Winston Timp

Cancer as a dysregulated epigenome allowing cellular growth advantage at the expense of the host

Although at the genetic level cancer is caused by diverse mutations, epigenetic modifications are characteristic of all cancers, from apparently normal precursor tissue to advanced metastatic disease, and these epigenetic modifications drive tumour cell heterogeneity. We propose a unifying model of cancer in which epigenetic dysregulation allows rapid selection for tumour cell survival at the expense of the host. Mechanisms involve both genetic mutations and epigenetic modifications that disrupt the function of genes that regulate the epigenome itself. Several exciting recent discoveries also point to a genome-scale disruption of the epigenome that involves large blocks of DNA hypomethylation, mutations of epigenetic modifier genes and alterations of heterochromatin in cancer (including large organized chromatin lysine modifications (LOCKs) and lamin-associated domains (LADs)), all of which increase epigenetic and gene expression plasticity. Our model suggests a new approach to cancer diagnosis and therapy that focuses on epigenetic dysregulation and has great potential for risk detection and chemoprevention.

Genome-scale epigenetic reprogramming during epithelial-to-mesenchymal transition

Epithelial-to-mesenchymal transition (EMT) is an extreme example of cell plasticity that is important for normal development, injury repair and malignant progression. Widespread epigenetic reprogramming occurs during stem cell differentiation and malignant transformation, but EMT-related epigenetic reprogramming is poorly understood. Here we investigated epigenetic modifications during EMT mediated by transforming growth factor beta. Although DNA methylation was unchanged during EMT, we found a global reduction in the heterochromatin mark H3 Lys9 dimethylation (H3K9Me2), an increase in the euchromatin mark H3 Lys4 trimethylation (H3K4Me3) and an increase in the transcriptional mark H3 Lys36 trimethylation (H3K36Me3). These changes depended largely on lysine-specific demethylase-1 (Lsd1), and loss of Lsd1 function had marked effects on EMT-driven cell migration and chemoresistance. Genome-scale mapping showed that chromatin changes were mainly specific to large organized heterochromatin K9 modifications (LOCKs), which suggests that EMT is characterized by reprogramming of specific chromatin domains across the genome.

Enhanced sensitivity to IGF-II signaling links loss of imprinting of IGF2 to increased cell proliferation and tumor risk.

Loss of imprinting (LOI) of the insulin-like growth factor-II gene (IGF2), leading to abnormal activation of the normally silent maternal allele, is a common human epigenetic population variant associated with a 5-fold increased frequency of colorectal neoplasia. Here, we show first that LOI leads specifically to increased expression of proliferation-related genes in mouse intestinal crypts. Surprisingly, LOI(+) mice also have enhanced sensitivity to IGF-II signaling, not simply increased IGF-II levels, because in vivo blockade with NVP-AEW541, a specific inhibitor of the IGF-II signaling receptor, showed reduction of proliferation-related gene expression to levels half that seen in LOI(−) mice. Signal transduction assays in microfluidic chips confirmed this enhanced sensitivity with marked augmentation of Akt/PKB signaling in LOI(+) cells at low doses of IGF-II, which was reduced in the presence of the inhibitor to levels below those found in LOI(−) cells, and was associated with increased expression of the IGF1 and insulin receptor genes. We exploited this increased IGF-II sensitivity to develop an in vivo chemopreventive strategy using the azoxymethane (AOM) mutagenesis model. LOI(+) mice treated with AOM showed a 60% increase in premalignant aberrant crypt foci (ACF) formation over LOI(−) mice. In vivo IGF-II blockade with NVP-AEW541 abrogated this effect, reducing ACF to a level 30% lower even than found in exposed LOI(−) mice. Thus, LOI increases cancer risk in a counterintuitive way, by increasing the sensitivity of the IGF-II signaling pathway itself, providing a previously undescribed epigenetic chemoprevention strategy in which cells with LOI are “IGF-II addicted” and undergo reduced tumorigenesis in the colon upon IGF-II pathway blockade.

Nanopore Sequencing: Electrical Measurements of the Code of Life

Sequencing a single molecule of deoxyribonucleic acid (DNA) using a nanopore is a revolutionary concept because it combines the potential for long read lengths (>5 kbp) with high speed (1 bp/10 ns), while obviating the need for costly amplification procedures due to the exquisite single molecule sensitivity. The prospects for implementing this concept seem bright. The cost savings from the removal of required reagents, coupled with the speed of nanopore sequencing places the

Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy

1000 genome within grasp. However, challenges remain: high fidelity reads demand stringent control over both the molecular configuration in the pore and the translocation kinetics. The molecular configuration determines how the ions passing through the pore come into contact with the nucleotides, while the translocation kinetics affect the time interval in which the same nucleotides are held in the constriction as the data is acquired. Proteins like ¿-hemolysin and its mutants offer exquisitely precise self-assembled nanopores and have demonstrated the facility for discriminating individual nucleotides, but it is currently difficult to design protein structure ab initio, which frustrates tailoring a pore for sequencing genomic DNA. Nanopores in solid-state membranes have been proposed as an alternative because of the flexibility in fabrication and ease of integration into a sequencing platform. Preliminary results have shown that with careful control of the dimensions of the pore and the shape of the electric field, control of DNA translocation through the pore is possible. Furthermore, discrimination between different base pairs of DNA may be feasible. Thus, a nanopore promises inexpensive, reliable, high-throughput sequencing, which could thrust genomic science into personal medicine.

Detecting DNA cytosine methylation using nanopore sequencing

We developed a deep-ultraviolet (UV) microscope capable of imaging cell mitosis and motility at 280 nm for 45 min with minimal UV-induced toxicity, and for 6 h before the onset of visible cell death in cultured human and mouse cells. Combined with computational methods that convert the intensity of each pixel into an estimate of mass, deep-UV microscopy images generate maps of nucleic acid mass, protein mass and fluorescence yield in unlabeled cells.

The ballistic nano-transistor

In nanopore sequencing devices, electrolytic current signals are sensitive to base modifications, such as 5-methylcytosine (5-mC). Here we quantified the strength of this effect for the Oxford Nanopore Technologies MinION sequencer. By using synthetically methylated DNA, we were able to train a hidden Markov model to distinguish 5-mC from unmethylated cytosine. We applied our method to sequence the methylome of human DNA, without requiring special steps for library preparation.

Large hypomethylated blocks as a universal defining epigenetic alteration in human solid tumors

We have achieved extremely high drive current performance and ballistic (T>0.8) transport using ultra-thin (<2 nm) gate oxides in sub-30 nm effective channel length nMOSFETs. The peak drive performance in an nMOSFET was observed at t/sub ox//spl ap/1.3 nm for a 1.5 V power supply voltage with T/sub n//spl ap/0.7, while the peak performance in a pMOSFET was observed at t/sub ox//spl ap/1.5 nm for a -1.5 V supply with T/sub p//spl ap/0.5. Since the carrier scattering in the channel is due predominately to interface roughness, reducing the transverse surface field, either by reducing the gate voltage or by increasing the oxide thickness, can be used to improve the transmittance T/sub n//spl rarr/0.85, T/sub p//spl rarr/0.6, while diminishing the drive current.

Nanoelectromechanics of methylated DNA in a synthetic nanopore.

BackgroundOne of the most provocative recent observations in cancer epigenetics is the discovery of large hypomethylated blocks, including single copy genes, in colorectal cancer, that correspond in location to heterochromatic LOCKs (large organized chromatin lysine-modifications) and LADs (lamin-associated domains).MethodsHere we performed a comprehensive genome-scale analysis of 10 breast, 28 colon, nine lung, 38 thyroid, 18 pancreas cancers, and five pancreas neuroendocrine tumors as well as matched normal tissue from most of these cases, as well as 51 premalignant lesions. We used a new statistical approach that allows the identification of large hypomethylated blocks on the Illumina HumanMethylation450 BeadChip platform.ResultsWe find that hypomethylated blocks are a universal feature of common solid human cancer, and that they occur at the earliest stage of premalignant tumors and progress through clinical stages of thyroid and colon cancer development. We also find that the disrupted CpG islands widely reported previously, including hypermethylated island bodies and hypomethylated shores, are enriched in hypomethylated blocks, with flattening of the methylation signal within and flanking the islands. Finally, we found that genes showing higher between individual gene expression variability are enriched within these hypomethylated blocks.ConclusionThus hypomethylated blocks appear to be a universal defining epigenetic alteration in human cancer, at least for common solid tumors.

DNA Base-Calling from a Nanopore Using a Viterbi Algorithm

Methylation of cytosine is a covalent modification of DNA that can be used to silence genes, orchestrating a myriad of biological processes including cancer. We have discovered that a synthetic nanopore in a membrane comparable in thickness to a protein binding site can be used to detect methylation. We observe a voltage threshold for permeation of methylated DNA through a <2 nm diameter pore, which we attribute to the stretching transition; this can differ by >1 V/20 nm depending on the methylation level, but not the DNA sequence.