A Review of Driver Genetic Alterations in Thyroid Cancers.

Thyroid cancer is a frequent endocrine-related malignancy with continuously increasing incidence, and recently the development in understanding its molecular pathogenesis is mainly through the explanation of the original role of several key signaling pathways and related molecular distributors. Central to these mechanisms are the genetic and epigenetic alterations in these pathways such as mutation and DNA rearrangements. However, it does not mean that all the somatic abnormalities in a cancer genome are involved in cancer development and just driver mutations are concerned in tumor initiation. By way of illustrations, MAPK pathway motivated by BRAF V600E and RAS and RET / PTC rearrangements are suggesting driver genetic alterations in follicular derived thyroid cancers considered in the current review.

Introduction

Thyroid cancer is the most common endocrine-related cancer that its incidence has continuously increased in the last three decades all over the world (1-5). Thyroid carcinomas are heterogeneous groups of neoplasm with typical histopathological features similar to other tumors (6).

The thyroid gland is composed of two main types of epithelial cells: the follicular cells, which convert iodine into thyroxine, also known as T4, and Triiodothyronine, also known as T3. The thyroid hormones, triiodothyronine (T3) and its prohormone, thyroxine (T4), are tyrosine-based hormones produced by the thyroid gland primarily responsible for regulation of metabolism. Another type of epithelial cells is paralfollicular or C-cells, which secrete calcitonin. Primary thyroid cancers mostly initiate from thyroid follicular cells (epithelial tumors) and develop three main pathological types of carcinomas: papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC) and anaplastic thyroid carcinoma (ATC) contrary to medullary thyroid carcinoma (MTC) that arises from thyroid parafollicular (C) cells (7-9). Due to well differentiation and indolent tumor growth, PTC and FTC are classified as differentiated thyroid cancer (DTC). PTC consists of 85%-90% of all thyroid cancer cases, followed by FTC (5%-10%) and MTC (about 2%), while ATC accounts for a smaller amount than 2% of thyroid cancers and usually happens in the aged people (10).

The classic treatment for thyroid cancer is thyroidectomy and adjuvant radioiodine ablation that most patients can be cured, but still surgically inoperative recurrence, refractoriness to radioiodine in DTC, poorly differentiated thyroid carcinoma and ATC are unsolved. Similar to other solid cancers, thyroid cancer is commenced by genetic alterations and epigenetic changes in driver oncogenes or tumor suppressor genes (11-14). Recent advancement of molecular technologies brought a new insight to the thyroid tumors diagnosis and prognosis. The current review mainly focused on the follicular thyroid cell derived cancers genetics in order to shed light on driver genetic alterations and their importance in thyroid tumor genesis.

Molecular genetics of thyroid cancer

Thyroid cancer comes up as a result of multiple genetic and epigenetic alterations in the DNA of cancer cells. There are numerous somatic point mutations and chromosomal rearrangements in different steps of follicular cell-derived thyroid cancer (Figure 1) (15,16) that mainly belong to the MAPK signaling pathway and RET/PTC rearrangements (17).

Figure 1

Stepwise dedifferentiation of follicular cell-derived thyroid cancer.

It should be kept in mind that not all the somatic abnormalities of a cancer genome are involved in initiation of the cancer since some are the consequences of carcinogenesis; hence, the terms “driver” and “passenger” mutation are made up. A driver mutation is an oncogenesis implication in cancer stem cells and is positively selected in the microenvironment of the tissue where the cancer begins and is not needed for maintenance of the final cancer (although it often is) (18,19).

A passenger mutation is not selected, is not given clonal increase and therefore does not contribute to cancer development. Since somatic mutations without functional consequences often happen during cell division, passenger mutations are initiated within cancer genomes (20). One of the problematic issues is the discrimination of driver from passenger mutations. Whole-genome sequencing, however, incorporating analysis of more than 20,000 proteincoding genes and unknown numbers of functional elements in intronic and intergenic DNA, presents a greater challenge. Investigation of the biological consequences of putative driver mutations often consolidate the evidence implicating them in oncogenesis and provide insight into the subverted biological processes by which they contribute to cancer development. Thyroid cancer is a genetically simple disease with a relatively low number of mutations in each tumor. Driver mutations and gene fusions are identified in most of thyroid cancers suggesting that two main cell signaling pathways, MAPK and PI3K-AKT, are involved in the development of thyroid tumors (17,21). The MAPK/ERK pathway, also known as the Ras-Raf-MEK-ERK pathway, is a transporter of a signal from a receptor on the cell surface to the nucleus (DNA) (Figure 2). After binding a signaling molecule to its target receptor on the cell surface, this signaling pathway initiates and when the DNA in the nucleus expresses a protein in order to make some changes in the cell, it is terminated (22). This pathway has lots of proteins, including MAPK (mitogen-activated protein kinases, originally called ERK, extracellular signal-regulated kinases) and is connected with the cell proliferation, differentiation, migration, and senescence; and apoptosis components of the MAPK/ ERK pathway were discovered when they were found in cancer cells (6, 22-24).

Figure 2

The MAPK and related pathways in thyroid cancer.

Nuclear factor-κB (NF-κB) pathway that is leading to activation of the inhibitor of κB (IκB) kinase (IKK), resulting in the phosphorylation of IκB and dissociation from NF-κB. Free NF-κB then enters the nucleus to promote the expression of tumor-promoting genes. On the right side of the figure is the RASSF1–mammalian STE20-like protein kinase 1 (MST1)– fork head box O3 (FOXO3) pathway and activated MST1 then phosphorylates FOXO3 on Ser207. Phosphorylated FOXO3 enters the nucleus to promote the expression of pro-apoptotic genes in the FOXO pathway. In the middle of the figure is a unique and powerful mechanism of thyroid tumor genesis driven by BRAF-V600E. DAPK1, death-associated protein kinase 1; HIF1A, hypoxia-inducible factor 1α; MMP, matrix metalloproteinase; NIS, sodium–iodide symporter; TGFB1, transforming growth factor β1; TIMP3, tissue inhibitor of metallo proteinases 3; TPO, thyroid peroxidase; TSHR, thyroid-stimulating hormone receptor; TSP1, thrombospondin 1; UPA, urokinase plasminogen activator; UPAR, urokinase plasminogen activator receptor; VEGFA, vascular endothelial growth factor A (25).

In early thyroid cancer, MAPK pathway is motivated by mutations in BRAF and RAS or by RET / PTC rearrangements. A key driver mutation upsetting MAPK pathway is the point mutation of BRAF, which makes the expression of BRAFV600E mutant protein resulting in constitutive activation of the serine/threonine kinase (26-31). In fact, amino acid substitution at posi-, tion 600 in BRAF, from a Valine (V) to a glutamic acid (E) is the result of V600E mutation. This mu-i tation occurs within the activation segment of the kinase domain (Figure 3). BRAF mutations are also frequently found in tumors with no driver mutations in NRAS, KIT, and other genes. BRAFV600E mutation is found in about 45% of PTCs (32, 33). However, some human PTC tumors show intra-tumors heterogeneity in the BRAF genotype — a minority of cells have BRAFV600E while the majority contain wild-type BRAF (34).

Figure 3

Schematic of BRAF V600E mutation. Functional domains of BRAF are depicted. CR1: conserved regions 1. CR2: conserved region 2.

After BRAF mutations in thyroid cancer, RAS mutations are the most important driver genetic alteration (35, 36). RAS is in bound with GTP and when intrinsic GTPase of RAS hydrolyses GTP and converts RAS into an inactive GDP-bound state the RAS signaling is terminated (Figure 4) (37). There are three isoforms of RAS: HRAS, KRAS, and NRAS, and NRAS is predominantly mutated in thyroid tumors, mostly involving codons 12 and 61(30,38). The RAS mutations in follicular thyroid adenoma (FTA), a supposed premalignant lesion, suggests that activated RAS may have a role in early follicular thyroid cell tumor genesis and higher aggressive tumor behaviors (38,39). The expression of mutant HRAS was induced and resulted in differentiated colonies (39-41). Moreover, in the thyroid gland of transgenic mouse studies with conditional physiological expression of a KRAS had no transformation, but simultaneous KRAS mutant expression and PTEN deletion induced a rapid occurrence of aggressive FTC (42-44).

Figure 4

The PI3K–AKT and related pathways in thyroid cancer (37).

Another main driver genetic alteration in thyroid cancer is the rearranged during transfusion (RET) proto-oncogene. RET (rearranged during transfecntion), is localized on chromosome 10 (10q11.2) and has 21 exons (45). The natural alternative splicing of the RET gene consequences in making three different isoforms of the protein RET; RET51, RET43, and RET9, which have 51, 43, and 9 amino acids in their C-terminal tail respectively (46). Each protein is divided into three domains: an N-terminal extra-i cellular domain with four cadherin-like repeats and a cysteine-rich region, a hydrophobic transmembrane domain and a cytoplasmic tyrosine kinase domain, which is split by an insertion of 27 amino acids (47). As a result of its capability to transform NIH/3T3 cells by DNA rearrangement, the RET proto-oncogene was first recognized in 1985 (48). The proteins that RET encodes is a cellular tyrosine kinase transmembrane receptor that is separated into the three main domains: an N-terminal extracellular domain containing fourcadherin-like regions; a cysteine-rich region with a transmembrane domain; and a cytoplasmic domain with tyrosine kinase activity (47, 49-51). Four diverse ligands are described: Glial Derived Neurotrophic (GDN) factors, Neurturin (NRTN), Artimin (ARTN), and Persepin (PSPN), respectively (47, 52, 53). DNA rearrangements are a result of homologous recombination, gene conversion, and illegitimate recombination. During homologous recombination in a cell containing more than one copy of a given chromosome, one copy can combine with corresponding segments of the other. This kind of recombination is ultimately dependent upon the DNA sequence homology between the two copies. Several types of RET/PTC rearrangements are reported (Table 1) (54,55). The presence of RET/PTC rearrangement in microcarcinoma powerfully supports the hypothesis of a driving role of this oncogene in the tumor transformation (56).

Table 1

Different types of RET/PTC rearrangements in thyroid tumors according to Nikiforov YE (57)

Oncogene	Donor gene	Chromosomal location
RET/PTC1	CCD6(formerly H4)	10q21
RET/PTC2	PRKAR1A	17q23
RET/PTC3	NCO4 (formerly Ele 1)	10q11.2
RET/PTC4	NCO4 (formerly Ele1)	10q11.2
RET/PTC5	Golgas	14q
RET/PTC6	TRIM24	7q32-34
RET/PTC7	TRIM33	1p13
RET/PTC8	KTN1	14q22.1
RET/PTC9	RFG9	18q21-22
ELKS-RET	ELKS	12p13.3
PCM1-RET	PCM1	8p21-22
RFP-RET	TRIM27	6p21
HOOK3-RET	HOOK3	8p11.21

The described RET/PTC prevalence in thyroid tumors varies greatly in different studies (58-65). However, this difference can be the consequence of tumor heterogeneity, ethnical and geographic variations, and dissimilar sensitivities of detection methods (66-68). RET/PTC rearrangements are more often in thyroid cancers after radiation exposure (50-80%) (69-72). The biological mechanisms of radiation carcinogenesis related to RET/PTC rearrangements are studied several times. It is observed that damage to cellular DNA is responsible for mutagenesis and carcinogenesis and those double-strand breaks are the most important event for the direct generation of gene translocations and rearrangements (21, 73-77). Thanks to the recent advanced next generation sequencing and whole genome sequencing, the number of candidate genetic changes in thyroid cancer has increased (78). But it is really important to discriminate between driver and passenger ones. Other genetic changes considered as passenger mutations include: PI3K (phosphatidylinositol-3 kinase), β-catenin (CTNNB1), TP53, is citrate dehydrogenase 1 (IDH1), anaplastic lymphoma kinase (ALK), and epidermal growth factor receptor (EGFR) (79-89). The preferential occurrences of these mutations in PDTC and ATC, which are the most aggressive thyroid cancers, indicate the fact that they may have a role in the progression and aggressiveness of thyroid cancer.

Conclusions

While diverse oncogenes are involved in thyroid tumor genesis, BRAF and RAS mutations, and RET/ PTC rearrangements are most frequently involved as a driver changes. Notwithstanding all these observations, no strong supporting data still show a classic prognostic role for BRAF and RAS mutations, and RET/PTC rearrangements. But it is clear that RET/ PTC rearrangements are correlated with radiation exposure and are more recurrent in patients with radio induced PTC.