Hypothalamic Hormones And Cancer.pdf

Frontiers in Neuroendocrinology 22, 248 –291 (2001) doi:10.1006/frne.2001.0217, available online at http://www.idealibrary.com on

Hypothalamic Hormones and Cancer Andrew V. Schally, Ana Maria Comaru-Schally, Attila Nagy, Magdolna Kovacs, Karoly Szepeshazi, Artur Plonowski, Jozsef L. Varga, and Gabor Halmos Endocrine, Polypeptide, and Cancer Institute, Veterans Affairs Medical Center, New Orleans, Louisiana 70112; and Section of Experimental Medicine, Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana 70112 Published online July 26, 2001

The use of peptide analogs for the therapy of various cancers is reviewed. Inhibition of the pituitary– gonadal axis forms the basis for oncological applications of luteinizing hormone-releasing hormone (LH-RH) agonists and antagonists, but direct effects on tumors may also play a role. Analogs of somatostatin are likewise used for treatment of various tumors. Radiolabeled somatostatin analogs have been successfully applied for the localization of tumors expressing somatostatin receptors. Studies on the role of tumoral LH-RH, growth hormone-releasing hormone (GH-RH), and bombesin/GRP and their receptors in the proliferation of various tumors are summarized, but the complete elucidation of all the mechanisms involved will require much additional work. Human tumors producing hypothalamic hormones are also discussed. Treatment of many cancers remains a major challenge, but new therapeutic modalities are being developed based on antagonists of GH-RH and bombesin, which inhibit growth factors or their receptors. Other approaches consist of the use of cytotoxic analogs of LH-RH, bombesin, and somatostatin, which can be targeted to receptors for these peptides in various cancers and their metastases. These new classes of peptide analogs should lead to a more effective treatment for various cancers. KEY WORDS: tumor therapy; agonists and antagonists of LH-RH; bombesin antagonists; GH-RH antagonists; targeted cytotoxic analogs; somatostatin analogs. © 2001 Academic Press

INTRODUCTION

This review examines the involvement of hypothalamic hormones in the processes of malignant growth and especially the use of various synthetic analogs of hypothalamic peptides for the treatment of various cancers. We summarize experimental and clinical studies performed so far, and discuss recent concepts regarding antiproliferative action of diverse classes of peptide analogs and their status as drug candidates for treatment of various cancers. This work has been proceeding for more than 20 years (128 –130, 133–135, 137, 138). Many patients with various neoplastic diseases have already benefited Address all correspondence and requests for reprints to Dr. Andrew V. Schally, Endocrine, Polypeptide and Cancer Institute, VA Medical Center, 1601 Perdido Str., New Orleans, LA 70112-1262. Fax: (504) 566-1625. 0091-3022/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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from modern therapies based on hypothalamic hormones (135). The availability of novel analogs of hypothalamic hormones may permit the clinicians to better diagnose and treat a variety of cancers. The original concept of hypothalamic control of secretion of the anterior pituitary gland was put forward by Harris (55) and confirmed by the isolation of hypothalamic chemotransmitters (10, 45, 124, 127, 129, 132, 136). Hypothalamic hormones influence various bodily functions such as growth, reproduction, lactation, metabolism, and gastrointestinal function, as well as pathological processes such as tumorogenesis through the anterior pituitary hormones and their target glands (118, 133). Some hypothalamic peptide hormones are also produced in the extrahypothalamic brain areas and in nonneural tissues. In addition, luteinizing hormone-releasing hormone (LH-RH), somatostatin, growth hormone-releasing hormone (GH-RH), their mRNAs, and their receptors are found in some tumors (35, 39, 65, 123, 133, 135, 138). Mammalian bombesin-like peptides, such as gastrin-releasing peptide (GRP) and neuromedin B, are likewise present in various tumors, and appear to produce mitogenic effects (28, 35, 129, 142, 146, 156). In this presentation we have attempted to summarize and review significant information collected so far on these topics. It is not within the scope of this review to survey all the available findings. The topics of this article have been selected because of their special interest to us. Although our choice may show a definite bias, we hope that the subjects selected will be of general interest.

DISTRIBUTION OF NEUROHORMONE-PRODUCING CELLS

Neurohormones produced by the hypothalamus and transported by the hypophyseal portal vessels play a pivotal role in regulating the secretion of pituitary trophic hormones, but their physiological effects on the peripheral tissues are not significant, due to a dilution in the systemic circulation. The transport of neurohormones at effective concentrations by the hypophysial portal vessels exclusively to the pituitary may also protect the body from potential adverse effects of the high concentrations of these neurohormones. Thus, for instance, the high blood concentration of somatostatin in the systemic circulation could have adverse effects by inhibiting insulin secretion from the ␤ cells of the pancreas (38). LH-RH neurons are located mainly in the medial preoptic area, from where they project to the median eminence as well as to the organon vasculosum of the lamina terminalis. The LH-RH neurons migrate to the medial preoptic area from the olfactory placode. Failure of this migration caused by a genetic defect underlies Kallmann’s syndrome, which results in anosmia, hypogonadism, and infertility (139). Within the hypothalamus, the majority of somatostatin (SST)-positive nerve perikarya are located close to the third ventricle in a few layers parallel to the periventricular wall and frontal to the ventromedial nucleus (118). Axons from these cells run caudally through the hypothalamus to the median eminence.

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The SST-like peptides are multifunctional, and synthesized and localized in most brain regions as well as in peripheral organs (128). GH-RH was first isolated, not from the hypothalamus, but from human pancreatic islet tumors, in which the ectopic secretion was associated with hypersecretion of GH and acromegaly (45, 124). GH-RH was subsequently identified in human and animal hypothalami. GH-RH is distributed in neuronal cell bodies in the arcuate and ventromedial nucleus. Morphological evidence indicates the existence of anatomic and functional interactions between GH-RH and SST neurons. Bombesin-like immunoreactivity is widely distributed in mammalian brain including the hypothalamus (142, 146). GRP, which is related to bombesin, is present in brain, especially the hypothalamus (142, 146). Ghrelin-immunoreactive neurons were localized in the hypothalamic arcuate nucleus by using immunohistochemical analyses (81). Considering that mRNA for receptors of GH secretagogues (GHS) exists in hypothalamic regions, including the arcuate nucleus and pituitary gland, ghrelin in the arcuate nucleus may act on the hypothalamus or be transported to the anterior pituitary (81). Several neurohormones, such as SST, LH-RH, GH-RH, bombesin/GRP, and Ghrelin are also synthesized in various peripheral tissues. SST is secreted by cells in the gastrointestinal tract and the endocrine pancreas (118, 128). There is virtually no SST in the stomach and duodenum, but lower down the gut, the SST-28 molecular form is predominant. SST is also secreted in the pancreatic islets where it inhibits insulin and glucagon secretion (128). LH-RH is present in the human placenta, and LH-RH-like peptides have been identified in ovary, testis, endometrial tissue, human mammary gland, pancreas, and submandibular gland. In high concentration LH-RH affects the gonads and has also been implicated in mating behavior. GH-RH is produced by the upper intestine, and GH-RH mRNA was found in placenta, ovary, testis, lymphocytes, and pancreas (65, 138). Bombesin is present in lung and gastrointestinal (GI) tract and GRP is found in human fetal lung and GI tract (142, 146). Although stomach is the primary source of Ghrelin (81), mRNA for Ghrelin is expressed in several other tissues, such as kidney, bone, cartilage, and human placenta. In addition to their primary neuroendocrine role in the regulation of pituitary hormone secretion, hypothalamic neuropeptides carry out multiple endocrine, paracrine, and neurotransmitter functions in various tissues (38, 118). The physiological role of most of the neuropeptides produced by peripheral tissues is assumed to be an autocrine and/or paracrine regulation of the local tissue functions (38, 118). Some of these peptides, like SST, GRP, or placental LH-RH, also appear to exert an endocrine function by entering the systemic circulation and thereby gaining access to various tissues (118). The levels of the neurohormones in the systemic circulation, however, are much lower than in the pituitary portal vessels. The mechanism of action of hypothalamic hormones on peripheral tissues also appears to be different from that on the pituitary. Work is proceeding on further understanding the possible functions of neuropeptides produced in peripheral tissues.

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LH-RH

In 1971, our laboratory achieved the isolation, elucidation of structure, and synthesis of hypothalamic LH-RH, winning the race with other groups (94, 131, 132, 136). We showed that both natural LH-RH and the synthetic decapeptide corresponding to its structure possesed major follicle-stimulating hormone (FSH)-releasing as well as LH-releasing activity (132). The concept formulated by one of us (A.V.S.), that LH-RH regulates the secretion of both FSH and LH from the pituitary gland, is upheld by much experimental and clinical evidence (23, 72, 127, 129, 130, 133, 136). Indeed, it was originally suggested that the name LH-RH be changed to GnRH for gonadotropinreleasing hormone (132, 136). However, this led to confusion with GH-RH, so we now prefer to return to the original name (130). In the past 30 years, more than 3000 analogs of LH-RH have been synthesized (71, 130, 133). Agonistic analogs, such as Decapeptyl, Leuprolide, Zoladex, and Buserelin, much more active than the LH-RH itself, and available in depot preparations, have important clinical applications in gynecology and oncology (130, 133). Potent antagonists of LH-RH such as Cetrorelix, Ganerelix, and Abarelix, suitable for clinical use, have been likewise synthesized (6, 101, 105). In addition to Decapeptyl and Cetrorelix, cytotoxic analogs of LH-RH consisting of doxorubicin or 2-pyrrolinodoxorubicin linked to the LH-RH agonist [D-Lys 6]LH-RH (AN-152 and AN-207, respectively), which can be targeted to LH-RH receptors on tumors, have been also developed in our laboratory (103, 137) (Fig. 1). The actions of LH-RH and its analogs are mediated by high-affinity receptors for LH-RH found on the membranes of the pituitary gonadotrophs (23). An acute administration of agonists of LH-RH induces a marked release of LH and FSH. However, continous stimulation of the pituitary by chronic administration of LH-RH agonists produces an inhibition of the hypophyseal– gonadal axis through the process of “down-regulation” of pituitary receptors for LHRH, desensitization of the pituitary gonadotrophs, and a suppression of circulating levels of LH and sex steroids (35, 130, 133, 135). This down-regulation of LH-RH receptors, produced by sustained administration of LH-RH agonists, provides the basis for clinical applications in gynecology and oncology of this class of compounds (130, 133, 135). Antagonists of LH-RH exibihit no intrinsic activity, but compete with LH-RH for the same receptor sites (133, 135). LH-RH antagonists produce a competitive blockade of LH-RH receptors and cause an immediate inhibition of the release of gonadotropins and sex steroids (133, 135). The principal mechanism of action of LH-RH antagonists was thought to be based only on a competitive occupancy of LH-RH receptors, but recently, we demonstrated that administration of the LH-RH antagonist Cetrorelix to rats also produces down-regulation of pituitary LH-RH receptors and a decrease in the levels of mRNA for LH-RH receptors (109, 135). Our work indicates that LH-RH antagonists exert their inhibitory effects on the gene expression of pituitary LH-RH receptors by counteracting the stimulatory effect of endogenous LH-RH (85). Specific LH-RH receptors are also found on

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FIG. 1. Molecular structure of cytotoxic analogs of SST (AN-238), bombesin (AN-215), and LH-RH (AN-152 and AN-207). Cytotoxic radicals doxorubicin (R ⫽ NH 2) and 2-pyrrolinodoxorubicin (R ⫽ 2-pyrrolino) are linked through a glutaric acid spacer to the free amino groups of amino acids (circled) in the peptide carriers (P). (From Proc Natl Acad Sci USA 2000; 97, 830. Reprinted with permission from National Academy of Sciences, USA. Copyright 2000 Natl. Acad. Sci. USA.)

breast, prostatic, ovarian, endometrial, and even pancreatic cancers (30, 32– 35, 36, 46, 47, 49, 56, 57, 91, 133, 135). These LH-RH receptors on tumor cells can mediate direct effects of LH-RH analogs (135). Thus, high-affinity binding sites for LH-RH and the expression of mRNA for LH-RH receptors were detected in human prostate cancer samples, human prostate cancer lines, and Dunning rat prostate cancers (30, 46, 84, 91, 134, 135). The presence of LH-RH receptors in various human mammary carcinoma cell lines was also reported. We detected high-affinity LH-RH binding sites in more than 50% of human breast cancer samples (36). LH-RH receptors were similarly found in human ovarian epithelial cancer specimens and human ovarian cancer lines (35, 56, 99, 100, 135). The presence of high-affinity membrane receptors for LH-RH

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was also established in nearly 80% of human endometrial carcinomas and in some endometrial cancer lines (35, 57, 135). LH-RH receptors on human cancers appear to be similar to pituitary LH-RH receptors (135). The expression of LH-RH receptor gene in human breast, endometrial, ovarian tumors, and respective cancer cell lines was also demonstrated by RT-PCR (56, 57, 66, 67, 99, 100, 135). The inhibition of growth of human mammary, ovarian, endometrial, and prostatic cancer cell lines by LH-RH agonists and antagonists in vitro strongly supports the concept of their direct effects (30, 91, 130, 133). The evidence for the production of an LH-RH-like peptide and/or expression of mRNA for LH-RH was also demonstrated in human prostatic, mammary, endometrial, and ovarian cancer lines (57, 91, 130). This suggests that local LH-RH may be involved in the growth of these tumors. The existence of functional regulatory systems consisting of locally produced LH-RH-like peptides and specific LH-RH receptors has also been postulated in prostate cancer and ovarian cancer (30, 32). Dondi et al. (30) and Emons et al. (32) suggested that this LH-RH, produced by tumor cells, might have an inhibitory function. However, the proliferation of various cancer cells in vitro is dose-dependently suppressed by LHRH antagonists and inhibitory effects of agonists might be explained by receptor down-regulation (135). Our studies in ES-2 human ovarian cancer lines suggest that locally produced LH-RH is stimulatory (135). Additional investigations are needed to resolve the role and the action of endogenous LH-RH-like peptides produced by various tumors. We will now summarize selected findings on the effects of LH-RH agonists, LH-RH antagonists typified by Cetrorelix and cytotoxic LH-RH analogs on various tumors.

Prostate Cancer

The greatest therapeutic impact of LH-RH analogs was in the field of prostate cancer, which is the most common noncutaneous malignant tumor in men (130, 133, 134). About 70% of human prostate cancers are testosteronedependent and the treatment of advanced prostate cancer is based upon androgen deprivation (130, 133, 134). Therapy with agonists of LH-RH with or without antiandrogens is currently the preferred treatment for men with advanced prostate cancer and in about 70% of cases LH-RH agonists are selected for primary treatment (130, 133, 134). Administration of antiandrogens prior to and during early therapy with agonists can prevent the disease flare. Clinical trials in patients with advanced prostate cancer show that the antagonist of LH-RH Cetrorelix can induce a clinical remission (42, 43, 133, 134). Cetrorelix and other LH-RH antagonists could be beneficial as a monotherapy for patients with prostate cancer and metastases in the brain, spine, liver, and bone marrow, in whom the LH-RH agonists cannot be used as single drugs, because of the possibility of flare-up (42, 43). LH-RH antagonists cause an immediate fall in the levels of gonadotropins and sex steroids and greatly reduce the time of the onset of therapeutic effects (42, 133, 134). In addition, treatment with Cetrorelix can produce long-term improvement in patients

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with symptomatic benign prostatic hyperplasia (BPH) (24, 43). Cetrorelix offers a therapeutic alternative to patients with BPH who are considered poor surgical risks (24). Clinical evidence indicates that in patients with advanced prostate cancer, medical castration produced by chronic administration of LH-RH analogs accounts for most benefits derived from the treatment (133, 134). However, LH-RH analogs and other peptides can also exert direct effects on tumor cells (91, 135). These direct effects of peptides and the presence of receptors for LH-RH, somatostatin, GH-RH, and bombesin on prostate cancers may be of critical importance for the development of methods for treatment of hormonerefractory prostate cancer (46, 50, 144). Since all hormonal therapies aimed at androgen deprivation, including LH-RH analogs, can provide only a remission of limited duration, most patients with metastatic prostatic carcinoma eventually relapse and die of androgen-independent prostatic cancer (133). This androgen-independent growth of prostate cancer cells is apparently caused by various growth factors such as insulin-like growth factor (IGF)-I and IGF-II, epidermal growth factor (EGF), and others (134). Interference with endogenous growth factors and their receptors on tumors by bombesin/GRP antagonists and GH-RH antagonists or the use of targeted cytotoxic peptide analogues could inhibit the growth of androgen-independent prostate cancers and improve the tumor treatment outcome (133–135). Recent investigation of a large number of specimens of human prostate adenocarcinomas showed that 86% of cancers exhibited high-affinity binding sites for LH-RH and expressed mRNA for LH-RH receptors (46). The expression of specific LH-RH receptors in a high percentage of human prostate cancers provides a rationale for the development of methods for therapy of this malignancy based on targeted cytotoxic LH-RH analogs (134, 137). Investigations with cytotoxic analogs of LH-RH were carried out in various models of prostate cancer. The effects of the cytotoxic analog of LH-RH, AN-207, were first evaluated in rats bearing hormone-dependent Dunning R-3327-H prostate carcinomas (62). After three injections of AN-207 on Days 1, 7, and 28, the prostate tumors regressed to about one-half of their initial volume (62). In another study, we investigated the effect of analog AN-207 on PC-82 human prostate cancers xenografted into nude mice. Eight weeks after a single administration of cytotoxic analog AN-207, there was a major reduction in tumor volume and a fall in serum prostate specific antigen (PSA) levels (84). RT-PCR analyses demonstrated the expression of mRNA for LH-RH receptors in PC-82 tumor samples. The recovery of LH-RH receptors after initial therapy will permit a repeated administration of cytotoxic LH-RH analogs. The side effects of targeted cytotoxic LH-RH analogs are expected to be minor, because the receptors for LH-RH are not widely distributed in normal tissues. In addition, DNA-intercalating cytotoxic radical 2-pyrrolinodoxorubicin (AN-201) is maximally cytotoxic to cells undergoing mitotic division. Thus, it was demonstrated that after administration of a high dose of AN-207, the pituitary gonadotropic function in rats is impaired only temporarily (137). Hormonal replacement

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therapy can also alleviate the undesirable side effects caused by the damage to other pituitary cells.

Breast Cancer

Breast cancer is the most common malignancy in women. About 30% of women with breast cancers have estrogen-dependent tumors and can be treated by hormonal manipulations such as Tamoxifen or oophorectomy (126, 130, 133). Experimental and clinical studies clearly demonstrated that agonists of LH-RH can be used for treatment of estrogen-dependent breast cancer (130, 133). Thus, initial investigations in rat and mouse models of breast adenocarcinoma showed that chronic administration of agonist [D-Trp 6]LH-RH decreased tumor weight and volume (129, 130, 133). This suggested that agonists of LH-RH should be considered for a new hormonal therapy for breast cancer in women. Various clinical trials, carried out since the early 1980s, demonstrated regression of tumor mass and disappearance of metastases in premenopausal and some postmenopausal women with breast cancer treated with [D-Trp 6]LH-RH, Buserelin, Zoladex, or Leuprolide (74, 126, 130, 133). These studies showed that LH-RH agonists are efficacious for the treatment of premenopausal women with estrogen-dependent, estrogen receptor-positive breast cancer (74). A recent clinical trial in 161 premenopausal women with advanced breast cancer revealed that combined treatment with LH-RH agonists and tamixofen was more effective and resulted in longer overall survival than treatment with either drug alone (80). The main effect of LH-RH agonists on mammary carcinomas is based on estrogen deprivation, but some direct antitumor effects of LH-RH analogs are also likely. LH-RH antagonists have been so far tested only in experimental models of breast cancer. Cetrorelix inhibited tumor growth of murine mammary carcinomas and of human breast cancers transplanted into nude mice and also exhibited strong antiproliferative activity in vitro (130, 133, 135). These findings indicate that Cetrorelix might be effective clinically for the treatment of breast cancer. Targeted cytotoxic analogs of LH-RH bind with high affinity to LH-RH receptors on human breast cancers (47). In mice bearing estrogen-independent MXT mouse mammary cancers, cytotoxic analogs AN-207 and AN-152 produced a 89 to 93% inhibition of tumor growth (153). The advantage of AN-207 is that it is effective in doses about 100 times smaller than AN-152 (153). In another investigation, one injection of AN-207 caused a complete regression of MX-1 hormone-independent doxorubicin-resistant human breast cancers in nude mice, which remained tumor-free for at least 60 days after treatment (66). A single dose of 250 nmol/kg of cytotoxic LH-RH analog AN-207 also inhibited significantly the growth of MDA-MB-231 in nude mice for 3 weeks (67). Three weeks after treatment binding sites for LH-RH were not detectable on tumors treated with AN-207, but reappeared 60 days after administration of the drug (67). This provides a rationale for using repeated treatments with

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cytotoxic LH-RH analogs. Treatment with AN-207 also prevented metastatic spread of orthotopically grown MDA-MB-435 estrogen-independent human mammary carcinoma (20). In recent studies we linked AN-152, the cytotoxic analog of LH-RH containing doxorubicin, to a two-photon fluorophore (C625) to study its cellular pathways in tumors (86). By using two-photon laser scanning microscopy, the AN-152 fluorophore conjugate could be observed directly as it interacted with LH-RH receptor-positive MCF-7 breast cancer cells. The receptor-mediated entry of AN-152 into the cell cytoplasm and subsequently into the nucleus was clearly demonstrated (86).

Epithelial Ovarian Cancer

Epithelial ovarian cancer is the fourth most frequent cause of cancer-related deaths in women (129, 133). The treatment based on surgery or chemotherapy is not very effective and new approaches are needed (133). Ovarian cancer may be dependent on LH and FSH and in experimental cancer models, the suppression of the secretion of gonadotropins produced by LH-RH analogs inhibits the growth of ovarian tumors (133, 159). Studies in vivo indicate that Cetrorelix inhibits growth of human OV-1063 and ES-2 epithelial ovarian cancers xenografted into nude mice better than agonist [D-Trp 6]LH-RH (133, 135). In clinical studies some patients with advanced ovarian carcinoma treated with agonists of LH-RH showed stabilization of disease (133), but in a multicenter trial no beneficial effects of therapy with [D-Trp 6]LH-RH could be found (34). Clinical trials with Cetrorelix are in progress. In addition, specific membrane receptors for LH-RH have been found in 78% of surgically removed human ovarian carcinomas and in EFO-21, EFO-27, and OV-1063 and ES-2 human ovarian cancer cell lines (33, 35, 135). These receptors mediate direct effects of LH-RH analogs on the growth of ovarian cell lines in vitro. The agonist [D-Trp 6]LH-RH and antagonist Cetrorelix reduced proliferation of EFO cell lines in culture (33, 35). The expression of mRNA for LH-RH and LH-RH receptors in these cell lines supports the view that local LH-RH-like substances may be involved in the proliferation of ovarian cancer (56). Our recent work shows that ES-2 human ovarian cancer cells secrete LH-RH and their growth can be inhibited by Cetrorelix (135). The function of this autocrine LH-RH might be stimulatory since the addition of an LH-RH antibody inhibits the proliferation of ES-2 cells (135). We have also demonstrated that therapy of nude mice bearing human ovarian OV-1063 tumors with agonist [D-Trp 6]LH-RH or LH-RH antagonist Cetrorelix produced a fall in the number of receptors for LH-RH on tumor membranes accompanied by an increase in levels of these receptors in the tumor cell nuclei (49). Our results indicate the translocation of membrane receptors for LH-RH to the nuclei of OV-1063 ovarian cancers after treatment with analogs of LH-RH (49). Doxorubicin-containing targeted cytotoxic LH-RH analog AN-152 was shown by confocal laser scanning microscopy to enter LH-RH receptor-positive

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human ovarian cancer cells producing an elevated concentration of doxorubicin in these cells compared with exposure to unconjugated doxorubicin (157). The effects of targeted cytotoxic LH-RH analogs on growth of ovarian cancers in vivo were also evaluated (99, 100). In the first study, we showed that a single injection of cytotoxic analog AN-152 inhibited the growth of LH-RH receptorpositive OV-1063 ovarian tumors in nude mice for at least 4 weeks (99). AN-152 did not inhibit the growth of LH-RH receptor-negative UCI-107 human ovarian carcinomas, showing that the presence of receptors is essential (99). In another study, we demonstrated that the growth of OV-1063 ovarian cancers could be suppressed by administration of cytotoxic LH-RH analog AN-207 in doses 100 times smaller than those of AN-152 (100). Targeted chemotherapy based on analogs such as AN-152 and AN-207 may improve the management of ovarian cancer (130, 133, 135).

Endometrial Cancer

Endometrial cancer is a common gynecologic malignancy in the Western world (129, 133). Surgery or radiotherapy is successful in 75% of cases, but new methods are needed for advanced or relapsed cancers (35). Endometrial carcinoma is estrogen-dependent and thus it should respond to therapy with LH-RH analogs (133). In addition, high-affinity receptors for LH-RH are present on nearly 80% of membranes of human endometrial cancers and endometrial cancer cell lines (57). Bioactive and immunoreactive LH-RH and the expression of mRNA for LH-RH were also found in these cells (57). Agonist [D-Trp 6]LH-RH and antagonist Cetrorelix inhibited the proliferation of endometrial cancer cell lines in vitro (130, 133, 135). In limited clinical studies carried out so far, administration of a depot preparation of LH-RH agonists produced a partial or complete remission in 35% of patients with recurrent endometrial cancer (40). In view of the presence of LH-RH receptors on endometrial cancers, targeted cytotoxic analogs are also being investigated.

Renal Cell Carcinoma (RCC)

The ethiology of RCC is poorly understood and the present methods of treatment of RCC must be improved (133). Sex steroids and growth factors may play a role in proliferation of kidney neoplasms (133). Consequently, we tested LH-RH antagonist Cetrorelix for its effects on the growth of the CAKI-I RCC line xenografted into nude mice (61). After 4 weeks of treatment, tumor volume in animals receiving Cetrorelix was significantly decreased. Cetrorelix also reduced serum testosterone and the number of receptors for EGF on CAKI-I tumors (61). Clinical trials are in progress.

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Receptors for LH-RH are present in hamster and human pancreatic cancers (133), but their function is not known. Pancreatic cancers may be sensitive to sex steroids. We have shown that the treatment with [D-Trp 6]LH-RH and antagonist Cetrorelix can inhibit growth of pancreatic cancers in hamsters (147, 148). However, clinical trials with LH-RH agonists in patients with pancreatic carcinoma were unsuccessful (133).

SOMATOSTATIN

Somatostatin is a hormonal neuropeptide that was isolated from ovine (10) and later from porcine hypothalami (127, 129, 133), and characterized as an inhibitor of growth hormone secretion from the pituitary gland. In mammals, SST exists in several active forms, a 14-amino acid peptide (SST-14), an amino terminally extended version consisting of 28 amino acids (SST-28), a fragment corresponding to the first 12 amino acids of SST-28, and still larger forms that vary in molecular size (10, 118, 127, 129, 133). Both SST-14 and SST-28 exhibit similar effects on a wide variety of cells, and appear to be endogenous growth inhibitors (128). SST is ubiquitous and inhibits the secretion of many hormones including insulin and glucagon under physiologic and pathologic conditions (128). SST can also suppress various exocrine secretions (118, 128). The targets of SST action are often the same tissues in which the peptide is localized. Thus, in addition to its endocrine effects SST can also serve as an autocrine/paracrine regulator and SST present in discrete cells of the pancreas, gastric mucosa, duodenum may, through paracrine control, regulate the endocrine pancreas and gastrointestinal tract (118, 133). Because of the short plasma half-life of SST-14 (⬍3 min), more stable synthetic analogs had to be developed to exploit its great therapeutic potential (128). As a result of intense research, several stable SST analogs were developed including octreotide (SMS 201–995, Sandostatin) (7) and vapreotide (RC-160) (16). These analogs have a plasma half-life of ⬃120 min and are about 50 times more potent than SST in inhibiting growth hormone release from the pituitary. It has been established that SST and its octapeptide analogs exert their effects through specific membrane receptors (133). So far, five distinct receptor subtypes (sst 1–5) have been cloned and characterized (108, 116, 119, 137). These receptors are widely distributed in normal and cancerous tissues, with cells often expressing more than one subtype (116, 123, 133). While native SST shows similar high affinity to sst 1–5, the synthetic octapeptides such as RC-160 and RC-121, developed in our institute (128), and octreotide bind preferentially to sst 2 and sst 5, and display moderate affinity to sst 3 and a low affinity to sst 1 and sst 4 (108, 116). RC-160 stimulates tyrosine phosphatase activity and inhibits the proliferation of the cells expressing the sst 2 gene. Thus, tyrosine phosphatase appears to be a transducer of the growth inhibition signal (12). In sst 5-expressing cells, the phosphatase pathway is not involved in the mechanism of action of RC-160 on

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cell growth and the phosphoinositide/calcium pathway could be implicated in the inhibitory effect (13). SST analogs are used for the treatment of acromegaly, endocrine tumors of the gastroenteropancreatic system, including carcinoid tumors, insulinomas, glucagonomas, gastrinomas, and VIPomas (89, 128). RC-160 is a powerful tumor growth suppressor in experimental models of various cancers including pancreatic, prostatic, renal, colorectal, gastric, and mammary cancer, brain tumors, and human small cell lung carcinomas (SCLC) and non-SCLC (reviewed in (128, 133)). Consequently, attempts have been made to use modern SST analogs for the therapy of human cancers, such as prostatic, breast, pancreatic, and lung, but relevant palliative benefits have been obtained only in hepatocellular carcinoma (133). Thus, RC-160 was used as a single drug in patients with inoperable pancreatic cancer and 30% of patients showed some tumor stabilization, but RC-160 could not induce an effective palliation in most patients (133). Poor therapeutic results with SST analogs are likely due to the fact that in human exocrine pancreatic cancers there is a loss of gene expression for sst 2, which is the preferred subtype for these analogs (14). However, the expression of sst 5 and sst 3 in pancreatic cancer should make possible the therapy with SST analogs labeled with various radioisotopes or cytotoxic SST analogs.

Localization of Tumors and Metastases by Scintigraphy with Radiolabeled SST Analogs

The presence of SST receptors permits the localization of some tumors and metastases using scanning techniques (87, 88, 123). Radiolabeled analogs of SST, such as [ 111In-DTPA-D-Phe 1]-octreotide (OctreoScan) are used clinically for the localization of tumors expressing receptors for SST. Krenning et al. (87) carried out SST receptor scintigraphy in more than 1000 patients and reported that various primary tumors and their metastases, both neuroendocrine or nonneuroendocrine, can be localized in vivo. Neuroendocrine tumors that could be localized with OctreoScan include pituitary tumors, gastrinomas, insulinomas, glucagonomas, medullary thyroid carcinoma, neuroblastomas, carcinoids, and SCLC (87). Nonneuroendocrine tumors that could be localized by scintigraphy included non-SCLC, meningiomas, breast cancer, and astrocytomas. A positive scintigram may predict a good response to treatment with octreotide (87).

Cytotoxic SST Analogs

The presence of receptors for SST on various neuroendocrine malignancies and many other solid tumors serves as a rationale to use SST octapeptides as carriers to deliver cytotoxic agents specifically to cancerous cells (50, 52, 123, 137). Thus, recently we developed a series of novel targeted cytotoxic SST conjugates that consist of carriers RC-121 and RC-160 coupled to doxorubicin

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(DOX) or its superactive derivative, 2-pyrrolino-DOX (AN-201) (104, 137). Of these hybrid cytotoxic conjugates, AN-238 containing AN-201 was demonstrated to be very effective on many human and rodent experimental cancer models (137). Although a wide variety of normal tissues such as those of the gastroenteropancreatic system, the kidneys, or the pituitary also express highaffinity receptors for SST, in our studies, no receptor-specific toxicity was observed after treatment with cytotoxic analog AN-238, possibly because it is used in relatively low doses (76, 83, 113, 114, 137). Moreover, AN-201, the cytotoxic radical in AN-238, affects mainly the neoplastic cells with high mitotic activity and the damage to well-differentiated cells with slow turnover ratio is probably smaller than that inflicted on neoplastic tissue. In addition, the resting cells in the GI tract can eventually replace the damaged cells, restoring the normal organ function (83). Appropriate supportive care and replacement therapy could further alleviate the symptoms of dysfunction of the endocrine and alimentary systems. In any case, targeted somatostatin analogs would be much less toxic than adjuvant chemotherapy.

ONCOLOGIC USE OF CYTOTOXIC SST ANALOGS Carcinoma of the Prostate

The prognosis of patients with androgen refractory prostate cancer is very poor, and no effective treatment exists at present. Recently, high-affinity binding of radiolabeled RC-160 was demonstrated in 50 of 80 (65%) primary prostate cancer specimens (50) and we found the expression of sst 2 on 14% and sst 5 on 64% of 22 samples tested. These findings suggest that targeted cytotoxic SST analog AN-238 may be useful for the treatment of hormone refractory prostate cancer patients. This theory was first evaluated on the very aggressive androgen-independent Dunning R-3327-AT-1 prostate carcinoma in Copenhagen rats (83). At a well-tolerated dose of 300 nmol/kg, AN-238 produced a ⬎80% decrease in tumor weight and significantly decreased tumor burden 4 weeks after therapy. In contrast, cytotoxic radical AN-201 showed only a weak effect at 110 nmol/kg and killed 9 of 10 animals at 115 nmol/kg within 12 days. AN-238 also showed high-affinity binding to Dunning R-3327AT-1 tumor membrane preparations (83). Similarly impressive results were obtained in nude mice bearing androgenindependent PC-3 human prostate cancers after treatment with a single dose or two consecutive injections of AN-238 (114). In these experiments, AN-238 reduced final tumor volumes, tumor weights, and tumor burden by more than 60%. Histological examination of tumors revealed that the effect of AN-238 is mainly due to a significant increase in the number of cells undergoing apoptosis. This is important because the growth of prostate cancer is associated with a low rate of apoptosis rather than a high rate of mitosis. In metastatic model of PC-3, the treatment with AN-238 had a strong effect on the weight of orthotopically grown tumors, producing a 77% reduction (114). In addition, no

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retroperitoneal or distant metastases could be observed 4 weeks after the initiation of the therapy with AN-238. Cytotoxic radical AN-201 was ineffective and more toxic in all these studies (114).

Breast Cancer

Estrogen-independent metastatic breast cancer is a major challenge to oncologists especially if the patients have chemoresistant tumors (68). The presence of SST octapeptide-preferring receptor subtypes on human breast cancer specimens has been established by several research groups (68). Thus, patients with breast cancer could benefit from treatment with analogs such as AN-238. To investigate the efficacy of SST receptor-targeting in human breast cancer, nude mice bearing xenografts of estrogen-sensitive MCF-7-MIII, estrogenindependent MDA-MB-231, and MX-1 (also DOX-resistant) tumors were treated with a single i.v. injection of AN-238 or cytotoxic radical AN-201 (68). In the MCF-7-MIII model, 3 of 8 tumors showed a regression, even 60 days after the administration of AN-238. In the MDA-MB-231 model, 4 of 13 tumors showed an apparent initial regression after treatment with AN-238 that lasted for about 2 weeks. In the MX-1 model 5 of 10 animals were tumor free 60 days after a single injection of AN-238. Because chemoresistance is a major problem in the management of breast cancers in patients, it is important to point out that AN-238 is active on DOX-resistant tumor cells. In all three studies, AN-201 was again more toxic and less effective than AN-238 (68).

Epithelial Ovarian Cancer

Recently, we reported that 13 of 17 (76%) surgical specimens of human epithelial ovarian cancer exhibited high-affinity binding sites for radiolabeled RC-160 and the expression of mRNA for sst 2, sst 3, and sst 5 was also demonstrated (52). Thus, in addition to cytotoxic LH-RH conjugates, ovarian cancers might be also targeted by cytotoxic SST analogs. This concept was tested in LH-RH receptor-negative, but SST receptor-positive UCI-107 human ovarian cancers xenografted into nude mice (A. Plonowski, A. V. Schally, and A. Nagy, submitted for publication). After two iv injections of AN-238 final tumor weights were reduced by 67.3% compared with controls. Treatment with AN201 produced only a slight, but nonsignificant effect, and caused the death of two of eight animals, while none died in the AN-238 group.

Renal Cell Carcinoma (RCC)

RCC is often diagnosed at an advanced stage, when metastatic spread cannot be prevented by surgical intervention. The prognosis for metastatic RCC is dismal because of its resistance to both chemotherapy and radiother-

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apy (113, 133). Because more than 70% of RCCs express high-affinity binding sites for SST (122), we evaluated the effects of AN-238 on growth of xenografts of human RCC lines in nude mice. Among these lines SW-839 was sst 2-positive, 786-0 was sst 5-positive, and CAKI-1 was SST receptor-negative (113). Highaffinity binding of AN-238 was found on membrane preparations of SW-839 and 786-0 and the growth of SW-839 and 786-0 tumors was inhibited significantly (113). Even more striking results were obtained with orthotopically grown 786-0 metastatic RCC. Three of seven mice treated with AN-238 were tumor-free at the end of the experiment and in three other animals the tumor mass was ⬍30 mg. The mean tumor weight in the group treated with AN-238 was 55.3 ⫾ 44.3 mg, representing an 87% reduction compared with controls, which measured 414.2 ⫾ 41.0 mg. More importantly, in the AN-238-treated group only one mouse with a large receptor-negative primary tumor developed lymphatic metastases. In contrast, metastases were observed in five of six animals in both the control and the AN-201-treated group (113). No significant antitumor effect of AN-238 was observed on CAKI-1 xenografts. In all these studies, AN-201 was again ineffective and more toxic than AN-238, indicating that targeting may help overcome the chemoresistance of RCCs, and extenuate the toxicity (113). Brain Tumors

Glioblastomas represent the most common form of primary brain tumors and are considered incurable (129, 133). Low-grade glioblastomas (astrocytomas) express sst 2 (76, 133) making these malignancies amenable for targeted chemotherapy based on SST analogs. Because U-87 MG human glioblastomas express receptors for SST, we tested AN-238 in both subcutaneous and orthotopic models in nude mice (76). Animals with large (⬎500 mm 3) subcutaneous xenografts received a single injection of AN-238. This treatment resulted in an 82% tumor growth inhibition compared with controls. In the same study, mice bearing very large tumors (⬃900 mm 3) were given two injections of AN-238 (76). Nineteen days after the first injection, a 30% shrinkage in the tumor volumes was observed. In both studies, AN-201 was ineffective and more toxic. We also tested the efficacy of AN-238 injected into the tail vein of mice bearing orthotopically grown U-87 MG tumors. Again, AN-238 produced a significant prolongation of survival time compared with controls, indicating that the tumor blood– brain barrier may be penetrable for cytotoxic SST analog AN238. AN-238 bound to the receptors present in U-87 MG tumors with high affinity and sst 2 was determined as the receptor subtype by mRNA analysis (76). Small Cell Lung Carcinoma and Non-SCLC

SCLC constitutes only about 20% of all lung cancers, but most cases are already metastatic at the time of diagnosis (133). Chemotherapy can be used

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for treatment, but long-term survival rate is low (133). A high percentage of primary SCLC tumors and metastatic lesions express SST receptors and can be localized in patients with OctreoScan scintigraphy (87). Non-SCLC can also be visualized by OctreoScan in patients, although the receptors for SST are not found on tumor cells, but only in peritumoral tissue (blood vessels, immune cells, etc.) (87). To evaluate the efficacy of SST receptor-targeting in SCLC, we treated nude mice bearing H-69 SCLC xenografts with AN-238 and a single dose produced a ⬎50% inhibition of tumor growth (77). H-69 tumors bound radiolabeled RC-160 and expressed high levels of mRNA for sst 2. Although we could not find mRNAs for sst 2, sst 5, or sst 3 in H-157 non-SCLC tumors or in cultured cells, radioreceptor assays indicated that four of five control tumors showed specific binding of radiolabeled RC-160. Treatment with AN-238 also produced a very impressive 91% growth inhibition of non-SCLC compared with controls (77). To determine whether tumor vasculature from the host could be the target for AN-238, we evaluated the RNA extract from the control tumor specimens for mRNA for the mouse sst 2 and sst 5 subtypes and found a strong expression of sst 2. These results are the first examples of a successful targeted therapy based on SST receptors in tumor vasculature (77).

Pancreatic Cancer

Because some studies indicated a binding of a radionuclide SST octapeptide to pancreatic cancers in patients and the expression of mRNA for sst 5 and sst 3 was also reported (14), we tested AN-238 on human pancreatic cancer lines xenografted in nude mice that express these subtypes (K. Szepeshazi, A. V. Schally et al., submitted for publication). Studies in progress demonstrate that AN-238 inhibits the growth of experimental pancreatic cancer, while AN-201 is much less effective. All these studies demonstrate major differences in efficacy between a “straight” analog such as RC-160 and a cytotoxic analog like AN-238. Thus, it is possible that very potent cytotoxic SST analogs such as AN-238 would be targeted even to tumors with a low concentration of SST receptors, producing effective clinical responses.

BOMBESIN AND GASTRIN-RELEASING PEPTIDE (GRP)

The family of bombesin-like peptides consists of a large number of peptides found in amphibians and mammals, including humans (129, 142, 146). The tetradecapeptide bombesin was the first family member isolated from frog skin (142). Subsequently, two mammalian bombesin-like peptides, GRP, which is related to bombesin, and neuromedin B, which is related to amphibian ranatensin, were isolated from porcine stomach and spinal cord (142). GRP is a 27-amino acid peptide and its carboxyl-terminal decapeptide is identical to

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that of bombesin, except for one amino acid (142). Bombesin-like peptides are not classical hypothalamic hypophyseal regulating hormones since they play only a perfunctory role in the release of pituitary hormones. However, bombesin-like immunoreactivity is widely distributed in mammalian brain, including the hypothalamus as well as in lung and GI tract (146). GRP is found in rat brain, especially the hypothalamus and in human fetal lung and GI tract (146). Bombesin and GRP may function as gastrointestinal hormones and neurotransmitters (133). In digestive tract, bombesin and GRP stimulate the contraction of smooth muscles, the growth of intestine and pancreas, the exocrine secretion of the stomach and pancreas, and the release of gastrin and other gastrointestinal hormones. From an oncologic point of view, the most important action of bombesin/GRP and neuromedin B is their ability to function as growth factors and, through autocrine or paracrine mechanisms, modulate the growth of tumoral tissues (28, 133, 156). Thus, in addition to SCLC, where they were originally discovered (28), bombesin-like peptides are produced also in other cancers, such as breast, prostatic, and pancreatic cancer, and could act as growth factors involved in tumor progression.

Receptors for Bombesin/GRP

Four receptor subtypes (BRS) associated with the bombesin-like peptide family have been identified and cloned so far (22, 102, 133, 137, 142, 144, 145). Receptor subtype 1 binds bombesin and GRP with high affinity. Subtype 2 prefers neuromedin B, and subtype 3 is classified as an orphan receptor because its natural ligand is yet to be identified. A fourth subtype has a higher affinity for the amphibian tetradecapeptide bombesin than for GRP (102). The bombesin/GRP receptor subtype 1 has been detected on a wide variety of human malignancies including SCLC and breast, prostate, pancreatic, gastric, and colon cancer as well as brain tumors.

Bombesin/GRP Antagonists

The finding that bombesin or GRP can function as an autocrine growth factor for various tumors stimulated several groups, including ours, to develop bombesin/GRP antagonists (15, 129, 133). During the past decade, a large number of bombesin/GRP antagonists was synthesized in our laboratory. Among these compounds was RC-3095 [D-Tpi 6, Leu 13⌿(CH 2NH)Leu 14]bombesin(6 –14), which showed strong inhibitory effect on several experimental cancers (15, 133). Later, modification of the C- and N-terminal amino acids led to new antagonists such as RC-3940-II [Hca 6, Leu 13⌿(CH 2N)Tac 14]bombesin(6 –14) with increased binding affinity to the receptors and stronger antitumor activity. The tumor-inhibitory mechanism of bombesin/GRP antagonists seems to be more complex than a simple competitive action on the receptor, and it is not completely understood. Bombesin/GRP antagonists

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affect intracellular second messengers causing changes in calcium concentrations (18). The main mechanism of tumor-inhibitory action of bombesin/GRP antagonists appears to be the reduction in concentration of EGF receptors on tumors (48, 147, 152). Although bombesin/GRP antagonists are rapidly eliminated from the bloodstream after a single administration, EGF receptors remain down-regulated for many hours. This explains how daily single injections of the antagonists can maintain tumor growth inhibition (152). Because bombesin receptors are widely expressed in the GI tract and other tissues, it is possible that long-term treatment with bombesin/GRP antagonists may have various side effects, but these could be controlled with appropriate medication. Toxicology studies on bombesin/GRP antagonists revealed no sign of systemic toxicity.

STUDIES WITH BOMBESIN/GRP ANTAGONISTS IN VARIOUS CANCERS Prostate Cancer

Neuroendocrine cells are widely distributed throughout acinar epithelium and urothelium of the normal prostate (2, 29). These cells produce a number of bioactive substances, including serotonin, chromogranin A, somatostatin, bombesin-like peptides, parathormone-related protein, vasoactive intestinal peptide, and others (reviewed in (29)). The network of neuroendocrine cells constitutes a complex mechanism that regulates growth and differentiation of developing prostate and secretory processes of the mature gland. Neuroendocrine differentiation occurs frequently in prostate cancer (1, 29). Although small cell prostatic carcinoma that exclusively contains neuroendocrine cells accounts for only 1% of all cases of prostate cancer, most often neuroendocrine cells are dispersed individually or in clusters among prevailing population of nonneuroendocrine malignant cells (1, 29). Because neuroendocrine cells do not express androgen receptors (8), small cell carcinoma of the prostate pursues a highly aggressive course and is not responsive to androgen withdrawal (154). Similarly, neuroendocrine cells scattered in the common type of prostate cancer continue to grow despite androgen suppression and are responsible for androgen-independent regrowth of the prostate cancer (154) and unfavorable prognosis (41). Neuropeptides such as bombesin, and growth factors produced by neuroendocrine cells are believed to interact with neighboring nonneuroendocrine cells and cross-activate androgen-dependent mitogenic pathways in the absence of testosterone (5, 27, 59). Thus, therapeutic approaches that suppress activity of neuroendocrine cells may greatly benefit patients by extending clinical response to antiandrogen treatment. Likewise, interference with autocrine/paracrine mitogenic activity of bombesin may improve management of androgen-refractory prostate cancer. High-affinity receptors for bombesin/GRP were detected on PC-3 and DU145 human prostate cancer cell lines (60, 134, 137). After binding of bombesin, the receptors were rapidly internalized and seemed to interact with G-protein

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(133). When 80 human prostate cancer samples were analyzed for bombesin/ GRP receptors, 50 showed high-affinity receptors for bombesin/GRP. RT-PCR analyses were performed on 22 samples and 20 were found to express GRPreceptor mRNA, 3 specimens neuromedin B-receptor mRNA, and 2 specimens BRS-3 mRNA (144). These receptors are rarely detected in benign prostatic lesions. The expression of GRP receptors in prostate can be correlated with neoplastic transformation of the tissue (93). GRP receptors are detected also in bone metastases of prostate cancers. Our early work showed that bombesin/GRP antagonist RC-3095 inhibited growth of PC-82, DU-145, and PC-3 human prostate cancers in nude mice (129, 133, 134). RC-3095 was also highly active on androgen-independent Dunning R-3327-AT-1 rat prostate cancers (134). Subsequent studies demonstrated that newer bombesin antagonists RC-3940-II and RC-3950-II strongly inhibit growth of DU-145 and PC-3 androgen-independent prostate cancers in nude mice and cause down-regulation of EGF receptors in the tumors (60, 134). Recent work indicates that bombesin/GRP antagonists can potentiate the inhibitory effects of GH-RH antagonists on the growth of PC-3 prostate cancers (134). The combination of both classes of analogs appears to interfere with both IGF and bombesin/EGF pathways and could be useful clinically for the management of androgen-independent prostate cancer (134).

Breast Cancer

Some breast cancer cell lines express receptors for bombesin/GRP and also synthesize GRP (133). Bombesin/GRP receptors were found by radioreceptor assay in about 33% of human breast cancer samples (53) and were also detected by autoradiography in a relatively high percentage of breast cancers and also in nontumorous breast tissue samples (44). Various studies in our laboratory clearly demonstrate that bombesin/GRP antagonists can inhibit growth of mammary cancers (140, 151, 158). In an early study, RC-3095 was shown to inhibit growth of both estrogen-dependent and -independent MXT mouse mammary cancers (151). Tumor inhibition was associated with a downregulation of EGF receptors in estrogen-independent cancers (151). We have also shown that bombesin can stimulate the proliferation of some human breast cancer lines in vitro and antagonist RC-3095 inhibits this effect (158). In studies in vivo in nude mice, bombesin antagonists RC-3095 and RC-3950-II inhibited the growth of MCF-7 MIII human breast cancer, an estrogen-independent, but estrogen-sensitive, tumor (140). Antagonists RC-3940-II and RC-3095 also suppressed growth of estrogen-independent MDA-MB-231 human breast cancers in nude mice, RC-3940-II being the more powerful. The inhibition of tumor growth was associated with a decrease in EGF receptors in the tumors (69, 98). Therapy with RC-3940-II and RC-3095 also resulted in a regression of MDA-MB-468 estrogen-independent human breast cancers in nude mice (69). Bombesin/GRP antagonists could provide a new treatment modality for breast tumors expressing bombesin receptors.

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Ovarian Cancer

Human epithelial ovarian cancers express three subtypes of bombesin/GRP receptors (145). These subtypes are also found in ES-2 and OV-1063 ovarian cancer lines (I. Chatzistamou and A. V. Schally, submitted for publication). Recent work shows that bombesin antagonists RC-3095 and RC-3940-II can inhibit growth of ES-2 and OV-1063 tumors in nude mice. The tumor inhibitory effect of the bombesin antagonist in OV-1063 tumors was associated with a down-regulation of c-jun and c-fos proto-oncogenes in the tumor cells. On the basis of these results, bombesin antagonists are being tested clinically in patients with ovarian cancer.

Renal Cell Carcinoma

Bombesin/GRP receptors are expressed in human RCC, but not in normal kidney (107). GRP receptors were also demonstrated in RCC lines such as A498, CAKI-1, CAKI-2, and ACHN. The growth of CAKI-2 cells can be stimulated by bombesin and GRP and this effect is abolished by a GRP receptor antagonist (107). Our work shows that bombesin/GRP antagonist RC-3940-II inhibits growth of CAKI-1 human renal cell cancers in nude mice (61). This effect is accompanied by a decrease in EGF receptors (61) on tumors. Based on these results bombesin antagonists could be considered in therapy for RCC.

Brain Tumors

About 13,000 deaths annually in the United States are attributed to brain tumors (133). Treatment of primary brain tumors such as malignant astrocytomas (glioblastomas) should be improved. Much evidence indicates that brain tumors are hormone sensitive and the presence of receptors for EGF in human brain tumors is well established (129). Various brain tumors also contain receptors for bombesin/GRP. Functional bombesin receptors are also detected in 85% of human glioblastoma cell lines and in U-87MG xenografts they are of subtype 1 and 2. Bombesin/GRP antagonists RC-3095 and RC-3940-II inhibit the proliferation of U-87MG and U-373MG human glioblastomas transplanted into nude mice or cultured in vitro (78, 133). This inhibition is associated with a down-regulation of EGF receptors. The survival time of animals inoculated with tumor cells orthotopically into the brain is also significantly prolonged by treatment with RC-3095 (133). GRP(14 –27) stimulated the expression of c-fos oncogene in the U-87MG tumors, but antagonist RC-3940-II inhibited it (78). Bombesin/GRP antagonists could be considered for the development of new approaches to the treatment of some brain tumors.

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Lung cancer is the leading cause of cancer-related deaths in the Western world and new therapeutic modalities are needed for both SCLC and nonSCLC (129, 133). SCLC can secrete bombesin-like peptides, including GRP and neuromedin B, which function as autocrine growth factors and stimulate tumor growth (28, 142). Receptors for bombesin-like peptides and mRNA for receptor subtypes 2 and 3 have been found in various SCLC lines (75, 129, 142). Bombesin/GRP receptor mechanisms may play a role in development of lung cancers in smokers. Recently, it was reported that the GRP receptor gene is activated in bronchial epithelial cells after long-term exposure to tobacco that makes the cells susceptible to development of cancer (141). New endocrine methods for treating SCLC, based on bombesin/GRP antagonists and aimed at interfering with bombesin/GRP receptors, are being developed (129, 133). In our first study we showed that bombesin/GRP antagonist RC-3095 inhibits growth of H-69 SCLC but not of H-157 non-SCLC xenografted into nude mice (129, 133). Subsequently we demonstrated that antagonist RC-3940-II also inhibits growth of H-69 SCLC and possesses greater antitumor activity than RC-3095 (82). The inhibition of tumor growth was associated with a decrease in the levels and mRNA expression of EGF receptors (82). In another human SCLC line, H-128, treatment with RC-3095 decreased the concentration of both bombesin/GRP and EGF receptors on the cells concomitantly with tumor suppression (48). Clinical trials with RC-3095 are in progress.

Pancreatic Cancer

The prognosis for patients with ductal carcinoma of the pancreas is very poor and it is essential to develop more effective therapies (133). Experimental findings suggest that the growth of exocrine pancreatic cancer may be influenced by GI hormones, growth factors such as EGF, IGF-I, and -II, and sex steroids (133). For nearly 20 years we have been attempting to develop a hormonal therapy for exocrine cancer of the pancreas based on diverse analogues including bombesin/GRP antagonists (129, 133). Numerous studies show that bombesin and GRP can influence the release of GI hormones, promote pancreatic secretion and growth, and stimulate pancreatic carcinogenesis (133). mRNAs for both bombesin and its receptors were detected in various human pancreatic cancer cell lines showing that bombesin/GRP may be autocrine growth factors in pancreatic cancer (156). Bombesin stimulates the proliferation of CFPAC-1 and other human pancreatic cancer cells in vitro through its receptors (117). This effect is abolished by antagonist RC-3095. The inhibitory effect of RC-3095 and RC-3940-II on nitrosamine-induced ductal pancreatic cancers in hamster was demonstrated in several studies and was accompanied by a major down-regulation of EGF receptors (147). Antagonists RC-3095 and RC-3940-II also inhibit the growth of CFPAC-1, SW-1990, and other human pancreatic cancer cells xenografted in nude mice (117, 133, 147).

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Phase I/II trials with RC-3095 in patients with exocrine cancers are in progress.

Gastric Cancer

Carcinoma of the stomach ranks seventh in the United States among causes of cancer-related deaths. However, stomach cancer is an international health problem, being the leading cause of mortality from cancer in Japan, China, and India and thus it ranks No. 2 overall in the world, second only to lung cancer. In patients with unresectable cancer of the stomach, the prognosis is dismal and new treatment modalities are needed for patients with locally advanced and metastatic gastric carcinoma (129, 133). Gastrointestinal hormones, especially gastrin and bombesin/GRP and growth factors such as EGF and transforming growth factor-␣ (TGF-␣) are implicated in the growth of human gastric adenocarcinoma (129, 133). The receptors for bombesin and GRP are present in human gastric cancers and various cell lines. We reported bombesin receptors in MKN-45 and Hs746T gastric cancer cell lines and demonstrated that bombesin stimulates the growth of Hs746T cells in vitro (129, 133). We also showed that the administration of bombesin/GRP antagonists inhibits the growth of MKN-45 and Hs746T human gastric cancers xenografted in nude mice and causes a down-regulation of EGF receptors on tumor cells (129, 133). Bombesin antagonists could be tried in patients with advanced gastric cancer.

Colorectal Cancer

Advanced colon cancer is difficult to treat (133). Gastrointestinal hormones, especially gastrin and bombesin/GRP and EGF may be involved in tumorigenesis of the colon (133). Approaches based on the use of antagonists of bombesin/ GRP and aimed at inhibiting GI hormones, and growth factors are being tried in colorectal cancer. Recent studies show that most colon cancers, but not normal colonocytes, express GRP, GRP receptor and mRNA for GRP receptor (17). Thus, GRP could be an autocrine growth factor in colorectal cancer (22). We have demonstrated that bombesin antagonists can inhibit the growth of HT-29 human colon cancers in nude mice and down-regulate EGF receptors (133). Bombesin/GRP antagonists could be considered for therapy of colon cancer.

CYTOTOXIC BOMBESIN ANALOGS

Since bombesin/GRP receptors are expressed in various tumors, the application of radiolabeled bombesin analogs for tumor detection has been proposed (93, 133, 137). 111In-labeled analogs of bombesin can be rapidly internalized

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and stored in the receptor-containing cells, allowing a more specific and longer lasting visualization of tumors. Because the bombesin antagonists developed by us have high binding affinity to the bombesin/GRP receptor subtype, we decided to synthesize targeted cytotoxic bombesin conjugates using these bombesin-like antagonists as carriers (102). Promising results with doxorubicin or AN-201 prompted us to use these cytotoxic agents and carriers such RC-3095 and its amino terminally truncated analog to form cytotoxic bombesin-like hybrids (Fig. 1). Thus, superactive cytotoxic bombesin conjugate AN-215 was prepared by linking AN-201-14-O-hemiglutarate to the amino terminal of des-D-Tpi-RC-3095. This analog showed high binding affinity to bombesin/ GRP receptors on Swiss 3T3 cells in vitro (102). We then demonstrated that bombesin receptors present on human H-69 SCLC could be used for targeting cytotoxic bombesin analogs in vivo. Thus, nude mice bearing xenografted H-69 SCLC cell line received injections of AN-215 or AN-201 (75). The growth of SCLC H-69 tumors was greatly inhibited by the treatment with AN-215, while equimolar doses of the cytotoxic radical AN-201 were toxic and produced only a minor tumor inhibition. The effectiveness of cytotoxic bombesin analog AN-215 was also evaluated in nude mice bearing PC-3 androgen-independent human prostate cancers (112). Treatment with AN-215 caused about 70% reduction in tumor volume and greatly extended tumor doubling time. Cytotoxic radical AN-201 was ineffective and more toxic. Because bombesin receptors are present on metastatic prostate cancers, targeted chemotherapy with AN-215 should benefit patients with advanced prostatic carcinoma who no longer respond to androgen deprivation. As in the case of the receptors for SST, the receptors for bombesin/GRP are present in various normal tissues, but again in our studies on the effects of cytotoxic bombesin analog AN-215, we did not observe any receptorspecific toxicity on GI function (75, 112).

GROWTH HORMONE-RELEASING HORMONE

The identification of GH-RH was facilitated by the demonstration of ectopic production of GH-RH by carcinoid and pancreatic cell tumors (39). The 44- and 40-amino acid forms of GH-RH were then isolated from human pancreatic tumors and subsequently identified in human hypothalamus (45, 124). The full intrinsic biological activity is retained by the NH 2-terminal 29-amino acid sequence [GH-RH(1–29)NH 2] (133, 138). Many agonistic analogs of GH-RH(1– 29)NH 2, intended for potential clinical and veterinary applications, have been synthesized by various groups (133). There is an even greater clinical need for antagonistic analogs of GH-RH, since somatostatin analogs do not adequately suppress GH and IGF-I levels in patients with neoplasms potentially dependent on IGF-I (115).

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Development of Potent GH-RH Antagonists

Robberecht et al. (125) showed that replacement of Ala 2 by D-Arg 2 within the NH 2-terminal 29 amino acid segment of GH-RH leads to antagonists. Subsequent work in our laboratory, aimed at the development of analogs with enhanced activity, yielded the potent GH-RH antagonists MZ-4-71 and MZ-5156 (138, 160), containing a hydrophobic N-acyl moiety at the NH 2 terminal and enzyme-resistant C terminal. Incorporation of positively charged amino acids into these GH-RH antagonists led to analogs JV-1-36, JV-1-38, and JV-1-42 with further increased bioactivity (155). These compounds show a high binding affinity to pituitary and tumoral GH-RH receptors, and strongly inhibit pituitary GH secretion, in vitro and in vivo (51, 120, 155). The antiproliferative activity of GH-RH antagonists was evaluated in many tumor models in vivo and also in vitro (138).

GH-RH Receptors in Human Cancers

Since the discovery of GH-RH production by carcinoid and pancreatic tumors in 1981 (39), much evidence has accumulated on the involvement of GH-RH and its receptors in the pathophysiology of cancer. The expression of mRNA for GH-RH and the presence of biologically or immunologically active GH-RH was demonstrated in several human malignant tumors, including cancers of the breast, endometrium, and ovary, and SCLC (65, 70, 79). These results suggest that GH-RH can function as an autocrine growth factor (79, 138). Peptide receptors on tumors that might mediate the effects of GH-RH and its antagonists were identified recently (51, 120). Employing a well-characterized radiolabeled GH-RH ligand for binding assays and RT-PCR, the pituitary form of GH-RH receptors could not be detected on any of the cancer models (21, 70). However, using 125I-labeled GH-RH antagonist JV-1-42 as a special ligand, we were able to demonstrate the presence of specific high-affinity binding sites for GH-RH and its antagonists on CAKI-1 renal, MiaPaCa-2 pancreatic, LNCaP and PC-3 prostatic, and OV-1063 ovarian cancers (21, 51, 120). The isolation and sequencing of cDNAs corresponding to the tumoral GH-RH receptor mRNAs revealed that they are splice variants (SV) of the pituitary GH-RH receptors (120). Tumoral SV 1, SV 2, and SV 4, have a retained intronic sequence at their 5⬘ end and lack the first three exons (51, 120). The major part of the cDNA sequence of SV 1 is identical with the corresponding sequence of pituitary GH-RH receptor cDNA, but the first 334 nucleotides of SV 1 are completely different (120). The deduced protein sequence of SV 1 differs from that of the pituitary GH-RH receptor only in the N-terminal extracellular domain (120). SV 2 may encode a GH-RH receptor isoform truncated after the second transmembrane domain. The short protein sequence corresponding to SV 4 lacks all transmembrane domains, implying that it is not expressed on the cell surface. RT-PCR analyses revealed the expression of receptor SV in several cancers, including LNCaP, PC-3, and MDA-PCa-2B prostatic, MiaPaCa-2 pancreatic,

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CAKI-1 renal, H-69 SCLC, MDA-MB-468 breast, and OV-1063 ovarian cancers (21, 51, 120). Additional studies are needed to confirm that mRNAs for tumoral SV receptors are translated into the GH-RH binding sites found by radioligand assay.

EFFECTS OF GH-RH ANTAGONISTS ON GROWTH OF EXPERIMENTAL CANCERS

Our initial investigations of oncological activities of the GH-RH antagonists were based only on the assumption that the blockade of the pituitary GH/ hepatic IGF-I axis might inhibit the growth of IGF-I dependent cancers (110). However, subsequently we discovered that GH-RH antagonists can also suppress the proliferation of diverse tumors that are influenced by autocrine and/or paracrine production of IGF-I and -II (90, 111). In addition, GH-RH antagonists can inhibit growth of some cancers such as SCLC by blocking the action of autocrine/paracrine GH-RH by a direct effect that may not involve IGFs. Hence, a wide range of cancers can be inhibited by GH-RH antagonists.

Prostate Cancer

Both IGF-I and IGF-II appear to be involved in the malignant transformation and the progression of many tumors, including prostate cancer (134). Androgen-independent PC-3 and DU-145 prostate cancer cell lines produce and secrete IGF-I or IGF-II and possess IGF-I receptors. In an attempt to develop a new approach to the treatment of androgen-independent prostate cancer, we evaluated the effects of GH-RH antagonist MZ-4-71 in nude mice bearing DU-145 and PC-3 prostate cancer cell lines (64). Therapy with MZ4-71 significantly decreased tumor growth, serum levels of GH and IGF-I, and liver IGF-I levels (64). The concentration of IGF-I and IGF-II in PC-3 tumor tissue was reduced to nondetectable values after treatment with MZ-4-71 (64, 134). To investigate the mechanisms involved, we treated male nude mice bearing xenografts of DU-145 prostate cancer with GH-RH antagonist MZ-5156 and again found significant reductions in the volume of DU-145 tumors, serum IGF-I levels, and the concentration of IGF-II in tumor tissue (90). RT-PCR analysis revealed that the expression of IGF-II mRNA in DU-145 tumors was greatly decreased. Thus, GH-RH antagonists may inhibit the growth of DU-145 prostate cancers also by reducing the production of IGF-II in the tumor tissue through a direct, GH-independent action (90, 134). These findings have clinical implications. It is well known that the duration of clinical response to androgen deprivation is limited, as cancer cells acquire the ability to grow in the absence of androgens (133, 134). One of the proposed mechanisms of progression to androgen-independent stage is cross-activation of androgen receptor-dependent pathways by intracellular cascades evoked by growth factors (26). Thus

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the inhibition of the IGF-dependent mitogenic loop by GH-RH antagonists would eliminate one of the factors supporting the survival of prostate cancer cells following androgen deprivation. Studies in nude mice implanted orthotopically with LNCaP human androgen-sensitive prostate cancer show that castration alone does not cause a lasting suppression of serum PSA or inhibition of tumor growth (121). After an initial arrest of about 1 week, serum PSA starts to rise rapidly, indicating that a relapse has occurred. GH-RH antagonist JV-1-38 alone similarly does not affect serum PSA or tumor growth in intact mice. However, treatment with JV-1-38 combined with castration can powerfully decrease PSA serum concentration (121) and reduce the weight of orthotopic LNCaP tumors (121). A marked inhibition of cultured LNCaP cells and reduction of PSA secretion by GH-RH antagonists can be likewise demonstrated in vitro. GH-RH antagonists might provide new approaches to therapy of patients with prostatic carcinoma who have relapsed following conventional androgen deprivation (134).

Breast Cancer

IGF-I and IGF-II might influence the growth of human breast cancers by endocrine, autocrine, or paracrine mechanisms (70, 133). In addition, the presence of biologically and immunologically active GH-RH and messenger ribonucleic acid for GH-RH in human breast cancers supports the hypothesis that locally produced GH-RH may play a role in the proliferation of these tumors (65). Antagonists of GH-RH may offer a new approach to treatment of breast cancer. GH-RH antagonists MZ-5-156 and JV-1-36 induced regression of estrogen-independent MDA-MB-468 human breast cancers xenografted into nude mice and in vitro inhibited the rate of proliferation of MDA-MB-468 cell line (70). The expression of mRNA for human GH-RH was found in some tumors. These results suggest that GH-RH antagonists inhibit MDA-MB-468 breast cancers also through mechanisms involving interference with locally produced GH-RH (70, 138). Various GH-RH antagonists including MZ-5-156, JV-1-36, and JV-1-38 likewise inhibit growth of MXT estrogen-independent mouse mammary cancers (K. Szepeshazi and A. V. Schally, unpublished). These investigations indicate that GH-RH antagonists could be potentially useful for treatment of breast cancer.

Ovarian Cancer

IGF-I and -II stimulate proliferation of human epithelial ovarian cancer cell lines (21). We investigated the effects of antagonists of GH-RH on the growth of human epithelial ovarian cancer cell line xenografted into nude mice. Treatment with GH-RH antagonists JV-1-36 or MZ-5-156 decreased growth of OV-1063 cancers and reduced the levels of mRNA for IGF-II in tumors (21). In vitro, JV-1-36 also inhibited OV-1063 cell proliferation (21). OV-1063 cancers

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expressed mRNA for tumoral GH-RH receptors and showed the presence of binding sites for GH-RH (21). Antagonist JV-1-36 also inhibited the growth of UCI-107 human ovarian cell carcinomas. These results indicate that antagonistic analogs of GH-RH inhibit growth of epithelial ovarian cancers.

Renal Cell Carcinoma

Since GH and IGF-I may play a role in the development of renal cancers, we investigated the effects of GH-RH antagonist MZ-4-71 on Caki-1 human RCC (63). Treatment with MZ-4-71 significantly decreased growth of Caki-1 tumors in nude mice, and reduced serum levels of GH and IGF-I, liver concentrations of IGF-I, and tumor levels of IGF-I and IGF-II (63). GH-RH antagonist JV-1-38 also inhibited the growth of orthotopic Caki-1 RCC and inhibited the development of metastases to lung and lymph nodes (51). Specific binding sites for GH-RH were found in Caki-1 tumors and RT-PCR revealed the expression of splice variants of hGH-RH receptor (51). These distinct receptors can mediate the inhibitory effect of GH-RH antagonists in RCC. Further investigations are needed to evaluate the use of GH-RH antagonists for the therapy of advanced RCC.

Brain Tumors

Gliomas contain IGF-I and IGF-II and express mRNA for IGF-I receptors. Thus, a potential therapy for gliomas could be based on the inhibition of the IGF system (133, 138). The effects of GH-RH antagonists MZ-5-156 and JV1-36 were investigated in nude mice bearing xenografts of U-87MG human glioblastomas (138). Therapy with GH-RH antagonists decreased the growth of U-87MG glioblastomas and the levels of mRNA for IGF-II in tumors (138). Treatment with MZ-5-156 also decreased telomerase activity in U-87MG glioblastomas (138). mRNA for GH-RH was detected in U-87MG tumors, suggesting that GH-RH might play a role in the pathogenesis of this tumor. Antagonistic analogs of GH-RH should be further developed for treatment of malignant glioblastoma.

Lung Cancer

Human SCLC and non-SCLC cell lines secrete and respond to IGF-I and -II, and express IGF-I and -II genes and receptors (111, 133, 138). We were able to demonstrate that GH-RH antagonists significantly inhibited growth of SCLC H69 and non-SCLC H157 tumors in nude mice and decreased the levels of IGF-I in serum and liver tissue (111). Treatment with MZ-4-71 also greatly reduced IGF-I and IGF-II concentration in H157 non-SCLC tumor tissue. In vitro the proliferation of H-69 SCLC cells and the H-157 non-SCLC line was

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inhibited by this antagonist (111). New GH-RH antagonist JV-1-36 also powerfully inhibits growth of H-69 SCLC in vivo (79). H-69 and H-510A SCLC lines, cultured in vitro, express mRNA for GH-RH and secrete immunoreactive GH-RH (79). GH-RH(1–29)NH 2 stimulates the proliferation of these cells in vitro and GH-RH antagonist JV-1-36 blocks this effect. Our results suggest that GH-RH can function as an autocrine growth factor in SCLCs (79). Collectively these studies show that GH-RH antagonists can inhibit the growth of SCLC as well as non-SCLC (138).

Pancreatic Cancer

IGF-I and IGF-II are implicated in the pathogenesis of pancreatic carcinoma (133, 138). The effects of GH-RH antagonists were evaluated in two experimental models of pancreatic cancer (150). MZ-4-71 and MZ-5-156 inhibited the growth of nitrosamine-induced pancreatic cancers in hamsters and SW-1990 human pancreatic cancers xenografted into nude mice and reduced IGF-II concentration in tumors (150). In vitro, MZ-5-156 decreased SW-1990 cell proliferation and IGF-II synthesis and secretion in the cells (150). These results suggest that the inhibitory effects of GH-RH antagonists on the growth of pancreatic cancers may result from a reduction in the production of IGF-II in the tumors (138, 150).

Colorectal Cancer

Various findings support the involvement of IGF-I and IGF-II in the growth of colorectal cancers (133). The incidence of colon cancer is increased in acromegalics, suggesting that excessive secretion of GH or IGF-I may be a factor (133). In an attempt to interfere with the production of IGFs, we treated nude mice bearing xenografts of HT-29 human colon cancer with GH-RH antagonists MZ-4-71, MZ-5-156, and JV-1-36 (149). These antagonists produced major decreases in the growth of HT-29 cancer and reduced IGF-II concentrations and IGF-II mRNA expression in tumors. In vitro MZ-5-156 diminished IGF-II production and the proliferation of HT-29 cells (149). These studies demonstrate that GH-RH antagonists inhibit the growth of HT-29 human colon cancers through a reduction in the production and secretion of IGF-II by cancer cells (138, 149).

Osteogenic Sarcomas

Osteogenic sarcomas are the most common primary bone tumors in children and young adults. The growth of osteosarcomas is stimulated by IGF-I and GH (110, 133). Consequently, we evaluated the effect of GH-RH antagonist MZ4-71 on human osteosarcoma cell lines SK-ES-1 and MNNG/HOS (110). MZ-

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4-71 inhibited growth of SK-ES-1 and MNNG/HOS tumors in nude mice and decreased IGF-I levels in serum and tumor tissue (110). The growth rate of the two cell lines in vitro was also suppressed by MZ-4-71 (110). These findings suggest that GH-RH antagonists could be considered for the treatment of osteogenic sarcomas (133, 138).

MECHANISM OF ACTION OF GH-RH ANTAGONISTS

GH-RH antagonists can inhibit tumor growth indirectly through suppression of the endocrine GH–IGF-I axis, and also by direct action on the tumor cells (138) (Fig. 2). The indirect mechanism is important for those cancers that depend on IGF-I as a growth factor (138). A strong positive association was reported between plasma IGF-I levels and the risk of prostate, breast, and colorectal cancers (133, 138). GH-RH antagonists decrease the levels of IGF-I in serum by inhibiting the release of GH from the pituitary, which results in a suppression of hepatic IGF-I production (138) (Fig. 2). In nude mice bearing prostatic (64, 90) or renal cancers (63), osteosarcomas (110), SCLCs and non-SCLCs (111), tumor inhibition was accompanied by a decrease in levels of serum IGF-I. In such cases the antiproliferative effect of GH-RH antagonists could be ascribed at least in part to the suppression of the GH–IGF-I endocrine axis (Fig. 2). However, this indirect mechanism alone cannot account for tumor inhibition observed in other cancer models in which GH-RH antagonists did not cause significant inhibition of serum IGF-I levels (21, 79, 149, 150). GH-RH antagonists also inhibit the proliferation of various cancer cell lines in vitro, where the involvement of the GH–IGF-I axis is clearly excluded (21, 70, 79, 138, 149, 150). This direct inhibitory effect appears to be mediated by the tumoral GH-RH receptors, by mechanisms dependent or independent of IGF-I and IGF-II (Fig. 2). Thus, GH-RH antagonists inhibit the production of IGF-I and IGF-II and the expression of IGF-II mRNA in many human cancer lines in vitro and in vivo, including prostatic (64, 90), renal (63), pancreatic (150), and colon cancers (149), glioblastomas (138), ovarian cancers (21), and non-SCLCs (111). Since IGF-II is a potent mitogen for many cancers, a suppression of its production would inhibit tumor growth. GH-RH antagonists can also exert their effects independently of IGF-II (Fig. 2), as in the case of H-69 SCLC where the tumor inhibition is not associated with a suppression of production of IGF-II, but due to the blockade of the stimulatory action of tumoral autocrine GH-RH (79, 138). The relative importance of these mechanisms could vary in different tumors. GH-RH antagonists may offer distinctive advantages over other classes of prospective antitumor agents. Because GH-RH antagonists inhibit IGF-II-dependent tumors, they should be superior to GH antagonists, as the synthesis of IGF-II is not controlled by GH. GH-RH antagonists could be also used for suppression of tumors that do not express somatostatin receptors, such as human osteogenic sarcomas or those that contain only low levels of SST receptors.

FIG. 2.

Schematic representation of the three known mechanisms by which GH-RH antagonists inhibit tumor growth.

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Synthetic hexapeptides related to Met-enkephalin and called growth hormone-releasing peptides or GH secretagogues can also stimulate the release of GH from the pituitary (9). GHSs act both in the hypothalamus and directly on the anterior pituitary (9). The actions of GHSs are mediated through GHS receptors, G-protein-coupled receptors for which the natural ligand was originally unknown. Recently, the isolation from the rat stomach of an endogenous ligand specific for GHS receptors has been achieved (81). The ligand was identified as a 28-amino acid peptide and designated the GH-releasing peptide, or ghrelin (81). Human ghrelin is homologous to rat ghrelin, apart from two amino acids. Ghrelin is present in human plasma at considerable levels. It is likely that this molecule, produced in and secreted from the stomach, can circulate in the bloodstream to act on the pituitary. RT-PCR amplification of mRNA for ghrelin revealed the presence of this transcript in brain. The occurrence of ghrelin in both stomach and hypothalamus gives a new dimension to the regulation of GH release. Recently, receptors for ghrelin were identified in some tumors including breast cancer (19).

TUMORS PRODUCING HYPOTHALAMIC HORMONES Somatostatin-Producing Tumors

Somatostatinomas are rare neuroendocrine tumors arising in the D cells of the gastrointestinal tract and brain. Only about 100 cases have been reported so far in the medical literature (3, 4, 54). Most somatostatinomas are malignant, the main sites of metastases being the liver, peripancreatic lymph nodes, and bone (3). The features of the somatostatinoma syndrome include diabetes mellitus, cholelithiasis, steatorrhea, postprandial fullness, weight loss, and anemia (3, 4, 54). Some studies suggest that somatostatinomas possess functioning SST receptors (4). Thus, somatostatin receptor scintigraphy may be used for tumor localization (4). The usual management of somatostatinoma is based on aggressive surgical approach, total pancreatectomy, and the Whipple procedure being frequently employed (3, 54). However, somatostatinomas can be rarely cured by surgery and there is no effective chemotherapy (3, 54). The mortality rate for patients with metastatic disease is high; only 35% will survive 2 years and 13% for more than 5 years (3). Palliative treatment based on somatostatin analogs such as Octreotide has been used in patients with somatostatinomas and extensive tumor dissemination (4, 54). Octreotide causes a decrease in plasma levels of somatostatin and attenuates related symptoms due to the syndrome in some patients (4, 54). It is possible that an analysis of the SST receptor subtypes in tumor samples of patients with somatostatinomas may help identify those patients who could respond to novel therapies based on somatostatin analogues linked to cytotoxic compounds or radionuclides.

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Corticotropin-Releasing Hormone (CRH)-Producing Tumors

Corticotropin-releasing hormone is produced and acts both within the central nervous system and at several peripheral sites. Ectopic secretion of CRH is a very rare disorder, having been proved only in a small number of cases, mostly in patients with well-differentiated bronchial carcinoid tumors (106). The majority of CRH-secreting tumors also contain ACTH, suggesting that ectopic CRH may represent a paracrine rather than an endocrine phenomenon (37). In addition, CRH-like bioactivity and CRH-like immunoreactivity have been also demonstrated in some nonpituitary tumors associated with Cushing’s syndrome and certain neoplasms that do not produce Cushing’s syndrome (37). Among these tumors which also secrete ACTH are SCLC, bronchial carcinoid, medullary thyroid carcinoma, pheochromocytoma, and paraganglioma (37). The prognosis for patients with ectopic CRH or ACTH syndrome is poor because in most cases, the tumor is not resectable at the time of diagnosis (106). It has been shown that Octreotide rapidly reduces ectopic ACTH secretion from some nonpituitary tumors, but does not usually decrease tumor size. Uptake of 111In-labeled pentetreotide by the tumor predicts a positive response to the drug. Thus, it is conceivable that the cytotoxic analogues of somatostatin might be useful for treating those patients with unresectable CRH or ACTHsecreting tumors, whose lesions can be localized by somatostatin receptor scintigraphy.

Growth Hormone-Releasing Hormone-Secreting Tumors

Eutopic and ectopic GH-RH hypersecretion by certain tumors may be associated with pituitary GH-cell hyperplasia or adenoma and GH hypersecretion (96, 143). GH-RH immunoreactivity is frequently demonstrated in neuroendocrine tumors from patients without acromegaly, usually at lower levels than in tumors associated with acromegaly (143). It has been shown that some hypothalamic tumors such as hamartomas or gliomas may secrete high levels of GH-RH (96). Furthermore, GH-RH receptors have been identified on pituitary adenomas and the response of these tumors is often not down-regulated during prolonged or repeated GH-RH stimulation (96). However, the hypersecretion of GH-RH occurs in fewer than 1% of cases of acromegaly (96, 143). In the great majority of these cases, patients have carcinoid tumors in the bronchus, gastrointestinal tract, or pancreas, which secrete high levels of GH-RH (96, 143). Occasionally, pancreatic islet cell tumors, small cell lung cancer, medullary thyroid carcinomas, adrenal adenoma, pheochromocytomas, and endometrial carcinomas synthesize GH-RH and may cause acromegaly (96, 143). Persistently elevated GH and IGF-I levels in acromegalics are a major medical problem. This hypersomatotropism is associated with significant morbidity and mortality from cardiovascular, cerebrovascular, and malignant disease. The prognosis for patients with GH-RH-producing tumors is generally poor, because many of these tumors are already metastatic at the time of diagnosis

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(31, 58, 88, 96, 143). The use of standard chemotherapy for GH-RH-producing tumors is generally unsuccessful (31). Because of the presence of elevated levels of high-affinity somatostatin receptors on endocrine tumors, including carcinoid tumors, somatostatin receptor scintigraphy is a useful tool for localizing metastases or primary lesions not detected by conventional imaging techniques (88). In addition, somatostatin analog octreotide has also been used in the past decade to manage ectopic acromegaly (96, 143). Recently a patient with acromegaly caused by disseminated GH-RH-secreting carcinoid was treated with a long-acting preparation of lanreotide (31). The treatment induced clinical improvement and biochemical remission (31). Administration of somatostatin analogues to patients with nonresectable GH-RH-secreting tumors improves some of the clinical manifestations of acromegaly and reduces GH levels (31, 96, 143). In about 50% of cases the levels of GH return to normal with octreotide treatment and most of the remaining patients have a partial response (103). The level of GH-RH is often reduced less than that of GH, suggesting that octreotide affects primarily the pituitary response to GH-RH (103). The administration of an early GH-RH antagonist was also reported to reduce GH hypersecretion in a patient with metastatic GH-RH-secreting carcinoid tumor, but the effect lasted only 3– 4 h (58). Unfortunately, none of these current therapeutic modalities produces a cure or causes prolonged remission in patients with nonresectable GH-RH-secreting tumors. New generations of GH-RH antagonists or cytotoxic somatostatin analogues might prove to be of benefit in patients with acromegaly due to metastatic GH-RH-secreting tumors.

Thyrotropin (TSH)-Secreting Pituitary Tumors

Thyrotroph tumors are aggressive, invasive macroadenomas that usually respond poorly to available surgical and medical treatments (11, 25). These tumors are characterized by an autonomous TSH secretion, high ␣-subunit, and high ␣-subunit/TSH ratio (11). Patients with TSH-secreting tumors show a lack of a TSH response to the TRH test and their TSH levels are not suppressed by T3 suppression test (11). The possible involvement of TRH in the pathogenesis of these tumors is not known. Palliative medical therapy with octreotide is used in patients with persistent, clinically significant tumoral secretion in spite of pituitary surgery and external radiation to the pituitary (11, 25). In patients with TSH-secreting pituitary tumors, treatment with octreotide can lower TSH and/or ␣-subunit levels and in some cases produce tumor shrinkage (11, 25). An analysis of SST receptor subtypes of TSHsecreting pituitary adenomas or carcinomas might facilitate new therapeutic approaches to the treatment of invasive TSH-secreting tumors, based on cytotoxic somatostatin analogs.

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Gonadotropin-Secreting Pituitary Tumors

Gonadotroph adenomas are usually clinically nonfunctioning pituitary tumors that often cause significant morbidity from mass effect, including hypopituitarism, visual impairment, and headache (73, 95). High levels of gonadotropins, usually FSH and free ␣- and ␤-subunits are frequently demonstrated in such tumors (73, 95). There is evidence that gonadotropin secretion from gonadotropin-secreting tumors is LH-RH dependent (97). The expression of LH-RH mRNA and mRNA for LH-RH receptors was also detected in gonadotroph tumors (97). The development of new modalities of medical treatment is needed for patients with gonadotroph pituitary macroadenomas. Treatment with octreotide can produce a decrease in serum ␣-subunit levels and a reduction in tumor size in some patients (73). Administration of LH-RH agonistic analogs to patients with such tumors usually either has an agonistic effect or produces no change on gonadotropin secretion and tumor size (95). In one study, an early LH-RH antagonist (Nal-Glu) also had no effect on the adenoma size, in spite of the reduction in FSH levels (95). More potent LH-RH antagonists such as Cetrorelix might be more effective in reducing gonadotroph adenoma size before transphenoidal surgery as well as in those patients who continue to have significant residual tumor after operation.

Hypothalamic Hamartoma Secreting LH-RH

Some hypothalamic hamartomas contain LH-RH neurosecretory neurons and cause true precocious puberty (92). Medical therapy with the LH-RH agonists is considered the preferred approach, because surgery carries an increased risk of morbidity and mortality and if the removal of the hamartoma is incomplete it does not arrest the sexual precocity (92). Chronic treatment of these patients with LH-RH agonists suppresses gonadotropin secretion and arrests pubertal maturation, but does not affect the tumor size (92). Future clinical trials with the LH-RH antagonist Cetrorelix, which causes immediate suppression of gonadotropin secretion, may also prove to be useful for the management of patients with precocious puberty due to pituitary hamartomas.

Perspectives for the Future

Some analogs of hypothalamic hormones already have major applications for the diagnosis and especially the treatment of diverse malignancies. These applications are certain to increase in the future. It is gratifying that the discovery of hypothalamic hormones has led to practical clinical use of their analogs for cancer treatment. New generations of peptide analogs should improve the treatment of various tumors considered untreatable by current therapeutic modalities.

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Some original experimental work described in this article was supported by the Medical Research Service of the Veterans Affairs Department, an award from CaP CURE (Association for the Cure of Prostate Cancer), and grants from ASTA Medica to Tulane University School of Medicine (all to A.V.S.). Some early oncological studies originating in this laboratory and cited in this article were also supported by U.S. Public Health Service Grants CA-40003, CA-40077, and CA-40004.

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