The Notch family in mammary gland development and tumorigenesis

Note: transgenic mice in which INT3 is expressed under control of the WAP gene promoter (WAP-INT3 mice) or MMTV-LTR (MMTV-INT3 mice) have been established and analyzed.

Background

The Drosophila N gene, its vertebrate homologs, and the related Lin12 and glp-1 genes of C. elegans encode a transmembrane receptor protein that is part of a signaling pathway which, through local cell interaction, plays a pivotal role in the determination of cell fates during development (reviewed in [1]). N is widely expressed in the Drosophila embryo and continues to be expressed in uncommitted and proliferating cell populations at later stages of development. Studies of Drosophila N mutants have shown that its function is required for normal development and formation of the nervous system, mesoderm, germ line, ovarian follicle cells, adult sensory bristles, and eye structure. It is thought that N affects these developmental programs in complex tissues through either a process termed "lateral inhibition or specification" between developmentally equivalent cells or "inductive signaling" between nonequivalent cells [1, 2]. Lateral specification occurs as a consequence of stochastic fluctuations in components of a signaling pathway within a cell or group of cells. Cell-autonomous amplification of these signals causes differences to arise between adjacent cells which are enhanced by local inhibitory signals and result in a spaced pattern of two different cell types. The pattern of cell fate determination by inductive signaling between nonequivalent cells is dependent on timing, spatial arrangement, and specificity of the signals between the two cell types.

The N protein is composed of an extracellular domain containing 36 tandem epidermal growth factor (EGF)-like repeats and 3 cysteine rich LIN12 repeats, a transmembrane domain, and a intracellular domain containing 6 tandem CDC10/Ankyrin repeats, an Opa sequence and a PEST sequence. Current molecular models of the N signaling pathway suggests that it is unlike the classical signaling pathways involving cascades of protein kinases [1, 3-5]. In the absence of a ligand-receptor interaction the N transmembrane receptor exists as monomer on the cell surface and the Suppressor of Hairless (Su(H)) protein is bound to the CDC10/Ankyrin repeats of the N intracellular domain. The Su(H) protein is closely related to the KBF-2/RBP-Jk family of mammalian DNA binding transcription factors [6, 7]. Activation of N function is initiated with the physical interaction of its extracellular domain with either of its two known ligands, DELTA (Dl)and SERRATE (Ser). In Drosophila these ligands play equivalent but complementary roles in compartment specific signaling for wing margin development [8]. The presence of EGF repeats 10 and 11 in the N extracellular domain are both necessary and sufficient for ligand binding to occur. It is thought that this interaction results in receptor multimerization, a process which is necessary for N activation and the translocation of Su(H) protein to the nucleus. Based on studies of the role murine N plays in myogenesis. Jarriault et al. [4] and Kopan et al. [3] postulate that for nuclear translocation to occur the activated N intracellular domain/ Su(H) complex must be proteolytically processed to release a protein complex capable of nuclear localization. Genetic studies in Drosophila have showed that the Enhancer of Split complex [E(spl)-C], a genetic locus composed of eight genes is the target of N/Su(H) action. Bailey and Posakony [5] have established that Drosophila Su(H) activates transcription of genes within E(spl)-C. in response to N activation and that three of these genes contain multiple specific binding sites for Su(H). E(spl)-C encodes seven functionally redundant basic helix-loop-helix (bHLH) proteins that are transcriptional repressors and another nuclear protein having WD-40 repeats. Up regulation of E(spl)-C suppresses the expression of achaete-scute gene complex (AS-C) which encodes tissue specific transcription factors involved neuroblastic differentiation. In addition, Heitzler et al. [9] have provided evidence for a regulatory loop between N and Dl that is under the transcriptional control of E(spl)-C and AS-C. such that within a cell or group of cells activation of N leads to suppression of Dl expression.

Activation of members of the N gene family in MMTV induced mouse mammary tumors

The first indication that members of the N gene family may play an important role in mammary gland development and tumorigenesis was the determination that a frequent common insertion site for MMTV in feral mouse mammary tumors, designate INT3 (Gallahan and Callahan, 1987, [10, 11]) is a member of the N gene family [12]. The transcriptional orientation of the integrated viral genome and the target gene are the same. MMTV integration into INT3 occurs within a 200 bp region of the genome and activates the expression of , a 2.4 kb RNA species corresponding to sequences 3' of the viral integration site, from within the 3' MMTV long terminal repeat sequence (LTR). Nucleotide sequence analysis demonstrated that the activated INT3 RNA species is 60% similar to the region of N encoding the intracellular domain of the gene product. INT3 is located on chromosome 17 [13] in the murine genome and on chromosome 6p21.3 at the junction of class II and classIII gene of the MHC locus in the human genome [14]. Recently, we (Gallahan and Callahan, manuscript in prep; and [15]) have completed the nucleotide sequence of the normal 6.5 kb INT3 RNA transcript. Since the overall sequence is 57-59% similar to that of murine or rat NOTCH1, 2, and 3; we have renamed INT3, NOTCH4/INT3. The Notch4/INT3 gene spans 24 kb of genomic DNA and is composed of 27 exons/introns (Gallahan and Callahan, man. in prep). The Notch4/INT3 protein, although similar to the other NOTCH proteins, has several unique characteristic. The extracellular domain of the Notch4/INT3 protein contains 29 instead of 36 EGF-like repeats. Four novel EGF-like repeats have been created as a consequence of small deletions which have occurred within the gene during evolution. Thus, the N-terminal end of EGF repeat 14 is fused with a portion of the C-terminus of repeat 15. Similar fusions have occurred between repeats 16 and 17, repeats 20 and 23, and repeats 31 and 32. Since N EGF repeats are known to be important for ligand binding [16, 17], it may be that Notch4/INT3 has one or more unique ligands that do not interact with other NOTCH receptors.

Members of the murine NOTCH gene family are all expressed at varying levels in adult tissue that have been tested [15, 18, 19]. However, within different tissues all possible combinations of NOTCH homologue expression has been observed in different cell types. It has been suggested that this may represent a combinatorial code for vertebrate NOTCH function [18]. In the normal mammary gland, RNA transcripts corresponding to NOTCH 1, 2 and 4/INT3 have been detected by RT-PCR in mammary tissue from virgin, pregnant, and lactating females (Callahan, Unpublished data). The distribution of NOTCH expression within the mammary gland is relatively uncharacterized. We have begun to address this issue by developing antibodies specific for the intracellullar domain of NOTCH4/INT3. Immunohistochemical analysis of mammary tissue from different stages of mammary gland development indicate that NOTCH4/INT3 expression is strongly associated with the cap cells in the advancing end buds of the developing ductal system and later during early pregnancy, strongly in the epithelium of the proliferating alveolar buds [20].

Deletion of the portion of Drosophila N which encodes the extracellular domain corresponds to a gain of function mutation [17]. Thus the intracellular domain functions as though the extracellular domain was interacting with its ligand. In human T lymphoblastic leukemia (T-ALL) the break points in t(7;9)(q34;q34.3) translocations occur in the human homolog of NOTCH1 (TAN-1) resulting in the expression of a truncated RNA species encoding the intracellular domain of the protein [21]. Expression of this truncated NOTCH1 protein is thought to play a role in in the pathogenesis of some T cell neoplasms. Nucleotide sequence analysis of host-viral junction fragments from 9 independent MMTV induced mouse mammary tumors, demonstrated that all of the viral integration events in NOTCH4/INT3 have occurred within a 174 bp region 3' of the Lin12 repeat encoding sequences and 5' of the transmembrane domain encoding sequences. In preliminary studies of mammary tumors in MMTV infected erB-2/neu transgenic mice, NOTCH1 was rearranged by MMTV integration in two tumors resulting in the expression of a truncated NOTCH1 RNA species (Paul Jolicoeur, Clinical Research Institute of Montreal, Montreal, Quebec, Canada, personal communication). In each case viral integration had occurred in the region of the gene encoding the transmembrane domain. These strongly suggests that loss of Lin12 repeats sequences are required for viral induced activation of NOTCH4/INT3 and NOTCH1. Data consistent with this conclusion have been reported by Kopan et al. [3]. They have shown that expression vectors containing mouse NOTCH1 expressing truncated forms of the receptor in which the EGF-like repeats are missing, but the Lin12 repeats are present as a contiguous part of the remainder of the protein, exhibit no gain of function. The presence of the Lin12 repeats appears to negatively regulate multimerization and proteolytic cleavage of the intracellular domain thereby providing a strong selection for viral integration events to occur 3' of the sequence encoding them to circumvent this level of control of NOTCH function.

In Vivo consequences of activated NOTCH4/INT3 on mammary gland development and tumorigenesis

To gain an understanding of the role of NOTCH4/INT3 in mammary gland development and tumorigenesis, we have developed two transgenic mouse strains in which the portion of NOTCH4/INT3 encoding the intracellular domain of the protein is under the transcriptional control of either the MMTV LTR or the whey acidic protein (WAP) promoter [22, 23]. Characteristics of these mouse strains are given in the transgenic model systems section. In the MMTV LTR NOTCH4/INT3 mice normal ductal branching morphogenesis is blocked in postpubertal females. After the first pregnancy ducts fill the mammary fat pad, but alveolar development is arrested. WAP NOTCH4/INT3 virgin females contain a full and complete ductal system in their mammary fat pads. However, like the MMTV LTR NOTCH4/INT3 mice, secretory lobular development is severely depressed during pregnancy, resulting in a nonlactational phenotype. In both mouse strains undifferentiated mammary carcinomas develop in 100% of the females, as early as 1.5 months of age. These tumors appear to arise from within focal displastic areas of the mammary gland. There is evidence that in the case of the MMTV LTR NOTCH4/INT3 mice that the tumors arise from the luminal epithelial cells or aberrantly differentiated cap cells, whereas in the WAP NOTCH4/INT3 mice the cells giving rise to the mammary tumors originate from the secretory epithelial cell progenitors. Our current model is that NOTCH4/INT3 normally functions in the presence of its ligand to maintain mammary epithelial cells in a proliferatively responsive state by limiting their response to differentiative environmental signals.

Future Directions

The potential complexity of the role the NOTCH family, its ligands and intracellular effectors play in mammary gland development is difficult to evaluate. For instance, the timing and location of expression of the NOTCH family within the mammary gland is mostly uncharacterized. Other questions include: If within a given mammary cell more than one NOTCH gene is expressed, does heterodimerization play a role in its function? Are the putative ligands and intracellular effectors for the NOTCH family promiscuous in their interactions with the different family members? Is this signaling pathway a target for mutation in human breast cancer?

Acknowledgements

I thank Dr. Gilbert H. Smith for reading the manuscript before submission and Dr. Paul Jolicoeur for communicating his unpublished data.

References

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23. Gallahan, D., et al., Expression of a truncated Int3 gene in developing secretory mammary epithelium specifically retards lobular differentiation resulting in tumorigenesis. Cancer Res., 1996. 56: p. 1775-1785.

Contributed by

Robert Callahan
in August 1996
Robert Callahan
tel. (301) 496-2367
FAX (301) 402-0711
(e-mail callahanr@ltiblp.nci.nih.gov)