Low levels of raf kinase inhibitory protein in growth hormone-secreting pituitary adenomas correlate with poor response to octreotide treatment.
ABSTRACT Excessive GH production by pituitary tumors causes acromegaly. Medical treatment of acromegaly with somatostatin analogs (SMSs), like octreotide, is well established, but the clinical effect is variable. One mechanism for octreotide effect is inhibition of the MAPK signaling pathway after binding to the G protein-coupled somatostatin receptor. Nonphosphorylated Raf kinase inhibitory protein (RKIP) binds to and inhibits Raf1 kinase, and thereby attenuates MAPK signaling, whereas phosphorylated RKIP inhibits G protein receptor internalization and degradation due to inhibition of G protein receptor kinase 2. Objective: Our objective was to study RKIP levels in pituitary somatotroph adenomas, and relate them to clinical characteristics and response to octreotide treatment in patients with acromegaly.
RKIP level was analyzed by Western blot of proteins extracted from somatotroph tumors frozen a short time after surgery in 51 patients with active acromegaly. An acute somatostatin test was performed in 46 of the patients, and in 21 the IGF-I level before and 6 months after SMS treatment was available.
The adenoma RKIP level correlated significantly to both the acute and the long-term octreotide responses on serum levels of GH and IGF-I, respectively. In multiple regression analyses, the RKIP level was a significant determinant for both the GH reduction in the acute test and the IGF-I reduction after approximately 6 months.
The RKIP level in somatotroph adenomas seems to be important for the clinical effect of SMS treatment, in which low levels of RKIP correlate to poor clinical response to SMSs.
- SourceAvailable from: Marily Theodoropoulou[Show abstract] [Hide abstract]
ABSTRACT: Somatostatin is a peptide with a potent and broad antisecretory action, which makes it an invaluable drug target for the pharmacological management of pituitary adenomas and neuroendocrine tumors. Somatostatin receptors (SSTR1, 2A and B, 3, 4 and 5) belong to the G protein coupled receptor family and have a wide expression pattern in both normal tissues and solid tumors. Investigating the function of each SSTR in several tumor types has provided a wealth of information about the common but also distinct signaling cascades that suppress tumor cell proliferation, survival and angiogenesis. This provided the rationale for developing multireceptor-targeted somatostatin analogs and combination therapies with signaling-targeted agents such as inhibitors of the mammalian (or mechanistic) target of rapamycin (mTOR). The ability of SSTR to internalize and the development of rabiolabeled somatostatin analogs have improved the diagnosis and treatment of neuroendocrine tumors.Frontiers in Neuroendocrinology 07/2013; · 7.99 Impact Factor
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ABSTRACT: Somatostatin (SRIF), an inhibitory polypeptide with two biologically active forms SRIF-14 and SRIF-28, inhibits growth hormone (GH), prolactin (PRL), thyrotropin (TSH), and adrenocorticotropin (ACTH) secretion in the anterior pituitary gland. Somatostatin also has an anti-proliferative effect inducing cell-cycle arrest and apoptosis. Such actions are mediated through five G-protein coupled somatostatin receptors (SSTR); SSTR1 to SSTR5. In GH-secreting adenomas, SSTR2 expression predominates, and somatostatin receptor ligands (SRLs; octreotide and lanreotide) directed to SSTR2 are presently the mainstays of medical therapy. However, about half of patients show incomplete biochemical remission, but the definition of resistance per se remains controversial. We summarize here the determinants of SRL resistance in acromegaly patients, including clinical, imaging features as well as molecular (mutations, SSTR variants and polymorphisms), and histopathological (granulation pattern, and proteins and receptor expression) predictors. The role of SSTR5 may explain the partial responsiveness to SRLs in patients with adequate SSTR2 density in the cell membrane. In patients with ACTH-secreting pituitary adenomas, i.e. Cushing disease (CD), SSTR5 is the most abundant receptor expressed and tumors show low SSTR2 density due to hypercortisolism-induced SSTR2-down regulation. Clinical studies with pasireotide, a multireceptor-targeted SRL with increased SSTR5 activity, lead to approval of pasireotide for treatment of patients with CD. Other SRL delivery modes (oral octreotide), multireceptor-targeted SRL (somatoprim) or chimeric compounds targeting dopamine D2 receptors and SSTR2 (dopastatin) are briefly discussed.Journal of Molecular Endocrinology 03/2014; · 3.58 Impact Factor
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ABSTRACT: INTRODUCTION: Only one study has evaluated Ki-67 as a predictor of the response to somatostatin analogues (SSA) therapy in acromegaly; however, other predictors like somatostatin receptor type 2 (SSTR2) and cytokeratin pattern expressions were not considered. OBJECTIVE: To evaluate whether the Ki-67 is a predictor of octreotide LAR (OCT-LAR) response in somatotropinomas independent of SSTR2 and cytokeratin expression patterns. METHODS: Protein expression was analyzed by immunohistochemistry. The percentage of cell nuclei that were immunolabeled for Ki-67 and the percentage of cells with positive SSTR2 staining was calculated. SSTR2 expression was considered high when ≥25%, and a cutoff of 2.3% was designated for Ki-67. Tumors were classified as densely or sparsely granulated according to the cytokeratin pattern. RESULTS: Thirty-one somatotropinomas were studied. Fourteen patients (45.2%) were controlled with OCT-LAR therapy. The median Ki-67 LI was higher in patients not controlled with OCT-LAR than in those controlled (1.63 and 0.15, respectively, p=0.002). Higher SSTR2 expression and densely granulated tumors were correlated with control as well (p=0.04 and 0.038, respectively). There was no difference in Ki-67 levels between patients with high and low SSTR2 expression (p=0.651). After multivariate analysis, both Ki-67 and SSTR2 remained statically significant as predictors of OCT-LAR response (p=0.017 and 0.012, respectively). The Ki-67 LI was higher in sparsely than in densely granulated tumors (p=0.047). CONCLUSIONS: Ki-67 is a predictor of response to OCT-LAR in acromegaly, independent of SSTR2 expression and relates to cytokeratin patterns.European Journal of Endocrinology 06/2013; · 3.14 Impact Factor
Low Levels of Raf Kinase Inhibitory Protein in Growth
Hormone-Secreting Pituitary Adenomas Correlate
with Poor Response to Octreotide Treatment
Stine Lyngvi Fougner, Jens Bollerslev, Fahim Latif, John K. Hald, Terje Lund, Jon Ramm-Pettersen,
and Jens Petter Berg
Research Institute for Internal Medicine (S.L.F.), University of Oslo, N-0027 Oslo, Norway; Hormone Laboratory, Aker University Hospital
(F.L., T.L., J.P.B.), University of Oslo, N-0027 Oslo, Norway; and Section of Endocrinology (S.L.F., J.B.), and Departments of Radiology
(J.K.H.) and Neurosurgery (J.R.-P.), Rikshospitalet Medical Centre, N-0027 Oslo, Norway
Context: Excessive GH production by pituitary tumors causes acromegaly. Medical treatment of
acromegaly with somatostatin analogs (SMSs), like octreotide, is well established, but the clinical
after binding to the G protein-coupled somatostatin receptor. Nonphosphorylated Raf kinase
whereas phosphorylated RKIP inhibits G protein receptor internalization and degradation due to
inhibition of G protein receptor kinase 2.
Objective: Our objective was to study RKIP levels in pituitary somatotroph adenomas, and relate
them to clinical characteristics and response to octreotide treatment in patients with acromegaly.
Patients and Methods: RKIP level was analyzed by Western blot of proteins extracted from so-
after SMS treatment was available.
Results: The adenoma RKIP level correlated significantly to both the acute and the long-term
octreotide responses on serum levels of GH and IGF-I, respectively. In multiple regression analyses,
the RKIP level was a significant determinant for both the GH reduction in the acute test and the
IGF-I reduction after approximately 6 months.
Conclusion: The RKIP level in somatotroph adenomas seems to be important for the clinical effect
of SMS treatment, in which low levels of RKIP correlate to poor clinical response to SMSs. (J Clin
Endocrinol Metab 93: 1211–1216, 2008)
GH is secreted from a somatotroph pituitary adenoma. The pri-
mary treatment is transsphenoidal surgery, which in a recent
large study, has been reported to give a total cure rate of 28–
39%, depending on cure definition (2). Therefore, a relatively
large proportion of patients are in need of additional treatment.
cromegaly is a clinical syndrome caused by excessive GH
able for clinical use, octreotide and lanreotide. Both bind with
high affinity to the somatostatin receptor (SSTR) subtype 2
(SSTR2), less affinity to subtype 5, and with low affinity to re-
ceptor subtype 3. Reduced tumor expression of SSTR2 is sug-
gested to explain the lack of response to SMSs in acromegaly.
Studies correlating SSTR2 mRNA levels to octreotide efficacy
have had contradictory results, but most have shown a correla-
tion between receptor mRNA level and octreotide response (3–
Printed in U.S.A.
Copyright © 2008 by The Endocrine Society
doi: 10.1210/jc.2007-2272 Received October 9, 2007. Accepted January 18, 2008.
First Published Online January 29, 2008
Abbreviations: GRK2, G protein-coupled receptor kinase 2; MRI, magnetic resonance im-
and posterior; SMS, somatostatin analog; SSTR2, somatostatin receptor subtype 2.
O R I G I N A LA R T I C L E
E n d o c r i n e C a r e
J Clin Endocrinol Metab, April 2008, 93(4):1211–1216jcem.endojournals.org
8). Recently, we have demonstrated a better octreotide response
in patients with adenomas containing a large proportion of im-
patients exhibited poor response despite a high proportion of
SSTR2a-positive adenoma cells and vice versa.
served and widely expressed protein. It inhibits the MAPK sig-
naling pathway Ras/Raf-1/MEK/ERK by inhibition of Raf-1
phosphorylation and activation (10–12). This intracellular sig-
C phosphorylates RKIP, which then dissociates from Raf-1, and
binds to and inhibits G protein-coupled receptor kinase 2
(GRK2). This inhibition leads to decreased internalization and
degradation of G protein-coupled receptors, and, therefore, a
the MAPK pathway (14). Internalization of the G protein-cou-
pled receptor SSTR2a is shown to be dependent of GRK2-me-
diated phosphorylation of the receptor (15). Moreover, RKIP
way (16). Low levels of RKIP have been associated with an in-
creased propensity of tumors to metastasize and were the stron-
gest predictor for reduced survival in colorectal cancer (17–19).
Loss of RKIP has also been linked to resistance to chemothera-
peutic drugs in prostate and breast cancer cell lines (20).
The purpose of this study was to examine the RKIP levels in
somatotroph pituitary adenomas, and relate this to tumor size
and invasiveness, hormone levels, and to the clinical response to
Subjects and Methods
A total of 51 patients with active acromegaly were included in the
tomy at Rikshospitalet Medical Centre in the period 1996–2006. The
by elevated serum IGF-I compared with the normal reference range of
their age and failure to suppress GH less than 2.5 mU/liter after a 75-g
oral glucose load. In all patients, a pituitary tumor was visualized on a
magnetic resonance imaging (MRI) scan.
One patient had previously undergone transsphenoidal surgery.
There were 15 patients who received treatment with octreotide before
octreotide treatment in ongoing clinical trials. One patient was treated
preoperatively with pegvisomant. None had received radiation therapy
before surgery. Table 1 provides an overview of the study population.
The study was approved by the local ethical committee and con-
ducted according to the Declaration of Helsinki II. Written informed
consent was obtained from all patients.
In 26 patients additional serum was stored at ?80 C until analyzed.
Serum IGF-I was measured by RIA (Nichols Institute, Nijmegen, The
Netherlands) except in the four last included patients (Immulite; Diag-
nostic Products Corp., Los Angeles, CA). The same method was used
when IGF-I levels were compared before and after SMS treatment. Over
methods) were used. At every change of method, cross-calibration was
performed and, if necessary, factorial adjustments introduced. In most
patients, an additional three to seven samples for measurement of GH
were drawn during daytime (0800–1500 h) for calculation of mean GH
level. The acute somatostatin test was performed in 46 of the patients
before medical treatment with a SMS. During the test, one to three mea-
surements of basal GH levels were performed, and two to three analyses
between 2 and 4 h after sc injection of octreotide. The percent reduction
of GH was calculated and nadir GH recorded for each patient.
In addition, serum IGF-I was measured in the stored samples in one
run to minimize between-assay variability and was compared with pre-
used for statistical analysis.
The MRI scan was available for renewed analysis in 46 patients, and
be reevaluated. The measurements were performed by two investigators
the coronal view before and after contrast injection was used for the
used for the measurement of maximal vertical and anteroposterior di-
ameter. For each tumor, the suprasellar, infrasellar, parasellar, anterior,
and posterior (SIPAP) grading score was determined (21). The formula
width ? height ? length ? 0.5 was used to estimate tumor volume (22).
used: noninvasive, maximum SIPAP 1 in the suprasellar extension; bor-
imum 1 in the parasellar extension; and invasive, all adenomas that did
not classify under the specifications for noninvasive or borderline
Shortly after pituitary surgery, adenoma tissue was frozen at ?70 C.
The tissue was homogenized in TRIZOL Reagent (Invitrogen Corp.,
Carlsbad, CA), and protein was extracted following the manufacturer’s
instructions, then precipitated and washed as described in detail previ-
binding procedure using Ultrospec 3300 pro spectrophotometer (Am-
ersham Biosciences, Buckinghamshire, UK).
4–12% BisTris gel (Invitrogen) and blotted onto a polyvinylidene fluo-
ride membrane. Nonfat dry milk in Tris-buffered saline with Tween 20
was used for blocking (5%) and antibody incubation (2.5%). The mem-
was incubated with rabbit polyclonal anti-RKIP antibody (1:2,000; Ab-
cam plc, Cambridge, UK) at 4 C overnight, washed three times, and
Baseline characteristics of the patients
No. of women/men
Tumor volume (cm3)
Serum GH (mU/liter)
Serum IGF-I (nmol/liter)
1.21 (0.59–2.48)1.18 (0.72–4.37)
1.35 (0.91–2.31)1.66 (0.68–2.60)
Unless stated, values are expressed as median (interquartile range).
Fougner et al.Adenoma RKIP Level and Octreotide EfficacyJ Clin Endocrinol Metab, April 2008, 93(4):1211–1216
incubated with the secondary antibody antigoat IgG (1:10,000; Calbio-
chem, San Diego, CA) for 1 h at room temperature. The same protocol
was used for the membrane part containing the largest proteins but with
Louis, MO) and as secondary antibody an antimouse IgG (1:10,000;
incubated with a mouse anti-?-tubulin antibody (1:1000; Sigma-
Aldrich) and with the antimouse IgG as secondary antibody. The
enhanced chemiluminescence Western Blotting Detection Reagents
(Amersham Biosciences) were used for visualization. Protein levels were
adenoma the mean of RKIP/?-actin and RKIP/?-tubulin was designated
the RKIP ratio and used in the analyses.
Most adenomas have previously been examined for SSTR2a protein
Laboratories, Schwabhausen, Germany), standardized against the ?-ac-
tin signal. This procedure has been described in detail previously (9).
Semiquantitative scores of SSTR2a immunohistochemical expres-
multiple regression analysis with RKIP in this study. The SSTR2a scores
were based on less than 75% (grade 1 or 2) or more than 75% (grade 3)
of the cells positively stained by immunohistochemistry (9).
Differences between groups were analyzed using the Mann-Whitney
rank sum test and Kruskal-Wallis test when more than two groups were
compared. Relationships between variables were tested by Spearman
correlation analysis. Multiple linear regression analysis was performed
with stepwise addition of the variables that had P values less than 0.1 in
the univariate analyses. P ? 0.05 was considered significant for all tests.
The statistical analyses were performed using SPSS software version 13
(SPSS, Inc., Chicago, IL).
Staining with anti-RKIP antibody gave a single band at ap-
proximately 22 kDa. As described in the Subjects and Methods
section, the mean of RKIP/?-actin and RKIP/?-tubulin was used
RKIP level and acute octreotide response
The RKIP level in the somatotroph adenomas correlated to
both the percent reduction of serum GH concentration during
the acute octreotide test (R ? 0.342; P ? 0.020) and the nadir
GH level during the test (R ? ?0.457; P ? 0.001) (Fig. 2). In a
multiple regression model, both SSTR2a immunohistochemical
expression scores of the adenoma and RKIP levels were signifi-
cant determinants of the acute octreotide response, defined as
The good responders, defined as patients with more than
75% GH reduction in the acute test, had a significantly higher
(P ? 0.004).
RKIP level and the long-term octreotide response
The RKIP level in the tumor correlated to both the percent
reduction in serum IGF-I concentration (R ? 0.674; P ? 0.001)
and the absolute IGF-I reduction (R ? 0.619; P ? 0.003) after
3). Both the RKIP level and the adenoma SSTR2a immunohis-
tochemical grading were significant determinants for the long-
term percent serum IGF-I reduction in the multiple regression
model (R20.722) (Table 2). The good responders, defined as
higher RKIP level (median 1.85) than the bad responders (me-
dian 0.73) (P ? 0.015).
The effect of octreotide on tumor shrinkage did not signifi-
cantly correlate to the level of RKIP in the adenoma (R ? 0.41;
P ? 0.116).
RKIP level and baseline patient characteristics
The RKIP level did not correlate to patient age or baseline
serum GH or IGF-I levels (P ? 0.41, P ? 0.70). No correlation
to preoperative tumor size or invasiveness could be demon-
strated (P ? 0.45, P ? 0.84).
There was no difference in RKIP levels among the patients
preoperatively treated with octreotide compared with those not
pretreated (P ? 0.98).
A Gs? mutation analysis was performed in 47 of the patients
positive. There was no difference in RKIP level between the gsp-
positive and gsp-negative adenomas (P ? 0.344).
RKIP level and adenoma SSTR2a status
There was no difference in RKIP level between tumors with
different immunohistochemical scores for SSTR2a (P ? 0.60),
and no correlation between the RKIP level and the SSTR2a level
measured by Western blot in a previous study (P ? 0.17) (9).
FIG. 1. A representative Western blot analysis of RKIP (lower panel; the band at
?22 kDa) and ?-actin (upper panel; the band at ?42 kDa) from tumors in six
patients. MW, Molecular weight.
J Clin Endocrinol Metab, April 2008, 93(4):1211–1216jcem.endojournals.org
The present study demonstrates a significant correlation be-
tween the adenoma RKIP level and the effect of octreotide in
tumors with low RKIP levels.
ment in acromegaly has been the tumor expression of SSTRs,
mainly the subtype 2 receptor. Generally, a low receptor level
correlates to a poor octreotide response with some exceptions
level, which may not necessarily correlate to the protein level of
the receptor (23, 24). In our recent study of adenoma SSTR2a
protein level, we demonstrated a correlation between octreotide
response and adenoma SSTR2a expression immunohistochemi-
cally, in which adenomas with a high proportion of SSTR2a-
positive cells responded significantly better to octreotide treat-
ern blot analysis of protein extracts from the tumors was found
(9). Nevertheless, exceptions were observed, suggesting that
other mechanisms downstream of the SSTRs may also be in-
volved in determining the clinical effect of SMSs in the soma-
totroph adenomas. In the present study, we found a strong cor-
level in tumor extracts or expressed immunohistochemically. In
the multiple regression models, both SSTR2a immunohisto-
chemical grading and the RKIP level were significant determi-
nants of the octreotide response. Patients with poor octreotide
response despite a high proportion of SSTR2a-positive cells
seemed to have low RKIP levels, and patients with few SSTR2a-
positive cells, but relatively good octreotide response, had high
RKIP levels. This suggests a model in which SSTR2a expression
is a permissive factor for octreotide response in acromegaly but
that RKIP is an independent modulator of the efficacy of SMSs.
Multiple regression analyses
Univariate analysesMultivariate analyses
For % GH reduction in acute octreotide test
SSTR2a to actin ratio
Log RKIP ratio
For % IGF-I reduction after approximately 6 months
SSTR2a to actin ratio
Log RKIP ratio
aSSTR2a grading represents the immunohistochemical grading of the adenomas in two categories: less than 75% of the cells positively stained (grade 1 and 2), and
more than 75% of the cells positively stained by immunohistochemistry (grade 3).
bThe Mann-Whitney rank sum test P value is presented. For the other variables, the Spearman correlations are presented in the univariate analyses.
FIG. 2. Correlation between tumor RKIP level and short-term octreotide response in patients with somatotrophinomas. Each symbol represents the logarithm of the
mean RKIP/?-actin and RKIP/?-tubulin ratios in the tumor, and the individual short-term octreotide response on the percent serum GH reduction (left panel) and the
nadir GH level (right panel) during the acute octreotide test (n ? 46). Correlation between variables was tested with Spearman correlation analysis.
Fougner et al. Adenoma RKIP Level and Octreotide EfficacyJ Clin Endocrinol Metab, April 2008, 93(4):1211–1216
No correlation between RKIP level and tumor shrinkage was
demonstrated, but MRI scan for this analysis was limited to 16
patients. Previous studies have not shown any correlation be-
tween biochemical effect and tumor size reduction during long-
term treatment of acromegaly (25, 26).
to medical treatment. Some prostate and breast cancer cell lines
low levels of RKIP. Increasing the levels by transfecting the cells
with an RKIP expression vector re-sensitized the cells to drug-
induced apoptosis. In drug-sensitive cell lines, down-regulation
of RKIP led to the resistance to the chemotherapeutic drug (20).
non-Hodgkin’s lymphoma cell lines to chemotherapeutic drug-
induced apoptosis (27). Both inhibition of the MEK-ERK path-
way and inhibition of the nuclear factor-?B pathway were sug-
gested as possible mechanisms for the drug-sensitizing effects.
Studies have indicated RKIP as a metastasis suppressor gene
in cancer. Loss of RKIP was associated with metastasis devel-
opment, and increased angiogenesis and vascular invasion were
suggested as possible mechanisms (18). Somatotroph pituitary
tumors rarely metastasize, but growth can be locally invasive
with erosion of surrounding bone. The tumor often expands
outside of the sella turcica at diagnosis, making radical surgery
difficult. In our study adenoma RKIP level did not correlate to
tumor size and invasiveness. This could be due to the general
benign nature of these adenomas.
29). Therefore, a synergistic inhibitory effect on the MEK-ERK
pathway is a possible mechanism for the association between
adenoma RKIP levels and the clinical efficacy of octreotide.
Phosphorylation of RKIP by protein kinase C activates RKIP
binding to and inhibition of GRK2. Therefore, internalization
correlation between total RKIP level and the level of SSTR2a in
We found a significant correlation between the adenoma
RKIP level and the clinical effect of octreotide, both in the acute
test situation and after long-term treatment. The RKIP level was
also a significant determinant of octreotide efficacy in the mul-
tiple regression models, in addition to the SSTR2a protein ex-
pression. However, potentially RKIP by itself might not be cru-
cial but could be related to an associating factor of an unknown
mechanism. Yet, there are several potential mechanisms for
RKIP to be of importance for the SMS effect, e.g. the effect of
RKIP on drug sensitivity of other cell types.
In conclusion, RKIP levels in somatotroph tumors correlated
ment, even in patients who expressed high levels of the main
octreotide receptor SSTR2a. The study suggests that RKIP is an
important modulator of the therapeutic effect of octreotide of
GH producing tumors. Further studies are needed to reveal the
mechanism for this and to evaluate the role of RKIP on SMS
response in other tumors.
We thank Dr. Olivera Casar Borota for the immunohistochemical anal-
yses of somatostatin receptor subtype 2a, and Mrs. Aase-Brith Jensen
and Mrs. Vigdis Enge for assistance with the protein analyses.
Fougner, Research Institute for Internal Medicine, Section of Endocri-
nology, Rikshospitalet Medical Centre, N-0027 Oslo, Norway. E-mail:
Disclosure Summary: S.L.F. has received lecture fees from Novartis
fees from Novartis and Pfizer, and J.P.B. from Pfizer. The other authors
have nothing to disclose.
kanen H, Loyttyniemi E, Ebeling T, Jaatinen P, Laine H, Nuutila P, Salmela P,
FIG. 3. Correlation between tumor RKIP level and the long-term octreotide response in patients with somatotrophinomas. Each symbol represents the logarithm of the
mean RKIP/?-actin and RKIP/?-tubulin ratios in the tumor and the individual change in serum-IGF-I levels presented as percent (left panel) or absolute (right panel)
reduction after approximately 6-month treatment with octreotide (n ? 21). Correlation between variables was tested with Spearman correlation analysis.
J Clin Endocrinol Metab, April 2008, 93(4):1211–1216jcem.endojournals.org