Prohibitin 1 Orchestrates Mitochondrial-ER Crosstalk via the VDAC1-GRP75-IP3R Axis to Drive Malignancy in Gastric Cancer

Prohibitin 1 Orchestrates Mitochondrial-ER Crosstalk via the VDAC1-GRP75-IP3R Axis to Drive Malignancy in Gastric Cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Prohibitin 1 Orchestrates Mitochondrial-ER Crosstalk via the VDAC1-GRP75-IP3R Axis to Drive Malignancy in Gastric Cancer Yu Miao, Xiaofei Wang, Ying Huang, Yuxia Zhang, Zhanchuan Liu, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7625265/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract Background : Mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), serving as critical hubs for mitochondrial- ER interactions, play a pivotal role in cancer progression. Prohibitin 1 (PHB1), an inner mitochondrial membrane protein, contributes to tumorigenesis by regulating energy metabolism and structural integrity. This study aimed to elucidate the molecular mechanism by which PHB1 regulates MAMs structural integrity and its cascade signaling in driving gastric cancer (GC) metastasis. Methods : We analyzed the expression profiles of PHB1/PHB2 in GC using the TCGA database. Immunohistochemistry was performed to detect PHB1/PHB2 protein expression in GC tissues, adjacent non-tumor tissues, and omental metastasis tissues, and to assess their correlation with clinicopathological features. Immunofluorescence was used to observe the co-localization of PHB1 with the IP3R-GRP75-VDAC1 complex. Live-cell Ca2+ imaging, ATP/ROS detection, and mitochondrial functional assays (mPTP opening/MMP) were employed to evaluate calcium flux and energy metabolism changes. Transmission electron microscopy and Western blot were utilized to analyze MAMs structural integrity. A nude mouse xenograft model was established to validate in vivo functions. Results : TCGA analysis confirmed that both PHB1 and PHB2 were overexpressed in GC. PHB1 expression significantly correlated with tumor stage, while PHB2 expression correlated with demographic characteristics. Mitochondrial PHB1 expression was significantly higher than PHB2 in omental metastasis tissues. Furthermore, PHB1 co-localized and interacted with the IP3R-GRP75-VDAC1 complex, regulating ER-to-mitochondria Ca2+ flux. PHB1 overexpression enhanced MAMs-mediated Ca2+ signaling, increased ATP production, and drove cytoskeletal remodeling, thereby promoting malignant progression. Conversely, PHB1 knockdown disrupted the IP3R-GRP75-VDAC1 axis, leading to MAMs structural dissociation and mitochondrial dysfunction. Notably, treatment with the VDAC1-specific inhibitor VBIT-12 in PHB1- overexpressing cells significantly reversed the pro-tumorigenic effects of PHB1, reduced PHB1 protein levels, and diminished its association with the GRP75-IP3R complex, demonstrating that PHB1 function depends on VDAC1 activity. In summary, PHB1 regulates the VDAC1-GRP75-IP3R complex in a VDAC1-dependent manner to maintain MAMs structural integrity. This subsequently modulates calcium homeostasis, mitochondrial energy metabolism, and cytoskeletal remodeling, ultimately driving GC progression. Conclusion : Our findings reveal a novel mechanism by which PHB1 promotes GC progression via regulating MAMs function through the VDAC1 complex, providing a theoretical basis for therapeutic targeting. PHB1 MAMs VDAC1 GRP75 Gastric cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The research on cancer metastasis has reached an intense stage, also regarded as a "bottleneck period." Clarifying the relationship between oncogenes and ultrastructural changes is crucial to uncovering the mechanisms underlying cancer metastasis[1]. Gastric cancer (GC) is a tumor characterized by high molecular and phenotypic heterogeneity, with a high incidence and mortality rate[2]. Despite substantial advancements in therapeutic approaches such as neoadjuvant chemotherapy and radiotherapy, clinical challenges such as high recurrence and metastasis potential persist[3, 4]. Recent years have witnessed growing interest in the communication between the endoplasmic reticulum (ER) and mitochondria (Mito), with its dysregulation implicated in diverse pathologies including cancer, diabetes, and neurodegenerative diseases[5]. Mitochondria-associated ER membranes (MAMs) play pivotal roles in key intracellular pathways such as calcium homeostasis, mitochondrial dynamics, and energy metabolism.[6, 7]. In cancer research, MAMs serve as a central hub regulating cell survival: MAMs-dependent calcium release from the ER and its selective uptake by Mito are closely linked to malignant behaviors such as proliferation and invasion in cancer cells when their homeostasis is disrupted[8]. Studies indicate that numerous proteins, including Drp1, PINK1, Mfn2, PERK, GRP75, IP3R, VDAC1, and REEP1, participate in the formation and regulation of MAMs[9] [10]. Specifically, GRP75 facilitates the interaction between the inositol trisphosphate receptor (IP3R) on the ER membrane and VDAC1 on the mitochondrial outer membrane, thereby maintaining MAMs structure by modulating calcium ion homeostasis and inter-organelle signaling [11]. Moreover, GRP75 acts as a critical tethering protein promoting MAMs formation. Notably, in ovarian cancer patients experiencing relapse, GRP75 enhances cisplatin resistance by promoting MAMs enrichment[11]. However, the role of MAMs structure in the metastatic progression of gastric cancer remains poorly understood. The prohibitin (PHB) family members PHB1 and its homolog PHB2 are multifunctional chaperone/scaffold proteins. Primarily localized to the mitochondrial inner membrane, they are also present in the plasma membrane and nucleus. PHB1 and PHB2 play crucial roles in fundamental cellular processes including proliferation, differentiation, and apoptosis, contributing to the maintenance of mitochondrial homeostasis and apoptotic machinery[12] [13]. Research indicates that PHB1 and PHB2 are involved in the development of various cancers, exhibiting a dual role in oncogenesis—functioning as either tumor suppressors or promoters depending on the cancer type and their interacting protein partners[14]. Studies suggest that PHB1, acting as a regulator of mitochondrial inner membrane permeability, mediates the survival and death of prostate cancer cells through Smad-dependent pathways and MAPK intracellular signaling[15]. Another study demonstrated that silencing the PHB1 gene in ovarian cancer disrupts mitochondrial integrity, alters the proportion in the G0/G1 phase of the cell cycle, and increases susceptibility to apoptosis[16]. Similarly, downregulation of PHB2 expression suppresses lung cancer cell proliferation by stabilizing RACK1[17]. Reduced PHB2 expression also delays the proliferation rate of renal cancer cells by inhibiting MNK/eIF4E signaling[18]. In summary, PHB1 and PHB2 play pivotal roles in the progression of multiple cancers; however, the precise mechanisms underlying their context-dependent contributions to cancer advancement warrant further investigation. Analysis of the TCGA database in this study revealed elevated expression of both PHB1 and PHB2 in gastric cancer tissues. Notably, PHB1 expression was found to be significantly higher than that of PHB2 in omental metastatic tissues. Our investigation uncovered a coupling between PHB1 and the MAMs structure (specifically, the IP3R-GRP75-VDAC1 complex) in both primary gastric cancer and its metastatic lesions. Furthermore, we demonstrated that PHB1 maintains the integrity of the MAMs structure by modulating the VDAC1-GRP75-IP3R complex in a VDAC1-dependent manner. This mechanism ultimately drives gastric cancer progression by regulating intracellular calcium homeostasis, mitochondrial energy metabolism, and cytoskeletal remodeling. These findings unveil a novel mechanism through which PHB1 promotes gastric cancer progression by regulating MAMs function via the VDAC1 complex, providing a theoretical foundation for therapeutic strategies targeting this pathway. Materials and methods Bioinformatical analyses For the expression of PHB1 and PHB2 in normal and gastric cancer tissues, we downloaded RNA-seq data for gastric cancer from The Cancer Genome Atlas Program (TCGA) and used R software to filter the PHB1 gene. Data visualization and plotting were performed using the limma, ggplot2, and ggplot2 packages in R software. For the generation of heatmaps for PHB1 and PHB2, we downloaded clinical data of gastric cancer patients from TCGA. The ComplexHeatmap and limma packages in R software were used to create heatmaps based on gene expression data and clinical information. Specimen of human pathological tissue Clinical specimens of gastric cancer tissues, adjacent tissues of the gastric cancer, and omental tissues with gastric cancer metastasis from 29 patients were collected from 79 gastric cancer patients in the North China University of Science and Technology Affiliated Hospital from 2020 to 2023. None of these patients had a history of chemotherapy or radiotherapy. This study has been approved by the Institutional Review Board of North China University of Science and Technology Affiliated Hospital (Ethics Approval Number: 2020405). Each patient signed a written informed consent form. All the procedures were conducted in strict accordance with the Declaration of Helsinki. Immunohistochemistry (IHC) and immunofluorescence (IF) staining Gastric cancer tissues, adjacent non-cancerous tissues, and metastatic gastric cancer tissues were fixed, embedded, sectioned, deparaffinized and repaired. For IHC, the sections were blocked with 3% hydrogen peroxide, while for IF, the sections were permeabilized for 15 min and then blocked with goat serum. The sections were incubated with primary antibodies PHB1(1:200; Cat No.10787-1-AP; Proteintech, China), VDAC1(1:100; Cat No.55259-1-AP; Proteintech, China), GRP75(1:200; Cat No.14887-1-AP; Proteintech, China), IP3R (1:100; DF3000; Affinity, China) were overnight at 4°C. The next day, after rewarming, for IHC, the sections were incubated with HRP-conjugated secondary antibody (PV-9000, ZSGB-BIO, China) at 37°C for 1 h. After washing with PBS three times, the sections were stained with DAB and hematoxylin, sealed with neutral resin, and observed under an inverted microscope. For IF, the sections were incubated with secondary antibody Alexa Fluor® 488 Conjugate (1:100; #ZF-0511; ZSGB-BIO) at 37°C for 1 h, counterstained with DAPI, and observed under a fluorescence microscope. Cell culture Human gastric adenocarcinomas cell lines (AGS; ZQ0240), human gastric cancer cells (HGC-27; ZQ0192) and human gastric mucosal cells (GES-1; ZQ0905) were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (www.zqxzbio.com).). According to the manufacturers’ instructions, AGS and HGC-27cells were grown in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin/streptomycin) at 37℃ and 5% CO 2 . GES-1 cells were grown in DMEM high glucose medium containing 10% FBS and 1% antibiotics (penicillin/streptomycin). Cell transfection and drug treatment Full-length of PHB1 (Sangon Biotech Co., Ltd.) genes was cloned into the pcDNA3.1(+) vector, separately. 2.5 µg of PHB1 overexpression (OE-PHB1) plasmid were transfected separately into AGS, HGC-27 and GES-1 with Lipo8000™ (cat no. C0533; Beyotime Biotechnology) at 37˚C for 24 h, with empty vector as a negative control (NC). The short hairpin (sh)RNA target PHB1 were designed by GenePharma for the knockdown of the corresponding genes. The sequences are as follows: PHB1 shRNA(5’- GCCGTTCTCGACCACGTAATG-3’)； and shRNA-NC (Sense 5’- TTCTCCGAACGTGTCACGT-3’). For the infection of lentivirus, when cell confluency reached 40-60%, the culture medium was removed, and fresh medium containing 5 µg/mL Polybrene and 15 µL of lentiviral solution (1×10 8 TU/mL) were added. After 24h of incubation, the fresh medium was replaced and the cell phone cells were cultured for another 3 days for experimental days. Transwell Following a 24-h serum starvation period, GES-1 cells were prepared into a cell suspension, and 200 µL of the cell suspension was added to the Transwell upper chamber. The culture was conventionally grown at 37°C and 5% CO 2 for 24 h. Subsequently, the cells were fixed with 4% paraformaldehyde solution for 10 min and stained with 0.1% crystal violet for 20 min. Subsequently, the cells were washed twice with PBS and gently wiped with a cotton swab to remove residual cells. Five visual fields were randomly selected from the center and surrounding areas; these fields were then observed, photographed, and counted with an inverted microscope. Skeleton remodeling The morphology and distribution of actin filaments were detected using Actin-Tracker Red-594 (C2205S, Beyotime Biotechnology, China). Transfected cells were cultured on coverslips for 24 h, washed, fixed, and the staining solution was added and incubated for 30 min. After the cells were washed and dried, the coverslips were blocked using DAPI and then imaged. Mitochondrial permeability transition pore assay The mPTP Assay Kit (C2009S, Beyotime Biotechnology, China) was utilized to assess the degree of mPTP opening. After the transfected cells were seeded on 6-well plates and cultured for 24 h, the medium was removed, and the cells were washed. Subsequently, 1mL of calcein AM staining solution, fluorescence quenching solution, or ionomycin was added and incubated for 30 min. After re-incubation with fresh medium, the medium was removed, detection buffer was added to incubate for 30 min, and the cells were observed under a fluorescence microscope. Mitochondrial membrane potential assay MMP was assessed using the JC-1 fluorescent probe (C2006, Beyotime Biotechnology, China). After the above seeding plates and culturing procedures were repeated for transfected cells, 1 mL of cell medium and 1 mL of JC-1 staining working solution were added and incubated for 20 min. The supernatant was removed, cells were washed twice, cell culture medium was added and imaged. ATP level assay Intracellular ATP levels in individual live cells were monitored in real-time using the ATP fluorescent probe (pCMV-AT1.03) (D2604, Beyotime Biotechnology, China). AGS, HGC-27, and GES-1 cells, successfully transfected were seeded in 12-well plates and cultured at 37℃ with 5% CO₂ for 24 h. Afterward, 1 µg of the ATP fluorescent probe was added to each well, and the cells were further incubated for 24 h. Fluorescence imaging was then performed using a fluorescence microscope. ROS production assay AGS, HGC-27, and GES-1 cells, successfully transfected were stained with 10 μM DCFH-DA (S0033M, Beyotime Biotechnology, China) in the dark for 30 min. Fluorescence images of intracellular ROS were then captured using a fluorescence microscope. Western blot Crude mitochondria from ten representative samples were isolated following the established protocols[11]. Total protein extraction kit (KGP2100; KeyGEN BioTECH, Nanjing, China) and BCA Protein Assay Kit (PC0020; Solarbio, Beijing, China) were used to extract proteins and determine protein concentrations. Protein was separated by 9% SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with blocking solution containing TBST (TBS with Tween-20) and 5% milk powder for 1 h at room temperature. After that, the membranes were incubated with primary antibodies VDAC1(1:1000; Cat No.55259-1-AP; Proteintech, Wuhan, China), PHB1(1:2000; Cat No. 10787-1-AP; Proteintech, Wuhan, China), PHB2 (1:2000; Cat No. 12295-1-AP; Proteintech, Wuhan, China), GRP75(1:5000; Cat No.14887-1-AP; Proteintech, China), IP3R (1:1000; DF3000; Affinity, China), Vimentin (1:1000; bs-8533R; Bioss, Beijing, China), β-catenin (1:2000; bs-1165R; Bioss, Beijing, China), ATP2A2 (1:3000; Cat No. 67248-1-Ig; Proteintech, Wuhan, China), ATP5A1 (1:5000; Cat No. 66037-1-Ig; Proteintech, Wuhan, China), β-actin (1:5000; Cat No.60004-1-Ig; Proteintech, Wuhan, China). After overnight incubation at 4℃, the secondary antibody Horseradish Peroxidase (HRP)-conjugated goat-anti-rabbit IgG (ZB-2301, ZSGB-Bio) or HRP-conjugated goat-anti-mouse IgG (ZB-2305, ZSGB-Bio) was used to incubate the PVDF membranes for 1 h at room temperature. Finally, the protein signals were detected signals with electrochemiluminescence solution. RT-qPCR Crude mitochondria from ten representative samples were isolated following the established protocols[11]. Total RNA and Mitochondrial RNA was extracted using the Trizol method. RNA was reverse-transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (00752219, Thermo Fisher Scientific, China). Quantitative real-time PCR (RT-qPCR) was performed using PowerUp™ SYBR™ Green Master Mix (01118369, Thermo Fisher Scientific, China) in a 20 μL reaction volume. The PCR cycling conditions consisted of an initial denaturation at 94°C for 2 min; followed by 30 cycles of denaturation at 94°C for 5 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. GAPDH was used as the internal reference gene. Primers were synthesized by Sangon Biotech (Shanghai, China; https://www.sangon.com/). The primer sequences were as follows: PHB1-F: 5’-CACTGGTAGCAAAGATTTACAG-3’ PHB1-R: 5’-ATAGTCCTCTCCGATGCTG-3’ PHB2- F: 5’-AATCTGTGTTCACCGTGGA -3’ PHB2- R: 5’- CCAGGATAGTGTCCTGCTG-3’ GAPDH-F: 5’-CAAGGTCATCCATGACAACTTTG-3’ GAPDH-R:5’ -GTCCACCACCCTGTTGCTGTAG-3’ Cellular immunofluorescence The cells were fixed with 4% paraformaldehyde for 10 min and then permeabilized with 0.5% Triton X-100 for 15 min at 37℃. After incubation with goat serum for 30 min, cells were incubated overnight with the following antibodies: VDAC1(1:100; Cat No.55259-1-AP; Proteintech, Wuhan, China), PHB1(1:200; Cat No. 10787-1-AP; Proteintech, Wuhan, China), GRP75(1:200; Cat No.14887-1-AP; Proteintech, China), IP3R (1:100; DF3000; Affinity, China). The second day, the cells were incubated with Alexa Fluor® 488 Conjugate (1:100; ZF-0511; ZSGB-BIO, China) secondary antibodies at 37˚C for 1 h and sealed with DAPI, observed under a fluorescence microscope. Transmission electron microscope detection Transmission electron microscopy (TEM) was used to examine the morphology and distribution of cellular Mito and ER. After digestion, centrifugation, and collection, the cells were fixed with 2.5% glutaraldehyde and 1% osmium tetroxide, respectively. After the cells were dehydrated and embedded, ultrathin sections were prepared using an ultramicrotomy mechanism. The sections were stained with lead citrate and uranyl acetate and subsequently observed with TEM. Co-staining of mitochondria and endoplasmic reticulum Mitochondrial and endoplasmic reticulum changes in cells were labeled using the MitoTracker® Red CMXRos and ER-Tracker Green fluorescent probe. After culturing the cells on coverslips for 24 h, the culture medium was removed, and the cells were incubated with pre-warmed MitoTracker® Red CMXRos and ER-Tracker Green staining solutions for 30 min. The staining solution was then removed, and the cells were washed twice with culture medium. Fluorescence microscopy was subsequently used for observation and imaging. Co‑immunoprecipitation Cells were rinsed with PBS, followed by the addition of 100 μL of lysis buffer. Of the extracted proteins, 50μl were served as the input control. 3μg of PHB1 antibody were used, with normal IgG of the same species serving as the negative control. The antibodies were diluted according to the manufacturer's instructions and added 100 µL of Protein A+G magnetic beads, and incubated at room temperature for 15 min. 500 μL protein sample was incubated with Protein A+G magnetic beads (Beyotime, Cat.no: P2179) bound with antibodies or normal IgG overnight at 4°C to allow binding of the proteins to the antibodies. After incubation, the samples were centrifuged at 1000 g for 5 minutes at 4°C, and the supernatant was removed. The beads were washed four times with PBS. Subsequently, 100 μL of 1X SDS-PAGE Sample Loading Buffer was added and the samples were heated at 95°C for 5 min, the supernatant was collected as the sample for the PHB1-IP group for Western blot analysis. For VDAC1-IP, repeat the above operation. Mitochondrial Ca 2+ imaging Mitochondrial Ca 2+ dynamics were monitored with 4mtD3cpv sensor, a genetically encoded Ca 2+ indicator targeted to the mitochondrial matrix. The cells were transfected with the 4mtD3cpv probe for 48 h. Coverslips were washed with Ca 2+ containing KRB solution and mounted on the microscope stage. Samples were illuminated at 420 nm and simultaneously acquired at 475 nm (donor, ECFP) and 530 nm (acceptor, circularly permuted (cp) Venus). CpVenus/ECFP ratio was calculated online using MetaFluor software. After acquisition of basal Ca 2+ levels (first 30 s of acquisition), the cells were stimulated with 100 μM ATP. Regions of interest (ROIs) were defined around individual Mito. Fura-2 Ca 2+ imaging The concentration of Ca²⁺ was determined using Fura-2 AM (S1052, Beyotime Biotechnology, China) as previously described in the literature[19]. In brief, the cells were plated onto coverslips (3×10 4 cell/coverslip), and loaded with 2.5 μM Fura-2 AM (S1052, Beyotime Biotechnology, China) in the presence of 0.005% Pluronic F-127 and 10 μM sulfinpyrazone in Ca 2+ -containing KRB solution. After loading (30 min in the dark at normal temperature), cells were washed once with KRB solution and allowed to de-esterify for 30 min. After this, the coverslips were mounted in an acquisition chamber and placed on the stage of the microscope, and cells were alternately excited at 340 and 380 nm. The fluorescent signal was collected through a 510/20 nm bandpass filter. The cells were stimulated with 100 μM ATP. For comparison of Ca 2+ dynamics, measured as an amplitude of Ca 2+ increase from the baseline level, Fura-2 ratio values were normalized using the formula (Fi-F0)/F0[referred to as Normalized (Norm.) Fura Ratio]. Endoplasmic reticulum Ca 2+ imaging ER Ca 2+ dynamics were monitored with D1ER, a genetically encoded Ca 2+ sensor, targeted to the ER lumen. The cells were transfected with the D1ER probe for 48 h. Coverslips were mounted in a chamber in KRB solution and placed on the stage of the microscope. Cells were alternately excited at 405 and 470 nm, and the fluorescent signal was acquired using a 510/20 nm bandpass filter. After recording basal signal for 30 s, KRB solution was removed and replaced with a Ca 2+ -free solution. After allowing the signal to stabilize for an additional 30 s, cells were stimulated with 100 µM ATP and 100 µM Tert-butylhydroquinone (TBHQ), and the response was recorded for 300 s. Animal experiments BALB/c nude mice (16–20 g, 4–5 weeks old; obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were randomly assigned into two groups (n = 5 per group). AGS cells transfected with shRNA-NC or shRNA-PHB1 (5 × 10⁶ cells in 0.1 mL) were intraperitoneally injected into the region surrounding the greater omentum. After a 4-week breeding period, the mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg) and subsequently euthanized by cervical dislocation. The greater omentum tumor tissues were excised to measure tumor width (W) and length (L), and the tumor volume (V) was calculated using the formula V = (L × W²)/2. The tumor tissues were reserved for further experiments as required. All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of North China University of Science and Technology Affiliated Hospital (Approval No. LAEC-NCST-2020036). Statistical analysis All data were expressed as mean ± standard error and analyzed with SPSS 27.0 software. Differences between groups were compared with one-way ANOVA followed by Bonferroni post hoc test. The P value less than 0.05 indicates a statistically significant difference. Results 1. Expression of PHB1 in gastric cancer and metastatic tissues and its coupling with the MAMs structure This project focused on investigating the mechanism by which PHB1 mediates gastric cancer metastasis ( Fig. 1A ). Analysis of the TCGA database revealed significantly elevated expression of both PHB1 and PHB2 in gastric cancer tissues (GCT) compared to normal gastric tissues (NGT). Notably, PHB1 expression correlated significantly with tumor stage, while PHB2 expression was associated with demographic factors such as sex and age ( Fig. 1B-E ). Based on these findings, we collected GCT and NGT from 79 gastric cancer patients, including greater omentum (GO) metastatic tissues from 29 of these patients. IHC, RT-qPCR, and Western blot analyses consistently demonstrated significantly higher expression of both PHB1 and PHB2 in GCT and GO compared to NGT. Crucially, PHB1 expression in GO, particularly its mitochondrial localization, was significantly higher than in GCT, underscoring its pivotal role in gastric cancer metastasis ( Fig. 1F-J ). Clinicopathological analysis of the 79 patients further revealed that PHB1 expression significantly correlated with TNM stage, depth of invasion, and metastasis, but showed no association with sex, age, Helicobacter pylori infection status, or overall survival ( Table 1 ), aligning with the TCGA findings. Immunofluorescence results indicated markedly enhanced co-localization (manifested as yellow areas/white arrows) of PHB1 with the mitochondrial channel protein VDAC1 and the ER calcium channel protein IP3R in GCT and GO compared to NGT, suggesting spatial coupling and functional association among PHB1, VDAC1, and IP3R in gastric cancer and metastatic tissues ( Fig. 1K-L ). TEM further revealed closer apposition between the ER and mitochondrial membranes, along with broader ER coverage over mitochondria in gastric cancer and metastatic tissues ( Fig. 1M-O ). Integrating these experimental results with the established central role of GRP75 in mediating IP3R-VDAC1 signaling[11], we propose a hypothetical model outlining the potential mechanism of PHB1 in gastric cancer metastasis ( Fig. 1P ). 2. PHB1 regulates MAMs-mediated cytoskeletal remodeling and energy metabolism in GES-1 cells, promoting the acquisition of malignant phenotypes Western blot analysis revealed that PHB1 overexpression significantly upregulated the protein expression levels of key MAMs components (VDAC1, GRP75, IP3R) and malignant phenotype markers (Vimentin, β-catenin) ( Fig. 2A-C ). Immunofluorescence co-localization analysis further confirmed that PHB1 overexpression enhanced its co-localization with VDAC1, GRP75, and IP3R within MAMs domains (manifested as yellow signal) ( Fig. 2D-F ). Functionally, PHB1 overexpression not only increased ATP production ( Fig. 2G-H ) but also induced cytoskeletal remodeling, as evidenced by elevated fluorescence intensity ( Fig. 2I-J ). Protein-protein interaction network analysis and subsequent Western blot validation both indicated that PHB1 interacts with and upregulates the expression of ATP synthase components ATP2A2 and ATP5A1 ( Fig. 2K-M ). TEM observations demonstrated that PHB1 overexpression significantly reduced the distance between Mito-ER and expanded the area of ER coverage over Mito ( Fig. 2N-P ). Based on these findings, we propose that PHB1 likely modulates MAMs structure by regulating the GRP75-IP3R complex via VDAC1. To test this hypothesis, we employed the VDAC1-specific inhibitor VBIT-12. Experimental results showed that VBIT-12 effectively attenuated the effects induced by PHB1 overexpression, including suppression of cytoskeletal remodeling, reduction in cell invasive capacity, and decreased ATP production ( Fig. 2R-U ). Concurrently, VBIT-12 treatment downregulated the protein expression of PHB1, GRP75, and IP3R ( Fig. 2V-W ). These results collectively demonstrate that PHB1, by regulating the VDAC1-GRP75-IP3R complex, influences MAMs structure and function, thereby mediating cytoskeletal remodeling and energy metabolic reprogramming, ultimately promoting the acquisition of malignant phenotypes (the efficacy of the PHB1 overexpression plasmid was validated in Supplementary Fig. 1A-B ). Furthermore, our study confirms that PHB1 plays a critical regulatory role in mitochondrial function. Overexpression of PHB1 promoted closure of the mPTP in GES-1 cells ( Fig. 2X-Y ) and enhanced MMP ( Fig. 2Z-A1 ). Using dual fluorescence probes co-labeling mitochondria (red) and the endoplasmic reticulum (green), we observed that PHB1 overexpression significantly increased their co-localization area (yellow signal), indicating enhanced mitochondria-ER contacts ( Fig. 2B1-C1 ). Calcium signaling detection using transfected specific indicators (4mtD3cpv, D1ER, Fura-2) revealed that PHB1 overexpression augmented mitochondrial calcium uptake capacity. This led to corresponding decreases in calcium levels within the ER and cytosol. Although this regulatory effect was transient, mitochondrial calcium signaling was significantly potentiated during its active phase ( Fig. 2D1-I1 ). Collectively, these results demonstrate that by enhancing the formation of MAMs, PHB1 effectively regulates mitochondrial calcium homeostasis and metabolic function, thereby maintaining mitochondrial hyperactivation and ultimately driving malignant transformation in gastric cancer cells. 3. PHB1 regulates ER-mitochondrial calcium flux homeostasis to influence mitochondrial function and bioenergetic metabolism, ultimately driving cancer cytoskeletal remodeling To further validate the mechanistic role of PHB1 in gastric cancer metastasis, we established PHB1 overexpression and knockdown models in AGS and HGC-27 cells ( Supplementary Fig. 1C-J ). Given that cytoskeletal remodeling represents a critical morphological hallmark of malignant progression in cancer cells, we observed that PHB1 overexpression significantly promoted filopodia formation and elongation in both cell lines, whereas PHB1 knockdown suppressed this process ( Fig. 3A-C ). This indicates that PHB1 drives cytoskeletal remodeling in gastric cancer cells. Regarding calcium homeostasis, PHB1 knockdown impaired mitochondrial calcium uptake capacity, resulting in calcium accumulation in the ER and cytosol, ultimately triggering calcium overload ( Fig. 3D-I ). Thus, PHB1 directly impacts mitochondrial function and cytoskeletal dynamics by regulating calcium flux between Mito-ER. Building upon this, we further investigated the central role of PHB1 in mitochondrial function. PHB1 deficiency induced excessive opening of the mPTP ( Fig. 3J-L ), reduced MMP ( Fig. 3M-O ), decreased ATP production ( Fig. 3P-R ), and increased ROS generation ( Fig. 3S-U ). Conversely, PHB1 overexpression produced the opposite effects. Collectively, these findings demonstrate that PHB1 serves as a central hub maintaining mitochondrial functional homeostasis. By regulating ER-mitochondrial calcium flux equilibrium, PHB1 drives cytoskeletal remodeling in gastric cancer cells, thereby promoting their malignant progression. 4. PHB1 drives cancer adaptation by enhancing MAMs to modulate calcium signaling and energy metabolism In cancer, ATP2A2-mediated calcium signaling activates mitochondrial metabolism, which cooperates with ATP5A1-driven ATP synthesis to promote oxidative phosphorylation (OXPHOS). This coordinated process collectively fulfills the high energy demands of cancer cells, supports apoptotic resistance, and enhances microenvironmental adaptation[20, 21]. Our study reveals that PHB1 regulates the expression of both ATP2A2 and ATP5A1, which is crucial for maintaining calcium homeostasis and ATP production ( Fig. 4A-D ). Furthermore, PHB1 overexpression significantly increased the number of mitochondria-associated endoplasmic reticulum membranes (yellow areas, Fig. 4E-G ), reduced the intermembrane distance, and expanded the ER coverage over mitochondria. Conversely, PHB1 knockdown resulted in fewer MAMs, increased intermembrane distance, and reduced ER coverage ( Fig. 4H-J ). These findings establish that PHB1 enhances MAM structure, thereby providing a critical platform for efficient calcium signal transduction and energy metabolism driven by OXPHOS. Given that VDAC1, GRP75, and IP3R are core constituents of MAMs and play pivotal roles in ER-Mito signaling, we observed that PHB1 overexpression not only expanded its contact sites with the VDAC1-GRP75-IP3R complex but also upregulated the expression levels of these proteins. Conversely, PHB1 knockdown dampened these effects ( Fig. 4K-Q ). Furthermore, Co-IP assays directly confirmed protein-protein interactions between PHB1 and VDAC1, VDAC1 and GRP75, and GRP75 and IP3R (Fig. 4R-W). Notably, treatment with the VDAC1 inhibitor VBIT-12 under PHB1-overexpressing conditions downregulated protein expression of PHB1, VDAC1, GRP75, and IP3R ( Fig. 4X-A1 ). Collectively, these results demonstrate a mutually dependent, functionally coupled bidirectional regulatory relationship between PHB1 and the VDAC1-GRP75-IP3R complex at MAMs. 5. PHB1 knockdown suppresses tumor growth and disrupts energy homeostasis by impairing MAMs integrity Using a peritoneal metastasis model established by intraperitoneal injection of gastric cancer cells ( Fig. 5A ), we demonstrated that PHB1 knockdown significantly suppressed tumor growth ( Fig. 5B-D ). Furthermore, PHB1 depletion led to a marked reduction in protein expression levels of the core MAMs components VDAC1, GRP75, and IP3R ( Fig. 5E-F ). Critically, a significant decrease in the overlapping co-localization signals between PHB1 and these proteins was observed ( Fig. 5G-I ). We also noted that PHB1 knockdown significantly downregulated the protein expression of ATP5A1 and ATP2A2. Collectively, PHB1 knockdown disrupted the VDAC1-GRP75-IP3R complex, impaired MAMs functionality, caused dysregulation of ATP5A1 and ATP2A2 protein expression, perturbed cancer cell bioenergetic homeostasis, and consequently inhibited in vivo tumor growth. Discussion In recent years, targeting key functional proteins involved in metastasis has proven effective in significantly suppressing cancer progression, including survival, metastasis, and drug resistance[22]. However, identifying critical proteins within the complex molecular interaction networks governing metastasis remains a challenge for clinicians[23]. Tight membrane contact sites between organelles, such as MAMs that coordinate ion homeostasis and cellular functions[24, 25]，participate in regulating cancer cell metabolism, metastasis, and drug resistance[26]. Their functionality is typically mediated through the modulation of ER proteins or the influence on mitochondrial proteins[27, 28]. This study revealed the specific expression of the mitochondrial inner membrane proteins PHB1 and PHB2 in gastric cancer tissues and their omental metastases, particularly highlighting the significance of PHB1 in omental metastasis. This implicates a coupling relationship between PHB1 and MAMs components (VDAC1, GRP75, IP3R). Mechanistically, PHB1 regulates the VDAC1-GRP75-IP3R complex in a VDAC1-dependent manner, maintaining MAM structure integrity. This, in turn, modulates calcium flux between the ER and Mito to sustain intracellular calcium homeostasis. Ultimately, this PHB1-mediated regulation facilitates the acquisition of malignant phenotypes by driving MAM-dependent cytoskeletal remodeling and alterations in energy metabolism, thereby promoting gastric cancer progression. Analysis of The Cancer Genome Atlas (TCGA) and The Human Protein Atlas indicates that prohibitins (PHBs) are overexpressed in various tumor tissues and are recognized as critical regulators of the cell cycle[13]. PHBs (including PHB1 and PHB2) participate in regulating cancer cell proliferation, apoptosis, metabolism, differentiation, and metastasis, yet exhibit dual roles—either promoting or suppressing tumorigenesis—in different cancer studies[22]. Specifically, PHB1 mediates cancer cell drug resistance and metastasis by regulating the Ras-mediated c-Raf-MEK-ERK signaling pathway[29, 30]；In non-small cell lung cancers (NSCLCs), high PHB1 expression significantly correlates with poor tumor differentiation, lymph node metastasis, and low survival rates[31]；Similarly, PHB2 expression is upregulated in liver cancer, and silencing PHB2 suppresses cancer cell growth and colony formation, induces G1 phase arrest, and enhances susceptibility to apoptosis[32]. These findings suggest that both PHB1 and PHB2 mediate adaptive survival in different cancer cells across various developmental stages. This study focuses on gastric cancer. We observed specific and differential expression patterns of PHB1 and PHB2 in GC tissues, adjacent non-tumorous tissues, and omental metastatic tissues, particularly highlighting the significance of PHB1 in omental metastasis. Furthermore, we found that PHB1 expression closely correlates with the tumor stage of GC, further emphasizing its critical role in GC metastasis. Notably, although reduced PHB1 expression has been reported to associate with tumor dedifferentiation and carcinogenesis, and alterations in PHB copy number and the rs6917 polymorphism may influence GC progression[33], both our study and data retrieved from the TCGA database revealed PHB1 overexpression in GC tissues. Our analysis of 79 GC patient tissues also aligned with the database findings. This discrepancy may reflect variations in the differentiation status of the collected samples, indicating that PHB1 function differs across GCs of varying differentiation grades. Additionally, this study reveals an association between PHB1 and components of MAMs. MAMs are closely implicated in cancer initiation and progression[34]. Within this context, the IP3R-GRP75-VDAC signaling axis plays a pivotal role, particularly by regulating calcium ion (Ca 2+ ) signaling, lipid metabolism, and cellular stress responses, thereby influencing cancer cell proliferation and growth[35]. Specifically, the IP3R-GRP75-VDAC complex facilitates Ca²⁺ transfer from the ER to Mito. This process is crucial for regulating glycolytic metabolism and survival in highly therapy-resistant glioma-initiating cells[36]. Our study revealed that overexpression of the PHB1 protein in GES-1 cells promoted cytoskeletal remodeling and enhanced Ca 2+ flux, ultimately leading to the acquisition of a malignant phenotype. Concurrently, PHB1 overexpression significantly upregulated the protein levels of key MAMs components—VDAC1, GRP75, and IP3R—reduced the distance between Mito and the ER, increased the number and extent of MAMs contact sites, and induced fusiform morphological changes in the ER, which appeared to wrap around mitochondria. Regarding these ER morphological alterations, we propose two potential explanations: First, a self-protective adaptive adjustment by the ER in response to altered ionic environments (e.g., active Ca 2+ flux). Second, an adaptive remodeling in response to changes in mitochondrial function. We posit that these PHB1-mediated structural alterations in MAMs are significant factors driving cytoskeletal remodeling and the acquisition of a malignant phenotype in GES-1 cells. However, the specific functional implications of the observed ER morphological changes, including those involving the lamellar ER[37], were not explored in depth in the current study. Maintaining mitochondrial function is a complex process involving numerous resident and non-resident mitochondrial proteins[38]. Mitochondrial dysfunction is associated with impaired mitochondrial OXPHOS, increased ROS production, mitochondrial permeability transition, and swelling induced by Ca 2+ overload[39]. PHB1, as an inner mitochondrial membrane protein, has garnered significant attention[40]. It has been found to participate in the degradation of mitochondrial respiratory chain subunits, the assembly and activity of the OXPHOS system, mitochondrial biogenesis, apoptosis, and autophagy[41]. Recent studies indicate that the role of PHB1 in cancer is closely linked to mitochondrial function and metabolism. Knockdown of PHB1 leads to significantly reduced mitochondrial integrity, increased ROS production, and calcium imbalance, subsequently resulting in mitochondrial dysfunction[42]. We observed similar outcomes through genetic manipulation of PHB1. Elevated PHB1 protein expression was detected in both human gastric cancer omental metastases and those in nude mice, potentially indicating that tumor cells within metastatic sites exhibit heightened energy metabolism (potentially influenced by hypoxic factors, though this study did not investigate this aspect). Depletion of PHB1 in cancer cells can induce mitochondrial dysfunction and disrupt energy output. More significantly, PHB1 was found to modulate the distance and contact area between Mito and the ER in gastric cancer cells. Based on the observed alterations in mitochondria-ER interactions, we speculate that this modulation may involve Ca²⁺ signaling contributing to increased energy output. Changes in the distance and contact area between Mito and the ER reflect the structural integrity of MAMs. MAMs integrity directly regulates mechanisms underlying cancer cell metastasis and drug resistance[11]. Indeed, PHB1 deficiency disrupts MAMs structural integrity, thereby impeding Ca 2+ exchange between Mito and the ER. This disruption leads to reduced mitochondrial Ca 2+ concentration and Ca 2+ accumulation within the ER. ER Ca 2+ accumulation acts as a dual-edged sword; excessive accumulation can induce apoptosis, depending on the magnitude and duration of the accumulation[43]. We propose that PHB1 may influence cancer cell biological behavior by regulating Ca 2+ homeostasis mechanisms between Mito and the ER, thereby altering MAMs structure and function. Notably, Ca 2+ accumulation in the ER might be transient, as the ER can activate other signaling pathways to restore Ca 2+ homeostasis. This hypothesis warrants further exploration in future studies. Notably, administration of the VDAC1-specific inhibitor VBIT-12 not only blocked the regulatory effects of PHB1 but also suppressed the protein expression of PHB1, VDAC1, GRP75, and IP3R. More importantly, this experiment demonstrated that disrupting VDAC1, a key component of MAMs, and its function effectively reversed the malignant phenotypes induced by PHB1 overexpression. This provides compelling causal evidence that PHB1 drives cytoskeletal remodeling and malignant transformation by remodeling MAMs structure, specifically by enhancing VDAC1-GRP75-IP3R-mediated Ca 2+ signaling. Given the characteristic enrichment of hypermetabolic mitochondria in metastatic cancer cells, research targeting the disruption of MAMs integrity to suppress cancer progression contributes to a deeper understanding of the roles of mitochondrial function and energy metabolism in human tumors. A key challenge addressed in this study was determining the specific role of PHB1 in maintaining MAM integrity. However, the function of PHB1 in cancer is complex and contentious: while it exhibits oncogenic properties in certain cell lines, opposing effects are observed in others. This paradoxical phenomenon may be attributed to the tissue of origin, differentiation status, microenvironmental conditions (including hypoxia), and distinct stages of cancer progression[44]. Notably, metabolic profiles undergo dynamic changes during cancer progression, exerting differential effects on primary and metastatic tumors[45]. Based on these observations, we propose that PHB1 mediates peritoneal metastasis in gastric cancer cells by regulating the structural integrity of MAMs. In summary, PHB1 maintains MAMs structural integrity by modulating the VDAC1-GRP75-IP3R complex in a VDAC1-dependent manner. This regulation governs calcium homeostasis, mitochondrial energy metabolism, and cytoskeletal remodeling, ultimately driving gastric cancer progression. These findings reveal that PHB1 promotes gastric cancer progression through VDAC1 complex-mediated regulation of MAMs function, providing a compelling rationale for targeted therapeutic strategies. Abbreviations PHB1, prohibitin 1; ER, endoplasmic reticulum; MAMs, mitochondria-associated ER membranes; Mito, mitochondria; mPTP, mitochondrial permeability transition pore; MMP, mitochondrial membrane potential; Drp1, dynamin-related protein 1; PINK, PTEN-induced putative kinase 1; Mfn2, mitofusin-2; PS1, presenilin-1, PKR, protein kinase R; PERK, PKR-like endoplasmic reticulum kinase; GRP75, glucose-regulated protein 75; VAPB: vesicle-associated membrane protein-associated protein B, VDAC1, voltage-dependent anion channel 1; REEP1, receptor expression-enhancing protein 1; HSP70, heat shock protein 70; IP3R, inositol 1,4,5-trisphosphate receptor; OXPHOS, oxidative phosphorylation; ROS, reactive oxygen species; GES-1, human gastric mucosal epithelial cells; TCGA, the cancer genome atlas program; OE-PHB1, PHB1 overexpression; TEM, transmission electron microscopy; FBS, fetal bovine serum; NC, negative control; sh, short hairpin; shRNA-PHB1, (sh)RNA target PHB1 Declarations Ethics approval and consent to participate This study was performed in line with the principles of the Declaration of Helsinki. All procedures were performed in compliance with relevant laws and institutional guidelines and have been approved by the Ethics Committee of North China University of Science and Technology Affiliated Hospital (No. 2020405, LAEC-NCST-2020036). Informed consent was obtained from the participants. Consent for publication Consent for the publication of our data was obtained from the patients. Availability of data and materials Not applicable Competing interests The authors declare that they have no competing interests. Funding This study was supported by Ningxia Natural Science Foundation (2024AAC03702、2023AAC03529), Leading Talents of Science and Technology in Ningxia Hui Autonomous Region (2023GKLRLX19), National 14th Five-Year Key Research and Development Plan Project (2022YFC3602101), Training Project of Top Young Talents in General Hospital of Ningxia Medical University. Authors' contributions Yu Miao: Conceptualization; Xiaofei Wang: Methodology; Ying Huang: Data curation; Yuxia Zhang: Formal analysis; Zhanchuan Liu: Resources; Hengtong Yin: Software; Caiyue Liu: Visualization; Yuanzhen Wang: Investigation; Hua Yin: Project administration; Yafang Lai: Supervision; Feixiong Zhang: Validation; Shaoqi Yang: Writing-review & editing and Funding acquisition; Weiqiang Li: Writing-original draft Acknowledgements Not applicable. References Stoletov K, Beatty PH, Lewis JD. Novel therapeutic targets for cancer metastasis. Expert review of anticancer therapy. 2020; 20: 97-109. Smyth EC, Nilsson M, Grabsch HI, van Grieken NC, Lordick F. Gastric cancer. Lancet (London, England). 2020; 396: 635-648. Song Z, Wu Y, Yang J, Yang D, Fang X. Progress in the treatment of advanced gastric cancer. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine. 2017; 39: 1010428317714626. Patel TH, Cecchini M. 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Bezwada D, Perelli L, Lesner NP, Cai L, Brooks B, Wu Z, et al. Mitochondrial complex I promotes kidney cancer metastasis. Nature. 2024; 633: 923-931. Table Table 1. Correlation of clinicpathological parameters and PHB1 expression in patients with gastric cancer Clinical parameters Total (n=79) PHB1 χ 2 p Positive（57） Negative（22） Gender 0.023 0.879 Male 42 30 12 Female 37 27 10 Age 1.475 0.225 ＜60 38 25 13 ≥60 41 32 9 Helicobacter pylori infection 0.142 0.706 No 35 26 9 Yes 44 31 13 TNM stage 6.286 0.012 Ⅰ-Ⅱ 36 21 15 Ⅲ-Ⅳ 43 36 7 Depth of invasion 4.318 0.038 T1/T2 39 24 15 T3/T4 40 33 7 Lymph metastasis 3.970 0.046 No 51 33 18 Yes 28 24 4 Peritoneal metastasis 4.780 0.029 No 58 38 20 Yes 21 19 2 Survival 0.065 0.799 No 27 19 8 Yes 52 38 14 Additional Declarations No competing interests reported. Supplementary Files SupplementaryFig.1.tif Supplementary Fig. 1 (A-B) Western blot for verifying the transfection efficiency of PHB1 in GES-1 cells transfected with OE-PHB1 plasmid. *** P <0.001 vs OE-NC. (C-D) Western blot for verifying the transfection efficiency of PHB1 in AGS cells transfected with OE-PHB1 plasmid. *** P <0.001 vs OE-NC. (E-F) Western blot for verifying the infection efficiency of PHB1 in AGS cells infected with shRNA-PHB1. ### P <0.001 vs shRNA-NC. (G-H) Western blot for verifying the transfection efficiency of PHB1 in HGC-27 cells transfected with OE-PHB1 plasmid. *** P <0.001 vs OE-NC. (I-J) Western blot for verifying the infection efficiency of PHB1 in HGC-27 cells infected with shRNA-PHB1. ### P <0.001 vs shRNA-NC. 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03:08:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7625265/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7625265/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":92681968,"identity":"4efc94ef-8ec9-4e86-9289-19da6f4f21c9","added_by":"auto","created_at":"2025-10-03 01:10:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":697454,"visible":true,"origin":"","legend":"\u003cp\u003ePHB1 is specifically expressed in gastric cancer and metastatic tissues and coupled with MAMs components. (A) Schematic illustration depicting the relationship between the mechanism of metastasis and the structure of MAMs in gastric cancer patients. (B-C) RNA-seq gastric cancer data were downloaded from TCGA database, PHB1 and PHB2 genes were screened by R software. Data visualization and plotting were performed using the limma, ggplot2, and ggplot2 packages in R software. (D-E) Clinical data of gastric cancer patients were downloaded from the TCGA database. The ComplexHeatmap and limma packages in R software were used to generate a heatmap based on gene expression data and clinical information. (F-G) Immunohistochemistry was used to detect the expression of PHB1 in normal gastric tissue (NGT), gastric cancer tissue (GCT) and greater omentum (GO). Scale bar, 100 μm. ** \u003cem\u003eP\u003c/em\u003e \u0026lt;0.01, *** \u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 vs NGT. ###\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs GCT. (H) RT-qPCR was used to detect the protein expression of PHB1 and PHB2 in the isolated total and mitochondrial proteins. *\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 VS NGT. #\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, ###\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 vs GCT. (I-J) Western blot was used to detect the protein expression of PHB1 and PHB2 in the isolated total and mitochondrial proteins. *\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 VS NGT. ###\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 vs GCT. (K-L) Immunofluorescence double staining was performed to detect the co-localization of PHB1 with VDAC1 and PHB1 with IP3R. Scale bar, 10 μm. *** \u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 vs NGT. #\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, ##\u003cem\u003eP\u003c/em\u003e \u0026lt;0.01 vs GCT. (M) Representative TEM images of Mito-ER contacts. Scale bar, 100 nm. Quantification of the mean distance between Mito and ER association (N), and the percentage of the Mito-surface close to the ER (O) in NGT, GCT and GO. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 vs NGT. (P) Schematic illustration of the subcellular location of PHB1, VDAC1, GRP75 and IP3R and their formation of MAMs structures. Created with BioRender.com.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7625265/v1/d11394e68233f74439837d83.png"},{"id":92681967,"identity":"1aab61b9-178f-47c7-90d5-1c88ce2f5ec9","added_by":"auto","created_at":"2025-10-03 01:10:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":499336,"visible":true,"origin":"","legend":"\u003cp\u003ePHB1 mediates MAMs changes in GES-1 cells, relying on oxidative phosphorylation to acquire a malignant phenotype. (A-C) Western blot analysis of PHB1, VDAC1, GRP75, IP3R, Vimentin, β-catenin protein expression in GES-1 cells transfected with OE-PHB1 plasmid. ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 vs OE-NC. (D-F) Immunofluorescence double staining was performed to detect the co-localization of PHB1 with VDAC1, PHB1 with GRP75 and PHB1 with IP3R in GES-1 cells transfected with OE-PHB1 plasmid. Scale bar, 25 μm. ***\u003cem\u003eP \u003c/em\u003e\u0026lt;0.001 vs OE-NC. (G-H) Representative images of GES-1 cells after transfection with pCMV-AT1.03 plasmid. Scale bar, 50 μm. *** \u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs OE-NC. (I-J) The effect of Phalloidin staining on microfilaments in GES-1 cells transfected with OE-PHB1 plasmid. Scale bar, 40 μm. ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 vs OE-NC. (K) Interaction network diagram illustrating the relationships between PHB1 and ATP2A2, ATP5F1A, obtained from Protein-Protein Interaction Networks. (L-M) Western blot analysis of ATP5A1 and ATP2A2 protein expression in GES-1 cells transfected with OE-PHB1 plasmid. ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 vs OE-NC. (N) Representative TEM images of Mito-ER contacts in GES-1 cells transfected with OE-PHB1 plasmid. Scale bar, 100 nm. Quantification of the mean distance between Mito and ER association (O), and the percentage of the Mito-surface close to the ER (P) in GES-1 cells.\u0026nbsp; ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001 vs OE-NC. (Q) Schematic diagram illustrating the acquisition of a malignant phenotype in GES-1 cells. Created with BioRender.com. (R-U) Actin-Tracker Red-594, Transwell and pCMV-AT1.03 were used to detect cytoskeletal remodeling, invasive ability, and ATP production, respectively. ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001 vs OE-NC, \u0026amp;\u0026amp;\u0026amp;\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001 vs OE-PHB1. \u0026nbsp;(V-W) Western blot analysis of PHB1, VDAC1, GRP75 and IP3R protein expression in GES-1 cells transfected with OE-PHB1 plasmid or treated with VBIT-12. ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001 vs OE-NC, \u0026amp;\u0026amp;\u0026amp;\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001 vs OE-PHB1. (X-Y) Detection of mPTP opening in GES-1 cells transfected with OE-PHB1 plasmid using Calcein AM staining. Scale bar, 50 μm. ***\u003cem\u003eP \u003c/em\u003e\u0026lt;0.001 vs OE-NC. (Z-A1) Detection of the level of MMP in GES-1 cells transfected with OE-PHB1 plasmid using JC-1 fluorescent probe staining. Scale bar, 50 μm. ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 vs OE-NC. (B1-C1) MitoTracker® Red CMXRos and ER-Tracker Green were used for dual staining of mitochondria and endoplasmic reticulum in GES-1 cells transfected with OE-PHB1 plasmid. Scale bar, 50 μm. ***\u003cem\u003eP \u003c/em\u003e\u0026lt;0.001 vs OE-NC. Representative traces and quantifications of Ca\u003csup\u003e2+\u003c/sup\u003e signals in the mitochondrial matrix (D1), ER lumen (F1), and cytosol (H1) in GES-1 cells. Whisker plots of data collected from 88–150 (E1), 92–260 (G1), and 130–192 (I1) seconds from at least three independent coverslips analyzed from 3 independent experiments in GES-1 cells.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7625265/v1/e06fdd50a371bf9b77929027.png"},{"id":92682599,"identity":"fb8c6b3e-5692-414e-9fa8-96c9a310314f","added_by":"auto","created_at":"2025-10-03 01:18:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":580360,"visible":true,"origin":"","legend":"\u003cp\u003ePHB1 affects mitochondrial function and energy metabolism, and mediates cytoskeleton remodeling in gastric cancer through calcium channel. (A-C) The effect of Phalloidin staining on microfilaments in AGS and HGC-27 cells transfected with OE-PHB1 plasmid and shRNA-PHB1, separately. Scale bar, 50 μm. ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 vs OE-NC, ###\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001, ##\u003cem\u003eP\u003c/em\u003e \u0026lt;0.01 vs shRNA-NC. Representative traces and quantifications of Ca\u003csup\u003e2+\u003c/sup\u003e signals in the mitochondrial matrix (D), ER lumen (F), and cytosol (H) in AGS cells. Whisker plots of data collected from 88–150 (E), 92–260 (G), and 130–192 (I) seconds from at least three independent coverslips analyzed from 3 independent experiments in AGS cells. (J-L) Detection of mPTP opening in AGS and HGC-27 cells transfected with OE-PHB1 plasmid and shRNA-PHB1 using Calcein AM staining. Scale bar, 50 μm. ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 vs OE-NC, ###\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001, ##\u003cem\u003eP\u003c/em\u003e \u0026lt;0.01 vs shRNA-NC. (M-O) Detection of the level of MMP in AGS and HGC-27 cells transfected with OE-PHB1 plasmid and shRNA-PHB1 using JC-1 fluorescent probe staining. Scale bar, 50 μm. ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 vs OE-NC, ###\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 vs shRNA-NC. (P-R) Representative images of AGS and HGC-27 cells following transfection with the pCMV-AT1.03 plasmid. Scale bar, 40 μm. ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 vs OE-NC, ###\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 vs shRNA-NC. (S-U) The fluorescent probe DCFH-DA was used to stain ROS in AGS and HGC-27 cells transfected with OE-PHB1 plasmid and shRNA-PHB1. Rosup was used as a positive control for ROS. Scale bar, 50 μm. **\u003cem\u003eP\u003c/em\u003e \u0026lt;0.01 vs OE-NC, ###\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 vs shRNA-NC. (V) Schematic diagram illustrating the subcellular localization of PHB1, VDAC1, GRP75, IP3R and their role in the composition of the MAMs structure. Created with BioRender.com.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7625265/v1/596405cd4e1d267955393411.png"},{"id":92681972,"identity":"ef305e8d-fdfb-4cfb-9888-985e63d745f1","added_by":"auto","created_at":"2025-10-03 01:10:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":547079,"visible":true,"origin":"","legend":"\u003cp\u003ePHB1 mediates the structure of MAMs to regulate mitochondrial and endoplasmic reticulum communication and affects ATP synthase production. (A-D) Western blot analysis of ATP2A2 and ATP5A1 protein expression in AGS and HGC-27 cells transfected with OE-PHB1 plasmid and shRNA-PHB1. ***\u003cem\u003eP \u003c/em\u003e\u0026lt;0.001 vs OE-NC. ###\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs shRNA-NC. (E-G) MitoTracker® Red CMXRos and ER-Tracker Green were used for dual staining of mitochondria and endoplasmic reticulum in AGS and HGC-27 cells transfected with OE-PHB1 plasmid and shRNA-PHB1. Scale bar, 30 μm. ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 vs OE-NC. ##\u003cem\u003eP\u003c/em\u003e \u0026lt;0.01, ###\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 vs shRNA-NC. (H) Representative TEM images of Mito-ER contacts in AGS cells transfected with OE-PHB1 plasmid and shRNA-PHB1. Scale bar, 100 nm. Schematic diagram illustrating the connections between mitochondria and endoplasmic reticulum in AGS cells transfected with OE-PHB1 plasmid and shRNA-PHB1. Created with BioRender.com. Quantification of the mean distance between Mito and ER association (I), and the percentage of the Mito-surface close to the ER (J) in AGS cells. ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 vs OE-NC. ###\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs shRNA-NC. (K-M) Immunofluorescence double staining was performed to detect the co-localization of PHB1 with VDAC1, PHB1 with GRP75 and PHB1 with IP3R in AGS and HGC-27 cells transfected with OE-PHB1 plasmid and shRNA-PHB1. Scale bar, 25 μm. **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 vs OE-NC. ##\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ###\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 vs shRNA-NC. (N-Q) Western blot analysis of PHB1, VDAC1, GRP75 and IP3R protein expression in AGS and HGC-27 cells transfected with OE-PHB1 plasmid and shRNA-PHB1. *** \u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 vs OE-NC. ##\u003cem\u003eP\u003c/em\u003e \u0026lt;0.01, ###\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 vs shRNA-NC. (R-T) Co‑IP assay verified the binding between PHB1 and VDAC1, VDAC1 and GRP75, GRP75 and IP3R in AGS cells. (U-W) Co‑IP assay verified the binding between PHB1 and VDAC1, VDAC1 and GRP75, GRP75 and IP3R in HGC-27 cells. (X-Z1) Western blot analysis of PHB1, VDAC1, GRP75 and IP3R protein expression in AGS and HGC-27 cells transfected with OE-PHB1 plasmid or treated with VBIT-12. *\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, * *\u003cem\u003eP\u003c/em\u003e \u0026lt;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs OE-NC. 888\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs OE-PHB1.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-7625265/v1/7c1328b3d12bb5d85ec0d32c.png"},{"id":92681974,"identity":"85eb0984-5f25-496d-bc9c-a49accae5afb","added_by":"auto","created_at":"2025-10-03 01:10:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":90697395,"visible":true,"origin":"","legend":"\u003cp\u003ePHB1 regulates the protein expression of VDAC1, GRP75, IP3R and ATP synthase in vivo. (A) Schematic illustration of the establishment of a nude mouse model. (B) The photographs of the nude mouse models established by intraperitoneal injection of AGS cells transfected with shRNA-NC and shRNA-PHB1, respectively. (C) Photographs of tumors from above two groups. (D) Tumor volume was quantified using Image J (n=5). ###\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 vs shRNA-NC. (E-F) Western blot was used to detect the protein expression levels of PHB1, VDAC1, GRP75, IP3R, ATP5A1, and ATP2A2 in nude mice. ##\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001, ###\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs shRNA-NC. (G-I) Immunofluorescence double staining was performed to detect the co-localization of PHB1 with VDAC1, PHB1 with GRP75 and PHB1 with IP3R in mice. Scale bar, 50 μm. ###\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 vs shRNA-NC.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-7625265/v1/1b416b8f7517e81124e881a7.png"},{"id":92683112,"identity":"39f59b2a-e911-49b4-a4e6-23c62f5f2a06","added_by":"auto","created_at":"2025-10-03 01:35:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":86822462,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7625265/v1/dec138ef-669e-461a-8035-cd6b607bcdf5.pdf"},{"id":92681971,"identity":"7c9c927d-e96c-4ac1-8e30-486ed9f48c8f","added_by":"auto","created_at":"2025-10-03 01:10:53","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4790692,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Fig. 1 (A-B) Western blot for verifying the transfection efficiency of PHB1 in GES-1 cells transfected with OE-PHB1 plasmid. ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 vs OE-NC. (C-D) Western blot for verifying the transfection efficiency of PHB1 in AGS cells transfected with OE-PHB1 plasmid. ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 vs OE-NC. (E-F) Western blot for verifying the infection efficiency of PHB1 in AGS cells infected with shRNA-PHB1. ###\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 vs shRNA-NC. (G-H) Western blot for verifying the transfection efficiency of PHB1 in HGC-27 cells transfected with OE-PHB1 plasmid. ***\u003cem\u003eP \u003c/em\u003e\u0026lt;0.001 vs OE-NC. (I-J) Western blot for verifying the infection efficiency of PHB1 in HGC-27 cells infected with shRNA-PHB1. ###\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 vs shRNA-NC.\u003c/p\u003e","description":"","filename":"SupplementaryFig.1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7625265/v1/37bdd346a2dea4106b53fb55.tif"},{"id":92681970,"identity":"538ccabd-4e70-41c6-817e-3ec0308a6fdb","added_by":"auto","created_at":"2025-10-03 01:10:53","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1409998,"visible":true,"origin":"","legend":"","description":"","filename":"Westernblots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7625265/v1/312b7aaefe25188d122bdbe2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Prohibitin 1 Orchestrates Mitochondrial-ER Crosstalk via the VDAC1-GRP75-IP3R Axis to Drive Malignancy in Gastric Cancer","fulltext":[{"header":"Introduction ","content":"\u003cp\u003eThe research on cancer metastasis has reached an intense stage, also regarded as a \"bottleneck period.\" Clarifying the relationship between oncogenes and ultrastructural changes is crucial to uncovering the mechanisms underlying cancer metastasis[1]. Gastric cancer (GC) is a tumor characterized by high molecular and phenotypic heterogeneity, with a high incidence and mortality rate[2]. Despite substantial advancements in therapeutic approaches such as neoadjuvant chemotherapy and radiotherapy, clinical challenges such as high recurrence and metastasis potential persist[3, 4].\u003c/p\u003e\n\u003cp\u003eRecent years have witnessed growing interest in the communication between the endoplasmic reticulum (ER) and mitochondria (Mito), with its dysregulation implicated in diverse pathologies including cancer, diabetes, and neurodegenerative diseases[5]. Mitochondria-associated ER membranes (MAMs) play pivotal roles in key intracellular pathways such as calcium homeostasis, mitochondrial dynamics, and energy metabolism.[6, 7]. In cancer research, MAMs serve as a central hub regulating cell survival: MAMs-dependent calcium release from the ER and its selective uptake by Mito are closely linked to malignant behaviors such as proliferation and invasion in cancer cells when their homeostasis is disrupted[8]. Studies indicate that numerous proteins, including Drp1, PINK1, Mfn2, PERK, GRP75, IP3R, VDAC1, and REEP1, participate in the formation and regulation of MAMs[9] [10]. Specifically, GRP75 facilitates the interaction between the inositol trisphosphate receptor (IP3R) on the ER membrane and VDAC1 on the mitochondrial outer membrane, thereby maintaining MAMs structure by modulating calcium ion homeostasis and inter-organelle signaling [11]. Moreover, GRP75 acts as a critical tethering protein promoting MAMs formation. Notably, in ovarian cancer patients experiencing relapse, GRP75 enhances cisplatin resistance by promoting MAMs enrichment[11]. However, the role of MAMs structure in the metastatic progression of gastric cancer remains poorly understood.\u003c/p\u003e\n\u003cp\u003eThe prohibitin (PHB) family members PHB1 and its homolog PHB2 are multifunctional chaperone/scaffold proteins. Primarily localized to the mitochondrial inner membrane, they are also present in the plasma membrane and nucleus. PHB1 and PHB2 play crucial roles in fundamental cellular processes including proliferation, differentiation, and apoptosis, contributing to the maintenance of mitochondrial homeostasis and apoptotic machinery[12] [13]. Research indicates that PHB1 and PHB2 are involved in the development of various cancers, exhibiting a dual role in oncogenesis—functioning as either tumor suppressors or promoters depending on the cancer type and their interacting protein partners[14]. Studies suggest that PHB1, acting as a regulator of mitochondrial inner membrane permeability, mediates the survival and death of prostate cancer cells through Smad-dependent pathways and MAPK intracellular signaling[15]. Another study demonstrated that silencing the PHB1 gene in ovarian cancer disrupts mitochondrial integrity, alters the proportion in the G0/G1 phase of the cell cycle, and increases susceptibility to apoptosis[16]. Similarly, downregulation of PHB2 expression suppresses lung cancer cell proliferation by stabilizing RACK1[17]. Reduced PHB2 expression also delays the proliferation rate of renal cancer cells by inhibiting MNK/eIF4E signaling[18]. In summary, PHB1 and PHB2 play pivotal roles in the progression of multiple cancers; however, the precise mechanisms underlying their context-dependent contributions to cancer advancement warrant further investigation.\u003c/p\u003e\n\u003cp\u003eAnalysis of the TCGA database in this study revealed elevated expression of both PHB1 and PHB2 in gastric cancer tissues. Notably, PHB1 expression was found to be significantly higher than that of PHB2 in omental metastatic tissues. Our investigation uncovered a coupling between PHB1 and the MAMs structure (specifically, the IP3R-GRP75-VDAC1 complex) in both primary gastric cancer and its metastatic lesions. Furthermore, we demonstrated that PHB1 maintains the integrity of the MAMs structure by modulating the VDAC1-GRP75-IP3R complex in a VDAC1-dependent manner. This mechanism ultimately drives gastric cancer progression by regulating intracellular calcium homeostasis, mitochondrial energy metabolism, and cytoskeletal remodeling. These findings unveil a novel mechanism through which PHB1 promotes gastric cancer progression by regulating MAMs function via the VDAC1 complex, providing a theoretical foundation for therapeutic strategies targeting this pathway.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eBioinformatical analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the expression of PHB1 and PHB2 in normal and gastric cancer tissues, we downloaded RNA-seq data for gastric cancer from The Cancer Genome Atlas Program (TCGA) and used R software to filter the PHB1 gene. Data visualization and plotting were performed using the limma, ggplot2, and ggplot2 packages in R software. For the generation of heatmaps for PHB1 and PHB2, we downloaded clinical data of gastric cancer patients from TCGA. The ComplexHeatmap and limma packages in R software were used to create heatmaps based on gene expression data and clinical information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpecimen of human pathological tissue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eClinical specimens of gastric cancer tissues, adjacent tissues of the gastric cancer, and omental tissues with gastric cancer metastasis from 29 patients were collected from 79 gastric cancer patients in the North China University of Science and Technology Affiliated Hospital from 2020 to 2023. None of these patients had a history of chemotherapy or radiotherapy. This study has been approved by the Institutional Review Board of North China University of Science and Technology Affiliated Hospital (Ethics Approval Number: 2020405).\u0026nbsp;Each patient signed a written informed consent form. All the procedures were conducted in strict accordance with the Declaration of Helsinki.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry (IHC) and immunofluorescence (IF) staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGastric cancer tissues, adjacent non-cancerous tissues, and metastatic gastric cancer tissues were fixed, embedded, sectioned, deparaffinized and repaired. For IHC, the sections were blocked with 3% hydrogen peroxide, while for IF, the sections were permeabilized for 15 min and then blocked with goat serum. The sections were incubated with primary antibodies PHB1(1:200; Cat No.10787-1-AP; Proteintech, China), VDAC1(1:100; Cat No.55259-1-AP; Proteintech, China), GRP75(1:200; Cat No.14887-1-AP; Proteintech, China), IP3R (1:100; DF3000; Affinity, China) were overnight at 4°C. The next day, after rewarming, for IHC, the sections were incubated with HRP-conjugated secondary antibody (PV-9000, ZSGB-BIO, China) at 37°C for 1 h. After washing with PBS three times, the sections were stained with DAB and hematoxylin, sealed with neutral resin, and observed under an inverted microscope. For IF, the sections were incubated with secondary antibody Alexa Fluor® 488 Conjugate (1:100; #ZF-0511; ZSGB-BIO) at 37°C for 1 h, counterstained with DAPI, and observed under a fluorescence microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman gastric adenocarcinomas cell lines (AGS; ZQ0240), human gastric cancer cells (HGC-27; ZQ0192) and human gastric mucosal cells (GES-1; ZQ0905) were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (www.zqxzbio.com).). According to the manufacturers’ instructions, AGS and HGC-27cells were grown in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin/streptomycin) at 37℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e. GES-1 cells were grown in DMEM high glucose medium containing 10% FBS and 1% antibiotics (penicillin/streptomycin). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell transfection and drug treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Full-length of PHB1 (Sangon Biotech Co., Ltd.) genes was cloned into the pcDNA3.1(+) vector, separately. 2.5 µg of PHB1 overexpression (OE-PHB1) plasmid were transfected separately into AGS, HGC-27 and GES-1 with Lipo8000™ (cat no. C0533; Beyotime Biotechnology) at 37˚C for 24 h, with empty vector as a negative control (NC). The short hairpin (sh)RNA target PHB1 were designed by GenePharma for the knockdown of the corresponding genes. The sequences are as follows: PHB1 shRNA(5’- GCCGTTCTCGACCACGTAATG-3’)；\u0026nbsp;and shRNA-NC (Sense 5’-\u0026nbsp;TTCTCCGAACGTGTCACGT-3’). For the infection of lentivirus, when cell confluency reached 40-60%, the culture medium was removed, and fresh medium containing 5 µg/mL Polybrene and 15 µL of lentiviral solution (1×10\u003csup\u003e8\u003c/sup\u003e TU/mL) were added. After 24h of incubation, the fresh medium was replaced and the cell phone cells were cultured for another 3 days for experimental days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranswell\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing a 24-h serum starvation period, GES-1 cells were prepared into a cell suspension, and 200 µL of the cell suspension was added to the Transwell upper chamber. The culture was conventionally grown at 37°C and 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 h. Subsequently, the cells were fixed with 4% paraformaldehyde solution for 10 min and stained with 0.1% crystal violet for 20 min. Subsequently, the cells were washed twice with PBS and gently wiped with a cotton swab to remove residual cells. Five visual fields were randomly selected from the center and surrounding areas; these fields were then observed, photographed, and counted with an inverted microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSkeleton remodeling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe morphology and distribution of actin filaments were detected using Actin-Tracker Red-594 (C2205S, Beyotime Biotechnology, China). Transfected cells were cultured on coverslips for 24 h, washed, fixed, and the staining solution was added and incubated for 30 min. After the cells were washed and dried, the coverslips were blocked using DAPI and then imaged.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMitochondrial permeability transition pore assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mPTP Assay Kit (C2009S, Beyotime Biotechnology, China) was utilized to assess the degree of mPTP opening. After the transfected cells were seeded on 6-well plates and cultured for 24 h, the medium was removed, and the cells were washed. Subsequently, 1mL of calcein AM staining solution, fluorescence quenching solution, or ionomycin was added and incubated for 30 min. After re-incubation with fresh medium, the medium was removed, detection buffer was added to incubate for 30 min, and the cells were observed under a fluorescence microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMitochondrial membrane potential assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMMP was assessed using the JC-1 fluorescent probe (C2006, Beyotime Biotechnology, China). After the above seeding plates and culturing procedures were repeated for transfected cells, 1 mL of cell medium and 1 mL of JC-1 staining working solution were added and incubated for 20 min. The supernatant was removed, cells were washed twice, cell culture medium was added and imaged.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eATP level assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntracellular ATP levels in individual live cells were monitored in real-time using the ATP fluorescent probe (pCMV-AT1.03) (D2604, Beyotime Biotechnology, China). AGS, HGC-27, and GES-1 cells, successfully transfected were seeded in 12-well plates and cultured at 37℃ with 5% CO₂ for 24 h. Afterward, 1 µg of the ATP fluorescent probe was added to each well, and the cells were further incubated for 24 h. Fluorescence imaging was then performed using a fluorescence microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eROS\u003c/strong\u003e \u003cstrong\u003eproduction assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAGS, HGC-27, and GES-1 cells, successfully transfected were stained with 10 μM DCFH-DA (S0033M, Beyotime Biotechnology, China) in the dark for 30 min. Fluorescence images of intracellular ROS were then captured using a fluorescence microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCrude mitochondria from ten representative samples were isolated following the established protocols[11]. Total protein extraction kit (KGP2100; KeyGEN BioTECH, Nanjing, China) and BCA Protein Assay Kit (PC0020; Solarbio, Beijing, China) were used to extract proteins and determine protein concentrations. Protein was separated by 9% SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with blocking solution containing TBST (TBS with Tween-20) and 5% milk powder for 1 h at room temperature. After that, the membranes were incubated with primary antibodies VDAC1(1:1000; Cat No.55259-1-AP; Proteintech, Wuhan, China), PHB1(1:2000; Cat No. 10787-1-AP; Proteintech, Wuhan, China), PHB2 (1:2000; Cat No. 12295-1-AP; Proteintech, Wuhan, China), GRP75(1:5000; Cat No.14887-1-AP; Proteintech, China), IP3R (1:1000; DF3000; Affinity, China), Vimentin (1:1000; bs-8533R; Bioss, Beijing, China), β-catenin (1:2000; bs-1165R; Bioss, Beijing, China), ATP2A2 (1:3000; Cat No. 67248-1-Ig; Proteintech, Wuhan, China), ATP5A1 (1:5000; Cat No. 66037-1-Ig; Proteintech, Wuhan, China), β-actin (1:5000; Cat No.60004-1-Ig; Proteintech, Wuhan, China). After overnight incubation at 4℃, the secondary antibody Horseradish Peroxidase (HRP)-conjugated goat-anti-rabbit IgG (ZB-2301, ZSGB-Bio) or HRP-conjugated goat-anti-mouse IgG (ZB-2305, ZSGB-Bio) was used to incubate the PVDF membranes for 1 h at room temperature. Finally, the protein signals were detected signals with electrochemiluminescence solution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRT-qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCrude mitochondria from ten representative samples were isolated following the established protocols[11]. Total RNA and Mitochondrial RNA was extracted using the Trizol method. RNA was reverse-transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (00752219, Thermo Fisher Scientific, China). Quantitative real-time PCR (RT-qPCR) was performed using PowerUp™ SYBR™ Green Master Mix (01118369, Thermo Fisher Scientific, China) in a 20 μL reaction volume. The PCR cycling conditions consisted of an initial denaturation at 94°C for 2 min; followed by 30 cycles of denaturation at 94°C for 5 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. GAPDH was used as the internal reference gene. Primers were synthesized by Sangon Biotech (Shanghai, China; https://www.sangon.com/). The primer sequences were as follows:\u003c/p\u003e\n\u003cp\u003ePHB1-F: 5’-CACTGGTAGCAAAGATTTACAG-3’\u003c/p\u003e\n\u003cp\u003ePHB1-R: 5’-ATAGTCCTCTCCGATGCTG-3’\u003c/p\u003e\n\u003cp\u003ePHB2- F: 5’-AATCTGTGTTCACCGTGGA -3’\u003c/p\u003e\n\u003cp\u003ePHB2- R: 5’- CCAGGATAGTGTCCTGCTG-3’\u003c/p\u003e\n\u003cp\u003eGAPDH-F: 5’-CAAGGTCATCCATGACAACTTTG-3’\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGAPDH-R:5’\u0026nbsp;-GTCCACCACCCTGTTGCTGTAG-3’\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCellular immunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cells were fixed with 4% paraformaldehyde for 10 min and then permeabilized with 0.5% Triton X-100 for 15 min at 37℃. After incubation with goat serum for 30 min, cells were incubated overnight with the following antibodies: VDAC1(1:100; Cat No.55259-1-AP; Proteintech, Wuhan, China), PHB1(1:200; Cat No. 10787-1-AP; Proteintech, Wuhan, China), GRP75(1:200; Cat No.14887-1-AP; Proteintech, China), IP3R (1:100; DF3000; Affinity, China). The second day, the cells were incubated with Alexa Fluor® 488 Conjugate (1:100; ZF-0511; ZSGB-BIO, China) secondary antibodies at 37˚C for 1 h and sealed with DAPI, observed under a fluorescence microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransmission electron microscope detection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTransmission electron microscopy (TEM) was used to examine the morphology and distribution of cellular Mito and ER. After digestion, centrifugation, and collection, the cells were fixed with 2.5% glutaraldehyde and 1% osmium tetroxide, respectively. After the cells were dehydrated and embedded, ultrathin sections were prepared using an ultramicrotomy mechanism. The sections were stained with lead citrate and uranyl acetate and subsequently observed with TEM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-staining of mitochondria and endoplasmic reticulum\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMitochondrial and endoplasmic reticulum changes in cells were labeled using the MitoTracker® Red CMXRos and ER-Tracker Green fluorescent probe. After culturing the cells on coverslips for 24 h, the culture medium was removed, and the cells were incubated with pre-warmed MitoTracker® Red CMXRos and ER-Tracker Green staining solutions for 30 min. The staining solution was then removed, and the cells were washed twice with culture medium. Fluorescence microscopy was subsequently used for observation and imaging.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo‑immunoprecipitation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were rinsed with PBS, followed by the addition of 100 μL of lysis buffer. Of the extracted proteins, 50μl were served as the input control. 3μg of PHB1 antibody were used, with normal IgG of the same species serving as the negative control. The antibodies were diluted according to the manufacturer's instructions and added 100 µL of Protein A+G magnetic beads, and incubated at room temperature for 15 min. 500 μL protein sample was incubated with Protein A+G magnetic beads (Beyotime, Cat.no: P2179) bound with antibodies or normal IgG overnight at 4°C to allow binding of the proteins to the antibodies. After incubation, the samples were centrifuged at 1000 g for 5 minutes at 4°C, and the supernatant was removed. The beads were washed four times with PBS. Subsequently, 100 μL of 1X SDS-PAGE Sample Loading Buffer was added and the samples were heated at 95°C for 5 min, the supernatant was collected as the sample for the PHB1-IP group for Western blot analysis. For VDAC1-IP, repeat the above operation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e imaging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e dynamics were monitored with 4mtD3cpv sensor, a genetically encoded Ca\u003csup\u003e2+\u003c/sup\u003e indicator targeted to the mitochondrial matrix. The cells were transfected with the 4mtD3cpv probe for 48 h. Coverslips were washed with Ca\u003csup\u003e2+\u003c/sup\u003e containing KRB solution and mounted on the microscope stage. Samples were illuminated at 420 nm and simultaneously acquired at 475 nm (donor, ECFP) and 530 nm (acceptor, circularly permuted (cp) Venus). CpVenus/ECFP ratio was calculated online using MetaFluor software. After acquisition of basal Ca\u003csup\u003e2+\u003c/sup\u003e levels (first 30 s of acquisition), the cells were stimulated with 100 μM ATP. Regions of interest (ROIs) were defined around individual Mito.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFura-2 Ca\u003csup\u003e2+\u003c/sup\u003e imaging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe concentration of Ca²⁺ was determined using Fura-2 AM (S1052, Beyotime Biotechnology, China) as previously described in the literature[19]. In brief, the cells were plated onto coverslips (3×10\u003csup\u003e4\u003c/sup\u003e cell/coverslip), and loaded with 2.5 μM Fura-2 AM (S1052, Beyotime Biotechnology, China) in the presence of 0.005% Pluronic F-127 and 10 μM sulfinpyrazone in Ca\u003csup\u003e2+\u003c/sup\u003e-containing KRB solution. After loading (30 min in the dark at normal temperature), cells were washed once with KRB solution and allowed to de-esterify for 30 min. After this, the coverslips were mounted in an acquisition chamber and placed on the stage of the microscope, and cells were alternately excited at 340 and 380 nm. The fluorescent signal was collected through a 510/20 nm bandpass filter. The cells were stimulated with 100 μM ATP. For comparison of Ca\u003csup\u003e2+\u003c/sup\u003e dynamics, measured as an amplitude of Ca\u003csup\u003e2+\u003c/sup\u003e increase from the baseline level, Fura-2 ratio values were normalized using the formula (Fi-F0)/F0[referred to as Normalized (Norm.) Fura Ratio].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEndoplasmic reticulum Ca\u003csup\u003e2+\u003c/sup\u003e imaging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eER Ca\u003csup\u003e2+\u003c/sup\u003e dynamics were monitored with\u0026nbsp;D1ER, a genetically encoded Ca\u003csup\u003e2+\u003c/sup\u003e sensor, targeted to the ER lumen. The cells were transfected with the D1ER probe for 48 h. Coverslips were mounted in a chamber in KRB solution and placed on the stage of the microscope. Cells were alternately excited at 405 and 470 nm, and the fluorescent signal was acquired using a 510/20 nm bandpass filter. After recording basal signal for 30 s, KRB solution was removed and replaced with a Ca\u003csup\u003e2+\u003c/sup\u003e-free solution. After allowing the signal to stabilize for an additional 30 s, cells were stimulated with 100 µM ATP and 100 µM Tert-butylhydroquinone (TBHQ), and the response was recorded for 300 s.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBALB/c nude mice (16–20 g, 4–5 weeks old; obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were randomly assigned into two groups (n = 5 per group). AGS cells transfected with shRNA-NC or shRNA-PHB1 (5 × 10⁶ cells in 0.1 mL) were intraperitoneally injected into the region surrounding the greater omentum. After a 4-week breeding period, the mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg) and subsequently euthanized by cervical dislocation. The greater omentum tumor tissues were excised to measure tumor width (W) and length (L), and the tumor volume (V) was calculated using the formula V = (L\u0026nbsp;× W²)/2. The tumor tissues were reserved for further experiments as required. All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of North China University of Science and Technology Affiliated Hospital (Approval No. LAEC-NCST-2020036).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data were expressed as mean ± standard error and analyzed with SPSS 27.0 software. Differences between groups were compared with one-way ANOVA followed by Bonferroni post hoc test. The \u003cem\u003eP\u003c/em\u003e value less than 0.05 indicates a statistically significant difference.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1.\u003c/strong\u003e \u003cstrong\u003eExpression of PHB1 in gastric cancer and metastatic tissues and its coupling with the MAMs structure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project focused on investigating the mechanism by which PHB1 mediates gastric cancer metastasis (\u003cstrong\u003eFig. 1A\u003c/strong\u003e). Analysis of the TCGA database revealed significantly elevated expression of both PHB1 and PHB2 in gastric cancer tissues (GCT) compared to normal gastric tissues (NGT). Notably, PHB1 expression correlated significantly with tumor stage, while PHB2 expression was associated with demographic factors such as sex and age (\u003cstrong\u003eFig. 1B-E\u003c/strong\u003e). Based on these findings, we collected GCT and NGT from 79 gastric cancer patients, including greater omentum (GO) metastatic tissues from 29 of these patients. IHC, RT-qPCR, and Western blot analyses consistently demonstrated significantly higher expression of both PHB1 and PHB2 in GCT and GO compared to NGT. Crucially, PHB1 expression in GO, particularly its mitochondrial localization, was significantly higher than in GCT, underscoring its pivotal role in gastric cancer metastasis (\u003cstrong\u003eFig. 1F-J\u003c/strong\u003e). Clinicopathological analysis of the 79 patients further revealed that PHB1 expression significantly correlated with TNM stage, depth of invasion, and metastasis, but showed no association with sex, age, Helicobacter pylori infection status, or overall survival (\u003cstrong\u003eTable 1\u003c/strong\u003e), aligning with the TCGA findings. Immunofluorescence results indicated markedly enhanced co-localization (manifested as yellow areas/white arrows) of PHB1 with the mitochondrial channel protein VDAC1 and the ER calcium channel protein IP3R in GCT and GO compared to NGT, suggesting spatial coupling and functional association among PHB1, VDAC1, and IP3R in gastric cancer and metastatic tissues (\u003cstrong\u003eFig. 1K-L\u003c/strong\u003e). TEM further revealed closer apposition between the ER and mitochondrial membranes, along with broader ER coverage over mitochondria in gastric cancer and metastatic tissues (\u003cstrong\u003eFig. 1M-O\u003c/strong\u003e). Integrating these experimental results with the established central role of GRP75 in mediating IP3R-VDAC1 signaling[11], we propose a hypothetical model outlining the potential mechanism of PHB1 in gastric cancer metastasis (\u003cstrong\u003eFig. 1P\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePHB1 regulates MAMs-mediated cytoskeletal remodeling and energy metabolism in GES-1 cells, promoting the acquisition of malignant phenotypes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWestern blot analysis revealed that PHB1 overexpression significantly upregulated the protein expression levels of key MAMs components (VDAC1, GRP75, IP3R) and malignant phenotype markers (Vimentin, \u0026beta;-catenin) (\u003cstrong\u003eFig. 2A-C\u003c/strong\u003e). Immunofluorescence co-localization analysis further confirmed that PHB1 overexpression enhanced its co-localization with VDAC1, GRP75, and IP3R within MAMs domains (manifested as yellow signal) (\u003cstrong\u003eFig. 2D-F\u003c/strong\u003e). Functionally, PHB1 overexpression not only increased ATP production (\u003cstrong\u003eFig. 2G-H\u003c/strong\u003e) but also induced cytoskeletal remodeling, as evidenced by elevated fluorescence intensity (\u003cstrong\u003eFig. 2I-J\u003c/strong\u003e). Protein-protein interaction network analysis and subsequent Western blot validation both indicated that PHB1 interacts with and upregulates the expression of ATP synthase components ATP2A2 and ATP5A1 (\u003cstrong\u003eFig. 2K-M\u003c/strong\u003e). TEM observations demonstrated that PHB1 overexpression significantly reduced the distance between Mito-ER and expanded the area of ER coverage over Mito (\u003cstrong\u003eFig. 2N-P\u003c/strong\u003e). Based on these findings, we propose that PHB1 likely modulates MAMs structure by regulating the GRP75-IP3R complex via VDAC1. To test this hypothesis, we employed the VDAC1-specific inhibitor VBIT-12. Experimental results showed that VBIT-12 effectively attenuated the effects induced by PHB1 overexpression, including suppression of cytoskeletal remodeling, reduction in cell invasive capacity, and decreased ATP production (\u003cstrong\u003eFig. 2R-U\u003c/strong\u003e). Concurrently, VBIT-12 treatment downregulated the protein expression of PHB1, GRP75, and IP3R (\u003cstrong\u003eFig. 2V-W\u003c/strong\u003e). These results collectively demonstrate that PHB1, by regulating the VDAC1-GRP75-IP3R complex, influences MAMs structure and function, thereby mediating cytoskeletal remodeling and energy metabolic reprogramming, ultimately promoting the acquisition of malignant phenotypes (the efficacy of the PHB1 overexpression plasmid was validated in Supplementary \u003cstrong\u003eFig. 1A-B\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eFurthermore, our study confirms that PHB1 plays a critical regulatory role in mitochondrial function. Overexpression of PHB1 promoted closure of the mPTP in GES-1 cells (\u003cstrong\u003eFig. 2X-Y\u003c/strong\u003e) and enhanced MMP (\u003cstrong\u003eFig. 2Z-A1\u003c/strong\u003e). Using dual fluorescence probes co-labeling mitochondria (red) and the endoplasmic reticulum (green), we observed that PHB1 overexpression significantly increased their co-localization area (yellow signal), indicating enhanced mitochondria-ER contacts (\u003cstrong\u003eFig. 2B1-C1\u003c/strong\u003e). Calcium signaling detection using transfected specific indicators (4mtD3cpv, D1ER, Fura-2) revealed that PHB1 overexpression augmented mitochondrial calcium uptake capacity. This led to corresponding decreases in calcium levels within the ER and cytosol. Although this regulatory effect was transient, mitochondrial calcium signaling was significantly potentiated during its active phase (\u003cstrong\u003eFig. 2D1-I1\u003c/strong\u003e). Collectively, these results demonstrate that by enhancing the formation of MAMs, PHB1 effectively regulates mitochondrial calcium homeostasis and metabolic function, thereby maintaining mitochondrial hyperactivation and ultimately driving malignant transformation in gastric cancer cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePHB1 regulates ER-mitochondrial calcium flux homeostasis to influence mitochondrial function and bioenergetic metabolism, ultimately driving cancer cytoskeletal remodeling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further validate the mechanistic role of PHB1 in gastric cancer metastasis, we established PHB1 overexpression and knockdown models in AGS and HGC-27 cells (\u003cstrong\u003eSupplementary Fig. 1C-J\u003c/strong\u003e). Given that cytoskeletal remodeling represents a critical morphological hallmark of malignant progression in cancer cells, we observed that PHB1 overexpression significantly promoted filopodia formation and elongation in both cell lines, whereas PHB1 knockdown suppressed this process (\u003cstrong\u003eFig. 3A-C\u003c/strong\u003e). This indicates that PHB1 drives cytoskeletal remodeling in gastric cancer cells. Regarding calcium homeostasis, PHB1 knockdown impaired mitochondrial calcium uptake capacity, resulting in calcium accumulation in the ER and cytosol, ultimately triggering calcium overload (\u003cstrong\u003eFig. 3D-I\u003c/strong\u003e). Thus, PHB1 directly impacts mitochondrial function and cytoskeletal dynamics by regulating calcium flux between Mito-ER. Building upon this, we further investigated the central role of PHB1 in mitochondrial function. PHB1 deficiency induced excessive opening of the mPTP (\u003cstrong\u003eFig. 3J-L\u003c/strong\u003e), reduced MMP (\u003cstrong\u003eFig. 3M-O\u003c/strong\u003e), decreased ATP production (\u003cstrong\u003eFig. 3P-R\u003c/strong\u003e), and increased ROS generation (\u003cstrong\u003eFig. 3S-U\u003c/strong\u003e). Conversely, PHB1 overexpression produced the opposite effects. Collectively, these findings demonstrate that PHB1 serves as a central hub maintaining mitochondrial functional homeostasis. By regulating ER-mitochondrial calcium flux equilibrium, PHB1 drives cytoskeletal remodeling in gastric cancer cells, thereby promoting their malignant progression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePHB1 drives cancer adaptation by enhancing MAMs to modulate calcium signaling and energy metabolism\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn cancer, ATP2A2-mediated calcium signaling activates mitochondrial metabolism, which cooperates with ATP5A1-driven ATP synthesis to promote oxidative phosphorylation (OXPHOS). This coordinated process collectively fulfills the high energy demands of cancer cells, supports apoptotic resistance, and enhances microenvironmental adaptation[20, 21]. Our study reveals that PHB1 regulates the expression of both ATP2A2 and ATP5A1, which is crucial for maintaining calcium homeostasis and ATP production (\u003cstrong\u003eFig. 4A-D\u003c/strong\u003e). Furthermore, PHB1 overexpression significantly increased the number of mitochondria-associated endoplasmic reticulum membranes (yellow areas, \u003cstrong\u003eFig. 4E-G\u003c/strong\u003e), reduced the intermembrane distance, and expanded the ER coverage over mitochondria. Conversely, PHB1 knockdown resulted in fewer MAMs, increased intermembrane distance, and reduced ER coverage (\u003cstrong\u003eFig. 4H-J\u003c/strong\u003e). These findings establish that PHB1 enhances MAM structure, thereby providing a critical platform for efficient calcium signal transduction and energy metabolism driven by OXPHOS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGiven that VDAC1, GRP75, and IP3R are core constituents of MAMs and play pivotal roles in ER-Mito signaling, we observed that PHB1 overexpression not only expanded its contact sites with the VDAC1-GRP75-IP3R complex but also upregulated the expression levels of these proteins. Conversely, PHB1 knockdown dampened these effects (\u003cstrong\u003eFig. 4K-Q\u003c/strong\u003e). Furthermore, Co-IP assays directly confirmed protein-protein interactions between PHB1 and VDAC1, VDAC1 and GRP75, and GRP75 and IP3R (Fig. 4R-W). Notably, treatment with the VDAC1 inhibitor VBIT-12 under PHB1-overexpressing conditions downregulated protein expression of PHB1, VDAC1, GRP75, and IP3R (\u003cstrong\u003eFig. 4X-A1\u003c/strong\u003e). Collectively, these results demonstrate a mutually dependent, functionally coupled bidirectional regulatory relationship between PHB1 and the VDAC1-GRP75-IP3R complex at MAMs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePHB1 knockdown suppresses tumor growth and disrupts energy homeostasis by impairing MAMs integrity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing a peritoneal metastasis model established by intraperitoneal injection of gastric cancer cells (\u003cstrong\u003eFig. 5A\u003c/strong\u003e), we demonstrated that PHB1 knockdown significantly suppressed tumor growth (\u003cstrong\u003eFig. 5B-D\u003c/strong\u003e). Furthermore, PHB1 depletion led to a marked reduction in protein expression levels of the core MAMs components VDAC1, GRP75, and IP3R (\u003cstrong\u003eFig. 5E-F\u003c/strong\u003e). Critically, a significant decrease in the overlapping co-localization signals between PHB1 and these proteins was observed (\u003cstrong\u003eFig. 5G-I\u003c/strong\u003e). We also noted that PHB1 knockdown significantly downregulated the protein expression of ATP5A1 and ATP2A2. Collectively, PHB1 knockdown disrupted the VDAC1-GRP75-IP3R complex, impaired MAMs functionality, caused dysregulation of ATP5A1 and ATP2A2 protein expression, perturbed cancer cell bioenergetic homeostasis, and consequently inhibited in vivo tumor growth.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiscussion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn recent years, targeting key functional proteins involved in metastasis has proven effective in significantly suppressing cancer progression, including survival, metastasis, and drug resistance[22]. However, identifying critical proteins within the complex molecular interaction networks governing metastasis remains a challenge for clinicians[23]. Tight membrane contact sites between organelles, such as MAMs that coordinate ion homeostasis and cellular functions[24, 25]，participate in regulating cancer cell metabolism, metastasis, and drug resistance[26]. Their functionality is typically mediated through the modulation of ER proteins or the influence on mitochondrial proteins[27, 28]. This study revealed the specific expression of the mitochondrial inner membrane proteins PHB1 and PHB2 in gastric cancer tissues and their omental metastases, particularly highlighting the significance of PHB1 in omental metastasis. This implicates a coupling relationship between PHB1 and MAMs components (VDAC1, GRP75, IP3R). Mechanistically, PHB1 regulates the VDAC1-GRP75-IP3R complex in a VDAC1-dependent manner, maintaining MAM structure integrity. This, in turn, modulates calcium flux between the ER and Mito to sustain intracellular calcium homeostasis. Ultimately, this PHB1-mediated regulation facilitates the acquisition of malignant phenotypes by driving MAM-dependent cytoskeletal remodeling and alterations in energy metabolism, thereby promoting gastric cancer progression.\u003c/p\u003e\n\u003cp\u003eAnalysis of The Cancer Genome Atlas (TCGA) and The Human Protein Atlas indicates that prohibitins (PHBs) are overexpressed in various tumor tissues and are recognized as critical regulators of the cell cycle[13]. PHBs (including PHB1 and PHB2) participate in regulating cancer cell proliferation, apoptosis, metabolism, differentiation, and metastasis, yet exhibit dual roles\u0026mdash;either promoting or suppressing tumorigenesis\u0026mdash;in different cancer studies[22]. Specifically, PHB1 mediates cancer cell drug resistance and metastasis by regulating the Ras-mediated c-Raf-MEK-ERK signaling pathway[29, 30]；In non-small cell lung cancers (NSCLCs), high PHB1 expression significantly correlates with poor tumor differentiation, lymph node metastasis, and low survival rates[31]；Similarly, PHB2 expression is upregulated in liver cancer, and silencing PHB2 suppresses cancer cell growth and colony formation, induces G1 phase arrest, and enhances susceptibility to apoptosis[32]. These findings suggest that both PHB1 and PHB2 mediate adaptive survival in different cancer cells across various developmental stages. This study focuses on gastric cancer. We observed specific and differential expression patterns of PHB1 and PHB2 in GC tissues, adjacent non-tumorous tissues, and omental metastatic tissues, particularly highlighting the significance of PHB1 in omental metastasis. Furthermore, we found that PHB1 expression closely correlates with the tumor stage of GC, further emphasizing its critical role in GC metastasis. Notably, although reduced PHB1 expression has been reported to associate with tumor dedifferentiation and carcinogenesis, and alterations in PHB copy number and the rs6917 polymorphism may influence GC progression[33], both our study and data retrieved from the TCGA database revealed PHB1 overexpression in GC tissues. Our analysis of 79 GC patient tissues also aligned with the database findings. This discrepancy may reflect variations in the differentiation status of the collected samples, indicating that PHB1 function differs across GCs of varying differentiation grades. Additionally, this study reveals an association between PHB1 and components of MAMs.\u003c/p\u003e\n\u003cp\u003eMAMs are closely implicated in cancer initiation and progression[34]. Within this context, the IP3R-GRP75-VDAC signaling axis plays a pivotal role, particularly by regulating calcium ion (Ca\u003csup\u003e2+\u003c/sup\u003e) signaling, lipid metabolism, and cellular stress responses, thereby influencing cancer cell proliferation and growth[35]. Specifically, the IP3R-GRP75-VDAC complex facilitates Ca\u0026sup2;⁺ transfer from the ER to Mito. This process is crucial for regulating glycolytic metabolism and survival in highly therapy-resistant glioma-initiating cells[36]. Our study revealed that overexpression of the PHB1 protein in GES-1 cells promoted cytoskeletal remodeling and enhanced Ca\u003csup\u003e2+\u003c/sup\u003e flux, ultimately leading to the acquisition of a malignant phenotype. Concurrently, PHB1 overexpression significantly upregulated the protein levels of key MAMs components\u0026mdash;VDAC1, GRP75, and IP3R\u0026mdash;reduced the distance between Mito and the ER, increased the number and extent of MAMs contact sites, and induced fusiform morphological changes in the ER, which appeared to wrap around mitochondria. Regarding these ER morphological alterations, we propose two potential explanations: First, a self-protective adaptive adjustment by the ER in response to altered ionic environments (e.g., active Ca\u003csup\u003e2+\u003c/sup\u003e flux). Second, an adaptive remodeling in response to changes in mitochondrial function. We posit that these PHB1-mediated structural alterations in MAMs are significant factors driving cytoskeletal remodeling and the acquisition of a malignant phenotype in GES-1 cells. However, the specific functional implications of the observed ER morphological changes, including those involving the lamellar ER[37], were not explored in depth in the current study.\u003c/p\u003e\n\u003cp\u003eMaintaining mitochondrial function is a complex process involving numerous resident and non-resident mitochondrial proteins[38]. Mitochondrial dysfunction is associated with impaired mitochondrial OXPHOS, increased ROS production, mitochondrial permeability transition, and swelling induced by Ca\u003csup\u003e2+\u003c/sup\u003e overload[39]. PHB1, as an inner mitochondrial membrane protein, has garnered significant attention[40]. It has been found to participate in the degradation of mitochondrial respiratory chain subunits, the assembly and activity of the OXPHOS system, mitochondrial biogenesis, apoptosis, and autophagy[41]. Recent studies indicate that the role of PHB1 in cancer is closely linked to mitochondrial function and metabolism. Knockdown of PHB1 leads to significantly reduced mitochondrial integrity, increased ROS production, and calcium imbalance, subsequently resulting in mitochondrial dysfunction[42]. We observed similar outcomes through genetic manipulation of PHB1. Elevated PHB1 protein expression was detected in both human gastric cancer omental metastases and those in nude mice, potentially indicating that tumor cells within metastatic sites exhibit heightened energy metabolism (potentially influenced by hypoxic factors, though this study did not investigate this aspect). Depletion of PHB1 in cancer cells can induce mitochondrial dysfunction and disrupt energy output. More significantly, PHB1 was found to modulate the distance and contact area between Mito and the ER in gastric cancer cells. Based on the observed alterations in mitochondria-ER interactions, we speculate that this modulation may involve Ca\u0026sup2;⁺ signaling contributing to increased energy output. Changes in the distance and contact area between Mito and the ER reflect the structural integrity of MAMs. MAMs integrity directly regulates mechanisms underlying cancer cell metastasis and drug resistance[11]. Indeed, PHB1 deficiency disrupts MAMs structural integrity, thereby impeding Ca\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003eexchange between Mito and the ER. This disruption leads to reduced mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e concentration and Ca\u003csup\u003e2+\u003c/sup\u003e accumulation within the ER. ER Ca\u003csup\u003e2+\u003c/sup\u003e accumulation acts as a dual-edged sword; excessive accumulation can induce apoptosis, depending on the magnitude and duration of the accumulation[43]. We propose that PHB1 may influence cancer cell biological behavior by regulating Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis mechanisms between Mito and the ER, thereby altering MAMs structure and function. Notably, Ca\u003csup\u003e2+\u003c/sup\u003e accumulation in the ER might be transient, as the ER can activate other signaling pathways to restore Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis. This hypothesis warrants further exploration in future studies.\u003c/p\u003e\n\u003cp\u003eNotably, administration of the VDAC1-specific inhibitor VBIT-12 not only blocked the regulatory effects of PHB1 but also suppressed the protein expression of PHB1, VDAC1, GRP75, and IP3R. More importantly, this experiment demonstrated that disrupting VDAC1, a key component of MAMs, and its function effectively reversed the malignant phenotypes induced by PHB1 overexpression. This provides compelling causal evidence that PHB1 drives cytoskeletal remodeling and malignant transformation by remodeling MAMs structure, specifically by enhancing VDAC1-GRP75-IP3R-mediated Ca\u003csup\u003e2+\u003c/sup\u003e signaling.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGiven the characteristic enrichment of hypermetabolic mitochondria in metastatic cancer cells, research targeting the disruption of MAMs integrity to suppress cancer progression contributes to a deeper understanding of the roles of mitochondrial function and energy metabolism in human tumors. A key challenge addressed in this study was determining the specific role of PHB1 in maintaining MAM integrity. However, the function of PHB1 in cancer is complex and contentious: while it exhibits oncogenic properties in certain cell lines, opposing effects are observed in others. This paradoxical phenomenon may be attributed to the tissue of origin, differentiation status, microenvironmental conditions (including hypoxia), and distinct stages of cancer progression[44]. Notably, metabolic profiles undergo dynamic changes during cancer progression, exerting differential effects on primary and metastatic tumors[45]. Based on these observations, we propose that PHB1 mediates peritoneal metastasis in gastric cancer cells by regulating the structural integrity of MAMs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn summary, PHB1 maintains MAMs structural integrity by modulating the VDAC1-GRP75-IP3R complex in a VDAC1-dependent manner. This regulation governs calcium homeostasis, mitochondrial energy metabolism, and cytoskeletal remodeling, ultimately driving gastric cancer progression. These findings reveal that PHB1 promotes gastric cancer progression through VDAC1 complex-mediated regulation of MAMs function, providing a compelling rationale for targeted therapeutic strategies.\u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePHB1, prohibitin 1; ER, endoplasmic reticulum; MAMs, mitochondria-associated ER membranes; Mito, mitochondria; mPTP, mitochondrial permeability transition pore; MMP, mitochondrial membrane potential; Drp1, dynamin-related protein 1; PINK, PTEN-induced putative kinase 1; Mfn2, mitofusin-2; PS1, presenilin-1, PKR, protein kinase R; PERK, PKR-like endoplasmic reticulum kinase; GRP75, glucose-regulated protein 75; VAPB: vesicle-associated membrane protein-associated protein B, VDAC1, voltage-dependent anion channel 1; \u0026nbsp; REEP1, receptor expression-enhancing protein 1; HSP70, heat shock protein 70; IP3R, inositol 1,4,5-trisphosphate receptor; OXPHOS, oxidative phosphorylation; ROS, reactive oxygen species; GES-1, human gastric mucosal epithelial cells; TCGA, the cancer genome atlas program; OE-PHB1, PHB1 overexpression; TEM, transmission electron microscopy; FBS, fetal bovine serum; NC, negative control; sh, short hairpin; shRNA-PHB1, (sh)RNA target PHB1\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki. All procedures were performed in compliance with relevant laws and institutional guidelines and have been approved by the Ethics Committee of North China University of Science and Technology Affiliated Hospital (No. 2020405, LAEC-NCST-2020036). Informed consent was obtained from the participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsent for the publication of our data was obtained from the patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Ningxia Natural Science Foundation (2024AAC03702、2023AAC03529), Leading Talents of Science and Technology in Ningxia Hui Autonomous Region (2023GKLRLX19), National 14th Five-Year Key Research and Development Plan Project (2022YFC3602101), Training Project of Top Young Talents in General Hospital of Ningxia Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYu Miao: Conceptualization; Xiaofei Wang: Methodology; Ying Huang: Data curation; Yuxia Zhang: Formal analysis; Zhanchuan Liu: Resources; Hengtong Yin: Software; Caiyue Liu: Visualization; Yuanzhen Wang: Investigation; Hua Yin: Project administration; Yafang Lai: Supervision; Feixiong Zhang: Validation; Shaoqi Yang: Writing-review \u0026amp; editing and Funding acquisition; Weiqiang Li: Writing-original draft\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eStoletov K, Beatty PH, Lewis JD. 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Frontiers in physiology. 2022; 13: 910452.\u003c/li\u003e\n\u003cli\u003eArtal-Sanz M, Tavernarakis N. Prohibitin and mitochondrial biology. Trends in endocrinology and metabolism: TEM. 2009; 20: 394-401.\u003c/li\u003e\n\u003cli\u003eSignorile A, Sgaramella G, Bellomo F, De Rasmo D. Prohibitins: A Critical Role in Mitochondrial Functions and Implication in Diseases. Cells. 2019; 8: 71.\u003c/li\u003e\n\u003cli\u003eQi A, Lamont L, Liu E, Murray SD, Meng X, Yang S. Essential Protein PHB2 and Its Regulatory Mechanisms in Cancer. Cells. 2023; 12: 1211.\u003c/li\u003e\n\u003cli\u003eZheng S, Wang X, Zhao D, Liu H, Hu Y. Calcium homeostasis and cancer: insights from endoplasmic reticulum-centered organelle communications. Trends in cell biology. 2023; 33: 312-323.\u003c/li\u003e\n\u003cli\u003eBarbier-Torres L, Lu SC. Prohibitin 1 in liver injury and cancer. Experimental biology and medicine (Maywood, NJ). 2020; 245: 385-394.\u003c/li\u003e\n\u003cli\u003eBezwada D, Perelli L, Lesner NP, Cai L, Brooks B, Wu Z, et al. Mitochondrial complex I promotes kidney cancer metastasis. Nature. 2024; 633: 923-931.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1. \u0026nbsp;Correlation of clinicpathological parameters and PHB1 expression in patients with gastric cancer\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003eClinical parameters\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003cp\u003e(n=79)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\"\u003e\n \u003cp\u003ePHB1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePositive（57）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNegative（22）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGender\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e0.023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e0.879\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e1.475\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e0.225\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e＜60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026ge;60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eHelicobacter pylori infection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e0.142\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e0.706\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTNM stage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e6.286\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e0.012\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eⅠ-Ⅱ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eⅢ-Ⅳ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eDepth of invasion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e4.318\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e0.038\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eT1/T2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eT3/T4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eLymph metastasis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e3.970\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e0.046\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePeritoneal metastasis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e4.780\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e0.029\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSurvival\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e0.065\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\"\u003e\n \u003cp\u003e0.799\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-communication-and-signaling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccas","sideBox":"Learn more about [Cell Communication and Signaling](http://biosignaling.biomedcentral.com/)","snPcode":"12964","submissionUrl":"https://submission.nature.com/new-submission/12964/3","title":"Cell Communication and Signaling","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"PHB1, MAMs, VDAC1, GRP75, Gastric cancer","lastPublishedDoi":"10.21203/rs.3.rs-7625265/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7625265/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), serving as critical hubs for mitochondrial- ER interactions, play a pivotal role in cancer progression. Prohibitin 1 (PHB1), an inner mitochondrial membrane protein, contributes to tumorigenesis by regulating energy metabolism and structural integrity. This study aimed to elucidate the molecular mechanism by which PHB1 regulates MAMs structural integrity and its cascade signaling in driving gastric cancer (GC) metastasis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: We analyzed the expression profiles of PHB1/PHB2 in GC using the TCGA database. Immunohistochemistry was performed to detect PHB1/PHB2 protein expression in GC tissues, adjacent non-tumor tissues, and omental metastasis tissues, and to assess their correlation with clinicopathological features. Immunofluorescence was used to observe the co-localization of PHB1 with the IP3R-GRP75-VDAC1 complex. Live-cell Ca2+ imaging, ATP/ROS detection, and mitochondrial functional assays (mPTP opening/MMP) were employed to evaluate calcium flux and energy metabolism changes. Transmission electron microscopy and Western blot were utilized to analyze MAMs structural integrity. A nude mouse xenograft model was established to validate in vivo functions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: TCGA analysis confirmed that both PHB1 and PHB2 were overexpressed in GC. PHB1 expression significantly correlated with tumor stage, while PHB2 expression correlated with demographic characteristics. Mitochondrial PHB1 expression was significantly higher than PHB2 in omental metastasis tissues. Furthermore, PHB1 co-localized and interacted with the IP3R-GRP75-VDAC1 complex, regulating ER-to-mitochondria Ca2+ flux. PHB1 overexpression enhanced MAMs-mediated Ca2+ signaling, increased ATP production, and drove cytoskeletal remodeling, thereby promoting malignant progression. Conversely, PHB1 knockdown disrupted the IP3R-GRP75-VDAC1 axis, leading to MAMs structural dissociation and mitochondrial dysfunction. Notably, treatment with the VDAC1-specific inhibitor VBIT-12 in PHB1- overexpressing cells significantly reversed the pro-tumorigenic effects of PHB1, reduced PHB1 protein levels, and diminished its association with the GRP75-IP3R complex, demonstrating that PHB1 function depends on VDAC1 activity. In summary, PHB1 regulates the VDAC1-GRP75-IP3R complex in a VDAC1-dependent manner to maintain MAMs structural integrity. This subsequently modulates calcium homeostasis, mitochondrial energy metabolism, and cytoskeletal remodeling, ultimately driving GC progression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: Our findings reveal a novel mechanism by which PHB1 promotes GC progression via regulating MAMs function through the VDAC1 complex, providing a theoretical basis for therapeutic targeting.\u003c/p\u003e","manuscriptTitle":"Prohibitin 1 Orchestrates Mitochondrial-ER Crosstalk via the VDAC1-GRP75-IP3R Axis to Drive Malignancy in Gastric Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-03 01:10:48","doi":"10.21203/rs.3.rs-7625265/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-30T17:09:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-08T14:16:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-06T07:54:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"6607061719381834250354509017770460047","date":"2025-09-27T12:48:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"40688894624321638492553534099502747597","date":"2025-09-22T01:09:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"75865127226079489588678706319111619890","date":"2025-09-22T00:50:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-21T18:48:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-19T09:52:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-19T09:50:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Communication and Signaling","date":"2025-09-16T02:55:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-communication-and-signaling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccas","sideBox":"Learn more about [Cell Communication and Signaling](http://biosignaling.biomedcentral.com/)","snPcode":"12964","submissionUrl":"https://submission.nature.com/new-submission/12964/3","title":"Cell Communication and Signaling","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a19c1c5d-262a-499a-a4c4-d54d66ca63d2","owner":[],"postedDate":"October 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-03-28T20:09:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-03 01:10:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7625265","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7625265","identity":"rs-7625265","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}