Anton Hittmair

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.

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.

Presence and possible significance of immunocytochemically demonstrable metallothionein over-expression in primary invasive ductal carcinoma of the breast

Metallothioneins (MTs) are ubiquitous low-molecular-weight proteins with a high affinity for heavy metal ions such as zinc, copper and cadmium. MT over-expression has been associated with resistance against anticancer drugs. In the present study we investigated 86 cases (45 cases of tumour category pT1 and 41 of category pT2) of routinely fixed and paraffin-embedded primary breast carcinomas immunohistochemically with a monoclonal antibody to an epitope of MT shared by its I and II isoforms. Immunohistochemically demonstrated MT over-expression was found in the invasive components of 7 of 32 pT1 and 17 of 28 pT2 invasive ductal carcinomas, whereas all 26 invasive lobular carcinomas gave weak or negative results. Fourteen of 17 pT2 and 2 of 7 pT1 invasive ductal carcinomas with MT over-expression developed metastases during follow-up with poor prognostic outcome. In contrast only 3 of 11 pT2 and none of the 25 pT1 cases without MT over-expression had a poor clinical course (P < 0.001). It is concluded that MT over-expression is associated with significantly poor prognosis particularly in pT2 invasive ductal breast carcinomas.

LAPAROSCOPIC RETROPERITONEAL LYMPH NODE DISSECTION FOR CLINICAL STAGE I NONSEMINOMATOUS TESTICULAR CARCINOMA: LONG-TERM OUTCOME

PURPOSE We report the long-term oncological efficacy and morbidity of laparoscopic retroperitoneal lymph node dissection for testicular carcinoma. MATERIALS AND METHODS From August 1992 to September 1999, 73 consecutive patients underwent laparoscopic retroperitoneal lymph node dissection with modified unilateral template dissection. All lumbar vessels within the template were routinely transected in the initial 29 cases only. Patients with positive lymph nodes received 2 cycles of chemotherapy. RESULTS Operative time ranged from 150 to 630 minutes (mean 221) in our most recent 28 cases. All but 2 operations were completed as planned for a conversion rate of 2.7%. Minor intraoperative complications developed in only 6.8% of cases. In our last 44 patients there was no major and only 1 minor (2.3%) postoperative complication. Mean postoperative hospitalization was 3.3 days. Ejaculation was preserved in all patients. Lymph nodes were positive in 19 cases (26%). Mean followup in 47 patients with pathological stage I disease was 43.3 months (range 7 to 84). We noted 1 retroperitoneal recurrence due to false-negative histological findings but there were no other relapses. At a mean followup of 42.7 months (range 6 to 86) 17 patients with pathological stage II carcinoma treated with 2 cycles of adjuvant chemotherapy were also free of disease. CONCLUSIONS In our hands laparoscopic retroperitoneal lymph node dissection has not only proved its surgical efficiency, but also its oncological efficacy. Patient satisfaction is high. During long-term followup of more than 3 years not a single recurrence developed due to surgical failure.

Immunohistochemically demonstrated metallothionein expression in malignant melanoma.

Metallothioneins are ubiquitous proteins with a high affinity for heavy metal ions, e.g. zinc, copper and cadmium. Experimentally, metallothionein over‐expression in cell lines derived from a variety of cancers has been associated with resistance to anticancer drugs and irradiation therapy. Using a monoclonal antibody (E9) to metallothionein we investigated immunoreactive expression in routinely fixed and paraffin‐embedded tissue from 63 cases of malignant melanoma and 13 secondary deposits. Whereas a variety of cells in normal skin showed metallothionein expression, all forms of benign naevi studied were uniformly negative. In contrast 13/30 ‘thin’ (≤1.5 mm; 0.7 ± 0.4). 25 29 ‘thick’ malignant melanoma (> 1.5 mm; 5.5 ± 3.9) and 12/13 metastases were positive. Six patients with thin and 19 with thick melanoma with metallothionein expression died during a mean observation period of 6.4 ± 1.8 and 3.6 ± 2.5 years, respectively, their survival distribution function analyses giving statistically significant results for both the vertical tumour thickness (P < 0.0001) and metallothionein expression (P < 0.0001). These immunohistochemical results, based on routinely processed paraffin‐embedded tissue, suggest that metallothionein expression in malignant melanoma is significantly associated with progressive disease and might therefore be a useful prognostic indicator.

Basic fibroblast growth factor levels in cancer cells and in sera of patients suffering from proliferative disorders of the prostate.

Both benign and malignant growth of the prostate depend on the induction of a microvasculature. Basic fibroblast growth factor (bFGF), a potent angiogenic factor, is thought to play an important role in this process.

Human prostatic smooth muscle cells in culture: Estradiol enhances expression of smooth muscle cell-specific markers

Smooth muscle cells (SMCs) constitute a major cellular component of prostatic stroma. SMC tension plays an important role in urethral obstruction secondary to benign prostatic hyperplasia (BPH). We have developed an in vitro procedure for the propagation of human prostatic SMCs. Tissue specimens from patients undergoing radical prostatectomy or cystectomy were enzymatically disaggregated and cultured in MCDB‐131 medium supplemented with horse serum, insulin, conditioned medium from the tumor cell line CRL‐5813, and steroid hormones. The medium was assembled on the basis of the effects these supplements have on the growth of SMC cultures and on the expression of the two markers desmin and smooth muscle myosin. Addition of 0.1 μM of estradiol to the growth medium dramatically increased expression of these SMC‐specific markers. Dihydrotestosterone (DHT) and hydrocortisone had a similar, albeit less pronounced effect. At three to five passages, about two thirds of the cells were immunohistologically positive for smooth muscle myosin or desmin. Almost all cells were positive for the myofibroblast marker smooth muscle α‐actin throughout 10 passages and more. In SMC cultures, cells staining for smooth muscle myosin and desmin were found to seek direct contact to myofibroblasts. They grew in aggregates on a layer of myofibroblasts which adhered to the surface of the culture vessel. As revealed by transmission electron microscopy the cultured cells exhibited morphological features of myofibroblasts. Characteristics of smooth muscle cells, such as prominent bundles of microfilaments associated with dense bodies, basal laminae investing the cells, and numerous caveolae at the cell surfaces were regularly observed in cultures of low passages. After several passages, these features were markedly decreased and organelles of the biosynthetic system became more prominent. In summary, we present an in vitro model of prostatic SMCs and demonstrate that steroid hormones have characteristic effects on these cells. SMC cultures are expected to facilitate investigation of the functions and properties of human prostatic SMCs. Prostate 30:117–129, 1997.