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

It is now appreciated that long non-coding RNAs (lncRNAs) are important players in orchestrating cancer progression. In this study we characterized GHSROS, 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 GHSROS alters the expression of cancer-associated genes. Functional analyses in vitro showed that GHSROS mediates tumor growth, migration and survival, and resistance to the cytotoxic drug docetaxel. Increased cellular proliferation of GHSROS-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, GHSROS modulates the expression of PPP2R2C, the loss of which may drive androgen receptor pathway-independent prostate tumor progression in a subset of prostate cancers. Collectively, our findings suggest that GHSROS can reprogram prostate cancer cells toward a more aggressive phenotype and that this lncRNA may represent a potential therapeutic target

The Rag1-/- mouse: Establishing links between obesity and prostate cancer

Obesity and metabolic syndrome are associated with increased risk of advanced prostate cancer, more aggressive high grade disease and increased risk of death from prostate cancer. Androgen targeted therapies can rapidly induce symptoms similar to metabolic syndrome in patients, affecting health and quality of life. The development of therapeutics which could target metabolic dysregulation in addition to cancer growth is urgently needed. This thesis established a new mouse model of metabolic syndrome and assessed components of the ghrelin axis as treatments of prostate cancer. Importantly, an association between the obesity hormone neurotensin, metabolic dysregulation, and androgen-independent prostate cancer was revealed

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

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

Limited short-term effects on human prostate cancer xenograft growth and epidermal growth factor receptor gene expression by the ghrelin receptor antagonist [D-Lys3]-GHRP-6

Purpose The ghrelin axis regulates many physiological functions (including appetite, metabolism, and energy balance) and plays a role in disease processes. As ghrelin stimulates prostate cancer proliferation, the ghrelin receptor antagonist [D-Lys3]-GHRP-6 is a potential treatment for castrate-resistant prostate cancer and for preventing the metabolic consequences of androgen-targeted therapies. We therefore explored the effect of [D-Lys3]-GHRP-6 on PC3 prostate cancer xenograft growth. Methods NOD/SCID mice with PC3 prostate cancer xenografts were administered 20 nmoles/mouse [D-Lys3]-GHRP-6 daily by intraperitoneal injection for 14 days and tumour volume and weight were measured. RNA sequencing of tumours was conducted to investigate expression changes following [D-Lys3]-GHRP-6 treatment. A second experiment, extending treatment time to 18 days and including a higher dose of [D-Lys3]-GHRP-6 (200 nmoles/mouse/day), was undertaken to ensure repeatability. Results We demonstrate here that daily intraperitoneal injection of 20 nmoles/mouse [D-Lys3]-GHRP-6 reduces PC3 prostate cancer xenograft tumour volume and weight in NOD/SCID mice at two weeks post treatment initiation. RNA-sequencing revealed reduced expression of epidermal growth factor receptor (EGFR) in these tumours. Further experiments demonstrated that the effects of [D-Lys3]-GHRP-6 are transitory and lost after 18 days of treatment. Conclusions We show that [D-Lys3]-GHRP-6 has transitory effects on prostate xenograft tumours in mice, which rapidly develop an apparent resistance to the antagonist. Although further studies on [D-Lys3]-GHRP-6 are warranted, we suggest that daily treatment with the antagonist is not a suitable treatment for advanced prostate cancer

Insights from engraftable immunodeficient mouse models of hyperinsulinaemia

Hyperinsulinaemia, obesity and dyslipidaemia are independent and collective risk factors for many cancers. The long‐term effects of a 23% high fat diet (HFD) in two immunodeficient mouse strains (NOD/SCID and Rag1−/−), and the effect of diet‐induced hyperinsulinaemia on human prostate cancer cell line xenograft growth, were investigated. Rag1−/− and NOD/SCID HFD‐fed mice demonstrated diet‐induced impairments in glucose tolerance at 16 and 23 weeks post weaning. Rag1−/− mice developed significantly higher fasting insulin levels (2.16 ± 1.01 ng/mL, P = .01) and increased insulin resistance (6.70 ± 1.68 HOMA‐IR, P = .01) compared to normal chow‐fed controls (0.71 ± 0.12 ng/mL and 2.91 ± 0.42 HOMA‐IR), however, alterations in insulin resistance were not observed in the NOD/SCID strain..

The lncRNA GHSROS mediates tumour growth and expression of genes associated with metastasis and adverse outcome

Long non-coding RNAs (lncRNAs) play key regulatory roles in cancer progression and are novel therapeutic targets. We recently discovered an lncRNA termed GHSROS (GHSR opposite strand) on the antisense strand of the ghrelin receptor gene (GHSR). Using quantitative RT-PCR, we demonstrated that GHSROS is highly expressed in a subset of high-grade prostate cancers. GHSROS over-expression significantly increased cell proliferation and migration in the PC3 (1.76 ± 0.18 fold, P<0.01; 1.54 ± 0.35 fold, P<0.05) and DU145 prostate cancer cell lines (1.74 fold ± 0.73 P<0.01; 1.94 ± 0.43 fold, P<0.01). Tumour volumes were significantly increased in both PC3 and DU145 GHSROS over-expressing prostate cancer cell line xenografts in NOD/SCID mice (P<0.05). Preliminary studies indicate that up-regulation of GHSROS confers resistance to the androgen receptor (AR) antagonist enzalutamide and the chemotherapeutic drug docetaxel, both of which are used to treat advanced prostate cancer. Through high-throughput transcriptome sequencing (RNA-seq) ~400 differentially expressed genes were identified in GHSROS over-expressing PC3 cells, demonstrating enrichment of genes associated with motility, survival, and regulation of cell growth. From this gene set, concept mapping and interrogation of publicly-available clinical prostate cancer data sets revealed a 34-gene signature associated with poorer disease outcome and metastatic progression in patients. Analysis of The Cancer Genome Atlas (TCGA) data suggests that the signature has potential as a prognostic indicator for disease free- or overall survival for numerous cancers. Finally, locked nucleic acid antisense oligonucleotide (LNA-ASO) inhibition of endogenous GHSROS expression reciprocally regulated cell growth (RNV124: -1.14 ± 0.06, P = 0.049 and RNV104L: -1.18 ± 0.05, P = 0.030), migration (RNV124: -1.96 ± 0.11, P = 0.004) and gene expression changes. Taken together, we therefore propose that GHSROS has clinical significance as a potential target for novel antisense therapy

The long non-coding RNA GHSROS mediates expression of genes associated with tumor growth, metastasis and adverse disease outcome

Long non-coding RNAs (lncRNAs) play key regulatory roles in cancer progression and are emerging therapeutic targets. The objective of this study was to investigate the expression and function of the lncRNA GHSROS in prostate cancer. Quantitative RT-PCR revealed that GHSROS is highly expressed in a subset of prostate cancers (Gleason score 8-10; z-score >1; Mann-Whitney-Wilcoxon test P=0.0021). Forced overexpression of the lncRNA stimulated cell migration in vitro in the PC3 (1.82 ± 0.35, P=0.006; Student’s t-test), DU145 (1.94 ± 0.34, P=0.017), and LNCaP (1.27 ± 0.02, P=0.0002) prostate cancer cell lines. Cell proliferation was increased in GHSROS overexpressing PC3 (3.36 ± 1.91, P=0.029), DU145 (1.749 ± 0.59, P=0.026), and LNCaP (1.39 ± 0.26, P=0.040) prostate cancer cell lines. These results were recapitulated in NOD/SCID mice, with increased tumor growth and Ki67 immunohistochemical staining in PC3 (P=0.0040) and DU145 (P = 0.036) xenografts overexpressing the lncRNA.High-throughput transcriptome sequencing (RNA-seq) identified 400 differentially expressed genes in GHSROS overexpressing PC3 cells, with enrichment of genes associated with motility, migration and regulation of cell growth. Further interrogation of the 400 gene set using Oncomine concept mapping, and interrogation of publicly-available clinical prostate cancer data sets, revealed a 34-gene signature associated with poorer disease outcome and metastatic progression. Preliminary analysis of The Cancer Genome Atlas (TCGA) data, suggest that the signature has potential as a prognostic indicator for disease free- or overall survival in numerous cancers. Finally, locked antisense oligonucleotide (LNA-ASO) inhibition of endogenous GHSROS reciprocally regulated cell growth (Student’s t-test; RNV124: -1.14 ± 0.06, P=0.049 and RNV104L: -1.18 ± 0.05, P=0.030, migration (RNV124: -1.96 ± 0.11, P=0.004) and gene expression changes, supporting the observations from forced GHSROS overexpression experiments.In summary, we provide evidence that GHSROS is a prostate cancer associated lncRNA that promotes a gene expression signature which enhances the propensity for metastasis and adverse disease outcomes. We also demonstrate that GHSROS can be targeted using antisense oligonucleotides. Further studies on this lncRNA may provide new prognostic and therapeutic opportunities

No effect of administration of unacylated ghrelin on subcutaneous PC3 xenograft growth in a Rag1−/− mouse model of metabolic dysfunction

Ghrelin is a peptide hormone which, when acylated, regulates appetite, energy balance and a range of other biological processes. Ghrelin predominately circulates in its unacylated form (unacylated ghrelin; UAG). UAG has a number of functions independent of acylated ghrelin, including modulation of metabolic parameters and cancer progression. UAG has also been postulated to antagonise some of the metabolic effects of acyl-ghrelin, including its effects on glucose and insulin regulation. In this study, Rag1-/- mice with high-fat diet-induced obesity and hyperinsulinaemia were subcutaneously implanted with PC3 prostate cancer xenografts to investigate the effect of UAG treatment on metabolic parameters and xenograft growth. Daily intraperitoneal injection of 100 μg/kg UAG had no effect on xenograft tumour growth in mice fed normal rodent chow or 23% high-fat diet. UAG significantly improved glucose tolerance in host Rag1-/- mice on a high-fat diet, but did not significantly improve other metabolic parameters. We propose that UAG is not likely to be an effective treatment for prostate cancer, with or without associated metabolic syndrome

Insights from engraftable immunodeficient mouse models of hyperinsulinaemia

Hyperinsulinaemia, obesity and dyslipidaemia are independent and collective risk factors for many cancers. Here, the long-term effects of a 23% Western high-fat diet (HFD) in two immunodeficient mouse strains (NOD/SCID and Rag1-/-) suitable for engraftment with human-derived tissue xenografts, and the effect of diet-induced hyperinsulinaemia on human prostate cancer cell line xenograft growth, were investigated. Rag1(-/-) and NOD/SCID HFD-fed mice demonstrated diet-induced impairments in glucose tolerance at 16 and 23 weeks post weaning. Rag1(-/-) mice developed significantly higher fasting insulin levels (2.16 +/- 1.01 ng/ml, P = 0.01) and increased insulin resistance (6.70 +/- 1.68 HOMA-IR, P = 0.01) compared to low-fat chow-fed mice (0.71 +/- 0.12 ng/ml and 2.91 +/- 0.42 HOMA-IR). This was not observed in the NOD/SCID strain. Hepatic steatosis was more extensive in Rag1(-/-) HFD-fed mice compared to NOD/SCID mice. Intramyocellular lipid storage was increased in Rag1(-/-) HFD-fed mice, but not in NOD/SCID mice. In Rag1(-/-) HFD-fed mice, LNCaP xenograft tumours grew more rapidly compared to low-fat chow-fed mice. This is the first characterisation of the metabolic effects of longterm Western HFD in two mouse strains suitable for xenograft studies. We conclude that Rag1(-/-) mice are an appropriate and novel xenograft model for studying the relationship between cancer and hyperinsulinaemia

No effect of unacylated ghrelin administration on subcutaneous PC3 xenograft growth or metabolic parameters in a Rag1-/- mouse model of metabolic dysfunction

Ghrelin is a peptide hormone which, when acylated, regulates appetite, energy balance and a range of other biological processes. Ghrelin predominately circulates in its unacylated form (unacylated ghrelin; UAG). UAG has a number of functions independent of acylated ghrelin, including modulation of metabolic parameters and cancer progression. UAG has also been postulated to antagonise some of the metabolic effects of acyl-ghrelin, including its effects on glucose and insulin regulation. In this study, Rag1-/- mice with high-fat diet-induced obesity and hyperinsulinaemia were subcutaneously implanted with PC3 prostate cancer xenografts to investigate the effect of UAG treatment on metabolic parameters and xenograft growth. Daily intraperitoneal injection of 100 μg/kg UAG had no effect on xenograft tumour growth in mice fed normal rodent chow or 23% high-fat diet. UAG significantly improved glucose tolerance in host Rag1-/- mice on a high-fat diet, but did not significantly improve other metabolic parameters. We propose that UAG is not likely to be an effective treatment for prostate cancer, with or without associated metabolic syndrome