Introduction

Cancer is a significant challenge that poses a threat to human life and health. The incidence and mortality rates are escalating year by year, and it is anticipated that the global cancer burden will increase by 50% by 20401. With the continuous advancement of medicine and scientific research, an increasing number of treatment approaches and factors influencing cancer progression have been uncovered. Currently, surgery, radiotherapy, and chemotherapy are conventional treatments2,3,4. while targeted therapy, photodynamic therapy, and photothermal therapy are also being developed and applied5,6. Tumor immunotherapy has offered new hope for cancer treatment. Immune checkpoint inhibitors (ICIs), such as antagonists of PD1, PD-L1, CTLA4, and LAG-3, have been successfully utilized in the treatment of various cancers and have achieved remarkable efficacy7,8,9. Nevertheless, only a small proportion of cancer patients respond to immune therapy, and immunotherapy resistance exists in the majority of cancer types10,11,12. Due to the complexity of the mechanisms of Tumor Microenvironment (TME) formation and alteration, the identification of cancer prognostic genes and immune biomarkers associated with TME can assist in overcoming the limitations of immunotherapy13. However, the intricate mechanisms regulating the formation and dynamics of TME remain unclear. Therefore, the identification of novel human cancer prognostic and TME-related genes and immune biomarkers will facilitate overcoming the shortcomings of tumor immunotherapy.

The Solute carrier family 2 (SLC2) gene encodes the GLUT protein, which pertains to major facilitator superfamily (MFS). SLC2A6, a member of this family, is governed by the NF-kB signaling pathway and participates in various disease processes14. An increasing number of studies have discovered that SLC2A6 is implicated in the occurrence and development of multiple cancers. SLC2A6 serves as a poor prognostic factor for disease-specific survival and progression-free survival in prostate cancer patients15. SLC2A6 influences the disease course by affecting insulin secretion in genetically obese mice16. Knockout of YBX1 in triple-negative breast cancer impacts tumor progression by influencing the expression of genes such as SLC2A617. Aberrant expression of SLC2A6 promotes the occurrence and advancement of endometrial cancer18. Members of the SLC2 family, such as SLC2A3, SLC2A6, SLC2A9, and other genes, are involved in the genesis of lung adenocarcinoma through the regulation of immune cells19. Slc2a6 affects muscle cell differentiation via the SLC2A6-LDHB axis and thereby influences the progression of diabetes20. Research indicates that SLC2A6 genes increase in gram-negative lymphocele virus infection of the tissue and cell and are regulated by the NF—kB signaling pathway to control the inflammation virus14. Nevertheless, the association between SLC2A6 and cancer immune responses remains insufficiently investigated; thus, a more profound analysis of the function of SLC2A6 in cancer is necessary.

In this study, a comprehensive pan-cancer analysis of SLC2A6 was conducted in terms of gene expression, genomic alterations, prognosis, immune checkpoints and immunotherapy, immune infiltration, and drug sensitivity through bioinformatics approaches. Additionally, in vitro and in vivo experiments further substantiated the role of SLC2A6 in HCC.

Materials and methods

Gene expression analysis of SLC2A6 in pan carcinoma tissues

The Tumor Immune Estimation Resource (TIMER) database is a network for analyzing tumor immune cell infiltration. It can not only analyze the infiltration level of tumor immune cells but also the differential gene expression between tumor tissue and normal tissue. As some tumors in the Cancer Genome Atlas (TCGA) lack normal tissue, RNA Sequencing (RNA-seq) data for 33 cancers were obtained from the TCGA database and the Genotypic Tissue Expression (GTEx) database. Additionally, the Human protein Atlas (HPA) database was utilized to study the protein expression level of SLC2A6 in some tumors, and the acronyms of 33 tumors are shown in Table 1.

Table 1 Abbreviations for 33 cancers.
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We selected overall survival (OS) to explore the correlation between SLC2A6 expression and the prognosis of 33 cancers. Based on the median expression value of SLC2A6, patients were classified into 50% high and 50% low cut-off values of SLC2A621. The “survminer” and “survival” software packages were employed for Kaplan–Meier survival analysis and Cox regression analysis, and the results of Cox regression were presented through “forest plot”. To evaluate the predictive ability of SLC2A6, we generated Receiver Operating Characteristic Curve (ROC) curves by using timeROC in R language. Meanwhile, the correlation between SLC2A6 expression and clinicopathological features was analyzed using the Wilcoxon test.

Mutation analysis of SLC2A6 gene

The Gene Set Cancer Analysis (GSCA) database was utilized to analyze the copy number variation and methylation of the SLC2A6 gene.

The relationship between SLC2A6 expression and immunity

The relationship between SLC2A6 and 24 types of immune infiltrating cells was investigated through single-sample Gene Set Enrichment (ssGSEA) using the Gene Set Variation Analysis (GSVA) package21. Meanwhile, the “ESTIMATE” package was employed to examine the association between SLC2A6 expression and tumor stroma as well as immune cells, and heat maps were created to illustrate the Spearman correlation between SLC2A6 and various immune cells. Additionally, the connection between SLC2A6 and the major histocompatibility complex (MHC), immune stimulating factors, chemokines and their receptors was analyzed based on the TCGA database, and the correlation between SLC2A6 and immune checkpoints was explored.

Correlation of SLC2A6 with TMB, MSI and MMR

Tumor Mutation Burden (TMB) reflects the capacity of tumors to generate neoantigens and can predict the efficacy of immunotherapy in various cancers22. Mismatch repair (MMR) is a genetic surveillance mechanism that plays a significant role in maintaining genetic stability. MMR maintains genomic stability, and microsatellite instability (MSI) is caused by abnormal MMR, which influences cancer prognosis22,23. Spearman correlation analysis was employed to assess the relationship between SLC2A6 and MMR-related genes (MLH1, MSH2, MSH6, PMS2, EPCAM), TMB and MSI.

Drug-susceptibility analysis

GSCALite is an integrated platform for gene expression analysis and drug sensitivity analysis.

Comprehensive analysis of SLC2A6-related genes and functions

The Xiantao tool was employed to identify the genes related to SLC2A6 from TCGA. Based on the screening criteria of |log2FoldChange|> 1 and FDR < 0.05, significant differential genes were screened out. KEGG24,25,26 and GO analyses of the selected genes were conducted using the Xiantao tool.

Immunohistochemical analysis (IHC)

To verify the differential expression of SLC2A6 in cancer and paracancerous tissues, immunohistochemical analysis was conducted using 20 pairs of clinical specimens collected from the First Affiliated Hospital of Guangxi Medical University.

Cell culture and transfection

Huh7 and Hep3B cells were acquired from the Shanghai Cell Bank of the Chinese Academy of Sciences, and the cells were cultivated in DMEM culture medium containing 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin–streptomycin at 5% CO2 and 37 °C in a culture incubator. The lentivirus containing SLC2A6-shRNA and shNC was purchased from Songon Biotech. The sh-1 sequence was 5′-GCUGUGUGCCCGAGACCAA-3′, and the sh-2 sequence was 5′-CGGUGUACGUGUCUGAGAU-3′. Stable cells transfected with SLC2A6-shRNA were obtained after purinamycin screening for 14 days (2 μg/ml). The interference effect was detected by PCR and WB at 24 and 48 h after transfection. Subsequently, diffusion, migration and invasion capabilities were determined.

Cell proliferation assay

After the shRNA was transfected into Huh7 and Hep3B cells, they were seeded at a density of 5 × 103/ml in 96-well plates. Once the cells adhered, 10 μl/well of CCK8 reagent was added, and the OD value was measured following 2-h incubation in the incubator. The OD values were recorded continuously for 5 days. Simultaneously, a certain quantity of cells was seeded in 6-well plates for cloning experiments.

Scratch and transwell assay

Huh7 and Hep3B cells were seeded in 6-well plates. When the cells reached 90% confluence, they were scratched using a 200 μl pipette tip and a ruler, and photographs were taken under a microscope at 0 and 48 h. For the Transwell assay, cells were seeded into the chambers, and 700 μl of culture medium containing serum was added to the lower chamber. After 48 h, the cells were fixed with 4% paraformaldehyde and stained with crystal violet, and photographs were taken for documentation.

Reverse transcription and quantitative real-time polymerase chain reaction (qRT-PCR) analysis

RNAsimple Total RNA Kit was used for total RNA. (TIANGEN Biotech, China) Isolation from cells and EC samples according to the manufacturer’s protocol; After cDNA was prepared, qRT-PCR was performed (TaKaRa, China). The primer specificity was SLC2A6, 5′-GAGTTCGAGCAGATCCAGGACAAC-3′ in the forward direction and 5′-CAGACATGAGCAGCCAGGTGATG-3′ in the reverse direction. β-actin, forward 5′TGATCTTCATTGTGCTGGGTG-3′, reverse 5′-CCTTCCTGGGCATGGAGTC-3′. The expression of target genes relative to β-actin was quantified by comparative Ct(2−ΔΔCt).

Western blot

RIPA and PMSF (Thermo Fisher Scientific, USA) lysates were added to the transfected cells to obtain cellular proteins. The protein concentration was determined, and then a fourfold volume of protein loading buffer was added. Proteins were electrophoresed on SDS-PAGE gels and transferred from the gels to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with skim milk powder for 1 h, washed three times with PBST, and incubated with primary antibodies overnight at 4 °C. The plates were washed again, incubated with secondary antibodies for 1 h at room temperature, and protein bands were visualized using a Bio-Rad ChemiDoc MP imaging system and analyzed by Image J software. The primary antibodies are against SLC2A6, NRF2, P53, SLC7A11, ACSL4, GPX4 and GAPDH (Proteintech, Wuhan, China).

Animal experiments

BALB/c mice (male, 4–5 weeks old, 15–20 g) were procured from the Animal Center of Guangxi Medical University, and all animal experiments were sanctioned by the Ethical Review Committee for Experimental Animal Welfare of Guangxi Medical University. Animal ethical review conformed to the national standard GB/T35892-2018 “Guidelines for the Treatment of Laboratory Animals” and “Guidelines for Ethical Review of Laboratory Animals—Animal Welfare” issued by the Ministry of Science and Technology of the People’s Republic of China, Ethics Number: 2024-E668-01. The mice were randomly divided into the control group and the experimental group, with 4 mice in each group. shSLC2A6-Huh7 cells were cultivated in large quantities and then digested and collected. BALB/c mice were subcutaneously inoculated with 0.2 ml cell suspension at a density of 1 × 107/ml near the back of the right forelimb. The tumor growth in mice was observed every two days for 2 weeks, tumor tissues were taken after anesthetically executing mice using the inhalational anesthetic Isoflurane.

Statistical analysis

R language, SPSS 22.0 and GraphPad Prism 9.5 software were used for statistical analysis. P < 0.05 was considered statistically significant.

Results

SLC2A6 expression in various human normal tissues

Analysis of the HPA database revealed that SLC2A6 mRNA was highly expressed in various tissues, particularly in the appendix and lymph nodes (Fig. 1A). The protein expression of SLC2A6 varied significantly among different tissues (Fig. 1B). Immunohistochemical analysis indicated that SLC2A6 was expressed in both the nucleus and cytoplasm, with distinct staining patterns across tissues with varying expression levels, including Colon and Rectal Cancer (COAD) and Kidney Clear Cell Carcinoma (KIRC) (high expression), stomach adenocarcinoma (STAD) and LIHC (moderate expression), Lung Adenocarcinoma (LUAD) (low expression), and Pancreatic Adenocarcinoma (PAAD) (no expression) (Fig. 1C–H).

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SLC2A6 Expression in Normal Human Tissues. (A) SLC2A6 mRNA Expression Profile in Normal Human Tissues; (B) SLC2A6 Protein Expression Profile in Human Normal Tissues; Representative Immunohistochemical Plot of (C–H) SLC2A6 Expression in Normal Kidney, Colon, Liver, Stomach, Lung, and Pancreas Tissues.

SLC2A6 expression in various tumor tissues

The TIMER 2.0 analysis showed that SLC2A6 expression exhibited significant differences between tumor and normal tissues across multiple cancer types. Notably, in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), COAD, Diffuse Large B-Cell Lymphoma (DLBC), Acute Myeloid Leukemia (LAML), Ovarian Cancer (OV), PADD, Melanoma (SKCM), Testicular Cancer (TGCT), and Uterine Carcinosarcoma (UCS), SLC2A6 showed a marked increase in expression, while others, including Glioblastoma (GBM) and Kidney Chromophobe (KICH), displayed decreased expression (Fig. 2A–B). Furthermore, Gene Expression Profiling Interactive Analysis (GEPIA) analysis revealed an association between SLC2A6 expression and the pathological stages of Bladder Cancer (BLCA), Breast Cancer (BRCA), KICH, KIRC, Lung Squamous Cell Carcinoma (LUSC), OV, SKCM, STAD, and UCS (Fig. 2C).

Fig. 2
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SLC2A6 Expression in Each Tumor Tissue. (A) TIMER2.0 was utilized to analyze the differential expression of SLC2A6 in pan-cancer tissues from the TCGA database; (B) Differential Expression of SLC2A6 in Pan-Cancer Tissues from the TCGA and GTEx Databases; (C) The GEPIA2 database was used to verify the relationship between SLC2A6 expression and different cancer pathological stages.

DNA methylation levels of SLC2A6 in cancer

We examined the DNA methylation levels of SLC2A6 in various tumors using the University of Alabama at Birmingham Cancer Data Analysis Portal (UALCAN) database and found that the DNA methylation levels of SLC2A6 were higher in BRCA, COAD, KIRC, PAAD, Esophageal Cancer (ESCA), and LIHC than in normal tissues, while they were decreased in STAD, PRAD, and CESC (Fig. 3A). In LIHC, the cg14082398 and cg25512587 fragments of SLC2A6 were weakly methylated, whereas the cg00411595 and cg02257517 fragments were highly methylated (Fig. 3B).

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Methylation Levels of SLC2A6 in Cancer. (A) DNA Methylation Levels of SLC2A6 in BRCA, COAD, KIRC, PAAD, ESCA, LIHC, STAD, PRAD, and CESC Cancers; (B) Heat Map of SLC2A6 in LIHC.

Expression in pan-cancer

Analysis using the GSCA database demonstrated that aberrant SLC2A6 expression was significantly positively correlated with gene copy number variation (CNV) in cancers such as Lower Grade Glioma (LGG), OV, SARC, LUAD, and SKCM. (Fig. 4A). Additionally, SLC2A6 DNA methylation levels were significantly correlated with mRNA expression in Thyroid Cancer (THCA), SKCM, Kidney Papillary Cell Carcinoma (KIRP), BRCA, and KIRC (Fig. 4B). Further investigation of the relationship between SLC2A6 and methyltransferase genes revealed significant correlations across STAD, BRCA, PRAD, Acute Myeloid Leukemia (LAML), and LIHC (Fig. 4C).

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CNA and Methylation Help Drive Aberrant SLC2A6 Expression in Pan-Cancer. (A) Correlation Between CNV and SLC2A6 mRNA Expression in GSCA Database; (B) Correlation Between SLC2A6 mRNA Expression and Methylation Level; (C) Correlation Between SLC2A6 mRNA and Four Methyltransferases DNMT1, DNMT3A, DNMT3B, and DNMT3L.

The expression level of SLC2A6 is related to the prognosis of cancer

To further elucidate the influence of SLC2A6 expression on the prognosis of cancer patients, we downloaded TCGA RNA-seq and clinical data and conducted univariate COX regression analysis of the association between SLC2A6 expression and overall survival (OS) for 33 cancer types (Fig. 5A–B). The results were basically consistent with the OS analysis. High SLC2A6 expression was significantly associated with a poor prognosis in patients with Adrenocortical Cancer (ACC), LIHC, COAD, KIRC, Mesothelioma (MESO), Ocular melanomas (UVM), BLCA, glioblastoma multiforme (GBM), LAML, Thymoma (THYM), UCEC and SARC. The relationship between LIHC and SLC2A6 was more significant. In contrast, high expression levels of SLC2A6 were positively correlated with better outcomes in ESCA, STAD, and LGG (Fig. 5C). These results indicate that SLC2A6 expression levels are significantly associated with the prognosis of patients with multiple cancer types.

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Survival Analysis of SLC2A6 in Different Types of Cancer in the TCGA Database. (A–B) Correlation Between SLC2A6 Expression and OS and DSS in Patients with Different Tumors; (C) Kaplan–Meier (KM) Analysis of Cancer in the TCGA Database.

Relevance of SLC2A6 expression to immune checkpoints and immunotherapy

Analysis of the TCGA database indicated that SLC2A6 was closely associated with several immune checkpoint genes, showing a strong positive correlation with genes such as LAIR1 and CD244 (Fig. 6A). The TIMER 2.0 analysis confirmed the relationship between SLC2A6 and specific immune checkpoint blockade genes, including PD1, PD-L1, CTLA-4, and LAG-3, with consistent results (Fig. 6B–E). Additionally, SLC2A6 was also associated with MMR genes in ACC, Head and Neck Cancer (HNSC), KIRC, LIHC, PRAD, and THYM, suggesting its potential role in immunotherapy (Fig. 6F). In THYM, CESC, Rectal Cancer (READ), GBM, LUAD, MESO, and SKCM, SLC2A6 expression was positively correlated with TMB but negatively correlated with UVM, Bile Duct Cancer (CHOL), and UCEC (Fig. 6G). Furthermore, SLC2A6 expression was positively correlated with MSI in BRCA, DLBC, HNSC, LUSC, OV, and SARC, but negatively correlated with MSI in ACC, STAD, LGG, and SKCM (Fig. 6H).

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Relevance of SLC2A6 Expression at Immune Checkpoints to Immunotherapy. (A) Correlation Between SLC2A6 and Immune Checkpoint Genes in Pan-Cancer Tissues; (B–E) Correlation Between SLC2A6 and PD-1, PD-L1, CTLA-4 and LAG-3; (F) Correlation Between SLC2A6 and MMR-Related Genes in Pan-Cancer Tissues; (G–H) Correlation of SLC2A6 with TMB and MSI in Pan-Cancer Tissues.

Correlation between SLC2A6 expression and immune infiltration

The TIMER database analysis revealed a significant correlation between SLC2A6 expression and tumor immune infiltration, particularly in LIHC,KIRP,HNSC,BRCA,LUAD and STAD. SLC2A6 expression was strongly associated with six types of immune cells, including B cells and CD4 + T cells, with the strongest correlation observed for CD4 + T cells (Fig. 7A). In conjunction with TCGA database analysis, SLC2A6 expression was found to be closely related to immune scores across multiple cancers, with the strongest correlations observed in cancers such as LIHC and TGCT (Fig. 7B–J).

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Relationship Between SLC2A6 Expression and Immune Infiltration. (A) The Association Between SLC2A6 Expression and the Degree of Immune Cell Infiltration in Multiple Malignancies; (B–J) The Top 9 Tumors with the Most Significant Correlation Between SLC2A6 Expression and Immune Score.

Role of SLC2A6-related genes in LIHC

We analyzed the potential function of SLC2A6 by utilizing the Linked Omics database. The Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of LIHC based on SLC2A6-related genes revealed that they were mainly enriched in biological processes such as organic acid catabolism, small molecule metabolism, fatty acid metabolism and signaling pathways such as cell cycle, P53 and ferroptosis (Fig. 8A–B). The correlation between SLC2A6 and 23 genes related to ferroptosis was explored and heat map was used to show that they were closely related to ferroptosis -related genes such as GPX4, SLC7A11, ACSL4, and SLC2A6 was positively correlated with ferroptosis negative regulatory genes such as SLC7A11, FANCD2, SLC1A5 and HSPA5. (Fig. 8C–D). These results imply that the possible mechanism by which the expression level of SLC2A6 affects the progression of HCC is related to ferroptosis, it may be a negative regulator of ferroptosis.

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Results of KEGG/GO Enrichment Analysis of SLC2A6-Related Genes in LIHC. (A–B) Bar Graph of DEGs Significantly Enriched in KEGG/GO; (C–D) Heat map and scatter map of ferroptosis -related Associated with SLC2A6.

Drug-susceptibility analysis

GSCALite was employed to detect the drug sensitivity of SLC2A6 expression in tumors. It was found that SLC2A6 expression was negatively correlated with the 50% inhibitory concentration (IC50) values of dasatinib, A-770041, AZD6482, BEZ235, CGP-60474, TW37, sirolimus lipides, TGX221 and Z-Llni-CHO. It was positively correlated with IC50 values of BMS-536924, BMS754807, CH5424802, GSK1070916, HG-5-113-01, Lisitinib, masitinib, MPS-1-IN-1, nilotinib, TL-2-105, and WZ3105 (Fig. 9A–B).

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Relationship between SLC2A6 Expression and Drug Sensitivity Based on GSCALite. (A) Correlation between CTRP Drug Sensitivity and mRNA Expression; (B) Correlation between GDSC Drug Sensitivity and mRNA Expression.

The SLC2A6 transfection efficiency was verified and its effect on the cell proliferation ability was evaluated

Immunohistochemical results indicated that SLC2A6 expression was higher in hepatocellular carcinoma tissues than in adjacent tissues (Fig. 10A).The transfection efficiency of SLC2A6 was verified by PCR and WB, and the results demonstrated that SLC2A6 expression was successfully knocked down (Fig. 10B–C).

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Transfection Efficiency of SLC2A6 and Its Impact on HCC Cell Proliferation. (A) Immunohistochemical Results; (B) PCR Verification Results; (C) WB Verification Results; (D) CCK8 Proliferation Curve; (E) Plate Cloning Results. (original blots/gels are presented in Supplementary Fig. 1).

Knockdown of SLC2A6 inhibited cell proliferation (Fig. 10D–E).

Effect of SLC2A6 knockdown on cell migration and invasion

Scratch and Transwell assays were employed to observe the effect of SLC2A6 Knockdown on cell migration and invasion, and it was discovered that cell migration and invasion were significantly decreased after SLC2A6 Knockdown (Fig. 11A–C).

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Effect of SLC2A6 Knockdown on Cell Migration and Invasion. (A) Results of cell scratch assay; (B) Results of cell migration assay; (C) Results of cell invasion assay.

SLC2A6 knockdown affects HCC development by promoting ferroptosis

To further explore the mechanism through which SLC2A6 expression influences liver cancer, we observed the effect of SLC2A6 knockdown on ferroptosis-related gene proteins via WB experiment and found that the expression of NRF2, SLC7A11, and GPX4 was decreased, while the expression of P53 and ACSL4 was increased (Fig. 12A), indicating that the mechanism of SLC2A6 influencing liver cancer is related to ferroptosis. The results were in accordance with the previous KEGG enrichment analysis. In addition, the in vivo tumor growth rate was inhibited in the knockdown mice (Fig. 12B).

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Effect of SLC2A6 Knockdown on Ferroptosis-Related Genes and tumor growth. (A) Protein expression levels of ferroptosis-related genes in Huh7 cells; (B) Curve of tumor volume change. (original blots/gels are presented in Supplementary Fig. 2).

Discussion

In mammalian cells, glucose transport across the plasma membrane is mediated by the solute carrier 2A (SLC2A) family (also known as the glucose transporter or GLUT family)27. The SLC2A protein family comprises 14 proteins containing 12 transmembrane domains, and these carrier proteins are key regulators of cellular energetics28. SLC2A6 is a member of the SLC2A family. Studies have indicated that SLC2A6 is implicated in the occurrence and development of breast cancer, prostate cancer, lung adenocarcinoma, endometrial cancer and other cancers, and is associated with the prognosis and immunotherapy of these tumors15,17,18,19. Additionally, it was discovered that SLC2A6 exhibited the strongest positive correlation with the infiltration level of Th1 cells, and SLC2A6 may be a crucial biomarker for predicting sepsis survival29. We employed TCGA, GTEx, UALCAN, cBioportal, TIMER and other databases to comprehensively disclose the molecular characteristics of SLC2A6 in 33 tumors in terms of gene expression, gene alterations, prognosis, immune infiltration, DNA methylation, and drug sensitivity. To illuminate its potential function and regulatory mechanism in the occurrence and development of different tumors.

Our study confirmed significant overexpression of SLC2A6 in 20 cancers from the TCGA and GTEx databases. We conducted immunohistochemical staining of HCC tissues to validate SLC2A6 protein expression in tumor and normal tissue samples. The expression of SLC2A6 protein was higher in LIHC compared to normal tissues, which is in line with the analysis presented herein. That is, the expression of SLC2A6 in LIHC and other tumors is higher than that in normal tissues. The high expression of SLC2A6 is associated with the clinical stage of LIHC and other cancers, and the high expression of SLC2A6 indicates a poor prognosis of LIHC, ACC, BLCA and other tumors. Additionally, we also discovered that abnormal SLC2A6 expression could exert a role by influencing immune cell infiltration, immunotherapy, and inflammatory response. The findings suggest that SLC2A6 plays a significant role in different cancers. These findings imply that SLC2A6 can be utilized as a potential marker for predicting tumor prognosis.

The tumor microenvironment (TME) encompasses immune cells, cancer-associated fibroblasts, endothelial cells, and various other tissue-resident cells, etc. Its dynamic alterations are a key factor influencing the occurrence and progression of tumors, including carcinogenesis, tumor growth, angiogenesis, metastasis, and invasion30,31,32,33. An increasing number of studies have demonstrated that immune cell infiltration is a crucial factor in tumor progression and immunotherapy34. Hence, it is of paramount importance to explore the relationship between SLC2A6 and tumor-associated immune cell infiltration. As one of the significant components of TME, immune cells play a vital role in tumor biology35. Studies have indicated that the immunoscore can be utilized as an indicator of survival, recurrence, metastasis, and drug resistance in tumor patients36. We discovered that SLC2A6 was correlated with the immune scores of various cancers, including LAML, READ, LIHC, BRCA, THCA, BRCA, READ, LGG, THYM, and UCEC, suggesting that SLC2A6 interacts with tumor cells and immune cells in tumors. This finding offers a novel perspective for the development of effective treatments. Immune checkpoint genes are important targets for immune checkpoint inhibitor (ICI) treatment of various cancers37, and ICIs are an effective immunotherapy approach38. Our study found that SLC2A6 expression was associated with a variety of common immune checkpoints such as PD-L1, PD-1, CTLA4, and LAG3, suggesting that SLC2A6 may be a new target for tumor immunotherapy. Additionally, our study revealed that SLC2A6 expression was significantly correlated with immunomodulatory-related genes and immune infiltration, and affected the prognosis of cancer patients. The above findings provide a new perspective for new directions and targets of tumor immunotherapy in the future.

DNA methylation is one of the most prevalent epigenetic modifications and plays a crucial role in gene expression, genome stability, and tumorigenesis39,40. Studies have revealed that aberrant DNA methylation can facilitate the development of LIHC through immune regulation, cell stemness, apoptosis, cell proliferation and invasion41. Utilizing the UALCAN tool, we observed significantly decreased SLC2A6 promoter methylation levels in BLCA, CHOL, GBM, LUAD, TGCT, THCA, PRAD, KIRP, Pheochromocytoma & Paraganglioma (PCPG), HNSC, and UCEC when compared with normal tissues. Nevertheless, SLC2A6 promoter methylation levels were elevated in KIRC, COAD, READ, and PAAD. This implies that SLC2A6 may promote tumorigenesis and progression via DNA methylation.

We validated the potential function of SLC2A6 using the Linked Omics database, and the analysis results indicated that SLC2A6 was mainly involved in cell cycle, P53, ferroptosis and other signaling pathways. We further investigated the correlation between SLC2A6 mRNA and anticancer drug sensitivity. The GDSC database demonstrated that SLC2A6 overexpression was negatively correlated with IC50 values of eight drugs, including A-770041, AZD6482, BEZ235, CGP-60474, TW37, sirolimus lipides, TGX221 and Z-Llni-CHO. These drugs may have the effect of inhibiting tumor development. This finding will assist in guiding clinical drug selection and enhancing patient outcomes.

Ferroptosis, a distinctive cell death modality driven by iron-dependent phospholipid peroxidation, has emerged as a promising approach for the treatment of HCC. P53-mediated ferroptosis is specifically triggered by ROS, and the levels of SLC7A11 are crucial for the p53-mediated ferroptosis response. SLC7A11 is a target of P53, and an increase in P53 can result in a decrease in SLC7A11 expression42. SLC7A11 is the upstream mediator of GPX4, and NRF2 inhibits ferroptosis by regulating SLC7A1143,44. We discovered a significant correlation between SLC2A6 and genes related to ferroptosis and then verified it by cell function experiments and WB experiments. The results demonstrated that the proliferation, migration, invasion, and other capabilities of HCC cells were suppressed after SLC2A6 was knocked down. It was found that the expression levels of NRF2, SLC7A11, and GPX4 proteins decreased, while the expression levels of P53 and ACSL4 proteins increased, indicating that the regulatory mechanism of SLC2A6 in HCC is associated with ferroptosis and the P53 signaling pathway. These findings offer new targets for therapeutic strategies for HCC.

In conclusion, increasing number of studies find potential prognostic and therapeutic value of genes of different origins in cancer45,46. we conducted a systematic exploration of the correlation between SLC2A6 expression and clinical characteristics, prognosis, mutational status, DNA methylation, immune checkpoints, immunotherapy, immunoinfiltration, and drug sensitivity in multiple cancers. It was discovered that SLC2A6 is implicated in the regulation of various cancers and plays a role in tumor prognosis and immune-related processes. The outcomes of in vivo and in vitro experiments demonstrate that knockdown of SLC2A6 can significantly inhibit the proliferation, invasion, and migration of HCC, and its mechanism might be associated with P53, ferroptosis, and other signaling pathways. These findings imply that SLC2A6 could potentially serve as a prognostic biomarker and therapeutic target for HCC.

However, this study has several limitations. First, a portion of the findings is derived from bioinformatics analysis of public databases. Additionally, only the biological function of SLC2A6 in LIHC was investigated, with no research on its role in other types of cancer. Furthermore, the study did not utilize real-world cohorts to validate the diagnostic and prognostic value of SLC2A6 across various cancers or its predictive capacity for immunotherapy.