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While a protein primary structure is determined by genetic code, its specific functional form is mostly achieved in a dynamic interplay that includes actions of many enzymes involved in post-translational modifications. This versatile repertoire is widely used by cells to direct their response to external stimuli, regulate transcription and protein localization and to keep proteostasis. Herein, post-translational modifications with evident potency to drive prostate cancer are explored. A comprehensive list of proteome-wide and single protein post-translational modifications and their involvement in phenotypic outcomes is presented. Specifically, the data on phosphorylation, glycosylation, ubiquitination, SUMOylation, acetylation, and lipidation in prostate cancer and the enzymes involved are collected. This type of knowledge is especially valuable in cases when cancer cells do not differ in the expression or mutational status of a protein, but its differential activity is regulated on the level of post-translational modifications. Since their driving roles in prostate cancer, post-translational modifications are widely studied in attempts to advance prostate cancer treatment. Current strategies that exploit the potential of post-translational modifications in prostate cancer therapy are presented.

Integrins are heterodimeric cell surface receptors composed of α and β subunits that control adhesion, proliferation and gene expression. The integrin heterodimer binding to ligand reorganises the cytoskeletal networks and triggers multiple signalling pathways that can cause changes in cell cycle, proliferation, differentiation, survival and motility. In addition, integrins have been identified as targets for many different diseases, including cancer. Integrin crosstalk is a mechanism by which a change in the expression of a certain integrin subunit or the activation of an integrin heterodimer may interfere with the expression and/or activation of other integrin subunit(s) in the very same cell. Here, we review the evidence for integrin crosstalk in a range of cellular systems, with a particular emphasis on cancer. We describe the molecular mechanisms of integrin crosstalk, the effects of cell fate determination, and the contribution of crosstalk to therapeutic outcomes. Our intention is to raise awareness of integrin crosstalk events such that the contribution of the phenomenon can be taken into account when researching the biological or pathophysiological roles of integrins.

Integrins are heterodimeric glycoproteins that bind cells to extracellular matrix. Upon integrin clustering, multimolecular integrin adhesion complexes (IACs) are formed, creating links to the cell cytoskeleton. We have previously observed decreased cell migration and increased sensitivity to microtubule (MT) poisons, paclitaxel and vincristine, in the melanoma cell line MDA-MB-435S upon transfection with integrin alpha V specific siRNA, suggesting a link between adhesion and drug sensitivity. To elucidate the underlying mechanism, we determined alpha V-dependent changes in IAC composition. Using mass spectrometry (MS)-based proteomics, we analyzed the components of isolated IACs of MDA-MB-435S cells and two MDA-MB-435S-derived integrin alpha V specific shRNA-expressing cell clones with decreased expression of integrin alpha V. MS analysis showed that cells preferentially use integrin alpha V beta 5 for the formation of IACs. The differential analysis between MDA-MB-435S cells and clones with decreased expression of integrin alpha V identified key components of integrin alpha V beta 5 adhesion complexes as talins 1 and 2, alpha-actinins 1 and 4, filamins A and B, plectin and vinculin. The data also revealed decreased levels of several components of the cortical microtubule stabilization complex, which recruits MTs to adhesion sites (notably liprins alpha and beta, ELKS, LL5b, MACF1, KANK1, and KANK2), following alpha V knockdown. KANK2 knockdown in MDA-MB-435S cells mimicked the effect of integrin alpha V knockdown and resulted in increased sensitivity to MT poisons and decreased migration. Taken together, we conclude that KANK2 is a key molecule linking integrin alpha V beta 5 IACs to MTs, and enabling the actin-MT crosstalk that is important for both sensitivity to MT poisons and cell migration.

Anticancer therapies mainly target primary tumor growth and little attention is given to the events driving metastasis formation. Metastatic prostate cancer, in comparison to localized disease, has a much worse prognosis. In the work presented here, groups of genes that are common to prostate cancer metastatic cells from bones, lymph nodes, and liver and those that are site-specific were delineated. The purpose of the study was to dissect potential markers and targets of anticancer therapies considering the common characteristics and differences in transcriptional programs of metastatic cells from different secondary sites. To that end, a meta-analysis of gene expression data of prostate cancer datasets from the GEO database was conducted. Genes with differential expression in all metastatic sites analyzed belong to the class of filaments, focal adhesion, and androgen receptor signaling. Bone metastases undergo the largest transcriptional changes that are highly enriched for the term of the chemokine signaling pathway, while lymph node metastasis show perturbation in signaling cascades. Liver metastases change the expression of genes in a way that is reminiscent of processes that take place in the target organ. Survival analysis for the common hub genes revealed involvements in prostate cancer prognosis and suggested potential biomarkers.

The Kank (kidney or KN motif and ankyrin repeat domain-containing) family of proteins has been described as essential for crosstalk between actin and microtubules. Kank1, 2, 3 and 4 arose by gene duplication and diversification and share conserved structural domains. KANK proteins are localised mainly to the plasma membrane in focal adhesions, indirectly affecting RhoA and Rac1 thus regulating actin cytoskeleton. In addition, Kank proteins are part of the cortical microtubule stabilisation complex regulating microtubules. Most of the data have been collected for Kank1 protein whose expression promotes apoptosis and cell- cycle arrest while Kank3 was identified as hypoxia- inducible proapoptotic target of p53. A discrepancy in Kanks role in regulation of cell migration and sensitivity to antitumour drugs has been observed in different cell models. Since expression of Kank1 and 3 correlate positively with tumour progression and patient outcome, at least in some tumour types, they are candidates for tumour suppressors.

Background: Many genome- and epigenome-wide association studies (GWAS and EWAS) and studies of promoter methylation of candidate genes for inflammatory bowel disease (IBD) have demonstrated significant associations between genetic and epigenetic changes and IBD. Independent GWA studies have identified genetic variants in the BACH2, IL6ST, LAMB1, IKZF1, and MGAT3 loci to be associated with both IBD and immunoglobulin G (IgG) glycosylation. Methods: Using bisulfite pyrosequencing, we analyzed CpG methylation in promoter regions of these five genes from peripheral blood of several hundred IBD patients and healthy controls (HCs) from two independent cohorts, respectively. Results: We found significant differences in the methylation levels in the MGAT3 and BACH2 genes between both Crohn's disease and ulcerative colitis when compared to HC. The same pattern of methylation changes was identified for both genes in CD19 + B cells isolated from the whole blood of a subset of the IBD patients. A correlation analysis was performed between the MGAT3 and BACH2 promoter methylation and individual IgG glycans, measured in the same individuals of the two large cohorts. MGAT3 promoter methylation correlated significantly with galactosylation, sialylation, and bisecting GlcNAc on IgG of the same patients, suggesting that activity of the GnT-III enzyme, encoded by this gene, might be altered in IBD. The correlations between the BACH2 promoter methylation and IgG glycans were less obvious, since BACH2 is not a glycosyltransferase and therefore may affect IgG glycosylation only indirectly. Conclusions: Our results suggest that epigenetic deregulation of key glycosylation genes might lead to an increase in pro-inflammatory properties of IgG in IBD through a decrease in galactosylation and sialylation and an increase of bisecting GlcNAc on digalactosylated glycan structures. Finally, we showed that CpG methylation in the promoter of the MGAT3 gene is altered in CD3 + T cells isolated from inflamed mucosa of patients with ulcerative colitis from a third smaller cohort, for which biopsies were available, suggesting a functional role of this glyco-gene in IBD pathogenesis. The authors would like to thank Stephanie Scott for her organizational and administrational contribution. The study has been funded by the EU FP7 grant European Commission IBD-BIOM (contract # 305479), EU FP7 Regional Potential Grant INTEGRA-Life (contract # 315997), European Structural and Investment Funds grant for the Croatian National Centre of Research Excellence in Personalized Healthcare (contract # KK.01.1.1.01.0010), and Croatian Science Foundation grant EpiGlycoIgG (contract # 3361). Financial support from Portugal (PI: SSP): FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia e Inovação in the framework of the project (POCI-01/0145-FEDER-016601; PTDC/DTP-PIC/0560/2014) was received. SSP also acknowledges the European Crohn’s and Colitis Organization (ECCO) and the “Broad Medical Research program at Crohn’s and Colitis Foundation of America-CCFA” for funding. SSP acknowledges the Portuguese Group of Study on IBD (GEDII) for funding. A.M.D. [PD/BD/105982/2014] also acknowledges FCT for funding. IBD-BIOM consortium: Daniel Kolarich (Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany), Manfred Wuhrer (Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands; Division of BioAnalytical Chemistry, VU University Amsterdam, Amsterdam, the Netherlands), Dermot P. B. McGovern (F. Widjaja Family Foundation Inflammatory Bowel and Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles), Iain K. Pemberton (IP Research Consulting SAS, Paris, France), Daniel IR Spencer (Ludger Ltd., Culham Science Centre, Oxford, UK, Daryl L. Fernandes (Ludger Ltd., Culham Science Centre, Oxford, UK), Rahul Kalla, Kate O’Leary, Alex T Adams, Hazel Drummond, Elaine Nimmo, Ray Boyapati, David C Wilson (Centre for Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK), Ray Doran (Ludger Ltd., Culham Science Centre, Oxford, UK), Igor Rudan (all, Centre for Population Health Sciences, University of Edinburgh, Edinburgh, UK), Paolo Lionetti (Paediatric Gastroenterology Unit, AOU Meyer, Viale Pieraccini, Florence, Italy), Natalia Manetti (Department of Medical and Surgical Sciences, Division of Gastroenterology, University Hospital Careggi, Florence, Italy), Fabrizio Bossa (Department of Medical Sciences, Division of Gastroenterology, IRCCS-CSS Hospital, Viale Cappuccini, Rotondo, Italy), Paola Cantoro, Anna Kohn (Division of Gastroenterology, S. Camillo Hospital, Rome, Italy), Giancarlo Sturniolo (Gastrointestinal Unit, University of Padua, Padua, Italy), Silvio Danese (IBD Unit, Humanitas Research Institute, Rozzano, Milan, Italy), Mariek Pierik (Maastricht University Medical Centre (MUMC), Maastricht, the Netherlands), and David C. Wilson (Centre for Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK). This independent research was generously supported by the following grants: EU FP7 research grant IBD-BIOM (contract # 305479) to JS, VA, GL, and VZ; EU FP7 Regional Potential Grant INTEGRA-Life (contract # 315997) to GL and VZ; European Structural and Investment Funds grant for the Croatian National Centre of Research Excellence in Personalized Healthcare (contract # KK.01.1.1.01.0010) to GL and VZ; Croatian Science Foundation grant EpiGlycoIgG (contract # 3361) to VZ; FEDER COMPETE 2020 POCI, Portugal 2020, and Portuguese funds through FCT (contracts # POCI-01/0145-FEDER-016601 and PTDC/DTP-PIC/0560/2014) to SP; and FTC (contract # PD/BD/105982/2014) to AMD.

Background The RAS association domain family protein 1a (RASSF1A) is a prominent tumor suppressor gene showing altered promoter methylation in testicular germ cell tumors (TGCT). RASSF1A promoter hypermethylation might represent an early event in TGCT tumorigenesis. We investigated whether the RASSF1A promoter methylation in peripheral blood of TGCT patients can be associated with testicular cancer risk. Materials and methods Following a meta-analysis, we performed a cohort study including 32 testicular cancer patients and 32 healthy controls. Promoter methylation of the RASSF1A and O6-methylguanine-DNA-methyltransferase (MGMT) genes was analyzed using bisulfite pyrosequencing of DNA from peripheral blood. Results Meta-analysis showed an odds ratio (OR) of 7.69 for RASSF1A promoter methylation as a risk factor for TGCT. Cohort study found altered methylation of the RASSF1A promoter in blood of TGCT patients. Methylation was higher in TGCT patients before BEP chemotherapy. Conclusion The meta-analysis indicates a role of the RASSF1A promoter hypermethylation from peripheral blood in TCGT. We confirmed that finding in our cohort study, which represents the first report of changed RASSF1A promoter methylation in peripheral blood TGCT.

Epigenetic alterations may provide important insights into gene-environment interaction in inflammatory bowel disease (IBD). Here we observe epigenome-wide DNA methylation differences in 240 newly-diagnosed IBD cases and 190 controls. These include 439 differentially methylated positions (DMPs) and 5 differentially methylated regions (DMRs), which we study in detail using whole genome bisulphite sequencing. We replicate the top DMP (RPS6KA2) and DMRs (VMP1, ITGB2 and TXK) in an independent cohort. Using paired genetic and epigenetic data, we delineate methylation quantitative trait loci; VMP1/microRNA-21 methylation associates with two polymorphisms in linkage disequilibrium with a known IBD susceptibility variant. Separated cell data shows that IBD-associated hypermethylation within the TXK promoter region negatively correlates with gene expression in whole-blood and CD8+ T cells, but not other cell types. Thus, site-specific DNA methylation changes in IBD relate to underlying genotype and associate with cell-specific alteration in gene expression., Epigenetic perturbations may be an important factor in diseases where both genes and environment play a role. Here, Ventham and colleagues show that DNA methylation changes in inflammatory bowel disease are related to the underlying genotype, and are associated with cell-specific changes to gene expression.

Background Most eukaryotic proteins are modified by covalent addition of glycan molecules that considerably influence their function. Aberrant glycosylation is profoundly involved in malignant transformation, tumor progression and metastasis. Some glycan structures are tumor-specific and reflect disturbed glycan biosynthesis pathways. Methods We analyzed DNA methylation and expression of 86 glyco-genes in melanoma, hepatocellular, breast and cervical cancers using data from publicly available databases. We also analyzed methylation datasets without the available matching expression data for glyco-genes in lung cancer, and progression of melanoma into lymph node and brain metastases. Results Ten glyco-genes (GALNT3, GALNT6, GALNT7, GALNT14, MGAT3, MAN1A1, MAN1C1, ST3GAL2, ST6GAL1, ST8SIA3) showing changes in both methylation and expression in the same type of cancer belong to GalNAc transferases, GlcNAc transferases, mannosidases and sialyltransferases, which is in line with changes in glycan structures already reported in the same type of tumors. Some of those genes were additionally identified as potentially valuable for disease prognosis. The MGAT5B gene, so far identified as specifically expressed in brain, emerged as a novel candidate gene that is epigenetically dysregulated in different cancers other than brain cancer. We also report for the first time aberrant expression of the GALNT and MAN genes in cancer by aberrant promoter methylation. Conclusions Aberrant expression of glyco-genes due to aberrant promoter methylation could be a way leading to characteristic glycosylation profiles commonly described in cancer. General significance Methylation status in promoters of candidate glyco-genes might serve as prognostic markers for specific tumors and point to potential novel targets for epigenetic drugs. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.

BACKGROUND AND AIMS: Causes of inflammatory bowel diseases are not well understood and the most prominent forms, Crohn's disease (CD) and ulcerative colitis (UC), are sometimes hard to distinguish. Glycosylation of IgG has been associated with CD and UC. IgG Fc-glycosylation affects IgG effector functions. We evaluated changes in IgG Fc- glycosylation associated with UC and CD, as well as with disease characteristics in different patient groups. METHODS: We analyzed 3441 plasma samples obtained from 2 independent cohorts of patients with CD (874 patients from Italy and 391 from the United States) or UC (1056 from Italy and 253 from the US and healthy individuals [controls] ; 427 in Italy and 440 from the United States). IgG Fc-glycosylation (tryptic glycopeptides) was analyzed by liquid chromatography coupled to mass spectrometry. We analyzed associations between disease status (UC vs controls, CD vs controls, and UC vs CD) and glycopeptide traits, and associations between clinical characteristics and glycopeptide traits, using a logistic regression model with age and sex included as covariates. RESULTS: Patients with CD or UC had lower levels of IgG galactosylation than controls. For example, the odds ratio (OR) for IgG1 galactosylation in patients with CD was 0.59 (95% confidence interval [CI], 0.51-0.69) and for patients with UC was 0.81 (95% CI, 0.71-0.92). Fucosylation of IgG was increased in patients with CD vs controls (for IgG1: OR, 1.27 ; 95% CI, 1.12-1.44), but decreased in patients with UC vs controls (for IgG23: OR, 0.72 ; 95% CI, 0.63-0.82). Decreased galactosylation associated with more severe CD or UC, including the need for surgery in patients with UC vs controls (for IgG1: OR, 0.69 ; 95% CI, 0.54-0.89) and in patients with CD vs controls (for IgG23: OR, 0.78 ; 95% CI, 0.66-0.91). CONCLUSIONS: In a retrospective analysis of plasma samples from patients with CD or UC, we associated levels of IgG Fc-glycosylation with disease (compared to controls) and its clinical features. These findings could increase our understanding of mechanisms of CD and UC pathogenesis and be used to develop diagnostics or guide treatment.