FNDC3B promotes gastric cancer metastasis via interacting with FAM83H and preventing its proteasomal degradation

Abstract

Background

Gastric cancer (GC) is one of the most prevalent digestive tract malignancies, with metastasis being a major contributor to poor prognosis in patients. Fibronectin type III domain-containing 3B (FNDC3B) plays pivotal roles in various tumors, yet its role in GC remains unknown.

Methods

We utilized a GC immunohistochemistry (IHC) chip and specimens to identify high expression of FNDC3B in GC. In vitro cellular experiments and in vivo nude mouse models were constructed to validate the biological functions of FNDC3B. Truncated mutants of FNDC3B and immunofluorescence (IF) assays were conducted to explore the critical domains of FNDC3B. Liquid chromatography-tandem mass spectrometry (LC–MS) and coimmunoprecipitation (Co-IP) were employed to screen and identify target proteins interacting with FNDC3B. Rescue experiments were performed to uncover interactions between FNDC3B and the binding protein.

Results

FNDC3B was found to function as an oncogene in GC. Clinically, FNDC3B was significantly upregulated in GC specimens and associated with poor survival in patients with GC. Functionally, elevated FNDC3B promoted GC metastasis both in vitro and in vivo. Structurally, the proline-rich N-terminus and a transmembrane (TM) domain at the C-terminus were crucial for maintaining the metastasis function of FNDC3B. Mechanistically, FNDC3B interacted with FAM83H, inhibiting the ubiquitin–proteasome degradation of FAM83H, which in turn enhanced GC progression through the FNDC3B/FAM83H/Snail/EMT axis.

Conclusions

Our results demonstrate that FNDC3B promotes GC metastasis and has the potential to serve as a therapeutic target for GC.

Supplementary Information

The online version contains supplementary material available at 10.1186/s11658-025-00741-7.

Introduction

Gastric cancer (GC) is a prominent and lethal malignancy within the digestive system, presenting high incidence and mortality rates [1]. The global burden of GC is projected to increase substantially, with forecasts predicting 1.8 million new cases and 1.3 million deaths annually by 2040 [2, 3]. This alarming situation is exacerbated by the fact that patients often receive a GC diagnosis at advanced stages and face recurrence due to distant metastasis to vital organs including the liver, peritoneum, and lungs [4–6]. Uncovering the pivotal mechanisms underpinning GC progression is crucial for identifying effective biomarkers for early diagnosis and devising innovative therapeutic strategies to optimize patient outcomes.

The fibronectin type III domain-containing (FNDC) protein family plays crucial roles in cell adhesion, migration, and differentiation processes [7, 8]. A hallmark of FNDC family members is the presence of at least one conserved fibronectin type III domain, which is necessary to maintain extracellular matrix integrity and cell adhesion [7, 9]. Through protein–protein interactions (PPIs) and signaling pathway activation, FNDC proteins serve as vital biomarkers and regulators in various tumors [10–12]. Notably, fibronectin type III domain-containing protein 1 (FNDC1) has emerged as a pivotal player in digestive system cancers, including GC [13, 14]. However, research on FNDC3B, another member of the FNDC family, presents contradictory findings in digestive system tumors, and its specific role in GC is yet to be elucidated [11, 15–18].

The family with sequence similarity 83 member H (FAM83H), a member of the FAM83 protein family characterized by a conserved DUF1669 motif that is involved in PPIs, has garnered attention for its potential function in GC progression [19, 20]. Initially, FAM83H was reported to participate in the regulation of cytoskeleton, cellular adhesion, and differentiation [21–23]. Recent studies have highlighted its role in facilitating GC progression through the regulation of the epithelial–mesenchymal transition (EMT) [19]. EMT is a fundamental biological process that enables epithelial cells to acquire mesenchymal properties, thereby enhancing their migratory and invasive capabilities [24, 25]. In the context of GC, EMT facilitates the intravasation of cancer cells into the bloodstream and lymphatic system, promoting their survival and colonization in distant organs [25, 26]. Consequently, unraveling the precise role of FAM83H and its interplay with the EMT pathway will provide new insights into GC pathogenesis and contribute to the development of targeted therapies.

In this study, we demonstrated that FNDC3B was upregulated in GC and promoted GC cell metastasis both in vitro and in vivo. Structurally, we emphasized the importance of the proline-rich N-terminus and a transmembrane (TM) domain encompassing the C-terminus in maintaining the biological function of FNDC3B. Mechanistically, our findings revealed that FNDC3B interacted with and inhibited the ubiquitin–proteasome degradation of FAM83H, thereby fostering GC progression through the FNDC3B/FAM83H/Snail/EMT axis.

Materials and methods

Cell cultures

The normal gastric epithelial cell GES-1 or GC-derived cell lines HGC-27 were procured from the American Type Culture Collection (ATCC) through the Army Medical University (Chongqing, China). AGS (no. CTCC-001-0038) and MKN-74 (no. CTCC-400-0252) cells were purchased from Meisen (Hangzhou, China). All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Basal Media, Shanghai, China) or RPMI 1640 (Basal Media, Shanghai, China) containing 10% fetal bovine serum (Lonsera, Montevideo, Uruguay) in an incubator at 37 ℃, 5% CO₂ atmosphere.

Clinical specimens

In total, 25 pairs of GC tissues and their adjacent normal tissue were obtained from Southwest Hospital of Army Medical University (Chongqing, China) as previously described in our published study [27]. Clinical characteristics are listed in Supplementary Material Table S1. All fresh specimens were preserved in RNAlater (Thermo Fisher, Massachusetts, USA) immediately at 4 ℃ overnight, followed by storage at −80 ℃. All specimens required for this study were approved by the Ethics Committee of Southwest Hospital of Army Medical University.

Plasmids, siRNA, and cell transfection

Transient overexpression of FNDC3B and FAM83H was constructed by inserting the CDS region into the pcDNA3.1 and pCMV3 vector, respectively; 3× Flag-tagged full-length and truncated FNDC3B plasmids were constructed in the pCMV vector. pCMV-HA-Ub plasmids were provided by Professor Lei Liu from the First Affiliated Hospital of Chongqing Medical University, Chongqing, China. Plasmids and Neofect™ (Zero-Customer Innovation Biotechnology, Beijing, China) were mixed at a 1:1 ratio and transfected into GC cells for 48 h.

Small interfering RNA (siRNA) targeting FNDC3B, FAM83H, and negative control (NC) were synthesized by GenePharma (Shanghai, China) (Supplementary Material Table S2). Generally, 2.5 × 10⁵ cells/well were seeded in six-well plates overnight, followed by transfection with mixture containing siRNA and Lipo2000™ (Invitrogen, CA, USA) according to the manufacture’s instruction. Fresh medium was replaced 4–6 h after transfection, and cells were collected at setting time points.

Lentivirus transduction

To construct GC cell lines with stable knockdown of FNDC3B and FAM83H, the pHBLV-luc-Puro and pLVX-luc-Puro vectors were used to produce lentivirus particles at HanBio (Shanghai, China). For the generation of GC cell lines with stable overexpression of FNDC3B, the pLVX-Puro-FNDC3B lentivirus (Oligobio, Beijing, China) was used to infect GC cells. AGS cells were infected with lentiviruses at a multiplicity of infection (MOI) of 125 and selected with puromycin (2 μg/ml) for 1 week. Western blot analysis was employed to ascertain the efficiency of transfection.

Cell counting kit-8 (CCK-8) test and colony formation assay

Cell viability was assessed using the CCK-8 (Biosharp, Hefei, China). Initially, 1.5 × 10³ cells/well were seeded into a 96-well plate overnight with 100 μL of complete growth medium. Subsequently, the CCK-8 reagent was diluted in complete culture medium at a 1:10 ratio, and 100 μL of this mixture was added to each well. After incubating the cells for 2 h, the absorbance values at 450 nm were measured using a Varioskan LUX microplate reader (Thermo Fisher Scientific, Massachusetts, USA).

Colony formation assay was employed to assess the proliferative capacity of GC cells. Briefly, 1.5 × 10³ cells/well were seeded into a six-well plate and maintained under standard conditions for approximately 2 weeks. The resulting colonies were fixed with 4% paraformaldehyde (Biosharp, Hefei, China) for 15 min, followed by staining with a 1% crystal violet (Biosharp, Hefei, China) for 5 min. Images were scanned using an Epson scanner (Suwa, Japan).

Transwell migration and wound healing assays

To assess the migration ability of GC cells, both Transwell migration and wound healing assays were conducted. Specifically, the 8-μm Transwell chamber (Corning, NY, USA) was filled with 150 µL of serum-free media containing 5 × 104 GC cells, while the corresponding 24-well plate was filled with 600 µL of media containing 10% serum. After incubation for 20 h, the Transwell chambers were fixed with 4% paraformaldehyde for 15 min, and then stained with 1% crystal violet for 5 min. Additionally, a wound healing assay was performed as previously described. Briefly, GC cells were scratched in six-well plate for 48 h of transfection. Following three washes with phosphate-buffered saline (PBS), the complete culture medium was replaced with medium containing 1% FBS. The Transwell chamber and wound images were captured using a microscope (Nexscope, Ningbo, China) and analyzed by ImageJ software.

Subcutaneous tumor xenograft model

Male Balb/c nude mice (4–6 weeks old) were purchased from Chengdu Yaokang Biotechnology Company. All animal studies were approved by the Ethics Review Committee of Chongqing Medical University, China. To construct subcutaneous xenografts, 1 × 10⁶ HGC-27 cells suspended in 100 μL of PBS were subcutaneously inoculated on the right back of each mouse. The experiments were performed when the tumor volume reached approximately 70 mm³. In brief, cholesterol modified siNC (negative control) or si-FNDC3B (GenePharma, Shanghai, China) was mixed with an in vivo transfection reagent (Engreen, Beijing, China) and dissolved in sterile saline solution to a total volume of 25 μL per mouse. Each nude mouse received multiple intratumoral injections at intervals of 1 day. Tumor volume was measured using calipers and recorded to plot growth curves. After approximately 3 weeks of treatment, the nude mice were euthanized to harvest the tumors tissues for photography, weighing, and western blot detection.

In vivo metastasis model

Male Balb/c nude mice (5 weeks old) were used for in vivo metastasis model constrction. Briefly, AGS cells (sh-NC and sh-FNDC3B) were intraperitoneally injected at a concentration of 2.0 × 10⁶ AGS cells in 200 μL of PBS. After 50 days, the mice were euthanized, and liver tissues were harvested for hematoxylin and eosin (H&E) staining. For the in vivo metastasis rescue experiment, 1.5 × 10⁶ AGS cells (sh-NC + Lv-Vector, sh-NC + Lv-FNDC3B, and sh-FAM83H + Lv-FNDC3B) were suspended in 100 μL PBS and injected into the spleens of the nude mice. Following a 50-day observation period, the liver tissues were collected for IHC staining.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Quantitative RT-PCR assays were performed as previously described [28, 29]. First, total cellular and tissue RNA were extracted by Trizol reagent (Takara, Kyoto, Japan). Next, reverse transcription process was conducted using the PrimeScript™ RT reagent Kit (Takara, Kyoto, Japan), followed by the quantitative PCR (qPCR) analysis of RNA expression levels using 2× SP qPCR Mix (Bioground, Chongqing, China). All primers utilized in this study were synthesized by Tsingke Biotechnology (Beijing, China). The sequences of the primers are detailed in Supplementary Material Table S3.

Western blot analysis

Western blot was conducted as mentioned earlier [30]. Total protein from tissue or cultured cells was extracted by radioimmunoprecipitation assay buffer (RIPA) (Beyotime, Shanghai, China) containing 1% phenylmethylsulfonyl fluoride (PMSF). Proteins were then separated by 8–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels and transferred to a polyvinylidene fluoride (PVDF) membrane (0.22 μm). The membrane was blocked with 5% skim milk at room temperature for 1 h and subsequently incubated with various primary antibodies (Supplementary Material Table S4) overnight at 4 ℃. The next day, the blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and visualized with the enhanced chemiluminescence (ECL) detection reagent (Bioscience, China).

Immunofluorescence (IF) assay

IF assay was employed to assess the subcellular localization of FNDC3B and FAM83H as previously reported [31]. Briefly, 2.5 × 10⁴ GC cells were seeded in u-Slide eight-well plates (Ibidi, Martinsried, Germany) and incubated overnight. On the following day, cells were fixed with 4% paraformaldehyde (Biosharp, Hefei, China) for 15 min. After fixation, the cells were permeabilized and blocked with 5% bovine serum albumin (BSA) containing 0.5% Triton X-100 at room temperature for 30 min. Subsequently, the primary antibody (Supplementary Material Table S4) was diluted to an appropriate concentration according to the manufacturer’s instructions and incubated with the cells at 4 ℃ overnight. On the next day, the corresponding fluorescent secondary antibody was used to incubate the cells at room temperature for 1 h, and the cell nuclei were stained with Hoechst (Beyotime, Shanghai, China) for 10 min. After being washed three times by PBS, images of the stained cells were obtained using a fluorescence microscope (Leica SP8, Wetzlar, Germany).

Immunoprecipitation–mass spectrometry (LC–MS) analysis

LC–MS detection was conducted by Jingzhou Gene Technology (Shanghai, China). Briefly, the protein samples were subjected to SDS–PAGE separation, followed by overnight tryptic digestion. Subsequently, the peptide solution resulting from enzymatic hydrolysis was desalted using a desalting column [32, 33]. The digested peptides were analyzed with a Q Exactive mass spectrometer (Thermo Fisher Scientific, Massachusetts, USA). The fragment spectra were analyzed using the Proteome Discoverer 2. 2.

Coimmunoprecipitation (Co-IP)

The Co-IP experiment was conducted following the manufacturer’s protocol for the Pierce Crosslink Magnetic IP/Co-IP Kit (Thermo Fisher Scientific, Massachusetts, USA). Generally, 30-μL protein A/G magnetic beads and either primary antibodies or control immunoglobulin (Ig)G (5 µg antibody) were added and incubated for 15 min at room temperature on a rotator respectively. Next, the beads were crosslinked with antibodies using 20 μM of sodium dextran sulfate (DSS) at room temperature for 45 min on a rotator. Concurrently, 1.5 × 10⁷ cells were lysed using 1 mL Pierce™ IP lysis buffer for 30 min on ice. Postlysis, supernatants were harvested by centrifugation at 13,000g for 15 min. The lysate supernatants were then incubated with the magnetic beads overnight at 4 ℃ with rotation. Finally, the antigens bound to the magnetic beads were eluted using 100 μL of elution buffer, and the samples were processed for SDS–PAGE or mass spectrometry (MS) analysis.

Immunohistochemistry (IHC) and H&E staining assay

IHC assays on 80 pairs of GC tissues and their corresponding adjacent noncancerous tissues were performed by Outdo Biotech Company (Shanghai, China), following standardized protocols. The specific information is listed in Supplementary Material Table S5. Image Pro Plus software was used to quantify the expression of FNDC3B. HE staining assays on mouse liver tissues were conducted by Servicebio (Wuhan, China). Images were obtained using a fluorescence microscope (Leica, Germany).

Cycloheximide (CHX) and proteasome inhibitor (MG132) assays

CHX, as an inhibitor of protein synthesis in eukaryotic organisms, is used for the detection of protein stability [34]. CHX (10 µM) (Genview, Beijing, China) was added to AGS cells to detect the half-life of FAM83H. MG132 is a proteasome inhibitor that can inhibit protein degradation by blocking the proteolytic activity of the 26S proteasome complex [35]. AGS cells were treated with 20 µM MG132 (MedChemExpress, New Jersey, USA) for the measurement of the FAM83H accumulation rate.

Molecular docking

AlphaFold-predicted structures of human FNDC3B (UniProt ID: Q53EP0) and FAM83H (UniProt ID: Q6ZRV2) were retrieved from the AlphaFold Database. Protein–protein docking simulations were conducted using AutoDock with default parameters optimized for macromolecular interactions. Binding affinity was evaluated using two metrics: (1) the docking score, where more negative values indicate stronger binding, and (2) the confidence score, with values > 0.7 denoting high-confidence interactions. Molecular interactions were visualized using surface representation.

Statistical analysis

Student’s t-tests were used to evaluate the difference between two groups. A one-way analysis of variance (ANOVA) analysis was chosen for comparisons among multiple groups. P-values < 0.05 were considered statistically significant. Statistical analysis was performed using GraphPad Prism 8 software (GraphPad, San Diego, USA).

Results

High expression of FNDC3B correlates with tumor stage and poor prognosis in GC

In our preliminary investigations, mRNA microarray for GC tissues and paired adjacent tissues underscored the pivotal of the FNDC protein family, with particular emphasis on the prominent ranking of FNDC1 [36]. To gain insights into the expression patterns of the FNDC family, we analyzed their expressions across diverse tumor types by using the TIMER 2.0 database (http://timer.cistrome.org/). As illustrated in Supplementary Material Fig. S1 a and b, our analysis revealed that FNDC3B displays significant expression variations in a wider array of tumors compared with FNDC1, particularly encompassing five distinct gastrointestinal cancer types (cholangiocarcinoma, colon adenocarcinoma, esophageal carcinoma, liver hepatocellular carcinoma, and stomach adenocarcinoma). This observation prompted us to shift our focus towards elucidating the expression patterns and functional roles of FNDC3B in GC.

To decipher the role of FNDC3B in GC, we initiated our investigation by assessing its expression levels. Data retrieved from Gene Expression Profiling Interactive Analysis 2.0 (GEPIA 2.0) (http://gepia2.cancer-pku.cn/#survival) revealed that FNDC3B expression was notably elevated in GC tissues compared with their normal counterparts (Fig. 1A). Importantly, this overexpression positively correlated with advanced tumor stages and was predictive of poorer overall survival rates among patients with GC (Fig. 1B, C). To confirm these findings, we examined FNDC3B expression in multiple GC cell lines, observing significant elevations in both mRNA and protein levels in GC cell lines (AGS, HGC-27, and MKN-74) compared with the normal gastric epithelial cell line GES-1 (Fig. 1D, E). These results were further validated in GC specimens by qRT-PCR and western blot assays (Fig. 1F, G).

Fig. 1.

High expression of FNDC3B correlates with tumor stage and poor prognosis in GC. A–C Analysis of FNDC3B expression in GC using GEPIA 2.0 database: (A) expression levels, (B) clinical staging, and (C) overall survival (high cutoff 55%, low cutoff 45%). D, E Relative mRNA and protein expression levels of FNDC3B in normal gastric epithelial cell line (GES-1) and GC cell lines. F, G Relative mRNA (n = 25) and protein (n = 16) expression levels of FNDC3B in GC biopsy specimens. H Representative images and analysis of FNDC3B staining in GC tissue microarrays (n = 80). I Analysis of FNDC3B expression levels in the nucleus and cytoplasm in GC tissue arrays (n = 80). J Subcellular localization of FNDC3B in AGS and HGC-27 cells detected by immunofluorescence (scale bar: 10 μm). P-values were calculated using Student’s t-test and one way ANOVA, ns means no significance, *P < 0.05, **P<0.01, ***P < 0.001 and ****P < 0.0001

Additionally, IHC assays performed on tissue arrays from 80 patients with GC corroborated the upregulation of FNDC3B in GC (Fig. 1H). Detailed analysis revealed that the heightened expression of FNDC3B was predominantly localized within the cytoplasm, with no discernible difference in nuclear expression between cancerous and adjacent noncancerous tissues (Fig. 1I). Immunofluorescence assays provided further support, demonstrating the predominant cytoplasmic localization of FNDC3B in AGS and HGC-27 cells (Fig. 1J). Collectively, these findings suggest that the highly expressed FNDC3B represents a potential novel oncogene in GC associated with poor prognosis and advanced tumor stages.

FNDC3B drives GC cell metastasis in vitro and in vivo

To comprehend the role of FNDC3B in GC carcinogenesis, we manipulated its expression levels in AGS and HGC-27 cells using specific siRNAs (si-FNDC3B1/2), a pcDNA3.1–FNDC3B overexpression plasmid, and a lentiviral overexpression system. As shown in Fig. 2A–C, successful modulation of FNDC3B protein levels was verified. Subsequent functional assays, including wound healing and Transwell migration tests, illustrated that FNDC3B silencing significantly impaired GC cell migration, whereas its overexpression markedly enhanced it (Fig. 2E–G). Additionally, proliferation assays revealed that FNDC3B promoted cell growth to some extent (Supplementary Material Fig. S2 a and b).

Fig. 2.

FNDC3B drives GC cell metastasis in vitro and in vivo. A, B Protein knockdown and overexpression efficiency of FNDC3B in AGS or HGC-27 cells. C Protein stable overexpression efficiency of FNDC3B in AGS cells. D Protein stable knockdown efficiency of FNDC3B in AGS cells. E Transwell assay for migration capacity of AGS and HGC-27 cells with FNDC3B knockdown or overexpression (scale bar: 200 μm). F, G Wound healing assay for migration capacity of AGS and HGC-27 cells with FNDC3B knockdown or overexpression (scale bar: 200 μm). H Wound healing (left) and Transwell (right) assays for migration capacity of AGS cells with stable FNDC3B knockdown or overexpression (scale bar: 200 μm). I Schematic diagram of the peritoneal dissemination model in nude mice. J Representative images (left) and quantitative statistics (right) of liver metastatic nodules. K Representative images of liver metastatic nodules after H&E staining (scale bar: 500 μm). P-values were calculated using Student’s t-test and one way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001

We next interrogated the impact of FNDC3B on GC cells in vivo. AGS cells were transfected with a FNDC3B-interfering lentivirus based on the si-FNDC3B-1 sequence, which showed superior inhibition efficacy on FNDC3B expression (Fig. 2D). The sh-FNDC3B group exhibited obvious inhibition of cell migration in vitro (Fig. 2H). Next, a peritoneal dissemination model in nude mice was established (Fig. 2I). Approximately 50 days later, the nude mice were euthanized, and major abdominal organs (liver, kidneys, spleen, and mesentery) were examined. GC metastatic foci were exclusively found in the liver, with fewer metastatic nodules in the sh-FNDC3B group compared with the sh-NC group (Fig. 2J). Histological analysis using H&E staining confirmed the presence of liver metastases, characterized by a high nuclear–cytoplasmic ratio, heterogeneity of tumor cells, mitosis, and necrosis (Fig. 2K). These data underscored the pivotal oncogene role of FNDC3B in facilitating GC cell migration in vivo.

Meanwhile, a subcutaneous xenograft model utilizing HGC-27 cells was established in Balb/c nude mice. Upon reaching a tumor volume of approximately 70 mm³, mice were randomly divided into two groups, and received intratumorally injections with cholesterol-modified si-FNDC3B-1 or si-NC, respectively (Supplementary Material Fig. S2 c). Although the difference was not statistically significant, the si-FNDC3B group exhibited a slower tumor growth rate compared with the control group, indicating a moderate suppression of tumor cell proliferation in vivo (Supplementary Material Fig. S2 d and e). Western blot analysis confirmed the effectiveness of the siRNA injections in downregulating FNDC3B expression (Supplementary Material Fig. S2 f). In summary, our findings demonstrated that FNDC3B significantly facilitates GC cell metastasis and modestly enhances cell proliferation, both in vitro and in vivo.

The N-terminal and TM domains are crucial for maintaining the metastasis-promoting function of FNDC3B

According to the NCBI database annotation (https://www.ncbi.nlm.nih.gov/protein/NP_001128567.1), human FNDC3B encodes a protein consisting of 1204 amino acids, featuring a proline-rich N-terminal region, nine FNIII domains, and a C-terminal region that harbors a transmembrane (TM) domain (Fig. 3A). Further analysis using DeepTMHMM and SignalP-5.0 revealed the presence of a C-terminal α-helical TM domain in FNDC3B, whereas a signal peptide domain was absent (Supplementary Material Fig. S3 a and b). On the basis of these structural insights, Flag-tagged FNDC3B truncated mutants were designed (Fig. 3A) and successfully transfected into AGS and HGC2-7 cells (Supplementary Material Fig. S3 c and d). Subsequently, migration assays revealed that the full-length (FL) FNDC3B significantly promoted the migration of GC cells, while the ΔFNIII mutant exhibited comparable migration-promoting effects to the FL group. In contrast, the ΔN-terminal mutant failed to augment GC cell migration, mirroring the control group’s migratory capabilities. Notably, the ΔTM mutant drastically inhibited GC cell migration, displaying a diametrically opposed function compared with the FL group (Fig. 3B–E).

Fig. 3.

The N-terminal and TM domains are crucial for maintaining the metastasis-promoting function of FNDC3B. A Schematic diagram of the construction of 3× Flag-tagged full-length FNDC3B and its truncated mutants. B, C Transwell assay to detect the migration ability of AGS and HGC-27 cells transfected with full-length and various truncated mutants (scale bar: 200 μm). D, E Wound healing assay to detect the migration ability of AGS and HGC-27 cells transfected with full-length and truncated mutants (scale bar: 200 μm). F Immunofluorescence detection of colocalization of FNDC3B with the ER, lysosomes, and mitochondria. G, H Immunofluorescence detection of the colocalization of full-length and truncated mutants of FNDC3B with the ER in AGS and HGC-27 cells. P-values were calculated using one way ANOVA, ns means no significance, *P < 0.05, **P < 0.01, and ***P < 0.001

The TM domain, a hydrophobic amino acid sequence within transmembrane proteins, serves to anchor these proteins to cellular or organelle membranes. Preliminary GeneCards analysis suggested a potential localization of FNDC3B in the endoplasmic reticulum (ER) and cell membrane (Supplementary Material Fig. S3 e), whereas Fig. 1J indicated a primary cytoplasmic localization. Therefore, we postulated that FNDC3B might reside on the ER membrane. To validate this hypothesis, we conducted coimmunostaining of HEK-293 T cells with antibodies against FNDC3B and various organelle markers, including the ER, mitochondria, and lysosomes. As shown in Fig. 3F, FNDC3B predominantly colocalized with ER protein marker calnexin and partially colocalized with mitochondria, but displayed minimal colocalization with lysosome.

Strikingly, upon deletion of the TM domain in GC cells, FNDC3B shifted its localization from the cytoplasm to the nucleus and ceased to colocalize with the ER. However, deletions of the N-terminal and FNIII domains did not alter the subcellular localization of the FNDC3B (Fig. 3G, H). These findings underscored the importance of the N-terminal and TM domains in maintaining FNDC3B’s prometastatic function. Furthermore, deletion of the TM domain not only reversed the biological function of FNDC3B but also altered its subcellular localization.

FNDC3B interacts with FAM83H protein at its N-terminal region

Existing studies have highlighted the critical role of PPIs in FNDC3B’s regulation of tumor progression [10–12]. To pinpoint downstream target proteins that specifically engage with FNDC3B, we devised rigorous criteria to screen potential binding partners following FNDC3B-IP and LC–MS analyses (Fig. 4A, B and Supplementary Material Table S6). Among the 13 candidates identified, four that play important biological roles in GC were selected, and the interactions were initially examined through FNDC3B-IP experiments. Remarkably, Supplementary Material Fig. S4 a illustrated that FAM83H exhibited specific and binding with FNDC3B in both AGS and MKN-74 cells, surpassing other candidates. Notably, as one of the top-ranking target proteins, FAM83H achieved a high confidence score of 0.9411 in molecular docking with FNDC3B (Fig. 4C), strongly suggesting a substantial interaction between these two proteins. This hypothesis was further confirmed using FNDC3B-IP and FAM83H-IP experiments in AGS and MKN-74 cells (Fig. 4D). Meanwhile, immunofluorescence colocalization assays indicated partial colocalization of FNDC3B and FAM83H in the cytoplasm (Fig. 4E). In addition, it was also confirmed that FAM83H, similar to FNDC3B, colocalized with the ER (Supplementary Material Fig. S4 b), further corroborating FNDC3B-FAM83H interaction.

Fig. 4.

FNDC3B interacts with FAM83H protein at its N-terminal region. A Strategy for screening candidate target proteins bind to FNDC3B using LC–MS. B In total, 13 proteins interacting with FNDC3B were screened by LC–MS, and their corresponding binding information was listed. C Protein–protein molecular docking model of FNDC3B and FAM83H. D Co-IP validation of the interaction between FNDC3B and FAM83H in AGS and MKN-74 cells. E Immunofluorescence detection of colocalization of FNDC3B and FAM83H in AGS and MKN-74 cells (scale bar: 10 μm). F Co-IP detection of the binding region between FAM83H and FNDC3B

To delineate the specific region of FNDC3B that interacts with FAM83H, we transfected HEK-293 T cells with truncated FNDC3B mutants. As depicted in Fig. 4F, the interaction between FNDC3B and FAM83H was abrogated upon deletion of the N-terminal region of FNDC3B. This observation was reinforced by protein–protein molecular docking results, revealing 13 interaction sites between FNDC3B and FAM83H, with 10 located within the N-terminal region of FNDC3B (Supplementary Material Fig. S4 c). In conclusion, our findings indicate that FAM83H primarily binds to the N-terminal region of FNDC3B.

FAM83H is upregulated and promotes GC metastasis

FAM83H, a member of the FAM83 protein family, is recognized for its regulatory function in diverse tumor types through modulation of cellular signaling, proliferation, and migration processes [19, 37–39]. Analysis from GEPIA 2.0 uncovered that FAM83H is significantly overexpressed in six gastrointestinal cancers, including GC (Supplementary Material Fig. S5 a), and its upregulation is closely associated with poor prognosis in patients with GC (Fig. 5A, B). The elevated expression of FAM83H was subsequently validated in both GC cell lines and clinical specimens (Fig. 5C, D). To explore the functional implications of FAM83H in GC, we evaluated the efficacy of both targeted knockdown and overexpression of FAM83H in AGS cells (Fig. 5E). Subsequent cell migration assays showed that the silencing of FAM83H significantly inhibited GC cell migration, whereas its overexpression promoted migration (Fig. 5F, G). Intriguingly, although FAM83H knockdown reduced the proliferative capacity and colony formation abilities of AGS cells, its overexpression did not significantly augment proliferation (Supplementary Material Fig. S5 b and c). This observation indicated that FAM83H’s role in regulating GC cell proliferation was less pronounced compared with its influence on migration. Taken together, these results suggest that FAM83H primarily exerts its effects by promoting GC cell migration.

Fig. 5.

FAM83H is upregulated and promotes GC metastasis. A, B Analysis of FAM83H expression in GC using GEPIA 2.0: (A) FAM83H expression, and (B) overall survival. C Western blot analysis of FAM83H expression in clinical GC tissue samples (n = 8). D Protein expression levels of FAM83H in GES-1 and GC cell lines. E The efficiency of FAM83H knockdown or overexpression on FAM83H protein levels in AGS cells. F Transwell assay to detect the migration ability of AGS cells with FAM83H knockdown or overexpression (scale bar: 200 μm). G Wound healing assay to detect the migration ability of AGS cells with FAM83H knockdown or overexpression (scale bar: 200 μm). P-values were calculated using Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001

FNDC3B modulates the stability of FAM83H via the ubiquitin–proteasome pathway

To interrogate the mechanism underlying FNDC3B’s regulation of FAM83H, we evaluated the effect of FNDC3B on FAM83H mRNA and protein levels. As shown in Fig. 6A and B, FNDC3B upregulated the expression of FAM83H protein without altering its mRNA levels. Analysis from the clinical specimens, we observed a positive correlation between FNDC3B and FAM83H (Supplementary Material Fig. S6 a).

Fig. 6.

FNDC3B modulates the stability of FAM83H via the ubiquitin–proteasome pathway. A, B Detection of FAM83H protein (left) and mRNA(right) levels after FNDC3B knockdown in MKN-74 and AGS cells. C Single-sample gene set enrichment analysis (ssGSEA) of the correlation between FNDC3B and the proteasome regulatory pathway. D Measurement of FAM83H accumulation rate after adding proteasome inhibitor MG-132 in AGS cells with FNDC3B overexpression (top) or knockdown (bottom). E Measurement of FAM83H degradation half-life after adding protein synthesis inhibitor CHX in AGS with FNDC3B overexpression (top) or knockdown (bottom). F Ubiquitination Co-IP assay using anti-FAM83H antibody to detect the ubiquitination level of FAM83H after FNDC3B overexpression (left) or knockdown (right) in AGS cells. P-values were calculated using Student’s t-test, ns means no significance, *P < 0.05, and ****P < 0.0001

Protein degradation primarily occurs through the autophagy–lysosome pathway or the ubiquitin–proteasome system [40–42]. However, as indicated in Supplementary Material Fig. S7 a and b, FNDC3B did not directly influence the autophagy marker LC3II/I, suggesting that FNDC3B might not be involved in autophagy regulation. Single-sample gene set enrichment analysis (ssGSEA) from the NCBI database implicated that FNDC3B may participate in protein regulation through the ubiquitin–proteasome pathway (Fig. 6C). To prove the above hypothesis, we treated AGS cells with the proteasome inhibitor MG-132, and found that the accumulation rate of FAM83H protein decreased with FNDC3B silencing, whereas it increased upon FNDC3B overexpression (Fig. 6D). Additionally, the influence of FNDC3B on the half-life of FAM83H in CHX-treated GC cells was characterized. The results indicated that the knockdown of FNDC3B shortened the half-life of the FAM83H protein, whereas the overexpression of FNDC3B prolonged it (Fig. 6E). Likewise, Co-IP analysis demonstrated that FNDC3B knockdown enhanced the ubiquitination level of FAM83H, while its overexpression lowered it (Fig. 6F). In summary, these findings uncover that FNDC3B positively regulates the stability of FAM83H protein in GC by inhibiting its degradation through the ubiquitin–proteasome pathway.

FAM83H is involved in FNDC3B-mediated promotion of GC metastasis

In the context of GC, EMT is recognized as a critical mechanism driving tumor progression and metastasis [43–45]. An analysis of the GEPIA 2.0 database revealed a positive correlation between FNDC3B and the EMT pathway (Fig. 7A). EMT involves a transformation of epithelial cells, wherein they lose their intercellular adhesion properties and adopt an invasive phenotype. This transition is intricately orchestrated by a complex interplay of signaling pathways and transcription factors, including Snail, Slug, and ZEB-1 [46, 47]. Notably, our findings demonstrated that FNDC3B upregulated the expression of EMT-associated transcription factors ZEB-1, Snail, and Slug (Fig. 7B, C and Supplementary Material Fig. S8 a), without significantly altering the expression of tight junction proteins such as Claudin-1 and ZO-1, or cytoskeletal proteins including vimentin and β-catenin (Supplementary Material Fig. S8 b). However, the low expression levels of E-cadherin and N-cadherin in GC cells posed challenges in elucidating the regulatory relationship between FNDC3B and these proteins.

Fig. 7.

FAM83H is involved in FNDC3B-mediated promotion of GC metastasis. A GEPIA 2.0 database analysis showing the positive regulatory relationship between FNDC3B and the EMT pathway. B–C Detection of transcription factors (Zeb-1, Snail, and Slug) in the EMT pathway in, (B) HGC-27 cells with FNDC3B knockdown and (C) AGS cells with FNDC3B overexpression. D Detection of Snail and Slug protein expression levels in AGS cells with FAM83H knockdown or overexpression. E GEPIA 2.0 database analysis showing the relationship between Slug (left) and Snail (right) with overall survival in patients with GC. F Detection of Snail protein expression changes in AGS cells with FNDC3B knockdown followed by FAM83H overexpression. G Transwell assay showing the migration ability of AGS cells with FNDC3B knockdown followed by FAM83H overexpression (scale bar: 200 μm). H Wound healing assay showing the migration ability of AGS cells with FNDC3B knockdown followed by FAM83H overexpression. (scale bar: 200 μm). I Representative images of liver tissues and H&E staining results (scale bar: 500 μm). P-values were calculated using Student’s t-test and one way ANOVA, ns meas no significance, *P < 0.05, **P < 0.01 and ***P < 0.001

Previous studies have underscored the role of FAM83H in modulating the EMT process in GC [19]. As shown in Fig. 7D, we consistently confirmed that FAM83H significantly upregulated the protein expression of Snail and Slug. Moreover, bioinformatics analysis revealed that high Snail expression correlated with poorer prognosis in patients with GC, whereas Slug expression did not show a significant prognostic association (Fig. 7E). These results suggest that both FNDC3B and FAM83H could positively regulate Snail protein levels. Intriguingly, it was found that overexpression of FAM83H rescued the Snail protein downregulation caused by silencing FNDC3B (Fig. 7F). Additionally, migration assays showed that FAM83H overexpression could partially counteract the inhibitory effect of FNDC3B knockdown on GC cell migration (Fig. 7G, H). Consistent with in vitro results, overexpression of FNDC3B led to an increase in liver metastases in vivo compared with the control group. However, when FNDC3B was overexpressed concurrently with the knockdown of FAM83H, the migration capability of GC cells was comparable to that of the control group (Fig. 7I). Hence, above results indicate that FNDC3B promotes the expression of Snail and GC cell migration in vitro and in vivo in a FAM83H-dependent manner.

Discussion

FNDC3B, initially designated as a factor for adipocyte differentiation 104 (FAD104), plays a crucial role as a positive regulator of adipogenesis, with expression levels rapidly increasing during the early stages of adipogenesis [48, 49]. In recent years, FNDC3B has garnered significant attention owing to its diverse functions in various malignancies [50–53]. Specifically, FNDC3BFNDC3B can transcribe into multiple circular RNAs (circRNAs) that regulate tumor development [54–56]. Additionally, FNDC3B has been widely reported as an oncogene in over ten cancer types, including cervical carcinoma, nasopharyngeal carcinoma, and tongue squamous cell carcinoma [50, 57–61]. However, findings regarding FNDC3B in digestive system tumors, particularly hepatocellular carcinoma (HCC), are contradictory. Some studies suggest that FNDC3B, regulated by transcription factors E2F1 and EYA1, or interacting with ANXA2, enhances metastatic ability in HCC [10, 11, 16]. Conversely, others propose that FNDC3B inhibits liver cancer metastasis by impeding lipogenesis and cholesterogenesis or through the NF-kappaβ/miR-143/FNDC3B axis [17, 18]. Bioinformatics analysis indicates that FNDC3B is significantly upregulated in most digestive system tumors, including GC. Importantly, our study is the first to comprehensively demonstrate the high expression pattern of FNDC3B in GC and its pivotal role in promoting GC metastasis via the FNDC3B/FAM83H/Snail/EMT axis.

Structurally, FNDC3B is distinguished by nine FNIII domains, a TM domain located at its C-terminus, and a proline-rich N-terminal region where proline constitutes 13.36%, notably exceeding the average amino acid occurrence rate of 5%. These domains establish a robust structural backbone, enabling effective interactions with downstream target proteins. Existing studies have shown that FNDC3B can bind to STAT3, ANXA2, and FAM46C in melanoma, liver cancer, and multiple myeloma, respectively, thereby influencing tumor progression [10, 62–64]. In our findings, FAM83H was identified as a novel binding partner of FNDC3B using Immunoprecipitation-mass spectrometry (IP-MS) technology. Elevated levels of FAM83H have been linked to poorer prognoses in digestive system tumors, specifically stomach, colon, pancreatic, and liver cancers [19, 22, 38, 65]. It is worth noting that FAM83H displays the highest mutation rate among the FAM83 family genes in GC, and its high expression correlates with lymph node metastasis and immune cell infiltration [65]. In line with this, we have verified that FAM83H is overexpressed in GC cells and specimens, significantly enhancing GC metastasis. In contrast to the experimental approach employing MKN-45 and NCI-N87 cell lines in the Hussein et al. study [19], our systematic investigation revealed that FAM83H overexpression in AGS GC cells failed to demonstrate statistically significant proliferative enhancement (P > 0.05). This fundamental divergence likely originates from inherent biological heterogeneity across distinct gastric carcinoma cellular models. Furthermore, our findings revealed that FAM83H binds to the N-terminal region of FNDC3B, which is reminiscent of the interaction observed between STAT3 and the N-terminal region of FNDC3B [62]. The proline-rich N-terminal region of FNDC3B exhibits structural rigidity due to the cyclization of its side chains with the amino backbone, reducing flexibility [66, 67]. This rigidity is instrumental in maintaining a stable three-dimensional structure, facilitating target protein binding. Regarding the structure of FAM83H, the DUF1669 [68–70] domain is crucial for comprehending the biological functions of the FAM83 protein family. All FAM83 family members interact with casein kinase 1 (CK1), a family of serine/threonine-specific protein kinases [68, 71]. Beyond the DUF1669 region, these proteins lack sequence similarity, and their C-termini consist of nonspherical, unstructured sequences [69]. To delve deeper into the interaction dynamics, we aim to pinpoint the precise region where FNDC3B interfaces with FAM83H in the future.

Functionally, our study uncovered the critical role of FNDC3B in promoting GC metastasis, highlighting the importance of its N-terminal region and C-terminal TM domain in preserving its metastatic capabilities. The removal of the N-terminal region prevented FNDC3B from interacting with FAM83H, thus eliminating its capacity to promote GC metastasis. Conversely, the deletion of the TM domain shifted the subcellular localization of FNDC3B from the ER in the cytoplasm to the nucleus, leading to a reversal of its metastasis-promoting function. In our previous published research, we identified circFNDC3B (circBase ID: hsa_circ_0003692), which is transcribed from the parental gene FNDC3B, can encode a novel protein FNDC3B-267-aa [72]. This protein, lacking the TM domain, was primarily nuclear-localized and demonstrated an inhibitory effect on GC cell migration. Collectively, these findings consistently underscore the critical importance of the TM domain in maintaining FNDC3B’s subcellular localization and its role of promoting metastasis.

Mechanistically, FNDC3B inhibited FAM83H protein degradation via the ubiquitin–proteasome pathway and regulated metastasis of GC cells through the FNDC3B/FAM83H/Snail/EMT axis. However, several unresolved issues remain regarding the regulatory process within the ubiquitin–proteasome pathway in our study. Key enzymes involved in this pathway include E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin ligase), responsible for ubiquitin activation, ubiquitin transfer, and ubiquitin attachment to specific substrate proteins, respectively [40, 73, 74]. It is interesting that ubiquitin-like modifier-activating enzyme 1 (UBA1), the most studied and widely recognized ubiquitin-activating enzyme initiating the ubiquitination cascade, was identified in our IP-MS analysis [75–77]. Additionally, PPIs analysis from the STRING (https://string-db.org/network/9606.ENSP00000338523) suggested that ligand of numb-protein X 1 (LNX1), functioning as an E3 ubiquitin-protein ligase, may interact with FNDC3B. In future studies, we aim to identify the crucial enzymes involved in the ubiquitin–proteasome pathway that interact with FNDC3B to further clarify this mechanism.

Conclusions

In summary, our study has identified FNDC3B as a novel oncogene that is significantly upregulated in GC and promotes GC cell metastasis both in vitro and in vivo. Specifically, FNDC3B binds to FAM83H at its N-terminus, and this interaction inhibits the ubiquitin–proteasome-mediated degradation of FAM83H, thereby facilitating the EMT process and facilitating GC metastasis (Fig. 8). The elucidation of the expression profiles and specific regulatory mechanisms of FNDC3B in GC offers new insights into the diagnosis and treatment of this malignancy.

Fig. 8.

Schematic diagram of FNDC3B’s role in promoting gastric cancer metastasis via interacting with FAM83H and preventing its proteasomal degradation (Fig. 8 was drawn by Figdraw)

Abbreviations

CHX

Cycloheximide

Co-IP

Coimmunoprecipitation

EMT

Epithelial–mesenchymal transition

FNDC1

Fibronectin type III domain-containing 1

FNDC3B

Fibronectin type III domain-containing 3B

FAM83H

Family with sequence similarity 83 member H

GC

Gastric cancer

IF

Immunofluorescence

IHC

Immunohistochemistry

LC–MS

Immunoprecipitation–mass spectrometry

ssGSEA

Single-sample gene set enrichment analysis

TM

Transmembrane

Author contributions

Yuying Zhang: conceptualization, investigation, methodology, data curation, software, writing—original draft, and funding acquisition. Lingyu Ran: conceptualization, investigation, and methodology. Yuying Liu: investigation and validation. Wei Li: validation. Ai Ran: validation. Haiping Li: validation. Bo Huang: investigation. Junwu Ren: investigation. Hao Ning: investigation. Qiang Ma: investigation. Xiaolin Wang: formal analysis. Feifei Yang: validation. Xiaojuan Pan: validation. Ce Liang: validation. Shimin Wang: validation. Changhong Qin: validation. Yan Jiang: validation. Kun Qian: conceptualization and project administration. Bin Xiao: conceptualization, writing—review and editing, project administration, and funding acquisition.

Funding

This study was supported by the National Natural Science Foundation of China (no. 82073254), Chongqing Talents-Exceptional Young Talents Project (nos. CQYC202005044 and cstc2021ycjh-bgzxm0094), Innovation and Development Joint Foundation of Chongqing Natural Science Foundation (no. CSTB2022NSCQ-LZX0043), Science and technology research project of Chongqing Municipal Education Commission (no. KJZD-K202100405), Future Medical Youth Innovation Team Project of Chongqing Medical University (no. W0042), Graduate Tutor Team Construction Project of Chongqing (no. CQMUDSTD202210), and Top Graduate Talent Cultivation Program of Chongqing Medical University (no. BJRC202230).

Availability of data and materials

The datasets used and analyzed in this paper are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

The project was conducted in compliance with the guidelines outlined in the Declaration of Helsinki and the Basel Declaration. Studies involving human tissues were approved by the Ethics Committee of Southwest Hospital, Army Medical University, Chongqing (approval number: KY2020089, dated 8 June 2020). The study was performed in accordance with the principles of the Declaration of Helsinki. Written informed consent was obtained from all participants for the use of their clinical information. The animal experiments were performed in accordance with the Basel Declaration and were approved by the Institutional Animal Care and Use Committee, Chongqing Medical University (approval number: 2022012, 20 May 2022). The ethics committee follows the guidelines of the International Council for Laboratory Animal Science (ICLAS) to ensure the ethical compliance of the experiments.

Consent for publication

Informed consent was obtained from all subjects involved in this study.

Competing interests

The authors declare no competing interests.