S100A6 Protein Negatively Regulates CacyBP/SIP-Mediated Inhibition of Gastric Cancer Cell Proliferation and Tumorigenesis

Xiaoxuan Ning,Yucai Chuai,Shiren Sun,Xiaoming Wang,Yuan Li,Kun Zhang,Jie Liang,Rossella Rota

doi:10.1371/journal.pone.0030185

Xiaoxuan Ning, Yucai Chuai + Show 6 more

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https://doi.org/10.1371/journal.pone.0030185

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Abstract

Calcyclin-binding protein (CacyBP/SIP), identified on the basis of its ability to interact with S100 proteins in a calcium-dependent manner, was previously found to inhibit the proliferation and tumorigenesis of gastric cancer cells in our laboratory. Importantly, the effects of S100 proteins on the biological behavior of CacyBP/SIP in gastric cancer remain unclear. Herein, we report the construction of eukaryotic expression vectors for wild-type CacyBP/SIP and a truncated mutant lacking the S100 protein binding domain (CacyBP/SIPΔS100). The expressions of the wild-type and truncated recombinant proteins were demonstrated by transfection of MKN45 gastric cancer cells. Co-immunoprecipitation assays demonstrated interaction between S100A6 and wild-type CacyBP/SIP in MKN45 cells. Removal of the S100 protein binding domain dramatically reduced the affinity of CacyBP/SIP for S100 proteins as indicated by reduced co-immunoprecipitation of S100A6 by CacyBP/SIPΔS100. The MTT assay, FACS assay, clonogenic assay and tumor xenograft experiment were performed to assess the effect of CacyBP/SIP on cell growth and tumorigenesis in vitro and in vivo. Overexpression of CacyBP/SIP inhibited the proliferation and tumorigenesis of MKN45 gastric cancer cells; the proliferation and tumorigenesis rates were even further reduced by the expression of CacyBP/SIPΔS100. We also showed that S100 proteins negatively regulate CacyBP/SIP-mediated inhibition of gastric cancer cell proliferation, through an effect on β-catenin protein expression and transcriptional activation of Tcf/LEF. Although the underlying mechanism of action requires further investigation, this study provides new insight into the interaction between S100 proteins and CacyBP/SIP, which might enrich our knowledge of S100 proteins and be helpful for our understanding of the development of gastric cancer.

Highlights

S100 proteins are the largest subgroup within the EF-hand Ca2+-binding protein family and are characterized by their celland tissue-specific expression patterns
S100A6 was selected as a model to observe the effect of S100 proteins on the biological function of CacyBP/SIP
Immunofluorescence staining indicates that S100A6 was mainly distributed in the nuclear membrane with very little found in the nuclear plasma of the MKN45 cells (Fig. 1B)

Summary

Introduction

S100 proteins are the largest subgroup within the EF-hand Ca2+-binding protein family and are characterized by their celland tissue-specific expression patterns. These proteins are known to regulate intracellular processes such as the cell cycle transition and cellular growth, differentiation and motility [1]. A unique feature of these proteins is that individual members are localized in specific cellular compartments from which some are able to relocate upon Ca2+ activation [2,3]. Upon translocation, these proteins may transduce the Ca2+ signal in a temporal and spatial manner by interacting with different S100 protein-specific targets. Several possible protein targets, including glyceraldehyde-3-phosphate dehydrogenase, annexin II, annexin VI, annexin XI, caldesmon and CacyBP/SIP, have been shown to interact with S100 proteins in vitro in a Ca2+-dependent manner [5,7]

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