STAT3 expression in activating EGFR-driven adenocarcinoma of the lung

Lung Cancer 75 (2012) 24–29

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STAT3 expression in activating EGFR-driven adenocarcinoma of the lung Saburo Takata a , Nagio Takigawa b,∗ , Yoshihiko Segawa c , Toshio Kubo a , Kadoaki Ohashi a , Toshiyuki Kozuki c , Norihiro Teramoto d , Motohiro Yamashita e , Shinichi Toyooka f , Mitsune Tanimoto a , Katsuyuki Kiura a a Department of Hematology, Oncology, and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences and Okayama University Hospital, 2-5-1 Shikata-cho, Okayama 700-8558, Japan b Department of General Internal Medicine 4, Kawasaki Medical School, 2-1-80 Nakasange, Kita-ku, Okayama 700-8505, Japan c Department of Thoracic Oncology, National Hospital Organization Shikoku Cancer Center, Matsuyama, Japan d Department of Pathology, National Hospital Organization Shikoku Cancer Center, Matsuyama, Japan e Department of Thoracic Surgery, National Hospital Organization Shikoku Cancer Center, Matsuyama, Japan f Department of Cancer and Thoracic Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan

a r t i c l e

i n f o

Article history: Received 3 December 2010 Received in revised form 16 May 2011 Accepted 20 May 2011 Keywords: Non-small cell lung cancer Bronchioloalveolar carcinoma Adenocarcinoma STAT3 EGFR JAK2

s u m m a r y Bronchioloalveolar carcinoma (BAC) pattern is often seen at the margin of invasive adenocarcinomas. We investigated EGFR signaling abnormalities involved in the progression of adenocarcinoma. Fifty tumors were obtained from patients who underwent surgery for lung adenocarcinoma seen as dense areas in ground glass opacity on computed tomography. Six, 18, and 26 tumors <1 cm, 1–2 cm, and ≥2 cm in diameter, respectively, were analyzed. Of the 24 tumors ≤2 cm in diameter, nine were preinvasive and 15 were invasive. EGFR, pAKT, and pMAPK were overexpressed in the center of the adenocarcinoma compared to the BAC component (p < 0.01) by immunohistochemistry, while pSTAT3 expression was reversed (p = 0.017). In the tumors ≤2 cm in diameter, pSTAT3 expression in the central area was higher in preinvasive tumors than in invasive tumors (p = 0.005). pSTAT3 was identified in the BAC component of 88% of the EGFR mutant (n = 17) and 82% of the wild-type tumors (n = 33). Transgenic mice expressing delE748-A752 EGFR and two lung cancer cell lines (PC-9 mutant and A549 wild-type EGFR) were also investigated. In transgenic mice, pSTAT3 was overexpressed in the BAC component around the adenocarcinoma center. Two lung cancer cell lines that overexpressed pSTAT3 were equally sensitive to a JAK2/STAT3 inhibitor (JSI-124). The role of STAT3 in the progression of adenocarcinoma should be further pursued. © 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Although the overall incidence rate of lung cancer decreased by 0.8% per year from 1999 to 2005, it remains the leading cause of death by malignant tumors worldwide [1]. Adenocarcinoma, the most prevalent histology, is present in 50% of all non-small cell lung cancers (NSCLCs) [2]. Pulmonary atypical adenomatous hyperplasia (AAH) is recognized as a premalignant lesion of adenocarcinoma [3]. Bronchioloalveolar carcinoma (BAC) pattern is often seen at the margin of invasive adenocarcinomas [4]. According to the Noguchi classification scheme (adenocarcinoma size ≤ 2 cm), types A (localized BAC) and B (localized BAC with foci of collapsed alveoli) are thought to be in situ peripheral adenocarcinomas (preinvasive tumors), whereas type C (localized BAC with foci of active fibroblastic proliferation; invasive tumors) are considered to be an advanced stage of types A and B [5].

∗ Corresponding author. Tel.: +81 86 225 2111; fax: +81 86 232 8343. E-mail address: [email protected] (N. Takigawa). 0169-5002/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2011.05.015

Screening by computed tomography (CT) found more asymptomatic early lung cancers [6]. Computed tomographic imaging of AAH and small-sized adenocarcinomas revealed the existence of three types: (1) pure ground glass opacity (GGO), non-solid type; (2) mixed GGO, partially solid type; (3) no GGO, solid type [7]. Following the rise of a pure GGO, a solid part appeared in the center (mixed GGO), which was interpreted as the progression from in situ peripheral adenocarcinoma (Noguchi A or B) to invasive adenocarcinoma (Noguchi C) [7]. Somatic activating mutations in epidermal growth factor receptor (EGFR) are more frequently observed in females, non-smokers, and adenocarcinoma patients, especially in Asian populations, where the mutation rate is between 30 and 50% [8]. The relationship between mutations in EGFR and adenocarcinoma carcinogenesis has been demonstrated in transgenic mice by several groups, including ours [9–11]. We previously reported that EGFR mutations were detected at the same frequency in both AAH and adenocarcinoma, and these mutations are thought to occur during the early stages of adenocarcinoma [12]. PI3K/AKT, RAS/MAPK, and STAT3 are three major downstream pathways activated by EGFR phos-

S. Takata et al. / Lung Cancer 75 (2012) 24–29

phorylation [8]. STAT3 is an oncogene that is expressed in alveolar type II epithelial cells [13,14]. STAT3 has also been reported to be a critical mediator of the oncogenic effects of EGFR mutations [15]. Non-receptor tyrosine kinases such as SRC and the JAKs (JAK1 and JAK2) also phosphorylate STAT3 [16]. In this study, we investigated several EGFR-related proteins (EGFR, HER3, STAT3, AKT, and MAPK) and an EGFR mutation to identify EGFR signaling abnormalities involved in the progression of adenocarcinoma. 2. Materials and methods 2.1. Patients Between June 2002 and August 2004, 50 adenocarcinoma tissue specimens were obtained from Japanese patients who underwent surgery for lung adenocarcinoma at the National Shikoku Cancer Center Hospital (Matsuyama, Japan). The patients all showed GGO with a solid central portion by CT. To avoid selection bias, sequentially resected adenocarcinomas that displayed a mixed GGO on CT were included. This study was approved by the Institutional Review Board. 2.2. Mouse model and cell lines Tumor tissues from transgenic mice expressing delE748-A752 mouse EGFR (corresponding to delE746-A750 human EGFR) [11] were embedded in paraffin then sectioned every 5 ␮m. Two lung adenocarcinoma cell lines, PC-9 carrying a 15-bp in-frame deletion in EGFR (delE746-A750) and A549 (wild-type EGFR), were cultured at 37 ◦ C under 5% CO2 in RPMI 1640 supplemented with 10% heatinactivated fetal bovine serum. 2.3. Immunohistochemistry Formalin-fixed paraffin-embedded tissue blocks from the clinical samples were cut to a thickness of 5 ␮m, placed on glass slides, then deparaffinized in xylene and graded alcohol. The antigen was incubated in 10 mM sodium citrate buffer, pH 6.0, for 10 min in a 95 ◦ C water bath. The sections were then blocked for endogenous peroxidase with 0.3% hydrogen peroxide in methanol for 10 min. The slides were rinsed with TBS containing 0.1% Tween 20 and the sections were blocked with goat normal serum for 60 min. The sections were incubated with anti-EGFR (EGFR PharmDxTM , DakoCytomation, Ely, UK) monoclonal antibodies for 60 min at 25 ◦ C, anti-HER3 (C-17, Santa Cruz Biotechnology, Santa Cruz, CA, USA) polyclonal antibodies, anti-phosphorylated-STAT3 (pSTAT3) (58E12, Cell Signaling, Danvers, MA, USA) monoclonal antibodies, anti-phosphorylated AKT (pAKT) (36E11, Cell Signaling) monoclonal antibodies, and anti-polyclonal phosphorylated MAPK (pMAPK) (20G11, Cell Signaling) antibodies overnight at 4 ◦ C. The sections were amplified using biotinylated anti-rabbit antibodies and avidin–biotinylated horseradish peroxidase conjugate for 10 min (LSABTM 2 Kit, DakoCytomation) then reacted with 3,3 diaminobenzidine. Finally, the sections were counterstained with hematoxylin. EGFR and HER3 expression was scored as positive if >10% of the tumor cells exhibited cytoplasmic or membrane staining. Similarly, pSTAT3, pMAPK, and pAKT were scored as positive if >10% of the tumor cells exhibited cytoplasmic or nuclear staining [17]. The primary monoclonal antibody against pSTAT3 used for the transgenic mice was D3A7 (Cell Signaling). All immunohistochemical analyses were completed independently by two investigators (S.T. and N.T.).

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mal tissue. Genomic DNA was extracted using DEXPATTM (TaKaRa, Kyoto, Japan) according to the manufacturer’s instructions. Active EGFR mutations (deletion in exon 19 and L858R in exon 21) were analyzed by mutant-enriched PCR [18]. 2.5. Sensitivity test A JAK2/STAT3 inhibitor, JSI-124 (Cucurbitacin I) [16], was purchased from Calbiochem (Darmstadt, Germany). Drug sensitivity was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay [19]. The cells were placed on 96-well plates at a density of 2,000 cells per well and exposed continuously to each drug for 96 h. Each assay was performed in triplicate or quadruplicate. 2.6. Immunoblotting Protein extracts were prepared from crushed tissue samples that had been incubated in lysis buffer (1% Triton X-100, 0.1% SDS, 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM ␤-glycerol phosphate, 10 mM NaF, and 1 mM Naorthovanadate) containing protease inhibitors (Roche Diagnostics, Basel, Switzerland) and centrifuged at 14,500 rpm for 20 min at 4 ◦ C. After quantification using a Bio-Rad protein assay, ∼50 ␮g of protein was subjected to 5–15% SDS-PAGE (Bio-Rad, Hercules, CA, USA) then transferred to nitrocellulose membranes. Specific proteins were detected by enhanced chemiluminescence (GE Healthcare, Buckinghamshire, UK) using antibodies against pEGFR (1:1,000 dilution; Cell Signaling), pSTAT3 (1:1,000 dilution; Cell Signaling), pSRC (1:1,000 dilution; Cell Signaling), pJAK2 (1:2,000 dilution; Cell Signaling), total EGFR, total JAK2, total SRC (1:1,000 dilution; Cell Signaling), total STAT3 (1:200 dilution; Santa Cruz Biotechnology), GAPDH (1:1,000 dilution; Cell Signaling), and actin (1:5,000 dilution; Chemicon, Temecula, CA, USA). The secondary antibodies were anti-rabbit and anti-mouse IgG (horseradish peroxidaselinked, species-specific whole antibodies; GE Healthcare), each of which was used at a 1:5,000 dilution. 2.7. Statistical analysis Statistical analysis was performed using the SPSS Base SystemTM and Advanced StatisticsTM programs (SPSS, Chicago, IL, USA). All relationships between categorical variables were assessed by chisquare testing; p-values ≤ 0.05 were considered to be statistically significant. 3. Results 3.1. Patient demographics The median age of the patients (18 males and 32 females) was 66 years (range, 42–83 years). Most patients (98%) exhibited BAC pattern at pathological stage IA or IB although we selected the tumors which displayed a mixed GGO on CT. One patient had pathological single N2 disease (stage IIIA). Table 1 summarizes the patients’ demographic characteristics. Six tumors <1 cm, 18 tumors between 1 and 2 cm, and 26 tumors ≥2 cm in diameter were observed. Of the 24 tumors ≤2 cm in diameter, nine were types A and B and 15 were type C. 3.2. Immunohistochemistry

2.4. Mutation analysis Tumor tissue was selectively dissected from the formalin-fixed paraffin-embedded sections to minimize contamination by nor-

EGFR, HER3, pSTAT3, pAKT, and pMAPK overexpression was observed in 15, 17, 42, 13, and 18 of the tumors, respectively (Fig. 1A and Table 2). Those tumors ≥1 cm overexpressed EGFR

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S. Takata et al. / Lung Cancer 75 (2012) 24–29

Table 1 Patient characteristics (n = 50). Variables

(p = 0.048), HER3 (p = 0.020), and pMAPK (p = 0.021) significantly more often than those <1 cm (Table 3). Consequently, we separately evaluated protein expression in cancer cells from the center and margins of the tumor tissues (corresponding to the dense area and GGO on CT, respectively). The ‘margin’ was defined as an adjacent tumor to normal bronchial epithelium. All evaluated margins were present within one-fifth of outer part in the tumor. EGFR expression was stronger in the center than in the margin (26% vs. 14%; p = 0.003), while greater pSTAT3 expression was observed in the margin than in the center (84% vs. 34%; p = 0.017) (Fig. 1A and Table 2). When pSTAT3 expression in the center was analyzed, tumors <1 cm overexpressed pSTAT3 to a greater extent than did those ≥1 cm (p = 0.007), while those of types A and B overexpressed pSTAT3 more than those of type C (p = 0.005) (Table 4).

n (%)

Pathological stage IA 42 (84) IB 7 (14) 1 (2) IIIA Tumor size 6 (12) <1 cm 18 (36) 1–2 cm 26 (52) >2 cm Noguchi classification (tumor size ≤ 2 cm) 9 (18) A+B 15 (30) C

Fig. 1. (A) Representative normal bronchial epithelium (NBE) at the margin and center of a lung adenocarcinoma. pSTAT3 was observed at the margins of the tumor, although it was weakly expressed in NBE and center of the tumor. EGFR was overexpressed in the margins and center of the tumor, as well as in the NBE. HER3 was found in the margins and center of the tumor. H.E.: hematoxylin and eosin staining. (B) Western blotting for EGFR, HER3, AKT, MAPK, and STAT3 expression in the lungs of 2-week-old transgenic (Tg) and control mice (WT). (C and D) Immunohistochemical staining of pSTAT3 in lung tumors from transgenic mice. pSTAT3 was more highly overexpressed at the margins (C) than in the center of the adenocarcinoma (D). (E) Western blotting for EGFR, HER3, AKT, MAPK, and STAT3 expression in the lungs of 7-week-old transgenic mice (Tg) treated with gefitinib. On days 2 and 7, the mice were sacrificed and their lungs were examined. pSTAT3 was decreased to a lesser degree than pEGFR. Table 2 Protein expression by tumor location. Variables

Total Margin Center * † ‡ §

No. showing protein overexpression (n = 50) EGFR

HER3

pSTAT3

pAKT

pMAPK

15 (30%) 7 (14%)* 13 (26%)*

17 (34%) 17 (34%) 17 (34%)

42 (84%) 42 (84%)† 17 (34%)†

13 (26%) 0‡ 13 (26%)‡

18 (36%) 0§ 18 (36%)§

p-Value (chi-square test): 0.003. p-Value (chi-square test): 0.017. p-Value (chi-square test): 0.000. p-Value (chi-square test): 0.000.

S. Takata et al. / Lung Cancer 75 (2012) 24–29

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Table 3 Protein expression by tumor size, Noguchi classification, and EGFR mutation status. Variables

No. showing protein overexpression (n = 50)

Tumor size <1 cm ≥1 cm Noguchi classification A+B C EGFR mutation status Positive/negative Exon 19 Exon 21 * † ‡ §

EGFR

HER3

pSTAT3

pAKT

pMAPK

0* 13 (26%)*

0† 17 (34%)†

5 (10%) 37 (74%)

1 (2%)‡ 12 (24%)‡

2 (4%)§ 16 (32%)§

1 (2%) 5 (10%)

1 (2%) 4 (8%)

7 (14%) 15 (30%)

3 (6%) 4 (8%)

3 (6%) 5 (10%)

5/8 2 3

6/11 5 1

14/28 6 8

3/10 1 2

7/11 5 2

p-Value (chi-square test): 0.048. p-Value (chi-square test): 0.020. p-Value (chi-square test): 0.309. p-Value (chi-square test): 0.021.

Table 4 pSTAT3 expression in the central area by tumor size and Noguchi classification. Category

pSTAT3 expression

Tumor size <1 cm ≥1 cm Noguchi classification A+B C * †

−

+

1 32

5* 12*

2 12

7† 3†

3.4. Mutated EGFR-transgenic mice

p-Value (chi-square test): 0.007. p-Value (chi-square test): 0.005.

3.3. Analysis of an activating EGFR mutation Seventeen of the 50 adenocarcinoma tissues had EGFR mutations (seven in exon 19 and ten in exon 21). No differences were

A

1.2 1

JSI124 JSI124+gefitinib 0.001

Surviving

0.8

detected in the expression of EGFR, HER3, pSTAT3, pAKT, or pMAPK between the mutant and wild-type EGFR tumors. pSTAT3 was found in the BAC component in 88% of the mutant tumors and 82% of the wild-type tumors.

JSI124+gefitinib 0.01

Protein expression in the lungs of 2-week-old transgenic mice was also examined. pSTAT3, pEGFR, pHER3, and pAKT overexpression was observed by Western blotting (Fig. 1B). Immunohistochemical staining of lung tumors from 8-week-old mice showed that pSTAT3 was overexpressed more in the BAC component than in the adenocarcinoma center (Fig. 1C and D, respectively). The overexpression of pSTAT3 was detected on the nuclei of the cells in the alveolar walls (Fig. S1). It was also detected on a substantial proportion of the exfoliated cells in the alveolar space. The high power view of the lesion showed that the pSTAT3-positive exfoliated cells were BAC cells with conspicuous nuclear atypia which were piled up in to the alveolar space and that pSTAT3-negative cells would be alveolar macrophages with bland nuclei (Fig. S2). Gefitinib was administered by gavage as a 5 mg/kg suspension to 7-week-old transgenic mice carrying lung tumors.

0.6 0.4 0.2 0 0.001

0.01

0.1

1

Concentration (μM)

B

1.2 1 JSI124

Surviving

0.8

JSI124+gefitinib 2 JSI124+gefitinib 20

0.6 0.4 0.2 0 0.001

0.01

0.1

1

Concentration (μM) Fig. 2. Drug–response curve by MTT assay. (A) The IC50 of JSI-124 in PC-9 cells was 0.026 ± 0.004 ␮M. The IC50 s of JSI-124 combined with gefitinib (0.001 or 0.01 ␮M) were 0.022 ± 0.003 (p = 0.11, compared with JSI-124 alone) and 0.016 ± 0.005 ␮M (p = 0.01, compared with JSI-124 alone), respectively. (B) The IC50 of JSI-124 in A549 cells was 0.027 ± 0.002 ␮M. The IC50 s of JSI-124 combined with gefitinib (2 or 20 ␮M) were 0.026 ± 0.002 (p = 0.33, compared with JSI-124 alone) and 0.031 ± 0.010 ␮M (p = 0.46, compared with JSI-124 alone), respectively.

Fig. 3. Western blotting for EGFR, JAK2, SRC, and GAPDH (control) in PC-9 and A549 cells. The cells were treated with G (gefitinib, 2 ␮M) and/or J (JSI-124, 5 ␮M) for 6 h. pSTAT3 was partially suppressed by J irrespective of the presence of G in both cell lines. In PC-9 cells, although pJAK2 and pSTAT3 expression seemed to be increased by treatment with G, J clearly suppressed the proteins.

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S. Takata et al. / Lung Cancer 75 (2012) 24–29

On day 2 or 7, the mice were sacrificed and their lungs were examined by Western blotting. pSTAT3 expression was decreased by a smaller amount than pEGFR (Fig. 1E). 3.5. In vitro experiments The mean ± standard deviation of the 50% inhibitory concentration (IC50 ) of JSI-124 in PC-9 and A549 cells was determined to be 0.026 ± 0.004 and 0.027 ± 0.002 ␮M, respectively (Fig. 2). When JSI124 was combined with gefitinib (0.01 ␮M) in PC-9 cells, the IC50 of JSI-124 was significantly decreased to 0.016 ± 0.005 ␮M (p = 0.01). However, the IC50 s of JSI-124 in combination with gefitinib (2 or 20 ␮M) in A549 cells were similar to that for JSI-124 alone. pSTAT3, pJAK2, and pSRC expression was observed in PC-9 and A549 cells by Western blotting (Fig. 3). Although pSTAT3 was preserved after gefitinib treatment (2 ␮M, 6 h), it was partially suppressed by JSI-124 irrespective of the presence of gefitinib. In PC-9 cells, although pJAK2 and pSTAT3 expression seemed to increase after treatment with gefitinib, JSI-124 suppressed expression of the protein. 4. Discussion The results presented here demonstrate that adenocarcinomas with a BAC component overexpress pSTAT3 at the margin compared to the center of the tumor (Fig. 1A and Table 2). Although the distinction between ‘center’ and ‘margin’ seemed difficult, the ‘margin’ was adjacent to normal bronchial epithelium and was actually located in the edge of the tumor. A positive correlation between the presence of pSTAT3 and a smaller tumor size was reported previously [20,21]. Our results confirm these data, as pSTAT3 was overexpressed more fully in preinvasive tumors <1 cm than in invasive tumors ≥1 cm (Table 4). Li et al. [22] showed that persistent STAT3 activation induced pulmonary tumorigenesis using STAT3-transgenic mice. Taken together, STAT3 seems to be involved in the tumorigenesis of human adenocarcinoma of the lung. Achcar Rde et al. reported that pSTAT3 was present sparingly in bronchial epithelium [23]. We could not detect pSTAT3 expression more than 10% of cells in normal bronchial epithelium (NBE) (Fig. 1A). In addition, pSTAT3 expression was less in normal mouse lungs than in tumors by Western blotting (Fig. 1B). Our data using clinical samples suggested that NBE had weak pSTAT3 expression, and in situ peripheral adenocarcinoma (Noguchi A/B) had more pSTAT3, however, Noguchi C which had the property of invasiveness had less pSTAT3 in the center of the tumors (Table 4). Once the tumor acquired invasiveness, pSTAT3 levels might be decreased. Further investigation was needed to clarify the reason of decrease of pSTAT3 in a larger size and a center of the tumor. The activation of STAT3 by receptor tyrosine kinases such as EGFR and MET, cytokine receptors such as IL-6, and non-receptor kinases such as SRC regulates survival pathways in certain lung cancer cells [24]. The mechanism by which STAT3 was activated in lung adenocarcinoma was through mutated EGFR regulating expression of the IL-6 cytokine, which activated the gp130/JAK pathway [21]. When we focused on the EGFR/STAT3 pathway using mutant EGFR-transgenic mice, pSTAT3 was also overexpressed in the BAC component around the central adenocarcinoma. STAT3 is activated downstream of EGFR; however, it was less suppressed compared to EGFR despite the administration of gefitinib (Fig. 1E). This suggested that signals from other upstream might activate STAT3 even in EGFR-driven lung cancer. PC-9 cells carrying the EGFR mutation were sensitive to JSI-124. The combination of gefitinib and JSI-124 in PC-9 cells seemed more effective at suppressing pEGFR and pSTAT3, respectively (Fig. 3). We supposed that JAK-dependent

phosphorylation of STAT3 existed even in EGFR-dependent tumors. Taken together, STAT3 inhibition may be effective against EGFR mutant tumors through different pathways downstream of EGFR. The median survival time of the gefitinib-treated transgenic mice was 35 weeks and they died due to pulmonary adenocarcinoma, which the resistant mechanisms remained to be unknown [11]. Now, we are investigating the role of pSTAT3 as one of the gefitinibresistant mechanisms. There was no significant difference in pSTAT3 expression between the EGFR-mutated (83%) and EGFR-wild-type tumors (56%), and the expression of pSTAT3 was not a result of EGFR kinase activity in NSCLC [25]. pSTAT3 was generally observed irrespective of the EGFR mutation status in the present study, confirming previously published data [25]. Although their analysis included squamous cell carcinoma at a more advanced stage, the researchers suggested that STAT3 activity contributes to the carcinogenic potential of NSCLC independent of EGFR mutations. STAT3-related carcinogenesis is initiated by pro-inflammatory cytokines (e.g., IL-6) and growth factors (e.g., EGF) in association with lymphocyte and macrophage infiltration [22]. In the clinical samples tested here, inflammatory cells such as lymphocytes and macrophages were not evident in or around the tumor (data not shown). Whole-genome allelic imbalance scanning showed that preinvasive tumors (Noguchi types A and B) progressed to invasive tumors due to subsequent alterations in several tumor suppressor genes, including those at 11p11–p12, 17p12–p13, 18p11, and TP53 [26]. Additional studies are needed to evaluate the relationship between these tumor suppressor genes and activated STAT3. Several inhibitors of JAK and SRC, which activate STAT3, have been developed and introduced in clinical trials [27,28]. JSI-124 was found to suppress pSTAT both in vitro (A549 cells) and in vivo [16]. The role of STAT3 inhibitors for the treatment of adenocarcinoma of the lung might be clarified in the clinical studies. Conflict of interest statement None declared. Acknowledgment Grant support: Ministry of Education, Culture, Sports, Science, and Technology, Japan (grant 21590995 to N. Takigawa). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.lungcan.2011.05.015. References [1] Jemal A, Thun MJ, Ries LA, Howe HL, Weir HK, Center MM, et al. Annual report to the nation on the status of cancer, 1975–2005, featuring trends in lung cancer, tobacco use, and tobacco control. J Natl Cancer Inst 2008;100:1672–94. [2] William WN, Lin HY, Lee JJ, Lippman SM, Roth JA, Kim ES. Revisiting stage IIIB and IV non-small cell lung cancer: analysis of the surveillance, epidemiology, and end results data. Chest 2009. [3] Kerr KM, Carey FA, King G, Lamb D. Atypical alveolar hyperplasia: relationship with pulmonary adenocarcinoma, p53, and c-erbB-2 expression. J Pathol 1994;174:249–56. [4] Okada M, Nishio W, Sakamoto T, Uchino K, Hanioka K, Ohbayashi C, et al. Correlation between computed tomographic findings, bronchioloalveolar carcinoma component, and biologic behavior of small-sized lung adenocarcinomas. J Thorac Cardiovasc Surg 2004;127:857–61. [5] Noguchi M, Morikawa A, Kawasaki M, Matsuno Y, Yamada T, Hirohashi S, et al. Small adenocarcinoma of the lung. Histologic characteristics and prognosis. Cancer 1995;75:2844–52. [6] Henschke CI, Yankelevitz DF, Libby DM, Pasmantier MW, Smith JP, Miettinen OS. Survival of patients with stage I lung cancer detected on CT screening. N Engl J Med 2006;355:1763–71.

S. Takata et al. / Lung Cancer 75 (2012) 24–29 [7] Travis WD, Garg K, Franklin WA, Wistuba II, Sabloff B, Noguchi M, et al. Evolving concepts in the pathology and computed tomography imaging of lung adenocarcinoma and bronchioloalveolar carcinoma. J Clin Oncol 2005;23:3279–87. [8] Mitsudomi T, Yatabe Y. Mutations of the epidermal growth factor receptor gene and related genes as determinants of epidermal growth factor receptor tyrosine kinase inhibitors sensitivity in lung cancer. Cancer Sci 2007;98:1817–24. [9] Politi K, Zakowski MF, Fan PD, Schonfeld EA, Pao W, Varmus HE. Lung adenocarcinomas induced in mice by mutant EGF receptors found in human lung cancers respond to a tyrosine kinase inhibitor or to down-regulation of the receptors. Genes Dev 2006;20:1496–510. [10] Ji H, Li D, Chen L, Shimamura T, Kobayashi S, McNamara K, et al. The impact of human EGFR kinase domain mutations on lung tumorigenesis and in vivo sensitivity to EGFR-targeted therapies. Cancer Cell 2006;9:485–95. [11] Ohashi K, Rai K, Fujiwara Y, Osawa M, Hirano S, Takata K, et al. Induction of lung adenocarcinoma in transgenic mice expressing activated EGFR driven by the SP-C promoter. Cancer Sci 2008;99:1747–53. [12] Kozuki T, Hisamoto A, Tabata M, Takigawa N, Kiura K, Segawa Y, et al. Mutation of the epidermal growth factor receptor gene in the development of adenocarcinoma of the lung. Lung Cancer 2007;58:30–5. [13] Yan C, Naltner A, Martin M, Naltner M, Fangman JM, Gurel O. Transcriptional stimulation of the surfactant protein B gene by STAT3 in respiratory epithelial cells. J Biol Chem 2002;277:10967–72. [14] Bromberg JF, Wrzeszczynska MH, Devgan G, Zhao Y, Pestell RG, Albanese C, et al. Stat3 as an oncogene. Cell 1999;98:295–303. [15] Alvarez JV, Greulich H, Sellers WR, Meyerson M, Frank DA. Signal transducer and activator of transcription 3 is required for the oncogenic effects of nonsmall-cell lung cancer-associated mutations of the epidermal growth factor receptor. Cancer Res 2006;66:3162–8. [16] Blaskovich MA, Sun J, Cantor A, Turkson J, Jove R, Sebti SM. Discovery of JSI-124 (cucurbitacin I), a selective Janus kinase/signal transducer and activator of transcription 3 signaling pathway inhibitor with potent antitumor activity against human and murine cancer cells in mice. Cancer Res 2003;63: 1270–9. [17] Cappuzzo F, Magrini E, Ceresoli GL, Bartolini S, Rossi E, Ludovini V, et al. Akt phosphorylation and gefitinib efficacy in patients with advanced non-smallcell lung cancer. J Natl Cancer Inst 2004;96:1133–41.

29

[18] Asano H, Toyooka S, Tokumo M, Ichimura K, Aoe K, Ito S, et al. Detection of EGFR gene mutation in lung cancer by mutant-enriched polymerase chain reaction assay. Clin Cancer Res 2006;12:43–8. [19] Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55–63. [20] Haura EB, Zheng Z, Song L, Cantor A, Bepler G. Activated epidermal growth factor receptor-Stat-3 signaling promotes tumor survival in vivo in non-small cell lung cancer. Clin Cancer Res 2005;11:8288–94. [21] Gao SP, Mark KG, Leslie K, Pao W, Motoi N, Gerald WL, et al. Mutations in the EGFR kinase domain mediate STAT3 activation via IL-6 production in human lung adenocarcinomas. J Clin Invest 2007;117:3846–56. [22] Li Y, Du H, Qin Y, Roberts J, Cummings OW, Yan C. Activation of the signal transducers and activators of the transcription 3 pathway in alveolar epithelial cells induces inflammation and adenocarcinomas in mouse lung. Cancer Res 2007;67:8494–503. [23] Achcar Rde O, Cagle PT, Jagirdar J. Expression of activated and latent signal transducer and activator of transcription 3 in 303 non-small cell lung carcinomas and 44 malignant mesotheliomas: possible role for chemotherapeutic intervention. Arch Pathol Lab Med 2007;131:1350–60. [24] Song L, Turkson J, Karras JG, Jove R, Haura EB. Activation of Stat3 by receptor tyrosine kinases and cytokines regulates survival in human non-small cell carcinoma cells. Oncogene 2003;22:4150–65. [25] Zimmer S, Kahl P, Buhl TM, Steiner S, Wardelmann E, Merkelbach-Bruse S, et al. Epidermal growth factor receptor mutations in non-small cell lung cancer influence downstream Akt, MAPK and Stat3 signaling. J Cancer Res Clin Oncol 2009;135:723–30. [26] Nakanishi H, Matsumoto S, Iwakawa R, Kohno T, Suzuki K, Tsuta K, et al. Whole genome comparison of allelic imbalance between noninvasive and invasive small-sized lung adenocarcinomas. Cancer Res 2009;69:1615–23. [27] Garber K. JAK2 inhibitors: not the next imatinib but researchers see other possibilities. J Natl Cancer Inst 2009;101:980–2. [28] Sen B, Saigal B, Parikh N, Gallick G, Johnson FM. Sustained Src inhibition results in signal transducer and activator of transcription 3 (STAT3) activation and cancer cell survival via altered Janus-activated kinase-STAT3 binding. Cancer Res 2009;69:1958–65.