Loss of Nrdp1 Enhances ErbB2/ErbB3–Dependent Breast Tumor Cell Growth Lily Yen, 1 Zhongwei Cao, 1 Xiuli Wu, 1 Ellen R.Q. Ingalla, 1 Colin Baron, 2 Lawrence J.T. Young, 3 Jeffrey P. Gregg, 2 Robert D. Cardiff, 2,3 Alexander D. Borowsky, 2,3 Colleen Sweeney, 1 and Kermit L. Carraway III 1 1 University of California Davis Cancer Center; 2 Department of Pathology and Laboratory Medicine, School of Medicine; and 3 Center for Comparative Medicine, University of California, Davis, Sacramento, California Abstract Dysregulation of ErbB receptor tyrosine kinases is thought to promote mammary tumor progression by stimulating tumor cell growth and invasion. Overexpression and aberrant activation of ErbB2/HER2 confer aggressive and malignant characteristics to breast cancer cells, and patients displaying ErbB2-amplified breast cancer face a worsened prognosis. Recent studies have established that ErbB2 and ErbB3 are commonly co-overexpressed in breast tumor cell lines and in patient samples. ErbB2 heterodimerizes with and activates the ErbB3 receptor, and the two receptors synergize in promoting growth factor–induced cell proliferation, transformation, and invasiveness. Our previous studies have shown that the neuregulin receptor degradation protein-1 (Nrdp1) E3 ubiq- uitin ligase specifically suppresses cellular ErbB3 levels by marking the receptor for proteolytic degradation. Here, we show that overexpression of Nrdp1 in human breast cancer cells results in the suppression of ErbB3 levels, accompanied by the inhibition of cell growth and motility and the attenuation of signal transduction pathways. In contrast, either Nrdp1 knockdown or the overexpression of a dominant- negative form enhances ErbB3 levels and cellular prolifera- tion. Additionally, Nrdp1 expression levels inversely correlate with ErbB3 levels in primary human breast cancer tissue and in a mouse model of ErbB2 mammary tumorigenesis. Our observations suggest that Nrdp1-mediated ErbB3 degradation suppressescellulargrowthandmotility,andthatNrdp1lossin breasttumorsmaypromotetumorprogressionbyaugmenting ErbB2/ErbB3 signaling. (Cancer Res 2006; 66(23): 11279-86) Introduction The ErbB family of receptor tyrosine kinases regulates a number of cellular processes, such as proliferation, differentiation, cell survival, migration, and invasion (1). Members of this family include the epidermal growth factor receptor (ErbB1/HER1), ErbB2 (HER-2/neu ), ErbB3 (HER3), and ErbB4 (HER4). Aberrant expres- sion of these receptors is commonly found in human cancers (1–3) and is associated with aggressive disease (1). The expression of multiple ErbB family members has been observed in a number of cancers, including breast (4, 5), and co-overexpression of ErbB2 or ErbB3 is significantly associated with decreased survival (6–10). Both the diversity and hence complexity of the ErbB signaling network is mediated by the existence of multiple ligands, each with specificity towards distinct members of the ErbB family. The ErbB receptors take part in a complex process of combinatorial interactions through the formation of ligand-induced homodimers and heterodimers between the different family members (11–13), which in turn activate distinct signaling pathways (11, 12). The ErbB3 receptor has impaired kinase activity (14), whereas the ErbB2 receptor has no known ligand. In this respect, both receptors must signal in the context of a receptor heterodimer. Binding of the neuregulin-1 (NRG1) growth factor to its cognate ErbB3 receptor results in preferential activation of ErbB2/ErbB3 heterodimers (15), thus initiating extracellular-related kinase/mitogen-activated protein kinase (ERK/MAPK) and phosphatidylinositol 3-kinase (PI3K) pathway activation (16–19). Both the ERK (20–22) and PI3K (23–26) pathways have been implicated in cellular motility and invasion, cell survival, and proliferation; thus, they are critical mediators of the aggressive and invasive breast cancer phenotypes resulting from ErbB receptor activation. Indeed, it is now clear that the ErbB2/ErbB3 receptor pair forms the most potent mitogenic (27) and transforming (17, 28) receptor complex. ErbB2-positive tumors were found to have elevated levels of ErbB3 phosphorylation (17), suggesting that ErbB2 recruitment of ErbB3 contributes to malignant growth. Furthermore, in tumors from transgenic mice generated by expressing an active mutant of Neu/ErbB2 (29) or in cell lines derived from tumors borne of transgenic mice overexpressing wild-type Neu/ErbB2 (30), ErbB3 overexpression and activation is also observed. Moreover, ErbB2 and ErbB3 coexpression has been found in human breast cancer cells lines (4, 19) and primary human breast cancer (6, 31), with coexpression correlating with even further reduced patient survival compared with expression of ErbB2 or ErbB3 alone (10). ErbB2/ ErbB3 heterodimers have also been shown to be involved in NRG- mediated motility and invasion (32–34). Taken together, these observations suggest that there may be an advantage for both receptors to be activated in tumor cells to promote breast tumor growth and progression. Although the mechanisms by which overexpressed and aber- rantly activated ErbB receptors contribute to tumor progression are coming into focus, cellular mechanisms controlling receptor protein levels in tumors remain largely unexplored. For example, the loss of specific protein degradation pathways could play a significant role in augmenting ErbB receptor levels in tumors (35). The RING finger E3 ubiquitin ligase neuregulin receptor degrada- tion protein-1 (Nrdp1) associates with ErbB3 in an activation- independent manner (36) and is believed to be involved in its trafficking or localization. Nrdp possesses ubiquitin ligase activity towards ErbB3 in vitro (36, 37), suggesting that it functions in the Requests for reprints: Kermit L. Carraway III, University of California Davis Cancer Center, University of California Davis Medical Center Research Building III, 4645 2nd Avenue, Sacramento, CA 95817-2305. Phone: 916-734-3114; Fax: 916-734-0190; E-mail: klcarraway@ucdavis.edu. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-2319 www.aacrjournals.org 11279 Cancer Res 2006; 66: (23). December 1, 2006 Research Article Research. on December 29, 2015. © 2006 American Association for Cancer cancerres.aacrjournals.org Downloaded from