RAC 3, one of the p160 family of co-activators is known to enhance the transcriptional activity of a number of steroid receptors. As co-activators are also known to enhance androgen receptor (AR) activity, we investigated the role of RAC 3 in the context of prostate cancer. In prostate cancer cell lines, we found variable levels of the RAC 3 protein with highest expression seen in AR-positive LNCaP cells, moderate expression in AR-negative PC 3 cells and low-level expression in AR-negative DU 145 cells. Immuno-precipitation studies showed that endogenous RAC 3 interacted with the AR in vivo and transfection assays confirmed that RAC 3 enhanced AR transcriptional activity. In clinical prostate tissue, we found strong RAC 3 mRNA expression and immuno-histochemistry demonstrated that in benign tissue, the protein was expressed predominantly in luminal cells, while in primary malignant epithelium it was more homogeneously expressed. In a series of 37 patients, the levels of RAC 3 expression correlated significantly with tumour grade ( P = 0.01) and stage of disease ( P = 0.03) but not with serum PSA levels. In addition moderate or high RAC 3 expression was associated with poorer disease-specific survival ( P = 0.03). We conclude that RAC 3 is an important co-activator of the AR in the prostate and may have an important role in the progression of prostate cancer. © 2001 Cancer Research Campaign http://www.bjcancer.com

Fibroblast growth factor receptors (FGFRs) mediate the tumourigenic effects of FGFs in prostate cancer. These receptors are therefore potential therapeutic targets in the development of inhibitors to this pathway. To identify the most relevant targets, we simultaneously investigated FGFR1-4 expression using a prostate cancer tissue microarray (TMA) and in laser capture microdissected (LCM) prostate epithelial cells. In malignant prostates (n = 138) we observed significant FGFR1 and FGFR4 protein over-expression in comparison with benign prostates (n = 58; p < 0.0001). FGFR1 was expressed at high levels in the majority of tumours (69% of grade 3 or less, 74% of grade 4 and 70% of grade 5), while FGFR4 was strongly expressed in 83% of grade 5 cancers but in only 25% of grade 1-3 cancers (p < 0.0001). At the transcript level we observed a similar pattern, with FGFR1 and FGFR4 mRNA over-expressed in malignant epithelial cells compared to benign cells (p < 0.0005 and p < 0.05, respectively). While total FGFR2 was increased in some cancers, there was no association between expression and tumour grade or stage. Transcript analysis, however, revealed a switch in the predominant isoform expressed from FGFR2IIIb to FGFR2IIIc among malignant epithelial cells. In contrast, protein and transcript expression of FGFR3 was very similar between benign and cancer biopsies. The functional effect of targeting FGFR4 in prostate cancer cells has not previously been investigated. In in vitro experiments, suppression of FGFR4 by RNA interference effectively blocked prostate cancer cell proliferation (p < 0.0001) and invasion (p < 0.001) in response to exogenous stimulation. This effect was evident regardless of whether the cells expressed the FGFR4 Arg388 or Gly388 allele. In parallel experiments, FGFR3 suppression had no discernible effect on cancer cell behaviour. These results suggest evidence of selective over-expression of FGFR1 and FGFR4 in clinical prostate cancer and support the notion of targeted inhibition of these receptors to disrupt FGF signalling.

The RNA-binding protein Sam68 has been reported to be up-regulated in clinical cases of prostate cancer (PCa), where it is thought to contribute to cell proliferation and survival. Consistent with this, we observed over-expression of Sam68 in a panel of clinical prostate tumours as compared with benign controls. Since Sam68 is implicated in a number of signalling pathways, we reasoned that its role in PCa may involve modulation of the androgen receptor (AR) signalling cascade, which drives the onset and progression of PCa. We found that Sam68 interacts with the AR in vivo in LNCaP cells, and is dynamically recruited to androgen response elements within the promoter region of the prostate-specific antigen (PSA) gene. Based on its known functions and nuclear location, Sam68 might either: (a) co-regulate AR-dependent transcription positively or negatively; or (b) modulate AR-dependent alternative splicing by enhancing incorporation of a Sam68-responsive exon transcribed under the control of an androgen-responsive promoter. We tested these possibilities using functional assays. Both wild-type Sam68 protein and the Sam68(V229F) mutant, which is impaired in RNA binding, functioned as a ligand-dependent AR co-activator on an androgen-regulated reporter gene. In contrast, splicing of a Sam68-responsive variable exon, transcribed under control of an androgen-responsive promoter, was strongly repressed in the presence of AR and androgens. This splicing inhibition was reversed by ectopic expression of Sam68 but enhanced by Sam68(V229F). These results demonstrate that Sam68 has separable effects on AR-regulated transcriptional activity and alternative splicing, both of which may affect PCa phenotypes.

Abnormal intracellular signaling contributes to carcinogenesis and may represent novel therapeutic targets. mitogen/extracellular signal-regulated kinase kinase-5 (MEK5) overexpression is associated with aggressive prostate cancer. In this study, we examined the role of extracellular signal-regulated kinase (ERK5, an MAPK and specific substrate for MEK5) in prostate cancer. ERK5 immunoreactivity was significantly upregulated in high-grade prostate cancer when compared to benign prostatic hyperplasia (Po0.0001). Increased ERK5 cytoplasmic signals correlated closely with Gleason sum score (Po0.0001), bony metastases (P ¼ 0.0044) and locally advanced disease at diagnosis (P ¼ 0.0023), with a weak association with shorter disease-specific survival (P ¼ 0.036). A subgroup of patients showed strong nuclear ERK5 localization, which correlated with poor diseasespecific survival and, on multivariant analysis, was an independent prognostic factor (Po0.0001). Analysis of ERK5 expression in matched tumor pairs (before and after hormone relapse, n ¼ 26) revealed ERK5 nuclear expression was significantly associated with hormoneinsensitive disease (P ¼ 0.0078). Similarly, ERK5 protein expression was increased in an androgen-independent LNCaP subline. We obtained the following in vitro and in vivo evidence to support the above expression data: (1) cotransfection of ERK5wt and MEK5D constructs in PC3 cells results in predominant ERK5 nuclear localization, similar to that observed in aggressive clinical disease; (2) ERK5-overexpressing PC3 cells have enhanced proliferative, migrative and invasive capabilities in vitro (Po0.0001), and were dramatically more efficient in forming tumors, with a shorter mean time for tumors to reach a critical volume of 1000 mm 3 , in vivo (Po0.0001); (3) the MEK1 inhibitor, PD184352, blocking ERK1/2 activation at low dose, did not suppress proliferation but did significantly decrease proliferation at a higher dose required to inhibit ERK5 activation. Taken together, our results establish the potential importance of ERK5 in aggressive prostate cancer.

Summary Prostatic adenocarcinoma commonly metastasizes to bone. Unlike most other bony secondaries, the majority of skeletal prostatic metastases are osteoblastic rather than osteolytic i nature. Several growth factors which are known to stimulate bone formation are expressed in benign and malignant prostate cells. bi none has been specifically linked to osteoscierotic metastases. Bone morphogenetic proteins (BMPs) induce ectopic bone formation in vivo. We have reported previously that BMP-6 mRNA and protein are expressed in the majority of primary prostatic carcinomas with established skeletal metastases but rarely in clinically organ-confined tumours. This study examines the expression of BMP-6 mRNA in matched prostatic primary and secondary bony lesions and in isolated skeletal metastases from prostatic adenocarcinomas, as well as other common human malignancies, by in situ hybridization. BMP-6 mRNA was detected in 11 out of 13 bone metastases from prostate carcinoma and in three paired samples of primary prostate carcinoma and matching skeletal metastasis. Weak signals for BMP-6 were also present in 5 out of 17 skeletal deposits from non-prostatic malignancies. BMP-6 mRNA appears to be strongly expressed in prostatic adenocarcinomas, both in the primary tumour and in bone metastases. It is also expressed, though less frequentty, in skeletal metastases from other human carcinomas. Our findings suggest that BMP-6 may hold potential as an attractive marker and possible mediator of skeletal metastases, particularly in prostate carcinoma.Keywords: bone morphogenetic protein-6; prostate carcinoma; skeletal metastasis; in situ hybridization: growth factors Metastases are the most common neoplastic lesions in bone. and more than 80%-7 of the tumours are accounted for by a limited number of primary malignancies (Orr et al. 1995). These include carcinomas of the breast, prostate. thyroid. kidney and lungy. Normal bone is being remodelled continuously with new bone formation by osteoblasts and bone degradation by osteoclasts. In the presence of skeletal metastases. there is a disturbance of the fine balance between the two processes. When bone destruction dominates there is net loss of bone mass and the lesion is described as osteolytic. W'hen excessise amounts of new bone formation takes place with less bone destruction. the lesion is described as osteoblastic or osteosclerotic. This type of increased ossification only occurs in relatively acellular skeletal metastases associated with the deselopment of a fibrous stroma. and is particularly common in metastases from prostatic carcinoma (Galasko. 1982). Human prostatic adenocarcinoma is one of the rare cancers that consistently produces osteoblastic metastases to bone. in approximately 90% of cases. Several growth factors which are known to stimulate osteoblast growth and bone matrix formation are also expressed in benign and malignant prostate cells. These include members of the fibroblast growth factor. insulin-lke growth factor. platelet-denried growth factor and tr...