GSTP1 DNA Methylation and Expression Status Is Indicative of 5-aza-2′-Deoxycytidine Efficacy in Human Prostate Cancer Cells

DNA methylation plays an important role in carcinogenesis and the reversibility of this epigenetic modification makes it a potential therapeutic target. To date, DNA methyltransferase inhibitors (DNMTi) have not demonstrated clinical efficacy in prostate cancer, with one of the major obstacles being the inability to monitor drug activity during the trial. Given the high frequency and specificity of GSTP1 DNA methylation in prostate cancer, we investigated whether GSTP1 is a useful marker of DNMTi treatment efficacy. LNCaP prostate cancer cells were treated with 5-aza-2′-deoxycytidine (5-aza-CdR) either with a single high dose (5–20 µM), every alternate day (0.1–10 µM) or daily (0.005–2.5 µM). A daily treatment regimen with 5-aza-CdR was optimal, with significant suppression of cell proliferation achieved with doses of 0.05 µM or greater (p<0.0001) and induction of cell death from 0.5 µM (p<0.0001). In contrast, treatment with a single high dose of 20 µM 5-aza-CdR inhibited cell proliferation but was not able to induce cell death. Demethylation of GSTP1 was observed with doses of 5-aza-CdR that induced significant suppression of cell proliferation (≥0.05 µM). Re-expression of the GSTP1 protein was observed only at doses of 5-aza-CdR (≥0.5 µM) associated with induction of cell death. Treatment of LNCaP cells with a more stable DNMTi, Zebularine required at least a 100-fold higher dose (≥50 µM) to inhibit proliferation and was less potent in inducing cell death, which corresponded to a lack of GSTP1 protein re-expression. We have shown that GSTP1 DNA methylation and protein expression status is correlated with DNMTi treatment response in prostate cancer cells. Since GSTP1 is methylated in nearly all prostate cancers, our results warrant its testing as a marker of epigenetic therapy response in future clinical trials. We conclude that the DNA methylation and protein expression status of GSTP1 are good indicators of DNMTi efficacy

Circulating Serum Exosomal miRNAs As Potential Biomarkers for Esophageal Adenocarcinoma

Author version made available in accordance with publisher policy.Abstract Background The poor prognosis and rising incidence of esophageal adenocarcinoma highlight the need for improved detection methods. The potential for circulating microRNAs (miRNAs) as biomarkers in other cancers has been shown, but circulating miRNAs have not been well characterized in esophageal adenocarcinoma. We investigated whether circulating exosomal miRNAs have potential to discriminate individuals with esophageal adenocarcinoma from healthy controls and non-dysplastic Barrett’s esophagus. Methods Seven hundred fifty-eight miRNAs were profiled in serum circulating exosomes from a cohort of 19 healthy controls, 10 individuals with Barrett’s esophagus, and 18 individuals with locally advanced esophageal adenocarcinoma. MiRNA expression was assessed using all possible permutations of miRNA ratios per individual. Four hundred eight miRNA ratios were differentially expressed in individuals with cancer compared to controls and Barrett’s esophagus (Mann-Whitney U test, P<0.05). The 179/408 ratios discriminated esophageal adenocarcinoma from healthy controls and Barrett’s esophagus (linear regression, P0.7, P<0.05). A multi-biomarker panel (RNU6-1/miR- 16-5p, miR-25-3p/miR-320a, let-7e-5p/miR-15b-5p, miR- 30a-5p/miR-324-5p, miR-17-5p/miR-194-5p) demonstrated enhanced specificity and sensitivity (area under ROC=0.99, 95 % CI 0.96–1.0) over single miRNA ratios to distinguish esophageal adenocarcinoma from controls and Barrett’s esophagus. Conclusions This study highlights the potential for serum exosomal miRNAs as biomarkers for the detection of esophageal adenocarcinoma

MicroRNA Profiling in Oesophageal Adenocarcinoma Cell Lines and Patient Serum Samples Reveals a Role for miR-451a in Radiation Resistance

Many patients with Oesophageal Adenocarcinoma (OAC) do not benefit from chemoradiotherapy treatment due to therapy resistance. To better understand the mechanisms involved in resistance and to find potential biomarkers, we investigated the association of microRNAs, which regulate gene expression, with the response to individual treatments, focusing on radiation. Intrinsic radiation resistance and chemotherapy drug resistance were assessed in eight OAC cell lines, and miRNA expression profiling was performed via TaqMan OpenArray qPCR. miRNAs discovered were either uniquely associated with resistance to radiation, cisplatin, or 5-FU, or were common to two or all three of the treatments. Target mRNA pathway analyses indicated several potential mechanisms of treatment resistance. miRNAs associated with the in vitro treatment responses were then investigated for association with pathologic response to neoadjuvant chemoradiotherapy (nCRT) in pre-treatment serums of patients with OAC. miR-451a was associated uniquely with resistance to radiation treatment in the cell lines, and with the response to nCRT in patient serums. Inhibition of miR-451a in the radiation resistant OAC cell line OE19 increased radiosensitivity (Survival Fraction 73% vs. 87%, p = 0.0003), and altered RNA expression. Pathway analysis of effected small non-coding RNAs and corresponding mRNA targets suggest potential mechanisms of radiation resistance in OAC

MicroRNA profiling in oesophageal adenocarcinoma cell lines and patient serum samples reveals a role for mir-451a in radiation resistance

Many patients with Oesophageal Adenocarcinoma (OAC) do not benefit from chemoradiotherapy treatment due to therapy resistance. To better understand the mechanisms involved in resistance and to find potential biomarkers, we investigated the association of microRNAs, which regulate gene expression, with the response to individual treatments, foc

The role of epigenetic modifications in prostate tumourigenesis.

Prostate cancer is the second-leading cause of cancer death in Australian men. Current therapies for advanced prostate cancer are not curative and most patients eventually develop castrate resistant prostate cancer. Epigenetic modifications are heritable and reversible biochemical changes of the chromatin that regulate gene expression and are important in prostate tumourigenesis. There is also evidence that excess foetal nutrition is associated with increased risk of developing prostate cancer. Hence, the aims of this thesis were to determine the involvement of epigenetic modifications in: the early origin of prostate cancer, prostate cancer progression, as prognostic and therapeutic targets in prostate cancer. The first aim of this thesis was to use a rodent model to determine if a maternal high fat diet (MHFD) is associated with increased risk of prostate cancer in offspring. Offspring exposed to a MHFD had increased incidence of prostate abnormalities compared to offspring exposed to a maternal control diet. GSTP1 is hypermethylated and silenced in human prostate cancer and was decreased in these offspring prostates. The MHFD altered the male offspring prostates microRNA expression and provided insights of possible underlying mechanisms that support a link between MHFD and risk of prostate cancer in adult offspring. The second aim was to investigate if specific histone modifications H3K18Ac and H3K4diMe were prognostic markers for prostate cancer. High levels of H3K18Ac and H3K4diMe were associated with increased risk of prostate cancer relapse respectively. To further investigate the underlying mechanisms, epigenetic genes were mined in microarray data, and an epigenetic gene signature was identified which distinguished non-malignant from tumour prostate tissues in an independent prostate cancer cohort. To investigate if the DNA methyltransferase inhibitor (DNMTi) 5-aza-CdR was a potential treatment agent for prostate cancer, proliferation assays were performed in prostate cancer cells. A daily low-dose and prolonged 5-aza-CdR treatment regime was the most effective treatment in prostate cancer cells compared to high doses administered less frequently. Furthermore, GSTP1 DNA methylation and protein status were good indicators of DNMTis efficacy in vitro, where demethylation indicated growth suppression and protein re-expression indicated cell death induction. To investigate if the Kruppel-like-factor 6 (KLF6) prostate cancer susceptibility gene is epigenetically altered during prostate cancer progression, DNA methylation analyses were performed in human and mouse (TRAMP) prostate cancers. Our results suggest that DNA hypermethylation is not responsible for decreased KLF6 expression in human and TRAMP prostate cancers in our study. Collectively, the findings of this thesis further support the importance of epigenetic modifications in prostate tumourigenesis. We demonstrated the potential of using epigenetic modifications as prognostic markers, therapeutic targets and as a marker of treatment efficacy. Lastly, we provide evidence, for the first time, that MHFD is a risk factor for prostate cancer and that miRNAs are involved. This finding is important and suggests the potential of early prevention/ intervention of prostate cancer by targeting epigenetic modifications and diet intervention.Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 201