A lincRNA connected to cell mortality and epigenetically-silenced in most common human cancers

- Gene expression changes linked to two step immortalization of human mammary epithelial cells (HMEC). - A lincRNA, MORT, associated with the mortal state, was identified - MORT is silenced by DNA methylation in immortal mammary epithelial cells Abstract: Immortality is an essential characteristic of human carcinoma cells. We recently developed an efficient, reproducible method that immortalizes human mammary epithelial cells (HMEC) in the absence of gross genomic changes by targeting two critical senescence barriers. Consistent transcriptomic changes associated with immortality were identified using microarray analysis of isogenic normal finite pre-stasis, abnormal finite post-stasis, and immortal HMECs from four individuals. 277 genes consistently changed in cells that transitioned from post-stasis to immortal. Gene ontology analysis of affected genes revealed biological processes significantly altered in the immortalization process. These immortalization-associated changes showed striking similarity to the gene expression changes seen in The Cancer Genome Atlas (TCGA) clinical breast cancer data. The most dramatic change in gene expression seen during the immortalization step was the down-regulation of an unnamed, incompletely annotated transcript that we called MORT, for mortality, since its expression was closely associated with the mortal, finite lifespan phenotype. We show here that MORT (ZNF667-AS1) is expressed in all normal finite lifespan human cells examined to date and is lost in immortalized HMEC. MORT gene silencing at the mortal/immortal boundary was due to DNA hypermethylation of its CpG island promoter. This epigenetic silencing is also seen in human breast cancer cell lines and in a majority of human breast tumor tissues. The functional importance of DNA hypermethylation in MORT gene silencing is supported by the ability of 5-aza-2’-deoxycytidine to reactivate MORT expression. Analysis of TCGA data revealed deregulation of MORT expression due to DNA hypermethylation in 15 out of the 17 most common human cancers. The epigenetic silencing of MORT in a large majority of the common human cancers suggests a potential fundamental role in cellular immortalization during human carcinogenesis. The aim of the study was to determine what genes change in expression during the individual steps of the targeted model immortalization of HMEC. Design: Post-Stasis vs Pre-Stasis, and Immortal vs Post-Stasis. Biological replicates: 4 HMEC genotypes