The positive correlation between gene expression of the two angiogenic factors: VEGF and BMP-2 in lung cancer patients

Lung Cancer 66 (2009) 319–326

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The positive correlation between gene expression of the two angiogenic factors: VEGF and BMP-2 in lung cancer patients Magdalena Bieniasz a,∗ , Katarzyna Oszajca a , Mak Eusebio a , Jacek Kordiak b , Jacek Bartkowiak a , Janusz Szemraj a a b

Department of Medical Biochemistry, Medical University of Lodz, 6/8 Mazowiecka, Lodz 92215, Poland Department of Surgery, Medical University of Lodz, 4 Zeromskiego, Lodz 93509, Poland

a r t i c l e

i n f o

Article history: Received 2 October 2008 Received in revised form 17 February 2009 Accepted 22 February 2009 Keywords: Lung cancer Non-small cell lung cancer Small cell lung cancer Angiogenesis VEGF BMP-2 BMP-4 Polymorphism

a b s t r a c t Lung cancer is a particular challenge in oncology. More than 1 million new cases occur worldwide every year and despite many clinical trials and modern diagnostic techniques, long-term survival rate has only marginally improved. The aim of the current research is to explore new molecular prognostic factors and identify new targets for anticancer therapy. Current evidence shows that angiogenesis is controlled by several angiogenic factors including VEGF and BMP-2. It has been also demonstrated that VEGF plays a key role in this process that is essential in carcinogenesis. Our study has shown that the expressions of the VEGF, BMP-2 and BMP-4 mRNAs were significantly higher (7.1-fold, 25.6-fold and 2.3-fold, respectively) in lung cancer samples than in adjacent normal lung tissues (real-time RT-PCR). Analysis based on the Pearson’s correlation coefficient indicated the positive correlation between VEGF and BMP-2 gene expression, whereas no significant correlation between VEGF and BMP-4 gene expression was found. The mean ± standard deviation serum level of VEGF was 423 ± 136 pg/ml. Significant differences in the serum levels of VEGF between patients with T1 tumors and patients with T2, T3 or T4 tumors were observed. Patients with T2, T3 and T4 tumors, respectively, had 1.6-fold, 1.8-fold and 2.3-fold greater serum levels of VEGF than their peers with T1 tumors. In current study patients homozygous for the 936T allele of the +936C/T VEGF gene polymorphism had 12-fold lower VEGF gene expression and 1.3-fold lower VEGF serum level than patients homozygous for the 936C allele. In conclusion, our findings underline the importance of the two angiogenic factors namely VEGF and BMP-2 as well as +936C/T VEGF gene polymorphism in the evaluation of lung cancer patients. © 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction 1.1. Lung cancer 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

∗ Corresponding author. Tel.: +48 42 678 06 20; fax: +48 42 678 24 65. E-mail address: [email protected] (M. Bieniasz). 0169-5002/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2009.02.020

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 [6–10]. 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 [11–18] and the serum level of VEGF [14,19–24] 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 [16,18,23,25–33], 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 [11,18,27–30,34,35]. 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

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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-␤ superfamily composed of growth and differentiation factors [39–41]. They bind serine kinase receptors (BMPR I and BMPR II) and regulate signaling predominately through Smad proteins [42–44]. 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 [55–61]. 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.

Table 1 Characteristics of patients with lung cancer. No. of cases

Percentage

Age (years) Mean age Range

65 51–82

N/A N/A

Sex Male Female

68 20

77.3% 22.7%

Histological type AD SCC SCLC

31 36 21

35.2% 41.0% 23.8%

T stage of the tumor T1 T2 T3 T4

16 37 31 4

18.2% 42.0% 35.2% 4.6%

Lymph nodes involvement N0 N1 N2

18 38 32

20.4% 43.2% 36.4%

2. Materials and methods 2.1. 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 microtubes and stored at −70 ◦ C until use. Fresh tissues from the carcinomas and non-neoplastic fragments from surrounding lung tissues were snap frozen in liquid nitrogen and stored at −70 ◦ C until use. All samples were collected before treatment. Tumor fragments were analyzed according to WHO histological classification (World Health Organization, 1982). Pathological stage was determined according to the TMN staging system of lung cancer [62]. Prior to sampling, all patients gave informed consent and the study has been reviewed and approved by the Bioethical Committee of the Medical University of Lodz, no.: RNN/211/07/KE. 2.2. RNA extraction and reverse transcription Total RNA was extracted from resected lung tissues using a RNA extraction reagent, TRIZOL (Invitrogen Life Technologies), according to the standard acid–guanidinium–phenol–chlorophorm method [63]. The extracted RNA was analyzed by agarose gel electrophoresis and only cases with preserved 28S, 18S and 5S ribosomal RNA bands indicating good RNA quality were used in the study. Total RNA was digested with DNase (GIBCO) at room temperature for 15 min. Five micrograms of digested RNA were reverse transcribed at 42 ◦ C for 60 min in a total of 20 ␮l reaction volume using the ImProm-IITM Reverse Transcription System kit (Promega, USA). Obtained cDNA was used in real-time PCR reaction. 2.3. Detection of gene expression using real-time RT-PCR method Real-time PCR based on TaqManTM technology was performed using master mix prepared according to the FastStart Universal Probe Master (ROX) from Roche Applied Science. Probes and primers were designed using the online Universal ProbeLibrary

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(www.universalprobelibrary.com). Primer sequences and probe numbers are as follow: VEGF (forward, 5-tgcccgctgctgtctaat-3, reverse, 5-tctccgctctgagcaagg-3, probe: #1), BMP-2 (forward, 5cggactgcggtctcctaa-3, reverse, 5-ggaagcagcaacgctagaag-3, probe: #49), BMP-4 (forward, 5-tccacagcactggtcttgag-3, reverse, 5tgggatgttctccagatgttct-3, probe: #31) and GAPDH (forward, 5agccacatcgctcagacac-3, reverse, 5-gcccaatacgaccaaatcc-3, probe: #60), which was used as internal control for real-time PCR. Real-time PCR was carried out in a final volume of 50 ␮l, with 0.05 ␮g cDNA, 25 ␮l FastStart Universal Probe Master (ROX) 2×, 250 nM probe and 1 ␮M of each primer. Amplification was performed for 10 min at 95 ◦ C to activate FastStart Taq DNA polimerase and 40 rounds of 15 s at 95 ◦ C and 1 min at 60 ◦ C for amplification and signal analysis. ABI Prism 7000 Sequence Detection System from Applied Biosystems was used to detect amplifications. Each sample was assayed in triplicate in independent reactions. Realtime PCR data were automatically calculated with the data analysis module. The results were analyzed according to the 2−Ct method [64]. Validation of PCR efficiency was performed with a standard curve. 2.4. Determination of serum VEGF level using enzyme-linked immunosorbent assay (ELISA) For the quantitative detection of circulating serum VEGF, the RayBio® Human VEGF ELISA from RayBiotech was used. Each serum sample was determined three times. Instructions and collection of results were performed according to the manufacturer’s recommendations. Standards and samples were pipetted into the wells with immobilized antibody specific for human VEGF and incubated. After incubation and washing biotinylated antihuman VEGF antibody was added. After washing away unbound substances, the horseradish peroxidase-conjugated streptavidin was pipetted to the wells. The wells were again washed and a tetramethylbenzidine (TMB) substrate solution was added to the wells and color developed in proportion to the amount of VEGF bound. The color development was stopped (Stop Solution) and the intensity of the color was measured by the Thermo Labsystems Multiskan Ascent 354 from Lab Recyclers at 450 nm. 2.5. Genotyping of VEGF gene polymorphisms using PCR-RFLP assay The isolation of genomic DNA from the peripheral blood lymphocytes was performed using proteinase K digestion and phenol/chloroform extraction. The VEGF gene +405G/C, and +936C/T variants were determined by polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) assay. The primers for amplifying the VEGF gene fragments were 5cgacggcttggggagattgc-3 (forward) and 5-gggcggtgtctgtctgtctg-3 (reverse) for +405G/C variant and 5-agggttcgggaaccagatc-3 (forward) and 5-ctcggtgatttagcagcaag-3 (reverse) for +936C/T variant. The PCR reactions were performed in a 25 ␮l volume containing 200 ng genomic DNA, 0.2 mM deoxynucleotide triphosphates, 2.5 ␮l buffer DyNAzymeTM II 10×, 1 unit of DyNAzymeTM II DNA polymerase (Finnzymes) and ether 1 ␮M of each primer (+936C/T polymorphism) or 0.4 ␮M of each primer (+405G/C polymorphism). The PCR cycle conditions consisted of an initial denaturation step at 94 ◦ C for 3 min, followed by 35 cycles of 30 s at 94 ◦ C, 30 s at 62 ◦ C, 30 s at 72 ◦ C, and a final elongation at 72 ◦ C for 10 min. Aliquots of 8 ␮l of PCR product were incubated for 10 min at 65 ◦ C with 1 ␮l of appropriate reaction buffer. After incubation PCR products were mixed with 1 unit of restriction enzyme from Fermentas (Faq I for +405G/C polymorphism and Hin1 II for +936C/T polymorphism) and incubated overnight at 37 ◦ C. Digestion products were separated by electrophoresis in a 7% polyacrylamide gel

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stained with ethidium bromide for visualization under UV light. As a result 405G alleles were represented by DNA bands with size of 304 bp, the 405C alleles were represented by DNA bands with size of 203 bp and 111 bp, whereas the heterozygotes displayed a combination of both alleles (304 bp, 203 bp and 111 bp). Alleles of the +936C/T polymorphism of the VEGF gene were observed as the 208 bp band (C allele) and 122 bp and 86 bp (T allele) bands. For quality control, 10% of the samples were genotyped twice. 2.6. Statistical analysis The data were shown as mean values ± standard deviation (S.D.). Wilcoxon’s rank-sum test was used to compare the differences in gene expression between lung cancer tissue and non-neoplastic lung tissue. A one-way ANOVA followed by the post hoc Tukey’s test was used to determine associations between gene and protein expression and the following clinical variables: histological type, T stage of the tumor and nodal invasion. The relationship between VEGF gene polymorphisms and mRNA and/or protein levels of VEGF and patient’s clinical variables was determined using Kruskal–Wallis analysis. The Pearson correlation analysis was used to establish the relationship between VEGF gene expression and circulating VEGF levels. P-Values less than 0.05 were considered to be significant. 3. Results 3.1. 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−Ct 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 tissues. However, part of the lung cancer fragments: 2/88 (2.3%), 5/88 (5.7%), 12/88 (13.6%) and non-neoplastic lung fragments: 13/88 (14.7%), 55/88 (62.5%), 28/88 (31.8%) present no expression or trace amounts of VEGF, BMP-2 and BMP-4 mRNA (2−Ct < 0.001), respectively. The mean gene expression values in lung cancer tissue were 0.099 (range: 0.000–0.604) for VEGF gene, 0.077 (range: 0.000–0.293) for BMP-2 gene and 0.009 (range: 0.000–0.021) for BMP-4 gene, whereas for non-neoplastic lung tissue mean gene expression values were 0.014 (range: 0.000–0.062) for VEGF, 0.003 (range: 0.000–0.018) for BMP-2 and 0.004 (range: 0.000–0.052) for BMP-4 (Table 2). As shown in Table 2, the expressions of VEGF, BMP-2 and BMP4 mRNAs were significantly higher (7.1-fold, P = 0.000; 25.6-fold, P = 0.000 and 2.3-fold, P = 0.001, respectively) in lung cancer samples than in adjacent normal lung tissues (Wilcoxon matched pairs test) (Fig. 1). The VEGF, BMP-2 and BMP-4 mRNA expressions were compared with the clinical and histological features of the tumors. The differences in gene expression between the pathological subtypes of lung cancer were significant only for the VEGF gene (P = 0.000). No association was found between BMP-2 and BMP-4 gene expression and histology of the tumor (ANOVA test). VEGF mRNA expression was 3-fold (P = 0.000) and 2-fold (P = 0.031) higher in squamous cell carcinoma than in adenocarcinoma and small cell lung cancer, respectively (Table 2). No significant differences in VEGF, BMP-2 and BMP-4 mRNA levels were found according to the T- and N-factors of the tumor. However, there was a tendency toward higher VEGF gene expression in patients with greater T stage of the tumor and lymph node metastasis (ANOVA test) (Table 2).

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Table 2 VEGF, BMP-2 and BMP-4 mRNA expression in lung cancer and corresponding non-neoplastic lung tissue. Mean VEGF gene expression (±S.D.)

Mean BMP-2 gene expression (±S.D.)

Mean BMP-4 gene expression (±S.D.)

Normal

Cancer

P

Normal

Cancer

P

Normal

Cancer

P

Total

0.014 (±0.014)

0.099 (±0.109)

0.000

0.003 (±0.004)

0.077 (±0.075)

0.000

0.004 (±0.005)

0.009 (±0.011)

0.001

Histology AD SCC SCLC

0.012 (±0.013) 0.016 (±0.016) 0.013 (±0.013)

0.052 (±0.050) 0.154 (±0.143) 0.075 (±0.066)

0.000 0.000 0.000

0.001 (±0.001) 0.006 (±0.006) 0.000 (±0.000)

0.061 (±0.058) 0.083 (±0.078) 0.091 (±0.090)

0.000 0.000 0.000

0.002 (±0.002) 0.004 (±0.004) 0.007 (±0.007)

0.011 (±0.011) 0.003 (±0.003) 0.015 (±0.014)

0.000 0.278 0.019

T stage of the tumor T1 0.010 (±0.007) T2 0.014 (±0.015) T3 0.016 (±0.017) T4 0.012 (±0.012)

0.050 (±0.076) 0.109 (±0.127) 0.112 (±0.106) 0.109 (±0.065)

0.019 0.000 0.000 0.068

0.001 (±0.002) 0.003 (±0.005) 0.003 (±0.004) 0.006 (±0.006)

0.077 (±0.072) 0.069 (±0.085) 0.085 (±0.067) 0.084 (±0.064)

0.001 0.000 0.000 0.067

0.005 (±0.004) 0.004 (±0.005) 0.004 (±0.005) 0.001 (±0.000)

0.007 (±0.008) 0.010 (±0.013) 0.009 (±0.010) 0.006 (±0.005)

0.026 0.000 0.085 0.273

Lymph nodes involvement N0 0.012 (±0.010) N1 0.013 (±0.014) N2 0.016 (±0.017)

0.065 (±0.084) 0.101 (±0.127) 0.117 (±0.102)

0.002 0.000 0.000

0.002 (±0.005) 0.003 (±0.005) 0.003 (±0.004)

0.072 (±0.069) 0.071 (±0.087) 0.087 (±0.063)

0.001 0.000 0.000

0.005 (±0.004) 0.004 (±0.005) 0.003 (±0.005)

0.006 (±0.008) 0.010 (±0.012) 0.009 (±0.009)

0.472 0.000 0.054

Pearson’s correlation coefficient was used to evaluate the relationship between gene expression of the VEGF, BMP-2 and BMP-4 in lung cancer tissue. The result showed a positive correlation between the VEGF and BMP-2 mRNA expressions (P = 0.010, r = 0.342), however, no significant correlations between VEGF and BMP-4 mRNA expressions were found (Fig. 2).

3.2. VEGF serum level The mean ± standard deviation serum level of VEGF of all 88 patients was 423 ± 136 pg/ml (VEGF range: 207–615 pg/ml). We found that in the squamous cell carcinoma group, VEGF serum levels were statistically significantly higher (1.2-fold) than in adenocarcinoma group (ANOVA test, P = 0.047). Between other histological types of lung cancer these differences were not significant (Table 3). The relationship between the VEGF serum level and tumor status or nodal involvement was also investigated. There were significant differences observed in the VEGF serum levels between patients with T1 tumors versus patients with T2, T3 or T4 tumors. Patients with T2, T3 and T4 tumors had 1.6-fold (P = 0.000), 1.8-fold (P = 0.000) and 2.3-fold (P = 0.000), respectively, greater serum levels of VEGF than patients with the T1 factor (Fig. 3). VEGF serum levels were not different among tumors of different N-factors. This current study also shows positive correlation between VEGF gene expression and VEGF serum level (P = 0.01, r = 0.353) based on the Pearson’s correlation coefficient. 3.3. VEGF gene polymorphisms

Fig. 1. The significantly higher expressions of VEGF, BMP-2 and BMP-4 mRNAs (7.1fold, P = 0.000; 25.6-fold, P = 0.000 and 2.3-fold, P = 0.001, respectively) in lung cancer samples than in adjacent normal lung tissues (Wilcoxon matched pairs test).

Genomic DNA from 88 lung cancer patients was analyzed to determine the distribution of the +405G/C and +936C/T VEGF gene polymorphisms. The frequencies of the genotypes and alleles are listed in Table 4. Analysis based on Kruskal–Wallis test revealed that patients homozygous for the 936T allele had 12-fold (P = 0.005)

Table 3 VEGF serum level (pg/ml).

Fig. 2. The statistically significant correlation between VEGF and BMP-2 gene expression in tumor tissue (P = 0.010, r = 0.342) according to Pearson’s correlation coefficient.

Patients

VEGF serum level ± S.D.

Total

88

423 ± 136

Histology AD SCC SCLC

31 36 21

374 ± 136 454 ± 139 441 ± 117

T stage of the tumor T1 T2 T3 T4

16 37 31 4

261 433 472 588

Lymph nodes involvement N0 N1 N2

18 38 32

429 ± 152 413 ± 142 430 ± 123

± ± ± ±

70 123 115 5

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Fig. 3. The statistically significant relationship between increased VEGF serum level and greater T stage of the tumor in patients with lung cancer. Patients with T2, T3 and T4 tumors had 1.6-fold (P = 0.000), 1.8-fold (P = 0.000) and 2.3-fold (P = 0.000), respectively, greater serum levels of VEGF than patients with the T1 factor (ANOVA test). Table 4 Frequencies of the VEGF gene polymorphisms in 88 lung cancer patients. Polymorphism

No. of cases

Percentage

G405C GG GC CC

10 38 40

11.3% 43.2% 45.5%

C936T CC CT TT

48 36 4

54.5% 41.0% 4.5%

lower VEGF gene expression and 1.3-fold (P = 0.0145) lower serum VEGF level compared with patients homozygous for the 936C allele (Figs. 4 and 5). There were no any significant correlations between VEGF gene expression and VEGF serum level and +405G/C VEGF gene polymorphism. 4. 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 pro-

Fig. 4. The statistically significant correlation between 936T allele and lower VEGF gene expression in lung tumor. Patients homozygous for the 936T allele had 12-fold (P = 0.005) lower VEGF gene expression compared with patients homozygous for the 936C allele (Kruskal–Wallis test).

Fig. 5. The statistically significant correlation between 936T allele and lower serum VEGF level. Patients homozygous for the 936T allele had 1.3-fold (P = 0.0145) lower serum VEGF level compared with patients homozygous for the 936C allele (Kruskal–Wallis test).

vided 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 comparison of expression levels of several molecules involved in angiogenesis pathways in non-neoplastic and malignant lung tissues. Our research demonstrated that VEGF gene expression is 7.1-fold up-regulated in lung carcinomas comparing to adjacent non-neoplastic lung tissue, which is in accordance with other findings [15–18]. In the current study, there was significantly higher VEGF mRNA expression in tumor tissue of the squamous cell carcinoma than in adenocarcinoma and small cell lung cancer. Several studies reported the lack of the differences between VEGF gene expression among different histological types of lung cancer [16,18,31] whereas, Yuan et al. demonstrated the higher VEGF mRNA expression in non-squamous cell carcinoma than in squamous cell carcinoma [17]. The discrepancy in our results that may originate from difference in ethnic background is still not fully understood. Studies examining the biological significance of BMP-2 and BMP-4 in cancer have just recently been reported. In the present study we demonstrated that the expression of BMP-2 and BMP-4 mRNAs was significantly higher (25.6-fold and 2.3-fold, respectively) in lung cancer samples than in adjacent normal lung tissues. There are only two reports (Langenfeld et al., 2003, 2005) in the medical literature that have examined BMP-2 and BMP-4 gene expression in lung tumors. The authors showed a 17-fold [43] and 26-fold [52] increase in BMP-2 gene expression and 4.9-fold increase in BMP-4 gene expression [43], which are in accordance with our study. On one hand some current reports show that BMP-2 promotes tumor growth, progression and neovascularization [43,46,52,66,67]. On the other Langenfeld et al. reported the dual effect of BMP-2 on proliferation of A549 cells. They prove that BMP2 stimulates the proliferation of A549 cells cultured in medium containing serum, while in serum free medium it inhibits growth [43]. Based on these findings, it has been determined that the in vitro and in vivo effects of BMP-2 on cells proliferation may vary based on the presence of cytokines [52] or intracellular and extracellular antagonists [68,69]. In our study we could not find any associations between BMP-2 gene expression, histology, T stage of the tumor and the involvement of lymph nodes, as supported by previous reports [43,52].

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Little is known about the impact of BMP-4 on lung cancer development and behavior. Buckley et al. showed that BMP-4 can induce senescence and thus negatively regulate the growth of A549 lung cancer cells [70]. We reported only an increase in BMP-4 gene expression in lung cancer tissue. We could not find any association between these molecules and the prognostic factors of lung cancer such as the histology, T stage of the tumor or lymph node involvement. We showed a positive correlation between VEGF and BMP-2 mRNA expression (P = 0.010, r = 0.342) in lung cancer tissue. To the best of our knowledge, there are no published reports regarding the correlation between VEGF and BMP-2 gene expression in lung cancer. BMP-2 may collaborate with VEGF in the promotion of tumor growth or invasion by stimulating angiogenesis. However, additional studies in larger populations are needed to confirm this hypothesis. During the past few years there has been an increasing interest in the investigations of the role of circulating levels of VEGF. In our study we used serum VEGF levels to examine its relationship with the clinicopathological characteristics of patients suffering from lung cancer. Since the serum level of VEGF is affected by blood platelets [4,71,72] it impacts tumor biology because platelets participate in tumor-induced angiogenesis by releasing and delivering VEGF to the tumor site [71,73–75]. The mean ± standard deviation serum level of VEGF 423 ± 136 pg/ml (VEGF range: 207–615 pg/ml) in the present study was compatible with the results from other studies [14,22–24,27,30,35,74]. When results were evaluated according to cancer histotype, we found that in squamous cell carcinoma group, VEGF serum levels were 1.2-fold higher than in adenocarcinoma group. Unfortunately, none of the other studies has observed the associations between VEGF serum levels and the different histological types of lung cancer [22,24,27,30,74]. Although the serum levels of VEGF are marginally higher in squamous cell carcinoma than in adenocarcinoma in current study and similar results have not been previously reported, this finding may not be significant. Our study also revealed that patients with T2, T3 and T4 tumors had 1.6-fold, 1.8-fold and 2.3-fold, respectively, greater serum levels of VEGF than patients with the T1 factor. There are a few studies showing positive correlations between VEGF serum levels and tumor volume [30,34], which support our findings. Moreover, the study performed by Jefferson et al. proved that tumor volume was an extremely significant independent predictor of prognosis in operable NSCLC patients, which confirm the importance of the evaluation of this clinicopathological parameter [76]. On the other hand, Suzuki et al. [24] did not find any correlations between VEGF serum level and T stage of the tumor. This inconsistency with our results could be explained by the difference in ethnicity or by the fact that Suzuki et al. included only NSCLC patients, whereas in our study there was a large proportion of patients with small cell lung cancer (24%). In the present study we could not find any association between VEGF serum level and lymph node involvement, which is in accordance with others [22]. In present study there was a positive correlation between VEGF gene expression and VEGF serum level (p = 0.010, r = 0.353), which suggested that the serum VEGF level may reflect VEGF synthesis at the tumor site. This finding is important because the assessment of the VEGF gene expression in tumor tissues is relevant to the understanding of the processes occurring in tumor. However, it is a less reliable approach than the more appealing non-invasive assessment of circulating VEGF in blood. There is only one study investigating the correlation between VEGF mRNA expression and serum level performed by Imoto et al. [34]. There are currently no reports demonstrating a significant association between the VEGF expression in tumor tissue of NSCLC patients and the serum VEGF levels. This inconsistency can arise from differences in our method-

ology and population as compared with Imoto’s [34]. The human VEGF gene is highly polymorphic and there is considerable variation between individuals in VEGF expression [77]. Furthermore, the correlation of VEGF production with a specific genotype may help to identify individuals exhibiting high or low levels of VEGF production that predispose them to different therapies [57]. Patients homozygous for the 936T allele had a 12-fold VEGF lower gene expression and 1.2-fold lower serum VEGF level compared with patients homozygous for the 936C allele. To the best of our knowledge, this is the first report showing the correlation between +936C/T VEGF gene polymorphism and mRNA level in lung cancer patients. Other authors have found a relationship between lower VEGF plasma levels and 936T allele of the VEGF gene polymorphism, which is confirmed by our study [78–80]. In contrast, in a Japanese study, no relation was found between the 936C/T polymorphism and VEGF serum levels [81]. The impact of +405G/C gene polymorphism of the VEGF gene on the VEGF serum level has been controversial based on results of several studies. Awata et al. reported that individuals with the 405C/C genotype had a higher fasting serum VEGF level than those with other genotypes [81]. In contrast, Watson et al. documented that the CC genotype for the +405G/C polymorphism correlates with lower VEGF production from stimulated peripheral blood mononuclear cells [57]. In our research, we could not find any significant correlations between VEGF gene expression or VEGF serum level and the +405G/C VEGF gene polymorphism. 5. 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 sequence variations are strong determinants of the molecular VEGF-downstream phenotype of lung cancer. Our findings also underline the importance of the evaluation of the two angiogenic factors such as VEGF and BMP-2 in the biological assessment of lung malignancy. In association with VEGF, BMP-2 may be a good target for therapy in lung cancer, because of its high expression in lung tumors with little or no expression in normal lung tissue, implying that anti-BMP-2 therapy would have minimal toxicity. However, further studies are needed to determine whether inhibition of both VEGF and BMP-2 signaling pathways will significantly alter tumor growth, invasion, and/or metastasis. 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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