title: Epigenetically regulated clonal heritability of CTA expression profiles in human melanoma.
abstract: The intratumoral heterogeneity of cancer testis antigens (CTA) expression, which is driven by promoter methylation status, may hamper the effectiveness of CTA-directed vaccination of melanoma patients. Thus, we investigated whether the intratumoral heterogeneity of CTA expression is inherited at cellular level, or evolves throughout cellular replication, leading to a phenotypically unstable tumor cell population with reduced immunogenicity and/or able to escape immune control. Utilizing a previously characterized ex vivo clonal model of intratumoral heterogeneity of CTA expression in melanoma, Mel 313 MAGE-A3-low clone 5 (clone 5(M3-low)) and MAGE-A3-high clone 14 (clone 14(M3-high)) were sub-cloned and analyzed for CTA profile. Molecular assays demonstrated that levels of MAGE-A3 expression were highly conserved among generated sub-clones, as compared to parental clones. A similar behavior was identified for an extensive panel of other CTA investigated. Inherited levels of MAGE-A3 expression correlated with the extent of promoter methylation among clone 5(M3-low) and clone 14(M3-high) sub-clones analyzed. Treatment of clone 5(M3-low) with a DNA hypomethylating agent (DHA) resulted in an up-regulated expression of MAGE-A3, which was inherited at single cell level, being still detectable at day 60 in its sub-clones. Bisulfite sequencing demonstrated that also MAGE-A3 promoter methylation status was inherited among sub-clones generated from DHA-treated clone 5(M3-low) and strictly correlated with MAGE-A3 expression levels in investigated sub-clones. Similar results were obtained for additional CTA studied. Altogether our findings demonstrate that constitutive and DHA-modified CTA profiles of melanoma cells are clonally inherited throughout cellular replications, thus providing relevant insights to improve the effectiveness of CTA-based immunotherapy.
authors.affiliation: Cancer Bioimmunotherapy Unit, Centro di Riferimento Oncologico, Istituto di Ricovero e Cura a Carattere Scientifico, Aviano, PN, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Fratta
authors.forename: Luca
authors.initials: L
authors.lastname: Sigalotti
authors.forename: Francesca
authors.initials: F
authors.lastname: Colizzi
authors.forename: Alessia
authors.initials: A
authors.lastname: Covre
authors.forename: Hugues J M G
authors.initials: HJ
authors.lastname: Nicolay
authors.forename: Riccardo
authors.initials: R
authors.lastname: Danielli
authors.forename: Ester
authors.initials: E
authors.lastname: Fonsatti
authors.forename: Maresa
authors.initials: M
authors.lastname: Altomonte
authors.forename: Luana
authors.initials: L
authors.lastname: Calabrò
authors.forename: Sandra
authors.initials: S
authors.lastname: Coral
authors.forename: Michele
authors.initials: M
authors.lastname: Maio
mesh_terms: D000951:Antigens, Neoplasm / Q000235:genetics*; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D002455:Cell Division / Q000235:genetics; D002999:Clone Cells / Q000187:drug effects / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D019175:DNA Methylation / Q000187:drug effects; D000077209:Decitabine; D044127:Epigenesis, Genetic / Q000187:drug effects / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D040582:Inheritance Patterns / Q000235:genetics*; D008545:Melanoma / Q000235:genetics* / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D014407:Tumor Cells, Cultured
chemical_list: D000951:Antigens, Neoplasm; D000964:Antimetabolites, Antineoplastic; C072013:MAGEA3 protein, human; D009363:Neoplasm Proteins; D000077209:Decitabine; D001374:Azacitidine