Cancer Letters

Cancer Letters

Volume 373, Issue 1, 1 April 2016, Pages 67-76
Cancer Letters

Original Articles
β2-adrenergic receptor signaling promotes pancreatic ductal adenocarcinoma (PDAC) progression through facilitating PCBP2-dependent c-myc expression

https://doi.org/10.1016/j.canlet.2016.01.026Get rights and content

Highlights

  • Activated β2-AR interacts with PCBP2 in pancreatic cancer cells.

  • β2-AR promotes c-myc expression in PDAC cells in a PCBP2-dependent manner.

  • β2-AR and PCBP2 are upregulated in human pancreatic cancer tissues.

  • High levels of β2-AR/PCBP2 predict poor prognosis in pancreatic cancer patients.

Abstract

The β2-adrenergic receptor (β2-AR) plays a crucial role in pancreatic ductal adenocarcinoma (PDAC) progression. In this report, we identified poly(rC)-binding protein 2 (PCBP2) as a novel binding partner for β2-AR using immunoprecipitation-mass spectrometry (IP-MS) approach. The association between β2-AR and PCBP2 was verified using reciprocal immunoprecipitation. Importantly, we found significant interaction and co-localization of the two proteins in the presence of β2-AR agonist in Panc-1 and Bxpc3 PDAC cells. β2-AR-induced recruitment of PCBP2 led to augmented protein level of c-myc in PDAC cells, likely as a result of enhanced internal ribosome entry segment (IRES)-mediated translation of c-myc. The activation of β2-AR accelerated cell proliferation and colony formation, while knockdown of PCBP2 or c-myc restrained the effect. Furthermore, overexpression of PCBP2 was observed in human PDAC cell lines and tissue specimens compared to the normal pancreatic ductal epithelial cells and the non-cancerous tissues respectively. Overexpression of β2-AR and PCBP2 was associated with advanced tumor stage and significantly worsened prognosis in patients with PDAC. Our results elucidate a new molecular mechanism by which β2-AR signaling facilitates PDAC progression through triggering PCBP2-dependent c-myc expression.

Introduction

Pancreatic cancer is one of the five most frequent causes of tumor-associated deaths in the western world with a 5-year survival rate of only 5% [1]. Pancreatic ductal adenocarcinoma (PDAC) accounts for 90% of pancreatic cancer cases. Although our understanding of the pathological and molecular mechanisms of PDAC has advanced significantly over the past decades, we are still far away from a remarkable success in the treatment of this deadly disease. PDAC remains one of the most aggressive tumor entities [2]. Recently, clinical studies indicate that stress, chronic depression and other psychological factors might influence pancreatic cancer onset and progression [3], [4]. Evidence suggests that patients with pancreatic cancer produce the severest symptoms of anxiety and depression among 14 investigated cancer types [5]. In addition, several laboratory models show that chronic stress accelerates PDAC growth and invasion in mice [6]. These findings point to a critical relevance of psychological stress in the regulation of PDAC progression.

β-adrenergic signaling plays a crucial role in psychological stress-induced physiological responses. Numerous studies have proved that chronic psychological stress could significantly up-regulate the level of catecholamines, including norepinephrine (NE) and epinephrine (E), in human bodies [7]. In addition, β-adrenergic receptors (β-ARs), especially β2-AR, have been reported to be widely expressed in various cancer cells, including PDAC cells [8]. β2-AR is a member of the G protein-coupled receptor family, and emerging experimental and clinical data indicate that β2-AR has a vital role in PDAC progression and metastasis [9], [10], [11]. Previous researches suggest that β2-AR exerts its effect on carcinogenesis through activating signaling via adenylyl cyclase and its downstream effectors cAMP, PKA, p-CREB and STAT3 as well as transactivation of the EGFR pathway [12], [13], [14]. However, the molecular mechanism underlying β2-AR-mediated PDAC progression remains to be clarified.

To further elucidate the role of β2-AR in pancreatic tumorigenesis, we determined the protein-protein interactions of β2-AR using immunoprecipitation-mass spectrometry (IP-MS) analysis. In this way, we identified poly(rC)-binding protein 2 (PCBP2) as a novel binding partner for β2-AR. PCBP2 belongs to the family of poly(rC)-binding proteins, which are characterized by the high-affinity and sequence-specific interaction with polycytosine [15]. PCBP2 can directly bind to mRNA, and plays a vital role in mRNA stabilization, translational silencing and enhancement [16], [17], [18]. PCBP2 has been documented to be involved in leukemia, glioma, gastric carcinoma and oral cancer, and may contribute to uncontrolled proliferation of cancer cells [19], [20], [21], [22]. However, the expression and significance of PCBP2 in PDAC remains obscure. In the present study, we demonstrated that PCBP2 was significantly overexpressed in human PDAC specimens and cell lines. We showed that β2-AR interacted with PCBP2 in the presence of β-AR agonist, and the interaction promoted the expression of c-myc protein in PDAC cells. Furthermore, the impact of β2-AR/PCBP2 interaction on cell proliferation was analyzed using PDAC cell cultures. Importantly, high expression levels of β2-AR and PCBP2 were associated with significantly worsened prognosis in patients with PDAC. On the basis of these data, we speculate that PCBP2 may play a crucial role in pancreatic carcinogenesis by linking β2-AR activation to enhanced c-myc expression and PDAC proliferation.

Section snippets

Mass spectrometry analysis

The fresh-frozen PDAC tissue was intensively homogenized with immunoprecipitation lysis buffer (25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, and 1% NP-40, pH 7.8) and precleared with protein G sepharose (Sigma) for 2 h. Then, 100 mg of protein lysate was immunoprecipitated overnight at 4 °C with 5 µg of anti-β2-AR antibody, or rabbit normal immunoglobulin G (IgG) (Bioworld Technology, MN). Immune complexes were recovered with protein G sepharose for 2 h. After three times of washes, the

The interaction between β2-AR and PCBP2

Using the mass spectrometry, 43 candidate proteins binding with β2-AR were identified (data not shown). To validate the interactions, immunoprecipitations in PDAC tissues and cells were performed. Among them, we characterized PCBP2 protein as a novel β2-AR-interacting protein. PCBP2 was brought down by an anti-β2-AR antibody from the fresh PDAC tissue lysate and vice versa (Fig. 1A). Because PCBP2 was expressed in partial normal pancreatic tissues (data not shown), we also analyzed whether

Discussion

A growing body of evidence has linked psychological factors to tumor initiation and progression in recent years [31], [32], [33], [34], [35]. The potential involvement of psychological stress and the β-adrenergic pathway in PDAC development has attracted significant attention in very recent years [6], [36], [37]. In the present study, we reported that activated β2-AR could recruit PCBP2 protein in PDAC cells. Additionally, we found that β2-AR-mediated recruitment of PCBP2 promoted the

Conflict of interest

None.

Acknowledgements

We thank Prof. Zhengfan Jiang of Peking University for kindly offering us the pCMV-HA-PCBP2 construct. This work was supported by grants from the National Basic Research Program of China (973 Program, No. 2012CB822104 and No. 2011CB910604), the National Natural Science Foundation of China (No. 81572397), Jiangsu Province's Outstanding Medical Academic Leader Program (No. LJ101135) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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