Cancer Letters

Cancer Letters

Volume 428, 1 August 2018, Pages 192-200
Cancer Letters

Original Articles
PA28γ acts as a dual regulator of IL-6 and CCL2 and contributes to tumor angiogenesis in oral squamous cell carcinoma

https://doi.org/10.1016/j.canlet.2018.04.024Get rights and content

Highlights

  • PA28γ enhanced the ability of OSCC cells to promote angiogenesis in vitro.

  • PA28γ promoted tumor-induced angiogenesis in xenografts mice models.

  • PA28γ contributes to tumor angiogenesis by regulating IL-6 and CCL2.

  • PA28γ may be a novel therapeutic target for treating PA28γ-positive OSCC.

Abstract

PA28γ promotes tumor development and progression and is suggested to play a role in tumor angiogenesis, but the molecular mechanisms have not been investigated. Here, we found that PA28γ enhanced the ability of OSCC cells to promote the migration, invasion, and tube formation of HUVECs and promoted tumor-induced angiogenesis in xenograft mice models in vivo. Then, a mechanism study revealed that the expression and secretion of IL-6 and CCL2 were dependent on PA28γ expression. Furthermore, blocking IL-6 or CCL2 or the transcription factor NF-κB induced the inhibition of tube formation in HUVECs co-cultured with PA28γ-overexpression OSCC cell supernatants. Moreover, we revealed that p-STAT3 and p-AKT, which are downstream of the IL-6 and CCL2 signaling axis, were downregulated in HUVECs co-cultured with the PA28γ-silenced supernatant and were upregulated with the PA28γ-overexpressing supernatant. In addition, IL-6, CCL2 and PA28γ expressions were correlated in a clinical OSCC cohort. Collectively, our study indicates that PA28γ contributes to tumor angiogenesis by regulating IL-6 and CCL2. PA28γ may be a novel therapeutic target as a dual regulator of IL-6 and CCL2 for treating PA28γ-positive OSCC.

Introduction

Oral squamous cell carcinoma (OSCC) is one of the most common human head and neck cancers. Currently, the morbidity and mortality rates of OSCC remains high in most countries. PA28γ (also known as REGγ, Ki or PSME3), a member of the 11S proteasome activator family, binds and activates 20S proteasomes [1,2]. Activated PA28γ degrades several important regulatory proteins, such as SRC-3 [3], PTTG1 [4], hepatitis C virus core protein [5] and p21/16/19 [2,6,7]. In addition, PA28γ inhibits the activity of the tumor suppressor p53 by promoting its cellular distribution and mouse double minute 2 homolog (MDM2)-mediated degradation [8,9]; meanwhile, p53 suppresses PA28γ expression, and mutant p53 (p53-R248Q and p53-R175H) upregulates PA28γ expression [10,11]. PA28γ affects multiple apoptotic hallmarks, including p53 phosphorylation and caspase activation [12,13]. Moreover, PA28γ is required for DNA repair and chromosomal stability [14,15].

PA28γ is reported to be overexpressed in OSCC [16,17], thyroid [18], colorectal [19], breast [20] and lung and liver cancers [21]. In addition, PA28γ is associated with metastasis and poor prognosis in patients with breast cancer [20]. Our previous study showed that PA28γ is a good predictor for the risk of death in OSCC and that PA28γ promotes the proliferation, migration and invasion of OSCC cells in vitro and in vivo [16]. In addition, we found that the tumor microvascular density in PA28γ-silenced nude mice with a subcutaneous transplantation of tumor cells was less than that in the control group, suggesting that PA28γ might be associated with OSCC tumor angiogenesis. However, the molecular mechanisms underlying this phenomenon have not been investigated.

In this study, we verified that PA28γ promoted tumor angiogenesis, and PA28γ silencing significantly suppressed tumor angiogenesis using a co-culture model and a subcutaneous xenotransplantation tumor model. Further experiments showed that PA28γ stimulated tumor angiogenesis via a dual regulation of IL-6 and CCL2 expression. This study not only helped us to understand the basic biological processes of OSCC tumorigenicity and metastasis but also provided some new ideas and lay the foundation for the development of new tumor immunotherapy strategies, which target PA28γ based on the “double” regulation of IL-6 and CCL2.

Section snippets

Cell culture

HSC-3 cells were purchased from the Cell Bank of Japanese Collection of Research Bioresource (JCRB, Shinjuku, Japan). HN31 cells were provided by Dr. J. Silvio Gutkind (Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health). The 293T lines were purchased from the American Type Culture Collection (Manassas, VA, USA). The HUVECs were provided by Dr. Xikun Zhou (State Key Laboratory of Biotherapy, Collaborative Innovation Center

PA28γ promotes OSCC cells to recruit endothelial cells and induce angiogenesis in vitro

To investigate the biological function of PA28γ during angiogenesis in OSCC, we established stable PA28γ-expressing HSC-3 and HN31 cell lines using lentivirus (Fig. 1A and Fig. S1). Then, we investigated endothelial cell migration and invasion after co-culturing with conditioned media derived from the indicated PA28γ differentially expressed OSCC cells. We observed that silencing PA28γ inhibited, whereas overexpressing PA28γ strongly provoked, the abilities of both the HSC-3 and HN31 cells to

Discussion

Tumor growth is a complicated biological process, and blood vessels are necessary for supplying nutrients and oxygen to the tumor [25]. Tumor angiogenesis is critical for tumor growth, maintenance, and metastasis [26,27]. Many studies demonstrate that angiogenesis inhibitors have a significant therapeutic value [28,29]. Recently, PA28γ was reported as a novel angiogenic factor that plays an important role in the VEGF-induced expression of VCAM-1 and E-Selectin by antagonizing PKA signaling [30

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Nos. 81672675, 81621062 and 81472533), the 111 Project of MOE China (Grant No. B14038), and the Open Foundation of State Key Laboratory of Oral Diseases, Sichuan University (SKLOD201802, SKLOD201701, and SKLOD201714).

Conflicts of interest

The authors declare no potential conflicts of interest.

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