RESUMEN
This study aimed to elucidate the role of ELF3, an ETS family member in normal prostate growth and prostate cancer. Silencing ELF3 in both benign prostate (BPH-1) and prostate cancer (PC3) cell lines resulted in decreased colony-forming ability, inhibition of cell migration and reduced cell viability due to cell cycle arrest, establishing ELF3 as a cell cycle regulator. Increased ELF3 expression in more advanced prostate tumours was shown by immunostaining of tissue microarrays and from analysis of gene expression and genetic alteration studies. This study indicates that ELF3 functions not only as a part of normal prostate epithelial growth but also as a potential oncogene in advanced prostate cancers.
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Proteínas de Unión al ADN , Próstata , Neoplasias de la Próstata , Proteínas Proto-Oncogénicas c-ets , Factores de Transcripción , Ciclo Celular/genética , Movimiento Celular/genética , Proteínas de Unión al ADN/genética , Humanos , Masculino , Próstata/metabolismo , Neoplasias de la Próstata/genética , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Proto-Oncogénicas c-ets/metabolismo , Factores de Transcripción/genéticaRESUMEN
Low Temperature Plasma (LTP) generates reactive oxygen and nitrogen species, causing cell death, similarly to radiation. Radiation resistance results in tumour recurrence, however mechanisms of LTP resistance are unknown. LTP was applied to patient-derived prostate epithelial cells and gene expression assessed. A typical global oxidative response (AP-1 and Nrf2 signalling) was induced, whereas Notch signalling was activated exclusively in progenitor cells. Notch inhibition induced expression of prostatic acid phosphatase (PAP), a marker of prostate epithelial cell differentiation, whilst reducing colony forming ability and preventing tumour formation. Therefore, if LTP is to be progressed as a novel treatment for prostate cancer, combination treatments should be considered in the context of cellular heterogeneity and existence of cell type-specific resistance mechanisms.
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Gases em Plasma/uso terapéutico , Neoplasias de la Próstata/radioterapia , Tolerancia a Radiación/efectos de la radiación , Receptores Notch/genética , Fosfatasa Ácida/genética , Muerte Celular/efectos de la radiación , Diferenciación Celular/efectos de la radiación , Línea Celular Tumoral , Proliferación Celular/efectos de la radiación , Células Epiteliales/efectos de la radiación , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Humanos , Masculino , Factor 2 Relacionado con NF-E2/genética , Gases em Plasma/efectos adversos , Próstata/patología , Próstata/efectos de la radiación , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Tolerancia a Radiación/genética , Especies de Nitrógeno Reactivo/efectos de la radiación , Especies Reactivas de Oxígeno/efectos de la radiación , Transducción de Señal/efectos de la radiación , Células Madre/efectos de la radiación , Factor de Transcripción AP-1/genéticaRESUMEN
BACKGROUND: Human prostate cancers display numerous DNA methylation changes compared to normal tissue samples. However, definitive identification of features related to the cells' malignant status has been compromised by the predominance of cells with luminal features in prostate cancers. METHODS: We generated genome-wide DNA methylation profiles of cell subpopulations with basal or luminal features isolated from matched prostate cancer and normal tissue samples. RESULTS: Many frequent DNA methylation changes previously attributed to prostate cancers are here identified as differences between luminal and basal cells in both normal and cancer samples. We also identified changes unique to each of the two cancer subpopulations. Those specific to cancer luminal cells were associated with regulation of metabolic processes, cell proliferation and epithelial development. Within the prostate cancer TCGA dataset, these changes were able to distinguish not only cancers from normal samples, but also organ-confined cancers from those with extraprostatic extensions. Using changes present in both basal and luminal cancer cells, we derived a new 17-CpG prostate cancer signature with high predictive power in the TCGA dataset. CONCLUSIONS: This study demonstrates the importance of comparing phenotypically matched prostate cell populations from normal and cancer tissues to unmask biologically and clinically relevant DNA methylation changes.
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Metilación de ADN , Fenotipo , Neoplasias de la Próstata/genética , Islas de CpG , Humanos , MasculinoRESUMEN
The PI3K/AKT/mTOR pathway is frequently activated in advanced prostate cancer, due to loss of the tumour suppressor PTEN, and is an important axis for drug development. We have assessed the molecular and functional consequences of pathway blockade by inhibiting AKT and mTOR kinases either in combination or as individual drug treatments. In established prostate cancer cell lines, a decrease in cell viability and in phospho-biomarker expression was observed. Although apoptosis was not induced, a G1 growth arrest was observed in PTEN null LNCaP cells, but not in BPH1 or PC3 cells. In contrast, when the AKT inhibitor AZD7328 was applied to patient-derived prostate cultures that retained expression of PTEN, activation of a compensatory Ras/MEK/ERK pathway was observed. Moreover, whilst autophagy was induced following treatment with AZD7328, cell viability was less affected in the patient-derived cultures than in cell lines. Surprisingly, treatment with a combination of both AZD7328 and two separate MEK1/2 inhibitors further enhanced phosphorylation of ERK1/2 in primary prostate cultures. However, it also induced irreversible growth arrest and senescence. Ex vivo treatment of a patient-derived xenograft (PDX) of prostate cancer with a combination of AZD7328 and the mTOR inhibitor KU-0063794, significantly reduced tumour frequency upon re-engraftment of tumour cells. The results demonstrate that single agent targeting of the PI3K/AKT/mTOR pathway triggers activation of the Ras/MEK/ERK compensatory pathway in near-patient samples. Therefore, blockade of one pathway is insufficient to treat prostate cancer in man.
RESUMEN
Radiation therapy is a major primary treatment option for both localized early stage prostate cancer, and for advanced, regionally un-resectable, cancer. However, around 30% of patients still experience biochemical recurrence after radiation therapy within 10 years. Thus, identification of better biomarkers and new targets are urgently required to improve current therapeutic strategies. The miR-99 family has been shown to play an important role in the regulation of the DNA damage response, via targeting of the SWI/SNF chromatin remodeling factors, SMARCA5 and SMARCD1 in cell line models. In the present study, we have demonstrated that low expression of miR-99a and miR-100 is present in cell populations which are relatively radiation insensitive, for example in prostate cancer stem cells and in castration-resistant prostate cancer. Additionally, treatment of cells with the synthetic glucocorticoid, Dexamethasone resulted in decreased miR-99a and 100 expression, suggesting a new mechanism of miR-99a and 100 regulation in androgen-independent prostate cells. Strikingly, treatment of prostate cells with the glucocorticoid receptor inhibitor, Mifepristone was found to sensitize prostate cells to radiation by increasing the levels of miR-99a and miR-100. These results qualify the miR99 family as markers of radiation sensitivity and as potential therapeutic targets to improve efficiency of radiotherapy.
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MicroARNs/metabolismo , Neoplasias de la Próstata/genética , Tolerancia a Radiación/genética , Receptores de Glucocorticoides/antagonistas & inhibidores , Línea Celular Tumoral , Dexametasona/farmacología , Regulación Neoplásica de la Expresión Génica/genética , Glucocorticoides/farmacología , Antagonistas de Hormonas/farmacología , Humanos , Masculino , Mifepristona/farmacología , Células Madre Neoplásicas/patología , Neoplasias de la Próstata/patologíaRESUMEN
UNLABELLED: Benign prostatic hyperplasia (BPH) treatments have changed little over many years and do not directly address the underlying cause. Because BPH is characterised by uncontrolled cell growth, the chromosomal telomeres should be eroded in the reported absence or low levels of telomerase activity, but this is not observed. We investigated the telomere biology of cell subpopulations from BPH patients undergoing transurethral resection of prostate (TURP). Measurement of TERC, TERT, and telomerase activity revealed that only the epithelial stem-like and progenitor fractions expressed high levels of telomerase activity (p<0.01) and individual enzyme components (p<0.01). Telomerase activity and TERT expression were not detected in stromal cells. Telomere length measurements reflected this activity, although the average telomere length of (telomerase-negative) luminal cells was equivalent to that of telomerase-expressing stem/progenitor cells. Immunohistochemical analysis of patient-derived BPH arrays identified distinct areas of luminal hyperproliferation, basal hyperproliferation, and basal-luminal hyperproliferation, suggesting that basal and luminal cells can proliferate independently of each other. We propose a separate lineage for the luminal and basal cell components in BPH. PATIENT SUMMARY: We unexpectedly found an enzyme called telomerase in the cells that maintain benign prostatic hyperplasia (BPH), suggesting that telomerase inhibitors could be used to alleviate BPH symptoms.
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Linaje de la Célula , Proliferación Celular , Próstata/enzimología , Hiperplasia Prostática/enzimología , Células Madre/enzimología , Telomerasa/metabolismo , Homeostasis del Telómero , Biomarcadores/metabolismo , Humanos , Masculino , Fenotipo , Próstata/patología , Próstata/cirugía , Hiperplasia Prostática/genética , Hiperplasia Prostática/patología , Hiperplasia Prostática/cirugía , ARN/metabolismo , Células Madre/patología , Telomerasa/genética , Resección Transuretral de la PróstataRESUMEN
In comparison to more differentiated cells, prostate cancer stem-like cells are radioresistant, which could explain radio-recurrent prostate cancer. Improvement of radiotherapeutic efficacy may therefore require combination therapy. We have investigated the consequences of treating primary prostate epithelial cells with gamma irradiation and photodynamic therapy (PDT), both of which act through production of reactive oxygen species (ROS). Primary prostate epithelial cells were cultured from patient samples of benign prostatic hyperplasia and prostate cancer prior to treatment with PDT or gamma irradiation. Cell viability was measured using MTT and alamar blue assay, and cell recovery by colony-forming assays. Immunofluorescence of gamma-H2AX foci was used to quantify DNA damage, and autophagy and apoptosis were assessed using Western blots. Necrosis and senescence were measured by propidium iodide staining and beta-galactosidase staining, respectively. Both PDT and gamma irradiation reduced the colony-forming ability of primary prostate epithelial cells. PDT reduced the viability of all types of cells in the cultures, including stem-like cells and more differentiated cells. PDT induced necrosis and autophagy, whereas gamma irradiation induced senescence, but neither treatment induced apoptosis. PDT and gamma irradiation therefore inhibit cell growth by different mechanisms. We suggest these treatments would be suitable for use in combination as sequential treatments against prostate cancer.
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Apoptosis/efectos de la radiación , Autofagia/efectos de la radiación , Senescencia Celular/efectos de la radiación , Células Epiteliales/metabolismo , Células Epiteliales/efectos de la radiación , Rayos gamma , Próstata/citología , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Senescencia Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Rayos gamma/efectos adversos , Humanos , Masculino , Estrés Oxidativo/genética , Fotoquimioterapia , Fármacos Fotosensibilizantes/farmacología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/terapia , Especies Reactivas de Oxígeno/metabolismo , Células MadreRESUMEN
BACKGROUND: Objective identification of key miRNAs from transcriptomic data is difficult owing to the inherent inconsistencies within miRNA target-prediction algorithms and the promiscuous nature of miRNA-mRNA target relationship. METHODS: An integrated database of miRNAs and their 'relevant' mRNA targets was generated from validated miRNA and mRNA microarray data sets generated from patient-derived prostate epithelial normal and cancer stem-like cells (SCs) and committed basal (CB) cells. The effect of miR-542-5p inhibition was studied to provide proof-of-principle for database utility. RESULTS: Integration of miRNA-mRNA databases showed that signalling pathways and processes can be regulated by a single or relatively few miRNAs, for example, DNA repair/Notch pathway by miR-542-5p, P=0.008. Inhibition of miR-542-5p in CB cells (thereby achieving miR-542-5p expression levels similar to SCs) promoted efficient DNA repair and activated expression of Notch reporters, HES1 and Survivin, without inducing dedifferentiation into SCs. CONCLUSIONS: Our novel framework impartially identifies therapeutically relevant miRNA candidates from transcriptomic data sets.
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Células Epiteliales/metabolismo , Células Epiteliales/patología , MicroARNs/genética , Próstata/metabolismo , Próstata/patología , ARN Mensajero/genética , Reparación del ADN/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Masculino , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Receptores Notch/genética , Transducción de Señal/genéticaRESUMEN
UNLABELLED: MicroRNA (miRNA) expression profiles were generated from prostate epithelial subpopulations enriched from patient-derived benign prostatic hyperplasia (n=5), Gleason 7 treatment-naive prostate cancer (PCa) (n=5), and castration-resistant PCa (CRPC) (n=3). Microarray expression was validated in an independent patient cohort (n=10). Principal component analysis showed that miRNA expression is clustered by epithelial cell phenotype, regardless of pathologic status. We also discovered concordance between the miRNA expression profiles of unfractionated epithelial cells from CRPCs, human embryonic stem cells (SCs), and prostate epithelial SCs (both benign and malignant). MiR-548c-3p was chosen as a candidate miRNA from this group to explore its usefulness as a CRPC biomarker and/or therapeutic target. Overexpression of miR-548c-3p was confirmed in SCs (fivefold, p<0.05) and in unfractionated CRPCs (1.8-fold, p<0.05). Enforced overexpression of miR-548c-3p in differentiated cells induced stemlike properties (p<0.01) and radioresistance (p<0.01). Reanalyses of published studies further revealed that miR-548c-3p is significantly overexpressed in CRPC (p<0.05) and is associated with poor recurrence-free survival (p<0.05), suggesting that miR-548c-3p is a functional biomarker for PCa aggressiveness. Our results validate the prognostic and therapeutic relevance of miRNAs for PCa management while demonstrating that resolving cell-type and differentiation-specific differences is essential to obtain clinically relevant miRNA expression profiles. PATIENT SUMMARY: We report microRNA (miRNA) expression profiles of epithelial cell fractions from the human prostate, including stem cells. miR-548c-3p was revealed as a functional biomarker for prostate cancer progression. The evaluation of miR-548c-3p in a larger patient cohort should yield information on its clinical usefulness.
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Biomarcadores de Tumor/genética , Perfilación de la Expresión Génica , MicroARNs/genética , Células Madre Neoplásicas , Hiperplasia Prostática/genética , Neoplasias de la Próstata Resistentes a la Castración/genética , Supervivencia sin Enfermedad , Células Epiteliales , Humanos , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/patología , Tolerancia a Radiación/genética , Regulación hacia ArribaRESUMEN
Interleukin (IL)-6 overexpression and constitutive STAT3 activation occur in many cancers, including prostate cancer. However, their contribution to prostate stem and progenitor cells has not been explored. In this study, we show that stem-like cells from patients with prostate cancer secrete higher levels of IL-6 than their counterparts in non-neoplastic prostate. Tumor grade did not influence the levels of expression or secretion. Stem-like and progenitor cells expressed the IL-6 receptor gp80 with concomitant expression of pSTAT3. Blockade of activated STAT3, by either anti-IL-6 antibody siltuximab (CNTO 328) or LLL12, a specific pSTAT3 inhibitor, suppressed the clonogenicity of the stem-like cells in patients with high-grade disease. In a murine xenograft model used to determine the in vivo effects of pSTAT3 suppression, LLL12 treatment effectively abolished outgrowth of a patient-derived castrate-resistant tumor. Our results indicate that the most primitive cells in prostate cancer require pSTAT3 for survival, rationalizing STAT3 as a therapeutic target to treat advanced prostate cancer.
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Quinasas Janus/antagonistas & inhibidores , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/genética , Anciano , Anciano de 80 o más Años , Animales , Antraquinonas/farmacología , Anticuerpos Monoclonales/farmacología , Línea Celular Tumoral , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Quinasas Janus/genética , Quinasas Janus/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Células Madre Neoplásicas/efectos de los fármacos , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismo , Factor de Transcripción STAT3/metabolismo , Sulfonamidas/farmacologíaRESUMEN
The outcome for patients with advanced metastatic and recurrent prostate cancer is still poor. Therefore, new chemotherapeutics are required, especially for killing cancer stem cells that are thought to be responsible for disease recurrence. In this study, we screened the effect of a novel palladium-based anticancer agent (Pd complex) against six different prostate cancer cell lines, and primary cultures from seven Gleason 6/7 prostate cancer, three Gleason 8/9 prostate cancer and four benign prostate hyperplasia patient samples, as well as cancer stem cells selected from primary cultures. MTT and ATP viability assays were used to assess cell growth and flow cytometry to assess cell cycle status. In addition, immunofluorescence was used to detect γH2AX nuclear foci, indicative of DNA damage, and Western blotting to assess the induction of apoptosis and autophagy. The Pd complex showed a powerful growth-inhibitory effect against both cell lines and primary cultures. More importantly, it successfully reduced the viability of cancer stem cells as first reported in this study. The Pd complex induced DNA damage and differentially induced evidence of cell death, as well as autophagy. In conclusion, this novel agent may be promising for use against the bulk of the tumour cell population as well as the prostate cancer stem cells, which are thought to be responsible for the resistance of metastatic prostate cancer to chemotherapy. This study also indicates that the combined use of the Pd complex with an autophagy modulator may be a more promising approach to treat prostate cancer. In addition, the differential effects observed between cell lines and primary cells emphasise the importance of the model used to test novel drugs including its genetic background, and indeed the necessity of using cells cultured from patient samples.
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Células Epiteliales/efectos de los fármacos , Células Madre Neoplásicas/efectos de los fármacos , Paladio/toxicidad , Próstata/citología , Próstata/efectos de los fármacos , Neoplasias de la Próstata/patología , Antineoplásicos/química , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Masculino , Paladio/químicaRESUMEN
While chromosomal translocations have a fundamental role in the development of several human leukaemias, their role in solid tumour development has been somewhat more controversial. Recently, it was shown that up to 80% of prostate tumours harbour at least one such gene fusion, and that the most common fusion event, between the prostate-specific TMPRSS2 gene and the ERG oncogene, is a critical, and probably early factor in prostate cancer development. Here we demonstrate the presence and expression of this significant chromosomal rearrangement in prostate cancer stem cells. Moreover, we show that in the prostate epithelial hierarchy from both normal and tumour tissues, TMPRSS2 transcription is subjected to tight monoallelic regulation, which is retained upon asymmetric division and relaxed during epithelial cell differentiation. The presence and expression of TMPRSS2/ERG in prostate stem cells would provide ERG-driven survival advantages, allowing maintenance of this mutated genotype.