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The chemokine receptor, CXCR4 signaling regulates cell growth, invasion, and metastasis to the bone-marrow niche in prostate cancer (PCa). Previously, we established that CXCR4 interacts with phosphatidylinositol 4-kinase IIIα (PI4KIIIα encoded by PI4KA) through its adaptor proteins and PI4KA overexpressed in the PCa metastasis. To further characterize how the CXCR4-PI4KIIIα axis promotes PCa metastasis, here we identify CXCR4 binds to PI4KIIIα adaptor proteins TTC7 and this interaction induce plasma membrane PI4P production in prostate cancer cells. Inhibiting PI4KIIIα or TTC7 reduces plasma membrane PI4P production, cellular invasion, and bone tumor growth. Using metastatic biopsy sequencing, we found PI4KA expression in tumors correlated with overall survival and contributes to immunosuppressive bone tumor microenvironment through preferentially enriching non-activated and immunosuppressive macrophage populations. Altogether we have characterized the chemokine signaling axis through CXCR4-PI4KIIIα interaction contributing to the growth of prostate cancer bone metastasis.
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Neoplasias Óseas , Neoplasias de la Próstata , Humanos , Masculino , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias Óseas/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Quimiocina CXCL12/metabolismo , Neoplasias de la Próstata/patología , Receptores CXCR4/metabolismo , Transducción de Señal , Microambiente TumoralRESUMEN
Prostate cancer commonly metastasizes to bone due to its favorable microenvironment for cell growth and survival. Currently, the standard of care for metastatic prostate cancer is medical castration in conjunction with chemotherapeutic agents and newer anti-androgen/androgen receptor therapies. While these therapies aim to improve the quality of life in patients with advanced disease, resistance to these therapies is inevitable prompting the development of newer therapies to contain disease progression. The CXCL12/CXCR4 axis has previously been shown to be involved in prostate cancer cell homing to bone tissue, and new investigations found a novel interaction of Phosphatidyl Inositol 4 kinase IIIa (PI4KA) downstream of chemokine signaling. PI4KA phosphorylates at the 4th position on phosphatidylinositol (PI), to produce PI4P and is localized to the plasma membrane (PM). At the PM, PI4KA provides precursors for the generation of PI(4,5)P2, and PI(3,4,5)P3 and helps maintain PM identity through the recruitment of lipids and signaling proteins. PI4KA is recruited to the PM through evolutionarily conserved adaptor proteins, and in PC cells, CXCR4 binds with adaptor proteins to recruit PI4KA to the PM. The objective of this review is to summarize our understanding of the role that phosphatidyl inositol lipid messengers in cancer cells.
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Prostate cancer is a highly heterogeneous disease, understanding the crosstalk between complex genomic and epigenomic alterations will aid in developing targeted therapeutics. We demonstrate that, even though snail family transcriptional repressor 2 (SNAI2) is frequently amplified in prostate cancer, it is epigenetically silenced in this disease, with dynamic changes in SNAI2 levels showing distinct clinical relevance. Integrative clinical data from 18 prostate cancer cohorts and experimental evidence showed that gene fusion between transmembrane serine protease 2 (TMPRSS2) and ETS transcription factor ERG (ERG) (TMPRSS2-ERG fusion) is involved in the silencing of SNAI2. We created a silencer score to evaluate epigenetic repression of SNAI2, which can be reversed by treatment with DNA methyltransferase inhibitors and histone deacetylase inhibitors. Silencing of SNAI2 facilitated tumor cell proliferation and luminal differentiation. Furthermore, SNAI2 has a major influence on the tumor microenvironment by reactivating tumor stroma and creating an immunosuppressive microenvironment in prostate cancer. Importantly, SNAI2 expression levels in part determine sensitivity to the cancer drugs dasatinib and panobinostat. For the first time, we defined the distinct clinical relevance of SNAI2 expression at different disease stages. We elucidated how epigenetic silencing of SNAI2 controls the dynamic changes of SNAI2 expression that are essential for tumor initiation and progression and discovered that restoring SNAI2 expression by treatment with panobinostat enhances dasatinib sensitivity, indicating a new therapeutic strategy for prostate cancer.
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Proteínas de Fusión Oncogénica , Neoplasias de la Próstata , Factores de Transcripción de la Familia Snail , Línea Celular Tumoral , Dasatinib/uso terapéutico , Humanos , Masculino , Proteínas de Fusión Oncogénica/genética , Panobinostat/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Factores de Transcripción de la Familia Snail/genética , Microambiente TumoralAsunto(s)
Antineoplásicos Hormonales/farmacología , Biomarcadores , Resistencia a Antineoplásicos , Metabolismo de los Lípidos , Neoplasias de la Próstata/metabolismo , Antineoplásicos Hormonales/uso terapéutico , Humanos , Lipidómica/métodos , Masculino , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/etiología , Receptores Androgénicos/metabolismoRESUMEN
Pro-inflammatory cytokine and chemokines genes drive prostate cancer progression and metastasis: molecular mechanism update and the science that underlies racial disparity. comprehensive review article. Isaac J. Powell, S. Chinni, S.S. Reddy, Alexander Zaslavsky, Navnath Gavande Introduction: In 2013 we reported that with the use of bioinformatics and ingenuity pathway network analysis we were able to identify functional driver genes that were differentially expressed among a large population of African American men (AAM) and European American men (EAM). Pro-inflammatory cytokine genes were found to be more interactive and more expressed among AAM and have been found to be functional drivers of aggressive prostate cancer (CaP) and aggressiveness in other solid tumors. We examined these genes and biological pathways initiated by these cytokines in primary CaP tissue. Method We unravel the gene network and identified biologic pathways that impacted activation of the androgen receptor, mesenchymal epithelial transition (invasion) and chemokines associated with metastasis in the CaP tissue from 639 radical prostatectomy specimens. Results Biologic pathways identified by unraveling pro-inflammatory genes from our network, more expressed among AAM compared to EAM, were tumor necrosis factor (TNF), IL1b, IL6, and IL8. IL6 and IL8 are downstream of TNF activity and are known activators of androgen receptor and through mediators promote CaP cell proliferation. TNF and IL1b mediate tumor cell invasiveness through the activation of MMP (matrix metalloproteinase) which down regulates E-Cadherin to initiate epithelial mesenchymal transition which allows cells to become invasive in the microenvironment. Ultimately our network analysis indicates that TNF and IL1b activate CXCR4 receptor on CaP cells, which facilitates metastatic progression reportedly by binding to CXCL12 on lipid rafts and tumor implantation in the bone marrow. Conclusion Our retrospective biologic mechanistic model reveals a set of pro-inflammatory cytokines and chemokines that drive CaP aggressiveness, tumor heterogeneity, progression and metastasis. A prospective multi-institutional study needs to be conducted for clinical validation as well consideration of targeted therapy.
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Negro o Afroamericano/genética , Quimiocinas/fisiología , Citocinas/fisiología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Población Blanca/genética , Proliferación Celular , Humanos , Masculino , Metástasis de la Neoplasia , Procesos NeoplásicosRESUMEN
BACKGROUND: Castrate Resistant Prostate Cancer (CRPC) is an advanced disease resistant to systemic traditional medical or surgical castration, and resistance is primarily attributed to reactivation of AR through multiple mechanisms. TMPRSS2-ERG fusions have been shown to regulate AR signaling, interfere with pro-differentiation functions, and mediate oncogenic signaling. We have recently shown that ERG regulates intra-tumoral androgen synthesis and thereby facilitates AR function in prostate cancer cells. We hypothesize that enzalutamide treatment will be more effective in cells/tumors with TMPRSS2-ERG translocations because these tumors have increased AR signaling. METHODS: ERG knockdown was performed with VCaP cells using lentiviral infections to generate VCaP ERGshRNA cells and control VCaP scr cells with scrambled shRNA. Cell-growth analysis was performed to determine the effect of enzalutamide. Reverse transcription, quantitative real-time PCR (RT-qPCR) was used to determine the expression of AR responsive genes. Luciferase tagged VCaP scr and shRNA infected cells were used in an intra-tibial animal model for bone tumor growth analysis and enzalutamide treatment used to inhibit AR signaling in bone tumors. Western blotting analyzed VCaP bone tumor samples for ERG, AR, AKR1C3 and HSD3B1 and HSD3B2 expression. RESULTS: Enzalutamide inhibited the growth of VCaP scr cells more effectively than shERG cells. Analysis of AR responsive genes shows that Enzalutamide treatment at 5 micromolar concentration inhibited by 85-90% in VCaP Scr cells whereas these genes were inhibited to a lesser extent in VCaP shERG cells. Enzalutamide treatment resulted in severe growth inhibition in VCaP scr shRNA cells compared to VCaP shERG cells. In bone tumor growth experiment, VCaP ERG shRNA cells grew at slower than VCaP scr shRNA cells. Androgen biosynthetic enzyme expression is lower VCaP shERG bone tumors compared to VCaP scr shRNA bone tumors and enzalutamide inhibited the enzyme expression in both types of tumors. CONCLUSIONS: These data suggest that ERG transcription factor regulates androgen biosynthetic enzyme expression that enzalutamide treatment is more effective against VCaP bone tumors with an intact ERG expression, and that knocking down ERG in VCaP cells leads to a lesser response to enzalutamide therapy. Thus, ERG expression status in tumors could help stratify patients for enzalutamide therapy.
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Antagonistas de Receptores Androgénicos , Neoplasias Óseas , Proteínas de Fusión Oncogénica , Feniltiohidantoína , Serina Endopeptidasas , Animales , Humanos , Masculino , Ratones , Antagonistas de Receptores Androgénicos/uso terapéutico , Benzamidas , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/secundario , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Resistencia a Antineoplásicos/genética , Técnicas de Silenciamiento del Gen , Ratones SCID , Nitrilos , Proteínas de Fusión Oncogénica/genética , Feniltiohidantoína/análogos & derivados , Feniltiohidantoína/uso terapéutico , Neoplasias de la Próstata Resistentes a la Castración/patología , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Transducción de Señal/efectos de los fármacos , Regulador Transcripcional ERG/genética , Regulador Transcripcional ERG/metabolismo , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chemokine signaling regulates cell migration and tumor metastasis. CXCL12, a member of the chemokine family, and its receptor, CXCR4, a G protein coupled receptor (GPCR), are key mediators of prostate-cancer (PC) bone metastasis. In PC cells androgens activate CXCR4 gene expression and receptor signaling on lipid rafts, which induces protease expression and cancer cell invasion. To identify novel lipid-raft-associated CXCR4 regulators supporting invasion/metastasis, we performed a SILAC-based quantitative proteomic analysis of lipid-rafts derived from PC3 stable cell lines with overexpression or knockdown of CXCR4. This analysis identified the evolutionarily conserved phosphatidylinositol 4-kinase IIIα (PI4KIIIα), and SAC1 phosphatase that dephosphorylates phosphatidylinositol-4-phosphate as potential candidate CXCR4 regulators. CXCR4 interacted with PI4KIIIα membrane targeting machinery recruiting them to the plasma membrane for PI4P production. Consistent with this interaction, PI4KIIIα was found tightly linked to the CXCR4 induced PC cell invasion. Thus, ablation of PI4KIIIα in CXCR4-expressing PC3 cells reduced cellular invasion in response to a variety of chemokines. Immunofluorescence microscopy in CXCR4-expressing cells revealed localized production of PI4P on the invasive projections. Human tumor studies documented increased PI4KIIIα expression in metastatic tumors vs. the primary tumor counterparts, further supporting the PI4KIIIα role in tumor metastasis. Furthermore, we also identified an unexpected function of PI4KIIIα in GPCR signaling where CXCR4 regulates PI4KIIIα activity and mediate tumor metastasis. Altogether, our study identifies a novel cross-talk between PI4KIIIα and CXCR4 in promoting tumor metastasis and suggests that PI4KIIIα pharmacological targeting may have therapeutic benefit for advanced prostate cancer patients.
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1-Fosfatidilinositol 4-Quinasa/fisiología , Proteínas de la Membrana/fisiología , Invasividad Neoplásica/patología , Metástasis de la Neoplasia/fisiopatología , Proteínas de Neoplasias/fisiología , Neoplasias de la Próstata/metabolismo , Receptores CXCR4/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , División Celular , Línea Celular Tumoral , Quimiocinas/farmacología , Humanos , Masculino , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Neoplasias de la Próstata/patología , Mapeo de Interacción de Proteínas , Transporte de Proteínas , Proteínas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Receptores CXCR4/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
BACKGROUND: The CXCL12/CXCR4 axis transactivates HER2 and promotes intraosseous tumor growth. To further explore the transactivation of HER2 by CXCL12, we investigated the role of small GTP protein Gαi2 in Src and HER2 phosphorylation in lipid raft membrane microdomains and the significance of CXCR4 in prostate cancer bone tumor growth. METHODS: We used a variety of methods such as lipid raft isolation, invasion assays, an in vivo model of intratibial tumor growth, bone histomorphometry, and immunohistochemistry to determine the role of CXCR4 signaling in lipid raft membrane microdomains and effects of targeting of CXCR4 for bone tumor growth. RESULTS: We determined that (a) CXCL12/CXCR4 transactivation of EGFR and HER2 is confined to lipid raft membrane microdomains, (b) CXCL12 activation of HER2 and Src is mediated by small GTP proteins in lipid rafts, (c) inhibition of the CXCL12/CXCR4 axis through plerixafor abrogates the initial establishment of tumor growth without affecting the growth of established bone tumors, and (d) inhibition of EGFR signaling through gefitinib leads to inhibition of established bone tumor growth. CONCLUSIONS: These data suggest that lipid raft membrane microdomains are key sites for CXCL12/CXCR4 transactivation of HER2 via small GTP binding protein Gαi2 and Src kinase. The initial establishment of prostate cancer is supported by the endosteal niche, and blocking the CXCL12/CXCR4 axis of this niche along with its downstream signaling severely compromises initial establishment of tumors in the bone microenvironment, whereas expanding bone tumors are sensitive only to the members of growth factor receptor inhibition.
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Neoplasias Óseas/metabolismo , Neoplasias Óseas/secundario , Quimiocina CXCL12/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Receptores CXCR4/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Bencilaminas , Neoplasias Óseas/tratamiento farmacológico , Línea Celular Tumoral , Ciclamas , Modelos Animales de Enfermedad , Receptores ErbB/agonistas , Receptores ErbB/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Compuestos Heterocíclicos/farmacología , Humanos , Masculino , Microdominios de Membrana/metabolismo , Ratones , Fosforilación , Neoplasias de la Próstata/tratamiento farmacológico , Receptor ErbB-2/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Familia-src Quinasas/metabolismoRESUMEN
PURPOSE: Intratumoral androgen synthesis in prostate cancer contributes to the development of castration-resistant prostate cancer (CRPC). Several enzymes responsible for androgen biosynthesis have been shown to be overexpressed in CRPC, thus contributing to CRPC in a castrated environment. The TMPRSS2-ERG transcription factor has been shown to be present in primary prostate cancer tumors as well as CRPC tumors. We hypothesize that TMPRSS2-ERG fusions regulate androgen biosynthetic enzyme (ABE) gene expression and the production of androgens, which contributes to the development of CRPC. EXPERIMENTAL DESIGN: We used a panel of assays, including lentivirus transduction, gene expression, chromatin immunoprecipitation and sequencing, liquid chromatography-mass spectrometric quantitation, immunocytochemistry, immunohistochemistry, and bioinformatics analysis of gene microarray databases, to determine ERG regulation of androgen synthesis. RESULTS: We found that ERG regulated the expression of the ABE AKR1C3 in prostate cancer cells via direct binding to the AKR1C3 gene. Knockdown of ERG resulted in reduced AKR1C3 expression, which caused a reduction in both DHT synthesis and PSA expression in VCaP prostate cancer cells treated with 5α-androstanedione (5α-Adione), a DHT precursor metabolite. Immunohistochemical staining revealed that ERG was coexpressed with AKR1C3 in prostate cancer tissue samples. CONCLUSIONS: These data suggest that AKR1C3 catalyzes the biochemical reduction of 5α-Adione to DHT in prostate cancer cells, and that ERG regulates this step through upregulation of AKR1C3 expression. Elucidation of ERG regulation of ABEs in CRPC may help to stratify TMPRSS2-ERG fusion-positive prostate cancer patients in the clinic for anti-androgen receptor-driven therapies; and AKR1C3 may serve as a valuable therapeutic target in the treatment of CRPC.
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3-Hidroxiesteroide Deshidrogenasas/biosíntesis , Hidroxiprostaglandina Deshidrogenasas/biosíntesis , Neoplasias de la Próstata Resistentes a la Castración/genética , Serina Endopeptidasas/genética , Transactivadores/genética , 3-Hidroxiesteroide Deshidrogenasas/genética , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas , Antagonistas de Andrógenos/administración & dosificación , Andrógenos/biosíntesis , Andrógenos/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Hidroxiprostaglandina Deshidrogenasas/genética , Masculino , Proteínas de Fusión Oncogénica/genética , Próstata/efectos de los fármacos , Próstata/patología , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Receptores Androgénicos/genética , Transducción de Señal/efectos de los fármacos , Transactivadores/biosíntesis , Regulador Transcripcional ERGRESUMEN
Isothiocyanates (ITCs) derived from cruciferous vegetables, including phenethyl isothiocyanate (PEITC) and sulforaphane (SFN), exhibit in vivo activity against prostate cancer in a xenograft and transgenic mouse model, and thus are appealing for chemoprevention of this disease. Watercress constituent PEITC and SFN-rich broccoli sprout extract are under clinical investigations but the molecular mechanisms underlying their cancer chemopreventive effects are not fully understood. The present study demonstrates that chemokine receptor CXCR4 is a novel target of ITCs in prostate cancer cells. Exposure of prostate cancer cells (LNCaP, 22Rv1, C4-2, and PC-3) to pharmacologically applicable concentrations of PEITC, benzyl isothiocyanate (BITC), and SFN (2.5 and 5 µmol/L) resulted in downregulation of CXCR4 expression. None of the ITCs affected secretion of CXCR4 ligand (stromal-derived factor-1). In vivo inhibition of PC-3 xenograft growth upon PEITC treatment was associated with a significant decrease in CXCR4 protein level. A similar trend was discernible in the tumors from SFN-treated TRAMP mice compared with those of control mice, but the difference was not significant. Stable overexpression of CXCR4 in PC-3 cells conferred significant protection against wound healing, cell migration, and cell viability inhibition by ITCs. Inhibition of cell migration resulting from PEITC and BITC exposure was significantly augmented by RNAi of CXCR4. This study demonstrates, for the first time, that cancer chemopreventive ITCs suppress CXCR4 expression in prostate cancer cells in vitro as well as in vivo. These results suggest that CXCR4 downregulation may be an important pharmacodynamic biomarker of cancer chemopreventative ITCs in prostate adenocarcinoma.
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Adenocarcinoma/prevención & control , Anticarcinógenos/uso terapéutico , Isotiocianatos/uso terapéutico , Terapia Molecular Dirigida , Neoplasias de la Próstata/prevención & control , Receptores CXCR4/antagonistas & inhibidores , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Línea Celular Tumoral , Quimioprevención/métodos , Humanos , Isotiocianatos/farmacología , Masculino , Ratones , Ratones Transgénicos , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , ARN Interferente Pequeño/farmacología , ARN Interferente Pequeño/uso terapéutico , Receptores CXCR4/genética , Sulfóxidos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Genomic rearrangement and overexpression of the ERG oncogene (also known as v-ets avian erythroblastosis virus E26 oncogene homolog) is estimated to occur at a rate of 40-50% in prostate cancer. Early evidence suggests that ERG overexpression may be associated with disease progression, and the utilization of ERG levels as a biomarker for prostate cancer is being strongly considered. However, the evidence is incomplete because it relies on studies that primarily focused on men of European ancestry, giving little consideration to African-American men even though African-American men bear a greater disease burden in the form of significantly greater incidence and worse outcomes. In this perspective article we bring to light the issue that the potential use of ERG expression as a biomarker is yet to be solidly established and may have limited utility or varied applicably for African-American men as compared with European-American men.
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BACKGROUND: CXCL12/CXCR4 transactivation of epidermal growth factor family receptors in lipid raft membrane microdomains on cell surface is thought to mediate tumor growth and subsequent development of metastatic disease. CTCE-9908 is a known inhibitor of CXCR4. Herein, we tested the efficacy of CTCE-9908 in inhibiting prostate cancer cell growth, invasion, and metastasis. METHODS: We used a panel of in vitro assays utilizing human prostate cancer cell lines and an in vivo orthotopic prostate cancer model to assess the anti-tumoral activity of CTCE-9908. RESULTS: We demonstrated that (a) CTCE-9908 treatment resulted in no significant change in the growth of PC-3 and C4-2B cells; (b) 50 µg/ml of CTCE-9908 inhibited the invasive properties of PC-3 cells; (c) 25 mg/kg of CTCE-9908 did not alter primary tumor growth but it did significantly reduce total tumor burden in the animal including the growth of prostate and soft tissue metastases to lymph node and distant organ tissues. Histological analysis showed that CTCE-9908 treatment resulted in tumor necrosis in primary prostate tumors and no significant change in proliferation of tumor cells as measured by Ki-67 staining; (d) CTCE-9908 inhibited the tumor angiogenesis as measured by CD34 positive vessels in tumors. CONCLUSIONS: These data suggest that CXCR4 inhibition by CTCE-9908 decreases the invasion potential in vitro, which then translated to a reduction of tumor spread with associated reduction in angiogenesis. Hence, CTCE-9908 may prove to be an efficacious novel agent to prevent and treat the spread of metastatic prostate cancer.
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Terapia Molecular Dirigida/métodos , Péptidos/administración & dosificación , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/secundario , Receptores CXCR4/antagonistas & inhibidores , Antineoplásicos/administración & dosificación , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Masculino , Neoplasias de la Próstata/fisiopatología , Receptores CXCR4/metabolismo , Resultado del TratamientoRESUMEN
INTRODUCTION: The chemokine CXCL12, also known as SDF-1, and its receptor, CXCR4, are overexpressed in prostate cancers and in animal models of prostate-specific PTEN deletion, but their regulation is poorly understood. Loss of the tumor suppressor PTEN (phosphatase and tensin homolog) is frequently observed in cancer, resulting in the deregulation of cell survival, growth, and proliferation. We hypothesize that loss of PTEN and subsequent activation of Akt, frequent occurrences in prostate cancer, regulate the CXCL12/CXCR4 signaling axis in tumor growth and bone metastasis. METHODS: Murine prostate epithelial cells from PTEN+/+, PTEN+/-, and PTEN-/- (prostate specific knockdown) mice as well as human prostate cancer cell lines C4-2B, PC3, and DU145 were used in gene expression and invasion studies with Akt inhibition. Additionally, HA-tagged Akt1 was overexpressed in DU145, and tumor growth in subcutaneous and intra-tibia bone metastasis models were analyzed. RESULTS: Loss of PTEN resulted in increased expression of CXCR4 and CXCL12 and Akt inhibition reversed expression and cellular invasion. These results suggest that loss of PTEN may play a key role in the regulation of this chemokine activity in prostate cancer. Overexpression of Akt1 in DU145 resulted in increased CXCR4 expression, as well as increased proliferation and cell cycle progression. Subcutaneous injection of these cells also resulted in increased tumor growth as compared to neo controls. Akt1 overexpression reversed the osteosclerotic phenotype associated with DU145 cells to an osteolytic phenotype and enhanced intra-osseous tumor growth. CONCLUSIONS: These results suggest the basis for activation of CXCL12 signaling through CXCR4 in prostate cancer driven by the loss of PTEN and subsequent activation of Akt. Akt1-associated CXCL12/CXCR4 signaling promotes tumor growth, suggesting that Akt inhibitors may potentially be employed as anticancer agents to target expansion of PC bone metastases.
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Quimiocina CXCL12/metabolismo , Fosfohidrolasa PTEN/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores CXCR4/metabolismo , Animales , Línea Celular Tumoral , Quimiocina CXCL12/genética , Humanos , Masculino , Ratones , Ratones Noqueados , Fosfohidrolasa PTEN/metabolismo , Neoplasias de la Próstata/metabolismo , Receptores CXCR4/genética , Transducción de SeñalRESUMEN
UNLABELLED: CXCR4 is a chemokine receptor that mediates invasion and metastasis. CXCR4 expression is transcriptionally regulated in cancer cells and is associated with aggressive prostate cancer phenotypes. Previously, we and others have shown that the transcription factor ERG regulates CXCR4 expression in prostate cancer cells and that androgens modulate CXCR4 expression via increasing ERG expression. Herein, the molecular mechanisms of ERG-mediated CXCR4 promoter activation, phosphorylation of ERG by intracellular kinases and subsequent CXCR4 expression, as well as the status of ERG and CXCR4 in human prostate cancer specimens were investigated. Using multiple molecular strategies, it was demonstrated that (i) ERG expressed in TMPRSS2-ERG fusion positive VCaP cells selectively binds to specific ERG/Ets bindings sites in the CXCR4 promoter; (ii) distal binding sites mediate promoter activation; (iii) exogenously expressed ERG promotes CXCR4 expression; (iv) ERG is phosphorylated at Serine-81 and -215, by both IKK and Akt kinases, and Akt mediates CXCR4 expression; (v) ERG-induced CXCR4 drives CXCL12-dependent adhesion to fibronectin; and (vi) ERG and CXCR4 were coexpressed in human prostate cancer tissue, consistent with ERG-mediated transcriptional activation of CXCR4. These data demonstrate that ERG activates CXCR4 expression by binding to specific ERG/Ets responsive elements and via intracellular kinases that phosphorylate ERG at discrete serine residues. IMPLICATIONS: These findings provide a mechanistic link between TMPRSS2-ERG translocations and intracellular kinase-mediated phosphorylation of ERG on enhanced metastasis of tumor cells via CXCR4 expression and function in prostate cancer cells.
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Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Neoplasias de la Próstata/genética , Receptores CXCR4/metabolismo , Transactivadores/metabolismo , Sitios de Unión/genética , Adhesión Celular/fisiología , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Masculino , Invasividad Neoplásica , Fosforilación , Regiones Promotoras Genéticas , Neoplasias de la Próstata/patología , Receptores CXCR4/genética , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Transducción de Señal/genética , Transactivadores/genética , Regulador Transcripcional ERG , Microambiente TumoralRESUMEN
TMPRSS2-Ets gene fusions were identified in prostate cancers where the promoter of transmembrane protease, serine 2 (TMPRSS2) fused with coding sequence of the erythroblastosis virus E26 (Ets) gene family members. TMPRSS2 is an androgen responsive transmembrane serine protease. Ets family members are oncogenic transcription factors that contain a highly conserved Ets DNA binding domain and an N-terminal regulatory domain.Fusion of these gene results in androgen dependent transcription of Ets factor in prostate tumor cells. The ERG is the most common fusion partner with TMPRSS2 promoter in prostate cancer patients. The high prevalence of these gene fusions, in particular TMPRSS2-ERG, makes them attractive as potential diagnostic and prognostic indicators, as well as making them a potential target for tailored therapies.This review focuses on the clinical and biological significance of TMPRSS2-ERG fusions and their role in PC development and progression.
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TMPRSS2-ERG fusion transcripts have been shown to be expressed in a majority of prostate cancer (PC) patients because of chromosomal translocations or deletions involving the TMPRSS2 gene promoter and the ERG gene coding sequence. These alterations cause androgen-dependent ERG transcription factor expression in PC patients. We and others have shown that chemokine receptor CXCR4 expression is upregulated in PC tumor cells, and its ligand, CXCL12, is expressed in bone stromal cells. The CXCL12/CXCR4 axis functions in PC progression to enhance invasion and metastasis. To address the regulation of CXCR4 expression, we identified several putative ERG consensus-binding sites in the promoter region of CXCR4. We hypothesized that androgen-dependent regulation of the ERG transcription factor could induce CXCR4 expression in PC cells. Results of the current study show that 1) prostate tumor cells coexpress higher ERG and CXCR4 compared with benign tissue, 2) CXCR4 expression is increased in the TMPRSS2-ERG fusion-positive cell line, 3) ERG transcription factor binds to the CXCR4 gene promoter, 4) synthetic androgen (R1881) upregulates both ERG and CXCR4 in TMPRSS2-ERG fusion-positive VCaP cells, 5) small interfering RNA-mediated down-regulation of ERG resulted in the loss of androgen-dependent regulation of CXCR4 expression in VCaP cells, and 6) R1881-activated TMPRSS2-ERG expression functionally activates CXCR4 in VCaP cells. These findings provide a link between TMPRSS2-ERG translocations and enhanced metastasis of tumor cells through CXCR4 function in PC cells.
RESUMEN
Hormone-refractory prostate cancer, its skeletal metastasis and complications remain a therapeutic challenge. Here we show that treatment with (S)-3-((R)-9-bromo-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-6,7-dimethoxyiso-benzofuran-1(3H)-one (EM011), the brominated analogue of a plant-derived non-toxic antitussive alkaloid, noscapine, achieved significant inhibition of hormone-refractory human prostate cancer implanted intratibially in the bone as shown by non-invasive, real-time bioluminescent imaging of tumour growth in nude mice. Mechanistically, in vitro data suggested that the antiproliferative and proapoptotic effects of EM011 in human prostate cancer cell lines were through blockade of cell-cycle progression by impairing the formation of a bipolar spindle apparatus. The G2/M arrest was accompanied by activation of the mitotic checkpoint, a pre-requisite for induction of optimal apoptosis. Attenuation of mitotic checkpoint by siRNA duplexes led to a reduction in mitotic arrest and subsequent apoptosis. Our results further demonstrated participation of an intrinsic mitochondrially mediated apoptotic pathway that ultimately triggered caspase-driven EM011-induced apoptosis. EM011 did not exert any detectable toxicity in normal tissues with frequently dividing cells such as the gut and bone marrow. Thus, these data warrant further evaluation of EM011 for the management of prostate cancer.
Asunto(s)
Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Dioxoles/farmacología , Isoquinolinas/farmacología , Mitocondrias/enzimología , Neoplasias de la Próstata/tratamiento farmacológico , Moduladores de Tubulina/farmacología , Animales , Apoptosis/fisiología , Línea Celular Tumoral , Fase G2/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Mitosis/efectos de los fármacos , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Chemokines and their receptors function in migration and homing of cells to target tissues. Recent evidence suggests that cancer cells use a chemokine receptor axis for metastasis formation at secondary sites. Previously, we showed that binding of the chemokine CXCL12 to its receptor CXCR4 mediated signaling events resulting in matrix metalloproteinase-9 expression in prostate cancer bone metastasis. A variety of methods, including lipid raft isolation, stable overexpression of CXCR4, cellular adhesion, invasion assays, and the severe combined immunodeficient-human bone tumor growth model were used. We found that (a) CXCR4 and HER2 coexist in lipid rafts of prostate cancer cells; (b) the CXCL12/CXCR4 axis results in transactivation of the HER2 receptor in lipid rafts of prostate cancer cells; (c) Src kinase mediates CXCL12/CXCR4 transactivation of HER2 in prostate cancer cells; (d) a pan-HER inhibitor desensitizes CXCR4-induced transactivation and subsequent matrix metalloproteinase-9 secretion and invasion; (e) lipid raft-disrupting agents inhibited raft-associated CXCL12/CXCR4 transactivation of the HER2 and cellular invasion; (f) overexpression of CXCR4 in prostate cancer cells leads to increased HER2 phosphorylation and migratory properties of prostate cancer cells; and (g) CXCR4 overexpression enhances bone tumor growth and osteolysis. These data suggest that lipid rafts on the cell membrane are the key site for CXCL12/CXCR4-induced HER2 receptor transactivation. This transactivation contributes to enhanced invasive signals and metastatic growth in the bone microenvironment.
Asunto(s)
Neoplasias Óseas/secundario , Quimiocina CXCL12/fisiología , Microdominios de Membrana/fisiología , Metástasis de la Neoplasia/patología , Neoplasias de la Próstata/patología , Receptor ErbB-2/metabolismo , Receptores CXCR4/fisiología , Biotinilación , Neoplasias Óseas/patología , División Celular , Línea Celular Tumoral , Membrana Celular/fisiología , Quimiocina CXCL12/genética , Humanos , Masculino , Invasividad Neoplásica , Receptor Cross-Talk , Receptores CXCR4/genética , Activación Transcripcional , TransfecciónRESUMEN
Membrane type 1-matrix metalloproteinase (MT1-MMP) is a major mediator of collagen I degradation. In human samples, we show that prostate cancer cells in skeletal metastases consistently express abundant MT1-MMP protein. Because prostate cancer bone metastasis requires remodeling of the collagen-rich bone matrix, we investigated the role of cancer cell-derived MT1-MMP in an experimental model of tumor-bone interaction. MT1-MMP-deficient LNCaP human prostate cancer cells were stably transfected with human wild-type MT1-MMP (MT1wt). Furthermore, endogenous MT1-MMP was down-regulated by small interfering RNA in DU145 human prostate cancer cells. Intratibial tumor injection in severe combined immunodeficient mice was used to simulate intraosseous growth of metastatic tumors. LNCaP-MT1wt cells produced larger osseous tumors than Neo control cells and induced osteolysis, whereas DU145 MT1-MMP-silenced transfectants induced osteogenic changes. In vitro assays showed that MT1wt overexpression enhanced collagen I degradation, whereas MT1-MMP-silencing did the opposite, suggesting that tumor-derived MT1-MMP may contribute directly to bone remodeling. LNCaP-MT1wt-derived conditioned medium stimulated in vitro multinucleated osteoclast formation. This effect was inhibited by osteoprotegerin, a decoy receptor for receptor activator of nuclear factor kappaB ligand, and by 4-[4-(methanesulfonamido) phenoxy] phenylsulfonyl methylthiirane, an MT1-MMP inhibitor. Our findings are consistent with the hypothesis that prostate cancer-associated MT1-MMP plays a direct and/or indirect role in bone matrix degradation, thus favoring intraosseous tumor expansion.
Asunto(s)
Neoplasias Óseas , Metaloproteinasa 14 de la Matriz/metabolismo , Neoplasias de la Próstata , Animales , Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Línea Celular Tumoral , Colágeno Tipo I/metabolismo , Humanos , Masculino , Metaloproteinasa 14 de la Matriz/genética , Ratones , Ratones SCID , Metástasis de la Neoplasia , Osteólisis , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Tibia/citología , Tibia/metabolismo , Tibia/patologíaRESUMEN
BACKGROUND: Experimental bone metastases are typically analyzed when the skeletal tumor burden is large enough to be detected by imaging or histology. By this time, the bone microenvironment is usually destroyed, preventing useful analysis of tumor-bone interactions. METHODS: Small intraosseous tumors generated by intratibial injection of C4-2B prostate cancer cells transfected with green fluorescent protein (GFP) were assessed using in vivo and ex vivo fluorescence imaging, radiography, histology, and fluorometric analysis of bone lysates. RESULTS: Ex vivo fluorescence imaging and fluorometric analysis were capable of detecting tiny bone tumors as early as 10 days after injection. Ex vivo fluorescence imaging allowed simple quantification of small skeletal tumor burden and was useful in measuring the effect of systemic therapy. CONCLUSIONS: Ex vivo fluorescence imaging is a sensitive and easy method to quantify small skeletal tumor burden. This technique allows investigation of tumor-bone interactions while the bone microanatomy is still intact.