FAM3B promoter hypermethylation. A) FAM3B promoter contains a CpG island, and its methylation levels correlate negatively with FAM3B expression in The Cancer Genome Atlas (TCGA) prostate cancers. Top, FAM3B CpG island (green bar) on the University of California Santa Cruz (UCSC) genome browser. Middle, CpG island and 5’ end of FAM3B in higher magnification. Bottom, methylated CpG sites from TCGA prostate cancer methylation array data and the Spearman correlation coefficients between the methylation levels of each CpG site and FAM3B mRNA levels in TCGA prostate cancers (n = 497). B. In both fusion-negative and -positive TCGA prostate cancers, high methylation of the cg18440523 CpG site is associated with fast disease progression. High and low groups are separated based on the median methylation level. C) Bisulfite amplicon sequencing shows that decitabine treatment of VCaP and C4-2 cells decreases cytosine methylation in cg18440523 (methylated cytosine remains intact after bisulfite conversion). D, E) Decitabine treatment increases FAM3B expression in VCaP (D) and C4-2 cells (E). RNA and protein were assessed by real-time quantitative PCR capillary Western analysis, respectively. F, G) Decitabine treatment inhibits the colony-forming ability of VCaP (F) and C4-2 cells (G). Decitabine = 1 uM. P values in panel B from log-rank test. P values in other panels from Welch t test. AR = androgen receptor; PC = prostate cancer; PFS = progression-free survival; DMSO = dimethyl sulfoxide; T = thymine; C = cytosine.