Oxygen Concentration Determines the Biological Effects by hse16929


									    Priority Report

Oxygen Concentration Determines the Biological Effects of
NOTCH-1 Signaling in Adenocarcinoma of the Lung
                       1                                   1                           1                      2                       3
Yuanbin Chen, Melissa A. De Marco, Irene Graziani, Adi F. Gazdar, Peter R. Strack,
            1                      1
Lucio Miele, and Maurizio Bocchetta
 Oncology Institute, Loyola University Chicago, Maywood, Illinois; 2Hamon Center for Therapeutic Oncology Research and Simmons
Cancer Center, University of Texas Southwestern, Dallas, Texas; and 3Department of Cancer Biology and Therapeutics,
Merck & Co., Inc., Boston, Massachusetts

Abstract                                                                                   signaling is mediated by a family of transmembrane receptors
NOTCH signaling is an evolutionarily conserved signaling                                   (NOTCH-1 to NOTCH-4) and ligands (JAGGED-1 and JAGGED-2
                                                                                           and DELTA-like 1, 3, and 4; ref. 3). NOTCH receptors consist
pathway that regulates cell fate during development and
                                                                                           of a modular NH2-terminal extracellular subunit (NEC) non-
postnatal life. It has been increasingly linked to carcinogen-
esis, although its role in cancer seems to be highly context and                           covalently bound to the COOH-terminal transmembrane domain
tissue specific. Although NOTCH signaling is required for lung                             (NTM) subunit (4).
development, little is known about its role in lung cancer. In                                NOTCH ligands are single-pass transmembrane proteins that are
this study, we show that NOTCH signaling, as measured by the                               presented to NOTCH receptors by a neighboring cell. On ligand
;-secretase cleavage product NIC-1, is active in both normal                               binding, NOTCH receptors undergo proteolytic modifications in
                                                                                           the NTM, which makes them susceptible to final cleavage by a
human and lung tumor samples; however, downstream
NOTCH readouts (i.e., HES-1 and HES-5) are elevated in lung                                presenilin-1–dependent g-secretase (3). This process leads to the
tumors. Levels of NOTCH signaling components in primary                                    release of the activated form of NOTCH (intracellular NOTCH or
human lung cells reflect observations in tissue samples, yet                               NIC), which translocates to the nucleus where it modulates gene
lung tumor cell lines showed little NOTCH signaling. Because                               expression primarily by binding to ubiquitous transcription factor
oxygen concentrations are important in normal lung physio-                                 CBF-1 in humans (3). NOTCH target genes include several helix-
logy and lung tumors are hypoxic, the effect of low oxygen                                 loop-helix transcription factors collectively named Hairy/enhancer
on these lung tumor cell lines was evaluated. We found that                                of split (HES) and HEY (3). Many of these are negative trans-
hypoxia dramatically elevates NOTCH signaling (especially                                  criptional regulators that inhibit differentiation-inducing factors
NOTCH-1) in lung tumor cell lines and concomitantly                                        during development contributing to the maintenance of a
sensitizes them to inhibition via small-molecule ;-secretase                               precommitted cell state for proper interpretation of differentiation
inhibitors or NOTCH-1 RNA interference. ;-Secretase inhibitor–                             or proliferation stimuli (3). Knockout mouse studies have shown
induced apoptosis of lung tumor cells grown under hypoxic                                  that NOTCH signaling is required for lung development (5). During
                                                                                           postnatal life, NOTCH regulation of cell proliferation and apoptosis
conditions could be rescued by reintroduction of active
NOTCH-1. Our data strengthen the role of NOTCH in lung                                     is context dependent (6) and although in certain tissues NOTCH is
cancer and as a therapeutic target for the treatment of lung                               suggested to play a tumor suppressor role (6), NOTCH signaling
and other hypoxic tumor types. [Cancer Res 2007;67(17):7954–9]                             is increasingly linked to oncogenicity (7). In light of mounting
                                                                                           evidence for a role of NOTCH in cancer, little is known about
                                                                                           NOTCH in lung cancer. Although expression of constitutively
Introduction                                                                               active NOTCH-1 caused growth arrest in small cell lung cancer
                                                                                           cells (8), NOTCH-3 seems overexpressed in 30% to 40% of non–
   Lung cancers are the most common malignancies in the United
                                                                                           small cell lung cancer (NSCLC; ref. 9). Moreover, a t(15:19)
States, accounting for 31% of male and 26% of female cancer-
                                                                                           chromosomal translocation has been detected in some lung cancer
related deaths (1). Adenocarcinoma of the lung (ACL) represents
                                                                                           and derived cell lines, suggesting that NOTCH-3 may be an
f50% of all lung cancers (2), and 15% to 20% of ACL cases occur in
                                                                                           oncogene in NSCLC (10). Here, we have studied the expression
nonsmokers (2). Genetic data in nonsmokers (2) suggest that
                                                                                           levels and the biological effects of NOTCH signaling in ACL using
unidentified carcinogens may be one cause of ACL in the
                                                                                           cell lines and frozen tumor biopsies.
nonsmoking population. Despite treatment, the 5-year survival
rate for ACL is 15% (1); thus, it is imperative that novel targets and
therapies are identified.                                                                  Materials and Methods
   NOTCH signaling is an evolutionarily conserved pathway that
                                                                                              Cell culture, hypoxia, and ;-secretase inhibitor. Human bronchial
regulates critical cell fate decisions (3). In humans, NOTCH
                                                                                           epithelial cells 4F0439 and 4F0624 and small airway epithelial cells 3F1584,
                                                                                           4F0001, and 4F0715 were cultured as recommended (Cambrex). Human
                                                                                           lung fibroblasts MRC5 and CRL-7285 [American Type Culture Collection
                                                                                           (ATCC)] were cultured in DMEM with 10% fetal bovine serum (FBS). We
   Note: Supplementary data for this article are available at Cancer Research Online       used NSCLC cell lines of different histologic subtypes [i.e., H226 and HCC95
(http://cancerres.aacrjournals.org/).                                                      (squamous cell carcinomas); HCC1171 (large cell carcinoma); and H1395,
   Requests for reprints: Maurizio Bocchetta, Oncology Institute, Loyola University        H1755, HCC2374, A549, HCC827, H1299, and H2347 (adenocarcinomas)]. All
Chicago, Maywood, IL 60153. Phone: 708-327-3362; Fax: 708-327-3228; E-mail:
                                                                                           lines were from ATCC. Cancer cell lines were grown in RPMI 1640 with 10%
   I2007 American Association for Cancer Research.                                         FBS. All cells were fingerprinted using the GenePrint fluorescent STR
   doi:10.1158/0008-5472.CAN-07-1229                                                       system (Promega). Cells grown in hypoxia were maintained in chambers

Cancer Res 2007; 67: (17). September 1, 2007                                       7954                                                      www.aacrjournals.org
                                                                                                             NOTCH-1 Confers a Survival Signal to NSCLC

(Stem Cell Technologies) filled with certified 1% O2, 5% CO2, and 94% N2              peroxidase, goat polyclonal anti-AKT (C20), rabbit polyclonal anti-BAX
(Airgas North Central) at 37jC. Oxygen concentration was measured with                (N-20), and mouse monoclonal anti-BCL-2 (C-2; all from Santa Cruz
MiniOX1 oxygen meters (Mine Safety Appliances Co.).                                   Biotechnology). Rabbit polyclonal anti-NOTCH-2 was from Abcam. Rabbit
   We used the g-secretase inhibitor MRK003 (11). This compound was                   polyclonal anti-cleaved NOTCH-1 (Val1744), rabbit monoclonal anti–
dissolved in DMSO to make 40 mmol/L stock solutions.                                  phospho-AKT (Ser473), rabbit polyclonal anti–c-Jun NH2-terminal kinase
   Plasmids and lentiviral vectors. The pNIC-1 plasmid expresses                      (JNK), and rabbit monoclonal anti–phospho-JNK (T183/Y185) were from
NOTCH-1 NIC cloned into the BamHI and EcoRI sites of pcDNA3.0                         Cell Signaling. Mouse monoclonal anti–glyceraldehyde-3-phosphate dehy-
(Invitrogen; ref. 12). This same cDNA was inserted into pLenti4/TO/V5-                drogenase (GAPDH) was from Chemicon. One hundred micrograms of
DEST (tetracycline-inducible ViraPower T-Rex Lentiviral system, Invitrogen)           total cell lysates were run onto 10% SDS-PAGE and assayed by Western blot
to obtain pNIC1-DEST plasmid. pNIC-1 and control vectors were transfected             following standard procedures. Immunohistochemistry on frozen biopsies
by electroporation. The vector expressing a short hairpin targeting                   was done following standard procedures.
NOTCH-1 (shN1) was constructed by annealing two oligonucleotides                          Real-time reverse transcription-PCR. Total RNA from cultured cells
(5¶-GATCCTCGAGAGCGACCGCTGCCTGGATCCAAGATCAATGGTGA-                                     was extracted with the RNeasy Mini kit, whereas RNA from frozen biopsies
AGCAGATGCATTGATCTTGTCCAGGCAGCGGCTGCCCTCGAG-3¶ and                                     was extracted with RNeasy Micro kit (Qiagen). cDNA was synthesized with
5¶-AATTCTCGAGGGCAGCCGCTGCCTGGACAAGATCAATGCATCTG-                                      First-Strand cDNA synthesis kit (Fermentas). Quantitative real-time PCR
CTTCACCATTGATCTTGGATCCAGGCAGCGGTCGCTCTCGAG-3¶). The                                   was done with SYBR Green PCR Master Mix (Applied Biosystems) in an
resulting dsDNA was ligated into the BamHI and EcoRI sites of pENTR-                  ABI 7300 thermal cycler (Applied Biosystems). Primer sequences are
gus (Invitrogen). The shN1 sequence was transferred to lentiviral expression          listed in Supplementary Table S1. For each sample, a serial dilution of
vector pLenti4/TO/V5-DEST to generate pshN1-DEST following the                        cDNA template was measured in triplicate. Non–reverse transcription
manufacturer’s instructions. To generate tetracycline-inducible, stable cell          reactions served as controls. All measurements were normalized for 18S
lines, ACL cells were first infected with tetracycline regulator lentivirus.          rRNA. Comparison between groups were analyzed by Student’s t test, with
Stable tetracycline regulator–expressing cells were then infected with                a = 0.05.
pNIC1-DEST virus, pshN1-DEST virus, or pDEST virus, respectively.                         Cell viability assays. Cell viability was determined using the 3-(4,5-
Lentiviral packaging and selection of transduced cells were done as                   dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity assay kit
recommended (Invitrogen). Doxycycline-inducible expression of NIC-1 was               (Roche). The results were verified by trypan blue assays. Results were
verified by Western blot.                                                             expressed as mean F SD of three independent experiments. Comparison
   Antibodies. We used the following antibodies: rabbit polyclonal anti-              between control and g-secretase inhibitor treatment was analyzed by
NOTCH-1 (C-20), rabbit polyclonal anti-NOTCH-3 (M134), rabbit polyclonal              Student’s t test.
anti-NOTCH-4 (H-225), goat polyclonal anti-JAGGED-1 (C-20), goat                          Apoptosis was measured by Annexin V-phycoerythrin/7-aminoactino-
polyclonal anti-DELTX (C-20), mouse monoclonal anti–hypoxia-inducible                 mycin D (7-AAD) fluorescence-activated cell sorting (FACS; BD FACSCanto;
factor (HIF-1a; 28b), mouse monoclonal anti-p53 (DO-1)-horseradish                    Becton Dickinson).

Figure 1. NOTCH signaling pathway is down-regulated in NSCLC cell lines compared with normal airway epithelial cells. A, Western blot analysis of NOTCH-1
expression levels in primary airway epithelial cultures (AEC ), lung fibroblasts (Fibr. ), and NSCLC cell lines. GAPDH was used as a load control. B, expression of
NOTCH ligands and other NOTCH receptors in cultured cells analyzed by Western blot. C, expression of NOTCH-1 mRNA in cultured cells measured by real-time
reverse transcription (RT-PCR). D, expression of HES-5 mRNA in cultured cells. In (C ) and (D ), expression levels of the genes of interest were normalized to 18S rRNA
as described in Materials and Methods with expression set relative to BE1 (100 arbitrary units). Columns, relative expression (six individual measurements); bars,
SD. BE, bronchoepithelial cell (BE1, 4F0439; BE2, 4F0624); SA, small airway epithelial cell (SA1, 4F0001; SA2, 4F0715; SA3, 3F1584); FB, fibroblast (FB1, MRC5;
FB2, CRL-7285); SC, squamous cell carcinoma cell line (SC1, H226; SC2, HCC95); LC, large cell carcinoma cell line (LC1, HCC1171); AD, adenocarcinoma cell
line (AD1, H1395; AD2, H1755; AD3, HCC2374; AD4, A549; AD5, HCC827; AD6, H1299; AD7, H2347).

www.aacrjournals.org                                                            7955                          Cancer Res 2007; 67: (17). September 1, 2007
Cancer Research

Results and Discussion                                                                   expression of antiapoptotic proteins BCL-2 and MCL-1 and by
   We measured the expression levels of the four NOTCH receptors                         increased expression of tumor necrosis factor–related apoptosis-
and ligands JAGGED-1 and DELTA-1 and DELTA-4 in 10 NSCLC                                 inducing ligand (Supplementary Fig. S3). These results suggested
cell lines compared with primary bronchoepithelial, small airway                         that NOTCH-1 could play a tumor-suppressive role in ACL. We
epithelial, and lung fibroblast by Western blot (Fig. 1A and B). All                     therefore tested the hypothesis that NOTCH signaling is reduced in
NSCLCs showed reduced expression of several components of the                            ACL compared with normal lung by measuring HES-1 mRNA expres-
NOTCH signaling pathway compared with primary bronchoepithe-                             sion in matched tumor lung biopsies. The results showed that mRNA
lial and small airway epithelial. The seven ACL lines displayed                          expression of this NOTCH downstream transcription factor was
reduced or undetectable amounts of NOTCH-1 (Fig. 1A), with                               either the same or increased in ACL biopsies compared with normal
similar observations made at the mRNA level (Fig. 1C). The mRNA                          lung (Supplementary Fig. S4). Similar results were obtained when
expression levels of NOTCH downstream effector HES-5 reflected                           measuring the HES-5 mRNA expression levels (data not shown).
what was observed at the protein level with exception of two NSCLC                       This suggested that the activation status of the pathway does not
lines (Fig. 1D). Similar results were obtained measuring other                           correlate with the levels of NIC-1 detected by immunohistochemistry
NOTCH targets, such as HES-1 and HEY-1 (data not shown). Overall,                        and that other factors contribute to regulating NOTCH pathway
the expression levels of NOTCH components in ACL cell lines were                         activity in ACL. Lung cancers are significantly hypoxic compared
more similar to those of lung fibroblasts than to those of primary                       with normal lung epithelia (13). A recent study has shown that
lung epithelial cells, suggesting epithelial to mesenchymal transi-                      HIF-1a potentiates CBF-1–mediated transcription through direct
tion. Because primary cells and cell lines are cultured in different                     association with NOTCH-1/CBF-1 transcriptional complexes (14).
media, we tested whether this could explain differences in expres-                          Thus, we investigated the NOTCH signaling pathway in two
sion. We compared NOTCH-1 expression levels in one ACL cell line                         ACL lines (A549 and H1755) under hypoxia. We found that, in 1%
and in one SA culture grown in RPMI 1640 and the media used for                          oxygen, mRNA expression levels of the NOTCH downstream factors
primary cells. We detected no differences in NOTCH-1 levels                              HES-1, HEY-1, and HEY-2 were increased compared with those
(Supplementary Fig. S1). Because NOTCH-1 expression seemed to                            measured in the same cells cultured in normoxia (Fig. 3A). Under
be specifically lost in ACL cell lines, we focused on this receptor.                     hypoxia, HIF-1a increased as did the total amount of NOTCH-1;
We tested 11 matched frozen biopsies of ACL and normal lung                              however, steady-state levels of NIC-1 showed only a modest increase
by immunohistochemistry using a highly specific antibody recog-                          (Fig. 3B), suggesting rapid activation followed by degradation. Other
nizing NIC-1 (active NOTCH-1; Supplementary Fig. S2A). Lung                              components of the NOTCH signaling pathway seemed unaffected by
epithelia showed a strong nuclear staining for NIC-1, whereas ACL                        hypoxia (Fig. 3B). In agreement with protein levels, hypoxia induced
samples displayed reduced NIC-1 expression, with some tumor areas                        NOTCH-1 mRNA expression (Fig. 3C), anticipated to further support
showing undetectable staining (Supplementary Fig. S2B and C).                            NOTCH signaling because NOTCH-1 expression is under a positive
   When we expressed NIC-1 in ACL lines either through transient                         feedback loop (15). To understand the biological function of NOTCH
transfection or via a doxycycline-inducible lentiviral vector, NIC-1                     signaling in ACL lines grown in hypoxia, we inhibited the pathway
induced apoptosis (Fig. 2) mediated at least in part by reduced                          using the g-secretase inhibitor MRK-003 (11). When ACL lines were

Figure 2. Expression of constitutively active NOTCH-1 in ACL cells causes cell death. Representative experiment. A, forced expression of NIC-1 in H1755 cell line
at the specified hour after transfection of control plasmid (lanes 1–4 ) and plasmid containing NIC-1 (lanes 5–8 ). After transfection, 100 Ag of whole-cell lysates
were analyzed by Western blot analysis. B, Annexin V/7-AAD double staining and FACS analysis to measure the rate of apoptosis of cells shown in [A, lane 4 (left) and
lane 8 (right )]. According to our FACS specialist (Patricia Simms, Loyola University Chicago, Maywood, IL), the positioning of the quadrants in left and right has been set
as the most biologically relevant. This is due to the significantly different number of death cells between the two samples, which affects the binding of Annexin V.
C, Western blot analysis was done to detect the expression of NIC-1 on lentivirus transduction (see Materials and Methods and text). Note the low levels of NIC-1
expression. After a series of doxycycline dilution experiments (data not shown), we determined that such level of NIC-1 was comparable with that of primary
airway epithelial cells. D, viability of A549 cells transduced with the control retroviral vector (DEST ) or with the vector expressing NIC-1 (NIC-1). Cells (105) were
transduced with the respective vectors; 4 wks after doxycycline treatment, cells were stained with crystal violet. The results showed complete absence of cells in the
NIC-1–transduced population.

Cancer Res 2007; 67: (17). September 1, 2007                                      7956                                                          www.aacrjournals.org
                                                                                                   NOTCH-1 Confers a Survival Signal to NSCLC

Figure 3. Hypoxia up-regulates the NOTCH
signaling pathway in ACL cell lines. A, up-
regulation of mRNA expression of NOTCH
downstream target genes by hypoxia. Cells were
cultured in regular incubator (21% O2) or in
hypoxic chambers (1% O2) for 48 h. Then, mRNA
expression was measured by real-time RT-PCR.
The expression levels of the mRNAs of the genes
of interest were normalized to 18S rRNA. Relative
expression is calculated as normalized level of
each mRNA in hypoxia-treated cells compared
with normoxic conditions (the latter are set as
1 arbitrary unit). Columns, relative expression
(six independent measurements); bars, SD.
We show the results obtained in ACL cell lines
A549 and H1755. Similar results were obtained
in other two ACL cell lines (data not shown).
B, hypoxia specifically up-regulates NOTCH-1
protein expression. Western blot analysis was
done 48 h of either normoxic or hypoxic
incubation. Note that the protein levels of total
NOTCH-1, cleaved NOTCH-1, and HIF-1a are
increased by hypoxia, whereas other components
of the NOTCH signaling pathway seem unaffected
by hypoxia. C, hypoxia up-regulates NOTCH-1
mRNA expression levels. The relative expression
of mRNA was determined as described in (A).

exposed to increasing concentration of MRK-003, we observed dose-         explain part of the controversy about the role of NOTCH signaling in
dependent accumulation of NTM-1 and corresponding loss of NIC-1           cancer (6). Furthermore, our data suggested that the role of hypoxia
(Fig. 4A), confirming that NOTCH-1 cleavage was inhibited. As a           should be carefully considered when reexamining earlier studies
further control, we measured HES-1 mRNA expression levels in cells        showing tumor suppressor activities for NOTCH (e.g., refs. 17–19).
treated with MRK-003 under normoxia and hypoxia. We found that               The effects of NOTCH signaling are notoriously dose dependent
in hypoxia, MRK-003 treatment reduced the expression of HES-1             (7). It is likely that mechanisms have evolved whereby cells with
mRNA 685-fold (Fig. 4B), further confirming MRK-003–mediated              excessive NOTCH activity die, as is the case for other well-known
NOTCH signaling inhibition. MRK-003 treatment caused a potent             oncogenes. An excess of NIC is toxic for numerous cell types
apoptotic response in ACL cells as early as 48 h after treatment          in vitro, and cells use multiple mechanisms to maintain optimal
(Fig. 4C, middle). This apoptotic response was reduced if NIC-1 was       levels of NOTCH activity (7). In normal human keratinocytes,
reexpressed in these cells through doxycycline-inducible lentivirus       NOTCH-1 causes growth arrest at high levels and transformation
(Fig. 4C, top right), whereas doxycycline did not affect cells            at low levels (7). Under hypoxia, which potentiates the strength of
transduced with control lentivirus (Fig. 4C, bottom right). Impor-        NOTCH signaling, the low levels of NIC-1 protein may reflect rapid
tantly, MRK-003 treatment specifically killed ACL cells under hypoxia     activation-degradation. Alternatively, ACL cells may reduce total
but had no effect under normoxia (Fig. 4D). Expression of NIC-1 in        NOTCH protein expression to prevent hyperactivation and
ACL cells exposed to MRK-003 in hypoxia did not rescue the totality       maintain NOTCH signaling to levels compatible with life. Our data
of cells. This can be explained by the fact that g-secretase inhibitors   do not exclude a participation of NOTCH-3 in the pathogenesis of
prevent the activation of all four NOTCH receptors (16) or to off-        ACL. Indeed, cross-talk between NOTCH-1 and NOTCH-3 has been
target effects. Thus, we used a genetic strategy by down-regulating       described is some systems (7). Nonetheless, NOTCH-1 signaling
NOTCH-1 under hypoxia using a RNA interference (RNAi) approach.           seems essential for survival of ACL cells under hypoxia. Our data
A shN1 was cloned into the pLenti4/TO/V5-DEST and the ACL cell            suggest that targeting NOTCH signaling using g-secretase inhib-
line A549 was transduced with this construct. When transcription of       itors may be an attractive therapeutic strategy to treat highly lethal
NOTCH-1 targeting small hairpin RNA was induced by doxycycline            ACL and possibly other commonly hypoxic malignancies (20).
in this cell line under hypoxia, apoptosis resulted (Supplementary
Fig. S5), further confirming that NOTCH-1 signaling is required for
ACL cell survival under hypoxia.                                          Acknowledgments
   Our data show that the biological outcome of NOTCH signaling           Received 4/2/2007; revised 6/6/2007; accepted 7/5/2007.
                                                                             Grant support: American Cancer Society grant RSG-05-077 (M. Bocchetta).
in lung cancer depends on oxygen concentrations. When ACL cell               Conflict of interest: Merck Research Labs Boston.
lines were studied under standard tissue culture conditions, NOTCH-          The costs of publication of this article were defrayed in part by the payment of page
1 seemed to have tumor-suppressive activities, whereas under              charges. This article must therefore be hereby marked advertisement in accordance
                                                                          with 18 U.S.C. Section 1734 solely to indicate this fact.
hypoxia (a condition that more closely reflects tumor physiology;            We thank Merck & Co. for providing MRK-003 compound and Dr. Michele Carbone
ref. 13), NOTCH seems to be essential for cell survival. This may         for providing the initial support for this research.

www.aacrjournals.org                                                  7957                          Cancer Res 2007; 67: (17). September 1, 2007
Cancer Research

                                                                                                                     Figure 4. Inhibition of NOTCH signaling results in cell
                                                                                                                     death under hypoxia. A, treatment with the g-secretase
                                                                                                                     MRK-003 leads to accumulation of the total (uncleaved)
                                                                                                                     NOTCH-1 protein in a dose-dependent fashion.
                                                                                                                     Cells were treated with MRK003 (at the specified
                                                                                                                     concentrations) under either normoxia or hypoxia for 48 h.
                                                                                                                     The transmembrane portion of NOTCH-1 was then
                                                                                                                     detected using an antibody specific for the total
                                                                                                                     (uncleaved) NOTCH-1 protein. B, treatment with MRK003
                                                                                                                     reduces HES-1 mRNA expression. A549-TR-N1 cells were
                                                                                                                     treated with 40 Amol/L g-secretase inhibitors (GSI )
                                                                                                                     under hypoxia for 48 h. Real-time RT-PCR was done and
                                                                                                                     quantitated as described in Fig. 3A . C, NOTCH inhibition
                                                                                                                     through MRK-003 treatment causes ACL cells death under
                                                                                                                     hypoxia. Cells were treated with 40 Amol/L g-secretase
                                                                                                                     inhibitors for 48 h, and then cell viability was determined by
                                                                                                                     Annexin V/7-AAD staining. Note that MRK003 treatment
                                                                                                                     leads to a f3.6 increase in the amount of dead cells.
                                                                                                                     Constitutively active NOTCH-1 reexpression in
                                                                                                                     these cells leads to a f2-fold reduction of dead cells
                                                                                                                     despite MRK-003 treatment. Similar results were obtained
                                                                                                                     using a RNAi-based strategy (see Supplementary Fig. S6).
                                                                                                                     D, inhibition of NOTCH signaling results in cell death
                                                                                                                     specifically under hypoxia. ACL cell lines A549 and H1755
                                                                                                                     were treated with 40 Amol/L MRK003 and then cultured
                                                                                                                     either in normoxia or in hypoxia for 48 h. Cell viability was
                                                                                                                     measured by trypan blue staining. Columns, viability
                                                                                                                     (four independent experiments); bars, SD. Note that the
                                                                                                                     g-secretase inhibitor MRK-003 does not affect ACL cell
                                                                                                                     viability in normoxia. Dox, doxycycline.

References                                                    4. Egan SE, St-Pierre B, Leow CC. Notch receptors,           8. Sriuranpong V, Borges MW, Ravi RK, et al. Notch
                                                               partners, and regulators: from conserved domains to          signaling induces cell cycle arrest in small cell lung
1. American Cancer Society. Key statistics about lung          powerful functions. Curr Top Microbiol Immunol 1998;         cancer cells. Cancer Res 2001;61:3200–5.
 cancer. Available from: http://www.cancer.org/docroot/        228:273–324.                                                9. Haruki N, Kawaguchi KS, Eichenberger S, et al.
 CRI/content/CRI_2_4_1x_What_Are_the_Key_Statistics_          5. Collins BJ, Kleeberger W, Ball DW. Notch in lung           Dominant-negative Notch3 receptor inhibits mitogen-
 About_Lung_Cancer_15.asp?sitearea.                            development and lung cancer. Semin Cancer Biol 2004;         activated protein kinase pathway and the growth of
2. Minna JD, Gazdar AF, Sprang SR, Herz J. Cancer. A           14:357–64.                                                   human lung cancers. Cancer Res 2005;65:3555–61.
 bull’s eye for targeted lung cancer therapy. Science 2004;   6. Radtke F, Raj K. The role of Notch in tumorigenesis:      10. Dang PT, Gazdar AF, Virmani AK, et al. Chromosome
 304:1458–61.                                                  oncogene or tumour suppressor? Nat Rev Cancer 2003;3:        19 translocation, overexpression of Notch3, and human
3. Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch               756–67.                                                      lung cancer.
 signaling: cell fate control and signal integration in       7. Miele L, Golde T, Osborne B. Notch signaling in cancer.   11. Lewis HD, Leveridge M, Strack PR, et al. Apoptosis in
 development. Science 1999;284:770–6.                          Curr Mol Med 2006;6:905–18.                                  T cell acute lymphoblastic leukemia cells after cell cycle

Cancer Res 2007; 67: (17). September 1, 2007                                           7958                                                             www.aacrjournals.org
                                                                                                                   NOTCH-1 Confers a Survival Signal to NSCLC

 arrest induced by pharmacological inhibition of Notch     requires notch signaling to maintain the undifferentiat-      18. Talora C, Cialfi S, Segatto O, et al. Constitutively
 signaling. Chem Biol 2007;14:209–19.                      ed cell state. Dev Cell 2005;9:617–28.                         active Notch1 induces growth arrest of HPV-positive
12. Aster JC, Robertson ES, Hasserjian RP, Turner JR,     15. Heitzler P, Simpson P. The choice of cell fate in the       cervical cancer cells via separate signaling pathways.
 Kieff E, Sklar J. Oncogenic forms of NOTCH1 lacking       epidermis of Drosophila . Cell 1991;64:1083–92.                Exp Cell Res 2005;305:343–54.
 either the primary binding site for RBP-Jn or nuclear    16. Das I, Craig C, Funahashi Y, et al. Notch oncoproteins     19. Qi R, An H, Yu Y, et al. Notch1 signaling inhibits
 localization sequences retain the ability to associate    depend on g-secretase/presenilin activity for processing       growth of human hepatocellular carcinoma through
 with RBP-Jn and activate transcription. J Biol Chem       and function. J Biol Chem 2004;279:30771–80.                   induction of cell cycle arrest and apoptosis. Cancer Res
 1997;272:11336–43.                                       17. Kunnimalaiyaan M, Vaccaro AM, Ndiaye MA, Chen               2003;63:8323–9.
13. Chen DL, Dehdashti F. Advances in positron             H. Overexpression of the NOTCH1 intracellular domain          20. Nickoloff BJ, Osborne BA, Miele L. Notch signaling as
 emission tomographic imaging of lung cancer. Proc         inhibits cell proliferation and alters the neuroendocrine      a therapeutic target in cancer: a new approach to the
 Am Thorac Soc 2005;2:541–4, 512.                          phenotype of medullary thyroid cancer cells. J Biol            development of cell fate modifying agents. Oncogene
14. Gustafsson MV, Zheng X, Pereira T, et al. Hypoxia      Chem 2006;281:39819–30.                                        2003;22:6598–608.

www.aacrjournals.org                                                               7959                                Cancer Res 2007; 67: (17). September 1, 2007

To top