LSD1 inhibition attenuates androgen receptor V7 splice variant activation in castration resistant prostate cancer models.

BACKGROUND: Castrate resistant prostate cancer (CRPC) is often driven by constitutively active forms of the androgen receptor such as the V7 splice variant (AR-V7) and commonly becomes resistant to established hormonal therapy strategies such as enzalutamide as a result. The lysine demethylase LSD1 is a co-activator of the wild type androgen receptor and a potential therapeutic target in hormone sensitive prostate cancer. We evaluated whether LSD1 could also be therapeutically targeted in CRPC models driven by AR-V7. METHODS: We utilised cell line models of castrate resistant prostate cancer through over expression of AR-V7 to test the impact of chemical LSD1 inhibition on AR activation. We validated findings through depletion of LSD1 expression and in prostate cancer cell lines that express AR-V7. RESULTS: Chemical inhibition of LSD1 resulted in reduced activation of the androgen receptor through both the wild type and its AR-V7 splice variant forms. This was confirmed and validated in luciferase reporter assays, in LNCaP and 22Rv1 prostate cancer cell lines and in LSD1 depletion experiments. CONCLUSION: LSD1 contributes to activation of both the wild type and V7 splice variant forms of the androgen receptor and can be therapeutically targeted in models of CRPC. Further development of this approach is warranted.

Cancer-associated fibroblasts predict poor outcome and promote periostin-dependent invasion in oesophageal adenocarcinoma.

Interactions between cancer cells and cancer-associated fibroblasts (CAFs) play an important role in tumour development and progression. In this study we investigated the functional role of CAFs in oesophageal adenocarcinoma (EAC). We used immunochemistry to analyse a cohort of 183 EAC patients for CAF markers related to disease mortality. We characterized CAFs and normal oesophageal fibroblasts (NOFs) using western blotting, immunofluorescence and gel contraction. Transwell assays, 3D organotypic culture and xenograft models were used to examine the effects on EAC cell function and to dissect molecular mechanisms regulating invasion. Most EACs (93%) contained CAFs with a myofibroblastic (α-SMA-positive) phenotype, which correlated significantly with poor survival [p = 0.016; HR 7. 1 (1.7-29.4)]. Primary CAFs isolated from EACs have a contractile, myofibroblastic phenotype and promote EAC cell invasion in vitro (Transwell assays, p ≤ 0.05; organotypic culture, p < 0.001) and in vivo (p ≤ 0.05). In vitro, this pro-invasive effect is modulated through the matricellular protein periostin. Periostin is secreted by CAFs and acts as a ligand for EAC cell integrins αvß3 and αvß5, promoting activation of the PI3kinase-Akt pathway. In patient samples, periostin expression at the tumour cell-stromal interface correlates with poor overall and disease-free survival. Our study highlights the importance of the tumour stroma in EAC progression. Paracrine interaction between CAF-secreted periostin and EAC-expressed integrins results in PI3 kinase-Akt activation and increased tumour cell invasion. Most EACs contain a myofibroblastic CAF-rich stroma; this may explain the aggressive, highly infiltrative nature of the disease, and suggests that stromal targeting may produce therapeutic benefit in EAC patients.

Relaxin modulates human and rat hepatic myofibroblast function and ameliorates portal hypertension in vivo.

UNLABELLED: Active myofibroblast (MF) contraction contributes significantly to the increased intrahepatic vascular resistance that is the primary cause of portal hypertension (PHT) in cirrhosis. We sought proof of concept for direct therapeutic targeting of the dynamic component of PHT and markers of MF activation using short-term administration of the peptide hormone relaxin (RLN). We defined the portal hypotensive effect in rat models of sinusoidal PHT and the expression, activity, and function of the RLN-receptor signaling axis in human liver MFs. The effects of RLN were studied after 8 and 16 weeks carbon tetrachloride intoxication, following bile duct ligation, and in tissue culture models. Hemodynamic changes were analyzed by direct cannulation, perivascular flowprobe, indocyanine green imaging, and functional magnetic resonance imaging. Serum and hepatic nitric oxide (NO) levels were determined by immunoassay. Hepatic inflammation was assessed by histology and serum markers and fibrosis by collagen proportionate area. Gene expression was analyzed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and western blotting and hepatic stellate cell (HSC)-MF contractility by gel contraction assay. Increased expression of RLN receptor (RXFP1) was shown in HSC-MFs and fibrotic liver diseases in both rats and humans. RLN induced a selective and significant reduction in portal pressure in pathologically distinct PHT models, through augmentation of intrahepatic NO signaling and a dramatic reduction in contractile filament expression in HSC-MFs. Critical for translation, RLN did not induce systemic hypotension even in advanced cirrhosis models. Portal blood flow and hepatic oxygenation were increased by RLN in early cirrhosis. Treatment of human HSC-MFs with RLN inhibited contractility and induced an antifibrogenic phenotype in an RXFP1-dependent manner. CONCLUSION: We identified RXFP1 as a potential new therapeutic target for PHT and MF activation status.

Enantioselective synthesis of tranylcypromine analogues as lysine demethylase (LSD1) inhibitors.

Asymmetric cyclopropanation of styrenes by tert-butyl diazoacetate followed by ester hydrolysis and Curtius rearrangement gave a series of tranylcypromine analogues as single enantiomers. The o,- m- and p-bromo analogues were all more active than tranylcypromine in a LSD1 enzyme assay. The m- and p-bromo analogues were micromolar growth inhibitors of the LNCaP prostate cancer cell line as were the corresponding biphenyl analogues prepared from the bromide by Suzuki crosscoupling.

Total synthesis of largazole and analogues: HDAC inhibition, antiproliferative activity and metabolic stability.

The total synthesis of largazole and four analogues is reported. All analogues were nanomolar HDAC inhibitors. The antiproliferative activity is driven by lipophilicity and cell permeability. In murine liver homogenates, largazole is rapidly metabolized (half-life ≤5 min) to the thiol which has a half-life of 51 min.

Fibroblasts derived from oesophageal adenocarcinoma differ in DNA methylation profile from normal oesophageal fibroblasts.

Oesophageal adenocarcinoma (OAC) is increasing in incidence and has a poor prognosis. Tumour derived fibroblasts (TDFs) differ functionally from normal fibroblasts (NDFs), and play a pivotal role in cancer. Many of the differences persist through subculture. We measured the DNA methylation profiles of 10 TDFs from OAC with 12 NDF from normal oesophageal mucosa using Infinium HumanMethylation450 Beadchips and found they differed in multidimensional scaling analysis. We identified 4,856 differentially methylated CpGs (DMCs, adjusted p < 0.01 and absolute difference in average ß-value > 0.15), of which 3,243 (66.8%) were hypomethylated in TDFs compared to NDFs. Hypermethylated DMCs were enriched at transcription start sites (TSSs) and in CpG islands, and depleted in transcriptional enhancers. Gene ontology analysis of genes with DMCs at TSSs revealed an enrichment of genes involved in development, morphogenesis, migration, adhesion, regulation of processes and response to stimuli. Alpha-smooth muscle actin (α-SMA) is a marker of activated fibroblasts and a poor prognostic indicator in OAC. Hypomethylated DMCs were observed at the TSS of transcript variant 2 of α-SMA, which correlated with an increase in α-SMA protein expression. These data suggest that DNA methylation may contribute to the maintenance of the TDF phenotype.