12 research outputs found
Insights into the transcriptome and ROS signaling of Arabidopsis thaliana root tips during Pi deficiency response
No full textFerroxidases LPR1, LPR2, and P5-type ATPase PDR2 mediate the accumulation of Fe, reactive oxygen species (ROS), and callose in the root tips leading to primary root growth inhibition upon Pi starvation (PS). Plasma membrane Intrinsic Proteins (PIPs) may translocate H2O2 to influence callose deposition. RNA-seq analysis in the Pi starved-root tips of wild-type (WT), hypersensitive pdr2 mutant, and insensitive lpr1lpr2 line showed that several ROS homeostasis-related genes and ROS signaling regulators were induced in WT and/or pdr2. A burst of ROS was detected in the Pi-starved stem cell niche of WT and pdr2. Moreover, PS increased the superoxide level in the root apical meristem of WT and pdr2. Although some PIPs were differentially expressed in Pi-starved root tips of WT and/or pdr2, no obvious root length phenotypes were observed in all 13 pip single mutants and pip1;3pip1;4 upon PS. The mechanism by which ROS works as signals upon PS needs further investigation.Die Ferroxidasen LPR1, LPR2 und die P5-Typ-ATPase PDR2 vermitteln die Anreicherung von Fe, reaktiven Sauerstoffspezies (ROS) und Kallose in den Wurzelspitzen, was zu einer primären Hemmung des Wurzelwachstums bei Pi-Mangel (PM) führt. Plasmamembran-intrinsische Proteine (PIPs) können H2O2 translozieren, um die Kalloseablagerung zu beeinflussen. RNA-Seq-Analysen von Wurzelspitzen des Wildtyps (WT), der hypersensitiven pdr2-Mutante und der insensitiven lpr1lpr2-Linie gewachsen auf Pi-Mangel-Medium zeigten, dass mehrere ROS-Homöostase-bezogene Gene und ROS-Signalregulatoren in WT und/oder pdr2 induziert wurden. In der Pi-armen Stammzellnische von WT und pdr2 konnte eine hohe Konzentration von ROS nachgewiesen werden. Darüber hinaus erhöhte PM den Superoxidspiegel im Wurzelapikalmeristem von WT und pdr2. Obwohl einige PIPs in Wurzelspitzen, gewachsen unter Pi-Mangel-Bedingungen, von WT und/oder pdr2 unterschiedlich exprimiert wurden, konnte in allen 13 Pip-Einzelmutanten und pip1;3pip1;4 unter gleichen Bedingungen kein offensichtlicher Wurzellängen-Phänotypen beobachtet werden
Functional and molecular analysis of a NF-kB pathway inhibitor, CYLD, in nasopharyngeal carcinoma
No full textNasopharyngeal carcinoma (NPC) is a malignant epithelial carcinoma of the head and neck area with a distinct gender, geographical and racial distribution across the world. It ranked as the tenth in cancer incidence with a high risk of mortality in Hong Kong. The pathogenesis of NPC is attributed to three main etiological factors: EBV infection, genetic factors and environmental factors.
NF-kB is an inducible transcriptional factor, regulating inflammation, angiogenesis, cell proliferation and metastasis in cancer. Its activation plays critical roles in NPC development. By our previous whole-exome sequencing and targeted sequencing of NPC patients, CYLD, a NF-kB inhibitor, was screened out as one of the top mutated candidate genes. As CYLD was downregulated in NPC clinical samples and NPC cell lines, it was expected to play an inhibitory role in NPC development. In order to investigate the potential functional effects of CYLD, the CRISPR-Cas9 system was used to knock out CYLD in NPC cell lines. Subsequent in vitro functional assays revealed CYLD inhibits NPC cell proliferation, survival and migration. More importantly, the in vivo assays showed that CYLD knockout contributed to enhanced NPC tumorigenicity and metastasis abilities. Staining of the xenografts showed an increased number of cells undergoing mitosis and microvessel formation after CYLD knockout. Mechanistic study also confirmed that the CYLD knockout increases p65 nuclear translocation, DNA binding activity, and NF-kB downstream target genes expression in vitro and in vivo.
As NF-kB mediates a crosstalk between inflammation and cancer in multiple aspects, hyperactive NF-kB is able to regulate the host immune response to recruit more stromal cells to support the tumor growth. By the method of single cell RNA-Seq analysis of NPC xenografts, CYLD was found to inhibit fibroblasts and endothelial stromal cells recruitment in the tumor microenvironment (TME).
Therefore, CYLD can regulate NPC via inhibition of three cancer hallmarks, cell proliferation, angiogenesis and metastasis and regulate TME via inhibition of fibroblast and endothelial stromal cells recruitment, by suppressing the NF-kB pathway.
Two truncation mutations (S323X and S371X) and one missense mutation (758Q) identified from our previous WES were further studied to enhance our understanding of how mutated CYLD contributes to cancer using a lentiviral transduction system to re-express these mutants in NPC cell lines. The in vitro assays showed the truncation mutations are associated with loss of the CYLD inhibitory roles in suppressing cell migration and NF-kB DNA binding activity. Further assays were applied to study the missense mutation, R758Q. It fails to inhibit cell proliferation and migration, NF-kB nucleus translocation, DNA binding activity and NF-kB downstream targets expression in vitro. While the in vivo assays for S323X, 371X and 758Q failed to significantly differ from the CYLD WT, further assays need to be performed in the future to have a better understanding of these mutations.published_or_final_versionClinical OncologyDoctoralDoctor of Philosoph
Abstract 3442: Functional and molecular analysis of the somatic mutations in the NF-κB pathway inhibitor, CYLD, in nasopharyngeal carcinoma
No full textAbstract 3442: Functional and molecular analysis of the somatic mutations in the NF-κB pathway inhibitor, CYLD, in nasopharyngeal carcinoma
No full textFunctional Characterization of BoaMYB51s as Central Regulators of Indole Glucosinolate Biosynthesis in Brassica oleracea var. alboglabra Bailey
No full textR2R3-MYB transcription factor MYB51 is known to control indole glucosinolate (indole GSL) biosynthesis in Arabidopsis. Here, two copies of BoaMYB51 have been isolated in Chinese kale (Brassica oleracea var. alboglabra Bailey), designated BoaMYB51.1 and BoaMYB51.2, which exhibit overlapping but distinct expression levels among different organs and respond to signaling molecules in a similar pattern. It has been demonstrated a structural and functional conservation between BoaMYB51s and AtMYB51 by phylogenetic analysis, complementation studies and transient expression assay. To further investigate the transcriptional mechanism, we identified the transcriptional activation domain (TAD) and putative interacting proteins of BoaMYB51s by means of yeast (Saccharomyces cerevisiae) two hybrid. Using tobacco (Nicotiana benthamiana) transient expression assay, we confirmed that the carboxy-end is required for transcriptional activation activity of BoaMYB51s. In addition, several BoaMYB51-interacting proteins have been identified by yeast two-hybrid screening. These results provide important insights into the molecular mechanisms by which MYB51 transcriptionally regulates indole GSL biosynthesis
Functional Characterization of BoaMYB51s as Central Regulators of Indole Glucosinolate Biosynthesis in Brassica oleracea var. alboglabra Bailey
No full text
Cylindromatosis Lysine 63 Deubiquitinase (CYLD) Regulates NF-kB Signaling Pathway and Modulates Fibroblast and Endothelial Cells Recruitment in Nasopharyngeal Carcinoma
No full textNasopharyngeal carcinoma (NPC) is a malignant epithelial carcinoma of the nasopharynx. Cylindromatosis lysine 63 deubiquitinase (CYLD), a NF-kB inhibitor, was reported as one of the top mutated candidate genes in NPC. NF-kB is an inducible transcription factor, contributing to cancer via regulating inflammation, angiogenesis, cell proliferation, and metastasis. In this study, the impact of CYLD on regulating the NF-kB signaling pathway and its contribution to NPC development was studied using in vitro and in vivo functional assays, together with single cell RNA sequencing to understand the NPC tumor microenvironment. CYLD was downregulated in NPC clinical specimens and multiple cell lines. Functional assays revealed CYLD inhibits NPC cell proliferation and migration in vitro and suppresses NPC tumorigenicity and metastasis in vivo by negatively regulating the NF-kB signaling pathway. Additionally, CYLD was able to inhibit fibroblast and endothelial stromal cell infiltration into the NPC tumor microenvironment. These findings suggest that CYLD inhibits NPC development and provides strong evidence supporting a role for CYLD inhibiting fibroblast and endothelial stromal cell infiltration into NPC via suppressing the NF-kB pathway
Data_Sheet_1_Functional Characterization of BoaMYB51s as Central Regulators of Indole Glucosinolate Biosynthesis in Brassica oleracea var. alboglabra Bailey.PDF
No full textR2R3-MYB transcription factor MYB51 is known to control indole glucosinolate (indole GSL) biosynthesis in Arabidopsis. Here, two copies of BoaMYB51 have been isolated in Chinese kale (Brassica oleracea var. alboglabra Bailey), designated BoaMYB51.1 and BoaMYB51.2, which exhibit overlapping but distinct expression levels among different organs and respond to signaling molecules in a similar pattern. It has been demonstrated a structural and functional conservation between BoaMYB51s and AtMYB51 by phylogenetic analysis, complementation studies and transient expression assay. To further investigate the transcriptional mechanism, we identified the transcriptional activation domain (TAD) and putative interacting proteins of BoaMYB51s by means of yeast (Saccharomyces cerevisiae) two hybrid. Using tobacco (Nicotiana benthamiana) transient expression assay, we confirmed that the carboxy-end is required for transcriptional activation activity of BoaMYB51s. In addition, several BoaMYB51-interacting proteins have been identified by yeast two-hybrid screening. These results provide important insights into the molecular mechanisms by which MYB51 transcriptionally regulates indole GSL biosynthesis.</p
DGKH-mediated phosphatidic acid oncometabolism as a driver of self-renewal and therapy resistance in HCC
No full textBackground and Aims: HCC is characterized by metabolic pathway aberrations, which enable cancer cells to meet their energy demands and accelerate malignant progression. Identifying novel metabolic players governing therapy resistance and self-renewal in HCC is crucial, as these properties are likely responsible for tumor recurrence. Approach and Results: Clinical traits and RNA-seq of patients with HCC in The Cancer Genome Atlas were used for weighted gene coexpression network analysis, where 1 module was significantly correlated with advanced pathological stage and stem cell population maintenance. Further analysis of this module by integrating data obtained from HCC patient nonresponders to tyrosine kinase inhibitors identified 361 commonly deregulated genes. Intriguingly, these genes are significantly enriched in the intracellular signal transduction pathway, with diacylglycerol kinase eta (DGKH) ranked as the most enriched gene in poorly differentiated HCC tumors. Clinically, DGKH was elevated in tumor tissues compared to nontumor tissues. Patients with higher DGKH expression exhibited a more undifferentiated state and were less responsive to tyrosine kinase inhibitors. Functional assays using DGKH-manipulated HCC cell lines demonstrated that DGKH augmented aggressive features, including cancer stemness, therapy resistance, and metastasis. Upstream of DGKH, we discovered that the E1A-associated protein p300 (EP300) binds to DGKH’s promoter region, thereby increasing its transcriptomic expression. Mechanistically, DGKH promotes mTOR signaling by producing phosphatidic acid. In an immunocompetent mouse model, cotreatment with sorafenib and liver-directed AAV8-mediated Dgkh depletion significantly reduced tumor burden, self-renewal, phosphatidic acid production, and mTOR signaling. Conclusions: Our research demonstrated that DGKH is a crucial oncometabolic regulator of cancer stemness and therapy resistance, suggesting that inhibiting DGKH may lead to more effective HCC treatment
