RESUMO
Each of us is a story. Mine is a story of doing science for 60 years, and I am honored to be asked to tell it. Even though this autobiography was written for the Annual Review of Immunology, I have chosen to describe my whole career in science because the segment that was immunology is so intertwined with all else I was doing. This article is an elongation and modification of a talk I gave at my 80th birthday celebration at Caltech on March 23, 2018.
Assuntos
Alergia e Imunologia/história , NF-kappa B/metabolismo , Vírus de RNA/fisiologia , Viroses/imunologia , Animais , Modelos Animais de Doenças , Rearranjo Gênico , História do Século XX , História do Século XXI , Humanos , Camundongos , Proteínas Tirosina Quinases/metabolismo , Transcrição Reversa , Estados UnidosRESUMO
Chinese guidelines recommend POF (paclitaxel, oxaliplatin, and 5-FU/levoleucovorin) as first-line treatment for advanced gastric cancer (AGC). Apatinib can augment the antitumor effect of paclitaxel, oxaliplatin, or fluorouracil in preclinical studies of AGC. A phase I clinical trial was conducted to evaluate the anticancer activity and maximum tolerated dose (MTD) of apatinib plus POF in treatment-naïve patients with AGC and to establish a recommended phase II dose. Participants received escalating doses of daily oral apatinib (250, 375, 500, 625, 750, and 850 mg) plus POF every 2 weeks using a conventional "3 + 3" study design. Among 21 treated patients, one experienced a dose-limiting toxicity (grade 3 skin ulceration at 850 mg). No MTD was reached. Apatinib 750 mg plus POF was recommended for phase II study. The most common grade 3-4 adverse events (AEs) were neutropenia (33.3%), mucositis (14.3%), and hand-foot syndrome (14.3%). Median progression-free and overall survival were 10.4 months (95% CI: 6.3, 14.6) and 18.4 months (95% CI: 9.8, 28.2), respectively. Apatinib up to 850 mg coadministered with POF was well tolerated with manageable AEs. The safety and anticancer activity of this regimen warrants its further investigation as first-line treatment for AGC in a larger study.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Fluoruracila , Leucovorina , Dose Máxima Tolerável , Oxaliplatina , Paclitaxel , Piridinas , Neoplasias Gástricas , Humanos , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologia , Pessoa de Meia-Idade , Masculino , Feminino , Piridinas/administração & dosagem , Piridinas/efeitos adversos , Piridinas/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Idoso , Paclitaxel/administração & dosagem , Paclitaxel/efeitos adversos , Paclitaxel/uso terapêutico , Adulto , Oxaliplatina/administração & dosagem , Oxaliplatina/uso terapêutico , Oxaliplatina/efeitos adversos , Fluoruracila/administração & dosagem , Fluoruracila/efeitos adversos , Fluoruracila/uso terapêutico , Leucovorina/administração & dosagem , Leucovorina/uso terapêutico , Leucovorina/efeitos adversosRESUMO
BACKGROUND: Interleukin 24 (IL-24) has been implicated in the nociceptive signaling. However, direct evidence and the precise molecular mechanism underlying IL-24's role in peripheral nociception remain unclear. METHODS: Using patch clamp recording, molecular biological analysis, immunofluorescence labeling, siRNA-mediated knockdown approach and behavior tests, we elucidated the effects of IL-24 on sensory neuronal excitability and peripheral pain sensitivity mediated by T-type Ca2+ channels (T-type channels). RESULTS: IL-24 enhances T-type channel currents (T-currents) in trigeminal ganglion (TG) neurons in a reversible and dose-dependent manner, primarily by activating the interleukin-22 receptor 1 (IL-22R1). Furthermore, we found that the IL-24-induced T-type channel response is mediated through tyrosine-protein kinase Lyn, but not its common downstream target JAK1. IL-24 application significantly activated protein kinase A; this effect was independent of cAMP and prevented by Lyn antagonism. Inhibition of PKA prevented the IL-24-induced T-current response, whereas inhibition of protein kinase C or MAPK kinases had no effect. Functionally, IL-24 increased TG neuronal excitability and enhanced pain sensitivity to mechanical stimuli in mice, both of which were suppressed by blocking T-type channels. In a trigeminal neuropathic pain model induced by chronic constriction injury of the infraorbital nerve, inhibiting IL-22R1 signaling alleviated mechanical allodynia, which was reversed by blocking T-type channels or knocking down Cav3.2. CONCLUSION: Our findings reveal that IL-24 enhances T-currents by stimulating IL-22R1 coupled to Lyn-dependent PKA signaling, leading to TG neuronal hyperexcitability and pain hypersensitivity. Understanding the mechanism of IL-24/IL-22R1 signaling in sensory neurons may pave the way for innovative therapeutic strategies in pain management.
Assuntos
Canais de Cálcio Tipo T , Proteínas Quinases Dependentes de AMP Cíclico , Receptores de Interleucina , Células Receptoras Sensoriais , Transdução de Sinais , Gânglio Trigeminal , Quinases da Família src , Animais , Canais de Cálcio Tipo T/metabolismo , Canais de Cálcio Tipo T/genética , Quinases da Família src/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Gânglio Trigeminal/metabolismo , Masculino , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/fisiologia , Receptores de Interleucina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Interleucinas/metabolismoRESUMO
BACKGROUND: Rivoceranib is an oral, selective tyrosine kinase inhibitor of vascular endothelial growth factor receptor-2. ANGEL (NCT03042611) was a global, randomized, double-blinded, placebo-controlled, phase 3 study evaluating rivoceranib as 3rd-line or ≥4th-line therapy in patients with advanced/metastatic gastric or gastroesophageal junction (GEJ) cancer. METHODS: Patients had failed ≥2 lines of chemotherapy and were randomized 2:1 to rivoceranib 700 mg once daily or placebo with best supportive care. PRIMARY ENDPOINT: overall survival (OS) in the intention-to-treat population. Secondary endpoints: progression-free survival (PFS), objective response rate (ORR), and disease control rate (DCR) by blinded independent central review (BICR). RESULTS: In total, 460 patients (rivoceranib n = 308, placebo n = 152) were enrolled. OS was not statistically different for rivoceranib versus placebo (median 5.78 vs. 5.13 months; hazard ratio [HR] 0.93, 95% CI 0.74-1.15; p = 0.4724). PFS by BICR (median 2.83 vs. 1.77 months; HR 0.58, 95% CI 0.47-0.71; p < 0.0001), ORR (6.5% vs. 1.3%; p = 0.0119), and DCR (40.3 vs. 13.2%; p < 0.0001) were improved with rivoceranib versus placebo. In patients receiving ≥4th-line therapy, OS (median 6.34 vs. 4.73 months; p = 0.0192) and PFS by BICR (median 3.52 vs. 1.71 months; p < 0.0001) were improved with rivoceranib versus placebo. The most common grade ≥ 3 treatment-emergent adverse events with rivoceranib were hypertension (17.9%), anemia (10.4%), aspartate aminotransferase increased (9.4%), asthenia (8.5%), and proteinuria (7.5%). CONCLUSIONS: This study did not meet its primary OS endpoint. Compared to placebo, rivoceranib improved PFS, ORR, and DCR. Rivoceranib also improved OS in a prespecified patient subgroup receiving ≥4th-line therapy.
Assuntos
Piridinas , Neoplasias Gástricas , Humanos , Neoplasias Gástricas/patologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular , Fator A de Crescimento do Endotélio Vascular , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Junção Esofagogástrica/patologia , Método Duplo-CegoRESUMO
Vascular endothelial growth factor receptor-targeted tyrosine kinase inhibitors (VEGFR-TKIs) are often used for treatment of several types of cancer; however, they are associated with an increased risk of proteinuria, sometimes leading to treatment discontinuation. We searched PubMed and Scopus to identify clinical studies examining the incidence and risk factors for proteinuria caused by VEGFR-TKIs in patients with renal cell carcinoma, thyroid cancer, and hepatocellular carcinoma. The global incidence of proteinuria ranged from 6% to 34% for all grades of proteinuria, and from 1% to 10% for grade ≥3 proteinuria. The incidence of proteinuria did not differ significantly by cancer type, but in all three cancer types, there was a trend toward a higher incidence of proteinuria with lenvatinib than with other VEGFR-TKIs. In terms of risk factors, the incidence of proteinuria was significantly higher among Asians (including Japanese) compared with non-Asian populations. Other risk factors included diabetes mellitus, hypertension, and previous nephrectomy. When grade 3/4 proteinuria occurs, patients should be treated according to the criteria for dose reduction or withdrawal specified for each drug. For grade 2 proteinuria, treatment should be continued when the benefits outweigh the risks. Referral to a nephrologist should be considered for symptoms related to decreased renal function or when proteinuria has not improved after medication withdrawal. These management practices should be implemented universally, regardless of the cancer type.
Assuntos
Carcinoma Hepatocelular , Carcinoma de Células Renais , Neoplasias Renais , Neoplasias Hepáticas , Proteinúria , Neoplasias da Glândula Tireoide , Humanos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/complicações , Incidência , Neoplasias Renais/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Prevalência , Proteinúria/epidemiologia , Quinolinas/uso terapêutico , Quinolinas/efeitos adversos , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fatores de Risco , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/complicações , Neoplasias da Glândula Tireoide/epidemiologia , /uso terapêuticoRESUMO
The phase 2, single-arm, multicenter, open-label J-ALTA study evaluated the efficacy and safety of brigatinib in Japanese patients with advanced ALK+ non-small-cell lung cancer (NSCLC). One expansion cohort of J-ALTA enrolled patients previously treated with ALK tyrosine kinase inhibitors (TKIs); the main cohort included patients with prior alectinib ± crizotinib. The second expansion cohort enrolled patients with TKI-naive ALK+ NSCLC. All patients received brigatinib 180 mg once daily (7-day lead-in at 90 mg daily). Among 47 patients in the main cohort, 5 (11%) remained on brigatinib at the study end (median follow-up: 23 months). In this cohort, the independent review committee (IRC)-assessed objective response rate (ORR) was 34% (95% CI, 21%-49%); median duration of response was 14.8 months (95% CI, 5.5-19.4); median IRC-assessed progression-free survival (PFS) was 7.3 months (95% CI, 3.7-12.9). Among 32 patients in the TKI-naive cohort, 25 (78%) remained on brigatinib (median follow-up: 22 months); 2-year IRC-assessed PFS was 73% (90% CI, 55%-85%); IRC-assessed ORR was 97% (95% CI, 84%-100%); the median duration of response was not reached (95% CI, 19.4-not reached); 2-year duration of response was 70%. Grade ≥3 adverse events occurred in 68% and 91% of TKI-pretreated and TKI-naive patients, respectively. Exploratory analyses of baseline circulating tumor DNA in ALK TKI-pretreated NSCLC showed associations between poor PFS and EML4-ALK fusion variant 3 and TP53. Brigatinib is an important treatment option for Japanese patients with ALK+ NSCLC, including patients previously treated with alectinib.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/induzido quimicamente , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/induzido quimicamente , População do Leste Asiático , Quinase do Linfoma Anaplásico/genética , Inibidores de Proteínas Quinases/efeitos adversosRESUMO
BACKGROUND: Epstein-Barr virus (EBV) affects more than 90% of global population. The role of the virus in causing infectious mononucleosis (IM) affecting B-cells and epithelial cells and in the development of EBV associated cancers is well documented. Investigating the associated interactions can pave way for the discovery of novel therapeutic targets for EBV associated lymphoproliferative (Burkitt's Lymphoma and Hodgkin's Lymphoma) and non-lymphoproliferative diseases (Gastric cancer and Nasopharyngeal cancer). METHODS: Based on the DisGeNET (v7.0) data set, we constructed a disease-gene network to identify genes that are involved in various carcinomas, viz. Gastric cancer (GC), Nasopharyngeal cancer (NPC), Hodgkin's lymphoma (HL) and Burkitt's lymphoma (BL). We identified communities in the disease-gene network and performed functional enrichment using over-representation analysis to detect significant biological processes/pathways and the interactions between them. RESULT: We identified the modular communities to explore the relation of this common causative pathogen (EBV) with different carcinomas such as GC, NPC, HL and BL. Through network analysis we identified the top 10 genes linked with EBV associated carcinomas as CASP10, BRAF, NFKBIA, IFNA2, GSTP1, CSF3, GATA3, UBR5, AXIN2 and POLE. Further, the tyrosine-protein kinase (ABL1) gene was significantly over-represented in 3 out of 9 critical biological processes, viz. in regulatory pathways in cancer, the TP53 network and the Imatinib and chronic myeloid leukemia biological processes. Consequently, the EBV pathogen appears to target critical pathways involved in cellular growth arrest/apoptosis. We make our case for BCR-ABL1 tyrosine-kinase inhibitors (TKI) for further clinical investigations in the inhibition of BCR-mediated EBV activation in carcinomas for better prognostic and therapeutic outcomes.
Assuntos
Linfoma de Burkitt , Carcinoma , Infecções por Vírus Epstein-Barr , Doença de Hodgkin , Neoplasias Nasofaríngeas , Neoplasias Gástricas , Humanos , Herpesvirus Humano 4/genética , Linfoma de Burkitt/genética , Linfoma de Burkitt/complicações , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/complicações , Doença de Hodgkin/complicações , Neoplasias Gástricas/complicações , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/complicações , Proteínas Tirosina Quinases , Carcinogênese/genética , TirosinaRESUMO
Jak3, a member of the Janus kinase family, is essential for the cytokine receptor common gamma chain (γc)-mediated signaling. During activation of Jak3, tyrosine residues are phosphorylated and potentially regulate its kinase activity. We identified a novel tyrosine phosphorylation site within mouse Jak3, Y820, which is conserved in human Jak3, Y824. IL-2-induced tyrosine phosphorylation of Jak3 Y824 in human T cell line HuT78 cells was detected by using a phosphospecific, pY824, antibody. Mutation of mouse Jak3 Y820 to alanine (Y820A) showed increased autophosphorylation of Jak3 and enhanced signal transducer and activator of transcription 5 (STAT5) tyrosine phosphorylation and transcriptional activation. Stably expressed Jak3 Y820A in F7 cells, an IL-2 responsive mouse pro-B cell line Ba/F3, exhibited enhanced IL-2-dependent cell growth. Mechanistic studies demonstrated that interaction between Jak3 and STAT5 increased in Jak3 Y820A compared to wild-type Jak3. These data suggest that Jak3 Y820 plays a role in negative regulation of Jak3-mediated STAT5 signaling cascade upon IL-2-stimulation. We speculate that this occurs through an interaction promoted by the tyrosine phosphorylated Y820 or a conformational change by Y820 mutation with either the STAT directly or with the recruitment of molecules such as phosphatases via a SH2 interaction. Additional studies will focus on these interactions as Jak3 plays a crucial role in disease and health.
Assuntos
Fator de Transcrição STAT5 , Tirosina , Animais , Interleucina-2/metabolismo , Interleucina-2/farmacologia , Janus Quinase 3 , Camundongos , Proteínas do Leite/metabolismo , Fosforilação , Fator de Transcrição STAT5/metabolismo , Transdução de SinaisRESUMO
The nonreceptor protein tyrosine kinase Fyn and protein Ser/Thr phosphatase 2A (PP2A) are major multifunctional signaling molecules. Deregulation of Fyn and altered PP2A methylation are implicated in cancer and Alzheimer's disease (AD). Here, we tested the hypothesis that the methylation state of PP2A catalytic subunit, which influences PP2A subunit composition and substrate specificity, can affect Fyn regulation and function. Using Neuro-2a (N2a) neuroblastoma cell models, we first show that methylated PP2A holoenzymes containing the Bα subunit coimmunoprecipitate and copurify with Fyn in membrane rafts. PP2A methylation status regulates Fyn distribution and Fyn-dependent neuritogenesis, likely in part by affecting actin dynamics. A methylation-incompetent PP2A mutant fails to interact with Fyn. It perturbs the normal partitioning of Fyn and amyloid precursor protein (APP) in membrane microdomains, which governs Fyn function and APP processing. This correlates with enhanced amyloidogenic cleavage of APP, a hallmark of AD pathogenesis. Conversely, enhanced PP2A methylation promotes the nonamyloidogenic cleavage of APP in a Fyn-dependent manner. Disturbances in one-carbon metabolic pathways that control cellular methylation are associated with AD and cancer. Notably, they induce a parallel loss of membrane-associated methylated PP2A and Fyn enzymes in N2a cells and acute mouse brain slices. One-carbon metabolism also modulates Fyn-dependent process outgrowth in N2a cells. Thus, our findings identify a novel methylation-dependent PP2A/Fyn signaling module. They highlight the underestimated importance of cross talks between essential metabolic pathways and signaling scaffolds that are involved in normal cell homeostasis and currently being targeted for cancer and AD treatment.
Assuntos
Precursor de Proteína beta-Amiloide/genética , Encéfalo/metabolismo , Proteína Fosfatase 2/genética , Processamento de Proteína Pós-Traducional/genética , Proteínas Proto-Oncogênicas c-fyn/genética , Doença de Alzheimer/genética , Animais , Encéfalo/patologia , Encéfalo/ultraestrutura , Domínio Catalítico/genética , Holoenzimas/química , Holoenzimas/genética , Humanos , Metilação , Camundongos , Neoplasias/genética , Neuritos/metabolismo , Fosforilação/genética , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Transdução de Sinais/genéticaRESUMO
CD40-activated CD40L reverse signaling is a major physiological regulator of axon and dendrite growth from developing hippocampal pyramidal neurons. Here we have studied how CD40L-mediated reverse signaling promotes the growth of these processes. Cultures of hippocampal pyramidal neurons were established from Cd40-/- mouse embryos to eliminate endogenous CD40/CD40L signaling, and CD40L reverse signaling was stimulated by a CD40-Fc chimera. CD40L reverse signaling increased phosphorylation and hence activation of proteins in the PKC, ERK, and JNK signaling pathways. Pharmacological activators and inhibitors of these pathways revealed that whereas activation of JNK inhibited growth, activation of PKC and ERK1/ERK2 enhanced growth. Experiments using combinations of pharmacological reagents revealed that these signaling pathways regulate growth by functioning as an interconnected and interdependent network rather than acting in a simple linear sequence. Immunoprecipitation studies suggested that stimulation of CD40L reverse signaling generated a receptor complex comprising CD40L, PKCß, and the Syk tyrosine kinase. Our studies have begun to elucidate the molecular network and interactions that promote axon and dendrite growth from developing hippocampal neurons following activation of CD40L reverse signaling.
Assuntos
Axônios/metabolismo , Ligante de CD40/metabolismo , Dendritos/fisiologia , Transdução de Sinais , Animais , Butadienos/farmacologia , Antígenos CD40/deficiência , Antígenos CD40/genética , Células Cultivadas , Dendritos/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/química , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/química , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Nitrilas/farmacologia , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Proteína Quinase C/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinase Syk/metabolismoRESUMO
Despite recent advances in diagnosis and treatment, osteosarcoma remains as the most common bone cancer in children and is associated with poor prognosis. Growing evidence has supported dysregulation of threonine and tyrosine protein kinase (TTK) expression as a hallmark of multiple cancers, however, its function in osteosarcoma remains to be elucidated. In the present study, we found that TTK was frequently overexpressed in osteosarcoma and associated with increased tumour growth and progression. Moreover, using both in vitro and in vivo assays, we provided evidence that TTK level was regulated by a molecular chaperone, heat shock protein 90 (Hsp90). Hsp90 directly interacted with TTK and prevents proteasome-dependent TTK degradation, leading to the accumulation of TTK in osteosarcoma cells. Elevated TTK promoted cancer cell proliferation and survival by activating cell-cycle progression and inhibiting apoptosis. Consistently, depletion of TTK by Hsp90 inhibition induced cell-cycle arrest, generated aneuploidy and eventually resulted in apoptotic cancer cell death. Together, our study revealed an important Hsp90-TTK regulatory axis in osteosarcoma cells to promote cancer cell growth and survival. These findings expand our knowledge on osteosarcoma pathogenesis and offer novel therapeutic options for clinical practice.
Assuntos
Carcinogênese , Regulação Neoplásica da Expressão Gênica/fisiologia , Proteínas de Choque Térmico HSP90/metabolismo , Osteossarcoma/enzimologia , Proteínas Tirosina Quinases/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Feminino , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas de Choque Térmico HSP90/genética , Humanos , Camundongos , Neoplasias Experimentais , Osteossarcoma/genética , Osteossarcoma/metabolismo , Proteínas Tirosina Quinases/genéticaRESUMO
The intracellular bacterial pathogen Coxiella burnetii is the etiological agent of the emerging zoonosis Q fever. Crucial to its pathogenesis is type 4b secretion system-mediated secretion of bacterial effectors into host cells that subvert host cell membrane trafficking, leading to the biogenesis of a parasitophorous vacuole for intracellular replication. The characterization of prokaryotic serine/threonine protein kinases in bacterial pathogens is emerging as an important strategy to better understand host-pathogen interactions. In this study, we investigated CstK (for Coxiella Ser/Thr kinase), a protein kinase identified in C. burnetii by in silico analysis. We demonstrate that this putative protein kinase undergoes autophosphorylation on Thr and Tyr residues and phosphorylates a classical eukaryotic protein kinase substrate in vitro This dual Thr-Tyr kinase activity is also observed for a eukaryotic dual-specificity Tyr phosphorylation-regulated kinase class. We found that CstK is translocated during infections and localizes to Coxiella-containing vacuoles (CCVs). Moreover, a CstK-overexpressing C. burnetii strain displayed a severe CCV development phenotype, suggesting that CstK fine-tunes CCV biogenesis during the infection. Protein-protein interaction experiments identified the Rab7 GTPase-activating protein TBC1D5 as a candidate CstK-specific target, suggesting a role for this host GTPase-activating protein in Coxiella infections. Indeed, CstK co-localized with TBC1D5 in noninfected cells, and TBC1D5 was recruited to CCVs in infected cells. Accordingly, TBC1D5 depletion from infected cells significantly affected CCV development. Our results indicate that CstK functions as a bacterial effector protein that interacts with the host protein TBC1D5 during vacuole biogenesis and intracellular replication.
Assuntos
Proteínas de Bactérias/metabolismo , Coxiella burnetii/enzimologia , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Quinases/metabolismo , Febre Q/metabolismo , Vacúolos/metabolismo , Proteínas de Bactérias/genética , Linhagem Celular Tumoral , Coxiella burnetii/genética , Proteínas Ativadoras de GTPase/genética , Humanos , Fosforilação , Proteínas Quinases/genética , Febre Q/genética , Vacúolos/genética , Vacúolos/microbiologiaRESUMO
Prostate cancer (PCa) cells heavily rely on an active androgen receptor (AR) pathway for their survival. Enzalutamide (MDV3100) is a second-generation antiandrogenic drug that was approved by the Food and Drug Administration in 2012 to treat patients with castration-resistant prostate cancer (CRPC). However, emergence of resistance against this drug is inevitable, and it has been a major challenge to develop interventions that help manage enzalutamide-resistant CRPC. Erythropoietin-producing human hepatocellular (Eph) receptors are targeted by ephrin protein ligands and have a broad range of functions. Increasing evidence indicates that this signaling pathway plays an important role in tumorigenesis. Overexpression of EPH receptor B4 (EPHB4) has been observed in multiple types of cancer, being closely associated with proliferation, invasion, and metastasis of tumors. Here, using RNA-Seq analyses of clinical and preclinical samples, along with several biochemical and molecular methods, we report that enzalutamide-resistant PCa requires an active EPHB4 pathway that supports drug resistance of this tumor type. Using a small kinase inhibitor and RNAi-based gene silencing to disrupt EPHB4 activity, we found that these disruptions re-sensitize enzalutamide-resistant PCa to the drug both in vitro and in vivo Mechanistically, we found that EPHB4 stimulates the AR by inducing proto-oncogene c-Myc (c-Myc) expression. Taken together, these results provide critical insight into the mechanism of enzalutamide resistance in PCa, potentially offering a therapeutic avenue for enhancing the efficacy of enzalutamide to better manage this common malignancy.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Neoplasias de Próstata Resistentes à Castração/metabolismo , Receptor EphB4/metabolismo , Receptores Androgênicos/genética , Animais , Antineoplásicos/uso terapêutico , Benzamidas , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Camundongos Nus , Nitrilas , Feniltioidantoína/análogos & derivados , Feniltioidantoína/uso terapêutico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptor EphB4/antagonistas & inibidores , Receptores Androgênicos/metabolismoRESUMO
Although signal transduction by immunoreceptors such as the T cell antigen receptor (TCR), B cell antigen receptor (BCR), and Fc receptors uses the same schematic and similar molecules, the threshold and the fine-tuning are set differently for each receptor. One manifestation of these differences is that inhibition of Src family kinases (SFK) blocks TCR but not BCR signaling. SFKs are key kinases phosphorylating immunoreceptor tyrosine-based activation motifs (ITAM) in both these receptors. However, it has been proposed that in B cells, downstream kinase SYK can phosphorylate ITAM sequences independently of SFK, allowing it to compensate for the loss of SFK activity, whereas its T cell paralog ZAP-70 is not capable of this compensation. To test this proposal, we examined signaling in SYK- and ZAP-70-deficient B and T cell lines expressing SYK or ZAP-70. We also analyzed signal transduction in T cells expressing BCR or B cells expressing part of the TCR complex. We show that when compared with ZAP-70, SYK lowered the threshold for SFK activity necessary to initiate antigen receptor signaling in both T and B cells. However, neither SYK nor ZAP-70 were able to initiate signaling independently of SFK. We further found that additional important factors are involved in setting this threshold. These include differences between the antigen receptor complexes themselves and the spatial separation of the key transmembrane adaptor protein LAT from the TCR. Thus, immunoreceptor sensing of SFK activity is a complex process regulated at multiple levels.
Assuntos
Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Quinases da Família src/metabolismo , Humanos , Células Jurkat , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos T/genética , Quinase Syk/genética , Quinase Syk/metabolismo , Proteína-Tirosina Quinase ZAP-70/genética , Proteína-Tirosina Quinase ZAP-70/metabolismo , Quinases da Família src/genéticaRESUMO
Treatment of patients with chronic lymphocytic leukemia (CLL) with inhibitors of Bruton's tyrosine kinase (BTK), such as ibrutinib, is limited by primary or secondary resistance to this drug. Examinations of CLL patients with late relapses while on ibrutinib, which inhibits BTK's catalytic activity, revealed several mutations in BTK, most frequently resulting in the C481S substitution, and disclosed many mutations in PLCG2, encoding phospholipase C-γ2 (PLCγ2). The PLCγ2 variants typically do not exhibit constitutive activity in cell-free systems, leading to the suggestion that in intact cells they are hypersensitive to Rac family small GTPases or to the upstream kinases spleen-associated tyrosine kinase (SYK) and Lck/Yes-related novel tyrosine kinase (LYN). The sensitivity of the PLCγ2 variants to BTK itself has remained unknown. Here, using genetically-modified DT40 B lymphocytes, along with various biochemical assays, including analysis of PLCγ2-mediated inositol phosphate formation, inositol phospholipid assessments, fluorescence recovery after photobleaching (FRAP) static laser microscopy, and determination of intracellular calcium ([Ca2+] i ), we show that various CLL-specific PLCγ2 variants such as PLCγ2S707Y are hyper-responsive to activated BTK, even in the absence of BTK's catalytic activity and independently of enhanced PLCγ2 phospholipid substrate supply. At high levels of B-cell receptor (BCR) activation, which may occur in individual CLL patients, catalytically-inactive BTK restored the ability of the BCR to mediate increases in [Ca2+] i Because catalytically-inactive BTK is insensitive to active-site BTK inhibitors, the mechanism involving the noncatalytic BTK uncovered here may contribute to preexisting reduced sensitivity or even primary resistance of CLL to these drugs.
Assuntos
Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia/metabolismo , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Fosfolipase C gama/genética , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Adenina/farmacologia , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática/efeitos dos fármacos , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Fosfolipase C gama/metabolismo , Mutação Puntual/efeitos dos fármacosRESUMO
The discovery of activating epidermal growth factor receptor (EGFR) mutations spurred the use of EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib, as the first-line treatment of lung cancers. We previously reported that differential degradation of TKI-sensitive (e.g. L858R) and resistant (T790M) EGFR mutants upon erlotinib treatment correlates with drug sensitivity. We also reported that SMAD ubiquitination regulatory factor 2 (SMURF2) ligase activity is important in stabilizing EGFR. However, the molecular mechanisms involved remain unclear. Here, using in vitro and in vivo ubiquitination assays, MS, and superresolution microscopy, we show SMURF2-EGFR functional interaction is important for EGFR stability and response to TKI. We demonstrate that L858R/T790M EGFR is preferentially stabilized by SMURF2-UBCH5 (an E3-E2)-mediated polyubiquitination. We identified four lysine residues as the sites of ubiquitination and showed that replacement of one of them with acetylation-mimicking glutamine increases the sensitivity of mutant EGFR to erlotinib-induced degradation. We show that SMURF2 extends membrane retention of EGF-bound EGFR, whereas SMURF2 knockdown increases receptor sorting to lysosomes. In lung cancer cell lines, SMURF2 overexpression increased EGFR levels, improving TKI tolerance, whereas SMURF2 knockdown decreased EGFR steady-state levels and sensitized lung cancer cells. Overall, we propose that SMURF2-mediated polyubiquitination of L858R/T790M EGFR competes with acetylation-mediated receptor internalization that correlates with enhanced receptor stability; therefore, disruption of the E3-E2 complex may be an attractive target to overcome TKI resistance.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Cloridrato de Erlotinib/farmacologia , Neoplasias Pulmonares/enzimologia , Mutação de Sentido Incorreto , Inibidores de Proteínas Quinases/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Substituição de Aminoácidos , Animais , Células CHO , Cricetulus , Resistencia a Medicamentos Antineoplásicos/genética , Estabilidade Enzimática/efeitos dos fármacos , Estabilidade Enzimática/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Células MCF-7 , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
BACKGROUND: Ibrutinib is a Bruton tyrosine kinase inhibitor with remarkable efficacy against B-cell cancers. Ibrutinib also increases the risk of atrial fibrillation (AF), which remains poorly understood. METHODS: We performed electrophysiology studies on mice treated with ibrutinib to assess inducibility of AF. Chemoproteomic analysis of cardiac lysates identified candidate ibrutinib targets, which were further evaluated in genetic mouse models and additional pharmacological experiments. The pharmacovigilance database, VigiBase, was queried to determine whether drug inhibition of an identified candidate kinase was associated with increased reporting of AF. RESULTS: We demonstrate that treatment of mice with ibrutinib for 4 weeks results in inducible AF, left atrial enlargement, myocardial fibrosis, and inflammation. This effect was reproduced in mice lacking Bruton tyrosine kinase, but not in mice treated with 4 weeks of acalabrutinib, a more specific Bruton tyrosine kinase inhibitor, demonstrating that AF is an off-target side effect. Chemoproteomic profiling identified a short list of candidate kinases that was narrowed by additional experimentation leaving CSK (C-terminal Src kinase) as the strongest candidate for ibrutinib-induced AF. Cardiac-specific Csk knockout in mice led to increased AF, left atrial enlargement, fibrosis, and inflammation, phenocopying ibrutinib treatment. Disproportionality analyses in VigiBase confirmed increased reporting of AF associated with kinase inhibitors blocking Csk versus non-Csk inhibitors, with a reporting odds ratio of 8.0 (95% CI, 7.3-8.7; P<0.0001). CONCLUSIONS: These data identify Csk inhibition as the mechanism through which ibrutinib leads to AF. Registration: URL: https://ww.clinicaltrials.gov; Unique identifier: NCT03530215.
Assuntos
Adenina/análogos & derivados , Antineoplásicos/toxicidade , Fibrilação Atrial/induzido quimicamente , Função do Átrio Esquerdo/efeitos dos fármacos , Proteína Tirosina Quinase CSK/antagonistas & inibidores , Átrios do Coração/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Piperidinas/toxicidade , Inibidores de Proteínas Quinases/toxicidade , Potenciais de Ação/efeitos dos fármacos , Adenina/toxicidade , Tirosina Quinase da Agamaglobulinemia/deficiência , Tirosina Quinase da Agamaglobulinemia/genética , Animais , Fibrilação Atrial/enzimologia , Fibrilação Atrial/fisiopatologia , Proteína Tirosina Quinase CSK/genética , Proteína Tirosina Quinase CSK/metabolismo , Bases de Dados Genéticas , Átrios do Coração/enzimologia , Átrios do Coração/fisiopatologia , Humanos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Medição de Risco , Fatores de RiscoRESUMO
MicroRNA24-2 (miR24-2) is associated with human tumorigenesis; however, its molecular mechanisms are poorly understood. Herein, our findings demonstrate that miR24-2 promotes the proliferation ability in vitro and the tumorigenic ability in vivo in human liver cancer stem cells (hLCSCs). Mechanically, the miR24-2 targets for 3' UTR (2,627-2,648) of protein arginine methyltransferase 7 (PRMT7) inhibit the translational ability of prmt7 gene. Moreover, miR24-2 inhibits the di-/tri-methylation of histone H4 arginine 3 by reducing PRMT7 and then promotes the expression of Nanog via long noncoding RNA HULC. Notably, miR24-2 inhibits histone deacetylase HDAC3 through miR675, which promotes the acetylation of histone H4 at lysine 16. Subsequently, miR24-2 enhances the interaction between LC3 and ATG4 dependent on PI3K and triggers cellular autophagy. Strikingly, miR24-2 inhibits the degradation of pyruvate kinase M1 via autophagosome-P62 in hLCSCs. Furthermore, miR24-2 enhances the activity of Src by promoting the binding of PKM1 to the Src promoter regions in hLCSCs. In particular, our results also indicate that src gene determines the oncogenic functions of miR24-2. These results provided a valuable theoretical basis for the discovery of liver cancer therapeutic targets and diagnosis markers based on miR24-2.
Assuntos
Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , MicroRNAs/genética , Células-Tronco Neoplásicas/metabolismo , Quinases da Família src/genética , Acetilação , Autofagia , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Histonas/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Proteínas de Membrana/metabolismo , Metilação , Proteína Homeobox Nanog/genética , Proteína-Arginina N-Metiltransferases/genética , Interferência de RNA , Hormônios Tireóideos/metabolismo , Proteínas de Ligação a Hormônio da TireoideRESUMO
Many endocrine disruptors may interfere with sperm motility, hyperactivation, and capacitation, thereby leading to male infertility. In the current study, we screened 14 endocrine disruptors, including plant ingredients, cigarette ingredients, minerals, insecticides and fungicides, plastics, and plasticizers, to inhibit human sperm motility and forward motility. Only ziram, a dithiocarbamate fungicide, can effectively inhibit sperm motility, forward motility, hyperactivation, capacitation, and spontaneous acrosome reaction of normal human spermatozoa. Its half maximum inhibitory concentration (IC50) values were less than 4 µM. Ziram also inhibited sperm motility and forward motility of asthenozoospermia spermatozoa and IC50 values were about 6-8 µM. In addition, ziram inhibited normal sperm motility, calcium influx, reactive oxygen species, and mitochondrial membrane potential at 2.5 and/or 5 µM, with IC50 values ââexceeding 100 µM, although it did not affect sperm DNA fragmentation up to 5 µM. Ziram-mediated inhibition of sperm motility and forward motility was irreversible. Forskolin, 8Br-cAMP, pentoxifylline, progesterone, vitamin E, and A23187 cannot prevent ziram-mediated inhibition of sperm motility and forward motility. Further studies have shown that ziram inhibited the level of tyrosine protein kinase with an IC50 value of about 10 µM, without affecting p21-activated kinase 4, and it caused damage to the mitochondrial structure of normal spermatozoa at 2.5 and 5 µM. In conclusion, ziram irreversibly inhibits human sperm motility, forward motility, and capacitation by reducing the level of tyrosine protein kinase and damaging the ultrastructure of mitochondria.
RESUMO
Ovarian cancer remains the leading cause of death due to gynecologic malignancy. Estrogen-related pathways genes, such as estrogen receptors (ESR1 and ESR2) and their coregulators, proline-, glutamic acid-, and leucine-rich protein 1 (PELP1), and proto-oncogene tyrosine-protein kinase c-Src (SRC) are involved in ovarian cancer induction and development, still they require in-depth study. In our study, tissue samples were obtained from 52 females of Caucasian descent (control group without cancerous evidence (n = 27), including noncancerous benign changes (n = 15), and the ovarian carcinoma (n = 25)). Using quantitative analyses, we investigated ESRs, PELP1, and SRC mRNA expression association with ovarian tumorigenesis. Proteins' presence and their location were determined by Western blot and immunohistochemistry. Results showed that PELP1 and SRC expression levels were found to differ in tissues of different sample types. The expression patterns were complex and differed in the case of ovarian cancer patients compared to controls. The most robust protein immunoreactivity was observed for PELP1 and the weakest for ESR1. The expression patterns of analyzed genes represent a potentially interesting target in ovarian cancer biology, especially PELP1. This study suggests that specific estrogen-mediated functions in the ovary and ovary-derived cancer might result from different local interactions of estrogen with their receptors and coregulators.