RESUMO
The complement system, especially the alternative pathway, plays essential roles in the induction of injury in collagen Ab-induced arthritis (CAIA) in mice. The goal of the current study was to directly compare the roles of receptors for C3a and C5a, as well as the membrane attack complex, as effector mechanisms in the pathogenesis of CAIA. Clinical disease activity in C3aR(-/-), C5aR(-/-), and C6-deficient (C6-def) mice was decreased by 52, 94, and 65%, respectively, as compared with wild-type mice. Decreases in histopathologic injury as well as in IgG and C3 deposition paralleled the clinical disease activity. A decrease in the percentage of synovial neutrophils was observed in C3aR(-/-), C5aR(-/-), and C6-def mice, and a decrease in macrophages was observed in C3aR(-/-) and C5aR(-/-), but not in C6-def, mice. Synovial mRNA obtained by laser capture microdissection exhibited a decrease in TNF-α in C5aR(-/-) mice and in IL-1ß in both C5aR(-/-) and C6-def mice, whereas C3aR(-/-) mice demonstrated no change in either cytokine. Our findings show that absent C3aR-, C5aR-, or membrane attack complex-initiated effector mechanisms each decrease susceptibility to CAIA, with clinical effects most pronounced in C5aR-deficient mice. Although the absence of C3aR, C5aR, or C6 led to differential deficiencies in effector mechanisms, decreased proximal joint IgG and C3 deposition was common to all three genotypes in comparison with wild-type mice. These data suggest the existence of positive-feedback amplification pathways downstream of all three effectors that promote additional IgG deposition and C3 activation in the joint.
Assuntos
Artrite Experimental/etiologia , Complemento C6/deficiência , Receptor da Anafilatoxina C5a/fisiologia , Receptores de Complemento/fisiologia , Animais , Ativação do Complemento , Complemento C3a/imunologia , Complemento C6/fisiologia , Citocinas , Suscetibilidade a Doenças , Imunoglobulina G , Macrófagos/patologia , Camundongos , Camundongos Knockout , Neutrófilos/patologia , Líquido Sinovial/imunologiaRESUMO
Rheumatoid arthritis is associated with the development of autoantibodies to citrullinated self-proteins. Citrullinated synovial proteins, which are generated via the actions of the protein arginine deiminases (PADs), are known to develop in the murine collagen-induced arthritis (CIA) model of inflammatory arthritis. Given these findings, we evaluated whether N-α-benzoyl-N5-(2-chloro-1-iminoethyl)-L-ornithine amide (Cl-amidine), a recently described pan-PAD inhibitor, could affect the development of arthritis and autoimmunity by treating mice in the CIA model with Cl-amidine on days 0-35. Cl-amidine treatment reduced total synovial and serum citrullination, decreased clinical disease activity by â¼50%, and significantly decreased IgG2a anti-mouse type II collagen Abs. Additionally, histopathology scores and total complement C3 deposition were significantly lower in Cl-amidine-treated mice compared with vehicle controls. Synovial microarray analyses demonstrated decreased IgG reactivity to several native and citrullinated epitopes compared with vehicle controls. Cl-amidine treatment had no ameliorative effect on collagen Ab-induced arthritis, suggesting its primary protective mechanism was not mediated through effector pathways. Reduced levels of citrullinated synovial proteins observed in mice treated with Cl-amidine are consistent with the notion that Cl-amidine derives its efficacy from its ability to inhibit the deiminating activity of PADs. In total, these results suggested that PADs are necessary participants in the autoimmune and subsequent inflammatory processes in CIA. Cl-amidine may represent a novel class of disease-modifying agents that modulate aberrant citrullination, and perhaps other immune processes, necessary for the development of inflammatory arthritis.
Assuntos
Amidinas/uso terapêutico , Artrite Experimental/tratamento farmacológico , Artrite Experimental/imunologia , Inibidores Enzimáticos/uso terapêutico , Hidrolases/antagonistas & inibidores , Imunossupressores/uso terapêutico , Ornitina/análogos & derivados , Animais , Artrite Experimental/patologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Autoanticorpos/biossíntese , Autoanticorpos/toxicidade , Citrulina/metabolismo , Colágeno Tipo II/antagonistas & inibidores , Colágeno Tipo II/imunologia , Hidrolases/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos DBA , Ornitina/uso terapêutico , Peptídeos Cíclicos/imunologia , Peptídeos Cíclicos/metabolismo , Desiminases de Arginina em Proteínas , Índice de Gravidade de Doença , Membrana Sinovial/imunologia , Membrana Sinovial/metabolismo , Membrana Sinovial/patologiaRESUMO
The complement cascade is an important part of the innate immune system, but pathological activation of this system causes tissue injury in several autoimmune and inflammatory diseases, including immune complex glomerulonephritis. We examined whether mice with targeted deletion of the gene for factor B (fB(-/-) mice) and selective deficiency in the alternative pathway of complement are protected from injury in the nephrotoxic serum (NTS) nephritis model of antibody-mediated glomerulonephritis. When the acute affects of the anti-glomerular basement membrane antibody were assessed, fB(-/-) mice developed a degree of injury similar to wild-type controls. If the mice were presensitized with sheep IgG or if the mice were followed for 5 mo postinjection, however, the fB(-/-) mice developed milder injury than wild-type mice. The immune response of fB(-/-) mice exposed to sheep IgG was similar to that of wild-type mice, but the fB(-/-) mice had less glomerular C3 deposition and lower levels of albuminuria. These results demonstrate that fB(-/-) mice are not significantly protected from acute heterologous injury in NTS nephritis but are protected from autologous injury in response to a planted glomerular antigen. Thus, although the glomerulus is resistant to antibody-initiated, alternative pathway-mediated injury, inhibition of this complement pathway may be beneficial in chronic immune complex-mediated diseases.
Assuntos
Via Alternativa do Complemento/fisiologia , Glomérulos Renais/imunologia , Nefrite/imunologia , Animais , Complemento C3/imunologia , Fator B do Complemento/genética , Modelos Animais de Doenças , Glomérulos Renais/patologia , Camundongos , Camundongos Knockout , Nefrite/patologia , Proteinúria/imunologia , Proteinúria/patologiaRESUMO
Gene-targeted mice deficient in the complement mannose-binding lectin-associated serine protease-1 and -3 (MASP1/3(-/-)) express only the zymogen of factor D (pro-factor D [pro-Df]), a necessary component of the alternative pathway (AP). We used the murine collagen Ab-induced arthritis (CAIA) model, in which the AP is unique among complement pathways in being both necessary and sufficient for disease induction, to determine whether MASP-1/3 are required in vivo for the development of tissue injury. Disease activity scores, complement C3 tissue deposition in the joint, and histopathologic injury scores were markedly decreased in MASP1/3(-/-) as compared with wild-type (WT) mice. MASP-1 protein was immunochemically localized to synovial cells of knees of WT mice with arthritis. Pro-Df was present in both synovial cells and chondrocytes of knees of WT and MASP1/3(-/-) mice without arthritis, with increased amounts present in synovial cells of WT mice with CAIA. No conversion of pro-Df to mature Df was detectable in the serum of MASP1/3(-/-) mice during the evolution of CAIA. C3 activation and deposition as well as C5a generation induced in vitro by adherent anti-type II collagen mAbs were absent using sera from MASP1/3(-/-) mice under conditions in which only the AP was active. The addition of human Df fully reconstituted in vitro C3 activation and C5a generation using sera from MASP1/3(-/-) mice. Our studies demonstrate for the first time, to our knowledge, the absolute requirement for the activity of MASP-1 protein in autoimmune-associated inflammatory tissue injury in vivo through activation of the AP of complement by cleavage of pro-Df to mature Df.
Assuntos
Artrite Experimental/imunologia , Via Alternativa do Complemento/imunologia , Serina Proteases Associadas a Proteína de Ligação a Manose/imunologia , Animais , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Western Blotting , Fator D do Complemento/imunologia , Fator D do Complemento/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da PolimeraseRESUMO
Ischemia/reperfusion (I/R) triggers a robust inflammatory response within the kidney. Numerous components of the immune system contribute to the resultant renal injury, including the complement system. We sought to identify whether natural Abs bind to the postischemic kidney and contribute to complement activation after I/R. We depleted peritoneal B cells in mice by hypotonic shock. Depletion of the peritoneal B cells prevented the deposition of IgM within the glomeruli after renal I/R and attenuated renal injury after I/R. We found that glomerular IgM activates the classical pathway of complement, but it does not cause substantial deposition of C3 within the kidney. Furthermore, mice deficient in classical pathway proteins were not protected from injury, indicating that glomerular IgM does not cause injury through activation of the classical pathway. We also subjected mice deficient in all mature B cells (µMT mice) to renal I/R and found that they sustained worse renal injury than wild-type controls. Serum IL-10 levels were lower in the µMT mice. Taken together, these results indicate that natural Ab produced by peritoneal B cells binds within the glomerulus after renal I/R and contributes to functional renal injury. However, nonperitoneal B cells attenuate renal injury after I/R, possibly through the production of IL-10.
Assuntos
Subpopulações de Linfócitos B/imunologia , Rim/imunologia , Traumatismo por Reperfusão/imunologia , Animais , Formação de Anticorpos , Western Blotting , Ativação do Complemento/imunologia , Ensaio de Imunoadsorção Enzimática , Imunoglobulina M/imunologia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Rim/lesões , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Traumatismo por Reperfusão/patologiaRESUMO
The alternative pathway (AP) of complement is required for the induction of collagen Ab-induced arthritis (CAIA) in mice. The objective of this study was to examine the effect of a recombinant AP inhibitor containing complement receptor 2 and factor H (CR2-fH) on CAIA in mice. CR2 binds to tissue-fixed activation fragments of C3, and the linked fH is a potent local inhibitor of the AP. CAIA was induced in C57BL/6 mice by i.p. injections of 4 mAb to type II collagen (CII) on day 0 and LPS on day 3. PBS or CR2-fH (250 or 500 microg) were injected i.p. 15 min after the mAb to CII on day 0 and 15 min after LPS on day 3; the mice were sacrificed on day 10. The disease activity score (DAS) was decreased significantly (p < 0.001) in both groups receiving CR2-fH compared with the PBS. Histology scores for inflammation, pannus, bone damage, and cartilage damage decreased in parallel with the DAS. C3 deposition in the synovium and cartilage was significantly reduced (p < 0.0001) in the mice treated with CR2-fH. In vitro studies with immune complexes containing type II collagen and mAb to CII showed that CR2-fH specifically inhibited the AP with minimal effect on the classical pathway (CP) and no effect on the lectin pathway (LP). The relative potency of CR2-fH in vitro was superior to mAbs to factor B and C5. Thus, CR2-fH specifically targets and inhibits the AP of complement in vitro and is effective in CAIA in vivo.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Artrite Experimental/imunologia , Colágeno Tipo II/imunologia , Fator H do Complemento/fisiologia , Proteínas Inativadoras do Complemento/fisiologia , Via Alternativa do Complemento/imunologia , Receptores de Complemento 3d/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , Animais , Artrite Experimental/patologia , Artrite Experimental/terapia , Bovinos , Fator H do Complemento/administração & dosagem , Proteínas Inativadoras do Complemento/administração & dosagem , Combinação de Medicamentos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Complemento 3d/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagemRESUMO
OBJECTIVE: The serum of most neuromyelitis optica (NMO) patients contains autoantibodies (NMO-IgGs) directed against the aquaporin-4 (AQP4) water channel located on astrocyte foot processes in the perivessel and subpial areas of the brain. Our objectives were to determine the source of central nervous system (CNS) NMO-IgGs and their role in disease pathogenesis. METHODS: Fluorescence-activated cell sorting and single-cell reverse transcriptase polymerase chain reaction were used to identify overrepresented plasma cell immunoglobulin (Ig) sequences in the cerebrospinal fluid (CSF) of an NMO patient after a first clinical attack. Monoclonal recombinant antibodies (rAbs) were generated from the paired heavy and light chain sequences and tested for target specificity and Fc effector function. The effect of CSF rAbs on CNS immunopathology was investigated by delivering single rAbs to rats with experimental autoimmune encephalomyelitis (EAE). RESULTS: Repertoire analysis revealed a dynamic, clonally expanded plasma cell population with features of an antigen-targeted response. Using multiple independent assays, 6 of 11 rAbs generated from CSF plasma cell clones specifically bound to AQP4. AQP4-specific rAbs recognized conformational epitopes and mediated both AQP4-directed antibody-dependent cellular cytotoxicity and complement-mediated lysis. When administered to rats with EAE, an AQP4-specific NMO CSF rAb induced NMO immunopathology: perivascular astrocyte depletion, myelinolysis, and complement and Ig deposition. INTERPRETATION: Molecular characterization of the CSF plasma cell repertoire in an early NMO patient demonstrates that AQP4-specific Ig is synthesized intrathecally at disease onset and directly contributes to CNS pathology. AQP4 is now the first confirmed antigenic target in human demyelinating disease.
Assuntos
Aquaporina 4/líquido cefalorraquidiano , Aquaporina 4/imunologia , Autoanticorpos/líquido cefalorraquidiano , Neuromielite Óptica/líquido cefalorraquidiano , Neuromielite Óptica/diagnóstico , Sequência de Aminoácidos , Animais , Aquaporina 4/genética , Biomarcadores/líquido cefalorraquidiano , Linhagem Celular , Células Cultivadas , Feminino , Feto , Humanos , Imunoglobulina G/líquido cefalorraquidiano , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Ratos , Ratos Endogâmicos Lew , Punção Espinal , Fatores de TempoRESUMO
Oncogenic kinase fusions of ALK, ROS1, RET, and NTRK1 act as drivers in human lung and other cancers. Residual tumor burden following treatment of ALK or ROS1+ lung cancer patients with oncogene-targeted therapy ultimately enables the emergence of drug-resistant clones, limiting the long-term effectiveness of these therapies. To determine the signaling mechanisms underlying incomplete tumor cell killing in oncogene-addicted cancer cells, we investigated the role of EGFR signaling in drug-naïve cancer cells harboring these oncogene fusions. We defined three distinct roles for EGFR in the response to oncogene-specific therapies. First, EGF-mediated activation of EGFR blunted fusion kinase inhibitor binding and restored fusion kinase signaling complexes. Second, fusion kinase inhibition shifted adaptor protein binding from the fusion oncoprotein to EGFR. Third, EGFR enabled bypass signaling to critical downstream pathways such as MAPK. While evidence of EGFR-mediated bypass signaling has been reported after ALK and ROS1 blockade, our results extended this effect to RET and NTRK1 blockade and uncovered the other additional mechanisms in gene fusion-positive lung cancer cells, mouse models, and human clinical specimens before the onset of acquired drug resistance. Collectively, our findings show how EGFR signaling can provide a critical adaptive survival mechanism that allows cancer cells to evade oncogene-specific inhibitors, providing a rationale to cotarget EGFR to reduce the risks of developing drug resistance. Cancer Res; 77(13); 3551-63. ©2017 AACR.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Receptores ErbB/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Proteínas de Fusão Oncogênica/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Nus , Proteínas de Fusão Oncogênica/genética , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Background: We have an incomplete understanding of the differences between cancer stem cells (CSCs) in human papillomavirus-positive (HPV-positive) and -negative (HPV-negative) head and neck squamous cell cancer (HNSCC). The PI3K pathway has the most frequent activating genetic events in HNSCC (especially HPV-positive driven), but the differential signaling between CSCs and non-CSCs is also unknown. Methods: We addressed these unresolved questions using CSCs identified from 10 HNSCC patient-derived xenografts (PDXs). Sored populations were serially passaged in nude mice to evaluate tumorigenicity and tumor recapitulation. The transcription profile of HNSCC CSCs was characterized by mRNA sequencing, and the susceptibility of CSCs to therapy was investigated using an in vivo model. SOX2 transcriptional activity was used to follow the asymmetric division of PDX-derived CSCs. All statistical tests were two-sided. Results: CSCs were enriched by high aldehyde dehydrogenase (ALDH) activity and CD44 expression and were similar between HPV-positive and HPV-negative cases (percent tumor formation injecting ≤ 1x10(3) cells: ALDH(+)CD44(high) = 65.8%, ALDH(-)CD44(high) = 33.1%, ALDH(+)CD44(high) = 20.0%; and injecting 1x10(5) cells: ALDH(-)CD44(low) = 4.4%). CSCs were resistant to conventional therapy and had PI3K/mTOR pathway overexpression (GSEA pathway enrichment, P < .001), and PI3K inhibition in vivo decreased their tumorigenicity (40.0%-100.0% across cases). PI3K/mTOR directly regulated SOX2 protein levels, and SOX2 in turn activated ALDH1A1 (P < .001 013C and 067C) expression and ALDH activity (ALDH(+) [%] empty-control vs SOX2, 0.4% ± 0.4% vs 14.5% ± 9.8%, P = .03 for 013C and 1.7% ± 1.3% vs 3.6% ± 3.4%, P = .04 for 067C) in 013C and 067 cells. SOX2 enhanced sphere and tumor growth (spheres/well, 013C P < .001 and 067C P = .04) and therapy resistance. SOX2 expression prompted mesenchymal-to-epithelial transition (MET) by inducing CDH1 (013C P = .002, 067C P = .01), followed by asymmetric division and proliferation, which contributed to tumor formation. Conclusions: The molecular link between PI3K activation and CSC properties found in this study provides insights into therapeutic strategies for HNSCC. Constitutive expression of SOX2 in HNSCC cells generates a CSC-like population that enables CSC studies.
Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias de Cabeça e Pescoço/genética , Células-Tronco Neoplásicas/metabolismo , Fosfatidilinositol 3-Quinase/genética , RNA Mensageiro/análise , Fatores de Transcrição SOXB1/genética , Aldeído Desidrogenase/metabolismo , Família Aldeído Desidrogenase 1 , Animais , Antígenos CD , Antineoplásicos/farmacologia , Caderinas/metabolismo , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/virologia , Divisão Celular , Proliferação de Células , Transformação Celular Neoplásica/efeitos dos fármacos , Receptores ErbB/metabolismo , Feminino , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Receptores de Hialuronatos/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Papillomaviridae/isolamento & purificação , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Retinal Desidrogenase , Fatores de Transcrição SOXB1/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Esferoides Celulares , Serina-Treonina Quinases TOR/metabolismo , Transcriptoma , Células Tumorais CultivadasRESUMO
BACKGROUND: This phase 1, dose-finding study determined the safety, maximum tolerated dose (MTD)/recommended phase 2 dose (RP2D), antitumor activity, and molecular correlates of IPI-926, a Hedgehog pathway (HhP) inhibitor, combined with cetuximab in patients with relapsed/metastatic squamous cell carcinoma of the head and neck. PATIENTS AND METHODS: Cetuximab was given with a 400mg/m(2) loading dose followed by 250mg/m(2) weekly. IPI-926 was given daily starting two weeks after cetuximab initiation. A "3+3" study design was used. Prior therapy with cetuximab was allowed. Tumor biopsies occurred prior to cetuximab initiation, prior to IPI-926 initiation, and after treatment with both drugs. RESULTS: Nine patients were enrolled. The RP2D was 160mg, the same as the single-agent IPI-926 MTD. Among 9 treated, 8 evaluable patients, the best responses were 1 partial response (12.5%), 4 stable disease (50%), and 3 disease progressions (37.5%). The median progression free survival was 77days (95% confidence interval 39-156). Decreases in tumor size were seen in both cetuximab-naïve patients (one HPV-positive, one HPV-negative). The most frequent treatment-emergent adverse events were fatigue, muscle cramps, and rash. No DLTs were observed. Tumor shrinkage and progression free survival were associated with intra-tumoral ErbB and HhP gene expression down-regulation during therapy, supporting the preclinical hypothesis. CONCLUSION: Treatment with IPI-926 and cetuximab yielded expected toxicities with signs of anti-tumor activity. Serial tumor biopsies were feasible and revealed proof-of-concept biomarkers.
Assuntos
Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Cetuximab/uso terapêutico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Intervalo Livre de Doença , Feminino , Expressão Gênica/efeitos dos fármacos , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/genética , Humanos , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Projetos Piloto , Resultado do TratamentoRESUMO
Triple-negative breast cancer (TNBC) is an aggressive disease with a poor prognosis. Advances in the treatment of TNBC have been hampered by the lack of novel effective targeted therapies. The primary goal of this study was to evaluate the efficacy of targeting Aurora kinase A (AurA), a key regulator of mitosis, in TNBC models. A secondary objective was to determine the role of the p53 family of transcriptional regulators, commonly mutated in TNBC, in determining the phenotypic response to the AurA inhibitor alisertib (MLN8237). Alisertib exhibited potent antiproliferative and proapoptotic activity in a subset of TNBC models. The induction of apoptosis in response to alisertib exposure was dependent on p53 and p73 activity. In the absence of functional p53 or p73, there was a shift in the phenotypic response following alisertib exposure from apoptosis to cellular senescence. In addition, senescence was observed in patient-derived tumor xenografts with acquired resistance to alisertib treatment. AurA inhibitors are a promising class of novel therapeutics in TNBC. The role of p53 and p73 in mediating the phenotypic response to antimitotic agents in TNBC may be harnessed to develop an effective biomarker selection strategy in this difficult to target disease.
Assuntos
Azepinas/administração & dosagem , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Pirimidinas/administração & dosagem , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Apoptose , Aurora Quinase A/antagonistas & inibidores , Azepinas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Camundongos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Proteína Tumoral p73 , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Local control and overall survival in patients with advanced head and neck squamous cell cancer (HNSCC) remains dismal. Signaling through the Hedgehog (Hh) pathway is associated with epithelial-to-mesenchymal transition, and activation of the Hh effector transcription factor Gli1 is a poor prognostic factor in this disease setting. Here, we report that increased GLI1 expression in the leading edge of HNSCC tumors is further increased by irradiation, where it contributes to therapeutic inhibition. Hh pathway blockade with cyclopamine suppressed GLI1 activation and enhanced tumor sensitivity to radiotherapy. Furthermore, radiotherapy-induced GLI1 expression was mediated in part by the mTOR/S6K1 pathway. Stroma exposed to radiotherapy promoted rapid tumor repopulation, and this effect was suppressed by Hh inhibition. Our results demonstrate that Gli1 that is upregulated at the tumor-stroma intersection in HNSCC is elevated by radiotherapy, where it contributes to stromal-mediated resistance, and that Hh inhibitors offer a rational strategy to reverse this process to sensitize HNSCC to radiotherapy.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Proteínas Hedgehog/metabolismo , Tolerância a Radiação/fisiologia , Células Estromais/metabolismo , Células Estromais/patologia , Animais , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/radioterapia , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/fisiologia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/radioterapia , Humanos , Camundongos , Camundongos Nus , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Carcinoma de Células Escamosas de Cabeça e Pescoço , Células Estromais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacos , Alcaloides de Veratrum/farmacologia , Proteína GLI1 em Dedos de ZincoRESUMO
The dual pathway inhibitor rigosertib inhibits phosphoinositide 3-kinase (PI3K) pathway activation as well as polo-like kinase 1 (PLK1) activity across a broad spectrum of cancer cell lines. The importance of PIK3CA alterations in squamous cell carcinoma of the head and neck (HNSCC) has raised interest in exploring agents targeting PI3K, the product of PIK3CA. The genetic and molecular basis of rigosertib treatment response was investigated in a panel of 16 HNSCC cell lines, and direct patient tumor xenografts from eight patients with HNSCC [four HPV-serotype16 (HPV16)-positive]. HNSCC cell lines and xenografts were characterized by pathway enrichment gene expression analysis, exon sequencing, gene copy number, Western blotting, and immunohistochemistry (IHC). Rigosertib had potent antiproliferative effects on 11 of 16 HPV(-) HNSCC cell lines. Treatment sensitivity was confirmed in two cell lines using an orthotopic in vivo xenograft model. Growth reduction after rigosertib treatment was observed in three of eight HNSCC direct patient tumor lines. The responsive tumor lines carried a combination of a PI3KCA-activating event (amplification or mutation) and a p53-inactivating event (either HPV16- or mutation-mediated TP53 inactivation). In this study, we evaluated the in vitro and in vivo efficacy of rigosertib in both HPV(+) and HPV(-) HNSCCs, focusing on inhibition of the PI3K pathway. Although consistent inhibition of the PI3K pathway was not evident in HNSCC, we identified a combination of PI3K/TP53 events necessary, but not sufficient, for rigosertib sensitivity.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Glicina/análogos & derivados , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Sulfonas/administração & dosagem , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/virologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Classe I de Fosfatidilinositol 3-Quinases , Glicina/administração & dosagem , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Mutação , Papillomaviridae/efeitos dos fármacos , Infecções por Papillomavirus/tratamento farmacológico , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Quinase 1 Polo-LikeRESUMO
The EGF receptor (EGFR)-directed monoclonal antibody cetuximab is the only targeted therapy approved for the treatment of squamous cell carcinoma of the head and neck (HNSCC) but is only effective in a minority of patients. Epithelial-to-mesenchymal transition (EMT) has been implicated as a drug resistance mechanism in multiple cancers, and the EGFR and Hedgehog pathways (HhP) are relevant to this process, but the interplay between the two pathways has not been defined in HNSCC. Here, we show that HNSCC cells that were naturally sensitive to EGFR inhibition over time developed increased expression of the HhP transcription factor GLI1 as they became resistant after long-term EGFR inhibitor exposure. This robustly correlated with an increase in vimentin expression. Conversely, the HhP negatively regulated an EGFR-dependent, EMT-like state in HNSCC cells, and pharmacologic or genetic inhibition of HhP signaling pushed cells further into an EGFR-dependent phenotype, increasing expression of ZEB1 and VIM. In vivo treatment with cetuximab resulted in tumor shrinkage in four of six HNSCC patient-derived xenografts; however, they eventually regrew. Cetuximab in combination with the HhP inhibitor IPI-926 eliminated tumors in two cases and significantly delayed regrowth in the other two cases. Expression of EMT genes TWIST and ZEB2 was increased in sensitive xenografts, suggesting a possible resistant mesenchymal population. In summary, we report that EGFR-dependent HNSCC cells can undergo both EGFR-dependent and -independent EMT and HhP signaling is a regulator in both processes. Cetuximab plus IPI-926 forces tumor cells into an EGFR-dependent state, delaying or completely blocking tumor recurrence.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma de Células Escamosas/metabolismo , Receptores ErbB/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Proteínas Hedgehog/metabolismo , Alcaloides de Veratrum/farmacologia , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Cetuximab , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Inativação Gênica , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Proteínas Hedgehog/genética , Humanos , Camundongos , Camundongos Nus , Receptor Cross-Talk , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e Pescoço , Alcaloides de Veratrum/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Targeted therapy development in head and neck squamous cell carcinoma (HNSCC) is challenging given the rarity of activating mutations. Additionally, HNSCC incidence is increasing related to human papillomavirus (HPV). We sought to develop an in vivo model derived from patients reflecting the evolving HNSCC epidemiologic landscape, and use it to identify new therapies. Primary and relapsed tumors from HNSCC patients, both HPV+ and HPV-, were implanted on mice, giving rise to 25 strains. Resulting xenografts were characterized by detecting key mutations, measuring protein expression by IHC and gene expression/pathway analysis by mRNA-sequencing. Drug efficacy studies were run with representative xenografts using the approved drug cetuximab as well as the new PI3K inhibitor PX-866. Tumors maintained their original morphology, genetic profiles and drug susceptibilities through serial passaging. The genetic makeup of these tumors was consistent with known frequencies of TP53, PI3KCA, NOTCH1 and NOTCH2 mutations. Because the EGFR inhibitor cetuximab is a standard HNSCC therapy, we tested its efficacy and observed a wide spectrum of efficacy. Cetuximab-resistant strains had higher PI3K/Akt pathway gene expression and protein activation than cetuximab-sensitive strains. The PI3K inhibitor PX-866 had anti-tumor efficacy in HNSCC models with PIK3CA alterations. Finally, PI3K inhibition was effective in two cases with NOTCH1 inactivating mutations. In summary, we have developed an HNSCC model covering its clinical spectrum whose major genetic alterations and susceptibility to anticancer agents represent contemporary HNSCC. This model enables to prospectively test therapeutic-oriented hypotheses leading to personalized medicine.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Alphapapillomavirus/patogenicidade , Animais , Western Blotting , Carcinoma de Células Escamosas/virologia , Cetuximab , Biologia Computacional , Receptores ErbB/genética , Receptores ErbB/metabolismo , Gonanos/farmacologia , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Imuno-Histoquímica , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptor Notch2/genética , Receptor Notch2/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mannose-binding lectin-associated serine proteases-1/3 (MASP-1/3) are essential in activating the alternative pathway (AP) of complement through cleaving pro-factor D (pro-Df) into mature Df. MASP are believed to require binding to mannose binding lectins (MBL) or ficolins (FCN) to carry out their biological activities. Murine sera have been reported to contain MBL-A, MBL-C, and FCN-A, but not FCN-B that exists endogenously in monocytes and is thought not to bind MASP-1. We examined some possible mechanisms whereby MASP-1/3 might activate the AP. Collagen antibody-induced arthritis, a murine model of inflammatory arthritis dependent on the AP, was unchanged in mice lacking MBL-A, MBL-C, and FCN-A (MBL(-/-)/FCN A(-/-) mice) in comparison to wild-type mice. The in vitro induction of the AP by adherent mAb to collagen II was intact using sera from MBL(-/-)/FCN A(-/-) mice. Furthermore, sera from MBL(-/-)/FCN A(-/-) mice lacked pro-Df and possessed only mature Df. Gel filtration of sera from MBL(-/-)/FCN A(-/-) mice showed the presence of MASP-1 protein in fractions containing proteins smaller than the migration of MBL-A and MBL-C in sera from C4(-/-) mice, suggesting possible binding of MASP-1 to an unknown protein. Lastly, we show that FCN-B was present in the sera of MBL(-/-)/FCN A(-/-) mice and that it was bound to MASP-1. We conclude that MASP-1 does not require binding to MBL-A, MBL-C, or FCN-A to activate the AP. MASP-1 may cleave pro-Df into mature Df through binding to FCN-B or to an unknown protein, or may function as an unbound soluble protein.
Assuntos
Artrite Experimental/metabolismo , Via Alternativa do Complemento/fisiologia , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Animais , Western Blotting , Fator D do Complemento/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Although current demands for therapeutic mAbs are growing quickly, production methods to date, including in vitro mammalian tissue culture and transgenic animals, provide only limited quantities at high cost. Several tumor-associated antigens in tumor cells have been identified as targets for therapeutic mAbs. Here we describe the production of mAb BR55-2 (IgG2a) in transgenic plants that recognizes the nonprotein tumor-associated antigen Lewis Y oligosaccharide overexpressed in human carcinomas, particularly breast and colorectal cancers. Heavy and light chains of mAb BR55-2 were expressed separately and assembled in plant cells of low-alkaloid tobacco transgenic plants (Nicotiana tabacum cv. LAMD609). Expression levels of plant-derived mAb (mAbP) were high (30 mg/kg of fresh leaves) in T1 generation plants. Like the mammalian-derived mAbM, the plant mAbP bound specifically to both SK-BR3 breast cancer cells and SW948 colorectal cancer cells. The Fc domain of both mAbP and mAbM showed the similar binding to FcgammaRI receptor (CD64). Comparable levels of cytotoxicity against SK-BR3 cells were also shown for both mAbs in antibody-dependent cell-mediated cytotoxicity assay. Furthermore, plant-derived BR55-2 efficiently inhibited SW948 tumor growth xenografted in nude mice. Altogether, these findings suggest that mAbP originating from low-alkaloid tobacco exhibit biological activities suitable for efficient immunotherapy.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Imunoterapia , Antígenos do Grupo Sanguíneo de Lewis/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Planticorpos/imunologia , Células 3T3 , Animais , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Citometria de Fluxo , Expressão Gênica , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Plantas Geneticamente Modificadas , Receptores de IgG/imunologia , Nicotiana/genética , Células Tumorais CultivadasRESUMO
The tumor-associated antigen EpCAM (GA733-2) is a highly expressed target on adenocarcinoma cells, as defined by murine mAb CO17-1A. We recently developed a transgenic plant system for the safe and inexpensive production of large quantities of mAb CO17-1A as a future source of clinical-grade protein. Although the glycosylation pattern of plant-derived mAb (mAb(P)) CO17-1A differs considerably from that of the mammalian-derived mAb (mAb(M)), we show here that the biological activity of both mAbs is quite similar. mAb(P) heavy and light chains assembled to bind the recombinant antigen GA733-2E and specifically bound to human SW948 colorectal carcinoma cells expressing the antigen GA733-2 to the same extent as mAb(M). mAb(P) was as effective as mAb(M) CO17-1A in inhibiting tumor growth of xenotransplanted SW948 cells in nude mice. These results suggest the promise of transgenic plants as a useful alternative way to produce full-size mAb for cancer immunotherapy.