Integrated Genomic And Functional Analyses Of Histone Lysine Demethylases (KDMs) Identify Oncogenic KDM2A Isoform In Breast Cancer

BackgroundThe breast cancer is a kind of malignant tumor from mammary terminal ductlobular units epithelium. The incidence increased slowly during the past few decades,now ranks first in the female malignant tumor. The breast cancer often occurs in40-60years old female. In recent years, the molecular subtypes of breast cancer isdivided into four categories depending on estrogen receptor (estrogen receptor, ER),progesterone receptor (progesterone receptor, PR), human epidermal growth factor(human epidermal growth factor2, HER2) and molecular detection of Ki-67throughthe combining application of microarray technology and immunohistochemicalmethods: Luminal A (ER+/PR+, HER2-); Luminal B (ER+/PR+, HER2+);HER2overexpression (ER-/PR-, HER2+); Basal-like (ER-/PR-, HER2-, CK5/6+or EGFR+), and normal-like breast cancers. Both Luminal A and Luminal B breastcancers are estrogen receptor positive. The prognosis of Luminal A is good,butLuminal B cancers have poorer outcomes. Basal-like breast cancer is highlyaggressive, marked by high rates of relapse, visceral metastases, and poor prognoses.The development of breast cancer is a complex, multifactorial process traditionallyviewed as the stepwise accumulation of genetic alterations. However, it has becomeincreasingly apparent that epigenetic alterations, including histone modifications, playfundamental roles in cancer initiation and progression.Histone lysine demethylases (KDMs) consist of24enzymes that catalyzesite-specific demethylation of lysine residues on histones and other proteins. Theyplay critical roles in controlling transcription, chromatin architecture, and epigeneticsignalling. Structurally, the KDMs can be broadly categorized into two functionalenzymatic families. The first family, with two members (KDM1A/LSD1andKDM1B/LSD2), can only remove mono-and dimethylated (me1/me2) lysine residues of histone through an amine oxidative reaction. The second family, referred to as theJumonji C (JmjC) domain-containing demethylases, employs an oxygenasemechanism to demethylate mono-, di-, and trimethylated (me1/me2/me3) lysineresidues. In humans,32proteins belong to the JmjC domain-containing proteinfamily. Among them,22proteins have been shown to demethylate histone lysineresidues in vitro and in vivo. Based on sequence homologies and structuralsimilarities, these22JmjC domain-containing demethylases can be categorized intoseven functionally divergent protein subfamilies (KDM2-8). Notably, each subfamilyof JmjC demethylases exhibits different substrate specificity toward different histonelysine residues. For example, the KDM2subfamily specifically targetsH3K36me2/me1methylation marks, the KDM4subfamily targets H3K9me3/me2andH3K36me3/me2, and the KDM5subfamily targets H3K4me3/me2marks. In general,H3K4and H3K36marks are associated with gene activation, whereas H3K9andH3K27marks are linked to gene repression. Given the correlation between particularmethyl marks and the transcriptional state of genes, it has been proposed that theactivity of specific KDMs contributes to different transcriptional and biologicaloutcomes, depending on the KDM substrate.Accumulating evidence indicates that genetic and epigenetic events in cancercells operate interdependently. Many lines of evidence have suggested that geneticalteration and dysregulation of KDMs are associated with breast cancer. TheKDM4C gene, originally termed GASC1(gene amplified in squamous cell carcinoma1), was identified and cloned from the9p24amplified region of esophageal cancercell lines. We previously demonstrated that KDM4C is significantly amplified andoverexpressed in aggressive basal-like breast cancers and functions as a transformingoncogene. KDM5A is amplified and overexpressed in breast cancer and is associatedwith a drug-resistant phenotype. Yet, many vital questions remain regarding thelandscape of genomic alterations, their clinical significance, and the molecularmechanisms of these histone-modifying enzymes in breast cancer. The lack of suchknowledge represents a major impediment to developing novel therapeutic inhibitorsthat selectively target these enzymes, which could provide substantial benefit for adistinct class of breast cancer patients.However, our knowledge regarding thespecificity of KDM demethylases in different subtypes of breast cancer remainslargely incomplete. The genomic profiles and clinical significance of KDMs in breast cancer is still not clear. This study integrate genomic and functional analyses ofhistone demethylases identify oncogenic KDM2A isoform in breast cancer.Methods and ResultsFirst, we conducted a meta-analysis of24KDMs in breast cancer in976casesfrom TCGA database and identified associations among recurrent DNA copy numberalterations, gene expression, breast cancer subtypes, and clinical outcome. Wedemonstrated that KDM2A and KDM5B had the higher frequency of geneticamplification and overexpression compared with other KDMs independent of subtype.Furthermore, we revealed that overexpression of KDM2A was significantlyassociated with shorter survival of breast cancer patients.Second，Detailed characterization of KDM2A in breast cancer revealed that theshort isoform of KDM2A is more abundant than the long isoform at DNA, mRNA,and protein levels through Comparative genomic hybridization (CGH) array data、Western Blot and Real time PCR methods. In addition, Knockdown of KDM2A byshRNA in breast cancer cell lines ZR75-1and T47D which overexpress KDM2A caninhibit cell proliferation and transforming phenotypes tested by MTT、Softagar andInvasion array. But it can enhance growth of the nontumorigenic MCF10A mammaryepithelial cells, and no significant effect on the growth in breast cancer cell linesSUM52and SUM225which have no amplification of KDM2A.At last, KDM2A has two isoforms and short isoform. We knockdown the longisoform and short isoform of KDM2A with separate SiRNA. The data indicate thatthe short isoform, but not long isoform, of KDM2A, has potential carcinogenicity andfunctions as an oncogenic factor.In addition, after knockdown KDM2A in the breast cancer cell lines ZR75-1withshRNA, we performed RNA sequencing and Real-time PCR and found severalKDM2A target genes, including BRAF and FABP5.ConclusionIn976cases of breast cancer specimens, KDM5B and KDM2A have higher geneamplification and overexpression compared with other KDMs. In addition, BetweenDNA copy number and mRNA expression of KDM2A has significantly positivecorrelation and there is more significant correlation between high expression ofmRNA of KDM2A and shorter survival of breast cancer patients. In a subset of breast cancers, the short isoform of KDM2A is more abundant than the long isoform at DNA,mRNA and protein levels. The genetic and functional analyses of KDM2A indicatethat its short isoform can maintain the malignant phenotype of breast cancer cells.