RESUMEN
Epidermal growth factor receptor (EGFR)-targeted therapy in head and neck squamous cell carcinoma (HNSCC) patients frequently results in tumor resistance to treatment. Autophagy is an emerging underlying resistance mechanism, however, the molecular autophagy machinery in HNSCC cells and potential biomarkers of patient response to EGFR-targeted therapy remain insufficiently characterized. Here we show that the EGFR blocking with cetuximab leads to varied autophagic responses, which modulate cancer cell susceptibility to EGFR inhibition. Inhibition of autophagy sensitizes HNSCC cells to EGFR blockade. Importantly, we identify a novel signaling hub centering on the NLRX1 (nucleotide-binding, lots of leucine-rich repeats-containing protein member X1)-TUFM (Tu translation elongation factor mitochondrial) protein complex, promoting autophagic flux. Defects in the expression of either NLRX1 or TUFM result in compromised autophagy when treated with EGFR inhibitors. As a previously undefined autophagy-promoting mechanism, we found that TUFM serves as a novel anchorage site, recruiting Beclin-1 to mitochondria, promoting its polyubiquitination, and interfering with its interaction with Rubicon. This protein complex is also essential for endoplasmic reticulum stress signaling induction, possibly as an additional mechanism to promote autophagy. Utilizing tumor specimens from a novel neoadjuvant clinical trial, we show that increased expression of the autophagy adaptor protein, SQSTM1/p62, is associated with poor response to cetuximab therapy. These findings expand our understanding of the components involved in HNSCC autophagy machinery that responds to EGFR inhibitors, and suggest potential combinatorial approaches to enhance its therapeutic efficacy.
Asunto(s)
Carcinoma de Células Escamosas/tratamiento farmacológico , Receptores ErbB/genética , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Proteínas Mitocondriales/genética , Factor Tu de Elongación Peptídica/genética , Anticuerpos Monoclonales/administración & dosificación , Autofagia/efectos de los fármacos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Cetuximab/administración & dosificación , Receptores ErbB/antagonistas & inhibidores , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Humanos , Terapia Molecular Dirigida , Proteína Sequestosoma-1/genética , Carcinoma de Células Escamosas de Cabeza y CuelloRESUMEN
Levels of prostaglandin E(2) (PGE(2)) and its processing enzyme, prostaglandin-endoperoxide-synthase-2/ cyclooxygenase-2 (PTGS2/COX-2), are elevated in actively progressing periodontal lesions, but suppressed in chronic disease. COX-2 expression is regulated through inflammatory signaling that converges on the mitogen-activated protein kinase (MAPK) pathway. Emerging evidence suggests a role for the inflammatory adaptor protein, ASC/Pycard, in MAPK activation. We postulated that ASC may represent a mediator of the MAPK-mediated regulatory network of PGE(2) production. Using RNAi-mediated gene slicing, we demonstrated that ASC regulates COX-2 expression and PGE(2) production in THP1 monocytic cells following infection with Porphyromonas gingivalis (Pg). Production of PGE(2) did not require the inflammasome adaptor function of ASC, but was dependent on MAPK activation. Furthermore, the MAP kinase kinase kinase CARD domain-containing protein RIPK2 was induced by Pg in an ASC-dependent manner. Reduced ASC and RIPK2 levels were revealed by orthogonal comparison of the expression of the RIPK family in ASC-deficient THP1 cells with that in chronic periodontitis patients. We show that pharmacological inhibition of RIPK2 represses PGE(2) secretion, and RNAi-mediated silencing of RIPK2 leads to diminished MAPK activation and PGE(2) secretion. These findings identify a novel ASC-RIPK2 axis in the generation of PGE(2) that is repressed in patients diagnosed with chronic adult periodontitis.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/metabolismo , Periodontitis Crónica/enzimología , Proteínas del Citoesqueleto/metabolismo , Dinoprostona/biosíntesis , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismo , Proteínas Bacterianas/metabolismo , Línea Celular Tumoral , Periodontitis Crónica/microbiología , Ciclooxigenasa 2/biosíntesis , Ciclooxigenasa 2/metabolismo , Proteínas del Citoesqueleto/genética , Regulación Enzimológica de la Expresión Génica , Humanos , Mediadores de Inflamación/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Sistema de Señalización de MAP Quinasas/fisiología , Porphyromonas gingivalis , Interferencia de ARN , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/genéticaRESUMEN
We constructed and analyzed six serial analysis of gene expression (SAGE) libraries to identify genes with previously uncharacterized roles in spleen or thymus development. A total of 625 070 tags were sequenced from the three spleen (embryonic day (E)15.5, E16.5 and adult) and three thymus (E15.5, E18.5 and adult) libraries. These tags corresponded to 83 182 tag types, which mapped unambiguously to 36 133 different genes. Genes over-represented in these libraries, compared to 115 mouse SAGE libraries (www.mouseatlas.org), included genes of known and unknown immunological or developmental relevance. The expression profiles of 11 genes with unknown roles in spleen and thymus development were validated using reverse transcription-qPCR. We further characterized the expression of one of these candidates, RIKEN cDNA 9230105E10 that encodes a murine homolog of Trim5alpha, in numerous adult tissues and immune cell types. In addition, we demonstrate that transcript levels are upregulated in response to TLR stimulation of plasmacytoid dendritic cells and macrophages. This work provides the first evidence of regulated and cell type-specific expression of this gene. In addition, these observations suggest that the SAGE libraries provide an important resource for further investigations into the molecular mechanisms regulating spleen and thymus organogenesis, as well as the development of immunological competence.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Biblioteca de Genes , Bazo/inmunología , Timo/inmunología , Factores de Transcripción , Animales , Células de la Médula Ósea/citología , Células Cultivadas , ADN Complementario , Etiquetas de Secuencia Expresada , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Embarazo , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/embriología , Células Madre/citología , Timo/embriologíaRESUMEN
Desoxyepothilone B (dEpoB), currently in clinical trials, is a novel microtubule inhibitor with similar mode-of-action to paclitaxel (Taxol). Intriguingly, it is effective in some cell lines and tumor xenografts refractory to Taxol. The purpose of this study is to compare signaling induced by the two drugs and identify a molecular basis for increased efficacy of dEpoB in resistant lines. The importance of ERK signaling, already established for Taxol, was shown for dEpoB and other G2-blocking agents. However, a role in differential sensitivity was not observed. Affymetrix analysis shows similar gene modulation by either agent, alone or in combination with MEK inhibitor. Differential sensitivity in a set of Taxol-resistant lines correlated to the expression of P-glycoprotein (P-gp), and its importance was demonstrated directly. These results suggest that Taxol and dEpoB elicit similar cell death pathways, and the increased efficacy of dEpoB in resistant tumor lines lies in differential susceptibility to P-gp.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Muerte Celular/efectos de los fármacos , Resistencia a Múltiples Medicamentos , Epotilonas/farmacología , Paclitaxel/farmacología , Transducción de Señal/efectos de los fármacos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Adenocarcinoma/patología , Neoplasias de la Mama/patología , Resistencia a Antineoplásicos , Femenino , Humanos , Neoplasias Pulmonares/patología , Células Tumorales CultivadasRESUMEN
Major histocompatibility complex (MHC) class II antigen presentation and subsequent CD4+ T-cell activation are critical for acquired immunity to Mycobacterium tuberculosis infection. MHC class II gene expression is primarily controlled by the master transactivator CIITA protein. Without functional CIITA protein, MHC class II expression is lost, impairing immune responses and increasing susceptibility to infection. In this study, we compared protective immune responses of CIITA-deficient mice and wild-type C57BL/6 controls with low dose aerosol M. tuberculosis infection. After aerogenic challenge, CIITA-/- mice failed to limit mycobacterial growth (2.5 and 2.0 log10 > WT lung and spleen CFUs, respectively, at day 58). Lung histopathology involved extensive necrosis, severe pneumonitis and overwhelming inflammation in the gene knockout mice. Mean survival time for CIITA-/- mice was significantly reduced (57 versus >300 days for WT). This extreme sensitivity to tuberculous infection was largely attributed to the absence of CD4+ cells. Flow cytometric studies detected virtually no CD4+ cells in CIITA-/- mouse spleens after infection versus elevated numbers in WT spleens. Failed CD4+ T-cell expansion markedly reduced interferon-gamma (IFN-gamma production in CIITA-/- mice versus WT controls. These results suggest the necessity of a functional CIITA pathway for controlling tuberculous infections and that interventions targeting CIITA expression may be useful antimycobacterial therapeutics.