Cancer Letters

Cancer Letters

Volume 360, Issue 2, 1 May 2015, Pages 141-150
Cancer Letters

Original Articles
Fibronectin promotes cell proliferation and invasion through mTOR signaling pathway activation in gallbladder cancer

https://doi.org/10.1016/j.canlet.2015.01.041Get rights and content

Highlights

Abstract

Fibronectin (FN), a heterodimeric glycoprotein overexpressed in several types of tumors, has been implicated in cancer progression via the activation of integrin-mediated pro-oncogenic pathways. The FN level in human bile fluid is dramatically increased in malignant biliary diseases; however, FN expression and its biological functions in gallbladder cancer (GBC) remain unknown. In this study, we found that FN was overexpressed in GBC tissues and was associated with a worse prognosis in GBC patients. In vitro experimental studies indicated that exogenous FN significantly enhanced cell proliferation, invasion and active MMP-9 secretion in human GBC cell lines (GBC-SD and NOZ). Moreover, the key kinases of the mTOR signaling pathway, including FAK, Akt, mTOR and 4E-BP1, were markedly activated in a time-dependent manner in FN-treated GBC-SD and NOZ cells. The IHC statistical analyses validated that FN expression was positively correlated with the phosphorylation levels of the 4E-BP1 protein in GBC tissues. Furthermore, rapamycin, a specific inhibitor of mTOR, almost completely blocked FN-induced phosphorylation of 4E-BP1 and also partially abrogated the stimulatory effects of FN on GBC cell proliferation and invasion. In vivo, FN treatment significantly promoted the proliferation and metastasis of GBC cells and markedly activated Akt/mTOR/4E-BP1 signaling cascade. These findings demonstrate that FN may play a critical role in the modulation of cell proliferation and invasion via mTOR signaling pathway activation during GBC progression.

Introduction

Tumor growth and invasion have been increasingly demonstrated to be regulated by the dynamic interactions between tumor cells and their surrounding extracellular matrix (ECM) [1], [2], [3]. FN, a heterodimeric glycoprotein overexpressed in several human cancers, has been shown to play essential roles in cancer development [4], [5], [6]. Through specifically binding to the integrin transmembrane receptors α5β1 or αvβ3, FN could trigger multiple signal transduction pathways, thereby regulating malignant cellular growth, invasive migration and metastasis [7], [8], [9]. These observations have promoted investigations into FN as a potential target for cancer therapy [10]. The diagnostic value of FN in human bile fluid for malignant biliary diseases has previously been documented [11], [12]; however, FN expression and its biological functions in GBC remain unclear.

The mammalian target of the rapamycin (mTOR) signaling pathway is frequently activated in human cancers and plays a prominent role in the regulation of multiple cellular functions in response to tumor microenvironmental stimuli [1], [13], [14]. As the two most characterized effectors downstream of mTOR, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and p70 ribosomal protein S6 kinase (p70S6K) are critically involved in the translational control of gene expression during cancer progression [15], [16], [17]. Recent studies have demonstrated that mTOR and p70S6K are substantially activated in GBC, especially in advanced tumors. Notably, highly expression of p-mTOR has been associated with a worse prognosis in GBC patients [18], [19]. Furthermore, the therapeutic targeting of mTOR was found to potently attenuate the migration and invasion of GBC cells via epithelial–mesenchymal transition inhibition [20]. These studies suggest that high activation of the mTOR signaling pathway may play a crucial role in GBC progression, which prompted our interest in exploring whether the mTOR pathway was involved in FN-mediated GBC biological behavior.

In the present study, we determined FN protein expression in human GBC tissue samples by using IHC and subsequently analyzed its clinicopathological significance. We also examined the effects of exogenous FN on the invasiveness of GBC cells in vitro. Furthermore, we investigated the potential involvement of the mTOR signaling pathway in this process. Our findings demonstrate that FN could promote GBC cell proliferation and invasion through mTOR signaling pathway activation.

Section snippets

Cell culture and chemicals

The two human GBC cell lines used in this study were as follows: GBC-SD (obtained from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China) and NOZ (obtained from the Health Science Research Resources Bank, Osaka, Japan). The GBC-SD cells were cultured in DMEM (Gibco, Gaithersburg, MD, USA), and the NOZ cells were maintained in William's medium E (Lonza, Belgium, WI, USA) at 37 °C in a humidified 5% CO2 incubator. Both media were supplemented with 10% fetal bovine serum (FBS). FN

FN expression is increased and associated with a poor outcome in GBC patients

To clarify the role of FN in GBC progression, we evaluated FN protein expression in 116 GBC and 35 chronic cholecystitis tissue samples by using IHC. As shown in Fig. 1A, FN was primarily expressed in the cytoplasm of the gallbladder epithelial cells from the cholecystitis tissues, whereas FN was localized in the membrane and cytoplasm of GBC cells and/or the peripheral stroma of malignant tumors. Furthermore, 64.7% (75/116) of the GBC samples exhibited FN-positive expression in the membrane

Discussion

The diagnostic value of FN in human bile fluid for malignant biliary diseases has been previously reported; however, FN expression and its biological functions in GBC remain uncharacterized. In this study, we examined FN protein expression in human GBC tissue samples by using IHC and found that FN was primarily overexpressed in the membrane and cytoplasm of GBC cells and was associated with histological grade and tumor stage. Furthermore, FN overexpression was associated with a worse prognosis

Conflict of interest

The authors declare no conflicts of interest.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (Nos. 81172026, 81272402, 81301816 and 81172029), National High Technology Research and Development Program (863 Program) (No. 2012AA022606), Foundation for Interdisciplinary Research of Shanghai Jiao Tong University (No. YG2011ZD07), Shanghai Science and Technology Commission Intergovernmental International Cooperation Project (No. 12410705900), Shanghai Science and Technology Commission Medical Guiding Project (No.

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