Histone Methylases and Demethylases Regulating Antagonistic Methyl Marks: Changes Occurring in Cancer.

Epigenetic regulation of gene expression is crucial to the determination of cell fate in development and differentiation, and the Polycomb (PcG) and Trithorax (TrxG) groups of proteins, acting antagonistically as complexes, play a major role in this regulation. Although originally identified in Drosophila, these complexes are conserved in evolution and the components are well defined in mammals. Each complex contains a protein with methylase activity (KMT), which can add methyl groups to a specific lysine in histone tails, histone 3 lysine 27 (H3K27), by PcG complexes, and H3K4 and H3K36 by TrxG complexes, creating transcriptionally repressive or active marks, respectively.

Cancer-associated hypersialylated MUC1 drives the differentiation of human monocytes into macrophages with a pathogenic phenotype.

The tumour microenvironment plays a crucial role in the growth and progression of cancer, and the presence of tumour-associated macrophages (TAMs) is associated with poor prognosis. Recent studies have demonstrated that TAMs display transcriptomic, phenotypic, functional and geographical diversity. Here we show that a sialylated tumour-associated glycoform of the mucin MUC1, MUC1-ST, through the engagement of Siglec-9 can specifically and independently induce the differentiation of monocytes into TAMs with a unique phenotype that to the best of our knowledge has not previously been described.

O-linked mucin-type glycosylation regulates the transcriptional programme downstream of EGFR.

Aberrant mucin-type O-linked glycosylation is a common occurrence in cancer where the upregulation of sialyltransferases is often seen leading to the early termination of O-glycan chains. Mucin-type O-linked glycosylation is not limited to mucins and occurs on many cell surface glycoproteins including EGFR, where the number of sites can be limited. Upon EGF ligation, EGFR induces a signaling cascade and may also translocate to the nucleus where it directly regulates gene transcription, a process modulated by Galectin-3 and MUC1 in some cancers.

O-linked mucin-type glycosylation in breast cancer.

Changes in mucin-type O-linked glycosylation are seen in over 90% of breast cancers where increased sialylation is often observed and a change from branched glycans to linear glycans is often seen. There are many mechanisms involved including increased/altered expression of glycosyltransferases and relocalisation to the endoplasmic reticulum of the enzymes responsible for the addition of the first sugar, -acetyl-d-galactosamine. It is now becoming clear that these changes can contribute to tumour growth and progression by modulating the micro-environment through glycan-sensing lectins expressed on immune cells, by modulating interactions with tumour surface receptors and by binding to selectins.

Latest developments in MUC1 immunotherapy.

Currently, there is renewed interest in attempting to recruit the host immune system to eliminate cancers, and within this renewed activity, MUC1 continues to arouse interest. MUC1 has been considered a possible therapeutic target for the past 30 years as it is up-regulated, aberrantly glycosylated and its polarization is lost in many adenocarcinomas. Moreover, MUC1 is expressed by some haematopoietic cancers, including acute myeloid leukaemia and myeloma.

Targeting of Tumor-Associated Glycoforms of MUC1 with CAR T Cells.

We read with interest the manuscript by June and colleagues published recently in Immunity in which they describe targeting of aberrantly glycosylated tumor-associated cell membrane mucin MUC1 using chimeric antigen receptor-engineered human T cells (Posey et al., 2016). In that study, the authors used a second generation 4-1BB costimulatory-molecule-based chimeric antigen receptor (CAR) (Imai et al.

The mucin MUC1 modulates the tumor immunological microenvironment through engagement of the lectin Siglec-9.

Siglec-9 is a sialic-acid-binding lectin expressed predominantly on myeloid cells. Aberrant glycosylation occurs in essentially all types of cancers and results in increased sialylation. Thus, when the mucin MUC1 is expressed on cancer cells, it is decorated by multiple short, sialylated O-linked glycans (MUC1-ST).

Elevated IgG4 in patient circulation is associated with the risk of disease progression in melanoma.

Emerging evidence suggests pathological and immunoregulatory functions for IgG4 antibodies and IgG4 B cells in inflammatory diseases and malignancies. We previously reported that IgG4 antibodies restrict activation of immune effector cell functions and impair humoral responses in melanoma. Here, we investigate IgG4 as a predictor of risk for disease progression in a study of human sera (n = 271: 167 melanoma patients; 104 healthy volunteers) and peripheral blood B cells (n = 71: 47 melanoma patients; 24 healthy volunteers).

The Breast Cancer-Associated Glycoforms of MUC1, MUC1-Tn and sialyl-Tn, Are Expressed in COSMC Wild-Type Cells and Bind the C-Type Lectin MGL.

Aberrant glycosylation occurs in the majority of human cancers and changes in mucin-type O-glycosylation are key events that play a role in the induction of invasion and metastases. These changes generate novel cancer-specific glyco-antigens that can interact with cells of the immune system through carbohydrate binding lectins. Two glyco-epitopes that are found expressed by many carcinomas are Tn (GalNAc-Ser/Thr) and STn (NeuAcα2,6GalNAc-Ser/Thr).

Targeting DNGR-1 (CLEC9A) with antibody/MUC1 peptide conjugates as a vaccine for carcinomas.

DCs are the most potent APCs and are the focus of many immunotherapeutic approaches for the treatment of cancer, although most of these approaches require the ex vivo generation and pulsing of DCs. We have targeted a subset of DCs in vivo using an Ab to DNGR-1, a C-type lectin dedicated to the cross-presentation of Ag expressed by subsets of DCs. HLA-A2 epitopes from the tumour-associated Ag, MUC1, were coupled to the anti-DNGR-1 Ab, and their efficacy in generating a Th1-cell response and inhibiting tumour growth was evaluated in a clinically relevant double transgenic mouse model expressing human MUC1 and A2K/b.

The human papillomavirus type 16 E7 oncoprotein induces a transcriptional repressor complex on the Toll-like receptor 9 promoter.

Human papillomavirus type 16 (HPV16) and other oncogenic viruses have been reported to deregulate immunity by suppressing the function of the double-stranded DNA innate sensor TLR9. However, the mechanisms leading to these events remain to be elucidated. We show that infection of human epithelial cells with HPV16 promotes the formation of an inhibitory transcriptional complex containing NF-κBp50-p65 and ERα induced by the E7 oncoprotein.

Autoantibodies to MUC1 glycopeptides cannot be used as a screening assay for early detection of breast, ovarian, lung or pancreatic cancer.

Background: Autoantibodies have been detected in sera before diagnosis of cancer leading to interest in their potential as screening/early detection biomarkers. As we have found autoantibodies to MUC1 glycopeptides to be elevated in early-stage breast cancer patients, in this study we analysed these autoantibodies in large population cohorts of sera taken before cancer diagnosis.

Methods: Serum samples from women who subsequently developed breast cancer, and aged-matched controls, were identified from UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS) and Guernsey serum banks to formed discovery and validation sets.

Cyclooxygenase-2 enzyme induces the expression of the α-2,3-sialyltransferase-3 (ST3Gal-I) in breast cancer.

Aberrant glycosylation is a common feature of malignant change. Changes in mucin-type O-linked glycosylation in breast cancer can result in the expression of truncated core 1-based sialylated glycans rather than the core 2-based glycans observed in normal mammary epithelium cells. This has been shown, in part, to be due to changes in the expression of glycosyltransferases, including the up-regulation of some sialyltransferases.

Selectin ligand sialyl-Lewis x antigen drives metastasis of hormone-dependent breast cancers.

The glycome acts as an essential interface between cells and the surrounding microenvironment. However, changes in glycosylation occur in nearly all breast cancers, which can alter this interaction. Here, we report that profiles of glycosylation vary between ER-positive and ER-negative breast cancers.

Identification of new cancer biomarkers based on aberrant mucin glycoforms by in situ proximity ligation.

Mucin glycoproteins are major secreted or membrane-bound molecules that, in cancer, show modifications in both the mucin proteins expression and in the O-glycosylation profile, generating some of the most relevant tumour markers in clinical use for decades. Thus far, the identification of these biomarkers has been based on the detection of either the protein or the O-glycan modifications. We therefore aimed to identify the combined mucin and O-glycan features, that is, specific glycoforms, in an attempt to increase specificity of these cancer biomarkers.

ST6GalNAc-I controls expression of sialyl-Tn antigen in gastrointestinal tissues.

Sialyl-Tn is a simple mucin-type carbohydrate antigen aberrantly expressed in gastrointestinal adenocarcinomas and in the precursor lesion intestinal metaplasia. Sialyl-Tn tumour expression is an independent indicator of poor prognosis. We have previously shown in vitro that ST6GalNAc-I and ST6GalNAc-II sialyltransferases can synthesize sialyl-Tn.

Transforming growth factor-β1 is constitutively secreted by Chinese hamster ovary cells and is functional in human cells.

Chinese hamster ovary (CHO) cells are widely used for the production of recombinant proteins for clinical use as well as academic research. They are particularly important for the production of glycoproteins where bacteria cannot be used. TGFβ1 is a potent cytokine highly conserved across species with multiple immunological and non-immunological effects.

Autoantibodies to aberrantly glycosylated MUC1 in early stage breast cancer are associated with a better prognosis.

Introduction: Detection of serum biomarkers for early diagnosis of breast cancer remains an important goal. Changes in the structure of O-linked glycans occur in all breast cancers resulting in the expression of glycoproteins that are antigenically distinct. Indeed, the serum assay widely used for monitoring disease progression in breast cancer (CA15.

PLU-1/JARID1B/KDM5B is required for embryonic survival and contributes to cell proliferation in the mammary gland and in ER+ breast cancer cells.

The four members of the JARID1/KDM5 family of proteins, a sub-group of the larger ARID (AT rich DNA binding domain) family, have been shown to demethylate trimethylated lysine 4 on histone 3 (H3K4me3), a chromatin mark associated with actively transcribed genes. In some lower organisms a single homologue of JARID1 is found, and functions of the four proteins found in mice and humans may be specific or overlapping. To investigate the function of the Jarid1B protein we examined the effects of deletion of the gene in mice.

T cells reactive with HLA-A*0201 peptides from the histone demethylase JARID1B are found in the circulation of breast cancer patients.

The nuclear protein PLU-1/JARID1B/KDM5 is widely expressed in breast cancers while showing highly restricted expression in normal adult tissues. To investigate whether JARID1B is a potential target antigen for immunotherapy of breast cancer, we have analyzed the responses of CD8(+) T cells to JARID1B HLA-A*0201 peptides in vitro and used peptide multimers to detect the presence of JARID1B reactive T cells in the circulation of breast cancer patients. Peptides were selected using two web-based algorithms: criteria for inclusion being a high score in both prediction algorithms, and nonhomology with retinoblastoma binding protein-2 (RBP2/JARID1A/KDM5A).

MUC1 immunotherapy.

The overexpression and aberrant glycosylation of MUC1 is associated with a wide variety of cancers, making it an ideal target for immunotherapeutic strategies. This review highlights the main avenues of research in this field, focusing on adenocarcinomas, from the preclinical to clinical; the problems and possible solutions associated with each approach; and speculates on the direction of MUC1 immunotherapeutic research over the next 5-10 years.

Over-expression of ST3Gal-I promotes mammary tumorigenesis.

Changes in glycosylation are common in malignancy, and as almost all surface proteins are glycosylated, this can dramatically affect the behavior of tumor cells. In breast carcinomas, the O-linked glycans are frequently truncated, often as a result of premature sialylation. The sialyltransferase ST3Gal-I adds sialic acid to the galactose residue of core 1 (Galbeta1,3GalNAc) O-glycans and this enzyme is over-expressed in breast cancer resulting in the expression of sialylated core 1 glycans.

Cancer biomarkers defined by autoantibody signatures to aberrant O-glycopeptide epitopes.

Autoantibodies to cancer antigens hold promise as biomarkers for early detection of cancer. Proteins that are aberrantly processed in cancer cells are likely to present autoantibody targets. The extracellular mucin MUC1 is overexpressed and aberrantly glycosylated in many cancers; thus, we evaluated whether autoantibodies generated to aberrant O-glycoforms of MUC1 might serve as sensitive diagnostic biomarkers for cancer.