MEX3A induces the development of thyroid cancer via targeting CREB1

Thyroid cancer is a prevalent form of endocrine cancer, and its global incidence has been steadily increasing. MEX3A is a protein that is known to be highly expressed in various human malignant tumors, including thyroid cancer, and it has been linked to patient prognosis. However, the molecular mechanisms underlying MEX3A's tumorigenic capabilities in thyroid cancer are not fully understood. In this study, we aimed to investigate the role of MEX3A in thyroid cancer. We confirmed that MEX3A was overexpressed in both thyroid cancer tissues and cell lines. Additionally, we found a positive correlation between high levels of MEX3A and the AJCC stage. To further understand the functional significance of MEX3A in thyroid cancer, we depleted MEX3A expression in B‐CPAP and TPC‐1 cells. Interestingly, we observed a significant reduction in thyroid cancer cell proliferation and migration, as well as ameliorated cell apoptosis and arrested tumor growth upon MEX3A depletion. These findings strongly suggested that MEX3A played a critical role in the development of thyroid cancer. Furthermore, our study uncovered an important interaction between MEX3A and CREB1 (cAMP response element‐binding protein 1). The interaction between MEX3A and CREB1 appeared to contribute to the tumor‐promoting effects of MEX3A in thyroid cancer by directly targeting CREB1. Silencing CREB1 was observed to alleviate the malignant phenotypes promoted by MEX3A in thyroid cancer cells. Together, this study highlighted the importance of the MEX3A‐CREB1 interaction in thyroid cancer development and suggested the therapeutic potential of targeting MEX3A for the treatment of this disease.

cancer, which constitute the majority of thyroid cancer cases (Cabanillas et al., 2016).Although ATC represents the most aggressive histological subtype, most deaths related to thyroid cancer occur in patients with WDTC (Ghossein et al., 2007;Silver et al., 2011).Surgical resection currently serves as an effective treatment for low-risk WDTC patients (Haugen et al., 2016).However, the therapeutic options for advanced and metastatic thyroid cancer are limited, and approved treatments mainly involve targeted therapies that are not curative (Brose et al., 2016;Kim et al., 2013;Rothenberg et al., 2015).Consequently, there is a pressing need for the identification of additional therapeutic targets and the development of novel treatment options for thyroid cancer.
MEX3A is a member of the human MEX3 protein family, consisting of four members (MEX3A-D).These proteins have two HNRNPK homology (KH) domains and a C-terminal really interesting new gene (RING) domain that mediates RNA binding and protein ubiquitination (Liang et al., 2020;Naef et al., 2020).MEX3 paralogs exhibit an oncofetal expression pattern, i.e. they are severely downregulated after birth but are re-expressed in various malignancies (Panzeri et al., 2021).High expression of MEX3 proteins in different cancers is strongly associated with poor prognosis, indicating their potential role as oncogenes.For example, MEX3A has been shown to regulate proliferation and migration of tumor cells in vitro and tumor growth in xenograft studies (Jiang et al., 2012;Yang, Jiao, Li, & Fang, 2020).Moreover, upregulation of MEX3A has been observed in thyroid cancer and is related to patients' prognosis (Ma et al., 2021).However, the molecular mechanisms underlying the tumorigenic properties of MEX3A have yet to be fully understood.
This study aimed to examine the expression pattern and functional importance of MEX3A in thyroid cancer and shed light on potential downstream mechanisms involved.The findings will contribute to our understanding of the pathogenesis of thyroid cancer.

| The Cancer Genome Atlas (TCGA) database analysis
In this study, we analyzed the expression profiles of thyroid cancer using RNAseq data from 560 samples, including 502 tumor samples and 58 normal samples obtained from TCGA database.
The infection efficiency was determined by observing the green fluorescence labels on the lentivirus under a microscope.An infection was considered effective when the fluorescence efficiency exceeded 80%.To overexpress MEX3A in B-CPAP and TPC-1 cell lines, we designed primer amplification sequences specific to MEX3A and constructed a MEX3A overexpression lentiviral vector.

| Western blot assay
After lentivirus infection, the total protein of B-CPAP and TPC-1 cells was collected and segregated using 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) (#P0015L, Beyotime Biotechnology Co., Ltd.).Polyvinylidene difluoride membranes were then blocked with TBST solution containing 5% skim milk at room temperature for 1 h.The membranes were incubated with primary antibodies and then with secondary antibodies for 2 h at room temperature.After that, the membranes were washed three times with TBST solution (10 min/time).
Finally, color rendering was conducted using the immobilon Western Chemiluminescent HRP Substrote kit (#WBKLS0500, Millipore).For endogenous and exogenous Co-IP analysis, 1.0-1.2mg of proteins from TPC-1 cells were incubated with IgG and anti-CREB1, or the proteins from 293T cells infected with MEX3A-Flag were incubated with Flag.
This was followed by 2 h of incubation with 20 μL beads at 4°C.The proteins in the immunocomplex were separated by 10% SDS-PAGE and used for MEX3A, CREB1 or Flag incubation.The details of the antibodies used in this section were shown in Table S1.

| Cell proliferation detection
After lentivirus infection, B-CPAP and TPC-1 cells were trypsinized and resuspended.Next, 100 μL cell suspension was added to each well of a 96-well plate, and cell images were captured using the Celigo image cytometer (Nexcelom Bioscience).A continuous 5-day cell proliferation curve was drawn.

| Colony forming assay
To perform a colony formation assay, the indicated cells were plated in a six-well plate at a density of 500 cells per well and cultured for 8 days.The colonies were then washed with PBS (#C3580-0500, Vivacell), fixed with 1 mL 4% paraformaldehyde (#AR Hu test, China National Pharmaceutical Group Co., Ltd.), and stained with 500 μL of Giemsa (#AR-0752, Dingguo).Visible colonies were recorded using fluorescence microscope (Olympus).

| Wound healing assay
B-CPAP and TPC-1 cells with indicated lentivirus were seeded in a 96-well plate at a density of 5 × 10 4 cells/well, followed by supplementation with low-concentration serum medium (#A1933, Sigma) on the next day.A scratch tester was used to create scratches on the plate by gently pushing upwards from the center.The cells were then washed with serum-free medium and treated with 0.5% FBS.The plate was placed in an incubator with 5% CO 2 at 37°C.The plate was scanned at appropriate time, and the migration area was analyzed using Cellomics (Thermo Fisher Scientific).

| Transwell assay
For the Transwell migration assay, the indicated cells (5 × 10 4 ) were seeded in 100 μL medium without FBS on a fibronectincoated polycarbonate membrane inserted in a Transwell apparatus (#354248, BioCoat).In the lower chamber, 500 μL medium with 10% FBS was added as a chemoattractant.After being cultured for 30 h, the cells adhering to the lower surface of the membrane were fixed, stained with 1% crystal violet (#KGA229, Kaiji) solution for 1 min, and counted under a microscope in three random fields.

| Detection of cell apoptosis by fluorescence activated cells sorting (FACS)
For the apoptosis assay, B-CPAP and TPC-1 cells with the indicated lentivirus were cultured in a six-well plate with a volume of 2 mL/well.When the cell confluence reached 85%, the cells were collected by centrifugation at 1300 rpm and the supernatant was  2.12 | Human Phospho-Kinase Array-membrane The expression of 39 phospho-kinases in TPC-1 cells following lentivirus infection was analyzed using the Human Phospho-Kinase Array Kit (#ARY003C, Bio-Techne China Co., Ltd.).To detect the phosphokinases, the cells were lysed and the Handling Array membranes were blocked with 2 mL 1× Wash Buffer II (#895478, Bio-Techne China Co., Ltd.).The cell lysates were then incubated with 1× biotin-conjugated anti-cytokines overnight at 4°C.After incubation, the handling array membranes were washed and developed using a chemiluminescence imaging system to detect the signals.

| Immunofluorescence experiment
The TPC-1 cells used for imaging had been silenced for MEX3A.To 22°C-25°C and a humidity of 50%-60%.A 12-h light/dark cycle was established to provide a regular light schedule for the mice.Adequate water and food supplies were provided to ensure the mice had free access.To establish xenograft models, either shMEX3A or shCtrl TPC-1 cells (1 × 10 7 cells/mice) were subcutaneously injected into the right axilla of the nude mice (10 mice/group).The tumors were allowed to grow for a period of 17 days, during which the length and width of the tumors were measured to calculate the tumor volume using the formula (tumor volume = π/6 × L × W × W).Finally, the mice were euthanized, and the tumors were removed, weighed, and frozen in liquid nitrogen for subsequent analysis.

| Statistical analysis
In this study, each assay was independently conducted three times to ensure the accuracy and reproducibility of the results.Data analysis was performed using GraphPad Prism 6 software, and the results were presented as mean ± standard deviation.Statistical differences between the experimental and control groups were evaluated using an unpaired t test, considering a p value of less than .05 to be statistically significant.

| MEX3A is frequently upregulated in thyroid cancer
To elucidate the roles of MEX3A in the pathogenesis of thyroid cancer, we initially examined its protein expression in thyroid cancer specimens and para-carcinoma tissues using a tissue microarray.
Consistent with previous findings, our results demonstrated a significant upregulation of MEX3A in thyroid cancer tissues compared to para-carcinoma tissues (p < .001, Figure 1a,b and Table 1).Immunohistochemical analysis revealed predominant nuclear expression of MEX3A in cells.Besides, we analyzed MEX3A mRNA level using RNA-seq datasets from the TCGA database, which confirmed a substantial upregulation of MEX3A in tumor tissues of thyroid cancer patients compared to the normal tissues of healthy volunteers (Figure 1c).Furthermore, we quantified the mRNA levels of MEX3A in thyroid cancer cell lines relative to the normal thyroid cell line Nthy-ori 3-1.Our findings indicated abundant expression of MEX3A mRNA in thyroid cancer cell lines (Figure 1d).Additionally, we investigated whether the expression of MEX3A was associated with clinicopathological parameters of thyroid cancer patients.
The results showed a significant association between increased MEX3A expression and higher AJCC stage (Table 2).Based on these findings, we concluded that MEX3A may serve as a potential tumorpromoting factor in the development of thyroid cancer.

| Depletion of MEX3A results in attenuated cell proliferation and migration in vitro
In this section, we aimed to investigate the effects of MEX3A group (Figure 2c).Additionally, the colony forming assay performed in B-CPAP and TPC-1 cells provided additional confirmation that loss of MEX3A expression diminished cell viability and proliferation (Figure 2d).Furthermore, MEX3A-deficient cells displayed reduced motility.The wound-healing experiment revealed attenuated migration ability in cells with MEX3A knockdown (Figure 2e).These results were further supported by the results of the transwell assay (Figure 2f).Moreover, MEX3A-deficient cells displayed a more than threefold increase in apoptosis potential (Figure 2g).Collectively, these results conclusively indicated that depletion of MEX3A caused diminished cell proliferation and migration, as well as enhanced cell apoptosis in vitro.

| Depletion of MEX3A results in impaired tumor growth in vivo
To determine the impact of MEX3A on tumor growth in vivo, a subcutaneous xenograft tumor model was constructed.Based on cell functional experiments, we found that MEX3A depletion had a stronger effect on the growth of TPC-1 cells compared to B-CPAP cells.Therefore, we selected TPC-1 cells for the xenograft experiments.We subcutaneously implanted TPC-1 cells infected with shCtrl/shMEX3A into BALB/c nude mice and closely monitored tumor growth (Figure 3a).The findings revealed that tumors derived from MEX3A-deficient cells grew slower compared to tumors derived from TPC-1 cells with shCtrl, which aligned with our in vitro proliferation assays showing a higher plateau in shMEX3A TPC-1 cells (Figure 3b-e).Moreover, IHC analysis of tumor tissues from the xenograft mice injected with MEX3Adepleted TPC-1 cells showed substantially decreased levels of Ki67, a marker of cell proliferation (Figure 3f,g).Together, these data demonstrated that MEX3A knockdown significantly delayed the growth of xenografted tumors, confirming the critical role of MEX3A in tumor progression.

| MEX3A moderates thyroid cancer via acting on CREB1
We proceeded to investigate the underlying mechanism through which MEX3A influences the malignant characteristics of thyroid cancer cells.Initially, we utilized the Human Phospho-Kinase Array-Membrane kit to analyze the levels of phosphorylated protein affected by MEX3A knockdown.The results showed a downregulation of several proteins, including CREB (S133), ERK1/ 2(T202/Y204, T185/Y187), GSK-3β (S9), Hsp27 (S78/S82), p53 (S15), p53 (S392), p70 S6K (T421/S424), STAT1 (Y701), and STAT6 (Y641) (Figure 4a).Furthermore, at the molecular level, we observed a decrease in CREB1, p-HSP27 and p-STAT1 upon MEX3A depletion (Figure 4b).Previous reports have highlighted the significance of CCDC6 in the dynamics of cAMP signaling by regulating CREB1 transcriptional activity in both normal and transformed thyroid cells (Luise et al., 2012).Notably, CCDC6 knock-in mice display thyroid hyperplasia associated with enhanced CREB1 activity (Leone et al., 2015).Considering these findings, we hypothesized that MEX3A might target CREB1, thereby influencing the development of thyroid cancer.To explore this hypothesis, we initially measured the levels of CREB1 in thyroid cancer cell lines and found that it was elevated compared to Nthy-ori 3-1 cells (Figure 4c).Additionally, immunofluorescence staining revealed a decrease in CREB1 levels upon MEX3A knockdown (Figure 4d).Immunoprecipitation experiments further demonstrated that MEX3A interacted with both endogenous and exogenous CREB1 (Figure 4e).Taken together, these findings provide evidence that CREB1 acts as a downstream factor of MEX3A, suggesting its involvement in the regulatory pathway of MEX3A-mediated effects in thyroid cancer development.
To investigate the functional significance of this interaction, we conducted experiments using cell models overexpressing MEX3A, knocking down CREB1, or both, and evaluated their impacts on cell

| DISCUSSION
The advancement in identifying new biomarkers for thyroid cancer have greatly contributed to personalized treatments for this disease (Laha et al., 2020).Currently, most targeted therapies focus on inhibiting known mechanisms that drive the growth and progression of thyroid cancer, such as the MAPK pathway (Ferrari et al., 2015;Kimura et al., 2003;Soares et al., 2003;Xing, 2013), PI3K/Akt-mTOR pathway (Hanna et al., 2018;Schneider et al., 2017), or VEGF (Brose et al., 2014;Matsui et al., 2008;Okamoto et al., 2013).Despite the discovery of new potentially effective therapeutic agents, there is still a deficiency of effective treatments for advanced and metastatic thyroid cancer.It is, therefore, crucial to identify more new drug targets and develop therapeutic agents specifically aimed at tackling advanced and metastatic thyroid cancer, particularly for patients at high risk of distant metastasis.By doing so, treatment outcomes for these patients could be significantly improved.
MEX3A is a widely recognized oncogene that has been implicated in various cancer types, including thyroid cancer.Previous studies have consistently demonstrated that upregulation of MEX3A expression was associated with unfavorable prognosis in patients with thyroid cancer (Jiang et al., 2012;Ma et al., 2021;Yang et al., 2020).In our study, we conducted MEX3A knockdown triggered by various external stimuli (Steven & Seliger, 2016).
Extensive research has shown that CREB1 was highly expressed in breast cancer and positively correlates with VASP expression.VASP, a protein involved in cell migration and proliferation, contains two CRE (cAMP response element) elements in its promoter region.
Activation or overexpression of CREB1 has been shown to significantly promote the expression of VASP, thereby promoting the proliferation and migration of breast cancer cells (Hu et al., 2019).
In our study, we explored the mechanistic link between MEX3A and CREB1 in thyroid cancer.We observed that depletion of MEX3A led to downregulation of CREB1 expression.Previous studies have also reported the involvement of CREB1 in the development of thyroid cancer (Leone et al., 2015;Luise et al., 2012).Importantly, we found that MEX3A could interact with CREB1 both endogenously and exogenously.Based on these findings, we hypothesized that MEX3A Overall, our findings highlighted the importance of MEX3A in modulating thyroid cancer development through its interaction with CREB1.Targeting this signaling axis could hold promise as a therapeutic strategy for the treatment of thyroid cancer.
In conclusion, this study shed light on the crucial role of MEX3A in the development of thyroid cancer, suggesting its potential as a therapeutic target for this disease.
removed.The cells were then washed with D-Hanks (4°C, pH = 7.2-7.4)and stained with 10 μL Annexin V-APC (#88-8007-74, eBioscience) in the dark.The stained cells were analyzed using a FACSCalibur (BD Biosciences) to evaluate the level of cell apoptosis.

FENG
prepare the cells for imaging, they were fixed in 4% paraformaldehyde and blocked with serum for 30 min to prevent nonspecific binding of the antibodies.The primary antibody was then applied to the cells and incubated overnight at 4°C to allow for specific binding to the antigen of interest.After washing away the primary antibody, a secondary antibody labeled with a fluorescent dye was added and incubated for 2 h at room temperature.The fluorescent label on the secondary antibody allowed visualization of the protein of interest.Finally, the nuclei of the cells were stained with DAPI (a fluorescent stain that binds to DNA, #ab104139, Abcam), and the cells were photographed using a fluorescent microscope.The detailed information of the antibodies used was described previously.2.14 | The construction of nude mouse tumor formation model All animal procedures were conducted in accordance with UK guideline.The experiments received approval from the Ethics Committee of Animal Experiments of Beijing Tiantan Hospital (No. 202002042) and were performed in accordance with the guidelines for the Care and Use of Laboratory Animals of the Ethics Committee of Animal Experiments, Beijing Tiantan Hospital.Twenty female BALB/c nude mice, aged 4 weeks, were obtained from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. and housed under specific pathogen-free conditions.The mice were kept in cages with five animals per cage, and the room maintained a temperature of F I G U R E 1 MEX3A was upregulated in thyroid cancer and MEX3A knockdown cell models were constructed.(a) The protein expression levels of MEX3A in thyroid cancer tumor tissues and para-carcinoma tissues were determined by immunohistochemical staining.(b) Quantitative analysis of MEX3A's IHC scores.(c) MEX3A mRNA levels were analyzed using RNA-seq datasets from TCGA database.(d) The MEX3A mRNA expression in thyroid cancer cell lines and normal thyroid cell line were detected by qRT-PCR.Results were presented as mean ± SD. *p < .05,**p < .01,***p < .001.IHC, immunohistochemistry; mRNA, messenger RNA; qRT-PCR, quantitative real-time PCR; TCGA, The Cancer Genome Atlas.

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I G U R E 2 MEX3A knockdown inhibited thyroid cancer cell proliferation and migration as well as enhanced cell apoptosis.(a, b) The knockdown efficiencies of MEX3A were detected by qRT-PCR (a) and western bloting (b).(c, d) After lentiviral infection, B-CPAP and TPC-1 cell proliferation was evaluated by Celigo cell counting assay (c) and Colony forming assay (d).The "/fold" represents the fold change compared with the cell count on the day 1. (e, f) After lentiviral infection, the changes of B-CPAP and TPC-1 cell migration were detected by wound-healing assay (e) and transwell assay (f).(g) The effects of MEX3A knockdown on B-CPAP and TPC-1 cell apoptosis were examined by fluorescence activated Cells Sorting (FACS).Results were presented as mean ± SD. *p < .05,**p < .01,***p < .001.qRT-PCR, quantitative real-time PCR.

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I G U R E 3 MEX3A knockdown suppressed thyroid cancer tumor growth in vivo.(a) A nude mice model of MEX3A knockdown was constructed.(b) The fluorescence intensity was obtained through injection of D-Luciferase before sacrificing the mice.(c) The volume of tumors was tested from feeding to sacrifice.(d) The weight of tumors was measured after sacrificing mice.(e) The photograph of tumors was taken after removing tumors.(f) The value of Ki67 was detected by IHC in tumor sections.(g) Quantitative analysis of Ki67's IHC scores.Results were presented as mean ± SD. **p < .01,***p < .001.IHC, immunohistochemistry.
proliferation and migration.The data indicated that MEX3A overexpression enhanced cell proliferation and migration, while CREB1 knockdown inhibited these processes.Partially reversing the effects of MEX3A overexpression, when combined with CREB1 knockdown, provided further evidence of the role of MEX3A in regulating CREB1 expression (Figure4f,g).Overall, these results suggested that MEX3A can target and regulate the expression of CREB1, thereby promoting the development of thyroid cancer.

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I G U R E 4 The exploration of molecular mechanism behind MEX3A regulating thyroid cancer.(a) Thirty-nine phosphorylated protein levels in TPC-1 cells with MEX3A depletion were measured using Human Phospho-Kinase Array-Membrane.(b) The expression of CREB1, HSP27, p-HSP27, STAT1, and p-STAT1 was detected in TPC-1 cells with MEX3A depletion by western blot.(c) The CREB1 mRNA expression in thyroid cancer cell lines and normal thyroid cell line were detected by qRT-PCR.(d) Immunofluorescence experiments showed the interaction between MEX3A and CREB1.(e) The endogenous and exogenous relationship between MEX3A expression and CREB1 level.(f, g) The changes in cell proliferation and migration of B-CPAP and TPC-1, in which MEX3A was overexpressed, CREB1 was silenced or MEX3A was overexpressed and CREB1 was silenced, were assessed by Celigo cell counting assay (f) and transwell assay (g).The "/fold" represents the fold change compared with the cell count on the day 1.Results were presented as mean ± SD. *p < .05,**p < .01,***p < .001.mRNA, messenger RNA.
experiments in two thyroid cancer cell lines, B-CPAP and TPC-1.The depletion of MEX3A resulted in a decrease in cell proliferation and migration abilities, improved cell apoptosis, and inhibited tumor growth in vivo.Moreover, we delved into the underlying mechanism through which MEX3A regulates thyroid cancer and identified CREB1 as a potential target gene.CREB1 (cAMP response element-binding protein 1) is a wellknown transcription factor belonging to the basic leucine zipper protein family.It becomes activated through phosphorylation at Ser133 in response to increased intracellular levels of cAMP or Ca 2+ plays a role in thyroid cancer development by interacting with CREB1.We validated this hypothesis through in vitro experiments, where elevation of MEX3A accelerated thyroid cancer cell proliferation and migration.To further elucidate the significance of the MEX3A-CREB1 interaction, we conducted additional experiments silencing CREB1 while overexpressing MEX3A in thyroid cancer cell models.The collective results revealed that silencing CREB1 impaired malignant progression-associated events in thyroid cancer cells and reversed the promotion of malignant phenotypes caused by elevated MEX3A expression.This evidence underscores the crucial role of the MEX3A-CREB1 axis in regulating thyroid cancer development.