Medullary Thyroid Carcinoma Cell Proliferation Research Articles

LncRNA ZFAS1 promotes invasion of medullary thyroid carcinoma by enhancing EPAS1 expression via miR-214-3p/UCHL1 axis.

lncRNA ZFAS1 was identified to facilitate thyroid cancer, but its role in medullary thyroid carcinoma (MTC) remains unknown. This study aimed to unravel the potential function of this lncRNA in MTC by investigating the involvement of the lncRNA ZFAS1 in a ceRNA network that regulates MTC invasion. Proliferation, invasion, and migration of cells were evaluated using EdU staining and Transwell assays. Immunoprecipitation (IP) assays, dual-fluorescence reporter, and RNA IP assays were employed to examine the binding interaction among genes. Nude mice were used to explore the role of lncRNA ZFAS1 in MTC in vivo. ZFAS1 and EPAS1 were upregulated in MTC. Silencing ZFAS1 inhibited MTC cell proliferation and invasion under hypoxic conditions, which reduced EPAS1 protein levels. UCHL1 knockdown increased EPAS1 ubiquitination. ZFAS1 positively regulated UCHL1 expression by binding to miR-214-3p. Finally, silencing ZFAS1 significantly repressed tumor formation and metastasis in MTC. LncRNA ZFAS1 promotes invasion of MTC by upregulating EPAS1 expression via the miR-214-3p/UCHL1 axis.

MiR‑592 acts as an oncogene and promotes medullary thyroid cancer tumorigenesis by targeting cyclin‑dependent kinase 8.

Medullary thyroid carcinoma (MTC) is a relatively rare subtype of thyroid cancer, accounting for 5–10% of all cases of thyroid cancer worldwide. Due to the current lack of knowledge regarding the tumorigenesis of MTC, the clinical treatment of MTC remains a challenge. It has been reported that microRNAs (miRNAs) regulate the progression of MTC; however, the regulatory network of miRNAs and the exact underlying mechanisms are not completely understood. In the present study, an miRNA expression profile (GSE40807), consisting of 80 samples, was downloaded and analyzed using Gene Expression Omnibus-2R to identify differentially expressed miRNAs between MTC and normal samples. miR-592 expression levels were significantly increased in MTC tissues and cell lines compared with normal tissues and cell lines. Patients with high miR-592 expression levels exhibited a less favorable prognosis compared with patients with low miR-592 expression. The results suggested that miR-592 overexpression promoted TT and MZ-CRC-1 cell proliferation in vitro. In addition, miR-592 negatively regulated cyclin-dependent kinase 8 (CDK8) via targeted binding in MTC cells. Moreover, co-transfection of CDK8 overexpression plasmid and miR-592 mimic reversed miR-592-mediated MTC cell proliferation. In conclusion, miR-592 may serve as an oncogene in MTC by decreasing the expression of CDK8, indicating that the miR-592/CDK8 axis might serve as a promising therapeutic target for MTC.

Genomic and Transcriptomic Characterization of Sporadic Medullary Thyroid Carcinoma.

Background: Sporadic medullary thyroid carcinoma (MTC) is a relatively uncommon neuroendocrine malignancy and the molecular tumorigenesis of its sporadic type (sMTC) is only partially understood. In this study, we performed a study focusing on the genomic and transcriptomic characterization of sMTC. Methods: Twenty-nine sMTC patients were included. Whole-exome sequencing (WES) was carried out in 18 patients, including both tumor samples and matched noncancerous tissues. Whole transcriptome sequencing (RNA-Seq) was performed in all 29 tumors. WES, RNA-Seq, and copy number alteration (CNA) data were analyzed. A Cell Counting Kit-8 (CCK-8) assay was used to evaluate cell proliferation. Results: Among the somatic mutations, RET was the only recurrently cancer-related mutated gene (5/18, 27.8%). In the germline, FAT1 and FAT4, two members of the FAT gene family, were identified as the two most common mutated genes. CNA analysis found that FAT1 and FAT4, both located on chromosome 4q, were also two of the genes most commonly affected by somatic chromosomal deletions (4/18, 22.2%). Using TT and MZ-CRC-1 cell lines, the CCK-8 assay showed that FAT1 and FAT4 knockdown could promote MTC cell proliferation. Based on the gene expression profile, patients were clustered into two molecular subtypes: the mesenchymal-like subtype is characterized by epithelial-mesenchymal transition, while the proliferative-like subtype is associated with enrichment of cell cycle pathways. Most events of structural recurrence (80%) occurred in the proliferative-like subtype. Conclusion: In addition to RET, these findings demonstrate that FAT1/FAT4 genomic alterations appear to be frequent in sMTC. Two molecular subtypes of sMTC with distinct biological behavior could be identified. However, these results need to be validated by larger samples and more comprehensive experiments in the future, especially for the frequency and function of FAT1/FAT4 germline variants.

Silencing of zinc finger protein 703 inhibits medullary thyroid carcinoma cell proliferation in vitro and in vivo.

Zinc finger protein 703 (ZNF703) is a new member of the zinc finger protein family of transcription factors that plays an important role during embryogenesis in metazoans. The overexpression of ZNF703 contributes to tumorigenesis and progression of a number of malignancies by activating the Akt/mammalian target of rapamycin (mTOR) signaling pathway. This pathway is activated in medullary thyroid cancer (MTC), but its mechanism of action is not yet fully understood. The aim of the present study was to examine the role of ZNF703 and its association with Akt/mTOR activation in MTC. The present study used the phosphorylation of Akt1 protein at serine 473 (pAkt473) as an indicator of signaling activation. Immunohistochemistry (IHC) staining and western blot analyses were performed in order to examine the expression of ZNF703 in 34 cases of MTC and 12 cases of corresponding normal thyroid tissues. ZNF703 expression in MTC was significantly higher compared with the corresponding normal thyroid tissues (P<0.05). Furthermore, expression of ZNF703 was associated with tumor size, lymph node metastasis and advanced stage of disease. IHC also demonstrated that the level of ZNF703 was positively correlated with p-Akt473 in the 34 cases of MTC. The human MTC cell line TT was selected for further investigation as TT cells exhibit Akt/mTOR activation. The biological effects of silencing ZNF703 in TT cells on proliferation and apoptosis, both in vitro and in vivo were investigated in the present study. ZNF703 silencing inhibited the proliferation of TT cells in vitro and inhibited xenograft tumor growth in vivo. These effects were accompanied by the substantial decrease of pAkt473 and the induction of p53 protein. These results demonstrate that ZNF703 may play a relevant role in MTC due to its association with the Akt/mTOR signaling pathway.

Somatostatin receptor subtype 1 might be a predictor of better response to therapy in medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC) is a malignant neoplasm of parafollicular cells. Because it is a neuroendocrine tumor, it has known somatostatin receptors (SSTRs). The actual frequencies of the SSTR subtypes and their potential influences (by binding with endogenous somatostatin) on MTC cell proliferation have not been fully elucidated to date. The present study evaluated the occurrence of SSTR subtypes 1, 2, 3 and 5 as well as the possible role that each subtype plays in the clinical evolution of patients with MTC. This retrospective, longitudinal study analyzed thyroid surgical material from 42 patients with MTC. Immunohistochemical staining was performed with monoclonal antibodies against subtypes 1, 2, 3 and 5 of SSTR. The histological material was classified as negative, focal positive or diffuse positive, in relation to each of the SSTR subtypes. The initial response to treatment, clinical course and patient mortality rate were assessed and related to the presence of SSTR subtypes. The most prevalent SSTR subtype was SSTR 3, which was found in 81% of the patients, when considering any pattern of positivity. However, subtype 2 had the lowest number of positive patients, with 28.6% demonstrating any positive pattern. Subtypes 1 and 5 had an intermediate prevalence of positivity, with subtype 1 present in 45.2% of the patients and subtype 5 positive in 54.8% of the patients, when considering any pattern of positivity. The presence of STR 1, in the form of diffuse positivity, independently predicted a better response to the initial therapy, with a hazard ratio (HR) of 4.80 (p = 0.03). This is the first study to show the correlation of the presence of SSTR1, detected by monoclonal immunohistochemical techniques, and better response to initial treatment and possibly better long-term clinical response in patients with MTC. In addition, these patients had low positivity rates for SSTR2, which might explain the low sensitivity of diagnostic and limited therapeutic response to octrotide based radioisotopes.

Protein Kinase C: A Putative New Target for the Control of Human Medullary Thyroid Carcinoma Cell Proliferation in Vitro

We investigate the role of protein kinase C (PKC) in the control of medullary thyroid carcinoma (MTC) cell proliferation by a PKC inhibitor, Enzastaurin, in human MTC primary cultures and in the TT cell line. We found that PKC inhibition reduces cell proliferation by inducing caspase-mediated apoptosis and blocks the stimulatory effect of IGF-I on calcitonin secretion. Enzastaurin reduces PKCβII (Thr500) phosphorylation, indicating a direct involvement of this isoform as well as the phosphorylated levels of Akt (Ser 473) and glycogen synthase kinase (Ser9), PKC pathway downstream targets and pharmacodynamic markers for PKC inhibition. PKCβII and PKCδ enzyme isoforms expression and localization were investigated. These data indicate that in vitro PKC is involved in the control of human MTC proliferation and survival by modulating apoptosis, with a mechanism that implicates PKCβII inhibition and translocation in different subcellular compartments. Targeting PKC may represent a useful therapeutic approach for controlling MTC proliferation.

Abstract 2181: Tumor suppressor role of Notch3 in medullary thyroid carcinoma

Abstract Introduction: NOTCH3 is a transmembrane receptor that appears to be absent in the neuroendocrine (NE) tumors. We have previously demonstrated that transient expression of the active portion of NOTCH3 (NICD3) alters the NE phenotype and inhibits the proliferation of carcinoid tumor cells in vitro. However, the long term effect of activation this pathway is not clearly understood in Medullary Thyroid Carcinoma (MTC). To elucidate the role of Notch3 in NE, we established a doxycycline inducible NOTCH3 intracellular domain in MTC TT cells. The aim of this study was to assess the long term effect of NOTCH3 and determine the mechanism of inhibition of TT cells growth. Methods: Doxycycline inducible TT-NOTCH3 cells were created by stable transfection of Tet-On vector followed by transfection of pRevTRE-NOTCH3 plasmid. Functional analysis of NICD3 was done by measuring the degree of luciferase activity by CBF binding assay. Quantification of HES and HEY gene expression (Notch signaling mediators) was done by real time RT-PCR. Effect of Notch3 on NE markers -chromogranin A (CgA) and achaete-scute complex-like1 (ASCL1) – was assessed by Western blot analysis. The influence of NOTCH3 overexpression on TT-NOTCH cell proliferation was measured by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) rapid colorimetric assay. The mechanism of growth inhibition was determined for apoptosis markers by Western blot and flow cytometry analyses. Results: Treatment of TT-NOTCH3 cells with doxycycline led to an induction of NOTCH3 protein in a dose-dependent manner. Increase in the CBF1 binding activity showed that the NOTCH3 protein is functional and this is associated with changes in transcriptional level of HES and HEY families. More importantly, NOTCH3 activation led to a dose dependant reduction of NE markers ASCL1 and CgA. In addition, NOTCH3 activity is required to suppress MTC cell proliferation and the level of growth regression depends on the amount of NOTCH3 protein expressed. Finally, Western blot analysis and flow cytometry experiments indicated that the growth inhibition is due to apoptosis. Conclusions: We demonstrate, for the first time, that overexpression of NOTCH3 in MTC cells suppresses tumor cells growth by promoting apoptosis. Moreover, Notch3 pathway is functional as active NOTCH3 triggers the CBF-dependent transcriptional network. Finally, the activation of Notch3 in the in vitro model resulted in down-regulation of NE markers indicated that this pathway is conserved in MTC. Therefore, activation of Notch3 could be a therapeutic strategy to treat patients with MTC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2181. doi:10.1158/1538-7445.AM2011-2181

The tyrosine kinase inhibitor ZD6474 blocks proliferation of RET mutant medullary thyroid carcinoma cells

Oncogenic conversion of the RET tyrosine kinase is a frequent feature of medullary thyroid carcinoma (MTC). ZD6474 (vandetanib) is an ATP-competitive inhibitor of RET, epidermal growth factor receptor (EGFR), and vascular endothelial growth factor receptors kinases. In this study, we have studied ZD6474 mechanism of action in TT and MZ-CRC-1 human MTC cell lines, carrying cysteine 634 to tryptophan (C634W) and methionine 918 to threonine (M918T) RET mutation respectively. ZD6474 blunted MTC cell proliferation and RET, Shc and p44/p42 mitogen-activated protein kinase (MAPK) phosphorylation. Single receptor knockdown by RNA interference showed that MTC cells depended on RET for proliferation. Adoptive expression of the ZD6474-resistant V804M RET mutant rescued proliferation of TT cells under ZD6474 treatment, showing that RET is a key ZD6474 target in these MTC cells. Upon RET inhibition, adoptive stimulation of EGFR partially rescued TT cell proliferation, MAPK signaling, and expression of cell-cycle-related genes. This suggests that simultaneous inhibition of RET and EGFR by ZD6474 may overcome the risk of MTC cells to escape from RET blockade through compensatory over-activation of EGFR.

Role of Pituitary Tumour Transforming Gene 1 in Medullary Thyroid Carcinoma

Background: Pituitary tumour transforming gene 1 (PTTG1) is over-expressed in a variety of endocrine-related tumours. We aimed at evaluatingPTTG1expression and function in human neoplastic parafollicular C-cells, represented by medullary thyroid carcinoma (MTC) and C-cell hyperplasia (CCH) samples and by the TT cell line.Methods: TT cells and tissues derived from human CCH (8 samples) and MTC (12 samples) were analyzed by northern blot, furthermore TT cells were subjected to PTTG gene silencing and cells were analyzed for DNA synthesis.Results:PTTG1expression was significantly higher (p&lt; 0.01) in CCH (3-fold), in papillary thyroid cancer and in MTC (5-fold) than in normal thyroid, and in MTC lymph-node metastases as compared to primary lesions (~2-fold; p &lt; 0.05).PTTG1mRNA and protein correlated with tumour diameter and TNM status (p &lt; 0.05). In TT cells,PTTG1silencing did not completely block DNA synthesis, but significantly reduced [3H]Thymidine incorporation (~50%; p &lt; 0.01) for up to 3 days.Conclusion:PTTG1levels correlate with tumour aggressiveness.PTTG1silencing causes reduced MTC cell proliferation, supporting the hypothesis thatPTTG1might have an important role in C-cell neoplastic proliferation.

Role of pituitary tumour transforming gene 1 in medullary thyroid carcinoma.

Background: Pituitary tumour transforming gene 1 (PTTG1) is over-expressed in a variety of endocrine-related tumours. We aimed at evaluating PTTG1 expression and function in human neoplastic parafollicular C-cells, represented by medullary thyroid carcinoma (MTC) and C-cell hyperplasia (CCH) samples and by the TT cell line. Methods: TT cells and tissues derived from human CCH (8 samples) and MTC (12 samples) were analyzed by northern blot, furthermore TT cells were subjected to PTTG gene silencing and cells were analyzed for DNA synthesis. Results: PTTG1 expression was significantly higher (p < 0.01) in CCH (3-fold), in papillary thyroid cancer and in MTC (5-fold) than in normal thyroid, and in MTC lymph-node metastases as compared to primary lesions (~2-fold; p < 0.05). PTTG1 mRNA and protein correlated with tumour diameter and TNM status (p < 0.05). In TT cells, PTTG1 silencing did not completely block DNA synthesis, but significantly reduced [3H]Thymidine incorporation (~50%; p < 0.01) for up to 3 days. Conclusion: PTTG1 levels correlate with tumour aggressiveness. PTTG1 silencing causes reduced MTC cell proliferation, supporting the hypothesis that PTTG1 might have an important role in C-cell neoplastic proliferation.

Search for Materials that Influence Human Medullary Thyroid Carcinoma Cell Proliferation

1 , ABSTRACT Background: Surgical excision is the only effective treatment of medullary thyroid carcinoma (MTC) and there is no certain treatment for recurrence or distant metastasis. Materials that influence MTC cell proliferation were recently reported. Presently, we evaluated the influence of dexamethasone, somatostatin, progesterone, estradiol-17-beta, forskolin and gastrin on MTC cell proliferation and calcitonin secretion. Methods: Genomic DNA was extracted and sequenced from untreated thyroid TT cells and cells treated with 10

Cdk5 Regulates STAT3 Activation and Cell Proliferation in Medullary Thyroid Carcinoma Cells

The biological behaviors of thyroid cancer are varied, and the pathological mechanisms remain unclear. Some reports indicated an apparent aggregation of amyloid accompanying medullary thyroid carcinoma (MTC). Amyloid aggregation in neurodegeneration leads to hyperactivation of Cdk5 and subsequent neuronal death. Based on the connection with amyloid, the role of Cdk5 in MTC is worthy of investigation. Initially, the expression of Cdk5 and its activator, p35, in MTC cell lines was identified. Cdk5 inhibition by specific inhibitors or short interfering RNA decreased the proliferation of MTC cell lines, which reveals the importance of Cdk5 in MTC cell growth. Although p35 cleavage has been considered as an important element in neurodegeneration, it seems that p35 cleavage was not a major cause in Cdk5 activity-dependent MTC cell proliferation because neither Cdk5 activity nor cell growth was affected by the inhibition of p35 cleavage. Clearance of amyloid by antibody neutralization indicated that MTC cell proliferation was supported by calcitonin-derived extracellular amyloid and subsequent Her2 and Cdk5 activation. Significantly, the STAT3 pathway was involved in Cdk5-dependent proliferation of MTC cells through Ser-727 phosphorylation. In addition, Cdk5 inhibition reduced nuclear distributions of both the Cdk5-p35 complex and phospho-STAT3 in MTC cells. Finally, Cdk5 inhibition retarded tumor formation in vivo accompanying the reduction of phospho-STAT3. Our findings suggest the first demonstration of a novel and specific role for Cdk5 kinase in supporting the proliferation of the medullary thyroid carcinoma cells and could shed light on a new field for diagnosis and therapy of thyroid cancer.

Inhibition of medullary thyroid carcinoma cell proliferation and RET phosphorylation by tyrosine kinase inhibitors

Background. Most medullary thyroid carcinomas (MTCs) result from gain-of-function mutations in the RET proto-oncogene, which encodes a transmembrane tyrosine kinase receptor. Systemic therapies have not been effective in treating this disease. We evaluated the effects of 3 tyrosine kinase inhibitors (TKIs) on MTC cell growth and RET tyrosine kinase activity by using an in vitro model. Methods. An MTC cell line (TT cells, RETc634 mutant) cultured in RPMI medium was exposed to varying concentrations of STI571, genistein, or allyl-geldanamycin with controls (no TKI) for 3 to 48 hours. Cellular protein was analyzed by immunoprecipitated Western blot analysis probing with a monoclonal antiphosphotyrosine antibody. Cell proliferation was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and 5-bromo-2′-deoxyuridine (BrdU) assays. Results. RET phosphorylation was inhibited at 24 hours of exposure to 5 to 20 μmol/L STI571 and 48 hours of exposure to genistein (200 μmol/L) and allyl-geldanamycin (6 μmol/L). RET protein was detected in equal concentrations in all experimental conditions. MTT and BrdU assays demonstrated a dose-dependent decrease in TT cell proliferation with exposure to the 3 TKIs. Conclusions. These TKIs selectively inhibit cell growth and RET tyrosine kinase activity of MTC cells in vitro in a dose manner. This study suggests the use of TKIs in human trials as a systemic therapy for MTC. (Surgery 2002;132:960-7.)