TERT-locus single nucleotide polymorphisms (SNPs) and leucocyte telomere measures are reportedly associated with risks of multiple cancers. Using the iCOGs chip, we analysed ~480 TERT-locus SNPs in breast (n=103,991), ovarian (n=39,774) and BRCA1 mutation carrier (11,705) cancer cases and controls. 53,724 participants have leucocyte telomere measures. Most associations cluster into three independent peaks. Peak 1 SNP rs2736108 minor allele associates with longer telomeres (P=5.8×10 −7 ), reduced estrogen receptor negative (ER-negative) (P=1.0×10 −8 ) and BRCA1 mutation carrier (P=1.1×10 −5 ) breast cancer risks, and altered promoter-assay signal. Peak 2 SNP rs7705526 minor allele associates with longer telomeres (P=2.3×10 −14 ), increased low malignant potential ovarian cancer risk (P=1.3×10 −15 ) and increased promoter activity. Peak 3 SNPs rs10069690 and rs2242652 minor alleles increase ER-negative (P=1.2×10 −12 ) and BRCA1 mutation carrier (P=1.6×10 −14 ) breast and invasive ovarian (P=1.3×10 −11 ) cancer risks, but not via altered telomere length. The cancer-risk alleles of rs2242652 and rs10069690 respectively increase silencing and generate a truncated TERT splicevariant.

Long noncoding RNAs (lncRNAs) are increasingly recognized to play major regulatory roles in development and disease. To identify novel regulators in breast biology, we identified differentially regulated lncRNAs during mouse mammary development. Among the highest and most differentially expressed was a transcript (Zfas1) antisense to the 59 end of the protein-coding gene Znfx1. In vivo, Zfas1 RNA is localized within the ducts and alveoli of the mammary gland. Zfas1 intronically hosts three previously undescribed C/D box snoRNAs (SNORDs): Snord12, Snord12b, and Snord12c. In contrast to the general assumption that noncoding SNORD-host transcripts function only as vehicles to generate snoRNAs, knockdown of Zfas1 in a mammary epithelial cell line resulted in increased cellular proliferation and differentiation, while not substantially altering the levels of the SNORDs. In support of an independent function, we also found that Zfas1 is extremely stable, with a half-life >16 h. Expression analysis of the SNORDs revealed these were expressed at different levels, likely a result of distinct structures conferring differential stability. While there is relatively low primary sequence conservation between Zfas1 and its syntenic human ortholog ZFAS1, their predicted secondary structures have similar features. Like Zfas1, ZFAS1 is highly expressed in the mammary gland and is down-regulated in breast tumors compared to normal tissue. We propose a functional role for Zfas1/ ZFAS1 in the regulation of alveolar development and epithelial cell differentiation in the mammary gland, which, together with its dysregulation in human breast cancer, suggests ZFAS1 as a putative tumor suppressor gene.

The entry of calcium into the mammary epithelial cell from the maternal plasma (i.e., calcium influx mechanisms) during lactation is poorly understood. As alterations in calcium channels and pumps are a key feature of some cancers, including breast cancer, understanding these calcium influx pathways may have significance beyond mammary biology. We show that the store-operated calcium influx protein, Orai1, is increased during lactation whereas the Orai1 activator Stim1, but not Stim2, is downregulated. Stim2 siRNA reduced basal calcium levels in a lactation model. Our results suggest that calcium influx is remodeled in mammary epithelial cells during lactation, with calcium influx increased through Orai1, activated by Stim2. Breast cancer cell lines had increased levels of ORAI1. ORAI1 siRNA in breast cancer cells reduced storeoperated calcium entry and remodeled the calcium influx associated with invasive stimuli. Analysis of microarray data from 295 breast cancers showed that the transcriptional breast cancer subtype with the poorest prognosis (basal) was associated with an altered relationship between the ORAI1 regulators STIM1 and STIM2, and that women with breast cancers with STIM1 high /STIM2 low tumors had a significantly poorer prognosis. Our studies show that during lactation there is a remodeling in the nature of calcium influx and that alteration in the ORAI1 influx pathway may be a feature of some breast cancers, particularly those with the poorest prognosis. Our studies suggest that this pathway may be a novel therapeutic target for breast cancer treatment in these women. Mol Cancer Ther; 10(3); 448-60. Ó2011 AACR.

The transcription factor SOX2 is essential for maintaining pluripotency in a variety of stem cells. It has important functions during embryonic development, is involved in cancer stem cell maintenance, and is often deregulated in cancer. The mechanism of SOX2 regulation has yet to be clarified, but the SOX2 gene lies in an intron of a long multi-exon non-coding RNA called SOX2 overlapping transcript (SOX2OT). Here, we show that the expression of SOX2 and SOX2OT is concordant in breast cancer, differentially expressed in estrogen receptor positive and negative breast cancer samples and that both are up-regulated in suspension culture conditions that favor growth of stem cell phenotypes. Importantly, ectopic expression of SOX2OT led to an almost 20-fold increase in SOX2 expression, together with a reduced proliferation and increased breast cancer cell anchorage-independent growth. We propose that SOX2OT plays a key role in the induction and/or maintenance of SOX2 expression in breast cancer.