RESUMO
BACKGROUND: Aberrant activation of the androgen receptor (AR) is a major factor highly relevant to castration-resistant progression of prostate cancer (PCa). FOXO1, a key downstream effector of PTEN, inhibits androgen-independent activation of the AR. However, the underlying mechanism remains elusive. METHODS: The inhibitory effect of FOXO1 on full-length and constitutively active splice variants of the AR was examined by luciferase reporter assays and real-time reverse transcription polymerase chain reaction (RT-qPCR). In vitro protein binding assays and western blot analyses were used to determine the regions in FOXO1 and AR responsible for their interaction. RESULTS: We found that a putative transcription repression domain in the NH2-terminus of FOXO1 is dispensable for FOXO1 inhibition of the AR. In vitro protein binding assays showed that FOXO1 binds to the transcription activation unit 5 (TAU5) motif in the AR NH2-terminal domain (NTD), a region required for recruitment of p160 activators including SRC-1. Ectopic expression of SRC-1 augmented transcriptional activity of some, but not all AR splice variants examined. Forced expression of FOXO1 blocked the effect of SRC-1 on AR variants' transcriptional activity by decreasing the binding of SRC-1 to the AR NTD. Ectopic expression of FOXO1 inhibited expression of endogenous genes activated primarily by alternatively spliced AR variants in human castration-resistant PCa 22Rv1 cells. CONCLUSIONS: FOXO1 binds to the TAU5 motif in the AR NTD and inhibits ligand-independent activation of AR splice variants, suggesting the PTEN/FOXO1 pathway as a potential therapeutic target for inhibition of aberrant AR activation and castration-resistant PCa growth.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Transdução de Sinais/fisiologia , Processamento Alternativo , Linhagem Celular Tumoral , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Humanos , Masculino , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Neoplasias da Próstata/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Androgênicos/genética , Transcrição Gênica/fisiologia , Ativação TranscricionalRESUMO
PURPOSE: DNA-dependent protein kinase catalytic subunit (DNA-PK) is a pleiotropic kinase involved in DNA repair and transcriptional regulation. DNA-PK is deregulated in selected cancer types and is strongly associated with poor outcome. The underlying mechanisms by which DNA-PK promotes aggressive tumor phenotypes are not well understood. Here, unbiased molecular investigation in clinically relevant tumor models reveals novel functions of DNA-PK in cancer.Experimental Design: DNA-PK function was modulated using both genetic and pharmacologic methods in a series of in vitro models, in vivo xenografts, and patient-derived explants (PDE), and the impact on the downstream signaling and cellular cancer phenotypes was discerned. Data obtained were used to develop novel strategies for combinatorial targeting of DNA-PK and hormone signaling pathways. RESULTS: Key findings reveal that (i) DNA-PK regulates tumor cell proliferation; (ii) pharmacologic targeting of DNA-PK suppresses tumor growth both in vitro, in vivo, and ex vivo; (iii) DNA-PK transcriptionally regulates the known DNA-PK-mediated functions as well as novel cancer-related pathways that promote tumor growth; (iv) dual targeting of DNA-PK/TOR kinase (TORK) transcriptionally upregulates androgen signaling, which can be mitigated using the androgen receptor (AR) antagonist enzalutamide; (v) cotargeting AR and DNA-PK/TORK leads to the expansion of antitumor effects, uncovering the modulation of novel, highly relevant protumorigenic cancer pathways; and (viii) cotargeting DNA-PK/TORK and AR has cooperative growth inhibitory effects in vitro and in vivo. CONCLUSIONS: These findings uncovered novel DNA-PK transcriptional regulatory functions and led to the development of a combinatorial therapeutic strategy for patients with advanced prostate cancer, currently being tested in the clinical setting.
Assuntos
Proteína Quinase Ativada por DNA/metabolismo , Neoplasias/metabolismo , Antagonistas de Receptores de Andrógenos/farmacologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Proteína Quinase Ativada por DNA/genética , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Receptores Androgênicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Transcrição Gênica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Purpose: Loss of cell-cycle control is a hallmark of cancer, which can be targeted with agents, including cyclin-dependent kinase-4/6 (CDK4/6) kinase inhibitors that impinge upon the G1-S cell-cycle checkpoint via maintaining activity of the retinoblastoma tumor suppressor (RB). This class of drugs is under clinical investigation for various solid tumor types and has recently been FDA-approved for treatment of breast cancer. However, development of therapeutic resistance is not uncommon.Experimental Design: In this study, palbociclib (a CDK4/6 inhibitor) resistance was established in models of early stage, RB-positive cancer.Results: This study demonstrates that acquired palbociclib resistance renders cancer cells broadly resistant to CDK4/6 inhibitors. Acquired resistance was associated with aggressive in vitro and in vivo phenotypes, including proliferation, migration, and invasion. Integration of RNA sequencing analysis and phosphoproteomics profiling revealed rewiring of the kinome, with a strong enrichment for enhanced MAPK signaling across all resistance models, which resulted in aggressive in vitro and in vivo phenotypes and prometastatic signaling. However, CDK4/6 inhibitor-resistant models were sensitized to MEK inhibitors, revealing reliance on active MAPK signaling to promote tumor cell growth and invasion.Conclusions: In sum, these studies identify MAPK reliance in acquired CDK4/6 inhibitor resistance that promotes aggressive disease, while nominating MEK inhibition as putative novel therapeutic strategy to treat or prevent CDK4/6 inhibitor resistance in cancer. Clin Cancer Res; 24(17); 4201-14. ©2018 AACR.
Assuntos
Quinase 4 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/genética , Fosfatase 1 de Especificidade Dupla/genética , MAP Quinase Quinase Quinases/genética , Neoplasias/tratamento farmacológico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fosfatase 1 de Especificidade Dupla/antagonistas & inibidores , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/genética , MAP Quinase Quinase Quinases/antagonistas & inibidores , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Neoplasias/genética , Neoplasias/patologia , Fosforilação/efeitos dos fármacos , Piperazinas/farmacologia , Piridinas/farmacologia , Proteína do Retinoblastoma/genética , Análise de Sequência de RNA , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PARP-1 holds major functions on chromatin, DNA damage repair and transcriptional regulation, both of which are relevant in the context of cancer. Here, unbiased transcriptional profiling revealed the downstream transcriptional profile of PARP-1 enzymatic activity. Further investigation of the PARP-1-regulated transcriptome and secondary strategies for assessing PARP-1 activity in patient tissues revealed that PARP-1 activity was unexpectedly enriched as a function of disease progression and was associated with poor outcome independent of DNA double-strand breaks, suggesting that enhanced PARP-1 activity may promote aggressive phenotypes. Mechanistic investigation revealed that active PARP-1 served to enhance E2F1 transcription factor activity, and specifically promoted E2F1-mediated induction of DNA repair factors involved in homologous recombination (HR). Conversely, PARP-1 inhibition reduced HR factor availability and thus acted to induce or enhance "BRCA-ness". These observations bring new understanding of PARP-1 function in cancer and have significant ramifications on predicting PARP-1 inhibitor function in the clinical setting.
Assuntos
Reparo do DNA , Fator de Transcrição E2F1/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Neoplasias da Próstata/patologia , Animais , Linhagem Celular , Progressão da Doença , Perfilação da Expressão Gênica , Recombinação Homóloga , Humanos , Imuno-Histoquímica , Masculino , Camundongos Endogâmicos BALB C , Análise Serial de TecidosRESUMO
The androgen receptor (AR) is a driver of prostate cancer (PCa) cell growth and disease progression. Therapies for advanced PCa exploit AR dependence by blocking the production or action of androgens, but these interventions inevitably fail via multiple mechanisms including mutation or deletion of the AR ligand binding domain (LBD). Thus, the development of new inhibitors which act through non-LBD interfaces is an unmet clinical need. EPI-001 is a bisphenol A-derived compound shown to bind covalently and inhibit the AR NH2-terminal domain (NTD). Here, we demonstrate that EPI-001 has general thiol alkylating activity, resulting in multilevel inhibitory effects on AR in PCa cell lines and tissues. At least one secondary mechanism of action associated with AR inhibition was found to be selective modulation of peroxisome proliferator activated receptor-gamma (PPARγ). These multi-level effects of EPI-001 resulted in inhibition of transcriptional activation units (TAUs) 1 and 5 of the AR NTD, and reduced AR expression. EPI-001 inhibited growth of AR-positive and AR-negative PCa cell lines, with the highest sensitivity observed in LNCaP cells. Overall, this study provides new mechanistic insights to the chemical biology of EPI-001, and raises key issues regarding the use of covalent inhibitors of the intrinsically unstructured AR NTD.
Assuntos
Antagonistas de Receptores de Andrógenos/farmacologia , Compostos Benzidrílicos/farmacologia , Cloridrinas/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Receptores Androgênicos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptores Androgênicos/biossíntese , Receptores Androgênicos/genética , TransfecçãoRESUMO
The androgen receptor (AR) is a master regulator transcription factor in normal and cancerous prostate cells. Canonical AR activation requires binding of androgen ligand to the AR ligand binding domain, translocation to the nucleus, and transcriptional activation of AR target genes. This regulatory axis is targeted for systemic therapy of advanced prostate cancer. However, a new paradigm for AR activation in castration-resistant prostate cancer (CRPC) has emerged wherein alternative splicing of AR mRNA promotes synthesis of constitutively active AR variants that lack the AR ligand binding domain (LBD). Recent work has indicated that structural alteration of the AR gene locus represents a key mechanism by which alterations in AR mRNA splicing arise. In this review, we examine the role of truncated AR variants (ARVs) and their corresponding genomic origins in models of prostate cancer progression, as well as the challenges they pose to the current standard of prostate cancer therapies targeting the AR ligand binding domain. Since ARVs lack the COOH-terminal LBD, the genesis of these AR gene rearrangements and their resulting ARVs provides strong rationale for the pursuit of new avenues of therapeutic intervention targeted at the AR NH2-terminal domain. We further suggest that genomic events leading to ARV expression could act as novel biomarkers of disease progression that may guide the optimal use of current and next-generation AR-targeted therapy.
Assuntos
Rearranjo Gênico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Receptores Androgênicos/genética , Androgênios/genética , Androgênios/metabolismo , Progressão da Doença , Variação Estrutural do Genoma , Humanos , Masculino , Neoplasias da Próstata/metabolismo , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores Androgênicos/metabolismoRESUMO
Persistent androgen receptor (AR) transcriptional activity underlies resistance to AR-targeted therapy and progression to lethal castration-resistant prostate cancer (CRPC). Recent success in retargeting persistent AR activity with next generation androgen/AR axis inhibitors such as enzalutamide (MDV3100) has validated AR as a master regulator during all stages of disease progression. However, resistance to next generation AR inhibitors limits therapeutic efficacy for many patients. One emerging mechanism of CRPC progression is AR gene rearrangement, promoting synthesis of constitutively active truncated AR splice variants (AR-V) that lack the AR ligand-binding domain. In this study, we show that cells with AR gene rearrangements expressing both full-length and AR-Vs are androgen independent and enzalutamide resistant. However, selective knock-down of AR-V expression inhibited androgen-independent growth and restored responsiveness to androgens and antiandrogens. In heterogeneous cell populations, AR gene rearrangements marked individual AR-V-dependent cells that were resistant to enzalutamide. Gene expression profiling following knock-down of full-length AR or AR-Vs showed that AR-Vs drive resistance to AR-targeted therapy by functioning as constitutive and independent effectors of the androgen/AR transcriptional program. Further, mitotic genes deemed previously to be unique AR-V targets were found to be biphasic targets associated with a proliferative level of signaling output from either AR-Vs or androgen-stimulated AR. Overall, these studies highlight AR-Vs as key mediators of persistent AR signaling and resistance to the current arsenal of conventional and next generation AR-directed therapies, advancing the concept of AR-Vs as therapeutic targets in advanced disease.