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, 36 (50), 6884-6894

Runx3 Plays a Critical Role in Restriction-Point and Defense Against Cellular Transformation

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Runx3 Plays a Critical Role in Restriction-Point and Defense Against Cellular Transformation

X-Z Chi et al. Oncogene.

Abstract

The restriction (R)-point decision is fundamental to normal differentiation and the G1-S transition, and the decision-making machinery is perturbed in nearly all cancer cells. The mechanisms underlying the cellular context-dependent R-point decision remain poorly understood. We found that the R-point was dysregulated in Runx3-/-mouse embryonic fibroblasts (MEFs), which formed tumors in nude mice. Ectopic expression of Runx3 restored the R-point and abolished the tumorigenicity of Runx3-/-MEFs and K-Ras-activated Runx3-/-MEFs (Runx3-/-;K-RasG12D/+). During the R-point, Runx3 transiently formed a complex with pRb and Brd2 and induced Cdkn1a (p21Waf1/Cip1/Sdi1; p21), a key regulator of the R-point transition. Cyclin D-CDK4/6 promoted dissociation of the pRb-Runx3-Brd2 complex, thus turning off p21 expression. However, cells harboring oncogenic K-Ras maintained the pRb-Runx3-Brd2 complex and p21 expression even after introduction of Cyclin D1. Thus, Runx3 plays a critical role in R-point regulation and defense against cellular transformation.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Runx3−/− MEFs bearing p53 mutation develop into tumors in nude mice. (a) Targeting of Runx3flox and K-RasLSL-G12D alleles by Ad-Cre in MEFs was verified by genomic PCR. PCR primers were described previously. (b) Expression of Runx3 mRNA. Total mRNA was obtained from Rx3+/+ (WT), Rx3+/− (Runx3f/+ treated with Ad-Cre), and Rx3−/− (Runx3f/f treated with Ad-Cre) MEFs, and expression of Runx3 was measured by RT-PCR using the primer pairs shown in Supplementary Figures 1A and B. Arrows indicate Runx3 mRNAs from WT alleles, and arrowheads indicate those from exon 4–deleted alleles. (c) Growth curves of Rx3+/+, Rx3+/−, and Rx3−/− MEFs cultivated according to a 3T3 protocol. The Y-axis indicates the estimated number of accumulated cells. Rx3−/−#1 and Rx3−/−#2 MEFs were derived from two independent embryos. (d) IB analysis of p53 and its phosphorylation at Ser-15 in immortalized or early passage (p3 = passage 3) MEFs, either not treated (−) or treated (+) with 1 μM doxorubicin (Doxo) for 6 h. (e) Immortalized Rx3+/− and Rx3−/− (#1 and #2) MEFs were injected into the left and right sides, respectively, of the back of each nude mouse. The mice were photographed 36 days later, with tumors indicated by circles. (f) IB analysis of ERT-Runx3 (ERT-Rx3) expression in Rx3−/−;ERT-Rx3 (#1 and #2) MEFs in the absence of inducer. Rx3−/− MEFs stably transfected with empty vector (Rx3−/−;Vec) were used as negative controls. (g) Subcellular localization of ERT-Rx3 in the presence or absence of inducer (4-OHT = 4-hydroxy tamoxifen), as determined by subcellular fractionation followed by IB. Very low levels of inducer-independent nuclear ERT-Rx3 are indicated by arrows. (h) Rx3−/−;Vec (#1 and #2) and Rx3−/−;ERT-Rx3 (#1 and #2) MEFs were injected into the left and right sides, respectively, of the back of each nude mouse. The mice were photographed 36 days later, with tumors indicated by circles. The same mice were viewed from the left, middle and right sides.
Figure 2
Figure 2
Expression of Runx3 abolished the tumorigenicity of Rx3−/−;K-RasG12D/+ MEFs. (a) Growth curves of Rx3+/−;K-Ras* (#1 and #2) and Rx3−/−;K-Ras* (#1 and #2) MEFs, cultivated according to a 3T3 protocol. The Y-axis indicates the estimated number of accumulated cells. (b) Rx3+/−;K-Ras* (#1 and #2) and Rx3−/−;K-Ras* (#1 and #2) MEFs were injected into the left and right sides, respectively, of the back of each nude mouse, and photographs were taken 36 days later. (c) Hematoxylin–eosin staining of tumors generated by Rx3−/−, Rx3−/−;K-Ras*, and Rx3+/−;K-Ras* MEFs. (d) Levels of ERT-Runx3 in the indicated MEFs, measured by IB. (e) Proliferation rates of MEFs of the indicated genotypes. (f) Rx3−/−;K-Ras*Vec (#1 and #2) and Rx3−/−;K-Ras*ERT-Rx3 (#1 and #2) MEFs were injected into the left and right sides, respectively, of the back of each nude mouse. (g) RT-PCR analysis of Runx1, Runx2, and Runx3 mRNA levels in Rx3+/+, Rx3+/−, Rx3−/−;K-Ras* (#1 and #2), and Rx3+/−;K-Ras* (#1 and #2) MEFs. Predicted sizes of the Runx3 cDNAs from wild-type Runx3 mRNA are indicated. The four Runx3 PCR products amplified from Rx3+/− MEFs using two different primer pairs are indicated by circles. These PCR products were clearly present in Rx3+/−, but not in Rx3+/−;K-Ras* (#1 and #2) MEFs. (h) Amplification of the CpG island region of Runx3 by unmethylated (UM) or methylated (M) DNA–specific PCR from Rx3+/+, Rx3+/−, Rx3−/−;K-Ras*, and Rx3+/−;K-Ras* MEFs. Mw = molecular size markers.
Figure 3
Figure 3
Deletion of Runx3 disturbs R-point in MEFs. (a) Immortalized MEFs were synchronized by serum deprivation for 24 h, stimulated with 10% serum for the indicated times, and then cultured for additional periods of time under serum starvation conditions. Eighteen hours after serum treatment, cells were harvested, and the proportion of S-phase cells was measured by FACS analysis. (b) Rx3+/+and Rx3−/− MEFs (three independent lines each) were synchronized by serum deprivation and treated with 10% serum for 2 h. RNA was extracted from the MEFs, and the gene expression pattern was analyzed by mRNA sequencing. Expression 2 h after serum stimulation was quantified as the log2 of the fold change relative to the average of control reactions (i.e., before serum stimulation) of Rx3+/+and Rx3−/− MEFs. Differential expression changes were analyzed by plotting log2(ERx3+2h/ERx3+0h) and log2(ERx3-2h/ERx3-0h). ERx3+0h, ERx3+2h, ERx3-2h, and ERx3-0h are the average expression levels of genes in Rx3+/+ or Rx3−/− MEFs 0 or 2 h after serum stimulation. Group A and group B indicate genes induced or suppressed by serum stimulation, respectively. Gray and red spots indicate genes regulated in a Runx3-independent and Runx3-dependent manner, respectively (FDR<0.001, P<0.05). (c) Runx3-dependent genes involved in major signaling pathways. (d) Expression levels of p21 and p27 before and 2 h after serum stimulation in Rx3+/+and Rx3−/− MEFs, obtained from RNA sequencing data. Relative expression levels of p21 and p27 at the indicated times are depicted by the bar graph. (e) Rx3+/+ and Rx3−/− MEFs were synchronized by serum deprivation and treated with 10% serum for the indicated times. The time course of p21 expression levels was determined by Northern blotting (NB). Gapdh mRNA was used as a loading control. (f) Levels or phosphorylation status of early G1–associated proteins in similarly treated Rx3+/+ and Rx3−/− MEFs, measured by IB. (g) Rx3−/− and Rx3−/−;ERT-Rx3 MEFs were synchronized by serum deprivation and treated with 10% serum for the indicated times. The time course of p21 expression levels was determined by Northern blotting.
Figure 4
Figure 4
RUNX3 and pRB form a complex in response to serum stimulation and induce p21. (a) WT MEFs were serum-starved for 40 h and treated with 10% serum. The expression levels of endogenous pRB and RUNX3, and the interaction between the two endogenous proteins, were measured by IP and IB with anti-RUNX3 antibody and anti-pRB antibody, respectively, at the indicated time points. (b) HEK293 cells were serum-starved for 24 h and treated with 10% serum. The expression levels of endogenous pRB and RUNX3, and the interaction between the two endogenous proteins, were measured as described above. (c) HEK293 cells were transfected with Myc-RX3 and HA-RB. Starting 24 h post-transfection, the cells were serum-starved for 24 h and stimulated with serum, and the expression levels of the transfected genes and the RUNX3–pRB interaction were measured by IP and IB, respectively. (d) Wild-type RUNX3 and RUNX3 deletion mutants were transfected into HEK293 cells either with or without RB. β-Gal was co-transfected as an internal control. The reporter activity of p21 promoter–driven luciferase was measured by luciferase assay. A schematic diagram of the RUNX3 deletion constructs is shown. Runt=Runt domain. (e) HEK293 cells were treated with si-RB RNA or si-control RNA for 24 h, cultured under serum-free conditions for 24 h, and then treated with serum for the indicated times. The expression levels of RB, RUNX3, Cyclin D1 (CycD1), and p21 were measured by IB. (f) HEK293 cells were treated with either control siRNA or RX3-specific siRNA for 24 h, and then serum-starved for 24 h. The cells were then treated with 10% serum, and the binding of pRB to the p21 promoter was measured by ChIP analysis at the indicated time points. One-thirtieth of the lysates were PCR amplified for input. Knockdown of RUNX3 by specific siRNA was verified by IB.
Figure 5
Figure 5
Cyclin D–CDK4/6 inhibits the RUNX3–pRB interaction. (a) HA-RB wild-type and HA-RB-M10-16RB (phosphorylation sites–mutated RB) (Supplementary Figure 5A) were transfected to HEK293 cells with Myc-RUNX3. pRB was immunoprecipitated with an anti-HA antibody, and RUNX3 was detected with an anti-Myc- antibody in the immunoprecipitates. (b) Wild-type RB and RB mutated at its phosphorylation sites (RB-M1-9, RB-M10-16, and RB-M1-16) (Supplementary Figure 5A) were transfected into HEK293 cells either with or without RUNX3. The reporter activities of WT p21-promoter-luciferase (p21-WT) or RUNX-binding site–mutated p21-promoter-luciferase (p21-mABC) were measured using a luciferase assay. A, B, and C indicate the three RUNX3-binding sites in p21 promoter. (c) HEK293 cells were transfected with Myc-RUNX3 and HA-RB. Starting 24 h post-transfection, the cells were serum-starved for 1 day, and then treated with 200 nM CDK4 inhibitor for 1 h. The cells were then stimulated with serum, and the RUNX3–pRB interaction was measured by IP and IB at the indicated time points. (d) HEK293 cells were transfected with a fixed amount of RUNX3 (0.2 μg) and RB (0.6 μg) and increasing amounts of Cyclin D1 (0, 0.2, and 0.4 μg, as indicated). Cyclin D1 (0.4 μg)–transfected cells were treated with a CDK4 inhibitor (200 nM) for 4 h, and the effect of Cyclin D1–CDK4 on RUNX3–pRB-mediated p21 promoter (WT-p21-promoter-luciferase) activation was measured using a luciferase assay.
Figure 6
Figure 6
Mitogenic signals trigger the interaction between RUNX3 and pRB. (a) HEK293 cells were transfected with HA-RB, Flag-BRD2, and Myc-RX3 (or Myc-RX3-KR-94-171). The cells were serum-starved for 24 h and treated with 10% serum, and then the interactions between pRB, BRD2, and RUNX3 were measured by IP and IB at the indicated time points. (b) HEK293 cells transfected with either empty vector or Myc-K-RasG12V were serum-starved for 24 h and treated with 10% serum, and then the expression levels of endogenous pRB, RUNX3, and BRD2, as well as the interactions between the three endogenous proteins, were measured by IP and IB at the indicated time points. (c) HEK293 cells were transfected with HA-RB, Flag-BRD2, and Myc-RX3 (or Myc-RX3-KR-94-171) and Myc-K-RasG12V(or MEK1-CA). Cells were serum-starved for 24 h, and then treated with 10% serum for 8 h. Expression level of p21 and interactions between pRB, BRD2, and RUNX3 were measured by IP and IB.
Figure 7
Figure 7
Schematic illustration of R-point regulation by the pRB–RUNX3–BRD2 complex. Formation of the pRB–RUNX3–BRD2 complex is triggered by the RAS-MEK pathway 1 h after serum stimulation. The complex binds to the p21 promoter through RUNX-binding sites and induces p21 expression. Four hours after stimulation, the RAS-MEK pathway activity is downregulated and Cyclin D1 is induced. The induced Cyclin D1 forms a complex with CDK4/6 with the help of the induced p21. Cyclin D1–CDK4/6 dissociates pRB by pRB phosphorylation, and Cyclin D1–HDAC4 dissociates BRD2 by RUNX3 deacetylation from RUNX3. As a result, p21 expression is turned off. Subsequently, the decrease in p21 expression allows activation of Cyclin E–CDK2 activation, which drives cells to pass through the R-point. However, oncogenic K-RAS inhibits destruction of the pRB–RUNX3–BRD2 complex and prolongs p21 expression, which inhibits further cell-cycle progression. This series of molecular events may enable cell to distinguish normal mitogenic signals from abnormal oncogenic K-RAS signals, and thus make context-dependent R-point commitments.

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