No abstract

The prostanoid thromboxane (TX) A2 plays a central role in haemostasis and is increasingly implicated in cancer progression. TXA2 signals through two T Prostanoid receptor (TP) isoforms termed TPα and TPβ, with both encoded by the TBXA2R gene. Despite exhibiting several functional and regulatory differences, the role of the individual TP isoforms in neoplastic diseases is largely unknown.This study evaluated expression of the TPα and TPβ isoforms in tumour microarrays of the benign prostate and different pathological (Gleason) grades of prostate cancer (PCa). Expression of TPβ was significantly increased in PCa relative to benign tissue and strongly correlated with increasing Gleason grade. Furthermore, higher TPβ expression was associated with increased risk of biochemical recurrence (BCR) and significantly shorter disease-free survival time in patients post-surgery. While TPα was more variably expressed than TPβ in PCa, increased/high TPα expression within the tumour also trended toward increased BCR and shorter disease-free survival time. Comparative genomic CpG DNA methylation analysis revealed substantial differences in the extent of methylation of the promoter regions of the TBXA2R that specifically regulate expression of TPα and TPβ, respectively, both in benign prostate and in clinically-derived tissue representative of precursor lesions and progressive stages of PCa. Collectively, TPα and TPβ expression is differentially regulated both in the benign and tumourigenic prostate, and coincides with clinical pathology and altered CpG methylation of the TBXA2R gene. Analysis of TPβ, or a combination of TPα/TPβ, expression levels may have significant clinical potential as a diagnostic biomarker and predictor of PCa disease recurrence.

The mediators of the DNA damage response (DDR) are highly phosphorylated by kinases that control cell proliferation, but little is known about the role of this regulation. Here we show that cell cycle phosphorylation of the prototypical DDR mediator Saccharomyces cerevisiae Rad9 depends on cyclin-dependent kinase (CDK) complexes. We find that a specific G2/M form of Cdc28 can phosphorylate in vitro the N-terminal region of Rad9 on nine consensus CDK phosphorylation sites. We show that the integrity of CDK consensus sites and the activity of Cdc28 are required for both the activation of the Chk1 checkpoint kinase and its interaction with Rad9. We have identified T125 and T143 as important residues in Rad9 for this Rad9/Chk1 interaction. Phosphorylation of T143 is the most important feature promoting Rad9/Chk1 interaction, while the much more abundant phosphorylation of the neighbouring T125 residue impedes the Rad9/Chk1 interaction. We suggest a novel model for Chk1 activation where Cdc28 regulates the constitutive interaction of Rad9 and Chk1. The Rad9/Chk1 complex is then recruited at sites of DNA damage where activation of Chk1 requires additional DDR–specific protein kinases.

The prostanoid thromboxane (TX)A signals through the TPα and TPβ isoforms of T Prostanoid receptor (TP) that are transcriptionally regulated by distinct promoters termed Prm1 and Prm3, respectively, within the TBXA2R gene. We recently demonstrated that expression of TPα and TPβ is increased in PCa, differentially correlating with Gleason grade and with altered CpG methylation of the individual Prm1/Prm3 regions within the TBXA2R. The current study sought to localise the sites of CpG methylation within Prm1 and Prm3, and to identify the main transcription factors regulating TPβ expression through Prm3 in the prostate adenocarcinoma PC-3 and LNCaP cell lines. Bisulfite sequencing revealed extensive differences in the pattern and status of CpG methylation of the individual Prm1 and Prm3 regions that regulate TPα and TPβ expression, respectively, within the TBXA2R. More specifically, Prm1 is predominantly hypomethylated while Prm3 is hypermethylated across its entire sequence in PC-3 and LNCaP cells. Furthermore, the tumour suppressors FOXP1 and NKX3.1, strongly implicated in PCa development, were identified as key transcription factors regulating TPβ expression through Prm3 in both PCa cell lines. Specific siRNA-disruption of FOXP1 and NKX3.1 each coincided with up-regulated TPβ protein and mRNA expression, while genetic-reporter and chromatin immunoprecipitation (ChIP) analyses confirmed that both FOXP1 and NKX3.1 bind to cis‑elements within Prm3 to transcriptionally repress TPβ in the PCa lines. Collectively these data identify Prm3/TPβ as a bona fide target of FOXP1 and NKX3.1 regulation, providing a mechanistic basis, at least in part, for the highly significant upregulation of TPβ expression in PCa.

The UCD community has made this article openly available. Please share how this access benefits you. Your story matters! (@ucd_oa) Some rights reserved. For more information, please see the item record link above. TitleTranscriptional regulation of the human thromboxane A2 receptor gene by Wilms' tumor (WT)1 and hypermethylated in cancer (HIC) 1 in prostate and breast cancers Author(s)Keating, Garret L.; Reid, Helen M.; Eivers, Sarah B.; Mulvaney, Eamon P.; Kinsella, B. Therese Publication date 2014-06 Publication informationBiochimica Biophysica Acta (Gene Regulator Mechanisms), 1839 (6): 476-492Publisher Elsevier Item record/more information http://hdl.handle.net/10197/5646 Publisher's statementThis is the author's version of a work that was accepted for publication in Biochimica Biophysica Acta (Gene Regulator Mechanisms) . Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Biochimica Biophysica Acta (Gene Regulator Mechanisms) (VOL1839, ISSUE 6, (2014 Abstract:The prostanoid thomboxane (TX) A 2 plays a central role in hemostasis and is increasingly implicated in neoplastic disease, including prostate and breast cancers. In humans, TXA 2 signals though the TP and TP isoforms of the T prostanoid receptor, two structurally related receptors transcriptionally regulated by distinct promoters, Prm1 and Prm3, respectively, within the TP gene. Focusing on TP, the current study investigated its expression and transcriptional regulation though Prm1 in prostate and breast cancers. Expression of TPα correlated with increasing prostate and breast tissue tumor grade while the TXA 2 mimetic U46619 promoted both proliferation and migration of the respective prostate (PC3) and breast (MCF-7 and MDA-MD-231) derived-carcinoma cell lines.Though 5' deletional and genetic reporter analyses, several functional upstream repressor regions (URRs) were identified within Prm1 in PC3, MCF-7 and MDA-MB-231 cells while site-directed mutagenesis identified the tumor suppressors Wilms' tumor (WT)1 and hypermethylated in cancer (HIC) 1 as the trans-acting factors regulating those repressor regions. Chomatin immunoprecipitation (ChIP) studies confirmed that WT1 binds in vivo to multiple GC-enriched WT1 cis-elements within the URRs of Prm1 in PC3, MCF-7 and MDA-MB-231 cells. Furthermore, ChIP analyses established that HIC1 binds in vivo to the HIC1 (b) cis-element within Prm1 in PC3 and MCF-7 cells but not in the MDA-MB-231 carcinoma line. Collectively, these data establish that WT1 and HIC1, both tumor suppressors implicated in prostate and breast cancers, transcriptionally repress TPα expression and thereby provide a strong genetic basis for understanding the role of TXA 2 in the progression of certain human cancers. Highlights: Upregulation of the thomboxane receptor (th...