Cancer Letters

Cancer Letters

Volume 432, 28 September 2018, Pages 38-46
Cancer Letters

Original Articles
IGFBP2 promotes salivary adenoid cystic carcinoma metastasis by activating the NF-κB/ZEB1 signaling pathway

https://doi.org/10.1016/j.canlet.2018.06.008Get rights and content

Highlights

  • High level of IGFBP2 correlates positively with perineural invasion or metastasis and indicates poor prognosis in SACC.

  • IGFBP2 promotes invasion-metastasis cascade of SACC cells in vitro and in vivo.

  • IGFBP2 activates NF-κB (p65)/ZEB1 pathway to improve the EMT process and metastasis.

  • Nuclear translocation of IGFBP2 is critical for its function on p65 nuclear entry, EMT process and metastasis in SACC.

Abstract

Metastasis is a major cause of poor prognosis in patients suffered with salivary adenoid cystic carcinoma (SACC), in which many factors are implicated. In this study, we identified that IGFBP2, overexpressed in SACC, correlated positively with perineural invasion or metastasis and indicated worse outcome. Moreover, IGFBP2 overexpression could dramatically improve motility and invasion capacity of SACC cells in vitro. Mechanically, IGFBP2 enhanced expression of ZEB1 in a NF-κB (p65)-dependent manner and then promoted epithelial-mesenchymal transition (EMT) in SACC. In addition, IGFBP2 mutation in the nuclear localization signal could impede nuclear translocation of p65, lower ZEB1 expression, and abrogate the EMT process. In xenograft models, IGFBP2 overexpression promoted lung and liver metastases of SACC cells; while if nuclear IGFBP2 was reduced, the formation of metastases in lung and liver was weakened. Together, these results for the first time demonstrate that IGFBP2 plays an important role in invasion and metastasis of SACC through the NF-κB/ZEB1 signaling pathway and IGFBP2 may be a novel biomarker and target for SACC.

Introduction

Salivary adenoid cystic carcinoma (SACC) is one of the most common malignant cancer of the salivary glands, and is characterized by high rates of recurrence and distant metastasis [1,2]. The poor prognosis of SACC is largely due to local invasion and metastasis [3,4], but the molecular mechanisms of the metastatic spread of SACC are poorly understood.

Insulin-like growth factors (IGFs) are regulatory peptides involved in a variety of biological processes, including apoptosis [5], proliferation [6], and cancer cell invasion [7]. Among these proteins, there are two IGF peptide ligands (IGF-I and IGF-II), six high-affinity IGF-binding proteins (IGFBP1 to IGFBP6), and two receptors (IGF-IR and IGF-IIR) [8,9]. Under normal physiological conditions, IGFs bind with IGFBPs, blocking its bioactivity [9]. When stimulated by kinases, IGFs are released from IGFBPs and bind to IGF receptors [9]. Studies showed that aberrant IGFBP function is involved in cancer progression [[10], [11], [12]]. Particularly, IGFBP2 participates in angiogenesis [13], cancer cell invasion and metastasis [14], chemo-resistance [15], and tumorigenesis [16]. Previous studies revealed that IGFBP2 is aberrantly expressed in several kind of carcinomas, including glioma [12,17], esophageal adenocarcinoma [15], colorectal adenocarcinoma [18], triple-negative breast cancer [19], lung cancer [20], and ovarian cancer [21,22]. In addition, aberrant IGFBP2 expression is associated with poor survival [23,24]. As a secretory protein, IGFBP2 translocates to the nucleus through its nuclear localization signal (NLS). Mutations in the NLS abolish its nuclear transport and hinder cancer invasion [13,25]. Nevertheless, the role of IGFBP2 in SACC remains unclear.

Epithelial-mesenchymal transition (EMT) is a physiopathological process characterized by the loss of epithelial cell-like features and the gradual development of mesenchymal cell-like features in cancer cells originating from the epithelium [26]. EMT plays an important role in the process of tumor metastasis and invasion [27]. The critical EMT regulator, zinc finger E-box binding homeobox 1 (ZEB1) protein is involved in the development and progression of several kinds of tumors such as osteosarcoma [28], lung cancer [29], and pancreatic cancer [30]. Previously, we had found that ZEB1 is aberrantly expressed in oral cavity squamous cell carcinoma specimens and correlates with lymph node metastasis and poor outcomes [31]. It is worth noting that ZEB1 is a downstream protein of NF-κB [29]. Moreover, IGFBP2 drives EMT by activating the NF-κB pathway in pancreatic ductal adenocarcinoma [14], indicating that NF-κB is an important mediator for IGFBP2. These results suggest that there may be a relationship between IGFBP2 and NF-κB/ZEB1 pathway.

Because of the uncertain roles of IGFBP2 and ZEB1 in SACC, the present study aimed to explore the role of IGFBP2 and its relation to NF-κB/ZEB1 signaling pathway in SACC. The results could provide some hints for novel molecular targets for the treatment of SACC.

Section snippets

Cell culture

The human SACC cell lines ACC-M, ACC-2, SACC-LM, and SACC-83 were kept by Tianjin Medical University (China). ACC-M and SACC-LM are highly metastatic cell lines, while ACC-2 and SACC-83 are cells lines of low metastatic potential. All cell lines were cultured in RPMI-1640 (Invitrogen Inc., Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS; GIBCO, Invitrogen Inc., Carlsbad, CA, USA) and 1% penicillin-streptomycin, and maintained in a humidified atmosphere containing 5% CO2 at

IGFBP2 positively correlates with perineural invasion and metastasis and indicates poor survival of SACC patients

Although overexpression of IGFBP2 has been reported in several kinds of cancers, its association with SACC remained unexplored. Therefore, we first examined IGFBP2 expression in SACC specimens. Immunohistochemistry (IHC) results showed that SACC tumor tissues expressed higher level of IGFBP2 compared with adjacent normal tissues (Fig. 1A), according to the criteria of IGFBP2 levels (Supplementary Fig. S1). Furthermore, we confirmed this finding both at the protein and mRNA levels in 8 pairs of

Discussion

The role of IGFBP2 has been studied in many types of cancers, but not in SACC. Therefore, in the present study, we demonstrated that IGFBP2 was highly expressed in metastatic SACC and associated with overall survival of patients with SACC. Hence, targeting IGFBP2 may contribute to the treatment of SACC.

Previous studies showed that IGFBP2 participates in a number of processes involved in the development, progression, and metastatic spread of cancer, including angiogenesis [13], invasion [14],

Acknowledgements

This project was funded by National Natural Science Foundation of China (81702420, 81572492, 81672684), and Committee of Tianjin Science and Technology (16JCZDJC34800). We are grateful to Dr. S. Gao from Tianjin Medical University for providing pLVX-IGFBP2 and pLVX-IGFBP2-ΔNLS constructs.

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