NEK1 deficiency affects mitochondrial functions and the transcriptome of key DNA repair pathways.

Previous studies have indicated important roles for NIMA-related kinase 1 (NEK1) in modulating DNA damage checkpoints and DNA repair capacity. To broadly assess the contributions of NEK1 to genotoxic stress and mitochondrial functions, we characterised several relevant phenotypes of NEK1 CRISPR knockout (KO) and wild-type (WT) HAP1 cells. Our studies revealed that NEK1 KO cells resulted in increased apoptosis and hypersensitivity to the alkylator methyl methanesulfonate, the radiomimetic bleomycin and UVC light, yet increased resistance to the crosslinker cisplatin. Mitochondrial functionalities were also altered in NEK1 KO cells, with phenotypes of reduced mitophagy, increased total mitochondria, elevated levels of reactive oxygen species, impaired complex I activity and higher amounts of mitochondrial DNA damage. RNA-seq transcriptome analysis coupled with quantitative real-time PCR studies comparing NEK1 KO cells with NEK1 overexpressing cells revealed that the expression of genes involved in DNA repair pathways, such as base excision repair, nucleotide excision repair and double-strand break repair, are altered in a way that might influence genotoxin resistance. Together, our studies underline and further support that NEK1 serves as a hub signalling kinase in response to DNA damage, modulating DNA repair capacity, mitochondrial activity and cell fate determination.

Clinical utility of the imunohistochemical co-expression of p53 and MDM2 in thyroid follicular lesions.

In order to investigate the possible correlation between p53 and MDM2 co-expression with clinicopathological features of differentiated thyroid cancer (DTC) and its use as diagnostic and/or prognostic markers, we used immunohistochemistry to evaluate 317 thyroid samples including 208 DTC and 94 benign nodules, in addition to 15 normal tissues. MDM2 and p53 expression were highly associated (r = 0.7161; p < 0.0001). The co-expression of p53-MDM2 was observed more frequently in malignant lesions (p < 0.0001) and helped characterize follicular patterned lesions distinguishing FVPTC from FA (p < 0.0001) and FVPTC from FTC (p < 0.0001). In addition, p53-MDM2 co-expression was associated with characteristics of less aggressiveness. It was more frequent in patients ≤45 years old (p = 0.0035), with unique tumors (p = 0.0095), tumors <2 cm (p < 0.0001), tumors without extrathyroid invasion (p = 0.0425), without metastasis at evolution (p = 0.0179), and in patients evolving free of disease after treatment (p = 0.0485). We suggest that p53-MDM2 co-expression profile analysis might help establishing diagnostic and determining prognostic of DTC patients.

Expression of the NEK family in normal and cancer tissue: an immunohistochemical study.

BACKGROUND: The NEK serine/threonine protein kinases are involved in cell cycle checkpoints, DNA damage repair, and apoptosis. Alterations in these pathways are frequently associated with cell malignant cellular transformations. Thyroid cancer is the most common malignant tumour in the endocrine system. Despite good treatment methods, the number of cases has increased significantly in recent years. Here, we studied the expression of NEK1, NEK2, NEK3, and NEK5 in different types of normal and malignant tissues, using tissue microarray analysis, and identified NEKs as potential markers in thyroid malignancy. METHODS: The studied cases comprised multiple cancer tissue microarrays, including breast, colon, esophagus, kidney, lung, pancreas, prostate, stomach, thyroid and uterine cervix, as well as 281 patients who underwent thyroid resection for thyroid cancer or thyroid nodules. The expression of NEK1, NEK2, NEK3, and NEK5 was analyzed by immunohistochemistry. The expression pattern was evaluated in terms of intensity by two methods, semiquantitative and quantitative, and was compared between normal and cancer tissue. RESULTS: We analysed the expression of each member of the NEK family in a tissue-dependent manner. Compared to normal tissue, most of the evaluated proteins showed lower expression in lung tumour. However, in the thyroid, the expression was higher in malignant tissue, especially for NEK 1, NEK3 and NEK5. Concerning characteristics of the thyroid tumour, such as aggressiveness, NEK1 expression was higher in tumours with multifocality and in patients with lymph node metastasis. NEK3 expression was stronger in patients with stage II, that involved metastasis. NEK5, on the other hand, showed high expression in patients with invasion and metastasis and in patients with tumour size > 4 cm. Furthermore, this work, demonstrated for the first time a high specificity and sensitivity of over-expression of NEK1 in classical and follicular variants of papillary thyroid cancer and NEK3 in tall-cell papillary thyroid cancer. CONCLUSION: Taken together, the NEK protein kinases emerge as important proteins in thyroid cancer development and may help to identify malignancy and aggressiveness features during diagnosis. TRIAL REGISTRATION: This study was retrospectively registered.  www.accamargo.org.br/cientistas-pesquisadores/comite-de-etica-em-pequisa-cep.

Polymorphisms of IL-4 and IL-4R are associated to some demographic characteristics of differentiated thyroid cancer patients but are not determinants of risk in the Brazilian population.

BACKGROUND: IL-4 is known to present abnormal expression in thyroid tumors and SNPs in the IL-4 and its receptor IL-4R genes are associated to risk and mortality of various types of cancer. METHODS: In order to evaluate their role in differentiated thyroid cancer (DTC), we investigated genetic frequencies of two IL-4 promoter SNPs (rs2070874 C>T, rs2243250 C>T) and four non-synonymous SNPs of the IL-4R gene (rs1805010 A>G, rs1805012 C>T, rs1805013 C>T, rs1801275 A>G) in 300 DTC patients matched to 300 controls. All patients were managed according to current guidelines and followed-up for a period of 12-252 months (69.20 ± 52.70 months). RESULTS: Although none of the six investigated SNPs showed association with risk of DTC, rs1805010 was associated with age of diagnosis and the SNPs rs1805012 and rs1801275 were associated to gender. Further, in-silico analysis showed that all these three SNPs were able to cause decreased stability of the protein. We were not able to demonstrate any other association to clinical features of aggressiveness or to patients' prognosis. CONCLUSIONS: These findings indicate that although genetic variants in IL-4 and IL-4R do not influence the risk or outcome of DTC patients, their influence on the behavior of thyroid tumors deserves further investigation.

On Broken Ne(c)ks and Broken DNA: The Role of Human NEKs in the DNA Damage Response.

NIMA-related kinases, or NEKs, are a family of Ser/Thr protein kinases involved in cell cycle and mitosis, centrosome disjunction, primary cilia functions, and DNA damage responses among other biological functional contexts in vertebrate cells. In human cells, there are 11 members, termed NEK1 to 11, and the research has mainly focused on exploring the more predominant roles of NEKs in mitosis regulation and cell cycle. A possible important role of NEKs in DNA damage response (DDR) first emerged for NEK1, but recent studies for most NEKs showed participation in DDR. A detailed analysis of the protein interactions, phosphorylation events, and studies of functional aspects of NEKs from the literature led us to propose a more general role of NEKs in DDR. In this review, we express that NEK1 is an activator of ataxia telangiectasia and Rad3-related (ATR), and its activation results in cell cycle arrest, guaranteeing DNA repair while activating specific repair pathways such as homology repair (HR) and DNA double-strand break (DSB) repair. For NEK2, 6, 8, 9, and 11, we found a role downstream of ATR and ataxia telangiectasia mutated (ATM) that results in cell cycle arrest, but details of possible activated repair pathways are still being investigated. NEK4 shows a connection to the regulation of the nonhomologous end-joining (NHEJ) repair of DNA DSBs, through recruitment of DNA-PK to DNA damage foci. NEK5 interacts with topoisomerase IIß, and its knockdown results in the accumulation of damaged DNA. NEK7 has a regulatory role in the detection of oxidative damage to telomeric DNA. Finally, NEK10 has recently been shown to phosphorylate p53 at Y327, promoting cell cycle arrest after exposure to DNA damaging agents. In summary, this review highlights important discoveries of the ever-growing involvement of NEK kinases in the DDR pathways. A better understanding of these roles may open new diagnostic possibilities or pharmaceutical interventions regarding the chemo-sensitizing inhibition of NEKs in various forms of cancer and other diseases.

Serum interleukin measurement may help identify thyroid cancer patients with active disease.

BACKGROUND: Investigate the clinical utility of serum interleukin dosages of IL-2, IL-2R, IL-4, IL-6, IL-6R, IL-8, IL-10 and IL-12 in the diagnosis and characterization of patients with DTC. In particular, verify ILs utility in the identification of individuals who are evolving disease-free or with the active disease. METHODS: We evaluated 200 patients with malignant nodules (100 patients disease-free and 100 patients with recurrence/active disease); 60 benign nodules and 100 healthy controls, serum levels were assessed by ELISA. RESULTS: All ILs, but not IL-4, differentiated these three groups. We observed that IL-2, 2R and 10 serum concentrations were associated with thyroglobulin levels. Serum IL-2 was able to differentiate patients with active disease from the disease-free with a sensitivity of 98%, specificity of 58%, positive predictive value (PPV) of 70% and negative predictive value (NPV) of 97% (p=0.0007). IL-6R levels differentiated patients with active disease from the disease-free patients with 56% sensitivity, 63% specificity, PPV of 60% and NPV of 59% (p<0.0001). IL-8 values also distinguished patients with active disease from the disease-free ones with sensitivity of 50%, specificity of 76%, PPV of 68% and NPV of 60% (p=0.0025); using IL-12, we obtained a sensitivity value of 73%, specificity of 66%, PPV of 68% and NPV of 71% (p<0.0001). Furthermore, interleukin levels showed association with some tumor characteristics of aggressiveness. CONCLUSION: We suggest that the serum concentration of ILs may assist in the diagnosis and characterization of tumor malignancy helping identify patients with active disease who deserve closer medical attention.

Polymorphism in LEP and LEPR May Modify Leptin Levels and Represent Risk Factors for Thyroid Cancer.

Purpose. To understand the role of polymorphisms in the LEP (rs7799039 and rs2167270) and LEPR (rs1137101 and rs1137100) genes in DTC susceptibility and their effect on leptin levels. Methods. We studied 153 patients with DTC and 234 controls through TaqMan SNP Genotyping and ELISA, comparing these data to the clinicopathological data of patients with DTC. Results. Patients with AA genotype of rs7799039 had higher levels of serum leptin (9.22 ± 0.98 ng/mL) than those with AG genotype (10.07 ± 0.60 ng/mL; P = 0.005). Individuals with AG genotype of rs2167270 also produced higher serum leptin levels (10.05 ± 0.59 ng/mL) than the subjects with GG genotype (9.52 ± 0.79 ng/mL; P < 0.05). A multivariate logistic regression adjusted for gender, age, and BMI showed that the AG genotype of rs7799039 was an independent risk for DTC (OR, 11.689; P = 0.0183; 95% CI, 1.516-90.119). Similarly, AG and GG genotypes of rs1137101 increased the susceptibility to DTC (OR, 3.747; P = 0.027; 95% CI, 1.161-12.092 and OR, 5.437; P = 0.013; 95% CI, 1.426-20.729). Conclusions. We demonstrated that rs7799039 and rs2167270 polymorphisms modify the serum leptin concentrations in patients with DTC. Furthermore, polymorphisms rs7799039 and rs1137101 increase the risk of DTC development, although they do not correlate with tumor aggressiveness.

CD8+ TIL recruitment may revert the association of MAGE A3 with aggressive features in thyroid tumors.

BACKGROUND: We aimed to investigate a possible role of MAGE A3 and its associations with infiltrated immune cells in thyroid malignancy, analyzing their utility as a diagnostic and prognostic marker. MATERIALS AND METHODS: We studied 195 malignant tissues: 154 PTCs and 41 FTCs; 102 benign tissues: 51 follicular adenomas and 51 goiter and 17 normal thyroid tissues. MAGE A3 and immune cell markers (CD4 and CD8) were evaluated using immunohistochemistry and compared with clinical pathological features. RESULTS: The semiquantitative analysis and ACIS III analysis showed similar results. MAGE A3 was expressed in more malignant than in benign lesions (P < 0.0001), also helping to discriminate follicular-patterned lesions. It was also higher in tumors in which there was extrathyroidal invasion (P = 0.0206) and in patients with stage II disease (P = 0.0107). MAGE A3+ tumors were more likely to present CD8+ TIL (P = 0.0346), and these tumors were associated with less aggressive features, that is, extrathyroidal invasion and small size. There was a trend of MAGE A3+ CD8+ tumors to evolve free of disease. CONCLUSION: We demonstrated that MAGE A3 and CD8+ TIL infiltration may play an important role in malignant thyroid nodules, presenting an interesting perspective for new researches on DTC immunotherapy.

Clinical utility of KAP-1 expression in thyroid lesions.

Although there are evidences of the involvement of KAP-1 in other tumors, data on differentiated thyroid carcinomas (DTC) are still lacking. We aimed to evaluate KAP-1 clinical utility in the diagnosis and prognosis of DTC. We used both visual immunohistochemistry and a semiquantitative analysis to evaluate KAP-1 expression in 230 thyroid carcinomas and 131 noncancerous thyroid nodules. There were 43 follicular carcinomas (FC) and 187 papillary thyroid carcinomas (PTC), including 130 classic (CPTC), 4 tall cells (TCPTC), and 53 follicular variants (FVPTC). Patients were followed up for 53.8 ± 41 months. They were classified as free-of-disease (142 cases) or poor outcome (25 cases--10 deaths), according to their serum Tg levels and image evidences. KAP-1 was identified in 78 % PTC, 75 % TCPTC, 74 % FC, 72 % FVPTC, 55 % FA, 44 % hyperplasia, and 11 % normal thyroid tissues. A ROC analysis identified malignant nodules with 69 % sensitivity and 75 % specificity, using a cutoff of 73.19. In addition to distinguishing benign from malignant thyroid tissues (p < 0.0001), KAP-1 expression differentiated CPTC from nodular hyperplasia (p < 0.0001), CPTC from FA (p = 0.0028), FVPTC from hyperplasia (p = 0.0039), and FC from hyperplasia (p = 0.0025). Furthermore, KAP-1 was more expressed in larger tumors (>4 cm; p = 0.0038) and in individuals who presented recurrences/metastases (p = 0.0130). We suggest that KAP-1 may help diagnose thyroid nodules, characterize follicular-patterned thyroid lesions, and identify individuals with poor prognosis.