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Journal of Clinical Oncology, Vol 20, Issue 4 (February), 2002: 900-910
© 2002 American Society for Clinical Oncology

Aberrant p53 Expression Correlates With Expression of Vascular Endothelial Growth Factor mRNA and Interleukin-8 mRNA and Neoangiogenesis in Non–Small-Cell Lung Cancer

By Ang Yuan, Chong-Jen Yu, Kwen-Tay Luh, Sow-Hsong Kuo, Yung-Chie Lee, Pan-Chyr Yang

From the Division of Chest Medicine, Department of Internal Medicine; Division of Chest Surgery, Department of Surgery; and Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, and Institute of Biomedical Sciences, Academia Sinica, Taiwan.

Address reprint requests to Pan-Chyr Yang, MD, PhD, National Taiwan University Hospital, No 7, Chung-Shan South Rd, Taipei 100, Taiwan; email: pcyang{at}ha.mc.ntu.edu.tw


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PURPOSE: To evaluate interactions between expressions of tumor suppressor gene p53 and angiogenic factors vascular endothelial cell growth factor (VEGF) and interleukin-8 (IL-8) and their effect on tumor angiogenesis and patient prognosis in non–small-cell lung cancer (NSCLC).

PATIENTS AND METHODS: p53, VEGF, IL-8, and the microvessel endothelium were immunostained, and VEGF and IL-8 mRNA expression were quantified using the real-time quantitative reverse-transcription polymerase chain reaction in 65 NSCLC surgical specimens. Aberrant p53 expression was correlated with VEGF and IL-8 mRNA expression, microvessel count (MVC), other clinical-pathologic variables, and patients’ survival.

RESULTS: Tumors with high aberrant p53 expression showed significantly higher VEGF and IL-8 mRNA expression and MVC than those with low aberrant p53 expression (P < .001). When tested as a continuous variable, aberrant p53 expression correlated strongly and positively with VEGF and IL-8 mRNA expression and MVC (P < .0001). Tumors with high aberrant p53 expression were associated with mediastinal or distant lymph node metastasis (P = .006). Survival and postoperative relapse time were significantly shorter in patients with high aberrant p53 expression tumors than in those with low aberrant expression tumors (P < .0001). A significant difference in survival was also seen between patients with high and low tumoral VEGF mRNA expression and between those with high and low tumoral IL-8 mRNA expression (P < .0001).

CONCLUSION: We report here for the first time that aberrant p53 expression is strongly positively correlated with VEGF mRNA and IL-8 mRNA expression in NSCLC. This result indicates that aberrant p53 expression may play a significant role in regulation of VEGF and IL-8 expression and be involved in controlling angiogenesis and explains the adverse prognosis of cancers with high aberrant p53 expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ANGIOGENESIS IS NECESSARY for tumor growth. Without developing new blood vessels, a tumor cannot exceed a volume of 1 to 2 mm2.1 New blood vessel formation (neovascularization) also provides a route for cancer cells to spread to distant organs.2 A high intratumor microvessel count (MVC) (an index of angiogenesis) is reported to be associated with rapid tumor progression and poor prognosis in melanoma3 and cancer of the breast,4 prostate,5 brain,6 colon,7 and lung.8,9

Many studies have demonstrated that angiogenesis is stimulated by a variety of tumor-secreted angiogenic factors and inhibited by angiogenic inhibitors.10 Vascular endothelial growth factor (VEGF) is one of the most potent endothelial cell–specific mitogens and can stimulate endothelial cell proliferation, migration, and tubular formation.11 VEGF overexpression has been noted in human cancers of the brain,12 colon,13 breast,14 and lung15 and is reported to correlate with a high MVC and an adverse prognosis in these cancers, including non–small-cell lung cancer (NSCLC).16-19 Interleukin-8 (IL-8), a C-X-C proinflammatory chemokine, has also recently been reported to have angiogenic activity and play an important role in tumor-associated angiogenesis in several solid cancers, namely melanoma,20 colorectal carcinoma,21 glioblastoma,22 and NSCLC.23

p53 is a well-known tumor suppressor gene in a wide variety of cancers, and its mutation is reported to be one of the most common genetic changes found in malignant tumors.24 Wild-type p53 protein can suppress tumorigenesis and promote apoptosis and is a transcriptional factor that influences the transcription of other genes.25,26 p53 gene mutation is reported to occur in 40% to 50% of NSCLCs, and aberrant p53 expression correlates with an adverse prognosis in lung cancers.27 Interestingly, several recent studies have indicated that p53 might be involved in angiogenesis. Dameron et al28 showed that in fibroblasts, wild-type p53 inhibits angiogenesis via upregulation of TSP-1, a potent inhibitor of angiogenesis. Kieser et al29 demonstrated that in the 293 cell line (an adenovirus-transformed human fetal kidney cell line) and the NIH 3T3 cell line, mutant p53 can enhance VEGF expression induced by 12-O-tetradecanoylphorbal-13-acetate, and Mukhopadhyay et al30 showed that wild-type p53 reduces endogenous VEGF mRNA levels and VEGF promotor activity in U-87M6 human glioblastoma-astrocytoma cells and the 293 cell line.

Several clinical studies have examined the relationship between p53 mutation and VEGF expression in human cancer in vivo with conflicting results. Some studies showed that aberrant p53 protein expression is associated with VEGF overexpression in human solid cancers, including colon cancer,31 gastric cancer,32 and NSCLC,33 whereas others found no correlation between mutated p53 and VEGF expression in brain cancer,12 oral squamous cell cancer,34 and NSCLC.35 In a recent study, wild-type p53 did not repress the hypoxia-induced transcription of VEGF.36

Whether p53 also plays a role in regulating IL-8 expression in cancer-associated angiogenesis is still unclear, because there have been few studies of the relationship between p53 mutation and IL-8 expression; in addition, this relationship has never been examined in NSCLC.

To answer these questions, we assessed aberrant p53 protein expression, the expression of VEGF and IL-8 proteins, and MVC by immunohistochemical staining and quantified VEGF mRNA and IL-8 mRNA expression by real-time quantitative (RTQ) reverse transcriptase polymerase chain reaction (RT-PCR) in tumors from 65 patients with NSCLC. The aims of this study were to determine whether there was a correlation between aberrant p53 expression and VEGF mRNA or IL-8 mRNA expression, intratumoral MVC, and the clinicopathologic characteristics and prognosis of patients with NSCLC.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Tissue Samples
Between May 1994 and December 1996, 76 sequential patients underwent surgical resection for NSCLC in this institute. Of these, three were excluded because of inappropriate specimen storage, five because of lack of clinical follow-up, and three because of mortality from complications after their operations; 65 patients were therefore included in the study. These consisted of 48 men and 17 women with a mean age of 61 ± 11 years. The histologic classification of the NSCLC was determined as recommended by the World Health Organization,37 and tumor staging was performed according to the tumor-node-metastasis staging system recommended by the American Joint Committee on Cancer.38 Paraffin-embedded, formalin-fixed surgical specimens were collected for immunohistochemical staining for p53, VEGF, and IL-8 proteins and intratumoral microvessel endothelial cells. The tumor tissue, obtained immediately after surgery, was placed in liquid nitrogen and stored frozen at -80°C for subsequent quantification of VEGF mRNA and IL-8 mRNA expression. The histopathology present in the archived frozen tissues was confirmed by a pathologist to be similar to that in the paraffin-embedded tissues.

The survival time of patients was calculated from the date of operation to the date of death. The relapse time was calculated from the date of operation to the date of local recurrence or distant metastasis. The follow-up period lasted up to 60 months.

Immunohistochemical Staining for p53 Protein, VEGF, IL-8, and Microvessels
Immunohistochemical staining of p53 protein, VEGF, IL-8, and microvessels was performed using the avidin-biotin peroxidase method, with modifications.8 Briefly, 5-µm sections were mounted on poly-L-lysine–coated slides, dewaxed, rehydrated, and predigested with protease for 10 minutes at 37°C. Antigen retrieval was performed by immersing the slides in 0.1 mol/L sodium citrate buffer, pH 6.0, and then heating them in a microwave oven for 10 minutes (600 wattage, 110 V). All subsequent stages were performed at room temperature. Endogenous peroxidase was blocked by 20-minute treatment with 0.3% hydrogen peroxide in methanol. After preincubation with fetal mouse serum, mouse monoclonal antibodies against p53 protein (DO-7; 1:100 dilution; Novocastra, Newcastle, United Kingdom), IL-8 (1:200 dilution; Endogen, Woburn, MA) or VEGF (1:50 dilution; Upstate Biotechnology, Lake Placid, NY) or a polyclonal mouse anti-CD34 antiserum (1:100 dilution; Novocastra; endothelial cell marker) were applied for 60 minutes. After washes, the slides were incubated for 30 minutes with rabbit anti-mouse Ig antibody (Zymed Laboratories, South San Francisco, CA) and then with a 1:20 dilution of avidin-biotin peroxidase complex (Zymed Laboratories) for 30 minutes. The color was developed by incubating the slides for 20 minutes with 3,3-diaminobenzidine (Zymed Laboratories). Counterstaining was performed using Mayer’s solution, giving a blue background. A breast cancer specimen with known p53 mutation was used as the positive control for aberrant p53 protein expression, and the capillary surrounding the alveoli of normal lung tissue was used as the positive control for anti-CD34 staining. Bronchial epithelium and alveolar macrophages were used, respectively, as positive controls for VEGF and IL-8. Negative controls were sections stained without the use of primary antibodies or using a control IgG instead of primary antibodies.

Analysis of Aberrant p53 Expression and Microvessel Counts
Brown staining of the nuclei of cancer cells was considered as positive for aberrant p53 protein expression (Fig 1). The percentage of cells with aberrant p53 expression was estimated by counting the number of immunoreactive cells in all cancer cells in at least 5 x 200 fields (x 20 objective with x 10 ocular, 0.785 mm2 per field). Aberrant p53 protein expression was considered both a continuous and a dichotomous variable. The median value for the percentage of positively stained cells (20%) was used as the cutoff to distinguish between tumors with low or high aberrant p53 expression.



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  		Fig 1. Immunohistochemical staining of p53. The aberrant p53 protein accumulation (brown color) is localized in the nuclei of cancer cells of a squamous cell carcinoma.

 
Brown immunostained endothelial cell clusters that were clearly separated from one another were considered a single microvessel.8 Microvessels in the area of most intense neovascularization were counted in three randomly chosen x 200 fields, and the average of the three readings was taken as the MVC. The MVC was considered both a continuous and dichotomous variable. The median value of 110 was used as the cutoff to distinguish between tumors with low and high MVCs.

Cells with aberrant p53 protein expression were counted by two independent observers who had no knowledge of the clinicopathologic variables, including the MVC. The MVC was similarly estimated without the observers having any knowledge of the aberrant p53 expression status. There was good interobserver correlation for both the counting of cells with aberrant p53 expression (r = .88, P < .001) and the MVC (r = .90, P < .001).

Quantification of VEGF mRNA and IL-8 mRNA Expression Using RTQ RT-PCR
An RNA extraction kit (Rneasy Minit kit; Qiagen, Valencia, CA) was used to extract total RNA from the frozen resected tumor tissue.

RTQ RT-PCR, a newly developed kinetic quantitative RT-PCR method, is considered to be one of the most sensitive and accurate methods for the quantification of nucleic acid (DNA and RNA) in tissue samples.39 This method (the TaqMan reaction) is based on the 5' nuclease activity of Taq polymerase, which cleaves a specific dual-labeled fluorogenic hybridization probe during the extension phase of the PCR. As long as this sequence-specific probe is intact, emission by a reporter dye at its 5' end is quenched by a second fluorescent dye at the 3' end. During the extension phase of the PCR, Taq polymerase hydrolyses the probe and releases the reporter dye, resulting in an increase in peak fluorescence emission that is directly proportional to the number of amplified copies and is detected and quantified by a detector in real time. A higher starting copy number of the nucleic acid results in an earlier increase in fluorescence. The threshold cycle (CT) is defined as the fractional cycle number at which the fluorescence generated by cleavage of the probe exceeds a fixed threshold above baseline. For a chosen threshold, a smaller starting copy number results in a higher CT value. In this study, we used RTQ RT-PCR for the relative quantification of VEGF mRNA and IL-8 mRNA in tumor specimens, with ß-actin mRNA as an internal control. The integrity of the RNA was estimated by real-time RT-PCR of mRNA for the TATA box-binding protein (TBP), a nonabundant housekeeping gene.40

Primers, Probes, and Reference Internal Control mRNA
Primers and probes were chosen using the computer program Primer Express (Perkin-Elmer Applied Biosystems, Forster City, CA). Primers and probes were synthesized by and purchased from Perkin-Elmer Applied Biosystems. On the basis of the cDNA sequence (gene bank accession no. m32977), the sequences of the primers and probe used for RTQ RT-PCR of VEGF mRNA were as follows: (1) forward primer, 5'-GCA CCC ATG GCA GAA GG-3' (in exon 2); (2) reverse primer, 5'-CTC GAT TGG ATG GCA GTA GCT-3' (in exon 3); and (3) probe, 5'-ACG AAG TGG TGA AGT TCA TGG ATG TCT ATC AC-3' (spanning the exon 2-exon 3 junction to avoid quantification of the PCR product of contaminating VEGF genomic DNA). The sequences of the primers and probe for IL-8 mRNA quantification, chosen using the IL-8 cDNA sequence data,41 were as follows: (1) forward primer, 5'- CTC TTG GCA GCC TTC CTG ATT-3' (exon 1); (2) reverse primer, 5'-TAT GCA CTG ACA TCT AAG TTC TTT AGCA-3' (exon 2); and (3) probe, 5'-CTT GGC AAA ACT GCA CCT TCA CAC AGA-3' (spanning the exon 1-exon 2 junction). The probe sequence is specific to IL-8 cDNA, and the probe does not hybridize with other chemokine family members42; it was chosen to span the exon 1-exon 2 junction to avoid quantification of the PCR product from contaminating IL-8 genomic DNA. ß-actin mRNA (internal control) in the tumor sample was quantified in the same way using forward and reverse primers and a probe designed for ß-actin mRNA analysis, the forward primer sequence being 5'-CGC CCA GCA CGA TGA AA-3', the reverse primer sequence being 5'-CCG CCG ATC CAC ACA GA-3', and the probe sequence being 5'-AAG ATC ATT GCT CCT CCT GAG CGC AAG T-3'.43 The sequences of the primers and probes used to assess RNA integrity were as previously described.40

Standard Curve Sample Preparation
The standard curve samples used for RTQ RT-PCR were prepared by serial dilution of a specific RNA sample to cover the range of 5 to 500 ng. The serially diluted samples were aliquoted and stored at -80°C until use.

RT-PCR Procedure
The amplification mixture (50 µL) contained 50 ng of sample RNA, x 5 TaqMan EZ buffer (10 µL), 25 mmol/L manganese acetate (6 µL), 300 µmol/L dATP, dCTP, and dGTP, 600 µmol/L dUTP, 5 units of rTth DNA polymerase, 0.5 units of AmpErase uracil N-glycosylase (UNG), 200 nmol/L VEGF (or IL-8) forward and reverse primers, and 100 nmol/L VEGF (or IL-8) probe (all from Perkin-Elmer Applied Biosystem). The rTth DNA polymerase had both RTase and Taq polymerase activity. The thermal cycling parameters were an initial step of 2 minutes at 50°C, 30 minutes at 60°C for reverse transcription, 5 minutes at 95°C for deactivation, and then 40 cycles at 94°C for 20 seconds and 62°C for 1 minute for the melting and combined annealing and extension phases of the PCR reaction. Each assay included duplicate standard curve samples, a no-template control, and triplicate total RNA samples. All samples with a coefficient of variation (CV) higher than 10% were retested.

Detection of Fluorescence Emission and Quantification of VEGF mRNA and IL-8 mRNA
Fluorescence emission from the reporter dye (FAM-6-carbosy-fluorescein, peak fluorescence emission at 518 nm) was detected online in real time using an ABI prism 7,700 sequence detection system (Perkin-Elmer Applied Biosystem). The amount of VEGF mRNA or IL-8 mRNA in the tissue, standardized to the ß-actin mRNA, was expressed as follows: -{Delta}CT = -[CT VEGF(or IL-8) - CT ß-actin]. The ratio of the amount of VEGF or IL-8 mRNA/amount of ß-actin mRNA was then calculated as 2-{Delta}CT x K (K indicates constant).

The -{Delta}CT was analyzed as both a continuous and dichotomous variable. The median value was used as the cutoff to distinguish between low and high levels of VEGF or IL-8 mRNA expression.

Statistical Analysis
All statistical analyses were performed using SPSS for Windows software (version 8.0) (SPSS Inc, Chicago, IL). The independent two-tailed t test was used for comparison of VEGF mRNA and IL-8 mRNA expression and of MVCs in tumors with high and low aberrant p53 expression. The {chi}2 test44 was used to compare categorical tumor variables. The correlation between aberrant p53 protein expression and VEGF mRNA or IL-8 mRNA expression was analyzed by linear regression.44 Linear regression was also used to analyze the correlation between aberrant p53 protein expression and MVC. The survival curve was obtained using the Kaplan-Meier method45 and the log-rank test44 was used to test the difference in survival between patients with tumors with high and low aberrant p53 expression. P < .05 was considered statistically significant. When appropriate, the data are presented as the mean ± SD.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Clinical-Pathologic Variables
The clinical-pathologic variables of the 65 patients with NSCLC are listed in Table 1. The cancer specimens consisted of 35 squamous cell carcinomas and 30 adenocarcinomas. Surgical-pathologic tumor staging showed that 24 patients had stage I, 10 had stage II, 28 had stage IIIA or IIIB, and three had stage IV cancer. Tumor status was T1 in 13 cases, T2 in 30, T3 in 21, and T4 in one. Thirty-one patients had regional or distant lymph node metastasis (N1 in 14, N2 in 15, and N3 in two) and 34 had no lymph node metastasis (N0).


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  		Table 1.  Clinocopathologic Characteristics and Aberrant p53Expression in 65 Patients With NSCLC
 
Aberrant p53 Expression
Immunostaining for aberrant p53 protein was localized in the nuclei of cancer cells (Fig 1). The percentage of cancer cells positive for aberrant nuclear p53 accumulation ranged from 0% to 92%, with a mean ± SD of 39.7% ± 36.5% and a median value of 20%. Using 20% as the cutoff value, 36 and 29 tumors showed, respectively, high and low aberrant p53 protein expression. When compared with the clinical-pathologic variables, higher aberrant p53 protein expression was associated with mediastinal or distant lymph node metastasis (N0 to N1 v N2 to N3; P = .006, independent t test). Aberrant p53 expression was not associated with histologic type, sex, age, tumor status, or tumor staging in this series (Table 1).

Expression of VEGF mRNA or IL-8 mRNA
Real-time RT-PCR of TBP mRNA showed the integrity of the tumoral RNA to be good (Fig 2). VEGF mRNA or IL-8 mRNA expression in lung cancer tissue (standardized to ß-actin mRNA) was expressed as -{Delta} CT = -(CT VEGF (or IL-8) -CTß-actin). Figs 3A and 3C show, respectively, the determination of the CT value for VEGF or IL-8 mRNA expression in tumor samples, whereas Fig 3B and 3D show the corresponding standard curve. The CT value represents the fractional cycle number at which a significant increase in Rn above a chosen threshold (horizontal black line) is first detected (Figs 3A and 3C). The -{Delta} CT values for VEGF mRNA expression in the 65 lung cancer tissue samples ranged from 2.45 to 14.0, with a mean ± SD of 9.46 ± 2.27 and a median value of 9.75, whereas the corresponding values for IL-8 mRNA expression ranged from 5.4 to15.71, with a mean ± SD of 9.71 ± 2.25 and a median value of 9.83. Using the median value as the cutoff point, 32 tumors showed high VEGF mRNA expression and 34 showed high IL-8 mRNA expression.



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  		Fig 2. Integrity of the total RNA isolated from tumor sample examined by real-time RT-PCR of TBP mRNA (lanes two through 11). Lane 1, 100-bp marker. The size of the TBP mRNA RT-PCR product was 89 bp.

 


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  		Fig 3. Quantification of VEGF and IL-8 mRNA expression in tumor samples by RTQ RT-PCR. (A) and (C), Determination of the CT values for VEGF mRNA and IL-8 mRNA expression, respectively; (B) and (D), respective standard curves.

 
Immunohistochemical Staining for VEGF and IL-8 Proteins
VEGF and IL-8 proteins were expressed predominantly in the cytoplasm of the lung cancer cells of the tumor specimens (Figs 4A and 4B). Little staining was found in the stromal cells or tissue.



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  		Fig 4. Immunohistochemical staining for VEGF and IL-8. VEGF (A) and IL-8 (B) protein are mainly located in the cytoplasm of cancer cells, and little is expressed by stroma cells and tissue (x 400 magnification).

 
Correlation Between Aberrant p53 Expression and VEGF mRNA or IL-8 mRNA Expression
VEGF mRNA expression in tumors with high aberrant p53 expression was significantly higher than that in tumors with low aberrant p53 expression (-{Delta} CT = 10.61 ± 1.84 v 8.03 ± 1.93, P < .001, independent t test). Similarly, IL-8 mRNA expression in tumors with high aberrant p53 expression was significantly higher than that in tumors with low aberrant p53 expression (-{Delta} CT = 10.59 ± 2.23 v 8.61 ± 1.77, P < .001, independent t test) (Table 2). Tumors with high aberrant p53 expression showed a higher frequency of high VEGF mRNA expression (>= 9.75, 26 of 36) (P = .00004, {chi}2 test) and high IL-8 mRNA expression (>= 9.83, 28 of 36) (P < .00001, {chi}2 test) than tumors with low aberrant p53 expression. When the percentage of cells with aberrant p53 expression or VEGF mRNA or IL-8 mRNA expression was treated as a continuous variable, linear regression showed that the percentage of cells with aberrant p53 expression correlated strongly with VEGF mRNA expression (r = .68, P < .0001) (Fig 5A) and with IL-8 mRNA expression (r = .58, P < .0001) (Fig 5B).


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  		Table 2.  VEGF mRNA, IL-8 mRNA Expressions, and Microvessel Counts in Tumors With High or Low Aberrant p53 Expression
 


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  		Fig 5. Correlation of aberrant p53 expression with VEGF mRNA expression, IL-8 mRNA expression, or intratumoral microvessel counts in NSCLC. (A) VEGF mRNA (r = .68, P < .0001; linear regression); (B) IL-8 mRNA (r = .58, P < .0001); (C) MVC (r = .65, P < .0001).

 
Correlation Between Aberrant p53 Expression and MVC
The MVC in tumors with high aberrant p53 expression was 150.6 ± 57.7, significantly higher than that in tumors with low aberrant p53 expression (83.6 ± 37.9, P < .001, independent t test) (Table 2). A high MVC was seen in 22 of the 36 tumors with high aberrant p53 expression, but in only six of the 29 tumors with low aberrant p53 expression (P < .00107, {chi}2 test). Linear regression showed that there was a significant positive correlation between the percentage of cells with aberrant p53 expression in the tumor and an intratumoral MVC (r = .65, P < .0001) (Fig 5C).

Aberrant p53 Protein, VEGF mRNA, and IL-8 mRNA Expressions and Prognosis
As shown in Table 2, the median survival time for patients with tumors with high aberrant p53 expression (>= 20%) was 20.0 ± 1.50 months, significantly shorter than that for patients with tumors with low aberrant p53 expression (< 20%) (45.0 ± 5.13 months, P < .0001, log-rank test) (Fig 6A). The relapse time after operation was significantly earlier in patients with tumors with high aberrant p53 expression than in those with low aberrant p53 expression (7.00 ± 1.19 months v 36.0 ± 5.71 months, P < .0001). The difference of survival was still significant after stratification by disease stage (P = .0006 for stage I and II and P = .0017 for stage III and IV).



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  		Fig 6. Kaplan-Meier survival plots for different categories of NSCLC patients. Patients are grouped according to high or low expression of p53 (A), VEGF mRNA (B), or IL-8 mRNA (C). The difference in survival for each group was statistically significant (P < .0001, log-rank test).

 
Tumoral expression of VEGF and IL-8 mRNAs also correlated with the patients’ prognosis. The survival of patients with high tumoral VEGF mRNA expression (-{Delta} CT >= 9.75) was significantly shorter than that of patients with low tumoral VEGF mRNA expression (median survival ± SE, 16.0 ± 1.41 months v 40.0 ± 4.64 months, P < .0001, log-rank test) (Fig 6B), and this difference was still significant after stratification by disease stage (P = .0023 for stage I and II and P < .0001 for stage III and IV). The median survival time for patients with high tumoral IL-8 mRNA expression (-{Delta} CT >= 9.83) was 17.0 ± 1.46 months, significantly shorter than that for patients with low tumoral IL-8 mRNA expression (45.0 ± 5.39 months, P < .0001, log-rank test) (Fig 6C), and, again, this difference was still significant after stratification by disease stage (P < .0001 for stage I and II and P = .0018 for stage III and IV).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
There is considerable evidence that angiogenesis is required for tumor growth and metastasis.1-3 In the prevascular phase of tumor development, the tumor is limited in growth and there is little or no metastasis. After angiogenic phenotype switching, the tumor grows rapidly and starts to metastasize systemically.2 A high degree of angiogenesis in the tumor is reported to be associated with rapid tumor progression and poor prognosis in a variety of human solid cancers,3-9 including NSCLC.

The process of angiogenesis is stimulated or inhibited by a variety of factors. VEGF is one of the most potent endothelium-specific angiogenic factors, and its overexpression is reported to be associated with high intratumoral MVC, rapid tumor progression, and poor prognosis in several human cancers.16-19 Another recently recognized important angiogenic factor is IL-8, a cytokine of the C-X-C chemokine family, recently shown to induce endothelial cell proliferation,22 and the expression of which is associated with angiogenesis and metastatic potential in melanoma in nude mice,20 human colon carcinoma,21 head and neck squamous cell carcinoma,46 and NSCLC.23 Several studies have shown that certain human oncogenes, such as ras, or tumor suppressor genes, such as p53, might be involved in the regulation of expression of angiogenic factors or angiogenic inhibitors and thus influence the subsequent angiogenesis process in various cancers.47,48

In this study, we have demonstrated for the first time that high aberrant p53 expression correlated positively and strongly with high expression of both VEGF mRNA and IL-8 mRNA in NSCLC. Aberrant p53 expression was also associated with a high tumoral MVC, shorter patient survival, and early disease relapse. These results suggest that p53, in addition to its role as a tumor suppressor and apoptosis inducer, might also inhibit angiogenesis by negatively regulating the transcription of VEGF mRNA and IL-8 mRNA in NSCLC. A relationship between p53 mutation and VEGF expression has been reported by other investigators in lung and oral squamous cell carcinoma.33-35 The results of the present study confirmed the findings in lung cancer of Fontanini et al33 but not those of Ambs et al,35 who found that expression of mutated p53 is not associated with increased VEGF expression. Another study by Giafromanolaki et al48 showed that loss of wild-type p53 is associated with VEGF switch-on and that there was a trend for mutant p53 protein to be positively correlated with VEGF expression (P = .10 and P = .06 for the 10% and 20% cutoff points of p53 mutation). The discrepancy between these results might be explained by differences in the methods used to assess p53 mutation and VEGF expression in cancer tissues, the antibodies used, and the patient populations. In some studies, including the one by Ambs et al, VEGF expression was assessed by immunohistochemical staining (IHC), which is used as a qualitative or semiquantitative method of evaluating protein expression in tissues and is frequently influenced by tissue preparation, the source and potency of the monoclonal or polyclonal antibodies used, and experience of staining.13

In the present study, we used RTQ RT-PCR, presently the most accurate PCR method for the quantification of gene expression, to quantitate VEGF mRNA expression in NSCLC, and the results showed a linear correlation between the two continuous variables of the percentage of tumor cells showing aberrant p53 expression and the -{Delta} CT value for VEGF mRNA expression. Our results are also supported by the in vitro studies of Kieser et al29 and Mukhopadhyay et al,30 who showed that transfection with a mutant p53 gene can induce VEGF expression and that transfection with the wild-type p53 gene can downregulate VEGF mRNA levels in cultured cell lines.

Angiogenesis phenotype switching in tumors has been considered to result from the accumulation of sequential mutation in multiple genes. It is important to investigate whether the activation of an oncogene or the ablation of a tumor suppressor gene can affect the expression of angiogenesis. This study and several recent studies28-33 have shown that in NSCLC, certain human oncogenes, such as ras, or tumor suppressor genes, such as p53, might be involved in the regulation of expression of angiogenic factors or angiogenic inhibitors and thus influence the subsequent angiogenesis process.

The mechanism involved in the regulation of VEGF expression by p53 is still unclear. p53 is a transcription factor, and overexpression of wild-type p53 can inhibit the transcription of many cellular and viral promoters.25,49 Wild-type p53 specifically represses the activity of TATA promoters that do not contain a wild-type p53 DNA binding sequence.50 In the case of the VEGF promotor, which does not contain a TATA box or a known wild-type p53 DNA binding sequence, it has been suggested that repression of VEGF mRNA expression by wild-type p53 occurs through an indirect pathway (TSP-1) or via an undefined new p53 binding site in the VEGF promoter and that, after p53 mutation, repression is decreased, resulting in upregulation of VEGF mRNA expression.30

Regulation of IL-8 expression by p53 protein in tumor-associated angiogenesis has not previously been reported. Michel et al51 found that the antipsoriatic agent Tacrolimus (FK506) causes decreased expression of IL-8 mRNA in cultured primary keratinocytes, with concomitant increased expression of wild-type p53 mRNA and protein. Mirmohammadsadegh et al52 showed that in epidermal cells in vitro, another antipsoriatic agent, N-(trifluoromethylphenyl)-2-cyano-3-hydroxy-crotonic acid amide, caused a dose-dependent reduction in IL-8 receptor A mRNA and induced the expression of anti-inflammatory cytokine IL-10 receptor mRNA, with concomitant upregulation of wild-type p53. Our study shows for the first time that in NSCLC, aberrant p53 expression correlates strongly with both IL-8 mRNA expression and the intratumoral MVC. This implies that altered p53 might also play an important role in the regulation of IL-8 mRNA expression in tumor angiogenesis in human cancers. The actual pathway by which p53 regulates IL-8 activity is unknown, and additional investigations are needed to elucidate this point.

Several studies have shown that aberrant p53 expression indicates a poor prognosis in human cancers.27 In addition to being an inducer of apoptosis,53 a check-point in the cell cycle,54 and a negative transcriptional regulator for oncoproteins,49,50 wild-type p53 might also inhibit the expression of angiogenic factors and affect the angiogenesis process in human cancer.30 p53 mutation and the resulting upregulation of tumor angiogenesis might explain in part the adverse prognosis of these cancers. In this study in NSCLC, high aberrant p53 expression was associated with high intratumoral MVC, early disease relapse, and short survival. The correlation between the high aberrant p53 expression and high VEGF or IL-8 mRNA expression in tumors and the association between shorter patient survival and high tumoral VEGF or IL-8 mRNA expression also provide indirect evidence that upregulation of angiogenic factors and angiogenesis might be one mechanism accounting for the association of aberrant p53 expression with adverse prognosis in NSCLC.

In this study, we used the monoclonal antibody Do-7 to assess aberrant p53 expression in NSCLC. This monoclonal antibody recognizes both mutant and wild-type p53 proteins, but because of the short half-life of wild-type p53 protein, the immunoreactivity seen in the nuclei of cancer cells was considered to show aberrant p53 expression.55 However, the accumulation of aberrant p53 protein in the nucleus assessed by IHC does not equate with p53 gene mutation assessed by molecular techniques, such as single-strand conformation polymorphism and DNA sequencing, the concordance between the two approaches being approximately 60% to 70% in NSCLC.56 p53 gene mutation occurs in approximately 40% to 50% of NSCLCs, and most of the p53 gene mutations in cancer cells are missense mutations.56 IHC cannot detect cells with truncated, splicing, or null mutation of the p53 gene, because no p53 protein will be expressed in the cell.56 Conversely, aberrant p53 protein expression (assessed by IHC) has been described in resected tumor specimens in the absence of p53 gene mutation, as assessed by single-strand conformation polymorphism and DNA sequencing.56 This discrepancy might result from the lack of detection of p53 gene mutation as a result of only analyzing exons 5 to 8 of the p53 gene57; the overexpression of p53 protein in cells with ongoing DNA damage58; the binding of wild-type p53 to a variety of cellular proteins, resulting in its conformational change, stabilization, and detection (by IHC staining) and also abnormal function59; or mutation analysis (direct sequencing) only being able to identify p53 gene mutation when wild-type alleles (from stromal and inflammatory cells) do not exceed two thirds of the material analyzed60 or when the cells carrying the mutated gene represent at least 5% to 10% of the neoplastic cell population.56 Recently, in addition to the loss of wild-type p53 function, mutant p53 has been considered an oncoprotein with a gain of oncogenesis function. The oncogenic effects of p53 mutants can result from either trans-dominant suppression of wild-type p53 or a wild-type p53-independent oncogenic gain of function.61 p53 Mutants increase mutation frequency,62 block differentiation,63 increase metastatic potential,64 and can transactivate or repress specific genes (eg, MDR-1 gene and c-myc).65,66 In this case, IHC can be used as a semiquantitative method to assess mutant p53 (oncoprotein) expression in cancer cells, whereas DNA sequencing is usually considered a qualitative analysis of gene mutation. Positive p53 immunostaining has recently been suggested to provide a more complete assessment of p53 abnormality than sequence analysis, because it reflects both the presence of most mutant p53 protein products and the accumulation of wild-type p53 protein product, which may relate to other deregulated cellular functions.67,68 Furthermore, accumulation of p53 protein, in the absence of p53 gene mutation, may actually represent a gain of function.56 Most previous investigations using IHC to assess aberrant p53 expression in NSCLC have shown a statistically significant negative correlation between aberrant p53 protein expression and patients’ survival.27,67,69-74 In the present study, we showed that high aberrant p53 protein expression, assessed by IHC, was not only associated with poor prognosis but also correlated with high expression of VEGF mRNA and IL-8 mRNA and a high MVC in NSCLC. However, our results contrast with those of a recent study by Lee et al,75 who reported a favorable prognostic influence of aberrant p53 expression in primary NSCLCs; these differences may result from differences in the immunohistochemical staining procedures, antibodies used, and patient population. In the study by Lee et al, the cutoff value for positive p53 staining was higher (50%) compared with previous studies (10% to 20%),71 and the prognostic significance was found mainly in a subset (mediastinal lymph node–positive group) of early-stage NSCLCs.

Desbaillets et al22 have shown that exposure of a human glioblastoma cell line to anoxic stress causes upregulation of the expression of both IL-8 and VEGF mRNAs but with different time courses. Using IHC, Ferrer et al76 showed coexpression of IL-8 and VEGF protein in prostate cancer cells, whereas little staining for either angiogenic factor was seen in benign prostatic hyperplasia and normal prostate cells; in addition, they showed that IL-8 and VEGF protein can be induced, respectively, by IL-1 and TNF in a prostate cancer cell line grown in culture. Using IHC and ELISA, Eisma et al46 also demonstrated coexpression of IL-8 and VEGF protein in 35 head and neck squamous cell carcinoma samples; as in our own unpublished results on VEGF mRNA expression in NSCLC, there was a good correlation between the expression of IL-8 mRNA and VEGF mRNA (r = .63, P < .0001). These results imply that, although it is likely that IL-8 and VEGF mRNA are upregulated by different pathways, these two angiogenic factors could be simultaneously upregulated in cancer cells by a common upstream inducer or by cross-talk of these two signal transduction pathways. p53 Might be one such factor regulating the coexpression of VEGF and IL-8 mRNA in NSCLC.

We conclude that in NSCLC, there is a strong association between high aberrant p53 expression and increased expression of both VEGF mRNA and IL-8 mRNA. Aberrant p53 expression might play a role in the regulation of VEGF mRNA and IL-8 mRNA expression and therefore be involved in controlling the angiogenesis process.


   NOTES
 
Supported in part by grant no. NSC-89-2314-B-002-425 from the National Science Council of the Republic of China.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
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Submitted May 14, 2001; accepted October 23, 2001.




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