Aberrant Expression And Association Of VEGF And Dll4/Notch Pathway Molecules Under Hypoxia In Patients With Lung Cancer

Background:Lung cancer is one of the most common cancers and has become the leading cause of cancer death. Its development is positively correlated with tumor vascular proliferation under hypoxia. VEGF plays an important role in tumor angiogenesis through binding its receptors, and inhibition of the VEGF pathway inhibits tumor growth in some preclinical tumor models. VEGFR1 and VEGFR2 are two primary receptors of VEGF, and are prominently expressed by vascular endothelial cells. VEGF released by tumor cells up-regulates the expression of VEGFR1/VEGFR2. Many data showed that VEGFR1 was a negative regulator of VEGF activity while VEGFR2 was the main mediator of VEGF biological effect. However, several studies suggest that expression of VEGF/VEGFR1/VEGFR2 in lung cancer does not necessarily mean a functional VEGF signaling pathway. Not all lung cancers are responsive to VEGF blockers, and some of them that are responsive initially may become resistant during the course of treatment, and produce gastrointestinal reactions, bleeding and other side effects. Therefore, it is very important to explore other angiogenesis signaling pathways as therapeutic targets.Delta-like ligand 4 (D114)/Notch signaling pathway is recognized as another important pathway in angiogenesis. Membrane D114 binds with Notch receptors on adjacent cells leading to Notch receptor cleavage and releasing its intracellular domain (Notch-IC), which is translocated into the nucleus. In the nucleus, Notch-IC forms a ternary complex with a highly conserved transcription factor, CSL (CBF1/Suppressor of Hairless/Lag1) and transcriptional coactivators of the mastermind-like (MAML) family. This complex activates target gene transcription including the hairy/enhancer-of-split (Hes1) and Hey1 and regulates vascular endothelial cell differentiation and proliferation.Some studies have shown that the expression of D114 is increased in tumor blood vessels of xenograft tumor in mice, human breast cancer and kidney cancer, while there is little or no expression in normal blood vessels. Patel showed that D114-inhibition can induce tumor vascular endothelial cell cycle arrest, apoptosis increase and tumor angiogenesis reduction, which further confirmed the promoting effect of D114 in tumor angiogenesis. However, another study showed that D114 may play an inhibitory effect in tumor angiogenesis. When D114 signaling is blocked by antibodies or siRNA, it may promote tumor vascular endothelial cell proliferation, sprouting and branching, and the tumor vascular density has a significant increase. However, the effect of the D114/Notch pathway in the angiogenesis of lung cancer remains to be elucidated.VEGF induces significantly higher D114 expression to promote tumor angiogenesis, and blocking the expression of VEGF causes sharp decline of D114 and endothelial cell growth inhibition, which indicates that D114 expression is regulated by VEGF. However, studies have shown that a high expression of D114 may inhibit VEGF. Overexpression of D114 in vascular endothelial cells could decrease the expression of VEGFR2, weaken vascular endothelial cell response to VEGF-induced angiogenesis and inhibit angiogenesis, while expression of VEGFR3 was increased and angiogenesis was promoted after D114 was inhibited. Therefore, it is speculated that there may exist a negative feedback loop between VEGF and D114/Notch pathway.Hypoxia has been another regulator of neo-angiogenesis and a hallmark in a number of solid tumors. Recent studies show that there is an important interaction between D114/Notch signaling pathway and HIFla. Several groups have demonstrated that the level of D114 is increased in low-02 tension (0.1%). Notchl appears to up-regulate HIFla expression, and HIFla binds and stabilizes activated Notchl leading to enhanced Notchl signaling.To gain insight into their roles in the pathogenesis, progression and prognosis of lung cancer, we examined VEGF and D114 signaling pathway molecules expression and evaluated their clinical relevance.Objective:To study aberrant expression and association of VEGF and D114/Notch pathway molecules under hypoxia in patients with lung cancer.Methods:1. Patients:A total of 36 patients with newly-diagnosed lung cancer (9 females and 27 males, age range 42-83 years, median 63 years) were enrolled in this study. Thirty-six lung cancer specimens and 27 normal specimens at the margin of tumor sections were obtained after lung cancer operation in Qilu Hospital of Shandong University, Jinan, China. Cancer and normal lung tissues were examined by a certified pathologist, and the normal lung samples were confirmed to be free from tumor deposits. The study was approved by the Institutional Review Boards of Qilu Hospital of Shandong University. Informed consent was obtained from each patient before being included in the study.2. Immunohistochemical analysis:Formalin-fixed, paraffin-embedded tissue sections were deparaffined in xylene, rehydrated in grade alcohols, and briefly microwaved in citrate buffer to optimize antigen retrieval. Nonspecific binding sites were blocked with diluted goat serum for 30 minutes at room temperature. Slides were incubated with rabbit polyclonal primary antibodies raised against human Notchl intra (ab8387), Hesl (ab49170), D114 (ab7280), VEGFR1 (ab2350), VEGFR2 (ab2349), HIF1 alpha (abl) from Abcam, or VEGF (ZA-0509) and CD31 (ZM-0044) from Zhongshan Co. Ltd, China at a 1:200 dilution at 4° C overnight. Phosphate buffered saline was used for all subsequent washes and for antiserum dilution. After extensive washing (3x5 min) to removeexcess antibody, the slides were incubated with diluted HRP-labeled goat anti-rabbit antibody (Jingmei Co. Ltd, Beijing, China) for 1 hour at room temperature. All the slides were then processed by the SP method (Zhongshan Co. Ltd, Beijing, China) for 30 minutes at room temperature. Non-immune IgG was used as negative controls instead of the primary antiserum. For measurement and scoring of each sample, all slides were stained in a single batch and thus received equal staining. All the sides were imaged digitally and evaluated by Image Pro Plus (IPP), a digitalized immunohistochemistry scoring program (Media Cybernetics, San Diego, CA).3. RNA extraction and reverse transcription:Total RNA was isolated by Trizol (Invitrogen) according to the manufacturer’s instructions. Approximately, one μg of total RNA from each sample was subjected to first-strand cDNA synthesis using RevertAidTM First Strand cDNA Synthesis Kit (MBI, Fermentas, USA). Reverse transcription reaction was done at 42° C for 1 h, followed by 95° C for 5 min. Real-time quantitative polymerase chain reaction (RQ-PCR) was performed using an ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA) in accordance with the manufacturer’s instructions. The real-time PCR contained, in a final volume of 20 μL,10 μL of 2xSYBR Green Real-time PCR Master Mix,1 μL of cDNA, and 1 μL of the forward and reverse primers. The thermal cycling profile consisted of a 95° C denaturation step for 5min, then 40 cycles at 95° C for 15 sec,65° C for 15 sec and 72° C for 45 sec. All experiments were conducted in triplicate. The PCR products were analyzed by melt curve analysis and agarose gel electrophoresis to determine product size and to confirm that no by-products were formed. The results were expressed relative to the number of GAPDH transcripts used as an internal control.4. Human umbilical vein endothelial cells (HUVECs) culture:HUVECs were isolated from fresh human umbilical vein according to methods previously published (Marin et al.,2001). Briefly, the umbilical vein was filled with collagenase. Then we incubated the cord for 7 min at 37° C . After incubation, the cord was kneaded gently to help cell detachment. We centrifuged the cells to obtain HUVECs. HUVECs were cultured in M199 media supplemented with 20% fetal bovine serum (FBS) (Gibco) at 37° C and 5% CO2. HUVECs were used for transfection at passages 3 to 7.5. D114 transfection into HUVECs:Transfection was performed in 24-well plates using Lipofectamine 2000 according to the manufacturer’s instructions. Briefly, HUVECs (5x105) in 500ul of growth medium without antibiotics were seeded into plates. Twenty-four hours later, 8 ug plasmid DNA pD114-IRES2-EGFP or its backbone control vector p-IRES2-EGFP (kindly provided by Dr. Andreas Fischer, University of Heidelberg, German) in 50 ul of opti-MEM I Medium was mixed with diluted Lipofectamine 2000 for twenty minutes. Then, the complexes were added into plates. The transfection media were removed and replaced by culture media 8 h later. HUVECs were harvested 3 days after transfection, and used for real-time PCR detection.6. Statistical analysis:Statistical analyses were performed using SAS version 9 software. For data with nonnormal distribution or heterogeneity of variance, median (range) was shown. Comparison between groups was analyzed by Wilcoxon rank-sum test or Student t test. Spearman’s test was used for correlation analysis. P-value <0.05 was considered statistically significant.Results:1. Aberrant expression profile of D114/Notch and VEGF pathway molecules in lung cancer patients:VEGFR1, VEGFR2 and HES1 of lung cancer tissues were statistically higher than those of controls, while the ratio of VEGFR1/VEGFR2 was significantly decreased in lung cancers. Though other molecules (Notchl, D114 and VEGF) were marginally higher in lung cancers, no statistical difference was observed. RQ-PCR method was performed to confirm the alternation of protein level of Notchl-IC and D114. In concordance with the immunohistochemical analysis, the mean levels of Notchl-IC and D114 mRNA in lung cancer group [2.49e-4 (4.23e-5～2.80e-3) or 1.7e-3 (3.75e-4～4.45e-2)] were marginally higher than those in control group [8.05e-5 (1.09e-5-2.35e-2) or 7.3e-4 (1.34e-5～5.6e-1)]. Spearman correlation coefficients have been calculated between these factors. VEGFR1 was negatively correlated with Notchl (r=-0.43356, p=0.0117) and positively correlated with D114 (r=0.35809, p=0.0442) in lung cancer tissues, while VEGFR2 was positively correlated with D114 in lung cancer tissues(r=0.25427, p=0.1405). Besides that, there were no correlations among other indicators.2. Elevated HIF1a and CD31 in lung cancer tissues:We determined the levels of CD31 and HIFla using the immunohistochemical method. The results demonstrated that HIFla and CD31 were significantly higher in lung tumor tissues compared with controls. In lung cancer tissues, CD31 was negatively correlated with VEGFR1 (r=-0.38055, p=0.0289) and positively correlated with HIF1a (r=0.35437, p=0.0340). Moreover, HIFla and HES1 nearly reached positive correlation in lung cancer tissues (r=0.31745, p=0.0767). No significant differences were found between other factors.3. D114 up-regulated Notch and down-regulated VEGF expression in HUVECs: To confirm the interaction of D114/Notch and VEGF pathway, HUVECs cultured on dishes were transfected with D114-expressing plasmid. Compared with HUVECs transfected with GFP control plasmid, D114, Notchl and VEGFR1 were up-regulated while VEGF and VEGFR2 were down-regulated in D114 transfected HUVECs (P<0.05).4. Clinical relevance of D114/Notch and VEGF pathway molecules in lung cancer patients:VEGFR2 was significantly higher in stage Ⅰ (Median: 116.59, range:50.512-396.853) than that in stage Ⅱ and stage Ⅲ (Median:79.413, range:5.078～176.993; p=0.0193). As for the differentiation status, VEGF expression was higher in poorly-differentiated lung cancers tissues (Median:92.188, range: 10.248～126.330) than that in well-differentiated lung cancers (Median: 51.266, range:16.745～109.059; p=0.0225). No significant relationship was found between the other molecules and clinical parameters in lung cancer tissues.Conclusions:1. There exists overexpression of the VEGF and the Notch signaling pathway molecules in lung cancers, which positively correlate with hypoxia (HIF1a) and angiogenesis (CD31).2. There might be a negative feedback loop between VEGF and the D114/Notch signaling pathway in lung tumor angiogenesis.