RESUMEN

Pancreatic adenocarcinoma (PDAC) is one the most intractable cancers, in part due to its highly inflammatory microenvironment and paucity of infiltrating dendritic cells (DCs). Here, we find that genetic ablation or antibody blockade of tumor necrosis factor receptor 1 (TNFR1) enhanced intratumor T cell activation and slowed PDAC growth. While anti-PD-1 checkpoint inhibition alone had little effect, it further enhanced intratumor T cell activation in combination with anti-TNFR1. The major cellular alteration in the tumor microenvironment in the absence of TNFR1 signaling was a large increase in DC number and immunostimulatory phenotype. This may reflect a direct effect on DCs, because TNF induced TNFR1-dependent apoptosis of bone-marrow-derived DCs. The therapeutic response to anti-TNFR1 alone was superior to the combination of DC-activating agonistic anti-CD40 and Flt3 ligand (Flt3L). These observations suggest that targeting TNFR1, perhaps in concert with other strategies that promote DC generation and mobilization, may have therapeutic benefits.

RESUMEN

Extramedullary erythropoiesis is not expected in healthy adult mice, but erythropoietic gene expression was elevated in lineage-depleted spleen cells from Cd47-/- mice. Expression of several genes associated with early stages of erythropoiesis was elevated in mice lacking CD47 or its signaling ligand thrombospondin-1, consistent with previous evidence that this signaling pathway inhibits expression of multipotent stem cell transcription factors in spleen. In contrast, cells expressing markers of committed erythroid progenitors were more abundant in Cd47-/- spleens but significantly depleted in Thbs1-/- spleens. Single-cell transcriptome and flow cytometry analyses indicated that loss of CD47 is associated with accumulation and increased proliferation in spleen of Ter119-CD34+ progenitors and Ter119+CD34- committed erythroid progenitors with elevated mRNA expression of Kit, Ermap, and Tfrc. Induction of committed erythroid precursors is consistent with the known function of CD47 to limit the phagocytic removal of aged erythrocytes. Conversely, loss of thrombospondin-1 delays the turnover of aged red blood cells, which may account for the suppression of committed erythroid precursors in Thbs1-/- spleens relative to basal levels in wild-type mice. In addition to defining a role for CD47 to limit extramedullary erythropoiesis, these studies reveal a thrombospondin-1-dependent basal level of extramedullary erythropoiesis in adult mouse spleen.

Asunto(s)

Antígeno CD47 , Eritropoyesis , Bazo , Trombospondina 1 , Animales , Antígeno CD47/metabolismo , Antígeno CD47/genética , Trombospondina 1/metabolismo , Trombospondina 1/genética , Bazo/metabolismo , Ratones , Ratones Noqueados , Regulación de la Expresión Génica , Ratones Endogámicos C57BL , Células Precursoras Eritroides/metabolismo

RESUMEN

Signal regulatory protein-α (SIRPα, SHPS-1, CD172a) expressed on myeloid cells transmits inhibitory signals when it engages its counter-receptor CD47 on an adjacent cell. Elevated CD47 expression on some cancer cells thereby serves as an innate immune checkpoint that limits phagocytic clearance of tumor cells by macrophages and antigen presentation to T cells. Antibodies and recombinant SIRPα constructs that block the CD47-SIRPα interaction on macrophages exhibit anti-tumor activities in mouse models and are in ongoing clinical trials for treating several human cancers. Based on prior evidence that engaging SIRPα can also alter CD47 signaling in some nonmalignant cells, we compared direct effects of recombinant SIRPα-Fc and a humanized CD47 antibody that inhibits CD47-SIRPα interaction (CC-90002) on CD47 signaling in cancer stem cells derived from the MDA-MB- 231 triple-negative breast carcinoma cell line. Treatment with SIRPα-Fc significantly increased the formation of mammospheres by breast cancer stem cells as compared to CC-90002 treatment or controls. Furthermore, SIRPα-Fc treatment upregulated mRNA and protein expression of ALDH1 and altered the expression of genes involved in epithelial/mesenchymal transition pathways that are associated with a poor prognosis and enhanced metastatic activity. This indicates that SIRPα-Fc has CD47-mediated agonist activities in breast cancer stem cells affecting proliferation and metastasis pathways that differ from those of CC-90002. This SIRPα-induced CD47 signaling in breast carcinoma cells may limit the efficacy of SIRPα decoy therapeutics intended to stimulate innate antitumor immune responses.

RESUMEN

Extramedullary erythropoiesis is not expected in healthy adult mice, but erythropoietic gene expression was elevated in lineage-depleted spleen cells from cd47-/- mice. Expression of several genes associated with early stages of erythropoiesis was elevated in mice lacking CD47 or its signaling ligand thrombospondin-1, consistent with previous evidence that this signaling pathway inhibits expression of multipotent stem cell transcription factors in spleen. In contrast, cells expressing markers of committed erythroid progenitors were more abundant in cd47-/- spleens but significantly depleted in thbs1-/- spleens. Single cell transcriptome and flow cytometry analyses indicated that loss of CD47 is associated with accumulation and increased proliferation in spleen of Ter119-CD34+ progenitors and Ter119+CD34- committed erythroid progenitors with elevated mRNA expression of Kit, Ermap, and Tfrc. Induction of committed erythroid precursors is consistent with the known function of CD47 to limit the phagocytic removal of aged erythrocytes. Conversely, loss of thrombospondin-1 delays the turnover of aged red blood cells, which may account for the suppression of committed erythroid precursors in thbs1-/- spleens relative to basal levels in wild type mice. In addition to defining a role for CD47 to limit extramedullary erythropoiesis, these studies reveal a thrombospondin-1-dependent basal level of extramedullary erythropoiesis in adult mouse spleen.

RESUMEN

Thymic epithelial cells (TECs) produce glucocorticoids, which antagonize negative selection of autoreactive thymocytes and promote a competent T cell antigen-specific repertoire. To characterize their source, we generated a knock-in reporter mouse in which endogenous Cyp11b1, the final enzyme in de novo production of active glucocorticoids, was fluorescently tagged with mScarlet. Here, we find that Cyp11b1 is expressed in medullary TECs (mTECs) but not cortical TECs or other cells in the thymus. A distinct characteristic of mTECs is the presence of Aire, a transcription factor that drives expression of tissue-restricted antigens (TRAs) important for establishing immune tolerance. Cyp11b1 expression was highest in Aire+ mTECs, lower in post-Aire mTECs, and absent in mTECs of Aire-deficient mice. Transcriptomic analyses found that multiple enzymatic biosynthetic pathways are expressed specifically in mTECs and are also Aire dependent. In particular, we found that the thymus expresses messenger RNA for enzymes that catalyze production of many bioactive steroids and that glucocorticoids and sex steroids were secreted by cultured thymi. Expression of the transcripts for these genes and production of their final steroid products were markedly reduced in the absence of Aire. Thus, in addition to its well-established role in inducing TRAs that promote negative selection, Aire has an additional and contrary function of inducing glucocorticoids that antagonize negative selection, which together may expand and enhance the TCR repertoire. Furthermore, because Aire drives expression of multiple enzymes responsible for production of other non-gene-encoded bioactive molecules, it might have yet other roles in thymus development and function.

Asunto(s)

Glucocorticoides , Esteroide 11-beta-Hidroxilasa , Factores de Transcripción , Animales , Ratones , Células Epiteliales , Perfilación de la Expresión Génica , Factores de Transcripción/metabolismo , Timo/metabolismo , Proteína AIRE

RESUMEN

Glucocorticoids are steroid hormones with potent immunosuppressive properties. Their primary source is the adrenals, where they are generated via de novo synthesis from cholesterol. In addition, many tissues have a recycling pathway in which glucocorticoids are regenerated from inactive metabolites by the enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1, encoded by Hsd11b1). Here, we find that multiple tumor types express Hsd11b1 and produce active glucocorticoids. Genetic ablation of Hsd11b1 in such cells had no effect on in vitro growth, but reduced in vivo tumor progression, which corresponded with increased frequencies of CD8+ tumor-infiltrating lymphocytes (TILs) expressing activation markers and producing effector cytokines. Tumor-derived glucocorticoids were found to promote signatures of Treg activation and suppress signatures of conventional T cell activation in tumor-infiltrating Tregs. Indeed, CD8+ T cell activation was restored and tumor growth reduced in mice with Treg-specific glucocorticoid receptor deficiency. Importantly, pharmacologic inhibition of 11ß-HSD1 reduced tumor growth to the same degree as gene knockout and rendered immunotherapy-resistant tumors susceptible to PD-1 blockade. Given that HSD11B1 expression is upregulated in many human tumors and that inhibition of 11ß-HSD1 is well tolerated in clinical studies, these data suggest that targeting 11ß-HSD1 may be a beneficial adjunct in cancer therapy.

Asunto(s)

Glucocorticoides , Neoplasias , Ratones , Humanos , Animales , Glucocorticoides/farmacología , Glucocorticoides/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Receptores de Glucocorticoides/genética , Técnicas de Inactivación de Genes

RESUMEN

The development of an effective vaccine to protect against HIV acquisition will be greatly bolstered by in-depth understanding of the innate and adaptive responses to vaccination. We report here that the efficacy of DNA/ALVAC/gp120/alum vaccines, based on V2-specific antibodies mediating apoptosis of infected cells (V2-ADCC), is complemented by efferocytosis, a cyclic AMP (cAMP)-dependent antiphlogistic engulfment of apoptotic cells by CD14+ monocytes. Central to vaccine efficacy is the engagement of the CCL2/CCR2 axis and tolerogenic dendritic cells producing IL-10 (DC-10). Epigenetic reprogramming in CD14+ cells of the cyclic AMP/CREB pathway and increased systemic levels of miRNA-139-5p, a negative regulator of expression of the cAMP-specific phosphodiesterase PDE4D, correlated with vaccine efficacy. These data posit that efferocytosis, through the prompt and effective removal of apoptotic infected cells, contributes to vaccine efficacy by decreasing inflammation and maintaining tissue homeostasis.

Asunto(s)

Vacunas contra el SIDA , Infecciones por VIH , Femenino , Animales , Eficacia de las Vacunas , Macaca mulatta , Vacunación , Citotoxicidad Celular Dependiente de Anticuerpos , Anticuerpos Anti-VIH , Infecciones por VIH/prevención & control , Proteína gp120 de Envoltorio del VIH/genética

RESUMEN

Elevated expression of CD47 in some cancers is associated with poor survival related to its function as an innate immune checkpoint when expressed on tumor cells. In contrast, elevated CD47 expression in cutaneous melanomas is associated with improved survival. Previous studies implicated protective functions of CD47 expressed by immune cells in the melanoma tumor microenvironment. RNA sequencing analysis of responses induced by CD3 and CD28 engagement on wild type and CD47-deficient Jurkat T lymphoblast cells identified additional regulators of T cell function that were also CD47-dependent in mouse CD8 T cells. MYCN mRNA expression was upregulated in CD47-deficient cells but downregulated in CD47-deficient cells following activation. CD47 also regulated alternative splicing that produces two N-MYC isoforms. The CD47 ligand thrombospondin-1 inhibited expression of these MYCN mRNA isoforms, as well as induction of the oncogenic decoy MYCN opposite strand (MYCNOS) RNA during T cell activation. Analysis of mRNA expression data for melanomas in The Cancer Genome Atlas identified a significant coexpression of MYCN with CD47 and known regulators of CD8 T cell function. Thrombospondin-1 inhibited the induction of TIGIT, CD40LG, and MCL1 mRNAs following T cell activation in vitro. Increased mRNA expression of these T cell transcripts and MYCN in melanomas was associated with improved overall survival.

Asunto(s)

Antígeno CD47 , Melanoma , Ratones , Animales , Antígeno CD47/metabolismo , Proteína Proto-Oncogénica N-Myc/genética , Linfocitos T CD8-positivos , Expresión Génica , Melanoma/genética , ARN Mensajero/genética , Trombospondinas/genética , Microambiente Tumoral

RESUMEN

CD47 has established roles in the immune system for regulating macrophage phagocytosis and lymphocyte activation, with growing evidence of its cell-intrinsic regulatory roles in natural killer and CD8+ T cells. CD47 limits antigen-dependent cytotoxic activities of human and murine CD8+ T cells, but its role in T cell activation kinetics remains unclear. Using in vitro and in vivo models, we show here that CD47 differentially regulates CD8+ T cell responses to short- versus long-term activation. Although CD47 was not required for T cell development in mice and early activation in vitro, short-term stimuli elevated pathogen-reactive gene expression and enhanced proliferation and the effector phenotypes of Cd47-deficient relative to Cd47-sufficient CD8+ T cells. In contrast, persistent TCR stimulation limited the effector phenotypes of Cd47 -/- CD8+ T cells and enhanced their apoptosis signature. CD8+ T cell expansion and activation in vivo induced by acute lymphocytic choriomeningitis virus (LCMV) infection did not differ in the absence of CD47. However, the frequency and effector phenotypes of Cd47-/- CD8+ T cells were constrained in chronic LCMV-infected as well as in mice bearing B16 melanoma tumors. Therefore, CD47 regulates CD8+ T cell activation, proliferation, and fitness in a context-dependent manner.

Asunto(s)

Activación de Linfocitos , Coriomeningitis Linfocítica , Animales , Antígeno CD47/genética , Linfocitos T CD8-positivos , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados

RESUMEN

PURPOSE: The standard treatment of patients with locally advanced rectal cancer consists of preoperative chemoradiotherapy (CRT) followed by surgery. However, the response of individual tumors to CRT is extremely diverse, presenting a clinical dilemma. This broad variability in treatment response is likely attributable to intratumor heterogeneity (ITH). EXPERIMENTAL DESIGN: We addressed the impact of ITH on response to CRT by establishing single-cell-derived cell lines (SCDCL) from a treatment-naïve rectal cancer biopsy after xenografting. RESULTS: Individual SCDCLs derived from the same tumor responded profoundly different to CRT in vitro. Clonal reconstruction of the tumor and derived cell lines based on whole-exome sequencing revealed nine separate clusters with distinct proportions in the SCDCLs. Missense mutations in SV2A and ZWINT were clonal in the resistant SCDCL, but not detected in the sensitive SCDCL. Single-cell genetic analysis by multiplex FISH revealed the expansion of a clone with a loss of PIK3CA in the resistant SCDCL. Gene expression profiling by tRNA-sequencing identified the activation of the Wnt, Akt, and Hedgehog signaling pathways in the resistant SCDCLs. Wnt pathway activation in the resistant SCDCLs was confirmed using a reporter assay. CONCLUSIONS: Our model system of patient-derived SCDCLs provides evidence for the critical role of ITH for treatment response in patients with rectal cancer and shows that distinct genetic aberration profiles are associated with treatment response. We identified specific pathways as the molecular basis of treatment response of individual clones, which could be targeted in resistant subclones of a heterogenous tumor.

Asunto(s)

Heterogeneidad Genética , Neoplasias del Recto/etiología , Neoplasias del Recto/patología , Análisis de la Célula Individual , Animales , Biomarcadores de Tumor , Línea Celular Tumoral , Terapia Combinada , Hibridación Genómica Comparativa , Modelos Animales de Enfermedad , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Ratones , Neoplasias del Recto/metabolismo , Neoplasias del Recto/terapia , Transducción de Señal , Resultado del Tratamiento , Secuenciación del Exoma , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Current cancer treatments are largely based on the genetic characterization of primary tumors and are ineffective for metastatic disease. Here we report that DNA methyltransferase 3B (DNMT3B) is induced at distant metastatic sites and mediates epigenetic reprogramming of metastatic tumor cells. Multiomics analysis and spontaneous metastatic mouse models revealed that DNMT3B alters multiple pathways including STAT3, NFκB, PI3K/Akt, ß-catenin, and Notch signaling, which are critical for cancer cell survival, apoptosis, proliferation, invasion, and colonization. PGE2 and IL6 were identified as critical inflammatory mediators in DNMT3B induction. DNMT3B expression levels positively correlated with human metastatic progression. Targeting IL6 or COX-2 reduced DNMT3B induction and improved chemo or PD1 therapy. We propose a novel mechanism linking the metastatic microenvironment with epigenetic alterations that occur at distant sites. These results caution against the "Achilles heel" in cancer therapies based on primary tumor characterization and suggests targeting DNMT3B induction as new option for treating metastatic disease. SIGNIFICANCE: These findings reveal that DNMT3B epigenetically regulates multiple pro-oncogenic signaling pathways via the inflammatory microenvironment at distant sites, cautioning the clinical approach basing current therapies on genetic characterization of primary tumors.

Asunto(s)

Neoplasias de la Mama/patología , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Dinoprostona/metabolismo , Interleucina-6/metabolismo , Neoplasias Pulmonares/secundario , Animales , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Mama/patología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Línea Celular Tumoral/trasplante , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa 2/farmacología , Inhibidores de la Ciclooxigenasa 2/uso terapéutico , Conjuntos de Datos como Asunto , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Epigénesis Genética/efectos de los fármacos , Epigénesis Genética/inmunología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/inmunología , Técnicas de Silenciamiento del Gen , Humanos , Interleucina-6/antagonistas & inhibidores , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Ratones , Receptor de Muerte Celular Programada 1/inmunología , Prueba de Estudio Conceptual , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/inmunología , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/inmunología , ADN Metiltransferasa 3B

RESUMEN

Elevated CD47 expression in some cancers is associated with decreased survival and limited clearance by phagocytes expressing the CD47 counterreceptor SIRPα. In contrast, elevated CD47 mRNA expression in human melanomas was associated with improved survival. Gene-expression data were analyzed to determine a potential mechanism for this apparent protective function and suggested that high CD47 expression increases recruitment of natural killer (NK) cells into the tumor microenvironment. The CD47 ligand thrombospondin-1 inhibited NK cell proliferation and CD69 expression in vitro Cd47 -/- NK cells correspondingly displayed augmented effector phenotypes, indicating an inhibitory function of CD47 on NK cells. Treating human NK cells with a CD47 antibody that blocks thrombospondin-1 binding abrogated its inhibitory effect on NK cell proliferation. Similarly, treating wild-type mice with a CD47 antibody that blocks thrombospondin-1 binding delayed B16 melanoma growth, associating with increased NK cell recruitment and increased granzyme B and interferon-γ levels in intratumoral NK but not CD8+ T cells. However, B16 melanomas grew faster in Cd47 -/- than in wild-type mice. Melanoma-bearing Cd47 -/- mice exhibited decreased splenic NK cell numbers, with impaired effector protein expression and elevated exhaustion markers. Proapoptotic gene expression in Cd47-/- NK cells was associated with stress-mediated increases in mitochondrial proton leak, reactive oxygen species, and apoptosis. Global gene-expression profiling in NK cells from tumor-bearing mice identified CD47-dependent transcriptional responses that regulate systemic NK activation and exhaustion. Therefore, CD47 positively and negatively regulates NK cell function, and therapeutic antibodies that block inhibitory CD47 signaling can enhance NK immune surveillance of melanomas.

Asunto(s)

Antígeno CD47/genética , Expresión Génica , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Microambiente Tumoral/genética , Microambiente Tumoral/inmunología , Animales , Apoptosis , Antígeno CD47/metabolismo , Modelos Animales de Enfermedad , Metabolismo Energético/efectos de los fármacos , Humanos , Células Asesinas Naturales/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Melanoma Experimental , Ratones , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/metabolismo , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/mortalidad , Pronóstico , ARN Mensajero , Especies Reactivas de Oxígeno/metabolismo , Estrés Fisiológico , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Trombospondina 1/farmacología

RESUMEN

Innate lymphoid cells (ILCs) play important functions in immunity and tissue homeostasis, but their development is poorly understood. Through the use of single-cell approaches, we examined the transcriptional and functional heterogeneity of ILC progenitors, and studied the precursor-product relationships that link the subsets identified. This analysis identified two successive stages of ILC development within T cell factor 1-positive (TCF-1+) early innate lymphoid progenitors (EILPs), which we named 'specified EILPs' and 'committed EILPs'. Specified EILPs generated dendritic cells, whereas this potential was greatly decreased in committed EILPs. TCF-1 was dispensable for the generation of specified EILPs, but required for the generation of committed EILPs. TCF-1 used a pre-existing regulatory landscape established in upstream lymphoid precursors to bind chromatin in EILPs. Our results provide insight into the mechanisms by which TCF-1 promotes developmental progression of ILC precursors, while constraining their dendritic cell lineage potential and enforcing commitment to ILC fate.

Asunto(s)

Linaje de la Célula/inmunología , Células Dendríticas/citología , Factor Nuclear 1-alfa del Hepatocito/inmunología , Células Progenitoras Linfoides/citología , Linfocitos T/citología , Animales , Diferenciación Celular/inmunología , Células Cultivadas , Regulación de la Expresión Génica/genética , Factor Nuclear 1-alfa del Hepatocito/genética , Ratones , Ratones Endogámicos C57BL , Transcripción Genética/genética

RESUMEN

Moxetumomab pasudotox (Moxe) is a chimeric protein composed of an anti-CD22 Fv fused to a portion of Pseudomonas exotoxin A and kills CD22-expressing leukemia cells. It is very active in hairy-cell leukemia, but many children with relapsed/refractory acute lymphoblastic leukemia (ALL) either respond transiently or are initially resistant. Resistance to Moxe in cultured cells is due to low expression of diphthamide genes (DPH), but only two of six ALL blast samples from resistant patients had low DPH expression. To develop a more clinically relevant model of resistance, we treated NSG mice bearing KOPN-8 or Reh cells with Moxe. More than 99.9% of the cancer cells were killed by Moxe, but relapse occurred from discrete bone marrow sites. The resistant cells would no longer grow in cell culture and showed major chromosomal changes and changes in phenotype with greatly decreased CD22. RNA deep sequencing of resistant KOPN-8 blasts revealed global changes in gene expression, indicating dedifferentiation toward less-mature B cell precursors, and showed an up-regulation of myeloid genes. When Moxe was combined with 5-azacytidine, resistance was prevented and survival increased to over 5 months in the KOPN-8 model and greatly improved in the Reh model. We conclude that Moxe resistance in mice is due to a new mechanism that could not be observed using cultured cells and is prevented by treatment with 5-azacytidine.

Asunto(s)

Azacitidina/uso terapéutico , Toxinas Bacterianas/uso terapéutico , Exotoxinas/uso terapéutico , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/uso terapéutico , Azacitidina/administración & dosificación , Toxinas Bacterianas/administración & dosificación , Médula Ósea , Línea Celular Tumoral , Regulación hacia Abajo , Resistencia a Antineoplásicos , Exotoxinas/administración & dosificación , Humanos , Leucemia , Ratones , Neoplasias Experimentales , Leucemia-Linfoma Linfoblástico de Células Precursoras , Recurrencia

RESUMEN

Understanding the mechanisms supporting tumor-initiating cells (TIC) is vital to combat advanced-stage recurrent cancers. Here, we show that in advanced ovarian cancers NFκB signaling via the RelB transcription factor supports TIC populations by directly regulating the cancer stem-like associated enzyme aldehyde dehydrogenase (ALDH). Loss of RelB significantly inhibited spheroid formation, ALDH expression and activity, chemoresistance, and tumorigenesis in subcutaneous and intrabursal mouse xenograft models of human ovarian cancer. RelB also affected expression of the ALDH gene ALDH1A2 Interestingly, classical NFκB signaling through the RelA transcription factor was equally important for tumorigenesis in the intrabursal model, but had no effect on ALDH. In this case, classical signaling via RelA was essential for proliferating cells, whereas the alternative signaling pathway was not. Our results show how NFκB sustains diverse cancer phenotypes via distinct classical and alternative signaling pathways, with implications for improved understanding of disease recurrence and therapeutic response. Cancer Res; 77(24); 6927-40. ©2017 AACR.

Asunto(s)

Carcinogénesis/genética , Proliferación Celular/genética , Isoenzimas/metabolismo , Células Madre Neoplásicas/fisiología , Neoplasias Ováricas/genética , Retinal-Deshidrogenasa/metabolismo , Factor de Transcripción ReIA/fisiología , Familia de Aldehído Deshidrogenasa 1 , Animales , Carcinogénesis/metabolismo , Carcinogénesis/patología , Línea Celular Tumoral , Femenino , Xenoinjertos , Humanos , Ratones , Ratones Desnudos , FN-kappa B/genética , FN-kappa B/fisiología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Transducción de Señal/genética , Factor de Transcripción ReIA/genética

RESUMEN

Intratumoral heterogeneity at the genetic, epigenetic, transcriptomic, and morphologic levels is a commonly observed phenomenon in many aggressive cancer types. Clonal evolution during tumor formation and in response to therapeutic intervention can be predicted utilizing reverse engineering approaches on detailed genomic snapshots of heterogeneous patient tumor samples. In this study, we developed an extensive dataset for a GBM case via the generation of polyclonal and monoclonal glioma stem cell lines from initial diagnosis, and from multiple sections of distant tumor locations of the deceased patient's brain following tumor recurrence. Our analyses revealed the tissue-wide expansion of a new clone in the recurrent tumor and chromosome 7 gain and chromosome 10 loss as repeated genomic events in primary and recurrent disease. Moreover, chromosome 7 gain and chromosome 10 loss produced similar alterations in mRNA expression profiles in primary and recurrent tumors despite possessing other highly heterogeneous and divergent genomic alterations between the tumors. We identified ETV1 and CDK6 as putative candidate genes, and NFKB (complex), IL1B, IL6, Akt and VEGF as potential signaling regulators, as potentially central downstream effectors of chr7 gain and chr10 loss. Finally, the differences caused by the transcriptomic shift following gain of chromosome 7 and loss of chromosome 10 were consistent with those generally seen in GBM samples compared to normal brain in large-scale patient-tumor data sets.

Asunto(s)

Biomarcadores de Tumor/genética , Neoplasias Encefálicas/genética , Cromosomas Humanos Par 10/genética , Cromosomas Humanos Par 7/genética , Glioma/genética , Recurrencia Local de Neoplasia/genética , Células Madre Neoplásicas/metabolismo , Animales , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Aberraciones Cromosómicas , Perfilación de la Expresión Génica , Genómica/métodos , Glioma/patología , Xenoinjertos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Recurrencia Local de Neoplasia/patología , Células Madre Neoplásicas/patología , Pronóstico

RESUMEN

BACKGROUND: Age changes in expression of inflammatory, synaptic, and neurotrophic genes are not well characterized during human brain development and senescence. Knowing these changes may elucidate structural, metabolic, and functional brain processes over the lifespan, as well vulnerability to neurodevelopmental or neurodegenerative diseases. HYPOTHESIS: Expression levels of inflammatory, synaptic, and neurotrophic genes in the human brain are coordinated over the lifespan and underlie changes in phenotypic networks or cascades. METHODS: We used a large-scale microarray dataset from human prefrontal cortex, BrainCloud, to quantify age changes over the lifespan, divided into Development (0 to 21 years, 87 brains) and Aging (22 to 78 years, 144 brains) intervals, in transcription levels of 39 genes. RESULTS: Gene expression levels followed different trajectories over the lifespan. Many changes were intercorrelated within three similar groups or clusters of genes during both Development and Aging, despite different roles of the gene products in the two intervals. During Development, changes were related to reported neuronal loss, dendritic growth and pruning, and microglial events; TLR4, IL1R1, NFKB1, MOBP, PLA2G4A, and PTGS2 expression increased in the first years of life, while expression of synaptic genes GAP43 and DBN1 decreased, before reaching plateaus. During Aging, expression was upregulated for potentially pro-inflammatory genes such as NFKB1, TRAF6, TLR4, IL1R1, TSPO, and GFAP, but downregulated for neurotrophic and synaptic integrity genes such as BDNF, NGF, PDGFA, SYN, and DBN1. CONCLUSIONS: Coordinated changes in gene transcription cascades underlie changes in synaptic, neurotrophic, and inflammatory phenotypic networks during brain Development and Aging. Early postnatal expression changes relate to neuronal, glial, and myelin growth and synaptic pruning events, while late Aging is associated with pro-inflammatory and synaptic loss changes. Thus, comparable transcriptional regulatory networks that operate throughout the lifespan underlie different phenotypic processes during Aging compared to Development.

Asunto(s)

Envejecimiento/metabolismo , Regulación de la Expresión Génica , Proteínas del Tejido Nervioso/biosíntesis , Corteza Prefrontal/crecimiento & desarrollo , Corteza Prefrontal/metabolismo , Transducción de Señal , Adolescente , Adulto , Anciano , Envejecimiento/patología , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Inflamación/metabolismo , Inflamación/patología , Masculino , Persona de Mediana Edad

RESUMEN

In vitro and in vivo models are widely used in cancer research. Characterizing the similarities and differences between a patient's tumor and corresponding in vitro and in vivo models is important for understanding the potential clinical relevance of experimental data generated with these models. Towards this aim, we analyzed the genomic aberrations, DNA methylation and transcriptome profiles of five parental tumors and their matched in vitro isolated glioma stem cell (GSC) lines and xenografts generated from these same GSCs using high-resolution platforms. We observed that the methylation and transcriptome profiles of in vitro GSCs were significantly different from their corresponding xenografts, which were actually more similar to their original parental tumors. This points to the potentially critical role of the brain microenvironment in influencing methylation and transcriptional patterns of GSCs. Consistent with this possibility, ex vivo cultured GSCs isolated from xenografts showed a tendency to return to their initial in vitro states even after a short time in culture, supporting a rapid dynamic adaptation to the in vitro microenvironment. These results show that methylation and transcriptome profiles are highly dependent on the microenvironment and growth in orthotopic sites partially reverse the changes caused by in vitro culturing.

Asunto(s)

Glioma/genética , Células Madre Neoplásicas/metabolismo , Animales , Metilación de ADN/genética , Metilación de ADN/fisiología , Femenino , Humanos , Técnicas In Vitro , Ratones , Ratones SCID , Polimorfismo de Nucleótido Simple/genética , Análisis de Componente Principal , Estudios Prospectivos , Células Tumorales Cultivadas

RESUMEN

Age is a powerful predictor of survival in glioblastoma multiforme (GBM) yet the biological basis for the difference in clinical outcome is mostly unknown. Discovering genes and pathways that would explain age-specific survival difference could generate opportunities for novel therapeutics for GBM. Here we have integrated gene expression, exon expression, microRNA expression, copy number alteration, SNP, whole exome sequence, and DNA methylation data sets of a cohort of GBM patients in The Cancer Genome Atlas (TCGA) project to discover age-specific signatures at the transcriptional, genetic, and epigenetic levels and validated our findings on the REMBRANDT data set. We found major age-specific signatures at all levels including age-specific hypermethylation in polycomb group protein target genes and the upregulation of angiogenesis-related genes in older GBMs. These age-specific differences in GBM, which are independent of molecular subtypes, may in part explain the preferential effects of anti-angiogenic agents in older GBM and pave the way to a better understanding of the unique biology and clinical behavior of older versus younger GBMs.

Asunto(s)

Envejecimiento/genética , Neoplasias Encefálicas/genética , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Adulto , Factores de Edad , Anciano , Envejecimiento/patología , Inhibidores de la Angiogénesis/uso terapéutico , Neoplasias Encefálicas/irrigación sanguínea , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/mortalidad , Variaciones en el Número de Copia de ADN , Metilación de ADN , Exones , Femenino , Glioblastoma/irrigación sanguínea , Glioblastoma/tratamiento farmacológico , Glioblastoma/mortalidad , Humanos , Masculino , MicroARNs , Persona de Mediana Edad , Neovascularización Patológica , Proteínas del Grupo Polycomb/genética , Proteínas del Grupo Polycomb/metabolismo , Polimorfismo de Nucleótido Simple , Análisis de Supervivencia

RESUMEN

Glioblastoma Multiforme (GBM) is a tumor with high mortality and no known cure. The dramatic molecular and clinical heterogeneity seen in this tumor has led to attempts to define genetically similar subgroups of GBM with the hope of developing tumor specific therapies targeted to the unique biology within each of these subgroups. Recently, a subset of relatively favorable prognosis GBMs has been identified. These glioma CpG island methylator phenotype, or G-CIMP tumors, have distinct genomic copy number aberrations, DNA methylation patterns, and (mRNA) expression profiles compared to other GBMs. While the standard method for identifying G-CIMP tumors is based on genome-wide DNA methylation data, such data is often not available compared to the more widely available gene expression data. In this study, we have developed and evaluated a method to predict the G-CIMP status of GBM samples based solely on gene expression data.

Asunto(s)

Neoplasias Encefálicas/genética , Islas de CpG/genética , Metilación de ADN/genética , Bases de Datos Genéticas , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Análisis por Conglomerados , Humanos , Estimación de Kaplan-Meier , Modelos Genéticos , Análisis de Componente Principal , ARN Mensajero/genética , Reproducibilidad de los Resultados