Ann Surg Oncol (2013) 20:1035–1043 DOI 10.1245/s10434-012-2680-0

ORIGINAL ARTICLE – TRANSLATIONAL RESEARCH AND BIOMARKERS

Overexpression of Forkhead Box M1 Transcription Factor (FOXM1) is a Potential Prognostic Marker and Enhances Chemoresistance for Docetaxel in Gastric Cancer Kaoru Okada, MD, Yoshiyuki Fujiwara, MD, PhD, Tsuyoshi Takahashi, MD, PhD, Yurika Nakamura, PhD, Shuji Takiguchi, MD, PhD, Kiyokazu Nakajima, MD, PhD, Hiroshi Miyata, MD, PhD, Makoto Yamasaki, MD, PhD, Yukinori Kurokawa, MD, PhD, Masaki Mori, MD, PhD, and Yuichiro Doki, MD, PhD Department of Gastroenterological Surgery, Graduate School of Medicine, Osaka University, Osaka, Japan

ABSTRACT Background. Mammalian forkhead box transcription factor 1 (FoxM1) has been overexpressed and correlated with pathogenesis in a variety of human malignancies. We investigated the expression status and clinical significance of its overexpression in gastric adenocarcinoma. Furthermore, we demonstrated correlations between FoxM1 overexpression and drug resistance to chemotherapeutic agents in gastric cancer cells and gastric cancer patients treated with chemotherapy. Methods. Fifty-three (69 %) of 77 tumors were diagnosed as positive for FoxM1 by immunohistochemistry. Multivariate analysis identified FoxM1 expression as a significant independent prognostic predictor for overall and disease-free survival in gastric cancer patients (hazard ratio 3.9 and 3.5, respectively). Furthermore, we investigated associations between FoxM1 overexpression and clinical response of chemotherapy for patients with advanced gastric cancer. Results. Our clinical results showed that FoxM1 overexpression was significantly associated with resistance in chemotherapy of docetaxel in addition to 5-fluorouracil (5-FU) plus S-1 plus cisplatin (CDDP) and was not significant in chemotherapy of 5-FU plus CDDP for patients with advanced gastric cancer. In vitro experiments showed that Mkn7 transfected FoxM1 siRNA significantly reduced chemoresistance to docetaxel over that with parental cell lines and Mkn45 transfected with FoxM1 significantly

Ó Society of Surgical Oncology 2012 First Received: 5 April 2012; Published Online: 2 October 2012 Y. Fujiwara, MD, PhD e-mail: [email protected]

enhanced chemoresistance to docetaxel over that with parental cell lines. Conclusions. Our study showed that FoxM1 was an independent prognostic factor in gastric cancer. Furthermore, we showed that FoxM1 was a critical molecule for chemoresistance to a microtubule-stabilizing anticancer agent, docetaxel. Taken together, those results suggest that inhibition of overexpressed FoxM1 will be a promising therapeutic strategy for advanced gastric cancer.

Gastric cancer is one of the most common malignancies in the digestive tract especially in Eastern countries including Japan. In spite of recent improvements in early diagnosis and treatment, gastric cancer is the second leading cause of cancer death worldwide.1,2 The prognosis of patients with advanced or recurrent gastric cancer remains poor and these patients should be treated with chemotherapy including taxanes (docetaxel and paclitaxel), fluoropyrimidines (S-1 and capecitabine), and irinotecan.3–10 Recently, it has been reported that a multitude of doublet and triplet regimens, especially taxane-based regimens, are useful in the treatment of advanced or recurrent gastric cancer.8–10 Therefore, identification of suitable biomarkers for predicting patient prognosis and chemosensitivity is important for improving therapeutic effects for patients with advanced gastric cancer. Forkhead box protein M1 (FoxM1) belongs to a member of the Forkhead family of transcription factors.11,12 FoxM1 plays important roles in the cell cycle by regulating both the transition from the G1 to S phase and progression to mitosis.12–14 FoxM1 is predominantly expressed in fetal tissues and its expression may be maintained in proliferating adult tissues.13,14 Recently, FoxM1 has been linked to tumorigenesis and progression of several kinds of malignancies.

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Overexpression of FoxM1 has been observed in various cancers of the liver, breast, prostate, brain, cervix, colon, and lung.15–21 Furthermore, down-regulation of FoxM1 leads to inhibition of cell growth, migration, and invasion in several cancer types.22–24 Moreover, overexpression of FoxM1 in tumor cell lines showed resistance to apoptosis or premature senescence induced by oxidative stress, showing strong implications in resistance to chemotherapy.25 It has been reported that FoxM1 overexpression is a prognostic factor in breast cancer and breast cancer cell lines overexpressing FoxM1 mediated resistance to trastuzumab, paclitaxel, and cisplatin.26,27 These results suggest that FoxM1 may have an important role in progression of human cancers and may be associated with drug resistance to anticancer agents. In gastric cancer, Zeng et al. showed that FoxM1 was overexpressed in gastric cancer and its inhibition led to cellular senescence, which depended on p27kip1.28 Furthermore, Li et al.29 reported that FoxM1 mediated promotion of human gastric cancer angiogenesis, growth, and metastasis. However, the clinical significance of FoxM1 overexpression in gastric cancer is still unclear. In this study, we examined the expression of FoxM1 protein in gastric cancer specimens and assessed correlations between FoxM1 overexpression and clinicopathological characteristics. In addition, we investigated the relationship between overexpression of FoxM1 and drug resistance to chemotherapeutic agents in gastric cancer cells and patients with gastric cancer treated with chemotherapy.

K. Okada et al.

patients were treated with combination chemotherapy with an oral-intake 5-FU derivative, S-1 plus cisplatin (CDDP).30 In DFP therapy, treatment consisted of docetaxel (60 mg/m2 on day 1), 5-FU (350 mg/m2/day continuous intravenous administration during days 1–5), and cisplatin (10 mg/m2/ day on days 1–5) and was repeated twice every 28 days before evaluating therapeutic responses with computed tomography and gastric endoscopy. In SP therapy, the treatment cycle consisted of S-1 (80 mg/m2/day on days 1–21) and cisplatin (60 mg/m2 on day 8) and was repeated twice every 35 days before evaluating chemotherapeutic responses. Tumor response was assessed by computed tomography after two cycles of each treatment and evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST) criteria.31 Complete response (CR) was defined as the disappearance of all evidence of cancer for more than four weeks. Partial response (PR) was defined as more than a 50 % reduction in the sum of the products of the perpendicular diameters of all lesions without any evidence of new regions or progression of any lesions. Stable disease (SD) was defined as less than a 50 % reduction or less than a 25 % increase in the sum of the products of the perpendicular diameters of all lesions, without any evidence of new lesions. Progressive disease (PD) was defined as more than a 25 % increase in more than one region or the appearance of new regions. Immunohistochemical (IHC) Examination

MATERIALS AND METHODS Patients and Tissue Specimens Gastric cancer tissues were obtained from 77 patients who underwent gastrectomy at the Department of Gastroenterological Surgery, Osaka University Hospital, from 2001 to 2008 and were subjected to immunohistochemistry. All tumors were histologically confirmed to be gastric adenocarcinoma. None had received preoperative treatment such as chemotherapy and/or radiotherapy. The median duration of follow-up was 65.8 months (range 2.0–134 months). Eightyone pretreatment specimens were obtained via biopsy under endoscopy from gastric cancer patients who underwent chemotherapy and were subjected to immunohistochemistry. Chemotherapeutic Regimens and Evaluation of Responses Thirty-six of 81 patients were treated with combination chemotherapy of docetaxel, 5-fluorouracil (5-FU), and cisplatin (modified DFP) before surgery.10 The remaining 45

The expression of FoxM1 was evaluated by IHC analyses with 4-lm-thick sections of 10 % formalin-fixed and paraffin-embedded blocks. For IHC staining, tissue slides were deparaffinized in xylene and then rehydrated through graded ethanol. For antigen retrieval, these slides were incubated by autoclave in 10 mM citrate buffer (pH 6.0) for 20 min. Endogenous peroxidase activity was blocked with 0.3 % hydrogen peroxide in methanol for 20 min. Nonspecific binding was blocked with 10 % normal serum for 20 min. Subsequently, tissue slides were incubated overnight with FoxM1 antibody (sc502, dilution 1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA) at 4 °C in a moist chamber. Sites of antibody binding were visualized with the ABC peroxidase detection system (Vector Laboratories, Burlingame, CA). Finally, sections were incubated in 3,30 -diaminobenzidine tetrahydrochloride with 0.05 % H2O2 for 1 min and counterstained with 0.1 % hematoxylin. The percentage of cancer cells stained with the antibody was evaluated. Expression of FoxM1 protein was defined as positive when more than 10 % of cancer cells stained positive for FoxM1.

Overexpression of FoxM1 in Gastric Cancer

Cell Lines and Culture Conditions Four gastric cancer derived cell lines, GC3, AGS, MKn7, and Mkn45 were obtained from the Riken Cell Bank (Tsukuba, Japan). AGS was cultured in Ham F12K (Wako Pure Chemical Industries, Osaka, Japan) and the other three cell lines were cultured in RPMI 1640 (SigmaAldrich, St. Louis, MO), supplemented with 10 % FBS (Thermo Fisher Scientific Inc., Waltham, MA) and 1 % penicillin/streptomycin (Invitrogen, Tokyo, Japan). These cell lines were incubated in 5 % CO2 at 37 °C. RNA Extraction Total cellular RNA was extracted from cell pellets of each cell line with Trizol (Invitrogen, Tokyo, Japan) reagent according to the protocol provided by the manufacturer. In brief, the mixture was minced with disposable homogenizers (IEDA, Tokyo, Japan), mixed with 0.2 ml chloroform, and centrifuged at 12,0009g for 15 min. The supernatant was transferred to a fresh tube and mixed with 0.5 ml 100 % isopropyl alcohol. After incubation for 10 min at room temperature, RNA was precipitated by centrifugation, washed with 75 % ethanol, and diluted with diethyl pyrocarbonate(DEPC)-treated water. Real-time Quantitative RT-PCR

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protocol. After transfection, cells were cultured for 72 h and intermediate samples were collected at 24 and 48 h and were analyzed by reverse-transcriptase PCR (RT-PCR), immunoblotting, and MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay. Immunoblotting Adherent cells were washed with ice-cold PBS and lysed in a radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific Inc., Waltham, MA). Lysates were spun and the supernatant was collected. Equal amounts of cell extracts (15 mg) were fractionated by SDS-PAGE (Bio-Rad Laboratories Inc. Hercules, CA) and transferred onto membranes (ImmobilonP, Millipore, Billerica, MA). After blockade induced through incubation with milk, membranes were incubated overnight at 4 °C with primary antibodies and with secondary antibodies for 1 hour at room temperature. The following antibodies were used in this study: anti-actin (dilution 1:1,000; SigmaAldrich) and anti-FoxM1 (dilution 1:1,000; Santa Cruz Biotechnology). Immune complexes were detected with a detection kit (GE Healthcare, Little Chalfont, UK). Cell Viability Assay and Chemotherapeutic Agents

The primer sequences for PCR amplification were as follows, FoxM1: 50 -CACCCCAGTGCCAACCGCTAC TTG-30 (forward) and 50 -AAAGAGGAGCTATCCCCTC CTCAG-30 (reverse), the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control: GAPDH: 50 -GGTCTCCTCTGACT TCAACA-30 (forward) and 50 -GTGAGGGTCTCTCTCTT CCT-30 (reverse). The integrity of all RNA samples was verified by quantitative RT-PCR for GAPDH in each sample. The emission intensity of SYBR Green was detected in realtime with a LightCycler 3.5 instrument (Roche Diagnostics, Mannheim, Germany). The value of FoxM1 expression was divided by that of GAPDH in each sample.

The MTT assay was used to assess cell viability. Cells were seeded onto 96-well plates at a concentration of 5 9 103 per well and incubated overnight under the usual culture conditions; they were exposed to each of docetaxel, cisplatin, and 5-FU at various concentrations. After 10 ll of MTT solution was added to each well, the plates were incubated for 4 h at 37 °C and formazan crystals were dissolved with 100 ll of 0.04 N HCl–isopropanol. The absorbance of individual wells was read at a 550-nm test wavelength and 655-nm reference wavelength with a microplate reader (Bio-Rad Laboratories Inc., Hercules, CA). Docetaxel (Wako Pure Chemical Industries, Osaka, Japan) was dissolved in DMSO. 5-FU and cisplatin (Wako Pure Chemical Industries, Osaka, Japan) were dissolved in RPMI 1640.

siFoxM1 and FoxM1 Transfection

Statistical Analysis and Ethical Considerations

Cells were cultured up to a 60–80 % confluence state and transfected with 5 nmol/L of siFoxM1 or negative control oligonucleotides (Life Technologies, Carlsbad, CA) with the siPORT NeoFX Transfection Agent (Life Technologies, Carlsbad, CA) in accordance with the manufacturer’s protocol. The FoxM1 cDNA plasmid was purchased from Kazusa DNA Res (Kisarazu, Japan) and transfected using Lipofectamine LTX agent (Invitrogen, Tokyo, Japan) in accordance with the manufacturer’s

Statistical analysis was performed with JMP software (JMP version 8.0.2, SAS Institute, Cary, NC). The relationship between FoxM1 expression and various clinicopathological parameters was assessed by the v2 test. Disease-free survival (DFS) and overall survival (OS) were assessed with the Kaplan–Meier method and compared by the log-rank test. All parameters that were found to be significant on univariate analysis by the Cox proportional hazard model were entered into multivariate survival

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K. Okada et al.

a

b

Normal gastric epithelium

Differentiated tumor

c

d

Undifferentiated tumor

Negative staining for FoxM1

f

e Log rank test P = 0.0229

Overall survival rate

1.0

1.0

0.8

0.8

0.6

0.6

0.4

0.4

0.2

0

0.2

FoxM1 positive (n=53) FoxM1 negative (n=24)

500

1000

1500

2000 2500

3000

3500

Time after surgery (days)

Log rank test P = 0.0162

Disease-free survival rate

0

FoxM1 positive (n=53) FoxM1 negative (n=24)

500

1000

1500

2000 2500

3000

3500

Time after surgery (days)

FIG. 1 Representative immunohistochemical staining for FoxM1 antibody (a–d). Kaplan–Meier analyses of overall survival (e) and diseasefree survival (f) according to the results of FoxM1 expression

analysis. p-values of \0.05 were considered significant. The study protocol was approved by the Human Ethics Review Committee of Osaka University and a signed informed consent form was obtained from each patient regarding the use of biopsy and surgical specimens.

cancer, 53 (69 %) were diagnosed as positive for FoxM1 immunohistochemistry and staining was mainly in the cytoplasm of tumor cells (Fig. 1b, c). The remaining 24 (31 %) negatively stained for FoxM1 (Fig. 1d). FoxM1positive cells were detected in various areas of tumor including the surface, central, and deep areas of gastric walls.

RESULTS

Correlations Between FoxM1 Expression in Gastric Cancer and Clinicopathological Parameters

FoxM1 Protein Expression in Gastric Cancer Tissues Normal gastric mucosa showed nonspecific weak staining for FoxM1 (Fig. 1a). Among the 77 specimens of gastric

Table 1 shows correlations between FoxM1 expression in gastric tumors and various clinicopathological parameters. The frequency of FoxM1 positive cases did not correlate with

Overexpression of FoxM1 in Gastric Cancer

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TABLE 1 Association between FoxM1 expression and various clinicopathological factors in 77 patients with gastric cancer Variable

Entire group (n = 77)

FoxM1 expression Negative (n = 24)

p value

Positive (n = 53)

correlated with DFS. Multivariate analysis identified pathological T stage and FoxM1 expression as significant independent prognostic predictors for DFS of gastric cancer patients (HR 5.2 and 3.5, respectively). FoxM1 Expression and Response to Chemotherapy

Age

0.6266

\70 years

45

13

32

C70 years

32

11

21

Male

52

13

39

Female

25

11

14

Gender

0.1174

Depth of tumor invasion

0.3303

pT1–2

32

12

20

pT3–4

45

12

33

Lymph node metastasis

0.4623

Negative

36

13

23

Positive

41

11

30

Lymphatic invasion

0.4235

No

21

8

13

Yes

56

16

40

Venous invasion No Yes

0.6076 50 27

17 7

33 20

Histopathology Differentiated

0.8066 39

13

26

Undifferentiated 38

11

27

any clinicopathological parameters. However, FoxM1 expression correlated significantly with poor OS and recurrence-free survival duration (Fig. 1e, f). Prognostic Significance of FoxM1 Expression in Gastric Cancer, and OS and Recurrence-free Survival Associations between various clinicopathological parameters and OS of patients were examined by Cox’s proportional hazard model (Table 2). Univariate analysis showed that pathological T stage, N stage, lymphovascular invasion, and FoxM1 expression correlated with OS. However, multivariate analysis identified FoxM1 expression as the only significant independent prognostic predictor for OS of gastric cancer patients (HR 3.9). Associations between various clinicopathological parameters and DFS were also examined by Cox’s proportional hazard model (Table 2). Univariate analysis showed that pathological T stage, N stage, vessel invasion, lymphovascular invasion, and FoxM1 expression significantly

An association between clinical response to chemotherapy and FoxM1 expression is shown in Table 3. Among the 36 specimens treated with DFP, 26 (72 %) were diagnosed as positive and 10 (28 %) were diagnosed as negative for FoxM1 immunohistochemistry. The response rate to DFP therapy in 26 patients with FoxM1 positive expression was 35 % (9 of 26), while the response rate to DFP therapy in ten patients diagnosed as negative was 80 % (8 of 10). There was a significant negative correlation between clinical response to DFP therapy and FoxM1 expression (p = 0.025). Among the 45 specimens treated with SP therapy, 29 (64 %) were diagnosed as positive and 16 (36 %) were diagnosed as negative for FoxM1 immunohistochemistry. The response rate to SP therapy in 29 patients diagnosed as positive for FoxM1 was 55 % and in 16 patients diagnosed as negative for FoxM1 was 38 %. There was no relationship between clinical response to SP therapy and FoxM1 expression (p = 0.353). FoxM1 mRNA Expression of Cell Lines and Sensitivity to Anticancer Drugs Expression of FoxM1 mRNA was the highest in Mkn7 and lowest in Mkn45 over that in other gastric cancer cell lines when we used quantitative RT-PCR (data not shown). To investigate the relationship between sensitivity to anticancer drugs and FoxM1 expression, we examined sensitivity to docetaxel, cisplatin, and 5-FU in MKn7 and Mkn45 cell lines by the MTT assay. Cells were treated continuously with docetaxel and cisplatin for 24 h, and 5-FU for 72 h. The MTT assay showed that MKn7 overexpressing FoxM1 showed higher resistance to docetaxel and 5-FU than that with Mkn45, in which FoxM1 expression was the lowest (data not shown).

Knockdown of FoxM1 Reduces Resistance to Docetaxel In order to elucidate whether FoxM1 is essential for resistance to docetaxel and 5-FU in gastric cancer cells, FoxM1 siRNA was transfected into the MKn7 cell line, which overexpressed FoxM1. RT-PCR and Western blot analysis indicated that FoxM1 siRNA reduced expression of FoxM1 mRNA and protein, whereas FoxM1 expression in mocktransfected and negative control-transfected cells remained unchanged (Fig. 2a). We examined chemosensitivity to

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TABLE 2 Results of univariate and multivariate analyses of overall survival and disease-free survival by Cox’s proportional hazard model Variable

n

Univariate analysis HR

95 % CI

Multivariate analysis p

HR

95 % CI

p

Overall survival Age (\70/C70 years)

45/32

2

0.8–4.7

0.1314

Gender (F/M)

25/52

2

0.7–7.0

0.1847

pT (1–2/3–4)

32/45

8

2.3–50

0.0004

4.6

0.9–38.4

0.0675

pN (0/1–3)

36/41

6.4

2.1–27.5

0.0004

4.4

0.9–80.8

0.0803

Vessel invasion (negative/positive)

50/27

1.9

0.8–4.7

0.1499

Lymphovascular invasion(negative/positive) Histopathology (differentiated/undifferentiated)

21/56 39/38

4.3 1.6

1.2–27 0.7–4.1

0.0181 0.2893

0.4

FoxM1 (negative/positive)

24/53

4.7

1.3–29.3

0.0119

3.9

1.1–24.7

0.0298

5.2

1.1–41.4

0.0433

0.02–4.7

0.4641

Disease-free survival Age (\70/C70 years)

45/32

1.5

0.7–3.3

0.3309

Gender (F/M)

25/52

2.1

0.8–6.2

0.122

pT (1–2/3–4)

32/45

10.4

3.1–65.1

\0.0001

pN (0/1–3)

36/41

6

2.3–20.7

0.0001

3

0.8–19.1

0.0999

Vessel invasion(negative/positive)

50/27

2.6

1.2–5.9

0.017

1.4

0.6–3.4

0.4051

Lymphovascular invasion (negative/positive)

21/56

5.4

1.6–34.1

0.0036

0.6

0.1–5.9

0.6784

Histopathology (differentiated/undifferentiated)

39/38

1.3

0.6–3.1

0.4493

FoxM1 (negative/positive)

24/53

3.9

1.4–16.6

0.0089

3.5

1.2–15.1

0.0183

TABLE 3 Association between FoxM1 expression and responses to DFP or SP chemotherapy Response FoxM1 expression in cases with DFP Negative (n = 10) CR

0

PR SD PD

Positive (n = 26)

FoxM1 expression in cases with SP Negative (n = 16)

Positive (n = 29)

0

0

1

8

9

6

15

2

15

7

9

0

2

3

4

DFP combination chemotherapy with docetaxel, 5-fluorouracil (5-FU), and cisplatin, SP combination chemotherapy with an oralintake 5-FU derivative, S-1 plus cisplatin

docetaxel, cisplatin, and 5-FU by the MTT assay using MKn7 cells transfected with FoxM1 siRNA for 48 h and parental cell lines. The MTT assay showed that chemoresistance to docetaxel was significantly lower in Mkn7 transfected FoxM1 siRNA than that in parental cell lines (IC50; 20 vs. 2.8 lg/ml), while resistance to cisplatin and 5-FU remained unchanged (Fig. 2b). Up-regulation of FoxM1 Increases Resistance to Docetaxel To further investigate where FoxM1 overexpression has any effect on resistance to docetaxel, the FoxM1 cDNA

plasmid was transfected into MKn45 cell lines, which showed down-regulation of FoxM1. Up-regulation of FoxM1 mRNA and protein was confirmed by quantitative RT-PCR and western blot analysis (Fig. 3a). Chemoresistance to docetaxel was significantly higher in Mkn45 with overexpressed FoxM1 than that in parental cell lines (IC50; 1.25 vs. [20 lg/ml) (Fig. 3b). DISCUSSION In this study, we examined the clinical significance of FoxM1 overexpression in human gastric cancer. Although there were no significant associations between FoxM1 overexpression and clinicopathological factors such as pathological T factor, nodal involvement, and histological differentiation, FoxM1 overexpression was an independent prognostic factor for DFS and OS in human gastric cancer (Fig. 1e, f). Recently, Li et al.29 also examined FoxM1 overexpression in gastric cancer tissues and classified three grades of immunostaining, negative, moderate, and strong, and showed that strong FoxM1 expression was significantly associated with shorter survival duration than negative cases. They also showed that overexpressed FoxM1 enhanced tumorigenicity and metastasis of human gastric cancer cells in animal models and that its overexpression directly regulated expression of the VEGF gene and had associations with microvessel density (MVD) in gastric tumors, which may be related to tumor development and

Overexpression of FoxM1 in Gastric Cancer FIG. 2 Knockdown of FoxM1 expression recovers chemosensitivity for docetaxel and overexpressed FoxM1 enhances chemoresistance in gastric cancer cells. a qRT-PCR analysis and western blot analysis for FoxM1 expression after siRNA transfection. P parental cells, M mock transfection, N transfection with negative control, Si siFoxM1 transfection. b MTT assay with Mkn7 cells transfected with siFoxM1 exposed to docetaxel, cisplatin, and 5-FU

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a FoxM1/GAPDH P M

140

N Si

48 H

120

P

M

N

72H Si

P

M

N

Si

100 FoxM1

80 60

actin

40 20 48 H

24 H

72 H

b Cell viability (%)

DTX

CDDP

5-FU

*P < 0.05

100

100

*

80

80

60

60

60

40

40

40

100 *

80

20

20

MKn7 MKn7+siFoxM1

0

0.3

0.6

2.5

20

MKn7 MKn7+siFoxM1

10

0

0.3

µg/ml

1.3

10

20

MKn7 MKn7+siFoxM1

0

0.4

µg/ml

1.6

6.3

25

µg/ml

a FoxM1/GAPDH

1.60E+02 1.40E+02

MKN45+FoxM1 cDNA

1.20E+02

P

1.00E+02

24H

48H

72H

FoxM1

8.00E+01 6.00E+01

actin

4.00E+01 2.00E+01 24H

P

48H

72H

b Cell viability (%)

DTX *P < 0.05

80 60 *

5-FU

CDDP

100

100

100

80

80

60

60

40

40

* *

40

*

20

20

MKn45 MKn45+siFoxM1

0

0.3

0.6

1.2

20

20

MKn7 MKn7+siFoxM1

0

0.3

µg/ml FIG. 3 FoxM1 cDNA plasmid was transfected into MKn45 cell lines, which showed down-regulation of FoxM1 (a) qRT-PCR and western blot analyses of FoxM1 expression in MKn45 cells

1.3

5

µg/ml

10 20

MKn7 MKn7+siFoxM1

0

0.4

1.6

6.3

25

µg/ml

transfected with the FoxM1 cDNA plasmid. b MTT assay with Mkn45 transfected with the FoxM1 cDNA plasmid exposed to docetaxel, cisplatin, and 5-FU

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progression. By means of gastric cancer cell lines, Zeng et al. showed that inhibition of overexpressed FoxM1 in gastric cancer cells triggered senescence and clonogenicity of tumor cells by up-regulating expression of the CDK inhibitor p27kip1 and by down-regulating the oncogene cMyc, which led to reductions in expression of the cMyc target, telomerase reverse transcriptase (hTERT). These findings indicated that inhibition of FoxM1 was a promising therapeutic strategy for gastric cancer treatment. Furthermore, we investigated associations between FoxM1 overexpression and the clinical response of chemotherapy for patients with advanced gastric cancer. Our clinical results showed that FoxM1 overexpression was significantly associated with resistance to chemotherapy of docetaxel in addition to 5-FU plus CDDP and was not significant in chemotherapy of 5-FU plus CDDP only for patients with advanced gastric cancer. Quantitative RT-PCR confirmed that FoxM1 was highly expressed in MKn7 cell lines and was expressed at lower levels in MKn45. MKn7 overexpressing FoxM1 showed higher resistance to docetaxel and 5-FU than that with Mkn45, which indicated that overexpression of FoxM1 may be involved in chemoresistance to docetaxel and 5-FU in gastric cancer cells. Further experiments showed that chemoresistance to docetaxel in Mkn7 transfected FoxM1 siRNA was significantly recovered comparing to that in parental cell lines; however, resistance to cisplatin and 5-FU remained unchanged. Chemoresistance to docetaxel in Mkn45 with overexpressed FoxM1 was significantly higher than that in parental cell lines. These results confirmed that a potential prognostic factor, FoxM1 was also associated with chemoresistance to docetaxel in human gastric cancer and not to 5-FU and CDDP. Using breast cancer cells, Carr et al. reported that FoxM1 functioned chemoresistance to a microtubulestabilizing anticancer drug, paclitaxel by direct transcriptional regulation of Stathmin, which was known as a regulator of microtubule dynamics.26,32 As docetaxel is also an anticancer agent, which binds to b-tubulin and stabilizes microtubules consisting a/b-tubulin dimers, resulting in mitotic failure like paclitaxel, the same mechanism should occur in gastric cancer cells. In our study, FoxM1 was not associated with chemoresistance to CDDP in gastric cancer, although Kwok et al. identified that FoxM1 was a critical factor for CDDP resistance in one breast cancer cell line, MCF-7.27 The mechanism of difference in chemoresistance to CDDP requires further investigation. It may depend on different cell lines or different originated organs, such as the breast or stomach. In conclusion, our study showed that FoxM1 was an independent prognostic factor for disease-free and OS in gastric cancer. Furthermore, we also showed that FoxM1 was a critical molecule for chemoresistance to a microtubulestabilizing anticancer agent, docetaxel. Taken together, these results suggest that inhibition of overexpressed

K. Okada et al.

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