Somatostatin Receptor Ligands and Resistance to Treatment in Pituitary Adenomas

D CUEVAS-RAMOS and M FLESERIU Somatostatin resistance in 52:3 R223–R240 Review tumor therapy

Somatostatin receptor ligands and resistance to treatment in pituitary adenomas

Daniel Cuevas-Ramos and Maria Fleseriu1 Correspondence should be addressed Department of Medicine, Pituitary Center, Cedars-Sinai Medical Center, Los Angeles, California, USA to M Fleseriu 1Northwest Pituitary Center, and Departments of Medicine and Neurological Surgery, Oregon Health and Email Science University, 3181 SW Sam Jackson Park Road (BTE 472), Portland, Oregon 97239, USA ﬂ[email protected]

Abstract

Somatostatin (SST), an inhibitory polypeptide with two biologically active forms SST14 and SST28, inhibits GH, prolactin (PRL), TSH, and ACTH secretion in the anterior pituitary gland. SST also has an antiproliferative effect inducing cell cycle arrest and apoptosis. Such actions are mediated through five G-protein-coupled somatostatin receptors (SSTR): SSTR1–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 Journal of Molecular Endocrinology cell membrane. In patients with ACTH-secreting pituitary adenomas, i.e. Cushing’s 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 Journal of Molecular SSTR2 (dopastatin), are briefly discussed. Endocrinology (2014) 52, R223–R240

Introduction

The discovery of somatostatin or somatotropin release- identification of key SST molecular structural charac- inhibiting factor (SST) as an inhibitory polypeptide teristics provided the rationale for its modification and hormone was soon followed by its therapeutic use. design of novel analogs that function as specific ligands to Understanding SST action at each of the five subtypes of cognate receptors. G-protein-coupled somatostatin receptors (SSTR), SSTR1– Somatostatin receptor ligands (SRLs) are structurally SSTR5, and the description of the downstream signaling similar to SST and have been used to treat growth and function highlighted both hormone secretion inhibi- hormone (GH)-secreting and gastroenteropancreatic tory activity and an antiproliferative role. Furthermore, neuroendocrine tumors. Subsequently, SRLs have been

http://jme.endocrinology-journals.org Ñ 2014 Society for Endocrinology Published by Bioscientiﬁca Ltd. DOI: 10.1530/JME-14-0011 Printed in Great Britain Downloaded from Bioscientifica.com at 10/04/2021 12:09:00PM via free access Review D CUEVAS-RAMOS and M FLESERIU Somatostatin resistance in 52:3 R224 tumor therapy

tested in other pituitary tumors such as thyrotropin (TSH) peripheral nervous system, pancreas, and gut, but also in and adrenocorticotropin (ACTH)-secreting adenomas. The the placenta, kidneys, thyroid, retina, adrenals, and currently approved formulations of SRLs are octreotide submandibular glands. In the CNS, SST acts as both a (s.c. and long-acting repeatable/release (LAR)) and lanreo- stimulatory and an inhibitory neurotransmitter. However, tide (slow release (SR) or aqueous gel formulation autogel it has a key inhibitory action in the secretion of GH (ATG)) for acromegaly, and pasireotide for Cushing’s (Brazeau et al. 1973), prolactin (PRL; Vale et al. 1974), TSH disease (CD). An oral octreotide formulation and a long- (Siler et al. 1974), and ACTH (Richardson & Schonbrunn acting pasireotide LAR are under clinical evaluation. 1981) from the anterior pituitary gland. At the peripheral Response to SRLs (control of hormonal hypersecretion nervous system level, SST plays a regulatory role in the and tumor shrinkage) is variable in both acromegaly and gastrointestinal tract inhibiting ﬂow from the gallbladder, CD patients. bowel motility and gastric emptying, smooth muscle This review summarizes SST structure, function, contraction, and nutrient absorption from the intestine receptor pathways, and various SRLs (clinically available as well as in the exocrine pancreas. It also inhibits the or being developed). Mechanisms (molecular, histopatho- release of glucagon (Boden et al. 1986), insulin (Alberti logical, and clinical) that are currently known to be et al. 1973), and pancreatic polypeptide (Koerker et al. associated with pituitary tumor resistance to SRL therapy 1974) as well as cytokine release from immune cells are also discussed. (van Hagen et al. 1994). Although the main effect of SST is to block exocytosis and hormone secretion, in vitro and in vivo studies SST structure, synthesis, and function have suggested suppression of GH and ACTH transcription through SSTR2 (Castillo et al. 2011, Ben-Shlomo SST is a cyclic peptide with a circulating half-life of et al. 2013). !3 min and two biologically active forms (Brazeau et al. 1973): a 14 amino acid (AA) structure – SST14 – and a polypeptide with 14 AAs and a NH2-terminal extension – Structure of SSTR SST28 (Fig. 1). SST is generated from larger precursor molecules called prepro-SST (116 AAs), which is then The biological actions of SST are mediated through ﬁve processed to pro-SST (92 AAs) and subsequently to SST14 seven-domain G-protein-coupled receptors, SSTR1–SSTR5. and SST28. The name SST originates from a supposed The gene encoding SSTR2 contains an intron; the

Journal of Molecular Endocrinology ‘speciﬁc’ function as an inhibitor of somatotropin (GH) transcribed mRNA is spliced to encode two variants, a

release, thus SST. SST can also directly stimulate GH long (SSTR2A) form and a shorter (SSTR2B) form, which secretion at low concentrations (Co´rdoba-Chaco´n et al. differ only in the length of their C-terminus tail (Patel et al.

2012a). SST production occurs not only in the CNS and 1993). The human pituitary expresses only SSTR2A

Ala Gly Cys Lys Asn Phe Phe D Phe Cys D βnal Cys Tr p Phe Tyr S D Trp D Trp SST14 S S S Lys Octreotide Lanreotide S S Thr Lys Lys Cys Ser Thr Phe Thr Cys Thr Cys Val H2N

Pro Ala Met Ala Pro Asn Ser Asn Ala Ser

Phg O= GABA Phe Tr p Arg H2N–NH O H-Pro D Trp D Trp Glu Arg Lys Ala Gly Cys Lys Asn Phe Phe O= Pasireotide Somatoprim (CH2)3 Trp Phe Lys Lys S SST28 Tyr Thr H2N Gly Phe S Lys Thr Cys Ser Thr Phe

Figure 1 Amino acid sequences of the two biologically active somatostatin (SST) forms, SST14 and SST28 (Brazeau et al. 1973), and other four somatostatin receptor ligands (Bauer et al. 1982, Taylor et al. 1988, Bruns et al. 2002, Shimon 2003).

(Ben-Shlomo & Melmed 2010). Truncated SSTR5 variants (Vieira Neto et al. 2009). High-dose SST induces SSTR1, in mice, rats, and humans from normal and tumoral SSTR2, and SSTR5 expression in normal primate somato- pituitary samples have also been characterized (Durań- tropes (Co´rdoba-Chacoń et al. 2012b). On the other hand, Prado et al. 2009, Co´rdoba-Chacoń et al. 2010). Native low-dose SST increases GH release and SSTR1 mRNA SST14 binds to SSTR1–SSTR4 with higher affinity, while expression, but decreases SSTR5 expression (Co´rdoba- SST28 is SSTR5 selective (Patel et al. 1994). Chacoń et al. 2012b). GH and TSH are inhibited by SSTR subtypes have 42–60% identical AA sequences SSTR1, SSTR2, and SSTR5, while PRL secretion is pre- (Reisine & Bell 1995). The extracellular loop 2, between dominantly inhibited by SSTR5 (Shimon et al. 1997a,b). domains 4 and 5, and the hydrophobic and charged AAs Increased ACTH in Sstr2 knockout mice indicates a within transmembrane domains 3, 6, and 7 are essential possible regulatory role for SSTR2. However, SSTR5 dis- for the interaction with SST and second messenger plays a more potent suppressive action on ACTH release system activation (Kaupmann et al. 1995, Strnad & (Table 1; Hofland et al. 2005). Hadcock 1995).

SSTR expression in pituitary adenomas SSTR expression in normal pituitary The same SSTR subtypes as in normal pituitary are also Fetal normal pituitary expresses all five SSTRs; however, found in pituitary adenomas; however, SSTRs show the adult human pituitary gland expresses SSTR1, SSTR2, different expression level and activity depending on the SSTR3, and SSTR5. SSTR4 is expressed in extremely low type of adenoma cell (Table 1; Taboada et al. 2007, 2008, levels (Reubi et al. 2001, Vieira Neto et al. 2009, Ben Shlomo & Melmed 2010). Ben-Shlomo & Melmed 2010). SSTR5 is the highly expressed SSTR subtype in normal human pituitaries SSTR expression in GH-secreting pituitary adeno- followed by SSTR2, SSTR1, SSTR3, and SSTR4 (Vieira mas Acromegaly is predominantly caused by a Neto et al. 2009). Normal human pituitaries express GH-secreting pituitary adenoma, resulting in high more SSTR1 and less SSTR2 when compared with circulating GH and insulin-like growth factor 1 (IGF1) somatotropinomas, whereas there are no significant concentrations (Melmed 2006). SSTR2 and SSTR5 are differences in the expression of SSTR5, SSTR3, and SSTR4 predominantly expressed, both at the mRNA level (Jaquet Journal of Molecular Endocrinology Table 1 Biochemical features of human somatostatin receptors. Data derived from Reubi et al. (2001), Hofland & Lamberts (2003), Florio (2008), Lania et al. (2008) and Ben-Shlomo et al. (2009)

SSTR1 SSTR2 SSTR3 SSTR4 SSTR5

Fetal pituitary CCCCC Adult pituitary CCCKC Pituitary adenomaa GH 60% 90% 45% !5% 90% ACTH 60% 75% 10% 30% 75% PRL 90% 60% 20% 0% 80% NFA 25% 55% 45% 0% 50% TSH 100% 100% 0% 0% 50% Chromosome 14q13 17q24 22q13.1 20p11.2 16p13.3 mRNA (kb) 4.3 8.5 5.0 4.0 4.0 Amino acids 391 2A: 369 418 388 364 2B: 356 Protein size (kDa) 43 41 46 42 39 Glycosylation sites 3 4 2 1 3 Internalization degree 4% 20% 78% 29% 66% Properties YAngiogenesis YHormone [Apoptosis Arrest cell cycle YHormone secretion secretion Arrest cell cycle Arrest cell cycle Arrest cell cycle

aThe values represent the percentage of tumors expressing the SSTR subtype among the tumors screened including studies using solution hybridization, ribonuclease protection assay, RT-PCR, quantitative RT-PCR, northern blotting, in situ hybridization, and immunohistochemistry. NFA, clinically non- functioning pituitary adenoma.

et al. 2000) and at the protein level (Reubi et al. 2001). expression appears to be relatively unaffected by high SSTR2 is expressed in w95% of GH-secreting adenomas, cortisol levels. Therefore, in patients with active CD, while SSTR5 is found in w85%. SSTR1 and SSTR3 are found SSTR5 is predominantly expressed over SSTR2 (de Bruin in much fewer cases (40%), while SSTR4 is almost never et al. 2009). After eucortisolemia restoration, SSTR2 identified (Table 1; Reubi et al. 1994, Nielsen et al. 2001). expression recovers, becoming similarly abundant as Moreover, SSTR5 was identified as the most abundant SSTR5, thus improving treatment responsiveness (Fig. 2; subtype, followed by SSTR2, SSTR3, SSTR1, and SSTR4 van der Pas et al. 2013). Human SSTR2 and SSTR5 using quantitative evaluation of the SSTR subtypes overexpression in mouse corticotrope adenoma AtT20 (Taboada et al. 2007). Co-secreting GH and PRL adenomas cells demonstrates SST regulation of ACTH secretion and have increased SSTR5 expression (Shimon et al. 1997a,b, the ligand-free effects and constitutive activity of such Hofland et al. 2004). receptors (Ben-Shlomo et al. 2009).

SSTR expression in ACTH-secreting pituitary ade- SSTR expression in other pituitary adeno- nomas CD is caused by a pituitary corticotrope mas PRL-secreting pituitary adenomas mainly express adenoma responsible for ACTH hypersecretion with SSTR1 and SSTR5, whereas SSTR2 is expressed at a lower subsequent adrenal overproduction of cortisol. The level. SSTR5 has a high PRL-suppressive effect (Table 1; majority of corticotrope adenomas (O85%) express Jaquet et al. 1999). SSTR2 and SSTR5 mRNAs, and to a lesser extent SSTR1 TSH-secreting adenomas are rare tumors, already mRNA (63%) (Miller et al. 1995). The membrane density of macroadenomas at diagnosis with mass effect symptoms SSTR subtypes, particularly SSTR2, is affected by hypercor- and signs of hyperthyroidism. SSTR2 seems to be the tisolism (de Bruin et al.2009). By contrast, SSTR5 predominant receptor, but co-expression with SSTR5 has

SSTR2 2+ NH2 Ca SST SRL K+

PTP Adenylyl Ras P13K cyclase Rap1 η Gβ α COOH G i Journal of Molecular Endocrinology Voltage-gated Src Gγ SHP2 SHP1 Ca2+ and K+ channels Akt

Caspases 2+ nNOS nNOS cAMP Ca MAPK GSKb p53 Bax TSC2/ TSC1 NO GC PKA ERK1/2 Apoptosis mTOR p38 GTP cGMP Zac1

p27 Hormone secretion p21 Elk1 ATF2 Cell cycle Cell growth arrest arrest = Phosphorylation

Figure 2 Hypercortisolemia in active Cushing’s disease (CD) hampers ACTH corticotrope adenomas is reversible after achieving eucortisolemia; use of suppression by somatostatin or SSTR2 receptor ligand treatment due to SSTR2 receptor ligands may induce improved biochemical responsiveness down-regulation of SSTR2 expression in corticotrope pituitary adenomas (van der Pas et al. 2013). (de Bruin et al. 2009). Cortisol-mediated SSTR2 down-regulation in

C also been reported (Table 1; Yoshihara et al. 2007, Gatto and calcium (Ca2 ) levels, with hyperpolarization of C C et al. 2012). potassium (K ) channels as well as inhibition of Ca2 C Non-functioning pituitary adenomas (NFAs) show a influx closing voltage-sensitive Ca2 channels (Patel et al. C variable SSTR subtype expression profile, whereby SSTR2 1994). The lack of intracellular Ca2 impedes intracellular and SSTR3 seem to be the predominant subtypes (Table 1; vesicle movement, exocytosis, and hormone secretion Zatelli et al. 2004, Vieira Neto et al. 2013). (Fig. 3). Depending on the specific SSTR subtype, the final pathway and effect on cellular function may be different. In addition, SSTR signaling is further modulated by SSTR signaling pathway receptor endocytosis and trafficking (Fig. 4; Hofland & A conformational change of the receptor after SST binding Lamberts 2003, Lesche et al. 2009). leads to activation of an associated heterotrimeric G-protein complex (consisting of a-, b-, and g-subunits) Mechanism of action for pituitary tumor growth and exchange of GTP for GDP on the a-subunit. Through inhibition pertussis toxin-sensitive inhibitory Gi protein (Gai) activation, adenylyl cyclase activity is hampered in all SST has a potent antiproliferative effect through both five SSTRs. This results in reduction of intracellular cAMP direct and indirect mechanisms.

SSTR5 Ca2+

NH2 SRL SST

PIP2 PIP3 PLC Adenylyl PKC cyclase

β COOH Voltage-gated G Gα i Ca2+ channel γ G DAG GDP GTP MAPK Gβ IP3 2+ 2+ Ca γ α Ca G G i PP3R ERK1/2

Journal of Molecular Endocrinology IP3R-gated Ca2+ channel Endoplasmic reticulum

Cell growth c-fos arrest

Figure 3 Schematic of the intracellular signaling pathways modulated by SSTR2 which in turn inhibits the mammalian target of rapamycin (mTOR) also after somatostatin (SST) or SRLs action. From left to right, the anti- inducing cell apoptosis. Additionally, SHP1 associates with nitric oxide proliferative pathway is activated through three different phosphotyrosine synthase (NOS) and dephosphorylates it, leading to NOS activation and phosphatases (PTPs), SHP1, SHP2, and PTPh (Pan et al. 1992). SHP2 activates nitric oxide (NO) production. NO activates soluble guanylate cyclase (GC), Src that directly interacts with PTPh inducing its phosphorylation and which converts GTP to cGMP. Increased cGMP inhibits cell growth activation (Lopez et al. 1997). Then, Ras and Rap1-GTP inhibits the (Ben-Shlomo & Melmed 2010). SST also induces ZAC1 expression through a phosphatidylinositide-3-kinase (PI3K) target Akt. This, in turn, activates the mechanism involving Gai, SHP1, GSK3b, and the Zac1 activator p53. Zac1 is MAPK pathway stimulating the ERK1/2, and also p38, which have as targets capable of inducing apoptosis and cell cycle arrest (Theodoropoulou et al. C Elk1, and the activating transcription factor 2 (ATF2) (Sellers et al. 2000). In 2010). Finally, SST has key anti-secretory effects increasing K efﬂux and addition, the glycogen synthase kinase 3 (GSK3b) is phosphorylated and membrane hyperpolarization, which in turn closes voltage-dependent inhibited. The results are upregulation of p21Cip1 and p27Kip1, which will Ca2C channels decreasing intracellular Ca2C inﬂux and concentration. arrest cell cycle at the G1/S transition phase (Ben-Shlomo & Melmed 2010, Adenylyl cyclase inhibition also lowers cAMP levels and PKA activity Theodoropoulou et al. 2010). Activated SHP1 also triggers intracellular hampering hormone secretion (Patel et al. 1994). Activated pathway, black pro-apoptotic signals involving the induction of caspases activation and arrows; inhibited pathway, red arrows. p53/Bax. GSK3b activates the tumor suppressor tuberin TSC2/TSC1 complex,

SSTR

SSTS

Recycling

Internalization Degradation

β-arrestin

β-arrestin Lysosome = Phosphorylation Desensitization

Figure 4 Schematic of SSTR desensitization through mechanisms involving (Lesche et al. 2009). Recently, lower expression of the scaffold protein internalization and recycling, or receptor degradation. Internalization b-arrestin 1 correlated with less SSTR2 desensitization and improved SRL degree varies depending on SSTR type (Table 1). It also depends whether responsiveness in GH-secreting pituitary adenomas (Gatto et al. 2013). the stimuli is through SST or a different somatostatin receptor ligand (SRL)

Direct antiproliferative mechanisms The Gai sub- cGMP levels and inhibit cell growth (Fig. 3; Ben-Shlomo &

Journal of Molecular Endocrinology unit is activated (after SST binding at SSTR2), inducing the Melmed 2010). antiproliferative second messenger pathway associated Although CDK inhibitor 1B (p27Kip1) is an important with protein tyrosine phosphatases (PTPs; Pan et al. 1992). target of the antiproliferative action of SST, the rat This pathway involves activation of the cytosolic Src pituitary tumor GH3 cells do not express p27Kip1 homology 2 (SH2) domain, SHP1 (PTPN6), and SHP2 (Cdkn1b), but shows decreased cell proliferation through (PTPN11), and the membrane-anchored PTPh (DEP1 SSTR2 activation. In these cells, SSTR2 induces ZAC1 (PTPRJ)) (Lopez et al. 1997; Fig. 3). This, in turn, activates (ZACN) expression through a mechanism involving Gai, the serine/threonine MAPK pathway. The MAPK pathway SHP1, GSK3b,andtheZAC1activatorp53(Fig. 3; usually mediates the mitogenic action of growth factors, Theodoropoulou et al. 2010). ZAC1 is a zinc ﬁnger protein, cytokines, and hormones. However, depending on the cell predominant in normal adenohypophysis, capable of system, it can also halt cell growth in order to promote inducing apoptosis and cell cycle arrest (Theodoropoulou differentiation. Thus, SSTR-induced MAPK activation et al. 2010). Loss of expression frequently occurs in NFAs causes cell cycle arrest with up-regulation of two key cell (Theodoropoulou et al. 2010, Vieria Neto et al. 2013). inhibitors: p21Cip1 and p27Kip1, which will prevent the ZAC1 target genes include the pituitary adenylate cyclase- formation of the cyclin-dependent kinase (CDK) activating polypeptide type 1 receptor (PAC1R), peroxi- complexes, arresting the cell cycle at the G1/S transition some proliferator-activated receptor g (PPARg), and phase (Fig. 3; Ben-Shlomo & Melmed 2010, Theodoropou- p21Cip1 and p57Kip2 via coactivation of p53 and p73 lou et al. 2010). Activated SHP1 and the glycogen synthase (Theodoropoulou et al. 2010). ZAC1 gene expression kinase 3b (GSK3b) also trigger intracellular pro-apoptotic increases if phosphoinositide 3-kinase (PI3K) is inhibited signals (Fig. 3). Additionally, SHP1 associates with nitric using octreotide treatment, leading to inhibition of oxide synthase (NOS) and dephosphorylates it to increase 3-phosphoinositide-dependent protein kinase 1 (PDK1)

and the protein Ser/Thr kinase (Akt) activities that, in turn, through receptor heterodimerization (Rocheville et al. result in GSK3b activation and increased p53 transcrip- 2000). Interestingly, SSTR5 truncated variants showed tional activity that up-regulates ZAC1 expression (Fig. 3; intracellular distribution and preserved functionality with C Theodoropoulou et al. 2010). ligand-induced Ca2 and cAMP despite being truncated, By activation of SHP1-mediated E-cadherin depho- suggesting distinct roles after SST action (Co´rdoba-Chacoń sphorylation, SSTR2 activity also suppresses tumor inva- et al. 2010). siveness restoring E-cadherin function, which is reduced with cell dedifferentiation (Benali et al. 2000). Indirect antiproliferative mechanisms IGF1 Antiproliferative effects of SSTR5 are mediated reduction after inhibition of GH secretion is the most through permeability transition pore (PTP)-independent important indirect mechanism of cell growth inhibition pathways (Fig. 5). SSTR5 uses its inhibitory action on by SST (Shimon et al. 1997b). Activation of hepatic phospholipase C (PLC) and inositol 1,4,5-trisphosphate SSTR2 and SSTR3 down-regulates IGF1 transcription by C (IP3) to inhibit the intracellular release of Ca2 as well as dephosphorylation and inactivation of GH-induced C the extracellular Ca2 influx (Fig. 5; Wilkinson et al. STAT5b (Murray et al. 2004). Additional suppression of 1997). In addition, SSTR5 blocks cell proliferation inhibit- the secretion or synthesis of epidermal growth factor, ing MAPK and c-fos (Fig. 5; Cordelier et al. 1997). Cells platelet-derived growth factor, and transforming growth expressing both SSTR2 and SSTR5 have more efficacious factor a by SST contributes to a decrease in tumor growth adenylate cyclase inhibition, ERK1/2 activation, and (Florio 2008). p27Kip1 induction after SSTR2 activation by a selective Further indirect antiproliferative effects are endo- agonist, when compared with cells expressing SSTR2 thelial cell proliferation reduction with inhibition of alone. This finding suggests that amplification of the cell angiogenesis. Three signaling pathways are identified proliferation pathway inhibition may be actually achieved as responsible for the antiangiogenic activity of SST:

SSTR22 SSTR5 Active Cushing’s disease Journal of Molecular Endocrinology

ACTH-secreting adenoma cell

= SSTR2 receptor ligand ACTH and/or cortisol lowering therapy = SSTR5 receptor ligand

Controlled Cushing’s disease

ACTH-secreting adenoma cell

Figure 5 Schematic of the intracellular signaling pathways modulated by SSTR5 binds to its receptor (IP3R) at endoplasmic reticulum and release C after somatostatin (SST) or SRLs action. In contrast to SSTR2, the intracellular Ca2 whereas DAG recruits and activates PKC, which in turn C antiproliferative mechanism is activated through PTP-independent open voltage-gated Ca2 channels. SSTR5 uses its inhibitory action on PLC pathways. After inhibition of adenylate cyclase by Gai subunit activation, and IP3 to inhibit intracellular release of Ca2C as well as extracellular Ca2C SSTR5 affects phospholipase C (PLC) which in turn cleaves phosphatidyl- inﬂux (Wilkinson et al. 1997). In addition, SSTR5 blocks cell proliferation inositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol inhibiting MAPK and c-fos (Cordelier et al. 1997). Activated pathway, black 1,4,5-trisphosphate (IP3). The latter is released into the cytosol, where it arrows; inhibited pathway, red arrows.

i) down-regulation of the vascular endothelial growth Single-receptor-targeted SRLs factor (VEGF) and basic fibroblast growth factor (bFGF) transcription. VEGF drives the development of new vessels Octreotide and lanreotide Essential structural in the growing tumor as well as inhibits endothelial nitric features of SST include the b-turn comprising 7–10 AAs, oxidase and the following attenuation of nitric oxide, a as well as the cysteine–cys–cysteine (Cys–Cys) bridge second messenger that plays a pivotal role in angiogenesis between position 3 and terminal position 14, with the (Florio et al. 2003). SST also decreases VEGF cell most critical cleavage at tryptophan on position 8, which secretion and viability (Zatelli et al. 2007). ii) Inhibition leads to completely inactive fragments (Fig. 1; Brazeau of endothelial cell activity (proliferation, migration, and et al. 1973). The clinically available synthetic SRLs, SMS invasion). The antiproliferative effect is mediated by 201-995 or octreotide (Bauer et al. 1982) and BIM-23014 inhibition of ERK1/2 activity and endothelial NOS or lanreotide (Taylor et al. 1988), were developed by (eNOS). The anti-invasive effect of SST is dependent on capturing the Cys–Cys bridge and stabilizing the b-turn the inhibition of MAPK, the small G-protein RAC (ATK1), incorporating a D-Trp (Fig. 1). Both peptides display and the expression of metalloproteases such as MMP2 selective high-affinity binding to SSTR2, with a moderate (Florio et al. 2003). iii) Inhibition of monocyte activation. affinity for SSTR3 and SSTR5, and no binding to SSTR1 and SST inhibits monocyte migration and their recruitment in SSTR4 (Table 2; Ben-Shlomo & Melmed 2008). They have the areas where new vessels are forming (Florio 2008). enhanced metabolic stability (half-life of 2 and !1h respectively), a small volume of distribution, and low clearance, all resulting in a longer duration of exposure Somatostatin receptor ligands and consequently a long-lasting biological activity compared with SST. Furthermore, rebound hypersecretion The potent antisecretory properties of SST have made it an does not occur, making these ligands optimal for clinical important pharmacological target for treatment of hor- use (Ben-Shlomo & Melmed 2008). monal hypersecretion. However, its short half-life, the LAR formulation results from mixing octreotide multiple and simultaneous actions in different organs, the with microspheres of biodegradable glucose polymers need of parenteral administration, and the post-infusion and diluent (carboxymethylcellulose sodium, mannitol, rebound in GH, insulin, and glucagon release hampered and water). Therapeutic concentrations of the drug in its clinical use. SRLs were developed after manipulation of this suspension can be maintained for 24–42 days key SST structural characteristics, which allowed for (Ben-Shlomo & Melmed 2008). Octreotide LAR is

Journal of Molecular Endocrinology several improvements over the above disadvantages. 41–65% protein bound and 11–32% of the administered

Table 2 Somatostatin receptor ligands and chimeric compounds

SRL SSTR Dose Formulation Route Reference

Single-receptor targeted Octreotide 2O5 50–500 mg/8 h 50, 100, 500 mgin1ml s.c. Ben-Shlomo & 200, 1000 mg/ml in 5 ml Melmed (2008) Octreotide LAR 2O5 10–40 mg/3–4 weeks 10, 20, 30 mg in 5 ml i.m. Fleseriu (2011) Octreolin 2O5 20–80 mg/day For clinical trials only Oral Tuvia et al. (2012) Lanreotide SR 2O5 30–60 mg/7–28 days 30, 60 mg in 1 ml i.m. Ben-Shlomo & Melmed (2008) Lanreotide autogel 2O5 60–120 mg/1–4 weeks 60, 90, 120 mg pre-ﬁlled s.c. Ben-Shlomo & syringe Melmed (2008) Multireceptor targeted Pasireotide 5O2O3O1 100–900 mg/12–24 h 300, 600, 900 mg in 1 ml s.c. Petersenn et al. (2010) and Colao et al. (2011) Pasireotide LAR 5O2O3O1 40–60 mg/28 days For clinical trials only i.m. Bronstein et al. (2012) Somatoprim 2O4O5 – For experimental research – Pio¨ ckinger et al. (2012) Chimeric Dopastatin 2 and DA2R – For experimental research – Ren et al. (2003a)

DA2R, dopamine D2 receptor; SR, slow release; LAR, long-acting release.

drug is eliminated unchanged in the urine. Within 1 h of a 2.5 times lower binding afﬁnity to SSTR2, compared with single i.m. dose of octreotide LAR of 10–30 mg, an initial octreotide (Bruns et al. 2002). Due to longer half-life peak in serum concentrations occurs presumably from (w24 h), the once- or twice-daily dosing regimen main- drug adsorbed to the carrier microspheres; then, concen- tains steady-state concentrations (Table 2; Boscaro et al. trations decline within 12 h of drug administration, 2009, 2013, Fleseriu & Petersenn 2013). remaining subtherapeutic until day 7, before increasing Pasireotide modulates SSTR trafﬁcking in a different in a dose-dependent manner to a plateau in about 14 days manner than octreotide, resulting in rapid recycling of (Table 2). The plateau remains stable until days 35–60 and SSTR2 to the plasma membrane after endocytosis (Lesche then steadily declines. Octreotide LAR (20 or 30 mg) at et al. 2009). While octreotide and SST14 phosphorylated at 4-week intervals reaches the steady state after three least six carboxy-terminal serine and threonine residues of

injections (BBen-Shlomo & Melmed 2008). Therapeutic SSTR2A, pasireotide stimulates selective phosphorylation drug concentration ranges between 1 and 3 mg/l. of S341 and S343 residues, which was followed by Lanreotide is also available in two long-acting its internalization (Nagel et al. 2011). This accelerated formulations. Sustained-release lanreotide (lanreotide recycling may counteract the desensitization of SSTR2 SR), prepared in microspheres of biodegradable lactide/ (Fig. 4). glycolide copolymers, is administered by i.m. injection at A LAR form of pasireotide was developed using a dose of 10–30 mg every 7–14 days, and 60 mg every biodegradable polymers similar to octreotide LAR 21–28 days. A second formulation is lanreotide autogel, (Table 2). Pasireotide LAR is undergoing clinical trials consisting of a solution that is a long-acting viscous (http://clinicaltrials.gov) for CD (NCT01374906), acrome- aqueous formulation of lanreotide, which is supplied galy (NCT01137682, NCT01673646, NCT00600886, and in ready-to-use preﬁlled syringes (does not require NCT00446082), and NFAs (NCT01283542). reconstitution) intended for deep s.c. injection. It has a linear pharmacokinetic proﬁle and is administered every Somatoprim DG3173 (somatoprim (Fig. 1)) is a novel 28–56 days (Table 2; Ben-Shlomo & Melmed 2008). SRL that binds to SSTR2, SSTR4, and SSTR5 (Table 2). In vitro studies reported a higher response to somatoprim Oral delivery An oral formulation for octreotide than octreotide, including an additional 38% of pituitary (Octreolin; Chiasma) became feasible with the transient adenomas resistant to octreotide. Furthermore, the lower permeability enhancer technology that enables the drug insulin-suppressing activity may be associated with to be absorbed by the intestine. A phase II study less hyperglycemic effect than the other SRLs (Pio¨ckinger

Journal of Molecular Endocrinology demonstrated equivalent pharmacokinetic parameters et al. 2012). using 20 mg oral octreotide or 100 mg s.c. octreotide, supporting oral octreotide as an alternative to parenteral Dopastatin The combination of individual SSTR2 and formulation to treat acromegaly patients (Table 2; Tuvia dopamine D2 receptor (DAR2) agonists shows an additive et al. 2012). A phase III trial is ongoing to investigate the effect on suppressing GH secretion in both rat and human efficacy and safety of octreolin in patients with acromegaly GH-secreting pituitary cells (Ren et al. 2003a). BIM- (http://clinicaltrials.gov/ct2/show/NCT01412424). 23A387 or dopastatin is a chimeric compound containing structural elements of both SST and dopamine in a single molecule, retaining potent and selective binding to both Multireceptor-targeted SRLs and chimeric molecules SSTR2 and DAR2 (Table 2; Ren et al. 2003a). However, Pasireotide Essential functional groups of the SST clinical studies with dopastatin have been halted due to peptide with high binding affinity to SSTRs were detected insufficient SST-like activity. using the alanine scanning technology. Incorporation of four synthetic and two essential AAs of SST in the form of a novel basic trans-(L)-hydroxyproline aminoethyl-urethane SRL effects in pituitary tumors extension, phenylglycine, O-benzyl-tyrosine, and D-Trp to GH-secreting pituitary tumors corresponding positions into a stable cyclohexapeptide template resulted in SOM230 (pasireotide), a multiligand Octreotide and lanreotide SRLs represent the main- SRL (Fig. 1; Bruns et al. 2002, Fleseriu & Petersenn 2013). stay of medical therapy for acromegaly (Melmed 2006, SOM230 displays a 40, 30, and 5 times higher binding Melmed et al. 2009). In contrast to the tachyphylaxis of affinity to SSTR5, SSTR1, and SSTR3 respectively, and a physiological GH secretion after continuous receptor

activation seen within hours to days (Hofland & Lamberts in 31 vs 19% of patients treated with octreotide LAR. 2003), therapeutic escape has not been observed in Notably, this study included a large majority of patients acromegaly, even after prolonged treatment. Several with no previous history of pituitary surgery (Bronstein clinical studies observed normalization of GH and IGF1 et al. 2012). Furthermore, switching patients who were not levels and amelioration of clinical symptoms in about controlled on octreotide LAR to pasireotide LAR enabled 60–70% of acromegaly patients (Ben-Shlomo & Melmed 21% of patients to achieve biochemical control (Fleseriu 2008, Melmed et al. 2009, Fleseriu 2013). However, et al.2012). However, the frequency and degree of prospective studies that included patients, not preselected hyperglycemia and diabetes in all pasireotide studies were forSRLefficacy,haveshownmuchlowerratesof significantly higher compared with octreotide. biochemical control (Mercado et al. 2007, Melmed et al. 2010, Bronstein et al. 2012). The efficacies of octreotide SRLs in ACTH-secreting pituitary adenomas LAR and lanreotide autogel seem to be similar (Ben- Shlomo & Melmed 2008, Murray & Melmed 2008, Melmed Octreotide and lanreotide Octreotide is able to 2009). Dose escalation provide additional biochemical suppress ACTH levels in corticotropinoma primary cell control in patients who were inadequately controlled with culture in vitro, but is without effect in vivo. Octreotide and conventional starting SRL doses, without significantly lanreotide are SSTR2-preferring ligands: their low efficacy changing safety and adverse events (Fleseriu 2011). in reducing ACTH levels is most probably due to low SSTR2 Tumor shrinkage occurs in about 33% (10–77%) of expression of these tumors in hypercortisolemic states patients treated with lanreotide autogel (Mazziott & (Fig. 2; de Bruin et al. 2009). Giustina 2010) and about 53% (45–61%) and 66% (57–74%) of patients with octreotide and octreotide LAR Pasireotide ACTH secretion in human corticotropi- respectively (Giustina et al. 2012). As expected, naı¨ve noma primary cell cultures as well as in mouse AtT20 patients experience a better response compared with corticotropinoma cells resistant to octreotide (due to those previously treated with radiotherapy and surgery glucocorticoid-induced SSTR2 down-regulation) is sup- (Mazziott & Giustina 2010). pressed by pasireotide. In stable AtT20 cells overexpressing Overall, the true efficacy of SRLs and all medical SSTR2 and SSTR5, short- and long-term enhanced pasireo- therapies in general are somewhat difficult to assess tide action and its potency were not affected by SSTR2 because of varying study entry criteria (de novo, post- abundance, SSTR2 antagonist, or octreotide co-treatment surgery, or preselected sensitivity to SRLs) and changing (Ben-Shlomo et al. 2009). Studies in dogs with CD showed

Journal of Molecular Endocrinology biochemical cutoff goals over time (Bronstein et al. 2012, both ACTH and cortisol reduction with symptoms Fleseriu 2013). However, only a small minority (!10%) ameliorated after 6 months of pasireotide treatment should be considered to be fully resistant to SRLs (Colao (Castillo et al. 2011). Of the 29 patients in the primary et al. 2011). efficacy analysis of the phase II study in CD, 22 (76%) Advances in combination medical therapy offer new showed a reduction in urine free cortisol (UFC) levels, after perspectives for those patients who are partially resistant 15 days of treatment with 600 mg pasireotide twice daily. to available SRLs. Of them, 17% achieved normal UFC levels; serum cortisol levels and plasma ACTH levels were also reduced. Steady- Pasireotide Pasireotide induces a dose-dependent state plasma concentrations of pasireotide were achieved decrease in GH and IGF1 levels in rats, dogs, and monkeys within 5 days of treatment; interestingly, responders (Bruns et al. 2002). In human mixed GH/PRL-secreting appeared to have higher exposure than non-responders adenomas, pasireotide suppressed both PRL and GH, which (Boscaro et al. 2009). A large phase III study showed was also confirmed in vitro using Hmga2 overexpressing normalization of UFC in 15 and 26% for the 600 and mice (Hofland et al. 2004, Fedele et al. 2007). In a phase II 900 mg doses respectively, including normalization of UFC study in acromegaly patients, after 4 weeks of 200–600 mg in w50% of the mild cases (defined as UFC less than s.c. pasireotide twice daily, 19% of patients achieved twofold of the upper limit of normal (ULN) at baseline) on biochemical control (GH %2.5 mg/l and normal IGF1). 900 mg twice a day treatment. However, pasireotide was This increased to 27% after 3 months and 39% of patients associated with hyperglycemia-related adverse events in had a significant (O20%) reduction in pituitary tumor 118 out of 162 cases (Colao et al. 2012). The therapeutic volume (Petersenn et al. 2010). In a large phase III clinical effect of pasireotide is through activation of SSTR5 and the trial, pasireotide LAR achieved disease control at 12 months drug also enhances ACTH and cortisol suppression via

restored SSTR2 expression (Fig. 2; van der Pas et al. 2013). ‘Tumor resistance’ to SRLs is usually defined as !20% In addition to clinical improvement and UFC reduction, shrinkage from baseline volume (Colao et al. 2011) and salivary cortisol can be a useful parameter to monitor could vary considerably between patients treated with treatment responsiveness (Biller et al. 2013). Pasireotide is first- or second-line therapy (Mazziotti & Giustina 2010, the first pituitary-targeted therapy available for treatment Giustina et al. 2012). of CD (Colao et al. 2012, Fleseriu & Petersenn 2012, However, before considering SRL resistance, both Fleseriu & Petersenn 2013, Tritos & Biller 2013). Further- duration (at least 12 months) and doses of SRL should be more, interaction between SRLs and dopamine receptor optimized (Fleseriu 2011). In recent studies, high-frequency agonists may allow for synergistic suppression of ACTH, (30 mg every 21 days) or high-dose (40 or 60 mg every but the role of combination therapy must be further 28 days) octreotide LAR achieved additional clinical, characterized (Fleseriu 2012, Fleseriu & Petersenn 2013). biochemical, and/or tumor shrinkage in 25% of patients with acromegaly (Giustina et al.2009, Fleseriu 2011). An algorithm proposed in March 2013 by the Acro- SRLs in other pituitary tumors megaly Consensus Group (Expert consensus document: Although long-term data are limited, most patients with a consensus on the medical treatment of acromegaly) for TSH-secreting pituitary adenomas respond well to either the medical management of acromegaly after surgery or as octreotide or lanreotide after surgery. Octreotide induces primary treatment strategy when surgery is inappropriate tumor shrinkage in 30–50% of TSH-secreting pituitary was recently published (Giustina et al.2014). Therapeutic adenomas (Ben-Shlomo & Melmed 2010). approaches for patients with SRL resistance were also Prolactinomas with predominant SSTR5 expression reviewed in the guidelines and elsewhere (Fleseriu 2013, respond weakly to the SSTR2-directed ligands octreotide Gadelha et al.2013, Giustina et al.2014). For patients or lanreotide; however, in vitro experiments show a high who do not have any response to SRL therapy, switching PRL-suppressive effect using a selective SSTR5 agonist to pegvisomant treatment is optimal. For patients with (Shimon et al. 1997a,b, Jaquet et al. 1999). inadequate response, administration of high-dose SRLs NFAs predominantly express SSTR2 (Table 1); however, alone or in combination with the GH receptor antagonist the efficacy of SRLs remains to be proven. Tumor shrinkage pegvisomant and/or the dopamine agonist cabergoline has been sporadically reported in 12% of patients under SRL should be considered (Colao et al.2011, Fleseriu 2013, treatment. The mechanism may be related to the sup- Gadelha et al.2013, Giustina et al.2014). Repeat pituitary pressive action of VEGF and PI3K inhibition. surgery for tumor debulking and/or radiotherapy could also

Journal of Molecular Endocrinology be entertained (Colao et al.2011, Giustina et al.2014). Definition of resistance to SRLs ACTH-secreting pituitary adenomas Definition of SRL resistance in pituitary tumor therapy should take treatment characteristics, biochemical con- The definition of remission in CD is controversial, as there trol, and tumor shrinkage response into consideration is no consensus on the most accurate assessment method (Colao et al. 2011). or exact timeline, either postoperative or during medical treatment. Persistent immediate postoperative morning serum cortisol levels of !2 mg/dl (w50 nmol/l) are GH-secreting pituitary adenomas associated with remission (Biller et al. 2008, Hameed Criteria for biochemical remission in acromegaly include et al. 2013), but delayed remission has also been noted. normal IGF1, random GH !1 mg/l, and a nadir GH after 75-g Normalization of hypercortisolemia (normal UFC and oral glucose tolerance test !0.4 mg/l (Giustina et al. 2010). salivary cortisol) remains the main goal of therapy in CD Use of optimal GH and IGF1 assays and correction for (Biller et al. 2008, 2013). gender, age, and BMI are essential. SRLs are rarely completely ineffective in GH-secreting pituitary tumors (Hofland & Lamberts 2003, Colao et al. 2011); however, there is no exact Determinants of SRL responsiveness definition of partial resistance. In those patients without GH-secreting pituitary adenomas biochemical remission after 6 months of first-line treatment with SRLs, surgical debulking and then a second course of Clinical predictors Age at diagnosis has been iden- SRL may improve biochemical control (Colao et al. 2011). tified as a possible clinical marker of tumor size and

aggressiveness (Table 3). Younger patients tend to have both after surgery and in response to SRL (Melmed 2006, larger and more aggressive tumors than older patients 2009). As expected, patients treated with ﬁrst-line with less SRL response. Female patients and lower therapy with SRLs achieved better shrinkage than those circulating IGF1 and GH levels at diagnosis have also treated with second-line therapy after unsuccessful surgery been associated with better SRL response (Brzana et al. (Mazziotti & Giustina 2010, Giustina et al. 2012). Tumor 2013, Gadelha et al. 2013). growth during SRL treatment is extremely rare (!2%).

Tumor characteristics on magnetic resonance Adenoma intensity by magnetic resonance imaging Adenoma imaging Tumor size and invasiveness Radiological evalu- hypointensity on T2-weighted MR images is associated ation usually reveals a lower percentage of microadenomas with densely granulated, less invasive adenomas, with (!10 mm in size, 20%) than macroadenomas (R10 mm, better SRL response (Heck et al. 2012). By contrast, T2 80%) (Melmed 2006). The surgical cure rate depends on the hyperintensity imaging suggests a sparse granulation tumor size at presentation and is higher for microadeno- pattern as assessed by immunohistochemistry, with mas (81%) than non-invasive (71%) or invasive (37%) more invasive and less SRL-responsive tumors (Heck macroadenomas. Tumor invasiveness was shown to et al. 2012). Therefore, T2 MR imaging signal may be hamper surgical resection and reduce cure probability able to identify responsiveness to SRL and prognosis

Table 3 Summary of the SRL resistance determinants

Parameter Determinant Description

Clinical Female gender Female patients showed better response to SRL probably due to SSTR2 up-regulation by estrogens Age Age at diagnosis is a clinical marker of tumor size and invasiveness. Younger patients potentially harbor more aggressive GH-secreting pituitary tumors Imaging Tumor size Tumor size and invasiveness hamper surgical resection and reduced cure probability both after surgery and in response to SRL MRI intensity T2 hyperintensity suggests sparse granulation pattern on immunohistochemistry, with more invasive and less SRL-responsive tumors Histopathology Granulation pattern Densely granulated GH-secreting pituitary adenomas are associated with better SRL response than those with sparsely granulated adenomas Journal of Molecular Endocrinology SSTR2 expression SSTR2 expression is positively correlated with SRL response at both mRNA and protein levels Ki-67 index Adenomas with high labeling index of the proliferation marker Ki-67 are more invasive and SRL resistant ZAC1 expression Positive correlation exists between treatment response and ZAC1 immunoreactivity b-arrestins Lower expression of b-arrestin 1 in GH- and PRL-secreting pituitary adenomas correlated with reduced recycling rate of SSTR2 and better SRL response RKIP RKIP in GH-secreting pituitary adenomas correlated with poor octreotide treatment response AIP expression AIP expression, even without AIP gene mutation, has been associated with octreotide LAR resistance Molecular SSTR5 mutation (Arg240Trp) Increases mitogenic pathways resulting in lack of GH inhibition and aberrant cell proliferation with octreotide treatment SSTR5TMD4 and SSTR5TMD5 These SSTR5-truncated isoforms correlated with reduced response to octreotide treatment in acromegalic tumors C1004T and T-461C Polymorphisms at the SSTR5 gene that have been associated with SRL resistance AIP gene mutation Familial and sporadic mutations of AIP gene predict an unfavorable SRL response SSTRs Desensitization Desensitization of SSTRs seems to have a minor, if any, role in determining different responsiveness to SRLs Heterogeneous expression In contrast to receptor desensitization, reduced SSTR density can explain a high proportion of patients partially resistant to SRLs

MRI, magnetic resonance imaging.

in patients with active acromegaly after unsuccessful recycling rate of SSTR2, a higher amount of biologically surgery (Table 3). active receptor exposed on the cell membrane, and better SRL response in terms of GH suppression, both in vitro and Histopathological predictors Granulation pattern in vivo (Table 3; Gatto et al. 2013). Interestingly, Densely granulated GH-secreting pituitary adenomas are pasireotide treatment resulted in lower b-arrestin recruit- associated with a better SRL response than those with ment with reduced SSTR internalization and trafficking sparsely granulated adenomas (Brzana et al. 2013, Gadelha in vitro (Lesche et al. 2009). et al. 2013). The increased responsiveness of densely Raf kinase inhibitory protein Raf kinase inhibitory protein (RKIP) granulated tumors to SRLs has been related to high functions as MAPK inhibitor in the SSTR2 signaling SSTR2A expression and high adenylate cyclase levels cascade. RKIP (PEBP1) expression is not correlated with associated with the GSP mutation. Factors associated SSTR2 expression; however, low levels of RKIP in with lower SRL responsiveness in sparsely granulated GH-secreting pituitary adenomas correlated with poor adenomas are lower SSTR2A immunoreactivity (Brzana octreotide treatment response (Fougner et al. 2008). et al. 2013), somatic mutation in GH receptor interfering with post-translational processing, maturation, ligand binding, and signaling (Asa et al. 2007) as well as decreased Molecular predictors E-cadherin immunoreactivity (Fougner et al. 2010). Mutations in SSTR genes and truncated SSTR5 var- SSTR2 expression SSTR2 expression is positively correlated iants The genes encoding SSTR1–SSTR5 are given in with SRL response at both mRNA and protein levels Table 1. Mutations in SSTR2 and SSTR5 genes are rarely (Table 3; Gadelha et al. 2013). Monoclonal anti-SSTR2 encountered (Ballare` et al. 2001), and their polymorph- antibody immunostaining of paraffin-embedded tissues isms may influence basal and IGF1 levels (Table 3). predicted SRL response in patients with acromegaly However, polymorphic variants in SSTR2 and SSTR5 (Brzana et al. 2013). A high SSTR2 to SSTR5 ratio correlates genes seem to have a minor role in determining with biochemical control under octreotide treatment SRL resistance in patients with acromegaly (Lania et al. (Gadelha et al. 2013). 2008). The SSTR5 truncated variants may account for Ki-67 proliferation index Tumors with a high proliferation of differential effects observed in different tissues upon SRL marker Ki-67 are more invasive and SRL resistant (Table 3). treatment (Table 3; Durań-Prado et al. 2009, Co´rdoba- Moreover, a lower Ki-67 index is identified in SRL- Chacoń et al. 2010). Journal of Molecular Endocrinology responsive treated patients, which could be due to their antiproliferative effects (Florio 2008). Mutations in aryl hydrocarbon receptor interact- ing protein gene Aryl hydrocarbon receptor interact- ZAC1 ZAC1 is a tumor suppressor gene that was cloned ing protein (AIP) is a tumor-suppressor gene, which has from immortalized GH-secreting adenoma cells and been shown to induce tumor shrinkage via ZAC1 (Gadelha mediates, at least in part, the antiproliferative action of et al. 2013). Patients with AIP gene germ line mutations octreotide (Theodoropoulou et al. 2010). A retrospective are usually male, with young-onset, sparsely granulated study of patients with acromegaly treated preoperatively GH-secreting pituitary tumors or GH/PRL mixed adeno- with SRLs revealed a positive correlation between mas (Gadelha et al. 2013). Familial and sporadic mutations treatment response (both IGF1 normalization and of the AIP gene predict an unfavorable SRL response. AIP tumor volume reduction) and ZAC1 immunoreactivity expression, even without an AIP gene mutation, has also (Theodoropoulou et al. 2010). been associated with octreotide LAR resistance (Table 3). b-arrestins b-arrestins (1 and 2) are scaffold proteins recruited The mechanism seems to be independent of SSTR2 on the cell membrane to regulate uncoupling of expression. However, SRL responsiveness when both G-protein-coupled receptors and are thought to partici- markers are used together is predicted with an accuracy pate in the desensitization process. In particular, b-arrestin higher than that when each is used individually (Gadelha 1 plays an important role in SSTR2 desensitization upon et al.2013). In addition to biochemical control, agonist activation (Fig. 4). Therefore, lower expression of tumor shrinkage after SRL treatment was significantly b-arrestin 1 mRNA in GH- and PRL-secreting pituitary higher in the control group (40%) than in the AIP-mutated adenomas, compared with NFA, correlates with a reduced patients (0%) (Gadelha et al. 2013). Furthermore, SSTR5

staining is slightly higher in AIP-mutated tumors than in decreased GH secretion in in vitro tumor cells derived sporadic ones. from patients partially responsive or resistant to SRLs. SSTR1 mRNA levels correlated with the degree of inhibition SSTR desensitization Many G-protein-coupled of GH secretion in vitro (Zatelli et al. 2003, Matrone et al. receptors, including SSTR1–SSTR5, show the ability to 2004), as well as SSTR1 mRNA expression was associated regulate their responsiveness to continued agonist with a higher proportion of patients with GH and IGF1 exposure with different degrees of receptor internalization normalization using SRLs (Casarini et al. 2009). and degradation (Table 1; Hofland & Lamberts 2003). Although the acute administration of SST produces ACTH-secreting pituitary adenomas In vitro, and multiple inhibitory effects, the initial response diminishes in vivo, studies show that hydrocortisone treatment as well with continued exposure to the peptide. However, in as endogenous hypercortisolemia hampers ACTH suppres- patients with acromegaly who respond well to SRLs, GH sion by SST or octreotide treatment. By contrast, ACTH secretion can be inhibited without escape after prolonged secretion was significantly suppressed in normal cortico- treatment periods (Hofland & Lamberts 2003). Therefore, trope cells cultured in glucocorticoid-free medium as well desensitization of SSTRs seems to have a minor role, if any, as in patients with adrenal insufficiency (Lamberts 1988). in determining different responsiveness to SRLs. A plausible explanation is the down-regulation of SSTR2 expression in corticotrope adenomas by glucocorticoids (Fig. 2). Interestingly, after achieving eucortisolemia with Heterogeneity of SSTR expression pasireotide in patients with CD, cortisol-mediated SSTR2 GH-secreting pituitary adenomas In contrast to down-regulation in corticotrope adenomas was reversible receptor desensitization, variable tumor expression or at the mRNA level, but not at the protein level. It is not reduced receptor density of SSTR subtypes can explain clear if sustained eucortisolemia induced by medical the high proportion of patients partially resistant to SRLs therapy can induce re-expression of functional SSTR2 (Reubi et al. 1994, 2001, Casarini et al. 2009). Positive protein (van der Pas et al. 2013). correlation exists among SSTR2 mRNA expression, SSTR2 immunohistochemistry positivity, or membrane receptor density with the in vivo GH suppression induced by SRLs Conclusions (Casarini et al. 2009, Brzana et al. 2013). Therefore, SSTR2 seems to be a predominant receptor in determining the SST and SSTR are broadly expressed in humans, including

Journal of Molecular Endocrinology inhibitory effect of octreotide or lanreotide on circulating normal pituitary tissue and pituitary adenomas. Thus, GH release in acromegalic patients. However, loss of SSTR2 SRLs have a therapeutic value in several pituitary is sometimes encountered in partially SRL-sensitive disorders; they are the mainstay of medical therapy in tumors, where SSTR5 mRNA expression is higher and acromegaly and have also been recently approved for CD seems to explain the incomplete resistance despite low patients after failure of surgery. However, a large pro- SSTR2 mRNA expression (Reubi et al. 1994, Nielsen et al. portion of patients are at least ‘partially resistant’ to 2001, Brzana et al. 2013). Interestingly, GH suppression treatment in both diseases. The term SRL ‘resistance’ per se with SSTR2 and SSTR5 bi-specific compound was better remains elusive. The exact mechanism is yet unclear, and achieved (73%) when compared with the use of SSTR2 it is likely that multiple factors are involved. (32%) or SSTR5 (34%)-selective agonists (Ren et al. 2003b). Clinical predictors of response in patients with Additionally, in co-secreting GH and PRL adenomas, PRL acromegaly include gender, age, initial GH and IGF1 secretion is preferentially inhibited by SSTR5-specific levels, and possibly tumor size. Several molecular pre- ligands (Shimon et al. 1997a,b, Jaquet et al. 2000). There- dictors such as reduced sensitivity of SSTRs, SST receptor fore, some adenomas show better response to SSTR2- internalization, heterogeneity of SSTR expression, and specific ligands, whereas in others, SSTR5-specific ones are signaling pathway disruption seem to also play an more potent. The additive inhibitory effects on GH release important role. following activation of both SSTR2 and SSTR5 are likely Development of novel multiligand SRLs, chimeric mediated via a functional interaction of both SSTR molecules, and combination therapies could further subtypes (Rocheville et al. 2000, Ren et al. 2003b). Finally, improve the response rates and/or decrease the incidence SSTR1 may also play a role in patients poorly sensitive to of adverse events in acromegaly, CD, and other hyper- SRLs. Activation of SSTR1 by a SSTR1-selective ligand secretory or non-functioning pituitary tumors.

We recommend to include both biochemical and Biller BM, Grossman AB, Stewart PM, Melmed S, Bertagna X, Bertherat J, tumor effects to define the concept of SRL resistance in a Buchfelder M, Colao A, Hermus AR, Hofland LJ et al. 2008 Treatment of adrenocorticotropin-dependent Cushing’s syndrome: a consensus better way. Further understanding of the clinical, molecu- statement. Journal of Clinical Endocrinology and Metabolism 93 lar, and histopathological predictors of resistance will help 2454–2462. (doi:10.1210/jc.2007-2734) optimize individualized treatment for each patient. Biller MB, Petersenn S, Pivonello R, Findling J, Fleseriu M, Trovato A, Hughes G, Ligueros-Saylan M & Newell-Price J 2013 Evaluation of late- night salivary cortisol during 12 months of pasireotide treatment in patients with Cushing’s disease. Endocrine Reviews 34 (03_Meeting- Abstracts): MON-89. Declaration of interest Boden G, Ryan IG, Eisenschmid BL, Shelmet JJ & Owen OE 1986 Treatment D C-R declare that there is no conflict of interest that could be perceived as of inoperable glucagonoma with the long-acting somatostatin prejudicing the impartiality of the review. analogue SMS 201-995. New England Journal of Medicine 314 1686–1689. (doi:10.1056/NEJM198606263142606) Boscaro M, Ludlam WH, Atkinson B, Glusman JE, Petersenn S, Reincke M, Funding Snyder P, Tabarin A, Biller BM, Findling J et al. 2009 Treatment of M F has received research grants to OHSU from Ipsen, Novartis, and Corcept pituitary-dependent Cushing’s disease with the multireceptor ligand Therapeutics and has been an ad hoc scientific advisor for Genentech, somatostatin analog pasireotide (SOM230): a multicenter, phase II trial. Journal of Clinical Endocrinology and Metabolism Ipsen, and Novartis. 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Received in ﬁnal form 5 March 2014 Accepted 18 March 2014 Accepted Preprint published online 18 March 2014