RESUMO
Approximately 50%-55% of high-grade serous ovarian carcinoma (HGSOC) patients have MYC oncogenic pathway activation. Because MYC is not directly targetable, we have analyzed molecular pathways enriched in MYC-high HGSOC tumors to identify potential therapeutic targets. Here, we report that MYC-high HGSOC tumors show enrichment in genes controlled by NRF2, an antioxidant signaling pathway, along with increased thioredoxin redox activity. Treatment of MYC-high HGSOC tumors cells with US Food and Drug Administration (FDA)-approved thioredoxin reductase 1 (TrxR1) inhibitor auranofin resulted in significant growth suppression and apoptosis in MYC-high HGSOC cells in vitro and also significantly reduced tumor growth in an MYC-high HGSOC patient-derived tumor xenograft. We found that auranofin treatment inhibited glycolysis in MYC-high cells via oxidation-induced GAPDH inhibition. Interestingly, in response to auranofin-induced glycolysis inhibition, MYC-high HGSOC cells switched to glutamine metabolism for survival. Depletion of glutamine with either glutamine starvation or glutaminase (GLS1) inhibitor CB-839 exerted synergistic anti-tumor activity with auranofin in HGSOC cells and OVCAR-8 cell line xenograft. These findings suggest that applying a combined therapy of GLS1 inhibitor and TrxR1 inhibitor could effectively treat MYC-high HGSOC patients.
Assuntos
Auranofina , Genes myc , Glutamina , Neoplasias Ovarianas , Tiorredoxina Dissulfeto Redutase , Feminino , Humanos , Auranofina/farmacologia , Auranofina/uso terapêutico , Linhagem Celular Tumoral , Genes myc/genética , Glutaminase/genética , Glutaminase/metabolismo , Glutamina/genética , Glutamina/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/antagonistas & inibidores , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMO
Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer lacking targeted therapy. Here, we evaluated the anti-cancer activity of APR-246, a P53 activator, and CX-5461, a RNA polymerase I inhibitor, in the treatment of TNBC cells. We tested the efficacy of individual and combination therapy of CX-5461 and APR-246 in vitro, using a panel of breast cancer cell lines. Using publicly available breast cancer datasets, we found that components of RNA Pol I are predominately upregulated in basal-like breast cancer, compared to other subtypes, and this upregulation is associated with poor overall and relapse-free survival. Notably, we found that the treatment of breast cancer cells lines with CX-5461 significantly hampered cell proliferation and synergistically enhanced the efficacy of APR-246. The combination treatment significantly induced apoptosis that is associated with cleaved PARP and Caspase 3 along with Annexin V positivity. Likewise, we also found that combination treatment significantly induced DNA damage and replication stress in these cells. Our data provide a novel combination strategy by utilizing APR-246 in combination CX-5461 in killing TNBC cells that can be further developed into more effective therapy in TNBC therapeutic armamentarium.
Assuntos
Benzotiazóis/farmacologia , Dano ao DNA , Replicação do DNA/efeitos dos fármacos , Naftiridinas/farmacologia , Quinuclidinas/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Replicação do DNA/genética , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , RNA Polimerase I/antagonistas & inibidores , RNA Polimerase I/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Triple-negative breast cancer (TNBCs) is a very aggressive and lethal form of breast cancer with no effective targeted therapy. Neoadjuvant chemotherapies and radiotherapy remains a mainstay of treatment with only 25-30% of TNBC patients responding. Thus, there is an unmet clinical need to develop novel therapeutic strategies for TNBCs. TNBC cells have increased intracellular oxidative stress and suppressed glutathione, a major antioxidant system, but still, are protected against higher oxidative stress. We screened a panel of antioxidant genes using the TCGA and METABRIC databases and found that expression of the thioredoxin pathway genes is significantly upregulated in TNBC patients compared to non-TNBC patients and is correlated with adverse survival outcomes. Treatment with auranofin (AF), an FDA-approved thioredoxin reductase inhibitor caused specific cell death and impaired the growth of TNBC cells grown as spheroids. Furthermore, AF treatment exerted a significant in vivo antitumor activity in multiple TNBC models including the syngeneic 4T1.2 model, MDA-MB-231 xenograft and patient-derived tumor xenograft by inhibiting thioredoxin redox activity. We, for the first time, showed that AF increased CD8+Ve T-cell tumor infiltration in vivo and upregulated immune checkpoint PD-L1 expression in an ERK1/2-MYC-dependent manner. Moreover, combination of AF with anti-PD-L1 antibody synergistically impaired the growth of 4T1.2 primary tumors. Our data provide a novel therapeutic strategy using AF in combination with anti-PD-L1 antibody that warrants further clinical investigation for TNBC patients.
Assuntos
Anticorpos/uso terapêutico , Auranofina/uso terapêutico , Antígeno B7-H1/imunologia , Inibidores Enzimáticos/uso terapêutico , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Auranofina/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Espécies Reativas de Oxigênio/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The DJ-1 protein was originally linked with Parkinson's disease and is now known to have antioxidant functions. The protein has three redox-sensitive cysteine residues, which are involved in its dimerisation and functional properties. A mildly oxidised form of DJ-1 is the most active form and protects cells from oxidative stress conditions. DJ-1 functions as an antioxidant through a variety of mechanisms, including a weak direct antioxidant activity by scavenging reactive oxygen species. DJ-1 also regulates a number of signalling pathways, including the inhibition of apoptosis signal-regulating kinase 1 (ASK1)-induced apoptosis under oxidative stress conditions. Other proteins regulated by DJ-1 include enzymes, chaperones, the 20S proteasome and transcription factors, including Nrf2. Once activated by oxidative stress, Nrf2 upregulates antioxidant gene expression including members of the thioredoxin and glutathione pathways, which in turn mediate an antioxidant protective function. Crosstalk between DJ-1 and both the thioredoxin and glutathione systems has also been identified. Thioredoxin reduces a cysteine residue on DJ-1 to modulate its activity, while glutaredoxin1 de-glutathionylates DJ-1, preventing degradation of DJ-1 and resulting in its accumulation. DJ-1 also regulates the activity of glutamate cysteine ligase, which is the rate-limiting step for glutathione synthesis. These antioxidant functions of DJ-1 are key to its role in protecting neurons from oxidative stress and are hypothesised to protect the brain from the development of neurodegenerative diseases such as Parkinson's disease (PD) and to protect cardiac tissues from ischaemic-reperfusion injury. However, DJ-1, as an antioxidant, also protects cancer cells from undergoing oxidative stress-induced apoptosis.
Assuntos
Antioxidantes/metabolismo , Glutationa/metabolismo , Proteína Desglicase DJ-1/metabolismo , Tiorredoxinas/metabolismo , Animais , Humanos , MAP Quinase Quinase Quinase 5/metabolismo , Modelos Biológicos , Fator 2 Relacionado a NF-E2/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Multiple myeloma (MM) is an incurable plasma B cell malignancy. Despite recent advancements in anti-MM therapies, development of drug resistance remains a major clinical hurdle. DJ-1, a Parkinson's disease-associated protein, is upregulated in many cancers and its knockdown suppresses tumor growth and overcomes chemoresistance. However, the role of DJ-1 in MM remains unknown. Using gene expression databases we found increased DJ-1 expression in MM patient cells, which correlated with shorter overall survival and poor prognosis in MM patients. Targeted DJ-1 knockdown using siRNAs induced necroptosis in myeloma cells. We found that Krüppel-like factor 6 (KLF6) is expressed at lower levels in myeloma cells compared to PBMCs, and DJ-1 knockdown increased KLF6 expression in myeloma cells. Targeted knockdown of KLF6 expression in DJ-1 knockdown myeloma cells rescued these cells from undergoing cell death. Higher DJ-1 levels were observed in bortezomib-resistant myeloma cells compared to parent cells, and siRNA-mediated DJ-1 knockdown reversed bortezomib resistance. DJ-1 knockdown increased KLF6 expression in bortezomib-resistant myeloma cells, and subsequent siRNA-mediated KLF6 knockdown rescued bortezomib-resistant myeloma cells from undergoing cell death. We also demonstrated that specific siRNA-mediated DJ-1 knockdown reduced myeloma cell growth under a hypoxic microenvironment. DJ-1 knockdown reduced the expression of HIF-1α and its target genes in hypoxic-myeloma cells, and overcame hypoxia-induced bortezomib resistance. Our findings demonstrate that elevated DJ-1 levels correlate with myeloma cell survival and acquisition of bortezomib resistance. Thus, we propose that inhibiting DJ-1 may be an effective therapeutic strategy to treat newly diagnosed as well as relapsed/refractory MM patients.
Assuntos
Apoptose , Fator 6 Semelhante a Kruppel/genética , Mieloma Múltiplo/fisiopatologia , Antineoplásicos/farmacologia , Bortezomib/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Fator 6 Semelhante a Kruppel/metabolismo , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Proteína Desglicase DJ-1/metabolismo , Regulação para CimaRESUMO
Although Clostridium novyi-NT is an anti-cancer bacterial therapeutic which germinates within hypoxic tumors to kill cancer cells, the actual germination triggers for C. novyi-NT are still unknown. In this study, we screen candidate germinants using combinatorial experimental designs and discover by serendipity that D-valine is a potent germinant, inducing 50% spore germination at 4.2 mM concentration. Further investigation revealed that five D-valine analogs are also germinants and four of these analogs are enantiomeric pairs. This stereoflexible effect of L- and D-amino acids shows that spore germination is a complex process where enantiomeric interactions can be confounders. This study also identifies L-cysteine as a germinant, and hypoxanthine and inosine as co-germinants. Several other amino acids promote (L-valine, L-histidine, L-threonine and L-alanine) or inhibit (L-arginine, L-glycine, L-lysine, L-tryptophan) germination in an interaction-dependent manner. D-alanine inhibits all germination, even in complex growth media. This work lays the foundation for improving the germination efficacy of C. novyi-NT spores in tumors.
Assuntos
Esporos Bacterianos , Valina , Valina/metabolismo , Valina/farmacologia , Esporos Bacterianos/metabolismo , Aminoácidos/metabolismo , Alanina , Esporos/metabolismoRESUMO
A common problem in the study of human malignancy is the elucidation of cancer driver mechanisms associated with recurrent deletion of regions containing multiple genes. Taking B-cell acute lymphoblastic leukaemia (B-ALL) and large deletions of 6q [del(6q)] as a model, we integrated analysis of functional cDNA clone tracking assays with patient genomic and transcriptomic data, to identify the transcription factors FOXO3 and PRDM1 as candidate tumour suppressor genes (TSG). Analysis of cell cycle and transcriptomic changes following overexpression of FOXO3 or PRDM1 indicated that they co-operate to promote cell cycle exit at the pre-B cell stage. FOXO1 abnormalities are absent in B-ALL, but like FOXO3, FOXO1 expression suppressed growth of TCF3::PBX1 and ETV6::RUNX1 B-ALL in-vitro. While both FOXOs induced PRDM1 and other genes contributing to late pre-B cell development, FOXO1 alone induced the key transcription factor, IRF4, and chemokine, CXCR4. CRISPR-Cas9 screening identified FOXO3 as a TSG, while FOXO1 emerged as essential for B-ALL growth. We relate this FOXO3-specific leukaemia-protective role to suppression of glycolysis based on integrated analysis of CRISPR-data and gene sets induced or suppressed by FOXO1 and FOXO3. Pan-FOXO agonist Selinexor induced the glycolysis inhibitor TXNIP and suppressed B-ALL growth at low dose (ID50 < 50 nM).
Assuntos
Fatores de Transcrição Forkhead , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Fatores de Transcrição Forkhead/metabolismo , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Cromossomos Humanos Par 6/metabolismo , Regulação da Expressão Gênica , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Fator 1 de Ligação ao Domínio I Regulador Positivo/genéticaRESUMO
BACKGROUND: Metastatic cancer cells exploit Epithelial-mesenchymal-transition (EMT) to enhance their migration, invasion, and resistance to treatments. Recent studies highlight that elevated levels of copper are implicated in cancer progression and metastasis. Clinical trials using copper chelators are associated with improved patient survival; however, the molecular mechanisms by which copper depletion inhibits tumor progression and metastasis are poorly understood. This remains a major hurdle to the clinical translation of copper chelators. Here, we propose that copper chelation inhibits metastasis by reducing TGF-ß levels and EMT signaling. Given that many drugs targeting TGF-ß have failed in clinical trials, partly because of severe side effects arising in patients, we hypothesized that copper chelation therapy might be a less toxic alternative to target the TGF-ß/EMT axis. RESULTS: Our cytokine array and RNA-seq data suggested a link between copper homeostasis, TGF-ß and EMT process. To validate this hypothesis, we performed single-cell imaging, protein assays, and in vivo studies. Here, we used the copper chelating agent TEPA to block copper trafficking. Our in vivo study showed a reduction of TGF-ß levels and metastasis to the lung in the TNBC mouse model. Mechanistically, TEPA significantly downregulated canonical (TGF-ß/SMAD2&3) and non-canonical (TGF-ß/PI3K/AKT, TGF-ß/RAS/RAF/MEK/ERK, and TGF-ß/WNT/ß-catenin) TGF-ß signaling pathways. Additionally, EMT markers of MMP-9, MMP-14, Vimentin, ß-catenin, ZEB1, and p-SMAD2 were downregulated, and EMT transcription factors of SNAI1, ZEB1, and p-SMAD2 accumulated in the cytoplasm after treatment. CONCLUSIONS: Our study suggests that copper chelation therapy represents a potentially effective therapeutic approach for targeting TGF-ß and inhibiting EMT in a diverse range of cancers.
RESUMO
BACKGROUND: High-grade serous ovarian carcinomas (HGSCs) are a heterogeneous subtype of epithelial ovarian cancers and include serous cancers arising in the fallopian tube and peritoneum. These cancers are now subdivided into homologous recombination repair (HR)-deficient and proficient subgroups as this classification impacts on management and prognosis. PARP inhibitors (PARPi) have shown significant clinical efficacy, particularly as maintenance therapy following response to platinum-based chemotherapy in BRCA-mutant or homologous recombination (HR)-deficient HGSCs in both the 1st and 2nd line settings. However, PARPi have limited clinical benefit in HR-proficient HGSCs which make up almost 50% of HGSC and improving outcomes in these patients is now a high priority due to the poor prognosis with ineffectiveness of the current standard of care. There are a number of potential lines of investigation including efforts in sensitizing HR-proficient tumors to PARPi. Herein, we aimed to develop a novel combination therapy by targeting SSRP1 using a small molecule inhibitor CBL0137 with PARPi in HR-proficient HGSCs. EXPERIMENTAL DESIGN: We tested anti-cancer activity of CBL0137 monotherapy using a panel of HGSC cell lines and patient-derived tumor cells in vitro. RNA sequencing was used to map global transcriptomic changes in CBL0137-treated patient-derived HR-proficient HGSC cells. We tested efficacy of CBL0137 in combination with PARPi using HGSC cell lines and patient-derived tumor cells in vitro and in vivo. RESULTS: We show that SSRP1 inhibition using a small molecule, CBL0137, that traps SSRP1 onto chromatin, exerts a significant anti-growth activity in vitro against HGSC cell lines and patient-derived tumor cells, and also reduces tumor burden in vivo. CBL0137 induced DNA repair deficiency via inhibition of the HR repair pathway and sensitized SSRP1-high HR-proficient HGSC cell lines and patient-derived tumor cells/xenografts to the PARPi, Olaparib in vitro and in vivo. CBL0137 also enhanced the efficacy of DNA damaging platinum-based chemotherapy in HGSC patient-derived xenografts. CONCLUSION: Our findings strongly suggest that combination of CBL0137 and PARP inhibition represents a novel therapeutic strategy for HR-proficient HGSCs that express high levels of SSRP1 and should be investigated in the clinic.
Assuntos
Neoplasias Ovarianas , Inibidores de Poli(ADP-Ribose) Polimerases , Feminino , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Reparo de DNA por Recombinação , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Carcinoma Epitelial do Ovário/tratamento farmacológico , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Fatores de Elongação da Transcrição/genéticaRESUMO
[This corrects the article DOI: 10.18632/oncotarget.3795.].
RESUMO
Chronic myeloid leukaemia (CML) is currently treated with inhibitors of the CML specific oncoprotein, bcr-abl. While this strategy is initially successful, drug resistance can become a problem. Therefore, new targets need to be identified to ensure the disease can be appropriately managed. The thioredoxin (Trx) system, comprised of Trx, thioredoxin reductase (TrxR), and NADPH, is an antioxidant system previously identified as a target for therapies aimed at overcoming drug resistance in other cancers. We assessed the effectiveness of TrxR inhibitors on drug resistant CML cells and examined links between TrxR and the bcr-abl cell-signalling pathway. Two TrxR inhibitors, auranofin and [Au(d2pype)2]Cl, increased intracellular ROS levels and elicited apoptosis in both sensitive and imatinib resistant CML cells. Inhibition of TrxR activity by these pharmacological inhibitors, or by specific siRNA, also resulted in decreased bcr-abl mRNA and protein levels, and lower bcr-abl downstream signalling activity, potentially enhancing the effectiveness of TrxR inhibitors as CML therapies. In addition, imatinib resistant CML cell lines showed upregulated expression of the Trx system. Furthermore, analysis of datasets showed that CML patients who did not respond to imatinib had higher Trx mRNA levels than patients who responded to treatment. Our study demonstrates a link between the Trx system and the bcr-abl protein and highlights the therapeutic potential of targeting the Trx system to improve CML patients' outcomes.
RESUMO
High expression of centrosomal protein CEP55 has been correlated with clinico-pathological parameters across multiple human cancers. Despite significant in vitro studies and association of aberrantly overexpressed CEP55 with worse prognosis, its causal role in vivo tumorigenesis remains elusive. Here, using a ubiquitously overexpressing transgenic mouse model, we show that Cep55 overexpression causes spontaneous tumorigenesis and accelerates Trp53+/- induced tumours in vivo. At the cellular level, using mouse embryonic fibroblasts (MEFs), we demonstrate that Cep55 overexpression induces proliferation advantage by modulating multiple cellular signalling networks including the hyperactivation of the Pi3k/Akt pathway. Notably, Cep55 overexpressing MEFs have a compromised Chk1-dependent S-phase checkpoint, causing increased replication speed and DNA damage, resulting in a prolonged aberrant mitotic division. Importantly, this phenotype was rescued by pharmacological inhibition of Pi3k/Akt or expression of mutant Chk1 (S280A) protein, which is insensitive to regulation by active Akt, in Cep55 overexpressing MEFs. Moreover, we report that Cep55 overexpression causes stabilized microtubules. Collectively, our data demonstrates causative effects of deregulated Cep55 on genome stability and tumorigenesis which have potential implications for tumour initiation and therapy development.
Assuntos
Proteínas de Ciclo Celular/genética , Transformação Celular Neoplásica/genética , Expressão Gênica , Instabilidade Genômica , Animais , Biomarcadores Tumorais , Biópsia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Transformação Celular Neoplásica/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Suscetibilidade a Doenças , Fibroblastos/metabolismo , Genótipo , Imuno-Histoquímica , Cariótipo , Linfonodos/metabolismo , Linfonodos/patologia , Camundongos , Camundongos Transgênicos , Microtúbulos/metabolismo , Mitose , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Estresse Fisiológico , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Multiple myeloma (MM), the second most common haematological malignancy, is a clonal plasma B-cell neoplasm that forms within the bone marrow. Despite recent advancements in treatment, MM remains an incurable disease. Auranofin, a linear gold(I) phosphine compound, has previously been shown to exert a significant anti-myeloma activity by inhibiting thioredoxin reductase (TrxR) activity. A bis-chelated tetrahedral gold(I) phosphine complex [Au(d2pype)2]Cl (where d2pype is 1,2-bis(di-2-pyridylphosphino)ethane) was previously designed to improve the gold(I) compound selectivity towards selenol- and thiol-containing proteins, such as TrxR. In this study, we show that [Au(d2pype)2]Cl significantly inhibited TrxR activity in both bortezomib-sensitive and resistant myeloma cells, which led to a significant reduction in cell proliferation and induction of apoptosis, both of which were dependent on ROS. In clonogenic assays, treatment with [Au(d2pype)2]Cl completely abrogated the tumourigenic capacity of MM cells, whereas auranofin was less effective. We also show that [Au(d2pype)2]Cl exerted a significant anti-myeloma activity in vivo in human RPMI8226 xenograft model in immunocompromised NOD/SCID mice. The MYC oncogene, known to drive myeloma progression, was downregulated in both in vitro and in vivo models when treated with [Au(d2pype)2]Cl. This study highlights the "proof of concept" that improved gold(I)-based compounds could potentially be used to not only treat MM but as an alternative tool to understand the role of the Trx system in the pathogenesis of this blood disease.
Assuntos
Ouro/química , Mieloma Múltiplo/tratamento farmacológico , Fosfinas/administração & dosagem , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mieloma Múltiplo/enzimologia , Mieloma Múltiplo/genética , Fosfinas/química , Fosfinas/farmacologia , Proteínas Proto-Oncogênicas c-myc/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Purpose: Lacking effective targeted therapies, triple-negative breast cancer (TNBCs) is highly aggressive and metastatic disease, and remains clinically challenging breast cancer subtype to treat. Despite the survival dependency on the proteasome pathway genes, FDA-approved proteasome inhibitors induced minimal clinical response in breast cancer patients due to weak proteasome inhibition. Hence, developing effective targeted therapy using potent proteasome inhibitor is required. Methods: We evaluated anti-cancer activity of a potent proteasome inhibitor, marizomib, in vitro using breast cancer lines and in vivo using 4T1.2 murine syngeneic model, MDA-MB-231 xenografts, and patient-derived tumor xenografts. Global proteome profiling, western blots, and RT-qPCR were used to investigate the mechanism of action for marizomib. Effect of marizomib on lung and brain metastasis was evaluated using syngeneic 4T1BR4 murine TNBC model in vivo. Results: We show that marizomib inhibits multiple proteasome catalytic activities and induces a better anti-tumor response in TNBC cell lines and patient-derived xenografts alone and in combination with the standard-of-care chemotherapy. Mechanistically, we show that marizomib is a dual inhibitor of proteasome and oxidative phosphorylation (OXPHOS) in TNBCs. Marizomib reduces lung and brain metastases by reducing the number of circulating tumor cells and the expression of genes involved in the epithelial-to-mesenchymal transition. We demonstrate that marizomib-induced OXPHOS inhibition upregulates glycolysis to meet the energetic demands of TNBC cells and combined inhibition of glycolysis with marizomib leads to a synergistic anti-cancer activity. Conclusions: Our data provide a strong rationale for a clinical evaluation of marizomib in primary and metastatic TNBC patients.
Assuntos
Antineoplásicos/uso terapêutico , Lactonas/uso terapêutico , Complexo de Endopeptidases do Proteassoma/metabolismo , Pirróis/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Camundongos , Fosforilação Oxidativa/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Inibidores de Proteassoma/uso terapêutico , Neoplasias de Mama Triplo Negativas/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Breast cancer (BC) is one of the most common cancers in women. TNBC (Triple-negative breast cancer) has limited treatment options and still lacks viable molecular targets, leading to poor outcomes. Recently, RNA-binding proteins (RBPs) have been shown to play crucial roles in human cancers, including BC, by modulating a number of oncogenic phenotypes. This suggests that RBPs represent potential molecular targets for BC therapy. Methods: We employed genomic data to identify RBPs specifically expressed in TNBC. NONO was silenced in TNBC cell lines to examine cell growth, colony formation, invasion, and migration. Gene expression profiles in NONO-silenced cells were generated and analyzed. A high-throughput screening for NONO-targeted drugs was performed using an FDA-approved library. Results: We found that the NONO RBP is highly expressed in TNBC and is associated with poor patient outcomes. NONO binds to STAT3 mRNA, increasing STAT3 mRNA levels in TNBC. Surprisingly, NONO directly interacts with STAT3 protein increasing its stability and transcriptional activity, thus contributing to its oncogenic function. Importantly, high-throughput drug screening revealed that auranofin is a potential NONO inhibitor and inhibits cell growth in TNBC. Conclusions: NONO is an RBP upstream regulator of both STAT3 RNA and protein levels and function. It represents an important and clinically relevant promoter of growth and resistance of TNBCs. NONO is also therefore a potential therapeutic target in TNBC.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Ligação a RNA/metabolismo , Fator de Transcrição STAT3/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Portadores de Fármacos/química , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genômica , Humanos , Nanopartículas/química , Medicina de Precisão/métodos , Proteínas de Ligação a RNA/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Nanomedicina Teranóstica/métodos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
: Claspin is essential for activating the DNA damage checkpoint effector kinase Chk1, a target in oncotherapy. Claspin functions are tightly correlated to Claspin protein stability, regulated by ubiquitin-dependent proteasomal degradation. Here we identify Glycogen Synthase Kinase 3-ß (GSK3-ß) as a new regulator of Claspin stability. Interestingly, as Chk1, GSK3-ß is a therapeutic target in cancer. GSK3-ß inhibition or knockdown stabilizes Claspin, whereas a GSK3-ß constitutively active form reduces Claspin protein levels by ubiquitination and proteasome-mediated degradation. Our results also suggest that GSK3-ß modulates the interaction of Claspin with ß-TrCP, a critical E3 ubiquitin ligase that regulates Claspin stability. Importantly, GSK3-ß knock down increases Chk1 activation in response to DNA damage in a Claspin-dependent manner. Therefore, Chk1 activation could be a pro-survival mechanism that becomes activated upon GSK3-ß inhibition. Importantly, treating triple negative breast cancer cell lines with Chk1 or GSK3-ß inhibitors alone or in combination, demonstrates that Chk1/GSK3-ß double inhibition restrains cell growth and triggers more apoptosis compared to individual treatments, thereby revealing novel possibilities for a combination therapy for cancer.
RESUMO
BACKGROUND: Despite the increasing progress in targeted and immune based-directed therapies for other solid organ malignancies, currently there is no targeted therapy available for TNBCs. A number of mechanisms have been reported both in pre-clinical and clinical settings that involve inherent, acquired and adaptive resistance to small molecule inhibitors. Here, we demonstrated a novel resistance mechanism in TNBC cells mediated by PDGFRß in response to JAK2 inhibition. METHODS: Multiple in vitro (subG1, western blotting, immunofluorescence, RT-PCR, Immunoprecipitation), in vivo and publically available datasets were used. RESULTS: We showed that TNBC cells exposed to MEK1/2-JAK2 inhibitors exhibit resistant colonies in anchorage-independent growth assays. Moreover, cells treated with various small molecule inhibitors including JAK2 promote PDGFRß upregulation. Using publically available databases, we showed that patients expressing high PDGFRß or its ligand PDGFB exhibit poor relapse-free survival upon chemotherapeutic treatment. Mechanistically we found that JAK2 expression controls steady state levels of PDGFRß. Thus, co-blockade of PDGFRß with JAK2 and MEK1/2 inhibitors completely eradicated resistant colonies in vitro. We found that triple-combined treatment had a significant impact on CD44+/CD24- stem-cell-like cells. Likewise, we found a significant tumor growth inhibition in vivo through intratumoral CD8+ T cells infiltration in a manner that is reversed by anti-CD8 antibody treatment. CONCLUSION: These findings reveal a novel regulatory role of JAK2-mediated PDGFRß proteolysis and provide an example of a PDGFRß-mediated resistance mechanism upon specific target inhibition in TNBC.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Janus Quinase 2/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Feminino , Humanos , Inibidores de Janus Quinases/farmacologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
The centrosomal protein, CEP55, is a key regulator of cytokinesis, and its overexpression is linked to genomic instability, a hallmark of cancer. However, the mechanism by which it mediates genomic instability remains elusive. Here, we showed that CEP55 overexpression/knockdown impacts survival of aneuploid cells. Loss of CEP55 sensitizes breast cancer cells to anti-mitotic agents through premature CDK1/cyclin B activation and CDK1 caspase-dependent mitotic cell death. Further, we showed that CEP55 is a downstream effector of the MEK1/2-MYC axis. Blocking MEK1/2-PLK1 signaling therefore reduced outgrowth of basal-like syngeneic and human breast tumors in in vivo models. In conclusion, high CEP55 levels dictate cell fate during perturbed mitosis. Forced mitotic cell death by blocking MEK1/2-PLK1 represents a potential therapeutic strategy for MYC-CEP55-dependent basal-like, triple-negative breast cancers.
Assuntos
Aneuploidia , Proteínas de Ciclo Celular/metabolismo , Citocinese , Mitose , Proteínas Nucleares/metabolismo , Neoplasias da Mama/patologia , Proteína Quinase CDC2/metabolismo , Caspases/metabolismo , Proteínas de Ciclo Celular/genética , Morte Celular , Linhagem Celular Tumoral , Ciclina B/metabolismo , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Modelos Biológicos , Proteínas Nucleares/genéticaRESUMO
Multiple myeloma (MM) is a B-cell malignancy characterized by an accumulation of abnormal clonal plasma cells in the bone marrow. Introduction of the proteasome-inhibitor bortezomib has improved MM prognosis and survival; however hypoxia-induced or acquired bortezomib resistance remains a clinical problem. This study highlighted the role of thioredoxin reductase 1 (TrxR1) in the hypoxia-induced and acquired bortezomib resistance in MM. Higher TrxR1 gene expression correlated with high-risk disease, adverse overall survival, and poor prognosis in myeloma patients. We demonstrated that hypoxia induced bortezomib resistance in myeloma cells and increased TrxR1 protein levels. Inhibition of TrxR1 using auranofin overcame hypoxia-induced bortezomib resistance and restored the sensitivity of hypoxic-myeloma cells to bortezomib. Hypoxia increased NF-Ðºß subunit p65 nuclear protein levels and TrxR1 inhibition decreased hypoxia-induced NF-Ðºß p65 protein levels in the nucleus and reduced the expression of NF-кß-regulated genes. In addition, higher TrxR1 protein levels were observed in bortezomib-resistant myeloma cells compared to the naïve cells, and its inhibition using either auranofin or TrxR1-specific siRNAs reversed bortezomib resistance. TrxR1 inhibition reduced p65 mRNA and protein expression in bortezomib-resistant myeloma cells, and also decreased the expression of NF-кß-regulated anti-apoptotic and proliferative genes. Thus, TrxR1 inhibition overcomes both hypoxia-induced and acquired bortezomib resistance by inhibiting the NF-Ðºß signaling pathway. Our findings demonstrate that elevated TrxR1 levels correlate with the acquisition of bortezomib resistance in MM. We propose considering TrxR1-inhibiting drugs, such as auranofin, either for single agent or combination therapy to circumvent bortezomib-resistance and improve survival outcomes of MM patients.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Tiorredoxina Redutase 1/genética , Apoptose/efeitos dos fármacos , Auranofina/administração & dosagem , Bortezomib/administração & dosagem , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Mieloma Múltiplo/patologia , NF-kappa B/genética , Inibidores de Proteassoma/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Tiorredoxina Redutase 1/biossínteseRESUMO
Multiple myeloma (MM) is characterized by an accumulation of abnormal clonal plasma cells in the bone marrow. Despite recent advancements in anti-myeloma therapies, MM remains an incurable disease. Antioxidant molecules are upregulated in many cancers, correlating with tumor proliferation, survival, and chemoresistance and therefore, have been suggested as potential therapeutic targets. This study investigated the cross-talk between two antioxidant molecules, thioredoxin reductase (TrxR) and heme oxygenase-1 (HO-1), and their therapeutic implications in MM. We found that although auranofin, a TrxR inhibitor, significantly inhibited TrxR activity by more than 50% at lower concentrations, myeloma cell proliferation was only inhibited at higher concentrations of auranofin. Inhibition of TrxR using lower auranofin concentrations induced HO-1 protein expression in myeloma cells. Using a sub-lethal concentration of auranofin to inhibit TrxR activity in conjunction with HO-1 inhibition significantly decreased myeloma cell growth and induced apoptosis. TrxR was shown to regulate HO-1 via the Nrf2 signaling pathway in a ROS-dependent manner. Increased HO-1 mRNA levels were observed in bortezomib-resistant myeloma cells compared to parent cells and HO-1 inhibition restored the sensitivity to bortezomib in bortezomib-resistant myeloma cells. These findings indicate that concurrent inhibition of HO-1 with either a TrxR inhibitor or with bortezomib would improve therapeutic outcomes in MM patients. Hence, our findings further support the need to target multiple antioxidant systems alone or in combination with other therapeutics to improve therapeutic outcomes in MM patients.