Stem Cell Reports Report The Luminal Progenitor Compartment of the Normal Human Mammary Gland Constitutes a Unique Site of Telomere Dysfunction Nagarajan Kannan,1,5 Nazmul Huda,2,5 LiRen Tu,2 Radina Droumeva,1 Geraldine Aubert,1 Elizabeth Chavez,1 Ryan R. Brinkman,1 Peter Lansdorp,1,3 Joanne Emerman,4 Satoshi Abe,2 Connie Eaves,1,5,* and David Gilley2,5,* 1Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC V5Z 1L3, Canada 2Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202-5251, USA 3European Research Institute for the Biology of Ageing, University Medical Center Groningen, and University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands 4Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC V6T 1Z3, Canada 5These authors contributed equally to this work. *Correspondence: [email protected] (C.E.), [email protected] (D.G.) http://dx.doi.org/10.1016/j.stemcr.2013.04.003 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Telomeres are essential for genomic integrity, but little is known about their regulation in the normal human mammary gland. We now demonstrate that a phenotypically defined cell population enriched in luminal progenitors (LPs) is characterized by unusually short telomeres independently of donor age. Furthermore, we find that multiple DNA damage response proteins colocalize with telomeres in >95% of LPs but in <5% of basal cells. Paradoxically, 25% of LPs are still capable of exhibiting robust clonogenic activity in vitro. This may be partially explained by the elevated telomerase activity that was also seen only in LPs. Interestingly, this potential telomere salvage mechanism declines with age. Our findings thus reveal marked differences in the telomere biology of different subsets of primitive normal human mammary cells. The chronically dysfunctional telomeres unique to LPs have potentially important implications for normal mammary tissue homeostasis as well as the development of certain breast cancers. INTRODUCTION normal human mammary epithelial cells with distinct bio- logical properties have markedly different telomere lengths Chromosome ends, referred to as telomeres, contain repeat and telomerase activities. Interestingly, a phenotype that sequences (TTAGGG)n and associated proteins that protect is highly enriched in luminal progenitors (LPs) is uniquely cells from the formation of chromosome end-to-end characterized by critically short telomeres, frequent evi- fusions (Artandi and DePinho, 2010). In normal tissues, dence of a telomere-specific DNA damage response (DDR), such as in the hematopoietic system, where cell turnover and an age-related decrease in telomerase activity. is high and continuous throughout life, telomeres are main- tained in the most primitive cells at relatively long lengths and then become progressively shorter as the cells differen- RESULTS AND DISCUSSION tiate through multiple amplifying divisions and with age (Aubert and Lansdorp, 2008). Epithelial tissues, including Normal Human Mammary LPs Possess Short the mammary gland in both humans and mice, also un- Telomeres dergo extensive turnover, and recent studies indicated To examine telomere length in different compartments of that this may involve a similarly organized hierarchical normal human breast tissue and possible age-related differentiation process (Eirew et al., 2008; Visvader, 2009). changes, we isolated four phenotypically distinct subsets To date, analysis of telomere length regulation in normal of cells at high purities (>95%) from different normal reduc- mammary epithelial cells has been limited to reports of tion mammoplasty tissue samples obtained from donors of shorter telomeres in luminal cells (Kurabayashi et al., different ages and analyzed them directly, without culture 2008; Meeker et al., 2004), and human telomerase reverse (Figure 1A; Table S1 available online). We examined transcriptase (hTERT) messenger RNA (mRNA; Kolquist four subsets of cells: (1) a basal epithelial cell (BC) subset et al., 1998) in histological sections. However, the presence that is highly enriched in cells with bipotent as well as of telomere fusions was noted in primary mammary epithe- myoepithelial clonogenic activity in vitro (Figure 1B), (2) lial cells after their extensive passage in vitro (Romanov an LP subset that is similarly enriched in cells with luminal et al., 2001), and we recently reported that telomere- clonogenic activity in vitro (Figure 1B), (3) a third mam- dysfunction-specific chromosomal fusions are common mary epithelial cell subset that contains exclusively mature in early-stage breast cancers (Tanaka et al., 2012). Here luminal cells (LCs) with no clonogenic activity (Figure 1B), we show that phenotypically separable compartments of and (4) a population consisting of nonepithelial stromal 28 Stem Cell Reports j Vol. 1 j 28–37 j June 4, 2013 j ª2013 The Authors Stem Cell Reports Telomere Dysfunction in Breast Luminal Progenitors A Depletion of dead/blood/ endothelial cells CFC assay Fix, stain & score ~10 days Single cell Staining Isolation LC LP Luminal colony dissociation of subsets Normal mammary SC EpCAM BC tissue /CD31/CD45 Snap freeze Basal colony DAPI for telomere analysis SSC CD49f B C D 12 BC 12 LP 12 LC 12 SC 100 12 10 10 R² = 0.002 10 R² = 0.5 10 P = 0.9 P < 0.01 (kb) RF (kb) R R C C 10 10 8 8 8 8 1 8 6 6 6 6 0.1 R² = 0.05 R² = 0.08 6 Percentage of Percentage CF Telomere length - length Telomere T P = 0.5 P = 0.3 R² = 0.75 P < 0.0001 Telomere length - length Telomere qPC 0.01 4 4 4 4 4 BC LP LC 0 255075 0 255075 0 255075 0255075 4681012 Telomere length - TRF (kb) Age (years) Age 463146 20 26 E H Unstained BC Unstained LP Stained BC Stained LP Thymocytes Thymocytes HT1080 ladder EEC EEC TC TC SC SC SC BC LP LC BC LP LC BC LP LC 100 BC LP LC 80 Age 46 20 26 60 e (kb) maximum e of 40 23.1 20 Percentag 12.0 0 10.0 0 200 400 600 800 1000 0 200 400 600 800 1000 9.0 8.0 Telomere-PNA-FITC probe 7.0 6.0 I 5.0 4.3 4.0 3.0 2.4 2.0 0 100 0100100 0 % Maximum 1.5 BC LP 1.0 J 200 BC mean 6.3 kb FGFRT RCPq LP mean 3.4 kb P = 0.004 150 12 P < 0.001 12 11 11 P = 0.004 P < 0.001 (kb) (kb) 10 10 h h h 100 9 9 8 8 Frequency 7 7 50 6 6 5 5 Telomere lengt Telomere lengt 4 4 0 BC LP LC SC BC LP LC SC 0 3 6 9 12 15 18 21 24 27 30 Telomere fluourescene units (x1000) Figure 1. Telomere Length Alterations in Different Subsets of Cells Present in Normal Human Breast Tissue (A) Process for isolating and characterizing the four subsets of cells studied (after removal of CD45+ hematopoietic and CD31+ endothelial cells), showing examples of colonies obtained from cells in the LP (above) and BC (below) fractions. BCs are defined by their CD49fhiEpCAMneg/low phenotype and contain bipotent and self-renewing mammary stem cells identified by in vivo transplantation assays (legend continued on next page) Stem Cell Reports j Vol. 1 j 28–37 j June 4, 2013 j ª2013 The Authors 29 Stem Cell Reports Telomere Dysfunction in Breast Luminal Progenitors cells (SCs) that are still prominent after the hematopoietic mammary epithelial cells (on average 9 kb by southern and endothelial cells are removed (Figure 1A). Extracts of blot analysis), and also do not show significant changes in each of these four highly purified cell populations were telomere length with age (Figures 1C and 1E–1G). analyzed for telomere length both by quantitative phos- The age-related changes in telomere length observed in phorimage scanning of telomere restriction fragment the isolated LCs were masked in southern analyses of (TRF) lengths in southern blots (Aviv et al., 2011; Cawthon, DNA extracts obtained from unseparated breast tissue 2009) and by quantitative PCR (qPCR; Aviv et al., 2011; (Figure 1E, lane marked TC). This likely reflects the low Cawthon, 2009; Figures 1C–1G). To evaluate telomere representation of the LCs in whole breast tissue relative length values for individually analyzed cells in the BC and to the predominance of other cells (Figure 1A) regardless LP subsets, we used flow-fluorescent in situ hybridization of donor age. The discovery of short telomeres specific to (Flow-FISH) to examine freshly isolated cells (Rufer et al., the LP compartment is notable given that a high propor- 1998; Figure 1H) and quantitative FISH (Q-FISH) to examine tion of cells with an LP phenotype (up to 48%; Figure 1B) metaphases obtained on the proliferating progeny of LPs can execute multiple divisions in vitro. and BCs present in 3-day adherent cultures of these cells (Poon et al., 1999; Figures 1I and 1J). The striking finding LPs Display Evidence of a Telomere-Associated DDR from all of these analyses is the very short average telomere without Telomere Fusions length that uniquely characterizes LP cells, regardless of the To determine whether the short telomeres characteristic age of the donor. The southern data imply that some chro- of normal human LPs are sufficient to elicit a DDR, we mosomes within the LP population likely contain TRFs <3 first performed microarray analyses on RNA extracted kb in length (Figure 1F) and this was also evident from the from BC, LP, and LC populations purified from young Q-FISH data (Figure 1J). This telomere length has been asso- (premenopausal, 20–49 years old, n = 6) and older donors ciated with telomere dysfunction and cell death in the (postmenopausal, 59–68 years old, n = 3).