Regulation of breast cancer metastasis by Runx2 and estrogen signaling: the role of SNAI2
© Chimge et al.; licensee BioMed Central Ltd. 2011
Received: 6 July 2011
Accepted: 9 December 2011
Published: 9 December 2011
In contrast to its role in breast cancer (BCa) initiation, estrogen signaling has a protective effect in later stages, where estrogen receptor (ER)α loss associates with aggressive metastatic disease. We asked whether the beneficial effect of estrogen signaling in late-stage BCa is attributable to the recently reported estrogen-mediated antagonism of the pro-metastatic transcription factor Runx2.
MCF7/Rx2dox breast cancer cells were engineered with a lentivirus expressing Runx2 in response to doxycycline (dox). Cells treated with dox and/or estradiol (E2) were subjected to genome-wide expression profiling, RT-qPCR analysis of specific genes, and Matrigel™ invasion assays. Knockdown of genes of interest was performed using lentiviruses expressing appropriate shRNAs, either constitutively or in response to dox. Gene expression in BCa tumors was investigated using a cohort of 557 patients compiled from publicly available datasets. Association of gene expression with clinical metastasis was assessed by dichotomizing patients into those expressing genes of interest at either high or low levels, and comparing the respective Kaplan-Meier curves of metastasis-free survival.
Runx2 induced epithelial-mesenchymal transition (EMT) evidenced by acquisition of a fibroblastic morphology, decreased expression of E-cadherin, increased expression of vimentin and invasiveness. Runx2 stimulated SNAI2 expression in a WNT- and transforming growth factor (TGF)β-dependent manner, and knockdown of SNAI2 abrogated the pro-metastatic activities of Runx2. E2 antagonized the pro-metastatic activities of Runx2, including SNAI2 upregulation. In primary BCa tumors, Runx2 activity, SNAI2 expression, and metastasis were positively correlated, and SNAI2 expression was negatively correlated with ERα. However, the negative correlation between SNAI2 and ERα in bone-seeking BCa cells was weaker than the respective negative correlation in tumors seeking lung. Furthermore, the absence of ERα in primary tumors was associated with lung- and brain- but not with bone metastasis, and tumor biopsies from bone metastatic sites displayed the unusual combination of high Runx2/SNAI2 and high ERα expression.
E2 antagonizes Runx2-induced EMT and invasiveness of BCa cells, partly through attenuating expression of SNAI2, a Runx2 target required for mediating its pro-metastatic property. That ERα loss promotes non-osseous metastasis by unleashing Runx2/SNAI2 is supported by the negative correlation observed in corresponding tumors. Unknown mechanisms in bone-seeking BCa allow high Runx2/SNAI2 expression despite high ERα level
Metastasis of primary tumors to distant sites is a complex process that involves a sequence of interdependent events including intravasation, survival within the circulation, extravasation, and colonization. Epithelial-mesenchymal transition (EMT) has been strongly implicated in metastasis [1, 2]. During EMT, epithelial cells dissociate from each other, in part due to loss of E-cadherin expression, upregulate mesenchymal markers, acquire a fibroblast-like morphology, reorganize their cytoskeleton, and become more motile and invasive [1, 2]. Several transcription factors, including members of the SNAI family have been shown to promote EMT and thus tumor dissemination [3–5]. Bone metastasis is a frequent complication of breast cancer (BCa), with distinct gene signatures defining bone-seeking tumors [6–9].
Prolonged exposure to estradiol (E2) is associated with an increased risk of BCa [10–14]. The mechanisms through which estrogens contribute to BCa initiation and progression are complex and implicate estrogen receptor (ER)-mediated genomic and nongenomic signaling as well as the action of genotoxic estrogen metabolites . In contrast to E2-mediated carcinogenesis, the presence of ERα is a favorable prognostic marker associated with less invasive tumors, and those negative for ERα are more aggressive . A randomized clinical trial of postmenopausal women receiving equine estrogen treatment revealed a decrease in BCa incidence  and low-dose estradiol treatment has been proposed as a treatment modality for advanced ERα-positive BCa that does not respond to aromatase inhibition . Indeed, introduction of ERα into ERα-negative BCa cells attenuated their pro-cancerous properties in vitro [18–21]. Thus, anti-estrogen therapy for BCa patients, while antagonizing the oncogenic properties of estrogens, may inadvertently result in loss of their anti-metastatic property. Better understanding of mechanisms underlying the beneficial role of estrogen signaling in advanced disease may inform the development of novel therapeutic approaches and improved treatment plans for BCa patients.
Runx2 is a lineage-specific transcription factor with crucial roles in both bone biology and carcinogenesis [22–24]. During development Runx2 is involved in the process of osteogenesis. Targeted disruption of Runx2 in mice leads to failure of osteoblast differentiation and bone formation [25, 26], and Runx2 haploinsufficiency in humans results in the skeletal disorder cleidocranial dysplasia, with a similar phenotype observed in Runx2 haploinsufficient mice . Although Runx proteins have tumor suppressor properties , recent studies assigned a role for Runx2 in promoting breast and prostate cancer metastasis [27–32]. Thus, Runx2 and E2 signaling play dual roles in BCa, with each functioning to either promote or suppress tumor progression. The mechanisms underlying these contrasting manifestations in cancer are poorly understood.
We previously showed that in the presence of ligand, ERα physically binds Runx2 and inhibits expression of several Runx2 target genes ; our recent study revealed that in MCF7 BCa cells E2 independently regulated about half of the Runx2-responsive genes . We therefore hypothesized that gene(s) stimulated by Runx2 and inhibited by E2 may contribute to manifestations of the pro-metastatic or tumor suppressor functions of Runx2 and E2, respectively. Using a combination of tissue culture modeling and bioinformatics analysis of gene expression in BCa biopsies, we present evidence suggesting that SNAI2/SLUG plays a role in mediating the pro- and anti-metastatic effects of Runx2 and E2, respectively.
Materials and methods
Cell culture assays and reagents
MCF7 and T47D BCa cells were obtained from the American Type Culture Collection (Rockville, MD, USA). MCF7/Rx2dox cells, engineered to express Runx2 in response to doxycycline (dox) , were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 50 μg/ml Hygromycin B (GIBCO, Carlsbad, CA). Data presented herein were obtained from cells cultured in phenol red-free DMEM containing 10% charcoal-stripped FBS (Hyclone, South Logan, UT), 0.5 μg/ml dox (Calbiochem, San Diego, CA) and/or 10 nM E2 (Sigma, St Louis, MO). For invasion assays, cells further engineered to constitutively express luciferase were placed in Matrigel™-containing inserts (BD Biosciences, Bedford, MA) and invasion was assessed based on luciferase activity in cells that had crossed the membrane . The Wnt inhibitor ICG-001 was synthesized as previously described . Mouse monoclonal anti-SNAI2 and anti-Runx2 antibodies were from Millipore (Billerica, MA) and Invitrogen (Grand Island, NY), respectively. Anti-TGF-β type I receptor (TGFBRI) blocking antibody (sc-398) and negative control antibody (sc-2027) were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).
Real-time quantitative RT-qPCR analysis
Total RNA was extracted using Aurum™ Total RNA Mini-Kit (BioRad, Hercules, CA) and cDNA was synthesized with iScript Reverse Transcription Kit (BioRad). RT-qPCR was carried out in triplicate using iQ™ SYBR Green Supermix (BioRad) and a CFX96 qPCR machine (BioRad). All transcript levels were normalized to that of GAPDH. Primers used for PCR are listed in Additional file 1.
T47D/shRx2dox cells, conditionally expressing shRNA to silence Runx2 in response to dox, were previously described . For SNAI2 silencing, specific Mission-shRNA lentiviral plasmids (SHCLNG) and a negative control shRNA plasmid (SHC002) were purchased from Sigma and packaged as described  using HEK293T cells and the helper plasmids pMD.G1 and pCMV.R8.91. Viral particles were used for transduction of MCF7/Rx2dox cells followed by selection with 1 μg/ml Puromycin (Invitrogen, Carlsbad, CA). Of five shRNAs tested, two were found to reduce SNAI2 expression by > 75% and the corresponding cells were propagated as described above. shRNA sequences used in this study are listed in Additional file 1.
For primary analysis of gene expression profiles in clinical samples, we compiled microarray data from 557 BCa patients by combining three Gene Expression Omnibus (GEO) datasets, GSE2034, GSE2603 and GSE12276 [37–39]. These datasets were generated using Affymetrix chips HG-U133A (GSE2034 and GSE2603) and HG-U133plus2 (GSE12276). The Affymetrix CEL files were downloaded into Partek, normalized using the RMA (Robust Multiarray Averaging) method and filtered for common probes using interplatform comparison. The data were standardized by centering and scaling and patients were dichotomized into low and high expressing groups with regard to the indicated genes of interest. Dichotomizing of the tumors according to the ESR1 Affymetrix probe '205225_at' was consistent with the histological definition of their ER status in 329 of 368 cases (89.4%) for which pathological annotation was available, and with the molecular definition of their ER status in 177 of 189 cases (93.7%) where it was based on different processing of the microarray data . Clinical features, including hormonal status, metastasis-free survival time and sites of metastasis, have been compiled from the supplementary information of the published studies [9, 37]. For each patient, metastasis-free survival was defined as the time interval between surgery and the diagnosis of metastasis. Only tumors with unambiguous information about sites of metastasis were included in the cohort. For metastatic tissue analyses we used the microarray data from 58 archival human breast carcinoma metastasis specimens deposited into GEO under accession number GSE14020 . Because all the data were from publicly available resources, we did not require ethical approval to carry out this study.
Survival analyses and hierarchical clustering were performed using Partek Genomics Suite 6.6 and the correlation study was performed using Graphpad. Functional annotation of gene groups was performed using both the web-based Database for Annotation, Visualization, and Integrated Discovery (DAVID) system  and the Ingenuity Pathways Analysis (IPA™) package.
Metastasis-related genes in BCa cells are stimulated by Runx2 and inhibited by E2
Functional Annotation Clustering of Genes Independently Regulated by Runx2 and E2 in MCF7/Rx2dox Cells
Annotation Cluster 1
Enrichment Score: 4.50
GO:0048514~blood vessel morphogenesis
GO:0001568~blood vessel development
Annotation Cluster 2
Enrichment Score: 4.48
GO:0051674~localization of cell
Genes Independently Regulated by Runx2 and E2 in MCF7/Rx2dox Cells
Functional link between Runx2, E2 and SNAI2 in BCa
E2 antagonizes Runx2-induced EMT and invasiveness in vitro
We further assessed expression of EMT and motility-related genes seven days after Runx2 induction in MCF7/Rx2dox cells. As shown in Figure 2B, this prolonged induction resulted in a more complete EMT. Not only were SNAI2 and S100A4 stimulated, CDH1 was downregulated and VIM was upregulated. Finally, microscopic examination of cultures seven days after Runx2 induction demonstrated a fibroblast-like and scattered morphology as compared to control cultures (Figure 2C). Thus, the partial EMT indicated by increased SNAI2 and S100A4 expression on day 2 (Figure 2A) developed into a more comprehensive EMT phenotype by day 7. More importantly, E2 significantly antagonized most of the Runx2-induced EMT characteristics. It blunted the Runx2-mediated effects on SNAI2 and CDH1 expression, and attenuated the Runx2-mediated effects on the expression of VIM and S100A4 (Figure 2B), as well as its effects on cell morphology (Figure 2C).
Finally, we measured the effects of Runx2 and E2 on BCa cell invasiveness. MCF7/Rx2dox cells were transduced with lentiviruses constitutively expressing firefly luciferase and placed on top of Matrigel™ membranes within BD Biocoat™ inserts. Luciferase activity associated with cells that had crossed the membrane, a measure of invasiveness , was two-fold higher in cultures treated with dox to induce Runx2 as compared to controls, while co-treatment with E2 attenuated the Runx2-mediated invasiveness (Figure 2D). E2 alone had little, if any, effect on MCF7/Rx2dox cell invasiveness through Matrigel™.
SNAI2 plays a critical role in Runx2-mediated MCF7 cell invasion
WNT and TGFβ signaling are required for Runx2-mediated stimulation of SNAI2 expression
Negative association of SNAI2 with metastasis-free survival in BCa patients
Anomalous high expression of SNAI2, Runx2 and ERα in bone-seeking BCa tumors
Suppression of SNAI2 expression in BCa cells by E2 (Figure 2A) may be related to the protective effect of ERα against metastasis. Indeed, consistent with previous reports [15, 17–21], tumors with high ESR1 expression were less likely to metastasize in our cohort, particularly during the initial 20 to 40 months after surgery (Figure 5E). Analysis of the association between ESR1 expression and metastasis to specific sites, however, revealed an interesting exception. Whereas high ESR1 expression was negatively correlated with lung and brain metastasis (Figure 5F-G), it positively correlated with bone metastasis (Figure 5H). Thus, unleashing SNAI2 gene expression by loss of ERα is a plausible mechanism promoting metastasis in general; however, high expression of SNAI2 in cells that metastasize to bone occurs despite high ESR1 expression. Furthermore, SNAI2 expression was associated with lung and brain metastasis only for tumors expressing low levels of ESR1 (Figure 5J-K), whereas bone metastasis was associated with SNAI2 for tumors expressing ESR1 at either low or high levels (Figure 5L), suggesting weaker control of SNAI2 by ERα in bone-seeking tumors. Consequently, the combination of low ESR1 and high SNAI2 expression was predominant within lung and brain-seeking tumors (Figure 5O-P), whereas bone-seeking tumors were mostly those expressing both ESR1 and SNAI2 at high levels (Figure 5Q). Consistent with these observations, the negative correlation between ESR1 and SNAI2 expression was weaker in bone compared to lung-seeking tumors (Figure 5R-U; brain-seeking tumors were not analyzed because only eight of them were ER-positive). Finally, we investigated Runx2, SNAI2 and ESR1 gene expression in 58 BCa tumors derived from metastatic sites: 16 from bone, 19 from brain, 18 from lung and 5 from liver . The standardized signal intensities plot showed preferential upregulation of Runx2, SNAI2 and ESR1 in the bone-derived biopsies (Figure 5V). The unusual concomitant high expression of ESR1 and Runx2/SNAI2 may reflect dominant regulation of Runx2/SNAI2 by E2-resistant signaling pathways that promote BCa bone metastasis.
Apart from promoting BCa progression, estrogen signaling has paradoxical anti-metastatic properties [15, 17, 19–21]. Potentially contributing to this, E2 antagonized the transcription factor Runx2, whose role in metastasis is being increasingly recognized [22, 24, 30–32]. Specifically, Runx2 promoted EMT and invasion of BCa cells in vitro, and this was antagonized by E2 (Figure 2). At the center stage of the opposing effects of Runx2 and E2 signaling on EMT and invasion was the transcription factor SNAI2. Consistent with previous reports [30, 48, 49], SNAI2 was stimulated by Runx2 and inhibited by E2 (Figure 1A and 2A); Runx2 no longer enhanced EMT and invasion after SNAI2 knockdown; SNAI2 expression in BCa biopsies positively correlated with a metagene that reports on Runx2 activity and negatively correlated with ERα mRNA (Figure 1B), and expression of SNAI2 was associated with BCa metastasis in general and bone metastasis in particular (Figure 5A-D).
SNAI2 likely plays different roles during various stages of BCa progression, and its ability to transcriptionally repress E-cadherin and induce EMT is well documented [3–5, 49–51]. The role of SNAI2 downstream of Runx2 in BCa is reminiscent of the role of its homologue, SNAI1, in EMT and metastasis of breast, ovarian, colon, lung and squamous cell carcinomas . However, SNAI1 is not stimulated by Runx2 in either MCF7 BCa  or C4-2B PCa cells . Instead, it is SNAI2 that is strongly stimulated by Runx2 in BCa and PCa cells (Figure 2). Furthermore, SNAI2 and not SNAI1 was repressed by E2 in our study (data not shown), and SNAI2 and not SNAI1 exhibited an inverse correlation with ERα and E-cadherin in human BCa tumors . Like Runx2, Twist1 has recently been shown to induce SNAI2 expression, and SNAI2 was essential for Twist1-mediated EMT of human mammary epithelial cells . Thus, depending on context, either SNAI1 or SNAI2 regulate BCa metastasis, with the latter mediating the pro-metastatic activity of Runx2 and Twist1 as well as the anti-metastatic activity of E2.
It remains to be investigated whether Runx2 directly regulates SNAI2 transcription. On the one hand, there are several Runx-binding motifs upstream of the SNAI2 transcription start site (Additional file 3: Supplemental Figure 1A). However, Runx2 ChIP-seq analysis in C4-2B cells, where SNAI2 expression is also stimulated by Runx2 (Figure 1B), did not suggest occupancy of Runx2 at these SNAI2 upstream Runx motifs . Instead, we believe that Runx2 indirectly stimulates SNAI2 expression via modulation of ETS signaling. This hypothesis is based on the observation that Runx2 down-regulated expression of SPDEF (Additional file 3: Supplemental Figure 1B and ), a transcriptional inhibitor belonging to the ETS family, which suppresses tumor progression and metastasis  in part through direct inhibition of SNAI2 expression . Indeed, our Runx2 ChIP-seq analysis in C4-2B/Rx2dox cells  demonstrated strong occupancy of the SPDEF transcription start site by Runx2 (Additional file 3: Supplemental Figure 1C). We are currently investigating how such Runx2 occupancy represses SPDEF transcription, and which of the many ETS sites upstream of the SNAI2 transcription start site (Additional file 3: Supplemental Figure 1A) mediate its repression by SPDEF and/or its activation by other members of the ETS family. Regardless of the precise mechanism by which Runx2 controls SNAI2, expression of the two is tightly correlated in BCa tumors (Figure 1D). In fact, much of the reported association between Runx2 expression and BCa metastasis  is attributable to SNAI2. Indeed, the results of our independent analysis validating the association of Runx2 with metastasis (Additional file 4) are remarkably similar to those demonstrating the association of SNAI2 with metastasis (Figure 5).
The anti-Runx2/SNAI2, anti-metastatic properties of E2 signaling in BCa cells may be relevant for early in vivo metastatic events (Figure 5E), and possibly to targeting BCa metastasis to non-osseous tissues (Figure 5F-G). However, they appear less relevant for bone metastasis. Whereas SNAI2 expression is associated with bone-seeking primary BCa tumors and BCa bone metastasis even more strongly than with lung or brain metastasis, ERα is negatively associated with only non-osseous metastasis (Figure 5). In fact, consistent with previous reports [9, 55], the correlation between ERα expression and bone metastasis was positive, not negative in our cohort (Figure 5H); BCa tumors that metastasized to bone were mostly ER-positive (Figure 5Q) and BCa bone metastases co-expressed high levels of Runx2/SNAI2 and ERα (Figure 5V). The bone-seeking property that ERα bestows on BCa cells may be counteracted during the first two years after surgery by the general anti-metastatic property of estrogens, resulting in a total neutral effect, but at the end it is the former that prevails (Figure 5H). Possibly, activation of bone-seeking pathways in primary BCa cells, such as Src1 [8, 9], results in the stimulation of SNAI2 and other Runx2-regulated genes despite high levels of ERα.
Runx2 stimulates EMT and invasiveness in MCF7 BCa cell cultures, adding to the mounting evidence for its role in metastasis. Our data suggest that E2 attenuates BCa metastasis by antagonizing Runx2. Indeed, EMT and invasiveness of MCF7 cells were severely compromised by E2 specifically after induction of Runx2. The linkage between the pro- and anti-metastatic properties of Runx2 and E2, respectively, is attributable in part to SNAI2, which is upregulated by Runx2 and downregulated by E2. Furthermore, we observe a strong positive association between Runx2 and SNAI2 expression, a negative association between ESR1 and SNAI2, and a positive correlation between SNAI2 expression and metastasis in BCa tumors. These observations are consistent with the hypothesis that unopposed stimulation of Runx2 target genes such as SNAI2 in ER-negative tumors contributes to their aggressive metastatic phenotype. However, the relationship between ER expression and metastasis is more complicated in bone-seeking tumors. Here, the negative correlation between SNAI2 and ER is weak, and the anti-metastatic property of ER is likely masked by an opposite property, which ultimately prevails. Mimicking ER signaling to specifically antagonize Runx2 and SNAI2 offers a research avenue towards the development of novel therapeutic approaches for the management of BCa patients who fail first line therapy.
Database for Annotation: Visualization and Integrated Discovery
estrogen receptor alpha
Ingenuity Pathway Analysis
Robust Multiarray Averaging
snail homolog 2
SAM pointed domain-containing ets transcription factor.
We thank USC's Dr. Susan Groshen at the Norris Cancer Center Biostatistics Services Core for guidance with data analysis, Ms. Meng Li at the Bioinformatics Service Program, Norris Medical Library for helpful discussions, and Ms. Yunfan Shi for excellent technical support. This work was funded by NIH grants RO1 DK071122 and DK071122S1 to BF, who holds the J. Harold and Edna L. LaBriola Chair in Genetic Orthopedic Research at USC. ZB is funded by NIH grant RO1 HL089445 and holds the Ralph Edgington Chair in Medicine, DT holds the Priscilla and Art Ulene Chair in Women's Cancer.
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