Supplementary Materials Supporting Information supp_293_8_2850__index. pathway. Selective blockade of STAT5 phosphorylation by pimozide, a small-molecule inhibitor, markedly reduced the production of the EGF family growth factors and inhibited PRL-induced tumor cell proliferation gene product with cell division, cell cycle, and cell proliferation (52). Structurally, CUZD1 is composed of two tandem CUB domains, a ZP domain, and a putative transmembrane domain (53, 54). Presently, little information exits describing the function of these domains, but they are often found in proteins that regulate developmental processes (55, 56). Studies illustrating the presence of CUZD1 in specific tissues Flunixin meglumine have provided some functional analysis. We previously reported that CUZD1, also known as estrogen-regulated gene 1 (ERG1), is an estrogen-regulated gene in the rodent reproductive tract and is important for mammary epithelial cell proliferation during pregnancy and lactation (51, 53). Additionally, expression of has been identified in the pancreas, epididymis, human ovarian cancer cells, and human embryonic stem cells (57,C61). Leong (61, 62) demonstrated the importance of CUZD1 in cell growth and Flunixin meglumine proliferation of a human ovarian cancer cell line and proposed a potential role of CUZD1 in chemotherapeutic resistance. Efforts have also been made to develop serum-based Rabbit Polyclonal to PDK1 (phospho-Tyr9) assays using CUZD1 as a biomarker for ovarian cancer and pancreatic cancer; however, controversial reports support the need for additional studies (61, 63,C69). Our recent work fills a gap in the body of knowledge surrounding CUZD1 by detailing the molecular signaling pathway of CUZD1-induced proliferation in mammary epithelial cells (51). The expression may lead to excessive proliferation of the mammary epithelium, leading to tumorigenesis. In this study, we tested the concept that overexpression of CUZD1 in mammary epithelial cells may drive constitutive activation of the STAT5 pathway and inappropriate stimulation of the EGF family growth factor pathways, leading to uncontrolled cell proliferation. We demonstrate that such dysregulation of CUZD1 and its downstream STAT5 and EGF receptor pathways indeed leads to Flunixin meglumine breast carcinoma. Furthermore, we provide evidence that pimozide, a selective inhibitor of STAT5 phosphorylation, can suppress CUZD1/STAT5-powered mammary epithelial tumorigenesis and proliferation, presenting it being a potential healing drug focus on in breast cancers in which the STAT5 pathway plays a major role. Results Overexpression of Cuzd1 leads to transformation of HC11 cells To test whether the overexpression of promotes transformation of mammary epithelial cells, we employed HC11 cells, a non-transformed mammary epithelial cell line derived from pregnant BALB/c mice. As described previously, a lentiviral expression vector harboring a full-length cDNA encoding or -galactosidase (control) was integrated into HC11 cells to generate stable cell lines which constitutively express elevated levels of (HC11-Cuzd1) or -galactosidase (HC11-LacZ) (51). Western blot analysis indicated that HC11-Cuzd1 cells overexpress CUZD1 about 2-fold over the HC11-LacZ control cells (51). These cells also expressed prolactin receptor and low levels of estrogen receptor and progesterone receptor. We then subjected these cells to a cell invasion assay using Boyden chambers. The HC11-Cuzd1 cells exhibited enhanced motility Flunixin meglumine and were able to migrate across a barrier, whereas control HC11-LacZ cells failed to penetrate the membrane (Fig. 1in HC11 mammary epithelial cells altered their growth and migratory properties, two important hallmarks of precancerous cells. Open in a separate window Physique 1. Overexpression of leads to enhanced motility and anchorage-independent growth of HC11 cells. leads to enhanced motility of HC11 cells. Serum-starved MDA-MB-231 cells (positive control), HC11-LacZ, or HC11-Cuzd1 cells were placed in Boyden chambers and allowed to migrate toward 10% FBS for 72 h. The number of invading cells was quantified using CyQuant fluorescence labeling and compared with corresponding cells.