RESUMO
We have reported previously that CD33hi myeloid-derived suppressor cells (MDSCs) play a direct role in the pathogenesis of myelodysplastic syndromes (MDSs) and that their sustained activation contributes to hematopoietic and immune impairment, including modulation of PD1/PDL1. MDSCs can also limit the clinical activity of immune checkpoint inhibition in solid malignancies. We hypothesized that depletion of MDSCs may ameliorate resistance to checkpoint inhibitors and, hence, targeted them with AMV564 combined with anti-PD1 in MDS bone marrow (BM) mononuclear cells (MNCs) enhanced activation of cytotoxic T cells. AMV564 was active in vivo in a leukemia xenograft model when co-administered with healthy donor peripheral blood MNCs (PBMCs). Our findings provide a strong rationale for clinical investigation of AMV564 as a single agent or in combination with an anti-PD1 antibody and in particular for treatment of cancers resistant to checkpoint inhibitors.
Assuntos
Anticorpos Biespecíficos , Antineoplásicos , Melanoma , Síndromes Mielodisplásicas , Células Supressoras Mieloides , Animais , Anticorpos Biespecíficos/farmacologia , Antineoplásicos/farmacologia , Humanos , Melanoma/tratamento farmacológico , Síndromes Mielodisplásicas/tratamento farmacológico , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico , Linfócitos TRESUMO
BACKGROUND: Increases in fatigue, depressive symptomatology, and cognitive impairment are common after the initiation of androgen deprivation therapy (ADT) for prostate cancer. To date, no studies have examined the potential role of inflammation in the development of these symptoms in ADT recipients. The goal of the current study was to examine circulating markers of inflammation as potential mediators of change in fatigue, depressive symptomatology, and cognitive impairment related to the receipt of ADT. METHODS: Patients treated with ADT for prostate cancer (ADT+; n = 47) were assessed around the time of the initiation of ADT and 6 and 12 months later. An age- and education-matched group of men without a history of cancer (CA-; n = 82) was assessed at comparable time points. Fatigue, depressive symptomatology, and cognitive impairment were assessed with the Fatigue Symptom Inventory, the Center for Epidemiological Studies Depression Scale, and a battery of neuropsychological tests, respectively. Circulating markers of inflammation included interleukin 1 receptor antagonist (IL-1RA), interleukin 6 (IL-6), soluble tumor necrosis factor receptor II (sTNF-RII), and C-reactive protein (CRP). RESULTS: Fatigue, depressive symptomatology, and serum IL-6 increased significantly over time in the ADT+ group versus the CA- group; rates of cognitive impairment also changed significantly between the groups. No significant changes in IL-1RA, sTNF-RII, or CRP over time were detected. Treatment-related increases in IL-6 were associated with worsening fatigue but not depressive symptomatology or cognitive impairment. CONCLUSIONS: Results of this preliminary study suggest that increases in circulating IL-6, perhaps due to testosterone inhibition, may play a role in fatigue secondary to receipt of ADT. Additional research is needed to determine whether interventions to reduce circulating inflammation improve fatigue in this population.
Assuntos
Antagonistas de Androgênios/uso terapêutico , Antineoplásicos Hormonais/uso terapêutico , Mediadores da Inflamação/sangue , Inflamação/sangue , Neoplasias da Próstata/tratamento farmacológico , Idoso , Antagonistas de Androgênios/efeitos adversos , Antineoplásicos Hormonais/efeitos adversos , Proteína C-Reativa/análise , Transtornos Cognitivos/diagnóstico , Transtornos Cognitivos/etiologia , Depressão/diagnóstico , Depressão/etiologia , Fadiga/diagnóstico , Fadiga/etiologia , Humanos , Inflamação/complicações , Proteína Antagonista do Receptor de Interleucina 1/sangue , Interleucina-6/sangue , Masculino , Testes Neuropsicológicos , Dados Preliminares , Neoplasias da Próstata/sangue , Receptores Tipo II do Fator de Necrose Tumoral/sangue , Avaliação de SintomasRESUMO
Dendritic cells (DCs) are professional antigen presenting cells with a great capacity for cross-presentation of exogenous antigens from which robust anti-tumor immune responses ensue. However, this function is not always available and requires DCs to first be primed to induce their maturation. In particular, in the field of DC vaccine design, currently available methodologies have been limited in eliciting a sustained anti-tumor immune response. Mechanistically, part of the maturation response is influenced by the presence of stimulatory receptors relying on ITAM-containing activating adaptor molecules like DAP12, that modulates their function. We hypothesize that activating DAP12 in DC could force their maturation and enhance their potential anti-tumor activity for therapeutic intervention. For this purpose, we developed constitutively active DAP12 mutants that can promote activation of monocyte-derived DC. Here we demonstrate its ability to induce the maturation and activation of monocyte-derived DCs which enhances migration, and T cell stimulation in vitro using primary human cells. Moreover, constitutively active DAP12 stimulates a strong immune response in a murine melanoma model leading to a reduction of tumor burden. This provides proof-of-concept for investigating the pre-activation of antigen presenting cells to enhance the effectiveness of anti-tumor immunotherapies.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Células Dendríticas/imunologia , Imunidade Celular/imunologia , Melanoma Experimental/imunologia , Proteínas de Membrana/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Vacinas Anticâncer/imunologia , Movimento Celular/genética , Proliferação de Células/genética , Humanos , Imunidade Celular/genética , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Proteínas de Membrana/imunologia , Camundongos , Monócitos/imunologia , Proteínas Mutantes/genética , Proteínas Mutantes/imunologia , Carga Tumoral/imunologiaRESUMO
Transforming growth factor ß1 (TGF-ß), enriched in the tumor microenvironment and broadly immunosuppressive, inhibits natural killer (NK) cell function by yet-unknown mechanisms. Here we show that TGF-ß-treated human NK cells exhibit reduced tumor cytolysis and abrogated perforin polarization to the immune synapse. This result was accompanied by loss of surface expression of activating killer Ig-like receptor 2DS4 and NKp44, despite intact cytoplasmic stores of these receptors. Instead, TGF-ß depleted DNAX activating protein 12 kDa (DAP12), which is critical for surface NK receptor stabilization and downstream signal transduction. Mechanistic analysis revealed that TGF-ß induced microRNA (miR)-183 to repress DAP12 transcription/translation. This pathway was confirmed with luciferase reporter constructs bearing the DAP12 3' untranslated region as well as in human NK cells by use of sense and antisense miR-183. Moreover, we documented reduced DAP12 expression in tumor-associated NK cells in lung cancer patients, illustrating this pathway to be consistently perturbed in the human tumor microenvironment.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Células Matadoras Naturais/imunologia , Proteínas de Membrana/antagonistas & inibidores , MicroRNAs/metabolismo , Neoplasias/imunologia , Receptores de Células Matadoras Naturais/antagonistas & inibidores , Fator de Crescimento Transformador beta/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Citometria de Fluxo , Humanos , Immunoblotting , Imuno-Histoquímica , Células Matadoras Naturais/metabolismo , Luciferases , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Receptores de Células Matadoras Naturais/metabolismo , Transdução de Sinais/imunologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Advances in the understanding of the tumor microenvironment have led to development of immunotherapeutic strategies, such as chimeric antigen receptor T cells (CAR-Ts). However, despite success in blood malignancies, CAR-T therapies in solid tumors have been hampered by their restricted infiltration. Here, we used our understanding of early cytotoxic lymphocyte infiltration of human lymphocytes in solid tumors in vivo to investigate the receptors in normal, adjacent, and tumor tissues of primary non-small-cell lung cancer specimens. We found that CX3CL1-CX3CR1 reduction restricts cytotoxic cells from the solid-tumor bed, contributing to tumor escape. Based on this, we designed a CAR-T construct using the well-established natural killer group 2, member D (NKG2D) CAR-T expression together with overexpression of CX3CR1 to promote their infiltration. These CAR-Ts infiltrate tumors at higher rates than control-activated T cells or IL-15-overexpressing NKG2D CAR-Ts. This construct also had similar functionality in a liver-cancer model, demonstrating potential efficacy in other solid malignancies.
RESUMO
NK cell migration and activation are crucial elements of tumor immune surveillance. In mammary carcinomas, the number and function of NK cells is diminished, despite being positively associated with clinical outcome. MicroRNA-155 (miR-155) has been shown to be an important regulator of NK cell activation through its interaction with SHIP-1 downstream of inhibitory NK receptor signaling, but has not been explored in regard to NK cell migration. Here, we explored the migratory potential and function of NK cells in subcutaneous AT3 in mice lacking miR-155. Without tumor, these bic/miR-155-/- mice possess similar numbers of NK cells that exhibit comparable surface levels of cytotoxic receptors as NK cells from wild-type (WT) mice. Isolated miR-155-/- NK cells also exhibit equivalent cytotoxicity towards tumor targets in vitro compared to isolated WT control NK cells, despite overexpression of known miR-155 gene targets. NK cells isolated from miR-155-/- mice exhibit impaired F-actin polymerization and migratory capacity in Boyden-chamber assays in response chemokine (C-C motif) ligand 2 (CCL2). This migratory capacity could be normalized in the presence of SHIP-1 inhibitors. Of note, miR-155-/- mice challenged with mammary carcinomas exhibited heightened tumor burden which correlated with a lower number of tumor-infiltrating NK1.1+ cells. Our results support a novel, physiological role for SHIP-1 in the control of NK cell tumor trafficking, and implicate miR-155 in the regulation of NK cell chemotaxis, in the context of mammary carcinoma. This may implicate dysfunctional NK cells in the lack of tumor clearance in mice.
Assuntos
Células Matadoras Naturais/metabolismo , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , MicroRNAs/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Quimiotaxia/genética , Feminino , Técnicas de Inativação de Genes , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Transdução de Sinais/genéticaRESUMO
Myeloid-derived suppressor cells (MDSC) represent a primary mechanism of immune evasion in tumors and have emerged as a major obstacle for cancer immunotherapy. The immunoinhibitory activity of MDSC is tightly regulated by the tumor microenvironment and occurs through mechanistic mediators that remain unclear. Here, we elucidated the intrinsic interaction between the expression of AMP-activated protein kinase alpha (AMPKα) and the immunoregulatory activity of MDSC in tumors. AMPKα signaling was increased in tumor-MDSC from tumor-bearing mice and patients with ovarian cancer. Transcription of the Ampkα1-coding gene, Prkaa1, in tumor-MDSC was induced by cancer cell-derived granulocyte-monocyte colony-stimulating factor (GM-CSF) and occurred in a Stat5-dependent manner. Conditional deletion of Prkaa1 in myeloid cells, or therapeutic inhibition of Ampkα in tumor-bearing mice, delayed tumor growth, inhibited the immunosuppressive potential of MDSC, triggered antitumor CD8+ T-cell immunity, and boosted the efficacy of T-cell immunotherapy. Complementarily, therapeutic stimulation of AMPKα signaling intrinsically promoted MDSC immunoregulatory activity. In addition, Prkaa1 deletion antagonized the differentiation of monocytic-MDSC (M-MDSC) to macrophages and re-routed M-MDSC, but not granulocytic-MDSC (PMN-MDSC), into cells that elicited direct antitumor cytotoxic effects through nitric oxide synthase 2-mediated actions. Thus, our results demonstrate the primary role of AMPKα1 in the immunosuppressive effects induced by tumor-MDSC and support the therapeutic use of AMPK inhibitors to overcome MDSC-induced T-cell dysfunction in cancer. SIGNIFICANCE: AMPKα1 regulates the immunosuppressive activity and differentiation of tumor-MDSC, suggesting AMPK inhibition as a potential therapeutic strategy to restore protective myelopoiesis in cancer.
Assuntos
Proteínas Quinases Ativadas por AMP/imunologia , Carcinoma Epitelial do Ovário/imunologia , Células Supressoras Mieloides/imunologia , Neoplasias Experimentais/imunologia , Microambiente Tumoral/imunologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Carcinoma Epitelial do Ovário/metabolismo , Diferenciação Celular/imunologia , Feminino , Humanos , Camundongos , Células Supressoras Mieloides/metabolismo , Neoplasias Experimentais/metabolismo , Evasão Tumoral/imunologiaRESUMO
Immune escape is a hallmark of cancer. In human lung cancer, we have identified a unique microRNA (miR)-based pathway employed by tumor cells to repress detection by immune cells via the NKG2D-MICA/B receptor-ligand system. MICA/B is readily induced by cell transformation and serves as a danger signal and ligand to alert NK and activated CD8+ T cells. However, immunohistochemical analysis indicated that human lung adenocarcinoma and squamous cell carcinoma specimens express little MICA/B while high levels of miR-183 were detected in both tumor types in a TCGA database. Human lung tumor cell lines confirmed the reverse relationship in expression of MICA/B and miR-183. Importantly, a miR-183 binding site was identified on the 3'untranslated region (UTR) of both MICA and MICB, suggesting its role in MICA/B regulation. Luciferase reporter constructs bearing the 3'UTR of MICA or MICB in 293 cells supported the function of miR-183 in repressing MICA/B expression. Additionally, anti-sense miR-183 transfection into H1355 or H1299 tumor cells caused the upregulation of MICA/B. Abundant miR-183 expression in tumor cells was traced to transforming growth factor-beta (TGFß), as evidenced by antisense TGFß transfection into H1355 or H1299 tumor cells which subsequently lost miR-183 expression accompanied by MICA/B upregulation. Most significantly, anti-sense miR-183 transfected tumor cells became more sensitive to lysis by activated CD8+ T cells that express high levels of NKG2D. Thus, high miR-183 triggered by TGFß expressed in lung tumor cells can target MICA/B expression to circumvent detection by NKG2D on immune cells.
RESUMO
BACKGROUND: The use of relaxation and guided imagery to reduce stress and improve immune function has great potential benefits for patients with breast cancer. METHODS: This pilot study used a pretest-posttest experimental design with 28 breast cancer patients, aged 25 to 75 years, with the diagnosis of stage 0, 1, or 2 breast cancer. The experimental group received a relaxation and guided imagery intervention and the control group received standard care. The effects of the intervention on immune function were measured by natural killer (NK) cell cytotoxicity and IL-2-activated NK cell activity prior to surgery and 4 weeks postsurgery. NK cell activity was measured using a 15-hr incubation chromium release assay. Cytotoxicity of NK cells was measured against chromium-labeled K-562 target cells. IL-2 was used to enhance reactivity of NK cells against tumor cells. After incubation for 15 hr, cytotoxicity was measured through the release of radioactive chromium. RESULTS: Significant differences between groups were found at 4 weeks postsurgery. T-tests showed increased NK cell cytotoxicity for the intervention group at 100:1, 50:1, and 25:1 effector cell: target cell ratios (E:T) (p < .01 to p < .05) and increased activation for IL-2 at 100:1, 50:1, 25:1, and 12.5:1 (E:T) (p < .01 to p < .05) for the intervention group as compared to the control group. DISCUSSION: These findings suggest that a relaxation intervention such as guided imagery could have an effect on NK cell cytotoxicity and NK cell cytotoxicity after activation with IL-2 in patients undergoing surgery for breast cancer.
Assuntos
Neoplasias da Mama/imunologia , Imagens, Psicoterapia , Adulto , Idoso , Neoplasias da Mama/psicologia , Neoplasias da Mama/terapia , Feminino , Humanos , Interleucina-2/imunologia , Células Matadoras Naturais/imunologia , Pessoa de Meia-IdadeRESUMO
One of the major obstacles in curing prostate cancer is the development of drug resistance to docetaxel, which is the gold standard for the treatment of this disease. It is not only imperative to discover the molecular basis of resistance but also to find therapeutic agents that can disrupt the resistant pathways. Based on initial findings that docetaxel-resistant PC3-DR and DU145-DR prostate tumor cell lines express tumor necrosis factor-related apoptosis inducing ligand (TRAIL) receptors, we examined whether TRAIL could be used as an alternative method to kill PC3-DR and DU145-DR cells. However, these tumor cells were found to be TRAIL resistant. Because PC3-DR and DU-145-DR cells were previously shown by us to be clusterin positive, we examined if clusterin could play a role in TRAIL resistance. We found that resveratrol could sensitize docetaxel-resistant tumor cells to TRAIL, and it worked by blocking clusterin expression. In particular, small interfering RNA clusterin expression in the cell lines was sufficient to produce apoptosis by TRAIL. Further analysis indicated that resveratrol functions as an effective tyrosine kinase inhibitor, similar to its analogue, piceatannol, and could inhibit Src and Jak kinases, thus resulting in loss of Stat1 activation. We have shown earlier that Stat1 is essential for gene transcription of clusterin. These results, taken together, show that resveratrol could be a useful new therapeutic agent to combat docetaxel resistance.
Assuntos
Clusterina/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias da Próstata/patologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Clusterina/genética , Docetaxel , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Janus Quinases/metabolismo , Masculino , Neoplasias da Próstata/enzimologia , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Resveratrol , Fatores de Transcrição STAT/metabolismo , Estilbenos/farmacologia , Taxoides/farmacologiaRESUMO
Myeloid-derived suppressor cells (MDSCs) constitute a key checkpoint that impedes tumor immunity against cancer. Chemotherapeutic intervention of MDSCs has gained ground as a strategy for cancer therapy but its mechanism remains obscure.We report here a unique mechanism by which monocytic (M)-MDSCs are spared, allowing them to polarize towards M1 macrophages for reactivation of immunity against breast cancer. We first demonstrated that curcumin, like docetaxel (DTX), can selectively target CD11b(+)Ly6G(+)Ly6C(low) granulocytic (G)-MDSCs, sparing CD11b(+)Ly6G(-)Ly6C(high) M-MDSCs, with reduced tumor burden in 4T1-Neu tumor-bearing mice. Curcumin treatment polarized surviving M-MDSCs toward CCR7(+) Dectin-1(-)M1 cells, accompanied by IFN-γ production and cytolytic function in T cells. Selective M-MDSC chemoresistence to curcumin and DTX was mediated by secretory/cytoplasmic clusterin (sCLU). sCLU functions by trapping Bax from mitochondrial translocation, preventing the apoptotic cascade. Importantly, sCLU was only found in M-MDSCs but not in G-MDSCs. Knockdown of sCLU in M-MDSCs and RAW264.7 macrophages was found to reverse their natural chemoresistance. Clinically, breast cancer patients possess sCLU expression only in mature CD68(+) macrophages but not in immature CD33(+) immunosuppressive myeloid cells infiltrating the tumors. We thus made the seminal discovery that sCLU expression in M-MDSCs accounts for positive immunomodulation by chemotherapeutic agents.
Assuntos
Antineoplásicos/administração & dosagem , Clusterina/metabolismo , Curcumina/administração & dosagem , Neoplasias Mamárias Experimentais/tratamento farmacológico , Células Supressoras Mieloides/efeitos dos fármacos , Animais , Antígenos Ly/metabolismo , Antineoplásicos/farmacologia , Antígeno CD11b/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Curcumina/farmacologia , Feminino , Interferon gama/metabolismo , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Células Supressoras Mieloides/citologia , Células Supressoras Mieloides/imunologia , Células RAW 264.7 , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Maintenance of T cell immune homeostasis is critical for adequate anti-tumor immunity. The transcription factor Ikaros is essential for lymphocyte development including T cells. Alterations in Ikaros expression occur in blood malignancies in humans and mice. In this study, we investigated the role of Ikaros in regulating T cell immune balance in pancreatic cancer mouse models. METHODOLOGY AND PRINCIPAL FINDINGS: Using our Panc02 tumor-bearing (TB) mouse model, western blot analysis revealed a reduction in Ikaros proteins while qRT-PCR showed no differences in Ikaros mRNA levels in TB splenocytes compared to control. Treatment of naïve splenocytes with the proteasomal inhibitor, MG132, stabilized Ikaros expression and prevented Ikaros downregulation by Panc02 cells, in vitro. Western blot analyses showed a reduction in protein phosphatase 1 (PP1) and protein kinase CK2 expression in TB splenocytes while CK2 activity was increased. Immunofluorescence microscopy revealed altered punctate staining of Ikaros in TB splenocytes. Flow cytometry revealed a significant decrease in effector CD4+ and CD8+ T cell percentages but increased CD4+CD25+ regulatory T cells in TB splenocytes. Similar alterations in T cell percentages, as well as reduced Ikaros and CK2 but not PP1 expression, were observed in a transgenic, triple mutant (TrM) pancreatic cancer model. Ikaros expression was also reduced in enriched TB CD3+ T cells. MG132 treatment of naïve CD3+ T cells stabilized Ikaros expression in the presence of Panc02 cells. Western blots showed reduced PP1 and CK2 expression in TB CD3+ T cells. CONCLUSIONS/SIGNIFICANCE: The results of this study suggest that the pancreatic tumor microenvironment may cause proteasomal degradation of Ikaros, possibly via dysregulation of PP1 and CK2 expression and activity, respectively. This loss of Ikaros expression may contribute to an imbalance in T cell percentages. Ikaros may potentially be a therapeutic target to restore T cell homeostasis in pancreatic cancer hosts, which may be critical for effective anti-tumor immunity.
Assuntos
Adenocarcinoma/genética , Adenocarcinoma/imunologia , Homeostase/genética , Fator de Transcrição Ikaros/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Adenocarcinoma/metabolismo , Animais , Complexo CD3/metabolismo , Caseína Quinase II/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica , Fator de Transcrição Ikaros/metabolismo , Contagem de Linfócitos , Camundongos , Neoplasias Pancreáticas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Receptores de Neuropeptídeo Y/genética , Receptores de Neuropeptídeo Y/metabolismo , Ubiquitina/metabolismoRESUMO
Rheumatoid arthritis (RA) is an autoimmune disease characterized by dysregulated and chronic systemic inflammatory responses that affect the synovium, bone, and cartilage causing damage to extra-articular tissue. Innate immunity is the first line of defense against invading pathogens and assists in the initiation of adaptive immune responses. Polymorphonuclear cells (PMNs), which include neutrophils, are the largest population of white blood cells in peripheral blood and functionally produce their inflammatory effect through phagocytosis, cytokine production and natural killer-like cytotoxic activity. TREM1 (triggering receptor expressed by myeloid cells) is an inflammatory receptor in PMNs that signals through the use of the intracellular activating adaptor DAP12 to induce downstream signaling. After TREM crosslinking, DAP12's tyrosines in its ITAM motif get phosphorylated inducing the recruitment of Syk tyrosine kinases and eventual activation of PI3 kinases and ERK signaling pathways. While both TREM1 and DAP12 have been shown to be important activators of RA pathogenesis, their activity in PMNs or the importance of DAP12 as a possible therapeutic target have not been shown. Here we corroborate, using primary RA specimens, that isolated PMNs have an increased proportion of both TREM1 and DAP12 compared to normal healthy control isolated PMNs both at the protein and gene expression levels. This increased expression is highly functional with increased activation of ERK and MAPKs, secretion of IL-8 and RANTES and cytotoxicity of target cells. Importantly, based on our hypothesis of an imbalance of activating and inhibitory signaling in the pathogenesis of RA we demonstrate that inhibition of the DAP12 signaling pathway inactivates these important inflammatory cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Artrite Reumatoide/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Neutrófilos/metabolismo , Receptores Imunológicos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Artrite Reumatoide/imunologia , Estudos de Casos e Controles , Regulação da Expressão Gênica , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Receptores Imunológicos/genética , Receptor Gatilho 1 Expresso em Células MieloidesRESUMO
Development of chemoresistance, especially to docetaxel (DTX), is the primary barrier to the cure of castration-resistant prostate cancer but its mechanism is obscure. Here, we report a seminal crosstalk between dying and residual live tumor cells during treatment with DTX that can result in outgrowth of a chemoresistant population. Survival was due to the induction of secretory/cytoplasmic clusterin (sCLU), which is a potent anti-apoptotic protein known to bind and sequester Bax from mitochondria, to prevent caspase 3 activation. sCLU induction in live cells depended on HMGB1 release from dying cells. Supernatants from DTX-treated DU145 tumor cells, which were shown to contain HMGB1, effectively induced sCLU from newly-plated DU145 tumor cells and protected them from DTX toxicity. Addition of anti-HMBG1 to the supernatant or pretreatment of newly-plated DU145 tumor cells with anti-TLR4 or anti-RAGE markedly abrogated sCLU induction and protective effect of the supernatant. Mechanistically, HMGB1 activated NFκB to promote sCLU gene expression and prevented the translocation of activated Bax to mitochondria to block cell death. Importantly, multiple currently-used chemotherapeutic drugs could release HMGB1 from tumor cells. These results suggest that acquisition of chemoresistance may involve the HMGB1/TLR4-RAGE/sCLU pathway triggered by dying cells to provide survival advantage to remnant live tumor cells.
Assuntos
Clusterina/metabolismo , Resistencia a Medicamentos Antineoplásicos , Proteína HMGB1/metabolismo , Neoplasias da Próstata/metabolismo , Apoptose/genética , Linhagem Celular Tumoral , Clusterina/genética , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteína HMGB1/farmacologia , Humanos , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Neoplasias da Próstata/genética , Proteínas Recombinantes/farmacologia , Receptor 4 Toll-Like/metabolismo , Fator de Transcrição RelA/metabolismoRESUMO
Cytotoxicity is a key function of natural killer (NK) and T cells; yet the molecular mechanism is unclear. We have biological, biochemical and molecular evidence to demonstrate that phosphatidyl-inositol (PI) 3-kinase is critical for direct NK lysis of tumor cells, via control of intracellular granule movement. Tumor cell engagement rapidly activated PI 3-kinase in NK cells within 5 min, as demonstrated by p85 subunit tyrosine phosphorylation and its ability to generate phosphatidylinositol 3-phosphate, PI(3)P, from PI. Wortmannin and LY294002 effectively inhibited NK cells to lyse 51Cr-labeled tumor cells at the same doses that blocked PI-phosphorylating function in tumor-activated NK cells. Immunostaining demonstrated that tumor engagement for only 5 min mobilized perforin and granzyme B from NK cells unidirectionally towards the target, and prior treatment of NK cells with either PI 3-kinase inhibitor effectively stopped this intracellular polarization. Lastly, ectopic expression of dominant-negative p85 or p110 mutant markedly suppressed NK lytic capacity. These results taken together demonstrate that PI 3-kinase may control NK lytic function via granule polarization towards the contacted target cell.
Assuntos
Citotoxicidade Imunológica , Células Matadoras Naturais/enzimologia , Células Matadoras Naturais/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/imunologia , Androstadienos/farmacologia , Comunicação Celular/imunologia , Cromonas/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Granzimas , Células HL-60 , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/patologia , Humanos , Células Jurkat , Leucemia , Glicoproteínas de Membrana/biossíntese , Morfolinas/farmacologia , Perforina , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Citotóxicas Formadoras de Poros , Serina Endopeptidases/biossíntese , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , WortmaninaRESUMO
PURPOSE: Myeloid-derived suppressor cells (MDSC) accumulate in tumor-bearing hosts and are associated with immune suppression. To date, there have only been few studies that evaluate the direct effect of chemotherapeutic agents on MDSCs. Agents that inhibit MDSCs may be useful in the treatment of patients with various cancers. EXPERIMENTAL DESIGN: We investigated the in vivo effects of docetaxel on immune function in 4T1-Neu mammary tumor-bearing mice to examine if a favorable immunomodulatory effect accompanies tumor suppression. Primary focus was on the differentiation status of MDSCs and their ability to modulate T-cell responses. RESULTS: Docetaxel administration significantly inhibited tumor growth in 4T1-Neu tumor-bearing mice and considerably decreased MDSC proportion in the spleen. The treatment also selectively increased CTL responses. Docetaxel-pretreated MDSCs cocultured with OT-II splenocytes in the presence of OVA(323-339) showed OT-II-specific CD4 activation and expansion in vitro. In characterizing the phenotype of MDSCs for M1 (CCR7) and M2 [mannose receptor (CD206)] markers, MDSCs from untreated tumor bearers were primarily MR(+) with few CCR7(+) cells. Docetaxel treatment polarized MDSCs toward an M1-like phenotype, resulting in 40% of MDSCs expressing CCR7 in vivo and in vitro, and macrophage differentiation markers such as MHC class II, CD11c, and CD86 were upregulated. Interestingly, docetaxel induced cell death selectively in MR(+) MDSCs while sparing the M1-like phenotype. Finally, inhibition of signal transducer and activator of transcription 3 may in part be responsible for the observed results. CONCLUSIONS: These findings suggest potential clinical benefit for the addition of docetaxel to current immunotherapeutic protocols.
Assuntos
Imunomodulação/efeitos dos fármacos , Células Mieloides/efeitos dos fármacos , Neoplasias/imunologia , Linfócitos T/efeitos dos fármacos , Taxoides/farmacologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica/efeitos dos fármacos , Docetaxel , Feminino , Tolerância Imunológica/imunologia , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Células Mieloides/imunologia , Células Mieloides/fisiologia , Transplante de Neoplasias , Neoplasias/tratamento farmacológico , Linfócitos T/imunologia , Taxoides/uso terapêuticoRESUMO
Clusterin (CLU), in its cytoplasmic form, is abundant in many advanced cancers and has been established to be cytoprotective against chemotherapeutic agents including docetaxel. However, little is known of the mechanism of its induction. Here, we provide evidence that AKT plays a critical role in upregulating cytoplasmic/secretory sCLU, which is responsible for docetaxel resistance. Western blot analysis indicated that docetaxel-resistant sublines derived from DU145 and PC3 prostate tumor cell lines displayed a markedly increased phospho-AKT level closely accompanied by heightened sCLU expression when compared with parental cells. To examine if AKT has a role in sCLU expression, AKT blockade was done by treatment with a specific inhibitor, API-2, or dominant-negative AKT transduction before analysis of sCLU gene expression. Loss of AKT function resulted in loss of sCLU and was accompanied by chemosensitization to docetaxel and increased cell death via a caspase-3-dependent pathway. To confirm that AKT affected resistance to docetaxel through sCLU and not through other mediators, tumor cells were first transfected with full-length CLU for overexpression and then treated with the AKT inhibitor API-2. We found that once sCLU was overexpressed, API-2 could not chemosensitize the tumor cells to docetaxel. Thus, the chemoresistance to docetaxel is mediated by sCLU and it can be induced by AKT. Lastly, AKT was found to mediate sCLU induction via signal transducer and activator of transcription 1 activation, which we have earlier shown to drive sCLU gene expression. These results identify a previously unrecognized pathway linking AKT to cytoprotection by sCLU in tumor cells.
Assuntos
Antineoplásicos/farmacologia , Clusterina/metabolismo , Citoproteção/efeitos dos fármacos , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Taxoides/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Clusterina/genética , Docetaxel , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Dominantes , Humanos , Masculino , Fenótipo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Fator de Transcrição STAT1/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Interleukin-2 (IL-2) prevents cell apoptosis and promotes survival, but the involved mechanisms have not been completely defined. Although phosphatidylinositide 3-kinase (PI 3-kinase) has been implicated in IL-2-mediated survival mechanisms, none of the 3 chains of the IL-2 receptor (IL-2R) expresses a binding site for PI 3-kinase. However, IL-2Rbeta does express a Syk-binding motif. By using an IL-2-dependent natural killer (NK) cell line, followed by validation of the results in fresh human NK cells, we identified Syk as a critical effector essential for IL-2-mediated prosurvival signaling in NK cells. Down-regulation of Syk by piceatannol treatment impaired NK cellular viability and induced prominent apoptosis as effectively as suppression of PI 3-kinase function by LY294002. Expression of kinase-deficient Syk or pretreatment with piceatannol markedly suppressed IL-2-stimulated activation of PI 3-kinase and Akt, demonstrating that Syk is upstream of PI 3-kinase and Akt. However, constitutively active PI 3-kinase reversed this loss of Akt function caused by kinase-deficient Syk or piceatannol. Thus, Syk appears to regulate PI 3-kinase, which controls Akt activity during IL-2 stimulation. More important, we observed Rac1 activation by IL-2 and found that it mediated PI 3-kinase activation of Akt. This conclusion came from experiments in which dominant-negative Rac1 significantly decreased IL-2-induced Akt activation, whereas constitutively active Rac1 reelevated Akt activity not only in Syk-impaired but also in PI 3-kinase-impaired NK cells. These results constitute the first report of a Syk --> PI3K --> Rac1 --> Akt signal cascade controlled by IL-2 that mediates NK cell survival.
Assuntos
Precursores Enzimáticos/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Células Sanguíneas , Linhagem Celular , Sobrevivência Celular , Inibidores Enzimáticos/farmacologia , Precursores Enzimáticos/antagonistas & inibidores , Precursores Enzimáticos/metabolismo , Humanos , Interleucina-2/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Quinase SykRESUMO
Emerging evidence suggests that NK-activatory receptors use KARAP/DAP12, CD3zeta, and FcepsilonRIgamma adaptors that contain immunoreceptor tyrosine-based activatory motifs to mediate NK direct lysis of tumor cells via Syk tyrosine kinase. NK cells may also use DAP10 to drive natural cytotoxicity through phosphoinositide 3-kinase (PI3K). In contrast to our recently identified PI3K pathway controlling NK cytotoxicity, the signaling mechanism by which Syk associates with downstream effectors to drive NK lytic function has not been clearly defined. In NK92 cells, which express DAP12 but little DAP10/NKG2D, we now show that Syk acts upstream of PI3K, subsequently leading to the specific signaling of the PI3K-->Rac1-->PAK1-->mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase-->ERK cascade that we earlier described. Tumor cell ligation stimulated DAP12 tyrosine phosphorylation and its association with Syk in NK92 cells; Syk tyrosine phosphorylation and activation were also observed. Inhibition of Syk function by kinase-deficient Syk or piceatannol blocked target cell-induced PI3K, Rac1, PAK1, mitogen-activated protein/ERK kinase, and ERK activation, perforin movement, as well as NK cytotoxicity, indicating that Syk is upstream of all these signaling events. Confirming that Syk does not act downstream of PI3K, constitutively active PI3K reactivated all the downstream effectors as well as NK cytotoxicity suppressed in Syk-impaired NK cells. Our results are the first report documenting the instrumental role of Syk in control of PI3K-dependent natural cytotoxicity.
Assuntos
Citotoxicidade Imunológica , Precursores Enzimáticos/fisiologia , Células Matadoras Naturais/enzimologia , Células Matadoras Naturais/imunologia , MAP Quinase Quinase Quinase 1 , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Tirosina Quinases/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Linhagem Celular , Grânulos Citoplasmáticos/imunologia , Grânulos Citoplasmáticos/metabolismo , Citotoxicidade Imunológica/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , Inibidores Enzimáticos/farmacologia , Precursores Enzimáticos/antagonistas & inibidores , Precursores Enzimáticos/metabolismo , Humanos , Imunossupressores/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Células K562 , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Ligantes , Glicoproteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Perforina , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Citotóxicas Formadoras de Poros , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico/imunologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Receptores Imunológicos/metabolismo , Transdução de Sinais/imunologia , Estilbenos/farmacologia , Quinase Syk , Células Tumorais Cultivadas , Quinases Ativadas por p21 , Proteínas rac1 de Ligação ao GTP/fisiologiaRESUMO
Elevated levels of mitogen-activated protein kinase/extracellular regulatory kinase (MAPK/ERK) activity are frequently found in some cancer cells. In efforts to reduce tumor growth, attempts have been made to develop cancer therapeutic agents targeting the MAPK. Here, by use of biologic, biochemical, and gene manipulation methods in human polymorphonuclear neutrophils (PMNs), we have identified a key pathway important in normal cell function involving MAPK/ERK in PMNs for growth inhibition of Candida albicans. Contact with C albicans triggered MAPK/ERK activation in PMNs within 5 minutes, and blocking of MAPK/ERK activation, either by the pharmacologic reagent PD098059 or by dominant-negative MAPK kinase (MEK) expression via vaccinia viral delivery, suppressed antimicrobial activity. Rac and Cdc42, but not Ras or Rho, were responsible for this MAPK/ERK activation. Expression of dominant-negative Rac (N17Rac) or Cdc42 (N17Cdc42) eliminated not only C albicans- mediated ERK phosphorylation but also phagocytosis and granule migration toward the ingested microbes, whereas dominant-negative Ras (N17Ras) and Rho (N19Rho) did not. PAK1 (p21-activated kinase 1) activation is induced by C albicans, suggesting that PAK1 may also be involved in the Rac1 activation of MAPK/ERK. We conclude from these data that Rac/Cdc42-dependent activation of MAPK/ERK is a critical event in the immediate phagocytic response of PMNs to microbial challenge. Therefore, use of MAPK pharmacologic inhibitors for the treatment of cancer may result in the interruption of normal neutrophil function. A balance between therapeutic outcome and undesirable side effects must be attained to achieve successful and safe anticancer therapy.