Background
Salivary adenoid cystic carcinoma (SACC) has unique characteristics, such as perineural and perivascular invasion and distant metastasis [
1]. Clinical investigations have shown that SACC has a higher incidence of distant metastasis at an early stage, ranging from 35% to 50% [
2]. To date, several studies have attempted to uncover the molecular mechanism underlying the distinctive biological behaviors of SACC. A number of growth factors, including vascular endothelial growth factor (VEGF), nerve growth factor (NGF) and epidermal growth factor (EGF) have been found to stimulate invasion of SACC cells [
3,
4]. Other signaling molecules have also been implicated in the metastasis of SACC, including mitogen-activated protein kinase 1/2 (MAPK1/2) and snail homolog 2 (Snail2, a.k.a., Slug) [
5,
6]. Although substantial progress has been made in defining the genes that contribute to the initiation and progression of SACC, the mechanistic rationale for the increased metastatic capacity of SACC is still ambiguous and needs to be further investigated.
MTUS1 is identified as an 8p22 candidate tumor suppressor gene encoding a family of angiotensin II (AT2) receptor-interacting proteins (ATIP) [
7]. Alternative exon utilization of this gene leads to 5 known transcript variants that code for 5 different protein isoforms of ATIP (ATIP1, ATIP2, ATIP3a, ATIP3b and ATIP4) [
8,
9]. The ATIP polypeptides exhibit distinct motifs in the amino-terminal portion for localization to the cytosol, nucleus or cell membrane (cytosol, nucleus and plasma membrane for ATIP1, ATIP3 and ATIP4, respectively). The down-regulation of the MTUS1 gene has been documented in many cancer types [
10-
20]; among the 5 isoforms, ATIP1 and ATIP3a/b have been found to exhibit tumor suppressor function [
11,
12]. Our recent studies also suggest that the down-regulation of MTUS1/ATIP is a frequent event in tongue squamous cell carcinoma (TSCC) and also correlated with poor differentiation and associated with reduced overall survival [
21,
22]. ATIP1, ATIP3a and ATIP3b are found to be the major isoforms of MTUS1 gene in tongue epithelial cells and significantly down-regulated in TSCC tissues [
22]. We also find that restoration of ATIP1 expression reduced cell proliferation in TSCC cell lines [
22].
To improve patient survival, a better understanding of tumor invasion and metastasis is required so that aggressive tumors can be detected early in the disease process and targeted therapeutic interventions can be developed. Although the tumor suppressor function of MTUS1/ATIP has been defined, its role in the initiation and progression of SACC has not been reported. In the present study, we aim to assess the clinical significance of MTUS1/ATIP deregulation in patients with SACC. Further we investigate the role of MTUS1/ATIP3a in the proliferation, migration and invasion ability of SACC. We found that MTUS1 down-regulation is associated with poor prognosis in SACC; MTUS1/ATIP3a down-regulation contributes to the proliferation, migration and the invasion abilities of SACC, which involved extracellular signal-regulated kinase 1/2 (ERK1/2)-Slug signaling.
Discussion
MTUS1/ATIP is one of the candidate tumor suppressor genes located in 8p22-p21.3, a region identified in our previous study as one of the most frequent LOH (87.9%) sites in oral cancer [
28]. The MTUS1/ATIP gene has been detected in a large panel of normal human tissues, with the exception of bone marrow and lymphocytes, which express very little or no ATIP mRNA. ATIP3a and ATIP3b are the major ATIP transcripts in all tissues except the brain, and ATIP1 is ubiquitously expressed and ATIP4 is brain-specific [
8]. Similar to Di Benedetto’s report [
8] and our previous report [
22], we found in the present study that ATIP1, ATIP3a and ATIP3b are the major ATIP transcripts in parotid granular tissue, whereas ATIP2 and ATIP4 are not expressed to any significant degree.
Many studies had found that the deregulation of MTUS1/ATIP is related to many types of cancer [
10-
15,
22,
29], such as hepatocellular carcinoma, bladder cancer, breast cancer, colon cancer, prostate cancer and head and neck cancer. Our recent study also found that the reduction of MTUS1 expression is associated with the development and prognosis of TSCC [
21,
22,
28]. In the present study, we also demonstrated that down-regulation of MTUS1/ATIP is a frequent event during the progression of SACC and associates with short overall survival of the SACC patients. Thus, these findings underscore the critical contribution of MTUS1/ATIP deregulation in the tumorigenesis of SACC. We also found that the expression of MTUS1/ATIP is significantly down-regulated in parotid ACC tissue samples compared to normal parotid epithelial samples. ATIP1, ATIP3a and ATIP3b are all significantly down-regulated in these SACC tissue samples.
ATIP3a polypeptides contain a nuclear localization signal in their N-terminal portion and may not colocalize with the seven-transmembrane domain AT2 receptor inside the cell, suggesting AT2-independent roles for ATIP3 proteins in most tissues. Rodrigues-Ferreira
et al. found that restoring ATIP3 expression in breast cancer led to reduced cancer cell proliferation, clonogenicity and anchorage-independent growth and reduced the incidence and size of xenografts grown in vivo [
12]. Molina
et al. also found that ATIP3a was related to cancer cell proliferation and metastasis [
30]. In the present study, we also found that ATIP3a overexpression in SACC-LM cells significantly inhibited cancer cell proliferation, the migration and invasion ability and knockdown of ATIP3a promoted cancer cell proliferation, the migration and invasion ability in SACC-83 cells. All these results reveal that ATIP3a plays an important role in the proliferation, migration and invasion of SACC.
ERK signaling pathway had been found to play a crucial role in almost all cell functions [
31]. ERK2⁄ERK1 are two isoforms of ERK that belong to the family of mitogen-activated protein kinases (MAPKs). Recent studies indicate that hundreds of proteins are under ERK-dependent control [
32].
Slug belongs to the Snail family of zinc-finger transcription factors, and is a well-established downstream target of the MAPK/ERK pathway in many cell types [
33,
34]. Our previous study had found that MAPK-Slug pathway plays an important role in salivary adenoid cystic carcinoma (SACC) metastasis; Slug is a downstream target of MAKP1 (ERK2); siRNA-mediated ERK2-knockdown suppressed the Slug gene promoter activity and reduced the Slug protein level in SACC cells [
26]. Slug gene is also best known for their roles in epithelial-mesenchymal transition (EMT) [
35]. In many human cancers, there is an inverse relationship between E-cadtherin and Slug expression [
34,
36,
37]. Wang et al. confirmed that Slug overexpression was correlated with reduced E-cadtherin expression and enhanced Vimentin expression in two independent TSCC patient cohorts. In vitro, knockdown of Slug suppressed the cell invasion and migration, in contrast, ectopic transfection of Slug led to enhanced cell invasion and migration [
38]. In the present study, we found that overexpression of ATIP3a in SACC-LM cells led to an increase in E-Cadtherin and a notable decrease in pERK1/2, Slug and Vimentin. However, after knockdown the expression of ATIP3a in SACC-83 cells, the protein levels of pERK1/2, Slug, Vimentin were obviously increased, and the protein levels of E-cadtherin were obviously decreased. These results indicate that ERK1/2-Slug signaling contributes to ATIP3a induced anti-proliferation, migration and invasion in SACC.
Conclusion
In this study, we described the expression pattern of MTUS1/ATIP in SACC for the first time. We demonstrated that down-regulation of MTUS1/ATIP plays an important role in the development of SACC and is correlated with poor prognosis. ATIP1, ATIP3a and ATIP3b are the major isoforms produced by the MTUS1 gene and are significantly decreased in SACC cells. MTUS1/ATIP3a has a notable anti-proliferation effect and inhibits the migration and invasion ability in SACC cells. Furthermore, the tumor suppressor function of ATIP3a is achieved by regulating the ERK-Slug pathway. Thus, our results provide evidence suggesting a critical role of MTUS1/ATIP in the tumorigenesis of SACC, and MTUS1/ATIP3a may serve as a biomarker or a novel therapeutic target for patients with SACC.
Acknowledgements
This work was supported in part by grants from National Nature Science Foundation of China (NSFC81472523, NSFC81272953), Guangdong Natural Science Foundation (S2011020002325, S2012010008665), research fund for the doctoral program of Ministry of Education (20120171110050), Program for New Century Excellent Talents in University (NCET-10-0857) and the Project of Science and Technology of Guangdong Province (2012B031800080) .
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
TZ, XD designed and performed experiments, analyzed data, and drafted the paper. AW, XZ designed experiments and analyzed data. BC collected the tumour samples. All authors contributed to the writing of and approved the paper.