Anaplastic Thyroid Carcinoma Samples Research Articles

Anaplastic and poorly differentiated thyroid carcinomas: genetic evidence of high-grade transformation from differentiated thyroid carcinoma.

Anaplastic thyroid carcinoma (ATC) is the most advanced and aggressive thyroid cancer, and poorly differentiated thyroid carcinoma (PDTC) lacks anaplastic histology but has lost architectural and cytologic differentiation. Only a few studies have focused on the genetic relationship between the two advanced carcinomas and coexisting differentiated thyroid carcinomas (DTCs). In the present study, we investigated clinicopathologic features and genetic profiles in 57 ATC and PDTC samples, among which 33 cases had concomitant DTC components or DTC history. We performed immunohistochemistry for BRAF V600E, p53, and PD-L1 expression, Sanger sequencing for TERT promoter and RAS mutations, and fluorescence in situ hybridization for ALK and RET rearrangements. We found that ATCs and PDTCs shared similar gene alterations to their coexisting DTCs, and most DTCs were aggressive subtypes harboring frequent TERT promoter mutations. A significantly higher proportion of ATCs expressed p53 and PD-L1, and a lower proportion expressed PAX-8 and TTF-1, than the coexisting DTCs. Our findings provide more reliable evidence that ATCs and PDTCs are derived from DTCs.

Immunological characteristics of immunogenic cell death genes and malignant progression driving roles of TLR4 in anaplastic thyroid carcinoma

Anaplastic thyroid carcinoma (ATC) was a rare malignancy featured with the weak immunotherapeutic response. So far, disorders of immunogenic cell death genes (ICDGs) were identified as the driving factors in cancer progression, while their roles in ATC remained poorly clear. Datasets analysis identified that most ICDGs were high expressed in ATC, while DE-ICDGs were located in module c1_112, which was mainly enriched in Toll-like receptor signalings. Subsequently, the ICD score was established to classify ATC samples into the high and low ICD score groups, and function analysis indicated that high ICD score was associated with the immune characteristics. The high ICD score group had higher proportions of specific immune and stromal cells, as well as increased expression of immune checkpoints. Additionally, TLR4, ENTPD1, LY96, CASP1 and PDIA3 were identified as the dynamic signature in the malignant progression of ATC. Notably, TLR4 was significantly upregulated in ATC tissues, associated with poor prognosis. Silence of TLR4 inhibited the proliferation, metastasis and clone formation of ATC cells. Eventually, silence of TLR4 synergistically enhanced paclitaxel-induced proliferation inhibition, apoptosis, CALR exposure and release of ATP. Our findings highlighted that the aberrant expression of TLR4 drove the malignant progression of ATC, which contributed to our understanding of the roles of ICDGs in ATC.

The Tyrosine Kinase Inhibitor Lenvatinib Inhibits Anaplastic Thyroid Carcinoma Growth by Targeting Pericytes in the Tumor Microenvironment.

Background: Anaplastic thyroid carcinoma (ATC) is a rapidly fatal cancer with a median survival of a few months. Enhanced therapeutic options for durable management of ATC will rely on an understanding of genetics and the role of the tumor microenvironment. The prognosis for patients with ATC has not improved despite more detailed scrutiny of underlying tumor genetics. Pericytes in the microenvironment play a key evasive role for thyroid carcinoma (TC) cells. Lenvatinib improves outcomes in patients with radioiodine-refractory well-differentiated TC. In addition to the unclear role of pericytes in ATC, the effect and mechanism of lenvatinib efficacy on ATC have not been sufficiently elucidated. Design: We assessed pericyte enrichment in ATC. We determined the effect of lenvatinib on ATC cell growth cocultured with pericytes and in a xenograft mouse model from human BRAFWT/V600E-ATC-derived cells coimplanted with pericytes. Results: ATC samples were significantly enriched in pericytes compared with normal thyroid samples. BRAFWT/V600E-ATC-derived cells were resistant to lenvatinib treatment shown by a lack of suppression of MAPK and Akt pathways. Moreover, lenvatinib increased CD47 protein (thrombospondin-1 [TSP-1] receptor) levels over time vs. vehicle. TSP-1 levels were downregulated upon lenvatinib at late vs. early time points. Critically, ATC cells, when cocultured with pericytes, showed increased sensitivity to this therapy and ultimately decreased number of cells. The coimplantation in vivo of ATC cells with pericytes increased ATC growth and did not downregulate TSP-1 in the microenvironment in vivo. Conclusions and Implications: Pericytes are enriched in ATC samples. Lenvatinib showed inhibitory effects on BRAFWT/V600E-ATC cells in the presence of pericytes. The presence of pericytes could be crucial for effective lenvatinib treatment in patients with ATC. Degree of pericyte abundance may be an attractive prognostic marker in assessing pharmacotherapeutic options. Effective durable management of ATC will rely on an understanding not only of genetics but also on the role of the tumor microenvironment.

Siglec-15 Silencing Inhibits Cell Proliferation and Promotes Cell Apoptosis by Inhibiting STAT1/STAT3 Signaling in Anaplastic Thyroid Carcinoma.

Thyroid cancer (THCA) represents a frequently seen endocrine cancer, which can be divided as anaplastic thyroid carcinoma (ATC), follicular thyroid carcinoma (FTC), and papillary thyroid carcinoma (PTC). A total of 362 IDEGs were obtained from TCGA-THCA and IMMPORT databases, which were found to be related to BP, CC, MF, and STAT signaling pathway upon GO functional annotation and KEGG analysis. This work identified 23 survival-related hub genes using WGCNA and uniCOX analysis. In addition, a risk prognosis model was constructed to obtain a signature involving fifteen IDEGs. According to survival and univariate along with multivariate analysis, high-risk patients had markedly dismal prognostic outcome compared with low-risk counterparts. Siglec-15 belongs to one of the fifteen IDEG signature, but the precise biological roles in diverse THCA subtypes are largely unclear. In this work, Siglec-15 expression evidently increased in ATC and FTC samples compared with matched surrounding PTC and THCA samples, which was used as a diagnostic biomarker for THCA. Siglec-15 RNAi significantly inhibited cell proliferation and promoted cell apoptosis. Meanwhile, Siglec-15 knockout suppressed the expression of STAT1, STAT3, and VEGF and promoted that of cleaved caspase-3. In vivo experiments revealed that transfection with vectors expressing STAT1 and STAT3 inhibited the Siglec-15 RNAi-induced inhibition on tumor growth and the increases in CD4+/CD8+ ratio. In conclusion, Siglec-15 expression increases in ATC and FTC, which promotes THCA occurrence via the STAT1/STAT3 signaling, in particular for FTC and ATC. Therefore, it is the possible marker that can be used to diagnose and treat THCA.

Association between mutation profiles and clinicopathological features in Chinese patients with thyroid cancer

AbstractRecently, mutation profiles provided new insights into comprehensive understanding of TC biology by Next Generation Sequencing (NGS). We explored association between mutation profiles and clinicopathological features in Chinese patients with thyroid cancer (TC). Two hundred and twenty‐five formalin‐fixed, paraffin‐embedded tissue specimens from surgically removed thyroid samples were detected with 15 target genes by NGS. Mutation profiles and clinicopathological features were analyzed. Two hundred and seven mutations including two hundred mutations in 81.40% papillary thyroid carcinoma samples, three mutations in 50.00% MTC samples, and four mutations in 100% anaplastic thyroid carcinoma samples were detected. There were 19.56% samples without any mutations in target genes, 69.78% samples harbored mutations in single gene, 9.78% samples carried two gene mutations, and 0.89% samples had triple different gene mutations. For PTC, BRAF mutations were predominant, TERT mutations are more prevalent in advanced PTC and RET fusion was only observed among the PTC. For MTC, RET point mutations were predominant. For samples carried more than one gene mutations, the allelic frequency of mutants were almost similar. Multiple mutations in TC patients were significantly more frequent in cases of patients aged 55 and over (p <.001) and advanced American Joint Committee on Cancer (AJCC) cancer stage (p <.001). Gender (p = .309) and pathological subtype (p = .121) did not show significant correlation with mutations. Analysis between mutation profiles and clinicopathological features provides new insights into the biology of TC and is expected to increase the accuracy of diagnosis and prognostication in TC, leading to improved precision treatment for TC patients.

Expression of fibroblast growth factor receptor 4 and clinical response to lenvatinib in patients with anaplastic thyroid carcinoma: a pilot study.

Fibroblast growth factor receptor 4 (FGFR4) expression has association with tumor malignancy. In thyroid cancers, FGFR4 has been reported to be characteristically expressed in aggressive thyroid tumors, such as anaplastic thyroid carcinoma (ATC). We investigated FGFR4 expression in patients with ATC and analyzed their clinical responses to lenvatinib. Primary tumor samples were obtained from 12 patients with ATC who underwent surgery or core needle biopsy. FGFR4 protein expression in all ATC samples was analyzed via immunohistochemistry, and the treatment efficacy of lenvatinib was evaluated. The proportion of FGFR4-positive cells in the samples ranged from 0 to 50%. Four patients had partial responses, and three patients had stable diseases as a best clinical response to lenvatinib. The median PFS durations of patients with none, weak, and moderate intensity were 0.5, 3.2 (95% CI 1.1-not estimable [NE]), and 4.6 (95% CI 1.1-NE) months, respectively (p= 0.003). Because FGFR4 was expressed in ATC tissues, the FGFR4 expression might be associated with the treatment efficacy of lenvatinib in a part of ATC patients. To clarify whether FGFR4 can serve as a prognostic or predictive factor for lenvatinib therapy, more cases must be accumulated.

The Long Non-Coding RNA Prader Willi/Angelman Region RNA5 (PAR5) Is Downregulated in Anaplastic Thyroid Carcinomas Where It Acts as a Tumor Suppressor by Reducing EZH2 Activity

Anaplastic thyroid carcinoma (ATC) represents one the most aggressive neoplasias in humans, and, nowadays, limited advances have been made to extend the survival and reduce the mortality of ATC. Thus, the identification of molecular mechanism underlying its progression is needed. Here, we evaluated the long non-coding RNA (lncRNA) expression profile of nine ATC in comparison with five normal thyroid tissues by a lncRNA microarray. By this analysis, we identified 19 upregulated and 28 downregulated lncRNAs with a fold change >1.1 or <−1.1 and p-value < 0.05, in ATC samples. Some of them were subsequently validated by qRT-PCR. Then, we investigated the role of the lncRNA Prader Willi/Angelman region RNA5 (PAR5), drastically and specifically downregulated in ATC. The restoration of PAR5 reduces proliferation and migration rates of ATC-derived cell lines indicating that its downregulation contributes to thyroid cancer progression. Our results suggest that PAR5 exerts its anti-oncogenic role by impairing Enhancer of Zeste Homolog 2 (EZH2) oncogenic activity since we demonstrated that PAR5 interacts with it in thyroid cancer cell lines, reducing EZH2 protein levels and its binding on the E-cadherin promoter, relieving E-cadherin from the negative regulation by EZH2. Consistently, EZH2 is overexpressed in ATC, but not in differentiated thyroid carcinomas. The results reported here define a tumor suppressor role for PAR5 in undifferentiated thyroid neoplasias, further highlighting the pivotal role of lncRNAs in thyroid carcinogenesis.

Targeting CD47 in Anaplastic Thyroid Carcinoma Enhances Tumor Phagocytosis by Macrophages and Is a Promising Therapeutic Strategy.

Background: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive human cancers, with a median survival of only three to six months. Standard treatment options and even targeted therapies have so far failed to improve long-term overall survival. Thus, novel treatment modalities for ATC, such as immunotherapy, are urgently needed. CD47 is a “don't eat me” signal, which prevents cancer cells from phagocytosis by binding to signal regulatory protein alpha on macrophages. So far, the role of macrophages and the CD47–signal regulatory protein alpha signaling axis in ATC is not well understood.Methods: This study analyzed 19 primary human ATCs for macrophage markers, CD47 expression, and immune checkpoints by immunohistochemistry. ATC cell lines and a fresh ATC sample were assessed by flow cytometry for CD47 expression and macrophage infiltration, respectively. CD47 was blocked in phagocytosis assays of co-cultured macrophages and ATC cell lines. Anti-CD47 antibody treatment was administered to ATC cell line xenotransplanted immunocompromised mice, as well as to tamoxifen-induced ATC double-transgenic mice.Results: Human ATC samples were heavily infiltrated by CD68- and CD163-expressing tumor-associated macrophages (TAMs), and expressed CD47 and calreticulin, the dominant pro-phagocytic molecule. In addition, ATC tissues expressed the immune checkpoint molecules programmed cell death 1 and programmed death ligand 1. Blocking CD47 promoted the phagocytosis of ATC cell lines by macrophages in vitro. Anti-CD47 antibody treatment of ATC xenotransplanted mice increased the frequency of TAMs, enhanced the expression of macrophage activation markers, augmented tumor cell phagocytosis, and suppressed tumor growth. In double-transgenic ATC mice, CD47 was expressed on tumor cells, and blocking CD47 increased TAM frequencies.Conclusions: Targeting CD47 or CD47 in combination with programmed cell death 1 may potentially improve the outcomes of ATC patients and may represent a valuable addition to the current standard of care.

Integrated Bioinformatics Analysis of Master Regulators in Anaplastic Thyroid Carcinoma

Anaplastic thyroid carcinoma (ATC) is one of the most aggressive and rapidly lethal tumors. However, limited advances have been made to prolong the survival and to reduce the mortality over the last decades. Therefore, identifying the master regulators underlying ATC progression is desperately needed. In our present study, three datasets including GSE33630, GSE29265, and GSE65144 were retrieved from Gene Expression Omnibus with a total of 32 ATC samples and 78 normal thyroid tissues. A total of 1804 consistently changed differentially expressed genes (DEGs) were identified from three datasets. KEGG pathways enrichment suggested that upregulated DEGs were mainly enriched in ECM-receptor interaction, cell cycle, PI3K-Akt signaling pathway, focal adhesion, and p53 signaling pathway. Furthermore, key gene modules in PPI network were identified by Cytoscape plugin MCODE and they were mainly associated with DNA replication, cell cycle process, collagen fibril organization, and regulation of leukocyte migration. Additionally, TOP2A, CDK1, CCNB1, VEGFA, BIRC5, MAPK1, CCNA2, MAD2L1, CDC20, and BUB1 were identified as hub genes of the PPI network. Interestingly, module analysis showed that 8 out of 10 hub genes participated in Module 1 network and more than 70% genes of Module 2 consisted of collagen family members. Notably, transcription factors (TFs) regulatory network analysis indicated that E2F7, FOXM1, and NFYB were master regulators of Module 1, while CREB3L1 was the master regulator of Module 2. Experimental validation showed that CREB3L1, E2F7, and FOXM1 were significantly upregulated in ATC tissue and cell line when compared with normal thyroid group. In conclusion, the TFs regulatory network provided a more detail molecular mechanism underlying ATC occurrence and progression. TFs including E2F7, FOXM1, CREB3L1, and NFYB were likely to be master regulators of ATC progression, suggesting their potential role as molecular therapeutic targets in ATC treatment.

The LAT1 inhibitor JPH203 reduces growth of thyroid carcinoma in a fully immunocompetent mouse model

BackgroundThe L-type amino acid transporter 1 (LAT1/SLC7A5) transports essential amino acids across the plasma membrane. While LAT1 is overexpressed in a variety of human neoplasms, its expression and its role in thyroid cancer is currently unknown. Anaplastic thyroid carcinoma (ATC) is a highly aggressive malignancy for which no effective therapy exists. The purpose of this study was to explore whether the inhibition of LAT1 in ATC would affect tumor growth both in vitro and in vivo.MethodsLAT1 was pharmacologically blocked by JPH203 in human ATC and papillary thyroid cancer (PTC) cell lines. The effects on proliferation and mTORC1 activity were addressed in vitro. A genetically engineered mouse model of ATC was used to address the effect of blocking LAT1 on tumor growth in vivo. SLC7A5 transcription was measured in patient-derived ATC samples to address the clinical relevance of the findings.ResultsLAT1 block by JPH203 reduced proliferation and mTORC1 signaling in human thyroid cancer cell lines. SLC7A5 transcription was upregulated in ATC tissues derived from a genetically engineered mouse model and in ATC samples recovered from patients. JPH203 treatment induced thyroid tumor growth arrest in vivo in a fully immunocompetent mouse model of thyroid cancer. Additionally, analysis of publicly available datasets of thyroid carcinomas revealed that high LAT1 expression is associated with potentially untreatable PTC presenting reduced NIS/SLC5A5 transcription and with ATC.ConclusionsThese preclinical results show that LAT1 inhibition is a novel therapeutic approach in the context of thyroid cancers, and more interestingly in untreatable thyroid cancers.

Identification of Key Pathways and Genes in Anaplastic Thyroid Carcinoma via Integrated Bioinformatics Analysis.

BackgroundTo provide a better understanding of anaplastic thyroid carcinoma (ATC) at the molecular level, this study aimed to identify the genes and key pathways associated with ATC by using integrated bioinformatics analysis.Material/MethodsBased on the microarray data GSE9115, GSE65144, and GSE53072 derived from the Gene Expression Omnibus, the differentially expressed genes (DEGs) between ATC samples and normal controls were identified. With DEGs, we performed a series of functional enrichment analyses. Then, a protein–protein interaction (PPI) network was constructed and visualized, with which the hub gene nodes were screened out. Finally, modules analysis for the PPI network was performed to further investigate the potential relationships between DEGs and ATC.ResultsA total of 537 common DEGs were screened out from all 3 datasets, among which 247 genes were upregulated and 275 genes were downregulated. GO analysis indicated that upregulated DEGs were mainly involved in cell division and mitotic nuclear division and the downregulated DEGs were significantly enriched in ventricular cardiac muscle cell action potential. KEGG pathway analysis showed that the upregulated DEGs were mainly enriched in cell cycle and ECM-receptor interaction and the downregulated DEGs were mainly enriched in thyroid hormone synthesis, insulin resistance, and pathways in cancer. The top 10 hub genes in the constructed PPI network were CDK1, CCNB1, TOP2A, AURKB, CCNA2, BUB1, AURKA, CDC20, MAD2L1, and BUB1B. The modules analysis showed that genes in the top 2 significant modules of PPI network were mainly associated with mitotic cell cycle and positive regulation of mitosis, respectively.ConclusionsWe identified a series of key genes along with the pathways that were most closely related with ATC initiation and progression. Our results provide a more detailed molecular mechanism for the development of ATC, shedding light on the potential biomarkers and therapeutic targets.

Inhibition of X-linked inhibitor of apoptosis protein suppresses tumorigenesis and enhances chemosensitivity in anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma (ATC) is one of the most lethal carcinoma with a poor prognosis; however, molecular mechanisms underlying the aggressiveness of ATC remain unclear. Our goal was to examine the expression of X-linked inhibitor of apoptosis protein (XIAP) in ATC, as well as its role in ATC tumorigenesis. This is a retrospective study of ATC patients from the Second Affiliated Hospital of Harbin Medical University during June 2003 to October 2013. The expression of XIAP in tumor specimens of ATC patients was examined by immunohistochemical staining. The roles of XIAP in proliferation, migration, invasion, and chemoresistance were investigated by shRNA mediated-knockdown of XIAP in human ATC cell lines. The effect of XIAP on tumorigenesis was evaluated using a xenograft tumor model with nude mice. XIAP expression was significantly higher in the invasive area of ATC samples, whereas XIAP expression was negative in either normal thyroid follicular epithelial cells or the differentiated papillary thyroid carcinoma. XIAP-depleted ATC cells showed a remarkable decrease in the proliferation, migration, and invasion compared with the scramble group. Knockdown of XIAP expression significantly enhanced the chemosensitivity of WRO and SW1736 cells to docetaxel or taxane. Moreover, knockdown of XIAP significantly suppressed ATC tumorigenesis in vivo. XIAP is highly expressed in ATC cells and tumors. XIAP play important roles in tumor behaviors and chemosensitivity of ATC cells. XIAP may function in ATC aggressiveness and may serve as a potential therapeutic target for ATC treatment.

Selinexor (KPT-330) has antitumor activity against anaplastic thyroid carcinoma in vitro and in vivo and enhances sensitivity to doxorubicin

Anaplastic thyroid carcinoma (ATC) is one of the most lethal malignancies having no effective treatment. Exportin-1 (XPO1) is the key mediator of nuclear export of many tumor suppressor proteins and is overexpressed in human cancers. In this study, we examined the therapeutic potential of selinexor (XPO1 inhibitor) against human ATC cells both in vitro and in vivo. Here, we showed that XPO1 is robustly expressed in primary ATC samples and human ATC cell lines. Silencing of XPO1 by either shRNA or selinexor significantly reduced cellular growth and induced cell cycle arrest, apoptosis of ATC cells by altering the protein expression of cancer-related genes. Moreover, selinexor significantly inhibited tumor growth of ATC xenografts. Microarray analysis showed enrichment of DNA replication, cell cycle, cell cycle checkpoint and TNF pathways in selinexor treated ATC cells. Importantly, selinexor decreased AXL and GAS6 levels in CAL62 and HTH83 cells and suppressed the phosphorylation of downstream targets of AXL signaling such as AKT and P70S6K. Finally, a combination of selinexor with doxorubicin demonstrated a synergistic decrease in the cellular proliferation of several ATC cells. These results provide a rationale for investigating the efficacy of combining selinexor and doxorubicin therapy to improve the outcome of ATC patients.

Hobnail variant of papillary thyroid carcinoma: molecular profiling and comparison to classical papillary thyroid carcinoma, poorly differentiated thyroid carcinoma and anaplastic thyroid carcinoma.

BackgroundAs a rare but aggressive papillary thyroid carcinoma (PTC) variant, the genetic changes of hobnail variant of PTC (HVPTC) are still unclear.ResultsThe prevalence of HVPTC was 1.69% (18/1062) of all PTC diagnosed in our cohort. 73 samples from 55 patients (17 HVPTC, 26 CPTC, 7 PDTC and 5 ATC) were successfully analyzed using targeted NGS with an 18-gene panel. Thirty-seven mutation variant types were identified among 11 genes. BRAF V600E mutation was the most common mutation, which is present in almost all HVPTC samples (16/17, 94%), most CPTC samples (20/26, 77%), and none of the ATC and PDTC samples. TERT promoter mutation (C228T) was identified in 2 ATC and one HVPTC patient. RAS and TP53 mutation are almost exclusively present among ATC and PDTC samples although TP53 mutation was also observed in 3 HVPTC patients. Six different GNAS mutations were identified among 8 CPTC patients (31%) and none of the HVPTC patients. The only patient who died of disease progression harbored concomitant TERT C228T mutation, BRAF V600E mutation and TP53 mutation.MethodsHVPTC cases were identified from a group of 1062 consecutive surgical specimens diagnosed as PTC between 2000 and 2010. Targeted next-generation sequencing (NGS) was applied to investigate the mutation spectrum of HVPTC, compared to classical PTC (CPTC), poorly differentiated thyroid carcinoma (PDTC) and anaplastic thyroid carcinoma (ATC).ConclusionAs an aggressive variant of PTC, HVPTC has relatively specific molecular features, which is somewhat different from both CPTC and ATC/PDTC and may underlie its relatively aggressive behavior.

Bioinformatics analysis of key genes and latent pathway interactions based on the anaplastic thyroid carcinoma gene expression profile.

Anaplastic thyroid carcinoma (ATC) is an aggressive malignant disease in older adults with a high mortality rate. The present study aimed to examine several key genes and pathways, which are associated with ATC. The GSE33630 gene expression profile was downloaded from the Gene Expression Omnibus database, which included 11 ATC and 45 normal thyroid samples. The differentially expressed genes (DEGs) in ATC were identified using the Limma package in R. The Gene Ontology functions and Kyoto Encyclopedia of Genes and Genomes pathways of the selected DEGs were enriched using the Database for Annotation, Visualization and Integrated Discovery. A protein-protein interaction (PPI) network of the DEGs was constructed to select significant modules. Furthermore, a latent pathway interactive network was constructed to select the significant pathways associated with ATC. A total of 665 DEGs in the ATC samples were screened, and four significant modules were selected from the PPI network. The DEGs in the four modules were enriched in several functions and pathways. In addition, 29 significant pathways associated with ATC were selected, and he Toll-like receptor (TLR) signaling pathway, extracellular matrix (ECM)-receptor interaction and cytokine-cytokine interaction pathway were identified as important pathways. FBJ murine osteosarcoma viral oncogene homolog (FOS), chemokine C-X-C motif ligand 10 (CXCL10), collagen type V α1 (COL5A1) and chemokine (C-C motif) ligand 28 (CCL28) were the key DEGs involved in these significant pathways. The data obtained in the present study revealed that the TLR signaling pathway, ECM-receptor interaction and cytokine-cytokine receptor interaction pathway, and the FOS, CXCL10, COL5A1, COL11A1 and CCL28 genes have different roles in the progression of ATC, and these may be used as therapeutic targets for ATC.

Targeted next-generation sequencing for TP53, RAS, BRAF, ALK and NF1 mutations in anaplastic thyroid cancer.

Anaplastic thyroid carcinoma (ATC) is the most aggressive thyroid cancer with a median survival of 4-6 months. Identification of mutations contributing to aberrant activation of signaling cascades in ATC may provide novel opportunities for targeted therapy. Thirty-nine ATC samples were studied by next-generation sequencing (NGS) with an established gene panel. High quality readout was obtained in 30/39 ATC. Twenty-eight ATC harbored a mutation in at least one of the studied genes: TP53 (18/30), NF1 (11/30), ALK (6/30), NRAS (4/30), ATRX (3/30), BRAF (2/30), HRAS (2/30), KRAS (1/30). In 17/30 ATC (54 %) mutations were found in two or more genes. Twenty-one of the identified variants are listed in COSMIC as somatic mutations reported in other cancer entities. In three ATC samples no mutations were detected and none of the ATCs was positive for BRAFV600E. The most frequent mutations were found in TP53 (60 %), followed by NF1 (37 %). ALK mutations were detected in 20 % of ATC and were more frequent than RAS or BRAF mutations. ATRX mutations were identified in 10 % of the ATC samples. These sequencing data from 30 ATC samples demonstrate the accumulation of genetic alterations in ATC because in 90 % of samples mutations were already found in the investigated nine genes alone. Mutations were found with high prevalence in established tumor suppressor and oncogenes in ATC, such as TP53 and H/K/NRAS, but also, although less frequent, in genes that may harbor the potential for targeted treatment in a subset of ATC patients, such as ALK and NF1.

TWIST1/miR-584/TUSC2 pathway induces resistance to apoptosis in thyroid cancer cells.

TWIST1, a transcription factor, plays a pivotal role in cancer initiation and progression. Anaplastic thyroid carcinoma (ATC) is one of the deadliest human malignancies; TWIST1 is overexpressed in ATC and increases thyroid cancer cell survival, migration and invasion. The molecular mechanisms underlying the effects of TWIST1 are partially known. Here, using miRNome profiling of papillary thyroid cancer cells (TPC-1) ectopically expressing TWIST1, we identified miR-584. We showed that TWIST1 directly binds miR-584 using chromatin immunoprecipitation. Importantly, miR-584 was up-regulated in human ATC compared to papillary thyroid carcinoma (PTC) and normal thyroid samples. Overexpression of miR-584 in TPC cells induced resistance to apoptosis, whereas stable transfection of anti-miR-584 in TPC-TWIST1 and 8505C cells increased the sensitivity to apoptosis. Using bioinformatics programs, we identified TUSC2 (tumor suppressor candidate 2) as a novel target of miR-584. TUSC2 mRNA and protein levels were decreased in TPC miR-584 and increased in TPC-TWIST1 anti-miR-584 cells. Luciferase assays demonstrated direct targeting. Restored expression of TUSC2 rescued the inhibition of apoptosis induced by miR-584. Finally, qRT-PCR and immunohistochemical analysis showed that TUSC2 was down-regulated in ATC and PTC samples compared to normal thyroids. In conclusion, our study identified a novel TWIST1/miR-584/TUSC2 pathway that plays a role in resistance to apoptosis of thyroid cancer cells.

Elucidation of the molecular mechanisms of anaplastic thyroid carcinoma by integrated miRNA and mRNA analysis.

To elucidate the complex molecular mechanisms of anaplastic thyroid carcinoma(ATC), the mRNA and miRNA expression profiles of ATC were systematically explored. A total of 55common differentially expressed genes(DEGs) were obtained from two mRNA expression datasets including 23ATC samples and 24paired normal samples. Gene expression levels of three randomly selected DEGs, VCAN, COL5A1 and KCNJ16, were examined using RT-PCR in 10 ATC samples. Notably, the ATC and normal samples were clearly classified into two groups based on their common DEGs. Moreover 23common DEGs, such as TG, NKX2-1, KCNJ16 and CTHRC1, were predicted to be the potential targets of 17identified miRNAs in ATC. Meanwhile, several miRNA target genes were associated with biological processes related to tumor progression such as angiogenesis, cell migration or growth and potassium channel regulation. In summary, the poor prognosis of ATC is possibly caused via complex biological processes. Firstly, angiogenesis was activated by the high expression of CTHRC1, VCAN and POSTN, providing necessary nutrition for tumor cells. Then tumor distant metastasis was induced via stimulation of cell migration and cell growth or regulation of cell-cell interaction. Moreover, intracellular potassium concentration changes promoted ATC progression indirectly. Hence, identification of these critical DEGs was valuable in understanding the molecular mechanisms of ATC.

Abstract 5570: Laminin-5 gamma-2 (LAMC2) is highly expressed in anaplastic thyroid carcinoma and is associated with tumor progression, migration and invasion by modulating signaling of EGFR

Abstract Context: Anaplastic thyroid carcinoma (ATC) is an aggressive malignancy having no effective treatment. Laminin subunit gamma-2 (LAMC2) is an epithelial basement membrane protein involved in cell migration and tumour invasion and might represent an ideal target for the development of novel therapeutic approaches for ATC. Objective: Study the role of LAMC2 in ATC tumorigenesis. Design: LAMC2 expression was evaluated by RT-PCR, western blotting and immunohistochemistry in tumor specimens, adjacent non-cancerous tissues and cell lines. shRNA approach was used to investigate the effect of LAMC2 knockdown on tumorigenesis of ATC. Results: LAMC2 was highly expressed in ATC samples and cell lines compared to normal thyroid tissues. Silencing LAMC2 by shRNA in ATC cells moderately inhibited cell growth in liquid culture and dramatically decreased growth in soft agar and in xenografts growing in immunodeficient mice. Silencing LAMC2 caused cell cycle arrest and significantly suppressed migration, invasion and wound healing of ATC cells. Rescue experiments by overexpressing LAMC2 in LAMC2 knockdown cells, reversed the inhibitory effects as shown by increased cell proliferation and colony formation. Microarray data demonstrated that LAMC2 shRNA significantly altered expression of genes associated with migration, invasion, proliferation and survival. Immunoprecipitation and co-localization experiments showed that LAMC2 bound to EGFR in ATC cells. Silencing LAMC2 partially blocked EGF-mediated activation of EGFR and its downstream pathway. Interestingly, cetuximab (EGFR blocking antibody) or EGFR siRNA additively enhanced the anti-proliferative activity of the LAMC2 knockdown ATC cells compared to control cells. Conclusions: To our knowledge, this is the first report investigating the effect of LAMC2 on cell growth, cell cycle, migration, invasion and EGFR signaling in ATC cells, suggesting that LAMC2 may be a potential therapeutic target for treatment of ATC. Citation Format: Manoj Garg, Deepika Kanojia, Ryoko Okamoto, Vikas Madan, Wenwen Chien, Abhishek Sampath, Ling-Wen Ding, Meng Xuan, Jonathan W Said, Ngan Doan, Li-Zhen Liu, Henry Yang, Sigal Gery, Gleen D Braunstein, H.Phillip Koeffler. Laminin-5 gamma-2 (LAMC2) is highly expressed in anaplastic thyroid carcinoma and is associated with tumor progression, migration and invasion by modulating signaling of EGFR. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5570. doi:10.1158/1538-7445.AM2014-5570

MiRNA expression in anaplastic thyroid carcinomas.

Anaplastic thyroid carcinoma (ATC) is the most lethal form of thyroid neoplasia and represents an end stage of thyroid tumor progression. No effective treatment exists so far. In this study, we analyzed the miRNA expression profiles of 11 ATC by microarrays and their relationship with the mRNA expression profiles of the same 11 ATC samples. ATC show distinct miRNA expression profiles compared to other less aggressive thyroid tumor types. ATC show 18 commonly deregulated miRNA compared to normal thyroid tissue (17 downregulated and 1 upregulated miRNA). First, the analysis of a combined approach of the mRNA gene expression and of the bioinformatically predicted mRNA targets of the deregulated miRNA suggested a role for these regulations in the epithelial to mesenchymal transition (EMT) process in ATC. Second, the direct interaction between one of the upregulated mRNA target, the LOX gene which is an EMT key player, and a downregulated miRNA, the miR-29a, was experimentally validated by a luciferase assay in HEK cell. Third, we confirmed that the ATC tissue is composed of about 50% of tumor associated macrophages (TAM) and suggested, by taking into account our data and published data, their most likely direct or paracrine intercommunication between them and the thyroid tumor cells, amplifying the tumor aggressiveness. Finally, we demonstrated by in situ hybridization a specific thyrocyte localization of 3 of the deregulated miRNA: let-7g, miR-29a and miR-30e and we pointed out the importance of identifying the cell type localization before drawing any conclusion on the physiopathological role of a given gene.