Tenascin-C Modulates Cell Cycle Progression to Enhance Tumour Cell Proliferation through AKT/FOXO1 Signalling in Pancreatic Cancer

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a disease with an extremely poor prognosis that is characterized by a rich extracellular matrix (ECM). Tenascin-C (TNC) is a component of the ECM and plays a role in tumour progression. In this study, we reported that TNC is overexpressed in PDAC tissues and is correlated with tumour stage and cyclin D1 expression. Cyclin D1 is key regulator of the cell cycle G1/S transition. Further experiments revealed that TNC promotes G1/S transition through AKT signalling. TNC/AKT increases the expression of cyclin D1 by enhancing the transcriptional activity of β-catenin, whereas the translocation of FOXO1 from the nucleus results in the downregulation of p27^Kip1. Cyclin D1 and p27^Kip1 regulate the activity of cyclin D1-CDK4 complexes and retinoblastoma (Rb), and then they stimulate the progression of G1/S transition and tumour cell proliferation. In conclusion, TNC exerts its activating effect on the proliferation of pancreatic cancer cells in vitro and in vivo through its functional target AKT/FOXO1/β-catenin. The molecular mechanisms that drive PDAC progression will be useful for the development of molecular markers and the evaluation of patient prognosis.

Keywords: FOXO1; TNC; cell cycle; pancreatic cancer; proliferation.

Figure 1

TNC and cyclin D1 are overexpressed in pancreatic cancer tissues. Immunohistochemical staining (A) and western blot (B) results of TNC and cyclin D1 in pancreatic adenocarcinoma and paracarcinoma tissues (n = 25) (original magnification ×40). (C) Percentage of different TNC/cyclin D1 expression levels (Negative, Weak, Moderate and Strong) in tissues representing different tumour stages [I - IV]. (D) The correlation between TNC and cyclin D1 protein levels in pancreatic cancer tissues (n = 91; r = 0.628; *P < 0.01). (E) The mRNA levels of TNC are shown in the tested cell lines.

Figure 2

TNC promotes pancreatic cancer cell proliferation through the acceleration of G1/S transition. (A) Pancreatic cancer cell lines Capan-2, MiaPaCa-2 and PANC-1 were transfected with the TNC plasmid or siTNC, and then cell proliferation was determined by CCK-8 assay. (B) Representative FACS results are shown from PANC-1 cells that were stained for 7-AAD and BrdU-FITC. (n = 3, *P < 0.05). (C) The proliferation curves of PANC-1 cell growth were evaluated after treatment with TNC or TNC+LY294002/PD98059/siAKT. Data represent the mean ± s.d. (n = 3, ^#P < 0.05, transfection with TNC versus the Vector treatment; *P < 0.05, treatment with TNC+LY294002/PD98059/siAKT versus transfection with TNC alone).

Figure 3

TNC promotes pancreatic cancer cell proliferation via the modulation of AKT/FOXO1 signals. (A) The flow cytometry cell cycle analyses of PANC-1 cell growth were evaluated after treatment with TNC or TNC+LY294002/siAKT/FOXO1. (B) Western blot analysis was performed to determine the expression of AKT, pAKT, CDK4 and cyclin D1 in PANC-1 cells after the indicated treatment. (C) PANC-1 cells were preincubated with LY294002 or DMSO for 1 h before exogenous TNC stimulation. Cells were harvested at the indicated time, and the levels of phosphorylated AKT, FOXO1 and the subcellular expression of pFOXO1 in the cell nuclear and cytoplasmic fractions were analysed by western blot. Data represent the mean ± the s.d. (n = 3, *P < 0.05).

Figure 4

TNC/AKT transactivates cyclin D1 but is dependent on β-catenin. (A) RT-qPCR was used to detect the expression of cyclin D1 in PANC-1 cells with the indicated treatments. (B) The subcellular expression of β-catenin in the cell nuclear and cytoplasmic fractions was analysed by western blot. (C) ChIP of β-catenin bound to the promoter of CCND1. The sequence and position of the TCF/LEF binding site in the CCND1 promoter are shown. PANC-1 cells were transfected with siCtrl, siTNC, vector or TNC plasmid. PCR amplification from the total chromatin (bottom) was used as a positive control, anti-IgG (middle) served as a negative control, and anti-β-catenin (top) showed the interaction between β-catenin and the CCND1 promoter after the indicated treatment. (D) Dual luciferase activity assays were performed using the CCND1 promoter containing the WT, deleted, or mutant TCF/LEF binding site in PANC-1 cells transfected with the indicated treatments. (E) Confocal microscopy images of PANC-1 cells coated with rhTNC and their controls. The green signal represents the distribution of FOXO1 or β-catenin in the cytoplasm and nucleus, and the blue signal represents the nuclear DNA staining by DAPI. Scale bar, 20 μm. (F) TOPflash and FOPflash luciferase expression vectors were co-transfected with TNC or/and FOXO1/siβ-catenin, and the luciferase activity was measured. Data represent the mean ± the s.d. (n = 3, *P < 0.05).

Figure 5

TNC regulates the formation of the cyclin D1-CDK4 complex via β-catenin and p27^Kip1. (A) ChIP of FOXO1 bound to the promoter of CDKN1B. The sequence and position of the FOXO1 binding site in the CDKN1B promoter are shown. PANC-1 cells were transfected with siCtrl, siTNC, vector or TNC plasmid. PCR amplification from the total chromatin (bottom) was used as a positive control, anti-IgG (middle) served as a negative control, and anti-FOXO1 (top) showed the interaction between FOXO1 and the CDKN1B promoter after the indicated treatment. (B) Western blot assay was used to detect the expression of FOXO1, pFOXO1 and p27^Kip1 in PANC-1 cells with the indicated treatments. (C) PANC-1 cell lysates were immunoprecipitated with anti-CDK4, and immunoblotting was performed with cyclin D1 and CDK4 antibodies. The expression levels of p27^Kip1, cyclin D1, CDK4, β-actin, E2F, total Rb and phosphorylated Rb were determined in PANC-1 cell lysates by western blot. (D) A schematic diagram shows the effect of TNC on proliferation via activation of AKT signalling. Data represent the mean ± the s.d. (n = 3, *P < 0.05).

Figure 6

TNC promotes tumourigenicity in vivo. (A) Tumour sizes were measured at the indicated times after PANC-1-Vector and TNC cells were injected into the mice. (B) The photograph shows representative images of tumour size. (C) IHC staining was performed to evaluate Ki67, TNC, pAKT, p27^Kip1, pFOXO1 and β-catenin expression in the indicated primary tumours. (original magnification ×40).