SRCIN1 promotes thyroid carcinoma growth by activating Wnt signaling pathway

Abstract

The adaptor protein SRCIN1 is a novel Src-binding protein that regulates Src activation through C-terminal Src kinase. SRCIN1 has been shown to promote the development of colorectal cancer, while acting as a tumor suppressor in breast cancer. However, its role in the development of thyroid cancer has remained unknown. In this study, we analyzed the biological characteristics of SRCIN1 in thyroid cancer using public data (HPA, TIMER, GEPIA and UALCAN). Additionally, we investigated the biological impact of SRCIN1 knockdown and overexpression on thyroid cancer cells both in vitro and in vivo. Our results revealed that the expression of SRCIN1 was higher (more than twice) in thyroid cancer tissues than in normal tissues and was positively correlated with tumor stage (stage1 vs. stage4 p < 0.05). Suppression of SRCIN1 promoted cell apoptosis (at least onefold) and inhibited cell proliferation and migration (at least 50%), whereas overexpression had the opposite effect. Furthermore, our analysis indicated that SRCIN1 activates the Wnt/β-catenin signaling axis, as evidenced by increased levels of β-catenin, DVL2, Cyclin D1, c-Myc, and Axin2. TOP/FOP experiments also revealed that Wnt/β-catenin signaling was activated by SRCIN1. It is noteworthy that the specific knockdown of SRCIN1 results in a reduction of key downstream gene targets within the Wnt/β-catenin axis, and tumors in mice treated with SRCIN1-targeting siRNA exhibit significantly smaller volumes (reduction exceeding 50%). Taken together, our study highlights the clinical and therapeutic relevance of SRCIN1 in thyroid cancer and suggests it as a potential therapeutic target for this disease.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-16674-2.

Introduction

Thyroid carcinoma (THCA) is the most prevalent malignant endocrine tumor globally, with a continuing increase in incidence. The estimated number of new cases of THCA was ranked fifth among cancer types in 2020¹, compared to sixth in 2019². The average age of patients with thyroid cancer is decreasing. In individuals aged 20–39 years, the incidence rate of thyroid cancer has been increasing by 3% annually³. THCA encompass various types, with papillary thyroid carcinoma (PTC) being the most common pathological type, accounting for approximately 80% of cases. Despite the rising incidence rate, the mortality rate remains stable over time^4–7. Due to regional variations and medical environments, it is estimated that between 50% (Japan, the Nordic countries, England, and Scotland) and 90% (South Korea) of newly diagnosed cases in women may be overdiagnosed⁵. Over the past decade, systematic screening, diagnosis, and targeted therapy have significantly improved clinical response and survival rates for cancer patients. Furthermore, molecular analysis not only enhances prognosis but also helps minimize treatment side effects^8,9.

Canonical Wnt signaling, also referred to as Wnt/β-catenin signaling, plays a crucial role in numerous physiological processes, such as embryonic development and tissue homeostasis. Dysregulation of this pathway has been associated with various human diseases, including multiple cancers^10,11. Activation of the Wnt pathway in tumor cells can lead to the stabilization of β-catenin and its translocation into the nucleus, where it serves as a coactivator for TCF-LEF transcription factors and regulates the expression of a range of genes, including AXIN2, DVL2, CCND1 (Cyclin D1), and c-Myc^12–14.

SRCIN1, an adaptor protein indirectly associated with BRCA1, has been demonstrated to play a role in integrin and epidermal growth factor (EGF)—dependent signal transduction¹⁵. It is encoded by SRCIN1, located on chromosome 17q12. One of its primary functions is to bind and activate C-terminal Src kinase (CSK), leading to SRC inhibition and downstream signaling with relevance to tumor characteristics¹⁶. Specifically, SRCIN1 stabilizes adhesion and inhibits EGF receptor (EGFR) and Ras signaling through dual control of SRC and Ras activity, thereby impacting critical tumor features such as growth and invasion¹⁷. However, several studies have demonstrated that SRCIN1 functions as a tumor suppressor protein, capable of disrupting the malignant nature of cancer and correlating with improved prognosis^16,18,19. This stands in stark contrast to its oncogenic role in colorectal carcinoma²⁰. Given the heterogeneity of SRCIN1’s function in tumors, elucidating its precise role in thyroid carcinoma is of paramount importance.

To investigate the pathogenesis of SRCIN1 in thyroid cancer, we conducted comprehensive research by integrating bioinformatics analysis, cell experiments, and animal studies. Utilizing online data, we analyzed the biological characteristics of SRCIN1 and its association with thyroid cancer. Through the generation of SRCIN1-enriched cells and subsequent in vitro and in vivo experiments, we observed that SRCIN1 enrichment significantly promotes cell proliferation and migration via the Wnt/β-catenin signaling axis as evidenced by TOP/FOP assay. Conversely, knockdown of SRCIN1 abolished these activities in both in vitro and in vivo models, underscoring the functional relevance of SRCIN1 in thyroid cancer and highlighting its potential as a therapeutic target for this disease.

Materials and methods

GEPIA, TIMER, UALCAN, and HPA analyses

Protein expression analysis of SRCIN1 in clinical datasets was performed using the HPA (https://www.proteinatlas.org/), TIMER (http://timer.cistrome.org/), GEPIA (http://gepia.cancer-pku.cn/), and UALCAN (http://ualcan.path.uab.edu/) databases.

Quantitative real-time PCR with reverse transcription

Total RNA was isolated using a TRIzol kit (Invitrogen). Reverse transcription was performed using the SuperScript III First-Strand Synthesis System for RT-PCR (Vazyme), according to the manufacturer’s instructions. Quantitative RT-PCR was performed using the Taq Pro Universal SYBR qPCR Master Mix (Vazyme) on the ABI PRISM 7500 system (Applied Biosystems, Waltham, MA, USA).

Cell culture and transfection

B-CPAP and TPC-1 human thyroid cancer cell lines were purchased from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). B-CPAP and TPC-1 cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; HyClone, GE Healthcare Life Sciences, Logan, UT, USA), 100 U/ml penicillin, and 100 mg/ml streptomycin, in a humidified atmosphere containing 5% CO 2 at 37 °C. Small interfering (si)RNA against SRCIN1 (si-SRCIN1-1; 5'-CAG AAC CTC TAT CCC TGT ATT-3'; si-SRCIN1-2; 5'-GCA GTA TTA TCA AGA TCT ACA-3'; si-SRCIN1-3; 5'-GAC TGA CTT CAA CAA GAG CGT-3') and siRNA negative control (si-NC; 5'-CCT AAG GTT AAG TCG CCC TCG-3') were obtained from Shanghai Saiheng Co., Ltd (Shanghai, China) and were transfected at a final concentration of 100 nM into B-CPAP and TPC-1 cells (1 × 10⁵ cells/well) in RPMI-1640 medium using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific Inc.) at room temperature according to the manufacturer’s protocol. Transfection efficiencies were determined in every experiment 48 h following transfection using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting.

Overexpression plasmids and transfection procedures

The sequences of human SRCIN1 (NM_025248.3) were amplified and cloned into the plasmid GV219 (Shanghai Genechem Co.,Ltd. , China). Constructs were verified by DNA sequencing. Cells were transfected with plasmids by LipofectamineTM 3000 (Invitrogen). The GenBank accession number of the plasmid is PV631213 in NCBI.

Cell viability assay

Cell viability was determined by CCK-8 assay to evaluate cell proliferation. Briefly, cells transfected with si-NC, si-SRCIN1, or OE-SRCIN1 were seeded into 96-well plates (3 × 10³ cells/well) in 100 μl medium. CCK-8 reagent was added to each well at 24 h, 48 h and 72 h by incubating at 37 °C. Optical density (OD) was measured at 450 nm using an enzyme-linked immunosorbent assay reader.

Migration assay

The migration properties of the cells were determined using Transwell chamber inserts (EMD Millipore). Briefly, 5 × 10⁴ transfected cells were added to the top chamber of the inserts and cultured in serum-free RPMI-1640 medium. The lower chamber was filled with RPMI‑1640 medium supplemented with 10% FBS as a chemoattractant. Following 48 h incubation, cells on the surface of the upper chamber were removed by scraping with a cotton swab, and the cells that had invaded the lower chamber of the filter were fixed with 70% ethanol for 30 min at room temperature and stained with 0.2% crystal violet for 10 min at room temperature. Photomicrographs of five randomly selected fields of fixed cells were captured, and cells were counted under a phase-contrast microscope (Olympus Corp.).

Apoptosis analysis

B-CPAP and TPC-1 cells (2 × 10⁵ cells) were seeded in 6‑well plates in RPMI‑1640 medium containing 10% FBS at 37 °C and then transfected with si-NC or si-SRCIN1 at 100 nM for 48 h. Subsequently, the cells were harvested by trypsin digestion, washed with PBS, and then processed according to the instructions of the apoptosis kit (V13242, Thermo Fisher Scientific, Inc.). Cell apoptosis was determined using an Annexin V Apoptosis Detection kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol using a BD FACSCanto flow cytometer (BD Biosciences, 2350 Qume and Drive, San Jose, CA 95,131 USA). The apoptosis ratio was calculated using the FlowJo software version 10.7.1 (BD Biosciences).

Western blot analysis

Cultured cells were collected, washed in ice-cold radioimmunoprecipitation assay buffer (JRDun Biotechnology, Co., Ltd.,Shanghai, China) containing a 0.01% protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA), and incubated on ice for 30 min. Protein concentration was determined using a bicinchoninic acid assay kit (Pierce; Thermo Fisher Scientific, Inc.). A total of 20 µg of protein was separated using 10% SDS-PAGE and then transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk for 2 h at room temperature to block nonspecific binding. The membranes were subsequently incubated overnight at 4 °C with polyclonal anti-SRCIN1 antibody (1:1,000 dilution; cat. no.55362–1-AP) and a monoclonal anti-GAPDH antibody (1:200,00 dilution; cat. no. 60004–1-Ig) (Proteintech). Membranes were then incubated with goat anti-mouse horseradish peroxidase-conjugated immunoglobulin G (1:10,000 dilution; cat. no. sc-2204; Santa Cruz Biotechnology,Inc.) at room temperature for 1 h. GAPDH was used as an internal control. Protein bands were visualized using the SuperSignal Chemiluminescence Detection ECL kit (Pierce; Thermo Fisher Scientific, Inc.).

In vivo xenograft assays

Mice were purchased from Shanghai Jiesijie Experimental Mobility Co., LTD. Six-week-old female nude mice were injected subcutaneously and intravenously with 4 × 10⁶ control and SRCIN1-enriched B-CPAP cells. The control group was divided into two groups, with three in each group. One group was injected with the control vector, and the other group was injected with SRCIN1 siRNA. The SRCIN1-enriched group also contained three mice. Mice injected subcutaneously were observed and given intra-tumor (50µL, 50 µM) Control and Ambion® In Vivo SRCIN1 siRNA (Thermo Fisher Scientific) injections post 2 weeks in 2 control groups respectively. Control and SRCIN1 siRNA injections were repeated twice a week for the next 4.5 weeks. Tumor sizes of in subcutaneously injected mice were measured twice a week on above time points. All observations were performed, updated, and discussed to avoid blinding on allocation/accessing the experimental outcome. This study was carried out in strict accordance with the recommendations of the Animal Experiment Committee, Safety and Environment Management Division, and Ethics Committee for Laboratory Animal Welfare of the Second Hospital of Shanxi Medical University. This study has been approved by the Ethics Committee of Shanxi Medical University, with the approval number DW2022064. All experimental protocols have been approved by Second Hospital of Shanxi Medical University Scientific Research Council. All methods are performed in accordance with relevant guidelines and regulations. The study was reported in full accordance with ARRIVE guidelines.

Instructions for Euthanasia: According to the American Veterinary Medical Association (AVMA) Guidelines for Animal Euthanasia, the nude mice at the end of the experiment were euthanized with carbon dioxide to ensure that the animals died quickly and without pain during carbon dioxide euthanasia. All animals were re-ventilated with CO2 for 2 min after cessation of respiration, and the animals were removed when respiratory arrest and eye color loss were observed, otherwise the animals should continue to be exposed to CO2.

Statistical analysis

All statistical analyses were performed using SPSS 25.0, R 3.5.2 (http://r-project.org/), and GraphPad Prism 8.0 (GraphPad Software, Inc.). Group differences were analyzed using the Wilcoxon test or Kruskal–Wallis test and expressed as mean ± standard deviation (SD). Correlation analysis was performed using the Pearson’s correlation coefficient. Statistical significance was set at p < 0.05. The comprehensive statistical analysis corresponding to Figure S2B-2D is presented in the supplementary materials under the designation "Figure S2 Statistical Data."

Results

SRCIN1 was overexpressed in thyroid papillary carcinoma tissues

The expression level of SRCIN1 in various cancers and the expression status of SRCIN1 in different cancer patients are shown in Figure S1A and S1B, respectively. The TIMER database shows that SRCIN1 is expressed at higher levels in various types of cancer than in normal tissues, such as thyroid cancer, breast cancer, and renal papillary cell carcinoma (Figure S1C). The UALCAN database indicated that the mean expression level of SRCIN1 in thyroid cancer tissues was approximately 11-fold higher than that in normal tissues (Figure S1D). Similarly, the GEPIA database also shows that the expression of SRCIN1 is significantly higher in thyroid cancer than in normal tissue (Figure S1E).

Expression of SRCIN1 and its relationship with clinicopathological characteristics

The HPA database revealed higher expression of SRCIN1 in cancer tissues compared to normal tissues (Figure S2A). Analysis of the UALCAN database showed a correlation between SRCIN1 expression and clinicopathological characteristics, indicating that patients with elevated levels of SCRIN1 had higher clinical stages (Figure S2B). Furthermore, further analysis of the UALCAN database indicated a positive correlation between protein expression of SRCIN1 and lymph node metastasis in thyroid cancer as well as patient age (Figure S2C-S2D). These findings provide additional evidence for the significant role of SRCIN1 in promoting the occurrence and progression of thyroid carcinoma.

Positive correlation between SRCIN1 and Wnt signal pathway

Previous research has established that the overexpression of SRCIN1 contributes to the proliferation and spread of colorectal cancer [20]. The canonical Wnt/β-catenin pathway plays a crucial role in the regulation of various types of cancer. We utilized the TIMER database to investigate the association between SRCIN1 and the Wnt/β-catenin signaling pathway. The findings from both TIMER and UALCAN databases indicated a positive correlation between SRCIN1 expression and DVL2, CCND1 (Cyclin D1), β-catenin (CTTNB1), CCNY, SMAD2, and c-Myc in THCA (Figure S3A-S3F). Given that these genes are closely linked to the Wnt/β-catenin signaling pathway, with some being target genes of this pathway, it can be concluded that there is a positive correlation between SRCIN1 and Wnt signaling.

SRCIN1 knockdown attenuated cancer cell growth and migration

Based on the previous analysis, we hypothesized that SRCIN1 plays an oncogenic role in the initiation and progression of thyroid cancer. Therefore, we conducted experiments to silence SRCIN1 expression in B-CPAP and TPC-1 cells using targeted siRNAs. The efficiency of SRCIN1 knockdown was determined by qPCR and western blotting (Fig. 1A). The results from CCK8 assays demonstrated that silencing SRCIN1 effectively inhibited the proliferation of thyroid cancer cells (Fig. 1B). Transwell assays showed a decrease in migration ability when SRCIN1 expression was reduced (Fig. 1C). Annexin-V/pi experiments revealed an increase in apoptosis rate in thyroid cancer cells with SRCIN1 knockdown compared to the control group (Fig. 1D). Furthermore, the parallel experiments of siRNA-4 also supported the above conclusion (Figure S4).

Fig. 1.

SRCIN1 knockdown attenuated cancer cell growth and migration. (A) Validation of SRCIN1 knockdown. (B) CCK8 test before and after knockdown in THCA cells. (C) Transwell assays before and after knockdown in THCA cells. (D) Apoptosis experiment before and after knockdown in THCA cells. All data were representative of at least three independent experiments (n = 3; error bar, SD).

SRCIN1 overexpression promotes thyroid cancer cell growth and migration

To further investigate the biological function of SRCIN1, we generated a plasmid for overexpressing SRCIN1. After 48 h of transfection, the efficiency of SRCIN1 overexpression was assessed using qPCR and western blotting (Fig. 2A). The CCK8 analysis results demonstrated that overexpression of SRCIN1 significantly enhanced the proliferation and clonogenicity of thyroid cancer cells (Fig. 2B). Transwell assays revealed that the migration ability of thyroid cancer cells was promoted upon overexpression of SRCIN1 (Fig. 2C). Furthermore, apoptosis in thyroid cancer cells was suppressed by SRCIN1 overexpression (Fig. 2D). Additionally, our experimental findings indicated that overexpression of SRCIN1 restored the biological function impaired by siRNAs (Fig. 2B–D).

Fig. 2.

SRCIN1 overexpression promotes thyroid cancer cell growth and migration. (A) Validation of SRCIN1 overexpression. (B) CCK8 test before and after overexpression in THCA cells. (C) Transwell assays before and after overexpression in THCA cells. (D) Apoptosis experiment before and after overexpression in THCA cells. All data were representative of at least three independent experiments (n = 3; error bar, SD).

SRCIN1 positively regulates Wnt/β-catenin signaling pathway in thyroid cancer

The canonical Wnt pathway regulates the proliferation, differentiation, migration, and invasion of thyroid cancer [20]. To investigate SRCIN1’s potential role in the canonical Wnt pathway, we assessed its impact on TOPFlash luciferase activity. Our findings indicate that overexpression of SRCIN1 enhances Wnt3a-induced reporter activity without affecting basal Wnt signaling (Fig. 3A). Consistent with this, knockdown of SRCIN1 resulted in decreased TOPFlash reporter activity (Fig. 3B). Furthermore, our qPCR analysis revealed that SRCIN1 knockdown negatively modulates the expression of Wnt target genes Axin2, c-Myc, CCND1, DVL2, and β-catenin (Fig. 3C), indicating a regulatory role for SRCIN1 in the Wnt/β-catenin signaling pathway. This was further supported by our observation that transfection of plasmids overexpressing SRCIN1 rescued the inhibitory effect of SRCIN1 knockdown on Wnt-promoting gene expression (Fig. 3C).

Fig. 3.

SRCIN1 promoted canonical Wnt signaling downstream of β-catenin accumulation. (A) TOPFlash luciferase activity after overexpression of SRCIN1. (B) TOPFlash luciferase activity after knockdown of SRCIN1. (C) SRCIN1 knockdown negatively modulates the expression of genes in Wnt/β-catenin signaling pathway.

SRCIN1 knockdown attenuated tumor growth in xenograft models

We conducted vivo xenograft assay to investigate the physiological relevance of SRCIN1-promoted thyroid cancer and its abrogation during suppression. Nude cells with B-CPAP were subcutaneously injected and treated with control and SRCIN1 siRNAs according to the treatment regime (Fig. 4A). On day 35, tumor size was observed, revealing that tumors administered intratumoral SRCIN1 siRNA injections harbored smaller-sized tumors than the control, while mice injected with SRCIN1-enriched cells had larger tumors (Fig. 4B). Tumor-weight analyses showed that SRCIN1 enrichment promoted tumor growth, whereas its knockdown led to suppression of their growth (Fig. 4A,B), with no difference in mouse body weight between mice administered intratumoral SRCIN1 siRNAs and control (Fig. 4C). HE staining images (magnification:200x) indicated a higher degree of tumor deterioration in the SRCIN1 enrichment group compared to the SRCIN1 knockdown group (Fig. 4D). These findings demonstrate the physiological relevance of SRCIN1 in tumor growth and its inhibition by SRCIN1 knockdown.

Fig. 4.

SRCIN1 knockdown attenuated tumor growth in xenograft models. (A) Flow chart of animal experiment. (B) Physical images of mice and tumors. (C) Tumor weight changes before and after knockdown or overexpression. (D) Tumor volume changes before and after knockdown or overexpression.

Discussion

In this study, initial analysis of public databases revealed a significant upregulation of SRCIN1 expression in thyroid carcinoma compared to normal thyroid tissues, suggesting its potential pivotal role in the pathogenesis and progression of thyroid cancer. Further analysis of its expression in relation to clinical tumor staging demonstrated that SRCIN1 levels were elevated across all stages (I-IV) compared to normal tissues, with higher expression observed in advanced stages (Stage IV) than in early stages (Stage I). Additionally, SRCIN1 expression was consistently higher than normal regardless of lymph node metastasis status or patient age, indicating its potential role in promoting thyroid cancer progression. To validate this hypothesis, gene editing and cellular functional assays demonstrated that SRCIN1 upregulation enhanced the proliferation and migration of B-CPAP and TPC-1 thyroid cancer cell lines. Animal experiments corroborated these findings. Mechanistic investigations revealed that amplified SRCIN1 in thyroid carcinoma was associated with increased expression of multiple markers in the Wnt/β-catenin signaling pathway, including DVL2, cyclin D1, c-Myc, Axin2, and β-catenin. Furthermore, TOP/FOP assays indicated that SRCIN1 overexpression activated the Wnt/β-catenin signaling pathway. Given the established association between Wnt signaling and tumorigenesis, this study provides preliminary evidence that SRCIN1 may promote thyroid cancer progression through the Wnt signaling pathway.

Previous research has demonstrated that overexpression of SRCIN1 in colorectal cancer can enhance the proliferation and invasion of colorectal cancer cells, as confirmed by cellular and animal experiments²⁰. While the study initially explored the relationship between SRCIN1 and the Wnt signaling pathway²⁰, there is currently no reported association between SRCIN1 and thyroid cancer. However, LU et al. and Grasso et al.^19,21. have documented that SRCIN1 suppresses the growth and invasion of breast cancer and neuroblastoma, respectively. The same gene may play completely opposite roles in different tumors or even in different subtypes of the same tumor, which may be attributed to the influence on different signaling pathways. This study identified SRCIN1 as a key factor in the progression of thyroid cancer and a promising candidate for targeted therapy.

In this study, although the functional validation of SRCIN1 in thyroid cancer has been conducted at both the cellular and animal levels, there are still some shortcomings. Firstly, it remains unclear whether SRCIN1 directly or indirectly regulates the Wnt signaling pathway. This requires first capturing the complexes of SRCIN1 with interacting proteins for mass spectrometry detection, then conducting in vivo and in vitro protein–protein interaction validations, and designing mutants to confirm the interaction domains. Secondly, the overexpression of some oncogenes can stimulate the senescence induced by the oncogenes themselves, which may lead to the situation that the overexpression cannot produce the opposite phenotype to that of knockdown. However, not all oncogene overexpressions will cause cellular senescence. This occurs under certain conditions, including activating related signaling pathways and reaching a certain level of overexpression. Thirdly, numerous previous studies have shown that many oncogenes are difficult to target (undruggable) precisely because of the lack of high-precision structural analysis. Currently, there are no reports on the structural analysis of SRCIN1, which might pose a serious challenge to its potential as a target. However, I also believe that with the continuous development of PROTAC technology, this problem will be solved in the future.

Conclusions

In conclusion, SRCIN1 is overexpressed in thyroid cancer and is closely associated with tumor stage, lymph node involvement, and patient age. SRCIN1 can modulate the growth of thyroid tumors through the Wnt/β-catenin signaling pathway and represents a potential target for therapeutic intervention.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

H.C.Y. conceived and designed the experiments, conducted the experiments, analyzed the data, prepared the charts, and completed the drafting of the paper.

Funding

The authors did not receive any financial support for this research.

Data availability

The datasets generated and/or analysed during the current study are available in the NCBI repository, accession nimber is PV631213.

Declarations

Competing interests

The authors declare no competing interests.