Domain and Functional Analysis of a Novel Breast Tumor Suppressor Protein, SCUBE2

Yuh-Charn Lin,Chun-Chuan Chen,Chien-Jui Cheng,Ruey-Bing Yang

doi:10.1074/jbc.m111.244418

Abstract

Signal peptide CUB (complement proteins C1r/C1s, Uegf, and Bmp 1)-EGF domain-containing protein 2 (SCUBE2) is a secreted, membrane-associated multidomain protein composed of five recognizable motifs: an NH(2)-terminal signal peptide sequence, nine copies of epidermal growth factor (EGF)-like repeats, a spacer region, three cysteine-rich repeats, and one CUB domain at the COOH terminus. Our previous clinical study showed that SCUBE2 may act as a novel breast tumor suppressor gene and serve as a useful prognostic marker. However, the specific domain responsible for its tumor suppressor activity and the precise mechanisms of its anti-tumor effect remain unknown. Using a combination of biochemical, molecular, and cell biology techniques, we further dissected the molecular functions and signal pathways mediated by the NH(2)-terminal EGF-like repeats or COOH-terminal CUB domain of SCUBE2. Independent overexpression of the NH(2)-terminal EGF-like repeats or COOH-terminal CUB domain resulted in suppression of MCF-7 breast cancer cell proliferation and reduced MCF-7 xenograft tumor growth in nude mice. Molecular and biochemical analyses revealed that the COOH-terminal CUB domain could directly bind to and antagonize bone morphogenetic protein activity in an autocrine manner, whereas the NH(2)-terminal EGF-like repeats could mediate cell-cell homophilic adhesions in a calcium-dependent fashion, interact with E-cadherin (a master tumor suppressor), and decrease the β-catenin signaling pathway. Together, our data demonstrate that SCUBE2 has growth inhibitory effects through a coordinated regulation of two distinct mechanisms: antagonizing bone morphogenetic protein and suppressing the β-catenin pathway in breast cancer cells.

Highlights

Signal peptide CUB-epidermal growth factor (EGF) domain-containing protein 2 (SCUBE2) belongs to a small, evolutionarily conserved SCUBE gene family
The COOHterminal CUB domain binds and antagonizes the proliferation signaling mediated by bone morphogenetic protein (BMP), whereas the NH2-terminal EGF-like repeats function as a Ca2ϩ-dependent homophilic adhesive module that interacts with E-cadherin and suppresses ␤-catenin signaling
Establishing Inducible MCF-7 Breast Cancer Cell Lines Expressing Signal peptide CUB-EGF domain-containing protein 2 (SCUBE2) Full-length, NH2, or COOH-terminal Mutant Protein—To further investigate the specific domain contributing to the breast tumor suppressor activity, we engineered three stable MCF-7 breast cancer cell lines under the control of an inducible promoter, the Tet-Off promoter, with the expression of: 1) the FL protein, 2) the NH2-terminal mutant encoding nine EGF-like repeats and the spacer region mimicking the ty97 allele in zebrafish [4, 5, 7], or 3) the COOH-terminal mutant (D4) containing the COOH-terminal fragment of three cysteine-rich motif repeats and the CUB domain

Summary

Introduction

Signal peptide CUB-EGF domain-containing protein 2 (SCUBE2) belongs to a small, evolutionarily conserved SCUBE gene family. Independent overexpression of the NH2-terminal EGF-like repeats or COOH-terminal CUB domain resulted in suppression of MCF-7 breast cancer cell proliferation and reduced MCF-7 xenograft tumor growth in nude mice.

Results

Conclusion