Elsevier

Regulatory Peptides

Volume 88, Issues 1–3, 17 March 2000, Pages 61-73
Regulatory Peptides

Somatostatin receptor gene expression in neuroblastoma

https://doi.org/10.1016/S0167-0115(99)00121-4Get rights and content

Abstract

Somatostatin receptor expression is a favorable prognostic factor in human neuroblastoma. Somatostatin receptors have been demonstrated in vitro by pharmacologic analysis of tumor tissue and in vivo by diagnostic radioreceptor scintigraphy. However, which receptor subtypes (sst1, sst2, sst3, sst4, and sst5) are expressed in these tumors has not yet been delineated. We used RT-PCR to analyze expression of the five somatostatin receptor genes in 32 neuroblastoma tumor specimens. All 32 tumor specimens expressed mRNA for c-abl and sst1; sst2 mRNA was detected in 27/32 samples and somatostatin mRNA was detected in 30/32 tumor specimens. The remaining receptor subtypes, sst3, sst4, and sst5 were variably expressed. Receptor protein for sst1 and sst2 was visualized in tumor neuroblasts as well as in endothelial cells of tumor vessels using immunostaining with specific anti-receptor antibodies. The effect of high expression of somatostatin receptors on cell proliferation was examined in SKNSH neuroblastoma cells transfected with sst1 and sst2. SS14 binding to wild-type SKNSH cells was undetectable; but the native peptide bound with high affinity to the SKNSH/sst1 and SKNSH/sst2 neuroblastoma cell lines. Pharmacologic analysis of binding with two long-acting analogues, CH275 and octreotide, confirmed selective expression of sst1 and sst2 in stably transfected SKNSH cells. Formation of neuroblastoma xenograft tumors in nude mice was significantly delayed for both SKNSH/sst1 (P<0.001) and SKNSH/sst2 (P<0.05) cells compared to wild-type SKNSH. We conclude that: (1) Somatostatin receptors, sst1 and sst2, are expressed in the majority of neuroblastomas at diagnosis; and (2) upregulation of functional sst1 or sst2 in neuroblastoma cell lines suppresses tumorigenicity in a xenograft model. These observations suggest that somatostatin receptors may be a useful therapeutic target in neuroblastoma.

Introduction

Neuroblastoma is a malignant tumor which arises in the peripheral nervous system during early childhood. The malignant cells may be either neuroblasts or Schwann cells, both of which are derivatives of the neural crest [1]. Neuroblastoma tumors have variable outcomes; stage IV-S tumors may spontaneously regress, while stage I tumors differentiate to ganglioneuroma. Stage II and most stage III neuroblastoma tumors can be cured with surgery and chemotherapy, while stage IV tumors are more aggressive, often becoming resistant to chemo- and radiotherapies. Recent investigations suggest that migration of normal Schwann cells into the tumors may contribute to the spontaneous regression and/or maturation of some neuroblastomas [2].

Currently used prognostic indicators in neuroblastoma include Shimada classification [3], MYCN amplification, patient age, and stage [4]. Several laboratories, including our own, have recently investigated the use of neuropeptide levels as prognostic factors in neuroblastoma [5]. Increased plasma levels of NPY have been demonstrated to correlate with a rapidly growing tumor and to predict early relapse [6]. Increased NPY mRNA expression has been demonstrated in stage IVS tumors [7]. Neuropeptides, including NPY, VIP and somatostatin are thought to play a role in neural crest differentiation. Tyrrell and Landis recently studied NPY and VIP in embryonic neurons, where transcriptional repression of peptide gene expression plays a role in the final neuropeptide profile of sympathetic neurons [8]. During embryonic development, NPY and VIP are detected in dividing neuroblasts and in postmitotic neurons of the rat superior cervical and stellate ganglial [8]. High plasma or tumor levels of VIP and somatostatin correlate with neuroblast differentiation and are favorable prognostic indicators [5].

Expression of somatostatin receptors has also been identified as a favorable prognostic indicator in neuroblastoma [9]. Somatostatin receptor (sst) expression has been demonstrated in neuroblastoma tumor tissue by several techniques, including scintigraphy [10], competitive binding to tumor membranes [11], ex vivo autoradiography of excised tumor tissue [12] and in vivo radioreceptor guided surgery [13]. Moertel et al. demonstrated a positive correlation between somatostatin receptor expression and a favorable prognosis; in addition, these investigators observed an inverse correlation between somatostatin receptors and MYCN amplification [12]. Similarly, Chen et al., demonstrated a positive correlation between high affinity binding of 125I-SS14 to tumor membranes, low clinical stage, and survival in children with neuroblastoma [9].

Five genes for somatostatin receptors (sst1–5) have recently been cloned [14], [15], [16], [17], [18], [19]; however, which receptor subtypes are expressed in neuroblastoma has not yet been delineated. Accordingly, our purpose was two-fold, first to analyze the expression of the five receptor subtype genes in neuroblastoma tissue and second to study the effect of expression of specific receptor subtypes on neuroblastoma cell growth in vivo. The results have important implications for the design of diagnostic and therapeutic protocols utilizing somatostatin analogues in neuroblastoma.

Section snippets

Tumor procurement

Human neuroblastoma tumor specimens were obtained through the Cooperative Human Tissue Network (CHTN). Tissues had been obtained at the time of biopsy and frozen to −80°C within 60 min of extraction. All available patient data, including age at diagnosis, sex, site of primary tumor, stage, Shimada classification, DNA analysis for MYCN content, and survival (Table 1) were provided by CHTN in accordance with guidelines established by the National Cancer Institute and the Children’s Hospital

Somatostatin receptor expression in human neuroblastoma tumors

Thirty-two neuroblastoma tumor specimens were provided by the CHTN (Table 1). The age of patients at diagnosis ranged from 1 day to 8 years. Tumor distribution was 50% adrenal, 19% mediastinal, 12% retroperitoneal, and 6% abdominal which is consistent with the frequency of primary sites in neuroblastoma [4]. Clinical staging is also indicated in Table 1: 50% were stage IV, 28% were stage III, 18% were stages I/II, and 1 patient was stage IV-S. Shimada classification was 41% unfavorable, 19%

Discussion

Our work presents the first molecular analysis of sst expression in human neuroblastoma tumors. We analyzed the expression of the somatostatin peptide gene as well as expression of five somatostatin receptors in 32 tumors from patients with neuroblastoma. All tumors analyzed expressed sst1 while 27/32 expressed sst2.

The lack of expression of sst2 in 16% of the tumor samples analyzed closely approximates the 15% false negative rate of somatostatin receptor based scintigraphy observed in

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