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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.

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.

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.

Objective: Pyruvate dehydrogenase kinase1 (PDK1), is a mitochondrial enzyme associated with metabolic pathways and known to play many roles in carcinogenesis. Genome wide association studies (GWAS) recently identified single nucleotide polymorphisms (SNPs) in PDK1 to be associated with prostate cancer (PCa) risk. Two SNPs in particular, rs1530865 (G>C) and rs2357637 (C>A), were predicted to alter miRNA binding to the PDK1 gene which led us to hypothesise that PDK1 is involved in PCa progression by regulating cellular metabolic pathways and is regulated via miRNAs in an allele specific manner. Methods: The effects of PDK1 knockdown on cell proliferation, migration and invasion were assessed. Luciferase reporter vector assays were performed to determine miRNA-SNP affinity. Results: Higher expression of PDK1 was observed in metastatic cell lines compared with normal prostate epithelial cells. While knockdown of PDK1 did not affect proliferation of LNCaP and PC3 cells, the rate of migration and invasion decreased significantly. Furthermore, suppression of PDK1 reduced aerobic glycolysis, in PCa cells indicating that aerobic glycolysis may promote PCa tumorigenesis. Computational predictions revealed that PCa risk associated SNP rs1530865 affects the binding site for various miRNAs (miR-877-5p, miR-3125, miR-3916 and miR-3928-3p). Similarly, rs2357637 SNPs miR-889-3p and miR-2116-3p miRNAs. Reporter gene assays confirmed the specific affinity of the identified miRNAs for SNPs in an allele dependent manner. Conclusions: Our findings demonstrate the role of PDK1 in prostate cancer and its regulation by miRNAs. We also show that PDK1-miRNA axis regulates PCa metabolism. Following exhaustive experimentation, this relationship can be explored for the development of novel therapeutic approaches.

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

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 Carica papaya 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 vitro, 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 IC50 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. Copyright © 2018 Pandey, Walpole, Shaw, Cabot, Hewavitharana and Batra.

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

Objective: Genome wide association studies (GWAS) recently identified single nucleotide polymorphisms (SNPs) in Pyruvate dehydrogenase kinase1 (PDK1), to be associated with prostate cancer (PCa) risk. This gene is activated under hypoxic conditions and encodes for mitochondrial enzymes associated with metabolic pathways. GWAS identified two SNPs,rs1530865 (G>C) and rs2357637 (C>A), in the 3'UTR of PDK1, which were predicted to alter miRNA binding to the PDK1 gene. We hypothesised that PDK1 is involved in PCa progression by regulating cellular metabolic pathways under normoxic and/or hypoxic conditions via miRNAs. Methods: PDK1 function in the PCa cells was determined using a knockdown approach and Seahorse Extracellular Flux(XF) Analyser. In addition, miRNA-SNP affinity was determined using luciferase reporter vector assay. Results: Higher expression of PDK1 was observed in PCa cell lines under hypoxic versus normoxic conditions. Under normoxic conditions, knockdown of PDK1did not affect proliferation of LNCaP andPC3 cells however, the rate of migration and invasion was decreased. Furthermore, suppression of PDK1 reduced aerobic glycolysis, in PCa cells indicating that aerobic glycolysis may promote PCa tumorigenesis. Computational predictions revealed that PCa risk associated rs1530865 SNP was predicted to affect the binding site formiR-877-5p, miR-3125, miR-3916 andmiR-3928-3p and rs2357637 for miR-889-3p and miR-2116-3p miRNAs. Reporter gene assays confirmed that miR-3125,miR-3916 and miR-2116-3p have a specific affinity for the rs1530865 SNP C-allele and rs2357637 SNP A allele respectively. Conclusions: Our findings demonstrated strong evidence for the role of miRNAs in the regulation of PDK1 in PCa in an allele dependent manner, which in turn regulated PCa metabolism.

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 (IC50=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. KEYWORDS: Anti-proliferative activity; Extraction; Papaya; Prostate cancer

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.

After publication of the original article [1], a reader noted that one reference cited in the main text had not been mentioned in the References section. The reference (Qin et al., [2]) was cited as Ref. 33 within the text, but mistakenly did not appear in the References. As such the total number of References was also incorrect – there should have been 36 in total. References 33 – 35 should have been numbered 34 – 36 in the main text and in the References section.

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.

Current translational and basic prostate cancer research is limited by the number of cell lines that truly reflect the spectrum of disease progression, with most commonly used cell lines being derived from metastatic lesions. There are essentially no prostate cancer cell lines derived from primary tumours or localised disease in wide use.