Search

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.

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.

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.

Long noncoding RNAs (lncRNAs) play key regulatory roles in cancer progression and are novel therapeutic targets. We recently discovered the lncRNA gene, GHSROS (GHSR opposite strand), on the antisense DNA strand of the ghrelin receptor gene (GHSR). Here, we studied the expression and function of GHSROS in prostate cancer. Interrogation of microarray and RNA-seq data sets revealed that (similar to other lncRNA oncogenes) GHSROS is actively transcribed, although expressed at very low levels in cancer cell lines and tissues. By quantitative RT-PCR we demonstrate that GHSROS is highly expressed in a subset of high-grade prostate cancers (~11.4%). Moreover, the lncRNA is upregulated in high Gleason-score prostate tumors in two clinical data sets. Forced GHSROS overexpression significantly increased in vitro cell proliferation and migration of PC3, DU145, and LNCaP prostate cancer cell lines (P ≤ 0.05, Student's t-test). Increased cell proliferation observed in GHSROS-overexpressing prostate cancer cell lines was recapitulated in PC3, DU145, and LNCaP prostate cancer xenografts in NOD/SCID mice. Cell survival was significantly increased in GHSROS-overexpressing LNCaP cells treated with the cytotoxic drug docetaxel (P ≤ 0.05, Student's t-test). Docetaxel treatment also increased GHSROS expression in native LNCaP and PC3 cells in a dose-dependent manner (P ≤ 0.05, Student's t-test). These data suggest that GHSROS mediates tumor survival and resistance to docetaxel. To identify fundamental drivers of the observed tumorigenic phenotype of GHSROS-overexpressing cell lines, high-throughput RNA-seq data from in vitro cultured PC3 cells and LNCaP xenografts were examined. A quarter of the genes differentially expressed by GHSROS-overexpressing PC3 cells were also differentially expressed by GHSROS-overexpressing LNCaP xenografts. These 101 genes include several transcription factors with established roles in prostate cancer (including the androgen receptor) and genes associated w

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 potenti

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.

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 summa

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 long-term 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.

The distribution, phenotype, and requirement of macrophages for fracture-associated inflammation and/or early anabolic progression during endochondral callus formation were investigated. A murine femoral fracture model [internally fixed using a flexible plate (MouseFix)] was used to facilitate reproducible fracture reduction. IHC demonstrated that inflammatory macrophages (F4/80+Mac-2+) were localized with initiating chondrification centers and persisted within granulation tissue at the expanding soft callus front. They were also associated with key events during soft-to-hard callus transition. Resident macrophages (F4/80+Mac-2neg), including osteal macrophages, predominated in the maturing hard callus. Macrophage Fas-induced apoptosis transgenic mice were used to induce macrophage depletion in vivo in the femoral fracture model. Callus formation was completely abolished when macrophage depletion was initiated at the time of surgery and was significantly reduced when depletion was delayed to coincide with initiation of early anabolic phase. Treatment initiating 5 days after fracture with the pro-macrophage cytokine colony stimulating factor-1 significantly enhanced soft callus formation. The data support that inflammatory macrophages were required for initiation of fracture repair, whereas both inflammatory and resident macrophages promoted anabolic mechanisms during endochondral callus formation. Overall, macrophages make substantive and prolonged contributions to fracture healing and can be targeted as a therapeutic approach for enhancing repair mechanisms. Thus, macrophages represent a viable target for the development of pro-anabolic fracture treatments with a potentially broad therapeutic window...