Estrogens have been implicated in prostatic cancerogenesis and tumor progression. The mechanisms underlying estrogen signaling in human prostate tissue, however, remain poorly understood. Using immunohistochemical and in situ hybridization (ISH) techniques, the present study demonstrates the classical estrogen receptor (ERalpha) in premalignant lesions and prostatic adenocarcinoma through the various stages of the disease. Conversely, the novel characterized ERbeta subtype was undetectable in human prostate tissue. High-grade prostatic intraepithelial neoplasia revealed ERalpha mRNA and protein expression in 28% and 11% of cases evaluated. Focal ER immunoreactivity was detected in a minority of low- to intermediate-grade adenocarcinoma. High-grade (primary Gleason grade 4 and 5) tumors revealed ER protein expression in 43% (62% respectively) of cases. The most significant ERalpha gene expression on mRNA and protein levels was observed in hormone refractory tumors and metastatic lesions, including lymph node and bone metastases. Results of the current study suggest that estrogens can affect prostatic cancerogenesis and neoplastic progression through an ER-mediated process in human prostate tissue.

The progressive emergence of the PR during tumor progression obviously reflects the ability of metastatic and androgen-insensitive tumors to use estrogens through a ERalpha-mediated pathway. The present data provide a theoretical background for studying the efficiency of antiestrogens and antigestagens in the medical treatment of advanced prostate cancer.

BACKGROUND The recent discovery of the classical estrogen receptor α (ERα) in androgen‐insensitive prostate cancer has shed new light on the role of estrogens in endocrine therapy failure. To get more information on downstream events of estrogen signaling in these tumors, we investigated the relation between ERα gene expression, and the estrogen‐inducible heat shock protein HSP27 in recurrent prostatic adenocarcinomas. METHODS Palliative transurethral resection specimens from 50 patients with androgen‐insensitive disease were submitted for study. Messenger RNA in situ hybridization for the ERα and immunohistochemistry of the HSP27 protein were performed on adjacent sections of an equal number of prostate cancer tissue with and without ERα protein expression. RESULTS Cancerous lesions lacking the nuclear ERα at the protein level revealed ERα mRNA expression in 15 of 25 cases (60%). A coordinate expression of ERα mRNA and HSP27 was observed in 33 of 40 cases (83%), although a significant correlation between ERα protein and HSP27 expression was not obtained. Conversely, 90% of neoplastic lesions without detectable levels of ERα mRNA and protein also lacked HSP27 immunoreactivity. CONCLUSIONS ERα gene expression at the mRNA level significantly correlated with the immunoprofile of the estrogen‐inducible HSP27 protein in androgen‐insensitive prostatic adenocarcinomas. This may indicate that these tumors harbor functional active estrogen receptors promoting transcriptional activity of the HSP27 gene. Determination of the receptor status by immunohistochemistry is unable to identify neoplastic lesions with established ERα mRNA expression in a substantial number of cases. HSP27 may be an additional surrogate biomarker for estrogen‐regulated growth in androgen‐insensitive prostate cancer. Prostate 45:36–41, 2000. © 2000 Wiley‐Liss, Inc.

In situ DNA fragmentation assays have proved to be particularly useful in the detection of apoptosis in routinely processed, paraffin-embedded tissue sections. In the present study, a triple-antigen labelling technique was performed to demonstrate DNA fragmentation (apoptosis), cell proliferation (MIB-1), and phenotypic markers in the same tissue section. The in situ apoptosis assay was conducted with the TUNEL method developed by a avidin-biotin alkaline phosphatase complex (ABcomplex/AP). The proliferation-associated MIB-1 antigen was demonstrated in the second staining sequence by the avidin-biotin peroxidase method (ABC). The phenotypic markers chromogranin A or prostate-specific antigen (PSA) were visualized by the alkaline phosphatase anti-alkaline phosphatase method (APAAP) in the third staining sequence. The feasibility of this triple-labelling technique was tested in formalin-fixed, paraffin-embedded tissue of prostatic adenocarcinomas from 8 patients with recurrent, hormone-refractory disease. Although these tumours revealed marked neuroendocrine differentiation, cell proliferation and apoptosis were detected exclusively in non-endocrine (chromogranin A-negative) tumour cells that expressed PSA variably. The triple-labelling protocol described here allows the phenotypic characterization of proliferating and apoptotic cell populations in the same tissue section. It may be useful in studies of tissue kinetics in physiological and pathological processes.

BACKGROUND. The recent discovery of the classical estrogen receptor ␣ (ER␣) in androgeninsensitive prostate cancer has shed new light on the role of estrogens in endocrine therapy failure. To get more information on downstream events of estrogen signaling in these tumors, we investigated the relation between ER␣ gene expression, and the estrogen-inducible heat shock protein HSP27 in recurrent prostatic adenocarcinomas. METHODS. Palliative transurethral resection specimens from 50 patients with androgeninsensitive disease were submitted for study. Messenger RNA in situ hybridization for the ER␣ and immunohistochemistry of the HSP27 protein were performed on adjacent sections of an equal number of prostate cancer tissue with and without ER␣ protein expression. RESULTS. Cancerous lesions lacking the nuclear ER␣ at the protein level revealed ER␣ mRNA expression in 15 of 25 cases (60%). A coordinate expression of ER␣ mRNA and HSP27 was observed in 33 of 40 cases (83%), although a significant correlation between ER␣ protein and HSP27 expression was not obtained. Conversely, 90% of neoplastic lesions without detectable levels of ER␣ mRNA and protein also lacked HSP27 immunoreactivity. CONCLUSIONS. ER␣ gene expression at the mRNA level significantly correlated with the immunoprofile of the estrogen-inducible HSP27 protein in androgen-insensitive prostatic adenocarcinomas. This may indicate that these tumors harbor functional active estrogen receptors promoting transcriptional activity of the HSP27 gene. Determination of the receptor status by immunohistochemistry is unable to identify neoplastic lesions with established ER␣ mRNA expression in a substantial number of cases. HSP27 may be an additional surrogate biomarker for estrogen-regulated growth in androgen-insensitive prostate cancer. Prostate 45: 36-41, 2000.