RESUMO
Alveolar bone loss is a main manifestation of periodontitis. Human periodontal ligament stem cells (PDLSCs) are considered as optimal seed cells for alveolar bone regeneration due to its mesenchymal stem cell like properties. Osteogenic potential is the premise for PDLSCs to repair alveolar bone loss. However, the mechanism regulating osteogenic differentiation of PDLSCs remain elusive. In this study, we identified Neuron-derived orphan receptor 1 (NOR1), was particularly expressed in PDL tissue in vivo and gradually increased during osteogenic differentiation of PDLSCs in vitro. Knockdown of NOR1 in hPDLSCs inhibited their osteogenic potential while NOR1 overexpression reversed this effect. In order to elucidate the downstream regulatory network of NOR1, RNA-sequencing was used. We found that downregulated genes were mainly enriched in TGF-ß, Hippo, Wnt signaling pathway. Further, by western blot analysis, we verified that the expression level of phosphorylated-SMAD2/3 and phosphorylated-SMAD4 were all decreased after NOR1 knockdown. Additionally, ChIP-qPCR and dual luciferase reporter assay indicated that NOR1 could bind to the promoter of TGFBR1 and regulate its activity. Moreover, overexpression of TGFBR1 in PDLSCs could rescue the damaged osteogenic potential after NOR1 knockdown. Taken together, our results demonstrated that NOR1 could activate TGF-ß/SMAD signaling pathway and positively regulates the commitment of osteoblast lineages of PDLSCs by targeting TGFBR1 directly.
Assuntos
Diferenciação Celular , Osteoblastos , Osteogênese , Ligamento Periodontal , Receptor do Fator de Crescimento Transformador beta Tipo I , Transdução de Sinais , Fator de Crescimento Transformador beta , Humanos , Diferenciação Celular/genética , Células Cultivadas , Osteoblastos/metabolismo , Osteoblastos/citologia , Osteogênese/genética , Ligamento Periodontal/citologia , Ligamento Periodontal/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Células-Tronco/metabolismo , Células-Tronco/citologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
BACKGROUND: The robustness and credibility of RT-qPCR results are critically dependent on the selection of suitable reference genes. However, the mineralization of the extracellular matrix can alter the intracellular tension and energy metabolism within cells, potentially impacting the expression of traditional reference genes, namely Actb and Gapdh. OBJECTIVE: To methodically identify appropriate reference genes for research focused on mouse cementoblast mineralization. MATERIALS AND METHODS: Time-series transcriptomic data of mouse cementoblast mineralization were used. To ensure expression stability and medium to high expression levels, three specific criteria were applied to select potential reference genes. The expression stability of these genes was ranked based on the DI index (1/coefficient of variation) to identify the top six potential reference genes. RT-qPCR validation was performed on these top six candidates, comparing their performance against six previously used reference genes (Rpl22, Ppib, Gusb, Rplp0, Actb, and Gapdh). Cq values of these 12 genes were analyzed by RefFinder to get a stability ranking. RESULTS: A total of 4418 (12.27%) genes met the selection criteria. Among them, Rab5if, Chmp4b, Birc5, Pea15a, Nudc, Supt4a were identified as candidate reference genes. RefFinder analyses revealed that two candidates (Birc5 and Nudc) exhibited superior performance compared to previously used reference genes. LIMITATIONS: RefFinder's stability ranking does not consider the influence of primer efficiency. CONCLUSIONS AND IMPLICATIONS: We propose Birc5 and Nudc as candidate reference genes for RT-qPCR studies investigating mouse cementoblast mineralization and cementum repair.
Assuntos
Cemento Dentário , Reação em Cadeia da Polimerase em Tempo Real , Survivina , Animais , Camundongos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Survivina/genética , Padrões de Referência , RNA-Seq/métodos , RNA-Seq/normas , Calcificação Fisiológica/genéticaRESUMO
RNA-binding proteins (RBPs) are critical regulators of transcription and translation that are often dysregulated in cancer. Although RBPs are increasingly recognized as being important for normal hematopoiesis and for hematologic malignancies as oncogenes or tumor suppressors, RBPs that are essential for the maintenance and survival of leukemia remain elusive. Here we show that YBX1 is specifically required for maintaining myeloid leukemia cell survival in an N6-methyladenosine (m6A)-dependent manner. We found that expression of YBX1 is significantly upregulated in myeloid leukemia cells, and deletion of YBX1 dramatically induces apoptosis and promotes differentiation coupled with reduced proliferation and impaired leukemic capacity of primary human and mouse acute myeloid leukemia cells in vitro and in vivo. Loss of YBX1 has no obvious effect on normal hematopoiesis. Mechanistically, YBX1 interacts with insulin-like growth factor 2 messenger RNA (mRNA)-binding proteins (IGF2BPs) and stabilizes m6A-tagged RNA. Moreover, YBX1 deficiency dysregulates the expression of apoptosis-related genes and promotes mRNA decay of MYC and BCL2 in an m6A-dependent manner, which contributes to the defective survival that results from deletion of YBX1. Thus, our findings have uncovered a selective and critical role of YBX1 in maintaining myeloid leukemia survival, which might provide a rationale for the therapeutic targeting of YBX1 in myeloid leukemia.
Assuntos
Adenosina/análogos & derivados , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo , Adenosina/metabolismo , Animais , Apoptose/genética , Sobrevivência Celular/genética , Deleção de Genes , Regulação Leucêmica da Expressão Gênica , Hematopoese/genética , Humanos , Leucemia Mieloide Aguda/genética , Camundongos Endogâmicos C57BL , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Neoplásico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína 1 de Ligação a Y-Box/genéticaRESUMO
The NLRP3 inflammasome is a critical innate immune pathway responsible for producing active interleukin (IL)-1ß, which is associated with tumor development and immunity. However, the mechanisms regulating the inflammatory microenvironment, tumorigenesis and tumor immunity are unclear. Herein, we show that the NLRP3 inflammasome was over-expressed in human HNSCC tissues and that the IL-1ß concentration was increased in the peripheral blood of HNSCC patients. Additionally, elevated NLRP3 inflammasome levels were detected in tumor tissues of Tgfbr1/Pten 2cKO HNSCC mice, and elevated IL-1ß levels were detected in the peripheral blood serum, spleen, draining lymph nodes and tumor tissues. Blocking NLRP3 inflammasome activation using MCC950 remarkably reduced IL-1ß production in an HNSCC mouse model and reduced the numbers of myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and tumor-associated macrophages (TAMs). Moreover, inhibiting NLRP3 inflammasome activation increased the numbers of CD4+ and CD8+ T cells in HNSCC mice. Notably, the numbers of exhausted PD-1+ and Tim3+ T cells were significantly reduced. A human HNSCC tissue microarray showed that NLRP3 inflammasome expression was correlated with the expression of CD8 and CD4, the Treg marker Foxp3, the MDSC markers CD11b and CD33, and the TAM markers CD68 and CD163, PD-1 and Tim3. Overall, our results demonstrate that the NLRP3 inflammasome/IL-1ß pathway promotes tumorigenesis in HNSCC and inactivation of this pathway delays tumor growth, accompanied by decreased immunosuppressive cell accumulation and an increased number of effector T cells. Thus, inhibition of the tumor microenvironment through the NLRP3 inflammasome/IL-1ß pathway may provide a novel approach for HNSCC therapy.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Linfócitos T CD8-Positivos/imunologia , Carcinogênese/efeitos dos fármacos , Carcinogênese/imunologia , Carcinoma de Células Escamosas/imunologia , Neoplasias de Cabeça e Pescoço/imunologia , Humanos , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
Leukemia stem cells (LSCs) are leukemia-initiating population with the capacity to self-renew, differentiate, and stay quiescent. Human hematopoietic malignancies such as chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) are derived from this cell population. LSCs are also responsible for disease relapse due to its resistance to drug treatment. This rare cell population is phenotypically and functionally heterogeneous. Increasing evidence indicates that this heterogeneous cellular state of LSCs might determine the different drug sensitivity and is the major reason for disease relapse. In here, focusing on myeloid leukemia stem cells, we describe the biological features including cellular and molecular state, heterogeneity of LSCs, and the dynamic cross talk between LSCs and bone marrow microenvironment. These specific features of LSCs highlight the dynamic cellular state of LSCs, and further exploring on it might provide potential therapeutic targets that are important for eliminating LSCs.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , Leucemia Mieloide Aguda , Células-Tronco Neoplásicas , Medula Óssea/fisiologia , Resistencia a Medicamentos Antineoplásicos , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/fisiopatologia , Leucemia Mieloide Aguda/fisiopatologia , Microambiente Tumoral/fisiologiaRESUMO
Epithelial-mesenchymal transition (EMT) is associated with metastasis formation, generation and maintenance of cancer stem cells (CSCs). However, the regulatory mechanisms of CSCs have not been clarified. This study aims to investigate the role of TNF receptor-associated factor 6 (TRAF6) on EMT and CSC regulation in squamous cell carcinoma of head and neck (SCCHN). We found TRAF6 was overexpressed in human SCCHN tissues, and high TRAF6 expression was associated with lymphatic metastasis and resulted in poor prognosis in patients with SCCHN. In addition, elevated TRAF6 expression was observed in several HNSCC cell lines, and wound healing and transwell assay results showed that TRAF6 knockdown inhibited the migration and invasion ability of the SCCHN cells. Moreover, the expression of Vimentin, Slug and N-cadherin was down-regulated and that of E-cadherin was elevated after TRAF6 knockdown but decreased by transforming growth factor beta 1 (TGF-ß1) and CAL27 similar to mesenchymal cells formed after TGF-ß1 induction. In addition, the expression levels of CD44, ALDH1, KLF4 and SOX2 were inhibited after TRAF6 knockdown, and the anchor-dependent colony formation number and sphere number were remarkably reduced. Flow cytometry showed TRAF6 knockdown reduced ALDH1-positive cancer stem cells. We also demonstrated that TRAF6 is closely associated with EMT process and cancer stem cells using a Tgfbr1/Pten 2cKO mice SCCHN model and human SCCHN tissue microarray. Our findings indicate that TRAF6 plays a role in EMT phenotypes, the generation and maintenance of CSCs in SCCHN, suggesting that TRAF6 is a potential therapeutic target for SCCHN.
Assuntos
Transição Epitelial-Mesenquimal , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Fator 6 Associado a Receptor de TNF/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Humanos , Fator 4 Semelhante a Kruppel , Metástase Linfática/patologia , Camundongos Knockout , Invasividade Neoplásica , Metástase Neoplásica , FenótipoRESUMO
The adenosine-induced immunosuppression hampers the immune response toward tumor cells and facilitates the tumor cells to evade immunosurveillance. CD73, an ecto-5-nucleotidase, is the ectoenzyme dephosphorylating extracellular AMP to adenosine. Here, using immunocompetent transgenic head and neck squamous cell carcinoma (HNSCC) mouse model, immune profiling showed high expression of CD73 on CD4+ and CD8+ T cells was associated with an "exhausted" phenotype. Further, treatment with anti-CD73 monoclonal antibody (mAb) significantly blunted the tumor growth in the mouse model, and the blockade of CD73 reversed the "exhausted" phenotype of CD4+ and CD8+ T cells through downregulation of total expression of PD-1 and CTLA-4 on T cells. Whereas the population of CD4+ CD73hi /CD8+ CD73hi T cells expressed higher CTLA-4 and PD-1 as compared to untreated controls. In addition, the human tissue microarrays showed the expression of CD73 is upregulated on tumor infiltrating immune cells in patients with primary HNSCC. Moreover, CD73 expression is an independent prognostic factor for poor outcome in our cohort of HNSCC patients. Altogether, these findings highlight the immunoregulatory role of CD73 in the development of HNSCC and we propose that CD73 may prove to be a promising immunotherapeutic target for the treatment of HNSCC.
Assuntos
5'-Nucleotidase/metabolismo , Linfócitos T CD8-Positivos/imunologia , Carcinoma de Células Escamosas/imunologia , Neoplasias de Cabeça e Pescoço/imunologia , Tolerância Imunológica/imunologia , Linfócitos do Interstício Tumoral/imunologia , 5'-Nucleotidase/antagonistas & inibidores , 5'-Nucleotidase/genética , Animais , Anticorpos Monoclonais/farmacologia , Apoptose , Biomarcadores Tumorais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Antígeno CTLA-4/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proliferação de Células , Seguimentos , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , PTEN Fosfo-Hidrolase/fisiologia , Fenótipo , Prognóstico , Receptor do Fator de Crescimento Transformador beta Tipo I/fisiologia , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
Immune evasion is a hallmark feature of cancer, and it plays an important role in tumour initiation and progression. In addition, tumour immune evasion severely hampers the desired antitumour effect in multiple cancers. In this study, we aimed to investigate the role of the Notch pathway in immune evasion in the head and neck squamous cell carcinoma (HNSCC) microenvironment. We first demonstrated that Notch1 signaling was activated in a Tgfbr1/Pten-knockout HNSCC mouse model. Notch signaling inhibition using a γ-secretase inhibitor (GSI-IX, DAPT) decreased tumour burden in the mouse model after prophylactic treatment. In addition, flow cytometry analysis indicated that Notch signaling inhibition reduced the sub-population of myeloid-derived suppressor cells (MDSCs), tumour-associated macrophages (TAMs) and regulatory T cells (Tregs), as well as immune checkpoint molecules (PD1, CTLA4, TIM3 and LAG3), in the circulation and in the tumour. Immunohistochemistry (IHC) of human HNSCC tissues demonstrated that elevation of the Notch1 downstream target HES1 was correlated with MDSC, TAM and Treg markers and with immune checkpoint molecules. These results suggest that modulating the Notch signaling pathway may decrease MDSCs, TAMs, Tregs and immune checkpoint molecules in HNSCC.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Carcinoma de Células Escamosas/imunologia , Diaminas/farmacologia , Modelos Animais de Doenças , Neoplasias de Cabeça e Pescoço/imunologia , Células Mieloides/imunologia , Linfócitos T Reguladores/imunologia , Tiazóis/farmacologia , Animais , Apoptose , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proliferação de Células , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Terapia de Imunossupressão , Camundongos , Camundongos Knockout , Células Mieloides/efeitos dos fármacos , PTEN Fosfo-Hidrolase/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Linfócitos T Reguladores/efeitos dos fármacos , Células Tumorais Cultivadas , Evasão Tumoral/efeitos dos fármacosRESUMO
Angiogenesis is an essential event in tumor growth and metastasis, and immune system also contributes to the tumor evasion. Emerging evidences have suggested the bidirectional link between angiogenesis and immunosuppression. Myeloid-derived suppressor cell (MDSC) is a kind of immunosuppressive cells and plays an important role in this process. However, the actual regulatory mechanisms of angiogenesis and MDSCs in head and neck squamous cell carcinoma (HNSCC) were unclear. In this study, through analyzing the immunohistochemistry staining of human HNSCC tissue microarray, we found that the microvascular density (MVD) was significantly increased in HNSCC patients. We also characterized angiogenic factors p-STAT3, VEGFA, CK2, and MDSCs marker CD11b in HNSCC tissue array, and found the close expression correlation among these markers. To determine the role of JAK2/STAT3 pathway in tumor microenvironment of HNSCC, we utilized AG490 (an inhibitor of JAK2/STAT3) for further research. Results showed that inhibition of JAK2/STAT3 suppressed angiogenesis by decreasing VEGFA and HIF1-α both in vitro and vivo. Moreover, in HNSCC transgenic mouse model, inhibiting JAK2/STAT3 not only suppressed angiogenesis but also reduced MDSCs in the tumor microenvironment through suppressing VEGFA and CK2. Our findings demonstrated the close relationship between angiogenesis and MDSCs in HNSCC, and inhibition of JAK2/STAT3 could reduce tumor-induced angiogenesis and decrease MDSCs.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Janus Quinase 2/antagonistas & inibidores , Células Supressoras Mieloides/efeitos dos fármacos , Neovascularização Patológica/tratamento farmacológico , Fator de Transcrição STAT3/antagonistas & inibidores , Tirfostinas/uso terapêutico , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinoma de Células Escamosas/irrigação sanguínea , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Neoplasias de Cabeça e Pescoço/irrigação sanguínea , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Janus Quinase 2/metabolismo , Camundongos Transgênicos , Células Supressoras Mieloides/metabolismo , Células Supressoras Mieloides/patologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Tirfostinas/farmacologiaRESUMO
Tumor necrosis factor receptor-associated factor 1, an adaptor protein of tumor necrosis factor 2, is involved in classical nuclear factor (NF)-κB activation and lymphocyte recruitment. However, less is known about the expression and association of tumor necrosis factor receptor-associated factor 1 with cancer stem cell markers in oral squamous cell carcinoma. This study aimed to investigate the expression of tumor necrosis factor receptor-associated factor 1 and stem cell characteristic markers (lin28 homolog B, B cell-specific Moloney murine leukemia virus integration site 1, and aldehyde dehydrogenase 1) in oral squamous cell carcinoma and analyze their relations. Paraffin-embedded tissues of 78 oral squamous cell carcinomas, 39 normal oral mucosa, and 12 oral dysplasia tissues were employed in tissue microarrays, and the expression of tumor necrosis factor receptor-associated factor 1, B cell-specific Moloney murine leukemia virus integration site 1, aldehyde dehydrogenase 1, and lin28 homolog B was measured by immunohistostaining and digital pathological analysis. The expression of tumor necrosis factor receptor-associated factor 1 was higher in the oral squamous cell carcinoma group as compared with the expression in the oral mucosa (p < 0.01) and oral dysplasia (p < 0.001) groups. In addition, the expression of tumor necrosis factor receptor-associated factor 1 was associated with those of B cell-specific Moloney murine leukemia virus integration site 1, aldehyde dehydrogenase 1, and lin28 homolog B (p = 0.032, r2 = 0.109; p < 0.0001, r2 = 0.64; and p < 0.001, r2 = 0.16) in oral squamous cell carcinoma. The patient survival rate was lower in the highly expressed tumor necrosis factor receptor-associated factor 1 group, although the difference was not significant. The clustering analysis showed that tumor necrosis factor receptor-associated factor 1 was most related to aldehyde dehydrogenase 1. These findings suggest that tumor necrosis factor receptor-associated factor 1 has potential direct/indirect regulations with the cancer stem cell markers in oral squamous cell carcinoma, which may help in further analysis of the cancer stem cell characteristics.
Assuntos
Carcinoma de Células Escamosas/química , Isoenzimas/análise , Neoplasias Bucais/química , Células-Tronco Neoplásicas/química , Complexo Repressor Polycomb 1/análise , Proteínas de Ligação a RNA/análise , Retinal Desidrogenase/análise , Fator 1 Associado a Receptor de TNF/análise , Família Aldeído Desidrogenase 1 , Biomarcadores , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Humanos , Neoplasias Bucais/mortalidade , Neoplasias Bucais/patologiaRESUMO
MicroRNAs (miRNAs) in oral squamous cell carcinoma (OSCC)-derived small extracellular vesicles (sEVs) play a pivotal role in modulating intercellular communications between tumor cells and other cells in the microenvironment, thereby influencing tumor progression and the efficacy of therapeutic interventions. However, a comprehensive inventory of these secretory miRNAs in sEVs and their biological and clinical implications remains elusive. This study aims to profile the miRNA content of OSCC cell line sEVs and computationally elucidate their biological and clinical relevance. We conducted miRNA sequencing to compare the miRNA profiles of OSCC cells and their corresponding sEVs. Our motif enrichment analysis identified specific sorting motifs that are implicated in either cellular retention or preferential sEV secretion. Target cell analysis suggested that the sEV miRNAs potentially interact with various immune cell types, including natural killer cells and dendritic cells. Additionally, we explored the clinical relevance of these miRNAs by correlating their expression levels with TNM stages and patient survival outcomes. Intriguingly, our findings revealed that a distinct sEV miRNA signature is associated with lymph node metastasis and poorer survival in patients in TCGA-HNSC dataset. Collectively, this research furthers our understanding of the miRNA sorting mechanisms in OSCC and underscores their clinical implications.
Assuntos
Carcinoma de Células Escamosas , Vesículas Extracelulares , Neoplasias de Cabeça e Pescoço , MicroRNAs , Neoplasias Bucais , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço , Carcinoma de Células Escamosas/patologia , Metástase Linfática , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Linhagem Celular Tumoral , Vesículas Extracelulares/metabolismo , Microambiente TumoralRESUMO
Despite the success of programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) inhibition in tumor therapy, many patients do not benefit. This failure may be attributed to the intrinsic functions of PD-L1. We perform a genome-wide CRISPR synthetic lethality screen to systematically explore the intrinsic functions of PD-L1 in head and neck squamous cell carcinoma (HNSCC) cells, identifying ferroptosis-related genes as essential for the viability of PD-L1-deficient cells. Genetic and pharmacological induction of ferroptosis accelerates cell death in PD-L1 knockout cells, which are also more susceptible to immunogenic ferroptosis. Mechanistically, nuclear PD-L1 transcriptionally activates SOD2 to maintain redox homeostasis. Lower reactive oxygen species (ROS) and ferroptosis are observed in patients with HNSCC who have higher PD-L1 expression. Our study illustrates that PD-L1 confers ferroptosis resistance in HNSCC cells by activating the SOD2-mediated antioxidant pathway, suggesting that targeting the intrinsic functions of PD-L1 could enhance therapeutic efficacy.
Assuntos
Antígeno B7-H1 , Ferroptose , Animais , Humanos , Camundongos , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Sistemas CRISPR-Cas , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Superóxido Dismutase/metabolismo , Superóxido Dismutase/genética , Mutações Sintéticas LetaisRESUMO
Only a minority of cancer patients benefit from immune checkpoint blockade therapy. Sophisticated cross-talk among different immune checkpoint pathways as well as interaction pattern of immune checkpoint molecules carried on circulating small extracellular vesicles (sEV) might contribute to the low response rate. Here we demonstrate that PD-1 and CD80 carried on immunocyte-derived sEVs (I-sEV) induce an adaptive redistribution of PD-L1 in tumour cells. The resulting decreased cell membrane PD-L1 expression and increased sEV PD-L1 secretion into the circulation contribute to systemic immunosuppression. PD-1/CD80+ I-sEVs also induce downregulation of adhesion- and antigen presentation-related molecules on tumour cells and impaired immune cell infiltration, thereby converting tumours to an immunologically cold phenotype. Moreover, synchronous analysis of multiple checkpoint molecules, including PD-1, CD80 and PD-L1, on circulating sEVs distinguishes clinical responders from those patients who poorly respond to anti-PD-1 treatment. Altogether, our study shows that sEVs carry multiple inhibitory immune checkpoints proteins, which form a potentially targetable adaptive loop to suppress antitumour immunity.
Assuntos
Antígeno B7-1 , Antígeno B7-H1 , Vesículas Extracelulares , Receptor de Morte Celular Programada 1 , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Humanos , Antígeno B7-1/metabolismo , Antígeno B7-H1/metabolismo , Antígeno B7-H1/imunologia , Animais , Camundongos , Linhagem Celular Tumoral , Feminino , Neoplasias/imunologia , Neoplasias/patologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Inibidores de Checkpoint Imunológico/farmacologia , Tolerância Imunológica , Camundongos Endogâmicos C57BL , Masculino , Microambiente Tumoral/imunologiaRESUMO
Periodontitis is a prevalent oral disease. It can cause tooth loss and has a significant impact on patients' quality of life. While existing treatments can only slow the progression of periodontitis, they are unable to achieve complete regeneration and functional reconstruction of periodontal tissues. As a result, regenerative therapies based on biomaterials have become a focal point of research in the field of periodontology. Despite numerous studies reporting the superiority of new materials in periodontal regeneration, limited progress has been made in translating these findings into clinical practice. This may be due to the lack of appropriate animal models to simulate the tissue defects caused by human periodontitis. This review aims to provide an overview of established animal models for periodontal regeneration, examine their advantages and limitations, and outline the steps for model construction. The objective is to determine the most relevant animal models for periodontal regeneration based on the hypothesis and expected outcomes.
RESUMO
Although Brassica juncea has demonstrated potential as a hyperaccumulator crop, it was not entirely clear how cadmium (Cd) accumulates in plants. Here, we found that BjNRAMP1 (Natural Resistance-Associated Macrophage Protein 1) plays a crucial role in the accumulation of Cd and manganese (Mn) through its expression in yeast and Arabidopsis thaliana. The high concentration of Cd exposure could induce the expression of BjNRAMP1. The ectopic expression of BjNRAMP1 in yeast led to higher accumulation of Cd and Mn compared to the vector control. BjNARAMP1 was localized to the plasma membrane and expressed in the vascular system of roots, leaves, and flowers. The overexpression of BjNRAMP1 in A. thaliana resulted in an increased accumulation of Cd in both roots and shoots, which inhibited the normal growth of transgenic lines. Moreover, Mn uptake in roots was activated by the increase in Cd stress. Together, our results indicated that BjNRAMP1 significantly contributes to the uptake of Mn and Cd in B. juncea.
RESUMO
In oral squamous cell carcinoma (OSCC), a highly aggressive and frequently lethal malignancy, the role and action mechanism of the microtubule regulatory protein CDK5RAP2 have not been fully understood. Here, we show that CDK5RAP2 is highly expressed in OSCC and its expression correlates with clinical stage and lymph node metastasis of the disease. The expression of CDK5RAP2 is regulated by the Wnt signaling pathway. Depletion of CDK5RAP2 inhibits the tumorigenesis and migration of OSCC cells and alters the OSCC cancer stem (-like) cell (CSC) signature. Notably, suppression of CDK5RAP2 expression disrupts spindle orientation during mitosis. Collectively, these results identify CDK5RAP2 as a potential CSC marker and reveal a mechanism that controls the CSC population in OSCC.
Assuntos
Proteínas de Ciclo Celular , Neoplasias Bucais , Proteínas do Tecido Nervoso , Carcinoma de Células Escamosas de Cabeça e Pescoço , Humanos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Bucais/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/genéticaRESUMO
Interferon-γ (IFN-γ), a key effector molecule in anti-tumor immune response, has been well documented to correlate with the intratumoral infiltration of immune cells. Of interest, however, a high level of IFN-γ has been reported in salivary adenoid cystic carcinoma (SACC), which is actually a type of immunologically cold cancer with few infiltrated immune cells. Investigating the functional significance of IFN-γ in SACC would help to explain such a paradoxical phenomenon. In the present study, we revealed that, compared to oral squamous cell carcinoma cells (a type of immunologically hot cancer), SACC cells were less sensitive to the growth-inhibition effect of IFN-γ. Moreover, the migration and invasion abilities of SACC cells were obviously enhanced upon IFN-γ treatment. In addition, our results revealed that exposure to IFN-γ significantly up-regulated the level of programmed death ligand 1 (PD-L1) on SACC cell-derived small extracellular vesicles (sEVs), which subsequently induced the apoptosis of CD8+ T cells through antagonizing PD-1. Importantly, it was also found that SACC patients with higher levels of plasma IFN-γ also had higher levels of circulating sEVs that carried PD-L1 on their surface. Our study unveils a mechanism that IFN-γ induces immunosuppression in SACC via sEV PD-L1, which would account for the scarce immune cell infiltration and insensitivity to immunotherapy.
Assuntos
Carcinoma Adenoide Cístico , Carcinoma de Células Escamosas , Neoplasias Bucais , Neoplasias das Glândulas Salivares , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Carcinoma Adenoide Cístico/metabolismo , Carcinoma Adenoide Cístico/patologia , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Humanos , Terapia de Imunossupressão , Interferon gama/metabolismo , Interferon gama/farmacologia , Neoplasias Bucais/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Neoplasias das Glândulas Salivares/patologiaRESUMO
N6-methyladenosine (m6A) is the most prevalent mRNA modification which plays crucial roles in various biological processes, but its role in cementogenesis remains largely unknown. Here, using time-series transcriptomic analysis, we reveal that mRNA m6A demethylase Fat mass and obesity-associated protein (FTO) is involved in cementogenesis. Knocking down FTO decreases cementoblast differentiation and mineralization in both OCCM-30 cellular model and murine ectopic bone formation model. Mechanistically, we find that FTO directly binds Runt-related transcription factor 2 (Runx2) mRNA, an important cementogenesis factor, thus protecting it from YTH domain-containing family protein 2 (YTHDF2) mediated degradation, when cementoblasts are differentiating. Knocking down YTHDF2 restores the expression of Runx2 in FTO-knockdown cells. Moreover, under inflammatory conditions, TNF-α inhibits cementoblast differentiation and mineralization partly through FTO/RUNX2 axis. Collectively, our study reveals an important regulatory role of FTO/RUNX2 axis in normal and pathological cementogenesis.
Assuntos
Fenômenos Biológicos , Subunidade alfa 1 de Fator de Ligação ao Core , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Animais , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Cemento Dentário/metabolismo , Camundongos , RNA Mensageiro/metabolismo , Fatores de Transcrição , Fator de Necrose Tumoral alfaRESUMO
CMTM6, a regulator of PD-L1 expression, also modulates tumor immunity. Little is known about the function of CMTM6 and its mechanism of action in head and neck squamous cell carcinoma (HNSCC). In this study, we found by IHC analysis that CMTM6 overexpression predicted a poor prognosis for patients with HNSCC. We discovered that CMTM6 expression was correlated with increased activity through the Wnt/ß-catenin signaling pathway, which is essential for tumorigenesis, maintenance of cancer stem cells (CSC), and the epithelial-to-mesenchymal transition (EMT) characteristic of multiple cancers. We used short hairpin RNA to eliminate expression of CMTM6, which led, in HNSCC cells, to reduced expression of nuclear ß-catenin as well as inhibition of stem cell-like properties, TGFß-induced EMT, and cell proliferation. Consistent with these results, we identified a significant positive correlation between expression of CMTM6 and EMT- and CSC-related genes in The Cancer Genome Atlas (TCGA). We found positive correlations for both RNA and protein between expression of CMTM6 and immune checkpoint components. CMTM6 silencing-induced PD-L1 downregulation delayed SCC7 tumor growth and increased CD8+ and CD4+ T-cell infiltration. The proportions of PD-1+, TIM-3+, VISTA+, LAG-3+, and B7-H3+ exhausted T cells were decreased significantly in the CMTM6 knockdown group. CMTM6 thus regulates stemness, EMT, and T-cell dysfunction and may be a promising therapeutic target in the treatment of HNSCC.
Assuntos
Neoplasias de Cabeça e Pescoço/imunologia , Proteínas de Membrana/antagonistas & inibidores , Células-Tronco Neoplásicas/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Animais , Antígeno B7-H1/imunologia , Antígeno B7-H1/metabolismo , Carcinogênese/genética , Carcinogênese/imunologia , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal , Feminino , Técnicas de Inativação de Genes , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Imunidade Celular/imunologia , Proteínas com Domínio MARVEL , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Proteínas da Mielina , Gradação de Tumores , Células-Tronco Neoplásicas/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Taxa de Sobrevida , Via de Sinalização WntRESUMO
N6-methyladenosine (m6A) is a commonly present modification of mammalian mRNAs and plays key roles in various cellular processes. m6A modifiers catalyze this reversible modification. However, the underlying mechanisms by which these m6A modifiers are regulated remain elusive. Here we show that expression of m6A demethylase ALKBH5 is regulated by chromatin state alteration during leukemogenesis of human acute myeloid leukemia (AML), and ALKBH5 is required for maintaining leukemia stem cell (LSC) function but is dispensable for normal hematopoiesis. Mechanistically, KDM4C regulates ALKBH5 expression via increasing chromatin accessibility of ALKBH5 locus, by reducing H3K9me3 levels and promoting recruitment of MYB and Pol II. Moreover, ALKBH5 affects mRNA stability of receptor tyrosine kinase AXL in an m6A-dependent way. Thus, our findings link chromatin state dynamics with expression regulation of m6A modifiers and uncover a selective and critical role of ALKBH5 in AML that might act as a therapeutic target of specific targeting LSCs.