Elsevier

Experimental Cell Research

Volume 316, Issue 3, 1 February 2010, Pages 466-476
Experimental Cell Research

Research Article
Stanniocalcin-2 is a HIF-1 target gene that promotes cell proliferation in hypoxia

https://doi.org/10.1016/j.yexcr.2009.09.018Get rights and content

Abstract

Stanniocalcin-2 (STC2), the paralog of STC1, has been suggested as a novel target of oxidative stress response to protect cells from apoptosis. The expression of STC2 has been reported to be highly correlated with human cancer development. In this study, we reported that STC2 is a HIF-1 target gene and is involved in the regulation of cell proliferation. STC2 was shown to be up-regulated in different breast and ovarian cancer cells, following exposure to hypoxia. Using ovarian cancer cells (SKOV3), the underlying mechanism of HIF-1 mediated STC2 gene transactivation was characterized. Hypoxia-induced STC2 expression was found to be HIF-1α dependent and required the recruitment of p300 and HDAC7. Using STC2 promoter deletion constructs and site-directed mutagenesis, two authentic consensus HIF-1 binding sites were identified. Under hypoxic condition, the silencing of STC2 reduced while the overexpression of STC2 increased the levels of phosphorylated retinoblastoma and cyclin D in both SKOV3 and MCF7 cells. The change in cell cycle proteins correlated with the data of the serial cell counts. The results indicated that cell proliferation was reduced in STC2-silenced cells but was increased in STC2-overexpressing hypoxic cells. Solid tumor progression is usually associated with hypoxia. The identification and functional analysis of STC2 up-regulation by hypoxia, a feature of the tumor microenvironment, sheds light on a possible role for STC2 in tumors.

Introduction

Human stanniocalcin-2 (STC2) is a glycoprotein hormone with limited sequence homology to its paralog STC1 [1], [2], [3], [4]. It has recently been suggested that the hormone may play a role in human carcinogenesis, as altered expression profiles in cancerous tissues were detected [5]. Intriguingly STC2 was identified as an estrogen responsive gene and was found to co-express with estrogen receptor in human breast cancers or breast cancer cell lines [6], [7]. As a result of all these findings, the use of STC2 as a prognostic marker to renal, ovarian and breast cancers has been suggested [8], [9], [10], [11], [12], [13].

Hypoxia is a common feature of most solid tumors. Solid tumor progression is usually associated with hypoxia and endoplasmic reticulum (ER) stress. This microenvironmental factor has profound consequence to tumor growth characteristics and to the responses in cancer therapy. The general hypoxia-driven responses in tumor cells include angiogenesis, anaerobic glycolysis and the reduction of macromolecule synthesis [14], [15]. This adaptative response is basically mediated by the activation of HIF-1 responsive genes and the phosphorylation of the translation initiation factors [14], [16], resulting in the development of resistance to apoptosis and an increased risk of metastasis. Therefore the identification of genes associated in hypoxic tolerance may be useful in diagnostic prediction and therapeutic treatment. In deciphering the regulation and function of STC2, it was reported that the gene was epigenetically regulated in cancer cells [17]. The stimulatory effects of hypoxia [17], [18] and ER stress [19] to STC2 expression were demonstrated, and functionally, STC2 was related to the unfold protein response (UPR).

Clinically, STC2 has been proposed to be a biomarker for ovarian cancers, in association with the formation of tumor neovessels [8]. The growth and the spread of the ovarian cancer cells throughout peritoneal cavity depend on the outcomes of hypoxic adaptation (i.e., angiogenesis and proliferative/invasive capability). Since hypoxic cells often demonstrate resistance to therapeutic treatment, peritoneal invasion of ovarian cancer cells generally leads to poor patient outcome. Hypoxia regulated STC2 expression was demonstrated and was found to be HIF-1 dependent [17], [18], [20]. However the direct transactivation role of HIF-1α and the location of the functional hypoxia responsive element (HRE) on STC2 gene promoter had not been characterized. Additionally, the clinical relevance of STC2 is not clear. In the present study, we demonstrated the direct transactivation role of HIF-1α and identified the functional HRE on human STC2 gene promoter. The HIF-1 transactivation of STC2 promoter was found to require two consecutive HRE binding sites and the recruitment of the coactivators p300 and HDAC7. We also identified that STC2 stimulated cell proliferation under hypoxia. The stimulation was correlated to the increasing cellular levels of phosphorylated form of retinoblastoma (Rb) and cyclin D.

Section snippets

Effects of hypoxia and drug treatments to the expression of STC2, and the binding of HIF-1α to STC2 promoter

The human breast cancer cells (i.e., MCF7, T47D and MDA-231) and the human ovarian cancer cell lines (SKOV3, OVCAR3 and CaOV3) were maintained in their respective media and exposed to hypoxic condition for 24 h. Total RNA was extracted for the determination of STC2 mRNA by real-time PCR. Significant inductions of STC2 were observed in all the tested cell lines. Among those SKOV3 produced the most significant response in STC2 expression at hypoxia. Therefore SKOV3 cells were used in the

The activation of STC2 expression in hypoxic breast and ovarian cancer cells

The exposure of various human cancer cell lines (i.e., breast cancers (T47D, MDA-231 and MCF7) and ovarian cancers (OVCAR3, CaOV3 and SKOV3)) to hypoxia caused significant induction of STC2 mRNA expression (Fig. 1). Among those, the detected response in SKOV3 was the most significant.

HIF-1α induced STC2 promoter-driven luciferase activity in normoxic SKOV3 cells

To illustrate the direct gene transactivation, we overexpressed the wild-type form of HIF-1α in normoxic condition and measured the STC2 promoter-driven luciferase activities. In Fig. 2A, the Western blot data

Discussion

STC2 has been shown to be an oxidative stress responsive gene that was induced by redox agents and hypoxia in multiple cell lines [17], [19]. However the molecular mechanism of HIF-1 transactivation of STC2 promoter was not clear. In the present study, we reported that hypoxia-mediated STC2 expression was transcriptionally activated by HIF-1. The data confirmed that STC2 is a HIF-1 target gene. Hypoxia caused HIF-1α stabilization and significantly induced STC2 in both mRNA and protein levels.

Acknowledgments

This work was supported by the Faculty Research Grant, Hong Kong Baptist University (C.K.C. Wong).

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