SERCA2b expression dramatically decreases in virally transformed, highly tumorigenic, PC Cl3 thyroid cells. These results suggest that, in the thyroid, SERCA2b, in addition to its housekeeping role, is linked to differentiation and is a regulated gene. We therefore sought to study the effect of TSH, the main regulator of thyroid function, on SERCA2b expression and activity. METHODS: PC Cl3 cells were hormone starved in low-serum medium and stimulated for long (48 h) or short (1, 2 and 4 h) times. SERCA2b expression and activity were evaluated by Northern and Western blots, Ca2+-ATPase activity and Ca2+ store content. RESULTS: In PC Cl3 cells, SERCA2b mRNA and protein were induced twofold by a 48-h long treatment with TSH. Long-term elevation (48 h) of intracellular cAMP levels, by forskolin or 8-Br-cAMP, had similar effects on SERCA2b mRNA and protein. We also measured Ca2+-ATPase activity and Ca2+ store content. Both long (48 h) and short (0.5-1 h) treatments with TSH, forskolin or 8-Br-cAMP induced a marked increase of SERCA2b activity. This effect was completely abolished by H89, a specific inhibitor of cAMP-dependent protein kinase A (PKA). TSH and 8-Br-cAMP increased Ca2+ store content after both long (48 h) and short (1-2 h) treatments. CONCLUSIONS: These data suggested that TSH/cAMP acts as an important regulator of both SERCA2b expression and activity in the thyroid system, through PKA activation.

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)-κB 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-κB 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-κB. 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-κB contributes to anaplastic thyroid cancer up-regulating the expression of miR-146a.

Perturbing the endoplasmic reticulum homeostasis of thyroid cell lines with thapsigargin, a specific inhibitor of the sarcoendoplasmic reticulum Ca(2+) adenosine triphosphatases, and tunicamycin, an inhibitor of the N-linked glycosylation, blocked Tg in the endoplasmic reticulum. This event was signaled outside the endoplasmic reticulum and resulted in activation of the c-Jun N-terminal kinase (JNK)/stress-activated protein kinase and nuclear factor-kappa B (NF-kappa B) stress response pathways. Activation of the JNK/stress-activated protein kinase signaling pathway was assessed by measuring the amount of phospho-JNK and the activity of JNK by kinase assays. Activation of the NF-kappa B signaling pathway was assessed by measuring the level of inhibitory subunit I kappa B alpha, DNA binding, and transcriptional activity of NF-kappa B. Cycloheximide treatment, at a dose able to profoundly inhibit protein synthesis in FRTL-5 cells, obliterated the decrease in the level of the inhibitory subunit I kappa B alpha produced by thapsigargin and tunicamycin. Therefore, protein synthesis was required to generate a signal from stressed endoplasmic reticulum. This substantiates the hypothesis that endoplasmic reticulum retention of newly synthesized Tg and other cargo (secretory and membrane) proteins functions upstream of signal activation. Dominant negative TNF receptor-associated factor 2 (TRAF2) inhibited activation of NF-kappa B, which was also inhibited in embryonic fibroblasts derived from TRAF2(-/-) mice, respect to their normal counterpart. These data extend the recent demonstration that TRAF2 mediated JNK activation in response to endoplasmic reticulum stress and strongly strengthened the idea that endogenous stress signals initiated in the endoplasmic reticulum proceed by a pathway similar to that initiated by plasma membrane receptors in response to extracellular signals.

Follicular thyroglobulin (TG) decreases expression of the thyroid-restricted transcription factors, thyroid transcription factor (TTF)-1, TTF-2, and Pax-8, thereby suppressing expression of the sodium iodide symporter, thyroid peroxidase, TG, and thyrotropin receptor genes (Suzuki, K., Lavaroni, S., Mori, A., Ohta, M., Saito, J., Pietrarelli, M., Singer, D. S., Kimura, S., Katoh, R., Kawaoi, A. , and Kohn, L. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 95, 8251-8256). The ability of highly purified 27, 19, or 12 S follicular TG to suppress thyroid-restricted gene expression correlates with their ability to bind to FRTL-5 thyrocytes and is inhibited by a specific antibody to the thyroid apical membrane asialoglycoprotein receptor (ASGPR), which is related to the ASGPR of liver cells. Phosphorylating serine/threonine residues of TG, by autophosphorylation or protein kinase A, eliminates TG suppression and enhances transcript levels of the thyroid-restricted genes 2-fold in the absence of a change in TG binding to the ASGPR. Follicular TG suppression of thyroid-restricted genes is thus mediated by the ASPGR on the thyrocyte apical membrane and regulated by a signal system wherein phosphorylation of serine/threonine residues on the bound ligand is an important component. These data provide a hitherto unsuspected role for the ASGPR in transcriptional signaling, aside from its role in endocytosis. They establish a functional role for phosphorylated serine/threonine residues on the TG molecule.

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-kappa B 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-kappa B in protecting cells from ER stress-induced apoptosis.

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 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.

We have recently identified a novel gene, named CIKS ( C onnection to IK K-complex and S APK), able to activate the transcription factor NF-κB, after interaction with the regulatory subunit NEMO/IKKγ 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)-α, Interleukin (IL)-1β and transforming growth factor (TGF)-β, 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.

During its initial folding in the endoplasmic reticulum (ER), newly synthesized thyroglobulin (Tg) is known to interact with calnexin and other ER molecular chaperones, but its interaction with calreticulin has not been examined previously. In the present study, we have investigated the interactions of endogenous Tg with calreticulin and with several other ER chaperones. We find that, in FRTL-5 and PC-Cl3 cells, calnexin and calreticulin interact with newly synthesized Tg in a carbohydrate-dependent manner, with largely overlapping kinetics that are concomitant with the maturation of Tg intrachain disulphide bonds, preceding Tg dimerization and exit from the ER. Calreticulin co-precipitates more newly synthesized Tg than does calnexin; however, using two different experimental approaches, calnexin and calreticulin were found in ternary complexes with Tg, making this the first endogenous protein reported in ternary complexes with calnexin and calreticulin in the ER of live cells. Depletion of Ca2+ from the ER elicited by thapsigargin (a specific inhibitor of ER Ca2+-ATPases) results in retention of Tg in this organelle. Interestingly, thapsigargin treatment induces the premature exit of Tg from the calnexin/calreticulin cycle, while stabilizing and prolonging interactions of Tg with BiP (immunoglobulin heavy chain binding protein) and GRP94 (glucose-regulated protein 94), two chaperones whose binding is not carbohydrate-dependent. Our results suggest that calnexin and calreticulin, acting in ternary complexes with a large glycoprotein substrate such as Tg, might be engaged in the folding of distinct domains, and indicate that lumenal Ca2+ strongly influences the folding of exportable glycoproteins, in part by regulating the balance of substrate binding to different molecular chaperone systems within the ER.

Maintaining a high Ca(2+) concentration in the lumen of the endoplasmic reticulum (ER), by the action of sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCAs), is important in many cellular processes, such as Ca(2+)-mediated cytosolic signaling in response to extracellular stimuli, cell growth and proliferation, and synthesis, processing and folding of ER-translated proteins. In the thyroid gland, SERCAs have not been studied yet, and there is little information available on general problems such as the expression of SERCAs following neoplastic transformation. In this study we investigated the expression of SERCA2b and SERCA3 in rat thyroid tIssue and, in addition, in normal and transformed rat thyroid cell lines. RT-PCR and Northern blot assays showed that SERCA2b is the SERCA form preferentially expressed in the thyroid. In rat thyroid, SERCA2b mRNA was expressed at a higher level than that of other non-muscle tIssues such as liver or spleen, but at much lower level than in brain. On the other hand, SERCA3 mRNA was not detected in thyroid by Northern blot analysis, or barely detected by RT-PCR assays. We also studied the SERCA2b expression pattern in PC Cl3 thyroid cells transformed by several oncogenes that induce different degrees of malignancy and dedifferentiation. RT-PCR and Northern blot assays showed that SERCA2b mRNA expression dramatically decreased in highly tumorigenic thyroid cells, while expression of glyceraldehyde-3-phosphate dehydrogenase mRNA, a housekeeping gene used as internal control, exhibited no variations. The dramatic down-regulation of SERCA2b expression in fully transformed thyroid cells was also evident by Western blot analysis. Also, following neoplastic transformation of thyroid cells, the enzymatic activity of SERCA2b was reduced in a measure which correlated with the mRNA and protein levels. Therefore, rat thyrocytes expressed intermediate levels of SERCAs, mostly the SERCA2b isoform. This pattern of expression was basically reproduced in fully differentiated thyroid cells in culture and was sensitive to neoplastic transformation.

We have recently shown that the rat hepatic lectin (RHL)-1 subunit of the asialoglycoprotein receptor (ASGPr) is expressed in the PC C13 differentiated thyroid cell line. To investigate in vivo the expression of RHL-1 and the ability of thyrotropin (TSH) to modulate its expression, reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blot assays have been performed on thyroid extracts from rats treated with thyroxine (T4) or propylthiouracil (PTU), each of which modulates TSH levels. It is shown that RHL-1 expression is down-regulated by T4 (which decreases serum TSH) and upregulated by PTU (which increases serum TSH), at both mRNA and protein levels. The sensitivity of RHL-1 to neoplastic transformation of thyroid cells has been investigated. The RHL-1 expression pattern has been studied in PC C13 thyroid cells transformed by several oncogenes that induce different degrees of malignancy and dedifferentiation. RT-PCR and Western blot assays show that RHL-1 expression progressively decreases as PC C13 cells acquire a more transformed phenotype. Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, a housekeeping gene used as internal control to normalize RHL-1 mRNA content, exhibits no variations in the different PC C13 cell lines used. In addition, we show that both native and asialo-thyroglobulin (Tg) bind RHL-1 in vitro, and native Tg binds RHL-1 on the surface of PC C13 cells. After thyroid cells transformation, the surface expression of RHL-1 is inhibited in a measure that correlates with the mRNA and protein levels. Therefore, the RHL-1 inhibition at the mRNA, protein and plasma membrane expression follows a gradient that parallels the progressive acquisition of the fully transformed phenotype in the PC C13 system. The results reported in the present article, together with our previous data, suggest that RHL-1 expression could be regulated, at least in part, by the same transcription factors involved in the expression of the other molecules characteristic of the thyroid differentiated state.

We have previously reported that the rat hepatic lectin-1 (RHL-1) subunit of rat asialoglycoprotein receptor (ASGPr), the endocytic receptor found on the basolateral surface of hepatocytes, was expressed in rat thyroid tissue and localized on the apical surface of polarized rat thyroid FRT cells. Here we show that PC Cl3 cells, a differentiated rat thyroid cell line, bound thyroglobulin (Tg) via ASGPr. In fact, both the bacterial recombinant carbohydrate recognition domain of RHL-1 (rCRD(RHL-1)) and the anti-rCRD(RHL-1) antibody markedly inhibited (125)I-Tg binding to the cell surface of PC Cl3 cells. Ligand blot assays with deglycosylated Tg show that the rCRD(RHL-1) was able to interact with Tg even after remotion of sugars. The region of Tg involved in the binding to RHL-1 was investigated by ligand blot assays with biotinylated rCRD(RHL-1) on thermolysin-digested native and desialated rat thyroglobulin. It is shown that the rCRD(RHL-1) specifically recognized a thyroglobulin fragment with an apparent M(r) of 68,000, corresponding to the amino-terminal part of the molecule. To our knowledge, this is the first report that attributes to the amino-terminal portion of Tg molecule, containing its earliest and major hormonogenic site, the function of binding to a cell surface receptor of the thyroid. Moreover, we show that oligosaccharides are not the only molecular signals for binding to RHL-1, but amino acidic determinants could also play a role.

Background: Previous studies indicate that platelets and leucocytes might contribute to the development of neointimal hyperplasia following arterial injury. The present study was aimed at further investigating the role of platelets and leucocytes, alone or in combination, in promoting vascular smooth muscle cell (SMC) proliferation in vitro, focusing on the relative contribution of different soluble growth factors released by these cells, and on the ability to induce proto-oncogene expression. such as c-fos. Methods: SMCs from rabbit aortas, made quiescent by serum deprivation, were stimulated with either activated platelets, leucocytes, or both, separated from SMCs by a membrane insert. SMC proliferation was evaluated by measuring the incorporation of H-3-thymidine. The relative contribution of different platelet-derived mediators to SMC growth was evaluated by adding either ketanserin, a 5-HT2 receptor antagonist, R68070, a TxA(2) receptor antagonist, BN52021, a platelet activating factor (PAF) receptor antagonist, and trapidil, a platelet derived growth factor (PDGF) receptor antagonist. The role of different leucocyte sub-populations (neutrophils and monocytes+lymphocytes) was also determined in additional experiments. Results: SMC proliferation was significantly increased by activated platelets to 360+/-9% of control values (P