RESUMO
As part of the classical renin-angiotensin system, the peptidase angiotensin-converting enzyme (ACE) makes angiotensin II which has myriad effects on systemic cardiovascular function, inflammation, and cellular proliferation. Less well known is that macrophages and neutrophils make ACE in response to immune activation which has marked effects on myeloid cell function independent of angiotensin II. Here, we discuss both classical (angiotensin) and nonclassical functions of ACE and highlight mice called ACE 10/10 in which genetic manipulation increases ACE expression by macrophages and makes these mice much more resistant to models of tumors, infection, atherosclerosis, and Alzheimer's disease. In another model called NeuACE mice, neutrophils make increased ACE and these mice are much more resistant to infection. In contrast, ACE inhibitors reduce neutrophil killing of bacteria in mice and humans. Increased expression of ACE induces a marked increase in macrophage oxidative metabolism, particularly mitochondrial oxidation of lipids, secondary to increased peroxisome proliferator-activated receptor α expression, and results in increased myeloid cell ATP. ACE present in sperm has a similar metabolic effect, and the lack of ACE activity in these cells reduces both sperm motility and fertilization capacity. These nonclassical effects of ACE are not due to the actions of angiotensin II but to an unknown molecule, probably a peptide, that triggers a profound change in myeloid cell metabolism and function. Purifying and characterizing this peptide could offer a new treatment for several diseases and prove potentially lucrative.
Assuntos
Células Mieloides , Peptidil Dipeptidase A , Animais , Humanos , Peptidil Dipeptidase A/metabolismo , Peptidil Dipeptidase A/genética , Células Mieloides/metabolismo , Células Mieloides/imunologia , Células Mieloides/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Camundongos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/efeitos dos fármacos , Sistema Renina-Angiotensina/efeitos dos fármacos , Angiotensina II/farmacologiaRESUMO
An upregulation of angiotensin-converting enzyme (ACE) expression strengthens the immune activity of myeloid lineage cells as a natural functional regulation mechanism in our immunity. ACE10/10 mice, possessing increased ACE expression in macrophages, exhibit enhanced anti-tumor immunity and anti-bactericidal effects compared to those of wild type (WT) mice, while the detailed molecular mechanism has not been elucidated yet. In this report, we demonstrate that peroxisome proliferator-activated receptor alpha (PPARα) is a key molecule in the functional upregulation of macrophages induced by ACE. The expression of PPARα, a transcription factor regulating fatty acid metabolism-associated gene expressions, was upregulated in ACE-overexpressing macrophages. To pinpoint the role of PPARα in the enhanced immune function of ACE-overexpressing macrophages, we established a line with myeloid lineage-selective PPARα depletion employing the Lysozyme 2 (LysM)-Cre system based on ACE 10/10 mice (named A10-PPARα-Cre). Interestingly, A10-PPARα-Cre mice exhibited larger B16-F10-originated tumors than original ACE 10/10 mice. PPARα depletion impaired cytokine production and antigen-presenting activity in ACE-overexpressing macrophages, resulting in reduced tumor antigen-specific CD8+ T cell activity. Additionally, the anti-bactericidal effect was also impaired in A10-PPARα-Cre mice, resulting in similar bacterial colonization to WT mice in Methicillin-Resistant Staphylococcus aureus (MRSA) infection. PPARα depletion downregulated phagocytic activity and bacteria killing in ACE-overexpressing macrophages. Moreover, THP-1-ACE-derived macrophages, as a human model, expressing upregulated PPARα exhibited enhanced cytotoxicity against B16-F10 cells and MRSA killing. These activities were further enhanced by the PPARα agonist, WY 14643, while abolished by the antagonist, GW6471, in THP-1-ACE cells. Thus, PPARα is an indispensable molecule in ACE-dependent functional upregulation of macrophages in both mice and humans.
RESUMO
Testis angiotensin-converting enzyme (tACE) plays a critical role in male fertility, but the mechanism is unknown. By using ACE C-domain KO (CKO) mice which lack tACE activity, we found that ATP in CKO sperm was 9.4-fold lower than WT sperm. Similarly, an ACE inhibitor (ACEi) reduced ATP production in mouse sperm by 72%. Metabolic profiling showed that tACE inactivation severely affects oxidative metabolism with decreases in several Krebs cycle intermediates including citric acid, cis-aconitic acid, NAD, α-ketoglutaric acid, succinate, and L-malic acid. We found that sperms lacking tACE activity displayed lower levels of oxidative enzymes (CISY, ODO1, MDHM, QCR2, SDHA, FUMH, CPT2, and ATPA) leading to a decreased mitochondrial respiration rate. The reduced energy production in CKO sperms leads to defects in their physiological functions including motility, acrosine activity, and fertilization in vitro and in vivo. Male mice treated with ACEi show severe impairment in reproductive capacity when mated with female mice. In contrast, an angiotensin II receptor blocker (ARB) had no effect. CKO sperms express significantly less peroxisome proliferators-activated receptor gamma (PPARγ) transcription factor, and its blockade eliminates the functional differences between CKO and WT sperms, indicating PPARγ might mediate the effects of tACE on sperm metabolism. Finally, in a cohort of human volunteers, in vitro treatment with the ramipril or a PPARγ inhibitor reduced ATP production in human sperm and hence its motility and acrosine activity. These findings may have clinical significance since millions of people take ACEi daily, including men who are reproductively active.
Assuntos
Fertilização , PPAR gama , Peptidil Dipeptidase A , Espermatozoides , Animais , Feminino , Humanos , Masculino , Camundongos , Trifosfato de Adenosina/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Fertilização/genética , PPAR gama/genética , PPAR gama/metabolismo , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Testículo/enzimologia , Camundongos Endogâmicos C57BL , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Proteínas Mitocondriais/genética , Técnicas de Inativação de Genes , Fosforilação OxidativaRESUMO
The pathogenesis of atherosclerosis is defined by impaired lipid handling by macrophages which increases intracellular lipid accumulation. This dysregulation of macrophages triggers the accumulation of apoptotic cells and chronic inflammation which contributes to disease progression. We previously reported that mice with increased macrophage-specific angiotensin-converting enzyme, termed ACE10/10 mice, resist atherosclerosis in an adeno-associated virus-proprotein convertase subtilisin/kexin type 9 (AAV-PCSK9)-induced model. This is due to increased lipid metabolism by macrophages which contributes to plaque resolution. However, the importance of ACE in peripheral blood monocytes, which are the primary precursors of lesional-infiltrating macrophages, is still unknown in atherosclerosis. Here, we show that the ACE-mediated metabolic phenotype is already triggered in peripheral blood circulating monocytes and that this functional modification is directly transferred to differentiated macrophages in ACE10/10 mice. We found that Ly-6Clo monocytes were increased in atherosclerotic ACE10/10 mice. The monocytes isolated from atherosclerotic ACE10/10 mice showed enhanced lipid metabolism, elevated mitochondrial activity, and increased adenosine triphosphate (ATP) levels which implies that ACE overexpression is already altered in atherosclerosis. Furthermore, we observed increased oxygen consumption (VO2), respiratory exchange ratio (RER), and spontaneous physical activity in ACE10/10 mice compared to WT mice in atherosclerotic conditions, indicating enhanced systemic energy consumption. Thus, ACE overexpression in myeloid lineage cells modifies the metabolic function of peripheral blood circulating monocytes which differentiate to macrophages and protect against atherosclerotic lesion progression due to better lipid metabolism.
Assuntos
Aterosclerose , Pró-Proteína Convertase 9 , Animais , Camundongos , Aterosclerose/patologia , Lipídeos , Células Mieloides/patologiaRESUMO
AIMS: The metabolic failure of macrophages to adequately process lipid is central to the aetiology of atherosclerosis. Here, we examine the role of macrophage angiotensin-converting enzyme (ACE) in a mouse model of PCSK9-induced atherosclerosis. METHODS AND RESULTS: Atherosclerosis in mice was induced with AAV-PCSK9 and a high-fat diet. Animals with increased macrophage ACE (ACE 10/10 mice) have a marked reduction in atherosclerosis vs. WT mice. Macrophages from both the aorta and peritoneum of ACE 10/10 express increased PPARα and have a profoundly altered phenotype to process lipids characterized by higher levels of the surface scavenger receptor CD36, increased uptake of lipid, increased capacity to transport long chain fatty acids into mitochondria, higher oxidative metabolism and lipid ß-oxidation as determined using 13C isotope tracing, increased cell ATP, increased capacity for efferocytosis, increased concentrations of the lipid transporters ABCA1 and ABCG1, and increased cholesterol efflux. These effects are mostly independent of angiotensin II. Human THP-1 cells, when modified to express more ACE, increase expression of PPARα, increase cell ATP and acetyl-CoA, and increase cell efferocytosis. CONCLUSION: Increased macrophage ACE expression enhances macrophage lipid metabolism, cholesterol efflux, efferocytosis, and it reduces atherosclerosis. This has implications for the treatment of cardiovascular disease with angiotensin II receptor antagonists vs. ACE inhibitors.
Assuntos
Aterosclerose , Pró-Proteína Convertase 9 , Humanos , Animais , Camundongos , Pró-Proteína Convertase 9/metabolismo , PPAR alfa/genética , PPAR alfa/metabolismo , Metabolismo dos Lipídeos , Colesterol/metabolismo , Macrófagos/metabolismo , Aterosclerose/genética , Aterosclerose/prevenção & controle , Angiotensinas/metabolismo , Trifosfato de Adenosina/metabolismo , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismoRESUMO
Head and neck squamous cell carcinoma (HNSCC) is a highly aggressive disease with poor prognosis, which is mainly due to drug resistance. The biology determining the response to chemo-radiotherapy in HNSCC is poorly understood. Using clinical samples, we found that miR124-3p and miR766-3p are overexpressed in chemo-radiotherapy-resistant (non-responder) HNSCC, as compared to responder tumors. Our study shows that inhibition of miR124-3p and miR766-3p enhances the sensitivity of HNSCC cell lines, CAL27 and FaDu, to 5-fluorouracil and cisplatin (FP) chemotherapy and radiotherapy. In contrast, overexpression of miR766-3p and miR124-3p confers a resistance phenotype in HNSCC cells. The upregulation of miR124-3p and miR766-3p is associated with increased HNSCC cell invasion and migration. In a xenograft mouse model, inhibition of miR124-3p and miR766-3p enhanced the efficacy of chemo-radiotherapy with reduced growth of resistant HNSCC. For the first time, we identified that miR124-3p and miR766-3p attenuate expression of CREBRF and NR3C2, respectively, in HNSCC, which promotes aggressive tumor behavior by inducing the signaling axes CREB3/ATG5 and ß-catenin/c-Myc. Since miR124-3p and miR766-3p affect complementary pathways, combined inhibition of these two miRNAs shows an additive effect on sensitizing cancer cells to chemo-radiotherapy. In conclusion, our study demonstrated a novel miR124-3p- and miR766-3p-based biological mechanism governing treatment-resistant HNSCC, which can be targeted to improve clinical outcomes in HNSCC.
RESUMO
Thyroid cancer is the most common endocrine malignancy. A multitargeted tyrosine kinase inhibitor, lenvatinib, has been used for the treatment of advanced thyroid cancer. To elucidate the mechanism of resistance to lenvatinib in thyroid cancer cells, we established lenvatinib-resistant sublines and analyzed the molecular mechanisms of resistance. Two thyroid cancer cell lines (TPC-1 and FRO) were used, and resistant sublines for lenvatinib (TPC-1/LR, FRO/LR) were established. In TPC-1/LR, the phosphorylation of epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK), and Akt was enhanced whereas in FRO/LR, the phosphorylation of EGFR and downstream signal transduction molecules was not enhanced. The addition of epidermal growth factor decreased sensitivity to lenvatinib in TPC-1 and FRO. The combination of EGFR inhibitors lapatinib and lenvatinib significantly inhibited the growth of TPC-1/LR in both in vitro and mouse xenograft models. Short-term exposure to lenvatinib enhanced the phosphorylation of EGFR in six thyroid cancer cell lines regardless of their histological origin or driver gene mutations; however, phosphorylation of ERK was enhanced in all cells except TPC-1. A synergistic growth-inhibitory effect was observed in three thyroid cancer cell lines, including intrinsically lenvatinib-resistant cells. The results indicate that signal transduction via the EGFR pathway may be involved in the development of lenvatinib resistance in thyroid cancer cells. The inhibition of the EGFR pathway simultaneously by an EGFR inhibitor may have therapeutic potential for overcoming lenvatinib resistance in thyroid cancer.
Assuntos
Antineoplásicos , Neoplasias da Glândula Tireoide , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB , Humanos , Camundongos , Compostos de Fenilureia/farmacologia , Compostos de Fenilureia/uso terapêutico , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Quinolinas , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/metabolismoRESUMO
Angiotensin converting enzyme (ACE) is well known for its role producing the vasoconstrictor angiotensin II and ACE inhibitors are commonly used for treating hypertension and cardiovascular disease. However, ACE has many different substrates besides angiotensin I and plays a role in many different physiologic processes. Here, we discuss the role of ACE in the immune response. Several studies in mice indicate that increased expression of ACE by macrophages or neutrophils enhances the ability of these cells to respond to immune challenges such as infection, neoplasm, Alzheimer's disease, and atherosclerosis. Increased expression of ACE induces increased oxidative metabolism with an increase in cell content of ATP. In contrast, ACE inhibitors have the opposite effect, and in both humans and mice, administration of ACE inhibitors reduces the ability of neutrophils to kill bacteria. Understanding how ACE affects the immune response may provide a means to increase immunity in a variety of chronic conditions now not treated through immune manipulation.
Assuntos
Hipertensão , Peptidil Dipeptidase A , Angiotensina I/metabolismo , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Animais , Hipertensão/tratamento farmacológico , Hipertensão/metabolismo , Macrófagos/metabolismo , Camundongos , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismoRESUMO
PURPOSE: Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are widely used for the treatment of advanced estrogen receptor (ER)-positive breast cancer. To develop a treatment strategy for cancers resistant to CDK4/6 inhibitors, here, we established palbociclib-resistant sublines and analyzed their resistance mechanisms. METHODS: Palbociclib-resistant sublines were established from T47D and MCF7 cells. Sensitivity to other drugs was assessed via the WST assay. Altered expression/phosphorylation of proteins related to signal transduction and cell cycle regulation was examined using western blotting. Copy number alterations and mutations in the retinoblastoma (RB1) gene were also analyzed. RESULTS: Although an increase in CDK6 and decrease in retinoblastoma protein (Rb) expression/phosphorylation were commonly observed in the resistant sublines, changes in other cell cycle-related proteins were heterogeneous. Upon extended exposure to palbociclib, the expression/phosphorylation of these proteins became altered, and the long-term removal of palbociclib did not restore the Rb expression/phosphorylation patterns. Consistently a copy number decrease, as well as RB1 mutations were detected. Moreover, although the resistant sublines exhibited cross-resistance to abemaciclib, their response to dinaciclib was the same as that of wild-type cells. Of note, the cell line exhibiting increased mTOR phosphorylation also showed a higher sensitivity to everolimus. However, the sensitivity to chemotherapeutic agents was unchanged in palbociclib-resistant sublines. CONCLUSION: ER-positive breast cancer cells use multiple molecular mechanisms to survive in the presence of palbociclib, suggesting that targeting activated proteins may be an effective strategy to overcome resistance. Additionally, palbociclib monotherapy induces mutations and copy number alterations in the RB1 gene.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Piperazinas/farmacologia , Piridinas/farmacologia , Receptores de Estrogênio/metabolismo , Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/biossíntese , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/biossíntese , Receptor alfa de Estrogênio/metabolismo , Humanos , Células MCF-7 , Inibidores de Proteínas Quinases/farmacologia , Receptor ErbB-2/biossíntese , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/biossíntese , Transdução de SinaisRESUMO
Sequential treatment with endocrine or chemotherapy is generally used in the treatment of estrogen receptor (ER)-positive recurrent breast cancer. To date, few studies have investigated the effect of long-term endocrine therapy on the response to subsequent chemotherapy in ER-positive breast cancer. We examined whether a preceding endocrine therapy affects the sensitivity to subsequent chemotherapy in ER-positive breast cancer cells. Three ER-positive breast cancer cell lines (T47D, MCF7, BT474) and tamoxifen-resistant sublines (T47D/T, MCF7/T, BT474/T) were analyzed for sensitivity to 5-fluorouracil, paclitaxel, and doxorubicin. The mRNA levels of factors related to drug sensitivity were analyzed by RT-PCR. MCF7/T cells became more sensitive to 5-fluorouracil than wild-type (wt)-MCF7 cells. In addition, the apoptosis induced by 5-fluorouracil was significantly increased in MCF7/T cells. However, no difference in sensitivity to chemotherapeutic agents was observed in T47D/T and BT474/T cells compared with their wt cells. Dihydropyrimidine dehydrogenase (DPYD) mRNA expression was significantly decreased in MCF7/T cells compared with wt-MCF7 cells. The expression of DPYD mRNA was restored with 5-azacytidine treatment in MCF7/T cells. In addition, DPYD 3'-UTR luciferase activity was significantly reduced in MCF7/T cells. These data indicated that the expression of DPYD mRNA was repressed by methylation of the DPYD promoter region and post-transcriptional regulation by miRNA in MCF7/T cells. In the mouse xenograft model, capecitabine significantly reduced the tumor volume in MCF7/T compared with MCF7. The results of this study indicate that endocrine therapy could alter the sensitivity to chemotherapeutic agents in a subset of breast cancers, and 5-fluorouracil may be effective in tamoxifen-resistant breast cancers.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fluoruracila/farmacologia , Receptores de Estrogênio/metabolismo , Tamoxifeno/farmacologia , Animais , Antimetabólitos Antineoplásicos/farmacologia , Antineoplásicos Hormonais/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Capecitabina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Estradiol/farmacologia , Estrogênios/farmacologia , Feminino , Humanos , Células MCF-7 , Camundongos Endogâmicos BALB C , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Covalent modification of disease-associated proteins with small molecules is a powerful approach for achieving an increased and sustained pharmacological effect. To reduce the potential risk of nonselective covalent modification, molecular design of covalent inhibitors is critically important. We report herein the development of a targeted covalent inhibitor for mutated epidermal growth factor receptor (EGFR) (L858R/T790M) using α-chlorofluoroacetamide (CFA) as the reactive group. The chemically tuned weak reactivity of CFA was suitable for the design of third-generation EGFR inhibitors that possess the pyrimidine scaffold. The structure-activity relationship study revealed that CFA inhibitor 18 (NSP-037) possessed higher inhibition selectivity to the mutated EGFR over wild-type EGFR when compared to clinically approved osimertinib. Mass-based chemical proteomics analyses further revealed that 18 displayed high covalent modification selectivity for the mutated EGFR in living cells. These findings highlight the utility of CFA as a warhead of targeted covalent inhibitors and the potential application of the CFA-pyrimidines for treatment of non-small-cell lung cancer.
RESUMO
Many diseases, including cancer, have been associated with impaired regulation of angiogenesis, of which vascular endothelial growth factor (VEGF)-A is a key regulator. Here, we test the contribution of N-myc downstream regulated gene 1 (NDRG1) to VEGF-A-induced angiogenesis in vascular endothelial cells (ECs). Ndrg1-/- mice exhibit impaired VEGF-A-induced angiogenesis in corneas. Tumor angiogenesis induced by cancer cells that express high levels of VEGF-A was also reduced in a mouse dorsal air sac assay. Furthermore, NDRG1 deficiency in ECs prevented angiogenic sprouting from the aorta and the activation of phospholipase Cγ1 (PLCγ1) and ERK1/2 by VEGF-A without affecting the expression and function of VEGFR2. Finally, we show that NDRG1 formed a complex with PLCγ1 through its phosphorylation sites, and the inhibition of PLCγ1 dramatically suppressed VEGF-A-induced angiogenesis in the mouse cornea, suggesting an essential role of NDRG1 in VEGF-A-induced angiogenesis through PLCγ1 signaling.
Assuntos
Indutores da Angiogênese/farmacologia , Neovascularização da Córnea/enzimologia , Células Endoteliais/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Fosfolipase C gama/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Neovascularização da Córnea/genética , Neovascularização da Córnea/patologia , Modelos Animais de Doenças , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de SinaisRESUMO
Nuclear expression of Y-box-binding protein (YBX1) is closely correlated with clinical poor outcomes and drug resistance in breast cancer. Nuclear translocation of YBX1 is facilitated by YBX1 phosphorylation at serine 102 by AKT, p70S6K, and p90RSK, and the phosphorylated YBX1 (pYBX1) promotes expression of genes related to drug resistance and cell growth. A forthcoming problem to be addressed is whether targeting the phosphorylation of YBX1 overcomes antiestrogen resistance by progressive breast cancer. Here, we found that increased expression of pYBX1 was accompanied by acquired resistance to antiestrogens, fulvestrant and tamoxifen. Forced expression of YBX1/S102E, a constitutive phosphorylated form, resulted in acquired resistance to fulvestrant. Inversely, YBX1 silencing specifically overcame antiestrogen resistance. Furthermore, treatment with everolimus, an mTORC1 inhibitor, or TAS0612, a novel multikinase inhibitor of AKT, p70S6K, and p90RSK, suppressed YBX1 phosphorylation and overcame antiestrogen resistance in vitro and in vivo IHC analysis revealed that expression of pYBX1 and YBX1 was augmented in patients who experienced recurrence during treatment with adjuvant endocrine therapies. Furthermore, pYBX1 was highly expressed in patients with triple-negative breast cancer compared with other subtypes. TAS0612 also demonstrated antitumor effect against triple-negative breast cancer in vivo Taken together, our findings suggest that pYBX1 represents a potential therapeutic target for treatment of antiestrogen-resistant and progressive breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Moduladores de Receptor Estrogênico/farmacologia , Everolimo/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteína 1 de Ligação a Y-Box/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células , Quimioterapia Combinada , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilação , Prognóstico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The development of potent and selective therapeutic approaches to glioblastoma (GBM), one of the most aggressive primary brain tumors, requires identification of molecular pathways that critically regulate the survival and proliferation of GBM. Previous studies have reported that deregulated expression of N-myc downstream regulated gene 1 (NDRG1) affects tumor growth and clinical outcomes of patients with various types of cancer including glioma. Here, we show that high level expression of NDRG1 in tumors significantly correlated with better prognosis of patients with GBM. Loss of NDRG1 in GBM cells upregulated GSK3ß levels and promoted cell proliferation, which was reversed by selective inhibitors of GSK3ß. In contrast, NDRG1 overexpression suppressed growth of GBM cells by decreasing GSK3ß levels via proteasomal degradation and by suppressing AKT and S6 cell growth signaling, as well as cell-cycle signaling pathways. Conversely, GSK3ß phosphorylated serine and threonine sites in the C-terminal domain of NDRG1 and limited the protein stability of NDRG1. Furthermore, treatment with differentiation inducing factor-1, a small molecule derived from Dictyostelium discoideum, enhanced NDRG1 expression, decreased GSK3ß expression, and exerted marked NDRG1-dependent antitumor effects in vitro and in vivo. Taken together, this study revealed a novel molecular mechanism by which NDRG1 inhibits GBM proliferation and progression. Our study thus identifies the NDRG1/GSK3ß signaling pathway as a key growth regulatory program in GBM, and suggests enhancing NDRG1 expression in GBM as a potent strategy toward the development of anti-GBM therapeutics. SIGNIFICANCE: This study identifies NDRG1 as a potent and endogenous suppressor of glioblastoma cell growth, suggesting the clinical benefits of NDRG1-targeted therapeutics against glioblastoma.
Assuntos
Neoplasias Encefálicas/patologia , Proteínas de Ciclo Celular/metabolismo , Glioblastoma/patologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Hexanonas/administração & dosagem , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Encéfalo/patologia , Encéfalo/cirurgia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/cirurgia , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/mortalidade , Glioblastoma/cirurgia , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Hexanonas/uso terapêutico , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Prognóstico , Estabilidade Proteica/efeitos dos fármacos , Piridinas/farmacologia , Pirimidinas/farmacologia , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiadiazóis/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Y-box binding protein-1 (YBX1), a multifunctional oncoprotein containing an evolutionarily conserved cold shock domain, dysregulates a wide range of genes involved in cell proliferation and survival, drug resistance, and chromatin destabilization by cancer. Expression of a multidrug resistance-associated ATP binding cassette transporter gene, ABCB1, as well as growth factor receptor genes, EGFR and HER2/ErbB2, was initially discovered to be transcriptionally activated by YBX1 in cancer cells. Expression of other drug resistance-related genes, MVP/LRP, TOP2A, CD44, CD49f, BCL2, MYC, and androgen receptor (AR), is also transcriptionally activated by YBX1, consistently indicating that YBX1 is involved in tumor drug resistance. Furthermore, there is strong evidence to support that nuclear localization and/or overexpression of YBX1 can predict poor outcomes in patients with more than 20 different tumor types. YBX1 is phosphorylated by kinases, including AKT, p70S6K, and p90RSK, and translocated into the nucleus to promote the transcription of resistance- and malignancy-related genes. Phosphorylated YBX1, therefore, plays a crucial role as a potent transcription factor in cancer. Herein, a novel anticancer therapeutic strategy is presented by targeting activated YBX1 to overcome drug resistance and malignant progression.
Assuntos
Núcleo Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos , Neoplasias/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes , Humanos , Masculino , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Fosforilação/efeitos dos fármacos , Prognóstico , Ativação TranscricionalRESUMO
Irreversible inhibition of disease-associated proteins with small molecules is a powerful approach for achieving increased and sustained pharmacological potency. Here, we introduce α-chlorofluoroacetamide (CFA) as a novel warhead of targeted covalent inhibitor (TCI). Despite weak intrinsic reactivity, CFA-appended quinazoline showed high reactivity toward Cys797 of epidermal growth factor receptor (EGFR). In cells, CFA-quinazoline showed higher target specificity for EGFR than the corresponding Michael acceptors in a wide concentration range (0.1-10 µM). The cysteine adduct of the CFA derivative was susceptible to hydrolysis and reversibly yielded intact thiol but was stable in solvent-sequestered ATP-binding pocket of EGFR. This environment-dependent hydrolysis can potentially reduce off-target protein modification by CFA-based drugs. Oral administration of CFA quinazoline NS-062 significantly suppressed tumor growth in a mouse xenograft model. Further, CFA-appended pyrazolopyrimidine irreversibly inhibited Bruton's tyrosine kinase with higher target specificity. These results demonstrate the utility of CFA as a new class warheads for TCI.
Assuntos
Acetamidas/síntese química , Cisteína/metabolismo , Quinazolinas/síntese química , Acetamidas/química , Acetamidas/farmacologia , Animais , Antineoplásicos , Linhagem Celular , Receptores ErbB , Humanos , Camundongos , Camundongos Nus , Neoplasias , Fosfotransferases/fisiologia , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/antagonistas & inibidores , Quinazolinas/química , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The enhanced expression of the Y-box binding protein YBX1 is consistently correlated with poor outcomes or reduced survival of breast cancer patients. However, the mechanism underlying the association between increased YBX1 expression and poor outcomes has yet to be revealed. We searched a database for the top 500 genes that are positively or negatively correlated with YBX1 and with ESR1 in breast cancer patients. We further examined the association between YBX1-correlated genes and breast cancer outcomes in patients at Kyushu University Hospital. More than 60% of genes that are positively correlated with YBX1 are also negatively correlated with ESR1. The enhanced expression levels of the top 20 positively correlated genes mostly predict negative outcomes, while the enhanced expression levels of the top 20 negatively correlated genes mostly predict positive outcomes. Furthermore, in breast cancer patients at Kyushu University Hospital, the expression levels of YBX1 and YBX1-positively correlated genes were significantly higher and the expression levels of genes negatively correlated with YBX1 were significantly lower in patients who relapsed after their primary surgery than in those who did not relapse. The expression of YBX1 together with the expression of its positively or negatively correlated genes may help to predict outcomes as well as resistance to endocrine therapies in breast cancer patients. Determining the expression of YBX1 and its closely correlated genes will contribute to the development of precision therapeutics for breast cancer.
RESUMO
Endocrine therapies effectively improve the outcomes of patients with estrogen receptor (ER)-positive breast cancer. However, the emergence of drug-resistant tumors creates a core clinical challenge. In breast cancer cells rendered resistant to the antiestrogen fulvestrant, we defined causative mechanistic roles for the transcription factor YBX1 and the levels of ER and the ERBB2 receptor. Enforced expression of YBX1 in parental cells conferred resistance against tamoxifen and fulvestrant in vitro and in vivo Furthermore, YBX1 overexpression was associated with decreased and increased levels of ER and ERBB2 expression, respectively. In antiestrogen-resistant cells, increased YBX1 phosphorylation was associated with a 4-fold higher degradation rate of ER. Notably, YBX1 bound the ER, leading to its accelerated proteasomal degradation, and induced the transcriptional activation of ERBB2. In parallel fashion, tamoxifen treatment also augmented YBX1 binding to the ERBB2 promoter to induce increased ERBB2 expression. Together, these findings define a mechanism of drug resistance through which YBX1 contributes to antiestrogen bypass in breast cancer cells. Cancer Res; 77(2); 545-56. ©2016 AACR.
Assuntos
Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Moduladores de Receptor Estrogênico/farmacologia , Receptor ErbB-2/biossíntese , Receptores de Estrogênio/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo , Adulto , Animais , Western Blotting , Linhagem Celular Tumoral , Estradiol/análogos & derivados , Estradiol/farmacologia , Feminino , Fulvestranto , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Fosforilação , Tamoxifeno/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Although a few cases with idiopathic horseshoe-like macular tear have been reported, the mechanism remains unknown and a standard treatment has yet to be determined. OBJECTIVE: To report the outcome for a patient with idiopathic horseshoe-like macular tear who underwent vitreous surgery. CASE REPORT: A 65-year-old man with no previous injury or ophthalmic disease presented with abnormal vision in his left eye. Best-corrected visual acuity was 0.8 in the right and 0.3 in the left, and the relative afferent pupillary defect was negative. Ophthalmoscopy revealed a horseshoe-like tear on the temporal side of the macula in the left eye. The tear size was 0.75 disc diameters (DD). Optical coherence tomography showed that the focal retinal detachment reached the fovea. A few days after the first visit, there was no longer adhesion of the flap of the tear to the retina and the tear size had increased to 1.5 DD. The patient underwent vitreous surgery similar to large macular hole surgery, with the tear closure repaired using the inverted internal limiting membrane flap technique with 20% SF6 gas tamponade. Although the tear decreased to 0.5 DD after the surgery, complete closure of the tear was not achieved. CONCLUSION: While cases with horseshoe-like macular tear following trauma and branch retinal vein occlusion have been reported, to the best of our knowledge, this is the first reported idiopathic case. In the present case, there was expansion of the tear until the patient actually underwent surgery. If vertical vitreous traction indeed plays a role in horseshoe-like macular tears, this will need to be taken into consideration at the time of the vitreous surgery in these types of cases.
RESUMO
Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Although recent studies facilitate the identification of crucial genes and relevant regulatory pathways, therapeutic approaches against advanced HCC are insufficiently effective. Therefore, we aimed here to develop potent therapeutics to provide a reliable biomarker for the therapeutic efficacy in patients with HCC. To this end, we first compared the cytotoxic effects of various anti-cancer drugs between well differentiated (HAK-1A) and poorly differentiated (HAK-1B) cell lines established from a single HCC tumor. Of various drug screened, HAK-1B cells were more sensitive by a factor of 2,000 to the mTORC1 inhibitors (rapalogs), rapamycin and everolimus, than HAK-1A cells. Although rapalogs inhibited phosphorylation of mTOR Ser2448 in HAK-1A and HAK-1B cells, phosphorylation of mTOR Ser2481 was specifically inhibited only in HAK-1B cells. Silencing of Raptor induced apoptosis and inhibited the growth of only HAK-1B cells. Further, three other cell lines established independently from the tumors of three patients with HCC were also approximately 2,000-fold times more sensitive to rapamycin, which correlated closely with the inhibition of mTOR Ser2481 phosphorylation by rapamycin. Treatment with everolimus markedly inhibited the growth of tumors induced by poorly differentiated HAK-1B and KYN-2 cells and phosphorylation of mTOR Ser2481 in vivo. To our knowledge, this is the first study showing that the phosphorylation of mTOR Ser2481 is selectively inhibited by rapalogs in mTORC1-addicted HCC cells and may be a potential reliable biomarker for the therapeutic efficacy of rapalogs for treating HCC patients.