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Forkhead box M1 expression in pulmonary squamous cell carcinoma: correlation with clinicopathologic features and its prognostic significance
Human Pathology (2009) 40, 464–470
www.elsevier.com/locate/humpath
Original contribution
Forkhead box M1 expression in pulmonary squamous cell carcinoma: correlation with clinicopathologic features and its prognostic significance☆ Doo Kyung Yang MD a , Choon Hee Son MD, PhD a , Soo Keol Lee MD, PhD a , Phil Jo Choi MD, PhD b , Kyung Eun Lee MSc c , Mee Sook Roh MD, PhD c,d,⁎ a
Department of Internal Medicine, Dong-A University College of Medicine, Busan 602-715, South Korea Department of Thoracic and Cardiovascular Surgery, Dong-A University College of Medicine, Busan 602-715, South Korea c Medical Research Center for Cancer Molecular Therapy, Dong-A University College of Medicine, Busan 602-715, South Korea d Department of Pathology, Dong-A University College of Medicine, Busan 602-715, South Korea b
Received 16 June 2008; revised 29 September 2008; accepted 1 October 2008
Keywords: Immunohistochemical expression; Forkhead box M1; Lung squamous cell carcinoma; Prognosis
Summary Forkhead box M1 (FoxM1) transcription factor has been shown to play important roles in regulating the expression of genes that are involved in cell proliferation, differentiation, and transformation by promoting both G1/S and G2/M transition. Although it has been reported that the FoxM1 signaling network is frequently deregulated with an up-regulated FoxM1 expression in human malignancies, the role of FoxM1 in lung cancer remains to be determined. We performed immunohistochemical detection of FoxM1 protein in 69 tissue samples from patients with primary pulmonary squamous cell carcinoma using a tissue microarray, and Western blotting was done to confirm the immunohistochemical observations. FoxM1 immunoreactivity was observed in 26 (37.7%) of the 69 squamous cell carcinoma cases. Analysis of the FoxM1 expression in 12 squamous cell carcinoma tissues and 2 normal lung tissues by Western blotting confirmed the immunohistochemical results. A FoxM1 expression was more frequently detected in the moderately or poorly differentiated squamous cell carcinomas than in the well-differentiated squamous cell carcinomas (P = .008). The tumors with a positive FoxM1 expression more frequently showed lymph node metastasis (P = .027) and an advanced American Joint Committee on Cancer stage (P = .049). The Kaplan-Meier survival curves demonstrated that patients with a positive FoxM1 expression had a significantly shorter survival time than those patients with a negative FoxM1 expression (P = .003). The multivariate analysis revealed that the FoxM1 expression was an independent poor prognostic factor (P = .018). A subset of pulmonary squamous cell carcinoma with a FoxM1 expression was associated with progressive pathologic features and an aggressive clinical course. © 2009 Elsevier Inc. All rights reserved.
☆ This work was supported by the Korea Science and Engineering Foundation through the Medical Research Center for Cancer Molecular Therapy (MRCCMT) at the Dong-A University, Busan, South Korea. ⁎ Corresponding author. Department of Pathology, Dong-A University College of Medicine, Busan 602-715, South Korea. E-mail address: [email protected] (M. S. Roh).
0046-8177/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2008.10.001
Forkhead box M1 expression in pulmonary squamous cell carcinoma
1. Introduction
2. Materials and methods
Non–small cell lung cancer (NSCLC) constitutes 80% to 85% of all the primary lung cancer cases, and adenocarcinoma (AC) and squamous cell carcinoma (SCC) are the 2 major histopathologic subtypes of this group [1]. Although many reports have described biologic markers for NSCLC, SCC and AC have different pathogenetic pathways and distinct biologic characteristics. Consistently, gefitinib was less effective against SCC than AC, despite the fact that SCC showed higher epidermal growth factor receptor expression [2]. Therefore, to improve the poor prognosis of patients with SCC, biologic markers that can predict prognosis and response toward a specific therapy should be established in the treatment of SCC. The development of pulmonary SCC is a multistep process that includes the gain of function mutations that activate the cell cycle-promoting Ras/mitogen-activated protein kinase signaling pathway [3]. Activated mitogenactivated protein kinase kinase has recently been shown to directly phosphorylate Forkhead box M1 (FoxM1) protein, and this contributes to its transcriptional activation [4]. The FoxM1 transcription factor has been shown to play important roles in regulating the expression of genes involved in cell proliferation, differentiation, and transformation [5]. It has recently been reported that alterations in FoxM1 signaling are associated with tumorigenesis in some cancers [6-14]. FoxM1 is ubiquitously expressed in all proliferating mammalian cells, whereas its expression is extinguished in cells that are undergoing terminal differentiation [15]. The up-regulated expression of FoxM1 prevents differentiation, and this ultimately guides undifferentiated cells toward malignant transformation [7]. Furthermore, Kim et al [9] showed that FoxM1 is essential for the proliferation and development of lung cancer in urethane-induced mouse lung tumors. Depletion of the FoxM1 levels in the A549 lung cancer cell line by short interfering RNA (siRNA) transfection caused diminished DNA replication and mitosis; it decreased the expression of the cell cyclepromoting cyclin A2 and cyclin B1 genes, and it reduced the anchorage-independent growth of cell colonies on soft agar. These data show that FoxM1 stimulates the proliferation of tumor cells during progression of lung cancer. Based on these published reports, we wanted to examine the role of FoxM1 in the development and proliferation of human pulmonary SCC, which is one of the most important members of NSCLC. We performed immunohistochemical detection of FoxM1 protein in pulmonary SCC tissue samples by a using tissue microarray (TMA) to determine whether the immunohistochemical expression of FoxM1 could provide useful information as a novel therapeutic or prognostic option for treating primary pulmonary SCC. This study is the first study to critically determine the clinicopathologic roles of the expression of FoxM1 in patients with pulmonary SCC.
2.1. Patients and tissues
465
Tissue samples were obtained from 69 Korean patients who underwent surgical resection for primary pulmonary SCC at Dong-A University Medical Center, Busan, South Korea, from 2000 to 2004. No preoperative chemotherapy or radiotherapy had been performed in any of these cases. Standard lobectomy and lymph node dissections were performed in every case. The cases having any other malignancies that occurred before or after the primary lung cancer were excluded from our study. At the time of performing thoracotomy, the mediastinal lymph nodes were dissected as completely as possible, and this included the ipsilateral, paratracheal, lower mediastinal, subcarinal, and N1 areas to arrive at an accurate pathologic staging. The clinical records, pathologic reports, and follow-up information were also obtained when available. The institutional review board at Dong-A University Medical Center approved our study, and written informed consent was obtained from all the patients for surgery and to use their resected samples for research. The hematoxylin and eosin– stained slides were reviewed in each case to confirm the original diagnosis, which was based on the World Health Organization criteria [16]. According to the location of the primary tumor site, the cases were classified into a central type and a peripheral type [17]. The postoperative pathologic staging was determined according to the guidelines of the American Joint Committee on Cancer (AJCC) [18].
2.2. Construction of TMA One-millimeter cores were removed from the SCCs that had previously been formalin fixed and paraffin embedded. For all the arrays, 3 cores of different areas of the tumor were removed from each case, and these were put in a new blank recipient paraffin block in a previously described manner [19], and 4-μm-thick sections were taken for all the immunohistochemical staining. Full cross-sections from the paraffin blocks were used for 5 of the SCCs along with the adjacent normal lung tissue to confirm the staining patterns seen on the TMA.
2.3. Immunohistochemistry Immunohistochemical staining for FoxM1 was performed on the TMA slides by using the avidin-biotin-peroxidase complex method. Deparaffinization of all sections was performed through a series of xylene baths, and rehydration was performed with a series of graded alcohol solutions. To enhance the immunoreactivity, we performed microwave antigen retrieval at 750 W for 30 minutes in citrate buffer (pH 6.0). After blocking the endogenous peroxidase activity with 5% hydrogen peroxidase for 10 minutes, incubation with the
466 primary antibody was performed for 1 hour at room temperature. The primary antibody was a rabbit polyclonal antibody directed against FoxM1 (clone K-19; Santa Cruz Biotechnology, Santa Cruz, CA) used at a 1:100 dilution. An Envision Chem Detection Kit (DakoCytomation, Carpinteria, CA) was used for the secondary antibody at room temperature for 30 minutes. After washing the tissue samples in Tris-buffered saline for 10 minutes, 3,3′-diaminobenzidine was used as a chromogen, and then Mayer's hematoxylin counterstain was applied.
2.4. Immunohistochemical assessment The percentage and intensity of the immunoreactive tumor cells in each core was recorded, and the final value of the positive tumor cells was determined as the mean of the immunoreactivity in 3 cores. The presence of tumor tissue in at least 2 interpretable cores was required to include a case in the statistical analysis. All the slides were evaluated independently by an experienced pathologist (M. S. Roh) and one of the authors (K. E. Lee) without knowledge of any of the clinicopathologic data. There were only minor discrepancies in the evaluation. Those slides with discrepant evaluation were reevaluated under a multihead microscope until a consensus evaluation was obtained. FoxM1 immunoreactivity was defined as those showing a nuclear with/ without cytoplasmic staining pattern of the lesional tissue with a minimal background. The percentage scoring of the immunoreactive tumor cells was as follows: 0 (0%), 1 (1%10%), 2 (11%-50%), and 3 (N50%). The staining intensity was visually scored and stratified as follows: 0 (negative), 1 (weak, if it was a blush), and 2 (strong, if it was obviously positive at original magnification ×20). A final score was obtained for each case by multiplying the percentage and the intensity score. Therefore, tumors with multiplied score exceeding 4 (ie, tumors with a strong intensity of N10% of the tumor cells) was recorded as positive immunoreactivity to FoxM1; all the other scores were considered to be negative.
2.5. Western blot analysis Twelve SCCs (5 cases with a negative FoxM1 expression and 7 cases with a positive FoxM1 expression) and 2 samples of adjacent normal lung tissue were analyzed. All the tissue specimens were snap frozen within 20 minutes after excision, and they were stored at −80°C. The cryofrozen tissue samples were lysed in PRO-PREP protein extraction solution (iNtRON Biotechnology Inc, Korea), and comparable amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; the proteins were then transferred to a nitrocellulose membrane. The membrane was blocked in 5% nonfat milk for 1 hour, and then it was incubated overnight with an antibody against FoxM1 (clone K-19, 1:200 dilution; Santa Cruz Biotechnology). Anti–β-actin (clone AC-15, 1:2000 dilution; Sigma Chemi-
D. K. Yang et al. cal, St Louis, MO) was used for the loading controls. The signals from the primary antibody were amplified by horseradish peroxidase-conjugated antirabbit immunoglobulin G (Chemicon, Temecula, CA) and detected by using Enhanced Chemiluminescence Plus (Amersham Pharmacia Biotech, Piscataway, NJ), followed by autoradiography.
2.6. Statistical analysis Associations between the FoxM1 expression and the clinicopathologic characteristics were analyzed using the χ2 test or Fisher exact test according to test condition. Student t test with Yates correction was used for the continuous variables. The survival probabilities were estimated using the Kaplan-Meier method, and they were compared using the log-rank test. The overall survival time was defined as the time from the date of surgery to the date of death as a result of any cause. The patients who were alive at the date of the last follow-up were censored on that date plus 1 day. Multivariate Cox proportional hazard regression analysis was used to assess the prognostic significance of the FoxM1 expression and the other clinicopathologic characteristics on survival. Overall, 95% confidence intervals were used throughout the analysis. Statistical significance was defined as P b .05. All the statistical tests were performed with the Statistical Package, SPSS 14.0 for Windows (SPSS Inc, Chicago, IL).
3. Results 3.1. Clinicopathologic characteristics The patients consisted of 65 men and 4 women, and they ranged in age from 43 to 84 years (median, 62 years). The tumor size ranged from 1.3 to 11 cm, with 20 cases involving tumors 3 cm or smaller, whereas 49 cases involved tumors larger than 3 cm. Histologically, the tumors were all SCCs, and they showed 21 in the well-differentiated subgroup, 35 in the moderately differentiated subgroup, and 13 in the poorly differentiated subgroup. According to location, there were 48 central types and 21 peripheral types. There were 57 negative cases and 12 positive cases of lymphovascular invasion. There were 41 negative cases and 28 positive cases of lymph node metastases. According to the AJCC staging system, 11 patients were stage IA, 26 were stage IB, 1 was stage IIA, 13 were stage IIB, 13 were stage IIIA, and 5 were stage IIIB.
3.2. Immunohistochemical findings of FoxM1 All the cores for each tumor demonstrated similar staining characteristics. Five cases had only 2 cores with enough tissue to evaluate. The staining patterns of the TMA cores
Forkhead box M1 expression in pulmonary squamous cell carcinoma
467
Table 1 Relationship between staining intensity and percentage of FoxM1 expression cells in 69 SCC tissues Intensity
Weak Strong
% of FoxM1 expression cells 0-10
11-50
N50
20 3
15 16
5 10
P
.002
adjacent normal lung from 5 full cross-sections; thus, the nonneoplastic bronchial, alveolar epithelial cells, and endothelial cells were randomly and heterogeneously reactive for FoxM1 (Fig. 1A). In the tumor tissue, FoxM1 immunoreactivity was observed in 26 (37.7%) of the 69 SCC cases. The expression was limited to only the tumor cells without any background labeling. The staining intensity and percentage were well concordant (P = .002) (Table 1).
3.3. Correlation between the FoxM1 immunoreactivity and the Western blot analysis To confirm the immunohistochemical observations, Western blotting was performed with equal amount of the total lysates from 7 SCCs with a positive FoxM1 expression, 5 SCCs with a negative FoxM1 expression, and 2 samples of normal lung tissue. Consistent with the immunohistochemical results, Western blot analysis showed that the total levels of FoxM1 protein were increased in the tumor tissues with a positive FoxM1 expression, as compared with the tumor tissues with a negative FoxM1 expression or the normal lung tissue (Fig. 2).
3.4. Correlation between FoxM1 immunoreactivity and the clinicopathologic characteristics The various clinicopathologic characteristics of the patients and their tumors were compared according to the
Fig. 1 Immunohistochemical staining of FoxM1 in lung tissue. In the adjacent normal lung tissue, the nonneoplastic alveolar epithelial cells were randomly and heterogeneously reactive for FoxM1 (arrows) (A). In pulmonary SCC, a well-differentiated SCC showed negative expression of FoxM1 except for a few nuclear staining (arrows) and nonspecific weak staining in keratin pearl (arrowhead) (B), whereas a poorly differentiated SCC showed positive expression of FoxM1 (C).
showed concordant results with those of the 5 full crosssections. The expression of FoxM1 protein was detected in the nuclei with/without cytoplasms of both the normal cells and tumor cells. The FoxM1 expression was limited in the
Fig. 2 Western blot detection of FoxM1 in tumor tissues with a positive FoxM1 expression (lanes 5-7) compared with a normal tissue (lane 1) and the tumor tissues with a negative FoxM1 expression (lanes 2-4). Equal amounts of protein were loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the proteins were subsequently transferred to a nitrocellulose membrane. Immunodetection was performed with anti-FoxM1 antibody. Anti–β-actin antibody was used to control for equal loading. Western blot analysis showed consistent results with the immunohistochemical study.
468
D. K. Yang et al.
Table 2 Correlations of the FoxM1 expression with the conventional clinicopathologic factors in 69 patients with SCC Clinicopathologic factors
Age ≤60 N60 Sex Male Female Tumor size ≤3 cm N3 cm Smoking history (pack-years) Differentiation Well Moderate/poor Location Central Peripheral Lymphovascular invasion Negative Positive Lymph node metastasis Negative Positive AJCC stage I II/III
FoxM1 expression Positive (n = 26)
Negative (n = 43)
7 19
23 20
24 2
41 2
9 17 27.8 ± 18.6
11 32 28.6 ± 18.5
3 23
18 25
18 8
30 13
P
.056
.629
.597
.855 .008
.823
.521 20 6
37 6
11 15
30 13
10 16
27 16
.027
Fig. 3 The overall survival curves after surgical therapy grouped by FoxM1 expression, calculated by the Kaplan-Meier method. The positive FoxM1 expression group (n = 26, green line) has significantly worse survival than the negative FoxM1 expression group (n = 43, blue line) (P = .003).
.049
FoxM1 immunoreactivity (Table 2). The FoxM1 expression was more frequently detected in the moderately or poorly differentiated SCCs than in the well-differentiated SCCs (P = 0.008) (Fig. 1B and C). The tumors with a positive FoxM1 expression more frequently showed lymph node metastasis (P = .027) and an advanced AJCC stage (P = .049). There was no significant association with age, sex, smoking history, tumor size, tumor location, or lymphovascular invasion.
3.5. The influence of FoxM1 expression on recurrence and survival Adequate clinical follow-up information was available for all 69 cases. The mean follow-up of the 35 cases was 47.2 months, ranging from 5.2 to 97.3 months. Forty-six (66.7%) were still alive, but 23 (33.3%) died during the follow-up period. Of the latter, 17 died of documented progressive lung cancer, 4 of respiratory dysfunction, and 2 of another diseases. Twenty-five patients (36.2%) had recurrences during the follow-up period: 6 had distant recurrences that included the liver, adrenal gland, and bone, and there were
19 locoregional recurrences. The mean duration from the surgery to the recurrence in these 25 patients was 30 months, ranging from 3.2 to 97.3 months. The 5-year survivals of the patients with a negative and positive FoxM1 expression were 79.1% and 36.2%, respectively. The Kaplan-Meier survival curves demonstrated that the patients with a positive FoxM1
Table 3 Cox multivariate analysis to determine the independent prognostic value of different variables in relation to the overall survival of 69 patients with SCC Covariate
Risk ratio (95% confidence interval)
P
Age (≤60 vs N60) Sex (male vs female) Tumor size (≤3 vs N3 cm) Smoking (− vs +) Differentiation (well/ moderate/poor) Location (central vs peripheral) Lymphovascular invasion (− vs +) Lymph node metastasis (− vs +) TNM stage (I/II/III) FoxM1 (− vs +)
0.603 (0.191-1.902) 1.56 (0.78-2.92) 1.692 (0.114-25.124) 6.042 (0.778-46.921) 1.422 (0.682-2.967)
.388 .982 .703 .085 .348
0.983 (0.435-1.452)
.642
1.282 (0.317-5.176)
.727
1.701 (0.333-3.687)
.075
1.341 (0.434-2.957) 3.972 (1.168-12.440)
.079 .018
Forkhead box M1 expression in pulmonary squamous cell carcinoma expression had a significantly shorter survival time than those patients with a negative FoxM1 expression (P = .003) (Fig. 3). The multivariate analysis using the Cox proportional hazard model revealed that a FoxM1 expression was an independent poor prognostic factor (P = .018) (Table 3).
4. Discussion In the present investigation, we have shown that FoxM1 expression occurred in pulmonary SCC and a subset of SCC cancers with a FoxM1 expression was correlated with progressive pathologic factors including poor histologic differentiation, lymph node metastasis, and an advanced tumor stage. The patients with a positive FoxM1 expression had a significantly shorter survival time than those patients with a negative FoxM1 expression. The multivariate analysis revealed that a positive FoxM1 expression was an independent poor prognostic factor for pulmonary patients with SCC. These findings suggest that a FoxM1 expression may contribute to the development or progression of pulmonary SCC in vivo, and so, it could be a novel prognostic factor for patients with pulmonary SCC. To the best of our knowledge, this is the first report about the relationship between the FoxM1 expression and the prognosis in patients with primary pulmonary SCC. FoxM1 is known to stimulate the transcription of genes that are essential for progression into DNA replication and mitosis [5,20,21]. Dysfunction of FoxM1 may prevent differentiation, and this ultimately guides undifferentiated cells toward malignant transformation [22]. Kalinichenko et al [23] developed transgenic mice in which the Rosa26 promoter was used to drive the ubiquitous expression of the human FoxM1 complementary DNA and revealed that transgenic overexpression of FoxM1 accelerated the onset of DNA replication and mitosis in mouse lung after butylated hydroxytoluene-mediated lung injury. In human NSCLC, increased levels of FoxM1 mRNA were confirmed by quantitative real-time reverse transcriptase polymerase chain reaction analysis with the use of primers specific for the FoxM1 gene [9]. However, the relationship of a FoxM1 expression with the clinicopathologic factors has hardly been analyzed in human cancer tissues, including lung cancer. A study on brain tumors showed that the levels of FoxM1 expression was significantly higher in glioblastoma multiforms than in anaplastic astrocytomas and low-grade astrocytomas, and an increased expression of FoxM1 in glioblastoma multiform was associated with decreased patient survival [8]. Consistent with these results, our study also showed that the subset of pulmonary SCC with a FoxM1 expression was associated with progressive pathologic features and an aggressive clinical course. Despite the recent advances in both diagnostic modalities and therapeutic strategies, pulmonary SCC remains one of the deadliest human cancers [1]. Therefore, identification of
469
new molecular targets that are essential for the proliferation of tumor cell will benefit both the treatment and chemoprevention of pulmonary SCC. Some studies [7,9] have provided a foundation for exploring the use of FoxM1targeted therapeutic strategies for the care of cancer patients. FoxM1 siRNA-transfected pancreatic cancer cells showed a decreased expression of cyclin B, cyclin D1, and cyclindependent kinase2, whereas the p27kip1 and p21cip1 expressions were increased. These cells also reduced the expression of matrix metalloproteinase-2, matrix metalloproteinase-9, and vascular endothelial growth factor, resulting in the inhibition of migration, invasion, and angiogenesis of pancreatic cancer cells [7]. FoxM1 siRNA-transfected U87MG glioblastoma cells showed a reduced expression of FoxM1, and these cells produced no brain tumors in most of the mice injected with them [8]. An induced expression of the Mx-cre recombinase transgene resulted in conditional deletion of the FoxM1 fl/fl-targeted allele, which caused a significant reduction in the proliferation of lung tumor cells, as well as in the number and size of lung adenomas after urethane treatment [9]. Therefore, further understanding of the biology and molecular mechanisms of FoxM1 in the development and progression of pulmonary SCC could provide advances for the treatment of pulmonary SCC, and the down-regulation of FoxM1 could potentially be an effective therapeutic approach for pulmonary SCC. In conclusion, our current study shows a role for FoxM1 in the development and progression of pulmonary SCC. This study indicates that FoxM1 immunoreactivity may identify a subset of pulmonary SCC that was associated with progressive pathologic features and an aggressive clinical course. However, we emphasize here that this study was not able to demonstrate the statistical significance of AJCC stage and lymph node metastasis, which are known in the literature to be of prognostic significance, on multivariate analysis. This was the result because our study is limited by the number of study cases with relatively small and heterogeneous stage subgroups and by retrospective study. Further prospective investigations with a large number of cases would allow us to evaluate FoxM1 in a variety of clinical settings to help us better understand its unique role in pulmonary SCC progression. These results further suggest that strategies to reduce the FoxM1 expression could represent a valuable therapeutic approach for the treatment of human pulmonary SCC.
References [1] Parkin DM, Bray F, Ferlay J, et al. Global cancer statistics, 2002. CA Cancer J Clin 2005;55:74-108. [2] Cappuzzo F, Magrini E, Ceresoli GL, et al. Akt phosphorylation and gefitinib efficacy in patients with advanced non–small-cell lung cancer. J Natl Cancer Inst 2004;96:1133-41. [3] McCormick F. Signaling networks that cause cancer. Trends Cell Biol 1999;9:M53-6.
470 [4] Ma RY, Tong TH, Cheung AM, et al. Raf/MEK/MAPK signaling stimulates the nuclear translocation and transactivating activity of FOXM1c. J Cell Sci 2005;118:795-806. [5] Katoh M, Katoh M. Human FOX gene family (review). Int J Oncol 2004;25:1495-500. [6] Teh MT, Wong ST, Neill GW, et al. FOXM1 is a downstream target of Gli1 in basal cell carcinomas. Cancer Res 2002;62:4773-80. [7] Wang Z, Banerjee S, Kong D, Li Y, Sarkar FH. Down-regulation of Forkhead Box M1 transcription factor leads to the inhibition of invasion and angiogenesis of pancreatic cancer cells. Cancer Res 2007; 67:8293-300. [8] Liu M, Dai B, Kang SH, et al. FoxM1B is overexpressed in human glioblastomas and critically regulates the tumorigenicity of glioma cells. Cancer Res 2006;66:3593-602. [9] Kim IM, Ackerson T, Ramakrishna S, et al. The Forkhead Box m1 transcription factor stimulates the proliferation of tumor cells during development of lung cancer. Cancer Res 2006;66:2153-61. [10] Kalin TV, Wang IC, Ackerson TJ, et al. Increased levels of the FoxM1 transcription factor accelerate development and progression of prostate carcinomas in both TRAMP and LADY transgenic mice. Cancer Res 2006;66:1712-20. [11] Yoshida Y, Wang IC, Yoder HM, Davidson NO, Costa RH. The Forkhead Box M1 transcription factor contributes to the development and growth of mouse colorectal cancer. Gastroenterology 2007;132: 1420-31. [12] Gusarova GA, Wang IC, Major ML, et al. A cell-penetrating ARF peptide inhibitor of FoxM1 in mouse hepatocellular carcinoma treatment. J Clin Invest 2007;117:99-111. [13] Madureira PA, Varshochi R, Constantinidou D, et al. The Forkhead box M1 protein regulates the transcription of the estrogen receptor α in breast cancer cells. J Bio Chem 2006;281:25167-76.
D. K. Yang et al. [14] Obama K, Ura K, Li M, et al. Genome-wide analysis of gene expression in human intrahepatic cholangiocarcinoma. Hepatology 2005;41:1339-48. [15] Ye H, Kelly TF, Samadani U, et al. Hepatocyte nuclear factor 3/fork head homolog 11 is expressed in proliferating epithelial and mesenchymal cells of embryonic and adult tissues. Mol Cell Biol 1997;17:1626-41. [16] Travis WD, Brambilla E, Muller-Hermelink HK, Harris CC. World Health Organization International Histological Classification of Tumours. Pathology and genetics of tumors of the lung, pleura, thymus and heart. Lyon: IARC Press; 2004. [17] Saijo T, Ishii G, Nagai K, et al. Differences in clinicopathological and biological features between central-type and peripheral-type squamous cell carcinoma of the lung. Lung Cancer 2006;52:37-45. [18] Mountain CF. Revisions in the international staging for lung cancer. Chest 1997;111:1711-7. [19] Hsu FD, Nielsen TO, Alkushi A, et al. Tissue microarrays are an effective quality assurance tool for diagnostic immunohistochemistry. Mod Pathol 2002;13:1374-80. [20] Wonsey DR, Follettie MT. Loss of the forkhead transcription factor FoxM1 causes centrosome amplification and mitotic catastrophe. Cancer Res 2005;65:5181-9. [21] Leung TW, Lin SS, Tsang AC, et al. Over-expression of FoxM1 stimulates cyclin B1 expression. FEBS Lett 2001;507:59-66. [22] Wang X, Quail E, hung NJ, et al. Increased levels of forkhead box M1B transcription factor in transgenic mouse hepatocytes prevent agerelated proliferation defects in regenerating liver. Proc Natl Acad Sci USA 2001;98:11468-73. [23] Kalinichenko VV, Gusarova GA, Tan Y, et al. Ubiquitous expression of the Forkhead Box M1B transgene accelerates proliferation of distinct pulmonary cell types following lung injury. J Biol Chem 2003; 278:37888-94.
www.elsevier.com/locate/humpath
Original contribution
Forkhead box M1 expression in pulmonary squamous cell carcinoma: correlation with clinicopathologic features and its prognostic significance☆ Doo Kyung Yang MD a , Choon Hee Son MD, PhD a , Soo Keol Lee MD, PhD a , Phil Jo Choi MD, PhD b , Kyung Eun Lee MSc c , Mee Sook Roh MD, PhD c,d,⁎ a
Department of Internal Medicine, Dong-A University College of Medicine, Busan 602-715, South Korea Department of Thoracic and Cardiovascular Surgery, Dong-A University College of Medicine, Busan 602-715, South Korea c Medical Research Center for Cancer Molecular Therapy, Dong-A University College of Medicine, Busan 602-715, South Korea d Department of Pathology, Dong-A University College of Medicine, Busan 602-715, South Korea b
Received 16 June 2008; revised 29 September 2008; accepted 1 October 2008
Keywords: Immunohistochemical expression; Forkhead box M1; Lung squamous cell carcinoma; Prognosis
Summary Forkhead box M1 (FoxM1) transcription factor has been shown to play important roles in regulating the expression of genes that are involved in cell proliferation, differentiation, and transformation by promoting both G1/S and G2/M transition. Although it has been reported that the FoxM1 signaling network is frequently deregulated with an up-regulated FoxM1 expression in human malignancies, the role of FoxM1 in lung cancer remains to be determined. We performed immunohistochemical detection of FoxM1 protein in 69 tissue samples from patients with primary pulmonary squamous cell carcinoma using a tissue microarray, and Western blotting was done to confirm the immunohistochemical observations. FoxM1 immunoreactivity was observed in 26 (37.7%) of the 69 squamous cell carcinoma cases. Analysis of the FoxM1 expression in 12 squamous cell carcinoma tissues and 2 normal lung tissues by Western blotting confirmed the immunohistochemical results. A FoxM1 expression was more frequently detected in the moderately or poorly differentiated squamous cell carcinomas than in the well-differentiated squamous cell carcinomas (P = .008). The tumors with a positive FoxM1 expression more frequently showed lymph node metastasis (P = .027) and an advanced American Joint Committee on Cancer stage (P = .049). The Kaplan-Meier survival curves demonstrated that patients with a positive FoxM1 expression had a significantly shorter survival time than those patients with a negative FoxM1 expression (P = .003). The multivariate analysis revealed that the FoxM1 expression was an independent poor prognostic factor (P = .018). A subset of pulmonary squamous cell carcinoma with a FoxM1 expression was associated with progressive pathologic features and an aggressive clinical course. © 2009 Elsevier Inc. All rights reserved.
☆ This work was supported by the Korea Science and Engineering Foundation through the Medical Research Center for Cancer Molecular Therapy (MRCCMT) at the Dong-A University, Busan, South Korea. ⁎ Corresponding author. Department of Pathology, Dong-A University College of Medicine, Busan 602-715, South Korea. E-mail address: [email protected] (M. S. Roh).
0046-8177/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2008.10.001
Forkhead box M1 expression in pulmonary squamous cell carcinoma
1. Introduction
2. Materials and methods
Non–small cell lung cancer (NSCLC) constitutes 80% to 85% of all the primary lung cancer cases, and adenocarcinoma (AC) and squamous cell carcinoma (SCC) are the 2 major histopathologic subtypes of this group [1]. Although many reports have described biologic markers for NSCLC, SCC and AC have different pathogenetic pathways and distinct biologic characteristics. Consistently, gefitinib was less effective against SCC than AC, despite the fact that SCC showed higher epidermal growth factor receptor expression [2]. Therefore, to improve the poor prognosis of patients with SCC, biologic markers that can predict prognosis and response toward a specific therapy should be established in the treatment of SCC. The development of pulmonary SCC is a multistep process that includes the gain of function mutations that activate the cell cycle-promoting Ras/mitogen-activated protein kinase signaling pathway [3]. Activated mitogenactivated protein kinase kinase has recently been shown to directly phosphorylate Forkhead box M1 (FoxM1) protein, and this contributes to its transcriptional activation [4]. The FoxM1 transcription factor has been shown to play important roles in regulating the expression of genes involved in cell proliferation, differentiation, and transformation [5]. It has recently been reported that alterations in FoxM1 signaling are associated with tumorigenesis in some cancers [6-14]. FoxM1 is ubiquitously expressed in all proliferating mammalian cells, whereas its expression is extinguished in cells that are undergoing terminal differentiation [15]. The up-regulated expression of FoxM1 prevents differentiation, and this ultimately guides undifferentiated cells toward malignant transformation [7]. Furthermore, Kim et al [9] showed that FoxM1 is essential for the proliferation and development of lung cancer in urethane-induced mouse lung tumors. Depletion of the FoxM1 levels in the A549 lung cancer cell line by short interfering RNA (siRNA) transfection caused diminished DNA replication and mitosis; it decreased the expression of the cell cyclepromoting cyclin A2 and cyclin B1 genes, and it reduced the anchorage-independent growth of cell colonies on soft agar. These data show that FoxM1 stimulates the proliferation of tumor cells during progression of lung cancer. Based on these published reports, we wanted to examine the role of FoxM1 in the development and proliferation of human pulmonary SCC, which is one of the most important members of NSCLC. We performed immunohistochemical detection of FoxM1 protein in pulmonary SCC tissue samples by a using tissue microarray (TMA) to determine whether the immunohistochemical expression of FoxM1 could provide useful information as a novel therapeutic or prognostic option for treating primary pulmonary SCC. This study is the first study to critically determine the clinicopathologic roles of the expression of FoxM1 in patients with pulmonary SCC.
2.1. Patients and tissues
465
Tissue samples were obtained from 69 Korean patients who underwent surgical resection for primary pulmonary SCC at Dong-A University Medical Center, Busan, South Korea, from 2000 to 2004. No preoperative chemotherapy or radiotherapy had been performed in any of these cases. Standard lobectomy and lymph node dissections were performed in every case. The cases having any other malignancies that occurred before or after the primary lung cancer were excluded from our study. At the time of performing thoracotomy, the mediastinal lymph nodes were dissected as completely as possible, and this included the ipsilateral, paratracheal, lower mediastinal, subcarinal, and N1 areas to arrive at an accurate pathologic staging. The clinical records, pathologic reports, and follow-up information were also obtained when available. The institutional review board at Dong-A University Medical Center approved our study, and written informed consent was obtained from all the patients for surgery and to use their resected samples for research. The hematoxylin and eosin– stained slides were reviewed in each case to confirm the original diagnosis, which was based on the World Health Organization criteria [16]. According to the location of the primary tumor site, the cases were classified into a central type and a peripheral type [17]. The postoperative pathologic staging was determined according to the guidelines of the American Joint Committee on Cancer (AJCC) [18].
2.2. Construction of TMA One-millimeter cores were removed from the SCCs that had previously been formalin fixed and paraffin embedded. For all the arrays, 3 cores of different areas of the tumor were removed from each case, and these were put in a new blank recipient paraffin block in a previously described manner [19], and 4-μm-thick sections were taken for all the immunohistochemical staining. Full cross-sections from the paraffin blocks were used for 5 of the SCCs along with the adjacent normal lung tissue to confirm the staining patterns seen on the TMA.
2.3. Immunohistochemistry Immunohistochemical staining for FoxM1 was performed on the TMA slides by using the avidin-biotin-peroxidase complex method. Deparaffinization of all sections was performed through a series of xylene baths, and rehydration was performed with a series of graded alcohol solutions. To enhance the immunoreactivity, we performed microwave antigen retrieval at 750 W for 30 minutes in citrate buffer (pH 6.0). After blocking the endogenous peroxidase activity with 5% hydrogen peroxidase for 10 minutes, incubation with the
466 primary antibody was performed for 1 hour at room temperature. The primary antibody was a rabbit polyclonal antibody directed against FoxM1 (clone K-19; Santa Cruz Biotechnology, Santa Cruz, CA) used at a 1:100 dilution. An Envision Chem Detection Kit (DakoCytomation, Carpinteria, CA) was used for the secondary antibody at room temperature for 30 minutes. After washing the tissue samples in Tris-buffered saline for 10 minutes, 3,3′-diaminobenzidine was used as a chromogen, and then Mayer's hematoxylin counterstain was applied.
2.4. Immunohistochemical assessment The percentage and intensity of the immunoreactive tumor cells in each core was recorded, and the final value of the positive tumor cells was determined as the mean of the immunoreactivity in 3 cores. The presence of tumor tissue in at least 2 interpretable cores was required to include a case in the statistical analysis. All the slides were evaluated independently by an experienced pathologist (M. S. Roh) and one of the authors (K. E. Lee) without knowledge of any of the clinicopathologic data. There were only minor discrepancies in the evaluation. Those slides with discrepant evaluation were reevaluated under a multihead microscope until a consensus evaluation was obtained. FoxM1 immunoreactivity was defined as those showing a nuclear with/ without cytoplasmic staining pattern of the lesional tissue with a minimal background. The percentage scoring of the immunoreactive tumor cells was as follows: 0 (0%), 1 (1%10%), 2 (11%-50%), and 3 (N50%). The staining intensity was visually scored and stratified as follows: 0 (negative), 1 (weak, if it was a blush), and 2 (strong, if it was obviously positive at original magnification ×20). A final score was obtained for each case by multiplying the percentage and the intensity score. Therefore, tumors with multiplied score exceeding 4 (ie, tumors with a strong intensity of N10% of the tumor cells) was recorded as positive immunoreactivity to FoxM1; all the other scores were considered to be negative.
2.5. Western blot analysis Twelve SCCs (5 cases with a negative FoxM1 expression and 7 cases with a positive FoxM1 expression) and 2 samples of adjacent normal lung tissue were analyzed. All the tissue specimens were snap frozen within 20 minutes after excision, and they were stored at −80°C. The cryofrozen tissue samples were lysed in PRO-PREP protein extraction solution (iNtRON Biotechnology Inc, Korea), and comparable amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; the proteins were then transferred to a nitrocellulose membrane. The membrane was blocked in 5% nonfat milk for 1 hour, and then it was incubated overnight with an antibody against FoxM1 (clone K-19, 1:200 dilution; Santa Cruz Biotechnology). Anti–β-actin (clone AC-15, 1:2000 dilution; Sigma Chemi-
D. K. Yang et al. cal, St Louis, MO) was used for the loading controls. The signals from the primary antibody were amplified by horseradish peroxidase-conjugated antirabbit immunoglobulin G (Chemicon, Temecula, CA) and detected by using Enhanced Chemiluminescence Plus (Amersham Pharmacia Biotech, Piscataway, NJ), followed by autoradiography.
2.6. Statistical analysis Associations between the FoxM1 expression and the clinicopathologic characteristics were analyzed using the χ2 test or Fisher exact test according to test condition. Student t test with Yates correction was used for the continuous variables. The survival probabilities were estimated using the Kaplan-Meier method, and they were compared using the log-rank test. The overall survival time was defined as the time from the date of surgery to the date of death as a result of any cause. The patients who were alive at the date of the last follow-up were censored on that date plus 1 day. Multivariate Cox proportional hazard regression analysis was used to assess the prognostic significance of the FoxM1 expression and the other clinicopathologic characteristics on survival. Overall, 95% confidence intervals were used throughout the analysis. Statistical significance was defined as P b .05. All the statistical tests were performed with the Statistical Package, SPSS 14.0 for Windows (SPSS Inc, Chicago, IL).
3. Results 3.1. Clinicopathologic characteristics The patients consisted of 65 men and 4 women, and they ranged in age from 43 to 84 years (median, 62 years). The tumor size ranged from 1.3 to 11 cm, with 20 cases involving tumors 3 cm or smaller, whereas 49 cases involved tumors larger than 3 cm. Histologically, the tumors were all SCCs, and they showed 21 in the well-differentiated subgroup, 35 in the moderately differentiated subgroup, and 13 in the poorly differentiated subgroup. According to location, there were 48 central types and 21 peripheral types. There were 57 negative cases and 12 positive cases of lymphovascular invasion. There were 41 negative cases and 28 positive cases of lymph node metastases. According to the AJCC staging system, 11 patients were stage IA, 26 were stage IB, 1 was stage IIA, 13 were stage IIB, 13 were stage IIIA, and 5 were stage IIIB.
3.2. Immunohistochemical findings of FoxM1 All the cores for each tumor demonstrated similar staining characteristics. Five cases had only 2 cores with enough tissue to evaluate. The staining patterns of the TMA cores
Forkhead box M1 expression in pulmonary squamous cell carcinoma
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Table 1 Relationship between staining intensity and percentage of FoxM1 expression cells in 69 SCC tissues Intensity
Weak Strong
% of FoxM1 expression cells 0-10
11-50
N50
20 3
15 16
5 10
P
.002
adjacent normal lung from 5 full cross-sections; thus, the nonneoplastic bronchial, alveolar epithelial cells, and endothelial cells were randomly and heterogeneously reactive for FoxM1 (Fig. 1A). In the tumor tissue, FoxM1 immunoreactivity was observed in 26 (37.7%) of the 69 SCC cases. The expression was limited to only the tumor cells without any background labeling. The staining intensity and percentage were well concordant (P = .002) (Table 1).
3.3. Correlation between the FoxM1 immunoreactivity and the Western blot analysis To confirm the immunohistochemical observations, Western blotting was performed with equal amount of the total lysates from 7 SCCs with a positive FoxM1 expression, 5 SCCs with a negative FoxM1 expression, and 2 samples of normal lung tissue. Consistent with the immunohistochemical results, Western blot analysis showed that the total levels of FoxM1 protein were increased in the tumor tissues with a positive FoxM1 expression, as compared with the tumor tissues with a negative FoxM1 expression or the normal lung tissue (Fig. 2).
3.4. Correlation between FoxM1 immunoreactivity and the clinicopathologic characteristics The various clinicopathologic characteristics of the patients and their tumors were compared according to the
Fig. 1 Immunohistochemical staining of FoxM1 in lung tissue. In the adjacent normal lung tissue, the nonneoplastic alveolar epithelial cells were randomly and heterogeneously reactive for FoxM1 (arrows) (A). In pulmonary SCC, a well-differentiated SCC showed negative expression of FoxM1 except for a few nuclear staining (arrows) and nonspecific weak staining in keratin pearl (arrowhead) (B), whereas a poorly differentiated SCC showed positive expression of FoxM1 (C).
showed concordant results with those of the 5 full crosssections. The expression of FoxM1 protein was detected in the nuclei with/without cytoplasms of both the normal cells and tumor cells. The FoxM1 expression was limited in the
Fig. 2 Western blot detection of FoxM1 in tumor tissues with a positive FoxM1 expression (lanes 5-7) compared with a normal tissue (lane 1) and the tumor tissues with a negative FoxM1 expression (lanes 2-4). Equal amounts of protein were loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the proteins were subsequently transferred to a nitrocellulose membrane. Immunodetection was performed with anti-FoxM1 antibody. Anti–β-actin antibody was used to control for equal loading. Western blot analysis showed consistent results with the immunohistochemical study.
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Table 2 Correlations of the FoxM1 expression with the conventional clinicopathologic factors in 69 patients with SCC Clinicopathologic factors
Age ≤60 N60 Sex Male Female Tumor size ≤3 cm N3 cm Smoking history (pack-years) Differentiation Well Moderate/poor Location Central Peripheral Lymphovascular invasion Negative Positive Lymph node metastasis Negative Positive AJCC stage I II/III
FoxM1 expression Positive (n = 26)
Negative (n = 43)
7 19
23 20
24 2
41 2
9 17 27.8 ± 18.6
11 32 28.6 ± 18.5
3 23
18 25
18 8
30 13
P
.056
.629
.597
.855 .008
.823
.521 20 6
37 6
11 15
30 13
10 16
27 16
.027
Fig. 3 The overall survival curves after surgical therapy grouped by FoxM1 expression, calculated by the Kaplan-Meier method. The positive FoxM1 expression group (n = 26, green line) has significantly worse survival than the negative FoxM1 expression group (n = 43, blue line) (P = .003).
.049
FoxM1 immunoreactivity (Table 2). The FoxM1 expression was more frequently detected in the moderately or poorly differentiated SCCs than in the well-differentiated SCCs (P = 0.008) (Fig. 1B and C). The tumors with a positive FoxM1 expression more frequently showed lymph node metastasis (P = .027) and an advanced AJCC stage (P = .049). There was no significant association with age, sex, smoking history, tumor size, tumor location, or lymphovascular invasion.
3.5. The influence of FoxM1 expression on recurrence and survival Adequate clinical follow-up information was available for all 69 cases. The mean follow-up of the 35 cases was 47.2 months, ranging from 5.2 to 97.3 months. Forty-six (66.7%) were still alive, but 23 (33.3%) died during the follow-up period. Of the latter, 17 died of documented progressive lung cancer, 4 of respiratory dysfunction, and 2 of another diseases. Twenty-five patients (36.2%) had recurrences during the follow-up period: 6 had distant recurrences that included the liver, adrenal gland, and bone, and there were
19 locoregional recurrences. The mean duration from the surgery to the recurrence in these 25 patients was 30 months, ranging from 3.2 to 97.3 months. The 5-year survivals of the patients with a negative and positive FoxM1 expression were 79.1% and 36.2%, respectively. The Kaplan-Meier survival curves demonstrated that the patients with a positive FoxM1
Table 3 Cox multivariate analysis to determine the independent prognostic value of different variables in relation to the overall survival of 69 patients with SCC Covariate
Risk ratio (95% confidence interval)
P
Age (≤60 vs N60) Sex (male vs female) Tumor size (≤3 vs N3 cm) Smoking (− vs +) Differentiation (well/ moderate/poor) Location (central vs peripheral) Lymphovascular invasion (− vs +) Lymph node metastasis (− vs +) TNM stage (I/II/III) FoxM1 (− vs +)
0.603 (0.191-1.902) 1.56 (0.78-2.92) 1.692 (0.114-25.124) 6.042 (0.778-46.921) 1.422 (0.682-2.967)
.388 .982 .703 .085 .348
0.983 (0.435-1.452)
.642
1.282 (0.317-5.176)
.727
1.701 (0.333-3.687)
.075
1.341 (0.434-2.957) 3.972 (1.168-12.440)
.079 .018
Forkhead box M1 expression in pulmonary squamous cell carcinoma expression had a significantly shorter survival time than those patients with a negative FoxM1 expression (P = .003) (Fig. 3). The multivariate analysis using the Cox proportional hazard model revealed that a FoxM1 expression was an independent poor prognostic factor (P = .018) (Table 3).
4. Discussion In the present investigation, we have shown that FoxM1 expression occurred in pulmonary SCC and a subset of SCC cancers with a FoxM1 expression was correlated with progressive pathologic factors including poor histologic differentiation, lymph node metastasis, and an advanced tumor stage. The patients with a positive FoxM1 expression had a significantly shorter survival time than those patients with a negative FoxM1 expression. The multivariate analysis revealed that a positive FoxM1 expression was an independent poor prognostic factor for pulmonary patients with SCC. These findings suggest that a FoxM1 expression may contribute to the development or progression of pulmonary SCC in vivo, and so, it could be a novel prognostic factor for patients with pulmonary SCC. To the best of our knowledge, this is the first report about the relationship between the FoxM1 expression and the prognosis in patients with primary pulmonary SCC. FoxM1 is known to stimulate the transcription of genes that are essential for progression into DNA replication and mitosis [5,20,21]. Dysfunction of FoxM1 may prevent differentiation, and this ultimately guides undifferentiated cells toward malignant transformation [22]. Kalinichenko et al [23] developed transgenic mice in which the Rosa26 promoter was used to drive the ubiquitous expression of the human FoxM1 complementary DNA and revealed that transgenic overexpression of FoxM1 accelerated the onset of DNA replication and mitosis in mouse lung after butylated hydroxytoluene-mediated lung injury. In human NSCLC, increased levels of FoxM1 mRNA were confirmed by quantitative real-time reverse transcriptase polymerase chain reaction analysis with the use of primers specific for the FoxM1 gene [9]. However, the relationship of a FoxM1 expression with the clinicopathologic factors has hardly been analyzed in human cancer tissues, including lung cancer. A study on brain tumors showed that the levels of FoxM1 expression was significantly higher in glioblastoma multiforms than in anaplastic astrocytomas and low-grade astrocytomas, and an increased expression of FoxM1 in glioblastoma multiform was associated with decreased patient survival [8]. Consistent with these results, our study also showed that the subset of pulmonary SCC with a FoxM1 expression was associated with progressive pathologic features and an aggressive clinical course. Despite the recent advances in both diagnostic modalities and therapeutic strategies, pulmonary SCC remains one of the deadliest human cancers [1]. Therefore, identification of
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new molecular targets that are essential for the proliferation of tumor cell will benefit both the treatment and chemoprevention of pulmonary SCC. Some studies [7,9] have provided a foundation for exploring the use of FoxM1targeted therapeutic strategies for the care of cancer patients. FoxM1 siRNA-transfected pancreatic cancer cells showed a decreased expression of cyclin B, cyclin D1, and cyclindependent kinase2, whereas the p27kip1 and p21cip1 expressions were increased. These cells also reduced the expression of matrix metalloproteinase-2, matrix metalloproteinase-9, and vascular endothelial growth factor, resulting in the inhibition of migration, invasion, and angiogenesis of pancreatic cancer cells [7]. FoxM1 siRNA-transfected U87MG glioblastoma cells showed a reduced expression of FoxM1, and these cells produced no brain tumors in most of the mice injected with them [8]. An induced expression of the Mx-cre recombinase transgene resulted in conditional deletion of the FoxM1 fl/fl-targeted allele, which caused a significant reduction in the proliferation of lung tumor cells, as well as in the number and size of lung adenomas after urethane treatment [9]. Therefore, further understanding of the biology and molecular mechanisms of FoxM1 in the development and progression of pulmonary SCC could provide advances for the treatment of pulmonary SCC, and the down-regulation of FoxM1 could potentially be an effective therapeutic approach for pulmonary SCC. In conclusion, our current study shows a role for FoxM1 in the development and progression of pulmonary SCC. This study indicates that FoxM1 immunoreactivity may identify a subset of pulmonary SCC that was associated with progressive pathologic features and an aggressive clinical course. However, we emphasize here that this study was not able to demonstrate the statistical significance of AJCC stage and lymph node metastasis, which are known in the literature to be of prognostic significance, on multivariate analysis. This was the result because our study is limited by the number of study cases with relatively small and heterogeneous stage subgroups and by retrospective study. Further prospective investigations with a large number of cases would allow us to evaluate FoxM1 in a variety of clinical settings to help us better understand its unique role in pulmonary SCC progression. These results further suggest that strategies to reduce the FoxM1 expression could represent a valuable therapeutic approach for the treatment of human pulmonary SCC.
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