FNDC5 enhances quercetin-induced anoikis in pancreatic adenocarcinoma cells via focal adhesion kinase-dependent mechanisms

Abstract

BACKGROUND

The role of FNDC5/irisin in regulating sensitivity to pancreatic ductal adenocarcinoma (PAAD) chemotherapy remains unclear.

AIM

To explore the role of FNDC5/irisin in regulating sensitivity to PAAD chemotherapy.

METHODS

The prognostic significance of FNDC5 was analyzed using bioinformatics. PAAD cells with FNDC5 overexpression, treated with quercetin, and control cells were compared in vitro. Cell proliferation was measured by real-time cellular analysis and colony formation assays, while apoptosis and anoikis were assessed flow cytometry. Protein expression was determined by western blotting, gene expression was determined by real-time quantitative polymerase chain reaction, and mitochondrial membrane potential was determined using the JC-1 method.

RESULTS

FNDC5 expression correlated with a good prognosis of PAAD. Overexpression of FNDC5 significantly enhanced the tumoricidal effects of quercetin, as evidenced by reduced proliferation and increased apoptosis and anoikis. Mechanistically, FNDC5 suppressed the focal adhesion kinase (FAK)/phosphatidylinositol 3-kinase/protein kinase B pathway, leading to mitochondrial-dependent apoptosis and anoikis through modulation of apoptosis-related proteins.

CONCLUSION

Our findings suggest that the FNDC5/FAK axis as a novel regulator of anoikis sensitivity in PAAD. Targeting this axis may represent a promising strategy to enhance the efficacy of quercetin and other natural compounds, offering a potential therapeutic approach to overcome chemoresistance in this aggressive malignancy.

Key Words: FNDC5; Irisin; Quercetin; Pancreatic ductal adenocarcinoma; Anoikis; Focal adhesion kinase; Mitochondrial apoptosis; Chemosensitivity

Core Tip: This study highlights that FNDC5 enhances quercetin-mediated growth inhibition, apoptosis, and anoikis through focal adhesion kinase/phosphatidylinositol 3-kinase/protein kinase B inhibition and mitochondrial apoptosis initiation. Our results provide further insight into the effect of irisin on pancreatic ductal adenocarcinoma (PAAD) cells and suggest a new strategy for PAAD treatment that combines quercetin and irisin.

INTRODUCTION

The five-year survival rate for pancreatic cancer (PC) is below 5%, accounting for its rank as the seventh most common cause of cancer-related mortality worldwide[1,2]. In the United States, about 66440 new cases and 51750 deaths are projected for 2024, making it a major contributor to cancer mortality[3-5]. Pancreatic ductal adenocarcinoma (PAAD), the most common and aggressive histological subtype of PC, represents the critical pathological category driving poor clinical outcomes in this malignancy. Given that it comprises the vast majority of cases, its biology drives collective prognostic outcomes[6]. The main causes of death in patients with PAAD are distant metastasis and treatment resistance, and its treatment mainly depends on surgical resection[2,7,8]. However, most patients present with locally advanced or metastatic disease at diagnosis and have lost the opportunity to undergo surgery[5,8,9]. Therefore, an in-depth understanding and intervention of key cellular biological processes related to PC metastasis remain a critical focus of contemporary research. Tumor metastasis is a complex multi-step process, and anoikis resistance is a crucial component. Anoikis is a specific form of programmed cell death triggered by loss of cell adhesion to the extracellular matrix. It is an important physiological barrier that maintains homeostasis of the epithelial tissue structure and inhibits the ectopic growth of abnormal cell[10]. During the progression of malignant tumors, tumor cells can acquire tolerance to anoikis by remodeling adhesion signals and survival pathways, which is considered one of the key prerequisites for tumor metastasis[10]. Anoikis resistance not only promotes the distant dissemination of tumor cells but is also intimately associated with the maintenance of tumor stemness, epithelial-mesenchymal transition (EMT), and therapeutic resistance[11,12]. Given the established significance of anoikis in cancer, reactivating or enhancing the sensitivity of PC cells to anoikis is considered a potential anti-metastatic treatment strategy. Nevertheless, studies on the regulation of anoikis are still mainly focused on few signaling pathways, and the specific regulatory network and intervention nodes in PAAD have not been fully elucidated.

Quercetin, a natural bioflavonoid primarily found in fruits and vegetables, has been extensively studied for its diverse biological properties, including well-documented anti-inflammatory, antiviral, and anti-fibrotic effects[13-17]. In addition to these pleiotropic protective properties, increasing evidence over the past decade has highlighted its potential anti-tumor activities. In PAAD, quercetin suppresses cell proliferation, induces apoptosis, inhibits migration[18]. However, its role in regulating anoikis a critical barrier to metastasis remains poorly characterized in this malignancy. While limited evidence suggests that quercetin may influence anoikis, the specific efficacy and underlying mechanism of anoikis induction in PAAD have yet to be elucidated[19]. Moreover, the moderate efficacy of quercetin as a single therapy emphasizes the urgent need for a strategy to make PAAD cells sensitive to quercetin-induced anoikis.

Discovered in 2002, FNDC5 is a transmembrane protein that is proteolytically processed to yield the exercise-induced myokine irisin[20,21]. In addition to metabolism, FNDC5/irisin has also attracted attention in oncology. Despite its different performance in different cancers, for example, in breast cancer, FNDC5/irisin is highly expressed, and patients with high expression of FNDC5/irisin show a better prognosis[22]. Functionally, elevated FNDC5 expression curbs the migratory and invasive capacities of gastric cancer cells, and clinically, lower levels of FNDC5 are linked to improved patient outcomes[23]. Notably, emerging evidence highlights the potential of FNDC5 to regulate the response of cancer cells to treatment, such as by enhancing doxorubicin-induced apoptosis in other cancers[24]. Recently, researchers explored the role of irisin in PAAD. For instance, irisin suppressed PAAD cell progression by regulating the adenosine 5-monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin pathway, which attenuates EMT in PC cells[25]. Furthermore, irisin enhances the tumoricidal effect of doxorubicin by activating the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/nuclear factor kappa-B signaling pathway, leading to increased expression of pro-apoptotic proteins and decreased expression of anti-apoptotic proteins[26]. However, in the field of PAAD, research on the function of FNDC5 is lacking, especially whether and how it regulates the sensitivity of tumor cells to treatment (such as quercetin) is completely unknown. Given that FNDC5 is known to regulate pathways such as cell metabolism, oxidative stress, and AMPK, which are closely related to the key anoikis regulator focal adhesion kinase (FAK) and cell survival, we speculate that FNDC5 may reshape the fate of tumor cells by interfering with key nodes such as FAK. Addressing this question was the key objective of the current study.

Therefore, this study aimed to investigate the role of FNDC5/irisin in regulating anoikis sensitivity in PAAD cells and to determine whether FNDC5 overexpression enhances quercetin-induced anti-tumor effects. Specifically, we sought to elucidate the underlying molecular mechanism, focusing on the FAK/PI3K/AKT signaling pathway and mitochondrial-dependent apoptosis. Our findings may provide new insights into the interplay between FNDC5 and natural compounds, offering a potential strategy to overcome anoikis resistance and improve treatment outcomes in this aggressive malignancy.

MATERIALS AND METHODS

Bioinformatics analysis

Using public databases, the prognostic value and expression pattern of FNDC5 were systematically analyzed. Based on The Cancer Genome Atlas (TCGA) database, we first evaluated the prognostic significance of FNDC5 across multiple cancers by including transcriptome and clinical data from 33 cancer types, including PAAD. To evaluate the prognostic value of FNDC5, its relationship with overall survival (OS) was determined using Cox regression and Kaplan-Meier survival analysis. According to the hazard ratio (HR)/Log-rank test P value, the prognostic role of FNDC5 in each cancer was classified as: “Risky” (HR > 1 and P < 0.05), “protective” (HR < 1 and P < 0.05), “no significant correlation” (nonsense, P ≥ 0.05) or “data unavailable” (NA). The results of the pan-cancer analysis were presented in the form of classified heat maps and visualized using the pheatmap package of R software (v4.2.0). Subsequently, the TCGA-PAAD dataset was analyzed using the gene expression profile interactive analysis platform to compare the differences in the expression of FNDC5 in tumor tissues (n = 179) and normal tissues (n = 171). The threshold was set as follows: Log₂fold change cut-off of 1 and Q value cut-off of 0.01. Additionally, the correlation between its expression and clinicopathological stage (stage I-IV) was analyzed. Furthermore, using the gene expression profile interactive analysis platform built-in tool, we classified PAAD patients into FNDC5-high/FNDC5-low groups depending on whether their expression level was above or below the median, and Kaplan-Meier curves of OS and progression-free survival were plotted.

Cell culture

Human PAAD cell lines (Capan-1, PANC-1, Su86.86, and MiaPaCa-2) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, United States). These cell lines differ in their differentiation, invasion potential, and adhesion-dependent survival characteristics, providing a representative model for studying anoikis regulation and FAK-dependent signaling in different PAAD backgrounds[27,28]. PANC-1 and MiaPaCa-2 cells were cultured in DMEM (Corning, NY, United States), Su86.86 cells in RPMI-1640 (Corning), and Capan-1 cells in IMDM (Corning). All cells were incubated at 37 °C under 5% carbon dioxide in a humidified atmosphere, using media containing 10% fetal bovine serum (Gibco, Grand Island, NY, United States) and 1% penicillin-streptomycin. All cell line experiments were conducted in accordance with institutional biosafety guidelines. No human or animal subjects were involved.

RNA extraction and real-time quantitative polymerase chain reaction

To determine the gene expression levels in PAAD cell lines, total RNA was isolated using TRIzol^® reagent (Invitrogen, Carlsbad, CA, United States). Subsequently, complementary DNA was synthesized from equal amounts of RNA using a dedicated kit (Invitrogen), strictly adhering to the supplier’s protocol. Quantitative polymerase chain reaction (qPCR) was performed using an ABI Prism 7900HT system (Applied Biosystems, Foster city, CA, United States). The 2^-ΔΔCt method was used to calculate relative gene expression. Primer sequences used in this study are listed in Supplementary Table 1.

Western blot

For western blot analysis, protein lysates were prepared from PAAD cell lines using radio immunoprecipitation assay buffer (EpiZyme, Shanghai, China) supplemented with both protease and phosphatase inhibitor cocktails. The lysates were electrophoretically resolved on sodium dodecyl sulfate-polyacrylamide gels (Beyotime, Shanghai, China) and subsequently transferred onto polyvinylidene difluoride membranes (Beyotime). To prevent nonspecific binding, the membranes were blocked with 5% bovine serum albumin for 2 hours at room temperature. The membranes were then incubated with specific primary antibodies, followed by incubation with the corresponding secondary antibodies. Immunoreactive bands were detected using a chemiluminescent substrate and signals were captured using a digital imaging system. Information regarding the primary antibodies used is provided in Supplementary Table 2.

Overexpression cell construction

A lentiviral system was used to establish stable FNDC5-overexpressing PAAD cell lines. Briefly, the complete coding sequence of human FNDC5 was inserted into a lentiviral shuttle plasmid (Hanbi; Shanghai, China). HEK293T cells were co-transfected with both recombinant FNDC5 plasmid and lentiviral packaging plasmids using lipofectamine 3000 (Invitrogen). The supernatant containing the virus was harvested 48 hours after transfection and was concentrated, purified, and titered. Subsequently, Capan-1 and MiaPaCa-2 cells were infected with the prepared lentivirus at a multiplicity of infection of 10 for 72 hours. To select a stable transductants, cells were cultured in a medium supplemented with 1.0 μg/mL puromycin for two weeks. The polyclonal populations with stable overexpression of FNDC5 were named Capan-1-F5 and MiaPaCa-2-F5. Following the same procedure, empty polyclonal populations were obtained as the control groups, which were Capan-1 and MiaPaCa-2, respectively.

Real-time monitoring of cell proliferation and long-term clonogenic survival

For proliferation analysis, real-time cellular analysis (RTCA) was performed using the xCELLigence system (ACEA Biosciences, San Diego, CA, United States). Culture medium (100 μL) was added to each well of an E-plate for baseline measurement, followed by incubation at 37 °C for 1 hour. Subsequently, Capan-1 and MiaPaCa-2 cells (including control and FNDC5-overexpressing lines) were seeded at a density of 2.0 × 10³ cells/well. After 24 hours of culturing, quercetin was added to the cells at concentrations of 0 nM, 50 nM, 100 nM and 200 nM. The concentration of quercetin here is set according to other people’s research[29,30]. During 120 hours of treatment, the cell index reflecting cell proliferation and adhesion was automatically recorded every 1 hour. Cell index values were analyzed using RTCA Software Pro (v2.6.0).

For colony formation analysis, cells (1.0 × 10³ cells/well) were plated in 6-well plates and continuously cultured in a medium containing 100 nM quercetin for 2 weeks, with the medium refreshed every 3 days. Following a 20-minute fixation with 4% paraformaldehyde, the colonies were subjected to staining with 0.1% crystal violet. Finally, cells were photographed under a microscope (Olympus, Tokyo, Japan). Colonies containing ≥ 50 cells were counted as viable.

Cell apoptotic analysis by flow cytometry

To ensure that the cells were in a suspended state to simulate an environment that was detached from the extracellular matrix, cells in the FNDC5 overexpression and control groups were seeded in 6-well plates with special treatment on the surface to prevent adherence. Following treatment with or without quercetin (100 nM; Sigma, St. Louis, MO, United States) for 24 hours, both floating and loosely attached cells were collected by gentle centrifugation. The cells were then washed with cold phosphate-buffered saline and stained with Annexin V and PI using a kit (Beyotime) according to the manufacturer’s instructions. Apoptotic cells were analyzed by flow cytometry (Beckman, Brea, CA, United States).

Mitochondrial membrane potential assay

FNDC5-overexpressing and control cells were seeded in 6-well plates and treated with quercetin (Sigma, St. Louis, MO, United States) or a drug solvent [dimethyl sulfoxide (DMSO)] for 24 hours. Following the manufacturer’s instructions, the cells were stained with a mitochondrial membrane potential assay kit (Beyotime). Finally, the cells were photographed using a confocal microscope purchased from Leica Microsystems (Wetzlar, Germany).

Statistical analysis

GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, United States) was used for statistical processing and mapping, and the data were expressed as mean ± SD. Statistical comparisons were conducted as follows: Inter-group differences were assessed with a two-tailed t-test, while differences across multiple groups were evaluated by one-way analysis of variance, followed by the Tukey test for pairwise comparisons. The significance of survival differences observed in the Kaplan-Meier analysis was assessed using the Log-rank test, with the HR and its corresponding 95% confidence interval also computed. Differences were considered significant at P < 0.05.

RESULTS

FNDC5 expression is correlate with better OS of PAAD patients

To systematically assess the role of FNDC5 in PAAD, we conducted a clinical correlation analysis using a public database. Analysis of TCGA data revealed a significant upregulation of FNDC5 in PAAD tissues relative to adjacent normal samples (Figure 1A, P < 0.05). Next, we analyzed the stage expression (stages I-IV) of FNDC5 in PAAD. The analysis indicated that FNDC5 expression varied significantly according to disease stage in PAAD (Figure 1B, P = 3.49 × 10^-5). Survival analysis further showed that FNDC5 had significant prognostic value in PAAD, and patients with high FNDC5 expression showed longer OS (P = 0.044) and DFS (P = 0.002) (Figure 1C and D). FNDC5 was classified as a protective factor against PAAD (Figure 1E).

Figure 1 FNDC5 expression is correlate with better overall survival of pancreatic adenocarcinoma patients. A: Based on the Cancer Genome Atlas database, the box plot showed that FNDC5 was highly expressed in pancreatic ductal adenocarcinoma (PAAD) tissues; B: Violin plot displaying FNDC5 expression levels stratified by clinical stages of PAAD (stage I-IV); C: Kaplan-Meier curve showed that FNDC5 expression was associated with overall survival in PAAD patients; D: Kaplan-Meier curve showed that FNDC5 expression was associated with progression-free survival in PAAD patients; E: Heat map showed that FNDC5 was a protective factor for PAAD by Cox regression analysis; F: Real-time quantitative polymerase chain reaction analysis showing gene expression of FNDC5 in PAAD cell lines (Capan-1, PANC-1, SU86.86, and MiaPaCa-2); G: Western blot analysis showing protein levels of irisin in PAAD cell lines. ^bP < 0.01. ^cP < 0.001. TPM: Transcripts per million; PAAD: Pancreatic ductal adenocarcinoma; HR: Hazard ratio; OS: Overall survival; KM: Kaplan-Meier; mRNA: Messenger RNA; ACC: Adrenocortical carcinoma; BLCA: Bladder urothelial carcinoma; BRCA: Breast invasive carcinoma; CESC: Cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL: Cholangiocarcinoma; COAD: Colon adenocarcinoma; DLBC: Lymphoid neoplasm diffuse large B-cell lymphoma; ESCA: Esophageal carcinoma; GBM: Glioblastoma multiforme; HNSC: Head and neck squamous cell carcinoma; KICH: Kidney chromophobe; KIRC: Kidney renal clear cell carcinoma; KIRP: Kidney renal papillary cell carcinoma; LAML: Acute myeloid leukemia; LGG: Brain lower grade glioma; LIHC: Liver hepatocellular carcinoma; LUAD: Lung adenocarcinoma; LUSC: Lung squamous cell carcinoma; MESO: Mesothelioma; OV: Ovarian serous cystadenocarcinoma; PCPG: Pheochromocytoma and paraganglioma; PRAD: Prostate adenocarcinoma; READ: Rectum adenocarcinoma; SARC: Sarcoma; SKCM: Skin cutaneous melanoma; STAD: Stomach adenocarcinoma; TGCT: Testicular germ cell tumors; THCA: Thyroid carcinoma; THYM: Thymoma; UCEC: Uterine corpus endometrial carcinoma; UCS: Uterine carcinosarcoma; UVM: Uveal melanoma.

To further verify these findings, we analyzed the expression of FNDC5/irisin in four PAAD cell lines (PANC-1, Capan-1, Su86.86, and MiaPaCa-2). The real-time qPCR (RT-qPCR) results showed that the expression of FNDC5 was different in these four cell lines. Capan-1 and MiaPaca-2 cells had lower FNDC5 expression than PANC-1 and Su86.86 (Figure 1F, P < 0.05). Consistent with the RT-qPCR findings, western blot results also indicated lower protein levels of irisin in Capan-1 and MiaPaCa-2 than in PANC-1 and Su86.86 (Figure 1G). In summary, these clinical and experimental data collectively suggest that FNDC5 may play a protective role in PAAD, and its reduced levels correlate with increased tumor aggressiveness.

FNDC5 enhances the inhibitory effects of quercetin in PAAD cells

We further investigated the underlying mechanism by which FNDC5 affects PAAD progression. Among the four PAAD cell lines we previously examined, Capan-1 and MiaPaca-2 cells showed lower expression of FNDC5 than the other two lines. Therefore, Capan-1 and MiaPaca-2 cells were chosen as in vitro models for further experiments. We constructed two FNDC5-upregulated stable cell lines and their control cell lines using a lentivirus system: Namely, MiaPaCa-2-F5, Capan-1-F5, MiaPaCa-2, and Capan-1. Successful overexpression of FNDC5/irisin was verified at both transcriptional and translational levels (Supplementary Figure 1).

Next, we used RTCA to study the cytotoxic effects of quercetin on FNDC5-overexpressing PAAD cells and their corresponding controls. Quercetin exerted a potent inhibitory effect on MiaPaCa-2 cell proliferation after 24 hours, in a concentration- and time-dependent manner. It is worth noting that this inhibitory effect was more pronounced in FNDC5-overexpressing cells (MiaPaCa-2-F5) than in the control cells (Figure 2A and B).

Figure 2 FNDC5 enhances the inhibitory effects of quercetin in pancreatic ductal adenocarcinoma cells. A-D: The cell proliferation of Capan-1-F5, MiaPaca-2-F5, and their control cells treated with 0 nM, 50 nM, 100 nM, and 200 nM quercetin was measured by real time cellular analysis; E-H: The cell inhibition rate of Capan-1-F5, MiaPaCa-2-F5, and their control cells treated with 0 nM, 50 nM, 100 nM quercetin; I: Colony formation assay of Capan-1-F5, MiaPaCa-2-F5, and their control cells treated with 100 nM quercetin. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. NS: Not significant; QUN: Quercetin.

We further calculated the relative inhibition rate of the cells over a representative time period. Given that almost all cells died when the concentration of quercetin was 200 nM in the cell index, we determined the cell inhibition rates at 0 nM, 50 nM, 100 nM (Figure 2C-H). Consistent with the cell index curve, the relative inhibition rate further confirmed that FNDC5 overexpression increased the sensitivity of PAAD cells to quercetin-induced growth inhibition (Figure 2E and F, P < 0.05). Similar results were observed in Capan-1 and Capan-1-F5 cells (Figure 2C, D, G and H, P < 0.05). Taken together, these data demonstrated that increased expression of FNDC5 enhanced the anti-proliferative response of PAAD cells to quercetin, which was further confirmed by colony formation experiments.

To further examine the effects of quercetin on the long-term clone formation ability of PAAD cells, a clonogenic assay was conducted. The results were consistent with the RTCA analysis: Quercetin treatment suppressed the number of cell clones in a concentration-dependent manner in both MiaPaCa-2 and Capan-1 cells. Notably, in FNDC5 overexpressing cells (MiaPaCa-2-F5 and Capan-1-F5), the suppressive effect of quercetin on clonogenicity was more pronounced (Figure 2I). Taken together, these results demonstrate that FNDC enhances the inhibitory effect of quercetin on the proliferation and colony formation of PAAD cells.

FNDC5 enhances quercetin-induced apoptosis in PAAD cells

Next, we evaluated the effect of FNDC5 overexpression on quercetin-induced apoptosis by flow cytometry. Compared to the DMSO group, 100 nM quercetin treatment significantly increased the apoptosis rates in both parental and control PAAD cells; Notably, FNDC5 overexpression cells (MiaPaCa-2-F5 and Capan-1-F5) exhibited markedly enhanced sensitivity to quercetin-induced apoptosis compared to their respective controls (Figure 3A and B, P < 0.01). These data suggest that increased FNDC5 expression renders PAAD cells sensitive to quercetin-induced apoptotic cell death.

Figure 3 FNDC5 enhances quercetin-induced apoptosis in pancreatic ductal adenocarcinoma cells. A: The apoptosis of pancreatic ductal adenocarcinoma cells treated with quercetin (100 nM) or dimethyl sulfoxide (DMSO) was determined by flow cytometry; B: Quantitative analysis of apoptotic cell percentages (early and late apoptosis) corresponding to (A). Compared to the DMSO group, 100 nM quercetin treatment increased apoptosis to 36% (MiaPaCa-2) and 25% (Capan-1). FNDC5 overexpression further enhanced quercetin-induced apoptosis to 56% (MiaPaCa-2-F5) and 52% (Capan-1-F5); C: The expression of apoptosis-related proteins was determined by western blot; D: After quercetin treatment, the intensity of green fluorescence in cells (Capan-1-F5 and MiaPaCa-2-F5) was significantly enhanced. The green fluorescence intensity of FNDC5 overexpressing cells in quercetin treatment group was further enhanced compared with control cells. Green fluorescence represented JC-1 monomer which means impaired mitochondrial function while red fluorescence represented JC-1 aggregated which means complete mitochondrial function; E: Statistical results of the proportion of green fluorescence in cells. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. QUN: Quercetin; FITC: Fluorescein isothiocyanate; PE: P-phycoerythrin; DMSO: Dimethyl sulfoxide.

The expression profiles of apoptosis-associated proteins were assessed by western blotting. Quercetin-treated cells showed reduced expression of anti-apoptotic markers (e.g., Bcl-2, B-cell lymphoma-extra large, and Cas9) and enhanced expression of pro-apoptotic markers (e.g., Bax, Bad, and cleaved-Cas9) compared to the DMSO group. MiaPaCa-2-F5 and Capan-1-F5 cells showed further reduced and elevated levels of anti-/pro-apoptotic proteins, respectively, when treated with quercetin (Figure 3C). Western blotting results demonstrated that FNDC5 had an enhanced effect on the apoptosis induction ability of quercetin.

The mitochondrial pathway is one of the key mechanisms of apoptosis, and quercetin is known to affect mitochondrial function through its antioxidant properties[31]. To test whether apoptosis induced by quercetin and FNDC5 overexpression is mitochondria-dependent, we treated Capan-1-F5, MiaPaCa-2-F5, and their control cells with quercetin or DMSO and stained them with JC-1. As shown in Figure 3D and E, after quercetin treatment, the intensity of green fluorescence (JC-1 monomer, representing a decrease in mitochondrial membrane potential) in the cells was significantly enhanced, indicating that quercetin induced mitochondrial dysfunction. More importantly, in the quercetin treatment group, the green fluorescence intensity of FNDC5 overexpressing cells (Capan-1-F5 and MiaPaCa-2-F5) was further enhanced relative to the control cells (P < 0.01), suggesting that FNDC5 overexpression aggravated quercetin-induced mitochondrial depolarization. Taken together, these results support the hypothesis that FNDC5 increases the sensitivity of PAAD cells to quercetin by bolstering the mitochondrial-dependent apoptosis pathway.

FNDC5 reduced FAK signaling pathway and promotes quercetin-induced anoikis in PAAD cells

Based on the above findings that FNDC5 affects PAAD cell apoptosis through the mitochondrial pathway, we further explored its upstream regulatory mechanism. Previous studies have demonstrated that FNDC5 could regulate FAK signaling and affect the growth phenotype of adipocyte progenitor cells[32]. The FAK/PI3K/AKT axis is centrally involved in the regulation of cell adhesion and anoikis[33,34]. We speculated that FNDC5 may enhance the pro-apoptotic effect of quercetin by targeting this signaling axis. To verify this hypothesis, we examined the expression of key FAK signaling proteins in the control group and FNDC5 overexpressing cells after quercetin (100 nM) or DMSO treatment.

As shown in Figure 4A, FAK, AKT, and PI3K were hyperphosphorylated in quercetin-treated cells. Quercetin treatment inhibited the FAK signaling pathway, and this inhibition was more pronounced in cells with high FNDC5 expression. To further clarify whether FNDC5 promotes anoikis by affecting cell proliferation, we used RTCA technology to monitor the adhesion ability of cells within 8 hours of quercetin treatment in real time. The results suggested that high FNDC5 expression resulted in decreased cell proliferation capacity (Figure 4B and C, P < 0.01). In summary, FNDC5 may exert its anti-proliferative effect by suppressing the FAK/PI3K/AKT signaling axis, thereby promoting anoikis through a mitochondria-dependent pathway and enhancing quercetin-induced cell death.

Figure 4 FNDC5 reduced focal adhesion kinase signaling pathway and promotes quercetin-induced anoikis in pancreatic ductal adenocarcinoma cells. A: Western blot detected the anoikis apoptosis related proteins in Capan-1-F5, MiaPaca-2-F5, and their control cells treated with 100 nM quercetin or dimethyl sulfoxide; B and C: Real-time cellular analysis assay showed that high FNDC5 led to a decrease in cell proliferation (Capan-1-F5, MiaPaca-2-F5) after quercetin treatment. ^bP < 0.01. ^cP < 0.001. NS: Not significant; QUN: Quercetin; DMSO: Dimethyl sulfoxide; FAK: Focal adhesion kinase; P-FAK: Phospho-focal adhesion kinase; AKT: Protein kinase B; P-AKT: Phospho-protein kinase B; PI3K: Phosphatidylinositol 3-kinase; P-PI3K: Phospho-phosphatidylinositol 3-kinase.

DISCUSSION

Our work contributes to the elucidation of the role and mechanism of FNDC5 as a sensitizer for quercetin chemotherapy in PAAD. The results demonstrated that FNDC5 overexpression significantly suppressed cell proliferation and enhanced apoptosis and anoikis in combination with quercetin. Combined with our previous studies, the molecular mechanism may be that irisin blocks the interaction between cells and the extracellular matrix by competitively binding to integrins, thereby inhibiting phosphorylation of the FAK/PI3K/AKT signaling pathway. This effect further induces mitochondrial dysfunction, reduces cell adhesion, and alters the expression pattern of anoikis-related proteins, eventually amplifying quercetin-induced anoikis. This discovery revealed the “FNDC5/irisin-FAK-mitochondria/adhesion” regulatory axis, providing a new strategy to overcome the limitations of quercetin monotherapy.

Our work confirmed that FNDC5 is a new sensitizer that enhances quercetin-induced anti-tumor effects in PAAD cells. Specifically, FNDC5 overexpression significantly enhanced quercetin-mediated apoptosis and anoikis, while inhibiting FAK/PI3K/AKT signaling pathway and subsequent mitochondrial dysfunction. Given that irisin is a known integrin-related secretory peptide, we propose that FNDC5/irisin may disrupt integrin-mediated cell extracellular matrix adhesion, thereby weakening FAK phosphorylation and downstream survival signals. Kim et al[35] showed that blocking αV integrin could significantly inhibit irisin-mediated signal transduction. Other studies have reported that irisin can bind to αVβ5 integrin to maintain skeletal muscle homeostasis[36]. He et al[37] found that physiological concentration of irisin can activate FAK signal through integrin-mediated pathway. These studies suggest that this interaction between FNDC5/irisin and FAK signal represents a possible mechanism axis. Of course, further studies, such as co-immunoprecipitation or surface plasmon resonance, are needed to confirm whether irisin directly interacts with specific integrin subunits in PAAD cells.

This study also found that the high expression of FNDC5 was related to the good prognosis of PAAD, suggesting that it had a tumor suppressor effect in PAAD. This finding is consistent with the report of breast cancer, but contrary to the observation of gastric cancer[22,23]. These results suggest that the effect of irisin is highly dependent on the specific tumor microenvironment and downstream signal background, rather than on a single, fixed tumor-promoting or tumor suppressor molecule. In addition, patients with high FNDC5 expression had better OS, suggesting that FNDC5 may be involved in limiting the malignant progression of tumor cells, especially in the process of cell survival and metastasis. Of course, this is our only speculation and needs to be further verified by systematic experimental investigations.

This study further confirmed that overexpression of FNDC5 significantly potentiated the anti-tumor effect of quercetin on PAAD cells. Through RTCA dynamic monitoring and colony formation experiments, we observed that FNDC5 overexpression augmented the capacity of quercetin to inhibit tumor cell proliferation relative to quercetin alone. This combination also triggered mitochondrial pathway-mediated apoptosis involving the regulation of key apoptosis-related proteins. There is a consensus on the therapeutic potential of quercetin in cancer. In diverse tumor models, quercetin inhibits cell proliferation and induces apoptosis, primarily through suppression of pivotal signaling pathways, including PI3K/AKT and nuclear factor kappa-B, but its single drug effect is often limited, and there are significant differences in the sensitivity of different tumor cells to it[38-41]. In addition, irisin has been reported to induce apoptosis in prostate cancer cells, which may regulate endogenous apoptosis by changing the amount of epidermal cells/β5 receptors and affecting key nodes related to mitochondrial pathways, such as Bcl-2/Bax and PI3K-AKT signaling pathways[42]. In cardiomyocytes, FNDC5 can affect the reactive oxygen species-driven endogenous mitochondrial apoptosis pathway, further confirming our findings[24]. Combined with our study, the high expression of FNDC5 may constitute an important molecular background factor that determines the anti-tumor effect of quercetin.

It is worth noting that the effect of FNDC5/irisin on enhancing chemosensitivity is not limited to quercetin. Studies have confirmed that irisin can enhance the chemosensitivity of PC cells to gemcitabine: Sugimoto et al[43] found that skeletal muscle-derived irisin enhances gemcitabine sensitivity by inhibiting the mitogen-activated protein kinase (MAPK) pathway and inhibits EMT by inhibiting the transforming growth factor-β/SMAD pathway. In the PAAD xenograft mouse model, irisin combined with gemcitabine treatment significantly inhibited tumor growth[43]. These findings suggest that irisin may act as a broad-spectrum chemotherapeutic sensitizer and have a synergistic effect with a variety of standard chemotherapeutic drugs, including gemcitabine and albumin paclitaxel. Given that gemcitabine combined with albumin paclitaxel is currently the first-line treatment for PAAD, exploring the combined application strategy of FNDC5/irisin with these drugs will have a broader clinical transformation prospect. In summary, FNDC5/irisin may reshape the sensitivity of tumor cells to chemotherapeutic drugs by regulating multiple signaling pathways such as FAK/PI3K/AKT and MAPK, providing a new intervention target for overcoming PAAD resistance.

We further studied the upstream regulatory mechanisms and found that FNDC5 overexpression reduced the adhesion ability of PAAD cells and enhanced quercetin-induced anoikis. It has been reported that the FAK signaling pathway and its downstream proteins, including PI3K, protein kinase B, and AKT are involved in anoikis in cells[33,34,44]. Tumor cells often obtain anoikis resistance by activating survival pathways such as PI3K/AKT and MAPK, so as to survive and metastasize after detachment of extracellular matrix[45]. Overexpression of integrins (such as β1 integrins) further exacerbates this resistance by maintaining anchorage-independent signaling[46,47]. Collectively, these studies indicate that both irisin and quercetin are involved in modulating the anoikis pathways in PC. Our current findings not only support these observations, but also propose a novel mechanism: FNDC5 can sensitize PC cells to quercetin-induced anoikis, offering potential insights into therapeutic strategies targeting metastatic progression.

Interestingly, research in non-tumor systems has indicated that quercetin can upregulate the expression of FNDC5 through the AMPK-peroxisome proliferator-activated receptor γ coactivator-1α pathway indicating that there may be a two-way functional relationship[48]. Although this regulatory axis remains to be verified in PAAD, it presents an interesting possibility that quercetin may enhance the expression of FNDC5, thereby rendering cells sensitive to quercetin-induced stress and apoptosis. This cycle can partially explain the synergistic effects observed in our study and provide a biologically reasonable framework for further exploration.

The translational potential of our findings is supported by emerging evidence. Recombinant irisin has been shown to enhance gemcitabine sensitivity and suppress migration in PAAD cells, and its combination with chemotherapy inhibited tumor growth in xenograft models[43,49]. These observations, together with our demonstration that FNDC5 overexpression sensitizes PAAD cells to quercetin-induced anoikis, suggest that targeting the FNDC5/FAK axis could represent a viable therapeutic strategy. Clinically, FNDC5/irisin may serve as a predictive biomarker for selecting patients who would benefit from quercetin-based regimens, or as a therapeutic agent itself either as recombinant protein or via gene therapy approaches to augment the efficacy of natural compounds in this refractory malignancy[50]. Collectively, these converging lines of evidence underscore the significance of FNDC5/irisin as a critical node in tumor biology, positioning its modulation as a promising paradigm to overcome anoikis resistance and enhance the efficacy of phytochemicals in PC treatment.

This study had some limitations. First, these conclusions are mainly based on in vitro models; the effects of FNDC5 on quercetin sensitivity and metastasis require further validation in appropriate in vivo models. Moreover, FNDC5 loss-of-function approaches, such as knockdown or clustered regularly interspaced short palindromic repeats/Cas9-mediated deletion, were not performed in the present study, and such experiments would be necessary to establish a more definitive causal relationship between FNDC5 expression and quercetin sensitivity under physiological conditions. Secondly, the precise molecular interaction of FNDC5 in regulating FAK activation and mitochondrial integrity remains to be elucidated. Thirdly, although quercetin concentrations of 0-200 nM were applied for mechanistic exploration in vitro, the clinical relevance of these doses remains uncertain in the absence of pharmacokinetic characterization and in vivo efficacy data. Finally, whether quercetin regulates the expression of FNDC5 in PAAD cells, as in other tissues, remains to be determined. Future studies should address these issues and evaluate the transformation potential of FNDC5 targeting strategies in combination with quercetin or other therapeutic drugs.

CONCLUSION

In conclusion, this study identifies the FNDC5/FAK axis as a novel regulator of the anti-tumor effects of quercetin in PAAD. Our results demonstrate that FNDC5 enhances quercetin-induced growth inhibition and anoikis by suppressing the FAK/PI3K/AKT pathway and triggering mitochondrial apoptosis (Figure 5). These findings establish a mechanistic link between FNDC5 and the adhesion-independent cell death pathway. The implication is that targeting the FNDC5/FAK axis may represent a viable strategy to potentiate the efficacy of natural compounds like quercetin, offering a potential therapeutic avenue for improving outcomes in this highly aggressive malignancy.

Figure 5 Diagram illustrating the underlying mechanism that FNDC5 enhanced the quercetin-induced anoikis in pancreatic ductal adenocarcinoma cell via FNDC5/irisin/focal adhesion kinase/phosphatidylinositol 3-kinase/protein kinase B axis. ECM: Extracellular matrix; FAK: Focal adhesion kinase; AKT: Protein kinase B; PI3K: Phosphatidylinositol 3-kinase; NF-κB: Nuclear factor kappa-B.