PES1 promotes the occurrence and development of papillary thyroid cancer by upregulating the ERα/ERβ protein ratio

PES1, a BRCT domain-containing protein, has been shown to play a role in modulating the balance and ratio between ERα and ERβ protein, which is involved in the occurrence and development of breast and ovarian cancer. However, its role in connection with the balance and ratio between ERα and ERβ protein in papillary thyroid cancer (PTC) remains unclear. Here, we found that ERα and ERβ were co-expressed in human PTC tissues and cells. ERα promoted and ERβ inhibited the proliferation, invasion and migration of PTC cells. PES1 modulated the balance between ERα and ERβ by elevating the ERα protein level and simultaneously reducing the ERβ protein level, then upregulating the ERα/ERβ protein ratio and promoting the proliferation, invasion and migration of PTC cells. In PTC tissues, PES1 protein level was positively correlated with the ERα protein level and negatively correlated with the ERβ protein level. The PES1 and ERα protein levels were gradually increased and the ERβ protein level was decreased by degree in the occurrence and development of PTC. Increased PES1 and ERα protein levels and decreased ERβ protein level were correlated with the aggressive behaviors of PTC patients such as large tumor size, extrathyroidal extension (ETE), lymph node metastasis (LNM), high BRAFV600E expression and high TNM stage. It is suggested that PES1 promotes the occurrence and development of PTC by elevating the ERα protein level and reducing the ERβ protein level, and then upregulating the ERα/ERβ protein ratio.


Results
Scientific RepoRts | (2019) 9:1032 | https://doi.org/10.1038/s41598-018-37648-7 gradually increased and ERβ protein level is decreased by degree in the occurrence and development of PTC. As shown in Table 1, the majority of normal thyroid tissues were negative or had an IHC score 1 for PES1 and were negative or had an IHC score 1, 2, 3 or 4 for ERα, whereas none of cases showed high expression (IHC score ≥5) of PES1 and only 19 cases displayed high expression of ERα. On the other hand, however, only 6 normal thyroid tissues were negative for ERβ and the majority of cases had an IHC score 1-8, whereas 112 cases exhibited high expression of ERβ. High expression rates in normal thyroid tissues were 0%, 9.5% and 56% for PES1, ERα and ERβ, respectively. By contraries, the majority of the PTC tissues were positive and had an IHC score 1-8 for PES1 and ERα, whereas 104 and 103 of the PTC cases exhibited high expression (IHC score ≥5) of PES1and ERα, respectively. On the other hand, however, a significant number of PTC cases were negative for ERβ and only 30 PTC cases showed high expression of ERβ. High expression rates in PTC tissues were 52%, 51.5% and 15% for PES1, ERα and ERβ, respectively. It was indicated that PES1 and ERα protein levels are significantly upregulated and ERβ protein level is significantly downregulated in PTC tissues when compared with those in normal thyroid tissues (P < 0.001) (  40 . As shown in Fig. 2A   DPN (ERβ-selective agonist) were used to stimulate ERα and ERβ, respectively. As shown in Fig. 3, ERα-shRNA and ERβ-shRNA expression vectors effectively silenced ERα and ERβ expression, respectively, in BCPAP, K1 and Nthy-ori3-1cells. Compared with scrambed shRNA, ERα-shRNA decreased and ERβ-shRNA increased the proliferation, invasion and migration of human PTC-derived BCPAP and K1 cells and normal thyroid-derived Nthy-ori3-1 cells. Compared with the control (vehicle, Veh), ERα agonist PPT increased and ERβ agonist DPN decreased the proliferation, invasion and migration of these cells. It was indicated that ERα promotes and ERβ inhibits the proliferation, invasion and migration of human PTC cells and normal thyroid cells. Interestingly, E2 increased the proliferation, invasion and migration of human PTC-derived BCPAP and K1 cells with an ERα/ ERβ protein ratio of >1, however, decreased the proliferation, invasion and migration of normal thyroid-derived Nthy-ori3-1 cells with an ERα/ERβ protein ratio of <1. It was indicated that the balance between ERα and ERβ protein levels and the ERα/ERβ protein ratio are crucial to the effects of E2 on the proliferation, invasion and migration of these thyroid cells.

PES1 upregulates the ERα/eRβ protein ratio and promotes the proliferation, invasion and migration of human PTC cells and normal thyroid cells.
To explore the effects of PES1 on the ERα/ ERβ protein ratio and the proliferation, invasion and migration of human PTC cells and normal thyroid cells, PES1-shRNA expression vector was used to generate the stable transfected PTC cells with knockdown of PES1 and PES1expression vector was used to generate the stable transfected normal thyroid cells with increased PES1 expression. As shown in Fig. 4, compared with scrambed shRNA, PES1-shRNA reduced the ERα protein level and simultaneously elevated the ERβ protein level, and then resulted in a decrease of the ERα/ERβ protein ratio and a decrease of the promotion effects of E2 on the proliferation, invasion and migration of human PTC-derived BCPAP and K1 cells. Conversely, compared with empty vector, PES1 expression vector elevated the ERα protein level and simultaneously reduced the ERβ protein level, and then resulted in an increase of the ERα/ERβ protein ratio and an inhibition-promotion transition of the effects of E2 on the proliferation, invasion and migration in normal thyroid-derived Nthy-ori3-1 cells. It was indicated that PES1 elevates the ERα protein level and simultaneously reduces the ERβ protein level, i.e., upregulates the ERα/ERβ protein ratio, and then promotes the proliferation, invasion and migration of human PTC cells and normal thyroid cells.

Discussion
Studies have shown that estrogen may be involved in the occurrence and development of PTC 1-5 , as has been shown in breast, endometrial and ovarian cancer 6 . Estrogen exerts its effects mainly via ERα and ERβ 7,8 . ERα and ERβ are often co-expressed and contribute to the physiological and pathophysiological responses of estrogen 42,43 .
In this study, we detected ERα and ERβ protein levels in PTC tissues and cells using IHC staining and Western blotting, respectively. Compared with normal thyroid tissues and cells, the ERα protein level was upregulated and the ERβ protein level was downregulated in PTC tissues and cells. PTC tissues had higher rates of high ERα and low ERβ protein expression. PTC cells had more than two times higher protein level of ERα than that of ERβ. This result is in line with the previous data showing that ERα and ERβ are co-expressed in normal and tumor tissues of the thyroid 20,21 , compared with normal thyroid tissues, the level of ERα is relatively higher than that of ERβ in PTC tissues [21][22][23][24] .
Although ERα and ERβ are architecturally similar with three functional domains of N-terminal domain (NTD), DNA binding domain (DBD) and ligand binding domain (LBD), there are obvious differences in their structures of NTD and LBD. The differences in their structures suggest that ERα and ERβ may have different functions 9 . Cell-based assays have shown that ERβ is generally less active than ERα and may influence ERα activity. Despite a small number of data associating ERβ with pro-growth and pro-survival when present alone  in ERα-negative estrogen related cancer tissues and cells, a large number of data both in vitro and in vivo support that ERβ acts as an anti-proliferative and pro-apoptotic factor, especially when co-expressed with ERα 44,45 .
Most studies have revealed that ERα promotes cell proliferation, invasion and migration and has been shown to have tumor-promoting effects, whereas ERβ, when co-expressed with ERα, may play an inhibitory role against the ERα-mediated tumor-promoting effects [14][15][16] . Thus, the complement of ER isoform could influence the biological responses and the balance and ratio between ERα and ERβ protein levels would be critical in defining the overall response [14][15][16][17][18] . In this study, we showed that compared with normal thyroid tissues, the ERα protein level was upregulated and the ERβ protein level was downregulated in PTC tissues. The ERα protein level was positively correlated with the aggressive behaviors of PTC patients, conversely, the ERβ protein level was negatively correlated with the aggressive behaviors of PTC patients. PTC patients with large tumor size, ETE, LNM, high BRAFV600E expression and high TNM stage (III-IV) had higher rates of high ERα and low ERβ protein expression. Moreover, cell-based assays showed that the ERα/ERβ protein ratio was greater than one (>1) in human PTC-derived BCPAP and K1 cells with the BRAFV600E mutation, conversely, smaller than one (<1) in normal thyroid-derived Nthy-ori3-1 cells without the BRAFV600E mutation. ERα promoted and ERβ inhibited the proliferation, invasion and migration of these PTC cells and normal thyroid cells. Interestingly, E2 increased the proliferation, invasion and migration of human PTC cells with an ERα/ERβ protein ratio of >1, however, decreased the proliferation, invasion and migration of normal thyroid cells with an ERα/ERβ protein ratio of <1. It was indicated that the ERα/ERβ protein ratio is crucial to the effects of E2 on the proliferation, invasion and migration of these thyroid cells. PES1, a BRCT domain-containing protein, is essential for ribosome biogenesis, nucleologenesis and cell growth, which are all important components that determine the cell proliferation rate 25,28 . The increased expression of PES1 is involved in the proliferation and malignant conversion of cells and may contribute to the occurrence and development of some human cancers such as prostate, head and neck, stomach, colon, breast and ovarian cancer [29][30][31][32][33][34][35][36][37][38][39] . Recently, Cheng et al. 33 and Li et al. 38 reported a novel function of PES1 that regulates the balance between ERα and ERβ protein levels. They found that PES1 enhances the stability of ERα while simultaneously targeting ERβ for proteasome degradation, thereby increasing the protein level of ERα and decreasing that of ERβ, which contributes to the occurrence and development of breast cancer and ovarian cancer. As some ERs regulators have been found to have tissue specificity that modulate their activities 46,47 , in this study, we examined PES1 protein level in PTC tissues and cells and assessed the correlations of its protein level with the ERα and ERβ protein levels and with the occurrence and development of PTC. We found that PES1 protein level was positively correlated with ERα protein level and negatively correlated with ERβ protein level in human PTC tissues and cells. Compared with normal thyroid tissues, PES1 protein level was significantly increased in PTC tissues and was associated with the aggressive behaviors of PTC patients. PTC patients with large tumor size, ETE, LNM, high BRAFV600E expression and high TNM stage (III-IV) had higher rates of high PES1 and ERα protein levels and low ERβ protein level. Increased PES1 and ERα protein levels and decreased ERβ protein level were associated with the occurrence and development of PTC. Cell-based assays showed that PES1 elevated ERα protein level and simultaneously reduced ERβ protein level and resulted in an upregulated ERα/ERβ protein ratio, and then promoted the proliferation, invasion and migration of human PTC cells.
In summary, ERα and ERβ were co-expressed in human PTC tissues and cells. ERα promoted and ERβ inhibited the proliferation, invasion and migration of PTC cells. PES1 modulated the balance between ERα and ERβ by elevating the ERα protein level and simultaneously reducing the ERβ protein level, then upregulated the ERα/ ERβ ratio and promoted the proliferation, invasion and migration of PTC cells. In PTC tissues, PES1 protein level was positively correlated with ERα protein level and negatively correlated with ERβ protein level. PES1 and ERα protein levels were gradually increased and ERβ protein level was decreased by degree in the occurrence and development of PTC. Increased PES1 and ERα protein levels and decreased ERβ protein level were correlated with the aggressive behaviors of PTC patients such as large tumor size, ETE, LNM, high BRAFV600E expression and high TNM stage. It was suggested that PES1 promotes the occurrence and development of PTC by elevating the ERα protein level and reducing the ERβ protein level, and then upregulating the ERα/ERβ protein ratio.

Materials and Methods
Case selection and tissue samples. Tumor tissue samples were obtained from 200 PTC patients who underwent initial thyroidectomy in the Department of Surgery, the First Affiliated Hospital, Chongqing Medical University, between January 2015 and January 2017. At initial thyroidectomy, cervical lymph node dissection (CLND) was performed, tumor size was assessed, histologic subtype was defined and extrathyroidal extension (ETE) and BRAFV600E mutation were evaluated. Of the 200 cases, 142 were classic PTC, 21 follicular variant of PTC, 24 tall cell variant of PTC and 13 oncocytic variant of PTC. There were 39 patients with ETE, 90 patients with lymph node metastasis (LNM), 133 patients with high BRAFV600E expression, 62 patients with tumor size of ≤2 cm, 71 patients with tumor size of >2 and ≤4 cm and 67 patients with tumor size of >4 cm. Of this patient cohort, 46 were men and 154 women; 24 patients were aged <45 years and 176 were aged ≥45 years. According to the seventh edition of thyroid cancer tumor-node-metastasis (TNM) staging system by American Joint Committee on Cancer, 50 patients were stage I, 50 patients stage II, 50 patients stage III and 50 patients 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, 200 normal thyroid tissues were taken from the contralateral lobes of PTC tissues. The study protocol was approved by the Ethics Committee of Chongqing Medical University and informed consent was obtained from all of the patients.

IHC scoring.
A semi-quantitative IHC scoring assessment was performed by two observers blinded to the diagnosis. IHC score was assigned based on the percentage of positive cells and the staining intensity. The percentage was evaluated and assigned a score of 0−4: 0, <5% positive cells; 1, 6-25% positive cells; 2, 26-50% positive cells; 3, 51-75% positive cells; and 4, >75% positive cells. The staining intensity was evaluated and assigned a score of 0−3: 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining. The IHC score was then assigned to each sample by multiplying the percentage score and the staining intensity score, and thus the score ranged from 0 to 12. For statistical analysis, a final IHC score of 0 (negative) or 1-4 (+) was defined as low expression and a final IHC score of 5-8 (++) or 9-12 (+++) as high expression.
BrdU assay. Cells were seeded in 96-well culture plates at a density of 5 × 10 3 cells per well, incubated for 24 h, serum-deprived for 48 h and treated as indicated in the figure legends. Then a 5-bromo-2′-deoxyuridine (BrdU) incorporation colorimetric ELISA kit (Roche Diagnostics) was used to assay the cell proliferation in accordance with the standard protocol of the manufacturer.
Cell count assay. Cells were seeded in 6-well culture plates at a density of 2 × 10 5 cells per well, incubated for 24 h, serum-deprived for 48 h and treated as indicated in the figure legends. Then cells were harvested using Trypsin-EDTA, resuspended in the fresh medium and diluted in 0.4% trypan blue at a volume ratio of 1:1. The cell number was manually counted using a haemocytometer under microscope.
In vitro migration and invasion assays. In vitro migration and invasion assays were performed using transwell chambers (8-μm pore size, 24-well insert, Corning Inc., Corning, NY, USA) according to the manufacturer's instructions. Briefly, 3 × 10 4 serum-starved cells were resuspended and seeded in the upper chambers in phenol-red free and serum-free medium supplemented with or without E2, PPT or DPN. The lower chambers were filled with phenol-red free medium supplemented with 10% charcoal-stripped FBS as a chemoattractant. For the invasion assay, the inserts were pre-coated with extracellular matrix gel (BD Biosciences, Bedford, MA, USA). Following 72 h incubation, MTT solution (0.5 mg/mL) was added to the upper and lower chambers and incubated for another 4 h. After this, the cells in the upper membrane surface (residual cells) and the cells in the lower membrane surface (migrated or invaded cells) were scraped off with a cotton swab and dissolved in 400 μl of DMSO, respectively. Then 100 μl of the dissolved solution was taken and the absorbance was measured at 570 nm. Data were expressed as a percentage of migrated and invaded cells, i.e., A/(A + B) × 100, where A is the absorbance of the migrated or invaded cells and B the absorbance of the residual cells.
Statistics. Statistical analyses were performed using the SPSS version 18.0 (SPSS Inc., Chicago, IL, USA).
Data are presented as percentages and mean plus standard deviation, according to distribution. Significance was assessed using Chi-square, Spearman rank and Student's t-tests, as appropriate, to compare groups. A P-value of < 0.05 was considered to be statistically significant.

Statements for Materials and Methods. The study protocol was approved by the Ethics Committee of
Chongqing Medical University and informed consent was obtained from all of the patients. All experiments were performed in accordance with the relevant guidelines and regulations.

Data Availability
All data generated or analyzed during this study are included in this article (and its Supplementary Information files).