Enhanced Expression of Tie2, Its Ligand Angiopoietin-1, Vascular Endothelial Growth Factor, and CD31 in Human Non-Small Cell Lung Carcinomas¹

Lung cancer is the leading cause of cancer-related death of males in both Japan³ and the United States (1). Surgical resection remains the most effective treatment for NSCLC,⁴ although, at the time of diagnosis, the majority of patients present with advanced-stage disease that is no longer amenable to curative therapy (2). Indeed, a large percentage of patients undergoing surgical resection ultimately die of recurrent NSCLC, due to the presence of occult metastatic sites at the time of diagnosis (3). Attempts to improve this situation are focused on eliminating the causes, preventing the disease in high-risk groups, diagnosing lesions at an early curable stage, and developing new adjuvant and neoadjuvant protocols. Each of these strategies has met with limited success. Recently, there have been intense studies of molecular abnormalities involving dominant and recessive oncogenes in clinically evident lung cancers (4, 5, 6), targeting biomarkers for early detection, due to chemotherapeutic response and gene therapy. Changes in angiogenic factors that contribute to NSCLC progression, some as independent predictors of prognosis, are particularly important in this context (7, 8, 9).

Tie1 and Tie2 (tek) are members of the endothelial cell-specific receptor tyrosine kinase family (10, 11), essential for formation of the embryonic vasculature (12). They are up-regulated in the vascular endothelium of metastatic melanomas (13), brain tumors (14) and mammary carcinomas (15). A ligand for the Tie1 receptor has not yet been identified, but ligands for Tie2, Ang-1, and Ang-2 have recently been shown to be essential for normal vascular development in the mouse (16, 17). Whether Tie2 and Ang-1 might be up-regulated in vessels of lung neoplasms has not been addressed, to our knowledge. The recently developed multiprobe RPA has advantages for simultaneous investigation of mRNA expression of several genes. In the present study, it was used to examine the expression of 10 angiogenesis-associated factors in human NSCLC. In addition to Tie1, Tie2, and Ang-1, VEGF, a well-established angiogenic factor (18, 19), and its receptors VEGF receptor-1/Flt-1 and VEGF receptor-2/fetal liver tyrosine kinase-1 were included because they have been reported to be up-regulated in human (7, 8, 20) and rat (21) NSCLCs. CD31 was also studied as a marker for vascularity (22).

Primary NSCLC tissues obtained from 32 patients undergoing surgery in Nara Medical University Hospital between April 1998 and December 1998 were frozen in liquid nitrogen and stored at −80°C until use. Data for histological types and details for sex, age distribution, and pathological stages are summarized in Table 1. The histology and pathological stage were classified according to General Rules for Clinical and Pathological Recording of Union International Contre le Cancer (23). Noncancerous tissues adjacent to tumors were also frozen in liquid nitrogen and stored at −80°C. Total RNA was extracted from each sample using an ISOGENE kit (Nippon Gene, Toyama, Japan), as described previously (21).

A panel of angiogenesis-associated mRNA species were detected using a multiprobe protection assay system (RiboQuant, PharMingen, San Diego, CA). Radiolabeled probes were synthesized from DNA templates containing a T7 RNA polymerase promoter (PharMingen), transcribed in the presence of 100 μCi [α-³²P] dCTP to yield radioactive probes of defined sizes. Probes were hybridized with 5 μg of total RNA, then samples were treated with RNase A and T1 to digest single-stranded RNA. Intact double-stranded RNA hybrids were resolved on 5% polyacrylamide/7 M urea gels at 50 W for 70 min. Dried gels were analyzed for band locations and PSL values with a BAS 1000 Phospho Imaging Analyzer (Fuji Photo Film Co., Ltd., Minato-ku, Tokyo, Japan). Within each sample, the intensity of each angiogenesis-associated mRNA band was divided by the sum of the L32 + GAPDH bands. The size of each band was analyzed in terms of its migration distance against a plotted standard curve of migration distance versus log nucleotide length for each undigested probe (RiboQuant, Instruction manual, 5th edition, April 1998, PharMingen). The resulting value for each mRNA species was then expressed as a percentage of the average for the parameter. Finally, dried gels were redeveloped overnight by traditional autoradiography.

Statistical analyses were performed using a personal computer, basically as described previously elsewhere (24). To assess the statistical significance of differences in angiogenesis-associated mRNA species between cancer and noncancer lung specimens, the Mann-Whitney nonparametric test was applied. To assess relationships between two specified factors, Pearson’s correlation coefficients were obtained with linear trends also determined using the data generated in the ANOVA. Significant differences from zero of the slope of each regression function were assessed using ANOVA tables. Lack of significant departure from linearity for each regression function was confirmed doubly by a Runs test and a test using the ANOVA table.

The results of the multiprobe RPA analysis are depicted in Fig. 1. The range of PSL values was from 7.29–979.3, therefore, all values quantified could be analyzed in the linear range. The PSL values for angiogenesis-associated gene expression are summarized in Table 2. Four angiogenesis-associated gene products, Tie2, Ang-1, CD31, and VEGF were increased in cancers, the average magnitudes being 1.3-fold for Tie2, 3.9-fold for Ang-1, 1.5-fold for CD31, and 1.4-fold for VEGF. Expression of VEGF-C mRNA was not detected in any of the samples examined. Correlations between Ang-1 mRNA and Tie2 mRNA, and Ang-1 mRNA and CD31 mRNA are shown in Fig. 2, A and B, respectively. A clear relationship was observed between Ang-1 mRNA and Tie2 mRNA expression (r = 0.98, P < 0.0001). The correlation coefficient between Ang-1 and CD31 mRNA expression was also significant statistically (r = 0.47, P < 0.0001), but with a significant departure from linearity (P < 0.0001). Correlations between VEGF mRNA and CD31 mRNA, and VEGF mRNA and Flt-1 mRNA are shown in Fig. 3, A and B, respectively. A clear relationship was observed between VEGF mRNA and CD31 mRNA expression (r = 0.43, P = 0.0003). Although the correlation coefficient between VEGF mRNA and Flt-1 mRNA expression was also significant statistically (r = 0.79, P < 0.0001), there was again a significant departure from linearity (P = 0.019). Overexpression of Tie2, Ang-1, CD31, and VEGF and the correlations among Tie2 and Ang-1 or VEGF and CD31 did not differ with the histological type or pathological stage of NSCLCs.

Angiogenesis, the sprouting of new blood vessels from preexisting ones, is a complex process involving the interactions of a magnitude of angiogenesis-associated genes (25, 26, 27, 28). It is well known that angiogenic factors contribute to development and progression of solid tumors (26, 29). The present finding of overexpression and correlation of mRNA levels of both Tie2 and Ang-1 in NSCLC is indicative of activation of the Tie2/Ang-1 system (16). The results for VEGF and CD31 are further suggestive of angiogenesis in NSCLC (18, 22).

Angiopoietins (Ang-1 and Ang-2) constitute a novel family of endothelial growth factors that are ligands for Tie2 (9, 10, 11, 12, 14, 30). Whereas Ang-1 can stimulate endothelial sprouting in vitro (31), however, exposure of Tie2 in cultured endothelial cells to either Ang-1 or Ang-2, unlike the case with other endothelial growth factors, does not produce a mitogenic response (9). Unfortunately, we could not analyze the expression of Ang-2 because of the lack of an appropriate probe, but this warrants further investigation.

VEGF, first detected as a factor inducing microvascular permeability (32) and extravasation of various proteins to metastases, also acts as an endothelial cell mitogen (33). Previously, we reported frequent expression of VEGF detected by immunohistochemistry in human NSCLCs (34). The present results, thus, provide confirmation at the mRNA level with a molecular biological technique.

The realization that multiple pathways are likely to be required for the assembly of a functional vasculature has broad implications for therapeutic modulation. For example, it has already been shown that blocking either the Tie2/Ang-1 pathway or the VEGF pathway can inhibit tumor angiogenesis (35, 36). Because Tie2/Ang-1 and VEGF function at different stages of vascular development, it seems likely that inhibiting both pathways may be more effective than inhibiting each individually (37). Moreover, our results suggest the possibility that antibody therapy against each of these may improve the outcome with this otherwise generally refractory cancer.

We express our gratitude to Dr. Malcolm Moore for English correction and careful reading of this manuscript; to Megumi Inagaki-Yamaguchi, Hiroko Masuda, and Sachiko Nakai for technical assistance; and to Yumi Horikawa, Rie Maeda, Nami Makimura, and Yuko Yoshinaka for editorial assistance.