Proximity-based activation of AURORA A by MPS1 potentiates error correction.

Faithfull cell division relies on mitotic chromosomes becoming bioriented with each pair of sister kinetochores bound to microtubules oriented toward opposing spindle poles. Erroneous kinetochore-microtubule attachments often form during early mitosis, but are destabilized through the phosphorylation of outer kinetochore proteins by centromeric AURORA B kinase (ABK) and centrosomal AURORA A kinase (AAK), thus allowing for re-establishment of attachments until biorientation is achieved. MPS1-mediated phosphorylation of NDC80 has also been shown to directly weaken the kinetochore-microtubule interface in yeast. In human cells, MPS1 has been proposed to transiently accumulate at end-on attached kinetochores and phosphorylate SKA3 to promote microtubule release. Whether MPS1 directly targets NDC80 and/or promotes the activity of AURORA kinases in metazoans remains unclear. Here, we report a novel mechanism involving communication between kinetochores and centrosomes, wherein MPS1 acts upstream of AAK to promote error correction. MPS1 on pole-proximal kinetochores phosphorylates the C-lobe of AAK thereby increasing its activation at centrosomes. This proximity-based activation ensures the establishment of a robust AAK activity gradient that locally destabilizes mal-oriented kinetochores near spindle poles. Accordingly, MPS1 depletion from Drosophila cells causes severe chromosome misalignment and erroneous kinetochore-microtubule attachments, which can be rescued by tethering either MPS1 or constitutively active AAK mutants to centrosomes. Proximity-based activation of AAK by MPS1 also occurs in human cells to promote AAK-mediated phosphorylation of the NDC80 N-terminal tail. These findings uncover an MPS1-AAK cross-talk that is required for efficient error correction, showcasing the ability of kinetochores to modulate centrosome outputs to ensure proper chromosome segregation.

CHEK2 germline variants identified in familial nonmedullary thyroid cancer lead to impaired protein structure and function.

Approximately 5 to 15% of nonmedullary thyroid cancers (NMTC) present in a familial form (familial nonmedullary thyroid cancers [FNMTC]). The genetic basis of FNMTC remains largely unknown, representing a limitation for diagnostic and clinical management. Recently, germline mutations in DNA repair-related genes have been described in cases with thyroid cancer (TC), suggesting a role in FNMTC etiology. Here, two FNMTC families were studied, each with two members affected with TC. Ninety-four hereditary cancer predisposition genes were analyzed through next-generation sequencing, revealing two germline CHEK2 missense variants (c.962A > C, p.E321A and c.470T > C, p.I157T), which segregated with TC in each FNMTC family. p.E321A, located in the CHK2 protein kinase domain, is a rare variant, previously unreported in the literature. Conversely, p.I157T, located in CHK2 forkhead-associated domain, has been extensively described, having conflicting interpretations of pathogenicity. CHK2 proteins (WT and variants) were characterized using biophysical methods, molecular dynamics simulations, and immunohistochemistry. Overall, biophysical characterization of these CHK2 variants showed that they have compromised structural and conformational stability and impaired kinase activity, compared to the WT protein. CHK2 appears to aggregate into amyloid-like fibrils in vitro, which opens future perspectives toward positioning CHK2 in cancer pathophysiology. CHK2 variants exhibited higher propensity for this conformational change, also displaying higher expression in thyroid tumors. The present findings support the utility of complementary biophysical and in silico approaches toward understanding the impact of genetic variants in protein structure and function, improving the current knowledge on CHEK2 variants' role in FNMTC genetic basis, with prospective clinical translation.

Identification of Germline FOXE1 and Somatic MAPK Pathway Gene Alterations in Patients with Malignant Struma Ovarii, Cleft Palate and Thyroid Cancer.

Germline variants in the FOXE1 transcription factor have been associated with thyroid ectopy, cleft palate (CP) and thyroid cancer (TC). Here, we aimed to clarify the role of FOXE1 in Portuguese families (F1 and F2) with members diagnosed with malignant struma ovarii (MSO), an ovarian teratoma with ectopic malignant thyroid tissue, papillary TC (PTC) and CP. Two rare germline heterozygous variants in the FOXE1 promoter were identified: F1) c.-522G>C, in the proband (MSO) and her mother (asymptomatic); F2) c.9C>T, in the proband (PTC), her sister and her mother (CP). Functional studies using rat normal thyroid (PCCL3) and human PTC (TPC-1) cells revealed that c.9C>T decreased FOXE1 promoter transcriptional activity in both cell models, while c.-522G>C led to opposing activities in the two models, when compared to the wild type. Immunohistochemistry and RT-qPCR analyses of patients' thyroid tumours revealed lower FOXE1 expression compared to adjacent normal and hyperplastic thyroid tissues. The patient with MSO also harboured a novel germline AXIN1 variant, presenting a loss of heterozygosity in its benign and malignant teratoma tissues and observable ß-catenin cytoplasmic accumulation. The sequencing of the F1 (MSO) and F2 (PTC) probands' tumours unveiled somatic BRAF and HRAS variants, respectively. Germline FOXE1 and AXIN1 variants might have a role in thyroid ectopy and cleft palate, which, together with MAPK pathway activation, may contribute to tumours' malignant transformation.

Preoperative serum inflammation-based scores in medullary thyroid cancer.

INTRODUCTION: Neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) and systemic immune-inflammation index (SII) are prognostic factors in several tumours, though little is known in medullary thyroid cancer (MTC). OBJECTIVE: To evaluate the association between preoperative NLR, PLR and SII with MTC clinicopathological and molecular features, and their predictive value for lymph node and distant metastasis. METHODS: We retrospectively analysed 75 patients with MTC who underwent surgery at our institution. The familial form of MTC was found in 12% of patients. RESULTS: In our cohort, 56% were females, the median age at diagnosis was 57 years (44-69), the median tumour diameter was 25mm (15-50); 21.3% were multifocal and 34.7% had extrathyroidal extension. Lymph node and distant metastasis were observed in 36 (48.0%) and 8 (10.7%) patients, respectively. Higher NLR was associated with preoperative calcitonin, angioinvasion, extrathyroidal extension, moderate/severe fibrosis; higher PLR was associated with extrathyroidal extension and advanced T stages; lower SII and NLR were associated with biochemical cure after surgery. Increased PLR, NLR and SII were associated with advanced MTC stages. In the univariate analysis, only NLR was associated with lymph node metastasis (odds ratio (OR)=2.69, 95% confidence interval (CI): 1.50-5.84; p=0.004); however, in the multivariate model, NLR was no longer a predictive factor for lymph node metastasis. None of these serum inflammatory markers predicted the occurrence of distant metastasis. CONCLUSION: In conclusion, NLR, PLR and SII are associated with aggressive MTC, but do not predict lymph node or distant metastasis.

Identification of SPRY4 as a Novel Candidate Susceptibility Gene for Familial Nonmedullary Thyroid Cancer.

Background: The molecular basis of familial nonmedullary thyroid cancer (FNMTC) is still poorly understood, representing a limitation for molecular diagnosis and clinical management. In this study, we aimed to identify new susceptibility genes for FNMTC through whole-exome sequencing (WES) analysis of leukocyte DNA of patients from a highly informative FNMTC family. Methods: We selected six affected family members to conduct WES analysis. Bioinformatic analyses were undertaken to filter and select the genetic variants shared by the affected members, which were subsequently validated by Sanger sequencing. To select the most likely pathogenic variants, several studies were performed, including family segregation analysis, in silico impact characterization, and gene expression (messenger RNA and protein) depiction in databases. For the most promising variant identified, we performed in vitro studies to validate its pathogenicity. Results: Several potentially pathogenic variants were identified in different candidate genes. After filtering with appropriate criteria, the variant c.701C>T, p.Thr234Met in the SPRY4 gene was prioritized for in vitro functional characterization. This SPRY4 variant led to an increase in cell viability and colony formation, indicating that it confers a proliferative advantage and potentiates clonogenic capacity. Phosphokinase array and Western blot analyses suggested that the effects of the SPRY4 variant were mediated through the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway, which was further supported by a higher responsiveness of thyroid cancer cells with the SPRY4 variant to a MEK inhibitor. Conclusions: WES analysis in one family identified SPRY4 as a likely novel candidate susceptibility gene for FNMTC, allowing a better understanding of the cellular and molecular mechanisms underlying thyroid cancer development.

Phosphatases in Mitosis: Roles and Regulation.

Mitosis requires extensive rearrangement of cellular architecture and of subcellular structures so that replicated chromosomes can bind correctly to spindle microtubules and segregate towards opposite poles. This process originates two new daughter nuclei with equal genetic content and relies on highly-dynamic and tightly regulated phosphorylation of numerous cell cycle proteins. A burst in protein phosphorylation orchestrated by several conserved kinases occurs as cells go into and progress through mitosis. The opposing dephosphorylation events are catalyzed by a small set of protein phosphatases, whose importance for the accuracy of mitosis is becoming increasingly appreciated. This review will focus on the established and emerging roles of mitotic phosphatases, describe their structural and biochemical properties, and discuss recent advances in understanding the regulation of phosphatase activity and function.

Protein Phosphatase 1 inactivates Mps1 to ensure efficient Spindle Assembly Checkpoint silencing.

Faithfull genome partitioning during cell division relies on the Spindle Assembly Checkpoint (SAC), a conserved signaling pathway that delays anaphase onset until all chromosomes are attached to spindle microtubules. Mps1 kinase is an upstream SAC regulator that promotes the assembly of an anaphase inhibitor through a sequential multi-target phosphorylation cascade. Thus, the SAC is highly responsive to Mps1, whose activity peaks in early mitosis as a result of its T-loop autophosphorylation. However, the mechanism controlling Mps1 inactivation once kinetochores attach to microtubules and the SAC is satisfied remains unknown. Here we show in vitro and in Drosophila that Protein Phosphatase 1 (PP1) inactivates Mps1 by dephosphorylating its T-loop. PP1-mediated dephosphorylation of Mps1 occurs at kinetochores and in the cytosol, and inactivation of both pools of Mps1 during metaphase is essential to ensure prompt and efficient SAC silencing. Overall, our findings uncover a mechanism of SAC inactivation required for timely mitotic exit.

Identification of somatic TERT promoter mutations in familial nonmedullary thyroid carcinomas.

OBJECTIVE: The genes causing familial nonmedullary thyroid carcinoma (FNMTC) identified to date are only involved in a small fraction of the families. Recently, somatic mutations in TERT promoter region and in EIF1AX gene were reported in thyroid tumours of undefined familial status. The aim of this study was to investigate the role of TERT and EIF1AX mutations in familial thyroid tumours. DESIGN: The promoter region of TERT was sequenced in leucocyte DNA of the probands from 75 FNMTC families. In thyroid tumours from 54 familial cases, we assessed somatic TERT promoter, RAS and BRAF hotspot mutations, and the whole EIF1AX gene. RESULTS: No potentially pathogenic germline variants were identified in TERT in the 75 FNMTC families' probands. In the 54 carcinomas, we identified five cases (9%) with hotspot somatic TERT promoter mutations. BRAF mutations were found in 41% of the tumours. All TERT-positive samples were also positive for BRAF p.Val600Glu, and this co-occurrence was found to be statistically significant (P=.008). RAS mutations were detected in four tumours wild-type for TERT (7%). Evaluation of tumour mutation data together with the patients' clinicopathological features revealed a significant correlation between TERT plus BRAF mutations and advanced tumour stage (T4) (P=.020). No mutations were identified in EIF1AX. CONCLUSIONS: The results of this study suggest that TERT promoter and EIF1AX mutations are not frequently involved in FNMTC aetiology. However, we show for the first time that TERT alterations are associated with familial thyroid tumour progression. Our data also suggest that TERT mutations are more often found in concomitance with BRAF mutations in advanced stages of FNMTC.

Osteopontin expression is correlated with differentiation and good prognosis in medullary thyroid carcinoma.

BACKGROUND: Osteopontin (OPN) or secreted phosphoprotein 1 (SPP1) is a matricellular glycoprotein whose expression is elevated in various types of cancer and has been shown to be involved in tumourigenesis and metastasis in many malignancies, including follicular cell-derived thyroid carcinomas. Its role in C-cell-derived thyroid lesions and tumours remains to be established. OBJECTIVE: The objective of this study is to clarify the role of OPN expression in the development of medullary thyroid carcinoma (MTC). METHODS: OPN expression was analysed in a series of 116 MTCs by immunohistochemistry and by qPCR mRNA quantification of the 3 OPN isoforms (OPNa, OPNb and OPNc) in six cases from which fresh frozen tissue was available. Statistical tests were used to evaluate the relationship of OPN expression and the clinicopathological and molecular characteristics of patients and tumours. RESULTS: OPN expression was detected in 91 of 116 (78.4%) of the MTC. We also observed high OPN expression in C-cell hyperplasia as well as in C-cells scattered in the thyroid parenchyma adjacent to the tumours. OPN expression was significantly associated with smaller tumour size, PTEN nuclear expression and RAS status, and suggestively associated with non-invasive tumours. OPNa isoform was expressed significantly at higher levels in tumours than in non-tumour samples. OPNb and OPNc presented similar levels of expression in all samples. Furthermore, OPNa isoform overexpression was significantly associated with reduced growth and viability in the MTC-derived cell line (TT). CONCLUSION: The expression of OPN in normal C-cells and C-cell hyperplasia suggests that OPN is a differentiation marker of C-cells, rather than a marker of biological aggressiveness in this setting. At variance with other cancers, OPN expression is associated with good prognostic features in MTC.

RAS proto-oncogene in medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC) is a rare malignancy originating from the calcitonin-secreting parafollicular thyroid C cells. Approximately 75% of cases are sporadic. Rearranged during transfection (RET) proto-oncogene plays a crucial role in MTC development. Besides RET, other oncogenes commonly involved in the pathogenesis of human cancers have also been investigated in MTC. The family of human RAS genes includes the highly homologous HRAS, KRAS, and NRAS genes that encode three distinct proteins. Activating mutations in specific hotspots of the RAS genes are found in about 30% of all human cancers. In thyroid neoplasias, RAS gene point mutations, mainly in NRAS, are detected in benign and malignant tumors arising from the follicular epithelium. However, recent reports have also described RAS mutations in MTC, namely in HRAS and KRAS. Overall, the prevalence of RAS mutations in sporadic MTC varies between 0-43.3%, occurring usually in tumors with WT RET and rarely in those harboring a RET mutation, suggesting that activation of these proto-oncogenes represents alternative genetic events in sporadic MTC tumorigenesis. Thus, the assessment of RAS mutation status can be useful to define therapeutic strategies in RET WT MTC. MTC patients with RAS mutations have an intermediate risk for aggressive cancer, between those with RET mutations in exons 15 and 16, which are associated with the worst prognosis, and cases with other RET mutations, which have the most indolent course of the disease. Recent results from exome sequencing indicate that, besides mutations in RET, HRAS, and KRAS, no other recurrent driver mutations are present in MTC.

Cell cycle deregulation and TP53 and RAS mutations are major events in poorly differentiated and undifferentiated thyroid carcinomas.

BACKGROUND: Anaplastic thyroid carcinomas (ATCs) are among the most lethal malignancies, for which there is no effective treatment. OBJECTIVE: In the present study, we aimed to elucidate the molecular alterations contributing to ATC development and to identify novel therapeutic targets. DESIGN: We profiled the global gene expression of five ATCs and validated differentially expressed genes by quantitative RT-PCR in an independent set of tumors. In a series of 26 ATCs, we searched for pathogenic alterations in genes involved in the most deregulated cellular processes, including the hot spot regions of RAS, BRAF, TP53, CTNNB1 (ß-catenin), and PIK3CA genes, and, for the first time, a comprehensive analysis of components involved in the cell cycle [cyclin-dependent kinase (CDK) inhibitors (CDKI): CDKN1A (p21(CIP1)); CDKN1B (p27(KIP1)); CDKN2A (p14(ARF), p16(INK4A)); CDKN2B (p15(INK4B)); CDKN2C (p18(INK4C))], cell adhesion (AXIN1), and proliferation (PTEN). Mutational analysis was also performed in 22 poorly differentiated thyroid carcinomas (PDTCs). RESULTS: Expression profiling revealed that ATCs were characterized by the underexpression of epithelial components and the up regulation of mesenchymal markers and genes from TGF-ß pathway, as well as, the overexpression of cell cycle-related genes. In accordance, the up regulation of the SNAI2 gene, a TGF-ß-responsive mesenchymal factor, was validated. CDKN3, which prevents the G1/S transition, was significantly up regulated in ATCs and PDTCs and aberrantly spliced in ATCs. Mutational analysis showed that most mutations were present in TP53 (42% of ATCs; 27% of PDTCs) or RAS (31% of ATCs; 18% of PDTCs). TP53 and RAS alterations showed evidence of mutual exclusivity (P = .0354). PIK3CA, PTEN, and CDKI mutations were present in 14%-20% of PDTCs, and in 10%-14% of ATCs. BRAF, CTNNB1, and AXIN1 mutations were rarely detected. CONCLUSION: Overall, this study identified crucial roles for TP53, RAS, CDKI, and TGF-ß pathway, which may represent feasible therapeutic targets for ATC and PDTC treatment.

TERT promoter mutations are a major indicator of poor outcome in differentiated thyroid carcinomas.

CONTEXT: Telomerase promoter mutations (TERT) were recently described in follicular cell-derived thyroid carcinomas (FCDTC) and seem to be more prevalent in aggressive cancers. OBJECTIVES: We aimed to evaluate the frequency of TERT promoter mutations in thyroid lesions and to investigate the prognostic significance of such mutations in a large cohort of patients with differentiated thyroid carcinomas (DTCs). DESIGN: This was a retrospective observational study. SETTING AND PATIENTS: We studied 647 tumors and tumor-like lesions. A total of 469 patients with FCDTC treated and followed in five university hospitals were included. Mean follow-up (±SD) was 7.8 ± 5.8 years. MAIN OUTCOME MEASURES: Predictive value of TERT promoter mutations for distant metastasization, disease persistence at the end of follow-up, and disease-specific mortality. RESULTS: TERT promoter mutations were found in 7.5% of papillary carcinomas (PTCs), 17.1% of follicular carcinomas, 29.0% of poorly differentiated carcinomas, and 33.3% of anaplastic thyroid carcinomas. Patients with TERT-mutated tumors were older (P < .001) and had larger tumors (P = .002). In DTCs, TERT promoter mutations were significantly associated with distant metastases (P < .001) and higher stage (P < .001). Patients with DTC harboring TERT promoter mutations were submitted to more radioiodine treatments (P = .009) with higher cumulative dose (P = .004) and to more treatment modalities (P = .001). At the end of follow-up, patients with TERT-mutated DTCs were more prone to have persistent disease (P = .001). TERT promoter mutations were significantly associated with disease-specific mortality [in the whole FCDTC (P < .001)] in DTCs (P < .001), PTCs (P = .001), and follicular carcinomas (P < .001). After adjusting for age at diagnosis and gender, the hazard ratio was 10.35 (95% confidence interval 2.01-53.24; P = .005) in DTC and 23.81 (95% confidence interval 1.36-415.76; P = .03) in PTCs. CONCLUSIONS: TERT promoter mutations are an indicator of clinically aggressive tumors, being correlated with worse outcome and disease-specific mortality in DTC. TERT promoter mutations have an independent prognostic value in DTC and, notably, in PTC.

High prevalence of RAS mutations in RET-negative sporadic medullary thyroid carcinomas.

CONTEXT: Sporadic medullary thyroid carcinomas (MTC) frequently harbor mutations in the RET protooncogene. We have earlier reported a series of 51 sporadic MTC with 64.7% of RET-positive and 35.3% of RET-negative cases. OBJECTIVE: In the present study, we investigated the possible involvement of RAS and BRAF protooncogenes in the development of sporadic RET-negative MTC. PATIENTS AND DESIGN: We performed PCR amplification and sequencing analysis of the three mutational hotspots (codons 12, 13, and 61) of the H-, K-, and N-RAS genes, and of the mutational hotspot (codon 600) and exon 11 of the BRAF gene in 65 sporadic MTC, of which 40 were RET positive and 25 were RET negative. RESULTS: Somatic H-RAS and K-RAS mutations were detected in 14 of 25 (56.0%) and three of 25 (12.0%) of RET-negative sporadic MTC, respectively. On the other hand, only one of 40 (2.5%) RET-positive sporadic MTC had a RAS mutation, namely in H-RAS. One of the H-RAS mutations was novel (c.32_37dupCCGGCG). No mutations of N-RAS or BRAF were detected in all assessed tumor samples. CONCLUSIONS: Overall, our results showed that RAS mutations were present in 68.0% (17 of 25) of the RET-negative MTC and in only 2.5% of the RET-positive MTC (P < 0.0001), suggesting that activation of the protooncogenes RAS and RET represents alternative genetic events in sporadic MTC tumorigenesis.