Mir-128 Regulates the Self-Renewal of Breast Tumor-Initiating Cells through HTERT.

Abstract

Abstract Background: hTERT (human telomerase reverse transcriptase), which maintains telomere ends by addittion of the telomere repeat sequence TTAGGG, is increased in cancer cells and embryonic stem cells. However, whether hTERT has a role in regulating breast tumor-initiating cells remains unknown. Therefore, the regulation of hTERT in breast tumor initiating cells (BT-IC) would potentially facilitate our understanding of the etiology of breast cancer.Material and Methods: Enrichment for BT-ICs was performed by mammospheres from suspension cultures. The capacity of self-renewal and differentiation of BT-ICs were determined by mammosphere formation, immunohistochemical analysis of epithelial markers and FACS, respectively. TRAP assay was used to detect telomerase activity. The expression levels of hTERT in BT-ICs, with or without lentivirus-mediated mir-128 transduction, were determined by qRT-PCR and western blot. hTERT siRNA or lentivirus-shTERT were used to silence the expression of hTERT in BT-ICs. Differentially expressed mir-128 in BT-ICs was quantified by qRT-PCR and Northern Blot. The expression of BMI1 was determined by Western Blot.Results: hTERT, as well as telomerase activity, had a higher expression in BT-ICs, compared to the differentiated breast cancer cells. BMI1, the target of mir-128, is increased in BT-ICs. Conversely, the expression level of mir-128 is lower in BT-ICs, compared to the differentiated cells. Mir-128 over-expressing reduced the level of BMI1,as well as hTERT, in BT-ICs. Silencing BMI1 by lentivirus-shRNA transfection in BT-ICs leads to the decrease of hTERT protein level. Furthermore, BT-ICs, with hTERT knockdown, lost its ability to form mammospheres and proliferated poorly under differentiating conditions.Conclutions: mir-128 miRNA is reduced in mammospheric BT-ICs, resulting in up-regulation of BMI1, which further brought on the up-regulation of hTERT. And hTERT promotes self-renewal and blocks epithelial differentiation of BT-ICs. Therefore, mir-128 regulates BT-IC stem-cell like properties by BMI1 and hTERT. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5157.