MUC1 is a large (>400 kDa), heavily glycosylated transmembrane protein that is aberrantly expressed on greater than 90% of human breast carcinomas and subsequent metastases. The precise function of MUC1 overexpression in tumorigenesis is unknown, although various domains of MUC1 have been implicated in cell adhesion, cell signaling, and immunoregulation. Stimulation of the MDA-MB-468 breast cancer line as well as mouse mammary glands with epidermal growth factor results in the co-immunoprecipitation of MUC1 with a tyrosine-phosphorylated protein of ϳ180 kDa. We have generated transgenic lines overexpressing full-length (MMF), cytoplasmic tail deleted (⌬CT), or tandem repeat deleted (⌬TR)-human MUC1 under the control of the mouse mammary tumor virus promoter to further examine the role of MUC1 in signaling and tumorigenesis. Immunoprecipitation experiments revealed that fulllength transgenic MUC1 physically associates with all four erbB receptors, and co-localizes with erbB1 in the lactating gland. Furthermore, we detected a sharp increase in ERK1/2 activation in MUC1 transgenic mammary glands compared with Muc1 null and wild-type animals. These results point to a novel function of increased MUC1 expression, potentiation of erbB signaling through the activation of mitogenic MAP kinase pathways.The transmembrane mucin MUC1 (DF3, CD227, episialin, PEM) is a heavily O-glycosylated protein expressed on most secretory epithelium, including the mammary gland as well as some hematopoetic cells. MUC1 is expressed abundantly in the lactating mammary gland in addition to being overexpressed in greater than 90% of human breast carcinomas and metastases (1). In the normal mammary gland, MUC1 is expressed mainly on the apical surface of glandular epithelium and is believed to play a role in anti-adhesion and immune protection (2-4). In breast cancer, MUC1 is overexpressed, underglycosylated, and apical localization is lost (2). Mice lacking Muc1 demonstrate no overt phenotypic developmental abnormalities in the mammary gland, but when crossed with the tumorigenic MMTV 1 -mTag transgenic line (5), mammary gland tumor growth was significantly slowed. Additionally, these Muc1-null/MMTVmTag transgenics demonstrated a trend toward decreased metastasis, showing that the absence of Muc1 results in both reduced tumor growth and spread (6). MUC1 is transcribed as a large precursor gene product, which, upon translation, is cleaved in the endoplasmic reticulum, yielding two separate proteins that form a heterodimeric complex, bound together by noncovalent interactions (7). The larger of the two components (the "mucin-like" subunit) contains most of the extracellular domain, including the signal sequence, tandem repeats, as well as some degenerate repeats. The tandem repeats consist of 30 to 90 repeat sequences of 20 amino acids, rich in serine and threonine residues. Approximately 50 -90% of the mass of MUC1 is derived from O-glycosylation that occurs on these amino acids (8)). The second component of the heterodimer consists of an extr...

MUC1 is aberrantly expressed in greater than 90% of all breast carcinomas, yet its function as a tumor antigen is not fully understood. Recently, studies have shown that MUC1 interacts with b-catenin, erbB receptors, src, GSK-3b and protein kinase Cd, possibly in a complex that promotes the disassembly of adherens junctions and the invasion of cells. Here we show that the deletion of Muc1 expression from MMTV-Wnt-1 transgenic mice results in a significant increase in the time to mammary gland tumor onset. Analysis of MMTV-Wnt-1 tumors on a wild-type Muc1 background shows a tumor-specific complex formation between Muc1 and b-catenin that can be observed in both the membrane and the cytoplasm of transformed epithelium. Analysis of primary human adenocarcinomas revealed that this MUC1/b-catenin interaction occurs in both primary and metastatic tumors, but is dramatically increased in metastatic lesions. Addition of MUC1-cytoplasmic domain peptides to the invasive MDA-MB-468 and MDA-MB-231 cell lines increases their invasive capability, and these peptides colocalize with both b-catenin and the focal adhesion protein vinculin, primarily at sites of membrane invasion into a collagen matrix. These data indicate a potential mechanism for MUC1 promotion of invasive tumorigenesis in the breast through the modulation of b-catenin localization and subsequent cytoskeletal dynamics.

MUC1 is a transmembrane mucin that was initially cloned from malignant mammary epithelial cells as a tumor antigen. More than 90% of human breast carcinomas overexpress MUC1. Numerous studies have demonstrated an interaction between MUC1 and other oncogenic proteins such as b-catenin, erbB receptors and c-Src, but a functional role for MUC1 in transformation has not been identified. We previously reported the development of transgenic mice that overexpress human MUC1 in the mouse mammary gland (MMTV-MUC1). Analysis of these transgenic mice at an early age demonstrated the ability of MUC1 to potentiate EGF-dependent activation of MAP kinase signaling pathways in the lactating mammary gland. We now report that multiparous MMTV-MUC1 transgenic mice stochastically develop unifocal mammary gland carcinomas late in life. Molecular analysis of these tumors shows a tumor-specific coimmunoprecipitation between MUC1 and b-catenin. Examination of the contralateral glands in MMTV-MUC1 transgenics demonstrates that the development of frank carcinomas is accompanied by a failure of multiparous glands to undergo postlactational involution. Furthermore, uniparous MMTV-MUC1 transgenic mice display decreased postlactational apoptosis, elevated whey acidic protein expression and aberrant pErk2 activation. These findings are the first to determine that MUC1 overexpression promotes in vivo transformation of the mammary gland.

No abstract

Anti-CD30 diabodies were engineered with two cysteine mutations for site-specific drug conjugation in each chain of these homodimeric antibody fragments. Diabodies were conjugated with f4 equivalents of the anti-tubulin drugs, monomethyl auristatin E or F, via a protease-cleavable dipeptide linker, to create the conjugates, diabody-vcE4 and diabody-vcF4, respectively. Diabody conjugation had only minor (<3-fold) effects on antigen binding. DiabodyvcF4 was potently cytotoxic against the antigen-positive cell lines, Karpas-299 (34 pmol/L IC 50 ) and L540cy (22 pmol/L IC 50 ), and was 8-and 21-fold more active than diabody-vcE4 against these cell lines, respectively. Clearance of diabody-vcF4 (99-134 mL/d/kg) was 5-fold slower than for the nonconjugated diabody in naive severe combined immunodeficient mice. Diabody-vcF4 had potent and dose-dependent antitumor activity against established Karpas-299 xenografts and gave durable complete responses at well-tolerated doses. Biodistribution experiments with diabody-[ 3 H]-vcF4 (0.72-7.2 mg/kg) in tumorbearing mice showed a dose-dependent increase in total auristatin accumulation in tumors (V520 nmol/L) and decrease in relative auristatin accumulation (V8.1 %ID/g), with peak localization at 4 to 24 h after dosing. Diabody-vcF4 had f4-fold lower cytotoxic activity than the corresponding IgG1-vcF4 conjugate in vitro. A similar potency difference was observed in vivo despite 25-to 34-fold faster clearance of diabody-vcF4 than IgG1-vcF4.