Questions and Controversies in the Clinical Application of Tyrosine Kinase Inhibitors to Treat Patients with Radioiodine-Refractory Differentiated Thyroid Carcinoma: Expert Perspectives.

Notwithstanding regulatory approval of lenvatinib and sorafenib to treat radioiodine-refractory differentiated thyroid carcinoma (RAI-R DTC), important questions and controversies persist regarding this use of these tyrosine kinase inhibitors (TKIs). RAI-R DTC experts from German tertiary referral centers convened to identify and explore such issues; this paper summarizes their discussions. One challenge is determining when to start TKI therapy. Decision-making should be shared between patients and multidisciplinary caregivers, and should consider tumor size/burden, growth rate, and site(s), the key drivers of RAI-R DTC morbidity and mortality, along with current and projected tumor-related symptomatology, co-morbidities, and performance status. Another question involves choice of first-line TKIs. Currently, lenvatinib is generally preferred, due to greater increase in progression-free survival versus placebo treatment and higher response rate in its pivotal trial versus that of sorafenib; additionally, in those studies, lenvatinib but not sorafenib showed overall survival benefit in subgroup analysis. Whether recommended maximum or lower TKI starting doses better balance anti-tumor effects versus tolerability is also unresolved. Exploratory analyses of lenvatinib pivotal study data suggest dose-response effects, possibly favoring higher dosing; however, results are awaited of a prospective comparison of lenvatinib starting regimens. Some controversy surrounds determination of net therapeutic benefit, the key criterion for continuing TKI therapy: if tolerability is acceptable, overall disease control may justify further treatment despite limited but manageable progression. Future research should assess potential guideposts for starting TKIs; fine-tune dosing strategies and further characterize antitumor efficacy; and evaluate interventions to prevent and/or treat TKI toxicity, particularly palmar-plantar erythrodysesthesia and fatigue.

Autochthonous tumors driven by Rb1 loss have an ongoing requirement for the RBP2 histone demethylase.

Inactivation of the retinoblastoma gene (RB1) product, pRB, is common in many human cancers. Targeting downstream effectors of pRB that are central to tumorigenesis is a promising strategy to block the growth of tumors harboring loss-of-function RB1 mutations. One such effector is retinoblastoma-binding protein 2 (RBP2, also called JARID1A or KDM5A), which encodes an H3K4 demethylase. Binding of pRB to RBP2 has been linked to the ability of pRB to promote senescence and differentiation. Importantly, genetic ablation of RBP2 is sufficient to phenocopy pRB's ability to induce these cellular changes in cell culture experiments. Moreover, germline Rbp2 deletion significantly impedes tumorigenesis in Rb1+/- mice. The value of RBP2 as a therapeutic target in cancer, however, hinges on whether loss of RBP2 could block the growth of established tumors as opposed to simply delaying their onset. Here we show that conditional, systemic ablation of RBP2 in tumor-bearing Rb1+/- mice is sufficient to slow tumor growth and significantly extend survival without causing obvious toxicity to the host. These findings show that established Rb1-null tumors require RBP2 for growth and further credential RBP2 as a therapeutic target in human cancers driven by RB1 inactivation.

Novel Inhibitor-Based Therapies for Thyroid Cancer-An Update.

Thyroid cancers (TCs) are the most common tumors of the endocrine system and a constant rise in the number of TC cases has been observed for the past few decades. TCs are one of the most frequent tumors in younger adults, especially in women, therefore early diagnosis and effective therapy are especially important. Ultrasonography examination followed by fine needle biopsy have become the gold standard for diagnosis of TCs, as these strategies allow for early-stage detection and aid accurate qualification for further procedures, including surgical treatment. Despite all the advancements in detection and treatment of TCs, constant mortality levels are still observed. Therefore, a novel generation line of targeted treatment strategies is being developed, including personalized therapies with kinase inhibitors. Recent molecular studies on TCs demonstrate that kinase inhibitor-based therapies might be considered as the most promising. In the past decade, new kinase inhibitors with different mechanisms of action have been reported and approved for clinical trials. This review presents an up-to-date picture of new approaches and challenges of inhibitor-based therapies in treatment of TCs, focusing on the latest findings reported over the past two years.

Membrane type 1 matrix metalloproteinase regulates anaplastic thyroid carcinoma cell growth and invasion into the collagen matrix.

Anaplastic thyroid carcinoma (ATC) is one of the most aggressive cancer types; however, the molecular mechanism contributing to the aggressive characteristics remain unclear. Membrane type 1 matrix metalloproteinase (MT1-MMP) plays an important role in cancer invasion and has been associated with a poor prognosis in various malignant neoplasms. In this study, we investigated the relationship between MT1-MMP expression and the proliferation and invasion of ATC cells, along with the association with clinicopathologic factors in patients with ATC. Suppression of MT1-MMP reduced the proliferation and invasion of ATC cells, and suppressed ERK activity, indicating a role in cancer cell proliferation in collagen matrix culture conditions. The expression of MT1-MMP was detected in 29 of 34 (85.3%) surgical specimens from ATC patients. In addition, the expression of MT1-MMP in the tumor lesion was higher than that of normal and stromal tissues. Collectively, these results suggest that elevated MT1-MMP expression plays a role in the pathogenesis of ATC, which may promote its aggressive characteristics such as proliferation and invasion, highlighting a potential new therapeutic target.

Upregulated PTPN2 induced by inflammatory response or oxidative stress stimulates the progression of thyroid cancer.

PTPN2 is one of the members of the protein Tyrosine Phosphatases (PTPs) family. To explore the promotive effect of upregulated PTPN2 induced by inflammatory response or oxidative stress on the progression of thyroid cancer. PTPN2 level in thyroid cancer tissues and cell lines was detected. Kaplan-Meier method was applied for evaluating the prognostic value of PTPN2 in thyroid cancer patients. After stimulation of inflammatory response (treatment of IFN-γ and TNF-α), or oxidative stress (treatment of H2O2), protein level of PTPN2 in K1 cells was measured by Western blot. Regulatory effects of PTPN2 on EdU-positive staining and Ki-67 positive cell ratio in K1 cells either with H2O2 stimulation or not were determined. PTPN2 was upregulated in thyroid cancer tissues and cell lines. Its level was higher in metastatic thyroid cancer patients than those of non-metastatic ones. High level of PTPN2 predicted worse prognosis of thyroid cancer. Treatment of either IFN-γ or TNF-α upregulated protein level of PTPN2 in K1 cells. Meanwhile, H2O2 stimulation upregulated PTPN2, which was reversed by NAC administration. With the stimulation of increased doses of H2O2, EdU-positive staining and Ki-67 positive cell ratio were dose-dependently elevated. Silence of PTPN2 attenuated proliferative ability and Ki-67 expression in K1 cells either with H2O2 stimulation or not. Inflammatory response or oxidative stress induces upregulation of PTPN2, thus promoting the progression of thyroid cancer.

Clinicopathologic features of kinase fusion-related thyroid carcinomas: an integrative analysis with molecular characterization.

The discovery of actionable kinase gene rearrangements has revolutionized the therapeutic landscape of thyroid carcinomas. Unsolved challenges include histopathologic recognition of targetable cases, correlation between genotypes and tumor behavior, and evolving resistance mechanisms against kinase inhibitors (KI). We present 62 kinase fusion-positive thyroid carcinomas (KFTC), including 57 papillary thyroid carcinomas (PTC), two poorly differentiated thyroid carcinomas (PDTC), two undifferentiated thyroid carcinomas (ATC), and one primary secretory carcinoma (SC), in 57 adults and 5 adolescents. Clinical records, post-operative histology, and molecular profiles were reviewed. Histologically, all KFTC showed multinodular growth with prominent intratumoral fibrosis. Lymphovascular invasion (95%), extrathyroidal extension, gross and microscopic (63%), and cervical lymph node metastasis (79%) were common. Several kinase fusions were identified: STRN-ALK, EML4-ALK, AGK-BRAF, CUL1-BRAF, MKRN1-BRAF, SND1-BRAF, TTYH3-BRAF, EML4-MET, TFG-MET, IRF2BP2-NTRK1, PPL-NTRK1, SQSTM1-NTRK1, TPR-NTRK1, TPM3-NTRK1, EML4-NTRK3, ETV6-NTRK3, RBPMS-NTRK3, SQSTM1-NTRK3, CCDC6-RET, ERC1-RET, NCOA4-RET, RASAL2-RET, TRIM24-RET, TRIM27-RET, and CCDC30-ROS1. Individual cases also showed copy number variants of EGFR and nucleotide variants and indels in pTERT, TP53, PIK3R1, AKT2, TSC2, FBXW7, JAK2, MEN1, VHL, IDH1, PTCH1, GNA11, GNAQ, SMARCA4, and CDH1. In addition to thyroidectomy and radioactive iodine, ten patients received multi-kinase and/or selective kinase inhibitor therapy, with 6 durable, objective responses and four with progressive disease. Among 47 cases with >6 months of follow-up (median [range]: 41 [6-480] months), persistent/recurrent disease, distant metastasis and thyroid cancer-related death occurred in 57%, 38% and 6%, respectively. In summary, KFTC encompass a spectrum of molecularly diverse tumors with overlapping clinicopathologic features and a tendency for clinical aggressiveness. Characteristic histology with multinodular growth and prominent fibrosis, particularly when there is extensive lymphovascular spread, should trigger molecular testing for gene rearrangements, either in a step-wise manner by prevalence or using a combined panel. Further, our findings provide information on molecular therapy in radioiodine-refractory thyroid carcinomas.

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.

Anti-Tumor Activity and Safety of Multikinase Inhibitors in Advanced and/or Metastatic Thyroid Cancer: A Systematic Review and Network Meta-Analysis of Randomized Controlled Trials.

Many trials have demonstrated prime antitumor activity of novel, small molecule multikinase inhibitors (MKIs) in advanced and/or metastatic thyroid cancer (TC). In this work, the PubMed, EMBASE, Cochrane Central Register of Controlled Trials, Web of Science, SCOPUS, and clinicaltrials.gov databases were searched. Quality/risk of bias were assessed using GRADE criteria. Randomized clinical trials (RCTs) comparing two or more systemic therapies in patients with advanced and/or metastatic thyroid cancer were assessed. A total of 1347 articles and 548 clinical trials in clinicaltrials.gov were screened. We included seven relevant RCTs comprising 1934 unique patients assigned to different MKIs. Two separate network meta-analyses included four RCTs in radioiodine refractory well-differentiated thyroid cancer (RR-WDTC) and three RCTs in medullary thyroid cancer (MTC), respectively; all with a low risk of bias. We identified three therapies for RR-WDTC: sorafenib [disease control rate (DCR) odds ratio (OR): 0.11 (95% CI: 0.03-0.40); progression-free survival (PFS) hazard ratio (HR): 1.99 (95% CI: 1.62-2.46)], vandetanib [DCR_OR:0.26 (95% CI: 0.06-1.24); PFS_HR: 0.99 (95% CI: 0.82-1.20)] and lenvatinib [DCR_OR: 0.26 (95% CI: 0.05-1.33); PFS_HR: 0.99 (95% CI: 0.81-1.22)]; and the following therapies for MTC: vandetanib 300 mg [objective response rate (ORR)_OR: 3.31 (95% CI: 0.68-16.22); vandetanib 150 mg ORR_OR: 0.60 (95% CI: 0.16-2.33)]; and cabozantinib [ORR_OR: 85.32 (95% CI: 5.22-1395.15)]. Serious side effect (SE) analysis per organ/system demonstrated a varying MKI SE profile across both RR-WDTC and MTC diagnoses, more commonly involving metabolic/nutritional disorders [OR: 2.07 [95% CI: 0.82-5.18)] and gastrointestinal SE [OR: 1.63 (95% CI: 1.0-2.66)]. This network meta-analysis on advanced and/or metastatic TC points towards a higher efficacy of lenvatinib in RR-WDTC. The included MKIs exhibit a varying SE profile across different organs/systems favoring a patient-tailored approach with the anticipated toxicities guiding clinicians' decisions.

Integrated computational and Drosophila cancer model platform captures previously unappreciated chemicals perturbing a kinase network.

Drosophila provides an inexpensive and quantitative platform for measuring whole animal drug response. A complementary approach is virtual screening, where chemical libraries can be efficiently screened against protein target(s). Here, we present a unique discovery platform integrating structure-based modeling with Drosophila biology and organic synthesis. We demonstrate this platform by developing chemicals targeting a Drosophila model of Medullary Thyroid Cancer (MTC) characterized by a transformation network activated by oncogenic dRetM955T. Structural models for kinases relevant to MTC were generated for virtual screening to identify unique preliminary hits that suppressed dRetM955T-induced transformation. We then combined features from our hits with those of known inhibitors to create a 'hybrid' molecule with improved suppression of dRetM955T transformation. Our platform provides a framework to efficiently explore novel kinase inhibitors outside of explored inhibitor chemical space that are effective in inhibiting cancer networks while minimizing whole body toxicity.

Prognostic Significance of COX-2 Overexpression in BRAF-Mutated Middle Eastern Papillary Thyroid Carcinoma.

The cyclooxygenase-2 (COX-2)-prostaglandin E2 (PGE2) pathway has been implicated in carcinogenesis, with BRAF mutation shown to promote PGE2 synthesis. This study was conducted to evaluate COX-2 expression in a large cohort of Middle Eastern papillary thyroid carcinoma (PTC), and further evaluate the prognostic significance of COX-2 expression in strata of BRAF mutation status. BRAF mutation analysis was performed using Sanger sequencing, and COX-2 expression was evaluated immunohistochemically using tissue microarray (TMA). COX-2 overexpression, noted in 43.2% (567/1314) of cases, was significantly associated with poor prognostic markers such as extra-thyroidal extension, lymph-node metastasis, and higher tumor stage. COX-2 was also an independent predictor of poor disease-free survival (DFS). Most notably, the association of COX-2 expression with DFS differed by BRAF mutation status. COX-2 overexpression was associated with poor DFS in BRAF-mutant but not BRAF wild-type PTCs, with a multivariate-adjusted hazard ratio of 2.10 (95% CI = 1.52-2.92; p < 0.0001) for COX-2 overexpressed tumors in BRAF-mutant PTC. In conclusion, the current study shows that COX-2 plays a key role in prognosis of PTC patients, especially in BRAF-mutated tumors. Our data suggest the potential therapeutic role of COX-2 inhibition in patients with BRAF-mutated PTC.

Current and Future Role of Tyrosine Kinases Inhibition in Thyroid Cancer: From Biology to Therapy.

Thyroid cancer represents a heterogenous disease whose incidence has increased in the last decades. Although three main different subtypes have been described, molecular characterization is progressively being included in the diagnostic and therapeutic algorithm of these patients. In fact, thyroid cancer is a landmark in the oncological approach to solid tumors as it harbors key genetic alterations driving tumor progression that have been demonstrated to be potential actionable targets. Within this promising and rapid changing scenario, current efforts are directed to improve tumor characterization for an accurate guidance in the therapeutic management. In this sense, it is strongly recommended to perform tissue genotyping to patients that are going to be considered for systemic therapy in order to select the adequate treatment, according to recent clinical trials data. Overall, the aim of this article is to provide a comprehensive review on the molecular biology of thyroid cancer focusing on the key role of tyrosine kinases. Additionally, from a clinical point of view, we provide a thorough perspective, current and future, in the treatment landscape of this tumor.

Knockdown of KDM1A suppresses tumour migration and invasion by epigenetically regulating the TIMP1/MMP9 pathway in papillary thyroid cancer.

Epigenetic dysregulation plays an important role in cancer. Histone demethylation is a well-known mechanism of epigenetic regulation that promotes or inhibits tumourigenesis in various malignant tumours. However, the pathogenic role of histone demethylation modifiers in papillary thyroid cancer (PTC), which has a high incidence of early lymphatic metastasis, is largely unknown. Here, we detected the expression of common histone demethylation modifiers and found that the histone H3 lysine 4 (H3K4) and H3 lysine 9 (H3K9) demethylase KDM1A (or lysine demethylase 1A) is frequently overexpressed in PTC tissues and cell lines. High KDM1A expression correlated positively with age <55 years and lymph node metastasis in patients with PTC. Moreover, KDM1A was required for PTC cell migration and invasion. KDM1A knockdown inhibited the migration and invasive abilities of PTC cells both in vitro and in vivo. We also identified tissue inhibitor of metalloproteinase 1 (TIMP1) as a key KDM1A target gene. KDM1A activated matrix metalloproteinase 9 (MMP9) through epigenetic repression of TIMP1 expression by demethylating H3K4me2 at the TIMP1 promoter region. Rescue experiments clarified these findings. Altogether, we have uncovered a new mechanism of KDM1A repression of TIMP1 in PTC and suggest that KDM1A may be a promising therapeutic target in PTC.

MicroRNA-520a-3p suppresses epithelial-mesenchymal transition, invasion, and migration of papillary thyroid carcinoma cells via the JAK1-mediated JAK/STAT signaling pathway.

Papillary thyroid cancer (PTC) is a kind of thyroid cancer and frequently presents with epithelial-mesenchymal transition (EMT). MicroRNAs (miRNAs) were previously reported to be associated with PTC. Thus, this study aims to define the role of microRNA-520a-3p (miR-520a-3p) in PTC through the JAK/STAT signaling pathway by targeting JAK1. The PTC and normal thyroid tissues of 137 PTC patients were collected. First of all, the expression pattern of miR-520a-3p, JAK1, JAK2, STAT3, E-cadherin, and vimentin in PTC was identified. The relationship between miR-520a-3p and JAK1 was predicted and analyzed. And a series of miR-520a-3p mimic or inhibitor, or siRNA JAK1 introduced into PTC cells were applied to examine the effect of miR-520a-3p on PTC cell viability, migration, invasion, cell cycle, apoptosis, and EMT. Meanwhile, the regulatory effect of miR-520a-3p and JAK1 on the JAK/STAT signaling pathway was also determined. The expression of JAK1, JAK2, STAT3, and vimentin increased yet miR-520a-3p and E-cadherin decreased in PTC tissue. JAK1 was negatively regulated by miR-520a-3p. Functionally, EMT induction was prevented by miR-520a-3p upregulation through downregulating JAK1. When upregulating miR-520a-3p or silencing JAK1 in PTC cells, PTC cell viability, migration, and invasion were inhibited yet cell apoptosis promoted with cells arrested at G1 phase, indicating that miR-520a-3p prevented PTC progression by downregulating JAK1. Moreover, miR-520a-3p elevation or JAK1 inhibition inactivated the JAK/STAT signaling pathway. Collectively, miR-520a-3p prevents cancer progression through inactivating the JAK/STAT signaling pathway by downregulating JAK1 in PTC.

PI3K inhibitors IC87114 inhibits the migration and invasion of thyroid cancer cell in vitro and in vivo.

Thyroid cancer is currently the most common endocrine malignancies, and PI3K/Akt pathway play an important role during its initiation and development. IC87114 is an inhibitor of PI3K/Akt pathway. Little is known about the role of IC87114 in the initiation and progression of thyroid cancer. Here, we demonstrated that IC87114 significantly inhibited both the migration and invasion of thyroid cancer cells, which can be mediated by inhibition of PI3K/Akt pathway. These results suggest that application of IC87114 may prove to be a particularly effective treatment for thyroid cancer.

Telomerase reverse transcriptase mutations are independent predictor of disease-free survival in Middle Eastern papillary thyroid cancer.

Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. Tumor recurrence occurs in ∼20% of PTCs and some reach advanced stages. Promoter mutation in the telomerase reverse transcriptase (TERT) gene is identified to be a prognostic marker in PTC. However, the contribution of TERT promoter mutation to cancer progression in PTC patients is still not fully understood. In this study, we investigated the incidence of TERT promoter mutations and TERT protein expression and their association with clinicopathological outcomes in a large cohort of PTC samples using direct sequencing technology and immunohistochemistry. Furthermore, two PTC cell lines were utilized to investigate role of TERT mutations in mediating metastasis. Two promoter hotspot mutations C228T and C250T were identified in 18.0% (167/927) of our cohort and were significantly associated with poor 5 years disease-free survival and distant metastasis of PTC. TERT protein overexpression was noted in 20.1% of our PTC cohort and was significantly associated with poor prognostic markers such as older age, extrathyroidal extension and Stage IV tumors. A significant association was also found between TERT overexpression and epithelial-mesenchymal transition (EMT) markers. Functional analysis showed that TERT inhibition reduced cell growth, invasion, migration and angiogenesis in PTC via suppression of EMT in PTC cells. Our results suggest that TERT promoter mutation is an independent predictor of disease-free survival and might drive the metastasis, and downregulation of TERT could potentiate antitumor and antimetastatic activities in PTC.

Overexpression of epidermal growth factor receptor and its downstream effector, focal adhesion kinase, correlates with papillary thyroid carcinoma progression.

Epidermal growth factor receptor (EGFR) and its downstream effector, focal adhesion kinase (FAK), have been shown to be overexpressed frequently in human malignancies and implicated in tumour aggressiveness. We aimed to investigate the relationship between EGFR and FAK expression and their possible correlation with the clinical phenotype of patients with papillary thyroid carcinoma (PTC). Expression profiles of EGFR and FAK were analysed in PTC tissue samples (n = 104) by immunohistochemistry and Western blotting. Additionally, EGFR and FAK were immunohistochemically analysed in 20 primary tumours paired with their metastatic tissue in lymph nodes. High expression of EGFR and FAK was found in 55.77% and 57.69% cases, respectively, with a strong positive association between them (P < 0.0001, Spearman's correlation coefficient = 0.844). Expression of each molecule and their coexpression correlated significantly with the presence of lymph node metastasis (LNM), degree of tumour infiltration, extrathyroid invasion and pT status of the patients. Western blot analysis confirmed that coexpression of high levels of EGFR and FAK correlated with adverse clinicopathological features. When compared to the corresponding primary tumour, increased or maintained high levels of EGFR and FAK were found in LNM, indicating their concordant expression during lymphatic spread. In conclusion, high levels of EGFR and its downstream effector, FAK, in association with lymphatic spread and tumour infiltration indicate their involvement in PTC progression and suggest that both molecules may predict its aggressive behaviour. Furthermore, FAK could be a potential target for anticancer therapy in patients with advanced thyroid cancer.

The role of matrix metalloproteinase-9 as a prognostic biomarker in papillary thyroid cancer.

BACKGROUND: The aim of the present study was to investigate the association between matrix metalloproteinase-9 (MMP-9) expression with BRAF V600E mutation and clinicopathological features, in Iranian papillary thyroid cancer (PTC) patients. METHODS: In total, 90 participants including 60 PTC patients (15 males and 45 females) and 30 individuals with benign multinodular goiter (MNG) (5 males and 25 females) which were confirmed by surgical pathology, were investigated. MMP-9 was evaluated at both mRNA and protein levels, using SYBR-Green Real-Time PCR and enzyme-linked immune sorbent assay (ELISA), respectively. BRAF V600E mutation was detected by sequencing. RESULTS: Mean age of PTC and MNG patients was 37.6 ± 12.6 and 48.1 ± 13.3 years, respectively (P = 0.001). BRAF V600E mutation was found in 24 of the 60 (40%) PTC cases, with mean tumor size of 1.59 ± 1.20 cm. MMP-9 mRNA levels were elevated in tumoral compared to the adjacent non-tumoral tissues (P = 0.039); moreover, this rise was also observed in PTC patients compared to MNG patients (P = 0.001). The mRNA levels of MMP-9 increased in patients aged≥45 years (P = 0.015), those with lymphovascular invasion (P = 0.003), and higher tumor stages (III and IV) (P = 0.011). The protein level of MMP-9 increased in tumoral compared to adjacent non-tumoral tissues (P < 0.001); this increase was also found in PTC patients compared to MNG participants (P = 0.004). MMP-9 protein level was higher in patients aged≥45 years (P = 0.001), those with lymphovascular invasion (P = 0.036) and higher TNM stages (III and IV) (P = 0.001). Area under the ROC curve (AUC) was 0.70 (95%CI: 0.57-0.83, P = 0.003), with 91.4% sensitivity and 51.9% specificity at the cutoff value of 0.50. CONCLUSION: The mRNA and protein levels of MMP-9 had no association with BRAF V600E mutation in Iranian PTC patients. These levels were associated with age, TNM stages, and lymphovascular invasion, being defined as malignant factors. Thus, elevated levels of MMP-9 in PTC patients compared to MNG participants illustrated that it can be used as a potential biomarker to differentiate PTC patients from those with MNG.

Overexpression of G Protein-Coupled Receptor Kinase 6 (GRK6) Is Associated with Progression and Poor Prognosis of Papillary Thyroid Carcinoma.

BACKGROUND Approximately 20% of patients with papillary thyroid carcinoma (PTC) will develop cancer recurrence, but no clinically available biomarker has been identified. Our study aimed to evaluate the prognostic value of G protein-coupled receptor kinase 6 (GRK6) in PTCs. MATERIAL AND METHODS We retrospectively enrolled 108 PTC patients in this study, and explored the expression of GRK6 in resected tumor samples by RT-qPCR and immunohistochemistry (IHC). The clinical data were interpreted by chi-square test, univariate analysis, and multivariate analysis. To investigate the functional mechanisms of GRK6 in regulating PTC progression, we also performed overexpression and silencing experiments in TPC-1 cells, a cell line generated from PTC tissues. RESULTS RT-qPCR results showed a higher level of GRK6-mRNA in PTCs than in adjacent thyroid tissues. IHC revealed a distinct protein expression pattern of GRK6 among PTCs. Accordingly, we classified patients into low-GRK6 and high-GRK6 groups. The chi-square test showed that a higher GRK6 was associated with larger tumor size (P=0.045) and advanced TNM stage (P=0.001). Kaplan-Meier survival curve and log rank test demonstrated that higher GRK6 predicted poor disease-free survival (DFS) in PTC patients (P=0.002). Furthermore, Cox regression analysis confirmed that GRK6 was an independent prognostic factor for a higher recurrence risk of PTCs (P=0.047). MTT assay and Transwell assay demonstrated that GRK6 overexpression can significantly enhance tumor proliferation and invasion, which was consistent with clinical findings. CONCLUSIONS Our data show the oncogenic effects of GRK6 in promoting PTC progression.

Shikonin Inhibites Migration and Invasion of Thyroid Cancer Cells by Downregulating DNMT1.

BACKGROUND Shikonin is a component of Chinese herbal medicine. The aim of this study was to investigate the effects of shikonin on cell migration of papillary thyroid cancer cells of the TPC-1 cell line in vitro and expression levels of the phosphate and tensin homolog deleted on chromosome 10 (PTEN) and DNA methyltransferase 1 (DNMT1) genes. MATERIAL AND METHODS The Cell Counting Kit-8 (CCK-8) assay was performed to evaluate the proliferation of TPC-1 papillary thyroid cancer cells, and the normal thyroid cells, HTori-3, in vitro. A transwell motility assay was used to analyze the migration of TPC-1 cells. Western blot was performed to determine the expression levels of PTEN and DNMT1 genes. A methylation-specific polymerase chain reaction (PCR) (MSP) assay was used to evaluate the methylation of PTEN. RESULTS Following treatment with shikonin, the cell survival rate of TPC-1 cells decreased in a dose-dependent manner; the inhibitory effects on HTori-3 cells were less marked. Shikonin inhibited TPC-1 cell migration and invasion in a dose-dependent manner. The methylation of PTEN was suppressed by shikonin, which also reduced the expression of DNMT1 in a dose-dependent manner, and increased the expression of PTEN. Overexpression of DNMT1 promoted the migration of TPC-1 cells and the methylation of PTEN. Levels of protein expression of PTEN in TPC-1 cells treated with shikonin decreased, and were increased by DNMT1 knockdown. CONCLUSIONS Shikonin suppressed the expression of DNMT1, reduced PTEN gene methylation, and increased PTEN protein expression, leading to the inhibition of TPC-1 cell migration.

NADPH oxidase DUOX1 promotes long-term persistence of oxidative stress after an exposure to irradiation.

Ionizing radiation (IR) causes not only acute tissue damage, but also late effects in several cell generations after the initial exposure. The thyroid gland is one of the most sensitive organs to the carcinogenic effects of IR, and we have recently highlighted that an oxidative stress is responsible for the chromosomal rearrangements found in radio-induced papillary thyroid carcinoma. Using both a human thyroid cell line and primary thyrocytes, we investigated the mechanism by which IR induces the generation of reactive oxygen species (ROS) several days after irradiation. We focused on NADPH oxidases, which are specialized ROS-generating enzymes known as NOX/DUOX. Our results show that IR induces delayed NADPH oxidase DUOX1-dependent H2O2 production in a dose-dependent manner, which is sustained for several days. We report that p38 MAPK, activated after IR, increased DUOX1 via IL-13 expression, leading to persistent DNA damage and growth arrest. Pretreatment of cells with catalase, a scavenger of H2O2, or DUOX1 down-regulation by siRNA abrogated IR-induced DNA damage. Analysis of human thyroid tissues showed that DUOX1 is elevated not only in human radio-induced thyroid tumors, but also in sporadic thyroid tumors. Taken together, our data reveal a key role of DUOX1-dependent H2O2 production in long-term persistent radio-induced DNA damage. Our data also show that DUOX1-dependent H2O2 production, which induces DNA double-strand breaks, can cause genomic instability and promote the generation of neoplastic cells through its mutagenic effect.