Betel quid extract promotes oral cancer cell migration by activating a muscarinic M4 receptor-mediated signaling cascade involving SFKs and ERK1/2

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Abstract

Betel quid (BQ) is a widely accepted etiological factor for oral squamous cell carcinoma (OSCC) in Southeast Asia, but how BQ chewing leads to oral carcinogenesis remains to be elucidated. We have previously demonstrated that the activation of Src family kinases (SFKs) is critical for BQ-induced oral cancer cell motility. Here we investigate whether this biological effect is mediated by specific membrane receptors in oral cancer cells. We found that BQ-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and cell migration could be inhibited by atropine, suggesting the involvement of the muscarinic receptor family. The enhanced activities of ERK1/2 and cell migration were significantly counteracted by PD102807, the selective antagonist of muscarinic M4 receptor. Moreover, cold BQ extract effectively competed with a known ligand, [3H]-N-methyl scopolamine, for binding to muscarinic M4 receptor in vitro, thereby implying that BQ could activate motility-promoting signaling pathways through direct interaction with the receptor. The requirement of muscarinic M4 receptor for BQ-induced oral cancer cell migration was demonstrated by knockdown of the receptor using RNA interference (RNAi). Remarkably, ectopic expression of muscarinic M4 receptor in two oral cancer cell lines, Ca9-22 and SCC-9, further augmented BQ-induced cell migration by 83% and 99%, respectively. Finally, we verified that BQ-induced oral cancer cell migration was mediated through a muscarinic M4 receptor  SFKs  ERK1/2 signaling pathway. Thus, our findings have identified a novel signaling cascade mediating BQ-induced oral cancer cell motility, which could be a therapeutic target for BQ-related oral malignancies.

Research highlights

► Muscarinic M4 receptor mediates betel quid-induced oral cancer cell migration. ► Betel quid can selectively bind to muscarinic M4 receptor in vitro. ► Muscarinic M4 receptor is required for betel quid-induced cancer cell migration. ► Betel quid-induced cell migration was mediated through M4 receptor  SFKs  ERK1/2.

Introduction

Betel quid (BQ) chewing prevails in Southeast Asia, largely accounting for a total of 600 million BQ users worldwide [1]. BQ usually contains areca nut, inflorescence of Piper betle and slaked lime. In Taiwan the incidence of oral cancer death has been steadily growing during the past decade. A recent report shows that oral squamous cell carcinoma (OSCC), the most widespread malignant neoplasm of the oral cavity, has become the sixth and the fourth common cancer in total and male population, respectively [2]. There is mounting epidemiological evidence indicating a close causal relationship between BQ chewing and OSCC [3], [4], [5]. In fact, approximately 85% of Taiwanese OSCC patients are BQ chewers [6], [7]. Despite the fact that smoking and alcohol drinking are also important risk factors for OSCC and may act synergistically with BQ as a carcinogen [8], studies conducted by the International Agency for Research on Cancer (IARC) suggest that BQ without tobacco or areca nut alone is sufficient to cause carcinogenic effects in human. However, the molecular mechanism behind BQ-induced oral carcinogenesis is not fully understood.

G protein-coupled receptors (GPCRs) have been shown to initiate signaling cascades modulating a multitude of physiological responses including cell migration [9], [10]. Muscarinic receptors are class I heptahelical GPCRs consisting of five subtypes, namely M1, M2, M3, M4 and M5 receptors [11]. When activated through binding to their canonical agonist, acetylcholine, muscarinic receptors have been shown to regulate many second messengers, such as cAMP/calcium-signaling pathway and ion channel activities, by coupling to heterotrimeric guanine nucleotide-binding proteins (G proteins) [12]. Despite the well-characterized functions in neurotransmission, there is emerging evidence indicating that muscarinic receptors play an oncogenic role in many cancer types. For example, previous studies have reported that muscarinic receptors in small cell lung carcinoma are activated by acetylcholine, stimulating cell proliferation in an autocrine manner [13], [14]. In addition, activation of M1, M3 or M5 receptors induces transformation of 3T3 cells [15], while activation of M3 and M5 receptors in murine adenocarcinoma cells induce cell growth [16]. The biological roles of muscarinic receptors in malignant tumors are relatively obscure.

We have previously shown that Src family kinases (SFKs) mediate BQ-induced oral cancer cell migration and invasion [17]. In this study, we focus on the identification of upstream membrane receptors that initiate such motility-promoting signaling pathway. Our results suggested that muscarinic M4 receptor mediated BQ-induced oral cancer cell migration through the activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Besides, we found that BQ could physically interact with M4 receptor in vitro. Most importantly, knockdown of M4 receptor by siRNA inhibited BQ-induced cell migration while ectopic expression of M4 receptor further enhanced the effect in two oral cancer cell lines. Furthermore, our data indicated that SFKs acted upstream of ERK1/2 in this BQ-stimulated signaling cascade via M4 receptor. Thus, these results have defined a novel signaling pathway responsible for BQ-induced oral cancer cell motility.

Section snippets

Antagonists, inhibitors and antibodies

PD102807 were purchased from Tocris Bioscience (Ellisville, MO) while SP600125, PP2, U0126 and PD98059 were from Calbiochem (San Diego, CA). Atropine was obtained from Sigma–Aldrich (St. Louis, MO). Anti-Myc and anti-ERK1/2 antisera were obtained from Cell Signaling Technology (Beverly, MA) whereas anti-M4 antiserum from Chemicon (Temecula, CA). Anti-Src (Src 2) antiserum was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-ERK1/2 and anti-phospho-Src family (pY416)

Muscarinic M4 receptor mediates BQ-induced oral cancer cell migration and activation of ERK1/2

Since the major alkaloids of BQ structurally resemble the classical agonist of muscarinic receptors, acetylcholine, we asked whether these receptors were involved in BQ-induced oral cancer cell migration. Ca9–22 cells were mock treated, treated with BQ extract (0.4 mg/ml) only or together with atropine (200 nM), the general antagonist of the muscarinic receptor family, while such treated cells were allowed to migrate in transwell inserts for 20 h. As shown in Fig. 1A, we found that treatment of

Discussion

Cumulative evidence suggests that lymph node metastasis contributes to poor prognosis and reduced 5 year survival rate in OSCC [23], [24]. Therefore a better understanding of the molecular mechanism behind tumor metastasis in OSCC is urgently needed. BQ chewing has long been linked to oral carcinogenesis, but the biological role of BQ in promoting malignant progress of OSCC has not been defined. Our previous studies reveal that BQ can promote oral cancer cell motility through the activation of

Acknowledgments

We thank Dr. Jau-Ling Suen for technical support on the receptor-binding assay and Dr. Yi-Hsin Yang for statistical assistance. This work was supported by National Science Council (98-2311-B-037-002-MY3) and Center of Excellence for Environmental Medicine at Kaohsiung Medical University (KMU-EM-98-1-3)

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