MUC5B Leads to Aggressive Behavior of Breast Cancer MCF7 Cells

The mucin MUC5B has a critical protective function in the normal lung, salivary glands, esophagus, and gallbladder, and has been reported to be aberrantly expressed in breast cancer, the second leading cause of cancer-related deaths among women worldwide. To understand better the implication of MUC5B in cancer pathogenesis, the luminal human breast cancer cell line MCF7 was transfected with a vector encoding a recombinant mini-mucin MUC5B and was then infected with a virus to deliver a short hairpin RNA to knock down the mini-mucin. The proliferative and invasive properties in Matrigel of MCF7 subclones and subpopulations were evaluated in vitro. A xenograft model was established by subcutaneous inoculation of MCF7 clones and subpopulations in SCID mice. Tumor growth was measured, and the tumors and metastases were assessed by histological and immunological analysis. The mini-mucin MUC5B promoted MCF7 cell proliferation and invasion in vitro. The xenograft experiments demonstrated that the mini-mucin promoted tumor growth and MCF7 cell dissemination. In conclusion, MUC5B expression is associated with aggressive behavior of MCF7 breast cancer cells. This study suggests that MUC5B may represent a good target for slowing tumor growth and metastasis.     

MUC1-specific immune responses in human MUC1 transgenic mice immunized with various human MUC1 vaccines

Analyses of MUC1-specific cytotoxic T cell precursor (CTLp) frequencies were performed in mice immunized with three different MUC1 vaccine immunotherapeutic agents. Mice were immunized with either a fusion protein comprising MUC1 and glutathione S-transferase (MUC1-GST), MUC1-GST fusion protein coupled to mannan (MFP) or with a recombinant vaccinia virus expressing both MUC1 and interleukin-2. Mouse strain variations in immune responsiveness have been observed with these vaccines. We have constructed mice transgenic for the human MUC1 gene to study MUC1-specific immune responses and the risk of auto-immunity following MUC1 immunization. Transgenic mice immunized with MUC1 were observed to be partially tolerant in that the MUC1-specific antibody response is lower than that observed in syngeneic but non-transgenic mice. However, a significant MUC1-specific CTLp response to all three vaccines was observed, indicating the ability to overcome T cell, but to a lesser extent B cell, tolerance to MUC1 in these mice. Histological analysis indicates no evidence of auto-immunity to the cells expressing the human MUC1 molecule. These results suggest that it is possible to generate an immune response to a cancer-related antigen without damage to normal tissues expressing the antigen. Received: 7 July 1999 / Accepted: 26 August 1999     

Distinct localization of MUC5B glycoforms in the human salivary glands

Salivary mucins, encoded by the MUC5B gene, make up a heterogeneous family of molecules, which are secreted by several glands, including the submandibular, sublingual, and palatine glands. Previous studies have shown that heterogeneity in the salivary mucin population is related to its multiglandular origin. In the present study we address the question to what extent the mucin (MUC5B) population from a single human salivary gland is made up of different glycoforms. Using monoclonal antibodies to defined protein and sulfated carbohydrate epitopes specific to MUC5B, we conduct an immunohistochemical study of different salivary gland types, including submandibular, sublingual, and labial glands. In all tissues studied we found a mosaic expression pattern of sulfo-Lewis a antigen, recognized by mAb F2, which in salivary glands is exclusively present on MUC5B. On the other hand, mucous acini were uniformly labeled by mAb EU-MUC5Bb, evoked against a peptide-stretch of the tandem repeat region of MUC5B. Double staining with both antibodies confirmed the presence of MUC5B-positive/sulfo-Lewis a-positive cells, as well as MUC5B-positive/sulfo-Lewis a-negative cells within one glandular unit. These results indicate that one and the same salivary gland synthesizes different MUC5B glycoforms.     

MDM2 is required for suppression of apoptosis by activated Akt1 in salivary acinar cells

Chronic damage to the salivary glands is a common side effect following head and neck irradiation. It is hypothesized that irreversible damage to the salivary glands occurs immediately after radiation; however, previous studies with rat models have not shown a causal role for apoptosis in radiation-induced injury. We report that etoposide and gamma irradiation induce apoptosis of salivary acinar cells from FVB control mice in vitro and in vivo; however, apoptosis is reduced in transgenic mice expressing a constitutively activated mutant of Akt1 (myr-Akt1). Expression of myr-Akt1 in the salivary glands results in a significant reduction in phosphorylation of p53 at serine(18), total p53 protein accumulation, and p21(WAF1) or Bax mRNA following etoposide or gamma irradiation of primary salivary acinar cells. The reduced level of p53 protein in myr-Akt1 salivary glands corresponds with an increase in MDM2 phosphorylation in vivo, suggesting that the Akt/MDM2/p53 pathway is responsible for suppression of apoptosis. Dominant-negative Akt blocked phosphorylation of MDM2 in salivary acinar cells from myr-Akt1 transgenic mice. Reduction of MDM2 levels in myr-Akt1 primary salivary acinar cells with small interfering RNA increases the levels of p53 protein and renders these cells susceptible to etoposide-induced apoptosis in spite of the presence of activated Akt1. These results indicate that MDM2 is a critical substrate of activated Akt1 in the suppression of p53-dependent apoptosis in vivo.     

MUC16 in the lacrimal apparatus

The aim of the present study was to determine the possible expression of the mucin MUC16 in the lacrimal apparatus. Expression and distribution of MUC16 in lacrimal gland, accessory lacrimal glands, and nasolacrimal ducts was monitored by RT-PCR and immunohistochemistry. MUC16 was expressed and detected in all tissues investigated. Comparable to conjunctiva and cornea it was membrane-anchored in accessory lacrimal glands whereas in lacrimal gland acinar cells and columnar cells of the nasolacrimal ducts it was stored in intracytoplasmic vesicles without membrane-association. Subepithelial serous glands of the nasolacrimal ducts revealed staining of the secretion product. Intracelluar production of MUC16 is present in lacrimal gland and epithelial cells of the nasolacrimal ducts but it is not clear whether this MUC16 is secreted. MUC16 seems to be shedded or secreted from the epithelial surface of subepithelial serous glands of the nasolacrimal ducts. Our results show that MUC16 is present in the whole lacrimal apparatus. Its distribution pattern suggests different physiological functions with regard to tear film physiology and tear outflow. Moreover, the results demonstrate the existence of so far not recognized qualitative differences in the secretion product of main lacrimal gland and accessory lacrimal glands (glands of Krause).     

Degradation of MUC7 and MUC5B in Human Saliva

Background

Two types of mucins, MUC7 and MUC5B constitute the major salivary glycoproteins, however their metabolic turnover has not been elucidated in detail to date. This study was conducted to examine turnover of MUC7 and MUC5B in saliva, by focusing on the relationship between their deglycosylation and proteolysis.

Methodology/Principal Findings

Whole saliva samples were collected from healthy individuals and incubated at 37°C in the presence of various protease inhibitors, sialidase, or a sialidase inhibitor. General degradation patterns of salivary proteins and glycoproteins were examined by SDS-polyacrylamide-gel-electrophoresis. Furthermore, changes of molecular sizes of MUC7 and MUC5B were examined by Western blot analysis. A protein band was identified as MUC7 by Western blot analysis using an antibody recognizing an N-terminal epitope. The MUC7 signal disappeared rapidly after 20-minutes of incubation. In contrast, the band of MUC7 stained for its carbohydrate components remained visible near its original position for a longer time indicating that the rapid loss of Western blot signal was due to the specific removal of the N-termimal epitope. Pretreatment of saliva with sialidase facilitated MUC7 protein degradation when compared with samples without treatment. Furthermore, addition of sialidase inhibitor to saliva prevented proteolysis of N-terminus of MUC7, suggesting that the desialylation is a prerequisite for the degradation of the N-terminal region of MUC7. The protein band corresponding to MUC5B detected in both Western blotting and glycoprotein staining showed little sign of significant degradation upon incubation in saliva up to 9 hours.

Conclusions/Significance

MUC7 was highly susceptible to specific proteolysis in saliva, though major part of MUC5B was more resistant to degradation. The N-terminal region of MUC7, particularly sensitive to proteolytic degradation, has also been proposed to have distinct biological function such as antibacterial activities. Quick removal of this region may have biologically important implication.     

Tumor-specific immunity in MUC1.Tg mice induced by immunization with peptide vaccines from the cytoplasmic tail of CD227 (MUC1)

Purpose: CD227 (MUC1), a membrane-associated glycoprotein expressed by many types of ductal epithelia, including pancreas, breast, lung, and gastrointestinal tract, is overexpressed and aberrantly glycosylated by malignant cells. We sought to define epitopes on MUC1 recognized by the different cell-mediated immune responses by an in vivo assay. Epitopes identified by this assay were evaluated for efficacy to protect mice transgenic for human MUC1 (MUC1.Tg) against MUC1-expressing tumor growth. Methods: We investigated contributions of the tandem repeat (TR) and the cytoplasmic tail (CT) of MUC1 to the MUC1-specific immunological rejection of tumor cells. MUC1 cDNA constructs, in which the TR region was deleted or the CT was truncated, were transfected into two different murine tumor cell lines (B16 and Panc02), which were used to challenge mice and evaluate immunological rejection of the tumors. We used tumor rejection in vivo to define epitopes on the TR and CT of MUC1 recognized by T cell–mediated immune responses in a preclinical murine model. Results: Our findings demonstrated that the TR and a portion of the MUC1 CT contributed to CD4⁺ T cell rejection of MUC1-expressing B16 tumor cells, but not rejection of MUC1-expressing Panc02 tumor cells. A separate epitope in the CT of MUC1 was necessary for CD8⁺ T cell rejection of Panc02 tumor cells. Based on these studies, we sought to evaluate the efficacy of immunizing mice transgenic for (and immunologically tolerant to) human MUC1 with peptides derived from the amino acid sequence of the CT of MUC1. Results showed that survival can be significantly prolonged in vaccinated MUC1.Tg mice challenged with MUC1-expressing tumor cells, without induction of autoimmune responses. Conclusions: These studies demonstrated that MUC1 peptides may be utilized as an effective anticancer immunotherapeutic, and confirmed the importance of immunogenic epitopes outside of the TR.Abbreviations aa Amino acid - CT Cytoplasmic tail - IFN- Interferon gamma - MUC1.Tg MUC1 transgenic mice - TR Tandem repeat - wt Wild-type C57BL/6 mice This work was supported by National Institutes of Health grants CA72712 and CA57362 (M.A.H.), National Institutes of Health training grant CA09476 (K.G.K., A.J.G, and M.L.V.), and fellowship awards from the University of Nebraska Medical Center (to K.G.K. and M.L.V).     

Local Effects of Regulatory T Cells in MUC1 Transgenic Mice Potentiate Growth of MUC1 Expressing Tumor Cells In Vivo

MUC1 transgenic (MUC1.Tg) mice have widely been used as model recipients of cancer immunotherapy with MUC1. Although MUC1.Tg mice have previously been shown to be immunologically tolerant to MUC1, the involvement of regulatory T (Treg) cells in this phenotype remains unclear. Here, we showed that numbers of Treg cells in MUC1-expressing tumors were greater in MUC1.Tg mice than in control C57BL/6 (B6) mice, and that the growth of tumor cells expressing MUC1, but not that of control cells, in MUC1. Tg mice was faster than in B6 mice. The MUC1.Tg mice appeared to develop MUC1-specific peripheral tolerance, as transferred MUC1-specific T cells were unable to function in MUC1.Tg mice but were functional in control B6 mice. The suppressive function of CD4⁺CD25^high cells from MUC1.Tg mice was more potent than that of cells from control B6 mice when Treg cell activity against MUC1-specific T cells was compared in vitro. Therefore, the enhanced growth of MUC1-expressing tumor cells in MUC1.Tg mice is likely due to the presence of MUC1-specific Treg cells.     

Divergent solid-phase synthesis and candidacidal activity of MUC7 D1, a 51-residue histidine-rich N-terminal domain of human salivary mucin MUC7.

Domain 1 of the low-molecular-weight human salivary mucin, designated MUC7 D1, spans the 51 N-terminal amino acid residues. This domain contains a 15-residue basic histidine-rich subdomain (R3-Q17) which has 53% sequence similarity to histatin 5 (Hsn-5), a salivary molecule known to exert potent in vitro cidal activity against Candida albicans and many other medically important fungi. The MUC7 D1-15mer and its derivatives have previously been synthesized in our laboratory and their candidacidal activities have been found to be inferior to that of Hsn-5. We were therefore intrigued to explore the candidacidal potency of the full-length MUC7 D1 (51-mer). Linear solid-phase synthesis of this domain has been accomplished following standard Fmoc chemistry. The problems of partial coupling, owing to the peptide chain length, at several stages of the solid-phase step-by-step synthesis were circumvented either by double-coupling techniques or efficient coupling procedures. The MUC7 D1 peptide was purified to homogeneity by conventional reverse-phase HPLC using two columns connected in series. Secondary structure of the purified peptide was assessed by circular dichroism (CD) spectroscopy in phosphate buffer and trifluoroethanol and compared to that of MUC7 D1-15mer and Hsn-5. The MUC7 D1 candidacidal activity was assessed against azole-sensitive and azole-resistant C. albicans strains and was found, unlike that of the MUC7 D1-15mer, to be comparable with that of Hsn-5, indicating that in addition to Hsn-5, MUC7 D1 could provide an attractive alternative to the classical antifungal agents. The candidacidal potency of MUC7 D1, like that of MUC7 D1-15mer, and of Hsn-5, appears to be largely dependent on peptide charge, irrespective of alpha-helical structure.     

Mice with spontaneous pancreatic cancer naturally develop MUC-1-specific CTLs that eradicate tumors when adoptively transferred

Pancreatic cancer is a highly aggressive, treatment refractory cancer and is the fourth leading cause of death in the United States. In humans, 90% of pancreatic adenocarcinomas overexpress altered forms of a tumor-specific Ag, mucin 1 (MUC1; an epithelial mucin glycoprotein), which is a potential target for immunotherapy. We have established a clinically relevant animal model for pancreatic cancer by developing a double transgenic mouse model (called MET) that expresses human MUC1 as self molecule and develops spontaneous tumors of the pancreas. These mice exhibit acinar cell dysplasia at birth, which progresses to microadenomas and acinar cell carcinomas. The tumors express large amounts of underglycosylated MUC1 similar to humans. Tumor-bearing MET mice develop low affinity MUC1-specific CTLs that have no effect on the spontaneously occurring pancreatic tumors in vivo. However, adoptive transfer of these CTLs was able to completely eradicate MUC1-expressing injectable tumors in MUC1 transgenic mice, and these mice developed long-term immunity. These CTLs were MHC class I restricted and recognized peptide epitopes in the immunodominant tandem repeat region of MUC1. The MET mice appropriately mimic the human condition and are an excellent model with which to elucidate the native immune responses that develop during tumor progression and to develop effective antitumor vaccine strategies.     

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.     

A soluble form of Siglec-9 provides an antitumor benefit against mammary tumor cells expressing MUC1 in transgenic mice

Tumor-associated MUC1 binds to Siglec-9, which is expected to mediate tumor cell growth and negative immunomodulation. We hypothesized that a soluble form of Siglec-9 (sSiglec-9) competitively inhibits a binding of MUC1 to its receptor molecules like human Siglec-9, leading to provide antitumor benefit against MUC1-expressing tumor, and generated transgenic mouse lines expressing sSiglec-9 (sSiglec-9 Tg). When mammary tumor cells expressing MUC1 were intraperitoneally transplanted into sSiglec-9 Tg, tumor proliferation was slower with the lower histological malignancy as compared with non-transgenic mice. The sSiglec-9 was detected in the ascites caused by the tumor in the sSiglec-9 Tg, and sSiglec-9 and MUC1 were often colocalized on surfaces of the tumor cells. PCNA immunohistochemistry also revealed the reduced proliferation of the tumor cells in sSiglec-9 Tg. In sSiglec-9 Tg with remarkable suppression of tumor proliferation, MUC1 expressions were tend to be reduced. In the ascites of sSiglec-9 Tg bearing the tumor, T cells were uniformly infiltrated, whereas aggregations of degenerative T cells were often observed in the non-transgenic mice. These results suggest that sSiglec-9 has an antitumor benefit against MUC1-expressing tumor in the transgenic mice, which may avoid the negative immunomodulation and/or suppress tumor-associated MUC1 downstream signal transduction, and subsequent tumor proliferation.     

The cooperation between two CD4 T cells induces tumor protective immunity in MUC.1 transgenic mice

Immunity and tumor protection in mice transgenic for human MUC.1, a glycoprotein expressed in the majority of cancers of epithelial origin in humans, were induced by vaccination with B lymphocytes genetically programmed to activate MUC.1-specific CD4 T cells. Their activation required a functional cooperation between two Th cells, one specific for a self (MUC.1) and the other for a nonself T cell determinant. The immunological switch provided by Th-Th cooperation was sufficient to induce MUC.1-specific CD4 and CD8 T cell responses in MUC.1-transgenic mice, and protect them permanently from tumor growth. CD4 T cells specific for MUC.1 lacked cytolytic function, but produced IFN-gamma upon restimulation with Ag. We conclude that immunity against tumor self-Ags and tumor protection can be regulated exploiting an inherent property of the immune system.     

Bile salt-stimulated lipase (BSSL) distribution in rat, mouse and transgenic mouse expressing human BSSL

 In some species, including man and mouse, bile salt-stimulated lipase (BSSL) in milk catalyzes the hydrolysis of triacylglycerides into glycerol and free fatty acids, a reaction that is of particular importance during suckling. The enzyme is also secreted by the pancreas (referred to as carboxyl-ester hydrolase, CEH). We wished to localize sources and storage sites for BSSL/CEH in rats, in wild-type mice, and in transgenic mice producing recombinant human BSSL in milk. Immunoreactivity against several BSSL fragments was strong in the pancreatic acinar cells and moderate in the absorptive cells of the small intestine and in salivary duct cells of the mice, as well as in rats. Sections from lactating mammary glands of mouse, but not rat, also showed immunoreactivity for BSSL; the signal was strongest in the transgenic mice. Radioactive riboprobes for BSSL mRNA hybridized on sections of rat and mouse pancreatic acinar cells, and mouse mammary glands (both wild-type and transgenic). Using RT-PCR, it was possible to amplify BSSL mRNA from wild-type mouse pancreas and mammary gland, from rat submandibular glands, and, in a few cases, from rat liver. In transgenic mice, the BSSL mRNA was highly expressed only in lactating mammary gland, but could be detected in a few other organs as well. Accepted: 31 March 1998     

Structure and Chromosomal Localization of the Human Salivary Mucin Gene, MUC7

We have isolated and characterized several MUC7 genomic clones encoding the human low-molecular-weight salivary mucin, MG2. The MUC7 gene spans ∼10.0 kb and comprises of three exons and two introns. Intron 1 is ∼1.7 kb long and is located in the 5′-untranslated region of the corresponding MUC7 cDNA. Intron 2 spans ∼6.0 kb and is located close to the boundary of the putative leader peptide and secreted protein. The entire region encoding the secreted peptide is located on exon 3, spanning ∼2.2 kb. The nucleotide sequence of sections of the MUC7 gene, including 1500 bp of the 5′-flanking region, was determined and analyzed for motifs identical or homologous to other known response elements. A modified RACE procedure was used to determine the 5′-end of the MUC7 mRNA. PCR, the human–hamster somatic cell hybrid panel PCRable DNAs kit, and anin situhybridization analysis on the complete metaphase chromosome spreads were used for the chromosomal localization of the MUC7 gene. It was mapped to chromosome 4q13–q21.