RÉSUMÉ
S100A10 (p11) is a plasminogen receptor that regulates cellular plasmin generation by cancer cells. In the current study, we used the MMTV-PyMT mouse breast cancer model, patient tumor microarray, and immunohistochemical (IHC) analysis to investigate the role of p11 in oncogenesis. The genetic deletion of p11 resulted in significantly decreased tumor onset, growth rate, and spontaneous pulmonary metastatic burden in the PyMT/p11-KO (knock-out) mice. This phenotype was accompanied by substantial reduction in Ki67 positivity, macrophage infiltration, decreased vascular density in the primary tumors, and decrease in invasive carcinoma and pulmonary metastasis. Surprisingly, IHC analysis of wild-type MMTV-PyMT mice failed to detect p11 expression in the tumors or metastatic tumor cells and loss of p11 did not decrease plasmin generation in the PyMT tumors and cells. Furthermore, tumor cells expressing p11 displayed dramatically reduced lung metastasis when injected into p11-depleted mice, further strengthening the stromal role of p11 in tumor growth and metastasis. Transcriptome analysis of the PyMT tumors from p11-KO mice showed marked reduction in genes such as Areg, Muc1, and S100a8 involved in breast cancer development, progression, and inflammation. The PyMT/p11-KO tumors displayed a remarkable increase in inflammatory cytokines such as interleukin (Il)-6, Il-10, and interferon (Ifn)-γ. Gene expression profiling and IHC of primary breast cancer samples showed that p11 mRNA and protein levels were significantly higher in tumor tissues compared to normal mammary tissue. P11 mRNA expression was significantly associated with poor patient prognosis and significantly elevated in high grade, triple negative (TN) tumors, and tumors with high proliferative index. This is the first study examining the crucial role of p11 in breast tumor development and metastasis, thus emphasizing its potential as a diagnostic and prognostic biomarker in breast cancer.
RÉSUMÉ
Plasma membrane carboxypeptidase-D (CPD) hydrolyzes C-terminal arginine (Arg) from extracellular substrates, and Arg is converted into nitric oxide (NO) in the cell. CPD is upregulated by prolactin (PRL) and androgens in breast cancer (BCa) cells, increasing NO production to promote cell survival. EDD E3 ubiquitin ligase, upregulated by PRL/androgens, is implicated in TORC1 signaling. This study investigated CPD and EDD in triple-negative (TNBC) and HER2+ BCa. Kaplan-Meier analysis showed a negative correlation between CPD or EDD mRNA expression in TNBC patients and relapse-free survival. Immunohistochemistry showed that benign and malignant breast tissues stained abundantly for the PRL receptor (PRLR) and androgen receptor (AR). CPD and EDD staining were elevated in TNBC and HER2+ tumors as compared to benign tissues. In TNBC/HER2+ cell lines, CPD and EDD protein expression were upregulated by PRL or synthetic androgen methyltrienolone (R1881) at 3-6 h. PRL/R1881-induced CPD in TNBC and HER2+ cells increased intracellular NO production, which was abolished by PRLR antagonist ∆1-9-G129R-hPRL and AR antagonist flutamide. In turn, treatment with NO increased viability and decreased apoptosis in Arg-deprived TNBC cells. Cell viability and apoptosis were also affected in HER2+ cells with CPD knockdown. Lastly, EDD knockdown decreased PRL/R1881-induced phosphorylation of initiation factor 4E binding protein-1 and decreased 4E release in TNBC cells. In summary, PRL/R1881-induced CPD promotes TNBC/HER2+ cell survival through production of NO, and EDD promotes TNBC cell survival by TORC1 activation. This study implicates CPD and EDD as useful therapeutic targets for TNBC/HER2+ tumors, and suggests that PRLR and AR blockade are also beneficial to these patients.
RÉSUMÉ
Previously, we identified a prolactin (PRL)-inducible gene encoding EDD E3 ubiquitin ligase in human breast cancer (BCa) cells. We reported that EDD binds the mTOR (TORC1)-associated α4 phosphoprotein-PP2Ac protein phosphatase complex that regulates initiation of translation and cell cycle progression, and that EDD targets PP2Ac for proteasomal degradation. The present study showed that EDD immunostaining was low in benign human breast tissues, but increased progressively in ductal carcinoma in-situ, low-grade, and high-grade BCa, and in triple-negative BCa (TNBC). EDD mRNA and protein levels varied in human BCa cell lines. In high-EDD expressing MCF-7 and T47D cells, siRNA knockdown of EDD arrested cells in the G2-phase of the cell cycle, decreased cell viability, and increased apoptosis. EDD siRNA-induced apoptosis in MCF-7 cells correlated with significantly increased levels of pro-apoptotic Bim and Bak mRNAs and proteins (P < 0.05, n = 3-6), and increased levels of pro-apoptotic Bax and MOAP-1 proteins (P < 0.001, n = 3-6), leading to increased cleavage of caspase-7 and caspase substrate poly-ADP-ribose polymerase-1 (PARP-1), as compared to control cells. Loss of EDD in MCF-7 cells decreased PRL-induced phosphorylation of eukaryotic initiation factor 4E-binding protein-1, a mediator of TORC1 signaling, resulting in decreased binding of 4E to γ-aminophenyl-m7GTP agarose in Cap-binding assays. In low-EDD expressing MDA-MB-436 TNBC cell line, gain of EDD following pCMV-Tag2B.EDD transfection increased cell resistance to chemotherapeutic drugs cisplatin and doxorubicin, TORC1 inhibitor rapamycin, and TORC1/TORC2 inhibitor INK128, as compared to controls. In contrast, loss of EDD in MCF-7 cells increased cell sensitivity to cisplatin, doxorubicin, rapamycin, and selective estrogen receptor modulator tamoxifen. In summary, EDD levels increase with BCa progression in vivo. PRL-inducible EDD in BCa cells promotes TORC1 signaling, anti-apoptotic protein expression, and drug resistance in vitro. These findings implicate EDD as a potential therapeutic target and support PRL receptor blockade as an additional therapy for BCa.
RÉSUMÉ
PURPOSE: Carboxypeptidase-D (CPD) cleaves C-terminal arginine (Arg) to produce nitric oxide (NO). Upregulation of CPD and NO by 17ß-estradiol, prolactin (PRL), and androgen increases survival of human breast cancer (BCa) cells in vitro. To demonstrate similar events in vivo, CPD, nitrotyrosine (NT, hallmark of NO action), androgen receptor (AR), prolactin receptor (PRLR), and phospho-Stat5a (for activated PRLR) levels were evaluated in benign and malignant human breast tissues, and correlated with cell proliferation (Ki67) and BCa progression (Cullin-3) biomarkers. METHODS: Paraffin-embedded breast tissues were analyzed by immunohistochemistry (IHC). BCa progression markers in human MCF-7 and T47D BCa cell lines treated with NO donor SIN-1 or PRL, ±CPD inhibitors were analyzed by RT-qPCR and immunoblotting. RESULTS: IHC showed progressive increases in CPD, NT, Ki67, and Cullin-3 from low levels in benign tissues to high levels in ductal carcinoma in situ, low-grade, high-grade, and triple-negative BCa. CPD and NT staining were closely associated, implicating CPD in NO production. Phospho-Stat5a increased significantly from benign to high-grade BCa and was mostly nuclear. AR and PRLR were abundant in benign breast and BCa, including triple-negative tumors. SIN-1 and PRL increased VEGF-C and Runx2, but not Cullin-3, in BCa cell lines. PRL induction of VEGF-C and Runx2 was inhibited partly by CPD inhibitors, implicating NO, produced by PRL-regulated CPD, in BCa progression. CONCLUSIONS: The CPD-Arg-NO pathway contributes to BCa progression in vitro and in vivo. PRL/androgen activation of the pathway support combined AR and PRLR blockade as an additional therapy for BCa.
Sujet(s)
Tumeurs du sein/traitement médicamenteux , Carboxypeptidases/génétique , Sous-unité alpha 1 du facteur CBF/génétique , Facteur de croissance endothéliale vasculaire de type C/génétique , Androgènes/métabolisme , Tumeurs du sein/génétique , Tumeurs du sein/métabolisme , Tumeurs du sein/anatomopathologie , Carboxypeptidases/antagonistes et inhibiteurs , Cullines/génétique , Oestradiol/administration et posologie , Femelle , Régulation de l'expression des gènes tumoraux/effets des médicaments et des substances chimiques , Humains , Antigène KI-67/génétique , Cellules MCF-7 , Monoxyde d'azote/métabolisme , Prolactine/métabolisme , Récepteurs aux androgènes/génétique , Récepteur prolactine/génétique , Tyrosine/analogues et dérivés , Tyrosine/métabolismeRÉSUMÉ
BACKGROUND: Carboxypeptidase-D (CPD) cleaves C-terminal arginine for conversion to nitric oxide (NO) by nitric oxide synthase (NOS). Prolactin (PRL) and androgens stimulate CPD gene transcription and expression, which increases intracellular production of NO to promote viability of prostate cancer (PCa) cells in vitro. The current study evaluated whether hormonal upregulation of CPD and NO promote PCa cell viabilty in vivo, by correlating changes in expression of CPD and nitrotyrosine residues (products of NO action) with proliferation marker Ki67 and associated proteins during PCa development and progression. METHODS: Fresh prostate tissues, obtained from 40 men with benign prostatic hyperplasia (BPH) or PCa, were flash-frozen at the time of surgery and used for RT-qPCR analysis of CPD, androgen receptor (AR), PRL receptor (PRLR), eNOS, and Ki67 levels. Archival paraffin-embedded tissues from 113 men with BPH or PCa were used for immunohistochemical (IHC) analysis of CPD, nitrotyrosines, phospho-Stat5 (for activated PRLR), AR, eNOS/iNOS, and Ki67. RESULTS: RT-qPCR and IHC analyses showed strong AR and PRLR expression in benign and malignant prostates. CPD mRNA levels increased â¼threefold in PCa compared to BPH, which corresponded to a twofold increase in Ki67 mRNA levels. IHC analysis showed a progressive increase in CPD from 11.4 ± 2.1% in benign to 21.8 ± 3.2% in low-grade (P = 0.007), 40.7 ± 4.0% in high-grade (P < 0.0001) and 50.0 ± 9.5% in castration-recurrent PCa (P < 0.0001). Immunostaining for nitrotyrosines and Ki67 mirrored these increases during PCa progression. CPD, nitrotyrosines, and Ki67 tended to co-localize, as did phospho-Stat5. CONCLUSIONS: CPD, nitrotyrosine, and Ki67 levels were higher in PCa than in benign and tended to co-localize, along with phospho-Stat5. The strong correlation in expression of these proteins in benign and malignant prostate tissues, combined with abundant AR and PRLR, supports in vitro evidence that the CPD-Arg-NO pathway is involved in the regulation of PCa cell proliferation. It further highlights a role for PRL in the development and progression of PCa.
Sujet(s)
Carboxypeptidases/métabolisme , Antigène KI-67/métabolisme , Prolactine/pharmacologie , Prostate/effets des médicaments et des substances chimiques , Hyperplasie de la prostate/métabolisme , Tumeurs de la prostate/métabolisme , Testostérone/pharmacologie , Tyrosine/analogues et dérivés , Humains , Mâle , Grading des tumeurs , Nitric oxide synthase type III/métabolisme , Phosphorylation/effets des médicaments et des substances chimiques , Prostate/métabolisme , Prostate/anatomopathologie , Hyperplasie de la prostate/anatomopathologie , Tumeurs de la prostate/anatomopathologie , Récepteurs aux androgènes/métabolisme , Récepteur prolactine/métabolisme , Facteur de transcription STAT-5/métabolisme , Transduction du signal/effets des médicaments et des substances chimiques , Tyrosine/métabolisme , Régulation positive/effets des médicaments et des substances chimiquesRÉSUMÉ
BACKGROUND: Carboxypeptidase-D (CPD) cleaves C-terminal arginine for nitric oxide (NO) production. CPD and NO levels are upregulated by testosterone (T) and prolactin (PRL) to promote survival of prostate cancer (pCa) cells. This study evaluated CPD immunostaining and T/PRL regulation of CPD and NO levels in benign and malignant prostate tissues/cells to determine the role of CPD in pCa. METHODS: Immunohistochemistry (IHC) and tissue microarrays (TMA) were used to determine CPD immunostaining in prostate specimens. QPCR and immunoblotting were used to quantify CPD mRNA/protein expression in prostate cells. NO production was measured using 4,5-diaminofluorescein diacetate assay. RESULTS: CPD staining increased from 8.9 ± 3.8% (Mean ± SEM, n = 15) of benign epithelial cell area to 30.9 ± 2.9% (n = 30) of tumor cell area in one set of TMAs (P = 0.0008) and from 5.9 ± 0.9% (n = 45) of benign epithelial cell area to 18.8 ± 1.9% (n = 55) of tumor area in another (P < 0.0001). IHC of prostate tissues (≥50 mm(2)) confirmed increased CPD staining, from 13.1 ± 2.9% in benign (n = 16) to 29.5 ± 4.4% in pCa (n = 31, P = 0.0095). T and/or PRL increased CPD expression in several pCa but not benign cell lines. T and PRL acted synergistically to increase NO production, which was abolished only when receptor antagonists flutamide and Δ1-9-G129R-hPRL were used together. CONCLUSIONS: CPD immunostaining and T/PRL-stimulated CPD expression were higher in pCa than benign tissues/cells. Elevated CPD increased NO production, which was abolished when both AR and PRLR were inhibited. Our study implicates a critical role for the T/PRL-stimulated CPD-Arg-NO pathway in pCa progression, and suggests that AR+PRLR inhibition is a more effective treatment for pCa.
Sujet(s)
Apoptose/physiologie , Carboxypeptidases/métabolisme , Prostate/métabolisme , Tumeurs de la prostate/métabolisme , Récepteurs aux androgènes/métabolisme , Récepteur prolactine/métabolisme , Antagonistes des androgènes/pharmacologie , Androgènes/pharmacologie , Apoptose/effets des médicaments et des substances chimiques , Carboxypeptidases/génétique , Lignée cellulaire tumorale , Flutamide/pharmacologie , Humains , Mâle , Monoxyde d'azote/biosynthèse , Prolactine/pharmacologie , Prostate/effets des médicaments et des substances chimiques , Prostate/anatomopathologie , Tumeurs de la prostate/génétique , Tumeurs de la prostate/anatomopathologie , Récepteurs aux androgènes/génétique , Récepteur prolactine/génétique , Transduction du signal/effets des médicaments et des substances chimiques , Testostérone/pharmacologie , Régulation positive/effets des médicaments et des substances chimiquesRÉSUMÉ
Plasma membrane-bound carboxypeptidase-D (CPD) cleaves C-terminal arginine from extracellular substrates. In the cell, arginine is converted to nitric oxide (NO). We have reported that up-regulation of CPD mRNA/protein levels by 17ß-estradiol and prolactin (PRL) in breast cancer cells, and by testosterone in prostate cancer cells, increased NO production and cell survival. The CPD promoter contains a consensus γ-interferon-activated sequence (GAS) and 3 putative androgen response elements (ARE.1, ARE.2, ARE.3) that could potentially bind PRL-activated transcription factor Stat5 (signal transducer and activator of transcription 5) and the liganded androgen receptor (AR), respectively. This study showed that synthetic androgen R1881 and PRL elevated CPD mRNA/protein levels in human MCF-7 and T47D breast cancer cells in a time-/dose-dependent manner. PRL/R1881-elevated CPD expression was blocked by actinomycin-D, and a CPD promoter construct containing these GAS and AREs was stimulated by PRL or R1881, indicating transcriptional regulation by both hormones. Luciferase reporter assays showed that GAS and the adjacent ARE.1 only were active. Mutation of GAS in the ΔGAS-CPD construct (ARE.1 intact) abolished CPD promoter activity in response to PRL and, surprisingly, to R1881 as well. ΔGAS-CPD promoter activity was restored by PRL+R1881 in combination, and enhanced by ectopic Stat5, but abolished by Stat5 gene knockdown. Chromatin immunoprecipitation analysis confirmed binding of activated Stat5 and liganded AR to GAS and ARE.1, respectively. Activated Stat5 also induced binding of unliganded AR to ARE.1, and liganded AR induced binding of unactivated Stat5 to GAS. In summary, PRL and R1881, acting through Stat5 and AR, act cooperatively to stimulate CPD gene transcription in breast cancer cells.
Sujet(s)
Androgènes/pharmacologie , Métribolone/pharmacologie , Prolactine/physiologie , Protéines/génétique , Facteur de transcription STAT-5/physiologie , Androgènes/physiologie , Séquence nucléotidique , Tumeurs du sein , Séquence consensus , Cycloheximide/pharmacologie , Dactinomycine/pharmacologie , Activation enzymatique , Induction enzymatique , Femelle , Régulation de l'expression des gènes tumoraux , Humains , Interféron gamma/physiologie , Cellules MCF-7 , Prolactine/pharmacologie , Régions promotrices (génétique) , Inhibiteurs de la synthèse protéique/pharmacologie , Protéines/métabolisme , Récepteurs aux androgènes/métabolismeRÉSUMÉ
Mammalian α4 phosphoprotein binds to the protein phosphatase 2A catalytic subunit (PP2Ac) to regulate PP2A activity, and to poly(A)-binding protein (PABP) and progestin-inducible EDD E3 ubiquitin ligase. This study showed induction of the EDD protein by progesterone, 17ß-estradiol and prolactin in breast cancer cells. Co-immunoprecipitation analyses, using lysates of COS-1 cells transfected with α4-deletion constructs, showed the α4 N-terminus binding to endogenous PP2Ac and PABP, and the C-terminus to EDD. Monoubiquitinated α4 in MCF-7 cells was unaffected by EDD-targeting siRNA (siEDD) nor by non-targetting siNT, thus, EDD does not ubiquitinate α4. PP2Ac is polyubiquitinated, and 36-kDa PP2Ac only was detected in siEDD- or siNT-transfected cells. However, treatment with proteasomal inhibitor MG132 showed polyubiquitinated-PP2Ac molecules (â¼65-250kDa) abundantly in siNT controls but low in siEDD-transfectants, implicating PP2Ac as an EDD substrate. Finally, progesterone induction of EDD in MCF-7 cells correlated with decreased PP2Ac levels, further implicating hormone-inducible EDD in PP2Ac turnover.
Sujet(s)
Protéines et peptides de signalisation intracellulaire/métabolisme , Progestines/pharmacologie , Protein Phosphatase 2/métabolisme , Protéolyse/effets des médicaments et des substances chimiques , Ubiquitin-protein ligases/biosynthèse , Ubiquitination/effets des médicaments et des substances chimiques , Protéines adaptatrices de la transduction du signal , Animaux , Tumeurs du sein/métabolisme , Tumeurs du sein/anatomopathologie , Cellules COS , Chlorocebus aethiops , Induction enzymatique/effets des médicaments et des substances chimiques , Femelle , Humains , Protéines et peptides de signalisation intracellulaire/composition chimique , Cellules MCF-7 , Chaperons moléculaires , Protéines mutantes/métabolisme , Protéines de liaison au poly(A)/métabolisme , Polyubiquitine/métabolisme , Liaison aux protéines/effets des médicaments et des substances chimiques , Spécificité du substrat/effets des médicaments et des substances chimiquesRÉSUMÉ
BACKGROUND: Plasma-membrane carboxypeptidase-D (CPD) releases arginine from extracellular substrates. Arginine is converted intracellularly to nitric oxide (NO). This study determined the effects of testosterone (T) and prolactin (PRL) on CPD expression, and the role(s) of CPD in NO production and survival of prostate cancer (PCa) cells. METHODS: LNCaP cells were treated with T and/or PRL. CPD expression was measured. Regulation by T (low doses) was determined using transfected cells overexpressing 5α-reductase type-1 (5αR1), which converts T to the more potent dihydrotestosterone. The effects of siRNAs targeting CPD (siCPDs) on NO production, cell viability, and apoptosis were determined using DAF2-DA, MTS, and Annexin-V assays. The effects of PRL/T on CPD/NO levels in PC-3, MDA-PCa-2b, and 22Rv1 cells were also evaluated. RESULTS: In LNCaP cells, 10 nM T and 10 ng/ml PRL-upregulated CPD mRNA/protein levels. In pTRE-transfectants, 1 nM T-upregulated CPD mRNA levels by â¼2-fold over controls, whereas 0.1 nM T caused similar upregulation in pTRE-5αR1-transfectants. In LNCaP cells cultured in arginine-free medium, addition of furylacryloyl-Ala-Arg (FAR; CPD substrate) increased NO levels. NO production, with FAR, was enhanced by PRL and/or T. siCPDs decreased NO production and cell viability, but increased apoptosis. QPCR analysis showed T/PRL-upregulation of CPD in 22Rv1, MDA-PCa-2b, and PC-3 cells. NO production was doubled by T/PRL in 22Rv1 cells, tripled by T in MDA-PCa-2b cells, and marginally increased by PRL in MDA-PCa-2b and PC-3 cells. CONCLUSIONS: T and PRL upregulate CPD and NO levels in PCa cells. CPD increases NO production to promote PCa cell survival.
Sujet(s)
Adénocarcinome/anatomopathologie , Carboxypeptidases/métabolisme , Monoxyde d'azote/métabolisme , Prolactine/pharmacologie , Tumeurs de la prostate/anatomopathologie , Testostérone/pharmacologie , Adénocarcinome/métabolisme , Apoptose/effets des médicaments et des substances chimiques , Arginine/métabolisme , Lignée cellulaire tumorale , Survie cellulaire/effets des médicaments et des substances chimiques , Humains , Mâle , Tumeurs de la prostate/métabolisme , Petit ARN interférent/pharmacologie , Transduction du signal/effets des médicaments et des substances chimiques , Régulation positive/effets des médicaments et des substances chimiquesRÉSUMÉ
Growth of the human MCF-7 breast cancer cell line is highly dependent on L-arginine. We have reported that L-arginine, released from extracellular substrates by prolactin (PRL)- and 17ß-estradiol (E2)-induced carboxypeptidase-D in the cell membrane, promotes nitric oxide (NO) production for MCF-7 cell survival. Arginine uptake is mediated by members of the cationic amino acid transporter (CAT) family and may coincide with induction of nitric oxide synthase (NOS) for the production of NO. The present study investigated the CAT isoforms and PRL/E2 regulation of CAT and NOS in breast cancer cell lines. Using RT-PCR analysis, CAT-1, CAT-2A, and CAT-2B transcripts were detected in MCF-7, T47D, and MDA-MB-231 cells. The CAT-4 transcript was detected in MDA-MB-231 only. CAT-3 was not detected in any of these cells. PRL and E2 did not significantly alter levels of CAT-1 mRNA and protein, nor CAT-2A and CAT-2B mRNAs in MCF-7 and T47D cells. PRL and E2 also had no effect on the overall uptake of L-[2,3,4,5-H(3)] arginine into these cells. However, confocal immunofluorescent microscopy showed that PRL and E2 upregulated eNOS and iNOS proteins, which distributed in the cytoplasm and/or nucleus of MCF-7 cells. Knockdown of CAT-1 gene expression using small interfering RNA significantly decreased L-[2,3,4,5-H(3)]-arginine uptake, decreased viability and increased apoptosis of MCF-7 and T47D cells. In summary, several CAT isoforms are expressed in breast cancer cells. The CAT-1 isoform plays a role in arginine uptake and, together with PRL/E2-induced NOS, contribute to NO production for the survival of MCF-7 and T47D cells.
Sujet(s)
Arginine/pharmacocinétique , Transporteur-1 d'acides aminés cationiques/métabolisme , Nitric oxide synthase/métabolisme , Apoptose , Arginine/métabolisme , Transport biologique/effets des médicaments et des substances chimiques , Technique de Western , Tumeurs du sein/génétique , Tumeurs du sein/métabolisme , Tumeurs du sein/anatomopathologie , Transporteur-1 d'acides aminés cationiques/génétique , Transporteur-2 d'acides aminés cationiques/génétique , Transporteur-2 d'acides aminés cationiques/métabolisme , Lignée cellulaire tumorale , Noyau de la cellule/effets des médicaments et des substances chimiques , Noyau de la cellule/enzymologie , Survie cellulaire , Cytoplasme/effets des médicaments et des substances chimiques , Cytoplasme/enzymologie , Oestradiol/pharmacologie , Régulation de l'expression des gènes tumoraux/effets des médicaments et des substances chimiques , Humains , Microscopie confocale/méthodes , Nitric oxide synthase type II/métabolisme , Nitric oxide synthase type III/métabolisme , Prolactine/pharmacologie , Interférence par ARN , RT-PCRRÉSUMÉ
INTRODUCTION: Conversion of testosterone to dihydrotestosterone (DHT) by the enzymes 5 alpha-reductase types 1 (5 alpha R1) and 2 (5 alpha R2) is important for normal and pathological growth of the prostate. The predominant isoenzyme in normal prostate is 5 alpha R2. However, prostate cancer (PCa) development is accompanied by a decrease in 5 alpha R2 and an increase in 5 alpha R1. The biological significance of increased 5 alpha R1 expression is not fully understood. Therefore, the aim of this study was to determine the effect of overexpression of 5 alpha R1 on growth and prostate-specific antigen (PSA) production in PCa cells. MATERIALS AND METHODS: LNGK-9 PCa cells, transiently transfected with pTRE-5 alpha R1 or pTRE alone, were cultured in the presence or absence of testosterone at varying concentrations. Cell growth and PSA secretion were measured after 4-6 days. Cyclin E1, Ki67, and PSA mRNA levels were evaluated using RT-PCR after 24 hr of treatment. RESULTS: 10 pM testosterone increased growth of pTRE-5 alpha R1 transfectants by 54.1% over cells grown in the absence of testosterone, compared to 25.0% in pTRE transfectants (P < 0.01). Likewise, PSA secretion was increased by 56-fold in pTRE-5 alpha R1 transfectants treated with 10 pM testosterone, compared to 26-fold in pTRE transfectants (P < 0.01). At concentrations of testosterone above 10 pM, the stimulatory effect on growth and PSA secretion was not distinguishable between pTRE-5 alpha R1 and pTRE transfectants. CONCLUSIONS: These results demonstrate that upregulation of 5 alpha R1 enhances the cellular response to low, but not high, concentrations of testosterone. This explains one mechanism by which castration-recurrent PCa can proliferate in the presence of castrate levels of circulating testosterone.
Sujet(s)
3-Oxo-5-alpha-Steroid 4-Dehydrogenase/génétique , Régulation de l'expression des gènes codant pour des enzymes , Régulation de l'expression des gènes tumoraux , Tumeurs de la prostate/génétique , Testostérone/pharmacologie , Lignée cellulaire tumorale , Amorces ADN , ADN complémentaire/génétique , ADN complémentaire/isolement et purification , ADN tumoral/génétique , ADN tumoral/isolement et purification , Humains , Isoenzymes/génétique , Mâle , Antigène spécifique de la prostate/génétique , Tumeurs de la prostate/enzymologie , Tumeurs de la prostate/anatomopathologie , ARN messager/génétique , ARN tumoral/génétique , RT-PCR , Transfection , Régulation positiveRÉSUMÉ
OBJECTIVES: Both normal and pathological growth of the prostate is dependent on dihydrotestosterone (DHT) synthesis, which is catalysed by two 5alpha-reductase (5alphaR) isoenzymes, 5alphaR1 and 5alphaR2, of which only 5alphaR2 has traditionally been viewed as important in the prostate. The objective of this study was to evaluate the role of both isoenzymes during development/progression of prostate cancer. METHODS: A thorough literature search was performed with the MEDLINE database to identify studies that have assessed expression of 5alphaR1/2 in prostate tissue. RESULTS: DHT suppression data for the 5alphaR2-specific inhibitor, finasteride, and the dual 5alphaR1/2 inhibitor, dutasteride, show that both isoenzymes are active in benign prostate. Furthermore, immunostaining studies have shown that 5alphaR1 expression increases and 5alphaR2 expression decreases in prostatic intraepithelial neoplasia (PIN) and prostate cancer, compared with nonmalignant prostate tissue. Both isoenzymes appear increased in high-grade compared with low-grade localised cancer. Dual inhibition of both isoenzymes with dutasteride may, therefore, be effective in preventing or delaying the growth of prostate cancer. The 4-yr REduction by DUtasteride of prostate Cancer Events (REDUCE) trial is underway to test this hypothesis. Androgen-withdrawal therapy can reverse prostate tumour growth by reducing circulating testosterone. However, 5alphaR-catalysed DHT synthesis within the prostate can continue and most tumours eventually develop resistance to androgen-deprivation therapy. Full assessment of the role of a 5alphaR inhibitor in this scenario is warranted. CONCLUSIONS: The consensus of evidence to date shows that 5alphaR1 is present in the prostate, and that levels are higher in malignant compared with benign prostate hyperplasia tissue.
Sujet(s)
3-Oxo-5-alpha-Steroid 4-Dehydrogenase/métabolisme , Tumeurs de la prostate/enzymologie , Azastéroïde/usage thérapeutique , Essais cliniques comme sujet , Évolution de la maladie , Dutastéride , Antienzymes/usage thérapeutique , Finastéride/usage thérapeutique , Humains , Isoenzymes/métabolisme , Mâle , Tumeurs de la prostate/traitement médicamenteux , Tumeurs de la prostate/anatomopathologieRÉSUMÉ
PURPOSE: In the prostate testosterone is converted to dihydrotestosterone by 5alpha-reductase type 1 and/or 2. Although 5alpha-reductase type 2 is predominant in normal prostates, type 1 is increased in cancer vs benign tissue. It is unclear whether 5alpha-reductase type 1/2 levels correlate with cancer grade. We compared the relative expression of 5alpha-reductase type 1 and 2 in localized high and low grade prostate cancer. MATERIALS AND METHODS: Immunostaining for 5alpha-reductase type 1/2 was evaluated in 64 prostate tissues from untreated men with localized prostate cancer. The percent of tumor area with moderate-high intensity staining was estimated for each Gleason pattern in the tissues. Adjacent benign tissue was evaluated in 26 prostate cancer specimens. RESULTS: Moderate-high staining for 5alpha-reductase type 1 increased from 18.8% +/- 2.9% (mean +/- SEM) in 34 Gleason pattern 3 cancers to 31.0% +/- 4.1% in 30 Gleason pattern 4/5 cancers (p = 0.016). Staining for 5alpha-reductase type 2 increased from 22.9% +/- 3.0% in 34 Gleason pattern 3 cancers to 39.2% +/- 4.1% in 30 Gleason pattern 4/5 cancers (p = 0.002). Compared to benign prostatic hyperplasia tissues staining for 5alpha-reductase type 1 was greater than 3-fold higher and staining for 5alpha-reductase type 2 was significantly lower in benign tissue adjacent to cancer (p = 0.006 and 0.0236, respectively). CONCLUSIONS: Levels of 5alpha-reductase type 1 and 2 are increased in localized high vs low grade prostate cancer. Levels of 5alpha-reductase type 1 are higher in benign tissue adjacent to cancer than in benign prostatic hyperplasia. These results raise the possibility that increased 5alpha-reductase type 1 in localized high grade cancers may contribute to the decreased effectiveness of the 5alpha-reductase type 2 selective inhibitor finasteride against high grade prostate cancer in the Prostate Cancer Prevention Trial.
Sujet(s)
3-Oxo-5-alpha-Steroid 4-Dehydrogenase/analyse , Tumeurs de la prostate/composition chimique , Tumeurs de la prostate/anatomopathologie , Sujet âgé , Humains , Mâle , Adulte d'âge moyenRÉSUMÉ
BACKGROUND: In the prostate, conversion of testosterone to dihydrotestosterone (DHT), by the enzymes 5alpha-reductase types 1 and 2 (5alphaR1, 5alphaR2) is required for normal growth and probably also for development of prostate cancer (PCa). Finasteride, a 5alphaR2 inhibitor, was shown to reduce the prevalence of PCa in the Prostate Cancer Prevention Trial. However, inhibition of both 5alphaR isoenzymes causes a greater decrease in serum DHT. The aim of this study was to assess differential expression of these enzymes at various stages of PCa development. METHODS: Immunostaining for 5alphaR1 and 5alphaR2, using specific, well-validated antibodies, was evaluated in 26 benign prostatic hyperplasia (BPH) (16 for 5alphaR2), 53 primary PCa (21 for 5alphaR2), 18 prostatic intraepithelial neoplasia (PIN), 12 primary PCa treated with neoadjuvant androgen ablation, 15 locally recurrent PCa specimens, and 18 PCa metastases. RESULTS: The mean area of moderate plus high intensity staining for 5alphaR1 increased from 4.8 +/- 2.8% of total epithelial area in BPH, to 18.9 +/- 5.7% in PIN, 17.0 +/- 3.2% in primary cancer, 38.0 +/- 7.3% in recurrent cancer, and 55.8 +/- 8.5% in PCa metastases. The mean staining area for 5alphaR2 decreased from 58.8 +/- 7.2% in BPH, to 21.1 +/- 5.5% in PIN and 34.8 +/- 6.7% in primary PCa. Staining for 5alphaR2 was increased in recurrent cancer and PCa metastases compared to primary PCa, at 58.7 +/- 5.2% and 69.2 +/- 8.7%, respectively. CONCLUSIONS: 5alphaR1 immunostaining is increased and 5alphaR2 immunostaining is decreased during development of PCa. In addition, there is increased expression of both 5alphaR isozymes in recurrent and metastatic cancers, suggesting that both isozymes may be important in the development and progression of PCa.