Ann Surg Oncol (2010) 17:2486–2493 DOI 10.1245/s10434-010-1040-1

ORIGINAL ARTICLE – THORACIC ONCOLOGY

Phosphorylated mTOR Expression is Associated with Poor Prognosis for Patients with Esophageal Squamous Cell Carcinoma Kotaro Hirashima, MD1,2, Yoshifumi Baba, MD, PhD1,2, Masayuki Watanabe, MD, PhD1, Ryu-ichi Karashima, MD1, Nobutaka Sato, MD1, Yu Imamura, MD, PhD1, Yukiharu Hiyoshi, MD, PhD1, Yohei Nagai, MD1, Naoko Hayashi, MD, PhD1, Ken-ichi Iyama, MD, PhD2, and Hideo Baba, MD, PhD1 1

Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan; 2Department of Surgical Pathology, Kumamoto University Hospital, Kumamoto, Japan

ABSTRACT Background. The mammalian target of rapamycin (mTOR) plays central roles in the regulation of cell growth and proliferation by monitoring nutrient availability, cellular energy level, oxygen level, and mitogenic signals. The aberrant activation of mTOR in relation to clinical outcome has been reported in several types of cancers. mTOR is increasingly important as a potential target for anticancer therapy. Nonetheless, a prognostic feature of mTOR activation in esophageal squamous cell carcinoma (ESCC) remains uncertain. Materials and Methods. First, in order to validate phospho-specific mTOR antibody (Ser2448), phosphorylated mTOR (p-mTOR) expression levels in five ESCC cell lines under cultural conditions with or without everolimus (mTOR inhibitor, also known as RAD001) were evaluated by in vitro immunohistochemistry and immunoblotting. Second, we examined p-mTOR expression by immunohistochemistry using 143 resected ESCC specimens. Prognostic significance of p-mTOR expression was examined by Cox regression and Kaplan–Meier analyses. Results. Among 143 patients, 71 (49.7%) were classified into p-mTOR-positive and 72 (50.3%) were classified into p-mTOR-negative. Compared with p-mTOR-negative patients, p-mTOR-positive patients experienced high overall mortality [hazard ratio (HR) 2.44; 95% confidence interval (CI), 1.24–4.83; P = 0.008], which persisted in multivariate analysis (multivariate HR 2.92; 95% CI, 1.48– 5.78; P = 0.002). A similar finding was observed for Ó Society of Surgical Oncology 2010 First Received: 3 December 2009; Published Online: 26 March 2010 H. Baba, MD, PhD e-mail: [email protected]

esophageal cancer-specific mortality. p-mTOR expression was not related with any clinical or pathologic variables including age, sex, tumor location, histological grading, operative procedure, T classification (tumor invasion), or lymph-node metastasis. Conclusions. p-mTOR overexpression was independently associated with poor prognosis in ESCC, supporting the potential for mTOR as a therapeutic target for ESCC.

Esophageal squamous cell carcinoma (ESCC), the major histological type of esophageal cancer in East Asian countries, is one of the most aggressive malignant tumors.1 Despite the development of multimodality therapies including surgery, chemotherapy, radiotherapy, and chemoradiotherapy, prognosis even of patients who underwent complete resection of their carcinomas remains poor.2–6 Limited improvement in treatment outcomes by conventional therapies urges us to seek innovative strategies for treating ESCC, especially those that are molecular targeted. In addition, identification of new prognostic molecular markers for ESCC could improve risk-adapted treatment strategies and help stratify patients in the future clinical trials for drugs targeting these molecules. The mammalian target of rapamycin [mTOR, mechanistic target of rapamycin (MTOR); the official symbol] is a serine/threonine kinase responsible for cellular growth and homeostasis.7–9 mTOR is activated by phosphorylation of Ser2448 through the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway and activates the eukaryotic translation factor 4E (elF4E) and the p70 ribosomal S6 kinase (p70S6 kinase).7–9 Previous studies on several types of cancers have shown that the aberrant activation of mTOR is associated with poor prognosis.10–13 Recently, mTOR has been recognized as an important and attractive

mTOR in Esophageal Squamous Cell Carcinoma

target for anticancer therapy.14–17 In particular, everolimus (RAD001), an oral mTOR inhibitor, has shown promising results in experimental studies, inhibiting tumor growth and having antiangiogenic effects.18–21 Thus, a better understanding of the mechanism and consequence of mTOR activation in human cancers is important for the purpose of cancer therapy targeting the PI3K/AKT/mTOR pathway. Nonetheless, mTOR activation status or its prognostic significance in ESCC remains uncertain. We therefore evaluated mTOR activity by immunohistochemistry for phosphorylated mTOR and determined clinical, pathologic, and prognostic features of mTOR activation among 143 ESCC patients. METHODS Patients The present study involved 143 consecutive patients with ESCC who underwent curative surgical resection at Kumamoto University Hospital from January 1996 to December 2006. A total of 120 patients underwent a transthoracic esophagectomy (TTE) with lymph node dissection, 14 patients underwent transhiatal esophagectomy (THE), and 9 patients underwent pharyngolaryngeal esophagectomy (PLE). None of these patients underwent endoscopic mucosal resection, palliative resection, preoperative chemotherapy, preoperative radiotherapy, or preoperative chemoradiotherapy. None of them had synchronous or metachronous multiple cancers in the other organs. Clinical data, including age at operation, sex, tumor location (Ut, upper thoracic; Mt, middle thoracic; Lt, lower thoracic), T classification (tumor invasion), N classification (lymph node metastasis), TNM stage, and histological grading were available for all 143 patients (Table 1). Patients were periodically (every 1–3 months) examined on an outpatient basis to make sure they did not have disease recurrence. Using our database with adequate patient follow-up, we previously demonstrated that molecular features in ESCC such as type IV collagen, p12CDK2-AP1, laminin-332, and type VII collagen were significantly associated with patient prognosis.22–25 Patients were observed until death or March 31, 2009, whichever came first. The mean follow-up period for 143 patients was 27.6 months (range, 1–133 months). Informed consent for the research was obtained from each patient. The study design was approved by the ethics review board of our university. Reagents and Antibodies Everolimus (RAD001) was provided from Novartis Pharma AG (Basel, Switzerland). Antibodies recognizing

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phospho-mTOR (Ser2448) and b-actin were purchased from Cell Signaling Technology (Boston, MA). Immunohistochemistry for Phosphorylated mTOR Tissue sections of 5 lm in thickness were deparaffinized and pretreated for antigen retrieval by autoclave heating (132°C) in 10 mM sodium citrate buffer (pH 6.0) for 5 min. These sections were blocked for endogenous peroxidase activity with 3% H2O2 in methanol for 60 min and then washed in phosphate-buffered saline (PBS). Thereafter, sections were immersed in 5% normal rabbit serum in PBS for 30 min, covered with primary rabbit monoclonal antibody specific for p-mTOR (Phospho-mTOR, Ser2448, dilution 1:50) and incubated overnight at 4°C. This antibody detects mTOR only when it is phosphorylated at Ser2448. Immunoreactions were performed using Vectastain peroxidase ABC kit (Vector Laboratories, Burlingame, CA). The antigenic sites were demonstrated by reacting the sections with a mixture of 0.05% 3,30 -diaminobenzidine tetrahydrochloride (Dojin Chemicals, Tokyo, Japan) in 0.05 mol/l Tris–HCl buffer, pH 7.6, containing 0.01% H2O2 for 7 min. The nuclei were stained with hematoxylin. The staining assessment was independently carried out by two pathologists (K. H. and K. I.) without any information about clinicopathological features or prognosis. The fraction of tumor cells with cytoplasmic p-mTOR expression (0–100%) was recorded, and the average value of 2 pathologists was calculated in each patient. There is currently no standardized cutoff (positive vs. negative expression) to evaluate mTOR activation status in human cancer by immunohistochemistry using paraffin-embedded specimens. Thus, in order to divide all cases (N = 143) in half, p-mTOR positivity was defined as the presence of at least cytoplasmic staining in C10% of tumor cells (N = 71). Either absent staining or staining in \10% of tumor cells was interpreted as negative (N = 72). Cell Culture Esophageal squamous cell cancer cell lines were obtained from the Cell Resource Center for Biomedical Research (Tohoku University, Japan). Cell cultures were grown in recommended medium with 10% fetal bovine serum and incubated in 5% CO2 at 37°C. For IHC staining, the cells were plated and grown on the slide glass (Matsunami Glass, Japan) in 6-well plates, fixed with 4% paraformaldehyde, and permeabilized with 0.2% PBST (Triton X-100). The similar staining protocol with tissue sections was used. The dilution for primary antibody was 1:25, and the incubation time was 1 h.

2488 TABLE 1 Clinical and pathologic features in relation to p-mTOR expression status

K. Hirashima et al.

Clinical and pathologic features

Total (n = 143)

Mean age

p-mTOR expression

P value

Positive (n = 71)

Negative (n = 72)

64.8 ± 9.5

62. 8 ± 9.8

0.23 0.08

Sex Male

126

59

67

Female

17

12

5

Tumor location Ut

20

8

12

Mt Lt

80 43

43 20

37 23

43

20

23

Histological grading Well

0.45

Moderately

65

30

35

Poor

18

12

6

Others

17

9

8

T1

86

44

42

T2

17

9

8

T3

40

18

22

T classification

0.50

N status

0.051

N0

81

46

35

N1

62

25

37

I IIA

62 19

34 12

28 7

IIB

33

14

19

III

29

11

18

PHE

9

3

6

THE

14

8

6

TTE

120

60

60

TNM staging

Ut upper thoracic, Mt middle thoracic, Lt lower thoracic, PLE pharyngolaryngeal esophagectomy, THE transhiatal esophagectomy, TTE transthoracic esophagectomy

0.23

Operative procedure

Immunoblotting Cultured cells were harvested and lysed in the lysis buffer [25 mM Tris–HCl (pH 7.4), 100 mM NaCl, 2 mM EDTA, 1% Triton X-100, Leupeptin, 1 mM Na3VO4, 1 mM PMSF] for 30 min. Lysates were centrifuged at 10,000 rev/min at 4°C for 5 min. Also, 10 lg of each protein sample was mixed with 59 sample buffer including 10% b-mercaptoethanol and boiled for 5 min. The total cellular protein extracts were separated on 7.5% SDSpolyacrylamide gels (SDS-PAGE) for p-mTOR, and 12.5% SDS-PAGE for b-actin, respectively. Next, these samples were transferred to PVDF membranes (Bio-Rad, Hercules, CA). The membranes were blocked in 5% skimmed milk in PBS containing 0.1% Tween 20 overnight at 4°C. The membranes were probed with each primary monoclonal antibody overnight at 4°C, followed by a 1:1,000 dilution of peroxidase-conjugated anti-rat IgG antibody (Sigma, St

0.48

0.53

Louis, MO). Detection was accomplished with an enhanced chemiluminescence (ECL) reagent (GE Healthcare, Piscataway, NJ). Statistical Analysis p-mTOR expression was assessed to identify any associations with clinicopathological parameters using v2 test or Fisher exact test. The Kaplan–Meier method was used to describe the distribution of overall survival time and esophageal cancer-specific survival time, and the log-rank test was used to evaluate the statistical significance of the differences. The prognostic significance of clinicopathological parameters was determined using univariate and multivariate Cox regression analysis. Multivariate Cox regression analysis included all variables: age at operation (continuous), sex (male vs. female), tumor location (Ut, Lt vs. Mt), T classification (T1, 2 vs. T3), N status (N0 vs.

mTOR in Esophageal Squamous Cell Carcinoma

N1), operative procedure (THE, PLE vs. TTE) and pmTOR expression (positive vs. negative). Statistical analysis was performed with Stat View-J 5.0 software (Abacus Concepts, Inc., Berkeley, CA). A two-sided significance level of P \ 0.05 was used for all statistical analysis. RESULT Validation of Phospho-Specific mTOR Antibody We performed immunoblotting and immunohistochemistry experiments in ESCC cell lines to validate phosphospecific mTOR antibody (Ser2448) to monitor activity of mTOR. Immunoblotting experiment showed that of the five ESCC cell lines tested, TE1, TE4, TE9, and TE13 cells had high levels of phosphorylated mTOR (p-mTOR) expression, and TE11 cells had low level of p-mTOR expression (Fig. 1a). Immunohistochemistry also showed a similar difference of p-mTOR expression in TE4 and TE11 cells (Fig. 1b). When TE4 cells were treated with everolimus (mTOR inhibitor, also known as RAD001), immunoblotting showed that everolimus could decrease p-mTOR expression in a dose-dependent manner in vitro assay (Fig. 1c). Immunohistochemistry using parallel samples from the everolimus experiment observed a similar decrease of p-mTOR expression (Fig. 1d). Similar results were observed in other cell lines with p-mTOR positive expression (TE1, 9, and 13). These studies confirm the reliability of this phospho-specific antibody for ESCC. p-mTOR Expression in Esophageal Squamous Cell Carcinoma We assessed p-mTOR expression (i.e., mTOR activation) using immunohistochemistry. Strong p-mTOR

FIG. 1 Immunohistochemistry and immunoblotting for p-mTOR in vitro. a Immunoblotting for p-mTOR and bactin in TE1, 4, 9, 11, and 13 cell lines. b Immunohistochemistry for p-mTOR in TE4 and 11 cell lines. c Immunoblotting for p-mTOR and bactin in TE4 cell lines under cultural conditions with everolimus or control. d Immunohistochemistry for p-mTOR in TE4 cell lines under cultural conditions with everolimus or control

2489

immunoreactivity was absent in normal squamous cell epithelium (Fig. 2a). p-mTOR immunoreactivity was observed in normal gastric gland and normal submucosal gland (Fig. 2b). Among 143 patients with ESCC, 71 patients (49.7%) showed positive for p-mTOR overexpression (Fig. 2c, d). p-mTOR expression was not associated with any clinical or pathologic features including age at operation, sex, tumor location, histological grading, operative procedure, T classification (tumor invasion), or N classification (lymph node metastasis) (Table 1). p-mTOR Expression and Clinical Outcome In Kaplan–Meier analysis, p-mTOR expression was significantly associated with shorter overall survival (log rank P = 0.008) (Fig. 3a). The 5-year overall survival rate was 44.4% in patients with p-mTOR positive tumors, while it was 77.5% in patients with p-mTOR negative tumors. In univariate Cox regression analysis, compared with p-mTOR-negative cases, p-mTOR-positive cases experienced a significantly higher overall mortality [hazard ratio (HR) 2.44; 95% confidence interval (CI), 1.24–4.83; P = 0.008] (Table 2). In the univariate analysis, other significant prognostic factors for overall survival included T classification (P = 0.003) and N status (P \ 0.0001). In multivariate Cox regression analysis, we included all variables: age at operation, sex, tumor location, T classification, N status, operative procedure, and p-mTOR expression. p-mTOR overexpression was independently associated with poor prognosis (multivariate HR 2.92; 95% CI, 1.48–5.78; P = 0.002) (Table 2). Similar finding was obtained for esophageal cancer-specific survival (log rank P = 0.05; Fig. 3b).

2490

K. Hirashima et al.

FIG. 2 Immunostaining for pmTOR. a Negative for p-mTOR expression in normal esophageal squamous epithelium (black arrows). b Positive for p-mTOR expression in submucosal gland (white arrow) and negative for pmTOR expression in normal esophageal squamous epithelium (black arrow). c Positive for pmTOR expression in esophageal squamous cell cancer cells (white arrowheads). d Negative for pmTOR expression in esophageal squamous cell cancer cells (black arrowheads). Original magnification: 9200

DISCUSSION We conducted this study to examine clinical, pathologic, and prognostic features of p-mTOR expression (i.e., mTOR activation) in ESCC. As mTOR is increasingly important as a therapeutic target for human cancers, better understanding mTOR activation in ESCC tissues may provide insights into new strategies for treating ESCC. Our current study using 143 resected ESCC tumors has shown that pmTOR expression is independently associated with poor prognosis, supporting the potential for mTOR as a therapeutic target for ESCC. There are currently no standardized methods (e.g., antibody, cutoff) to evaluate mTOR activity by immunohistochemistry using paraffin-embedded specimens. First, we validated the phospho-specific mTOR antibody (Ser2448) to assess mTOR activation in ESCC using 5 ESCC cell lines and everolimus (mTOR inhibitor, also known as RAD001). Interestingly, each cell line had a different p-mTOR expression level in vitro, suggesting the variousness of mTOR activity in ESCC. Next, immunohistochemistry was performed using 143 resected ESCC tissues. Normal esophageal squamous epithelium showed negative for p-mTOR expression, which was in agreement with previous reports.26,27 As normal gastric gland and normal esophageal submucosal gland showed positive for p-mTOR expression, we could use them as a positive

control in every run of immunohistochemistry. Our cutoff assessing cytoplasmic p-mTOR expression in C 10% of tumor cells yielded a significant association between pmTOR expression and poor prognosis in ESCC. Thus, this simple assessment method may be reasonable when one evaluates the activation level of mTOR pathway in human cancers, although we of course acknowledge that it might not be the best cutoff. Our intriguing findings and methods need to be confirmed by independent studies in the future. It remains controversial whether mTOR expression correlates with patient prognosis in human cancer. A previous study on ESCC has shown that p-mTOR expression is unrelated with clinical outcome, which is not consistent with our current study.27 This discrepancy might be due to a difference in the patient population tested or the method used to evaluate p-mTOR expression, or simply due to a chance variation between different studies. Regarding other types of human cancers, studies on cervical, hepatic, gastric, and biliary tract cancers have shown that mTOR expression is an adverse prognostic factor.10–13 On the other hand, studies on ovarian cancer and lung adenocarcinoma have shown that mTOR expression is associated with better prognosis.28,29 It is possible that a difference in mTOR status in each organ system may contribute to apparently discrepant results between studies on different organ systems. Anyway, our current study on ESCC supports that mTOR likely affects aggressive phenotype of

mTOR in Esophageal Squamous Cell Carcinoma

2491

a Overall Survival 1.0

P-mTOR-negative (n = 72) P-mTOR-positive (n = 71) p = 0.0008

0.8

0.6

0.4

0.2

0

2

4 6 8 Years after Operation

12

10

b Cancer-Specific Survival 1.0

P-mTOR-negative (n = 72) P-mTOR-positive (n = 71) p = 0.05

0.8

0.6

0.4

0.2

0

2

4 6 8 Years after Operation

10

12

FIG. 3 Kaplan–Meier curves according to p-mTOR status. a Overall survival curves according to p-mTOR status. b Esophageal cancerspecific survival curves according to p-mTOR status

ESCC and represents an attractive therapeutic target for ESCC. Interestingly, we also found the inverse relationship

TABLE 2 Univariate and multivariate analysis of overall survival in ESCC

Features

Total (n = 143)

between p-mTOR expression and lymph node metastasis, although this was not statistically significant (P = 0.051). This finding may suggest that mTOR expression contributes to patient poor prognosis by distant metastasis rather than by lymph node metastasis. Future studies are needed to confirm our findings as well as to elucidate exact mechanism of the possible pathogenic link between mTOR activation and tumor aggressiveness. The PI3K/AKT/mTOR signaling pathway is frequently altered in human cancers and increasingly crucial as a target for anticancer therapy.30–34 Rapamycin and its analogs including everolimus (RAD001), temsirolimus (CCI-779), and deforolimus (AP23573) are small molecules that selectively inhibit mTOR activity.34,35 These molecules have been shown to inhibit cell proliferation and induce apoptosis in cell lines derived from several types of human cancers.36,37 Among these molecules, everolimus is considered a promising candidate for clinical use, and several trials for different types of cancers have revealed the clinical effectiveness of this drug.38–41 Considering that several experimental studies have reported the therapeutic efficacy of mTOR inhibitors on ‘‘squamous cell carcinoma’’ of other organs, the clinical trial using these drugs may be conducted for ESCC in the near future.42–46 Our results identify a patient population that is characterized by high levels of mTOR activity, providing a potential patient pool that may be distinctly responsive to mTOR inhibitor therapy. Future studies focusing on the ability of mTOR inhibitors will be particularly useful in assessing whether p-mTOR expression can serve as a therapeutic biomarker for clinical trials involving mTOR inhibitory molecules. In this respect, our finding may have useful clinical implications. In summary, p-mTOR expression is independently associated with poor prognosis in ESCC. Our finding may support mTOR as a promising therapeutic target for ESCC. We currently plan further in vitro and in vivo study to clarify the antitumor effect of everolimus on ESCC.

Univariate analysis HR

95% CI

Multivariate analysis P value

HR

95% CI

P value

T classification T1

86

1.00

T2–3

57

2.74

81

1.00

N1 62 p-mTOR expression

5.46

1.00 1.41–5.29

0.003*

1.69

2.58–11.63

\0.0001*

5.26

1.24–4.83

0.008*

0.84–3.37

0.12

N status N0 HR hazard ratio, CI confidence interval * Significant at the significance level \0.05

Negative

72

1.00

Positive

71

2.44

1.00 2.33–11.11

\0.0001*

1.48–5.78

0.002*

1.00 2.92

2492

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