TD-19, an erlotinib derivative, induces EGFR wild-type NSCLC...

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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

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

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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:

[email protected]

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

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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TD-19, an erlotinib derivative, induces EGFR wild-type NSCLC...

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