Elevated levels of the steroidogenic factor 1 are associated with over-expression of CYP19 in an oestrogen-producing testicular Leydig cell tumour.

Abstract

Testicular Leydig cell tumours (LCTs) are rare, steroid-secreting tumours. Elevated levels of aromatase (CYP19 or CYP19A1) mRNA have been previously described in LCTs; however, little is known about the mechanism(s) causing CYP19 over-expression. We report an LCT in a 29-year-old male with elevated plasma oestradiol caused by enhanced CYP19 transcription.
First, we measured the intra-tumour expression of CYP19 and determined the use of CYP19 promoters by qPCR. Secondly, we explored CYP19 and promoter II (PII) for gene amplifications and activating mutations in PII by sequencing. Thirdly, we analysed intra-tumour expression of steroidogenic factor 1 (SF-1 (NR5A1)), liver receptor homologue-1 (LRH-1 (NR5A2)) and cyclooxygenase-2 (COX2 (PTGS2)). Finally, we analysed SF-1 for promoter mutations and gene amplifications.
Similar to what has been recorded in normal Leydig cells, we first found the bulk of tumour CYP19 transcripts to be PII derived, excluding promoter shift as a cause of enhanced transcription. Secondly, we excluded CYP19 and PII gene amplifications, and activating mutations in PII, as causes of elevated CYP19 mRNA. We found SF-1 mRNA to be up-regulated in the tumour, while LRH-1 and COX2 were down-regulated. The finding of elevated SF-1 levels in the tumour was confirmed by immunohistochemistry. The elevated level of SF-1 was not due to promoter mutations or amplifications of the SF-1 gene.
Our results strongly suggest that the elevated levels of SF-1 have induced PII-regulated CYP19 transcription in this tumour. These findings are of relevance to the understanding of CYP19 up-regulation in general, which may occur in several tissues, including breast cancer.

Full text

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CASE REPORT
Elevated levels of the steroidogenic factor 1 are associated with
over-expression of CYP19 in an oestrogen-producing testicular
Leydig cell tumour
Anne Hege Straume1,2, Kristian Løva ˚s3,4, Hrvoje Miletic5,6, Karsten Gravdal5, Per Eystein Lønning1,2
and Stian Knappskog1,2
1Section of Oncology, Institute of Medicine, University of Bergen, Bergen, Norway,2Department of Oncology, Haukeland University Hospital, Bergen,
Norway,3Section of Endocrinology, Institute of Medicine, University of Bergen, Bergen, Norway,4Department of Medicine and5Section of Pathology,
Haukeland University Hospital, Bergen, Norway and6Department of Biomedicine, University of Bergen, Bergen, Norway
(Correspondence should be addressed to S Knappskog who is now at Mohn Cancer Research Laboratory (1M), Haukeland University Hospital,
5021 Bergen, Norway; Email: stian.knappskog@med.uib.no)
Abstract
Background and objectives: Testicular Leydig cell tumours (LCTs) are rare, steroid-secreting tumours.
Elevated levels of aromatase (CYP19 or CYP19A1) mRNA have been previously described in LCTs;
however, little is known about the mechanism(s) causing CYP19 over-expression. We report an LCT in
a 29-year-old male with elevated plasma oestradiol caused by enhanced CYP19 transcription.
Design and methods: First, we measured the intra-tumour expression of CYP19 and determined the use
of CYP19 promoters by qPCR. Secondly, we explored CYP19 and promoter II (PII) for gene
amplifications and activating mutations in PII by sequencing. Thirdly, we analysed intra-tumour
expression of steroidogenic factor 1 (SF-1 (NR5A1)), liver receptor homologue-1 (LRH-1 (NR5A2))
and cyclooxygenase-2 (COX2 (PTGS2)). Finally, we analysed SF-1 for promoter mutations and gene
amplifications.
Results: Similar to what has been recorded in normal Leydig cells, we first found the bulk of tumour
CYP19 transcripts to be PII derived, excluding promoter shift as a cause of enhanced transcription.
Secondly, we excluded CYP19 and PII gene amplifications, and activating mutations in PII, as causes
of elevated CYP19 mRNA. We found SF-1 mRNA to be up-regulated in the tumour, while LRH-1 and
COX2 were down-regulated. The finding of elevated SF-1 levels in the tumour was confirmed by
immunohistochemistry. The elevated level of SF-1 was not due to promoter mutations or
amplifications of the SF-1 gene.
Conclusions: Our results strongly suggest that the elevated levels of SF-1 have induced PII-regulated
CYP19 transcription in this tumour. These findings are of relevance to the understanding of CYP19
up-regulation in general, which may occur in several tissues, including breast cancer.
European Journal of Endocrinology 166 941–949
Introduction
Leydig cells are found adjacent to the seminiferous
tubules in the testicles. These cells constitute the main
androgen-synthesising compartment in adult males
and are also capable of oestrogen production (reviewed
in (1, 2, 3, 4)).
Leydig cell tumours (LCTs) of the testis are rare,
representingabout 1–3% of all testicular tumours. They
are most frequently diagnosed in pre-pubertal boys
between 5 and 10 years and adult men aged 30–60
years (5, 6). While usually benign, about 10% of LCTs
in adult patients reveal a malignant phenotype (7).
LCTs are steroid-secreting tumours and con-
sequently associated with endocrine disturbances.
In testosterone-secreting LCTs, boys usually present
symptoms of precocious puberty, whereas excess
androgen rarely causes notable effects in adults. About
one-fourth of all LCTs are oestrogen secreting, and
the most common symptoms include gynaecomastia
in addition to sexual dysfunction and infertility in
adults (6, 8, 9, 10). There have also been described
cases of LCTs in pre-pubertal boys, revealing symptoms
of both oestrogen and androgen production, causing
gynaecomastia and precocious puberty in concert
(reviewed in (4)).
Local oestrogen synthesis plays a functional role
in the testis (reviewed in (11, 12)). In resemblance
with other oestrogen-producing compartments, andro-
gens are converted into oestrogens by the aromatase
European Journal of Endocrinology (2012) 166 941–949ISSN 0804-4643
q 2012 European Society of Endocrinology DOI: 10.1530/EJE-11-0849
Online version via www.eje-online.org
This is an Open Access article distributed under the terms of the European Journal of Endocrinology’s Re-use Licence which permits unrestricted
non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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(CYP19) enzyme. CYP19 is encoded by the CYP19A1
gene, localised at chromosome 15q21.2 (13, 14, 15).
The unusually large regulatory region of this gene
contains several tissue-specific promoters, and each of
the promoters give rise to mRNAs with distinct
untranslated first exons (16). The first exon is spliced
onto a common splice junction in exon II, immediately
upstream of the coding region. Thus, the different
mRNA species expressed from this gene contains an
identical open reading frame (exons II–X), and the
translated protein is the same, regardless of the
promoter used.
Elevated CYP19 expression with subsequent high
plasma oestradiol (E2) levels has been reported in a few
cases of testicular LCTs (17, 18, 19, 20, 21). The main
regulator of normal testicular cell aromatization is the
CYP19 proximal promoter II (PII), which has also been
reported as the main active promoter in LCTs (18, 20).
Different response elements have been identified in
PII, and both steroidogenic factor 1 (SF-1 (encoded by
NR5A1)) and liver receptor homologue-1 (LRH-1
(encoded by NR5A2)) have been reported to be involved
in PII-regulated CYP19 expression (22, 23, 24, 25).
Moreover, several cAMP-response element (CRE)-like
sequences have been identified in PII (26, 27), and
cyclooxygenase-2 (COX2 (encoded by PTGS2)) was
recently reported to be involved in the phosphorylation
ofCRE-binding proteins
CYP19 expression and the proliferation of Leydig
tumour cells (28).
While over-expression of SF-1 was reported in Fisher
rat testicular tumours (22), so far we lack information
regarding what mechanism(s) may cause CYP19 over-
expression in human testicular LCTs. Here, we report
an oestrogen-producing LCT in a 29-year-old man. The
finding of SF-1 over-expression in concert with elevated
levels of the PII-derived CYP19 transcripts suggests
increased SF-1 levels to play a key role in stimulating
oestrogen synthesis in these tumour cells.
andthus stimulate
Materials and methods
Patient
A 29-year-old male with severe gynaecomastia and
elevated plasma E2levels (248 pM, normal upper range
130 pM) was referred to the Endocrine Unit. He was
subsequently diagnosed with an oestrogen-producing
LCT on the right testis. The tumour was not palpable
but visualised on testicular ultrasound. He had normal
pubertal growth, had developed normal secondary sex
characteristics and was the father of one child. There
was no clinical, biochemical (HCG and a-fetoprotein
negative) or radiological sign of malignant disease.
The high serum E2levels returned to normal after
surgical removal of the tumour. Although E2, testoster-
one and SHBG levels normalised rapidly, LH and FSH
remained elevated at follow-up, indicating impaired
function of the left testicle. All serum hormone levels are
summarised in Table 1. At 1-year follow-up, he felt well
and the gynaecomastia had resolved.
Subject to patient consent, part of the tumour
specimen obtained at orchidectomy was snap-frozen
and stored in liquid nitrogen for research purposes.
Histology
A part of the testis specimen was fixed in 4% formalin
and representative samples including both tumour and
normal tissues were embedded in paraffin. Standard
haematoxylin- and eosin (H&E)-stained sections (5 mm)
were made for microscopic histology evaluation.
Immunohistochemistry
Sections (5 mm) from formalin-fixed, paraffin-embedded
tumours were prepared. Immunohistochemical aroma-
tase enzyme staining was performed using the MAB
Aro677 (gift from Dr Dean Evans, Novartis AG). SF-1
was stained with the mouse antihuman MAB 434200
(Invitrogen), while the oestrogen and progesterone
receptors (PRs) were stained using the MABs M7047
and M3659 (Dako, Glostrup, Denmark) respectively.
For the staining procedure, the DAKO Envision HRP
mouse kit (Dako) with DAB as detection method
was used.
RNA extraction and cDNA synthesis
Total RNA was extracted from snap-frozen biopsies
using Trizol reagent (Life Technologies) according to
the manufacturer’s procedure and dissolved in DEPC-
treated deionised water as described by Knappskog
et al. (29). The RNA concentration was measured on a
Nanodrop ND1000 spectrophotometer and adjusted to
1 mg/ml.
Single-strand cDNA was synthesised from 4 mg total
RNA using Transcriptor reverse transcriptase (Roche)
according to the manufacturer’s procedure. Both oligoT
(16-mers) and random hexamers were used as primers
in the cDNA synthesis reaction mix.
Table 1 Patient serum hormone levels (pmol/l).
Normal
Before
operation
1 Month
after
operation
1 Year
follow-up
S-oestradiol
(pmol/l)
S-testosterone
(nmol/l)
S-SHBG
(nmol/l)
S-LH (IE/l)
S-FSH (IE/l)
20–130 248 112124
6.7–31.9 7.815.219.0
13–71 504041
0.8–7.6
0.7–11.1
2.8
0.9
26.7
25.4
25.9
29.6
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Table 2 Primers and probes.
Primers/probesSequences
mRNA expression
Total CYP19 F
Total CYP19 R
Total CYP19 probe
RPLP2 F
RPLP2 R
RPLP2 probe
CYP19_exon1.3 F
CYP19_exon1.4 F
CYP19_exon1.7 F
CYP19_exon1 common R
CYP19_exon1 common probe
CYP19_exonII F
CYP19_exon3 R
CYP19_exonII probe
COX2 F
COX2 R
COX2 probe
SF-1 F
SF-1 R
SF-1 probe
LRH-1 F
LRH-1 R
LRH-1 probe
Gene copy number analysis
CYP19 gDNA F1
CYP19 gDNA R1
CYP19 gDNA probe 1
CYP19 gDNA F2
CYP19 gDNA R2
CYP19 gDNA probe 2
CYP19 PII gDNA F1
CYP19 PII gDNA R1
CYP19 PII gDNA F2
CYP19 PII gDNA R2
CYP19 PII gDNA probes 1 and 2
CYP19 PII gDNA F3
CYP19 PII gDNA R3
CYP19 PII gDNA probe 3
B2M gDNA F
B2M gDNA R
B2M gDNA probe
PCR/sequencing of CYP19 PII and SF-1 promoter area
CYP19 PII PCR F1
CYP19 PII PCR R1
CYP19 PII Seq F2
CYP19 PII Seq R2
CYP19 PII Seq F3
CYP19 PII Seq R3
SF-1 F2
SF-1 R8
SF-1 F4
SF-1 R7
SF-1 F6
SF-1 R2
SF-1 R9 (seq)
SF-1 R10 (seq)
SF-1 F6 (seq)
SF-1 F8 (seq)
SF-1 F9 (seq)
SF-1 F10 (seq)
50-ATCCTCAATACCAGGTCCTGGC
50-AGAGATCCAGACTCGCATGAATTCT
50-6FAM-ACCCGGTTGTAGTAGTTGCAGGCACT-BBQ
50-GACCGGCTCAACAAGGTTAT
50-CCCCACCAGCAGGTACAC
50-Cy5-AGCTGAATGGAAAAAACATTGAAGACGTC-BBQ
50-GTCTAAAGGAACCTGAGACTCTACC
50-CACTGGTCAGCCCATCAA
50-AGAAAGGGGTGAAATCAGCAA
50-ACGATGCTGGTGATGTTATAATGT
50-6FAM-TCGGGTTCAGCATTTCCAAAACCAT-BBQ
50-CCCTTTGATTTCCACAGGAC
50-CCCATGCAGTAGCCAGGAC
50-6FAM-CATGCCAGTCCTGCTCCTCA-BBQ
50-ATCACAGGCTTCCATTGACC
50-CAGGATACAGCTCCACAGCA
50-6FAM-CCGCAAACGCTTTATGCTGA-BBQ
50-TCATCCTCTTCAGCCTGGAT
50-AGGTACTCCTTGGCCTGCAT
50-6FAM-ACGCTCAGGAGAAGGCCAAC-BBQ
50-GCACAGGAGTTAGTGGCAAA
50-CTGCTGCGGGTAGTTACACA
50-6FAM-AACAAGTCAATGCCGCCCT-BBQ
50-TTATGGGAGCCTGGTAGTGG
50-AGGGTGGGGTTTTCTATTGG
50-6FAM-TTGGAACATCATACCCCGGG-BBQ
50-GGTGGCTTTTGCTGATCAAT
50-AGAAAATGGGCACGAAACTG
50-6FAM-AGCTCCTGCCAGCTCCCTTT-BBQ
50-TTGGTCAAAAAAGGGGAGTTG
50-ATCATCTTGCCCTTGAGTGG
50-TCCACCTCTGGAATGAGCTT
50-TTGCAGCATTTCTGACCTTG
50-6FAM-CTTTCAATTGGGAATGCACG-BBQ
50-GGGCTTCCTTGTTTTGACTT
50-GAGGGGGCAATTTAGAGTCC
50-6FAM-CACCCTCTGAAGCAACAGGA-BBQ
50-CATCCAGCAGAGAATGGAAAG
50-GAAAGACCAGTCCTTGCTGAA
50-Cy5-TGGGTTTCATCCATCCGACA-BBQ
CTCAACGATGCCCAAGAAAT
CATGGACCAAAATCCCAAGT
TTGGTCAAAAAGGGGAGTTG
CTTACCTGGTATTGAGGATGTGC
GGCAAGAAATTTGGCTTTCA
GAGGGGGCAATTTAGAGTCC
TCTGCCTCCCAGGTTCAAGC
GGTGACTGGATCTTTTGTGTGTTCCTC
GAGGAACACACAAAAGATCCAGTCACC
CTTGGCCAGCTGGCTGTTG
TGTCCTGACTCTACTCCAATGTCCG
AACCCCTGAGAAACAAGAGCATCTG
TGTGTGTGTTCCTCTGTTTGTCTCAC
GGGCTTGATTTATGGGCTGCTC
TGTCCTGACTCTACTCCAATGTCCG
CACCAACAAAGAAGGCGAGAGG
TCTGTCCCCCACCTGAGTTTC
CCACTGCCACCCTCATCC
Elevated SF-1 and CYP19 in testicular LCT
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DNA extraction
Genomic DNA was extracted from snap-frozen biopsies
using QIAamp DNA Mini Kit (Qiagen) according to the
manufacturer’s procedure.
Quantitative PCR
Transcript levels of total CYP19, promoter-specific
CYP19 transcript variants, COX2, SF-1 and LRH-1,
were quantified using the Lightcycler 480 instrument
(Roche). Ribosomal protein P2 (RPLP2) mRNA level
was used as reference. The amplification primers and
BlackBerry-quenched hydrolysis probes used are listed
in Table 2.
Amplification was performed in a 20 ml reaction
solution using the LC480 Probes Master (Roche)
reaction mix, 0.5 mM of each primer, 0.125 mM
hydrolysis probe and 0.5 ml cDNA synthesised from
4 mg total RNA. The following thermocycling conditions
were used: initial denaturation/enzyme activation at
95 8C for 5 min and then 50 cycles of 95 8C for 10 s
(denaturation) and 55 8C for 20 s (annealing/elonga-
tion) before a final cooling step at 40 8C for 5 s. Negative
controls (water) were included in each run.
The results were converted into relative concen-
trations using an in-run standard curve, and then
normalised for RPLP2 mRNA levels. All gene-specific
data from the tumour sample were compared with the
corresponding data from a reference sample consisting
of pooled total RNA from healthy testis of five donors
(BioChain, Newark, CA, USA).
The expression levels of the promoter-specific tran-
script variants were assessed using different primers and
assay-specific standard curves (i.e. different qPCR
efficiencies), and the data are therefore not directly
comparable. However, an estimate of the differences in
expression of these promoters can be made by assuming
that all reactions run with the same efficacy (effi-
ciencyZ2) and comparing the delta crossing points of
the different assays.
Gene copy number analysis (qPCR)
In order to investigate possible gene copy number
changes of the CYP19 gene and the CYP19 PII, levels of
genomic DNA were quantified in duplex reactions with
beta-2-microglobulin (B2M) as reference using the
Lightcycler 480 instrument (Roche). Two distinct
CYP19 genomic areas and three distinct areas
covering the CYP19 PII region were amplified using
the primers and BlackBerry-quenched hydrolysis
probes listed in Table 2. Amplification was performed
in a 20 ml reaction solution using the LC480 Probes
Master (Roche) reaction mix, 0.5 mM of each primer,
0.125 mM of each hydrolysis probe and 2 ml gDNA as
template. The following thermocycling conditions were
used: initial denaturation/enzyme activation of 95 8C
for 5 min and then 50 cycles of 95 8C for 15 s
(denaturation) and 55 8C for 20 s (annealing/elonga-
tion) before a final cooling step at 40 8C for 5 s. Negative
controls (water) were included in each run. The data
obtained through quantification were normalised by
adjusting for B2M levels. These normalised values were
divided by the corresponding values from a reference
sample (pooled DNA from six healthy donors). The
concentration of the reference was set to 1.0, and we
considered the samples to have reduced copy number if
the sample/reference ratio was !0.65 and increased
copy number if the ratio was O1.35.
PCR amplification and sequencing
The CYP19 PII and SF-1 promoter regions were
amplified and sequenced using primers listed in
Table2.TheSF-1genehastwoalternative,untranslated
first exons. Relative to the transcription start sites of
thesetwoexons,wesequencedtheareaK1475/C1053
(alternative 1) and K1650/C858 (alternative 2).
PCR amplification was performed using the Kod XL
DNA polymerase system (Merck) in a 50 ml reaction mix
containing 1! PCR buffer, 0.2 mM of each deoxynu-
cleotide triphosphate, 0.2 mM of each primer, 1.25 U
Kod XL DNA polymerase and 1 ml genomic DNA. The
thermocycling conditions used were an initial
denaturation step of 5 min at 94 8C, followed by 30
cycles of denaturation (94 8C for 1 min), annealing
(CYP19 PII: 54.9 8C and SF-1: 63.2 8C for 10 s) and
elongation (72 8C for 30 s) and a final elongation step of
7 min at 72 8C. Following amplification, the PCR
product was treated with ExoSAP-IT (USB Products,
Affymetrix, Inc., Santa Clara, CA, USA) at 37 8C for
30 minand80 8Cfor15 minaccordingtotheprocedure
as outlined by the manufacturer. DNA sequencing
was performed in a 10 ml reaction mix containing 1!
Figure 1 Standard H&E-stained section (5 mm) of testicular Leydig
cell tumour. The intra-testicular tumour (largest diameter of 1.8 cm)
was sharply delimited from the testicular parenchyma. The tumour
cells had abundant, deeply acidophilic, finely vacuolised cytoplasm
with focally deposited brownish yellow lipochrome pigment and
intracytoplasmic Reinke’s crystalloids. Scale barZ50 mm.
944
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sequencing buffer, 1 mM primer and 1! BigDye v.1.1
(Applied Biosystems, Carlsbad, CA, USA). Capillary
electrophoresis was performed on an automated DNA
sequencer (ABI 3730).
Multiplex ligation-dependent probe
amplification
The multiplex ligation-dependent probe amplification
(MLPA) method and the SALSA MLPA kit P185-B1 lot
0308 (MRC Holland, Amsterdam, The Netherlands)
were used according to the manufacturer’s instructions
to identify deletions or amplifications in the SF-1 gene
(NR5A1) localised on 9q33.3. Capillary electrophoresis,
data collection and peak analysis were performed on an
automated DNA sequencer (ABI 3730). In the patient
sample, the peak areas of all MLPA products resulting
from SF-1-specific probes were first normalised by the
average of peak areas resulting from control probes
specific for locations other than on chromosome
9q33.3. A ratio was then calculated where this
normalised value was divided by the corresponding
value from a sample consisting of pooled DNA from six
healthy individuals. A sample was scored as having a
reduced copy number at a specific location if this ratio
was below 0.75 and as having an increased copy
number if the ratio was above 1.25.
Results
Histology
The intra-testicular tumour (largest diameter of 1.8 cm)
was sharply delimited from the testicular parenchyma
with a non-infiltrative ‘pushing’ border and a solid/
nodular growth pattern. The tumour cells had
abundant, deeply acidophilic, finely vacuolised cyto-
plasm with focally deposited brownish yellow lipo-
chrome pigment and intracytoplasmic Reinke’s
crystalloids, as displayed by H&E staining in Fig. 1. No
malignant histological features were observed, i.e.
tumour diameter !5 cm, lack of infiltrating border,
few mitoses (mean 1 per 10 high power fields), absence
of necrosis, no vascular invasion and low tumour cell
proliferation assessed with Ki-67/MIB-1 (1–2%). Immu-
nohistochemically, vimentin and inhibin were strongly
co-expressed and cytokeratin AE1/3 showed weak and
focal positivity, typical for this entity. The epithelial
marker (EMA) and germ cell markers (AFP, PLAP) were
negative. PRwas weakly positive in about 5% of tumour
nuclei and oestrogen receptor alpha (ERa) was negative.
CYP19 expression level in the tumour tissue
We examined the CYP19 expression level and found it
to be strongly up-regulated (125-fold) in the tumour
sample compared with normal testis tissue (Fig. 2A).
Then, we determined promoter usage by quantifying
CYP19 transcript variants specific for promoter PII, 1.3,
1.4 and 1.7 in both tumour and normal tissues (Fig. 2B,
C, D and E). The expression level of the PII-specific
CYP19 transcript in the tumour sample was 59-fold
elevated relative to normal testis tissue. In addition, we
found the transcript variants specific for promoters 1.3
and 1.7 to be up-regulated in the tumour tissue. The
transcript related to promoter 1.3 displayed an 11-fold
increase from normal to tumour tissue while the levels
of the variant specific for promoter 1.7 became
detectable in the tumour sample contrasting undetect-
able levels (after 50 amplification cycles) in the normal
sample. Interestingly, the transcript variant specific for
CYP19 Total
A
TN
0.0
2.0×10–1
4.0×10–1
6.0×10–1
8.0×10–1
1.0
1.2
1.4
1.6
1.2×10–2
CYP19 PII
BC
2.5×10–5
CYP19 P1.3
TNTN
1.0×10–2
8.0×10–3
6.0×10–3
4.0×10–3
2.0×10–3
0.0
0.0
5.0×10–6
1.0×10–5
1.5×10–5
2.0×10–5
1.4×10–11
1.2×10–11
1.0×10–11
8.0×10–12
6.0×10–12
4.0×10–12
2.0×10–12
0.0
CYP19 P1.4 CYP19 P1.7
DE
TNTN
1.4×10–5
1.2×10–5
1.0×10–5
8.0×10–6
6.0×10–6
4.0×10–6
2.0×10–6
0.0
Figure 2 mRNA levels of total CYP19 and CYP19 promoter-specific
transcript variants. Transcript levels of total aromatase (CYP19) and
promoter-specificCYP19transcript variants weredeterminedinboth
tumour (T) and normal (N) testes with qPCR. Triplicate runs were
performed, and expression of the ribosomal protein P2 (RPLP2) was
used as reference in all the reactions. Individual in-run standard
curves were used in the five different assays and the relative
concentrations as displayed on the y-axes are therefore not
comparable between the assays A, B, C, D and E. We observed
an increased transcript level of (A) total CYP19 (125-fold),
(B) promoter II-specific CYP19 (59-fold), (C) promoter 1.3-specific
CYP19(11-fold)and(E)promoter1.7-specificCYP19(notdetectedin
the normal sample). (D) We observed a down-regulation of promoter
1.4-specific CYP19 transcript level (tumour to normal ratio: 0.4).
Elevated SF-1 and CYP19 in testicular LCT
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promoter 1.4 was down-regulated in the tumour tissue
(tumour to normal tissue ratio: 0.4).
Three runs were performed per promoter assay, and the
curves shown in Fig. 3 are representative for these runs.
ThecurvesforRPLP2wereremovedfromFig.3forclarity.
WhenassumingthattheqPCRsrunwiththesameefficacy,
we found the tumour CYP19 mRNA levels deriving from
promoter PII to be 77-, 1233- and 7236-fold higher than
from promoters 1.3, 1.7 and 1.4 respectively.
This finding strongly indicates that PII-transcribed
mRNA accounts for the majority of the total CYP19
mRNA detected. Moreover, this finding reveals PII to be
the principal promoter in both normal and tumour
tissue, arguing against the use of an alternative
promoter as an explanation for elevated CYP19 levels.
CYP19 coding region and PII gene copy
number
Addressing the potential causes for CYP19 over-
expression, we analysed the gene copy number for
CYP19 using qPCR. No amplifications within the
CYP19 locus were observed.
In addition, due to the large contribution from PII, we
speculated whether CYP19 up-regulation could be due
to either a selective amplification of the PII area or to an
activating mutation located in this promoter area.
Hence, we performed a copy number analysis speci-
fically for the region harbouring this promoter and
sequenced the PII region. No promoter amplification,
mutations or polymorphisms were detected.
CYP19 protein staining
In order to validate the data obtained by qPCR, we
analysed CYP19 at the protein level by immunohis-
tochemistry (IHC). Anti-CYP19 (Aro 677)-stained
sections revealed focal strong cytoplasmic staining of
tumour Leydig cells and negative staining of normal
tissue sections (Fig. 4).
The transcription factor SF-1 is up-regulated
in the tumour
Based on the lack of copy number changes within the
CYP19 gene and PII, and/or mutations in PII, we
hypothesised that the CYP19 over-expression could be
9.154
8.254
7.354
6.454
5.554
4.654
3.754
2.854
1.954
1.054
0.154
A
B
8.174
7.374
6.574
5.774
4.974
4.174
3.374
2.574
1.774
0.974
0.174
Fluorescence (498–580)
Fluorescence (498–580)
5 10 152025
Cycles
Cyp19 total
Cyp19 total
pII
pII
p1.3
p1.3
p1.4
p1.7
p1.7
p1.4
30354045
510152025
Cycles
303540 45
Figure 3 qPCR-crossing points (Cps) for total CYP19 and CYP19
promoter-specific transcript variants. The qPCR-Cp values for each
of the promoters examined were compared in a common run. Three
runs were performed per promoter assay, and the curves shown
here are representative for these runs. All CYP19 assays were run
with RPLP2 as a reference; however, the curves for RPLP2 were
removed from the figure for clarity. The Cps strongly indicated that
the main contribution to the total CYP19 mRNA level was from PII.
(A) Tumour sample, (B) normal tissue sample.
A
B
Figure 4 Anti-CYP19 staining of tumour and normal tissue. Anti-
CYP19 (Aro 677) stained sections showed (A) strong cytoplasmic
staining for tumour Leydig cells, and (B) negative staining in normal
tissue. Scale bar: 100 mm.
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caused by increased levels of a trans-acting factor.
Regarding the structure of PII and its known binding
sites for different transcription factors, we analysed the
mRNA levels of three potential ligand candidates: COX2,
LRH-1 and SF-1. COX2 and LRH-1 were down-
regulated in the tumour compared with normal tissue
(tumour to normal ratios of 0.04 and 0.08 respectively).
In contrast, we found the mRNA level of SF-1 to be 6.6-
fold up-regulated in the tumour tissue compared with
the normal sample (Fig. 5).
In order to validate the data obtained by qPCR, we
analysed SF-1 at the protein level by IHC. Anti-SF-1-
stained sections showed strong nuclear staining in
tumour tissue and negative staining in normal tissue
(Fig. 6).
Finally, we explored potential causes of SF-1 over-
expression by analysing the copy number of this gene
using MLPA, as well as sequencing the promoter region
for potential activating mutations. No promoter
mutations, amplifications or deletions of SF-1 were
observed.
Discussion
While most LCTs reveal benign characteristics, the
elevated steroid production may cause disturbing
virilising or feminising symptoms.
In this report, we describe a patient with an
oestrogen-producing LCT revealing elevated CYP19
and SF-1 mRNA levels. While some previous papers
reported elevated oestrogen production in LCTs to be
related to enhanced CYP19 mRNA levels (17, 18, 19,
20, 21), the underlying cause of elevated CYP19 mRNA
in these tumours has not been reported so far. The
results presented here provide further insight into the
mechanism(s) of CYP19 up-regulation in LCTs.
First, we ruled out gene amplification of the CYP19
gene as a potential cause of mRNA up-regulation.
Secondly, we evaluated the use of CYP19 promoters.
While the CYP19 enzyme is coded for by a single gene,
it is subject to tissue-specific regulation due to several
alternative promoters (16). Further, there is evidence
that CYP19 expression may be differentially regulated
in tumours compared with their normal tissue of origin.
Taking breast cancer as an example, CYP19 transcrip-
tion in normal breast tissue is mainly regulated by
promoter 1.4, while transcription in breast cancer
tissue is regulated by the promoters II, 1.3, 1.7 and 1.4
(30). Here, we found that the majority of the CYP19
mRNA transcripts in both normal and tumour tissue
originate from PII. Thus, the elevated CYP19 mRNA
level observed in the tumour tissue was not due to
alternative promoter use, in conformity with what has
been described earlier in a few other cases (18, 20).
Thirdly, we measured intra-tumour mRNA levels of
the ligands SF-1, LRH-1 and COX2. We found elevated
mRNA levels of SF-1 in tumour compared with normal
tissue, a finding validated by the observation of elevated
SF-1 protein staining by IHC. While our results are in
accordance with findings in experimental systems (22),
to the best of our knowledge, SF-1 up-regulation in
LCTs has not been demonstrated in humans earlier.
8.00
7.00
6.00
5.00
4.00
mRNA-radtio:
Tumour/normal relative to RPLP2
3.00
2.00
1.00
0.00
SF-1COX2LRH-1
Figure 5 The mRNA levels of SF-1, COX2 and LRH-1 (tumour/
normal tissue ratio) were analysed by qPCR. COX2 and LRH-1
were down-regulated in the tumour compared with normal tissue
(tumour to normal ratios of 0.04 and 0.08 respectively). In contrast,
we found the mRNA level of SF-1 to be 6.6-fold up-regulated in the
tumour tissue compared with the normal sample.
A
B
Figure 6 Anti-SF-1 staining of tumour and normal tissue. Anti-SF-1-
stained sections showed strong nuclear staining in tumour tissue
(A) and negative staining in normal tissue (B). Scale bar: 100 mm.
Elevated SF-1 and CYP19 in testicular LCT
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Notably, the SF-1 mRNA up-regulation was neither
due to NR5A1 gene amplification nor due to activating
mutations in the promoter area. Moreover, we found the
expression of LRH-1 and COX2 to be significantly down-
regulated in tumour tissue compared with normal
tissue. LRH-1, like SF-1, has been demonstrated to bind
a nuclear receptor half site in PII (31) and regulate
CYP19 expression in Leydig cells (23, 25). Our
observation of SF-1 up-regulation in concert with
LRH-1 down-regulation might indicate that there is a
negative feedback loop controlling the expression of
these genes.
In previous studies, local COX2 up-regulation has
been discussed as a potential mechanism of elevated
oestrogen synthesis in inflammation and some cancers
(reviewed in (32)), but we lack evidence confirming this
in vivo. To the best of our knowledge, this is the first
report to demonstrate a similar effect on oestrogen
synthesis through local SF-1 up-regulation.
In summary, we found elevated oestrogen synthesis
in an LCT to be caused by elevated PII-transcribed
CYP19 levels. Moreover, we found the enhanced CYP19
transcription to be most likely caused by elevated levels
of the transcription factor SF-1. Interestingly, CYP19
up-regulation in oestrogen-dependent breast cancer has
been attributed to enhanced PII activity (30). Our
findings reveal pathological SF-1 up-regulation to be a
potential mechanism enhancing local oestrogen
synthesis in LCTs, suggesting this mechanism to be
explored as a potential cause of CYP19 up-regulation in
other pathological conditions as well.
Declaration of interest
The authors declare that there is no conflict of interest that could be
perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by grants from the Norwegian Cancer
Society and the Norwegian Health Region West. A H Straume is the
recipient of a PhD grant and S Knappskog received his postdoc
fellowship from the Norwegian Cancer Society. All the laboratory work
was performed in Mohn Cancer Research Laboratory.
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Received 3 October 2011
Revised version received 20 January 2012
Accepted 1 February 2012
Elevated SF-1 and CYP19 in testicular LCT
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