RESUMO
The tumor-suppressor breast cancer 1 (BRCA1) in complex with BRCA1-associated really interesting new gene (RING) domain 1 (BARD1) is a RING-type ubiquitin E3 ligase that modifies nucleosomal histone and other substrates. The importance of BRCA1-BARD1 E3 activity in tumor suppression remains highly controversial, mainly stemming from studying mutant ligase-deficient BRCA1-BARD1 species that we show here still retain significant ligase activity. Using full-length BRCA1-BARD1, we establish robust BRCA1-BARD1-mediated ubiquitylation with specificity, uncover multiple modes of activity modulation, and construct a truly ligase-null variant and a variant specifically impaired in targeting nucleosomal histones. Cells expressing either of these BRCA1-BARD1 separation-of-function alleles are hypersensitive to DNA-damaging agents. Furthermore, we demonstrate that BRCA1-BARD1 ligase is not only required for DNA resection during homology-directed repair (HDR) but also contributes to later stages for HDR completion. Altogether, our findings reveal crucial, previously unrecognized roles of BRCA1-BARD1 ligase activity in genome repair via HDR, settle prior controversies regarding BRCA1-BARD1 ligase functions, and catalyze new efforts to uncover substrates related to tumor suppression.
Assuntos
Neoplasias , Proteínas Supressoras de Tumor , Humanos , Proteínas Supressoras de Tumor/metabolismo , Proteína BRCA1/metabolismo , Ubiquitinação , Histonas/genética , Histonas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Reparo de DNA por Recombinação , DNA , Reparo do DNARESUMO
Ovarian cancer (OCa) is the deadliest of all gynecological cancers. The standard treatment for OCa is platinum-based chemotherapy, such as carboplatin or cisplatin in combination with paclitaxel. Most patients are initially responsive to these treatments; however, nearly 90% will develop recurrence and inevitably succumb to chemotherapy-resistant disease. Recent studies have revealed that the epigenetic modifier lysine-specific histone demethylase 1A (KDM1A/LSD1) is highly overexpressed in OCa. However, the role of KDM1A in chemoresistance and whether its inhibition enhances chemotherapy response in OCa remains uncertain. Analysis of TCGA datasets revealed that KDM1A expression is high in patients who poorly respond to chemotherapy. Western blot analysis show that treatment with chemotherapy drugs cisplatin, carboplatin, and paclitaxel increased KDM1A expression in OCa cells. KDM1A knockdown (KD) or treatment with KDM1A inhibitors NCD38 and SP2509 sensitized established and patient-derived OCa cells to chemotherapy drugs in reducing cell viability and clonogenic survival and inducing apoptosis. Moreover, knockdown of KDM1A sensitized carboplatin-resistant A2780-CP70 cells to carboplatin treatment and paclitaxel-resistant SKOV3-TR cells to paclitaxel. RNA-seq analysis revealed that a combination of KDM1A-KD and cisplatin treatment resulted in the downregulation of genes related to epithelial-mesenchymal transition (EMT). Interestingly, cisplatin treatment increased a subset of NF-κB pathway genes, and KDM1A-KD or KDM1A inhibition reversed this effect. Importantly, KDM1A-KD, in combination with cisplatin, significantly reduced tumor growth compared to a single treatment in an orthotopic intrabursal OCa xenograft model. Collectively, these findings suggest that combination of KDM1A inhibitors with chemotherapy could be a promising therapeutic approach for the treatment of OCa.
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PURPOSE: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. Oncogenic PELP1 is frequently overexpressed in TNBC, and it has been demonstrated that PELP1 signaling is essential for TNBC progression. The therapeutic utility of targeting PELP1 in TNBC, however, remains unknown. In this study, we investigated the effectiveness of SMIP34, a recently developed PELP1 inhibitor for the treatment of TNBC. METHODS: To ascertain the impact of SMIP34 treatment, we used seven different TNBC models for testing cell viability, colony formation, invasion, apoptosis, and cell cycle analysis. Western blotting and RT-qPCR were used to determine the mechanistic insights of SMIP34 action. Using xenograft and PDX tumors, the ability of SMIP34 in suppressing proliferation was examined both ex vivo and in vivo. RESULTS: TNBC cells' viability, colony formation, and invasiveness were all decreased by SMIP34 in in vitro cell-based assays, while apoptosis was increased. SMIP34 treatment promoted the degradation of PELP1 through the proteasome pathway. RT-qPCR analyses confirmed that SMIP34 treatment downregulated PELP1 target genes. Further, SMIP34 treatment substantially downregulated PELP1 mediated extranuclear signaling including ERK, mTOR, S6 and 4EBP1. Mechanistic studies confirmed downregulation of PELP1 mediated ribosomal biogenesis functions including downregulation of cMyc and Rix complex proteins LAS1L, TEX-10, and SENP3. The proliferation of TNBC tumor tissues was decreased in explant experiments by SMIP34. Additionally, SMIP34 treatment markedly decreased tumor progression in both TNBC xenograft and PDX models. CONCLUSIONS: Together, these findings from in vitro, ex vivo, and in vivo models show that SMIP34 may be a useful therapeutic agent for inhibiting PELP1 signaling in TNBC.
Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Linhagem Celular Tumoral , Proliferação de Células , Proteínas Correpressoras , Cisteína Endopeptidases/metabolismo , Transdução de Sinais , Fatores de Transcrição , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
BACKGROUND: Methyltransferase SETDB1 is highly expressed in breast cancer (BC), however, the mechanisms by which SETDB1 promotes BC progression to endocrine therapy resistance remains elusive. In this study, we examined the mechanisms by which SETDB1 contribute to BC endocrine therapy resistance. METHODS: We utilized therapy sensitive (MCF7 and ZR75), therapy resistant (MCF7-TamR, MCF7-FR, MCF7-PELP1cyto, MCF7-SETDB1) estrogen receptor alpha positive (ER+)BC models and conducted in vitro cell viability, colony formation, 3-dimensional cell growth assays to investigate the role of SETDB1 in endocrine resistance. RNA-seq of parental and SETDB1 knock down ER+ BC cells was used to identify unique pathways. SETDB1 interaction with PELP1 was identified by yeast-two hybrid screen and confirmed by immunoprecipitation and GST-pull down assays. Mechanistic studies were conducted using Western blotting, reporter gene assays, RT-qPCR, and in vitro methylation assays. Xenograft assays were used to establish the role of PELP1 in SETDB1 mediated BC progression. RESULTS: RNA-seq analyses showed that SETDB1 regulates expression of a subset of estrogen receptor (ER) and Akt target genes that contribute to endocrine therapy resistance. Importantly, using yeast-two hybrid screen, we identified ER coregulator PELP1 as a novel interacting protein of SETDB1. Biochemical analyses confirmed SETDB1 and PELP1 interactions in multiple BC cells. Mechanistic studies confirmed that PELP1 is necessary for SETDB1 mediated Akt methylation and phosphorylation. Further, SETDB1 overexpression promotes tamoxifen resistance in BC cells, and PELP1 knockdown abolished these effects. Using xenograft model, we provided genetic evidence that PELP1 is essential for SETDB1 mediated BC progression in vivo. Analyses of TCGA datasets revealed SETDB1 expression is positively correlated with PELP1 expression in ER+ BC patients. CONCLUSIONS: This study suggests that the PELP1/SETDB1 axis play an important role in aberrant Akt activation and serves as a novel target for treating endocrine therapy resistance in breast cancer.
Assuntos
Neoplasias da Mama , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Proteínas Correpressoras/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/farmacologia , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Tamoxifeno/farmacologia , Fatores de Transcrição/genéticaRESUMO
Glioblastoma (GBM) is the most common and deadliest tumor of the central nervous system. GBM has poor prognosis and glioma stem cells (GSCs) are implicated in tumor initiation and therapy resistance. Estrogen receptor ß (ERß) is expressed in GBM and exhibit tumor suppressive function. However, the role of ERß in GSCs and the therapeutic potential of ERß agonists on GSCs remain largely unknown. Here, we examined whether ERß modulates GSCs stemness and tested the utility of two ERß selective agonists (LY500307 and Liquiritigenin) to reduce the stemness of GSCs. The efficacy of ERß agonists was examined on GSCs isolated from established and patient derived GBMs. Our results suggested that knockout of ERß increased the proportion of CD133+ and SSEA+ positive GSCs and overexpression of ERß reduced the proportion of GSCs in GBM cells. Overexpression of ERß or treatment with ERß agonists significantly inhibited the GSCs cell viability, neurosphere formation, self-renewal ability, induced the apoptosis and reduced expression of stemness markers in GSCs. RNA sequencing analysis revealed that ERß agonist modulate pathways related to stemness, differentiation and apoptosis. Mechanistic studies showed that ERß overexpression or agonist treatment reduced glutamate receptor signaling pathway and induced apoptotic pathways. In orthotopic models, ERß overexpression or ERß agonists treatment significantly reduced the GSCs mediated tumor growth and improved the mice overall survival. Immunohistochemical studies demonstrated that ERß overexpression decreased SOX2 and GRM3 expression and increased expression of GFAP in tumors. These results suggest that ERß activation could be a promising therapeutic strategy to eradicate GSCs.
Assuntos
Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Receptor beta de Estrogênio/genética , Glioma/genética , Células-Tronco Neoplásicas/metabolismo , Antígeno AC133/genética , Animais , Apoptose/efeitos dos fármacos , Benzopiranos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Receptor beta de Estrogênio/agonistas , Flavanonas/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/genética , Glioma/tratamento farmacológico , Glioma/patologia , Humanos , Camundongos , Células-Tronco Neoplásicas/efeitos dos fármacos , Receptores de Glutamato/genética , Fatores de Transcrição SOXB1/genética , Transdução de Sinais/efeitos dos fármacos , Antígenos Embrionários Estágio-Específicos/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ovarian cancer (OCa) is the deadliest gynecologic cancer. Emerging studies suggest ovarian cancer stem cells (OCSCs) contribute to chemotherapy resistance and tumor relapse. Recent studies demonstrated estrogen receptor beta (ERß) exerts tumor suppressor functions in OCa. However, the status of ERß expression in OCSCs and the therapeutic utility of the ERß agonist LY500307 for targeting OCSCs remain unknown. OCSCs were enriched from ES2, OV90, SKOV3, OVSAHO, and A2780 cells using ALDEFLUOR kit. RT-qPCR results showed ERß, particularly ERß isoform 1, is highly expressed in OCSCs and that ERß agonist LY500307 significantly reduced the viability of OCSCs. Treatment of OCSCs with LY500307 significantly reduced sphere formation, self-renewal, and invasion, while also promoting apoptosis and G2/M cell cycle arrest. Mechanistic studies using RNA-seq analysis demonstrated that LY500307 treatment resulted in modulation of pathways related to cell cycle and apoptosis. Western blot and RT-qPCR assays demonstrated the upregulation of apoptosis and cell cycle arrest genes such as FDXR, p21/CDKN1A, cleaved PARP, and caspase 3, and the downregulation of stemness markers SOX2, Oct4, and Nanog. Importantly, treatment of LY500307 significantly attenuated the tumor-initiating capacity of OCSCs in orthotopic OCa murine xenograft models. Our results demonstrate that ERß agonist LY500307 is highly efficacious in reducing the stemness and promoting apoptosis of OCSCs and shows significant promise as a novel therapeutic agent in treating OCa.
Assuntos
Receptor beta de Estrogênio , Neoplasias Ovarianas , Animais , Linhagem Celular Tumoral , Proliferação de Células , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Estrogênios/metabolismo , Feminino , Humanos , Camundongos , Células-Tronco Neoplásicas/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologiaRESUMO
Hemoglobin (Hb) is the oxygen transport protein in erythrocytes. In blood, Hb is a tetramer consisting of two Hb-alpha (Hb-α) chains and two Hb-beta (Hb-ß) chains. A number of studies have also shown that Hb-α is also expressed in neurons in both the rodent and human brain. In the current study, we examined for age-related regulation of neuronal Hb-α and hypoxia in the hippocampus and cerebral cortex of intact male and female mice. In addition, to confirm the role and functions of neuronal Hb-α, we also utilized lentivirus CRISPR interference-based Hb-α knockdown (Hb-α CRISPRi KD) in the non-ischemic and ischemic mouse hippocampus and examined the effect on neuronal oxygenation, as well as induction of hypoxia-inducible factor-1α (HIF-1α) and its downstream pro-apoptotic factors, PUMA and NOXA, and on neuronal survival and neurodegeneration. The results of the study revealed an age-related decrease in neuronal Hb-α levels and correlated increase in hypoxia in the hippocampus and cortex of intact male and female mice. Sex differences were observed with males having higher neuronal Hb-α levels than females in all brain regions at all ages. In vivo Hb-α CRISPRi KD in the mouse hippocampus resulted in increased hypoxia and elevated levels of HIF-1α, PUMA and NOXA in the non-ischemic and ischemic mouse hippocampus, effects that were correlated with a significant decrease in neuronal survival and increased neurodegeneration. As a whole, these findings indicate that neuronal Hb-α decreases with age in mice and has an important role in regulating neuronal oxygenation and neuroprotection.
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Hemoglobinas , Neurônios , Animais , Córtex Cerebral/metabolismo , Feminino , Hemoglobinas/metabolismo , Hipocampo/metabolismo , Hipóxia/metabolismo , Masculino , Camundongos , Neurônios/metabolismoRESUMO
Expression of the 17ß-estradiol (E2) synthesis enzyme aromatase is highly upregulated in astrocytes following brain injury. However, the precise role of astrocyte-derived E2 in the injured brain remains unclear. In the current study, we generated a glial fibrillary acidic protein (GFAP) promoter-driven aromatase knock-out (GFAP-ARO-KO) mouse model to deplete astrocyte-derived E2 in the brain and determine its roles after global cerebral ischemia (GCI) in male and female mice. GFAP-ARO-KO mice were viable and fertile, with normal gross brain structure, normal morphology, intensity and distribution of astrocytes, normal aromatase expression in neurons, and normal cognitive function basally. In contrast, after GCI, GFAP-ARO-KO mice: (1) lacked the normal elevation of astrocyte aromatase and hippocampal E2 levels; (2) had significantly attenuated reactive astrogliosis; and (3) displayed enhanced neuronal damage, microglia activation, and cognitive deficits. RNA-sequencing (RNA-seq) analysis revealed that the ischemic GFAP-ARO-KO mouse hippocampus failed to upregulate the "A2" panel of reactive astrocyte genes. In addition, the JAK-STAT3 pathway, which is critical for the induction of reactive astrogliosis, was significantly downregulated in the GFAP-ARO-KO hippocampus following GCI. Finally, exogenous E2 administration fully rescued the compromised JAK-STAT3 pathway and reactive astrogliosis, and reversed the enhanced neuronal damage and microglial activation in the GFAP-ARO-KO mice after GCI, suggesting that the defects in the KO mice are because of a loss of E2 rather than an increase in precursor androgens. In conclusion, the current study provides novel genetic evidence for a beneficial role of astrocyte-derived E2 in reactive astrogliosis, microglial activation, and neuroprotection following an ischemic injury to the brain.SIGNIFICANCE STATEMENT Following cerebral ischemia, reactive astrocytes express the enzyme aromatase and produce 17ß-estradiol (E2), although the precise role of astrocyte-derived E2 is poorly understood. In this study, we generated a glial fibrillary acidic protein (GFAP) promoter-driven aromatase knock-out (GFAP-ARO-KO) mouse to deplete astrocyte-derived E2 and elucidate its roles after global cerebral ischemia (GCI). The GFAP-ARO-KO mice exhibited significantly attenuated reactive astrogliosis, as well as enhanced microglial activation, neuronal damage, and cognitive dysfunction after GCI. Transcriptome analysis further revealed that astrocyte-derived E2 was critical for the induction of the JAK-STAT3 signaling pathway, as well as the A2 reactive astrocyte phenotype after ischemia. Collectively, these findings indicate that astrocyte-derived E2 has a key role in the regulation of reactive astrogliosis, microglial activation, and neuroprotection after cerebral ischemia.
Assuntos
Aromatase/genética , Astrócitos/metabolismo , Isquemia Encefálica/metabolismo , Estradiol/metabolismo , Gliose/metabolismo , Hipocampo/metabolismo , Animais , Aromatase/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Isquemia Encefálica/genética , Isquemia Encefálica/patologia , Condicionamento Clássico/fisiologia , Modelos Animais de Doenças , Estradiol/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/genética , Gliose/patologia , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Camundongos , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neuroproteção/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
17ß-Estradiol (E2) is produced from androgens via the action of the enzyme aromatase. E2 is known to be made in neurons in the brain, but the functions of neuron-derived E2 in the ischemic brain are unclear. Here, we used a forebrain neuron-specific aromatase KO (FBN-ARO-KO) mouse model to deplete neuron-derived E2 in the forebrain and determine its roles after global cerebral ischemia. We demonstrated that ovariectomized female FBN-ARO-KO mice exhibited significantly attenuated astrocyte activation, astrocytic aromatization, and decreased hippocampal E2 levels compared with FLOX mice. Furthermore, FBN-ARO-KO mice had exacerbated neuronal damage and worse cognitive dysfunction after global cerebral ischemia. Similar results were observed in intact male mice. RNA-seq analysis revealed alterations in pathways and genes associated with astrocyte activation, neuroinflammation, and oxidative stress in FBN-ARO-KO mice. The compromised astrocyte activation in FBN-ARO-KO mice was associated with robust downregulation of the astrocyte-derived neurotrophic factors, BDNF and IGF-1, as well as the astrocytic glutamate transporter, GLT-1. Νeuronal FGF2, which acts in a paracrine manner to suppress astrocyte activation, was increased in FBN-ARO-KO neurons. Interestingly, blocking FGF2 signaling by central injection of FGFR3-neutralizing antibody was able to reverse the diminishment in neuroprotective astrocyte reactivity, and attenuate neuronal damage in FBN-ARO-KO mice. Moreover, in vivo E2 replacement suppressed FGF2 signaling and rescued the compromised reactive astrogliosis and cognitive deficits. Collectively, our data provide novel genetic evidence for a beneficial role of neuron-derived E2 in astrocyte activation, neuroprotection, and cognitive preservation following ischemic injury to the brain.SIGNIFICANCE STATEMENT Following cerebral ischemia, astrocytes become highly reactive and can exert neuroprotection through the release of neurotrophic factors and clearance of neurotoxic glutamate. The current study advances our understanding of this process by demonstrating that neuron-derived 17ß-estradiol (E2) is neuroprotective and critical for induction of reactive astrocytes and their ability to produce astrocyte-derived neurotrophic factors, BDNF and IGF-1, and the glutamate transporter, GLT-1 after ischemic brain damage. These beneficial effects of neuron-derived E2 appear to be due, at least in part, to suppression of neuronal FGF2 signaling, which is a known suppressor of astrocyte activation. These findings suggest that neuron-derived E2 is neuroprotective after ischemic brain injury via a mechanism that involves suppression of neuronal FGF2 signaling, thereby facilitating astrocyte activation.
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Astrócitos/metabolismo , Isquemia Encefálica/metabolismo , Estrogênios/metabolismo , Gliose/metabolismo , Neurônios/metabolismo , Comunicação Parácrina , Animais , Aromatase/genética , Aromatase/metabolismo , Isquemia Encefálica/patologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/metabolismo , Células Cultivadas , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Transportador 2 de Aminoácido Excitatório/metabolismo , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Camundongos , Estresse OxidativoRESUMO
PURPOSE: The majority of breast cancers are estrogen receptor (ERα) positive making endocrine therapy a mainstay for these patients. Unfortunately, resistance to endocrine therapy is a common occurrence. Fatty acid synthase (FASN) is a key enzyme in lipid biosynthesis and its expression is commensurate with tumor grade and resistance to numerous therapies. METHODS: The effect of the FASN inhibitor TVB-3166 on ERα expression and cell growth was characterized in tamoxifen-resistant cell lines, xenografts, and patient explants. Subcellular localization of ERα was assessed using subcellular fractionations. Palmitoylation and ubiquitination of ERα were assessed by immunoprecipitation. ERα and p-eIF2α protein levels were analyzed by Western blotting after treatment with TVB-3166 with or without the addition of palmitate or BAPTA. RESULTS: TVB-3166 treatment leads to a marked inhibition of proliferation in tamoxifen-resistant cells compared to the parental cells. Additionally, TVB-3166 significantly inhibited tamoxifen-resistant breast tumor growth in mice and decreased proliferation of primary tumor explants compared to untreated controls. FASN inhibition significantly reduced ERα levels most prominently in endocrine-resistant cells and altered its subcellular localization. Furthermore, we showed that the reduction of ERα expression upon TVB-3166 treatment is mediated through the induction of endoplasmic reticulum stress. CONCLUSION: Our preclinical data provide evidence that FASN inhibition by TVB-3166 presents a promising therapeutic strategy for the treatment of endocrine-resistant breast cancer. Further clinical development of FASN inhibitors for endocrine-resistant breast cancer should be considered.
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Neoplasias da Mama , Inibidores Enzimáticos/uso terapêutico , Ácido Graxo Sintase Tipo I/antagonistas & inibidores , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/genética , Ácido Graxo Sintase Tipo I/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Tamoxifeno/farmacologiaRESUMO
PURPOSE: Cancer stem cells (CSCs) are highly tumorigenic, spared by chemotherapy, sustain tumor growth, and are implicated in tumor recurrence after conventional therapies in triple negative breast cancer (TNBC). Lysine-specific histone demethylase 1A (KDM1A) is highly expressed in several human malignancies and CSCs including TNBC. However, the precise mechanistic role of KDM1A in CSC functions and therapeutic utility of KDM1A inhibitor for treating TNBC is poorly understood. METHODS: The effect of KDM1A inhibition on cell viability, apoptosis, and invasion were examined by Cell Titer Glo, Caspase 3/7 Glo, and matrigel invasion assays, respectively. Stemness and self-renewal of CSCs were examined using mammosphere formation and extreme limiting dilution assays. Mechanistic studies were conducted using RNA-sequencing, RT-qPCR, Western blotting and reporter gene assays. Mouse xenograft and patient derived xenograft models were used for preclinical evaluation of KDM1A inhibitor. RESULTS: TCGA data sets indicated that KDM1A is highly expressed in TNBC. CSCs express high levels of KDM1A and inhibition of KDM1A reduced the CSCs enrichment in TNBC cells. KDM1A inhibition reduced cell viability, mammosphere formation, self-renewal and promoted apoptosis of CSCs. Mechanistic studies suggested that IL6-JAK-STAT3 and EMT pathways were downregulated in KDM1A knockdown and KDM1A inhibitor treated cells. Importantly, doxycycline inducible knockout of KDM1A reduced tumor progression in orthotopic xenograft models and KDM1A inhibitor NCD38 treatment significantly reduced tumor growth in patient derived xenograft (PDX) models. CONCLUSIONS: Our results establish that KDM1A inhibition mitigates CSCs functions via inhibition of STAT3 and EMT signaling, and KDM1A inhibitor NCD38 may represent a novel class of drug for treating TNBC.
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Histona Desmetilases , Neoplasias de Mama Triplo Negativas , Animais , Linhagem Celular Tumoral , Proliferação de Células , Histona Desmetilases/antagonistas & inibidores , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Camundongos , Recidiva Local de Neoplasia , Células-Tronco Neoplásicas , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In addition to being a steroid hormone, 17ß-estradiol (E2) is also a neurosteroid produced in neurons in various regions of the brain of many species, including humans. Neuron-derived E2 (NDE2) is synthesized from androgen precursors via the action of the biosynthetic enzyme aromatase, which is located at synapses and in presynaptic terminals in neurons in both the male and female brain. In this review, we discuss evidence supporting a key role for NDE2 as a neuromodulator that regulates synaptic plasticity and memory. Evidence supporting an important neuromodulatory role of NDE2 in the brain has come from studies using aromatase inhibitors, aromatase overexpression in neurons, global aromatase knockout mice, and the recent development of conditional forebrain neuron-specific knockout mice. Collectively, these studies demonstrate a key role of NDE2 in the regulation of synapse and spine density, efficacy of excitatory synaptic transmission and long-term potentiation, and regulation of hippocampal-dependent recognition memory, spatial reference memory, and contextual fear memory. NDE2 is suggested to achieve these effects through estrogen receptor-mediated regulation of rapid kinase signaling and CREB-BDNF signaling pathways, which regulate actin remodeling, as well as transcription, translation, and transport of synaptic proteins critical for synaptic plasticity and function.
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Estradiol/metabolismo , Neurônios/metabolismo , Memória Espacial/fisiologia , Sinapses/fisiologia , Animais , Aromatase/genética , Aromatase/metabolismo , Feminino , Humanos , Masculino , Plasticidade Neuronal , Transdução de SinaisRESUMO
17ß-estradiol (E2) is produced from androgens via the action of the enzyme aromatase. E2 is known to be made in neurons in the brain, but its precise functions in the brain are unclear. Here, we used a forebrain-neuron-specific aromatase knock-out (FBN-ARO-KO) mouse model to deplete neuron-derived E2 in the forebrain of mice and thereby elucidate its functions. FBN-ARO-KO mice showed a 70-80% decrease in aromatase and forebrain E2 levels compared with FLOX controls. Male and female FBN-ARO-KO mice exhibited significant deficits in forebrain spine and synaptic density, as well as hippocampal-dependent spatial reference memory, recognition memory, and contextual fear memory, but had normal locomotor function and anxiety levels. Reinstating forebrain E2 levels via exogenous in vivo E2 administration was able to rescue both the molecular and behavioral defects in FBN-ARO-KO mice. Furthermore, in vitro studies using FBN-ARO-KO hippocampal slices revealed that, whereas induction of long-term potentiation (LTP) was normal, the amplitude was significantly decreased. Intriguingly, the LTP defect could be fully rescued by acute E2 treatment in vitro Mechanistic studies revealed that FBN-ARO-KO mice had compromised rapid kinase (AKT, ERK) and CREB-BDNF signaling in the hippocampus and cerebral cortex. In addition, acute E2 rescue of LTP in hippocampal FBN-ARO-KO slices could be blocked by administration of a MEK/ERK inhibitor, further suggesting a key role for rapid ERK signaling in neuronal E2 effects. In conclusion, the findings provide evidence of a critical role for neuron-derived E2 in regulating synaptic plasticity and cognitive function in the male and female brain.SIGNIFICANCE STATEMENT The steroid hormone 17ß-estradiol (E2) is well known to be produced in the ovaries in females. Intriguingly, forebrain neurons also express aromatase, the E2 biosynthetic enzyme, but the precise functions of neuron-derived E2 is unclear. Using a novel forebrain-neuron-specific aromatase knock-out mouse model to deplete neuron-derived E2, the current study provides direct genetic evidence of a critical role for neuron-derived E2 in the regulation of rapid AKT-ERK and CREB-BDNF signaling in the mouse forebrain and demonstrates that neuron-derived E2 is essential for normal expression of LTP, synaptic plasticity, and cognitive function in both the male and female brain. These findings suggest that neuron-derived E2 functions as a novel neuromodulator in the forebrain to control synaptic plasticity and cognitive function.
Assuntos
Estradiol/fisiologia , Memória/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Sinapses/fisiologia , Animais , Ansiedade/genética , Ansiedade/psicologia , Aromatase/genética , Cognição , Espinhas Dendríticas , Estradiol/metabolismo , Estradiol/farmacologia , Feminino , Hipocampo , Potenciação de Longa Duração/genética , Potenciação de Longa Duração/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Prosencéfalo/enzimologia , Prosencéfalo/metabolismo , Desempenho Psicomotor/fisiologia , Aprendizagem EspacialRESUMO
Medulloblastoma (MB) is the most common and deadliest brain tumor in children. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) is a scaffolding protein and its oncogenic signaling is implicated in the progression of several cancers. However, the role of PELP1 in the progression of MB remains unknown. The objective of this study is to examine the role of PELP1 in the progression of MB. Immunohistochemical analysis of MB tissue microarrays revealed that PELP1 is overexpressed in the MB specimens compared to normal brain. Knockdown of PELP1 reduced cell proliferation, cell survival, and cell invasion of MB cell lines. The RNA-sequencing analysis revealed that PELP1 knockdown significantly downregulated the pathways related to inflammation and extracellular matrix. Gene set enrichment analysis confirmed that the PELP1-regulated genes were negatively correlated with nuclear factor-κB (NF-κB), extracellular matrix, and angiogenesis gene sets. Interestingly, PELP1 knockdown reduced the expression of NF-κB target genes, NF-κB reporter activity, and inhibited the nuclear translocation of p65. Importantly, the knockdown of PELP1 significantly reduced in vivo MB progression in orthotopic models and improved the overall mice survival. Collectively, these results suggest that PELP1 could be a novel target for therapeutic intervention in MB.
Assuntos
Neoplasias Cerebelares/metabolismo , Proteínas Correpressoras/metabolismo , Meduloblastoma/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Proteínas Correpressoras/análise , Proteínas Correpressoras/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Meduloblastoma/genética , Meduloblastoma/patologia , Camundongos , Camundongos Nus , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Fatores de Transcrição/análise , Fatores de Transcrição/genéticaRESUMO
17-ß estradiol (E2) has been implicated as neuroprotective in a variety of neurodegenerative disorders. However, the underlying mechanism remains unknown. Here, we provide genetic evidence, using forebrain-specific knockout (FBKO) mice, that proline-, glutamic acid-, and leucine-rich protein 1 (PELP1), an estrogen receptor coregulator protein, is essential for the extranuclear signaling and neuroprotective actions of E2 in the hippocampal CA1 region after global cerebral ischemia (GCI). E2-mediated extranuclear signaling (including activation of extracellular signal-regulated kinase and Akt) and antiapoptotic effects [such as attenuation of JNK signaling and increase in phosphorylation of glycogen synthase kinase-3ß (GSK3ß)] after GCI were compromised in PELP1 FBKO mice. Mechanistic studies revealed that PELP1 interacts with GSK3ß, E2 modulates interaction of PELP1 with GSK3ß, and PELP1 is a novel substrate for GSK3ß. RNA-seq analysis of control and PELP1 FBKO mice after ischemia demonstrated alterations in several genes related to inflammation, metabolism, and survival in PELP1 FBKO mice, as well as a significant reduction in the activation of the Wnt/ß-catenin signaling pathway. In addition, PELP1 FBKO studies revealed that PELP1 is required for E2-mediated neuroprotection and for E2-mediated preservation of cognitive function after GCI. Collectively, our data provide the first direct in vivo evidence, to our knowledge, of an essential role for PELP1 in E2-mediated rapid extranuclear signaling, neuroprotection, and cognitive function in the brain.
Assuntos
Região CA1 Hipocampal/metabolismo , Estrogênios/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuroproteção/genética , Animais , Isquemia Encefálica/patologia , Região CA1 Hipocampal/patologia , Proteína Tirosina Quinase CSK , Cognição , Receptor alfa de Estrogênio/metabolismo , Feminino , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Inflamação , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oligonucleotídeos Antissenso , Fosforilação , Transdução de Sinais , Quinases da Família src/metabolismoRESUMO
Triple-negative breast cancer (TNBC), the most aggressive breast cancer subtype, occurs in younger women and is associated with poor prognosis. Gain-of-function mutations in TP53 are a frequent occurrence in TNBC and have been demonstrated to repress apoptosis and up-regulate cell cycle progression. Even though TNBC responds to initial chemotherapy, resistance to chemotherapy develops and is a major clinical problem. Tumor recurrence eventually occurs and most patients die from their disease. An urgent need exists to identify molecular-targeted therapies that can enhance chemotherapy response. In the present study, we report that targeting PELP1, an oncogenic co-regulator molecule, could enhance the chemotherapeutic response of TNBC through the inhibition of cell cycle progression and activation of apoptosis. We demonstrate that PELP1 interacts with MTp53, regulates its recruitment, and alters epigenetic marks at the target gene promoters. PELP1 knockdown reduced MTp53 target gene expression, resulting in decreased cell survival and increased apoptosis upon genotoxic stress. Mechanistic studies revealed that PELP1 depletion contributes to increased stability of E2F1, a transcription factor that regulates both cell cycle and apoptosis in a context-dependent manner. Further, PELP1 regulates E2F1 stability in a KDM1A-dependent manner, and PELP1 phosphorylation at the S1033 residue plays an important role in mediating its oncogenic functions in TNBC cells. Accordingly, depletion of PELP1 increased the expression of E2F1 target genes and reduced TNBC cell survival in response to genotoxic agents. PELP1 phosphorylation was significantly greater in the TNBC tumors than in the other subtypes of breast cancer and in the normal tissues. These findings suggest that PELP1 is an important molecular target in TNBC, and that PELP1-targeted therapies may enhance response to chemotherapies.
Assuntos
Proteínas Correpressoras/metabolismo , Fator de Transcrição E2F1/metabolismo , Mutação , Fatores de Transcrição/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Proteína Supressora de Tumor p53/genética , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Proteínas Correpressoras/antagonistas & inibidores , Proteínas Correpressoras/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Humanos , Fosforilação , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Proteína Supressora de Tumor p53/metabolismoRESUMO
Sacituzumab Govitecan (SG) is an antibody-drug conjugate that has demonstrated efficacy in patients with TROP-2 expressing epithelial cancers. In a xenograft model of intracranial breast cancer, SG inhibited tumor growth and increased mouse survival. We conducted a prospective window-of-opportunity trial (NCT03995706) at the University of Texas Health Science Center at San Antonio to examine the intra-tumoral concentrations and intracranial activity of SG in patients undergoing craniotomy for breast cancer with brain metastases (BCBM) or recurrent glioblastoma (rGBM). We enrolled 25 patients aged ≥18 years diagnosed with BCBM and rGBM to receive a single intravenous dose of SG at 10 mg/kg given one day before resection and continued on days 1 and 8 of 21-day cycles following recovery. The PFS was 8 months and 2 months for BCBM and rGBM cohorts, respectively. The OS was 35.2 months and 9.5 months, respectively. Grade≥3 AE included neutropenia (28%), hypokalemia (8%), seizure (8%), thromboembolic event (8%), urinary tract infection (8%) and muscle weakness of the lower limb (8%). In post-surgical tissue, the median total SN-38 was 249.8 ng/g for BCBM and 104.5 ng/g for rGBM, thus fulfilling the primary endpoint. Biomarker analysis suggests delivery of payload by direct release at target site and that hypoxic changes do not drive indirect release. Secondary endpoint of OS was 35.2 months for the BCBM cohort and 9.5 months for rGBM. Non-planned exploratory endpoint of ORR was 38% for BCBM and 29%, respectively. Exploratory endpoint of Trop-2 expression was observed in 100% of BCBM and 78% of rGBM tumors. In conclusion, SG was found to be well tolerated with adequate penetration into intracranial tumors and promising preliminary activity within the CNS. Trial Registration: Trial (NCT03995706) enrolled at Clinical Trials.gov as Neuro/Sacituzumab Govitecan/Breast Brain Metastasis/Glioblastoma/Ph 0: https://clinicaltrials.gov/study/NCT03995706?cond=NCT03995706 .
Assuntos
Anticorpos Monoclonais Humanizados , Neoplasias Encefálicas , Neoplasias da Mama , Glioblastoma , Imunoconjugados , Recidiva Local de Neoplasia , Humanos , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Neoplasias Encefálicas/secundário , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Pessoa de Meia-Idade , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Monoclonais Humanizados/administração & dosagem , Adulto , Idoso , Imunoconjugados/uso terapêutico , Camptotecina/análogos & derivados , Camptotecina/uso terapêutico , Estudos Prospectivos , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/metabolismoRESUMO
Ovarian cancer (OCa) is the most lethal form of gynecologic cancer, and the tumor heterogeneities at the molecular, cellular, and tissue levels fuel tumor resistance to standard therapies and pose a substantial clinical challenge. Here, we tested the hypothesis that the heightened basal endoplasmic reticulum stress (ERS) observed in OCa represents an exploitable vulnerability and may overcome tumor heterogeneity. Our recent studies identified LIPA as a novel target to induce ERS in cancer cells using the small molecule ERX-41. However, the role of LIPA and theutility of ERX-41 to treat OCa remain unknown. Expression analysis using the TNMplot web tool, TCGA data sets, and immunohistochemistry analysis using a tumor tissue array showed that LIPA is highly expressed in OCa tissues, compared to normal tissues. ERX-41 treatment significantly reduced the cell viability and colony formation ability and promoted the apoptosis of OCa cells. Mechanistic studies revealed a robust and consistent induction of ERS markers, including CHOP, elF2α, PERK, and ATF4, upon ERX-41 treatment. In xenograft and PDX studies, ERX-41 treatment resulted in a significant reduction in tumor growth. Collectively, our results suggest that ERX-41 is a novel therapeutic agent that targets the LIPA with a unique mechanism of ERS induction, which could be exploited to treat heterogeneity in OCa.
RESUMO
Of all gynecologic cancers, epithelial-ovarian cancer (OCa) stands out with the highest mortality rates. Despite all efforts, 90% of individuals who receive standard surgical and cytotoxic therapy experience disease recurrence. The precise mechanism by which leukemia inhibitory factor (LIF) and its receptor (LIFR) contribute to the progression of OCa remains unknown. Analysis of cancer databases revealed that elevated expression of LIF or LIFR was associated with poor progression-free survival of OCa patients and a predictor of poor response to chemotherapy. Using multiple primary and established OCa cell lines or tissues that represent five subtypes of epithelial-OCa, we demonstrated that LIF/LIFR autocrine signaling is active in OCa. Moreover, treatment with LIFR inhibitor, EC359 significantly reduced OCa cell viability and cell survival with an IC50 ranging from 5-50 nM. Furthermore, EC359 diminished the stemness of OCa cells. Mechanistic studies using RNA-seq and rescue experiments unveiled that EC359 primarily induced ferroptosis by suppressing the glutathione antioxidant defense system. Using multiple in vitro, ex vivo and in vivo models including cell-based xenografts, patient-derived explants, organoids, and xenograft tumors, we demonstrated that EC359 dramatically reduced the growth and progression of OCa. Additionally, EC359 therapy considerably improved tumor immunogenicity by robust CD45+ leukocyte tumor infiltration and polarizing tumor-associated macrophages (TAMs) toward M1 phenotype while showing no impact on normal T-, B-, and other immune cells. Collectively, our findings indicate that the LIF/LIFR autocrine loop plays an essential role in OCa progression and that EC359 could be a promising therapeutic agent for OCa.
RESUMO
INTRODUCTION: Lysine-specific histone demethylase 1A (KDM1A/LSD1) has emerged as an important therapeutic target in various cancer types. LSD1 regulates a wide range of biological processes that influence cancer development, progression, metastasis, and therapy resistance. However, recent studies have revealed novel aspects of LSD1 biology, shedding light on its involvement in immunogenicity, antitumor immunity, and DNA damage response. These emerging findings have the potential to be leveraged in the design of effective LSD1-targeted therapies. AREAS COVERED: This paper discusses the latest developments in the field of LSD1 biology, focusing on its role in regulating immunogenicity, antitumor immunity, and DNA damage response mechanisms. The newfound understanding of these mechanisms has opened possibilities for the development of novel LSD1-targeted therapies for cancer treatment. Additionally, the paper provides an overview of LSD1 inhibitor-based combination therapies for the treatment of cancer. EXPERT OPINION: Exploiting LSD1 role in antitumor immunity and DNA damage response provides cues to not only understand the LSD1-resistant mechanisms but also rationally design new combination therapies that are more efficient and less toxic than monotherapy. The exploration of LSD1 biology and the development of LSD1-targeted therapies hold great promise for the future of cancer treatment.