TWIST1 Plays a Pleiotropic Role in Determining the Anaplastic Thyroid Cancer Phenotype

10 páginas, 5 figuras, 1 tabla.-- et al.[Context]:Anaplastic thyroid carcinoma (ATC) is one of the most aggressive human tumors; it is characterized by chemoresistance, local invasion, and distant metastases. ATC is invariably fatal. [Objective]:The aim was to study the role of TWIST1, a basic helix-loop-helix transcription factor, in ATC. [Design]:Expression of TWIST1 was studied by immunohistochemistry and real-time PCR in normal thyroids and well-differentiated, poorly differentiated, and ATC. The function of TWIST1 was studied by RNA interference in ATC cells and by ectopic expression in well-differentiated thyroid carcinoma cells. [Results]:ATCs up-regulate TWIST1 with respect to normal thyroids as well as to poorly and well-differentiated thyroid carcinomas. Knockdown of TWIST1 by RNA interference in ATC cells reduced cell migration and invasion and increased sensitivity to apoptosis. The ectopic expression of TWIST1 in thyroid cells induced resistance to apoptosis and increased cell migration and invasion. [Conclusions]:TWIST1 plays a key role in determining malignant features of the anaplastic phenotype in vitro.This work was supported by the Associazione Italiana per la Ricerca sul Cancro, the Istituto Superiore di Oncologia, the Italian Ministero della Salute, the Ministero dell’Università e della Ricerca, the European Community Contract FP6-36495, and the Programa Ramón y Cajal–Ministerio de Ciencia e Innovación, Social EU Funds, Universidad de Valladolid, Spain.Peer reviewe

Functional relevance of particular residues within the BRAF kinase domain in anaplastic thyroid carcinoma

Resumen del trabajo presentado al 103rd Annual Meeting of the American Association for Cancer Research celebrado en Chicago (US) del 31 de marzo al 4 de abril de 2012.-- et al.The BRAFV600E point mutation represents the most common oncogenic event in sporadic papillary thyroid cancer (PTC) pathogenesis. Although at a much lower frequency, different missense point mutations (K601E, G474R, G469R) and complex genetic alterations, such as rearrangements (AKAP9-BRAF) or frameshift mutations (V600E-K601del, V599ins, V600D-FLAGT601-605ins, T599I-VKSRdel), have also been reported in PTC. In contrast with PTC, no other alteration different from the V600E substitution has been shown in anaplastic thyroid carcinomas (ATC). In this study, we sought to determine the functional relevance of particular amino acid residues, within the BRAF kinase domain, that we found mutated in some ATC. Fifty-four ATC were investigated for the existence of mutations at the activation loop, the glicine-rich phosphate-binding loop and the AKT binding sites of BRAF by means of PCR-SSCP and direct sequence. The structural-conformational changes behind punctual, unreported BRAF mutations and their effect on BRAF catalytic activity, substrate binding and downstream MAPK signalling were evaluated. Besides the V600E mutation, two additional point mutations (G615E and G474R) were detected within the BRAF kinase domain. The prevalence of both mutations, G615E (11% = 6 ATC) and G474R (7.4% = 4 ATC), was much lower than that of V600E (20% = 11 ATC). Interestingly, structural biology studies showed that G615E and G474R induced profound conformational changes within the BRAF kinase domain that resulted in reduced catalytic activity, decreased substrate binding and impaired downstream signalling. The G474R mutant, by preventing the N lobe from folding into a well ordered beta sheet structure, caused a broken or distorted phosphorylation-loop (P-loop). Because the P-loop is pivotal for ATP coordination and phosphotransfer reactions, the kinase activity of the BRAF G474R mutant was severely compromised and BRAF was unable to phosphorylate the downstream substrate. The G615E mutant by disrupting the structural integrity of the activation segment in the C lobe could in addition inhibit substrate binding. Significantly, none of the ATC bearing the G615E or the G474R mutation expressed the phospho-p44/42 MAPK (Thr202/Tyr204), which specifically recognizes the dually phosphorylated and active forms of ERK1 and ERK2. This observation not only supported the existence of impaired BRAF kinase activity in vivo but also suggested the abrogation of intracellular MEK/ERK signalling in those tumours. In summary, this study demonstrates that G615E and G474R behave similarly to D594V and the kinase-dead BRAFK483M mutant, knocking-down BRAF catalytic activity and downstream MEK/ERK signalling. These results should significantly impact on the rationale of future treatment modalities for patients with deadly ATC. Clinical trials involving the inhibitor of threonine/serine kinases Sorafenib or kinase specific inhibitors targeting constitutively active BRAF will prove inefficient therapeutic strategies in patients with G615E or G474R mutations.Peer Reviewe

Anaplastic thyroid carcinoma: Molecular tools for diagnosis and therapy

This is an open access article distributed under the Creative Commons Attribution License.-- Editorial.Anaplastic thyroid carcinoma (ATC) is one of the most lethal human malignancies, with a median overall survival of less than six months. While most of the genetic mutations occurring in papillary thyroid carcinoma (PTC) were recently discovered through an integrated genomic approach, molecular genetics of ATC is still in part unknown.Peer Reviewe

FOXM1 is a molecular determinant of the mitogenic and invasive phenotype of anaplastic thyroid carcinoma

PMID:22919068.-- et al.Anaplastic thyroid carcinoma (ATC) is a very aggressive thyroid cancer. forkhead box protein M1 (FOXM1) is a member of the forkhead box family of transcription factors involved in control of cell proliferation, chromosomal stability, angiogenesis, and invasion. Here, we show that FOXM1 is significantly increased in ATCs compared with normal thyroid, well-differentiated thyroid carcinomas (papillary and/or follicular), and poorly differentiated thyroid carcinomas (P=0.000002). Upregulation of FOXM1 levels in ATC cells was mechanistically linked to loss-of-function of p53 and to the hyperactivation of the phosphatidylinositol-3-kinase/AKT/FOXO3a pathway. Knockdown of FOXM1 by RNA interference inhibited cell proliferation by arresting cells in G2/M and reduced cell invasion and motility. This phenotype was associated with decreased expression of FOXM1 target genes, like cyclin B1 (CCNB1), polo-like kinase 1 (PLK1), Aurora B (AURKB), S-phase kinase-associated protein 2 (SKP2), and plasminogen activator, urokinase: uPA (PLAU). Pharmacological inhibition of FOXM1 in an orthotopic mouse model of ATC reduced tumor burden and metastasization. All together, these findings suggest that FOXM1 represents an important player in thyroid cancer progression to the anaplastic phenotype and a potential therapeutic target for this fatal cancer.This study was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC), the Ministero dell'Università e della Ricerca (MiUR), and by the grant MERIT of MIUR. G Garcia-Rostan is supported by Programa Ramón y Cajal, Ministerio de Ciencia e Innovación, Social EU Funds, Universidad de Valladolid, Spain. C Nucera (Principal Investigator, Program: Human Thyroid Cancers Preclinical and Translational Research) was funded by the NIHR21CA165039-01A1 and the American Thyroid Association for Thyroid Cancer Research.Peer Reviewe

Poorly differentiated thyroid carcinoma: an evolving entity

10 páginas, 3 figuras.-- Mini-Symposium: Thyroid Pathology.The term Poorly Differentiated Thyroid Carcinoma (PDTC) was first proposed in the early eighties to describe a heterogeneous group of neoplasms, of follicular cell origin, dwelling in an intermediate clinicopathological position between well-differentiated follicular or papillary carcinomas and undifferentiated carcinomas. Over the following two decades defenders and sceptics have sustained an unsurmountable debate pertaining the nature of PDTC, its morphological diagnostic features, its clinical relevance and the most suitable management. In 2004, the WHO Committee on Thyroid Tumours agreed to recognize PDTCs as a distinct entity in thyroid follicular cell tumourigenesis. The proposed diagnostic criteria resulted however difficult to apply on daily practice. Four years ago, in an attempt to streamline the diagnosis of PDTC, a panel of experts met in Turin and developed a simple algorithmic approach for PDTC identification. This paper aims to review the state of the art on PDTC from a morphological, clinical and molecular standpoint.This work was supported by grant from Programa Ram ón y Cajal - Ministerio de Ciencia e Innovación – Social EU Funds - Universidad de Valladolid, Spain to G. G-R and grant from Fundaçâo Calouste Gulbenkian to M. S-S.Peer reviewe

CD97 amplifies LPA receptor signaling and promotes thyroid cancer progression in a mouse model

et al.CD97, a member of the adhesion family of G-protein-coupled receptors (GPCRs), complexes with and potentiates lysophosphatidic acid (LPA) receptor signaling to the downstream effector RHOA. We show here that CD97 was expressed in a majority of thyroid cancers but not normal thyroid epithelium and that the level of CD97 expression was further elevated with progression to poorly differentiated and undifferentiated carcinoma. Intratumoral progression also showed that CD97 expression correlates with invasiveness and dedifferentiation. To determine the functional role of CD97, we produced a transgenic model of thyroglobulin promoter-driven CD97 expression. Transgenic CD97 in combination with Thrb(PV), an established mouse model of thyroid follicular cell carcinogenesis, significantly increased the occurrence of vascular invasion and lung metastasis. Expression of transgenic CD97 in thyroid epithelium led to elevated ERK phosphorylation and increased numbers of Ki67+ cells in developing tumors. In addition, tumor cell cultures derived from CD97 transgenic as compared with non-transgenic mice demonstrated enhanced, constitutive and LPA-stimulated ERK activation. In human thyroid cancer cell lines, CD97 depletion reduced RHO-GTP and decreased LPA-stimulated invasion but not EGF-stimulated invasion, further suggesting that CD97 influences an LPA-associated mechanism of progression. Consistent with the above, CD97 expression in human thyroid cancers correlated with LPA receptor and markers of aggressiveness including Ki67 and pAKT. This study shows an autonomous effect of CD97 on thyroid cancer progression and supports the investigation of this GPCR as a therapeutic target for these cancers.This work was supported by the Intramural Research Program, Center for Cancer Research, National Cancer Institute and the Programa Ramón y Cajal—Ministerio de Ciencia e Innovación, Social EU Funds, Universidad de Valladolid, Spain.Peer Reviewe

Usefulness of VE1 immunohistochemical detection of BRAFV600E in agggressive thyroid cancers (PDCs and UCs)

Resumen del póster presentado al 107th Annual Meeting of the American Associationfor Cancer Research, celebrado en New Orleans, LA (US) del 16 al 20 de abril de 2016.-- et al.Activating BRAF mutations are frequent in thyroid follicular cell carcinogenesis. The BRAFV600E point mutation represents the most common oncogenic event in sporadic papillary thyroid cancer (PTC). Although at a much lower frequency the BRAFV600E mutation is also present among less differentiated, more aggressive, I131 resistant forms of thyroid cancer as poorly differentiated carcinomas (PDCs) and undifferentiated carcinomas (UCs). Small molecule inhibitors targeting either the BRAF V600E protein or upstream or downstream kinases involved in MAPK signalling are currently under pre-clinical or clinical investigation in advanced, metastatic, I131 resistant thyroid cancers. Recently, immunohistochemical (IHC) studies on PTCs, using the VE1 mouse anti-human BRAF V600E antibody have shown to be a reliable means for detecting the BRAFV600E mutation in a clinical setting without molecular genotyping units. In this study we sought to determine the usefulness of the VE1 antibody for detecting the BRAF V600E mutant protein in a series of 103 aggressive thyroid cancers (59 PDCs and 44 UCs) previously characterized by PCR-SSCP for the presence of BRAF mutations in exons 11 and 15. The BRAFV600E mutation was present in 10/59 PDCs (17%) and 11/44 UCs (25%). Immunohistochemistry revealed 9 mutated PDCs (sensitivity 90%) and 9 mutated UCs (82% sensitivity). The staining intensity in BRAF V600E mutated samples ranged from weak to strong. Non valuable results were found in 3 PDCs and 4 UCs. Overall the results indicate that immunohistochemistry with VE1 antibody may be an alternative to molecular biology approaches for the routine detection of BRAFV600E point mutations in clinical settings without molecular genotyping facilities. It may help clinicians in targeted therapy decision making.Peer Reviewe

A new role of the Rac-GAP ß2-chimaerin in cell adhesion reveals opposite functions in breast cancer initiation and tumor progression

β2-chimaerin is a Rac1-specific negative regulator and a candidate tumor suppressor in breast cancer but its precise function in mammary tumorigenesis in vivo is unknown. Here, we study for the first time the role of β2-chimaerin in breast cancer using a mouse model and describe an unforeseen role for this protein in epithelial cell-cell adhesion. We demonstrate that expression of β2-chimaerin in breast cancer epithelial cells reduces E-cadherin protein levels, thus loosening cell-cell contacts. In vivo, genetic ablation of β2-chimaerin in the MMTV-Neu/ErbB2 mice accelerates tumor onset, but delays tumor progression. Finally, analysis of clinical databases revealed an inverse correlation between β2-chimaerin and E-cadherin gene expressions in Her2+ breast tumors. Furthermore, breast cancer patients with low β2-chimaerin expression have reduced relapse free survival but develop metastasis at similar times. Overall, our data redefine the role of β2-chimaerin as tumor suppressor and provide the first in vivo evidence of a dual function in breast cancer, suppressing tumor initiation but favoring tumor progression.This work was initially supported by a grant from the Spanish Ministry of Economy and Competitiveness to MJC (BFU2009-08051), but could be finished only thanks to the support from the Castilla-León Autonomous Government to MJC (grants BIO103/VA44/11, BIO/VA22/14, CSI090U14 and BIO/VA34/15).Peer Reviewe