notch signaling in mammary development and oncogenesis

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Journal of Mammary Gland Biology and Neoplasia (JMGBN) pp1270-jmgbn-490063 August 2, 2004 12:12 Style file version June 22, 2002

Journal of Mammary Gland Biology and Neoplasia, Vol. 9, No. 2, April 2004 ( C© 2004)

Notch Signaling in Mammary Developmentand Oncogenesis

Robert Callahan1 and Sean E. Egan2

With the discovery of an activated Notch oncogene as a causative agent in mouse mammarytumor virus induced breast cancer in mice, the potential role for Notch signaling in normaland pathological mammary development was revealed. Subsequently, Notch receptors havebeen found to regulate normal development in many organ systems. In addition, inappropriateNotch signaling has been implicated in cancer of several tissues in humans and animal modelsystems. Here we review important features of the Notch system, and how it may regulatedevelopment and cancer in the mammary gland. A large body of literature from studies inDrosophila and C. elegans has not only revealed molecular details of how the Notch proteinssignal to control biology, but shown that Notch receptor activation helps to define how othersignaling pathways are interpreted. In many ways the Notch system is used to define thecontext in which other pathways function to control proliferation, differentiation, cell survival,branching morphogenesis, asymmetric cell division, and angiogenesis—all processes which arecritical for normal development and function of the mammary gland.

KEY WORDS: Notch; mammary development; breast cancer; Int3.

INTRODUCTION

In 1919 Otto Mohr described a mutation in fruitflies that caused wing Notching in heterozygous fe-males (1,2). Later D. F. Poulson carefully analyzed thephenotypic consequences of the responsible chromo-somal deficiency, termed Notch8 (3,4). Interestingly,male Notch mutants, without a normal copy of the X-chromosome to supply the Notch-gene-encoded pro-tein, had a number of dramatic developmental defectsaffecting many embryonic tissues, including deriva-tives of all three germ layers. The most dramatic de-velopmental defect observed in Notch null mutants in-volves an almost complete transformation of surfaceectoderm into cells of an expanded nervous system.Indeed, this so-called “Neurogenic” phenotype was

1 Mammary Biology and Tumorigenesis Laboratory, National Can-cer Institute, Bethesda, Maryland 20892; e-mail: [email protected].

2 Program in Developmental Biology, The Hospital for Sick Chil-dren, 555 University Avenue, Toronto, Ontario, M5G 1 × 8,Canada and the Department of Molecular and Medical Genetics,The University of Toronto; e-mail: [email protected].

subsequently found in flies with mutations in othergenes, now collectively known as the Neurogenicgenes (5–7). It is this historical context that has led to astrong neurobiological focus of studies following fromPoulson’s work. Interestingly, the molecular charac-terization of Neurogenic gene products has led to theidentification of a “Notch signaling transduction path-way” by which many proteins function together to reg-ulate development in the nervous system, and in otherectoderm derived, as well as mesoderm and endodermderived, tissues in invertebrates and vertebrates (6).

NOTCH RECEPTOR PROTEINS

In the mid-1980s the fly Notch gene was clonedand found to encode a large transmembrane receptor(8,9). At about the same time, research on the ne-matode worm C. elegans resulted in cloning of twoNotch-related genes, lin-12 and glp-1, which controldevelopment of multiple tissues in this organism (10–12). In each case, the Notch receptor was a single

1451083-3021/04/0400-0145/0 C© 2004 Plenum Publishing Corporation

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146 Callahan and Egan

pass transmembrane protein with multiple EGF-likerepeats and cdc10/ankyrin repeats. Specifically, theDrosophila Notch protein contains 36 extracellularEGF-like repeats, followed by three cysteine-richLin12/Notch repeats (LNR), a transmembrane do-main, a “RAM23” region, seven cdc10/ankyrin re-peats (13), a nuclear localization sequence, and a C-terminal extension with PEST sequences (14). Theworm proteins are highly related in structure, al-though somewhat smaller with 13 and 10 EGF-likerepeats for Lin-12 and Glp-1, respectively. The fourmammalian Notch proteins (Notch1, 2, 3, and 4) areclosely related to the Drosophila protein (Fig. 1)(14). Notch1 and Notch2 contain 36 EGF-like repeats,whereas Notch3 has 34 EGF-like repeats and Notch4has 29 EGF-like repeats. Notch receptors are pro-teolytically processed and glycosylated prior to ex-pression on the cell surface in most systems. This firstcleavage reaction occurs in the golgi complex, is me-diated by Furin or a related preprotein convertase(15,16), and results in the expression of a noncova-lently linked heterodimer on the surface of cells (17).

NOTCH ACTIVATION SYSTEM: DELTA ANDSERRATE/JAGGED LIGANDS

Genetic analysis in flies also led to the identifica-tion of two related families of Notch ligands (Fig. 1)(18). The first described was a transmembrane pro-tein, Delta. Delta functions to activate Notch recep-tors on an adjacent cell and to inhibit Notch activa-tion in the same cell (6,19). The Delta extracellulardomain contains a single cysteine-rich motif relatedto the EGF-like repeat, termed DSL on the basisof its identification in the Notch-ligands Delta, Ser-rate (see below), and Lag-2 (from C. elegans) (20).These domains have only been found in Notch ligands.The DSL domain of Delta is followed by nine EGF-like repeats and a transmembrane domain. Mammalshave three Delta genes, Delta-like 1, 3, and 4 (or Dll1,Dll3, and Dll4). Each protein has a single DSL andmultiple EGF-like repeats. Dll1 has eight EGF-likerepeats, Dll3 has six, and Dll4 has eight. While notformally considered a Delta-family ligand by mostauthors, there is a fourth mammalian gene that en-codes a protein with similarity to Delta ligands (21).This gene is know as Pref-1 or Delta-like (Dlk), and itencodes a protein with an N-terminal EGF/DSL-likedomain followed by five EGF-like repeats, a trans-membrane domain, and a cytoplasmic domain with-out obvious domain homology. Like the more widelystudied Delta ligands listed above, Pref-1/Dlk can also

block differentiation and can induce expression ofHes-family proteins (see below) (22).

The second Notch ligand discovered in fliesis termed Serrate. The mammalian homologues ofSerrate are Jagged1 and Jagged2 (18,20). The Ser-rate/Jagged proteins are also single pass transmem-brane proteins. The extracellular domain of each hasa DSL domain, followed by 14–16 EGF-like repeats(Serrate has 14 and mammalian Jagged proteins have16), and a von Willebrand factor type C domain (likelyinvolved in oligomerization). Sequences at the ex-treme N and C termini of Delta and Serrate lig-ands are less conserved. N-terminal to the DSL do-mains in both Delta and Serrate-family ligands isa cysteine-containing sequence which likely controlsNotch receptor-binding specificity (18). An artificialDelta–Serrate chimeric molecule, with the N termi-nus from Delta but DSL, EGF-like repeat region, vonWillebrand factor type C domain, transmembrane do-main, and C terminus from Serrate, behaves like Deltaduring fly wing development (see discussion of Fringemodification of ligand specificity below). The intracel-lular sequences of Delta and Serrate ligands are alsosomewhat related, although neither contains any rec-ognizable domain structure.

ALTERNATIVE FORMS OF NOTCH AND ITSLIGANDS: TRANSCRIPTION, SPLICING,AND GLYCOSYLATION

Several Notch receptor and ligand genes inmammals are subject to alternative transcriptionalinitiation or splicing to generate protein isoforms withdistinct domain organization. For example, Imataniand Callahan have described an isoform of Notch4 ex-pressed in breast cancer cell lines, which is transcribedfrom an alternative promoter (see below) (23). Thepredicted gene product from this novel mRNA codesfor an N-terminally truncated Notch4 product, con-sisting of most of the intracellular domain. The Dll3ligand gene can generate multiple protein isoformsthrough alternative splicing (24). In this case, the alter-native isoforms vary at their extreme C termini. TheJagged2 gene is subject to alternative splicing wherebyEGF-like repeat 6 can be included, or not (hJAG2.del-E6), depending on the presence of a specificin-frame exon (see Genbank). The extent to whichalternative promoter usage and alternative splicingare used to generate multiple Notch receptors andligands is not clear. Indeed, biological functions foreach Notch4, Dll3, and Jagged2 isoform have yet to bedetermined.

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Notch Signaling in Mammary Development and Oncogenesis 147

Fig. 1. The Notch activation system. Fringe proteins modify DSL ligands and Notch receptors to control ligand-receptorspecificity (top). Note that the positions of carbohydrate structures on Notch and DSL ligands are arbitrary in this figure.Mammalian DSL ligands and Notch receptors (bottom).

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As with most large-cell surface proteins, theNotch receptors and ligands are subject to N-linkedglycosylation (17,25). The regulation and functionof this form of glycosylation in the Notch systemhas not been determined, although minimally N-linked glycosylation is thought to be important forprotein folding. In 1998, Haltiwanger and cowork-ers found that Notch1 is also modified through ad-dition of O-linked Fucose and O-linked Glucoseresidues (26), each found as a monosaccharide mod-ification of specific Serine or Threonine residueson Notch, or as part of a Serine/Threonine-Fucose-GlcNAc-Galactose-Sialic acid tetrasaccharide or aSerine/Threonine-Glucose-Xylose-Xylose trisaccha-ride, respectively (27,28). These modifications are un-usual, having been previously been described on onlya limited set of proteins, including the EGF-like re-peat containing coagulation factors.

During the mid- to late 1990s, Irvine and cowork-ers characterized a gene in flies, termed Fringe, thatfunctions to control whether Delta or Serrate activateNotch in specific regions of the developing wing disc(25,29). The Fringe protein had homology to bacterialglycosyltransferases, suggesting that it might functionby regulating glycosylation of Notch or its ligands.These two observations were connected when severallabs collaborated to show that Fringe proteins add aβ1,3-linked N-acetyl-Glucosamine (GlcNAc) to theO-linked Fucose residues on Notch (30,31). It has sub-sequently been shown that Delta and Serrate ligandscontain O-linked Fucose and that these can be furthermodified by Fringes through addition of β1,3-linkedGlcNAc (32). In 1997, the mammalian Fringe-familywas identified and found to consist of three relatedgenes, termed Lunatic Fringe, Manic Fringe, andRadical Fringe (33,34). The Lunatic Fringe and ManicFringe gene products, like Drosophila Fringe, areGlcNAc transferases that elongate O-linked Fucoseresidues on Notch receptors and ligands (30). Abiochemical function for Radical Fringe has yet tobe described. Genetic studies published to date havehighlighted a role for Lunatic Fringe in regulation ofNotch activation during somitogenesis (35,36). Thebiological role of Manic Fringe and Radical Fringeare less clear (37). One intriguing possibility is thatthe mammalian Fringe genes function redundantlyin development. While Lunatic and Radical Fringegenes do not overlap in expression or function (37),it will be important to test for redundancy betweenLunatic and Manic Fringe, as well as between Manicand Radical Fringe. Using tissue culture assays and aseries of mutant CHO cell lines, Pam Stanley’s group

has shown that the trisaccharide structure Fucose-GlcNAc-Galactose must be generated throughfurther elongation of the Fringe-generated disac-charides on Notch, in order for Fringe to effectivelyblock activation of Notch1 by Jagged1 in vitro (38).

Interestingly, Fringe proteins do not functionin all Notch-dependent developmental systems, andtherefore are not always required for Notch activa-tion or inhibition. In contrast, the Fucose transferaseenzyme protein O-fucosyltransferase 1 (O-FUT1or Neurotic) is required for most, if not all, Notchfunctions in flies (39,40). Deletion of this gene in themouse gives rise to Notch loss-of-function phenotypesand early embryonic lethality (41). In fact, O-FUT1-mediated fucosylation of Notch is required to createhigh-affinity Delta- and Serrate-binding sites (42).The Fringe-mediated elongation of these O-Fucoseresidues then affects specificity of Notch for eitherligand. For example, addition of both O-Fucose andGlcNAc to Notch EGF-like repeat 12 is requiredfor Fringe to inhibit Serrate binding to Notch invitro (43).

ALTERNATIVE FORMS OF NOTCH AND ITSLIGANDS; PROTEOLYTIC PROCESSING ANDβ-HYDROXYLATION DURING SYNTHESIS

Notch receptors are subject to proteolytic cleav-age as they move through the secretory pathway (17).This cleavage occurs at cleavage site 1 (S1), betweenthe LNR repeats and the transmembrane domain, andgenerates two polypeptides that remain associatedthrough a noncovalent interaction requiring divalentcations (44). A furin-like convertase enzyme is likelyresponsible for this cleavage, at least in mammaliantissue culture cells (16). Interestingly, full-lengthuncleaved Notch can also be found at the cell surface,as can Notch proteins that have been cleaved withinthe EGF-like repeat containing domain and Notchproteins that have been cleaved to remove C-terminalsequences (45–48). Ligand-induced Notch signalingoccurs in the absence of furin processing of Notch1p300 (45). Bush et al. (45) were able to show thatinteraction of ligands with the uncleaved form ofNotch1 can still block mouse C2C12 myoblast dif-ferentiation. The nature and function of alternativeproteolytic processing of Notch is unclear. It is impor-tant to note that proteolytic removal of N-terminalEGF-like repeat containing sequences is sometimesassociated with loss of Delta and Serrate/Jaggedligand binding sites (46). In contrast, removal of

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C-terminal sequences is associated with removalof disheveled-binding sites and expected to pro-foundly affect Notch signal transduction (see below)(48). Given the nature of, and variability of, Notchprocessing, this will remain an active area of research.

Many EGF-repeats contain a consensus se-quence for β-hydroxylation of aspartic acid and as-paragine residues. Dinchuk et al. recently generatedaspartyl β-hydroxylase (BAH) mutant mice. Thesemice had undetectable β-hydroxylation activity inliver preparations. Surprisingly, they displayed phe-notypes similar to Jagged2 loss-of-function mutants(49). The authors went on to show that Jagged pro-teins are subject to β-hydroxylation in tissue culturecells, strongly suggesting that this modification playsa significant role in Notch activation, at least throughalterations of Jagged2-Notch signaling.

ALTERNATIVE NOTCHACTIVATION SYSTEMS

The extracellular domain of Notch receptors islarge and conserved, suggesting that they may inter-act with a number of proteins beside Delta and Ser-rate/Jagged ligands and Fringes. In a screen to identifyDrosophila gene products with affinity for the Notchextracellular domain, Cedric Wesley identified a num-ber of extracellular and cell surface proteins that canbind to Notch (50). For example, he found that thepoorly characterized Pecanex protein seemed to havehigh affinity for Notch. Perhaps most provocatively,he identified Wingless (Wg), a Drosophila Wnt pro-tein, in this screen. Interestingly, Wg bound to an ar-tificially truncated Notch protein lacking the first 18EGF-like repeats, indicating that this protein inter-acts with Notch through a surface, or surfaces, thatis distinct from those used to bind DSL ligands. Wgalso bound to naturally expressed forms of Notchthat were significantly smaller than the predicted full-length Notch protein (46,50). This binding was depen-dent on the presence of extracellular calcium. Morerecent data suggest that the Wingless–Notch interac-tion may activate alternative forms of signal transduc-tion and play an important role in regulating develop-mental processes that are dependent on Delta/Serrateligands and Wnt proteins (see below) (51).

A novel Notch ligand, F3/Contactin, was recentlyshown to activate Notch signaling in oligodendrocytes(52). This cell surface protein has six immunoglobulin-like domains followed by four fibronectin type-threedomains and a GPI-link. Interestingly, F3/Contactin

activates the disheveled-signaling pathway, a path-way distinct from that activated by Delta and Serrate-family ligands (see below) (52).

There are many other complexities in the Notchactivation system, some of which have only recentlycome to light. For example, extracellular calcium con-centrations control the affinity of Notch ligand/Notchreceptor interactions (53). This phenomenon isexploited to restrict Notch activation during earlyembryonic development. In addition, the secretedDrosophila protein Scabrous is thought to controlendocytic trafficking of Delta and/or Notch in cellsthat internalized this fibrinogen-related protein, andtherefore it is thought to alter the threshold forNotch activation (54). Finally, the recently describedDrosophila cell adhesion protein Echinoid is asso-ciated with cis-endocytosis of Delta and facilitatesefficient activation of Notch by Delta (55).

THE DELTA-/SERRATE-INDUCEDACTIVATION EVENT

The activation of Notch receptors by Delta orSerrate ligands is a complex cell–cell communicationevent, which is only partially understood (56). To startwith, a Delta or Serrate ligand binds to the extracel-lular domain of Notch. In some cases this can oc-cur through interaction of ligand on the surface ofDelta-induced filopodia with Notch on cells that arenot even direct neighbors of the ligand-expressingcell (57). In response to DSL ligand bindingto Notch, two related events occur: internalizationof a ligand/Notch extracellular domain (ECD) com-plex into the ligand expressing cell, and cleavageof the Notch extracellular domain at cleavage site2 (S2). It is not known whether internalization ofDelta–Notch ECD or S2 cleavage occurs first, butboth are required and presumably occur together.The highly related ADAM metalloproteases TACEand/or Kuzbanian are responsible for S2 cleavage.The S2 cleavage event targets a sequence just out-side of the Notch transmembrane domain, and thisleaves a membrane-spanning Notch protein fragmentwith a tiny stub projecting into the extracellular space.Fragments of this sort are targeted for further pro-teolytic cleavage by a presenilin containing multi-subunit protease commonly known as γ -secretase.γ -Secretase cleaves Notch at cleavage site 3 (S3),near the C-terminal end of the transmembrane do-main, thus releasing Notch ICD from its membranetether (58). Notch ICD translocates into the nucleus,

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where its RAM23 sequences bind to a transcriptionalrepressor complex through direct contact with theCSL DNA-binding protein (for CBF1, Suppressorof Hairless, or Lag-1). Notch ICD–CSL interactiondisrupts the transcriptional repressor complex sittingon DNA, recruits transcriptional activating proteins,and turns on transcription of genes that were re-pressed prior to Notch activation (see below) (6,56).Interestingly, DSL ligands are also cleaved by Kuzba-nian/TACE ADAM proteases (59) and ultimately arecleaved by γ -secretase to liberate ligand ICD frag-ments that have transcriptional activation properties(60–62). These data suggest that DSL ligand–Notchinteraction may activate bidirectional signaling. Muchof what has been learned about Notch activation andregulation of transcription has been stimulated by thediscovery of activated alleles of Notch receptors incancer, including the discovery of activated Notch4in mouse mammary tumor virus- (MMTV) inducedbreast cancer (63–65).

MMTV-/IAP-INDUCEDNOTCH4/INT3 TUMORS

The potential importance of aberrant Notch sig-naling in mammary tumor development was first rec-ognized when Notch4 was found to be a common inte-gration site (CIS) for MMTV in 18% of virus-inducedmouse mammary tumors (9 out of 45 tumors) of thehigh-incidence CzechII strain of feral Mus musculusmusculus. MMTV is not an acute transforming virus.Instead the virus, as a consequence of integration ofits genome into the host genome, activates expressionof adjacent cellular genes. In the case of Notch4, all(n = 9) of the viral insertions occur within exons 21and 22 that span a 174-bp region within the gene. Thisregion encodes a portion of the extracellular domain(ECD) adjacent to the transmembrane domain (TM).

The “activated” RNA transcript is initiatedwithin the MMTV long terminal repeat (LTR) and en-codes a small portion of ECD, TM, and the completeICD. The frequency with which Notch4 is activated byMMTV in mouse mammary tumors appears to be in-fluenced by the genetic background of the host mousestrain. In CzechII mice the frequency of integrationevents in Notch4 was 18% (n = 45), whereas in thehigh-incidence BR6 inbred and M. m. jyg mousestrains the frequency was 7% (n = 30) and 43% (n =23), respectively. A similar phenomenon has beennoted for the MMTV CISs, Wnt1, and FGF3/FGF4(reviewed in (66)). These studies strongly implythat alterations fixed in the genetic background

of the host mouse strain affect the frequency withwhich particular MMTV integration sites will beselected. Investigators have taken advantage of thisconsideration to expand studies to MMTV-infectedtransgenic mouse strains (reviewed in (66)). In thesestudies the goal was to use the MMTV genome as amolecular tag to identify CIS-associated genes thatcomplement the transgene during the evolution ofmalignant progression. This approach was takenwith erbB2 transgenic mice infected with MMTV(67). In that setting, 2 out of 24 mouse mammarytumors contained MMTV-induced rearrangements ofNotch1. In both tumors the viral genome integratedwithin sequences coding for the last Notch/lin-12repeats and TM. In one case the viral genome wasin the same transcriptional orientation as Notch1,while in the other tumor it was in the reverse orien-tation. As with MMTV-activated Notch4, the geneproduct was a protein composed of the Notch1 TMand ICD.

MMTV integration within Notch4 is not the onlymutagenic event that leads to the expression of “ac-tivated” Notch4 ICD. In addition, two studies havereported that intracisternal A-particle (IAP) trans-posable elements have integrated into Notch4 nearMMTV CIS in a spontaneous Balb/c tumor and aCzechII mouse mammary tumor (68,69). In the Balb/ctumor, a fragment of an IAP genome (LTR and gagregion) was linked to an extra copy of genomic se-quences containing exons 23 and 24 that were in-serted in the same transcription orientation as Notch4in intron 24. Two alternatively spliced transcripts ini-tiated from IAP LTR were detected. The longer ofthe two coded for the complete intracellular domainof Notch4 from a cryptic translation start signal in theIAP gag region. Translation of the shorter transcriptbegan in exon 24 and therefore coded for a smallerICD polypeptide that was missing the RAM23 region.In the CzechII mammary tumor, the IAP genome wasintegrated in the reverse transcriptional orientationfrom Notch4. In this case transcription of the Notch4ICD was initiated from a cryptic transcription pro-moter in the reverse LTR. Translation of a Notch4 TMand ICD containing polypeptide was initiated from anin-frame methionine in the IAP reverse LTR.

THE BIOLOGICAL CONSEQUENCES OF“ACTIVATED” NOTCH4 EXPRESSION

The biological consequences of “activated”Notch4 expression on mammary gland developmentand tumorigenesis have been studied in vitro and in

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vivo. The HC11 mouse mammary epithelial cell line(70) is a clonal derivative of the COMMA D cell linethat was derived from normal midpregnant BALB/cmammary epithelium (71). HC11 cells are notcapable of anchorage-independent growth but haveretained the capability to differentiate and expressmilk proteins in response to lactogenic hormones.Taking advantage of these properties, Robbins et al.(72) and Dievart et al. (67) showed that expression ofNotch4 and Notch1 ICDs, respectively, in these cellsconferred the capability for anchorage-independentgrowth in soft agar. In addition, expression of Notch4ICD in these cells blocks their ability to expressmilk proteins in response to lactogenic hormones(Callahan, unpublished data). Thus, expressionof Notch ICD blocks the ability of HC11 cells todifferentiate and confers on them growth propertiesassociated with malignant transformation.

TAC-2 is another mouse mammary epithelialcell line that has been useful for studying the ef-fect of Notch4 ICD signaling on normal epithelialarchitecture. These cells have the ability to formwell-polarized histotypic structures when grown incollagen gels (73), and when treated with hepatocytegrowth factor (HGF) they form branching tubulesin culture. Uyttendaele et al. (74) have shown thatNotch4 ICD expression inhibits tubule formationby TAC-2 cells. In a subsequent study, Soriano et al.(75) showed that Notch4 ICD signaling blocks theformation of glucocorticoid-induced alveolar-likestructures in TAC-2 cells grown in collagen gels andcauses loss of contact inhibition in TAC-2 cells grownon collagen-coated dishes. These tissue culture sys-tems offer a means to molecularly dissect the effectsof different Notch signaling pathway components onmammary epithelial biology, but do they reflect theconsequences of Notch4 ICD expression in vivo?

Two transgenic mouse strains have been devel-oped which express the Notch4 ICD from either theMMTV LTR or the whey acidic protein (WAP) pro-moter (65,76–78). The common phenotype in thesestrains is that 100% of females are blocked in theirability to lactate, and all develop mammary tumors.In virgin females containing the MMTV LTR-Notch4ICD transgene, there is minimal ductal developmentin the mammary gland. After the first pregnancy,ducts do fill the mammary fat pad, but there is littlelobular development or cellular differentiation. Incontrast, WAP-Notch4 ICD females exhibit normalductal development, but during pregnancy secretorylobular development is severely impaired. Recipro-cal transplantation studies of mammary epitheliumfrom females of each of the transgenic strains

with normal FVB/N demonstrated that transgenicmammary epithelium was unable, either to grow inepithelium-divested FVB/N mammary fat pads fromvirgin mice (from MMTV LTR-Notch4 ICD), orto functionally differentiate in epithelium-divestedFVB/N mammary fat pads from parous mice (fromWAP-Notch4 ICD). In contrast, FVB/N mammaryepithelium was able to grow and differentiate inepithelium-divested transgenic mammary fat pads.Coincident with limited lobular development ofthe WAP-Notch4 ICD mammary gland duringpregnancy, dysplastic lesions appear throughout thegland that do not regress after weaning and progressto frank carcinoma. Immunohistochemical analysisof this tissue indicates that WAP-Notch4 ICD mam-mary tumors originate from secretory epithelial cellprogenitors, whereas the MMTV LTR-Notch4 ICDtumors appear early and arise from ductal progenitorcells or from individual epithelial stem cells.

COMPONENTS OF “ACTIVATED”NOTCH4 SIGNALING

The Notch ICD is composed of three regions(reviewed in (79)). The RAM23 region, adjacent toTM, contains a nuclear localization signal sequenceand is responsible for binding the CSL transcrip-tion repressor/factor. Adjacent to RAM23 is theCDC10/Ankryn region, which is composed of sevenconsecutive 32 amino acid repeat residues (13). TheCDC10/Ankryn repeats region binds several proteinsthat modulate or regulate Notch4 signaling, and alsoa number of transcriptional coactivators includingMastermind/Maml (see below and reviewed in(79)). Notch1 and Notch2 have a third region that isC-terminal to the CDC10/Ankryn and is defined byits ability to transactivate transcription from GAL4fusion constructs (80).

MMTV integration in Notch4 represents again-of-function mutation, since it releases expres-sion and activity of the Notch4 ICD from negativeregulatory effects of the Notch4 ECD. All but one ofthe viral insertions occur 5′ to the sequence encodinga methionine at amino acid residue 1411 (81). Theexception occurred 5′ of the sequence encodinga methionine at residue 1381. Although there arethree more potential translation start signals 3′ tothe Notch4 CIS, none have been used as translationstart sites by MMTV integration in viral-inducedmammary tumors. This suggests that aberrant ex-pression of the entire ICD may be required for therapid development of mammary tumors.

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The complex series of interactions involvingNotch ICD and other proteins involved in transmit-ting the Notch signal, or modifying the nature of thissignal during mammary gland development and tu-morigenesis, are not yet well characterized. In mostdevelopmental contexts studied to date, the majorNotch signaling pathway is dependent on interactionof the CSL transcription repressor/factor with theRAM23 region of the Notch ICD. This interactiondisplaces corepressors SMRT and HDAC1, replacingthem with coactivators (82).

PRIMARY TARGETS OF CSL-DEPENDENTNOTCH SIGNALING ARE MEMBERS OF THEHES AND HERP GENE FAMILIES

Hes (Hairy-Enhancer of Split) and Herp (Hes-related repressor protein; a.k.a. Hey, Hesr, and Hrt)gene families encode basic helix-loop-helix (bHLH)proteins that are transcriptional repressors of geneexpression, functioning downstream of Notch signal-ing (reviewed by (83,84)). Their expression patternsin different tissues and during development are notcompletely overlapping. Hes has been shown to act asa transcriptional repressor by three different mech-anisms. One of these occurs through formation ofa complex with mammalian TLE/Grg (mammalianhomologues of Groucho) corepressor proteins. Thiscomplex binds to E-box and/or N-box elements inresponsive promoters (85). Another is through for-mation of nonfunctional heterodimers with bHLHtranscription activating factors, such as MyoD andH/Mash1. A third, less characterized mechanism de-scribed for Hes-1 requires its Orange or helix3–helix4domain to repress transcription of its own promoter,as well as transcription of the p21waf promoter (83).Herp, in addition to passive repression of transcrip-tion by sequestration, can bind a heterologous setof corepressors, N-CoR/mSin3A/HDAC. For the sixHes-family proteins and three Herp proteins, only alimited number of target genes are known. For ex-ample, Hes1 represses its own expression, as well asexpression of H/Mash, CD4, and acid α-glucosidase,whereas Herp1 is known to repress its own promoter.

OTHER GENES DIRECTLY REGULATEDBY NOTCH SIGNALING

In addition to members of the Hes and Herp genefamilies, a number of genes directly implicated in can-

cer are known to be upregulated as a direct conse-quence of CSL-dependent Notch signaling. One ofthese genes is erbB2. Chen et al. (86), while study-ing the transcriptional control of ErbB2, identifieda palindrome binding protein that bound the ErbB2promoter. When its sequence was determined, it wasfound to be identical to CBF-1/CSL. In 293 tissueculture cells, Notch1 ICD and CBF-1/CSL cooper-ated to activate transcription from a wild-type, butnot mutant, erbB2 promoter. In experiments to studythe mechanism by which activated Notch1 transformsHC11 mouse mammary epithelial cells, Dievant et al.were unable to detect upregulation of erbB2 geneexpression (67), suggesting that regulation of erbB2by Notch ICD may depend on cell-type-specific tran-scription factor expression or be subject to regulationby other signaling pathways. It will be interesting tocompare erbB2 expression in tumors with MMTV in-serted into Notch1, with erbB2 expression in tumorswhere Notch1 is not activated.

Another gene whose expression is upregulatedby CSL-dependent Notch signaling is cyclin D1. Ron-chini and Capobianco (87) have previously shownthat Notch1 ICD activates transcription of the cyclinD1 gene and CDK2 activity with rapid kinetics in anE1A-immortalized baby rat kidney cell line (RKE).This resulted in stimulation of cell cycle progressionfrom G1 to S-phase. Notch1 ICD mutations that wereincapable of activating cyclin D1 transcription failedto transform RKE cells, suggesting that Notch1 ICDtransformation of RKE cells requires induction ofcyclinD1 expression. Next, this group showed thatCSL binds, in a DNA sequence specific manner, tothe cyclin D1 promoter, consistent with the idea thatNotch1 ICD activates cyclinD1 transcription throughthe CSL-dependent signaling pathway. Interestingly,Notch1 ICD expression did not significantly increaseDNA synthesis in 0.1% serum, indicating that induc-tion of cyclinD1 expression and activation of CDK2 inRKE cells are not sufficient to induce proliferation orcellular transformation, and that other factors are re-quired for oncogenic transformation by Notch1 in thiscontext. There are at least two points to keep in mindin evaluating the role of Notch signaling and cyclinD1expression. First, at the present time it is not knownwhich of the Notch gene(s) normally affect cyclin D1expression in the mammary gland. Second is the con-text in which cyclin D1 expression is being evaluated.For instance, it is known that transgenic mice thatoverexpress cyclin D1 in the mammary epithelium de-velop mammary hyperplasias and subsequently mam-mary tumors (88). However, in the HC11 mammary

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epithelial cell line, which is frequently used to studythe effects of oncogene expression on cell growth anddifferentiation, a different result is obtained. In thesecells, overexpression of cyclin D1 causes inhibition ofcell cycle progression and suppression of cell growth(89). In addition, overexpression of cyclin D1 in HC11cells induces differentiation as defined by increasedβ-casein expression.

A third gene upregulated by Notch1 signaling insome contexts is in fact Notch4, but not Notch2 orNotch3 (90). It is likely that this too is a consequenceof CSL-dependent signaling, since the Notch4 pro-moter contains a CSL-responsive element. In this re-gard it would be of interest to know if the mammarytumors in which Notch1 is rearranged by MMTV, aswell as the HC11 mammary epithelial cells expressingactivated Notch ICD (67), also express the completeNotch4 receptor protein. We have found that HC11cells express three Notch ligands, and that when trans-fected with a vector expressing the complete Notch4receptor, these cells acquire the ability to grow in softagar (Callahan, unpublished data). The role of Notch4in Notch1 signaling during mammary gland develop-ment and tumorigenesis merits further study. Finally, afourth gene which may function downstream of Notchsignaling to control oncogenic behavior in some con-texts is NFκB2. This gene is strongly repressed by CSLin the absence of Notch activation (91). It will be im-portant to test whether NFκB2 is regulated by Notchin mammary cells.

DISTINGUISHING BETWEENCSL-DEPENDENT AND-INDEPENDENT SIGNALING

Initial deletion analysis of activated Notch ICDsuggested that signaling detected in the absenceof RAM23 sequences reflected CSL-independentNotch signaling (80,92–96). For example, Jeffries andCapobianco (95) showed that RAM23 sequences inICD were not necessary for transformation of RKE.Moreover, they could not detect a physical interactionbetween CSL and the RAM23-deleted variant ICD(δRAM23), nor could they detect CSL-dependent re-porter gene induction in response to δRAM23 ICDexpression. The general conclusion from these stud-ies was that there was a CSL-independent componentof Notch signaling that did not require the RAM23domain.

Recently, however, Jeffries et al. (97) have pro-vided evidence that deletion of the RAM23 do-

main does not necessarily disrupt CSL-dependentNotch signaling. They showed that a complex ofδRAM23 Notch1 ICD, Mastermind-Like-1 (Maml),and CSL can form, and that this complex can stimu-late CSL/Notch-dependent transcription. They sug-gest that Maml acts as a tether between CSL andδRAM23. Further, they speculate that cell lines ex-pressing Maml can support CSL-dependent transcrip-tional activation with δRAM23 Notch ICD proteins,whereas cell lines that express low levels of Mamlcannot. In this regard, Dievart et al. (67) found thatonly vectors expressing the complete Notch1 ICDwere capable of stimulating HC11 mouse mammaryepithelial cells to grow in soft agar, suggesting thatHC11 cells have low levels of Maml. Imatani andCallahan (23) identified a novel 1.8-kb Notch4/Int3mRNA species (designated h-Int3sh) that is normallyexpressed in human testis and is aberrantly expressedin some tumor cell lines. Translation of the productbegins at a methionine in the first CDC10/Ankrynrepeat of ICD and terminates at the normal trans-lation stop site. It thus represents a naturally occur-ring δRAM23 Notch4/Int3 ICD. Expression of Int3shin the MCF10A human mammary epithelial cell linestimulates anchorage-independent growth. It will beinteresting to see if MCF10A cells express high levelsof Maml, and whether transformation of these cellsby h-Int3sh is dependent on Maml expression.

Recently we have developed three founder linesof transgenic mice expressing h-Int3sh under con-trol of the WAP promoter (Raafat, Callahan, et al.,manuscript in preparation). The phenotype of allthree lines with regard to mammary gland develop-ment and tumorigenesis is the same. Unlike WAP-Int3mice, where pregnancy-associated mammary glanddevelopment is blocked, lobulo/alveolar develop-ment and lactogenic differentiation is normal in WAP-h-Int3sh mice. However, like WAP-Int3 females,WAP-Int3sh females develop mammary tumors (butat a lower frequency and with a longer latency). Thus,in our transgenic WAP-h-Int3sh model, mammary tu-mor development could still be a consequence of CSL-dependent Notch signaling. At the present time we areinvestigating why mammary gland development oc-curs normally in WAP-h-Int3sh females as comparedto WAP-Int3 females. This could reflect the existenceof a previously unappreciated regulatory protein thatinteracts with the RAM23 portion of Notch4 ICD toinhibit mammary gland development, or could indi-cate that the level/extent of h-Int3sh-CSL interactionmediated by Maml is not sufficient to block mam-mary gland development and differentiation, but is

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sufficient to contribute to mammary tumorigenesis.Consistent with the latter possibility, Lin et al. (98)have shown that Notch4/Int3 CSL-dependent signal-ing is relatively resistant to augmentation by Maml1,-2, and -3. Alternatively, Notch4/Int3-induced mam-mary tumorigenesis could be a consequence of CSL-independent Notch signaling.

In vertebrates, additional evidence for non-canonical Notch signaling exists. For example,CSL-independent signaling is found in the devel-oping avian neural crest, where a Delta-1-activatedNotch signal is required to control Slug expression.In this case, regulation of Slug is not altered byexpression of a dominant negative CSL protein inthe neuroectoderm (99). In addition, Timmermanet al. have shown that Notch signaling can induce anepithelial to mesenchymal transition in a number ofcellular contexts, and that this is mediated throughupregulation of the Slug relative Snail (100). It willbe important to test whether these latter two genesare targets of Notch in mammary epithelium. In-terestingly, Slug and Snail expression are associatedwith invasion and metastasis in human breast cancer(101,102). Perhaps Notch-mediated regulation ofSlug or Snail occurs in some of these tumors.

Mediator(s) of the many speculated CSL-independent Notch signaling pathways remainlargely unknown. However, there are several knowntranscription factors such as Mef2 (myocyte enhancerfactor 2) and LEF1 (lymphocyte enhancer factor 1)that have been shown to interact with Notch ICD(103,104). Mef2 is a member of the MADS-boxtranscription factors (reviewed in (105)), whichinteract with the CDC10/Ankryn repeats of Notch1ICD. This interaction blocks DNA binding by Mef2and blocks its ability to cooperate with MyoD andmyogenin to activate myogenic differentiation (104).LEF1 is a member of the high-mobility-group-boxDNA binding protein family (103). This proteinbinds to the TAD region of Notch1 and Notch2 (butnot Notch3), which is located just C-terminal to theCDC10/Ankryn in ICD. The Notch ICD acts as acoactivator of LEF-1 transcription factor activity inthis context.

Another set of candidates to mediate CSL-independent Notch signaling is the Deltex (mam-malian DTX) proteins (106,107). The E47 transcrip-tion factor activates B-cell-specific immunoglobulingene transcription and is required for early B-celldevelopment (108,109). Ordentlich et al. (110)showed that overexpression of Deltex inhibits E47transcription factor independent of CSL-dependentsignaling and that this probably occurs through inhi-

bition of Ras signaling. In another study, Yamamotoet al. (111) have shown that DTX-1 mediates a Notchsignal to block differentiation of neural progeni-tor cells. They found that a significant fraction ofDeltex1 is localized in the nucleus and physicallyinteracts with the transcriptional coactivator p300.Deltex competitively inhibits the binding of theneural-specific helix-loop-helix-type transcriptionfactor Mash1, thereby inhibiting its transcriptionalactivity and neural differentiation. It seems likelythat additional transcription factors and cytoplasmicadaptor proteins will be identified as mediators ofCSL-independent oncogenic Notch signaling. Inaddition, it should be anticipated that these factorswill show specificity for individual Notch receptors.

THE UBIQUITINATION PATHWAYAND NOTCH SIGNALING

Several studies have demonstrated the impor-tance of ubiquitination in the regulation of Notchsignaling (reviewed by Lai (112)). The ubiquitinationpathway is a critical and conserved pathway forregulation of many cellular processes includingprotein turnover, trafficking, and transcription. Thebasic pathway starts with the ubiquitin-activatingenzyme (E1) which transfers ubiquitin, a 76 aminoacid peptide, to a ubiquitin-conjugating enzyme (E2).An E3 ubiquitin ligase combines with an E2 proteinto transfer ubiquitin to a target substrate, or to theend of a polyubiquitin chain. As a consequence ofthis pathway, soluble target proteins are markedwith ubiquitin for degradation in the proteasome. Inaddition, ubiquitination of transmembrane proteinsat the plasma membrane targets them for endocyticinternalization, whereas ubiquitination of transmem-brane proteins in the sorting endosome targets themfor degradation in the lysosome.

There are seven classes of E3 ubiquitin ligaseenzymes that have been implicated in Notch signaltransduction, or in regulation of the strength or du-ration of Notch signal transduction. These E3 pro-teins are Su(dx)/Itch, Deltex (DTX), Cbl, Neuralized,Mindbomb, Sel-10/Cdc-4 (a substrate recognitionsubunit in a multi-subunit E3 ligase), and Lnx. First,Su(dx)/Itch, a Nedd4 class E3 ligase with an N-terminal C2 domain, central WW domains, and a C-terminal Hect domain, binds to the Notch ICD andubiquitinates it. Interestingly, Su(dx)/Itch can transferubiquitin to Notch proteins that are still attached tothe plasma membrane, and may therefore control thelevels of Notch available for activation at the cell

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surface (113). Deltex (Dtx) proteins are ring-fingerclass E3 ligases, capable of self-ubiquitination (114).In mammals there are four DTX proteins (DTX-1,-2, -2δE, and -3) that influence myogenesis, neuroge-nesis, and lymphogenesis. The N terminus of DXT-1,DXT-2, and DXT-2δE binds to the Ankryn/CDC10 ofNotch ICD, whereas this domain is missing in DXT-3.The role of Deltex (DTX) E3 ligase activity is un-clear, since the ring-finger motif is conserved in evo-lution but is not required for rescue of Deltex loss-of-function phenotypes in flies. Cbl is an E3 ligase,most studied in the context of tyrosine kinase signal-ing. Jehn et al. recently reported that when Notch1 isphosphorylated on tyrosine residues in C2C12 cells, itbinds to c-Cbl, and that the tyrosine-phosphorylatedform of Notch1 accumulates when cells are treatedwith the lysosomal inhibitor chloroquine. Thus, Cblappears to target membrane-bound Notch for de-struction in the lysosome, at least in some cells whereit is tyrosine-phosphorylated. Neuralized and Mind-bomb are unrelated and conserved ring-finger classE3 ligases, which function to ubiquitinate the cyto-plasmic domain of Delta-family ligands in order tostimulate their endocytic internalization (115). Thisinternalization of Delta, together with Notch ECD,into signaling cells is required for activation of Notchreceptors in neighboring signal-receiving cells (seeabove). It is not yet clear why both Neuralized andMindbomb have been conserved to perform the sameor similar functions during Notch activation. Sel-10/Cdc-4 is an F-box WD40 domain protein in a largemulti-subunit E3 ligase known as SCF. Sel-10 binds tothe PEST domain of a specific phosphorylated form ofNotch ICD found in the nucleus, perhaps in complexwith the negative feedback regulator Nrarp (116).Once bound, nuclear phospho-Notch ICD is ubiq-uitinated and targeted to the proteosome for degra-dation. The Sel-10 E3 ligase therefore functions tolimit Notch-dependent transcriptional regulation af-ter nuclear translocation of Notch ICD (117–119). In-terestingly, the Sel-10 gene has recently been deletedfrom the mouse germline, and this causes a dramaticelevation of Notch4 ICD without increasing expres-sion of Notch1, 2, or 3 ICDs (120). Perhaps theseICDs are ubiquitinated by the related E3 ligase Skp2,or by Sel-10 and Skp2, in vivo (121). In 2001, SteveReed’s group found that Sel-10 also targets the cy-clin E protein for ubiquitination, and therefore forproteolytic degradation (122). In addition, this groupfound that Sel-10 is a tumor-suppressor gene, mutatedin the SUM149PT breast cancer cell line, and in a largenumber of endometrial carcinomas (122,123). Finally,LNX (ligand of Numb-protein X) is involved in regu-

lating Notch signaling during asymmetric cell division(reviewed in (112)). Numb is asymmetrically localizedin sibling cells whose fate is decided by Notch signal-ing (reviewed in (124)). McGill and McGlade (125)have shown that Numb interacts with the WW do-main of Su(dx)/Itch, and suggested that Numb actsas an adaptor protein that recruits the Itch E3 ligaseand the ubiquitination machinery for the ubiquitina-tion of the Notch ICD complexed with Numb. LNXis an E3 ligase that targets Numb for ubiquitination.Nie et al. (126) suggest that asymmetric distributionof LNX would establish an asymmetric distributionof Numb. It has been proposed that LNX augmentsNotch signaling by lowering the level of Numb inthe daughter cell destined to respond to Notchsignaling.

It will be important to determine what role thesemany E3 ligases play in regulating Notch signaling inthe mammary gland and breast cancer, and for whatbiological outcome. Some recent data on mammalianNeuralized speak to this issue (see below).

CROSS-TALK BETWEEN THE NOTCHPATHWAY AND OTHER SIGNALINGPATHWAYS

Many examples of cross-talk between the Notchpathway and other signaling pathways have been doc-umented. For instance, cooperative and antagonisticeffects between Receptor Tyrosine Kinase/Ras path-ways and the Notch pathway have been reported inDrosophila (127) and C. elegans (128–130). In theDrosophila eye, erbB and Notch signaling are usedtogether to control development of cone cells. In thiscase, activation of erbB signaling in the neuronal pho-toreceptor cells is required for expression of Deltaligand in these cells (131). Delta then functions to-gether with erbB signaling to activate cone cell dif-ferentiation in neighboring cells (132). An antago-nistic interaction between erbB and Notch signalinghas been seen during C. elegans vulval development(128). In this case, erbB signaling and Notch signalingare known to interact at multiple levels (133). First,strong activation of erbB signaling in the P6.p vulvalprecursor cell leads to expression of three Notch lig-ands (including the secreted Notch ligand, Dsl-1), andto endocytosis and destruction of the worm Notch re-ceptor, Lin-12, in this cell. Once P6.p expresses Notchligands, these ligands activate Lin-12 in neighboringP5.p and P7.p cells. Lin-12/Notch signaling in P5.pand P7.p activates expression of several redundantinhibitors of erbB signaling in these cells, including

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Mapk kinase phosphatase, Ack-1 kinase, and a num-ber of proteins implicated in endocytic downregula-tion of erbB receptors (129,130,133–135). The com-plex interplay of Notch signaling and erbB signalingin the mammary gland and breast cancer remains to beinvestigated. However, as discussed above, Notch sig-naling can upregulate erbB2 expression (86), and acti-vation of Notch1 cooperates with activation of erbB2to induce breast cancer in mice (67). Notch signalingis also used together with FGF receptor signaling toregulate development in many contexts. In flies, Notchsignaling is used to suppress MAP kinase activationdownstream of FGF receptor signaling in the devel-oping and branching trachea (136). This interactionbetween Notch and FGF receptor signaling is found inmammalian limb patterning and tooth development,indeed in many contexts (137,138). In vitro, an anti-sense oligonucleotide against Jagged1 was found tobe a potent enhancer of FGF-induced angiogenesis(139). This result led to the finding that Notch sig-naling even inhibits FGF-induced transformation offibroblasts, confirming the antagonistic relationshipbetween Notch and FGF signaling (140).

Fitzgerald et al. (78) have attempted to iden-tify which signaling pathways collaborate with Notch4ICD in mammary tumor development. Using cell linesderived from transgenic MMTV LTR-Notch4 ICD,they showed that inhibition of Erk/MAP kinase andPI-3 kinase pathways, downstream of Ras, blockedanchorage-independent growth of cells in soft agar.Inhibitors of other signaling pathways including Src-like kinases (Lck and Fyn) and protein kinases Aand C had no effect in this assay. So, in this set-ting, there is a synergistic relationship between Notchand Ras signaling pathways in Notch4 ICD initiatedmammary tumors. In another experimental system,Weijzen et al. (90) show that oncogenic Ras activatesNotch signaling and that wild-type Notch1 is neces-sary to maintain the neoplastic phenotype in Ras-transformed human cells, in vitro and in vivo. In theseRas-transformed cells, expression of Notch1, Delta-1,and presenilin-1 are upregulated, and Ras influencesNotch-1 signaling through a p38-mediated pathway.

Interactions between Notch and TGFβ signalingpathways have also been reported. For example,Blokzijl et al. (141) have described a synergistic in-teraction between Notch1 and TGFβ1 to upregulateHes-1 in myogenic cells. This effect was blocked byexpression of a dominant-negative form of CSL. Themechanism underlying the integration of TGFβ1and Notch signaling is likely through protein–proteininteraction between SMAD3, the intracellular trans-

ducer of TGFβ1 signals, and Notch1 ICD. SMAD4 isrequired for the functional interaction between theNotch1 and TGFβ1 pathways. In addition, a SMAD3point mutation that is not able to bind DNA is stillcapable of potentiating the Notch1 ICD/TGFβ1signal at the Hes-1 promoter. In reciprocal experi-ments, using the PAI-1 promoter that is specific forSMAD3 and highly responsive to TGFβ1, Notch1ICD could further enhance transcription activity ofthe promoter in a concentration-dependent manner.Whether this functional relationship between Notch1and SMAD3 can be generalized to other membersof the Notch family and to other cellular contextsremains to be determined.

As discussed briefly above, the fly Wnt proteinWingless can bind to Notch extracellular domainsequences (50). The function of this interaction wasinitially very puzzling. More recent studies in fliesand mammals have revealed that Wnt and Notchsignaling pathways interact at multiple levels, sincethese two pathways frequently function together toregulate development (51,142). Indeed, Wingless notonly binds to Notch, but also activates a cytoplasmicsignaling pathway that functions to inhibit DSL-ligand activated Notch signaling. Wingless activatesthe cytoplasmic Dishevelled protein, which in turnbinds to the Notch cytoplasmic domain to inhibitsignaling through Deltex. Wingless signaling alsoinhibits glycogen-synthase kinase-3 (GSK-3) activity,which is required to stabilize Notch ICD. Uyttendaeleet al. (74,143) have presented evidence for negativecross-talk between the Wnt-1 and Notch4 signalingpathways in the TAC-2 mammary epithelial cell linesuspended in collagen gels. In this setting Wnt1, likeHGF and TGF-β2, induces branching morphogen-esis by TAC-2 cells. TAC-2 cells expressing Notch4ICD did not respond to HGF or to TGF-β2, butunderwent branching morphogenesis in the presenceof Wnt-1, suggesting that Wnt1 signaling is dominantto Notch4 ICD signaling in TAC-2 cells in thebranching morphogenesis assay. Deletion analysisof Notch4 ICD demonstrated that the RAM23 andAnkryn/CDC10 repeats are required for inhibitionof ductal morphogenesis.

NOTCH IN NORMAL AND PATHOLOGICALMAMMARY DEVELOPMENT:MANY QUESTIONS

What is the role of Notch signaling in devel-opment of the mammary gland? This is a difficult

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question to address with our current state of knowl-edge. From what is known about the function ofNotch signaling in flies (6) and worms (144), wecould imagine a role for Notch signaling in ep-ithelial/mesenchymal transitions, such as those inmammary bud formation (145). Notch signalingmight regulate mammary epithelial growth andbranching during puberty (146). Notch signalingcould be involved in cell-fate specification, perhapscontrolling the rationing of cell types within themammary gland (6). Perhaps Notch signaling isinvolved in inductive interactions in the mammarygland (6,25,131). For example, Notch signaling couldcontrol induction of mammary bud formation orexpansion. Maybe Notch signaling controls lacto-genic differentiation (6,147–149), cell proliferation(150,151), stem cell self-renewal (152), or even apop-tosis/involution (153). The problem here is that thereare so many documented biological functions forNotch signaling in both invertebrate and vertebratesystems that we cannot even begin to guess what itsmost important roles will be in the mammary glandin the absence of some basic information, such asNotch receptor, ligand, and Fringe gene expressionduring development. In addition, the gain-of-functionexperiments such as those performed with activatedNotch ICD proteins in tissue culture cells and intransgenic mice must be complemented with Notchgene loss-of-function experiments to determine whatNotch signaling does in the normal mammary gland.

In any case, we have learned some significantlessons already that can hint at Notch functions inthis context (see above). For example, in TAC-2mammary epithelial cells in vitro, activated Notch4suppressed ductal growth and branching, as well asformation of glucocorticoid-induced alveolar-likestructures (74,75). In addition, expression of Notch4ICD blocked lactogenic differentiation (milk geneexpression) in HC11 cell cultures (Callahan, un-published data). In vivo, expression of Notch4 ICDinhibited ductal elongation and branching, as well asalveolar development and differentiation in MMTV-Notch4 ICD transgenic mice. Alveolar development,cellular polarity, and lactogenic differentiation wereseverely impaired in WAP-Notch4 ICD transgenicmice. Multiple Notch receptors and ligands areexpressed in HC11 mammary epithelial cells in vitro(see above), and multiple Notch receptors, ligands,and Fringes are expressed during mouse mammarygland development in vivo (Keli Xu and Egan, un-published data). Interestingly, the expression patternof Notch receptors, ligands, and Fringes changes dra-

matically with each stage of mammary development,suggesting that Notch signaling may indeed be usedfor distinct functions during sequential stages. Pre-liminary loss-of-function experiments on a numberof Notch receptors, ligands, and Fringes support thisview (Keli Xu and Egan, unpublished data).

In 2001, Vollrath et al. reported a mammaryphenotype in mice with a targeted mutation in amammalian homologue of Drosophila Neuralized.Unfortunately, this phenotype has not been charac-terized in detail, but appears to represent a defect inalveolar development since the mutant gland is stillcomposed primarily of adipocytes during lactation.In addition, lactogenic differentiation of alveolarcells may be somewhat impaired. Since Neuralizedis required for Notch activation, this result stronglysuggests that Notch activation may play an importantrole during pregnancy. Interestingly, Notch signalingcontrols differentiation of adipocytes (154) and devel-opment/remodeling of the vascular system (155), twocritical stromal elements (156–158) regulating devel-opment in the mammary gland. Perhaps Notch regu-lates epithelial and stromal elements in this context.

NOTCH IN HUMAN BREAST CANCER

In many ways, it is also very difficult to addressthe role, or potential role(s), played by Notch signal-ing in human breast cancer with the current state ofknowledge. As a starting point, it will be importantto determine which Notch receptors, ligands, andFringes are expressed in breast cancer, and whetherspecific expression profiles correlate with specificpathological or clinical features. To this end, suchexpression analysis has been performed by in situhybridization on a collection of 25 human breasttumors (Michael Reedijk and Egan, manuscript inpreparation). These data have revealed that all fourNotch receptors, four Notch ligands, and one ofthree Fringes are expressed at varying frequencieswithin this collection of tumors. For example, Notch3is expressed in approximately half of the tumorsand highly expressed in three tumors. Interestingly,Notch3 is very highly expressed in neovessels thathave been recruited to many of the tumors, suggest-ing that this receptor may play an important role inbreast tumor angiogenesis (Fig. 2). One of the biggestchallenges to assessing the role of Notch signalingin human breast cancer is the heterogeneity of thisdisease. Clearly a large collection of tumors mustbe studied to establish any correlation between theNotch system expression profile and tumor pathology

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Fig. 2. Notch-3 expression in human breast cancer samples. In situ hybridization using Notch-3antisense (A & C) or sense (B & D) RNA probes. Images are shown at 20× magnificationusing either brightfield or darkfield (insets) microscopy. Tumor 13 (C) demonstrates a fivefoldgreater level of Notch-3 expression than Tumor 11 (A) as assessed by quantifying silver grains.Solid arrows identify Notch-3-expressing VSMC.

or outcomes. Despite these challenges, our prelimi-nary data indicate that Notch receptors, ligands, andFringes are expressed in human breast cancers andsupporting stromal elements, including tumor vessels.Studies on Notch1 and Notch4 ICD in mammaryepithelial cell transformation have been extremelyinformative and have guided our understanding ofoncogenic Notch signaling. It will also be important tokeep in mind the recent finding that Notch genes canfunction to suppress tumor growth (159). Perhaps thelack of Notch receptor, ligand, or Fringe expressionwill be important in some human breast cancers.Clearly, there are more experiments to do.

ACKNOWLEDGMENTS

We apologize to those whose work has notbeen directly cited because of space limitations.Sean Egan thanks Drs Michael Reedijk and KeliXu for permission to cite unpublished results.Robert Callahan thanks Dr Ahmed Raafat andSharon Bargo for permission to cite unpublished

results. S.E.E. is supported by grants from theTerry Fox Foundation, The Canadian Institutes forHealth Research, and the U.S. Army/Department ofDefense.

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notch signaling in mammary development and oncogenesis

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