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Collaboration of MYC and RUNX2 in lymphoma simulates T-cell receptor signaling and attenuates p53 pathway activity.

ABSTRACT: MYC and RUNX oncogenes each trigger p53-mediated failsafe responses when overexpressed in vitro and collaborate with p53 deficiency in vivo. However, together they drive rapid onset lymphoma without mutational loss of p53. This phenomenon was investigated further by transcriptomic analysis of premalignant thymus from RUNX2/MYC transgenic mice. The distinctive contributions of MYC and RUNX to transcriptional control were illustrated by differential enrichment of canonical binding sites and gene ontology analyses. Pathway analysis revealed signatures of MYC, CD3, and CD28 regulation indicative of activation and proliferation, but also strong inhibition of cell death pathways. In silico analysis of discordantly expressed genes revealed Tnfsrf8/CD30, Cish, and Il13 among relevant targets for sustained proliferation and survival. Although TP53 mRNA and protein levels were upregulated, its downstream targets in growth suppression and apoptosis were largely unperturbed. Analysis of genes encoding p53 posttranslational modifiers showed significant upregulation of three genes, Smyd2, Set, and Prmt5. Overexpression of SMYD2 was validated in vivo but the functional analysis was constrained by in vitro loss of p53 in RUNX2/MYC lymphoma cell lines. However, an early role is suggested by the ability of SMYD2 to block senescence-like growth arrest induced by RUNX overexpression in primary fibroblasts.

SUBMITTER: Hay J

PROVIDER: S-EPMC6772115 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Collaboration of MYC and RUNX2 in lymphoma simulates T-cell receptor signaling and attenuates p53 pathway activity.

Hay Jodie J Gilroy Kathryn K Huser Camille C Kilbey Anna A Mcdonald Alma A MacCallum Amanda A Holroyd Ailsa A Cameron Ewan E Neil James C JC

Journal of cellular biochemistry 20190630 10

MYC and RUNX oncogenes each trigger p53-mediated failsafe responses when overexpressed in vitro and collaborate with p53 deficiency in vivo. However, together they drive rapid onset lymphoma without mutational loss of p53. This phenomenon was investigated further by transcriptomic analysis of premalignant thymus from RUNX2/MYC transgenic mice. The distinctive contributions of MYC and RUNX to transcriptional control were illustrated by differential enrichment of canonical binding sites and gene o ...[more]

PMID: 31257681

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Project description:Runt-related transcription factor 2 (RUNX2) is the best known as an essential protein for osteoblast differentiation. In this study, we have found for the first time that RUNX2 acts as a negative regulator for p53 in response to DNA damage. On DNA damage mediated by adriamycin (ADR) exposure, p53 as well as RUNX2 was induced at protein and mRNA level in human osteosarcoma-derived U2OS cells in association with a significant upregulation of various p53-target genes. Indirect immunostaining and co-immunoprecipitation experiments demonstrated that RUNX2 colocalizes with p53 in cell nucleus and forms a complex with p53 following ADR treatment. Chromatin immunoprecipitation assays revealed that RUNX2/p53 complex is efficiently recruited onto p53-target promoters in response to ADR, suggesting that RUNX2 might be involved in the regulation of transcriptional activation mediated by p53. Indeed, forced expression of RUNX2 resulted in a remarkable downregulation of p53-target genes. Consistent with these observations, knockdown of RUNX2 enhanced ADR-mediated apoptosis and also elevated p53-target gene expression in response to ADR. On the other hand, depletion of RUNX2 in p53-deficient human lung carcinoma-derived H1299 cells had an undetectable effect on p53-target gene expression regardless of ADR treatment, indicating that RUNX2-mediated downregulation of p53-target genes is dependent on p53. Furthermore, RUNX2/p53 complex included histone deacetylase 6 (HDAC6) and HDAC6 was also recruited onto p53-target promoters following ADR exposure. Of note, HDAC6-specific chemical inhibitor tubacin treatment enhanced ADR-mediated upregulation of p53-target gene expression, indicating that deacetylase activity of HDAC6 is required for RUNX2-mediated downregulation of p53-target gene. Taken together, our present findings strongly suggest that RUNX2 inhibits DNA damage-induced transcriptional as well as pro-apoptotic activity of p53 through the functional collaboration with HDAC6 and therefore might be an attractive therapeutic target for cancer treatment.

Project description:BackgroundC-Myc aberrations confer a more aggressive clinic behavior in diffuse large B-cell lymphoma (DLBCL). Matrine is an alkaloid extracted from Sophora flavescens Ait. It possesses anti-cancer property through inhibiting the cell proliferation and inducing the apoptosis. The present study aimed to explore the underlying mechanisms of matrine in suppressing the cell growth of DLBCL.MethodsThe influence of matrine on the viability of cultured DLBCL cell lines SU-DHL-16 and OCI-LY3 cells were determined by CCK-8. Apoptosis and cell cycle were measured by flow cytometry after matrine exposure. Western blot was taken to investigate the expression of activated Caspase-3, cleaved PARP, c-Myc, phospho-c-Myc (Ser62), CaMKIIγ, phospho-CaMKIIγ (Thr287), CDK4 and CDK6 after matrine treatment. Cycloheximide chase analysis was used to determine the c-Myc protein half-lives before and after matrine treatment. Growth salvage analysis was taken by ectopic expression of c-Myc.ResultsIn cultured DLBCL cells, matrine suppressed cell viability in a concentration and time dependent fashion. Matrine treated SU-DHL-16 and OCI-LY3 cells for 48 h with IC50 value of 1.76 mM and 4.1 mM, respectively. Matrine induced apoptosis through a caspase-independent pathway and caused G0/G1 cell cycle arrest in a concentration dependent manner in DLBCL cells. The protein expression of c-Myc was inhibited while the transcription of c-Myc was not reduced by matrine. c-Myc protein half-lives were decreased from 30.4, 69.4 min to 16.6, 15.9 min after matrine treatment in SU-DHL-16 and OCI-LY3, respectively. As a critical protein kinase of c-Myc, CaMKIIγ phosphorylation at Thr287 was found to be down-regulated and c-Myc phosphorylation at Ser62 was reduced together after matrine treatment in DLBCL. The growth suppression of SU-DHL-16 cells induced by matrine was rescued by over-expression of c-Myc achieved by recombinant adenovirus infection. The decreased expression of CDK6, not CDK4, induced by matrine was rescued by ectopic expression of c-Myc protein.ConclusionsThis study has shown for the first time that matrine suppresses cell growth of DLBCL via inhibiting CaMKIIγ/c-Myc/CDK6 signaling pathway.

Dataset Information

Collaboration of MYC and RUNX2 in lymphoma simulates T-cell receptor signaling and attenuates p53 pathway activity.

Publications

Collaboration of MYC and RUNX2 in lymphoma simulates T-cell receptor signaling and attenuates p53 pathway activity.

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