Gastrin-releasing Peptide Receptor Expression Research Articles

How gastrin-releasing peptide receptor (GRPR) and \u03b1v\u03b23 integrin expression reflect reorganization features of tumors after hyperthermia treatments

The outcome of tumor treatment via hyperthermia in the clinic has been reported to be heterogeneous. Here, we assessed how the presence of gastrin-releasing peptide receptor (GRPR) and αvβ3 integrin together with the morphology of the vascularization reflects the growth behavior of tumors after hyperthermia treatment. MDA-MB-231 tumor bearing mice were treated either with high (46 °C) or low dose (42 °C) water hyperthermia for 60 min. Changes of GRPR and αvβ3 integrin expression were assessed via multiplexed optical imaging. Vascularization was reconstructed and quantified by µCT imaging after contrast agent injection. We found that high dose hyperthermia is capable of increasing the expression of GRPR, αvβ3 integrin, CD31, and Ki67 in tumors. Also the morphology of tumor vasculature changed (increased relative blood volume and small-diameter vessel density, decreased expression of α-SMA). Low dose hyperthermia induced comparatively moderate effects on the investigated protein expression pattern and vascular remodeling. We conclude that under defined circumstances, specific temperature doses affect the reorganization of tumor regrowth, which is triggered by residual “dormant” cells even though tumor volumes are transiently decreasing. Further on, GRPR, αvβ3 integrin expression are versatile tools to surveil potential tumor regrow during therapy, beyond the conventional determination of tumor volumes.

Expression of IL-22 in the Skin Causes Th2-Biased Immunity, Epidermal Barrier Dysfunction, and Pruritus via Stimulating Epithelial Th2 Cytokines and the GRP Pathway.

Increased expression of Th22 cytokine IL-22 is a characteristic finding in atopic dermatitis (AD). However, the specific role of IL-22 in the pathogenesis of AD in vivo has yet to be elucidated. Consistent with observations in human AD, IL-22 was significantly increased in the AD skin of mice after epicutaneous sensitization to house dust mite allergen. Utilizing a skin-specific inducible transgenic system, we show in the present study that expression of IL-22 in the skin of mice caused an AD-like phenotype characterized by chronic pruritic dermatitis associated with Th2-biased local and systemic immune responses, downregulation of epidermal differentiation complex genes, and enhanced dermatitis upon epicutaneous allergen exposure. IL-22 potently induced the expression of gastrin-releasing peptide (GRP), a neuropeptide pruritogen, in dermal immune cells and sensory afferents and in their skin-innervating sensory neurons. IL-22 also differentially upregulated the expression of GRP receptor (GRPR) on keratinocytes of AD skin. The number of GRP+ cells in the skin correlated with the AD severity and the intensity of pruritus. IL-22 directly upregulated the expression of epithelial-derived type 2 cytokines (thymic stromal lymphopoietin and IL-33) and GRP in primary keratinocytes. Furthermore, GRP not only strongly induced thymic stromal lymphopoietin but it also increased the expression of IL-33 and GRPR synergistically with IL-22. Importantly, we found that the expression of GRP was strikingly increased in the skin of patients with AD. These results indicate that IL-22 plays important pathogenic roles in the initiation and development of AD, in part through inducing keratinocyte production of type 2 cytokines and activation of the GRP/GRPR pathway.

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.

High Contrast PET Imaging of GRPR Expression in Prostate Cancer Using Cobalt-Labeled Bombesin Antagonist RM26.

High gastrin releasing peptide receptor (GRPR) expression is associated with numerous cancers including prostate and breast cancer. The aim of the current study was to develop a 55Co-labeled PET agent based on GRPR antagonist RM26 for visualization of GRPR-expressing tumors. Labeling with 57Co and 55Co, stability, binding specificity, and in vitro and in vivo characteristics of 57Co-NOTA-PEG2-RM26 were studied. NOTA-PEG2-RM26 was successfully radiolabeled with 57Co and 55Co with high yields and demonstrated high stability. The radiopeptide showed retained binding specificity to GRPR in vitro and in vivo. 57Co-NOTA-PEG2-RM26 biodistribution in mice was characterized by rapid clearance of radioactivity from blood and normal non-GRPR-expressing organs and low hepatic uptake. The clearance was predominantly renal with a low degree of radioactivity reabsorption. Tumor-to-blood ratios were approximately 200 (3 h pi) and 1000 (24 h pi). The favorable biodistribution of cobalt-labeled NOTA-PEG2-RM26 translated into high contrast preclinical PET/CT (using 55Co) and SPECT/CT (using 57Co) images of PC-3 xenografts. The initial biological results suggest that 55Co-NOTA-PEG2-RM26 is a promising tracer for PET visualization of GRPR-expressing tumors.

Shriver SP, Bourdeau HA, Gubish CT, Tirpak DL, Davis AL, Luketich JD, Siegfried JM. Sex-specific expression of gastrin-releasing peptide receptor : relationship to smoking history and risk of lung cancer. J Natl Cancer Inst 2000 ; 92 : 24-33

Shriver SP, Bourdeau HA, Gubish CT, Tirpak DL, Davis AL, Luketich JD, Siegfried JM. Sex-specific expression of gastrin-releasing peptide receptor : relationship to smoking history and risk of lung cancer. J Natl Cancer Inst 2000 ; 92 : 24-33

Targeting Gastrointestinal Stromal Tumor with 68Ga-Labeled Peptides: An In Vitro Study on Gastrointestinal Stromal Tumor-Cell Lines.

The gastrointestinal stromal tumor (GIST) is a rare disease with limited therapeutic options when resistance to tyrosine kinase inhibitor (TKI) treatment occurs. The authors investigated binding of various 68Ga-labeled peptides, targeting receptors reported to be overexpressed in GIST, in different cell lines. For this purpose, three GIST cell lines were tested: GIST-T1, GIST882 (Imatinib sensitive), and GIST430 (Imatinib resistant). DOTA-NT 8-13 (targeting NTR1), DOTA-TATE (targeting SSTR2), CP04 (a minigastrin derivative targeting CCK2-R), VIP-DOTA (targeting VPAC2-R), and 2 DOTA-bombesin derivatives [targeting gastrin releasing peptide receptors (GRPR)] were radiolabeled with 68Ga and incubated with the respective tumor cell and control cell lines. Membrane-bound and internalized activity was measured. Very low or no specific binding to GIST cells was found for all 68Ga-labeled DOTA peptides except for bombesin derivatives indicating no or very low expression of respective receptors. Related to GRPR a pronounced specific binding to all GIST cell lines with no preference related to TKI resistance status was found, both for an agonist (AMBA) with high internalization and for an antagonist (NeoBOMB1) with mainly membrane-bound activity (with up to >80% bound/mg protein). GRPR expression was confirmed by immunohistochemistry. The results show that radiolabeled bombesin analogues, especially antagonists are very promising candidates for targeting GIST.

Investigation of bombesin peptide as a targeting ligand for the gastrin releasing peptide (GRP) receptor

Gastrin releasing peptide (GRP) receptor (GRPR), a bombesin family receptor, is overexpressed in many cancers including breast, prostate, pancreatic and lung. The targeting of therapeutics to GRPR can be achieved using the full-length (14 amino acid) GRP analogue Bombesin (BBN) or the truncated BBN(6–14) sequence, both of which bind GRPR with high affinity and specificity. In this study, we have investigated the level of GRPR expression in various cancerous (Caco-2, HeLa, LNCap, MDA-MB-231, and PC-3) and non-cancerous (WPMY-1) cell lines using a western blotting approach. Such information is currently lacking in the literature, and is therefore of importance for the in vitro assessment of GRPR targeted therapeutics. Of the cell lines assessed, the PC-3 (prostate cancer) and Caco-2 (colon cancer) cell lines demonstrated the highest and lowest levels of GRPR expression respectively. Using this information, we further investigated the cellular uptake of carboxyfluorescein-labelled BBN and BBN(6–14) peptides by flow cytometry and confocal microscopy using cell lines that express GRPR (Caco-2, HeLa, PC-3). The uptake of each of these peptides was similar, suggesting that the shorter BBN(6–14) peptide is sufficient for GRPR targeting. Further, the uptake of these peptides could be inhibited by competition with unlabelled BBN peptides, suggesting their cellular uptake is GRPR-mediated, while the level of BBN uptake (as measured by flow cytometry) was found to be directly proportional to the level of GRPR expression. Overall, the information obtained from these studies provides useful information for the in vitro assessment of GRPR targeted therapeutics.

Dual-Modality Imaging of Prostate Cancer with a Fluorescent and Radiogallium-Labeled Gastrin-Releasing Peptide Receptor Antagonist.

We designed and synthesized an IRDye 650 and DOTA-conjugated GRPr antagonist, HZ220 (DOTA-Lys(IRDye 650)-PEG4-[D-Phe6, Sta13]-BN(6-14)NH2), by reacting DOTA-Lys-PEG4-[D-Phe6, Sta13]-BN(6-14)NH2 (HZ219) with IRDye 650 N-hydroxysuccinimide (NHS) ester. Receptor-specific binding of gallium-labeled HZ220 was characterized in PC-3 prostate cancer cells (PC-3), and tumor uptake in mice was imaged with PET/CT and fluorescence imaging. Receptor binding affinity, in vivo tumor uptake, and biodistribution were compared with the GRPr antagonists HZ219, DOTA-PEG4-[D-Phe6, Sta13]-BN(6-14)NH2 (DOTA-AR), and DOTA-(4-amino-1-carboxymethyl-piperidine)-[D-Phe6, Sta13]-BN(6-14)NH2 (DOTA-RM2). After hydrophilic-lipophilic balance cartridge purification, 68Ga-HZ220 was obtained with a radiochemical yield of 56% ± 8% (non-decay-corrected), and the radiochemical purity was greater than 95%. Ga-HZ220 had a lower affinity for GRPr (inhibitory concentration of 50% [IC50], 21.4 ± 7.4 nM) than Ga-DOTA-AR (IC50, 0.48 ± 0.18 nM) or Ga-HZ219 (IC50, 0.69 ± 0.18 nM). Nevertheless, 68Ga-HZ220 had an in vivo tumor accumulation similar to 68Ga-DOTA-AR (4.63 ± 0.31 vs. 4.07 ± 0.29 percentage injected activity per mL [%IA/mL] at 1 h after injection) but lower than that of 68Ga-DOTA-RM2 (10.4 ± 0.4 %IA/mL). The tumor uptake of 68Ga-HZ220 was blocked significantly with an excessive amount of GRP antagonists. IVIS spectrum imaging also visualized PC-3 xenografts in vivo and ex vivo with a high-contrast ratio. Autoradiography and fluorescent-based microscopic imaging with 68Ga-HZ220 consistently colocated the expression of GRPr. 68Ga-HZ220 displayed a higher kidney uptake than both 68Ga-DOTA-AR and 68Ga-DOTA-RM2 (16.9 ± 6.5 vs. 4.48 ± 1.63 vs. 5.01 ± 2.29 %IA/mL). 68Ga-HZ220 is a promising bimodal ligand for noninvasive PET imaging and intraoperative optical imaging of GRPr-expressing malignancies. Bimodal nuclear/fluorescence imaging may not only improve cancer detection and guide surgical resections, but also improve our understanding of the uptake of GRPr ligands on the cellular level.

NPR-9 regulates the innate immune response in Caenorhabditis elegans by antagonizing the activity of AIB interneurons.

npr-9 encodes a homologue of the gastrin-releasing peptide receptor (GRPR) and is expressed in AIB interneurons. In this study, we investigated the role of NPR-9 in the neuronal control of innate immunity using the model system Caenorhabditis elegans. After exposure to Pseudomonas aeruginosa PA14, npr-9(tm1652) mutants showed resistance to infection, decreased PA14 colonization and increased expression of immunity-related genes. Nematodes overexpressing NPR-9 exhibited increased susceptibility to infection, increased PA14 colonization and reduced expression of immunity-related genes. In nematodes, ChR2-mediated AIB interneuron activation strengthened the innate immune response and decreased PA14 colonization. Overexpression of NPR-9 suppressed the innate immune response and increased PA14 colonization in nematodes with the activation of AIB interneurons mediated by ChR2 or by expressing pkc-1(gf) in AIB interneurons. We, therefore, hypothesize that NPR-9 regulates the innate immune response by antagonizing the activity of AIB interneurons. Furthermore, expression of GRPR, the human homologue of NPR-9, could largely mimic NPR-9 function by regulating innate immunity in nematodes. Our results provide insight into the pivotal role of interneurons in controlling innate immunity and the complex biological functions of GRPRs.

GRPR-targeted Protein Contrast Agents for Molecular Imaging of Receptor Expression in Cancers by MRI.

Gastrin-releasing peptide receptor (GRPR) is differentially expressed on the surfaces of various diseased cells, including prostate and lung cancer. However, monitoring temporal and spatial expression of GRPR in vivo by clinical MRI is severely hampered by the lack of contrast agents with high relaxivity, targeting capability and tumor penetration. Here, we report the development of a GRPR-targeted MRI contrast agent by grafting the GRPR targeting moiety into a scaffold protein with a designed Gd3+ binding site (ProCA1.GRPR). In addition to its strong binding affinity for GRPR (Kd = 2.7 nM), ProCA1.GRPR has high relaxivity (r1 = 42.0 mM−1s−1 at 1.5 T and 25 °C) and strong Gd3+ selectivity over physiological metal ions. ProCA1.GRPR enables in vivo detection of GRPR expression and spatial distribution in both PC3 and H441 tumors in mice using MRI. ProCA1.GRPR is expected to have important preclinical and clinical implications for the early detection of cancer and for monitoring treatment effects.

Clinical Relevance of Targeting the Gastrin-Releasing Peptide Receptor, Somatostatin Receptor 2, or Chemokine C-X-C Motif Receptor 4 in Breast Cancer for Imaging and Therapy.

Imaging and therapy using radioligands targeting receptors overexpressed on tumor cells is successfully applied in neuroendocrine tumor patients. Because expression of the gastrin-releasing peptide receptor (GRPR), somatostatin receptor 2 (SSTR2), and chemokine C-X-C motif receptor 4 (CXCR4) has been demonstrated in breast cancer, targeting these receptors using radioligands might offer new imaging and therapeutic opportunities for breast cancer patients. The aim of this study was to correlate messenger RNA (mRNA) expression of GRPR, SSTR2, and CXCR4 with clinicopathologic and biologic factors, and with prognosis and prediction to therapy response, in order to identify specific breast cancer patient groups suited for the application of radioligands targeting these receptors. First, we studied GRPR and SSTR2 expression in 13 clinical breast cancer specimens by in vitro autoradiography and correlated this with corresponding mRNA levels to investigate whether mRNA levels reliably represent cell surface expression. Next, GRPR, SSTR2, and CXCR4 mRNA levels were measured by quantitative reverse transcriptase polymerase chain reaction in 915 primary breast cancer tissues and correlated with known clinicopathologic and biologic factors, disease-free survival, distant metastasis-free survival, and overall survival (DFS, MFS, and OS, respectively). In 224 adjuvant hormonal treatment-naïve estrogen receptor (ER, ESR1)-positive patients who received tamoxifen as first-line therapy for recurrent or metastatic disease, the expression levels of the receptors were correlated with progression-free survival. Our results showed a significant positive correlation between GRPR and SSTR2 expression analyzed by in vitro autoradiography and by quantitative reverse transcriptase polymerase chain reaction (Spearman's rank correlation coefficient [Rs]=0.94, P<0.001, and Rs=0.73, P=0.0042, respectively). Furthermore, high GRPR and SSTR2 mRNA levels were observed more frequently in ESR1-positive specimens, whereas high CXCR4 expression was associated with ESR1-negative specimens. Also, high mRNA expression of CXCR4 was associated with a prolonged DFS, MFS, and OS (multivariate hazard ratio MFS=0.76 [95% confidence interval, 0.64-0.90], P=0.001), whereas high mRNA levels of GRPR were associated with a prolonged progression-free survival after the start of first-line tamoxifen treatment (multivariate hazard ratio=0.68 [95% confidence interval, 0.48-0.97], P=0.031). Our data indicate that imaging and therapy using GRPR or SSTR2 radioligands might especially be beneficial for ESR1-positive breast cancer and CXCR4 radioligands for ESR1-negative breast cancer.

A central role for spinal dorsal horn neurons that express neurokinin-1 receptors in chronic itch.

We investigated roles for spinal neurons expressing the neurokinin-1 receptor (NK1R) and/or gastrin-releasing peptide receptor (GRPR) in a mouse model of ovalbumin (OVA)-induced chronic atopic dermatitis. Mice receiving repeated topical application of OVA exhibited atopic-like skin lesions and behavioral signs of chronic itch including spontaneous scratching, touch-evoked scratching (alloknesis), and enhancement of chloroquine-evoked scratching (hyperknesis). Substance P-saporin (SP-SAP) and bombesin-saporin (BB-SAP) were intrathecally injected into OVA-sensitized mice to neurotoxically ablate NK1R- or GRPR-expressing spinal neurons, respectively. SP-SAP diminished the expression of NK1R in the superficial spinal dorsal horn and significantly attenuated all behavioral signs of chronic itch. BB-SAP reduced the spinal dorsal horn expression of GRPR and significantly attenuated hyperknesis, with no effect on spontaneous scratching or alloknesis. To investigate whether NK1R-expressing spinal neurons project in ascending somatosensory pathways, we performed a double-label study. The retrograde tracer, Fluorogold (FG), was injected into either the somatosensory thalamus or lateral parabrachial nucleus. In the upper cervical (C1-2) spinal cord, most neurons retrogradely labeled with FG were located in the dorsomedial aspect of the superficial dorsal horn. Of FG-labeled spinal neurons, 89% to 94% were double labeled for NK1R. These results indicate that NK1R-expressing spinal neurons play a major role in the expression of symptoms of chronic itch and give rise to ascending somatosensory projections. Gastrin-releasing peptide receptor-expressing spinal neurons contribute to hyperknesis but not to alloknesis or ongoing itch. NK1R-expressing spinal neurons represent a potential target to treat chronic itch.

GRPR-selective PET imaging of prostate cancer using [18F]-lanthionine-bombesin analogs

The gastrin-releasing peptide receptor (GRPR) is overexpressed in a variety of human malignancies, including prostate cancer. Bombesin (BBN) is a 14 amino acids peptide that selectively binds to GRPR. In this study, we developed two novel Al18F-labeled lanthionine-stabilized BBN analogs, designated Al18F-NOTA-4,7-lanthionine-BBN and Al18F-NOTA-2,6-lanthionine-BBN, for positron emission tomography (PET) imaging of GRPR expression using xenograft prostate cancer models. (Methyl)lanthionine-stabilized 4,7-lanthionine-BBN and 2,6-lanthionine-BBN analogs were conjugated with a NOTA chelator and radiolabeled with Al18F using the aluminum fluoride strategy. Al18F-NOTA-4,7-lanthionine-BBN and Al18F-NOTA-2,6-lanthionine-BBN was labeled with Al18F with good radiochemical yield and specific activity>30GBq/μmol for both radiotracers. The logD values measured for Al18F-NOTA-4,7-lanthionine-BBN and Al18F-NOTA-2,6-lanthionine-BBN were −2.14±0.14 and −2.34±0.15, respectively. In athymic nude PC-3 xenografts, at 120min post injection (p.i.), the uptake of Al18F-NOTA-4,7-lanthionine-BBN and Al18F-NOTA-2,6-lanthionine-BBN in prostate cancer (PC-3) mouse models was 0.82±0.23% ID/g and 1.40±0.81% ID/g, respectively. An excess of unlabeled ɛ-aminocaproic acid-BBN(7–14) (300-fold) was co-injected to assess GRPR binding specificity. Tumor uptake of Al18F-NOTA-4,7-lanthionine-BBN and Al18F-NOTA-2,6-lanthionine-BBN in PC-3 tumors was evaluated by microPET (μPET) imaging at 30, 60 and 120min p.i. Blocking studies showed decreased uptake in PC-3 bearing mice. Stabilized 4,7-lanthionine-BBN and 2,6-lanthionine-BBN peptides were rapidly and successfully labeled with 18F. Both tracers may have potential for GRPR-positive tumor imaging.

In vitro and in vivo application of radiolabeled gastrin-releasing peptide receptor ligands in breast cancer.

Breast cancer (BC) consists of multiple subtypes defined by various molecular characteristics, for instance, estrogen receptor (ER) expression. Methods for visualizing BC include mammography, MR imaging, ultrasound, and nuclear medicine-based methods such as (99m)Tc-sestamibi and (18)F-FDG PET, unfortunately all lacking specificity. Peptide receptor scintigraphy and peptide receptor radionuclide therapy are successfully applied for imaging and therapy of somatostatin receptor-expressing neuroendocrine tumors using somatostatin receptor radioligands. On the basis of a similar rationale, radioligands targeting the gastrin-releasing peptide receptor (GRP-R) might offer a specific method for imaging and therapy of BC. The aim of this study was to explore the application of GRP-R radioligands for imaging and therapy of BC by introducing valid preclinical in vitro and in vivo models. GRP-R expression of 50 clinical BC specimens and the correlation with ER expression was studied by in vitro autoradiography with the GRP-R agonist (111)In-AMBA. GRP-R expression was also analyzed in 9 BC cell lines applying (111)In-AMBA internalization assays and quantitative reverse transcriptase polymerase chain reaction. In vitro cytotoxicity of (177)Lu-AMBA was determined on the GRP-R-expressing BC cell line T47D. SPECT/CT imaging and biodistribution were studied in mice with subcutaneous and orthotopic ER-positive T47D and MCF7 xenografts after injection of the GRP-R antagonist (111)In-JMV4168. Most of the human BC specimens (96%) and BC cell lines (6/9) were found to express GRP-R. GRP-R tumor expression was positively (P = 0.026, χ(2)(4) = 12,911) correlated with ER expression in the human BC specimens. Treatment of T47D cells with 10(-7) M/50 MBq of (177)Lu-AMBA resulted in 80% reduction of cells in vitro. Furthermore, subcutaneous and orthotopic tumors from both BC cell lines were successfully visualized in vivo by SPECT/CT using (111)In-JMV4168; T47D tumors exhibited a higher uptake than MCF7 xenografts. Targeting GRP-R-expressing BC tumors using GRP-R radioligands is promising for nuclear imaging and therapy, especially in ER-positive BC patients.

Expression of Gastrin-releasing Peptide Receptor in Epidermoid Carcinoma of the Anal Canal

Gastrin-releasing peptide is a neuroendocrine homolog of bombesin that demonstrated important growth-stimulatory effects in various types of cancer. High levels of expression of gastrin-releasing peptide receptors (GRPR) has been found in different malignancies, and the studies exploring the therapeutic use of GRPR antagonists have shown promising results. Our aim was to determine the GRPR expression in epidermoid carcinoma of the anal canal and discuss its potential clinical applications. We performed immunohistochemical analysis for GRPR on formalin-fixed and paraffin-embedded tumor samples obtained from 35 patients with anal cancer. As a control group, we analyzed 24 samples of nonmalignant anal tissues (hemorrhoidectomy specimens). GRPR expression was evaluated using a semiquantitative approach according to the intensity and distribution of staining. All analyzed tissues, except 1 control sample, showed positive GRPR immunoexpression. GRPR was strongly expressed in 54% of cancer specimens as compared with only 12% of the control specimens (P<0.003). In tumors, the receptor showed a diffuse and homogenous pattern of distribution within the specimens. In contrast, control specimens showed a focal pattern of staining restricted to the basal half of the epithelium. In conclusion, we demonstrated that GRPR is highly expressed in epidermoid carcinoma of the anal canal, suggesting this receptor might have a role in anal carcinogenesis. Our results provide a basis for exploiting GRPR as a target for diagnostic and therapeutic purposes in the anal cancer.

Gα13/PDZ-RhoGEF/RhoA Signaling Is Essential for Gastrin-Releasing Peptide Receptor–Mediated Colon Cancer Cell Migration

Gastrin-releasing peptide receptor (GRPR) is ectopically expressed in over 60% of colon cancers. GRPR expression has been correlated with increased colon cancer cell migration. However, the signaling pathway by which GRPR activation leads to increased cancer cell migration is not well understood. We set out to molecularly dissect the GRPR signaling pathways that control colon cancer cell migration through regulation of small GTPase RhoA. Our results show that GRP stimulation activates RhoA predominantly through G13 heterotrimeric G-protein signaling. We also demonstrate that postsynaptic density 95/disk-large/ZO-1 (PDZ)-RhoGEF (PRG), a member of regulator of G-protein signaling (RGS)-homology domain (RH) containing guanine nucleotide exchange factors (RH-RhoGEFs), is the predominant activator of RhoA downstream of GRPR. We found that PRG is required for GRP-stimulated colon cancer cell migration, through activation of RhoA-Rho-associated kinase (ROCK) signaling axis. In addition, PRG-RhoA-ROCK pathway also contributes to cyclo-oxygenase isoform 2 (Cox-2) expression. Increased Cox-2 expression is correlated with increased production of prostaglandin-E2 (PGE2), and Cox-2-PGE2 signaling contributes to total GRPR-mediated cancer cell migration. Our analysis reveals that PRG is overexpressed in colon cancer cell lines. Overall, our results have uncovered a key mechanism for GRPR-regulated colon cancer cell migration through the Gα13-PRG-RhoA-ROCK pathway.

Gastrin-Releasing Peptide Receptor Expression in Lung Cancer

Gastrin-releasing peptide receptors (GRPRs) activate mitogen-activated protein kinase signaling pathway primarily through epidermal growth factor receptor activation and are under investigation as a molecular target because they are overexpressed in several solid tumors. To determine GRPR expression in both non-small cell lung carcinoma and small cell lung carcinoma, comparing results with clinical stages and demographic data. We analyzed the immunohistochemical expression of GRPR in 200 non-small cell lung carcinoma and 38 small cell lung carcinoma archival cases from 2004 to 2008. Non-small cell lung carcinoma cases tended to be higher GRPR expressers at a rate of 62.5% (weak, moderate, and strong expression in 41.5%, 13.5%, and 7.5%, respectively), compared with 52.62% in small cell lung carcinoma cases (weak, moderate, and strong expression in 34.21%, 15.78%, and 2.63%, respectively; P = .30). In non-small cell lung carcinoma there was a trend for higher percentages of strong expression in adenocarcinoma cases (10%; P = .67), and in patients with advanced stages (III and IV; 9.43% and 6.9%; P = .01). To the best of our knowledge, this is the first study to demonstrate GRPR tissue expression in a large population of patients with lung cancer. Although GRPR expression was similar in small cell and non-small cell carcinoma, the expression was more pronounced in an advanced-stage lung cancer, particularly in adenocarcinoma cases, and may represent a potential target for the development of new treatment approaches in this population.

Early over‐expression of GRP receptors in prostatic carcinogenesis

The GRP receptor shows high over-expression in prostatic adenocarcinoma and high grade PIN, but low expression in normal prostate glands. This represents the molecular basis for GRP receptor imaging of prostate cancer with radioactive compounds. However, a focal, high density GRP receptor expression can be observed in hitherto uncharacterized prostate glands. GRP receptors were quantitatively measured with in vitro receptor autoradiography using ¹²⁵I-Tyr⁴ -bombesin in samples from 115 prostates. On successive tissue sections, ¹²⁵I-Tyr⁴ -bombesin autoradiography was compared with H&E staining and MIB-1 and 34βE12 immunohistochemistry. On one hand, it was confirmed that GRP receptors were expressed in adenocarcinoma and high grade PIN in high density and high incidence (77% and 73%, respectively), but in normal prostate glands in low density and low frequency (18%). On the other hand, a novel and intriguing observation was the existence of focal non-invasive prostate glands with high GRP receptor density, characterized by low grade nuclear atypia and increased proliferation, compatible with lower grade PIN. There was a significant GRP receptor density gradient (P ≤ 0.005), increasing from normal prostate glands (mean relative optical density, ROD, of ¹²⁵I-Tyr⁴ -bombesin binding: 0.17) over atypical glands without increased MIB-1 labeling (0.28) and atypical glands with increased MIB-1 expression (0.44) to high grade PIN and adenocarcinoma (0.64 and 0.58, respectively). GRP receptor over-expression may be a novel, specific marker of early prostatic neoplastic transformation, arising in low grade PIN, and progressively increasing during malignant progression. This should be considered when interpreting in vivo GRP receptor imaging in males.

Chronic itch development in sensory neurons requires BRAF signaling pathways

Chronic itch, or pruritus, is associated with a wide range of skin abnormalities. The mechanisms responsible for chronic itch induction and persistence remain unclear. We developed a mouse model in which a constitutively active form of the serine/threonine kinase BRAF was expressed in neurons gated by the sodium channel Nav1.8 (BRAF(Nav1.8) mice). We found that constitutive BRAF pathway activation in BRAF(Nav1.8) mice results in ectopic and enhanced expression of a cohort of itch-sensing genes, including gastrin-releasing peptide (GRP) and MAS-related GPCR member A3 (MRGPRA3), in nociceptors expressing transient receptor potential vanilloid 1 (TRPV1). BRAF(Nav1.8) mice showed de novo neuronal responsiveness to pruritogens, enhanced pruriceptor excitability, and heightened evoked and spontaneous scratching behavior. GRP receptor expression was increased in the spinal cord, indicating augmented coding capacity for itch subsequent to amplified pruriceptive inputs. Enhanced GRP expression and sustained ERK phosphorylation were observed in sensory neurons of mice with allergic contact dermatitis– or dry skin–elicited itch; however, spinal ERK activation was not required for maintaining central sensitization of itch. Inhibition of either BRAF or GRP signaling attenuated itch sensation in chronic itch mouse models. These data uncover RAF/MEK/ERK signaling as a key regulator that confers a subset of nociceptors with pruriceptive properties to initiate and maintain long-lasting itch sensation.

Expression of gastrin-releasing peptide receptor in patients with cutaneous malignant melanoma

Gastrin-releasing peptide (GRP) is a neuroendocrine peptide shown to possess growth-stimulatory effects in many types of human cancers. High levels of GRP receptors have been found in various types of human cancers, and preclinical studies exploring the therapeutic use of GRP receptor (GRPR) antagonists have been reported, with promising results. Data on GRPR expression in human malignant melanoma (MM) are scanty. To determine GRPR expression in biopsy material obtained from patients diagnosed with cutaneous MM. Immunohistochemistry was performed on formalin-fixed, paraffin wax-embedded tissue samples obtained from 51 patients with cutaneous MM. The relationship between GRPR expression and the clinicopathological features was analysed using the Fisher exact test. GRPR immunoexpression was found in 42/51 cutaneous melanoma samples (82.4%). It was strongly expressed in 30 cases (58.9%). There was no significant difference in the levels of GRPR expression between primary or metastatic lesions. We correlated the GRPR expression score with pathological features associated with prognosis in cutaneous MM. There was no significant difference in GRPR expression in relation to Clark level (CL; P = 0.35) or Breslow Index (BI; P = 0.17). GRPR expression levels were high in tissue specimens of MM (82.4%), but did not correlate with pathological features related to prognosis, such as CL or BI. Further studies, preferably in a larger patient population, are warranted.