The positive correlation between gene expression of the two angiogenic factors: VEGF and BMP-2 in lung cancer patients
Introduction
Lung cancer is the leading cause of death among malignant tumors worldwide [1]. Unfortunately, its prognosis is poor, the disease is rarely curable with an overall 5-year survival rate of about 15% [2]. The cure rates of lung cancer have been relatively unaltered during the past 40 years. Therefore, new strategies for screening and treatment of this disease are necessary for the improvement of patients’ outcome [3], [4].
It has been shown that angiogenesis, a process whereby new blood vessels are formed by sprouting from a preexisting vasculature, is a relatively early event of carcinogenesis [5], [6]. Neovascularization is necessary for tumors to be able to growth beyond 2 mm3, and is essential for adequate supply of oxygen and nutrients to tissues [7]. VEGF is the most important growth factor controlling angiogenesis in normal and tumor cells. It binds to different vascular endothelial growth factor receptors (VEGFR) that belong to the tyrosine-kinase receptor family [6], [7], [8], [9], [10]. VEGF gene expression is regulated by several factors, including hypoxia, growth factors, cytokines and other extracellular molecules [7], [8].
It is suggested that VEGF activates several critical gene products, which are involved in VEGF-induced progression and metastasis of lung cancer [9], [11]. Several studies have demonstrated that the VEGF mRNA expression [11], [12], [13], [14], [15], [16], [17], [18] and the serum level of VEGF [14], [19], [20], [21], [22], [23], [24] are increased in patients with lung cancer as compared to healthy individuals. Other studies have shown the association between increased tumor or serum VEGF levels and poorer survival [16], [18], [23], [25], [26], [27], [28], [29], [30], [31], [32], [33], higher stage of the lung cancer [9], [23], [25] and greater tumor size [30], [34]. Furthermore, VEGF serum level is considered to be a prognostic factor in patients with lung malignancy [11], [18], [27], [28], [29], [30], [34], [35]. It has been also reported that tumor angiogenesis, tumor growth and metastases are suppressed by the inhibition of VEGF signal transduction [36]. The expression of VEGF may therefore reflects the angiogenic potential and biological aggressiveness of a tumor and may be an effective target for therapy to improve the prognosis of lung cancer [37], [38].
Bone morphogenetic proteins (BMP) represent about one-third of the TGF-β superfamily composed of growth and differentiation factors [39], [40], [41]. They bind serine kinase receptors (BMPR I and BMPR II) and regulate signaling predominately through Smad proteins [42], [43], [44]. BMPs have been recognized as critical in the control of multiple organogenic processes including the development of lungs where BMP-2 and BMP-4 play an important role [40], [41], [45]. Several studies also claimed that BMP-2 promotes angiogenesis by activating endothelial cells through the stimulation of Smad 1/5, Erk 1/2, and Id expression [43], [46]. The Id transcription factors which are very important in neovascularization have been identified as one of the main targets of the BMP-2 signaling pathway [47]. Other studies have shown that BMP-2 and BMP-4 induce the production of VEGF by different cell lines or tumor cells, which contributes to the angiogenic response [46], [48], [49]. Furthermore, BMP-2 may enhance angiogenesis by serving as a chemotactic factor for monocytes [50]. Monocytes are present in lung tumors, which can secrete cytokines that promote blood vessels formation [51].
It has been demonstrated that the BMP-2 gene is highly expressed in non-small cell lung cancer (NSCLC) when compared to non-neoplastic lung tissue [43], [52]. Several reports provide evidence that BMP-2 stimulates the growth and progression of lung tumor [46]. BMP-4, a close relative of BMP-2, has not been extensively studied in lung cancer and its role is not established in this malignancy. Further studies are needed to clarify the role of bone morphogenetic proteins in lung cancer development and establish a probable relationship between these molecules and vascular endothelial growth factor in the promotion of tumor angiogenesis.
Although cigarette smoking is the major cause of lung cancer [2], only a small fraction of smokers suffer from this disease, which suggests the influence of some genetic factors in lung cancer development. Several studies indicate that the genetic susceptibility to lung cancer may result from a combination of low-penetrance gene polymorphisms [53], [54]. DNA sequence variations in the VEGF gene may lead to altered VEGF production and/or activity, thereby causing interindividual variability in the susceptibility to lung cancer development and progression [55]. VEGF gene polymorphisms such as +405G/C localized in the 5′-untranslated region (5′-UTR) and +936C/T localized in the 3′-untranslated region (3′-UTR) (transcription start continued as +1) have been associated with variations in VEGF protein production. These polymorphisms have been involved in the susceptibility to several disorders, including lung cancer, in which angiogenesis may play a critical role [55], [56], [57], [58], [59], [60], [61].
The purposes of this study were: (1) to evaluate gene expression of angiogenic factors such as VEGF, BMP-2 and BMP-4 in lung cancer tissue and its surrounding healthy tissue and establish probable association between these factors in 88 preoperative lung cancer patients; (2) to determine the correlation between VEGF serum level and clinicopathological characteristics including T stage of the tumor, the involvement of lymph nodes and the histological type of lung cancer; (3) to determine the link between any of these two polymorphisms (+405G/C and +936C/T) and VEGF gene or VEGF protein expression in patients with lung cancer.
Gene and/or protein expression of VEGF and BMP-2, BMP-4 associated with common polymorphisms (+405G/C and +936C/T) of the VEGF gene may be useful genetic markers of angiogenesis-linked pathologic processes leading to the progression of the lung cancer. Furthermore, the results of our study may contribute to a better understanding of the role of VEGF, BMP-2 and BMP-4 in angiogenesis and in the biological behavior of this tumor. In addition it will provide potential molecular targets for the management of lung malignancies.
Section snippets
Patient characteristic
The study included 88 non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) patients diagnosed between 2005 and 2007 in the Chest and Surgical Oncology Clinic of the Medical University of Lodz. There were 68 men and 20 women. The median age of the lung cancer patients was 65 years (range: 51–82 years). Details of the patient characteristics are listed in Table 1. Venous blood was taken from each patient and centrifuged at 3000 × g for 5 min at 4 °C. Supernatant was transferred into
VEGF, BMP-2 and BMP-4 gene expression
In this study, we used real-time RT-PCR method to compare VEGF, BMP-2 and BMP-4 mRNA expressions in pairs of lung cancer fragments and non-neoplastic lung tissues from 88 patients. The mean values for mRNA levels of studied genes were standardized by the mRNA levels of GAPDH of the same sample. Relative gene expression was calculated using the 2−ΔΔCt method. The method could detect VEGF, BMP-2 and BMP-4 mRNA expression in most of the lung cancer fragments and corresponding non-neoplastic lung
Discussion
Tumor vascularity is an independent prognostic factor in lung cancer, and is a critical step in the development of systemic metastasis [6], [65]. Several strategies of blocking angiogenesis by affecting the VEGF/VEGF receptor pathways have been evaluated and provided promising results [3]. However, because of the heterogeneous clinical behavior of lung cancers, more individualized treatment strategies will need to be developed.
Using the real-time RT-PCR method, this study has enabled the
Conclusion
In conclusion, our study has demonstrated a number of novel findings, since no published data on the positive correlation between the VEGF and BMP-2 gene expression and relationship between the 936T allele of the VEGF gene and decreased expression of mRNA of this gene in lung cancer exist in the current literature. Knowledge about the +936C/T VEGF gene polymorphism leading to decreased VEGF gene expression and serum levels could be of diagnostic value suggesting that inherited VEGF gene
Conflict of interest statement
I, Magdalena Bieniasz, Katarzyna Oszajca, Mak Eusebio, Jacek Kordiak, Jacek Bartkowiak, Janusz Szemraj, declare that we have no proprietary, financial, professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the reviewer of, the manuscript entitled, “The positive correlation between gene expression of the two angiogenic factors: VEGF and BMP-2 in lung cancer patients”.
Acknowledgments
This work was supported by Ministry of Science and Higher Education (MNiSW) grant number 2PO5A14129 and Medical University of Lodz grant number 50216807.
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