Inhibition of Bromodomain Proteins Enhances Oncolytic HAdVC5 Replication and Efficacy in Pancreatic Ductal Adenocarcinoma (PDAC) Models.

Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive type of pancreatic cancer, which rapidly develops resistance to the current standard of care. Several oncolytic Human AdenoViruses (HAdVs) have been reported to re-sensitize drug-resistant cancer cells and in combination with chemotherapeutics attenuate solid tumour growth. Obstacles preventing greater clinical success are rapid hepatic elimination and limited viral replication and spread within the tumour microenvironment. We hypothesised that higher intratumoural levels of the virus could be achieved by altering cellular epigenetic regulation. Here we report on the screening of an enriched epigenetics small molecule library and validation of six compounds that increased viral gene expression and replication. The greatest effects were observed with three epigenetic inhibitors targeting bromodomain (BRD)-containing proteins. Specifically, BRD4 inhibitors enhanced the efficacy of Ad5 wild type, Ad∆∆, and Ad-3∆-A20T in 3-dimensional co-culture models of PDAC and in vivo xenografts. RNAseq analysis demonstrated that the inhibitors increased viral E1A expression, altered expression of cell cycle regulators and inflammatory factors, and attenuated expression levels of tumour cell oncogenes such as c-Myc and Myb. The data suggest that the tumour-selective Ad∆∆ and Ad-3∆-A20T combined with epigenetic inhibitors is a novel strategy for the treatment of PDAC by eliminating both cancer and associated stromal cells to pave the way for immune cell access even after systemic delivery of the virus.

Expression of two WFDC1/ps20 isoforms in prostate stromal cells induces paracrine apoptosis through regulation of PTGS2/COX-2.

BACKGROUND: WFDC1/Prostate stromal 20 (ps20) is a small secreted protein highly expressed within the prostate stroma. WFDC1/ps20 expression is frequently downregulated or lost in prostate cancer (PCa) and ps20 has demonstrated growth-suppressive functions in numerous tumour model systems, although the mechanisms of this phenomenon are not understood. METHODS: Ps20 was cloned and overexpressed in DU145, PC3, LNCaP and WPMY-1 cells. Cellular growth, cell cycle and apoptosis were characterised. WPMY-1 stromal cells expressing ps20 were characterised by transcriptome microarray and the function of WPMY-1 conditioned media on growth of PCa cell lines was assessed. RESULTS: Prostrate stromal 20 expression enhanced the proliferation of LNCaP cells, whereas stromal WPMY-1 cells were inhibited and underwent increased apoptosis. Prostrate stromal 20-expressing WPMY-1 cells secrete a potently proapoptotic conditioned media. Prostrate stromal 20 overexpression upregulates expression of cyclooxygenase-2 (COX-2) in LNCaP and WPMY-1 cells, and induces expression of a growth-suppressive phenotype, which inhibits proliferation of PCa cells by ps20-expressing WPMY-1 conditioned media. This growth suppression was subsequently shown to be dependent on COX-2 function. CONCLUSIONS: This work posits that expression of ps20 in the prostate stroma can regulate growth of epithelial and other tissues through the prostaglandin synthase pathway, and thereby restricts development and progression of neoplasms. This provides a rational for selective pressure against ps20 expression in tumour- associated stroma.

Cathepsin-L and transglutaminase dependent processing of ps20: A novel mechanism for ps20 regulation via ECM cross-linking.

Whey-acidic-protein (WAP) four-disulphide core (WFDC) proteins have important roles in the regulation of innate immunity, anti-microbial function, and the inhibition of inflammatory proteases at mucosal surfaces. It was recently demonstrated that the WFDC protein, prostate stromal 20 (ps20), encoded by the WFDC1 gene, is a potent growth inhibitory factor, and shares with other WFDC proteins the ability to modulate wound healing processes and immune responses to viral infections. However, ps20 remains relatively uncharacterised at the protein level. Using a panel of ps20 antibodies for western-blotting (WB), ELISA and immunoaffinity purification, we isolated, biochemically characterised and tested ps20 preparations for three biological properties: (i) interactions with glycosaminoglycans (GAG) (ii) inhibition of cell proliferation, and (iii) transglutaminase2 (TG2) mediated crosslinking of ps20 to fibronectin, a process implicated in wound healing. We show herein that ps20 preparations contain multiple molecular forms including full-length ps20 (resolving at ≈27 kDa), an exon 3 truncated form (≈22 kDa) that lacks aa113-140, and variable amounts of a putatively cleaved lower MW (≈15-17 kDa) species. Untagged purified ps20 preparations containing a mixture of these forms are biologically active in significantly suppressing prostate cell proliferation. We show that one mechanism by which lower LMW forms of ps20 arise is through cathepsin L (CL) cleavage, and confirm that CL cleaves ps20 at the C-terminus, but this does not inhibit its growth inhibitory function. However, CL cleavage abrogated the interaction between ps20 and solid-phase fibronectin. Therefore, we demonstrate for the first time that LMW forms of ps20 that lack a C-terminal immunogenic epitope can arise through CL cleavage and this cleavage impairs multimerisation and potential capacity to cross-link to ECM, but not the capacity of ps20 to inhibit cell proliferation. We propose that ps20 like other WFDC proteins can become associated with GAGs and the ECM. Furthermore, we suggest post-translational processing and cleavage of ps20 is required to generate functional protein species, and TG2 mediated crosslinking and CL cleavage form components of a ps20 regulatory apparatus.