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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.