Copine III interacts with ERBB2 and promotes tumor cell migration

Breast cancer is the most prevalent form of cancer in females: one of nine women develops breast cancer during her lifetime and it is predicted that one in 27 women will die as a result of this disease. Moreover, it is anticipated that with almost 30 % of females affected, breast cancer will be the most frequently diagnosed cancer in 2009 (www.cancer.org). Given these facts, much time and resources have been provided to research in the breast cancer area.
The ErbB2 receptor tyrosine kinase is one of the most-studied oncogenes in breast cancer as amplification and overexpression of the ERBB2 gene is known to occur in up to 25 % of all affected patients and is correlated with a highly aggressive disease and poor patient prognosis.
Our study focused on signaling molecules interacting with the C-terminal regulatory region of the ErbB2 receptor. We used T47D breast cancer cells metabolically labeled with SILAC to identify binding partners of the pTyr1248 site of ErbB2. Using a peptide affinity pull-down approach followed by quantitative mass spectrometry, we identified Copine III as a novel interaction partner of ErbB2-pTyr1248.
Copine III belongs to a family of Ca2+-dependent phospholipid binding proteins that is conserved from plants to humans. All copines carry two C2 domains followed by an A domain, similar to the von Willebrand A domain of integrins, in their C-terminus. Although Copine III is ubiquitously expressed, to date it has not been assigned a function downstream of ErbB2.
In this study we first analyzed the biochemical properties of Copine III and its interaction with ErbB2. We show that Copine III is a cytoplasmic protein that localizes to the nucleus and the plasma membrane in a Ca2+-dependent manner and upon stimulation of the cells with the ErbB ligand heregulin (HRG). We used FRET acceptor photobleaching to show that Copine III and ErbB2 not only co-localize in HRG-stimulated breast cancer cells, but also interact at the plasma membrane. This co-localization is blocked when the cells are treated with the ErbB2 inhibitor AEE788, implying that Copine III only interacts with phosphorylated active ErbB2.
The second goal of my studies was to place Copine III within a signaling pathway downstream of ErbB2. For this, we again used SILAC together with quantitative mass spectrometry and identified the scaffolding protein RACK1 as a binding partner of Copine III. We were able to show that Copine III, RACK1 and the adaptor molecule Shc form a complex with ErbB2 in HRG-stimulated cells. RACK1 has been implicated in focal-adhesion mediated cell migration and here we demonstrate that Copine III localizes to focal adhesions and is required for ErbB2-dependent cell migration. Moreover, knock-down of Copine III affects Src kinase activity and the subsequent phosphorylation of focal adhesion kinase, resulting in the observed defects in cellular migration. Thus Copine III is an important effector molecule in ErbB2-mediated cell migration.
Finally, we analyzed Copine III expression in the broader context of cancer, looking at carcinomas of the breast, prostate and ovary. In a set of 49 breast cancer tumor samples, 10 of the 11 cases with ERBB2 amplification display elevated levels of Copine III. This connected well with the protein expression levels of Copine III in a panel of breast cancer cell lines that also correlated with ErbB2 amplification. In published ovarian and prostate transcriptome studies, Copine III mRNA levels are upregulated in cancer as compared to normal tissue. Based on these findings, we performed immunohistochemistry (IHC) stainings of Copine III on breast, prostate and ovarian tissue microarrays. While some Copine III staining was evident in normal breast, normal prostate and ovarian tissues have very low levels of Copine-III. Strikingly, tumors of all three types showed higher Copine III levels.
To summarize, we present Copine III here for the first time as an interaction partner of the ErbB2 receptor. Copine III interacts with ErbB2 in a Ca2+- and HRG-dependent manner and is required for tumor cell migration. Furthermore, Copine III levels were found to be upregulated in tissue microarrays of breast, ovarian and prostate tumor tissue as compared to normal tissue.
Together, these findings imply a biological function for Copine III in cancer progression and suggest that further studies into the functions of Copine III are merited.