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ARTICLE |
Section of Endocrinology, Department of Biomedical Sciences and Advanced Therapies, University of Ferrara, Via Savonarola 9, 44100 Ferrara, Italy
(Correspondence should be addressed to M C Zatelli; Email: ztlmch{at}unife.it)
This paper was presented at a symposium held at the Erasmus Medical Center, Rotterdam, The Netherlands, 2005. The symposium was jointly organized by LJ Hofland, Erasmus Medical Center, and A Colao, Federico II University of Naples, Italy. Ipsen partially supported the publication of these proceedings.
Abstract
The antisecretory effects of somatostatin (SRIH) and its analogs are widely recognized and provide the basis for treatment of hormonal hypersecretion in patients with pituitary adenomas, especially in the settings of acromegaly. Dopamine (DA) agonists have also been used for medical treatment of prolactin and/or GH hypersecretion, and recent evidence points to an even greater antisecretory effect for a chimeric molecule, having high affinity for both SRIH and DA receptors. Evidence for an antiproliferative effect of these compounds has also been provided. This review focuses on the antiproliferative effects of SRIH and its analogs, of DA and chimeric compounds on pituitary adenomas, and on the clinical consequences on tumor volume of pharmacological treatment of pituitary adenomas with these drugs.
Since the discovery of the antisecretory properties of somatostatin (SRIH) analogs, these compounds have been widely employed in the medical therapy of pituitary adenomas. The natural peptide, SRIH, is secreted by hypothalamic neurons into the hypophyseal portal circulation and regulates pituitary hormone secretion (1). It inhibits growth hormone (GH) and thyroid-stimulating hormone secretion (2), and also prolactin (PRL) and adrenocorticotropic hormone (ACTH) secretion under some circumstances (3). SRIH transduces its inhibitory effects by interacting with five different somatostatin receptor (SSTR) subtypes, in normal pituitary cells as well as in pituitary adenomas (4). The evidence that SRIH and its analogs have antisecretory effects prompted many studies aimed at investigating the possible antiproliferative effects of these compounds on pituitary adenomas. Indeed, it has been demonstrated that native SRIH inhibits the proliferation of both normal and neoplasic cells by inducing cell cycle arrest or apoptosis, depending on the receptor subtype and the target tissue (5–7). As a consequence, many studies have been performed and octreotide, one of the first SRIH analogs being synthesized and used in clinical practice, has been demonstrated to significantly reduce not only PRL secretion, but also DNA synthesis in a rat prolactinoma cell line (8). Moreover, in these settings, evidence for desensitization was shown due to loss of SSTR expression, which was restored by withdrawal from long-term exposure to octreotide. In addition, a dissociation between antiproliferative effects of octreotide, being apparent when SSTRs are highly expressed, and antisecretory effects, appearing for much lower SSTR expression levels in the same rat prolactinoma cell line, was suggested (9). Therefore, many studies subsequently explored SSTR subtype expression in pituitary adenomas, demonstrating that SSTR2 is the most frequently expressed receptor (10). These studies provided the basis for the use of SRIH analogs in the medical therapy of pituitary adenomas, in the attempt to restrain pituitary tumor growth and function. Evidence for octreotide’s directly antiproliferative effects was provided by a multicenter randomized trial recruiting 32 acromegalic patients with pituitary macroadenoma. A group of 16 patients underwent octreotide treatment for 4 months before surgical resection, showing a 83% reduction in the mean growth fraction in comparison with the untreated surgical controls (11), indicating that octreotide pretreatment significantly suppressed pituitary adenoma cell growth. In addition, another study showed that apoptosis was not significantly altered by the octreotide pretreatment (12), suggesting that a reduction in cell cycling, rather than an increase in apoptotic rate, is the basis for the antiproliferative action of octreotide in somatotrope adenomas. This evidence was further confirmed by a clinical study where 14 acromegalic patients underwent a 3-month pretreatment with SRIH analogs, showing more prominent regressive changes and increased perivascular fibrosis when compared with 17 surgical controls. Moreover, the Ki-67 labeling index was lower in treated than in untreated patients, supporting the hypothesis that SRIH analogs have antiproliferative effects on somatotrope adenomas (13). These effects, however, are not mirrored by significant morphological changes, since octreotide pretreatment does not cause necrosis, whereas it induces acidophilia and interstitial fibrosis, with a slight reduction in cell size (14). This picture greatly differs from that observed in prolactinomas after treatment with a dopamine (DA) agonist, where marked reduction in cellular, cytoplasmic and nuclear volume as well as marked perivascular and interstitial fibrosis and effects on the endoplasmic reticulum and Golgi have been observed (15). A recent review summarized the results of SRIH analog treatment on the size of pituitary adenomas in 921 acromegalic patients (16), showing that 42% of the patients displayed a reduction in tumor volume. This effect was more evident in patients in primary therapy (52%), when compared with patients who had already undergone surgery (21%). In addition, only 5% of the patients experienced further tumor enlargement, suggesting that treatment with SRIH analogs may contribute to the control of tumor growth in the majority of patients, at least for the treatment periods of up to 3 years.
The in vivo antiproliferative effects of octreotide are in agreement with previous in vitro studies, showing a significant reduction in cell number in three out of six primary cultures from GH-secreting pituitary adenomas exposed for 3 days to 1–10 µM octreotide (17). Similar effects were shown in primary cultures from clinically nonfunctioning pituitary adenomas (NFA), where significant inhibition of cell number was obtained for higher octreotide concentrations (10 µM), suggesting a lower SSTR expression level in these pituitary tumors (17). More insights into the antiproliferative effects of SRIH and its analogs have been provided by a more recent study analyzing 15 GH-secreting pituitary adenomas. SSTR2 and SSTR5 mRNAs were expressed in all tumors, while SSTR1 and SSTR3 mRNAs were expressed in 11 and 12 tumors respectively. Primary cultures were performed and tested with 1 µM SRIH or lanreotide (Lan), a SRIH analog interacting with both SSTR2 and SSTR5. After 24 h, no effect was observed in some cultures, but in others (as well as in the GH3 rat cell line) a parallel reduction in cell viability and DNA synthesis was observed. Moreover, tumors that were responsive to SSTR2- and SSTR5-specific analogs were also responsive to Lan. This study also highlighted a dissociation between the in vitro effects of SRIH analogs on tumor cell proliferation and GH secretion, suggesting that the inhibition of cell proliferation occurs independently of the effects on GH secretion (18).
The mechanism by which SRIH inhibits pituitary cell proliferation has been investigated in the GH3 rat pituitary cell line, where octreotide and a chimeric compound, formed by GH-releasing peptide linked to SRIH, significantly inhibited cell proliferation by activating protein tyrosine phosphatases (PTP), involving a cGMP-dependent pathway (19). The involvement of PTPs was then confirmed by further studies performed in GH-secreting pituitary adenoma primary cultures, where SRIH and Lan inhibited cell proliferation induced by phorbol esters (PMA). This inhibitory effect was blocked by incubation with vanadate, a nonspecific PTP inhibitor, suggesting that this enzyme family is involved in mediating the antiproliferative effects of SRIH and its analogs. Moreover, this study showed that the inhibition of GH secretion was mediated by blockade of calcium channels and adenylyl cyclase activity, further supporting the hypothesis that the inhibition of cell proliferation occurs independently of the effects on GH-secretory pathways (20). It has been recently demonstrated that octreotide can induce a dose-dependent increase in caspase-3 activity, indicating that this compound can promote apoptosis in somatotrope tumors. A similar effect was also observed after treatment with a selective SSTR2 agonist, BIM-23120, which induces apoptosis by a phosphatase-dependent mechanism and causes growth arrest by an increase in p27 and a decrease in cyclin D1 expression (21). This finding confirmed the previous studies by Luciani et al. (22), showing that octreotide treatment induces apoptosis in GH-secreting adenomas, where the expression level of the antiapoptotic gene seladin-1 is significantly lower when compared with NFA. In addition, SRIH analogs have been shown to produce their antiproliferative action by acting on the PI3K/Akt signaling pathway and increasing Zac1 gene expression. Zac1 induces cell cycle arrest and apoptosis and is highly expressed in normal pituitary, mammary and ovarian glands but is downregulated in pituitary, breast and ovarian tumors, suggesting that it might act as an oncosuppressor gene (23).
Previous studies demonstrated SSTR expression in a group of NFA, where SRIH and Lan inhibited PMA-stimulated cell proliferation in most NFA primary cultures. This effect was once again blocked by vanadate incubation, providing further evidence for PTP involvement in SRIH signal transduction in pituitary adenomas (24). Indeed, PTPs are listed among the pathways activated by SSTRs triggering cell proliferation inhibition, since the phosphatase family, and especially SHP-1, is involved in the control of apoptosis and in cell cycle regulation (25). We have previously demonstrated that constitutive SHP-1 activity controls cell proliferation in a human medullary thyroid carcinoma cell line, providing also evidence that, upon SSTR2 activation, SHP-1 activity can be further upregulated leading to cell proliferation inhibition, despite constitutive activation of potent proliferative stimuli (26). Taken together, these results not only confirm that SSTR2 is important in transducing the antiproliferative signals of SRIH, but also suggest that more than one SSTR is able to induce antiproliferative effects. At the same time, multiple subtypes may cooperate to induce growth arrest, also in the light of the recent advances on SSTR biology, indicating the possibility for these receptors to homo-and heterodimerize with other G-protein coupled receptors (27). The availability of SSTR-selective analogs therefore provides the opportunity to study the effects of activating single versus multiple SSTRs, since it now appears that the concept of a single dominant SSTR subtype controlling a specific biological function is too simplistic (28).
Trying to elucidate which other SSTR might be involved in transducing SRIH antiproliferative effects, we investigated SSTR1 activation in pituitary adenomas. For this purpose, we selected a group of 15 GH-secreting pituitary adenomas expressing SSTR1 and performed primary cultures that underwent treatment with SRIH or with a SSTR1-selective agonist, BIM-23926 at 10 nM. As expected, we observed a significant inhibition in GH and PRL secretion not only under SRIH treatment, but also under treatment with the SSTR1-selective agonist. Interestingly, this compound was as potent as SRIH in inhibiting pituitary cell viability in vitro. Moreover, we found a significant positive correlation between the antiproliferative effects of BIM-23926 on GH and PRL secretion and SSTR1 mRNA expression. However, such correlation was not evident for the antiproliferative effect, suggesting that even very low SSTR1 expression might induce the antiproliferative effects of the SSTR1-selective agonist (29).
These results also indicate that SSTR1 may mediate SRIH actions at pituitary level, suggesting that this receptor may represent a target for new therapies attempting to control growth and hormonal secretion of pituitary tumors. This hypothesis is further supported by the finding that a SSTR1-selective agonist significantly inhibits GH secretion in vitro also in tumors from patients resistant to octreotide or Lan therapy (30).
The importance of SSTR1 as a possible target for medical therapy of pituitary adenomas was further underlined by the study we performed on a selected group of 12 NFA, all expressing SSTR1, SSTR2 and SSTR5, as well as chromogranin A (CgA) and 
-subunit. We showed that SSTR1- and SSTR2-selective agonists inhibit both -subunit and CgA secretion by NFA primary cultures. Moreover, we showed that only the SSTR1-selective agonist was capable of significantly reducing in vitro cell viability, while the SSTR5-selective agonist enhanced pituitary cell viability. Again, the inhibitory effects of the SSTR1-selective agonist did not correlate with the extent of SSTR1 expression, further supporting the hypothesis that even very low SSTR1 expression might induce the inhibitory effects of the SSTR1-selective agonist (31). These results suggest that SRIH analogs with enhanced potency for SSTR1 could offer useful tools for the medical therapy of NFA. To the contrary, our results show that the treatment with a SSTR5-selective agonist significantly promoted NFA cell viability. This effect might account for the lack of NFA volume reduction observed in some patients treated with octreotide or Lan, which mainly interact not only with SSTR2, but also with SSTR5 (32–35). Moreover, our findings could explain the increase in tumor volume observed in some cases during such therapy (33), suggesting that SSTR5-interacting drugs might fail to control NFA tumor growth or, on the contrary, might facilitate pituitary cell survival in these adenomas. Therefore, the indication for medical therapy with octreotide or Lan in NFA patients with evidence for tumor SSTR5 expression should be carefully evaluated. On the other hand, it has been demonstrated that growth reduction of the mouse thyrotrope tumor cell line TtT-97 by thyroid hormone treatment is associated with increased expression of SSTR5 (36). Further experiments led to the hypothesis that thyroid hormones, acting through increased SSTR5, could activate the ERK pathway, resulting in a reduced expression of critical cell cycle proteins and a subsequent arrest of S-phase progression. These processes were reversed upon withdrawal of thyroid hormones, resulting in an increase in the fraction of S-phase cells (37). In addition, it has been recently demonstrated that treatment of dispersed GH-secreting pituitary adenoma cells with octreotide, as well as pasireotide (SOM230, a novel SRIH analog interacting with all SSTRs except for SSTR4), upregulates the expression of a cyclin-dependent kinase inhibitor, p27, possibly resulting in cell cycle inhibition (38).
Besides the direct actions on pituitary cells, SRIH and its analogs also have indirect effects, since they inhibit production and secretion of many angiogenic factors, therefore reducing tumor growth rate (39). Neoangiogenesis is fundamental for tumor growth and one of the main factors promoting this process is vascular endothelial growth factor (VEGF). It has been recently demonstrated that VEGF is overexpressed in the pituitary of DA receptor subtype 2 (DR2) knockout mice (40), indicating that this growth factor is under dopaminergic control. DR2 is extensively expressed in all pituitary tumor types, as demonstrated by in situ hybridization and immunohistochemistry (41). It has also been demonstrated that DR2 knockout mice develop prolactinomas with age (42, 43), DA transporter gene knockout mice show arrested lactotrope development (44), and transgenic mice bearing the short isoform of DR2 (DR2s) display lactotrope hypoplasia (45). This evidence provides the basis for the use of DA agonists in the medical treatment of prolactinomas and highlights the importance of DA agonists in the control of lactotrope proliferation. Indeed, it has been recently demonstrated in vivo and in vitro that lactotrope treatment with DA and its agonist, bromocriptine, induces transforming growth factor (TGF)-ß1 expression and secretion, which, in turn, can mediate the antiproliferative effects of DA on lactotropes. In addition, constitutive expression of the DR2s receptor upregulated the TGFß1 expression and action, and inhibited cell proliferation in transformed lactotropes (46). DR2 activation in lactotropes leads to the alteration of G-protein coupling, inhibition of adenylyl cyclase, and reduction of intracellular cAMP (47, 48). In addition, DRs have been shown to regulate other transduction pathways that lead to alteration of intracellular calcium, protein kinase C, and the MAPK pathway (45, 47–50), resulting in reduced cell proliferation in a number of cellular models.
However, the effects of DA agonists on pituitary tumors different from prolactinomas are still a matter of debate. Indeed, some clinical studies showed that treatment with DA agonists has no effect on NFA volume (51), while others documented tumor shrinkage of up to 11% (52). In vitro studies have shown that, depending on the dose, bromocriptine is capable of significantly reducing the cell number in NFA, an effect reversed by a DR2 antagonist, haloperidol (17). Further studies showed that DR2 expression is variable in NFA, and that recurrent tumors lack expression of DR2s (53). Moreover, three out of nine NFA cultures responded to bromocriptine in terms of DNA synthesis reduction, and all responder adenomas expressed DR2s. In all cases, haloperidol, a DR2-selective antagonist, blocked the inhibitory effects of bromocriptine, demonstrating that this action was mediated by DR2. These data suggest that DR2s expression in NFA favors the growth suppressive effect of bromocriptine. This hypothesis is further supported by the finding that DR2s expression is lower in resistant prolactinomas when compared with responsive adenomas (54), and that increased DR2s expression in lactotrope cells results in pituitary hypoplasia (45). The mechanisms underlying the antiproliferative effects of DA agonists have been explored in the GH3 cell line, where the specific inhibitors of p38 MAPK and ERK pathways completely abrogated such effect, indicating that DA induces antiproliferative effects and cell death via DR2, involving p38 MAPK and ERK pathways (55).
Moreover, it has been recently demonstrated that approximately 80% of corticotrope pituitary tumors express functional DR2 and that treatment with cabergoline is effective in controlling the cortisol hypersecretion associated with Cushing’s disease (56). This study confirms the previous reports, showing that cabergoline treatment caused remission of Nelson’s syndrome (57) and normalization of PRL, ACTH, and cortisol levels in a macroprolactinoma associated with Cushing’s disease (58), suggesting that the DA agonist may be useful in restraining hormone secretion by neoplastic corticotrope cells. In addition, a previous in vitro study demonstrated that bromocriptine inhibits murine ACTH-secreting pituitary adenoma (AtT-20) cell proliferation, likely inducing apoptosis (59), further supporting the therapeutic use of DA agonists in the management of Cushing’s disease. And indeed, three cases of persistent Cushing’s disease have been described, where treatment with cabergoline induced a decrease or normalization in hypercortisolism, with stabilization of tumor volume in one case (60). Besides the DA agonists, SRIH analogs have been studied as possible pharmacological agents to treat ACTH-secreting pituitary adenomas. It has been recently demonstrated that human corticotrope tumors express multiple SSTR subtypes, and that the multiligand SRIH analog SOM230 significantly suppresses cell proliferation, as well as ACTH secretion, in primary cultures of human corticotrope tumors. These data suggest that SOM230 may have a role in the medical therapy of corticotrope tumors (61).
Together with SRIH analogs and DA agonists, chimeric compounds interacting with SSTR2 or SSTR5 and DR2 have been explored for their effects on cell viability. In a recent study, these compounds were capable of reducing cell viability in most (but not all) NFA primary cultures, having an inhibitory effect similar to selective SSTR or DR agonists. However, bromocriptine was the most effective agonists in reducing NFA cell viability (62).
In conclusion, the antiproliferative effect of SRIH analogs, DA agonists and chimeras, both in vitro (Table 1
) and in vitro (Table 2), depends on tissue SSTR and DR expression pattern, the different binding profiles of the various agonists and SSTR and DR effector coupling. Further studies are therefore needed to elucidate the mechanisms underlying such antiproliferative effects and to verify the possible clinical applications.
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This work was supported by grants from the Italian Ministry of University and Scientific and Technological Research (University of Ferrara: 60% – 2006 and MIUR 2005060839-004), Fondazione Cassa di Risparmio di Ferrara, Associazione Italiana per la Ricerca sul Cancro (AIRC) and the Associazione Ferrarese dell’Ipertensione Arteriosa to the University of Ferrara.
References
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