Deciphering the crucial role of IGF2BP3 in modulating stemness traits of salivary adenoid cystic carcinoma

Abstract

Salivary adenoid cystic carcinoma (SACC) is a rare yet clinically vexing malignancy characterized by perineural invasion, late lung-dominant metastasis, and limited systemic options, underscoring the need for mechanistic targets specific to SACC. This study employs a systematic experimental approach to elucidate the functional role and regulatory mechanisms of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) in SACC cells. Initial validation confirmed elevated IGF2BP3 expression in SACC tumor tissue, with knockdown of IGF2BP3 markedly suppressing cell proliferation, clonogenicity, and migratory capacity in SACC cells. Subsequent RNA sequencing analysis revealed the downregulation of stemness pathways upon IGF2BP3 knockdown, corroborating its regulatory role in SACC stemness. Leveraging single-cell sequencing data, we corroborated the association between IGF2BP3 and stemness traits within SACC cells. In vivo experiments further demonstrated that IGF2BP3 knockdown attenuated SACC tumor growth. In summary, this study provides a comprehensive understanding of IGF2BP3’s function and regulatory mechanisms in SACC, offering vital theoretical support for future targeted therapeutic strategies involving IGF2BP3.

Introduction

Salivary adenoid cystic carcinoma (SACC) stands as a rare yet markedly aggressive and highly recurrent malignancy of the head and neck region [1–3], accounting for approximately 5–10% of all head and neck tumors [4]. Despite some advancements in clinical interventions, SACC remains a formidable clinical challenge due to its inherent resistance to complete eradication, propensity for metastasis, and proclivity for relapse [5]. Consequently, deeper mechanistic investigation is urgently warranted to address the therapeutic impasse that continues to beset SACC management.

Cancer stem cells (CSCs), a distinct subset of cells characterized by their self-renewal and differentiation potential, are pivotal in critical oncological processes such as tumor initiation, progression, recurrence, and therapeutic resistance [6–8]. However, a comprehensive understanding of the regulatory mechanisms underlying CSCs and their precise roles in SACC development remains relatively scant. This is particularly true concerning the intricate interplay between the pathological biology of SACC and the presence and function of CSCs.

Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) is an RNA-binding protein that stabilizes and enhances the translation of target transcripts; across multiple cancers, it has been linked to tumor growth, metastasis, and therapy resistance [9–14], providing a rationale to examine its role in SACC. While IGF2BP3’s involvement in multiple cancer contexts has garnered substantial investigation, its functional repertoire and mechanistic roles in SACC have remained relatively underexplored. This holds particularly true for the intricate landscape of IGF2BP3 expression patterns, its distribution profiles, and its contribution to regulatory pathways that underpin SACC progression. Notably, in SACC, it remains unknown whether IGF2BP3 sustains CSC-like stemness via post-transcriptional regulation, which downstream programs are engaged, and whether these effects translate in vivo.

Through a comprehensive integration of high-throughput sequencing techniques and analytical approaches, our study endeavors to systematically investigate IGF2BP3 expression dynamics, its target signaling pathway, and its intricate connections with SACC development. By leveraging rigorous experimentation and meticulous data analysis, we aspire to unveil the mechanistic underpinnings of IGF2BP3 in SACC, thereby furnishing critical theoretical foundations to guide the future development of targeted therapeutic strategies tailored toward IGF2BP3 in the SACC milieu.

Materials and methods

Cell culture and treatment

Two established SACC cell lines (SACC-LM and SACC-83), obtained from the China Center for Type Culture Collection (Shanghai, China), were cultured in RPMI-1640 medium (GIBCO, RPMI 1640-C11875500BT), maintained at a controlled temperature of 37 °C, and an atmosphere containing 5% CO₂ in a dedicated cell culture incubator.

For our experimental design, we categorized the study into two pivotal groups: the knockdown group and the overexpression group. In the knockdown group, targeted silencing of the IGF2BP3 gene was achieved through transfection with specific IGF2BP3 shRNA sequences (shRNA-IGF2BP3-1: 5′-GACCAGCUUGUUUGGCUAUTT-3′, shRNA-IGF2BP3-2: 5′-GAAGCUAGCGAUUAGGUUUTT-3′). Conversely, full-length IGF2BP3 coding sequences were cloned in the pCMV vector (pCMV-IGF2BP3) for genetic enhancement. Both interventions were conducted using Lipofectamine™ 2000 (ThermoFisher, 11,668,019) according to the manufacturer’s guidelines.

Clinical samples

Tumor specimens from patients diagnosed with salivary adenoid cystic carcinoma were obtained at Shenzhen People’s Hospital. All procedures were approved by the Institutional Review Board of Shenzhen People’s Hospital, and written informed consent was obtained from all participants.

Cell proliferation assay

Cell counting kit-8 (CCK-8) reagent (DOJINDO) was used to comprehensively evaluate the cell proliferation ability. Cells were seeded in 96-well plates and cultured under standard conditions. Cell proliferation ability was evaluated by the CCK-8 assay (Dojindo, CCK8-500) according to a standard procedure. Briefly, 100 μL of a cell suspension containing 2 × 10³ cells was seeded into each well of a 96-well plate and cultured in a cell incubator. At equal time intervals (every 24 h), 10 μL of CCK-8 reagent was added to each well and cultured at 37 °C for 1 h. The absorbance of the cell culture medium was then read at 450 nm using an Infinite 200 PRO microplate reader. This procedure was repeated at 24, 48, 72, and 96 h after seeding to monitor changes in absorbance over time and provide insight into the proliferation dynamics of the cells.

Clonogenic assay

To evaluate the clonogenic potential of the cells, 500 cells per well were seeded uniformly on a 6-well plate containing growth medium. After 2 weeks of careful incubation, the cells were carefully fixed with methanol and then stained with 0.1% crystal violet solution. After staining, colonies were precisely quantified using ImageJ software to ensure accurate determination of colony numbers. Each experimental group was repeated in triplicate to ensure robustness and reliability of results.

Cell migration assay

Utilizing transwell cell migration chambers with 8 μm pore-size inserts (Corning), we performed the cell migration assay. The upper chamber, containing the cell suspension, was placed atop the lower chamber replete with culture medium supplemented with 10% fetal bovine serum. The transwell chambers were assembled in a 12-well plate format for the assay. Following 24 h, the upper chamber was removed. The migrated cells that adhered to the lower filter surface were fixed with ice-cold methanol, followed by crystal violet staining for visualization and quantification. The average number of cells was calculated based on random visual fields using ImageJ software for quantitative analysis.

RT-qPCR analysis

To investigate the gene expression changes induced by IGF2BP3 modulation, reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed. Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Subsequently, cDNA synthesis was performed using the RevertAid RT kit (ThermoFisher, K1691). Real-time quantitative PCR (qPCR) was performed using an Applied Biosystems StepOnePlus™ Real-Time PCR System. Amplification curves were analyzed to determine Ct (threshold cycle) values for each gene. The relative expression levels of IGF2BP3 were calculated using the comparative Ct method (2^−ΔΔCt), normalized to the expression of the reference gene GAPDH, and compared between experimental groups. The primer sequences used for RT-qPCR were as follows:

IGF2BP3 Forward Primer: 5′-AGCGTGGAGAAAGGACGAGA-3′

IGF2BP3 Reverse Primer: 5′-GGCCATCTTGGTGTCACTGA-3′

GAPDH Forward Primer: 5’-ACTCTTCCACCTTCGATGC-3′

GAPDH Reverse Primer: 5′-CCGTATTCATTGTCATACCAGG-3’.

RNA sequencing analysis

Total RNA extraction was performed on both the knockdown and control groups of cells. Following cellular lysis using Trizol reagent, total RNA was extracted, and subsequent assessment ensured the quality and concentration of the RNA. To gain insights into the transcriptional landscape, we subjected the extracted total RNA to ribosomal RNA (rRNA) depletion, followed by cDNA synthesis and library construction. For the high-throughput sequencing analysis, we used the Illumina sequencing platform. Indexed libraries were sequenced on an Illumina NovaSeq 6000 with paired-end 2 × 150 bp reads to ≥ 40 million read pairs per sample. Adapters and low-quality bases were trimmed with fastp v0.23; QC was summarized by FastQC. Reads were aligned to GRCh38 using STAR v2.7.10a.

Gene ontology (GO) and KEGG pathway analysis

In order to gain a deeper understanding of the functional implications of the differentially expressed genes identified through RNA-seq, we performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. For GO analysis, we employed the "clusterProfiler" R package, setting a significance threshold of adjusted p-value < 0.05 to identify the enriched biological processes, molecular functions, and cellular components associated with the differentially expressed genes. Additionally, for KEGG pathway analysis, we utilized the same package to uncover pathways that were significantly enriched based on the adjusted p-value threshold.

Gene set enrichment analysis (GSEA)

To further unravel enriched gene sets within our experimental data, we conducted gene set enrichment analysis (GSEA) using the GSEA software tool. This analysis was performed on pre-ranked gene lists, with the enrichment scores calculated using 1000 permutations. We applied a false discovery rate (FDR) correction to the p-values, and gene sets with FDR-corrected p-values < 0.05 were considered significantly enriched.

Single-cell sequencing data analysis

The SACC single-cell sequencing data were obtained from the publicly available gene expression omnibus (GEO) database (GSE217084) [15]. Matrices were analyzed in Seurat v5. Cells with nFeature_RNA < 200 or > 6,000, nCount_RNA > 50,000, or percent.mt > 15% were excluded and genes detected in < 3 cells were removed. Doublets were removed with DoubletFinder. Data were normalized with SCTransform. We computed principal component analysis (PCA) and uniform manifold approximation and projection (UMAP) for dimensionality reduction. Employing Seurat tools, we conducted a comprehensive analysis of the single-cell data to ascertain the expression patterns of IGF2BP3 within the SACC single-cell population. Based on their expression levels, cells were stratified into two distinct subgroups: IGF2BP3-positive and IGF2BP3-negative. Then DESeq2 was utilized to identify genes exhibiting significant expression disparities linked to stemness attributes.

Western blot

Total protein was extracted from cells in the knockdown and control groups. After cell collection, RIPA buffer (Beyotime, P0013D) supplemented with protease and phosphatase inhibitors was utilized for cell lysis and subsequent protein extraction. Extracted proteins were subjected to SDS-PAGE gel electrophoresis for separation, following which proteins were transferred onto polyacrylamide gel membranes. After the membrane transfer, the blocking step was implemented, followed by incubation with anti-IGF2BP3 (Proteintech, IGF2BP3), anti-SOX2 (Abcam, ab97959), anti-BMI1 (Cell Signaling Technology, 6964S), anti-NOTCH1 (Proteintech, 20687-1), and anti-GAPDH antibodies (Abcam, ab181602). Subsequently, secondary antibodies (Cell Signaling Technology, 7076S) were used for detection, with chemiluminescence employed to assess the expression of IGF2BP3.

Tumor sphere formation assay

Cells seeded in 6-well ultralow attachment plates at 3,000 cells/mL were cultivated in TSCM serum-free medium (Shanghai QiDa, P2401) specifically formulated to promote the enrichment of tumor stem cells. The formation of tumor spheres was monitored, and the quantification of tumor sphere count was carried out.

Xenograft tumor experiment

5 × 10⁶ SACC-LM cells, transfected with either control shRNA or IGF2BP3-targeting shRNA, were resuspended in 100 µL PBS and injected subcutaneously into the right flank of 6-week-old female BALB/c nude mice (n = 6 per group). Mice were housed in standard polystyrene cages under SPF conditions. Tumor growth was monitored every 3 days using calipers, and volume was determined using the formula: (length × width²)/2. After 2 weeks, the mice were euthanized, and tumors were excised using carbon dioxide (CO₂) asphyxiation. The tumors were then excised and weighed. All animal experiments were conducted in accordance with ethical guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) of Shenzhen People’s Hospital.

Immunohistochemistry

Tumors were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at 5 μm. After deparaffinization and rehydration, antigen retrieval was performed in citrate buffer pH 6.0. Endogenous peroxidase was quenched with 3% H₂O₂, and sections were blocked with Biocare blocking reagent. Primary antibodies were incubated overnight at 4 °C: Ki67 (Novus, NB500-170) and SOX2 (Abcam, ab97959). Slides were then incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibodies for 30 min at room temperature, treated with 3,3′‐diaminobenzidine and counterstained with hematoxylin.

Statistical analysis

For two-group comparisons, Student’s t-test was used (Welch’s correction applied when variances were unequal); for three or more groups, one-way ANOVA with Tukey’s post hoc test was employed. All tests were two-sided, and statistical significance was set at p < 0.05. Data are presented as mean ± s.e.m.; exact n and the test used are reported in the figure legends. Analyses and visualization were performed in GraphPad Prism v8.0.

Results

Knockdown of IGF2BP3 suppresses proliferation, clonogenic formation, and cellular migration of SACC cells

To investigate the expression level of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) in samples from patients with salivary adenoid cystic carcinoma (SACC), we collected surgical resection specimens (including tumor tissue and corresponding tumor-adjacent normal tissue) from three patients with salivary adenoid cystic carcinoma at Shenzhen People’s Hospital. Notably, we ascertained a pronounced upregulation of IGF2BP3 expression within tumor tissues compared to adjacent non-tumor tissues (Fig. 1A). In pursuit of unraveling the functional implications of IGF2BP3 in SACC, we adeptly employed a gene silencing strategy employing siRNA technology, effectively attenuating the expression of IGF2BP3 within the SACC cell line SACC-LM. Western blot and RT-qPCR analysis distinctly indicated a substantial reduction in IGF2BP3 expression within the knockdown group (Fig. 1B, C). In utilizing the CCK-8 assay, we definitively observed a significant attenuation in the proliferative rate of SACC cells within the IGF2BP3 knockdown group. After sustaining culture for a duration of 96 h, the absorbance values of cells within the IGF2BP3 knockdown cohort exhibited a marked reduction in comparison with the control group (Fig. 1D, n = 3, p < 0.05). Progressing further, we embarked on clonogenic formation assays, a robust evaluation of the influence of IGF2BP3 on the proliferative and clonogenic potential of SACC cells. Encouragingly, our findings incontrovertibly demonstrated a significant diminution in the number of colonies formed upon IGF2BP3 knockdown (Fig. 1E, F, n= 3, p < 0.05). Additionally, transwell cell migration assay results showed that IGF2BP3 knockdown substantially repressed the migratory capacity of SACC cells (Fig. 1G, H, n = 3, p < 0.05). Synthesizing these comprehensive findings, the attenuation of IGF2BP3 profoundly curbs the proliferative, clonogenic, and migratory capabilities of SACC cells.

Fig. 1.

IGF2BP3 modulation in SACC cells. A Western blot analysis of protein expression in tumor and adjacent tissue samples from three patients with SACC. B Validation of IGF2BP3 knockdown in SACC-LM cell line by Western blot. NC stands for negative control, while sh1 and sh2 represent different shRNA constructs used for IGF2BP3 knockdown. C qPCR validation of IGF2BP3 knockdown (n = 3). *p < 0.05 by one-way ANOVA with Tukey’s multiple comparison test. D CCK-8 assay showing cell proliferation upon IGF2BP3 knockdown (n = 4). *p < 0.05 by one-way analysis of variance, Dunnett’s test. E and F Colony formation assay results: representative images (E) and quantification (F) after IGF2BP3 knockdown (n = 3). *p < 0.05 by one-way ANOVA with Tukey’s multiple comparison test. G and H Cell migration assay results: representative images (G) and quantification (H) following IGF2BP3 knockdown (n = 3). *p < 0.05 by one-way ANOVA with Tukey’s multiple comparison test

IGF2BP3 overexpression enhances proliferation and migration abilities of SACC cells

To elucidate the role of IGF2BP3 in salivary adenoid cystic carcinoma (SACC) cells, we conducted IGF2BP3 overexpression experiments. Through transfection of SACC-83 cells, successful overexpression of IGF2BP3 was achieved. Western blot and RT-qPCR results demonstrated a significant upregulation of IGF2BP3 expression in the overexpression group compared to the control group (Fig. 2A, B). We employed cell proliferation assays to assess the impact of IGF2BP3 overexpression on SACC cell growth. CCK-8 assay results revealed a noticeable increase in cell proliferation in cells with IGF2BP3 overexpression (Fig. 2C, n = 3, p < 0.05). Furthermore, compared to the control group, the IGF2BP3 overexpression group exhibited a significant increase in colony numbers (Fig. 2D, E, n = 3, p < 0.05). The results from transwell cell migration experiments indicated that IGF2BP3 overexpression significantly augmented the migration capacity of SACC cells (Fig. 2F, G, n = 3, p < 0.05). Our findings suggest that IGF2BP3 overexpression promotes both proliferation and migration capabilities of SACC cells.

Fig. 2.

IGF2BP3 overexpression effects in SACC-83 cells. A Western blot validation of IGF2BP3 overexpression in SACC-83 cell line. B qPCR validation of IGF2BP3 overexpression (n = 3). *p < 0.05, Student’s t-test. C Cell proliferation assay using CCK-8, illustrating the impact of IGF2BP3 overexpression (n = 4). *p < 0.05 by one-way analysis of variance, Dunnett’s test. D and E Representative images (D) and statistical analysis (E) of colony formation assay after IGF2BP3 overexpression (n = 3). *p < 0.05, Student’s t-test. F and G Cell migration assay results: representative images (F) and corresponding quantification (G) upon IGF2BP3 overexpression (n = 3). *p < 0.05, Student’s t-test

Downregulation of IGF2BP3 leads to suppression of stemness pathways

In order to further elucidate the molecular mechanisms of IGF2BP3 in SACC, we conducted RNA sequencing (RNA-seq) and employed comprehensive analytical methods such as gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA) to analyze the gene expression changes upon IGF2BP3 knockdown. We initially performed RNA-seq on SACC cells from both the IGF2BP3 knockdown group and the control group. Differential expression analysis revealed significant alterations in gene expression levels within the IGF2BP3 knockdown group, with 22 genes upregulated and 43 genes downregulated (Fig. 3A). We subsequently subjected the downregulated differentially expressed genes to GO and KEGG analyses to further comprehend the impact of IGF2BP3 knockdown on the biological functions and pathways of SACC cells. The results indicated that the downregulated genes were primarily enriched in multiple functional categories and pathways related to stemness pathways, including cell differentiation, self-renewal of stem cells, and fate determination of stem cells (Fig. 3B, C). To comprehensively explore the influence of IGF2BP3 knockdown on SACC cells, we additionally conducted GSEA analysis to identify enriched gene sets associated with stemness pathways. The results revealed a significant increase in the enrichment of the stemness-related gene set in the IGF2BP3 knockdown group, further corroborating the regulatory role of IGF2BP3 in stemness pathways within SACC (Fig. 4D, E). In summary, we assert that the depletion of IGF2BP3 results in the attenuation of stemness pathways within SACC cells.

Fig. 3.

Suppression of stemness pathways upon IGF2BP3 downregulation. A Volcano plot illustrating the differential expressed genes (DEGs) in SACC-LM cells with IGF2BP3 knockdown compared to the control group using RNA-seq data. B GO-BP enrichment analysis of downregulated DEGs. C KEGG pathway enrichment analysis of downregulated DEGs. D Enrichment of the wnt beta catenin signaling pathway in RNA-seq results as determined by gene set enrichment analysis (GSEA). E Enrichment of the Notch signaling pathway in RNA-seq results by GSEA

Fig. 4.

Single-cell insights and expression patterns of IGF2BP3. A UMAP plot representing the cell types within the SACC single-cell dataset. B UMAP plot highlighting the expression distribution of IGF2BP3 in acinar cells. C Volcano plot displaying differential gene expression between IGF2BP3-positive and IGF2BP3-negative cells. D Enriched GO-BP terms associated with downregulated genes in IGF2BP3-negative cells. E Enriched KEGG pathways linked to downregulated genes in IGF2BP3-negative cells. F Violin plot showing expression levels of stemness markers in IGF2BP3-positive and IGF2BP3-negative cells

Validation of IGF2BP3’s association with stemness through single-cell sequencing analysis

In order to delve deeper into the correlation between IGF2BP3 and stemness pathways, we conducted an analysis utilizing an online dataset of single-cell sequencing for SACC (GSE217084). Through meticulous exploration of this dataset, we identified the expression pattern of IGF2BP3 within SACC cells, followed by a thorough investigation of the relationship between IGF2BP3 expression and the stemness characteristics of SACC cells. From the available online single-cell sequencing data, we observed predominant IGF2BP3 expression in acinar cell populations, which underscores its potential significance within SACC cells (Fig. 4A, B). To gain a more nuanced understanding of IGF2BP3’s expression pattern in SACC, we further stratified cells into IGF2BP3-positive and IGF2BP3-negative groups based on IGF2BP3 expression levels. Results unveiled a series of downregulated gene expressions within IGF2BP3-negative cells (Fig. 4C). To gain deeper insights into the functionality of these differentially expressed genes, we performed enrichment analyses using GO and KEGG annotations. In IGF2BP3-negative cells, the downregulated genes were notably enriched in stemness-associated pathways and functional categories, such as stem cell differentiation, self-renewal, and fate determination (Fig. 4D, E). To validate the relationship between IGF2BP3 and stemness characteristics, we employed violin plots to compare the expression levels of stemness marker genes in IGF2BP3-positive and IGF2BP3-negative cells. The results demonstrated a consistency between IGF2BP3 expression and the expression of stemness marker genes, thereby providing further support for the potential association of IGF2BP3 with stemness characteristics in SACC (Fig. 4F). In summary, at the single-cell level of SACC analysis, the expression of IGF2BP3 closely correlates with stemness attributes.

Regulation of SACC tumor stemness by IGF2BP3: in vivo validation and functional analysis

To validate the findings from earlier experiments and further elucidate the role of IGF2BP3 in the tumor stemness of SACC, we conducted a series of in vitro and in vivo experiments. First, we performed tumor sphere assays in SACC cells to assess the impact of IGF2BP3 on tumor stemness. The results revealed that IGF2BP3 knockdown significantly reduced the tumor sphere-forming ability of SACC cells (Fig. 5A, B, n = 3, p < 0.05). Moreover, we investigated the expression of stemness-associated marker genes in SACC cell lines to validate whether the effects of IGF2BP3 were correlated with tumor stemness characteristics. The findings demonstrated that IGF2BP3 knockdown led to a marked decrease in the expression of stemness marker genes in tumor tissues, while IGF2BP3 overexpression significantly enhanced the expression of these markers (Fig. 5C, D). This further confirmed the critical role of IGF2BP3 in regulating tumor stemness characteristics in SACC.

Fig. 5.

Impact of IGF2BP3 modulation on tumor stemness and in vivo tumorigenicity. A and B Representative images (A) and corresponding quantification (B) of tumor sphere formation in SACC-LM cells following IGF2BP3 knockdown and control conditions (n = 3). *p < 0.05 by Student’s t-test. C Western blot analysis depicting the expression levels of stemness markers in SACC-LM cells after IGF2BP3 knockdown and control treatments. D Western blot analysis revealing the expression levels of stemness markers in SACC-83 cells with IGF2BP3 overexpression and control conditions. E Macroscopic images of tumor xenografts generated by injecting IGF2BP3 knockdown and control SACC-LM cells into nude mice (n = 6). F and G Statistical analysis of tumor volume (F) and tumor weight (G) in nude mice carrying IGF2BP3 knockdown and control SACC-LM xenografts. *p < 0.05 by one-way analysis of variance, Dunnett’s test and Student’s t-test. H Representative IHC for Ki67 and SOX2 in control versus IGF2BP3-KD tumors. Scale bar, 50 μm

To verify the role of IGF2BP3 in SACC tumor growth, we conducted a nude mouse xenograft experiment. By transplanting IGF2BP3 knockdown and control SACC cells into nude mice, we observed a significant inhibition of tumor growth rate in the IGF2BP3 knockdown group (Fig. 5E, n  = 6). The tumor volume and mass were notably reduced in the IGF2BP3 knockdown group compared to the control, with statistically significant differences (Fig. 5F, G, p < 0.05). Immunohistochemistry on xenograft tumors showed a lower Ki67 index and SOX2 staining in IGF2BP3 knockdown versus control tumors (Fig. 5H). In summary, these results underscore the significance of IGF2BP3 in the regulation of SACC tumor stemness characteristics, as validated through in vivo experiments and functional analyses.

Discussion

IGF2BP3, as an RNA-binding protein, has garnered significant attention for its regulatory role across various tumor types [9, 10, 16, 17]. Prior studies consistently report elevated IGF2BP3 expression during tumor development and its association with adverse prognosis. In lung cancer investigations, the upregulation of IGF2BP3 correlates tightly with enhanced proliferation, migration, and treatment resistance of tumor cells [18]. Similarly, in breast cancer, heightened expression of IGF2BP3 is significantly linked to invasive growth and unfavorable prognosis [19]. These research findings further corroborate the observations in our study within the context of SACC: IGF2BP3 promotes processes such as cell proliferation, clonal formation, and migration.

Cancer stem cells play a pivotal role in the progression of tumors, and the relationship between IGF2BP3 and tumor stem cell characteristics has garnered significant attention [20–22]. In breast cancer, the upregulation of IGF2BP3 is closely associated with elevated expression of stem cell markers and the enhancement of cancer cell stemness features [23, 24]. In our study, the loss of IGF2BP3 was accompanied by the attenuation of stemness-related programs in bulk RNA sequencing, and single-cell analyses associated higher IGF2BP3 with elevated stemness signatures, together supporting a model in which IGF2BP3 contributes to stemness regulation in SACC. These converging results provide a rationale to probe the precise mechanisms by which IGF2BP3 modulates stemness-linked pathways.

Although our data underscore the importance of IGF2BP3 in SACC, the exact regulatory mechanisms remain to be defined. As an RNA-binding protein, IGF2BP3 may exert selective control over target genes through specific RNA sequence structures and interactions with other proteins [14, 25, 26]. This mechanism of action likely holds significant relevance in the development and transformation of tumors. For instance, in colorectal cancer, IGF2BP3 engages in interactions with the COPS7B protein, participating in gene regulation and subsequently influencing tumor cell proliferation and metastasis [27]. Future work integrating molecular biology (e.g., RIP-qPCR/eCLIP to map direct RNA targets; mRNA stability and polysome profiling to dissect decay vs. translation) with bioinformatics will help delineate the regulatory architecture of IGF2BP3 in SACC.

Furthermore, our study delved into the functional role of IGF2BP3 in SACC through animal experiments. Employing a nude mouse transplantation model, we validated the tumor-promoting effect of IGF2BP3, furnishing direct evidence of its biological impact within the in vivo context. Through these animal experiments, we corroborated the favorable impact of IGF2BP3 on SACC cell proliferation and tumor formation in vivo, thereby bolstering our findings from cellular experiments. These results strengthen the biological relevance of IGF2BP3 in SACC and motivate exploration of therapeutic strategies that perturb IGF2BP3 or its downstream programs.

Despite these advances, several limitations should be acknowledged. First, clinical validation is preliminary, and larger multi-center cohorts are needed to establish the prevalence, prognostic value, and stemness association of IGF2BP3 in SACC. Second, while our bulk and single-cell datasets suggest stemness-linked functions, we have not yet identified direct RNA targets or resolved the causal chain from binding to phenotype; in situ co-localization is also lacking. Future work will prioritize RIP-seq/eCLIP with RIP-qPCR validation, together with mRNA stability (actinomycin D) and translation assays (polysome/reporter), and multiplex IF/ISH or spatial transcriptomics in independent patient cohorts. Third, our cell line and xenograft models may not fully capture patient heterogeneity; patient-derived organoids and PDX models will improve translational fidelity. Finally, our in vivo endpoints focused on growth rather than hallmark features of SACC such as perineural invasion and distant metastasis; future studies will evaluate these dimensions in IGF2BP3-perturbed models.

Synthesizing the cumulative research findings with our experimental results, our comprehension of the multifaceted role of IGF2BP3 in SACC has grown more profound. While certain mechanisms and limitations continue to elude us, our research provides pivotal leads for unraveling the regulatory mechanisms of IGF2BP3, exploring its potential in clinical applications, and elucidating its associations with distinct tumor types and subtypes.

Author contributions

Hongliang Xie: Conceptualization, Methodology, Investigation, Formal analysis, Writing—Original Draft, and Funding acquisition. Lu Lu: Investigation, Data curation, Validation, and Writing—Review & Editing. Shiqin Wang: Resources, Visualization, and Project administration. Li Feng: Software, Validation, and Formal analysis. Bohan Li: Investigation and Methodology. Jianming Tang: Supervision. Guoquan Zhang: Conceptualization, Supervision, Writing—Review & Editing, and Funding acquisition. All authors have read and approved the final manuscript.

Funding

This study was funded by the Shenzhen Science and Technology Project (JCYJ20220530152406015).

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Conflicts of interest

The authors declare no competing interests.