INTRODUCTION

Thyroid cancer is a malignancy of the endocrine system that has exhibited an increasing occurrence rate over recent decades, triggering extensive investigation into its molecular foundations and possible therapeutic targets.[1-6] Despite the generally favorable prognosis of thyroid cancer, a subset of patients exhibits aggressive disease and resistance to conventional therapies; in this regard, novel molecular pathways and biomarkers should be identified for improved treatment strategies.[7,8]

A promising research area in thyroid cancer focuses on the investigation of apoptosis, which is essential to maintain cellular balance and remove damaged or malignant cells.[6,7,9-11] Dysregulation of apoptotic pathways is a hallmark of cancer, and it enables uncontrolled cell proliferation and tumor growth.[12,13] Thus, understanding the apoptosis in thyroid cancer cells is essential for the development of targeted therapies that can restore apoptotic mechanisms and inhibit tumor progression.

Cortactin-binding protein 2 N-terminal-like CTTNBP2NL has emerged as an important factor in the apoptosis of various cancer cells.[14] Although its role in thyroid cancer has not been extensively studied, initial findings suggest that CTTNBP2NL may influence key apoptotic pathways. CTTNBP2NL interacts with components of the cytoskeleton and signal transduction pathways and potentially affects cell survival and apoptosis. Investigating the function of CTTNBP2NL in thyroid cancer could, therefore, provide valuable evidence in molecular underpinnings.

TEA domain family member 1 (TEAD1) is another essential element in cancer biology that is involved in the Hippo signaling cascade.[15-20] TEAD1 functions as a transcription factor that governs genes that are important in cell growth, survival, and programmed cell death. Considering the significance of TEAD1 in regulating cellular activities, its interaction with other proteins, such as CTTNBP2NL, may be crucial in influencing apoptosis in thyroid cancer cells.

The potential regulatory relationship between CTTNBP2NL and TEAD1 in thyroid cancer apoptosis is a novel and intriguing field of study. Previous studies suggested that TEAD1 can influence cell growth-related genes, suggesting that it may interact with CTTNBP2NL to control cell apoptosis in thyroid cancer cells. Understanding how CTTNBP2NL and TEAD1 interact to regulate apoptosis could reveal new mechanisms of tumor suppression and identify targets for therapeutic intervention.

Analysis of the functions of CTTNBP2NL and TEAD1 in thyroid cancer has broad implications for cancer research. Apoptosis is a fundamental process in many types of cancer, and insights gained from thyroid cancer studies can be applied to other malignancies. Understanding the interactions among CTTNBP2NL, TEAD1, and apoptotic pathways could provide a framework for exploring similar mechanisms in other cancers, potentially leading to widespread therapeutic advances. Therefore, identifying the roles of CTTNBP2NL and TEAD1 in regulating thyroid cancer cell apoptosis represents a critical and promising area of research. In papillary thyroid cancer (PTC), the TEAD1-CTTNBP2NL axis could become a novel therapeutic target because manipulating this pathway may restore apoptotic processes and inhibit tumor growth. Translating these findings into clinical strategies poses several challenges, including the need for specific inhibitors or modulators of the TEAD1-CTTNBP2NL interaction. However, early evidence from preclinical models suggests that targeting this axis could significantly reduce tumor cell proliferation and enhance sensitivity to conventional therapies.

MATERIAL AND METHODS Cancer genome atlas database analysis

Data on gene expression levels, clinical parameters, and survival outcomes were extracted from the Cancer Genome Atlas (TCGA). Bioinformatics tools, namely, cBioPortal (https://www.cbioportal.org/) and GEPIA (https://gepia.cancer-pku.cn/) were employed to analyze the correlation between TEAD1 and CTTNBP2NL expression and their association with patient prognosis. Differential expression analysis was conducted to compare tumor tissues with normal thyroid tissues.

Cell culture

TPC1 cells were obtained from BeNa Culture Collection Company (BNCC337912, BeNa Culture Collection, Beijing, China) and used in STR testing and mycoplasma testing. The cells were grown in RPMI-1640 medium (11875-093, Gibco, Beijing, China), which was added with 1% penicillin-streptomycin solution (15140-122, Gibco, Beijing, China) to prevent bacterial contamination. The cultures were maintained at 37°C in a 5% CO2 incubator. For experimental setups, the cells were carefully plated at an optimal density on tissue culture-treated dishes and allowed to adhere overnight before further experimental treatments.

Colony formation assay

Cells were harvested through trypsinization (TrypsinEthylenediaminetetraacetic acid 0.25%, 25200-056, Gibco, Beijing, China), counted, and seeded into six-well plates (140675, Thermo Fisher Scientific, Beijing, China), with each well receiving 500 cells. The plates were then placed in an incubator at 37°C for 10–14 days until the emergence of visible colonies. The colonies were fixed in 4% paraformaldehyde (158127, Sigma–Aldrich, St. Louis, Beijing, China) for 15 min, rinsed with phosphate-buffered saline (PBS), and stained with 0.5% crystal violet for 30 min. After thorough washing with water and air-drying, the colonies were enumerated either manually or with the assistance of an automated colony counter (ZX-400 automatic colony counter, Ze xi biotechnology, Hangzhou, China).

Flow cytometry

According to the manufacturer’s guidelines of annexin V-allophycocyanin/propidium iodide (V-APC/PI) fluorescent double stain apoptosis detection kit (P-CA-207, Pricella, Wuhan, China), the cells were rinsed with PBS, resuspended in 100 µL of binding buffer, stained using PI and annexin V-APC dye, and added with 5 µL of APC and PI staining solution to each tube. The cells were incubated for 30 min at room temperature under light protection. After staining, the cells were washed before being resuspended in 500 µL of binding buffer for further analysis. Flow cytometry evaluation was conducted using BD FACSCanto II (BD Biosciences, San Jose, CA, USA). Data were processed through FlowJo software (Treestar FlowJo 10.10.0, Becton, Dickinson and Company, New York, USA), where precise gating strategies were employed to accurately identify and quantify various cell populations.

Dual-luciferase reporter assay

Cells were seeded in 24-well plates and transfected with 500 ng firefly luciferase reporter plasmid and 50 ng Renilla luciferase control plasmid using Lipofectamine 3000 (L3000001, Thermo Fisher). After transfection, cells were treated for 24–48 h, then lysed in 1X Passive Lysis Buffer (E1941, Promega). Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (E1910, Promega), with luminescence detected using the Glomax Multi+ Detection System (GM2000, Promega).

Chromatin immunoprecipitation (ChIP)

Cells were cross-linked with 1% formaldehyde for 10 min, and the reaction was quenched with 125 mM glycine for 5 min. Cells were then lysed in sodium dodecyl sulfate (SDS) buffer with protease inhibitors (11836170001, Roche). Chromatin was fragmented by sonication into 200–500 bp pieces. The chromatin was incubated overnight at 4°C with 2–5 µg specific antibodies (ab8580, Abcam) pre-bound to Protein A/G magnetic beads (88802, Thermo Fisher). Beads were washed with low-salt, high-salt, lithium chloride, and tris- ethylene diamine tetraacetic acid buffers. Chromatin was eluted, and cross-links reversed by heating at 65°C for 4 hours. DNA was purified using a PCR kit (DP214-02, Tiangen), and enriched regions were quantified by quantitative polymerase chain reaction (qPCR) and normalized to input DNA.

RNA extraction

Total RNA was isolated from TPC1 cells by utilizing the TRIzol reagent (15596018, Invitrogen, Beijing, China) following the protocol provided by the manufacturer. The cells were directly lysed in the culture dish by adding the TRIzol reagent, after which the lysate was thoroughly homogenized. Chloroform was then added to achieve phase separation. The aqueous phase containing RNA was meticulously transferred into a fresh tube. Subsequently, RNA was precipitated using isopropanol, washed with 75% ethanol, and dissolved in RNase-free water (10977015, Invitrogen, Carlsbad, USA).

qPCR

Reverse transcription of 1 µg of RNA into complementary DNA (cDNA) was conducted using the iScript cDNA synthesis kit (1708891, Bio-Rad, China) to efficiently convert RNA into cDNA and ensure high-quality templates for subsequent qPCR. SYBR Green Master Mix (CW3360H, Cwbio, Jiangsu, China) containing fluorescent dye was used for DNA detection. The mix was added to the reaction setup, which was then loaded into the StepOnePlus Real-Time PCR System (Applied Biosystems), which is known for its precision and accuracy in quantifying nucleic acids in real time. The following primers were used:

TEAD1 forward, 5’-TGGCTACTTCCTGGAAGACC-3’;

TEAD1 reverse, 5’-CCTTCTGCTGCTGTAGTCCT-3’;

CTTNBP2NL forward, 5’-AGGAAGGAGGAGGAGGAAGG-3’;

CTTNBP2NL reverse, 5’-CCTTCTGGGAGGAGGTAGTG-3’;

GAPDH forward, 5’-CAAGGTCATCCATGACAACTTTG-3’;

GAPDH reverse, 5’-GTCCACCACCCTGTTGCTGTAG-3’.

Cell transfection

To modulate the expression of TEAD1 and CTTNBP2NL, we used plasmid-based overexpression and small interfering RNA-mediated knockdown approaches. For TEAD1 overexpression, the cells were transfected with the pCMVTEAD1 plasmid (Plasmid 33109, Addgene, Watertown, MA, USA). The sequence information is shown in Table S1.

Cell proliferative capacity assay

Cell proliferation was assessed using the CCK-8 kit (CK04, Dojindo, Kumamoto, Japan) following the manufacturer’s protocol. Absorbance at 450 nm was recorded with a BioRad microplate reader (SpectraMax i3X, Molecular Devices, Shanghai, China) to determine the proliferation rate relative to the control group.

Protein extraction and western blot analysis

Protein was extracted using radioimmunoprecipitation assay buffer (89900, Thermo Fisher Scientific, Waltham, USA). The cells were lysed on ice to ensure that the proteins remained intact during extraction. The lysates were clarified by spinning them at 12,000 rpm for 15 min in a refrigerated centrifuge set to 4°C.

Equal amounts of protein (30 µg/sample) were loaded onto an SDS-polyacrylamide gel electrophoresis gel for electrophoresis to separate proteins by molecular weight. Following electrophoresis, the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Massachusetts, USA), which were selected for their high protein-binding capacity.

The membranes were blocked in a 5% non-fat milk solution prepared in tris-buffered saline (TBS-T) buffer. They were then incubated with primary antibodies specific to TEAD1 (diluted 1:1000, ab221367, Abcam), CTTNBP2NL (diluted 1:1000, 25523-1-AP, Proteintech), and loading control GAPDH (diluted 1:5000, ab8245, Abcam), BAX monoclonal antibody (diluted 1:1000, 50599-2-Ig, Proteintech), caspase 3/p17/p19 monoclonal antibody (diluted 1:1000,66470-2-Ig, Proteintech), P53 monoclonal antibody (diluted 1:1000, 60283-2-Ig, Proteintech), and human BCL2 polyclonal antibody (diluted 1:1000, 12789-1-AP, Proteintech). After thorough washing to remove unbound primary antibodies, the membranes were incubated with HRP-conjugated secondary antibodies (diluted 1:5000, ab205719, Abcam) for 1 h at room temperature.

The protein bands were detected using the Enhanced Chemiluminescence detection system from Thermo Fisher Scientific to visualize protein bands through a chemiluminescent reaction.

Statistical analysis

Each experiment was conducted in triplicate, unless specified otherwise. Data are expressed as mean ± standard deviation. Significance was assessed using Student’s t-test for comparisons between two groups. One-way analysis of variance followed by Tukey’s post hoc test was applied for analyzing differences across multiple groups. These analyses were performed using GraphPad Prism 9.0 software (9.0, GraphPad Software, San Diego, USA). P < 0.05 was deemed statistically significant.

RESULTS CTTNBP2NL plays a role in thyroid cancer

We utilized the TCGA database to evaluate the expression of CTTNBP2NL in tumor and normal tissues. High expression levels of CTTNBP2NL were observed in several cancers within the TCGA database, including thyroid cancer (THCA). Conversely, CTTNBP2NL exhibited low expression in many cancers, including breast invasive carcinoma [Figure 1a and Supplemental Figure 1].

Figure 1: CTTNBP2NL has function in thyroid cancer. (a) CTTNBP2NL was highly expressed in several cancers, including THCA. (b) PDIA3 expression was positively correlated with TMB in skin cutaneous melanoma but negatively correlated with TMB in cholangiocarcinoma, acute myeloid leukemia, lung squamous cell carcinoma, and thyroid cancer. (c) In thyroid cancer, CTTNBP2NL expression showed a strong positive correlation with the gene TEAD1, indicating a potential regulatory relationship. (d) CTTNBP2NL expression was positively correlated with immune cells in THCA, suggesting it may influence tumor development, prognosis, and treatment through immune cell interactions. ✶P < 0.05; ✶✶P < 0.01; ✶✶✶P < 0.001. CTTNBP2NL: Cortactin-binding protein 2 N-terminal-like, TMB: Tumor mutational burden, THCA: Thyroid carcinoma, TGCT: Testicular germ cell tumors, STAD: Stomach adenocarcinoma, SKCM: Skin cutaneous melanoma, SARC: Sarcoma, READ: Rectum adenocarcinoma, PRAD: Prostate adenocarcinoma, PCPG: Pheochromocytoma and paraganglioma, PAAD: Pancreatic adenocarcinoma, OV: Ovarian serous cystadenocarcinoma, MESO: Mesothelioma, LUSC: Lung squamous cell carcinoma, LUAD: Lung adenocarcinoma, LIHC: Liver hepatocellular carcinoma, LGG: Lower-grade glioma, LAML: Acute myeloid leukemia, KIRP: Kidney renal papillary cell carcinoma, KIRC: Kidney renal clear cell carcinoma, KICH: Kidney chromophobe, HNSC: Head and neck squamous cell carcinoma, GBM: Glioblastoma multiforme, ESCA: Esophageal carcinoma, DLBC: Diffuse large B-cell lymphoma, COAD: Colon adenocarcinoma, CHOL: Cholangiocarcinoma, CESC: Cervical squamous cell carcinoma and endocervical adenocarcinoma, BRCA: Breast invasive carcinoma, BLCA: Bladder urothelial carcinoma, ACC: Adrenocortical carcinoma, UVM: Uveal melanoma, UCS: Uterine carcinosarcoma, UCEC: Uterine corpus endometrial carcinoma, THYM: Thymoma.

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To determine the importance of CTTNBP2NL in predicting immune checkpoint inhibitors, we analyzed the association between the expression levels of CTTNBP2NL and two crucial cancer-related factors, namely, tumor mutational burden and microsatellite instability [Figure 1b].

To explore the regulatory role of CTTNBP2NL in THCA, we further evaluated genes correlated with CTTNBP2NL expression [Figure 1c]. TEAD1 was found to have a high Pearson correlation coefficient of 0.7 with CTTNBP2NL. To elucidate the interplay between CTTNBP2NL and the immune environment within tumors, we investigated the correlation between the expression levels of CTTNBP2NL and the infiltration of diverse immune cells [Figure 1d and Supplemental Figure 2]. The analysis accounted for the infiltration levels of several immune cell types with THCA.

Figure 2: Silencing CTTNBP2NL in TPC1 papillary thyroid carcinoma cells inhibits cell growth. (a) Silencing CTTNBP2NL in TPC1 papillary thyroid carcinoma cells led to a twofold reduction in protein levels. (b-d) This resulted in increased apoptosis and reduced cell proliferation and colony formation, as shown by flow cytometry, CCK8, and clonogenic assays. (e) Western blot detection of apoptosis marker. These findings highlight the crucial role of CTTNBP2NL in promoting cell survival and proliferation, suggesting it as a potential therapeutic target in PTC. ✶✶P < 0.01; ✶✶✶P < 0.001. CTTNBP2NL: Cortactin-binding protein 2 N-terminal-like, PTC: Papillary thyroid carcinoma.

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Silencing CTTNBP2NL inhibits TPC1 cell growth

In this study, we investigated the role of CTTNBP2NL in TPC1 cells by silencing its expression and evaluating the subsequent effects on cell behavior. We confirmed that silencing CTTNBP2NL significantly downregulated the protein expression by approximately twofold compared with the control group [Figure 2a].

Flow cytometry analysis was performed to assess the impact of CTTNBP2NL silencing on cell apoptosis [Figure 2b]. A marked increase in apoptosis in the CTTNBP2NL-silenced group compared with that in the control group indicates that CTTNBP2NL may play a protective role against apoptosis in TPC1 cells.

Further investigation into cell proliferation was conducted using CCK8 assay [Figure 2c]. Cell proliferation was significantly reduced in the CTTNBP2NL-silenced group relative to that in the control group, suggesting that CTTNBP2NL is critical for the proliferative capacity of TPC1 cells. A clonogenic assay was also performed to evaluate the long-term proliferative potential of TPC1 cells [Figure 2d]. The Western blot analysis results of apoptosis marker proteins BAX and Bcl-2 were consistent with those obtained from flow cytometry [Figure 2e]. The number of colonies formed by the CTTNBP2NL-silenced cells significantly decreased compared with that in the control group.

In summary, our findings indicate that CTTNBP2NL plays a crucial role in regulating apoptosis and proliferation in TPC1 cells.

Overexpression of CTTNBP2NL induces TPC1 cell growth

In this study, we aimed to elucidate the functional role of CTTNBP2NL in TPC1 cells by overexpressing the gene and examining the resultant cellular effects. Western blot analysis confirmed that the overexpression of CTTNBP2NL led to an approximately two-fold increase in its protein levels compared with that in the control group [Figure 3a].

Figure 3: Overexpression of CTTNBP2NL in TPC1 papillary thyroid carcinoma cells induced cell growth. (a) Overexpression of CTTNBP2NL in TPC1 papillary thyroid carcinoma cells resulted in a two-fold increase in protein levels. (b-d) Flow cytometry showed reduced apoptosis, while CCK8 and clonogenic assays demonstrated enhanced cell proliferation and colony formation. (e) Western blot detection of apoptosis marker. These findings suggest that CTTNBP2NL promotes cell growth and survival, making it a potential therapeutic target in PTC. ✶✶P < 0.01; ✶✶✶P < 0.001. CTTNBP2NL: Cortactin-binding protein 2 N-terminal-like, PTC: Papillary thyroid carcinoma.

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To determine the impact of CTTNBP2NL overexpression on apoptosis, we conducted a flow cytometry analysis [Figure 3b]. Apoptosis was significantly reduced in CTTNBP2NL-overexpressing cells compared with that in the control group. Hence, CTTNBP2NL may have an anti-apoptotic role in TPC1 cells and potentially contribute to cell survival.

We further evaluated cell proliferation using the CCK8 assay [Figure 3c]. Cell proliferation increased in the CTTNBP2NLoverexpressing group relative to that in the control group. This finding indicates that CTTNBP2NL is likely involved in promoting the proliferative capacity of TPC1 cells.

In addition, we performed a clonogenic assay to assess the long-term proliferative potential of TPC1 cells [Figure 3d]. The results of the Western blot analysis for apoptosis marker proteins BAX and Bcl-2 were consistent with those obtained from flow cytometry [Figure 3e]. The number of colonies formed by CTTNBP2NL-overexpressing cells significantly increased compared with that in the control group. This finding further supports the role of CTTNBP2NL in enhancing cell proliferation and survival in TPC1 cells.

In summary, our findings demonstrate that CTTNBP2NL overexpression in TPC1 cells leads to a reduction in apoptosis and a significant enhancement of cell proliferation and clonogenic potential.

TEAD1 regulates CTTNBP2NL expression in TPC1

We explored the regulatory relationship between TEAD1 and CTTNBP2NL in TPC1 cells. A dual-luciferase reporter assay was conducted to investigate whether TEAD1 directly influences the expression of CTTNBP2NL [Figure 4a]. The expression of CTTNBP2NL was significantly regulated by TEAD1. Specifically, the luciferase activity was higher in cells co-transfected with a TEAD1 expression vector and a CTTNBP2NL promoter-luciferase construct compared with that in the controls. This finding indicates that TEAD1 upregulates CTTNBP2NL transcription.

Figure 4: TEAD1 regulates CTTNBP2NLin TPC1. (a and b) TEAD1 regulates CTTNBP2NL expression. (a and b) TEAD1 regulates CTTNBP2NL expression. (c-e) In TPC1 papillary thyroid carcinoma cells, overexpression of TEAD1 combined with CTTNBP2NL silencing led to increased cell proliferation, reduced apoptosis, and enhanced clonogenic potential compared with CTTNBP2NL silencing alone. These results suggest that TEAD1 can compensate for the loss of CTTNBP2NL, highlighting its crucial role in cell survival and proliferation. ✶✶P < 0.01; ✶✶✶P < 0.001. CTTNBP2NL: Cortactin-binding protein 2 N-terminal-like, TEAD1: Transcriptional enhanced associate domain transcription factor 1.

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To further substantiate these findings, we performed ChIP followed by PCR (ChIP-PCR) to determine if TEAD1 physically binds to the promoter region of the CTTNBP2NL gene [Figure 4b]. The ChIP-PCR results confirmed the direct interaction between TEAD1 and the CTTNBP2NL promoter. DNA fragments immunoprecipitated with TEAD1 antibodies showed significant enrichment of the CTTNBP2NL promoter region compared with those immunoprecipitated with control immunoglobulin G antibodies. Hence, TEAD1 directly binds to the promoter of the CTTNBP2NL gene to regulate its transcription.

Given the previously observed roles of CTTNBP2NL in promoting cell proliferation and inhibiting apoptosis, understanding its regulation by TEAD1 could have significant implications for therapeutic strategies targeting TEAD1-CTTNBP2NL signaling in PTC. Future studies should focus on delineating the broader transcriptional network involving TEAD1 and CTTNBP2NL and exploring the potential of targeting this pathway to inhibit tumor growth and improve patient outcomes in PTC.

CTTNBP2NL-controlled TPC1 cell growth is regulated by TEAD1

In this study, we aimed to elucidate the interplay between TEAD1 and CTTNBP2NL in regulating cellular behavior in TPC1 cells. Specifically, we examined the effects of TEAD1 overexpression combined with CTTNBP2NL silencing on cell proliferation, apoptosis, and clonogenic potential.

To assess the impact on cell proliferation, we performed a CCK8 assay [Figure 4c]. The results demonstrated a notable increase in cell proliferation in the TEAD1 overexpression and CTTNBP2NL silencing groups compared with that in the CTTNBP2NL silencing alone group. This finding suggests that TEAD1 overexpression can partially rescue the proliferation deficit caused by CTTNBP2NL knockdown, highlighting a compensatory mechanism. We also evaluated apoptosis using flow cytometry [Figure 4d]. Apoptosis was significantly reduced in the TEAD1 overexpression and CTTNBP2NL silencing groups compared with that in the CTTNBP2NL silencing alone group. Hence, TEAD1 may exert anti-apoptotic effects even when CTTNBP2NL is silenced, suggesting that TEAD1 plays a crucial role in cell survival.

In addition, we performed a clonogenic assay to investigate the long-term proliferative capacity of the cells [Figure 4e]. The number of colonies formed by cells with TEAD1 overexpression and CTTNBP2NL silencing significantly increased compared with that formed by cells with CTTNBP2NL silencing only. This finding further supports the notion that TEAD1 can enhance clonogenic potential and promote cell survival and proliferation despite the absence of CTTNBP2NL.

In summary, our findings indicate that TEAD1 overexpression can compensate for the loss of CTTNBP2NL by promoting cell proliferation, reducing apoptosis, and enhancing clonogenic potential in TPC1 cells. These results suggest a complex regulatory relationship between TEAD1 and CTTNBP2NL. This understanding could open new avenues for therapeutic strategies targeting the TEAD1-CTTNBP2NL axis in PTC to disrupt tumor growth and improve patient outcomes.

DISCUSSION

This study investigated the relationship between TEAD1 and CTTNBP2NL in PTC cells by focusing on how TEAD1 overexpression and CTTNBP2NL silencing affect cellular behavior, including proliferation, apoptosis, and clonogenic potential. Our findings suggest a significant compensatory mechanism where TEAD1 mitigates the adverse effects of CTTNBP2NL loss, highlighting its critical role in promoting cell survival and growth.

The dual-luciferase reporter assay and ChIP-PCR experiments established that TEAD1 directly regulates CTTNBP2NL expression by binding to its promoter region. This regulatory interaction underscores the importance of TEAD1 in maintaining the transcriptional activity of CTTNBP2NL. However, when CTTNBP2NL is silenced, TEAD1 overexpression can compensate for this loss. The CCK8 assay results showed a notable increase in cell proliferation in cells with TEAD1 overexpression and CTTNBP2NL silencing compared with those with CTTNBP2NL silencing alone. Hence, TEAD1 can partially rescue the proliferation deficit, possibly through its role in activating alternative signaling pathways or target genes involved in cell cycle progression.

Flow cytometry analysis indicated a significant reduction in apoptosis in the TEAD1 overexpression and CTTNBP2NL silencing groups compared with that in the CTTNBP2NL silencing-only group. This finding reveals that TEAD1 possesses intrinsic anti-apoptotic properties independent of CTTNBP2NL. TEAD1 could activate downstream effectors that inhibit apoptotic pathways, including the upregulation of anti-apoptotic proteins or the suppression of proapoptotic factors. This anti-apoptotic role is crucial in cancer biology because it allows cancer cells to evade programmed cell death, thereby contributing to tumor progression and resistance to therapies.

The clonogenic assay further supported the role of TEAD1 in cell survival and proliferation. The increased number of colonies formed in the TEAD1 overexpression and CTTNBP2NL silencing groups compared with that in the CTTNBP2NL silencing alone group indicates that TEAD1 enhances the long-term proliferative capacity of PTC cells. This enhanced clonogenic potential suggests that TEAD1 not only supports immediate cell proliferation but also maintains the cells’ ability to proliferate over extended periods, which is a critical feature of cancer stem cells and tumorigenicity.

The interplay between TEAD1 and CTTNBP2NL opens new avenues for therapeutic interventions in PTC. Targeting the TEAD1-CTTNBP2NL axis could potentially disrupt the compensatory mechanisms that cancer cells employ to survive and proliferate.[21-23] For instance, inhibiting TEAD1 in conjunction with silencing CTTNBP2NL might significantly reduce cell viability and tumor growth because the cells would lose a primary survival signal and a compensatory pathway.

Understanding the broad network of genes regulated by TEAD1 could help identify additional therapeutic targets. TEAD1 interacts with various cofactors and participates in multiple signaling pathways, including the Hippo pathway, which plays a pivotal role in regulating cell proliferation and apoptosis.[24,25] Disrupting these interactions could provide a multifaceted approach to cancer therapy by potentially overcoming resistance mechanisms faced by single-target therapy.

In addition to the direct regulation of CTTNBP2NL expression by TEAD1, the compensatory effects observed on TEAD1 overexpression may involve alternative signaling pathways or factors. For instance, TEAD1 interacts with several effector proteins in the Hippo pathway, which plays a crucial role in regulating cell proliferation, migration, and apoptosis. TEAD1 may activate genes involved in cell cycle progression, anti-apoptotic responses, and metastasis through interaction with other transcription factors or co-activators. Moreover, TEAD1 could potentially cross-talk with classical signaling pathways, such as MAPK and PI3K/ Akt, which are integral to cancer cell growth and survival. Thus, beyond the TEAD1-CTTNBP2NL axis, other signaling networks associated with TEAD1 could provide additional therapeutic targets. Targeting these pathways in combination with TEAD1 inhibition might overcome the resistance mechanisms that often limit the effectiveness of single-target therapy, thereby offering a comprehensive approach to treatment. For example, concurrent inhibition of TEAD1 and disruption of the Hippo or PI3K/Akt pathways could effectively suppress tumor growth and induce apoptosis in cancer cells. Future studies should focus on exploring the specific roles of these alternative pathways and factors and evaluate their potential as therapeutic targets in clinical settings.

Further studies are warranted to explore the detailed molecular mechanisms underlying the TEAD1-CTTNBP2NL interaction. Downstream targets of TEAD1 that contribute to its compensatory effects should be identified by comprehensive transcriptomic analyses using RNA sequencing to pinpoint gene expression changes and proteomic profiling to uncover affected protein networks. Functional studies, such as gene knockdown or overexpression experiments, could validate the roles of these downstream targets in PTC cell survival and apoptosis. In addition, in vivo studies using orthotopic or transgenic animal models of PTC would be critical to confirm the therapeutic potential of modulating the TEAD1-CTTNBP2NL axis in reducing tumor growth and metastatic potential. These models could also facilitate the testing of small molecule inhibitors or gene-editing approaches, such as Clustered Regularly Interspaced Short Palindromic Repeats Cas9 (CRISPR/Cas9), targeting TEAD1 or CTTNBP2NL. Clinical studies should aim to evaluate the expression levels of TEAD1 and CTTNBP2NL in patients with PTC through immunohistochemistry or transcription analysis and correlate the findings with clinical outcomes such as tumor stage, recurrence, or resistance to therapy. Moreover, patient-derived xenograft models could provide an excellent platform to investigate the efficacy of personalized therapeutic approaches targeting the TEAD1-CTTNBP2NL pathway, paving the way for tailored treatments based on individual molecular profiles.

SUMMARY

Our study highlights the significant role of TEAD1 in compensating for the loss of CTTNBP2NL in PTC cells. TEAD1 promotes cell proliferation, reduces apoptosis, and enhances clonogenic potential, underscoring its critical role in sustaining cellular growth and survival. The TEAD1-CTTNBP2NL axis represents a promising therapeutic target in PTC. Further research into this regulatory network could lead to the development of novel treatments aimed at improving patient outcomes in PTC.