Abstract
Induction of angiogenic responses by multiple factors, a crucial step in tumor growth and metastasis, is not completely understood. Recently, involvement of the cytoskeletal actin-binding proteins in angiogenesis has been suggested as a target for anti-neovascular cancer therapy in vitro. In this study, the expression of filamin A (FLNA) and vascular endothelial growth factor (VEGF) in paraffin-embedded tumor samples from patients with well-characterized lung tumors was immunohistochemically analyzed and compared with clinical variables and survival outcome. A positive expression of FLNA and VEGF was detected in the cytoplasm of tumor cells in 66 (48.2%) and 69 (50.4%) of the 137 patients with lung cancer, respectively (p<0.0001). A significant difference was observed between FLNA expression and VEGF expression. Although our findings do not suggest that the expression of FLNA alone plays an independent prognostic role, the angiogenesis pathway mediated by FLNA appears to be responsible for controlling the growth of lung tumors.
Lung cancer is the leading cause of cancer-related deaths worldwide. Despite a large amount of multidisciplinary patient management and research, the prognosis for patients with lung cancer remains dismal (1). A more fundamental knowledge of the molecular basis of lung cancer is required to accurately predict patient outcome, select the optimal therapy, and identify novel molecular targets for future therapy. Therefore, it is important to evaluate the biological and molecular characteristics of lung cancer regarding the factors related to a poor prognosis (2, 3).
Angiogenesis, which is primarily mediated through VEGF, is one of the key steps in tumor growth and metastasis (4). Furthermore, cancer cell motility is an important characteristic that facilitates the multistep process of tumor growth (5). In addition, it is known that remodeling of the actin cytoskeleton accompanies alterations in cell shape and motility during processes such as angiogenesis (6). FLNA is a large cytoskeletal actin-binding protein that organizes filamentous actin into networks and stress fibers and also provides a scaffold for a wide range of cytoplasmic proteins involved in signal transduction (5). Recently, the role of cytoskeletal actin-binding proteins in angiogenesis have been discussed for the development of anti-vascular cancer therapies in vitro (7). These findings encouraged us to examine whether VEGF expression, which is implicated in the malignant lung cancer, correlates with FLNA. FLNA may interact with many signal transduction molecules that possibly regulate oncogenic microenvironmental changes (5). Therefore, the detection of FLNA-positive cells may help us to identify those patients at a high risk for lung cancer progression. This is the first retrospective cohort study examining the relationship between FLNA and VEGF expression and the clinicopathological characteristics of patients with lung cancer.
Materials and Methods
Patients, clinical features, and follow-up. The institutional review board approved this study and informed consent for the use of the tumor specimens was obtained either from all patients or their legal guardians. Tumor samples were obtained from 262 consecutive patients with primary lung cancer who had undergone a surgical resection between April 1993 and July 1996 in the Second Department of Surgery, University of Occupational and Environmental Health, Kitakyushu, Japan. Tumor samples from 125 patients were too small to evaluate by immunohistochemical (IHC) staining for FLNA and VEGF status, and were thus excluded from the study. As a result, 137 tumor specimens were evaluated. The clinicopathological data were obtained based on a retrospective chart review. The tumor stage was classified according to the Revisions in the International System for Staging Lung Cancer (8).
For the postoperative follow-up, the patients were examined every month within the first year and at approximately 2- to 4-month intervals thereafter. The evaluations included a physical examination, chest roentgenography, an analysis of blood chemistry, and measurements of tumor markers such as carcinoembryonic antigen (CEA), squamous cell carcinoma-related antigen (SCC), and cytokeratin fragment (CYFRA). Chest and abdominal computed tomographic scans, brain magnetic resonance imaging, and a bone scintiscan were performed every 6 months for 3 years after surgery. If any symptoms or signs of recurrence appeared based on these follow-up studies, additional examinations were performed to evaluate the precise site of recurrence. Follow-up was performed for all patients. The median observation period was 52.4 months. At the last follow-up, 66 patients were alive and free of cancer, while 27 patients had died of other causes without evidence of cancer, 2 patients were alive with recurrent cancer, and 42 patients had succumbed to the cancer. A total of 44 (32.1%) of the 137 patients had disease recurrence after surgery. Twenty and eighteen patients had hematogenous and locoregional metastases, respectively, and six patients had both.
Immunohistochemical (IHC) staining. IHC staining was conducted using serial sections from the same paraffin-embedded blocks by previously described methods (2, 3, 9). Briefly, all tissue specimens were formalin-fixed and similarly processed, according to the standard histology practices. A 3 μm-thick formalin-fixed, paraffin-embedded tissue section was prepared from each specimen. All specimens were stained with hematoxylin and eosin for the histopathologic diagnosis. IHC staining was performed by the streptavidin-biotin-peroxidase complex method as previously described (9). The sections were briefly immersed in citrate buffer (0.01 mol/l citric acid: pH 6.0) and incubated for two 5-minute intervals at 100 °C in a microwave oven for antigen retrieval. The sections were then incubated with polyclonal FLNA antibody (Santa Cruz Biotechnology, Inc., CA, USA) or VEGF antibody (A20, Santa Cruz Biotechnology) (10) diluted at 1:100 and 1:200, respectively, in phosphate-buffered saline (PBS) for 18 h in a cold room using a Labeled Streptavidin Biotin kit (DAKO LSAB kit, CA930 13, Dako Corp., Carpinteria, CA, USA) (11). Positive controls for FLNA and VEGF were macrophages (12) and previously reported positive specimens (3, 10), respectively. Negative controls were processed by the exclusion of the primary antibody.
Evaluation of the stained specimens. The percentage of positive cells was calculated by counting more than 1000 cells in randomly-chosen high-powered fields (10×40), and was scored according to the percentage of positive FLNA cells as follows. Score 0, 0%; score 1, 1-20%; score 2, 21-40%; score 3, 41-60%; score 4, 61-80%; or score 5, 81-100%. To evaluate any correlations with clinicopathological characteristics, the FLNA expression scores were divided into two groups. Specimens with expression scores of 0 were considered to have a negative expression for FLNA, and specimens with scores of 1-5 were considered to have a positive expression for FLNA. We considered a specimen to be VEGF positive if unequivocal staining of the membrane or cytoplasm was seen in more than 5% of the tumor cell in the slide of the largest section of the tumor as previously described (3, 10).
Statistical analysis. Regarding the statistical analyses, Fisher's exact test was used. Survival curves were plotted according to the Kaplan-Meier method, and differences between the curves were analyzed by the log-rank test. The multivariate logistic regression was used to evaluate independent associations. The Cox proportional hazards model was applied to a multivariate survival analysis. Differences were considered to be statistically significant for p-values of less than 0.05. The odds ratio (OR) and 95% confidence interval (95% CI) were calculated for each variable. The data were analyzed using the Stat View software package (Abacus Concepts, Inc., Berkeley, CA, USA).
Results
All of the patients were Japanese, consisting of 96 male patients and 41 female patients in this series, with a mean age of 65.9 years (range, 34 to 84 years of age). The pathological types included 81 adenocarcinomas, 43 squamous cell carcinomas, 2 adenosquamous cell carcinomas, 4 carcinoid, and 5 large cell carcinomas, and 2 small cell carcinomas. According to the pathological staging, 29 patients were stage IA, 41 stage IB, 3 stage IIA, 17 stage IIB, 31 stage IIIA, and 16 stage IIIB.
Of all 137 specimens, 66 (48.2%) and 69 (50.4%) were positively stained for FLNA and VEGF in the cytoplasm of tumor cells, respectively (p<0.0001). Typical staining for FLNA is demonstrated in Figure 1. The relationships between FLNA and VEGF expression and various clinicopathological characteristics of the patients are summarized in Tables I and II, respectively. No significant differences were observed between the expression of FLNA and VEGF and the gender, age at operation, histological type, pathological stage, pathological T factor, or pathological N factor. The incidence of positive FLNA expression was 73.9% and 22.1% in the patients with VEGF-positive and negative expression, respectively (p<0.0001).
FLNA expression was identified in 23 (34.8%) out of 68 patients and 22 (29.6%) out of 71 patients with and without recurrence, respectively (p=0.509). VEGF expression was not significantly correlated with postoperative recurrence (p=0.299). The multivariate logistic regression models indicated that neither the FLNA nor VEGFR expression were an independent predictors for recurrence (data not shown).
The overall 5-year survival rate for patients with positive and those with negative FLNA expression was 43.7% and 54.9%, respectively (p=0.06) (Figure 2A). The overall 5-year survival rate for patients with positive and those with a negative VEGF expression was 46.0% and 51.7%, respectively (p=0.16) (Figure 2B). Although univariate and multivariate survival analyses demonstrated that two variables (pathological T status and pathological N status) were independently associated with the survival of the patients, positive expression of FLNA and VEGF in the cytoplasm of tumor cells was not found to be associated with an increased risk of death (Tables III and IV).
Filamin immunohistochemical detection in lung tumors. An immunohistochemical analysis of FLNA expression with brown stained cytoplasm is shown in a case of adenocarcinoma (original magnification A: ×100, B: ×400).
Discussion
The present study demonstrated three significant findings. Firstly, we provided clinical evidence that FLNA is frequently overexpressed in lung cancer specimens. Few reports have suggested the potential implications of FLNA in human cancer (13). Recently, FLNA was reported to be essential for the locomotion of human melanoma cells (14), and strong positivity was also seen in stromal cells of adenocarcinoma, compared with the normal colonic mucosa (15). Our findings are consistent with such studies. Loss of FLNA might cause both the inhibition of cell migration and the induction of apoptosis (16).
Secondly, there was a significant positive relationship between FLNA and VEGF in patients with lung cancer. The targeted expression of filamin-interacting protein 1-like (FILIP1L), which binds to FLNA and induces its degradation (17), has been reported to inhibit tumor growth (7). Recently, our findings showed that the expression of E-cadherin, which is a key regulator of epithelial mesenchymal transition (18), was found less frequently in tumors with a positive expression of VEGF in resected stage I NSCLCs (3).
A: The survival curves of cancer patients with positive (heavy line) or negative (narrow line) FLNA expression. The overall 5-year survival rate for patients with positive expression and negative expression was 43.7% and 54.9%, respectively (p=0.06). B: The survival curves of cancer patients with positive (heavy line) or negative (narrow line) VEGF expression. The overall 5-year survival rate for patients with positive expression and negative expression was 46.0% and 51.7%, respectively (p=0.16).
As a result, positive FLNA expression tended to predict a better patient prognosis, although FLNA expression levels may not be an independent marker for survival and an accurate predictor of recurrence in lung cancer patients. Shieh et al. reported that FLNA did not provide prognostic information, which is consistent with the present data (12). FLNA may mediate the effects of growth factors in human lung cancer cell migration and invasion (19). In addition, FLNA is coincidently overexpressed with several oncogenes and growth factors, such as c-MET, in activated cancer cells in vitro (20). Significantly, the expression of gelsolin, which regulates actin filament organization through complex effects on the dynamics of actin assembly into filaments, was reported to be a significant prognostic factor for cancer recurrence in patients with stage I NSCLC (12). During sexual differentiation regulated by gender hormones, FLNA co-localizes with the androgen receptor (AR) into the nucleus (21). The female gender, in which less AR is supposedly expressed, was a significant factor predicting improved patient survival (22). In cases of metastatic breast cancer, the majority of which consisted of females, the plasma level of a FLNA variant was reported to be a specific and sensitive marker (23).
Relationship between FLNA expression and clinicopathological characteristics.
Relationship between VEGF expression and clinicopathological characteristics.
In conclusion, we herein demonstrated, for the first time, the potential involvement of FLNA in VEGF expression in lung cancer. Inhibiting angiogenesis has therefore become a major therapeutic strategy, and a monoclonal antibody directed against VEGF has shown promise in treating lung cancer (24). Our findings suggest that expression of FLNA may be used as a prognostic marker for lung tumor development and may be targeted as anti-neovascular agent for lung cancer therapy. Recently, we reported that FLNA is an important regulator of c-MET (25), which has been correlated with poor clinical outcome and drug resistance (26, 27). However, further work is still required to predict patient outcome and to select the most appropriate patients who may best respond to therapy.
Univariate analysis using a proportional hazard model for the overall survival.
Multivariate analysis of prognostic factors.
Acknowledgements
We thank Gotoh Yukiko for her valuable technical assistance. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.
Footnotes
-
Competing Financial Interests
The Authors declare that they have no competing financial interests.
- Received June 16, 2010.
- Revision received July 23, 2010.
- Accepted August 18, 2010.
- Copyright© 2010 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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