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The transcription factor Runt-related transcription factor 1 (RUNX1) is critical for the earliest steps of hematopoiesis. RUNX1 was originally identified as a gene fusion in acute myeloid leukemia (AML) and thus has garnered heavy attention as a tumor suppressor in hematopoietic malignancies. However, RUNX1 is also strongly expressed in breast epithelia and may be misregulated during tumorigenesis. Here, I discuss our recent work implicating RUNX1 in proliferation control during breast epithelial-acinar morphogenesis. My goal is to place these findings in the context of a handful of other reports, which together argue that RUNX1 could act as a tumor suppressor gene in breast cancer. Testing this hypothesis requires focused in vivo studies, because the major commercial platform for global mRNA expression profiling does not reliably reflect RUNX1 levels. Our in vitro results indicate that hyperproliferation in RUNX1-deficient breast epithelia relies on another family of transcription factors, the Forkhead box O (FOXO) proteins. FOXOs could, therefore, represent a synthetic-lethal target for RUNX1-deficient tumors if the hypothesized link to breast cancer is correct.
The RUNX gene family is composed of three transcription factors: RUNX1 (also known as AML1, PEBP2αB, CBFA2), RUNX2 (also known as AML3, PEBP2αA, CBFA1) and RUNX3 (also known as AML2, PEBP2αC, CBFA3). All three RUNX isoforms bind a common TG(T/C)GGT consensus binding site1 and regulate important developmental gene-expression programs. However, gene targeting of individual isoforms in mice yields strikingly different phenotypes, and only the Runx1−/− genotype is embryonic lethal.2–4 This suggests that there is minimal redundancy between RUNX family members, and one cannot compensate for loss of the other during development.
Each RUNX family member has been strongly linked to cancer,5,6 but the precise roles and cellular contexts reported thus far differ depending on the isoform: RUNX1 is a tumor suppressor in AML,7,8 RUNX2 promotes bone metastasis in breast cancer,9,10 and RUNX3 is a tumor suppressor in gastric cancer.4 Remarkably little work has been done to determine whether certain RUNX isoforms are important for cancers unrelated to the site of their knockout phenotype.
One prime location to explore such a possibility is in the breast.11 Ductal outgrowths in mouse mammary glands do not develop until ~3 weeks after birth,12 which means that germline knockouts causing early lethality will overlook potential phenotypes in the expanding ductal tree. Recently, we stumbled upon RUNX1 as an important regulator of breast tissue morphogenesis while studying the single-cell regulation of another important class of tumor suppressors, the FOXOs.13–16 By monitoring the cell-to-cell fluctuations in expression for a panel of FOXO-regulated genes,17 we found that roughly half of the measured FOXO targets were coregulated by RUNX1.14 Another RUNX isoform, RUNX2, had been previously examined in the same 3D culture model that our study used.18 However, RUNX2 is more important for luminal cells that express milk proteins,9 and the 3D culture model involves a breast epithelial cell line (MCF10A) with a basal-like molecular signature.19,20 Accordingly, we found that endogenous RUNX1 mRNA was expressed at > 15-fold higher levels than RUNX2,14 suggesting it was the dominant isoform for this subtype.
In 3D culture, knockdown of RUNX1 caused a hyperproliferative phenotype that required normal FOXO function. Reducing RUNX1 levels created a more oxidative cellular environment that could only be withstood through the adaptive responses of FOXO-mediated gene expression.21,22 Adaptive epigenetic changes have been described in other cancer contexts, prompting the suggestion that such changes represent good therapeutic targets.23,24 Indeed, we found that compound deficiency in RUNX1 and FOXO signaling caused an “oxidative catastrophe,” which induced a proliferation arrest that restored normal acinar size and shape. RUNX1 knockdown did not cause a compensatory increase in RUNX2 levels, but instead seemed to create a synthetic-lethal dependency on FOXOs. Importantly, in a retrospective analysis of estrogen receptor-, progesterone receptor- and HER2-negative (“triple-negative”) breast cancers, we observed that RUNX1 mRNA expression was strongly anticorrelated with FOXO1 expression. This suggests that breast cancers with reduced RUNX1 levels may rely on increased FOXO activity to support tumor progression.
Our work adds to a smattering of reports that implicate RUNX1 downregulation or loss as a critical event for certain subtypes of breast cancer. RUNX1 protein is strongly detected in both luminal and basal cells in normal breast tissue (Fig. 1A), but expression is weak or absent in many breast cancers (Fig. 1B).25 Early work with various microarray platforms identified RUNX1 downregulation as part of a 17-gene signature that predicts breast cancer metastasis.26 The predictive ability of RUNX1 was originally suggested to reflect decreased tumor surveillance by inflammatory cells. However, this interpretation should be reevaluated given that inflammation is now known to promote various tumors,27 and many of the mostaggressive breast cancers have a strong inflammatory component.28 Algorithms predicting breast-cancer metastasis may instead use RUNX1 because of downregulation in the primary tumor.
It has been strongly argued that a key step in metastasis of epithelial cancers is loss of E-cadherin (CDH1).29 RUNX1 binds to the CDH1 promoter and acts as a positive regulator of E-cadherin expression.30 When RUNX1 expression is partly retained in primary breast tumors, its expression coincides with KRT5,31 a basal cytokeratin that is used as a marker for basal-like carcinomas.32 Accordingly, basal-like carcinoma cell lines, such as MDA-MB-231,19 express low, but detectable, levels of RUNX1.30 Kadota et al. recently showed that the RUNX1 genomic locus was specifically lost in a variant of Ras-transformed MCF10A cells that formed malignant, poorly differentiated tumors in mice. This group went on to show in clinical specimens that RUNX1 expression levels decrease with increasing breast tumor grade. However, the overall effect size was modest, possibly because the authors did not separate the cases based on the known molecular subtypes of breast cancer.34–36 Taken together, the evidence above predicts that the most-striking difference in RUNX1 levels would be observed in basal-like carcinomas, the subtype with the worst clinical prognosis.35 This prediction is supported by our retrospective analysis of RUNX1 expression in triple-negative breast cancers,14 which are enriched in basal-like carcinomas.37
Considering the thousands of breast tumors that have been molecularly characterized,36 why has RUNX1 not emerged as a gene of interest? Part of the problem may be subset selection: only ~10% of all breast-cancer cases are triple-negative,38 and so most expression-profiling studies are underpowered to detect differences within this subgroup. Yet, a more realistic (and, perhaps, troublesome) explanation may lie in the details of the commercial microarrays that dominate the expression-profiling literature.
The Affymetrix Human Genome U133 Set monitors RUNX1 with seven different probesets, all of which are retained on the modern GeneChip® Human Genome U133 Plus 2.0 Array. One probe-set (209360_s_at, “Probe209”) consistently yields fluorescence readings that are ~10-fold higher than any other RUNX1 probeset. RUNX1 measurements based on Probe209 are reproducible between biological replicates and show variation across different conditions (for example, Fig. 2A, left). On the surface, Probe209 appears to be the best RUNX1 probeset. The only problem is that Probe209 does not measure RUNX1 expression.
I came across this difficulty during our study of RUNX1 and FOXOs.14 Using an earlier microarray data set characterizing the time course of gene expression during 3D culture,39 I observed an interesting time-dependent pattern for Probe209 (Fig. 2A, left). Measurements with the probeset increased acutely on the days immediately before our single-cell experiments on day 10. This appeared to create an opportunity for a transient collaboration with FOXOs, whose expression progressively increased during morphogenesis (Fig. 2B and not shown).
When I attempted to validate the Probe209-based data by quantitative PCR (qPCR), however, I observed a qualitatively different pattern of RUNX1 expression (Fig. 2A, right). A similar discrepancy was not observed with FOXO1, where the brightest and most-reproducible probeset (202724_s_at, “Probe202”) largely agrees with independent qPCR data (Fig. 2B). No other RUNX1 probe-set matched the Probe209 pattern, but these alternate probesets were not reliably detected because of their dramatically lower sensitivity. Thus, without qPCR verification, I would have been inclined to believe that Probe209 was the best microarray-based measurement of RUNX1 levels.
For this Extra View, I further explored the validity of Probe209 by analyzing its general performance in a recent expression-profiling study of AML samples (GEO #GSE14471).40 RUNX1 is abundantly expressed in peripheral blood,41 and regulation of RUNX1 is sporadically disrupted in AML because of a recognized chromosomal translocation.8 The AML samples thus provided a source of true RUNX1 expression whose sample-to-sample variations could be used as a test bed to gauge concordance among RUNX1 probesets.
Across 111 independent AML samples, I found that Probe209 did not significantly correlate with any other RUNX1 probeset (median RPearson = 0.11, range: 0.02–0.28; Bonferroni-corrected α = 0.05). Conversely, the other six RUNX1 probesets were all positively correlated with one another (median RPearson = 0.53, range: 0.26–0.73). Taken together with the qPCR discrepancy, I conclude that Probe209 does not accurately reflect RUNX1 levels despite reproducible variation and strong hybridization signals across samples.
Nonetheless, we managed to extract a clear anticorrelation between RUNX1 and FOXO1 expression14 by using clinical breast cancer samples (GEO #GSE6861) measured with another Affymetrix platform. The GeneChip® Human X3P Array is designed for archival tissue samples and contains many additional RUNX1 probesets. One in particular (g1932819_3p_a_at, “ProbeX3P”) yields fluorescence intensities several-fold greater than the weak probesets on the U133 arrays. Importantly, ProbeX3P correlates strongly with many other new RUNX1 probesets (not shown). ProbeX3P may, therefore, represent the high-sensitivity probeset of choice for tracking RUNX1 in tissues where expression levels are modest.
ProbeX3P was also positively correlated with one weak probeset that is common to both the X3P and U133 platforms (210805_3p_x_at on the X3P Array and 210805_x_at on the U133 Array, “Probe210”) (Fig. 2C). The observed correlation provided the opportunity to extend our reported RUNX1-FOXO1 anticorrelation14 to other breast cancer data sets that had been collected on the U133 platform. Using an independent set of 45 triple-negative tumors (GEO #GSE2109), I observed hints of an anticorrelation between RUNX1 Probe210 and FOXO1 Probe202 (Fig. 2D). I predict that the anticorrelation would have been stronger if more of the samples had not plateaued at the Probe210 detection limit. (Note the cluster of samples in Fig. 2D with log2 relative expression of Probe210 between −4 and −5, which is essentially background fluorescence). Strikingly, using the same data set, one would have reached precisely the opposite conclusion by selecting RUNX1 Probe209 for analysis (Fig. 2E).
In the future, the peculiarities of individual probesets may become progressively less relevant with the emergence of new technologies, such as RNA-seq.42 However, until then, the reliability of genome-wide hybridization probes should not be taken for granted. Neither should the RUNX1 Probe209 discrepancy be forgotten, considering that there are currently over 85,000 microarrays deposited on NCBI GEO that contain the probeset. This has important implications for retrospective and meta-analyses that seek to mine existing data sets or fuse them together for new insights.26,36
More in vivo work should be done to explore the importance of RUNX1 in non-hematopoietic contexts. Many tissues express RUNX1 transcripts,41 but functional studies have largely focused on its role during early hematopoiesis and in various hematologic malignancies.5,6,43,44 Mice harboring floxed Runx1 alleles have been available for some time,45,46 and tissue-specific knockouts have thus far revealed important roles for Runx1 in skin,47 muscle46 and nociceptive sensory neurons.48 Of great interest would be to cross Runx1F/F mice with transgenic strains expressing MMTV-Cre, which drives recombination in the mammary gland.49 Normal gland development in this context would raise the possibility that Runx1 is specifically important during tumorigenesis, which could be explored in a number of murine breast-cancer models.50
If Runx1 loss in the mammary gland were to accelerate cancer progression in mice, it would warrant a more careful analysis of RUNX1 expression in human basal-like carcinomas. The results from our recent publication suggest that RUNX1-deficient tumors (Fig. 1B) would specifically require FOXOs for continued proliferation.14 In support of this prediction, a report that appeared immediately after ours showed that FOXO signaling may be essential for up to 40% of human AML cases.51 Such reliance could create an opportunity to develop cytostatic therapies that block FOXO-dependent oxidative stress pathways. There is already clinical precedent for this type of non-oncogene addiction in breast cancer.52–55
K.A.J. is supported by the National Institutes of Health Director's New Innovator Award Program (1-DP2-OD006464), the American Cancer Society (120668-RSG-11-047-01-DMC), the Pew Scholars Program in the Biomedical Sciences, and the David and Lucile Packard Foundation.