Publications by authors named "Carina Walpole"

24 Publications

  • Page 1 of 1

Human CD141 dendritic cells (cDC1) are impaired in patients with advanced melanoma but can be targeted to enhance anti-PD-1 in a humanized mouse model.

J Immunother Cancer 2021 Mar;9(3)

Mater Research, The University of Queensland, Woolloongabba, Queensland, Australia

Background: The conventional type 1 dendritic cell subset (cDC1) is indispensable for tumor immune responses and the efficacy of immune checkpoint inhibitor (ICI) therapies in animal models but little is known about the role of the human CD141 DC cDC1 equivalent in patients with melanoma.

Methods: We developed a flow cytometry assay to quantify and characterize human blood DC subsets in healthy donors and patients with stage 3 and stage 4 metastatic melanoma. To examine whether harnessing CD141 DCs could improve responses to ICIs in human melanoma, we developed a humanized mouse model by engrafting immunodeficient NSG-SGM3 mice with human CD34 hematopoietic stem cells (HSCs) from umbilical cord blood followed by transplantation of a human melanoma cell line and treatment with anti-programmed cell death protein-1 (anti-PD-1).

Results: Blood CD141 DC numbers were significantly reduced in patients with stage 4 melanoma compared with healthy controls. Moreover, CD141 DCs in patients with melanoma were selectively impaired in their ability to upregulate CD83 expression after stimulation with toll-like receptor 3 (TLR3) and TLR7/8 agonists ex vivo. Although DC numbers did not correlate with responses to anti-PD-1 and/or anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) ICIs, their numbers and capacity to upregulate CD83 declined further during treatment in non-responding patients. Treatment with anti-PD-1 was ineffective at controlling tumor growth in humanized mice but efficacy was enhanced by indirectly expanding and activating DCs in vivo with -like tyrosine kinase-3 ligand (Flt3L) and a TLR3 agonist. Moreover, intratumoral injections of CD141 DCs resulted in reduced tumor growth when combined with anti-PD-1 treatment.

Conclusions: These data illustrate quantitative and qualitative impairments in circulating CD141 DCs in patients with advanced melanoma and that increasing CD141 DC number and function is an attractive strategy to enhance immunogenicity and response rates to ICIs.
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http://dx.doi.org/10.1136/jitc-2020-001963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7978242PMC
March 2021

The long non-coding RNA GHSROS reprograms prostate cancer cell lines toward a more aggressive phenotype.

PeerJ 2021 1;9:e10280. Epub 2021 Feb 1.

Ghrelin Research Group, Translational Research Institute, School of Biomedical Sciences, Queensland University of Technology, Brisbane, Queensland, Australia.

It is now appreciated that long non-coding RNAs (lncRNAs) are important players in orchestrating cancer progression. In this study we characterized , a human lncRNA gene on the opposite DNA strand (antisense) to the ghrelin receptor gene, in prostate cancer. The lncRNA was upregulated by prostate tumors from different clinical datasets. Transcriptome data revealed that alters the expression of cancer-associated genes. Functional analyses in vitro showed that mediates tumor growth, migration and survival, and resistance to the cytotoxic drug docetaxel. Increased cellular proliferation of -overexpressing PC3, DU145, and LNCaP prostate cancer cell lines in vitro was recapitulated in a subcutaneous xenograft model. Conversely, in vitro antisense oligonucleotide inhibition of the lncRNA reciprocally regulated cell growth and migration, and gene expression. Notably, modulates the expression of , the loss of which may drive androgen receptor pathway-independent prostate tumor progression in a subset of prostate cancers. Collectively, our findings suggest that can reprogram prostate cancer cells toward a more aggressive phenotype and that this lncRNA may represent a potential therapeutic target.
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http://dx.doi.org/10.7717/peerj.10280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7860111PMC
February 2021

KLK4 Induces Anti-Tumor Effects in Human Xenograft Mouse Models of Orthotopic and Metastatic Prostate Cancer.

Cancers (Basel) 2020 Nov 24;12(12). Epub 2020 Nov 24.

Australian Prostate Cancer Research Centre-Queensland, Institute of Health and Biomedical Innovation, Queensland University of Technology, Translational Research Institute, Brisbane, QLD 4102, Australia.

Recent reports have suggested the role of kallikrein-related peptidase 4 (KLK4) to be that of remodeling the tumor microenvironment in many cancers, including prostate cancer. Notably, these studies have suggested a pro-tumorigenic role for KLK4, especially in prostate cancer. However, these have been primarily in vitro studies, with limited in vivo studies performed to date. Herein, we employed an orthotopic inoculation xenograft model to mimic the growth of primary tumors, and an intracardiac injection to induce metastatic dissemination to determine the in vivo tumorigenic effects of KLK4 overexpressed in PC3 prostate cancer cells. Notably, we found that these KLK4-expressing cells gave rise to smaller localized tumors and decreased metastases than the parent PC-3 cells. To our knowledge, this is the first report of an anti-tumorigenic effect of KLK4, particularly in prostate cancer. These findings also provide a cautionary tale of the need for in vivo analyses to substantiate in vitro experimental data.
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http://dx.doi.org/10.3390/cancers12123501DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761350PMC
November 2020

Human CLEC9A antibodies deliver NY-ESO-1 antigen to CD141 dendritic cells to activate naïve and memory NY-ESO-1-specific CD8 T cells.

J Immunother Cancer 2020 07;8(2)

Mater Research Institute, University of Queensland, Woolloongabba, Queensland, Australia

Background: Dendritic cells (DCs) are crucial for the efficacy of cancer vaccines, but current vaccines do not harness the key cDC1 subtype required for effective CD8 T-cell-mediated tumor immune responses. Vaccine immunogenicity could be enhanced by specific delivery of immunogenic tumor antigens to CD141 DCs, the human cDC1 equivalent. CD141 DCs exclusively express the C-type-lectin-like receptor CLEC9A, which is important for the regulation of CD8 T cell responses. This study developed a new vaccine that harnesses a human anti-CLEC9A antibody to specifically deliver the immunogenic tumor antigen, NY-ESO-1 (New York esophageal squamous cell carcinoma 1), to human CD141 DCs. The ability of the CLEC9A-NY-ESO-1 antibody to activate NY-ESO-1-specific naïve and memory CD8 T cells was examined and compared with a vaccine comprised of a human DEC-205-NY-ESO-1 antibody that targets all human DCs.

Methods: Human anti-CLEC9A, anti-DEC-205 and isotype control IgG4 antibodies were genetically fused to NY-ESO-1 polypeptide. Cross-presentation to NY-ESO-1-epitope-specific CD8 T cells and reactivity of T cell responses in patients with melanoma were assessed by interferon γ (IFNγ) production following incubation of CD141 DCs and patient peripheral blood mononuclear cells with targeting antibodies. Humanized mice containing human DC subsets and a repertoire of naïve NY-ESO-1-specific CD8 T cells were used to investigate naïve T cell priming. T cell effector function was measured by expression of IFNγ, MIP-1β, tumor necrosis factor and CD107a and by lysis of target tumor cells.

Results: CLEC9A-NY-ESO-1 antibodies (Abs) were effective at mediating delivery and cross-presentation of multiple NY-ESO-1 epitopes by CD141 DCs for activation of NY-ESO-1-specific CD8 T cells. When benchmarked to NY-ESO-1 conjugated to an untargeted control antibody or to anti-human DEC-205, CLEC9A-NY-ESO-1 was superior at ex vivo reactivation of NY-ESO-1-specific T cell responses in patients with melanoma. Moreover, CLEC9A-NY-ESO-1 induced priming of naïve NY-ESO-1-specific CD8 T cells with polyclonal effector function and potent tumor killing capacity in vitro.

Conclusions: These data advocate human CLEC9A-NY-ESO-1 Ab as an attractive strategy for specific targeting of CD141 DCs to enhance tumor immunogenicity in NY-ESO-1-expressing malignancies.
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http://dx.doi.org/10.1136/jitc-2020-000691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7394304PMC
July 2020

Human CLEC9A antibodies deliver Wilms' tumor 1 (WT1) antigen to CD141 dendritic cells to activate naïve and memory WT1-specific CD8 T cells.

Clin Transl Immunology 2020 12;9(6):e1141. Epub 2020 Jun 12.

Cancer Immunotherapies Laboratory Mater Research Institute - The University of Queensland Translational Research Institute Woolloongabba Australia 4102 Australia.

Objectives: Vaccines that prime Wilms' tumor 1 (WT1)-specific CD8 T cells are attractive cancer immunotherapies. However, immunogenicity and clinical response rates may be enhanced by delivering WT1 to CD141 dendritic cells (DCs). The C-type lectin-like receptor CLEC9A is expressed exclusively by CD141 DCs and regulates CD8 T-cell responses. We developed a new vaccine comprising a human anti-CLEC9A antibody fused to WT1 and investigated its capacity to target human CD141 DCs and activate naïve and memory WT1-specific CD8 T cells.

Methods: WT1 was genetically fused to antibodies specific for human CLEC9A, DEC-205 or β-galactosidase (untargeted control). Activation of WT1-specific CD8 T-cell lines following cross-presentation by CD141 DCs was quantified by IFNγ ELISPOT. Humanised mice reconstituted with human immune cell subsets, including a repertoire of naïve WT1-specific CD8 T cells, were used to investigate naïve WT1-specific CD8 T-cell priming.

Results: The CLEC9A-WT1 vaccine promoted cross-presentation of WT1 epitopes to CD8 T cells and mediated priming of naïve CD8 T cells more effectively than the DEC-205-WT1 and untargeted control-WT1 vaccines.

Conclusions: Delivery of WT1 to CD141 DCs via CLEC9A stimulates CD8 T cells more potently than either untargeted delivery or widespread delivery to all Ag-presenting cells via DEC-205, suggesting that cross-presentation by CD141 DCs is sufficient for effective CD8 T-cell priming in humans. The CLEC9A-WT1 vaccine is a promising candidate immunotherapy for malignancies that express WT1.
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http://dx.doi.org/10.1002/cti2.1141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7292901PMC
June 2020

The long non-coding RNA GHSROS facilitates breast cancer cell migration and orthotopic xenograft tumour growth.

Int J Oncol 2019 Dec 4;55(6):1223-1236. Epub 2019 Oct 4.

Ghrelin Research Group, Translational Research Institute-Institute of Health and Biomedical Innovation, School of Biomedical Sciences, Queensland University of Technology, Brisbane, Queensland 4102, Australia.

Recent evidence suggests that numerous long non‑coding RNAs (lncRNAs) are dysregulated in cancer, and have critical roles in tumour development and progression. The present study investigated the ghrelin receptor antisense lncRNA growth hormone secretagogue receptor opposite strand (GHSROS) in breast cancer. Reverse transcription‑quantitative polymerase chain reaction revealed that GHSROS expression was significantly upregulated in breast tumour tissues compared with normal breast tissue. Induced overexpression of GHSROS in the MDA‑MB‑231 breast cancer cell line significantly increased cell migration in vitro, without affecting cell proliferation, a finding similar to our previous study on lung cancer cell lines. Microarray analysis revealed a significant repression of a small cluster of major histocompatibility class II genes and enrichment of immune response pathways; this phenomenon may allow tumour cells to better evade the immune system. Ectopic overexpression of GHSROS in the MDA‑MB‑231 cell line significantly increased orthotopic xenograft growth in mice, suggesting that in vitro culture does not fully capture the function of this lncRNA. This study demonstrated that GHSROS may serve a relevant role in breast cancer. Further studies are warranted to explore the function and therapeutic potential of this lncRNA in breast cancer progression.
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http://dx.doi.org/10.3892/ijo.2019.4891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6831199PMC
December 2019

Simple, rapid and inexpensive typing of common HLA class I alleles for immunological studies.

J Immunol Methods 2019 02 8;465:72-76. Epub 2018 Dec 8.

University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Woolloongabba, Queensland, Australia. Electronic address:

Current HLA-typing methods are typically designed to provide exquisitely-detailed identification of multiple HLA-alleles to satisfy the requirements for organ and bone marrow transplantation or genetic studies. Many human immunological studies, on the other hand, focus around only a small number of HLA alleles that are abundant or of relevance to specific diseases. Consequently, for such studies, many HLA typing approaches are not cost-effective and are potentially complicated, slow and not easily performed in-house. Work-flow would be streamlined by a simple, inexpensive and rapid typing method able to be performed in-house. We outline a straightforward approach that provides appropriate data for much immunological research. In a predominantly Caucasian population, flow cytometry using anti-HLA-A2, -B8 and -B7 antibodies consistently and accurately screened for samples carrying the highly-abundant HLA class I alleles HLA-A*02:01, -B*08:01 and -B*07:02 that form the focus of immunological studies. Next, we describe a straightforward and simple strategy for design and use of allele-specific PCR primers to identify, at high-resolution, alleles of interest. When combined with a simple gDNA extraction technique this provides reliable, simple and inexpensive in-house HLA typing demonstrated here for highly-abundant HLA class I alleles.
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http://dx.doi.org/10.1016/j.jim.2018.12.002DOI Listing
February 2019

Bio-Guided Fractionation of Papaya Leaf Juice for Delineating the Components Responsible for the Selective Anti-proliferative Effects on Prostate Cancer Cells.

Front Pharmacol 2018 16;9:1319. Epub 2018 Nov 16.

School of Biomedical Sciences, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, QLD, Australia.

Alternative therapies against cancer cells with minimal or no effect on healthy tissues are highly sought after. Prostate cancer (PCa) is the second most frequently diagnosed malignancy in males. The L. leaf extract has been traditionally used by Australian aboriginal people for anticancer properties. In this study, medium polar fraction of papaya leaf extract that had shown anti-proliferative activity in PCa cell lines , in earlier studies, was further fractionated to 28 fractions by semi-preparative HPLC. Nine of these fractions were identified to possess selective anti-proliferative responses on PCa cells in comparison to non-cancerous cells of prostate gland origin. When these nine sub-fractions were mixed in various combinations, a combination containing six of the specific fractions (FC-3) showed the best potency. FC3 inhibited the growth of BPH-1, PC-3, and LNCaP cells in a concentration-dependent manner with an IC value <20 μg/mL, while (unlike paclitaxel, the positive control) minimal effect was observed on the proliferation of non-cancerous, WPMY-1 and RWPE-1cells. Furthermore, synergistic interaction of FC-3 with paclitaxel was observed with combination index values in the range of 0.89-0.98 and 0.85-1.10 on PC-3 and LNCaP cells, respectively. Untargeted qualitative analysis using UHPLC (Ultra High-Performance Liquid Chromatography)-QToF (Quadrupole Time of-Flight) mass spectrometry and screening against the METLIN database indicated presence of multiple known anticancer compounds in the FC-3 extract. These outcomes show that the potent and selective anti-proliferative effects are due to a range of bio-active compounds within the medium polar fraction of papaya leaf juice.
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http://dx.doi.org/10.3389/fphar.2018.01319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6250729PMC
November 2018

A microsatellite repeat in PCA3 long non-coding RNA is associated with prostate cancer risk and aggressiveness.

Sci Rep 2017 12 4;7(1):16862. Epub 2017 Dec 4.

Australian Prostate Cancer Research Centre - Queensland, Translational Research Institute, Brisbane, 4102, Australia.

Short tandem repeats (STRs) are repetitive sequences of a polymorphic stretch of two to six nucleotides. We hypothesized that STRs are associated with prostate cancer development and/or progression. We undertook RNA sequencing analysis of prostate tumors and adjacent non-malignant cells to identify polymorphic STRs that are readily expressed in these cells. Most of the expressed STRs in the clinical samples mapped to intronic and intergenic DNA. Our analysis indicated that three of these STRs (TAAA-ACTG2, TTTTG-TRIB1, and TG-PCA3) are polymorphic and differentially expressed in prostate tumors compared to adjacent non-malignant cells. TG-PCA3 STR expression was repressed by the anti-androgen drug enzalutamide in prostate cancer cells. Genetic analysis of prostate cancer patients and healthy controls (N > 2,000) showed a significant association of the most common 11 repeat allele of TG-PCA3 STR with prostate cancer risk (OR = 1.49; 95% CI 1.11-1.99; P = 0.008). A significant association was also observed with aggressive disease (OR = 2.00; 95% CI 1.06-3.76; P = 0.031) and high mortality rates (HR = 3.0; 95% CI 1.03-8.77; P = 0.045). We propose that TG-PCA3 STR has both diagnostic and prognostic potential for prostate cancer. We provided a proof of concept to be applied to other RNA sequencing datasets to identify disease-associated STRs for future clinical exploratory studies.
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http://dx.doi.org/10.1038/s41598-017-16700-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5715103PMC
December 2017

Selective anti-proliferative activities of Carica papaya leaf juice extracts against prostate cancer.

Biomed Pharmacother 2017 May 27;89:515-523. Epub 2017 Feb 27.

School of Pharmacy, The University of Queensland, Brisbane, Queensland, Australia. Electronic address:

Background: Prostate cancer (PCa) is the leading cause of cancer related deaths in men. Carica papaya is a popular tropical plant that has been traditionally used for its nutritional and medicinal properties.

Methods: We investigated the anti-proliferative responses of papaya leaf juice (LJP) and its various extracts ("biological"- in vitro digested, "physical"- size exclusion, and "chemical"-solvent extraction) on a range of cell lines representing benign hyperplasia, tumorigenic and normal cells of prostate origin.

Results: Time course analysis (by 24h, 48h and 72h) of LJP (1-0.1mg/mL) before and after in vitro digestion, and of molecular weight based fractions of LJP showed anti-proliferative responses. The medium polarity fraction of LJP (0.03-0.003mg/mL) after 72h exposure showed potent growth inhibitory (IC=0.02-0.07mg/mL) and cytotoxic activities on all prostate cells, with the exception of the normal (RWPE-1 and WPMY-1) cells. Flow cytometry analysis showed S phase cell cycle arrest and apoptosis as a possible mechanism for these activities. Medium polar fraction of LJP also inhibited migration and adhesion of metastatic PC-3 cells.

Conclusion: This is the first report suggesting selective anti-proliferative and anti-metastatic attributes of LJP extract against prostatic diseases, including PCa.
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http://dx.doi.org/10.1016/j.biopha.2017.02.050DOI Listing
May 2017

Multi-species sequence comparison reveals conservation of ghrelin gene-derived splice variants encoding a truncated ghrelin peptide.

Endocrine 2016 Jun 20;52(3):609-17. Epub 2016 Jan 20.

Comparative and Endocrine Biology Laboratory, Translational Research Institute-Institute of Health and Biomedical Innovation (TRI-IHBI), Queensland University of Technology, Woolloongabba, QLD, 4102, Australia.

The peptide hormone ghrelin is a potent orexigen produced predominantly in the stomach. It has a number of other biological actions, including roles in appetite stimulation, energy balance, the stimulation of growth hormone release and the regulation of cell proliferation. Recently, several ghrelin gene splice variants have been described. Here, we attempted to identify conserved alternative splicing of the ghrelin gene by cross-species sequence comparisons. We identified a novel human exon 2-deleted variant and provide preliminary evidence that this splice variant and in1-ghrelin encode a C-terminally truncated form of the ghrelin peptide, termed minighrelin. These variants are expressed in humans and mice, demonstrating conservation of alternative splicing spanning 90 million years. Minighrelin appears to have similar actions to full-length ghrelin, as treatment with exogenous minighrelin peptide stimulates appetite and feeding in mice. Forced expression of the exon 2-deleted preproghrelin variant mirrors the effect of the canonical preproghrelin, stimulating cell proliferation and migration in the PC3 prostate cancer cell line. This is the first study to characterise an exon 2-deleted preproghrelin variant and to demonstrate sequence conservation of ghrelin gene-derived splice variants that encode a truncated ghrelin peptide. This adds further impetus for studies into the alternative splicing of the ghrelin gene and the function of novel ghrelin peptides in vertebrates.
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http://dx.doi.org/10.1007/s12020-015-0848-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4879156PMC
June 2016

Fusion transcript loci share many genomic features with non-fusion loci.

BMC Genomics 2015 Dec 1;16:1021. Epub 2015 Dec 1.

Australian Prostate Cancer Research Centre - Queensland, Translational Research Institute, Brisbane, Australia.

Background: Fusion transcripts are found in many tissues and have the potential to create novel functional products. Here, we investigate the genomic sequences around fusion junctions to better understand the transcriptional mechanisms mediating fusion transcription/splicing. We analyzed data from prostate (cancer) cells as previous studies have shown extensively that these cells readily undergo fusion transcription.

Results: We used the FusionMap program to identify high-confidence fusion transcripts from RNAseq data. The RNAseq datasets were from our (N = 8) and other (N = 14) clinical prostate tumors with adjacent non-cancer cells, and from the LNCaP prostate cancer cell line that were mock-, androgen- (DHT), and anti-androgen- (bicalutamide, enzalutamide) treated. In total, 185 fusion transcripts were identified from all RNAseq datasets. The majority (76%) of these fusion transcripts were 'read-through chimeras' derived from adjacent genes in the genome. Characterization of sequences at fusion loci were carried out using a combination of the FusionMap program, custom Perl scripts, and the RNAfold program. Our computational analysis indicated that most fusion junctions (76%) use the consensus GT-AG intron donor-acceptor splice site, and most fusion transcripts (85%) maintained the open reading frame. We assessed whether parental genes of fusion transcripts have the potential to form complementary base pairing between parental genes which might bring them into physical proximity. Our computational analysis of sequences flanking fusion junctions at parental loci indicate that these loci have a similar propensity as non-fusion loci to hybridize. The abundance of repetitive sequences at fusion and non-fusion loci was also investigated given that SINE repeats are involved in aberrant gene transcription. We found few instances of repetitive sequences at both fusion and non-fusion junctions. Finally, RT-qPCR was performed on RNA from both clinical prostate tumors and adjacent non-cancer cells (N = 7), and LNCaP cells treated as above to validate the expression of seven fusion transcripts and their respective parental genes. We reveal that fusion transcript expression is similar to the expression of parental genes.

Conclusions: Fusion transcripts maintain the open reading frame, and likely use the same transcriptional machinery as non-fusion transcripts as they share many genomic features at splice/fusion junctions.
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http://dx.doi.org/10.1186/s12864-015-2235-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4667522PMC
December 2015

Fine-mapping of the HNF1B multicancer locus identifies candidate variants that mediate endometrial cancer risk.

Hum Mol Genet 2015 Mar 6;24(5):1478-92. Epub 2014 Nov 6.

Department of Genetics, Portuguese Oncology Institute, Porto, Portugal, Biomedical Sciences Institute (ICBAS), University of Porto, Porto, Portugal.

Common variants in the hepatocyte nuclear factor 1 homeobox B (HNF1B) gene are associated with the risk of Type II diabetes and multiple cancers. Evidence to date indicates that cancer risk may be mediated via genetic or epigenetic effects on HNF1B gene expression. We previously found single-nucleotide polymorphisms (SNPs) at the HNF1B locus to be associated with endometrial cancer, and now report extensive fine-mapping and in silico and laboratory analyses of this locus. Analysis of 1184 genotyped and imputed SNPs in 6608 Caucasian cases and 37 925 controls, and 895 Asian cases and 1968 controls, revealed the best signal of association for SNP rs11263763 (P = 8.4 × 10(-14), odds ratio = 0.86, 95% confidence interval = 0.82-0.89), located within HNF1B intron 1. Haplotype analysis and conditional analyses provide no evidence of further independent endometrial cancer risk variants at this locus. SNP rs11263763 genotype was associated with HNF1B mRNA expression but not with HNF1B methylation in endometrial tumor samples from The Cancer Genome Atlas. Genetic analyses prioritized rs11263763 and four other SNPs in high-to-moderate linkage disequilibrium as the most likely causal SNPs. Three of these SNPs map to the extended HNF1B promoter based on chromatin marks extending from the minimal promoter region. Reporter assays demonstrated that this extended region reduces activity in combination with the minimal HNF1B promoter, and that the minor alleles of rs11263763 or rs8064454 are associated with decreased HNF1B promoter activity. Our findings provide evidence for a single signal associated with endometrial cancer risk at the HNF1B locus, and that risk is likely mediated via altered HNF1B gene expression.
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http://dx.doi.org/10.1093/hmg/ddu552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4321445PMC
March 2015

Cloning and tissue distribution of novel splice variants of the ovine ghrelin gene.

BMC Vet Res 2014 Sep 6;10:211. Epub 2014 Sep 6.

Background: The ghrelin axis is involved in the regulation of metabolism, energy balance, and the immune, cardiovascular and reproductive systems. The manipulation of this axis has potential for improving economically valuable traits in production animals, and polymorphisms in the ghrelin (GHRL) and ghrelin receptor (GHSR) genes have been associated with growth and carcass traits. Here we investigate the structure and expression of the ghrelin gene (GHRL) in sheep, Ovis aries.

Results: We identify two ghrelin mRNA isoforms, which we have designated Δex2 preproghrelin and Δex2,3 preproghrelin. Expression of Δex2,3 preproghrelin is likely to be restricted to ruminants, and would encode truncated ghrelin and a novel C-terminal peptide. Both Δex2 preproghrelin and canonical preproghrelin mRNA isoforms were expressed in a range of tissues. Expression of the Δex2,3 preproghrelin isoform, however, was restricted to white blood cells (WBC; where the wild-type preproghrelin isoform is not co-expressed), and gastrointestinal tissues. Expression of Δex2 preproghrelin and Δex2,3 preproghrelin mRNA was elevated in white blood cells in response to parasitic worm (helminth) infection in genetically susceptible sheep, but not in resistant sheep.

Conclusions: The restricted expression of the novel preproghrelin variants and their distinct WBC expression pattern during parasite infection may indicate a novel link between the ghrelin axis and metabolic and immune function in ruminants.
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http://dx.doi.org/10.1186/s12917-014-0211-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4172912PMC
September 2014

Ghrelin O-acyltransferase (GOAT) is expressed in prostate cancer tissues and cell lines and expression is differentially regulated in vitro by ghrelin.

Reprod Biol Endocrinol 2013 Jul 23;11:70. Epub 2013 Jul 23.

Ghrelin Research Group, Translational Research Institute - Institute of Health and Biomedical Innovation, Queensland University of Technology, 37 Kent St, Woolloongabba, Queensland, 4102, Australia.

Background: Ghrelin is a 28 amino acid peptide hormone that is expressed in the stomach and a range of peripheral tissues, where it frequently acts as an autocrine/paracrine growth factor. Ghrelin is modified by a unique acylation required for it to activate its cognate receptor, the growth hormone secretagogue receptor (GHSR), which mediates many of the actions of ghrelin. Recently, the enzyme responsible for adding the fatty acid residue (octanoyl/acyl group) to the third amino acid of ghrelin, GOAT (ghrelin O-acyltransferase), was identified.

Methods: We used cell culture, quantitative real-time reverse transcription (RT)-PCR and immunohistochemistry to demonstrate the expression of GOAT in prostate cancer cell lines and tissues from patients. Real-time RT-PCR was used to demonstrate the expression of prohormone convertase (PC)1/3, PC2 and furin in prostate cancer cell lines. Prostate-derived cell lines were treated with ghrelin and desacyl ghrelin and the effect on GOAT expression was measured using quantitative RT-PCR.

Results: We have demonstrated that GOAT mRNA and protein are expressed in the normal prostate and human prostate cancer tissue samples. The RWPE-1 and RWPE-2 normal prostate-derived cell lines and the LNCaP, DU145, and PC3 prostate cancer cell lines express GOAT and at least one other enzyme that is necessary to produce mature, acylated ghrelin from proghrelin (PC1/3, PC2 or furin). Finally, ghrelin, but not desacyl ghrelin (unacylated ghrelin), can directly regulate the expression of GOAT in the RWPE-1 normal prostate derived cell line and the PC3 prostate cancer cell line. Ghrelin treatment (100nM) for 6 hours significantly decreased GOAT mRNA expression two-fold (P < 0.05) in the PC3 prostate cancer cell line, however, ghrelin did not regulate GOAT expression in the DU145 and LNCaP prostate cancer cell lines.

Conclusions: This study demonstrates that GOAT is expressed in prostate cancer specimens and cell lines. Ghrelin regulates GOAT expression, however, this is likely to be cell-type specific. The expression of GOAT in prostate cancer supports the hypothesis that the ghrelin axis has autocrine/paracrine roles. We propose that the RWPE-1 prostate cell line and the PC3 prostate cancer cell line may be useful for investigating GOAT regulation and function.
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http://dx.doi.org/10.1186/1477-7827-11-70DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3724588PMC
July 2013

Identification of a long non-coding RNA gene, growth hormone secretagogue receptor opposite strand, which stimulates cell migration in non-small cell lung cancer cell lines.

Int J Oncol 2013 Aug 30;43(2):566-74. Epub 2013 May 30.

Ghrelin Research Group, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia.

The molecular mechanisms involved in non‑small cell lung cancer tumourigenesis are largely unknown; however, recent studies have suggested that long non-coding RNAs (lncRNAs) are likely to play a role. In this study, we used public databases to identify an mRNA-like, candidate long non-coding RNA, GHSROS (GHSR opposite strand), transcribed from the antisense strand of the ghrelin receptor gene, growth hormone secretagogue receptor (GHSR). Quantitative real-time RT-PCR revealed higher expression of GHSROS in lung cancer tissue compared to adjacent, non-tumour lung tissue. In common with many long non-coding RNAs, GHSROS is 5' capped and 3' polyadenylated (mRNA-like), lacks an extensive open reading frame and harbours a transposable element. Engineered overexpression of GHSROS stimulated cell migration in the A549 and NCI-H1299 non-small cell lung cancer cell lines, but suppressed cell migration in the Beas-2B normal lung-derived bronchoepithelial cell line. This suggests that GHSROS function may be dependent on the oncogenic context. The identification of GHSROS, which is expressed in lung cancer and stimulates cell migration in lung cancer cell lines, contributes to the growing number of non-coding RNAs that play a role in the regulation of tumourigenesis and metastatic cancer progression.
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http://dx.doi.org/10.3892/ijo.2013.1969DOI Listing
August 2013

Paclitaxel resistance and multicellular spheroid formation are induced by kallikrein-related peptidase 4 in serous ovarian cancer cells in an ascites mimicking microenvironment.

PLoS One 2013 25;8(2):e57056. Epub 2013 Feb 25.

Cancer Program, Institute of Health and Biomedical Innovation and Faculty of Sciences and Technology, Queensland University of Technology, Kelvin Grove, Queensland, Australia.

High tumor kallikrein-related-peptidase 4 (KLK4) levels are associated with a poor outcome for women with serous epithelial ovarian cancer (EOC), for which peritoneal dissemination and chemoresistance are key events. To determine the role of KLK4 in these events, we examined KLK4-transfected SKOV-3 and endogenous KLK4 expressing OVCA432 cells in 3-dimensional (3D) suspension culture to mimic the ascites microenvironment. KLK4-SKOV-3 cells formed multicellular aggregates (MCAs) as seen in ascites, as did SKOV-3 cells treated with active KLK4. MCA formation was reduced by treatment with a KLK4 blocking antibody or the selective active site KLK4 sunflower trypsin inhibitor (SFTI-FCQR). KLK4-MCAs formed larger cancer cell foci in mesothelial cell monolayers than those formed by vector and native SKOV-3 cells, suggesting KLK4-MCAs are highly invasive in the peritoneal microenvironment. A high level of KLK4 is expressed by ascitic EOC cells compared to matched primary tumor cells, further supporting its role in the ascitic microenvironment. Interestingly, KLK4 transfected SKOV-3 cells expressed high levels of the KLK4 substrate, urokinase plasminogen activator (uPA), particularly in 3D-suspension, and high levels of both KLK4 and uPA were observed in patient cells taken from ascites. Importantly, the KLK4-MCAs were paclitaxel resistant which was reversed by SFTI-FCQR and to a lesser degree by the general serine protease inhibitor, Aprotinin, suggesting that in addition to uPA, other as yet unidentified substrates of KLK4 must be involved. Nonetheless, these data suggest that KLK4 inhibition, in conjunction with paclitaxel, may improve the outcome for women with serous epithelial ovarian cancer and high KLK4 levels in their tumors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0057056PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3581584PMC
September 2013

The ghrelin axis--does it have an appetite for cancer progression?

Endocr Rev 2012 Dec 23;33(6):849-91. Epub 2012 Jul 23.

Ghrelin Research Group, Institute of Health and Biomedical Innovation, Queensland University of Technology and Australian Prostate Cancer Research Centre-Queensland, Brisbane, Queensland 4001, Australia.

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.
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http://dx.doi.org/10.1210/er.2011-1007DOI Listing
December 2012

Ghrelin and cancer.

Mol Cell Endocrinol 2011 Jun 25;340(1):65-9. Epub 2011 May 25.

Queensland University of Technology, Brisbane, Australia.

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.
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http://dx.doi.org/10.1016/j.mce.2011.04.013DOI Listing
June 2011

The expanding roles of the ghrelin-gene derived peptide obestatin in health and disease.

Mol Cell Endocrinol 2011 Jun 1;340(1):111-7. Epub 2011 Apr 1.

Queensland University of Technology, Queensland, Brisbane, Australia.

Obestatin is a 23 amino acid, ghrelin gene-derived peptide hormone produced in the stomach and a range of other tissues throughout the body. While it was initially reported that obestatin opposed the actions of ghrelin with regards to appetite and food intake, it is now clear that obestatin is not an endogenous ghrelin antagonist, but it is a multi-functional peptide hormone in its own right. In this review we will discuss the controversies associated with the discovery of obestatin and explore emerging central and peripheral roles of obestatin, which includes adipogenesis, pancreatic homeostasis and cancer.
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http://dx.doi.org/10.1016/j.mce.2011.03.018DOI Listing
June 2011

Kallikrein-related peptidase 7 promotes multicellular aggregation via the alpha(5)beta(1) integrin pathway and paclitaxel chemoresistance in serous epithelial ovarian carcinoma.

Cancer Res 2010 Apr 23;70(7):2624-33. Epub 2010 Mar 23.

Hormone Dependent Cancer Program, Institute of Health and Biomedical Innovation and School of Life Sciences, Queensland University of Technology, Kelvin Grove, Queensland, Australia.

Kallikrein-related peptidase 7 (KLK7) is upregulated in epithelial ovarian carcinoma (EOC) with high levels correlated with poor prognosis. However, the mechanisms underlying this relationship and the role of KLK7 in EOC progression are unknown. We report that two different KLK7 transcripts, KLK7-253 and KLK7-181, are simultaneously expressed in high-grade serous EOC. Multicellular aggregates (MCA), which promote cell survival and chemoresistance, were observed in SKOV-3 cells stably overexpressing KLK7-253 in particular. Importantly, these MCAs invade into a monolayer of mesothelial cells and form cancer cell foci. Blocking MCA using antibodies against KLK7 and alpha(5)beta(1) and beta(1) integrins confirmed the involvement of KLK7 and integrin-regulated cell adhesion. Increased levels of alpha(5)/beta(1) integrins and enhanced attachment to fibronectin and vitronectin, which was blocked with an anti-beta(1) integrin antibody, were also observed. Finally, Western blot and immunohistochemistry showed higher KLK7 and alpha(5)/beta(1) integrin levels in serous EOC cells from ascites and tumor samples from chemotherapy nonresponders with short postsurvival times. Additionally, both KLK7-253 and KLK7-181 clones were more resistant to paclitaxel treatment in vitro. These findings suggest a mechanism for the association of high KLK7 levels with chemoresistance and poor prognosis for serous EOC patients by promotion of peritoneal dissemination and reinvasion via increased MCA and alpha(5)beta(1) integrin-dependent cell adhesion.
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http://dx.doi.org/10.1158/0008-5472.CAN-09-3415DOI Listing
April 2010

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

Clin Exp Pharmacol Physiol 2010 Jan 29;37(1):125-31. Epub 2009 Jun 29.

Institute of Health and Biomedical Innovation and School of Life Sciences, Queensland University of Technology, Brisbane, Queensland, Australia.

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. 2. The present article reviews recent findings in the study of ghrelin and its receptor that suggest that the ghrelin gene locus may give rise to a number of functional molecules (peptides and RNA transcripts) in addition to ghrelin. 3. The ghrelin gene encodes a precursor protein, preproghrelin, from which ghrelin and other potentially active peptides are derived by alternative mRNA splicing and/or proteolytic processing. The metabolic role of the peptide obestatin, derived from the preproghrelin C-terminal region, is contentious. However, obestatin has direct effects on cell proliferation. 4. The regulation of ghrelin expression and the mechanisms through which the peptide products arise are unclear. We have recently re-examined the organization of the ghrelin gene and identified several novel exons and transcripts. One transcript, which lacks the ghrelin-coding region of preproghrelin, contains the coding sequence of obestatin. 5. Furthermore, we have identified an overlapping gene on the antisense strand of ghrelin, namely GHRLOS, which generates transcripts that may function as non-coding regulatory RNAs or code for novel, short bioactive peptides. 6. The identification of these novel ghrelin-gene related transcripts and peptides raises critical questions regarding their physiological function and their potential role in obesity, diabetes and cancer.
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http://dx.doi.org/10.1111/j.1440-1681.2009.05241.xDOI Listing
January 2010

Substrate-guided design of a potent and selective kallikrein-related peptidase inhibitor for kallikrein 4.

Chem Biol 2009 Jun;16(6):633-43

Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland 4059, Australia.

Human kallikrein-related peptidase 4 (KLK4/prostase), a trypsin-like serine protease, is a potential target for prostate cancer treatment because of its proteolytic ability to activate many tumorigenic and metastatic pathways including the protease activated receptors (PARs). Currently there are no KLK4-specific small-molecule inhibitors available for therapeutic development. Here we re-engineer the naturally occurring sunflower trypsin inhibitor to selectively block the proteolytic activity of KLK4 and prevent stimulation of PAR activity in a cell-based system. The re-engineered inhibitor was designed using a combination of molecular modeling and sparse matrix substrate screening.
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http://dx.doi.org/10.1016/j.chembiol.2009.05.008DOI Listing
June 2009