Cancer Letters

Cancer Letters

Volume 325, Issue 1, 1 December 2012, Pages 80-88
Cancer Letters

STAT3 activation by IL-6 from mesenchymal stem cells promotes the proliferation and metastasis of osteosarcoma

https://doi.org/10.1016/j.canlet.2012.06.006Get rights and content

Abstract

We previously demonstrated that human mesenchymal stem cells (MSCs) promote the growth of osteosarcoma in the bone microenvironment. The aim of the present study was to further determine the effect of IL-6/STAT3 signaling on the progression of osteosarcoma. First, conditioned medium from MSCs was used to stimulate the growth of osteosarcoma cells (Saos-2) in vitro. We found that STAT3 was activated and that the activation could be blocked by an IL-6-neutralizing antibody. The inhibition of STAT3 in Saos-2 cells by siRNA or AG490 decreased cell proliferation, migration and invasion, down-regulated the mRNA expression of Cyclin D, Bcl-xL and Survivin and enhanced the apoptotic response. Furthermore, a nude mouse osteosarcoma model was established by injecting luciferase-labeled Saos-2 cells into the tibia, and the effect of STAT3 on tumor growth was determined by treating the mice with AG490. In vivo bioluminescence images showed that tumor growth was dramatically reduced in the AG490 group. In addition, STAT3 inhibition decreased the lung metastasis rate and prolonged the survival of these mice. After treatment with AG490, the protein levels of IL-6, p-STAT3 and PCNA were decreased, and the level of apoptosis in the tumor was increased. Altogether, these data indicate that MSCs in the bone microenvironment might promote the progression of osteosarcoma and protect tumor cells from drug-induced apoptosis through IL-6/STAT3 signaling.

Introduction

Osteosarcoma is the most common primary malignant tumor of the skeleton and the third most prevalent childhood and adolescent cancer [1]. Appropriate surgical resection and chemotherapy are the most important treatments for patients with osteosarcoma [2], [3]. Lesions often occur in the metaphyses of long bones [4], which represent the major pool of mesenchymal stem cells (MSCs). Previous reports have provided evidence that the bone marrow, especially MSCs, contribute significantly to the growth of various tumors [5], [6], [7], [8]. Some studies have shown that MSCs exert pro-inflammatory effects by constitutively producing soluble factors in the bone marrow microenvironment [9], [10].

Of these factors and cytokines, interleukin-6 (IL-6) may be one of the most important inflammatory factors [11]. IL-6 is a pro-proliferative and anti-apoptotic cytokine that is up-regulated during injury, inflammation and infection [12]. IL-6 protein and mRNA are often overexpressed in serum and tumor samples from breast, bone, liver, and colon cancers in humans and mice. IL-6 has a direct stimulatory effect on the growth of many tumors by increasing the expression of pro-proliferation and survival proteins [13]. The inhibition of IL-6 signaling was shown to slow the growth of colon and lung cancers [14]. Additionally, IL-6-ablated mice were found to be more resistant to the development of colorectal cancer [15]. IL-6 exerts its effects by binding to the IL-6 receptor complex (IL-6R and gp130) or the soluble form of IL-6R (sIL-6R) [16]. The interaction between IL-6 and its receptors produces conformational changes in the gp130 subunit, which then activates signal transducer and activator of transcription 3 (STAT3) via Janus kinases (JAKs). This activation depends on STAT3 phosphorylation of at Y705 by kinases, including JAK2 [17].

The dysregulation of STAT3 activation has been shown to directly promote tumor growth directly by tumor-autonomous mechanisms. STAT3 mediates a wide spectrum of cellular responses, including cell proliferation and apoptosis [18]. Accumulating evidence indicates that persistently activated STAT3 contributes to the development and progression of tumors in numerous organs. In some tumors, constitutively activated STAT3 is associated with resistance to chemotherapeutics and decreased survival [19], [20]. Consequently, STAT3 may serve as a novel target for the therapy of some tumors [21]. Consistent with this hypothesis, a variety of STAT3 inhibitors have been shown to prevent tumor cell growth and induce apoptosis both in vitro and in vivo [22]. Although the hyperactivation of STAT3 in cancer may have important prognostic and therapeutic value, and a recent study has shown that the level of STAT3 activation directly correlates with prognosis in osteosarcoma patients [23], the correlation between STAT3 expression and osteosarcoma progression is not well understood.

We have previously shown that bone marrow-derived MSCs promote the proliferation of osteosarcoma in mice and that the high level of IL-6 secreted by MSCs plays a critical role in this process [24]. However, the mechanisms of this activity have not been fully explored. Although it is the major transcription factor target of IL-6, the effect of STAT3 on the interaction between MSCs and osteosarcoma is unclear. In the present study, we aimed to determine the role of the IL-6-dependent STAT3 signaling pathway in MSCs-driven osteosarcoma cell growth both in vitro and in vivo.

Section snippets

Cell culture and preparation of conditioned medium

Human bone marrow-derived MSCs were obtained from the proximal femur during orthopedic surgery as previously described [25], according to the ethical guidelines of the Shanghai Ninth People’s Hospital, Shanghai, China. The human osteosarcoma cell line Saos-2 was purchased from the Chinese Academy of Sciences (Shanghai, China). Luciferase-labeled Saos-2 cells were generated in our lab. Briefly, luciferase was sub-cloned from the phFL-cmv plasmid and inserted into a lentiviral vector system

MSCs activate STAT3 in Saos-2 cells via IL-6

Our previous study showed that MSCs could promote the osteosarcoma growth in mice by secreting IL-6. We hypothesized that the activation of STAT3 is a critical event in this process. We first explored the response of Saos-2 cells to MSCs by examining the effects of MSCs CM on STAT3 activation. The results showed that activation occurred 10 min after exposure to MSCs CM, and the maximal activation was observed at 30 min (Fig. 1A). Because IL-6 is the main activator of STAT3, the IL-6 levels in MSC

Discussion

The aberrant activation of STAT3 in the absence of JAK2 mutations is a recurring theme in many human tumors [29]. However, the implications of STAT3 activation for osteosarcoma remain unclear. In this study, we identify important functions of IL-6 and STAT3 and their implications for multiple aspects of osteosarcoma development. We show that IL-6 produced by MSCs has a paracrine effect on Saos-2 cells and activates STAT3 in these tumor cells. The IL-6/STAT3 signaling pathway stimulates cell

Acknowledgments

This work was supported by a Grant from the National Natural Science Foundation of China (81172549) and Grants from the Shanghai Science and Technology Development Fund (10410711100, 11XD1403300), the key disciplines program of the Shanghai Municipal Education Commission (J50206), the program for innovative research team of the Shanghai Municipal Education Commission (phase I) and a Grant from Ph.D. Innovation Fund of Shanghai Jiaotong University School of Medicine (BXJ201126).

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