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: Breast cancer remains the most common malignant disease in women worldwide. Despite advances in detection and therapies, studies are still needed to understand the mechanisms underlying this cancer. Cancer stem cells (CSC) play an important role in tumor formation, growth, drug resistance, and recurrence. Here, it is demonstrated that the transcription factor RUNX1, well known as essential for hematopoietic differentiation, represses the breast cancer stem cell (BCSC) phenotype and suppresses tumor growth . The current studies show that BCSCs sorted from premalignant breast cancer cells exhibit decreased RUNX1 levels, whereas ectopic expression of RUNX1 suppresses tumorsphere formation and reduces the BCSC population. RUNX1 ectopic expression in breast cancer cells reduces migration, invasion, and tumor growth (57%) in mouse mammary fat pad. Mechanistically, RUNX1 functions to suppress breast cancer tumor growth through repression of CSC activity and direct inhibition of ZEB1 expression. Consistent with these cellular and biochemical results, clinical findings using patient specimens reveal that the highest RUNX1 levels occur in normal mammary epithelial cells and that low RUNX1 expression in tumors is associated with poor patient survival. IMPLICATIONS: The key finding that RUNX1 represses stemness in several breast cancer cell lines points to the importance of RUNX1 in other solid tumors where RUNX1 may regulate CSC properties.

Pur␤ is a gene regulatory factor belonging to a family of highly conserved nucleic acid-binding proteins related by their ability to preferentially bind single-stranded DNA or RNA sequences rich in purine nucleotides. In conjunction with Pur␣, Pur␤ has been implicated in transcriptional and translational repression of genes encoding contractile proteins found in the heart and vasculature. Although several models of sequence-specific DNA recognition, strand separation, and activator inhibition by oligomeric Pur␣ and Pur␤ have been proposed, it is currently unclear whether protein-protein interaction is a prerequisite to, or a consequence of nucleic acid binding. In this study, a recombinant protein purification scheme was devised to yield homogenous mouse Pur␤ devoid of nucleic acid. Recombinant Pur␤ was then subjected to light scattering and analytical ultracentrifugation analyses to assess the size, shape, and oligomeric state of the purified protein in solution. Results of laser light scattering and sedimentation velocity experiments indicated that Pur␤ reversibly self-associates in the absence of nucleic acid. Both approaches independently showed that the hydrodynamic shape of the Pur␤ homodimer is markedly asymmetric and non-spherical. Sedimentation velocity analyses indicated that dimeric Pur␤ has a sedimentation coefficient of 3.96 Svedberg, a frictional coefficient ratio (ƒ/ƒ 0 ) of 1.60, and a hydrodynamic radius of 4.43 nm. These values were consistent with those determined by independent dynamic light scattering studies. Sedimentation equilibrium analyses confirmed that Pur␤ self-associates in a reversible monomer-dimer equilibrium characterized by a K d ‫؍‬ 1.13 ؎ 0.27 M.Pur␣ and Pur␤ are members of a highly conserved family of nucleic acid-binding proteins related by primary structure and a propensity to interact with single-stranded DNA (ssDNA) 2 or RNA sequences rich in purine nucleotides (for review, see Ref.

Expression of the smooth muscle α-actin gene in growth-activated vascular smooth muscle cells and stromal fibroblasts is negatively regulated by members of the Pur family of single-stranded DNA/RNA-binding proteins. In particular, Purα and Purβ are postulated to repress transcription by forming helix-destabilizing complexes with the sense strand of an asymmetric polypurine-polypyrimidine tract containing a canonical MCAT enhancer motif in the 5′ region of the gene. Herein, we establish the mechanism of Purβ binding to the purine-rich strand of the enhancer using quantitative methods and purified components. Initial evaluation of DNA-binding specificity and equilibrium stoichiometry via colorimetric-, autoradiographic-, and fluorescence-based assays suggested that Purβ interacts with two distinct G/A-rich sites within the nominal single-stranded enhancer element to form a high affinity 2:1 protein:DNA complex. Statistical mechanical analyses of band shift titrations of the nominal element in conjunction with DNase I footprint titrations of the extended smooth muscle α-actin 5′-flanking region demonstrated that assembly of the nucleoprotein complex likely occurs in a sequential, cooperative, and monomer-dependent fashion. Resolution of the microscopic energetics of the system indicated that monomer association with two non-identical sites flanking the core MCAT motif accounts for the majority of the intrinsic binding affinity of Purβ with intersite cooperativity contributing a ~12-fold increase to the stability of the nucleoprotein complex. These findings offer new insights into the mechanism, energetics, and sequence determinants of Purβ repressor binding to a biologically relevant, contractile phenotype-regulating cis-element while also revealing the thermodynamic confines of putative Purβ-mediated effects on DNA structure.

Significant limitations exist in our ability to predict breast cancer risk at the individual level. Circulating microRNAs (C-miRNAs) have emerged as measurable biomarkers (liquid biopsies) for cancer detection. We evaluated the ability of C-miRNAs to identify women most likely to develop breast cancer by profiling miRNA from serum obtained long before diagnosis. 24 breast cancer cases and controls (matched for risk and age) were identified from women enrolled in the High-Risk Breast Program at the UVM Cancer Center. Isolated RNA from serum was profiled for over 2500 human miRNAs. The miRNA expression data were input into a stepwise linear regression model to discover a multivariable miRNA signature that predicts long-term risk of breast cancer. 25 candidate miRNAs were identified that individually classified cases and controls based on statistical methodologies. A refined 6-miRNA risk-signature was discovered following regression modeling that distinguishes cases and controls (AUC0.896, CI 0.804-0.988) in this cohort. A functional relationship between miRNAs that cluster together when cases are contrasted against controls was suggested and confirmed by pathway analyses. The discovered 6 miRNA risk-signature can discriminate high-risk women who ultimately develop breast cancer from those who remain cancer-free, improving current risk assessment models. Future studies will focus on functional analysis of the miRNAs in this signature and testing in larger cohorts. We propose that the combined signature is highly significant for predicting cancer risk, and worthy of further screening in larger, independent clinical cohorts.

The mammalian runt-related factor 1 (RUNX1) is a master transcription factor that regulates lineage specification of hematopoietic stem cells. RUNX1 translocations result in the development of myeloid leukemias. Recently, RUNX1 has been implicated as a tumor suppressor in other cancers. We postulated RUNX1 expression may be associated with lung adenocarcinoma etiology and/or progression. We evaluated the association of RUNX1 mRNA expression with overall survival data from The Cancer Genome Atlas (TCGA), a publically available database. Compared to high expression levels, Low RUNX1 levels from lung adenocarcinomas were associated with a worse overall survival (Hazard Ratio = 2.014 (1.042-3.730 95% confidence interval), log-rank p = 0.035) compared to those that expressed high RUNX1 levels. Further immunohistochemical examination of 85 surgical specimens resected at the University of Vermont Medical Center identified that low RUNX1 protein expression was associated with larger tumors (p = 0.038). Gene expression network analysis was performed on the same subset of TCGA cases that demonstrated differential survival by RUNX1 expression. This analysis, which reveals regulatory relationships, showed that reduced RUNX1 levels were closely linked to upregulation of the transcription factor E2F1. To interrogate this relationship, RUNX1 was depleted in a lung cancer cell line that expresses high levels of RUNX1. Loss of RUNX1 resulted in enhanced proliferation, migration, and invasion. RUNX1 depletion also resulted in increased mRNA expression of E2F1 and multiple E2F1 target genes. Our data implicate loss of RUNX1 as driver of lung adenocarcinoma aggression, potentially through deregulation of the E2F1 pathway.

Previous studies from our laboratories have implicated two members of the Pur family of single-stranded DNA/RNA-binding proteins, Pur␣ and Pur␤, in transcriptional repression of the smooth muscle ␣-actin gene in vascular cell types. Although Pur␣ and Pur␤ share substantial sequence homology and nucleic acid binding properties, genomic promoter and cis-element occupancy studies reported herein suggest that Pur␤ is the dominant factor in gene regulation. To dissect the molecular basis of Pur␤ repressor activity, site-directed mutagenesis was used to map amino acids critical to the physical and functional interaction of Pur␤ with the smooth muscle ␣-actin promoter. Of all the various acidic, basic, and aromatic residues studied, mutation of positionally conserved arginines in the class I or class II repeat modules significantly attenuated Pur␤ repressor activity in transfected vascular smooth muscle cells and fibroblasts. DNA binding and protein-protein interaction assays were conducted with purified recombinant Pur␤ and selected mutants to reveal the physical basis for loss-of-function. Mutants R57E, R57E/R96E, and R57A/R96A each exhibited reduced single-stranded DNA binding affinity for an essential promoter element and diminished interaction with corepressor YB-1/MSY1. Structural analyses of the R57A/R96A and R57E/R96E double mutants in comparison to the wildtype Pur␤ homodimer revealed aberrant self-association into higher order oligomeric complexes, which correlated with decreased ␣-helical content and defective DNA and protein binding in vitro. These findings point to a previously unrecognized structural role for certain core arginine residues in forming a conformationally stable Pur␤ protein capable of physical interactions necessary for smooth muscle ␣-actin gene repression.