Modulation of Notch signaling by mastermind-like (MAML) transcriptional co-activators and their involvement in tumorigenesis

Seminars in Cancer Biology 14 (2004) 348–356

Modulation of Notch signaling by mastermind-like (MAML) transcriptional co-activators and their involvement in tumorigenesis Lizi Wu a,b , James D. Griffin a,b,∗ a b

Department of Medical Oncology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA

Abstract Notch signaling is mediated by cell–cell interactions and is critical for cell fate determination in many species. Recently, a family of mastermind-like (MAML) transcriptional co-activator genes was identified that encode proteins that cooperate with Notch and CSL to activate transcription. Here, we review our current understanding of the roles of the MAML proteins in Notch signaling, and their involvement in certain human cancers. The mounting biochemical and functional evidence indicate that the MAML genes are critical components of the Notch signaling pathway, likely regulating cellular events involved in both normal development and oncogenesis. © 2004 Elsevier Ltd. All rights reserved. Keywords: Notch signaling; Mastermind; Transcriptional co-activator; Mucoepidermoid carcinoma; Cervical cancer

1. Introduction Notch signaling is critical for determination of cell fates within multiple tissues, and contributes to self-renewal and survival of undifferentiated, multipotent cells throughout development and adulthood [1]. The Notch signaling pathway is involved in strikingly diverse biological processes including hematopoiesis, neurogenesis, myogenesis, vascular development, skin differentiation and the immune response [2–7]. The cellular basis for this extensive range of functions lies in the ability of the Notch signaling pathway to influence cellular proliferation, differentiation and apoptosis [8–11]. Although characterization of this pathway, its specific components, and its target genes is a field of intense current research, the mechanisms responsible for the differential effects in distinct tissues remain unclear. Notch genes encode single-pass, heterodimeric type I transmembrane proteins that serve as receptors for the DSL (Delta, Serrate, Lag-2) family of type I transmembrane ligands, which are expressed on neighboring cells. The Notch receptors are produced by cleavage of a single precursor peptide by a furin-like convertase at the S1 site [12], and contain distinct structural domains (Fig. 1). The Abbreviations: MAML, mastermind-like; ICN, intracellular domain of the Notch receptor; CSL, CBF1/RBP-J␬ in mammals, Su(H) in Drosophila, and Lag-1 in C. elegans ∗ Corresponding author. Tel.: +1-617-632-3360; fax: +1-617-632-4388. E-mail address: james [email protected] (J.D. Griffin). 1044-579X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcancer.2004.04.014

extracellular domains contain multiple epidermal growth factor (EGF)-like repeats which bind to ligands, and three membrane proximal Lin12/Notch repeats that have negative regulatory activities. The intracellular domains of Notch receptors are composed of a RAM domain, ankyrin repeats, a transcriptional activation domain (TAD), and the C-terminal PEST (proline, glutamate, serine, threonine) sequence. In mammals, there are four Notch receptors (Notch1, Notch2, Notch3 and Notch4) and six ligands (Jagged1, Jagged2, Delta1, Delta-like 1(Dll1), Dll3 and Dll4), in contrast to one Notch receptor and two ligands (Delta and Serrate) in Drosophila. The four mammalian Notch receptors are highly homologous, but contain different number of EGF repeats and distinct transcriptional activity in their intracellular domains. These multiple Notch receptors and ligands seem to have both independent and overlapping activities in mammalian cells. Numerous biochemical and genetic studies have led to a model of Notch activation, despite difficulties in detecting nuclear Notch protein in normal cells [13,14] (Fig. 2). When Notch signaling is initiated by receptor–ligand interactions between adjacent cells, the Notch receptor undergoes at least two successive proteolytic cleavages at the S2 and S3 sites. The first cleavage (S2), mediated by a member of the ADAM metalloprotease family, TNF-␣ converting enzyme (TACE), occurs external to and near the transmembrane domain: a membrane-tethered product is generated that is sensitive to the second (S3) cleavage [15]. The S3 cleavage occurs within the membrane,

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TM

EGF-Like Repeats

N1

349

Ankyrin Repeats

L

RAM LNR

N2

PEST

Fig. 1. Structure of a Notch receptor. Notch receptors are heterodimeric transmembrane proteins, each consisting of an extracellular ligand-binding domain, transmembrane domain, and intracellular domain. The intracellular domain contains a RAM domain involved in CSL binding; ankyrin repeats (the most conserved domain among different Notch receptors); nuclear localization signals (N1 and N2); a PEST sequence (possibly involved in stability of the Notch receptor). Transcriptional activation domain (TAD) differs among the four receptors. The extracellular domain contains multiple EGF-like repeats and LIN12/Notch repeats (LNR).

and requires the gamma secretase activity of presenilins 1 and 2 to produce the free intracellular domain of Notch (ICN) [16,17]. The ICN translocates to the nucleus and binds to the CSL family of DNA-binding transcription factors (CBF1/RBP-J␬ in mammals, Su(H) in Drosophila, and Lag-1 in C. elegans). Through several mechanisms that are just beginning to be understood, the CSL then is transformed into a transcriptional activator. This occurs by displacement of transcriptional co-repressors, including CIR (CBF1-interacting co-repressor) [18], SMRT/N-CoR (silencing mediator for retinoic acid and thyroid hormone receptor/nuclear repressor) [19], and KyoT2 [20]. Also, transcriptional co-activators are recruited, including those with a general role in transcriptional regulation such as CBP/p300 [21], pCAF and GCN5 [22], and more importantly, the newly identified mastermind-like proteins

Cytosol

DSL Notch

S2 S3

Extracellular Cytosol

ICN

MAML HATs CoR/HDAC CSL

ICN CSL

Fig. 2. Model for the roles of MAML proteins in Notch signaling. Ligand binding induces at least two successive proteolytic cleavages at the S2 and S3 sites, producing the free intracellular domain of Notch (ICN). ICN translocates to the nucleus and binds to a transcription factor, CSL. An active Notch transcriptional complex, consisting of the ICN/MAML/CSL core components, is formed through displacement of transcriptional co-repressor complex, and recruitment of transcriptional co-activators. Notch target genes are then activated.

(MAML family: see further for description) that appear to be specifically recruited in response to Notch signaling [21–23]. Interestingly, CSL-independent Notch signaling also has been documented [24], suggesting that some Notch effects can be mediated by other unidentified DNA-binding transcription factor(s). In light of the diverse roles of Notch signaling in distinct cellular, developmental and oncogenic contexts, it previously was expected that a diverse set of target genes would be activated. Intriguingly, however, only a limited number of target genes currently are identified. The most well-characterized of these is the HES gene family (mammalian homologues of Drosophila Hairy and Enhancer of Split genes) including HES-1 and HES-5 [25]. HES genes encode basic helix–loop–helix (bHLH) transcription factors and repress transcription of lineage specific transcription factors such as those involved in neurogenesis. A related but distinct bHLH protein family HERP (HES-related repressor protein), recently was discovered to be a Notch target, and is able to form heterodimers with HES and cooperate for transcriptional repression [25]. Other identified target genes include: MAP kinase phosphatase LIP-1 [26]; and the cell cycle regulators, p21WAF1/Cip1 [10]; cyclin D1; and CDK2 [27]. Several studies support the idea that Notch target gene expression varies with cell context, and may have opposing functions [10,27]. Therefore, further identification of the specific target genes in different cell types (for example, by gene profiling) is important to understand the diverse functions of Notch signaling in cellular proliferation, differentiation and apoptosis. Consistent with diverse effects of Notch effects in multiple tissues, mutations of Notch receptors and components of its signaling pathway are associated with a number of cancers [28,29]. For example, truncated activated forms of Notch1 (TAN1) resulting from a recurring t(7;9)(q34;q34.3) translocation cause a subset of T cell leukemias [30–32]. The transforming proteins essential for Epstein–Barr virus transformation of B cells (EBNA2, EBNA3a, EBNA3c) activate CSL factors independently of Notch ligand, thus modifying normal Notch signaling [33,34]. Additionally, the Notch4 gene is an integration site of mouse mammary tumor virus (Int3), resulting in constitutive activation of Notch4 and breast carcinoma [35]. Further, abnormal expression of Notch receptors, ligands and targets has been observed in a

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number of cancers including cervical, endometrial, mesothelioma, lung, breast, renal and hematological malignancies [36–43]. To this growing list, our recent studies linked the deregulation of Notch signaling via the MAML family to mucoepidermoid carcinoma, and cervical cancer (see further for discussion) [44,45]. Taken together, these studies support the concept that aberrant Notch signaling has widespread implications in many cancer types, and it is likely that many more associations of Notch signaling and human cancer will be recognized in the future. Therefore, investigations into the regulatory mechanisms of this pathway will provide important insights into its role in both normal development and in cancers, as well as methods for modulating Notch signaling for therapeutic intervention.

2. Mastermind-like transcriptional co-activators for Notch signaling 2.1. MAML family structure and function Currently, it is still unclear how the active Notch transcriptional complex is assembled, resulting in chromosomal remodeling and activation of the transcriptional machinery to induce specific target gene expression. Likewise, little is known concerning how this transcription complex is terminated, which dictates the duration of the Notch signal and also has profound effects on the specific signaling outcomes. Identification of the components of the Notch transcriptional complex, and investigating their interactions with cell-context specific factors, are crucial to understand how the Notch complex is regulated. The MAML family, mammalian counterparts of the Drosophila mastermind gene, was recently identified and appears to function specifically in Notch signaling [23,45–47]. The mastermind gene in Drosophila encodes a glutamine-rich nuclear protein [48,49]. It was identified as one of the original group of “neurogenic” loci in Drosophila along with Notch, because the loss of function mutation results in overproduction of neural cells at the expense of epidermal cells. The mastermind gene was repeatedly identified in multiple genetic screens for modifiers of Notch mutations [48,50,51], and has extensive genetic interactions with other Notch components such as Deltex [52] and Su(H) [52]. The mastermind protein is associated with specific chromosome sites [53], and the expression of truncated forms interferes with Notch function [54]. These studies in Drosophila demonstrated that the mastermind gene is an essential component of Notch signaling and may regulate Notch signaling at the transcriptional level. In C. elegans, the LAG-3 gene has been reported to have mastermind-like activity [55]. The LAG-3 gene encodes a glutamine-rich protein that forms a ternary complex with the LAG-1 DNA-binding protein and the intracellular domain of the receptor (GLP-1). It was a potent transcriptional activator, and therefore was proposed to function as tran-

Basic nls Acidic I

Acidic II 1016

MAML1 1 ICN p300 binding binding MAML1 MAML2 MAML3

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Fig. 3. Structure of the prototypic MAML1 protein. The MAML proteins all have an N-terminal basic domain, and two acidic domains (I and II) in the middle region and C-terminus. ICN binding sites are located in the basic domains of the three MAML protein. A p300 binding site is located in TAD1 of MAML1 (aa 75–300) of MAML1, but the site has not been mapped on MAML2 and MAML3. TAD2 is located in the C-terminal region (from aa 303 to 1016). Sequence alignments of the most conserved basic domains of three MAML proteins are shown.

scriptional co-activator. However, the sequence homology to mastermind genes in both Drosophila and human is very limited. The Xenopus homologue of the mastermind gene was recently cloned, which has high homology with human MAML1 [56] and is required for primary neurogenesis. It is unknown if there are other forms of mastermind genes. The human MAML family currently consists of three members, MAML1, MAML2 and MAML3 [23,45–47]. The mouse maml1 homologue also was recently identified and characterized by our group. The full-length cDNAs encoding mouse Maml2 and Maml3 have not yet been cloned; however, a reported short peptide sequence [57], and available EST sequences reveal that there are highly homologous sequences to the human counterparts, MAML2 and MAML3, demonstrating the presence of three mastermind-like genes in the mouse as well. Although the in vivo roles of these MAML genes in mammals have not been established, biochemical and cell-based studies have revealed the roles of MAML genes as transcriptional co-activators for Notch receptors [23,45,58–61]. Structurally, the three human MAMLs each contain an N-terminal basic domain and two acidic domains (I and II) in their middle region and C-terminus (Fig. 3). The highest sequence homology exists within the basic domain, which contains binding sites for all four mammalian ICNs, while the two acidic domains likely contribute to transcriptional activation. Although detailed mapping studies are needed, two TAD domains have been proposed. TAD1 (aa 75–300) contains a p300/CBP binding site, and recruits this molecule to the Notch transcriptional complex. TAD2 is a glutamine-rich region that is required for transcription in vivo. The MAML proteins directly bind to the most conserved domain of all four Notch receptors (ICN1–ICN4), ankyrin repeats, through their N-terminal conserved basic domains. They interact with CSL only in the presence of ICN, forming a stable DNA-binding ternary complex. Importantly, the MAML proteins are able to activate transcription of Notch target genes when Notch receptors are stimulated by ligands, or when constitutively active forms of Notch ICN are expressed. These data provide a model in which the

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N-terminal basic domain of MAML proteins forms a stable complex with ICN and CSL, and activates transcription of Notch-dependent genes through their C-terminal TAD. In support of this model, MAML1 mutants that are either deficient in transcriptional activities or incapable of binding ICN1 interfere with the ability of MAML1 to activate either ICN or ligand-induced Notch signaling [45]. Additionally, MAML1 is essential for chromatin-dependent transactivation via the recombinant Notch ICN-CSL enhancer complex. Structural analyses further demonstrate that the interaction of ICN and CSL creates the recruitment site for MAML protein [59]. The isolation of a large, stable multiprotein complex containing the endogenous MAML1, ICN and CSL, also is consistent with MAML proteins being core components of a Notch transcriptional complex [62]. Importantly, the three MAML proteins exhibit differential effects on Notch activation, in cooperation with distinct Notch receptors and ligands [23]. In reporter assays, MAML1 and MAML2 cooperate with all four Notch receptors to activate Notch target genes; however, MAML3 acts as a more effective transcriptional co-activator for ICN4 than other forms of ICN. Also, MAML1 and MAML2 are able to activate Notch target genes when the ligands Jagged2 and Delta1 were the stimuli, while MAML3 displayed little effect. While the mechanisms for such specific activities are not completely understood, it seems that it is due, in part, to the differential binding between MAML and various ICNs, and perhaps to unidentified components interacting with other MAML regions [23]. Currently, the mechanisms involved in MAML activation and recruitment remain undefined. A candidate molecule for activating this family is p300. p300 binds to MAML1, and enhances Notch-mediated transcription via MAML1 in an in vitro transcription assay [61]. However, the recruitment of p300 to MAML1 alone is not sufficient to account for the transcriptional activity of MAML1 protein, because a C-terminal truncation mutant of MAML1 that retains p300 binding activity failed to activate Notch target genes and also interfered with Notch signaling in vivo [45,61]. Therefore, undefined proteins that interact with the internal and C-terminal region of MAML proteins might be essential for Notch activation. Intriguingly, MAML proteins may also be involved in modulating the duration of the Notch signal. Although little is known concerning the termination of Notch signaling, four E3 ubiquitin ligases are implicated in this process [63], suggesting that ubiquitination and proteolytic steps are involved. For example, Sel-10 stimulates phosphorylation-dependent ubiquitination of nuclear ICN1. Further, Numb protein promotes the ubiquitination and degradation of membrane-bound Notch1 receptor following activation [64]. In vitro data recently revealed that MAML1 stimulates phosphorylation and turnover of ICN1 [61]. Therefore, MAML proteins also might function as regulatory proteins to regulate the stability of Notch protein, acting to terminate Notch signaling.

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2.2. MAML family expression patterns The biochemical and structural studies described earlier have been valuable in elucidating the MAML family’s mechanism of action in Notch signaling. However, studies also are required to investigate further their interesting localization and expression patterns. Sub-cellularly, the MAML family members are all detected in nuclear “dots” of heterogeneous sizes [23]. Although the molecular composition of the dots remains unclear, mounting evidence suggests that they have several important functions. MAML1 is able to relocate ICN, CSL, and p300 to its nuclear structure as shown by immunofluorescence studies [23,45,61]. Also, full-length MAML1, but not the MAML1 mutants that do not exhibit the nuclear dot localization, can increase the phosphorylation of p300 and ICN. It is therefore tempting to speculate that the dot structures recruit unknown kinase(s) to modify p300 and ICN, perhaps for proper assembly, function and degradation of the Notch transcriptional complex, and possibly also affect the transcription that depends on p300/CBP as co-activators. The identification of the components of the MAML nuclear dots, as well as mutational analysis of the domain(s) that are required for MAML nuclear dot localization, will shed light on the role of these structures in Notch signaling. Finally, our preliminary data has revealed considerable diversity in expression patterns of the three MAML genes [23]. For example, expression of three MAML genes seems dynamic and spatially specific in the early developing spinal cord of mice. Taken together, the differential activity of the MAML family members (discussed earlier) and their non-overlapping expression patterns suggest that three MAML proteins are not functionally redundant. Further studies are needed to determine whether these factors increase the diversity of signals from individual Notch receptors and ligands in distinct cell types.

3. MAML family members linked to cancer Since the MAML proteins play essential and positive regulatory roles in Notch signaling, it is not surprising that they are being linked to cancers: specifically, mucoepidermoid carcinoma and cervical cancer. Also, through the MAML family, Notch signaling can be modulated to achieve cell growth inhibition in the cancer cells that depend on this pathway for survival. 3.1. MAML2 and mucoepidermoid carcinoma Mucoepidermoid carcinoma (MEC) arises from major and minor salivary glands throughout the upper aerodigestive tract [65]. Although the incidence is rare, MEC represents the most common human malignant salivary and bronchial gland tumors. The name represents the two main histological components of the tumor: mucous cells that contain intra-

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11q21 breakpoint MAML2

1

intron1

exons 2-5

19p12-13 breakpoint 1

MECT1

MECT1-MAML2

intron1

1

exons 2-18

exons 2-5

Altered Notch signaling ??? MEC tumorigenesis Fig. 4. MECT1–MAML2, resulting from a t(11;19) chromosomal translocation in the MEC, alters Notch signaling and possibly contributes to tumorigenesis.

cellular mucin, and epidermoid (squamous type) epithelial cells. A third type of cells, sharing the features between mucous and epidermoid cells, are also present, therefore, MEC shows an unusual pattern of mixed cell differentiation. The diagnosis of these salivary gland tumors can be quite difficult, and so a molecular diagnostic tool would be valuable. Cytogenetic analysis showed that a recurring t(11;19) (q14–21;p12–13) translocation was associated with MEC, and occasionally, is the sole cytogenetic alteration [66–72]. To understand the biological and genetic basis for mucoepidermoid carcinoma, we cloned the chromosomal breakpoints of the t(11;19)(q14–21;p12–13) translocation and identified the genes involved in this translocation, MAML2 and MECT1 [44]. MECT1 was characterized very recently as a CREB binding protein [73]. The major consequence of the chromosomal translocation is the creation of a fusion protein: exon 1 of MECT1 gene fused to the exons 2–5 of MAML2 genes. The resulting MECT1–MAML2 molecule contains the first 42 aa of the N-terminus of the MECT protein (MECT1) and the 982 aa of the C-terminus of the MAML2 protein (Fig. 4). MECT1–MAML2 mRNA transcripts are detectable only in primary tumor biopsy samples from patients with either a bronchopulmonary, lingual, or parotid MEC and the MEC tumor cell lines, but not other non-MEC tumors. These data suggest that the expression of MECT1–MAML2 might be used as a molecular marker for diagnosis of this cancer. It would be interesting to determine whether MECT1–MAML2 is expressed in all histologic subtypes. The transforming potential of the MECT1–MAML2 fusion product was demonstrated using an E1A immortalized RK3E baby rat kidney cell assay, which has been used to assess the oncogenic activity of a truncated Notch receptor allele [74]. The MECT1–MAML2 chimera is able to efficiently induce foci formation in RK3E cells while wild-type MAML2 and vector alone have no activity, suggesting that

this fusion protein is able to cooperate with E1A for cell transformation. However, the biological consequences of MECT1–MAML2 in MEC require further studies in vivo. Also, it is important to determine whether MECT1–MAML2 is required for maintenance and growth of MEC cells. If specific inhibition of the MECT1–MAML2 expression leads to growth suppression of MEC, this fusion protein will provide a useful target for therapeutic intervention. Further studies of the MECT1–MAML2 gene in animal models will provide important insights into its biological activities. In the MECT1–MAML2 fusion, the N-terminal basic domain of MAML2 (required for binding to the Notch ICN) is replaced with an N-terminal domain of MECT1. Interestingly, MECT1–MAML2 still co-localizes with the Notch ICN in the nuclear dots, and immunocoprecipitates with ICN, although the interaction appears weaker in comparison with the MAML2 protein. However, MECT1–MAML2 was not immunocoprecipitated with CSL even in the presence of ICN, suggesting that possibility that MECT1–MAML2 and CSL might interact with the same domain in ICN. The significance of the interaction of the fusion protein and ICN is unknown. However, MECT1–MAML2 fusion seems to be able to inhibit ICN-induced HES-1 promoter activity, suggesting that the fusion protein might have inhibitory effects on the CSL-dependent Notch signaling. One hypothesis is that MECT1–MAML2 might compete with endogenous MAML protein for ICN, and sequester ICN from activating its target genes. One important function of MECT1–MAML2 is to activate Notch target genes in both the CSL and Notch ligand independent fashion, because activation of Notch target HES-1 reporter occurred independent of Notch ligand stimulation and with the mutated CSL binding sites. The ability to bypass ligand binding was confirmed by the observation that MECT1–MAML2 was able to activate Notch signaling in the presence of a gamma secretase inhibitor, which prevents photolytic cleavage and nuclear localization. Consistent with the ability of MECT1–MAML2 to activate Notch target genes in the reporter assay, increased levels of Notch target transcripts including HES-1, HERP1, and HERP2 were observed in several MEC cell lines, suggesting that Notch signaling is upregulated in MEC cell lines. Moreover, overexpression of MECT1–MAML2 in an immortalized parotid cell line induced the expression of these target genes. The mechanisms underlying the activation of Notch target genes by MECT1–MAML2 are still unclear. Preliminary studies suggest that induction of HES-1 by MECT1–MAML2 is independent of CSL, implying that other DNA-binding protein(s) are involved. Since MECT1 binds CREB [73], this could represent a potential mechanism for activation of Notch targets. The MECT1 portion of MECT1–MAML2, 1–42 aa, was found to be part of the coiled coil domain that mediates interaction of MECT1 (TORC1) with CREB. Also, MECT1–MAML2 was able to bind to CREB and activate a CRE-containing luciferase reporter. In fact, activation of CRE-containing reporters

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by both MECT1–MAML2 and MECT1 seems to require CREB. However, these activities alone cannot explain the unique activity of the fusion protein. We constructed two chimeric proteins, MECT1–MAML1 and MECT1–VP16. Both contain the coiled coil domain of MECT1 (1–2 aa), and strong transcriptional activity. Yet, MECT1–MAML1 can directly activate Notch target genes, while MECT1–VP16 has no effects. Therefore, the oncogenic potential of the fusion protein is likely to require functional domains from both MECT1 and a MAML2 genes. Comparing the activation of Notch target genes and cAMP induced genes by both MECT1–MAML2 and MECT1 will provide understanding of how MECT1–MAML2 activates Notch target genes. Since salivary gland cells undergo constant cellular renewal, it is likely that Notch signaling has a role in modulating salivary gland differentiation. Notch signaling has been implicated in salivary gland development in Drosophila [75]. Therefore, it is possible that altered Notch signaling is a significant mediator of MECT1–MAML2 oncogenic activity. Currently, it is still unclear if the transforming activity of MECT1–MAML2 is mediated by altered Notch activity, or other cellular signaling pathways, or in combination. Future studies looking for the target genes of MECT1-MAML2 will provide us the clues as to the signaling pathways with which this fusion protein might interfere. 3.2. Cervical cancer and MAML proteins Cervical cancer is the second leading cause of cancer death in women worldwide. The human papillomavirus (HPV) is strongly implicated as a causative agent in the etiology of over 90% of cervical cancer cases [76,77], although its mechanism of action is not completely understood. HPVs are DNA tumor viruses that induce proliferative lesions in cutaneous and mucosal epithelia [78]. More than 90 types of HPV have been identified and are categorized into the high-risk and low-risk type based on their clinical association and risk for malignant progression. In fact, the high-risk HPV type 16 alone causes more than 50% of all cervical cancer. Two small transforming oncogenes of high-risk HPV, E6 and E7, are selectively expressed in cervical cancers. A major component of the transforming activities of E6 is related to its ability to interact with an important cellular tumor suppressor protein, p53, through its interaction with another cellular protein, E6 associated protein (E6-AP). This interaction results in the degradation of p53 via the ubiquitin-mediated degradation pathway [79,80]. However, abundant evidence indicates E6 also has p53-independent mechanisms of transformation [81–86]. For example, certain forms of E6 that transform epithelial cells are unable to degrade p53. Conversely, other E6 forms that can degrade p53 fail to transform epithelial cells. The efforts to understand the E6 p53-independent mechanisms have led to identification of a number of E6 binding proteins [87]. However, the biological consequences of these interactions are undefined,

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and the mechanisms responsible for E6-inhibitory effect on differentiation are still unclear. Notch signaling induces epidermal differentiation [6,10,88]. Therefore, disruption of Notch signaling would disrupt normal epithelial differentiation and this could contribute to the development of cervical cancers. However, the exact role of Notch signaling in cervical cancer is currently unclear. Expression of Notch receptor (Notch1) and its ligand (Jagged1) are up-regulated in cervical cancers [37,38,89]. Detection of nuclear Notch expression seems to correlate with the transition from cervical intraepithelial lesions to invasive cervical carcinoma [36]. The activated Notch1 is synergistic with papillomavirus oncogene E6 in transformation of an immortalized epithelial cell line in vitro [90]. The above evidence suggests that upregulation of Notch signaling is possibly required at certain stage of cervical cancer. We speculate that Notch signaling may have different roles during early and late stages of cervical cancer development. In the early stage, interference of Notch signaling by HPV infection might lead to a block of differentiation, which commits cells in the path for further transformation, and thus is likely to be required for the pathogenesis of cervical cancer. During the late stage, other cellular events occur such as Ras activation, which might lead to high level of expression of Notch or nuclear Notch. In fact, Notch-1 has been found upregulated in Ras-transformed epithelial cells [91]. The enhanced Notch signaling in the oncogenic context might have growth promoting activities, and could contribute to tumorigenesis. Interestingly, we originally cloned MAML1 as a binding protein for high-risk HPV type 16 E6 in a yeast two-hybrid screening [45]. Our preliminary studies indicate that transforming variants of E6 interact with human mastermind-like proteins (unpublished). These interactions possibly interfere with the functions of MAML proteins as the transcriptional co-activator for Notch receptors. Therefore, we propose that E6–MAML complex provides a potential molecular mechanism whereby E6 could interfere with Notch signaling and inhibit epithelial differentiation in the early stage of cervical cancer. Studies that examine the significance of E6/MAML interactions in keratinocyte differentiation are needed to test this hypothesis. 3.3. Growth suppression of Notch-dependent leukemia cells by modulating Notch signaling via the MAML family Previous studies have shown that two types of dominant negative mutants of MAML1, one containing ICN binding domain but defective in transactivation, and the other, defective in ICN binding but with the TAD region, act as dominant negatives in reducing the activation of Notch target HES-1 expression. These results point to the possibility of a modulation of Notch signaling through the MAML proteins. The feasibility of this idea was affirmed by a recent study in which a dominant negative mutant was used to down-

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regulate Notch signaling in leukemia cells transformed with activated form of Notch1 (TAN1) [60]. TAN1 was initially identified through analysis of a recurring t(7;9)(q34;q34.3) chromosomal translocation in a subset of human acute lymphoblastic leukemia (T-ALL) [30]. This t(7;9) translocation fuses the 3 end of Notch1 to T cell receptor ␤ promoter/enhancer locus, which results in expression of 100–125 kDa truncated Notch proteins composed of most of the intracellular domain. Multiple studies have demonstrated that the TAN1 protein functions as a constitutively active Notch receptor, and behaves as an oncogene since infection of murine marrow cells with a retrovirus encoding TAN1 cDNA induces lethal T cell leukemia in mice [31,32]. Weng et al. employed a MAML1 dominant negative peptide that contains MAML1 (13–74 aa) fused to GFP, which was able to form a DNA-binding complex with RAM-ANK and CSL, and strongly inhibit CSL-dependent transcription [60]. Interestingly, the growth-suppressive effects of this dominant negative mutant seems to be restricted to those Notch1-transformed cell lines, because it caused a significant growth suppression of Notch1-transformed lymphoid cell lines expressing membrane-tethered Notch1, or ICN1, or TAN1 from t(7;9) translocation, but not the growth of other non-Notch1 transformed cell lines. This finding demonstrated that persistent Notch1 signaling is necessary for tumor growth of Notch1-transformed T cell leukemia. Moreover, the growth-suppressive effects were similar to the small-molecule presenilin inhibitors developed to prevent proteolysis and nuclear translocation of Notch receptors. While presenilin inhibitors only affect Notch signaling requiring proteolytic cleavage of the membrane-tether form of the Notch receptor, the MAML1 dominant negative mutant has a more general effect downstream by forming an inactive ICN transcriptional complex. This study clearly demonstrates that the MAML proteins are important targets for modulation of Notch signaling. The disruption or inactivation of the ICN/CSL/MAML transcriptional complex will present an important avenue to inhibit Notch signaling in those cancer cells requiring deregulated activated Notch signaling for maintenance and survival. Currently, we do not know if upregulation of Notch signaling is required for other cancer types, except the subset of TALL with t(7;9)(q34;q34.3). However, upregulation of the nuclear form of Notch has been observed in several cancers, including cervical cancers at the advanced stage. Besides dominant negative MAML1 peptides, small compounds that are able to disrupt the ternary ICN/CSL/MAML complex may prove useful for achieving growth inhibitory effect on Notch-dependent cancer cells.

4. Conclusions The family of mastermind-like genes encodes transcriptional co-activators required for Notch signaling. When

Notch receptors are activated through cell–cell interactions, MAML proteins are recruited to stabilize the Notch/CSL transcriptional complex on the target gene promoters, and activate transcription. In addition, MAML genes might have regulatory roles in controlling the duration of Notch signaling. However, many questions remain to be addressed regarding the mechanisms underlying the co-activator function of MAML proteins, other pathways with which the MAML proteins may potentially interact, and individual contributions of the MAML proteins to Notch signaling in distinct cell types. The involvements of MAML proteins in cancer development are implicated by studies that link them with two types of human epithelial cancers. Further understanding of altered Notch signaling in cancer development is required. Furthermore, modulation of Notch signaling via MAML proteins in cancer cells requiring Notch signaling for survival may prove to be an important therapeutic strategy.

Acknowledgements This work was supported in part by NIH RO1 CA36167 (J.D.G.), NIH R01 CA097148 (L.W.) and a Scholar Award of General Motors cancer research foundation (L.W.).

References [1] Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling: cell fate control and signal integration in development. Science 1999;284:770– 6. [2] Kojika S, Griffin JD. Notch receptors and hematopoiesis. Exp Hematol 2001;29:1041–52. [3] Wang S, Barres BA. Up a notch: instructing gliogenesis. Neuron 2000;27:197–200. [4] Kuroda K, Tani S, Tamura K, Minoguchi S, Kurooka H, Honjo T. Delta-induced Notch signaling mediated by RBP-J inhibits MyoD expression and myogenesis. J Biol Chem 1999;274:7238–44. [5] Iso T, Hamamori Y, Kedes L. Notch signaling in vascular development. Arterioscler Thromb Vasc Biol 2003;23:543–53. [6] Lowell S, Jones P, Le Roux I, Dunne J, Watt FM. Stimulation of human epidermal differentiation by delta-notch signalling at the boundaries of stem-cell clusters. Curr Biol 2000;10:491–500. [7] Hoyne GF. Notch signaling in the immune system. J Leukoc Biol 2003;74:971–81. [8] Miele L, Osborne B. Arbiter of differentiation and death: Notch signaling meets apoptosis. J Cell Physiol 1999;181:393–409. [9] Morimura T, Goitsuka R, Zhang Y, Saito I, Reth M, Kitamura D. Cell cycle arrest and apoptosis induced by Notch1 in B cells. J Biol Chem 2000;275:36523–31. [10] Rangarajan A, Talora C, Okuyama R, Nicolas M, Mammucari C, Oh H, et al. Notch signaling is a direct determinant of keratinocyte growth arrest and entry into differentiation. EMBO J 2001;20:3427– 36. [11] Schroeder T, Just U. mNotch1 signaling reduces proliferation of myeloid progenitor cells by altering cell-cycle kinetics. Exp Hematol 2000;28:1206–13. [12] Logeat F, Bessia C, Brou C, LeBail O, Jarriault S, Seidah NG, et al. The Notch1 receptor is cleaved constitutively by a furin-like convertase. Proc Natl Acad Sci USA 1998;95:8108–12.

L. Wu, J.D. Griffin / Seminars in Cancer Biology 14 (2004) 348–356 [13] Mumm JS, Kopan R. Notch signaling: from the outside in. Dev Biol 2000;228:151–65. [14] Weinmaster G. Notch signal transduction: a real rip and more. Curr Opin Genet Dev 2000;10:363–9. [15] Brou C, Logeat F, Gupta N, Bessia C, LeBail O, Doedens JR, et al. A novel proteolytic cleavage involved in Notch signaling: the role of the disintegrin-metalloprotease TACE. Mol Cell 2000;5:207–16. [16] De Strooper B, Annaert W, Cupers P, Saftig P, Craessaerts K, Mumm JS, et al. A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain. Nature 1999;398:518–22 [see comments]. [17] Taniguchi Y, Karlstrom H, Lundkvist J, Mizutani T, Otaka A, Vestling M, et al. Notch receptor cleavage depends on but is not directly executed by presenilins. Proc Natl Acad Sci USA 2002;99:4014–9. [18] Hsieh JJ, Zhou S, Chen L, Young DB, Hayward SD. CIR, a corepressor linking the DNA binding factor CBF1 to the histone deacetylase complex. Proc Natl Acad Sci USA 1999;96:23–8. [19] Kao HY, Ordentlich P, Koyano-Nakagawa N, Tang Z, Downes M, Kintner CR, et al. A histone deacetylase corepressor complex regulates the Notch signal transduction pathway. Genes Dev 1998;12:2269–77. [20] Taniguchi Y, Furukawa T, Tun T, Han H, Honjo T. LIM protein KyoT2 negatively regulates transcription by association with the RBP-J DNA-binding protein. Mol Cell Biol 1998;18:644–54. [21] Oswald F, Tauber B, Dobner T, Bourteele S, Kostezka U, Adler G, et al. p300 Acts as a transcriptional coactivator for mammalian notch-1. Mol Cell Biol 2001;21:7761–74. [22] Kurooka H, Honjo T. Functional interaction between the mouse notch1 intracellular region and histone acetyltransferases PCAF and GCN5. J Biol Chem 2000;275:17211–20. [23] Wu L, Sun T, Kobayashi K, Gao P, Griffin JD. Identification of a family of mastermind-like transcriptional coactivators for mammalian notch receptors. Mol Cell Biol 2002;22:7688–700. [24] Martinez Arias A, Zecchini V, Brennan K. CSL-independent Notch signalling: a checkpoint in cell fate decisions during development? Curr Opin Genet Dev 2002;12:524–33. [25] Iso T, Kedes L, Hamamori Y. HES and HERP families: multiple effectors of the Notch signaling pathway. J Cell Physiol 2003;194:237– 55. [26] Berset T, Hoier EF, Battu G, Canevascini S, Hajnal A. Notch inhibition of RAS signaling through MAP kinase phosphatase LIP-1 during C. elegans vulval development. Science 2001;291:1055–8. [27] Ronchini C, Capobianco AJ. Induction of cyclin D1 transcription and CDK2 activity by Notch(ic): implication for cell cycle disruption in transformation by Notch(ic). Mol Cell Biol 2001;21:5925–34. [28] Joutel A, Tournier-Lasserve E. Notch signalling pathway and human diseases. Semin Cell Dev Biol 1998;9:619–25. [29] Maillard I, Pear WS. Notch and cancer: best to avoid the ups and downs. Cancer Cell 2003;3:203–5. [30] Ellisen LW, Bird J, West DC, Soreng AL, Reynolds TC, Smith SD, et al. TAN-1, the human homolog of the Drosophila notch gene, is broken by chromosomal translocations in T lymphoblastic neoplasms. Cell 1991;66:649–61. [31] Pear WS, Aster JC, Scott ML, Hasserjian RP, Soffer B, Sklar J, et al. Exclusive development of T cell neoplasms in mice transplanted with bone marrow expressing activated Notch alleles. J Exp Med 1996;183:2283–91. [32] Aster J, Pear W, Hasserjian R, Erba H, Davi F, Luo B, et al. Functional analysis of the TAN-1 gene, a human homolog of Drosophila notch. Cold Spring Harb Symp Quant Biol 1994;59:125–36. [33] Hofelmayr H, Strobl LJ, Stein C, Laux G, Marschall G, Bornkamm GW, et al. Activated mouse Notch1 transactivates Epstein–Barr virus nuclear antigen 2-regulated viral promoters. J Virol 1999;73:2770– 80. [34] Strobl LJ, Hofelmayr H, Stein C, Marschall G, Brielmeier M, Laux G, et al. Both Epstein–Barr viral nuclear antigen 2 (EBNA2) and

[35]

[36]

[37]

[38]

[39] [40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

355

activated Notch1 transactivate genes by interacting with the cellular protein RBP-J kappa. Immunobiology 1997;198:299–306. Jhappan C, Robinson G, Hennighausen L, Sharp R, Kordon E, Callahan R, et al. Expression of a truncated Int3 gene in developing secretory mammary epithelium specifically retards lobular differentiation resulting in tumorigenesis. Cancer Res 1996;56:1775– 85. Daniel B, Rangarajan A, Mukherjee G, Vallikad E, Krishna S. The link between integration and expression of human papillomavirus type 16 genomes and cellular changes in the evolution of cervical intraepithelial neoplastic lesions. J Gen Virol 1997;78:1095– 101. Gray GE, Mann RS, Mitsiadis E, Henrique D, Carcangiu ML, Banks A, et al. Human ligands of the Notch receptor. Am J Pathol 1999;154:785–94. Zagouras P, Stifani S, Blaumueller CM, Carcangiu ML, Artavanis-Tsakonas S. Alterations in Notch signaling in neoplastic lesions of the human cervix. Proc Natl Acad Sci USA 1995;92:6414– 8. Sattler M, Salgia R. Molecular and cellular biology of small cell lung cancer. Semin Oncol 2003;30:57–71. Rae FK, Stephenson SA, Nicol DL, Clements JA. Novel association of a diverse range of genes with renal cell carcinoma as identified by differential display. Int J Cancer 2000;88:726–32. Suzuki T, Aoki D, Susumu N, Udagawa Y, Nozawa S. Imbalanced expression of TAN-1 and human Notch4 in endometrial cancers. Int J Oncol 2000;17:1131–9. Bocchetta M, Miele L, Pass HI, Carbone M. Notch-1 induction, a novel activity of SV40 required for growth of SV40-transformed human mesothelial cells. Oncogene 2003;22:81–9. Imatani A, Callahan R. Identification of a novel NOTCH-4/INT-3 RNA species encoding an activated gene product in certain human tumor cell lines. Oncogene 2000;19:223–31. Tonon G, Modi S, Wu L, Kubo A, Coxon AB, Komiya T, et al. t(11;19)(q21;p13) translocation in mucoepidermoid carcinoma creates a novel fusion product that disrupts a Notch signaling pathway. Nat Genet 2003;33:208–13. Wu L, Aster JC, Blacklow SC, Lake R, Artavanis-Tsakonas S, Griffin JD. MAML1, a human homologue of Drosophila mastermind, is a transcriptional co-activator for NOTCH receptors. Nat Genet 2000;26:484–9. Kitagawa M, Oyama T, Kawashima T, Yedvobnick B, Kumar A, Matsuno K, et al. A human protein with sequence similarity to Drosophila mastermind coordinates the nuclear form of notch and a CSL protein to build a transcriptional activator complex on target promoters. Mol Cell Biol 2001;21:4337–46. Lin SE, Oyama T, Nagase T, Harigaya K, Kitagawa M. Identification of new human mastermind proteins defines a family that consists of positive regulators for notch signaling. J Biol Chem 2002;277:50612– 20. Smoller D, Friedel C, Schmid A, Bettler D, Lam L, Yedvobnick B. The Drosophila neurogenic locus mastermind encodes a nuclear protein unusually rich in amino acid homopolymers. Genes Dev 1990;4:1688–700. Yedvobnick B, Smoller D, Young P, Mills D. Molecular analysis of the neurogenic locus mastermind of Drosophila melanogaster. Genetics 1988;118:483–97. Go MJ, Artavanis-Tsakonas S. A genetic screen for novel components of the notch signaling pathway during Drosophila bristle development. Genetics 1998;150:211–20. Xu T, Rebay I, Fleming RJ, Scottgale TN, Artavanis-Tsakonas S. The Notch locus and the genetic circuitry involved in early Drosophila neurogenesis. Genes Dev 1990;4:464–75. Busseau I, Diederich RJ, Xu T, Artavanis-Tsakonas S. A member of the Notch group of interacting loci, deltex encodes a cytoplasmic basic protein. Genetics 1994;136:585–96.

356

L. Wu, J.D. Griffin / Seminars in Cancer Biology 14 (2004) 348–356

[53] Bettler D, Pearson S, Yedvobnick B. The nuclear protein encoded by the Drosophila neurogenic gene mastermind is widely expressed and associates with specific chromosomal regions. Genetics 1996;143:859–75. [54] Helms W, Lee H, Ammerman M, Parks AL, Muskavitch MAT, Yedvobnick B. Engineered truncations in the Drosophila mastermind protein disrupt Notch pathway function. Dev Biol 1999;215:358–74. [55] Petcherski A, Kimble J. LAG-3 is a putative trancriptional activator in the C. elegans Notch pathway. Nature 2000;405:364–8. [56] Katada T, Kinoshita T. Xmam1, the Xenopus homologue of mastermind, is essential to primary neurogenesis in Xenopus laevis embryos. Int J Dev Biol 2003;47:397–404. [57] Petcherski AG, Kimble J. Mastermind is a putative activator for Notch. Curr Biol 2000;10:R471–3. [58] Wallberg AE, Pedersen K, Lendahl U, Roeder RG. p300 and PCAF act cooperatively to mediate transcriptional activation from chromatin templates by notch intracellular domains in vitro. Mol Cell Biol 2002;22:7812–9. [59] Nam Y, Weng AP, Aster JC, Blacklow SC. Structural requirements for assembly of the CSL/intracellular Notch1/mastermind-like 1 transcriptional activation complex. J Biol Chem 2003;278:21232–9. [60] Weng AP, Nam Y, Wolfe MS, Pear WS, Griffin JD, Blacklow SC, et al. Growth suppression of pre-T acute lymphoblastic leukemia cells by inhibition of notch signaling. Mol Cell Biol 2003;23:655–64. [61] Fryer CJ, Lamar E, Turbachova I, Kintner C, Jones KA. Mastermind mediates chromatin-specific transcription and turnover of the Notch enhancer complex. Genes Dev 2002;16:1397–411. [62] Jeffries S, Robbins DJ, Capobianco AJ. Characterization of a high-molecular-weight Notch complex in the nucleus of Notch(ic)-transformed RKE cells and in a human T-cell leukemia cell line. Mol Cell Biol 2002;22:3927–41. [63] Lai EC. Protein degradation: four E3s for the notch pathway. Curr Biol 2002;12:R74–78. [64] McGill MA, McGlade CJ. Mammalian numb proteins promote Notch1 receptor ubiquitination and degradation of the Notch1 intracellular domain. J Biol Chem 2003;278:23196–203. [65] Cawson RA, Gleeson MJ, Eveson JW. Pathology and surgery of the salivary glands. 1st ed. ISIS Medical Media; 1997. [66] Dahlenfors R, Wedell B, Rundrantz H, Mark J. Translocation (11;19)(q14–21;p12) in a parotid mucoepidermoid carcinoma of a child. Cancer Genet Cytogenet 1995;79:188. [67] Horsman DE, Berean K, Durham JS. Translocation (11;19)(q21; p13.1) in mucoepidermoid carcinoma of salivary gland. Cancer Genet Cytogenet 1995;80:165–6. [68] Johansson M, Mandahl N, Johansson L, Hambraeus G, Mitelman F, Heim S. Translocation 11;19 in a mucoepidermoid tumor of the lung. Cancer Genet Cytogenet 1995;80:85–6. [69] El-Naggar AK, Lovell M, Killary AM, Clayman GL, Batsakis JG. A mucoepidermoid carcinoma of minor salivary gland with t(11;19)(q21;p13.1) as the only karyotypic abnormality. Cancer Genet Cytogenet 1996;87:29–33. [70] Stenman G, Petursdottir V, Mellgren G, Mark J. A child with a t(11;19)(q14–21;p12) in a pulmonary mucoepidermoid carcinoma. Virchows Arch 1998;433:579–81. [71] Dahlenfors R, Lindahl L, Mark J. A fourth minor salivary gland mucoepidermoid carcinoma with 11q14–21 and 19p12 rearrangements. Hereditas 1994;120:287–8. [72] Nordkvist A, Gustafsson H, Juberg-Ode M, Stenman G. Recurrent rearrangements of 11q14–22 in mucoepidermoid carcinoma. Cancer Genet Cytogenet 1994;74:77–83.

[73] Conkright MD, Canettieri G, Screaton R, Guzman E, Miraglia L, Hogenesch JB, et al. TORCs: transducers of regulated CREB activity. Mol Cell 2003;12:413–23. [74] Capobianco AJ, Zagouras P, Blaumueller CM, Artavanis-Tsakonas S, Bishop JM. Neoplastic transformation by truncated alleles of human NOTCH1/TAN1 and NOTCH2. Mol Cell Biol 1997;17:6265–73. [75] Haberman AS, Isaac DD, Andrew DJ. Specification of cell fates within the salivary gland primordium. Dev Biol 2003;258:443–53. [76] Severson J, Evans TY, Lee P, Chan T, Arany I, Tyring SK. Human papillomavirus infections: epidemiology, pathogenesis, and therapy. J Cutan Med Surg 2001;5:43–60. [77] Alani RM, Munger K. Human papillomaviruses and associated malignancies. J Clin Oncol 1998;16:330–7. [78] Howley PM. In: Fields, BN, Howley, PM, Knipe, DM, editors. Virology. Philadephila, PA: Lippincott–Raven; 1996. p. 2045–76. [79] Scheffner M, Werness BA, Huibregtse JM, Levine AJ, Howley PM. The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 1990;63:1129–36. [80] Huibregtse JM, Scheffner M, Howley PM. E6-AP directs the HPV E6-dependent inactivation of p53 and is representative of a family of structurally and functionally related proteins. Cold Spring Harb Symp Quant Biol 1994;59:237–45. [81] Sherman L, Schlegel R. Serum- and calcium-induced differentiation of human keratinocytes is inhibited by the E6 oncoprotein of human papillomavirus type 16. J Virol 1996;70:3269–79. [82] Sherman L, Jackman A, Itzhaki H, Stoppler MC, Koval D, Schlegel R. Inhibition of serum- and calcium-induced differentiation of human keratinocytes by HPV16 E6 oncoprotein: role of p53 inactivation. Virology 1997;237:296–306. [83] Klingelhutz AJ, Foster SA, McDougall JK. Telomerase activation by the E6 gene product of human papillomavirus type 16. Nature 1996;380:79–82. [84] Liu Y, Chen JJ, Gao Q, Dalal S, Hong Y, Mansur CP, et al. Multiple functions of human papillomavirus type 16 E6 contribute to the immortalization of mammary epithelial cells. J Virol 1999;73:7297– 307. [85] Kiyono T, Foster SA, Koop JI, McDougall JK, Galloway DA, Klingelhutz AJ. Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells. Nature 1998;396:84–8. [86] Song S, Pitot HC, Lambert PF. The human papillomavirus type 16 E6 gene alone is sufficient to induce carcinomas in transgenic animals. J Virol 1999;73:5887–93. [87] Fehrmann F, Laimins LA. Human papillomaviruses: targeting differentiating epithelial cells for malignant transformation. Oncogene 2003;22:5201–7. [88] Nicolas M, Wolfer A, Raj K, Kummer JA, Mill P, van Noort M, et al. Notch1 functions as a tumor suppressor in mouse skin. Nat Genet 2003;33:416–21. [89] Tewari KS, Taylor JA, Liao SY, DiSaia PJ, Burger RA, Monk BJ, et al. Development and assessment of a general theory of cervical carcinogenesis utilizing a severe combined immunodeficiency murine–human xenograft model. Gynecol Oncol 2000;77:137–48. [90] Rangarajan A, Syal R, Selvarajah S, Chakrabarti O, Sarin A, Krishna S. Activated Notch1 signaling cooperates with papillomavirus oncogenes in transformation and generates resistance to apoptosis on matrix withdrawal through PKB/Akt. Virology 2001;286:23–30. [91] Weijzen S, Rizzo P, Braid M, Vaishnav R, Jonkheer SM, Zlobin A, et al. Activation of Notch-1 signaling maintains the neoplastic phenotype in human Ras-transformed cells. Nat Med 2002;8:979–86.