Departamento de Patologia e Oncologia

Mitochondria are central organelles for cellular metabolism. In cancer cells, mitochondrial oxidative phosphorylation (OXPHOS) dysfunction has been shown to promote migration, invasion, metastization and apoptosis resistance. With the purpose of analysing the effects of OXPHOS dysfunction in cancer cells and the molecular players involved, we generated cybrid cell lines harbouring either wild-type (WT) or mutant mitochondrial DNA (mtDNA) [tRNAmut cybrids, which harbour the pathogenic A3243T mutation in the leucine transfer RNA gene (tRNAleu)]. tRNAmut cybrids exhibited lower oxygen consumption and higher glucose consumption and lactate production than WT cybrids. tRNAmut cybrids displayed increased motility and migration capacities, which were associated with altered integrin-β1 N-glycosylation, in particular with higher levels of β-1,6-N-acetylglucosamine (GlcNAc) branched N-glycans. This integrin-β1 N-glycosylation pattern was correlated with higher levels of membrane-bound integr...

The presence of somatic mitochondrial DNA (mtDNA) mutations in cancer cells has been interpreted in controversial ways, ranging from random neutral accumulation of mutations, to positive selection for high pathogenicity, or conversely to purifying selection against high pathogenicity variants as occurs at the population level. Methods: Here we evaluated the predicted pathogenicity of somatic mtDNA mutations described in cancer and compare these to the distribution of variations observed in the global human population and all possible protein variations that could occur in human mtDNA. We focus on oncocytic tumors, which are clearly associated with mitochondrial dysfunction. The protein variant pathogenicity was predicted using two computational methods, MutPred and SNPs&GO. Results: The pathogenicity score of the somatic mtDNA variants were significantly higher in oncocytic tumors compared to non-oncocytic tumors. Variations in subunits of Complex I of the electron transfer chain were significantly more common in tumors with the oncocytic phenotype, while variations in Complex V subunits were significantly more common in non-oncocytic tumors.

GRIM-19, a gene associated with retinoid interferoninduced mortality, was originally identified as a critical regulatory protein for interferon-b and retinoic acid-induced cell death. It was also demonstrated that GRIM-19 is involved in mitochondrial metabolism, as an integrant component of complex I of the mitochondrial respiratory chain. GRIM-19 appears, therefore, as a dual function protein involved in cell death and mitochondrial metabolism. GRIM-19 knock out leads to Complex I assembly disruption and embryonic lethality in mice, showing that it is a crucial component of the mitochondrial respiratory chain essential for early embryonic development. Recently, mutations in GRIM-19 were described in Hu¨rthle cell (mitochondrion-rich) tumors of the thyroid and down-regulation or loss of its expression were found in renal cell carcinomas, suggesting a role for GRIM-19 in tumorigenesis. As GRIM-19 binds and inhibits the signal transducer and activator of transcription-3 (STAT3), which has been shown to be activated in several human tumors it is tempting to advance that GRIM-19 may function as a tumor suppressor gene in tumors in which STAT3 plays a major role.

In an attempt to progress in the understanding of the relationship of mitochondrial DNA (mtDNA) alterations and thyroid tumorigenesis, we studied the mtDNA in 79 benign and malignant tumors (43 Hürthle and 36 non-Hürthle cell neoplasms) and respective normal parenchyma. The mtDNA common deletion (CD) was evaluated by semiquantitative polymerase chain reaction. Somatic point mutations and sequence variants of mtDNA were searched for in 66 tumors (59 patients) and adjacent parenchyma by direct sequencing of 70% of the mitochondrial genome (including all of the 13 OXPHOS system genes). We detected 57 somatic mutations, mostly transitions, in 34 tumors and 253 sequence variants in 59 patients. Follicular and papillary carcinomas carried a significantly higher prevalence of nonsilent point mutations of complex I genes than adenomas. We also detected a significantly higher prevalence of complex I and complex IV sequence variants in the normal parenchyma adjacent to the malignant tumors. Every Hürthle cell tumor displayed a relatively high percentage (up to 16%) of mtDNA CD independently of the lesion's histotype. The percentage of deleted mtDNA molecules was significantly higher in tumors with D-loop mutations than in mtDNA stable tumors. Sequence variants of the ATPase 6 gene, one of the complex V genes thought to play a role in mtDNA maintenance and integrity in yeast, were significantly more prevalent in patients with Hürthle cell tumors than in patients with non-Hürthle cell neoplasms. We conclude that mtDNA variants and mtDNA somatic mutations of complex I and complex IV genes seem to be involved in thyroid tumorigenesis. Germline polymorphisms of the ATPase 6 gene are associated with the occurrence of mtDNA CD, the hallmark of Hürthle cell tumors.

In order to investigate the cell death-inducing effects of rotenone, a plant extract commonly used as a mitochondrial complex I inhibitor, we studied cancer cell lines with different genetic backgrounds. Rotenone inhibits cell growth through the induction of cell death and cell cycle arrest, associated with the development of mitotic catastrophe. The cell death inducer staurosporine potentiates the inhibition of cell growth by rotenone in a dose-dependent synergistic manner. The tumor suppressor p53 is involved in rotenone-induced cell death, since the drug treatment results in increased expression, phosphorylation and nuclear localization of the protein.

Purpose: Germline SDHB, SDHC, and/or SDHD mutations have been reported in familial and apparently sporadic paragangliomas (PGLs). There is, however, some variation in the prevalence, penetrance, and phenotypic expression of the succinate dehydrogenase (SDH) mutated gene among different populations. We sought to determine whether germline mutations in SDHB, SDHC, and/or SDHD play a role in cervical PGLs from northern Spain, where this disorder is particularly frequent, and whether there is any difference with respect to the data published in other populations.

The BRAF gene has been shown to be a major target for mutations in papillary thyroid carcinoma (PTC) (36 -69%), which forms almost all of the over 2000 cases of thyroid carcinoma that have occurred in Chernobyl. BRAF is activated by point mutation, and were it to occur at a high frequency in Chernobyl-related tumors, it would challenge the dominant role of double-strand breaks in radiation-induced PTC. In a previous study, we detected the BRAF V600E mutation in 46% (23 of 50) of sporadic adult PTC. Using the same methodology, we have analyzed 34 post-Chernobyl PTC and detected RET/PTC rearrangements in 14 (41%) and BRAF mutations (V600E) in four (12%). These two alterations did not coexist in any PTCs. The mean age at exposure of patients with PTC showing BRAF mutation was higher than that of patients with tumors without BRAF mutation irrespective of their RET status. We have also analyzed 17 sporadic cases of childhood PTC and found that only one (6%) harbored the BRAF V600E mutation. We conclude that the frequency of BRAF mutations is significantly lower (P ‫؍‬ 0.0008) in post-Chernobyl PTC than in adult sporadic PTC, whereas no significant difference was found between post-Chernobyl and sporadic childhood PTCs. (J Clin Endocrinol Metab 89: 4267-4271, 2004) Abbreviations: FA, Follicular adenoma; PTC, papillary thyroid carcinoma; SSCP, single strand conformation polymorphism. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

Despite the numerous studies describing a high frequency of mitochondrial DNA (mtDNA) somatic mutations in many types of human primary tumors the mechanisms that generate such mutations and the role of mtDNA mutations in tumor development remain unclear. We present the results obtained in the study of mtDNA displacement-loop (D-Loop) region in a series of 66 thyroid tumors, and respective adjacent parenchyma, including benign (adenomas, nZ30) and malignant tumors (follicular carcinomas, nZ17 and papillary carcinomas, nZ19). Three repetitive regions were analyzed [two mononucleotide repetitive (D310 and D568) and one dinucleotide repetitive ]. Thirty-two (48.5%) of the 66 tumors [15/30 (50.0%) adenomas, 8/17 (47.1%) follicular carcinomas and 9/19 (47.4%) papillary carcinomas] harbored somatic insertions in D-Loop repetitive regions. Twenty (30.3%) of the 66 tumors [12/30 (40%) adenomas, 3/17 (17.6%) follicular carcinomas and 5/19 (26.3%) papillary carcinomas] harbored somatic insertions at the D310 mononucleotide repeat. Three (4.6%) of the 66 tumors [1/30 (3.3%) adenomas and 2/17 (11.8%) follicular carcinomas] harbored somatic insertions at the D568 mononucleotide repeat. Fifteen (22.7%) of the 66 tumors [3/30 (10.0%) adenomas, 5/17 (29.4%) follicular carcinomas and 7/19 (36.8%) papillary carcinomas] harbored somatic insertions at the D514 dinucleotide repeat. Five (7.6%) of the 66 tumors [1/30 (3.3%) adenomas, 1/17 (5.9%) follicular carcinomas and 2/19 (10.5%) papillary carcinomas] harbored somatic insertions in more than one region, and in one of them (a carcinoma) alterations were detected in the three regions. We conclude that mutations in the mtDNA D-Loop region are frequent in benign and malignant thyroid tumors and cannot be considered a marker of malignancy. Our study shows, furthermore, two repetitive regions (D310 and D514) that appear to be susceptible to mutation in thyroid tumors. q

Rearrangement of RET proto-oncogene is the major event in the etiopathogenesis of papillary thyroid carcinoma (PTC). We report a high prevalence of BRAF V599E mutation in sporadic PTC and in PTC-derived cell lines. The BRAF V599E mutation was detected in 23 of 50 PTC (46%) and in three of four PTC-derived cell lines. The prevalence of the BRAF V599E mutation in PTC is the highest reported to date in human carcinomas, being only exceeded by melanoma. PTC with RET/PTC rearrangement as well as the TPC-1 cell line (the only one harboring RET/PTC rearrangement) did not show the BRAF V599E mutation. BRAF V599E mutation was not detected in any of 23 nodular goiters, 51 follicular adenomas and 18 follicular carcinomas. A distinct mutation in BRAF (codon K600E) was detected in a follicular adenoma. Activating mutations in RAS genes were detected in 15% of FA, 33% of FTC and 7% of PTC. BRAF V599E mutation did not coexist with alterations in any of the RAS genes in any of the tumors. These results suggest that BRAF V599E mutation is frequent in the etiopathogenesis of PTC. The BRAF V599E mutation appears to be an alternative event to RET/PTC rearrangement rather than to RAS mutations, which are rare in PTC. BRAF V599E may represent an alternative pathway to oncogenic MAPK activation in PTCs without RET/PTC activation.

Type and prevalence of BRAF mutations are closely associated with papillary thyroid carcinoma histotype and patients' age but not with tumour aggressiveness Abstract A high prevalence of the BRAF V600E somatic mutation was recently reported in several series of papillary thyroid carcinomas (PTC). This mutation appears to be particularly prevalent in PTC with a predominantly papillary architecture. Another BRAF mutation (K601E) was detected in a follicular adenoma and in some cases of the follicular variant of PTC. The few studies on record provided controversial data on the relationship between the occurrence of BRAF mutations and clinicopathologic parameters such as gender, age and tumour staging. In an attempt to clarify such controversies we decided to enlarge our previous series to 315 tumours or tumour-like lesions diagnosed in 280 patients, including a thorough analysis of several clinicopathologic features. The BRAF V600E mutation was exclusively detected in PTC with a papillary or mixed follicular/papillary architecture both of the conventional type (46%) and of other histotypes, such as microcarcinoma (43%), Warthin-like PTC (75%) and oncocytic variant of PTC (55%). The BRAF K601E mutation was detected in four of the 54 cases of the follicular variant of PTC (7%). The mean age of patients with conventional PTC harbouring BRAF V600E (46.7 years) was significantly higher (P<0.0001) than that of patients with conventional PTC without BRAF V600E (29.5 years). The BRAF (BRAF V600E ) mutated PTC did not exhibit signs of higher aggressiveness (size, vascular invasion, extra-thyroid extension and nodal metastasis) and were in fact less often multicentric than PTC without the mutation.

C cell hyperplasia is associated with medullary carcinoma of the thyroid in the inherited MEN2 syndromes, in which the great majority of cases have been shown to be due to a mutation in the RET oncogene. We report a study of a family with C cell hyperplasia and hypercalcitoninemia in which no cases of medullary carcinoma have yet occurred and which lacked an identifiable causative RET mutation. Four of the family members showed hypercalcitoninemia, and marked C cell hyperplasia was present in each of the three in whom thyroidectomy has been performed. We investigated the possible involvement of the SDHD gene, because somatic and germline mutations in this gene have been found in a variety of tumors of neural crest-derived tissue. A germline mutation in exon 2 of the SDHD gene (c149 A-G, His 50 Arg) was found in six members of the family; all the four available members with hypercalcitoninemia possessed the mutation. One of the five available members without hypercalcitoninemia, an 1...

Our findings both support and extend those of Nikiforova et al. [M.N. Nikiforova, R. Ciampi, G. Salvatore, M. Santoro, M. Gandhi, J.A. Knauf, et al., Low prevalence of BRAF mutations in radiation-induced thyroid tumors in contrast to sporadic papillary carcinomas, Cancer Lett. 209 (2004) 1–6]: BRAF mutations are rare in childhood PTC, both in an irradiation setting and in sporadic tumors.

Papillary thyroid carcinoma (PTC) is the most prevalent type of endocrine cancer and, in recent epidemiological surveys, one of the types of human cancer whose incidence is growing. Despite the favourable outcome and long survival rates of most patients, some tumours display an aggressive behaviour and may progress to the highly aggressive and lethal, anaplastic thyroid carcinoma. In recent years, several progresses have been made on the molecular characterization of PTC, in general, and in the genetic alterations underlying the histotype diversity of this type of cancer, in particular. This holds true regarding alterations on nuclear DNA as well as mitochondrial DNA. In this review we have summarized the most recent findings in the genetic characterization of PTC, giving a particular emphasis to the genotype-phenotype associations, the prognosis implications, and the diagnostic and therapeutic value of the newly identified genetic markers. (Arq Bras Endocrinol Metab 2007;51/5:643-653) RESUMO Genética Molecular do Carcinoma Papilífero de Tireóide -Grandes Esperanças… O carcinoma papilífero de tireóide (CPT) é o tipo mais prevalente de câncer endócrino e, em pesquisas epidemiológicas recentes, um dos tipos de câncer humano cuja incidência vêm crescendo. A despeito do prognóstico favorável e da longa taxa de sobrevivência da maioria dos pacientes, alguns tumores mostram um comportamento agressivo e podem progredir para o altamente agressivo e letal carcinoma anaplásico de tireóide.

Mutations in the PKLR gene responsible for pyruvate kinase (PK)-deficient anaemia are mainly located in the coding regions: 11 are in the splicing sites and, recently, three mutations have been described in the promoter region. We now report a novel point mutation A3G on nucleotide 72, upstream from the initiation codon of the PKLR gene, in four Portuguese PK-deficient patients. This new regulatory mutation occurs within the most proximal of the four GATA motifs (GATA-A element) in the R-type promoter region. In two patients who were homozygous for this mutation, a semiquantitative reverse transcription polymerase chain reaction (PCR) procedure was used to evaluate the amount of R-PK mRNA transcript in the reticulocytes. The mRNA level was about five times