Reverse-engineering the anti-MUC1 antibody 139H2 by mass spectrometry-based de novo sequencing.

Mucin 1 (MUC1) is a transmembrane mucin expressed at the apical surface of epithelial cells at mucosal surfaces. MUC1 has a barrier function against bacterial invasion and is well known for its aberrant expression and glycosylation in adenocarcinomas. The MUC1 extracellular domain contains a variable number of tandem repeats (VNTR) of 20 amino acids, which are heavily -linked glycosylated.

R-spondin-3 promotes proliferation and invasion of breast cancer cells independently of Wnt signaling.

We recently identified R-spondin-3 (RSPO3) as a novel driver of breast cancer associating with reduced patient survival, expanding its clinical value as potential therapeutic target that had been recognized mostly for colorectal cancer so far. (Pre)clinical studies exploring RSPO3 targeting in colorectal cancer approach this indirectly with Wnt inhibitors, or directly with anti-RSPO3 antibodies. Here, we address the clinical relevance of RSPO3 in breast cancer and provide insight in the oncogenic activities of RSPO3.

R-spondin-3 is an oncogenic driver of poorly differentiated invasive breast cancer.

R-spondins (RSPOs) are influential signaling molecules that promote the Wnt/β-catenin pathway and self-renewal of stem cells. Currently, RSPOs are emerging as clinically relevant oncogenes, being linked to cancer development in multiple organs. Although this has instigated the rapid development and testing of therapeutic antibodies targeting RSPOs, functional evidence that RSPO causally drives cancer has focused primarily on the intestinal tract.

Insertional mutagenesis in a HER2-positive breast cancer model reveals ERAS as a driver of cancer and therapy resistance.

Personalized medicine for cancer patients requires a deep understanding of the underlying genetics that drive cancer and the subsequent identification of predictive biomarkers. To discover new genes and pathways contributing to oncogenesis and therapy resistance in HER2+ breast cancer, we performed Mouse Mammary Tumor Virus (MMTV)-induced insertional mutagenesis screens in ErbB2/cNeu-transgenic mouse models. The screens revealed 34 common integration sites (CIS) in mammary tumors of MMTV-infected mice, highlighting loci with multiple independent MMTV integrations in which potential oncogenes are activated, most of which had never been reported as MMTV CIS.

Interaction of galectin-3 with MUC1 on cell surface promotes EGFR dimerization and activation in human epithelial cancer cells.

Epidermal growth factor receptor (EGFR) is an important regulator of epithelial cell growth and survival in normal and cancerous tissues and is a principal therapeutic target for cancer treatment. EGFR is associated in epithelial cells with the heavily glycosylated transmembrane mucin protein MUC1, a natural ligand of galectin-3 that is overexpressed in cancer. This study reveals that the expression of cell surface MUC1 is a critical enhancer of EGF-induced EGFR activation in human breast and colon cancer cells.

Insulin receptor substrate 4 (IRS4) is a constitutive active oncogenic driver collaborating with HER2 and causing therapeutic resistance.

Insulin receptor substrate 4 (IRS4) belongs to a family of cytoplasmic docking proteins mediating signals from cell surface receptors to downstream effectors. While IRS1 and IRS2 mediate signals from an active receptor, we found that IRS4 hyperactivates the phosphatidylinositol phosphate kinase (PI3K)-pathway independent of upstream signals and is irresponsive to feedback regulation causing cancer and resistance to human epidermal growth factor receptor 2 (HER2) targeted therapy.

IRS4 induces mammary tumorigenesis and confers resistance to HER2-targeted therapy through constitutive PI3K/AKT-pathway hyperactivation.

In search of oncogenic drivers and mechanisms affecting therapy resistance in breast cancer, we identified Irs4, a poorly studied member of the insulin receptor substrate (IRS) family, as a mammary oncogene by insertional mutagenesis. Whereas normally silent in the postnatal mammary gland, IRS4 is found to be highly expressed in a subset of breast cancers. We show that Irs4 expression in mammary epithelial cells induces constitutive PI3K/AKT pathway hyperactivation, insulin/IGF1-independent cell proliferation, anchorage-independent growth and in vivo tumorigenesis.

MUC1-ARF-A Novel MUC1 Protein That Resides in the Nucleus and Is Expressed by Alternate Reading Frame Translation of MUC1 mRNA.

Translation of mRNA in alternate reading frames (ARF) is a naturally occurring process heretofore underappreciated as a generator of protein diversity. The MUC1 gene encodes MUC1-TM, a signal-transducing trans-membrane protein highly expressed in human malignancies. Here we show that an AUG codon downstream to the MUC1-TM initiation codon initiates an alternate reading frame thereby generating a novel protein, MUC1-ARF.

RSPO3 expands intestinal stem cell and niche compartments and drives tumorigenesis.

Objective: The gross majority of colorectal cancer cases results from aberrant Wnt/β-catenin signalling through adenomatous polyposis coli ( or mutations. However, a subset of human colon tumours harbour, mutually exclusive with and mutations, gene fusions in or leading to enhanced expression of these R-spondin genes. This suggested that activation can substitute for the most common mutations as an alternative driver for intestinal cancer.

MUC1 extracellular domain confers resistance of epithelial cancer cells to anoikis.

Anoikis, a special apoptotic process occurring in response to loss of cell adhesion to the extracellular matrix, is a fundamental surveillance process for maintaining tissue homeostasis. Resistance to anoikis characterises cancer cells and is a pre-requisite for metastasis. This study shows that overexpression of the transmembrane mucin protein MUC1 prevents initiation of anoikis in epithelial cancer cells in response to loss of adhesion.

Chromatin landscapes of retroviral and transposon integration profiles.

The ability of retroviruses and transposons to insert their genetic material into host DNA makes them widely used tools in molecular biology, cancer research and gene therapy. However, these systems have biases that may strongly affect research outcomes. To address this issue, we generated very large datasets consisting of ~ 120,000 to ~ 180,000 unselected integrations in the mouse genome for the Sleeping Beauty (SB) and piggyBac (PB) transposons, and the Mouse Mammary Tumor Virus (MMTV).

Analysis of tumor heterogeneity and cancer gene networks using deep sequencing of MMTV-induced mouse mammary tumors.

Cancer develops through a multistep process in which normal cells progress to malignant tumors via the evolution of their genomes as a result of the acquisition of mutations in cancer driver genes. The number, identity and mode of action of cancer driver genes, and how they contribute to tumor evolution is largely unknown. This study deployed the Mouse Mammary Tumor Virus (MMTV) as an insertional mutagen to find both the driver genes and the networks in which they function.

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

Background: Formation 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.

Results: It 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 (Galbeta1,3GalNAcalpha-, 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.

AsialoGM1 and integrin alpha2beta1 mediate prostate cancer progression.

The most lethal aspect of cancer is the metastatic spread of primary tumors to distant sites. Any mechanism revealed is a target for therapy. In our previous studies, we reported that the invasive activity of the bone metastatic C4-2B prostate cancer cells could be ascribed to the reorganization of the alpha2beta1 integrin receptor and the alpha2 subunit-mediated association and activation of downstream signaling towards the activation of MMPs.

Circulating galectin-3 promotes metastasis by modifying MUC1 localization on cancer cell surface.

Adhesion of circulating tumor cells to the blood vessel endothelium is a critical step in cancer metastasis. We show in this study that galectin-3, the concentration of which is greatly increased in the circulation of cancer patients, increases cancer cell adhesion to macrovascular and microvascular endothelial cells under static and flow conditions, increases transendothelial invasion, and decreases the latency of experimental metastasis in athymic mice. These effects of galectin-3 are shown to be a consequence of its interaction with cancer-associated MUC1, which breaks the "protective shield" of the cell-surface MUC1 by causing MUC1 polarization, leading to exposure of smaller cell-surface adhesion molecules/ligands including CD44 and ligand(s) for E-selectin.

Ether lipid 1-O-octadecyl-2-O-methyl-3-glycero-phosphocholine inhibits cell-cell adhesion through translocation and clustering of E-cadherin and episialin in membrane microdomains.

The ether lipid 1-O-octadecyl-2-O-methyl-3-glycero-phosphocholine (ET-18-OMe) inhibits cell-cell adhesion and induces invasiveness of breast cancer cells. Previously, we showed that a loss of cell-cell adhesion was due to sterical hindrance of E-cadherin by the anti-adhesive properties of the cell surface mucin episialin. Here, we demonstrated that the ether lipid ET-18-OMe induced the translocation of E-cadherin and episialin to membrane microdomains, enriched in glycosphingolipids, known to be involved in cell-cell adhesion and cell signaling.

The expression pattern of MUC1 (EMA) is related to tumour characteristics and clinical outcome of invasive ductal breast carcinoma.

Aims: To clarify MUC1 patterns in invasive ductal breast carcinoma and to relate them to clinicopathological parameters, coexpression of other biological markers and prognosis.

Methods And Results: Samples from 243 consecutive patients with primary ductal carcinoma were incorporated into tissue microarrays (TMAs). Slides were stained for MUC1, oestrogen receptor (ER), progesterone receptor (PR), Her2/neu, p53 and cyclin D1.

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.

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.

Recent translational research: oncogene discovery by insertional mutagenesis gets a new boost.

Knowledge of the genes and genetic pathways involved in onco-genesis is essential if we are to identify novel targets for cancer therapy. Insertional mutagenesis in mouse models is among the most efficient tools to detect novel cancer genes. Retrovirus-mediated insertional mutagenesis received a tremendous boost by the availability of the mouse genome sequence and new PCR methods.

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.

Tumor-host interactions regulate glucocorticoid-mediated epiglycanin expression in TA3Ha murine mammary carcinoma cells.

The mucin-type glycoprotein epiglycanin is highly expressed on the cell surface of TA3Ha mouse mammary carcinoma cells if the cells are maintained by serial intraperitoneal passage in mice. We found that upon transfer of the cells to growth in vitro in medium supplemented with fetal bovine serum, the expression level of epiglycanin slowly diminished to less than 15% of that in vivo. Repassage of the cells in vivo fully restored the expression of epiglycanin.

Altered expression and allelic association of the hypervariable membrane mucin MUC1 in Helicobacter pylori gastritis.

Background & Aims: Infection with Helicobacter pylori causes chronic gastritis, and this confers a risk of gastric cancer. Short alleles of the membrane-bound mucin MUC1, which has a large extracellular highly glycosylated domain and is highly polymorphic due to variation in the number of tandemly repeated (TR) 20-amino acid units, have been shown to be associated with gastric cancer. Our aim was to investigate the involvement of MUC1 in chronic gastritis and, by implication, gastric cancer.

Alkyl-lysophospholipid 1-O-octadecyl-2-O-methyl- glycerophosphocholine induces invasion through episialin-mediated neutralization of E-cadherin in human mammary MCF-7 cells in vitro.

1-O-octadecyl-2-O-methyl-glycerophosphocholine (ET-18-OMe) is an analogue of the naturally occurring 2-lysophosphatidylcholine belonging to the class of antitumor lipids. Previously, we demonstrated that ET-18-OMe modulates cell-cell adhesion of human breast cancer MCF-7 cells. In the present study, we tested the effect of ET-18-OMe on adhesion, invasion and localisation of episialin and E-cadherin in MCF-7/AZ cells expressing a functional E-cadherin/catenin complex.

Biosynthesis and shedding of epiglycanin: a mucin-type glycoprotein of the mouse TA3Ha mammary carcinoma cell.

Epiglycanin is a mucin-type glycoprotein present at the surface of TA3Ha mouse mammary tumour cells. It is a long rod-like glycoprotein with a molecular mass of 500 kDa. Its function has not yet been established but its overexpression can affect cell-cell and cell-matrix adhesion.