ABSTRACT
Current endocrine treatment for advanced prostate cancer does not result in a complete ablation of adrenal androgens. Adrenal androgens can be metabolized by prostate cancer cells, which is one of the mechanisms associated with progression to castration-resistant prostate cancer (CRPC). Aldo-keto reductase family 1 member C3 (AKR1C3) is a steroidogenic enzyme that plays a crucial role in the conversion of adrenal androgen dehydroepiandrosterone (DHEA) into high-affinity ligands for the androgen receptor (testosterone [T] and dihydrotestosterone [DHT]). The aim of this study was to examine whether AKR1C3 could be used as a marker and therapeutic target for CRPC. AKR1C3 mRNA and protein levels were upregulated in CRPC tissue, compared with benign prostate and primary prostate cancer tissue. High AKR1C3 levels were found only in a subset of CRPC patients. AKR1C3 can be used as a biomarker for active intratumoral steroidogenesis and can be measured in biopsy or transurethral resection of the prostate specimens. DuCaP (a CRPC cell line that has high AKR1C3 expression levels) used and converted DHEA under hormone-depleted conditions into T and DHT. The DHEA-induced growth of DuCaP could be antagonized by indomethacine, an inhibitor of AKR1C3. This study indicates that AKR1C3 can be considered a therapeutic target in a subgroup of patients with high AKR1C3 expression.
Subject(s)
3-Hydroxysteroid Dehydrogenases/metabolism , Biomarkers, Tumor/metabolism , Hydroxyprostaglandin Dehydrogenases/metabolism , Molecular Targeted Therapy , Orchiectomy , Prostatic Neoplasms/enzymology , Prostatic Neoplasms/surgery , Aldo-Keto Reductase Family 1 Member C3 , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Dehydroepiandrosterone/pharmacology , Dihydrotestosterone/metabolism , Humans , Indomethacin/pharmacology , Male , Prostatic Neoplasms/pathology , Testosterone/metabolismABSTRACT
Considerable levels of testosterone and dihydrotestosterone (DHT) are found in prostate cancer (PCa) tissue after androgen deprivation therapy. Treatment of surviving cancer-initiating cells and the ability to metabolize steroids from precursors may be the keystones for the appearance of recurrent tumors. To study this hypothesis, we assessed the expression of several steroidogenic enzymes and stem cell markers in clinical PCa samples and cell cultures during androgen depletion. Gene expression profiles were determined by microarray or qRT-PCR. In addition, we measured cell viability and analyzed stem cell marker expression in DuCaP cells by immunocytochemistry. Seventy patient samples from different stages of PCa, and the PCa cell line DuCaP were included in this study. The androgen receptor (AR) and enzymes (AKR1C3, HSD17B2, HSD17B3, UGT2B15 and UGT2B17 ) that are involved in the metabolism of adrenal steroids were upregulated in castration resistant prostate cancer (CRPC). In vitro, some DuCaP cells survived androgen depletion, and eventually gave rise to a culture adapted to these conditions. During and after this transition, most of the steroidogenic enzymes were upregulated. These cells also are enriched with stem/progenitor cell markers cytokeratin 5 (CK5) and ATP-binding cassette sub-family G member 2 (ABCG2). Similarly, putative stem/progenitor cell markers CK5, c-Kit, nestin, CD44, c-met, ALDH1A1, α2-integrin, CD133, ABCG2, CXCR4 and POU5F1 were upregulated in clinical CRPC. The upregulation of steroidogenic enzymes and stem cell markers in recurrent tumors suggests that cancer initiating cells can expand by adaptation to their T/DHT deprived environment. Therapies targeting the metabolism of adrenal steroids by the tumor may prove effective in preventing tumor regrowth.
Subject(s)
Biomarkers, Tumor/metabolism , Enzymes/genetics , Neoplastic Stem Cells/metabolism , Prostatic Neoplasms/genetics , 3-Hydroxysteroid Dehydrogenases/genetics , 3-Hydroxysteroid Dehydrogenases/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/metabolism , Aldo-Keto Reductase Family 1 Member C3 , Androgen Antagonists/therapeutic use , Cell Line, Tumor , Enzymes/metabolism , Estradiol Dehydrogenases/genetics , Estradiol Dehydrogenases/metabolism , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Glucuronosyltransferase/genetics , Glucuronosyltransferase/metabolism , Humans , Hydroxyprostaglandin Dehydrogenases/genetics , Hydroxyprostaglandin Dehydrogenases/metabolism , Immunohistochemistry , Keratin-5/metabolism , Male , Minor Histocompatibility Antigens , Neoplasm Proteins/metabolism , Oligonucleotide Array Sequence Analysis , Orchiectomy , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Reverse Transcriptase Polymerase Chain Reaction , Steroids/metabolism , Up-Regulation/drug effectsABSTRACT
BACKGROUND: Even though patients with prostate cancer commonly respond to endocrine treatment, in most cases the disease progresses to castration resistant prostate cancer (CRPC). Our objective was to generate a novel cell line model representing the endocrine treatment naive prostate cancer for testing treatments that target the androgen receptor (AR) and androgen metabolism. METHODS: After culturing DuCaP cells 20 passages with additional 1 nM R1881, DuCaP-N(aive) cell line was developed and validated for testing endocrine therapy combinations. Cell viability, apoptosis and cell cycle distribution were assessed in DuCaP and DuCaP-N when interfering with the hormonal content. RESULTS: Addition of 1 nM R1881 to DuCaP reduces cell viability and induces cell cycle inhibition and apoptosis. Eventually, an androgen accustomed DuCaP-N cell line developed. An antiandrogen (bicalutamide), a histone deacetylase (HDAC) inhibitor (trichostatin A) and a 5alpha-reductase (SRD5A) inhibitor (finasteride) reduce cell viability, and their combinations give a synergistic response in inducing apoptosis. CONCLUSIONS: The TMPRSS2-ERG expressing DuCaP-N cell line represents human prostate cancer prior to endocrine treatment, and its parental DuCaP cell line is a model for CRPC. These cell lines can be used for preclinical evaluation of compounds that target the androgenic pathway.
Subject(s)
Androgen Antagonists/therapeutic use , Finasteride/therapeutic use , Prostatic Neoplasms/drug therapy , Androgens/pharmacology , Androgens/therapeutic use , Animals , Apoptosis , Cell Cycle , Cell Line, Tumor , Cell Survival , Cholesterol Side-Chain Cleavage Enzyme/genetics , DNA Primers , Disease Progression , Humans , In Situ Hybridization, Fluorescence , Male , Mice , Mice, SCID , Polymerase Chain Reaction/methods , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , RNA, Messenger/genetics , Serine Endopeptidases/genetics , Steroids/therapeutic useABSTRACT
MicroRNAs (miRNA) are small, endogenously expressed noncoding RNAs that negatively regulate expression of protein-coding genes at the translational level. Accumulating evidence, such as aberrant expression of miRNAs, suggests that they are involved in the development of cancer. They have been identified in various tumor types, showing that different sets of miRNAs are usually deregulated in different cancers. To identify the miRNA signature specific for prostate cancer, miRNA expression profiling of 6 prostate cancer cell lines, 9 prostate cancer xenografts samples, 4 benign prostatic hyperplasia (BPH), and 9 prostate carcinoma samples was carried out by using an oligonucleotide array hybridization method. Differential expression of 51 individual miRNAs between benign tumors and carcinoma tumors was detected, 37 of them showing down-regulation and 14 up-regulation in carcinoma samples, thus identifying those miRNAs that could be significant in prostate cancer development and/or growth. There was a significant trend (P=0.029) between the expression of miRNAs and miRNA locus copy number determined by array comparative genomic hybridization, indicating that genetic aberrations may target miRNAs. Hierarchical clustering of the tumor samples by their miRNA expression accurately separated the carcinomas from the BPH samples and also further classified the carcinoma tumors according to their androgen dependence (hormone naive versus hormone refractory), indicating the potential of miRNAs as a novel diagnostic and prognostic tool for prostate cancer.
Subject(s)
Gene Expression Profiling , Gene Expression Regulation, Neoplastic , MicroRNAs , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Carcinoma/metabolism , Cell Line, Tumor , Genes, Neoplasm , Humans , Male , MicroRNAs/metabolism , Models, Genetic , Nucleic Acid Hybridization , Oligonucleotides/chemistry , Prognosis , Prostate/metabolismABSTRACT
BACKGROUND: Recent studies indicate the presence of a small, stem-like cell population in several human cancers that is crucial for the tumour (re)population. OBJECTIVE: Six established prostate cancer (PCa) cell lines-DU145, DuCaP, LAPC-4, 22Rv1, LNCaP, and PC-3-were examined for their stem cell properties in vitro. DESIGN, SETTINGS, AND PARTICIPANTS: The colony-forming efficiency and self-renewal ability of morphologically distinguishable holoclones and paraclones were tested with low-density plating and serial passaging. Expression of the putative stem cell marker CD133 and breast cancer resistance protein (BCRP) was examined with flow cytometry, and immunohistochemical stainings were made for CD133, alpha2-integrin, nestin, BCRP, cytokeratin 5 (CK5), and cytokeratin 18 (CK18). RESULTS AND LIMITATIONS: Five out of six cell lines formed clear holo-, mero-, and paraclones. Unlike paraclones, we can maintain DU145 holoclones in culture for several passages, which is indicative of self-renewal ability. Using fluorescence-activated cell sorting (FACS) analysis only in DU145 cells, a small fraction (0.01%) of CD133(+) cells was detected. CD133(+) cells; however, like DU145 BCRP(+) (0.15%) cells, they were not more clonogenic, and they did not show more holoclone formation than the marker-negative cells or unselected cells. Immunohistochemistry revealed alpha2-integrin and BCRP as potential stem cell markers and CK5 with the combination of CK18 to distinguish transient amplifying cells. CONCLUSIONS: These results indicate the possible presence of stem-like cells in several established PCa cell lines. CD133 selection does not enrich for stem-like cells in PCa cell lines.