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Seminars in Cell & Developmental Biology 23 (2012) 606– 612

Contents lists available at SciVerse ScienceDirect

Seminars in Cell & Developmental Biology

j ourna l ho me pag e: www.elsev ier .com/ locate /semcdb

eview

he biology of human breast epithelial progenitors

fshin Raouf ∗, Yujia Sun, Sumanta Chatterjee, Pratima Basakepartment of Immunology, Faculty of Medicine, University of Manitoba and Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, Manitoba, Canada

r t i c l e i n f o

rticle history:vailable online 17 May 2012

eywords:ierarchy of human breast cells

a b s t r a c t

Current evidence suggests that similar to other tissues in the human body mammary epithelia cells arebeing maintained by the unique properties of stem cells, undifferentiated as well as lineage-restrictedprogenitors. Because of their longevity, proliferation and differentiation potentials these primitive breastepithelial cells are likely targets of transforming mutations that can cause them to act as cancer initiating

uman breast progenitorsipotent progenitorsuminal-restricted progenitorsyoepithelial-restricted progenitors

cells. In this context, understanding the molecular mechanisms that regulate the normal functions ofthe human breast epithelial stem cells and progenitors and how alterations to these same mechanismscan confer a cancer stem cell phenotype on these rare cell populations is crucial to the development ofnew and more effective therapies again breast cancer. This review article will examine the current stateof knowledge about the isolation and characterization of human breast epithelial progenitors and theirrelevance to breast cancer research.

© 2012 Elsevier Ltd. All rights reserved.

ontents

1. Isolation and characterization of breast epithelial progenitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6062. Role of progenitor subtypes in mammary gland biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6073. The molecular determinants of the bipotent and luminal progenitor cell functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608

3.1. Oncogenes and transcription factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6083.2. Endocrine factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6093.3. Non-protein coding RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610

4. Role of progenitors in breast carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6105. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611

. Isolation and characterization of breast epithelialrogenitors

Human mammary gland is a dynamic organ in that it under-oes major changes post puberty and throughout life. Becausef difficulties in obtaining breast tissue at different developmen-al stages, much of what is known about the human mammaryland development is extrapolated from rodent studies. For theurposes of this review article, every effort has been made to sum-

filled with an elaborated network of ducts and alveolar structures.The functional cells in these mammary structures are luminal andmyoepithelial cells [1–3]. While the luminal cells in the alveolarstructures have the ability to further differentiate into milk pro-ducing cells, the myoepithelial cells are muscle-like cells that inthe presence of oxytocin, contract to force the movement of themilk from the alveolar structures into the ducts to be ejected fromthe nipples [1–4].

Interestingly, during each menstrual cycle, these breast epithe-

arize human breast development. Pre-pubertal male and femaleammary glands are rather analogous in anatomy and cellularity

1]. Post puberty however, under the influence of sex hormonesuch as Estrogen and Progesterone, the female mammary gland is

∗ Corresponding author at: 471 Apotex Centre, 750 McDermot Avenue, Winnipeg,anitoba, Canada R3E 0T5.

E-mail address: raouf@cc.umanitoba.ca (A. Raouf).

084-9521/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.semcdb.2012.04.009

lial cells expand in number (up to 2 fold expansion) andsubsequently retract [5–7]. During pregnancy however, thisepithelium compartment can significantly expand. When the pro-liferation index (PI) of non-pregnant human mammary gland(1.6–4.4) was compared to the pregnant gland (at 15th week PI

as high as 17.6 has been recorded) a 9–10 fold expansion of theepithelial cells can be observed [8]. This increased is mostly due tothe proliferation of the luminal cells since only 2% of the myoep-ithelial cells were reported to be proliferating [8]. Upon weaning

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A. Raouf et al. / Seminars in Cell & D

he gland regresses back to its pre-pregnant stage through a pro-ess know as involution [2,3]. These multiple cycles of expansionsnd regression of the mammary epithelium suggests that humanreast epithelium has an enormous regenerative potential. Theegenerative capability of mammary gland is further demonstratedhrough experiments performed by DeOme and et al. where theyhowed that different parts of the mouse mammary tree havehe ability to regenerate the entire mammary gland [9]. Sub-equent experiments through retro-viral marking of the mouseammary epithelial cells showed the clonal origin of these mam-ary structures [10]. More recently, the single-cell injection of

D24low �6 integrin (CD49f)bright mouse mammary epithelial cellsnto epithelium-cleared mammary fat pads of mice showed thebility to recapitulate the entire mammary gland [11]. Similaresults were found when �1 integrin (CD29)bright CD24low mousereast epithelial cells were injected into epithelium-free mouseammary fat pads as single-cell [12]. Serial transplantation of these

ngrafted cells (up to 3 times) suggests that these cells are haveelf-renewal capacity. Together these experiments suggested thatammary epithelial cells are being continuously replenished by

are subpopulation of cells with stem cell properties. Recently pub-ished data obtained from the mouse mammary gland challengeshe common origin of the luminal and the myoepithelial cells, sug-esting that these cells each may be continuously derived fromistinct and self-renewing populations of stem cells. The biolog-

cal relevance of this finding and whether it is also applicable tohe hierarchical structure of the human mammary epithelial cellsemains to be seen [13].

The notion that the human breast epithelial cells are clonaln origin was initially based on X-chromosome inactivation pat-erns of the mammary gland where large localized clones thatontained both the luminal and the myoepithelial cells wherebserved [14]. Current evidence indicates that similar to mouse,he human mammary epithelial cells are also derived from a pop-lation of cells with stem cell properties. The experiments thatrovided proof to the existence of human breast epithelial stemells however, proved difficult due to the differences in the require-ent for extracellular matrix that surrounds the breast structures

n mice compared to the humans mammary glands [3,15,16].ndeed, the detection and characterization of human breast epithe-ial stem cells was made possible when in a kidney xenograft

odel where the Epithelial Cell Adhesion Molecule (EpCAM)low

D49fbright subpopulation of human breast cells were placed in col-agen gels filled with irradiated human breast fibroblasts [3,17,18].n such highly vascularized and matrix-enriched environment,he EpCAMlow CD49fbright cells were able to produce bi-layereductal and alveolar structures [17,18]. The parallel transplantsf these xenografts demonstrated that the EpCAMlow CD49fbright

ave self-renewal capacity [17]. Such experiments suggested thatpCAMlow CD49fbright subpopulation of human breast epithelialells contain cells with bi-lineage differentiation, proliferation andelf-renewal capacities that are hallmark of breast epithelial stemells.

The proof that human breast epithelial cells are organizedn a lineage hierarchy was provided by in vitro experiments

here low (clonal)-density cultures of normal adult human breastells lead to the detection of 3 distinct primitive cell phe-otype: a myoepithelial-restricted colony forming cell (CFC), a

uminal-restricted CFC, and an uncommitted bipotential CFC thatenerated colonies consisting of both luminal and the myoepithe-ial cells [19,20]. These colony-forming cells with the exceptionf myoepithelial-restricted CFCs, were shown to be phenotypicaly

istinct suggesting that these CFCs represent different populationsf breast epithelial progenitors that correspond to earlier stagesf human breast development [21]. The molecular characteriza-ion of these distinct progenitor subtypes however requires them

mental Biology 23 (2012) 606– 612 607

to be isolated at high and specific purities. To this end, it was laterdemonstrated that the unique progenitors of the human breast canbe isolated at high purities from EpCAM+CD49f+ subpopulationsof pre-cultured (up to 54 h) reduction mammoplasty samples [22].Interestingly the CD49− cells from the same preps were devoid ofany cells capable of colony formation, suggesting that � 6 integrinplays a crucial role regulating the undifferentiated cell func-tions. In these experiments the basal (Muc1-AC133−(CD10/Thy1)+)subset of EpCAM+ CD49f+ cells allowed the bipotent progeni-tors to be isolated at nearly 45% purity. Similarly the luminal((Muc1/AC133)+CD10−Thy1−) subset of the EpCAM+ CD49f+ cellsprovided a 33% pure luminal-restricted progenitor population[22]. These progenitor subpopulations thus isolated, included lessthan 5% contaminants of other progenitors, which made theircharacterization at a molecular level possible. It is noteworthythat the relationship between the purified bipotent progenitor(EpCAM+CD49f+ Muc1−AC133−(CD10/Thy1)+) and the bipotentialbreast epithelial stem cells (EpCAM(low)CD49f(bright)) cells is notclear and needs to be examined (Fig. 1).

The comparative transcriptome profiles of the bipotent andthe luminal-restricted progenitors and their differentiated progenyidentified unique molecular signatures for each of these purifiedsubpopulations and revealed the differential expression of severalgenes involved with important signaling pathways that have beenimplicated in breast cancer such as the NF-kappaB and the Notchand the WNT signaling pathways [22]. Interestingly these stud-ies also reveal the expression of a number of non-protein codingRNA such as H19 that also have been associated with breast car-cinogenesis. These findings suggest that elucidating the molecularmechanisms that control normal mammary gland development isessential to the understanding of how their perturbed expressioncan confer a cancer stem cell phenotype on these rare but biologi-cally relevant cells.

2. Role of progenitor subtypes in mammary gland biology

The in vivo and in vitro evidence discussed thus far suggeststhat human breast epithelial cells are arranged in a hierarchi-cal structure where the differentiated luminal and myoepithelialcells are continuously replaced by a population of uncommittedbipotential progenitors that are themselves produced by a self-renewing population of breast epithelial stem cells. These bipotentprogenitors differentiate into lineage-restricted progenitors thatultimately produce the differentiated luminal and the myoepithe-lial cells that make up the functional elements of the breast tissue.Interestingly, the mature luminal cells can further differentiateinto milk producing cells during the second phase of pregnancy[1–3]. It is noteworthy that to date no in vivo or in vitro dataexist to support the notion that bipotential progenitors can dif-ferentiate into lineage-restricted progenitors. Also at the momentthe isolation and characterization of the myoepithelial-restrictedprogenitors remains elusive due to lack of specific markers to dis-tinguish these cells from the bipotential progenitors. This may bein part due to the fact the bipotent progenitors and the differ-entiated myoepithelial cells (CD49f−(CD10/THY1)+) share a greatdeal of overlap in terms of their transcriptome profiles [22]. More-over, the experimental evidence to suggest that self-renewingpopulation of breast stem cells can indeed differentiate into bipo-tential progenitors is presently lacking. The hierarchical structureof the human breast epithelial cells therefore, is based on infer-

ences drawn from in vivo xenograft data that demonstrated theself-renewal capacity of EpCAMlowCD49fbright cells and in vitrodata that proved the existence of bi-lineage and lineage restrictedcells.

608 A. Raouf et al. / Seminars in Cell & Developmental Biology 23 (2012) 606– 612

Fig. 1. A diagrammatic depiction of the presumed hierarchical structure of the human mammary epithelial cells. This hierarchical structure is maintained by self-renewingpopulation of stem cells. Under the normal physiological conditions breast stem cells can undergo asymmetric self-renewal to produce a single bipotential progenitorcell as well as a copy of the parental stem cells. However under certain circumstances, breast stem cells can symmetrically divide to expand the stem cell pool (i.e. pre-pubertal mammary gland development). Bipotential progenitors appear to share many features of the mammary epithelial stem cells in that both can be purified by isolatingEpCAM+CD49f+ subsets of the human breast cells. However, in vivo xenograft assays have demonstrated that EpCAM+CD49f+ subset of uncultured human breast epithelialcells are enriched for cells with stem cell phenotype while isolating this subset from pre-cultured cells enriches for bipotent progenitors as well as the luminal-restrictedprogenitors. In subpopulation of cells thus isolated, the use of myoepithelial cell makers such as Thy1 or CD10 provides further enrichment towards the isolation of bipotentialprogenitors and the use of luminal cell markers such as Muc1 or CD133 provides enrichment for the luminal-restricted progenitors. Due to the lack of markers to furtherpurify breast epithelial stem cells or the bipotential progenitors, the exact relationship between to the bipotential progenitors and the breast stem cells cannot be determined.T thatd

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Deciphering the precise relationship between stem cells andhe different progenitor cells in the human breast is essential innderstanding their role in the normal breast development andheir possible contribution to breast cancer pathology (Fig. 1).

In the human mammary gland the luminal cells of the alveolartructures can further differentiate into milk producing cellshile the luminal cells that reside in the ducts do not. While it

s attractive to hypothesize that the ducts and alveolar structuresf the mammary gland originate from distinct progenitors, thexistence of such progenitors in vivo remains uncertain. Mammaryucts and alveolar structures both consist of the luminal andyoepithelial cells, though the luminal cells in the alveolar struc-

ures are surrounded by a discontinuous layer of myoepithelialells (Fig. 2) [23,24]. In such an environment, the luminal cellsre exposed to the extracellular matrix (ECM) that surroundsach alveolar structure [23,24]. The exposure of the luminal cellso the ECM may provide the necessary signals to facilitate theirurther differentiation into milk producing cells. In contrast, theuctal luminal epithelial cells are not exposed to the ECM signalss they are shielded by a contiguous layer of myoepithelial cells23]. It is therefore, possible to postulate that the ductal epithelialells are not exposed to the environmental signals that are con-ucive to lactogenesis. We are of the opinion that in the humanammary gland, the luminal cells of the ductal and the alveolar

tructures both originate from the luminal-restricted progeni-ors and that the environmental signals determine their furtherifferentiation abilities. This hypothesis needs to be validatedut the ability to isolated luminal-restricted progenitor-enrich

regulate the biological processes (i.e. lineage restriction, or self-renewal) that are

subpopulations from the human breast epithelial cells may proveuseful.

3. The molecular determinants of the bipotent and luminalprogenitor cell functions

Much is known about the signaling molecules that are involvedin the breast cancer survival and proliferation. However, very littleis known about the genes that are involved in breast cancer initi-ation. Such information can be used toward the detection of early,premalignant disease and is therefore highly desirable. To addressthis need we must first understand the molecular mechanisms thatregulate the unique functions (i.e. proliferation, differentiation andself-renewal) of the breast stem cells and their immediate progenyin the normal breast tissue. Such understanding then can be used toascertain how alteration to these same mechanisms can cause theprimitive cells of the breast tissue to act as cancer initiating cellsand later as tumor maintaining cells (i.e. cancer stem cells).

3.1. Oncogenes and transcription factors

The ability to isolate the bipotent and luminal-restricted pro-genitors at very high and specific purities from human breast tissueallowed their unique messenger RNA (mRNA) signatures to be

obtained. These signatures revealed the differential expression ofa number of genes involved in important signaling pathways thathave been implicated in breast cancer. Some of these pathwaysinclude; the Notch, the NF-�B and the Wnt signaling cascades. A

A. Raouf et al. / Seminars in Cell & Developmental Biology 23 (2012) 606– 612 609

Fig. 2. Myoepithelial cell coverage of the duct and aveolar structures in the human mammary gland. Discared tissue from the breast reduction surgeries where formalinfi specia el (A)l myoel lveola

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xed and embedded in paraffin. Cross sections from these blocks were stained withntibodies in red) or the luminal epithelial cells (anti MUC1 antibodies in green). Panayer around the luminal epithelial cells. Panel (B) shows the discontiguous layer ofayer of the myoepithelial cell surrounding the alveolar structures. The ducts and a

etailed analysis of the purified progenitor subtypes brought toight the differential expression of the different NOTCH receptors

here NOTCH4 receptor (NR4), a potent mammary oncogene, wasound to highly expressed in the undifferentiated bipotent progen-tors while NOTCH3 receptor (NR3) was highly expressed in theuminal-restricted progenitors.

Further experiments using the highly purified populationsreast epithelial progenitors showed that the differentiation of theommitted luminal progenitors is not dependent on the NOTCHignaling while lineage restriction of the bipotential progenitors tohe luminal cells required the Notch signaling [22]. In the sametudy, loss of function assays using short hairpin RNA speciesemonstrated that NR3 alone was sufficient to regulate luminalell fate during the differentiation of the bipotent progenitors. Suchon-redundant function of NR3 is a departure from the presentaradigm of NOTCH signaling and suggests that each receptor mayegulate the expression of unique target gene. The role of Notchignaling in regulating luminal cell-fate is corroborated by datarom the mouse models where complete loss of Notch signalinged to decreased luminal cell differentiation [25,26]. Interestingly,he loss of Notch 3 or Notch 4 Receptors in the mammary gland hasot resulted in distinct mammary phenotype in animal models [27],uggests that there are appreciable differences between human andouse mammary gland development in terms of Notch signaling.

t is interesting that Notch1 and Notch 3 receptors were found to beifferentially regulated by estrogen signaling in mice while Notch

and the Notch 4 receptors levels were unaffected [27]. It woulde interesting to determine if the expression of NOTCH receptors inuman breast epithelial cells are also regulated by estrogen signal-

ng and how alterations to estrogen signaling may related NOTCHignaling to estrogen responsive breast tumors.

In addition to the Notch signaling pathway, the mRNA signa-ures of the different subpopulations of the human breast epithelialells also revealed the differential expression of members of the

NT and the NF-�B signaling cascades. Interestingly the transcriptsor the NF-�B inhibitors such as NF-�B1A, NF-�B1Z, and IKBKEhere found to be highly present in the luminal-restricted pro-

enitors while the WNT signaling molecules (WNT4, WNT5B, and-Catenin) where highly expressed by the bipotential progenitors

22]. Given the significant cross talk between these important sig-aling molecules, it is imperative to ascertain their precise role in

ineage-determination, proliferation, and self-renewal capacity ofhe primitive breast cells.

.2. Endocrine factors

Steroid hormones such as estrogen and progesterone playssential roles in the development of pubertal mammary gland

fic antibodies to detect the myoepithelial cells (anti � smooth muscle actin [�SMA] shows the cross section of a duct where the myoepithelial cells exhibit a contiguouspithelial cells that surround the aveolar structures. Arrows show the discontiguousr structures where photographed at 40× magnification.

[1,3]. The role of these sex hormones is extensively studied in thecontext of mouse mammary gland development. Loss of estrogensignaling achieved through targeted gene knockout or utilizingOophorectomy surgeries to decrease systemic estrogen levels inmice, revealed that estrogen signaling is involved in ductal elon-gation and overall growth of the mammary gland [28,29]. Similarstudies showed that progesterone signaling is needed for branchingmorphogenesis and alveolar development [4,30–32].

The role of estrogen signaling in the human breast developmentis evidenced by the developmental differences in the male andfemale mammary glands. Up until puberty the male and femalesmammary gland development is very similar. However, at pubertydue to the surge of estrogen and progesterone, the female mam-mary gland develops into an integrate system of ducts and alveolarstructures [3,4,33]. During puberty the male mammary gland willremain at prepubertal state and contain rudimentary breast struc-tures only. In some physiologic or pathologic condition such asGynecomastia the unusually high levels of estrogen in combinationwith low levels of androgens lead to enlargement of male breast tis-sue [34,35], which suggest that estrogen plays a similar role in thehuman mammary gland development as in mice.

The biological effects of estrogen and progesterone hormonesare mediated through Estrogen Receptors (ER) and ProgesteroneReceptors (PR), which belong to the nuclear receptor superfamilyof transcription factors. Two estrogen receptors have been iden-tified thus far ER� and ER� [36–38]. Interestingly, these estrogenreceptors show very different expression patterns in the breast tis-sue. ER� shows the widest expression pattern, as it is present in theluminal and the myoepithelial cells as well as the stromal fibroblastcells of the human breast tissue [39,40]. ER� on the other hand ismuch more discrete in it expression pattern [39]. Depending on themenstrual cycle, 4–20% of the luminal epithelial cells in the humanbreast tissue express ER� [41,42]. Interestingly, these ER�+ cellsare only found in isolation surrounded by ER�− cells [41,43].

Progesterone signaling is transduced through activation ofPRA and PRB that are transcribed from the same gene throughalternative promoters [44]. While both PRA and PRB can activateexpression of target genes, signaling through PRB has been shownto regulate the development of mammary gland [45,46] andknockout animal studies have shown that PRB regulates branchmorphogenesis [47,48]. Similar to ER�, PRB is mainly expressedin the luminal epithelial cells surrounding the ER�+ cells in thehuman mammary gland [41,42,44]. The expression pattern ofER� and PR are very different in the mouse breast tissue in that

majority of the epithelial cells express both receptors [49]. Thislack of discreteness in the expression of ER� and PR suggeststhat potentially important differences exists between human andmouse with respect to the estrogen and progesterone signaling

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nd their regulation of mammary gland development, differenceshat may be pertinent to the understanding of how ER�+ breastumors develop. Interestingly, in mouse mammary gland proges-erone signaling regulates breast stem cell proliferation throughsteoclast differentiation factor the Rank ligand [50,51].

It is noteworthy that because of the restricted expression ofR�+ cells, much of the information regarding the regulation andctivation ER� is obtained from studies of ER�+ breast cancer cellines and may not be reflective of estrogen signaling in the normaluman breast tissue.

Interestingly, the recent efforts in the isolation and characteri-ation of the primitive human breast epithelial cells demonstratedhat the undifferentiated bi-potential progenitors have high PRranscript expression and nearly undetectable ER� transcripts [22].n contrast, luminal-restricted progenitors had high ER� tran-cript expression and very little PR transcript expression. Curiously,R� transcript can be detected in the myoepithelial cells [22].his peculiar transcript expression of the estrogen and proges-erone receptors needs to be validated by examining the proteinxpression of these steroid receptors in the mammary glandsing immunofluorescent techniques to achieve better sensitiv-

ty than the traditional immunohistochemical technique. It is wellstablished that PR expression is under the regulation of estro-en signaling. Therefore, the biological significance of the distinctxpression pattern exhibited by these steroid hormone receptorsithin the hierarchy of the human breast epithelial cells is not clear

nd needs to be ascertained. To this end, detailed study of estrogenignaling and ER activation in the ER�+ luminal-restricted progeni-ors might provide some answers toward the differential activationf ER in the healthy breast tissue compared to ER�+ breast tumors.he understanding of estrogen signaling and ER activation in theormal breast epithelial cells compared to ER�+ breast cancer cellsay potentially reveal indicators of early events in breast cancer

nitiation and potentially lead to the identification of biomarkers toiagnose early premalignant breast cancer cells.

.3. Non-protein coding RNA

The completion of the Human Genome Project in combinationith the recent advances in sequencing technologies have revealed

hat large majority of the human genome does not code for pro-eins and are therefore referred to as non-protein coding (npc)NA [52,53]. npcRNAs are subject of intense investigation as theyave been shown to play essential roles in important biologicalrocesses such as proliferation and differentiation. As well, a num-er of npcRNA species have been shown to be over expressed inany human cancers and regulate cancer cell proliferation and sur-

ival [54–56]. Small npcRNAs such as microRNAs (miRs) referrero short RNA species that are typically 17–22 nucleotides capa-le of inducing posttranscriptional silencing of target genes [57].iRs regulate expression of their target transcripts by base par-

ng and thereby creating a double stranded RNA which can causeranslation blockage or destablization of the transcript [57].

The studies done in the context of the mouse mammary glandevelopment suggests that some miR species have been implicated

n mammary gland development. A recent study, examined thexpression of a set of 102 microRNA species during the mouseammary gland development and found that the expression of

hese miRs and their putative target transcripts were reduced dur-ng lactation and early stages of involution [58]. Other studies that

iR-212 and miR-132 were found to be indispensible to mam-ary gland development through regulation of stromal epithelial

nteraction by regulating MMP9 gene expression in the mouseammary gland [59]. In addition, miR-205 is expressed in the

reast stem cell-enriched population of mouse breast epithelialells and its forced expression can enhance the colony formation

mental Biology 23 (2012) 606– 612

potential of a murine breast epithelia cell line, COMMA-DbetaGeo[60]. These data suggest that micro RNAs can play a role in regulat-ing the normal proliferation and differentiation of mammary cells.However, further experiments are required to investigate if theseand other miRs play essential roles in the human mammary glanddevelopment and breast carcinogenesis.

Long non-coding (lnc)RNAs are another regulatory form ofnpcRNAs. lncRNAs refer to RNA species that range from 200to over 100 kilobase pairs. lncRNAs have been implicated indiverse biological processes including genomic imprinting, chro-matin modification, and transcriptional interference [61,62]. SomelncRNA species are polyadenylated and can also act as precursorsfor miRs while others act independently as long transcripts [61,62].Because some of the lncRNAs are polyadenylated, their expressionprofile can be examined using the conventional transcriptome pro-filing methods such as oligonucleotide arrays or the Serial Analysisof Gene Expression (SAGE). Indeed, when the transcriptome signa-tures of the human bipotent and the luminal-restricted progenitorswere compared to their differentiated progeny [22], lncRNA genesH19 and MALAT1 were found to be differentially expressed. WhileH19 transcripts were abundantly expressed by the undifferentiatedbipotential progenitors, MALAT1 transcripts were highly expressedin the luminal-restricted progenitors. It is curious that H19 tran-scripts were at limit detection in the luminal subset of the humanbreast epithelial cells [22]. These findings are interesting becauseH19 and MALAT1 genes have been found to be highly expressed inhuman breast tumors and cancers of other tissues [63–68]. H19 is amaternally expressed gene that acts as part of a genomic imprint-ing network to regulate IGF2 expression [69,70]. H19 transcript alsoharbors an exonic microRNA, miR-675 that has been shown to reg-ulate the expression of the tumor suppresser retinoblastoma gene[71,72]. While both MALAT1 and H19 have been shown to enhancethe proliferation of breast cancer cell lines, their potential role in theregulating the proliferation and differentiation potential of bipo-tent and luminal-restricted progenitors is not clear and needs to bestudied.

4. Role of progenitors in breast carcinogenesis

Current evidence suggests that human breast cancers developthrough a multi-step process. This process could take a long timewhere the accumulation of a series of rare mutations and/orepigenetic changes generate clonal populations of fully malig-nant cells [73–75]. These clonal populations of malignant breastcells may arise from a rare subpopulation of cells with stem cellphenotype. In this context, the proliferative potential and thelongevity of the breast stem cells make them the likely cellulartargets for accumulating transforming mutations that could leadto development of breast cancer. In addition, the dysregulationof the molecular mechanisms that regulate the high proliferativepotential the normal mammary progenitors may confer malignantproperties on these cells. Moreover, the recent finding that fullydifferentiated adult cells can acquire a pluripotent stem cellphenotype through defined molecular alterations [76] (inducedpluripotent cells), suggests that even the differentiated luminaland/or myoepithelial cells of the breast tissue can accumulateenough mutational events to acquire a cancer stem cell phenotype.However, these differentiated cells would probably represent aless likely source of cancer initiating cells populations. Therefore,while it is inviting to hypothesize that the normal breast stem cellsare the sources of cancer initiating cells, it should be noted that any

breast cell has the potential to act as cancer initiating cells. Furthergenetic changes to these cancer initiating can cause them to act ascancer stem cells. Because this process may be heavily influencedby genomic instability, the resultant tumor phenotype may not

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epresent the tumor initiating cell population. Interestingly, recenttudies demonstrated that Brca1 inactivating-mutation on P53utant mice causes luminal progenitors to act as cancer initiating

ells. However these luminal progenitors produced basal-likearcinomas, suggesting that the tumor initiating cell populationnd the resultant tumor may have very different phenotypes80].

Regardless of the their source, the molecular mechanisms thategulate the proliferation, differentiation, and the self-renewal ofreast cancer stem cells may be shared with the normal stemells and progenitors. These ideas then suggest that elucidatinghe molecular mechanisms that control normal mammary glandevelopment is paramount to identifying pathways that may be

nvolved in breast carcinogenesis. Such understanding can provide framework to hypothesize more effective ways to categorize andreat, or even prevent the development or progression of breastancer.

Recently, the expression of CD44 or Aldehyde dehydrogenase 1nzyme has been used to enrich for breast cancer stem cells [77,78].hile the expression of these genes may provide some enrichment

or tumor cells with stem cell phenotype, the role of these in tumorells maintenance has not been demonstrated. Furthermore, rela-ionship between these markers and the normal human breast stemells and progenitors needs further investigation.

We are of the opinion that the development of breast cancertem cell specific therapies necessitates the understanding of theelationship between the normal primitive cells of the breast andreast cancer stem cells.

. Conclusions

Mounting evidence now suggests that breast tumors, much likehe normal breast tissue, are organized in a lineage hierarchy where

self-renewing stem cell is responsible for maintaining the dif-erentiated cell populations. The origin of these cancer stem cellsi.e. the tumor initiating cell population) however, is not limitedo the normal breast stem cells. Current data suggests that breastancer stem cells can arise from the progenitor cells as well ashe differentiated luminal and myoepithelial cells if they accu-

ulate enough pertinent mutations over a long period of timend the resultant tumor may or may not have similar pheno-ypes as the tumor initiating cell population [79,80]. Interestinglyowever, recent evidence suggests that within a tumor microen-ironment, the breast cancer stem cells may arise as the resultf spontaneous and stochastic mechanisms where differentiatedumor cells can acquire cancer stem cell-like phenotypes and con-ribute to the maintenance of that tumor [81,82]. These conceptso far have been identified and demonstrated in cultured breastpithelial cells or breast cancer cell lines and whether they cane replicated in primary human breast tumors and their biological

mplication is yet to be examined. These observations however, doaise an interesting question; do early premalignant breast lesionsontain cancer stem cells or only malignant breast tumors con-ain cancer stem cells that play a role in the maintenance of theseumors?

In the long run understanding the signaling molecules that arenique to cancer stem cells will allow them to be targeted specifi-ally while sparing the normal stem cells. Such treatments will beore effective and may potentially be curative. In the short run the

lucidation of mechanisms that regulate the biology and functionsf the breast stem cells and progenitors will provide clues as to how

lterations to their functions can cause disease. Such understand-ngs can form the bases for development of preventative therapiess well as more effective ways to diagnose the early premalignantreast lesions.

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