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

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.

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.

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.

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.

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.