Japanese Journal of Clinical Oncology, 2015, 45(9) 854–859 doi: 10.1093/jjco/hyv094 Advance Access Publication Date: 16 July 2015 Original Article
Original Article
Fibulin-1 functions as a prognostic factor in lung adenocarcinoma Yuan Cui1, Jian Liu2, Hai-Bing Yin4, Yi-Fei Liu3,*, and Jun-Hua Liu1,* 1
Downloaded from http://jjco.oxfordjournals.org/ at EPFL Lausanne on September 26, 2015
Department of Thoracic Surgery, Affiliated Hospital of Nantong University, Nantong, Jiangsu, 2Department of Oncology, Affiliated Hospital of Nantong University, Nantong, Jiangsu, 3Department of Pathology, Affiliated Hospital of Nantong University, Nantong, Jiangsu, and 4Department of Pathology, Nantong Tumor Hospital, Nantong, Jiangsu, China *For reprints and all correspondence: Jun-Hua Liu, Department of Thoracic Surgery, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu, China. E-mail: [email protected] Received 26 January 2015; Accepted 1 June 2015
Abstract Objective: Fibulin-1 is a member of the fibulin gene family, characterized by tandem arrays of epidermal growth factor-like domains and a C-terminal fibulin-type module. Fibulin-1 plays important roles in a range of cellular functions including morphology, growth, adhesion and mobility. It acts as a tumor suppressor gene in cutaneous melanoma, prostate cancer and gastric cancer. However, whether fibulin-1 also acts as a tumor suppressor gene in lung adenocarcinoma remains unknown. We also determined the association of fibulin-1 expression with various clinical and pathological parameters, which would show its potential role in clinical prognosis. Methods: We investigated and followed up 140 lung adenocarcinoma patients who underwent lung resection without pre- and post-operative systemic chemotherapy at the Affiliated Hospital of Nantong University from 2009 to 2013. Western blot assay and immunohistochemistry were used to evaluate the expression of fibulin-1 in lung adenocarcinoma tissues. We then analyzed the correlations between fibulin-1 expression and clinicopathological variables as well as the patients’ overall survival rate. Results: Both western blot assay and immunohistochemistry demonstrated that the level of fibulin-1 was downregulated in human lung adenocarcinoma tissues compared with that of normal lung tissues. Fibulin-1 expression significantly correlated with histological differentiation (P = 0.046), clinical stage (P < 0.01), lymph node status (P = 0.038) and expression of Ki-67 (P = 0.013). More importantly, multivariate analysis revealed that fibulin-1 was an independent prognostic marker for lung adenocarcinoma, and high expression of fibulin-1 was significantly associated with better prognosis of lung adenocarcinoma patients. Conclusions: The results supported our hypothesis that fibulin-1 can act as a prognostic factor in lung adenocarcinoma progression. Key words: fibulin-1, lung adenocarcinoma, prognosis, tumor suppressor gene
Introduction Lung cancer is the leading cause of cancer-related deaths worldwide (1). Non-small-cell lung cancer (NSCLC) constitutes 80% of all lung cancers (2) and includes adenocarcinoma, squamous cell carcinoma (SqCC), adenosquamous cell carcinoma and large cell carcinoma
(3). Despite the advances in lung cancer therapies over the years, prognosis of NSCLC has not been improved significantly, with an overall 5-year survival rate of only 15% (4), which may be because patients with NSCLC are usually found in the advanced stages. Previous studies have reported that lung adenocarcinoma (LAC) tended to grow and spread faster than lung SqCC and accounts for almost half of all lung
© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
854
Jpn J Clin Oncol, 2015, Vol. 45, No. 9
Patients and methods Patients and tissue samples Tumor specimens were obtained from 140 LAC patients who underwent lung resection without pre- and post-operative systemic chemotherapy at the Affiliated Hospital of Nantong University from 2009 to 2013. After surgical removal, a portion of paired tissue samples were snap-frozen in liquid nitrogen and maintained at −80°C before protein extraction, and the other portion of tissue samples were immediately fixed in formalin and embedded in wax for immunohistochemistry (IHC). The LAC diagnosis was confirmed histologically in all cases, based mainly on hematoxylin and eosin staining of the sections. The main clinicopathological variables of the patients are shown in Table 1. The follow-up time was 1– 66 months. Permission to use the tissue sections for research purposes was obtained from the Ethics Committee of the Affiliated Hospital of Nantong University and written informed consent was obtained from every patient.
Western blot analysis Frozen lung tissues were used for western blot assay. Tissue proteins were promptly homogenized in a homogenization buffer containing 1 M Tris HCl pH 7.5, 1% Triton X-100, 1% Nonidet p-40 10% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 0.5 M EDTA, leupeptin 10 μg/ml, aprotinin 10 μg/ml and 1 mM PMSF, and then centrifuged at 12,000×g for 30 min to collect the supernatant liquid. The protein concentration was measured by the BCA Protein Assay Kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. The supernatant was diluted in 2× SDS loading buffer and boiled at 100°C for 15 min. The proteins were separated with SDS polyacrylamide gel electrophoresis (SDS-PAGE) at 250 V for 80 min and transferred to polyvinylidene difluoride filter (PVDF) membranes (Millipore, Bedford, MA, USA) using the wet transfer method. The membranes were blocked with 5% dried skim milk in TBS-T (20 mM Tris, 150 mM NaCl and 0.05% Tween-20) for 2 h at room temperature. The membranes were incubated overnight at 4°C with antifibulin-1 antibody (sc-25281, 1:800; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and glyceraldehyde-3-phosphate dehydrogenase
Table 1. Expression of fibulin-1 in 140 human lung adenocarcinoma carcinoma (LAC) tissues Clinicopathological parameters
Gender Male Female Age (years)
Original Article
Fibulin-1 functions as a prognostic factor in lung adenocarcinoma Yuan Cui1, Jian Liu2, Hai-Bing Yin4, Yi-Fei Liu3,*, and Jun-Hua Liu1,* 1
Downloaded from http://jjco.oxfordjournals.org/ at EPFL Lausanne on September 26, 2015
Department of Thoracic Surgery, Affiliated Hospital of Nantong University, Nantong, Jiangsu, 2Department of Oncology, Affiliated Hospital of Nantong University, Nantong, Jiangsu, 3Department of Pathology, Affiliated Hospital of Nantong University, Nantong, Jiangsu, and 4Department of Pathology, Nantong Tumor Hospital, Nantong, Jiangsu, China *For reprints and all correspondence: Jun-Hua Liu, Department of Thoracic Surgery, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu, China. E-mail: [email protected] Received 26 January 2015; Accepted 1 June 2015
Abstract Objective: Fibulin-1 is a member of the fibulin gene family, characterized by tandem arrays of epidermal growth factor-like domains and a C-terminal fibulin-type module. Fibulin-1 plays important roles in a range of cellular functions including morphology, growth, adhesion and mobility. It acts as a tumor suppressor gene in cutaneous melanoma, prostate cancer and gastric cancer. However, whether fibulin-1 also acts as a tumor suppressor gene in lung adenocarcinoma remains unknown. We also determined the association of fibulin-1 expression with various clinical and pathological parameters, which would show its potential role in clinical prognosis. Methods: We investigated and followed up 140 lung adenocarcinoma patients who underwent lung resection without pre- and post-operative systemic chemotherapy at the Affiliated Hospital of Nantong University from 2009 to 2013. Western blot assay and immunohistochemistry were used to evaluate the expression of fibulin-1 in lung adenocarcinoma tissues. We then analyzed the correlations between fibulin-1 expression and clinicopathological variables as well as the patients’ overall survival rate. Results: Both western blot assay and immunohistochemistry demonstrated that the level of fibulin-1 was downregulated in human lung adenocarcinoma tissues compared with that of normal lung tissues. Fibulin-1 expression significantly correlated with histological differentiation (P = 0.046), clinical stage (P < 0.01), lymph node status (P = 0.038) and expression of Ki-67 (P = 0.013). More importantly, multivariate analysis revealed that fibulin-1 was an independent prognostic marker for lung adenocarcinoma, and high expression of fibulin-1 was significantly associated with better prognosis of lung adenocarcinoma patients. Conclusions: The results supported our hypothesis that fibulin-1 can act as a prognostic factor in lung adenocarcinoma progression. Key words: fibulin-1, lung adenocarcinoma, prognosis, tumor suppressor gene
Introduction Lung cancer is the leading cause of cancer-related deaths worldwide (1). Non-small-cell lung cancer (NSCLC) constitutes 80% of all lung cancers (2) and includes adenocarcinoma, squamous cell carcinoma (SqCC), adenosquamous cell carcinoma and large cell carcinoma
(3). Despite the advances in lung cancer therapies over the years, prognosis of NSCLC has not been improved significantly, with an overall 5-year survival rate of only 15% (4), which may be because patients with NSCLC are usually found in the advanced stages. Previous studies have reported that lung adenocarcinoma (LAC) tended to grow and spread faster than lung SqCC and accounts for almost half of all lung
© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
854
Jpn J Clin Oncol, 2015, Vol. 45, No. 9
Patients and methods Patients and tissue samples Tumor specimens were obtained from 140 LAC patients who underwent lung resection without pre- and post-operative systemic chemotherapy at the Affiliated Hospital of Nantong University from 2009 to 2013. After surgical removal, a portion of paired tissue samples were snap-frozen in liquid nitrogen and maintained at −80°C before protein extraction, and the other portion of tissue samples were immediately fixed in formalin and embedded in wax for immunohistochemistry (IHC). The LAC diagnosis was confirmed histologically in all cases, based mainly on hematoxylin and eosin staining of the sections. The main clinicopathological variables of the patients are shown in Table 1. The follow-up time was 1– 66 months. Permission to use the tissue sections for research purposes was obtained from the Ethics Committee of the Affiliated Hospital of Nantong University and written informed consent was obtained from every patient.
Western blot analysis Frozen lung tissues were used for western blot assay. Tissue proteins were promptly homogenized in a homogenization buffer containing 1 M Tris HCl pH 7.5, 1% Triton X-100, 1% Nonidet p-40 10% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 0.5 M EDTA, leupeptin 10 μg/ml, aprotinin 10 μg/ml and 1 mM PMSF, and then centrifuged at 12,000×g for 30 min to collect the supernatant liquid. The protein concentration was measured by the BCA Protein Assay Kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. The supernatant was diluted in 2× SDS loading buffer and boiled at 100°C for 15 min. The proteins were separated with SDS polyacrylamide gel electrophoresis (SDS-PAGE) at 250 V for 80 min and transferred to polyvinylidene difluoride filter (PVDF) membranes (Millipore, Bedford, MA, USA) using the wet transfer method. The membranes were blocked with 5% dried skim milk in TBS-T (20 mM Tris, 150 mM NaCl and 0.05% Tween-20) for 2 h at room temperature. The membranes were incubated overnight at 4°C with antifibulin-1 antibody (sc-25281, 1:800; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and glyceraldehyde-3-phosphate dehydrogenase
Table 1. Expression of fibulin-1 in 140 human lung adenocarcinoma carcinoma (LAC) tissues Clinicopathological parameters
Gender Male Female Age (years)
