RESUMO
BACKGROUND: Medulloblastoma (MB) patients with MYC oncogene amplification or overexpression exhibit extremely poor prognoses and therapy resistance. However, MYC itself has been one of the most challenging targets for cancer treatment. Here, we identify a novel marinopyrrole natural derivative, MP1, that shows desirable anti-MYC and anti-cancer activities in MB. METHODS: In this study, using MYC-amplified (Group 3) and non-MYC amplified MB cell lines in vitro and in vivo, we evaluated anti-cancer efficacies and molecular mechanism(s) of MP1. RESULTS: MP1 significantly suppressed MB cell growth and sphere counts and induced G2 cell cycle arrest and apoptosis in a MYC-dependent manner. Mechanistically, MP1 strongly downregulated the expression of MYC protein. Our results with RNA-seq revealed that MP1 significantly modulated global gene expression and inhibited MYC-associated transcriptional targets including translation/mTOR targets. In addition, MP1 inhibited MYC-target metabolism, leading to declined energy levels. The combination of MP1 with an FDA-approved mTOR inhibitor temsirolimus synergistically inhibited MB cell growth/survival by downregulating the expression of MYC and mTOR signaling components. Our results further showed that as single agents, both MP1 and temsirolimus, were able to significantly inhibit tumor growth and MYC expression in subcutaneously or orthotopically MYC-amplified MB bearing mice. In combination, there were further anti-MB effects on the tumor growth and MYC expression in mice. CONCLUSION: These preclinical findings highlight the promise of marinopyrrole MP1 as a novel MYC inhibition approach for MYC-amplified MB.
Assuntos
Neoplasias Cerebelares , Meduloblastoma , Sirolimo/análogos & derivados , Humanos , Animais , Camundongos , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Pontos de Checagem da Fase G2 do Ciclo Celular , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/genética , Serina-Treonina Quinases TORRESUMO
Lung cancer is the leading cause of cancer deaths worldwide. We found that the cytochrome P450 isoform CYP4F11 is significantly overexpressed in patients with lung squamous cell carcinoma. CYP4F11 is a fatty acid ω-hydroxylase and catalyzes the production of the lipid mediator 20-hydroxyeicosatetraenoic acid (20-HETE) from arachidonic acid. 20-HETE promotes cell proliferation and migration in cancer. Inhibition of 20-HETE-generating cytochrome P450 enzymes has been implicated as novel cancer therapy for more than a decade. However, the exact role of CYP4F11 and its potential as drug target for lung cancer therapy has not been established yet. Thus, we performed a transient knockdown of CYP4F11 in the lung cancer cell line NCI-H460. Knockdown of CYP4F11 significantly inhibits lung cancer cell proliferation and migration while the 20-HETE production is significantly reduced. For biochemical characterization of CYP4F11-inhibitor interactions, we generated recombinant human CYP4F11. Spectroscopic ligand binding assays were conducted to evaluate CYP4F11 binding to the unselective CYP4A/F inhibitor HET0016. HET0016 shows high affinity to recombinant CYP4F11 and inhibits CYP4F11-mediated 20-HETE production in vitro with a nanomolar IC 50 Cross evaluation of HET0016 in NCI-H460 cells shows that lung cancer cell proliferation is significantly reduced together with 20-HETE production. However, HET0016 also displays antiproliferative effects that are not 20-HETE mediated. Future studies aim to establish the role of CYP4F11 in lung cancer and the underlying mechanism and investigate the potential of CYP4F11 as a therapeutic target for lung cancer. SIGNIFICANCE STATEMENT: Lung cancer is a deadly cancer with limited treatment options. Cytochrome P450 4F11 (CYP4F11) is significantly upregulated in lung squamous cell carcinoma. Knockdown of CYP4F11 in a lung cancer cell line significantly attenuates cell proliferation and migration with reduced production of the lipid mediator 20-hydroxyeicosatetraenoic acid (20-HETE). Studies with the unselective inhibitor HET0016 show a high inhibitory potency of CYP4F11-mediated 20-HETE production using recombinant enzyme. Overall, our studies demonstrate the potential of targeting CYP4F11 for new transformative lung cancer treatment.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Carcinoma de Células Escamosas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Ácidos Graxos , Sistema Enzimático do Citocromo P-450/metabolismo , Citocromo P-450 CYP4A , Eicosanoides , Ácidos Hidroxieicosatetraenoicos/metabolismo , Família 4 do Citocromo P450/genéticaRESUMO
Cancer is a heterogeneous disease, comprising of a mixture of different cell populations. Cancer stem cells (CSCs), also known as tumor-initiating cells (TICs), are a subpopulation of multipotent cells within the cancer that has self-renewing capability, tumor-initiating ability, multi-differentiation potential, and an inherent capacity for drug and chemoresistance. Sphere-formation assay is commonly used for enrichment and analysis of CSC properties in vitro and is typically used as a metric for testing the viability of tumor cells to anticancer agents. This model is based on the ability of CSCs to grow under ultralow-attachment conditions in serum-free medium supplemented with growth factors. In contrast to the adherent 2D culture of cancer cells, the 3D culture of tumorsphere assay exploits inherent biologic features of CSCs such as anoikis resistance and self-renewal. We describe here the detailed methodology for the generation and propagation of spheres generated from pediatric brain tumor medulloblastoma (MB) cells. As signal transducer and activator of transcription (STAT3) is known to play an important role in maintaining cancer stem cell properties, we accessed the effect of depleting or inhibiting STAT3 on MB-sphere sizes, numbers, and integrity. This may serve as a promising platform for screening potential anti-CSC agents and small-molecule inhibitors.
Assuntos
Neoplasias Encefálicas , Neoplasias Cerebelares , Meduloblastoma , Criança , Humanos , Meduloblastoma/patologia , Esferoides Celulares , Neoplasias Encefálicas/patologia , Neoplasias Cerebelares/metabolismo , Células-Tronco Neoplásicas/metabolismo , Linhagem Celular Tumoral , Proliferação de CélulasRESUMO
Aim: To codeliver an anticancer drug (doxorubicin) and siRNA in the form of nanoparticles into CD44-overexpressing colon cancer cells (HT-29) using an anionic, amphiphilic biopolymer comprising modified hyaluronic acid (6-O-[3-hexadecyloxy-2-hydroxypropyl]-hyaluronic acid). Materials & methods: Characterization of nanoparticles was performed using dynamic light scattering, scanning electron microscopy, transmission electron microscopy, molecular docking, in vitro drug release and gel mobility assays. Detailed in vitro experiments, including a gene silencing study and western blot, were also performed. Results: A 69% knockdown of the target gene was observed, and western blot showed 5.7-fold downregulation of the target protein. The repulsive forces between siRNA and 6-O-(3-hexadecyloxy-2-hydroxypropyl)-hyaluronic acid were overcome by hydrogen bonding and hydrophobic interactions. Conclusion: The authors successfully codelivered a drug and siRNA by anionic vector.
Mutations in genes are the main reason for the development of cancer. siRNA can selectively downregulate the key genes responsible for a particular cancer; however, this depends on successful delivery of siRNA into cancer cells. Usually, a negatively charged cell membrane repels negatively charged siRNA, which hinders siRNA entry into cancer cells. Usually, positively charged (cationic) polymers are used to mask siRNA for crossing the cancer cell membrane. However, cationic polymers are usually toxic to the body and show several adverse effects. By contrast, negatively charged polymers are safe alternatives for delivering siRNA into cancer cells. However, the main challenge of using anionic polymer lies behind the repulsive interaction between siRNA and negatively charged vectors, as both are negative. Herein we report successful codelivery of siRNA and an anticancer drug into cancer cells using a negatively charged polymer.
Assuntos
Ácido Hialurônico , Nanopartículas , RNA Interferente Pequeno/química , Ácido Hialurônico/química , Eletricidade Estática , Simulação de Acoplamento Molecular , Nanopartículas/química , Biopolímeros , Linhagem Celular TumoralRESUMO
MB is a common childhood malignancy of the central nervous system, with significant morbidity and mortality. Among the four molecular subgroups, MYC-amplified Group 3 MB is the most aggressive type and has the worst prognosis due to therapy resistance. The present study aimed to investigate the role of activated STAT3 in promoting MB pathogenesis and chemoresistance via inducing the cancer hallmark MYC oncogene. Targeting STAT3 function either by inducible genetic knockdown (KD) or with a clinically relevant small molecule inhibitor reduced tumorigenic attributes in MB cells, including survival, proliferation, anti-apoptosis, migration, stemness and expression of MYC and its targets. STAT3 inhibition attenuates MYC expression by affecting recruitment of histone acetyltransferase p300, thereby reducing enrichment of H3K27 acetylation in the MYC promoter. Concomitantly, it also decreases the occupancy of the bromodomain containing protein-4 (BRD4) and phosphoSer2-RNA Pol II (pSer2-RNAPol II) on MYC, resulting in reduced transcription. Importantly, inhibition of STAT3 signaling significantly attenuated MB tumor growth in subcutaneous and intracranial orthotopic xenografts, increased the sensitivity of MB tumors to cisplatin, and improved the survival of mice bearing high-risk MYC-amplified tumors. Together, the results of our study demonstrate that targeting STAT3 may be a promising adjuvant therapy and chemo-sensitizer to augment treatment efficacy, reduce therapy-related toxicity and improve quality of life in high-risk pediatric patients.
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BACKGROUND: Medulloblastoma (MB) patients with MYC oncogene amplification or overexpression exhibit extremely poor clinical outcomes and respond poorly to current therapies. Epigenetic deregulation is very common in MYC-driven MB. The bromodomain extra-terminal (BET) proteins and histone deacetylases (HDACs) are epigenetic regulators of MYC transcription and its associated tumorigenic programs. This study aimed to investigate the therapeutic potential of inhibiting the BET proteins and HDACs together in MB. METHODS: Using clinically relevant BET inhibitors (JQ1 or OTX015) and a pan-HDAC inhibitor (panobinostat), we evaluated the effects of combined inhibition on cell growth/survival in MYC-amplified MB cell lines and xenografts and examined underlying molecular mechanism(s). RESULTS: Co-treatment of JQ1 or OTX015 with panobinostat synergistically suppressed growth/survival of MYC-amplified MB cells by inducing G2 cell cycle arrest and apoptosis. Mechanistic investigation using RNA-seq revealed that co-treatment of JQ1 with panobinostat synergistically modulated global gene expression including MYC/HDAC targets. SYK and MSI1 oncogenes were among the top 50 genes synergistically downregulated by JQ1 and panobinostat. RT-PCR and western blot analyses confirmed that JQ1 and panobinostat synergistically inhibited the mRNA and protein expression of MSI1/SYK along with MYC expression. Reduced SYK/MSI expression after BET (specifically, BRD4) gene-knockdown further confirmed the epigenetic regulation of SYK and MSI1 genes. In addition, the combination of OTX015 and panobinostat significantly inhibited tumor growth in MYC-amplified MB xenografted mice by downregulating expression of MYC, compared to single-agent therapy. CONCLUSIONS: Together, our findings demonstrated that dual-inhibition of BET and HDAC proteins of the epigenetic pathway can be a novel therapeutic approach against MYC-driven MB.
Assuntos
Neoplasias Cerebelares , Meduloblastoma , Humanos , Camundongos , Animais , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Histona Desacetilases/metabolismo , Proteínas Nucleares/metabolismo , Panobinostat/farmacologia , Panobinostat/uso terapêutico , Azepinas/farmacologia , Epigênese Genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Fatores de Transcrição/metabolismo , Triazóis/farmacologia , Apoptose , Proliferação de Células , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
Pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive types of cancer, is characterized by aberrant activity of oncogenic KRAS. A nuclease-hypersensitive GC-rich region in KRAS promoter can fold into a four-stranded DNA secondary structure called G-quadruplex (G4), known to regulate KRAS expression. However, the factors that regulate stable G4 formation in the genome and KRAS expression in PDAC are largely unknown. Here, we show that APE1 (apurinic/apyrimidinic endonuclease 1), a multifunctional DNA repair enzyme, is a G4-binding protein, and loss of APE1 abrogates the formation of stable G4 structures in cells. Recombinant APE1 binds to KRAS promoter G4 structure with high affinity and promotes G4 folding in vitro. Knockdown of APE1 reduces MAZ transcription factor loading onto the KRAS promoter, thus reducing KRAS expression in PDAC cells. Moreover, downregulation of APE1 sensitizes PDAC cells to chemotherapeutic drugs in vitro and in vivo. We also demonstrate that PDAC patients' tissue samples have elevated levels of both APE1 and G4 DNA. Our findings unravel a critical role of APE1 in regulating stable G4 formation and KRAS expression in PDAC and highlight G4 structures as genomic features with potential application as a novel prognostic marker and therapeutic target in PDAC.
Assuntos
Carcinoma Ductal Pancreático , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Quadruplex G , Neoplasias Pancreáticas , Proteínas Proto-Oncogênicas p21(ras) , Carcinoma Ductal Pancreático/genética , DNA/química , Endonucleases/metabolismo , Humanos , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Neoplasias PancreáticasRESUMO
Genomic DNA in the nucleus is wrapped around nucleosomes, a repeating unit of chromatin. The nucleosome, consisting of octamer of core histones, is a barrier for several cellular processes that require access to the naked DNA. The FAcilitates Chromatin Transcription (FACT), a histone chaperone complex, is involved in nucleosome remodeling via eviction or assembly of histones during transcription, replication, and DNA repair. Increasing evidence suggests that FACT plays an important role in multiple DNA repair pathways including transcription-coupled nucleotide excision repair (TC-NER) of UV-induced damage, DNA single- and double-strand breaks (DSBs) repair, and base excision repair (BER) of oxidized or alkylated damaged bases. Further, studies have shown overexpression of FACT in multiple types of cancer and its association with drug resistance and patients' poor prognosis. In this review, we discuss how FACT is accumulated at the damage site and what functions it performs. We describe the known mechanisms by which FACT facilitates repair of different types of DNA damage. Further, we highlight the recent advances in a class of FACT inhibitors, called curaxins, which show promise as a new adjuvant therapy to sensitize multiple types of cancer to chemotherapy and radiation.
Assuntos
Montagem e Desmontagem da Cromatina , Reparo do DNA , Chaperonas de Histonas/metabolismo , Nucleossomos/metabolismo , Animais , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genéticaRESUMO
BACKGROUND: Neuroblastoma (NB) patients with MYCN amplification or overexpression respond poorly to current therapies and exhibit extremely poor clinical outcomes. PI3K-mTOR signaling-driven deregulation of protein synthesis is very common in NB and various other cancers that promote MYCN stabilization. In addition, both the MYCN and mTOR signaling axes can directly regulate a common translation pathway that leads to increased protein synthesis and cell proliferation. However, a strategy of concurrently targeting MYCN and mTOR signaling in NB remains unexplored. This study aimed to investigate the therapeutic potential of targeting dysregulated protein synthesis pathways by inhibiting the MYCN and mTOR pathways together in NB. METHODS: Using small molecule/pharmacologic approaches, we evaluated the effects of combined inhibition of MYCN transcription and mTOR signaling on NB cell growth/survival and associated molecular mechanism(s) in NB cell lines. We used two well-established BET (bromodomain extra-terminal) protein inhibitors (JQ1, OTX-015), and a clinically relevant mTOR inhibitor, temsirolimus, to target MYCN transcription and mTOR signaling, respectively. The single agent and combined efficacies of these inhibitors on NB cell growth, apoptosis, cell cycle and neurospheres were assessed using MTT, Annexin-V, propidium-iodide staining and sphere assays, respectively. Effects of inhibitors on global protein synthesis were quantified using a fluorescence-based (FamAzide)-based protein synthesis assay. Further, we investigated the specificities of these inhibitors in targeting the associated pathways/molecules using western blot analyses. RESULTS: Co-treatment of JQ1 or OTX-015 with temsirolimus synergistically suppressed NB cell growth/survival by inducing G1 cell cycle arrest and apoptosis with greatest efficacy in MYCN-amplified NB cells. Mechanistically, the co-treatment of JQ1 or OTX-015 with temsirolimus significantly downregulated the expression levels of phosphorylated 4EBP1/p70-S6K/eIF4E (mTOR components) and BRD4 (BET protein)/MYCN proteins. Further, this combination significantly inhibited global protein synthesis, compared to single agents. Our findings also demonstrated that both JQ1 and temsirolimus chemosensitized NB cells when tested in combination with cisplatin chemotherapy. CONCLUSIONS: Together, our findings demonstrate synergistic efficacy of JQ1 or OTX-015 and temsirolimus against MYCN-driven NB, by dual-inhibition of MYCN (targeting transcription) and mTOR (targeting translation). Additional preclinical evaluation is warranted to determine the clinical utility of targeted therapy for high-risk NB patients.
Assuntos
Acetanilidas/farmacologia , Azepinas/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Proteína Proto-Oncogênica N-Myc/antagonistas & inibidores , Neuroblastoma/tratamento farmacológico , Sirolimo/análogos & derivados , Serina-Treonina Quinases TOR/antagonistas & inibidores , Triazóis/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Regulação para Baixo , Sinergismo Farmacológico , Fator de Iniciação 4E em Eucariotos/efeitos dos fármacos , Fator de Iniciação 4E em Eucariotos/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Esferoides Celulares/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismoRESUMO
Medulloblastoma (MB) is a malignant pediatric brain tumor with a poor prognosis. Post-surgical radiation and cisplatin-based chemotherapy have been a mainstay of treatment, which often leads to substantial neurocognitive impairments and morbidity, highlighting the need for a novel therapeutic target to enhance the sensitivity of MB tumors to cytotoxic therapies. We performed a comprehensive study using a cohort of 71 MB patients' samples and pediatric MB cell lines and found that MB tumors have elevated levels of nucleosome remodeling FACT (FAcilitates Chromatin Transcription) complex and DNA repair enzyme AP-endonuclease1 (APE1). FACT interacts with APE1 and facilitates recruitment and acetylation of APE1 to promote repair of radiation and cisplatin-induced DNA damage. Further, levels of FACT and acetylated APE1 both are correlate strongly with MB patients' survival. Targeting FACT complex with CBL0137 inhibits DNA repair and alters expression of a subset of genes, and significantly improves the potency of cisplatin and radiation in vitro and in MB xenograft. Notably, combination of CBL0137 and cisplatin significantly suppressed MB tumor growth in an intracranial orthotopic xenograft model. We conclude that FACT complex promotes chemo-radiation resistance in MB, and FACT inhibitor CBL0137 can be used as a chemo-radiation sensitizer to augment treatment efficacy and reduce therapy-related toxicity in high-risk pediatric patients.
Assuntos
Cisplatino/administração & dosagem , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Proteínas de Ligação a DNA/genética , Proteínas de Grupo de Alta Mobilidade/genética , Meduloblastoma/tratamento farmacológico , Fatores de Elongação da Transcrição/genética , Adolescente , Adulto , Animais , Carbazóis/administração & dosagem , Carbazóis/efeitos adversos , Criança , Pré-Escolar , Cisplatino/efeitos adversos , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Xenoenxertos , Proteínas de Grupo de Alta Mobilidade/antagonistas & inibidores , Chaperonas de Histonas/genética , Humanos , Masculino , Meduloblastoma/genética , Meduloblastoma/patologia , Meduloblastoma/radioterapia , Camundongos , Fatores de Elongação da Transcrição/antagonistas & inibidores , Adulto JovemRESUMO
Medulloblastoma (MB) is the most common malignant central nervous system tumor in pediatric patients. Mainstay of therapy remains surgical resection followed by craniospinal radiation and chemotherapy, although limitations to this therapy are applied in the youngest patients. Clinically, tumors are divided into average and high-risk status on the basis of age, metastasis at diagnosis, and extent of surgical resection. However, technological advances in high-throughput screening have facilitated the analysis of large transcriptomic datasets that have been used to generate the current classification system, dividing patients into four primary subgroups, i.e., WNT (wingless), SHH (sonic hedgehog), and the non-SHH/WNT subgroups 3 and 4. Each subgroup can further be subdivided on the basis of a combination of cytogenetic and epigenetic events, some in distinct signaling pathways, that activate specific phenotypes impacting patient prognosis. Here, we delve deeper into the genetic basis for each subgroup by reviewing the extent of cytogenetic events in key genes that trigger neoplastic transformation or that exhibit oncogenic properties. Each of these discussions is further centered on how these genetic aberrations can be exploited to generate novel targeted therapeutics for each subgroup along with a discussion on challenges that are currently faced in generating said therapies. Our future hope is that through better understanding of subgroup-specific cytogenetic events, the field may improve diagnosis, prognosis, and treatment to improve overall quality of life for these patients.
RESUMO
The MYC oncogene is frequently amplified in patients with medulloblastoma, particularly in group 3 patients, who have the worst prognosis. mTOR signaling-driven deregulated protein synthesis is very common in various cancers, including medulloblastoma, that can promote MYC stabilization. As a transcription factor, MYC itself is further known to regulate transcription of several components of protein synthesis machinery, leading to an enhanced protein synthesis rate and proliferation. Thus, inhibiting enhanced protein synthesis by targeting the MYC and mTOR pathways together may represent a highly relevant strategy for the treatment of MYC-driven medulloblastoma. Here, using siRNA and small-molecule inhibitor approaches, we evaluated the effects of combined inhibition of MYC transcription and mTOR signaling on medulloblastoma cell growth/survival and associated molecular mechanism(s) in MYC-amplified (group 3) medulloblastoma cell lines and xenografts. Combined inhibition of MYC and mTOR synergistically suppressed medulloblastoma cell growth and induced G1 cell-cycle arrest and apoptosis. Mechanistically, the combined inhibition significantly downregulated the expression levels of key target proteins of MYC and mTOR signaling. Our results with RNA-sequencing revealed that combined inhibition synergistically modulated global gene expression including MYC/mTOR components. In addition, the combination treatment significantly delayed tumor growth and prolonged survival of MYC-amplified medulloblastoma xenografted mice by downregulating expression of MYC and the key downstream components of mTOR signaling, compared with single-agent therapy. Together, our findings demonstrated that dual inhibition of MYC (transcription) and mTOR (translation) of the protein synthesis pathway can be a novel therapeutic approach against MYC-driven medulloblastoma.
Assuntos
Azepinas/farmacologia , Neoplasias Cerebelares/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Imidazóis/farmacologia , Meduloblastoma/tratamento farmacológico , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Quinolinas/farmacologia , Triazóis/farmacologia , Animais , Antineoplásicos/farmacologia , Apoptose , Ciclo Celular , Proliferação de Células , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Feminino , Humanos , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Proto-Oncogênicas c-myc/genética , Serina-Treonina Quinases TOR/antagonistas & inibidores , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Fluorouracil (5-FU) remains a first-line chemotherapeutic agent for colorectal cancer. However, a subset of colorectal cancer patients who have defective mismatch-repair (dMMR) pathway show resistance to 5-FU. Here, we demonstrate that the efficacy of 5-FU in dMMR colorectal cancer cells is largely dependent on the DNA base excision repair (BER) pathway. Downregulation of APE1, a key enzyme in the BER pathway, decreases IC50 of 5-FU in dMMR colorectal cancer cells by 10-fold. Furthermore, we discover that the facilitates chromatin transcription (FACT) complex facilitates 5-FU repair in DNA via promoting the recruitment and acetylation of APE1 (AcAPE1) to damage sites in chromatin. Downregulation of FACT affects 5-FU damage repair in DNA and sensitizes dMMR colorectal cancer cells to 5-FU. Targeting the FACT complex with curaxins, a class of small molecules, significantly improves the 5-FU efficacy in dMMR colorectal cancer in vitro (â¼50-fold decrease in IC50) and in vivo xenograft models. We show that primary tumor tissues of colorectal cancer patients have higher FACT and AcAPE1 levels compared with adjacent nontumor tissues. Additionally, there is a strong clinical correlation of FACT and AcAPE1 levels with colorectal cancer patients' response to chemotherapy. Together, our study demonstrates that targeting FACT with curaxins is a promising strategy to overcome 5-FU resistance in dMMR colorectal cancer patients.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carbazóis/farmacologia , Neoplasias do Colo/tratamento farmacológico , Proteínas de Ligação a DNA/antagonistas & inibidores , Fluoruracila/farmacologia , Proteínas de Grupo de Alta Mobilidade/antagonistas & inibidores , Fatores de Elongação da Transcrição/antagonistas & inibidores , Animais , Carbazóis/administração & dosagem , Cromatina/metabolismo , Neoplasias do Colo/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fluoruracila/administração & dosagem , Células HCT116 , Células HEK293 , Proteínas de Grupo de Alta Mobilidade/metabolismo , Chaperonas de Histonas/antagonistas & inibidores , Chaperonas de Histonas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Fatores de Elongação da Transcrição/metabolismo , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: MYC amplification or overexpression is common in Group 3 medulloblastoma and is associated with the worst prognosis. Recently, protein arginine methyl transferase (PRMT) 5 expression has been closely associated with aberrant MYC function in various cancers, including brain tumors such as glioblastoma. However, the role of PRMT5 and its association with MYC in medulloblastoma have not been explored. Here, we report the role of PRMT5 as a novel regulator of MYC and implicate PRMT5 as a potential therapeutic target in MYC-driven medulloblastoma. METHODS: Expression and association between PRMT5 and MYC in primary medulloblastoma tumors were investigated using publicly available databases. Expression levels of PRMT5 protein were also examined using medulloblastoma cell lines and primary tumors by western blotting and immunohistochemistry, respectively. Using MYC-driven medulloblastoma cells, we examined the physical interaction between PRMT5 and MYC by co-immunoprecipitation and co-localization experiments. To determine the functional role of PRMT5 in MYC-driven medulloblastoma, PRMT5 was knocked-down in MYC-amplified cells using siRNA and the consequences of knockdown on cell growth and MYC expression/stability were investigated. In vitro therapeutic potential of PRMT5 in medulloblastoma was also evaluated using a small molecule inhibitor, EPZ015666. RESULTS: We observed overexpression of PRMT5 in MYC-driven primary medulloblastoma tumors and cell lines compared to non-MYC medulloblastoma tumors and adjacent normal tissues. We also found that high expression of PRMT5 is inversely correlated with patient survival. Knockdown of PRMT5 using siRNA in MYC-driven medulloblastoma cells significantly decreased cell growth and MYC expression. Mechanistically, we found that PRMT5 physically associated with MYC by direct protein-protein interaction. In addition, a cycloheximide chase experiment showed that PRMT5 post-translationally regulated MYC stability. In the context of therapeutics, we observed dose-dependent efficacy of PRMT5 inhibitor EPZ015666 in suppressing cell growth and inducing apoptosis in MYC-driven medulloblastoma cells. Further, the expression levels of PRMT5 and MYC protein were downregulated upon EPZ015666 treatment. We also observed a superior efficacy of this inhibitor against MYC-amplified medulloblastoma cells compared to non-MYC-amplified medulloblastoma cells, indicating specificity. CONCLUSION: Our results reveal the regulation of MYC oncoprotein by PRMT5 and suggest that targeting PRMT5 could be a potential therapeutic strategy for MYC-driven medulloblastoma.
Assuntos
Neoplasias Cerebelares/metabolismo , Meduloblastoma/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/genética , Humanos , Isoquinolinas/farmacologia , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Ligação Proteica , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/genética , Proteínas Proto-Oncogênicas c-myc/genética , Pirimidinas/farmacologia , Interferência de RNA , Análise de SobrevidaRESUMO
Medulloblastoma (MB) is a highly aggressive, malignant brain tumor in children with poor prognosis. Cyclin-dependent kinase 9 (CDK9), a serine-threonine kinase, is widely implicated in the control of basal gene expression by phosphorylating Serine 2 (Ser2) of the heptad repeat in the RNA Polymerase II (RNA Pol II) C-terminal domain (CTD). Although CDK9 plays a pathogenic role in various cancers, its function in MB remains unknown. Here, we show that CDK9 is highly expressed in MB tumors and increased CDK9 expression is correlated with high risk MB patients. CDK9 expression along with phospho-Ser2 RNA Pol II (pRNA Pol II ser2) and bromodomain-binding protein 4 (BRD4), which recruits CDK9, were elevated in multiple MB cell lines and in MB tumors originated spontaneously from Ptch1+/-p53-/- mice. Inhibition of CDK9 with LDC067 suppressed MB cell growth, reduced pRNA Pol II ser2 level and expression of oncogenic markers, including MYC. Moreover, LDC067 treatment synergistically sensitizes MB cells to chemotherapeutic agent cisplatin. Further, LDC067 in combination with BRD4 inhibitor decreased MB cells growth, delayed cell migration and attenuated pRNA Pol II ser2 occupancy to CCND1 and BCL2 gene promoters as revealed by chromatin immunoprecipitation assay (ChIP). Together, these findings highlight the importance of CDK9 in MB pathogenesis and suggest that it may serve as a promising therapeutic target for the treatment of MB.
Assuntos
Neoplasias Cerebelares/tratamento farmacológico , Quinase 9 Dependente de Ciclina/metabolismo , Meduloblastoma/tratamento farmacológico , Pirimidinas/farmacologia , Sulfonamidas/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Cisplatino/administração & dosagem , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Humanos , Meduloblastoma/genética , Meduloblastoma/patologia , Camundongos Mutantes , Terapia de Alvo Molecular , Neoplasias Experimentais , Pirimidinas/administração & dosagem , RNA Polimerase II/metabolismo , Serina/metabolismo , Sulfonamidas/administração & dosagem , Fatores de Transcrição/metabolismoRESUMO
Aberrant activation and interactions of hedgehog (HH) and PI3K/AKT/mTOR signaling pathways are frequently associated with high-risk medulloblastoma (MB). Thus, combined targeting of the HH and PI3K/AKT/mTOR pathways could be a viable therapeutic strategy to treat high-risk patients. Therefore, we investigated the anti-MB efficacies of combined HH inhibitor Vismodegib and PI3K-mTOR dual-inhibitor BEZ235 together or combined individually with cisplatin against high-risk MB. Using non-MYC- and MYC-amplified cell lines, and a xenograft mouse model, the in vitro and in vivo efficacies of these therapies on cell growth/survival and associated molecular mechanism(s) were investigated. Results showed that combined treatment of Vismodegib and BEZ235 together, or with cisplatin, significantly decreased MB cell growth/survival in a dose-dependent-fashion. Corresponding changes in the expression of targeted molecules following therapy were observed. Results demonstrated that inhibitors not only suppressed MB cell growth/survival when combined, but also significantly enhanced cisplatin-mediated cytotoxicity. Of these combinations, BEZ235 exhibited a significantly greater efficacy in enhancing cisplatin-mediated MB cytotoxicity. Results also demonstrated that the MYC-amplified MB lines showed a higher sensitivity to combined therapies compared to non-MYC-amplified cell lines. Therefore, we tested the efficacy of combined approaches against MYC-amplified MB growing in NSG mice. In vivo results showed that combination of Vismodegib and BEZ235 or their combination with cisplatin, significantly delayed MB tumor growth and increased survival of xenografted mice by targeting HH and mTOR pathways. Thus, our studies lay a foundation for translating these combined therapeutic strategies to the clinical setting to determine their efficacies in high-risk MB patients.
RESUMO
Medulloblastoma (MB) is a malignant pediatric brain tumor with poor prognosis. Signal transducers and activators of transcription-3 (STAT3) is constitutively activated in MB where it functions as an oncoprotein, mediating cancer progression and metastasis. Here, we have delineated the functional role of activated STAT3 in MB, by using a cell permeable STAT3-NH2 terminal domain inhibitor (S3-NTDi) that specifically perturbs the structure/function of STAT3. We have implemented several biochemical experiments using human MB tumor microarray (TMA) and pediatric MB cell lines, derived from high-risk SHH-TP53-mutated and MYC-amplified Non-WNT/SHH tumors. Treatment of MB cells with S3-NTDi leads to growth inhibition, cell cycle arrest, and apoptosis. S3-NTDi downregulated expression of STAT3 target genes, delayed migration of MB cells, attenuated epithelial-mesenchymal transition (EMT) marker expressions and reduced cancer stem-cell associated protein expressions in MB-spheres. To elucidate mechanisms, we showed that S3-NTDi induce expression of pro-apoptotic gene, C/EBP-homologous protein (CHOP), and decrease association of STAT3 to the proximal promoter of CCND1 and BCL2. Of note, S3-NTDi downregulated microRNA-21, which in turn, de-repressed Protein Inhibitor of Activated STAT3 (PIAS3), a negative regulator of STAT3 signaling pathway. Furthermore, combination therapy with S3-NTDi and cisplatin significantly decreased highly aggressive MYC-amplified MB cell growth and induced apoptosis by downregulating STAT3 regulated proliferation and anti-apoptotic gene expression. Together, our results revealed an important role of STAT3 in regulating MB pathogenesis. Disruption of this pathway with S3-NTDi, therefore, may serves as a promising candidate for targeted MB therapy by enhancing chemosensitivity of MB cells and potentially improving outcomes in high-risk patients.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , MicroRNAs/genética , Chaperonas Moleculares/genética , Peptídeos/farmacologia , Proteínas Inibidoras de STAT Ativados/genética , Fator de Transcrição STAT3/genética , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cisplatino/farmacologia , Humanos , Meduloblastoma/genética , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Chaperonas Moleculares/metabolismo , Peptídeos/síntese química , Proteínas Inibidoras de STAT Ativados/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismoRESUMO
CYP4Z1 is an "orphan" cytochrome P450 (P450) enzyme that has provoked interest because of its hypothesized role in breast cancer through formation of the signaling molecule 20-hydroxyeicosatetraenoic acid (20-HETE). We expressed human CYP4Z1 in Saccharomyces cerevisiae and evaluated its catalytic capabilities toward arachidonic and lauric acids (AA and LA). Specific and sensitive mass spectrometry assays enabled discrimination of the regioselectivity of hydroxylation of these two fatty acids. CYP4Z1 generated 7-, 8-, 9-, 10-, and 11-hydroxy LA, whereas the 12-hydroxy metabolite was not detected. HET0016, the prototypic CYP4 inhibitor, only weakly inhibited laurate metabolite formation (IC50 â¼15 µM). CYP4Z1 preferentially oxidized AA to the 14(S),15(R)-epoxide with high regioselectivity and stereoselectivity, a reaction that was also insensitive to HET0016, but neither 20-HETE nor 20-carboxy-AA were detectable metabolites. Docking of LA and AA into a CYP4Z1 homology model was consistent with this preference for internal fatty acid oxidation. Thus, human CYP4Z1 has an inhibitor profile and product regioselectivity distinct from most other CYP4 enzymes, consistent with CYP4Z1's lack of a covalently linked heme. These data suggest that, if CYP4Z1 modulates breast cancer progression, it does so by a mechanism other than direct production of 20-HETE.
Assuntos
Neoplasias da Mama/metabolismo , Família 4 do Citocromo P450/metabolismo , Ácidos Hidroxieicosatetraenoicos/metabolismo , Ácidos Láuricos/metabolismo , Amidinas/farmacologia , Família 4 do Citocromo P450/antagonistas & inibidores , Família 4 do Citocromo P450/química , Família 4 do Citocromo P450/isolamento & purificação , Progressão da Doença , Humanos , Hidroxilação/efeitos dos fármacos , Quinases Associadas a Receptores de Interleucina-1 , Espectrometria de Massas , Microssomos Hepáticos , Simulação de Acoplamento Molecular , Oxirredução/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiaeRESUMO
Apurinic/apyrimidinic (AP) sites are frequently generated in the genome by spontaneous depurination/depyrimidination or after removal of oxidized/modified bases by DNA glycosylases during the base excision repair (BER) pathway. Unrepaired AP sites are mutagenic and block DNA replication and transcription. The primary enzyme to repair AP sites in mammalian cells is AP endonuclease (APE1), which plays a key role in this repair pathway. Although overexpression of APE1 in diverse cancer types and its association with chemotherapeutic resistance are well documented, alteration of posttranslational modification of APE1 and modulation of its functions during tumorigenesis are largely unknown. Here, we show that both classical histone deacetylase HDAC1 and NAD+-dependent deacetylase SIRT1 regulate acetylation level of APE1 and acetylation of APE1 enhances its AP-endonuclease activity both in vitro and in cells. Modulation of APE1 acetylation level in cells alters AP site repair capacity of the cell extracts in vitro. Primary tumor tissues of diverse cancer types have higher level of acetylated APE1 (AcAPE1) compared to adjacent non-tumor tissue and exhibit enhanced AP site repair capacity. Importantly, in the absence of APE1 acetylation, cells accumulate AP sites in the genome and show increased sensitivity to DNA damaging agents. Together, our study demonstrates that elevation of acetylation level of APE1 in tumor could be a novel mechanism by which cells handle the elevated levels of DNA damages in response to genotoxic stress and maintain sustained proliferation.
Assuntos
Dano ao DNA , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Acetilação , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Histona Desacetilase 1/metabolismo , Humanos , NAD/metabolismo , Neoplasias/patologia , Ligação Proteica , Sirtuína 1/metabolismoRESUMO
BACKGROUND: The ribosome, which acts as a platform for mRNA encoded polypeptide synthesis, is also capable of assisting in folding of polypeptide chains. The peptidyl transferase center (PTC) that catalyzes peptide bond formation resides in the domain V of the 23S rRNA of the bacterial ribosome. Proper positioning of the 3' -CCA ends of the A- and P-site tRNAs via specific interactions with the nucleotides of the PTC are crucial for peptidyl transferase activity. This RNA domain is also the center for ribosomal chaperoning activity. The unfolded polypeptide chains interact with the specific nucleotides of the PTC and are released in a folding competent form. In vitro transcribed RNA corresponding to this domain (bDV RNA) also displays chaperoning activity. RESULTS: The present study explores the effects of tRNAs, antibiotics that are A- and P-site PTC substrate analogs (puromycin and blasticidin) and macrolide antibiotics (erythromycin and josamycin) on the chaperoning ability of the E. coli ribosome and bDV RNA. Our studies using mRNA programmed ribosomes show that a tRNA positioned at the P-site effectively inhibits the ribosome's chaperoning function. We also show that the antibiotic blasticidin (that mimics the interaction between 3'-CCA end of P/P-site tRNA with the PTC) is more effective in inhibiting ribosome and bDV RNA chaperoning ability than either puromycin or the macrolide antibiotics. Mutational studies of the bDV RNA could identify the nucleotides U2585 and G2252 (both of which interact with P-site tRNA) to be important for its chaperoning ability. CONCLUSION: Both protein synthesis and their proper folding are crucial for maintenance of a functional cellular proteome. The PTC of the ribosome is attributed with both these abilities. The silencing of the chaperoning ability of the ribosome in the presence of P-site bound tRNA might be a way to segregate these two important functions.