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. Author manuscript; available in PMC: 2013 Jul 1.
Published in final edited form as: Oncogene. 2011 Jun 27;31(4):527–534. doi: 10.1038/onc.2011.252

RUNX3 acts as a tumor suppressor in breast cancer by targeting estrogen receptor α

Bo Huang 1, Zhaoxia Qu 2, Chee Wee Ong 3,4, Ying-Hung Nicole Tsang 1,5, Gutian Xiao 2, David Shapiro 1, Manuel Salto-ellez 3,4, Kosei Ito 6,†, Yoshiaki Ito 4,5, Lin-Feng Chen 1,7,†
PMCID: PMC3697905  NIHMSID: NIHMS364345  PMID: 21706051

Abstract

Transcription factor RUNX3 is inactivated in a number of malignancies, including breast cancer, and is suggested to function as a tumor suppressor. How RUNX3 functions as a tumor supressor in breast cancer remains undefined. Here, we show that about 20% of female Runx3+/− mice spontaneously developed ductal carcinoma at an average age of 14.5 months. Additionally, RUNX3 inhibits the estrogen-dependent proliferation and transformation potential of ERα-positive MCF-7 breast cancer cells in liquid culture and in soft agar and suppresses the tumorigenicity of MCF-7 cells in severe combined immunodeficiency (SCID) mice. Furthermore, RUNX3 inhibits ERα-dependent transactivation by reducing the stability of ERα. Consistent with its ability to regulate the levels of ERα, expression of RUNX3 inversely correlates with the expression of ERα in breast cancer cell lines, human breast cancer tissues and Runx3+/− mouse mammary tumors. By destabilizing ERα, RUNX3 acts as a novel tumor suppressor in breast cancer.

Keywords: breast cancer, degradation, ERα, RUNX3, tumor suppressor

Introduction

Breast carcinoma is a leading female malignancy and cause of cancer death in developed countries. Estrogen plays a critical role in normal mammary gland development and is also involved in the pathogenesis of breast cancer (LaMarca and Rosen, 2007). The actions of estrogen are mediated by estrogen receptors α and β. ERα functions as a transcriptional regulator, stimulating proliferation and suppressing apoptosis through the regulation of genes involved in the cell cycle and apoptosis (Katzenellenbogen and Katzenellenbogen, 2000). Abnormal estrogen signaling through ERα is associated with initiation and progression of breast cancer (Cheskis et al., 2007). ERα is overexpressed in about 75% of breast cancers (Katzenellenbogen and Katzenellenbogen, 2000). Deregulated ERα expression in mammary epithelial cells leads to the development of ductal carcinoma in situ (Frech et al., 2005). Therefore, tight control of the level of ERα is important in maintaining normal estrogen responsiveness. Proteasome-mediated degradation of ERα plays an important role in regulating ERα function by controlling its cellular levels (Duong et al., 2007; Fan et al., 2004; Nawaz et al., 1999; Tateishi et al., 2004).

RUNX3 belongs to the Runt-related gene family, whose members are all developmental regulators and have been shown to be involved in carcinogenesis (Ito, 2004). The Runx3 gene is located in 1p36, a region often deleted in a wide variety of human carcinomas, including breast cancer. A possible role for RUNX3 as a tumor suppressor in breast cancer is suggested by its frequent inactivation by protein mis-localization and promoter hypermethylation in breast cancer cell lines and breast cancer tissues, and this inactivation constitutes an early event in breast cancer progression (Jiang et al., 2008; Lau et al., 2006; Subramaniam et al., 2009a). Furthermore, expression of RUNX3 is associated with a more favorable prognosis in breast cancer patients, and RUNX3 expression generally decreases during breast cancer progression (Jiang et al., 2008). In mice, loss of Runx3 affects ovulation and estrogen-induced endometrial cell proliferation in female mice (Sakuma et al., 2008). These studies highlight RUNX3's potential role as a tumor suppressor in breast cancer. However, there was no direct evidence that RUNX3 functions as a tumor suppressor in breast cancer and a plausible mechanism by which RUNX3 could act as a breast cancer tumor suppressor had not been described.

Here, we report that Runx3+/− female mice spontaneously develop mammary ductal carcinoma. Expression of RUNX3 suppresses estrogen-dependent proliferation and tumorigenesis of ERα-positive MCF-7 cells. Furthermore, RUNX3 inhibits the transcriptional activation of ERα by inducing its proteasome-mediated degradation. These data suggest that RUNX3 functions as a tumor suppresser in breast cancer by regulating the stability of ERα.

Results and discussion

Disruption of Runx3 led to the development of ductal adenocarcinoma in mammary gland epithelium

While maintaining Runx3+/− mice, we observed that some female mice developed abnormalities in the mammary gland after a year. About one fifth [11 of 52 (21.2%)] of the Runx3+/− female mice developed mammary tumors at the age of 15 months while none of the 32 wild-type (WT) mice developed mammary tumors at this age (Figure 1a). The tumors in the mice ranged from 10 mm to 20 mm in diameter and all the primary tumors were relatively well differentiated. Histochemical staining reveals that these spontaneous tumors are ductal adenocarcinoma with solid pattern of growth (Figure 1b, E and F). In these Runx3+/− tumors, expression of RUNX is markedly reduced (Figure 1b, G & H) compared to the highly expressed RUNX3 in the mammary gland epithelial cells of the WT mice (Figure 1b, C & D).

Figure 1.

Figure 1

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Histopathology of mammary tumors from female Runx3+/− mice. (a) Incidence of mammary tumors in Runx3+/− mice. The generation of Runx3+/− BALB/c mice was described previously (Ito et al., 2008). The mice were maintained in pathogen-free conditions and monitored daily. All procedures involving animals were performed in accordance with the guidelines of Nagasaki University Animal Care and Use Committee. (b) Mammary gland specimens from wild-type and Runx3+/− BALB/c mice were fixed with 4% paraformaldehyde in PBS, embedded in paraffin, and sectioned at 4 μm. Sections were stained with hematoxylin and eosin (HE). HE sections were evaluated by a pathologist (M.S.-T.). Representative HE staining and RUNX3-immunohistochemical staining of a normal mammary gland from a WT mouse (left 4 panels, A-D) and an adenocarcinoma of mammary gland from a Runx3+/− mouse (right 4 panels, E-H) are shown. Boxed regions are enlarged to the right of each image. Scale bars: 100 (lower magnification) and 50 (higher magnification) μm. (c) (d) Immunohistochemical staining of Ki-67 (c) and ERα (d) in serial sections from (b). For the detection of RUNX3, Ki-67 and ERα protein expression, rehydrated specimens were treated for 40 min at 96°C with an antigen retrieval solution (S1700; DAKO). The specimens were incubated with antibodies against RUNX3 (R3-3F12 as previously described (Ito et al., 2009)), Ki-67 (M7249; DAKO) or ERα (SC-542; Santa Cruz Biotech). The EnVision+ system (DAKO) was used for visualization of signals in (b), (c) and (d).

Examining the proliferation marker Ki-67 revealed that Ki-67 was quite abundant in the Runx3+/− tumors whereas Ki-67 levels were significantly lower in the mammary gland epithelial cells of WT mice (Figure 1c). These data suggest that the mammary tumors in Runx3+/− mice are due to increased proliferation and that RUNX might act as a tumor suppressor by inhibiting mammary gland epithelial cell proliferation.

To further characterize these Runx3+/− tumors, we examined the expression of ERα, PR (progesterone receptor) and Her2/neu (human epidermal growth factor receptor 2), three different prognostic biomarkers of breast cancer. In the mammary gland epithelial cells of the WT mice, the expression of ERα, PR and Her2/neu was low and barely detectable (Figure 1d and Supplementary Figure S1). Interestingly, the expression of ERα was significantly enhanced in Runx3+/− tumors (Figure 1d, C and D), while the expression of PR was moderately increased and the expression of Her2/neu remained the same compared to WT mice (Supplementary Figure S1). Since ERα regulates the expression of PR (Anderson, 2002), the enhanced PR expression might result from the enhanced expression of ERα. Therefore, these Runx3+/− tumors are ERα-positive and likely PR-positive, but are Her2/neu-negative.

RUNX3 inhibits the proliferation and tumorigenesis of MCF-7 cells

Since Runx3+/− tumors are ERα positive and excessive signaling through ERα has been shown to be the primary mechanism for breast tumorigenesis (Jerry et al., 2010), we hypothesized that RUNX3 might target ERα signaling for its tumor suppressor function. To test this hypothesis, we examined the effect of RUNX3 on the proliferation of an ERα-positive MCF-7 cell line. Since RUNX3 is not expressed in MCF-7 cells due to hypermethylation of the Runx3 promoter (Lau et al., 2006), we re-introduced RUNX3 into MCF-7 cells using a retroviral expression vector and generated MCF-7 cells stably expressing RUNX3 (designated RUNX3-MCF-7). Vector-MCF-7 and RUNX3-MCF-7 cells exhibited very slow growth over 3 days without 17β-estradiol (E2) (Figure 2a). The vector-MCF-7 cells started to grow quickly when E2 was added to the media at day 3 (Figure 2a). In contrast, the growth of RUNX3-MCF-7 cells was significantly reduced in the presence of E2 compared to the vector-MCF-7 cells (Figure 2a) and the reduced growth of RUNX3-MCF-7 cells was not due to increased cell death (Supplementary Figure S2). Together, these data suggest that RUNX3 inhibits estrogen-dependent proliferation of MCF-7 cells.

Figure 2.

Figure 2

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RUNX3 suppresses the proliferation and tumorigenesis of MCF-7 cells. (a) Both vector-MCF-7 and RUNX3-MCF-7 cells were plated in 96-well plates in 0.1 ml phenol red-free MEM with double-stripped calf serum and cultured for three days. Cells were then stimulated with 100 pM E2 and cultured for up to 4 days. Cell proliferation was measured by OD 490 nm using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) (Promega) and converted to cell number according to the standard curve. Data represent the average of three independent experiments +/−SD. (b) A total of 5000 cells from vector-MCF-7 or RUNX3-MCF-7 were suspended in DMEM containing 0.35% SELECT Agar® (Invitrogen) and then plated in 6-well plates coated with an initial underlay of 0.5% SELECT Agar® (Invitrogen) in culture medium. Colony growth was scored after 14 days of cell incubation at the normal condition. Representative photographs were taken at day 1 and day 14 to show colonies. All the colony formation assays presented in this study were repeated in at least 3 independent experiments. (c) Six-week old female severe combined immunodeficient (SCID) mice (C.B-17/IcrCrl-scidBR) (Charles River Laboratories) were subcutaneously implanted with a slow release pellet of 25 μg E2. Four days later, the mice were injected in the inguinal mammary fat pads with vector-MCF-7 or RUNX3-MCF-7 stable cell lines (5×106). The recipient mice were monitored daily by palpation, and killed and dissected for tumor evaluation 30 days post-injection. Pictures were taken at day 30 after cell inoculation. Mouse injection was performed essentially as previously described (Yan et al., 2009). All experiments involving mice were approved by the Institutional Animal Care and Use Committee (IACUC). (d) Summary of the average weight of tumors from (c). Data represent mean ± SEM (n = 3). * P < 0.05 (paired t test).

Next, we investigated the effect of RUNX3 on the transformation potential of MCF-7 cells by anchorage-independent growth assay. As expected, vector-MCF-7 cells formed colonies in soft agar (Figure 2b). Conversely, both the number and size of the colonies formed by the RUNX3-MCF-7 cells were significantly reduced (Figure 2b), indicating that RUNX3 impairs the transformation potential of MCF-7 cells.

We then determined the effect of RUNX3 expression on breast cancer formation and growth in an animal model. Compared with vector-MCF-7 cells, RUNX3-MCF-7 cells produced smaller tumors in SCID mice (Figure 2c). The average weight of the tumors formed by the RUNX3-MCF-7 cells was >80% lower than those formed by the vector-MCF-7 cells (Figure 2d). These results indicate that RUNX3 inhibits the ability of ERα -positive breast cancer cells to grow as tumors.

RUNX3 inhibits the transcriptional activity of ERα

Since RUNX3 inhibits E2-dependent proliferation and tumorigenesis of MCF-7 cells (Figure 2), and ERα plays a key role in mediating the action of estrogen, we next examined the effect of RUNX3 on the activity of ERα. MCF-7 cells were transfected with RUNX3 expression vector and a reporter plasmid containing four copies of the estrogen response element (ERE). Co-expression of RUNX3 inhibited basal as well as E2-activated ERE reporter activity (Figure 3a, left panel). Conversely, RUNX3 has no effect on the luciferase reporter activity when the ERE sites are deleted (Supplementary Figure S3), suggesting a direct effect of RUNX3 on ERα. Similarly, RUNX3 inhibited the basal and E2-activated PI-9 promoter driven-luciferase reporter activity in both MCF-7 cells and normal mammary epithelial cells (Figure 3a, right panel and Supplementary Figure S4), ruling out the possibility that the inhibitory response reflects a selective inhibition of the 4×-ERE reporter in cancer cells.

Figure 3.

Figure 3

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RUNX3 represses transcriptional activity of ERα. (a) MCF-7 cells were cultured in phenol red-free MEM with 10% double-stripped calf serum for three days followed by transfection with 4×ERE- (left panel) or PI-9-luciferase (right panel) reporter and RUNX3 plasmid DNA. Twenty-four hours after transfection, cells were stimulated with vehicle or E2 (100 nM) for 24 hr. Luciferase activity was measured and expressed as fold induction after normalization with Renilla luciferase. Results represent the average of three independent experiments +/− SD. (b) MCF-7 cells stably expressing control vector or RUNX3 were stimulated with E2 (100 nM) for 24 hr. The expression levels of pS2 and NRIP1 mRNA were measured with quantitative RT-PCR using Qiagen SYBR green PCR kit by 7300 real-time PCR system (ABI). The primers for pS2, NRIP1, and actin were purchased from Qiagen. Expression levels of RUNX3 and ERα protein in the MCF-7 stable cell lines are shown in the right panels. Intensity of each band was measured and analyzed by ImageJ 1.40g. Mean of three measurements is indicated beneath each band image. Data are representative of three independent experiments. (c) MDA-MB-361 cells were transfected with control or RUNX3 siRNA using Lipofectamine 2000 (Invitrogen). Sixty hours post-transfection, cells were left untreated or stimulated with E2 (100 nM) for 24 hr. The expression levels of pS2 and NRIP1 mRNA were measured by quantitative real-time PCR as in (b). Efficiency of siRNA knock-down as well as ERα expression levels are shown in the right panels. The predesigned siRNA targeting RUNX3 was purchased from Santa Cruz Biotech (SC-37679).

When we assessed the effect of RUNX3 on the expression of ERα target genes, we found that E2-stimulated expression of two ERα-responsive genes, pS2 and NRIP1, was significantly reduced in the RUNX3-MCF-7 cells compared to vector-MCF-7 cells (Figure 3b, left two panels). Interestingly, the expression level of ERα in RUNX3-MCF-7 cells was decreased compared to that of the vector-MCF-7 cells (Figure 3b, right panel). Similar down-regulation of ERα activity and ERα expression by stably expressed RUNX3 were observed in another ERα-positive breast cancer cell line, T47D (Supplementary Figure S5), further confirming that RUNX3 negatively regulates the activity of ERα.

To further confirm the inhibitory effect of RUNX3, we knocked down the expression of RUNX3 using small interfering RNA (siRNA) in MDA-MB-361 cells, which express both RUNX3 and ERα. Depletion of RUNX3 enhanced the E2-induced expression of both pS2 and NRIP1 genes (Figure 3c). Consistent with the decreased expression of ERα in RUNX3-MCF-7 and RUNX3-T47D cells (Figure 3b and Supplementary Figure S5), depletion of RUNX3 resulted in an increased level of ERα (Figure 3c). These data together with the data from the Runx3+/− mice (Figure 1d), indicate that RUNX3 down-regulates the level of ERα.

RUNX3 induces the proteasome-mediated degradation of ERα

To further explore the possible relationship between the expression levels of ERα and RUNX3, we examined the expression of RUNX3 and ERα in a panel of cell lines including non-tumorigenic breast epithelial cells and breast cancer cell lines. While the levels of ERα vary in different cell lines (Figure 4a), the overall expression of ERα appears to be inversely correlated with the expression of RUNX3 (Figure 4a). Furthermore, when we examined the possible pathological relationship of the expression of RUNX3 and ERα in human normal breast and breast cancer samples, we also observed a close inverse correlation between the expression of RUNX3 and ERα (Figure 4b). Importantly, when we examined the expression of RUNX3 and ERα in a cohort of 80 human breast ductal carcinoma samples, we observed that 70% of samples (16 out of 23) with low expression of RUNX3 display high expression of ERα (Figure 4c). Conversely, 65% of samples (37 out of 57) with high expression of RUNX3 have low levels of ERα. Statistic analysis reveals an inverse correlation between the expression of RUNX3 and ERα in these cancer samples with a Spearman coefficient for correlation (RUNX3 and ERα) of −0.314 (p=0.005) (Figure 4c).

Figure 4.

Figure 4

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Expression of RUNX3 inversely correlated with ERα expression. (a) Expression levels of RUNX3 and ERα in various normal mammary and breast cancer cells. (b) Representative immunohistochemical staining of RUNX3 and ERα in normal and tumor breast tissues. Boxed regions are enlarged below. The detection of the expression of RUNX3 and ERα of the human tissue microarray was performed as previously described (Zhang et al., 2003). (d) Tissue sections of 80 breast cancer samples were immunostained with anti-RUNX3 or anti-ERα antibodies for the expression of RUNX3 and ERα and their correlation was analyzed by Sperman rank correlation test (p = 0.005). RUNX3 staining intensity was graded as previously described with a score 0 to 3 (Subramaniam et al., 2009a). Samples with a score 0 or 1 were graded as Low, samples with a score 2 or 3 were graded as High. For ERα, staining occurred in more than 50% of tumor cells was graded as High whereas staining occurred in less than 50% of tumor cells was graded as Low (Zhang et al., 2003). (d) MCF-7 cells were transfected with increasing amounts of Myc-RUNX3 as indicated. Forty-two hours post-transfection, cells were treated with vehicle or MG-132 (20 μM) for 6 hrs, and whole cell lysates were immunoblotted as indicated. (e) Vector-MCF-7 and RUNX3-MCF-7 cells were plated in 6 cm dishes. Twenty-four hours later, cells were incubated with DMEM lacking methionine and cysteine (Invitrogen) for 1hr, and then labeled with 35S-methionine (PerkinElmer) in a final concentration of 20 μCi/ml for 1 hr. The media was replaced with DMEM and cells were cultured (chased) for indicated time points. ERα was immunoprecipitated using an anti-ERα antibody and separated on SDS-page gel. 35S-labelled ERα was detected by autoradiography. Representative gels are shown in the upper panel. Quantification of the results from three independent experiments is shown in the lower panel.

The inverse relationship between ERα and RUNX3 expression prompted us to investigate whether RUNX3 might regulate ERα stability. We transfected increasing amounts of RUNX3 into MCF-7 cells and examined the effect of RUNX3 on the expression of endogenous ERα. Expression of increasing levels of RUNX3 decreased the levels of endogenous ERα in a dose-dependent manner (Figure 4d). The decreased expression of endogenous ERα was reversed by treatment with proteasome inhibitor MG-132 (Figure 4d), and RUNX3 had no effect on the level of ERα mRNA (Supplementary Figure S6), suggesting that RUNX3 reduces the expression of ERα at the protein level rather than at the mRNA level and that RUNX3 regulates the stability of ERα.

To more quantitatively evaluate the effect of RUNX3 on the stability of ERα, we compared the half-life of endogenous ERα in the absence and presence of RUNX3 using vector-MCF-7 and RUNX3-MCF-7 cells. We performed the pulse-chase experiment using 35S-methionine to label endogenous ERα or cycloheximide to inhibit the synthesis of new ERα (Figure 4e and Supplementary Figure S7). The levels of ERα gradually decreased in vector-MCF-7 cells and ERα was relatively stable in the absence of RUNX3 with a half-life of about 6 hr (Figure 4e, right panel, and Supplementary S7). However, in RUNX3-MCF-7 cells, the degradation of ERα was accelerated and the half-life of ERα was reduced to about 2 hr (Figure 4e, right panel and Supplementary Figure S7). Importantly, RUNX3 has no effect on the half-life of Her2/neu (Supplementary Figure S8), indicating a specific effect of RUNX3 on ERα stability. Together, these findings demonstrate that RUNX3 reduces the stability of ERα by inducing its proteasome-mediated degradation.

Inactivation of RUNX3 by protein mis-localization and hypermethylation of the Runx3 promoter is often found in breast cancer cells and breast tumors (Subramaniam et al., 2009b), suggesting a tumor suppressor function of RUNX3 in breast cancer. However, there was not clear and direct evidence for its tumor suppressor activity in breast cancer. Here, we demonstrate that Runx3+/− mice developed spontaneous mammary tumors (Figure 1), supporting the notion that RUNX3 is a tumor suppressor in breast cancer. Moreover, we show that RUNX3 elicits its tumor suppressor activity by targeting ERα signaling. RUNX3 inhibits ERα activity by inducing the proteolytic degradation of ERα (Figures 3 &4).

It has been well documented that ubiquitination and degradation of ERα regulate ERα activity. Ubiquitination and degradation of ligand-bound ERα and its co-activators have been shown to be involved in the transcriptional activation or inactivation of ERα, depending on the experimental conditions (Fan et al., 2004; Lonard et al., 2000; Nawaz et al., 1999; Reid et al., 2003). Nevertheless, ubiquitination and degradation of ligand-free receptor plays a clear role in down-regulating the basal levels of ERα (Duong et al., 2007; Fan et al., 2004; Tateishi et al., 2004). Although RUNX3 inhibits the transcriptional activity of ERα by inducing its degradation in the presence of E2 (Figure 3 and Supplementary Figure S9), it appears that degradation of ERα by RUNX3 could be ligand-independent precede the binding of ERα to DNA since RUNX3 inhibits the basal ERα activity and degrades ERα in the absence of E2 (Figure 3 and Supplementary Figure S9). Consistent with this notion, RUNX3 induced the degradation of a ligand binding-deficient mutant of ERα (ERα-L525A) (Ekena et al., 1996) to the same level as WT ERα (Supplementary Figure S10). Currently, it is unclear how RUNX3 induces the degradation of ERα. But it appears that the transcriptional activity of RUNX3 is not essential for the degradation since a transcriptionally incompetent mutant of RUNX3, RUNX3-R122C (Chi et al., 2005), was still able to induce the degradation of ERα (Supplementary Figure S11). It is possible that RUNX3 might recruit an E3 ubiquitin ligase or directly function as an E3 ubiquitin ligase for the ubiquitination and degradation of ERα, but the detailed molecular mechanism needs to be further explored.

Our data define a role for RUNX3 as a tumor suppressor in ERα-positive breast cancers and show that the expression of ERα and RUNX3 display an inverse correlation in ERα-positive cancer cells (Figure 4a). However, it has to be noted that expression of RUNX3 is also down-regulated in some ERα-negative breast cancer cells like MDA-MB-231 and that RUNX3 might also exert tumor suppressor activity in these breast cancer cells (Lau et al., 2006). Although the mechanism remains unknown, in ERα-negative MDA-MB-231 cells, RUNX3 reduced the invasiveness and tumor forming potential of these cells (Lau et al., 2006), indicating a tumor suppressor role for RUNX3 in ERα-negative breast cancer. While down-regulation of the stability of ERα by RUNX3 likely is the major factor in its tumor suppressor activity in ERα-positive cancer cells, in ERα-negative cancer cells RUNX3 might cooperate with other transcription factors, such as FOXO3a or receptor-regulated Smads, to induce apoptosis by activating pro-apoptotic genes (Yamamura et al., 2006; Yano et al., 2006). Thus, it is likely that RUNX3, using different mechanisms, possesses tumor suppressor activity in both ERα-positive and -negative breast cancers,.

Enhanced ERα expression in normal breast epithelium is associated with increased risk of breast cancer (Khan et al., 1994; Shoker et al., 2000). RUNX3 might function as a “gate-keeper” and play a role in the onset of breast cancer by controlling the response to circulating estrogens through regulation of the cellular level of ERα. Inactivation of RUNX3 results in increased stability of ERα and enhanced cell proliferation in response to circulating estrogens. Since RUNX3 is frequently inactivated in primary breast tumors, restoring RUNX3 expression represents an attractive new therapeutic target with potential for treatment of ERα-positive breast cancer.

Supplementary Material

Figure S1 Immunohistochemical staining of PR and Her2/neu in serial sections were performed as described Figure 1c using anti-PR (A0098, Dako) or anti-Her/neu (MS-325, Thermo-Fisher) antibodies.

Figure S2 RUNX3 does not induce the cell death of MCF-7 cells. Cell death was assessed using the Multitox-Fluor cytotoxicity assay (G9200, Promega) according to the manufacturer's protocol. Vector-MCF-7 and RUNX3-MCF-7 cells were seeded in 96-well plate in hormone-free MEM media for 3 days and then treated with 100 pM E2 for another four days. Dead cells and total cells were measured at indicated time points. The ratio of relative dead cells to total cells at each time point was calculated and presented as percentage of the ratio to vector-MCF-7 cells at day 2 (as 100%).

Figure S3 RUNX3 does not inhibit ΔERE-luciferase reporter activity. MCF-7 cells were cultured in phenol red-free MEM with 10% double-stripped calf serum for three days followed by transfection with ΔERE-luciferase reporter and RUNX3 plasmid DNA. Twenty-four hours after transfection, cells were stimulated with vehicle or E2 (100 nM) for 24 hr. Luciferase activity was measured as described in Figure 3a.

Figure S4 RUNX3 inhibits E2-induced ERα activity in MCF-12A cells. MCF-12A cells were transfected with PI-9 luciferase reporter plasmid DNA and increasing amounts of plasmid DNA encoding RUNX3. Twenty-four hours after transfection, cells were stimulated with vehicle or E2 (100 nM) for 24 hr. Luciferase activity was measured and expressed as fold induction after normalization with Renilla luciferase.

Figure S5 RUNX3 inhibits the transcriptional activity of ERα in T47D cells. T47D cells stably expressing control vector or RUNX3 were stimulated with E2 (100 nM) for 24 hr. The expression levels of pS2 and NRIP1 mRNA were measured as described in Figure 3b. Expression levels of RUNX3 and ERα protein in the MCF-7 stable cell lines are shown in the right panels.

Figure S6 RUNX3 does not affect the level of ERα mRNA. MCF-7 cells were transfected with expression vector for RUNX3 as indicated. Quantitative real-time PCR with primers (Qiagen) spanning the cDNA of ERα was performed to measure mRNA levels of endogenous ERα.

Figure S7 RUNX3 shortens the half-life of ERα. Vector-MCF-7 and RUNX3-MCF-7 cells were treated with cycloheximide (CHX) (10 μg/ml) for various time points and immunoblotted for endogenous ERα (upper panel). Quantification of the results from three independent experiments is shown in the lower panel.

Figure S8 RUNX3 does not affect the half-life of Her2-/neu. Vector-MCF-7 and RUNX3-MCF-7 cells were treated with cycloheximide (CHX) (10 μg/ml) for various time points and immunoblotted for endogenous ERα (upper panel). Quantification of the results from three independent experiments is shown in the lower panel.

Figure S9 RUNX3 induces the degradation of ERα in the absence and presence of E2. MCF-7 cells were cultured in the presence or absence of estrogen (10−8M) and transfected with Myc-RUNX3. Forty-eight hours later, the expression level of ERα and RUNX3 were detected by immunoblotting with anti-ERα or anti-Myc antibodies. Tubulin is a loading control.

Figure S10 RUNX3 induces the degradation of ligand binding-deficient mutant of ERα. HEK293T cells were transfected with expression vectors for Flag-tagged WT or ligand binding defective mutant ERα (L525A) and increasing amounts of RUNX3. Forty hours after transfection, whole cell lysates were immunoblotted for indicated proteins.

Figure S11 Transcriptionally incompetent RUNX3 mutant induces the degradation of ERα. HEK293T cells were transfected with Flag-tagged ERα and Myc-tagged WT RUNX3 or RUNX3-R122C as indicated. Forty hours after transfection, whole cell lysates were immunoblotted for indicated proteins.

Acknowledgments

We thank W Xu for reagents; W Xu, A Nardulli and members in the Chen lab for discussion. This work is supported in part by fund provided by UIUC (to L.F.C.) and NIH grants DK-085158 (to L.F.C), CA116616 (to G.T. X.) and DK-071909 (to D. S.).

This project is supported in part by fund provided by UIUC (to L.F.C.) and NIH grants DK-085158 (to L.F.C), CA116616 (to G.T. X.) and DK-071909 (to D. S.). Y.H.T. is an A*STAR-Illinois Partnership fellow.

Footnotes

Conflict of interest: The authors declare no conflict of interest

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 Immunohistochemical staining of PR and Her2/neu in serial sections were performed as described Figure 1c using anti-PR (A0098, Dako) or anti-Her/neu (MS-325, Thermo-Fisher) antibodies.

Figure S2 RUNX3 does not induce the cell death of MCF-7 cells. Cell death was assessed using the Multitox-Fluor cytotoxicity assay (G9200, Promega) according to the manufacturer's protocol. Vector-MCF-7 and RUNX3-MCF-7 cells were seeded in 96-well plate in hormone-free MEM media for 3 days and then treated with 100 pM E2 for another four days. Dead cells and total cells were measured at indicated time points. The ratio of relative dead cells to total cells at each time point was calculated and presented as percentage of the ratio to vector-MCF-7 cells at day 2 (as 100%).

Figure S3 RUNX3 does not inhibit ΔERE-luciferase reporter activity. MCF-7 cells were cultured in phenol red-free MEM with 10% double-stripped calf serum for three days followed by transfection with ΔERE-luciferase reporter and RUNX3 plasmid DNA. Twenty-four hours after transfection, cells were stimulated with vehicle or E2 (100 nM) for 24 hr. Luciferase activity was measured as described in Figure 3a.

Figure S4 RUNX3 inhibits E2-induced ERα activity in MCF-12A cells. MCF-12A cells were transfected with PI-9 luciferase reporter plasmid DNA and increasing amounts of plasmid DNA encoding RUNX3. Twenty-four hours after transfection, cells were stimulated with vehicle or E2 (100 nM) for 24 hr. Luciferase activity was measured and expressed as fold induction after normalization with Renilla luciferase.

Figure S5 RUNX3 inhibits the transcriptional activity of ERα in T47D cells. T47D cells stably expressing control vector or RUNX3 were stimulated with E2 (100 nM) for 24 hr. The expression levels of pS2 and NRIP1 mRNA were measured as described in Figure 3b. Expression levels of RUNX3 and ERα protein in the MCF-7 stable cell lines are shown in the right panels.

Figure S6 RUNX3 does not affect the level of ERα mRNA. MCF-7 cells were transfected with expression vector for RUNX3 as indicated. Quantitative real-time PCR with primers (Qiagen) spanning the cDNA of ERα was performed to measure mRNA levels of endogenous ERα.

Figure S7 RUNX3 shortens the half-life of ERα. Vector-MCF-7 and RUNX3-MCF-7 cells were treated with cycloheximide (CHX) (10 μg/ml) for various time points and immunoblotted for endogenous ERα (upper panel). Quantification of the results from three independent experiments is shown in the lower panel.

Figure S8 RUNX3 does not affect the half-life of Her2-/neu. Vector-MCF-7 and RUNX3-MCF-7 cells were treated with cycloheximide (CHX) (10 μg/ml) for various time points and immunoblotted for endogenous ERα (upper panel). Quantification of the results from three independent experiments is shown in the lower panel.

Figure S9 RUNX3 induces the degradation of ERα in the absence and presence of E2. MCF-7 cells were cultured in the presence or absence of estrogen (10−8M) and transfected with Myc-RUNX3. Forty-eight hours later, the expression level of ERα and RUNX3 were detected by immunoblotting with anti-ERα or anti-Myc antibodies. Tubulin is a loading control.

Figure S10 RUNX3 induces the degradation of ligand binding-deficient mutant of ERα. HEK293T cells were transfected with expression vectors for Flag-tagged WT or ligand binding defective mutant ERα (L525A) and increasing amounts of RUNX3. Forty hours after transfection, whole cell lysates were immunoblotted for indicated proteins.

Figure S11 Transcriptionally incompetent RUNX3 mutant induces the degradation of ERα. HEK293T cells were transfected with Flag-tagged ERα and Myc-tagged WT RUNX3 or RUNX3-R122C as indicated. Forty hours after transfection, whole cell lysates were immunoblotted for indicated proteins.

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