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Although CRISPR-Cas9 technology is poised to revolutionize the treatment of diseases with underlying genetic mutations, it faces some significant issues limiting clinical entry. They include low-efficiency in vivo systemic delivery and undesired off-target effects. Here, we demonstrate, by modifying Cas9 with phosphorothioate-DNA oligos (PSs), that one can efficiently deliver single and bi-specific CRISPR-Cas9/guide RNA (gRNA) dimers in vitro and in vivo with reduced off-target effects. We show that PS-Cas9/gRNA-mediated gene knockout preserves chimeric antigen receptor T cell viability and expansion in vitro and in vivo. PS-Cas9/gRNA mediates gene perturbation in patient-derived tumor organoids and mouse xenograft tumors, leading to potent tumor antitumor effects. Further, HER2 antibody-PS-Cas9/gRNA conjugate selectively perturbs targeted genes in HER2+ ovarian cancer xenografts in vivo. Moreover, we created bi-specific PS-Cas9 with two gRNAs to target two adjacent sequences of the same gene, leading to efficient targeted gene disruption ex vivo and in vivo with markedly reduced unintended gene perturbation. Thus, the cell-penetrating PS-Cas9/gRNA can achieve efficient systemic delivery and precision in gene disruption.
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BACKGROUND: Ovarian cancer is the most lethal gynecological malignancy, with limited treatment options after failure of standard therapies. Despite the potential of poly(ADP-ribose) polymerase inhibitors in treating DNA damage response (DDR)-deficient ovarian cancer, the development of resistance and immunosuppression limit their efficacy, necessitating alternative therapeutic strategies. Inhibitors of poly(ADP-ribose) glycohydrolase (PARG) represent a novel class of inhibitors that are currently being assessed in preclinical and clinical studies for cancer treatment. METHODS: By using a PARG small-molecule inhibitor, COH34, and a cell-penetrating antibody targeting the PARG's catalytic domain, we investigated the effects of PARG inhibition on signal transducer and activator of transcription 3 (STAT3) in OVCAR8, PEO1, and Brca1-null ID8 ovarian cancer cell lines, as well as in immune cells. We examined PARG inhibition-induced effects on STAT3 phosphorylation, nuclear localization, target gene expression, and antitumor immune responses in vitro, in patient-derived tumor organoids, and in an immunocompetent Brca1-null ID8 ovarian mouse tumor model that mirrors DDR-deficient human high-grade serous ovarian cancer. We also tested the effects of overexpressing a constitutively activated STAT3 mutant on COH34-induced tumor cell growth inhibition. RESULTS: Our findings show that PARG inhibition downregulates STAT3 activity through dephosphorylation in ovarian cancer cells. Importantly, overexpression of a constitutively activated STAT3 mutant in tumor cells attenuates PARG inhibitor-induced growth inhibition. Additionally, PARG inhibition reduces STAT3 phosphorylation in immune cells, leading to the activation of antitumor immune responses, shown in immune cells cocultured with ovarian cancer patient tumor-derived organoids and in immune-competent mice-bearing mouse ovarian tumors. CONCLUSIONS: We have identified a novel antitumor mechanism underlying PARG inhibition beyond its primary antitumor effects through blocking DDR in ovarian cancer. Furthermore, targeting PARG activates antitumor immune responses, thereby potentially increasing response rates to immunotherapy in patients with ovarian cancer.
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Glicósido Hidrolasas , Neoplasias Ováricas , Factor de Transcripción STAT3 , Animales , Femenino , Humanos , Ratones , Línea Celular , Inmunidad , Neoplasias Ováricas/tratamiento farmacológico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Factor de Transcripción STAT3/efectos de los fármacos , Factor de Transcripción STAT3/metabolismo , Glicósido Hidrolasas/antagonistas & inhibidores , Glicósido Hidrolasas/metabolismoRESUMEN
Reprogrammed metabolism is a hallmark of cancer. However, the metabolic dependency of cancer, from tumour initiation through disease progression and therapy resistance, requires a spectrum of distinct reprogrammed cellular metabolic pathways. These pathways include aerobic glycolysis, oxidative phosphorylation, reactive oxygen species generation, de novo lipid synthesis, fatty acid ß-oxidation, amino acid (notably glutamine) metabolism and mitochondrial metabolism. This Review highlights the central roles of signal transducer and activator of transcription (STAT) proteins, notably STAT3, STAT5, STAT6 and STAT1, in orchestrating the highly dynamic metabolism not only of cancer cells but also of immune cells and adipocytes in the tumour microenvironment. STAT proteins are able to shape distinct metabolic processes that regulate tumour progression and therapy resistance by transducing signals from metabolites, cytokines, growth factors and their receptors; defining genetic programmes that regulate a wide range of molecules involved in orchestration of metabolism in cancer and immune cells; and regulating mitochondrial activity at multiple levels, including energy metabolism and lipid-mediated mitochondrial integrity. Given the central role of STAT proteins in regulation of metabolic states, they are potential therapeutic targets for altering metabolic reprogramming in cancer.
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Neoplasias , Factores de Transcripción STAT , Transducción de Señal , Humanos , Citocinas/metabolismo , Lípidos/uso terapéutico , Neoplasias/patología , Factor de Transcripción STAT3/metabolismo , Microambiente TumoralRESUMEN
Recently, poly(ADP-ribosyl)ation polymerase inhibitors (PARPis), which induce synthetic lethality of tumor cells with DNA damage repair defects, have emerged as a promising therapy for ovarian, breast, and pancreatic cancer. Although the PARPi Olaparib is limited to treating cancer patients with DNA repair deficiencies, the PARPi Niraparib is FDA approved to treat ovarian cancer patients regardless of their status in DNA repair pathways. Despite differences in the affinity to PARP enzymes, the rationale behind the clinical use of Niraparib in patients without DNA repair deficiencies is still lacking. Moreover, only Olaparib has been approved for pancreatic ductal adenocarcinoma (PDAC) patients with BRCA mutations, accounting for only 5-7% of total PDACs. It remains unclear whether Niraparib could be beneficial to PDACs without BRCA mutations. We found that Niraparib inhibits ovarian and PDAC tumor cell growth, regardless of BRCA mutational status, more effectively than Olaparib. Unlike Olaparib, which is known to activate STAT3, Niraparib inhibits STAT3 activity in ovarian and PDAC cancer cell lines and patient tumors. Moreover, Niraparib regulates the expression of several STAT3 downstream genes involved in apoptosis. Overexpression of a constitutively activated STAT3 mutant rescues Niraparib-induced cancer cell apoptosis. Our results suggest that Niraparib inhibits pSTAT3 by interfering with SRC tyrosine kinase. Collectively, our studies provide a mechanism underlying Niraparib's ability to induce tumor cell apoptosis without BRCA mutations, suggesting the potential use of Niraparib for treating PDAC patients regardless of BRCA status.
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If the tigernut meal left after oil extraction is used as a material for starch resources instead of being wasted, the industrial value of tigernut would be improved. Thus, we investigated the effect of extrusion before oil extraction on the yield, structure and function of starches within tigernut meals (TMS). Compared with the yield of native starch, the yield of TMS-130-11 (barrel temperature: 130 °C; feed moisture: 11 %) was increased by 1.97 %, and that of TMS-140-11 (barrel temperature: 140 °C; feed moisture: 11 %) was decreased by 7.82 %. The starches cannot be obtained when the barrel temperature is above 140 °C with 11 % feed moisture. Extrusion slightly decreased the relative crystallinity and increased the ratio of B2-chains in amylopectin. These changes resulted in reductions in peak viscosity while improving the elastic properties of the starch gel. These results will provide useful information regarding the use of starch isolated from tigernut meal.
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Amilopectina , Almidón , Amilopectina/química , Amilosa , Comidas , Almidón/química , Temperatura , ViscosidadRESUMEN
The key point in facing the demand for the disposal of waste storage in rural areas of China is to manage informal landfills. However, limited studies have been conducted to evaluate the phytoremediation efficiency of heavy metal and polycyclic aromatic hydrocarbon (PAHs) co-contaminated dumpsite soil with high ammonia nitrogen content. In this study, we selected the tolerant plant legume alfalfa (Medicago sativa L.) for a pot experiment to investigate the effects of nitrogen (N) (0, 10, and 50 mg·kg-1) on plant growth, the removal of pollutants, and soil bacterial community structure in Cd-PAHs co-contaminated soil, so as to evaluate the role of N in the process of phytoremediation of dumpsite soil. The results showed that the biomass of alfalfa under high co-contamination conditions (Cd:10 mg·kg-1 and PAHs:400 mg·kg-1) increased with N supply and was 6.0 and 6.3 times higher than that of the treatment without N supply, respectively. Furthermore, the lower N level promoted the growth of alfalfa in the low-contamination group (Cd:1 mg·kg-1 and PAHs:100 mg·kg-1), but the difference was not significant, and a high concentration of N significantly inhibited its growth. In addition, the phytoremediation efficiency for Cd in the low-contamination group ranged from 5.58% to 7.49%, and N significantly increased the efficiency in the high co-contamination group from 0.95% to 3.02%. Compared with the removal of phenanthrene, N had a stronger influence on the removal of pyrene. Meanwhile, alfalfa could promote the removal of them in soil, among which the degradation of PAHs by microorganisms was dominant, whereas the contribution of the plant uptake pathway was less than 0.21%. As reflected by distance-based redundancy analysis (db-RDA), PAHs and Cd were the main factors affecting the structure of the microbial community; moreover, N had a greater effect on bacterial community composition in the single Cd-contamination and high co-contamination groups, promoting genera with bioremediation effects as the dominant soil bacterial communities, including Arthrobacter, Microbacterium, and Novosphingobium. This study will provide a theoretical basis for the remediation of dumpsites as well as informal landfills with contaminated soil.
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Metales Pesados , Fenantrenos , Hidrocarburos Policíclicos Aromáticos , Contaminantes del Suelo , Amoníaco/metabolismo , Biodegradación Ambiental , Cadmio/análisis , Medicago sativa , Metales Pesados/análisis , Nitrógeno/análisis , Fenantrenos/análisis , Hidrocarburos Policíclicos Aromáticos/análisis , Pirenos/análisis , Suelo/química , Microbiología del Suelo , Contaminantes del Suelo/análisisRESUMEN
Overcoming resistance to chemotherapies remains a major unmet need for cancers, such as triple-negative breast cancer (TNBC). Therefore, mechanistic studies to provide insight for drug development are urgently needed to overcome TNBC therapy resistance. Recently, an important role of fatty acid ß-oxidation (FAO) in chemoresistance has been shown. But how FAO might mitigate tumor cell apoptosis by chemotherapy is unclear. Here, we show that elevated FAO activates STAT3 by acetylation via elevated acetyl-coenzyme A (CoA). Acetylated STAT3 upregulates expression of long-chain acyl-CoA synthetase 4 (ACSL4), resulting in increased phospholipid synthesis. Elevating phospholipids in mitochondrial membranes leads to heightened mitochondrial integrity, which in turn overcomes chemotherapy-induced tumor cell apoptosis. Conversely, in both cultured tumor cells and xenograft tumors, enhanced cancer cell apoptosis by inhibiting ASCL4 or specifically targeting acetylated-STAT3 is associated with a reduction in phospholipids within mitochondrial membranes. This study demonstrates a critical mechanism underlying tumor cell chemoresistance.
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Membranas Mitocondriales , Neoplasias de la Mama Triple Negativas , Acetilcoenzima A/metabolismo , Apoptosis , Ácidos Grasos/metabolismo , Humanos , Lípidos de la Membrana/metabolismo , Membranas Mitocondriales/metabolismo , Oxidación-Reducción , Fosfolípidos/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
BACKGROUND: The aim of this study is to test if the newly proposed 45 mm size criterion for ascending aortic replacement (AAR) in bicuspid aortic valve (BAV) patients undergoing aortic valve replacement (AVR) is predictive of improved early outcomes. METHODS: Data of 306 BAV patients with an aortic diameter of ≥45 mm undergoing AVR alone or with AAR were retrospectively analyzed. Patients were divided into groups of AVR + AAR (n = 220) and AVR only (n = 86) based on if surgery was performed according to the 45 mm criterion. End point was early adverse events, including 30-day and in-hospital mortality, cardiac events, acute renal failure, stroke, and reoperation for bleeding. Cox regression was used to assess if conformance to 45 mm criterion could predict fewer early adverse events. RESULTS: AVR + AAR group had significantly higher postoperative left ventricular ejection fraction (LVEF) (0.59 ± 0.09 vs. 0.55 ± 0.11, p = 0.006) and longer cardiopulmonary bypass (CPB) time (128 vs. 111 minutes, p = 0.002). Early adverse events occurred in 45 patients (14.7%), which was more prevalent in the AVR-only group (22.1% vs. 11.8%, p = 0.020). Conformance to the 45 mm criterion predicted lower rate of early adverse events (hazard ratio [HR]: 0.53, 95% confidence interval [CI]: 0.28-0.98, p = 0.042). After adjustment for gender, age, AAo diameter, sinuses of Valsalva diameter, preoperative LVEF, Sievers subtypes, BAV valvulopathy, and CPB and cross-clamp times, conformance to the 45 mm size criterion still predicted lower incidence of early adverse events (HR: 0.37, 95% CI: 0.15-0.90, p = 0.028). CONCLUSIONS: This study shows that conformance to 45 mm size cutoff for preemptive AAR during aortic valve replacement in patients with BAV was not associated with increased risk for adverse events and may improve early surgical outcomes.
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Estenosis de la Válvula Aórtica , Enfermedad de la Válvula Aórtica Bicúspide , Implantación de Prótesis de Válvulas Cardíacas , Válvula Aórtica/diagnóstico por imagen , Válvula Aórtica/cirugía , Estenosis de la Válvula Aórtica/diagnóstico por imagen , Estenosis de la Válvula Aórtica/etiología , Estenosis de la Válvula Aórtica/cirugía , Implantación de Prótesis de Válvulas Cardíacas/efectos adversos , Humanos , Estudios Retrospectivos , Volumen Sistólico , Resultado del Tratamiento , Función Ventricular IzquierdaRESUMEN
Despite the promising activity of poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) in many cancer types with defects in the DNA damage response the majority of the treated patients acquire PARPi resistance and succumb to their diseases. Consequently, there is an urgent need to identify the mechanisms of PARPi resistance. Here, we show that PARPi treatment promotes STAT3 activation in ovarian cancer cells, tumor-associated immune cells and fibroblasts, resulting in PARPi resistance and immunosuppression. Comparison of ovarian cancer patient-matched tumor biopsies before and after PARPi therapy revealed that STAT3 activity was significantly higher in tumor cells and tumor-associated immune cells and fibroblasts post PARPi treatment. Moreover, one-time PARPi treatment activated STAT3 both in tumor cells as well as diverse immune subsets and fibroblasts. PARPi-treated immune cells exhibited decreased expression of immunostimulatory interferon (IFN)-γ and Granzyme B while increasing immunosuppressive cytokine IL-10. Finally, we demonstrate that the acquisition of PARPi resistance in ovarian cancer cells was accompanied by increased STAT3 activity. Ablating STAT3 inhibited PARPi-resistant ovarian tumor cell growth and/or restored PARPi sensitivity. Therefore, our study has identified a critical mechanism intrinsic to PARPi that promotes resistance to PARPi and induces immunosuppression during PARPi treatment by activating STAT3 in tumor cells and tumor-associated immune cells/fibroblasts.
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Clinically, immune cell function is correlated with pathogenesis of endometrial polyp (EP) and infertility of women of reproductive-age. However, the underlying immune cell hallmark in EP patients remains unclear. Here, we focused on analyzing circulating immune cells, and attempted to reveal the correlation between peripheral immune cell functional phenotypes and fertility in EP patients. Through comparison of circulating CD4+/CD8+ T cells, NK cells, and γδ T cells between 64 EP patients and 68 healthy females, we found that γδ T cells, but not CD4+/CD8+ T cells and NK cells, were immunologically correlated with conception rate and conception interval time. Specifically, total γδ T cells and the Vδ1+PD1+ γδ T subpopulation decreased whereas the Vδ1/Vδ2 ratio increased in EP patients compared to healthy controls. Moreover, the patients with the higher Vδ1/Vδ2 ratio (median value equals 1.04) had a poorer fertility and longer interval time of conception (210 days versus 158 days for control). Meanwhile, higher Vδ1+PD1+ γδ T cell proportion (median equals 15.7) was positively correlative with both higher conception rate and shortened median conception interval time (130 days for Vδ1+PD1high group versus 194 days for Vδ1+PD1low group). Notably, in healthy controls, both Vδ1/Vδ2 ratio and Vδ1+PD1+ γδ T cell proportion correlated with pregnancy rate oppositely, comparing to EP patients. Together, our results suggested that imbalanced γδ T cell population occurred in EP patients, and that Vδ1/Vδ2 ratio and PD-1 expression of Vδ1+ γδ T cells could be potentially developed into valuable predictors for fertility in EP patients.
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Endometrio/inmunología , Fertilidad/inmunología , Linfocitos Intraepiteliales/inmunología , Pólipos/sangre , Pólipos/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Adulto , Estudios de Casos y Controles , Femenino , Estudios de Seguimiento , Humanos , Masculino , Embarazo , Adulto JovenRESUMEN
To date, there are no inhibitors that directly and specifically target activated STAT3 and c-Myc in the clinic. Although peptide-based inhibitors can selectively block activated targets, their clinical usage is limited because of low cell penetration and/or serum stability. Here, we generated cell-penetrating acetylated (acet.) STAT3, c-Myc, and Gp130 targeting peptides by attaching phosphorothioated (PS) polymer backbone to peptides. The cell-penetrating peptides efficiently penetrated cells and inhibited activation of the intended targets and their downstream genes. Locally or systemically treating tumor-bearing mice with PS-acet.-STAT3 peptide at low concentrations effectively blocked STAT3 in vivo, resulting in significant antitumor effects in 2 human xenograft models. Moreover, PS-acet.-STAT3 peptide penetrated and activated splenic CD8+ T cells in vitro. Treating immune-competent mice bearing mouse melanoma with PS-acet.-STAT3 peptide inhibited STAT3 in tumor-infiltrating T cells, downregulating tumor-infiltrating CD4+ T regulatory cells while activating CD8+ T effector cells. Similarly, systemic injections of the cell-penetrating c-Myc and Gp130 peptides prevented pancreatic tumor growth and induced antitumor immune responses. Taken together, we have developed therapeutic peptides that effectively and specifically block challenging cancer targets, resulting in antitumor effects through both direct tumor cell killing and indirectly through antitumor immune responses.
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Antineoplásicos/farmacología , Péptidos de Penetración Celular/farmacología , Factor de Transcripción STAT3/antagonistas & inhibidores , Acetilación , Animales , Antineoplásicos/química , Línea Celular Tumoral , Péptidos de Penetración Celular/química , Receptor gp130 de Citocinas/química , Diseño de Fármacos , Células HCT116 , Humanos , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/inmunología , Melanoma Experimental/metabolismo , Ratones , Ratones Endogámicos C57BL , Neoplasias Pancreáticas/tratamiento farmacológico , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Multimerización de Proteína , Proteínas Proto-Oncogénicas c-myc/química , Proteínas Proto-Oncogénicas c-myc/farmacología , Factor de Transcripción STAT3/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Depression is a prevalent mental disorder that is associated with aging and contributes to increased mortality and morbidity. The overall prevalence of geriatric depression with clinically significant symptoms is currently on the rise. Recent studies have demonstrated that altered expressions of long non-coding RNAs (lncRNAs) in the brain affect neurodevelopment and manifest modulating functions during the depression. However, most lncRNAs have not yet been studied. Herein, we analyzed the transcriptome of dysregulated lncRNAs to reveal their expressions in a mouse model exhibiting depressive-like behaviors, as well as their corresponding response following antidepressant fluoxetine treatment. A chronic unpredictable mild stress (CUMS) mouse model was applied. A six-week fluoxetine intervention in CUMS-induced mice attenuated depressive-like behaviors. In addition, differential expression analysis of lncRNAs was performed following RNA-sequencing. A total of 282 lncRNAs (134 up-regulated and 148 down-regulated) were differentially expressed in CUMS-induced mice relative to non-stressed counterparts ( P<0.05). Moreover, 370 differentially expressed lncRNAs were identified in CUMS-induced mice after fluoxetine intervention. Gene Ontology (GO) analyses showed an association between significantly dysregulated lncRNAs and protein binding, oxygen binding, and transport activity, while the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that these dysregulated lncRNAs might be involved in inflammatory response pathways. Fluoxetine effectively ameliorated the symptoms of depression in CUMS-induced mice by regulating the expression of lncRNAs in the hippocampus. The findings herein provide valuable insights into the potential mechanism underlying depression in elderly people.
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Trastorno Depresivo/tratamiento farmacológico , Fluoxetina/uso terapéutico , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , ARN Largo no Codificante/metabolismo , Animales , Antidepresivos de Segunda Generación/uso terapéutico , Masculino , Ratones , Ratones Endogámicos BALB C , ARN Largo no Codificante/genética , Estrés PsicológicoRESUMEN
Despite significant progress in cancer therapy over the last decades, ovarian cancer remains the most lethal gynecologic malignancy worldwide with the five-year overall survival rate less than 30% due to frequent disease recurrence and chemoresistance. CD44 is a non-kinase transmembrane receptor that has been linked to cancer metastatic progression, cancer stem cell maintenance, and chemoresistance development via multiple mechanisms across many cancers, including ovarian, and represents a promising therapeutic target for ovarian cancer treatment. Moreover, CD44-mediated signaling interacts with other well-known pro-tumorigenic pathways and oncogenes during cancer development, such as signal transducer and activator of transcription 3 (STAT3). Given that both CD44 and STAT3 are strongly implicated in the metastatic progression and chemoresistance of ovarian tumors, this review summarizes currently available evidence about functional crosstalk between CD44 and STAT3 in human malignancies with an emphasis on ovarian cancer. In addition to the role of tumor cell-intrinsic CD44 and STAT3 interaction in driving cancer progression and metastasis, we discuss how CD44 and STAT3 support the pro-tumorigenic tumor microenvironment and promote tumor angiogenesis, immunosuppression, and cancer metabolic reprogramming in favor of cancer progression. Finally, we review the current state of therapeutic CD44 targeting and propose superior treatment possibilities for ovarian cancer.
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Although obesity is known to be critical for cancer development, how obesity negatively impacts antitumor immune responses remains largely unknown. Here, we show that increased fatty acid oxidation (FAO) driven by activated STAT3 in CD8+ T effector cells is critical for obesity-associated breast tumor progression. Ablating T cell Stat3 or treatment with an FAO inhibitor in obese mice spontaneously developing breast tumor reduces FAO, increases glycolysis and CD8+ T effector cell functions, leading to inhibition of breast tumor development. Moreover, PD-1 ligation in CD8+ T cells activates STAT3 to increase FAO, inhibiting CD8+ T effector cell glycolysis and functions. Finally, leptin enriched in mammary adipocytes and fat tissues downregulates CD8+ T cell effector functions through activating STAT3-FAO and inhibiting glycolysis. We identify a critical role of increased oxidation of fatty acids driven by leptin and PD-1 through STAT3 in inhibiting CD8+ T effector cell glycolysis and in promoting obesity-associated breast tumorigenesis.
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Neoplasias de la Mama/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Carcinogénesis/inmunología , Ácidos Grasos/metabolismo , Obesidad/metabolismo , Factor de Transcripción STAT3/metabolismo , Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Carcinogénesis/genética , Carcinogénesis/metabolismo , Línea Celular , Proliferación Celular/genética , Inmunoprecipitación de Cromatina , Femenino , Glucólisis/genética , Glucólisis/fisiología , Humanos , Interferón gamma/metabolismo , Leptina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Obesidad/inmunología , Oxidación-Reducción/efectos de los fármacos , Receptor de Muerte Celular Programada 1/metabolismo , Factor de Transcripción STAT3/genéticaRESUMEN
Despite their well-recognized success in the clinic, antibodies generally do not penetrate cellular membranes to target intracellular molecules, many of which underlie incurable diseases. Here we show that covalently conjugating phosphorothioated DNA oligonucleotides to antibodies enabled their efficient cellular internalization. Antibody cell penetration was partially mediated by membrane potential alteration. Moreover, without an antigen to bind, intracellular levels of the modified antibodies underwent cellular clearance, which involved efflux and lysosomal degradation, enabling detection of intended intracellular molecules as tested in fibroblasts, tumor cells, and T cells. This target-dependent cellular retention of modified antibodies extended to in vivo studies. Both local and systemic administrations of low doses of modified antibodies effectively inhibited intracellular targets, such as transcription factors Myc, interferon regulatory factor 4, and tyrosine-protein kinase SRC, and expression of their downstream genes in tumors, resulting in tumor cell apoptosis and tumor growth inhibition. This simple modification enables the use of antibodies to detect and modulate intracellular molecules in both cultured living cells and in whole animals, forming the foundation for a new paradigm for antibody-based research, diagnostics, and therapeutics.
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Anticuerpos Monoclonales/administración & dosificación , Permeabilidad de la Membrana Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos/métodos , Inmunoconjugados/administración & dosificación , Oligodesoxirribonucleótidos/farmacología , Animales , Anticuerpos Monoclonales/química , Línea Celular Tumoral , Humanos , Inmunoconjugados/química , Inmunoconjugados/farmacología , Melanoma/tratamiento farmacológico , Melanoma/patología , Ratones , Oligodesoxirribonucleótidos/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Ubiquitin-like (Ubl) post-translational modifications are potential targets for therapeutics. However, the only known mechanism for inhibiting a Ubl-activating enzyme is through targeting its ATP-binding site. Here we identify an allosteric inhibitory site in the small ubiquitin-like modifier (SUMO)-activating enzyme (E1). This site was unexpected because both it and analogous sites are deeply buried in all previously solved structures of E1s of ubiquitin-like modifiers (Ubl). The inhibitor not only suppresses SUMO E1 activity, but also enhances its degradation in vivo, presumably due to a conformational change induced by the compound. In addition, the lead compound increased the expression of miR-34b and reduced c-Myc levels in lymphoma and colorectal cancer cell lines and a colorectal cancer xenograft mouse model. Identification of this first-in-class inhibitor of SUMO E1 is a major advance in modulating Ubl modifications for therapeutic aims.
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Sumoilación , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Regulación Alostérica , Sitio Alostérico , Animales , Línea Celular Tumoral , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Ratones SCID , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Sumoilación/efectos de los fármacos , Trasplante Heterólogo , Ubiquitina/metabolismo , Enzimas Activadoras de Ubiquitina/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
The miR-34 family of microRNAs suppresses the expression of proteins involved in pluripotency and oncogenesis. miR-34 expression is frequently reduced in cancers; however, the regulation of their expression is not well understood. We used genome-wide miRNA profiling and mechanistic analysis to show that SUMOylation regulates miR-34b/c expression, which impacts the expression of c-Myc and other tested miR-34 targets. We used site-directed mutagenesis and other methods to show that protein kinase B (also known as Akt) phosphorylation of FOXO3a plays an important role in SUMOylation-dependent expression of miR-34b/c. This study reveals how the miR-34-targeted gene expression program is regulated by SUMOylation and shows that SUMOylation need not regulate target proteins through direct modification, but instead can act through the expression of their targeting miRNAs.