Yin Xiong

MiR-886-3p Regulates Cell Proliferation and Migration, and Is Dysregulated in Familial Non-Medullary Thyroid Cancer

Background The molecular basis and characteristics of familial non-medullary thyroid cancer are poorly understood. In this study, we performed microRNA (miRNA) profiling of familial and sporadic papillary thyroid cancer tumor samples. Methodology/Principal Findings Genome wide miRNA profiling of sporadic and familial papillary thyroid cancer was performed. Differentially expressed miRNAs were validated by quantitative RT-PCR. Ectopic expression of miR-886-3p in thyroid cancer lines was performed to identify pathways targeted by the miRNA, as well as, to determine its effect on tumor cell biology. We found four differentially expressed miRNAs between familial and sporadic papillary thyroid cancer tumor samples. MiR-886-3p and miR-20a were validated to be differentially expressed by 3- and 4-fold, respectively. Pathway analysis of genome-wide expression data on cells overexpressing miR-886-3p and target prediction analysis showed genes involved in DNA replication and focal adhesion pathways were regulated by miR-886-3p. Overexpression of miR-886-3p in thyroid cancer cell lines significantly inhibited cellular proliferation, the number and size of spheroids and cellular migration. Additionally, overexpression of miR-886-3p increased the number of cells in S phase. Conclusions/Significance Our findings for the first time suggest that miR-886-3p plays an important role in thyroid cancer tumor cell biology and regulates genes involved in DNA replication and focal adhesion. Thus, miR-886-3p may play a role in the initiation and or progression of papillary thyroid cancer.

miR-145 suppresses thyroid cancer growth and metastasis and targets AKT3.

The expression and function of miR-145 in thyroid cancer is unknown. We evaluated the expression and function of miR-145 in thyroid cancer and its potential clinical application as a biomarker. We found that the expression of miR-145 is significantly downregulated in thyroid cancer as compared with normal. Overexpression of miR-145 in thyroid cancer cell lines resulted in: decreased cell proliferation, migration, invasion, VEGF secretion, and E-cadherin expression. miR-145 overexpression also inhibited the PI3K/Akt pathway and directly targeted AKT3. In vivo, miR-145 overexpression decreased tumor growth and metastasis in a xenograft mouse model, and VEGF secretion. miR-145 inhibition in normal primary follicular thyroid cells decreased the expression of thyroid cell differentiation markers. Analysis of indeterminate fine-needle aspiration samples showed miR-145 had a 92% negative predictive value for distinguishing benign from malignant thyroid nodules. Circulating miR-145 levels were significantly higher in patients with thyroid cancer and showed a venous gradient. Serum exosome extractions revealed that miR-145 is secreted. Our findings suggest that miR-145 is a master regulator of thyroid cancer growth, mediates its effect through the PI3K/Akt pathway, is secreted by the thyroid cancer cells, and may serve as an adjunct biomarker for thyroid cancer diagnosis.

MiR-20a Is Upregulated in Anaplastic Thyroid Cancer and Targets LIMK1

Background There have been conflicting reports regarding the function of miR-20a in a variety of cancer types and we previously found it to be dysregulated in sporadic versus familial papillary thyroid cancer. In this study, we studied the expression of miR-20a in normal, benign and malignant thyroid samples, and its effect on thyroid cancer cells in vitro and in vivo. Methodology/Principal Findings The expression of miR-20a in normal, benign and malignant thyroid tissue was determined by quantitative RT-PCR. Thyroid cancer cells were transfected with miR-20a and the effect on cellular proliferation, tumor spheroid formation, and invasion was evaluated. Target genes of miR-20 were determined by genome-wide mRNA expression analysis with miR-20a overexpression in thyroid cancer cells and target prediction database. Target genes were validated by quantitative PCR and immunoblotting, and luciferase assays. MiR-20a expression was significantly higher in anaplastic thyroid cancer than in differentiated thyroid cancer, and benign and normal thyroid tissues. MiR-20a significantly inhibited thyroid cancer cell proliferation in vitro (p<0.01) and in vivo (p<0.01), tumor spheroid formation (p<0.05) and invasion (p<0.05) in multiple thyroid cancer cell lines. We found that LIMK1 was a target of miR-20a in thyroid cancer cell lines and direct knockdown of LIMK1 recapitulated the effect of miR-20a in thyroid cancer cells. Conclusions/Significance To our knowledge, this is the first study to demonstrate that miR-20a plays a role as a tumor suppressor in thyroid cancer cells and targets LIMK1. Our findings suggest the upregulated expression of miR-20a in anaplastic thyroid cancer counteracts thyroid cancer progression and may have therapeutic potential.

An In Vivo Mouse Model of Metastatic Human Thyroid Cancer

BACKGROUND Mouse models of metastatic human cancers are important tools in preclinical studies for testing new systematic therapies and studying effectors of cancer metastasis. The major drawbacks of current mouse models for metastatic thyroid cancer are that they have low metastasis rates and do not allow in vivo tumor detection. Here, we report and characterize an in vivo detectable metastasis mouse model of human thyroid cancer using multiple thyroid cancer cell lines. METHODS Human anaplastic thyroid cancer cell lines 8505C, C-643, SW-1736, and THJ-16T; follicular thyroid cancer cell lines FTC-133, FTC-236, and FTC-238; and Hürthle cell carcinoma cell line XTC-1 were transfected with a linearized pGL4.51[luc2/CMV/Neo] vector or transduced with lentivirus containing Luc2-eGFP reporter genes. The stably transfected cells were injected intravenously into NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ mice. Tumors were detected with an in vivo imaging system-Xenogen IVIS. Vemurafenib, a BRAF inhibitor, was used to treat lung metastases generated from 8505C-Luc2 cells with a BRAF(V600E) mutation to test the accuracy of the model to evaluate response to therapy. RESULTS Intravenous injection of as few as 30,000 8505C-Luc2 cells produced lung metastases in 100% of the injected mice, and many of these mice also developed bone metastases at a later stage of the disease. Similarly, metastatic tumors also developed in all mice injected with C-643-Luc2, THJ-16T-Luc2, FTC-133-Luc2, FTC-236-Luc2, FTC-238-Luc2, and XTC-1-Luc2 cells. The metastases were easily detectable in vivo, and tumor progression could be dynamically and accurately followed and correlated with the actual tumor burden. Furthermore, disease progression could be easily controlled by adjusting the number of injected cells. The in vivo treatment of 8505C xenograft lung metastases with vemurafenib dramatically reduced the growth and signal intensity with good correlation with actual tumor burden. CONCLUSIONS Herein we report an in vivo detectable mouse model of metastatic human thyroid cancer that is reliable and reproducible. It will serve as a useful tool in the preclinical testing of alternative systematic therapies for metastatic thyroid cancer, and for functional studies of thyroid cancer tumor biology in vivo.

miR-126-3p Inhibits Thyroid Cancer Cell Growth and Metastasis, and Is Associated with Aggressive Thyroid Cancer

Background Previous studies have shown that microRNAs are dysregulated in thyroid cancer and play important roles in the post-transcriptional regulation of target oncogenes and/or tumor suppressor genes. Methodology/Principal Findings We studied the function of miR-126-3p in thyroid cancer cells, and as a marker of disease aggressiveness. We found that miR-126-3p expression was significantly lower in larger tumors, in tumor samples with extrathyroidal invasion, and in higher risk group thyroid cancer in 496 papillary thyroid cancer samples from The Cancer Genome Atlas study cohort. In an independent sample set, lower miR-126-3p expression was observed in follicular thyroid cancers (which have capsular and angioinvasion) as compared to follicular adenomas. Mechanistically, ectopic overexpression of miR-126-3p significantly inhibited thyroid cancer cell proliferation, in vitro (p<0.01) and in vivo (p<0.01), colony formation (p<0.01), tumor spheroid formation (p<0.05), cellular migration (p<0.05), VEGF secretion and endothelial tube formation, and lung metastasis in vivo. We found 14 predicted target genes, which were significantly altered upon miR-126-3p transfection in thyroid cancer cells, and which are involved in cancer biology. Of these 14 genes, SLC7A5 and ADAM9 were confirmed to be inhibited by miR-126-3p overexpression and to be direct targets of miR-136-3p. Conclusions/Significance To our knowledge, this is the first study to demonstrate that miR-126-3p has a tumor-suppressive function in thyroid cancer cells, and is associated with aggressive disease phenotype.

Diagnosis of thyroid cancer: state of art

INTRODUCTION Thyroid cancer is the most common endocrine cancer in the USA and its incidence is increasing worldwide. Thyroid fine-needle aspiration biopsy (FNA) and cytologic analysis is the most cost-effective approach to distinguish between malignant and benign thyroid nodules. However, up to 30% of thyroid FNA biopsy results are inconclusive. AREAS COVERED In this article, the authors provide an update on the current status and emerging approaches for improving thyroid cancer diagnosis. This review covers imaging, genetic and genomic approaches being used or in development to help distinguish between malignant and benign thyroid nodules. EXPERT OPINION There has been considerable progress in improving thyroid cancer diagnosis. The molecular markers analysis to avoid diagnostic surgeries seems to be promising. However, the clinical utility and accuracy of some markers reported in this review are not conclusive and need to be validated as clinical diagnostic tool.

Testosterone regulates thyroid cancer progression by modifying tumor suppressor genes and tumor immunity

Cancer gender disparity has been observed for a variety of human malignancies. Thyroid cancer is one such cancer with a higher incidence in women, but more aggressive disease in men. There is scant evidence on the role of sex hormones on cancer initiation/progression. Using a transgenic mouse model of follicular thyroid cancer (FTC), we found castration led to lower rates of cancer in females and less advanced cancer in males. Mechanistically, less advanced cancer in castrated males was due to increased expression of tumor suppressor (Glipr1, Sfrp1) and immune-regulatory genes and higher tumor infiltration with M1 macrophages and CD8 cells. Functional study showed that GLIPR1 reduced cell growth and increased chemokine secretion (Ccl5) that activates immune cells. Our data demonstrate that testosterone regulates thyroid cancer progression by reducing tumor suppressor gene expression and tumor immunity.

Abstract 3098: MiRNA-145 is a master regulator of the hallmarks of thyroid cancer.

microRNA-145 (miR-145) in an important member of the family of microRNAs which are downregulated in several types of tumors. However, miR-145 expression and function in thyroid cancer and in cellular differentiation is unknown. In our study, we performed miRNA profiling in normal, benign and malignant thyroid cancers including undifferentiated tumors with validation of differentially expressed miRNAs by quantitative RT PCR. We found significant downregulation of miR-145 in differentiated and undifferentiated thyroid cancer. We performed functional studies in thyroid cancer cell lines and found that miR-145 decreases cell proliferation, induces cell cycle arrest, decreases cell invasion and targets the PI3K/Akt pathway, one of the most commonly activated pathways in thyroid cancer and which is associated with epithelial-mesenchymal transition (EMT). Overexpression of miR-145 in cell lines also decreased the expression of EMT markers vimentin and N-cadherin. Furthermore, inhibition of miR-145 in normal primary thyroid cultured cells decreased NIS and PAX8 expression, key regulators of thyroid cell differentiation. miRNA-145 expression in tumor tissue was inversely correlated with VEGF serum levels in the same patients and overexpression of miR-145 in vitro decreased angiogenesis and VEGF secretion. MiRNA-145 has important regulatory effects on the hallmarks of thyroid cancer cells and thus may be involved in thyroid cancer progression. Citation Format: Myriem Boufraqech, Lisa Zhang, Meenu Jain, Neelam Gulati, Naris Nilubol, Mio Kitano, Yin Xiong, Electron Kebebew. MiRNA-145 is a master regulator of the hallmarks of thyroid cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3098. doi:10.1158/1538-7445.AM2013-3098

Abstract 4191: MiR-20a inhibits thyroid cancer cell growth and invasion through LIMK1.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC There have been conflicting reports regarding the function of miR-20a in a variety of cancers. In this study, we evaluated the expression and function of miR-20a in thyroid cancer tissue and cell lines in vitro and in vivo. We found miR-20a was significantly downregulated in undifferentiated and aggressive thyroid cancer (p<0.05). Ectopic overexpression of miR-20a significantly inhibited thyroid cancer cell proliferation in vitro (p < 0.01) and in vivo (p < 0.01), tumor spheroid formation (p < 0.05), and invasion (p < 0.05) in multiple thyroid cancer cell lines. We performed integrated transcriptome analysis with and without miR-20a overexpression and target prediction analysis, and identified LIMK1 as a target of miR-20a. Luciferase assay demonstrated that LIMK1 was a direct target of miR-20a and that direct knockdown of LIMK1 recapitulated the effects of miR-20a in multiple thyroid cancer lines. Our findings suggest that miR-20a has a tumor suppressive function in thyroid cancer which is mediated through LIMK1 and that loss of miR-20a expression may be involved in thyroid cancer progression. Citation Format: Yin Xiong, Lisa Zhang, Electron Kebebew. MiR-20a inhibits thyroid cancer cell growth and invasion through LIMK1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4191. doi:10.1158/1538-7445.AM2013-4191

Abstract 3041: Differential expression of microRNAs between sporadic and hereditary nonmedullary thyroid cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: MicroRNAs (miRNAs) are small noncoding RNAs, which regulate gene expression and play a significant role in many biological processes, including tumorigenesis. Approximately 5% of all non-medullary thyroid cancers are hereditary and may have more aggressive tumor biology than sporadic cases. The susceptibility gene for hereditary non-medullary thyroid cancer has not been identified. We hypothesized that miRNA expression profile between sporadic and hereditary non-medullary thyroid cancer are different and could help identify candidate suspceptibility genes. Methods: Twenty nine tumor tissue samples were analyzed using miRNA microarrays for 1190 miRNAs with genotyping for common somatic mutations (BRAF V600E, NRAS, KRAS, RET/PTC1, RET/PTC3) present in non-medullary thyroid cancer. The samples were matched for age, gender and stage of cancer. Differences in miRNA expression profile were validated by quantitative RT-PCR and correlated with clinicopathologic data. Results: We found 5 miRNAs significantly differentially expressed between sporadic and hereditary non-medullary thyroid cancer cases; two miRNAs were upregulated and three miRNAs were downregulated in hereditary non-medullary thyroid cancer. Two miRNAs were validated by quantitative RT-PCR; miR-20a (p=0.025) and miR-886-3p (p=0.028) were upregulated in hereditary non-medullary thyroid cancer samples. Higher expression level of miR-886-3p was significantly associated with higher TNM stage (p<0.05). There was no significant difference in somatic mutation rates between sporadic and hereditary cases (9 BRAF V600E, 1 RET/PTC1), and there was no significant difference in miR-20a and miR-886-3p expression by mutation status. Conclusions: To our knowledge, this is the first study to show different expression profile of miRNAs between sporadic and hereditary cancer using non-medullary thyroid cancer as a model. These differences in miRNAs may have an important role in tumor cell biology and clinical manifestation of non-medullary thyroid cancer in sporadic versus hereditary cases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3041.