Angiotensin converting enzymes ACE and ACE2 in thyroid cancer progression.

Angiotensin-converting enzymes, ACE and ACE2, play not only a pivotal role in the regulation of blood pressure, but are involved in the processes of pathophysiology, including thyroid dysfunction or progression of several neoplasia such as cancers of skin, lungs, pancreas and leukemia. However, their role in the thyroid carcinogenesis remains unknown. We examined in this study the expression of ACE and ACE2 in thyroid tissues and their possible employment as biomarkers for thyroid cancer progression. Thyroid tissues, including 14 goiters (G), 12 follicular adenomas (FA), 10 follicular thyroid carcinomas (FTC), 14 papillary thyroid carcinomas (PTC) and 11 undifferentiated thyroid carcinomas (UTC), were subjected to RT-PCR and protein analyses with primers or antibodies specific for ACE and ACE2, respectively. FA revealed significantly increased ACE compared to other groups and FTC was significantly higher than UTC. ACE2 was significantly increased in PTC in comparison to G, FA and UTC, and in FTC as compared to G. The ratio ACE/ACE2 decreased, while ACE2/ACE increased with the differentiation grade of thyroid carcinoma. ACE was significantly diminished in individuals older than 50. Both ACEs were significantly diminished in M1 patients, ACE2 additionally in higher tumor masses. ACE and ACE2 are regulated within thyroid benign and malignant tissues. As the transcript ratio between both enzymes correlate proportional with the differentiation status of thyroid cancer, ACE and ACE2 may serve as new markers for thyroid carcinoma.

Expression of ARE-binding proteins AUF1 and HuR in follicular adenoma and carcinoma of thyroid gland.

Both adenylate-uridylate rich elements binding proteins AUF1 and HuR may participate in thyroid carcinoma progression. In this study we investigated the expression of both factors on a protein level with a special focus on follicular adenoma and follicular thyroid carcinoma. By employment of immunofluorescence and western blot on 68 thyroid tissues including 7 goiter, 16 follicular adenoma (4 adenomatous hyperplasia), 19 follicular thyroid carcinomas, 13 papillary thyroid carcinomas and 14 undifferentiated thyroid carcinomas we investigated protein expression of AUF1 and HuR. In addition to previous results we demonstrated that AUF1 and HuR are significantly up-regulated in carcinoma tissues as compared with follicular adenoma or goiter tissues. Furthermore, by evaluation of AUF1 or HuR expression, or combination of both proteins on total tissue lysates, we were able to demonstrate a significant difference between follicular adenoma and follicular thyroid carcinoma. Overexpression of AUF1 and HuR is a common finding observed in thyroid malignancy. Analysis of the tissues obtained by surgical resection as demonstrated in this study is comparable to a fine needle aspiration and in combination with AUF1/HuR immuno-analysis may support the conventional immunohistological investigations. The promising results of this study were performed on relatively small collective, but justify future development of a quick thyroid diagnostic test on larger cohort of the patients, especially for thyroid samples which are inadequate for histological examinations.

Downregulation of microRNAs directs the EMT and invasive potential of anaplastic thyroid carcinomas.

Anaplastic thyroid carcinomas (ATCs) arise from epithelial thyroid cells by mesenchymal de-/transdifferentiation and rapidly invade the adjacent tissue. Specific microRNA signatures were suggested to distinguish ATCs from normal thyroid tissue and other thyroid carcinomas of follicular origin. Whether distinct microRNA patterns correlate with de-/transdifferentiation and invasion of ATCs remained elusive. We identified two significantly decreased microRNA families that unambiguously distinguish ATCs from papillary and follicular thyroid carcinomas: miR-200 and miR-30. Expression of these microRNAs in mesenchymal ATC-derived cells reduced their invasive potential and induced mesenchymal-epithelial transition (MET) by regulating the expression of MET marker proteins. Supporting the role of transforming growth factor (TGF)beta signaling in modulating MET/epithelial-mesenchymal transition (EMT), expression of SMAD2 and TGFBR1, upregulated in most primary ATCs, was controlled by members of the miR-30 and/or miR-200 families in ATC-derived cells. Inhibition of TGFbeta receptor 1 (TGFBR1) in these cells induced MET and reduction of prometastatic miR-21, but caused an increase of the miR-200 family. These findings identify altered microRNA signatures as potent markers for ATCs that promote de-/transdifferentiation (EMT) and invasion of these neoplasias. Hence, TGFBR1 inhibition could have a significant potential for the treatment of ATCs and possibly other invasive tumors.

Expression of hypoxia-inducible factor 1 alpha in thyroid carcinomas.

Hypoxia-inducible factor 1 alpha (HIF-1 alpha) is upregulated by hypoxia and oncogenic signalling in many solid tumours. Its regulation and function in thyroid carcinomas are unknown. We evaluated the regulation of HIF-1 alpha and target gene expression in primary thyroid carcinomas and thyroid carcinoma cell lines (BcPAP, WRO, FTC-133 and 8505c). HIF-1 alpha was not detectable in normal tissue but was expressed in thyroid carcinomas. Dedifferentiated anaplastic tumours (ATCs) exhibited high levels of nuclear HIF-1 alpha staining. The HIF-1 target glucose transporter 1 was expressed to a similar level in all tumour types, whereas carbonic anhydrase-9 was significantly elevated in ATCs. In vitro studies revealed a functionally active HIF-1 alpha pathway in thyroid cells with transcriptional activation observed after graded hypoxia (1% O(2), anoxia) or treatment with a hypoxia mimetic cobalt chloride. High basal and hypoxia-induced expression of HIF-1 alpha in FTC-133 cells that harbour a phosphatase and tensin homologue (PTEN) mutation was reduced by introduction of wild-type PTEN. Similarly, pharmacological inhibition of the phosphoinositide 3-kinase (PI3K) pathway using LY294002 inhibited HIF-1 alpha and HIF-1 alpha targets in all cell lines, including those with B-RAF mutations (BcPAP and 8505c). In contrast, the effects of inhibition of the RAF/MEK/extracellular signal-regulated kinase pathway were restricted by environmental condition and B-RAF mutation status. HIF-1 is functionally expressed in thyroid carcinomas and is regulated not only by hypoxia but also via growth factor signalling pathways and, in particular, the PI3K pathway. Given the strong association of HIF-1 alpha with an aggressive disease phenotype and therapeutic resistance, this pathway may be an attractive target for improved therapy in thyroid carcinomas.

AhR-agonist-induced transcriptional changes of genes involved in thyroid function in primary porcine thyrocytes.

The Ah receptor (AhR) is a ligand transcription factor mediating toxic effects of chemicals such as dioxins. The 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) and the coplanar polychlorinated biphenyl 126 (PCB 126) are member of the polyhalogenated aromatic hydrocarbons family exerting a variety of toxic effects in a tissue-specific and species-specific manner including thyroid function. In the present study, we aimed to investigate the effects of TCDD (1 and 10 nM) and dioxin-like PCB 126 (306 nM) on the AhR signaling pathway and on the gene expression profiles of key factors involved in thyroid function, including thyroglobulin (TG), thyroid peroxidase (TPO), the sodium iodide symporter (NIS), TSH receptor (TSHR), and cathepsins (Cat B and L), using a primary porcine thyrocyte culture as the experimental model. AhR and ARNT expression was detected both as mRNA and on the protein level. Expression did not vary upon treatment with either TCDD or PCB 126. However, treatment with TCDD and PCB 126 induced an AhR signaling response, as indicated by the expression of the AhR-target gene cytochrome P-450 1A1 (CYP1A1). Both 10 nM TCDD and PCB 126 treatment induced a significant downregulation in the expression of NIS and cathepsin B without affecting any of the other parameters investigated. In conclusion, these data indicate that (a) thyrocytes are targets of TCDD and TCDD-like compounds and (b) there is evidence for two independent most likely AhR-mediated molecular mechanisms, by which these compounds negatively interfere with thyroid function.

Role of CD97(stalk) and CD55 as molecular markers for prognosis and therapy of gastric carcinoma patients.

OBJECTIVES: To explore the mechanism of development and aggressiveness in gastric carcinomas by investigating the expression and role of CD97 and its cellular ligand CD55 in gastric carcinomas. METHODS: Tumor and corresponding normal mucosal tissue, collected from 39 gastric carcinoma patients, were examined by immunohistochemistry and RT-PCR for the expression of CD97 and CD55. RESULTS: CD97(stalk) was strongly stained on scattered tumor cells or small tumor cell clusters at the invasion front of gastric carcinomas. The expression of CD97(stalk) was frequently observed in tumors of stage I and T1 gastric carcinoma patients. The expression of CD97(stalk) between Stage I and Stage II, III, IV specimens showed significant difference (P<0.05), between T1 and T2, T3, T4 specimens also showed significant difference (P<0.05). Specimens with tumor invasion depth limited in mucosa of T1 specimens showed higher positive CD55 expression than specimens with the same tumor invasion depth in T2, T3, T4 specimens, the expression of CD55 between T1 and T2, T3, T4 specimens was significantly different (P<0.05). There was strong correlation between the distribution patterns of CD97(stalk) and CD55 on tumor tissues (r=0.73, P<0.05). Signet ring cell carcinomas frequently contained strong CD97(stalk) and CD55-staining. CONCLUSIONS: Our results suggest that CD97(stalk) is probably involved in the growth, invasion and aggressiveness of gastric carcinomas by binding its cellular ligand CD55. CD97(stalk) and CD55 could be useful as molecular markers for prognosis and therapy of gastric carcinoma patients.

Frequent promoter methylation of tumor-related genes in sporadic and men2-associated pheochromocytomas.

Hypermethylation of CpG island promoters is associated with transcriptional inactivation of tumor suppressor genes in neoplasia. Inactivation of p16 and Pten was related to the development of pheochromocytomas. In this report, we investigated the methylation status of the p16INK4a cell cycle inhibitor gene and other prominent tumor-related genes ( PTEN, RASSF1 A, CDH1, MSH2, MLH1, VHL, and TIMP3) in sporadic and multiple endocrine neoplasia type 2 (MEN2) pheochromocytomas by methylation-specific PCR. Hypermethylation was detected in 48 % of pheochromocytomas for RASSF1 A, 24 % for p16, 36 % for MSH2, 16 % for CDH1, and 8 % for PTEN. No VHL, MLH1, and TIMP3 methylation was observed. Interestingly, the frequency of p16 inactivation in familial tumors was higher (5 out of 12, 42 %) than in sporadic tumors (1 out of 13, 8 %; p = 0.047) and RASSF1 A inactivation was more common in the hereditary tumors (58 %) compared to the sporadic tumors (38 %). Combined methylation of RASSF1 A and p16 was found only in MEN2-related pheochromocytomas. Thus, a subset of hereditary pheochromocytomas displays preferential methylation of p16 and RASSF1 A.

[Role of the class II tumor suppressor gene maspin in thyroid carcinogenesis]. / Die rolle des Klasse II Tumorsuppressorgens Maspin in der thyreoidalen Karzinogenese.

The presented study was aimed to investigate new mechanisms of carcinogenesis in thyroids at the molecular level and to find potential protein markers involved in the initiation of the different histological subtypes of thyroid carcinoma. For this, we performed differential proteome analysis on primary cultured thyrocytes and transformed thyrocytes derived from 238Pu alpha-particle irradiation using two-dimensional electrophoresis (2-DE) and peptide mass fingerprinting (PMF) with matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS). Proteome analysis identified a strong upregulation of maspin, a serine protease inhibitor and class II tumor suppressor, in irradiated thyrocytes. To clarify the role of maspin in thyroid carcinogenesis, we searched for mRNA/protein expression in 30 normal (tumor-free) thyroid tissues, 35 follicular adenomas, 68 papillary carcinomas, 38 follicular carcinomas, 25 poorly differentiated carcinomas, and 34 undifferentiated carcinomas and compared the results with maspin promoter methylation status, p53 expression, clinicopathological data and prognosis. Maspin expression was detectable in 48 of 68 papillary carcinomas exclusively. There was a low methylation rate of 28% in papillary carcinomas in contrast to the other tissues (89-100%). p53 was positive in 2% of maspin positive cases, and in 80% of maspin negative cases. After 110 month follow-up 83% of the maspin positive patients had recurrence-free disease, whereas only 40% of the maspine negative patients were recurrence-free. Our data suggest: (1) maspin expression is a special feature of papillary thyroid carcinomas, (2) promotor methylation-caused maspin repression plays a major role in gene balance and in the process of tumor determination, (3) maspin protein possibly functions as a clinically relevant inhibitor of tumor progression, (4) our data delivers the hints for a p53-depentent regulatory pathway of the maspin protein in human cancer.

HRPT2 mutations are associated with malignancy in sporadic parathyroid tumours.

BACKGROUND: Hyperparathyroidism is a common endocrinopathy characterised by the formation of parathyroid tumours. In this study, we determine the role of the recently identified gene, HRPT2, in parathyroid tumorigenesis. METHODS: Mutation analysis of HRPT2 was undertaken in 60 parathyroid tumours: five HPT-JT, three FIHP, three MEN 1, one MEN 2A, 25 sporadic adenomas, 17 hyperplastic glands, two lithium associated tumours, and four sporadic carcinomas. Loss of heterozygosity at 1q24-32 was performed on a subset of these tumours. RESULTS: HRPT2 somatic mutations were detected in four of four sporadic parathyroid carcinoma samples, and germline mutations were found in five of five HPT-JT parathyroid tumours (two families) and two parathyroid tumours from one FIHP family. One HPT-JT tumour with germline mutation also harboured a somatic mutation. In total, seven novel and one previously reported mutation were identified. "Two-hits" (double mutations or one mutation and loss of heterozygosity at 1q24-32) affecting HRPT2 were found in two sporadic carcinomas, two HPT-JT-related and two FIHP related tumours. CONCLUSIONS: The results in this study support the role of HRPT2 as a tumour suppressor gene in sporadic parathyroid carcinoma, and provide further evidence for HRPT2 as the causative gene in HPT-JT, and a subset of FIHP. In light of the strong association between mutations of HRPT2 and sporadic parathyroid carcinoma demonstrated in this study, it is hypothesised that HRPT2 mutation is an early event that may lead to parathyroid malignancy and suggest intragenic mutation of HRPT2 as a marker of malignant potential in both familial and sporadic parathyroid tumours.

Nuclear factor-kappaB is constitutively active in C-cell carcinoma and required for RET-induced transformation.

Specific point mutations of the RET proto-oncogene have been demonstrated to be responsible for multiple endocrine neoplasia (MEN) types 2A and 2B, for familial medullary thyroid carcinoma (MTC) syndromes, as well as for sporadic MTC. Here we show that nuclear factor (NF)-kappaB is activated in RET-associated C-cell carcinoma specimens. TT cells, a human MTC cell line expressing MEN 2A type RET, display transcriptionally active RelA(p65) in the nucleus. NF-kappaB activity in these cells is attributable to constitutive IkappaB kinase (IKK) activity and high turn over of IkappaBalpha. RET harboring the mutations C634R (MEN 2A) or M918T (MEN 2B), in contrast to wild-type RET, activates a NF-kappaB-dependent reporter construct upon transient transfection in HeLa cells. We show that the prototype RET mutation C634R enhances phosphorylation of IkappaBalpha by IKKbeta but not by IKKalpha. RET-induced NF-kappaB and IKKbeta activity requires Ras function but does neither involve the classical mitogen-activated protein kinase kinase/extracellular signal-regulated kinase nor the phosphoinositide 3-kinase/Akt pathways. In contrast, RET-induced NF-kappaB activity is dependent on Raf and MEKK1. Inhibition of constitutive NF-kappaB activity results in cell death of TT cells and blocks focus formation induced by oncogenic forms of RET in NIH 3T3 cells. These results suggest that RET-mediated carcinogenesis critically depends on IKK activity and subsequent NF-kappaB activation.

Over-representation of a germline variant in the gene encoding RET co-receptor GFRalpha-1 but not GFRalpha-2 or GFRalpha-3 in cases with sporadic medullary thyroid carcinoma.

In contrast to the hereditary form of medullary thyroid carcinoma (MTC), little is known about the etiology of sporadic MTC. Somatic gain-of-function mutations in the RET proto-oncogene, encoding a receptor tyrosine kinase, are found in an average of 40% of sporadic MTC. We analysed 31 sporadic MTC for somatic and germline variants in GFRA1, GFRA2 and GFRA3 which encode the co-receptors of RET. Although there were no somatic mutations in any of the three genes, a sequence variant (-193C>G) in the 5'-UTR of GFRA1 was found in 15% of cases. Three patients were heterozygous (het); another three patients homozygous (hom) for the G variant. The G allele was not observed in 31 race-matched normal controls. Hence, the relative frequency of this variant in sporadic MTC cases and controls differed significantly (P<0.05). Since this variant lies in the 5' UTR, likely at the transcriptional start site, we analysed for differential expression of GFRalpha-1 at the transcript and protein levels. At the mRNA level, GFRA1 was over-expressed in tumors harboring the rare variant (P=0.06). The presence of the G polymorphic allele seemed to be associated with increased expression by immunostaining for GFRalpha-1. Interestingly, cytoplasmic staining was stronger in intensity for het patients and nuclear staining predominant in hom cases. In conclusion, mutation analysis of GFRA1, GFRA2 and GFRA3 revealed over-representation of a rare variant in GFRA1 (-193C>G) in the germline of sporadic MTC cases. Our data suggest that the mechanism is related to over-expression of GFRalpha-1 and differential subcellular compartmentalization but the precise mechanism as to how it acts as a low penetrance susceptibility allele for the development of sporadic MTC remains to be elucidated.

Mutation analysis of NTRK2 and NTRK3, encoding 2 tyrosine kinase receptors, in sporadic human medullary thyroid carcinoma reveals novel sequence variants.

Somatic mutations in the proto-oncogene RET are found in 25% to 80% of sporadic medullary thyroid carcinomas (MTCs). The significance of somatic RET mutation in MTC initiation and progression, however, remains unknown. Like RET, TRK is a neurotrophic receptor tyrosine kinase. Immunostaining has shown that only a subset of normal C cells expresses Trk family receptors, but in C-cell hyperplasia, they consistently express NTRK2, with variable expression of NTRK1 and NTRK3. In later stages of MTC, NTRK2 expression was reduced while NTRK3 expression was increased. In the context of these data, we sought to determine whether sequence variants in NTRK2 and NTRK3 are responsible for these differences in protein expression. We determined the genomic structure of NTRK2 and found that it consists of at least 17 exons varying in size from 36 to 306 bp. Mutation analysis of sporadic MTC did not reveal any sequence variants in NTRK2 but did reveal 3 variants in NTRK3, c.573C >T (N191N, exon 5), c.678T > C (N226N, exon 6) and c.1488C > G (A496A, exon 12) occurring among 19 chromosomes (31%), 1 chromosome (2%) and 24 chromosomes (39%), respectively. Corresponding germline also harbored these variants. There was a trend toward excess association of the NTRK3 variant c.1488C > G (A496A) in cases (24/62 chromosomes, 39%) compared to controls (18/62, 29%), but this difference did not reach significance (p > 0.05). The remaining 2 NTRK3 variants occurred with similar frequencies between MTC cases and population-matched controls (19 vs. 17 and 1 vs. 0, p > 0.05). We conclude that sequence variants in NTRK2 and NTRK3 are not likely to be responsible for large differences in expression at the protein level, but we cannot exclude very low penetrance effects.