Differential Effects of Octreotide and Pasireotide on Somatostatin Receptor Internalization and Trafficking in Vitro

Home , Lanreotide, Pasireotide, Somatostatin receptor 1, Somatostatin receptor 2

ORIGINAL ARTICLE

Endocrine Research

Sarah Lesche, Diana Lehmann, Falko Nagel, Herbert A. Schmid, and Stefan Schulz

Department of Pharmacology and Toxicology (S.L., D.L.), Otto-von-Guericke-University, D-39120 Magdeburg,

Germany; Department of Pharmacology (F.N.), Julius-Maximilians-University, D-97078 Wu¨ rzburg, Germany; Novartis Downloaded from https://academic.oup.com/jcem/article/94/2/654/2598625 by guest on 28 September 2021 Institutes for Biomedical Research (H.A.S.), CH-4057 Basel, Switzerland; and Department of Pharmacology and Toxicology (F.N., S.S.), Friedrich-Schiller-University, D-07743 Jena, Germany

Objective: The clinically used somatostatin analogs, octreotide and lanreotide, act primarily by

binding to somatostatin receptor 2 (sst2). In contrast, the novel multireceptor ligand pasireotide

(SOM230) binds with high affinity to somatostatin receptor subtypes sst1, sst2, sst3, and sst5. SOM230 is currently under clinical evaluation for treatment of acromegaly, Cushing’s disease, and octreotide-resistant carcinoid tumors. However, the effects of SOM230 on internalization and postendosomal sorting of individual human somatostatin receptor subtypes have not been determined so far.

Results: Here we show that SOM230 was less potent than octreotide in inducing internalization and

signaling of sst2 receptors expressed in human embryonic kidney cells. In contrast, SOM230 was

more potent than octreotide in inducing internalization and signaling of sst3 and sst5 receptors.

Both SOM230 and octreotide stimulated a rapid down-regulation of sst3 but not of sst2 or sst5 ␤ receptors. SOM230 and octreotide profoundly differed in their patterns of sst2-stimulated -arrestin mobilization. Whereas octreotide-mediated receptor activation led to the formation of ␤ stable complexes facilitating the internalization of sst2 and -arrestin-2 into the same endocytic vesicles, SOM230-mediated receptor activation led to the formation of unstable complexes that

dissociated at or near the plasma membrane. Consequently, sst2 receptors recycled rapidly to the plasma membrane after endocytosis in SOM230-treated cells, but not in octreotide-treated cells.

Conclusion: We show that SOM230 modulates somatostatin receptor trafficking in a manner clearly distinct from octreotide and somatostatin. These findings may provide an explanation for the differential regulation of somatostatin receptor responsiveness during long-term administration of stable somatostatin analogs. (J Clin Endocrinol Metab 94: 654–661, 2009)

he peptide hormone somatostatin is widely distributed of which, octreotide and lanreotide, were approved for clinical T throughout the brain and periphery where it regulates the use. Octreotide and lanreotide bind with high subnanomolar release of many hormones such as GH, TSH, ACTH, glucagon, affinity to sst2 only, have moderate affinity to sst3 and sst5, and insulin, gastrin, and ghrelin. The biological actions of soma- show very low or absent binding to sst1 and sst4. tostatin are mediated by five G protein-coupled receptors named In clinical practice, octreotide and lanreotide are used as first somatostatin receptor 1 (sst1) through sst5. Natural somatosta- choice medical treatment of neuroendocrine tumors such as GH- tin binds with high affinity to all five somatostatin receptors. secreting adenomas and carcinoid (1, 2). Octreotide initially con- However, the clinical utility of somatostatin is limited by its rapid trols symptoms caused by hormonal overproduction in about degradation in human plasma. In the past, a number of meta- 90% of carcinoid patients. After 1 yr of treatment, however, bolically stable somatostatin analogs have been synthesized, two some 50% of patients show an escape of response (2, 3). In

ISSN Print 0021-972X ISSN Online 1945-7197 Abbreviations: EGFP, Enhanced green fluorescent protein; HA, hemagglutinin; HEK, hu- Printed in U.S.A. man embryonic kidney; SDS, sodium dodecyl sulfate; sst, somatostatin receptor. Copyright © 2009 by The Endocrine Society doi: 10.1210/jc.2008-1919 Received September 2, 2008. Accepted October 30, 2008. First Published Online November 11, 2008

654 jcem.endojournals.org J Clin Endocrinol Metab. February 2009, 94(2):654–661 J Clin Endocrinol Metab, February 2009, 94(2):654–661 jcem.endojournals.org 655 acromegaly, octreotide normalizes GH levels in only 65% of response in one third of acromegalic patients occurs due to di- patients (1). Octreotide response in these patients clearly de- minished expression of sst2, whereas expression of sst5 persists (4). pends on the presence of sst2 receptors (4). Loss of octreotide Octreotide has no suppressive effect on ACTH and cortisol levels in patients with Cushing’s disease, a condition

with predominant sst5 expression (5–7). Recently, a novel multireceptor somatostatin analog, pasireotide (SOM230), has been synthesized (8, 9). SOM230 is a cyclo- hexapeptide that binds with high affinity to

all somatostatin receptors except sst4 (10). In contrast to octreotide, SOM230 exhib-

its particular high subnanomolar affinity Downloaded from https://academic.oup.com/jcem/article/94/2/654/2598625 by guest on 28 September 2021

to sst5 and an improved metabolic stabil- ity (11). Preclinical studies suggest that SOM230 is a promising candidate for clinical applications where octreotide and lanreotide were shown to be weakly active or even ineffective (12–21). In fact, SOM230 is currently under clinical evaluation for treatment of ACTH-adenomas and octreotide-resistant GH-adenomas (11, 22, 23). Given that the responsiveness of individual target cells to long-term administration of somatostatin analogs is likely to be regulated by distinct modes of somatostatin receptor trafficking, we here examined the effects of SOM230 on the internalization, down-regulation, and recycling of human somatostatin receptors in a heterologous expression system.

Materials and Methods

Materials SOM230 and octreotide were provided by Dr. H. Schmid (Novartis, Basel, Switzerland). Soma- tostatin (SS-14) was obtained from Bachem (Weil

am Rhein, Germany). Anti-sst1{9498}, anti- sst2A{0786}, anti-sst3{4823}, anti-sst5{0917}, and anti-hemagglutinin (HA) antibodies were generated and extensively characterized as previously de-

scribed (4, 24). Plasmids encoding the human sst1,

sst2, sst3, and sst5 receptors as well as plasmids encoding human HA-tagged sst2, sst3, and sst5 receptors were obtained from University of Mis- souri-Rolla cDNA Resource Center (Rolla, MO).

Cell culture and transfection Human embryonic kidney (HEK) 293 cells were obtained from the American Type Culture Collec-

FIG. 1. Comparison of SOM230- and octreotide-induced endocytosis of human sst2, sst3, and sst5 tion (Manassas, VA) and grown in DMEM supple- receptors. HEK 293 cells stably expressing sst2 (upper panel), sst3 (middle panel), or sst5 (lower panel) mented with 10% fetal calf serum in a humidified either were not exposed (0 nM) or were exposed to SS-14, SOM230, or octreotide in concentrations atmosphere containing 10% CO2. Transfections ranging from 0.1 nM to 1000 nM for 30 min. Cells were subsequently fixed and fluorescently labeled with were performed using LipofectAMINE 2000 (In- anti-sst2A{0786}, anti-sst3{4823}, or anti-sst5{0917} antibodies, and the subcellular distribution of receptor vitrogen, San Diego, CA) according to the instruc- proteins was examined by confocal microscopy. Note that in untreated cells, sst , sst , and sst receptors 2 3 5 tions of the manufacturer. Stable transfectants were almost exclusively confined to the plasma membrane. SS-14 induced robust internalization of sst 2 were selected in the presence of 500 ␮g/ml G418. and sst3 and to a lesser extent of sst5. Although SOM230 was less potent in inducing internalization of HEK 293 cells stably expressing somatostatin re- sst2, it was more potent in inducing internalization of sst3. SOM230 stimulated only limited endocytosis of sst5, whereas octreotide failed to promote any detectable redistribution of sst5. Shown are representative ceptors were characterized using radioligand-bind- results from one of three independent experiments performed in duplicate. Scale bar,20␮m hsst, Human sst. ing assays, Western blot analysis, and immunocy- 656 Lesche et al. Pharmacological Characterization of SOM230 J Clin Endocrinol Metab, February 2009, 94(2):654–661

tochemistry as described previously (24–26). The level of somatostatin priate treatment with SS-14, SOM230, or octreotide, cells were lysed in receptorexpressionwasbetween800and1200fmol/mgmembraneprotein. detergent buffer ͓50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl Immunocytochemistry sulfate (SDS), 0.2 mM phenylmethylsulfonyl fluoride, 10 ␮g/ml leupep- tin, 1 ␮g/ml pepstatin A, 1 ␮g/ml aprotinin, and 10 ␮g/ml bacitracin͔. Cells were grown on poly-L-lysine-coated coverslips overnight. After the appropriate treatment with SS-14, SOM230, or octreotide, cells were Glycosylated proteins were partially enriched using wheat germ-lectin- fixed with 4% paraformaldehyde and 0.2% picric acid in phosphate agarose beads as described. Proteins were eluted from the beads using buffer (pH 6.9) for 40 min at room temperature and washed several SDS-sample buffer for 20 min at 60 C and then resolved on 8% SDS- times. Specimens were permeabilized and then incubated with 1 ␮g/ml polyacrylamide gels. After electroblotting, membranes were incubated ␮ with 1 g/ml anti-sst2A{0786}, anti-sst3{4823}, or anti-sst5{0917} anti- anti-sst1{9498}, anti-sst2A{0786}, anti-sst3{4823}, or anti-sst5{0917} antibodies followed by cyanine 3.18-conjugated secondary antibodies bodies followed by detection using an enhanced chemiluminescence de- (Amersham, Braunschweig, Germany). Specimens were mounted and tection system (Amersham) (28). examined using a Leica TCS-NT laser scanning confocal microscope

(Leica Microsystems, Nussloch, Germany) (25). Measurements of cAMP accumulation Downloaded from https://academic.oup.com/jcem/article/94/2/654/2598625 by guest on 28 September 2021 Transfected cells were seeded onto poly-L-lysine-treated 22-mm 12- Quantification of receptor internalization by ELISA well dishes. On the next day, cells were washed and then incubated with ␮ ␮ Cells expressing HA-tagged sst , sst ,orsst receptors were seeded 0.5 ml of serum-free RPMI medium containing 25 M forskolin or 25 M 2 3 5 forskolin plus SS-14, SOM230, or octreotide in concentrations ranging onto poly-L-lysine-treated 24-well plates. The next day, the cells were Ϫ12 Ϫ6 preincubated with 1 ␮g/ml anti-HA antibody for2hat4C.After the from 10 to 10 M for 15 min at 37 C. The reaction was terminated appropriate treatment with SS-14, SOM230, or octreotide, cells were by removal of the culture medium and subsequent addition of 1 ml of fixed and incubated with peroxidase-conjugated antirabbit antibody for ice-cold HCl/ethanol (1 volume of 1 N HCl, 100 volumes of ethanol). 2 h at room temperature. After washing, the plates were developed with After centrifugation, the supernatant was evaporated, the residue was 2,2Ј-azino-di[3-ethyl-benzthiazoline sulfate (6)] (Roche Diagnostics, dissolved in TE buffer (50 mM Tris-EDTA, pH 7.5), and the cAMP Manheim, Germany) solution and analyzed at 405 nm using a microplate content was determined using a RIA as described (25). reader (24). ERK assays ␤-Arrestin-EGFP mobilization assay Cells were seeded onto poly-L-lysine-treated 22-mm 12-well dishes HEK 293 cells were seeded into 35-mm glass-bottom culture dishes and grown for2dinDMEM containing 0.5% fetal calf serum. Cells were (Mattek, Ashland, MA). The next day, cells were transiently cotrans- then exposed to SS-14, SOM230, or octreotide in concentrations ranging Ϫ12 Ϫ6 fected with 2 ␮g ␤-arrestin-2-enhanced green fluorescent protein (EGFP) from 10 to 10 M for 5 min in RPMI medium without fetal calf serum and 8 ␮g sst . After 48 h, cells were transferred onto a temperature- at 37 C. Incubation was terminated by removal of the culture medium 2 ␮ controlled microscope stage set at 37 C of a Leica TCS-NT laser scanning and subsequent addition of 300 l of boiling SDS-sample buffer. Samples confocal microscope. Images were collected sequentially using single line were heated to 95 C for an additional 5 min period. Equal amounts of excitation at 488 nm with 515–540-nm band pass emission filters. Sat- protein of each sample were separated on 10% SDS-polyacrylamide gels urating concentrations of SS-14, SOM230, or octreotide (1 ␮M) were and electroblotted onto nitrocellulose membranes. Membranes were in- applied directly into the culture medium immediately after the initial cubated with mouse monoclonal phosphospecific anti-ERK1/2 antibody image was taken (27). clone E10 (1:1000; New England Biolabs, Beverly, MA) or phosphorylation-independent rabbit polyclonal anti-ERK2 antibody (1:1000; Western blot analysis Santa Cruz Biotechnology, Santa Cruz, CA). Blots were developed using peroxidase-conjugated secondary antibodies and enhanced chemilu- Stably transfected HEK 293 cells were plated onto poly-L-lysine- minescence (Amersham). Densitometric analysis of total ERK2 and coated 100-mm dishes and grown to 80% confluence. After the appro- phospho-ERK1/2 levels on Western blots exposed in the linear range of the x-ray film was performed using National Institutes of Health TABLE 1. Functional properties of SS-14, SOM230, and Image 1.57 software (24). octreotide Data analysis

Ligand hsst2 hsst3 hsst5 Data from cAMP, ERK, and internalization assays were analyzed by nonlinear regression curve fitting using GraphPad Prism 4.0 software ERK1/2 activation EC50 (nM) SS-14 0.39 0.25 0.85 (GraphPad Software, San Diego, CA). SOM230 1.45 0.47 0.53 Octreotide 0.22 7.50 15.88 Inhibition of cAMP Results accumulation IC50 (nM) SS-14 0.46 0.22 0.41 SOM230 1.78 0.57 0.58 SOM230 exhibits high binding affinity to the somatostatin re- Octreotide 0.36 4.24 20.19 ceptors sst1, sst2, sst3, and sst5. To study the SOM230-stimulated

Internalization EC50 (nM) membrane trafficking of these receptors, we first generated HEK SS-14 3.26 2.71 4.8 293 cells stably expressing the human somatostatin receptors. Ͼ SOM230 31.78 5.62 1000 Immunocytochemical examination of these cells revealed that in Octreotide 6.46 16.57 Ͼ1000 the absence of agonist sst , sst and sst receptors were almost ERK, cAMP, and internalization assays were carried out as described under 2 3 5 exclusively confined to the plasma membrane (Fig. 1, left panel). Materials and Methods. The half-maximal inhibitory concentrations (IC50) for cAMP assays and the half-maximal effector concentration (EC50) for ERK Similar to that previously observed (4), a large proportion of sst1 activation and internalization were analyzed by nonlinear regression curve receptors resided within the cytoplasm, which prevented us from fitting using the computer program GraphPad Prism 4.0. Data are presented as the mean of three or four independent experiments. SE values were smaller studying the SOM230-induced sst1 internalization in this sys- than 15%. hsst, Human sst. tem. When sst2-expressing cells were exposed to different con- J Clin Endocrinol Metab, February 2009, 94(2):654–661 jcem.endojournals.org 657 Downloaded from https://academic.oup.com/jcem/article/94/2/654/2598625 by guest on 28 September 2021

FIG. 2. Comparison of SOM230- and octreotide-induced ERK activation, adenylyl cyclase inhibition, and internalization of human sst2, sst3, and sst5 receptors. A, Time- ␮ ␮ ␮ dependent ERK1/2 activation. HEK 293 cells expressing sst2, sst3,orsst5 were exposed to 1 M SS-14, 1 M SOM230, or 1 M octreotide for 0, 2, 5, 10, 20, or 30 min. Cells were lysed, equal amounts of protein were resolved by SDS-PAGE, and levels of phosphorylated ERK1/2 were determined by immunoblotting. The positions of phospho-ERK1/2 are indicated on the right. Three additional experiments gave similar results. Note that maximal ERK1/2 activation for all ligands and receptors

occurred between 2 and 5 min. B–D, Dose-dependent ERK1/2 activation. HEK 293 cells expressing sst2, sst3,orsst5 were exposed to SS-14, SOM230, or octreotide in Ϫ12 Ϫ6 concentrations ranging from 10 to 10 M for 5 min. Cells were lysed, equal amounts of protein were resolved by SDS-PAGE, and levels of phosphorylated ERK1/2 were determined by immunoblotting. Results were quantified by densitometric analysis. Data were normalized to total ERK2 and expressed as fold over ERK1/2 phosphorylation over the basal value in untreated cells. The maximum agonist-induced activation of ERK1/2 was defined as 100%. Values represent means of three independent experiments performed in duplicate. SE values were smaller than 15%. E–G, Dose-dependent inhibition of forskolin-stimulated cAMP. HEK 293 cells ␮ Ϫ12 Ϫ6 expressing sst2, sst3,orsst5 were exposed to 25 M forskolin plus SS-14, SOM230, or octreotide in concentrations ranging from 10 to 10 M for 15 min. After washing, cAMP levels were determined using a RIA. The maximum forskolin-stimulated cAMP formation in the absence of agonist was defined as 100%. Values represent means from three separate measurements performed in triplicate. SE values were smaller than 10%. H–J, Dose-dependent internalization. HEK 293 cells Ϫ12 Ϫ6 expressing N-terminally HA-tagged sst2, sst3,orsst5 receptors were exposed to SS-14, SOM230, or octreotide in concentrations ranging from 10 to 10 M for 30 min. Cell surface receptors were labeled with anti-HA antibodies followed by a peroxidase-conjugated secondary antibody. Receptor sequestration, quantified as the percent loss of cell-surface receptors in agonist-treated cells, was measured by ELISA. Data are presented as the mean of four independent experiments performed in quadruplicate. SE values were smaller than 15%. SS-14 (), Solid lines; SOM230 (●), dashed lines; octreotide (Œ), dotted lines. hsst, Human sst. 658 Lesche et al. Pharmacological Characterization of SOM230 J Clin Endocrinol Metab, February 2009, 94(2):654–661

centrations of SS-14, SOM230, and octreotide, it became ap- We therefore assessed the down-regulation of sst2, sst3, and sst5 parent that SOM230 was less potent than octreotide in inducing receptors during prolonged agonist exposure by Western blot

endocytosis of sst2 receptors (Fig. 1, upper panel). Whereas a analysis. As shown in Fig. 3, left panel, we did not observe any

30-min treatment with octreotide or SS-14 in concentrations as detectable change in the level of cellular sst2 receptors during the

low as 100 nM promoted a nearly complete translocation of sst2 22-h treatment with SS-14, SOM230, or octreotide. In contrast,

receptors from the plasma membrane into the cytosol, SOM230 the sst3 receptor underwent pronounced down-regulation,

induced only a partial redistribution of sst2 receptors under the which became clearly detectable as soon as3hofcontinued

same conditions. Internalization of sst2 receptors was still de- exposure to SS-14, SOM230, or octreotide (Fig. 3, middle panel). tectable in cells treated with 1 nM SS-14 or 1 nM octreotide, but During prolonged treatment with SS-14, SOM230, or oct-

not in cells treated with 1 nM SOM230. Examination of inter- reotide, cellular sst5 receptor content was transiently increased

nalization in sst3-expressing cells revealed that SOM230 was but did not show a dramatic down-regulation (Fig. 3, right

more potent than octreotide in inducing endocytosis of sst3 re- panel). Downloaded from https://academic.oup.com/jcem/article/94/2/654/2598625 by guest on 28 September 2021

ceptors (Fig. 1, middle panel). Similar to that observed in sst2- Given the different capacities of SOM230 and octreotide to

expressing cells, a 30-min treatment with 100 nM SS-14 pro- stimulate sst2 receptor endocytosis, we employed functional ␤ moted a nearly complete translocation of sst3 receptors from the -arrestin-2 conjugated to enhanced green fluorescent protein to ␤ plasma membrane into the cytosol. Whereas 100 nM SOM230 visualize the patterns of sst2-stimulated -arrestin mobilization ␤ stimulated clearly visible sst3 redistribution, administration of in live HEK 293 cells. In the absence of agonist, -arrestin-2- octreotide in concentrations as high as 1000 nM was required to EGFP was uniformly distributed throughout the cytoplasm of

induce detectable sst3 internalization. In contrast to that ob- the cells (Fig. 4, 0 min). The addition of saturating concentrations

served in sst2- and sst3-expressing cells, a 30-min treatment with of SS-14, SOM230, or octreotide induced a rapid redistribution ␤ SS-14 promoted only a partial translocation of sst5 receptors of -arrestin-2 from the cytoplasm to the plasma membrane, from the plasma membrane into the cytosol (Fig. 1, lower panel). resulting in fluorescence outlining the shape of the cells (Fig. 4,

Although subnanomolar sst5 binding affinity has been reported 1 min). After extended agonist exposure, internalization of for SOM230, it stimulated only very limited internalization of ␤-arrestin-2-EGFP into endocytic vesicles was observed only this receptor. Moreover, octreotide did not induce any detectable in SS-14- and octreotide-treated cells, but not in SOM230-

endocytosis in sst5-expressing cells. treated cells (Fig. 4, 30 min). To analyze SOM230-induced somatostatin receptor inter- The pattern of ␤-arrestin trafficking is believed to dictate the

nalization quantitatively, we stably expressed HA-tagged sst2, rate of receptor recycling and resensitization. We therefore ex-

sst3, and sst5 receptors in HEK 293 cells. Insertion of the N- terminal epitope tag allowed us to determine the agonist-stimulated loss of cell surface receptors using anti-HA antibodies in an ELISA. The results depicted in Table 1 and Fig. 2, H–J, con- firmed that SOM230 was less potent than octreotide in inducing

internalization of sst2 but more potent than octreotide in induc-

ing internalization of sst3 and sst5. All somatostatin receptors are coupled to inhibitory G proteins, thereby mediating an inhibition of adenylyl cyclase. In our heterologous cell system, stimulation of somatostatin receptors also leads to an activation of ERK1/2 with a maximum between 2 and 5 min (Fig. 2A). In an effort to compare SOM230-stimulated somatostatin receptor endocytosis and signaling, we then used inhibition of forskolin-stimulated cAMP accumulation and ERK activation as read-out for receptor activation. The results depicted in Table 1 and Fig. 2, B–G, clearly demonstrated that SOM230 was less potent than octreotide in inducing signaling

via the sst2 receptor, but more potent than octreotide in inducing

signaling via the sst3 and sst5 receptors. For all ligands, higher concentrations were required to stimulate receptor internalization, which may reflect a high receptor reserve present in this system. In general, there was a good correlation between endocytosis and signaling. The only notable exception was the effect of SOM230 on sst . Like SS-14, SOM230 stimulated sst sig- 5 5 FIG. 3. Comparison of SOM230- and octreotide-induced down-regulation of naling at subnanomolar concentrations. Unlike SS-14, however, human sst2, sst3, and sst5 receptors. HEK 293 cells stably expressing sst2 (left ␮ SOM230 did not stimulate substantial sst5 internalization. panel), sst3 (middle panel), or sst5 (right panel) were exposed to 1 M SS-14, After internalization, somatostatin receptors are either resen- 1 ␮M SOM230, or 1 ␮M octreotide for 0, 1, 3, 6, or 22 h. The cellular receptor content was then determined by Western blot analysis. Three sitized and recycled to plasma membrane or transferred into the additional experiments gave similar results. The positions of molecular mass lysosomal compartment and subject to proteolytic degradation. markers are indicated on the right (in kilodaltons). hsst, Human sst. J Clin Endocrinol Metab, February 2009, 94(2):654–661 jcem.endojournals.org 659

lesser extent in SOM230-treated cells. In the absence of agonist, it became apparent that

nearly the entire pool of internalized sst2 receptors recycled to the plasma membrane during the 60-min agonist-free interval in SOM230-treated cells (Fig. 5, middle panel).

In contrast, only part of the internalized sst2 receptors were redistributed to the plasma membrane in SS-14- and octreotide-treated

cells, whereas a large proportion of sst2 receptors was still confined to intracellular

vesicles (Fig. 5, upper and lower panels). Downloaded from https://academic.oup.com/jcem/article/94/2/654/2598625 by guest on 28 September 2021

Discussion

␤ The qualitative and quantitative internaliza- FIG. 4. Differential sst2-mediated -arrestin-2 mobilization. HEK 293 cells were transiently transfected with 2 ␮ ␤ ␮ ␤ g -arrestin-2-EGFP and 8 g sst2. The distribution of -arrestin-2 was visualized sequentially in the same live tion assays carried out in the present study cells before (0 min) and after (1, 3, 10, and 30 min) the addition of 1 ␮M SS-14, 1 ␮M SOM230, or 1 ␮M clearly demonstrate that short-term admin- octreotide to the culture medium. Shown are representative images from one of four independent experiments istration of SOM230 modulates somatosta- performed in duplicate. Arrowheads, Translocation of ␤-arrestin-2-EGFP to the plasma membrane; arrows, trafficking of ␤-arrestin-2-EGFP into early endosomes. Scale bar,20␮m. hsst2, Human sst2. tin receptor trafficking in a manner clearly distinct from octreotide. SOM230 was less amined the redistribution of sst2 receptors after internalization potent than octreotide in inducing signaling and internalization stimulated by SS-14, SOM230, or octreotide. Cells were treated of the sst2 receptor. Whereas octreotide-activated sst2 receptors for 30 min, washed extensively, and subjected to an additional cointernalized with ␤-arrestin-2 into the same endocytic vesicles, incubation in the absence of agonist for 30 or 60 min. As depicted SOM230-mediated sst2 activation led to the formation of un- in Fig. 5, sst2 receptors were almost exclusively confined to the stable complexes that dissociated at or near the plasma mem- plasma membrane in untreated cells. After 30 min of agonist brane. The pattern of ␤-arrestin trafficking is believed to dictate exposure, sst2 receptors were extensively sequestered into intra- the rate of receptor recycling and resensitization. In fact, sst2 cellular vesicles in SS-14- and octreotide-treated cells and to a receptors recycled faster to the plasma membrane in SOM230- than in octreotide- treated cells. It is conceivable that the accel- erated recycling of SOM230-activated receptors may counteract homologous desensitization in sst2-expressing cells and, hence, result in longer lasting functional responses of SOM230. However, virtually no

sst2 receptor down-regulation was detected after administration of both SOM230 and octreotide in our cell system. The clinical utility of octreotide as a suppressor of hormonal hypersecretion in neuroendocrine tumors depends on the presence of functional

sst2 receptors (3, 4, 29). SOM230 is currently under clinical evaluation for treatment of acromegaly, Cushing’s disease, and octreotide-resistant carcinoid tumors. It re- mains to be seen to what extent the observed differences in internalization, ␤-arrestin trafficking, and recycling may relate to distinct clinical responses during long-term administration of SOM230 and octreotide in conditions with predominant sst expression. FIG. 5. Internalization and recycling of sst2. HEK 293 cells expressing sst2 were exposed for 30 min to 2 1 ␮M SS-14, 1 ␮M SOM230, or 1 ␮M octreotide. Cells were washed extensively, followed by an agonist-free Loss of octreotide response in acrome- interval of 0, 30, or 60 min. Cells were subsequently fixed, processed for immunofluorescence, and galic patients occurs due to diminished ex- examined by confocal microscopy. Representative images from one of three independent experiments pression of sst , whereas expression of sst performed in duplicate are shown. Note that sst2 receptors recycled completely within 60 min only in 2 5 SOM230-treated cells, but not in octreotide-treated cells. Scale bar,20␮m. hsst2, human sst2. persists (4). Octreotide has no suppressive 660 Lesche et al. Pharmacological Characterization of SOM230 J Clin Endocrinol Metab, February 2009, 94(2):654–661

effect on ACTH levels in patients with Cushing’s disease, a con- Schiller-University, Nonnenplan 4, 07743 Jena, Nonnenplan 4, Ger- many. E-mail: [email protected]. dition with predominant sst5 expression (14). Although SOM230-stimulated internalization was limited, it was clearly This work was supported by Grant SCHU924/10–1 from the Deutsche Forschungsgemeinschaft, Grant 107721 from the Deutsche more potent than octreotide in inducing signaling and endocy- Krebshilfe, and a grant from Novartis. tosis of the sst5 receptor. Thus, SOM230 is a promising candi- Author Disclosure Summary: S.L., D.L., and F.N. have nothing to date for clinical applications where octreotide and lanreotide disclose. H.A.S. is employed by Novartis Institutes for Biomedical Re- were shown to be weakly active or even ineffective. SOM230 search (Basel, Switzerland). S.S. has received a research grant from Novartis. indications primarily targeting sst5 receptors may include ACTH-adenomas, octreotide-resistant GH-adenomas, and octreotide-resistant carcinoid tumors.

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SOM230 stimulated sst5 signaling at subnanomolar concen- Front Horm Res 35:169–178 6. Silva AP, Schoeffter P, Weckbecker G, Bruns C, Schmid HA 2005 Regulation trations without inducing substantial internalization of the sst5 of CRH-induced secretion of ACTH and corticosterone by SOM230 in rats. receptor. This is similar to that previously observed with other Eur J Endocrinol 153:R7–R10 potent sst5 agonists such as KE108, BIM-23244, or L-817,818 7. van der Hoek J, Lamberts SW, Hofland LJ 2004 The role of somatostatin (26). So far only SS-14 and SS-28 have been found to stimulate analogs in Cushing’s disease. Pituitary 7:257–264 8. Bruns C, Lewis I, Briner U, Meno-Tetang G, Weckbecker G 2002 SOM230: a robust sst5 endocytosis. a novel somatostatin peptidomimetic with broad somatotropin release inhib- Radiolabeled octreotide analogs are increasingly used for di- iting factor (SRIF) receptor binding and a unique antisecretory profile. Eur J agnostic and therapeutic in vivo targeting of somatostatin re- Endocrinol 146:707–716 9. Weckbecker G, Briner U, Lewis I, Bruns C 2002 SOM230: a new somatostatin ceptor-expressing tumors. Depending on the radioisotope, these peptidomimetic with potent inhibitory effects on the growth hormone/insulin-like compounds can be used for tumor imaging or receptor radio- growth factor-I axis in rats, primates, and dogs. Endocrinology 143:4123–4130 therapy. In these clinical applications, not only the binding of the 10. Lewis I, Bauer W, Albert R, Chandramouli N, Pless J, Weckbecker G, Bruns C 2003 A novel somatostatin mimic with broad somatotropin release inhib- radiopeptide to its receptor but also the internalization of the itory factor receptor binding and superior therapeutic potential. J Med Chem receptor-ligand complex is important for successful in vivo tar- 46:2334–2344 geting (30–32). Based on our qualitative and quantitative inter- 11. Ma P, Wang Y, van der Hoek J, Nedelman J, Schran H, Tran LL, Lamberts SW 2005 Pharmacokinetic-pharmacodynamic comparison of a novel multiligand nalization assays, we would expect radiolabeled octreotide de- somatostatin analog, SOM230, with octreotide in patients with acromegaly. rivatives to be more efficient in targeting sst2-expressing tumors, Clin Pharmacol Ther 78:69–80 whereas radiolabeled SOM230 derivatives would be more effi- 12. Batista DL, Zhang X, Gejman R, Ansell PJ, Zhou Y, Johnson SA, Swearingen B, Hedley-Whyte ET, Stratakis CA, Klibanski A 2006 The effects of SOM230 cient in targeting tumors predominantly expressing sst3.Incon- on cell proliferation and adrenocorticotropin secretion in human corticotroph trast, none of the currently available somatostatin peptides pituitary adenomas. J Clin Endocrinol Metab 91:4482–4488 would be expected to efficiently target tumors with predominant 13. Ben-Shlomo A, Melmed S 2007 Pasireotide—a somatostatin analog for the potential treatment of acromegaly, neuroendocrine tumors and Cushing’s dis- or isolated sst5 expression. ease. IDrugs 10:885–895 SOM230 was designed to imitate the broad sst binding pattern 14. Hofland LJ, van der Hoek J, Feelders R, van Aken MO, van Koetsveld PM, of SS-14. However, the present results indicate that SOM230 mod- Waaijers M, Sprij-Mooij D, Bruns C, Weckbecker G, de Herder WW, Beckers A, Lamberts SW 2005 The multi-ligand somatostatin analogue SOM230 in- ulates sst2 and sst5 receptor trafficking in a manner clearly distinct hibits ACTH secretion by cultured human corticotroph adenomas via soma- from SS-14 and octreotide, suggesting that it exhibits a different tostatin receptor type 5. Eur J Endocrinol 152:645–654 mode of binding and activating these receptors. 15. Hofland LJ, van der Hoek J, van Koetsveld PM, de Herder WW, Waaijers M, Sprij-Mooij D, Bruns C, Weckbecker G, Feelders R, van der Lely AJ, Beckers A, Lamberts SW 2004 The novel somatostatin analog SOM230 is a potent inhibitor of hormone release by growth hormone- and prolactin-secreting pituitary adenomas in vitro. J Clin Endocrinol Metab 89:1577–1585 Acknowledgments 16. Murray RD, Kim K, Ren SG, Lewis I, Weckbecker G, Bruns C, Melmed S 2004 The novel somatostatin ligand (SOM230) regulates human and rat anterior pituitary hormone secretion. J Clin Endocrinol Metab 89:3027–3032 We thank Dana Mayer for skillful technical assistance. 17. Pasquali D, Rossi V, Conzo G, Pannone G, Bufo P, De Bellis A, Renzullo A, Bellastella G, Colao A, Vallone G, Bellastella A, Sinisi AA 2008 Effects of Address all correspondence and requests for reprints to: Stefan somatostatin analog SOM230 on cell proliferation, apoptosis, and catecholamine Schulz, Department of Pharmacology and Toxicology, Friedrich- levels in cultured pheochromocytoma cells. J Mol Endocrinol 40:263–271 J Clin Endocrinol Metab, February 2009, 94(2):654–661 jcem.endojournals.org 661

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