Exercise inhibits proliferation and induces apoptosis of lung adenocarcinoma cells via the POU2F2-SPOCD1-PI3K/AKT signaling axis

Abstract

Background

Lung adenocarcinoma (LUAD) is a prevalent and deadly form of lung cancer. Exercise has been shown to inhibit LUAD progression, yet the underlying molecular mechanisms remain unclear. The transcription factor POU2F2 has been implicated in LUAD tumorigenesis, but its precise role and regulatory targets have not been fully elucidated.

Methods

POU2F2 expression in LUAD cell lines (HCC4006, Calu-3, NCI-H2009) and normal lung epithelial cells (BEAS-2B) was assessed by qRT-PCR and Western blot. Functional assays (CCK-8, EdU, flow cytometry, TUNEL) were performed following shRNA-mediated knockdown of POU2F2 or SPOCD1 and upregulation of POU2F2. Mechanistic studies included bioinformatics, dual-luciferase assay, ChIP, DNA pull-down, and Western blot. In vivo tumor growth was evaluated via xenograft models with Ki67 IHC.

Results

Exercise suppressed POU2F2 expression, which was otherwise elevated in LUAD cells. Knockdown of POU2F2 or its downstream target SPOCD1 reduced proliferation and increased apoptosis. Mechanistically, POU2F2 directly bound to and activated the SPOCD1 promoter, thereby enhancing PI3K/AKT pathway signaling. Rescue experiments confirmed that SPOCD1 mediates POU2F2’s oncogenic effects. In vivo, POU2F2 knockdown inhibited tumor growth and Ki67 expression.

Conclusions

Exercise suppresses LUAD progression by downregulating POU2F2, thereby disrupting the POU2F2–SPOCD1–PI3K/AKT axis. This pathway plays a critical role in LUAD cell survival and may serve as a promising therapeutic target.

Key points

Exercise inhibits LUAD progression by downregulating the oncogenic transcription factor POU2F2. The POU2F2-SPOCD1 axis exerts its oncogenic function by activating the PI3K/AKT pathway. The POU2F2-SPOCD1 axis may represent a promising therapeutic target for LUAD.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s12094-026-04273-0.

Introduction

Lung cancer remains the leading cause of cancer-related mortality worldwide [1], with lung adenocarcinoma (LUAD) constituting the most prevalent histological subtype [2]. Despite significant advances in diagnosis and treatment, the 5-year survival rate for LUAD remains unsatisfactory [3], primarily due to tumor metastasis [4], intratumoral heterogeneity [5], and therapy resistance [6]. A deeper understanding of the molecular mechanisms driving LUAD progression is urgently needed to identify novel therapeutic targets. Accumulating evidence has demonstrated that physical exercise confers anti-cancer benefits and improves outcomes in lung cancer treatment and recovery [7–10]. However, the precise anti-tumor mechanisms of exercise in LUAD remain incompletely elucidated.

Transcription factors (TFs) play a central role in regulating gene expression programs that govern cancer cell proliferation [11], apoptosis [12], and metastasis [13]. Among them, the POU domain class 2 transcription factor 2 (POU2F2, also known as OCT2) is a B-cell-restricted member of the POU domain family [14, 15]. It is primarily expressed in B lymphocytes and B-cell-derived tumors, where it modulates immunoglobulin synthesis, cell proliferation, and the expression of differentiation-related genes [16–19]. Emerging evidence suggests that POU2F2 is aberrantly expressed in lung cancer and may promote oncogenesis through the transcriptional activation of downstream targets [20]. Nevertheless, its specific function, underlying mechanisms, and potential regulation by exercise in LUAD remain poorly characterized.

Our preliminary data showed that POU2F2 expression was downregulated in lung tissues from exercise-trained mice and upregulated in LUAD cells. Functional assays demonstrated that POU2F2 promoted LUAD cell proliferation, suppressed apoptosis in vitro, and facilitated tumor growth in vivo. To elucidate its downstream mechanisms, we identified SPOCD1 as a key effector. Here, we demonstrate that POU2F2 transcriptionally activates SPOCD1, which in turn enhances PI3K/AKT pathway activation. Furthermore, we comprehensively investigate the role of this exercise-mediated POU2F2–SPOCD1–PI3K/AKT signaling axis in LUAD tumorigenesis. Our findings provide novel mechanistic insights into LUAD pathophysiology and suggest that targeting this axis may offer therapeutic benefits.

Materials and methods

Human tissue samples and immunohistochemistry

Human lung adenocarcinoma (LUAD) specimens and paired adjacent non-tumorous tissues were obtained from Nanjing First Hospital. The study protocol was approved by the hospital’s Ethics Committee, and written informed consent was obtained from all patients. All procedures were conducted in accordance with the ethical principles of the Declaration of Helsinki.

For immunohistochemical (IHC) staining, tissue specimens were fixed, embedded in paraffin, and sectioned into 5-µm slices. After deparaffinization and rehydration, antigen retrieval was performed by microwave heating in sodium citrate buffer (pH 6.0) using the following cycle: medium–high power for 10 min, standing for 5 min, medium–low power for 5 min, standing for 2 min, and finally medium–low power for 5 min. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 15 min. Sections were then blocked with 2% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) in PBS for 30 min at room temperature.

Subsequently, sections were incubated overnight at 4°C with a rabbit monoclonal anti-Ki67 primary antibody (Servicebio, Wuhan, China; catalog no. GB111499) at a dilution of 1:600. After washing, sections were incubated for 1 h at room temperature with an HRP-conjugated goat anti-rabbit IgG secondary antibody (Servicebio, catalog no. GB23303) at a dilution of 1:500. Diaminobenzidine (DAB) was used as the chromogen for signal development, followed by counterstaining with hematoxylin. Finally, sections were dehydrated, cleared, and mounted. Stained sections were examined and imaged under a light microscope (Nikon Eclipse Ci-L) at 200 × magnification.

Cell culture

The human lung adenocarcinoma (LUAD) cell lines HCC4006, Calu-3, and NCI-H2009, the immortalized normal human bronchial epithelial cell line BEAS-2B, and the human embryonic kidney 293T cells were all purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

HCC4006 cells were maintained in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco). Calu-3 cells were cultured in Eagle’s Minimum Essential Medium (EMEM; ATCC) supplemented with 10% FBS. BEAS-2B cells were grown in Bronchial Epithelial Cell Growth Medium (BEGM; Lonza, Basel, Switzerland) supplemented with the BEGM SingleQuots growth factor kit (Lonza) according to the manufacturer’s protocol. NCI-H2009 cells were cultured in a 1:1 mixture of Dulbecco’s Modified Eagle Medium and Ham’s F12 nutrient mixture (DMEM/F12; Gibco) supplemented with 5% FBS, 2 mM L-glutamine, 0.005 mg/mL insulin, 0.01 mg/mL transferrin, 30 nM sodium selenite, 10 nM hydrocortisone, and 10 nM β-estradiol. 293T cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% FBS.

All culture media contained 1% penicillin–streptomycin (Gibco) unless otherwise specified. All cell lines were cultured at 37°C in a humidified incubator with 5% CO₂.

Cell transfection

LUAD cells were used to generate stable knockdown lines via lentiviral transduction of shRNAs targeting POU2F2 (sh-POU2F2-1/2/3) or SPOCD1 (sh-SPOCD1-1/2/3) (GenePharma, Shanghai, China). A non-targeting shRNA (sh-NC) was included as the negative control. Cells were infected with the corresponding lentiviral particles in the presence of polybrene (when applicable to enhance transduction efficiency). After infection, cells were subjected to puromycin selection to obtain stable knockdown cell populations. Knockdown efficiency was verified by qRT–PCR and Western blotting before subsequent functional assays.

For overexpression experiments, LUAD cells were transfected with pcDNA3.1-SPOCD1 or the empty pcDNA3.1 vector (Obio Technology, Shanghai, China) using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

Western blot

Protein lysates from LUAD cells were extracted using RIPA lysis buffer (Solarbio, Beijing, China), and protein concentrations were determined using a BCA kit (Solarbio). Equal amounts of protein were separated by SDS-PAGE and subsequently transferred onto PVDF membranes (Millipore, Billerica, MA, USA). To minimize nonspecific binding, the membranes were blocked with 5% skim milk at 4°C for 2 h. Following blocking, the membranes were probed with primary antibodies overnight at 4°C, then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. β-Actin served as the internal control. Protein bands were visualized using an enhanced chemiluminescence (ECL) substrate (Thermo Fisher Scientific, Waltham, MA, USA).

Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR)

Total RNA was isolated from LUAD cells and tissues using TRIzol reagent (Thermo Fisher Scientific), followed by cDNA synthesis with the PrimeScript RT reagent kit (Takara, Tokyo, Japan). qRT-PCR was performed using SYBR Premix Ex Taq II (Takara) on a real-time PCR system. β-actin served as the endogenous control for normalization, and relative gene expression levels were calculated using the 2^−ΔΔCt method.

Cell counting Kit-8 (CCK-8) assay

LUAD cells were plated in a 96-well culture plate at an initial density of 2 × 10³ cells per well, and were cultured for 24, 48, and 72 h. At each predetermined time point, 10 µL of CCK-8 solution (Dojindo, Kumamoto, Japan) was added to each well, followed by an additional 2-h incubation. The absorbance of the samples was subsequently measured at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).

5-ethynyl-2’-deoxyuridine (EdU) assay

The proliferative capacity of LUAD cells was evaluated through EdU assay employing the Click-iT EdU Imaging Kit (Invitrogen). Briefly, cells were incubated with EdU solution for 2 h. After incubation, cells were washed twice with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. Then cells were permeabilized with 0.5% Triton X-100 for 20 min. For nuclear counterstaining, cells were treated with 4’,6-diamidino-2-phenylindole (DAPI; Beyotime, Shanghai, China) for 5 min at room temperature, protected from light. Fluorescent images were captured using an inverted fluorescence microscope (Olympus IX-71, Tokyo, Japan).

Flow cytometry

For apoptosis detection, LUAD cells were harvested, washed with ice-cold PBS, and resuspended in 200 µL of binding buffer at a density of 1 × 10⁶ cells/mL. Subsequently, the cells were dual-stained with 5 µL of Annexin V-Alexa Fluor488 and 10 µL of propidium iodide (PI) for 15 min at room temperature in the dark. Flow cytometric analysis was performed after staining, and the acquired data were processed using FlowJo software (Ashland, OR, USA).

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay

Apoptosis in LUAD cells was assessed using the TUNEL Assay Kit (Beyotime). Following fixation with 4% paraformaldehyde for 30 min and permeabilization with 0.3% Triton X-100 for 5 min, cells were incubated with 50 μl TUNEL reaction mixture at 37°C for 60 min. Nuclei were counterstained with DAPI for 10 min before imaging. Fluorescence images were acquired from five random fields using a Nikon microscope.

Dual-luciferase reporter assay

The wild-type or mutant SPOCD1 promoter sequences were cloned into PGL3 vectors (GenePharma). 293T cells were co-transfected with pcDNA3.1 or pcDNA3.1-POU2F2 along with wild-type or mutant luciferase constructs using Lipofectamine 3000. Luciferase activity was quantified 48 h post-transfection using the Dual-Luciferase Reporter System (Promega).

Chromatin immunoprecipitation (ChIP)

ChIP analysis was conducted using the EZ-ChIP kit (Millipore) according to the manufacturer’s protocol. Briefly, LUAD cells were formaldehyde-crosslinked and chromatin was fragmented by sonication to 200–500 bp segments. Immunoprecipitation was performed using either anti-POU2F2 or control IgG antibodies, followed by incubation with protein A agarose beads. Precipitated DNA was purified and analyzed by qPCR.

DNA pull-down assay

Biotinylated DNA probes, including wild-type (Bio-SPOCD1 promoter WT) and mutant (Bio-SPOCD1 promoter MUT) promoter fragments, along with a negative control (Bio-NC), were incubated with cell lysates to allow DNA–protein binding. The input lysate served as the positive control. After capturing the DNA–protein complexes with streptavidin-coated magnetic beads, bound proteins were eluted and analyzed by Western blot.

Construction of animal models

A total of six SPF-grade male BALB/c nude mice (4–6 weeks old, approximately 20 g) were obtained from Ziyuan Laboratory Animal Technology Co., Ltd. (Hangzhou, China) and randomly divided into two groups (n = 3 per group): an exercise group and a non-exercise control group. Exercise training was performed according to previously described methods [8]. Briefly, mice underwent a 5-day acclimation period (1 m/min, 0° incline) followed by a formal training protocol (17 m/min, 0° incline, 60 min/day, 6 days/week with 1 rest day) for 30 days. Equipment parameters were calibrated every 15 days, and a 10-min warm-up (1 m/min) preceded each training session. Subsequently, 4 × 10⁶ HCC4006 cells were subcutaneously injected into the right axillary region of each mouse (Day 0). The same treadmill regimen was continuously maintained for an additional 30 days after tumor inoculation in the exercise group, while the control group remained sedentary. Mice were euthanized on Day 30 post-inoculation, and tumors were harvested for RNA-seq and qRT-PCR.

To establish LUAD xenograft models, an additional twelve mice were randomly allocated into four groups receiving equal numbers (4 × 10⁶ cells) of the following LUAD cells: HCC4006 cells transfected with sh-NC, HCC4006 cells transfected with sh-POU2F2-1, Calu-3 cells transfected with sh-NC, and Calu-3 cells transfected with sh-POU2F2-1. Tumors were successfully established one week later, and thereafter, tumor volume was measured every three days. After four weeks of injection, all tumors were excised, tumor growth curves were generated, and tumor weights were recorded.

Statistical analysis

All statistical analyses were performed using GraphPad Prism 9.0. Data are presented as mean ± standard deviation (SD). For two-group comparisons, Student’s t-test was applied, whereas one-way ANOVA was used for multi-group analyses. A p value of < 0.05 was considered statistically significant.

Results

Exercise downregulates POU2F2 expression, and POU2F2 knockdown suppresses proliferation and induces apoptosis in LUAD cells

To explore the effects of exercise on LUAD, we established an in vivo exercise mouse model. RNA sequencing (RNA-seq) analysis of lung tissues from exercised and sedentary mice revealed differential gene expression profiles. Among the downregulated genes, POU2F2, a member of the POU transcription factor family, was identified (Fig. S1). This downregulation was validated by qRT-PCR in independent samples (Fig. 1a).

Fig. 1.

POU2F2 is downregulated by exercise and promotes proliferation and survival in LUAD cells. a POU2F2 expression is downregulated in lung tissues from exercise-trained mice. qRT-PCR analysis of POU2F2 mRNA levels in lung tissues from sedentary and exercise-trained mice (n = 6 per group). b POU2F2 is overexpressed in LUAD cell lines. qRT-PCR and Western blot analysis of POU2F2 expression in the normal lung epithelial cell line BEAS-2B and three LUAD cell lines. c Elevated POU2F2 protein expression in human LUAD tissues. Representative IHC images of POU2F2 in paired tumor (T) and adjacent normal (N) tissues from three patients. Scale bar, 50 μm. d POU2F2 knockdown inhibits cell proliferation. CCK-8 assay of HCC4006 and Calu-3 cells expressing sh-NC or sh-POU2F2-1/2. e EdU incorporation assay confirms reduced proliferation upon POU2F2 knockdown. Representative images and quantification are shown. Scale bar, 50 μm. f,POU2F2 knockdown induces apoptosis. TUNEL staining of HCC4006 and Calu-3 cells (green: TUNEL signal; blue: DAPI). Scale bar, 50 μm. g Flow cytometric analysis of apoptosis by Annexin V/PI staining. The quantitation of apoptotic cells is shown

We next examined POU2F2 expression in human LUAD contexts. Compared to the normal lung epithelial cell line BEAS-2B, POU2F2 mRNA and protein levels were significantly elevated in multiple LUAD cell lines, with the highest expression observed in HCC4006 and Calu-3 cells (Fig. 1b). Immunohistochemical analysis of patient tissues confirmed that POU2F2 protein expression was markedly stronger in LUAD tumor tissues than in matched adjacent non-tumor tissues (Fig. 1c).

To investigate the functional role of POU2F2, we performed loss-of-function studies in HCC4006 and Calu-3 cells. Stable knockdown of POU2F2 was achieved using shRNAs, with sh-POU2F2-1 and sh-POU2F2-2 demonstrating high knockdown efficiency (Fig. S2a, b). CCK-8 and EdU incorporation assays revealed that POU2F2 knockdown significantly impaired the proliferative capacity of both cell lines (Fig. 1d, e). Furthermore, TUNEL assay and flow cytometry analysis of Annexin V/PI staining showed that POU2F2 depletion markedly increased the rate of apoptosis (Fig. 1f, g).

POU2F2 overexpression enhances proliferation and inhibits apoptosis in LUAD cells

To corroborate the findings from loss-of-function studies, we performed gain-of-function experiments. POU2F2 was overexpressed in HCC4006 and Calu-3 cells via transfection with a POU2F2 expression plasmid. Successful overexpression was verified at both mRNA and protein levels (Fig. S2c, d).

Functional assays revealed that POU2F2 overexpression significantly enhanced the proliferative capacity of both HCC4006 and Calu-3 cells, as measured by CCK-8 (Fig. 2a) and EdU incorporation assays (Fig. 2b). Conversely, POU2F2 overexpression exerted an anti-apoptotic effect. TUNEL staining (Fig. 2c) and flow cytometric analysis of Annexin V/PI staining (Fig. 2d) both showed a significant decrease in apoptotic cells upon POU2F2 overexpression compared to the vector control groups.

Fig. 2.

POU2F2 expression promotes the proliferation and anti-apoptotic capacities of LUAD cells a-b.CCK-8 (a) and EdU (b) assays were performed to evaluate the proliferation capability of LUAD cells (HCC4006 and Calu-3) transfected with plv-NC or plv-POU2F2-1/2. c-d.TUNEL assay (c) and flow cytometry (d) were used to assess the apoptosis capability of LUAD cells (HCC4006 and Calu-3) transfected with plv-NC or plv-POU2F2-1/2

Taken together, these complementary gain-of-function data reinforce the conclusion that POU2F2 functions as an oncogene in LUAD by promoting cell proliferation and conferring resistance to apoptosis.

POU2F2 regulates the expression of SPOCD1 in lung cancer cells

To identify downstream effectors of POU2F2, we utilized the hTFtarget database to predict its transcriptional targets. The top three candidate genes (PEX14, BAI2, and SPOCD1) were selected for experimental validation (Fig. 3a). qRT-PCR analysis revealed that knockdown of POU2F2 in HCC4006 and Calu-3 cells led to the most pronounced decrease in SPOCD1 mRNA levels among the three candidates (Fig. 3b), implicating SPOCD1 as a potential direct target.

Fig. 3.

POU2F2 transcriptionally activates SPOCD1 expression in LUAD. a Prediction of POU2F2 downstream targets using the hTFtarget database, identifying three candidate genes (PEX14, BAI2, SPOCD1). b qRT-PCR validation of candidate gene expression in HCC4006 and Calu-3 cells transfected with sh-NC or sh-POU2F2. c Putative POU2F2 binding motif within the SPOCD1 promoter predicted by the JASPAR database. The core sequence and position relative to the transcription start site are shown. d Dual-luciferase reporter assay comparing wild-type (WT) and mutant (Mut) SPOCD1 promoter activity in response to POU2F2 overexpression in 293T cells. e ChIP-qPCR analysis of POU2F2 binding to the WT SPOCD1 promoter in LUAD cells. IgG served as a negative control. f DNA pull-down assay demonstrating direct binding of recombinant POU2F2 protein to the biotin-labeled WT, but not Mut, SPOCD1 promoter probe

Bioinformatic analysis using the JASPAR database predicted a high-affinity POU2F2 binding motif within the SPOCD1 promoter region (Fig. 3c). The top-scoring site, located at positions -249 to -261 relative to the transcription start site (core sequence: AGTATTTACATAA), was selected for further analysis.

To test whether POU2F2 transcriptionally regulates SPOCD1 via this site, we performed dual-luciferase reporter assays. In 293T cells, co-transfection of a POU2F2 expression plasmid significantly increased the luciferase activity driven by the wild-type SPOCD1 promoter, but not by a promoter containing a mutation in the predicted binding site (Fig. 3d).

We next examined the direct binding of POU2F2 to the endogenous SPOCD1 promoter in LUAD cells. Chromatin immunoprecipitation (ChIP) assays using an anti-POU2F2 antibody demonstrated specific enrichment of the SPOCD1 promoter fragment containing the predicted motif in POU2F2-immunoprecipitated chromatin (Fig. 3e). This physical interaction was further validated by DNA pull-down assays, which showed that in vitro transcribed POU2F2 protein could be pulled down by a biotinylated oligonucleotide probe corresponding to the wild-type, but not the mutant, SPOCD1 promoter sequence (Fig. 3f).

SPOCD1 mediates the oncogenic effects of POU2F2 via PI3K/AKT pathway activation

Given prior evidence that SPOCD1 activates the PI3K/AKT pathway in other cancers [22] and the established oncogenic role of this pathway in LUAD [23], we investigated whether SPOCD1 similarly regulates PI3K/AKT signaling in our context. Western blot analysis revealed that knockdown of SPOCD1 in HCC4006 and Calu-3 cells significantly decreased the phosphorylation levels of PI3K (p-PI3K) and AKT (p-AKT) without affecting total protein expression, indicating suppression of pathway activity (Fig. 4a).

Fig. 4.

SPOCD1 mediates the oncogenic effects of POU2F2 via PI3K/AKT pathway activation a After transfecting LUAD cells with sh-NC or sh-SPOCD1-1/2, Western blot was performed to detect the protein expression levels of key molecules in the PI3K/AKT pathway. b Western blot analysis of the PI3K/AKT/mTOR signaling pathway in cells treated with different conditions: SPOCD1 knockdown, SPOCD1 knockdown with AKT activator SC79, SPOCD1 knockdown with PI3K inhibitor LY294002, and respective controls. β-actin serves as a loading control. c-d, CCK-8 (c) and EdU (d) assays were performed to evaluate the proliferation capability of LUAD cells (HCC4006 and Calu-3) with different conditions. e–f,TUNEL assay (e) and flow cytometry (f) were used to assess the apoptosis capability of LUAD cells (HCC4006 and Calu-3) with different conditions

To determine whether the PI3K/AKT pathway functionally mediates the effects of SPOCD1, we performed rescue experiments using pharmacological modulators. In SPOCD1-knockdown cells, treatment with the AKT activator SC79 restored p-PI3K and p-AKT levels, while the PI3K inhibitor LY294002 further suppressed their phosphorylation (Fig. 4b). Functionally, SC79 treatment partially reversed the proliferation inhibition (CCK-8, Fig. 4c; EdU, Fig. 4d) and increased apoptosis (TUNEL, Fig. 4e; flow cytometry, Fig. 4f) caused by SPOCD1 knockdown. In contrast, LY294002 treatment synergized with SPOCD1 knockdown, leading to more pronounced suppression of proliferation and induction of apoptosis (Fig. 4c-f).

Collectively, these results demonstrate that SPOCD1 exerts its pro-tumorigenic effects in LUAD cells primarily through activation of the PI3K/AKT signaling pathway. This positions SPOCD1 as a critical downstream mediator of the transcription factor POU2F2 within this oncogenic axis.

Independent validation of the essential role of SPOCD1 in LUAD cell proliferation and survival

The aforementioned rescue experiments implicated the PI3K/AKT pathway in mediating the functional output of the POU2F2-SPOCD1 axis. To independently confirm that SPOCD1 itself is a crucial node regulating LUAD cell fate, we performed direct loss-of-function studies. SPOCD1 expression was stably knocked down in HCC4006 and Calu-3 cells using specific shRNAs (knockdown efficiency verified in Fig. S2e).

Consistent with the phenotype observed upon POU2F2 inhibition, SPOCD1 knockdown alone was sufficient to robustly suppress cell proliferation, as measured by CCK-8 (Fig. 5a) and EdU incorporation assays (Fig. 5b). Moreover, SPOCD1 depletion significantly induced apoptosis, evidenced by increased TUNEL-positive cells (Fig. 5c) and elevated Annexin V/PI staining in flow cytometry (Fig. 5d).

Fig. 5.

SPOCD1 knockdown inhibits proliferation and promotes apoptosis of LUAD cells in vitro. a-b, CCK-8 (a) and EdU (b) assays were conducted to evaluate the proliferation capability of LUAD cells transfected with sh-NC or sh-SPOCD1-1/2. c-d, TUNEL assay (c) and flow cytometry (d) were used to assess the apoptosis capability of LUAD cells transfected with sh-NC or sh-SPOCD1-1/2

These results provide direct genetic evidence that SPOCD1 is indispensable for the proliferation and survival of LUAD cells. Combined with the mechanistic data presented in Sects. "POU2F2 regulates the expression of SPOCD1 in lung cancer cells" and "SPOCD1 mediates the oncogenic effects of POU2F2 via PI3K/AKT pathway activation", they solidify the model wherein POU2F2 drives LUAD progression by transcriptionally activating SPOCD1, which in turn executes its oncogenic functions primarily through activating the PI3K/AKT signaling pathway.

POU2F2 promotes the proliferation and anti-apoptotic capacities of LUAD cells via targeting SPOCD1

To validate that POU2F2 exerts its oncogenic effects via SPOCD1, we performed rescue experiments after confirming the overexpression efficiency of SPOCD1 in HCC4006 cells (Fig. S2f). CCK-8 (Fig. 6a) and EdU (Fig. 6b) assays demonstrated that POU2F2 knockdown significantly suppressed HCC4006 cell proliferation. Notably, this anti-proliferative effect was reversed by SPOCD1 overexpression. In addition, flow cytometry (Fig. 6c) and TUNEL assay (Fig. 6d) analyses revealed that knockdown of POU2F2 enhanced apoptosis in HCC4006 cells, while SPOCD1 overexpression effectively attenuated this pro-apoptotic effect. These results collectively establish SPOCD1 as a critical downstream effector of POU2F2, through which POU2F2 mediates its regulatory effects on LUAD cell proliferation and apoptosis.

Fig. 6.

POU2F2 promotes the proliferation and anti-apoptotic capacities of LUAD cells via targeting SPOCD1. HCC4006 cells were transfected with sh-NC, sh-POU2F2-1, sh-POU2F2-1 + pcDNA3.1 or sh-POU2F2-1 + pcDNA3.1-SPOCD1, respectively. a-b, CCK-8 (a) and EdU (b) assays were performed to evaluate the proliferation of HCC4006 cells under different conditions. c-d, Flow cytometry (c) and TUNEL assay (d) were used to assess the apoptosis of HCC4006 cells under different conditions

POU2F2 knockdown inhibits tumor growth in a xenograft mouse model

To validate the oncogenic role of POU2F2 in a physiological setting, we established a subcutaneous xenograft model using nude mice inoculated with control or POU2F2-knockdown HCC4006 cells.

Tumors derived from POU2F2-deficient cells grew significantly slower and were markedly smaller at the endpoint compared to control tumors (Fig. 7a). Quantitative analysis confirmed a substantial reduction in both final tumor weight and the rate of tumor growth over time in the POU2F2 knockdown group (Fig. 7b).

Fig. 7.

POU2F2 promotes tumor growth in vivo. a-b, Tumor images (a), weights, and growth curves (b) for the control group and the POU2F2 knockdown group. c, Representative IHC images of tumors from the control group and the POU2F2 knockdown group

To mechanistically link the observed growth impairment to cell proliferation, we performed immunohistochemical analysis for Ki67, a canonical proliferation marker. Consistent with the functional growth data, tumors from the POU2F2 knockdown group exhibited a significant decrease in the percentage of Ki67-positive cells (Fig. 7c).

In summary, these in vivo experiments provide compelling evidence that POU2F2 is essential for LUAD tumor growth. The suppression of tumor progression and proliferation upon POU2F2 loss underscores its critical role as a pro-tumorigenic driver and reinforces the potential therapeutic relevance of targeting the POU2F2-SPOCD1-PI3K/AKT axis in LUAD.

Discussion

Our study proposes and supports an exercise-linked transcriptional mechanism in LUAD, in which exercise is associated with reduced activity of the oncogenic transcription factor POU2F2 and downstream suppression of SPOCD1, ultimately dampening PI3K/AKT signaling. Conceptually, these data suggest a plausible route by which a systemic physiological intervention could converge on tumor-intrinsic transcriptional programs and reshape oncogenic signaling outputs. This framework provides a testable molecular bridge between exercise and LUAD progression.

POU2F2 (OCT2) has been increasingly implicated in cancer biology, with accumulating evidence that it can exhibit pro-tumorigenic functions in a context-dependent manner [24]. Elevated POU2F2 expression has been reported in several malignancies, including gastric and liver cancers [25]. However, its functional contribution to LUAD has remained insufficiently characterized. Our data support an oncogenic role of POU2F2 in LUAD models, consistent with the broader view that transcriptional regulators can become repurposed in epithelial tumors when embedded within permissive signaling networks.

Mechanistically, we identify SPOCD1 as a key downstream effector of POU2F2. SPOCD1 has been linked to epigenetic regulation and genome stability [26] and has recently been associated with tumorigenesis across multiple cancer types [22, 27, 28]. In our system, multi-layered evidence supports a direct POU2F2 → SPOCD1 regulatory relationship, and the functional rescue by SPOCD1 overexpression argues that SPOCD1 is not merely correlated with, but contributes to, the phenotypic outputs of POU2F2 perturbation. Given the multi-target nature of transcription factors, it is also likely that SPOCD1 represents one of several biologically relevant effectors, and systematic mapping of direct POU2F2 targets will be important in future work.

A notable downstream consequence of SPOCD1 perturbation is altered PI3K/AKT pathway activity. PI3K/AKT is a central oncogenic axis controlling survival, growth, and metabolism in cancer cells [29, 30], and its inhibition commonly shifts the balance toward reduced proliferation and increased apoptosis. While reduced phosphorylation of pathway components supports involvement of PI3K/AKT, phosphorylation changes alone do not establish functional mediation. Therefore, pathway-focused rescue experiments using AKT activators and/or PI3K/AKT inhibitors are important to demonstrate causality and to define whether PI3K/AKT is required for the POU2F2–SPOCD1-driven phenotypes.

Our in vivo findings further strengthen the biological relevance of this axis. Reduced tumor growth and lower Ki-67 labeling in xenografts derived from POU2F2-silenced cells are consistent with a proliferation-restricting effect in the tumor context. These results support the translational premise that targeting POU2F2 or its downstream effectors may have therapeutic value, particularly if the pathway dependency is confirmed by functional rescue in both in vitro and in vivo settings.

How exercise regulates tumor transcriptional programs remains an important question. Exercise induces systemic changes in hormones, metabolites, inflammatory tone, and immune activity, any of which may influence tumor cell gene regulation directly or indirectly through the microenvironment. In this context, an exercise-associated decrease in POU2F2 may reflect either tumor-intrinsic transcriptional reprogramming or secondary effects mediated by altered immune–endocrine signaling. Dissecting these possibilities will require time-resolved sampling and integrated analyses that can separate tumor-cell-intrinsic signals from microenvironmental contributions.

Several limitations should be acknowledged. First, the current mechanistic evidence is largely based on LUAD cell lines and xenograft models, which cannot fully recapitulate the heterogeneity and immune contexture of human LUAD. Second, clinical validation remains essential. Although public datasets can provide supportive evidence, expression estimates may be influenced by cohort composition, tumor purity, and immune infiltration, and independent validation using human LUAD specimens (e.g., IHC/WB and associations with clinicopathologic features) would strengthen translational relevance. Third, because transcription factors often regulate multiple targets, additional downstream effectors beyond SPOCD1 may contribute to the observed phenotypes and should be explored using integrated transcriptomic and chromatin-based approaches.

In summary, our study highlights a putative exercise-regulated POU2F2–SPOCD1 axis with downstream impact on PI3K/AKT signaling and LUAD cell survival and growth. This work provides a mechanistic basis for further investigation of how physiological interventions intersect with tumor transcriptional networks, and it motivates future studies to validate clinical relevance and establish pathway dependency through functional rescue.

Conclusion

Our study demonstrated that exercise downregulates POU2F2 expression and identified POU2F2 as a crucial oncogenic transcription factor in LUAD. We established that POU2F2 knockdown inhibits tumor cell proliferation and promotes apoptosis in both in vitro and in vivo settings. Furthermore, we elucidated the molecular mechanism by which POU2F2 binds to the SPOCD1 promoter region and transcriptionally activates its expression, thereby mediating downstream PI3K/AKT signaling pathway activation and contributing to POU2F2-driven tumorigenesis. These findings revealed the pivotal roles of both exercise and the POU2F2-SPOCD1-PI3K/AKT singling axis in LUAD progression. Our results not only provide novel insights into the molecular pathogenesis of LUAD but also suggest that POU2F2 and SPOCD1 represent promising therapeutic targets for LUAD treatment.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contribution statement

W.H, Z.Y. and W.G. designed the study; Y.L, B.Z, H.T. and F.W. performed the experiments. Y.L, B.Z, H.T, F.W and J.P. analyzed the data. Y.L, W.H, Z.Y. and W.G. interpreted results of the experiments; H.T. and B.Z. prepared figures. Y.L. drafted the manuscript. W.H, Z.Y. and W.G. revised the manuscript. All authors read and approved the final version of the manuscript.

Funding

This research was supported by General Project of Philosophical and Social Sciences Research in Jiangsu Universities (Grant No. 2025SJYB0272) and Nanjing Pukou District Health and Health Talent Development Center (Grant No.PWK2022-3).

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Declarations

Ethical approval

This study has been approved by the Ethics Committee of Nanjing First Hospital, Nanjing Medical University.(NMUL 2210205).

Conflict of interest

The authors declare no conflict of interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Consent to participate

Not applicable.

Consent for publication

Not applicable.