notch signaling in mammary gland tumorigenesis

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Journal of Mammary Gland Biology and Neoplasia (JMGBN) PP067-295935 February 1, 2001 18:41 Style file version Nov. 07, 2000

Journal of Mammary Gland Biology and Neoplasia, Vol. 6, No. 1, 2001

Notch Signaling in Mammary Gland Tumorigenesis

Robert Callahan1,2 and Ahmed Raafat1

The Notch receptor protein and its signaling pathway have been well conserved throughoutevolution and appear to be pivotal components in cell fate decisions during development.Recent studies suggest that, depending on the cellular and developmental context, Notchsignaling may also affect cell proliferation and programmed cell death. Mammals have fourrelated Notch genes. One of these, designated Notch-4, was found to be a common integrationsite for the mouse mammary tumor virus in mouse mammary tumors. One consequence ofthis type of viral integration event is the ectopic expression of the intracellular domain ofNotch-4 that corresponds to a gain-of-function mutation. Expression of “activated” Notch-4in mammary epithelium has profound effects on mammary gland development and tumori-genesis. In this review, we briefly summarize the structure and function of the Notch receptor,as well as the components that comprise and modify the signaling pathway. Finally we discussthe potential role of Notch in mammary gland development and tumorigenesis.

KEY WORDS: Notch signaling pathway; mouse mammary tumor virus; transgenic mice; mousemammary tumorigenesis.

INTRODUCTION

The gene encoding the Notch receptor was dis-covered almost 80 years ago, and gained its name be-cause partial loss of Notch function resulted in notchesin the wing margins of Drosophila (1). It only becameapparent some years later that the Notch signalingpathway has been conserved throughout evolution.The Notch signaling pathway plays a pivotal role inseveral cell functions; such as cell fate decisions, cellproliferation, differentiation, and cell death duringdevelopment and postnatal life in species as diverse asDrosophila, worms, and vertebrates [reviewed in (2–6)]. The Notch gene encodes a transmembrane recep-tor protein whose function(s) becomes activated uponthe interaction of its extracellular domain (NotchEC)3

1 Laboratory of Tumor Immunology and Biology, National CancerInstitute, Bethesda, Maryland 20892.

2 To whom correspondence should be addressed. e-mail: [email protected]

3 Abbreviations: Mouse mammary tumor virus (MMTV); NOTCHextracellular domain (NotchEC); Notch intracellular domain(NotchIC); Delta, Serrate and Lag ligands (DSL); Delta extra-cellular (DIEC); Hairy enhancer of split (HES); tumor necrosis

with one of its ligands. The particular consequencesof “activated” Notch expression on development ofcomplex tissues are dependent on the cellular contextin which it occurs. What is known of the Notch signal-ing pathway in several species suggests that there hasbeen an expansion in the copy number of homologuesof each of the components in the signaling pathwayduring evolution.

In mammals, the Notch gene family is com-prised of four members (Notch–1–4) with approxi-mately 60% sequence homology to each other and toDrosophila Notch (7,8). Interest in the role of Notchsignaling during mammary gland development stemsfrom the identification of mouse Notch-4 as a com-mon integration site for the mouse mammary tumorvirus (MMTV) in mammary tumors of certain strainsof feral mice (9–11). The purpose of this review isto briefly summarize the current knowledge of thestructure and function of mammalian Notch and the

factor α-converting enzyme (TACE); suppressor of Hairless(Su(H)); Alagille syndrome (AGS); cerebral autosomal dominantarteriopathy with subcortical infarcts and leukoencephalopathy(CADASIL); Drosophila wingless (Wg); disheveled (DSH).

231083-3021/01/0100-0023$19.50/0 C© 2001 Plenum Publishing Corporation

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24 Callahan and Raafat

components of its signaling pathway for researchers inthe field of mammary gland biology. This is a challeng-ing task, since much of the research done on Notchfunction and signaling has been done in Drosophila.Therefore, in this review we will highlight the workdone in mammalian systems and discuss it relativeto the findings in Drosophila. Additional discussionsof Notch biology in Drosophila, C.elegans, Xenopus,Zebra fish and mammals can also be found in severalrecent reviews (2–6).

NOTCH GENES, COMMON INTEGRATIONSITES FOR MMTV

Activation of the Notch-4 locus (9,11) was firstdetected in feral CzechII mouse mammary tumors(10,12). The locus was defined by the integration ofMMTV proviral genomes within a 500-bp region ofthe cellular genome of ten independent mammary tu-mors. The Notch-4 locus is located in the class II re-gion of the major histocompatibility (MHC) locus onchromosome 17 (12,13). In each case the transcrip-tional orientation of the integrated viral genome wasin the same direction as that of the target gene. A2.3 kb Notch-4 RNA species that initiated from the 3′

MMTV long terminal repeat (LTR) of the integratedMMTV proviral genome was detected in tumors con-taining a viral-induced rearrangement of Notch-4.This RNA species was not detected in tumors wherethe locus was intact or in the normal mammary gland.All of the viral integration events within Notch-4 oc-curred within one of three exons encoding amino acidresidues just N-terminal to the transmembrane do-main (NotchTM) of the encoded protein. These viralintegration events resulted in the constitutive overex-pression of a truncated portion of the gene productcorresponding to the intracellular domain (NotchIC)of the protein (10). Experiments in which the same re-gion of the Drosophila Notch gene is overexpressedin vivo demonstrated that expression of this truncatedprotein represents a gain-of-function mutation, mim-icking the consequences of the interaction betweenthe Notch protein and its ligand (14,15).

Since Notch-4 is expressed during normal mam-mary gland development, activation of this gene byMMTV appears either to deregulate normal devel-opmental controls leading to hyperplasia and tumori-genesis. Interestingly, Notch-4 is not rearranged byMMTV in a panel of viral induced mammary glandhyperplastic outgrowth lines (HOGs) [E. Kordon andG.H.Smith, personal communication]. It has been

found to be rearranged by MMTV only in feral mice(16,17) and two BR6 inbred mouse mammary tumors(18). Dievart et al. (19) found Notch-1 to be rear-ranged by MMTV in 2 out of 24 mammary tumorsof MMTV LTR-neu transgenic mice. In each caseMMTV integrated into Notch-1 in a manner similar tothat observed in Notch-4. So far Notch-2 and Notch-3have not been found to be rearranged by MMTV inviral induced mouse mammary tumors (our unpub-lished data). We speculate that the effect of truncatedNotch-4 protein on mammary gland development andtumorigenesis is exquisitely dependent on the geneticbackground of the host and on the timing of its ex-pression relative to mammary gland development.

THE NOTCH RECEPTOR PROTEIN

The four mammalian Notch receptor proteins(Notch-1, -2, -3, and -4) traverse the cellular mem-brane once (NotchTM), have an extracellular(NotchEC) ligand-binding domain and an intracel-lular domain (NotchIC) required for signal trans-duction (Fig. 1) (7,8,20). The NotchEC contains tan-dem epidermal growth factor-like (EGFL) repeatsequences that are thought to be involved in lig-and binding. A cysteine-rich region termed the LNRrepeats, is located C-terminal of the EGFL do-main. The NotchIC contains three domains that af-fect Notch function: a Ram23 protein binding re-gion located C-terminal to NotchTM that interactswith RBP-Jκ/CBF1, six tandem CDC10/Ankyrin re-peats that are sufficient for Notch activity, and at theC-terminus of the receptor, a polyglutamine region(OPA) and a proline-glutamate-serine-threonine richregion (PEST), which is thought to be involved inNotch turnover (20,21).

In mammals the Notch-1 and Notch-2 proteinsare organizationally similarly to Drosophila Notch,whereas during the course of evolution Notch-3 andNotch-4 have lost, by interstitial deletions, specificEGFL-repeats (7,9,11). In the case of Notch 3, theequivalent of EGFL-repeat 21 has been lost. Notch-3also contains a novel EGFL-repeat composed of por-tions of repeats 2 and 3. Similarly, Notch-4 is missingseveral of the equivalent EGFL-repeats. In each caseportions of adjacent EGFL-repeats have been fusedin register to form five novel EGFL-repeats; these in-clude: EGFL-repeats 14 and 15, 16 and 17, 20 and23, and 31 and 32. At present the functional signifi-cance of these differences in Notch-3 and Notch-4 areunknown.

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Notch Signaling in Mammary Gland Tumorigenesis 25

Fig. 1. Schematic diagram of Notch protein structure and its interacting proteins. (A) Notch family members have the same overall structureand arrangement of conserved motifs. The Notch extracellular domain (NotchEC) contains multiple EGF-like repeats (EGFL) and threecysteine-rich LIN-1/Notch repeats (LNR). The Notch transmembrane domain (NotchTM region is followed by the NotchIC which is composedof the RAM23 region which binds CBF-1/Su(H)/LAG-1 interacting proteins, six Ankyrin/CDC10 repeats, followed by the PEST motif whichis rich in proline, glutamic acid, serine and threonine. (B) Proteins that interact with NotchIC. The “Effect” column indicates whether theprotein exerts a positive (+) or negative (−) effect on Notch signaling. Binding affinity of each protein to the receptor is indicated in termsof high affinity (+), low affinity (±) and no binding (−).

The Notch receptor is synthesized as a sin-gle polypeptide precursor which during maturationis cleaved by a furin-like convertase into a extra-cellular subunit (NotchEC) and a transmembrane-intracellular subunit (NotchTM) (Fig. 2) (22). TheNotchEC and NotchTM subunits are recombined as aheterodimer in the trans-golgi. This event seems to benecessary for cell surface expression of Notch, but notfor ligand-induced release of the NotchIC (5).

THE NOTCH LIGANDS

Ligands that bind and activate the mammalianNotch receptors, which are related to the two inver-tebrate Notch ligands, Delta (Dl) and Serrate (Ser),have been identified in genetic and molecular inter-action studies. In C. elegans a similar gene codes fornumerous related ligands including Lag-2 and Apx-1.Notch ligands are collectively known as DSL lig-ands(for Delta, Serrate and Lag-2)[reviewed (23)].In mammals there are two Delta-like homologues(Dll-1 and Dll-3) and two serrate-like homologues(Jagged-1 and Jagged-2) (24–27). Each of these is atransmembrane protein whose extracellular domain

contains a characteristic number of contiguous EGFLrepeats and a cysteine-rich N-terminal DSL domain.Signaling in the Notch pathway is thought to be pri-marily mediated through ligand-receptor interactionsbetween contiguous cells, since the Notch ligands arepredominantly cell membrane associated (Fig. 2) (re-viewed in (20). However, one complication with thisscenario is that a soluble form of Drosophila Deltaextracellular (DlEC) has been detected both in vivoand in cell cultures. Expression of soluble DlEC canbe blocked by inactivation of the ADAM metallopro-tease Kuzbanian (Kuz) (Fig. 2). In addition, mutationsof Kuz resulting in a loss of function are associatedwith a phenotype that is similar to Notch loss of func-tion in Drosophila (28,29). These observations implythat DlEC must be cleaved and therefore must besoluble to be active.

NOTCH RECEPTOR-LIGAND INTERACTION

Activation of Notch signaling begins upon theinteraction of the DSL domain of the ligand withEGFL repeats #11 and #12 of the NotchEC (Fig. 1)(30). In Drosophila Notch, these two EGFL repeats

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Fig. 2. Elements of Notch signal transduction. (1) In the trans-Golgi network, full length Notch is cleaved by a Furin-like convertase.(2) Mature Notch receptor assembled by the adherence of the Notch extracellular domain (NotchEC) to the Notch intracellular domain(NotchIC). (3) The hetrodimeric receptor is transferred to the cell membrane. (4) Extracellular regions of Notch ligands, such as Delta (Dl),bind to the EGFL repeats of NotchEC. Upon ligand-receptor interaction, two cleavage events of the Notch heterodimer are initiated. (5A)The disintegrin-metalloprotease tumor necrosis factor-converting enzyme (TACE) cleaves at a site leaving only 12 extracellular amino acidsattached to the Notch transmembrane domain (NotchTM). (5B) The other site, within NotchTM, is subsequently cleaved in a Presenilin-dependent event that releases the NotchIC. (6) NotchIC nuclear localization signal sequence facilitates the NotchIC translocation to thenucleus. (7) In the nucleus, NotchIC replaces the CSL repressors and binds to CSL (details of this interaction are shown in Fig. 3). Thus,converting CSL to a transcriptional activator instead of repressor. (8) Activated CSL up-regulates transcription of the bHLH gene family,Hairy enhancer of Split (HES). (9) HES gene products repress the transcription of the mammalian Achaete-Scute Homolog (Mash-1) and,as a result, block cell differentiation.

have been shown to be both necessary and suffi-cient for the binding of Dl and Ser. Ligand-receptorinteraction, initiates two cleavage events of theNotch heterodimer (Fig. 2) (31,32). The disintegrin-metalloprotease tumor necrosis factor α-convertingenzyme (TACE) cleaves the extracelluar domain ata S2 site (Fig. 2) that leaves only 12 extracellularamino acids attached to the NotchTM. A second site(S3) within NotchTM, is subsequently cleaved in aPresenilin-dependent event that releases the NotchIC.The NotchIC fragment is hypothesized to enter the nu-

cleus and initiate downstream signaling events. How-ever, as Artavanis-Tsakonas et al. (5) have pointedout, except for some mammalian transformed and ter-minally differentiated cells, there is little immunocyto-chemical evidence for the existence of Notch proteinin the nucleus during development.

CBF1-DEPENDENT NOTCH SIGNALING

The transcription factor Suppressor of Hair-less (Su(H)) is well known as a mediator of Notch

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Fig. 3. Model of CBF-1 activation by Notch. CBF-1 acts as a transcription repressor as long as the repression complex (SMRT and HDAC-1)is bound to it. Upon Notch activation, NotchIC migrates to the nucleus and competes with SMART for contacts with SKIP and CBF-1.Release of the repression complex (SMRT and HDAC1) from CBF-1, results in transcription activation of the CBF1 bound genes.

signaling during Drosophila development (20). Themammalian and C. elegans homologues are known asCBF1 (or RBP-Jκ and Lag-1, respectively and collec-tively are known as the CSL family (CBF-1, Su (H),Lag-1) of transcription factors (23). As a consequenceof ligand dependent activation of mammalian Notch,the cleaved NotchIC is thought to interact with CBF1to initiate the cascade of the Notch signaling path-way. The RAM23 region is the main point of inter-action with CBF1 (Fig. 1) (21). Sequences within theAnkyrin repeats also interact with CBF1 with loweraffinity and positively enhance Notch signaling (33).

In mammals the repressor activity of CBF1is part of a large transcription repressing complexwhich also includes SMRT (silencing mediator ofretinoid and thyroid hormone receptors), HDAC-1(histone deacetylase) and SKIP (Ski-interacting pro-tein) (Fig. 3) (34,35). It has been hypothesized that“activated” NotchIC displaces the SMRT-HDAC-1complex from the CBF-1-SKIP complex. Thus, a newcomplex is formed in which the Ankyrin/CDC10 re-peats of NotchIC interact with SKIP, and the RAM23region with CBF-1 (35). This complex is thought topromote transcription of the target genes. In mam-mals the HES (Hairy Enhancer of Split) gene familyare known to be targets for up-regulation by activatedNotch (Fig. 2) (36,37). HES expression represses theexpression of MASH-1, the mammalian homologueof Drosophila Achaete-Scute Complex (AS-C) (38).Another target for up-regulation of transcription by“activated” Notch in mammals is the p100/NF-κB2

promoter (39). A common theme of the Notch-effector genes is that they encode transcription regulatorsthat affect the function of tissue-specific bHLH tran-scription factors (HES) or affect cell fate throughother molecular targets, for example NF-κB2 (2).

Genetic studies in Drosophila have shown thatthe Enhancer of Split complex (E(spl)-C), a ge-netic locus composed of eight genes is the target ofNotch Su (H) action (Fig. 2) [reviewed (20)] Sevenof these genes encode functionally redundant ba-sic helix-loop-helix (bHLH) proteins that are tran-scriptional repressors. The eighth gene (Groucho)encodes another nuclear protein that has WD-40-like repeats. Bailey and Posakony (40) have estab-lished that Drosophila Su (H) activates transcriptionof E (spl)-C in response to Notch activation and thatthree of these genes contain multiple specific bind-ing sites for Su (H). Up regulation of E (spl)-C sup-presses the expression of the AS-C which encodes tis-sue specific transcription factors involved in neuronaldifferentiation (Fig. 2). In addition, Heitzler et al. (41)have provided evidence for a regulatory loop betweenNotch and Dl that is under the transcriptional controlof E(spl)-C and AS-C such that within a cell or groupof cells activation of Notch leads to suppression of Dlexpression.

CBF1-INDEPENDENT PATHWAYS

Studies that were designed to dissect, at themolecular level, the function of different regions of

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the NotchIC have provided substantial evidence forCBF1-independent components of Notch signaling.For instance, Shawber et al. (42) have shown that ex-pression of the six Notch-1IC Ankyrin/CDC10 repeatsis sufficient to suppress myogenesis in murine C2C12myoblasts. This occurs in spite of the inability of thisportion of NotchIC to bind CBF-1 or the lack of up-regulation of HES-1. Similarly, Matsuno et al. (43)have shown in Drosophila that expression of the sixAnkyrin/CDC10 repeats directed by the cell specificSevenless (sev) promoter has a characteristic pheno-type in which the expression of the Mδ gene of the E(Sp)-C is not activated. These findings raise the ques-tion of what mediates the CSL-independent pathwayof Notch signaling.

Ordentlich et al. (44) provide evidence that, incertain situations, Deltex may mediate another Notchsignaling pathway (Fig. 1). Deltex is a cytoplasmic pro-tein that binds to the Ankyrin/CDC10 repeats of theNotchIC (45). It is evolutionarily well conserved andappears not to relocate to the nucleus upon bindingto NotchIC. Ordentlich et al. (44) showed that the ba-sic helix-loop-helix (bHLH) transcription factor E47,which mediates activation of mammalian B-cell spe-cific immunoglobin gene transcription, is inhibitedby activated Notch-1 and Notch-2. Notch inhibitionof E47 activity correlated with the binding of Del-tex to the Ankyrin/CDC10 repeats of the NotchIC.Based on this and other results, they hypothesize thatNotch/Deltex signaling represses E47 activity througha mechanism involving Ras and c-JunN-terminal ki-nase (JNK).

OTHER NOTCHIC BINDING PROTEINS

The regulatory network mediated by NF-κB isimportant in the immune response as well as prolifer-ation and differentiation of hemopoietic cells, apop-tosis, and embryogensis. Guan et al. (46) have shownthat human Notch-1IC binds to the p50 subunit of nu-clear factor (NF) κB/REL transcription factor. Thisinteraction specifically blocks p50NF-κB binding toDNA and blocks NF-κB transactivation of a reportergene in tissue culture cells. The authors further spec-ulate that in certain circumstances Notch-1 may acti-vate NF-κB dependent transcription by binding andthereby attenuating the inhibitory effect of p50NF-κBhomodimers.

Other examples of NotchIC binding proteins arethe orphan nuclear receptor Nur77 and Disabled.Nur77 is expressed in mouse T cell hybridomas. Acti-

vated Notch-1 represses Nur77 induced transcriptionand inhibits Nur77 dependent apoptosis (47). In ad-dition, to cell fate decisions, Notch also plays a role inaxon guidance in neurons (48). Disabled binds to theRAM23 region of the NotchIC in vitro and geneticallyinteracts with Abl to produce axon guidance defects(Fig. 1) (48). Drosophila Abl encodes a cytoplasmictyrosine kinase which is homologous to the vertebratec-Abl proto-oncogene.

ROLES OF NOTCH SIGNALING DURINGDEVELOPMENT

The role of Notch signaling during vertebrate de-velopment is being approached in tissue culture, exvivo systems and transgenic animals (reviewed (2–6)]. In these systems the increased genetic complex-ity of the components of Notch pathway, relative toDrosophila, as well as the partially overlapping tis-sue distribution of their expression and function, helpto explain the context dependence of Notch signal-ing effects. For instance, in some situations activatedNotch signaling blocks differentiation along a partic-ular pathway until the cell is able to respond to a sig-nal which will determine its fate. Carlesso et al. (49)have shown that activated Notch-1 affects cell cyclecontrol in HL-60 promyelocytic leukemia cells andCD34 bone marrow stem cells in such a way that pro-gression is accelerated through the G1 phase. Sincea G1 lag precedes commitment to terminal differen-tiation in many cell types, Notch induced cell prolif-eration may be linked to delayed differentiation. Inother situations, such as in mammalian models of thy-mocyte cell lineage decisions (50,51) and adipocytedifferentiation of mouse 3T3-L1 cells (52), expressionof Notch seems necessary for proper interpretationof differentiation stimuli. More recently it has beenshown that Notch signaling may also regulate cell sur-vival. Thus data from two groups suggests Notch sig-naling blocks apoptosis during thymocyte maturation(47,53).

In Drosophila, Notch signaling affects devel-opmental programs through a process termed “lat-eral inhibition or specification” between developmen-tally equivalent cells or “inductive signaling” betweennonequivalent cells or “cell-autonomous effects,” inwhich a cell determines its own cell fate through Notchsignaling. Each of these processes has been describedrecently in several excellent reviews (20,54). Over-all, the role of Notch signaling during developmentseems to be evolutionarily conserved and aimed at

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adjusting cellular responses to differentiation stimulirather than to specify a particular cell fate.

NOTCH MODIFYING FACTORS

In addition to the core components of the Notchpathway there is another group of factors which ei-ther positively or negatively influence Notch signalingor processing at particular stages of development oranatomic locations [reviewed (55)]. Secretory glyco-proteins encoded by Drosophila Fringe and the mam-malian Lunatic, Radical and Manic Fringe genes havebeen shown to modulate Notch-ligand interactions.In the Drosophila wing, Fringe inhibits the ability ofNotch to be activated by Ser and enhances the abil-ity of Notch to be activated by Dl (56,57). The bio-chemical mechanism by which this is accomplished issuggested by the weak sequence similarity betweenFringe and the Lex1 family of glycosyltransferases(58). Thus Fringe may enzymatically modify NotchEC.The location of the target region for Fringe activityseems to correspond to certain EGFL-repeats whichcontain gain-of-function missense mutations (Ax, seelater) that render Notch insensitive to Fringe (59).

A fundamental mechanism that contributes tothe generation of diverse cell types is asymmetriccell division and both intrinsic and extrinsic factorscan influence the cell fate decisions of the progeny(60). One example that is driven by an intrinsic factoroccurs during neurogenesis when the intrinsic factorNumb is distributed asymmetrically among daughtercells. Higher levels of Numb inhibit Notch activity inone daughter cell, biasing Notch mediated signaling(61,62). Numb encodes a cytoplasmic membrane as-sociated protein with a phosphotyrosine-binding do-main (63). It suppresses Notch signaling by bindingto the RAM23 and c-terminal regions of the NotchIC

(Fig. 1) (62).Some cell divisions in the mouse are symmetric,

giving rise to equivalent progeny in which Numb isequally distributed among daughter cells. Whetherthe cell division, and the distribution of Numb, isasymmetric or symmetric depends on whether theorientation of the mitotic spindle is horizontal orvertical (64). Similarly, Notch is also asymmetricallydistributed in horizontal cell divisions during mam-malian neurogenesis (65). Together these observa-tions suggest that asymmetric distribution of Numband/or Notch in stem cell progeny may result in dif-ferential Notch activity that establishes an initial sig-naling bias between otherwise equivalent progenitors.

In the development of the Drosophila peripheral ner-vous system (PNS) Notch and Numb are required andfunction antagonistically during three asymmetric celldivisions, however, Su (H) is required for only two ofthese divisions (66). This finding suggests that Numbcan negatively regulate both CSL-dependent and-independent Notch signaling pathways.

BIOLOGICAL CONSEQUENCES OF“ACTIVATED” NOTCH-4 ON MAMMARYGLAND DEVELOPMENT

Expression of each of the murine Notch genescan be detected in mammary glands of virgin, preg-nant and lactating mice (our unpublished data). Trans-genic mice in which expression of Notch-4IC is drivenby the MMTV LTR develop a profoundly alteredmammary gland and within 4 to 6 months 100% ofthe females have focal mammary tumors (67,68). Invirgin females the ductal epithelium minimally pene-trates the mammary fat pad (Fig. 4B). The hormonalstimulation of the first pregnancy overcomes the duc-tal growth deficiency (Fig. 4D). Thus the mammaryfat pad fills with ductal epithelium, but there is littlelobular-alveolar development and no expression ofmilk or milk proteins. Reciprocal transplantation oftransgenic MMTV LTR- Notch-4IC to the epithelium-divested mammary fat pads of normal FVB/N micedemonstrated that the transgenic mammary epithe-lium was unable to grow ducts except in hormonally-stimulated hosts or to produce functionally differ-entiated secretory lobules. However normal FVB/Nmammary epithelium grows and fully differentiates intransgenic MMTV LTR-activated Notch-4 transgenicfat pads. These results indicate that the dysfunctionof transgenic MMTV LTR-activated Notch-4 mam-mary glands is cell autonomous and due to Notch-4-expression within the epithelium. Furthermore, elec-tron microscopy reveals that MMTV LTR- Notch-4IC

mammary epithelial cells do not form intercellularjunctional complexes particularly in the terminal endbuds of the developing subadult mammary gland (68).This deficiency may affect the extension of the grow-ing ducts in the glands of young, nulliparous females.The tumors appear as focal outgrowths derived fromeither the termini of growing ducts or from intraductalhyperplasias, which are common within the mammaryglands of virgin and parous females.

To confirm and extend these findings, Notch-4IC

was expressed from the whey acidic protein (WAP)promoter whose activity, unlike the MMTV LTR,

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Fig. 4. Photomicrographs of mammary gland wholemounts from MMTV LTR-“activated” Notch-4 (B,D), WAP-“activated” Notch-4 (F)and FVB control (A,C,E) mice. Mammary glands of virgin MMTV LTR-“activated” Notch-4 mice (B) showed impaired ductal growth andelongation in comparison to mammary glands of virgin control FVB mouse (A). With pregnancy, the MMTV LTR-“activated” Notch-4transgenic mammary gland (D) showed ductal extension but failed to show lobular development compared to the mammary gland of thecontrol pregnant mice (C). Conversely, mammary glands from virgin WAP-Notch-4 transgenic mice were similar to the wild type, whereductal structures filled the mammary fat pad (data not shown). However, lactating mammary glands from WAP-Notch-4 showed impairedlobular development and growth (F) when compared to mammary glands of control lactating mice (E).

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is restricted primarily to late pregnant and lactat-ing mammary epithelial populations (69). In trans-genic mice carrying the WAP- Notch-4IC construct,mammary ductal growth was unaffected in virgin fe-males, but growth and differentiation of alveoli dur-ing gestation was profoundly inhibited (Fig. 4F). Co-incidental with the block in alveolar development,mammary dysplasia and tumorigenesis occurred in allbreeding females by 25 weeks of age. In nonbreed-ing WAP-Notch-4IC females mammary tumor inci-dence also reached 100% but only after 70 weeks. TheWAP- Notch-4IC mammary tumors were highly ma-lignant and most tumor-bearing females, irrespectiveof breeding history developed metastatic lung lesions.These results suggest that WAP-promoter targetedNotch-4IC function is associated with inhibition ofalveolar development and maintenance in this trans-genic model. Consistent with this conclusion, trans-plantation of WAP-Notch-4IC mammary gland intonontransgenic mammary fat pads produced completemammary ductal outgrowths in virgin FVB/N micebut failed to develop alveoli when the females wereimpregnated. Taken together the effects of deregu-lated expression of Notch-4IC in mammary epithelialcells, suggest that overexpression of Notch-4IC lim-its their capacity to perform the cell fate decisionsrequired for alveolar morphogenesis and functionaldifferentiation during mammary gland development.

To begin to understand the consequences of “ac-tivated” Notch-4 expression on gene expression dur-ing pregnancy and tumorigenesis at a molecular levelwe have used differential display and microarray(ClonTech, Inc.) analysis of RNA prepared from nor-mal pregnant, transgenic WAP-Notch-4IC pregnant,and transgenic malignant mammary tissues. Our pre-liminary results show WAP-Notch-4IC tumors couldbe divided into three groups based on their patternof milk protein expression: WAP only, β-casein only,or neither milk protein. Other apparently differen-tially expressed genes probably represent a “signa-ture expression pattern” of genes of Notch-4 express-ing mammary stem cells that have been malignantlytransformed and clonally expanded in the tumors(70).

NOTCH GERMLINE MUTATIONS

There is abundant genetic evidence that viablemutant alleles of Drosophila Notch bearing singleamino acid changes that cluster to particular EGFL-repeats, are associated with specific altered devel-

opmental phenotypes. Three classes of these Notchmutations (Split [Spl], Abruptex [Ax], and temper-ature sensitive [Notchts]) are potentially relevant tounderstanding the role of members of the mam-malian Notch gene family in development [reviewed(71)]. The Spl mutation is located in EGFL-repeat14 and is associated with an altered eye phenotypein Drosophila. By developmental and genetic criteriaAx mutations, depending on the particular mutation,can either suppress or enhance the nicked wing phe-notype produced by the Notch allele. These mutationsare located within EGFL-repeats 24 to 29. Furtherthe suppresser and enhancer Ax alleles are locatedin the proximal and distal halves of the Ax region,respectively. In addition, Ax mutations when geneti-cally located cis to the Spl mutation enhance the ex-pression of the Spl-phenotype. The conditional mu-tant, Notchts, is the consequence of a point mutationin EGFL-repeat 32, which causes lethality, and lossof Notch function at the nonpermissive temperature.It has been suggested that the lack of viable mutantsmapping to other EGFL repeats probably indicatesthat most missense mutations give rise to recessivelethal or dominant phenotypes.

In humans, there are two hereditary diseases,CADASIL (cerebral autosomal dominant arteriopa-thy with subcortical infarcts and leukoencephalopa-thy) and Alagille syndrome (AGS) that are associ-ated with point mutations in Notch-3 and Jagged-1,respectively [reviewed (72,73)]. The key features ofCADASIL are recurrent subcortical ischemic strokes,usually in the absence of any vascular risk factor, andprogressive dementia. Notch-3 is located on chromo-some 19. Analysis of Notch-3 from a large panel ofunrelated CADASIL patients showed that the major-ity had missense mutations affecting an EGFL repeatin the region of repeats 1 through 5. Many of these mu-tations resulted in the gain or loss of a cysteine residuewithin the repeat. These mutations could have multi-ple consequences on Notch-3 conformation, process-ing, and activity. AGS is an autosomal dominant con-dition associated with chronic childhood liver diseaseas well as developmental abnormalities of the skele-ton, kidney, eye, heart, and face. The AGS locus hasbeen assigned to chromosome 20p12. The majorityof AGS patients contain either frameshift, nonsenseor splice site mutations of the Notch ligand Jagged-1,that result in the expression of a truncated protein.Although the functional consequences of AGS muta-tions are uncertain, two recent studies in Drosophilamay provide a clue. Truncated forms of Delta andSerrate that are similar to those created by AGS

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mutations have a dominant-negative effect and thusact as antagonists of Notch signaling (74,75).

It may be significant that in Notch-4, 3 out ofthe 5 novel EGFL-repeats (repeats 14–15, 26–27,and 31–32) are the equivalent EGFL-repeats inwhich each class of these missense mutations oc-curs (9,11). This observation suggests that duringevolution the same selective constraints imposed onthe location of viable missense mutations have, atleast in part, defined the location and extent of thedeletions in Notch-4. In this scenario, the creationof novel EGFL-repeats 16–17 and 20–23 suggestsa more limited or specialized role for Notch-4 inmammalian development. A similar argument couldbe made for Notch-3 which has lost the equivalent ofEGFL-repeat 21 and contains a novel EGFL-repeatcomposed of portions of repeats 2 and 3 (76).

Gene targeting has been used to introduce muta-tions of endogenous Notch genes into the germline ofmice to investigate the requirements for Notch signal-ing during mammalian development. Mouse embryosthat contain homozygous null mutations for Notch-1, Notch-2, CBF-1, Dll-1, Jagged-1, or Jagged-2 dieduring embryonic development or shortly after birth(35,77–84). Each mouse strain has characteristic de-velopmental deficiencies that affect different aspectsof somite formation, anterior-posterior somite orien-tation, or vascularization. In contrast, homozygousNotch-4 null mice have no apparent phenotype. How-ever, Notch-1−/− Notch-4−/− double homozygousembryos displayed a more severe phenotype thanthe Notch-1 null embryos (85). Therefore Notch-4expression is not essential for embryonic develop-ment, but Notch-4 and Notch-1 have partially over-lapping roles during both embryogenesis and postna-tal development in mice.

TUMOR SPECIFIC EXPRESSION PATTERNSOF NOTCH

Several members of the Notch gene family areconstitutively activated in various types of malig-nancies. For instance, the chromosomal translocationt(7;9)(q34;q34.3) in human T-cell acute lymphoblasticleukemia leads to the expression of a truncated Notch-1/TAN-1 gene product that is missing most of the ex-tracellular domain (86,87). Notch-1 is also activatedby retroviral insertional mutagenesis in certain strainsof transgenic mice in a manner that is similar to thoseobserved at Notch-4 in MMTV infected feral mice.Thus, thymic lymphomas in murine leukemia virus

(MuLV) infected c-myc transgenic mice contain viralinduced rearrangements of Notch-1 (88). Expressionof genetically activated Notch-2 is also thought to con-tribute to the induction of thymic lymphomas in cats(89). In these cases the feline leukemia virus (FeLV)has been shown to transduce the region of Notch-2which is analogous to the portion of Notch-1 whoseexpression is activated by chromosomal translocationor viral induced rearrangement. In each case, whetherby chromosomal translocation or viral induced re-arrangement, expression of truncated Notch-1 andNotch-2 RNA appears to represent a gain-of-functionmutation. Further evidence for this conclusion is thedemonstration that expression of Notch-1 or Notch-2IC in E1A-immortalized baby rat kidney cells conferson them the ability to grow in soft agar and form tu-mors in nude mice (90).

In two studies the expression of Notch-1, Notch-2, Delta-1, Jagged-1 and Jagged-2 was analyzed inhuman cervical tissue specimens (26,91). Both Notchproteins and each of the ligands were detected in thestratum spinosum, which is composed of proliferatingcells committed to the fate of normal squamous cer-vical epithelium, but not in the undifferentiated basaland keratinized cells located above this stratum. Sim-ilarly, both Notch proteins, Delta-1 and Jagged-1 areexpressed in the “reserve” precursor cell but not inthe fully differentiated columnar epithelial cells of thecervix. Jagged-2 expression was not detected in any ofthese cells. It was concluded that Notch activity is asso-ciated with proliferative cell populations in the cervixthat are responsive to developmental signals. Thesecell populations are thought to be the targets for neo-plastic transformation. Notch-1, Notch-2 and each ofthe ligands were all up-regulated in in situ and in in-vasive cervical squamous cell carcinomas. Likewise,they were also up-regulated in in situ and in invasivecervical adenocarcinomas. Thus it seems likely thatin cervical cancer that the Notch signaling pathway isactivated through receptor-ligand interactions.

To evaluate gain-of-function Notch-4 activity inhuman cancers we have surveyed human breast, lung,and colon carcinoma tissue culture cell lines for ev-idence of increased Notch-4 RNA expression (92).High levels of a 1.8 kb Notch-4 mRNA species aredetected in normal human testis but not in other tis-sues where a 6.5 kb species is prevalent. Transformedhuman cancer cell lines express the 1.8 kb Notch-4mRNA species. We show that this RNA species en-codes a truncated form of the Notch-4IC. This novelNotch-4 protein is comprised of the CDC10/ANKRand the amino acid residues C-terminal to them, but

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is missing the CSL-binding region. A transgene thatexpresses the 1.8 kb Notch-4 RNA species in the“normal” human mammary epithelial cell line MCF-10A enables these cells to grow in soft agar (92). Wespeculate that this effect on MCF-10A growth prop-erties is a consequence of CSL-independent Notch-4signaling.

INTERACTION OF NOTCH WITH OTHERSIGNALING PATHWAYS

“Cross talk” between the Notch pathway andother signaling pathways indicates that extrinsic fac-tors can influence Notch signaling. An example is thenegative influence of the Wnt (Wingless) pathway onNotch signaling. Genetic analysis of the DrosophilaWingless (Wg) and Notch signaling pathways hasprovided evidence that they both function in manyof the same patterning events during developmentand that their expression antagonistically influencesthese events [reviewed (93)] Activation of DrosophilaNotch by its ligands or by the action of gain-of-function mutations leads through CSL-dependent sig-naling to the down-regulation of Ac-Sc transcriptionfactors (Fig. 2). In the presence of Wnt signaling,Notch signaling is repressed by the binding of the cyto-plasmic protein, Disheveled (Dsh), to the C-Terminusof NotchIC [reviewed (93,94)] (Fig. 1). Therefore itcould be expected that, in a situation where activatedNotch and Wg are co-expressed, the Wg phenotypewould be dominant.

Uyttendaele et al. (95) have addressed this is-sue in studies of the effect of “activated” Notch-4and Wnt1 expression on branching morphogenesisof the mouse mammary epithelial TAC-2 cell line.They have shown that, in this setting, Wnt1 expressioninduces elongation and branching of TAC-2 epithe-lial tubules whereas “activated” Notch4 expressioninhibits branching morphogenesis and disrupts theformation of organized, lumen containing structures(96). In addition, they have shown that these observa-tions are consequence of CSL-dependent Notch sig-naling. Thus, as predicted from the Drosophila stud-ies, TAC-2 cells co-expressing activated Notch-4 andWnt1 exhibited the Wnt1 branching ductal morpho-genesis.

Based on studies in Drosophila it seems likelythat the relationship between Notch and Wg (Wnt)signaling pathways is more complex. Wesley (97,98)has demonstrated that Wg binds to the NotchEC andthat EGFLR 19–36 are both necessary and sufficient

for binding. In fact, within this region there are twohighly conserved regions comprised of EGFLR 23–27 and EGFLR 31–34 whose functional significanceis suggested by the presence of at least one lethal mu-tation in each region. Drosophila S2 cells expressingeither full-length Notch or NotchδEGF1–18 (Notch lack-ing EGFLR 1–18) were compared for the effect of Wgsignaling on the expression of genes downstream ofNotch and Wg. In cells expressing full-length Notch,Wg up-regulates the expression of patched (Ptc, a neg-ative regulator of Wg expression) and shaggy (Sgg,a negative regulator of engrailed expression). In theabsence of Wg, cells expressing NotchδEGF1–18 are upregulated for Ptc, Sgg, hairy (h, a negative regula-tor of achaete expression) and Dfrizzled-2 (DFz1, awg receptor). In the presence of Wg each of thesegenes is repressed. The signaling pathway used byNotch to transduce signals in response to Wg is notknown. Thus in Drosophila novel signaling pathwaysseem to be used to transduce signals to the nucleus ofS2-Notch and S2-NotchδEGF1–18 cells in the presenceand absence of Wg.

At the present time it is not known whether mam-malian Wnt1 and other members of the Wnt fam-ily interact similarly with the extracellular domain ofmembers of the Notch family. It is interesting to note,however, that in the case of Notch-4, EGFLR 14–15, EGFLR 16–17, EGFLR 20–23, EGFLR 26–27,and EGFLR 31–32 have been fused to form novelEGFLRs (9,11). Since Drosophila Notch EGFLR23–27 and EGFLR 31–34 are required for bindingWg, it may be that Notch-4 has lost the capability tobind Wnt proteins or that it interacts with other novelligand(s).

CONCLUSIONS

It seems clear that Notch signaling during mam-malian development and the components that com-prise the signaling pathway are bound to be highlycomplex. For instance, since the four-mammalianNotch genes show an overlapping expression pat-tern, does this mean that, like the EGF receptorfamily, there is heterodimer formation between dif-ferent Notch proteins? Similarly, the extent of speci-ficity in ligand-receptor interactions is unknown noris it known whether there are additional, as yet, un-known ligands. What other signaling pathways inter-act with Notch during mammary gland development?It seems reasonable to expect that as the human andmouse genomic sequences become available, genes

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that are regulated by Notch signaling will becomeknown. With respect to the mammary gland, it willbe important to develop an atlas that depicts, as afunction of time and cellular location in the gland,the expression patterns of different components com-prising the Notch signaling pathway. It seems likely,therefore, that unraveling the complexity of Notchsignaling in the mammary gland is likely to representa fertile area for research in the near future.

ACKNOWLEDGMENTS

We thank Dr. Gilbert H. Smith and Dr. BarbaraVonderhaar for critical reading of the manuscript.

REFERENCES

1. O. L. Moohr (1919). Character changes caused by mutationof an entire region of a chromosome in Drosophila. Genetics4:275–282.

2. L. Miele and B. Osborne (1999). Arbiter of differentiation anddeath: Notch signaling meets apoptosis. J. Cell Physiol. 181:393–409.

3. B. Osborne and L. Miele (1999). Notch and the immune system.Immunity 11:653–663.

4. N. E. Baker (2000). Notch signaling in the nervous system.Pieces still missing from the puzzle. Bioessays 22:264–273.

5. S. Artavanis-Tsakonas, M. D. Rand, and R. J. Lake (1999).Notch signaling: Cell fate control and signal integration in de-velopment. Science 284:770–776.

6. L. A. Milner and A. Bigas (1999). Notch as a mediator of cellfate determination in hematopoiesis: Evidence and specula-tion. Blood 93:2431–2448.

7. M. Lardelli, R. Williams, and U. Lendahl (1995). Notch-relatedgenes in animal development. Int. J. Dev. Biol. 39:769–780.

8. E. M. Maine, J. L. Lissemore, and W. T. Starmer (1995). A phy-logenetic analysis of vertebrate and invertebrate Notch-relatedgenes. Mol. Phylogenet. Evol. 4:139–149.

9. D. Gallahan and R. Callahan (1997). The mouse mammary tu-mor associated gene INT-3 is a unique member of the NOTCHgene family (NOTCH4). Oncogene 14:1883–1890.

10. J. Robbins, B. J. Blondel, D. Gallahan, and R. Callahan (1992).Mouse mammary tumor gene Int-3: A member of the Notchgene family transforms mammary epithelial cells. J. Virol.66:2594–2599.

11. H. Uyttendaele, G. Marazzi, G. Wu, Q. Yan, D. Sassoon, andJ. Kitajewski (1996). Notch4/int-3, a mammary proto-oncogene,is an endothelial cell-specific mammalian Notch gene. Devel-opment 122:2251–2259.

12. D. Gallahan, C. Kozak, and R. Callahan (1987). A new commonintegration region (int-3) for mouse mammary tumor virus onmouse chromosome 17. J. Virol. 61:218–220.

13. L. D. Siracusa, M. H. Rosner, M. A. Vigano, D. J. Gilbert, L. M.Staudt, N. G. Copeland, and N. A. Jenkins. (1991). Chromoso-mal location of the octamer transcription factors, Otf-1, Otf-2,and Otf-3, defines multiple Otf-3-related sequences dispersedin the mouse genome. Genomics 10:313–326.

14. I. Rebay, R. G. Fehon, and S. Artavanis-Tsakonas (1993). Spe-cific truncations of Notch define dominant activated and dom-inant negative forms of the receptor. Cell 74:319–329.

15. G. Struhl, K. Fitzgerald, and I. Greenwald (1993). Intrinsic ac-tivity of the lin-12 and Notch intracellular domains in vivo. Cell74:331–345.

16. D. Gallahan and R. Callahan (1987). Mammary tumorigenesisin feral mice: Identification of a new int locus in mouse mam-mary tumor virus(CzechII)-induced mammary tumors. J. Virol.61:66–74.

17. N. H. Sarkar, S. Haga, A. F. Lehner, W. Zhao, S. Imai, andK. Moriwaki (1994). Insertional mutation of int protoonco-genes in the mammary tumors of a new strain of mice derivedfrom the wild in China: Normal- and tumor- tissue-specific ex-pression of int-3 transcripts. Virology 203:52–62.

18. G. Peters (1990). Oncogenes at viral integration sites. CellGrowth Differ. 1:503–510.

19. A. Dievart, N. Beaulieu, and P. Jolicoeur (1999). Involvement ofNotch1 in the development of mouse mammary tumors. Onco-gene 18:5973–5981.

20. S. Artavanis-Tsakonas, K. Matsuno, and M. E. Fortini (1995).Notch signaling. Science 268:225–232.

21. K. Tamura, Y. Taniguchi, S. Minoguchi, Y. Sakai, T. Tun, T.Furukawa, and T. Honjo (1995). Physical interaction betweena novel domain of the receptor Notch and the transcriptionfactor RBP-Jκ/Su(H). Curr. Biol. 5:1416–1423.

22. F. Logeat, C. Bessia, C. Brou, O. LeBail, S. Jarriault, N. G.Seidah, and A. Israel (1998). The Notch1 receptor is cleavedconstitutively by a furin-like convertase. Proc. Natl. Acad. Sci.U.S.A. 95:8108–8112.

23. G. Weinmaster (1997). The ins and outs of notch signaling. Mol.Cell Neurosci. 9:91–102.

24. S. L. Dunwoodie, D. Henrique, S. M. Harrison, and R. S.Beddington (1997). Mouse Dll3: A novel divergent Delta genewhich may complement the function of other Delta homo-logues during early pattern formation in the mouse embryo.Development 124:3065–3076.

25. B. Bettenhausen, M. Hrabe de Angelis, D. Simon, J. L. Guenet,and A. Gossler (1995). Transient and restricted expression dur-ing mouse embryogenesis of Dll1, a murine gene closely relatedto Drosophila Delta. Development 121:2407–2418.

26. G. E. Gray, R. S. Mann, E. Mitsiadis, D. Henrique, M. L.Carcangiu, A. Banks, J. Leiman, D. Ward, D. Ish-Horowitz, andS. Artavanis-Tsakonas (1999). Human ligands of the Notch re-ceptor. Am. J. Pathol. 154:785–794.

27. L. Li, L. A. Milner, Y. Deng, M. Iwata, A. Banta, L. Graf,S. Marcovina, C. Friedman, B. J. Trask, L. Hood, and B. Torok-Storb (1998). The human homolog of rat Jagged1 expressedby marrow stroma inhibits differentiation of 32D cells throughinteraction with Notch1. Immunity 8:43–55.

28. K. M. Klueg, T. R. Parody, and M. A. Muskavitch (1998). Com-plex proteolytic processing acts on Delta, a transmembraneligand for Notch, during Drosophila development. Mol. Biol.Cell. 9:1709–1723.

29. H. Qi, M. D. Rand, X. Wu, N. Sestan, W. Wang, P. Rakic, T. Xu,and S. Artavanis-Tsakonas (1999). Processing of the notch lig-and delta by the metalloprotease Kuzbanian. Science 283:91–94.

30. I. Rebay, R. J. Fleming, R. G. Fehon, L. Cherbas andS. Artavanis-Tsakonas (1991). Specific EGF repeats of Notchmediate interactions with Delta and Serrate: Implications forNotch as a multifunctional receptor. Cell 67:687–699.

P1: VENDOR/GCR



31. C. Brou, F. Logeat, N. Gupta, C. Bessia, O. LeBail, J. R.Doedens, A. Cumano, P. Roux, R. A. Black, and A. Israel(2000). A novel proteolytic cleavage involved in Notch signal-ing: The role of the disintegrin-metalloprotease TACE. Mol.Cell. 5:207–216.

32. J. S. Mumm, E. H. Schroeter, M. T. Saxena, A. Griesemer,X. Tian, D. J. Pan, W. J. Ray and R. Kopan (2000). A ligand-induced extracellular cleavage regulates gamma-secretase-likeproteolytic activation of Notch1. Mol. Cell. 5:197–206.

33. J. C. Aster, E. S. Robertson, R. P. Hasserjian, J. R. Turner,E. Kief, and J. Sklar (1997). Oncogenic forms of NOTCH1 lack-ing either the primary binding site for RBP-Jkappa or nuclearlocalization sequences retain the ability to associate with RBP-Jkappa and activate transcription. J. Biol. Chem. 272:11336–11343.

34. H. Y. Kao, P. Ordentlich, N. Koyano-Nakagawa, Z. Tang,M. Downes, C. R. Kintner, R. M. Evans, and T. Kadesch (1998).A histone deacetylase corepressor complex regulates the Notchsignal transduction pathway. Genes Dev. 12:2269–2277.

35. S. Zhou, M. Fujimuro, J. J. Hsieh, L. Chen, A. Miyamoto,G. Weinmaster, and S. D. Hayward (2000). SKIP, a CBF1-associated protein, interacts with the ankyrin repeat domainof NotchIC To facilitate NotchIC function. Mol. Cell Biol.20:2400–2410.

36. S. Stifani, C. M. Blaumueller, N. J. Redhead, R. E. Hill,and S. Artavanis-Tsakonas (1992). Human homologs of aDrosophila Enhancer of split gene product define a novelfamily of nuclear proteins [published erratum appears in Nat.Genet. 2(4):343]. Nat. Genet. 2:119–127.

37. S. Jarriault, C. Brou, F. Logeat, E. H. Schroeter, R. Kopan,and A. Israel (1995). Signaling downstream of activated mam-malian NOTCH. Nature 377:355–358.

38. S. Goodbourn (1995). Signal transduction. Notch takes a shortcut [news; comment]. Nature 377:288–289.

39. F. Oswald, S. Liptay, G. Adler, and R. M. Schmid (1998).NF-κB2 is a putative target gene of activated Notch-1 via RBP-Jκ . Mol. Cell Biol. 18:2077–2088.

40. A. M. Bailey and J. W. Posakony, (1995). Suppressor of Hair-less directly activates transcription of enhancer of slit complexgenes in response to NOTCH receptor activity. Genes Dev.9:2609–2622.

41. P. Heitzler, M. Bourouis, L. Ruel, C. Carteret, and P. Simpson(1996). Genes of the Enhancer of split and achaete-scutecomplexes are required for a regulatory loop between Notchand Delta during lateral signaling in Drosophila. Development122:161–171.

42. C. Shawber, D. Nofziger, J. J. Hsieh, C. Lindsell, O. Bogler,D. Hayward, and G. Weinmaster (1996). Notch signaling in-hibits muscle cell differentiation through a CBF1-independentpathway. Development 122:3765–3773.

43. K. Matsuno, M. J. Go, X. Sun, D. S. Eastman, and S. Artavanis-Tsakonas (1997). Suppressor of hairless-independent events inNotch signaling imply novel pathway elements. Development124:4265–4273.

44. P. Ordentlich, A. Lin, C. P. Shen, C. Blaumueller, K. Matsuno,S. Artavanis-Tsakonas, and T. Kadesch (1998). Notch inhibitionof E47 supports the existence of a novel signaling pathway. Mol.Cell Biol. 18:2230–2239.

45. K. Matsuno, D. Eastman, T. Mitsiades, A. M. Quinn, M. L.Carcanciu, P. Ordentlich, T. Kadesch, and S. Artavanis-Tsakonas (1998). Human deltex is a conserved regulator ofNotch signaling. Nat. Genet. 19:74–78.

46. E. Guan, J. Wang, J. Laborda, M. Norcross, P. A. Baeuerle,and T. Hoffman (1996). T cell leukemia-associated humanNotch/translocation-associated Notch homologue has IκB-likeactivity and physically interacts with nuclear factor-κB proteinsin T cells. J. Exp. Med. 183:2025–2032.

47. B. M. Jehn, W. Bielke, W. S. Pear, and B. A. Osborne (1999).Cutting edge: Protective effects of notch-1 on TCR-inducedapoptosis. J. Immunol. 162:635–638.

48. E. Giniger (1998). A role for Abl in Notch signaling. Neuron.20:667–681.

49. N. Carlesso, J. C. Aster, J. Sklar and D. T. Scadden (1999). Notch-1-induced delay of human hematopoietic progenitor cell dif-ferentiation is associated with altered cell cycle kinetics. Blood93:838–848.

50. T. Washburn, E. Schweighoffer, T. Gridley, D. Chang, B. J.Fowlkes, D. Cado and E. Robey (1997). Notch activity influ-ences the αβ versus γ δ T cell lineage decision. Cell 88:833–843.

51. E. Robey, D. Chang, A. Itano, D. Cado, H. Alexander, D. Lans,G. Weinmaster, and P. Salmon (1996). An activated form ofNotch influences the choice between CD4 and CD8 T cell lin-eages. Cell 87:483–492.

52. C. Garces, M. J. Ruiz-Hidalgo, J. F. de Mora, C. Park, L. Miele,J. Goldstein, E. Bonvini, A. Porras, and J. Laborda (1997).Notch-1 controls the expression of fatty acid-activated tran-scription factors and is required for adipogenesis. J. Biol. Chem.272:29729–29734.

53. M. L. Deftos, Y. W. He, E. W. Ojala, and M. J. Bevan (1998).Correlating notch signaling with thymocyte maturation. Immu-nity 9:777–786.

54. P. Simpson (1997). Notch signaling in development. PerspectDev. Neurobiol. 4:297–304.

55. V. M. Panin and K. D. Irvine (1998). Modulators of Notch sig-naling. Semin. Cell Dev. Biol. 9:609–617.

56. V. M. Panin, V. Papayannopoulos, R. Wilson and K. D. Irvine(1997). Fringe modulates Notch-ligand interactions. Nature387:908–912.

57. R. J. Fleming, Y. Gu, and N. A. Hukriede (1997). Serrate-mediated activation of Notch is specifically blocked by theproduct of the gene fringe in the dorsal compartment of theDrosophila wing imaginal disc. Development 124:2973–2981.

58. Y. P. Yuan, J. Schultz, M. Mlodzik, and P. Bork (1997). Secretedfringe-like signaling molecules may be glycosyltransferases[letter]. Cell 88:9–11.

59. J. F. de Celis and S. J. Bray (2000). The Abruptex domain ofNotch regulates negative interactions between Notch, its lig-ands and Fringe. Development 127:1291–1302.

60. B. Lu, L. Y. Jan, and Y. N. Jan (1998). Asymmetric cell division:Lessons from flies and worms. Curr. Opin. Genet. Dev. 8:392–399.

61. E. P. Spana and C. Q. Doe (1996). Numb antagonizes Notchsignaling to specify sibling neuron cell fates. Neuron. 17:21–26.

62. M. Guo, L. Y. Jan, and Y. N. Jan (1996). Control of daughtercell fates during asymmetric division: Interaction of Numb andNotch. Neuron. 17:27–41.

63. P. Bork and B. Margolis (1995). A phosphotyrosine interactiondomain [letter]. Cell 80:693–694.

64. W. Zhong, J. N. Feder, M-M. Jiang, L. Y. Jan, and Y. N. Jan(1996). Asymmetric localization of a mammalian numb ho-molog during mouse cortical neurogenesis. Neuron. 17:43–53.

65. A. Chenn and S. K. McConnell (1995). Cleavage orientationand the asymmetric inheritance of Notch-1 immunoreactivityin mammalian neurogenesis. Cell 82:631–641.

P1: VENDOR/GCR



66. S. Wang, S. Younger-Shepherd, L. Y. Jan, and Y. N. Jan (1997).Only a subset of the binary cell fate decisions mediated byNumb/Notch signaling in Drosophila sensory organ lineage re-quires Suppressor of Hairless. Development 124:4435–4446.

67. C. Jhappan, D. Gallahan, C. Stahle, E. Chu, G. H. Smith,G. Merlino, and R. Callahan (1992). Expression of an activatedNotch-related int-3 transgene interferes with cell differentia-tion and induces neoplastic transformation in mammary andsalivary glands. Genes Dev. 6:345–355.

68. G. H. Smith, D. Gallahan, F. Diella, C. Jhappan, G. Merlino,and R. Callahan (1995). Constitutive expression of a truncatedINT3 gene in mouse mammary epithelium impairs differenti-ation and functional development. Cell Growth Differ . 6:563–577.

69. D. Gallahan, C. Jhappan, G. Robinson, L. Hennighausen,R. Sharp, E. Kordon, R. Callahan, G. Merlino, and G. H. Smith(1996). Expression of a truncated Int3 gene in developing se-cretory mammary epithelium specifically retards lobular differ-entiation resulting in tumorigenesis. Cancer Res. 56:1775–1785.

70. G. Chepko and G. H. Smith (1997). Three division-competent,structurally-distinct cell populations contribute to murinemammary epithelial renewal. Tissue Cell 29:239–253.

71. P. Simpson (1994). The Notch Receptors. R. G. Landes Com-pany, Austin, Texas.

72. S. Artavanis-Tsakonas (1997). Alagille syndrome—a notch upfor the Notch receptor [news; comment]. Nat Genet. 16:212–213.

73. A. Joutel and E. Tournier-Lasserve (1998). Notch signallingpathway and human diseases. Semin. Cell Dev. Biol. 9:619–625.

74. N. A. Hukriede, Y. Gu and R. J. Fleming (1997). A dominant-negative form of Serrate acts as a general antagonist of Notchactivation. Development 124:3427–3437.

75. X. Sun and S. Artavanis-Tsakonas (1997). Secreted forms ofDELTA and SERRATE define antagonists of Notch signalingin Drosophila. Development 124:3439–3448.

76. M. Lardelli, J. Dahlstrand, and U. Lendahl (1994). The novelNotch homologue mouse Notch 3 lacks specific epidermalgrowth factor-repeats and is expressed in proliferating neuroep-ithelium. Mech. Dev. 46:123–136.

77. P. J. Swiatek, C. E. Lindsell, F. F. del Amo, G. Weinmaster,and T. Gridley (1994). Notch1 is essential for postimplantationdevelopment in mice. Genes Dev. 8:707–719.

78. Y. Xue, X. Gao, C. E. Lindsell, C. R. Norton, B. Chang,C. Hicks, M. Gendron-Maguire, E. B. Rand, G. Weinmaster,and T. Gridley (1999). Embryonic lethality and vascular de-fects in mice lacking the Notch ligand Jagged1. Human Mol.Genet. 8:723–730.

79. C. Oka, T. Nakano, A. Wakeham, J. L. de la Pompa, C. Mori,T. Sakai, S. Okazaki, M. Kawaichi, K. Shiota, T. W. Mak, andT. Honjo (1995). Disruption of the mouse RBP-J kappa generesults in early embryonic death. Development 121:3291–3301.

80. R. Jiang, Y. Lan, H. D. Chapman, C. Shawber, C. R. Norton,D. V. Serreze, G. Weinmaster, and T. Gridley (1998). Defects inlimb, craniofacial, and thymic development in Jagged2 mutantmice. Genes Dev. 12:1046–1057.

81. M. Hrabe de Angelis, J. McIntyre, 2nd, and A. Gossler (1997).Maintenance of somite borders in mice requires the Delta ho-mologue DII1. Nature 386:717–721.

82. Y. Hamada, Y. Kadokawa, M. Okabe, M. Ikawa, J. R. Coleman,and Y. Tsujimoto (1999). Mutation in ankyrin repeats of themouse Notch2 gene induces early embryonic lethality. Devel-opment 126:3415–3424.

83. J. L. de la Pompa, A. Wakeham, K. M. Correia, E. Samper,S. Brown, R. J. Aguilera, T. Nakano, T. Honjo, T. W. Mak,J. Rossant, and R. A. Conlon (1997). Conservation of the Notchsignaling pathway in mammalian neurogenesis. Development124:1139–1148.

84. R. A. Conlon, A. G. Reaume, and J. Rossant (1995). Notch1 isrequired for the coordinate segmentation of somites. Develop-ment 121:1533–1545.

85. L. T. Krebs, Y. Xue, C. R. Norton, J. R. Shutter, M. Maguire, J. P.Sundberg, D. Gallahan, V. Closson, J. Kitajewski, R. Callahan,G. H. Smith, K. L. Stark, and T. Gridley (2000). Notch signal-ing is essential for vascular morphogenesis in mice. [in processcitation]. Genes Dev. 14:1343–1352.

86. J. Aster, W. Pear, R. Hasserjian, H. Erba, F. Davi, B. Luo,M. Scott, D. Baltimore, and J. Sklar (1994). Functional analysisof the TAN-1 gene, a human homolog of Drosophila Notch,Cold Spring Harbor Symp. Quant. Biol. 59:125–136.

87. L. W. Ellison, J. Bird, D. C. West, A. L. Soreng, T. C. Reynolds,S. D. Smith, and J. Sklar (1991). TAN-1, the human homolog ofthe Drosophila Notch gene, is broken by chromosomal translo-cations in T Lymphoblastic neoplasms. Cell 66:649–661.

88. L. Girard, Z. Hanna, N. Beaulieu, C. D. Hoemann, C. Simard,C. A. Kozak, and P. Jolicoeur (1996). Frequent provirus inser-tional mutagenesis of Notch1 in thymomas of MMTVD/myctransgenic mice suggests a collaboration of c-myc and Notch1for oncogenesis. Genes Dev. 10:1930–1944.

89. J. L. Rohn, A. S. Lauring, M. L. Linenberger, and J. Overbaugh(1996). Transduction of Notch2 in feline leukemia virus-induced thymic lymphoma. J. Virol. 70:8071–8080.

90. A. J. Capobianco, P. Zagouras, C. M. Blaumueller, S. Artavanis-Tsakonas, and J. M. Bishop (1997). Neoplastic transformationby truncated alleles of human NOTCH1/TAN1 and NOTCH2.Mol. Cell Biol. 17:6265–6273.

91. P. Zagouras, S. Stifani, C. M. Blaumueller, M. L. Carcangiu, andS. Artavanis-Tsakonas (1995). Alterations in Notch signaling inneoplastic lesions of the human cervix. Proc. Natl. Acad. Sci.U.S.A. 92:6414–6418.

92. A. Imatani and R. Callahan (2000). Identification of a novelNOTCH-4/INT-3 RNA species encoding an activated geneproduct in certain human tumor cell lines. Oncogene 19:223–231.

93. J. D. Axelrod, K. Matsuno, S. Artavanis-Tsakonas, andN. Perrimon (1996). Interaction between wingless and Notchsignaling pathways mediated by dishevelled. Science 271:1826–1832.

94. H. Dierick and A. Bejsovec (1999). Cellular mechanisms ofwingless/Wnt signal transduction. Curr. Top. Dev. Biol. 43:153–190.

95. H. Uyttendaele, J. V. Soriano, R. Montesano, and J. Kitajewski(1998). Notch4 and Wnt-1 proteins function to regulate branch-ing morphogenesis of mammary epithelial cells in an opposingfashion. Dev. Biol. 196:204–217.

96. J. V. Soriano, H. Uyttendaele, J. Kitajewski, and R. Monte-sano (2000). Expression of an activated Notch4(int-3) onco-protein disrupts morphogenesis and induces an invasive pheno-type in mammary epithelial cells in vitro. Int. J. Cancer. 86:652–659.

97. C. S. Wesley (1999). Notch and wingless regulate expression ofcuticle patterning genes. Mol. Cell Biol. 19:5743–5758.

98. C. S. Wesley and L. Saez (2000). Notch responds differently toDelta and wingless in cultured Drosophila cells. J. Biol. Chem.275:9099–9101.

notch signaling in mammary gland tumorigenesis

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