The Isoforms of Estrogen Receptor Alpha and Beta in Thyroid Cancer

The incidence of thyroid cancer was predominant in women, indicating that the sex hormone may have a role in thyroid cancer development. Generally, the sex hormone exerts its function by binding to the correspondent nuclear receptors. Therefore, aberrant of these receptors may be involved in the development of thyroid cancer. Estrogen receptor alpha (ERα) and beta (ERβ), two main estrogen receptors, have been reported to have an important role in the pathogenesis of thyroid cancer. When the ERα and ERβ genes undergo the alternative RNA splicing, some ERα and ERβ isoforms with incomplete functional domains may be formed. To date, several isoforms of ERα and ERβ have been identified. However, their expression and roles in thyroid cancer are far from clear. In this review, we summarized the expressions and roles of ERα and ERβ isoforms in thyroid cancer, aiming to provide the perspective of modulating the alternative RNA splicing of ERα and ERβ against thyroid cancer.


INTRODUCTION
The morbidity of thyroid cancer has been rapidly increased during the past decades (1,2). The increased rate in women was particularly pronounced (2)(3)(4)(5). The biased occurrence of thyroid cancer between males and females suggests that the sex hormone may play a central role in the initiation of thyroid cancers or certain types of thyroid cancers. Traditionally, estrogen is the primary female sex hormone mainly responsible for the control of functions of the female reproductive system. In the genomic pathway, estrogen exerts its physiological functions by binding to specific nuclear receptors, the estrogen receptors (ERs), which activate transcriptional processes and/or signaling events and thus control the gene expression (6). ERs can express in both male and female organs/tissues. Therefore, the ERs are critical in the maintenance of health.
Numerous studies have shown that the critical and opposite roles of ERa and ERb in the development and progression of thyroid cancer. For example, Maura Di Vito et al. reported that the mRNA and protein of ERa, but not ERb, was upregulated in thyroid cancer, suggesting that ERa has a vital role in thyroid cancer (7). In addition, ERa positive and ERb negative were associated with a more aggressive phenotype of T1 and T2 thyroid cancer (8,9). Estrogen induced the metastatic potential of thyroid cancer through ERa and ERb (10). Yanhong Huang et al. evaluated the expression of ERa and ERb by immunohistochemical staining, and they reported that estrogen-activated ERa might mediate the stimulatory effect on thyroid cancer growth and progression (11). However, ERb was negatively correlated with mutant P53, suggesting that ERb has some inhibitory actions in thyroid cancer (11). ERa is significantly correlated with distant metastases and poorly differentiated thyroid cancer with multicentricity cases, whereas ERb is significantly associated with lymph node metastases in follicular thyroid cancer (12). These studies have all suggested that ERa and ERb play an important role in thyroid cancer.
Our previous studies have also illustrated the significance of ERs in thyroid cancer. We found crosstalk between ERs and peroxisome proliferator-activated receptor gamma (PPAR-g). The interaction between PPAR-g and ERb inhibited the proliferation and migration of thyroid cancer (13). ERa induced prosurvival autophagy through generating the reactive oxygen species and activating ERK1/2 in thyroid cancer (14). In addition, we also reported that the differential role of ERa and ERb in thyroid cancer mediated the production of endogenous PPAR-g ligand (15). Upregulated ERa/ ERb ratio by PES1 will promote the occurrence and development of papillary thyroid cancer (PTC) (16).
The roles of ERa and ERb in thyroid cancer appear to be convincing, and the signal pathway of estrogen and estrogen receptors in the development of thyroid cancer has been well reviewed (17). However, there is a controversial result showing the association of the expression of ERa with a good outcome in thyroid cancer. Giacomo sturniolo et al. (18) evaluated the expression of ERa in 203 PTC, and they observed an association between ERa expression and a favorable outcome in their cohort. The cause of such a controversial result remains unknown.
However, it is possible that controversial results are related to ERa antibodies used. For example, the antibody used by Giacomo sturniolo and colleagues is ERa SP1 clone, which was a synthetic peptide derived from the C-terminal of human estrogen receptor (18). The antibody used by Yanhong Huang and colleagues is ERa 1D5 clone, which is a recombinant human estrogen receptor protein (11). The antibodies recognized different regions of ERa protein might result in different expression patterns since several isoforms of ERa have been identified.
The signaling mechanism of ERs and their expression and roles in thyroid cancer have been well-reviewed (6,17,19,20). Therefore, this review focused on the alternative splicing of ERs or isoforms of ERa and ERb in thyroid cancer ( Table 1).

ALTERNATIVE SPLICING AND THYROID CANCER
Alternative splicing of protein-coding mRNAs is an essential regulatory mechanism in eukaryotic gene expression that controls the proper function of proteins. The alternative is a fundamental biological process that allows for considerable proteomics diversity and complexity from the limited approximately 20,000 genes (24). However, aberrant alternative splicing may lead to cancer development, and understanding aberrant alternative splicing can facilitate cancer diagnosis and therapy (25,26). Overall, The abnormal regulation of alternative splicing that can produce multiple different isoforms and diversify protein expression may lead to development of tumors.
Alternative splicing events frequently occur in thyroid cancer. Zenghong Wu et al. found that 45150 alternative splicing events in 10446 thyroid cancer cells derived from 506 patients (27). Furthermore, they found that the alternative splicing signatures were significantly associated with thyroid cancer patients' overall survival (27). Baoai Han et al. showed that abnormal alternative splicing events might play critical roles in the development and progression of thyroid cancer by participating in changes in molecular structure, homeostasis of the cell environment (28). To date, several isoforms of ERa and ERb have been reported, given the significance of alternative splicing and ERs isoforms in thyroid cancer, indicating that the expression and role of ERa and ERb isoforms in thyroid cancer are important. Therefore, in the following section, we would discuss the ERa and ERb isoforms in thyroid cancer.

ERa AND ITS ISOFORMS IN THYROID CANCER
According to the national center for biotechnology information database (https://www.ncbi.nlm.nih.gov/), ERa is located in 6q25.1-q25.2. The ERa protein contains an N-terminal ligandindependent transactivation domain, a central DNA binding domain, a hinge domain, and a C-terminal ligand-dependent transactivation domain. The N-terminal ligand-independent transactivation domain encompassed a ligand-independent activation function (AF1) domain involved in the transcriptional activation of target genes. The DNA binding domain mediates sequence-specific binding of ERs to DNA sequences in the target gene denoted estrogen-responsive elements (EREs). The C-terminal ligand-dependent transactivation domain contains a ligand-dependent activation domain (AF2) (29,30). The protein localizes to the nucleus, where it may form either a homodimer or a heterodimer with ERb.
Several alternative splicing isoforms of ERa have been identified, including ERa wild type/full length (ERa66), ERa46, and ERa36 ( Figure 1). The isoforms of ERa have incomplete function domains that may alter their roles in thyroid cancer. The expression and role of ERa66 in thyroid cancer have been described in the previous section. Therefore, this section would focus on the ERa46, ERa36 and exon-deleted ERa isoforms.

ERa46 and Thyroid Cancer
ERa46 was first identified and characterized in osteoblasts (31). ERa46 is generated by alternative splicing of an ERa66 gene product, which results in exon 1 being skipped with a start codon in exon 2 used to initiate translation of the protein. Consequently, compared to ERa66, the ERa46 protein lacks amino acids 1-173, which codes N-terminal ligand-independent transactivation domain (AF1). Therefore, ERa46 has an incomplete AF1 domain. Functional analysis revealed that ERa46 could heterodimerize with the ERa66 as well as the ERb (31). However, the expression and role of ERa46 in thyroid cancer remain largely unknown. In another ER-related cancer, breast cancer, the expression of ERa46 was observed in over 70% of breast tumors among 116 ERa66 positive human breast tumors (32). In addition, ERa46 decreased the proliferation rate of breast cancer MCF7 cells in response to 17b estradiol (32). The data suggested that ERa46 inhibited tumor cell functions, which is different from ERa66.
Furthermore, the reduced expression of ERa46 was found in tamoxifen-resistant breast cancer cells, and the force overexpression of ERa46 in these tamoxifen-resistant breast cancer cells restored growth inhibition by tamoxifen (33). A study reported that the enhanced expression of ERa in breast cancer was associated with thyroid cancer occurrence, suggesting that ERa may have a role in the link between breast cancer and thyroid cancer (34). However, no data shows the expression and the role of ERa46 in thyroid cancer. Further studies are necessary.

ERa36 and Thyroid Cancer
ERa36 isofrom is shorter than ERa46. ERa36 lacks both AF-1 and C-terminal ligand-dependent transactivation domain (AF2), and the last 138 amino acids are replaced with a unique 22 amino acid sequence. It was first identified and cloned by Zhaoyi Wang and colleagues, and ERa36 is predicted to function as a dominant-negative effector of ERa66 mediated estrogenresponsive gene pathways and has the potential to trigger membrane-initiated mitogenic estrogen signaling (35,36). Structurally, ERa36 has an incomplete AF1 domain and an AF2 domain. Therefore, understanding the role of ERa36 in thyroid cancer is vital for us to develop ERs as therapeutic targets.
There are limited studies on the ERa36 in thyroid cancer. The expression of ERa36 proteins was analyzed in 218 primary PTC by immunohistochemistry staining and it was found that its expression was upregulated in thyroid cancer (21). The functional study showed that upregulation of ERa36 by E2 enhanced the proliferation, invasion, and migration of PTC cells. The results suggested that increased expression of ERa36 is associated with aggressive thyroid cancer (21). Given the significance of ERa36 in cancer development and progression (37), further investigation of ERa36 in thyroid cancer may provide us with novel insight into the pathogenesis of thyroid cancer.

Exon-Deleted ERa Isoforms
In addition to ERa46 and ERa36, several exon-deleted ERa isoforms have been reported in breast cancer, such as exon 3 deleted ERa (ERaD3), exon 5 deleted ERa (ERaD5), exon 7 deleted ERa (ERaD7) (38). As shown in Figure 1, exon 3 codes for the DNA binding domain, exon 5 and exon 7 codes for part of the AF2 domain. Therefore, each exon-deleted ERa isoform may alter the function of ERa due to the alteration in functional domains, and their roles in thyroid cancer is warrant further studying.
To date, the expression and function of ERa isoforms in thyroid cancer were far from clear. Further studies were warranted to investigate it.

ERb AND ITS ISOFORMS IN THYROID CANCER
According to the national center for biotechnology information database (https://www.ncbi.nlm.nih.gov/), the ERb gene is located at 14q23.2-q23.3. The ERb protein contains an N- terminal ligand-binding domain, DNA binding domain, and Cterminal ligand-binding domain. ERb is classified as the nuclear receptor, and mainly located in the nucleus. However, The expression of ERb also can be observed in the cytoplasm and mitochondrial (20,39). The impact of subcellular localization on the ERb function remains unclear. Structurally, there is only a 16% similarity between the Nterminal ligand-binding domain of ERa and ERb. In contrast, the DNA binding domain is highly conserved between ERa and ERb with 97% amino acid identity. The C-terminal ligandbinding domains of ERa and ERb show a 59% overall amino acid sequence identity (29).
Generally, the function of ERb is opposite to ERa and it may act as a tumor suppressor in thyroid cancer (40). Downregulation of ERb will decrease its inhibitory role in thyroid cancer. Our previous study has found that the methylation of the ERb 5'-untranslated region will attenuate its inhibitory effect on ERa gene transcription and promote the initiation and progression of PTC (41). However, controversial results reported that the expression of ERb was upregulated by lncRNA-H19 to promote cancer stem-like properties in thyroid cancer, suggesting that ERb may exert its oncogenic role in thyroid cancer (22).
Similar to ERa, several isoforms of ERb have been identified in human cells. In 1998, 5 isoforms of ERb were cloned and characterized, and named from ERb1 (Erb full length) to ERb5. All these five ERb isoforms have novel C-terminus (42). Another splicing isoform of ERb was identified in 2001, and exon 3 was deleted from ERb, named ERbD3 (43). Missing exon 3 altered the subnuclear localization and capacity for transcriptional activation (43). Therefore, alternative splicing will change the function domain of ERb (Figure 2), subsequently affecting its function in thyroid cancer. This section would discuss the expression and roles of ERb and its isoforms in thyroid cancer.
Though several isoforms of ERb have been identified for many years, limited studies have been performed to analyze ERb isoforms in thyroid cancer. Wenwu Dong et al. (23) evaluated the expression of ERb2 in 106 PTC tissues. They reported that the expression of ERb2 was positively associated with Ki-67 expression in female patients with advanced reproductive age (>45 years, in low-estrogen status) and with VEGF expression in male PTC patients with reproductive age (18~45 years, in lowestrogen status) (P=0.005 and P=0.044, respectively). There was no association between ERb2 expression and tumor size, extrathyroidal extension, and tumor-node-metastasis stage in PTC patients. In addition, the expression of ERb2 was lower in female patients of reproductive age (18~45 years, in relatively high-estrogen status) with lymph node metastasis than in those patients without lymph node metastasis (P=0.035). The results suggested that the expression of ERb2 in PTC is associated with the progression of the disease (23).
Overall, the role of ERb in cancer is important. It has been proposed as a promising marker and potential target in cancer metastases (44). ERb was also correlated with the tumor microenvironment (45). However, the expression and roles of ERb isoforms remain largely unknown. The functional domains of ERs will respond to different modulators and degraders (46,47). Modulations of different ERs domains may have therapeutic impacts (48). The alternative splicing of ERs can result in an incomplete domain, thus affecting the treatment's efficiency. Therefore, the investigation should focus on the isoforms of ERs in thyroid cancer.

CONCLUSION AND PERSPECTIVE
The ERa and ERb in thyroid cancer are multifaced and complicated. This review has focused on the ERa and ERb isoforms in thyroid cancer. Given the significance of ERa and ERb in the development of thyroid cancer and the perspective potential of estrogen receptor modulators and degraders in the treatment of thyroid cancer, the investigation of ERa and ERb isoforms in the development and progression of thyroid cancer will provide us with a new avenue for the understanding and treatment of thyroid cancer.