Overexpression Of Gastrin-releasing Peptide Receptor Research Articles

Radiopharmaceuticals Targeting Gastrin-Releasing Peptide Receptor for Diagnosis and Therapy of Prostate Cancer.

The high incidence and heavy disease burden of prostate cancer (PC) require accurate and comprehensive assessment for appropriate disease management. Prostate-specific membrane antigen (PSMA) positron emission tomography (PET) cannot detect PSMA-negative lesions, despite its key role in PC disease management. The overexpression of gastrin-releasing peptide receptor (GRPR) in PC lesions reportedly performs as a complementary target for the diagnosis and therapy of PC. Radiopharmaceuticals derived from the natural ligands of GRPR have been developed. These radiopharmaceuticals enable the visualization and quantification of GRPR within the body, which can be used for disease assessment and therapeutic guidance. Recently developed radiopharmaceuticals exhibit improved pharmacokinetic parameters without deterioration in affinity. Several heterodimers targeting GRPR have been constructed as alternatives because of their potential to detect tumor lesions with a low diagnostic efficiency of single target detection. Moreover, some GRPR-targeted radiopharmaceuticals have entered clinical trials for the initial staging or biochemical recurrence detection of PC to guide disease stratification and therapy, indicating considerable potential in PC disease management. Herein, we comprehensively summarize the progress of radiopharmaceuticals targeting GRPR. In particular, we discuss the impact of ligands, chelators, and linkers on the distribution of radiopharmaceuticals. Furthermore, we summarize a potential design scheme to facilitate the advancement of radiopharmaceuticals and, thus, prompt clinical translation.

GRP/GRPR signaling pathway aggravates hyperuricemia-induced renal inflammation and fibrosis via ABCG2-dependent mechanisms

The gastrin-releasing peptide receptor (GRPR) binds to ligands such as gastrin-releasing peptide (GRP) and plays a variety of biological roles. In this study, we investigated the therapeutic effect of a novel gastrin-releasing peptide receptor antagonist RH-1402 in hyperuricemia-induced kidney fibrosis and its underlying mechanisms. We conducted enzyme linked immunosorbent assay (ELISA) and immunohistochemical analyses and found that proGRP and GRPR expression levels were significantly increased in patients with hyperuricemic nephropathy (HN) and HN mice. GRPR knockdown significantly attenuated inflammatory and fibrotic responses in adenosine-treated human proximal tubule epithelial cells. GRPR knockout or GRPR conditional knockout in renal tubular epithelial cells significantly alleviated the decline in renal function and fibrosis in HN mice in vivo. RNA-seq and String database analysis revealed that GRP/GRPR promoted HN by suppressing the ABCG2/PDZK1 and increasing TGF-β/Smad3 levels by activating the NF-κB pathway. Overexpression of GRPR increased TGF-β/Smad3 levels, where as it reduced ABCG2/PDZK1 levels in adenosine-treated HK2 cells, which was reversed by the NF-κB inhibitor. Furthermore, we evaluated the therapeutic effects of the novel GRPR inhibitor RH-1402 on hyperuricaemia-induced renal injury and evaluated the inflammatory and fibrosis responses in vivo and in vitro. Pre-treatment with RH-1402 attenuated hyperuricaemia-induced renal injury, restored renal function, and suppressed renal inflammation and fibrosis. Taken together, GRPR enhances hyperuricaemia-induced tubular injury, inflammation, and renal fibrosis via ABCG2-dependent mechanisms and may serve as a promising therapeutic target for HN treatment.

GRP/GRPR enhances alcohol-associated liver injury through the IRF1-mediated Caspase-1 inflammasome and NOX2-dependent ROS pathway.

The common characteristics of alcohol-associated liver injury (ALI) include abnormal liver function, infiltration of inflammatory cells, and generation of oxidative stress. The gastrin-releasing peptide receptor (GRPR) is activated by its neuropeptide ligand, gastrin-releasing peptide (GRP). GRP/GRPR appears to induce the production of cytokines in immune cells and promotes neutrophil migration. However, the effects of GRP/GRPR in ALI are unknown. We found high GRPR expression in the liver of patients with alcohol-associated steatohepatitis and increased pro-GRP levels in peripheral blood mononuclear cells of these patients compared with that of the control. Increased expression of GRP may be associated with histone H3 lysine 27 acetylation induced by alcohol, which promotes the expression of GRP and then GRPR binding. Grpr-/- and Grprflox/floxLysMCre mice alleviated ethanol-induced liver injury with relieved steatosis, lower serum alanine aminotransferase, aspartate aminotransferase, triglycerides, malondialdehyde, and superoxide dismutase levels, reduced neutrophil influx, and decreased expression and release of inflammatory cytokines and chemokines. Conversely, the overexpression of GRPR showed opposite effects. The pro-inflammatory and oxidative stress roles of GRPR might be dependent on IRF1-mediated Caspase-1 inflammasome and NOX2-dependent reactive oxygen species pathway, respectively. In addition, we verified the therapeutic and preventive effects of RH-1402, a novel GRPR antagonist, for ALI. A knockout or antagonist of GRPR during excess alcohol intake could have anti-inflammatory and antioxidative roles, as well as provide a platform for histone modification-based therapy for ALI.

64Cu-SAR-Bombesin PET-CT imaging in the staging of ER+/PR+/HER2- metastatic breast cancer: Safety, dosimetry, and feasibility in a phase I trial.

3092 Background: Breast cancers are most frequently oestrogen receptor (ER) and progesterone receptor (PR) positive and 18F-Fluorodeoxyglucose PET-CT (FDG) used in conventional staging of breast cancer has lower sensitivity for these subtypes. Gastrin releasing peptide receptors (GRPR) are a potential alternative diagnostic and theranostic target for ER+/PR+ breast cancers due to their overexpression of GRPR. This phase 1 study aims to assess the safety and potential of the novel radiotracer 64Cu-Sarcophagine(SAR)-Bombesin (BBN) in the re-staging of recurrent metastatic ER+/PR+/human epidermal growth factor 2 negative (HER2-) breast cancer. Methods: Patients with confirmed recurrent or primary metastatic ER+/PR+/HER2- breast cancer undergoing staging prior to a new treatment underwent 64Cu-SAR-BBN PET-CT with imaging at 1, 3 and 24 hours post-injection. Bloods and vital signs were acquired for patients at baseline, 1, 3 and 24 hours post-injection timepoints, and electrocardiogram (ECG) performed 1 hour pre and 1 hour post injection. Blood tracer-clearance and dosimetry was performed. GRPR receptor status was assessed in 4/7 patients from metastatic-site biopsy samples. Staging of the patients was assessed by conventional imaging (FDG, bone scan and diagnostic CT) within 3 weeks of 64Cu-SAR-BBN imaging. All PET scans were assessed visually, and quantitatively using MIM Software. Results: 9 patients were enrolled. 7/9 patients underwent all imaging modalities, while 2/9 did not undergo BBN imaging. 1/7 patient who underwent all imaging had de- novo metastatic ER+/PR+/Her 2- breast cancer and 6/7 recurrent metastatic disease. 2/7 had lobular subtype. There were no adverse events reported, and ECG, vitals and haematological, biochemical and coagulation markers remained unchanged. All 7 patients were positive on conventional imaging, while 6/7 were positive on FDG. BBN was positive in 5/7 patients. Both BBN negative patients had disease identified on FDG. Conversely, 1 patient was BBN positive but FDG negative. 4/7 patients were BBN positive and FDG positive. In these 4 patients, mean SUVmax was higher for BBN than FDG (15 vs. 12). In classical lobular subtype (2/7), BBN was highly avid compared to FDG (SUV max 20 vs 11, and 20 vs <3) and with a higher tumor volume compared to FDG (2034 vs 504, and 634 mL vs FDG negative). Conclusions: 64Cu-SAR-Bombesin is a novel tracer which appears safe and may have a diagnostic and theranostic role in patients with metastatic ER+/PR+/HER2- breast cancer, particularly lobular subtype. Further evaluation appears warranted.

Localization of 99mTc-GRP Analogs in GRPR-Expressing Tumors: Effects of Peptide Length and Neprilysin Inhibition on Biological Responses.

The overexpression of gastrin-releasing peptide receptors (GRPRs) in frequently occurring human tumors has provided the opportunity to use bombesin (BBN) analogs as radionuclide carriers to cancer sites for diagnostic and therapeutic purposes. We have been alternatively exploring human GRP motifs of higher GRPR selectivity compared to frog BBN sequences aiming to improve pharmacokinetic profiles. In the present study, we compared two differently truncated human endogenous GRP motifs: GRP(14–27) and GRP(18–27). An acyclic tetraamine was coupled at the N-terminus to allow for stable binding of the SPECT radionuclide 99mTc. Their biological profiles were compared in PC-3 cells and in mice without or with coinjection of phosphoramidon (PA) to induce transient neprilysin (NEP) inhibition in vivo. The two 99mTc-N4-GRP(14/18–27) radioligands displayed similar biological behavior in mice. Coinjection of PA exerted a profound effect on in vivo stability and translated into notably improved radiolabel localization in PC-3 experimental tumors. Hence, this study has shown that promising 99mTc-radiotracers for SPECT imaging may indeed derive from human GRP sequences. Radiotracer bioavailability was found to be of major significance. It could be improved during in situ NEP inhibition resulting in drastically enhanced uptake in GRPR-expressing lesions.

Expression of Gastrin-Releasing Peptide Receptor in Breast Cancer and Its Association with Pathologic, Biologic, and Clinical Parameters: A Study of 1,432 Primary Tumors.

A growing body of evidence suggests that gastrin-releasing peptide receptor (GRPR) might be a valuable target in breast cancer. To understand which patients can be potential candidates for GRPR-based imaging or targeted therapy, we screened invasive breast cancers by immunohistochemistry for the presence and intensity of GRPR expression. Methods: We explored a tissue microarray of 1,432 primary breast tumors from patients who underwent surgery between 2000 and 2005 at Institut Bergonié, without prior neoadjuvant treatment. We studied associations between GRPR expression and clinical, pathologic, and biologic parameters. The association between GRPR expression and distant metastasis-free interval was also examined. Results: GRPR overexpression was found in 75.8% of the 1,432 tumors and was most strongly associated with estrogen receptor (ER) positivity (GRPR was high in 83.2% of ER-positive and 12% of ER-negative tumors; P < 0.00001). When molecular subtypes of breast cancer were considered, GRPR was overexpressed in 86.2% of luminal A-like tumors, 70.5% of luminal B-like human epidermal growth factor receptor 2 (HER2)-negative tumors, 82.8% of luminal B-like HER2-positive tumors, 21.3% of HER2-enriched tumors, and 7.8% of triple-negative tumors. Importantly, when breast tumors overexpressed GRPR, high GRPR expression was also found in metastatic lymph nodes in 94.6% of cases. Primary tumors with high GRPR expression were associated with lower risk of distant metastases at follow-up in univariate analysis (Log-rank P = 0.0084) but not in multivariate analysis. Hence, the prognostic impact of GRPR was lost when examined within specific molecular subtypes. Conclusion: Because GRPR is overexpressed in a high percentage of ER-positive tumors, GRPR targeting offers wide perspectives for imaging and treatment in patients with ER-positive breast cancer, using recently developed radiolabeled GRPR ligands.

Synthesis of a novel long-circulating dual-receptor targeting and dual-modal molecular probe: an in-vitro study

Objective To explore a novel long-circulating dual-receptor targeting and dual-modal molecular probe and investigate its physicochemical properties and targeting effect on breast cancer cells in vitro. Methods Dual-receptor targeting and dual-modal molecular probe RGD@BBN-lipo(QDs)-SPIO was synthesized in the following steps: long-circulating liposome was prepared by film dispersion method; water-soluble superparamagnetic iron oxide (SPIO) nanoparticles and Quantum dots (QDs) were loaded in the hydrophilic and hydrophobic layer of liposome, respectively; RGD and BBN polypeptides were coupled on the former functional magnetic/fluorescent liposomes. Stability of the probe in different physiological solutions was investigated. Transmission electron microscopy (TEM) and particle size analyzer were used to measure nanoparticle sizes and the Zeta potential. Characterization of RGD and BBN was investigated through 1H-NMR and elemental analysis. The MRI T2 relaxivities (1/T2) of RGD@BBN-lipo(QDs)-SPIO was measured through T2 map scanning on 3.0 T MR system. HUV-EC-C cells were used for assessment of cells viability by MTS assay. Prussian blue staining and fluorescence imaging were carried out to determine the targeted breast cellular uptake of RGD@BBN-lipo(QDs)-SPIO nanoparticles. Results The targeting magnetic/fluorescent dual-model molecular probes appeared spherical or para-spherical, with a mean diameter of (118.2±3.9) nm,Zeta potential of (-24.78±1.68) mV,MR T2 magnetic relaxation rate of 0.498 1× 106 M -1·s -1.RGD and BBN polypeptides were successfully coupled on the former functionally magnetic/fluorescent liposomes with the bind rates of 33.05 % and 45.06 %, respectively. There was low cytotoxity of the molecular probe on human umbilical vein endothelical cells (HUV-EC-C) by MTS study. Prussian blue staining and fluorescence imaging studies showed that the RGD@BBN-lipo(QDs)-SPIO nanoparticles could target any αvβ3 or gastrin releasing peptide receptor overexpression breast cancer. Conclusions RGD@BBN-lipo (QDs)-SPIO is a novel long-circulating dual-receptor targeting and dual-modal molecular probe and has excellent physicochemical properties and stability, high T2 relaxivities and strong targeting effect on cancer cells and has laid a solid foundation for early diagnosis of breast cancer. Key words: Molecular probes; Magnetic resonance imaging; Breast neoplasms

Uncovering potential downstream targets of oncogenic GRPR overexpression in prostate carcinomas harboring ETS rearrangements.

Gastrin-releasing peptide receptor (GRPR) is known to be overexpressed in several human malignancies, including prostate cancer, and has been implicated in multiple important neoplastic signaling pathways. We recently have shown that GRPR is an ERG and ETV1 target gene in prostate cancer, using a genome-wide scale and exon-level expression microarray platform. Due to its cellular localization, the relevance of its function and the availability of blocking agents, GRPR seems to be a promising candidate as therapeutic target. Our present work shows that effective knockdown of GRPR in LNCaP and VCaP cells attenuates their malignant phenotype by decreasing proliferation, invasion and anchorage-independent growth, while increasing apoptosis. Using an antibody microarray we were able to validate known and identify new targets of GRPR pathway, namely AKT1, PKCε, TYK2 and MST1. Finally, we show that overexpression of these GRPR targets is restricted to prostate carcinomas harboring ERG and/or ETV1 rearrangements, establishing their potential as therapeutic targets for these particular molecular subsets of the disease.

The effect of mini-PEG-based spacer length on binding and pharmacokinetic properties of a 68Ga-labeled NOTA-conjugated antagonistic analog of bombesin.

The overexpression of gastrin-releasing peptide receptor (GRPR) in cancer can be used for peptide-receptor mediated radionuclide imaging and therapy. We have previously shown that an antagonist analog of bombesin RM26 conjugated to 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA) via a diethyleneglycol (PEG2) spacer (NOTA-PEG2-RM26) and labeled with 68Ga can be used for imaging of GRPR-expressing tumors. In this study, we evaluated if a variation of mini-PEG spacer length can be used for optimization of targeting properties of the NOTA-conjugated RM26. A series of analogs with different PEG-length (n = 2, 3, 4, 6) was synthesized, radiolabeled and evaluated in vitro and in vivo. The IC50 values of natGa-NOTA-PEGn-RM26 (n = 2, 3, 4, 6) were 3.1 ± 0.2, 3.9 ± 0.3, 5.4 ± 0.4 and 5.8 ± 0.3 nM, respectively. In normal mice all conjugates demonstrated similar biodistribution pattern, however 68Ga-NOTA-PEG3-RM26 showed lower liver uptake. Biodistribution of 68Ga-NOTA-PEG3-RM26 was evaluated in nude mice bearing PC-3 (prostate cancer) and BT-474 (breast cancer) xenografts. High uptake in tumors (4.6 ± 0.6%ID/g and 2.8 ± 0.4%ID/g for PC-3 and BT-474 xenografts, respectively) and high tumor-to-background ratios (tumor/blood of 44 ± 12 and 42 ± 5 for PC-3 and BT-474 xenografts, respectively) were found already at 2 h p.i. of 68Ga-NOTA-PEG3-RM26. Results of this study suggest that variation in the length of the PEG spacer can be used for optimization of targeting properties of peptide-chelator conjugates. However, the influence of the mini-PEG length on biodistribution is minor when di-, tri-, tetra- and hexaethylene glycol are compared.

FAK is a Critical Regulator of Neuroblastoma Liver Metastasis

Neuroblastomas express increased levels of gastrin-releasing peptide receptor (GRP-R). However, the exact molecular mechanisms involved in GRP-R-mediated cell signaling in neuroblastoma growth and metastasis are unknown. Here, we report that focal adhesion kinase (FAK), as a critical downstream target of GRP-R, is an important regulator of neuroblastoma tumorigenicity. We found that FAK expression correlates with GRP-R expression in human neuroblastoma sections and cell lines. GRP-R overexpression in SK-N-SH cells increased FAK, integrin α3 and β1 expressions and cell migration. These cells demonstrated flatter cell morphology with broad lamellae, in which intense FAK expression was localized to the leading edges of lamellipodia. Interestingly, FAK activation was, in part, dependent on integrin α3 and β1 expression. Conversely, GRP-R silencing decreased FAK as well as Mycn levels in BE(2)-C cells, which displayed a denser cellular morphology. Importantly, rescue experiments in GRP-R silenced BE(2)-C cells showed FAK overexpression significantly enhanced cell viability and soft agar colony formation; similarly, FAK overexpression in SK-N-SH cells also resulted in increased cell growth. These effects were reversed in FAK silenced BE(2)-C cells in vitro as well as in vivo. Moreover, we evaluated the effect of FAK inhibition in vivo. FAK inhibitor (Y15) suppressed GRP-induced neuroblastoma growth and metastasis. Our results indicate that FAK is a critical downstream regulator of GRP-R, which mediates tumorigenesis and metastasis in neuroblastoma.

Abstract 169: Inhibition of focal adhesion kinase suppresses gastrin-releasing peptide receptor-mediated liver metastasis in neuroblastoma

Abstract We have previously reported that high expression of gastrin-releasing peptide receptors (GRP-R), a member of the G-protein coupled receptor family, is observed in more undifferentiated human neuroblastoma. We have also found that GRP-R-mediated cell signaling plays a critical role in neuroblastoma cell transformation; however, its molecular mechanisms are unknown. Here, we sought to determine the role of focal adhesion kinase (FAK), a non-receptor protein tyrosine kinase, in GRP-R-mediated neuroblastoma cellular transformation. METHODS. Using human neuroblastoma tissue sections, the correlation of GRP-R and FAK expression was examined. Human neuroblastoma cell line, BE(2)-C and SK-N-SH were used for our experiments. Using transfection with Lipofectamine 2000, the level of knockdown and overexpression of GRP-R and FAK were modulated. MTT assay was carried out to measure the cell viability of neuroblastoma cells, and soft agar colony formation assay was performed to determine the capacity of cell transformation. Transwell migration and wound healing assay were employed to study cell motility. Rescue experiments by overexpression of FAK in GRP-R silencing cells were conducted to further validate the role of FAK in neuroblastoma cell transformation. Using FAK inhibitor and GRP-R agonist, animal experiment was performed. RESULTS. FAK expression correlated with GRP-R expression in BE(2)-C and SK-N-SH human neuroblastoma cells. GRP treatment induced phosphorylation of FAK at Y397 in both cell lines. Overexpression of GRP-R in SK-N-SH cells, which are known to express low endogenous levels of GRP-R, increased the levels of phosphorylated and total FAK. Moreover, overexpression of FAK increased cell viability and colony formation of SK-N-SH cells. These effects were reversed when cells were co-transfected with FAK siRNA in BE(2)-C cells, which endogenously express higher levels of GRP-R. Conversely, silencing of GRP-R decreased the levels of phosphorylated and total FAK in BE(2)-C cells. Interestingly, in cells with GRP-R silencing, FAK overexpression significantly enhanced cell viability and colony formation. GRP-R-mediated liver metastasis suppressed by FAK inhibitor in vivo. CONCLUSIONS. Our results suggest that FAK is a critical downstream regulator of GRP-R-mediated tumorigenesis. Furthermore, GRP-R and FAK expression correlate with morphological changes implicated in the malignant transformation of human neuroblastoma cells. Importantly, inhibition of FAK can suppress GRP-R-induced tumor progression in neuroblastoma in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 169. doi:1538-7445.AM2012-169

Small-Animal PET of Tumors with 64Cu-Labeled RGD-Bombesin Heterodimer

The overexpression of gastrin-releasing peptide receptor (GRPR) in various tumor types suggests that GRPR is an attractive target for cancer imaging and therapy with radiolabeled bombesin analogs. We recently reported the ability of (18)F-labeled RGD-bombesin heterodimer to be used for dual integrin alpha(v)beta(3)- and GRPR-targeted imaging. To further investigate the synergistic effect of the dual-receptor targeting of peptide heterodimers, we evaluated (64)Cu-labeled RGD-bombesin for PET imaging of tumors. RGD-bombesin was coupled with 1,4,7,10-tetraazacyclododecane-N, N', N'', N'''-tetraacetic acid (DOTA) and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and the conjugates were labeled with (64)Cu. The in vitro and in vivo characteristics of (64)Cu-NOTA-RGD-bombesin were compared with those of (64)Cu-NOTA-RGD, (64)Cu-NOTA-bombesin, and (64)Cu-DOTA-RGD-bombesin. (64)Cu-NOTA-RGD-bombesin and (64)Cu-DOTA-RGD-bombesin had comparable dual integrin alpha(v)beta(3)- and GRPR-binding affinities in vitro, both of which were slightly lower than RGD for integrin binding and bombesin for GRPR binding. (64)Cu-NOTA-RGD-bombesin possessed significantly higher tumor uptake than did (64)Cu-NOTA-RGD, (64)Cu-NOTA-bombesin, the mixture of (64)Cu-NOTA-RGD and (64)Cu-NOTA-bombesin, or (64)Cu-DOTA-RGD-bombesin in PC-3 prostate cancer. (64)Cu-NOTA-RGD-bombesin also showed improved in vivo kinetics such as lower liver and intestinal activity accumulation than did the bombesin tracers. (64)Cu-NOTA-RGD-bombesin also outperformed (64)Cu-NOTA-RGD in a 4T1 murine mammary carcinoma model that expresses integrin on tumor vasculature but no GRPR in tumor tissue, which had no uptake of (64)Cu-NOTA-bombesin. Compared with other tracers, (64)Cu-NOTA-RGD-bombesin showed favorable in vivo kinetics and enhanced tumor uptake, which warrants its further investigation for targeting tumors that express integrin or GRPR or that coexpress integrin and GRPR for imaging and therapeutic applications. The synergistic effect of RGD-bombesin heterodimers observed in this study also encourages further investigations of novel heterodimers recognizing other cell surface receptors for tumor targeting.

Overexpression of Gastrin-Releasing Peptide Receptors in Tumor-Associated Blood Vessels of Human Ovarian Neoplasms

Background: Peptide receptors, overexpressed in specific cancers, represent new diagnostic and therapeutic targets. In this study, receptors for the gastrin-releasing peptide (GRP), and other members of the bombesin-family of peptides, were evaluated in ovarian neoplasms. Methods: 75 primary, secondary and metastatic ovarian tumors were investigated for their bombesin-receptor subtype expression, incidence, localization and density using in vitro autoradiography on tissue sections with the universal radioligand I-[D-Tyr, ß-Ala, Phe, Nle14]-bombesin(6-14) and the GRP-receptor subtype-preferring I-[Tyr]-bombesin. Results: GRP-receptors were detected in 42/61 primary ovarian tumors; other bombesin-receptor subtypes (BB1, bb3) were rarely present (3/61). Two different tissue compartments expressed GRP-receptors: the tumoral vasculature was the predominant site of GRP-receptor expression (38/61), whereas neoplastic cells more rarely expressed GRP-receptors (14/61). GRP-receptor positive vessels were present in the various classes of ovarian tumors; generally, malignant tumors had a higher incidence of GRP-receptor positive vessels compared to their benign counterparts. The prevalence of such vessels was particularly high in ovarian carcinomas (16/19) and their metastases (5/5). The GRP-receptors were expressed in high density in the muscular vessel wall. Normal ovary (n=10) lacked GRP-receptors. Conclusions: The large amounts of GRP-receptors in ovarian tumor vessels suggest a role in tumoral vasculature and possibly angiogenesis. Further, these vessels might be targeted in vivo with bombesin analogs for diagnosis or for therapy.

Comparative in vitro and in vivo evaluation of two 64Cu-labeled bombesin analogs in a mouse model of human prostate adenocarcinoma

Bombesin (BBN), an analog of human gastrin-releasing peptide (GRP), binds to the GRP receptor (GRPR) with high affinity and specificity. Overexpression of GRPR has been discovered in mostly androgen-independent human prostate tissues and, thus, provides a potential target for prostate cancer diagnosis and therapy. We have previously demonstrated the feasibility of the positron emission tomography (PET) imaging using 64Cu-1,4,7,10-tetraazadodecane-N,N',N'',N'''-tetraacetic acid (DOTA)-[Lys3]BBN to detect GRPR-positive prostate cancer. In this study, we compared the receptor affinity, metabolic stability, tumor-targeting efficacy, and pharmacokinetics of a truncated BBN analog 64Cu-DOTA-Aca-BBN(7-14) with 64Cu-DOTA-[Lys3]BBN. Binding of each DOTA conjugate to GRPR on PC-3 and 22Rv1 prostate cancer cells was evaluated with competitive binding assay using 125I-[Tyr4]BBN as radioligand. In vivo pharmacokinetics was determined on male nude mice subcutaneously implanted with PC-3 cells. Dynamic microPET imaging was performed to evaluate the systemic distribution of the tracers. Metabolic stability of the tracers in blood, urine, tumor, liver and kidney was studied using high-performance liquid chromatography. The results showed that 125I-[Tyr4]BBN has a K(d) of 14.8+/-0.4 nM against PC-3 cells, and the receptor concentration on PC-3 cell surface is approximately 2.7+/-0.1 x 10(6) receptors per cell. The 50% inhibitory concentration value for DOTA-Aca-BBN(7-14) is 18.4 +/- 0.2 nM, and that for DOTA-[Lys3]BBN is 2.2 +/- 0.5 nM. DOTA-[Lys3]BBN shows a better tumor contrast and absolute tumor activity accumulation compared to DOTA-Aca-BBN(7-14). Studies on metabolic stability for both tracers on organ homogenates showed that 64Cu-DOTA-[Lys3]BBN is relatively stable. This study demonstrated that both tracers are suitable for targeted PET imaging to detect the expression of GRPR in prostate cancer, while 64Cu-DOTA-[Lys3]BBN may have a better potential for clinical translation.

Overexpression of gastrin-releasing peptide receptor upregulates integrin expression in neuroblastoma cells

Gastrin-releasing peptide (GRP) acts as an autocrine growth factor for neuroblastoma cells. Overexpression of the GRP receptor (GRP-R), which increases binding capacity for its ligand GRP, has been shown to stimulate cell growth; however, the exact role of GRP on tumor invasion/metastatic properties is unknown. Integrin is a heterodimer composed of α- and β-subunits; 24 integrin isoforms exist, from different pairings among 18 α- and 8 β-subunits. Integrins bind to components of extracellular matrix (ECM) such as collagen, fibronectin, and laminin to mediate cell adhesion, spread, or migration. The purpose of our study was to investigate the effect of GRP on integrin expression in neuroblastoma cells. Methods: To determine the effects of autocrine growth factor, GRP, on integrin expression, human neuroblastoma cell line, SK-N-SH, was stably transfected with pEGFP (control vector) and pEGFP-GRP-R plasmids. We then performed a metastasis specific gene array (SuperArray) assay. The expression of ECM receptor integrins was analyzed by Western blot analysis. The expression of cell membrane integrin protein levels was determined by fluorescence-activated cell sorter analysis (FACS). Results: Human neuroblastoma cells overexpressing GRP-R, which we had previously shown to increase GRP binding capacity, demonstrated marked changes in their cell morphology; cells appeared flat and more adhesive to culture plates. Gene array showed increased expression of integrins β1, α2, and α3 when compared to control cells. Consistent with these data, Western blot analysis showed increased protein expression of integrins β1, α2, and α3 in GRP-R overexpressing cells. These findings were further confirmed by FACS, where GRP-R overexpression resulted in increased expression of functional cell surface membrane integrins β1, α2, and α3. Conclusions: GRP-R overexpression resulted in enhanced mRNA and protein expression of integrins, suggesting that GRP-induced neuroblastoma cell proliferation may also enhance factors responsible for tumor invasion/metastatic potential. A better understanding of this process may allow us to develop novel therapy targeted at inhibiting GRP-induced neuroblastoma cell proliferation and metastasis.

Overexpression of gastrin-releasing peptide receptor induced layer disorganization in brain

Gastrin-releasing peptide-preferring and neuromedin B-preferring receptors, members of the bombesin-like peptide receptor subfamily, are reported to regulate proliferation, migration and differentiation. Since they are expressed in developing brain, we postulated that the gastrin-releasing peptide-preferring and neuromedin B-preferring receptors might be involved in normal brain development. Here we examined the effects of the overexpressions of the gastrin-releasing peptide-preferring and neuromedin B-preferring receptors on chick brain development in vivo using a retrovirus. In the overexpressed exogenous gastrin-releasing peptide-preferring receptor brain, we found laminar disorganization in the telencephalon, tectum and particularly in the cerebellum with severe atrophy. Processes of the radial glial cells in the telencephalon and optic tectum, as well as the projections of the Bergmann glia in the cerebellum were distorted, which might disturb normal cell migration. Despite the atrophy of the cerebellum, densely-stained proliferating cell nuclear antigen- and phospho-histone H3-positive cells increased in number. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells also increased in the cerebellum, suggesting that the ectopically proliferating cells were subjected to apoptosis. Glial fibrillary acidic protein-positive cells also increased in the hyperpallium accessorium and in the outer layers of the tectum. We also found smaller and spindle-shaped cells which resembled undifferentiated embryonic tumor cells. On the other hand, the layer structures of the neuromedin B-preferring receptors overexpressed brain were well organized and developed, and the size of brain was generally enlarged. These results indicated that although the gastrin-releasing peptide-preferring and neuromedin B-preferring receptors are involved in normal brain development, both receptors contribute and exert their effects differently.