Genetic deletion of osteopontin in TRAMP mice skews prostate carcinogenesis from adenocarcinoma to aggressive human-like neuroendocrine cancers.

Osteopontin (OPN) is a secreted glycoprotein, that belongs to the non-structural extracellular matrix (ECM), and its over expression in human prostate cancer has been associated with disease progression, androgen independence and metastatic ability. Nevertheless, the pathophysiology of OPN in prostate tumorigenesis has never been studied. We crossed TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice with OPN deficient (OPN-/-) mice and followed tumor onset and progression in these double mutants. Ultrasound examination detected the early onset of a rapidly growing, homogeneous and spherical tumor in about 60% of OPN-/- TRAMP mice. Such neoplasms seldom occurred in parental TRAMP mice otherwise prone to adenocarcinomas and were characterized for being androgen receptor negative, highly proliferative and endowed with neuroendocrine (NE) features. Gene expression profiling showed up-regulation of genes involved in tumor progression, cell cycle and neuronal differentiation in OPN-deficient versus wild type TRAMP tumors. Down-regulated genes included key genes of TGFa pathway, including SMAD3 and Filamin, which were confirmed at the protein level. Furthermore, NE genes and particularly those characterizing early prostatic lesions of OPN-deficient mice were found to correlate with those of human prostate NE tumours. These data underscore a novel role of OPN in the early stages of prostate cancer growth, protecting against the development of aggressive NE tumors.

Osteoblast-derived factors induce an expression signature that identifies prostate cancer metastasis and hormonal progression.

Identification of gene expression signatures associated with metastases provides a tool to discern mechanisms and potential therapeutic targets and may lead toward a molecular classification system in pathology. Prostate cancer (CaP) frequently metastasizes to the bone to form osteoblastic lesions. Correlative clinical data and in vitro evidence have led to the hypothesis that osteoblast-derived factors promote hormonal progression of CaP cells. Here, the gene expression signature of CaP exposed to osteoblast-derived factors was identified. This signature included known androgen-regulated genes, oncogenes, tumor suppressors, and genes whose products are involved in apoptosis and cell cycle. A comparative functional genomic approach involved the application of this responsive gene expression signature to clinical samples of human CaP, melanomas, and oral cancers. Cluster analysis revealed that this gene expression signature had specificity for CaP and could resolve clinical specimens according to stage (benign, localized, and metastatic) and androgen sensitivity with an accuracy of 100% and 80%, respectively. Together, these results suggest that factors derived from osteoblasts induce a more advanced phenotype of CaP and promotes hormonal progression.

Proteomic analyses to identify novel therapeutic targets for the treatment of advanced prostate cancer.

At present there is no cure for advanced prostate cancer once it progresses to an androgen independent stage. Hormonal therapy, radiotherapy, and chemotherapy all have limited durations of efficacy for men diagnosed with androgen independent disease and patients will succumb over a period of several months to two years. The androgen receptor (AR) has been suspected to play an important role in the mechanism of progression to androgen independence. This is because the AR is a transcription factor that 'normally' mediates the effects of androgen to regulate expression of genes involved in proliferation and survival of prostate cells. Thus identifying and characterizing the proteins that interact with the AR to facilitate an activated receptor is of critical importance. Proteomic approaches such as isotope-coded affinity tags (ICAT), isotope Tags for Relative and Absolute Quantification (iTRAQ)(TM), Stable Isotope Labeling with Amino acids in Cell culture (SILAC), Tandem Affinity Purification (TAP) of tagged proteins (TAP-tag) and Multidimensional Protein Identification Technology (MudPIT) provide large scale unbiased strategies and have not been previously applied to identify proteins that interact with the AR. Here an example of the power of these proteomic approaches to identify potential therapeutic targets for prostate cancer is provided. Application of MudPIT identified 82 peptides in endogenous complexes immunoprecipitated with the AR from prostate cancer cells. Identification of these novel proteins may ultimately lead to the development of better therapies for the treatment or prevention of advanced prostate cancer.

The androgen receptor pathway is by-passed in prostate cancer cells generated after prolonged treatment with bicalutamide.

BACKGROUND: Experimental work in various prostate cancer models revealed that the androgen receptor is frequently upregulated and implicated in tumor progression. However, little attention has been paid to the androgen receptor-signaling pathway in the development of therapy resistance in patients who receive chronic treatment with a non-steroidal anti-androgen. METHODS: We have generated a novel subline, LNCaP-Bic, after prolonged treatment with androgen and bicalutamide in vitro. Proliferation of LNCaP-Bic cells in the absence or presence of androgen, tocopherol succinate, and/or bicalutamide was assessed by cell counting. Androgen receptor expression was determined by Western blot. Luciferase activity was measured in cells transfected with an androgen-responsive reporter. RESULTS: In basal conditions, proliferation of LNCaP-Bic cells increased more than threefold over that of control LNCaP cells. Neither synthetic androgen R1881 nor bicalutamide showed any effect on LNCaP-Bic growth in vitro. Androgen receptor expression did not differ between the cell subline generated in the presence of bicalutamide and parental LNCaP cells. The ability of R1881 to induce reporter gene activity in LNCaP-Bic cells was reduced by 56%. Tocopherol succinate caused inhibition of proliferation only in the parental cell line although the androgen receptor and prostate-specific antigen were down regulated by the vitamin E derivative in both parental LNCaP and LNCaP-Bic cells. CONCLUSIONS: Androgen receptor-mediated signal transaction is not enhanced in cells selected in the presence of bicalutamide. Our data may suggest that a more differentiated approach in targeting the androgen receptor is needed in prostate cancers that become resistant to classic endocrine treatment.

p300 regulates androgen receptor-independent expression of prostate-specific antigen in prostate cancer cells treated chronically with interleukin-6.

Prostate cancer is the most frequent non-skin cancer in men. Although the mechanisms involved in the progression of prostate cancer are not entirely understood, androgen receptor has been shown to play an important role. Androgen receptor is expressed in both early and late-stage prostate cancer. Also, androgen-regulated pathways are thought to be active as evidenced by elevated levels of prostate-specific antigen (PSA). In addition, several androgen receptor coactivators and cytokines are involved in prostate cancer progression. In this regard, we have shown previously that the coactivator p300 plays a major role in the androgen-independent activation of PSA by interleukin 6 (IL-6), a cytokine involved in late-stage prostate cancer. In this study, we investigated the role of p300 and its homologue CREB-binding protein in prostate cancer cells treated chronically with IL-6. We found that p300 but not CREB-binding protein induced activation of PSA in these cells and that the histone acetyltransferase activity of p300 was critical. This effect was independent of the presence of androgens or antiandrogens. Moreover, we found markedly reduced levels of androgen receptor in these cells and p300 transfection did not affect those levels, suggesting that the p300 effect on PSA could be bypassing the androgen receptor. Transfection with exogenous androgen receptor showed minimal response of PSA to androgens but higher response to p300. We found similar effects of p300 on the androgen response element III, which mediates the androgen receptor-dependent activation of PSA. Finally, we showed that p300 alone regulates expression of the endogenous PSA gene in the IL-6-treated cells. These findings reveal a new insight in the progression of prostate cancer, suggesting that coactivators, such as p300, play more important roles in late-stage prostate cancer, and could regulate androgen-dependent genes in the absence or with very low levels of androgen receptor.

The androgen receptor co-activator CBP is up-regulated following androgen withdrawal and is highly expressed in advanced prostate cancer.

The androgen receptor co-activator CREB (cAMP-response element binding protein)-binding protein (CBP) enhances androgen receptor activity after stimulation by androgenic hormones and androgen receptor antagonists. The aim of the present study was to investigate the regulation of CBP expression by steroid and peptide hormones in prostate cancer. For this purpose, LNCaP cells were treated with the synthetic androgen methyltrienolone (R1881), epidermal growth factor, insulin-like growth factor-I or interleukin-6 (IL-6). CBP protein and mRNA expression were studied by western blotting and real-time PCR, respectively. CBP expression was also investigated in tissue specimens obtained from 26 patients with therapy-resistant carcinoma of the prostate. In LNCaP cells, CBP protein was down-regulated by R1881 or IL-6. The non-steroidal anti-androgen bicalutamide antagonized the effects of R1881 and the Janus kinase inhibitor AG 490 reversed the effects of IL-6. In contrast, neither R1881 nor IL-6 caused any effect on CBP expression in the PC-3 cell line. In LNCaP cells, the inhibition of CBP expression by R1881 or IL-6 was also observed at the mRNA level. CBP protein was detected in all 26 specimens by immunohistochemistry. The results suggest that up-regulation of CBP during androgen ablation may be relevant to the failure of endocrine therapy in patients with prostate carcinoma.

Expression and function of androgen receptor coactivators in prostate cancer.

Human androgen receptor (AR) associates with coactivator or corepressor proteins that modulate its activation in the presence of ligand. Early studies on AR coactivators in carcinoma of the prostate were hampered because of lack of respective antibodies. Investigations at mRNA level revealed that most benign and malignant prostate cells express common coactivators. AR coactivators SRC-1 and TIF-2 are up-regulated in tissue specimens obtained from patients who failed prostate cancer endocrine therapy. Increased expression of these coactivators is associated with enhanced activation of the AR by the adrenal androgen dehydroepiandrosterone. Similar association between AR coactivator expression and high prostate cancer grade and stage was reported for RAC-3 (SRC-3). The transcriptional integrator CBP was detected in clinical specimens representing organ-confined prostate cancer, lymph node metastases and tumour cell lines. Agonistic effect of the nonsteroidal antiandrogen hydroxyflutamide was strongly potentiated in prostate cells transfected with CBP cDNA. A functional homologue of CBP, p300, is implicated in ligand-independent AR activation by interleukin-6. The AR coactivator Tip60, which is up-regulated by androgen ablation, is recruited to the promoter of the prostate-specific antigen gene in the absence of androgen in androgen-independent prostate cancer sublines. It was proposed that the cofactor ARA70 is a specific enhancer of AR action. However, research from other laboratories has demonstrated interaction between ARA70 and other steroid receptors. Although in some cases dominant-negative coactivator mutants inhibited proliferation of prostate cancer cells in vitro, confirmation from in vivo tumour models is missing. In summary, several abnormalities in AR coactivator expression and function are associated with prostate cancer progression.

The transcriptional co-activator cAMP response element-binding protein-binding protein is expressed in prostate cancer and enhances androgen- and anti-androgen-induced androgen receptor function.

Progression of human prostate cancer toward therapy resistance occurs in the presence of wild-type or mutated androgen receptors (ARs) that, in some cases, exhibit aberrant activation by various steroid hormones and anti-androgens. The AR associates with a number of co-activators that possess histone acetylase activity and act as bridging molecules to components of the transcription initiation complex. In previous reports, it was shown that the transcriptional co-activator CREB (cAMP response element-binding protein)-binding protein (CBP) enhances AR activity in a ligand-dependent manner. In the present study, we have investigated whether CBP modifies antagonist/agonist balance of the nonsteroidal anti-androgens hydroxyflutamide and bicalutamide. In prostate cancer DU-145 cells, which were transiently transfected with CBP cDNA, hydroxyflutamide enhanced AR activity to a greater extent than bicalutamide in the presence of either wild-type or the mutated AR 730 val-->met. In two sublines of LNCaP cells that contain the mutated AR 877 thr-->ala and overexpressed CBP, increase in AR activity was observed after treatment with hydroxyflutamide but not with bicalutamide. Anti-androgens did not influence AR expression in cells transfected with CBP cDNA, as judged by Western blot analysis. Endogenous CBP protein was detected by Western blot in nuclear extracts from the three prostate cancer cell lines, LNCaP, PC-3, and DU-145, all derived from therapy-resistant prostate cancer. In addition, CBP was expressed in both basal and secretory cells of benign prostate epithelium, high-grade prostate intraepithelial neoplasia, and prostate cancer clinical specimens, as evidenced by immunohistochemical staining. Taken together, our findings demonstrate the selective enhancement of agonistic action of the anti-androgen hydroxyflutamide by the transcriptional co-activator CBP, which is a new, potentially relevant mechanism contributing to the acquisition of therapy resistance in prostate cancer.

Accelerated in vivo growth of prostate tumors that up-regulate interleukin-6 is associated with reduced retinoblastoma protein expression and activation of the mitogen-activated protein kinase pathway.

Interleukin-6 (IL-6) is a multifunctional cytokine that activates the signaling pathways of Janus kinases-signal transducers and activators of transcription (STAT) and/or mitogen-activated protein kinases (MAPK) in various tumors. Thus, it modulates cell growth and apoptosis. IL-6 levels are elevated in tissues and sera from prostate cancer patients and IL-6 receptor expression has been detected in prostate cancer cell lines and clinical specimens. Continuous exposure of prostate cancer cells to IL-6 might alter their responsiveness to this cytokine. To gain more insight into the function of IL-6 in prostate carcinoma, we have inoculated LNCaP-IL-6+ cells, generated after prolonged treatment with IL-6, into nude mice (total n = 16, two independent experiments). Controls included animals bearing LNCaP-IL-6- cells, passaged at the same time as LNCaP-IL-6+ cells without supplementation of IL-6. LNCaP-IL-6+ tumor volumes were larger than those of their counterparts at all time points. There were no signs of cachexia in any of the experimental animals and all mice were free of metastases. To better understand the mechanisms responsible for accelerated growth of LNCaP-IL-6+ tumors, we have investigated the expression of cell-cycle regulatory molecules by Western blot analysis. The levels of cyclin-dependent kinase 2 were elevated in LNCaP-IL-6+ cells. There was a strong down-regulation of cyclins D1 and E in the LNCaP-IL-6+ subline. The cell-cycle inhibitor p27 was expressed at a low level in LNCaP-IL-6+ cells and could not be up-regulated by addition of IL-6. Most notably, LNCaP-IL-6+ cells exhibited a reduced expression of the hypophosphorylated form of the retinoblastoma protein (pRb). Accelerated tumor growth in our model system was also associated with alterations in IL-6-signaling pathways. The ability of IL-6 to induce tyrosine phosphorylation of STAT3 was abolished in the LNCaP-IL-6+ subline. In contrast, the levels of the MAPK extracellular signal-regulated kinases 1/2 increased in cells generated after long-term IL-6 treatment. The inhibitor of MAPK kinase PD 98059 retarded the proliferation of LNCaP-IL-6+ but not that of control cells. In summary, we show in the present study that chronic exposure of prostate cancer cells to IL-6 facilitates tumor growth in vivo by abolishment of the growth control by pRb and activation of the MAPK signaling pathway. These findings could be relevant to understand the role of IL-6 in prostate cancer progression.