RESUMEN
PRL-3 (PTP4A3), a metastasis-associated phosphatase, is also upregulated in patients with acute myeloid leukemia (AML) and is associated with poor prognosis, but the underlying molecular mechanism is unknown. Here, constitutive expression of PRL-3 in human AML cells sustains leukemogenesis in vitro and in vivo Furthermore, PRL-3 phosphatase activity dependently upregulates LIN28B, a stem cell reprogramming factor, which in turn represses the let-7 mRNA family, inducing a stem cell-like transcriptional program. Notably, elevated levels of LIN28B protein independently associate with worse survival in AML patients. Thus, these results establish a novel signaling axis involving PRL-3/LIN28B/let-7, which confers stem cell-like properties to leukemia cells that is important for leukemogenesis.Implications: The current study offers a rationale for targeting PRL-3 as a therapeutic approach for a subset of AML patients with poor prognosis. Mol Cancer Res; 15(3); 294-303. ©2016 AACR.
Asunto(s)
Carcinogénesis/genética , Leucemia Mieloide Aguda/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Células Madre/metabolismo , Animales , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células HL-60 , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Fosfatasas/genética , Proteínas de Unión al ARN/genética , Transducción de Señal , TransfecciónRESUMEN
Overexpression of protein-tyrosine phosphatase of regenerating liver 3 (PRL-3) has been identified in about 50% of patients with acute myeloid leukemia (AML). The mechanism of regulation of PRL-3 remains obscure. Signal transducer and activator of transcription 3 (STAT3), a latent transcriptional factor, has also been often found to be activated in AML. We first identified STAT3-consensus-binding sites in the promoter of PRL-3 genes. Then we experimentally validated the direct binding and transcriptional activation. We applied shRNA-mediated knockdown and overexpression approaches in STAT3(-/-) liver cells and leukemic cells to validate the functional regulation of PRL-3 by STAT3. A STAT3 core signature, derived through data mining from publicly available gene expression data, was employed to correlate PRL-3 expression in large AML patient samples. We discovered that STAT3 binds to the -201 to -210 region of PRL-3, which was conserved between human and mouse. Importantly, PRL-3 protein was significantly reduced in mouse STAT3-knockout liver cells compared with STAT3-wild type counterparts, and ectopic expression of STAT3 in these cells led to a pronounced increase in PRL-3 protein. We demonstrated that STAT3 functionally regulated PRL-3, and STAT3 core signature was enriched in AML with high PRL-3 expression. Targeting either STAT3 or PRL-3 reduced leukemic cell viability. Silencing PRL-3 impaired invasiveness and induced leukemic cell differentiation. In conclusion, PRL-3 was transcriptionally regulated by STAT3. The STAT3/PRL-3 regulatory loop contributes to the pathogenesis of AML, and it might represent an attractive therapeutic target for antileukemic therapy.
Asunto(s)
Proteínas Inmediatas-Precoces/fisiología , Leucemia Mieloide Aguda/enzimología , Proteínas de Neoplasias/fisiología , Proteínas Tirosina Fosfatasas/fisiología , Factor de Transcripción STAT3/fisiología , Animales , Sitios de Unión , Diferenciación Celular , Línea Celular Tumoral , Secuencia Conservada , Análisis Mutacional de ADN , ADN de Neoplasias/genética , Dosificación de Gen , Regulación Leucémica de la Expresión Génica , Genes Reporteros , Humanos , Proteínas Inmediatas-Precoces/biosíntesis , Proteínas Inmediatas-Precoces/genética , Leucemia Mieloide Aguda/genética , Hígado/metabolismo , Ratones , Mutagénesis Sitio-Dirigida , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Regiones Promotoras Genéticas/genética , Proteínas Tirosina Fosfatasas/biosíntesis , Proteínas Tirosina Fosfatasas/genética , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Factor de Transcripción STAT3/deficiencia , Transducción de Señal , Especificidad de la Especie , TransfecciónRESUMEN
PRL-3, an oncogenic dual-specificity phosphatase, is overexpressed in 50% of acute myelogenous leukemia (AML) and associated with poor survival. We found that stable expression of PRL-3 confers cytokine independence and growth advantage of AML cells. However, how PRL-3 mediates these functions in AML is not known. To comprehensively screen for PRL3-regulated proteins in AML, we performed SILAC-based quantitative proteomics analysis and discovered 398 significantly perturbed proteins after PRL-3 overexpression. We show that Leo1, a component of RNA polymerase II-associated factor (PAF) complex, is a novel and important mediator of PRL-3 oncogenic activities in AML. We described a novel mechanism where elevated PRL-3 protein increases JMJD2C histone demethylase occupancy on Leo1 promoter, thereby reducing the H3K9me3 repressive signals and promoting Leo1 gene expression. Furthermore, PRL-3 and Leo1 levels were positively associated in AML patient samples (N=24; P<0.01). On the other hand, inhibition of Leo1 reverses PRL-3 oncogenic phenotypes in AML. Loss of Leo1 leads to destabilization of the PAF complex and downregulation of SOX2 and SOX4, potent oncogenes in myeloid transformation. In conclusion, we identify an important and novel mechanism by which PRL-3 mediates its oncogenic function in AML.
Asunto(s)
Carcinogénesis/genética , Leucemia Mieloide Aguda/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Oncogenes , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Carcinogénesis/metabolismo , Carcinogénesis/patología , Línea Celular Tumoral , Regulación hacia Abajo/genética , Femenino , Regulación Leucémica de la Expresión Génica , Células HEK293 , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Regiones Promotoras Genéticas , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción SOXC/genética , Factores de Transcripción SOXC/metabolismo , Regulación hacia ArribaRESUMEN
Multiple myeloma (MM) is characterized by recurrent chromosomal translocations. MMSET, identified by its fusion to the IgH locus in t(4;14) MM, is universally overexpressed in t(4;14) MM. In order to identify cell surface biomarkers associated with t(4;14) MM for small molecule or antibody based therapies, we knocked down MMSET expression with shRNA and generated a cell line pair from KMS11, a t(4;14) MM cell line. We used quantitative mass spectrometry to identify plasma membrane proteins associated with MMSET overexpression. Using this approach, 50 cell surface proteins were identified as differentially expressed between KMS11 and KMS11/shMMSET. Western blot and flow cytometry analysis indicated SLAMF7 was over-expressed in t(4;14) MM cell lines and down-regulated by MMSET shRNAs. SLAMF7 expression was also confirmed in primary t(4;14) MM samples by flow cytometry analysis. Quantitative RT-PCR and ChIP analysis indicated MMSET might regulate the transcription level of SLAMF7 and be an important functional element for SLAMF7 promoter activity. Furthermore, SLAMF7 shRNA could induce G1 arrest or apoptosis and reduce clonogenetic capacity in t(4;14) MM cells. Overall, these results illustrated SLAMF7 might be a novel cell surface protein associated with t(4;14) MM. It is potential to develop t(4;14) MM targeted therapy by SLAMF7 antibody mediated drug delivery.
Asunto(s)
Biomarcadores de Tumor/metabolismo , N-Metiltransferasa de Histona-Lisina/biosíntesis , Mieloma Múltiple/metabolismo , Proteínas Represoras/biosíntesis , Línea Celular Tumoral , Membrana Celular/genética , Membrana Celular/metabolismo , Cromosomas Humanos Par 14 , Cromosomas Humanos Par 4 , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Células HEK293 , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Mieloma Múltiple/genética , Proteómica/métodos , Receptores Inmunológicos/biosíntesis , Receptores Inmunológicos/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Transfección , Translocación GenéticaRESUMEN
BACKGROUND: Resistance to tyrosine kinase inhibitors (TKIs) remains a challenge in management of patients with chronic myeloid leukemia (CML). A better understanding of the BCR-ABL signalling network may lead to better therapy. FINDINGS: Here we report the discovery of a novel downstream target of BCR-ABL signalling, PRL-3 (PTP4A3), an oncogenic tyrosine phosphatase. Analysis of CML cancer cell lines and CML patient samples reveals the upregulation of PRL-3. Inhibition of BCR-ABL signalling either by Imatinib or by RNAi silencing BCR-ABL reduces PRL-3 and increases cleavage of PARP. In contrast, the amount of PRL-3 protein remains constant or even increased in response to Imatinib treatment in drug resistant cells expressing P210 T315I. Finally, analysis with specific shRNA shows PRL-3 involvement in the proliferation and self-renewal of CML cells. CONCLUSIONS: These data support a role for PRL-3 in BCR-ABL signalling and CML biology and may be a potential therapeutic target downstream of BCR-ABL in TKI resistant mutant cells.
Asunto(s)
Proteínas de Fusión bcr-abl/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Proteínas de Neoplasias/genética , Proteínas Tirosina Fosfatasas/genética , Antineoplásicos/farmacología , Benzamidas , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/genética , Resistencia a Antineoplásicos/genética , Expresión Génica , Regulación Leucémica de la Expresión Génica , Silenciador del Gen , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Metástasis de la Neoplasia/genética , Proteínas de Neoplasias/antagonistas & inhibidores , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Pirimidinas/farmacología , Factores de Transcripción STAT/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Recent studies have shown that 3-Deazaneplanocin A (DZNep), a histone methyltransferase inhibitor, disrupts polycomb-repressive complex 2 (PRC2), and preferentially induces apoptosis in cancer cells, including acute myeloid leukemia (AML). However, the underlying molecular mechanisms are not well understood. The present study demonstrates that DZNep induces robust apoptosis in AML cell lines, primary cells, and targets CD34(+)CD38(-) leukemia stem cell (LSC)-enriched subpopulations. Using RNA interference (RNAi), gene expression profiling, and ChIP, we identified that TXNIP, a major redox control molecule, plays a crucial role in DZNep-induced apoptosis. We show that disruption of PRC2, either by DZNep treatment or EZH2 knockdown, reactivates TXNIP, inhibits thioredoxin activity, and increases reactive oxygen species (ROS), leading to apoptosis. Furthermore, we show that TXNIP is down-regulated in AML and is a direct target of PRC2-mediated gene silencing. Consistent with the ROS accumulation on DZNep treatment, we also see a signature of endoplasmic reticulum (ER) stress-regulated genes, commonly associated with cell survival, down-regulated by DZNep. Taken together, we uncover a novel molecular mechanism of DZNep-mediated apoptosis and propose that EZH2 may be a potential new target for epigenetic treatment in AML.
Asunto(s)
Adenosina/análogos & derivados , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/metabolismo , Adenosina/farmacología , Animales , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proliferación Celular , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteína Potenciadora del Homólogo Zeste 2 , Epigenómica , Femenino , Perfilación de la Expresión Génica , Silenciador del Gen , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Análisis de Secuencia por Matrices de Oligonucleótidos , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Células Tumorales CultivadasRESUMEN
The 3-Deazaneplanocin A (DZNep), one of S-adenosylhomocysteine (AdoHcy) hydrolase inhibitors, has shown antitumor activities in a broad range of solid tumors and acute myeloid leukemia. Here, we examined its effects on multiple myeloma (MM) cells and found that, at 500 nM, it potently inhibited growth and induced apoptosis in 2 of 8 MM cell lines. RNA from un-treated and DZNep treated cells was profiled by Affymetrix HG-U133 Plus 2.0 microarray and genes with a significant change in gene expression were determined by significance analysis of microarray (SAM) testing. ALOX5 was the most down-regulated gene (5.8-fold) in sensitive cells and was expressed at low level in resistant cells. The results were corroborated by quantitative RT-PCR. Western-blot analysis indicated ALOX5 was highly expressed only in sensitive cell line H929 and greatly decreased upon DZNep treatment. Ectopic expression of ALOX5 reduced sensitivity to DZNep in H929 cells. Furthermore, down-regulation of ALOX5 by RNA interference could also induce apoptosis in H929. Gene expression analysis on MM patient dataset indicated ALOX5 expression was significantly higher in MM patients compared to normal plasma cells. We also found that Bcl-2 was overexpressed in DZNep insensitive cells, and cotreatment with DZNep and ABT-737, a Bcl-2 family inhibitor, synergistically inhibited growth and induced apoptosis of DZNep insensitive MM cells. Taken together, this study shows one of mechanisms of the DZNep efficacy on MM correlates with its ability to down-regulate the ALOX5 levels. In addition, DZNep insensitivity might be associated with overexpression of Bcl-2, and the combination of ABT-737 and DZNep could synergistically induced apoptosis. These results suggest that DZNep may be exploited therapeutically for a subset of MM.
Asunto(s)
Adenosina/análogos & derivados , Apoptosis/efectos de los fármacos , Mieloma Múltiple/patología , Adenosina/farmacología , Araquidonato 5-Lipooxigenasa/genética , Araquidonato 5-Lipooxigenasa/metabolismo , Compuestos de Bifenilo/farmacología , Caspasas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Proteína Potenciadora del Homólogo Zeste 2 , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Mieloma Múltiple/enzimología , Mieloma Múltiple/genética , Nitrofenoles/farmacología , Piperazinas/farmacología , Complejo Represivo Polycomb 2 , Sulfonamidas/farmacología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Combination with other small molecule drugs represents a promising strategy to improve therapeutic efficacy of FLT3 inhibitors in the clinic. We demonstrated that combining ABT-869, a FLT3 inhibitor, with SAHA, a HDAC inhibitor, led to synergistic killing of the AML cells with FLT3 mutations and suppression of colony formation. We identified a core gene signature that is uniquely induced by the combination treatment in 2 different leukemia cell lines. Among these, we showed that downregulation of PTP4A3 (PRL-3) played a role in this synergism. PRL-3 is downstream of FLT3 signaling and ectopic expression of PRL-3 conferred therapeutic resistance through upregulation of STAT (signal transducers and activators of transcription) pathway activity and anti-apoptotic Mcl-1 protein. PRL-3 interacts with HDAC4 and SAHA downregulates PRL-3 via a proteasome dependent pathway. In addition, PRL-3 protein was identified in 47% of AML cases, but was absent in myeloid cells in normal bone marrows. Our results suggest such combination therapies may significantly improve the therapeutic efficacy of FLT3 inhibitors. PRL-3 plays a potential pathological role in AML and it might be a useful therapeutic target in AML, and warrant clinical investigation.