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Cell, Tumor, and Stem Cell Biology |
1 Wellcome Trust Centre for Cell Matrix Research, Faculty of Life Sciences, and 2 Cancer Research UK Department of Medical Oncology, University of Manchester, Christie Hospital NHS Trust, Manchester, United Kingdom
Requests for reprints: Keith Brennan, Wellcome Trust Centre for Cell Matrix Research, Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, United Kingdom. Phone: 44-161-275-1517; Fax: 44-161-275-1505; E-mail: keith.brennan{at}manchester.ac.uk.
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Abstract |
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–dependent Notch signaling is sufficient to transform normal breast epithelial cells and that the mechanism of transformation is most likely through the suppression of apoptosis. More significantly, we show that attenuation of Notch signaling reverts the transformed phenotype of human breast cancer cell lines, suggesting that inhibition of Notch signaling may be a therapeutic strategy for this disease. (Cancer Res 2006; 66(3): 1517-25) |
Introduction |
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The Notch pathway has been associated with several human cancers, including cervical and lung carcinoma, and neuroblastoma (1, 2). Most notably, it has been linked to T-cell acute lymphoblastic leukemia (T-ALL), where activating mutations within Notch1 have been identified in >50% of tumors (3). Furthermore, growth arrest occurs in several T-ALL–derived cell lines when Notch signaling is blocked, suggesting that modulating the Notch pathway may be an effective treatment strategy for this tumor type.
Within the murine mammary gland, aberrant activation of the Notch pathway leads to adenocarcinoma (4–8). In addition, overexpression of active forms of the Notch1 and Notch4 receptors transform both normal human and murine mammary epithelial cells (8–10). Despite this, the role Notch signaling plays in human breast cancer has received little attention. Initial hints of a role came from two studies showing increased expression of Notch1 protein in four breast tumors that overexpress H-ras (11) and expression of a truncated Notch4 mRNA encoding an active form in two breast cancer cell lines (9). A much clearer indication has come from two surveys examining Numb expression (12), a negative regulator of Notch pathway, and the mRNA levels of Notch receptors and their ligands (13) in breast carcinoma samples. Numb was lost in >50% of tumors due to ubiquitination and proteosomal degradation, and its levels were inversely correlated with grade and proliferation rate (12). Furthermore, the authors showed that colony formation was reduced by reintroducing Numb into epithelial cells derived from Numb-negative tumors. On the other hand, elevated levels of Notch1 and Jagged1 mRNA correlated with poor prognosis (13).
During normal mammalian development and homeostasis, Notch signaling plays an essential role in regulating cell fate, apoptosis, proliferation, and migration (14, 15). This is largely thought to be through the regulation of the expression of the Hes/Hey family of transcriptional repressors, although several other genes including p21 and cyclin D1 have been suggested to be direct targets (16, 17). The pathway is activated through the interaction of Notch receptors with Delta-like and Jagged ligands on neighboring cells (18). This leads to two proteolytic cleavages, which release the Notch intracellular domain (NICD) allowing it to enter the nucleus. Once within the nucleus, NICD interacts with RBP-J/CBF1 and Mastermind to generate a large transcriptional activator complex (19).
The loss of Numb and elevation of Notch1 and Jagged1 mRNA levels in breast carcinomas has suggested that increased Notch signaling may occur in this tumor type. However, this is not necessarily the case as Numb only limits signaling through the pathway and does not alter its activation by ligands, whereas elevated mRNA levels does not necessarily equate to increased protein expression. Here, we provide direct evidence that Notch signaling is activated in a wide variety of human breast carcinomas as we observe the accumulation of NICD and the expression of known downstream target genes. More significantly, we show that attenuation of Notch signaling reverts the transformed phenotype of human breast cancer cell lines, suggesting that inhibition of Notch signaling may be a therapeutic strategy for this disease. Our data also indicate that the transformation of normal breast epithelial cells caused by increased RBP-J–dependent Notch signaling is due to the repression of apoptosis.
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Materials and Methods |
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/VP16 fusion protein were obtained from Dr. Martin Baron (University of Manchester) and Dr. Tasuku Honjo (Kyoto University, Japan), respectively. cDNAs encoding murine Numb (Image clone 3991630), Puma (Image clone 6310857), and Noxa (Image clone 6517820) were obtained from the Medical Research Council Geneservice (Hinxton, Cambridge, United Kingdom). The full-length Numb cDNA was recloned into pcDNA3.1(–) vector to permit selection of cell lines stably expressing the protein. The complete Puma and Noxa open reading frames were cloned by PCR into the pEYFP-C1 vector to generate cDNAs encoding YFP/Puma and YFP/Noxa fusion proteins.
Cell culture and transfection conditions. Four normal (MCF 10F, MCF 10A, MTSV1-7, HB4A) and eight tumorigenic human mammary epithelial cell lines (Hs578T, MDA-MB-468, MCF7, ZR75T, CAL51, MDA-MB-231, SK-BR-3, PMC-42) were obtained from Dr. Neal Anderson (University of Manchester). MCF 10A and MCF 10F cells were grown in a 1:1 mixture of Ham's F12 medium and DMEM with 2 mmol/L L-glutamine. The medium was supplemented with 5% horse serum, 10 µg/mL insulin, 20 ng/mL epidermal growth factor (EGF), 500 ng/mL hydrocortisone, and 10 µg/mL cholera toxin. MTSV1-7 and HB4A cells were grown in DMEM with 2 mmol/L L-glutamine supplemented with 10% fetal bovine serum (FBS) and 5 µg/mL hydrocortisone. Hs578T, MDA-MB-468, MCF7, ZR75T, CAL51, MDA-MB-231, SK-BR-3, and PMC-42 cells were grown in DMEM with 2 mmol/L L-glutamine and 10% FBS. All cells were maintained in a humidified incubator at 37°C and 5% CO2. MCF 10A, MDA-MB-231, and MCF7 cells were transfected using the standard calcium phosphate coprecipitation method. Cell lines stably expressing RBP-J/VP16, NICD, or Numb were selected by adding 600 µg/mL geneticin (Invitrogen, San Diego, CA) to the normal medium.
Reverse transcription coupled PCR analysis. Cells were grown to near confluence in T-75cm2 flasks before cell lysis and RNA extraction using Stat-60 (Tel-Test) according to manufacturer's instructions. cDNA was prepared from 5 µg total RNA using Superscript II reverse transcriptase (Invitrogen). Control reactions were done without Superscript II. Prepared cDNA was subsequently treated with RNase H (Invitrogen) to remove RNA/DNA hybrids.
The PCR primers that specifically recognize Notch1-Notch4, Delta-like1, Delta-like3, and Delta-like4, and Jagged1 and Jagged2 were designed using Vector NTI suite 7.0 sequence analysis software (sequences available on request). The specificity of the primers was confirmed using plasmid cocktails that encoded either all four Notch, three Delta-like, or two Jagged proteins and control cocktails that lacked a specific plasmid, which corresponded to the primer pair being analyzed. To determine expression of the genes, one tenth of the prepared cDNA was used in subsequent PCRs containing 0.2 µmol/L of each primer, 100 µmol/L deoxynucleotide triphosphates, 1.5 mmol/L MgCl2 0.02 unit/µL of Taq DNA polymerase (Roche, Indianapolis, IN).
Immunohistochemistry and tissue samples. Approval to remove normal and tumorigenic human breast tissues during reduction mammoplasty and from pathologic samples respectively was obtained by Dr. Rob Clarke from the Manchester Local Research Ethics Committees. Subsequently written informed consent was obtained from the women before surgery. These tissue samples were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned on a microtome (Leica RM 2135). Tissue sections were mounted onto glass slides, dewaxed in xylene, and rehydrated in graded alcohols. Endogenous peroxidases were quenched with 0.2% H2O2 in methanol for 10 minutes. To recover antigen sites, sections were incubated in citrate buffer under high pressure (Tefal Delicio) for 60 seconds. Nonspecific binding was blocked with 10% normal serum diluted in PBS; the serum used was from the same species as the secondary antibody was raised in. Primary antibodies were added in PBS containing 1% normal serum, and the slides were incubated overnight at 4°C. Sections were then washed in PBS, incubated with secondary antibody (diluted 1:400 in PBS containing 1% normal serum), and washed again before incubation with avidin-biotin complex Vectastain reagent (Vector Laboratories, Burlingame, CA). Immunoreactivity was detected using diaminobenzidine staining (Sigma, St. Louis, MO). Sections were counterstained with 5% Harris-modified hematoxylin to reveal morphology and washed in warm tap water. Samples were then dehydrated and cleared in histoclear before mounting. Controls were prepared for all sections without primary antibody.
Primary antibodies used were as follows: anti-Notch1 (Rockland, Gilbertsville, PA and Santa Cruz Biotechnology, Santa Cruz, CA), anti-Notch3 (R&D Systems, Minneapolis, MN), anti-Jagged1 (Santa Cruz Biotechnology), anti-Jagged2 (Santa Cruz Biotechnology), and anti-Muc1 (DAKO, Carpinteria, CA). Secondary antibodies used were obtained from Jackson ImmunoResearch Laboratory (West Grove, PA) and DAKO.
Soft agar assays. A solution of 2.7% low melting MetaPhor agarose (FMC, Rockland, ME) in distilled water was prepared, autoclaved and cooled in a water bath at 55°C for 2 hours. For the base layer, the 2.7% agar solution was mixed with the appropriate culture medium to reach a final concentration of 0.9%. One milliliter of this mixture was transferred to each 35-mm dish and allowed to cool and solidify for 1 hour. The cells were diluted to a final concentration of either 15,000/mL (MCF 10A) or 25,000/mL (MCF7, MDA-MB-231) and 0.3% agar. Two milliliters of the cell suspensions were layered on top of the base layer and allowed to cool and solidify for 1 hour. Plates were transferred to a humidified incubator at 37°C and 5% CO2 and were grown for 5 weeks (MCF 10A) or 2 weeks (MCF7, MDA-MB-231). The day before counting, colonies were stained with 1 mL/well of nitroblue tetrazolium (0.5 mg/mL NBT in PBS). Colonies were counted using an Olympus IMT-2 inverted microscope.
Drug treatment and apoptosis analysis. MCF 10A cells were treated for 4 hours with 0.1 µmol/L staurosporine (Calbiochem, La Jolla, CA) or 16 hours with 0.75 µmol/L melephalan (Sigma), 0.37 µmol/L mitoxantrone (Sigma), or 0.1 µmol/L thapsigargin (Sigma). Apoptosis was monitored by nuclear fragmentation and cytochrome c release from mitochondria. This method detects accumulated apoptosis over the time period of the experiment and consequently background levels will seem higher than with techniques that measure the rate of apoptosis at any one particular time point. After treatment with the different drugs, both attached and detached cells were harvested, spun down onto positively charged slides using a Cytospin 2 (Shandon), washed with PBS, fixed with 4% paraformaldehyde in PBS, and permeabilized with 0.5% Tween in PBS. Cells were then washed with PBS and incubated with anti–cytochrome c primary antibody (PharMingen, San Diego, CA) diluted in PBS containing 10% horse serum for 1 hour at room temperature. Following further washing, cells were incubated with Cy3-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratory) and 0.1 µg/mL 4',6-diamidino-2-phenylindole diluted in the same buffer for 1 hour. The cells were washed again and mounted in DakoCytomation Fluorescent mounting medium. The percentage of cells exhibiting apoptosis, as judged by chromatin condensation, nuclear fragmentation, and cytochrome c release from mitochondria, was assessed using a Hamamatsu ORCA-ER digital camera on a Zeizz Axioplan2 microscope driven by Openlab image analysis software (Improvision, Lexington, MA).
Western blotting. Cells were washed twice in PBS, and total protein was extracted in boiling SDS lysis buffer [2 % SDS, 50 mmol/L Tris-HCl (pH 7.4)]. Frozen tissue samples were diced with a clean razor blade before protein extraction in twice the volume of boiling SDS lysis buffer. Genomic DNA was sheared by passing the lysate through a 23-gauge syringe needle 10 times. Total protein concentrations were determined using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Twenty micrograms of protein were resolved by SDS-PAGE, and the separated proteins were transferred to nitrocellulose membrane. The subsequent Western blots were blocked in TBS-T buffer [10 mmol/L Tris-HCl (pH 8), 150 mmol/L NaCl, 0.05% (v/v) Tween 20] containing 5% nonfat dry milk and were probed with the primary antibody in blocking buffer at 4°C overnight. Following washing in TBS-T buffer, the blots were incubated in secondary antibodies diluted in TBS-T at room temperature and developed after further washing with Super Signal West Pico and Femto Chemiluminescent substrates (Pierce).
Primary antibodies used were as follows: anti-cleaved Notch1 (Rockland), anti-Numb (Abcam, Cambridge, MA), anti-Noxa (Abcam), anti-Puma (Novus Biologicals, Littleton, CO), anti-cleaved Caspase3 (Cell Signaling Technology, Beverly, MA), anti-Desmoplakin (kind gift of Prof. David Garrod, University of Manchester), anti-Hey1 (Santa Cruz Biotechnology), anti-E-cadherin (BD Biosciences, San Jose, CA), anti-tubulin (kind gift of Prof. Keith Gull, University of Oxford), anti–phospho-c-Jun NH2-terminal kinase (JNK) pT183pY185 (Biosource, Camarillo, CA), anti–phopho-p53 pT81 (Cell Signaling Technology), anti-Keratin18 (NeoMarkers, Fremont, CA), anti-Actin (Sigma), and anti-focal adhesion kinase (kind gift of Dr. Andrew Ziemiecki, University of Berne, Switzerland). Secondary antibodies used were obtained from Jackson ImmunoResearch Laboratory and DAKO.
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Results |
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and the other via Deltex (20), we investigated whether signaling via RBP-J is sufficient to cause transformation. To do this, we generated MCF 10A cell lines stably expressing NICD or a RBP-J/VP16 fusion protein (data not shown); expression of the RBP-J/VP16 fusion protein specifically activates signaling through the RBP-J pathway (21). In addition, we generated a control cell line containing the empty vector. As expected, expression of RBP-J/VP16 or NICD led to the expression of target genes, including Hey1 and Hes5 (Fig. 2A; data not shown). In addition, the activation of RBP-J–dependent signaling led to growth beyond confluence (data not shown) and a marked change in cell shape (Fig. 2B). This change in cell shape may have occurred due to a cell fate change from luminal to myoepithelium. However, this is unlikely to be the case as there was no change in the expression of keratin 18 and smooth muscle actin, which are markers of these cell types (Fig. 2C; data not shown). In contrast, we did observe a marked reduction in E-cadherin expression (Fig. 2C), suggesting that the change in cell shape is due to a loss of cell-cell adhesion and possibly cellular transformation. To test this latter possibility, we plated the different cell lines in soft agar. Significantly more colonies developed in the soft agar from the MCF 10A/RBP-J and MCF 10A/NICD cell lines demonstrating that activation of RBP-J–dependent signaling did indeed lead to cellular transformation (Fig. 2D).
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and MCF 10A/NICD cells in the soft agar (data not shown). This suggested to us that these cell lines were resistant to apoptosis induced by extracellular matrix withdrawal or anoikis. It also raised the possibility that the transformation of MCF 10A caused by increased Notch signaling is due to inhibition of apoptosis. To examine whether MCF 10A/RBP-J and MCF 10A/NICD cell lines were more generally resistant to apoptosis, we treated the cell lines with the kinase inhibitor staurosporine, the DNA-damaging agents melphalan and mitoxantrone, and the Ca2+-mobilizing compound thapsigargin, which induce apoptosis through different mechanisms. Treatment of parental cells or the control cell line carrying the empty vector with any of the four compounds induced apoptosis (Fig. 3A). In contrast, apoptosis was not induced in MCF 10A/RBP-J and MCF 10A/NICD cells following treatment with staurosporine, melphalan, or mitoxantrone (Fig. 3A). Apoptosis did, however, occur following thapsigargin treatment, showing that the apoptotic machinery downstream of the mitochondria is functional in these cell lines (Fig. 3A).
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and MCF 10A/NICD cells, leading to a failure to express Noxa and Puma (Fig. 3B; data not shown). We also found that Puma and Noxa were expressed in response to staurosporine treatment (data not shown). As Puma and Noxa are largely responsible for p53-induced apoptosis, the failure to express these proteins could explain the absence of apoptosis in MCF 10A/RBP-J and MCF 10A/NICD cells following melephalan or mitoxantrone treatment. This is likely to be the case as the deliberate overexpressed YFP-Noxa and YFP-Puma fusion proteins led to apoptosis in all four cell lines (Fig. 3C).
Together, these experiments indicate that the p53-mediated response to cellular damage is abolished in normal breast epithelial cells by increased Notch signaling. Furthermore, they suggest that the transformed phenotype observed in MCF 10A/RBP-J and MCF 10A/NICD cells may be due to the attenuation of several apoptotic mechanisms.
Notch signaling is activated in human breast cancer. Because Notch signaling can transform normal human breast epithelial cells, we next sought evidence of unregulated signaling in breast cancer. We initially surveyed a panel of normal and tumor cell lines. The tumor cell lines are derived from a wide range of breast cancers and include HER2/erbB2–positive, EGF receptor (EGFR)–positive, p53-negative, and estrogen receptor (ER)–positive, and ER-negative cell lines. In all of the cancer cell lines, we observed a clear accumulation of NICD and overexpression of Hey1, showing increased Notch signaling (Fig. 4A). As previously reported, we also saw a loss of Numb expression (Fig. 4A).
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Attenuation of Notch signaling reverts the transformed phenotype of human breast cancer cell lines. The clear aberrant activation of Notch signaling in both breast cancer cell lines and tumor tissue samples suggests that it may play a significant role in tumor development. Consequently, inhibition of Notch signaling may revert the transformed phenotype of breast cancer cell lines. To test this, we overexpressed Numb, a negative regulator of the pathway (22), in the ER-positive cell line MCF7 and the metastatic cell line MDA-MB-231 (Fig. 5A; data not shown). In both cell lines, this prevented NICD accumulation (Fig. 5A; data not shown). In MCF7 cells, it also led to an accumulation of E-cadherin and a change in cell morphology so that the cells resemble normal breast epithelial cells (Fig. 5A and B). To determine whether the inhibition of Notch signaling had reverted the transformed phenotype of both cell lines, we plated them in soft agar. As expected, the parental cell line and control lines carrying empty vector formed multiple colonies, with nearly all cells forming one (Fig. 5C and D; data not shown). In contrast, <10% of MCF7/Numb and MDA-MB-231/Numb cells produced colonies (Fig. 5C and D; data not shown).
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Discussion |
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–dependent Notch signaling transforms breast epithelial cells by preventing apoptosis in response to many different stimuli, including anoikis and p53-mediated, drug-induced apoptosis. Together, this suggests that targeting Notch signaling may represent a novel therapeutic strategy for the treatment of breast cancer.
RBP-J–dependent Notch signaling is sufficient to transform breast epithelial cell lines. Several lines of evidence suggest that there is a second distinct intracellular signaling pathway, which requires Deltex function, downstream of the Notch receptors (20). However, the molecular mechanism of this pathway is not well understood. The pathway may be activated by the F3/Contactin family of GPI-linked ligands (23). Intracellularly, it has been suggested that signaling may occur through either sequestration of the transcriptional coactivator p300 (24) or inhibition of JNK (25).
Like the RBP-J signaling cascade, this Deltex-dependent pathway can be activated by the overexpression of NICD (24, 25). Consequently, this raises the question of whether one or both of these pathways are required for cellular transformation by the overexpression of NICD. To address this question, deleted forms of the Notch1 and Notch4 intracellular domains have been overexpressed in normal mammary epithelial cell lines to determine the regions required for transformation (8, 9). However, these experiments have yielded conflicting results. For example, the transformation of HC11 cells by Notch1 requires a Notch molecule that can activate both Deltex-dependent and RBP-J–dependent signaling (8). In contrast, overexpression of a deleted form of Notch4 protein that cannot apparently interact with RBP-J and therefore may only activate Deltex-dependent signaling is sufficient to transform MCF 10A cells (9). However, recent experiments have indicated that a similarly deleted Notch1 protein can interact with RBP-J in the presence of Mastermind, and that overexpression of this deleted form of Notch4 can weakly activate the RBP-J–dependent Hes1 promoter (5, 19).
RBP-J–dependent Notch signaling can be specifically activated by overexpressing a RBP-J/VP16 fusion protein (21). We find that this mimics the effects of overexpressing NICD in MCF 10A cells (Figs. 2 and 3). In parallel experiments, we also attempted to generate a cell line in which only the Deltex-dependent Notch pathway was activated by overexpressing the Deltex protein (data not shown; refs. 24, 25). We were unable to isolate this cell line, but we could identify geneticin-resistant individual cells. These cells contained a large number of lipid droplets, suggesting that they had undergone terminal differentiation. Together, this suggests that activation of RBP-J–dependent Notch signaling on its own is sufficient to cause transformation. However, it should be noted that NICD can regulate signaling through a number of other signaling pathways, including the Wnt and transforming growth factor-ß pathways (20, 26), and that this crosstalk may contribute to the transforming potential of the NICD protein.
Multiple apoptotic mechanisms are regulated by Notch signaling. Notch signaling has been shown to protect a wide range of cell types from apoptosis, including glial, neural, endothelial cells, cervical keratinocytes, and leukocytes (27–34). Furthermore, Notch can protect these cells against diverse apoptotic stimuli. For example, expression of an active form of Notch1 in cervical keratinocytes protects them against anoikis and apoptosis induced by p53 overexpression (33, 34), whereas its expression protects T cells, erythroleukemia, and endothelial cells from drug-induced apoptosis (27, 28, 30, 32). However, it is not clear that there is one single protective mechanism activated by which Notch signaling in these different systems. In cervical keratinocytes and T cells, overexpression of NICD leads to the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, which has been linked to several antiapoptotic mechanisms (32–34). These include phosphorylation and inactivation of the proapoptotic BH3-only protein Bad and phosphorylation of FOXO3, preventing it from inducing transcription of the BH3-only protein Bim (35). This activation of Akt may be mediated through the formation of a large protein complex consisting of a NICD, a Src family kinase and PI3K (32). In contrast, reduced JNK activity and up-regulation of the antiapoptotic proteins Bcl-2 and Bcl-xL are seen in erythroleukemia and endothelial cells when Notch signaling is increased (27, 28). The expression of Bcl-2 and Bcl-xL can directly prevent apoptosis by limiting the activity of the proapoptotic members of the Bcl-2 family (36). Reducing JNK signaling is also protective, as JNK can directly phosphorylate and regulate the activity of a number of proapoptotic Bcl-2 family members and p53 (37, 38). In the latter case, JNK phosphorylation increases p53 function.
The results of our experiments suggest that in breast epithelial cells Notch signaling may protect them through one single mechanism. Melephalan and mitoxantrone are both DNA-damaging agents and, as expected, increased p53 activity, leading to the expression of Puma and Noxa (Fig. 3). We also observed up-regulation of Puma and Noxa following staurosporine but not thaspigargin treatment (data not shown). This suggests that Notch signaling is able to regulate apoptosis that is specifically caused by Puma and Noxa expression but not other mechanisms. This possibility can also explain our failure to observe apoptosis in MCF 10A/RBP-J and MCF 10A/NICD cells plated in soft agar. Growth in soft agar induces apoptosis (or anoikis) due to the loss of survival signals from the surrounding extracellular matrix. This has been shown in both fibroblasts and squamous cell carcinoma cell lines to be due to the activation of p53, suggesting that anoikis, like apoptosis following DNA damage, is in part mediated by Puma and Noxa expression (39, 40).
Aberrant Notch signaling in human breast cancer. The accumulation of NICD in a wide range of breast cancers and breast cancer cell lines provides direct evidence that Notch signaling is activated in this tumor type (Fig. 4). The tumors analyzed include those which express ER and PR, respond well to tamoxifen treatment and have good clinical outcome, and tumors that lack ER, overexpress erbB2 or EGFR, and have poor prognosis. The data also suggest that Notch signaling may be activated at an early stage of tumor development as it occurs in such a wide range of tumors. This possibility is supported by the fact that we also found increased Notch signaling in the one ductal carcinoma in situ sample, a preinvasive form of breast cancer, in our collection of 20 tumor samples. Furthermore, we show that Notch may have a causative role in breast cancer as inhibition of the signaling pathway reverts the transformed phenotype of two very different breast cancer cell lines (Fig. 5).
Finally, the ability of Notch signaling to regulate several different apoptotic mechanisms makes its regulation an attractive possibility for cancer treatment. Many tumors become resistant to conventional chemotherapeutic drugs due to the inactivation of several apoptotic mechanisms present within normal cells. If these pathways can be reactivated, then the tumor cells can be made sensitive again to these drugs. However, the possibility of reactivating several different apoptotic mechanisms by regulating Notch signaling has a significant advantage.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Martin Baron, Tasuku Honjo, Keith Gull, Neal Anderson, David Garrod, and Andrew Ziemiecki for providing plasmids, cell lines, and antibodies for this work; Allison Frost for her technical assistance in generating the YFP/Puma and YFP/Noxa expression plasmids; Anthony Valentijn and Andrew Gilmore for their suggestions on the analysis of apoptosis; Charles Streuli and Anthony Brown for helpful comments on the article.
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Footnotes |
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Received 8/26/05. Revised 11/ 4/05. Accepted 12/ 5/05.
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 Y. Xiao, Y. Ye, K. Yearsley, S. Jones, and S. H. Barsky The Lymphovascular Embolus of Inflammatory Breast Cancer Expresses a Stem Cell-Like Phenotype Am. J. Pathol., August 1, 2008; 173(2): 561 - 574. [Abstract] [Full Text] [PDF]
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P. Rizzo, H. Miao, G. D'Souza, C. Osipo, J. Yun, H. Zhao, J. Mascarenhas, D. Wyatt, G. Antico, L. Hao, et al. Cross-talk between Notch and the Estrogen Receptor in Breast Cancer Suggests Novel Therapeutic Approaches Cancer Res., July 1, 2008; 68(13): 5226 - 5235. [Abstract] [Full Text] [PDF]
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C. W. Lee, C. M. Raskett, I. Prudovsky, and D. C. Altieri Molecular Dependence of Estrogen Receptor-Negative Breast Cancer on a Notch-Survivin Signaling Axis Cancer Res., July 1, 2008; 68(13): 5273 - 5281. [Abstract] [Full Text] [PDF]
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 C. Sahlgren, M. V. Gustafsson, S. Jin, L. Poellinger, and U. Lendahl Notch signaling mediates hypoxia-induced tumor cell migration and invasion PNAS, April 29, 2008; 105(17): 6392 - 6397. [Abstract] [Full Text] [PDF]
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 P. Rizzo, C. Osipo, K. Foreman, J. Yun, T. Golde, B. Osborne, and L. Miele Rational Targeting of Notch in Breast Cancer Am. Assoc. Cancer Res. Educ. Book, April 12, 2008; 2008(1): 491 - 495. [Abstract] [Full Text] [PDF]
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N. Yamaguchi, T. Oyama, E. Ito, H. Satoh, S. Azuma, M. Hayashi, K. Shimizu, R. Honma, Y. Yanagisawa, A. Nishikawa, et al. NOTCH3 Signaling Pathway Plays Crucial Roles in the Proliferation of ErbB2-Negative Human Breast Cancer Cells Cancer Res., March 15, 2008; 68(6): 1881 - 1888. [Abstract] [Full Text] [PDF]
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 K. G. Leong, K. Niessen, I. Kulic, A. Raouf, C. Eaves, I. Pollet, and A. Karsan Jagged1-mediated Notch activation induces epithelial-to-mesenchymal transition through Slug-induced repression of E-cadherin J. Exp. Med., November 26, 2007; 204(12): 2935 - 2948. [Abstract] [Full Text] [PDF]
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C. F. O'Neill, S. Urs, C. Cinelli, A. Lincoln, R. J. Nadeau, R. Leon, J. Toher, C. Mouta-Bellum, R. E. Friesel, and L. Liaw Notch2 Signaling Induces Apoptosis and Inhibits Human MDA-MB-231 Xenograft Growth Am. J. Pathol., September 1, 2007; 171(3): 1023 - 1036. [Abstract] [Full Text] [PDF]
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 V. Bolos, J. Grego-Bessa, and J. L. de la Pompa Notch Signaling in Development and Cancer Endocr. Rev., May 1, 2007; 28(3): 339 - 363. [Abstract] [Full Text] [PDF]
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 G. Farnie, R. B. Clarke, K. Spence, N. Pinnock, K. Brennan, N. G. Anderson, and N. J. Bundred Novel Cell Culture Technique for Primary Ductal Carcinoma In Situ: Role of Notch and Epidermal Growth Factor Receptor Signaling Pathways J Natl Cancer Inst, April 18, 2007; 99(8): 616 - 627. [Abstract] [Full Text] [PDF]
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G. Chapman, L. Liu, C. Sahlgren, C. Dahlqvist, and U. Lendahl High levels of Notch signaling down-regulate Numb and Numblike J. Cell Biol., November 20, 2006; 175(4): 535 - 540. [Abstract] [Full Text] [PDF]
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