Sandra J. Gendler

MUC1, The Renaissance Molecule

MUC1 is a large, heavily glycosylated mucin expressed on the apical surfaces of most simple, secretory epithelia including the mammary gland, gastrointestinal, respiratory, urinary and reproductive tracts. Although MUC1 was thought to be an epithelial-specific protein, it is now known to be expressed on a variety of hematopoietic cells as well. Mucins function in protection and lubrication of epithelial surfaces. Transmembrane mucins, which contain cytoplasmic tail domains, appear to have additional functions through their abilities to interact with many proteins involved in signal transduction and cell adhesion. The goal of this review is to highlight recent discoveries that suggest that MUC1 may be a multifunctional protein, located on the surfaces of cells as a sensor of the environment, poised to signal to the interior when things go awry.

Abnormal Heart Rate Regulation in GIRK4 Knockout Mice

Acetylcholine (ACh) released from the stimulated vagus nerve decreases heart rate via modulation of several types of ion channels expressed in cardiac pacemaker cells. Although the muscarinic-gated potassium channel I(KACh) has been implicated in vagally mediated heart rate regulation, questions concerning the extent of its contribution have remained unanswered. To assess the role of I(KACh) in heart rate regulation in vivo, we generated a mouse line deficient in I(KACh) by targeted disruption of the gene coding for GIRK4, one of the channel subunits. We analyzed heart rate and heart rate variability at rest and after pharmacological manipulation in unrestrained conscious mice using electrocardiogram (ECG) telemetry. We found that I(KACh) mediated approximately half of the negative chronotropic effects of vagal stimulation and adenosine on heart rate. In addition, this study indicates that I(KACh) is necessary for the fast fluctuations in heart rate responsible for beat-to-beat control of heart activity, both at rest and after vagal stimulation. Interestingly, noncholinergic systems also appear to modulate heart activity through I(KACh). Thus, I(KACh) is critical for effective heart rate regulation in mice.

The epithelial mucin, MUC1, of milk, mammary gland and other tissues

MUC1 is a mucin-type glycoprotein that is integrally disposed in the apical plasma membrane of the lactating epithelial cell and protrudes from the cell surface into the alveolar lumen where milk is stored. Envelopment of milk fat globules by this membrane accomplishes their secretion and conveys MUC1 into milk. The human form of this mucin has been detected in many other organs, tissues and body fluids. It projects from the cell surface as long filaments. In the human and a number of other species, MUC1 is polymorphic due to variable numbers of a tandemly repeated segment 20 amino acids in length. The individual codominantly expresses two alleles for the mucin so that differences in its size among individuals and between the two forms of an individual are observed. The tandem repeats are rich in serines and threonines which serve as O-glycosylation sites. Carbohydrate content of MUC1, as isolated from milk of human, bovine and guinea pig, is approximately 50%. The oligosaccharides carry substantial sialic acid at their termini and this accounts for two putative functions of this mucin, i.e., to keep ducts and lumens open by creating a strong negative charge on the surface of epithelial cells which would repel opposite sides of a vessel, and to bind certain pathogenic microorganisms. MUC1 is protease resistant (trypsin, chymotrypsin and pepsin) and large fragments of it can be found in the feces of some but not all breast-fed infants. MUC1 has a highly varied structure because of its polymorphism, qualitative and quantitative variations in its glycosylation between tissues, individuals and species, and differences due to divergence in the nucleotide sequences among species. Sequencing of the MUC1 gene for various species is showing promise of revealing unique evolutionary relationships and has already indicated conserved aspects of the molecule that may be functionally important. Among these are positions of serine, threonine and proline in the tandem repeats and a high degree of homology in the transmembrane and cytoplasmic segments of the molecule.

Induction of Antitumor Immunity by Vaccination of Dendritic Cells Transfected with MUC1 RNA

Dendritic cells (DC) are potent APCs. In this study, murine bone marrow-derived DC were transfected with RNA encoding the MUC1 Ag that is aberrantly overexpressed in human breast and other carcinomas. The MUC1 RNA-transfected DC exhibited cell surface expression of MUC1 and costimulatory molecules. After injection at the base of the tail, the transfected DC were detectable in inguinal lymph nodes by dual immunochemical staining. Vaccination of wild-type mice with MUC1 RNA-transfected DC induced anti-MUC1 immune responses against MUC1-positive MC38/MUC1, but not MUC1-negative, tumor cells. Mice immunized with the transfected DC were protected against challenge with MC38/MUC1 tumor cells. Furthermore, mice with established MC38/MUC1 tumors were eliminated after receiving the vaccination. CTLs isolated from mice immunized with the transfected DC exhibited specific cytolytic activity against MC38/MUC1 tumor cells. In contrast to these findings, there was little if any anti-MUC1 immunity induced with the transfected DC in MUC1 transgenic (MUC1.Tg) mice. However, coadministration of the transfected DC and IL-12 reversed the unresponsiveness to MUC1 Ag in MUC1.Tg mice and induced MUC1-specific immune responses. These findings demonstrate that vaccination of DC transfected with MUC1 RNA and IL-12 reverses tolerance to MUC1 and induces immunity against MUC1-positive tumors.

Importance and regulation of the colonic mucus barrier in a mouse model of colitis

The colonic mucus layer serves as an important barrier and prevents colonic bacteria from invading the mucosa and cause inflammation. The regulation of colonic mucus secretion is poorly understood. The aim of this study was to investigate the role of the mucus barrier in induction of colitis. Furthermore, regulation of mucus secretion by luminal bacterial products was studied. The colon of anesthetized Muc2(-/-), Muc1(-/-), wild-type (wt), and germ-free mice was exteriorized, the mucosal surface was visualized, and mucus thickness was measured with micropipettes. Colitis was induced by DSS (dextran sodium sulfate, 3%, in drinking water), and disease activity index (DAI) was assessed daily. The colonic mucosa of germ-free and conventionally housed mice was exposed to the bacterial products LPS (lipopolysaccharide) and PGN (peptidoglycan). After DSS induction of colitis, the thickness of the firmly adherent mucus layer was significantly thinner after 5 days and onward, which paralleled the increment of DAI. Muc2(-/-) mice, which lacked firmly adherent mucus, were predisposed to colitis, whereas Muc1(-/-) mice were protected with significantly lower DAI by DSS compared with wt mice. The mucus barrier increased in Muc1(-/-) mice in response to DSS, whereas significantly fewer T cells were recruited to the inflamed colon. Mice housed under germ-free conditions had an extremely thin adherent colonic mucus layer, but when exposed to bacterial products (PGN or LPS) the thickness of the adherent mucus layer was quickly restored to levels observed in conventionally housed mice. This study demonstrates a correlation between decreasing mucus barrier and increasing clinical symptoms during onset of colitis. Mice lacking colonic mucus (Muc2(-/-)) were hypersensitive to DSS-induced colitis, whereas Muc1(-/-) were protected, probably through the ability to increase the mucus barrier but also by decreased T cell recruitment to the afflicted site. Furthermore, the ability of bacteria to regulate the thickness of the colonic mucus was demonstrated.

Immune recognition of tumor-associated mucin MUC1 is achieved by a fully synthetic aberrantly glycosylated MUC1 tripartite vaccine

The mucin MUC1 is typically aberrantly glycosylated by epithelial cancer cells manifested by truncated O-linked saccharides. The resultant glycopeptide epitopes can bind cell surface major histocompatibility complex (MHC) molecules and are susceptible to recognition by cytotoxic T lymphocytes (CTLs), whereas aberrantly glycosylated MUC1 protein on the tumor cell surface can be bound by antibodies to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). Efforts to elicit CTLs and IgG antibodies against cancer-expressed MUC1 have not been successful when nonglycosylated MUC1 sequences were used for vaccination, probably due to conformational dissimilarities. Immunizations with densely glycosylated MUC1 peptides have also been ineffective due to impaired susceptibility to antigen processing. Given the challenges to immuno-target tumor-associated MUC1, we have identified the minimum requirements to consistently induce CTLs and ADCC-mediating antibodies specific for the tumor form of MUC1 resulting in a therapeutic response in a mouse model of mammary cancer. The vaccine is composed of the immunoadjuvant Pam3CysSK4, a peptide Thelper epitope and an aberrantly glycosylated MUC1 peptide. Covalent linkage of the three components was essential for maximum efficacy. The vaccine produced CTLs, which recognized both glycosylated and nonglycosylated peptides, whereas a similar nonglycosylated vaccine gave CTLs which recognized only nonglycosylated peptide. Antibodies elicited by the glycosylated tripartite vaccine were significantly more lytic compared with the unglycosylated control. As a result, immunization with the glycosylated tripartite vaccine was superior in tumor prevention. Besides its own aptness as a clinical target, these studies of MUC1 are likely predictive of a covalent linking strategy applicable to many additional tumor-associated antigens.

MUC1 alters β-catenin-dependent tumor formation and promotes cellular invasion

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 β-catenin, erbB receptors, src, GSK-3β and protein kinase Cδ, 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 β-catenin that can be observed in both the membrane and the cytoplasm of transformed epithelium. Analysis of primary human adenocarcinomas revealed that this MUC1/β–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 β-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 β-catenin localization and subsequent cytoskeletal dynamics.

MUC1 enhances invasiveness of pancreatic cancer cells by inducing epithelial to mesenchymal transition

Increased motility and invasiveness of pancreatic cancer cells are associated with epithelial to mesenchymal transition (EMT). Snai1 and Slug are zinc-finger transcription factors that trigger this process by repressing E-cadherin and enhancing vimentin and N-cadherin protein expression. However, the mechanisms that regulate this activation in pancreatic tumors remain elusive. MUC1, a transmembrane mucin glycoprotein, is associated with the most invasive forms of pancreatic ductal adenocarcinomas (PDA). In this study, we show that over expression of MUC1 in pancreatic cancer cells triggers the molecular process of EMT, which translates to increased invasiveness and metastasis. EMT was significantly reduced when MUC1 was genetically deleted in a mouse model of PDA or when all seven tyrosines in the cytoplasmic tail of MUC1 were mutated to phenylalanine (mutated MUC1 CT). Using proteomics, RT–PCR and western blotting, we revealed a significant increase in vimentin, Slug and Snail expression with repression of E-Cadherin in MUC1-expressing cells compared with cells expressing the mutated MUC1 CT. In the cells that carried the mutated MUC1 CT, MUC1 failed to co-immunoprecipitate with β-catenin and translocate to the nucleus, thereby blocking transcription of the genes associated with EMT and metastasis. Thus, functional tyrosines are critical in stimulating the interactions between MUC1 and β-catenin and their nuclear translocation to initiate the process of EMT. This study signifies the oncogenic role of MUC1 CT and is the first to identify a direct role of the MUC1 in initiating EMT during pancreatic cancer. The data may have implications in future design of MUC1-targeted therapies for pancreatic cancer.

Reduced T-Cell and Dendritic Cell Function Is Related to Cyclooxygenase-2 Overexpression and Prostaglandin E2 Secretion in Patients With Breast Cancer

Background: In several neoplastic diseases, including breast cancer, immunosuppression correlates with disease stage, progression, and outcome. Thus, thorough analysis of immune parameters in breast cancer patients may be beneficial in designing effective anticancer immune-based therapies.Methods: We investigated dendritic cell and T-cell function in breast cancer patients at various stages of the disease and in age-matched controls. We also evaluated cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) levels within the tumor milieu and in the circulation.Results: T cells from cancer patients showed decreased proliferation in response to CD3 antibody stimulation. Analysis of T-cell helper type 1 and 2 cytokines revealed reduced levels of interferon-γ, tumor necrosis factor-α, interleukin (IL)-12, and IL-2 and increased levels of IL-10 and IL-4. Dendritic cells from these patients showed significantly reduced expression of co-stimulatory molecules (B7 and CD40) and demonstrated reduced phagocytic ability, reduced antigen presentation to T cells, and reduced ability to mature in response to lipopolysaccharide. Data revealed increased synthesis of PGE2, an immune suppressor, along with increased expression of COX-2, a key regulator of PGE2 synthesis.Conclusions: COX-2–induced PGE2 may contribute to immunosuppression and may directly block antitumor immunity while promoting tumor growth, providing us with the rationale for using COX-2 inhibition combined with immunotherapy.

MUC1 overexpression results in mammary gland tumorigenesis and prolonged alveolar differentiation

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 β-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 β-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.