Epigenetic silencing of miR-296 and miR-512 ensures hTERT dependent apoptosis protection and telomere maintenance in basal-type breast cancer cells.

Roberto Dinami,Valentina Buemi,Andrea Sacconi,Antonina Zappone,Eleonora Petti,Mauro Giacca,Silvano Piazza,Roberta Benetti,Stefan Schoeftner,Giovanni Blandino,Miguel Mano,Rosanna Sestito,Yari Ciani

doi:10.18632/oncotarget.21180

Abstract

The catalytic subunit of the telomerase complex, hTERT, ensures unlimited proliferative potential of cancer cells by maintaining telomere function and protecting from apoptosis. Using a miRNA screening approach we identified miR-296-5p and miR-512-5p as miRNAs that target hTERT in breast cancer cells. Ectopic miR-296-5p and miR-512-5p reduce telomerase activity, drive telomere shortening and cause proliferation defects by enhancing senescence and apoptosis in breast cancer cells. In line with the relevance of hTERT expression for human cancer we found that miR-296-5p and miR-512-5p expression is reduced in human breast cancer. Accordingly, high expression of miR-296-5p and miR-512-5p target genes including hTERT is linked with significantly reduced distant metastasis free survival and relapse free survival of basal type breast cancer patients. This suggests relevance of the identified miRNAs in basal type breast cancer. Epigenetic silencing of miR-296 and miR-512 encoding genes is responsible for low levels of miR-296-5p and miR-512-5p expression in basal type breast cancer cells. Disrupting gene silencing results in a dramatic upregulation of miR-296-5p and miR-512-5p levels leading to reduced hTERT expression and increased sensitivity to the induction of apoptosis. Altogether, our data suggest that epigenetic regulatory circuits in basal type breast cancer may contribute to high hTERT levels by silencing miR-296-5p and miR-512-5p expression, thereby contributing to the aggressiveness of basal type breast cancer.

Highlights

Vertebrate telomeres consist of TTAGGG tandem repeats that are localized at chromosome ends and protect linear chromosomes from eliciting a DNA damage response [1, 2]
We found that among candidate Micro RNAs (miRNAs) that mediate at least 50% reduction of hTERT 3’UTR reporter activity, only miR-296-5p, miR-512-5p and miR1207-5p showed significant down-regulation in breast cancer when compared to healthy tissue (Figure 1A, 1C, Supplementary Figure 2A)
This is consistent with the requirement of telomerase expression in cancer cells and anticipates clinical relevance for miR296-5p and miR-512-5p. miR-296-5p is encoded by the lincRNA Nespas that is transcribed from parental allele of the imprinted GNAS cluster located at chromosome 20q13.3 [53]. miR-296-5p has been shown to suppress cancer progression, metastasis, and neo-vascularization by targeting the expression of multiple genes including HMGA1, PUMA and SCRIB [35, 37, 54]

Summary

Introduction

Vertebrate telomeres consist of TTAGGG tandem repeats that are localized at chromosome ends and protect linear chromosomes from eliciting a DNA damage response [1, 2]. Performing a high-throughput luciferase reporter screen we show that miR-296-5p and miR-512-5p efficiently target the 3’UTR of hTERT Both miRNAs are donwnregulated in breast cancer. Ectopic miR-296-5p and miR-512-5p reduce telomerase activity, impair telomere maintenance and promote senescence and apoptosis in basal breast cancer cells. Disrupting miRNA gene silencing by the use of epigenetic drugs causes a dramatic miR-296-5p and miR-512-5p upregulation and concomitant reduction of hTERT expression that reduces the resistance to apoptotic stimuli. This suggests that miR-296-5p and miR-5125p execute epigenetic programs that control hTERT expression in breast cancer cells

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Conclusion