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. 2025 Jul 14;77(4):146. doi: 10.1007/s10616-025-00814-7

SLC35F2 promotes the progression of NSCLC via regulating CREB1 expression

Du Wei 1, Ge Wenyu 2, Yu Ling 4, Chen Hongzhe 3, Wang Dongmei 4, Xu Xinglu 4,✉
PMCID: PMC12259521  PMID: 40667522

Abstract

SLC35F2 has emerged as a potential oncogenic driver in non-small cell lung cancer (NSCLC), yet its mechanistic role in tumor progression remains poorly understood. This study aimed to explore the mechanism of SLC35F2 in mediating non-small cell lung cancer (NSCLC) progression through the cAMP signaling pathway. By analyzing TCGA and GEPIA databases, the present research found that SLC35F2 expression was significantly elevated in NSCLC tissues compared to normal lung tissues, with high SLC35F2 levels correlating with poor patient prognosis (P < 0.05). Functional enrichment analysis using R language revealed significant alterations in multiple pathways, including cAMP signaling, in SLC35F2-high NSCLC. Experimental validation through RT-qPCR and Western blot confirmed upregulated SLC35F2 expression in NSCLC cell lines. Knockdown of SLC35F2 inhibited cell proliferation, migration, and invasion while promoting apoptosis (P < 0.05), as demonstrated by CCK-8, EdU, colony formation, flow cytometry, TUNEL, scratch, and Transwell assays. Mechanistically, SLC35F2 suppression activated the cAMP signaling pathway, particularly through upregulation of the transcription factor CREB1. These findings suggest that SLC35F2 drives NSCLC progression by modulating the cAMP/CREB1 axis, highlighting its potential as a therapeutic target.

Keywords: Non-small cell lung cancer (NSCLC), SLC35F2, CREB1, cAMP signaling pathway

Introduction

Currently, lung cancer, as one of the most common malignant tumors, poses a serious threat to human health (Bray et al. 2024; Li et al. 2023a). According to global cancer statistics, lung cancer has gradually become the leading cause of cancer-related deaths, with its incidence and mortality rates continuously rising. It ranks second in the incidence rate among various types of cancer and first in the mortality rate (Dela Cruz et al. 2011; Siegel et al. 2024). The majority of lung cancer patients are diagnosed with NSCLC, which is mainly divided into subtypes such as lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), and large-cell carcinoma (Nicholson et al. 2022; Js and Boyle 2022). Currently, the treatment strategies for NSCLC mainly include surgical resection, radiotherapy, chemotherapy, or combined therapies (Miller and Hanna 2021; Warth 2015). Although the treatment methods for lung cancer have been continuously improving, unfortunately, these interventions often fail to bring satisfactory results for patients. The overall survival prognosis of NSCLC patients still faces challenges, especially for those with late-stage diagnosis (Wu 2022; Ramnath et al. 2013). The main reasons for the poor prognosis of NSCLC patients include high tumor heterogeneity, easy metastasis, and resistance to treatment (chemotherapy, targeted therapy, immunotherapy) (Suhail et al. 2019; Frost and Reck 2024; Xiang et al. 2024). Therefore, in-depth research on the malignant biological characteristics and pathological molecular mechanisms of NSCLC is of great clinical significance for finding new, safe, and effective therapeutic targets.

SLC35F2 (Solute Carrier Family 35 Member F2) belongs to the nucleotide sugar- transferase family (Stankovic et al. 1997; He et al. 2009). It is one of the post-translational modifications of proteins and is closely related to carcinogenesis (Pan and Chen 2022). Protein glycosylation modification can affect the phosphorylation state of proteins, thereby regulating protein functions and signaling transduction pathways (Chang et al. 2020). Studies have shown that SLC35F2 is highly expressed in various cancers, such as breast cancer (Chandrasekaran et al. 2021), bladder cancer (Kotolloshi et al. 2021), and papillary thyroid cancer (He et al. 2018), and is positively correlated with tumor progression, affecting the biological characteristics of tumor cells, including proliferation, invasion, metastasis, and apoptosis (Liu et al. 2017). Previous studies have already demonstrated the tumor promoting effect of SLC35F2 in various cancers (He et al. 2018; Liu et al. 2017). However, until now, the specific mechanism of action of SLC35F2 in NSCLC has not been thoroughly studied. Regarding the specific functions of the SLC35F2 protein and its intracellular mechanism of action, further research and exploration may be required.

The cAMP signaling pathway is one of the widely studied and recognized important pathways and is considered to play an important role in regulating the biological processes of tumor cells (Chen et al. 2020; Kwiecinska et al. 2012; Jiang et al. 2020). cAMP regulates various physiological processes, including cell proliferation, metabolic regulation, and secretory function, by activating protein kinase A (PKA) and phosphorylating corresponding substrate proteins (such as CREB) (Taylor et al. 2013; Fujishita et al. 2022; Shan et al. 2023). According to the KEGG enrichment analysis results, the cAMP signaling pathway is significantly enriched in NSCLC with low expression of SL35F2. The activation of the cAMP signaling pathway in NSCLC may lead to the alleviation of tumor cell proliferation, migration, and invasion. SL35F2 may glycosylate the key proteins in the downstream pathway of cAMP, thereby increasing their phosphorylation levels (Li et al. 2023b). This indicates that SLC35F2 may participate in the occurrence and development of NSCLC by regulating the cAMP signaling pathway. Protein molecular docking experiments and immunoprecipitation (IP) experiments have also demonstrated that SLC35F2 can bind to CREB1, the key enzyme of cAMP. Therefore, this study aims to reveal the expression of SLC35F2 in NSCLC and its relationship with the survival prognosis of patients, and to explore the molecular mechanism of how SLC35F2 affects the biological behavior of NSCLC by regulating the cAMP signaling pathway. This will lay a theoretical foundation and provide new treatment strategies for the clinical treatment of NSCLC.

Materials and methods

Bioinformatics analysis

Functional enrichment analysis: to further confirm the potential biological functions of the target genes, DESeq2 was used to screen for differentially expressed genes (DEGs) in NSCLC, and KEGG pathway enrichment analysis was performed on the screened DEGs. The “ClusterProfiler” package in R was adopted.

Main materials and reagents

RPMI-1640 and DMEM media were purchased from Gibco. PBS was purchased from Solarbio. The rabbit anti-human monoclonal antibody of SLC35F2 was purchased from MERCK (China). The rabbit anti-human monoclonal antibodies of p -PKA, t-PKA, p-CREB1, and t-CREB1 were purchased from ABclona. The rabbit anti -human monoclonal antibody of GAPDH was purchased from Cell Signaling Technology (CST), China. The CCK-8 kit, EdU kit, and TUNEL kit were purchased from Beyotime, China. The short-hairpin RNA fragments sh-SLC35F2#1, sh- SLC35F2#2, and sh-SLC35F2#NC were completed by MERCK (China). Primer synthesis was completed by Sangon Biotech.

Cell culture

The cell lines used in this study mainly include three types of cell lines: human non-small-cell lung cancer cell lines (H1299, A549) and human normal lung epithelial cells (BEAS-2B), which were purchased from the Cell Bank of the Chinese Academy of Sciences in Shanghai. According to the instructions, BEAS-2B and H1299 cells were cultured in RPMI-1640 T25 culture flasks containing 10% FBS and placed in a cell incubator at 37 °C with 5% CO₂ for culture. A549 and H1299 cells were cultured in RPMI-1640 medium containing 10% FBS in a cell incubator at 37 °C with 5% CO₂.

Establishment of NSCLC cell lines with SLC35F2 knockdown

Before transfection, H1299 and A549 cells need to be digested and centrifuged, and then inoculated into six-well plates respectively. When the cell confluence reaches 70%-80%, transfection operation is performed. 48 h after transfection, the medium containing shRNA is discarded, the cells are washed with PBS 2–3 times, and then replaced with a complete medium. RNA is extracted for subsequent experimental analysis. After culturing the cells (H1299, A549) for 72 h, the cells are collected and proteins are extracted for subsequent experimental detection. The experimental groups include a negative control group (shNC) and two knockdown groups (shSLC35F2#1 and shSLC35F2#2). The sequenceS of sh-SLC35F2 are showing as following: shRNA#1, 5′-GCCTACCAGTACACAACTCTA-3′; SLC35F2 -shRNA#2, 5′-CCTGTACAGCTTCATGCCATT-3′; shRNA#NC,5'-TCTCCGAACGTGTCACGT-3′.

Western Blot analysis

After collecting the cells, lyse the cells with a mixed solution containing PMSF and RIPA protein lysis buffer (1:100). Measure the protein concentration according to the instructions of the BCA kit and perform boiling denaturation treatment. Separate the target proteins by SDS–PAGE electrophoresis and transfer them to a PVDF membrane. Block the membrane with skim milk on a shaker for 1 h. After washing with TBST, place the PVDF membrane in the primary antibody solution and incubate it overnight at 4 °C on a shaker. After washing with TBST, incubate it with the secondary antibody solution for 1 h. Finally, develop the washed PDVF membrane with ECL developer according to the instructions and analyze protein expression with Image J software. 1.6 Real-time Quantitative Fluorescence PCR Collect the cells in an Eppendorf (EP) centrifuge tube and extract the total RNA of the cells according to the instructions of the Trizol reagent. Perform reverse transcription reaction using the TAKARA reverse transcription kit. Conduct qPCR reaction using the TAKARA qPCR kit, with GAPDH as the internal reference. The relative expression level of the target gene is calculated by the 2−ΔΔCT method. The experimental design includes two groups: (1) comparison between normal cell lines and non-small-cell lung cancer tissues; (2) comparison between the empty vector group and the SLC35F2 knockdown group. The primers used for amplifying the coding sequence are as follows: SLC35F2 forward:5′-TGGGACTGGAAAATTGCCCT-3′, reverse:5′-TAGAGGTCCGCTGTCAGGAT-3′; GAPDH forward: 5′-GGTGTGAACCATGAGAAGTATGA-3′.

reverse: 5′-GAGTCCTTCCACGATACCAAAG-3′.

CCK 8 assay

After transfection treatment, the cells were digested, centrifuged, and collected, and then inoculated into a 96-well plate at a density of 5 × 103 cells/mL. The plate was placed in a cell incubator at 37 °C with 5% CO₂ for culture, with 6 replicate wells for each group. The viability of the cells within 0, 24, 48, and 72 h was detected. After adding the CCK-8 reagent to the transfection control group and the SLC35F2 knockout group and continuing incubation for 1–2 h, the absorbance was measured at a wavelength of 450 nm using a microplate reader, and statistical analysis was performed.

Transwell assay

To evaluate the migration and invasion potential of tumor cells, the transfected H1299 and A549 cells were divided into a transfection control group (Control) and an SLC35F2 knockout group (shRNA-SLC35F2) and inoculated into the upper chamber of the Transwell, allowing them to migrate through the membrane pores, and complete medium was added to the lower chamber. For invasion assay, an appropriate amount of Matrigel was added to the upper layer of the Transwell chamber. After 24 h, the migrated cells were fixed with methanol for 10 min and then washed three times with PBS. Subsequently, the membrane was stained with 0.5% crystal violet solution for 10 min, washed three times with PBS again, and the non-migrated cells on the upper side of the membrane surface were removed. Finally, pictures were taken using a bright-field microscope.

Colony—forming assay of cells

The cells were cultured in a cell incubator (37 °C, 5% CO₂) for 10–14 days. 1 × 103 cells were inoculated into each well of a six-well plate until visible cell colonies were formed. After removing the medium, the cells were first washed three times with PBS, fixed with 4% paraformaldehyde for 30 min, and stained with an appropriate amount of 0.1% crystal violet. After 30 min, the dye solution was washed off with deionized water, and the plate was dried in the air, and pictures were taken with a digital camera.

EdU incorporation assay for measuring cell proliferation

The transfected H1299 and A549 cells were inoculated in a six-well plate and cultured in an incubator until an appropriate density was reached. According to the instructions of the kit, the Click-iT EdU kit was used to perform EdU assays on H1299 and A549 cells. H1299 and A549 cells were incubated with 2 × EdU for 2 h. After EdU- labeled cells were completed, they were fixed with 1 ml of 4% paraformaldehyde at room temperature for 15 min, then permeabilized with Triton X-100 for 10–15 min, and incubated with 5001.12 Flow Cytometry for Detecting Cell Apoptosis.

After culturing the cells of the non-specific sequence-transfected group (Control) and the SLC35F2 knockout group (shSLC35F2) in a six-well plate for 48–72 h, the culture medium was discarded, and the adherent cells were washed with PBS 1–2 times. An appropriate amount of trypsin was added to digest the cells (H1299, A549). After incubation at room temperature for 2–3 min, an appropriate amount of complete medium was added to terminate the digestion. After mixing, the cells were transferred to a centrifuge tube and centrifuged at 2200 rpm for 5 min. The supernatant was discarded, and the cells were collected. The cells were resuspended in PBS, centrifuged at 2200 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in 1× Annexin V Binding Buffer. The cells were stained with Annexin V–APC/PI working solution in the dark at room temperature for 15 min. Finally, the samples were analyzed using a flow cytometer.

TUNEL assay for detecting cell apoptosis

The cells of the non-specific sequence-transfected group (Control) and the SLC35F2 knockout group (sh-SLC35F2) were inoculated in culture dishes and cultured in an incubator until an appropriate density was reached. The cells were fixed with 4% PFA at room temperature for 15 min and then permeabilized with Triton X-100 for 15 min. The cells were collected by centrifugation and washed twice with 200 μl PBS. Using the TUNEL kit, the TdT reaction buffer was coated on the cells according to the manufacturer's instructions and incubated at 37 °C for 30 min. Finally, the cells were stained with dUTP-labeled TdT dye in the dark for 30 min. Finally, pictures were taken under a fluorescence microscope, and the apoptosis situation was analyzed using Image J.

Wound-healing (cell scratch) assay

H1299 and A549 cells were cultured in cell culture dishes to form a monolayer. A straight line was scratched on the cell monolayer using a cell scraper or a 200 μl pipette tip to form a scratch model. The free cells were removed by washing with PBS 1–3 times. After adding serum free medium, the dishes were placed in a 37 °C, 5% CO₂ incubator for culture. The migration and filling of the cells at the scratch site were observed at 0, 6, 12, and 24 h, the images were recorded, and the data were analyzed using Image J software. The experiment was divided into two groups: non-specific knockout NC and shSLC35F2. The cells were stained with Hoechst 33342 and fixed for microscopic imaging.

Molecular docking

Rigid protein–protein docking of SLC35F2 and CREB1 was performed using GRAMM-X (http://gramm.compbio.ku.edu/) to study the relationship between them. The protein domains of SLC35F2 and CREB1 were obtained from the Protein Data Bank (PDB) database (http://www.rcsb.org/). Pymol (version 2.4) and PDBePISA (https://www.ebi.ac.uk/pdbe/pisa/) were utilized to study the protein–protein interactions and for further visual analysis.

Immunoprecipitation (IP)

Brifly, collect the lyse of cells in different groups using a lysis buffer (Beyotime Technology, Shanghai, China) containing protease inhibitors to prevent protein degradation. Centrifuge the lysate to clear and transfer the supernatant to a new tube. Next, add specific antibodies against SLC35F2 and p-CREB1 to separate reactions and incubate overnight at 4 °C, allowing the antibodies to bind to their respective target proteins. Subsequently, add protein A/G agarose beads to pull down the antibody-protein complexes, incubating for 1–2 h at 4 °C with gentle agitation. Afterward, wash the beads multiple times with lysis buffer to remove non-specific bindings. Finally, elute the bound proteins by adding an appropriate elution buffer or offloading the beads, and analyze the samples using Western blotting to confirm the combination relationship between SLC35F2 and p-CREB1.

Statistical analysis

Graphpad Prime 8.0.1 and Image J were used for data analysis and the production of statistical charts. All experiments were independently repeated three times, and the results are presented as the mean ± standard deviation [x(± s) (SD)]. The chi-square test (χ2) was used to evaluate the different expression levels of SLC35F2. Survival curves were drawn using Kaplan–Meier, and long-rank test was performed. For pairwise comparisons between two groups, Student’s two-tailed t-test was used. For multiple group comparisons, one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was performed. P < 0.05 was considered to be statistically significant.

Result

SLC35F2 is highly expressed in non-small cell lung cancer (NSCLC) and is associated with poor prognosis in patients.

Using the Timer 2.0 platform, we analyzed the expression of SLC35F2 across various cancers. The results revealed that SLC35F2 is highly expressed in the majority of tumors (Fig. 1A). Further analysis using the GEPIA platform and TCGA datasets showed that SLC35F2 expression levels in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) were significantly higher than in normal tissues (Fig. 1B–G, P < 0.05). Subsequently, we assessed the SLC35F2 mRNA and protein expression in normal lung epithelial cells (BEAS-2B) and NSCLC cell lines (H1299, A549) using qPCR and Western blot analyses. The results indicated that SLC35F2 was upregulated in NSCLC cell lines compared to BEAS-2B (Fig. 2J–L, P < 0.05).

Fig. 1.

Fig. 1

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Relationship between SLC35F2 expression and survival prognosis in non-small cell lung cancer patients. A The mRNA expression levels of SLC35F2 in pan-cancers; B, C GEPIA database analysis showed that the expression of SLC35F2 in NSCLC tumor tissues was significantly higher than that in paracancerous normal tissues; D, E Analysis of TCGA database data showed that the expression level of SLC35F2 was significantly higher in NSCLC patients; F–I Survival and prognosis analysis of NSCLC patients with different SLC35F2 expression levels showed that high expression levels were associated with poor survival prognosis. The log-rank test was used for comparison (P < 0.05). J Quantitative PCR (qPCR) assay revealed that the mRNA expression level of SLC35F2 was significantly up-regulated in H1299 and A549 cells compared with BEAS-2B cells; K, L Western blot analysis showed that the expression level of SLC35F2 protein in H1299 and A549 NSCLC cells was significantly higher than that in the normal lung cell line BEAS-2B. N = 6, data analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was performed

Fig. 2.

Fig. 2

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The correlation analysis between SLC35F2 expression and clinical features in NSCLC patients. A Heatmap showing the different distributions of clinical features between low expression of SLC35F2 and high expression of SLC35F2. The expression of SLC35F2 in NSCLC patients differed according to different clinical features, including (B) gender, C age, D T stage (one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test), E N stage (one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test), F M stage, G stage (one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test), H group; I area under the ROC curve (AUC) of SLC35F2 expression in NSCLC for evaluating its diagnostic value in the diagnosis of NSCLC; J, K accuracy of ROC curve assessment in predicting OS during 1-, 3-, and 5-year follow-ups. N = 6

Additionally, to investigate the relationship between SLC35F2 expression and clinical prognosis in patients, we performed a Kaplan–Meier analysis. The results showed that patients with high SLC35F2 expression had significantly worse prognoses, with consistent findings in both LUAD and LUSC analyses (Fig. 1G-J, P < 0.05). Based on the above evidence, SLC35F2 was selected as the candidate for further investigation.

The diagnostic value of SLC35F2 for patient prognosis in relation to clinical pathological features

To evaluate the impact of SLC35F2 expression on the clinical characteristics of non-small cell lung cancer (NSCLC) patients, we categorized patients into SLC35F2 low-expression and high-expression groups based on the median expression level of SLC35F2 (33.213; patients with expression levels greater than 33.213 were classified as the high-expression group, while those with levels less than 33.213 were classified as the low-expression group). We then conducted a correlation analysis of the clinical and pathological features of the two groups (Fig. 2A). Our findings revealed that SLC35F2 expression levels were significantly associated with tumor T staging and clinical staging, while showing no significant correlation with N staging, M staging, age, or gender (Fig. 2B–H).

To further assess whether SLC35F2 could serve as a prognostic indicator for patients, we performed a receiver operating characteristic (ROC) curve analysis to evaluate its diagnostic value. The results indicated that SLC35F2 had high predictive efficacy for distinguishing between normal and tumor patients (AUC = 0.93, Fig. 2I). We further integrated these clinical features to evaluate their accuracy in predicting overall survival (OS) during 1-year, 3-year, and 5-year follow-ups (see Fig. 2J–K). The results demonstrated that SLC35F2 has moderate predictive efficacy, with high accuracy in distinguishing tumor patients from normal individuals and moderate accuracy in predicting the prognosis of tumor patients.

Knocking down SLC35F2 inhibits the proliferation of NSCLC cells while promoting their apoptosis

Numerous studies have shown that SLC35F2 is aberrantly expressed in various tumors and is closely associated with tumor proliferation, invasion, and migration. To investigate the specific regulatory role of SLC35F2 in non-small cell lung cancer (NSCLC), we used shRNA (shSLC35F2) plasmids to knock down SLC35F2 in NSCLC cells and assessed the transfection efficiency using RT-PCR and Western blot experiments (Fig. 3A–F, one-way ANOVA with Tukey’s post-hoc test was performed, *P < 0.05, *P < 0.01 vs. Sh#ctrl group). The results indicated that silencing SLC35F2 significantly reduced both mRNA and protein expression levels of SLC35F2 (P < 0.05), confirming successful construction of the shRNA knockdown. The SLC35F2#1 plasmid demonstrated the highest knockdown efficiency and was used for subsequent experiments.

Fig. 3.

Fig. 3

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Effect of SLC35F2 knockdown on NSCLC proliferation and apoptosis. A–F The knockdown efficiency of SLC35F2 was detected by Western blot and real-time quantitative PCR (qPCR); G, H The proliferation of transfected H1299 and A549 cell lines detected by CCK 8; I–L The proliferative ability of H1299 and A549 determined by cell clone formation assay and Edu assay. M, N The apoptosis of H1299 and A549 cells detected by Tunel staining and flow cytometric assay. N = 6

We measured cell viability over a continuous 3-day period using the CCK-8 assay. The results showed that at 0, 24, 48, and 72 h, the number of viable cells in the sh-SLC35F2 group was significantly lower than that in the sh-NC group (Fig. 3G, H, P < 0.01, one-way ANOVA with Tukey’s post-hoc test was performed, *P < 0.05, *P < 0.01 vs. Sh#ctrl group). Colony formation assays further revealed that the clonogenic ability of SLC35F2 knockdown cells was significantly reduced compared to the control group (NC) (Fig. 3I, J, P < 0.01). Additionally, the EdU incorporation assay provided further confirmation that knocking down SLC35F2 inhibits cell proliferation (Fig. 3K, L, P < 0.01).

To gain deeper insight into the effect of SLC35F2 on NSCLC cell apoptosis, we conducted TUNEL assays and flow cytometry analysis. The experiments confirmed that knocking down SLC35F2 significantly induced apoptosis in non-small cell lung cancer cells (H1299, A549) (see Fig. 3M–P, P < 0.01).

Knockdown of SLC35F2 can inhibit the migration and invasion of NSCLC cells

Tumor invasion and metastasis are complex and continuous processes regulated at the molecular level by adhesion molecules, proteolytic enzymes, growth factors, and various angiogenic factors. It is essential to identify effective biomarkers for evaluating tumor development and metastatic behavior to predict cancer patient prognosis and improve treatment strategies. To further assess the impact of SLC35F2 knockdown on the invasion and metastasis of NSCLC cells, we utilized a wound healing assay to investigate the effect of SLC35F2 on cell migration. The results showed that, compared to control cells, knocking down SLC35F2 significantly inhibited the rate of wound closure (Fig. 4A, B). Next, we conducted invasion assays to evaluate the effect of SLC35F2 on cell invasion. Following SLC35F2 knockdown, the number of NSCLC cells (H1299, A549) that passed through the basement membrane of Transwell chambers was markedly reduced. When matrigel was added to the Transwell chambers for invasion analysis, crystal violet staining results revealed that the number of cells that could pass through the matrigel and the basement membrane was significantly decreased after SLC35F2 knockdown (Fig. 4C, D). ShRNA-mediated reduction of SLC35F2 expression resulted in weakened migration and invasion abilities of NSCLC cells (H1299, A549), indicating that knocking down SLC35F2 has an inhibitory effect on the invasion and migration of NSCLC cells.

Fig. 4.

Fig. 4

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Effect of silencing SLC35F2 on the migration and invasion of NSCLC. A, B The migration of H1299 and A549 cells after knockdown of SLC35F2; C, D The migration and invasion ability of H1299 and A549 cells detected by Transwell analysis

SLC35F2 participates in the progression of NSCLC by regulating the cAMP signaling pathway.

The cAMP signaling pathway has been associated with tumor growth, apoptosis, and other processes in many studies. To explore the role of SLC35F2 in the progression of NSCLC and the signaling pathways it regulates, we utilized the Cancer Genome Atlas (TCGA) dataset to conduct differential gene expression (DEGs) screening and performed GO and KEGG enrichment analyses on the DEGs. The results showed that the cAMP signaling pathway was significantly enriched in NSCLC with low SLC35F2 expression (Fig. 5A). For the downregulated SLC35F2, the biological process (BP) was primarily enriched in the G protein-coupled receptor signaling pathway regulated by adenylate cyclase. The cellular components (CC) mainly involved the apical part of the cell and the apical plasma membrane, while the molecular functions (MF) were primarily enriched for metal ion transmembrane transport proteins and hormone activity (Fig. 5B). To further investigate the specific impact of SLC35F2 on downstream proteins in the cAMP pathway, we analyzed key proteins in the cAMP signaling pathway of shSLC35F2 NSCLC cell lines using Western blotting. The results indicated that knocking down SLC35F2 promoted the expression of p-PKA and p-CREB1 in NSCLC cells (Fig. 5C–F). To validate the relationship between SLC35F2 and CREB1, we performed rigid protein docking analysis and identified binding sites between SLC35F2 and CREB1 (Fig. 5G). The immunoprecipitation (IP) experiment also verified the binding relationship between SLC35F2 and p-CREB1 within the NSCLC cells (Fig. 5H). These findings suggest that SLC35F2 regulates the cAMP signaling pathway, particularly through the activation of the transcription factor CREB1.

Fig. 5.

Fig. 5

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Molecular mechanism of SLC35F2 promoting NSCLC progression. A, B KEGG and GO enrichment analysis as performed to explore the key signaling pathways involved in SLC35F2; C–F The expression of key proteins in the cAMP signaling pathway was detected by Western blot to reveal the regulation of the cAMP signaling pathway by SLC35F2, data analyzed by one-way ANOVA with Tukey’s post-hoc test was performed, *P < 0.05, *P < 0.01 vs. Sh#ctrl group; G Surface diagram of the docking model and their interfacing residues between SLC35F2 and CREB1 protein (SLC35F2-yellow CREB1-blue hydrogen bond interaction-dotted line); (H) IP experiments verified the binding relationship between SLC35F2 and p-CREB1. N = 6

Discussion

With the widespread application of targeted therapies and immunotherapies in patients with advanced non-small cell lung cancer (NSCLC), significant progress has been made in the treatment of NSCLC, resulting in notable improvements in patient survival outcomes. However, several challenges remain: compared to small cell lung cancer, NSCLC grows more slowly and exhibits lower responsiveness to chemotherapy and radiation therapy, leading to generally unsatisfactory overall prognosis. Therefore, there is still a considerable amount of research dedicated to enhancing treatment efficacy and improving survival rates for NSCLC patients. Investigating key regulatory molecules and biomarkers in NSCLC is crucial for developing effective therapeutic strategies.

SLC35F2, identified as an oncogene, plays a significant role in tumor progression. However, the mechanisms underlying its abnormal expression in NSCLC remain unclear. Previously, only one article reported that the gene transcript levels of SLC35F2 are significantly correlated with pathological staging in non-small cell lung cancer (NSCLC). Our study demonstrates that SLC35F2 expression is significantly elevated in NSCLC and closely correlates with clinical staging and prognosis. This suggests that SLC35F2 may play an important role in the pathogenesis of NSCLC and influence the biological behaviors of NSCLC cells, such as proliferation, migration, invasion, and apoptosis, through the regulation of the cAMP signaling pathway. Mechanistically, we found that SLC35F2 knockdown activated the cAMP pathway, as evidenced by upregulated p-PKA and p-CREB1 expression (Fig. 5C–F). Molecular docking and IP experiments further revealed direct binding between SLC35F2 and CREB1 (Fig. 5G, H), suggesting that SLC35F2 may promote CREB1 phosphorylation by facilitating its glycosylation. These findings not only underscore the critical role of SLC35F2 in NSCLC tumorigenesis but also position it as a promising and actionable therapeutic target for the development of novel intervention strategies against NSCLC.

This research is the first to reveal the mechanistic role of SLC35F2 in NSCLC, further confirming the significance of the cAMP signaling pathway in this disease and highlighting SLC35F2's role as a regulatory factor. However, a limitation of our study is that it does not delve deeply into the specific mechanisms by which SLC35F2 regulates the cAMP signaling pathway, especially the key enzyme CREB1 and its role in mediating the biological characteristics of NSCLC. Future research should investigate the molecular mechanisms underlying the regulation of the cAMP signaling pathway by SLC35F2, further elucidating the relationship between SLC35F2 and the cAMP signaling pathway and revealing its exact regulatory mechanisms in the development of NSCLC.

There are several limitations that should be addressed. First, although we validated SLC35F2 function in vitro using NSCLC cell lines, the in vivo tumorigenesis model was not performed, limiting the translational significance. Second, the mechanism by which SLC35F2 regulates CREB1 glycosylation remains unclear, such as the exact catalytic sites requires biochemical validation. Third, while we observed moderate survival predictive efficacy of SLC35F2, the model lacks integration of other clinical biomarkers (such as EGFR mutation status), which may improve prognostic accuracy. Lastly, the study focuses on NSCLC as a whole, without dissecting subtype-specific roles of SLC35F2 in LUAD vs. LUSC.

In summary, our findings illuminate the significant role of SLC35F2 in NSCLC, providing new theoretical insights for the treatment of this disease. While SLC35F2 shows promise as a diagnostic biomarker (AUC = 0.936), its prognostic utility may be enhanced by integrating clinical parameters. The observed moderate survival prediction efficacy (AUC = 0.553–0.735) highlights the need for multi-omics approaches to develop comprehensive prognostic models. Additionally, exploring the heterogeneity of SLC35F2 expression and function within different NSCLC subtypes can offer valuable perspective for personalized therapy strategies, as well as pave the way for combination therapies that exploit the vulnerabilities created by targeting SLC35F2 and other key molecular players in the tumor microenvironment.

Author contributions

Du Wei: conceptualization, writing and editing and investigation. Ge Wenyu, Yu Ling, Chen Hongzhe, Wang Dongmei: investigation, methodology. Xu Xinglu: writing—reviewing, editing and providing the fund.

Funding

Research project of Heilongjiang Provincial Health Commission: 20211111000145.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Declarations

Consent for publication

Written informed consent for publication was obtained from all participants.

Competing interests

The authors declare that there is no conflict of interest in the manuscript.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

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