Vito Rodolico

In vitro identification and characterization of CD133pos 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 CD133pos 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/CD133pos cells exhibited higher proliferation, self-renewal, colony-forming ability in comparison with ARO/CD133neg. Furthermore, ARO/CD133pos showed levels of thyroid transcription factor TTF-1 similar to the fetal thyroid cell line TAD-2, while the expression in ARO/CD133neg was negligible. The expression of the stem cell marker OCT-4 detected by RT-PCR and flow cytometry was markedly higher in ARO/CD133pos in comparison to ARO/CD133neg 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/CD133pos when compared with ARO/CD133neg cells. Conclusions/Significance We describe CD133pos cells in ATC cell lines. ARO/CD133pos 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.

BRAFV600E mutation and p27kip1 expression in papillary carcinomas of the thyroid ≤1 cm and their paired lymph node metastases

BRAFV600E mutation and p27kip1 expression have been introduced as novel indicators that may predict prognosis in different tumors, as well as in papillary thyroid carcinomas.

Lymph node metastasis in lower lip squamous cell carcinoma in relation to tumour size, histologic variables and p27KiP1 protein expression

We studied a consecutive series of 95 patients undergoing radical surgical resection of lower lip squamous cell carcinoma (LLSCC) to assess the correlation between lymph node status and several prognostic variables, such as sex and age, tumour size, histologic grading, maximal microscopic tumour thickness, perineural infiltration and p27Kip1 protein status, to see which of these might be predictive of the development of lymph node metastases. Statistical analysis demonstrated a significant association between node status and tumour size, histological grading, maximal thickness, perineural invasion and p27Kip1 protein expression; additionally to node metastasis, low p27Kip1 protein expression was significant correlated with high microscopic thickness. These results indicate that lower lip squamous cell carcinomas of >2 cm, with G3-G4 histological grading, maximal thickness of >6 mm, perineural invasion and low p27Kip1 protein expression (LI<19.7%) are at high risk for the development of lymph node metastases.

IL-34 is overexpressed in the inflamed salivary glands of patients with Sjögren’s syndrome and is associated with the local expansion of pro-inflammatory CD14brightCD16+ monocytes

OBJECTIVES To investigate the expression of IL-34 in labial salivary glands (LSGs) of patients with primary SS (p-SS) and its role in inducing a pro-inflammatory monocyte phenotype. METHODS LSG biopsies were obtained from 20 patients with p-SS and 10 patients with non-Sjögrens sicca syndrome (n-SS). The expression of IL-34, IL-1β, TNF-α, IL-17 and IL-23 was assessed by real-time PCR. IL-34 expression was also investigated in LSGs by immunohistochemistry. The frequencies of subpopulations of CD14(+) monocytes were evaluated by flow cytometry among isolated mononuclear cells from peripheral blood and salivary glands from both patients and controls. The role of recombinant IL-34 on isolated peripheral blood mononuclear cells was also evaluated. RESULTS IL-34 m-RNA was overexpressed in the inflamed salivary glands of p-SS and associated with increased expression of TNF-α, IL-1β, IL-17 and IL-23p19. The increased expression of IL-34 was confirmed by immunohistochemistry in paraffin-embedded salivary glands from p-SS patients. IL-34 expression was accompanied by the expansion of pro-inflammatory CD14(bright)CD16(+) monocytes in the salivary glands. In vitro stimulation of peripheral blood mononuclear cells with IL-34 induced the expansion of both CD14(+)CD16(-) cells and CD14(bright)CD16(+) cells in p-SS and non-SS subjects. CONCLUSION IL-34 seems to be involved in the pathogenesis of salivary gland inflammation in p-SS.

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

Hypoxia-inducible factor-1α 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-1α expression was found increased in differentiated, poorly differentiated, medullary and anaplastic variants. Hypoxia represents the principal stimulus responsible for hypoxia-inducible factor-1α induction. Other nonhypoxic stimuli increase hypoxia-inducible factor-1α 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 BRAFV600E mutation in papillary thyroid cancer cells as a factor that facilitates tumor cell growth and progression. In this study, we tested the hypothesis that BRAFV600E mutation influences hypoxia-inducible factor-1α expression in papillary thyroid carcinoma cells. We analyzed 27 papillary thyroid carcinomas, 13 of which presented BRAFV600E mutation. In tumor tissues, immunoreactivity for hypoxia-inducible factor-1α was detected in the majority of analyzed BRAFV600E mutated cases. Transcriptional analyses revealed elevated hypoxia-inducible factor-1α levels with significant differences between wild-type and mutated group. A BRAF wild-type papillary thyroid carcinoma cell line and a BRAFV600E mutated papillary thyroid carcinoma cell line were selected to study the effects of BRAF mutation on hypoxia-inducible factor-1α expression in vitro. Knockdown of mutant BRAFV600E or both the wild type and the BRAFV600E by RNA interference induced a significant reduction of hypoxia-inducible factor-1α expression at mRNA and protein levels. Pharmacological inhibition of BRAF significantly reduces hypoxia-inducible factor-1α expression levels in papillary thyroid carcinoma cell line harboring BRAFV600E mutation. Our results suggest that hypoxia-inducible factor-1α is expressed in papillary thyroid carcinomas and is regulated not only by hypoxia but also by BRAFV600E-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.

Interleukin‐9 Overexpression and Th9 Polarization Characterize the Inflamed Gut, the Synovial Tissue, and the Peripheral Blood of Patients With Psoriatic Arthritis

To investigate the expression and tissue distribution of Th9‐related cytokines in patients with psoriatic arthritis (PsA).

Multiple pluripotent stem cell markers in human anaplastic thyroid cancer: the putative upstream role of SOX2.

BACKGROUND Anaplastic thyroid carcinoma (ATC) is a rare and aggressive endocrine tumor with highly undifferentiated morphology. It has been suggested that cancer stem cells (CSCs) might play a central role in ATC. The objectives of this study were (i) to characterize CSCs from ex vivo ATC specimens by investigating the expression of several pluripotent stem cell markers, and (ii) to evaluate in vitro drug resistance modifications after specific CSC transcription factor switch-off. METHODS In ex vivo experiments, eight formalin-fixed, paraffin-embedded ATC specimens were analyzed by reverse-transcription and real-time quantitative PCR and immunohistochemistry. In in vitro experiments using ATC SW1736 cells, the expression levels of OCT-4, NANOG, and ABCG2 and the sensitivity to either cisplatin or doxorubicin were evaluated after silencing. RESULTS OCT-4, KLF4, and SOX2 transcription factors and C-KIT and THY-1 stem surface antigens showed variable up-regulation in all ATC cases. The SW1736 cell line was characterized by a high percentage of stem population (10.4±2.1% of cells were aldehyde dehydrogenase positive) and high expression of several CSC markers (SOX2, OCT4, NANOG, C-MYC, and SSEA4). SOX2 silencing down-regulated OCT-4, NANOG, and ABCG2. SOX2 silencing sensitized SW1736 cells, causing a significant cell death increase (1.8-fold) in comparison to control cells with 10 μM cisplatin (93.9±3.4% vs. 52.6±9.4%, p<0.01) and 2.7 fold with 0.5 μM doxorubicin (45.8±9.9% vs. 17.1±3.4% p<0.01). ABCG2 silencing caused increased cell death with both cisplatin (74.9±1.4%) and doxorubicin treatment (74.1±0.1%) vs. no-target-treated cells (respectively, 45.8±1.0% and 48.6±1.0%, p<0.001). CONCLUSIONS The characterization of CSCs in ATC through the analysis of multiple pluripotent stem cell markers might be useful in identifying cells with a stem-like phenotype capable of resisting conventional chemotherapy. In addition, our data demonstrate that SOX2 switch-off through ABCG2 transporter down-regulation has a major role in overcoming CSC chemotherapy resistance.

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

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.

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.