Concomitant high expression of ERα36, EGFR and HER2 is associated with aggressive behaviors of papillary thyroid carcinomas

ERα, ERβ, PR, ERα36, EGFR and HER2 mRNA and protein expression in papillary thyroid carcinoma (PTC) were examined by real time RT-PCR and immunohistochemical staining. The mRNA and protein expression of ERα and PR were gradually increased and those of ERβ were gradually decreased from normal thyroid tissues to nodular hyperplasias (P < 0.05) and to PTCs (P < 0.05). However, the mRNA and protein expression of ERα36, EGFR and HER2 were only significantly increased in PTCs when compared with those in normal thyroid tissues (P < 0.001) and nodular hyperplasias (P < 0.001). There was some correlation between ERα, ERβ and PR, and between ERα36, EGFR and HER2 protein expression in PTCs. As for ERα, ERβ and PR, there was a significant positive correlation between ERα and PR, and a significant negative correlation between ERα and ERβ and between PR and ERβ protein expression. As for ERα36, EGFR and HER2, there was a significant positive correlation between ERα36, EGFR and HER2 protein expression in PTCs. Concomitant high expression of ERα36, EGFR and HER2 was strongly associated with aggressive behaviors including extrathyroidal extension (ETE), lymph node metastasis (LNM) and high TNM stage in PTCs (P < 0.001).


Results
mRNA expression of ERα, ERβ, PR, ERα36, EGFR and HER2 in PTCs, nodular hyperplasias and normal thyroid tissues. To compare gene expression of ERα, ERβ, PR, ERα36, EGFR and HER2 in PTCs, nodular hyperplasias and normal thyroid tissues, 10 PTCs, 10 nodular hyperplasias and 10 normal thyroid tissues were collected to detect the mRNA levels of these molecules using real-time RT-PCR. As shown in Table 1, ERα, ERβ and PR mRNA were expressed in PTCs, nodular hyperplasias and normal thyroid tissues. ERα and PR mRNA levels were significantly higher in PTCs than in nodular hyperplasias (P < 0.001 for both ERα and PR) and normal thyroid tissues (P < 0.001 for both ERα and PR). However, ERβ mRNA level was significantly lower in PTCs than in nodular hyperplasias (P < 0.001) and normal thyroid tissues (P < 0.001). Furthermore, nodular hyperplasias had higher ERα and PR and lower ERβ mRNA levels when compared with normal thyroid tissues (P < 0.001 for ERα, ERβ and PR). These results indicated that ERα and PR mRNA levels were gradually increased and ERβ mRNA level was gradually decreased from normal thyroid tissues to nodular hyperplasias and to PTCs. As for ERα36, EGFR and HER2, the mRNA levels of the three molecules were significantly higher in PTCs than in nodular hyperplasias (P < 0.001 for ERα36, EGFR and HER2) and normal thyroid tissues (P < 0.001 for ERα36, EGFR and HER2). There were no statistically significant differences in mRNA levels of ERα36, EGFR and HER2 between nodular hyperplasias and normal thyroid tissues (P = 0.137, 0.117, 0.157 for ERα36, EGFR and HER2, respectively). These results indicated that increased mRNA expression of ERα36, EGFR and HER2 was only associated with the occurrence of PTC, but not with that of nodular hyperplasia.
Immunohistochemical expression of ERα, ERβ, PR, ERα36, EGFR and HER2 in PTCs, nodular hyperplasias and normal thyroid tissues. ERα, ERβ, PR, ERα36, EGFR and HER2 protein expression were examined by immunohistochemical staining and the representatives of immunostaining for the six molecules were illustrated in Fig. 1. As shown in Fig. 1, ERα, ERβ and PR protein were expressed in PTCs, nodular hyperplasias and normal thyroid tissues. Compared with normal thyroid tissues and nodular hyperplasias, PTCs had more follicular epithelial cells with staining for ERα and PR, however, less for ERβ. Furthermore, nodular hyperplasias had more follicular epithelial cells with staining for ERα and PR, however, less for ERβ when compared with normal thyroid tissues. As shown in Tables 2 and 3, in PTCs, high expression (≥5) was present in 109 (50%), 33 (15.1%) and 116 (53.2%) of 218 cases for ERα, ERβ and PR, respectively. High expression rates of ERα and PR were significantly higher in PTCs than in nodular hyperplasias (37.2%, P = 0.014 and 39.7%, P = 0.010 for ERα and PR, respectively) and normal thyroid tissues (6.3%, P < 0.001 and 8.6%, P < 0.001 for ERα and PR, respectively). However, high expression rate of ERβ was significantly lower in PTCs than in nodular hyperplasias (42.3%, P < 0.001) and normal thyroid tissues (56%, P < 0.001). Obviously, nodular hyperplasias had higher rates of high ERα and PR expression (P < 0.001 for both ERα and PR) and lower rate of high ERβ expression Groups(n = 10) ERα (ΔCT, P value) ERβ (ΔCT, P value) PR (ΔCT, P value) ERα36 (ΔCT, P value) EGFR (ΔCT, P value) HER2 (ΔCT, P value)  Stands for significant difference between PTCs and normal thyroid tissues; c Stands for significant difference between PTCs and nodular hyperplasias; P < 0.05 was considered to be statistically significant.
(P = 0.013) when compared with normal thyroid tissues. As for ERα36, EGFR and HER2, there were almost no follicular epithelial cells with staining for ERα36, EGFR and HER2 in normal thyroid tissues and nodular hyperplasias. However, in PTCs, there were a lot of follicular epithelial cells with staining for the three molecules. As shown in Table 2, like normal thyroid tissues, the majority of nodular hyperplasias had negative or 1 IHC score for ERα36, EGFR and HER2, no cases showed high expression (≥5) of the three molecules. However, in PTCs, the majority of cases had ≥2 IHC score for the three molecules, high expression (≥5) was present in 112 (51.4%), 132 (60.6%) and 135 (61.9%) of 218 cases for ERα36, EGFR and HER2, respectively. The differences in ERα36, EGFR and HER2 protein expression between PTCs and normal thyroid tissues as well nodular hyperplasias were statistically significant (P < 0.001) ( Table 3).  Table 2. Immunohistochemical analysis of ERα, ERβ, PR, ERα36, EGFR and HER2 expression in 218 PTCs, 156 nodular hyperplasias and 175 normal thyroid tissues according to the scoring system. The immunohistochemical scores in PTCs, nodular hyperplasias and normal thyroid tissues were determined as the multiplication of proportion score and intensity score.

Correlation of ERα
clinicopathological features analyzed. Notably, ERα36 protein expression was significantly correlated with ETE (P < 0.001), LNM (P < 0.001) and TNM stage (P < 0.001). PTCs with ETE, LNM and high TNM stage (III-IV) had higher rates of high ERα36 protein expression. However, there were no statistically significant differences in ERα36 protein expression between patients with different histologic subtype (P = 0.230), between older (≥45) and younger (<45) patients (P = 0.143), between male and female patients (P = 0.240), and between patients with large and small tumor size (P = 0.150). As for the two epidermal growth factor receptors, EGFR and HER2, no correlation was found to be present between the protein expression of the two molecules and histologic subtype, age, gender and tumor size of PTC patients (P = 0.160, 0.179, 0.132, 0.175 for EGFR and P = 0.166, 0.194, 0.151, 0.143 for HER2, respectively). However, EGFR and HER2 protein expression were significantly correlated with ETE (P < 0.001), LNM (P < 0.001) and TNM stage (P < 0.001). PTCs with ETE, LNM and high TNM stage (III-IV) had higher rates of high EGFR and HER2 protein expression than those with low TNM stage (I-II) and without ETE and LNM.
Correlation of ERα, ERβ, PR, ERα36, EGFR and HER2 protein expression with one another in PTCs. The correlation of ERα, ERβ, PR, ERα36, EGFR and HER2 protein expression with one another in PTCs was assessed by Spearman rank test. As shown in Table 4, there was no correlation between the protein expression of ERα, ERβ or PR and the protein expression of ERα36, EGFR or HER2. However, there was some correlation between ERα, ERβ and PR protein expression, and between ERα36, EGFR and HER2 protein  Table 3. Correlation of ERα, ERβ, PR, ERα36, EGFR and HER2 protein expression with clinicopathological parameters in 218 PTCs. P-values derived using Chi-square test to compare the expression of ERα, ERβ, PR, ERα36, EGFR and HER2 between subgroups defined by each clinicopathological parameter; a stands for significant difference between normal thyroid tissues and nodular hyperplasias; b stands for significant difference between PTCs and normal thyroid tissues; c stands for significant difference between PTCs and nodular hyperplasias. P < 0.05 was considered to be statistically significant.
expression. As for ERα, ERβ and PR, there was a significant positive correlation between ERα and PR protein expression (r s = 0.607, P < 0.001) and a significant negative correlation between ERα and ERβ protein expression (r s = −0.294, P < 0.001) and between PR and ERβ protein expression (r s = −0.245, P < 0.001). As for ERα36, EGFR and HER2, there was a significant positive correlation between ERα36 and EGFR (r s = 0.285, P < 0.001), between ERα36 and HER2 (r s = 0.352, P < 0.001) and between EGFR and HER2 (r s = 0.160, P = 0.018) protein expression.

Association of concomitant high expression of ERα36, EGFR and HER2 with ETE, LNM and high TNM stage in PTCs.
Given that ERα36, EGFR and HER2 protein expression were positively correlated with one another and statistical analysis showed that PTCs with ETE, LNM and high TNM stage had higher rates of high protein expression of the three molecules than those with low TNM stage and without ETE and LNM, we further evaluated the association of ETE, LNM and high TNM stage with concomitant high expression of ERα36, EGFR and HER2. As shown in Table 5

Discussion
Clinical and epidemiological studies have suggested that estrogen may be involved in the occurrence and development of PTC [1][2][3][4] , as largely demonstrated in breast, endometrial and ovarian carcinomas 5 . It is widely accepted that estrogen acts via ERα and ERβ, both of which function indisputably as hormone-dependent transcription factors to induce transactivation of a series of estrogen-dependent target genes 6,7 . PR is a paragon of estrogen-induced protein and its presence is a marker of a functional ERα 8,9 . Traditionally, ERα and PR expression are employed as biomarkers of endocrine therapy sensitivity and prognosis in breast cancer 10 . However, in the past 10 years, ERα36 (a novel 36 kDa variant of ERα) has been identified as a new member of the ER family and was found to be mainly located in the cytoplasm, as well as on the cell surface where it mediates non-genomic estrogen signaling through cross-talk with growth factor receptors and other signaling molecules (such as MAPK/ERK, PI3K/AKT    (7) and group (8), respectively; h stands for significant difference between groups with and without concomitant high expression of all the three molecules. P < 0.05 was considered to be statistically significant. and PKC) and promotes cell growth, invasion, migration and resistance to endocrine therapy [13][14][15][16][17] . EGFR and HER2 are two well-studied epidermal growth factor receptors and have been shown to be prognostic relevance in a variety of human malignancies including PTC [27][28][29][30][31][32] . To date, studies have shown that ERα36 is overexpressed in breast cancer stem cells [18][19][20][21] , ER-positive and -negative human breast carcinomas 22 , endometrial carcinomas 23 and gastric carcinomas 24 , which is associated with malignancy, invasion, metastasis, drug resistance and poor prognosis of these tumors. However, no study dealt with the expression of ERα36 together with ERα, ERβ, PR, EGFR and HER2 and systematically assessed the correlation of their expression with clinicopathological features in PTC. In our present study, we simultaneously examined ERα, ERβ, PR, ERα36, EGFR and HER2 mRNA and protein expression in PTCs, nodular hyperplasias and normal thyroid tissues using real time RT-PCR and immunohistochemical staining and demonstrated that ERα, ERβ and PR mRNA and protein were expressed in PTCs, nodular hyperplasias and normal thyroid tissues. Obviously, the mRNA and protein expression of ERα and PR were gradually increased and those of ERβ were gradually decreased from normal thyroid tissues to nodular hyperplasias and to PTCs. This result is in line with the previous studies showing that increased ERα and PR expression and decreased ERβ expression are associated with the occurrence of nodular hyperplasia and PTC [33][34][35] . As for ERα36, EGFR and HER2, the mRNA expression levels of the three molecules were significantly higher in PTCs than in nodular hyperplasias as well as normal thyroid tissues. There were no statistically significant differences in mRNA expression of ERα36, EGFR and HER2 between nodular hyperplasias and normal thyroid tissues. Consistent with the mRNA expression of ERα36, EGFR and HER2, no cases of normal thyroid tissue and nodular hyperplasia showed high protein expression of ERα36, EGFR and HER2. However, in PTCs, high protein expression was present in 51.4%, 60.6% and 61.9% for ERα36, EGFR and HER2, respectively. The differences in ERα36, EGFR and HER2 protein expression between PTCs and normal thyroid tissues as well nodular hyperplasias were statistically significant (P < 0.001). These results suggested that the increased mRNA and protein expression of ERα36, EGFR and HER2 are only associated with the occurrence of PTC, but not with that of nodular hyperplasia. Then we assessed the correlation of ERα, ERβ, PR, ERα36, EGFR and HER2 protein expression with clinicopathological features. We found that ERα and PR protein expression were positively correlated and ERβ protein expression was negatively correlated with tumor size, whereas the protein expression of them was not correlated with the other clinicopathological features analyzed. These results are in line with our and other researcher's previous studies showing that ERα and PR exert proliferative action and ERβ has an anti-proliferative function in breast cancer cells and PTC cells [36][37][38][39][40] . Notably, ERα36 protein expression was significantly correlated with ETE, LNM and TNM stage, whereas there was no correlation between the protein expression of ERα36 and the histologic subtype, age, gender and tumor size of PTC patients. PTCs with ETE, LNM and high TNM stage (III-IV) had higher rates of high ERα36 protein expression than those with low TNM stage (I-II) and without ETE and LNM. These results are in line with the previous studies in other tumor types such as breast, endometrial and gastric tumors [22][23][24] , indicating that ERα36 may also play important roles in progression and metastasis of PTC. As for EGFR and HER2, no correlation was found to be present between the protein expression of EGFR and HER2 and the histologic subtype, age, gender and tumor size of PTC patients. However, EGFR and HER2 protein expression were significantly correlated with ETE, LNM and TNM stage. PTCs with ETE, LNM and high TNM stage (III-IV) had higher rates of high EGFR and HER2 protein expression than those with low TNM stage (I-II) and without ETE and LNM. These results are consistent with the previous studies showing that EGFR and HER2 high expression are associated with some aggressive behaviors of PTC 31,32 .
Subsequently, we assessed the correlation of ERα, ERβ, PR, ERα36, EGFR and HER2 protein expression with one another. No correlation was found between the protein expression of ERα, ERβ or PR and the protein expression of ERα36, EGFR or HER2. However, we found that there was some correlation between ERα, ERβ and PR protein expression, and between ERα36, EGFR and HER2 protein expression. As for ERα, ERβ and PR, there was a significant positive correlation between ERα and PR protein expression (r s = 0.607, P < 0.001) and a significant negative correlation between ERα and ERβ protein expression (r s = −0.294, P < 0.001) and between PR and ERβ protein expression (r s = −0.245, P < 0.001). These correlations are in line with the previous studies indicating that PR is a typical estrogen dependent target gene which is positively regulated by ERα and negatively regulated by ERβ 41,42 . Furthermore, ERα and ERβ expression levels were reversely regulated by several mechanisms such as proteasome pathway 43,44 and some microRNAs 45,46 . As for ERα36, EGFR and HER2, there was a significant positive correlation between ERα36, EGFR and HER2 protein expression in PTCs. ERα36 expression was positively correlated with EGFR expression (r s = 0.285, P < 0.001) and HER2 expression (r s = 0.352, P < 0.001). Moreover, a significant positive correlation (r s = 0.160, P = 0.018) was also present between EGFR and HER2 expression. The existence of these positive correlations could be supported by the following data. A positive feedback loop between ERα36 and EGFR/HER2 was reported to promote malignant growth. EGFR signaling activated transcription of ERα36 through an activator-protein-1-binding site in the promoter of ERα36. In turn, ERα36 interacted with the EGFR/Src/Shc complex to strengthen the EGFR signaling pathway and stabilize EGFR protein 17,47 . A similar positive feedback loop between ERα36 and HER2 was also reported 47,48 .
Given that ERα36, EGFR and HER2 protein expression were positively correlated with one another and the expression of these individual molecules was related to ETE, LNM and TNM stage, we subsequently evaluated the association of concomitant expression of ERα36, EGFR and HER2 with ETE, LNM and TNM stage in PTCs. The results showed that ERα36 high expression combined with both EGFR and HER2 high expression had stronger correlation with ETE, LNM and high TNM stage when compared with ERα36 high expression combined with either EGFR or HER2 high expression (P = 0.016, 0.001 for ETE, P ≤ 0.001 for LNM, P < 0.001 for high TNM stage, respectively) and only ERα36 high expression (P < 0.001 for ETE, LNM and high TNM stage). It was indicated that concomitant high expression of ERα36, EGFR and HER2 was strongly associated with ETE, LNM and high TNM stage, and may be used as a predictive indicator for malignant behaviors such as ETE, LNM and high TNM stage in PTCs.
In summary, in the present study, we simultaneously examined ERα, ERβ, PR, ERα36, EGFR and HER2 expression, systematically assessed the association of their expression with clinicopathological features and evaluated the potential usefulness of these molecules in prediction for aggressive behaviors of PTCs. The results demonstrated that the mRNA and protein expression of ERα and PR were gradually increased and those of ERβ were gradually decreased from normal thyroid tissues to nodular hyperplasias and to PTCs. Increased ERα and PR and decreased ERβ mRNA and protein expression were associated with the occurrence of nodular hyperplasia and PTC. Remarkably, the mRNA and protein expression levels of ERα36, EGFR and HER2 were significantly higher in PTCs than in nodular hyperplasias and normal thyroid tissues. There were no significant differences in the mRNA and protein expression of ERα36, EGFR and HER2 between nodular hyperplasias and normal thyroid tissues. Increased mRNA and protein expression of ERα36, EGFR and HER2 were only associated with the occurrence of PTC, but not with that of nodular hyperplasia. There was no correlation between the protein expression of ERα, ERβ or PR and the protein expression of ERα36, EGFR or HER2. However, there was some correlation between ERα, ERβ and PR protein expression, and between ERα36, EGFR and HER2 protein expression in PTCs. As for ERα, ERβ and PR, there was a significant positive correlation between ERα and PR, and a significant negative correlation between ERα and ERβ and between PR and ERβ protein expression. As for ERα36, EGFR and HER2, there was a significant positive correlation between ERα36, EGFR and HER2 protein expression in PTCs. Concomitant high expression of ERα36, EGFR and HER2 was strongly associated with aggressive behaviors including ETE, LNM and high TNM stage, and may be used as a predictive indicator for ETE, LNM and high TNM stage in PTCs.

Materials and Methods
Case selection and tissue sample preparation. Tumor specimens for immunohistochemical analysis were obtained from 218 PTC patients who underwent initial thyroidectomy in the Department of Surgery, the First Affiliated Hospital, Chongqing Medical University, between Jan 2010 and Jan 2015. At the initial thyroid surgery for the 218 PTC patients, cervical lymph node dissection (CLND) was performed, tumor size was assessed, histologic subtype, extrathyroidal extension (ETE) and distant metastasis were confirmed. There were 135 patients confirmed to be classic PTC, 36 patients confirmed to be follicular variant of PTC, 26 patients confirmed to be tall cell variant of PTC and 21 patients confirmed to be oncocytic variant of PTC. There were 48 patients confirmed to have ETE, 105 patients confirmed to have lymph node metastasis (LNM), 61 patients confirmed to have distant metastasis, 85 PTCs with tumor size of ≤2 cm, 81 with tumor size of >2 and ≤4 cm, 52 with tumor size of >4 cm. There were 54 men and 164 women, 60 patients with the age of <45 years and 158 with the age of ≥45 years. According to TNM classification, there were 73 patients with stage I, 38 with stage II, 18 with stage III, and 89 with stage IV. For statistical analysis, stage I and II were combined into low TNM stage (I-II), and stage III and IV were combined into high TNM stage (III-IV). Besides, benign thyroid disease specimens were obtained from 156 patients with nodular hyperplasia. 175 normal thyroid tissues were taken from the contralateral lobe of PTC specimens, which exhibited apparently normal morphology as a control. The study protocol was approved by the Ethics Committee of Chongqing Medical University and informed consent was obtained from all patients.
Tumor specimens for real-time RT-PCR were obtained from 10 PTC patients between Jan 2017 and June 2017. The benign thyroid disease specimens were obtained from 10 patients with nodular hyperplasia. For controls, 10 normal thyroid tissue specimens were used. All specimens were immediately snap-frozen in liquid nitrogen and stored at −80 °C up to subsequent RNA extraction, reverse transcription and real-time PCR.
Tissue microarray (TMA). Formalin-fixed, paraffin-embedded blocks were routinely prepared from surgical specimens of PTC, nodular hyperplasia and normal thyroid tissue. Representative areas containing tumor, nodular hyperplasia or normal thyroid tissue were identified by a pathologist. Duplicate tissue cores with a diameter of 0.6 mm were taken from each specimen (Beecher Instruments, Silver Springs, USA) and arrayed on a recipient paraffin block using standard procedures. Serial 5-μm-thick sections were cut with a Leica microtome (Leica Microsystems, Wetzlar, Germany) and mounted onto polylysine-coated slides.
RNA Extraction, Reverse Transcription and Real-Time PCR. Total RNA was extracted from frozen PTC, nodular hyperplasia and normal thyroid tissue specimens using TRIzol reagent (Invitrogen, Camarillo,