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. 2017 Oct;31(10):2219-2227.
doi: 10.1038/leu.2017.40. Epub 2017 Jan 25.

RUNX3 is oncogenic in natural killer/T-cell lymphoma and is transcriptionally regulated by MYC

V Selvarajan  1 , M Osato  2   3 , G S S Nah  2 , J Yan  2 , T-H Chung  2 , D C-C Voon  4   5 , Y Ito  2 , M F Ham  6 , M Salto-Tellez  2   7 , N Shimizu  8 , S-N Choo  1 , S Fan  1 , W-J Chng  2   9 , S-B Ng  1   2   10
Affiliations

RUNX3 is oncogenic in natural killer/T-cell lymphoma and is transcriptionally regulated by MYC

V Selvarajan et al. Leukemia. 2017 Oct.

Abstract

RUNX3, runt-domain transcription factor, is a master regulator of gene expression in major developmental pathways. It acts as a tumor suppressor in many cancers but is oncogenic in certain tumors. We observed upregulation of RUNX3 mRNA and protein expression in nasal-type extranodal natural killer (NK)/T-cell lymphoma (NKTL) patient samples and NKTL cell lines compared to normal NK cells. RUNX3 silenced NKTL cells showed increased apoptosis and reduced cell proliferation. Potential binding sites for MYC were identified in the RUNX3 enhancer region. Chromatin immunoprecipitation-quantitative PCR revealed binding activity between MYC and RUNX3. Co-transfection of the MYC expression vector with RUNX3 enhancer reporter plasmid resulted in activation of RUNX3 enhancer indicating that MYC positively regulates RUNX3 transcription in NKTL cell lines. Treatment with a small-molecule MYC inhibitor (JQ1) caused significant downregulation of MYC and RUNX3, leading to apoptosis in NKTL cells. The growth inhibition resulting from depletion of MYC by JQ1 was rescued by ectopic MYC expression. In summary, our study identified RUNX3 overexpression in NKTL with functional oncogenic properties. We further delineate that MYC may be an important upstream driver of RUNX3 upregulation and since MYC is upregulated in NKTL, further study on the employment of MYC inhibition as a therapeutic strategy is warranted.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Endogenous RUNX3 expression in NKTL cells (KHYG1, NK-YS, SNK-1, SNK-6 and HANK-1), NKTL patient samples and normal NK cells (NK01-NK03). (a) mRNA expression profiles of RUNX3 in NKTL cells, normal NK cells and NKTL patient samples. cDNA was converted from total RNA of normal NK, NKTL cells and NKTL patient samples, subsequently assayed by quantitative real-time PCR (RT-PCR). RUNX3 mRNA is upregulated in NKTL cells and NKTL patient samples compared to normal NK cells. (b) Protein profiles of RUNX3 across normal and NKTL cells. NKTL cell lysates were probed with RUNX3 (5G4) antibody. There is overexpression of RUNX3 protein in NKTL cells compared to normal NK cells. (c) Immunohistochemistry showing overexpression of RUNX3 in NKTL patient sample (left) and NKTL cell line (SNK-6, middle) and negative expression in normal NK cells (right).
Figure 2
Figure 2
RUNX3 inhibition in NKTL cells. Silenced RUNX3 NKTL cells (siRUNX3) showed deregulation in cell proliferation. (a) NKTL cells were transiently transfected with control siRNA (siControl) and with siRNA against RUNX3 (siRUNX3). Seventy two hours post transfection, total RNA was prepared and subjected to quantitative RT-PCR. A significant reduction of RUNX3 mRNA was observed across all NKTL cells. (b) Whole-cell lysates were prepared and processed for immunoblotting, probed with anti-RUNX3 antibody. β-Actin was used as a loading control. RUNX3 expression was efficiently silenced after 72 h of transfection using siRUNX3. (c) Transfected NKTL cells were stained with Annexin-V and propidium iodide and analyzed for the induction of apoptosis by flow cytometry. Silenced RUNX3 NKTL cells showed an induction of apoptosis compared to control siRNA (siControl). (d) Transfected NKTL cells were stained with BrdU and analyzed for the effects on the cell proliferation rate. The cell proliferation rate was disrupted in RUNX3 silenced NKTL cells (siRUNX3).
Figure 3
Figure 3
Immunohistochemical expression of MYC and RUNX3 protein in NKTL patient samples. (a) Cases with high MYC protein expression (MYC⩾10%) also showed a significantly higher median expression of RUNX3 using Student’s t-test (P=0.006). (b) MYC and RUNX3 protein expression showed moderate correlation using Spearman correlation analysis (r=0.5, P=0.001).
Figure 4
Figure 4
RUNX3 is transcriptionally activated by MYC. (a) The enhancer activity in cells expressing the RUNX3 enhancer (eR3) and the construct with the mutated MYC binding sites (ΔeR3) co-transfected with c-MYC and RUNX3 was measured 48 h post transfection. RUNX3 was co-transfected due to the presence of RUNX binding sites in the enhancer. The co-transfected c-MYC construct showed substantially higher luciferase activity than the reference (eR3). This activity was reduced back to baseline by mutating the MYC binding sites in the enhancer region, suggesting that the transcriptional activity is due to specific binding by MYC. Black arrowheads on the luciferase constructs indicate the position of MYC binding sites. (b) Chromatin immunoprecipitation (ChIP)–qPCR for endogenous MYC binding to RUNX3 enhancer (eR3) in KHYG-1 and SNK-1 cells. Fold enrichment in the ChIP experiment represents the signal obtained after MYC immunoprecipitation followed by qPCR amplified by primer pairs that spanned the enhancer region. Fold enrichments were calculated by determining the IP efficiency (ratios of the amount of immunoprecipitated DNA to that of the input sample, i.e. percentage recovery) and normalized to the level observed at a control region, which was defined as 1. Control-1 and Control-2 denote the non-targeting negative controls while miR26a-2 and miR101 act as positive controls for the ChIP assay. There is a 3.3-fold (KHYG-1) and 6-fold (SNK-1) enrichment of genomic DNA fragments at the MYC binding sites compared to control pull-down sample, indicating binding of MYC to eR3. (c) Protein expressions of MYC and RUNX3 after MYC knockdown. NKTL cells were treated for 72 h and cell lysates were prepared for immunoblots for the MYC protein levels. RUNX3 expression profiles were analyzed post 96 h after MYC knockdown. Silencing of MYC sequentially deregulated RUNX3 expression in NKTL cells.
Figure 5
Figure 5
Effects of MYC inhibition in NKTL cells using BET bromodomain inhibitor (JQ1). RUNX3 downregulation was observed in a dose-dependent manner upon treatment with a small-molecule inhibitor (JQ1) that downregulates MYC. Downregulation of MYC and RUNX3 by JQ1 lead to apoptosis in NKTL cells. (a) Protein expression of MYC and RUNX3 in JQ1 treated NKTL cells at the respective concentrations. Increasing doses of JQ1 effectively reduced MYC and RUNX3 expression levels in NKTL cells. (b) JQ1-treated NKTL cells were stained with Annexin-V and propidium iodide and analyzed for the induction of apoptosis by flow cytometry. The effect of JQ1 treatment on the NKTL cells was a distinct increase in apoptosis in a dose-dependent manner in all NKTL cells.
Figure 6
Figure 6
Effects of MYC ectopic expression in NKTL cells using BET bromodomain inhibitor (JQ1). Overexpression of c-MYC in NKTL cells (SNK-1 and SNK-6) by transfection rescues the cell viability observed with JQ1 treatment, arguing that MYC downregulation by JQ1 contributes functionally to cell growth in NKTL. (a) mRNA were prepared after treating with JQ1 (0.25 μm, 48 h) or DMSO control. MYC expression was rescued (MYC treated) in NKTL cells. (b) Protein profiles from cell lysates of empty and MYC overexpression vector after treating with JQ1 (0.25 μm, 48 h). Immunoblotting showed MYC expression was upregulated in part upon MYC overexpression (MYC-T) compared to empty vector (Vector-T). (c) Cell viability analysis of empty and MYC-overexpressing NKTL cells treated with JQ1 (0.25 μm, 48 h). Cell viability increased in MYC transfected cells (MYC-treated) compared to Vector (treated).
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