REVIEW The clinical–molecular interface of somatostatin ...

361

REVIEW

The clinical–molecular interface of somatostatin, dopamineand their receptors in pituitary pathophysiology

Diego Ferone, Federico Gatto, Marica Arvigo, Eugenia Resmini, Mara Boschetti,Claudia Teti, Daniela Esposito and Francesco MinutoDepartment of Endocrinological & Medical Sciences (DiSEM) and Center of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV, 6, 16132 Genova, Italy

(Correspondence should be addressed to D Ferone; Email: [email protected])

Abstract

The role of somatostatin and dopamine receptors as molecular targets for the treatment of patients with pituitary

adenomas is well established. Indeed, dopamine and somatostatin receptor agonists are considered milestones for the

medical therapy of these tumours. However, in recent years, the knowledge of the expression of subtypes of somatostatin

and dopamine receptors in pituitary adenomas, as well as of the coexpression of both types of receptors in tumour cells,

has increased considerably. Moreover, recent insights suggest a functional interface of dopamine and somatostatin

receptors, when coexpressed in the same cells. This interaction has been suggested to occur via dimerisation of these

G-protein-coupled receptors. In addition, there was renewed interest around the concept of cell specificity in response to

ligand-induced receptor activation. New experimental drugs, including novel somatostatin analogues, binding to multiple

somatostatin receptor subtypes, as well as hybrid somatostatin–dopamine compounds have been generated, and

recently a completely novel class of molecules has been developed. These advances have opened new perspectives for

the medical treatment of patients with pituitary tumours poorly responsive to the present clinically available drugs, and

perhaps also for the treatment of other categories of neuroendocrine tumours. The aim of the present review is to

summarise the novel insights in somatostatin and dopamine receptor pathophysiology, and to bring these new insights

into perspective for the future strategies in the medical treatment of patients with pituitary adenomas.

Journal of Molecular Endocrinology (2009) 42, 361–370

Introduction

Somatostatin (SRIF) and dopamine (DA) are twocritical regulators of pituitary cells function, beinginvolved in the negative control of hormonal secretionof the anterior pituitary (Ben-Jonathan & Hnasko 2001,Guillemin 2005). The actions of SRIF and DA aremediated by specific G-protein-coupled receptors(GPCR) that are present on the cells of both thenormal gland and of the pituitary adenomas: SRIFreceptor (SSR) subtypes 1, 2, 3 and 5 (sst1–5; Miller et al.1995, Hofland & Lamberts 2001); and the subtype 2(D2) of DA receptors (DR; Missale et al. 1998).Conversely, the expression of sst4 is rather infrequent,whereas D4 receptor has also been found expressed inthe anterior pituitary and in a small subset of pituitarytumours (Missale et al. 1998, Pivonello et al. 2004b);however, its role in the pathophysiology of the pituitaryhas not been examined yet. Two different isoforms ofsst2 and D2 have also been found and characterised: thetwo forms of the sst2 (sst2A and sst2B) and of D2, the long

Journal of Molecular Endocrinology (2009) 42, 361–3700952–5041/09/042–361 q 2009 Society for Endocrinology Printed in Great Britain

(D2long) and short (D2short) isoforms, are generated viaalternative splicing (Missale et al. 1998, Hofland &Lamberts 2003). While sst2B is almost unexpressed inhumans, the two D2 isoforms may be coexpressed andassociated with different intracellular signalling trans-duction mechanisms, and may, therefore, elicitdifferent effects after binding with DA agonists (Missaleet al. 1998).

As for the physiological lineaments, SRIF, interactingwith SSRs, inhibits the secretion of a wide range ofhormones, including the pituitary GH, PRL and TSH.Signalling through SSRs is multifaceted. Indeed,binding of SRIF or SRIF analogues to SSRs initiates acomplex set of signalling events triggered by theinteraction of the activated receptors with a largenumber of different protein partners, involving firstlyspecific G-protein activation (Moller et al. 2003). As aconsequence, the activities of several key enzymes,including adenylyl cyclase, phosphotyrosine phospha-tases (PTPases) and mitogen-activated protein kinase(MAPK) are modulated along with changes in the

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D FERONE and others . Somatostatin and dopamine in the pituitary362

intracellular levels of calcium and potassium ions(Florio 2008).Gproteins, via the stimulation of PTPases,may also produce some of the cytostatic actions of SRIF(Fig. 1A). For example, in pituitary tumour cells, SRIFanalogues produce their anti-proliferative action byacting on the phosphatidylinositol 3-kinase (PI3K)/AKT signalling pathway (Theodoropoulou et al. 2006).Whereas, more recently, apoptosis has been alsoobserved upon binding of SRIF and SRIF analogues tosst3, and possibly to sst2 as well. (Fig. 1A). Which type ofsignalling prevails in given cells depends on the cell-specific distribution of SSR subtypes and signallingelements, as well as on SSRs internalisation, desensitisa-tion and/or receptor crosstalk (Lahlou et al. 2004,Schonbrunn 2008). Finally, the subcellular expressionpattern of SSR subtypes and their activity in response toagonist treatment may also be affected by intracellularcomplements, such as proteins involved in intracellularvesicle trafficking (e.g. b-arrestin). Indeed, differentSRIF analogues may induce distinct conformations of

Figure 1 (A) Principal intracellular signalling cascades associat(or somatostatin analogues) binding to somatostatin receptors inhibchannels. Phosphotyrosine phosphatases and mitogen-activated prphosphotyrosine phosphatase, may also produce cytostatic actions.well. Most of these effects are mediated by G proteins. (B) Principareceptors in pituitary cells. Dopamine (or dopamine agonists) bindinphosphatidylinositol metabolism, activates voltage-activated potassT-type calcium currents, modulates the activity of phospholipase C,signal-regulated kinase pathway. The expression of POU1F1 transcnegative control on PRL gene expression. Most of these effects are mreticulum; ERK, extracellular signal-regulated kinase; GSK3b, glycokinase; Ga, Gb and Gg, G-protein subunit; PDPK1, 3-phosphoinosphospholipase C; PKA, protein kinase A; PTPase, phosphotyrosine


the receptor/ligand complex, preferentially coupled toeither receptor signalling or receptor endocytosis(Tulipano & Schulz 2007).

TheD2 receptor is important formediating the effectsof DA to controlmovement, certain aspects of behaviourin the brain and PRL secretion from the anteriorpituitary. Multiple transduction mechanisms are acti-vated by D2 receptors in the pituitary. In addition toinhibition of adenylyl cyclase, pituitary D2 receptorsinhibit PI metabolism, stimulate voltage-activated pot-assium channels and decrease voltage-activated L-typeand T-type calcium currents (Fig. 1B). All these effectsare mediated by G proteins (Missale et al. 1998). Studieson D2 gene expression in different types of pituitaryadenomas showed a variable D2 expression localised inthe cytoplasm and nuclei of a large number ofadenomas. However, the significance of nuclear locali-sation of D2 remains unclear (Ferone et al. 2007a).Moreover, both isoforms of D2 receptor are relevant tothe signalling pathways involved in the proliferation and

ed to somatostatin receptors in pituitary cells. Somatostatinits adenylyl cyclase, activates K channels and/or inhibits Caotein kinase are modulated as well and along the stimulation ofMore recently, increase in apoptosis via p53 has been shown asl signal transduction associated with the activation of dopamineg to pituitary D2 receptors inhibits adenylyl cyclase,ium channels and decreases voltage-activated L-type andactivates the mitogen-activated protein kinase and extracellularription factor is inhibited by activation of D2 receptors, exerting aediated by G proteins. AC, adenylyl cyclase; ER, endoplasmicgen synthase kinase 3b; MAPK, mitogen-activated proteinitide-dependent protein kinase 1; pHi, intracellular pH; PLC,phosphatase.

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Somatostatin and dopamine in the pituitary . D FERONE and others 363

cell death of pituitary tumour cells, possibly throughp38MAPK and ERK activation (Ferone et al. 2007a).

Along the last decades, the knowledge on thepathophysiology of these two families of GPCRs inthe pituitary has progressively increased due to thesynthesis and availability of specific subtypes receptoragonists and antagonists, useful not only in experi-mental settings, but also of incredible value in clinicalpractice as potent therapeutic agents. However, untilrecently, each receptor molecule was believed tointeract separately with G proteins, while a series ofobservations have challenged this conclusion. Indeed,the emerging concept that these GPCRs may alsofunction as homo- and heterodimers has opened acomplete new scenario for those trying to interpretconfusing results obtained in vivo or in isolated tissuesthat do not seem to obey the classical binding andactivation patterns described for individual clonedreceptors.

The clinical–molecular interface of SRIF, DA andtheir receptors in pituitary pathophysiology will bediscussed in this review, because it represents an idealmodel of the possibility of a whole new level of complexinteractions that couldmultiply the range of activities ofthe already large family of GPCRs.

Heterogeneity of somatostatin and D2

dopamine receptor distribution in pituitaryadenomas

Both SSRs and D2 receptors are significantly expressedin pituitary adenomas, even if there is a large interstudyvariability reflecting not only the different sensitivity ofthe techniques employed, but also the compellingheterogeneity of these tumours (Stefaneanu et al. 2001,Moller et al. 2003, Saveanu et al. 2008).

In most GH adenomas, sst2, sst5 and D2 coexpressionhas been found, at both mRNA and protein levels,however, half of GH tumours also coexpress sst3 andsst1, particularly mixed GH/PRL adenomas (Panetta &Patel 1995, Stefaneanu et al. 2001, Zatelli et al. 2005,Taboada et al. 2007, Ferone et al. 2008, Saveanu et al.2008). The vast majority of prolactinomas express highnumber of D2 receptor; however, sst1, and particularlysst5, are also notably present, while sst2 is only expressedin a minority of them (Miller et al. 1995, Panetta & Patel1995, Stefaneanu et al. 2001, Fusco et al. 2007, Saveanu& Jaquet 2008). Non-functioning pituitary adenomas(NFPAs), including gonadotrophinomas, as well asa-subunit producing tumours, express mainly sst3 and,at a lesser degree, sst2, seldom associated with sst1(Zatelli et al. 2004, Taboada et al. 2007). The D2 receptoris expressed in most clinically NFPAs, and, interestingly,heterogeneous D2 isoform expression has been demon-strated in these tumours, in which D2short seems more


favourable than D2long expression for both in vitro andin vivo growth inhibitory response to DA agonists(Renner et al. 1998, Pivonello et al. 2004b, Taboadaet al. 2007). In Cushing’s disease, corticotroph adeno-mas mainly express sst5 and D2 receptors, whereas sst2 isexpressed at lower levels together with sst1 and sst3(Stefaneanu et al. 2001, Pivonello et al. 2004a, Batistaet al. 2006, de Bruin et al. 2009). Both sst5 and D2

receptors seem to play an important role in theregulation of ACTH release, at least in a subset ofspecific pituitary area-derived corticotroph tumours(Pivonello et al. 2004a, de Bruin et al. 2009). Interest-ingly, SSRs and D2 receptor seem differentiallyregulated by glucocorticoids in neuroendocrine cells(de Bruin et al. 2008a). This aspect should be taken intoaccount because cortisol-lowering therapy couldindirectly affect the responsiveness of tumour’s cellsto drugs targeting these receptors. Moreover, recently, ithas been proposed that canine corticotroph adenomasmay provide a model to study corticotroph cellpathophysiology (de Bruin et al. 2008b). However,since distinct differences do exist between human andcanine corticotroph adenomas in terms of SSR and D2

receptor expression and regulation, as well as therelative responses to SRIF and DA agonists, thesedifferences should be carefully considered when usingdogs as a model to evaluate efficacy of novel SRIF andDA agonists in human Cushing’s disease (de Bruin et al.2008b). D2 receptors have been found at very lowdensity and with a different distribution of the twoisoforms in the rare TSH-secreting tumours as well,where, on the contrary, sst1, and especially sst2 andsst5, are significantly represented (Panetta & Patel 1995,Stefaneanu et al. 2001, Yoshihara et al. 2007, Saveanuet al. 2008).

Although the above-mentioned high variability maybe due in part to the different techniques used in thesestudies (mainly mRNA analysis by northern blot, in situhybridisation and real-time PCR, or protein assays, suchas radioactive-binding studies, and, more recently,immunohistochemistry), an apparently rather complexsituation came out because of the great heterogeneityof these tumours.

However, when we examine more deeply these data, areliable pattern apparently emerges. The D2 receptor isthe GRCP mostly represented in the pituitary tumours,and is overall associated with two or more SSR subtypesin the different adenoma categories, possible followingpreferential outline in certain subclasses: sst2 and sst5are linked with D2 in the majority of somatotrophtumours, D2 is coexpressed with sst1 and sst5 inprolactinomas, and with sst3 and sst2 in NFPAs, finallyD2 and sst5 are almost equally represented in cortico-troph tumours (Zatelli et al. 2005, de Bruin et al. 2009,Ferone et al. 2008, Saveanu et al. 2008; Fig. 2).



Figure 2 Distribution of somatostatin and D2 dopamine receptors in the different type of pituitary adenomas.The D2 is the receptor mostly represented in the pituitary tumours and is preferentially associated with sst2and sst5 in somatotroph tumours, with sst1 and sst5 in prolactinomas, with sst3 and sst2 in clinically non-functioning adenomas and with sst5 in corticotroph tumours. In mixed and plurihormonal adenomas, ahighly heterogeneous pattern is observed.


Functional role of somatostatin and D2

dopamine receptors in pituitary adenomasand implications for therapeutic strategies

It is, however, intriguing that the observations arisingfrom studies of characterisation of the receptor profilein pituitary tumours did not always correlate with theefficacy expected for the corresponding medicaltherapy.

In fact, while dopaminergic drugs are efficient inmore than 90% of prolactinomas (Gillam et al. 2006),SRIF analogues, octreotide and lanreotide controlabout 80% of TSH-secreting adenomas (Beck-Peccoz& Persani 2002, Ness-Abramof et al. 2007), but only 60%of GH-producing tumours (Freda 2002, Burt & Ho2006). Moreover, clinically available SRIF analogues orDA agonists have a minor role in the medical treatmentof NFPAs, since they have been shown to control thegrowth of less than 20% of this tumour type (Colao et al.2008). However, it has been also shown that DA agonisttherapy is associated with a decreased prevalence ofresidual tumour enlargement in patients with NFPA,particularly when treatment is instituted before tumourremnant growth is detected (Greenman 2007). On theother hand, although still in lower percentagecompared with other pituitary adenomas, at leastcabergoline, a D2-selective second-generation DAagonist, seems able to control a specific subset ofcorticotroph pituitary tumours (Pivonello et al. 2004a,


de Bruin et al. 2009). Similarly, a new SRIF agonist,pasireotide, displaying a broader receptor-bindingprofile compared with octreotide and lanreotide,appears promising for the medical treatment ofCushing’s disease (Ben-Shlomo & Melmed 2007).

The item becomes even more interesting when weare faced with the plurihormonal adenomas of theacidophilic cell line, namely the pure somatotroph,somatomammotroph tumours, the truly mixedGH/PRL adenomas and even the very rare stem celltumour (Asa et al. 1992). This assorted family representsthe 30% of pituitary tumours in acromegaly. Theplurihormonal adenomas have been shown to befunctionally different, displaying a different sensitivityto the treatment with SRIF and/or DA agonists, alsowhen administered in combination. Indeed, althoughthe target cell could contribute to drug sensitivity, againin this case the specific receptor distribution on tumourcells plays the major role in determining the sensitivityto both SRIF and DA analogues (Ferone et al. 2001). Infact, it has been recently demonstrated that the reallymixed GH/PRL tumours seem more resistant to SRIFanalogues, whereas almost no difference in sensitivity,not only to SRIF analogues but also to DA agonists, hasbeen observed in somatotroph and somatomammo-troph adenomas (Ferone et al. 2008). This seems to bemainly due to the lower density of sst2 in mixedtumours. However, an interfering role of other receptorsubtypes cannot be ruled out. Indeed, previously, in an




experimental model, a full concordance betweentumour response to the DA agonist bromocriptineand the expression of D2 receptors by tumour cells wasfound in five lineages of a spontaneous transplantablerat pituitary tumour (SMtTW) exhibiting differentPRL/GH phenotypes (Trouillas et al. 1999). Thismodel resembles the various tumour phenotypesencountered in human pituitary pathology. However,Zatelli et al. (2005) elegantly investigated the in vitroresponse of a heterogeneous group of human somato-troph adenomas to experimental SRIF analogues. Theauthors suggested that adenomas expressing D2 are lesslikely to respond to SRIF analogues in terms ofinhibition of GH secretion (Zatelli et al. 2005).

The modern view of the concept of resistance tomedical therapy must take into account the effect ontumour mass as well. Indeed, until a few years ago, insecreting adenomas, the responsiveness or resistance tothe medical therapy was defined according to thehormonal response. In general, the cases biochemicallyresponsive were those experiencing also significanttumour size reduction during long-term treatments.However, at least for somatotroph adenomas treatedwith SRIF analogues, it is becoming clear that manyadenomas may undergo impressive tumour shrinkage,although the hormonal hyperproduction is not fullycontrolled (Bevan 2005, Casarini et al. 2006, Cozzi et al.2006, Resmini et al. 2007).

To overcome the resistance to single-agent treatment,the use of a combined SRIF analogue and DA agonisttreatment schedule has been explored, particularly inGH-secreting adenomas, which are known to bepotentially responsive to both classes of compounds(Colao et al. 2007). More recently, NFPAs were shown tobe sensitive to a combined treatment as well, however,

Table 1 Principal studies reporting results of combined treatment withadenomas

Dopamine agonist

Author (years)

Colao et al. (R) (2007) VariousSelvarajah et al. (2005) BRC/CABCozzi et al. (2004) CABFerone et al. (2001) CV/CABAndersen et al. (2001) CABLi et al. (2000) BRCMarzullo et al. (1999) CABMinniti et al. (1997) CABFløgstad et al. (1994) BRCFredstorp et al. (1994) BRCSadoul et al. (1992) BRCCremonini et al. (1992) BRCWagenaar et al. (1991) BRCPopovic et al. (1990) BRCLamberts et al. (1986) BRC

R, review; BRC, bromocriptine; CAB, cabergoline; OCT, octreotide; LAN, lanreo


the response rate seems limited (Colao et al. 2007). Nostudy with combined therapy has been reported in PRL-and ACTH-secreting adenomas so far, and no rationaleexists today for such an approach with the presentlyavailable compounds (Table 1).

The emerging concept of somatostatin andD2 dopamine receptors dimerisation inpituitary adenomas: implications forpharmacology and drug discovery

The experience of combination therapy has paved theway for new studies that have provided the first evidencefor a new challenge in the field of receptor pathophys-iology and therapeutic strategies. Indeed, the discrep-ancy between the ‘presence’ of a given profile ofSSR/D2 and the limited efficacy of agonist drugs may bepartially explained by the crosstalk on the cellmembranes, or even at post-membrane level, of theGPCRs (Ferone et al. 2007b, Saveanu & Jaquet 2008).The preclinical use of new experimental compounds iscrucial for understanding the new picture. In fact, SSRdistinct profiles may correspond to different responsesto SRIF subtypes preferential ligands. For example, thesst5 preferential ligand, BIM-23268, suppresses PRLsecretion in prolactinomas, as well as ACTH secretionin corticotroph tumours (Saveanu & Jaquet 2008). Therelationship between SSR expression and theirfunctionality may be more complex in GH-secretingadenomas expressing low levels of sst2, where thesst2-preferential ligand octreotide is ineffective insuppressing GH release. In such cases, an sst5 selectiveagonist may be of value because this SSR subtype is

dopaminergic agents and somatostatin analogues in pituitary

Somatostatin analogue Disease

Various Pituitary adenomasOCT/LAN AcromegalyOCT/LAN AcromegalyOCT AcromegalyOCT NFPAOCT AcromegalyLAN AcromegalyOCT AcromegalyOCT AcromegalyOCT AcromegalyOCT AcromegalyOCT McCune-AlbrightOCT AcromegalyOCT AcromegalyOCT Acromegaly

tide; CV, quinagolide; NFPA, clinically non-functioning pituitary adenoma.




often highly expressed (Saveanu et al. 2001). However,in this specific setting, a bi-specific analogue, such asBIM-23244, which can activate both receptors, couldachieve a better control of GH hypersecretion (Saveanuet al. 2001). Moreover, a selective sst1 ligand, BIM-23926,has been shown to inhibit in vitro hormone secretionand cell viability in GH- and PRL-secreting adenomasand in a subset of NFPAs (Zatelli et al. 2003, 2004).Whereas in corticotroph adenomas, the predominantmRNA expression of sst5 over sst2 makes these adeno-mas highly sensitive to multireceptor ligands, such asthe new SRIF analogue pasireotide (de Bruin et al.2009). Recent data have showed that pasireotidemodulates SSR trafficking in a manner clearly distinctfrom other SRIF analogues, providing an alternativeexplanation for the differential regulation of SSRresponsiveness during long-term administration of stableSRIF analogues (Lesche et al. 2008). Moreover, SSRsact not just as monomers but may display a differentialtendency to homo- and heterodimerise, depending onthe subtype involved, and perhaps also on the celltype in which they are expressed (Duran-Prado et al.2008). Indeed, pairs of distinct SSRs, such as sst2–sst3and sst1–sst5, may interact and establish a physicalinteraction, resulting in altered pharmacologicalor/and functional properties (Duran-Prado et al. 2008).

Figure 3 Somatostatin/dopamine chimera-induced dimPotential intracellular signal transduction pathways linkresult in phosphorylation of c-Jun N-terminal kinase thatranscription of the cyclin, KI67. This combination resultsdepending on the hybrid receptor complex, P21WAF1/Cfrom Kidd et al. 2008). JNK, c-Jun N-terminal kinase.


In this view, while the first chemical approach, follow-ing the discovery of the five SSRs, was to constructligands with a high affinity for each subtype, morerecently the pharmaceutical companies have driventhe research toward new compounds interacting withmore than one SSR subtype. Moreover, knowing theassociation of SSRs and D2 receptor in the majority ofpituitary adenomas, another chemical approach con-sisted in the synthesis of chimeric molecules containingstructural elements of both SRIF and DA and directedagainst both the superfamilies of GPCRs (Ferone et al.2007a,b, Saveanu & Jaquet 2008). The first molecules ofthis class, BIM-23A387 and BIM-23A760, were charac-terised by their sst2 and D2 and sst2, sst5 and D2 affinityrespectively, and resulted effective in controllinghormone hypersecretion in vitro in somatotrophadenomas that were partial responders to octreotide(Saveanu et al. 2006). These chimeric compounds wereeffective at lower concentrations compared with ana-logues directed toward a single receptor, and wereeven more efficient than octreotide in combinationwith cabergoline (Saveanu et al. 2006).

In addition to the binding properties of the hybridcompounds, other mechanisms may explain theirgreater efficiency in suppressing hormone release: thepharmacokinetics characteristics, the longer stability

erisation of somatostatin and dopamine receptors.ed to the heterodimer. Receptor activation mayt up-regulates P21WAF1/CIP1, while inhibitingin growth arrest of neuroendocrine cells. However,IP1 transcription can also be decreased (adapted




and, very intriguing, the demonstrated capacity intransfected cell lines, in which SRIF and DA ligandscould induce or maintain receptor homo- andheterodimerisation and increase functional activity(Rocheville et al. 2000). This latter observation suggeststhat at least a portion of the high activity of thesechimeric molecules may be due to promoting theformation of dimers between GPCRs.

Presently, the experience with the so-called ‘dopa-statin’ molecules has been extended to the NFPAs aswell (Gruszka et al. 2006, Ferone et al. 2007b, Florio et al.2008). Although in vitro chimeric agonists were effectivein inhibiting a-subunit secretion (Ferone et al. 2007b) aswell as cell proliferation (Florio et al. 2008) in thisheterogeneous class of pituitary tumours, the resultsappear less convincing so far. From a certain point ofview, it seems that also the chimeric molecules may actdifferently in various tissues tested, and the effect coulddiffer according to cell types. Actually, this hypothesiswas already explored with SSR subtype-specific ana-logues. In fact, another in vitro study on NFPAs, whilerevealing the potential importance of sst1 in mediatingan inhibitory effect of a selective agonist on chromo-granin A and a-subunit secretion as well as on cellviability, also demonstrated that the incubation of thesecells with a sst5-selective agonist may enhance pituitarycell viability (Zatelli et al. 2004). Therefore, it seemsthat the coexpression of SRIF and D2 receptors isnot sufficient to improve the responsiveness of pituitarytumours to the anti-secretory and/or the anti-proliferative actions of the new class of hybridcompounds, indicating that once again the cell typeshould also be taken into account. In support of thishypothesis is the experience in testing these drugs withother cell systems than pituitary cells. For example,dopastatins resulted more effective in inhibiting cellgrowth in a non-small lung carcinoma cell linecompared with subtype-specific SSR analogues andDA agonists, demonstrating in this cell system, con-stitutively expressing SSRs and DRs, a clear additivity ofthe hybrid compounds (Ferone et al. 2005). Conversely,in gastric enterochromaffin-like cell, the dopastatinBIM-27A760 did not displayed an additive effect onhistamine secretion and cell proliferation (Kidd et al.2007). On the other hand, the activation of multipleSSRs by pasireotide significantly reduced cell prolifer-ation in the neuroendocrine tumour cell lineNCI-H727 (Ono et al. 2007), and inhibited cell growthand catecholamine secretion in cell cultures ofphaeochromocytoma, also inducing apoptosis(Pasquali et al. 2008). Indeed, a differential cytotoxicityof chimeric compounds was recently observed inbronchopulmonary and small intestinal neuro-endocrine tumour cell lines (Kidd et al. 2008). Theresponses of each individual cell line suggested thatneuroendocrine tumours from diverse locations arising


from different neuroendocrine cells may require cell-specific anti-proliferative agents based on the uniquereceptor profile of individual lesions (Kidd et al. 2008).Pituitary and ectopic corticotroph tumours, expressingsst2, sst5 and D2 receptors, are promising potentialtarget for dopastatin molecules. In fact, although up tonow no data have been issued in this field, pre-clinicalresults and single preliminary clinical experiencesencouraged the research in this group (Pivonello et al.2005, de Bruin et al. 2009), while in prolactinomas,where sst5 and D2 are coexpressed, only a slightadditivity of specific agonists was demonstrated in cellcultures (Saveanu & Jaquet 2008).

Finally, GPCR dimerisation seems to influence notonly pituitary cell functions, but may also provide amechanism for the lack of tolerance seen with currentlyavailable SRIF analogues and their ability in controllingpituitary tumour growth. Indeed, heterodimerisationbetween sst2 and sst5 has been recently found involvedin this important phenomenon (Grant et al. 2008).The next important step will be the accurate charac-terisation of the post-receptor signalling pathways,which certainly depend on the SRIF and DR profile;however, it could be significantly affected by thespecific neuroendocrine (and non-neuroendocrine)cell type (Fig. 3).

In conclusion, the lack of clinical response of a ratherhigh percentage of patients with pituitary adenomas tothe currently available drugs could be due not only toan inadequate characterisation of the pituitary tumourreceptor profile, but also to the fact that a sort of cellspecificity in the response may affect the final outcome.Moreover, in this scenario, we should also consider thenew insight of receptor dimerisation. Dimerisation isfairly common in the GPCR superfamily, and over-whelming amounts of data suggest that many GPCRs,including SSRs and DRs, exhibit functional propertiesthat require direct or indirect interactions betweenclustered receptors. The integration of this property ofthe SSRs and DRs in the know-how is crucial toelucidate the physiological mechanism of action ofcertain hormones, while an understanding of themechanisms involved in these events could offer arationale in next drug design, also for tumours arisingform other cells expressing GPCRs.

Declaration of interest

The authors declare that there is no conflict of interest that wouldprejudice the impartiality of this work.

Funding

This study was partially supported by grants from Italian Ministry ofUniversity and Scientific and Technological Research (MIUR2007RFFFFN_005) and from University of Genova.




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Received in final form 8 January 2009Accepted 13 January 2009Made available online as an Accepted Preprint 13 January 2009



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