Elsevier

Gene

Volume 587, Issue 1, 1 August 2016, Pages 91-97
Gene

Research paper
SHCBP1 is over-expressed in breast cancer and is important in the proliferation and apoptosis of the human malignant breast cancer cell line

https://doi.org/10.1016/j.gene.2016.04.046Get rights and content

Highlights

  • SHCBP1 is frequently up-regulated in breast cancer tissues. The over-expression of SHCBP1 is associated with advanced clinical stage and poor prognosis.

  • SHCBP1 is a pro-proliferative oncogene for breast cancer.

  • SHCBP1 regulates breast cancer cell proliferation through apoptosis and cell cycle regulation.

Abstract

Background

SHC SH2-binding protein 1, a member of Src homolog and collagen homolog (Shc) family, has been recently identified in different contexts in unbiased screening assays. It has been reported to be over-expressed in several malignant cancers.

Methods

Immunohistochemistry of SHCBP1 on 128 breast cancer tissues and adjacent normal tissues were used to evaluate the prognostic significance of SHCBP1. Survival analyses were performed by Kaplan–Meier method. CRISPR/CAS9 method was used to knockout SHCBP1 expression. CRISPR/CAS9 technology was used to knockout SHCBP1 in 2 breast cancer cell lines. MTT assay, BrdU assay, colony formation assay, cell cycle assay and apoptosis analysis in MCF-7 and MDA-MB-231 cell lines were carried out to evaluate the effects of SHCBP1 on breast cancer in vitro.

Results

Immunohistochemical analysis revealed SHCBP1 was significantly up-regulated in breast cancer tissues compared with adjacent normal tissues (82 of 128, 64%). Over-expressed SHCBP1 was correlated with advanced clinical stage and poorer survival. Ablation of SHCBP1 inhibited the proliferation in vitro. SHCBP1 knockout increased cyclin-dependent kinase inhibitor p21, and decreased the Cyclin B1 and CDK1.

Conclusion

Our study suggests SHCBP1 is dysregulated expressed in breast cancer and plays a critical role in cancer progression, which can be a potential prognosis predictor of breast cancer.

Introduction

Breast cancer is one of the leading aggressive and lethal malignancies worldwide (Torre et al., 2015). In the past decades, due to the development in diagnosis and systemic therapy, including surgery, radiation and chemotherapy, the prognosis of breast cancer is improving. A great amount of evidence showed that multiple proteins are dysregulated in primary tumors and are associated with the development and progression of breast cancers (Wan et al., 2013, Guo et al., 2011, Zeng et al., 2012, Lu, 2012). To enable the development of novel and effective anticancer therapeutics, understanding the roles and molecular mechanisms of these proteins is of great importance.

Src homolog and collagen homolog (Shc), a kind of cell surface receptors adaptor protein, can activate growth factor receptors to signaling pathways, such as insulin receptor (IR), insulin growth factor receptor (IGFR), epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR) (Chen et al., 2008, Ornitz and Itoh, 2015, Koul et al., 2013, Qu et al., 2012). SHCBP1 (SHC SH2-binding protein 1), one of the partner of Shc family, has been recently identified in different contexts. Asano et al. showed that Aurora B-mediated SHCBP1 phosphorylation was critical for proper ingression of the cleavage furrow (Asano et al., 2014). SHCBP1 was also identified as a binding partner of central spindlin by proteomics analysis (Buckley et al., 2014). Due to the ability of growth factor receptors activating, SHCBP1 was shown to be play critical role in cell proliferation regulation. SHCBP1 was dysregulated in several diseases especially cancer. SHCBP1 was up-regulated in both human and mouse leukemia and lymphoma (Eckerle et al., 2009, Piccaluga et al., 2007, Kupershmidt et al., 2010, Truffinet et al., 2007). SHCBP1 was also found to be over-expressed inhuman hepatocellular carcinoma (HCC) samples, and inhibition of SHCBP1 reduced cell proliferation and colony formation in HCC cell lines (Tao et al., 2013). However, the exact role of SHCBP1 in cancer especially in breast cancer remains unknown.

In this study, we attempt to unveil the clinical significance and role of SHCBP1 in breast cancer. We found that SHCBP1 was over-expressed in breast cancer tissues compared with adjacent normal tissues (82 of 128, 64%). Over-expressed SHCBP1 was correlated with advanced clinical stage and poorer survival. Ablation of SHCBP1 inhibited the proliferation in vitro. SHCBP1 knockout decreased Ras levels and AKT activity, up-regulated cyclin-dependent kinase inhibitor p21, and down-regulated the Cyclin B1 and CDK1. Our study showed that SHCBP1 may play a role in cell growth and may be a potential diagnosis biomarker and therapeutic target for breast cancer.

Section snippets

Reagents

The antibodies used were anti-SHCBP1 was from ABCAM Biotechnology. Anti-Cyclin B1, anti-p-AKT, anti-p21, anti-CDK1 and anti-GAPDH were all from Santa Cruz Biotechnology (Santa Cruz, USA). All other chemicals were from Sigma-Aldrich Corporation (San Luis, USA) unless otherwise stated.

Cell lines

Breast cancer cell lines MDA-MB-231 and MCF-7 were purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco's Modified Eagle's Medium (Gibco)

SHCBP1 over-expression correlates with progression and poor prognosis in breast cancer

To investigate the clinical significance of SHCBP1, 128 human breast cancer tissue samples were analyzed by IHC. IHC analysis indicated that SHCBP1 was markedly up-regulated in breast cancer samples compared with adjacent normal tissues. Moreover, statistical analysis of the results revealed that SHCBP1 expression was strongly associated with the clinical stage (p < 0.001), T classification (p < 0.001), M classification (p < 0.001) and HER2 expression (p < 0.001) (Table 2). Kaplan–Meier survival curves

Discussion

In this study, we showed that SHCBP1 is over-expressed in breast cell tissues. The up-regulation of SHCBP1 is significantly correlated with the clinical stage, T classification, M classification, HER2 expression and poor survival rate in breast cancer. CRISPR/CAS9 lentivirus-mediated knockout technology was performed to specifically knockout SHCBP1 protein expression and demonstrated that loss of SHCBP1 function significantly inhibited cell proliferation and colony formation, and promoted

Acknowledgments

This work was supported by Putuo District of Shanghai Science and Technology Commission research project (No. 2011PTKW007), the Innovation Team of Shanghai Traditional Chinese Medicine and the Key Medical Discipline Project of Shanghai Putuo Distinct.

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1

These authors contributed to the study equally.

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