Interactome Analysis Reveals that C1QBP (complement component 1, q subcomponent binding protein) Is Associated with Cancer Cell Chemotaxis and Metastasis

Xiaofang Zhang,Fei Zhang,Lin Guo,Yanping Wang,Peng Zhang,Ruirui Wang,Ning Zhang,Ruibing Chen

doi:10.1074/mcp.m113.029413

Abstract

The complement component 1, q subcomponent binding protein (C1QBP/p32/HABP1) is a ubiquitously expressed and multicompartmental cellular protein involved in various biological processes. In order to further understand its biological functions, we conducted proteomics analysis of its interactome in this study. An improved sample preparation and mass spectrometric identification strategy was developed combining high-speed centrifugation, formaldehyde labeling, and two-dimensional reverse-phase liquid chromatography. Using this approach, we identified 187 interacting proteins and constructed a highly connected interacting network for C1QBP. Moreover, we explored the interaction between C1QBP and protein kinase C ζ, a key regulator of cell polarity and migration. The results indicated that C1QBP regulated the activity of protein kinase C ζ and modulated EGF-induced cancer cell chemotaxis. In addition, C1QBP was required for breast cancer metastasis in a severe combined immunodeficiency mouse model. Furthermore, C1QBP was observed to be overexpressed in breast cancer tissues, and its expression level was closely linked with distant metastasis and TNM stages. In summary, C1QBP was identified as a novel regulator of cancer metastasis that may serve as a therapeutic target for breast cancer treatment.

Highlights

From the ‡Research Center of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China; §Key Laboratory of Breast Cancer Prevention and Therapy, Laboratory of Cancer Cell Biology, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300070, China
The results demonstrated that epidermal growth factor (EGF) induced an interaction between C1QBP and PRKCZ, and that C1QBP has a great effect on the biological activity of PRKCZ
The results suggest that C1QBP knockdown cells failed to form tumors in SCID mouse lungs and that C1QBP was required for breast cancer metastasis

Summary

EXPERIMENTAL PROCEDURES

Antibodies, Reagents, and Animals—Dithiothreitol (DTT), iodoacetamide, urea, formaldehyde, deuterated formaldehyde, sodium cyanoborohydride, monoclonal antibody against ␥-tubulin, monoclonal antibody against Flag, and anti-Flag antibody-conjugated agarose beads were purchased from Sigma; antibodies against C1QBP, PRKCZ, actin, and phosphor-cofilin (Ser3) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); and antibodies against phosphor-PRKCZ (Thr410), GSK3␤, phosphor-GSK3␤ (Ser9), and cofilin were bought from Cell Signaling Technology (Danvers, MA). C1QBP and its interacting proteins were pulled down with anti-Flag antibody-conjugated agarose beads, and mass spectrometric analysis or Western blotting analysis was conducted on the samples. The peptides were eluted using a 7%– 45% B gradient (0.1% formic acid in acetonitrile) over 90 min into a nano-electrospray ionization LTQ Orbitrap mass spectrometer (Thermo Electron Corp.). For the cell polarity study, cells were allowed to migrate for 8 h in a wound-healing assay, fixed with 4% paraformaldehyde, and stained with anti-PRKCZ, anti-Giantin, and anti-␥-tubulin antibodies. After antigen retrieval with sodium citrate buffer, the sections were incubated with mouse anti-human C1QBP monoclonal antibody at 4 °C overnight and stained with HRP-conjugated secondary antibody. The relationships between C1QBP expression status and clinical parameters were analyzed via chi-squared test, and a p value less than 0.05 was considered statistically significant

RESULTS

Gene expression

Distant metastasisb

DISCUSSION