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JPET #215418
1
TD-19, an erlotinib derivative, induces EGFR wild-type NSCLC apoptosis
through CIP2A-mediated pathway
Ting-Ting Chao, Cheng-Yi Wang, Chih-Cheng Lai, Yen-Lin Chen, Yi-Ting Tsai,
Pao-Tzu Chen, Hen-I Lin, Yuh-Chin T. Huang, Chung-Wai Shiau, Chong-Jen Yu,
Kuen-Feng Chen
Medical Research Center (TTC, CYW, YTT, PTC), Department of Internal Medicine
(CYW, HIL), Department of Pathology (YLC), Cardinal Tien Hospital, School of
Medicine, Fu Jen Catholic University, New Taipei City, Taiwan; Graduate Institute of
Clinical Medicine (CYW), College of Medicine, National Taiwan University, Taipei,
Taiwan; Department of Intensive Care Medicine (CCL), Chi Mei Medical Center,
Liouying, Tainan, Taiwan; Department of Medicine (YCH), Duke University Medical
Center, Durham, North Carolina, USA; Institute of Biopharmaceutical Sciences
(CWS), National Yang-Ming University, Taipei, Taiwan; and Department of Internal
Medicine (CJY), Department of Medical Research (KFC), National Center of
Excellence for Clinical Trial and Research (KFC), National Taiwan University
Hospital and National Taiwan University, Taipei, Taiwan
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Running Title: TD-19 induces EGFR wild-type NSCLC apoptosis
Corresponding authors: Chung-Wai Shiau, Institute of Biopharmaceutical Sciences,
National Yang-Ming University, Taipei 11221, Taiwan. Tel:886-2-28267930; E-mail:
Chong-Jen Yu, Department of Internal Medicine, National Taiwan University Hospital,
Taipei 10002, Taiwan. Tel:886-2-23562905; E-mail: [email protected]
Kuen-Feng Chen, Department of Medical Research, National Taiwan University
Hospital, Taipei, 10002, Taiwan. Tel: 886-2-23123456 ext63548; Fax:
886-2-23225329; E-mail: [email protected]
Number of text pages:39
Number of tables:0
Number of figures:6
Number of references:39
Number of words in Abstract:196
Number of words in Introduction:440
Number of words in Discussion:594
ABBREVIATIONS: NSCLC, non-small cell lung cancer; EGFR, epidermal growth
factor receptor; CIP2A, cancerous inhibitor of protein phosphatase 2A; PP2A, protein
phosphatase 2A; HCC, hepatocellular carcinoma; BAC, bronchioloalveolar carcinoma;
DMSO, dimethyl sulfoxide; WST-1, water-soluble tetrazolium; OD, optical density;
IHC, immunohistochemistry; DAB, 3,3' diaminobenzidine; PBS, phosphate buffered
solution; HRP, horseradish peroxidase; ANOVA, analysis of variance; SD, standard
deviation; PARP, poly (ADP-ribose) polymerase; Akt, protein kinase B (PKB); OA,
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Okadaic acid; c-myc, cellular homolog of the retroviral v-myconcogene; KRAS,
V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog; TP53, tumor protein p53;
CDKN2A, cyclin-dependent kinase inhibitor 2A; STK11, serine/threonine kinase 11;
CHX, cycloheximide; TNM stage, primary tumor, lymph node involvement, and
distant metastasis; ATP, adenosine triphosphate;
PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase
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ABSTRACT
Some patients with non-small cell lung cancer (NSCLC) without EGFR mutations
still respond to gefitinib and erlotinib, suggesting that there may be mechanism(s)
other than the EGFR-pathway that mediates the tumoricidal effects. In the current
study, we tested the efficacy of TD-19, a novel compound chemically modified from
erlotinib, which has more potent apoptotic effects than erlotinib in EGFR wild-type
NSCLC cell lines. TD-19 induced significant cell death and apoptosis in H358, H441,
H460 and A549 cells, as evidenced by increased caspase 3 activity and cleavage of
pro-caspase 9 and PARP. The apoptotic effect of TD-19 in H460 cells, which were
resistant to erlotinib, was associated with downregulation of CIP2A, increased PP2A
activity and decreased AKT phosphorylation, but minimal effects on EGFR
phosphorylation. Overexpression of CIP2A partially protected the H460 cells from
TD-19-induced apoptosis. Okadaic acid, a known PP2A inhibitor, significantly
reduced the TD-19-induced apoptosis while forskolin, which increased PP2A activity,
increased apoptosis effect of TD-19. TD-19 inhibited the growth of H460 xenograft
tumors by approximately 80%. We conclude that TD-19 exerted its tumoricidal effects
on NSCLC cells. TD-19 provides proof that CIP2A pathway may be a novel approach
for the treatment of EGFR wild-type NSCLC.
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INTRODUCTION
Lung cancer is the leading cause of cancer-related deaths worldwide and 80% of
lung cancers are diagnosed as non-small cell lung cancer (NSCLC) (Jemal et al.,
2008). Epidermal growth factor receptor (EGFR) gene mutations are identified in
10-15% of Caucasian NSCLC patients and in even higher percentages have been
observed in Asian patients (Shigematsu et al., 2005). Patients with certain EGFR
mutations, such as L858R and exon 19 deletion have a higher response rate to the
EGFR targeted drugs, such as gefitinib (Iressa) and erlotinib (Tarceva) (Lynch et al.,
2004; Paez et al., 2004; Huang et al., 2004; Pao et al., 2004). Some NSCLC patients
without EGFR mutations, however, still respond to gefitinib and erlotinib (Cappuzzo
et al., 2010; Ciuleanu et al., 2012), suggesting that there may be mechanism(s) other
than the EGFR-pathway that mediates the tumoricidal effects of gefitinib and
erlotinib.
Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a cellular PP2A
inhibitor that inhibits proteolytic degradation of c-MYC (Junttila et al., 2007), and is
overexpressed in several human epithelial malignancies including non-small cell lung
cancer (Junttila et al., 2007; Soo et al., 2002; Vaarala, et al., 2010; Khanna et al., 2009;
Katz et al., 2010; Côme et al., 2009; Dong et al., 2011 ; Xu et al., 2012; Ma et al.,
2011). Overexpression of CIP2A in NSCLC correlates with poor prognosis (Dong et
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al., 2011 ; Xu et al., 2012; Ma et al., 2011). Decrease in CIP2A expression inhibits
proliferation and induces apoptosis in a variety of lung cancer cells (Ma et al., 2011).
In our previous study, we found that erlotinib could also suppress CIP2A and
induced apoptosis in vivo and in vitro in hepatocellular carcinoma (HCC) (Yu et al.,
2013) and lung cancer (Wang et al., 2014). Erlotinib is a quinazoline derivative with
amino substitutes at position 4 (Hennequin et al., 1996; van Muijlwijk-Koezen et al.,
2000; Shreder et al., 2004; Morphy, 2010). With various chemical modifications, we
have previously developed a series of erlotinib analogs that had stronger CIP2A
suppression effects (Chen et al., 2012). TD-19 is one such compound modified from
erlotinib that has 4-phenoxyaniline added at 2-position of quinazoline. The changes in
the chemical structure impede the hydrogen bond interaction between erlotinib and
the ATP binding site of EGFR. Without an amide functional group or a pyridine ring,
TD-19 exhibits low binding affinity to the ATP binding site of the EGFR tyrosine
kinase domain. These changes, however, increased the potency in suppressing CIP2A
compared to erlotinib. In this study, we tested the efficacy of TD-19 in EGFR
wild-type NSCLC cells and verified that the anti-tumor activity of TD-19 was
mediated by attenuating CIP2A.
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MATERIALS and METHODS
Cell culture
Four NSCLC cell lines were used in this study. H358 (bronchioloalveolar carcinoma
[BAC], mutant KRAS), H441 (papillary adenocarcinoma, mutant KRAS, and TP53),
and A549 (BAC, mutant KRAS, CDKN2A, and STK11) cell lines were obtained from
the American Type Culture Collection (Manassas, VA) and H460 (large cell lung
cancer, mutant KRAS, PIK3CA, STK11, and CDKN2A) cell line was from the
Bioresource Collection and Research Center (Hsinchu, Taiwan). The NSCLC cell
lines were kept in RPMI1640 (Invitrogen, Life Technologies, Saint Aubin, France)
supplemented with 10% FBS (GIBO/Life Techologies, Grand Island, NY), 100
units/mL penicillin G, and 100 µg/mL streptomycin sulfate in a 37°C humidified
incubator with 5% CO2 in air.
Reagents and antibodies
Erlotinib (Tarceva®) was purchased from Selleck chemicals (Houston, TX). TD-19
was synthesized by Dr. Chung-Wai Shiau. For in vitro studies, erlotinib and TD-19 at
various concentrations were dissolved in DMSO and then added to the cells in
serum-free RPMI1640. PP2A inhibitor and activator were purchased from Sigma
(Sigma-Aldrich, St. Louis, Missouri) and Merck Millipore (Billerica, MA),
respectively. Antibodies for immunoblotting such as anti-CIP2A, AKT and PARP
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were purchased from Santa Cruz Biotechnology (San Diego, CA). Other antibodies
such as anti-PP2A, EGFR, phospho-EGFR, and p-AKT (Ser473) were from Cell
Signaling (Danvers, MA).
Cell viability assay and apoptosis analysis
Four NSCLC cell lines were seeded in 96-well plates (5 × 103 cells/well), and 10%
WST-1 agent (water-soluble tetrazolium monosodium salt) (Cell Proliferation Reagent
WST-1; Roche applied science, Indianapolis, IN) was added to the cell suspension in
each well, Cells were then incubated for 1-2 h, and cell viability and proliferation was
quantified by measuring the absorbance at 450 nm using a Biotek Synergy HT ELISA
reader (Bioteck, Winooski, VT). Apoptotic cells were measured by flow cytometry
(sub-G1) and cell death was detected by Western blot.
Overexpression of CIP2A
CIP2A cDNA (KIAA1524) was purchased from Origene (Rockville, MD). Briefly,
following transfection, H460 cells were incubated in the presence of G418 (0.78
mg/mL) (Sigma-Aldrich; St. Louis, MO). After 8 weeks of selection, surviving
colonies, i.e., those arising from stably transfected cells were selected and
individually amplified. H460 cells with stable overexpression of CIP2A were then
treated with erlotinib or TD-19 respectively, harvested, and processed for Western blot
analysis.
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PP2A phosphatase activity
Protein phosphatase 2A (PP2A) activity was measured in fresh cells as described
previously (Yu et al., 2013) using PP2A DuoSet IC activity assay kit according to the
manufacturer’s description (R&D Systems, Minneapolis, MN). Briefly, an
immobilized capture antibody specific for the catalytic subunit of PP2A that binds
both active and inactive PP2A was used. After washing, a substrate was added that
was dephosphorylated by active PP2A to generate free phosphate, which was detected
by a sensitive dye-binding assay using malachite green and molybdic acid.
Quantification of CIP2A gene expression
Total RNA was extracted from TD-19-treated H460 cells (approximately 5 × 106)
using RNeasy mini kit (Qiagen, Gaithersburg, MD) and then reversely transcribed
using QuantiTect Reverse Transcription Kit (Qiagen, Gaithersburg, MD). The real
time quantitative PCR was performed on an Applied Roter-Gene 3000 detector
(Qiagen, Gaithersburg, MD) with a specific primer set for each target gene and SYBR
Green dye (Qiagen, Gaithersburg, MD) for detection as described in the
manufacturer’s guidelines. The PCR primer sets for target genes were as follows:
human CIP2A (Hs_KIAA1524 QuantiTect Primer Assay (NM_020890)) and human
actin (Hs_ACTB QuantiTect Primer Assay (NM_001101)). An aliquot of each sample
was analyzed by quantitative PCR for β-actin to normalize for inefficiencies in cDNA
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synthesis and RNA input amounts. For each sample, the average threshold (Ct) value
was determined from quadruplicate assays, and the ΔCt value was determined by
subtracting the average β-actin Ct value from the average CIP2A Ct value. Three
independent experiments were performed to measure the levels of CIP2A in H460
cells.
Xenograft tumor growth
Male NCr nude mice (5-7 weeks of age) were used. All experimental procedures were
performed according to protocols approved by the Institutional Laboratory Animal
Care and Use Committee of Cardinal Tien Hospital. Each mouse was inoculated
subcutaneously in the dorsal flank with 1 × 107 H460 cells suspended in 0.1 ml of
serum-free medium containing 50% Matrigel (BD Biosciences, Bedford, MA). When
tumors reached 100-200 mm3, the mice received erlotinib (10 mg/kg) p.o. once daily,
or TD-19 (10 mg/kg) p.o. once daily. The controls received vehicle. The tumors were
measured twice weekly using calipers and their volumes calculated using the
following standard formula: width × length × height × 0.523 (Yu et al., 2013).
Immunohistochemistry and quantitative histological measurement
Immunohistochemical (IHC) stains were performed, using the Ventana BenchMark
XT automated stainer (Ventana, Tucson, AZ). Briefly, 4 μm-thick sections were cut
consecutively from formalin-fixed, paraffin-embedded human tissues. Sections were
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mounted and allowed to dry overnight at 37°C. After deparaffinization and
rehydratation, slides would be incubated with 3% hydrogen peroxide solution for 5
min. After washing with buffer, tissue sections were repaired for 40 min with
ethylenediamine tetraacetic acid. The slides were incubated with the primary antibody
for overnight at 4°C. The primary antibodies used in the study were anti-p-AKT (1:50,
Genetex, Irvine, CA), anti-CIP2A (1:50, Novus Biologicals; Littleton, CO) and
anti-AKT (1:25, Santa Cruz; San Diego, CA) were performed. After three rinses in
buffers, the slides were incubated a secondary antibody (unbiotinylated antibody,
EnVisionTM System, HRP, anti-mouse/rabbit, DakoCytomation, Glostrup, Denmark).
Tissue staining was visualized with a DAB substrate chromogen solution
(DakoCytomation, Glostrup, Denmark). Slides were counterstained with hematoxylin,
dehydrated, and mounted. Each run included phosphate buffered solution (PBS) as the
negative control, and samples known to express these markers strongly as the positive
controls. The quantitative protein expression level by IHC stain was using the NIH
ImageJ program (National Institute of Health, Bethesda, USA) to obtain the mean
expression level from ten random fields (400X) of each sample.
Statistical analysis
Statistical analysis was performed using analysis of variance (ANOVA) followed by
the Tukey’s subtest. The results were expressed as mean ± standard deviation (SD).
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Differences were considered significant at P < 0.05.
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RESULTS
TD-19, an Erlotinib Derivative Lacking the Inhibitory Function of EGFR
The chemical structure of TD-19 is shown in Figure 1A. It is modified from erlotinib
by adding 4-phenoxyaniline at the 2-position of quinazoline which impedes the
hydrogen bond interaction between erlotinib and the ATP binding site of the EGFR
(Hennequin et al., 1996). TD-19 does not change the phosphorylation status of EGFR
in the EGFR wild-type H358 H441 H460 and A549 cells (Figure 1B). In contrast to
erlotinib, TD-19 had no effect on different EGFR phosphorylation sites in EGFR
mutation PC9 cells and EGFR wild-type H358 and H460 cells (Supplemental Figure
1).
TD-19 Showed Cell Death Effect in NSCLC Cell Lines. TD-19 decreased the
viability of H358, H441, H460 and A549 cells in a dose-dependent (Figure 2A), and a
time-dependent manner (Figure 2B). Since H460 cells are resistant to erlotinib, we
further tested the effects of TD-19 on this cell line. TD-19 treatment for 24 hours
increased sub-G1 phase population in H460 cells (Figure 3A). TD-19 decreased
CIP2A and p-Akt protein levels and induced apoptosis in H460 cells in a
dose-dependent (Figure 3B) and a time-dependent manner (Figure 3C and
Supplemental Figure 2). The data indicates that TD-19 exhibited more potent
anti-tumor activity than erlotinib in association with CIP2A and p-Akt
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downregulation in NSCLC cells independent of EGFR activation.
Sensitization by TD-19 in NSCLC Cell Lines via the CIP2A-PP2A-AKT Pathway
To confirm the role of the CIP2A signaling reduction as a determinant molecular
mechanism mediated by TD-19-induced apoptosis, we overexpressed CIP2A in H460
cells (CIP2A-myc in Figure 4A). Overexpression of CIP2A partially protected the
cells from apoptosis induced by TD-19 (Figure 4A). Addition of okadaic acid, a
known PP2A inhibitor, also significantly reduced the TD-19-induced apoptosis in
H460 cells (Figure 4B). Forskolin activates a variety of adenylate cyclases and
increases cyclic AMP production (Tang et al., 1998), which results in activation of
protein kinase A and increased PP2A activity. Forskolin also has other cAMP
independent effects, including inhibition of the Hedgehog (Hh) signaling pathway
(Yamanaka et al., 2010), inhibition of the binding of platelet-activating factor (PAF)
(Wong et al., 1993), and inhibition of glucose transport in erythrocytes, adipocytes,
platelets, and other cells (Mills et al., 1984). Moreover, downregulating of p-Akt and
promote of the apoptosis which has synergistic effects in combination with TD-19 and
forskolin (Figure 4C). These results indicate that the CIP2A/PP2A/p-Akt pathway
plays a role in mediating the apoptotic effect of TD-19 in erlotinib-resistant H460
cells.
To examine the mechanisms by which TD-19 inhibited CIP2A protein expression, we
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investigated whether TD-19 affected CIP2A protein degradation. After protein
translation was blocked by cycloheximide, the rate of CIP2A degradation did not
change significantly with or without TD-19 treatment in H460 cells (Figure 5A). We
next investigated whether TD19 affected CIP2A transcription. Figure 5B showed that
the mRNA levels of CIP2A decreased in a time-dependent and a dose-dependent
manner in H460 cells. Since this finding suggestes that TD-19 suppressed
transcription of CIP2A, we further investigated whether TD-19 affected CIP2A
promoter activity. TD-19 significantly down-regulated the activity of CIP2A promoter
in a dose-dependent manner in H460 cells (Figure 5C, right) while erlotinib had little
effect (Figure 5C, left). From these results we concluded that CIP2A reduction by
TD-19 treatment through diminishing the transcription of CIP2A subsequently
enhancing PP2A activity and downregulating of p-Akt, leading to NSCLC cell
apoptosis.
Evaluation of the Therapeutic Effect of TD-19 on H460-bearing Mice
To determine whether or not the in vitro effects of TD-19 on H460 cells could be
reproduced in vivo, mice were implanted with H460 xenograft. Treatment with TD-19
decreased H460 xenograft tumor growth by approximately 80% compared to the
control (Figure 6A, left). TD-19 was more potent than erlotinib in inhibiting H460
xenograft tumor growth. No apparent differences in body weight or toxicity were
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found in any mice (Figure 6A, right).
To determine whether or not the anti-tumor effect of TD-19 correlated with CIP2A
dependent pathway in vivo, tumor extract from vehicle-, TD-19- and erlotinib-treated
mice were immunoblotted for CIP2A, Akt and p-Akt. PP2A activity in TD-19- and
erlotinib-treated H460 xenograft was also examined. TD-19-treated tumors showed
downregulation of CIP2A and p-Akt expression (Figure 6B, top) compared to vehicle-
and erlotinib-treated tumors. TD-19-treated tumors also showed significant increase in
PP2A activity (Figure 6C). To assess the expression level of CIP2A, p-Akt and Akt,
IHC staining were performed in H460 xenograft tumor specimens. All the tumor
specimens showed a cytoplasmic staining pattern in CIP2A, p-Akt or total Akt. The
CIP2A and p-Akt expression levels were significantly decreased in TD-19-treated
sample when compared with vehicle- and erlotinib-treated samples. Moreover, there
were no significant differences between vehicle- and erlotinib-treated samples. The
CIP2A expression level was 62.8±7.2% in the TD-19-treated sample and 96±8.7% in
the erlotinib-treated sample. There was about a 33% decrease in the expression level.
Similarly, the p-Akt expression level was 71.2±5.9% in TD-19-treated sample and
91±11.3% in erlotinib-treated sample. There was about 19% of decrease in the
expression level. In contrast, total Akt expression level did not show significant
changes between vehicle-, erlotinib- and TD-19-treated samples (Figure 6D).
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DISCUSSION
Most of the NSCLC patients with various EGFR-mutantstions respond to EGFR
inhibitors, and many quinazoline derivatives are strong inhibitors of EGFR. TD-19 is
a compound modified from erlotinib, a quinazoline derivative, that has
4-phenoxyaniline added at the 2-position of quinazoline. These modifications
minimize the effects of erlotinib against EGFR while increasing the potency against
the CIP2A-dependent pathway (Chen et al., 2012). In this study, we showed that
TD-19 induced cell death in a lung cancer cell line (H460) that was resistant to
erlotinib. This inhibitory effect in vitro could be reproduced in vivo. Since TD-19 had
minimal effect against EGFR (Figure 1B and S1), the anti-tumor effects of TD-19
were likely mediated by its attenuation of the CIP2A-PP2A-Akt pathway.
That the TD-19-induced cell death was mediated by the CIP2A pathway was
supported by the following results. First, TD-19 inhibited the RNA and protein
expression of CIP2A. Second, overexpression of CIP2A, which upregulated p-Akt,
partially protected the H460 cells against TD-19 induced apoptosis. Third, a PP2A
inhibitor, okadaic acid, significantly reduced the TD-19-induced apoptosis and a
PP2A enhancer, forskolin, increased apoptosis effect of TD-19 in H460 cells. Taken
together, these results indicated that the inhibition of CIP2A with downstream
activation of PP2A and inhibition of p-Akt mediated the anti-tumor effects of TD-19
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(Yu et al., 2013 ; Liu et al., 2013; Tseng et al., 2012; Lin et al., 2012 ; Huang et al.,
2012; Chen et al., 2011 ; Chen et al., 2010).
Whether or not TD-19 interacts directly on CIP2A remains unclear. Although we
showed TD-19 inhibited CIP2A RNA and protein levels, we did not measure its direct
effect on CIP2A activity. The precise molecular target of TD-19 remains unknown
and may be a kinase that acts upstream of CIP2A. Future studies are needed to
elucidate the exact mechanisms. CIP2A is overexpressed in NSCLC and its
expression correlates with poor prognosis (Dong et al., 2011 ; Xu et al., 2012; Ma et
al., 2011) and TNM stage (Ma et al., 2011). Besides being a prognostic biomarker,
CIP2A may also act a novel therapeutic target (Yu et al., 2013). Liang Ma et al.
demonstrated that Rabdocoetsin B, a diterpenoid isolated from Isodon coetsa,
inhibited proliferation and induced apoptosis in a variety of lung cancer cells by
down-regulating CIP2A and inactivating the Akt pathway (Ma et al., 2011). In this
study, we used a new erlotinib derivative TD-19 to demonstrate its potent anti-tumor
efficacy on EGFR wild-type NSCLC cells. We discovered that TD-19 enhanced PP2A
activity by suppressing CIP2A subsequently reduced p-Akt expression through
depleted of CIP2A transcriptional activity. Several reports previously demonstrated
that there were different ways to modulate of CIP2A expression, such as, upregulation
CIP2A by Src and Ras/MAPK/ERK kinase pathways (Jung etal., 2013), repression of
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CIP2A by miRNA that bound to the coding region of CIP2A (Zhao et al.,2010) or
transcription factors interacting with CIP2A proximal promoter to regulate of CIP2A
expression (Khanna et al., 2011; Pallai et al., 2012). Our study demonstrated that
TD-19 induced cell death and apoptosis by attenuation of CIP2A signaling through
decreased the transcription of CIP2A. However, exactly how TD-19 actually
modulates CIP2A transcription will require further elucidation.
In conclusion, TD-19 induced apoptotic cell death in NSCLC cells that were resistant
to the EGFR inhibitor, erlotinib. The anti-tumor effects were mediated by enhancing
of PP2A-mediated p-Akt downregulation by inhibition of CIP2A. Thus this
compound may be a novel therapy for patients who have NSCLC without EGFR
mutations. The therapeutic efficacy of TD-19 needs to be tested and examined more
closely in future clinical trials in patients with NSCLC.
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AUTHORSHIP CONTRIBUTIONS
Participated in research design: T.-T. Chao, C.-Y. Wang, C.-C. Lai,Y.-L. Chen, and
H.-I. Lin
Conducted experiments: T.-T. Chao, Y.-L. Chen, Y.-T. Tsai, and P.-T. Chen
Contributed new reagents or analytic tools: C.-W. Shiau
Performed data analysis: T.-T. Chao, C.-Y. Wang, Y.-L. Chen and K.-F. Chen
Wrote or contributed to the writing of the manuscript: T.-T. Chao, C.-Y. Wang, Y.-C.
Y.-L. Chen, Huang, C.-J. Yu, and K.-F. Chen
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FOOTNOTES
This study was supported by the Cardinal Tien Hospital [Grants CTH-102-1-2A29.,
CTH-101-2-2A01; CTH-102-1-2A20] and National Science Council of Taiwan
[Grants NSC 102-2314-B-567-001-MY2; NSC 101–2314-B-567–001-MY3].
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FIGURE LEGENDS
Figure 1. (A) Chemical structure of erlotinib (left) and TD-19 (right). (B) EGFR
phosphorylation activity of TD-19. H358 H411 H460 and A549 cells were exposed to
TD-19 at 10 μM for 24 h and cell lysates were analyzed for EGFR phosphorylation.
Figure 2. Comparison of effects of TD-19 and erlotinib on cell death in the four
human NSCLC cells. (A) Dose-dependent effects of TD-19 and erlotinib on cell
viability in the four human NSCLC cell lines. Data are shown as mean ± SD. n=3, **,
P < 0.01; ***, P < 0.001, for each concentration for 48h. (B) Time-dependent effects
of TD-19 and erlotinib on cell viability in the four human NSCLC cell lines. Data are
shown as mean ± SD. n=3, **, P < 0.01; ***, P < 0.001, for 24h, 48h, and 72h at 5
μM.
Figure 3. Anticancer activity of TD-19 in NSCLC cells. (A) Dose-dependent effects
of TD-19 and erlotinib on apoptosis in H460 cells. H460 cells were exposed to TD-19
and erlotinib at various concentrations (0, 2, 4, 8, 10, 20, or 50 μM) in 6 cm dish for
48 h. Apoptotic cells were assessed by flow cytometry. Data are shown as mean ± SD.
n=3 for each concentration. (B) Effects of TD-19 and erlotinib on p-AKT, CIP2A,
caspase-9 and PARP in H460 cells. H460 cells were exposed to TD-19 at the
indicated doses for 24 h. Cell lysates were assayed by western blotting. CF, cleaved
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form. Data are shown as mean ± SD. n=3. Ratio of CIP2A to actin is shown below
each western blot data. (C) Time-dependent effects of TD-19 on CIP2A, p-AKT and
apoptosis-related proteins. H460 cells were exposed to 10 μM of TD-19 for the
indicated time intervals, cell lysates were prepared and assayed by western blotting.
Ratio of CIP2A to actin is shown below each western blot data.
Figure 4. Effects of CIP2A expression on TD-19-induced apoptosis. (A) Ectopic
expression of CIP2A (CIP2A-myc) reversed TD-19-induced apoptosis in H460 cells.
H460 cells over-expressing CIP2A were treated with 10 μM of TD-19 for another 24
h. (B) Okadaic acid (OA), a PP2A inhibitor, restores the effects of TD-19 by
increasing p-AKT and inhibited the effect of TD-19 on apoptosis in H460 cells. (C)
The combination of TD-19 with forskolin has a synergistic apoptosis effect in
erlotinib-resistant H460 cells.
Figure 5. Mechanisms of TD-19-induced downregulation of CIP2A in NSCLC cells.
(A) TD-19 inhibits CIP2A transcription. H460 cells were treated with 100 μg/ml
cycloheximide (CHX), in the presence or absence of 10 μM of TD-19 for the
indicated time period, and the cell lysates were probed for CIP2A. CIP2A mRNA was
quantified using real-time PCR. (B) H460 cells were treated with 6 μM or 10 μM of
TD-19 for the indicated time period and then total RNA was isolated. Data are shown
as mean ± SD. n=3, *, P < 0.05; **, P < 0.01. (C) CIP2A promoter activity was
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decreased by TD-19 but not by erlotinib. H460 cells were transfected by CIP2A
reporter and Renilla vectors for 24 h and then treated with 10 μM or 20 μM erlotinib
or TD-19 for another 24 h. Cell lysates were prepared for analysis by Dual-Glo
luciferase Assay. Data are shown as mean ± SD. n=3, **, P < 0.01.
Figure 6. Effect of erlotinib or TD-19 on H460 xenograft tumor growth in nude mice.
(A) Mice were treated with vehicle, erlotinib or TD-19 p.o at 10 mg/kg daily for 3
weeks. TD-19 inhibited tumor growth by approximately 80% (left). There was no
difference in body weight (right). Data are shown as mean ± SD. n=6, *, P < 0.05; **,
P < 0.01. Statistical analyzed by ANOVA. (B) Western blot analysis of CIP2A,
p-AKT and AKT in H460 tumors. Ratio of CIP2A to actin is shown below each
western blot data set. Immunoblots were quantitated using VisionWork LS software.
* Represents the p value < 0.05 when comparing the mean percentage of the
erlotinib-treated group (no. 4, 5, 6) with the mean percentage of the vehicle group
(no.1, 2, 3) by ANOVA. †† Represents the p value < 0.01 when comparing the mean
percentage of the TD19-treated group (no. 10,11,12) with the mean percentage of the
vehicle group (no.7, 8 ,9) by ANOVA. (C) Analysis of PP2A activity in tumors. Data
are shown as mean ± SD. n = 6; *, P < 0.05; **, P < 0.01. All data are representative
of three independent experiments. Statistically analyzed by ANOVA. (D)
Immunohistochemical stain and quantitative analyses of CIP2A, p-AKT and AKT in
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H460 xenografts tumors (400X magnification). Data are shown as mean ± SD. n = 6;
*, P < 0.05. Statistical analyzed by ANOVA.
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Figure 1
N
N
H N
O
O N H
O
TD-19
A
B
TD-19 (10 M) + - + - + - + -
H358
p-EGFR(Y1068)
EGFR
H441 H460 A549
Actin
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Figure 2
A
B
H358
Dose (uM)
Ce
ll v
iab
ilit
y (
%)
0
20
40
60
80
100
120
0 2.5 5 10 20
Erlotinib
TD-19
★★★ ★★★ ★★★ ★★★
H441
Dose (uM)
0
20
40
60
80
100
120
0 2.5 5 10 20
Erlotinib
TD-19★★ ★★★
★★★ ★★★
H460
Dose (uM)
0
20
40
60
80
100
120
0 2.5 5 10 20
Erlotinib
TD-19★★★ ★★★
★★★ ★★★
A549
Dose (uM)
0
20
40
60
80
100
120
0 2.5 5 10 20
Erlotinib
TD-19
★★ ★★★
★★★
★★★
H358
Ce
ll v
iab
ilit
y (
%)
Time (h)
0
20
40
60
80
100
120
0 24 48 72
Erlotinib
TD-19
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★★★
H441
Time (h)
0
20
40
60
80
100
120
0 24 48 72
Erlotinib
TD-19★★★
★★★
★★★
H460
Time (h)
0
20
40
60
80
100
120
0 24 48 72
Erlotinib
TD-19★★★
★★★
★★★
A549
Time (h)
0
20
40
60
80
100
120
0 24 48 72
Erlotinib
TD-19★★★
★★★
★★★
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Figure 3
A B
24 0 2 6 9 16
PARP
CIP2A
p-AKT
AKT
actin
CF PARP
pro-caspase 9
0 2 4 8 10 10
TD-19 (M)
CIP2A
p-AKT
AKT
actin
CIP
2A/a
ctin
rat
io
(% o
f c
on
tro
l)
H460
TD-19
(M)
( M)
H460
Ap
op
toti
c c
ell
s (%
)
30
20
10
0
40
caspase 9 CF
(hr)
C TD-19 (10M)
0
40
80
120
0 2 4 8 10 100
40
80
120
1 2 3 4 5 6
CIP
2A/a
ctin
rat
io
(% o
f c
on
tro
l)
24 0 2 6 9 16 (hr)
TD-19 (10 M)
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Figure 4
A
CIP2A-myc
TD-19 (10M)
CIP2A
p-AKT
AKT
actin
H460
myc-tag
Ap
op
tos
is
(%)
- + -
- +
+
+
-
0
10
20
30
40
1 2 3 4
★★
TD-19 (10M)
OA
-
-
+
+
+
-
p-AKT
AKT
actin
0
50
100
150
200
1 2 3
PP
2A
acti
vit
y
(% o
f co
ntr
ol)
★★
TD-19 (5M)
forskolin
-
-
+
+
+
-
p-AKT
AKT
actin
0
5
10
15
20
1 2 3
Ap
op
toti
c
cell
s (
%)
★★
PP
2A
acti
vit
y
(% o
f co
ntr
ol)
A
po
pto
tic
cell
s
(%)
B C H460 H460
★★
0
50
100
150
200
1 2 3
0
5
10
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20
1 2 3
★★ ★
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Figure 5
A
B
C
H460
CHX
CIP2A
actin
0 4 8 12 24 48
CHX+ TD-19
0 4 8 12 24 48
CIP
2A
mR
NA
(% o
f co
ntr
ol)
Time (hr)
★ ★★
★★
★★
★★
★★
★★
Time (hr)
★★
CIP
2A L
ucif
era
se
Act
ivit
y (%
of
con
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l)
Erlotinib (µM)
★★
★★
TD-19 (µM)
CIP
2A L
ucif
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se
Act
ivit
y (%
of
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tro
l)
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TD-19 Erlotinib Vehicle
Erlotinib Vehicle TD-19 Vehicle
Figure 6
A
B
Days of Treatment
Tu
mo
r S
ize
(m
m3)
0
300
600
900
1200
1500
1800
0 2 5 8 11 14 16 18 21
Vehicle
Erlotinib
TD2-2
0
10
20
30
40
0 2 5 8 11 14 16 18 21
Vehicle
Erlotinib
TD2-2
Days of Treatment
Bo
dy W
eig
ht
(g)
C
★★
★★
★
★★ ★★
★★ ★★
★★ ★★
★★
★★
PP
2A a
ctiv
ity
(% o
f co
ntr
ol)
CIP2A
p-AKT
AKT
actin
Erlotinib Vehicle TD-19 Vehicle
CIP
2A/a
ctin
(% o
f co
ntr
ol)
150
100
50
0
200
D
CIP2A
p-AKT
TD-19 TD-19
7 8 9 10 11 12
AKT
7 8 9 10 11 12 1 2 3 4 5 6
0
20
40
60
80
100
120
CIP2A p-AKT AKT
Vehicle Erlotinib TD-19
★
★
★
★
Rel
ativ
e ex
pre
ssio
n le
vel
(% o
f ve
hic
le)
This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on September 3, 2014 as DOI: 10.1124/jpet.114.215418
at ASPE
T Journals on A
pril 12, 2020jpet.aspetjournals.org
Dow
nloaded from