The oncoprotein HBXIP up-regulates Skp2 via activating transcription factor E2F1 to promote proliferation of breast cancer cells

doi:10.1016/j.canlet.2013.01.029

Cancer Letters

Volume 333, Issue 1, 1 June 2013, Pages 124-132

https://doi.org/10.1016/j.canlet.2013.01.029 Get rights and content

Abstract

Hepatitis B X-interacting protein (HBXIP) is a novel oncoprotein. In this study, we found that the expression levels of HBXIP were positively associated with those of S-phase kinase-associated protein 2 (Skp2) in clinical breast cancer tissues and cell lines. Moreover, we found that HBXIP was able to stimulate the promoter of Skp2 through binding to the −640/−443 region in Skp2 promoter involving activating E2F transcription factor 1 (E2F1). Skp2 plays crucial roles in HBXIP-enhanced proliferation of breast cancer cells in vitro and in vivo. We conclude that HBXIP up-regulates Skp2 via activating E2F1 to promote proliferation of breast cancer cells.

Introduction

The hepatitis B virus X-interacting protein (HBXIP), encoding a 18 kDa protein, was originally identified by its interaction with the C-terminus of the hepatitis B virus X protein (HBx) and located at human chromosome 1 p13.3 [1]. HBXIP inhibited the apoptosis induced by HBx in hepotoma cells [2]. HBXIP could form complex with survivin, an anti-apoptotic protein that was overexpressed in most human cancers [1], [3], resulting in the suppression of apoptosis through the mitochondrial/cytochrome pathway. HBXIP was also required for bipolar spindle formation and was a regulator of centrosome dynamics and cytokinesis in cells [4]. Our previous studies reported that HBXIP could promote cell proliferation and migration through S100A4 and IL-8 [5], [6]. However, the mechanism by which HBXIP enhances the proliferation of breast cancer cells remains unclear.

S-phase kinase-associated protein 2 (Skp2) belongs to the family of the F-box proteins. It was originally discovered by Beach and colleagues in 1995, because of its ability to interact with the cell cycle protein cyclin A [7]. Skp2 contains the N-terminal domain, F-box domain, and C-terminal leucine-rich repeats (LRRs) [8]. The Skp2 protein levels changes during the cell cycle, which is low in early G₁ phase, while it is high during G₁/S transition [9]. This alteration in the Skp2 protein level during cell cycle progression is partly due to a change in its gene expression and protein stability [10]. Co-transfection of cyclin E and Skp2 synergistically promoted cell cycle progression in cultured primary hepatocytes in the absence of mitogen or in the presence of growth inhibitors. Furthermore, transfection of hepatocytes in vivo with cyclin E and Skp2 promoted abundant hepatocyte replication and hyperplasia of the liver [11]. Subsequent experiments revealed that Skp2 was involved in cell cycle progression. Overexpression of Skp2 was frequently observed in numerous human cancers, such as colorectal, gastric, breast, prostate, lung, sarcoma, ovarian and other cancers [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23]. These observations suggest that Skp2 may contribute to the development of human cancers. Accumulated evidence suggests that Skp2 displays a proto-oncogenic role in vitro and in vivo.

In the present study, we try to gain insight into the effect of HBXIP on regulation of Skp2 in promotion of proliferation of breast cancer cells. Our data indicate that HBXIP is able to up-regulate Skp2 through E2F1 in breast cancer cells, resulting in the promotion of cell proliferation. Our findings provide insights into the mechanisms by which HBXIP enhances the proliferation of breast cancer cells.

Section snippets

Immunohistochemistry (IHC)

Breast cancer tissue array (No. 08C14), comprising 49 breast tumors, was purchased from Xi’an Aomei Biotechnology (Xi’an, China). Immunohistochemistry assay was performed as described previously [6]. The slides were incubated with rabbit anti-HBXIP (Proteintech Group, Chicago, USA) (or rabbit anti-Skp2, Boster Group, Wuhan, China) antibody at 4 °C for overnight. After incubation at room temperature for 30 min with biotinylated secondary antibody, the slides were incubated with

The expression levels of HBXIP are positively associated with those of Skp2 in clinical breast cancer tissues

Our previous report showed that 75% clinical breast cancer tissues were positive for HBXIP by IHC staining [6]. It has been reported that Skp2 is overexpressed in breast cancer tissues and cell lines [14]. Thus, we supposed that Skp2 might be correlated with HBXIP in breast cancer. Then, we try to investigate the expression correlation between HBXIP and Skp2 by IHC using tissue arrays which are from the same tissue paraffin block. Our data showed that the positive rate of HBXIP was 77.55%

Discussions

Our studies show that HBXIP is a novel oncoprotein. HBXIP was highly expressed in breast cancer tissues and metastatic lymph node tissues and significantly associated with the growth and metastasis of breast cancer cells [6], [32]. However, the underlying mechanism is poorly understood. Skp2 has an established role in tumors. Many studies have shown that the over-expression of Skp2 is observed in a variety of human cancers, including colorectal cancer, gastric cancer, breast cancer, prostate

Acknowledgements

This work was supported by grants from the National Basic Research Program of China (973 Program, Nos. 2011CB512113, and 2009CB521702) and National Natural Science Foundation of China (Nos. 81071623, 81071624 and 81272217).

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Tumor metastasis depends on the dynamic balance of the actomyosin cytoskeleton. As a key component of actomyosin filaments, non-muscle myosin-IIA disassembly contributes to tumor cell spreading and migration. However, its regulatory mechanism in tumor migration and invasion is poorly understood. Here, we found that oncoprotein hepatitis B X-interacting protein (HBXIP) blocked the myosin-IIA assemble state promoting breast cancer cell migration. Mechanistically, mass spectrometry analysis, co-immunoprecipitation assay and GST-pull down assay proved that HBXIP directly interacted with the assembly-competent domain (ACD) of non-muscle heavy chain myosin-IIA (NMHC-IIA). The interaction was enhanced by NMHC-IIA S1916 phosphorylation via HBXIP-recruited protein kinase PKCβII. Moreover, HBXIP induced the transcription of PRKCB, encoding PKCβII, by coactivating Sp1, and triggered PKCβII kinase activity. Interestingly, RNA sequencing and mouse metastasis model indicated that the anti-hyperlipidemic drug bezafibrate (BZF) suppressed breast cancer metastasis via inhibiting PKCβII-mediated NMHC-IIA phosphorylation in vitro and in vivo. We reveal a novel mechanism by which HBXIP promotes myosin-IIA disassembly via interacting and phosphorylating NMHC-IIA, and BZF can serve as an effective anti-metastatic drug in breast cancer.
N-Myc transcriptionally activates Skp2 to suppress p27 expression in small cell lung cancer
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Small cell lung cancer (SCLC) is characterized by a high proliferative rate, a strong predilection for early metastasis and poor prognosis. Novel SCLC biomarkers are urgently required to improve current diagnostic and treatment modalities. MYCN encodes the proto-oncogene N-Myc that is overexpressed in SCLC, but its downstream effectors are poorly characterized. Here, we investigated the role of the N-Myc/Skp2/p27 axis during SCLC progression.
Immunohistochemistry (IHC) and western blotting were performed to evaluate N-Myc/Skp2/p27 expression. SCLC cell apoptosis was investigated through TUNEL staining. Wound healing and transwell assays were performed to detect the migratory and invasive potential of SCLC cells. N-Myc and Skp2 binding was confirmed through luciferase reporter and ChIP assays. Xenograft models were developed to investigate the function of Skp2 during SCLC tumor growth in vivo.
N-Myc and Skp2 were overexpressed in SCLC, whilst p27 expression was suppressed. Skp2 facilitated SCLC progression by protecting cells from apoptosis and facilitating cell migration and invasion. N-Myc was found to bind to the promoter region of Skp2 to enhance its expression. Skp2 enhanced tumor growth in vivo through the suppression of p27. Skp2 silencing reversed the pro-oncogenic effects of N-myc in SCLC tumors.
We show that N-Myc enhances Skp2 to regulate p27 expression during SCLC progression. We therefore highlight the N-Myc/Skp2/p27 axis as a novel diagnostic and much-needed therapeutic target in SCLC.
HBXIP accelerates glycolysis and promotes cancer angiogenesis via AKT/mTOR pathway in bladder cancer
2021, Experimental and Molecular Pathology
Citation Excerpt :
The PI3K/AKT/mTOR pathways regulate cell growth, differentiation, migration, and survival, as well as angiogenesis and metabolism (Chamcheu et al., 2019; Zheng et al., 2019). Related studies have shown that HBXIP induces the activation of the PI3K/AKT signaling pathway in HepG2 HCC cells and increases the expression of cyclin D1 (Xu et al., 2013). HBXIP up-regulates several membrane-bound complement regulatory proteins through p-ERK1/2/NF-κB signaling, thereby promoting the growth of breast tumors (Liu et al., 2012a, 2012b; Zhang et al., 2014).
Abnormal metabolism and uncontrolled angiogenesis are two important characteristics of malignant tumors. Although HBXIP is known to be associated with a poor prognosis for bladder cancer (BC), its effects on glycolysis and angiogenesis in BC have not been investigated. BC prognosis and relative gene expression of HBXIP were analyzed using the GEPIA, UALCAN, and STRING databases. BC cell angiogenesis and glycolysis were assessed by vasculogenic mimicry and glycolysis assay. Human umbilical vein endothelial cell (HUVEC) viability, migration, and angiogenesis were assessed by CCK8, transwell, wound healing, and tube formation assays. The results showed that HBXIP was highly expressed in BC tissues and cells. Knockdown of HBXIP expression decreased the levels of glucose uptake, lactate production, and glycolytic enzyme expression in BC cells, and decreased cell viability and migration of HUVECs. Additionally, silencing HBXIP reduced the total length of tubes and number of intersections, and EPO and VEGF protein expression in BC cells and HUVECs. Furthermore, knockdown of HBXIP expression reversed cell viability, migration, tube formation, and vasculogenic mimicry under high glucose and lactate conditions. Mechanistically, silencing of HBXIP reduced the protein expression levels of pAKT-ser473 and pmTOR, and inhibition of HBXIP, AKT, and mTOR expression decreased glycolytic enzyme protein expression. Our findings suggest that HBXIP reduces glycolysis in BC cells via regulation of AKT/mTOR signaling, thereby blocking BC angiogenesis. Collectively, this study provides a potential strategy to target HBXIP and AKT/mTOR for regulating glycolysis progression concurrently with anti-angiogenesis effects, and thereby develop novel therapeutics for the treatment of BC.
HBXIP contributes to radioresistance through NF-κB-mediated expression of XIAP in breast cancer
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Hepatitis B X-interacting protein (HBXIP) plays an important role in breast tumorigenesis, tumor growth and metastasis, but its functional contribution in radioresistance remains poorly understood. As radiotherapy served as an essential adjuvant treatment, uncovering the role of HBXIP as well as its downstream molecular XIAP in radioresistance could benefit for the development of individual therapy strategy.
Immunohistochemistry of 42 breast cancer tissue samples and Western blot analysis of proteins from MCF-7 and MDA-MB-231 cells exposed to fractioned doses (γ-rays) were used to identify the expression of HBXIP/XIAP in breast cancer. To verify the radioresistance effects and potential mechanism, the cells were treated with designed pCMV and siRNA of targeting genes, and then measured with MTT assay, clonogenic survival assay and flow cytometry. Furthermore, a subcutaneous xenotransplanted tumor model of breast cancer was established in nude mice to validate the radioresistization effect of HBXIP in vivo.
HBXIP and XIAP expression levels in breast cancer tissues were positively correlated with chemo-radiotherapy resistance of breast cancer. Overexpression of HBXIP could desensitize MCF-7 and MDA-MB-231 cells to irradiation by inhibiting radiation-induced cell apoptosis, and knockdown of HBXIP in these cells had the converse response. Moreover, up-regulation of HBXIP resulted in the increase of XIAP and NF-κB levels in vitro and in vivo, while down-regulation of HBXIP led to the opposite effects. In addition, inhibition of XIAP and NF-κB abrogated the HBXIP overexpression induced radioresistization and increased cell apoptosis (25.8% augment for siRNA XIAP and 28.1% for NF-κB in MDA-MB-231 cells; 25.4% augment for siRNA XIAP and 27.2% for NF–κB in MCF-7 cells).
HBXIP enhances radioresistance of human breast cancer cells via upregulating XIAP, and targeting the HBXIP–NF–κB-XIAP pathway may be a potentially effective strategy to enhance the efficacy of radiotherapy for human breast cancer.
The oncogenic role of HBXIP
2021, Biomedicine and Pharmacotherapy
Hepatitis B X-interacting protein (HBXIP) is a conserved protein of 19 kDa that was originally identified as a binding partner of hepatitis B virus X protein. Emerging evidence indicates that HBXIP is highly expressed in a variety of cancers and is correlated with poor clinical outcomes in cancer patients. HBXIP plays a critical role in cancer progression, but the underlying mechanisms are still unclear. In this review, we primarily focus on publications investigating HBXIP in cancer research, including its expression and clinical significance in cancer patients, its role as a coactivator of transcription factors in cancer cells, its inhibitory effects on the mitochondrial cytochrome c-caspase apoptotic pathway, as well as its roles in promoting mitosis and drug resistance in cancer cells, its regulatory effects on cancer metabolism, and its relationships with other signaling pathways or microRNAs in cancer. This review aims to compile and summarize existing knowledge of the functions of HBXIP in cancer, which provides a comprehensive reference for future studies on the oncogenic mechanisms of HBXIP.
HBXIP up-regulates ACSL1 through activating transcriptional factor Sp1 in breast cancer
2017, Biochemical and Biophysical Research Communications
Citation Excerpt :
Growing evidence shows that HBXIP is an important oncoprotein in the carcinogenesis of breast cancer. Our recent researches strongly demonstrate that HBXIP functions as an important oncoprotein contributing to the development of breast cancer [11–16,18,23]. HBXIP acts as a co-activator of transcriptional factors to play crucial roles in breast cancer [12,14,15].
The oncoprotein hepatitis B X-interacting protein (HBXIP) results in the dysregulation of lipid metabolism to enhance the development of breast cancer. Acyl-CoA synthetase long-chain family member 1 (ACSL1) is required for thioesterification of long-chain fatty acids into their acyl-CoA derivatives. In this study, we present a hypothesis that HBXIP might be involved in the regulation of ACSL1 in breast cancer. Interestingly, we found that the overexpression of HBXIP was able to up-regulate ACSL1 at the levels of mRNA and protein in a dose-dependent manner in breast cancer cells. Conversely, silencing of HBXIP led to the opposite results. Mechanistically, HBXIP as a coactivator interacted with transcriptional factor Sp1 through binding to the promoter of ACSL1 by ChIP assays analysis, leading to the transcription of ACSL1 in breast cancer cells. Immunohistochemistry staining revealed that the positive rate of ACSL1 was 71.4% (35/49) in clinical breast cancer tissues, HBXIP 79.6% (39/49), in which the positive rate of ACSL1 was 76.9% (30/39) in the HBXIP-positive specimens. But, few positive rate of ACSL1 10% (1/10) was observed in normal breast tissues. The mRNA levels of ACSL1 were significantly higher in clinical breast cancer tissues than those in their corresponding peritumor tissues. The mRNA levels of ACSL1 were positively associated with those of HBXIP in clinical breast cancer tissues. Thus, we conclude that the oncoprotein HBXIP is able to up-regulate ACSL1 through activating the transcriptional factor Sp1 in breast cancer.

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¹: These authors contributed equally to the work.

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The oncoprotein HBXIP up-regulates Skp2 via activating transcription factor E2F1 to promote proliferation of breast cancer cells

Abstract

Introduction

Section snippets

Immunohistochemistry (IHC)

The expression levels of HBXIP are positively associated with those of Skp2 in clinical breast cancer tissues

Discussions

Acknowledgements

J. Biol. Chem.

J. Biol. Chem.

Cell

Cancer cell

Cancer Lett.

J. Biol. Chem.

Blood

Hum. Pathol.

Cancer cell

J. Biol. Chem.

Cloning and characterization of a novel hepatitis B virus X binding protein that inhibits viral replication

J. Virol.

Human ATP-dependent RNA/DNA helicase hSuv3p interacts with the cofactor of survivin HBXIP

FEBS. J.

HBXIP functions as a cofactor of survivin in apoptosis suppression

EMBO. J.

HBXIP, cellular target of hepatitis B virus oncoprotein, is a regulator of centrosome dynamics and cytokinesis

Cancer Res.

Deregulated proteolysis by the F-box proteins SKP2 and beta-TrCP: tipping the scales of cancer

Nat. Rev. Cancer

The amelioration of renal damage in Skp2-deficient mice canceled by p27 Kip1 deficiency in Skp2-/-p27-/-mice

PLoS. One.

Induction of hepatocyte proliferation and liver hyperplasia by the targeted expression of cyclin E and skp2

Oncogene

Skp2 overexpression is a prognostic factor in patients with ovarian adenocarcinoma

Clin. Cancer Res.

Clinical and biological significance of S-phase kinase-associated protein 2 (Skp2) gene expression in gastric carcinoma: modulation of malignant phenotype by Skp2 overexpression, possibly via p27 proteolysis

Cancer Res.

Significance of skp2 expression in primary breast cancer

Clin. Cancer Res.