Jon E. Ramsey

Nucleoprotein Interactions Governing Cell Type-dependent Repression of the Mouse Smooth Muscle α-Actin Promoter by Single-stranded DNA-binding Proteins Purα and Purβ

Purα and Purβ are structurally related single-stranded DNA/RNA-binding proteins implicated in the control of cell growth and differentiation. The goal of this study was to determine whether Purα and Purβ function in a redundant, distinct, or collaborative manner to suppress smooth muscleα-actin gene expression in cell types relevant to wound repair and vascular remodeling. RNA interference-mediated loss-of-function analyses revealed that, although Purβ was the dominant repressor, the combined action of endogenous Purα and Purβ was necessary to fully repress the full-length smooth muscle α-actin promoter in cultured fibroblasts but to a lesser extent in vascular smooth muscle cells. The activity of a minimal core enhancer containing a truncated 5′ Pur repressor binding site was unaffected by knockdown of Purα and/or Purβ in fibroblasts. Conversely, gain-of-function studies indicated that Purα or Purβ could each independently repress core smooth muscle α-actin enhancer activity albeit in a cell type-dependent fashion. Biochemical analyses indicated that purified recombinant Purα and Purβ were essentially identical in terms of their binding affinity and specificity for GGN repeat-containing strands of several cis-elements comprising the core enhancer. However, Purα and Purβ exhibited more distinctive protein interaction profiles when evaluated for binding to enhancer-associated transcription factors in extracts from fibroblasts and vascular smooth muscle cells. These findings support the hypothesis that Purα and Purβ repress smooth muscle α-actin gene transcription by means of DNA strand-selective cis-element binding and cell type-dependent protein-protein interactions.

Hydrodynamic Studies on the Quaternary Structure of Recombinant Mouse Purβ

Purβ 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 Purα, Purβ 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 Purα and Purβ 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 Purβ devoid of nucleic acid. Recombinant Purβ 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 Purβ reversibly self-associates in the absence of nucleic acid. Both approaches independently showed that the hydrodynamic shape of the Purβ homodimer is markedly asymmetric and non-spherical. Sedimentation velocity analyses indicated that dimeric Purβ has a sedimentation coefficient of 3.96 Svedberg, a frictional coefficient ratio (f/f0) 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 Purβ self-associates in a reversible monomer-dimer equilibrium characterized by a Kd = 1.13 ± 0.27 μm.

Structure-Function Analysis of Mouse Purβ II CONFORMATION ALTERING MUTATIONS DISRUPT SINGLE-STRANDED DNA AND PROTEIN INTERACTIONS CRUCIAL TO SMOOTH MUSCLE α-ACTIN GENE REPRESSION

Previous studies from our laboratories have implicated two members of the Pur family of single-stranded DNA/RNA-binding proteins, Purα and Purβ, in transcriptional repression of the smooth muscle α-actin gene in vascular cell types. Although Purα and Purβ share substantial sequence homology and nucleic acid binding properties, genomic promoter and cis-element occupancy studies reported herein suggest that Purβ is the dominant factor in gene regulation. To dissect the molecular basis of Purβ repressor activity, site-directed mutagenesis was used to map amino acids critical to the physical and functional interaction of Purβ with the smooth muscle α-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 Purβ repressor activity in transfected vascular smooth muscle cells and fibroblasts. DNA binding and protein-protein interaction assays were conducted with purified recombinant Purβ 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 wild-type Purβ homodimer revealed aberrant self-association into higher order oligomeric complexes, which correlated with decreased α-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 Purβ protein capable of physical interactions necessary for smooth muscle α-actin gene repression.

Isolation and characterization of the core single-stranded DNA-binding domain of purine-rich element binding protein B (Purβ).

Purβ is a single-stranded nucleic acid-binding protein implicated in the injury-induced repression of genes encoding certain muscle-restricted isoforms of actin and myosin expressed in the heart, skeletal muscle, and vasculature. To better understand how the modular arrangement of the primary sequence of Purβ affects the higher order structure and function of the protein, purified recombinant Purβ was subjected to partial proteolysis in an attempt to identify a well-folded truncation protein that retained purine-rich single-stranded DNA-binding activity. Limited tryptic digestion of Purβ liberated a core ∼30kDa fragment corresponding to residues 29-305 as determined by epitope mapping and mass spectrometry. Size exclusion chromatography indicated that the isolated core fragment retains the ability to self-associate while circular dichroism analysis confirmed that the Purβ core domain is stably folded in the absence of glycine-rich N- and C-terminal sequences. Comparative DNA-binding assays revealed that the isolated core domain interacts with purine-rich cis-elements from the smooth muscle α-actin gene with similar specificity but increased affinity compared to full-length Purβ. These findings suggest that the highly conserved modular repeats of Purβ fold to form a core functional domain, which mediates the specific and high affinity binding of the protein to single-stranded DNA.

Successful Treatment of Non-Small Cell Lung Cancer With Erlotinib Throughout Pregnancy.

Successful Treatment of Non–Small Cell Lung Cancer With Erlotinib Throughout Pregnancy Erlotinib is the standard of care for epidermal growth factor receptor (EGFR) mutated lung adenocarcinomas in the United States. However, in pregnant patients with lung cancer, chemotherapy is recommended, irrespective of EGFR mutations, given the lack of experience and uncertainty for fetus’s safety with erlotinib.

Development of a predictive miRNA signature for breast cancer risk among high-risk women

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.

RUNX1 suppresses breast cancer stemness and tumor growth

Recent studies have revealed that mutations in the transcription factor Runx1 are prevalent in breast tumors. Yet, how loss of Runx1 contributes to breast cancer (BCa) remains unresolved. We demonstrate for the first time that Runx1 represses the breast cancer stem cell (BCSC) phenotype and consequently, functions as a tumor suppressor in breast cancer. Runx1 ectopic expression in MCF10AT1 and MCF10CA1a BCa cells reduces (60%) migration, invasion and in vivo tumor growth in mouse mammary fat pad (P<0.05). Runx1 is decreased in BCSCs, and overexpression of Runx1 suppresses tumorsphere formation and reduces the BCSC population. Furthermore, Runx1 inhibits Zeb1 expression, while Runx1 depletion activates Zeb1 and the epithelial-mesenchymal transition. Mechanistically Runx1 functions as a tumor suppressor in breast cancer through repression of cancer stem cell activity. This key regulation of BCSCs by Runx1 may be shared in other epithelial carcinomas, highlighting the importance of Runx1 in solid tumors.

Suppression of Breast Cancer Stem Cells and Tumor Growth by the RUNX1 Transcription Factor

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 in vivo. 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 in vivo 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.

Loss of RUNX1 is associated with aggressive lung adenocarcinomas

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.

Novel organometallic chloroquine derivative inhibits tumor growth

Autophagy has emerged as a mechanism critical to both tumorigenesis and development of resistance to multiple lines of anti‐cancer therapy. Therefore, targeting autophagy and alternative cell death pathways has arisen as a viable strategy for refractory tumors. The anti‐malarial 4‐aminoquinoline compounds chloroquine and hydroxychloroquine are currently being considered for re‐purposing as anti‐cancer therapies intended to sensitize different tumors by targeting the lysosomal cell death pathway. Here, we describe a novel organometallic chloroquine derivative, cymanquine, that exhibits enhanced bioactivity compared to chloroquine in both normal, and reduced pH tumor microenvironments, thus overcoming a defined limitation of traditional 4‐aminoquinolines. In vitro, cymanquine exhibits greater potency than CQ in a diverse panel of human cancer cell lines, including melanoma, in both normal pH and in reduced pH conditions that mimic the tumor microenvironment. Cymanquine treatment results in greater lysosomal accumulation than chloroquine and induces lysosomal dysfunction leading to autophagy blockade. Using a mouse model of vemurafenib‐resistant melanoma, cymanquine slowed tumor growth greater than hydroxychloroquine, and when used in combination with vemurafenib, cymanquine partially restored sensitivity to vemurafenib. Overall, we show that cymanquine exhibits superior lysosomal accumulation and autophagy blockade than either chloroquine or hydroxychloroquine in vitro; and in addition to its high level of tolerability in mice, exhibits superior in vivo efficacy in a model of human melanoma.