KAT5 Inhibitor NU9056 Suppresses Anaplastic Thyroid Carcinoma Progression through c-Myc/miR-202 Pathway

Background Anaplastic thyroid carcinoma (ATC) is considered to be one of the most aggressive cancers. Our previous study proved that highly expressed lysine acetyltransferase 5 (KAT5) in ATC is associated with a poorer prognosis. Here, this study examined the effects of a KAT5 inhibitor (NU9056) in human ATC cells. Methods First, the Cancer Genome Atlas (TCGA) dataset was used to detect the relationship between KAT5 expression and outcomes of thyroid carcinoma patients. Then, both in vitro and in vivo experiments were conducted to investigate the effects of NU9056 on normal and ATC human thyroid cells. Finally, microRNA sequencing, qPCR, and dual-luciferase reporter assay were performed to explore potential mechanisms by identifying downstream microRNA related to NU9056. Results KAT5 dysregulation correlated with more advanced-stage and poorer outcomes of thyroid carcinoma patients. Endogenous KAT5 protein and mRNA levels were much higher in ATC cells than in normal thyroid cells. Suppression of KAT5 by NU9056 inhibited survival, growth, migration, invasion, and tube formation, and increased radiosensitivity and chemosensitivity in ATC cells but showed no impact on normal thyroid cells. Mechanistically, microRNA-202-5p (miR-202) was identified as the most significantly decreased miRNA after NU9056 treatment. Knockdown of miR-202 suppressed ATC cell progression, while forced expression of miR-202 partially blocked the inhibitory effect of NU9056 on ATC cells. Furthermore, c-Myc was validated as the transcription factor of miR-202, and NU9056 decreased the c-Myc protein level by shortening its half-life. Finally, we proved that NU9056 inhibited ATC proliferation in vivo. Conclusions Our results indicated that NU9056 targets KAT5, shortens c-Myc half-life, subsequently downregulates miR-202 expression, and results in the suppression of ATC cells. Overall, KAT5 could be a potential target for clinical treatment for ATC.


Introduction
Anaplastic thyroid carcinoma (ATC) is highly malignant due to its proliferative capacity and invasive nature [1,2]. Te prognosis of ATC patients still remains extremely poor, with a median survival of only around 5-6 months [1,2]. Tus, in spite of its rarity, ATC accounts for around half of thyroid cancer-related death [1,2]. Upon diagnosis, more than half of ATC patients already present with local invasion and distant metastasis, and only in 10% of cases the disease is confned to the thyroid gland [1,2]. Terefore, to change this dismal situation it is vital to understand the molecular mechanism that drives ATC progression.
Although there have been several suggestions as to mechanisms driving ATC progression, treatment with agents that target these mechanisms does not improve the prognosis of ATC patients [3][4][5]. Since ATC is uncommon, making most studies is harder to recruit a sufcient number of participants to draw conclusions [2].
Based on 82 ATC clinical samples, our previous study found a strong correlation of lysine acetyltransferase 5 (KAT5, also known as Tip60) overexpression with poorer prognosis and tumor metastasis of ATC [6]. KAT5 belongs to the MYSTfamily of acetyltransferases [7], and its substrate includes both histone proteins (histone H4 and H2A) and nonhistone proteins (ATM, p53, p21, estrogen receptor, and so on) [7]. KAT5 is, therefore, involved in regulating many important physiological processes. Recently, increasing evidence showed that KAT5 also functions as activators of oncogenes in various types of cancers. Wang et al. found that KAT5-regulated SPZ1-TWIST1 complex leads to invasion and metastasis of liver cancer [8]. Meanwhile, Li et al. proved that KAT5-mediated SMAD3 acetylation promotes melanoma progression and invasion [9]. We also showed that KAT5 acetylates and stabilizes c-Myc protein by blocking the proteasome system [6]. Te Myc proteins are the products that are encoded by the well-known MYC family of proto-oncogenes [10]. Among them, transcription factor c-Myc constitutes the central transcriptional regulator network that regulates the expression of around one-sixth of all human genes and plays key roles in several cellular processes including metabolism, proliferation, diferentiation, apoptosis, and so on [11]. Expression of c-Myc is enhanced and deregulated in many human tumors [11], suggesting a possibility that highly expressed KAT5 in ATC contributes to the progression of tumor by modulating c-Myc. Tese fndings also suggest that aberrant high expression of KAT5 may drive ATC progression and, therefore, that targeting KAT5 could provide a novel and efcient clinical strategy against ATC.
NU9056 is a cell-permeable disulfane compound that is a potent and highly selective KAT5 acetyltransferase inhibitor [12]. NU9056 displays favorable physicochemical and pharmacokinetic properties and a good safety profle, which makes it suitable for potential clinical development.
MicroRNA (miRNA) is a type of short, single-stranded, noncoding RNA widely expressed in mammalian cells. It regulates cell gene expression via blocking mRNA translation or causing mRNA decay [13]. At present, there is a lot of evidence to show that abnormally regulated miRNAs contribute to the occurrence, development, and treatment resistance of several malignant tumors, including thyroid carcinoma, lung cancer, colorectal cancer, and others [14]. It is plausible that in ATC, miRNA is regulated by KAT5 and contributes to tumor progression. However, KAT5-mediated regulation of miRNA in ATC has not yet been investigated.
Since KAT5 represents a novel potential oncotarget in ATC treatment, we focused on investigating the potential antitumor activity of NU9056 in human ATC cells and the underlying mechanisms through miRNA sequencing. In this study, we found that NU9056 blocks KAT5 activity leading to an antitumoral efect. Te underlying mechanism is based on c-Myc blockage and consequent miR-202 downregulation. Altogether, our data suggested that KAT5 could be a potential target for clinical treatment for ATC. . Human normal thyroid cell Nthy-ori 3-1 was purchased from Antonio Corp (Suzhou, China). All the cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin and streptomycin, in a humidifed incubator at 37°C with 5% CO2.

Materials and Methods
For the cell transfection, vector, or KAT5 plasmid, con-shRNA, or KAT5-shRNA were designed and synthesized by GeneChem Corp (Shanghai, China). Transfection was accomplished using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instruction: ATC cells were seeded into 12-well plates (2 × 10 5 cells/ well). Ten, 30 pmol of con-shRNA or KAT5-shRNA, or 3ug of vector or KAT5 plasmid was transfected. After 48 h, KAT5 expression was determined by Western blot (WB) and qPCR.

NU9056 Was Provided by Absin (Shanghai, China).
As for the concentration, according to a previous study [12], in growth inhibition assay, the concentration required to inhibit cell growth by 50% of NU9056 for human prostate cancer cells was about 24 uM. Terefore, in the pre-experiment NU9056 concentrations were chosen from 1 uM to 100 uM. Finally, in the normal experiment NU9056 concentrations were chosen from 2.5 uM to 40 Um.
To maintain drug concentration during the colony formation assay, mediums containing doses of NU9056 were changed every 3 days.

Cell
Viability. Cells were plated into 96-well plates (4 × 10 3 cells/well), cultured overnight, and treated with NU9056 for the indicated time. Cell counting kit-8 (CCK-8) was purchased from Beyotime. Ten, CCK-8 solution (10 ul/ well) was added and incubated for 2 h at 37°C. Next, the optical density (OD) value was measured using a microplate reader at a wavelength of 450 nm.
2.6. Transwell Migration and Invasion Assay. As described previously [6], cells cultured without FBS were seeded to the upper chamber of each Transwell with (invasion) or without (migration) Corning Matrigel matrix (diluted by 1 : 8), and a culture medium containing 10% FBS was added to the lower chambers for chemoattracion. Ten, cells on the bottom were fxed by 4% paraformaldehyde, dyed by violet crystal, and then pictured and counted.

Colony Formation Assay.
Cells were seeded into 6-well tissue culture plates and incubated overnight (2000 cells/ well). Ten, NU9056 was added at indicated doses and continued to incubate for around 2 weeks. Furthermore, the colonies were washed, fxed, stained crystal violet, and counted. Colonies consisting of 50 or more cells were counted.

Chemosensitivity Experiment.
According to our previous unpublished data, the half-maximal inhibitory concentration of cisplatin for human thyroid cancer cells for the CCK-8 assay was around 1 µg/ml. Terefore, we applied cisplatin concentrations ranging from 0.1 to 3 µg/ml. NU9056 was added with a concentration of 10 µM. Cells were then plated into 96-well plates at a density of 4 × 10 3 cells/well and were subsequently treated with the indicated doses of cisplatin for 48 h. Following this, 10 µl of CCK-8 solution was added to each well and incubated for 2 h at 37°C. Next, the OD value was measured using a microplate reader at a wavelength of 450 nm.
According to our previous unpublished data, for the transwell migration assay (described above), 0.5 µg/ml of cisplatin caused no apparent inhibitory efect on the viability of human thyroid cancer cells and was chosen for further study.
2.9. Radiosensitivity Experiment. Cells were seeded into a 6well plate (the cell numbers were as follows: 400 for 0 Gy, 600 for 1 Gy, 800 for 2 Gy, 1200 for 3 Gy, and 1600 for 4 Gy), and NU9056 was added at the indicated doses the next day. Meanwhile, cells were irradiated and incubated for another 14 days. Following this, the cells were fxed and dyed with crystal violet. Ten, colonies consisting of 50 or more cells were counted.
2.10. Immunofuorescence. As previously described [15], cells were seeded into a 6-well plate (1.5 × 10 5 cells/well) over a glass slide. At the corresponding time points after NU9056 treatment, cells were fushed with ice-cold PBS and fxed with 4% paraformaldehyde. Immunofuorescence for c-Myc (Santa Cruz, 1 : 400) was processed according to the previously published protocol. Te next day, the glass slides were washed and the secondary antibody was incubated (anti-mouse IgG (H + L)F(ab')2 Fragment (#4408), Cell Signaling Technology, 1 : 500). Ten, cells were pictured under an inverted fuorescence microscope (Olympus). Finally, the fuorescence intensity was analyzed by Image J.
2.11. Tube Formation Assay. ATC or normal thyroid cells were treated with indicated doses of NU9056. After 24 h, 1 × 10 4 HUVEC cells and 1 × 10 4 ATC or normal thyroid cells were seeded to the 96-well plate that were coated with Corning Matrigel matrix (in a 1 : 8 dilution with DMEM), and each dose exposure of NU9056 was replicated 4 times. After coculture for 24 h, the tube structures were pictured by Olympus inverted microscope (X 100). Ten, the lengths of tubes were counted by Image J.

Dual-Luciferase Reporter
Assay. PGL4-miR-202-5ppromoter (2000 bp; TSS −2000 to 0) and pcDNA3.1-c-Myc were designed and synthesized by GenePharma (Shanghai, China). 293T cells were seeded to a 24-well plate (2 × 10 5 cells/ well). Ten, pcDNA3.1, PGL4+RL-TK, PGL4-miR-202-5ppromoter, or pcDNA3.1-c-Myc was transfected into the 293Tcells using Lipofectamine 2000. Te luciferase activity was then measured by a dual-luciferase reporter assay. Briefy, 100 µl of passive lysis bufer was added into each well and incubated for 15 min. Next, 20 µl of LARII reagent was added into each well, and the frefy luciferase was determined by a and then held at 4°C. Finally, the cDNA construct was purifed and recovered. Te libraries were sequenced using the Illumina HiSeq X Ten platform, and 150 bp paired-end reads were generated. After high-throughput sequencing and subsequent quality control, high-quality clean reads were obtained for analysis. Te number of clean reads of all samples ranged from 10, 290, 533 to 14, 625, 392. Te length of all clean reads was between 20 and 24 nucleotides, with the largest population of reads being 23 nucleotides. Small RNAs were classifed and annotated on the basis of the Rfam database, species reference transcript, and repeat sequence database. Te known miRNAs were annotated using the miRBase v.21 database. Tose with a p value < 0.05 were defned as differentially expressed miRNAs.

In Vivo Assay.
Te animal experimental protocol was approved by the ethics committee of Soochow University (ECSU-2019000200). Male BALB/c SPF nude mice (n � 20, 4-6 weeks old, weight 18-22 g) were purchased from the Soochow University. Injections of 5 × 10 6 CAL-62 cells were subcutaneously administered into the right fank. Mice were then randomly allocated into either the control group or the NU9056 group (both n � 10). Te NU9056 group received intraperitoneal injection of NU9056 diluted in saline with a concentration of 0.5 µg/µl every 2 days. Te NU9056 injection dose was 200 µg/100 g. Te tumor weights were then measured. Te long and short diameters of tumors were measured using calipers every 3 days, and tumor volumes were calculated using the formula: volume � 0.52 × long diameter × (short diameter) 2 . After 21 days, mice were killed, and tumors were removed. Next, the total tumor RNA was extracted using the TRIzol method, and RT-qPCR was conducted for miRNA.

Statistics
Analysis. Data were expressed as mean ± SD. Comparisons were conducted using unpaired Student's t-test. A p < 0.05 was considered to be statistically signifcant. Statistical analysis was performed using SPSS 22.0.

Aberrantly Expressed KAT5 Correlated with More Advanced-Stage and Poor Prognosis in Tyroid Carcinoma and Was Overexpressed in ATC.
In this study, we frst assessed the expression levels of KAT5 in human thyroid carcinoma, and the TCGA database was downloaded. In a dataset that contains 500 papillary thyroid cancer patients (TCGA, PanCancer Atlas), we found that KAT5 alteration cases (mostly were KAT5 mRNA high expression) had more advanced T stages and poorer survival than KAT5 normally expressed ones (defned as KAT5 mRNA normal expression) (Figures 1(a)-1(c)).

NU9056 Specifcally Inhibits Cell Viability and Proliferation in KAT5
Overexpressed Human ATC Cells. We then found that endogeneous KAT5 expressions were much higher in ATC cell lines (8505C and CAL-62) than in a normal thyroid cell line (Nthy-ori 3-1) at both protein and mRNA levels (Figures 2(a), 2(b)).
NU9056 is a highly selective KAT5 acetyltransferase inhibitor, in order to prove that NU9056 is efective in blocking KAT5 activity, and human ATC cells CAL-62 were left untreated or treated with NU9056 (2.5-40uM) for 48h. Ten, the levels of KAT5's downstream, histone H2A, and histone H4 acetylation were detected by Western blot. Te results showed that NU9056 decreased the expression levels of acetyl-histone H2A and acetyl-histone H4 in a concentration-dependent manner but had no efect on the expression levels of total histone H2A and total histone H4 (Figure 2(c)).
Next, we studied the efects of elevated KAT5 expression on ATC viability and proliferation by treating human ATC cells with NU9056 at a range of concentrations (2.5-40 µM). Te results of the CCK8 assay (Figures 2(d), 2(f ), and 2(h)) showed that NU9056 potently inhibited ATC cell viability in a concentration-dependent manner but had no inhibitory efect on normal thyroid cells. Similarly, in the colony formation assay (Figures 2(e), 2(g), and 2(i)), NU9056 potently inhibited ATC cell proliferation in a concentration-dependent manner but had a weakened or no inhibitory efect on normal thyroid cells. Furthermore, after con-shRNA or KAT5-shRNA was   transfected into CAL-62 cells (Figures 2(j) and 2(k)), and the inhibitory efect of NU9056 on the viability of ATC cells was totally blocked after downregulation of KAT5 (Figure 2(l)).

NU9056 Specifcally Inhibits Migration, Invasion, and Tube Formation in KAT5 Overexpressed Human ATC Cells.
Next, the efect of NU9056 on migration and invasion ability of ATC cells was evaluated by Transwell assay. We found Next, cell radiosensitivity was tested by colony formation assay (E) F, (I). * p < 0.05 vs. "Ctrl" group, * * * p < 0.01 vs. "Ctrl" group. 8 International Journal of Endocrinology that NU9056 signifcantly inhibited ATC cell migration and invasion in a dose-dependent way (Figures 3(a)-3(d)). Since the angiogenic activity is also vital for tumor invasion and metastasis, a tube formation assay was conducted. Te results showed that NU9056 also remarkably inhibited tube formation of microvascular endothelial cells that were cocultured with ATC cells (Figures 3(e), 3(f )). Interestingly, compared with KAT5 overexpressed human ATC cell lines, NU9056 showed no or weakened inhibitory efect on migration, invasion, and tube formation ability of KAT5 lowexpressed normal thyroid cells (Figures 3(g)-3(i)).

NU9056 Specifcally Increases Radio-and Chemosensitivities of KAT5 Overexpressed Human ATC Cells.
Te impact of NU9056 treatment on radiosensitivity and chemosensitivity of ATC cells was evaluated. Te CCK-8 and Transwell assays both showed that NU9056 treatment increased the chemosensitivity of ATC cells to cisplatin (Figures 4(a)-4(d)). Te radiosensitivity of ATC cells was also increased after NU9056 treatment (Figures 4(e) and 4(f )). Interestingly, NU9056 treatment showed no radio-or chemosensitization efects on normal thyroid cells with low KAT5 expression (Figures 4(g)-4(i)).

NU9056 Inhibits ATC Proliferation In Vivo.
To investigate the potential antitumor activity of NU9056 on ATC in vivo, CAL-62 cells were injected into SPF mice to form subcutaneous xenografts. Ten, these mice were randomly assigned to receive an intraperitoneal injection of either DMSO (as control) or NU9056. Tumor growth curves, in Figure 5(a), showed that xenografts derived from the NU9056 treatment group grew remarkedly slower. After 21 days, mice were killed and tumors were removed ( Figures 5(b) and 5(c)). We found that the weights of tumors from the NU9056 treatment group were much lighter than those from the control group ( Figure 5(d)).

NU9056 Inhibits miR-202-5p Expression in Human ATC
Cells. To identify the potential downstream miRNAs of KAT5 and NU9056 treatment, transfection of empty vector or KAT5 plasmid into 8505C cells was conducted. Ten, miRNAs sequencing was conducted. We identifed 37 miRNAs that showed upregulation or downregulation for at least twofold after overexpression of KAT5 in ATC cells (Figures 6(a)-6(c), Supplementary Table 1). Among them, the level of miR-202-5p dramatically increased, and this fnding was further confrmed by qRT-PCR (Figure 6(d)). Data from qRT-PCR also consistently showed that inhibition of KAT5 by NU9056 signifcantly decreased the level of miR-202-5p ( Figure 6(e)). Meanwhile, in animal study NU9056 also signifcantly decreased the level of miR-202-5p in CAL-62 xenograft tissue (Figure 6(f )). To detect the function of miR-202-5p in ATC progression, CAL-62 cells were transfected with inhibitor NC or miR-202-5p antagomir ( Figure 6(g)). We found that knockdown of miR-202-5p inhibited proliferation and invasion of ATC cells  Figure 5: NU9056 inhibits ATC xenograft growth in mice. Te SPF mice were injected ATC cells at the right fanks and were divided into the control or NU9056 group. Te tumor growth curves were calculated (A). After 21 days, mice were killed and tumors were removed (B, C). Ten, the tumor weights of each group were measured (D). N � 10 mice per group. * * * p < 0.01. . Te SPF mice were injected with Cal-62 cells at the right fanks and were divided into control or NU9056 group. After 21 days, mice were killed and the expression of miR-202-5p of the tumor was detected by qRT-PCR (F). Cal-62 cells were transiently transfected with either control or miR-202-5p antagomir, and the knockdown efect was detected by RT-PCR (G). Ten, cells were left untreated or treated with NU9056, and cell viability (H) and invasion ability (I) were tested by the appropriate assay. Cal-62 cells were transiently transfected with either control or miR-202-5p mimic, and the transfection efect was detected by RT-PCR (J). Ten, cells were treated with NU9056, and cell viability (K) was tested by the appropriate assay. * p < 0.05 vs. "Ctrl" group, * * * p < 0.01vs. "Ctrl" group.

NU9056 Inhibits miR-202-5p Expression via Transcription
Factor c-Myc. To predict the transcription factors of miR-202-5p, the prediction program JASPAR was used. We found several potential c-Myc binding sites on the upstream promoter region of miR-202-5p, indicating that transcription factor c-Myc may regulate the level of miR-202-5p (Figure 7(a)). Next, dual-luciferase reporter assay showed that transfection of c-Myc signifcantly increased the luciferase activity of the PGL4-miR-202-5p promoter compared with the control (Figure 7(b)).
Our previous study proved that KAT5 promotes c-Myc expression via inhibition of its ubiquitin-mediated degradation in ATC cells [6]. We, therefore, sought to determine whether NU9056 treatment can afect c-Myc expression via regulation of ubiquitination. Oncomine database showed that c-Myc protein levels were most upregulated in ATC than in other types of thyroid carcinomas (Figure 7(c)). Next, Western blotting and immunofuorescence results both showed that NU9056 decreased the level of c-Myc expression in a dose-dependent way (Figures 7(d)-7(f )). Furthermore, after using cycloheximide to block protein synthesis, we found that NU9056 remarkably decreased the c-Myc half-life (Figures 7(g), 7(h)).

Discussion
Here, we examined the efects of NU9056, a highly selective KAT5 acetyltransferase inhibitor, on human ATC cells. Although tumors are typically considered to be a genetic disease, increasing evidence has shown that they initiated and progressed through both genetic and epigenetic alterations [16,17]. Terefore, novel drugs that target these epigenetic alterations could potentially provide a more tailored and specifc response, which could have a signifcant clinical impact on tumor treatment [16,17]. Of these epigenetic alterations, reversible acetylation/deacetylation regulates several aspects of key cellular activities and has been the subject of much research [18,19]. Increasing evidence is being produced to suggest that the imbalance of acetylation/deacetylation leads to tumorigenesis and progression in various common cancers, including lung, breast, and esophageal cancer [16,[20][21][22][23][24].
Our previous study reported that overexpression of KAT5 is associated with poorer survival and tumor metastasis in ATC and showed that KAT5 acetylates and stabilizes c-Myc by blocking proteasome [6]. KAT5 is the catalytic subunit of the evolutionarily conserved mammalian multi-subunit nucleosome acetyltransferase of the histone H4 complex, which mainly acetylates histone H4 and H2A. KAT5 is also recruited by several transcription factors and acetylates of several nonhistone substrates including androgen receptors, ATM, estrogen receptors, β-catenin, and E2F [7]. Moreover, KAT5 also plays important role in double-strand DNA break repair and apoptosis [7]. Terefore, KAT5 is involved in several pivotal cellular physiological and pathological processes, including carcinogenesis and tumor progression, and could be a novel potential therapeutic target for ATC treatment [6].
In this study, we showed that NU9056, a potent and highly selective KAT5 acetyltransferase inhibitor, inhibited survival, proliferation, migration, invasion, and tube formation and increased radiosensitivity and chemosensitivity in established human ATC lines (8505C and CAL-62) but showed no efect in human normal thyroid cells (Nthy-ori 3-1) and KAT5 downregulation ATC cell line. Tese results suggested that NU9056 selectively inhibited progression in KAT5 overexpressed ATC cells.
MiRNAs are single-stranded noncoding RNAs that include 21-25 nucleotides and play critical roles in several biological and pathological processes [13]. Dysregulation of miRNA plays a pivotal role in cancer formation and is commonly observed in several areas of human cancer studies including thyroid carcinoma, tumorigenesis, progression, therapy resistance, and prognosis predication [25][26][27]. Te results of our miRNA sequencing and qRT-PCR experiments showed that upregulation of KAT5 increased levels of miR-202-5p, while inhibition of KAT5 by NU9056 decreased miR-202-5p expression. We also found that in ATC cells, knockdown of miR-202 suppressed progression, while forced expression of miR-202 partially blocked the inhibitory efect of NU9056. Tese results indicate that miR-202-5p could be the primary downstream target of KAT5. Previous studies have reported miR-202-5p to have tumor-suppressing qualities for many types of cancers including ovarian cancer, colorectal cancer, and bladder cancer [28,29]. Other study also showed that miR-202-5 is upregulated and associated with oncogene properties in breast cancer, lymphomagenesis, and osteosarcoma [30][31][32][33]. Tese fndings bring into question whether the function of miR-202-5p as an oncogene/tumor suppressor is context dependent.
To investigate the mechanism of how KAT5 modulates miR-202-5p expression, we searched the JASPAR database. Based on the resultant prediction of the transcription factor and the binding sites, we suspect that c-Myc may be the transcription factor for miR-202-5p. Te results of this study support our speculation that forced overexpression of c-Myc signifcantly increases the luciferase activity of the miR-202-5p promoter. Furthermore, we observed that the inhibitory efect of NU9056 on levels of miR-202-5p was partially rescued in c-Myc knockdown ATC cells. Te powerful transcription factor c-Myc belongs to one of the most frequently overexpressed oncogenes, and its pivotal role in tumorigenesis and metastasis has been highly studied [11,12]. Interestingly, our previous study showed that KAT5-mediated acetylation stabilizes c-Myc via the inhibition of the ubiquitin-proteasome system [6]. Te results of the current study are consistent with this, and in that we found that NU9056 inhibits c-Myc expression by decreasing protein stability. Taken together, these data indicate that KAT5 upregulates miR-202-5p expression at a transcriptional level through c-Myc.   Our results supported a model in which the aberrant overexpression of KAT5 stabilizes c-Myc protein, subsequently increases miR-202-5p level, and may cause ATC progression ( Figure 8). Tus, inhibition of KAT5 by NU9056 could be a promising strategy for the clinical treatment of ATC ( Figure 8). Meanwhile, the establishment of the KAT5/c-Myc/miR-202-5p axis will lead to a better understanding of molecular pathways involved in ATC.

Data Availability
Te data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te authors declare no conficts of interest.