Salvatore Sciacchitano

The Loss of the p53 Activator HIPK2 Is Responsible for Galectin-3 Overexpression in Well Differentiated Thyroid Carcinomas

Background Galectin-3 (Gal-3) is an anti-apoptotic molecule involved in thyroid cells transformation. It is specifically overexpressed in thyroid tumour cells and is currently used as a preoperative diagnostic marker of thyroid malignancy. Gal-3 expression is downregulated by wt-p53 at the transcriptional level. In well-differentiated thyroid carcinomas (WDTCs) there is an unexplained paradoxical concomitant expression of Gal-3 and wt-p53. HIPK2 is a co-regulator of different transcription factors, and modulates basic cellular processes mainly through the activation of wt-p53. Since we demonstrated that HIPK2 is involved in p53-mediated Gal-3 downregulation, we asked whether HIPK2 deficiency might be responsible for such paradoxical Gal-3 overexpression in WDTC. Methodology/Principal Findings We analyzed HIPK2 protein and mRNA levels, as well as loss of heterozygosity (LOH) at the HIPK2 locus (7q32-34), in thyroid tissue samples. HIPK2 protein levels were high in all follicular hyperplasias (FHs) analyzed. Conversely, HIPK2 was undetectable in 91.7% of papillary thyroid carcinomas (PTCs) and in 60.0% of follicular thyroid carcinomas (FTCs). HIPK2 mRNA levels were upregulated in FH compared to normal thyroid tissue (NTT), while PTC showed mean HIPK2 mRNA levels lower than FH and, in 61.5% of cases, also lower than NTT. We found LOH at HIPK-2 gene locus in 37.5% of PTCs, 14.3% of FTCs and 18.2% of follicular adenomas. To causally link these data with Gal-3 upregulation, we performed in vitro experiments, using the PTC-derived K1 cells, in which HIPK2 expression was manipulated by RNA interference (RNAi) or plasmid-mediated overexpression. HIPK2 RNAi was associated with Gal-3 upregulation, while HIPK2 overexpression with Gal-3 downregulation. Conclusions/Significance Our results indicate that HIPK2 expression and function are impaired in WDTCs, in particular in PTCs, and that this event explains Gal-3 overexpression typically observed in these types of tumours. Therefore, HIPK2 can be considered as a new tumour suppressor gene for thyroid cancers.

Thyroid carcinoma in children and adolescents

Abstract A clinical and pathological study was undertaken to define the prevalence, clinical presentation and outcome of thyroid carcinoma in children and adolescents. Clinical records from 48 patients under 20 years of age at diagnosis, out of 372 patients with thyroid cancer examined between 1980 and 1994, were retrospectively reviewed. Female/male ratio was 3.8/1. None had a previous positive history of head and neck irradiation. Patients underwent near-total (44 patients) or partial (4 patients) thyroidectomy followed by 131I ablation of residual thyroid tissue. The mean follow up period was 58.4 months, ranging between 2 and 190 months. Clinically a thyroid mass was present in 41 patients, 28 of whom also showed neck lymph node involvement. Node metastases were present in 50% of the patients and lung metastases in 4.2%. Histological type was papillary in 41, follicular in 6, and medullary in 1 case. Surgical complications were observed in 19 patients (40%). In 3 patients papillary thyroid cancer was associated with chronic lymphocytic thyroiditis. All patients were treated with l-thyroxine suppressive therapy. Recurrence of cancer after surgical and radio-iodine treatment was observed only in one patient 8 months after surgery. Conclusion Our experience demonstrates that thyroid carcinoma in childhood cannot be considered a rare occurrence, since it represents about 13% of all thyroid cancers, and is frequently associated with lymph node but rarely with distant metastases. Nevertheless, the prognosis of thyroid carcinoma in childhood is fairly good.

Repression of the Antiapoptotic Molecule Galectin-3 by Homeodomain-Interacting Protein Kinase 2-Activated p53 Is Required for p53-Induced Apoptosis

ABSTRACT Galectin 3 (Gal-3), a member of the β-galactoside binding lectin family, exhibits antiapoptotic functions, and its aberrant expression is involved in various aspects of tumor progression. Here we show that p53-induced apoptosis is associated with transcriptional repression of Gal-3. Previously, it has been reported that phosphorylation of p53 at Ser46 is important for transcription of proapoptotic genes and induction of apoptosis and that homeodomain-interacting protein kinase 2 (HIPK2) is specifically involved in these functions. We show that HIPK2 cooperates with p53 in Gal-3 repression and that this cooperation requires HIPK2 kinase activity. Gene-specific RNA interference demonstrates that HIPK2 is essential for repression of Gal-3 upon induction of p53-dependent apoptosis. Furthermore, expression of a nonrepressible Gal-3 prevents HIPK2- and p53-induced apoptosis. These results reveal a new apoptotic pathway induced by HIPK2-activated p53 and requiring repression of the antiapoptotic factor Gal-3.

Large needle aspiration biopsy and galectin-3 determination in selected thyroid nodules with indeterminate FNA-cytology.

Thyroid fine-needle aspiration biopsy (FNA)-cytology is widely used for the preoperative characterisation of thyroid nodules but this task is difficult for follicular lesions, which often remain undefined. We propose a strategy for improving the preoperative characterisation of selected follicular thyroid proliferations, which is based on large needle aspiration biopsy (LNAB) and galectin-3 expression analysis. Eighty-five thyroid specimens were obtained by LNAB (20-gauge needles) from thyroid nodules with indeterminate follicular FNA-cytology. Aspirated material was processed as a tissue microbiopsy to obtain cell blocks for both cyto/histo-morphological evaluation and galectin-3 expression analysis, by using a purified monoclonal antibody to galectin-3 and a biotin-free immunoperoxidase staining method. Preoperative diagnosis was compared to the final histology. LNAB and cell-block technique allow a preliminary distinction between nodules with a homogeneous microfollicular/trabecular structure, as frequently observed in tumours, and lesions with mixed normo–micro–macrofollicular architecture, as observed in goitre. Furthermore, LNAB provides optimal substrates for galectin-3 expression analysis. Among 85 cases tested, 14 galectin-3-positive cases were discovered preoperatively (11 thyroid cancers and three adenomas confirmed at the final histology), whereas galectin-3-negative cases were 71 (one carcinoma and 70 benign proliferations at the final histology). Sensitivity, specificity and diagnostic accuracy of this integrated morphologic and phenotypic diagnostic approach were 91.6, 97.2 and 95.3%, respectively. In conclusion, LNAB plus galectin-3 expression analysis when applied preoperatively to selected thyroid nodules candidate to surgery can potentially reduce unnecessary thyroid resections.

3,5,3′‐Triiodo‐L‐thyronine enhances the differentiation of a human pancreatic duct cell line (hPANC‐1) towards a β‐cell‐Like phenotype

The thyroid hormone, 3,5,3′‐Triiodo‐L‐thyronine (T3), is essential for growth, differentiation, and regulation of metabolic functions in multicellular organisms, although the specific mechanisms of this control are still unknown. In this study, treatment of a human pancreatic duct cell line (hPANC‐1) with T3 blocks cell growth by an increase of cells in G0/G1 cell cycle phase and enhances morphological and functional changes as indicated by the marked increase in the synthesis of insulin and the parallel decrease of the ductal differentiation marker cytokeratin19. Expression analysis of some of the genes regulating pancreatic β‐cell differentiation revealed a time‐dependent increase in insulin and glut2 mRNA levels in response to T3. As last step of the acquisition of a β‐cell‐like phenotype, we present evidence that thyroid hormones are able to increase the release of insulin into the culture medium. In conclusion, our results suggest, for the first time, that thyroid hormones induce cell cycle perturbations and play an important role in the process of transdifferentiation of a human pancreatic duct line (hPANC‐1) into pancreatic‐β‐cell‐like cells. These findings have important implications in cell‐therapy based treatment of diabetes and may provide important insights in the designing of novel therapeutic agents to restore normal glycemia in subjects with diabetes.

Analysis of adenomatous polyposis coli gene in thyroid tumours

Familial adenomatous polyposis (FAP) is known to be associated with neoplasia of various tissues, including thyroid carcinoma. Germline mutations of the tumour-suppressor gene APC, responsible for the predisposition to FAP, may therefore be involved in the pathogenesis of these tumours. In this report the structure of the APC gene has been investigated in 26 thyroid tumours, at different stages of dedifferentiation, that were surgically excised from patients with a negative history of FAP. Approximately 35% of the APC gene coding region, where most of the mutations are clustered, has been analysed by a combination of single-strand conformation polymorphism and direct sequencing. No significant alterations could be demonstrated in any sample examined. It is concluded that, at least in patients not affected by FAP, APC gene abnormalities do not seem to play a relevant role in the pathogenesis of thyroid carcinoma.

PCR amplification and analysis of ras oncogenes from thyroid cytologic smears

Fine needle aspiration (FNA) cytology represents a reliable diagnostic procedure for preoperative identification of thyroid carcinoma. For improving its diagnostic accuracy, a technique that allows the analysis of cancer-related gene abnormalities on thyroid FNA smears has been developed. Cells were collected by scraping from thyroid smears, DNA-extracted and ras proto-oncogene sequences amplified by the polymerase chain reaction (PCR). Preliminary destaining of cytologic smears was essential for efficient PCR amplification. Twelve thyroid FNA cytologic smears, characterized by the indeterminate pattern of follicular neoplasia, were analysed for the presence of ras mutations known to confer an oncogenic potential. The same nucleotide substitution at codon 12 of the H-ras proto-oncogene was detected in two different thyroid nodules among six cases that, at final histology, were identified as follicular carcinomas. No ras mutations were observed in the remaining six cases that were diagnosed as follicular adenoma at histologic examination. Molecular analysis of FNA smears may provide additional information on the nature of the lesion underlying thyroid neoplasia, thus improving diagnostic accuracy of conventional FNA cytology.

Detection and molecular characterisation of thyroid cancer precursor lesions in a specific subset of Hashimoto's thyroiditis.

Hashimotos thyroiditis (HT) represents the most common cause of hypothyroidism and nonendemic goiter, but its clinical and pathological heterogeneity opens the question if this disease should be more properly considered as a spectrum of different thyroid conditions rather than as a single nosological entity. In this study, we analysed 133 cases of HT for the expression of galectin-3, a lectin molecule involved in malignant transformation, apoptosis and cell cycle control. An unexpected expression of galectin-3 was demonstrated in a subset of HT together with the presence of HBME-1, c-met and cyclin-D1 that are also involved in malignant transformation and deregulated cell growth. Furthermore, a loss of allelic heterozygosity in a specific cancer-related chromosomal region was demonstrated in some HT harbouring galectin-3-positive follicular cells, by using laser capture microdissection. On the basis of the morphological and molecular findings we identified four subsets of HT: (a) HT with classic features of chronic autoimmune thyroiditis; (b) HT associated to hyperplastic/adenomatous lesions; (c) HT harbouring thyroid cancer precursors; (d) HT associated to unequivocal thyroid microcarcinomas. Our findings provide a well-substantiated morphological and molecular demonstration that HT may include a spectrum of different thyroid conditions ranging from chronic autoimmune thyroiditis to thyroiditis triggered by specific immune-response to cancer-related antigens.

Gal-3 is stimulated by gain-of-function p53 mutations and modulates chemoresistance in anaplastic thyroid carcinomas.

Galectin‐3 (Gal‐3) is an anti‐apoptotic molecule of the β‐galactoside‐binding lectin family. Gal‐3 is down‐regulated by wt‐p53 and this repression is required for p53‐induced apoptosis. Since poorly differentiated thyroid carcinomas (PDTCs) and anaplastic thyroid carcinomas (ATCs) frequently harbour p53 mutations, we asked whether Gal‐3 expression and activity could be influenced by such mutations in these tumours. We found a positive correlation between Gal‐3 expression and p53 mutation in human thyroids and in thyroid carcinoma cell lines (TCCLs) harbouring different p53 mutations. Gal‐3 was over‐expressed in most ATCs and TCCLs, especially those with the most frequently detected p53 mutation (p53R273H). Over‐expression of p53R273H in two p53‐null cells (SAOS‐2 and SW‐1736) as well as in two wt‐p53‐carrying TCCLs (TPC‐1 and K1), stimulated Gal‐3 expression, while interference with p53R273H endogenous expression in ARO cells down‐regulated Gal‐3 expression. Conversely, over‐expression of wt‐p53 in ARO cells restored the inhibitory effect on Gal‐3 expression. ARO cells are highly resistant to apoptosis and express both p53 and Gal‐3, which are increased upon cisplatin treatment. Interference with Gal‐3 expression in these cells stimulated their chemosensitivity. In conclusion, gain‐of‐function p53 mutant acquires the de novo ability to stimulate Gal‐3 expression and to increase chemoresistance in ATCs. Copyright

Prevalence of Thyroid Cancer in Hyperthyroid Patients Treated by Surgery

A retrospective study has been carried out to evaluate the prevalence of malignant thyroid tumors in 202 patients submitted to surgery for hyperthyroidism. Thyroid cancer was diagnosed in 12 cases (5.9%); the final histologic examination revealed nine papillary carcinomas, one follicular carcinoma, and two Hürthle cell carcinomas. Concurrence of hyperthyroidism and thyroid cancer was more frequent in patients with single toxic adenomas than in those with toxic diffuse or multinodular goiters. In five cases thyroid malignancy was detected in the context of the hyperthyroid lesion (three toxic adenomas and two diffuse goiters). In eight patients the malignant lesion showed a maximum diameter of less than 1 cm, although in five of these cases unfavorable histologic features, such as minimal capsular invasion or multifocality, were present. All patients presenting with thyroid cancer are currently alive and apparently free of residual disease. It is concluded that hyperthyroid patients, particularly those with single toxic adenomas, should be carefully evaluated to exclude the presence of an associated malignancy and to plan the most appropriate therapeutic options.