Expression of the Angiogenesis Markers Vascular Endothelial Growth FactorA, Thrombospondin-1, and Platelet-Derived Endothelial Cell Growth Factor

in Human Sporadic Adrenocortical Tumors: Correlation with Genotypic Alterations

Abstract
Several studies have supported the hypothesis that adrenocortical tumor formation is the result of a multistep process. The angiogenic switch has been proposed to be a key step in tumor progression from adenoma to carcinoma. In this study we measured the cytosolic concentrations of three proteins involved in angiogenesis [namely platelet-derived endothelial cell growth factor vascular endothelial cell growth factor A (VEGF-A), and thrombospondin-1 (TSP1)] in a series of 43 human sporadic adrenocortical tumors. The tumors were classified as adenomas (n = 18), transitional tumors (n = 12), or carcinomas (n = 13) according to the histological criteria defined by Weiss. Platelet-derived endothelial cell growth factor/thymidine phosphorylase levels were not significantly different among these three groups. One hundred percent of the adenomas and 73% of the transitional tumors showed VEGF-A concentrations under the threshold value of 107 ng/g protein, whereas 75% of the carcinomas had VEGF-A concentrations above this threshold value. Similarly, 89% of the adenomas showed TSP1 concentrations above the threshold value of 57 g/g protein, whereas only 25% of the carcinomas and 33% of the transitional tumor samples did so. Insulin-like growth factor II overexpression, a common genetic alteration of adrenocortical carcinomas, was significantly correlated with higher VEGF-A and lower TSP1 concentrations. The tumors from the 6 patients with tumor recurrence after surgical ablation showed significantly higher VEGF-A values than the carcinomas and the transitional tumors from patients that did not relapse. Taken together, these data suggest that a decrease in TSP1 expression is an event that precedes an increase in VEGF-A expression during adrenocortical tumor progression. The population of premalignant tumors with low TSP1 and normal VEGF-A levels could represent a selective target for antiangiogenic therapies. THE ANALYSIS OF large autopsy series and the recent advances in sensitive imaging techniques have shown that more than 3% of people over 50 yr of age have adrenal nodules; however, only a small proportion of these tumors secrete steroids, and less than 1% are malignant (1, 2). Steroid-secreting adrenocortical carcinomas thus represent rare tumors with very poor prognosis. Despite some recent progress in the understanding of the pathogenesis of adrenocortical tumors, reliable markers to predict their natural evolution are still missing. Moreover, in many cases the benign or malignant nature of a localized tumor cannot be established with certainty, reflecting the lack of absolute clinical, biological, or histological criteria. Interestingly, the recent characterization of specific genetic rearrangements [uniparental disomy at the 11p15 locus, loss of heterozygosity at the 17p13 locus, and major overexpression of the insulin-like growth factor II (IGF-II) gene], which are highly frequent in carcinomas (>80%), but are observed at a low frequency in adenomas (<12.5%), offers new potential prognostic criteria that remain to be evaluated in a prospective study with long-term follow-up of patients (2, 3, 4, 5).

Angiogenesis, the biological process by which new blood capillaries are formed from preexisting microvessels and venules, is an essential step in the progression of a variety of solid tumors (6). It has been proposed from the results of experimental animal tumor models and from the observation of human breast and cervical carcinomas that induction of angiogenesis (often termed the angiogenic switch) is a discrete component of tumor evolution activated during the early premalignant stages of tumor development (7). Angiogenesis appears to be a limiting step for subsequent tumor growth beyond the size of a few cubic millimeters (7) and for metastasis formation (8). Under physiological conditions, the vasculature is quiescent in the adult, except for some hormonally controlled processes, such as ovulation, menstruation, implantation, and pregnancy. The biochemical characterization of a number of peptides and proteins that stimulate or inhibit angiogenesis has led to the concept of angiogenic balance (9, 10). The angiogenic status of a given cell type under any physiological or pathological situation results from the balance between the biological activities of both angiogenic and angiostatic factors. This concept is supported by the observation that tumor angiogenesis may be induced by either increased expression of angiogenic factors (10, 11) or decreased expression of angiostatic factors (10, 12, 13, 14). The angiogenic status of adrenocortical tumors has not been studied in detail to date. The normal adrenal gland is a highly vascularized tissue irrigated centripetally by a network of fenestrated capillaries (called sinusoids) originating from capsular arterioles (15, 16). Early, but still valid, histological observations of the adrenal cortex have established that each endocrine cell from the glomerulosa, the fasciculata, or the reticularis zone is in direct contact with a capillary sinusoid (17). A recent comparison of the vascular patterns of adrenocortical tumors with that of the normal adrenal cortex showed that the sinusoidal network in adenomas was indistinguishable from that in the normal gland, whereas carcinomas showed a disorganized vasculature, with large vessels interspersed with irregular networks of microcapillaries (18). In the present study we measured the abundance of three angiogenesis markers [namely vascular endothelial cell growth factor A (VEGF-A), platelet-derived endothelial cell growth factor (PDECGF), and thrombospondin-1 (TSP1)], in a series of 43 benign and malignant adrenocortical tumors. These markers were selected on the basis of their reported implication in cancer angiogenesis. VEGF-A is a potent and selective growth factor for micro- and macrovascular endothelial cells (19) and an inducer of vascular permeability (20). The dramatic alterations of yolk sac and embryonic vascularization observed in mouse embryos heterozygous for a disrupted VEGF-A gene (21, 22) have definitely established VEGF-A as a pivotal molecule in angiogenesis. Overexpression of VEGF has been reported in a number of tumor types and appears of significant prognostic value in breast, stomach, and colon cancers (23, 24). Although it was initially purified from platelet extracts as an inducer of thymidine incorporation into endothelial cells (25), PD-ECGF does not stimulate the proliferation of endothelial cells (26). However, it is angiogenic and promotes tumor growth in vivo (25). Its sequencing revealed complete identity with the enzyme thymidine phosphorylase that converts thymidine to thymine (27), an enzyme overexpressed in various neoplastic tissues (28, 29, 30, 31, 32). TSP1 is a multimodular secreted protein that associates with the extracellular matrix and possesses a variety of biological functions, including a potent antiangiogenic activity (33). TSP1 expression has been shown to be down-regulated in a number of experimental tumor models, and this regulation has been correlated with the decreased expression of tumor suppressor genes (34, 35).

On the other hand, TSP1 has been reported to be overexpressed in a number of malignant tissues and to be present in higher than normal levels in the plasma of cancer patients (36, 37). This discrepancy may be related to different contributions of the stroma to the tumor mass from one type of tumor to the other; a detailed immunohistochemical study of the localization of TSP1 in human breast neoplasms has shown that TSP1 is overexpressed in the stroma, but is absent from the tumor epithelial cells (38). The data reported here indicate that the mean cytosolic concentration of VEGF-A in the population of malignant tumors is higher than that in benign or transitional tumors. Conversely, the mean cytosolic TSP1 concentration is lower in malignant and transitional tumors than in adenomas. Thymidine phosphorylase activity does not vary significantly among the three populations. The pertinence of these markers as prognostic factors for localized or metastatic tumor recurrence has been evaluated.
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Subjects and Methods

Patients

Forty-three adult patients (10 men and 33 women), 2379 yr old, with sporadic adrenocortical tumors surgically removed between 1990 and 1998 were included in this study. The median postsurgery follow-up of these patients was 44 months (range, 5.2104 months). The hormonal status and the stage of the tumors as localized, regional, or metastatic were evaluated as previously described (39). Seventy-two percent of the patients (n = 31) were referred because of endocrine symptoms, and 86% (n = 37) exhibited an abnormal hormonal profile. Histological features, including high mitotic rate, atypical mitoses, high nuclear grade, low percentage of clear cells, necrosis, diffuse architecture of tumor, capsular invasion, sinusoidal invasion, and venous invasion, were carefully analyzed according to the method of Weiss (40, 41). Eighteen tumors without any of these histological features were classified as benign, 5 of them presenting with bilateral adenomas (macronodular hyperplasia). Twelve localized tumors with 13 of these histological features were classified as transitional. Thirteen tumors with more than 3 of these features or with documentation of metastasis were classified as malignant tumors (41). Clinical, pathological, and hormonal data are summarized in Table 1
Preparation of tumor cytosols

Liquid nitrogen-frozen tumors were homogenized at 4 C in 10 mmol/L Tris-HCl buffer, pH 7.4, containing 1 mmol/L ethylenediamine tetraacetate, 1.5 mmol/L MgCl2, 0.5 mmol/L dithiothreitol, 1 mmol/L phenylmethylsulfonylfluoride, 10 g/ml leupeptin, and 10% glycerol. The homogenate was centrifuged at 800 g for 10 min to pellet the nuclei, and the supernatant was then centrifuged at 100,000 g for 1 h. The protein content of the cytosols (100,000 g supernatants) was determined using the bicinchoninic acid method (Pierce Chemical Co., Rockford, IL).

Assessment of thymidine phosphorylase activity

Thymidine phosphorylase activity in tumor cytosols was determined by spectrophotometric methods as described previously (42). Briefly, 50100 L of cytosolic fractions were incubated for 3 h at 37 C with 10 mmol/L thymidine in a final volume of 1 mL 50 mmol/L potassium phosphate buffer, pH 7.4. At the beginning and the end of the reaction, 0.5-mL aliquots of each sample were mixed with 1 mL 0.5 mol/L NaOH. The amount of thymine formed was determined spectrophotometrically at 300 nm. One unit of activity is defined as the formation of 1 mol thymine/h. Parallel analysis of seven samples of human breast tumors by Western blotting using a monoclonal antibody to PD-ECGF (a gift from Dr. Roy Bicknell) showed a statistically valid correlation between the values of enzymatic activity and the intensities of immunoreactive signals (regression coefficient = 0.98).
Determination of TSP1 and VEGF-A protein expression

TSP1 concentrations in the tumor cytosols were determined by a sandwich enzyme-linked immunosorbent assay (ELISA) developed in the laboratory. The immobilized antibody was a commercial rabbit polyclonal antibody raised against human TSP1 (Calbiochem, La Jolla, CA). The mouse monoclonal antihuman TSP1 antibody used in the second step was from Roche (Meylan, France). The detection limit of the assay was 5 ng/ml. VEGF-A was quantitated using the Quantikine kit from R and D Systems, Inc. (Oxon, UK). This commercial ELISA does not cross-react with placenta growth factor. Moreover, it detects the different isoforms of VEGF-A, among which VEGF121 and VEGF165 are the most abundantly expressed.
Analysis at the chromosomal 11p15 locus and evaluation of IGF-II messenger RNA (mRNA) content

Allelic loss at the 11p15 locus in tumors was investigated by Southern blot analysis as previously described (3). Tumor IGF-II mRNA contents were evaluated by dot-blot analysis as previously described (3, 4) and compared with normal adrenal content. When the tumor IGF-II mRNA content was more than 10-fold larger than that in normal adrenals, the tumors were classified as overexpressing IGF-II.
Statistical analysis

Differences in angiogenic or angiostatic factor concentrations among the groups were analyzed by the Mann-Whitney U test.
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Results
Enzymatic assay of thymidine phosphorylase

An enzymatic assay was used to measure the thymidine phosphorylase activity of the angiogenic factor PD-ECGF in tumor cytosols. Thymidine phosphorylase activity of the adenomas (mean, 4.4 U/g; range, 115 U/g) was not significantly different from that of the carcinomas (mean, 5.1

U/g; range, 38 U/g) or that of the transitional tumors (mean, 4.4 U/g; range, 38 U/g; Table 2 and Fig. 1A).
Analysis of VEGF-A and TSP1 by ELISA

Concentrations of TSP1 were significantly higher in the cytosols of adenomas (mean, 142 g/g; range, 40390 g/g) than in carcinomas (mean, 69 g/g; range, 8344 g/g) or transitional tumors (mean, 53 g/g; range, 0161 g/g; Table 2 and Fig. 1B). The mean cytosolic TSP1 concentration determined from two normal human adrenals was 52 12 g/g. Using a threshold value of 57 g/g (corresponding to the 75th percentile of the carcinoma values), 89% of the adenoma samples presented TSP1 concentrations above threshold, whereas only 25% of the carcinoma and 33% of the transitional tumor samples did so. Conversely, levels of VEGF-A were significantly lower in the cytosols of adenomas (mean, 44.3 ng/g; range, 981 ng/g) and transitional tumors (mean, 83.2 ng/g; range, 3141 ng/g) than in those of carcinomas (mean, 404 ng/g; range, 301370 ng/g; Table 2 and Fig. 1C). The mean cytosolic VEGF-A concentration determined from two normal human adrenals was 111 23 ng/g. Using a threshold value of 107 ng/g (corresponding to the 25th percentile of the carcinoma values), 100% of the adenomas and 72% of the transitional tumors showed VEGF-A concentrations under threshold, whereas 75% of the carcinomas had VEGF-A concentrations above threshold. However, no significant correlation was observed between tumor weight and VEGF-A concentrations.
Correlations between genotypic and biochemical features of adrenocortical tumors

The population of tumors was split into two subgroups according to either one of the following two genotypic features: overexpression of the IGF-II gene (Fig. 2, A and B) or allelic loss at the 11p15 locus (Fig. 2, C and D), and the distribution of cytosolic VEGF-A and TSP1 concentrations in each of these subpopulations was determined. It clearly appeared that tumors with IGF-II overexpression had significantly higher VEGF-A (P < 0.01) and lower TSP1 (P < 0.001) concentrations than tumors without IGF-II overexpression. A similar, although less significant (P < 0.05), cosegregation was observed between 11p15 allelic loss and either high VEGF-A or low TSP1 values.
Correlation between VEGF-A expression and susceptibility to tumor recurrence

Of the 19 patients with transitional and malignant tumors who were regularly followed-up, 6 showed tumor recurrence within a median time period of 14.3 months (range, 3.232.1 months), whereas the other 12 did not recur over a median follow-up period of 58.9 months (range, 40.5 97 months). As shown in Fig. 3A, tumors from the patients (with a carcinoma or a transitional tumor) that showed recurrent tumor growth contained higher VEGF-A concentrations than those that did not, although the low number of recurrence events makes this difference barely significant. In contrast, TSP1 did not appear to be a useful marker to discriminate between recurring and nonrecurring tumors Discussion To date, it has not been clearly established whether adrenocortical adenomas and carcinomas represent different stages of a common multistep process or if they derive from distinct pathogenetic events (2). X-Chromosome inactivation analyses have shown that adrenocortical carcinomas have a monoclonal origin, whereas adenomas may be monoclonal or polyclonal (43). As adrenocortical adenomas occur frequently, whereas carcinomas are rare, it is likely that the

switch to malignancy results from more than one key alteration. Angiogenesis has been proposed to represent a switch between the benign and malignant states of numerous tumor types (7). This prompted us to investigate whether molecular actors of this biological process (namely the angiogenic factors VEGF-A and PD-ECGF and the angiostatic protein TSP1) may represent possible markers of the transition of adrenocortical adenomas toward malignancy. Until now, histochemical characterization remains the best method for tumor grading, and relevant biological markers are still unavailable. In this study the 43 sporadic adrenocortical tumors excised from human adults (age, 2379 yr) were classified into adenomas, carcinomas, and a third group of poorly defined tumors (with 13 abnormal Weiss criteria) that we called transitional tumors. PD-ECGF is a misnamed angiogenic factor that stimulates angiogenesis in vivo although it is not mitogenic for endothelial cells in vitro (26). PD-ECGF is thymidine phosphorylase, and its enzymatic activity was not significantly different among the three groups of adrenocortical tumors examined: adenomas, transitional tumors, and carcinomas. In contrast to other tumor types (30, 32), it did not appear to be a relevant marker of adrenocortical tumors and was not further studied. Comparison of TSP1 and VEGF-A concentrations in adenomas and carcinomas revealed statistically significant differences. Mean VEGF-A and TSP1 concentrations in carcinomas were, respectively, increased and decreased compared with those in adenomas, suggesting that overexpression of this angiogenic factor and decreased expression of this antiangiogenic protein are associated with the malignant phenotype. No major difference in the concentrations of these factors was observed between localized and more invasive carcinomas (either regional or metastatic at diagnosis), suggesting that these alterations in the angiogenic status of the tumors precede the invasive step. More interestingly, the analysis of transitional tumors (i.e. tumors presenting histological alterations intermediate between those of benign and malignant tumors) revealed that the mean TSP1 concentration was significantly lower than that in adenomas (and similar to that in carcinomas), whereas VEGF-A concentrations were not statistically different from those in adenomas (and were lower than those in carcinomas). Although these variations were not observed in 100% of the tumors of each population, 78% (7 of 9) of the carcinomas with low TSP1 concentrations also had elevated VEGF concentrations, whereas this correlation was observed in only 14% (1 of 7) of the transitional tumors. This suggested that the decrease in TSP1 expression may be an earlier event than the increase in VEGF-A expression during tumor progression from adenomas to carcinomas. Several genetic studies addressing tumor clonality or search for chromosomal aberrations have supported the hypothesis that the formation of adrenocortical carcinomas is the result of a multistep tumorigenic process (43, 44). To our knowledge, the early decrease in tumor TSP1 concentration observed in this study is the first biochemical alteration to be characterized that would allow discrimination between premalignant and malignant stages of adrenocortical tumors. Decreased TSP1 expression has been observed in several tumor cell lines, such as human glioblastoma, breast, and lung carcinoma cell lines (12, 34, 45), as well as in p53-deficient fibroblasts from Li-Fraumeni patients (46) and in the cytosols of bladder cancers (35). In several of these examples, a correlation has been established between the loss of wild-type p53 expression and decreased TSP1 expression (47). p53 mutations have

been identified in approximately 30% of sporadic adrenal carcinomas, but have not been detected in adenomas from Caucasian patients (48). A Taiwanese group identified mutations within exon 4 of the p53 gene (an unusual hotspot, as 90% of p53 mutations in human tumors are located within exons 58) in 60% of benign adrenocortical adenomas (49), but this was not confirmed in a larger series of 27 tumors from Europe and the U.S. (50). It would be informative to determine whether the decreased expression of TSP1 observed in 60% of the carcinomas and 50% of the transitional tumors in our study correlates with p53 mutations. Abnormalities of the IGF-II locus at chromosome 11p15 resulting in overexpression of this maternally imprinted gene are frequently observed in transitional and localized malignant adrenocortical tumors but rarely in adenomas (4). It was therefore not surprising to observe that the tumors with these alterations also had higher VEGF-A and lower TSP1 contents than tumors with normal IGF-II levels. Among the 38 patients with localized or regional tumor who were included in this study, 6 showed tumor recurrence after primary tumor resection. Although these 6 tumors had significantly higher VEGF-A concentrations (P < 0.05) than the nonrecurrent tumors, the size of this cohort needs to be increased to definitely establish the prognostic value of VEGF-A concentrations with a higher statistical confidence. Such a correlation between high VEGF-A concentrations and tumor recurrence has been established in a number of other tumor types, including primary breast cancer (51) and gastric carcinoma (52). The alterations in TSP1 and VEGF-A expression observed in adrenocortical tumors suggest that a change in the angiogenic phenotype accompanies tumor progression from adenomas to carcinomas. This is in agreement with the histological observations showing a normal vasculature in the adenomas and a more disorganized and irregular microvasculature in the carcinomas (18). This also supports the possibility of using inhibitors of angiogenesis such as TSP1 or angiostatic fragments of this large multimodular protein to block neovascularization and, therefore, tumor growth. Overexpression of TSP1 in a number of tumor cell lines, including breast and skin carcinomas and glioblastomas, has been shown to revert their transformed phenotype and their ability to form tumors in nude mice (14, 34, 53, 54). It will be worth investigating whether TSP1-based antiangiogenic therapy may represent a more efficient alternative or a complement to the classically used antimitotic treatments such as mitotane (55). In particular, our study allows definition of a subset of tumors showing low TSP1 and normal VEGF-A concentrations as a transitional group that may be particularly responsive to antiangiogenic therapy. References
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55.Gicquel C, Baudin E, Lebouc Y, Schlumberger M. 1997 Adrenocortical carcinoma. Ann Oncol. 8:423427. FREE Full Text Web: http://jcem.endojournals.org/content/85/12/4734.full Angiogenesis, proses biologis di mana kapiler darah baru terbentuk dari microvessels and venules yang sudah ada sebelumnya, yang merupakan langkah penting dalam perkembangan berbagai tumor solid. Itu telah diusulkan dari hasil percobaan model tumor hewan dan dari observasi kanker serviks dan payudara pada manusia yg menginduksi angiogenesis (sering disebut angiogenik) komponen diskrit evolusi tumor diaktifkan selama tahap premaligna awal perkembangan tumor. Angiogenesis sepertinya menjadi langkah yang membatasi untuk pertumbuhan tumor berikutnya yg melampaui ukuran beberapa milimeter kubik dan untuk pembentukan metastasis. Dalam kondisi fisiologis, pembuluh darah tersebut tdk bergerak pada orang dewasa, kecuali untuk beberapa proses hormonal yg terkontrol, seperti ovulasi, menstruasi, implantasi, dan kehamilan. Karakterisasi biokimia dari sejumlah peptida dan protein yang merangsang atau menghambat angiogenesis telah menyebabkan konsep keseimbangan angiogenik. Dalam penelitian ini kami mengukur kelimpahan tiga angiogenesisi marker [yaitu faktor pertumbuhan sel endotel vaskular A (VEGF-A), platelet-derived endothelial cell growth factor (PD-ECGF), dan thrombospondin-1 (TSP1)]. Penanda ini dipilih berdasarkan implikasi dalam angiogenesis kanker. VEGFA adalah faktor pertumbuhan potensial dan selektif untuk mikro dan makrovaskular sel endotel dan penginduksi permeabilitas vaskular.