Cancer Letters

Cancer Letters

Volume 322, Issue 2, 28 September 2012, Pages 169-176
Cancer Letters

SDF-1/CXCR4 signaling induces pancreatic cancer cell invasion and epithelial–mesenchymal transition in vitro through non-canonical activation of Hedgehog pathway

https://doi.org/10.1016/j.canlet.2012.02.035Get rights and content

Abstract

In our previous study, we found that blockade of SDF-1/CXCR4 signaling inhibits pancreatic cancer cell migration and invasion in vitro. However, the mechanism governing the downstream regulation of SDF-1/CXCR4-mediated invasion remains unclear. Here we report the role of SDF-1/CXCR4 in pancreatic cancer and the possible mechanism of SDF-1/CXCR4-mediated pancreatic cancer invasion. We show that there is a cross-talk between SDF-1/CXCR4 axis and non-canonical Hedgehog (Hh) pathway in pancreatic cancer. Furthermore, our data demonstrate that the ligand of CXCR4, SDF-1 induces CXCR4-positive pancreatic cancer invasion, epithelial–mesenchymal transition (EMT) process and activates the non-canonical Hh pathway. Moreover, we also demonstrate that the invasion of a pancreatic cancer and EMT resulting from the activation of SDF-1/CXCR4 axis is effectively inhibited by Smoothened (SMO) inhibitor cyclopamine and siRNA specific to Gli-1. Collectively, these data demonstrate that SDF-1/CXCR4 modulates the non-canonical Hh pathway by increasing the transcription of SMO in a ligand-independent manner. Taken together, SDF-1/CXCR4 axis may represent a promising therapeutic target to prevent pancreatic cancer progression.

Introduction

Pancreatic cancer is one of the most aggressive malignancies in the world and its 5-year survival rate is less than 5% [1]. Due to the lack of early symptoms and reliable diagnostic markers for early detection, 80% of patients with pancreatic cancers are diagnosed at a locally advanced or metastatic stage. The exact molecular mechanisms which are responsible for this dismal clinical course remain largely unknown. Recently, multiple genes have been identified to be involved in the process of pancreatic cancer invasion and metastasis [2], [3].

The stromal-derived factor-1 (SDF-1)/CXCR4 axis is deregulated in multiple human cancers, including pancreatic cancer [4], [5]. Elevated levels of SDF-1 in patients with pancreatic cancer have been linked to a poor outcome [6]. CXCR4 has also been reported to be highly expressed in pancreatic cancer cells [7] and its elevated level is correlated with a poor clinical outcome in pancreatic carcinoma patients [8]. In our previous study [9], we found that blockade of SDF-1/CXCR4 signaling inhibits pancreatic cancer cell migration and invasion in vitro. However, the mechanism governing the downstream regulation of SDF-1/CXCR4-mediated invasion is still the focus of investigators.

The Hedgehog (Hh) pathway, which is known to control cell proliferation and differentiation in developing embryos [10], is over-expressed in many extracutaneous cancers, including pancreatic cancers [11], [12]. The Hh signaling protein functions by binding to a 12-pass-transmembrane receptor called Patched 1 (Ptch1). The binding of Hh to Ptch1 results in the release of the inhibitory effects of Ptch1 on the Smoothened (SMO), a 7-transmembrane spanning protein. The activated SMO then re-localizes to the primary cilia and initiates an intracellular signaling cascade that eventually leads to the activation of the Gli-1 transcription factor and the up-regulation of the downstream target genes, including Ptch1 [13]. Another study has examined the expression of Ptch and Gli-1 in 54 pancreatic cancer surgical specimens and seven available pancreatic cancer cell lines. This study demonstrated that Hh signaling activation is a very common event in pancreatic cancer [14]. The activation of the Hh pathway in pancreatic cancer could induce an epithelial–mesenchymal transition (EMT), which results in invasion and metastasis through up-regulating the expression of E-cadherin and down-regulating the expression of vimentin [2], [15].

In the present study, we aimed to study the role of SDF-1/CXCR4 in pancreatic cancer and the possible mechanism of SDF-1/CXCR4-mediated pancreatic cancer invasion. We found that CXCR4 is expressed in pancreatic cancer cells and demonstrated that activation of CXCR4 by its ligand SDF-1 leads to increased expression of SMO, resulting in Hh pathway activation and cancer cell invasion.

Section snippets

Cell culture and reagents

The human pancreatic cancer cell lines BxPc-3, MiaPaCa-2, and Panc-1 were obtained from the American Type Culture Collection (Manassas, VA). All cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM) (HyClone, Logan, USA) supplemented with 10% fetal bovine serum (FBS), 100 μg/ml ampicillin, and 100 μg/ml streptomycin. The cultures were incubated at 37 °C in a humidified atmosphere containing 5% CO2. Recombinant human SDF-1 was purchased from PeproTech (Rocky Hill, USA). The

Expression of CXCR4 and SMO in pancreatic cancer cells

To explore the possible role of CXCR4 signaling in activation of Hh pathway in pancreatic cancer cells, we first explored the expression of CXCR4 and SMO in three human pancreatic cancer cell lines. According to the cell characteristics, MiaPaCa-2 is undifferentiated. Panc-1 is poorly differentiated while BxPc-3 is moderately differentiated. As shown in Fig. 1, the CXCR4 protein was detected in MiaPaCa-2 and Panc-1 cells, but not in BxPc-3. Similar results were observed while detecting the

Discussion

SDF-1 is broadly expressed in a variety of tissue types and acts as a potent chemo-attractant for immature and mature hematopoietic cells [19], [20]. Recently, there is increased evidence that SDF-1/CXCR4 signaling plays an important role in cancer [21], [22]. We and others have corroborated the role of CXCR4 signaling in the local invasion and distant metastasis of pancreatic cancer [9], [23]. CXCR4 expression was detected in pancreatic carcinoma cell lines and cancer tissues in earlier

Acknowledgments

This work was financially supported by Grants from the National Natural Science Foundation of China (No. 81172195), the Fundamental Research Funds for the Central Universities in Xi’an Jiaotong University, and Pilot Project Grant from the Centers of Biomedical Research Excellence (COBRE) Grant NIH P20 RR020151 from the National Center for Research Resources (NCRR). NCRR is a component of the National Institutes of Health (NIH). The contents of this report are solely the responsibility of the

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