Abstract
Background/Aim: The prognosis of anaplastic thyroid carcinoma (ATC) is poor, and there is currently no established treatment to improve its outcome. We previously reported that enhancer of zeste homolog 2 (EZH2) was highly expressed in ATC, and may be a therapeutic target; however, the effects of EZH2 on ATC growth currently remain unknown. Materials and Methods: We investigated the effects of an EZH2 inhibitor (DZNep) on four ATC cell lines (8305C, KTA1, TTA1 and TTA2). We performed a gene panel analysis of all ATC cell lines to identify differences in DZNep sensitivity between the cell lines. To investigate the effects of DZNep on the recovery of differentiation, we assessed changes in thyroid differentiation markers (TDMs) before and after the DZNep treatment using PCR. Results: EZH2 was expressed in all ATC cell lines. The cell-reducing effects of DZNep were detected in all ATC cell lines, and were the strongest in KTA1 cells followed by TTA2 cells. The TTA1 and 8305C cell lines, which showed weak cell-reducing effects, had TP53 mutations. No changes in TDMs were observed in any ATC cell line. Conclusion: DZNep, an EZH2 inhibitor, exerted suppressive effects on the growth of ATC cell lines and has potential as a therapeutic strategy; however, its effects may be attenuated in ATC with TP53 mutations.
Thyroid carcinoma is the most common malignant tumor of the endocrine organs. Thyroid carcinoma originating from follicular cells is histopathologically classified into differentiated carcinoma (papillary or follicular carcinoma), poorly differentiated carcinoma, and anaplastic carcinoma (1). The prognosis of differentiated thyroid carcinoma is good, whereas that of anaplastic thyroid carcinoma (ATC) is poor. Median survival after the diagnosis of ATC was previously reported to be 3-4 months, with a 1-year survival rate of approximately 18-20% (2, 3). Although molecular-targeted therapies have been developed, they have not effectively improved the prognosis of ATCs (4).
Enhancer of zeste homolog 2 (EZH2) regulates the expression of various genes by specifically catalyzing the methylation of the 27th lysine residue of the histone protein H3 (H3K27). EZH2 dysfunction promotes carcinogenesis, which includes gain-of-function mutations and overexpression. The expression of EZH2 has been associated with a poor prognosis in various carcinomas, such as breast, prostate, bladder, renal, lung, and gastric carcinomas (5-10). Although we previously reported that EZH2 expression levels were higher in ATC, followed by poorly differentiated and differentiated thyroid carcinoma, the effects of EZH2 on ATC growth currently remain unknown (11). Therefore, the present study investigated the effects of an EZH2 inhibitor on ATC cell lines and whether it improved the degree of differentiation.
Materials and Methods
Cell culture and reagents. The 8305c, KTA1, TTA1 and TTA2 cell lines were obtained from the RIKEN CELL BANK (Ibaraki, Japan). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (Fujifilm Wako Chemicals, Osaka, Japan) supplemented with 10% fetal bovine serum (Hyclone, Cytiva, Marlborough, MA, USA), 100 U/ml penicillin, and 100 mg/ml streptomycin, and were then incubated at 37°C in a humidified 5% CO2 atmosphere. DZNep was purchased from Fujifilm Wako Chemicals.
Cell viability. Cell viability was measured by the addition of Cell Titer-Glo 3D (#G9683, Promega, Madison, WI, USA) to each well followed by an incubation at room temperature for 30 minutes. Luminescence was assessed using the SpectraMax L Plate Reader (Molecular Devices, San Jose, CA, USA). Data were normalized to control wells for each cell culture. Data analysis was performed by two-way ANOVA followed by Tukey’s test using GraphPad Prism 9.3.1.
RNA isolation, reverse transcription and PCR. Total RNA was isolated from the ATC cell lines and normal thyroid tissue using TRIzol (Thermo Fisher Scientific, Waltham, MA, USA) and subjected to reverse transcription with SuperScriptIV (Thermo Fisher Scientific) and random primers. Reverse transcription products were then subjected to PCR using KOD one (TOYOBO, Osaka, Japan) and the following primers: Pax8 sense, 5′-AGCATTGACTCACAGAGCA-3′, and antisense 5′-GTCTGGTGAGTCGAGAGGTT-3′; TSHR sense, 5′-TATAGATGTGACTCTGCAGC-3′, and antisense 5′-GTCAGGTCAGGGAACATT-3′; DUOX1 sense, 5′-AGAAGGAGATTGAAGAAATC-3′, and antisense 5′-ATAGTCACGAACAACAGA-3′; TG sense, 5′-AATGGCGACTATCAGGCGGTGCA-3′, and antisense 5′-AATCTGGCTACTCTTGGAGAG-3′; DIO2 sense, 5′-ATGGACAATAACGCCAACATA-3′, and antisense 5′-GACTTCTTGAAGGTTGTAGGA-3′; NIS sense, 5′-ACACTGACTGCGACCCTCTCCT-3′, and antisense 5′-GACTGCAGCCATAGCATTGA-3′; GAPDH sense, 5′-AAGGCTGAGAACGGGAAGCTTGTCATCAAT-3′, and antisense 5′-TTCCCGTCTAGCTCAGGGATGACCTTGCCC-3′.
Immunoblotting. Cells were lysed with ice-cold lysis buffer containing 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton-X, and protease inhibitor cocktail (Calbiochem, Merck KGaA). According to the manufacturer’s instructions, protein concentrations were assessed using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein were denatured by heating at 95°C for 5 minutes with Laemmli sample buffer, separated by SDS-PAGE (Atto, Tokyo, Japan), and transferred to polyvinylidene fluoride membranes (0.45 μm, Millipore, Merck KGaA). After blocking membranes with 5% skim milk, blots were incubated with the following primary antibodies for 1 hour: anti-EZH2 [#5246; Cell Signaling Technology (CST), Danvers, MA, USA] and anti-Tubulin (E7; DSHB, Iowa City, IA, USA). The blot was then incubated with goat anti-rabbit or goat anti-mouse secondary antibodies (CST) and visualized with Amersham ECL Prime (Cytiva), followed by detection with chemiluminescence using the ChemiDoc Touch imaging system (Bio-Rad).
Examination of hotspot driver mutations with a gene panel. The next-generation sequencing (NGS) method that we used in our previous studies (12-14) was also employed in the present study. Ten nanograms of DNA was applied to library construction with Ion AmpliSeq™ Cancer Hotspot Panel v2 (CHPv2) (Thermo Fisher Scientific) based on Ion Torrent AmpliSeq™ technology (Thermo Fisher Scientific) and subjected to sequencing with the Ion PGM next-generation sequencer (Thermo Fisher Scientific). CHPv2 for NGS is a gene panel to analyze well-characterized mutation hotspots in the following 50 cancer-related genes: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53 and VHL. Torrent Suite 5.12.2 and Ion Reporter version 5.16 (Thermo Fisher Scientific) were used to process and analyze sequenced data.
Results
DZNep inhibits ATC cell growth and sensitivities correlate with TP53 mutation status. We first confirmed the expression of EZH2 in all ATC cell lines by immunoblotting (Figure 1A). We next examined the effects of DZNep, an EZH2 inhibitor, on ATC growth (Figure 1B). Sensitivity to DZNep differed among the 4 cell lines tested, with the strongest effects being observed in KTA1 cells followed by TTA2 cells. The cell growth reduction rate at 1.0 μM was >50% in KTA1 cells and approximately 30% in TTA2 cells. To clarify differences in DZNep sensitivity between the cell lines, we performed a gene panel analysis of all ATC cell lines (Table I). A Braf mutation was detected in KTA1 cells, in which the cell-reducing effects of DZNep were the strongest. NRas and STK11 mutations were detected in TTA2 cells, in which the cell-reducing effects of DZNep were the second strongest. Among the 4 cell lines examined in the present study, TTA1 and 8305C, which showed weak cell-reducing effects, had TP53 and CDH1 mutations and TP53, Braf, and CDKN2A mutations, respectively. The two cell lines had TP53 mutations that were statistically significantly lower efficacy than TP53 wild-type cell lines on day 6 (Figure 1B).
(A) EZH2 expression status by Western blotting in anaplastic thyroid carcinoma cell lines (upper panel). Tubulin was used as a loading control (lower panel). (B) Drug sensitivity test of DZNep. Cells were treated with dimethyl sulfoxide (DMSO) or DZNep (1, 2 and 4 μM) on day 0. Cell viability was measured on day 6. The data are presented as means±standard deviation (n=4). **p<0.01. Data were analyzed using two-way ANOVA followed by Tukey’s test.
Gene panel analysis of anaplastic thyroid carcinoma cell lines.
DZNep does not affect the expression of thyroid differentiation markers (TDMs). To investigate the effects of DZNep on the recovery of differentiation, we examined changes in TDMs before and after the DZNep treatment using PCR. Figure 2 shows the expression of TDMs in normal thyroid cells as a positive control and ATC cell lines before and after the DZNep treatment. GAPDH was used as an internal control. Pax8 is a transcription factor for differentiation and is positive in the normal epithelium, including thyroid tissues. Although no significant differences were observed in Pax8 expression levels before and after the DZNep treatment, all ATC cell lines were positive. TG, TSHR, NIS, DUOX1 and DIO2 were other established TDMs (15) and were negative in the ATC cell lines tested herein.
Expression patterns of thyroid differentiation markers in anaplastic thyroid carcinoma cell lines in the presence or absence of DZNep by PCR. Normal thyroid tissue (NT) was used as the positive control. TG, Thyroglobulin; TSHR, thyroid-stimulating hormone receptor; NIS, sodium-iodine symporter; DUOX1, dual oxidase 1; DIO2, iodothyronine deiodinase 2.
Discussion
The overexpression of EZH2 in malignant tumors of various organs has been associated with tumor aggression and poor prognosis (5-10). We previously reported that EZH2 was highly expressed in ATC tissues and has potential as a therapeutic target for patients (11). As shown in Figure 1B, the cell-reducing effects of DZNep, an EZH2 inhibitor, were observed in all ATC cell lines, thereby supporting its potential as a novel therapeutic strategy. However, no significant changes were noted in cell-suppressing effects even when the dose of the drug was increased. We attributed this phenomenon to the cell lines’ heterogeneity and some cells’ insensitivity to DZNep. Therefore, it may be necessary to consider the combination of drugs with other mechanisms of action. Furthermore, as shown in Figure 2, no changes were detected in differentiation markers in any cell line. These results indicate that the suppressive effects of the EZH2 inhibitor on ATC cell lines were not due to the induction of differentiation.
The suppressive effects of DZNep on cell growth were stronger in cell lines without TP53 mutations. Recently, it was reported that EZH2 blockade is associated with a favorable pancreatic ductal carcinoma prognosis mainly through the cell death response in TP53 wild-type but not in TP53 mutants (16). Consistent with the above report, we confirmed that the suppressive effects of DZNep on cell growth were stronger in ATC cell lines without TP53 mutations. TP53 is a tumor suppressor gene, the most frequent genetic abnormality in human cancer, and the most critical gene for carcinogenesis and its progression. Although TP53 mutations are rare in differentiated thyroid carcinoma, they are more frequently detected in ATC (17, 18). TP53 is involved in many cell functions, such as DNA damage repair pathways, cell proliferation, arrest and apoptosis. The loss of these functions makes it impossible to stop cell proliferation, and thus, they are strongly associated with the malignancy of ATC. The expected effects of DZNep on ATC cell lines with TP53 mutations suggest that if TP53, which is involved in this widespread cell function, is mutated, cell proliferation cannot be sufficiently suppressed by EZH2 inhibition alone. Therefore, in ATC, which is considered to have a high frequency of TP53 mutations, cell proliferation may be suppressed by the concomitant use of other drugs in addition to EZH2 inhibitors.
Conclusion
In conclusion, we confirmed that EZH2 is expressed in all ATC cell lines, and DZNep, an EZH2 inhibitor, exerted inhibitory effects on the growth of ATC cell lines, but these effects may be attenuated in ATC with TP53 mutations. These results indicate that DZNep has potential as a therapeutic strategy; however, its effects may be attenuated in the presence of TP53 mutations.
Acknowledgements
This work was supported by JSPS KAKENHI Grant Numbers JP19K09052 to H.N., JP19K09087 to No.S., and 21K07209 to D.H.
Footnotes
Authors’ Contributions
Conceptualization, H.N., No.S., Y.R., H.I., and D.H.; Immunoblotting analysis, Na.S.; Drug analysis, H.N. and Na.S.; Data Curation, H.N., K.M., H.Y., S.T., and K.S.; Gene panel analysis, R.K.; Writing original draft preparation, H.N.; Review and Editing, D.H.
Conflicts of Interest
The Authors have no conflicts of interest to declare regarding this study.
- Received October 20, 2022.
- Revision received November 25, 2022.
- Accepted December 5, 2022.
- Copyright © 2023 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
