| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Studies |
Prince Henry’s Institute of Medical Research, Monash University, and Department of Obstetrics and Gynecology, Monash Medical Center, Clayton, Victoria 3168, Australia
Address all correspondence and requests for reprints to: Dr. Peter J. Fuller, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail: peter.fuller{at}med.monash.edu.au
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Heterotrimeric G proteins couple cell surface receptors for hormones,
including FSH, to intracellular second messenger systems. Activating
mutations of both cell surface receptors and G proteins have been
reported in various tumors (7, 8). Mutations of the G protein
-subunit gene causing inhibition of the intrinsic guanosine
triphosphatase activity and thereby yielding an activating oncogene,
gsp, were first described in GH-secreting pituitary adenomas
(9). The gsp oncogene is found in 40% of somatotroph
adenomas as well as less frequently in nonfunctioning pituitary
adenomas, corticotroph adenomas, thyroid adenomas, and the
McCune-Albright syndrome (7, 8, 10). In a retrospective study of
archival material from many tumor types, Lyons et al. (10)
reported the presence of analogous mutations in the Gi-2
subunit gene (gip2) in 3 of 10 sex cord stromal tumors
and 3 of 11 adrenocortical tumors. In a recent study we were unable to
find activating mutations at codon 179 of the Gi-2 gene
in ovarian granulosa cell tumors (11), and Reincke et al.
(12) were similarly unsuccessful when they examined 18 adrenocorticoid
tumors.
The FSH receptor is a member of the seven-transmembrane domain or G protein-coupled receptor (GPCR) superfamily of cell surface receptors. Activating mutations of these receptors have been described in a range of conditions (8). Activating mutations of the TSH receptor have been implicated in the pathogenesis of malignancy in hyperfunctioning thyroid adenomas (13, 14) and well differentiated thyroid carcinoma (15, 16). Recently, a virally encoded constitutively active GPCR of the chemokine subfamily has been implicated in the pathogenesis of both Kaposi’s sarcoma and a rare ß-cell lymphoma (17).
The possibility that activation of the FSH-dependent signaling pathways may play a role in the pathogenesis of granulosa cell tumors of the ovary, in a manner analogous to the role of the TSH receptor in the pathogenesis of thyroid tumors, encouraged us to analyze a series of granulosa cell tumors for activating mutations of the FSH receptor.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Frozen granulosa cell tumor tissue (n = 3) or paraffin-embedded tissue sections (n = 12) were studied (11) as part of a much larger Melbourne-wide investigation of serum inhibin levels in ovarian tumors (3, 4). DNA extraction from fresh-frozen tissue was performed as previously described (11). Briefly, approximately 0.5 g tissue was homogenized in 5 mL extraction buffer (10 mmol/L Tris, 0.1 mol/L ethylenediamine tetraacetate, 20 µg/mL ribonuclease, and 0.5% SDS), digested with 100 µg/mL proteinase K at 37 C for 4 h, extracted twice with an equal volume of phenol and once with chloroform, precipitated with 0.2 vol 10 mol/L ammonium acetate and 2 vol 100% ethanol, and washed with 70% ethanol. DNA pellets were diluted in distilled water and stored at 4 C. For isolation of DNA from paraffin-embedded tissue, two or three 10-µm sections were cut for each sample, and excess paraffin was removed using a disposable scalpel. Tissue sections were scraped into a 1.5-mL microcentrifuge tube. Paraffin was removed from the samples by two 20-min extractions with 1 mL xylene. Xylene was removed by two washes with 1 mL 100% ethanol and one wash with 70% ethanol. Tissue sections were vacuum-dried and digested overnight at 50 C with 400 ng/µL proteinase K in 100–200 µL 50 mmol/L Tris, pH 8.3, as previously described (11). Samples were then boiled for 8 min to inactivate proteinase K, cooled on ice, and spun briefly to pellet out debris. The supernatant was transferred into a fresh tube for storage at -20 C.
Amplification of genomic DNA
PCR was used to amplify five overlapping amplicons covering exon
10 of the FSH receptor gene. The primer sequences are shown in Table 1
, and the positions of the amplicons are
shown schematically in Fig. 1. The
amplicons ranged from 226–275 bp in length. The PCR reaction mixture
consisted of 100 ng DNA extracted from frozen tissue or 5 µL tissue
section lysate, 30 pmol of each primer, 100 µmol/L deoxy-NTPs, 1.8
mmol/L MgCl2, 2.5 U Taq polymerase (Boehringer
Mannheim, Mannheim, Germany), and 1 x PCR buffer [10 x PCR
buffer is 100 mmol/L Tris (pH 8.5), 500 mmol/L KCl, and 1% Triton
X-100] in a total volume of 100 µL. PCR was initiated at 94 C for 5
min, followed by 35 cycles of 94 C for 30 s, 55 C for 40 s,
and 72 C for 60 s and a final extension at 72 C for 8 min. In all
PCR experiments, reactions containing no DNA were included as negative
controls.
|
|
To confirm the above findings, the PCR products were sequenced directly using a Sequenase 2.0 kit (U.S. Biochemical Corp., Cleveland, OH), [35S]deoxy-ATP, 15 pmol primer, and 500 ng of the PCR products for each sample.
Single strand conformational polymorphisms (SSCP)
The SSCP analyses were performed using minor modifications of
the method described originally by Orita et al. (18). PCR
reactions to be used for the SSCP analysis were performed in the
presence of 0.5 µCi [-33P]deoxy-ATP [2000 Ci/mmol;
DuPont (Australia), North Sydney, Australia] in a total volume of 50
µL. The products were run on standard denaturing (7 mol/L urea) or
nondenaturing 6% polyacrylamide sequencing gels at 28, 20, and 10 C
with or without 10% glycerol in the gel. Constant temperature was
maintained using a Strata Therm Temperature Controller (Stratagene, La
Jolla, CA). The conditions of the PCR are described above.
Reverse transcriptase-PCR
RNA was extracted from the three frozen granulosa cell tumors
(11), seven mucinous cystadenocarcinomas of the ovary, and normal human
colon using the guanidine thiocyanate/cesium chloride method. One
microgram of total RNA was reverse transcribed for 90 min at 42 C in a
total volume of 20 µL using AMV reverse transcriptase (Boehringer
Mannheim) with the FSH receptor antisense primer at position 1769
(Table 1
). Two microliters of this reaction were added to a PCR
reaction as described above using the sense and antisense primers for
amplicon 3 (Table 1). Ten microliters of each product were analyzed on
ethidium bromide-stained 1% agarose gels. Reactions containing either
no input RNA or no reverse transcriptase were included as negative
controls.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). Direct
sequencing revealed this to be a previously described (19) polymorphism
where a G to A transition results in either a serine or an asparagine
residue, respectively, at codon 680 (Fig. 3). We further examined DNA from 7
mucinous cystadenocarcinomas of the ovary and 22 randomly selected
normal DNA samples for this polymorphism (Fig. 2). The distribution of
the codon 680 alleles is tabulated in Table 2. No clear association of the codon 680
allele with the source of the DNA was observed. In their original
description of this polymorphism, Aittomäki et al.
(19) found that neither allele was linked to hereditary
hypergonadotropic ovarian failure, the phenotype being examined. In
addition, direct sequencing was performed on amplicon 4 from 10 of the
granulosa cell tumors, given that this region encompasses a previously
reported activating mutation (20) of the FSH receptor and corresponds
to a "hot spot" for activating mutations of the TSH receptor
(13, 14, 15, 16) and has very recently been reported to contain an inactivating
mutation in ovarian sex cord tumors (21). As with the SSCP analysis of
this amplicon, direct sequencing failed to identify any nucleotide
changes.
|
|
|
). Curiously, lower levels of expression
than in the granulosa cell tumors were noted in most of the mucinous
cystadenocarcinomas.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Recent clinical reports suggest that hyperstimulation of this signal
transduction pathway during gonadotropin therapy for the treatment of
infertility increases the incidence of granulosa cell tumors (22, 23);
however, the validity of these studies is uncertain (24, 25). Granulosa
cell tumors also arise in transgenic mice, in which the inhibin
-subunit gene has been deleted. The pathogenesis of these tumors is
unclear; however, the mice have markedly elevated levels of FSH and
also activin (26). Up-regulation of the signal transduction pathways
initiated by FSH might be postulated to play a role in the pathogenesis
of these tumors. Lyons et al. (10) reported the presence of
the gip2 oncogene in 3 of 10 ovarian sex cord stromal
tumors. The relationship of Gi-2 to FSH-stimulated
signal transduction is not clear; indeed, we were unable to confirm the
presence of the gip2 oncogene in the granulosa cell tumors
used in this current study (11).
In addition to mutations of G proteins, mutations of GPCR play a role in a range of inherited and acquired conditions (7, 8, 17). Inactivating mutations of these receptors have been extensively reported, including an alanine to valine substitution in the extracellular region of the FSH receptor first reported by Aittomäki et al. (19) in hereditary hypergonadotropic ovarian failure.
Activating mutations of GPCR were first identified in artificial constructs, but naturally occurring mutations have subsequently been identified in a range of receptors, including those for chemokines (17), calcium (8), PTH/PTH-related peptide (27), and gonadotropins. Activating mutations of the TSH receptor have been identified in both inherited and sporadic conditions. Inherited mutations of the TSH receptor cause familial nonautoimmune hyperthyroidism (28), whereas somatic mutations occur in both adenomas (13, 14) and well differentiated carcinomas (15, 16) of the thyroid. Activating mutations of the LH receptor have been reported in gonadotropin-independent male-limited precocious puberty (29, 30). A heterozygous A to G change at nucleotide 1700 in the human FSH receptor, causing an aspartate to glycine transition in codon 507, has been recently reported in a subject with pituitary-independent spermatogenesis (20). The substituted residue lies in the third intracytoplasmic loop. The majority of activating mutations of GPCR reported lie in this loop, the sixth transmembrane domain, or, less commonly, the second transmembrane domain.
In the present study we sought to address the hypothesis that activation of some component of the FSH-stimulated signal transduction pathway in granulosa cells may contribute to the pathogenesis of granulosa cell tumors of the ovary. We examined the FSH receptor not only because it represents the most proximal component of the pathway, but also because of the reports of activating mutations in the three gonadotropin receptors. Our inability to identify any mutations in exon 10 of the FSH receptor in the DNA from 15 granulosa cell tumors would seem to argue strongly against a major role for FSH receptor mutations in the pathogenesis of these tumors. It does not exclude the possibility that mutations may occur in a subset of cells or a minority or tumors, and it remains possible, although unlikely, that our exhaustive search using SSCP analysis under a range of conditions has missed a specific mutation. It is reassuring that a previously reported polymorphism was clearly identified. Limited direct sequencing was also performed; although this should be 100% sensitive, it may still miss mutations of one allele where a degree of contamination of the tumor by normal tissue, such as stromal elements, dilutes the abnormal allele.
Subsequent to the submission of this study, Kotlar et al. (21) reported a similar study in which they examined a smaller region of exon 10 of the FSH receptor gene in 13 ovarian sex cord tumors. They amplified the third intracytoplasmic loop together with parts of the fifth and sixth transmembrane domains. Using direct sequencing of PCR products from archival material amplified by nested PCR, they identified a missense mutation at nucleotide 1777 that converts codon 591 from phenylaline to serine (F5915) in 69% of the sex cord tumors (21). Functional studies suggest that this is an inactivating mutation (21), which runs counter to their and our initial hypothesis. We have been unable to identify this mutation in our tumors from the SSCP analysis, by direct sequencing of both strands of this amplicon from 10 of our tumors, or by allele-specific hybridization with the primers described by Kotlar et al. (21). It is difficult to explain the discrepancy between our study and that of Kotlar et al. (21); the methodology is fundamentally similar, except, perhaps, that we did not need to use nested PCR. The main difference, therefore, lies in the tumor populations studied. Our tumor sample represents essentially consecutive granulosa cell tumors identified in Melbourne as part of a larger study of inhibin levels in ovarian tumors (3, 4), whereas Kotlar et al. (21) examined archival material "accumulated by referral of specific blocks of tumors over several decades for confirmation of difficult histological diagnosis." They include a large number of juvenile granulosa cell tumors. The mutation was also observed in a curious subgroup of ovarian tumors, small cell carcinomas of the hypercalcemia type. In their discussion, Kotlar et al. (21) note that in two additional, presumably relatively unselected, cases, the mutation was not observed. An exciting conclusion that might be drawn from this dichotomy is that distinct subgroups of sex cord tumors might be defined on the basis of the F591S mutation in the FSH receptor gene.
The polymorphism at codon 680 was first recognized as such by Aittomäki et al. (19). Although the polymorphism would clearly seem to have no correlation to ovarian cancer and to be fairly evenly distributed, it represents a nonconservative substitution. This region of the receptor is not well conserved compared to the TSH or LH receptor, but is reasonably well conserved across species (31). Asparagine is present at this position in seven other species of FSH receptor (31). We know of no studies that examine the significance of this substitution. The cytoplasmic tail is known to be important in G protein coupling (7, 8), and thus changes in this region might alter the strength or specificity of such interactions. A serine at this position also creates a potential phosphorylation site (32).
FSH binding and stimulation of adenylyl cyclase activity in granulosa cell tumors have previously been reported (33, 34), and our demonstration of FSH receptor gene expression confirms this finding. The presence of FSH receptor messenger RNA, albeit at low levels, in mucinous cystadenocarcinomas is of interest given that these tumors also secrete inhibin (3).
The lack of activating mutations of the FSH receptor in granulosa cell tumors in this study, although in contrast to the situation with the TSH receptor, parallels the findings of studies of adrenal tumors in which activating mutations of the ACTH receptor were not identified (35). In conclusion, it would seem that the FSH receptor, like the gip2 oncogene (11) or the gsp oncogene (10), is unlikely to play a major role in the pathogenesis of granulosa cell tumors of the ovary, although an inactivating mutation may play a role in the pathogenesis of a subset of sex cord tumors (21). The pathogenesis of ovarian sex cord stromal tumors, therefore, remains largely unexplained, and perhaps because of their relative rarity, few studies have specifically examined the molecular genetics of these tumors. The evidence, however, that up-regulation of this signaling pathway occurs remains compelling, and future studies will need to examine molecules distal to the FSH receptor but proximal to cyclin D2 up-regulation.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Recipient of a Principal Research Fellowship from the National
Health and Medical Research Council of Australia.
3 Supported by a Monash University Graduate Research
Scholarship.
Received April 22, 1997.
Revised August 22, 1997.
Accepted September 28, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-chain
of Gs and stimulate adenylyl cyclase in human pituitary tumours. Nature. 340:692–696.[CrossRef][Medline]
in human thyroid cancers: low prevalence of mutations
predicts infrequent involvement in malignant transformation. J
Clin Endocrinol Metab. 81:3898–3901.-Inhibin is a tumour-suppressor gene with gonadal
specificity in mice. Nature. 360:313–319.[CrossRef][Medline]
This article has been cited by other articles:
 |
|
 |
|
  M. Zhang, Y.-X. Tao, G. L. Ryan, X. Feng, F. Fanelli, and D. L. Segaloff Intrinsic Differences in the Response of the Human Lutropin Receptor Versus the Human Follitropin Receptor to Activating Mutations J. Biol. Chem., August 31, 2007; 282(35): 25527 - 25539. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. M Dorward, K. L Shultz, and W. G Beamer LH analog and dietary isoflavones support ovarian granulosa cell tumor development in a spontaneous mouse model Endocr. Relat. Cancer, June 1, 2007; 14(2): 369 - 379. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-H. Choi, A. S. T. Wong, H.-F. Huang, and P. C. K. Leung Gonadotropins and Ovarian Cancer Endocr. Rev., June 1, 2007; 28(4): 440 - 461. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.Q. Yang, K.Y.K. Chan, H.Y.S. Ngan, U.S. Khoo, P.M. Chiu, Q.K.Y. Chan, W.C. Xue, and A.N.Y. Cheung Single nucleotide polymorphisms of follicle-stimulating hormone receptor are associated with ovarian cancer susceptibility Carcinogenesis, July 1, 2006; 27(7): 1502 - 1506. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Kalfa, A. Ecochard, C. Patte, P. Duvillard, F. Audran, C. Pienkowski, E. Thibaud, R. Brauner, C. Lecointre, D. Plantaz, et al. Activating Mutations of the Stimulatory G Protein in Juvenile Ovarian Granulosa Cell Tumors: A New Prognostic Factor? J. Clin. Endocrinol. Metab., May 1, 2006; 91(5): 1842 - 1847. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Anttonen, L. Unkila-Kallio, A. Leminen, R. Butzow, and M. Heikinheimo High GATA-4 Expression Associates with Aggressive Behavior, whereas Low Anti-Mullerian Hormone Expression Associates with Growth Potential of Ovarian Granulosa Cell Tumors J. Clin. Endocrinol. Metab., December 1, 2005; 90(12): 6529 - 6535. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Boerboom, M. Paquet, M. Hsieh, J. Liu, S. P. Jamin, R. R. Behringer, J. Sirois, M. M. Taketo, and J. S. Richards Misregulated Wnt/{beta}-Catenin Signaling Leads to Ovarian Granulosa Cell Tumor Development Cancer Res., October 15, 2005; 65(20): 9206 - 9215. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Sudo, M. Kudo, S.-i. Wada, O. Sato, A. J.W. Hsueh, and S. Fujimoto Genetic and functional analyses of polymorphisms in the human FSH receptor gene Mol. Hum. Reprod., October 1, 2002; 8(10): 893 - 899. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-X. Tao, D. Mizrachi, and D. L. Segaloff Chimeras of the Rat and Human FSH Receptors (FSHRs) Identify Residues that Permit or Suppress Transmembrane 6 Mutation-Induced Constitutive Activation of the FSHR via Rearrangements of Hydrophobic Interactions Between Helices 6 and 7 Mol. Endocrinol., August 1, 2002; 16(8): 1881 - 1892. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. E. Owens, R. A. Keri, and J. H. Nilson Ovulatory Surges of Human CG Prevent Hormone-Induced Granulosa Cell Tumor Formation Leading to the Identification of Tumor-Associated Changes in the Transcriptome Mol. Endocrinol., June 1, 2002; 16(6): 1230 - 1242. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Chu, S. Rushdi, E.T. Zumpe, P. Mamers, D.L. Healy, T. Jobling, H.G. Burger, and P.J. Fuller FSH-regulated gene expression profiles in ovarian tumours and normal ovaries Mol. Hum. Reprod., May 1, 2002; 8(5): 426 - 433. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. Fuller, E. T. Zumpe, S. Chu, P. Mamers, and H. G. Burger Inhibin-Activin Receptor Subunit Gene Expression in Ovarian Tumors J. Clin. Endocrinol. Metab., March 1, 2002; 87(3): 1395 - 1401. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. S. Choong, P. J. Fuller, S. Chu, Y. Jeske, F. Bowling, R. Brown, P. Borzi, N. D. Balazs, R. Suppiah, A. M. Cotterill, et al. Sertoli-Leydig Cell Tumor of the Ovary, a Rare Cause of Precocious Puberty in a 12-Month-Old Infant J. Clin. Endocrinol. Metab., January 1, 2002; 87(1): 49 - 56. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. P. N. Themmen and I. T. Huhtaniemi Mutations of Gonadotropins and Gonadotropin Receptors: Elucidating the Physiology and Pathophysiology of Pituitary-Gonadal Function Endocr. Rev., October 1, 2000; 21(5): 551 - 583. [Abstract] [Full Text]
|
|
|
|
 |
|
 J. L. Juengel, L. D. Quirke, D. J. Tisdall, P. Smith, N. L. Hudson, and K. P. McNatty Gene Expression in Abnormal Ovarian Structures of Ewes Homozygous for the Inverdale Prolificacy Gene Biol Reprod, June 1, 2000; 62(6): 1467 - 1478. [Abstract] [Full Text]
|
|
|
|
|
|
S. Chu, P. Mamers, H. G. Burger, and P. J. Fuller Estrogen Receptor Isoform Gene Expression in Ovarian Stromal and Epithelial Tumors J. Clin. Endocrinol. Metab., March 1, 2000; 85(3): 1200 - 1205. [Abstract] [Full Text]
|
|
|
|
|
|
H. Zhang, M. Vollmer, M. De Geyter, Y. Litzistorf, A. Ladewig, M. Durrenberger, R. Guggenheim, P. Miny, W. Holzgreve, and C. De Geyter Characterization of an immortalized human granulosa cell line (COV434) Mol. Hum. Reprod., February 1, 2000; 6(2): 146 - 153. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Hussein, S. Chu, and P. J. Fuller Comment on Analysis of Mutations in Genes of the Follicle-Stimulating Hormone Receptor in Ovarian Granulosa Cell Tumors J. Clin. Endocrinol. Metab., October 1, 1999; 84(10): 3852 - 3853. [Full Text] [PDF]
|
|
|
|
|
|
M. J. Ligtenberg, M. Siers, A. P. N. Themmen, T. G. Hanselaar, W. Willemsen, and H. G. Brunner Analysis of Mutations in Genes of the Follicle-Stimulating Hormone Receptor Signaling Pathway in Ovarian Granulosa Cell Tumors J. Clin. Endocrinol. Metab., June 1, 1999; 84(6): 2233 - 2234. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
