Oncogene (2011) 30, 287–300 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE The lymphovascular embolus of inflammatory exhibits a Notch 3 addiction

Y Xiao1,YYe2, X Zou1, S Jones1, K Yearsley1, B Shetuni1, J Tellez2 and SH Barsky2,3

1Department of Pathology, The Ohio State University College of Medicine, Columbus, OH, USA; 2Department of Pathology, University of Nevada School of Medicine, Reno, NV, USA and 3Nevada Cancer Institute, Las Vegas, NV, USA

Inflammatory breast (IBC) is characterized by Introduction exaggerated lymphovascular invasion (LVI), recapitulated in our human xenograft, MARY-X. This model exhibited Inflammatory breast carcinoma (IBC) is an aggressive lymphovascular emboli in vivo and corresponding sphe- form of human breast cancer characterized by florid roids in vitro. Owing to the morphological and profile lymphovascular invasion (LVI) and early resemblance of these spheroids to embryonal blastocysts, (Palangie et al., 1994; Cariati et al., 2005). LVI is we wondered whether they might exhibit embryonic stem considered a rate-limiting step in the metastatic process cell signaling. Specifically we investigated Notch and and is characterized by tumor emboli within lympho- observed selective Notch 3 activation by expression vascular channels. These emboli are resistant to radio- profiling, reverse transcriptase– and real-time PCR, therapy and (Alpaugh and Barsky, 2002; western blot and immunofluorescence in vitro, and Alpaugh et al., 2002a, b; Xiao et al., 2008). The IBC immunohistochemistry in vivo. Notch 3 intracellular phenotype has been successfully recapitulated in a domain (N3icd) and six target , HES-5, HEY-1, human xenograft model of IBC termed MARY-X c-Myc, Deltex-1, NRARP and PBX1, markedly in- (Tomlinson et al., 2001). This model exhibits florid creased in MARY-X. In addition, a significant percentage LVI in vivo, which generates tight tumor cell aggregates of MARY-X cells expressed aldehyde dehydrogenase (spheroids) in vitro. These spheroids resemble the human (ALDH), a stem cell marker. Only the ALDH þ cells blastocyst. Both structures overexpress E-cadherin, and were capable of secondary spheroidgenesis, tumorigenicity have a deficiency of fucosyltransferases and surface and self-renewal. Inhibiting Notch 3 activation in vitro fucosylation (Alpaugh et al., 2002b), but are efficient at with c-secretase inhibitors (GSIs) or small interfering organ implantation. On account of these similarities, RNA resulted in a downregulation of Notch target genes, we wondered whether the spheroids might express a including CD133, and an induction of caspase 3-mediated blastocyst-like expression profile. Based on microarray apoptosis. Transfection of N3icd but not Notch 1 intracellular data, we observed that certain embryonic pathways, domain into normal human mammary epithelial cells like Notch 3, appeared activated and so we decided to resulted in increased expression of Notch target genes investigate this further. and induction of spheroidgenesis. GSI in vivo resulted in inhibitory but diffusion-limited effects on Notch 3 signaling, resulting in xenograft growth reduction. The lymphovascular emboli of human IBC exhibited dual Results N3icd and ALDH1 immunoreactivities independently of molecular subtype. This Notch 3 addiction of lympho- MARY-X exhibits a ‘blastocyst-like’ phenotype with vascular emboli might be exploited in future therapeutic exaggerated Notch 3 signaling strategies. The MARY-X xenograft gave rise to tight spheroids in Oncogene (2011) 30, 287–300; doi:10.1038/onc.2010.405; suspension culture. When re-injected into immunodefi- published online 13 September 2010 cient mice, these spheroids manifested lymphovascular emboli within dermal lymphatics. Screening of the Keywords: inflammatory breast cancer; lymphovascular spheroids by expression profiling revealed increased emboli; Notch 3 signaling; g-secretase inhibitors; Notch Notch, specifically Notch 3. The downstream genes, 3 siRNA; apoptosis HES-5 and HEY-2, were noted by expression profiling also to be overexpressed (Po0.0001), suggesting the possibility of Notch signaling. More detailed reverse transcriptase (RT)–PCR, real-time RT–PCR, western blot and immunocytochemical studies looking at Notch Correspondence: Dr SH Barsky, Department of Pathology, University 3 (Xiao et al., 2008) and expanding the number of Notch of Nevada School of Medicine and Nevada Cancer Institute, 1 Manville targets confirmed these findings (Figures 1a–e). Medical Building, Reno, NV 89557-0350, USA. E-mail: [email protected] Although other Notch receptors were expressed in Received 1 May 2009; revised and accepted 8 July 2010; published online some of the non-IBC and normal lines, Notch 3 13 September 2010 dominated in MARY-X (Figure 1a), even when the Notch 3 dependency of IBC embolus Y Xiao et al 288

Figure 1 Notch 3 activation in MARY-X. (a) Western blot indicated strong expression of Notch 3 (N3icd) compared with Notch 1, Notch 2 and Notch 4 in MARY-X spheroids. Although other Notch family members were expressed in non-IBC breast carcinoma lines, and normal epithelial and myoepithelial lines (Sternlicht et al., 1996; Barsky, 2003), it was the dominant expression of Notch 3 that stood out in MARY-X. (b) Notch targets HEY-2 and HES-5, detected by RT–PCR, were prominent in MARY-X and MARY-X spheroids. (c) Evidence of Notch 3 signaling was also seen in the MARY-X xenograft by the presence of N3icd nuclear immunoreactivity (upper panel), and downstream HES-5 nuclear immunoreactivity (middle panel) compared with IgG control (lower panel). (d) Relative expression levels by real-time RT–PCR of select downstream targets of Notch, c-Myc, Deltex-1, NRARP and PBX1 in MARY-X compared with other breast carcinoma lines. All were significantly increased in MARY-X. (e) Relative expression levels by real-time RT–PCR of another downstream target of Notch, GATA3, which was expressed at higher levels in MCF-7 than MARY-X. Still, MARY-X expressed higher levels of GATA3 than the estrogen receptor-negative lines. (f) Cellular compartmentalization of N3icd and N1icd. In MARY-X spheroids, N3icd was located predominately in the nuclear compartment, whereas N1icd was located mainly in the membrane. The former normally signaled and the latter did not. When the cells were treated with g-secretase inhibitors (GSI treatment), the N3icd remained confined to the membrane, which is evidence of inhibition of Notch 3 signaling. As Notch 1 levels were low to begin with, with no appreciable signaling, its cellular compartmentalization was unaffected by GSI treatment. DMSO, dimethylsulfoxide.

non-IBC carcinoma and normal lines were induced to tested breast cancer cell lines (Figures 1b and d). grow as spheroids. MARY-X spheroids expressed the Actually, in nine Notch target genes (including HES-1, highest levels of six different Notch target genes: HES-5, HEY-2 and GATA3), MARY-X exhibited significantly HEY-1, c-Myc, Deltex-1, NRARP and PBX1, among all higher expression levels than MDA-MB-468 and MDA-

Oncogene Notch 3 dependency of IBC embolus Y Xiao et al 289 MB-231 (Figures 1b, d and e), but MCF-7 showed Initially we screened a series of secretase inhibitors higher level of HES-1 and GATA3 expression than (Table 1), peptides that mimicked the binding structure MARY-X. MCF-7, in fact, exhibited much higher of the amyloid precursor protein (APP) and peptides (417-fold) expression levels of GATA3 than MARY- that are able to inhibit APP processing, for their ability X. Notch 3 signaling manifested itself not only in the to inhibit Notch cleavage and nuclear translocation. MARY-X spheroids but also in the MARY-X xeno- Both b-secretase inhibitors and GSIs were initially graft. In the latter settings, prominent nuclear Notch 3 screened with immunohistochemistry studies for their intracellular domain (N3icd) and HES-5 immuno- ability to inhibit N3icd nuclear translocation over a reactivities were present, which was evidence that the range of concentrations. We used both classes of Notch 3 receptor had been cleaved, translocated to secretase inhibitors initially, even though b-secretase is the nucleus and engaged in downstream signaling not thought to be involved in the cleavage of Notch, to (Figure 1c). MARY-X spheroids also exhibited a loss provide a negative control for g-secretase cleavage. The of Numb expression (data not shown). The anti-N3icd GSIs, specifically a particular GSI-I (Z-Leu–Leu–Nle– antibody used, which recognized the cleaved intracellular CHO), were the more potent (Table 1). In as little as 4 h domain of Notch 3, demonstrated strong nuclear and and with as low a concentration as 0.8 mM, GSI-I totally cytoplasmic cellular compartmentalization (Figure 1f). abolished N3icd nuclear immunoreactivity and kept the In contrast, anti-Notch 1 intracellular domain (N1icd) Notch 3 immunoreactivity confined to the plasma antibody demonstrated only weak membrane compart- membrane of the MARY-X spheroids (Figure 2a). The mentalization. Other Notch members (Notch 2, Notch 4) inhibition of Notch 3 signaling was also manifested by exhibited no nuclear immunoreactivity in the MARY-X dramatic decrease in the expression levels of the down- spheroids (data not shown). stream genes HES-5 and HEY-2, measured by RT–PCR As many of the non-IBC lines expressed Notch (Figure 2b). The inhibition of the downstream genes receptors but did not demonstrate robust Notch began as early as 2 h and peaked at 10 h following GSI-I signaling (Figures 1b and d), we investigated possible treatment. Activated caspase 3 was noted as early as 8 h reasons for the selective Notch 3 dominance in MARY- following treatment and peaked at 24 h (Figure 2c). X. The entire Notch 1–4 receptors were sequenced in Endonuclease-mediated nucleosome excision manifest- MARY-X and found to be normal. Specifically there ing as a DNA apoptosis ladder was also noted at 8 h and were no mutations or deletions. Although array peaked at 24 h (Figure 2d). As the inhibitory actions of comparative genomic hybridization revealed a moder- the GSIs may not have been limited to only g-secretases, ately amplified Notch 2 (1p13-11), the Notch 3 especially considering recent studies that revealed that locus was not amplified. We did note, however, that the aldehyde group on the GSI-I peptide was able to Jagged-1 and DLL1 in MARY-X were slightly in- covalently bind to and inhibit certain serine proteases creased. However, knockdown of Jagged-1 with small (Curry et al., 2005), we had to exclude the possibility interfering RNA (siRNA) had no effect on the MARY- that the results we observed on the inhibition of Notch X spheroids (data not shown) compared with the signaling were due to the inhibition of serine proteases pronounced effect of Notch 3 knockdown on apoptosis and not g-secretases. To accomplish this, we performed (see the next section). Knockdown of DLL1 was experiments with GSI XIX, a noncovalent peptidomi- attempted but could not be technically achieved. metic inhibitor of g-secretases that is not able to bind serine proteases. Similar results on notch cleavage (Table 1), upregulation of activated caspase 3 and Directly inhibiting Notch 3 signaling in MARY-X apoptosis were observed with this inhibitor as were spheroids induces apoptosis observed with GSI-I (data not shown). As g-secretases We sought to inhibit Notch signaling by two different may cleave not only Notch but also ErbB4, syndecan experimental manipulations: inhibition of its enzymatic and other substrates (Shih and Wang, 2007), we had to cleavage (Figure 2) and Notch 3 knockdown with also exclude the possibility that these other g-secretase siRNA (Figure 3). Notch signaling is thought to be substrates might be mediating the apoptosis response dependent on two proteolytic cleavage events in the when g-secretase was inhibited. Notch receptor, an initial extracellular domain cleavage To do this, we attempted to specifically inhibit Notch by the ADAM metallopeptidase domain 10, which is signaling by knockdown of the Notch receptor itself thought to be triggered by ligand binding, and a using a siRNA approach. More than 90% knockdown subsequent intramembranous cleavage by g-secretase, of both mRNA as measured by real-time PCR which releases the Nicd to the nucleus, where it activates (Figure 3a) as well as protein as measured by western a number of downstream genes, including the HES and blot (Figure 3b) was achieved. Both control NCSI as HEY family of basic helix–loop–helix transcriptional well as luciferase siRNA had no effect. repressors. The effects of Notch 3 knockdown with siRNA were We then sought to initially inhibit Notch signaling by virtually identical to the effects with GSI-I: inhibition directly targeting the g-secretase with g-secretase in- of the Notch downstream genes, HEY-2 and HES-5, hibitors (GSIs; Figure 2). As the actions of g-secretase activation of caspase 3 and a DNA ladder of apoptosis and g-secretase inhibition were not specific for Notch 3, (data not shown). Notch 1 and Jagged-1 knockdown we subsequently targeted Notch 3 with an siRNA were effective at achieving specific knockdown of their knockdown approach as well (Figure 3). respective targets, but they did not have any effects on

Oncogene Notch 3 dependency of IBC embolus Y Xiao et al 290 Notch downstream signaling or the induction of and Notch 3 interference, apoptosis preceded the apoptosis (data not shown). disadherence. With both the g-secretase inhibition and Notch 3 interference, morphological changes within the spher- oids became noticeable within 12–24 h (Figure 3c). Inhibiting Notch 3 signaling in the MARY-X xenograft These morphological changes suggested that apoptosis has similar but diffusion-limited effects was occurring initially around the periphery of the We extended these Notch signaling inhibition experi- spheroids. In the setting of both g-secretase inhibition ments in vivo to the MARY-X xenografts. Use of GSI-I

Oncogene Notch 3 dependency of IBC embolus Y Xiao et al 291 in vivo resulted in similar inhibiting effects on Notch MARY-X spheroids exhibit high aldehyde dehydrogenase signaling, caspase 3 activation and apoptosis (Figure 2e), (ALDH) activity, which in turn exhibits concordance resulting in a reduction in the growth rate of the with Notch 3 signaling xenografts but not frank tumor regression (Figure 2f). A high activity of ALDH, a marker for both normal and These effects were more pronounced near the injection cancer stem cells, was observed in the cells comprising site of the drug and became less pronounced further the MARY-X spheroids. When the spheroids were away from the injection site, suggesting diffusion-limiting disadhered into single cells, approximately 23% of the effects. cells exhibited ALDH activity by the ALDEFLUOR

Figure 3 Notch 3 knockdown with small interfering RNA achieved 490% mRNA knockdown (a) and a significant protein knockdown (b). Notch 3 control NCSI and luciferase siRNA had no effect. (c) Induction of apoptosis by different manipulations. Two different experimental manipulations resulted in apoptosis in the spheroids of MARY-X. The intact spheroid (upper left panel) exhibited peripheral apoptosis with GSI-I treatment (upper right panel). Notch 3 RNA knockdown with siRNA similarly resulted in peripheral apoptosis (lower central). Note that the central portion of the spheroid remained intact with these strategies.

Figure 2 Inhibition of Notch 3 signaling in vitro with GSI-I. (a) The MARY-X spheroids, untreated, constitutively exhibited Notch 3 signaling as evidenced by nuclear N3icd fluorescence. Treatment with GSI-I resulted in inhibition of Notch 3 signaling, with the resulting abolishment of nuclear fluorescence and the emergence of membranous fluorescence after 4 h. The top row pertains to the primary antibody, anti-N3icd, and fluorescein isothiocyanate-labeled secondary antibody, the middle row Hoechst 33342 nuclear counterstain, and the bottom row the composite image. (b) RT–PCR showed a decrease in HES-5 and HEY-2 following GSI-I treatment over 10 h. (c) Treatment of the spheroids with GSI-I also produced activated caspase 3 as early as 8 h, which peaked at 24 h. The top row is fluorescein isothiocyanate-labeled anti-activated caspase 3 as primary antibody, the middle row Hoechst 33342 nuclear counterstain, and bottom row the composite image. (d) A prominent DNA fragment ladder of apoptosis emerged over the indicated time periods of GSI-I treatment. (e) Inhibition of Notch 3 signaling in vivo with GSI-I. GSI in vivo inhibited Notch 3 signaling, stimulated activated caspase 3 expression and induced apoptosis. Lower row: The white solid circle indicates the injection site and the GSI-I diffusion-limiting effects produced a gradient of Notch signaling inhibition (decreased nuclear N3icd immunoreactivity near the injection site); a gradient of caspase 3 activation (increased signal near the injection site) with fluorescein isothiocyanate-anti-activated caspase 3 with Hoechst 33342 nuclear counterstain and illustrated composite image; and a gradient of apoptosis (increased terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) chromogenic activity near the injection site). Upper row: No injections or dimethylsulfoxide (DMSO) only injections showed no such effects and no such gradients. (f) The net result of these diffusion-limiting effects of GSI-I was a reduction in growth rate of the xenografts but not tumor regression.

Oncogene Notch 3 dependency of IBC embolus Y Xiao et al 292 Table 1 Effects of secretase inhibitors on Notch signaling in MARY-X spheroidsa

Agent name 100 mM 20 mM 4 mM 0.8 mM

b-Secretase inhibitor II (Z-Val–Leu–Leu–CHO) þþþÀÀ b-Secretase inhibitor III (H-Glu–Val–Asn) ÀÀÀÀ g-Secretase inhibitor I (Z-Leu–Leu–Nle–CHO) þþþ þþþ þþþ þþþ g-Secretase inhibitor III (Z-Leu–Leu–CHO) þþþ þþ À À g-Secretase inhibitor IV (Z-Naphthoyl–Val–Phe–CHO) þþþÀÀ g-Secretase inhibitor XIX þþþ þþþ þþþ þþ

aThe effects of each of these inhibitors on Notch3 signaling (nuclear translocation) were measured immunocytochemically on a semi-quantitative ordinal scale and scored subjectively.

flow cytometric assay (Figure 4a). In contrast, the non- many genes in common that were overexpressed IBC lines and normal mammary epithelial cells exhib- (Table 3). Specifically there were a total of 766 genes ited very low percentages (1–5%) of ALDH activity overexpressed in the MARY-X spheroids compared (data not shown). We then investigated the secondary with MDA-MB-231, MDA-MB-468 and HMEC spheroidgenesis and tumorigenicity capacities of the (Po0.001). There were a total of 291 genes overexpressed sorted ALDH þ subpopulation. Our studies indicated in CD44 þ human breast cancer cells compared with that (1) only ALDH þ cells were capable of forming both CD24 þ breast cancer cells (Po0.01). There were a total secondary spheroids as well as tumors (Table 2) and (2) of 102 genes overexpressed in both the human mammo- ALDH þ cells were able to give rise to both ALDH þ and spheres and neurospheres (Po0.01). We identified 36 ALDHÀ cells, that is, a heterogeneous cell population overexpressed genes in common between the MARY-X (Figure 4a). These results suggested that ALDH þ cells spheroids and CD44 þ breast cancer cells. There were 12 in the MARY-X spheroids exhibited a stem cell-like overexpressed genes in common among MARY-X phenotype. spheroids and the human mammospheres and neuro- The presence of ALDH activity was corroborated by spheres. We found three overexpressed genes, C1R, the presence of similar levels of ALDH1 immunoreac- CHI3L1 and Notch 3, in common in all three groups. tivity within the lymphovascular emboli of MARY-X, which also exhibited concordant N3icd immunoreactiv- Observations made in MARY-X are applicable to human ity by dual colorimetric immunocytochemical methods cases of IBC of all molecular subclasses (Figure 4b). The MARY-X spheroids also demonstrated To test whether the MARY-X observations were increased Notch 3 signaling, by real-time RT–PCR, of applicable to actual human cases of IBC, we conducted HES-5 and HEY-2 compared with the levels exhibited Notch 3 and ALDH1 colocalization immunohistochem- by non-IBC lines and normal mammary epithelial cells istry studies of 25 cases of IBC, 25 cases of non-IBC and (Figure 4c). When the single cells of MARY-X were þ 5 normal breast tissues, of which frozen tissues had been sorted on the basis of ALDH , Notch 3 signaling was collected in the past and banked anonymously. In the further enhanced (Figure 4c). IBC cases, we also looked for evidence of Notch 3 downstream signaling by HES-5 and HEY-2 immuno- Transfection of N3icd into human mammary epithelial reactivities. The distribution of the IBC and the non-IBC cells (HMEC) induces Notch signaling subtypes is depicted in Table 4. Out of 25 IBC cases, 20 To examine this Notch 3 signaling further but from a (80%) expressed Notch 3 nuclear immunoreactivity and different perspective, we conducted a set of ‘reverse’ 23 (92%) expressed ALDH1 cytoplasmic immunoreac- experiments: transfection of N3icd into HMEC that tivity; there were no significant differences among the normally lack Notch 3 signaling, CD133 expression and subclasses of IBC for expression of either stem cell a Notch 3 addiction. Introduction of N3icd into HMEC marker (Table 4). The IBC cases with positive N3icd achieved a level of Notch 3 signaling equal to that in nuclear immunoreactivity failed to show equivalent MARY-X (Figure 5a). N3icd but not N1icd induced in N1icd or N4icd nuclear immunoreactivities (Figure 6). HMEC CD133 expression (Figures 5b and c) and Non-IBC cases, except for triple negative, were largely numerous other notch targets (Figure 5d) and morpho- negative for N3icd nuclear immunoreactivity and logical changes reflecting spheroidgenesis (anchorage- exhibited a lower overall percentage of cytoplasmic independent growth) (Figure 5e). Inhibition of notch ALDH1 positivity. The expression of N3icd and signaling by g-secretase inhibition or Notch 3 inter- ALDH1 in the cases overall was highly concordant ference in the MARY-X spheroids decreased CD133 (weighted Kappa (95% confidence interval) ¼ 0.76 (0.71, (Figure 5f) and abolished spheroidgenesis. 0.81)) (Table 4).

Gene expression profiles of MARY-X spheroids share common overexpressions with CD44 þ immunosorted cells Discussion and CD44 þ mammospheres/neurospheres The profiles of each of these acknow- Recent experimental evidence has supported the concept ledged models of stem cells revealed of cancer stem cells that retain the embryonal cell

Oncogene Notch 3 dependency of IBC embolus Y Xiao et al 293

Figure 4 (a) The ALDEFLUOR assay in MARY-X. This assay was applied to identify the ALDEFLUOR þ population exhibiting high aldehyde dehydrogenase (ALDH) enzymatic activity within the MARY-X spheroids. A single-cell suspension of disadhered cells was incubated with ALDEFLUOR substrate (BAAA) without DEAB and with DEAB, the specific inhibitor of ALDH, to establish the baseline fluorescence of these cells (left) and to define the ALDEFLUOR þ region (right). In all experiments, cells were first gated on PI-negative cells (viable cells). The ALDEFLUOR þ population is apparent (upper left panel) but abolished with the inhibitor (upper right panel). Approximately 23% of the cells of the MARY-X spheroids were ALDEFLUOR þ . The sorted ALDEFLUOR þ population was both tumorigenic in vivo and gave rise to secondary spheroids in vitro, which subsequently showed an enriched ALDEFLUOR þ population (lower left panel), again abolished with the inhibitor (lower right panel). However, the sorted ALDEFLUOR þ population, after secondary spheroidgenesis, consisted of a mixed cell population of both ALDEFLUOR þ and ALDEFLUORÀ cells. (b) Dual labeling colorimetric immunocytochemical studies using anti-human ALDH1 (red) and anti-N3icd (brown) in the MARY-X lymphovascular emboli confirmed dual nuclear (N3icd) and cytoplasmic (ALDH1) immunoreactivity. (c) Real-time RT–PCR studies in MARY-X spheroids of downstream Notch 3 activation: HES-5 and HEY-2 confirmed significantly increased signaling compared with that exhibited by non-IBC lines and normal mammary epithelial cells. Sorting of the ALDEFLUOR þ (ALDH þ ) population of the MARY-X spheroids revealed an even higher amount of Notch 3 signaling. properties of self-renewal and developmental potential Table 2 Selective tumorigenicity of ALDH cells þ (Bonnet and Dick, 1997; Rachel et al., 2001; Reya et al., Cell type Injected tumor Tumor incidence 2001; Kubota et al., 2003; Clark et al., 2004; Sheila et al., cell number within 3 months 2004; Laurie et al., 2005; Wicha et al., 2006). Notch ALDH þ 10 000 11/12 signaling is an example of a highly conserved pathway 2000 7/12 that is involved in multiple fundamental processes in 200 4/12 both embryogenesis and oncogenesis (Androutsellis- Theotokis et al., 2006; Bray, 2006; Chiba, 2006). ALDHÀ 10 000 0/12 2000 0/12 MARY-X grows as tight spheroids, whereas the cell 200 0/12 lines (breast cancer, and normal epithelial and myo- epithelial) to which it is compared normally grow as Abbreviation: ALDH, aldehyde dehydrogenase. monolayers. The differences observed with respect to

Oncogene Notch 3 dependency of IBC embolus Y Xiao et al 294

Figure 5 (a) Stable transfection of N3icd into HMEC followed by pooled clones selected for spheroidgenesis revealed N3icd expression by western blot at levels comparable to those expressed by MARY-X spheroids. (b) Analysis of these pooled clones revealed a dramatic increase in relative mRNA levels of CD133 by real-time RT–PCR. Stable transfection of N1icd or vehicle only exerted no effects. (c) Analysis of pooled clones selected for spheroidgenesis revealed a dramatic increase in CD133 protein levels. Transfection of N1icd or vehicle only exerted no effects. (d) The effects of N3icd transfection on many downstream targets of Notch, except for c-Myc, revealed strikingly increased levels of expression by RT real-time PCR. (e) Effects of N3icd transfection in HMEC revealed induction of spheroidgenesis (right panel) compared with vehicle-only transfection, which remained as a monolayer (left panel). (f) Inhibition of Notch 3 signaling in vitro with GSI-I resulted in decreased CD133 relative mRNA levels by RT real-time PCR.

Notch 3 and its downstream genes could simply be a structures derived from normal mammary stem cells, function of the three-dimensional spheroid growth vs that bear a resemblance to the spheroids of MARY-X, two-dimensional monolayer culture. For this reason we have been reported to show increased Notch signaling induced spheroidgenesis in each of the monolayer cell (Dontu et al., 2003, 2004), but the Notch signaling was lines. The induced spheroids showed only a minimal mainly Notch 1 and Notch 4. increase in Notch 3 signaling, especially in MCF-7 cells. Previous studies have revealed that Notch activation This has been noted previously (Sansone et al., is capable of promoting self-renewal of breast stem cells 2007a, b). Interestingly, mammospheres, which are and proliferation of early progenitor cells (Dontu et al.,

Oncogene Notch 3 dependency of IBC embolus Y Xiao et al 295 Table 3 Comparison of the gene expression profile of MARY-X spheroidsa with CD44 þ immunosortedb cells and CD44 þ mammospheres/ neurospheresc Gene symbol Gene name Gene function

(a) Overexpressed genes in common in MARY-X spheroids and CD44 þ human breast cancer cellsb ANGPT2 Angiopoietin 2 Angiogenesis G0S2 G0/G1switch 2 Cell cycle TNFSF13 Tumor necrosis factor (ligand) superfamily, member 13 Cytokine CCR1 Chemokine (C–C motif) receptor 1 Cytokine receptor CSF3 Colony stimulating factor 3 (granulocyte) Cytokine receptor FSTL1 Follistatin-like 1 Cytokine receptor MSN MOESIN Cytoskeleton BIN1 Bridging integrator 1 Development CHI3L1 Chitinase 3-like 1 (cartilage glycoprotein-39) Extracellular matrix COL4A2 Collagen, type IV, alpha 2 Extracellular matrix COL6A1 Collagen, type VI, alpha 1 Extracellular matrix SPON2 Spondin 2, extracellular matrix protein Extracellular matrix CRYAB Crystallin, alpha B Heat-shock like protein C1QL1 Complement component 1, q subcomponent-like 1 Immune system C1R Complement component 1, r subcomponent Immune system C1S Complement component 1, s subcomponent Immune system SRPX Sushi-repeat-containing protein, X-linked Cell adhesion UPK3B Uroplakin 3B Function unknown ITGA5 Integrin, alpha 5 (fibronectin receptor, alpha polypeptide) Membrane receptor EMP1 Epithelial membrane protein 1 Signal transduction NOTCH3 Notch homolog 3 Membrane receptor PROCR Protein C receptor, endothelial (EPCR) Signal transduction AKR1B1 Aldo-keto reductase family 1, member B1 (aldose reductase) Metabolism NT5E 50-Nucleotidase, ecto (CD73) Metabolism enzyme SAA2 Serum amyloid A2 Secretory protein SERPINE1 Serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1 Secretory peptidase inhibitor SLPI Secretory leukocyte peptidase inhibitor Secretory peptidase inhibitor DPYSL2 Dihydropyrimidinase-like 2 Signal transduction IGFBP6 Insulin-like growth factor binding protein 6 Signal transduction S100A9 S100 calcium binding protein A9 Signal transduction TP53I11 Tumor protein p53 inducible protein 11 Apoptosis ID2 Inhibitor of DNA binding 2, dominant negative helix–loop–helix protein Transcriptional factor ID3 Inhibitor of DNA binding 3, dominant negative helix–loop–helix protein Transcriptional factor ID4 Inhibitor of DNA binding 4, dominant negative helix–loop–helix protein Transcriptional factor SLC2A14 Solute carrier family 2 (facilitated glucose transporter), member 14 Transporter PDLIM4 PDZ and LIM domain 4 Function unknown

(b) Overexpressed genes in common in MARY-X spheroids and human mammospheres and neurospheresc FHL1 Four and a half LIM domains 1 Development CHI3L1 Chitinase 3-like 1 (cartilage glycoprotein-39) Extracellular matrix FBLN2 Fibulin 2 Extracellular matrix LAMC1 Laminin, gamma 1 (formerly LAMB2) Extracellular matrix C1R Complement component 1, r subcomponent Immune system NOTCH3 Notch homolog 3 Membrane receptor ADSS Adenylosuccinate synthase Metabolism enzyme DHCR7 7-Dehydrocholesterol reductase Metabolism enzyme PROS1 Protein S (alpha) Secretory peptidase inhibitor FXYD3 FXYD domain-containing ion transport regulator 3 Transporter SLC16A1 Solute carrier family 16 (monocarboxylic acid transporters), member 1 Transporter ABCA1 ATP-binding cassette, sub-family A (ABC1), member 1 Transporter

(c) Overexpressed genes in common in MARY-X spheroids, CD44 þ human breast cancer cells, human mammospheres and neurospheresc C1R Complement component 1, r subcomponent Immune system CHI3L1 Chitinase 3-like 1 (cartilage glycoprotein-39) Extracellular matrix Notch3 Notch homolog 3 Membrane receptor aGene expression profile was derived from an Affymetric chip of MARY-X spheroids compared with common breast carcinoma lines and normal mammary epithelial cells. bThe gene expression profile of CD44 þ immunosorted cells was obtained from a published study (Shipitsin et al., 2007). cThe gene expression profile of CD44 þ mammospheres/neurospheres was obtained from a published study (Dontu et al., 2003).

2004; Politi et al., 2006). Aberrant Notch signaling has Purow et al., 2005), and has also been seen in animal been observed in a variety of cancers, including breast models (Gallahan and Callahan, 1997; Dievart et al., (Zagouras et al., 1995; Miyamoto et al., 2003; Santagata 1999; Callahan and Egan, 2004; Hu et al., 2006). et al., 2004; Weng et al., 2004; Cuevas et al., 2005; Overexpression of active forms of murine Notch 1–4

Oncogene Notch 3 dependency of IBC embolus Y Xiao et al 296 Table 4 Notch 3 and ALDH1 IHC profile in the major molecular subtypes of IBC and non-IBCa Type IBC Non-IBC

Subtype Triple negative Her-2/neu þ ER þ Total Triple negative Her-2/neu þ ER þ Total (n ¼ 8) (n ¼ 11) (n ¼ 6) (n ¼ 25) (n ¼ 5) (n ¼ 6) (n ¼ 14) (n ¼ 25)

Marker ALDH1 þ 8 (100) 10 (91) 5 (83) 23 (92) 3 (60) 1 (17) 2 (14) 6 (24) Notch 3 þ 7 (88) 9 (82) 4 (67) 20 (80) 2 (40) 0 (0) 0 (0) 2 (08)

aAbsolute numbers of cases and (% positivity) are listed. Differences for both Notch3 and ALDH1 in IBC vs non-IBC were highly significant (Po0.001); differences among subclasses of IBC were not significant (P40.1). Strong agreement between Notch3 and ALDH1 was present (weighted kappa (95% confidence interval) ¼ 0.76 (0.71, 0.81)).

Figure 6 Studies of Notch 3, Notch 1 and Notch 4 immunoreactivities in the different molecular subtypes of human IBC, all showing LVI. Studies revealed the presence of significant nuclear immunoreactivities for Notch 3 but not Notch 1 or Notch 4, independent of molecular subtype. Triple-negative IBC (a) exhibited strong N3icd nuclear immunoreactivity (b), but weak-to-absent nuclear N1icd (c) and N4icd (d) immunoreactivities. Her-2/neu-positive IBC (e) similarly exhibited strong N3icd nuclear immunoreactivity (f), but weak-to-absent nuclear N1icd (g) and N4icd (h) immunoreactivities. Even ER-positive IBC (i) exhibited strong N3icd nuclear immunoreactivity (j) but weak-to-absent nuclear N1icd (k) and N4icd (l) immunoreactivities.

inhibited mammary differentiation and caused tumors Notch family members (Farnie et al., 2007). The MDA- in transgenic mice (Dievart et al., 1999). However, with MB-231 line expresses Notch 1 and Notch 4. The MDA- human Notch, the story has become more complicated. MB-468 line expresses Notch 1 (Stylianou et al., 2006) Notch, especially Notch 1, has been shown to signal in a and Notch 3 (Yamaguchi et al., 2008). The MCF-7 line number of different human breast cancer cell lines and expresses Notch 1 (Stylianou et al., 2006; Rizzo et al., to induce EMT through slug-mediated repression of 2008), Notch 3 (Yamaguchi et al., 2008) and Notch 4 E-cadherin (Stylianou et al., 2006; Leong et al., 2007). (Sun et al., 2005). However, Notch 3 selectively Some studies have revealed, however, that Notch was dominates in MARY-X. not sufficient for transformation when it was activated MARY-X also demonstrated a much greater activa- alone (Ayyanan et al., 2006) but needed to synergize tion of downstream Notch target genes than the other with additional molecular alterations to promote non-IBC lines. These target genes included HES-5, neoplastic transformation (Leong and Karsan, 2006). HEY-1, c-Myc, Deltex-1, NRARP and PBX1. The last- The cross-talk between Notch and other oncogenetic mentioned Notch target, PBX1, was recently identified signaling pathways could then form a feed-forward loop as a specific Notch 3 target gene in ovarian cancer (Park that promoted tumor cell growth (Girard et al., 1996; et al., 2008). But MCF-7 showed higher levels of HES-1 Palomero et al., 2006). and GATA3 expression than MARY-X. Clearly, not The overall pattern of Notch expression and its every Notch target has to be increased in MARY-X to downstream genes is indeed complicated. Clearly, other argue that overall MARY-X exhibits increased Notch 3 non-IBC and ductal carcinoma in situ cell lines express signaling. In fact, it has been argued that the cell type

Oncogene Notch 3 dependency of IBC embolus Y Xiao et al 297 and specific tumoral microenvironment may influence decreased expression of Notch 3 targets and profound which specific Notch target genes are induced by Notch apoptosis. It had been shown previously that Notch signaling (Iso et al., 2003). signaling can regulate apoptosis (Jhappan et al., 1992; The expression levels of the downstream Notch Hu et al., 2006; Klinakis et al., 2006). targets are probably a better indication of Notch Results of our in vitro studies were cleaner than our signaling than Notch levels per se. The literature also in vivo results that relied on the diffusion of a drug, in suggests that Notch 3 exhibits features distinct from this case, GSI-I. Intratumor injection studies generally other Notch family receptor members. For example, are imprecise and messy. We have attempted to Notch 3 contains a specialized TAD domain in the lentivirally transfect in vitro both a short hairpin RNA C-terminal region, which would be expected to prefer- Notch 3 construct as well as a DNAmastermind entially activate promoters with zinc-finger binding sites construct in MARY-X spheroids and see the effects of near a CSL-binding site, such as contained within the these maneuvers on the growing xenograft. DNAmas- HES-5 promoter (Bellavia et al., 2008). As one might termind is an inhibitor of notch signaling (Giraldez predict from this, MARY-X exhibited the highest level et al., 2002). However, emerging clones with either of HES-5 (148-fold greater than all other breast transfection did not exhibit sustained growth in vitro carcinoma cell lines tested). Furthermore, not all Notch and could not be used for an in vivo study. We are targets are specific for Notch. For example, HEY-1 can contemplating the use of a conditional knockdown be a downstream target of transforming growth factor system like cre/lox so that the effects of Notch 3 short beta as well. hairpin RNA or DNA mastermind can be studied The mechanisms of increased Notch 3 signaling in in vivo. MARY-X remain unknown. Some studies have sug- Our MARY-X model exhibited relatively high ex- gested that in some cancers increased Notch gene copy pression of ALDH enzymatically and ALDH1 immu- number can correlate with increased Notch signaling nocytochemically. ALDH1 has been shown to be a (Bray, 2006; Leong and Karsan, 2006; Park et al., 2006). marker for normal and malignant human mammary In our study, although we noted that the Notch 2 locus stem cells and a predictor of poor clinical outcome in (1p13-11) in Mary-X was moderately amplified on array human breast cancer (Ginestier et al., 2007). The sorted comparative genomic hybridization, the Notch 3 locus ALDH þ subpopulation of MARY-X exhibited enriched was not amplified. It had also been demonstrated that Notch 3 downstream signaling, increased spheroidgen- mutations within Notch receptors can activate signaling esis and tumorigenicity (Table 2). Interestingly, coloca- in the absence of a ligand (Sanchez-Irizarry et al., 2004; lization of Notch 3 and ALDH1 immunoreactivities Weng et al., 2004; Gordon et al., 2007). However, we did could be demonstrated within the lymphovascular not find these gain-of-function mutations in Notch 3 in emboli of MARY-X. Mary-X. Although the loss of Numb is thought to The gene expression profile of the MARY-X sphe- enable Notch signaling, the loss of Numb in MARY-X roids was compared with the published gene expression does not fully explain Notch 3 activation. As Notch profile of acknowledged models of mammary gland stem signaling begins with ligand binding, it was reasonable cells: CD44 þ mammospheres/neurospheres and CD44 þ for us to hypothesize that Notch activation in MARY-X immunosorted cells (Dontu et al., 2003; Shipitsin et al., is triggered by the close juxtaposition of ligand with 2007). Several genes in common, including Notch 3 receptors on neighboring cells within the tight spheroid (Sansone et al., 2007a, b), were overexpressed in all three aggregate. We did observe reasonable levels of expres- models (Table 3). One gene that was overexpressed only sion of the Notch ligand, Jagged-1, in the intact MARY- in the MARY-X spheroids was Il-6. It is interesting to X spheroids. However, Jagged-1 knockdown with postulate that interleukin-6 may be responsible for the siRNA did not decrease Notch 3 signaling and DLL1 clinical findings of IBC: a reddened, swollen and tender knockdown could not be technically achieved. These breast in the absence of inflammatory cells. findings all show that the mechanism of Notch 3 It is important to verify that the insights gained by activation in MARY-X spheroids is still a mystery. studying Notch 3 signaling and ALDH1 positivity in Another hypothesis we can consider to explain why MARY-X are applicable to human IBC. It could be the MARY-X spheroids exhibit Notch 3 rather than argued, however, that the stem cell-like properties of Notch 1 activation is that these spheroids are deficient in Notch 3 signaling and ALDH positivity exhibited by fucosyl transferases (Alpaugh et al., 2002a, b). Fucosyl MARY-X (as it is triple negative) could be a manifesta- transferases, specifically Pofut-1, are required for the tion of its basal origin rather than its IBC origin processing of Drosophila Notch and mammalian Notch (Bertucci et al., 2006; Stylianou et al., 2006; Yamaguchi 1 (Kopan and LLagan, 2009). One possibility then is et al., 2008; Sansone et al., 2007a, b). IBC is a that the MARY-X cells cannot adequately process heterogeneous disease and it has been reported that Notch 1, leading to a relative loss of Notch 1 signaling the same molecular subtypes present in non-IBC are and a compensatory increase in Notch 3 activity. also present in IBC (Bertucci et al., 2005). The stem cell As the effects of g-secretase inhibition were neither markers observed in MARY-X, namely Notch 3 and Notch 3 specific nor even g-secretase specific, we decided ALDH1, were not limited to triple-negative IBC, but to carry out specific Notch 3 knockdown experiments. were observed in ER-positive and Her-2/neu-positive The effects of Notch 3 knockdown were identical to IBC as well. The IBC stem cell-like phenotype trans- the effects of g-secretase inhibition in vitro, namely cended all the molecular subtypes of IBC.

Oncogene Notch 3 dependency of IBC embolus Y Xiao et al 298 The MARY-X spheroids, in particular, and IBC, in Inhibition of Notch3 signaling in vitro and in vivo general, resist chemotherapy and radiation-induced Inhibition of Notch 3 signaling in vitro and in vivo was apoptosis. The mechanisms underlying this resistance to performed on the MARY-X spheroids and xenograft using apoptosis remain unknown. However, the present ob- two approaches: RNA interference and secretase inhibition servations would suggest that the presence of Notch (see Supplementary Information). signaling in the intact spheroid might be one mechanism. The therapeutic efficacy of notch inhibitors has been Apoptosis measurements observed in other human cancer cell lines (Haruki et al., DNA was extracted from the MARY-X spheroids treated with 2005; Park et al., 2006). Although pharmacological GSI-I using the Suicide Track DNA Ladder isolation kit therapy with GSI-I was very effective in vitro, it was less (Calbiochem Inc, San Diego, CA, USA). A terminal deoxy- effective in vivo, probably because of diffusion-limited nucleotidyl transferase dUTP nick end labeling assay was also effects of either tissue solubility or half-life. These limi- performed (see Supplementary Information). tations might be circumvented by designing a more polar GSI. Notch has certainly become an attractive target for ALDEFLUOR assay anti-cancer drug development (Leong and Karsan, 2006; The ALDEFLUOR kit (StemCell Technologies, Durham, NC, Shih and Wang, 2007). In IBC we have demonstrated the USA) was applied to identify a cell population with high addiction of the lymphovascular embolus to Notch 3 ALDH enzymatic activity (see Supplementary Information). activation and suggest that targeting that pathway might provide a therapeutic advantage. N3icd transfection To examine the effects of N3icd on cells that do not express Notch 3 signaling, N3icd was transfected into HMEC and the Materials and methods effects on target gene expression and morphogenesis was examined (see Supplementary Information). Institutional approvals and human tissues Use of human tissues was approved by the Ohio State Human IBC cases University Cancer Institutional Review Board (IRB) under In all, 25 cases of IBC were studied immunocytochemically for protocol 2006C0042. Details of case selection and approvals Notch 3, Notch 1, Notch 4 and ALDH1 (see Supplementary are provided in Supplementary Information. Information).

Cell lines and xenograft studies MARY-X, established from an IBC patient, exhibited florid Statistical analysis LVI with tumor emboli formation in nude/severe combined Standard tests of significance were used (see Supplementary immunodeficiency mice (Alpaugh et al., 1999; Tomlinson Information). et al., 2001; Alpaugh and Barsky, 2002) (see Supplementary Information). Conflict of interest Gene expression profile studies and array comparative genomic hybridization Details of these methods (Jain et al., 2002; Krzywinski et al., The authors declare no conflict of interest. 2004) are provided in Supplementary Information.

RNA isolation, RT–PCR, real time PCR and sequencing studies Acknowledgements Total RNA was isolated using RNeasy and treated with DNase (Qiagen, Valencia, CA, USA). Details of RNA This study was supported by the American Airlines-Susan G isolation, PCR and sequencing studies are provided in Komen for the Cure Promise Grant KGO81287, Department Supplementary Information. of Defense Breast Cancer Research Program Grants BC990959, BC024258, BC053405, the Strategic Initiative Grant Program at Immunocytochemical, immunoprecipitation, cell fractionation Ohio State and The Donald A Senhauser Endowment. Figures 1a, and western blot studies b, c and 2e were, in part, reprinted from Am J Pathol 2008, Details of these methods are provided in Supplementary 173: 561–574 with permission from the American Society For Information. Investigative Pathology.

References

Alpaugh ML, Barsky SH. (2002). Reversible model of spheroid Alpaugh ML, Tomlinson JS, Kasraeian S, Barsky SH. formation allows for high efficiency of gene delivery ex vivo (2002a). Cooperative role of E-cadherin and sialyl-Lewis and accurate gene assessment in vivo. Hum Gene Ther 13: X/A-deficient MUC1 in the passive dissemination of tumor 1245–1258. emboli in inflammatory breast carcinoma. Oncogene 21: Alpaugh ML, Tomlinson JS, Shao ZM, Barsky SH. (1999). A novel 3631–3643. human xenograft model of inflammatory breast cancer. Cancer Res Alpaugh ML, Tomlinson JS, Ye Y, Barsky SH. (2002b). Relationship 59: 5079–5084. of sialyl-Lewis x/a underexpression and E-cadherin overexpression

Oncogene Notch 3 dependency of IBC embolus Y Xiao et al 299 in the lymphovascular embolus of inflammatory breast carcinoma. Gordon WR, Vardar-Ulu D, Histen G, Sanchez-Irizarry C, Aster JC, Am J Pathol 161: 619–628. Blacklow SC. (2007). Structural basis for autoinhibition of notch. Androutsellis-Theotokis A, Leker RR, Soldner F, Hoeppner DJ, Nat Struct Mol Biol 14: 295–300. Ravin R, Poser SW et al. (2006). Notch signaling regulates stem cell Haruki N, Kawaguchi KS, Eichenberger S, Massion PP, Olson S, numbers in vitro and in vivo. Nature 442: 823–826. Gonzalez A et al. (2005). Dominant-negative notch3 receptor Ayyanan A, Civenni G, Ciarloni L, Morel C, Mueller N, Lefort K inhibits mitogen-activated protein kinase pathway and the growth et al. (2006). Increased Wnt signaling triggers oncogenic conversion of human cancers. Cancer Res 65: 3555–3561. of human breast epithelial cells by a notch-dependent mechanism. Hu C, Dievart A, Lupien M, Calvo E, Tremblay G, Jolicoeur P. Proc Natl Acad Sci USA 103: 3799–3804. (2006). Overexpression of activated murine notch1 and notch3 in Barsky SH. (2003). Myoepithelial mRNA expression profiling reveals a transgenic mice blocks mammary gland development and induces common tumor suppressor phenotype. Exp Mol Pathol 74: 113–122. mammary tumors. Am J Pathol 168: 973–990. Bellavia D, Checquolo S, Campese AF, Felli MP, Gulino A, Screpanti Iso T, Kedes L, Hamamori Y. (2003). HES and HERP families: I. (2008). Notch3: from subtle structural differences to functional multiple effectors of the notch signaling pathway. J Cell Physiol 194: diversity. Oncogene 27: 5092–5098. 237–255. Bertucci F, Finetti P, Cervera N, Charafe-Jauffret E, Mamessier E, Jain AN, Tokuyasu TA, Snijders AM, Segraves R, Albertson DG, Adelaide J et al. (2006). Gene expression profiling shows medullary Pinkel D. (2002). Fully automatic quantification of microarray breast cancer is a subgroup of basal breast cancers. Cancer Res 66: image data. Genome Res 12: 325–332. 4636–4644. Jhappan C, Gallahan D, Stahle C, Chu E, Smith GH, Merlino G et al. Bertucci F, Finetti P, Rougemont J, Charafe-Jauffret, Cervera N, (1992). Expression of an activated Notch-related int-3 transgene Tarpin C et al. (2005). Gene expression profiling identifies molecular interferes with cell differentiation and induces neoplastic transfor- subtypes of inflammatory breast cancer. Cancer Res 65: 2170–2178. mation in mammary and salivary glands. Genes Dev 6: 345–355. Bonnet D, Dick J. (1997). Human acute myeloid leukemia is organized Klinakis A, Szabolcs M, Politi K, Kiaris H, Atavanis-Tsakonas S, as a hierarchy that originates from a primitive hematopoietic cell. Efstratiadis A. (2006). Myc is a Notch1 transcriptional target and a Nat Med 3: 730–737. requisite for Notch1-induced mammary tumorigenesis in mice. Proc Bray SJ. (2006). Notch signaling: a simple pathway becomes complex. Natl Acad Sci USA 103: 9262–9267. Nat Rev Mol Cell Biol 7: 678–689. Krzywinski M, Bosdet I, Smailus D, Chiu R, Mathewson C, Wye N Callahan R, Egan SE. (2004). Notch signaling in mammary development et al. (2004). A set of BAC clones spanning the . and oncogenesis. J Mammary Gland Biol Neoplasia 9: 145–163. Nucelic Acids Res 32: 3651–3660. Cariati M, Bennett-Britton TM, Pinder SE, Purushotham AD. (2005). Kopan R, Llagan MXG. (2009). The canonical notch signaling ‘Inflammatory’ breast cancer. Surg Oncol 14: 133–143. pathway: unfolding the activation mechanism. Cell 137: 216–233. Chiba S. (2006). Notch signaling in stem cell systems. Stem Cells 24: Kubota H, Avarbock MR, Brinster R. (2003). Spermatogonial stem 2437–2447. cells share some, but not all, phenotypic and functional characteri- Clark AT, Rodriguez RT, Bodnar MS. (2004). Human STELLAR, stics with other stem cells. Proc Natl Acad Sci USA 100: 6487–6492. NANOG, and GDF3 genes are expressed in pluripotent cells and Laurie AB, Tong IL, Megan FC, Sarah EJ, Stuart SL, Jacob PZ et al. map to 12p13, a hotspot for teratocarcinoma. Stem (2005). Core transcriptional regulatory circuitry in human embryo- Cells 22: 169–179. nic stem cells. Cell 122: 947–956. Cuevas IC, Slocum AL, Jun P, Costello JF, Bollen AW, Riggins GJ Leong KG, Karsan A. (2006). Recent insights into the role of notch et al. (2005). Meningioma transcript profiles reveal deregulated signaling in tumorigenesis. Blood 107: 2223–2233. notch signaling pathway. Cancer Res 65: 5070–5075. Leong KG, Niessen K, Kulic I, Raouf A, Eaves C, Pollet I et al. Curry CL, Reed LL, Golde TE, Miele L, Nickoloff BJ, Foreman KE. (2007). Jagged1-mediated notch activation induces epithelial- (2005). Gamma-secretase inhibitor blocks notch activation and induces to-mesenchymal transition through slug-induced repression of apoptosis in Kaposi’s tumor cells. Oncogene 24: 6333–6344. E-cadherin. J Exp Med 204: 2935–2948. Dievart A, Beaulieu N, Jolicoeur P. (1999). Involvement of notch1 in the Miyamoto Y, Maitra A, Ghosh B, Zechner U, Argani P, Iacobuzio- development of mouse mammary tumors. Oncogene 18: 5973–5981. Donahue CA et al. (2003). Notch mediates TGF alpha-induced Dontu G, Abdallah WM, Foley JM, Jackson KW, Michael FC, changes in epithelial differentiation during pancreatic tumori- Kawamura MJ et al. (2003). In vitro propagation and transcrip- genesis. Cancer Cell 3: 565–576. tional profiling of human mammary stem/progenitor cells. Genes Palangie T, Mosseri V, Mihura J, Campana F, Beuzeboc P, Dorval T Dev 17: 1253–1270. et al. (1994). Prognostic factors in inflammatory breast cancer and Dontu G, Jackson KW, McNicholas E, Kawamura MJ, Abdallah therapeutic implications. Eur J Cancer 30A: 921–927. WM, Wicha MS. (2004). Role of notch signaling in cell-fate Palomero T, Lim WK, Odom DT, Sulis ML, Real PJ, Margolin A determination of human mammary stem/progenitor cells. Breast et al. (2006). Notch1 directly regulates c-MYC and activates a feed- Cancer Res 6: R605–R615. forward-loop transcriptional network promoting leukemic cell Farnie G, Clarke RB, Spence K, Pinnock N, Brennan K, Anderson growth. Proc Natl Acad Sci USA 103: 18261–18266. NG et al. (2007). Novel cell culture technique for primary ductal Park JT, Li M, Nakayama K, Mao TL, Davidson B, Zhang Z et al. carcinoma in situ: role of notch and epidermal growth factor (2006). Notch 3 gene amplification in ovarian cancer. Cancer Res 66: receptor signaling pathways. J Natl Cancer Inst 99: 616–627. 6312–6318. Gallahan D, Callahan R. (1997). The mouse mammary tumor Park JT, Shih IM, Wang TL. (2008). Identification of Pbx1, a potential associated gene INT3 is a unique member of the NOTCH gene oncogene, as a notch 3 target gene in ovarian cancer. Cancer Res 68: family (NOTCH4). Oncogene 14: 1883–1890. 8852–8860. Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Politi K, Feirt N, Kitajewski J. (2006). Notch in mammary gland Brown M et al. (2007). ALDH1 is a marker of normal and development and breast cancer. Semin Cancer Biol 14: 341–347. malignant human mammary stem cells and a predictor of poor Purow BW, Haque RM, Noel MW, Su Q, Burdick MJ, Lee J et al. clinical outcome. Cell Stem Cell 1: 555–567. (2005). Expression of Notch-1 and its ligands, delta-like-1 and Giraldez AJ, Perez L, Cohen SM. (2002). A naturally occurring jagged-1, is critical for glioma cell survival and proliferation. Cancer alternative product of the mastermind locus that represses notch Res 65: 2353–2363. signaling. Mech Dev 115: 101–105. Rachel E, Maya S, Micha D, Ofra Y, Joseph IE, Nissim B. (2001). Girard L, Hanna Z, Beaulieu N, Hoemann CD, Simard C, Kozak CA Establishment of human embryonic stem cell-transfected clones et al. (1996). Frequent provirus insertional mutagenesis of notch1 in carrying a marker for undifferentiated cells. Curr Biol 11: 514–518. thymomas of MMTVD/myc transgenic mice suggests a collabora- Reya T, Morrison SJ, Clarke MF, Weissman IL. (2001). Stem cells, tion of c-myc and notch1 for oncogenesis. Genes Dev 10: 1930–1944. cancer, and cancer stem cells. Nature 414: 105–111.

Oncogene Notch 3 dependency of IBC embolus Y Xiao et al 300 Rizzo P, Osipo C, Foreman K, Golde T, Osborne B, Miele L. (2008). Sternlicht M, Safarians S, Calcaterra TC, Barsky SH. (1996). Rationale targeting of notch signaling in cancer. Oncogene 27: Establishment and characterization of a novel human myoepithelial 5124–5131. cell line and matrix-producing xenograft from a parotid basal cell Sanchez-Irizarry C, Carpenter AC, Weng AP, Pear WS, Aster JC, . In vitro Cell Dev Biol 32: 550–563. Blacklow SC. (2004). Notch subunit heterodimerization and Stylianou S, Clarke RB, Brennan K. (2006). Aberrant activation of prevention of ligand-independent proteolytic activation depend, notch signaling in human breast cancer. Cancer Res 66: 1517–1525. respectively, on a novel domain and the LNR repeats. Mol Cell Biol Sun Y, Lowther W, Kato K, Bianco C, Kenney N, Strizzi L et al. 24: 9265–9273. (2005). Notch4 intracellular domain binding to Smad3 and Sansone P, Storci G, Giovannini C, Pandolfi S, Pianetti S, Taffurelli M inhibition of the TGF-b signaling. Oncogene 24: 5365–5374. et al. (2007a). p66Shc/Notch-3 interplay controls self-renewal Tomlinson JS, Alpaugh ML, Barsky SH. (2001). An intact over- and hypoxia survival in human stem/progenitor cells of the expressed E-cadherin/alpha, beta-catenin axis characterizes the mammary gland expanded in vitro as mammospheres. Stem Cells lymphovascular emboli of inflammatory breast carcinoma. Cancer 25: 807–815. Res 61: 5231–5241. Sansone P, Storci G, Tavolari S, Guarnieri T, Giovannini C, Taffurelli Weng AP, Ferrando AA, Lee W, Morris JP, Silverman LB, Sanchez- M et al. (2007b). Il-6 triggers malignant features in mammospheres Irizarry C et al. (2004). Activating mutations of NOTCH1 in human from human ductal breast carcinoma and normal mammary gland. T cell acute lymphoblastic leukemia. Science 306: 269–271. J Clin Invest 117: 3988–4002. Wicha MS, Liu SL, Dontu G. (2006). Cancer stem cells: an old idea-a Santagata S, Demichelis F, Riva A, Varambally S, Hofer MD, Kutok paradigm shift. Cancer Res 66: 1883–1890. JL et al. (2004). JAGGED1 expression is associated with prostate Xiao Y, Yearsley K, Jones S, Barsky SH. (2008). The lymphovascular cancer metastasis and recurrence. Cancer Res 64: 6854–6857. embolus of inflammatory breast cancer expresses a stem cell-like Sheila K, Cynthia H, Clarke ID, Jeremy A, Jane B, Takuichiro H et al. phenotype. Am J Pathol 173: 561–574. (2004). Identification of human brain tumour initiating cells. Nature Yamaguchi N, Oyama T, Ito E, Satoh H, Azuma S, Hayashi M et al. 432: 396–401. (2008). Notch3 signaling pathway plays crucial roles in the Shih IeM, Wang TL. (2007). Notch signaling, gamma-secretase proliferation of erbB2-negative human breast cancer cells. Cancer inhibitors, and cancer therapy. Cancer Res 67: 1879–1882. Res 68: 1881–1888. Shipitsin M, Campbell LL, Argani P, Weremowicz S, Bloushtain- Zagouras P, Stifani S, Blaumueller CM, Carcangiu ML, Srtavanis- Qimron N, Yao J et al. (2007). Molecular definition of breast tumor Tsakonas A. (1995). Alterations in notch signaling in neoplastic heterogeneity. Cancer Cell 11: 259–273. lesions of the human cervix. Proc Natl Acad Sci USA 92: 6414–6418.

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene