SuFu somatic mutations are recognized as novel markers for genomic instability and tumor growth in lung adenocarcinoma

Introduction

Lung cancer remains the most lethal malignancy globally. The International Agency for Research on Cancer (IARC) 2020 Global Cancer Report documented 2.2 million new cases (11.4% of total cancers) and 1.8 million deaths (18.0% of cancer mortality), underscoring its urgent status as a clinical and research priority (1). Among histological subtypes, lung adenocarcinoma (LUAD) has emerged as the predominant form, accounting for approximately 39% of male and 57% of female lung cancer cases (2,3). This gender disparity, coupled with LUAD’s characteristically insidious onset and aggressive progression, highlights the critical need to elucidate novel molecular mechanisms for earlier diagnosis and targeted therapeutic strategies (4).

The Hedgehog (Hh) signaling pathway is essential for embryonic development and tissue homeostasis (5-7). In adults, aberrant Hh pathway activation is mechanistically linked to diverse malignancies, including medulloblastoma, rhabdomyosarcoma, and basal cell carcinoma (8,9). Mounting evidence also implicates the Hh pathway in lung cancer pathogenesis (10-13). For instance, Hh signaling contributes to targeted drug resistance in LUAD, and the smoothened (SMO) inhibitor sonidegib has shown promise in LUAD treatment (14). The pathway is identified as a stemness regulator in small cell lung cancer, with its activation level positively correlated with tumor malignancy (15), driving progression and serving as a diagnostic and therapeutic target. Additionally, Hh signaling plays a pivotal role in lung cancer bone metastasis, and its inhibition can protect against bone destruction (16). Despite increasing recognition of the Hh pathway’s role in lung cancer progression, the specific regulatory mechanisms of its full-pathway effector proteins, particularly in LUAD, are incompletely understood.

Suppressor of fused (SuFu) is generally considered an inhibitory factor of the Hh pathway in most tumors, influencing prognosis through mutations (13). Although the pan-cancer incidence of SuFu mutations is relatively low (about 1.2%), they significantly increase the risk of developing medulloblastoma, gonadal tumors, and meningiomas by age 50, emphasizing their clinical relevance (17). Paradoxically, SuFu mutations could sometimes inhibit tumor development, as seen in basal cell carcinoma, where carriers exhibit lower incidence (18). The biological effects of SuFu mutations are complex, influenced by tissue-specific functions and crosstalk with other signaling pathways like mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), and wingless-type MMTV integration site family (WNT), and even modulation of vitamin D receptor activity affecting tumor cell metabolism (19-21). These interactions may contribute to SuFu’s context-dependent tumor-regulatory effects.

However, there is a lack of systematic reports on the regulatory role of SuFu in LUAD. Our study herein aimed to explore the impact of SuFu on LUAD, demonstrating its role as a tumor suppressor gene through multifaceted approaches including cell and animal experiments, clinical data analysis, and interacting protein studies. This work laid a theoretical foundation for further investigation into SuFu’s regulatory mechanism in LUAD. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-723/rc).

Methods

Cell culture

A549 and H1975 cell lines were provided by the Respiratory and Critical Care Medicine Department of The First Hospital Affiliated to Army Medical University. Cells were cultured in Dulbecco’s Modified Eagle Medium (C0226, Beyotime, Beijing, China) supplemented with 10% fetal bovine serum, penicillin G (100 U/mL), streptomycin (100 µg/mL), and mycoplasma prevention/clearance agent (0.2 µg/mL, C0292, Beyotime) at 37 ℃ under 5% CO₂.

Bioinformatics analysis

Gene expression data from the TCGA_LUAD dataset in the platform University of California Santa Cruz (UCSC, https://xena.ucsc.edu/) and the GSE32474 dataset in the platform of Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/gds) were analyzed. Lung epithelial cell data were obtained from the platform of Genotype-Tissue Expression (GTEx, https://www.gtexportal.org/). Differential gene expression and tumor mutational burden (TMB) analysis were performed in R4.3.2 using Limma, ggpubr, ggExtra, and ggplot2 packages. SuFu mutation analysis in 385 LUAD samples utilized Maftools and Dplyr packages. The survival analysis of SuFu mutations was conducted on the cBioPortal website (http://www.cbioportal.org/). This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Construction of lentiviral plasmids

A wild type (WT) SuFu-overexpression (OE) vector was donated by our research group. sgRNA targeting the SuFu exon (5'-CACCGGTGCAGACACCAACGATCTG-3') was designed using CRISPRDB (https://crisprdb.org/). Three mutant (MT) SuFu-OE vectors including substitution of arginine for cysteine at SuFu residue 123 (R123C), substitution of serine for phenylalanine at SuFu residue 377 (S377F), and substitution of threonine for methionine at SuFu residue 411 (T411M), were constructed from the WT SuFu vector using a site-specific mutagenesis kit (D0602S, Beyotime). Lentiviral particles were generated by co-transfecting these vectors with packaging plasmids into 293T cells, concentrated by ultracentrifugation. Target cells were infected with concentrated virus in the presence of Polybrene (C0351, Beyotime), followed by selection with puromycin (2 µg/mL for 72 h, then 1 µg/mL maintenance).

Transcriptome analysis and data processing

Cells were digested, collected, washed, and lysed in Trizol on ice for 15 minutes. Lysates were flash-frozen in liquid nitrogen and stored at −80 ℃. RNA sample quality control, transcriptome library construction, sequencing, and higher-level analysis were performed by Chongqing Life Know-Origin Technology Co., Ltd. (China). Differentially expressed genes (|Padj| ≤0.05, |Log₂foldchange| ≥1) were visualized using volcano plots.

Dual-luciferase assay

293T cells were co-transfected with Glioma associated oncogene homolog 1 (Gli1) plasmid and either one of the three MT SuFu plasmids or a control vector. After 8 hours, medium was replaced. After 48 hours total incubation, luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega, Madison, WI, USA).

Co-immunoprecipitation (Co-IP)

To assess binding strength between SuFu variants and Gli1, 293T cells were co-transfected with Gli1 and either WT SuFu (negative control) or one of the three MT SuFu constructs (R123C, S377F, T411M). After 24 hours, cells were lysed. Lysates were pre-cleared with IgG magnetic beads (P2173, Beyotime), then incubated overnight at 4 ℃ with anti-SuFu antibody (2522S, Cell Signaling Technology, Danvers, MA, USA) and protein A/G magnetic beads (P2108, Beyotime). Bound proteins were eluted by heating at 95 ℃ for 10 minutes. SuFu enrichment was detected by Western blotting.

Colony formation assay

A549 or H1975 cells (3×10³ cells/dish) were seeded in culture dishes. After 14 days, cells were washed with phosphate buffer saline (PBS), fixed with 4% paraformaldehyde (37 ℃, 30 min), and stained with crystal violet solution (15 min). Colonies containing >50 cells were counted under a microscope.

Cell viability assay

LUAD cells (2×10³ cells/well) were seeded in 96-well plates. After 5 days of culture (37 ℃, 5% CO₂), 10 µL of Cell Counting Kit-8 (CCK-8) solution (C0037, Beyotime) was added per well. After 1 hour incubation, absorbance (optical density at 450 nm) was measured to calculate cell viability.

Immunohistochemistry (IHC)

LUAD tissue microarrays were purchased from Shanghai Zhuoli Biotechnology Co., Ltd. (China), and the production was approved by its ethics committee (No. SHLLS-BA-22101102).

Paraffin-embedded tissue microarrays were deparaffinized, rehydrated, and subjected to antigen retrieval using Tris-EDTA buffer. Endogenous peroxidase was blocked with 3% H₂O₂ for 30 min. Sections were blocked with 10% normal goat serum (Boster Co., Ltd, Pleasanton, CA, USA) for 1 h at room temperature, then incubated overnight at 4 ℃ with rabbit anti-human SuFu antibody (1:400 dilution, Abclonal, Wuhan, China). After washing, sections were incubated with an anti-mouse/rabbit polymer kit (Envision Plus; Dako, Glostrup, Denmark) for 30 min at room temperature, followed by detection with Diaminobenzidine. Stained sections were imaged using a microscope. IHC scores were calculated based on the percentage of positive cells (area score) and staining intensity. The median score was used to distinguish high and low expression groups.

RNA extraction and real-time quantitative polymerase-chain reaction (RT-qPCR)

Total RNA was extracted from cells (about 80% confluency) using trypsin digestion, PBS washing, and centrifugation. Reverse transcription was performed using a kit (RR047Q, Takara, Kusatsu, Shiga, Japan) according to product instruction and a thermal cycler (C1000 Touch, Bio-Rad, Hercules, CA, USA). cDNA was stored at −20 ℃. RT-qPCR for Gli1 used primers as below: F: 5'-AGCCTTCAGCAATGCCAGTGAC-3' and R: 5'-GTCAGGACCATGCACTGTCTTG-3').

Western blotting

Western blotting was performed as previously described (22). Antibodies used: anti-SuFu (2522S, Cell Signaling Technology), anti-Vinculin (1:2,000, Cell Signaling Technology) as loading control. Bands were detected using a ChemiDoc Touch system (Bio-Rad) and analyzed using ImageJ software.

Mouse model

32 C-NKG mice (Cyagen Biosciences, Suzhou, China) aged 4–6 weeks, weighing 17–21 grams at the time of purchase, were randomly divided into 4 groups, with 8 mice in each group, half male and half female, were housed under specific pathogen-free conditions (4 mice per cage). The experiment began after 1 week of adaptation to the environment. All animal experiments were performed under a project license (No. AMUWEC20211915) granted by Laboratory Animal Welfare and Ethics Committee of Third Military Medical University, in compliance with institutional guidelines for the care and use of animals. A protocol was prepared before the study without registration. A549 cells (4.5×10⁶ cells) or H1975 cells (7×10⁶ cells) were injected into the left axillary region (n=8 per group). Tumor dimensions were measured with calipers every 5 days starting from day 5. Tumor volume (V) was calculated as V = (a×b²)/2 (a: maximum diameter, b: minimum diameter). Mice were sacrificed on day 15, and tumor weights were recorded.

Statistical analysis

Data were summarized using appropriate descriptive statistics. Statistical significance was assessed using GraphPad Prism 10 (San Diego, CA, USA) and SPSS (version 10) software (Armonk, NY, USA). Pearson’s chi-squared test, Fisher’s exact test and Student’s t-test were applied as appropriate. Overall survival (OS) of LUAD patients was analyzed using the Kaplan-Meier method with log-rank test for comparisons. All experiments were performed at least three times with triplicate samples.

Results

SuFu predicted improved prognosis in LUAD

To investigate the regulatory role of SuFu in LUAD, we analyzed a dataset of SuFu expression in LUAD. This analysis revealed a significant negative correlation between endogenous SuFu expression levels and TMB (Spearman’s ρ=−0.15, P=0.001) (Figure 1A). Furthermore, analysis of clinical data from 340 LUAD patients (23) demonstrated that high TMB status (threshold =10 mutations/Mb) was significantly associated with advanced node stage (N stage) and overall stage, indicating a positive correlation between high TMB and increased tumor malignancy (Figure 1B). Based on these findings, we hypothesized that SuFu exerted a tumor-suppressive effect in LUAD. Supporting this hypothesis, Kaplan-Meier analysis of 1,411 LUAD patients stratified by endogenous SuFu expression (high vs. low) showed that higher SuFu expression was significantly associated with prolonged OS [hazard ratio (HR), 0.73, P<0.001] (Figure 1C), suggesting the clinical significance of SuFu as a prognostic regulator in LUAD. To further validate this hypothesis, we performed IHC on 201 LUAD tissue sections to assess SuFu protein expression (Figure 1D). Two blind evaluators independently and objectively scored tissue sections. The Tables S1-S6 showed that among 201 samples, the lowest survival period was 1 month and the highest was 152 months. Additionally, the lowest staining score is 0.52, and the highest is 2.98. The 201 samples were dichotomized into high-level (n=100) and low-level groups (n=101) based on the median score of 1.65.Consistent with the bioinformatics findings, Kaplan-Meier survival analysis (Figure 1E) based on IHC staining intensity (low vs. high SuFu expression) also revealed that the HR of the SuFu high expression group was 0.469 (95% CI: 0.348–0.631), and higher SuFu expression significantly correlated with improved OS, P<0.001. In addition, Table 1 showed that in patients with LUAD, high SuFu expression is significantly associated with advanced T stage, N stage, M stage, and higher overall stage. Collectively, these results established SuFu as a clinically significant gene with a protective role in LUAD.

Figure 1 Regulatory role and prognostic value of SuFu in LUAD. (A) Analysis of UCSC and GEO datasets revealed a significant negative correlation between TMB and SuFu expression levels in LUAD (Spearman’s ρ=−0.15, P=0.001). (B) The patients with high TMB status had a significantly higher proportion of advanced N stage and overall stage compared to the low TMB group. (C) Kaplan-Meier Plotter database analysis indicated that higher endogenous SuFu expression levels were significantly associated with improved OS in LUAD patients. (D) Representative IHC images of SuFu staining in low-grade and high-grade LUAD sections (Scale bar =100 μm for main images; 50 μm for insets). (E) Kaplan-Meier survival curves for LUAD patients with high vs. low SuFu IHC score. GEO, Gene Expression Omnibus; HR, hazard ratio; IHC, immunohistochemistry; LUAD, lung adenocarcinoma; OS, overall survival; TMB, tumor mutational burden; UCSC, University of California Santa Cruz.

SuFu loss led to activation of oncogenic signaling pathways

To identify tumor signaling pathways functionally associated with SuFu in LUAD, we achieved SuFu knockout (KO) in A549 cells through Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein 9 (Figure 2A). Transcriptome sequencing of A549-sgRNA (SuFu-KO) and control (A549-Ctrl) cells was conducted, followed by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and differential gene expression visualization via volcano plot (Figure 2B,2C). KEGG analysis revealed that SuFu-KO significantly upregulated numerous tumor-associated signaling pathways with the Hh pathway emerging as a significant one. Given SuFu’s established role as a negative regulator within the canonical Hh pathway, where it inhibits Gli1 transcription, we specifically examined Gli1 expression. Consistent with this mechanism, differential gene analysis identified Gli1 as significantly upregulated following SuFu-KO. Subsequent RT-qPCR validation confirmed a 5.77-fold increase in Gli1 mRNA levels in A549-sgRNA cells compared to controls (P<0.001) (Figure 2D). These results demonstrated that SuFu suppressed Gli1 transcription in LUAD cells.

Figure 2 SuFu-KO activated the Hh pathway in LUAD cells. (A) SuFu protein level was significantly reduced in A549-sgRNA (SuFu-knockout) compared to the A549-sgCtrl group. (B) Cancer-related pathways, including the Hh pathway, represented the most significantly enriched KEGG terms. (C) Gli1 was identified as the most significantly upregulated gene among differentially expressed genes. (D) Gli1 mRNA expression was significantly elevated in the A549-sgRNA group versus the A549-Ctrl group. ***, P<0.001. FDR, false discovery rate; Hh, Hedgehog; KEGG, Kyoto Encyclopedia of Genes and Genomes; KO, knockout; LUAD, lung adenocarcinoma.

SuFu was associated increased gene mutation during progression of LUAD

Having established the functional link between SuFu and Gli1 in LUAD, we next investigated SuFu’s role in LUAD malignancy. Analysis of endogenous SuFu expression revealed no significant decrease in LUAD tumors (n=515) compared to normal lung tissue (n=59) (Figure 3A). This finding was corroborated by GEO data, showing comparable SuFu expression levels between LUAD (n=261) and normal tissues (n=58) (Figure 3B). These results suggested that SuFu promoted LUAD progression through genetic mutation rather than transcriptional downregulation. Kaplan-Meier analysis demonstrated significantly reduced OS in patients (Table S7 and https://cdn.amegroups.cn/static/public/10.21037jtd-2025-386-1.pdf) with SuFu mutations (altered group, n=22) versus those without (unaltered group, n=3,435) (HR =2.198, 95% CI: 0.877–5.509, P=0.01) (Figure 3C). While SuFu mutation frequency in LUAD was low (~1%), it carried significant clinical implications and increased TMB (Figure 3D). This identified SuFu mutation as a clinically significant prognostic risk factor in LUAD.

Figure 3 SuFu mutation drove the malignant progression of LUAD. (A) Analysis of TIMER2.0 data revealed no significant difference in SuFu expression between LUAD and normal lung tissues. (B) GEO platform analyses corroborated TIMER findings, showing comparable SuFu expression in LUAD and normal tissues. (C) Kaplan-Meier survival curves for LUAD patients with altered vs. unaltered SuFu from cBioPortal. (D) The mutation frequency of SuFu in 478 LUAD samples was approximately 1%. ns, P>0.05; *, P<0.05; **, P<0.01; ***, P<0.001. CI, confidence interval; GEO, Gene Expression Omnibus; HR, hazard ratio; LUAD, lung adenocarcinoma.

Mutated SuFu lost affinity to Gli1 with increased Gli1 transcription

Building upon our finding that SuFu-KO upregulates Gli1 transcription in LUAD, we investigated whether SuFu mutations exert similar effects. We generated three single-base MT SuFu plasmid vectors (R123C, S377F, T411M) via site-directed mutagenesis (Figure 4A). Dual-luciferase reporter assays in 293T cells demonstrated that while WT SuFu significantly suppressed Gli1 transcriptional activity, all kinds of MT SuFu lost this inhibitory capacity (Figure 4B). To determine whether impaired binding underlies this functional loss, Co-IP assays revealed reduced SuFu-Gli1 binding affinity for all mutants (Figure 4C,4D). Furthermore, we established H1975 cell lines stably expressing EGFP-tagged MT SuFu proteins. Immunoblotting confirmed equivalent MT-SuFu-EGFP expression (Figure 4E), while RT-qPCR showed significantly elevated Gli1 transcription in all MT SuFu lines versus WT controls (Figure 4F).

Figure 4 MT SuFu promoted Gli1 transcription and impaired SuFu-Gli1 binding. (A) MT SuFu vectors (R123C, S377F, T411M) were generated using site-directed mutagenesis. (B) Dual-luciferase reporter assays revealed significantly reduced suppression of Gli1 transcriptional activity by MT SuFu (R123C, S377F, T411M) compared to WT SuFu. ***P<0.001. (C,D) Co-IP assays demonstrated decreased binding affinity between MT SuFu and Gli1. (E) Immunoblot confirming expression of MT SuFu-EGFP fusion proteins. (F) RT-qPCR confirmed upregulated Gli1 mRNA in LUAD cells expressing MT SuFu versus WT SuFu. ns, P>0.05; *, P<0.05; **, P<0.01; ***, P<0.001. Co-IP, Co-immunoprecipitation; LUAD, lung adenocarcinoma; MT, mutant; RT-qPCR, real-time quantitative polymerase-chain reaction; WT, wild type.

WT SuFu inhibited the proliferation of LUAD cells in vitro and in vivo

To validate the tumor-suppressive role of SuFu in LUAD, we performed functional assays comparing WT SuFu-overexpressing cells with control cells. CCK-8 assays demonstrated significantly inhibited proliferation in WT SuFu-OE A549 cells versus controls (Figure 5A). Colony formation assays revealed a significant reduction in clone numbers in WT SuFu-OE groups compared to controls (Figure 5B,5C). Subcutaneous xenograft models using nude mice showed that tumors derived from WT SuFu-OE cells exhibited significantly reduced volume and weight compared to control tumors (Figure 5D,5E). Tables S8,S9 recorded the subcutaneous tumor formation results of A549/SuFu and H1975/SuFu and corresponding control groups. After 15 days, the median tumor volume of A549/SuFu group was 23.058 mm³ (vs. control group 70.948 mm³, P<0.001), and the median weight was 26.713 mg (vs. control group 87.200 mg, P<0.001), indicating that SuFu-OE could reduce tumor volume and weight of A549 by 67.500% and 69.366%, respectively. Additionally, the median tumor volume and weight of the H1975/SuFu group were 98.291 mm³ (vs. control group 179.321 mm³, P=0.01) and 111.938 mg (vs. control group 159.775 mg, P=0.049), indicating that SuFu-OE could reduce the tumor volume and weight of H1975 by 45.187% and 29.940%, respectively. Collectively, these results demonstrated that WT SuFu suppressed LUAD cell proliferation in vitro and tumor growth in vivo, confirming its tumor-suppressive function.

Figure 5 SuFu-OE suppressed LUAD cell proliferation. (A) CCK-8 assay demonstrated suppressed proliferation in SuFu-overexpressing (SuFu-OE) A549 cells versus controls. (B,C) Colony formation assays revealed reduced colony numbers in SuFu-OE groups after 14 days (crystal violet staining). (D,E) Xenograft studies showed significantly decreased tumor volume (D) and weight (E) in SuFu-OE groups compared to controls. *, P<0.05; **, P<0.01; ***, P<0.001. CCK-8, Cell Counting Kit-8; LUAD, lung adenocarcinoma; OE, overexpression.

Discussion

SuFu is generally regarded as a tumor suppressor gene, which increases cancer risk by relying on the canonical Hh pathway after loss of function (24). However, SuFu has been reported as a therapeutic target in colorectal cancer or pancreatic ductal adenocarcinoma (25). More importantly, SuFu has been reported to reduce the sensitivity of tumor cells to ferroptosis by regulating the Hippo/YAP pathway, thereby promoting tumor progression in breast cancer (26). Unfortunately, the underlying mechanisms by which SuFu functions in LUAD remain underreported in a systematic manner.

To investigate the regulatory effect of SuFu in LUAD, this study firstly searched relevant databases for LUAD and found a significant negative correlation between SuFu and TMB level in this cancer, as well as a marked inverse relationship with clinical N stage and overall stage (23). We speculated that SuFu might serve as a clinically significant regulatory gene for LUAD. To further clarify the direct relationship between SuFu and the survival of LUAD patients, we conducted an IHC experiment and collected clinical data corresponding to 201 samples. The Kaplan-Meier curve analyses confirmed that SuFu prolonged the OS of LUAD patients, and the integration and analysis of clinical data revealed a significant correlation between SuFu and T stage, N stage, M stage as well as overall stage. We knocked out SuFu in LUAD cells and found that many cancer-related signaling pathways were activated, among which the Hh signaling pathway attracted our attention. Subsequent RT-qPCR results showed that SuFu-KO could increase the transcription level of Gli1 in LUAD. This upregulation of Gli1 transcription level could be regarded as evidence that the Hh signaling pathway was activated and exerted a carcinogenic effect (27). Combined with the observation that WT SuFu-OE could inhibit the proliferative capacity of LUAD cells, these results further supported the conclusion that SuFu could act as a tumor suppressor gene in LUAD.

So far, we have determined that the regulatory effect of SuFu on LUAD tends to be tumor-suppressive. Tumor suppressor genes could typically participate in the malignant progression of tumors in two ways. One method involves weakening its anti-cancer regulatory effect by reducing transcription level (28). Another approach entails expressing new proteins through gene mutations, thereby generating new regulatory effects (29). To clarify the primary way in which SuFu contributes to malignant progression of LUAD, we searched TIMER and GEO databases, and the results showed that there was no significant change in the endogenous expression level of SuFu in LUAD compared to normal tissue. These results indicated that SuFu contributed to the malignant progression of LUAD via mutations.

We identified using the cBioportal website that the SuFu mutation had a tendency to reduce the OS of LUAD patients. Although the low sample size of the mutation group limited statistical efficacy, this result could at least provide a reference for exploratory research on the prognostic value of new biomarkers in future studies, and we looked forward to larger sample sizes to verify their exact regulatory effects. To further investigate the biological consequences of SuFu mutations, we constructed MT SuFu cell lines (R123C, S377F, T411M), before conducting dual-luciferase reporter assays and RT-qPCR analyses, which confirmed that MT SuFu enhanced the transcriptional activity of Gli1 and upregulated its expression levels. In addition, we performed Co-IP experiments to further demonstrate that MT SuFu decreased its binding affinity for Gli1, thereby attenuating the pro-oncogenic regulatory effect mediated by Gli1. This suggested that further investigation into SuFu mutations may hold significant clinical relevance.

Finally, we constructed a WT SuFu-OE LUAD cell line and conducted in vitro and in vivo functional experiments on its proliferation ability. The results showed that WT SuFu could significantly inhibit the proliferation ability of LUAD cells. We have previously reported the impact of SuFu on the invasive ability of LUAD, which was consistent with the anti-cancer effect of SuFu reported in this study (30). Based on these results, this study comprehensively demonstrated the completely opposite LUAD regulatory effects of WT SuFu and MT SuFu, laying a theoretical foundation for establishing the clinical value of SuFu.

Compared to previous analyses of SuFu in pan cancer, this study demonstrated some unique advantages. One was to focus on the contradictory regulatory effects of WT SuFu and MT SuFu in LUAD, filling the gap in clinical value research of SuFu in LUAD. The second was to reveal that the MT SuFu promoted the malignant progression of LUAD by upregulating Gli1, the driver gene of the Hh signaling pathway, which was a specific mechanism that had not been revealed in pan cancer analysis. The third was to demonstrate the regulatory effect of SuFu in LUAD from multiple dimensions such as cell experiments, in vivo experiments, SuFu-OE experiments, SuFu-KO experiments, and clinical data analysis, which was a resolution that was difficult to achieve in pan cancer analysis. To guide future research to fully consider the potential functional connections between SuFu and other LUAD driver genes, we shared partial RNA sequencing data of A549-SuFu-KO cells in Table S10.

Despite this, there are certain limitations in this study. The low incidence of mutations in LUAD by SuFu slightly limited its broad applicability as a novel biomarker, but it still showed a high level of attractiveness in some research directions of precision medicine for tumors. Firstly, it may be involved in the formation of resistance to novel targeted drugs such as sunvozertinib (31). Furthermore, it may form a combined biomarker with other LUAD driver genes or immune checkpoints to enhance clinical applicability (32). Lastly, further studies should focus on familial genetic predisposition and specific clinical features (e.g., smoking, advanced age) to enrich the higher incidence of SuFu in the population through targeted cohort studies. The multidimensional experimental results of this study aimed to establish the clinical value of SuFu in advance and lay a theoretical foundation for future research on transforming SuFu from a basic research molecule to a clinically available diagnostic marker and therapeutic target.

Conclusions

In summary, this study innovatively demonstrated that SuFu functioned as a tumor suppressor gene in LUAD from multiple perspectives, explained the carcinogenic effect of rare mutations in SuFu, and enhanced the understanding of SuFu’s regulatory role in this malignancy, providing a theoretical foundation for exploring novel targets related to SuFu in future studies.

Acknowledgments

We would like to express our gratitude to Beijing Tsingke Biotech Co., Ltd. for providing technical guidance on vector design and construction, and to Hangzhou PTM BioLab, Inc. (China) for guiding the data analysis of Co-IP experimental results.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-723/rc

Data Sharing Statement: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-723/dss

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-723/prf

Funding: This work was supported by The National Key Research and Development Program of China (2022YFA1104303), National Major Science and Technology Program for Four Major Chronic Diseases (2024ZD0533100).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-723/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. All animal experiments were performed under a project license (No. AMUWEC20211915) granted by Laboratory Animal Welfare and Ethics Committee of Third Military Medical University, in compliance with institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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