The ANK repeats of Notch-4/Int3 activate NF-κB canonical pathway in the absence of Rbpj and causes mammary tumorigenesis

Transgenic mice expressing the Notch-4 intracellular domain (designated Int3) in the mammary gland have two phenotypes exhibited with 100% penetrance: arrest of mammary alveolar/lobular development and mammary tumorigenesis. Notch-4 signaling is mediated primarily through the interaction of Int3 with the transcription repressor/activator Rbpj. Interestingly, WAP-Int3/Rbpj knockout mice have normal mammary gland development but still developed mammary tumors with a slightly longer latency than the WAP-Int3 mice. Thus, Notch-induced mammary tumor development is Rbpj-independent. Here, we show that Int3 activates NF-κB in HC11 cells in absence of Rbpj through an association with the IKK signalosome. Int3 induced the canonical NF-κB activity and P50 phosphorylation in HC11 cells without altering the NF-κB2 pathway. The minimal domain within the Int3 protein required to activate NF-κB consists of the CDC10/Ankyrin (ANK) repeats domain. Treatment of WAP-Int3 tumor bearing mice with an IKK inhibitor resulted in tumor regression. In a soft agar assay, treatment of HC11-Int3 cells with P50-siRNA caused a significant decrease in colony formation. In addition, Wap-Int3/P50 knockout mice did not develop mammary tumors. This data indicates that the activation of NF-κB canonical signaling by Notch-4/Int3 is ANK repeats dependent, Rbpj-independent, and is mediated by IKK activation and P50 phosphorylation causing mammary tumorigenesis.

development and the development of mammary tumors 17,18 . Recently we have shown that these two phenotypes are a consequence of at least two different components of the Notch-4/Int3 signaling pathway 13 . Rbpj is a transcription repressor/activator that is a major partner in Notch signaling during development 19,20 . In the absence of Notch-ICD it acts as a transcription repressor. In the presence of Notch-ICD it binds to the Notch-ICD and becomes a transcription activator 20 . When we genetically crossed WAP-Int3 mice with Rbpj −/− knockout mice we developed a WAP-Int3/Rbpj −/− mouse strain that exhibited normal mammary gland development which could lactate. However, these mice still developed mammary tumors 13 . This is consistent with WAP-Int3 mammary tumor development being independent of Notch-4/Int3-Rbpj signaling.
The specific roles and the underlying mechanisms of Notch-4 signaling pathway on the malignant behavior of breast cancer are poorly understood. To date, the Rbpj-independent mechanism whereby Notch-4 promotes cell transformation is not clear. The NF-κB family is among the targets of activated Notch [21][22][23][24][25] . NF-κB is a family of transcription factors that play a critical role in regulating cell survival, inflammation, differentiation, and proliferation. The NF-κB subunits exist in inactive form in the cytoplasm due to binding to inhibitory proteins of the IκB family (IκBs). Upon stimulation, an IKB kinase (IKK) complex is activated by IKK kinases 26,27 . This activated IKK complex phosphorylates IκBs, leading to the degradation of IκB and release of NF-κB, enabling NF-κB subunits to translocate to the nucleus 26 .
Several reports documented the cross talk between Notch signaling and NF-κB. Song et al. 22 , showed that Notch-1 associates with IKKα and regulates IKK activity in cervical cancer cells. Also, Notch-1 regulates NF-κB activity in hematopoietic progenitor cells 28 . Notch augments NF-κB activity by facilitating its nuclear retention 24 . In Notch-3 transgenic mice, NF-κB is constitutively active due to Notch-3 interaction with IKKα 29 . The activation of NF-κB signaling pathway by Notch signaling 11,22,25 suggests that Notch tumorigenic signaling pathway may be mediated through the activation of NF-κB signaling pathway. Transcripts of NF-κB -regulated genes were found elevated in tumor initiating cells 30,31 and breast tumors, as compared to normal tissue 31,32 . Several studies revealed high levels of constitutive NF-κB activity in many breast cancer cells 30,[33][34][35] . Other studies showed NF-κB2 overexpression in breast and colon carcinomas [35][36][37] . NF-κB1 (P50) is overexpressed in colon, lung and breast cancers 35,37 . Therefore it has been considered a potential anti-cancer target 38,39 . In the current study we analyzed the Notch-4/ Int3-Rbpj-independent tumorigenic signaling pathway. The data shows that Notch-4/Int3 activation of NF-κB canonical signaling complex is Rbpj-independent. This activation is dependent on the CDC10/Ankyrin repeats in Int3 which induces mammary tumors but not blockage of mammary gland development.

Mice, experiment design and preparation of tissue for morphology and histology analysis.
WAP-Int3 female mice used in this study are maintained in our colony and have been described previously 18 . To delete P50 in mammary epithelial cells we crossed the WAP-Int3 mice with the P50 −/− mice (Jacksons lab, Bar Harbor, ME, USA). Tail DNA genotyping was performed by PCR for P50 −/− knockout using the following primers: forward, 5′-GCAAACCTGGGAATACTTCATGTGACTAAG-3′; and reverse 5′-ATAGGCAAGGTCAG AATGCACCAGAAGTCC-3′. DNA was amplified (94 C for 30 sec., 68 C for 30 sec., 72 C for 30 sec; 35 cycles, 72 C for 2 min) with a product size of 192 bp. To treat WAP-Int3-tumor-bearing mice with IMD-0354 39 , primary mammary tumors were palpated weekly. Tumor weight was determined as described previously 12 . When mammary tumors reached 400 mg, mice were euthanized and mammary tumors were collected as viable tissue. To reduce inter-tumor variations, nulliparous FVB/N female mice from our colony were used at 10 weeks of age and the inguinal mammary glands of these FVB/N mice served as the transplantation site of the primary WAP-Int3 viable tumor tissue. Viable tissue from each WAP-Int3 mammary tumor was placed in the inguinal mammary gland of two separate FVB/N mice. Once tumors reached the desired weight, one mouse received IMD-0354 and the matching control mouse received saline. Alzet mini osmotic pumps (Model 2001, pumping rate 1 µl/h, Durect Corp., Cupertino, CA, USA), implanted subcutaneously on the dorsal side of the mouse, were used to deliver a subcutaneous dose of IMD-0354 (5, 10 or 20 mg/mouse/week) or saline (control). Mammary whole mounts were prepared from the fourth abdominal gland, as previously described 13 . Mice were kept under standard laboratory conditions per the guidelines of the National Cancer Institute. All methods were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The National Cancer Institute (NCI) Animal Care and Use Committee approved all experimental procedures.
Cell culture and gene silencing. HC11 40,41 and HC11-Int3 mouse mammary epithelial cells were grown in RPMI medium. The HC11-Int3 cell line was generated as described previously 42 . Gene silencing using siRNA was done as previously described 43 Briefly, knockdown of proteins was achieved by the treatment of cells with siRNA (siGenome SMART pool, Dharmacon/Thermo Scientific Lafayette, CO) according to the manufactures protocol. The siRNA transient transfections were done with a final concentration of 50 nM in six-well plate or scaled up 10 cm-plate formats.
Evaluation of NF-κB activity. The Active Motif (Carlsbad, CA, USA) TransAM TM NF-κB family ELISA kit was used to facilitate the study of the NF-κB family transcription factors (P50, P52, P65 and Rel-B) in extracts of HC11 and HC11-Int3 cells treated with Int3-siRNA or Rbpj-siRNA according to the manufacturer's specifications. NF-κB activity was measured in nuclear protein extracts. The assay was performed according to the manufacturer's protocol as described previously 22 . Samples were analyzed using a microplate absorbance reader Bio-rad680 (Bio-Rad, Hercules, CA, USA).

Construction of Int3 deletion mutants.
The murine NICD-4 cDNA corresponding to a truncated Notch4/Int3 cDNA (residues 4382-6043) has been described 15,17 . An oligonucleotide encoding hemagglutinin (HA) tag was added to the 3′ end of NICD-4/Int3 cDNA. The HA-tagged NICD-4/Int3, RAM 23, CDC10/ANK, RAM-ANK and PB-PEST expression vectors were generated through PCR using Pfu polymerase from StrataGene (La Jolla, CA, USA) and cloned into the eukaryotic expression vector pcDNA3 as described previously 44 . The nucleotide sequence of all NICD-4/Int3 deletion plasmids have been determined and their sequences verified. The Int3 PCR deletion products were cloned into the NheI and XhoI site of the pcDNA3.1(−) (Invitrogen, Carlsbad, CA, USA). The protein encoded by the PCR product was synthesized using TNT Coupled Transcription/Translation System (Promega, Madison, WI, USA) according to the manufacturer's specifications and as described previously 45 . The synthesized protein was analyzed by electrophoresis on a 10% SDS-polyacrylamide gel (SDS-PAGE) and autoradiography. To measure the ability of the Int3 deletion mutants to activate NF-κB P50 signaling in HC11 cells containing an NF-κB reporter gene (Qiagen, Valencia, CA), cells were transiently transfected with expression vectors containing the Int3 deletions.
Colony formation in soft agar and luciferase assay. The soft agar assay utilizes an agar medium to assess the transformability of cells in an anchorage-independent manner. Soft agar assay was done as described previously 12 . For luciferase Assays, cells were grown in DMEM medium containing 10% fetal bovine Serum. Transfection and luciferase assays were conducted as previously described 12 . Cell migration and invasion assays. Cell invasion and migration across a basement membrane matrix were evaluated using a commercially available 12-well plate cell invasion/migration assay kit (Chemicon International, Temecula, CA, USA) and following the manufacturer's instructions 46 . Briefly, 2 × 10 5 cells were seeded into individual invasion chambers, and subsequently placed in 12-well plates containing serum (10% FBS) culture medium in the lower chamber and incubated for 24 and 48 h. Non-invading cells were carefully wiped off the upper surface of the filters with a swab. Cells that invaded and migrated through the matrix-containing membrane and reached the lower surface of the invasion chamber were stained with crystal violet and counted in at least three different high power fields using a light microscope.
Immunoblotting and Immunoprecipitation. Cells were harvested with trypsin-EDTA (1x) (Gibco BRL, San Francisco, CA, USA) and collected as a pellet by centrifugation at 4 °C. Cells were lysed and processed for Western blot analysis as described previously 43  Statistics. Quantitative values are represented as the mean of at least three experiments. All in vivo experiments were repeated at least three times, and at least five mice were used in each experiment. The statistical significance of the difference between groups was determined by the Wilcoxon rank sum test. Comparisons resulting in P-values ≤ 0.01 were considered statistically significant and identified in the figures with an asterisk ( * ) or (**).

Results
Involvement of NF-κB signaling in Notch induced tumorigenesis has been suggested from studies that demonstrate its activation in Notch-1 tumors 11,22 . To investigate whether Notch-4/Int3 signaling activates NF-κB and whether this activation is Rbpj-dependent or -independent, we used Notch-4-siRNA ( Fig. 1A) and Rbpj-siRNA ( Fig. 1B) to inhibit Notch-4 and Rbpj-RNA expression in HC11-Int3 cells after incubating these cells with TNF-α. TNF-α was added to stimulate the NF-κB pathway, however the used dose (5 ng) did not induce NF-κB in absence of Int3 (Fig. 1A). The respective siRNA did knock down the levels of the target mRNA (Fig. 1A,B). We investigated the effects of Notch4/Int3 on NF-κB activation/signaling by analyzing the effects of Int3 on activation of NF-κB promoter driven-reporter gene in HC11-Int3 cells in presence of increasing doses of Int3-siRNA (Fig. 1C). Int3 significantly induced NF-κB promoter-driven luciferase. This activity is suppressed in presence of Int3-siRNA in a dose dependent manner. Previous data showed that Int3 mammary tumorigenesis is Rbpj-independent 13 . To investigate if NF-κB -reporter activation is Rbpj-dependant/independent, HC11-Int3 cells were transfected with the NF-κB -reporter in presence of increasing concentrations of Rbpj-siRNA (Fig. 1C). Rbpj-siRNA treatment did not affect the NF-κB -driven luciferase activity, indicating that Notch4/Int3 activation of NF-κB is Rbpj-independent.
To verify that Int3 activation of NF-κB is Rbpj-independent and to investigate the activation of NF-κB canonical (P50 and RelA/P65) and non-canonical (P52 and RelB) pathway transcription factors by Int3; we quantified NF-κB transcription factors (P50, P52, P65 and Rel-B) activation when Int3 is knocked down with Int3-siRNA using TransAm NF-κB kit. Binding of P50 to NF-κB binding site decreased significantly in absence of Int3 in a dose dependent manner (Fig. 1D). Similarly, but to a lesser extent, P65 binding to the NF-κB binding site decreased (Fig. 1D). P52 and Rel-B binding to the NFκB binding site do not appear to be affected by Int3 expression. In addition, we performed experiments where HC11-Int3 cells are treated with increasing doses of Rbpj-siRNA. Reduction of Rbpj expression in HC11-Int3 cells did not affect the binding of P50 or P65 to the NF-κB binding site (Fig. 1E) indicating that the Int3/NFκB-P50 or P65 interaction/activation is Rbpj-independent. These results demonstrate that modulation of Int3 levels affects NF-κB activation, in Rbpj-independent fashion. Since P65 binding to the NF-κB binding site was not significant we have focused on Int3 interaction with the NF-κB1/P50. Although various homo-and heterodimeric combinations possibly function in the NF-κB/Rel family, the heterodimers of NF-κB1/P50 and RelA/P65 in the canonical pathway as well as the heterodimers of NF-κB2/ P52 and RelB in the non-canonical pathway are considered to be predominant components of the respective pathways 26,27,47 . To examine the effects of Notch-4-Int3 on the status of P105/P50 we employed P50 antibody. To test the specificity of the P50 antibody Western blots analysis of HC11 and HC11-Int3 nuclear and cytoplasmic extracts were reacted with the antibody. Major bands were detected at 105 KD and 50 KD ( Fig. 2A). Reactivity with these bands was blocked by the P50 blocking peptide (Fig. 2B). We have further characterized this interaction in the mammary glands of late pregnant FVB and WAP-Int3 mice, the P105 precursor of P50 is more abundant in the FVB mammary gland whereas in the WAP-Int3 gland total p50 and phosphorylated P50 are more abundant than in the FVB gland (Fig. 2C), indicating activation of P105 by Int3. This data is in agreement with previous studies 11,21,23,48,49 .
To obtain evidence that Int3 and P50 physically interact, an immune-precipitate (IP) and Western blot (wb) analysis was performed on TNF-α treated HC11-Int3-HA cell. cytoplasmic and nuclear protein extracts (Fig. 2D). Cytoplasmic and nuclear protein extracts were subjected to immunoprecipitation with HA antibody. Western blot of the immunoprecipitates were probed with the anti-P50 (Fig. 2D) antibody. Immunostaining analysis revealed a predominant Int3 binding to P50 in both cytoplasmic and nuclear extracts. Western blot analysis of HC11-Int3-HA cytoplasmic and nuclear extracts showed that the predominant species in the cytoplasmic fraction was P105 precursor and in the nuclear fraction the predominant species was P50 (Fig. 2E), indicating activation of P105 in presence of Int3.
The capability for anchorage-independent growth by tissue culture cells in soft agar is accepted as a measure of their tumor-inducing potential. We have shown previously 13,16 that the HC11 mouse mammary epithelial cells cannot grow in soft agar, whereas HC11-Int3 cells do have this capability 13 . To investigate the effect of NF-κB canonical pathway knockout on HC11-Int3 soft agar growth capability, we treated HC11 and HC11-Int3 cells with, P50-siRNA or Int3-siRNA (Fig. 2F). If the Int3/P50 complex is required to activate NF-κB pathway and confer the capability for Int3 anchorage-independent growth of HC11-Int3 cells, then blocking the expression of P50 should block growth in soft agar of these cells. As shown in Fig. 2F, soft agar growth significantly decreased in absence of P50 compared to HC11-Int3. Knocking down Rbpjk with siRNA has little or no effect on the ability of HC11-Int3 cells to form colonies in soft agar, indicating that Int3 colony formation is Rbpj-independent 13 . These results are compatible with the conclusion that the ability of HC11-Int3 cells for anchorage-independent growth in soft agar is independent of an Int3/Rbpj signaling pathway 13 and is NF-κB dependent. As a positive control we used Int3-siRNA to block Int3 expression in the HC11-Int3 cells and as expected colony formation was knocked down (Fig. 2F).
Others have shown that Notch-1 binds to the IKKα signalosome 22 . Therefore, to better understand the cross-talk between Noth-4-Int3 and NF-κB we investigated the binding of Int3 to IKKα and IKKβ. Protein extracts were prepared from HC11-HA and HC11-Int3-HA cells treated with either Int3-siRNA or Rbpj-siRNA. Immunoprecipitates were prepared using an antibody to the HA tag and electrophoretically separated on a polyacrylamide gel followed by blotting on a filter. The filters were reacted with an antibody to either IKKα or IKKβ. As shown in Fig. 3A (lane c) when HC11-Int3-HA cells were treated with Int3-siRNA the level of reactive IKKα and IKKβ was decreased to similar levels as seen in HC11 extracts (Fig. 3A, lane a). Whereas in extracts from cells treated with Rbpj-siRNA (Fig. 3A, lane d) the levels of bound IKKα and IKKβ were similar to that found in immunoprecipitates from untreated HC11-Int3-HA cells (Fig. 3A, lane b). We conclude that both IKKα and IKKβ interact with Int3 independent of Rbpj.
IKK signalosome phosphorylation of IκB leads to its proteosomal degradation and the translocation of P50/ P65 hetrodimers to the nucleus. IMD-0354 is a specific IκB kinase-β (IKKβ) inhibitor 39 that blocks NF-κB nuclear translocation. To ascertain whether members of the NF-κB family are involved in Notch-4/Int3-induced mammary tumorigenesis, first we treated HC11, HC11-Int3 with IMD-0354 in the presence and absence of Int3-siRNA (Fig. 3B). Blocking of NF-κB nuclear translocation by IMD-0354 significantly reduced Notch-4/Int3 transformation capacity to levels similar to those observed when HC11-Int3 cells were treated with Int3-siRNA. To further investigate the role of NF-κB in Int3 tumorigenesis, we undertook a dose-response study to determine the effects of IMD-0354 on WAP-Int3 tumor-bearing mice. Mice treated with a 20 mg/week dose showed significant reduction in the tumor weight compared to the saline treated WAP-Int3 tumor bearing mice (Fig. 3C). Therefore, a dose of 20 mg/week was used in all subsequent experiments. Histological analysis of hematoxylin In each assay 15,000 cells were plated in soft agar to measure their capability for anchorage independent growth. *P < 0.01 compared to HC11, **P < 0.01 compared to HC11-Int3. Each bar represents the mean ± SEM of a minimum of a duplicate of three independent experiments for each experimental group. and eosin (H&E)-stained liver and kidney sections from IMD-0354-treated mice did not show any evident morphological alterations such as necrosis or inflammatory changes due to toxicity (data not shown). These results demonstrate that WAP-Int3 mammary tumors are sensitive to IMD-0354 and further provide a strong rationale that the activation of one or more of the NF-κB target transcription proteins, in the context of Int3 signaling, likely contributes to tumor growth. To determine if this tumor regression by IKKβ inhibition is affected by Rbpj status, we treated WAP-Int3/Rbpj −/− tumor-bearing mice with 20 mg/week IMD-0354 for one week and monitored tumor weight (TW) daily. At the end of the treatment period, TW was significantly reduced (Fig. 3D) in all mice. When the pumps were depleted of IMD-0354 after day 6 ( Fig. 3E), there was a resumption of tumor growth, suggesting that continuous inhibition of IMD-0354 targets (IKKβ) is needed to effectively inhibit tumor growth. These results demonstrate that Notch4/Int3 mammary tumors are sensitive to IKKβ/NF-κB inhibition and further provide a strong evidence that the Notch-4/Int3 mammary tumorigenesis is NF-κB dependent and Rbpj independent.
To further define the minimal region of Int3 required for activating NF-κB, we have created a series of Int3-HA tagged deletion mutants that encode the RAM, ANK/CDC10, RAM-ANK and PB-PEST regions. Shown in Fig. 5A is a map of the regions of Int3 remaining in the deletion mutants. In Fig. 5B is shown the in vitro translated gene products expressed by the different deletion mutants. We have assayed HC11 cells transfected with expression vectors expressing these Int3 deletion mutants for NF-κB reporter activity (Fig. 5C). The HC11-RAM and HC11-PB-PEST cells were unable to activate the NF-κB reporter gene whereas HC11-ANK and HC11-RAM-ANK cells were capable of activating the reporter gene (Fig. 5C). In addition, HC11-ANK and HC11-RAM-ANK cells were capable of forming colonies in soft agar assay (Fig. 5D). Similarly, HC11-ANK and HC11-RAM-ANK cells showed significant increase in the number of invading cells in an invasion assay (Fig. 5E). These results are consistent with the Int3-ANK region being responsible for the activation of NF-κB.

Discussion
The WAP-Int3 transgenic mice exhibit two phenotypes with 100% penetrance 18 . One phenotype is the blockage of mammary gland lobulo-alveolar development resulting in an inability of WAP-Int3 females to lactate. We have previously shown that this phenotype is dependent on the interaction of the Notch4-ICD/Int3 with the transcription repressor/activator Rbpj 13 . The other phenotype is the development of mammary tumors in all females that is independent of Int3/Rbpj signaling 13 . The aim of the present study is to investigate and dissect the Notch/Rbpj independent signaling pathway. A limited microarray analysis of HC11-Int3 RNAs revealed high steady-state levels of IL1, IL6, IL-8, CXCL1 and CXCL2, as compared to control HC11 cells (our unpublished data). These genes are targets for the NF-κB transcription factors 51,52 . NF-κB pathways are important for normal mammary gland development as well as for breast cancer tumorigenesis and cancer stem cell biology 31,53 . In response to multiple stimuli, IKB kinase (IKK) complex is activated by IKK kinases 26 . Activated IKK complex consisting of two catalytic subunits (IKKα and IKKβ) and an IKKγ regulatory subunit in turn phosphorylates IκBs at two conserved serine residues in the N-terminal domain, leading to its proteasome mediated degradation. As a result, NF-κB translocates to the nucleus and induce a variety of genes encoding matrix metalloproteinases, inflammatory and chemotactic cytokines, and antiapoptotic proteins 26,47,54 . Several animal model studies linked NF-κB canonical and non-canonical pathway to inflammation and cancer. The canonical pathway is activated by IκB kinase (IKK α/β/γ complex), activation of RelA/P50 and the non-canonical pathway activated by NF-κB -inducing kinase (NIK) and IKKα, activation of RelB/ P52 pathways 26 .
A reporter assay showed that activation of NF-κB by Int3 is Rbpj-independent (Fig. 1C). The activation of NF-κB by Notch-4/Int3 indicates that in addition to a role for Notch in proliferation, differentiation, and survival of tumor cells 44,55 , Notch-4/Int3 may also have critical functions in the immune response, inflammation, viral infection, and apoptosis through control of NF-κB-mediated gene expression. Int3 enhanced P50, and to a lesser extent P65 (Rel-A), binding to the NF-κB DNA binding site but did not affect the binding of P52 or Rel-B to this site. High levels of P50 in presence of Notch-4/Int3, can alter the ratio of p50/p50 versus p65/p50 NF-κB dimers, thereby affecting the selectivity of the canonical NF-κB-dependent transcription. In addition, we have shown that Int3 binding to P50 is Rbpj-independent (Fig. 1E). Furthermore, knocking down P50 prevents anchorage independent growth by HC11-Int3 cells in soft agar (Fig. 2E) regardless of Rbpj presence. This suggests that the ability of Int3 to confer on HC11 cells the capability of anchorage independent growth is a consequence of Int3 activating the canonical NF-κB signaling pathway. In contrast, it has been reported that Notch-1-ICD interacts with the P50 subunit of NF-κB 56 , but not P65 22 , and blocks the formation of P50/ P65 heterodimers to NF-κB binding sites, thereby interfering with NF-κB-induced transcriptional regulation. Song et al. 22 have shown that Notch-1 activation of NF-κB in CaSki cells is Rbpj dependent. The crosstalk between NF-κB and Notch pathways is quite Others have shown that Notch-ICD Interacts with NF-κB-P50 and the IKK signalosome 22 . Therefore, our observation that Int3 binds to IKKα/IKKβ raised the possibility that inhibition of IKK could inhibit the ability of Int3 to induce tumor growth. Therefore, we treated mice bearing WAP-Int3 tumors with the IκB kinase-β (IKKβ) inhibitor IMD-0354 39 that blocks NF-κB nuclear translocation 39,57 . In each case using 20 mg/kg/day the tumors totally regressed and the effect of the drug was independent of presence or absence of Rbpj. When the drug was removed the tumor grew back. These results, along with the inhibition of HC11-Int3 cells anchorage independent growth in soft agar by P50-siRNA, are consistent with the hypothesis that it is the canonical NF-κB signaling that Int3 activates during mammary tumorigenesis. To test this hypothesis, we have developed a WAP-Int3/NF-κB-P50 −/− mouse strain. It should be noted that the parental NF-κB-P50 −/− mouse strain has no mammary gland developmental phenotype 50 . Like WAP-Int3 mice, WAP-Int3/NF-κB-P50 −/− can't lactate due to the absence of lobulo-alveolar development that we have previously shown to be a consequence of Int3/Rbpj signaling 13 . 80% of WAP-Int3 females develop tumors after the second pregnancy 13 , however, WAP-Int3/NF-κB-P50 −/− mice did not develop mammary tumors after 4-5 pregnancies (Table 1). These results are consistent with the conclusion that the consequence of the Int3/NF-κB1-P50 interaction is mouse mammary tumorigenesis. Interestingly, we were also able to obtain two WAP-Int3/NF-κB-P50 −/− /Rbpj −/− females which could lactate and did not develop mammary tumors i.e. no phenotype (data not shown). Again, consistent with the conclusion that Int3/Rbpj signaling affects mammary gland development and Int3/NF-κB canonical signaling results in mammary tumorigenesis.
Previously MacKenzie et al. demonstrated that the Notch-4 induced inhibition of endothelial sprouting requires the ANK repeats in the ICD-for Rbpj dependent and independent signaling 58 . Subsequently we showed that the Tacc3 protein binds specifically to the Notch-4 ANK repeats and inhibits Notch-4/Rbpj signaling 59 . Having established that Int3 binds to P50 we have genetically dissected, using deletion analysis, the region(s) of Int3 that are responsible for binding to P50. We found that Int3-ANK and RAM-ANK, but not RAM or PB-PEST, activates the expression of NF-κB reporter gene in HC11 cells. We conclude that only the ANK region is required for activation of NF-κB, since RAM by itself did not induce NF-κB reporter activity. Dumont E., et al., showed that the ANK region of Notch1-ICD is required for neoplastic transformation in collaboration with Adenovirus E1A protein and is independent of transcription activation by Rbpj signaling 60 . In contrast Wang et al. reported that Notch-1 inhibits NF-κB mediated gene expression through a region N-terminal to the ANK repeat, namely, aa 1773-1881 56 . The differences between Notch-1 and Notch-4 regions that bind to NF-κB may reflect the divergence between the amino acid sequences of their ICDs.
Several reports have described the crosstalk between NF-κB and Notch. Most of these studies investigated interaction between Notch-1 and NF-κB. The reports have been conflicting, numerous studies indicated that Notch receptors activate NF-κB. This activation involves a group of components along the signal transduction pathway at multiple levels. For example, Notch activates NF-κB by increasing transcription expression of NF-κB subunits and NF-κB dependent genes 24,28,61 . Two different mechanisms have been elucidated in the physical interaction of Notch with NF-κB. First, physical interaction between Notch and P50/Rel-A 21,24 , trapping activated NF-κB in the nucleus. Second, interaction with the components of the signalosome. Recently, it has been shown that overexpression of Notch-1-ICD activates NF-κB by interacting with the IKKα/β signalosome and enhancing IKKα/β kinase activity in Caski cells 22 . Notch-3 can increase the association between IKKα and NIK, resulting in P50/P65 heterodimer nuclear entry and subsequent cell proliferation 62 . Notch signaling activation mediated by Jagged-1 is reported to induce IKK kinase activity in human keratinocytes 63 and murine erythroleukemia 64 . In terms of Notch-4-Int3, it activates NF-κB by both mechanisms, interaction with P50 and interaction with the signalosome.
Several factors drive breast cancers to estrogen independence including down regulation of estrogen receptor (ER) expression, modulation of regulation of signal transduction pathways and ER mutations. It has been suggested that in ER independent breast tumors NF-κB expression contributes to a highly invasive and metastatic tumor that is chemotherapy resistant [64][65][66] . NF-κB was overexpressed in a majority of ER negative primary breast tumors and breast cancer cell lines compared to ER positive breast tumors and tumor cell lines 64,67,68 . Nakshatri et al. 64 proposed that breast cancers that lack functional ER overexpress NF-κB-regulated genes. Also in the ERα positive cells, estrogen inhibited Notch signaling 5 . Indicating a cross-talk between ER, Notch and NF-κB signaling. This proposal is supported by our observations in the MMTV-Int3 transgenic mice, where endogenous ovarian estrogen secretion in the post-pubertal MMTV-Int3 mice blocked Int3 inhibitory effects on normal mammary development, namely ductal elongation 13,19 . Because of these considerations NF-κB has been regarded as a potential target for therapeutic intervention of ER negative breast cancer 65,69,70 . This inhibition can be achieved by blocking Notch signaling in the ER negative breast cancers.
Inhibition of NF-κB enhanced the sensitivity of tumor cells to apoptosis induced by chemo drugs and radiation [71][72][73] . However, there is no specific NIK inhibitor for pharmaceutical use yet. Because of its important role in both canonical and non-canonical NF-κB pathways in the immunity and the severe phenotype of NIK-deficient mice 74 , inhibiting NIK may also cause severe side effects. A good alternative would be the agents that targets Notch signaling, such as tyrosine kinase inhibitors (Gleevec). Previously we showed that expression of activated Notch-4 -ICD (Int3) led to the up regulation of c-Kit and PDGFR-RNA expression 12 . Further, we showed that treating cells with the c-Kit/PDGR inhibitor Gleevec, leads to the activation of GSK3β 43 (non-phosphorylated form of GSK3β). We have demonstrated that activated GSK3β was associated with the phosphorylation of Int3, its ubiquitination and subsequent degradation in the proteasomes 43 . Interestingly Foltz at al., have reported that Notch-1-ICD is also a target for GSK3β phosphorylation 75 , however in this case the phosphorylated Notch-1-ICD is stabilized from proteasome-mediated degradation. This is consistent with the differential modulation of some components of Notch signaling by GSK3β. Demarchi et al., have reported that GSK3β also regulates the stability of the NF-κB-P50 precursor P105 76 . In the absence of GSK3β activity or the GSK3β protein, P105 is constitutively processed to P50. Phosphorylation of P105 by GSK3β and subsequently by IKK leads to proteasome-mediated degradation of P105 77,78 . Using this model (Fig. 6) we propose that Notch-4-Int3 activates c-Kit and PDGFR, leading to activation of GSK3β and NF-κB signaling. In presence of Gleevec, GSK3β is dephosphorylated and as a result it will phosphorylate Int3 leading to its ubiquitination and degradation 43 . As a result, the ability of Int3 to induce P105 processing and NF-κB activation is diminished. Thus, Gleevec treatment of a Notch-4-ICD or Int3 positive mammary tumors/estrogen negative would be expected to be associated with proteasome-mediated degradation of P105, inhibition of NF-κB canonical pathway and subsequent remission of mammary tumor growth.