Zoran Culig

Switch from antagonist to agonist of the androgen receptor blocker bicalutamide is associated with prostate tumour progression in a new model system

SummaryAdvanced prostate cancer is treated by androgen ablation and/or androgen receptor (AR) antagonists. In order to investigate the mechanisms relevant to the development of therapy-resistant tumours, we established a new tumour model which closely resembles the situation in patients who receive androgen ablation therapy. Androgen-sensitive LNCaP cells were kept in androgen-depleted medium for 87 passages. The new LNCaP cell subline established in this manner, LNCaP-abl, displayed a hypersensitive biphasic proliferative response to androgen until passage 75. Maximal proliferation of LNCaP-abl cells was achieved at 0.001 nM of the synthetic androgen methyltrienolone (R1881), whereas 0.01 nM of this compound induced the same effect in parental cells. At later passages (> 75), androgen exerted an inhibitory effect on growth of LNCaP-abl cells. The non-steroidal anti-androgen bicalutamide stimulated proliferation of LNCaP-abl cells. AR protein expression in LNCaP-abl cells increased approximately fourfold. The basal AR transcriptional activity was 30-fold higher in LNCaP-abl than in LNCaP cells. R1881 stimulated reporter gene activity in LNCaP-abl cells even at 0.01 nM, whereas 0.1 nM of R1881 was needed for induction of the same level of reporter gene activity in LNCaP cells. Bicalutamide that acts as a pure antagonist in parental LNCaP cells showed agonistic effects on AR transactivation activity in LNCaP-abl cells and was not able to block the effects of androgen in these cells. The non-steroidal AR blocker hydroxyflutamide exerted stimulatory effects on AR activity in both LNCaP and LNCaP-abl cells; however, the induction of reporter gene activity by hydroxyflutamide was 2.4- to 4-fold higher in the LNCaP-abl subline. The changes in AR activity were associated neither with a new alteration in AR cDNA sequence nor with amplification of the AR gene. Growth of LNCaP-abl xenografts in nude mice was stimulated by bicalutamide and repressed by testosterone. In conclusion, our results show for the first time that the non-steroidal anti-androgen bicalutamide acquires agonistic properties during long-term androgen ablation. These findings may have repercussions on the natural course of prostate cancer with androgen deprivation and on strategies of therapeutic intervention.

Regulation of prostatic growth and function by peptide growth factors

Polypeptide growth factors are positive and negative regulators of prostatic growth and function. Expression and biological effects of epidermal growth factor (EGF), transforming growth factors (TGFs) α and β, fibroblast growth factors (FGFs), and insulin‐like growth factors (IGFs) in the prostate have been extensively studied. EGF and TGFα, which share the same receptor, are strong mitogens for prostatic epithelial and stromal cells. Their paracrine mode of action in normal tissue and early‐stage tumors is apparently altered towards an autocrine stimulation in hormone‐independent tumors, which gain the ability to produce TGFα by themselves. TGFβ has a dual role in the regulation of prostatic growth. It inhibits growth of prostatic epithelial cells in culture and mediates programmed cell death after androgen withdrawal. However, advanced prostatic carcinomas become insensitive to the inhibitory effect of TGFβ. Several members of the FGF family have been identified in the prostate. They are mainly or exclusively expressed in the stromal cells, and stimulate the epithelial cells. In the rat Dunning tumor model, progression is accompanied by distinct changes in the expression of FGFs and their receptors. In the hyperplastic tissue, basic FGF (bFGF) is accumulated. This growth factor is also a potent angiogenic inducer, expression of which may determine the metastatic capability of a tumor. IGFs are paracrine growth stimulators in the normal and hyperplastic prostate. It is still under consideration whether prostatic cancer cells gain the ability to produce IGF‐I by themselves and thus shift to an autocrine mode of IGF‐I stimulation. Growth factors also interact with the androgen‐signaling pathway. IGF‐I in particular, other growth factors as well, can activate the androgen receptor.

Interleukin-6 and prostate cancer progression

Prostate cancer, while initially dependent on androgens for proliferation, progresses to an androgen-independent state. Evidence has been accumulating that interleukin-6 (IL-6) may contribute to prostate cancer progression. Serum levels of IL-6 correlate with prostate tumor burden and patient morbidity. The prostate tissue itself appears to be a source of IL-6 and its receptor. Furthermore, experimental data suggest that IL-6 is an autocrine and paracrine growth factor for androgen-independent prostate cancer cell lines. For example, inhibition of IL-6, with anti-IL-6 antibody, sensitizes androgen-independent prostate cancer cells to chemotherapeutic agents in vitro. Finally, IL-6 activates a variety of signal transduction cascades, some which stimulate androgen receptor activity, in prostate cancer cells. These data suggest that targeting IL-6 may have multiple benefits in prostate cancer patients.

Interleukin‐6 regulation of prostate cancer cell growth

Interleukin‐6 (IL‐6) is involved in regulation of immune reaction and cell growth and differentiation. It causes multifunctional responses ranging from inhibition of proliferation to promotion of cell survival. IL‐6 effects may depend on experimental conditions such as passage numbers and serum composition. IL‐6 signals in target tissues through the receptor that is composed of the ligand‐binding and signal‐transducing subunits. IL‐6 is expressed in benign and malignant prostate tissue and the levels of the cytokine and its receptor increase during prostate carcinogenesis. IL‐6 is considered a positive growth factor for most prostate cells. The only exemption seems to be the LNCaP cell line, in which IL‐6 causes growth arrest and induces differentiation function. In contrast, IL‐6 acts as an autocrine growth factor in the subline LNCaP‐IL‐6+ established after chronic treatment with IL‐6. IL‐6 is a candidate for targeted therapy in prostate cancer because of its association with morbidity. Activation of signaling pathways of Janus kinase/signal transducers and activators of transcription factors, mitogen‐activated protein kinase (MAPK), and phosphatidylinositol 3‐kinase has been reported in various prostate cancer cell lines. IL‐6 and the related cytokine oncostatin M induce activation of the androgen receptor (AR) in the absence of androgen. IL‐6 is also involved in regulation of vascular endothelial growth factor expression as well as neuroendocrine differentiation in prostate. Anti‐IL‐6 antibodies showed an inhibitory effect on the PC‐3 xenograft. However, the development of this therapy in prostate cancer is in early stages.

Inhibition of LncaP prostate cancer cells by means of androgen receptor antisense oligonucleotides.

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 ∼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.

Immunohistochemical localization of interleukin‐6 and its receptor in benign, premalignant and malignant prostate tissue

Interleukin‐6 (IL‐6) is a pleiotropic cytokine that interacts with its receptor in prostate cells, thus regulating proliferative response and differentiation. It also activates the human androgen receptor in prostate cancer cell lines. In order to assess the significance of these findings in vivo, the expression of key elements of the IL‐6 signalling pathway, IL‐6 and its receptor, was investigated in tissue samples obtained on radical prostatectomy from prostate cancer patients. IL‐6 immunohistochemistry was performed on 17 frozen prostate cancer specimens. IL‐6 receptor immunostaining was evaluated in 21 paraffin‐embedded prostate tumour specimens. In both groups, adjacent areas of high‐grade prostatic intraepithelial neoplasia and benign tissue were also investigated. In benign prostatic epithelium, IL‐6 was localized predominantly in basal cells, whereas in prostate cancer tissues more IL‐6‐positive glandular cells were identified. No IL‐6 expression was detected in stromal cells on immunohistochemistry, although IL‐6 protein was measured in the supernatants obtained from cultured stromal cells by ELISA. IL‐6 receptor was expressed in benign prostatic tissue in both epithelial and stromal cells. Furthermore, IL‐6 receptor expression was observed in all tumour specimens investigated and the majority of Gleason patterns analysed had more than 50% of cells showing a positive reaction. IL‐6 and IL‐6 receptor expression patterns in high‐grade prostatic intraepithelial neoplasia lesions were similar to those observed in tumour tissues. Taken together, the results of the present study imply that there are paracrine and autocrine IL‐6 loops in benign and neoplastic prostate. Copyright

Androgen receptor status of lymph node metastases from prostate cancer.

To date androgen receptor (AR) expression and structure in human prostatic cancer have been studied in primary tumor specimens and in cell lines. Investigation of alterations in the androgen‐signalling transduction cascade in prostatic carcinoma metastases is important to improve our understanding of tumor progression towards androgen insensitivity. In the present study we have collected data comparing AR expression in both the primary tumors and the respective pelvic lymph node metastases. Formalin‐fixed and paraffin‐embedded tissues derived from the primary tumors and positive lymph nodes of 12 patients undergoing radical prostatectomy were immunostained for the AR and prostate‐specific antigen (PSA). AR expression was evaluated with the polyclonal antibody PG‐21, which is directed against amino acids 1–21 in the N‐terminal region of the AR. All primary tumors stained for the AR. In 8 of the 12 lymph nodes examined more than 50% of the tumor cells were AR positive and displayed a uniform staining pattern; in one lymph node metastasis remarkable heterogeneity in AR expression was observed. In two cases less than 10% of the tumor cells stained for the AR. In one case the lymph node metastasis was immunohistochemically negative for the AR, whereas the primary tumor obtained from the same patient displayed intense staining for the AR. PSA was expressed in all metastases and primary tumors. Our data demonstrate that loss of the AR in lymph node metastases from prostatic carcinoma is a rare event.

Expression, structure, and function of androgen receptor in advanced prostatic carcinoma

Endocrine therapy for prostate cancer aims to reduce the levels of circulating androgen or to inhibit androgen action by blocking the androgen receptor in the prostate, or both. Studies in various animal and human prostate cancer models suggested that there may be a downregulation of androgen receptor during prostate cancer progression. Recent work, however, showed androgen receptor expression in all stages of prostate cancer. The presence of mutant androgen receptors in a portion of prostate cancers and receptor activation in the absence of androgen or in the presence of low androgen concentrations is discussed within this context.

Prevention and early detection of prostate cancer

Prostate cancer is a common malignancy in men and the worldwide burden of this disease is rising. Lifestyle modifications such as smoking cessation, exercise, and weight control offer opportunities to reduce the risk of developing prostate cancer. Early detection of prostate cancer by prostate-specific antigen (PSA) screening is controversial, but changes in the PSA threshold, frequency of screening, and the use of other biomarkers have the potential to minimise the overdiagnosis associated with PSA screening. Several new biomarkers for individuals with raised PSA concentrations or those diagnosed with prostate cancer are likely to identify individuals who can be spared aggressive treatment. Several pharmacological agents such as 5α-reductase inhibitors and aspirin could prevent development of prostate cancer. In this Review, we discuss the present evidence and research questions regarding prevention, early detection of prostate cancer, and management of men either at high risk of prostate cancer or diagnosed with low-grade prostate cancer.

Inhibition of the acetyltransferases p300 and CBP reveals a targetable function for p300 in the survival and invasion pathways of prostate cancer cell lines

Inhibitors of histone deacetylases have been approved for clinical application in cancer treatment. On the other hand, histone acetyltransferase (HAT) inhibitors have been less extensively investigated for their potential use in cancer therapy. In prostate cancer, the HATs and coactivators p300 and CBP are upregulated and may induce transcription of androgen receptor (AR)-responsive genes, even in the absence or presence of low levels of AR. To discover a potential anticancer effect of p300/CBP inhibition, we used two different approaches: (i) downregulation of p300 and CBP by specific short interfering RNA (siRNA) and (ii) chemical inhibition of the acetyltransferase activity by a newly developed small molecule, C646. Knockdown of p300 by specific siRNA, but surprisingly not of CBP, led to an increase of caspase-dependent apoptosis involving both extrinsic and intrinsic cell death pathways in androgen-dependent and castration-resistant prostate cancer cells. Induction of apoptosis was mediated by several pathways including inhibition of AR function and decrease of the nuclear factor kappa B (NF-κB) subunit p65. Furthermore, cell invasion was decreased upon p300, but not CBP, depletion and was accompanied by lower matrix metalloproteinase (MMP)-2 and MMP-9 transcriptions. Thus, p300 and CBP have differential roles in the processes of survival and invasion of prostate cancer cells. Induction of apoptosis in prostate cancer cells was confirmed by the use of C646. This was substantiated by a decrease of AR function and downregulation of p65 impairing several NF-κB target genes. Taken together, these results suggest that p300 inhibition may be a promising approach for the development of new anticancer therapies. Mol Cancer Ther; 10(9); 1644–55. ©2011 AACR.