KRT23 promotes proliferation invasion and metastasis of gastric cancer through epithelial-mesenchymal transition mediated by the PI3K/AKT/mTOR signaling pathway

Introduction

Gastric cancer (GC), a highly heterogeneous malignant tumor derived from the gastric epithelium, ranks fifth in global incidence and fourth in mortality, presenting a substantial public health challenge (1,2). Despite the progress made in early endoscopic screening, imaging optimization, and targeted/immunotherapy, the majority of patients are diagnosed at the advanced stage accompanied by invasion and metastasis due to the non-specificity of early symptoms such as dull upper abdominal pain and dyspepsia, resulting in limited therapeutic efficacy and unfavorable prognosis (3,4). An in-depth exploration of the pathogenesis of GC is of paramount importance for developing effective therapeutic targets and improving the survival of patients. The genesis and development of GC is a complex process involving genetic, epigenetic, and environmental factors, such as Helicobacter pylori infection and a high-salt diet (5). Research has indicated that the abnormal activation of key signaling pathways, like epidermal growth factor receptor (EGFR), Wnt/β-catenin, and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR), is closely associated with the progression of GC (6-8). Moreover, the regulation of epithelial-mesenchymal transition (EMT) and the tumor microenvironment (TME) play a crucial role in the invasion and metastasis of GC (9-11). These discoveries offer potential targets for precise treatment; however, key mechanisms such as EMT still need in-depth exploration. EMT, a key process where epithelial cells transform into mesenchymal phenotypes, significantly promotes tumor invasion and metastasis (8). In GC, EMT is characterized by the downregulation of epithelial cadherin (E-cadherin) and the upregulation of N-cadherin and vimentin, leading to weakened cell adhesion and enhanced motility (9). Studies have demonstrated that EMT is closely related to the high metastatic potential and poor prognosis of GC (9,11), suggesting that targeting EMT regulation might constitute a new strategy for improving the treatment of GC.

Keratin 23 (KRT23), a kind of intermediate filament protein belonging to the keratin family, is a type of cytoskeletal protein. It is mainly expressed in epithelial cells and participates in maintaining the structural integrity and mechanical stability of cells, as well as the interactions between cells and between cells and the matrix (12). In recent years, studies have revealed that KRT23 is aberrantly expressed in multiple tumors and is closely associated with tumorigenesis, development, invasion, and metastasis (13-15). Nevertheless, the tumor-promoting mechanism of KRT23 in GC remains undefined, and whether it regulates EMT via the PI3K/AKT/mTOR pathway to influence the progression of GC still requires verification. Based on the above background, this study intends to focus on the function and molecular mechanism of KRT23 in regulating EMT of GC cells. Through cell experiments, the role and mechanism of KRT23 in GC are explored, especially whether it mediates the EMT process through the PI3K/AKT/mTOR signaling pathway to promote the malignant behavior of GC cells. The effects of KRT23 on EMT marker proteins E-cadherin, N-cadherin, and transcription factors Snail and Twist are clarified. By revealing the molecular mechanism of KRT23 in GC, this study not only helps deepen the understanding of the pathogenesis of GC but may also provide new molecular targets for the early diagnosis, treatment, and prognosis assessment of GC. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-872/rc).

Methods

Correlation analysis of KRT23 expression with prognosis and chemosensitivity in GC via bioinformatics

The expression of KRT23 in GC was analyzed using the Gene Expression Profiling Interactive Analysis 2 (GEPIA2) database (http://gepia2.cancer-pku.cn/). To investigate the correlation between KRT23 expression and the prognosis of patients with stomach cancer, the Kaplan-Meier (KM) Plotter database (https://kmplot.com/analysis/) was utilized to generate KM survival curves. Additionally, the Gene Set Cancer Analysis (GSCA; https://guolab.wchscu.cn/GSCA/) was used to analyze the effects of the expression of these key genes on the sensitivity to the Genomics of Drug Sensitivity in Cancer (GDSC) chemotherapeutic drugs (16).

Patient information and immunohistochemistry

Clinical specimens were obtained from 10 GC patients (all from The Affiliated Hospital of Guizhou Medical University). Each patient provided a tumor sample and a sample from adjacent normal tissue. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. All patients signed informed consent, and the protocol was approved by the Ethics Committee of The Affiliated Hospital of Guizhou Medical University (No. 2020[252]). Immunohistochemical detection of KRT23 protein expression and localization of tissue samples was performed. After antigen repair in paraffin sections, endogenous peroxidase activity was blocked using 3% hydrogen peroxide. After blocking with 5% bovine serum albumin (BSA), the sections were treated with primary antibody KRT23 (1:200, DF9000, Affinity, Changzhou, China) and incubated overnight at 4 ℃. The next day, the sections were incubated with horseradish peroxidase (HRP) (1:200, S0001, Affinity) labeled secondary antibody at room temperature for 1 hour, followed by diaminobenzidine (DAB) coloration and counterstaining with hematoxylin. The expression location of the target protein was observed by the brown and yellow positive labeling under the optical microscope.

Cell culture and intervention

The normal gastric mucosal epithelial cell line GES-1, along with human GC cell lines AGS (RRID: CVCL_EQ22), MGC803 (RRID: CVCL_5334), HGC27 (RRID:CVCL_1279), and SGC7901 (RRID: CVCL_0520), were procured from Wuhan Shangen Biological Technology Co., Ltd. (Wuhan, China). All cell lines were maintained in either Roswell Park Memorial Institute 1640 Medium (RPMI-1640) or Dulbecco’s Modified Eagle Medium (DMEM) (11965118, Gibco, Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS) (Gibco), and cultured at 37 ℃ in a humidified atmosphere containing 5% CO₂. For MGC803 and SGC7901 cells, lentiviral vectors carrying KRT23, obtained from GENERAL (Chuzhou, China), were used for infection at a multiplicity of infection (MOI) of 10. Following 48 hours of infection, stable transfectants were selected using 2 µg/mL puromycin. Additionally, cells were treated with 30 µg/mL 740Y-P and 25 µM PI3K-IN-1 (HY-12068, MedChemExpress, Monmouth Junction, NJ, USA) for 48 hours to investigate the effects of PI3K pathway modulation.

Cell Counting Kit-8 (CCK-8) assay

Cell proliferation was assessed using the CCK-8 assay. Cells were seeded uniformly in 96-well plates and cultured for 0, 24, 48, and 72 hours. Following incubation, 10 µL of CCK-8 reagent was added to each well, and the plates were further incubated to allow formazan formation. Cell viability was quantified by measuring the optical density (OD) at 450 nm using a microplate reader (Multiskan FC, Thermo Fisher, Waltham, MA, USA).

Invasion assay

Cell invasion ability was evaluated using Matrigel-coated Transwell chambers (3422, Corning Inc., Corning, NY, USA). Briefly, Matrigel was diluted in serum-free medium at a 1:5 ratio and applied to the upper chamber, while the lower chamber was filled with medium containing 20% FBS as a chemoattractant. After Matrigel polymerization, 1×10⁵ cells suspended in 200 µL serum-free medium were seeded into the upper chamber. Following 24 hours of incubation at 37 ℃ with 5% CO₂, non-invading cells on the upper membrane surface were carefully removed. The invaded cells on the lower surface were fixed with 4% paraformaldehyde, stained with 1% crystal violet, and quantified under an inverted microscope (Shanghai Metro-Wei Optoelectronics, Shanghai, China). The number of invasive cells was analyzed using ImageJ software.

Real-time quantitative polymerase chain reaction (RT-qPCR) analysis

Total RNA was extracted from cells using TRIzol reagent (R1100, Solarbio, Beijing, China) following the manufacturer’s protocol. Reverse transcription was performed with the PrimeScript RT kit (R123-01, Vazyme Biotech, Nanjing, China) to synthesize complementary DNA (cDNA) from RNA. Quantitative polymerase chain reaction (qPCR) amplification was then carried out using SYBR Premix Ex Taq (Q111-02, Vazyme Biotech) in accordance with the manufacturer’s instructions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal reference gene, and relative gene expression levels were calculated using the 2^−ΔΔCt method. The specific primer sequences used for qPCR are listed in Table 1.

Western blot analysis

Protein samples were extracted from cells and tissues using radioimmunoprecipitation assay buffer (RIPA) lysis buffer (R0010, Solarbio), with protein concentrations determined by bicinchoninic acid (BCA) assay (PC0020, Solarbio). Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to polyvinylidene fluoride (PVDF) membranes. Following transfer, membranes were blocked with 5% non-fat milk and incubated overnight at 4 ℃ with the following primary antibodies: KRT23 (1:1,000, DF9000), PI3K (1:1,000, AF6241), phospho- (p-)PI3K (1:1,000, AF3242), AKT (1:1,000, AF6259), p-AKT (1:1,000, AF0016), E-cadherin (1:1,000, BF0219), N-cadherin (1:1,000, AF5239), p-mTOR (1:1,000, AF3308), Snail (1:1,000, AF6032), Twist1 (1:1,000, AF4009), GAPDH (1:1,000, AF7021), and β-actin (1:1,000, AF7018) (all from Affinity). After thorough washing with TBST, membranes were incubated with HRP-conjugated secondary antibody (1:1,000, S0001, Affinity) for 1 hour at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL) plus chemiluminescent substrate (PE0010, Solarbio) and detected using a chemiluminescence imaging system.

Statistical analysis

Experimental data were presented as mean ± standard deviation (SD) from at least three independent replicates. Statistical significance was determined using GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA). Differences between groups were considered statistically significant when the P value was less than 0.05 (P<0.05). All statistical tests were two-tailed and performed with appropriate controls for multiple comparisons where applicable.

Results

The relationship between KET23 expression and the prognosis of GC and the sensitivity to chemotherapy drugs

The pathogenesis of GC is complex, and individual prognosis varies significantly. Accurate screening of potential genes related to the prognosis and chemosensitivity of GC is of great significance for optimizing treatment strategies and improving the prognosis of patients. Bioinformatics analysis showed that KRT23 was highly expressed in GC (Figure 1A), and the prognosis of patients with high KRT23 expression was worse (Figure 1B). Combined with GDSC chemosensitivity analysis, KRT23 expression was positively correlated with most drugs and negatively correlated with afatinib, erlotinib, and cetuximab only (Figure 1C). These results suggest that the use of these drugs in patients with high KRT23 expression may have a better tumor inhibition effect, which needs to be further studied.

Figure 1 Correlation analysis of KRT23 expression with prognosis and chemotherapy response. (A) The KM survival curve. (B) Correlation between KRT23 expression and the prognosis of GC patients. (C) Correlation between GDSC drug sensitivity and KRT23 gene mRNA expression. *, P<0.05. CI, confidence interval; FDR, false discovery rate; GC, gastric cancer; GDSC, Genomics of Drug Sensitivity in Cancer; HR, hazard ratio; KM, Kaplan-Meier; KRT23, keratin 23; mRNA, messenger RNA; N, normal; STAD, stomach adenocarcinoma; T, tumor.

Differential expression patterns of KRT23 in GC cell lines

To investigate KRT23 expression patterns in GC, we performed immunohistochemical analysis on clinical specimens comprising both gastric tumor tissues and adjacent normal tissues. As demonstrated in Figure 2A, KRT23 exhibited minimal expression in healthy gastric mucosa, with only faint immunostaining observed. In striking contrast, GC tissues displayed markedly intensified KRT23 expression, as evidenced by prominent brown staining. Through comprehensive analysis using RT-qPCR and Western blot, we found that as shown in Figure 2B-2D, significant high expression of KRT23 was identified in multiple GC cell lines (AGS, MGC803, HGC27, and SGC7901) compared to normal gastric mucosal epithelial cells (GSE-1). Notably, the results showed that KRT23 expression was highest in SGC7901 cells, followed by MGC803 cells. In this study, we selected these two KRT23 cell lines for subsequent experiments to amplify the phenotypic effect of KRT23 by using a high-expression model, to cover core GC subsets with different differentiation statuses and molecular types, and to rely on the adequate representation of classical models to support the reliability of the results. To investigate KRT23’s biological role, we established stable knockdown models through lentiviral transduction followed by puromycin selection. As demonstrated in Figure 2E-2H, the resulting cell lines exhibited >70% silencing efficiency. This robust experimental system enabled precise evaluation of KRT23’s functional significance in GC pathogenesis.

Figure 2 The expression of KRT23 in GC cells. (A) The expression of KRT23 was detected by immunohistochemistry staining method (magnification ×400). (B) RT-qPCR was used to detect the mRNA expression of KRT23 in different GC cells. (C,D) Western blot detected the expression of KRT23 protein in different GC cells. (E,F) RT-qPCR was used to detect the mRNA silencing level of KRT23 in MGC803 and SGC7901 cells. (G,H) Western blots detected the protein silencing level of KRT23 in MGC803 and SGC7901 cells. **, P<0.01; ***, P<0.001. GC, gastric cancer; KRT23, keratin 23; mRNA, messenger RNA; NC, normal control; RT-qPCR, real-time quantitative polymerase chain reaction; shKRT23, short hairpin RNA KRT23; shNC, short hairpin RNA NC.

KRT23 promotes GC cell proliferation

To elucidate the functional role of KRT23 in GC progression, we first assessed its impact on cell proliferation using CCK-8 assays in MGC803 and SGC7901 cells. As shown in Figure 3A,3B, KRT23 knockdown significantly suppressed the proliferative capacity of both cell lines (MGC803, P=0.01; SGC7901, P=0.001). Given the established oncogenic role of PI3K/AKT signaling in GC, we next investigated whether KRT23’s proliferative effects were mediated through this pathway. Treatment with the PI3K activator 740Y-P in KRT23-deficient cells revealed an attenuated proliferative response. While 740Y-P treatment partially restored proliferation compared to KRT23 knockdown alone [short hairpin RNA KRT23 (shKRT23) + 740Y-P vs. shKRT23], this increase did not reach statistical significance. Importantly, the proliferative capacity of 740Y-P-treated, KRT23-deficient cells remained significantly lower than control cells (MGC803, P=0.001; SGC7901, P=0.001). These findings demonstrate that KRT23 depletion substantially diminishes the tumor-promoting effects of PI3K/AKT activation, suggesting that KRT23 may function upstream of this pathway to drive GC cell proliferation.

Figure 3 Effect of KRT23 knockdown on proliferation of GC cells detected by CCK-8. (A) MGC803 cells. (B) SGC7901 cells. shKRT23 vs. shNC: MGC803, P=0.01; SGC7901, P=0.001. shKRT23 + 740Y-P vs. shKRT23: MGC803, P=0.001; SGC7901, P=0.001. CCK-8, Cell Counting Kit-8; GC, gastric cancer; KRT23, keratin 23; NC, normal control; OD, optical density; shKRT23, short hairpin RNA KRT23; shNC, short hairpin RNA NC.

KRT23 promotes the invasion of GC cells

Given the critical role of tumor cell invasion and metastasis in cancer progression and patient prognosis (15), we investigated whether KRT23 regulates the migration and invasive potential of GC cells. The result is shown in Figure 4, we found that KRT23 knockdown significantly reduced the migration capacity of both MGC803 and SGC7901 cells compared to control groups (P<0.001). Notably, this inhibitory effect was partially reversed by treatment with the PI3K/AKT activator 740Y-P (shKRT23 + 740Y-P vs. shKRT23: MGC803, P=0.007; SGC7901, P=0.004), though the migration potential remained lower than control levels [shKRT23 + 740Y-P vs. short hairpin RNA normal control (shNC): MGC803, P=0.02; SGC7901, P=0.03]. The result was shown in Figure 5, complementary gain-of-function experiments demonstrated that KRT23 overexpression (oeKRT23) markedly enhanced GC cell invasion (oeKRT23 vs. KRT23-NC: MGC803, P=0.001; SGC7901, P=0.002). Importantly, this pro-invasive effect was effectively abolished by PI3K/AKT inhibition (oeKRT23 + PI3K-IN-1 vs. oeKRT23: MGC803, P=0.001; SGC7901, P<0.001). These comprehensive results establish that KRT23 significantly promotes the migration and invasive behavior of GC cells, and that this oncogenic function is mechanistically dependent on PI3K/AKT pathway activation. The bidirectional modulation achieved through both genetic and pharmacological interventions provides compelling evidence for a functional interaction between KRT23 and PI3K/AKT signaling in regulating GC cell invasion. These findings position KRT23 as a promising therapeutic target for inhibiting GC metastasis.

Figure 4 Detection of the effect of KRT23 down-regulation and 740Y-P intervention on the invasion ability of GC cells by crystal violet staining method (magnification ×100). shKRT23 vs. shNC: MGC803, P<0.001; SGC7901, P<0.001. shKRT23 + 740Y-P vs. shKRT23: MGC803, P=0.007; SGC7901, P=0.004. shKRT23 + 740Y-P vs. shNC: MGC803, P=0.02; SGC7901, P=0.03. GC, gastric cancer; KRT23, keratin 23; NC, normal control; shKRT23, short hairpin RNA KRT23; shNC, short hairpin RNA NC.

Figure 5 Detection of the effect of oeKRT23 and PI3K-IN-1 intervention on the invasion ability of GC cells by crystal violet staining method (magnification ×100). oeKRT23 vs. KRT23-NC: MGC803, P=0.001; SGC7901, P=0.002. oeKRT23 + PI3K-IN-1 vs. oeKRT23: MGC803, P=0.001; SGC7901, P<0.001. oeKRT23 + PI3K-IN-1 vs. KRT23-NC: MGC803, P<0.001; SGC7901, P=0.001. GC, gastric cancer; KRT23, keratin 23; NC, normal control; oeKRT23, KRT23 overexpression; oeNC, NC overexpression.

KRT23 promotes the EMT process of GC by activating the PI3K/AKT/mTOR signaling pathway

EMT serves as a pivotal mechanism driving GC initiation and progression. The activated PI3K/AKT/mTOR signaling pathway can regulate the EMT process through a variety of ways (7). In this study, Western blot was used to determine the association between KRT23 expression and PI3K/AKT/mTOR and EMT. As shown in Figure 6, consistent results were found in both MGC803 and SGC7901 GC cells, namely, KRT23 knockdown significantly up-regulated E-Cadherin expression [shKRT23 vs. normal control (NC): MGC803, P=0.006; SGC7901, P=0.007]. The expression levels of N-cadherin, Snail, Twist1, and p-PI3K/p-AKT/p-mTOR were down-regulated [shKRT23 vs. NC: MGC803, N-cadherin (P=0.04), Snail (P=0.04), Twist1 (P=0.03), p-PI3K (P=0.009), p-AKT (P=0.005), and p-mTOR (P=0.009); SGC7901, N-cadherin (P=0.009), Snail (P=0.008), Twist1 (P=0.004), p-PI3K (P=0.009), p-AKT (P<0.001), and p-mTOR (P=0.01)], and these effects were reversed by the PI3K activator 740Y-P. In addition, oeKRT23 combined with PI3K inhibitor PI3K-IN-1 intervention experiments were performed in this study. As shown in Figure 7, overexpression of KRT23 in both GC cells activated the signaling pathway and significantly inhibited the expression of E-Cadherin protein [oeKRT23 vs. NC overexpression (oeNC): MGC803, P=0.04; SGC7901, P=0.005], but significantly promoted the expression of N-Cadherin, Snail, Twist1, p-PI3K, p-AKT, and p-mTOR proteins [oeKRT23 vs. NC: MGC803, N-cadherin (P=0.004), Snail (P=0.003), Twist1 (P=0.009), p-PI3K (P=0.009), p-AKT (P=0.008), and p-mTOR (P=0.005); SGC7901, N-cadherin (P=0.006), Snail (P=0.004), Twist1 (P=0.001), p-PI3K (P=0.009), p-AKT (P=0.009), and p-mTOR (P=0.008)]. This effect was blocked by the PI3K inhibitor PI3K-IN-1. These results confirmed that KRT23 regulates the key factors of EMT through the PI3K/AKT/mTOR pathway, thereby affecting the invasion and metastasis of GC cells. This study reveals the key role of KRT23 in regulating the EMT process and the biological behavior of GC cells by regulating this signaling pathway.

Figure 6 The effects of Western blots detection of knocking down KRT23 and 740Y-P intervention on EMT, PI3K/AKT/mTOR signaling pathways. (A) EMT, PI3K/AKT/mTOR pathways, and EMT transcription factor protein bands. (B-H) Expression levels of E-cadherin, N-cadherin, p-PI3K, p-AKT, p-mTOR, Snail, and Twistl in each group. E-cadherin expression: shKRT23 vs. NC: MGC803, P=0.006; SGC7901, P=0.007. N-cadherin, Snail, Twist1, and p-PI3K/p-AKT/p-mTOR were down-regulated: shKRT23 vs. NC: MGC803, N-cadherin (P=0.04), Snail (P=0.04), Twist1 (P=0.03), p-PI3K (P=0.009), p-AKT (P=0.005), and p-mTOR (P=0.009); SGC7901, N-cadherin (P=0.009), Snail (P=0.008), Twist1 (P=0.004), p-PI3K (P=0.009), p-AKT (P<0.001), and p-mTOR (P=0.01). AKT, protein kinase B; EMT, epithelial-mesenchymal transition; KRT23, keratin 23; mTOR, mammalian target of rapamycin; NC, normal control; p-, phospho-; PI3K, phosphatidylinositol 3-kinase; shKRT23, short hairpin RNA KRT23.

Figure 7 Western blot detected the effects of oeKRT23 and PI3K-IN-1 intervention on EMT and PI3K/AKT/mTOR signaling pathway. (A) EMT, PI3K/AKT/mTOR pathways, and EMT transcription factor protein bands. (B-H) Expression levels of E-cadherin, N-cadherin, p-PI3K, p-AKT, p-mTOR, Snail, and Twistl in each group. E-cadherin protein: oeKRT23 vs. oeNC: MGC803, P=0.04; SGC7901, P=0.005. N-cadherin, Snail, Twist1, p-PI3K, p-AKT, and p-mTOR proteins: oeKRT23 vs. NC: MGC803, N-cadherin (P=0.004), Snail (P=0.003), Twist1 (P=0.009), p-PI3K (P=0.009), p-AKT (P=0.008), and p-mTOR (P=0.005); SGC7901, N-cadherin (P=0.006), Snail (P=0.004), Twist1 (P=0.001), p-PI3K (P=0.009), p-AKT (P=0.009), and p-mTOR (P=0.008). AKT, protein kinase B; EMT, epithelial-mesenchymal transition; KRT23, keratin 23; mTOR, mammalian target of rapamycin; NC, normal control; oeKRT23, KRT23 overexpression; oeNC, NC overexpression; p-, phospho-; PI3K, phosphatidylinositol 3-kinase.

Discussion

GC represents a highly heterogeneous malignancy characterized by complex molecular mechanisms and dysregulated signaling pathways that drive its development and progression (1). Despite advancements in diagnostic and therapeutic approaches, the prognosis for advanced GC patients remains poor, underscoring the critical need to elucidate novel molecular targets for more effective therapeutic strategies. Emerging evidence has implicated KRT23 as a pro-tumorigenic factor in various malignancies, including colorectal, breast, and cervical cancers (15,17,18), yet its functional role in GC pathogenesis remains incompletely understood. In this study, bioinformatics analysis revealed that KRT23 is highly expressed in GC and associated with poor patient prognosis, with its high expression enhancing tumor cell sensitivity to afatinib and erlotinib. These findings not only preliminarily confirm KRT23 as a prognostic marker for GC but also suggest its potential role in treatment response via regulating chemotherapy sensitivity, providing a theoretical basis for further exploring its functional mechanisms and signaling pathway regulation in GC pathogenesis.

Recent investigations have demonstrated that KRT23 deficiency potentiates melatonin’s anti-tumor effects in GC by suppressing colony formation and inducing cell cycle arrest (13). Building upon these findings, our study systematically investigated KRT23’s functional mechanisms in GC. We identified significant high expression of KRT23 across multiple GC cell lines (AGS, MGC803, HGC27, and SGC7901), and through comprehensive gain- and loss-of-function experiments, established that KRT23 critically regulates the proliferative and invasive capacities of MGC803 and SGC7901 cells. This result was consistent with previous reports (13), suggesting the potential of KRT23 as a therapeutic target for GC. The pathogenesis of GC involves complex interplay among multiple signaling pathways, including PI3K/AKT/mTOR, Wnt/β-catenin, transforming growth factor-β (TGF-β), and so on (8-10), which collectively regulate fundamental oncogenic processes such as proliferation, metabolic reprogramming, and EMT (9-11). EMT, as a central mechanism underlying tumor metastasis, is characterized by E-cadherin downregulation and N-cadherin upregulation, processes tightly controlled by various signaling cascades (12,19). Furthermore, these pathways contribute to TME remodeling, angiogenesis, and immune evasion, thereby cooperatively promoting GC invasion and metastasis (20,21).

The PI3K/AKT/mTOR signaling axis serves as a central regulator of fundamental cellular processes including growth, proliferation, survival, and metabolic homeostasis (22,23). In GC pathogenesis, dysregulation of this pathway is frequently observed and recognized as a critical driver of tumorigenesis and progression (24). Substantial evidence demonstrates that PI3K/AKT/mTOR activation promotes key oncogenic processes in GC, including cellular invasion, migration, proliferation, and EMT (25-27). Notably, keratin 17 (KRT17), a structural homolog of KRT23 within the keratin family, has been shown to mediate E-cadherin suppression, EMT induction, and metastatic progression in diffuse GC (28). KRT23 and KRT17 belong to the keratin family and share certain structural similarities. They are important members of the intermediate filament protein family and play a key role in maintaining cell structure and function (12). Therefore, this study speculates that KRT23 may affect GC progression through the PI3K/AKT/mTOR signaling pathway. The results showed that the levels of p-AKT, p-PI3K, and p-mTOR were significantly decreased in MGC803 and SGC7901 cells with KRT23 knockdown compared with the control group. This suggests that KRT23 may regulate the activity of the PI3K/AKT/mTOR signaling pathway by affecting its phosphorylation level, thereby influencing the occurrence and development of GC. Subsequently, through PI3K pathway activator and inhibitor intervention, the PI3K/AKT activator 740Y-P could reverse the inhibitory effect of KRT23 knockdown and activate the PI3K/AKT/mTOR pathway, indicating that KRT23 may affect the behavior of GC cells by regulating the pathway. Overexpression of KRT23 enhanced the invasion ability of GC cells. The PI3K-IN-1 inhibitor inhibited this pathway activated by oeKRT23, confirming that KRT23 promoted the proliferation of GC cells in a manner dependent on the PI3K signaling pathway. Our findings position KRT23 as a novel regulator within this complex signaling network, offering new insights into GC pathogenesis and potential therapeutic avenues.

The PI3K/AKT/mTOR signaling cascade serves as a pivotal regulator of multiple oncogenic processes, including tumor cell proliferation, migration, and invasion. This pathway exerts its biological effects primarily through downstream activation of AKT and mTOR, which coordinately drive cell cycle progression and enhance protein synthesis (29-31). Mechanistically, AKT-mediated phosphorylation inhibits GSK3β activity, leading to β-catenin stabilization and subsequent nuclear translocation. This process activates transcription of proliferative genes, thereby promoting tumor growth (32). Parallel to AKT signaling, mTOR orchestrates ribosome biogenesis and protein translation initiation, establishing the biosynthetic foundation required for cellular expansion and division (33). Cell membrane tyrosine kinase receptors, especially EGFR, play a crucial role in the progression of various cancers, including GC. A study has shown that EGFR can activate the PI3K/AKT signal transduction pathway, thereby promoting the malignant proliferation, invasion, and metastasis of GC cells (34). It is worth noting that keratin 1 (KRT1) and KRT17, which are also cytoskeletal proteins, have been confirmed to be closely related to tyrosine kinase receptors on the surface of the cell membrane, and they can affect receptor signal transduction by regulating the stability of the membrane microenvironment and changing the spatial conformation of receptors (35). However, as a member of the keratin family, the relationship between KRT23 and tyrosine kinase receptors is still unclear, which needs to be further studied. In terms of upstream regulation of KRT23, a recent study in colorectal cancer (CRC) has revealed a novel regulatory mechanism of microRNA miR-195-5p on keratin intermediate filaments; that is, miR-195-5p can target KRT23 mRNA and inhibit its translation process. It can effectively inhibit the progression of cancer cells (15). This provides a new direction for exploring the molecular regulatory mechanism of KRT23 in GC.

In GC, hyperactivation of the PI3K/AKT/mTOR axis has been shown to upregulate key cell cycle regulators, including CyclinD1, facilitating G1 to S phase transition and ultimately driving uncontrolled proliferation (36). While our findings demonstrate KRT23’s ability to modulate PI3K/AKT/mTOR pathway activity, the precise mechanistic link between KRT23 expression and cell cycle regulation in GC remains to be fully elucidated. Further investigation is warranted to determine whether KRT23 influences cell cycle progression through direct interaction with this signaling cascade or via alternative regulatory mechanisms.

EMT is a key biological event in the progression of GC, a process in which epithelial cells transform into cells with a mesenchymal phenotype through specific molecular mechanisms, which is closely related to tumor cell invasion, migration, and metastasis (11,12). In the process of EMT, the expression of the epithelial cell marker E-cadherin is down-regulated, while the expression of mesenchymal cell markers N-cadherin and vimentin is up-regulated, which usually leads to the reduction of intercellular adhesion and the enhancement of cell motility and invasion (12). The PI3K/AKT/mTOR pathway plays a key role in the regulation of EMT (13). Peritoneal metastasis is a common malignant event in patients with advanced GC, and its survival rate is low, while EMT has a non-negligible regulatory role (37). This is essential for the metastasis of GC cells. Relevant studies have found that the induction of EMT can promote peritoneal metastasis of GC cells through the PI3K/AKT/mTOR pathway (38,39). In this study, we found that KRT23 expression significantly regulated the EMT process: after KRT23 knockdown, the expression of E-cadherin was significantly increased, and the levels of N-cadherin, Snail, and Twist1 were decreased, which were reversed by the PI3K/AKT activator 740Y-P. Conversely, overexpression of KRT23 resulted in the down-regulation of E-cadherin and the up-regulation of N-cadherin, Snail, and Twist1, which were blocked by the PI3K-IN-1 inhibitor. In addition, a study has confirmed that KRT23 expression in ovarian cancer can promote cancer cell migration through EMT mediated by the TGF-β/Smad axis (40). This idea is further supported by the present study, which shows that KRT23 expression has a regulatory role in EMT. These results fully prove that KRT23 can regulate the EMT process of GC cells through the PI3K/AKT/mTOR signaling pathway, and then have an important impact on the biological behavior of GC cells. The results of this study are consistent with the previously reported roles of some oncogenes in GC (41-43). In the progression of a variety of cancers, the signaling pathways related to EMT are highly complex. Classical pathways such as TGF-β/Smad and Wnt/β-catenin play a key role in tumor invasion and metastasis by precisely regulating the EMT process (40,44). However, this study only focused on the effect of KRT23 on the EMT of GC cells through the PI3K/AKT/mTOR pathway, and the potential correlation between KRT23 expression levels and other important signaling pathways, as well as the synergistic regulatory mechanisms in the occurrence and development of GC, still need to be further explored and clarified.

Notably, this study demonstrated that KRT23 mediates EMT to drive the malignant phenotype of GC cells through the PI3K/AKT/mTOR pathway, but its effect in the TME in vivo remains to be explored. The interaction between the TME and KRT23 may affect the progression of GC, and KRT23 expression can distinguish “hot/cold tumors”, suggesting its immunomodulatory potential (18). Mechanistically, KRT23 may remodel the extracellular matrix or regulate immune checkpoints by activating the PI3K/AKT/mTOR pathway. However, the direct interaction between KRT23 and TME components, and whether it affects the TME through cancer-associated fibroblasts (CAFs) paracrine or hypoxia inducible factor-1α (HIF-1α)-mediated hypoxia response, needs to be verified in vivo.

In addition, although KRT23 promotes malignant phenotypes in both cells representing different differentiation states, GC is highly molecularly heterogeneous, with Epstein-Barr virus (EBV)⁺/high microsatellite instability (MSI-H)/chromosomal instability (CIN)/genomically stable (GS) subtypes in The Cancer Genome Atlas (TCGA) classification. Different subtypes may vary in the degree of KRT23 downstream pathway activation or treatment sensitivity due to differences in driver mutations or the microenvironment. Therefore, the conclusions of the current study are mainly applicable to the subgroups represented by the models involved in this study. In the future, hierarchical validation according to molecular typing is needed in organoids and patient-derived xenograft (PDX) models to accurately identify the potential benefit population of KRT23 targeted therapy.

In conclusion, the present study strongly supports that KRT23 promotes the proliferation, migration, and invasion of GC by mediating EMT through the PI3K/AKT/mTOR signaling pathway. Given the close connection between KRT23 and the PI3K/AKT/mTOR signaling pathway, combined targeting of KRT23 and PI3K signaling pathways is expected to be an effective strategy for the treatment of GC. For patients with high KRT23 expression and excessive activation of the PI3K signaling pathway, combined targeted therapy is expected to achieve better efficacy for the treatment of GC patients.

Conclusions

In summary, the present study confirmed that KRT23 promotes EMT progression in GC by activating the PI3K/AKT/mTOR signaling pathway, thereby driving tumor malignant progression. Despite the clear results obtained in vitro, the following limitations remain. First, validation data from in vivo animal models are insufficient. Second, the mechanism by which KRT23 may affect EMT through other signaling pathways besides the established PI3K/AKT/mTOR pathway still needs to be explored in depth. Future studies need to construct more complete animal models to clarify the mechanism of KRT23 in the TME. In conclusion, the present study systematically revealed the key role of KRT23 in the regulation of EMT in GC for the first time and proposed its potential clinical value as a novel therapeutic target for GC.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-872/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-872/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-872/prf

Funding: This work was supported by Science and Technology Plan Project of Guizhou Province (No. TS21011).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-872/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The protocol was approved by the Ethics Committee of The Affiliated Hospital of Guizhou Medical University (No. 2020[252]). All patients signed informed consent.

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

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