S100A4, frequently overexpressed in various human cancers, accelerates cell motility in pancreatic cancer cells

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Abstract

S100A4, a member of the Ca2+ dependent S100 protein family, is reported to associate with metastasis through regulation of the motility and invasiveness of cancer cells. A high level of S100A4 protein has been reported in a variety of cancers, including pancreatic cancer. However, its biological role in pancreatic carcinogenesis is largely unknown. We previously reported that S100A4 is frequently overexpressed and that RNAi-mediated knockdown induces apoptosis and suppression of cell growth, motility, and invasiveness. In this study, we analyzed the effects of forced expression of S100A4 in pancreatic cancer cell lines without S100A4-upregulation. We used two cell lines without upregulation of S100A4 (PCI-35 and PCI-43) as well as two cell lines with highly upregulated S100A4 as the control (MIA PaCa-2 and PAN-07-JCK). Cells did not show acceleration of their growth and invasiveness after forced expression of S100A4, but remarkable acceleration of cell motility was observed only in PCI-35 and PCI-43. We further performed microarray analyses using PCI-35 and PCI-43 with and without forced expression of S100A4 and identified 72 and 18 genes that were 2-fold or more upregulated or downregulated, respectively, in both cell lines after forced expression of S100A4. Our results suggest that S100A4 is crucial for cell motility in pancreatic cancer and that some downstream genes may play important roles in cell motility.

Highlights

► We analyzed the effects of forced expression of S100A4 in pancreatic cancer cell lines. ► Cell migration was accelerated after forced expression of S100A4. ► Candidate downstream genes were selected by microarray analysis. ► IFI27 was found to be one of the important downstream genes of S100A4.

Introduction

Pancreatic cancer is one of the most life-threatening diseases, and its invasive capacity and metastatic potential are crucial causes for the difficulty and/or impossibility of surgical resection. In addition, pancreatic cancers are largely resistant to both chemotherapy and radiation therapy. Diagnosis at the early stages of the disease is difficult because this disease does not show any particular symptoms. Therefore, most patients are diagnosed at an advanced disease stage, resulting in a poor prognosis [1]. Establishment of more effective methods for both diagnosis and treatment are necessary for improving the prognoses of pancreatic cancer patients. Curative treatments are limited by invasion and metastasis in many patients; thus, elucidation of molecular pathways is particularly important.

Recently, overexpression of S100A4 in various tumors has been reported [2], and its function as one of the key players in metastatic process has been gradually elucidated [3]. The S100 family members are Ca2+-binding proteins characterized by the EF-hand motif. Twenty-one different human S100 genes have been identified; most of the family member genes are clustered in chromosome 1q21. The physiological properties of these S100 proteins implicate their involvement in diverse cellular functions, including cell proliferation, differentiation, metabolism, motility, and signal transduction; therefore, targeting these molecules should be an effective strategy for cancer therapy [4]. We previously demonstrated the frequent overexpression of S100A4 in pancreatic cancer cell lines; siRNA-mediated knockdown of S100A4 suppressed cell proliferation and invasiveness in only those pancreatic cancer cell lines with a high level of S100A4 expression [5]. Hence it is of great interest to analyze the changes after the introduction of S100A4 into pancreatic cancer cells with low-level expression. Herein we report that cell motility is activated by induction of S100A4 in human pancreatic cancer.

Section snippets

Pancreatic cancer cell lines and culture conditions

In this study, pancreatic cancer cell lines Panc-1, BxPC-3, MIA PaCa-2, AsPC-1, PK-1, PK-8, PK-9, PK-45P, PK-45H, PK-59, PCI-35, PCI-43, PCI-64, PAN-03-JCK, PAN-07-JCK, and PAN-08-JCK were used. The first four cell lines were purchased from American Type Culture Collection (Manassas, VA), and the remaining 12 were obtained from the original developers; they were analyzed in our previous report [5]. The immortalized normal human pancreatic ductal epithelial cell, HPDE [6], was kindly provided by

Expression analyses of S100A4 before and after the introduction by RT-PCR and Western blotting

An association between poor prognosis and S100A4 overexpression has been reported in a variety of cancers, including pancreatic cancer [2]. We first examined the expression of S100A4 in 16 pancreatic cancer cell lines as well as in HPDE, an immortalized normal human pancreatic ductal epithelial cell line, and the results as shown in Supplementary Fig. 1 were in good agreement with our previous report [5]. Then we selected PCI-43 and PCI-35 as the representative cell lines without overexpression

Acknowledgments

We are grateful to Dr. B. L. S. Pierce (University of Maryland University College) for editorial work in the preparation of this manuscript and to Biomedical Research Core (Tohoku University School of Medicine) for technical support. This work was supported in part by Grants-in-Aid (Grant # 17015003, 22591512, and 23590452) and by the Academic Frontier Project for Private Universities: matching fund subsidy 2006–2010 from the Ministry of Education, Culture, Sports, Science and Technology of

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