The oncoprotein HBXIP up-regulates FGF4 through activating transcriptional factor Sp1 to promote the migration of breast cancer cells

https://doi.org/10.1016/j.bbrc.2016.01.174Get rights and content

Highlights

  • The highly expression of FGF4 is relevant to that of HBXIP in clinical breast cancer tissues.

  • FGF4 is up-regulated by HBXIP at transcriptional level in breast cancer cells.

  • HBXIP increases the expression level of FGF4 via activating transcriptional factor Sp1.

  • HBXIP enhances breast cancer cells migration through FGF4.

Abstract

We have reported that the oncoprotein hepatitis B X-interacting protein (HBXIP) is able to promote migration of breast cancer cells. Fibroblast growth factor 4 (FGF4) is a multipotent growth factor and is highly expressed in various human cancers. However, the regulatory mechanism of FGF4 in breast cancer remains poorly understood. In the present study, we report that HBXIP is able to up-regulate FGF4 to enhance the migration of breast cancer cells. Immunohistochemistry staining showed that HBXIP and FGF4 were highly expressed in clinical metastatic lymph nodes of breast tumor. The expression levels of HBXIP were positively related to those of FGF4 in clinical breast cancer tissues. Then, we validated that HBXIP up-regulated the expression of FGF4 at the levels of promoter, mRNA and protein by luciferase reporter gene assays, reverse transcription-polymerase chain reaction and Western blot analysis. Moreover, we found that HBXIP was able to activate FGF4 promoter through transcriptional factor Sp1 by luciferase reporter gene assays. Chromatin immunoprecipitation assays confirmed that HBXIP coactivated Sp1 to stimulate FGF4 promoter. In function, we showed that HBXIP promoted breast cancer cell migration through FGF4 by wound healing and transwell cell migration assays. Thus, we conclude that the oncoprotein HBXIP up-regulates FGF4 through activating transcriptional factor Sp1 to promote the migration of breast cancer cells. Therapeutically, HBXIP may serve as a novel target in breast cancer.

Introduction

Mammalian hepatitis B X-interacting protein (HBXIP) is originally identified due to its interaction with the C terminus of the hepatitis B virus X protein [1]. It can bind to survivin to control cell apoptosis and division [2], as well as modulate centrosome dynamics and cytokinesis in cell growth [3]. Additionally, HBXIP functions as a regulator component in amino acids-induced activation of mTORC1 [4]. Our group has pointed out that HBXIP functions as an oncoprotein and acts as a coactivator of transcription factors, such as TFIID, E2F1 and CREB, in facilitation of cancer cell proliferation [5], [6], [7]. HBXIP can markedly enhance angiogenesis and growth of breast cancer through modulating FGF8 and VEGF [7]. Recently, we have reported that HBXIP promotes the migration of breast cancer cells through increasing filopodia formation involving MEKK2/ERK1/2/Capn4 signaling [8]. However, the underling mechanism of HBXIP in the development of breast cancer is not well documented.

Fibroblast growth factors (FGFs) and their receptors tightly regulate a variety of biological processes including cell proliferation, differentiation, migration and survival, and play a fundamental role in embryonic development, regulation of angiogenesis, as well as wound healing in adults [9], [10]. The mammalian FGF family comprises 18 ligands, which exert their actions through 4 highly conserved transmembrane tyrosine kinase receptors [11]. Dysregulation of the FGF/FGFR signaling pathway has been associated with many developmental disorders and carcinogenesis [12], [13]. FGF8, as a member of FGF family, can be modulated by HBXIP in breast cancer [7]. FGF4, as another member of FGF family, appears to be the first FGF expressed during mammalian embryogenesis [14] and it plays a crucial role in embryonic development [15], [16], cell differentiation [17] and cell migration [18]. However, whether FGF4 is involved in HBXIP-mediated breast cancer remains unclear.

In this study, we are interested in the relationship between HBXIP and FGF4 in the development of breast cancer. We find that HBXIP is able to up-regulate FGF4 expression through coactivating transcriptional factor Sp1 to promote the migration of breast cancer cells. Thus, our finding provides new insights into the mechanism by which HBXIP enhances migration of breast cancer cells.

Section snippets

Immunohistochemistry (IHC)

Immunohistochemistry assay was performed as described previously [5]. Breast cancer tissue array (No. 08C14), comprising duplicates of 49 cases of infiltrating primary carcinoma and 38 metastatic tumors, was purchased from Xi'an Aomei Biotechnology (Xi'an, China). The slides were incubated with anti-HBXIP (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-FGF4 (BOSTER, Wuhan, China) antibody at 4 °C overnight. After incubation at room temperature for 30 min with biotinylated secondary antibody,

The expression levels of HBXIP are positively associated with those of FGF4 in clinical metastatic lymph nodes and breast cancer tissues

Previously, we reported that FGF8 was up-regulated by HBXIP in breast cancer [7]. FGF4 is highly expressed in breast cancer cells [21]. Therefore, we concern whether HBXIP is involved in the regulation of FGF4. Then, we investigated the expression of HBXIP and FGF4 in clinical metastatic lymph nodes and breast cancer tissues. Immunohistochemistry staining showed that the positive rate of HBXIP was 77.6% (38/49) in clinical primary breast cancer tissue samples, in which the positive rate of FGF4

Discussion

Our group has reported that the oncoprotein HBXIP acting as a coactivator of transcription factors is able to promote proliferation and migration of breast cancer cells. FGF4 gene was initially identified as an oncogene from human stomach cancers and Kaposi's sarcoma by a NIH3T3 transforming assay [24] and it is a multipotent growth factor that elicits diverse biological actions on various types of cells and tissues [10]. FGF4 gene amplification has been reported in various human cancers [25]

Conflict of interests

Hui Shi, Yinghui Li and Guoxing Feng designed the research methods, performed the experiments and prepared the manuscript, Leilei Li and Runping Fang analyzed the data, Jie Qu, Peijian Ding and Zhen Wang participated in the experiments. Lihong Ye and Xiaodong Zhang designed and revised the manuscript. All authors declare that there is no competing financial interest exists.

Acknowledgments

This work was supported by the grants of the National Basic Research Program of China (973 Program, No. 2015CB553905, 2015CB553703), the National Natural Scientific Foundation of China (No. 81372186, 81272218, 31470756), and Tianjin Natural Scientific Foundation (No. 14JCZDJC32800).

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      Citation Excerpt :

      The luciferase reporter gene assay was performed according to manufacturer's instructions and previous study [26]. Western blot analysis was carried out with standard protocols and based on previous report [27]. Male athymic (nu/nu) BALB/c mice of 4–6 weeks of age with an average body weight of 18 g were obtained from Beijing HFK Bioscience Co., Ltd. (Beijing, China) and housed in a certified, specific pathogen-free level animal facility (with individually ventilated cages and independent ventilation system) at the Institute of Radiation Medicine (IRM), Chinese Academy of Medical Sciences (CAMS).

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    1

    Contribute equally.

    2

    Present address: Chengde Medical College, Chengde 067000, Hebei Province, PR China.

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