BRAFV600E mutation, TIMP-1 upregulation, and NF-κB activation: closing the loop on the papillary thyroid cancer trilogy.

BRAF(V600E) is the most common mutation found in papillary thyroid carcinoma (PTC). Tissue inhibitor of metalloproteinases (TIMP-1) and nuclear factor (NF)-κB have been shown to play an important role in thyroid cancer. In particular, TIMP-1 binds its receptor CD63 on cell surface membrane and activates Akt signaling pathway, which is eventually responsible for its anti-apoptotic activity. The aim of our study was to evaluate whether interplay among these three factors exists and exerts a functional role in PTCs. To this purpose, 56 PTC specimens were analyzed for BRAF(V600E) mutation, TIMP-1 expression, and NF-κB activation. We found that BRAF(V600E) mutation occurs selectively in PTC nodules and is associated with hyperactivation of NF-κB and upregulation of both TIMP-1 and its receptor CD63. To assess the functional relationship among these factors, we first silenced BRAF gene in BCPAP cells, harboring BRAF(V600E) mutation. We found that silencing causes a marked decrease in TIMP-1 expression and NF-κB binding activity, as well as decreased invasiveness. After treatment with specific inhibitors of MAPK pathway, we found that only sorafenib was able to increase IκB-α and reduce both TIMP-1 expression and Akt phosphorylation in BCPAP cells, indicating that BRAF(V600E) activates NF-κB and this pathway is MEK-independent. Taken together, our findings demonstrate that BRAF(V600E) causes upregulation of TIMP-1 via NF-κB. TIMP-1 binds then its surface receptor CD63, leading eventually to Akt activation, which in turn confers antiapoptotic behavior and promotion of cell invasion. The recognition of this functional trilogy provides insight on how BRAF(V600E) determines cancer initiation, progression, and invasiveness in PTC, also identifying new therapeutic targets for the treatment of highly aggressive forms.

Glut-1 expression and in situ CD1a/CD57 immunologic deficit in keratoacanthoma and squamous cell carcinoma of immunocompetent patients.

It is not easy to reach a differential diagnosis between keratoacanthoma (KA) and squamous cell carcinoma (SCC) and furthermore there is still considerable discussion about the relationship of these 2 tumors with immunity. To facilitate such a diagnosis, we assessed the Glut-1 antibody, reported to be strongly and diffusely expressed in SCC but never assessed in KA. We studied 43 lesions of immunocompetent patients: 17 SCCs, 13 typical KAs (tKAs), and 13 atypical KAs (aKAs), with histologic features of SCC in less than 30% of the lesions. In tKA, Glut-1 stained only the basal layers of the squamous nests (basal pattern) whereas in SCC the squamous nests were randomly and diffusely stained (diffuse pattern). In aKA, a biphasic pattern was observed, with the typical KA areas showing the basal pattern and the SCC-like areas showing the diffuse pattern. Glut-1, therefore, helps to distinguish tKAs from SCCs and highlights the intermediate aKA group, supporting the hypothesis of a progression from KA to SCC. Finally, we used CD1a, CD57, CD4, CD8, CD3, and CD20 antibodies to assess whether or not the progression might be related to an in situ immunologic deficit. Significant differences were found both in CD1a+ cells, more numerous in tKA than in SCC and in CD57+ cells, more numerous in tKA than in aKA and in SCC. This suggests a local immunological failure in aKA and SCC, probably related to the action of UV rays, leading us to consider KA as a model for the study of the interaction of skin cancer and immunity.

Changes in immunohistochemical levels and subcellular localization after therapy and correlation and colocalization with CD68 suggest a pathogenetic role of Hsp60 in ulcerative colitis.

In an earlier work, the role of heat shock protein (Hsp60) in the pathogenesis of ulcerative colitis (UC) was suggested by its significant increase in the pathological mucosa parallel with an increase in inflammatory cells. More data in this direction are reported in this work. We analyzed by immunohistochemistry biopsies of colon tissue from 2 groups of patients with UC and treated with either 5-aminosalicylic acid (5-ASA) alone or in combination with a probiotic. We looked for inflammatory markers and Hsp60. Both the treatments were effective in reducing symptoms but the group treated with both 5-ASA and probiotics showed better clinical results. Amelioration of symptoms was associated with reduction of both inflammation and Hsp60, a reduction that was most marked in the group treated with 5-ASA and probiotics. The levels of Hsp60 positively correlated with those of CD68-positive cells, and double immunofluorescence showed a high index of colocalization of the chaperonin and CD68 in lamina propria. Immunoelectron microscopy showed that Hsp60-classically a mitochondrial protein-was abundantly also present in cytosol in biopsies taken at the time of diagnosis, but not after the treatment. Our data suggest that Hsp60 is an active player in pathogenesis of UC and it can be hypothesized that the chaperonin is responsible, at least in part, for initiation and maintenance of disease.

BRAF(V600E) mutation influences hypoxia-inducible factor-1alpha expression levels in papillary thyroid cancer.

Hypoxia-inducible factor-1alpha is found frequently overexpressed in solid tumors cells, exerting an important role in angiogenesis, glucose metabolism, cell proliferation, survival and invasion. In thyroid carcinomas, hypoxia-inducible factor-1alpha expression was found increased in differentiated, poorly differentiated, medullary and anaplastic variants. Hypoxia represents the principal stimulus responsible for hypoxia-inducible factor-1alpha induction. Other nonhypoxic stimuli increase hypoxia-inducible factor-1alpha synthesis through the activation of phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways in a cell-type-specific manner. We have previously shown the role of BRAF(V600E) mutation in papillary thyroid cancer cells as a factor that facilitates tumor cell growth and progression. In this study, we tested the hypothesis that BRAF(V600E) mutation influences hypoxia-inducible factor-1alpha expression in papillary thyroid carcinoma cells. We analyzed 27 papillary thyroid carcinomas, 13 of which presented BRAF(V600E) mutation. In tumor tissues, immunoreactivity for hypoxia-inducible factor-1alpha was detected in the majority of analyzed BRAF(V600E) mutated cases. Transcriptional analyses revealed elevated hypoxia-inducible factor-1alpha levels with significant differences between wild-type and mutated group. A BRAF wild-type papillary thyroid carcinoma cell line and a BRAF(V600E) mutated papillary thyroid carcinoma cell line were selected to study the effects of BRAF mutation on hypoxia-inducible factor-1alpha expression in vitro. Knockdown of mutant BRAF(V600E) or both the wild type and the BRAF(V600E) by RNA interference induced a significant reduction of hypoxia-inducible factor-1alpha expression at mRNA and protein levels. Pharmacological inhibition of BRAF significantly reduces hypoxia-inducible factor-1alpha expression levels in papillary thyroid carcinoma cell line harboring BRAF(V600E) mutation. Our results suggest that hypoxia-inducible factor-1alpha is expressed in papillary thyroid carcinomas and is regulated not only by hypoxia but also by BRAF(V600E)-mediated signaling pathway.

Hsp60 and Hsp10 increase in colon mucosa of Crohn's disease and ulcerative colitis.

The purpose of this work was to determine in colon mucosa of Crohn's disease (CD) and ulcerative colitis (UC) in relapse: a) the levels of the chaperonins Hsp60 and Hsp10; b) the quantity of inflammatory cells; and c) if the levels of chaperonins parallel those of inflammation cells. Twenty cases of CD and UC and twenty normal controls (NC) were studied using immunohistochemistry, Western blotting and immunofluorescence. Immunohistochemically, Hsp60 and Hsp10 were increased in both inflammatory bowel diseases (IBD) compared to NC. These results were confirmed by Western blotting. Hsp60 and Hsp10 occurred in the cytoplasm of epithelial cells in CD and UC but not in NC. Hsp60 and Hsp10 co-localised to epithelial cells of mucosal glands but not always in connective tissue cells of lamina propria, where only Hsp60 or, less often, Hsp10 was found. Cells typical of inflammation were significantly more abundant in CD and UC than in NC. Since chaperonins are key factors in the activation of the immune system leading to inflammation, we propose that they play a central role in the pathogenesis of the two diseases, which, consequently, ought to be studied as chaperonopathies.

Diagnostic and prognostic value of CD10 in peripheral nerve sheath tumors.

BACKGROUND: Neurofibromas are sporadic or associated with type 1 neurofibromatosis (NF1), with a higher risk of malignant progression. MATERIALS AND METHODS: We investigated CD10 immunoexpression in 39 peripheral nerve sheath lesions. They were 18 typical, solitary, sporadic neurofibromas (group A) and 21 cases (group B) consisting of 11 NF1-associated cases, 3 malignant peripheral sheath tumors (MPNST) and 8 atypical neurofibromas. RESULTS: CD10 immunopositivity was absent or very weak and focal in group A. On the contrary, CD10 was strongly expressed in group B, including all the MPNST and their metastases, with 95% sensitivity and 72% specificity in distinguishing between the two groups. CONCLUSION: CD10 is useful in the assessment of peripheral sheath tumors and could give evidence that atypical myxoid and/or diffuse neurofibromas, sometimes histologically difficult to distinguish from low-grade MPNST, represent not only a histological but also an immunohistochemical continuum with MPNST.

In vitro identification and characterization of CD133(pos) cancer stem-like cells in anaplastic thyroid carcinoma cell lines.

BACKGROUND: Recent publications suggest that neoplastic initiation and growth are dependent on a small subset of cells, termed cancer stem cells (CSCs). Anaplastic Thyroid Carcinoma (ATC) is a very aggressive solid tumor with poor prognosis, characterized by high dedifferentiation. The existence of CSCs might account for the heterogeneity of ATC lesions. CD133 has been identified as a stem cell marker for normal and cancerous tissues, although its biological function remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: ATC cell lines ARO, KAT-4, KAT-18 and FRO were analyzed for CD133 expression. Flow cytometry showed CD133(pos) cells only in ARO and KAT-4 (64+/-9% and 57+/-12%, respectively). These data were confirmed by qRT-PCR and immunocytochemistry. ARO and KAT-4 were also positive for fetal marker oncofetal fibronectin and negative for thyrocyte-specific differentiating markers thyroglobulin, thyroperoxidase and sodium/iodide symporter. Sorted ARO/CD133(pos) cells exhibited higher proliferation, self-renewal, colony-forming ability in comparison with ARO/CD133(neg). Furthermore, ARO/CD133(pos) showed levels of thyroid transcription factor TTF-1 similar to the fetal thyroid cell line TAD-2, while the expression in ARO/CD133(neg) was negligible. The expression of the stem cell marker OCT-4 detected by RT-PCR and flow cytometry was markedly higher in ARO/CD133(pos) in comparison to ARO/CD133(neg) cells. The stem cell markers c-KIT and THY-1 were negative. Sensitivity to chemotherapy agents was investigated, showing remarkable resistance to chemotherapy-induced apoptosis in ARO/CD133(pos) when compared with ARO/CD133(neg) cells. CONCLUSIONS/SIGNIFICANCE: We describe CD133(pos) cells in ATC cell lines. ARO/CD133(pos) cells exhibit stem cell-like features--such as high proliferation, self-renewal ability, expression of OCT-4--and are characterized by higher resistance to chemotherapy. The simultaneous positivity for thyroid specific factor TTF-1 and onfFN suggest they might represent putative thyroid cancer stem-like cells. Our in vitro findings might provide new insights for novel therapeutic approaches.

Increased expression of transketolase-like-1 in papillary thyroid carcinomas smaller than 1.5 cm in diameter is associated with lymph-node metastases.

BACKGROUND: Patients with small papillary thyroid carcinoma (PTC) may have a high incidence of regional lymph-node (LN) metastases at presentation, and these are considered to be an independent risk factor for tumor recurrence. A mutated transketolase transcript (TKTL1) has been found up-regulated in different human malignancies, and strong TKTL1 protein expression has been associated with aggressiveness and poor patient survival in several epithelial cancers. METHODS: TKTL1 protein expression was analyzed in 256 consecutive cases of PTCs

MUC1 oncoprotein promotes refractoriness to chemotherapy in thyroid cancer cells.

Overexpression of MUC1 oncoprotein is frequently observed in cancer and contributes to confer resistance to genotoxic agents. Papillary, follicular, and anaplastic thyroid carcinomas are the three forms of thyroid epithelial cancer. Anaplastic tumors are less differentiated and extremely aggressive, characterized by a poor prognosis. Little is known about the role of MUC1 in thyroid cancer. We recently showed that autocrine production of interleukin (IL)-4 and IL-10 controls thyroid cancer cell survival, growth, and resistance to chemotherapy through activation of Janus-activated kinase/signal transducers and activators of transcription (JAK/STAT) and phosphatidylinositide 3'-OH kinase (PI3K)/Akt pathways. In the present study, we showed that MUC1 COOH-terminal subunit (MUC1-C) is overexpressed in all the histologic variants of thyroid cancer cells and localizes to mitochondria where it interferes with the release of mitochondrial proapoptotic proteins. Moreover, IL-4 and IL-10 promote the increase of MUC1-C expression levels in normal thyroid cells, whereas blockage of both cytokines or neutralization of JAK/STAT and PI3K/Akt pathways through the exogenous expression of SOCS-1 and Akt(K179M) leads to a significant decrease of MUC1-C in primary thyroid cancer cells. Interestingly, down-regulation of MUC1 expression by direct targeting with RNA interference sensitizes anaplastic thyroid cancer cells to chemotherapy-induced apoptosis in vitro. Thus, MUC1 is a main component of the survival network acting in thyroid cancer and could be considered a key molecular target for sensitizing cancer cells to conventional or novel treatments.

The antiapoptotic protein BAG3 is expressed in thyroid carcinomas and modulates apoptosis mediated by tumor necrosis factor-related apoptosis-inducing ligand.

CONTEXT: We previously showed that BAG3 protein, a member of the BAG (Bcl-2-associated athanogene) co-chaperone family, modulates apoptosis in human leukemias. The expression of BAG3 in other tumor types has not been extensively investigated so far. OBJECTIVE: The objective of this study was to analyze BAG3 expression in thyroid neoplastic cells and investigate its influence in cell apoptotic response to TNF-related apoptosis-inducing ligand (TRAIL). DESIGN, SETTING, AND PATIENTS: We investigated BAG3 expression in human thyroid carcinoma cell lines, including NPA, and the effect of BAG3-specific small interfering RNA on TRAIL-induced apoptosis in NPA cells. Subsequently, we analyzed BAG3 expression in 30 benign lesions and 56 carcinomas from patients of the Naples Tumor Institute Fondazione Senatore Pascale. MAIN OUTCOME MEASURES: The main outcome measures were: analysis of BAG3 protein in NPA cells by Western blot and immunocytochemistry; analysis of apoptosis in TRAIL-stimulated NPA cells by flow cytometry; and evaluation of BAG3 expression in specimens from thyroid lesions by immunohistochemistry. RESULTS: BAG3 was expressed in human thyroid carcinoma cell lines; small interfering RNA-mediated downmodulation of its levels significantly (P < 0.0195) enhanced NPA cell apoptotic response to TRAIL. The protein was not detectable in 19 of 20 specimens of normal thyroid or goiters, whereas 54 of 56 analyzed carcinomas (15 follicular, 28 papillary, and 13 anaplastic) were clearly positive for BAG3 expression. CONCLUSIONS: BAG3 downmodulates the apoptotic response to TRAIL in human neoplastic thyroid cells. The protein is specifically expressed in thyroid carcinomas and not in normal thyroid tissue or goiter.

Autocrine production of interleukin-4 and interleukin-10 is required for survival and growth of thyroid cancer cells.

Although CD95 and its ligand are expressed in thyroid cancer, the tumor cell mass does not seem to be affected by such expression. We have recently shown that thyroid carcinomas produce interleukin (IL)-4 and IL-10, which promote resistance to chemotherapy through the up-regulation of Bcl-xL. Here, we show that freshly purified thyroid cancer cells were completely refractory to CD95-induced apoptosis despite the consistent expression of Fas-associated death domain and caspase-8. The analysis of potential molecules able to prevent caspase-8 activation in thyroid cancer cells revealed a remarkable up-regulation of cellular FLIP(L) (cFLIP(L)) and PED/PEA-15, two antiapoptotic proteins whose exogenous expression in normal thyrocytes inhibited the death-inducing signaling complex of CD95. Additionally, small interfering RNA FLIP and PED antisense sensitized thyroid cancer cells to CD95-mediated apoptosis. Exposure of normal thyrocytes to IL-4 and IL-10 potently up-regulated cFLIP and PED/PEA-15, suggesting that these cytokines are responsible for thyroid cancer cell resistance to CD95 stimulation. Moreover, treatment with neutralizing antibodies against IL-4 and IL-10 or exogenous expression of suppressor of cytokine signaling-1 of thyroid cancer cells resulted in cFLIP and PED/PEA-15 down-regulation and CD95 sensitization. More importantly, prolonged IL-4 and IL-10 neutralization induced cancer cell growth inhibition and apoptosis, which were prevented by blocking antibodies against CD95 ligand. Altogether, autocrine production of IL-4 and IL-10 neutralizes CD95-generated signals and allows survival and growth of thyroid cancer cells. Thus, IL-4 and IL-10 may represent key targets for the treatment of thyroid cancer.

PED mediates AKT-dependent chemoresistance in human breast cancer cells.

Killing of tumor cells by cytotoxic therapies, such as chemotherapy or gamma-irradiation, is predominantly mediated by the activation of apoptotic pathways. Refractoriness to anticancer therapy is often due to a failure in the apoptotic pathway. The mechanisms that control the balance between survival and cell death in cancer cells are still largely unknown. Tumor cells have been shown to evade death signals through an increase in the expression of antiapoptotic molecules or loss of proapoptotic factors. We aimed to study the involvement of PED, a molecule with a broad antiapoptotic action, in human breast cancer cell resistance to chemotherapeutic drugs-induced cell death. We show that human breast cancer cells express high levels of PED and that AKT activity regulates PED protein levels. Interestingly, exogenous expression of a dominant-negative AKT cDNA or of PED antisense in human breast cancer cells induced a significant down-regulation of PED and sensitized cells to chemotherapy-induced cell death. Thus, AKT-dependent increase of PED expression levels represents a key molecular mechanism for chemoresistance in breast cancer.

NF-kappaB protects Behçet's disease T cells against CD95-induced apoptosis up-regulating antiapoptotic proteins.

OBJECTIVE: To determine whether prolongation of the inflammatory reaction in patients with Behçet's disease (BD) is related to apoptosis resistance and is associated with the up-regulation of antiapoptotic factors. METHODS: The percentage of cell death was evaluated by flow cytometry in peripheral blood mononuclear cells from 35 patients with BD and 30 healthy volunteers. The expression levels of antiapoptotic factors and NF-kappaB regulatory proteins were measured using Western blotting and immunohistochemical analyses. To down-regulate NF-kappaB nuclear translocation, BD T lymphocytes were exposed in vitro to thalidomide and subjected to transfection with NF-kappaB small interfering RNA. RESULTS: Although CD95 is highly expressed in BD T cells, the absence of sensitivity to CD95-induced apoptosis observed may be attributable to the inhibitory action of antiapoptotic genes. Immunoblot analysis for major antiapoptotic proteins showed considerable up-regulation of the short form of cellular FLIP (cFLIP) and Bcl-x(L) in BD activated T cells, while levels of Bcl-2, caspase 3, and caspase 8 in activated T cells from patients with BD were comparable with those in activated T cells from normal donors. Moreover, expression of IKK and IkappaB was up-regulated, whereas NF-kappaB translocated to the nucleus in BD T cells, suggesting that NF-kappaB activation may modulate the expression of antiapoptotic genes. Interestingly, thalidomide and NF-kappaB small interfering RNA down-regulated cFLIP and Bcl-x(L) expression levels and sensitized BD activated T cells to CD95-induced apoptosis. CONCLUSION: Taken together, these results indicate that NF-kappaB contributes to the regulation of the apoptosis-related factors and death receptors leading to apoptosis resistance in BD T cell subsets. Our results suggest that NF-kappaB plays a crucial role in the pathogenesis of BD, and that its pharmacologic control could represent a key strategy in modulating specific immune-mediated disease.

IL-4 protects tumor cells from anti-CD95 and chemotherapeutic agents via up-regulation of antiapoptotic proteins.

We recently proposed that Th1 and Th2 cytokines exert opposite effects on the pathogenesis and clinical outcome of organ-specific autoimmunity by altering the expression of genes involved in target cell survival. Because a Th2 response against tumors is associated with poor prognosis, we investigated the ability of IL-4 to protect tumor cells from death receptor- and chemotherapy-induced apoptosis. We found that IL-4 treatment significantly reduced CD95 (Fas/APO-1)- and chemotherapeutic drug-induced apoptosis in prostate, breast, and bladder tumor cell lines. Analysis of antiapoptotic protein expression revealed that IL-4 stimulation resulted in up-regulation of cellular (c) FLIP/FLAME-1 and Bcl-x(L). Exogenous expression of cFLIP/FLAME-1 inhibited apoptosis induced by CD95 and to a lesser extent by chemotherapy, while tumor cells transduced with Bcl-x(L) were substantially protected both from CD95 and chemotherapeutic drug stimulation. Moreover, consistent IL-4 production and high expression of both cFLIP/FLAME-1 and Bcl-x(L) were observed in primary prostate, breast, and bladder cancer in vivo. Finally, primary breast cancer cells acquired sensitivity to apoptosis in vitro only in the absence of IL-4. Thus, IL-4 protects tumor cells from CD95- and chemotherapy-induced apoptosis through the up-regulation of antiapoptotic proteins such as cFLIP/FLAME-1 and Bcl-x(L). These findings may provide useful information for the development of therapeutic strategies aimed at restoring the functionality of apoptotic pathways in tumor cells.

Thyroid cancer resistance to chemotherapeutic drugs via autocrine production of interleukin-4 and interleukin-10.

We investigated the mechanisms responsible for the widespread refractoriness to chemotherapeutic drugs observed in thyroid cancers. We show that malignant epithelial cells from papillary, follicular, and anaplastic thyroid carcinomas express high levels of Bcl-2 and Bcl-xL. Exogenous expression of either Bcl-2 or Bcl-xL in normal thyrocytes was sufficient to prevent chemotherapeutic drug-induced cytotoxicity. All of the histological thyroid cancer variants examined produced interleukin-4 (IL-4) and interleukin-10 (IL-10), which increased Bcl-2 and Bcl-xL levels and protected thyroid cells from chemotherapeutic agents. Exposure to neutralizing antibodies against IL-4 and IL-10 resulted in down-modulation of Bcl-2 and Bcl-xL, death of a considerable percentage of thyroid cancer cells, and sensitization of the residual tumor population to cytotoxic drug-induced apoptosis. In conclusion, autocrine production of IL-4 and IL-10 promotes thyroid tumor cell progression and resistance to chemotherapy through the up-regulation of antiapoptotic proteins. Thus, IL-4 and IL-10 may represent new therapeutic targets for the treatment of thyroid cancer.