Epigenetic modifications in poorly differentiated and anaplastic thyroid cancer

Accepted Manuscript Epigenetic modifications in poorly differentiated and anaplastic thyroid cancer Thanyawat Sasanakietkul, Timothy D. Murtha, Mahsa Javid, Reju Korah, Tobias Carling PII:

S0303-7207(17)30291-5

DOI:

10.1016/j.mce.2017.05.022

Reference:

MCE 9953

To appear in:

Molecular and Cellular Endocrinology

Received Date: 16 March 2017 Revised Date:

12 May 2017

Accepted Date: 21 May 2017

Please cite this article as: Sasanakietkul, T., Murtha, T.D., Javid, M., Korah, R., Carling, T., Epigenetic modifications in poorly differentiated and anaplastic thyroid cancer, Molecular and Cellular Endocrinology (2017), doi: 10.1016/j.mce.2017.05.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Review Article Epigenetic Modifications in Poorly Differentiated and Anaplastic

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Thyroid Cancer

Thanyawat Sasanakietkul1,2, Timothy D. Murtha1,2, Mahsa Javid1,2, Reju Korah1,2, Tobias Carling1,2*

Yale Endocrine Neoplasia Laboratory, 2Department of Surgery, Section of Endocrine Surgery,

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Yale School of Medicine, New Haven, CT 06520, USA

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1

*To whom correspondence should be addressed

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Tobias Carling, MD, PhD, FACS Section of Endocrine Surgery

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Yale Endocrine Neoplasia Laboratory Department of Surgery

Yale University School of Medicine 333 Cedar Street, FMB130A New Haven, CT 06520

+1-203-737-2036 (phone) +1-203 737-4067 (fax) E-Mail: [email protected]

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ABSTRACT Well-differentiated thyroid cancer accounts for the majority of endocrine malignancies and, in general, has an excellent prognosis. In contrast, the less common poorly differentiated thyroid carcinoma (PDTC) and anaplastic thyroid carcinoma (ATC) are two of the most

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aggressive human malignancies. Recently, there has been an increased focus on the epigenetic alterations underlying thyroid carcinogenesis, including those that drive PDTC and ATC. Dysregulated epigenetic candidates identified include the Aurora group, KMT2D, PTEN,

RASSF1A, multiple non-coding RNAs (ncRNA), and the SWI/SNF chromatin-remodeling

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complex. A deeper understanding of the signaling pathways affected by epigenetic dysregulation may improve prognostic testing and support the advancement of thyroid-specific epigenetic

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therapies. This review outlines the current understanding of epigenetic alterations observed in PDTC and ATC and explores the potential for exploiting this understanding in developing novel therapeutic strategies.

Keywords: Epigenetics, Poorly differentiated thyroid carcinoma, Anaplastic thyroid carcinoma,

1. Introduction

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MicroRNA, Non-coding RNA

Thyroid cancer is the most common endocrine malignancy and has had a consistently increasing incidence for the past four decades (1-3). The vast majority (> 95%) of thyroid

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cancers are derived from follicular epithelial cells while the remainder arises from parafollicular cells (3). Follicular cell-based thyroid cancer is divided into three major categories: well-

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differentiated thyroid carcinoma (WDTC), poorly differentiated thyroid carcinoma (PDTC), and undifferentiated or anaplastic thyroid carcinoma (ATC). WDTC includes follicular thyroid carcinoma (FTC) and papillary thyroid carcinoma (PTC). Among the major thyroid cancer types, ATC is the most rare and aggressive variant (4). Because of its high proliferative index and invasive behavior, the disease-specific mortality of ATC is 69.4% at 6 months and 80.7% at 12 months (5). The dismal prognosis of ATC is partially due to its inherent resistance to both radioactive iodine and conventional chemotherapy. As the tumor cells of PDTC and ATC lack appreciable expression of the sodium-iodide symporter (NIS), they are incapable of iodine uptake (6-8). Moreover, ATC cells may not secrete thyroglobulin and are potentially refractory

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to thyroid stimulating hormone (TSH) because of thyrotropin receptor deficiency on their plasma cell membrane. The combination of these tumor characteristics significantly limits the efficacy

2. Poorly differentiated and anaplastic thyroid carcinoma

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of conventional radioactive iodine therapy (6, 7).

Since Sakamoto et al. (1983) first described PDTC, there has been contention regarding its characterization and diagnosis (9). Currently, several distinct criteria for diagnosing PDTC are in use. The 2004 World Health Organization Classification of Tumors describes PDTC as,

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“…follicular-cell neoplasms that show limited evidence of structural follicular cell

differentiation and occupy both morphologically and behaviorally an intermediate position

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between differentiated (follicular and papillary) and undifferentiated (anaplastic) carcinomas.” (10). The histologic features described are in part due to the dedifferentiation typically observed in PDTC.

Alternatively, the Turin proposal described PDTC as having, “(1) a solid/trabecular/insular pattern of growth, (2) absence of conventional nuclear features of PTC, and (3) presence of at least one of the following features: convoluted nuclei, mitotic activity

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(≥3×10 HPF), necrosis.” (11). Similarly, a Memorial Sloan-Kettering Cancer Center (MSKCC) study defined PDTC based on, “…the presence of ≥ 5 mitosis per 10 high-power microscopic fields (HPF)(x400) and/or fresh tumor necrosis in thyroid carcinomas demonstrating definite evidence of follicular cell differentiation on routine microscopy and/or by immunohistochemistry

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(i.e., thyroglobulin positivity).” (12). These definitions highlight PDTC’s association with necrosis and high mitotic indices, consistent with their aggressive phenotype. It is important to

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note that the MSKCC diagnostic criteria distinguish PDTC from other thyroid cancer subtypes including ATC (Table 1), irrespective of tumor growth patterns and nuclear features. PDTC accounts for approximately 0.5–7% of all thyroid cancers and has a mean age at

diagnosis of 60.6 years (13). ATC accounts for less than 1% of thyroid cancers and is predominately found in elderly patients with a mean age of 71 years (5). ATC also has a distinct female preponderance (60–70%) (5). On gross examination, ATCs are typically large, necrotic, and exhibit widespread hemorrhagic changes (14). Individual tumor morphology is influenced by the unique combination of spindle, giant, and squamoid cells (14). High mitotic rates and atypical mitoses are generally present, and necrosis is often observed in ATCs (Figure 1). On

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immunohistochemical analysis, the majority of ATCs are negative for characteristic transcription factors, such as thyroid transcription factor-1 (TTF-1), and thyroglobulin (14, 15). In contrast, PDTC is often positive for thyroglobulin and may display nuclear positivity for TTF-1 (13). In the diagnosis of equivocal cases, these immunohistochemistry features aid in differentiating

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between PDTC and ATC. However, molecular profiling using next generation genetic/epigenetic analysis could improve the process of establishing a definitive diagnostic distinction between these aggressive tumors. PDTC exhibits the intermediate aggressive clinical characteristics

between WDTC and ATC, such as tumor growth rate, lymph node metastasis (WDTC = 47%,

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PDTC = 64%, ATC = 100%), distant metastatic (WDTC = 0%, PDTC = 50%, ATC = 87%) 5year disease free survival (WDTC = 91%, PDTC = 51%, ATC = 0%) and overall survival in 43

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months (WDTC = 100%, PDTC = 70%, ATC = 0%) (16, 17). Currently, treatment for both PDTC and ATC are still challenging. Surgery (total thyroidectomy and cervical lymph node dissection) is the principle therapeutic option. Although PDTC and ATC are very aggressive subtypes of thyroid cancer, a unified single therapy usually will not achieve the desired curative goals to these thyroid cancers that run distinct clinical courses. The multimodality treatments (radioactive iodine ablation, external beam radiation, chemotherapy, and targeted therapy) have

17).

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been in use to control the extent of disease but the outcomes remain unsatisfactory (8, 9, 12, 16,

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Figure 1. Histopathologic pictures (H&E) of (A) PDTC and (B) ATC

Table 1. Characteristics of PDTC and ATC

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Disease Characteristics

Poorly differentiated thyroid carcinoma

Anaplastic thyroid carcinoma

Proportion of all thyroid

0.5-7%

< 1%

Mean age at diagnosis

60 years

71 years

Histopathology

Follicular cell origin

Follicular cell origin

- Limited evidence of structural follicular cell

- Severe nuclear atypia

differentiation a

- Numerous mitotic figures, atypical mitoses, extensive necrosis and, often mixed morphology

- Morphologically and behaviorally intermediate between differentiated and undifferentiated carcinoma b

with spindle cells and giant cells. - Three patterns:

- Absence of conventional nuclear features of PTC

b

- Presence of at least one of the following: convoluted b

1. Spindle cells resembling sarcoma 2. Large, pleomorphic giant cells resembling osteoclasts with cellular connective tissue septa

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nuclei, mitotic activity (≥3×10 HPF), or necrosis

a

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- A solid/trabecular/insular pattern of growth

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cancers

- Presence of ≥ 5/10 (HPF)(x400) and/or fresh tumor

3. Squamoid pattern

necrosis in demonstrating definite evidence of follicular cell differentiation on routine microscopy and/or by immunohistochemistry c

Positive: Cytokeratins (CK7, CK18, CK 19), TTF-1,

Positive: Cytokeratins (CK7, CK18, CK 19, CK10/13),

markers

Thyroglobulin, Ki-67, Cyclin D1, IMP3

Ki-67, p53, Vimentin, Cyclin D1, EMA

Negative: Calcitonin, Chromogranin, CEA,

Negative: Calcitonin, Chromogranin, TTF-1,

Synaptophysin

RET/PTC oncoprotein, Thyroglobulin, Synaptophysin

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Immunohistochemical

a. World Health Organization classification; b. Turin conference; c. Memorial Sloan-Kettering Cancer Center

3. PDTC and ATC’ alterated pathways and genetic alterations

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3.1 Dysregulated signaling pathways in PDTC and ATC WDTCs commonly exhibit dysregulated activity of the mitogen-activated protein kinase

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(MAPK) pathway, the phosphoinositide 3-kinase (PI3K) signaling pathway, or a combination of the two (18-20). These alterations occur through direct genomic/epigenomic mutations or indirectly via epigenetic deregulation (21-23). Although the molecular basis for disease progression from WDTC to PDTC and/or ATC is debatable, the overlap of dysregulated signaling pathways among these entities may support a continuum of PDTC and ATC from WDTC (18, 24-26). Another commonality between WDTC and PDTC/ATC is the observation that the dysfunctions identified in the ERK1/2-MEK1/2 and PI3K-Akt signaling pathways are due to common genetic alterations such as BRAFV600E (18, 19, 27). Similarly, mutations in NRAS and HRAS, which have the potential to activate the ERK1/2-MEK1/2 and the PI3K-Akt

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pathway, are often found in both FTC and ATC (18, 24, 28-30). Furthermore, PTEN silencing, found in both PTC and ATC, may result in constitutive activation of PI3K-Akt anti-apoptotic pathways (18, 19, 24, 27, 31). Despite the scarcity of studies focusing on signaling disarray in PDTC, the available data

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suggest a clear continuum of constitutively active MAPK signaling—potentially through the disease progression route or de novo activation—in PDTC (26, 32). In addition to the commonly dysregulated RAS-RAF-MAPK pathways, PDTC and ATC were also found to harbor aberrant PIK3CA-PTEN-Akt-mTOR pathways (24). Alternatively, in undifferentiated ATC, there is

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evidence of deregulated WNT and NOTCH pathways, in addition to the constitutively active MAPK and PI3K pathways, potentially contributing to their highly aggressive behavior (26, 33-

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37).

Both PDTC and ATC also exhibit deregulated expression of a variety of genes involved in cell cycle regulation, focal adhesion signaling, actin cytoskeleton dynamics, and the TGF-β signaling pathways (38). Moreover, the interruption of the thyroid-stimulating hormone receptor (TSHR) pathway and the consequent dedifferentiation signaling in PDTC and ATC has been found mediated by the silencing of hematopoietically-expressed homeobox (HHEX) protein (38).

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Similarly, nuclear factor kappa B (NF- B) activation has been observed in the majority of ATCs. In vitro inhibition of NF- B in ATC cell lines appears to block cellular proliferation and invasive behavior (39, 40) suggesting its potential contribution to the invasive phenotype of ATC. Genetic disruption of the isocitrate dehydrogenase (IDH1) pathway through mutation of IDH1 has been

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shown to be associated with a variety of aggressive tumors including ATC (41-43).

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3.2 Genetic alterations in PDTC and ATC The major genetic alterations described in PDTC and ATC include frequent somatic

mutations in BRAF and the RAS family, as well as additional recurrent mutations in TP53, CTNNB1, PI3KCA, ALK, PTEN, and TERT promoter (14, 25, 30, 37, 44-48). The BRAFV600E mutation, found in > 70% of well-differentiated PTCs, is also found in 33% of PDTCs and 1945% of ATCs. Presence of the BRAFV600E mutation in PDTC and ATC is associated with worse prognosis, more aggressive phenotype, a higher rate of recurrence, and an increased likelihood of treatment resistance (37, 48-50). Interestingly, mutations in the ubiquitous tumor suppressor gene TP53 are found in only 8% of PDTC and a variable number of ATCs (29–73%) (37, 48, 51, 52).

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Activating mutations of CTNNB1, found in 65% of ATCs, may stimulate the WNT-β-catenin signaling pathway (33, 34). Similarly, mutational activation of the p110α catalytic subunit of PI3K is reported in 18–23% of ATCs. Both WNT and PI3K signaling activation has been shown to have the potential to manipulate cancer cell cycle progression and cell motility (37, 53). Other

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somatic gene mutations reported in PDTC, include anaplastic lymphoma kinase (ALK; 4%), PTEN (4%), NRAS (21%), HRAS (5%), and KRAS (2%), while mutations in ATC include those in ALK gene (0–11%), PTEN (10–20%), NRAS (17–18%) and HRAS (5–6%) (28, 37, 46, 48, 51, 52). The promoter mutations in human telomerase reverse transcriptase (hTERT) have been

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recently reported in ATC as well (54). Of the two common TERT promoter mutations (C228T and C250T), 33–50% of hTERT promoter mutations in PDTC and ATC occur in C228T. This

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mutation is believed to increase telomerase activity, possibly contributing to the aggressive behavior of PDTC and ATC (54). The first comprehensive whole-exome sequencing study of ATC revealed that its genetic landscape is highly heterogeneous. Somatic mutations were detected in 64% of ATC cases which included multiple novel mutations including those in mTOR, NF1, NF2, ERBB2, EIF1AX, MLH1, MLH3, MSH5, MSH6 and USH2A (48). Other genomic alterations that may influence PDTC and ATC tumorigenesis include

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copy number variations (CNV), gene fusions, and chromosomal translocations (Table 2). The receptor tyrosine kinase group of genes, composed of EGFR, PDGFRα, PDGFRβ, PIK3Ca, PIK3Cb, PDK1 VEGFR1, VEGFR2, KIT, and MET, has been reported to have substantial copy number gains in ATC, implicating its potential role in promoting ATC tumorigenesis (18). Large

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chromosomal gains involving 7p, 8q, 9q, 3p13-14, and 11q13 were found in ATC, while chromosomal losses involving 5q11-31 were frequently observed (17, 55, 56). Gene fusions,

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such as RET-PTC and STRN/ALK (9% of PDTCs and 4% of ATCs), are found in a small proportion of PDTCs and ATCs and may also contribute to their aggressive behaviors (30, 57, 58).

Table 2. Major Signaling and Mutational landscapes of PDTC and ATC Poorly differentiated thyroid cancer

Anaplastic thyroid cancer

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MAPK

•

MAPK

•

PI3K-Akt

•

PI3K-Akt

•

PI3K/Akt/mTOR inactivation

•

PI3K/Akt/mTOR inactivation

•

WNT-β-catenin

•

WNT-β-catenin

•

TSHR

•

NOTCH*

•

p53

•

TSHR

•

NF B*

•

p53

•

IDH1*

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Gene alterations

•

BRAF, RAS, TP53, CTNNB1, PIK3CA, PTEN,

BRAF, RAS, TP53, CTNNB1, PI3KCA, PTEN, AKT1, TERT,

AKT1, TERT, TSHR, ALK, STRN/ALK

TSHR, ALK, mTOR*, NF1*, NF2*, MLH1*, MLH3*,

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Signaling pathways

MSH5*, MSH6*, ERBB2*, EIF1AX*, USH2A*, RTK*, IDH1*, STRN/ALK, RET-PTC*

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*Identified only in ATC, not in PDTC

4. Epigenetic mechanisms and classifications in cancer

Tightly controlled epigenetic regulation is essential for normal growth, development, acquisition, and maintenance of unique functions of organs and organ systems. Deregulation of epigenetic controls, on the other hand, may play crucial roles in the origin and/or progression of

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various disease processes including cancer. Cancer-specific, as well as global epigenetic aberrations, are reported in a variety of malignancies including thyroid cancers (59). The four major epigenetic mechanisms that are found deregulated during various stages of carcinogenesis are: (1) DNA methylation (the tumor suppressor genes or immune response genes are aberrantly

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silenced by DNA methylation in their promoter regions), (2) histone modification (a posttranslational modification of histone proteins that regulates gene expression which includes

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methylation, phosphorylation, acetylation, ubiquitylation, and sumoylation), (3) chromatin remodeling (the chromatin architectural rearrangement or modification that allow transcription factors to have atypical access to the condensed genomic DNA resulting in aberrant control of gene expression), and (4) deregulation of ncRNAs which constitutes small ncRNAs (microRNAs, Piwi-interacting RNA, transcription initiation RNA, tRNA-derived small RNA, etc.) and long ncRNAs (lncRNAs) (60-62). MicroRNAs (miRNAs) can impair RNA translation by hybridizing to specific domains of the untranslated region of target mRNAs (63, 64) while lncRNAs can play a role in tumorigenesis by aberrantly controlling the transcription and/or posttranscriptional regulation of gene expression (65).

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Based on their specific roles, epigenetic deregulators can be classified into: (a) epigenetic modifiers, (b) mediators, and (c) modulators (66). Epigenetic modifiers are genes that abrogate gene expression control designed for structural and/or functional differentiation of the cells through DNA methylation, histone modification, or chromatin structure alterations. Epigenetic

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mediators are genes and their products that can target epigenetic modifiers and mediate the dedifferentiation process of tumor cells or their progenitors towards a stem cell-like pre-

differentiated status. Epigenetic modulators are genes and effectors that can influence the

epigenetic modifiers and epigenetic mediators in executing their respective signaling pathways.

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Environmental factors, such as injury, inflammation, or other forms of stress, are examples of

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epigenetic modulators that can push tissues to acquire cancer-promoting alterations (66).

5. Epigenetic modifications in thyroid cancer

Aberrant DNA methylation leading to the activation of proto-oncogenes or silencing of tumor suppressor genes, is common in thyroid tumors. Tumor suppressor genes that are epigenetically inactivated in thyroid cancers include DAPK, PTEN, RASSF1A, RAPβ2, RAP1GAP, SLC5A8, and TIMP3 (23). Aberrant promotor methylation of TIMP3, SLC5A8,

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DAPK, and RARb2 also overlap with BRAF mutations in PTC and may contribute to the tumor behavior (67, 68). The PTEN promoter hypermethylation rate in PTC and FTC are 50% and 100% respectively, suggesting their role in abrogating the PI3K-Akt pathway and leading to the development of thyroid tumors (31). RAS association domain family 1A (RASSF1A) promotor

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hypermethylation is seen in approximately 30% of thyroid cancers and may alter the RASSF1MST1-FOXO3 apoptosis pathway (30, 69, 70). In PTC, promoter hypermethylation of RASSF1A

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is significantly correlated with tumor multifocality, while RASSF1A hypomethylation appears to be compatible with extracapsular extension (71). The tissue inhibitor of metalloproteinase 3 (TIMP3) gene, silenced by promoter hypermethylation in a large number of PTCs, results in the promotion of tumor aggressiveness, including lymph node metastasis and tumor multifocality (21, 67). Similarly, downregulation of the death-associated protein kinase (DAPK), a calcium/calmodulin-dependent serine-threonine kinase which controls cell death, has been observed in 34% of PTCs, potentially increasing cell survival and invasive behavior (21, 67). Although not as extensively studied as promoter methylation, the potential contributions of histone modifications toward thyroid carcinogenesis, are gaining attention (22). Based on the

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reported efficacy of histone deacetylase (HDAC) inhibitors in other cancer types, recent exploratory studies have demonstrated significant therapeutic potential of HDAC inhibitors in the treatment of thyroid cancers as well (72-75). HDAC inhibitors partially restored radioactive iodine uptake and, combined with chemotherapy, radiation therapy, and surgery, showed

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improvement in the curative rate of ATC and increased ATC cell death in vivo and in vitro (73, 76-79).

Several recent studies suggest an association between deregulated miRNA expression and thyroid cancers. Upregulation of the miR221-222 cluster, located on the X chromosome, has

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been implicated to regulate cell cycle progression and apoptosis in differentiated thyroid cancers (80, 81). MicroRNA miR-146 is involved in multiple cancer types and has been found to be

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overexpressed in all follicular cell-derived cancers (80). Other miRNAs that are commonly overexpressed in PTC include miR-21 and miR-181 (23, 82-84). In FTC, miR-192, miR-197, miR-328, and miR-346 expression have been reported to be downregulated when compared to follicular adenoma (85). These dysregulated miRNAs may contribute individually or in combination with other genetic or epigenetic events to promote thyroid tumorigenesis (64, 86, 87).

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The important roles played by lncRNAs in the origin and progression of various tumor types is slowly being unraveled (88-92). Three recent studies strongly suggest a role for various lncRNAs in thyroid carcinogenesis (93-95). Zheng et al. (2016) used quantitative reverse transcription polymerase chain reaction to demonstrate differential expression of three lncRNAs.

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Namely, (a) BRAF-activated lncRNA (BANCR), (b) PTC susceptibility candidate 3 (PTCSC3), and (c) lncRNA associated with MAPK pathway and cell cycle arrest (NAMA) in PTC (94).

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BANCR, which exhibited the potential to regulate PTC proliferation through the cyclin D1 and TSHR pathways, showed upregulated expression while both of PTCSC3 and NAMA displayed downregulated expression in PTC (94). Downregulation of lncRNA NONHSAT037832 has been observed in PTC cell lines and was found to correlate with aggressive tumor characteristics in PTC, such as larger tumor size or lymph node metastasis (93). Plasmacytoma variant translocation 1 (PVT1), a recent addition to lncRNAs, was found upregulated in PTC, FTC, and ATC. Intriguingly, the same study implicated PVT1 as a promotor of thyroid cancer cell growth via EZH2 and TSHR (95). In addition to WDTCs, recent findings also clearly suggest important roles for epigenetic regulation in the initiation, promotion, and progression of PDTC and ATC

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(21-23, 96-100). The discussion below focuses on reviewing potential roles of various epigenetic candidates and mechanisms in promoting de novo dedifferentiation of thyroid follicular cells into PDTC’s and ATC’s, as well as continued dedifferentiation of WDTCs to PDTC’s and ATC’s.

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6. Characterization of PDTC and ATC epigenomes

Recent comprehensive genetic and epigenetic analyses of PTC and FTC helped to

generate a better understanding of their respective epigenomes (20-23, 30, 48, 49, 59, 66, 96-99, 101). Although next-generation genetic analysis of ATC revealed aberrations in multiple genes

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that could compromise the integrity of thyroid epigenome (46), a clear understanding of a defined and distinctive ATC epigenome is still missing. As a new entrant into the field of

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genetic/epigenetic analysis, epigenome information on PDTCs is also lacking. The PDTC/ATC epigenome analysis outlined below focuses on alterations identified in epigenetic candidates and mechanisms in comparison to normal thyroid follicle cells and other follicular cell-derived neoplasias including PTC and FTC. Alterations reported to have dominant roles in defining PDTC and ATC epigenomes are grouped into deregulated (a) genes, (b) miRNAs, and (c) lncRNAs (Table 3). The discussed alterations in this review—individually or in tandem—are

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suggested to promote dedifferentiation de novo or to accentuate existing neoplasia-associated dedifferentiation depending on the context of the study reported (Figure 1). Dysregulated gene expressions associated with epigenetic alterations in PDTCs and ATCs are further grouped into (1) epigenetic modifiers, (2) epigenetic mediators, and (3) epigenetic modulators based on their

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implicated roles in the overall dedifferentiation process (66).

Table 3. Epigenetic alterations in PDTC and ATC Epigenetic modifiers

Poorly differentiated thyroid cancer

Anaplastic thyroid cancer

Histone modification: HMTs (KMT2A,

•

DNA methylation: MECP2

KMT2C, KMT2D, SETD2)

•

Histone modification: Aurora group, EZH2, HDAC1 & 2, HMTs (KMT2A, KMT2C, KMT2D, SETD2)

•

Chromatin remodeling: SWI/SNF complex, EP300

Epigenetic mediators

Maspin

RASSF1A, RASSF2, REC8, TTF-1, Maspin, MAP17

Epigenetic modulators

-

PTEN, p16, DAPK, UCHL1, TSHR, RASAL1, TCL1B, NOTCH4

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Downregulated

miR-23, miR-26a, miR-125b, miR-130b,

miR-1, miR-7, miR-15b, miR-17, miR-19b, miR19a-3p, miR-

miRNA

miR-139-5p, miR-150, miR-193a-5p, miR-

23b, miR-24, miR-25, miR-26a, miR-26b, miR-27b, miR-29

219-5p, miR-451, miR-455-3p, miR-886-3p,

family, miR-30 family, miR-34-b-3p, miR-98, miR -99a, miR-

let7c

99b, miR-100, miR-101, miR-106b, miR-107, miR-125a, miR125b, miR-126-3p, miR-129-3p, miR-130a, miR -135a-5p,

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miR-138, miR-141, miR-143, miR-144-3p, miR-145, miR148b, miR-151, miR-152, miR-181a, miR-191, miR-195, miR199 family, miR-200 family, miR-204, miR-206, miR-301a, miR-331-3p, miR-361, miR-486, miR-497, miR-513a-3p, miR -514-3p, miR-618, miR-708, let7 family miR-15a-3p, miR-125a-5p, miR-129, miR-

miR-17-92 cluster, miR-18a, miR-20a, miR-21, miR-92, miR-

miRNA

146b, miR-182, miR-183, miR-187, miR-221,

125a-3p, miR-130b, miR-137, miR-138-3p, miR-142, miR-

miR-222, miR-339

146a, miR-146b, miR-149-3p, miR-150, miR-205, miR-212,

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Upregulated

miR-221, miR-222, miR-223, miR-302c, miR-371, miR-422a,

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miR-487b, miR-491-3p, miR-494, miR-526b-3p, miR-584, miR-659-3p, miR-638, miR-665, miR-923, miR-4295, let7f-5p

Downregulated

MALAT1

MALAT1

lncRNA Upregulated lncRNA

-

MALAT1, LOC100507661, PVT1

6.1 Dysregulated genes

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Bold miRNAs are the miRNAs that can be identified in both PDTC and ATC.

6.1.1 Epigenetic modifiers of PDTC and ATC 6.1.1.1 Aurora group

Contrary to the lack of expression in normal thyroid tissue, Aurora group members A, B,

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and C are found overexpressed in ATC (102, 103). The Aurora group acts as the regulators of mitotic events by controlling the histone H3 phosphorylation and chromatin remodeling process

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(104, 105). Moreover, Aurora B silencing by RNA interference or inhibition of Aurora kinase activity has been shown to significantly reduce the growth of ATC cells in vivo, suggesting a tumor promoting role for Aurora proteins in ATC (103).

6.1.1.2 Histone lysine-methyltransferases Enhancer of Zeste homolog 2 (EZH2), a member of the polycomb family of proteins, is a histone lysine-methyltransferase that is overexpressed in ATC. In contrast, no significant change in expression was observed in WDTC compared to normal thyroid tissue (106). Silencing of EZH2 in ATC cell lines resulted in cell growth inhibition, loss of anchorage-independent growth

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capacity, cell migration, and invasion through Matrigel. Overexpression of EZH2 on the other hand, contributed to altered histone methylation, transcriptional silencing of thyroid-specific transcription factor paired-box gene 8 (PAX8), and ATC dedifferentiation (106).

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6.1.1.3 Histone deacetylases

Reduced acetylation of histones leads to altered expression of proteins involved in cell proliferation and cell cycle control. Specifically, dysregulation of both the ERK1/2-MEK1/2 and PI3K-Akt pathways were affected by HDAC’s overexpression (107). The overexpression of

6.1.1.4 Histone methyltransferases

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deregulation of cell cycle control observed in ATC’s (73).

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Histone deacetylases (HDACs) 1 and 2 has been reported in ATC and may contribute to the

KMT2D (Lysine Methyltransferase 2D), previously known as myeloid/ lymphoid or mixed-lineage leukemia protein 2 (MLL2), is a histone methyltransferase (HMT) that regulates gene transcription by modulating chromatin accessibility (108). Jeon et al. (2016) reported an association between KMT2D mutation status and shorter disease-specific survival in patients

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with ATC (51). Recent transcriptome analysis of PDTC and ATC showed a greater increase in HMTs (KMT2A, KMT2C, KMT2D, and SETD2) expression in ATC than in PDTC. In addition, the rate of KMT2A, KMT2C, KMT2D, and SETD2 mutation has been found to be higher in ATCs (24%) than PDTCs (7%) (37). Whether the increased expression of HMTs is associated with the

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pattern of dedifferentiation observed in PDTC and ATC needs to be further investigated.

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6.1.1.5 SWI/SNF chromatin remodeling complex Switch/Sucrose Non-Fermentable (SWI/SNF) is an important multi-subunit nucleosome-

remodeling complex found in both eukaryotes and prokaryotes. Landa et al. (2016) reported multiple mutations in SWI/SNF chromatin remodeling complex genes (ARID1A, ARID1B, ARID2, ARID5B, ATRX, SMARCB1, and PBRM1) in advanced thyroid tumors (37). SWI/SNF genes have a mutation rate in PDTC and ATC of 6% and 36% respectively (37), roughly aligning with their dedifferentiation status and potentially suggesting an active role in contributing to their aggressive behaviors.

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6.1.2 Epigenetic Mediators of PDTC and ATC 6.1.2.1 RAS association domain family members Epigenetic silencing of negative RAS effector, RASSF1A (Ras association domain family

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1 isoform A) has been detected in multiple thyroid cancer types including ATC and medullary thyroid carcinoma (MTC) (96, 109). Nakamura et al. (2005) reported methylation rates of

RASSF1A in PTC, MTC, and FTC as 32%, 40%, and 100% respectively (70). The RASSF1A methylation range reported in ATC was 33–78% (70, 96, 109). Analysis of the promoter

methylation status of RASSF2 (Ras Association (RalGDS/AF-6) Domain Family Member 2),

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another negative RAS effector, showed RASSF2 promoter methylation rates in MTC, PTC, FTC, and ATC as 0%, 54%, 80%, and 83% respectively (98). It remains to be seen whether the

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frequent RASSF family promoter methylation observed in ATCs deregulates the RAS pathway specifically or are part of global methylation changes prior to targeting RAS effectors as potential therapeutic candidates (98).

6.1.2.2 Meiosis-specific component of the cohesin complex

ATC samples have the highest methylation level of REC8 (REC8 Meiotic Recombination

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Protein), one of the members of the cohesion complex, followed by FTC, PTC, and benign thyroid tumors. Hypermethylation of REC8 is associated with advanced disease stage and thyroid cancer-related mortality, potentially by abrogating the PI3K pathway as suggested by the

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experiments using ATC cell lines (100).

6.1.2.3 Thyroid transcription factor 1

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Kondo et al. (2009) investigated DNA methylation status of thyroid transcription factor 1 (TTF-1) and found the lack of TTF-1 expression in ATC (6/10 cases) consistent with promoterhypermethylated TTF-1 gene. On the contrary, normal thyroid tissue and PTC displayed positive expression of TTF-1 with the nonmethylated TTF-1 promoter, leading to the conclusion that TTF-1 expression is regulated by an epigenetic promotor methylation process (97).

6.1.2.4 Serpin superfamily The methylation status of the promoter region of SERPINB5 (Serine (Or Cysteine) Proteinase Inhibitor, Clade B (Ovalbumin), Member 5), a mammary serine protease inhibitor

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(Maspin) was investigated in 6 human ATC cell lines, 17 PDTCs, and 13 ATCs by Ogasawara et al. (2004)(110). They found promoter hypomethylation in 41% of PDTCs and 62% of ATCs. The promotor hypomethylation of Maspin, causing Maspin overexpression is associated with

desirable therapeutic target for PDTC and ATC treatments.

6.1.2.5 Membrane-associated protein 17

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PDTC and ATC. With established metastatic promoting function, SERPINB5 could become a

Hypomethylation and overexpression of the membrane-associated protein 17 (MAP17), a

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reactive oxygen species dependent oncogene, was detected in 33% of ATCs. Overexpression of MAP17 promoted thyroid tumor cell growth in vitro and in vivo (111). It is currently unclear

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whether the MAP17 pathway is associated with the metabolic alterations observed in ATC.

6.1.3 Epigenetic modulators of PDTC and ATC 6.1.3.1 Phosphatase and tensin homolog

PTEN (phosphatase and tensin homolog) is frequently silenced via promoter methylation in ATC (112). Hou et al. (2008) suggest a positive correlation between PTEN hypermethylation

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and progression from benign thyroid adenoma to FTC or ATC (112). This concept is consistent with the model of silencing PTEN by aberrant methylation and activating genetic aberrations of the PI3K/Akt pathways in multiple cancer types (112-114).

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6.1.3.2 Thyroid-stimulating hormone receptor

Hypermethylation of the thyroid-stimulating hormone receptor (TSHR) is frequently

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observed in follicular adenoma, PTC, and ATC (109, 115). Hypermethylation and the resultant silencing of TSHR, the thyroidiodide-metabolizing gene, lead to the failure of iodine concentration, and consequent resistance to radioiodide therapy (68).

6.1.3.3 RAS protein activator like 1 The promoter sequence of RAS protein activator like 1 (RASAL1) gene coding for RASGTPase-activating protein was found hypermethylated in 33% of ATCs (116). Liu et al. (2013) concluded that RASAL1 silencing from promoter hypermethylation could influence ATC tumorigenesis via the RAS-coupled MAPK and PI3K pathways (116).

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6.1.3.4 T-cell leukemia/lymphoma 1B Dysregulated promoter methylation of T-cell leukemia/lymphoma 1B (TCL1B) oncogene, an established tumor driver gene of B-cell chronic lymphocytic leukemia, was found

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hypomethylated in 64% of ATCs (111). However, a definitive cancer driver role for TCL1B in thyroid tumorigenesis is yet to be established (117).

6.1.3.5 Neurogenic locus notch homolog protein 4

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The promoter of the neurogenic locus notch homolog protein 4 (NOTCH4) oncogene was found hypomethylated in 45% of ATC’s (111). Further, Geers et al. (2011) analyzed NOTCH4

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gene expression by immunohistochemistry, qRT-PCR, and Western-blot in normal thyroid tissue, benign nodules, and malignant thyroid cancer (PTC, FTC). This study determined that the overexpression of NOTCH4 in carcinoma has potential roles in thyroid dedifferentiation and angiogenesis (118).

Table 4. Genes epigenetically regulated in PDTC and ATC Full gene name

Type of

Mechanism

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Gene

Regulation

Poorly differentiated thyroid cancer Maspin

Mammary serine

Histone

(KMT2

methyltransferase

SETD2)

Dysregulated

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HMTs

family,

Upregulated

EP

protease inhibitor

Hypomethylation

Involved signaling

Outcome

References

pathway

Regulate the

Morphological

breakdown of proteins

dedifferentiation

Ogasawara (2004)

by inhibiting the catalytic activity of proteinases

Mutation

Histone

Deregulated gene

methyltransferase

expression

Landa (2016)

Dysregulated the

Morphological

methylation status

dedifferentiation

Histone

Important roles in

Impair of cell cycle

Sorrentino (2005),

modification

mitosis, Regulate

progression and

Wiseman (2007),

chromosome

maintenance of

Wunderlich (2011)

segregation and

genomic stability

Anaplastic thyroid cancer MECP2

Methyl CpG binding

Dysregulated

Gene fusion

protein 2

Aurora group

-

Upregulated

cytokinesis

Kasaian (2015)

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EZH2

Enhancer of Zeste

Upregulated

homolog 2

HDAC

Histone deacetylases

Upregulated

Histone

Transcriptional

Dysregulation of cell

Borbone (2011),

methylation

silencing of PAX8

proliferation and

Catalano (2012)

gene

differentiation

Histone

ERK1/2-MEK1/2 and

Condensation of

Borbone (2010),

acetylation

PI3K-Akt pathways

chromatin, preventing

Lin (2013)

transcription factor

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accessibility and function

HMTs

Histone

(KMT2

methyltransferase

Dysregulated

Mutation

Histone methyltransferase

family,

Switch/Sucrose Non-

complex

Fermentable

Dysregulated

Mutation

Chromatin structure

Dysregulated gene

Kasaian (2015),

modifier

expression

Kunstman (2015),

complex Histone

(EP300)

acetyltransferase

Dysregulated

RASSF1A

RAS association

Downregulated

Downregulated

Hypermethylation

-

Downregulated

Thyroid transcription

Downregulated

Mammary serine

Upregulated

Hypermethylation

Hypermethylation

Hypomethylation

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protease inhibitor

MAP17

Cyclin D1 and MST1

Interact with the

Dysregulated cell

Membrane-

Upregulated

Landa (2016) Kasaian (2015)

growth and division Increased tumor cell

Schagdarsurengin

proliferation

(2002), Nakamura (2005), Schagdarsurengin (2006)

Reducing apoptosis

proapoptotic kinases

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Maspin

Hypermethylation

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family 2

factor-1

Chromatin remodeling

deletion

family 1A

RAS association

Frame-shift

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HATs

TTF-1

Landa (2016),

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SWI/SNF

REC8

Kunstman (2015),

expression

Jeon (2016)

SETD2)

RASSF2

Dysregulated gene

Schagdarsurengin (2010)

MST1 and MST2 PI3K pathway

Associated with

Liu (2015)

advanced disease stages of thyroid cancer Regulate the

Dysregulating the

expression of TSH,

organogenesis and

NIS

thyroid function

Regulate the

Morphological

breakdown of proteins

dedifferentiation

Kondo (2009)

Ogasawara (2004)

by inhibiting the catalytic activity of proteinases Hypomethylation

PI3K/Akt pathway

associated protein 17

Promote thyroid tumor

Rodriguez-Rodero

growth in vitro and in

(2013)

vivo PTEN

Phosphatase and tensin homolog

Downregulated

Hypermethylation

Dephosphorylating

Reducing cell-cycle

Frisk (2002), Hou

PI(3,4,5)P3 to reduce

arrest and apoptosis

(2008)

the downstream activity of PKB/Akt kinase, PI3K/Akt

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pathway

TSHR

Thyroid-stimulating

Downregulated

Hypermethylation

TSHR pathway

hormone receptor

Tumor resistant to

Catalano (2012),

radioiodide therapy,

Schagdarsurengin

Dysregulation of

(2006)

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thyrocyte function and growth

RASAL1

RAS protein

Downregulated

Hypermethylation

activator like 1 TCL1B

T-cell

MAPK and PI3K pathways

Not associated

Hypomethylation

-

Rodriguez-Rodero

(2013)

Not associated

Hypomethylation

notch homolog

Notch pathway

Induce thyroid

Geers (2011),

carcinogenesis and

Rodriguez-Rodero

angiogenesis.

(2013)

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protein 4

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1B Neurogenic locus

Liu (2013)

development of ATC -

leukemia/lymphoma

NOTCH4

Promote the

6.2 MicroRNA deregulation in PDTC and ATC

MicroRNAs (miRNAs) are a group of small (19–25 nucleotide) RNA molecules that can negatively regulate their target gene expression by binding to the 3'- untranslated regions of the corresponding mRNAs and thereby inhibiting mRNA translation (119). They are involved in

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regulating a variety of molecular processes including apoptosis, tumor dedifferentiation, and metastasis (99, 120). It is well established that improper epigenetic regulation by miRNAs contributes to tumor progression in multiple cancer types including PDTC and ATC (64, 86, 121-124). Up- or downregulation of miRNA can influence the tumorigenic outcome depending

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on the role(s) of the target genes on vital signaling processes (Table 5) (64, 86, 121-124).

6.2.1 Downregulated miRNAs and their targets in PDTC and ATC

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While aberrant expression of hundreds of miRNAs has been identified in multiple types of thyroid cancers, very few were found to be exclusively dysregulated in either PDTC or ATC (64, 80, 86, 121-128). To date, approximately 10–15 miRNAs have been found to be downregulated in PDTC compared to 75–80 miRNAs in ATC. Three downregulated miRNAs (miR-26, miR-125, and let-7) were detected in both PDTC and ATC . There are some downregulated miRNAs in PDTC (for example; miR-23 and miR-150) and ATC (for example; miR-1, miR-26, miR-125, miR-141 and miR-618) that were not mentioned in this paragraph but we displayed them in (Table 3 and Table 5) (64, 121, 124-127, 129-132). The miR-30 family of miRNAs, composed of miR-30a, miR-30b, miR-30c, miR-30d, and miR-30e, is implicated to play tumor suppressor roles in multiple cancer types, and has been

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found to have reduced expression in ATC (64, 121, 125-127, 133, 134). Moreover, overexpression of miR-30a suppresses ATC migration, tumor spreading, and metastasis in vitro and in vivo, most likely mediated by modified LOX-ECM interactions (133).

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miR-138 potentially targets the human telomerase reverse transcriptase (hTERT) transcript and has been found significantly downregulated in ATC cell lines (130). This finding suggests an epigenetic regulatory mechanism for increasing hTERT expression in ATCs in addition to the noted TERT promoter mutations (54).

The inverse relationship observed between the expression levels of both miR-25 & miR-

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30d and EZH2 protein levels in ATC cell lines suggests a critical role for miR-25 and miR-30d in regulating EZH2 expression and potentially ATC carcinogenesis (135). Moreover, enforced

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upregulation of miR-25 expression in thyroid cells resulted in the reduction of two miR-25 protein targets, TNF-related apoptosis-inducing ligand (TRAIL) and mitogen-activated protein kinase kinase 4 (MEK4), resulting in aberrant cell adhesion and apoptosis, underscoring their potential roles in anaplastic dedifferentiation of the thyroid follicular cells (136). The miR-200 family consists of miR-200a, miR-200b, and miR-200c (124). Braun et al. (2010) demonstrated that downregulation of the miR-200 family can be useful in distinguishing

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ATC from WDTCs (127). Examination of their potential role in regulating mesenchymalepithelial transition revealed not only the inhibition of TGFβ receptor 1 (TGFBR1) but also the upregulation of metastasis-promoting miR-21 in miR-200-silenced ATC cells. Zhang et al. (2012) further studied the molecular mechanism underlying miR-200 regulation of EGF/EGFR

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signaling in epithelial–mesenchymal transition (EMT) in ATC cells and showed Rho/ROCKmediated EMT signaling as potential targets for miR-200 in ATC (137).

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A novel metastasis-specific role has been implicated for miR-206 in ATC. Zhang et al. (2015) demonstrated that miR-206 is downregulated in ATC leading to the upregulation of metastasis-related MRTF-A in metastatic ATC (138). Further, they showed that induced silencing of miR-206 resulted in promoting cellular migration and invasion of ATC cells in vitro. Let-7 family of miRNAs (let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, and let-7i) are

abundantly expressed in normal thyroid tissue (123). Let-7 displays the potential to overexpress the thyroid transcription factor-1 (TTF1/NKX2-1), which is an important component in regulating the sodium-iodine symporter gene expression. Multiple studies showed a marked decrease in the expression of let-7a, let-7c, let-7d, let-7f, let-7g, and let-7i in ATC (125-127). Incidentally,

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decreased expression of TTF-1 is frequently reported in PDTCs and almost universally reported in ATCs (15), suggesting a direct role for the let-7 family of miRNAs in the progression of

6.2.2 MicroRNA upregulation in PDTC and ATC

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thyroid cancer dedifferentiation.

Upregulation of miRNAs in PDTC and ATC are less frequent than downregulation. There are approximately 10–15 upregulated miRNAs in PDTC and 35–40 in ATC. Some

miRNAs, such as miR-146, miR-221, and miR-222, were found overexpressed in multiple

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thyroid cancer types including PTC, FTC, PDTC, and ATC (64). Although the target genes are not clearly understood, the most frequently upregulated miRNAs in PDTC are miR-129, miR-

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146b, miR-183 miR-187, miR-221, miR-222, and miR-339 (122, 128, 139) while ATC showed increased expression of miR-17-92 cluster, miR-137, miR-146 family, miR-205, miR-221/222 families, miR-302c, miR-584, and miR-4295 (64, 122, 124, 125, 140-142). Other upregulated miRNAs in PDTC (for example; miR-146b, miR-221 and miR-222) and ATC (for example; miR-20a) that were not mentioned in this paragraph, we demonstrated them in (Table 3 and Table 5) (64, 122, 128, 139, 143).

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The miR-17-92 cluster transcribes a polycistron that yields seven different mature miRNA’s including miR-17-5p, miR-17-3p, miR-18a, miR-19a, miR-19b, miR-20a, and miR92a (124). Two of the seven cluster components, miR-17-3p and miR-17-5p, were found overexpressed in three out of the six ATC specimens tested. Inhibition of miR-17-5p, miR-17-

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3p, and miR-19a in vitro resulted in blocking of tumor proliferation and apoptosis, demonstrating a role for the miR-17-92 cluster in ATC progression (141).

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The miR-146 family, including miR-146a and miR-146b, was found overexpressed in ATC, possibly regulated by the transcription factor NF- B (122, 144). Overexpression of miR146b promoted cell proliferation in ATC cells by inhibiting p21 (CDKN1A) cell cycle regulators (140). Upregulated expression of the miR-221/miR-222 cluster in ATC is associated with poor clinicopathologic features (122). Similar to miR-146b, miR-221/miR-222 also influenced cell proliferation in vitro by targeting the p27kip1 (CDKN1B) protein (83). Compared to PTC and normal thyroid samples, miR-584 is overexpressed in ATC (145). Induced expression of tumor suppressor candidate 2 (TUSC2), a target of miR-584, was shown to rescue the inhibition of apoptosis induced by miR-584 through a novel TWIST1/miR-584/TUSC2

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pathway in thyroid cancer cells, in vitro (145). In a similar fashion, overexpression of miR-4295 promoted ATC cell proliferation and invasion through direct targeting and inhibition of cyclindependent kinase inhibitor 1A (CDKN1A) (146). Damanakis et al. (2016) analyzed expression profiles of hsa-let7b-5p and hsa-let7f-5p

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and their predicted targets SLC5A5 (NIS) and high-mobility group AT-hook 2 (HMGA2) in

thyroid cancer and reported contrasting expression patterns in various subtypes of thyroid cancer (147). In ATC, hsa-let7f-5p was overexpressed compared to other types of thyroid cancers but

(147).

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6.3 Role of long non-coding RNAs in PDTC and ATC

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NIS was found stable or upregulated. There was no correlation between has-let7f-5p and NIS

lncRNAs are functionally defined as ‘non-protein coding transcripts longer than 200 nucleotides’ (65). Deregulation of lncRNA expression has been noted to regulate tumor proliferation, metastasis, and recurrence of breast, liver, lung, and thyroid cancer (65, 89, 95, 148-150). However, the limited numbers of studies addressing lncRNA’s regulation in thyroid cancer are mostly limited to PTC, PDTC, and ATC (Table 5) (93-95, 151, 152).

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Zhang et al. (2017) studied the expression pattern of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a nuclear-abundant lncRNA, in 10 normal thyroid tissue, 28 PTC, 21 PDTC and 35 ATC samples using tissue microarrays and found slightly upregulation in PDTC and ATC if compared with normal thyroid tissue (152). In the same study,

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qRT-PCR analysis of MALAT1 expression demonstrated that PTC is the most upregulated among other thyroid cancer subtype (FTC, PDTC and ATC) and normal thyroid tissue (152).

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MALAT1 and its suspected downstream target IQ Motif Containing GTPase Activating Protein 1 (IQGAP1), a scaffold protein involved in regulating the actin cytoskeleton, cellular adhesion, transcription, and migration, were found overexpressed in FTC samples (153). Additionally, the overexpression of MALAT1 was shown to induce ATC cell proliferation and invasion in vitro through the upregulation of IQGAP1 (153). LOC100507661 expression was analyzed in various thyroid cancer cell lines, that include PTC (TPC1, BCPAP), FTC (FTC133), and ATC (C643, 8505C) cells (151). PTC and ATC cells displayed LOC100507661 overexpression, with especially high levels in C643 and 8505C. However, LOC100507661 was undetectable in the FTC cell line FTC133. Moreover, elevated

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expression of LOC100507661 was found significantly correlated with lymph node metastasis (p = 0.035) and BRAF mutation status in PTCs. Additionally, stable overexpression of LOC100507661 induced proliferation, migration, and invasion in thyroid cancer cell lines in vitro, suggesting a potential role for LOC100507661 in promoting a more aggressive phenotype

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(151).

A comparative expression analysis of PVT1, a lncRNA encoded by the human PVT1 gene, was performed in 84 thyroid cancer cell lines including 66 PTC, 11 FTC, and 7 ATC cells, which showed universal overexpression of PVT1. Furthermore, silencing of PVT1 suppressed

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the expression of TSHR, the major regulator of thyroid proliferation, in the PTC, FTC, and ATC cell lines, supporting the proposed function of PVT1 in promoting thyroid cancer cell

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tumorigenesis including that of ATC’s via TSHR suppression (95).

Table 5. Dysregulated micro and long noncoding RNAs in PDTC and ATC ncRNA

Type of

Chromosomal

Regulation

Location

Poorly differentiated thyroid cancer Downregulated

miR-150

Downregulated

miR-146b

Upregulated

miR-221, miR-

Upregulated

222

19p13.12

19q13.33

Affected/Involved

Targets

signaling pathway

FGFR3

TP53

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miR-23

Potential/Predicted

10q24.32

SMAD4

Xp11.3

PTEN, CDK1A,

11q13.1

IQGAP1

MAPK pathway

PI3K-Akt pathway

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Downregulated

References

Promote tumor

Dettmer (2014),

relapse

Zhang (2016)

Enhance cancer-

Dettmer (2014),

specific mortality

Zhang (2016)

TGF- β pathway,

Promote thyroid

Nikiforova (2008),

NF- B pathway

tumorigenesis

Geraldo (2012)

MAPK pathway

Induce the tumor

Nikiforova (2008),

angiogenesis

Dettmer (2014)

MAPK/ERK-

Dysregulated cell

Huang (2016),

Pathway

cycle, cell

Damanakis (2016),

proliferation, and

Zhang (2017)

CDK1B, TIMP3, KIT

MALAT1

Outcome

cell migration

Anaplastic thyroid cancer miR-1

miR-25

Downregulated

Downregulated

21q21.1

7q22.1

CXCR4 and SDF-1α

CXCR4/SDF-1α

Dysregulated

protein

pathway

cellular proliferation and migration

EZH2, TRAIL, BIM,

MAPK pathway

Dysregulated

Esposito (2012),

chromatin

Zhang (2016),

condensation

Aherne (2016)

NF- B pathway,

Slow the cell

Visone (2007),

TGF-β pathway

proliferation rate

Schwertheim (2009),

KLF4

miR-26a, miR26b

Downregulated

3p22.3, 2q35

Leone (2011)

HMGA

Braun (2010), Hebrant (2014),

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Zhang (2016)

miR-30 family

Downregulated

6q13, 8q24.22,

SMAD2, TGFBR1,

1q34.2

LOX, beclin 1

Autophagy pathway

Protect ATC cells

Zhu (2009),

from apoptosis and

Schwertheim (2009),

autophagy

Braun (2010), Zhang (2014), Hebrant

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(2014), Boufraqech (2015), Zhang (2016)

miR-125a, miR-

Downregulated

125b

19q13.41,

MMP1, HMGA2

p53-dependent

Promote tumor

Visone (2007),

11q24.1

LIN28A, p53

pathway

invasion, histone

Schwertheim (2009),

modification

Braun (2010), Furizawa (2014),

miR-141

Downregulated

miR-200 family

Downregulated

12p13.31

Downregulated

let7 family

Downregulated

Wnt pathway

TGFBR1, SMAD2

TGF-β pathway

Zhang (2016)

Increase with the

Mitomo (2008),

progression of

Zhang (2016)

histological dedifferentiation, and malignant behavior Increase the

Braun (2010)

invasive potential of tumor

ZEB1, ZEB2, EZH2,

TGF-β pathway,

Increase the

Braun (2010),

12p13.31

TGFBR1, SMAD2

EGF pathway

invasive potential of

Esposito (2012),

tumor

Zhang (2012),

6p12.2

12q21.31

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miR-618

hTERT

1p36.33,

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miR-206

Downregulated

3p21.32

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Downregulated

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miR-138

SC

Hebrant (2014),

9q22.32,

MRTF-A

XIAP

Hebrant (2014), Zhang (2016) MRTF pathway

Promote invasion,

Zhang (2015)

migration

PI3K/Akt pathway

Dysregulated cell

Cheng (2014), Yi

growth, cell cycle

(2015)

arrest

RAS pathways, Let-

Induce tumor

Johnson (2005),

19q13.41,

7-independent

proliferation,

Visone (2007),

3p21.2,

pathways

histone

Schwertheim (2009),

modification

Braun (2010),

12q14.1, 21q21.1

RAS, HMGA2, LIN28

Furizawa (2014), Hebrant (2014), Zhang (2016)

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Upregulated

miR-17-92

13q31.3

cluster

p21, TIMP3, PTEN,

PI3K growth

Promote tumor

Takakura (2008),

E2F1

pathway

growth and invasion

Smallridge (2009),

LIMK1

Rho pathway

Dysregulated

Xiong (2014), Zhang

cellular

(2016)

Furizawa (2014) miR-20a

Upregulated

13q31.3

proliferation and

miR‑146a, miR-

Upregulated

146b

miR-221, miR-

Upregulated

5q33.3,

p21, NF- B, THRB,

10q24.32

SMAD4

Xp11.3

p27, RECK, PTEN

222

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invasion

Dysregulated cell

Nikiforova (2008),

differentiation, and

Furizawa (2014),

invasion

Wang (2016)

TGF-β pathway,

Promote tumor

Visone (2007),

MAPK pathway

growth and invasion

Furizawa (2014),

NF- B pathway

Upregulated

5q32

TUSC2

SC

Zhang (2016)

miR-584

TWIST1/miR-

Induced resistance

584/TUSC2

to apoptosis

Orlandella (2016)

pathway Upregulated

10q25.2

CDKN1A

-

let7f-5p

Upregulated

9q22.3

SLC5A5 (NIS),

MAPK pathway

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miR-4295

HMGA2

Increased cell migration and invasion

Shao (2015)

Dysregulated cell

Marini (2011),

proliferation, and

Damanakis (2016)

differentiation

MALAT1

Downregulated

11q13.1

-

-

-

Zhang (2017)

MALAT1

Upregulated

11q13.1

IQGAP1

MAPK/ERK-

Dysregulated cell

Liu (2010), Huang

Pathway

cycle, cell

(2016)

LOC100507661

Upregulated

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proliferation, and

3q26.2

Correlated with BRAF

cell migration -

mutation

8q24.21

EP

Upregulated

TSHR

Kim (2016)

proliferation, migration, and invasion Recruiting EZH2

Dysregulated tumor

Zhou (2016)

proliferation and arrested cell cycle at G0/G1 stage

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PVT1

Promote tumor

7. Current status of epigenetic-targeted therapy in thyroid cancer The drugs targeting epigenetic alterations in tumors can be classified according to the

mechanism(s) that they target. HDAC inhibitors and DNA methylation inhibitors (DNMTi) are two main classes of epigenetic pharmaceuticals. To date, the US Food and Drug Administration (FDA) has approved four HDAC inhibitors including belinostat, panobinostat, romidepsin, and vorinostat for the treatment of hematologic malignancies (154-157). These drugs inhibit histone deacetylase, the enzyme that detaches acetyl group from an ε-N-acetyl lysine on histones, which

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regulates gene expression. Belinostat (PXD101) has shown effective clinical usage by improving the overall response rate of peripheral T-cell lymphoma patients (154, 158, 159). Panobinostat acts as a pan-HDAC inhibitor and showed clinical benefit for treating relapsed and refractory multiple myeloma patients (160). Panobinostat combined with bortezomib and dexamethasone

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showed enhanced tumor response rate compared to placebo (161). Romidepsin and vorinostat show clinical efficacy in the treatment of cutaneous T-cell lymphoma by increasing the treatment response rate (162-164).

Promoter hypermethylation leading to tumor suppressor silencing is a common epigenetic

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mechanism observed to drive a variety of tumor types. Consequently, multiple studies have shown improved treatment responses in multiple cancer types with methylation inhibitors. A

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widely used example of DNMTi is azacitidine or 5-azacytidine, which has been approved by both European Medicine Agency (EMA) and FDA for treatment of myelodysplastic syndrome (MDS) (165). Azacitidine displayed significantly prolonged overall median survival compared to the conventional care regimens (best supportive care, low-dose cytarabine, and intensive chemotherapy) in MDS patients (165, 166). Another DNMTi, decitabine (5-aza-2'deoxycytidine) efficiently inhibited DNA methyltransferase leading to a hypomethylation status

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of target genes (168). Similar to azacitidine, decitabine also showed the potential to treat MDS by improving progression-free survival when compared to best supportive care (167).

8. Future prospects for epigenetic-targeted therapy in PDTC and ATC

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Currently, there are no thyroid-specific epigenetic medications available for the treatment of WDTC, PDTC, or ATC. However, multiple studies have recently demonstrated a progressive

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understanding of the nature of the epigenetic alterations in PDTC and ATC, potentially paving the way to the development of targeted epigenetic pharmaceuticals in the near future (22-24, 37, 48, 59, 96, 97, 99, 142, 168). Aurora kinase inhibitors have also shown promise in laboratory tests. Wunderlich et al.

(2011) evaluated the functions of MLN8054, an Aurora serine ⁄ threonine kinases inhibitors, on ATC cells in vitro and in vivo. MLN8054 was found to be effective in inhibiting ATC cell proliferation and promoting apoptosis in vitro and tumor growth in vivo (169). Similarly, in vitro use of SNS-314 mesylate, a pan-inhibitor of the Aurora kinases, has also been shown to be effective in inhibiting ATC cell growth (170).

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BET inhibitors are immunosuppressive epigenetic anti-cancer drugs that bind to the extra-terminal motif (BET) proteins that prevent interaction between BET proteins and acetylated histones/transcription factor complexes (171). Mio et al. (2016) reported that BET inhibitors caused decreased viability in ATC cells by causing MCM5 silencing and blocking

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ATC cell proliferation (172). Epigenetic-driven usage of conventional chemotherapeutic agents alone has shown limited promise in improving the treatment of ATC. The use of cisplatin in a subset of ATCs that expressed miR-30 led to a decrease in tumor size. It was postulated that

cisplatin-activated autophagy response (134).

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beclin1, an autophagy gene, could be negatively regulated as it affects the repression of the

Both PDTC and ATC are very complex diseases driven by a multitude of independent

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and overlapping genetic and epigenetic events. This diversity also highlights the potential opportunity for unique therapeutic approaches including those that combine both genetic and epigenetic targets. Towards this aim, various epigenetic approaches are currently being explored. For instance, the combined use of HDAC and proteasome inhibitors in rat thyroid cell line in vitro resulted in an additive apoptotic response, potentially by interfering with the cell’s dominant survival and ubiquitin-dependent pathways (73). This early evidence supports the

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notion that combined deacetylase-proteasome inhibitors would be potentially effective in treating ATCs where knock-down of multiple complimentary pathways supporting cell survival is likely to yield favorable therapeutic outcomes. In a similar approach, Catalano et al. (2012) used the non-selective HDAC inhibitor panobinostat to initiate ATC cell death both in cell culture and

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xenograft (78). Solitary use of PXD101, another HDAC inhibitor, showed inhibition of ATC cell proliferation but acted synergistically when used in combination with doxorubicin and paclitaxel

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(107).

In summary, PDTC and ATC are aggressive and fatal malignancies characterized by

complex genetic and epigenetic modifications. A lack of efficient genetic targeting and the persistent failure of conventional therapeutic approaches make epigenetic targeting an attractive prospect for treating these lethal diseases. Although there are no epigenetic-driven thyroidtargeted therapies currently available, a detailed understanding of the signaling events that are compromised in PDTC’s and ATC’s via epigenetic modifications will dictate the development of precise epigenetic pharmaceuticals to repair the pathways that are being appropriated during the process of PDTC and ATC dedifferentiation.

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Acknowledgement: The authors thank Dr Ngoentra Tantranont for providing the histopathologic pictures of PDTC and ATC.

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Conflict of interest: The authors have no actual or potential conflicts of interest to declare.

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Epigenetic alterations may play potentially decisive roles in the origin and the agrressive clinical course of PDTC and ATC. Mechanisms of epigenetic deregulations in PDTC and ATC include DNA methylation, histone modification, chromatin remodeling, and ncRNA aberration. Understanding the underpinnings of PDTC and ATC epigenetic dysregulations may lead to the development of novel epigenetics-based therapeutic strategies.

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