Androgen receptor expression in meningiomas

J Neurosurg 82:453–460, 1995

Androgen receptor expression in meningiomas RONA S. CARROLL, PH.D., JIANPING ZHANG, M.S., KATHLEEN DASHNER, B.A., MADHABANANDA SAR, PH.D., ELIZABETH M. WILSON, PH.D., AND PETER MCL. BLACK, M.D., PH.D. The Brain Tumor Center, Brigham and Women’s Hospital, Children’s Hospital, and Dana Farber Cancer Institute; Department of Surgery, Harvard Medical School, Boston, Massachusetts; and Department of Cell Biology and Anatomy and Department of Pediatrics and Biochemistry, University of North Carolina School of Medicine, Chapel Hill, North Carolina U The predominance of meningiomas in females, the accelerated growth of these tumors during the luteal phase of the menstrual cycle and during pregnancy, and the association between meningiomas and breast cancer have led to a number of studies examining the potential role of steroids in the growth of meningiomas. The possibility that androgens play a role in meningioma proliferation has been suggested by a small number of investigators. The aim of this study was to examine the expression of androgen receptor messenger ribonucleic acid (mRNA) and correlate it using immunochemistry with the nuclear localization of androgen receptor in a large number of meningiomas. Thirty-nine meningiomas were examined by Northern blot analysis for the presence of measurable amounts of androgen receptor mRNA and eight of these were analyzed by immunohistochemistry for receptor protein. Sixty-seven percent of the meningiomas expressed androgen receptor mRNA. There was a marked predominance of women among the patients whose tumors expressed androgen receptor; 69% were women and 31% were men. The immunohistochemical data correlated with Northern blot analysis of mRNA. The staining was predominantly nuclear, suggesting that the androgen receptor resides in a location that can activate gene expression.

KEY WORDS • meningioma • androgen receptor • neoplasm • Northern blot analysis • immunocytochemistry

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incidence of meningioma is twice as high in females as in males.8,52 In women the growth of meningiomas appears to be accelerated during the luteal phase of the menstrual cycle and during pregnancy.3,51 There is also an association between meningioma and breast cancer: three separate studies have demonstrated a positive correlation between these two entities.18,44,55 These observations suggested that meningiomas are a target for gonadal steroids. Numerous studies have been performed that examined the role of steroid receptors in meningioma proliferation.2,4,22–27,32,34,36,38,40,49,56,62 The potential roles of estrogens, progesterone, and androgens on the growth of meningiomas have been investigated both by determining whether their receptors are present in meningiomas and by assessing the effect of incubation of tumor cell cultures with agonists and/or antagonists of these agents. Although controversy remains concerning the presence of estrogen receptors in meningiomas,5,9,17,35,39,45,65 most hormone binding studies have demonstrated the presence of progesterone and androgen receptors. We recently showed that meningiomas express messenger ribonucleic acid HE

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(mRNA) for the progesterone receptor and its expression correlates with nuclear localization of progesterone receptor by immunohistochemistry.10 A major question that remains is whether these receptors are functional. Schwartz, et al.,57 suggested that the hormone binding data for putative progesterone and estrogen receptors in meningiomas may not meet the specific criteria for true steroid receptor proteins. The role of the androgen receptor in meningioma proliferation has been the focus of a small number of papers.6,32,33,45,48,50 In most of these studies the concentration of androgen receptor is equal to or exceeds that of progesterone receptor;6,50 by Scatchard analysis, 65% to 100% of meningiomas have the capacity to bind androgens with high affinity. Two separate studies33,45 have suggested that there is a good correlation between concentrations of cytosolic progesterone receptor and androgen receptor, suggesting that androgen receptors may modulate progesterone receptor activity.45 Studies that investigated the effects of androgens on meningioma proliferation have produced mixed results. Gibelli, et al.,20 showed that the addition of low concen453

R. S. Carroll, et al. of craniotomy for meningioma resection and was immediately snap frozen in liquid nitrogen and subsequently stored in liquid nitrogen. Normal arachnoid tissue was also obtained from a 43-year-old woman undergoing surgery for a meningioma. Each sample was taken from a specimen that was also used by the neuropathologist for diagnosis. These tumors were reviewed by a neuropathologist from Brigham and Women’s Hospital and were classified as syncytial, transitional, or fibroblastic, using standard criteria.8 The MCF-7 breast carcinoma cells* maintained in Dulbecco’s modified Eagle’s medium/10% fetal bovine serum were used as a positive control on some Northern blots (data not shown). Isolation of RNA and Northern Blot Analysis

trations of testosterone acetate inhibited cell growth in one of two cell cultures examined. Dihydrotestosterone addition has been shown to have a small stimulatory effect in two separate studies.48,66 Further investigations into gonadal steroid receptors should increase our understanding of the biology of meningiomas. In addition, the study of androgen and progesterone receptors may add valuable information concerning hormone therapy for these tumors. The aim of this study was to examine the expression of androgen receptor mRNA and using immunochemistry correlate it with the nuclear localization of androgen receptor in a large number of meningiomas. Materials and Methods Tissue Samples and Cell Culture Meningioma samples were obtained from 21 women and 18 men ranging in age from 23 to 84 years (Table 1). For Northern blot analysis, tissue was collected at the time 454

Total RNA was isolated by the method of Chirgwin, et al.13 Tissue samples were placed in 4 M guanidinium isothiocyanate and homogenized with the use of a Polytron until they were totally disrupted. For MCF-7 breast carcinoma cells, medium was aspirated and cells were washed twice in an ice-cold solution of phosphate buffered saline (PBS) (0.06 M NaH2PO4, 0.15 M NaCl/PBS; pH 7.3) and 3.3 ml guanidinium isothiocyanate was added to each flask. The cells were scraped into the guanidinium isothiocyanate and deoxyribonucleic acid (DNA) was sheared by passage through a No. 21 needle. After centrifugation for 10 minutes at 20˚C and 3000 rpm, the supernatant was layered over 5.7 M cesium chloride and ultracentrifuged at 38,000 rpm and 22˚C for 16 hours.† The RNA pellet was dissolved in 0.3 M sterile sodium acetate, and the RNA was ethanol precipitated. Twenty micrograms (A260) of total RNA from each sample was subjected to electrophoresis and diffusion blotted onto a nylon membrane.61 The RNA was cross-linked to the nylon membrane using ultraviolet light.‡ Blots were prehybridized for 2 hours at 42˚C in a mixture of 50% formamide, 5 X standard sodium citrate (SSC): 0.15 M NaCl, 0.015 M sodium citrate, pH 7, 10 3 Denhardt’s solution, 50 mM NaPO4, 1% sodium dodecylsulfate (SDS), 10 mg/ml Sigma free acid, and degraded herring sperm DNA and then hybridized overnight at 42˚C in a mixture of 50% formamide, 5 3 SSC, 1 3 Denhardt’s solution, 20 mM NaPO4, 0.5% SDS, 5% dextran sulfate, 20 mg/ml Sigma free acid, and degraded herring sperm with 106 cpm/ml of 32P-labeled complementary (c)DNA probe. Northern blots were sequentially hybridized to detect androgen receptor mRNA and then b-actin. Blots were washed in 1 3 SSC/1% SDS at room temperature for 15 minutes, 0.5% SSC/0.5% SDS at room temperature for 15 minutes, twice in 0.1 3 SSC/0.1% SDS at room temperature, and 0.1% SSC/0.1% SDS at 50˚C for 30 minutes and then subjected to autoradiography. DNA Fragments

The probes used for hybridization were a 718-bp _____________ * MCF-7 breast carcinoma cells obtained from American Type Culture Collection, Rockville, Maryland. † Centrifuge, Model RT 6000, and ultracentrifuge, SW 50.1 rotor, manufactured by Beckman Instruments, Irvine, California. ‡ Duralon nylon membrane and Stratalinker ultraviolet light obtained from Stratagene, La Jolla, California.


Androgen receptor expression in meningiomas tion) for 30 minutes.\ The avidin–biotin peroxidase staining procedure has been described previously.53 After a 10minute wash in PBS the sections were treated for 10 minutes in diaminobenzidine tetrahydrochloride solution (75 mg diaminobenzidine tetrahydrochloride in 100 ml 0.05 M Tris, pH 7.6, and 8.5 ml fresh 30% hydrogen peroxide). All incubations except those with the primary antibody were conducted at room temperature. After washing in PBS the sections were dried, mounted with Permount, and coverslipped. Results Northern Blot Analysis

FIG. 1. Northern blots. Upper: Representative blot showing the level of expression of androgen receptor in eight meningiomas. Lower: The same blot reprobed with b-actin showing that there is RNA loaded equivalently in each lane.

EcoRI-HindIII human androgen receptor insert, which spans the DNA-binding domain and part of the steroidbinding domain,37 and an 1800-bp human b-actin fragment. The cDNA fragments were labeled with phosphorus-32 using random primer translation to achieve a specific activity of 0.5 to 1.0 3 109 cpm/mg DNA.19 Immunohistochemistry for Androgen Receptor

Meningioma tissue obtained at the time of craniotomy was immediately embedded in ornithine carbamoyltransferase§ and frozen in isopentane precooled with liquid nitrogen. Frozen sections 8 mm thick were cut at 230˚C, air dried, and fixed for 10 minutes at 220˚C in 95% ethanol. Sections were washed twice with 0.01 M PBS (pH 7.4) for 5 minutes each time and then treated with 3% hydrogen peroxide in PBS for 2 to 3 minutes at room temperature to reduce endogenous peroxidase activity. Sections were blocked by 5% nonfat milk or bovine serum albumin in PBS for 30 minutes followed by 2% normal goat serum in PBS for 20 minutes to reduce nonspecific background staining. The sections were incubated overnight at 4˚C with an immunoglobulin (Ig)G fraction of immune serum, the antipeptide antibody AR52,59 or preimmune serum at a concentration of 5 to 10 mg/ml. The specificity of AR52 has previously been established.54 After incubation the sections were washed in PBS for 10 minutes and then treated with biotinylated goat anti–rabbit IgG (1:400 dilution in PBS) for 60 minutes, followed by incubation with avidin–biotin peroxidase (1:400 dilution) for 60 minutes or avidin–biotin complex (1:200 dilu§ Ornithine carbamoyltransferase obtained from Miles, Inc., Elkhart, Indiana.


Total RNA samples from 39 meningiomas were examined by Northern blot analysis for the presence of androgen receptor mRNA. Poly(A)+ mRNA was further isolated from two meningiomas, as indicated in Table 1. Total RNA was also isolated from MCF-7 cells, which were used as a positive control on some Northern blots (data not shown). We observed an androgen receptor mRNA species of approximately 9.6 kb in all meningiomas under stringent washing conditions (Fig. 1). This pattern was similar to that previously reported for human, rat, and mouse tissues known to contain androgen receptors.12,37 In some meningiomas (for example, lanes 3, 4, and 5 in Fig. 1) we also observed a band of approximately 9.4 kb in size. The band at 5.0 kb most likely represents crosshybridization with 28S RNA, because this band disappeared with the purification of poly(A)+ RNA. Loading of RNA was verified by reprobing with b-actin cDNA (Fig. 1). The level of expression of androgen receptor mRNA varied dramatically (Fig. 2, Table 1). Twenty-six of the tumors examined (67%) showed some level of androgen receptor mRNA expression; of these, 18 (69%) were from women and eight (31%) from men. By contrast, of the 13 tumors (33%) that were negative, three (23%) were from women and 10 (77%) from men. Using chi-square analysis there was a significant gender difference in the origin of tumors that were positive versus those that were negative for androgen receptor mRNA expression (p , 0.05); women had significantly more tumors with androgen receptor mRNA than did men. Eighteen of the 21 women (86%) had meningiomas expressing androgen mRNA receptor, whereas only eight of the 18 men (44%) had tumors expressing the receptor. There was no evident relationship between androgen receptor mRNA expression and histological subtype of the meningiomas. A human leptomeningeal cell line47 expressed a low level of androgen receptor mRNA by Northern blot analysis (data not shown). Immunohistochemical Studies

Five meningiomas (Cases 1, 5, 6, 7, and 8) that were positive for androgen receptor mRNA analysis were also positive for androgen receptor by immunohistochemistry under the conditions described in the Materials and \ Vectastain avidin-biotin complex kit obtained from Vector Laboratories, Burlingame, California.

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FIG. 2. Graph showing androgen receptor messenger ribonucleic acid (mRNA) expression in 26 meningiomas. The meningioma numbers correlate with case numbers in Table 1. ADU = arbitrary densitometric units.

Methods section. The cases included two men and three women ranging in age from 44 to 78 years. There was no difference in the immunostaining between men and women; there was intense nuclear staining with a small amount of staining observed in the cytoplasm. Figure 3A shows a tumor section incubated with preimmune serum which demonstrates little or no specific staining. Figures 3B and C illustrate the same tumor (Case 5) incubated with the AR52 primary antibody. The majority of the cells that were positive for androgen receptor were meningotheliomatous cells, which are identified by their histological type. Three meningiomas (Cases 27, 29, and 38, two women and one man ranging in age from 35 to 76 years) that were negative for androgen receptor by mRNA analysis were also negative for androgen receptor by immunochemistry (data not shown). Normal arachnoid membrane from a 43-year-old woman expressed low levels of androgen receptor protein by immunohistochemistry. A touch smear of normal arachnoid membrane incubated with preimmune serum showed no nuclear staining (Fig. 4A) whereas a smear from the same specimen revealed specific nuclear immunostaining with the AR52 primary antibody (Fig. 4B). Discussion The role of steroid hormones in the progression of meningioma remains controversial. A number of re456

searchers2,4,25,40,56 have used competitive binding studies to examine the presence of estrogen and progesterone receptors. When the data from these studies are combined, 28% of meningiomas demonstrate estrogen receptor and 72% demonstrate progesterone receptor. However, using solidphase immunoassay, in situ hybridization, immunohistochemistry, and Northern blot analysis, all methods that provide additional criteria over the steroid binding assays, recent studies have failed to demonstrate the presence of the estrogen receptor.56 There has been a small number of studies that examined meningiomas for the presence of androgen-binding sites.6,32,48 In these studies some level of androgen binding was detected in 65% to 100% of the tumors examined. We found similar results; in our study 67% of the tumors had detectable levels of androgen receptor mRNA. Investigators have addressed the functionality of the receptor by examining the effect of dihydrotestosterone or testosterone acetate on meningioma growth in culture.20,48,66 Most of the studies demonstrated some effect of testosterone agonists in meningioma proliferation, suggesting that the receptors that are present can be activated by ligand. We have demonstrated the presence of androgen receptors in meningiomas by two independent methods, Northern blot analysis for mRNA and immunohistochemistry for the receptor protein. The size of the predominant androgen receptor mRNA (9.6 kb) in meningiomas is very J. Neurosurg. / Volume 82 / March, 1995

Androgen receptor expression in meningiomas

FIG. 4. Photomicrographs showing immunocytochemical localization of AR52 in human arachnoid membrane. Touch smears of human arachnoid membrane from a 43-year-old woman were immunostained with an AR52 immunoglobulin G fraction of preimmune serum (A) and immune serum (B). Note the absence of nuclear staining in A and its presence in B. Avidin–biotin peroxidase, original magnification 3 720.

FIG. 3. Case 5. Photomicrographs showing immunocytochemical localization of AR52 in human meningioma tissue. Frozen sections 8 mm thick were immunostained with an AR52 immunoglobulin G fraction of preimmune serum (A) and immune serum (B and C). Immunostaining over background controls is predominantly in the nuclei of meningiotheliomatous cells. Note the absence of nuclear staining in A and intense nuclear staining (arrowheads) in B and C. Avidin–biotin peroxidase. Original magnification, 3 720 (A and B), original magnification 3 360 (C).

similar to that observed in breast carcinoma MCF-7 cells (data not shown) and other androgen-positive tissues such as human prostate.12 However, it is different in size from that reported by Maxwell, et al.,42 (3.0 and 4.5 kb). These differences could be accounted for by the use of different cDNAs for Northern blot analysis. We used a 718-bp fragment that spans the DNA-binding domain and part of the steroid-binding of the human androgen receptor. In conJ. Neurosurg. / Volume 82 / March, 1995

trast, Maxwell, et al., used a 1700-bp fragment that spans the 59 end of the hAR cDNA. The transcripts they observed were specific for androgen receptor and were observed in other tissues, including LNCaP, a prostate carcinoma cell line.1 The smaller transcripts are thought to represent alternate splicing of the first exon.1 In the rat slightly different sized mRNA species are observed, depending on the location of the cDNA probe used.43 When a probe from the 59 untranslated region of rat cDNA was used, a single mRNA species of approximately 11 kb was observed in Northern blots of poly(A)+ RNA from reproductive tissues, kidney, liver, and muscle. Using a probe from the 59 end of the coding region in addition to the 11-kb band, a novel transcript of approximately 9.3 kb in size was seen in whole brain. In poly(A)+ RNA from dissected brain regions, the 9.3-kb transcript was predominant in the cortex, cerebellum, and brain stem, whereas in the hippocampus, both transcripts were expressed to a similar degree.43 In our study, 67% of the tumors examined showed some level of androgen receptor expression. It is well documented that the incidence of meningioma is two times higher in women than in men for intracranial tumors and nine times higher for spinal meningiomas. Our study also demonstrates a sex difference in the expression of both androgen mRNA levels and receptor protein, in that a 457

R. S. Carroll, et al. higher percentage of women with meningiomas have androgen receptor mRNA expression than do men. A sex difference in androgen-binding capacity has previously been reported.32 The incidence and pattern of expression of androgen receptor mRNA and proteins are similar to those observed earlier for progesterone receptor.10 Several studies have shown a positive correlation between androgen-binding activity and progesterone-binding activity, suggestive of androgen rather than estrogen regulation of the progesterone receptor.45,50 These results implicate a functional relationship for androgen and progesterone receptors in meningiomas, as has been described for benign prostatic hyperplasia, prostate carcinoma, and breast tumors.31 Antiandrogenic actions of progesterone on the central nervous system have been noted in other systems, such as a decline in reproductive behavior in male guinea pigs treated with progesterone. This may be explained by the interference of progesterone with the retention of androgen receptor in the nucleus.14 Cloning of cDNA for the steroid receptors revealed that these receptors are members of a superfamily of DNAbinding proteins and potentially oncogenic regulatory genes. Control of gene expression by steroid hormone receptors is dependent on the binding of their specific ligand. Activation of transcription results from the specific binding of the hormone–receptor complex to promoter “enhancer” elements of target genes. It may be possible that alterations of specific enhancer elements of key cellular genes and/or their binding factors correspond to crucial steps in many types of carcinogenesis and cell transformation.21 In support of this hypothesis, few alterations of steroid receptor–related genes have been reported in human cancers such as hepatocellular carcinoma and small lung carcinoma.15,16,42 Alterations in the restriction pattern of the DNA-binding domain of the estrogen receptor have been reported in human meningiomas,63 as have aberrant estrogen receptor mRNA species.30 Protooncogenes, including c-sis/platelet-derived growth factor,41 epidermal growth factor (EGF) receptor,64 myc,28 and k-ras,11 have been found in meningiomas. There may be a link between the steroid and oncogenic systems in these tumors. For example, recently it has been shown that androgens can induce the synthesis of EGF-receptor in prostate tumor cells.46 In the second part of our study we examined the expression and location of the androgen receptor protein. Immunohistochemistry allowed us to examine both the cytoplasm and nucleus for the presence of androgen receptor. Androgen receptor immunostaining was largely confined to the nucleus, but a small amount of staining was observed in the cytoplasm. These results are not surprising, because in COS cells transfected with androgen receptor the receptor resides in the cytoplasm in the absence of ligand.29,58,67 Our results are in agreement with those of other studies42 that show the presence of nuclear androgen receptor in meningiomas by the use of the same highly specific monoclonal antipeptide antibody (AR52) used in our studies. Tumors that were negative for androgen receptor by Northern blot analysis were also negative by immunohistochemistry, showing a positive correlation between mRNA levels and protein levels. These results rule out the possibility that in men higher levels of circu458

lating androgens could down-regulate the androgen receptor mRNA and not the protein. Normal arachnoid membrane showed a low level of androgen receptor expression. These results suggest that in meningioma tumorgenesis the expression of androgen receptor may be increased. In our study the majority of the tumors were syncytial or transitional (Table 1), and there was no clear relationship between the presence of androgen receptor and tumor histological type. Some studies have suggested a relationship between androgen receptor binding and histological type, with higher androgen receptor levels observed in syncytial tumors.50 The present study shows that androgen receptor mRNA is present in meningiomas, with some distinct differences in regard to the sex of the patient. The androgen receptors are likely to be functional because they are found in the nucleus, although a formal in vitro or in vivo assay of androgen receptor functionality remains to be done. In breast carcinoma, the expression of androgen receptor appears to be preserved during the process of metastasis.60 Moreover, Teuling, et al.,60 and Bryan, et al.,7 have reported that data on androgen receptor expression added significantly to the prediction of survival and the response to hormone therapy. Acknowledgments The authors thank Dr. Matthew Frosch of Brigham and Women’s Hospital for classification of the tumor samples and Larry Kedes of the University of Southern California for providing the human bactin fragment. References 1. Antoniades HN, Maxwell M: Androgen and progesterone receptors in meningiomas—response. J Neurosurg 79: 635–636, 1993 (Letter) 2. Blaauw G, Blankenstein MA, Lamberts SWJ: Sex steroid receptors in human meningiomas. Acta Neurochir 79:42–47, 1986 3. Bickerstaff ER, Small JM, Guest IA: The relapsing course of certain meningiomas in relation to pregnancy and menstruation. J Neurol Neurosurg Psychiatry 21:89–91, 1958 4. Blankenstein MA, Blaauw G, Lamberts WJ, et al: Presence of progesterone receptors and absence of oestrogen receptors in human intracranial meningioma cytosols. Eur J Cancer Clin Oncol 19:365–370, 1983 5. Blankenstein MA, Blaauw G, Lamberts WJ: Progestin and estrogen receptors in human meningiomas. Clin Neuropharmacol 7:363–367, 1984 6. Brentani MM, Lopes MTP, Martins VR, et al: Steroid receptors in intracranial tumors. Clin Neuropharmacol 7:347–350, 1984 7. Bryan RM, Mercer RJ, Bennett RC, et al: Androgen receptors in breast cancer. Cancer 54:2436–2440, 1984 8. Burger PC, Scheithauer B, Vogel FS: Surgical Pathology of the Nervous System and Its Coverings. New York: Churchill Livingstone, 1991, pp 67–68 9. Cahill DW, Bashirelahi N, Solomn LW, et al: Estrogen and progesterone receptors in meningiomas. J Neurosurg 60: 985–993, 1984 10. Carroll RS, Glowacka D, Dashner K, et al: Progesterone receptor expression in meningiomas. Cancer Res 53:1312–1326, 1993 11. Carstens C, Messe E, Zang KD, et al: Human KRAS oncogene


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