RESUMO
Malignant peripheral nerve sheath tumors (MPNSTs) are chemotherapy resistant sarcomas that are a leading cause of death in neurofibromatosis type 1 (NF1). Although NF1-related MPNSTs derive from neural crest cell origin, they also exhibit intratumoral heterogeneity. TP53 mutations are associated with significantly decreased survival in MPNSTs, however the mechanisms underlying TP53-mediated therapy responses are unclear in the context of NF1-deficiency. We evaluated the role of two commonly altered genes, MET and TP53, in kinome reprograming and cellular differentiation in preclinical MPNST mouse models. We previously showed that MET amplification occurs early in human MPNST progression and that Trp53 loss abrogated MET-addiction resulting in MET inhibitor resistance. Here we demonstrate a novel mechanism of therapy resistance whereby p53 alters MET stability, localization, and downstream signaling leading to kinome reprogramming and lineage plasticity. Trp53 loss also resulted in a shift from RAS/ERK to AKT signaling and enhanced sensitivity to MEK and mTOR inhibition. In response to MET, MEK and mTOR inhibition, we observed broad and heterogeneous activation of key differentiation genes in Trp53-deficient lines suggesting Trp53 loss also impacts lineage plasticity in MPNSTs. These results demonstrate the mechanisms by which p53 loss alters MET dependency and therapy resistance in MPNSTS through kinome reprogramming and phenotypic flexibility.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Neurofibromatose 1 , Inibidores de Proteínas Quinases , Proteína Supressora de Tumor p53 , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Camundongos , Humanos , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Neurofibromatose 1/genética , Neurofibromatose 1/patologia , Neurofibromina 1/genética , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Neoplasias de Bainha Neural/genética , Neoplasias de Bainha Neural/patologia , Neoplasias de Bainha Neural/tratamento farmacológico , Linhagem Celular Tumoral , Transdução de Sinais , Linhagem da Célula/genética , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Neurofibrossarcoma/genética , Neurofibrossarcoma/patologia , Neurofibrossarcoma/tratamento farmacológico , Plasticidade Celular/efeitos dos fármacos , Plasticidade Celular/genéticaRESUMO
Purpose: To determine if proteasome inhibition using MG132 increased the efficiency of FIV vector-mediated transduction in human trabecular meshwork (TM)-1 cells and monkey organ-cultured anterior segments (MOCAS). Methods: TM-1 cells were pretreated for 1 hour with 0.5% dimethyl sulfoxide (DMSO; vehicle control) or 5 to 50 µM MG132 and transduced with FIV.GFP (green fluorescent protein)- or FIV.mCherry-expressing vector at a multiplicity of transduction (MOT) of 20. At 24 hours, cells were fixed and stained with antibodies for GFP, and positive cells were counted, manually or by fluorescence-activated cell sorting (FACS). Cells transduced with FIV.GFP particles alone were used as controls. The effect of 20 µM MG132 treatment on high- and low-dose (2 × 107 and 0.8 × 107 transducing units [TU], respectively) FIV.GFP transduction with or without MG132 was also evaluated in MOCAS using fluorescence microscopy. Vector genome equivalents in cells and tissues were quantified by quantitative (q)PCR on DNA. Results: In the MG132 treatment groups, there was a significant dose-dependent increase in the percentage of transduced cells at all concentrations tested. Vector genome equivalents were also increased in TM-1 cells treated with MG132. Increased FIV.GFP expression in the TM was also observed in MOCAS treated with 20 µM MG132 and the high dose of vector. Vector genome equivalents were also significantly increased in the MOCAS tissues. Increased transduction was not seen with the low dose of virus. Conclusions: Proteasome inhibition increased the transduction efficiency of FIV particles in TM-1 cells and MOCAS and may be a useful adjunct for delivery of therapeutic genes to the TM by lentiviral vectors.
Assuntos
Inibidores de Cisteína Proteinase/farmacologia , Vetores Genéticos , Vírus da Imunodeficiência Felina/genética , Leupeptinas/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Malha Trabecular/metabolismo , Transdução Genética , Animais , Segmento Anterior do Olho/metabolismo , Células Cultivadas , Citometria de Fluxo , Regulação da Expressão Gênica/fisiologia , Proteínas de Fluorescência Verde/genética , Humanos , Macaca mulatta , Técnicas de Cultura de Órgãos , Reação em Cadeia da Polimerase em Tempo Real , TransfecçãoRESUMO
Immunotherapy is a promising strategy for targeting tumors. One emerging approach is to harness the immune effector functions of natural antibodies to destroy tumor cells. Dinitrophenyl (DNP) and the galactose-α-1,3-galactose (αGal) epitope are two haptens that bind endogenous antibodies. One potential alternative is the deoxysugar L-rhamnose. We compared these candidates by using a biosensor assay to evaluate human sera for endogenous antibody concentration, antibody isotype distribution, and longevity of antibody-hapten interactions. Antibodies recognizing α-rhamnose are of equal or greater abundance and affinity as those recognizing αGal. Moreover, both rhamnose and αGal epitopes are more effective than DNP at recruiting the IgG antibody subtype. Exposure of tumor cells to rhamnose-bearing glycolipids and human serum promotes complement-mediated cytotoxicity. These data highlight the utility of α-rhamnose-containing glycoconjugates to direct the immune system to target cells.
Assuntos
Anticorpos/imunologia , Glicoconjugados/química , Glicoconjugados/farmacologia , Neoplasias/terapia , Ramnose/análogos & derivados , Ramnose/farmacologia , Linhagem Celular Tumoral , Galactose/química , Galactose/imunologia , Glicoconjugados/imunologia , Haptenos/química , Haptenos/imunologia , Humanos , Imunoglobulina G/imunologia , Imunoterapia , Neoplasias/imunologia , Ramnose/imunologia , Ressonância de Plasmônio de SuperfícieRESUMO
Integrins play myriad and vital roles in development and disease. They connect a cell with its surroundings and transmit chemical and mechanical signals across the plasma membrane to the cell's interior. Dissecting their roles in cell behavior is complicated by their overlapping ligand specificity and shared downstream signaling components. In principle, immobilized synthetic peptides can mimic extracellular matrix proteins by supporting integrin-mediated adhesion, but most short peptide sequences lack selectivity for one integrin over others. In contrast, synthetic integrin antagonists can be highly selective. We hypothesized that this selectivity could be exploited if antagonists, when immobilized, could support cellular adhesion and activate signaling by engaging specific cell-surface integrins. To investigate this possibility, we designed a bifunctional (RGD)-based peptidomimetic for surface presentation. Our conjugate combines a high affinity integrin ligand with a biotin moiety; the former engages the α(v)ß(3) integrin, and the latter allows for presentation on streptavidin-coated surfaces. Surfaces decorated with this ligand promote both cellular adhesion and integrin activation. Moreover, the selectivity of these surfaces for the α(v)ß(3) integrin can be exploited to capture a subset of cells from a mixed population. We anticipate that surfaces displaying highly selective small molecule ligands can reveal the contributions of specific integrin heterodimers to cell adhesion and signaling.