Vascular endothelial growth factor (VEGF) is a critical proangiogenic factor in solid tumors. However, its expression and role in human neuroendocrine tumor development and progression remains unclear.
Vascular endothelial growth factor (VEGF) is a critical proangiogenic factor in solid tumors. However, its expression and role in human neuroendocrine tumor development and progression remains unclear.
Using immunohistochemistry, VEGF and Sp1 expression patterns were investigated in 50 cases of human gastrointestinal neuroendocrine tumor having various clinicopathologic characteristics.
It was found that strong VEGF expression was detected in tumor cells, whereas no or very weak VEGF expression was detected in stromal cells surrounding or within the tumors. The levels of VEGF expression directly correlated with the expression levels of Sp1 and microvessel density. Strong, weak, and negative VEGF expression was observed in 32%, 54%, and 14% of cases, respectively. Compared with the group with negative VEGF expression, VEGF (weak/strong) expression was associated with metastasis (14% versus 58%; P = .03). The median progression-free survival (PFS) durations of patients with strong and weak VEGF expression were 29 months and 81 months, respectively. With a median follow-up duration of 50 months, the median PFS duration for the group with negative VEGF expression has not been reached. Compared with the log-rank test, VEGF expression was associated with poor PFS (P = .02). Using in vitro and in vivo models, human carcinoid cell lines were treated with bevacizumab, a monoclonal antibody targeting VEGF. Bevacizumab did not inhibit the growth of carcinoid cells in vitro but significantly reduced tumor angiogenesis and impaired tumor growth in animals.
The data suggest that overexpression of VEGF promotes the growth of human neuroendocrine tumors in part through up-regulation of angiogenesis. Cancer 2007. © 2007 American Cancer Society.
Carcinoid derives from the term “Karzinoide,” which Obendorfer1 first used in 1907 to describe tumors arising in the gastrointestinal tract that had a more indolent clinical course than the more common adenocarcinomas. These are low-grade neuroendocrine tumors that originate from the neuroendocrine cells throughout the body and are capable of producing a variety of hormones and biogenic amines. Those arising from the pancreas are also sometimes called islet cell carcinoma. Our recent analyses of the SEER database have shown steady increases in the diagnosed incidence of carcinoid tumors. Incidence has risen from 0.8 per 100,000 in 1973 to 2.9 per 100,000 in 1999.2 This is likely at least in part due to improved pathologic classification and an increase in the diagnosis of rectal carcinoids during screening colonoscopy.
Neuroendocrine tumors have a wide range of aggressiveness. For locoregional disease, a unified staging system is missing. Disease is incurable in the metastatic setting. Whereas some patients live for months after the diagnosis of metastatic disease, others live beyond 8 years. Somatostatin analogs are commonly used to control hormonal-related symptoms, but tumor regression is rare. Carcinoids are generally resistant to cytotoxic chemotherapy. Systemic therapy options are lacking.
Inherited mutations in several tumor suppressor genes including MEN1, vHL, TS2, and NF1 have been associated with the development of carcinoid tumors.3–6 The genetic abnormalities associated with carcinogenesis and progression in sporadic gastrointestinal neuroendocrine tumors are less well understood; however, allelic deletions at chromosome 11q13 and 3p25 (the sites of the MEN1 and vHL genes) have been observed.7–10 Gastrointestinal neuroendocrine tumors are highly vascular tumors with often accompanying desmoplastic reaction. Previous studies indicated the role of several growth factor families in carcinoid development and progression, including basic fibroblast growth factor (bFGF), transforming growth factor (TGF), endothelial growth factor receptor (EGFR), insulin-like growth factor receptor (IGFR), and vascular endothelial growth factor (VEGF).11–16 The molecular mechanisms behind the abnormal expression of multiple molecules remain unclear. Our recent study of human gastric and pancreatic cancer suggested that abnormal Sp1 activation augments the angiogenic and metastatic capacity of tumor cells through overexpression of multiple Sp1 downstream genes, including the key angiogenic factor VEGF.17, 18 However, it is unknown whether and how Sp1 signaling pathways contribute to neuroendocrine tumor development and progression.
In the present study we examined VEGF expression in human neuroendocrine tumors. We found that elevated VEGF expression occurred in human neuroendocrine tumors and was inversely correlated with progression-free survival (PFS), suggesting that abnormally elevated VEGF expression may represent a potential molecular marker for poor prognosis and directly contribute to neuroendocrine tumor development and progression.
Patients undergoing surgical resection of low-grade neuroendocrine carcinoma at the University of Texas M. D. Anderson Cancer Center were identified through our Neuroendocrine Database. Fifty patients with representative tumor type and stage distribution were selected. Low-grade neuroendocrine histology was confirmed in all cases. Selected cases consisted of 15 islet cell and 35 carcinoids. Primary tumors from resection were used for these analyses. PFS duration was determined by review of medical records and images from computed tomography (CT) or magnetic resonance imaging (MRI) by a physician.
The human bronchial carcinoid cancer cell line NCI-H727 (American Type Culture Collection, Manassas, Va, ATCC, CRL-5815) was purchased from the ATCC. Human pancreatic carcinoid cell line BON-119 was provided by Dr. Kjell Oberg (Uppsala University, Uppsala, Sweden). The cell lines were maintained in plastic flasks as adherent monolayers in minimal essential medium supplemented with 10% fetal bovine serum, sodium pyruvate, nonessential amino acids, L-glutamine, and a vitamin solution (Flow Laboratories, Rockville, Md).
Female athymic BALB/c nude mice were purchased from the Jackson Laboratory (Bar Harbor, Me). The mice were housed in laminar flow cabinets under specific pathogen-free conditions and used when they were 8 weeks old. The animals were maintained in facilities approved by the Association for Assessment and Accreditation of Laboratory Animal Care in accordance with the current regulations and standards of the US Department of Agriculture, Department of Health and Human Services, and National Institutes of Health. All experiments involving use of animals were performed at the M. D. Anderson Cancer Center with approval from its Institutional Animal Care and Use Committee.
Whole cell lysates were prepared from cell cultures. Standard Western blotting was performed using polyclonal rabbit antibody against human VEGF (Santa Cruz Biotechnology, Santa Cruz, Calif) and antirabbit IgG, a horseradish peroxidase-linked F(ab′)2 fragment obtained from a donkey (Amersham Life Sciences, Arlington Heights, Ill). Equal protein sample loading was monitored by hybridizing the same membrane filter with an anti-GAPDH antibody.17, 18 The probe proteins were detected using the Amersham enhanced chemiluminescence system according to the manufacturer's instructions.
Sections (5 μm thick) of formalin-fixed, paraffin-embedded tumor specimens were deparaffinized in xylene and rehydrated in graded alcohol. Antigen retrieval was performed with 0.05% saponin for 30 minutes at room temperature. Endogenous peroxidase was blocked using 3% hydrogen peroxide in phosphate-buffered saline (PBS) for 12 minutes. The specimens were incubated for 20 minutes at room temperature with a protein-blocking solution consisting of PBS (pH 7.5) containing 5% normal horse serum and 1% normal goat serum and then incubated at 4°C with rabbit polyclonal antibodies against human VEGF, CD34, and Sp1 (clone PEP2). The samples were then rinsed and incubated for 1 hour at room temperature with appropriate peroxidase-conjugated secondary antibodies. Next, the slides were rinsed with PBS and incubated for 5 minutes with diaminobenzidine (Research Genetics, Huntsville, Ala). The sections were washed 3 times with distilled water, counterstained with Mayer's hematoxylin (Biogenex Laboratories, San Ramon, Calif), and washed once each with distilled water and PBS. Afterward, the slides were mounted using Universal Mount (Research Genetics) and examined by using a brightfield microscope. A positive reaction was indicated by a reddish-brown precipitate in the nuclei and/or cytoplasm.17, 18 Depending on the percentage of positive cells and staining intensity, staining was classified into 3 groups: negative, weak positive, and strong positive. Specifically, the percentage of positive cells was divided into 5 grades (percentage scores): <10% (0), 10% to 25% (1), 25% to 50% (2), 50% to 75% (3), and >75% (4). The intensity of staining was divided into 4 grades (intensity scores): no staining (0), light brown (1), brown (2), and dark brown (3). Sp1 staining positivity was determined by the formula: overall scores = percentage score × intensity score. An overall score of ≤3 was defined as negative, of >3 to ≤6 as weak positive, and of >6 as strong positive.
Tumor cells (3 × 106) were cultured in 1 mL serum-free medium for 24 hours and the medium was collected and centrifuged to remove any cell debris before its use as a conditioned medium. The tube formation assay was performed as described previously.20 Briefly, 250 μL of growth factor-reduced Matrigel (Becton-Dickinson, San Jose, Calif) was pipetted into each well of a 24-well plate and polymerized for 30 minutes at 37°C. Human umbilical vein endothelial cells (HUVECs) was harvested and suspended in conditioned medium from either control cells or genetically altered cells cultured for 48 hours in modified Eagle's medium containing 1% fetal bovine serum. Then 2 × 104 HUVECs in 300 μL of conditioned medium was added to each well and incubated at 37°C, 5% CO2, for 20 hours. The cultures were photographed with brightfield microscopy using a Sony digital camera equipped with an Optimas 6.2 program. To obtain optimal contrast and visual effect the color scheme of the original photos was inverted so that the white color represents endothelial cells on black background. The degree of tube formation was assessed as the percentage of cell surface area versus total surface area. For the proliferation assay, 2 × 104 HUVECs in 200 μL of conditioned medium was added to 96-well plates and incubated at 37°C, 5% CO2 for 24 hours and [3H]-TdR was added at 0.1 mCi/mL. [3H]-TdR incorporation was determined 12 hours after the addition of [3H]-TdR.
To prepare tumor cells for inoculation, cells in the exponential growth phase were harvested by brief exposure to a 0.25% trypsin / 0.02% ethylenediaminetetraacetic acid solution (wt/vol). Cell viability was determined by using Trypan blue exclusion, and only single-cell suspensions that were more than 95% viable were used. Tumor cells (1.5 × 106 cells/mouse) were then injected into the subcutis of nude mice in groups of 5. Tumor sizes (longest and shortest diameters of each tumor) were measures once a week and tumor volume was calculated as described previously.
For VEGF staining, sections (5 μm thick) of formalin-fixed, paraffin-embedded tumor specimens were deparaffinized in xylene and rehydrated in graded alcohol. For CD31 staining, frozen sections (5 μm thick) were fixed with acetone. Endogenous peroxidase was blocked using 3% hydrogen peroxide in PBS for 12 minutes. The sections were incubated for 20 minutes at room temperature with a protein-blocking solution consisting of PBS (pH 7.5) containing 5% normal horse serum and 1% normal goat serum and then incubated overnight at 4°C in anti-CD31or anti-VEGF antibodies. VEGF expression and microvessel density (MVD) status were assessed as described above.17, 18
Each experiment was performed independently at least twice with similar results; 1 representative experiment is presented. The significance of the in vitro data was determined using a Student t-test (2-tailed), whereas that of the in vivo data was determined using the 2-tailed Mann-Whitney U-test. χ2 and the Fisher exact test were used to assess the relation between VEGF expression and clinical-pathologic parameters. One-way analysis of variance (ANOVA) analyses were used to compare mean of continuous variables. PFS duration was calculated from the date of surgical resection using the Kaplan-Meier method. Two-sided P-values ≤0.05 were considered statistically significant.
We performed immunostaining of VEGF and MVD on primary tumors resected from 50 patients with low-grade neuroendocrine tumor (Table 1). Classified into 3 groups, strong, weak, and negative, VEGF expression was observed in 16 (32%), 27 (54%), and 7 (14%) of cases, respectively. PFS duration was estimated by the Kaplan-Meier method and compared by VEGF expression. Strong VEGF expression was associated with a poor prognosis. The median PFS durations of patients with strong and weak VEGF expression were 29 months and 81 months, respectively (P = .02). The median PFS of patients with negative VEGF expression has not been reached. In this group, at last follow-up 6 (86%) patients, with median follow-up of 50 months, are alive and free from disease progression (Fig. 1A). We then stratified the cases into carcinoid and islet cell groups and compared the PFS duration by VEGF expression. VEGF expression remained a predictor of outcome (P = .05). Moreover, increased VEGF expression predicted an elevated angiogenic phenotype (Fig. 1B), which was observed in the majority of cases (Fig. 1C).
|Parameter||All||VEGF (−)||VEGF (+)||VEGF (+)|
|Site of primary|
|Small bowel NOS||4||1||3||0|
|Size of lesion|
Among the 50 cases included in this study, 24 had local-regional disease and 26 had metastasis. We compared the group with negative VEGF expression to the group with positive (weak or strong) VEGF expression (Table 2). The presence of VEGF expression was associated with metastasis (14% versus 58%; P = .03). We next examined the effect of VEGF expression among carcinoid and islet cell subgroups. Among 35 carcinoids, 71% of cases with positive VEGF expression had metastasis; 25% of cases with negative expression had metastasis. Similarly, among 15 islet cell cases 25% of cases with positive VEGF expression had metastasis; none of the cases with negative VEGF expression had metastasis. However, due to the smaller number of cases in the subgroups, these differences were not statistically significant.
|Locoregional, n = 24 No. (%)||Metastatic, n = 26 No. (%)|
|Negative, n = 7||6 (86)||1 (14)|
|Positive, n = 43||18 (42)||25 (58)|
Interestingly, a correlation between VEGF and Sp1 expression was observed. Among cases with strong (10 cases, 20%), weak (27 cases, 54%), and negative (13, 25%) Sp1 expression, VEGF expression (strong and weak) were observed in 13 of 13 (100%), 23 of 27 (85%), and 7 of 10 (70%) of cases, respectively (Table 3, P = .03). When Sp1 expression was analyzed relative to tumor size, larger tumor size correlated with strong expression (P = .04). The mean tumor sizes among patients with strong, weak, and negative Sp1 expression were 5.5, 2.6, and 3.2 cm.
|Negative, n = 10 No. (%)||Weak, n = 27 No. (%)||Strong, n = 13 No. (%)|
|Negative, n = 7||3 (30)||4 (15)||0 (0)|
|Weak, n = 27||4 (40)||18 (67)||5 (38)|
|Strong, n = 16||3 (30)||5 (19)||8 (61)|
To determine whether VEGF is an autocrine growth factor for human carcinoid cells, H727 and BON cells were incubated for 48 hours in a medium or medium containing 0.001 to 100 μg/mL bevacizumab. Viable cells were determined by MTT assay. Also, total protein lysates were harvested from the cell cultures and VEGF protein expression was determined by Western blot analysis. We found that bevacizumab treatment did not affect the growth of and VEGF protein expression in either BON and H727 cells (Fig. 2).
Next, culture supernatants were harvested from H727 cells, H727 cells treated with control IgG, H727 cells treated with bevacizumab, or H727 cells treated with bevacizumab and then addition of VEGF. The angiogenic potentials of the supernatants were determined by an endothelial cell tube formation assay and endothelial cell proliferation assay. We found that neutralization of VEGF in the culture supernatants of carcinoid cells almost totally blocked their ability to stimulate endothelial cells tube formation and proliferation, i.e., suppression of angiogenic potential, which was substantially reversed by addition of recombinant VEGF (Fig. 3). These data suggested that VEGF secreted from human carcinoid cells plays a pivotal role in angiogenic phenotype.
Finally, we determined the effect of neutralizing carcinoid cells-derived VEGF on their angiogenesis and growth in animal models. The H727 and BON cells (1 × 106/mouse) were injected into the subcutis of nude mice. Groups of mice (5 each) received either an intraperitoneal injection (twice a week) of PBS or bevacizumab when tumors reached 3 to 4 mm in diameter. Tumors sizes were monitored once every week. We found that bevacizumab exhibited significant antitumor activity (Fig. 4A,B) consistent with decreased MVD (Fig. 4C). However, an increased VEGF expression was consistently found in all bevacizumab-treated tumors as determined by immunohistochemistry.
Angiogenesis is required for tumor growth beyond a small size and is crucial in the process of metastasis. In the present study we provide clinical, experimental, and mechanistic evidence of VEGF overexpression and its important role in human neuroendocrine tumors. Specifically, we found that in human neuroendocrine tumors there is a strong correlation between expression of transcriptional factor Sp1, VEGF, and tumor size. In turn, VEGF expression was also associated with metastasis. In experimental models we found VEGF neutralizing antibody bevacizumab inhibited tumor growth in a human carcinoid xenograft model but not in tissue culture. This suggests the activity of bevacizumab in this model is mediated predominantly through an antiangiogenic mechanism.
In a previous report, Couvelard et al21 had reported that higher microvascular density and VEGF expression in islet cell tumors predicted a more favorable prognosis. However, it is important to note that there are significant differences between that study and our study. In our study only low-grade or well-differentiated neuroendocrine tumors were included. Thus, among patients with uniform histologic grade, VEGF expression predicted a poor outcome. In the previous study, the investigators included tumors with varying histologic grade. They found that VEGF expression and MVD were higher among benign islet cell tumors and well-differentiated islet cell carcinoma compared with poorly differentiated cases. However, similar to small cell carcinoma of the lung, poorly differentiated or high-grade neuroendocrine tumors are known to have different biology, pattern of growth, and may be less dependent on VEGF for growth and metastasis. Our findings are also supported by other investigators who have found plasma VEGF levels to correlate with disease progression in patients with neuroendocrine tumors.22
Our findings in human neuroendocrine tumors are also supported by prior studies in xenograft. Treating BON-1 implanted in nude mice with interferon, investigators found that the suppression of VEGF gene transcription by interferon alpha was mediated through an Sp1 and/or Sp3 dependent mechanism.23 In a separate study, investigators implanted duodenal carcinoid in nude mice. Treatment with neutralizing antibody to VEGF resulted in decreased tumor size and inhibition of liver metastasis.24 These findings support our findings and suggest that Sp1 and VEGF may be crucial in human neuroendocrine tumor progression. However, we acknowledge that in vitro and in vivo experiments may not necessarily be completely reflective of carcinoid biology in humans.
In the current study we also found that in human carcinoid xenograft, whereas treatment with bevacizumab inhibited tumor growth, it also led to increased VEGF expression. Our finding is consistent with the result observed in a Phase II study of the VEGF tyrosine kinase inhibitor sunitinib in patients with neuroendocrine tumors.25 In that study the investigators found a greater than 3-fold in plasma VEGF levels after 1 cycle of therapy. These data suggest up-regulation of VEGF transcription may be potential mechanism of resistance among neuroendocrine tumor patients treated with VEGF inhibitors.
The notion that abnormal Sp1 and VEGF expression contributes to neuroendocrine tumor development and progression is well supported not only by our study of pancreatic cancer18 and gastric cancer17 but also by other lines of evidence showing that Sp1 may regulate many aspects of cancer biology, including cell growth, survival, invasion, and angiogenesis. Early studies indicated that Sp1 regulates multiple growth-regulated genes, arguing that Sp1 may be important for cell-growth regulation. Direct evidence of the ability of Sp1 to modulate transcription during changes in cell growth came with the demonstration that Sp1 is involved in the effects of serum stimulation of quiescent cells at the rep3a promoter26 as well as at the hamster dihydrofolate reductase27, 28 and ornithine decarboxylase promoters.29 More recently, several studies established the ability of Sp1 to mediate the growth induction of a variety of promoters, including those of the genes encoding FGFR1, EGFR, IGFR1, HGFR,28, 30–32 IGF-binding protein 2,33 VEGF,18, 34 thymidine kinase,35 and serum response factor.36 Many of these are also proangiogenic. Accumulating evidence has suggested that Sp1 family members are involved in multiple aspects of angiogenesis.37
The exact mechanism for Sp1 overactivation in neuroendocrine tumors is currently unknown. Altered oncogenes and suppressor genes may have a particularly important role. Loss of the vHL gene has been associated with the development of islet cell carcinoma.4 Allelic deletion at chromosome 3p, the site of the vHL gene, has also been described to occur frequently in sporadic carcinoid and islet cell tumors.8–10, 38 Tumor cells harboring the inactivated vHL gene are known to have increased VEGF expression and angiogenesis.39–41 Also, vHL-mediated repression of VEGF expression has convincingly been shown to be mediated by transcriptional and posttranscriptional mechanisms.42, 43 At the transcriptional level, vHL forms a complex with the Sp1 transcription factor and inhibits Sp1-mediated VEGF expression.44 Whether oncogenes and/or tumor suppressor genes are involved in constitutive Sp1 activation in human cancer cells and how their status affects that activation remains to be fully elucidated.
Whereas genomic deletions may confer a proliferative advantage for by inactivating tumor suppressor genes such as MEN1 and vHL, they may also create weakness resulting in potential therapeutic targets. For example, the VEGF-B gene activates the same receptors as VEGF-A. Up-regulation of VEGF-B expression represents a potential mechanism of resistance to agents targeting VEGF-A. However, VEGF-B is located at 11q13 near the MEN1 gene, a site of frequent allelic deletion in low-grade neuroendocrine tumors.38, 45, 46 Indeed, VEGF-A itself is located on 6p12, a region where 1 copy is frequently deleted in islet cell.38 NRP-2, a nontyrosine kinase receptor for VEGF, appears to be expressed in normal neuroendocrine cells lining the digestive tract.47 Loss of expression has been reported in carcinoid tumors arising from the appendix, colon, and rectum.47 Thus, deletions and loss of expression of genes in the VEGF pathway may partially explain why neuroendocrine tumors are among the few diseases where there is single-agent activity for VEGF inhibitors.48, 49
In summary, the VEGF pathway is crucial for neuroendocrine tumor carcinogenesis and progression. VEGF expression may have prognostic value in neuroendocrine patients undergoing resection. We are currently investigating the molecular mechanism by which the Sp1 signaling pathway regulates neuroendocrine tumor development and progression and whether this Sp1 pathway is a potential therapeutic target for controlling cancer growth and metastasis. Human trials targeting VEGF are under development in clinics.
We thank Cindi Tomlin for expert help in the preparation of the article and Don Norwood for editorial comments