RESUMO
The triple-negative breast cancer (TNBC) subtype is enriched in cancer stem cells (CSCs) and clinically correlated with the highest rate of recurrence. Several studies implicate the RSK pathway as being pivotal for the growth and proliferation of CSCs, which are postulated to drive tumor relapse. We now address the potential for the newly developed RSK inhibitor LJI308 to target the CSC population and repress TNBC growth and dissemination. Overexpression of the Y-box binding protein-1 (YB-1) oncogene in human mammary epithelial cells (HMECs) drove TNBC tumor formation characterized by a multi-drug resistance phenotype, yet these cells were sensitive to LJI308 in addition to the classic RSK inhibitors BI-D1870 and luteolin. Notably, LJI308 specifically targeted transformed cells as it had little effect on the non-tumorigenic parental HMECs. Loss of cell growth, both in 2D and 3D culture, was attributed to LJI308-induced apoptosis. We discovered CD44+/CD49f+ TNBC cells to be less sensitive to chemotherapy compared to the isogenic CD44-/CD49f- cells. However, inhibition of RSK using LJI308, BI-D1870, or luteolin was sufficient to eradicate the CSC population. We conclude that targeting RSK using specific and potent inhibitors, such as LJI308, delivers the promise of inhibiting the growth of TNBC.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Pteridinas/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Células-Tronco Neoplásicas/patologia , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
There is growing evidence that cancer-initiation could result from epigenetic changes. Y-box binding protein-1 (YB-1) is a transcription/translation factor that promotes the formation of tumors in transgenic mice; however, the underlying molecular events are not understood. To explore this in a human model system, YB-1 was expressed in mammary epithelial cells under the control of a tetracycline-inducible promoter. The induction of YB-1 promoted phenotypes associated with malignancy in three-dimensional breast acini cultures. This was attributed to YB-1 enhancing the expression and activity of the histone acetyltransferase p300 leading to chromatin remodeling. Specifically, this relaxation of chromatin allowed YB-1 to bind to the BMI1 promoter. The induction of BMI1 engaged the Polycomb complex resulting in histone H2A ubiquitylation and repression of the CDKN2A locus. These events manifested functionally as enhanced self-renewal capacity that occurred in a BMI1-dependent manner. Conversely, p300 inhibition with anacardic acid prevented YB-1 from binding to the BMI1 promoter and thereby subverted self-renewal. Despite these early changes, full malignant transformation was not achieved until RSK2 became overexpressed concomitant with elevated human telomerase reverse transcriptase (hTERT) activity. The YB-1/RSK2/hTERT expressing cells formed tumors in mice that were molecularly subtyped as basal-like breast cancer. We conclude that YB-1 cooperates with p300 to allow BMI1 to over-ride p16(INK4a) -mediated cell cycle arrest enabling self-renewal and the development of aggressive breast tumors.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Mama/patologia , Transformação Celular Neoplásica/metabolismo , Montagem e Desmontagem da Cromatina , Células Epiteliais/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo , Células Acinares/metabolismo , Células Acinares/patologia , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Reprogramação Celular/genética , Montagem e Desmontagem da Cromatina/genética , Proteína p300 Associada a E1A/metabolismo , Células Epiteliais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Transcrição Gênica , Regulação para Cima/genéticaRESUMO
BACKGROUND: Molecular subtyping has allowed for the beginning of personalized treatment in children suffering from medulloblastoma (MB). However, resistance inevitably emerges against these therapies, particularly in the Sonic Hedgehog (SHH) subtype. We found that children with SHH subtype have the worst outcome underscoring the need to identify new therapeutic targets. PROCEDURE: High content screening of a 129 compound library identified agents that inhibited SHH MB growth. Lead molecular target levels, p90 ribosomal S6 kinase (RSK) were characterized by immunoblotting and qRT-PCR. Comparisons were made to human neural stem cells (hNSC). Impact of inhibiting RSK with the small molecule BI-D1870 or siRNA was assessed in growth assays (monolayer, neurosphere, and soft agar). NanoString was used to detect RSK in a cohort of 66 patients with MB. To determine BI-D1870 pharmacokinetics/pharmacodynamics, 100 mg/kg was I.P. injected into mice and tissues were collected at various time points. RESULTS: Daoy, ONS76, UW228, and UW426 MB cells were exquisitely sensitive to BI-D1870 but unresponsive to SHH inhibitors. Anti-tumor growth corresponded with inactivation of RSK in MB cells. BI-D1870 had no effect on hNSCs. Inhibiting RSK with siRNA or BI-D1870 suppressed growth, induced apoptosis, and sensitized cells to SHH agents. Notably, RSK expression is correlated with SHH patients. In mice, BI-D1870 was well-tolerated and crossed the blood-brain barrier (BBB). CONCLUSIONS: RSK inhibitors are promising because they target RSK which is correlated with SHH patients as well as cause high levels of apoptosis to only MB cells. Importantly, BI-D1870 crosses the BBB, acting as a scaffold for development of more long-lived RSK inhibitors.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Cerebelares/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Meduloblastoma/genética , Pteridinas/farmacologia , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Animais , Antineoplásicos/farmacocinética , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Cerebelares/enzimologia , Criança , Cromatografia Líquida , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Proteínas Hedgehog/antagonistas & inibidores , Humanos , Immunoblotting , Masculino , Espectrometria de Massas , Meduloblastoma/enzimologia , Camundongos , Pteridinas/farmacocinética , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Distribuição Tecidual , Transcriptoma , TransfecçãoRESUMO
Medulloblastoma is the most common malignant brain tumor in children. This disease is heterogeneous and is composed of four subtypes of medulloblastoma [WNT, Sonic Hedgehog (SHH), Group 3, and Group 4]. An immediate goal is to identify novel molecular targets for the most aggressive forms of medulloblastoma. Polo-like kinase 1 (PLK1) is an oncogenic kinase that controls cell cycle and proliferation, making it a strong candidate for medulloblastoma treatment. In this study, pediatric medulloblastomas were subtyped in two patient cohorts (discovery cohort, n = 63 patients; validation cohort, n = 57 patients) using NanoString nCounter analysis and PLK1 mRNA was assessed. We determined that the SHH and Group 3 subtypes were independently associated with poor outcomes in children as was PLK1 using Cox regression analyses. Furthermore, we screened a library of 129 compounds in clinical trials using a model of pediatric medulloblastoma and determined that PLK1 inhibitors were the most promising class of agents against the growth of medulloblastoma. In patient-derived primary medulloblastoma isolates, the PLK1 small-molecule inhibitor BI2536 suppressed the self-renewal of cells with high PLK1 but not low PLK1 expression. PLK1 inhibition prevented medulloblastoma cell proliferation, self-renewal, cell-cycle progression, and induced apoptosis. In contrast, the growth of normal neural stem cells was unaffected by BI2536. Finally, BI2536 extended survival in medulloblastoma-bearing mice with efficacy comparable with Headstart, a standard-of-care chemotherapy regimen. We conclude that patients with medulloblastoma expressing high levels of PLK1 are at elevated risk. These preclinical studies pave the way for improving the treatment of medulloblastoma through PLK1 inhibition.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Proteínas de Ciclo Celular/antagonistas & inibidores , Meduloblastoma/tratamento farmacológico , Terapia de Alvo Molecular , Medicina de Precisão/métodos , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Pteridinas/uso terapêutico , Adolescente , Animais , Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Proteínas de Ciclo Celular/genética , Criança , Pré-Escolar , Estudos de Coortes , Humanos , Lactente , Meduloblastoma/genética , Meduloblastoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Fatores de Risco , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Quinase 1 Polo-LikeRESUMO
Glioblastomas (GBM) are associated with high rates of relapse. These brain tumors are often resistant to chemotherapies like temozolomide (TMZ) and there are very few treatment options available to patients. We recently reported that polo-like kinase-1 (PLK1) is associated with the proliferative subtype of GBM; which has the worst prognosis. In this study, we addressed the potential of repurposing disulfiram (DSF), a drug widely used to control alcoholism for the past six decades. DSF has good safety profiles and penetrates the blood-brain barrier. Here we report that DSF inhibited the growth of TMZ resistant GBM cells, (IC90=100 nM), but did not affect normal human astrocytes. At similar DSF concentrations, self-renewal was blocked by ~100% using neurosphere growth assays. Likewise the drug completely inhibited the self-renewal of the BT74 and GBM4 primary cell lines. Additionally, DSF suppressed growth and self-renewal of primary cells from two GBM tumors.These cells were resistant to TMZ, had unmethylated MGMT, and expressed high levels of PLK1. Consistent with its role in suppressing GBM growth, DSF inhibited the expression of PLK1 in GBM cells. Likewise, PLK1 inhibition with siRNA, or small molecules (BI-2536 or BI-6727) blocked growth of TMZ resistant cells. Our studies suggest that DSF has the potential to be repurposed for treatment of refractory GBM.