Octreotide and Pasireotide (Dis)Similarly Inhibit Pituitary Tumor Cells in Vitro

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a ibáñez-costa and others Octreotide vs pasireotide in 231:2 135–145 Research pituitary tumors

Octreotide and pasireotide (dis)similarly inhibit pituitary tumor cells in vitro

Alejandro Ibáñez-Costa1,2,3,4, Esther Rivero-Cortés1,2,3,4, Mari C Vázquez-Borrego1,2,3,4, Manuel D Gahete1,2,3,4, Luis Jiménez-Reina1,5, Eva Venegas-Moreno6, Andrés de la Riva7, Miguel Ángel Arráez8, Inmaculada González-Molero9, Herbert A Schmid10, Silvia Maraver-Selfa11, Inmaculada Gavilán-Villarejo12, Juan Antonio García-Arnés9, Miguel A Japón13, Alfonso Soto-Moreno6, María A Gálvez1,14, Raúl M Luque1,2,3,4,* and Justo P Castaño1,2,3,4,*

1Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Córdoba, Spain 2Department of Cell Biology, Physiology and Immunology, Universidad de Córdoba, Córdoba, Spain 3Hospital Universitario Reina Sofía, Córdoba, Spain 4CIBER Fisiopatología de la Obesidad y Nutrición (CIBERObn), Córdoba, Spain 5Department of Morphological Sciences, Universidad de Córdoba, Córdoba, Spain 6Metabolism and Nutrition Unit, Hospital Universitario Virgen del Rocío, Instituto de Biomedicina de Sevilla (IBIS), Seville, Spain 7Service of Neurosurgery, Hospital Universitario Reina Sofía, Córdoba, Spain 8Neurosurgical Department, Carlos Haya Hospital, Málaga, Spain 9Department of Endocrinology and Nutrition, Carlos Haya Hospital, Málaga, Spain 10Novartis Pharma AG, Novartis Institutes for Biomedical Research, Oncology, CH-4057 Basel, Switzerland

Endocrinology 11Service of Endocrinology and Nutrition, Hospital Clínico Universitario Virgen de la Victoria, Málaga, Spain

of 12 Endocrinology and Nutrition Unit, Hospital Universitario Puerta del Mar, Cádiz, Spain Correspondence 13 Department of Pathology, Hospital Universitario Virgen del Rocío, Seville, Spain should be addressed 14 Service of Endocrinology and Nutrition, Hospital Universitario Reina Sofía, Córdoba, Spain to R M Luque or J P Castaño Journal *(R M Luque and J P Castaño co-directed this study) Email [email protected] or justo@ uco.es

Abstract

Somatostatin analogs (SSA) are the mainstay of pharmacological treatment for pituitary Key Words adenomas. However, some patients escape from therapy with octreotide, a somatostatin ff pituitary receptor 2 (sst2)-preferring SSA, and pasireotide, a novel multi-sst-preferring SSA, may ff pituitary adenoma help to overcome this problem. It has been proposed that correspondence between ff somatostatin sst1-sst5 expression pattern and SSA-binding profile could predict patient’s response. ff gene expression To explore the cellular/molecular features associated with octreotide/pasireotide ff cell signaling response, we performed a parallel comparison of their in vitro effects, evaluating sst1- 2+ 2+ sst5 expression, intracellular Ca signaling ([Ca ]i), hormone secretion and cell viability, in a series of 85 pituitary samples. Somatotropinomas expressed sst5>sst2, yet octreotide 2+ reduced [Ca ]i more efficiently than pasireotide, while both SSA similarly decreased growth hormone release/expression and viability. Corticotropinomas predominantly expressed sst5, but displayed limited response to pasireotide, while octreotide reduced functional endpoints. Non-functioning adenomas preferentially expressed sst3 but, surprisingly, both SSA increased cell viability. Prolactinomas mainly expressed sst1 2+ but were virtually unresponsive to SSA. Finally, both SSA decreased [Ca ]i in normal pituitaries. In conclusion, both SSA act in vitro on pituitary adenomas exerting both

http://joe.endocrinology-journals.org © 2016 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JOE-16-0332 Printed in Great Britain Downloaded from Bioscientifica.com at 09/29/2021 01:29:03AM via free access

10.1530/JOE-16-0332 Research a ibáñez-costa and others Octreotide vs pasireotide in 231:2 136 pituitary tumors

similar and distinct effects; however, no evident correspondence was found with the sst1-sst5 profile. Thus, it seems plausible that additional factors, besides the simple abundance of a given sst, critically influence the SSA response. Journal of Endocrinology (2016) 231, 135–145

Introduction

Pituitary tumors are generally benign adenomas which likely relates to the specific presence, abundance, from a monoclonal/oligoclonal origin, which display availability and/or signaling properties of sst1-sst5 heterogeneous clinical manifestations derived from expression in each particular tumor. To circumvent this oversecretion of a single hormone and/or size effects due problem, novel analogs with multireceptor-binding to excess growth (Melmed 2011). Surgery is the first-line profiles have been developed, such as pasireotide. This treatment for most pituitary tumors, except for prolactin SSA binds with high affinity not only to sst5, but also to (PRL)-secreting lactotrope tumors (prolactinomas), which sst2 and sst3 (and less potently to sst1), and is already often respond favorably to medical treatment with being applied to treat pituitary tumors in clinical practice, dopamine agonists. Synthetic somatostatin (SST) analogs as it controls a relevant proportion of corticotropinomas (SSA) represent a valuable first-line medical treatment in contrast to octreotide (Boscaro et al. 2009). In addition, for various types of pituitary tumors, particularly in the superiority in clinical efficacy of pasireotide vs GH- and thyrotrophin (TSH)-secreting tumors (called octreotide has been shown in treatment-naïve acromegaly somatotropinomas and thyrotropinomas, respectively), patients (Colao et al. 2014) as well as in patients who were and, potentially also, in adrenocorticotrophin (ACTH)- resistant to octreotide or lanreotide (Gadelha et al. 2014). secreting tumors (corticotropinomas), owing to their Despite recent advances in clinical SSA development, abundant expression of SST receptors (sst1, sst2, sst3, key aspects of the mechanisms mediating the effects of

Endocrinology sst4 and sst5; codified by SSTR1, SSTR2, SSTR3, SSTR4 different SSA on pituitary tumors remain incompletely of and SSTR5 genes). These receptors are commonly understood. Indeed, few studies have explored the actual co-expressed, simultaneously, at different levels in normal differences/similarities of the direct actions of sst2-preferring

Journal pituitary cells, where they convey SST actions to regulate and multi-sst SSA on the different pituitary tumor types, hormone release (Theodoropoulou & Stalla 2013). In which may not solely depend on their distinct sst-binding pituitary tumors, previous studies (Taboada et al. 2007, profile. Besides, a number of different factors substantially Neto et al. 2009, Hofland et al. 2010a, Chalabi et al. 2014) influence the functional capacities of a given analog, have reported different expression patterns of sst1-sst5 from receptor internalization, recycling, degradation or depending on the type of tumor: high sst2-sst5 expression interaction, to selectivity in signaling pathway activation, is typical in GH- and TSH-secreting adenomas; sst5 thereby defining precisely their actions, which would predominates in corticotropinomas; sst1-sst5 expression also depend on the target cell type (Schonbrunn 2008). in prolactinomas; and sst3-sst2 expression in gonadotrope Accordingly, we have herein implemented a systematic lineage-derived tumors, which frequently lack hormone analysis of the direct in vitro actions of octreotide and oversecretion and are thus known as non-functioning pasireotide in a representative series of the main classes pituitary adenomas (NFPAs). The ability of ssts to activate of pituitary tumors, as well as in non-tumoral human various, often overlapping and cross-talking signaling pituitary. Unlike previous reports comparing the in vitro pathways provides the basis for SSA inhibition of hormone actions of these SSA, which mostly studied separately a secretion, cell proliferation and tumor growth. Indeed, single type of pituitary adenoma (Stalla et al. 1994, Hofland widely used sst2-preferring SSA (octreotide, lanreotide) et al. 2004, Hofland et al. 2005, van der Hoek et al. 2005, represent versatile therapeutic tools in acromegaly and Zatelli et al. 2007, van der Pas et al. 2013), here we deploy an thyrotropinomas (Theodoropoulou & Stalla 2013). integrative methodology to evaluate, in parallel, the in vitro Unfortunately, a relevant proportion of patients are response of human primary pituitary tumor cell cultures to (or become) partially or totally resistant to these drugs, both SSA by assessing several functional parameters, which while other pituitary tumors are largely unresponsive to include key aspects in pituitary tumor pathology. Thus, ssts SSA (Colao et al. 2011, Theodoropoulou & Stalla 2013), expression analysis using quantitative real-time PCR (qPCR)

Figure 1 Hormone expression profile. mRNA expression profile of pituitary hormones: growth hormone (GH), prolactin (PRL), pro-opiomelanocortin (POMC), follicle-stimulating hormone (FSH), luteinizing hormone (LH), thyrotrophin (TSH) and α-subunit of glycoproteins (CGA); adjusted by ACTB of somatotropinomas (A; n = 32), corticotropinomas (B; n = 15), non-functioning pituitary adenomas (C; n = 28), prolactinomas (D; n = 4) and normal pituitaries (E; n = 6). Values are expressed as mean ± s.e.m.

was performed on the tissue samples, wherein we measured adenoma were also used. In all cases, tissue phenotype SSA-induced kinetics of free cytosolic calcium concentration confirmation was supported by three separate methods 2+ ([Ca ]i), a key signaling pathway that is effected upon ligand as described previously (Luque et al. 2013): detailed binding to sst receptors to control hormone exocytosis and histological examination by an anatomopathologist, cell proliferation/apoptosis, as well as the two main outputs testing the hormonal phenotype using single-cell in endocrine tumors, hormone release and cell viability, as secretion on cells seeded onto PVDF membranes to suitable markers for the secretory and growth response of evaluate the secretion of all pituitary hormones using the tumors. specific antibodies, as previously reported (Vazquez- Martinez et al. 2008, Luque et al. 2013, Diaz-Rodriguez et al. 2014); and molecular screening by qPCR, as shown Endocrinology

of Materials and methods in Fig. 1. Normal pituitary tissues were collected during the resection of microadenomas (size<1 cm) derived from Reagents five corticotropinomas and one somatotropinoma, and Journal Unless otherwise indicated, reagents and products were were classified as normal tissue by these three methods, purchased from Sigma-Aldrich. Octreotide was obtained and confirmed by the presence of the adenoma in post- from GP-Pharm (Barcelona, Spain) and pasireotide was surgical imaging studies and biochemical analyses. provided by Novartis. Both compounds were administered Available demographic, clinical data and pretreatment at 100 nM as reported previously in a comparative study therapies are summarized in Table 1. Particularly, each (van Hoek et al. 2009), in which other concentrations pituitary piece was placed after surgery in sterile cold were unable to exert any effect in vitro. S-MEM medium (Gibco) complemented with 0.1% BSA, 0.01% l-glutamine, 1% antibiotic-antimycotic solution and 2.5% HEPES and dispersed into single cells within the Patients, tissue collection and pituitary cell culture following 1–3 h as reported previously (Martínez-Fuentes All experimental protocols were approved by the et al. 2006, Luque et al. 2013, Ibáñez-Costa et al. 2015). University of Cordoba/Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC) RNA isolation, RT and qPCR licensing committee. This study has been performed in accordance with the Declaration of Helsinki. Pituitary Total RNA extraction, quantification, RT, qPCR procedure specimens were obtained by transsphenoidal surgery and primer sequences used to measure mRNA expression from 79 pituitary adenomas (32 somatotropinomas, 15 of the genes included in this study (SSTR1-SSTR5, GH, corticotropinomas, 28 NFPAs and 4 prolactinomas), after PRL, pro-opiomelanocortin (POMC), follicle-stimulating informed consent was provided by each patient and with hormone (FSH), luteinizing hormone (LH), TSH, CGA, the approval of the University of Córdoba/IMIBIC and POU1F1, ACTB) have been reported previously (Taboada Hospital Ethics Committees. Additionally, six normal et al. 2007, Neto et al. 2009, Luque et al. 2013, Ibáñez- pituitaries resected during surgical removal of a pituitary Costa et al. 2015).

Table 1 Demographic and clinical data of patients included in the study.

Variable GH-omas ACTH-omas NFPAs PRL-omas Normal pituitary N 32 15 28 4 6 Sex/age (min–max) Male n = 15/44 (12–75) n = 2/33 (23–43) n = 14/57 (22–74) n = 3/35 (18–47) n = 0 Female n = 17/43 (12–62) n = 13/41 (25–68) n = 14/51 (27–75) n = 1/21 n = 6/43 (21–69) Pretreatment Untreated 6 1 20 – – SSA 20 – – – – SSA/DA 5 1 – – – DA – – 1 4 – Ketoconazole – 12 – – – Missing data 1 1 7 – – Size Macroadenoma 28 5 21 4 – Microadenoma 2 9 – – – Missing data 2 1 7 – – Cure after surgery Cured 3 7 7 – – Biochemically 3 – – – – controlled Cured after 3 – – – – radiotherapy Cured after – 1 – – – adrenalectomy Active disease 14 2 11 3 – Missing data 9 5 10 1 –

DA, dopamine agonists; SSA, somatostatin analogs.

Measurement of free cytosolic calcium Analysis of cell viability concentration ([Ca2+] ) i As described previously (Durán-Prado et al. 2009, Luque Endocrinology

of Cells were plated on coverslips at a density of 50,000 cells et al. 2013, 2015, Ibáñez-Costa et al. 2015), cell viability 2+ per well and changes in [Ca ]i after treatment with was estimated using alamar-blue reagent (10,000 cells per octreotide and pasireotide were tracked in single cells using well plate; Biosource International, Camarillo, CA, USA) Journal fura-2/AM (Molecular Probes) as described previously in response to octreotide/pasireotide. (Martínez-Fuentes et al. 2006, Durán-Prado et al. 2009, Luque et al. 2013, Ibáñez-Costa et al. 2015). Statistical analysis

Statistical analyses were performed by unpaired Evaluation of hormone release parametric t-test and non-parametric Mann–Whitney To examine the effects of SSA on pituitary hormone U test according to normality, assessed by Kolmogorov– release, 100,000–150,000 cells per well were used. Smirnov test. We compared the effect of octreotide or First, cells were plated in supplemented DMEM + 10% pasireotide vs vehicle-treated control (set at 100%). Data fetal bovine serum (FBS) for 24–36 h, and then were were expressed as mean ± s.e.m. A P value of <0.05 was washed and serum starved for 1 h and incubated with considered to be significant. All statistical differences were octreotide or pasireotide (somatotropinomas: 4 and assessed using GraphPad Prism 6. 24 h; corticotropinomas: 24 h incubation) in absence of FBS. After treatment, media were collected and Results stored at −20°C until hormone measurement using commercial ELISAs (GH and ACTH (EIA-3552 and The main aim of this study was to perform a systematic, EIA-3647, respectively; DRG, Marburg, Germany)). comparative analysis of the effects of two SSA with distinct In somatotropinomas, whenever possible, cells were sst1-sst5 binding profile, octreotide and pasireotide on recovered for RNA analysis (see the ‘RNA isolation, RT basic functional parameters in the main types of pituitary and qPCR’ section above). All the information regarding adenomas after determining the particular expression each assay can be accessed at the company website. profile of sst.

Figure 2 GH-secreting adenomas. (A) sst mRNA expression profile adjusted by ACTB n( = 32). (B) Summarized 2+ table of [Ca ]i kinetics assay: n stands for ‘responsive samples/total samples analyzed’; PRC, proportion of responsive cells showing changes 2+ in [Ca ]i in response to SSA; PMR, percentage of maximum response; and time, time of response to SSA administration. (C) Effect of SSA on GH release after 4 h (octreotide and pasireotide: n = 2) and 24 h (octreotide: n = 7; pasireotide: n = 5) incubation. Values are expressed as percent of vehicle-treated controls, set at 100% within experiment. (D) Cell viability (24–72 h treatment) in response to octreotide and pasireotide (n = 13/9/8 at 24/48/72 h, respectively). (E) GH, PRL, POU1F1, sst2 and sst5 mRNA expression profile in response to SSA (octreotide and pasireotide: n = 3). Values are expressed as percent of vehicle-treated controls, set at 100% within experiment. Data are presented as mean ± s.e.m. Asterisks show significant differences between SSA- and vehicle-treated controls (*P < 0.05, **P<0.01,***P < 0.001).

Effects of octreotide and pasireotide on on sst5 levels (P = 0.07), octreotide did not significantly somatotropinomas alter the expression levels of any ssts (Fig. 2E, right panel). In addition, octreotide slightly, but significantly, Regarding their hormonal phenotype, somatotropinomas decreased cell viability in 13/13 cell cultures after 24 h, (n = 32) expressed GH exclusively and not PRL (only in 9/9 after 48 h and 8/9 after 72 h, whereas pasireotide present in 20% of adenomas; Fig. 1A). They displayed, on decreased cell viability in 13/13 after 24 h, in 9/9 after average, high expression levels of sst5 and sst2, followed

Endocrinology 48 h and 8/9 after 72 h (Fig. 2D). by sst3 and sst1 (Fig. 2A), which suggests a putative of effectiveness of SSA, especially pasireotide, on these tumors. Calcium is a pivotal second messenger involved

Journal Effects of octreotide and pasireotide on in the regulation of pituitary cell pathophysiology, corticotropinomas which is required to trigger and sustain hormone release and other cell processes (e.g., apoptosis), and thus, it Corticotropinomas (n = 15) expressed, on average, high is widely used as an indicator to assess pituitary cell levels of sst5, followed by sst2, which would enable a response to pharmacological treatment (Martínez- positive response to SSA, especially pasireotide (Fig. 3A). Fuentes et al. 2006, Ibáñez-Costa et al. 2015). Octreotide In terms of hormone expression, corticotropinomas and pasireotide similarly decreased, on average, expressed almost exclusively high POMC levels, with 2+ [Ca ]i (Fig. 2B). However, adenomas appeared to be only some tumors moderately expressing PRL (Fig. 1B). 2+ more responsive to octreotide than to pasireotide, in Both SSA were able to decrease [Ca ]i kinetics in vitro terms of the overall proportion of tumors showing a in roughly half of the corticotropinomas (4/10 and 5/9, 2+ [Ca ]i response to each drug, and in the percentage of respectively); yet, unexpectedly, octreotide appeared responsive cells within each tumor (Fig. 2B). Similarly, to be more efficient than pasireotide, as it exerted a both SSA decreased GH release in 2/2 cultures to a greater degree of inhibition in a higher percentage similar extent after 4 h treatment (Fig. 2C, left panel), to of cells (Fig. 3B), and in a shorter period. Accordingly, octreotide in 7/7, and to pasireotide in 5/5 cell cultures 24 h incubation with octreotide inhibited ACTH release after 24 h (Fig. 2C, right panel), compared with vehicle- in two of the three tumors studied, while pasireotide treated cells. Likewise, both SSA decreased GH, but not only induced a non-significant reduction in two of the PRL mRNA levels after 24 h incubation without altering four tumors (Fig. 3C). Similarly, octreotide induced a POU1F1 levels, the transcription factor responsible for moderate, albeit significant reduction in cell viability in GH and PRL expression (Fig. 2E, left panel). In contrast, two of the three tumors (P < 0.05), whereas pasireotide while pasireotide treatment drastically reduced sst2 did not evoke any reduction in cell viability in the two levels and induced an apparent, non-significant increase tumors studied (Fig. 3D).

Figure 3 ACTH-secreting adenomas. (A) sst mRNA expression profile adjusted by ACTB n( = 15). 2+ (B) Summarized table of [Ca ]i kinetics assay. (C) Effect of SSA on ACTH release after 24-h incubation (octreotide: n = 4; pasireotide: n = 5). (D) Cell viability in response to octreotide (n = 4/2/1 at 24/48/72 h, respectively) and pasireotide (n = 4/3/2 at 24/48/72 h, respectively). For further details see Fig. 2 legend.

Effects of octreotide and pasireotide on NFPAs 72 h. Likewise, pasireotide significantly augmented cell viability in 11/15 NFPA cell cultures after 24 h, in 6/7 after The NFPAs analyzed in this study were characterized 48 h and in 3/4 after 72 h (Fig. 4D). by high sst3 expression, followed by sst2, sst5, sst1 and sst4 (n = 28; Fig. 4A), which might suggest that pasireotide could be more effective than octreotide. As Effects of octreotide and pasireotide on prolactinomas expected, CGA (α-subunit of the glycoproteins), FSH and LH were highly expressed (Fig. 1C). Cultured cells Prolactinomas exhibited high sst1 expression, with lower derived from NFPAs were rarely and poorly responsive levels of other ssts (n = 4; Fig. 5A), and very high expression to both SSA, nevertheless, octreotide appeared more of PRL, exclusively (Fig. 1D). Cells derived from these effective than pasireotide, although it only inhibited prolactinomas, which were resistant to in vivo treatment 2+ with cabergoline, displayed poor responses to SSA in vitro. Endocrinology [Ca ]i kinetics moderately, in a discrete subset of cells 2+ of (Fig. 4B). Conversely, both compounds exerted clear Specifically, octreotide decreased [Ca ]i in 2/4 cultures, and divergent responses in terms of cell viability, which but only in a very low percentage of cells (7.0%), whereas 2+ it moderately increased [Ca ]i in 1/4 cell cultures, also in

Journal were consequently classified as ‘inhibitory responders’ or ‘stimulatory responders’. Specifically, octreotide tended a low percentage of cells (Fig. 5B). In contrast, pasireotide 2+ to moderately (non-significantly) decrease cell viability did not evoke any appreciable [Ca ]i response (Fig. 5B). in 5/16 cell cultures after 24 h, in 2/8 after 48 h and in Moreover, both SSA decreased cell viability only in 1/3 2/7 after 72 h; while pasireotide only appeared to exert tumors after 48–72 h (Fig. 5C). comparable, also non-significant actions on cell viability in 4/15 cell cultures after 24 h, in 1/7 after 48 h, and in 1/4 Effects of octreotide and pasireotide on normal pituitary after 72 h (Fig. 4C). Conversely, octreotide consistently induced moderate, albeit significant increases in cell Normal pituitaries featured high sst5 expression, with viability compared with vehicle-treated cells in 11/16 lower sst2 and very low sst3 and sst1 levels (n = 6; Fig. 6A). cell cultures after 24 h, in 5/8 after 48 h and in 2/6 after In terms of hormonal phenotype, as expected, GH and

Figure 4 Non-functioning pituitary adenomas. (A) sst mRNA expression profile adjusted by ACTB 2+ (n = 28). (B) Summarized table of [Ca ]i kinetics. (C) Inhibitory response in cell viability assay (24–72 h treatment) in response to octreotide (n = 5/2/2 at 24/48/72 h, respectively) and pasireotide (n = 4/1/1 at 24/48/72 h, respectively). (D) Stimulatory response in cell viability assay in response to octreotide and pasireotide (n = 11/5/3 at 24/48/72 h, respectively). For further details see Fig. 2 legend.

that, in general, they compare well with those described previously for each of the tumor types examined (Taboada et al. 2007, Neto et al. 2009, Hofland et al. 2010a), supporting the notion that the samples investigated provide a representative picture of the pituitary tumor types under study. Although mRNA levels may not always reflect the precise functional protein levels in a cell (and in this case protein determination was a technically challenging assay that we could not perform given the limited tissue availability) the use of qPCR to assess mRNA levels has been shown as a good alternative approach, as a proxy for protein presence, according to recent studies (Vogel & Marcotte 2012). Nevertheless, it is important to note that the precise sst1-sst5 profile in each tumor Figure 5 PRL-secreting adenomas. (A) sst mRNA expression profile adjusted by did not always match, necessarily, that observed, on 2+ ACTB (n = 4). (B) Summarized table of [Ca ]i kinetics assay. average, for its corresponding tumor type. It is widely and (C) Cell viability in response to octreotide and pasireotide (n = 3/3/3 at reasonably assumed that the actions of a specific SSA in 24/48/72 h, respectively). For further details see Fig. 2 legend. a given pituitary tumor would result from the functional PRL were highly expressed, followed by POMC, CGA and correspondence between the sst1-sst5-binding profile of LH/FSH, with faint TSH expression (Fig. 1E). Octreotide that SSA and the pattern of sst1-sst5 expression in the appeared less effective than pasireotide in decreasing tumor. Accordingly, it has been suggested that, in the 2+ future, recommendations to select a specific SSA could be [Ca ]i. However, in responsive cultures, octreotide affected more cells and caused reductions of comparable done on the basis of sst expression pattern, for example, amplitude to pasireotide (Fig. 6B). Besides, pasireotide, octreotide/lanreotide would be recommendable when

Endocrinology but not octreotide, significantly decreased cell viability sst2 is highly and predominantly expressed, whereas of after 24, 48 and 72 h (Fig. 6C). high expression of sst5, and sst2, sst3 or sst1 in pituitary adenomas would predict pasireotide responsiveness (Chalabi et al. 2014). However, this logical contention, Journal Discussion which likely applies in general terms when large numbers of tumors or patients are considered on average, might Quantitative assessment of hormonal and sst1-sst5 not be similarly evident on an individual tumor basis, expression profiles in the present tumor series revealed and, anyhow, such a theoretical assumption has not been unequivocally demonstrated through experimental testing so far. In fact, previous studies have indicated that sst2 presence in somatotropinomas positively correlated with in vivo SSA response (Taboada et al. 2008, Gatto et al. 2013), and a recent study revealed that octreotide-responsive patients were characterized by high sst2 and low sst5 presence, while octreotide-resistant patients showed high sst5 expression (Gonzalez et al. 2014). However, in this study, although somatotropinomas were characterized by sst5>>sst2 expression, as described previously (Taboada et al. 2007, 2008, Hofland et al. 2010a), we observed that both SSA induced in vitro comparable inhibition of both GH release and cell viability. Moreover, it was noteworthy that pasireotide evoked less responses from cells than 2+ Figure 6 octreotide in terms of [Ca ]i kinetics, which would argue Normal pituitary. (A) sst mRNA expression profile adjusted by ACTB against the theoretical superiority of pasireotide predicted 2+ (n = 6). (B) Summarized table of [Ca ]i kinetics assay. (C) Cell viability in response to octreotide and pasireotide (n = 2/1/1 at 24/48/72 h, by the higher sst5 levels observed in these GH tumors. respectively). For further details see Fig. 2 legend. The reason for these latter differences is still unknown,

but could relate to the existence of distinct, tumor-specific cortisol levels would be ineffective as previously reported sst1-sst5 distribution patterns, receptor–agonist and in two small series of untreated patients (Ambrosi et al. receptor–receptor interactions and dynamics, resulting 1990, Stalla et al. 1994). Additionally, a recent study, in activation of different signaling pathways. In any case, using corticotropinoma cell cultures demonstrated that in our data suggest that the mere abundance of a given sst cortisol-normalized patients, sst2 expression is increased would not provide sufficient support to explain the level at mRNA (van der Pas et al. 2013), which led to suggest of response of a somatotropinoma to a SSA with strong that a pharmacological treatment to decrease cortisol affinity for that specific receptor, and, thus, additional levels (e.g., ketoconazole), followed by octreotide may be mechanisms are required to explain the precise actions of effective. The reason for the clear responses observed in this each SSA in a target cell. study to octreotide may relate to a recovery of sst2-related Nevertheless, our in vitro results are in line with the responsiveness, but the role of other ssts, particularly recent comparative in vivo analysis of octreotide and sst5, should not be discarded. In contrast, pasireotide pasireotide treatment (Colao et al. 2014), where both did not induce significant responses in terms of ACTH compounds suppressed similarly GH levels, in that release or cell viability in the corticotropinomas studied, 2+ case, being pasireotide more efficient than octreotide, where only a faint effect on [Ca ]i kinetics was observed. since more patients were biochemically controlled These results are not in line with previous in vitro studies after 12 month treatment (Colao et al. 2014, Gadelha (Hofland et al. 2005, 2010b, van der Hoek et al. 2005, van et al. 2014, Samson 2015). Interestingly, in our study, der Pas et al. 2013), wherein pasireotide reduced ACTH differences between the actions of both SSA were found release. However, the lack of effect of pasireotide on these in the control of gene expression, since both analogs corticotropinomas despite their high expression levels of similarly decreased GH mRNA, but, while octreotide did sst5 and also sst2, and their clear response to octreotide, not appear to alter sst expression, pasireotide decreased strongly suggest that additional mechanisms distinct sst2 levels and moderately augmented sst5 expression, from the mere presence of a given receptor are required a receptor-specific positive feedback effect that could for a drug to achieve its desired functional effect.

Endocrinology potentially contribute to enhance in vivo responsiveness The sst expression profile observed in NFPAs of to this SSA (Colao et al. 2014). Thus, our results illustrate (sst3>>sst2>sst5) is similar to that reported previously a substantial similitude between octreotide and (Taboada et al. 2007), and suggested a possible pasireotide actions on GH secretion and cell survival responsiveness to pasireotide. However, only a small Journal in somatotropinomas in vitro, with some differences proportion of NFPA was responsive, in vitro, to either SSA, in regulation of Ca2+ signaling and sst expression. This at least in the parameters evaluated. Moreover, some of suggests that both analogs may exert comparable effects the effects observed could be considered paradoxical and, at pituitary level in somatotropinomas, thus raising the from a clinical perspective, unexplainable. Indeed, both possibility that some of the differences observed in vivo SSA might increase cell viability compared with vehicle- may relate to extrapituitary effects, which could help to treated cells in the majority of responsive tumors. Similar explain the stronger inhibitory effect of pasireotide vs stimulatory actions of pasireotide (Zatelli et al. 2007) and octreotide on insulin-like growth factor (IGF1) in patients of an sst5-specific agonist have been described previously with acromegaly in vivo (Colao et al. 2014). in NFPAs, but the precise underlying mechanisms and Analysis of corticotropinomas showed the typical specific ssts involved remain unclear. A reduction in cell 2+ sst5-predominant profile, yet, results on [Ca ]i kinetics, viability can result from decreased cell growth and/or ACTH release and cell survival demonstrated that these activation of apoptosis, whereas an increase of cell viability tumors are significantly responsive, in vitro, to octreotide, indicates that these compounds may protect primary similar to that previously reported on primary cultures cells from natural cell death and/or may activate survival/ (Hofland et al. 2005, 2010b) and in AtT-20 cells (Hofland proliferative mechanisms. In the tumors examined, et al. 2005, 2010b, van der Hoek et al. 2005). Our data also there were no statistical differences in sst expression support the notion that octreotide exerts divergent actions profile between inhibitory responders and stimulatory in vivo and in vitro on corticotropinomas. Indeed, it is well responders. However, the fact that sst3 is uniquely known that glucocorticoids downregulate sst2, affecting expressed at high levels in NFPAs and that pasireotide octreotide response (van der Hoek et al. 2005, van der seems more effective in stimulating cell viability in these Pas et al. 2013), which suggests that octreotide therapy in tumors invites speculation that this receptor may be untreated Cushing’s disease patients presenting with high involved in such responses. Nevertheless, further studies

are required to elucidate the precise role of sst3 and the to better understand the functional actions of the two rest of ssts in this unique response of NFPAs, which, SSA investigated here as well as novel SSA, wherein the already adds potentially useful information for the relevant endpoints and underlying mechanisms should current understanding of SSA effects on these tumors. be tested preferentially in the same ultimate targets for Actually, despite promising in vivo experiences with these drugs, that is, the primary pituitary tumor cells, as octreotide treatment in patients not cured after surgery they may provide a more precise and realistic portrait of (Fusco et al. 2012), which showed stabilized tumor size in the actual response that can be expected, and, hopefully, most cases (but not tumor volume reduction), the poor predicted, for a given type of tumor. in vitro response and the paradoxical increases in cell viability reported in response to both SSA discourages the use of SSA as first-line treatment in NFPAs. Declaration of interest Prolactinomas are often responsive to dopamine H A Schmid is an employee of Novartis. R M Luque and J P Castaño have agonists. However, 10% of patients fail to normalize received grants and lecture fees from Ipsen and Novartis. E Venegas- PRL levels and tumor growth (Fusco et al. 2008). The Moreno and A Soto-Moreno received grants and lecture fees from Ipsen, Novartis and Pfizer. The rest of the authors have nothing to disclose. SSA therapy has been proposed as an alternative, since prolactinomas express ssts and several studies demonstrated that SSA are able to inhibit PRL release in primary cultures (Shimon et al. 1997, Hofland et al. Funding 2004) and germane cell lines (Gruszka et al. 2007). Our Junta de Andalucía (BIO-0139, CTS-1406, PI-639-2012); Ministerio de Economía y Competitividad, Gobierno de España (BFU2013-43282); results showed that octreotide decreased cell viability in Proyectos de Investigación en Salud FIS, funded by Instituto de Salud 2+ Carlos III (PI13-00651); Instituto de Salud Carlos III, Ministerio de Sanidad, one prolactinoma and only inhibited [Ca ]i in a very low proportion of cells, with pasireotide being even Servicios Sociales e Igualdad ‘CIBERobn’ (to A I-C, M D G, R M L and J P C); Instituto de Salud Carlos III, Ministerio de Sanidad, Servicios Sociales less effective despite high sst1 expression levels in these e Igualdad ‘Sara Borrell Program’ (CD11/00276 to M D G) Ayuda Merck tumors. These results are in keeping with in vivo studies, Serono 2013 (to A I-C, E R-C, M D G, R M L and J P C).

Endocrinology which did not find consistent inhibitory effects (Fusco of et al. 2011, Colao & Savastano 2011). Finally, we also evaluated octreotide and pasireotide Author contribution statement effects on a limited set of normal pituitary cell cultures. Journal Conception and design: R M L and J P C; development of methodology: This revealed that, in line with in vivo analyses on healthy A I-C, E R-C, M C V-B, M D G, H A S, R M L and J P C; acquisition of clinical data: L J R, E V-M, A d l R, M A A, I G-M, S M-S, I G-V, J A G-A, M A J, A S-M volunteers treated with octreotide (Tuvia et al. 2012) or and M A G; analysis and interpretation of data: A I-C, E R-C, M A V-B, M D pasireotide (Golor et al. 2012), where both SSA decreased G, R M L and J P C; writing of the manuscript: A I-C, R M L and J P C; review hormone levels, particularly GH, both SSA directly acted and/or revision of the manuscript: A I-C, E R-C, M C V-B, M D G, L J R, E V-M, A d l R, M A A, I G-M, H A S, S M-S, I G-V, J A G-A, M A J, A S-M, M A G, R on pituitary cells to decrease [Ca2+] kinetics. Additionally, i M L and J P C; study supervision: R M L and J P C. we found that pasireotide, but not octreotide, also reduced cell viability in normal pituitary cell cultures. In summary, our results indicate that octreotide and References pasireotide act on the main types of pituitary adenomas Ambrosi B, Bochicchio D, Fadin C, Colombo P & Faglia G 1990 Failure 2+ by exerting similar and distinct effects on [Ca ]i kinetics, of somatostatin and octreotide to acutely affect the hypothalamic- hormone release, gene expression and cell viability. pituitary-adrenal function in patients with corticotropin hypersecretion. Journal of Endocrinological Investigation 13 257–261. However, we did not observe any evident correspondence (doi:10.1007/BF03349555) between the effects observed and the specific sst1- Boscaro M, Ludlam WH, Atkinson B, Glusman JE, Petersenn S, sst5 profile of the target tumors. Hence, the emerging Reincke M, Snyder P, Tabarin A, Biller BM, Findling J, et al. 2009 Treatment of pituitary-dependent Cushing’s disease with the picture is that there might not be a simple predictive multireceptor ligand somatostatin analog pasireotide (SOM230): correspondence between the presence of a sole receptor a multicenter, phase II trial. Journal of Clinical Endocrinology and and the response to a given SSA with high affinity for Metabolism 94 115–122. (doi:10.1210/jc.2008-1008) Colao A & Savastano S 2011 Medical treatment of prolactinomas. Nature this receptor, but that other factors may substantially Reviews Endocrinology 7 267–278. (doi:10.1038/nrendo.2011.37) influence the response of pituitary tumor cells, such as Colao A, Auriemma RS, Lombardi G & Pivonello R 2011 Resistance to the proportion of other ssts for which the SSA may not somatostatin analogs in acromegaly. Endocrine Reviews 32 247–271. (doi:10.1210/er.2010-0002) have high affinity, the signaling status of the target Colao A, Bronstein MD, Freda P, Gu F, Shen CC, Gadelha M, Fleseriu M, cell and so on. Therefore, further studies are warranted van der Lely AJ, Farrall AJ, Hermosillo Resendiz K, et al. 2014

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Received in final form 26 August 2016 Accepted 1 September 2016 Accepted Preprint published online 1 September 2016 Endocrinology of Journal