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Institute of Endocrine Sciences, University of Milano 20122, Ospedale Maggiore IRCCS (E.B., A.G.L., M.F., S.C., S.M., M.A., P.B.-P., G.F., A.S.), 20122 Milan; and Istituto Auxologico Italiano IRCCS (L.P., E.G.), 20145 Milano, Italy
Address all correspondence and requests for reprints to: Anna Spada, M.D., Istituto di Scienze Endocrine Ospedale Maggiore, IRCCS, Via Francesco Sforza 35, 20122 Milano, Italy. E-mail: anna.spada{at}unimi.it
Abstract
Introduction of somatostatin analogs has greatly contributed to
improving the prognosis of acromegaly. Although the majority of
patients are effectively treated by these agents, resistance occurs in
a subset of patients. So far, resistance to somatostatin has never been
associated with mutations of the somatostatin receptor subtypes (sst2
and sst5) that inhibit GH secretion. Molecular analysis of genomic DNA
from pituitary tumor and peripheral blood obtained from an acromegalic
resistant to octreotide showed a somatic activating mutation of Gs
(Arg201Cys), no mutation in sst2, and one polymorphism (Pro109Ser) and
one germ line mutation (Arg240Trp) in sst5. Wild-type (WT) and mutant
sst5 PCR products were cloned and transfected into Chinese hamster
ovary K1 cells. In Chinese hamster ovary K1 cells stably expressing
mutant sst5, somatostatin-28 was less potent in inhibiting cyclic AMP
levels than in WT cells. Proliferation of mutant cells exceeded that of
WT by 50%. Moreover, somatostatin reduced cell growth and MAPK
activity in WT but not in mutant cells in which the peptide even
increased MAPK activity. We suggest that this mutation that abrogates
the antiproliferative action of somatostatin and activates mitogenic
pathways may be involved in the resistance to somatostatin
treatment.
ACROMEGALY IS A chronic disease characterized by elevated GH and IGF-I levels, generally owing to a GH-secreting pituitary adenoma. When inadequately treated, acromegaly results in significant morbidity and greatly increased mortality (1, 2, 3). Because only about 50% of patients are effectively cured by the surgical removal of the adenoma (4, 5), medical therapy has an important role in the management of these patients (6, 7). The introduction of new drugs, especially somatostatin analogs, has greatly contributed to improve the prognosis of acromegaly (6, 7, 8, 9, 10).
Somatostatin is the hypothalamic peptide that physiologically inhibits GH secretion. The peptide binds to a family of five specific G protein-coupled receptor subtypes (sst1–5) with important differences in tissue distribution, coupling to second messengers, and affinity for somatostatin and its analogs (11, 12, 13). The commercially available long-acting somatostatin analogs, such as octreotide and lanreotide, bind with high affinity to sst2, sst5, and to a lesser extent sst3 (11, 12, 13). Activation of these receptors generates a series of intracellular events, including inhibition of adenylyl cyclase and reduction of cytosolic calcium (11, 13). Moreover, somatostatin exerts direct antiproliferative effects on cell lines by triggering still not completely defined signals (10, 13, 14, 15, 16, 17, 18, 19). In particular, in cells expressing sst2 and sst5, somatostatin reduces the activity of MAPK, a family of protein serine/threonine kinases considered key molecules in transducing growth factor proliferative signals (13, 17, 18).
Somatostatin analogs lower GH levels in about 90% of acromegalic
patients, although IGF-I and GH levels low enough to be considered safe
in respect to normalization of life expectancy are achieved in only
about 60% of patients (7, 8, 9, 10). In addition, somatostatin
analogs block the growth of the tumoral mass and may indeed slightly
reduce tumor size in up to 50% of patients (10). The
molecular basis of the different sensitivity to somatostatin remains
largely unknown. There is a general agreement that patients harboring
tumors with mutations of the Gs gene that constitutively activate
adenylyl cyclase (referred to as gsp mutations)
(20) are highly sensitive to the inhibitory action of
somatostatin (21, 22). Conversely, because no mutations of
sst2 and sst5 genes have so far been reported (23),
resistance has been attributed to a reduced expression of sst2 or sst5
(24, 25, 26).
In the present study, we report the first mutation of the sst5 gene that abrogates the antiproliferative action of somatostatin and activates mitogenic pathways in one acromegalic patient resistant to somatostatin analog octreotide and carrying gsp oncogene.
Case report
The patient, a 49-yr-old women, was admitted to our clinic in 1997
because of signs and symptoms of acromegaly that was confirmed by
hormonal data (Table 1
). Magnetic
resonance imaging revealed a 10-mm pituitary adenoma. The patient
underwent pituitary surgery with only partial removal of the tumor. The
patient was then treated with the depot long-acting somatostatin analog
octreotide LAR 20 mg every 28 d for 6 months and then increased to
30 mg every 28 d for another 6 months. During this period multiple
hormonal determinations according to standard protocols revealed no
modifications of GH and IGF-I levels (Table 1). Also, serum insulin,
whose concentrations are known to be reduced by somatostatin analogs,
did not change during treatment, both in fasting state (Table 1) and
after oral glucose tolerance test (3-h AUC: before therapy 5196
vs. 4743 µU/min per milliliter during therapy). After 6
months of therapy, no shrinkage of the tumor remnant was observed at
magnetic resonance imaging. Thus, octreotide therapy was interrupted,
and the patient was then given cabergoline, a long-acting
dopaminergic agonist, that caused a clear reduction of GH and IGF-I
levels after 6 months at the dose of 3 mg/wk. The patient is actually
under dopaminergic treatment. Written informed consent was obtained
from the patient after the study had been approved by the local ethical
committee.
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DNA and RNA extraction and sequencing
Genomic DNA and RNA were extracted according to standard
procedures, as previously described (27). The sst2 and
sst5 genes were amplified by PCR using the following primers pairs:
5'-CTGGCTGGAACTAGCCTAAGACT-3' and 5'-GACAGAGCATGGCTTGGTTCTTT-3',
5'-TCTTCTCTTGCAGAGCCTGA-3' and 5'-CGCAGTGCAACCTCCGCTCCTGGGGGTGA-3',
respectively. These primers identified sequences of sst2 and sst5
genes of 1184 and 1194 bp, respectively. A 50-µl reaction mix (0.5
µg of DNA sample, 50 nmol/l KCl, 50 nmol/l Tris-KCl (pH 8.3), 1
nmol/l MgCl2, 25 pmol of each primer, and 2.5 U Taq DNA
polymerase AmpliTaq (Perkin-Elmer Corp., Foster City, CA)
was subjected to denaturation at 94 for 3 min, followed by 35 cycles of
94 C for 60 s, 62 C for 60 s, and 72 C for 1 min. A final
cycle at 72 C for 10 min was carried out to allow complete extension of
the amplified fragments. The PCR products were sequenced from both
strands with an automatic technique. Total RNA was reverse transcribed
and PCR performed on the entire cDNA product with Taq DNA
polymerase and specific primers for sst5. Genomic DNA from the tumor
was analyzed for the presence of gsp mutations by amplifying
the hot spots of the Gs gene, as previously described
(27).
Cloning and transfection of the human sst5 gene
WT and mutant sst5 PCR products were directly cloned using the
Eukaryotic TA cloning kit (Invitrogen, Carlsbad, CA) in
two mammalian expression vectors pCR3.1. The two resulting vectors were
separately transfected into Chinese hamster ovary (CHO) cells (CHOK1
strain) using the calcium phosphate precipitation method. Stably
transfected cells were selected in modified Eagle’s medium (MEM)
with 10% FCS using geneticin 500 µg/ml (Sigma, St.
Louis, MO) and surviving colonies were subcloned and expanded. For each
vector transfected, three clones expressing the WT (WTsst5/15,
WTsst5/20, and WTsst5/25) and mutant (R240Wsst5/6, R240Wsst5/26, and
R240Wsst5/27) receptors were selected by binding studies. CHOK1 cells
were concomitantly transfected with the vector devoid of sst5 and used
as control clone.
Semiquantitative determination of sst2 and sst5 gene transcripts
Total RNA was extracted from the tumoral tissue and the levels of sst5 RNA transcripts were evaluated by semiquantitative RT-PCR using appropriately selected primer pairs, as previously described (27). The sequences of the oligonucleotides used for the amplification of sst2 and sst5 genes are available on request to the author. Data were compared with those of eight GH-secreting adenomas removed from patients responsive to octreotide. The glyceraldehyde phosphate dehydrogenase (GAPDH) gene was used as internal standard. Preliminary experiments were conducted to determine the PCR cycles corresponding to the exponential phase for both genes. Results were expressed as the ratio of the radioactivity of each fragment for sst genes to that of GAPDH.
Membrane preparation and binding studies
For membrane preparation, CHOK1 cells were grown in 10-cm
dishes, and binding studies were performed on cell lysates as
previously described, with minor modifications (28).
Briefly, membrane preparations (20 µg/tube) resuspended in
50-mM HEPES, 0.1 mM CaCl2, 10 mM
MgCl2, protease inhibitors, and 0.5% BSA were incubated with
(125ITyr11) somatostatin-14 (Amersham Pharmacia Biotech,
Aylesbury, United Kingdom) at 25 C for 120 min with and without 1
µM somatostatin-28 (Sigma) in triplicate.
Bound ligand was trapped by vacuum filtration with a Millipore Corp. (Bedford, MA) cell harvester through Watman GF/C filters
(Clifton, NJ). The filters were immediately washed with a 15-ml buffer
and then counted on a 
-counter.
cAMP measurement
For intracellular cAMP assay, CHO cells (at 8 x 105 in 6-well plastic plates) were preincubated with isotonic HBSS (Sigma) containing 0.4% BSA and 0.5 mM 3-isobutyl-1-methylxanthine, washed, and subsequently exposed for 30 min at 37 C to 1 µM forskolin with or without somatostatin-28. Cells were extracted in cold ethanol after 12–24 h at -20 C, lyophilized, and assayed by enzymatic immunoassay (Amersham Pharmacia Biotech) as previously described (29).
Cell growth assay
CHO (1 x 105) were cultured in
MEM containing 10% FCS and plated in 35-mm dishes. After 24-, 48-,
and 72-h incubation, the number of WT or mutant cells was evaluated
using a Coulter counter. To study the effect of somatostatin on cell
growth, cells were cultured in MEM containing 10% FCS with or
without 10 nM octreotide (provided by Novartis Spa, Basel, Switzerland) and counted after 24-h incubation.
Western blot analysis of phospho-44/42- MAPK and MAPK activity
Cells (1 x 106) serum starved for
18 h were incubated with MEM containing 10% FCS with or
without 1 nM somatostatin-28 for 10 min. Lysates were
collected in the presence of protease and phosphatase inhibitors and
protein concentrations determined by BCA protein assay kit
(PierceChemical, Rockford, IL). Western blot of
phospho-44/42- MAPK was performed using an anti-phospho-44/42- MAPK
(ERK-1/2) polyclonal antibody diluted 1:1000 (New England Biolabs, Inc., Beverly, MA) and detected by chemiluminescent
method. MAPK activity was measured on lysates immunoprecipitated with
anti-phospho-44/42MAPK antibody and incubated with Elk-1 fusion protein
and kinase buffer for 30 min. Western blot was performed using
anti-phospho-Elk1 antibody. Bands were evaluated by an imaging
densitometer (GS-700, Bio-Rad Laboratories, Inc.,
Richmond, CA).
Statistics
The results are presented as the mean ± SD. Statistical significance was determined by t test. Statistical significance is considered for P < 0.05.
Results
Genomic DNA and cDNA from the adenoma were analyzed for mutations
in the genes encoding Gs, sst2, and sst5. By analyzing the hot spots
of the Gs gene, a heterozygote point mutation at codon 201 (CGT
> TGT/Arg201Cys), known to constitutively activate adenylyl cyclase
(20), was identified. By fully sequencing the sst2 gene,
no nucleotide substitution in the coding or bordering regions was
found, whereas two heterozygote C-to-T substitutions were detected in
the sst5 gene. One replaced Pro with Ser (CCC > TCC) at codon 109
in the first extracellular loop, and the other replaced Arg with Trp
(CGG > TGG) at codon 240 in the third intracellular loop of the
receptor (Fig. 1). Direct sequencing of
PCR-amplified cDNA from the tumor revealed WT and mutant bases at about
equal intensities. These two substitutions were germ line because they
were also identified in the peripheral DNA of the patient. By direct
sequencing PCR products generated from 50 unaffected control subjects,
Pro109Ser substitution resulted to be an unreported polymorphism
because it was found in 3 of 100 normal alleles, and Arg240Trp resulted
to be a mutation because it was detected only in the patient.
Subcloning of the patient genomic DNA showed that the polymorphism was
present in cells expressing sst5 with the Arg240Trp mutation and not in
cells expressing the WT receptor, indicating that one sst5 allele had
both the mutation and the polymorphism. Owing to the presence of a germ
line mutation, we planned to screen the patient’s family.
Unfortunately, she was the only child of two unrelated parents who died
of cardiovascular diseases. Genomic DNA from peripheral leukocytes of
her only daughter, a healthy 25-yr-old woman, showed no substitutions
in the sst5 gene.
|
).
Cells expressing WT or mutant receptors showed a different growth rate,
because the proliferation of mutant cells exceeded that of WT by 50%
after 72-h incubation (Fig. 3A).
Incubation with the somatostatin analog octreotide (1 nM
for 24 h) caused a significant reduction of FCS-induced
proliferation of WT cells (25 ± 2% inhibition, P
< 0.001), that was completely prevented by pretreatment with pertussis
toxin (100 ng/ml for 24 h). Conversely, in mutant cells octreotide
did not cause any reduction of growth rate (Fig. 3B). Moreover, in
cells with WT sst5, 1 nM somatostatin-28 induced
a significant inhibition of FCS-stimulated MAPK activities, whereas in
mutant cells the peptide did not reduce but even increased this
activity by 2- to 3-fold. These data were confirmed by detecting the
levels of phosphorylated p44 and p42MAPK by Western blotting (Fig. 4).
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Discussion
In this study we describe the first mutation of a receptor of the
sst family in one acromegalic patient resistant to treatment with the
somatostatin analog octreotide. This mutation, replacing Arg 240 with
Trp in the sst5 gene, is located in the 22-residue region of Lys224 to
Arg245 within the third cytoplasmic loop of the receptor. This region
represents the cytoplasmic extension of the -elix 6 and is a
consensus sequence identifying a potential site for coupling with G
proteins (12, 13). In human and rat sst3–5, Arg is
present at codon 240, whereas in sst1 and sst2, it is replaced by Lys,
another hydrophilic and positively charged amino acid. The replacement
of Arg240 with Trp, an aromatic and highly hydrophobic amino acid, is
predicted to alter critical electrostatic interhelical interactions,
leading to altered G protein coupling. Indeed, several single amino
acid substitutions in other G protein-coupled receptors resulting in
altered signaling have been localized in this motif of the third
cytoplasmic loop (30, 31).
The expression of the mutant receptor in CHOK1 cells resulted in
profound alterations of somatostatin signaling. Although both the WT
and mutant receptors appeared to be effective in inhibiting cAMP
accumulation, the mutant sst5 required 10-fold higher somatostatin
concentrations than the WT to induce the same inhibitory effect. As
already reported (12, 13), in WT cells somatostatin analog
caused a reduction of cell growth by interacting with a pertussis
toxin-sensitive G protein coupling. The antiproliferative effect
mediated by sst5, which has been reported to be independent from cAMP
inhibition and phosphatase stimulation, was associated with an
inhibition of MAPK activity (12, 13, 18). The expression
of mutant receptor totally abrogated the inhibitory action of
somatostatin on cell growth and MAPK activity and was associated with a
higher rate of cell proliferation. Indeed, the mutant receptor
stimulated the serum stimulated MAPK activity, suggesting that
replacement of Arg 240 by Trp confers to the receptor the ability to
couple with G proteins different from those activated by the WT sst5
and able to signal to the MAPK cascade via G (such as Gq) or ß
subunits (32, 33). To the best of our knowledge, this is
the first example of how receptor mutations may alter G protein
coupling, thus changing an inhibitory signal into a stimulatory
input.
The relative role of sst2 and sst5 in the control of GH secretion in
acromegalic patients is still unclear, and both receptors are probably
required for hormonal control because the activation of sst2 and sst5
results in a synergistic effect on GH release (26, 34).
This view is also supported by the observation that these receptors may
form heterodimers with enhanced functionality (35) As far
as the expression of receptor subtypes in GH-secreting adenomas is
concerned, it is well documented that sst5 is the most abundantly
expressed receptor, sst5 mRNA levels being 10-fold higher than sst2
mRNA (26). This ratio was even higher in the present
adenoma, which showed a selective loss of sst2 mRNA, as frequently
occurs in poorly responsive tumors (24, 25, 26). Therefore,
although the phenotype resulting from the expression of mutant sst5 has
been characterized in cells without secretory properties, such as
CHOK1, we suggest that the mutation of sst5 gene together with the low
expression of sst2, may be responsible for the resistance to octreotide
observed in this patient. The presence of a germ-line mutation is
consistent with the phenotype of the patient because the expression of
this receptor is restricted to specific cell types. Indeed, the patient
was resistant to somatostatin at the two target organs in which sst5 is
mainly expressed and involved in hormonal control, that is somatotrophs
and pancreatic ß cells (36). In conclusion, in this
patient we identified two molecular abnormalities occurring on
different loci; the first is the germ-line mutation of sst5 that
abrogates the antiproliferative action of somatostatin and activates
mitogenic pathways, and the second is the somatic mutation of Gs
that constitutively activates cAMP formation, this effect being poorly
counteracted by the mutant receptor.
Acknowledgments
We wish to thank Dr. M. Losa and P. Mortini (Department of Neurosurgery, Scientific Institute San Raffaele, Milan, Italy) for the supply of the tumor and Dr. G. Pietrini (Department of Pharmacology, University of Milan, Milan, Italy) for her advice in procedures relating to transfection and cloning techniques.
Footnotes
This work was supported in part by MURST Grant 9906153187, the Ricerca Corrente Funds of Ospedale Maggiore IRCCS, and Istituto Auxologico Italiano IRCCS. Presented at the 11th International Congress of Endocrinology, October 29–November 2, 2000, Sydney, Australia.
1 E.B. and L.P. contributed equally to this work and should both be
considered first authors. 
Abbreviations: CHO, Chinese hamster ovary; GAPDH, glyceraldehyde phosphate dehydrogenase; sst, somatostatin receptor subtype; WT, wild-type.
Received January 26, 2001.
Accepted April 26, 2001.
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