Role of vascular endothelial growth factor in non‑small cell lung cancer pathogenesis

Krupnova E.V.1, Shapetska M.N.2, Mikhalenko E.P.1, Chebotaryova N.V.1, Shchayuk A.N.1, Pissarchik S.N.3, Prokhorov A.V.2

Summary. The angiogenesis is an important process in the pathogenesis of malignancies. It is regulated by various growth factors, with the vascular endothelial growth factor (VEGF) playing the central role. The aim of the present study was to evaluate possible associations of functional VEGF −2578C>A, −634G>C, and +936C>T polymorphisms with the risk for occurrence and progression of non-small cell lung cancer (NSCLC) in patients living in Republic of Belarus. Materials and Methods: A total of 202 patients (147 males and 55 females) diagnosed as having the NSCLC. The control group consisted of 336 individuals (245 males and 91 females) without an oncopathology. The total DNA was isolated from peripheral blood. We investigated the single nucleotide polymorphisms of VEGF (rs 2010963), (rs 699947), (rs 3025039). The genotyping was performed by PCR-RFLP analysis. Results: Our results revealed a marginally significant association of the –2578CC genotype (p=0.002) with a greater degree of tumor spread (Т2–Т4). Heterozygous genotypes –2578СА and +936СT carriers were included into the follow-up group significantly more often (р=0.021 and р=0.012, respectively). Our study demonstrate that VEGF –2578A/C and +936C/T polymorphisms are among the factors determining the individual peculiarities of NSCLC course in this population and can be used for clarifying the prognosis of the disease.

Submitted: January 20, 2015.
*Correspondence: E-mail: ekrupnova@inbox.ru
Fax: (+375 17) 284-19-17
Abbreviations used: 3´-UTR — 3´-untranslated region; 5´-UTR — 5´-untranslated region; NSCLC — non-small cell lung cancer; VEGF — vascular endothelial growth factor.

The angiogenesis is an important process in the pa­t­hogenesis of malignancies. It is regulated by various growth factors, with the vascular endothelial growth factor (VEGF-A or VEGF) playing the central role [1]. The VEGF gene expression is closely related to the de­gree of vascularization and the prognosis for the occurrence of numerous solid tumors and is considered to be a predictor of resistance to the chemo- and radiotherapy [2]. An elevated VEGF expression is associated with tumor growth and metastatic process, while the inhibited VEGF expression results in suppressed tumor growth [3].

The VEGF gene also triggers the activation of the protease cascade involved in the degradation of extracellular matrix, suppressing apoptosis, stimulates the endothelial cells survival, increases vascular permeability, inhibits the dendritic cells differentiation, regulates the hexose transport in endothelial cells as well as activates tissue factors and monocytes migration [1]. Clinical studies have shown that the high level of VEGF expression and, respectively, the increased number of microvessels in the tumor correlate with the disease stage and the unfavorable prognosis for many tumor types, including the  non-small cell lung cancer (NSCLC) [4–8].

The VEGF gene is located on the short arm of chromosome 6 (6p21.3) and consists of eight exons and seven introns [7]. VEGF is a diametric glycoprotein, acting via tyrosine kinase receptors VEGFR1 and VEGFR2 located predominantly on endothelial cells [9].

Polymorphic sites of VEGF gene determining the level of VEGF production are found in the promoter — 5´-untranslated region (5´-UTR) as well as in 5´- and 3´-untranslated regions of the gene (3´-UTR).

The VEGF gene polymorphism is associated with differentiated VEGF expression and protein production. VEGF-2578C/C, −634 C/-genotypes are related to high level of VEGF expression [5, 9–12], while +936T allele correlates with low VEGF expression and its low levels in blood plasma [13, 14].

Recent studies show that individual polymorphic variants of VEGF influence the development of certain cancers. In particular, VEGF C-634G, C-2578A, C+936T polymorphisms are associated with an increased lung cancer risk in Asian population [15]. Based on the above-mentioned, it can be assumed that these single nucleotide substitutions of the VEGF gene related to angiogenesis can affect the risk of tumour occurrence and progression as well as the patients’ survival.

The aim of the present study was to evaluate possible associations of functional VEGF −2578C>A, −634G>C, and +936C>T polymorphisms with the risk for occurrence and progression of NSCLC in patients living in the territory of the Republic of Belarus.

MATERIALS AND METHODS

202 patients (147 males and 55 females) diagnosed as having the NSCLC and treated at the Minsk City Oncology Dispensary during the period from 2003 to 2012 were included in the study. The control group consisted of 336 individuals (245 males and 91 females) without an oncopathology who were age-, gender- and comorbidity matched with NSCLC patients. Clinical characteristics of patients with NSCLC and controls are presented in Table 1.

Table 1. Characteristics of study population
Characteristics Patients with NSCLC
(n = 202)
Controls
(n = 336)
n (%) n (%)
Gender:
female 55 (27.2) 91 (27.1)
male 147 (72.8) 245 (72.9)
Smoking status:
smokes 67 (33.2) 201 (59.8)
does not smoke 119 (58.9) 135 (40.2)
no information 16 (7.9)
Stage:
I 108 (53.5)
II 27 (13.4)
III 55 (27.2)
IV 12 (5.9)
Histology:
squamous-cell carcinoma 106 (52.5)
adenocarcinoma 96 (47.5)
Surgery:
lobectomy/bilobectomy 121 (65.1)
pneumonectomy 42 (22.6)
lung resection 13 (7.0)
biopsy 10 (5.4)
Therapy:
chemotherapy 65 (32.2)
radiotherapy 39 (19.3)
no therapy 98 (48.5)

The study was performed in compliance with the principles of voluntary participation and confidentiality, in accordance with the questioning of patients and the approval from the local Ethics Committee to study tissue samples and biological fluids.

The diagnosis of lung cancer in patients has been established on the basis of clinical signs of the disease, history data, bronchoscopy, X-ray examination and computed tomography, cytomorphology of sputum and tumour tissue biopsies. In the group of NSCLC patients, the mean age was 61.6±0.6, in the control group — 61.6±0.8 years.

All cases of lung malignancies have been identified according to the International Classification TNM/pTNM (7th edition, 2009). The histological type of lung carcinoma was determined according to the WHO histological criteria (3rd edition, 1999). The group of NSCLC included the most common neoplasms — squamous-cell carcinoma and ade­nocarcinoma. The treatment was mostly surgical: lobectomy — 65.1%, pneumonectomy — 22.6%, lung resection — 7.0%. Only a biopsy was performed in 5.4% of cases, and in 7.9% of patients the operation was not done because of the process spread or severe comorbidity.

The total DNA from the peripheral blood was isolated using the Mathew method [16]. VEGF ­C-634G (rs 2010963), C-2578A (rs 699947), C+936T (rs 3025039) polymorphisms were genotyped by the polymerase chain reaction method and the restriction fragment length polymorphism analysis (PCR-RFLP analysis) using specific primers and restriction endonucleases [15, 17]. Primers for the analysis were synthesized by “Primetekh”, Minsk. The reagents for PCR and PCR-RFLP were manufactured by “Fermentas”, Vilnius.

Statistical analysis. Statistical analyses were performed using Excel and Statistica 7.0. When compa­ring genotype frequencies, Pearson’s chi-squared test (χ2) was used. The association between the genotypes and the disease course was assessed by the value of odds ratio (OR).

RESULTS AND DISCUSSION

An elevated VEGF expression was revealed while studying the variety of malignancies: cancers of the colon, rectum, liver, lung, thyroid, digestive tract; breast, kidney and bladder adenocarcinoma; ovary and uterus carcinoma; angiosarcoma, multiform glioblastoma [18–21]. In turn, the level of VEGF expression depends largely on the polymorphic variants of the gene.

Therefore, the first phase of our work was to analyses the association between VEGF C-634G, C-2578A and C+936T polymorphisms and the risk of NSCLC.

The analysis of data on VEGF genotyping in patients with NSCLC and controls is presented in Table 2.

Table 2. Frequency distribution of polymorphic variants of VEGF in patients with NSCLC and control groups in different populations

Table 2. Frequency distribution of polymorphic variants of VEGF in patients with NSCLC and control groups in different populations
Genotype Belarus (original data) Germany [22] Sweden [22]
Patients Controls OR (95% CI) Patients Controls OR (95% CI) Patients Controls OR (95% CI)
VEGF (G634C) N=186; n (%) N=364; n (%) N, % N, % N=936; n (%) N=941; n (%)
GG 83 (44.6) 186 (51.1) 0.77 (0.54–1.10) 488 (52.1) 492 (52.3) 1.00
GC 88 (47.3) 154 (42.3) 1.22 (0.86–1.75) 363 (38.8) 367 (39.0) 1.00 (0.82–1.21)
CC 15 (8.1) 24 (6.6) 1.24 (0.64–2.43) 85 (9.1) 82 (8.7) 1.05 (0.74–1.47)
G allele 254 (68.3) 526 (72.3) 0.83 (0.63–1.09) 1339 (71.5) 1351 (72.0) 0.99 (0.86–1.14)
С allele 118 (31.8) 202 (27.7) 1.21 (0.92–1.59) 533 (28.5) 531 (28.0) 1.01 (0.86–1.17)
VEGF (С2578А) N=162; n (%) N=360; n (%) N=153; n (%) N=162; n (%) N=939; n (%) N=940; n (%)
СС 41 (25.3) 83 (23.0) 1.13 (0.74–1.74) 44 (28.8) 50 (30.9) 1.00 258 (27.5) 257 (27.3) 1.00
СА 90 (55.6) 186 (51.7) 1.17 (0.81–1.70) 75 (49.0) 72 (44.4) 1.18 (0.68–2.06) 449 (47.8) 451 (48.0) 0.99 (0.79–1.24)
АА 31 (19.1) 91 (25.3) 0.70 (0.44–1.11) 34 (22.2) 40 (24.7) 0.97 (0.50–1.86) 232 (24.7) 232 (24.7) 1.00 (0.82–1.23)
C allele 172 (53.1) 352 (48.9) 1.18 (0.91–1.54) 163 (53.3) 172 (53.1) 1.01 (0.74–1.38) 965 (51.4) 965 (51.2) 0.78 (0.68–0.89)
A allele 152 (46.9) 368 (51.1) 0.85 (0.65–1.10) 143 (46.7) 152 (46.9) 0.99 (0.73–1.36) 913 (48.6) 915 (48.8) 1.05 (0.93–1.20)
VEGF (С936T) N=161; n (%) N=360; n (%) N=153; n (%) N=163; n (%) N=924; n (%) N=934; n (%)
CC 115 (71.4) 267 (74.2) 0.87 (0.57–1.32) 120 (78.4) 128 (78.5) 1.00 708 (76.6) 720 (77.1) 1.00
CT 38 (23.6) 80 (22.2) 1.08 (0.70–1.68) 31 (20.3) 31 (19.0) 1.07 (0.59–0.93) 204 (22.1) 203 (21.7) 1.02 (0.81–1.28)
TT 8 (5.0) 13 (3.6) 1.40 (0.57–3.44) 2 (1.3) 4 (2.5) 0.53 (0.07–3.46) 12 (1.3) 11 (1.2) 1.11 (0.45–2.71)
С allele 268 (83.2) 614 (85.3) 0.86 (0.60–1.23) 271 (59.8) 287 (88.0) 0.20 (0.14–0.30) 1620 (87.7) 1643 (88.0) 0.97 (0.80–1.18)
T allele 54 (16.8) 106 (14.7) 1.17 (0.82–1.67) 182 (40.2) 39 (12.0) 4.94 (3.37–7.25) 228 (12.3) 225 (12.0) 1.03 (0.84–1.25)

Frequency distribution of alleles and genotypes of functionally significant VEGF polymorphisms in the population of patients living in the territory of the Republic of Belarus was close to the occurrence of the same polymorphisms in the control group. Statistically significant differences in the frequency of the studied genotypes between the group of patients with NSCLC and the control group were not found. Similar results on the frequency of the studied VEGF single nucleotide substitutions were obtained for the populations in Germany and Sweden.

Based on the results, it can be assumed that there is no connection between the studied VEGF polymorphisms and the risk of NSCLC in this population.

Since VEGF is a major mediator of angiogenesis, it can be supposed that VEGF С-634G, C-2578A and C+936T polymorphisms will rather affect the phenotype and tumor biological behavior than the risk for tumor occurrence.

The next stage of our study was to find the relationship between the polymorphisms under study and the clinical course of this disease. The comparative analysis of genotypes distribution of three functionally significant VEGF polymorphisms and tumor size, metastasis and clinical outcomes in the group of patients with NSCLC has been carried out.

VEGF triggers the neoplastic angiogenesis, ­resulting in increased microvascular density, and malignant tissue receives more nutrients [23, 24]. VEGF secretion by tumor cells leads to the synthesis of proangiogenic factors. The newly formed vessels begin to supply malignant tissue with oxygen and nutrients; the tumor is growing and producing more VEGF. VEGF increases the level of VEGFR2 receptor expression by endotheliocytes of tumor microvessels, thus stimulating the cell growth and endothelial cells proliferation [25].

Structurally and functionally the neoplastic vessels differ from the normal ones, with high permeability, chaotic branching, multiple loops, weaves, dead-end branches, lack of structured vasculature being typical for them [2]. Chaotic arrangement of tumor vessels results in uneven oxygen supply to the surrounding tissues, formation of local hypoxic foci, and is followed by tumor resistance to the radio-and chemotherapy.

With an elevated VEGF expression, the vascular permeability increases leading to a higher interstitial and intratumoural pressure, facilitating the penetration of tumor cells into the bloodstream [26].

The relationship between the tumor angiogenesis level, tumor size and metastatic process is confirmed by the correlation between the course of the disease and microvascular density of the primary tumor [27–32]. Different VEGF polymorphisms are associated with lymphogenic and hematogenous metastases in a variety of malignancies [28, 29].

The performed analysis of the relationships between three VEGF gene polymorphisms and tumor size (Table 3) has shown that genotype –2578СС carriers displayed a greater degree of tumor spread (Т2–4)significantly more frequently (р = 0,002) compared to the primary focus spread (Т1). On the contrary, the carriers of –2578СА genotype more often presented with small non-invasive cancer (Т1). However, in this group of NSCLC patients, no statistically significant associations between the studied polymorphic allele variants and regional and/or distant metastases were found (Table 4).

Table 3. Distribution of frequencies of polymorphic variants of VEGF and tumor size in patients with NSCLC
Tumour size N G634C N C2578A N C936T
GG GC CC CC CA AA CC CT TT
n % n % n % n % n % n % n % n % n %
T1 70 32 45.7 33 47.1 5 7.1 57 6 10.5* 40 70.2** 11 19.3 41 29 70.7 11 26.8 1 2.4
T2–4 99 40 40.4 51 51.5 8 8.1 89 31 34.8* 44 49.4** 14 15.7 57 42 73.7 12 21.0 3 5.3

Note: *р = 0.002, OR = 4.54 (1.75–11.77); **p = 0.021, OR = 0.42 (0.21–0.84).

Table 4. Frequency distribution of genotype of VEGF and metastasis in patients with NSCLC
Metastasis N G634C N C2578A N C936T
GG GC CC CC CA AA CC CT TT
n % n % n % n % n % n % n % n % n %
T1–4N0M0 99 37 37.4 54 54.5 8 8.1 86 20 23.3 50 58.1 16 18.6 84 61 72.6 20 23.8 3 3.6
T1–4N1–3M0–1 69 35 50.7 29 42.0 5 7.3 59 17 28.8 33 55.9 9 15.3 60 43 71.7 13 21.7 4 6.7

The in vitro studies conducted by M. Mohammadi and M.С. Shahbazi has shown that -2578C allele correlated with higher VEGF expression compared to A allele [30, 31]. Similar results were obtained by M. Perrot-Applanat who proved that –2578С allele was associated with an elevated VEGF expression and pointed to the possible involvement of VEGF proteins in the autocrine regulation of the tumor growth [32]. Perhaps, this is likely to explain our findings, where the homozygous carriers (–2578СС) had higher degree of the tumor spread. The lower level of VEGF expression in the presence of –2578А allele seems to have a protective effect in heterozygous carriers (–2578СА) associated with a lesser degree of the neoplasm spread.

In the population under study, a significant relationship between the –2578СА variant and the degree of spread of the primary tumor as well as the outcome of the disease was detected (Table 5). Heterozygous genotype –2578СА carriers were included into the follow-up group significantly more often (р = 0.021). It should be noted that a similar results was observed for another VEGF polymorphism — the heterozygous variant +936СT located in the 3´-UTR (р = 0.012). Moreover, C. Oliveira et al. have shown that heterozygous +936СT genotype was increasingly associated with a non-recurrent disease [11]. No associations between the VEGF –634G>C polymorphism and the survival of patients with NSCLC have been found.

Table 5. Distribution of frequency of polymorphic variants of VEGF and survival in patients with NSCLC
NSCLC survival VEGF (G634C) VEGF (С2578А) VEGF (С936T)
N GG GC CC N CC CA AA N CC CT TT
n % n % n % n % n % n % n % n % n %
Alive 88 42 47.7 40 45.5 6 6.8 79 15 19.0 52 65.8 12 15.2 77 51 66.2 24 31.2* 2 2.6
Progression 16 9 56.2 7 43.8 16 7 43.8 7 43.8 2 12.4 16 11 68.8 3 18.8 2 12.4
Death 68 24 35.3 37 54.4 7 10.3 54 15 28.8 25 46.3 14 25.9 55 45 81.8 6 10.9* 4 7.2
Alive 88 42 47.7 40 45.5 6 6.8 79 15 19.0 52 65.8** 12 15.2 77 51 66.2 24 31.2# 2 2.6
Progression + death 84 33 39.3 44 52.4 7 8.3 70 22 31.4 32 45.7** 16 22.8 71 56 78.9 9 12.7# 6 8.4
Note: *р = 0.001, OR = 0.17 (0.06–0.48); **p = 0.021, OR =0.44 (0.23–0.85); #p = 0.012, OR = 0.32 (0.14–0.75).

It has been demonstrated that the polymorphism of a single nucleotide located in the promoter region or 3´- and 5´-UTR affects the protein expression at the transcriptional level [33]. Initially, the hypoxia induced factor (HIF-1) joins VEGF gene in the promoter region (–2578СА polymorphism) and increases its expression. Gene polymorphism in this region can weaken or strengthen this interaction, and, therefore, alter the VEGF expression (–2578СА) [34] that, in turn, will affect the formation and permeability of microvessels and, finally, the tumor size, metastasis and survival.

Based on the results of the study on the correlation between the polymorphisms and development of the di­sease in this population, it has been established that the carriers of –2578СС genotype displayed a greater degree of tumor spread (Т2–4) significantly more often (р = 0.002) while the carriers of –2578СА genotype presented with small non-invasive cancers (Т1) more frequently (р = 0.021). The carriers of –2578C/A genotype were significantly often observed (р = 0.021) in the “follow-up” group. Given that the high level of VEGF expression is related to an increased risk of a recurrent disease and shorter survival of subjects with different cancers [35], one may suggest the VEGF (–2578С/-) genotype to contribute to the high expression of the corresponding protein product in the development of NSCLC.

The mechanism of most associations between single nucleotide substitutions and clinical behavior of tumors is still largely unclear. It should be taken into account that 5´- and 3´-UTR contain key regulatory elements sensitive to hypoxia [36–38] and contribute to high variability of VEGF production [39]. For instance, VEGF-634G>C and –2578A>C polymorphisms in the 5´-UTR affect the efficiency of protein translation [33], and 936C>T polymorphism in the 3´-UTR influences the VEGF circulating plasma concentrations [13] and tumor tissue expression of VEGF [5]. This can explain the protective effect of the VEGF +936T allele in breast cancer metastasis (due to the reduced VEGF expression) [4, 11].

Thus, the results of our study demonstrate that VEGF –2578A/C and +936C/T polymorphisms are among the factors determining the individual peculiarities of NSCLC course in this population and can be used for clarifying the prognosis of the disease.

REFERENCES

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