TCHP regulates the proliferation and apoptosis of gastric cancer cells via AKT/BCL2 signaling pathway

Abstract

BACKGROUND

Gastric cancer (GC) imposes a substantial global health burden because of its high incidence and mortality. Its marked heterogeneity and poor prognosis have spurred intensive research. Therefore, identifying driver genes and elucidating the molecular mechanisms underlying GC are essential for developing more effective therapeutic strategies.

AIM

To evaluate the involvement of Trichoplein (TCHP) in controlling cellular proliferation and apoptotic processes, and to elucidate the underlying molecular mechanisms with a focus on the AKT/BCL2 signaling axis.

METHODS

TCHP expression was examined in GC cell lines and a normal gastric epithelial cell line. The Cancer Genome Atlas data analysis was performed using UALCAN, and survival analyses were conducted using KM Plotter. TCHP was knocked down using shRNA lentivirus, and the effects were analyzed using CCK-8, colony formation assay, flow cytometry, and Ki67 immunofluorescence. Proteomic profiling and Western blotting were used to study apoptotic mechanisms. Tumor growth and histopathological changes were assessed using a nude mice subcutaneous xenograft model.

RESULTS

Both in vitro experiments and analyses comparing tumor tissues with adjacent normal tissues showed increased TCHP expression in GC, and higher TCHP expression was significantly associated with poorer survival. TCHP knockdown markedly inhibited GC cell proliferation. In GC cells, reducing TCHP expression caused G1 phase accumulation and facilitated apoptotic progression. Proteomic profiling suggested that TCHP-associated changes were enriched in apoptosis-related pathways, and further pathway analysis highlighted PI3K-AKT signaling. Mechanistically, TCHP knockdown suppressed AKT/BCL2 signaling, inhibited tumor growth, and enhanced apoptosis. In vivo, TCHP knockdown significantly reduced tumor growth in xenograft models.

CONCLUSION

TCHP functions to facilitate proliferation and suppress apoptosis in GC cells through the AKT/BCL2 axis; thus, TCHP is suggested to be a promising therapeutic target for GC.

Key Words: Gastric cancer; TCHP; Proliferation; Apoptosis; AKT; BCL2

Core Tip: The function and molecular mechanism of Trichoplein (TCHP) are poorly understood in gastric cancer (GC). This study investigated the role of the centrosomal protein TCHP in GC progression. TCHP was highly expressed in GC cells, and it significantly inhibited cell proliferation, arrested the cell cycle at G1 phase, and promoted apoptosis. Proteomic analysis revealed that TCHP influenced apoptosis via AKT/BCL2 pathway. In vivo experiments further confirmed that TCHP knockdown suppressed tumor growth. These results demonstrate that TCHP promotes GC proliferation and inhibits apoptosis by activating the AKT/BCL2 pathway, suggesting its potential as a therapeutic target in GC.

INTRODUCTION

Characterized by high incidence and dismal survival outcomes, gastric cancer (GC) stands as a major global health problem[1]. Even with significant progress in surgery and chemotherapy, the management of advanced GC remains a significant clinical challenge, as patient outcomes are often unsatisfactory[2,3], largely attributable to its aggressive tumor biology, characterized by rapid proliferation, a high propensity for metastasis, and frequent development of drug resistance[4]. Therefore, elucidating the key genes and understanding the molecular mechanisms driving GC evolution and identifying novel therapeutic targets remain a pressing priority in GC research.

Trichoplein (TCHP) is a centriole-localized centrosomal protein that regulates fundamental processes, including cytoskeletal dynamics, ciliogenesis, and cell cycle progression[5,6]. This protein regulates primary cilium assembly by binding to and activating the Aurora a kinase (AURKA), a key centrosomal kinase, thereby suppressing aberrant ciliogenesis in proliferating cells[7,8]. Clinical studies have revealed the downregulation of TCHP expression in bladder and breast cancers[9]. TCHP gene deletion in mice inhibits the insulin/AKT signaling pathway[10]. Growing evidence suggests that TCHP may act as a cancer-related gene that contributes to malignant phenotypes by modulating oncogenic signaling pathways[11]. However, the potential role of TCHP in GC and the exact mechanisms remain to be elucidated. Here, we focused on the physiological functions of the centrosomal protein TCHP, aiming to elucidate how TCHP influences cell fate decisions and regulates GC initiation and progression.

Identifying the key molecules that drive tumor cell proliferation and survival is essential for developing innovative therapies for GC. The serine/threonine kinase AKT is a prime candidate that functions as a central hub regulating cell survival, proliferation, metabolism, and apoptosis. Hyperactivation of the AKT pathway is a common feature in various human cancers, encompassing GC[12,13]. Consequently, activated AKT phosphorylates downstream substrates, including BCL2 family proteins, thereby suppressing apoptosis and promoting tumor cell survival[14]. Given that AKT/BCL2 signaling is critical in GC, we hypothesized that TCHP may act as an upstream regulator to promote tumor cell proliferation and survival through sustained activation of this pathway. Elucidating this mechanism would provide important insights into GC pathogenesis and position TCHP as a promising biomarker and therapeutic target.

This study revealed that TCHP is highly expressed in GC. Following TCHP knockdown, proteomic analysis revealed the enrichment of apoptosis-related pathways, with secondary enrichment specifically in the PI3K-AKT pathway. Within this downregulated module, Western blotting demonstrated a reduction in phosphorylated AKT level at its activation site (S473). Additionally, BCL2, a key anti-apoptotic protein known to be transcriptionally regulated by AKT, was significantly downregulated. Together, these data outline a clear hierarchical regulatory model: TCHP serves as an important factor in sustaining PI3K-AKT pathway activity, and activated AKT promotes the expression of downstream targets such as BCL2, thereby collectively suppressing apoptosis. TCHP loss disrupts this survival signaling axis, leading to the overall activation of apoptotic pathways.

MATERIALS AND METHODS

TCHP gene expression profiling and survival evaluation

This work was to examine the expression and therapeutic implications of TCHP in GC. To assess TCHP differential expression, we first employed the UALCAN online tool, interrogating The Cancer Genome Atlas (TCGA) data for comparison of its levels in GC vs normal adjacent tissue. Expression levels were visualized using a box plot, and group differences were evaluated by the Mann-Whitney U test. Subsequently, we used the KM Plotter online platform to draw Kaplan-Meier survival curves and evaluated the prognostic value of TCHP expression, as well as its HR and 95%CI, through the Cox proportional hazards model. Derived from the median TCHP expression, patients were classified as having high or low TCHP expression using the median expression level to be the cutoff and a survival comparison was performed between groups applying the Log-rank test.

Cell culture

AGS and HGC-27 GC cell lines and the GES-1 gastric epithelial cell line were obtained from Wuhan Pricella Biotechnology Co., Ltd. (Wuhan, China). All cell lines were cultured in RPMI-1640 medium (Gibco, Carlsbad, CA, United States) and maintained at 37 °C in a humidified incubator with 5% CO₂. Cells were passaged at 70%-80% confluence using trypsin-EDTA and subcultured at a 1:3 ratio, with medium refreshed routinely according to cell growth state.

TCHP knockdown by shRNA lentivirus transfection

GC cells were seeded at an appropriate density. After removal of the spent medium, cells were incubated with transduction medium containing TCHP-knockdown shRNA lentivirus (GeneChem Co., Ltd., Shanghai, China). At 72 hours post-transduction, puromycin selection was applied to generate stable TCHP-knockdown cell lines, which were then used for downstream experiments.

Western blotting assay

To assess TCHP protein expression, Western blotting assay was employed. For Western blot analysis, lysed the cell samples using the RIPA buffer (Solarbio, Beijing, China), and determine the protein concentration using the BCA protein quantification kit (Beyotime, Shanghai, China). Subsequently, proteins were separated by SDS-PAGE and transferred to PVDF membranes. Following a 2-hour block at room, the membrane was placed at 4 °C and incubated with the appropriately diluted primary antibody overnight. The main antibodies used in the experiment were: TCHP (1:3000, 25931-1-AP, Proteintech), P21 (1:5000, 10355-1-AP, Proteintech), CDK4 (1:4000, 11026-1-AP, Proteintech), cyclin D1 (1:5000, 26939-1-AP, Proteintech), BCL2 (1:2000, CY5032, Abways), BAX (1:2000, CY5059, Abways), AKT (1:1500, CY5551, Abways), p-AKT (Ser473; 1:1500, CY5885, Abways), GAPDH (1:10000, 60004-1-Ig, Proteintech), and β-actin (1:2000, ZB15001-HRP-100, Servicebio). Subsequently, incubation with the corresponding secondary antibodies was carried out for 1 hour at ambient temperature. Immunoreactive bands were visualized using ECL reagents (Share-Bio, Shanghai, China). The resulting chemiluminescent signals were display using a gel imaging system (Amersham ImageQuant 800; Amersham Biosciences, United States). Image analysis was performed using ImageJ software.

CCK-8 assay

GC cells were trypsinized, counted, and seeded into 96-well plates. At the indicated time points (24, 48, 72, and 96 hours), 10 μL CCK-8 reagent was added. Absorbance was then measured at 450 nm (optical density = 450) using a microplate reader (EPOCH-SN, BioTek Instruments, United States).

Colony formation assay

Place the cells in a 6-well plate at a density of 1000 cells per well. After cell attachment, colony formation was monitored every 2 days. When colonies became visible and reached an appropriate size, colonies were fixed with 4% PFA (500 μL per well) for 20 minutes. Colonies were stained with 1% crystal violet (500 μL per well; Beyotime, Shanghai, China) for 20 minutes in the dark. After removal of the staining solution, stained colonies were then counted for quantitative analysis.

Flow cytometric analysis of the cell cycle

We first treated the cells, washed the cells with pre-cooled​ PBS, then fixed the cells with 70% ethanol, and placed them in a 4 °C refrigerator overnight. Subsequently, we took out the samples, centrifuged to remove the ethanol, washed the samples with PBS, incubated with RNase A (100 μg/mL) at room temperature for 30 minutes, and​ stained with PI (Elabscience, Wuhan, China) for 30 minutes in the dark. Finally, data were collected using a BD FACSLyric^TM flow cytometer (BD Biosciences, United States), and analyzed using ModFit LT software (Verity Software House, United States).

Apoptosis detection by flow cytometry

We collected the cells and washed them with PBS, resuspended the cells using 1 × binding buffer according to the kit instructions, and adjusted the cell concentration. Subsequently, the cells were stained with Annexin V-Elab Fluor 647 and PI for 15 minutes (Annexin V-Elab Fluor 647/PI, Elabscience, Wuhan), and all steps were performed under dark conditions. The samples were immediately analyzed using a BD FACSLyri^TM flow cytometer (BD Biosciences, United States), and were analyzed using FlowJo software (BD Biosciences, United States).

Ki67 immunofluorescence staining

AGS and HGC-27 cells were trypsinized, collected by centrifugation, and fixed with 4% paraformaldehyde. After fixation, cells were resuspended in 500 μL PBS, and 200 μL of the suspension was placed onto glass slides and air-dried. Slides were permeabilized with 0.1% Triton X-100 and then blocked with 5% BSA (GC305010, Servicebio, Wuhan, China) for 1 hour at room temperature. Slides were incubated with rabbit anti-Ki67 antibody (1:300, Servicebio, Wuhan, China) at 4 °C overnight, followed by incubation with a CoraLite594-conjugated secondary antibody (1:500, Proteintech) for 1 hour, in the dark. Nuclei were labeled with DAPI (G1012, Servicebio, Wuhan, China), and the slides were coverslipped using an anti-fade mounting medium. Slides were scanned using a Pannoramic MIDI scanner (3DHISTECH). For quantitative analysis, two investigators blinded to group allocation independently evaluated at least five randomly selected fields per slide. ImageJ was used to calculate the proliferation index as the proportion of Ki67-positive nuclei relative to total DAPI-stained nuclei.

Gene and pathway analysis using proteomic sequencing

AGS cells were divided into two groups: Control group and shTCHP3 group. Three independent biological replicates per group were included for proteomic analysis (n = 3 per group). Cells were cultured to 70%-90% confluence, trypsinized, and collected by centrifugation at 500 × g. Protein samples were analyzed with an iST sample preparation kit (PreOmics). Following desalting via an iST cartridge, the peptides were purified by elution (2 × 100 µL of buffer), vacuum-concentrated.

For analysis using liquid chromatography-tandem mass spectrometry, the peptides were taken up in mobile phase A and then treated with liquid chromatography-mass spectrometry/mass spectrometry. An UltiMate 3000 nano-LC system was used, and the eluate was analyzed in real-time by a timsTOF Pro2 mass spectrometer (Bruker Daltonics). Peptide samples (200 ng) were subjected to chromatographic separation on an AUR3-15075C18 column (150 mm × 75 μm, 1.7 μm, 120 Å; IonOpticks) using a 60-minutes linear gradient at 400 nL/min and 50 °C. The mobile phase composition (B: 80% acetonitrile, 0.1% formic acid) was programmed as follows: 4% B (initial), solvent B was raised from the starting conditions to 28% at 25 minutes, further to 44% over the next 10 minutes, and to 90% over an additional 10 minutes. The system was held at 90% B for 7 minutes before returning to 4% B for an 8-minute re-equilibration. Spectra were collected in diaPASEF mode using an m/z range of 349-1229 and a 40 Da isolation window.

DIA information were analyzed in Spectronaut 18 using BGS factory settings. Carbamidomethylation (C) was set as a fixed modification, and oxidation (M) was set as a variable modification. Retention time alignment and m/z calibration were performed using the iRT peptides. Protein and precursor false discovery rates were controlled at 1.0%. A decoy database was generated by random shuffling, and the data were normalized locally. MaxLFQ was used for labelfree quantification at the protein group level. Proteins with an absolute fold change > 1.2 and P < 0.05 between groups were differentially expressed.

Animal experiment

The animals were supplied by Beijing SPF Biotechnology Laboratory Animal Technology Co., Ltd. Female BALB/c nude mice, aged 4-6 weeks, were used in accordance with institutional guidelines for laboratory animal care and use. Mice were randomly assigned to two groups (n = 4 per group): NC and shTCHP3. AGS cells stably expressing NC or shTCHP3 were injected subcutaneously into the corresponding groups of mice. Tumor dimensions were measured with calipers at 7-day intervals, and tumor volumes were calculated as 0.5 × length × width². At the endpoint, tumors were excised and weighed.

Histological analysis by H&E staining

For histological examination, the tissue samples were fixed using a standard protocol in 4% paraformaldehyde. The subsequent histological processing involved dehydrating the tissues, clearing them in xylene, and embedding them in paraffin wax, all following standard protocols. Thin sections (4-5 μm) were then microtomed for H&E staining to visualize nuclear morphology (Servicebio, Wuhan, China). After hematoxylin staining and differentiation in 1% acid alcohol, the sections were counterstained with eosin, dehydrated, and finally mounted with neutral resin. Finally, sections were imaged using a light microscope (Nikon Eclipse E100, Japan).

Immunohistochemical staining for Ki67 expression

Immunohistochemical (IHC) staining for Ki67 was performed on formalin-fixed, paraffin-embedded (FFPE) tumor sections. After performing deparaffinization, rehydration, and antigen unmasking (citrate buffer, pH = 6.0), we treated the specimens with a rabbit anti-Ki67 antibody overnight at 4 °C (1:300, Servicebio). After washing, sections were incubated with the corresponding secondary antibody (1:500, Proteintech) for 1 hour in the dark. Signal detection was carried out using an HRP-polymer system with DAB chromogen, followed by hematoxylin counterstaining. Ki67 expression was evaluated in a blinded manner by two independent pathologists. The proliferation index was derived from the proportion of Ki67-positive tumor nuclei relative to a minimum count of 500 tumor cells in representative high-power fields. Cases were classified as Ki67-high (≥ 20% positivity) or Ki67-low (< 20%) according to a predefined cutoff for GC.

TUNEL staining for apoptosis detection in animal tissues

Apoptosis in the tissue sections was quantified using a commercial TUNEL assay kit (G1502, Servicebio, China). Following standard deparaffinization and rehydration procedures, which included dewaxing in xylene, gradual rehydration in an ethanol gradient, and a final rinse with distilled water, the TUNEL assay was performed. After epitope retrieval with proteinase K (20 minutes for paraffin sections) at 37 °C, the sections were treated with the TUNEL reaction mixture under humidified parameters and following a 10-minute DAPI counterstain. Imaging was performed using a fluorescence microscope (Nikon, Tokyo, Japan), the slides were mounted with an anti-fade mounting medium. Apoptotic cells (TUNEL positive and red fluorescence) and total nuclei (DAPI stained and blue fluorescence) were quantified.

Statistical analysis

Statistical analysis was carried out with GraphPad Prism (version 10.1.2) software. All dataset were presented as mean ± SD from at least three independent replicates. The normality of the data distribution was verified using the Shapiro-Wilk test. For comparisons between the two groups, an unpaired two-tailed Student's t-test was applied. For comparisons among more than two groups, one-way analysis of variance (ANOVA) was used with Tukey’s HSD test for post-hoc multiple comparisons. When the effects of two independent categorical parameters were analyzed, a two-way ANOVA was conducted, proceeding to Šídák's multiple comparisons test. Results were deemed significant when P < 0.05.

RESULTS

TCHP was upregulated in GC and promoted cell proliferation

Western blotting was employed to examine TCHP expression in AGS cells and HGC-27 cells vs the normal gastric epithelial cell line GES-1. Notably, TCHP protein was upregulated in the GC cells (Figure 1A). Given their high TCHP expression and favorable growth characteristics, AGS cells and HGC-27 cells served as in vitro models for the following investigations.

Figure 1 TCHP is upregulated in gastric cancer and promotes tumor cell proliferation. A: Protein expression levels of TCHP in the gastric mucosal epithelial cell line (GES-1) and two gastric cancer (GC) cell lines (AGS and HGC-27) were detected by Western blotting; B: TCHP expression was significantly higher in GC tissues compared with adjacent non-tumor tissues; C: High TCHP expression was significantly associated with a lower overall survival rate in patients with GC; D: Western blotting confirmed the knockdown efficiency of TCHP in AGS and HGC-27 cells transfected with shTCHP3, compared to the negative control group; E: Cell proliferation following TCHP knockdown was assessed in AGS and HGC-27 cells using CCK-8 assay; F: Cell proliferation following TCHP knockdown was assessed in AGS and HGC-27 cells using colony formation assay. Data are presented as mean ± SD of three independent experiments. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. NC: Negative control; STAD: Stomach adenocarcinoma; TCGA: The Cancer Genome Atlas.

TCHP expression was evaluated in TCGA samples using UALCAN, with tumor-adjacent comparisons performed accordingly. Analysis showed that TCHP was elevated in GC tissues, which complemented and extends our experimental observations (Figure 1B). To examine the clinical correlation between TCHP expression and prognosis, we performed a survival analysis. Kaplan-Meier analysis showed that high TCHP expression was linked to poor overall survival (Figure 1C). The survival curves separated early during follow-up and maintained a consistent divergence, suggesting that high TCHP expression may have prognostic value in GC patients.

To investigate TCHP’s role in cell proliferation, we knocked down its expression using shRNA. Western blotting confirmed efficient TCHP knockdown in GC cells (Figure 1D). The CCK-8 assay demonstrated a marked decrease in proliferative capacity following TCHP knockdown (Figure 1E). Consistent with this finding, a marked reduction in colony formation was observed after TCHP knockdown (Figure 1F). Thus, TCHP functioned as a critical regulator of GC cell proliferation.

TCHP knockdown triggered G0/G1 arrest and promoted apoptosis

Immunofluorescence revealed a marked reduction in Ki67-positive cells upon TCHP knockdown, indicating impaired proliferation (Figure 2A). Flow cytometry further demonstrated pronounced G0/G1 phase arrest, with an increased proportion of cells in G0/G1 and a decreased S phase fraction (Figure 2B). Moreover, TCHP knockdown significantly promoted apoptosis (Figure 2C). The detailed flow cytometry gating diagram is provided in the Supplementary Figure 1. These data indicated that TCHP knockdown suppressed proliferation by leading to G0/G1 phase arrest and activating apoptosis in GC cells.

Figure 2 TCHP knockdown suppresses gastric cancer cell proliferation by inducing G1 phase cell cycle arrest and apoptosis. A: Effect of TCHP knockdown on cell proliferation was assessed by Ki67 immunofluorescence staining; B: Analysis by flow cytometry showed that TCHP knockdown induced G1 phase cell cycle arrest in AGS and HGC-27 cells; C: Apoptosis in AGS and HGC-27 cells following TCHP knockdown was analyzed using flow cytometry. Data are presented as mean ± SD based on three independent experiments. ^bP < 0.01; ^cP < 0.001. NC: Negative control.

Functional enrichment analysis of proteomics-driven differentially expressed proteins

To determine the mechanistic basis of TCHP in controlling GC cell proliferation, we performed quantitative proteomic profiling of AGS cells following TCHP knockdown. Principal component analysis distinguished NC and shTCHP3 groups, indicating substantial proteomic alterations upon TCHP downregulation (Figure 3A). Volcano plot analysis identified 845 differentially expressed proteins, comprising 601 upregulated and 244 downregulated proteins (adjusted P < 0.05; Figure 3B). TCHP knockdown disrupted metabolic homeostasis, antioxidant defense, and survival signaling, as suggested by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, which highlighted apoptosis-related signaling as a major pathway linked to TCHP (Figure 3C). Notably, apoptosis was the most significantly enriched pathway identified. Also, gene set enrichment analysis showed that TCHP knockdown markedly activated the apoptotic pathway (Figure 3D), supporting the conclusion that TCHP loss activated pro-apoptosis signaling pathways. TCHP knockdown significantly altered the expression profile of a cluster of apoptosis and autophagy-related genes (BAD, BAK1, CASP7, and others), as visualized in the hierarchical clustering heatmap (Figure 3E). The PI3K/AKT signaling pathway was identified through additional enrichment analysis (Figure 3F). Given its central role in regulating cell survival and apoptosis, this finding suggests that the phenotypic effects observed upon TCHP depletion may be PI3K/AKT-dependent.

Figure 3 Proteomic analysis identifies differentially expressed proteins following TCHP knockdown. A: Principal component analysis demonstrates distinct clustering between negative and shTCHP3 groups; B: Volcano plot shows the distribution of differentially expressed proteins (DEPs); C: Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis reveals significant enrichment in metabolic and apoptosis pathways; D: Gene set enrichment analysis indicates a significant upregulation of the apoptosis pathway in TCHP-knockdown gastric cancer cells; E: A heatmap shows the expression levels of key DEPs in the apoptosis pathway; F: KEGG secondary enrichment analysis identifies significant enrichment of proteins in the PI3K/AKT pathway.

Based on proteomic profiling, TCHP was implicated in regulating metabolic balance and survival signaling in GC cells. TCHP knockdown disrupted homeostasis, thereby inducing programmed cell death and subsequent proliferation arrest.

TCHP regulated AKT/BCL2 signaling pathway in GC cells

Considering the potential role of TCHP in cell cycle regulation by G1/S checkpoint control, we hypothesized that it modulates key cyclins or kinases. To examine if TCHP regulates cell cycle progression, we examined expression levels of critical G1 regulators, including p21, cyclin D1, and CDK4. Western blotting revealed that TCHP knockdown significantly upregulated p21 and downregulated cyclin D1 and CDK4 (Figure 4A). This concerted downregulation indicates that TCHP silencing induces G1 arrest, thereby suppressing GC cell proliferation.

Figure 4 TCHP knockdown modulates the expression of cell cycle- and apoptosis-related proteins and affects associated signaling pathways. A: Western blotting shows that TCHP knockdown upregulates p21 and downregulates cyclin D1 and CDK4. This pattern indicates the induction of G1 phase cell cycle arrest; B: Western blotting of AKT signaling and apoptosis-related proteins shows that TCHP knockdown reduces p-AKT and BCL2 expression, while increasing BAX levels. These changes suggest an inhibition of AKT-mediated survival signaling and promotion of apoptosis. Data are presented as mean ± SD from three independent experiments. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. NC: Negative control.

To identify TCHP downstream mediators, proteomic sequencing followed by KEGG pathway analysis was performed, revealing apoptosis as a key pathway associated with TCHP. As AKT is a central signaling hub that regulates cell survival, proliferation, metabolism, and apoptosis, we next examined the AKT/BCL2 signaling axis. Western blotting demonstrated that TCHP knockdown consistently suppressed p-AKT and BCL2 while increasing the pro-apoptotic protein BAX in AGS cells and HGC-27 cells (Figure 4B). Collectively, these data indicate that the AKT/BCL2 pathway is a critical downstream signaling axis of TCHP in GC cells.

Therefore, the knockdown of TCHP led to arrest of the cell cycle and promoted apoptosis, culminating in suppressed proliferation. Moreover, TCHP appeared to promote G1/S progression and suppress apoptosis via the AKT/BCL2 axis.

TCHP knockdown suppressed GC cell growth in vivo

We assessed the function of TCHP in vivo using a subcutaneous xenograft GC model in BALB/c nude mice. Relative to the negative control (NC) group, the shTCHP3 group showed markedly inhibited tumor growth, evidenced by a considerable decrease in tumor volume (P < 0.05; Figure 5A and B). The mean tumor weight in the NC control group was higher than that in the shTCHP3 group (NC: 0.45 ± 0.11 g, shTCHP3: 0.18 ± 0.02 g, P < 0.05; Figure 5C). Histopathological analysis revealed distinct morphological differences between the NC control and the shTCHP3 group. Tumors in the NC group exhibited hyperchromatic nuclei with marked heterogeneity in size and shape, prominent nucleoli, poorly defined cell boundaries, and overall features indicative of high malignancy. Conversely, tumors from the shTCHP3 group displayed round or oval cells with enlarged blue-purple nuclei and pink cytoplasm. The background consisted of an evenly distributed pink stroma accompanied by fibrous and vascular structures (Figure 5D).

Figure 5 TCHP knockdown suppresses gastric cancer cell growth in vivo. A: Tumor volumes in mouse xenograft models were compared between negative control (NC) and shTCHP3 groups; B: Tumor growth curves showed the tumor volumes measured every seven days; C: Final tumor weights were significantly lower in the shTCHP3 group compared to the NC group; D: Representative images of H&E staining of tumor tissues; E: Western blotting confirmed the knockdown of TCHP in the harvested tumors; F: The expression of Ki67 protein was detected by immunohistochemistry; G: Apoptosis in the subcutaneous xenograft model was assessed by the TUNEL assay. Data are presented as mean ± SD; n = 4 mice per group. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. NC: Negative control.

Moreover, Western blotting confirmed the efficient knockdown of TCHP expression in the xenograft tumors (Figure 5E). To elucidate how this growth inhibitory effect was mediated, we conducted IHC analysis for Ki67 and TUNEL assays using xenograft tumor tissue sections. The data indicated that TCHP knockdown significantly reduced the Ki67-positive proliferative index (Figure 5F), while concurrently increasing the quantity of TUNEL-positive apoptotic cells (Figure 5G), in comparison with the NC group. Therefore, TCHP knockdown reduced the development of GC tumors in vivo and reduced their malignant potential.

In summary, our findings indicate that TCHP knockdown attenuates GC progression via suppression of the AKT/BCL2 pathway, thereby impeding tumor advancement (Figure 6).

Figure 6  Schematic diagram of the mechanisms by which TCHP regulates the proliferation and apoptosis of gastric cancer cells through the AKT/BCL2 signaling pathway.

DISCUSSION

TCHP has been identified as a centrosomal protein. Centrosomal abnormalities disrupt cell division, exacerbate genomic instability, and impair the signaling pathways that regulate cell proliferation and differentiation. These alterations are closely associated with cancer initiation and progression[15]. Consequently, elucidating the regulatory mechanisms and tumorigenic roles of centrosomes is critical for advancing basic biology and clinical oncology research. In China, GC continues to pose a major health burden due to its high incidence and mortality[16]. The lack of early symptoms often results in advanced-stage diagnosis, and resistance to conventional therapies further limits the treatment options. Considering these challenges, the centrosome has recently been identified as a critical node in GC studies, providing promising insights into tumor pathogenesis and therapeutic resistance[17]. Our study demonstrated that the centriolar protein TCHP was notably elevated in GC cells and that TCHP regulated GC cell multiplication and apoptosis via the AKT/BCL2 signaling pathway.

Uncontrolled cell proliferation is a hallmark of cancer[18]. Unlike cells in culture, most normal cells in vivo reside in a state of quiescence (G0 phase) and only re-enter the cell cycle (G1/S/G2/M) upon mitogenic stimulation[19]. In cancer, including GC, these regulatory circuits are disrupted, leading cells to evade quiescence and undergo continuous cell division, which is a pivotal driver of tumorigenesis. In addition to cell cycle arrest, the dysregulation of apoptosis can affect cellular proliferation. This process is governed by a conserved programmed cell-death pathway that limits excessive cell accumulation and thereby controls cell proliferation, regulating homeostasis and survival[20]. Apoptotic dysregulation is a defining feature of cancer that fundamentally supports uncontrolled growth[21]. Apoptosis may be activated by the intrinsic mitochondrial pathway[22]. As a key hub in oncology, signaling through AKT governs cellular viability and expansion by regulating key effector molecules, including the BCL2 family[23]. Activated AKT signaling promotes an anti-apoptotic state by transcriptionally upregulating BCL2 and repressing BAX expression[24]. Previous studies have established that TCHP regulates tumor cell survival and migration by influencing mitochondrial function and energy metabolism[25]. Notably, the AKT pathway is a well-known core regulator of mitochondrial homeostasis and anti-apoptotic signaling, suggesting a potential mechanistic link for TCHP function. Therefore, TCHP may act as an upstream regulator or cofactor that sustains constitutive AKT/BCL2 signaling in GC.

Our findings establish a functional association between TCHP and the AKT/BCL2 signaling pathway, with functional studies demonstrating that TCHP is significantly upregulated in GC and promotes tumor cell proliferation. TCHP knockdown impairs colony-forming ability, decreases Ki67-positive cells, leads to G1 phase cell-cycle arrest, and triggers apoptosis[26]. Proteomic analysis reveals that TCHP is considerably enriched in apoptosis-related pathways, with key protein expression differences involved in mitochondrial apoptosis including the pro-apoptotic executors BAX, BAK1, and CYCS (cytochrome C), exhibiting a marked shift toward apoptosis promotion, along with persistent upregulation of the effector caspase CASP7[27]. Concurrently, key anti-apoptotic and survival molecules are downregulated. Notably, the expression of MCL1 (a crucial BCL2 family protein) and PIK3R2 (a regulatory subunit of PI3K) is reduced. PIK3R2 downregulation provides a plausible explanation for the observed attenuation of AKT phosphorylation. Secondary enrichment analysis based on KEGG further identifies significant enrichment in the PI3K/AKT pathway, suggesting that TCHP may occupy a critical upstream position in tumor survival signaling by modulating apoptotic programs and potentially influencing PI3K/AKT signaling[28]. Aberrant TCHP expression correlates with tumor progression, and TCHP knockdown effectively blocks this pathway, positioning TCHP as a promising therapeutic target.

At the molecular level, we verify that TCHP depletion upregulates p21 and downregulates CDK4 and cyclin D1. Furthermore, TCHP depletion elevates the BAX/BCL2 ratio and reduces AKT phosphorylation at Ser473, indicating that the AKT/BCL2 pathway mediates TCHP-regulated GC cell proliferation and apoptosis[29]. This perspective is further supported by in vivo evidence: Tumors formed by TCHP knockdown cells in nude mice are significantly smaller and lighter. H&E staining further reveals disorganized tumor architecture and reduced mitotic figures after TCHP silencing, indicating suppressed proliferation. These in vivo results validate our in vitro data and provide evidence confirming the tumor-promoting function of TCHP in GC.

Despite substantial evidence that TCHP is implicated in promoting GC progression, it is also important to note the limitations of this study. First, although we delineate the role of TCHP in regulating the AKT/BCL2 axis, the precise molecular connection, whether through direct protein-protein interactions or via upstream regulators, remains incompletely defined. Second, in vivo validation used a relatively small sample size (e.g., n = 4 per group). Future studies aimed at elucidating the exact mechanism and incorporating larger experimental cohorts will strengthen these conclusions and further advance TCHP targeted precision oncology studies in GC.

CONCLUSION

Our study demonstrates that TCHP is upregulated in GC, and knockdown of TCHP exerts a tumor-suppressive effect by inhibiting the AKT pathway, which subsequently downregulates BCL2 expression and promotes BAX mediated apoptosis, thereby suppressing tumor growth and promoting cell death. These findings highlight TCHP as a prognostic marker and therapeutic target in GC.

ACKNOWLEDGEMENTS

We sincerely thank Dr. Mu-Qing Cao from the Key Laboratory of Cell Differentiation and Apoptosis (Ministry of Education), Department of Pathophysiology, Shanghai Jiao Tong University School of Medicine (Shanghai, China), for expert guidance in conducting the experiments.