TFAP2A Upregulates SKA3 to Promote Glycolysis and Reduce the Sensitivity of Lung Adenocarcinoma Cells to Cisplatin

Abstract Introduction: Studies have shown that glycolysis metabolism affects the resistance or sensitivity of tumors to chemotherapy drugs. Emerging from recent research, a paradigm-shifting revelation has unfolded, elucidating the oncogenic nature of SKA3 within the context of lung adenocarcinoma (LUAD). Consequently, this work was designed to delve into the effects of SKA3 on glycolysis and cisplatin (CDDP) resistance in LUAD cells and to find new possibilities for individualized treatment of LUAD. Methods: LUAD mRNA expression data from the TCGA database were procured to scrutinize the differential expression patterns of SKA3 in both tumor and normal tissues. GSEA and Pearson correlation analyses were employed to elucidate the impact of SKA3 on signaling pathways within the context of LUAD. In order to discern the upstream regulatory mechanisms, the ChEA and JASPAR databases were utilized to predict the transcription factors and binding sites associated with SKA3. qRT-PCR and Western blot were implemented to assay the mRNA and protein expression levels of SKA3 and TFAP2A. Chromatin immunoprecipitation and dual-luciferase assays were performed to solidify the binding relationship between the two. Extracellular acidification rate, glucose consumption, lactate production, and glycolysis-related proteins (HK2, GLUT1, and LDHA) were used to evaluate the level of glycolysis. Cell viability under CDDP treatment was determined utilizing the CCK-8, allowing for the calculation of IC50. The expression levels of SKA3 and TFAP2A proteins were detected by immunohistochemistry (IHC). Results: SKA3 exhibited upregulation in LUAD tissues and cell lines, establishing a direct linkage with glycolysis pathway. Overexpression of SKA3 fostered glycolysis in LUAD, resulting in reduced sensitivity toward CDDP treatment. The upstream transcription factor of SKA3, TFAP2A, was also upregulated in LUAD and could promote SKA3 transcription. Overexpression of TFAP2A also fostered the glycolysis of LUAD. Rescue assays showed that TFAP2A promoted glycolysis in LUAD cells by activating SKA3, reducing the sensitivity of LUAD cells to CDDP. The IHC analysis revealed a positive correlation between high expression of SKA3 and TFAP2A and CDDP resistance. Conclusion: In summary, TFAP2A can transcriptionally activate SKA3, promote glycolysis in LUAD, and protect LUAD cells from CDDP treatment, indicating that targeting the TFAP2A/SKA3 axis may become a plausible and pragmatic therapeutic strategy for the clinical governance of LUAD.


Introduction
Lung cancer (LC) is an extensively prevalent malignancy, representing one of the most frequently diagnosed cancers and the primary cause of cancer-related fatalities on a global scale [1].Non-small cell lung cancer (NSCLC) covers a large proportion of newly diagnosed LC cases [2], with lung adenocarcinoma (LUAD) as the main subtype of NSCLC, covering 40% of LC cases [3].Despite advances in surgical, targeted, and immunotherapy treatments for LUAD, the prognoses for LUAD individuals are unsatisfactory due to issues such as treatment resistance, tumor heterogeneity, and metastasis, with a mean 5-year survival rate of less than 26% [4,5].Cisplatin (CDDP) is one of the widely used drugs for treating various solid tumors [6].It can induce DNA damage by interacting with purine bases on cancer cell DNA, thereby inducing cancer cell apoptosis [7].However, the decreasing sensitivity of many malignant tumor cells to CDDP has led to a decline in treatment efficacy.Contemporary research has put forth compelling indications regarding the potential implication of glycolysis in the underlying mechanisms of CDDP-associated resistance.For example, Gong et al. [8] found that KLF5 knockdown could inhibit hypoxia-induced CDDP resistance in NSCLC cells, potentially by inactivating PI3K/Akt/ mTOR pathway and inhibiting HIF-1α-dependent glycolysis.Hence, acquiring a comprehensive comprehension of the molecular regulatory mechanisms governing glycolysis and CDDP resistance in LUAD is critical for identifying potential targets for the treatment of CDDPresistant LUAD patients and improving their survival rates.
Unlike normal cells that obtain energy through mitochondrial oxidative phosphorylation, most cancer cells utilize glycolysis instead of aerobic cycling to acquire energy, known as "Warburg effect" [9].Therefore, aerobic glycolysis is also considered a pivotal characteristic of cancer metabolism [10] and is linked with tumors' malignant progression.For example, Zhao et al. [11] found that lncRNA MIR17HG can promote invasion and liver metastasis of colorectal cancer (CRC) cells via a glycolysis-mediated positive feedback loop.Tan et al. [12] reported that IL-1β fosters glycolysis in LUAD cells via the p38 pathway, thereby facilitating tumor cell migration and invasion.Kim et al. [13] found that enhancing glycolysis can inhibit autophagy-mediated EGFR degradation, thereby maintaining the survival of EGFRmutant LUAD cells.Changes in glycolytic activity have also been reported to affect the sensitivity or resistance of certain types of malignant tumors to chemotherapy drugs.For example, exosome-derived circRNA was ascertained by Wang et al. [14] to trigger oxaliplatin resistance in CRC cells via miR-122/PKM2 axis-mediated promotion of glycolysis.Dai et al. [15] found that purpurin can enhance the sensitivity of NSCLC cells to CDDP by downregulating pyruvate kinase M2 (PKM2) and inhibiting glycolysis.Therefore, further understanding the mechanisms related to glycolysis and chemoresistance may provide new directions for the treatment of LUAD.
Spindle and kinetochore associated complex subunit 3 (SKA3) is a protein-encoding gene located in the chromosomal region 13q12.11[16].SKA3 can mediate interactions between kinetochores and microtubules [17].SKA3 is an essential gene for normal chromosome segregation and cell division and assumes a crucial role in various tumorigenic processes, thereby exerting a significant impact on the initiation and progression of diverse tumor types, such as gastric cancer [18], breast cancer [19], hepatocellular carcinoma [20], cervical cancer [21], laryngeal squamous cell carcinoma [22], and oral squamous cell carcinoma [23].The compelling findings presented strongly support the notion that SKA3 functions as an oncogene.However, the existing literature on SKA3 in the context of LUAD remains limited, with a scarcity of studies investigating the association between SKA3 and both the glycolysis pathway and chemoresistance in LUAD.
Transcription factor AP-2 alpha (TFAP2A), a member of AP-2 transcription factors, is located on the negative strand of chromosome 6 [24].The protein encoded by this gene functions as a transcription factor, exhibiting the ability to bind to the consensus sequence 5′-GCCNNNGGC-3′.Through its interaction with enhancer elements, TFAP2A orchestrates the regulation of target gene transcription [25].Previous reports have demonstrated that TFAP2A exhibits either oncogenic or tumor-suppressive properties, with its functional role being contingent upon the specific cancer type and its interaction with distinct molecular entities [26,27].Furthermore, TFAP2A has been implicated in chemotherapy resistance and sensitivity in patients.Studies by Wu et al. [28] suggest that variations in the 3′-UTR region of TFAP2A gene may serve as potential prognostic markers for CDDP therapy.Therefore, investigating the regulatory mechanisms of TFAP2A in conjunction with other molecules can contribute to the identification of biomarkers for the treatment of LUAD.
This study mined the role of upregulated SKA3 by TFAP2A in LUAD cell glycolysis and chemoresistance.High expression of SKA3 was found in LUAD, which was linked with unfavorable prognosis.Knockdown of SKA3 inhibited glycolysis in LUAD, while overexpression of SKA3 decreased the sensitivity of LUAD cells to CDDP.Further investigation revealed that the upstream transcription factor TFAP2A can activate the expression of SKA3, promoting the glycolytic process in LUAD and protecting LUAD cells from CDDP treatment.Overall, our study elucidated the mechanism of action of the TFAP2A/SKA3 axis in LUAD, indicating that targeting the TFAP2A/SKA3 axis may be a potential therapeutic approach for LUAD.

Bioinformatics Methods
LUAD mRNA expression data were downloaded from TCGA (https://portal.gdc.cancer.gov/).Differential expression analysis of tumor and normal tissues was performed using the "edgeR" package [29] to acquire differentially expressed mRNAs (DEmRNAs) with |logFC|>1.0and FDR <0.05.The target genes for the study were determined by combining the DEmRNAs with literature citations, and their relationship with patient prognosis was analyzed.GSEA and Pearson correlation analyses were performed to investigate pathways affected by target genes.
The upstream potential transcription factors for the target genes were forecasted using ChEA database (https://maayanlab.cloud/chea3/).The differentially expressed transcription factors were obtained by taking the intersection of the predicted transcription factors and upregulated mRNAs.Pearson correlation analysis was performed between the transcription factors and target genes, and highly correlated transcription factors were selected as the study objects.The motifs binding sites in the upstream 2000 bp of the target genes were forecasted utilizing JASPAR (https://jaspar.genereg.net/).

qRT-PCR
Total RNA was isolated from cells using TRIzol reagent (Invitrogen, USA).To obtain cDNA, reverse transcription was fulfilled utilizing PrimeScript™ RT Master Mix (TaKaRa, Japan).qRT-PCR was performed on the cDNA using SYBR Premix EX Taq (TaKaRa, Japan) on a 7,500 Fast Real-time PCR System (Applied Biosystems, USA).β-actin was employed as an endogenous reference gene, and the relative gene expression was determined using 2 −ΔΔCt method.The primer sequences are listed in Table 1.

Chromatin Immunoprecipitation
Cells (1 × 10 5 cells/mL) were put in culture dishes, and proteins were cross-linked to DNA utilizing 1% formaldehyde.Chromatin was fragmented by sonication, and the chromatin mixture was incubated with the target protein and IP-grade anti-TFAP2A antibody (1:20, ab108311) at 4°C overnight utilizing agarose magnetic beads (Millipore, USA).Anti-IgG antibody (1:200, ab172730) was utilized as a negative control.Protein-DNA crosslink was reversed by heating with NaCl, and the protein and RNA were degraded with proteinase K and RNase A. The remaining DNA was purified and recovered utilizing a DNA purification kit (Invitrogen, USA), and specific DNA fragments were analyzed by qPCR.The purified DNA was subjected to qPCR using the primers listed in Table 2.

Glycolytic Capacity Assay
Extracellular acidification rate (ECAR), which indirectly reflects glycolytic capacity, was assayed utilizing the Seahorse XF Glycolytic Stress Test Kit (Agilent, USA), according to a previously described method [30].Briefly, cells (4 × 10 4 cells/well) were put in a Seahorse XF microplate and incubated overnight.Seahorse buffer consisting of DMEM, phenol red, 25 mM glucose, 2 mM sodium pyruvate, and 2 mM L-glutamine was used.The next day, 10 mM glucose, 2 μM oligomycin, and 2-deoxy-D-glucose (2-DG) were sequentially added to the injection ports of the Seahorse XF Extracellular Flux Analyzers (Agilent, USA) to measure ECAR.
Glucose consumption and lactate production were measured as per a previously described method [31].Cells (5 × 10 4 cells/well) were put in a 24-well plate, digested with trypsin, and counted.Cell supernatant was collected.Glucose and lactate levels were assayed utilizing glucose and lactate assay kits (Solarbio, China), respectively.

CCK-8 Assay
Cell viability was ascertained utilizing CCK-8 assay kit (Beyotime, China).Cells (5 × 10 3 cells/well) were put in a 96-well plate and preincubated for 24 h.Cells underwent different concentrations of CDDP (0, 2, 4, 6, 8 μg/mL) for 48 h [32].Oligomycin or 2-DG was added as required.Then, 10 μL of CCK-8 solution was added to each well, followed by an incubation period of 2 h.The absorbance of each well was quantified at 450 nm utilizing a microplate reader.IC 50 of CDDP-treated cells was calculated using GraphPad 8.0 software.

Clinical Samples
The tissue samples were obtained from individuals diagnosed with LUAD, and five CDDP-sensitive and five CDDP-resistant samples were collected.Patients underwent initial surgery followed by CDDP-based chemotherapy.The patients were put into chemoresistant group and chemosensitive group.The definitions for chemoresistant group were patients who experienced disease progression during postoperative chemotherapy; patients whose CA125 levels failed to the normalize after completing 6 cycles of chemotherapy; or those who suffered a recurrence within 6 months after the initial chemotherapy.The definition for chemosensitive group was patients who experienced recurrence after a period extending beyond 6 months or those who did not experience any recurrence.This research protocol was approved by the Ethics Committee of The Second Affiliated Hospital, Hengyang Medical School, University of South China, and all patients signed informed consent forms.

Immunohistochemistry
Following deparaffinization and rehydration, paraffinembedded sections were immersed in a 3% H 2 O 2 solution for 20 min at 37°C to eliminate endogenous peroxidase.Subsequently, the sections were exposed to a sodium citrate buffer (pH 6.0) and heated at 121°C for 2 min.The sections were then incubated overnight at 4°C with primary antibodies SKA3 (1:500, orb459269) or TFAP2A (1:1,000, ab52222).After that, the sections were incubated at 37°C for 30 min with the secondary antibody anti-IgG (1:200, ab172730), followed by staining with 3,3′-diaminobenzidine and counterstaining with hematoxylin for 1 min.The sections were dehydrated in a series of ethanol gradients and then cleared with xylene.The anti-SKA3 antibody was procured from Biorbyt (UK), while the anti-TFAP2A antibody and anti-IgG antibody were sourced from Abcam (UK).

Statistical Analysis
GraphPad Prism 8.0 software was utilized for data analysis, and the results were presented as mean ± SD.Wilcoxon test was employed for the analysis of differential gene expression.Student's t test and one-way analysis of variance (ANOVA) were utilized to evaluate differences between groups.Kaplan-Meier analysis was used to generate survival curves, and differences between survival curves were evaluated utilizing log-rank test.The experiments were conducted in triplicate to ensure reproducibility and reliability of the results.Statistical significance was defined as a p value of less than 0.05.

SKA3 Is Highly Expressed in LUAD and Associated with Poor Prognosis
To identify new therapeutic targets for LUAD, we first performed differential analysis of mRNA expression data and obtained 5,542 DEmRNAs, including 3,758 upregulated genes and 1,784 downregulated genes (online suppl.Table 1; for all online suppl.material, see https:// doi.org/10.1159/000536557).Based on literature research and data from TCGA-LUAD, we selected SKA3 as the main subject of our study [33].Analysis of TCGA-LUAD data showed high-expression of SKA3 in LUAD tissues (Fig. 1a), and LUAD individuals with high SKA3 expression possessed a tellingly lower overall survival rate than those with low SKA3 expression (Fig. 1b).Subsequently, we ascertained the mRNA and protein levels of SKA3 in various LUAD cell lines using qRT-PCR and Western blot (WB), respectively, and found that SKA3 was notably upregulated in LUAD cell lines in contrast with BEAS-2B cell line (Fig. 1c, d).

SKA3 Promotes Glycolysis in LUAD Cells
To investigate the mechanism by which SKA3 affects LUAD progression, we first did GSEA enrichment analysis and found that SKA3 was enriched in the GLYCOLYSIS GLUCONEOGENESIS pathway (Fig. 2a).From Pearson correlation analysis, SKA3 was positively linked with key genes (MYC, SLC2A1, HK2, PDK1, ENO1, LDHA) in the glycolysis pathway (Fig. 2b).Based on previous literature reports [34], we speculated that SKA3 could mediate aerobic glycolysis in LUAD.To validate this hypothesis, we constructed A549 cells transfected with si-NC and si-SKA3, and Calu-3 cells transfected with oe-NC and oe-SKA3.We evaluated the role of SKA3 in LUAD glycolysis process by measuring ECAR, glucose consumption, lactate production, and glycolysis-related proteins, with results showing that SKA3 expression was notably reduced in A549 cells after knockdown, and tellingly increased in Calu-3 cells after overexpression, indicating the effective transfection of si-SKA3 and oe-SKA3, respectively (Fig. 2c).Seahorse XF analysis showed that si-SKA3 could decrease the level of

TFAP2A/SKA3 Axis Promotes Glycolysis in LUAD
ECAR in cells, while oe-SKA3 could increase the level of ECAR in cells (Fig. 2d).The assay kit showed that in contrast with si-NC group, si-SKA3 significantly decreased glucose consumption and lactate production, while oe-SKA3 group had the opposite effect (Fig. 2e, f).WB results showed that si-SKA3 significantly reduced the expression of glycolysis-related proteins (HK2, GLUT1, and LDHA) in cells, while the oe-SKA3 group had the opposite effect (Fig. 2g).Ground on these outcomes, we concluded that SKA3 could promote glycolysis in LUAD cells.

SKA3 Promotes Glycolysis and Reduces Sensitivity of LUAD Cells to CDDP
Previous studies have reported that increased glycolytic activity affects the resistance or sensitivity of malignant tumors to chemotherapy [14,15].Therefore, we dived into the possible linkage between SKA3, glycolysis, and chemoresistance in LUAD.First, to investigate whether glycolysis contributed to chemoresistance in LUAD, we treated cells with increasing doses of CDDP (µg/mL) alone or in combination with the glycolysis activator Oligomycin or the inhibitor 2-DG.We found that compared to treatment with CDDP alone, CDDP+Oligomycin increased cell viability and IC 50 , while CDDP+2-DG decreased cell viability and IC 50 (Fig. 3a), indicating that glycolysis enhances the resistance of A549 and Calu-3 cells to CDDP.Next, we investigated whether SKA3 modulated the sensitivity of LUAD cells to CDDP and found that knockdown of SKA3 decreased the IC 50 value of A549 cells, while overexpression of SKA3 in Calu-3 cells increased their IC 50 value, indicating that high expression of SKA3 reduced the sensitivity of LUAD cells to CDDP (Fig. 3b).In addition, we treated cells with si-NC+PBS, si-SKA3+PBS, si-SKA3+Oligomycin, and si-SKA3+2-DG and analyzed their CCK-8 results.We found that the IC 50 value of cells treated with CDDP decreased due to knockdown of SKA3, but this effect was restored by the addition of Oligomycin.Conversely, overexpression of SKA3 increased the IC 50 value of cells treated with CDDP, but this effect was restored by the addition of 2-DG (Fig. 3c).In summary, our in vitro experiments suggested that SKA3 protected LUAD cells from CDDP treatment by accelerating glycolysis.

TFAP2A Is an Upstream Transcription Factor of SKA3
To mine the mechanism of SKA3 action, we used the ChEA database to predict upstream transcription factors of SKA3 (online suppl.Table 2) and obtained four po-tential transcription factors by intersecting them with differentially upregulated genes (Fig. 4a).Based on literature, we selected TFAP2A as the upstream transcription factor of SKA3 [35].Pearson correlation analysis revealed positive correlation between SKA3 and TFAP2A (cor = 0.24) (Fig. 4b).Bioinformatics analysis, qRT-PCR, and WB outcomes illustrated high-expression of TFAP2A in both LUAD tissues and cell lines (Fig. 4c-e).Using the JASPAR database, we predicted the binding site between the SKA3 upstream 2000 bp region and TFAP2A (Fig. 4f).Further validation of the interaction between the two was performed using a dualluciferase assay and chromatin immunoprecipitation experiment in A549 cells, which displayed that oe-TFAP2A significantly increased the luciferase activity of SKA3-WT (Fig. 4g), and anti-TFAP2A antibody enriched SKA3 sequences (Fig. 4h).These findings illustrated that TFAP2A was an upstream transcription factor of SKA3.

TFAP2A Activates SKA3 and Reduces Sensitivity of LUAD Cells to CDDP by Promoting Glycolysis
First, to verify the regulatory role of TFAP2A on SKA3 as well as glycolysis, we overexpressed TFAP2A in A549 cells.We checked the transfection efficiency of oe-TFAP2A and found that TFAP2A expression was significantly upregulated in oe-TFAP2A group in contrast with oe-NC group, indicating successful transfection (Fig. 5a).We examined the expression of SKA3 in the oe-NC group and oe-TFAP2A group using qRT-PCR and WB and found that the overexpression of TFAP2A upregulated the expression of SKA3 (Fig. 5b, c).Similarly, we evaluated the role of TFAP2A in the glycolysis process of LUAD by measuring ECAR, glucose consumption, lactate production, and glycolysis-related proteins.The experimental results showed that oe-TFAP2A increased the ECAR levels in the cells (Fig. 5d) and significantly enhanced glucose consumption and lactate production (Fig. 5e, f).WB results demonstrated that the oe-TFAP2A group significantly promoted the expression of glycolysisrelated proteins (HK2, GLUT1, and LDHA) in the cells (Fig. 5g).These findings indicated that TFAP2A can promote glycolysis in LUAD cells.
To mine the effect of the TFAP2A/SKA3 axis on glycolysis and chemoresistance in LUAD, we established the following treatment groups: oe-NC+si-NC, oe-NC+si-SKA3, and oe-TFAP2A+si-SKA3.We then detected the expression of SKA3 in different treatment groups (oe-NC+si-NC, oe-NC+si-SKA3, and oe-TFAP2A+si-SKA3) by qRT-PCR and WB.We found that SKA3 expression was downregulated in the oe-NC+si-SKA3 group but was restored in the oe-TFAP2A+si-SKA3 group (Fig. 5h, i), implying that TFAP2A can upregulate SKA3 expression.In addition, glycolysis-related assays showed that oe-TFAP2A offset the inhibitory effect of si-SKA3 on ECAR, glucose consumption, and lactate production (Fig. 5j-l).As WB analysis revealed, knockdown of SKA3 significantly decreased the expression of HK2, GLUT1, and LDHA, while oe-TFAP2A restored their expression levels to those of the oe-NC+si-NC group (Fig. 5M), suggesting that TFAP2A can foster glycolysis in LUAD cells by activating SKA3 expression.We also explored whether TFAP2A regulates the chemoresistance of LUAD cells to CDDP and found that overexpression of TFAP2A increased the IC 50 value of A549 cells, indicating that overexpression of TFAP2A reduced the sensitivity of A549 cells to CDDP (Fig. 5n).What's more, we calculated the IC 50 of the different treatment groups (oe-NC+si-NC, oe-NC+si-SKA3, and oe-TFAP2A+si-SKA3) using the CCK-8 assay after CDDP treatment and found that knockdown of SKA3 decreased the IC 50 value, which was restored by overexpression of TFAP2A (Fig. 5o).This study also validated the findings in clinical tumor samples from CDDP-sensitive and CDDP-resistant LUAD patients.Through immunohistochemistry analysis, it was discovered that the levels of SKA3 and TFAP2A in the tissues of CDDP-resistant patients were significantly higher compared to the CDDP-sensitive group (Fig. 5p).
Moreover, we also compared the effects of different doses of si-SKA3 on SKA3 expression.Using WB, we observed that in the oe-NC+si-SKA3 group and oe-TFAP2A+si-SKA3 group, the transfected cells with 2 μg si-SKA3 exhibited lower levels of SKA3 protein expression compared to the cells transfected with 0.5 μg si-SKA3 (Fig. 6a).The results of the CCK-8 assay showed that increasing the dosage of transfected si-SKA3 to  reduce SKA3 expression after TFAP2A overexpression significantly decreased the IC 50 value of A549 cells, indicating enhanced sensitivity of A549 cells to CDDP (Fig. 6b).These results strongly demonstrated that TFAP2A indeed fostered glycolysis and reduces sensitivity to CDDP through its downstream target SKA3.Combined, these findings implied that TFAP2A activated SKA3 and reduced sensitivity of LUAD cells to CDDP by promoting glycolysis.

Discussion
Here, we unveiled that both SKA3 and TFAP2A were upregulated in LUAD tissues and cell lines.Overexpression of SKA3 fostered LUAD cell glycolysis and reduced sensitivity to CDDP.Additionally, we identified the transcription factor TFAP2A binding site in the upstream 2000 bp region of the SKA3 promoter.TFAP2A overexpression upregulated SKA3 expression and promoted LUAD cell glycolysis, affecting the chemotherapeutic efficacy of CDDP.Our findings confirmed the involvement of the TFAP2A/SKA3 axis in the molecular mechanisms regulating glycolysis and chemoresistance in LUAD, and suggested that targeting the TFAP2A/SKA3 axis could be a potential therapeutic strategy for LUAD.
Metabolic activity in cancer cells is widely recognized as a well-established phenomenon, unlike normal cells, largely relies on glycolysis to produce energy [36].Therefore, targeting aerobic glycolysis in cancer cells and correcting abnormal cell metabolism has become a new approach for preventing and treating tumor development.For example, in a study by Kim et al. [13], the carcinogenic EGFR-mediated enhancement of glycolysis was shown to be necessary for maintaining EGFR levels, making glucose metabolism a potential attractive therapeutic approach for EGFR-mutant NSCLC.Ancey et al. [37] ascertained that in LUAD, the absence of glucose transporter GLUT1 expression in tumor-associated neutrophils leads to reduced tumor growth and in-creases radiotherapy efficacy, indicating that targeting metabolic vulnerabilities may be advantageous against neutrophils in tumors.Here, we assessed LUAD cell glycolysis flux under different treatments by measuring ECAR, glucose consumption, lactate production, and glycolysis-related enzymes (GLUT1, HK2, LDHA), and found that overexpression of SKA3 and TFAP2A could promote glycolysis in LUAD cells.Furthermore, we demonstrated that the TFAP2A/SKA3 regulatory axis can reduce the sensitivity of LUAD cells to CDDP by fostering glycolysis.Reports on the increase in glycolytic activity regulating tumor drug resistance are not uncommon in published literature.For example, Wang et al. [14] found that extracellular vesicle-derived ciRS-122 can reverse resistance to oxaliplatin by inhibiting glycolysis by governing miR-122/PKM2 pathway in CRC cells.Lin et al. [38] found that activating POU2F1/ALDOA axis can enhance metabolic reprogramming in colon cancer cells, fostering cancer progression and reducing sensitivity to oxaliplatin.This study focused on the molecular mechanisms of the TFAP2A/SKA3 axis in regulating glycolysis and CDDP chemotherapy in LUAD, providing a new potential target for glycolysis-induced chemoresistance therapy in LUAD.
SKA3 is a common tumor-promoting factor that has been shown to participate in promoting the malignant phenotype of tumors in multiple studies, including LUAD [18,39].This gene encodes a component of the spindle and kinetochore-associated protein complex, which mediates microtubule-kinetochore interactions [17].Here, we explored the expression levels of SKA3 in LUAD tissues and cells based on public databases and molecular experiments.Our analysis, which encompassed data from public databases and molecular experiments, revealed a significant upregulation of SKA3 expression in LUAD tissues and cells.Survival analysis also confirmed that high expression of SKA3 was linked with poor prognosis in LUAD individuals, consistent with previous research results [33].Through bioinformatics analysis, we identified the transcription factor Fig. 5. TFAP2A activates SKA3 and reduces sensitivity of LUAD cells to CDDP by promoting glycolysis.a qRT-PCR detection of the transfection efficiency of oe-TFAP2A.b, c qRT-PCR and WB analysis of SKA3 mRNA and protein levels in the different treatment groups.d ECAR measurements of the different treatment groups using the Seahorse XF cell energy metabolism analyzer.e, f Levels of glucose consumption and lactate production in the different treatment groups measured using a kit.g WB analysis of the expression of glycolysis-related proteins (GLUT1, HK2, and LDHA) in the different treatment groups.h, i qRT-PCR and WB analysis of SKA3 mRNA and protein levels in the different treatment groups.j ECAR measurements of the different treatment groups using the Seahorse XF cell energy metabolism analyzer.k, l Levels of glucose consumption and lactate production in the different treatment groups measured using a kit.m WB analysis of the expression of glycolysis-related proteins (GLUT1, HK2, and LDHA) in the different treatment groups.n CCK-8 assay to measure cell viability and IC 50 of A549 cells treated with different concentrations of CDDP.o CCK-8 assay to measure IC 50 of the different treatment groups after CDDP treatment.p IHC detection of SKA3 and TFAP2A expression levels in CDDP-sensitive and CDDP-resistant samples.*p < 0.05.IHC, immunohistochemistry.
TFAP2A upstream of SKA3.TFAP2A's role in tumor development has been extensively established, with its protein expression level being capable of influencing cellular functions and survival outcomes [25].For instance, Su et al. [40] revealed that upregulation of TFAP2A can inhibit progression of gliomas, while in a study by Huang et al. [26].TFAP2A is upregulated in gallbladder cancer and can inhibit ferroptosis in gallbladder cancer cells.This also indicates that TFAP2A can play a carcinogenic or cancersuppressive role in different tumor types.In Zhou et al.'s study, high-expression of TFAP2A was found in LUAD and could activate the transcription of ITPKA, promoting LUAD metastasis.In our work, unveiling TFAP2A as a transcription factor acting upstream of SKA3, actively engaged in the regulation of metabolism and chemoresistance in LUAD, signified the intricate array of regulatory mechanisms in which TFAP2A exerted its influence as a critical gene within the context of LUAD.This breakthrough revelation served as a significant reference point in the quest for identifying pertinent biomarkers pertaining to the diagnosis and therapeutic management of LUAD.
In light of the aforementioned, this study was founded upon the utilization of bioinformatics analysis techniques and in vitro cellular experiments, substantiating the transcriptional upregulation of SKA3 by TFAP2A and its consequential promotion of glycolysis in LUAD cells, consequently leading to a diminished sensitivity of LUAD cells toward CDDP.Nonetheless, the absence of validation of the experimental outcomes at both animal and clinical levels imposed certain limitations on the scope of the paper.Going forward, our research endeavors will be directed toward delving deeper into the downstream signaling pathways modulated by the TFAP2A/SKA3 axis, as well as investigating the mechanistic interplay of this axis in LUAD glycolysis and chemoresistance at animal and clinical levels.In summation, the outcomes of this study dawned on the involvement of the TFAP2A/ SKA3 axis in LUAD glycolysis and chemoresistance, thus positioning it as a promising molecular target for therapeutic intervention in LUAD.

Fig. 1 .Fig. 2 .
Fig. 1.SKA3 is highly expressed in LUAD and associated with poor prognosis.a Expression of SKA3 in normal (n = 59) and tumor tissues (n = 539) of LUAD patients in TCGA database.b Kaplan-Meier analysis of the relationship between SKA3 expression and overall survival time based on TCGA database.c, d Expression of SKA3 mRNA and protein in LUAD cell lines (A549, PC-9, and Calu-3) and bronchial epithelial cells (BEAS-2B) detected by qRT-PCR and WB; *p < 0.05.

Fig. 3 .
Fig. 3. SKA3 promotes glycolysis and reduces the sensitivity of LUAD cells to CDDP. a In A549 and Calu-3 cells, inhibition of glycolysis increased sensitivity to CDDP (A549 and Calu-3 cells were treated with different concentrations of CDDP (0, 2, 4, 6, 8 μg/mL) alone or in combination with Oligomycin or 2-DG.After 48 h, cell viability was measured using the CCK-8 assay).b CCK-8 assay to measure cell viability and IC 50 of si-SKA3 or oe-SKA3 cells treated with different concentrations of CDDP.c CCK-8 assay to measure cell viability and IC 50 of different treatment groups after CDDP treatment.*p < 0.05.

Fig. 4 . 5 (
Fig. 4. TFAP2A is an upstream transcription factor of SKA3. a Upset plot showing potential upstream transcription factors of SKA3 predicted by the ChEA database and differentially upregulated mRNA.b Pearson correlation analysis between SKA3 and TFAP2A.c Expression of TFAP2A in normal tissue (green) and tumor tissue (red).d, e qRT-PCR and WB analysis of TFAP2A mRNA and protein levels in human normal bronchial epithelial cell lines and LUAD cell lines.f Predicted binding site between the SKA3 promoter region and TFAP2A using the JASPAR database.g Dual-luciferase assay showing the luciferase activity of A549 cells in different treatment groups.h ChIP experiment showing the interaction between SKA3 and TFAP2A.*p < 0.05.ChIP, chromatin immunoprecipitation.

Fig. 6 .
Fig. 6.TFAP2A alters sensitivity to CDDP by regulating SKA3 expression.a WB analysis of SKA3 protein levels in the different treatment groups.b CCK-8 assay to measure IC 50 of the different treatment groups after CDDP treatment *p < 0.05.