RESUMO
Enzalutamide is a potent second-generation androgen receptor (AR) antagonist with activity in metastatic castrate-resistant prostate cancer (CRPC). Although enzalutamide is initially effective, disease progression inevitably ensues with the emergence of resistance. Intratumoral hypoxia is also associated with CRPC progression and treatment resistance. Given that both AR and hypoxia inducible factor-1 α (HIF-1α) are key regulators of these processes, dual targeting of both signaling axes represents an attractive therapeutic approach. Crosstalk of the AR and HIF-1α signaling pathways were examined in prostate cancer cell lines (LNCaP, 22Rv1) with assays measuring the effect of androgen and hypoxia on AR-dependent and hypoxia-inducible gene transcription, protein expression, cell proliferation, and apoptosis. HIF-1α inhibition was achieved by siRNA silencing HIF-1α or via chetomin, a disruptor of HIF-1α-p300 interactions. In prostate cancer cells, the gene expression of AR targets (KLK3, FKBP5, TMPRSS2) was repressed by HIF-signaling; conversely, specific HIF-1α target expression was induced by dihydrotestosterone-mediated AR signaling. Treatment of CRPC cells with enzalutamide or HIF-1α inhibition attenuated AR-regulated and HIF-1α-mediated gene transcription. The combination of enzalutamide and HIF-1α inhibition was more effective than either treatment alone. Similarly, the combination also reduced vascular endothelial growth factor protein levels. HIF-1α siRNA synergistically enhanced the inhibitory effect of enzalutamide on cell growth in LNCaP and enzalutamide-resistant 22Rv1 cells via increased enzalutamide-induced apoptosis. In conclusion, the combination of enzalutamide with HIF-1α inhibition resulted in synergistic inhibition of AR-dependent and gene-specific HIF-dependent expression and prostate cancer cell growth.
Assuntos
Antineoplásicos/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Feniltioidantoína/análogos & derivados , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Receptores Androgênicos/metabolismo , Benzamidas , Hipóxia Celular , Linhagem Celular Tumoral/efeitos dos fármacos , Cobalto/farmacologia , Di-Hidrotestosterona/farmacologia , Dissulfetos/farmacologia , Sinergismo Farmacológico , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Alcaloides Indólicos/farmacologia , Masculino , Nitrilas , Feniltioidantoína/farmacologia , Neoplasias de Próstata Resistentes à Castração/metabolismo , RNA Interferente Pequeno/genética , Receptores Androgênicos/genética , Transdução de Sinais , Transcrição GênicaRESUMO
The downstream targets of hypoxia inducible factor-1 alpha (HIF-1α) play an important role in tumor progression and angiogenesis. Therefore, inhibition of HIF-mediated transcription has potential in the treatment of cancer. One attractive strategy for inhibiting HIF activity is the disruption of the HIF-1α/p300 complex, as p300 is a crucial coactivator of hypoxia-inducible transcription. Several members of the epidithiodiketopiperazine (ETP) family of natural products have been shown to disrupt the HIF-1α/p300 complex in vitro; namely, gliotoxin, chaetocin, and chetomin. Here, we further characterized the molecular mechanisms underlying the antiangiogenic and antitumor effects of these ETPs using a preclinical model of prostate cancer. In the rat aortic ring angiogenesis assay, gliotoxin, chaetocin, and chetomin significantly inhibited microvessel outgrowth at a GI50 of 151, 8, and 20 nM, respectively. In vitro co-immunoprecipitation studies in prostate cancer cell extracts demonstrated that these compounds disrupted the HIF-1α/p300 complex. The downstream effects of inhibiting the HIF-1α/p300 interaction were evaluated by determining HIF-1α target gene expression at the mRNA and protein levels. Dose-dependent decreases in levels of secreted VEGF were detected by ELISA in the culture media of treated cells, and the subsequent downregulation of VEGFA, LDHA, and ENO1 HIF-1α target genes were confirmed by semi-quantitative real-time PCR. Finally, treatment with ETPs in mice bearing prostate tumor xenografts resulted in significant inhibition of tumor growth. These results suggest that directly targeting the HIF-1α/p300 complex with ETPs may be an effective approach for inhibiting angiogenesis and tumor growth.
Assuntos
Antineoplásicos/farmacologia , Proteína p300 Associada a E1A/genética , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dissulfetos/farmacologia , Proteína p300 Associada a E1A/antagonistas & inibidores , Proteína p300 Associada a E1A/metabolismo , Células Endoteliais/efeitos dos fármacos , Gliotoxina/farmacologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Alcaloides Indólicos/farmacologia , Isoenzimas/genética , Isoenzimas/metabolismo , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Lactato Desidrogenase 5 , Masculino , Transplante de Neoplasias , Neovascularização Patológica/prevenção & controle , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Piperazinas/farmacologia , Neoplasias da Próstata/irrigação sanguínea , Neoplasias da Próstata/patologia , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Transdução de Sinais , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Calcium/calmodulin-dependent protein kinase IV (CaMKIV) is a serine/threonine kinase that is important in synaptic plasticity and T cell maturation. Activation of CaMKIV requires calcium/calmodulin binding and phosphorylation at T200 by CaMK kinase. Our previous work has shown that protein serine/threonine phosphatase 2A (PP2A) forms a complex with CaMKIV and negatively regulates its activity. Here we demonstrate that PP2A tightly regulates T200 phosphorylation of endogenous CaMKIV, but has little effect on the phosphorylation of the ectopically-expressed kinase. This differential regulation of endogenous versus exogenous CaMKIV is due to differences in their ability to associate with PP2A, as exogenous CaMKIV associates poorly with PP2A in comparison to endogenous CaMKIV. The inability of exogenous CaMKIV to associate with PP2A appears to be due to limiting amounts of endogenous PP2A regulatory B subunits, since coexpression of Balpha or Bdelta causes the recruitment of PP2Ac to ectopic CaMKIV, leading to formation of a CaMKIV.PP2A complex. Together, these data indicate that the B subunits are essential for the interaction of PP2A with CaMKIV.
Assuntos
Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/metabolismo , Proteína Fosfatase 2/metabolismo , Subunidades Proteicas/metabolismo , Anticorpos Fosfo-Específicos , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/genética , Linhagem Celular , Holoenzimas/genética , Holoenzimas/metabolismo , Humanos , Mutação , Fosforilação , Proteína Fosfatase 2/genética , Subunidades Proteicas/genética , Transdução de SinaisRESUMO
Protein-protein interactions between the hypoxia inducible factor (HIF) and the transcriptional coactivators p300/CBP are potential cancer targets due to their role in the hypoxic response. A natural product based screen led to the identification of indandione and benzoquinone derivatives that reduce the tight interaction between a HIF-1α fragment and the CH1 domain of p300. The indandione derivatives were shown to fragment to give ninhydrin, which was identified as the active species. Both the naphthoquinones and ninhydrin were observed to induce Zn(II) ejection from p300 and the catalytic domain of the histone demethylase KDM4A. Together with previous reports on the effects of related compounds on HIF-1α and other systems, the results suggest that care should be taken in interpreting biological results obtained with highly electrophilic/thiol modifying compounds.
Assuntos
Proteína p300 Associada a E1A/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Indanos/farmacologia , Compostos Organometálicos/farmacologia , Quinonas/farmacologia , Zinco/farmacologia , Relação Dose-Resposta a Droga , Humanos , Indanos/química , Modelos Moleculares , Estrutura Molecular , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Ligação Proteica/efeitos dos fármacos , Quinonas/química , Relação Estrutura-Atividade , Zinco/químicaRESUMO
Protein phosphatase 2A (PP2A) is a ubiquitous phospho-serine/threonine phosphatase that controls many diverse cellular functions. The predominant form of PP2A is a heterotrimeric holoenzyme consisting of a scaffolding A subunit, a variable regulatory B subunit, and a catalytic C subunit. The C subunit also associates with other interacting partners, such as α4, to form non-canonical PP2A complexes. We report visualization of PP2A complexes in mammalian cells. Bimolecular fluorescence complementation (BiFC) analysis of PP2A subunit interactions demonstrates that the B subunit plays a key role in directing the subcellular localization of PP2A, and confirms that the A subunit functions as a scaffold in recruiting the B and C subunits to form a heterotrimeric holoenzyme. BiFC analysis also reveals that α4 promotes formation of the AC core dimer. Furthermore, we demonstrate visualization of specific ABC holoenzymes in cells by combining BiFC and fluorescence resonance energy transfer (BiFC-FRET). Our studies not only provide direct imaging data to support previous biochemical observations on PP2A complexes, but also offer a promising approach for studying the spatiotemporal distribution of individual PP2A complexes in cells.
Assuntos
Proteína Fosfatase 2/metabolismo , Animais , Imunofluorescência , Camundongos , Células NIH 3T3 , Multimerização Proteica , Proteína Fosfatase 2/análise , Subunidades Proteicas/análise , Subunidades Proteicas/metabolismoRESUMO
OBJECTIVES: Although it does not alter the ERCC1 phenotype, the ERCC1 500C>T (rs11615) polymorphism has undergone a myriad of investigations into its role as a marker for nucleotide excision repair (NER) function in different races, diseases and treatment outcomes. The goal of our study was to test the hypothesis that 500C>T is in linkage disequilibrium (LD) with causative alleles, and that these haplotypes are more frequent in Caucasians with melanoma than in healthy Caucasians or African Americans. DESIGN: In this case-control study, we selected race-specific ERCC1 single-nucleotide polymorphism (SNPs), conducted LD analysis with ERCC1 500C>T and compared the frequency of ERCC1 diplotypes in Caucasians with melanoma (n=165), healthy Caucasians (n=150) and healthy African Americans (n=159). The haplotype was further studied using a fusion gene containing multiple ERCC1 SNPs. SETTING: Large cancer institute in the USA. PARTICIPANTS: A total of 165 Caucasian melanoma patients, 159 healthy Caucasian controls and 159 African American healthy controls. Men and women were enrolled in the clinical trial; however, since the screening trial included prostate cancer screening in addition to screening for other cancers, only male controls were available. OUTCOME MEASURES: The outcome measures were melanoma risk in Caucasians, and LD between ERCC1 SNP, N118N and other race-specific allelic variants. RESULTS: When compared to ERCC1 500C>T alone, a race-specific three-SNP variant haplotype in ERCC1 (comprised of rs11615, rs3212950 and rs3212948) was even more frequent in Caucasians with melanoma than in healthy Caucasians (p=0.0034) or African Americans (p<0.0001). A plasmid containing the variant haplotype was not differentially expressed. CONCLUSIONS: We demonstrate that ERCC1 500C>T participates in a previously characterised cancer-risk haplotype found more frequently in Caucasians, while LD is weak in African Americans; this haplotype appears to also be related to melanoma. It is therefore likely that ERCC1 500C>T is only a valid NER, disease or treatment outcome marker in Caucasians.