Helix-Loop-Helix Proteins in Mammary Gland Development and Breast Cancer

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Journal of Mammary Gland Biology and Neoplasia (JMGBN) pp944-jmgbn-470725 September 11, 2003 19:48 Style file version June 22, 2002

Journal of Mammary Gland Biology and Neoplasia, Vol. 8, No. 2, April 2003 ( C© 2003)

Helix-Loop-Helix Proteins in Mammary GlandDevelopment and Breast Cancer

Pierre-Yves Desprez,1,2 Tomoki Sumida,1 and Jean-Philippe Coppe1

The basic helix-loop-helix (bHLH) family of transcription factors functions in the coordi-nated regulation of gene expression, cell lineage commitment, and cell differentiation in mostmammalian tissues. Helix-loop-helix Id (Inhibitor of DNA binding) proteins are distinct frombHLH transcription factors in that they lack the basic domain necessary for DNA binding.Id proteins thus function as dominant negative regulators of bHLH transcription factors. Theinhibition of bHLH factor activity by forced constitutive expression of Id proteins is closelyassociated with the inhibition of differentiation in a number of different cell types, includingmammary epithelial cells. Moreover, recent literature suggests important roles of HLH pro-teins in many normal and transformed tissues, including mammary gland. Therefore, futuredirections for prognosis or therapeutic treatments of breast cancer may be able to exploitbHLH and Id genes as useful molecular targets. The purpose of this review is to summarizethe evidence implicating HLH proteins in the regulation of normal and transformed mammaryepithelial cell phenotypes.

KEY WORDS: Id proteins; proliferation; differentiation; invasion; metastasis.

INTRODUCTION

Helix-loop-helix (HLH) transcription factors (1)are important components in the transcriptional net-work regulating cell growth and differentiation dur-ing many essential developmental processes in bothvertebrates and invertebrates (2–4). The 240 knownmembers of the HLH family (2,5) coordinate cell-type-specific gene expression implicated in tissue em-bryogenesis and cell lineage determination and over-all regulate normal or abnormal cell fate withinmost mammalian tissues (2,6–16). The highly con-served HLH domain enables these proteins to specif-ically homo- or hetero-dimerize, a prerequisite forHLH proteins to achieve any transcriptional activity

1 California Pacific Medical Center, Cancer Research Institute, SanFrancisco, California.

2 To whom correspondence should be addressed at CaliforniaPacific Medical Center, Cancer Research Institute, 2330 ClayStreet, Stern Building, San Francisco, California 94115; e-mail:[email protected].

(2,7,17). Most HLH proteins belong to the basic helix-loop-helix (bHLH) family and act as transcriptionalenhancers or inhibitors of various genes through di-rect DNA binding to the canonical E-box sequence(18) (Fig. 1). One HLH subfamily is composed of Idproteins (inhibitor of differentiation/DNA binding)(19,20). While capable of dimerizing with ubiquitousand tissue-specific bHLH factors, the four mammalianId members lack a common region necessary for DNAbinding. Consequently, Id-bHLH heterodimers areunable to bind DNA, and Id proteins function as dom-inant negative regulators of bHLH transcription fac-tors (19) (Fig. 1).

The expression, as well as the segregation andfunctionality of Id proteins and their bHLH tar-gets, vary with the cell type and stage of develop-ment, differentiation, or growth. Eventually, the bal-ance of bHLH/Id activity is critical to specific gene

Abbreviations used: ECM, extracellular matrix; MEC, mammaryepithelial cell; HLH, helix-loop-helix proteins; bHLH, basic helix-loop-helix proteins; Id, inhibitor of DNA binding.

2251083-3021/03/0400-0225/0 C© 2003 Plenum Publishing Corporation

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226 Desprez, Sumida, and Coppe

Fig. 1. Id proteins as dominant negative regulators of bHLH transcription factors. (The basic helix-loop-helix (bHLH) proteinsare transcription factors acting through direct DNA binding, and Id proteins are dominant negative regulators of these bHLHfactors).

expression. Additionally, Ids can interact with non-HLH proteins, such as Rb, Ets, Pax, MIDA-1, orSREBP-1c (4,8,11,13,15,21), and inhibit their activ-ity. In short, the potential for Ids to interfere withbHLHs and crucial non-HLH proteins confers themwith central coordinating roles. Ultimately, Ids or-

Fig. 2. Implication of Id proteins as regulators of various biological events.

chestrate in a synergistic manner combinations of pro-tein networks and gene expression; this critical bal-ance is disrupted during tumorigenesis (Fig. 2). In-deed, strong evidence now suggests that Ids behaveas pro-oncogenic factors (7,8,11,13,15,21,22). Ids aregenerally overexpressed in tumor cells, correlating

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Helix-Loop-Helix Proteins in Mammary Gland Development and Breast Cancer 227

with dedifferentiated, proliferative, and invasive cellphenotypes (Fig. 2). Variations in expression levels ofIds have to be integrated into the whole cellular equi-librium, so that upstream dysregulation of Ids activity,from Id gene expression to Id protein segregation andfunctionality, can result in the downstream dysregu-lation of the expression of diverse genes.

While involved in lymphocyte, muscular, neu-ronal, adipocyte, and epithelial homeostasis (22),HLH transcriptional regulators are also primary fac-tors in mammary gland biology and neoplasia. Specif-ically, this review addresses how the Id/bHLH net-work regulates the mammary epithelial cell (MEC)phenotype. MECs undergo important changes in mor-phology and function, which underlie the striking re-modelling phenomena affecting the mammary glandas it evolves through female embryonic developmentand puberty into adulthood and the menstrual cycle,pregnancy, lactation, involution, and postmenopausalregression (23). Mammary cells undergo tightly coor-dinated modifications in growth, differentiation, in-vasion, and apoptosis. Interestingly, these dramaticchanges also drive mammary gland tumorigenesis, butspatial and temporal controls are lost during the tu-mor progression (24–26).

The breast is the source of the most frequentlydiagnosed malignancy among women and remainsa leading cause of female morbidity and mortalityworldwide (27). Breast cancer mostly manifests itselfwithin tissues of the ductal and glandular epithelia.During mammary gland neoplastic transformationand progression, some fundamental mechanisms, de-rived from dysregulated normal processes, promotebreast cancer malignancy. First, de-novo cancerouscells undergo transformation (oncogenic alterationsand dedifferentiation), a process related to cell sur-vival (avoiding apoptosis, senescence, hypoxia, theimmune system, and recognition by normal surround-ing cells), cell proliferation (loss of cell cycle con-trol and potential immortalization), and cell migra-tion and invasion (loss of cell–cell and cell–matrixcontact). Second, the expanding tumor requires aparticular vascularization, neo-angiogenesis drivenmainly by the tumor itself, to grow, invade, and metas-tazise. Finally, stromal cells of the microenvironmentsurrounding transformed MECs are also believedto participate and play critical roles during tumordevelopment.

In this review, we will attempt to demonstratethat HLH proteins are able to coordinate the sequen-tial events driving normal mammary gland develop-ment and breast tumor progression. This review cov-

ers this research field through two main aspects. First,we describe the HLH family in a general way andsome specifics of Id proteins and genes. Then, in thesecond part, we directly address the roles of Ids, as cor-related with their bHLH targets, in mammary glandepithelial cell biology and neoplasia.

HLH FAMILY AND ID PROTEINS

E-Box and HLH Classification

Originally, a cis-acting DNA element, µE, wasidentified as an intronic enhancer in the immunoglob-ulin heavy chain (IgH) (18). The core of this DNAsequence was shown to be a highly conserved hex-anucleotide among diverse promoters (CANNTG).This E-box element was shown to mediate cell-type-specific gene transcription. E12, E47, myc, MyoD, andDaughterless were the first transcription factors iden-tified as recognizing the E-box sites (1,28). Their dis-covery led to the identification of a common motifin their protein sequence, described as a HLH, giv-ing rise to the HLH protein family. These proteinsact as obligate dimers. Most HLH proteins possess abasic region amino terminal to the HLH motif, per-mitting HLH dimers to bind to the consensus E-box(Fig. 1).

The HLH transcriptional regulator family isgrouped into seven classes (I–VII), based upon tis-sue distribution, dimerization capabilities, and DNA-binding specificities (2) (Table I), or four classes(A–D) (5) based upon phylogenetic analyses as well asamino acid patterns of the basic domain, leucine zip-per motif, and E-box binding specificities. The largestHLH subfamilies are class I and class II bHLH. Class IHLH, or E-proteins, are ubiquitous bHLHs, includingE2A products (E12 and E47/ITF-1), E2-2 (or ITF-2),Daughterless (Da), or HEB (or HTF4). Class II HLHmembers are tissue-specific bHLHs, such as MyoD(MyoD1, myogenin), ATONAL (NeuroD/BETA-2,Neurogenin), Achaete–Scute complex (Mash, ac, as),or Twist. Class V HLH includes the four knownmammalian Id genes, Id1 (19,54), Id2 (55,56), Id3(57,58), and Id4 (59,60), and their homologue genes inDrosophila (Extramacrochaetae or Emc (20)), Xeno-pus (XIdx (61)), and Zebrafish (ZId (62)). This re-view mainly concerns interactions between class VHLH proteins (Id) and class I/II HLH factors (bHLH)within mammary gland tissues. Id proteins do notinteract with other HLH proteins, such as bHLH-LeuZip, e.g. Myc.

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Table I. Classification of the HLH Transcriptional Regulators

Tissue distribution, dimerization,Class Protein families Function DNA-binding, and other features Reference

Class I E12, E47, HEB, E2-2 orITF-2 (E proteins)

Myogenesis, lymphogenesis,neurogenesis, sexdetermination,lymphomagenesis, etc.

Ubiquitous pattern of expressionand capable of forming eitherhomo- or heterodimer

29–31, 32,33

Class II MyoD, MyogeninNeuroD/BETA2

dHAND, eHANDMashTwist

MistSCL/Tal1

MyogenesisNeurogenic differentiation,

pancreatic developmentCardiac morphogenesisPositive regulator of neurogenesisInhibition of myogenic and

neurogenic differentiationExocytosis of serous secretionsHematopoiesis

Tissue-restricted pattern ofexpression and incapable offorming homodimers andpreferentially heterodimerizewith the E proteins; Class I andClass II heterodimers can bindboth canonical andnoncanonical E-box sites

34,3536,3738394041,4243

Class III Myc

TFE

SREBP(bHLH-Zip)

Cell proliferation, differentiation,oncogenesis, apoptosis, etc.

Transcription in immunoglobulinheavy chain enhancer

Sterol synthesis, adipocytedetermination

Presence of leucine zipperadjacent to the HLH motif

444546

Class IV Mad, Max, Mxi(bHLH-Zip)

Interaction with Myc familyproteins and regulation of cellproliferation

Dimerization with the Mycprotein or with each other

47

Class V Id Inhibition of DNA binding, cellproliferation, differentiation,etc.

Lack the basic DNA-bindingdomain and act as dominantnegative regulators of Class Iand Class II bHLH proteins

11,19

Class VI HES, HESR1 Notch signaling pathway, cellproliferation

Presence of proline in their basicregion

48,49

Class VII AHR, ARNT, Sim

HIF

Biological responses to planararomatic hydrocarbons

Regulator of O2 homeostasis

Presence of a bHLH-PAS domain 50,5152,53

HLH Protein Structure and Specificity

The HLH motif is a bipartite domain neces-sary for protein–protein interactions. The HLHdimerization process results in the formation of aparallel, four-helix bundle in which dimerizationcontacts derive from conserved hydrophobic residuesclustered within a shielded core. Some nonconservedhydrophilic residues within the HLH domain, as wellas residues additional to the HLH region, probablycontribute to HLH dimerization specificity (63,64).These residues could explain the intrinsic structuraldifferences and dimerization specificities of ClassI E-proteins versus Class II tissue-specific bHLHfactors (64).

Almost all HLH proteins possess a basicDNA-binding region adjacent to the N-terminalHLH domain, critical for specific DNA-binding andtranscription-promoting activity (65). The basic do-main makes contact with the major groove of the

DNA (66). The DNA binding activity is also drivenby additional residues in the “loop” and the second“helix” of the HLH domain (66). The crucial residuesfor DNA interaction are conserved over the bHLHsubfamily. Each bHLH monomer interacts with halfof the canonical E-box site. Some HLH proteins alsobind to the related DNA sequence named N-box,CACNAG (2). Unlike bHLH proteins, Ids lack thebasic domain, thus naturally acting as dominant neg-ative transregulators of bHLH transcription factors(Fig. 1).

Ids and Modulation of Their Protein Activity

The predicted encoded Id proteins (13–20 kDa)share five boxes of homology, the central one corre-sponding to the HLH motif and extending over 50–55residues. The “loop” sequence varies and appears tobe essential for the dimerization specificity (67,68).Apart from the four smaller homology boxes sur-

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rounding the HLH domain, both the NH2 and COOHregion sequences vary greatly. The nonconserved re-gions may play important roles in some interactionswith bHLH or non-bHLH proteins. The first aminoterminal box is of particular interest since Id2, Id3 andId4, but not Id1, share a phosphorylation consensussite (SPVR), which is phosphorylated by Cyclin-A/E-CDK2 during cell cycle progression and is functionalin Id2 and Id3 (69,70). The phosphorylation state ofIds has been implicated in their degree of specificityof interaction with E2A products (71), thereby pro-viding a fast mechanism of regulation in response tosignaling pathways.

Various cellular signals may affect the stability,localization, and activity of the cellular Id pool. Amajor posttranslational modification is the phospho-rylation in the N-terminal region of Id2 and Id3. Theubiquitin–proteasome degradation pathway also reg-ulates Id levels, though no exact destruction boxeshave been identified that could destabilize Id proteins(72,73). The dimerization state is known to be essen-tial to the Id degradation process. Id3 half-life wasshown to be extended when heterodimerized with abHLH (72). Id turnover also depends on Id accessi-bility. Additionally, Id regulation is related to intra-cellular localization. Unlike bHLH proteins, Ids lacka functional nuclear localization signal. In the cyto-plasm, the bHLH can heterodimerize with Id and actas a nuclear chaperone (74). The cytoplasmic or nu-clear distribution of each Id protein is likely to beregulated by their different targets. Collectively, thesecomplex regulatory processes provide a fine controlof the final balance between levels of Ids and theirinteracting proteins, principally bHLH transcriptionfactors, and monitor the activity of Id partners.

Id Genes and Their Upstream Regulation

An alternative open reading frame in the 3′ cod-ing region of Id1 (75,76) and Id3 (77,78) genes wasidentified, potentially resulting in a drastic attenua-tion in the heterodimerization properties of Id vari-ants by a read-through of a small intron. An Id2pseudogene (Id2B) was identified which encodes anmRNA closely related to Id2, except for a nonsensemutation in the beginning of the amino-terminal cod-ing region (codon 37 (79)). While the intron–exon or-ganization of different Id genes shows a strong simi-larity and probably evolved from a common ancestralgene (77), Id variants may have different properties.Another type of regulation could occur at the RNA

level. The half-life of Id mRNA is short in serum- orPDGF-induced proliferation of arrested cells (33,80).Additionally, Id mRNA can be bound and regulatedby ELAV/Hu proteins (ribonucleoproteins), resultingin a posttranscriptional regulation (81,82).

Like most cell cycle regulators, Id genes are im-mediate early genes, mitogen-responsive, and highlyexpressed during serum treatments. In cell typessuch as fibroblasts, serum coordinately induced bothId1 and Id2 mRNAs, with two peaks of expressionin early and late stages of the G1 phase (11,75).The time-dependent profile of expression is a veryintegrated clock-controlled mechanism through thecis-regulating sequences present in Id promoters.Throughout cell cycle progression, the normal vari-ations of protein type, quantity, and activity differ-entially regulate Id promoters. Interestingly, some E-boxes are present in certain Id genes; therefore Ids canpotentially be regulated by bHLH factors themselves.Ids were shown to be downregulated at the transcrip-tional level upon differentiation (19,55). In fact, awide variety of extracellular mitogens and growth fac-tors regulate Id gene transcription in normal as well astumor cells (Fig. 2). Even though still poorly under-stood, the dysregulation of Id gene and protein ac-tivity is the key to understanding how HLH proteinscontribute to mammary gland malignant progression.

Id AND bHLH PROTEINS IN BREAST CELLS

HLH proteins have been identified as key regu-lators of MEC differentiation, growth, and tumorige-nesis in vitro as well as in vivo. The control of thesecellular mechanisms is summarized in Table II. Themammary gland provides a model for studying spe-cific upstream regulation as well as downstream ef-fects of Id proteins and their bHLH partners, throughdevelopment as well as during tumorigenesis. In-deed, breast tissue is distinct from most other verte-brate tissues in that it continually changes in structurethroughout the adult lifetime of reproductively activefemales (97). Moreover, many of the transcriptionalregulators vital for mammary development are alsoimplicated in breast cancer, including HLH proteins.

Mammary Epithelial Cell Growth, Invasion,and Differentiation

Id1

Id1 is expressed at high levels in proliferat-ing mammary epithelial SCp2 cells in vitro, and its

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Table II. Expression of Id Proteins in Breast Epithelial Cells

Cell type Function, expression, and phenotype Reference

Non transformed mammary epithelial cellsId-1 Induction of cell proliferation

Induction of cell migration and invasionInduction of 120 kDa gelatinase

Suppression of mammary epithelial celldifferentiation

Regulation of cell–cell interactionsTight junction sealing

Mammary gland developmentHighly expressed during mammary development

in virgin mice, early pregnancy, and involutionInduction of cell proliferation or apoptosis

depending on the cell density

83,8485

86

87

84

Id-2 Highly expressed in differentiated mammaryepithelial cells in culture

Mammary gland developmentHighly expressed during the second part of

pregnancyNecessary for normal architectureLactation defects in Id2−/− mice

84

84

8889

Transformed mammary epithelial cellsId-1 Induction of cell proliferation and invasion

Correlation with tumor grade (overexpressed inaggressive breast tumors)

Mediator of the effects of sex steroid hormonesLoss of regulation of Id-1 promoter in aggressive

breast cancer cells (NF-1/RB/HDAC-1)Strong unfavorable prognostic marker for node

negative breast cancer patientsInduction by BMP-2Repression of aggressive and metastatic phenotype

by Id-1 targeting

9090

9091

92

9394

Id-2 Inverse correlation with tumor grade (suppressed inaggressive breast tumors)

Decrease cell migration and invasionInduction by BMP-2

95

9593

Id-3 Induction by BMP-2 93Id-4 Regulation of BRCA1 expression 96

expression declines in quiescent and differentiatingcells (86). Indeed, when given lactogenic hormonesand extracellular matrix (ECM), the SCp2 murineMECs (98) aggregate, growth arrest, form alveolus-like structures, secrete milk proteins (99,100) andrapidly down-regulate Id1 (86). Interestingly, the ec-topic expression of Id1 in the SCp2 cells blocks differ-entiation and stimulates SCp2 cell proliferation (86);SCp2 cells stably expressing Id1 can eventually mi-grate and become invasive (85). Constitutive expres-sion of Id1 in SCp2 cells is also related to upreg-ulation of membrane-type matrix metalloproteinase(MT1-MMP) expression (unpublished data) and se-cretion of a 120-kDa gelatinase (85). Therefore, Id1appears to render cells refractory to differentiation

signals, but receptive to growth signals, potentiallyby inactivating one or more critical bHLH proteins(86).

It was shown that Id1 upregulates Zfp289 (83),a cytoplasmic protein, which is probably the humanhomologue of the yeast Gcs-1. Gcs-1 is a GAP (GT-Pase activating protein) necessary for the transitionfrom stationary to proliferative phase in yeast (re-entry into cell cycle) and is involved in vesicular traf-ficking and in the regulation of the actin cytoskele-ton network. The Zfp289 mRNA expression patternis closely related to that of Id1 during normal mousemammary gland development. Additionally, the ex-pression of Id1 in vitro (and also in vivo as shownfurther) was reported to be linked to and to induce

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proliferation and/or apoptosis (84). Proliferation is fa-vored in sparse SCp2 cell cultures, which more closelyresemble virgin or early pregnant gland, whereasapoptosis is favored in dense cultures, which closelyresemble the gland at the beginning of involution.Thus, depending on extracellular signals and cell–cell interactions, Id1 potentially assumes dual but op-posite functions. Id1 may therefore play a role inapoptosis (84).

Interestingly, some genes involved in cell–cellinteractions as well as in cell–matrix interactionsappear to be under Id1 regulation. Id1 downreg-ulates the clusterin gene, which encodes a glyco-protein regulating cell–cell interactions (83). Clus-terin is also involved in regulation of apoptosis. Innonconfluent SCp2 cells, constitutive expression ofId1 enhances the glucocorticoid stimulation of tightjunction sealing (87). The glucocorticoid signalingpathway controls tight junction dynamics, targetingthe adhesive and permeability properties of MECs.Id1 is an important element in the glucocorticoid-activated signalling pathway. Id1 establishes a com-petent state for MECs to form normal cell–cellcontacts (87).

Id1 expression was also examined during normalmouse mammary gland development in vivo (84). Theexpression of β-casein, a marker of MEC differen-tiation, is inversely correlated with Id1 expression.During the early stages of pregnancy, when epithe-lial cells of the ductal trees proliferate and invadethe stroma, Id1 is highly expressed. Using an Id1-specific antibody recognizing only one band on west-ern blots (84,90), Id1 was detected in the epithelialcells of growing ductal structures. This expression wasrecently confirmed at the mRNA level using the tech-nique of in situ hybridization (unpublished data). Dur-ing mid-pregnancy, Id1 is downregulated and remainsundetectable through much of the lactating period,corresponding to a highly differentiated stage (84).Surprisingly, during in vivo mammary gland involu-tion, a time corresponding to the last stage of glanddevelopment and to extensive apoptosis, Id1 mRNAis re-expressed, again suggesting a potential role ofId1 in apoptosis (84).

Recently, another research group used immuno-histochemistry to determine the distribution of Id1expression in the mammary gland (101). Immunore-activity was detected in the cytoplasm of myoep-ithelial cells and in vascular endothelial cells. Thisreport claimed that Id1 was not expressed in lu-minal epithelial cells in mouse mammary gland.However, the staining observed in the myoepithe-

lial cells did not correspond to Id1, because it wasalso observed in myoepithelial cells of mammarygland tissue from Id1 null mice. This report there-fore exemplifies the complications resulting from im-munohistochemistry using antibodies that give non-specific signals on western blots, and highlights the ur-gent need for reliable antibodies which work on tissuesections.

Id2

Id2 also plays an important role during mam-mary gland development. It has been recently re-ported that Id2-deficient female mice display severelactation defects (88,89). Indeed, Id2 null MECs losethe capacity to form alveoli, show a disorganized ar-chitecture, and are not able to undergo pregnancy-dependent differentiation (88). Only very early mark-ers of differentiation are expressed in MECs of Id2null mice. Moreover, the ductal marker Na-K-Cl co-transporter is locally downregulated (88). Therefore,Id2 is probably indispensable for mammary gland de-velopment and differentiation from early stages ofpregnancy.

In addition, the phenotype of Id2-deficient fe-male mice (89) is correlated with impaired prolifer-ation and enhanced apoptosis of mammary epitheliaat early and late stages of pregnancy, enabling theexpansion of epithelial cells during pregnancy (89).Even if Id2 in the pregnant mammary gland stimu-lates cell cycle progression by negative regulation ofsome bHLH important for differentiation, Id2 may al-ternatively promote differentiation of the mammarygland by inhibiting the activity of bHLH involved instimulation of cell proliferation (84,89). Indeed, un-like Id1, Id2 appears to be associated in vivo withMEC differentiation (84). This finding was confirmedin vitro with a mammary epithelial cell culture model.Id2 ectopic expression in SCp2 cells accelerates dif-ferentiation, while Id2 antisense blocks differentia-tion and milk production (84). Under differentiationconditions, Id2 proteins are detected only when milkproteins are first produced and increase throughoutlate pregnancy and the lactation period (84). More-over, Id2 upregulation is necessary for the functionalpathway of MEC differentiation (84).

The mammary glands of Id2−/− mice exhibitnormal development during embryogenesis andin the adult virgin animals, but the final phasesof differentiation occurring during pregnancy areblocked (89). Therefore Id2 may be considered to bean activator of mammary gland differentiation.

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Fig. 3. Id-bHLH network as a transcriptional control of mammary epithelial cell phenotypes. (Model representing a potential linkbetween Id-1 down-regulation and Id-2 up-regulation in normal mammary epithelial cells as well as in breast cancer cells.)

HLH Network

Both Id1 and Id2 appear to play an essentialrole in the regulation of breast epithelial cell differ-entiation and proliferation. The coordination of theId1/Id2 balance may be important for normal mam-mary gland development. As demonstrated by thescreening of a yeast two-hybrid library from differ-entiating mammary epithelial cells, Id1 interacts withthe ubiquitously expressed Class I bHLH ITF-2 (84).ITF-2 overexpression counterbalances the effects ofId1 on differentiation, proliferation, and apoptosis inmammary epithelial SCp2 cells (84). We propose thatthe effects of Id1 on mammary epithelial cell phe-notypes are mediated through Id1 interaction withITF-2, and most probably ITF-2A. ITF-2A is an alter-natively spliced form of mouse ITF-2 and forms activeheterodimers with MyoD, a muscle-specific bHLHtranscription factor, during muscle differentiation(102).

Id2 gene contains several E-boxes within its pro-moter, and bHLH factors regulate Id2 expression inF9 cells (103). Since Id2 also appears to be involvedin the normal process of differentiation and the ex-pression of Id2 is upregulated when Id1 is absent,Id1 could be the inhibitor of Id2 gene expressionthrough interaction with a specific bHLH. Therefore,Id1, Id2 and ITF-2 are acting within a network govern-ing MEC phenotypes and potentially acting as molec-ular switches of the differentiation.

Taking these results together, it is possible to pro-pose a model for mammary epithelial cell growth anddifferentiation (Fig. 3). The initial downregulation ofId1 would lead to the release of ITF-2A. ITF-2A as ahomodimer, or as a heterodimer with another bHLH(another E-protein), would then induce the upregu-lation of Id2 gene expression, leading to the seques-tration of ITF-2B by Id2 and the release of a putativetissue-specific (TS) bHLH. ITF-2B, a splice variantof the ITF-2 transcript, encodes a transcription fac-tor that inhibits MyoD activity (102). This inhibitoryactivity requires the N-terminal 83 amino acids, sinceITF-2A showed no inhibitory activity and a mutantMITF-2B with deletion of the N-terminal 83 aminoacids failed to inhibit MyoD-mediated transcriptionalactivation. We propose that, as in the muscle, ITF-2B inhibits the tissue-specific (TS) bHLH activity byforming an inactive heterodimer. After its releasefrom ITF-2B, this TS bHLH, then dimerizes with ITF-2A and consequently activates the transcription ofgenes involved in the functional and morphologicaldifferentiation of mammary epithelial cells (Fig. 3).

Mammary Gland Tumorigenesis

Id1

The finding that Id1 can induce an invasive phe-notype in mouse mammary epithelial cells implies

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that Id1 could contribute to human breast cancerprogression. To explore this possibility, Id1 expres-sion was examined in cell lines with varying degreesof invasiveness in culture. When cells were culturedin serum free medium for 2 days, Id1 mRNA ex-pression was almost undetectable in the noninvasivebreast cancer cells T47D and MCF-7, but was con-stitutively expressed at a high level in invasive cellsMDA-MB231 and MDA-MB436 (85). T47D cells sta-bly transfected with Id1 were invasive by BoydenChamber analysis, whereas T47D with low levels ofId1 remained largely noninvasive. Id1 overexpressionalso conferred a growth advantage in T47D culturedin serum free medium (90).

Id1 also appears to be an important mediator ofthe effects of sex steroid hormones on T47D cell pro-liferation (90). Estrogen stimulates proliferation andinduces Id1 expression, whereas progesterone inhibitsproliferation and represses Id1 expression. Proges-terone represses Id1 expression at least in part by re-pressing transcription. An antisense oligonucleotidethat reduces Id1 protein levels reduces the ability ofestrogen to stimulate cell proliferation, whereas con-stitutive Id-1 expression renders cells refractory togrowth inhibition by progesterone.

Recently, it has been reported that Id1 is a crit-ical target of c-myc in T47D and MCF-7 cells (104).Inducible overexpression of c-myc in these cells re-sulted in a rapid increase in Id1 protein, precedingentry into DNA synthesis. Cells pretreated with Id1antisense oligonucleotides failed to proliferate afterc-myc induction, suggesting that upregulation of Id1is required for c-myc-dependent proliferation. Inter-estingly, the expression of cyclin D1 is greatly andrapidly diminished in cells transfected with Id1 anti-sense oligonucleotides, indicating a direct regulationof cyclin D1 by Id1.

Id1 protein expression was also examined inbreast cancer biopsies. Using a limited number ofbiopsies, it was shown that almost all DCIS (duc-tal carcinoma in situ) were negative for Id1 stain-ing, and the majority of infiltrating grade III carci-nomas of ductal origin were strongly positive for Id1(90). Further, a total of 58 cases of infiltrating carci-nomas were analyzed for Id1 expression (94). Onlyabout 20% of the grade I invasive carcinoma biop-sies showed strong Id1 staining, whereas more than60% of the grade III invasive carcinoma investigatedshowed strong Id1 expression. These studies there-fore indicated that Id1 expression might be a reli-able prognostic marker for breast cancer invasion andmetastasis.

This hypothesis was confirmed recently by an-other laboratory. Id1 protein expression was inves-tigated in 191 patients with lymph node negativebreast cancer (92). Patients with strong or moder-ate Id1 expression had a significant shorter over-all (p = 0.003) and disease-free survival (p = 0.01)compared to those with absent or low expression.Thus, aberrant expression of Id1 protein representsa strong independent prognostic marker in node neg-ative breast cancer. Consistent with this hypothesisthat Id1 is a key regulator in human breast cancerprogression, it has been proposed that Id1 could bea promising candidate for future therapy and that in-hibiting Id1 expression might be of benefit for patientswith breast cancer. In agreement with this hypothe-sis, it has recently been shown that human metastaticbreast cancer cells become significantly less invasivein vitro and less metastatic in vivo when Id1 is down-regulated by stable transduction with Id1 antisense(94).

Recent studies on the regulation of the Id1 pro-moter in human breast cancer cells identified a small31 bp region at the 3′ end of the Id1 promoter whichappears to be responsible for the constitutive expres-sion of Id1 in highly aggressive cells (91). This elementcontains recognition sites for SP-1 and NF1 transcrip-tion factors. Data indicate that NF1 is the key media-tor for the control of promoter activity, as it is proba-bly able to recruit transcriptional repressors HDAC-1and Rb proteins to the site and to repress Id1 expres-sion in nonaggressive breast cancer cells. In contrast,highly invasive cell lines do not express any NF1 pro-tein (using an antibody reactive to all NF1 isoforms)and therefore are unable to recruit transcriptional re-pressors to the promoter region. This scenario resultsin constitutive expression of Id1 in the aggressive andmetastatic cells.

Another mechanism of regulation of Id1 geneexpression in breast cancer cells may be related tothe BMP factors, and thus to the TGF-β superfam-ily of cytokines which have dual roles in normal andabnormal cell homeostasis (105). Id1 appears to be aBMP-2 dependent gene (93,106,107). In fact, BMP-2is expressed in a wide range of breast cancer celllines and may be an enhancer of Id1 expression dur-ing oncogenic progression. BMP-2 induces sequentialchanges and overexpression of Id1, 2 and 3 in MCF-7 cells (93). BMP-specific response elements in theId1 promoter have been identified (108,109), provid-ing important insights into the BMP/Smad pathway.The BMP effect is likely to be mediated by Smad-1/-4directly binding to different motifs and activating

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Id1 transcription. About 1 kb upstream of the tran-scription start, some SBEs (Smad binding element),GCCGCC palindromic sequences and GC boxes, aswell as CAGAC boxes, are found to form primar-ily two independent DNA binding elements neces-sary for a Smad transcriptional complex binding. Thisrepresents a specific pathway to activate the targetgene.

Id2

In contrast to Id1, Id2 mRNA and protein areup-regulated in vitro and in vivo as mammary epithe-lial cells lose proliferative capacity and initiate differ-entiation (84). Id2 expression is inversely correlatedwith the rate of proliferation in murine MECs, andId2 is expressed at a higher level in differentiated hu-man breast cancer cells than in very aggressive andmetastatic cells (95). When re-introduced into aggres-sive breast cancer cells, Id2 is able to reduce their pro-liferative and invasive phenotypes. Moreover, littleId2 protein expression is detectable in human biop-sies from aggressive and invasive carcinomas in com-parison to in situ carcinomas. Therefore, Id2 expres-sion not only follows a pattern opposite to that of Id1during mammary gland development and breast can-cer progression, but also appears to act as an impor-tant factor for the maintenance of a differentiated andnoninvasive phenotype in normal and transformedbreast cells.

Id4

Id4 is upregulated and involved in breast cancerdevelopment through down-regulation of BRCA1 ex-pression, and its overexpression leads to anchorage-independent cell growth. Indeed, via a very ele-gant promoter-driven reporter system, Id4 is nowidentified as an upstream regulator of BRCA1 genein breast and ovarian cancer (96). An “inverse ge-nomics” approach based on a randomized ribozymegene library transduced into cells derived from humanbreast and ovarian cancer cells (SK-BR-3, T47-D,PA-1) demonstrates that Id4 is capable of modulat-ing BRCA1 promoter-driven reporter gene expres-sion and endogenous BRCA1 expression. Id4 andBRCA1 expression are inversely related in sporadicbreast cancer. Id4 is upregulated, while BRCA1 isdownregulated. Use of Id4 sense or antisense vectors,inducing respectively Id4 overexpression or repres-

sion, demonstrate that Id4 is potentially a crucial fac-tor in breast tumorigenesis and invasiveness. Id4 mayalso be involved in the hormone-dependent regula-tion of BRCA1 expression, since its expression is in-versely estrogen-dependent from BRCA1 (as shownby in vitro experiments using MCF-7 cells). There-fore, Id4 appears to be involved in the BRCA1 regu-lation pathway occurring in breast cancer pathogene-sis. As for Id1, Id4 upregulation might be a potentialmarker of breast cancer through its action on theBRCA1 gene. On the other hand, BRCA-2 may alsobe regulated by Ids, since some E-boxes, E2F, andEts recognition motifs, are present in its promoter(110).

HLH Network in Breast Cancer

ITF-2 is now established as a downstream tar-get of the Wnt/TCF pathway in human cancers (111).More specifically, ITF-2B rather than ITF-2A, canpromote neoplastic transformation and is upregu-lated in tumors with defective β-catenin. Mutationalactivation of Wnt signaling is implicated in many can-cers (112), and elevated levels of β-catenin are asso-ciated with poor prognosis in carcinoma of the breast(113). Additionally, inactivation of the tumor sup-pressor Apc perturbs mammary gland development(114), Apc being involved in the degradation of freeβ-catenin (115). Since the ITF-2A/B balance is as-sumed to be essential to the cancerous phenotype ofMECs (84) (Fig. 3), it is tempting to correlate Wntsignaling with Id gene expression.

Other bHLHs are probably implicated in MECmalignant transformation. E2A products are identi-fied procarcinogenic factors of epithelial cells. E12/47are implicated in Epithelial–Mesenchymal Transi-tions (EMT) (116) as repressors of E-cadherin (117).E47-expressing MECs become aggressive concomi-tant with a down-regulation in E-cadherin expression,an important event leading to tumor cell invasivenessand migration.

Id1 is most probably implicated in the pro-gression of breast cancer rather than in the initialtransformation of mammary epithelial cells (85,90).Indeed, some of the late events in the progressiontowards an aggressive malignant stage may be conse-quences of dysregulation of the entire HLH network.As previously described, Id1, Id2 and ITF-2A, andITF-2B are potential interacting molecular switcheswhich regulate MEC phenotypes (84) (Fig. 3). Thedominant expression of Id2 and ITF-2A over the

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expression of Id1 and ITF-2B may play a critical rolein the prevention of breast cancer progression. Thederegulation in this balance of HLH proteins couldtherefore be responsible for the appearance of aninvasive and metastatic phenotype in breast cancercells.

FINAL PERSPECTIVES

HLH proteins have now been identified as im-portant regulators of cell growth, apoptosis, senes-cence, cell survival, oncogenesis, tumor progression,and malignancy in many different types of tissues. Inthis review we have attempted to highlight the roleof HLH proteins in general, and Id proteins in par-ticular, in the regulation of mammary epithelial phe-notypes. Interestingly, in other female reproductiveorgans and tissues, Id genes have also been found tobe up-regulated during cancer progression. Indeed,studies show that Id1 overexpression in cervical, en-dometrial, and ovarian cancer correlates with the clin-ical stage and outcome, Id1 expression being associ-ated with higher tumor cell aggressiveness (118–121).Therefore, it is possible to speculate that hormonalimbalance may affect normal tissue functions throughthe disruption of proper Id gene expression. Ids couldthus represent independent markers for diagnosis andprognosis of hormone-related cancers in women. Idscould also represent promising targets against breastcancer progression, as well as for other types of femalecancers.

Much more work is required to fully understandthe upstream regulation of HLH genes, as well asthe downstream effects of Id and bHLH proteins.The regulation of HLH protein stability, degradation,and segregation may represent interesting subjectsof studies that could lead to breakthroughs in can-cer therapy. Future in vitro and in vivo studies willcertainly bring important new information about theroles of HLH proteins in molecular cell physiology;the mammary gland represents a valuable tool for un-derstanding HLH functions and participation in themolecular mechanisms of development, differentia-tion, and carcinogenesis.

ACKNOWLEDGMENTS

The authors thank Dr Andrew P. Smith for edit-ing. P. Y. Desprez is supported by grants from theUniversity of California Breast Cancer Research Pro-

gram (7WB-0026) and from the National Institutes ofHealth—National Cancer Institute (RO1 CA82548).

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P1: GCR



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Helix-Loop-Helix Proteins in Mammary Gland Development and Breast Cancer