Shari A. Price-Schiavi

AN INTRAMEMBRANE MODULATOR OF THE ERBB2 RECEPTOR TYROSINE KINASE THAT POTENTIATES NEUREGULIN SIGNALING

The ErbB2 receptor tyrosine kinase plays a critical role in a variety of developmental processes, and its aberrant activation may contribute to the progression of some breast and ovarian tumors. ASGP2, a transmembrane glycoprotein found on the surface of the highly metastatic ascites 13762 rat mammary adenocarcinoma cell line, is constitutively associated with ErbB2 in these cells and in mammary tissue from pregnant rats. Expression studies indicate that ASGP2 interacts directly and specifically with ErbB2 through one of its epidermal growth factor-like domains and that the co-expression of the two proteins in the same cell dramatically facilitates their direct stable interaction. Ectopic expression of ASGP2 in human melanoma tumor cells potentiates the response of endogenous ErbB2 to the neuregulin-1 growth factor. These observations point to a novel intramembrane mechanism for the modulation of receptor tyrosine kinase activity.

Rat Muc4 (sialomucin complex) reduces binding of anti‐ErbB2 antibodies to tumor cell surfaces, a potential mechanism for herceptin resistance

Muc4 (also called sialomucin complex), the rat homolog of human MUC4, is a heterodimeric glycoprotein complex that consists of a peripheral O‐glycosylated mucin subunit, ASGP‐1, tightly but noncovalently linked to a N‐glycosylated transmembrane subunit, ASGP‐2. The complex is expressed in a number of normal, vulnerable epithelial tissues, including mammary gland, uterus, colon, cornea and trachea. Muc4/SMC is also overexpressed or aberrantly expressed on a number of human tumors including breast tumors. Overexpression of Muc4/SMC has been shown to block cell‐cell and cell‐matrix interactions, protect tumor cells from immune surveillance and promote metastasis. In addition, as a ligand for ErbB2, Muc4/SMC can potentiate phosphorylation of ErbB2 and potentially alter signals generated from this receptor. Using A375 human melanoma cells and MCF7 human breast adenocarcinoma cells stably transfected with tetracycline regulatable Muc4, we have investigated whether overexpression of Muc4/SMC can repress antibody binding to cell surface‐expressed ErbB2. Overexpression of Muc4/SMC does not affect the level of ErbB2 expression in either cell line, but it does reduce binding of a number of anti‐ErbB2 antibodies, including Herceptin. Interestingly, overexpression of ErbB2 does not block binding of other unrelated antibodies of the same isotype, suggesting that the reduction in ErbB2 antibody binding is due to complex formation of Muc4/SMC and ErbB2. Furthermore, capping of Muc4/SMC with anti‐Muc4/SMC antibodies reduces antibody binding to ErbB2 instead of increasing binding, again suggesting that reduced antibody binding to ErbB2 is due to steric hindrance from complex formation of Muc4/SMC and ErbB2. Thus, overexpression of Muc4/SMC on tumor cells may have both prognostic and therapeutic relevance.

Sialomucin Complex, a Heterodimeric Glycoprotein Complex EXPRESSION AS A SOLUBLE, SECRETABLE FORM IN LACTATING MAMMARY GLAND AND COLON

Ascites 13762 rat mammary adenocarcinoma cells express abundantly on their cell surfaces a heterodimeric glycoprotein complex composed of a sialomucin ascites sialoglycoprotein (ASGP)-1 and a transmembrane subunit ASGP-2. The latter, which contains two epidermal growth factor-like domains, binds the receptor tyrosine kinase p185neu, suggesting that the complex is bifunctional as well as heterodimeric. Immunoblot analyses using monoclonal antibodies prepared against the complex demonstrate high levels of expression in rat lactating mammary gland and colon. Immunolocalization studies with anti-ASGP-2 indicate that ASGP-2 is present in these two tissues in the apical regions of secretory epithelial cells. Both mammary gland and colon contain a soluble, secretable form of ASGP-2, which is not found in the ascites cells; milk and mammary gland also have the membrane form. Immunoblot analyses using a COOH-terminal-specific polyclonal antibody indicate that the soluble form of ASGP-2 is missing its COOH-terminal domains. Both the soluble and membrane forms of ASGP-2 are similar to the membrane-associated form from the 13762 adenocarcinoma with respect to Mr, antigenicity, and association with ASGP-1. The presence of ASGP-1 in milk suggests that it is a candidate for the uncharacterized high Mr milk mucin, MUCX. ASGP-2 expression is up-regulated in mammary gland during pregnancy, because it is undetectable in virgin and early pregnant rats but abundant in the gland from late pregnant and lactating animals. However, compared with the lactating mammary gland, the 13762 ascites cells overexpress ASGP-2 by more than 100-fold, which may contribute to their malignancy. These combined results indicate that sialomucin complex is a unique secreted product in the mammary gland and colon, whose behavior is different from that in the mammary ascites tumors, and which may play important roles in mammary and intestinal physiology.

Muc4/sialomucin complex, the intramembrane Er6B2 ligand, in cancer and epithelia: To protect and to survive

The membrane mucin Muc4, also called sialomucin complex (SMC), is a heterodimeric complex of two subunits, ASGP-1 and ASGP-2, derived from a single gene. It is produced by multiple epithelia in both membrane and soluble forms and serves as a protective agent for the epithelia. The membrane form of Muc4 acts as a steric barrier to the apical cell surface of epithelial or tumor cells. An important example is the uterus of the rat, in which Muc4 expression is downregulated for blastocyst implantation. The soluble form facilitates the protection and lubrication of epithelia by mucous gels composed of gel-forming mucins, as in the airway, where Muc4 is proposed to participate in mucociliary transport as a constituent of the periciliary fluid. The soluble form is also found in body fluids, such as milk, tears, and saliva. The transmembrane subunit ASGP-2 acts as an intramembrane ligand and activator for the receptor tyrosine kinase ErbB2. Formation of this ligand-receptor complex is proposed to repress apopotosis in epithelial and cancer cells in which the ligand-receptor complex is formed, providing a second type of cell protective mechanism. Muc4 expression is regulated in epithelial tissues in a cell- and tissue-specific manner during epithelial differentiation. In stratified epithelia, it is predominantly in the most superficial, differentiated layers, often coincident with ErbB2. Dysregulation of Muc4 expression may contribute to cell and tissue dysfunction, such as the proposed contribution of Muc4 to mammary tumor progression. These observations clearly show that Muc4 has multiple roles in epithelia, which may provide insights into aberrant behaviors of these tissues and their derivative carcinomas.

Muc4/Sialomucin Complex in the Mammary Gland and Breast Cancer

MUC4 is a one of the membrane mucins of the mucin gene (MUC) family, characterized by mucin tandem repeat domains and a transmembrane domain which associates it with the cell plasma membrane. Although MUC4 is encoded by a single gene, it is produced by epithelial cells as a heterodimer through a proteolytic cleavage mechanism. This heterodimer is found in both membrane and soluble forms associated with epithelia. Functionally, MUC4 is proposed to provide a protective mechanism for vulnerable epithelia, such as those of the airway, eye, female reproductive tract and mammary gland. The protective mechanism(s) may be highjacked by some carcinomas, such as those of the breast, to increase tumor progression. Two mechanisms are proposed to contribute to the MUC4 functions. First, MUC4 acts as an anti-adhesive or anti-recognition barrier at epithelial or tumor cell surfaces. Second, MUC4 can bind the receptor tyrosine kinase ErbB2 and alter its cellular signaling. Expression of MUC4 in mammary gland is repressed by posttranscriptional mechanisms involving basement membrane and TGF-β, which are relieved during pregnancy to permit secretion of MUC4 into milk. These mechanisms are also abrogated in some breast cancers, providing a scenario for promotion of tumor progression. These observations imply important functions for MUC4 in both normal mammary function and in breast cancer.

Sialomucin complex (rat Muc4) is regulated by transforming growth factor β in mammary gland by a novel post-translational mechanism.

Sialomucin complex (SMC, rat Muc4) is a heterodimeric glycoprotein complex consisting of a mucin subunit ASGP-1 (for ascitessialoglycoprotein-1) and a transmembrane subunit ASGP-2, produced from a single gene and precursor. SMC expression is tightly regulated in mammary gland; the level in lactating mammary gland is about 100-fold that in virgin gland. In rat mammary epithelial cells, SMC is post-transcriptionally regulated by Matrigel by inhibition of SMC precursor synthesis. SMC is also post-transcriptionally regulated by transforming growth factor-β (TGFβ). The repression of SMC expression by TGFβ is rapid, is independent of TGFβ-induced cell cycle arrest, and does not require new protein synthesis. Unlike Matrigel, TGFβ does not reduce SMC protein synthesis, as SMC precursor accumulation is equivalent in TGFβ-treated and untreated cells. Instead, SMC precursor in TGFβ-treated cells is more persistent and does not become processed as rapidly into mature ASGP-1 and ASGP-2, indicating that TGFβ disrupts processing of SMC precursor. These results indicate that SMC, a product of normal mammary gland and milk, is regulated by TGFβ by a novel post-translational mechanism. Thus, SMC is regulated by multiple post-transcriptional mechanisms, which serve to repress potential deleterious effects of overexpression.

Expression, location, and interactions of ErbB2 and its intramembrane ligand Muc4 (sialomucin complex) in rat mammary gland during pregnancy

Muc4 (also called Sialomucin complex) is a heterodimeric glycoprotein complex consisting of a peripheral O‐glycosylated subunit ASGP‐1 (ascites sialoglycoprotein‐1) tightly but non‐covalently bound to an N‐glycosylated transmembrane subunit ASGP‐2. Muc4/SMC can act as an intramembrane ligand for ErbB2 via an EGF‐like domain present in the transmembrane subunit. The complex is developmentally regulated in normal rat mammary gland and overexpressed in a number of mammary tumors. Overexpression of Muc4/SMC has been shown to block cell–cell and cell–matrix interactions, protect tumor cells from immune surveillance, promote metastasis, and protect from apoptosis. We have investigated whether Muc4/SMC and ErbB2 are co‐expressed and co‐localized in normal rat mammary gland and whether Muc4/SMC–ErbB2 complex formation is developmentally regulated in this tissue. Muc4/SMC and ErbB2 have different expression patterns and regulatory mechanisms in the developing rat mammary gland, but both are maximally expressed during late pregnancy and lactation. The two proteins form a complex in lactating mammary gland which is not detected in the virgin gland. Moreover, this complex does not contain ErbB3. ErbB2 is co‐localized with Muc4/SMC at the apical surfaces of ductal and alveolar cells in lactating gland; however, another form of ErbB2, recognized by a different antibody, localizes to the basolateral surfaces of these cells. ErbB2 phosphorylated on Tyr 1248 co‐localized with Muc4/SMC at the apical surface but not at the basolateral surfaces of these cells. To investigate the function of Muc4 in the mammary gland, transgenic mice were derived using an MMTV‐Muc4 construct. Interestingly, mammary gland development in the transgenic mice was aberrant, exhibiting a bifurcated pattern, including invasion down the blood vessel, similar to that exhibited by transgenic mice inappropriately expressing activated ErbB2 in the mammary gland. These data provide further evidence of the ability of Muc4/SMC to interact with ErbB2 and influence its behavior in normal epithelia.

Extracellular regulated kinase (ERK)-dependent regulation of sialomucin complex/rat Muc4 in mammary epithelial cells.

Sialomucin complex (SMC, rat Muc4) is a membrane mucin implicated in the protection of epithelia and the metastasis of some tumors. It is a heterodimeric complex, containing a mucin subunit with anti-adhesive activity and a transmembrane subunit with epidermal growth factor-like domains, one of which acts as an intramembrane ligand for ErbB2. Serum, insulin and insulin-like growth factor, but not epidermal growth factor, induce the expression of sialomucin complex in mammary epithelial cells. Induction correlates with sustained, but not transient, activation of extracellular-regulated protein kinase (ERK). MEK inhibitor U0126 blocked the induction, while activated MEK-1 transfected into a rat mammary adenocarcinoma cell line induced a sustained activation of ERK and up-regulated SMC/Muc4 expression. Northern and Western blotting indicated that up-regulation occurred concomitantly at the transcript and protein levels, both of which could be blocked by U0126. These results suggest that expression of SMC/Muc4 in mammary epithelial cells is regulated by selected growth factors through an ERK-dependent pathway at the transcript level.

Membrane Mucins and Breast Cancer.

Cancer Control 613 Membrane mucins have been implicated in a number of carcinomas. The prototype MUC1 is overexpressed in most breast cancers and is the target for both diagnostic assays and immunotherapy. Two attributes of membrane mucins contribute to the interest in their expression in tumors. First, their rigid structures provide an antirecognition function that alters cell adhesiveness and blocks killing by cells of the immune system. Second, they are proposed to be involved in cellular signaling processes that regulate cell behavior. These properties contribute to two of the most important aspects of tumor cell progression.

Sialomucin Complex (rat Muc4) Transmembrane Subunit Binds the Differentiation Marker Peanut Lectin in the Normal Rat Mammary Gland

Sialomucin complex (SMC, rat Muc4) is a heterodimeric glycoprotein composed of two subunits, the mucin component ASGP‐1 and the transmembrane subunit ASGP‐2. SMC/Muc4 is highly expressed on the surface of 13762 rat mammary adenocarcinoma cells at approximately 100 times the level found in the lactating mammary gland. Immunocytochemical staining of SMC/Muc4 in the developing rat mammary gland is localized to the apical membrane of the ductal epithelium. This staining pattern is similar to that for peanut lectin, a differentiation marker, which binds to cells expressing the disaccharide Thomsen–Friedenreich or TF antigen. Blotting of glycoproteins expressing the TF antigen from mammary tissues with peanut lectin detects a protein matching the migration of ASGP‐2. Analysis of immunoprecipitated SMC/Muc4 by peanut lectin blotting shows that the TF antigen is abundantly present on the ASGP‐2 subunit, hence the similarity of staining pattern with SMC/Muc4 antisera and peroxidase‐conjugated lectin in mammary tissues. The TF antigen is also present on ASGP‐2 of SMC/Muc4 produced by confluent cultures of Rama 37 rat mammary epithelial stem cells after their induction to an alveolar‐like phenotype with prolactin. These results indicate that the TF antigen is present on the ASGP‐2 transmembrane subunit of SMC/Muc4 from phenotypically normal tissues and cells, in contrast to malignant cells whose peanut lectin‐binding disaccharide is located on ASGP‐1.