Endocrine-Related Cancer (2011) 18 C1–C14 COMMENTARY and calcitonin -related peptide receptors in endocrine-related cancers: opportunities and challenges

Debbie L Hay, Christopher S Walker and David R Poyner1

School of Biological Sciences and Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland 1142, New Zealand 1School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UK (Correspondence should be addressed to D R Poyner; Email: [email protected])

Abstract Adrenomedullin (AM), adrenomedullin 2 (AM2/intermedin) and calcitonin gene-related peptide (CGRP) are members of the calcitonin family of peptides. They can act as growth or survival factors for a number of tumours, including those that are endocrine-related. One mechanism through which this occurs is stimulating angiogenesis and lymphangiogenesis. AM is expressed by numerous tumour types and for some cancers, plasma AM levels can be correlated with the severity of the disease. In cancer models, lowering AM content or blocking AM receptors can reduce tumour mass. AM receptors are complexes formed between a seven transmembrane , -like receptor and one of the two accessory , receptor activity-modifying proteins (RAMPs) 2 or 3 to give the AM1 and AM2 receptors respectively. AM also has affinity at the CGRP receptor, which uses RAMP1. Unfortunately, due to a lack of selective pharmacological tools or antibodies to distinguish AM and CGRP receptors, the precise receptors and signal transduction pathways used by the peptides are often uncertain. Two other membrane proteins, RDC1 and L1/G10D (the ‘ADMR’), are not currently considered to be genuine CGRP or AM receptors. In order to properly evaluate whether AM or CGRP receptor inhibition has a role in cancer therapy, it is important to identify which receptors mediate the effects of these peptides. To effectively distinguish AM1 and AM2 receptors, selective receptor antagonists need to be developed. The development of specific CGRP receptor antagonists suggests that this is now feasible. Endocrine-Related Cancer (2011) 18 C1–C14

Introduction or CRLR). The pharmacology of this receptor is Adrenomedullin (AM) is a 52 amino acid peptide, determined by three accessory proteins known as which acts as a paracrine factor in many tissues in the receptor activity-modifying proteins (RAMPs). body, most notably the cardiovascular system where it Although AM first attracted interest as a potent is produced by endothelial cells and causes vasodilation vasodilator, it soon became apparent that it had a (Garcia et al. 2006). It belongs to the same peptide number of other actions, such as to promote cell growth family as amylin, calcitonin and calcitonin gene-related and survival and also to encourage both blood and peptide (CGRP), a 37 amino acid neuropeptide that is lymph vessel formation. Together, these activities especially abundant in the sensory nervous system suggest that AM might be an important factor in (Brain & Grant 2004). Another closely related peptide tumour growth (Zudaire et al. 2003b, Garcia et al. is adrenomedullin 2 (AM2; Takei et al. 2004), also 2006, Nakamura et al.2006, Nikitenko et al. known as intermedin (Roh et al. 2004). These peptides 2006). There is now considerable support for this utilise the same G-protein-coupled receptor (GPCR), hypothesis; however, there are areas of confusion, known as the calcitonin receptor-like receptor (CLR especially regarding the receptors that mediate the

Endocrine-Related Cancer (2011) 18 C1–C14 Downloaded from Bioscientifica.comDOI: 10.1677/ERC-10-0244 at 10/02/2021 09:04:26PM 1351–0088/11/018–C1 q 2011 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.orgvia free access D L Hay et al.: AM and CGRP in endocrine-related cancers effects of AM. This review will particularly focus on administration to adult mice increased the number the challenges associated with deciphering how AM as of blood and lymphatic vessels at the site of an injury well as CGRP and AM2 act in endocrine-related and (Jin et al. 2008). In vitro, AM can act upon endothelial other cancers. cells derived from both blood and lymphatic vessels to promote neovascularisation, proliferation and migration (Miyashita et al. 2003b, Schwarz et al. AM and CGRP receptors 2006, Jin et al. 2008). It has been proposed that AM has AM exerts its actions through two cell-surface a role in vascular maturation (Iwase et al. 2005). Thus, receptors (Poyner et al. 2002). These are multimeric there is great interest in the AM system as a novel and complexes of RAMPs with CLR, a family B GPCR. important angiogenic pathway (Kahn 2008). Mice with CLR in complex with RAMP2 forms an AM1 receptor disruption of the encoding either AM, CLR or whereas CLR complexed with RAMP3 forms an AM2 RAMP2 die in utero with vascular defects, and are receptor (Fig. 1). These are both potently activated by characterised by embryonic lethality, thin blood vessel AM; CGRP binds to the AM2 receptor with around walls and significant abnormalities in their vascular 50-fold lower affinity than AM but has a much lower systems (Caron & Smithies 2001, Shindo et al. 2001, affinity at AM1 receptors. The RAMP1/CLR com- Ando & Fujita 2003, Dackor et al. 2006, 2007). bination forms the CGRP receptor, which is potently The strikingly similar phenotype of all three of activated by CGRP; AM can also signal via this these mouse strains may be explained by the receptor, binding with around 10-fold lower affinity abnormalities in the blood and lymphatic vasculature than CGRP (Poyner et al. 2002, Bailey & Hay 2006). (Fritz-Six et al. 2008, Ichikawa-Shindo et al. 2008). AM2 may have similar pharmacology to AM but this is Interestingly, mice that lack RAMP3 do not show poorly explored (Roh et al. 2004, Takei et al. 2004, obvious changes in their vasculature and live to old age Qi et al. 2008). These receptors predominantly signal but have abnormalities in weight control (Dackor et al. via activation of adenylate cyclase and increase 2007). This indicates that the two AM receptors have intracellular cAMP. Other receptors for AM and different functions. CGRP have been proposed in the past; these will be discussed later in this review. AM in cancer AM expression is widely distributed throughout the Physiological actions of AM on body, principally due to its production by endothelial angiogenesis and lymphangiogenesis cells. In several different tumour types, it has been AM has profound actions on the growth and reported that AM levels are strongly upregulated; development of both the blood and lymphatic vascular especially in hypoxic environments (Table 1). These systems. Mice deficient in AM show reduced vascular- include endocrine or endocrine-related tumours isation (Iimuro et al. 2004). Furthermore, AM where the tumours involve hormone-secreting cells

CGRP>AM=AM2 AM>AM2>>CGRP AM=AM2>CGRP

ECD

TM

ICD

CGRP AM1 AM2

Figure 1 Composition of CGRP and AM receptors. CLR is the seven transmembrane (TM) protein. RAMP1 is shown in black, RAMP2 in dark grey and RAMP3 in white. ECD, extracellular domain; ICD, intracellular domain.

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Table 1 The role of adrenomedullin (AM) in endocrine-related and other cancers

AM Cancer type expression Receptor expression References

Hepatic cancer C ND Nakata et al. (2008) Renal cancer C ND Michelsen et al. (2006) Thyroid (CGRP)a ND Zajac et al. (1986) and Pacini et al. (1991) Lung C ND Martinez et al. (1995) Miller et al. (1996) and Buyukberber et al. (2007) Breast C AM bindingb, RAMP3 Martinez et al. (1995), Miller et al. (1996), Oehler et al. (2003) and Brekhman et al. (2010) Gastro-entero pancreatic C ND Pavel et al. (2006) neuroendocrine Pancreatic C CLR, RAMPs 1, 2, 3 Ishikawa et al. (2003) and Ramachandran et al. (2007) Pancreatic insulinoma C ND Letizia et al. (2001) Ovarian C ND Martinez et al. (1995) and Liu et al. (2009) Endometrial C CLR, RAMP1 (myometrium, Michishita et al. (1999) and Nikitenko et al. vascular smooth muscle) (2001) CLR, RAMP2 (uterine smooth muscle) Prostate C CLR, RAMPs 2, 3 Rocchi et al. (2001), Calvo et al. (2002) and Berenguer et al. (2008) Pituitary adenoma C ND Knerr et al. (2001) Somatotropinoma C ND Knerr et al. (2001) Prolactinoma C ND Knerr et al. (2001) Adrenocortical carcinoma C CLR, RAMP3 Albertin et al. (2005) Pheochromocytoma C CLR, RAMPs 1, 2, 3 Zudaire et al. (2003a,b) and Thouennon et al. (2010) Aldosteronoma C CLR, RAMPs 1, 2, 3 Albertin et al. (2001) and Forneris et al. (2001)

This table is based on data from Zudaire et al. (2003b) but has been updated and modified with additional data. ND, not done. aCGRP expression. b[125I]AM radioligand binding in MCF-7 cells.

(e.g. pancreatic insulinoma) or develop a neuroendo- stimulate angiogenesis (Zudaire et al. 2006). AM crine phenotype (prostate cancer; see below). AM does may also reduce the effectiveness of the immune not cause cancer but can contribute to its pathogenesis system to destroy tumours, by decreasing the in several ways. The overall scheme of activity of AM expression of pro-inflammatory cytokines and inhibit- in the tumour microenvironment is shown in Fig. 2. ing the activation of the alternative complement AM can act directly to stimulate cell growth and inhibit pathway by binding to complement factor H (Zudaire apoptosis in a variety of tumour cells (Forneris et al. et al. 2003a). 2001, Martinez et al. 2002, Albertin et al. 2005). AM The major sources of AM relevant to tumours are can change the phenotype of cells, leading them to from the tumour cells themselves (Table 1) and the exhibit tumourigenic behaviour, for example, in vascular endothelium. However, other cells that may prostate cancer, AM promotes the appearance of a be found associated with tumours such as mast cells neuroendocrine phenotype in cells (Martinez et al. can also produce the peptide (Zudaire et al. 2006). 2002, Berenguer et al. 2008). AM may promote tumour Furthermore, AM signalling is enhanced under blood and lymphatic angiogenesis, providing a supply hypoxic conditions, chiefly via hypoxia-inducible ofnutrientsandoxygentothetumourbutalso factor-1a (HIF-1a). This is a transcription factor providing a means for tumour cells to spread (Garayoa produced in response to hypoxia; it stimulates et al. 2000, Fritz-Six et al. 2008, Ichikawa-Shindo synthesis of both AM and CLR via hypoxic response et al. 2008). It is likely that this effect is mainly elements located on their respective genes (Nikitenko mediated by AM receptors on endothelial cells, et al. 2006). As solid tumours are typically hypoxic, although it has been reported that AM can activate they will be encouraged to produce even more AM and mast cells in a receptor-independent manner to to respond to it with greater sensitivity.

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Blood vessels Lymphatic vessels AM over-expression by tumours and in many cases the data are unavailable. This is because the majority of tissues normally express AM and appropriate compari- sons between normal tissue or cells and tumours can + AM AM + seldom be made.

Tumour cells Disease models AM AM Xenografts or the injection of tumour cells, which have been engineered to over-express AM into susceptible + AM + mice, can lead to tumour formation (Martinez et al. Hypoxic environment 2002, Oehler et al. 2002). Furthermore, agents that + reduce the activity of AM such as the peptide antagonist AM22–52 and agents that bind directly to Figure 2 AM and tumour proliferation and blood and lymphatic AM reduce endothelial cell proliferation, migration, vascular angiogenesis. AM, released from the tumour (particularly under hypoxia) and endothelial cells in vessels, angiogenesis, tumour vascularisation and tumour acts in an autocrine/paracrine manner to enhance tumour cell growth (Ishikawa et al. 2003, Miyashita et al. 2003a, proliferation and survival as well as promoting vascular Iimuro et al. 2004, Schwarz et al. 2006, Roldos et al. (blood or lymphatic) endothelial cell proliferation/migration and vessel growth. 2008, Tsuchiya et al. 2010). For example, tumour weight was reduced compared with control following Clinical evidence intratumoural injection of AM22–52 in a mouse model of in vivo pancreatic cancer cell growth (Ishikawa et al. There are now many studies that show an association 2003). This suggests that the antagonist was able to between AM expression and cancer. These are reduce the activity of endogenous AM produced by the predominantly studies where AM mRNA and/or tumour or local blood vessels (Ishikawa et al. 2003). protein expression have been determined in different Moreover, antibodies to the AM1 and AM2 receptors tumour types and compared with normal tissue. In inhibited migration and capillary tube formation of some studies, plasma AM concentrations have also endothelial cells and the growth of human tumour been measured. These studies suggest that AM is over- xenografts but it is important to note that the specificity expressed in numerous endocrine-related and other of these antibodies has not been properly reported tumours including renal cell carcinoma, granuloma cell (Fernandez-Sauze et al. 2004, Kaafarani et al. 2009). tumours, hepatocellular carcinoma, non-small cell lung carcinoma, bulky invasive squamous cell carcinoma and neuroendocrine tumours as shown in Table 1 (Li The roles of AM1 and AM2 receptors in et al. 2003, Michelsen et al. 2006, Pavel et al. 2006, tumour progression

Buyukberber et al. 2007, Nakata et al. 2008, Liu et al. As noted above, the AM1 and AM2 receptors have 2009). It is also possible that this apparent over- distinct roles. Where information is available, it is the expression could occur through decreased disposal or AM1 receptor that is most often implicated in the direct enhanced processing of mature AM. Furthermore, in effects on growth and survival of tumour cells. RAMP2 some tumours it is possible to correlate AM expression is expressed by clear cell renal carcinoma cells, with disease progression. In breast cancer, AM plasma hepatocellular carcinoma cells and pancreatic tumour concentrations correlate with the presence of lymph cells (Keleg et al. 2007, Park et al. 2008, Deville et al. node metastasis and in hepatocellular carcinoma, AM 2009); it is also expressed in a number of endocrine- expression was positively correlated with invasion related and other cancers (Table 1). In some cases there (Oehler et al. 2003, Park et al. 2008). In neuroendo- is also expression of RAMP3, making it difficult to crine cancers, AM expression correlates with tumour know which receptor is mediating the effects of AM; progression (Pavel et al. 2006). Interestingly, in however, neither renal nor pancreatic cancer cells primary aldosteronism due to adenoma, elevated AM express RAMP3. CLR knockout mice, lacking the levels return to normal after corrective surgery (Letizia GPCR component of AM receptors, die in utero et al. 1998). Thus, there is a significant body of and display severe vascular defects (Dackor et al. evidence showing association of AM and its receptors 2006), whereas mice haplo-insufficent for CLR are with cancer. However, it should be noted that it is relatively normal (Dackor et al. 2007). Only the frequently very difficult to ascertain the true level of RAMP2 knockout mouse shows a vascular phenotype

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(Fritz-Six et al. 2008, Ichikawa-Shindo et al. 2008). (Zheng et al. 2010). In vitro, CGRP can increase RAMP3 cannot compensate for RAMP2’s absence cellular proliferation (Kawase et al. 2005, Kawanami (Dackor et al. 2007). Aortic rings from mice with et al. 2009), endothelial cell tube formation and reduced RAMP2 expression show reduced angiogen- migration (Zheng et al. 2010) and the invasiveness of esis (Ichikawa-Shindo et al. 2008). In endothelial cells prostate-cancer-derived cells by enhancing both cell over-expressing either RAMP2 or RAMP3, only the mobility and haptotactic migration to fibronectin RAMP2 over-expressing cells show increased capil- (Nagakawa et al. 1998, 2001). Further evidence for a lary formation (Ichikawa-Shindo et al. 2008). In a role of CGRP in cancer comes from the study of CGRP separate study, RAMP2 gene silencing in human knockout mice; implanted tumours displayed reduced saphenous vein endothelial cells reduced AM- tumour size and vascularisation compared to control stimulated proliferation and tube formation (Guidolin mice (Toda et al. 2008). Furthermore, infusion of the et al. 2008). Although the AM receptor seems to be 1 CGRP receptor antagonist, CGRP8–37, mirrored the the likely candidate mediating the vascular effects of CGRP knockout model, with an implanted tumour AM, the AM2 receptor also appears to have actions in displaying reduced growth and angiogenesis (Toda tumour biology. As noted above, in some tumours et al. 2008). RAMP3 is expressed alongside RAMP2. RAMP3 Elevated CGRP expression has been identified in mRNA expression is elevated in prostate carcinoma both plasma and tumours from specific cancers; cells as opposed to those from prostate hypertrophy including small cell lung carcinomas (Kelley et al. (Mazzocchi et al. 2004). In a study of renal tumours, 1994), prostate cancer (Suzuki et al.2006), breast RAMP3 elevation was reported in inflammatory cells cancer (Papantoniou et al. 2007, 2010) and thyroid associated with the tumour, whereas RAMP2 was cancer (Pacini et al. 1991, 1992). In prostate cancer, found in the tumour cells themselves (Deville et al. serum CGRP correlated with high-grade/stage disease 2009). This is consistent with a complex role of AM (Suzuki et al. 2006), however, hormone replacement and its receptors, acting directly on the tumour itself therapy may interfere with CGRP’s use as a diagnostic and on its environment. In SW-13 cells, derived marker (Suzuki et al. 2009). Interestingly, CGRP has from an adrenocortical carcinoma, only CLR and been associated with several symptoms related to RAMP3 are expressed, compared to all three RAMPs specific cancers. For example, in medullary thyroid in normal adrenocortical cells; in this case, the cancer patients, elevated plasma CGRP is associated growth-promoting effects of AM must be mediated with increased heart rate (Rubinstein et al. 1992). via the AM receptor (Albertin et al.2005). It 2 Tumour-associated pain is one of the most debilitating remains unresolved as to whether the loss of RAMP1 and 2 expressions in SW-13 cells is typical of symptoms of cancer and is typically associated with adrenocortical tumour cells as a whole. Recently, metastatic invasion of bone tissue. In a model of bone inhibition of RAMP3 expression in breast cancer cells cancer invasion, hyperalgesia is associated with was shown to reduce tumour development (Brekhman increased spinal cord CGRP content and subsequent et al. 2010). irradiation treatment results in reduced hyperalgesia and spinal cord CGRP content (Park et al. 2005). In a Typically, both AM1 and AM2 receptors signal via mouse model, tumours associated with mechanical stimulation of Gs and production of cAMP. It is perhaps not surprising that a number of studies have hyperalgesia were more densely innervated with shown that AM can increase the activity of members of CGRP expressing nerve fibres and less vascularised the MAP kinase family and protein kinase B/Akt. In than tumours from non-hyperalgesic mice (Wacnik some cases this is probably due to an initial increase in et al. 2005). Furthermore, intratumoural injection of cAMP (Ouafik et al. 2009), but this cannot be assumed CGRP8–37 partially blocked tumour-associated pain to apply to all situations. (Wacnik et al. 2005), suggesting that treatment with one of a new class of small molecule CGRP receptor antagonist (Doods et al. 2007), developed for migraine CGRP in cancer treatment, may reduce pain and improve the quality of Although CGRP itself is not believed to cause cancer, life for some cancer sufferers. Interestingly, in one it may also contribute to disease progression. In vivo, study, tumour implantation was shown to increase CGRP infusion increased implanted tumour volume dorsal root ganglion (DRG) CGRP expression; (Toda et al. 2008). Furthermore, in a rat hindlimb following denervation, DRG CGRP expression was model, ischaemic conditions increased CGRP content normalised in association with reduced tumour growth and CGRP over-expression enhanced angiogenesis and angiogenesis (Toda et al.2008).

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The role of CGRP receptors in cancer femtomolar range and its antagonist, PAMP12–20, can reduce tumour growth in xenograph models (Martinez Although information is limited, several lines of et al. 2004). The PAMP sequence is distinct from that evidence indicate that CGRP receptors may play of AM and it appears to act through distinct receptors distinct roles in cancer. The expression of CGRP (Kamohara et al. 2005). As such, a detailed consider- receptor components and CGRP activity has been ation of it is beyond the scope of this review. However, examined in several tumours and tumour-derived cell its probable co-release with AM suggests that a lines. For example, RAMP1 mRNA expression has combination of AM and PAMP antagonists may be been detected in benign and malignant pheochromo- useful in anti-angiogenesis therapy. cytomas (Thouennon et al. 2010), Conn’s adenoma (Forneris et al. 2001) and pancreatic cancers (Ramachandran et al. 2007). Identification of AM and CGRP receptors CGRP receptors typically signal via stimulation of G s involved in tumour progression and production of cAMP, however, several other pathways can be activated (Walker et al. 2010). From what has been said above, it is clear that AM and In several studies, CGRP has been shown to increase related peptides may promote tumour growth by a proliferation by activating members of the MAP kinase number of mechanisms and receptors. It is important to family (Kawase et al.2005, Kawanami et al.2009)and identify the receptors that are involved at each step, increase vasodilation via cGMP and NO production both to understand the biological processes involved (Brain & Grant 2004). The functional significance of the and also to inform drug discovery programmes, given CGRP receptor in cancer is less clear than AM receptors. the potential of the AM and CGRP receptors as therapeutic targets. Unfortunately, this is far from simple. The main issues are considered below. AM2 (intermedin) and cancer Since the identification of AM2 there has been What constitutes an AM or a CGRP receptor? considerable interest in elucidating its biological func- Although it is clear that the AM and CGRP receptors tions. As AM2 is capable of interacting with CGRP, AM1 are complexes between CLR and RAMPs, the early and AM2 receptors (Roh et al. 2004), it is perhaps literature on this topic was misleading, due to the unsurprising that several actions attributed to CGRP and mistaken identification of two orphan GPCRs, RDC1 AM have been replicated by AM2. Although it is unlikely and L1/G10D as receptors for CGRP and AM that AM2 causes cancer directly, AM2 may contribute to respectively (Kapas & Clark 1995, Kapas et al. cancer progression through several mechanisms. AM2 1995). Regrettably, it has proved impossible to repeat has been shown to promote differentiation of cells; pro- the initial observations with either of these receptors angiogenic effects were observed in human umbilical (Kennedy et al.1998, McLatchie et al.1998). vein endothelial cells (HUVECs), where AM2 increased Consequently, the International Union of Basic and vascular endothelial growth factor (VEGF) expression Clinical Pharmacology (IUPHAR) nomenclature sub- and VEGFR-2 activity (Albertin et al. 2010). Further- committee for receptors of the calcitonin family has more, AM2 stimulated cell migration and tube formation determined that neither of these receptors should be via extracellular signal related kinase (ERK) activation considered as CGRP or AM receptors respectively in cultured endothelial cells and increased capillary/ (Poyner et al. 2002). We will consider some pertinent arteriole density in an ischaemic hindlimb model (Smith aspects of this matter further in this section. et al.2009). AM2 also displays anti-apoptotic properties L1/G10D, now known as GPR182, remains an (Pearson et al. 2009, Song et al. 2009). Interestingly, orphan GPCR with no known ligand (http://www. AM2 is also upregulated under hypoxic conditions in iuphar-db.org/DATABASE/ObjectDisplayForward? mouse lung, hepatocytes and endothelial cells at least in objectIdZ146). Unfortunately, the original mis- part via HIF-1a (Copple et al.2009, Pfeil et al. 2009). identification of this protein as an AM receptor continues to confuse a number of workers who refer to an ‘ADMR’ (AM receptor) based on L1/G10D Proadrenomedullin N-terminal 20 peptide (Ramachandran et al.2007, 2009). It is particularly and cancer regrettable that the official gene name for L1/G10D Proadrenomedullin N-terminal 20 peptide (PAMP) is has until recently been ADMR, perpetuating the produced along with AM by processing of a precursor unsubstantiated concept that this is a receptor for polypeptide. It is angiogenic at concentrations in the AM. The official gene name is now GPR182.

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RDC1 was initially reported as a CGRP receptor; it both antagonists should be compared to determine their has been reported that this observation could not be relative potency. There are now non-peptide CGRP repeated (McLatchie et al. 1998). In the original antagonists available such as BIBN4096BS and publication, it was reported that RDC1 could also bind MK0974, these show much greater selectivity for the AM with low affinity (200 nM). This seems to have CGRP over AM receptors and provide a simpler generated the notion that RDC1 is a putative AM approach to distinguish between the CGRP and AM receptor (Thouennon et al. 2010). There has been no receptors (Hay et al. 2002, Salvatore et al. 2008). The independent verification that RDC1 can bind AM and pharmacological discrimination between the AM1 and until it can be demonstrated that RDC1 can produce a AM2 receptors is much harder. Although AM22–52 functional response inside a cell on binding AM, it shows a small degree of selectivity, the size of this cannot be considered as an AM receptor. There are makes it difficult to exploit (Hay et al. 2003). reports in the literature that AM signalling and the If the pharmacological tools to distinguish between presence of RDC1 are associated but this correlation the AM1 and AM2 receptors are not currently available, does not mean that RDC1 is a receptor for AM how else may they be identified? The most reliable (Autelitano & Tang 1999, Chakravarty et al. 2000, technique is to identify the mRNA, by any of the Ladoux & Frelin 2000, Sierro et al. 2007, Thouennon standard techniques that are available to do this. et al. 2010). There are many possible explanations, However, it is important to identify the receptor such as regulation of CLR and/or RAMP expression, components in the specific cell types of interest to for example. RDC1 has been considered a receptor for ensure that there is likely to be a direct relevance with the chemokine SDF1 and has been renamed as CXCR7 tumour progression. Of course, there is not always a (Balabanian et al. 2005), although the IUPHAR sub- simple relationship between mRNA and protein committee on nomenclature do not expression. Indeed, it has sometimes been argued currently recommend this (http://www.iuphar-db.org/ that low expression of CLR or RAMPs is a sign that latestPairings.jsp). RDC1 was also once considered as these are not involved in an AM-mediated response a vasoactive intestinal peptide receptor and therefore (Thouennon et al. 2010). However, even if low mRNA there have clearly been problems with identifying its can be equated with low expression of protein, it does true function. not need high expression of a GPCR to produce a There remains the problem that some of the earlier response. These receptors are capable of considerable literature on AM and cancer predates the resolution of signal amplification; thus, very low expression does the AM receptor problem (Miller et al. 1996, Martinez not mean that the receptor could not function. There et al. 1997); there is a need to reconsider the nature of can be considerable variation in the amount of CLR the AM receptors that were identified in some of these expression that nevertheless produces CGRP or AM reports. It is recommended that IUPHAR guidelines binding and function (Choksi et al. 2002). should be followed when considering receptors for An alternative to identifying the mRNA transcript is CGRP or AM; if possible, the molecular composition to use an antibody against the protein components, should be specified (i.e. CLR/RAMP1) to avoid either CLR or the RAMPs. These are commercially ambiguity. In particular, the phrase ‘adrenomedullin available. However, the experience of the authors receptors’ should not be used in overarching statements is that it is necessary to carry out rigorous controls such as ‘adrenomedullin receptors were upregulated to confirm their specificity. In particular, it is in cancer’ without defining the precise molecular recommended that any antibody be compared on a components involved. western blot in control cells and cells that have been transfected with the desired component, to ensure that a product of the correct molecular weight is only Discriminating between the AM1,AM2 present in the transfected cells. Furthermore, and CGRP receptors antibodies should ideally be tested in knockout tissues AM at pharmacological concentrations (O10 nM) can to confirm their specificity. Unfortunately, many activate the AM1,AM2 and CGRP receptors. It is published studies using commercially available possible to distinguish between CGRP and AM antibodies do not appear to have performed these receptors pharmacologically. Traditionally, this has essential controls. When considering using any been done using peptide antagonists; CGRP8–37 is antibody that has been generated using a peptide, it is selective for the CGRP receptors, AM22–52 is selective important to consider where this epitope is found in the for the AM1 and AM2 receptors (Poyner et al. 2002, intact protein complex (Zhao et al. 2006, Eftekhari Hay et al. 2003). If this approach is used, then ideally et al. 2010).

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The fact that RAMPs can also associate with growth or survival of the cancer cells themselves is other receptors must not be neglected (Christopoulos directly stimulated by AM or its relatives. In these et al. 2003). This adds an additional complication to cases, AM is likely to be promoting tumour the situation. progression both directly and indirectly (via effects As an alternative to the use of chemical antagonists, on angiogenesis); thus, the antagonists will be able to knockdown with siRNA may be used, if the system block multiple processes. It is also important to lends itself to this approach (Bouschet et al. 2005, understand exactly how AM might interact with other Thouennon et al. 2010). This is most convincing when angiogenic molecules, to determine whether it can combined with one of the techniques described above usefully be applied as part of a combination therapy. to identify the component in the cells. Given the number of factors that are involved in blood and lymph vessel growth this may be the most realistic Signal transduction pathways used by option for AM antagonists. In parallel with these AM-related peptides in tumour biology studies, the safety of AM antagonists needs to be evaluated in early phase clinical trials. The knockout There is a need to further delineate the signal studies have given us some pointers as to the transduction pathways used by AM and its relatives, physiological role of AM in mice; however, it is not in particular, to establish how far cAMP-independent always obvious how this might apply to humans. mechanisms may be used. This may ultimately become Good antagonists would provide the necessary experi- important if pathway-selective antagonists for AM and mental evidence. CGRP are developed (Kenakin 2008). This work will Thus, the key to confirming the role of AM in cancer need careful use of inhibitors of the different second is likely to be the availability of appropriate messenger pathways. antagonists. There are grounds for thinking that the discovery of these compounds might be feasible. A number of high-affinity CGRP antagonists are now Future perspectives available; these exploit a binding groove between CLR There seems little doubt the AM and related peptides and RAMP1. A crystal structure of a bound antagonist are important factors in a number of endocrine-related at the CLR/RAMP1 complex has been solved (ter Haar tumours. There is now considerable evidence to et al. 2010). Although no structures of CLR/RAMP2 implicate both direct actions on the tumour cells or RAMP3 are available, it is likely that these show themselves and also indirect actions, particularly similar folds to the CLR/RAMP1 complex, in related to angiogenesis. More work is needed to particular, there are likely to be homologous pockets establish the receptor mechanisms that are used by that can be exploited by antagonists. these peptides. Most work has been done for AM. The Special comment is needed as to the role of CGRP majority of therapeutic agents work at cell-surface in cancer. It is particularly intriguing that symptoms receptors and so it is likely that this will be the most associated with certain tumours, especially pain, seem fruitful approach to blocking AM action, although it is to correlate with elevated CGRP levels. Selective possible to target AM itself through small molecule CGRP antagonists are available and are in clinical inhibitors or appropriate antibodies (Roldos et al. trials for migraine (Durham & Vause 2010); it would 2008). To target the individual receptors, there is an be particularly interesting to see whether these could urgent need for high-affinity, selective AM receptor also be used in cancer therapy. antagonists suitable for in vivo studies. Ideally, these should include the AM1 and AM2 receptor antagonists; there is also a case for combining these with another Summary molecule that blocks the CGRP receptor activity to There is significant evidence for a feed-forward treat cancer. Antagonists that block the AM1,AM2 and mechanism involving AM in cancer. AM can be CGRP receptors simultaneously may also prove useful produced by tumours, especially under hypoxic as cancer therapies. Peptide antagonists of this nature conditions and stimulates blood and lymph vessel have been developed (Robinson et al. 2009). Such angiogenesis, promoting tumour growth and spread. antagonists are also likely to block any action of AM2. To a greater or lesser extent, its actions may be shared Initially, these agents need to be used in a wide range of between CGRP and AM2. The receptors that mediate in vivo models to examine the effectiveness of AM the actions of CGRP and AM are complexes of CLR inhibition in treating tumours. It might be expected that and RAMPs. The AM1 receptor (CLR/RAMP2) is they will be most effective in solid tumours where particularly important in angiogenesis; the receptor

Downloaded from Bioscientifica.com at 10/02/2021 09:04:26PM via free access C8 www.endocrinology-journals.org Endocrine-Related Cancer (2011) 18 C1–C14 type(s) involved in the other tumourigenic actions of Berenguer C, Boudouresque F, Dussert C, Daniel L, the peptides is less clear. There are significant technical Muracciole X, Grino M, Rossi D, Mabrouk K, Figarella- challenges associated with identifying these receptors, Branger D, Martin PM et al. 2008 Adrenomedullin, an although these are surmountable with careful experi- autocrine/paracrine factor induced by androgen with- mentation and the inclusion of the appropriate controls. drawal, stimulates ‘neuroendocrine phenotype’ in LNCaP If new antagonists can be developed in the near future, prostate tumor cells. Oncogene 27 506–518. 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