Identification of a novel functional androgen response element withinhPar1promoter: implications to prostate cancer progression

Abstract

Human protease-activated receptor-1 (hPar1) plays a role in malignant and physiological invasion processes. We have identified a functional androgen response element (ARE) located in the hPar1 promoter upstream of the transcription start site at -1791 to -1777. Dihydrotestosterone treatment of the prostate cancer cell line LNCaP increased endogenous hPar1 mRNA levels, consistent with the threefold increase in promoter activity of hPar1-luciferase reporter construct. Specific binding of the hPar1-derived ARE to LNCaP nuclear extracts was demonstrated by electrophoretic mobility shift assay. This binding was abrogated by antiandrogen receptor (anti-AR) antibodies or excess cold oligonucleotide but not by a mutated oligonucleotide. Moreover, using chromatin immunoprecipitation assays, we confirm the in vivo interaction between the AR and ARE domain of the hPar1 promoter. In parallel, we show that hormone ablation therapy markedly reduces the otherwise high hPar1 expression levels in prostate cancer biopsy specimens. We suggest that the hPar1 gene is regulated transcriptionally by androgens, representing one of several target genes effectively reduced during hormone ablation therapy. A major limitation of hormonal deprivation is that it causes only a temporary remission, and the cancer eventually reappears in a more malignant, androgen-independent form. hPar1 is also overexpressed in CL1 cells, an aggressively metastasizing, hormone-independent subclone of LNCaP, and in PC3 prostate adenocarcinoma lacking AR in a mechanism yet to be fully elucidated. These data may imply that hPar1 expression correlates with prostate cancer progression in androgen-dependent and -independent phases and therefore, provides an instrumental, therapeutic target for treatment in prostate cancer.

p53 controls hPar1 function and expression

“…Among the activators are Sp1 (Tellez et al, 2003), androgen/ hormone response elements (Salah et al, 2005) and Egr-1 (Salah et al, 2007a). The repressors are AP-2 (Tellez et al, 2003) and wt p53 (this study).…”

“…Rather, it is because of increased hPar1 transcription as evaluated by amplified transcription rates in aggressive versus non-aggressive prostate cancer lines. Indeed, we have identified Egr-1 as a critical regulator of hPar1 gene overexpression in the hormoneindependent prostate carcinoma (Salah et al, 2005(Salah et al, , 2007b.…”

Prostatic trypsin-like kallikrein-related peptidases (KLKs) and other prostate-expressed tryptic proteinases as regulators of signalling via proteinase-activated receptors (PARs)

“…Consistent with the prostatic expression of PAR 1 , androgen regulation of the encoding gene has been recently reported (Salah et al, 2005). These workers showed that stimulation of LNCaP cells with dihydrotestosterone (DHT) increased the transcription of PAR 1 , whereas PC-3 cells, which lack an androgen receptor, were unresponsive.…”

“…However, as PAR 1 levels are elevated in late, androgen independent stage disease it is apparent that other mechanisms must also transcriptionally regulate the encoding gene. Consistent with this proposal, an aggressive, hormone resistant sub-line of LNCaP cells (CL1 cells) generated through in vitro androgen deprivation, has high levels of PAR 1 mRNA and protein and small amounts of AR protein; a phenomenon that is also seen in PC-3 cell which lack androgen receptor (Salah et al, 2005). More recently, Salah and colleagues employed fluorescence in situ chromosome hybridisation to indicate that differences in PAR 1 mRNA levels in cell lines of high (CL1 or PC-3) and low (LNCaP) expression is not due to gene amplification.…”

PAR1 plays a role in epithelial malignancies: Transcriptional regulation and novel signaling pathway

“…In addition to the well-established role of PAR 1 in thrombosis, hemostasis, and vascular biology, its role in tumor biology is currently emerging (1)(2)(3). We, as well as several independent laboratories, have highlighted the central involvement of PAR 1 in epithelial malignancies (4)(5)(6)(7)(8)(9). In fact, PAR 1 serves as a delicate cell sensor to mediate the function of classical enzyme/s centrally involved in thrombosis and hemostasis.…”