Lisa K. Chopin

Ghrelin and a novel preproghrelin isoform are highly expressed in prostate cancer and ghrelin activates mitogen-activated protein kinase in prostate cancer.

Purpose: There is evidence that the hormone ghrelin stimulates proliferation in the PC3 prostate cancer cell line although the underlying mechanism(s) remain to be determined. A novel, exon 3–deleted preproghrelin isoform has previously been detected in breast and prostate cancer cells; however, its characterization, expression, and potential function in prostate cancer tissues are unknown. Experimental Design: Expression of ghrelin and exon 3–deleted preproghrelin was investigated in prostate cancer cell lines and tissues by reverse transcription-PCR and immunohistochemistry. Proliferation and apoptosis assays were done in the LNCaP prostate cancer cell line to determine if ghrelin stimulates proliferation and/or cell survival. Stimulation of mitogen-activated protein kinase (MAPK) pathway activation by ghrelin was determined in PC3 and LNCaP cells by immunoblotting with antibodies specific for phosphorylated MAPKs. Results: Prostate cancer tissues display greater immunoreactivity for ghrelin and exon 3–deleted preproghrelin than normal prostate tissues, and prostate cancer cell lines secrete mature ghrelin into conditioned medium. Treatment with ghrelin (10 nmol/L), but not the unique COOH-terminal peptide derived from exon 3–deleted preproghrelin, stimulates proliferation in the LNCaP cells (45.0 ± 1.7% above control, P < 0.01) and rapidly activates the extracellular signal-regulated kinase-1/2 MAPK pathway in both PC3 and LNCaP cell lines. Ghrelin, however, does not protect prostate cancer cells from apoptosis induced by actinomycin D (1 μg/mL). The MAPK inhibitors PD98059 and U0126 blocked ghrelin-induced MAPK activation, as well as proliferation, in both cell lines. Conclusions: These data suggest that these components of the ghrelin axis may have potential as novel biomarkers and/or adjunctive therapeutic targets for prostate cancer.

The potential autocrine/paracrine roles of ghrelin and its receptor in hormone-dependent cancer.

Ghrelin is a recently identified 28 amino acid peptide capable of stimulating pituitary growth hormone release in humans. The actions of ghrelin are mediated via the naturally occurring ghrelin receptor, also known as the growth hormone secretagogue receptor (GHS-R). Ghrelin and its receptors are now being recognized as components of the growth hormone axis and are therefore potentially involved in tissue growth and development. As is the case for other members of this axis, evidence is rapidly emerging to indicate that ghrelin/GHS-R may play an important autocrine/paracrine role in some cancers. This review highlights the evidence for the expression, regulation and potential functional role of ghrelin and its receptor in hormone-dependent cancers, such as prostate and breast cancer.

Developmental, Stage-Specific, and Hormonally Regulated Expression of Growth Hormone Secretagogue Receptor Messenger RNA in Rat Testis

Abstract Recent evidence from our research suggested the direct role of ghrelin in the control of testicular function. However, the pattern of expression and hormonal regulation of the gene encoding its cognate receptor (i.e., the growth hormone-secretagogue receptor [GHS-R]) in the male gonad remains to be fully elucidated. In this paper, overall expression of GHS-R mRNA in rat testis was compared with that of the functional receptor form, namely GHS-R type 1a, in different developmental and experimental settings. In addition, cellular distribution of GHS-R within adult testis tissue was assessed. Our analyses demonstrated persistent expression of the GHS-R gene in rat testis throughout postnatal development. In contrast, testicular expression of GHS-R type 1a mRNA remained undetectable before puberty and sharply increased thereafter. In adult testis, GHS-R1a mRNA expression presented a scattered pattern of cellular distribution, including Sertoli and Leydig cells that also showed specific GHS-R1a immunoreactivity. Expression of total GHS-R and specific GHS-R1a mRNAs was detected in isolated seminiferous tubule preparations, with varying levels throughout the defined stages of the spermatogenic cycle. In addition, testicular expression of total GHS-R and GHS-R1a mRNAs was up-regulated by exposure to ghrelin in vitro and after stimulation with FSH in vivo. In conclusion, our data demonstrate that expression of the GHS-R gene in rat testis takes place in a developmental, stage-specific, and hormonally regulated manner. Divergent expression of total GHS-R and type 1a specific mRNAs was detected at certain stages of postnatal development and spermatogenic cycle, thus raising the possibility that, in addition to net changes in GHS-R gene expression, the balance between receptor subtypes may represent a novel mechanism for the tuning of ghrelin sensitivity in rat testis.

The Ghrelin Axis—Does It Have an Appetite for Cancer Progression?

Ghrelin, the endogenous ligand for the GH secretagogue receptor (GHSR), is a peptide hormone with diverse physiological roles. Ghrelin regulates GH release, appetite and feeding, gut motility, and energy balance and also has roles in the cardiovascular, immune, and reproductive systems. Ghrelin and the GHSR are expressed in a wide range of normal and tumor tissues, and a fluorescein-labeled, truncated form of ghrelin is showing promise as a biomarker for prostate cancer. Plasma ghrelin levels are generally inversely related to body mass index and are unlikely to be useful as a biomarker for cancer, but may be useful as a marker for cancer cachexia. Some single nucleotide polymorphisms in the ghrelin and GHSR genes have shown associations with cancer risk; however, larger studies are required. Ghrelin regulates processes associated with cancer, including cell proliferation, apoptosis, cell migration, cell invasion, inflammation, and angiogenesis; however, the role of ghrelin in cancer is currently unclear. Ghrelin has predominantly antiinflammatory effects and may play a role in protecting against cancer-related inflammation. Ghrelin and its analogs show promise as treatments for cancer-related cachexia. Further studies using in vivo models are required to determine whether ghrelin has a role in cancer progression.

Ghrelin and cancer.

Ghrelin is a peptide hormone that was originally isolated from the stomach as the endogenous ligand for the growth hormone secretagogue receptor (GHSR). Ghrelin has many functions, including the regulation of appetite and gut motility, growth hormone release from the anterior pituitary and roles in the cardiovascular and immune systems. Ghrelin and its receptor are expressed in a number of cancers and cancer cell lines and may play a role in processes associated with cancer progression, including cell proliferation, apoptosis, and cell invasion and migration.

Estimating cell diffusivity and cell proliferation rate by interpreting IncuCyte ZOOM™ assay data using the Fisher-Kolmogorov model

BackgroundStandard methods for quantifying IncuCyte ZOOM™ assays involve measurements that quantify how rapidly the initially-vacant area becomes re-colonised with cells as a function of time. Unfortunately, these measurements give no insight into the details of the cellular-level mechanisms acting to close the initially-vacant area. We provide an alternative method enabling us to quantify the role of cell motility and cell proliferation separately. To achieve this we calibrate standard data available from IncuCyte ZOOM™ images to the solution of the Fisher-Kolmogorov model.ResultsThe Fisher-Kolmogorov model is a reaction-diffusion equation that has been used to describe collective cell spreading driven by cell migration, characterised by a cell diffusivity, D, and carrying capacity limited proliferation with proliferation rate, λ, and carrying capacity density, K. By analysing temporal changes in cell density in several subregions located well-behind the initial position of the leading edge we estimate λ and K. Given these estimates, we then apply automatic leading edge detection algorithms to the images produced by the IncuCyte ZOOM™ assay and match this data with a numerical solution of the Fisher-Kolmogorov equation to provide an estimate of D. We demonstrate this method by applying it to interpret a suite of IncuCyte ZOOM™ assays using PC-3 prostate cancer cells and obtain estimates of D, λ and K. Comparing estimates of D, λ and K for a control assay with estimates of D, λ and K for assays where epidermal growth factor (EGF) is applied in varying concentrations confirms that EGF enhances the rate of scratch closure and that this stimulation is driven by an increase in D and λ, whereas K is relatively unaffected by EGF.ConclusionsOur approach for estimating D, λ and K from an IncuCyte ZOOM™ assay provides more detail about cellular-level behaviour than standard methods for analysing these assays. In particular, our approach can be used to quantify the balance of cell migration and cell proliferation and, as we demonstrate, allow us to quantify how the addition of growth factors affects these processes individually.

Ghrelin and the Brain-gut Axis as a Pharmacological Target for Appetite Control

Appetite regulation is highly complex and involves a large number of orexigenic and anorexigenic peptide hormones. These are small, processed, secreted peptides derived from larger prepropeptide precursors. These peptides are important targets for the development of therapeutics for obesity, a global health epidemic. As a case study, we consider the ghrelin axis. The ghrelin axis is likely to be a particularly useful drug target, as it also plays a role in energy homeostasis, adipogenesis, insulin regulation and reward associated with food intake. Ghrelin is the only known circulating gut orexigenic peptide hormone. As it appears to play a role in diet-induced obesity, blocking the action of ghrelin is likely to be effective for treating and preventing obesity. The ghrelin peptide has been targeted using a number of approaches, with ghrelin mirror-image oligonucleotides (Spiegelmers) and immunotherapy showing some promise. The ghrelin receptor, the growth hormone secretagogue receptor, may also provide a useful target and a number of antagonists and inverse agonists have been developed. A particularly promising new target is the enzyme which octanoylates ghrelin, ghrelin O-acyltransferase (GOAT), and drugs that inhibit GOAT are likely to circumvent pharmacological issues associated with approaches that directly target ghrelin or its receptor.

Ghrelin axis genes, peptides and receptors: recent findings and future challenges

The ghrelin axis consists of the gene products of the ghrelin gene (GHRL), and their receptors, including the classical ghrelin receptor GHSR. While it is well-known that the ghrelin gene encodes the 28 amino acid ghrelin peptide hormone, it is now also clear that the locus encodes a range of other bioactive molecules, including novel peptides and non-coding RNAs. For many of these molecules, the physiological functions and cognate receptor(s) remain to be determined. Emerging research techniques, including proteogenomics, are likely to reveal further ghrelin axis-derived molecules. Studies of the role of ghrelin axis genes, peptides and receptors, therefore, promises to be a fruitful area of basic and clinical research in years to come.

Expression and In Vitro Functions of the Ghrelin Axis in Endometrial Cancer

Ghrelin is a peptide hormone produced in the stomach and a range of other tissues, where it has endocrine, paracrine and autocrine roles in both normal and disease states. Ghrelin has been shown to be an important growth factor for a number of tumours, including prostate and breast cancers. In this study, we examined the expression of the ghrelin axis (ghrelin and its receptor, the growth hormone secretagogue receptor, GHSR) in endometrial cancer. Ghrelin is expressed in a range of endometrial cancer tissues, while its cognate receptor, GHSR1a, is expressed in a small subset of normal and cancer tissues. Low to moderately invasive endometrial cancer cell lines were examined by RT–PCR and immunoblotting, demonstrating that ghrelin axis mRNA and protein expression correlate with differentiation status of Ishikawa, HEC1B and KLE endometrial cancer cell lines. Moreover, treatment with ghrelin potently stimulated cell proliferation and inhibited cell death. Taken together, these data indicate that ghrelin promotes the progression of endometrial cancer cells in vitro, and may contribute to endometrial cancer pathogenesis and represent a novel treatment target.

Ghrelin gene-related peptides: multifunctional endocrine / autocrine modulators in health and disease.

1. Ghrelin is a multifunctional peptide hormone that affects various processes, including growth hormone and insulin release, appetite regulation, gut motility, metabolism and cancer cell proliferation. Ghrelin is produced in the stomach and in other normal and pathological cell types. It may act as an endocrine or autocrine/paracrine factor.