Gastrin-releasing peptide receptor (GRPr) promotes EMT, growth, and invasion in canine prostate cancer

Abstract

BACKGROUND The gastrin-releasing peptide receptor (GRPr) is upregulated in early and late-stage human prostate cancer (PCa) and other solid tumors of the mammary gland, lung, head and neck, colon, uterus, ovary, and kidney. However, little is known about its role in prostate cancer. This study examined the effects of a heterologous GRPr agonist, bombesin (BBN), on growth, motility, morphology, gene expression, and tumor phenotype of an osteoblastic canine prostate cancer cell line (Ace-1) in vitro and in vivo. METHODS The Ace-1 cells were stably transfected with the human GRPr and tumor cells were grown in vitro and as subcutaneous and intratibial tumors in nude mice. The effect of BBN was measured on cell proliferation, cell migration, tumor growth (using bioluminescence), tumor cell morphology, bone tumor phenotype, and epithelial-mesenchymal transition (EMT) and metastasis gene expression (quantitative RT-PCR). GRPr mRNA expression was measured in primary canine prostate cancers and normal prostate glands. RESULTS Bombesin (BBN) increased tumor cell proliferation and migration in vitro and tumor growth and invasion in vivo. BBN upregulated epithelial-to-mesenchymal transition (EMT) markers (TWIST, SNAIL, and SLUG mRNA) and downregulated epithelial markers (E-cadherin and β-catenin mRNA), and modified tumor cell morphology to a spindle cell phenotype. Blockade of GRPr upregulated E-cadherin and downregulated VIMENTIN and SNAIL mRNA. BBN altered the in vivo tumor phenotype in bone from an osteoblastic to osteolytic phenotype. Primary canine prostate cancers had increased GRPr mRNA expression compared to normal prostates. CONCLUSION These data demonstrated that the GRPr is important in prostate cancer growth and progression and targeting GRPr may be a promising strategy for treatment of prostate cancer.

Development of an orthotopic canine prostate cancer model expressing human GRPr

“…20 receptor on their surfaces and the receptor functioned biologically as a growth factor, grew reliably in nude mice, and could be optically imaged in vivo and microscopically with IR800-G-Abz4-t-BBN. [12][13][14] The histopathology results reported herein definitively demonstrate the growth of these cells in the prostate glands and other tissues of immunosuppressed dogs, as well as the cells' ability to seed to regional lymph nodes. The new canine model reported herein is thus the first time a human cancer growth factor expressing cell has been grown in dogs.…”

“…Starting with a known isolated Ace‐1 canine prostate cancer cell line that expresses very little mRNA for GRPr and does not bind GRPr targeting peptides, our prior study demonstrated that the Ace‐1 cell line could be transfected with human GRPr. The resulting Ace‐1 huGRPr cells expressed the GRPr receptor on their surfaces and the receptor functioned biologically as a growth factor, grew reliably in nude mice, and could be optically imaged in vivo and microscopically with IR800‐G‐Abz4‐t‐BBN . The histopathology results reported herein definitively demonstrate the growth of these cells in the prostate glands and other tissues of immunosuppressed dogs, as well as the cells’ ability to seed to regional lymph nodes.…”

“…A canine model has been developed that uses cultured Ace‐1 canine prostate cancer cells implanted into prostate glands of immune‐suppressed beagles . We previously reported development of a novel Ace‐1 cell line that was transfected with the human (hu) gastrin‐releasing peptide receptor (GRPr) gene, producing a highly GRPr expressing and biologically functioning cell line, Ace‐1 huGRPr , that grows stably in culture and in nude mice . Nude mice xenografts were imaged with a 10 nmol intravenous dose of a GRPr‐targeted optical imaging agent, IR800‐G‐Abz4‐AMBA .…”

“…11 We previously reported development of a novel Ace-1 cell line that was transfected with the human (hu) gastrin-releasing peptide receptor (GRPr) gene, producing a highly GRPr expressing and biologically functioning cell line, Ace-1 huGRPr , that grows stably in culture and in nude mice. 12,13 Nude mice xenografts were imaged with a 10 nmol intravenous dose of a GRPr-targeted optical imaging agent, IR800-G-Abz4-AMBA. 14 Herein we report the successful translation of these developments to dogs.…”

A human GRPr-transfected Ace-1 canine prostate cancer model in mice

“…Canine bombesin peptides show high sequence homology with human and terminate in the same sequence [47], suggesting that the canine Ace-1 cell line expresses few GRPr. In addition, quantitative RT-PCR showed that the Ace-1 cells had very low levels of canine GRPr mRNA in contrast to the primary carcinoma tissues from which the Ace-1 were derived, which had abundant GRPr mRNA [48]. Stable transfection generated 10 canine Ace-1 clones with the plasmid containing human GRPr (Ace-1 huGRPr ), and eight clones with the empty vector (Ace-1 CMV ).…”