RESUMO
Adoptive cell transfer (ACT) is a therapeutic strategy to augment antitumor immunity. Here, we report that ex vivo treatment of mouse CD8+ T cells with dimethyloxalylglycine (DMOG), a stabilizer of hypoxia-inducible factors (HIFs), induced HIF binding to the genes encoding the costimulatory receptors CD81, GITR, OX40, and 4-1BB, leading to increased expression. DMOG treatment increased T cell killing of melanoma cells, which was further augmented by agonist antibodies targeting each costimulatory receptor. In tumor-bearing mice, ACT using T cells treated ex vivo with DMOG and agonist antibodies resulted in decreased tumor growth compared to ACT using control T cells and increased intratumoral markers of CD8+ T cells (CD7, CD8A, and CD8B1), natural killer cells (NCR1 and KLRK1), and cytolytic activity (perforin-1 and tumor necrosis factor-α). Costimulatory receptor gene expression was also induced when CD8+ T cells were treated with three highly selective HIF stabilizers that are currently in clinical use.
Assuntos
Linfócitos T CD8-Positivos , Imunoterapia Adotiva , Animais , Camundongos , Imunoterapia Adotiva/métodos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Aminoácidos Dicarboxílicos/farmacologia , Linhagem Celular Tumoral , Receptores OX40/metabolismo , Proteína Relacionada a TNFR Induzida por Glucocorticoide/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Camundongos Endogâmicos C57BL , Melanoma Experimental/terapia , Melanoma Experimental/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Citotoxicidade Imunológica/efeitos dos fármacosRESUMO
Hypoxia-inducible factor 1 (HIF-1) is a transcriptional activator that mediates cellular adaptation to decreased oxygen availability. HIF-1 recruits chromatin-modifying enzymes leading to changes in histone acetylation, citrullination, and methylation at target genes. Here, we demonstrate that hypoxia-inducible gene expression in estrogen receptor (ER)-positive MCF7 and ER-negative SUM159 human breast cancer cells requires the histone H2A/H2B chaperone facilitates chromatin transcription (FACT) and the H2B ubiquitin ligase RING finger protein 20/40 (RNF20/40). Knockdown of FACT or RNF20/40 expression leads to decreased transcription initiation and elongation at HIF-1 target genes. Mechanistically, FACT and RNF20/40 are recruited to hypoxia response elements (HREs) by HIF-1 and stabilize binding of HIF-1 (and each other) at HREs. Hypoxia induces the monoubiquitination of histone H2B at lysine 120 at HIF-1 target genes in an HIF-1-dependent manner. Together, these findings delineate a cooperative molecular mechanism by which FACT and RNF20/40 stabilize multiprotein complex formation at HREs and mediate histone ubiquitination to facilitate HIF-1 transcriptional activity.
Assuntos
Proteínas de Ligação a DNA , Fator 1 Induzível por Hipóxia , Ubiquitina-Proteína Ligases , Humanos , Hipóxia Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Histonas/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Células MCF-7 , Ligação Proteica , Elementos de Resposta , Fatores de Transcrição/metabolismo , Ativação Transcricional , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Intratumoral hypoxia is a microenvironmental feature that promotes breast cancer progression and is associated with cancer mortality. Plexin B3 (PLXNB3) is highly expressed in estrogen receptor-negative breast cancer, but the underlying mechanisms and consequences have not been thoroughly investigated. Here, we report that PLXNB3 expression is increased in response to hypoxia and that PLXNB3 is a direct target gene of hypoxia-inducible factor 1 (HIF-1) in human breast cancer cells. PLXNB3 expression is correlated with HIF-1α immunohistochemistry, breast cancer grade and stage, and patient mortality. Mechanistically, PLXNB3 is required for hypoxia-induced MET/SRC/focal adhesion kinase (FAK) and MET/SRC/STAT3/NANOG signaling as well as hypoxia-induced breast cancer cell migration, invasion, and cancer stem cell specification. PLXNB3 knockdown impairs tumor formation and lung metastasis in orthotopic breast cancer mouse models.
Assuntos
Neoplasias da Mama , Neoplasias Pulmonares , Animais , Feminino , Humanos , Camundongos , Neoplasias da Mama/patologia , Hipóxia Celular/genética , Linhagem Celular Tumoral , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Pulmonares/patologia , Células-Tronco Neoplásicas/metabolismoRESUMO
Hypoxia is a key characteristic of the breast cancer microenvironment that promotes expression of the transcriptional activator hypoxia-inducible factor 1 (HIF-1) and is associated with poor patient outcome. HIF-1 increases the expression or activity of stem cell pluripotency factors, which control breast cancer stem cell (BCSC) specification and are required for cancer metastasis. Here, we identify nuclear prelamin A recognition factor (NARF) as a hypoxia-inducible, HIF-1 target gene in human breast cancer cells. NARF functions as an essential coactivator by recruiting the histone demethylase KDM6A to OCT4 bound to genes encoding the pluripotency factors NANOG, KLF4, and SOX2, leading to demethylation of histone H3 trimethylated at lysine-27 (H3K27me3), thereby increasing the expression of NANOG, KLF4, and SOX2, which, together with OCT4, mediate BCSC specification. Knockdown of NARF significantly decreased the BCSC population in vitro and markedly impaired tumor initiation capacity and lung metastasis in orthotopic mouse models.
Assuntos
Neoplasias da Mama , Fator 1 Induzível por Hipóxia , Animais , Feminino , Humanos , Camundongos , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Histonas/metabolismo , Hipóxia/genética , Hipóxia/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Microambiente Tumoral/genética , Microambiente Tumoral/fisiologia , Fator 1 Induzível por Hipóxia/genética , Fator 1 Induzível por Hipóxia/metabolismoRESUMO
Pediatric high-grade gliomas (pHGGs), including glioblastoma multiforme (GBM) and diffuse intrinsic pontine glioma (DIPG), are morbid brain tumors. Even with treatment survival is poor, making pHGG the number one cause of cancer death in children. Up to 80% of DIPGs harbor a somatic missense mutation in genes encoding histone H3. To investigate whether H3K27M is associated with distinct chromatin structure that alters transcription regulation, we generated the first high-resolution Hi-C maps of pHGG cell lines and tumor tissue. By integrating transcriptome (RNA-seq), enhancer landscape (ChIP-seq), genome structure (Hi-C), and chromatin accessibility (ATAC-seq) datasets from H3K27M and wild-type specimens, we identified tumor-specific enhancers and regulatory networks for known oncogenes. We identified genomic structural variations that lead to potential enhancer hijacking and gene coamplification, including A2M, JAG2, and FLRT1 Together, our results imply three-dimensional genome alterations may play a critical role in the pHGG epigenetic landscape and contribute to tumorigenesis.
Assuntos
Neoplasias do Tronco Encefálico , Glioma , Neoplasias do Tronco Encefálico/genética , Neoplasias do Tronco Encefálico/patologia , Criança , Cromatina/genética , Epigenômica , Glioma/genética , Glioma/patologia , Humanos , MutaçãoRESUMO
Histone H3.3 mutation (H3F3A) occurs in 50% of cortical pediatric high-grade gliomas. This mutation replaces glycine 34 with arginine or valine (G34R/V), impairing SETD2 activity (H3K36-specific trimethyltransferase). Consequently, reduced H3K36me3 is observed on H3.3G34V nucleosomes relative to wild-type, contributing to genomic instability and driving a distinct gene expression signature associated with tumorigenesis. However, it is not known if this differential H3K36me3 enrichment is due to H3.3G34V mutant protein alone. Therefore, we set to elucidate the effect of H3.3G34V mutant protein in pediatric glioma on H3K36me3, H3K27me3 and H3.3 enrichment in vitro. We found that the doxycycline-inducible shRNA knockdown of mutant H3F3A encoding the H3.3G34V protein resulted in loss of H3.3G34V enrichment and increased H3K36me3 enrichment throughout the genome. After knockdown, H3.3G34V enrichment was preserved at loci observed to have the greatest H3.3G34V and H3K36me3 enrichment prior to knockdown. Induced expression of mutant H3.3G34V protein in vitro was insufficient to induce genomic H3K36me3 enrichment patterns observed in H3.3G34V mutant glioma cells. We also observed strong co-enrichment of H3.3G34V and wild-type H3.3 protein, as well as greater H3K27me3 enrichment, in cells expressing H3.3G34V. Taken together, our study demonstrates the effects of H3.3G34V mutant protein on genomic H3K36me3, H3K27me3 and H3.3 enrichment patterns in isogenic cell lines.
Assuntos
Neoplasias Encefálicas/genética , Glioma/genética , Código das Histonas/genética , Histonas/genética , Astrócitos , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Criança , Imunoprecipitação da Cromatina , Sequenciamento de Cromatina por Imunoprecipitação , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glioma/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Metilação , Mutação de Sentido IncorretoRESUMO
Immune checkpoint blockade (ICB) has led to therapeutic responses in some cancer patients for whom no effective treatment previously existed. ICB acts on T lymphocytes and other immune cells that are inactivated due to checkpoint signals that inhibit their infiltration and function within tumors. But for more than 80% of patients, immunotherapy has not been effective. Here, we demonstrate a cancer-cell-intrinsic mechanism of immune evasion and resistance to ICB mediated by baculoviral IAP repeat-containing 2 (BIRC2). Knockdown of BIRC2 expression in mouse melanoma or breast cancer cells increases expression of the chemokine CXCL9 and impairs tumor growth by increasing the number of intratumoral activated CD8+ T cells and natural killer cells. Administration of anti-CXCL9 neutralizing antibody inhibits the recruitment of CD8+ T cells and natural killer cells to BIRC2-deficient tumors. Most importantly, BIRC2 deficiency dramatically increases the sensitivity of mouse melanoma and breast tumors to anti-CTLA4 and/or anti-PD1 ICB.
Assuntos
Imunidade Inata/imunologia , Imunoterapia/métodos , Proteínas Inibidoras de Apoptose/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Humanos , Camundongos , Microambiente TumoralRESUMO
BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem tumor. Most DIPGs harbor a histone H3 mutation, which alters histone post-translational modification (PTM) states and transcription. Here, we employed quantitative proteomic analysis to elucidate the impact of the H3.3K27M mutation, as well as radiation and bromodomain inhibition (BRDi) with JQ1, on DIPG PTM profiles. METHODS: We performed targeted mass spectrometry on H3.3K27M mutant and wild-type tissues (n = 12) and cell lines (n = 7). RESULTS: We found 29.2 and 26.4% of total H3.3K27 peptides were H3.3K27M in mutant DIPG tumor cell lines and tissue specimens, respectively. Significant differences in modification states were observed in H3.3K27M specimens, including at H3K27, H3K36, and H4K16. In addition, H3.3K27me1 and H4K16ac were the most significantly distinct modifications in H3.3K27M mutant tumors, relative to wild-type. Further, H3.3K36me2 was the most abundant co-occurring modification on the H3.3K27M mutant peptide in DIPG tissue, while H4K16ac was the most acetylated residue. Radiation treatment caused changes in PTM abundance in vitro, including increased H3K9me3. JQ1 treatment resulted in increased mono- and di-methylation of H3.1K27, H3.3K27, H3.3K36 and H4K20 in vitro. CONCLUSION: Taken together, our findings provide insight into the effects of the H3K27M mutation on histone modification states and response to treatment, and suggest that H3K36me2 and H4K16ac may represent unique tumor epigenetic signatures for targeted DIPG therapy.