SPINK4 modulates inhibition of glycolysis against colorectal cancer progression

Dysregulation of glycolysis


INTRODUCTION
Colorectal cancer (CRC) is one of the common malignant tumors of the digestive tract and originates from the transformation of adenomatous polyps (1,2).Its complicated etiology involves genetic, environmental, and lifestyle factors (3).Causative factors include age, family history, certain genetic abnormalities, and inflammatory bowel disease (4).Despite improvements in treatment approaches and early detection, CRC remains a significant global health concern because of its high morbidity and mortality (5).The need for targeted preventative measures and improved treatment options is highlighted by the variations in incidence and death rates among regions (6).Presently, the objectives of CRC research include elucidating the molecular pathways of tumor genesis and development and developing new prognostic indicators, diagnostic markers, and therapeutics (7,8).This is essential for formulating personalized treatment regimens and developing innovative precision medicine techniques, as well as for gaining a thorough understanding of the pathophysiology of CRC (9).
Glycolysis is a basic metabolic process in cells that arranges the breakdown of glucose into smaller molecules and is necessary for the production of ATP (10).Glycolysis is a cytoplasmic process that starts with the phosphorylation of glucose and proceeds to fructose-1,6bisphosphate, which is subsequently converted to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate (11).Further reactions with these three-carbon molecules produce pyruvate, NADH, and ATP (12).Remarkably, glycolysis, operating independently of oxygen, initiates both aerobic and anaerobic cellular respiration.The end products, like pyruvate, adapt to various metabolic pathways based on cellular conditions.In the context of CRC, glycolysis plays a critical role.According to a study by Zuo S et al., vitamin D-activated long non-coding RNA MEG3 reduces glycolysis in CRC via encouraging c-Myc degradation, pointing to vitamin D as a possible treatment target (13).Additionally, the study by Zhu Y et al. linked tumorigenesis in Dectin-3-lacking mice to commensal fungus Candida albicans, emphasizing the interconnected roles of macrophage-induced glycolysis and interleukin-22 secretion by innate lymphoid cells in CRC, thus emphasizing the influence of the fungal flora (14).
Additionally, the work by Zhu W et al. emphasized the crucial function of PTBP1 in CRC as a critical regulator of glycolysis and other important cellular processes (15).Thus, a thorough understanding of glycolysis is crucial for CRC research, as it is for defining cellular metabolic pathways and developing targeted therapeutics for a variety of illnesses.
Serine Peptidase Inhibitor, Kazal Type 4 (SPINK4) is a gene found on chromosome 19 of the human genome.Its protein product is an important regulator of serine proteases engaged in several physiological processes, and it belongs to the family of serine protease inhibitors (Kazal-type) (16).Although the precise roles of SPINK4 are unidentified, it is usually linked to the inhibition of serine peptidases, which are vital enzymes involved in the degradation of proteins.Notably, SPINK4 has led to investigations into its expression levels and regulatory role in cellular processes in cancer research, particularly in CRC (17).According to the research of Xie M et al., increasing serum SPINK4 levels in CRC, particularly in patients who are about to undergo surgery, indicate diagnostic potential with improved sensitivity and specificity (18).
The higher SPINK4 levels are related to the advancement of CRC as they correspond with the site of cancer and distant metastases.According to an analysis of comprehensive gene expression, survival data, and immunohistochemistry validation, research by Wang X et al.
showed that downregulated SPINK4 expression is an independent predictor of reduced survival in CRC patients (19).Moreover, alpha-1 antitrypsin (A1AT) and elevated genes REG4 and SPINK4 are related to cancer-associated thrombosis (CAT) in CRC according to research by Buijs JT et al.This suggests that these genes might be used as biomarkers to identify CRC patients (20).In addition to opening up new possibilities for diagnostic and treatment approaches, an understanding of the roles and regulation of SPINK4 offers insightful insights into the molecular mechanisms behind the development and progression of cancer in CRC.
This study investigated the impact of SPINK4 on glycolytic metabolism and CRC cell proliferation to investigate the critical role of glycolysis and the importance of this biomarker for CRC.The effects of SPINK4 expression on the prognosis, glycolytic enzyme levels, cellular energy balance, and cell cycle of CRC patients will be clarified in turn below.By integrating these molecular findings, we aim to improve our understanding of CRC pathophysiology and provide fresh perspectives into targeted therapeutic strategies.

Download and processing of GSE32323 and GSE141174 datasets
Two CRC-related datasets, namely GSE32323 and GSE141174, were obtained from the Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/gds/)repository.The GSE32323 dataset consisted of 44 samples, of which 17 tumor samples were chosen as the case group and 17 normal samples as the control group.The GSE141174 dataset includes 16 samples, from which 3 CRC primary tumor samples were selected as the case group and 3 normal samples were selected as the control group.The Limma package in R language was used for the differential gene expression study.Fold changes (FC) greater than 2 were considered upregulated differentially expressed genes (DEGs), whereas genes with an FC less than 0.5 were regarded as downregulated DEGs.The significance criterion for the P value was set at < 0.05.

Survival analysis of differential expression of SPINK4
The Bioinformatic platform (http://www.bioinformatics.com.cn/) was used to analyze the intersection DEGs in the GSE32323 and GSE141174 datasets, including SPINK4, and visualized through the Venn diagram.Next, in various groups of the GSE32323 and GSE141174 datasets, the differential expression of SPINK4 was compared.The effect of the differential expression of SPINK4 on the overall survival (OS) probability of CRC patients was then assessed using the Kaplan-Meier plotter (https://kmplot.com/analysis/),and the significance of the difference was determined using the log-rank test.

Cell lines and culture
The Chinese Academy of Sciences Cell Collection Center (Shanghai, China) provided four CRC cell lines (LoVo, HCT-116, RKO, and SW480) as well as normal colorectal epithelial cells (NCM460).After that, the cells were cultivated in DMEM and kept at 37°C in a humidified atmosphere with 5% CO2.DMEM was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

Cell transfection
For transient transfection, 2 × 10 5 CRC cells were planted per well in 24-well plates.
Transfection of the plasmid encoding SPINK4 into CRC cells using an appropriate transfection method will allow the transfected cells to express the SPINK4 protein for a specific period to achieve overexpression.Then, two specific small interfering RNAs (siRNAs), si-SPINK4-1 and si-SPINK4-2, were introduced into the CRC cells to achieve knockdown of SPINK4 expression, and cells were incubated for a specific time to allow efficient knockdown of SPINK4.Cells were transfected using Lipofectamine 3000 (Invitrogen, USA) by the manufacturer's instructions.

Cell treatment
CRC cells were seeded at a density of 2 × 10 5 cells/well in 24-well plates.After 24 hours of incubation to facilitate cell adhesion, cells underwent treatment with different substances for 48 hours as part of the specific study.Treatments include 0.5 mM Glucose (Glu), to assess basic cellular metabolic responses; 20 μM Cytochalasin B (Cyto-B), to study effects on cytoskeletal dynamics and cell motility; 10 μg/ml 3-Bromopyruvate (3-BrPA), to study the effects on glycolytic pathways and energy metabolism; 50 μg/ml 5-Fluorouracil (5-FU), to investigate the impact of chemotherapy on the survival and proliferation of cells; and 10 μM XAV -939 to examine the effects on Wnt/β-catenin signaling and related cellular processes.Without any active ingredient, cells in the control group were treated with an equivalent amount of dimethyl sulfoxide (DMSO).After treatment, cells were subjected to subsequent analysis.

Quantitative real-time polymerase chain reaction (qRT-PCR) assay
The TRIzol reagent (Thermo Fisher Scientific, USA) was used to extract the total RNA of CRC cells by following the manufacturer's instructions.For cDNA synthesis, we utilized a PrimeScript RT kit (Takara, Japan).SYBR Green PCR Master Mix (Applied Biosystems, USA) was used to perform qRT-PCR using the StepOnePlus Real-Time PCR System (Applied Biosystems, USA).Gene expression levels of SPINK4, β-catenin, PKM2, GLUT1, LDHA, and HK2 were quantified and normalized to GAPDH.All target expression levels were computed with the 2 -ΔΔCT technique.A set of primer sequences was found in Table 1.

Western blotting (WB) assay
Protease and phosphatase inhibitors were added to the RIPA lysis solution (Thermo Fisher Scientific, USA) to produce protein lysates from CRC cells.The BCA Protein Assay Kit (Thermo Fisher Scientific, USA) was utilized to ascertain the protein content.Proteins in equal quantities were separated using 10% SDS-PAGE and then put onto PVDF membranes from Millipore, USA.After membrane sealing with 5% skimmed milk, primary antibodies targeting SPINK4, TP53, Cyclin D1, P21, P18, CDK4, CDK6, Caspase 3, Caspase 9, Bcl-2, β-catenin, PKM2, GLUT1, LDHA, and HK2 were applied at a 1:1000 dilution for incubation.After that, the membranes were incubated with suitable horseradish peroxidase (HRP)-conjugated secondary antibodies.As an internal reference, GAPDH (1:5000) was employed.Thermo Fisher Scientific, USA, provided an enhanced chemiluminescence (ECL) kit for visualizing protein bands, and Bio-Rad, USA, provided ChemiDoc imaging equipment for capturing the images.

Cell viability assay
The viability of the cells was assessed using the Cell counting kit-8 (CCK-8) assay (Dojindo, Japan).CRC cells were added at a density of 5 × 10 3 cells per well in 96-well plates.After treatment, each well was filled with CCK-8 reagent, and the absorbance at 450 nm was measured using a microplate reader (Thermo Fisher Scientific, USA) at 24h, 48h, 72h, 96h, and 120h.Additionally, a focused analysis at the 48-hour mark was conducted to determine the effects of different treatments on cell viability.Treatments included exposure to 0.5 mM Glu, 20 μM/L Cyto-B, and 10 μg/ml 3-BrPA.

Transwell assays
Transwell system was used to assess cell invasion and migration.Transfected CRC cells were placed in the upper chamber of the Transwell using serum-free media.This was followed by the addition of 10% FBS to the medium in the lower chamber of the Transwell.After a designated incubation period, cells with motile membranes were stained with crystal violet and fixed with 4% paraformaldehyde.Lastly, the quantity of migrating cells in the field of view was counted using inverted microscopy.The cell invasion studies were carried out as described previously, with matrigel covered in the upper chamber.

Flow cytometry
CRC cells were initially collected using trypsin-EDTA (Gibco, USA) and rinsed in phosphatebuffered saline (PBS) to undertake apoptosis and cell cycle analysis.The manufacturer's instructions were followed while staining the cells with Annexin V and propidium iodide (PI) to detect apoptosis and distinguish between viable, apoptotic, and necrotic cells.Cells were frozen in ethanol, subjected to RNase A treatment, and stained with PI to analyze the DNA content to evaluate the cell cycle.Evaluation of the patterns of cell death and proliferation was made easier by these approaches.Flow cytometry was performed using a BD Biosciences instrument, and FlowJo software was used for data analysis.

Glucose uptake assay
To assess glucose uptake, 2-DG was employed as a glucose tracer.Briefly, a 6-well plate was seeded with around 1 × 10 5 cells in quadruplicate, and the plate was then incubated at 37°C with 5% CO2 for an entire night.On the following day, cells were subjected to a 4-hour glucose deprivation, followed by a 2-hour incubation with 25μM 2-DG for three wells and 25μM glucose-containing CRC medium for the remaining well as a negative control.Cells were taken out of the incubation phase and given two PBS washes.The cellular uptake of 2-DG was quantified by measuring the average fluorescence intensity of the cells using flow cytometry with excitation light.

Lactate and ATP production measurements
CRC cells were plated at a density of 1 × 10 5 cells per well in 24-well plates for lactate assays and 1 × 10 4 cells per well in 96-well plates for ATP assays to quantify the generation of lactate and ATP.The cells were seeded in triplicate for lactate assays and quintuplets for ATP assays and allowed to incubate for 24 h.After the first incubation, RPMI 1640 with 1 mM Glu was added to the media and left overnight.The culture media was taken for the ATP and lactate tests the next day.Lactate concentration was measured using commercial assay kits (Abcam) according to the manufacturer's instructions.Simultaneously, relative ATP concentration was measured using commercial assay kits (Abcam).This combined approach allowed for the comprehensive assessment of both lactate and ATP production in CRC cells under the specified conditions.

Xenograft model
Four-week-old naked mice had their flanks subcutaneously implanted with 1.0 × 10 6 stable transfected HCT-116 cells.Nude mice were purchased from Fudan University, and all experimental procedures complied with the guidelines and standards of the Animal Welfare and Ethics Group of the Department of Laboratory Animal Science, Fudan University.Upon the onset of visible tumors in every animal following 10 days of subcutaneous injection, the drug delivery experiments were initiated.Progression of tumor growth was monitored systematically, and the standard formula, Tumor Volume = (L × W 2 )/2, was used to calculate the tumor volume (mm 3 ), with W representing the width and L representing the length of the tumor.Harvesting of tumors was executed upon reaching an approximate diameter of 2.0 cm.counterstained with 1% hematoxylin.Images were captured using a microscope (Leica DMI3000B), and the staining intensity of positively stained cells was assessed.

Ethical statement
All animal experiments in this study were conducted in compliance with the "Regulations for the Administration of Affairs Concerning Experimental Animals" (China) and adhered to the ethical guidelines provided in the "Guidelines for the Care and Use of Laboratory Animals" (China).This study was approved by the Animal Welfare and Ethics Group, Department of Laboratory Animal Science, Fudan University (Approval Number: 2023JS MZX-313).All necessary measures were taken to minimize animal suffering and ensure animal welfare.

Statistical analysis
The statistical analysis was performed using the R language package (version 4.0.3).Each experiment was repeated three times and the mean ± standard deviation (SD) of the data is presented.One-way analysis of variance (ANOVA) was used, and Tukey's post hoc test was conducted for multiple comparisons.A P value less than 0.05 indicates statistical significance.

Analysis of SPINK4 expression in CRC and its association with patient prognosis
In this study, we employed the R package to screen 919 upregulated DEGs and 956 downregulated DEGs from the GSE32323 dataset (Figure 1A).Additionally, from the GSE141174 dataset, 982 upregulated DEGs and 919 downregulated DEGs were identified (Figure 1B).Through the bioinformatics platform, 507 overlapping genes were identified from the DEGs of both datasets, indicating the existence of common regulatory mechanisms (Figure 1C).Among these overlapping genes, SPINK4 showed significant downregulation in tumor samples of GSE32323 and GSE141174 datasets (Figures 1D and 1E), and the findings of the survival study demonstrated that increased SPINK4 expression was linked to good prognosis of CRC patients (Figure 1F), highlighted the possible contribution of SPINK4 in CRC development.

Overexpression of SPINK4 inhibits the proliferation, migration, and invasion of CRC cells
qRT-PCR revealed a significant downregulation of SPINK4 expression in CRC cell lines compared to normal colorectal epithelial cells.This finding was further confirmed by WB analysis, demonstrating a corresponding decrease in protein levels of the gene (Figures 2A and     2B).Notably, among the CRC cell lines, HCT-116 and LoVo exhibited the lowest SPINK4 expression levels, prompting their selection for subsequent experiments.After identifying low SPINK4 expression, overexpression experiments were conducted, and overexpression efficiency was assessed (Figures 2C and 2D).CCK-8 assays demonstrated a significant reduction in proliferative activity upon overexpression of SPINK4 in CRC cells (Figures 2E     and 2F).Consistent with the CCK-8 results, Transwell assays revealed a notable decrease in invasion and migration capabilities in CRC cells overexpressing SPINK4 (Figures 2G and 2H).
These findings collectively suggested that the upregulation of SPINK4 exerts suppressive effects on the proliferation, migration, and invasion abilities of CRC cells.

Knockdown of SPINK4 induces CRC cell cycle arrest
Flow cytometry analysis revealed that si-SPINK4-2 induction led to G1 phase cell cycle arrest in CRC cells (Figures 4A and 4B).The alterations in the protein levels of important cell cycle regulators were examined using WB analysis.The findings showed that, following si-SPINK4-2 induction, CRC cells exhibited a considerable rise in Cyclin D1, CDK4, and CDK6, concomitant with a notable decrease in the protein levels of TP53, P21, and P18 in CRC cells (Figures 4C-4E).These findings suggested that SPINK4-induced G1 arrest may be closely related to changes in the degrees of expression of important cell cycle proteins.

SPINK4 regulates apoptosis and cell proliferation in CRC cells under glucose-limited conditions
Analysis using flow cytometry revealed that following si-SPINK4-2 induction, the apoptotic rate of CRC cells was lowered.Furthermore, under low glucose conditions (0.5 mM Glu), the apoptosis rate of CRC cells was reduced.Interestingly, the combination of si-SPINK4-2 induction and 0.5 mM Glu resulted in a significant further reduction in the apoptotic rate of CRC cells (Figures 5A and 5B).To examine the impact of glucose consumption on cell survival, SPINK4-overexpressing CRC cells were treated with low glucose (0.5mM Glu), a glucose transport inhibitor (Cyto-B, 20μM/L), and a hexokinase-2 inhibitor (3-BrPA, 10μg/ml) for 48 hours.The CCK-8 test results showed a notable increase in cell proliferation activity compared to the control group after 48 hours of treatment (Figures 5C and 5D).These findings suggest that SPINK4 regulates CRC cell survival, potentially through its control of apoptotic mechanisms, in a glucose-dependent manner.

SPINK4 exerts anti-CRC effects by inhibiting glycolysis
5-FU is an anticancer medication that is frequently used to treat different types of cancer.It plays a critical role in cancer treatment, which aims to stop the growth of cancer cells, by compromising DNA integrity, influencing gene expression, and regulating apoptosis.
Apoptosis rates of LoVo and HCT-116 cell lines were determined using flow cytometry analysis under different treatment conditions.In LoVo cells, treatment with either overexpression of SPINK4 or 5-FU (50 µg/mL) alone increased apoptosis compared to vector control.However, apoptosis was significantly increased when both were combined (Figure 6A).
In addition, HCT-116 cells showed a similar trend (Figure 6B).Further, WB experiments detected the expression levels of key apoptotic and anti-apoptotic proteins, including Bcl-2, caspase-3, and caspase-9 (Figures 6C-6E).SPINK4 overexpression results in the upregulation of cleaved (active) forms of caspase-3 and caspase-9 in the presence or absence of 5-FU, indicating activation of the apoptotic pathway.In contrast, SPINK4 overexpression in conjunction with 5-FU therapy resulted in a downregulation of the levels of the anti-apoptotic protein Bcl-2.In HCT-116 and LoVo cells, overexpression of SPINK4 significantly reduced uptake of 2-DG, indicating altered glucose metabolism (Figures 6F and 6G).Conversely, overexpression of SPINK4 led to increased uptake of 2-DG (Figures 6H and 6I).Furthermore, overexpression of SPINK4 resulted in increased lactate production and ATP levels in both cell lines, indicating a shift towards aerobic glycolysis.Knockdown of SPINK4 led to the opposite trend, further confirming the effect of this gene on metabolism (Figures 6J and 6K).

SPINK4 regulates glycolysis in CRC by inhibiting the β-catenin pathway
The overexpression of SPINK4 dramatically reduced the expression of glycolytic-related proteins (β-catenin, PKM2, GLUT1, LDHA, and HK2) in CRC cells, as shown by qRT-PCR and WB studies (Figures 7A-7E).Subsequently, the expression of glycolytic-related proteins in CRC cells was evaluated by WB analysis after SPINK4 knockdown, either with or without the β-catenin inhibitor (XAV-939, 10μM, 48 h).The findings indicated that induction of si-SPINK4-2 significantly increased the levels of glycolytic-related proteins.However, the addition of 10μM XAV-939 reversed this elevation, causing a notable reduction in the amounts of glycolytic-related proteins (Figures 7F-7H).These results emphasize the regulatory function of SPINK4 in modulating the expression of key glycolytic proteins in CRC cells.

SPINK4 overexpression inhibits subcutaneous xenograft tumor growth
Tumor specimens collected from mice showed that tumor cells overexpressing SPINK4 were significantly smaller in size compared with vehicle controls (Figure 8A).Quantitative analysis of tumor volume further confirmed this observation, with tumors overexpressing SPINK4 significantly reduced in size compared with vehicle controls (Figure 8B).These results indicated that SPINK4 overexpression has an effective inhibitory effect on tumor growth after xenotransplantation.

SPINK4 enhances glycolytic-related protein expression in CRC
Histopathological features corresponding to CRC were observed through HE staining (Figure 9A).Compared to normal tissue, tumor samples exhibited disrupted tissue architecture and heterogeneous staining patterns, indicating tumorigenic alterations.Subsequent IHC staining of CRC tissues and normal tissues revealed increased expression of SPINK4, HK2, GLUT1, LDHA, and PKM2 compared to normal tissues (Figure 9B).This further supported the notion that SPINK4 may co-regulate CRC tissues along with glycolysis.

DISCUSSION
The importance of biomarkers in CRC treatment is crucial.By identifying specific molecular signatures of cancer, biomarkers enable more precise and effective treatment strategies (21).
For example, NEO1 expression levels decline as CRC advances, and low expression levels are linked to a poor prognosis, indicating the possibility of NEO1 as a prognostic marker (22).Furthermore, functional studies further highlighted the significance of NEO1 overexpression in therapeutic intervention by demonstrating that it suppressed the proliferation, migration, and invasion of CRC cells.Other study had also shown that CREPT is overexpressed in CRC, contributes to tumor progression and cell proliferation, is positively correlated with the efficacy of 5-Fluorouracil (5-FU) treatment, and can serve as a valuable prognostic biomarker for CRC patients (23).Current diagnostic modalities, targeted gene therapies, and five-year survival rates from CRC have been key aspects of research.This discussion highlights the search for more effective biomarkers for the diagnosis, treatment, and prognosis of CRC.The exploration of key biomarkers not only improves diagnostic accuracy but also holds the promise of improving treatment strategies and predicting patient outcomes.In this study, based on our bioinformatics analysis, we found that SPINK4 showed downregulation in CRC and was linked to a favorable prognosis for CRC patients.This observation provides valuable molecular insights into dysregulated processes associated with CRC and highlights the need for further studies to elucidate the functional implications of SPINK4 alterations and their potential as a diagnostic or prognostic indicator for CRC.
The importance of SPINK4 in previous studies on cancer also lies in its involvement in tumor proliferation, migration, and invasion (24).This multifaceted role emphasizes the key impact of SPINK4 on the invasiveness of cancer cells, further emphasizing its role in oncology research.Supported by the research results of Li T et al., the increased expression of SPINK4 in CRC is related to HCT-116 and DXH-1 cell invasion, migration, proliferation, and cell cycle progression (25).ELF-1, binding to the SPINK4 promoter, influenced its expression, and elevated ELF-1 levels were associated with poorer prognosis in colon cancer.Suppression of SPINK4 counteracted ELF-1 overexpression effects, suggesting a therapeutic avenue targeting ELF-1/SPINK4 expression for colon cancer treatment.Correspondingly, Hu BL et al. demonstrated that SPINK4 downregulation in CRC tissues was linked to enhanced proliferation and metastasis, while SPINK4 overexpression promoted CRC cell proliferation, metastasis, and tumor growth, primarily localized in the nucleoplasm and nucleus (26).Moreover, SPINK4 inhibited ferroptosis in CRC cells, emphasizing its multifaceted role in CRC pathogenesis by influencing cell proliferation, metastasis, and ferroptosis.Our in vitro studies revealed that downregulation of SPINK4 stimulates these activities and causes cell cycle stasis in CRC cells during the G1 phase, whereas overexpression of SPINK4 blocks the proliferation, invasion, and migration of CRC cells.Together, these results highlight the potential of SPINK4 as a therapeutic target for CRC treatment approaches and imply that it plays a dual function in CRC.
Glucose, cytochalasin B (Cyto-B), and 3-bromopyruvate (3-BrPA) are related to each other through their effects on cellular processes (27,28).Glucose, a fundamental carbohydrate and primary energy source, is central to cellular metabolism.Cyto-B disrupts actin polymerization, influencing cellular structure, while 3-BrPA inhibits glycolysis, a key metabolic pathway involving glucose breakdown.Their applications in biological studies and therapeutic interventions stem from their effects on cellular functions and metabolism.Subsequently, exemplifying the role of glucose in CRC, the research of Zhu Y et al. demonstrates that ketone bodies in CRC cells modulate MondoA phosphorylation, affecting glucose uptake, apoptosis, and proliferation, with MEK1 identified as a novel influencer (29).Zhu H explores a novel approach, combining glucose oxidase and gold nanorods in a nanoreactor for colon cancer treatment (30).Meanwhile, the study of Zhou X highlights Glucose-regulated protein 78 (GRP78) overexpression in CRC, impacting cancer cell proliferation, tumorigenesis, and resistance to 5-FU through AKT and ERK pathways, establishing a significant association between CRC progression and glucose regulation (31).In conclusion, our research findings indicate that SPINK4 inhibits aerobic glycolysis in CRC, providing valuable insights into potential therapeutic strategies for CRC treatment.
5-FU is a chemotherapeutic agent used in cancer treatment, inhibiting thymidylate synthase and disrupting DNA synthesis.2-Deoxy-D-glucose (2-DG) is a glucose analog that interferes with glycolysis, impacting cellular energy metabolism.Lactic acid (lactate) is a byproduct of anaerobic glycolysis, produced when cells generate energy without oxygen.ATP (adenosine triphosphate) is the cell's primary energy currency, generated through various cellular processes, including glycolysis and oxidative phosphorylation.The relationship between these compounds lies in their involvement in cellular metabolism and energy production.5-FU disrupts DNA synthesis, affecting cell division (32).2-DG interferes with glycolysis, reducing glucose utilization and ATP production (33).Lactic acid is produced as a result of anaerobic glycolysis, and ATP is the end product of cellular energy metabolism, essential for various cellular functions (34).These compounds are interconnected through their roles in cellular metabolism and energy production, impacting CRC progression.Zykova T et al. demonstrated that elevated expressions of phosphorylated p53-related protein kinase (p-PRPK) in colon adenocarcinomas correlate with advanced metastatic stages (35).PRPK, a known phosphorylator of survivin, promotes colon cancer metastasis, suggesting its potential as a (hexokinase 2).It is possible to gain a better understanding of the complex regulation of glycolysis and its potential therapeutic uses in many diseases, including cancer, by understanding the functions and interactions of these proteins.Research on CD36 and NDRG2 provides crucial information on glycolysis-related proteins in CRC.Found by Xu X et al., NDRG2 suppresses glycolysis and glutaminolysis in CRC cells by blocking c-Myc via βcatenin mediation (42).In addition, CD36, as described by Fang Y et al., inhibits GPC4 ubiquitination and β-catenin/c-myc signaling to reduce glycolysis as a tumor suppressor in CRC (43).Both results illustrate the intricate regulatory networks that include CD36 and NDRG2, as well as their roles in regulating metabolic pathways and safeguarding against cancer.Due to our research, β-catenin is regulated by SPINK4 and inhibits glycolysis.Notably, xenograft research demonstrates that SPINK4 can prevent CRC cell growth in vivo.After HE staining, the histopathological features of CRC tissues were revealed, while IHC staining exhibited elevated expression levels of SPINK4, HK2, GLUT1, LDHA, and PKM2 within CRC tissues.All of these findings contributed to our growing knowledge of the complex dynamics of glycolytic regulation and emphasize the potential use of these pathways in the treatment of CRC.

Limitations
Despite the valuable insights gained from our study, it is important to acknowledge that there are several limitations that may affect the interpretation and generalization of our findings.Our study was constrained by limited resources, including funding and specialized equipment.This limitation hindered our ability to perform exhaustive experiments and additional analyses such as disclosure of whole xenograft samples and histological examinations such as haematoxylin and eosin (HE) staining and immunohistochemistry (IHC) of representative proteins.The timelimited nature of our study limits the depth to which we can investigate complex mechanisms.
As a result, certain aspects may have been overlooked, thus limiting our full understanding of the regulatory role of SPINK4.Although our study provides compelling evidence that SPINK4 regulation is involved in CRC progression, it remains a preliminary exploration.The intricacies of the SPINK4-mediated signaling pathways need to be further elucidated through advanced experimental techniques and interdisciplinary collaborations.In conclusion, despite the substantial insights gained from our study, its limitations must be acknowledged.Addressing these limitations in future research endeavors will pave the way for a deeper understanding of the regulatory mechanisms of SPINK4 in colorectal cancer.

CONCLUSION
In conclusion, a detailed analysis of SPINK4 in CRC showed that it is markedly downregulated in tumor tissues and is associated with a poor prognosis.Functional analysis demonstrates that SPINK4 acts as a tumor suppressor by inhibiting the growth, invasion, and migration of CRC cells and inducing cell cycle arrest in the G1 phase.Notably, it was shown that glucose availability influenced how it affected cell proliferation and that SPINK4 decreased apoptosis in CRC cells.It is significant to note that SPINK4 inhibited glycolysis to produce ATP, change lactate release, and alter 2-DG absorption, all of which were indicators of its anti-CRC activities.Mechanistically, SPINK4 modulated the level of glycolytic-related proteins, including β-catenin, PKM2, LDHA, GLUT1, and HK2.Additionally, SPINK4 overexpression suppressed subcutaneous xenograft tumor growth in vivo.These findings underscored the intricate regulatory role of SPINK4 in CRC, shedding light on its potential as a therapeutic target for inhibiting tumorigenesis and modulating glycolytic pathways in CRC patients.Magnification: ×20.

Figure 1 .
Figure 1.Analysis of SPINK4 expression and survival in CRC patients.(A and B) Volcano

Figure 2 .
Figure 2. SPINK4 overexpression inhibits the proliferation, invasion, and migration of

Figure 3 .
Figure 3. Knockdown of SPINK4 promotes the proliferation, invasion, and migration of

Figure 5 .
Figure 5. SPINK4 inhibits aerobic glycolysis in CRC.(A and B) Flow cytometry was used

Figure 9 .
Figure 9. Increased expression of glycolysis-related proteins in CRC tissues.(A)