John Hilkens

Cell membrane-associated mucins and their adhesion-modulating property.

A class of highly sialylated glycoproteins with very large mucin-like domains that protrude high above the plasma membrane have been shown to strongly reduce cellular adhesion. In normal epithelial cells, where the expression is restricted to the luminal side of the cell, these molecules may prevent inadvertent closing of the lumen as a result of weak, non-specific protein-protein interactions between opposite luminal membranes. In malignant tumors cellular polarization is often lost, which can lead to the entire cell surface being covered by these molecules. The resulting strongly reduced adhesion and immune recognition properties may play an important role during invasion and metastasis.

Galectin-3 Interaction with Thomsen-Friedenreich Disaccharide on Cancer-associated MUC1 Causes Increased Cancer Cell Endothelial Adhesion *

Patients with metastatic cancer commonly have increased serum galectin-3 concentrations, but it is not known whether this has any functional implications for cancer progression. We report that MUC1, a large transmembrane mucin protein that is overexpressed and aberrantly glycosylated in epithelial cancer, is a natural ligand for galectin-3. Recombinant galectin-3 at concentrations (0.2-1.0 μg/ml) similar to those found in the sera of patients with metastatic cancer increased adhesion of MUC1-expressing human breast (ZR-75-1) and colon (HT29-5F7) cancer cells to human umbilical vein endothelial cells (HUVEC) by 111% (111 ± 21%, mean ± S.D.) and 93% (93 ± 17%), respectively. Recombinant galectin-3 also increased adhesion to HUVEC of MUC1 transfected HCA1.7+ human breast epithelial cells that express MUC1 bearing the oncofetal Thomsen-Friedenreich antigen (Galβ1,3 GalNAc-α (TF)) but did not affect adhesion of MUC1-negative HCA1.7-cells. MUC1-transfected, Ras-transformed, canine kidney epithelial-like (MDE9.2+) cells, bearing MUC1 that predominantly carries sialyl-TF, only demonstrated an adhesive response to galectin-3 after sialidase pretreatment. Furthermore, galectin-3-mediated adhesion of HCA1.7+ to HUVEC was reduced by O-glycanase pretreatment of the cells to remove TF. Recombinant galectin-3 caused focal disappearance of cell surface MUC1 in HCA1.7+ cells, suggesting clustering of MUC1. Co-incubation with antibodies against E-Selectin or CD44H, but not integrin-β1, ICAM-1 or VCAM-1, largely abolished the epithelial cell adhesion to HUVEC induced by galectin-3. Thus, galectin-3, by interacting with cancer-associated MUC1 via TF, promotes cancer cell adhesion to endothelium by revealing epithelial adhesion molecules that are otherwise concealed by MUC1. This suggests a critical role for circulating galectin-3 in cancer metastasis and highlights the functional importance of altered cell surface glycosylation in cancer progression.

MMTV insertional mutagenesis identifies genes, gene families and pathways involved in mammary cancer

We performed a high-throughput retroviral insertional mutagenesis screen in mouse mammary tumor virus (MMTV)-induced mammary tumors and identified 33 common insertion sites, of which 17 genes were previously not known to be associated with mammary cancer and 13 had not previously been linked to cancer in general. Although members of the Wnt and fibroblast growth factors (Fgf) families were frequently tagged, our exhaustive screening for MMTV insertion sites uncovered a new repertoire of candidate breast cancer oncogenes. We validated one of these genes, Rspo3, as an oncogene by overexpression in a p53-deficient mammary epithelial cell line. The human orthologs of the candidate oncogenes were frequently deregulated in human breast cancers and associated with several tumor parameters. Computational analysis of all MMTV-tagged genes uncovered specific gene families not previously associated with cancer and showed a significant overrepresentation of protein domains and signaling pathways mainly associated with development and growth factor signaling. Comparison of all tagged genes in MMTV and Moloney murine leukemia virus–induced malignancies showed that both viruses target mostly different genes that act predominantly in distinct pathways.

Development of mouse mammary gland: identification of stages in differentiation of luminal and myoepithelial cells using monoclonal antibodies and polyvalent antiserum against keratin.

The development of the mouse mammary gland was studied immunohistochemically using monoclonal antibodies against cell surface and basement membrane proteins and a polyclonal antibody against keratin. We have identified three basic cell types: basal, myoepithelial, and epithelial cells. The epithelial cells can be subdivided into three immunologically related cell types: luminal type I, luminal type II, and alveolar cells. These five cell types appear at different stages of mammary gland development and have either acquired or lost one of the antibody-defined antigens. The cytoplasmic distribution of several of these antigens varied according to the location of the cells within the mammary gland. Epithelial cells which did not line the lumen expressed antigens throughout the cytoplasm. These antigens were demonstrated on the apical site in situations where the cells lined the lumen. One antigen became increasingly basolateral as the cells became attached to the basement membrane. The basal cells synthesize laminin and deposit it at the cell base. They are present in endbuds and ducts and are probably the stem cells of the mammary gland. Transitional forms have been demonstrated which developmentally link these cells with both myoepithelial and (luminal) epithelial cells.

MUC1/episialin: a critical barrier in the female reproductive tract.

The female reproductive tract must resist microbial infections as well as support embryonic development, implantation and placentation. Reproductive tract mucins, in general, and Muc1/episialin, in particular, play key roles in implantation related events and in protection from microbial infection. High levels of mucin expression in the lower reproductive tract presumably affords protection against infection while down-regulation of uterine mucins has been suggested to provide access to the uterine surface. The present studies demonstrate that mucins, particularly Muc1, are effective barriers to embryo attachment. Furthermore, a strain of female Muc1 null mice in normal housing displays chronic infection and inflammation of the lower reproductive tract and markedly reduced fertility rates. This phenotype is not observed when Muc1 nulls are housed in a pathogen-free environment indicating that this phenotype results from chronic microbial exposure. Only normal endogenous flora were isolated from the reproductive tracts of affected Muc1 null mice, suggesting that these bacterial species become opportunistic with loss of the mucin barrier. Staphylococcal adherence to lower reproductive tract epithelia was found to be mediated by cell surface mucin carbohydrates. Collectively, these studies demonstrate a critical barrier role for Muc1 in various aspects of female reproductive tract physiology.

Interaction between circulating galectin-3 and cancer-associated MUC1 enhances tumour cell homotypic aggregation and prevents anoikis.

BackgroundFormation of tumour cell aggregation/emboli prolongs the survival of circulating tumour cells in the circulation, enhances their physical trapping in the micro-vasculature and thus increases metastatic spread of the cancer cells to remote sites.ResultsIt shows here that the presence of the galactoside-binding galectin-3, whose concentration is markedly increased in the blood circulation of cancer patients, increases cancer cell homotypic aggregation under anchorage-independent conditions by interaction with the oncofetal Thomsen-Friedenreich carbohydrate (Galβ1,3GalNAcα-, TF) antigen on the cancer-associated transmembrane mucin protein MUC1. The galectin-3-MUC1 interaction induces MUC1 cell surface polarization and exposure of the cell surface adhesion molecules including E-cadherin. The enhanced cancer cell homotypic aggregation by galectin-MUC1 interaction increases the survival of the tumour cells under anchorage-independent conditions by allowing them to avoid initiation of anoikis (suspension-induced apoptosis).ConclusionThese results suggest that the interaction between free circulating galectin-3 and cancer-associated MUC1 promotes embolus formation and survival of disseminating tumour cells in the circulation. This provides new information into our understanding of the molecular mechanisms of cancer cell haematogenous dissemination and suggests that targeting the interaction of circulating galectin-3 with MUC1 in the circulation may represent an effective therapeutic approach for preventing metastasis.

Is episialin/MUC1 involved in breast cancer progression?

Episialin, also designated MUC1, CA 15-3 antigen and PEM, is an established serum marker for breast cancer. Its function and possible involvement in tumor progression has not yet been completely established. The molecule is an extended rod-like molecule protruding high above the cell surface. It is often highly overexpressed in breast cancer relative to normal breast epithelium cells. Overexpression of episialin on cells in vitro reduces cell-cell and cell-extracellular matrix adhesion, because the rod-like molecule masks the adhesion receptors. Episialin also exerts its anti-adhesion effect in vivo. In certain human tumors, where episialin was present at the basal side of the cell, abnormal contacts between the plasma membrane and the stroma were observed. As a consequence of its anti-adhesion properties, episialin overexpression reduces the sensitivity of the cells for cytotoxic lymphocytes. This might be one of the reasons why episialin transfected cells are more potent to form experimental metastases after i.v. injection into nude mice.

Cloning of novel mammary tumor progression and metastasis genes

Microsatellite instability (MSI) and frameshift mutations in genes containing nucleotide repeats have been reported in a subset of colorectal and gastric carcinomas. This study describes the analysis of MSI-positive colorectal (39 cases) and gastric carcinomas (36 cases) for the presence of frameshift mutations of the six genes known to be involved in DNA repair and containing mononucleotide repeats in their coding region. Our mutational study of the 75 MSI-positive tumors revealed frequent mutations in hRAD50 (23 cases, 31%), BLM (16 cases, 21%), and hMSH6 (16 cases, 21%); rare mutations in BRCA1 (1 case, 1%) and ATM (3 cases, 4%); and no mutation in NBS1. In contrast, no frameshift mutation was found in 60 MSI-negative colorectal and gastric carcinomas. The mutation of hRAD50, a gene that is involved in the response to cellular DNA damage and forms a complex with hMRE11 and NBS1, has not been reported previously. Our results suggest that frameshift mutations of hRAD50, BLM, and hMSH6 are selected and play a role in the tumorigenesis of colorectal and gastric carcinomas with MSI. The MSI targeting of the hRAD50 and BLM genes represents an additional link between MSI and DNA repair because alteration of these genes could accelerate defective DNA repair.

Fgf10 is an oncogene activated by MMTV insertional mutagenesis in mouse mammary tumors and overexpressed in a subset of human breast carcinomas

Mouse mammary tumor virus (MMTV) infection causes a high incidence of murine mammary carcinomas by insertion of its proviral DNA in the genome of mammary epithelial cells. Retroviral insertion can activate flanking proto-oncogenes by a process called insertional mutagenesis. By sequencing the DNA adjacent to MMTV proviral insertions in mammary tumors from BALB/c mice infected with C3H-MMTV, we have found a common MMTV insertion site in the Fgf10 locus. RT–PCR studies showed that Fgf10 is expressed only in those tumors harboring a MMTV proviral insertion in this locus, suggesting that Fgf10 is a proto-oncogene. The oncogenicity of Fgf10 was evaluated in vivo by subcutaneous transplantation of retrovirally transduced HC11 mammary epithelial cells into BALB/c mice. Highly vascularized invasive subcutaneous tumors developed indicating that Fgf10 can act as an oncogene. A survey of primary human breast carcinomas revealed strongly elevated Fgf10 mRNA levels in approximately 10% of the tumors tested, suggesting that Fgf10 may also be involved in oncogenicity of a subset of human breast cancers.

The mouse episialin (Muc1) gene and its promoter: Rapid evolution of the repetitive domain in the protein

We have cloned the Muc1 gene of the mouse, encoding the murine equivalent of human episialin (also known as EMA or PEM), a mucin-like glycoprotein that is overexpressed in carcinoma cells. The extracellular domain of the mouse protein, that mainly consists of tandem repeats, contains 16 repeats of variable length and sequence, whereas the human protein usually contains between 30 and 90 nearly identical repeats. The murine repeats contain more potential O-glycan side chains and this may result in a more extended conformation of the murine protein. The transmembrane and cytoplasmic domains of the protein show about 90% conservation. The promoter region shows many conserved regions that could function as transcription factor binding sites.