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BACKGROUND In advanced stages of prostate cancer, the phosphatidylinositol-3′ kinase (PI3K)/Akt signaling cascade, one of the major survival pathways in the cell, is frequently constitutively activated due to mutation or loss of the tumor suppressor protein phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Using cell culture models representing different tumor stages, we explored the effect of inhibition of this survival pathway on the induction of apoptosis. METHODS Inhibition of the survival kinase Akt and induction of apoptosis was analyzed in androgen-insensitive DU145 and PC-3 cells, in androgen-responsive LNCaP, and in androgen-independent long-term androgen-ablated LNCaP-abl cells representing therapy-resistant prostate cancer cells. Activated Akt was determined by immunoblotting using a phospho-Akt specific antibody. Induction of apoptosis was analyzed employing annexing V and propidium iodide staining and flow cytometry and measurement of cleavage of the caspases substrate poly-ADP-ribose polymerase (PARP). RESULTS IGF-1, EGF, and heregulin but not PDGF or activators of protein kinase A induced phosphorylation of Akt in DU145 cells and activation was completely blocked by the PI3K inhibitor LY294002. In the hormone-responsive prostate cancer cell line LNCaP that has a constitutively switched-on Akt kinase, LY294002 caused a dose- and time-dependent Akt inhibition, which was absent in long-term androgen-ablated LNCaP sublines. In agreement with the resistance to inhibition of the PI3K/Akt pathway, long-term androgen-ablated LNCaP sublines remained relatively resistant to induction of cell death by LY294002 or the cytotoxic drug etoposide. Inhibition of the PI3K/Akt pathway restored the sensitivity of long-term androgen-ablated cells to induction of apoptosis by a cytotoxic drug almost completely. CONCLUSION These results suggest that long-term androgen ablation therapy for prostate cancer reinforces the PI3K/Akt pathway and impedes its inhibition thus contributing to increased resistance of tumor cells to induction of apoptosis. With regard to treatment of therapy-refractory prostate cancer, these findings suggest effectiveness of a combination of cytotoxic treatment and inhibition of the PI3K-Akt survival pathway in tumor cells after failure of androgen-ablation therapy. © 2003 Wiley-Liss, Inc.

The androgen receptor (AR) is the key regulatory element of androgen signaling in the cell. It mediates action of androgens and is therefore essential for growth, function and differentiation of the human male urogenital tract. Genetic alterations in the AR gene may cause impaired development resulting in androgen insensitivity syndromes (AIS) or in neurodegenerative diseases like Kennedy syndrome. Besides the crucial role in the process of virilization during embryogenesis and puberty, the AR also plays an important role in the adult man as the intracellular mediator of androgen action. Androgen withdrawal and/or AR blockade is the main choice of treatment of nonorgan-confined prostate cancer. Unfortunately, this treatment is only palliative and a majority of these tumors recur and progress to an androgen-independent and therapy-resistant stage. Recent findings gave new insight into the molecular structure and function of the AR and improved our understanding about prostate cancer progression, consequently resulting in the development of novel treatments. It has become evident that the AR is a nuclear transcription factor that can be activated ligand-dependently by androgens as well as ligand-independently by other hormones and various growth factors, respectively. Moreover, it was shown that the interaction of the AR with other proteins of the intracellular signal transduction cascade may promote prostate tumor growth. This review will summarize the most important findings about the AR and the androgen signaling pathway to improve the understanding of prostate diseases and novel treatment strategies that may be useful in the clinic.

Currently available methods for treatment of human prostatic carcinoma aim to inactivate the androgen receptor (AR) by androgen deprivation or blockade with anti-androgens. Failure of endocrine therapy and tumor progression is characterized by androgen-independent growth despite high levels of AR expression in metastatic disease. We inhibited AR expression in LNCaP prostate tumor cells by using antisense AR oligodeoxynucleotides (ODNs) and explored whether antisense AR treatment would be conceivable as a therapy for advanced prostate cancer. Among the various AR antisense ODNs tested, a 15-base ODN targeting the CAG repeats encoding the poly-glutamine region of the AR (as750/15) was found to be most effective. Treatment of LNCaP cells with as750/15 reduced AR expression to approximately 2% within 24 hours compared with mock-treated controls. AR down-regulation resulted in significant cell growth inhibition, strongly reduced secretion of the androgen-regulated prostate-specific antigen, reduction of epidermal growth factor receptor expression, and an increase in apoptotic cells. Mis-sense and mismatched control ODNs had no or only slight effects. Antisense inhibition was also very efficient in LNCaP-abl cells, a subline established after long-term androgen ablation of LNCaP cells, resulting in inhibition of AR expression and cell proliferation that was similar to that seen for parental LNCaP cells. This study shows that inhibition of AR expression by antisense AR ODNs may be a promising new approach for treatment of advanced human prostate cancer.

BACKGROUND Androgen receptor (AR) transcriptional activity is modulated by cofactor proteins. They act as costimulators, corepressors, or bridging proteins, and a disbalanced expression may contribute to the altered activity of the AR in advanced prostate cancer. We investigated the expression of a series of steroid receptor cofactors in prostate cancer cell lines, including several LNCaP sublines, and in prostate stromal cells. METHODS Expression of cofactors was analyzed by means of RT-PCR in PC-3, Du-145, LNCaP, three sublines of LNCaP established after long-term androgen deprivation, and two strains of primary prostate stroma cells. Expression in LNCaP and LNCaP-abl cells (which represented an advanced tumor cell) was analyzed employing semiquantitative RT-PCR. RESULTS Ten of the 12 cofactors tested were expressed in all cells analyzed (AIB1, ARA54, ARA70, CBP, cyclin D1, Her2/neu/erbB2, BAG-1/M/L, SRC-1, SMRT, and TIF2). Only ARA55 and FHL2 mRNAs were not detected in all cells. ARA55 mRNA was absent in LNCaP cells, LNCaP sublines, and DU-145 cells; FHL2 was not expressed in LNCaP cells and its derivatives. The expression pattern was identical in LNCaP cells, and the long-term androgen ablated LNCaP sublines. Moreover, comparison of expression levels in LNCaP and LNCaP-abl cells revealed a slight reduction in LNCaP-abl cells but no gross differences. CONCLUSIONS Prostatic cells express a great number of steroid receptor cofactors. AR activity thus seems to be modulated in a very complex way in prostate cells. Prostate 45:124–131, 2000. © 2000 Wiley-Liss, Inc.

We describe the generation of monocyte-derived dendritic cells (MoDC) from leukapheresis products using the CliniMACS system from Miltenyi Biotec. In a clinical setting, the method turned out to be feasible for the generation of clinical-grade MoDC from patients with metastatic renal-cell carcinoma. MoDC generated with this system exhibited a fully mature phenotype as well as high migratory and T-cell stimulatory capacity.

Androgens are pivotal regulators of prostate cell growth, differentiation and function, and their actions are believed to be involved in prostate cancer development. The androgen-signaling pathway in the prostate gland is therefore one of the possible sites of intervention in prostate cancer prevention efforts. The central element of androgen signaling in the cell is the androgen receptor (AR), a member of the superfamily of nuclear receptors. Binding of androgen to its ligand-binding domain transforms the receptor to an active transcription factor that regulates gene expression by interacting with specific regulatory elements in the promoters of genes. In addition to this genomic action, the AR also interacts with other signaling pathways through protein-protein interaction, for example with AP-1 or Ets transcription factors. It is not only the action of androgenic hormones, but also the interactions with growth factor and protein kinase A-signaling pathways that can induce activation of AR. Moreover, these ligand-independent activators act synergistically together with low concentrations of androgens. The effects of long-term androgen deprivation on androgen signaling have been investigated in the LNCaP cell culture system. Long-term culture in a steroid-free medium results in a subline showing a hyperreactive AR characterized by increased AR expression and enhanced AR transcriptional activity in an environment with low levels of androgen hormones. It is not yet clear if similar changes also occur in normal or premalignant prostate epithelial cells and are thus relevant for prevention trials which interfere with androgen hormone signaling.