AKT/GSK-3β regulates stability and transcription of snail which is crucial for bFGF-induced epithelial–mesenchymal transition of prostate cancer cells

doi:10.1016/j.bbagen.2014.07.018

Biochimica et Biophysica Acta (BBA) - General Subjects

Volume 1840, Issue 10, October 2014, Pages 3096-3105

https://doi.org/10.1016/j.bbagen.2014.07.018 Get rights and content

Highlights

•
bFGF can promote EMT and motility of human prostate cancer PC-3 cells.
•
bFGF increases both the protein and mRNA expression of Snail.
•
Snail is crucial for bFGF induced EMT in PC-3 cells.
•
bFGF regulates the stability, localization and transcription of Snail.
•
bFGF also regulates the transcription of Snail.

Abstract

Background

Epithelial–mesenchymal transition (EMT) plays a pivotal role in the development of metastatic cancers. Basic fibroblast growth factor (bFGF) is significantly elevated in metastatic prostate cancers, which has been mentioned mainly to induce EMT in normal cells. However, there is no description about bFGF induced EMT and its underlying mechanism in prostate cancer cells.

Methods

Western blotting, immunofluorescence and qRT-PCR assays were used to study protein or mRNA expression profiles of the EMT. Wound healing scratch, migration and invasion assays were used to test the motility of cells undergoing EMT. More methods were used to explore the underlying mechanisms.

Results

We demonstrated that bFGF promoted EMT and motility of human prostate cancer PC-3 cells. Both protein and mRNA expression of Snail were rapidly increased after bFGF treatment. Ectopic expression of Snail triggered EMT and enhanced cell motility in PC-3 cells, and knockdown of Snail almost abolished bFGF induced EMT, suggesting the critical role of Snail. Mechanistic study demonstrated that bFGF promoted the stability, nuclear localization and transcription of Snail by inhibiting the activity of glycogen synthase kinase 3 beta (GSK-3β) through phosphatidylinositide 3 kinases (PI3K)/protein kinase B (AKT) signaling pathway.

Conclusions

It is concluded that bFGF can promote EMT and motility of PC-3 cells, and AKT/GSK-3β signaling pathway controls the stability, localization and transcription of Snail which is crucial for this bFGF induced EMT.

General significance

To our knowledge, this is the first study to demonstrate that bFGF can induce EMT via AKT/GSK-3β/Snail signaling pathway in prostate cancer cells.

Introduction

Basic fibroblast growth factor (bFGF) is a growth factor that belongs to a large FGF family [16], and fulfills its functions mainly through activation of receptors [49]. bFGF activation of its receptors initiates FGF signaling cascades which are susceptible to hijack by cancer cells [32]. Recently, there is evidence from multiple cancer types to implicate FGF signaling in several oncogenic behaviors, including invasion and migration [31]. This promotes therapeutic targeting of FGFs and their receptors becoming a major area of drug development research [43].

Epithelial–mesenchymal transition (EMT) is one of multi-step events for cancer cell invasion and migration which occurs at the invasive front of many metastatic cancers [11], [42]. Cells undergoing EMT exhibit a fibroblastic-like phenotype, acquire mesenchymal components and motile features, loss of epithelial components and cell adhesion [28]. Several transcription factors have been implicated in the control of EMT, and Snail, a zinc finger transcription factor has been proved as the key EMT regulator [30], [35]. Snail binds to the promoter of E-cadherin gene and represses its transcription, which is one of the hallmark events of EMT and thought to suppress metastasis [17]. The important role of Snail in EMT regulation has been described in many cancer types [3], [9], [23], [36]. Ectopic expression of Snail alone can trigger EMT and enhance cell motility in cancer cells [3], [45]. Meanwhile, knockdown of Snail at least partially inhibits EMT and motility triggered by different stimuli [33], [45], [46].

Prostate cancer continues to be one of the most commonly diagnosed cancers in men in recent years [39]. The majority of deaths associated with prostate cancer are attributed to the failure to cure metastatic disease [41]. There are ample evidences that EMT plays a pivotal role in the development of metastatic prostate cancer [25], [29]. Significantly increased plasma level of bFGF is found in metastatic prostate cancer patients [15]. In addition, bFGF is mentioned to induce EMT, but mainly in normal epithelial cells [8], [24], [37], [40]. Thus, we wondered if bFGF is able to induce EMT in human prostate cancer cells and what are the underlying mechanisms. In this study, we revealed that bFGF can induce EMT in human prostate cancer PC-3 cells, and AKT/GSK-3β/Snail signaling pathway is crucial for this process.

Section snippets

Chemicals and reagents

PI3K inhibitor LY294002, p38 MAPK inhibitor SB203580, TGF-β/Smad2 inhibitor SB431542 and JAK/Stat3 inhibitor AG490 and proteasome inhibitor MG132 were obtained from Sigma-Aldrich (St Louis, MO). Primary antibodies against E-cadherin, Snail, p-GSK-3β (Ser9), GSK-3β, p-Akt (Ser473), Akt, p-p38 (Thr180/Tyr182), p38, p-Smad2 (Ser465/467), Smad2, p-Stat3 (Tyr705), Stat3 and β-catenin were obtained from Cell Signaling Technology (MA, USA). Primary antibody against H2A.X was obtained from Bioworld

bFGF induces EMT and promotes motility of PC-3 cells

Prostate cancer cells preferentially metastasize to bone to form bone metastases, which are a major cause of morbidity for men with prostate cancer [21]. As one of the three most used cell lines (PC-3, DU145 and LNCap) in prostate cancer research, PC-3 cells were initially isolated from the bone metastases of prostate cancer while the others were not [26]. Thus, PC-3 cells were used in the following studies. After treatment with bFGF (20 ng/ml) for 24 h, PC-3 cells became scattered and showed a

Discusion

Prostate cancer is the most common malignancy and the second leading cause of cancer deaths in men in the USA [22]. It underscores the need for a more thorough molecular understanding of this resilient disease [1]. The lethal consequences of prostate cancer are related to its metastasis to other organ sites. It has been suggested that EMT is co-opted by prostate cancer cells during their metastatic dissemination from a primary organ to secondary sites [25], [29]. bFGF is recorded to induce EMT

Acknowledgements

This study was supported by grants from the National Program on Key Basic Research Project (973 Program) (No.2011CB935800), National Natural Science Foundation of China (No.81272311 and No. 81071712).

References (50)

V.D. Acevedo et al.
Inducible FGFR-1 activation leads to irreversible prostate adenocarcinoma and an epithelial-to-mesenchymal transition
Cancer Cell
(2007)
K.-C. Chen et al.
Luteolin attenuates TGF-β1-induced epithelial–mesenchymal transition of lung cancer cells by interfering in the PI3K/Akt–NF-κB–Snail pathway
Life Sci.
(2013)
M.-C. Delfini et al.
The timing of emergence of muscle progenitors is controlled by an FGF/ERK/SNAIL1 pathway
Dev. Biol.
(2009)
R. Fang et al.
Nodal promotes aggressive phenotype via Snail-mediated epithelial–mesenchymal transition in murine melanoma
Cancer Lett.
(2013)
D. Hanahan et al.
Hallmarks of cancer: the next generation
Cell
(2011)
G.-M. Jiang et al.
Histone deacetylase inhibitor induction of epithelial–mesenchymal transitions via up-regulation of Snail facilitates cancer progression
Biochim. Biophys. Acta (BBA) - Mol. Cell Res.
(2013)
P. Mak et al.
ERβ impedes prostate cancer EMT by destabilizing HIF-1α and inhibiting VEGF-mediated snail nuclear localization: implications for gleason grading
Cancer Cell
(2010)
V. Masola et al.
Heparanase and syndecan-1 interplay orchestrates fibroblast growth factor-2-induced epithelial-mesenchymal transition in renal tubular cells
J. Biol. Chem.
(2012)
M. Okada-Ban et al.
Fibroblast growth factor-2
Int. J. Biochem. Cell Biol.
(2000)
F. Strutz et al.
Role of basic fibroblast growth factor-2 in epithelial–mesenchymal transformation
Kidney Int.
(2002)

H. Wang et al.

Stabilization of Snail through AKT/GSK-3β signaling pathway is required for TNF-α-induced epithelial–mesenchymal transition in prostate cancer PC3 cells

Eur. J. Pharmacol.

(2013)

Y. Wu et al.

Stabilization of Snail by NF-kB is required for inflammation-induced cell migration and invasion

Cancer Cell

(2009)

X. Zhang et al.

Receptor specificity of the fibroblast growth factor family: the complete mammalian FGF family

J. Biol. Chem.

(2006)

A. Albini et al.

The chemoinvasion assay: a method to assess tumor and endothelial cell invasion and its modulation

Nat. Protoc.

(2007)

G. Argast et al.

Inducible expression of TGFβ, Snail and Zeb1 recapitulates EMT in vitro and in vivo in a NSCLC model

Clin. Exp. Metastasis

(2011)

R.E. Bachelder et al.

Glycogen synthase kinase-3 is an endogenous inhibitor of Snail transcription: implications for the epithelial–mesenchymal transition

J. Cell Biol.

(2005)

M.J. Barbera et al.

Regulation of Snail transcription during epithelial to mesenchymal transition of tumor cells

Oncogene

(2004)

M. Brandl et al.

IKKα controls canonical TGFβ–SMAD signaling to regulate genes expressing SNAIL and SLUG during EMT in Panc1 cells

J. Cell Sci.

(2010)

J. Chen et al.

TGF-β1 and FGF2 stimulate the epithelial–mesenchymal transition of HERS cells through a MEK-dependent mechanism

J. Cell. Physiol.

(2014)

C. Dong et al.

G9a interacts with Snail and is critical for Snail-mediated E-cadherin repression in human breast cancer

J. Clin. Invest.

(2012)

J.-C. Cheng et al.

Hydrogen peroxide mediates EGF-induced down-regulation of E-cadherin expression via p38 MAPK and Snail in human ovarian cancer cells

Mol. Endocrinol.

(2010)

G. Christofori

New signals from the invasive front

Nature

(2006)

V. Flati et al.

Endothelial cell anergy is mediated by bFGF through the sustained activation of p38-MAPK and NF-kappaB inhibition

Int. J. Immunopathol. Pharmacol.

(2006)

D. Giri et al.

Alterations in expression of basic fibroblast growth factor (FGF) 2 and its receptor FGFR-1 in human prostate cancer

Clin. Cancer Res.

(1999)

D. Gospodarowicz

Purification of a fibroblast growth factor from bovine pituitary

J. Biol. Chem.

(1975)

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Altered expression of microRNA (miRNA) is associated with the occurrence and metastasis of various tumors. We previously found that miR-218 inhibits tumor angiogenesis through the RICTOR/VEGFA axis in prostate cancer (PCa). In this study, we determined that miR-218 also had a negative effect on cell growth, migration, and invasion ability in PCa. Our data showed that miR-218 bound to the Grb2-associated binding protein 2 (GAB2) 3'-UTR region and inhibited GAB2 expression. As a novel downstream target of miR-218, GAB2 has been reported to be involved in the occurrence and development of various human tumors, but its role in the progression and metastasis of PCa has not been addressed. We demonstrated for the first time that the expression of GAB2 in the PCa cell lines was increased, while knocking down GAB2 significantly inhibited cell growth, metastatic ability and EMT process in PCa. In addition, the recovery of GAB2 could reverse the changes in the biological function of PCa cells caused by the ectopic expression of miR-218. Mechanistically, miR-218-mediated GAB2 transcriptional suppression significantly inhibited the activity of the PI3K/AKT/GSK-3β pathway, whose abnormal activation was found to be related to the malignant progression of PCa. Taken together, our findings suggest that the miR-218/GAB2 axis may become a novel prognostic indicator and potential therapeutic target in PCa.
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The adaptive activation of alternative signaling pathways contributes to acquired resistance against targeted cancer therapies. Our previous research has shown that blocking Ras/ERK signaling promotes PI3K/AKT signaling in the lung metastatic derivative of MDA-MB-231 (LM2). Because AKT activation was required to drive sustained cell motility following MEK suppression, we extend our research to elucidate how activation of the PI3K/AKT signaling drives sustained motility following MEK inhibition. Reverse phase protein array (RPPA) revealed that SNAIL (SNAI1) was upregulated in U0126 (MEK inhibitor)-treated LM2 cells. Importantly, LM2 cells simultaneously treated with U0126 and PI3K inhibitor LY294002 exhibited reduced expression of SNAIL. Furthermore, depletion of SNAIL led to reduced cell motility in U0126-treated LM2 cells. In addition, we identified AXL as another downstream effector of AKT. These results suggest that SNAIL and AXL are key factors mediating sustained motility of LM2 cells following MEK suppression. Because AKT mediates motile behavior under MEK suppression, our results suggest that AKT and AXL may be targeted to overcome resistance against drugs targeting the Ras/ERK pathway.
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¹: Equal contribution to this manuscript.

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AKT/GSK-3β regulates stability and transcription of snail which is crucial for bFGF-induced epithelial–mesenchymal transition of prostate cancer cells

Highlights

Abstract

Background

Methods

Results

Conclusions

General significance

Introduction

Section snippets

Chemicals and reagents

bFGF induces EMT and promotes motility of PC-3 cells

Discusion

Acknowledgements

Cancer Cell

Life Sci.

Dev. Biol.

Cancer Lett.

Cell

Biochim. Biophys. Acta (BBA) - Mol. Cell Res.

Cancer Cell

J. Biol. Chem.

Int. J. Biochem. Cell Biol.

Kidney Int.

Eur. J. Pharmacol.

Cancer Cell

J. Biol. Chem.

The chemoinvasion assay: a method to assess tumor and endothelial cell invasion and its modulation

Nat. Protoc.

Inducible expression of TGFβ, Snail and Zeb1 recapitulates EMT in vitro and in vivo in a NSCLC model

Clin. Exp. Metastasis

Glycogen synthase kinase-3 is an endogenous inhibitor of Snail transcription: implications for the epithelial–mesenchymal transition

J. Cell Biol.

Regulation of Snail transcription during epithelial to mesenchymal transition of tumor cells

Oncogene

IKKα controls canonical TGFβ–SMAD signaling to regulate genes expressing SNAIL and SLUG during EMT in Panc1 cells

J. Cell Sci.

TGF-β1 and FGF2 stimulate the epithelial–mesenchymal transition of HERS cells through a MEK-dependent mechanism

J. Cell. Physiol.

G9a interacts with Snail and is critical for Snail-mediated E-cadherin repression in human breast cancer

J. Clin. Invest.

Hydrogen peroxide mediates EGF-induced down-regulation of E-cadherin expression via p38 MAPK and Snail in human ovarian cancer cells

Mol. Endocrinol.

New signals from the invasive front

Nature

Endothelial cell anergy is mediated by bFGF through the sustained activation of p38-MAPK and NF-kappaB inhibition

Int. J. Immunopathol. Pharmacol.

Alterations in expression of basic fibroblast growth factor (FGF) 2 and its receptor FGFR-1 in human prostate cancer

Clin. Cancer Res.

Purification of a fibroblast growth factor from bovine pituitary

J. Biol. Chem.