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Address reprint requests to Jean Claude Reubi, M.D., Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, University of Berne, PO Box 62, Murtenstrasse 31, CH-3010 Berne, Switzerland
Somatostatin analogues, which are used to treat neuroendocrine tumors, target the high levels of somatostatin receptor subtype 2 (SSTR1; alias sst2) expressed in these cancers. However, some tumors are resistant to somatostatin analogues, and it is unknown whether the defect lies in sst2 activation or downstream signaling events. Because sst2 phosphorylation occurs rapidly after receptor activation, we examined whether sst2 is phosphorylated in neuroendocrine tumors. The sst2 receptor phosphorylation was evaluated by IHC and Western blot analysis with the new Ra-1124 antibody specific for the sst2 receptor phosphorylated at Ser341/343 in receptor-positive neuroendocrine tumors obtained from 10 octreotide-treated and 7 octreotide-naïve patients. The specificity, time course, and subcellular localization of sst2 receptor phosphorylation were examined in human embryo kinase–sst2 cell cultures by immunofluorescence and confocal microscopy. All seven octreotide-naïve tumors displayed exclusively nonphosphorylated cell surface sst2 expression. In contrast, 9 of the 10 octreotide-treated tumors contained phosphorylated sst2 that was predominantly internalized. Western blot analysis confirmed the IHC data. Octreotide treatment of human embryo kinase–sst2 cells in culture demonstrated that phosphorylated sst2 was localized at the plasma membrane after 10 seconds of stimulation and was subsequently internalized into endocytic vesicles. These data show, for the first time to our knowledge, that phosphorylated sst2 is present in most gastrointestinal neuroendocrine tumors from patients treated with octreotide but that a striking variability exists in the subcellular distribution of phosphorylated receptors among such tumors.
Somatostatin receptors are overexpressed in most neuroendocrine tumors.
Placebo-controlled, double-blind, prospective, randomized study on the effect of octreotide LAR in the control of tumor growth in patients with metastatic neuroendocrine midgut tumors: a report from the PROMID Study Group.
As the predominantly expressed somatostatin receptor subtype, the somatostatin receptor subtype 2 (SSTR1; alias sst2) has been detected immunohistochemically (IHC) in neuroendocrine tumors, usually at the tumor cell membrane.
This histopathological diagnosis has permitted somatostatin receptor detection with clinically relevant implications for the diagnosis and therapeutic management of tumors. Although the sst2 receptors found in the tumors are usually membrane bound when patients are not receiving somatostatin analogue treatment, it has recently been possible to detect intracellular sst2 in tumor cells of patients treated with octreotide.
However, it is not known whether intracellular sst2 represents activated sst2 internalized after octreotide therapy or newly synthesized receptors that are unable to traffic to the cell surface. Similarly, it is not known whether membrane-bound sst2 can be found in an activated form in human tumor tissues.
Activation of G-protein–coupled receptors by agonist binding at the plasma membrane causes rapid phosphorylation of the activated receptors by G-protein–coupled receptor kinases, a critical step for subsequent receptor internalization.
In fact, somatostatin treatment of cultured cells stimulates the phosphorylation of the sst2A receptor within seconds and receptor phosphorylation is maintained for at least 2 hours in the continued presence of hormone.
Nevertheless, little is known about the phosphorylation of sst2A receptors, and G-protein–coupled receptors more generally, in humans.
It would be of great interest to be able to specifically identify sst2 in its activated form in tumor samples (namely, as phosphorylated sst2). Such IHC detection of sst2 after agonist activation in patients' tumors could have potential clinical implications (eg, to understand how tumors escape octreotide responsiveness). The residues in the sst2A receptor that become phosphorylated on hormone stimulation have been recently identified, and antibodies specific for the phosphorylated forms of the receptor have been developed.
to investigate the effect of octreotide treatment on receptor phosphorylation in patient tumors. Therefore, we compared the phosphorylation of the sst2 receptor in a broad sample of receptor-expressing tumors obtained from octreotide-treated and untreated patients with gastroenteropancreatic tumors.
Materials and Methods
Formalin-fixed and fresh-frozen tissue samples from surgically resected neuroendocrine tumors expressing sst2 receptors were used. The tumor samples originated from patients who had received an i.v. octreotide infusion (200 μg/hour) during surgical resection plus 20 mg of octreotide, long-acting release (LAR), <3 weeks before surgery, from patients who had received 200 μg of octreotide s.c. at the start of surgery, or, as controls, from patients who had not been in contact with octreotide before or during surgery. Protocol variability occurred because of patient-based and surgery-dependent situations and included a variable route of octreotide administration (i.v. injection, s.c. injection, or LAR) and variable intervals between octreotide injection and tumor removal. Written informed consent was available for all patients. The present study conformed to the ethical guidelines of the Institute of Pathology, University of Berne and University Hospital of Berne, Berne, Switzerland, and was reviewed by the Institutional Review Board.
sst2 IHC Data
The sst2 receptors were detected using IHC, as previously described.
Formalin-fixed, paraffin-embedded tissue sections (4 μm thick) were used. The antigen retrieval method for UMB-1 IHC was boiling in the microwave in the presence of a 5% urea buffer (pH 9.5). The antibody was used at a dilution of 1:100. The secondary antibody was a biotinylated goat anti-rabbit Ig (Dako, Baar, Switzerland) (1:200 dilution). Antibody binding was visualized using the VECTASTAIN Elite ABC Kit (Vector, Burlingame, CA). Staining was performed with diaminobenzidine, and counterstaining was performed with hemalum. For specificity control, the primary antibody was preadsorbed with 100 nmol/L of the corresponding antigen peptide.
Phosphorylated sst2 IHC
Ser341/343-phosphorylated sst2 receptors were detected by IHC, using the Ra-1124 antibody, as recently reported.
using 125Iodine-Tyrosine3-octreotide (125I-Tyr3-octreotide) as the radioactive ligand.
Receptor Purification and Immunoblot Analysis
Frozen tumor samples were homogenized using a Wheaton Tenbroeck Tissue Grinder in homogenization buffer (10 mmol/L Tris-HCl, 5 mmol/L EDTA, 3 mmol/L EGTA, and 250 mmol/L sucrose; pH 7.6) with protease and phosphatase inhibitors (1 mmol/L phenylmethylsulfonyl fluoride, 10 μg/mL soybean trypsin inhibitor, 10 μg/mL leupeptin, 50 μg/mL bacitracin, 10 mmol/L sodium pyrophosphate, 10 mmol/L sodium fluoride, 0.1 mmol/L sodium vanadate, and 100 nmol/L okadaic acid). After a low-speed centrifugation at 600 × g for 10 minutes, the supernatant was centrifuged at 100,000 × g for 60 minutes. The pelleted membranes were solubilized in cold lysis buffer (150 mmol/L NaCl and 20 mmol/L HEPES, pH 7.4; 5 mmol/L EDTA, 3 mmol/L EGTA containing 4 mg/mL dodecyl β maltoside, and protease and phosphatase inhibitors) for 60 minutes at 4°C. After centrifugation at 10,000 × g for 10 minutes, the solubilized receptors were purified by wheat germ agglutinin agarose adsorption, as previously described.
The entire preparation of purified receptors from each sample was subjected to SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and immunoblotted with the phospho-sst2A receptor antibody Ra-1124. The blots were then stripped and reprobed with a phosphorylation-independent sst2A antibody (NB100-74537; Novus Biologicals, Littleton, CO) to demonstrate the presence of the receptor.
Immunofluorescence microscopy using the human embryonic kidney cell line HEK293 expressing the T7-epitope–tagged human sst2 receptor (HEK-sst2) was performed as previously described.
Briefly, HEK-sst2 cells were grown overnight on poly-D-lysine (20 μg/mL) (Sigma-Aldrich, St. Louis, MO) coated 35-mm four-well plates (Cellstar; Greiner Bio-One GmbH, Frickenhausen, Germany). The cells were treated at 37°C in growth medium with 1 μmol/L octreotide or Coy-14 (BIM-23A180)
(provided by Dr. Jean Rivier, Salk Institute, San Diego, CA) for the times indicated, and subsequently processed for immunofluorescence microscopy. The sst2-specific antibody UMB-1 (SS-8000RM; Biotrend GmbH, Cologne, Germany), used at a dilution of 1:25, or the phospho-site-specific sst2 antibody Ra-1124,
used at a dilution of 1:500, were used as primary antibodies. As specificity control for Ra-1124, the antibody was preincubated with an excess of either the phosphorylated antigen peptide or with the nonphosphorylated antigen peptide before applying to the cells. The Alexa Fluor488 goat anti-rabbit IgG (H+L) antibody (Molecular Probes, Inc., Eugene, OR), diluted 1:600, was used as secondary antibody. Immunofluorescence staining of the cells was imaged using a Leica DM RB immunofluorescence microscope (Leica, Wetzlar, Germany) and an Olympus DP10 camera (Olympus, Tokyo, Japan).
Immunofluorescence Confocal Microscopy
HEK293 cells were transfected with hemagglutinin-tagged human-sst2A plasmid DNA and a clonal cell line that stably expressed the receptor (HEK-hsst2A cells) was selected by serial dilution. The localization of human sst2A receptors was examined in these cells before and after treatment with somatostatin, following a modified protocol that was previously described.
Briefly, HEK-sst2A cells were incubated with mouse monoclonal anti-HA antibody (1:1000; Covance, Berkeley, CA) at room temperature for 1 hour to label surface receptors and then treated with 100 nmol/L SS14 or octreotide at 37°C for various time intervals. After agonist stimulation, cells were incubated for 1 hour with the Ra-1124 antibody (1:1000) to label phosphorylated receptor. Subsequently, Alexa Fluor568–conjugated goat anti-mouse IgG (1:300; Invitrogen, Eugene, OR) and dichlorotriazinyl amino fluorescein–conjugated AffiniPure F(ab')2 fragment donkey anti-rabbit IgG (H+L) (1:300; Jackson ImmunoResearch Labs, West Grove, PA) were used as secondary antibodies. Cells were further stained with the nucleic acid stain, DAPI (Invitrogen), and mounted with ProLong Gold antifade mounting reagent (Invitrogen). Fluorescent images were viewed with a ×60 objective using an A1R confocal microscope (Nikon, Melville, NY) with an excitation filter of 488 nm and an emission filter of 500 to 550 nm for dichlorotriazinyl amino fluorescein, an excitation filter of 561 nm and an emission filter of 570 to 620 nm for Alexa Fluor568, and an excitation filter of 358 nm and an emission filter of 461 nm for DAPI. Images from at least 8 to 10 cells were recorded in each treatment group in each experiment. The images shown are representative of observations in three independent experiments. The specificity of the antibodies was validated by testing nontransfected HEK293 cells stimulated with 100 nmol/L somatostatin for 30 minutes and somatostatin stimulated HEK-hsst2A cells incubated with Ra-1124 antibody that had been pretreated with 100 nmol/L antigen peptide. No staining was detected with Ra-1124 in either condition.
The autoradiographic and IHC data are summarized in Table 1. Tumor tissue was obtained from a total of 17 patients. Ten of them were treated with octreotide during the surgical resection of their tumors, two receiving a perioperative infusion of 200 μg/hour octreotide plus 20 mg octreotide LAR 3 weeks before surgery and the other eight receiving 200 μg octreotide s.c. perioperatively. Furthermore, there were seven control patients who had not received octreotide at any time before or during surgery. All patients, treated and nontreated, had tumors with a high expression of sst2 receptors. In all those patients in which receptor autoradiography was feasible because of the availability of fresh-frozen tissue, the tumors showed high somatostatin binding. In addition, all tumors were strongly positive for sst2 IHC, as performed with the sst2-specific UMB-1 antibody. Although all seven control samples showed strong membranous sst2 staining, the 10 octreotide-treated tumors had internalized sst2 to varying degrees, ranging from mostly internalized sst2 to mostly membranous sst2, as previously reported.
IHC using Ra-1124 specific for Ser341/343-phosphorylated sst2 showed absence of staining in all tumors from octreotide-naïve patients, whereas Ra-1124 staining was visible to varying degrees in all but one of the tumors from treated patients. Phosphorylated sst2 was seen as mostly internalized in five cases, equally internalized and membranous in two samples from the same patient, and predominantly membranous in three cases. Figure 1 illustrates the results. First, it shows two typical octreotide-treated cases: one case shows predominantly internalized sst2 receptors identified as activated, phosphorylated receptors with Ra-1124; and the other case shows membranous and internalized sst2, also identified in both locations as Ser341/343-phosphorylated sst2 with Ra-1124. Second, it also shows a more rare condition of an octreotide-treated case with a predominance of membranous sst2, also detectable in the phosphorylated state at the membrane. Phospho-antibody specificity was demonstrated by showing lack of staining in the presence of the phosphorylated antigen peptide but no blockage in presence of the nonphosphorylated antigen peptide. Figure 2 shows absence of Ser341/343-phosphorylated sst2 in tumors of octreotide-naïve patients who express a high density of membranous sst2.
Interestingly, in tumor samples containing adjacent nonneoplastic pancreas or gastrointestinal tissue, we also observed phosphorylated sst2 in these normal tissues of octreotide-treated patients. Figure 3 shows phosphorylated sst2 in a pancreatic islet from an octreotide-treated patient, whereas no phosphorylated sst2 is detected in sst2-expressing islets in an octreotide-naïve patient. The same result is found in duodenal neuroendocrine cells: the normal duodenum of an octreotide-treated patient reveals numerous neuroendocrine cells positive for the phosphorylated sst2, whereas no such cells are seen in a naïve patient. As a reference, the pancreas and duodenum of octreotide-naïve patients always had sst2-expessing cells, as detected by UMB-1 staining (Figure 3).
Figure 4 shows a Western blot analysis of tumors from four octreotide-treated and one naïve patient. Although sst2 is detected in all five cases with the sst2-specific phosphorylation-independent antibody (NB100-74537), the phosphorylated sst2 is detected with Ra-1124 only in the four treated patients but not in the octreotide-naïve patient.
Immunofluorescence microscopy studies were performed with the phospho-site-specific sst2 antibody Ra-1124
using HEK293 cells stably expressing the sst2 receptor to compare with the data in patient tissues and to have a cellular basis to understand them. Figure 5A shows double labeling with the Ra-1124 antibody for the phosphorylated receptor and the HA antibody, which detects both phosphorylated and nonphosphorylated receptors. Ra-1124 produced no signal without agonist stimulation, although the nonphosphorylated cell surface receptors were readily visualized with anti-HA antibody. This observation is consistent with the absence of Ser341/343 sst2A receptor phosphorylation in unstimulated cells.
After treatment with SS14 for 30 minutes, Ra-1124 and anti-HA reactivity are colocalized in intracellular vesicles, showing that the phosphorylated receptor has been internalized. The Ra-1124 staining is specific because the signal is abolished with the phosphorylated antigen peptide, whereas nonphosphorylated peptide does not eliminate the signal (data not shown).
To investigate the distribution of phosphorylated receptors after different times of stimulation, cells were incubated with or without 1 μmol/L octreotide for 10 seconds, 10 minutes, and 30 minutes (Figure 5B). Staining with UMB-1 shows that, without agonist stimulation, the receptor is localized at the plasma membrane; and after stimulation with octreotide, the receptor internalizes into the cell, detectable after 10 minutes as a perinuclear dot. By using Ra-1124, no Ser341/343-phosphorylated receptors are detected without agonist stimulation. However, already 10 seconds after octreotide stimulation, the phosphorylated sst2 is detectable at the plasma membrane; and after 10 minutes, it is localized mostly within the cell and is detected as a perinuclear dot. Moreover, the established sst2 antagonist Coy-14
To our knowledge, this is the first study showing that octreotide treatment leads to agonist-induced phosphorylation of sst2 receptors in patients with sst2-expressing neuroendocrine tumors. The main findings are as follows: i) In the absence of stimulation, the receptor is not phosphorylated and is found exclusively at the cell surface, whereas octreotide treatment produces both sst2 phosphorylation and internalization. ii) Activated, phosphorylated sst2 can be detected both at the plasma membrane and intracellularly. The intracellular receptors must originally have been at the cell surface for octreotide to have stimulated their phosphorylation. iii) In vitro data show that sst2 activation occurs by 10 seconds of octreotide exposure. iv) At high magnification, the phosphorylated, internalized sst2 shows an intracellular distribution pattern corresponding to localization in endosomal structures.
The degree of receptor phosphorylation and the cellular localization of the phosphorylated sst2 varies substantially between patients. A good correlation was found between sst2 internalization and Ser341/343 phosphorylation of the receptor. A high-dose octreotide treatment combining octreotide LAR and octreotide infusion
during surgical resection consistently leads to the accumulation of intracellular, phosphorylated receptors that can be detected by IHC with Ra-1124. However, with only 200 μg octreotide given s.c. at the start of surgery, a variety of sst2 phosphorylation/internalization patterns are observed, ranging from almost complete receptor phosphorylation/internalization in some cases to only focal or even absent phosphorylation/internalization. Also, a heterogeneous distribution of tumor cells containing the phosphorylated receptors can sometimes be observed, with cells containing phosphorylated sst2 receptors located next to cells without such receptors. Similar findings were previously reported for cells with membrane-bound sst2.
and may not be sufficient to trigger a complete and long-lasting Ser341/343 phosphorylation/internalization effect on the sst2 in all tumors or may be the result of other aspects of tumor function. The octreotide concentration at the target will depend on numerous systemic and local factors, including the performance of the cardiovascular system and local tumor perfusion, and the time elapsing between s.c. application and tumor resection.
The specificity of immunostaining is confirmed by using Western blot analysis in selected tumor cases. Immunoblotting with the Ra-1124 showed a specific band corresponding to the sst2 receptor also detected by a phosphorylation-independent receptor antibody. Moreover, the phosphorylated sst2 immunostaining is always abolished in the presence of an excess of the phosphorylated antigen peptide but not with the nonphosphorylated antigen peptide. These results further show that the intracellular staining seen by IHC represents specific activated sst2 receptors.
Interestingly, there are a few tumor cases clearly showing cell surface staining of the phosphorylated sst2, paralleling our observations in vitro: using immunofluorescence microscopy, HEK-sst2 cells show phosphorylation of the sst2 receptors at the plasma membrane after 10 seconds of octreotide exposure. Later, the phosphorylated receptor is internalized, with little phosphorylated sst2 left on the membrane. Thus, although receptor internalization follows receptor phosphorylation in most instances, receptor internalization does not occur efficiently in all tumors, despite strong receptor phosphorylation. Previous studies
have shown that reversal of Ser341/343 phosphorylation after removal of ligand occurs only after the receptor has internalized. Because receptors must be dephosphorylated to be able to respond again to hormone stimulation, the inability to internalize receptors may lead to a resistance to further agonist stimulation.
Some patients present a phenomenon of escape after long-term therapy with somatostatin analogues (ie, they stop responding to somatostatin analogue stimulation).
The underlying mechanisms are not established. By assessing the localization and activation state of sst2 receptors resistant to somatostatin analogues in tissue samples from sensitive and resistant patients, it may be possible to determine whether receptor activation or internalization is impaired in this situation.
The novel approach described in this study will increase our understanding of the molecular behavior of somatostatin receptors in tumors and provide insights into the mechanisms of receptor activation in vivo that are likely to be helpful for the use of somatostatin analogues in the diagnosis and treatment of neuroendocrine tumors. Although, in the past decade, the detection by IHC of nonactivated tumoral sst2 has proved an important parameter for the design of diagnostic and therapeutic strategy, the assessment of sst2 activation by phospho-sst2 IHC likely in the future is to provide important insights into the nature and causes of variability in sst2 activation either on agonist stimulation or, alternatively, constitutive phosphorylation, as reported for estrogen receptors.
Placebo-controlled, double-blind, prospective, randomized study on the effect of octreotide LAR in the control of tumor growth in patients with metastatic neuroendocrine midgut tumors: a report from the PROMID Study Group.