Epigenetic reversion of breast carcinoma phenotype is accompanied by DNA sequestration

The importance of microenvironment and context in regulation of tissue-specific genes is finally well established. DNA exposure to, or sequestration from, nucleases can be used to detect differences in higher order chromatin structure in intact cells without disturbing cellular or tissue architecture. To investigate the relationship between chromatin organization and tumor phenotype, we utilized an established 3-D assay where normal and malignant human breast cells can be easily distinguished by the morphology of the structures they make (acinus-like vs tumor-like, respectively). We show that these phenotypes can be distinguished also by sensitivity to AluI digestion where the malignant cells are resistant to digestion relative to non-malignant cells. Reversion of the T4-2 breast cancer cells by either cAMP analogs, or a phospatidylinositol 3-kinase (P13K) inhibitor not only reverted the phenotype, but also the chromatin sensitivity to AluI. By using different cAMP-analogs, we show that the cAMP-induced phenotypic reversion, polarization, and shift in DNA organization act through a cAMP-dependent-protein-kinase A-coupled signaling pathway. Importantly, inhibitory antibody to fibronectin also reverted the malignant phenotype, polarized the acini, and changed chromatin sequestration. These experiments show not only that modifying the tumor microenvironment can alter the organization of tumor cells but also that architecture of the tissues and the global chromatin organization are coupled and yet highly plastic

Cellular automata modelling of slime mould actin network signalling

© 2016, The Author(s). Actin is a cytoskeletal protein which forms dense, highly interconnected networks within eukaryotic cells. A growing body of evidence suggests that actin-mediated intra- and extracellular signalling is instrumental in facilitating organism-level emergent behaviour patterns which, crucially, may be characterised as natural expressions of computation. We use excitable cellular automata modelling to simulate signal transmission through cell arrays whose topology was extracted from images of Watershed transformation-derived actin network reconstructions; the actin networks sampled were from laboratory experimental observations of a model organism, slime mould Physarum polycephalum. Our results indicate that actin networks support directional transmission of generalised energetic phenomena, the amplification and trans-network speed of which of which is proportional to network density (whose primary determinant is the anatomical location of the network sampled). Furthermore, this model also suggests the ability of such networks for supporting signal-signal interactions which may be characterised as Boolean logical operations, thus indicating that a cell’s actin network may function as a nanoscale data transmission and processing network. We conclude by discussing the role of the cytoskeleton in facilitating intracellular computing, how computation can be implemented in such a network and practical considerations for designing ‘useful’ actin circuitry

Actomyosin and vimentin cytoskeletal networks regulate nuclear shape, mechanics and chromatin organization

This work was supported in part by a Marie Curie CIG grant (PCIG14-GA-2013-631011 CSKFingerprints) and a BBSRC grant (BB/P006108/1). MCK is supported by a PhD studentship from the Life Sciences Initiative at QMUL

Vascular phenotype in angiogenic and non-angiogenic lung non-small cell carcinomas

We have previously described a group of non-small cell lung carcinomas without morphological evidence of neo-angiogenesis. In these tumours neoplastic cells fill up the alveoli and the only vessels present appear to belong to the trapped alveolar septa. In the present study we have characterised the phenotype of the vessels present in these non-angiogenic tumours, in normal lung and in angiogenic non-small cell lung carcinomas. The vessels, identified by the expression of CD31, were scored as mature when expressing the epitope LH39 in the basal membrane and as newly formed when expressing αVβ3 on the endothelial cells and/or lacking LH39 expression. In the nine putative non-angiogenic cases examined, the vascular phenotype of all the vessels was the same as that of alveolar vessels in normal lung: LH39 positive and αVβ3 variable or negative. Instead in 104 angiogenic tumours examined, only a minority of vessels (mean 13.1%; range 0–60%) expressed LH39, while αVβ3 (in 45 cases) was strongly expressed on many vessels (mean 55.5%; range 5–90%). We conclude that in putative non-angiogenic tumours the vascular phenotype is that of normal vessels and there is no neo-angiogenesis. This type of cancer may be resistant to some anti-angiogenic therapy and different strategies need to be developed

Molecular biology of breast cancer metastasis Molecular expression of vascular markers by aggressive breast cancer cells

During embryogenesis, the formation of primary vascular networks occurs via the processes of vasculogenesis and angiogenesis. In uveal melanoma, vasculogenic mimicry describes the 'embryonic-like' ability of aggressive, but not nonaggressive, tumor cells to form networks surrounding spheroids of tumor cells in three-dimensional culture; these recapitulate the patterned networks seen in patients' aggressive tumors and correlates with poor prognosis. The molecular profile of these aggressive tumor cells suggests that they have a deregulated genotype, capable of expressing vascular phenotypes. Similarly, the embryonic-like phenotype expressed by the aggressive human breast cancer cells is associated with their ability to express a variety of vascular markers. These studies may offer new insights for consideration in breast cancer diagnosis and therapeutic intervention strategies

Cytoskeletal Signaling: Is Memory Encoded in Microtubule Lattices by CaMKII Phosphorylation?

Memory is attributed to strengthened synaptic connections among particular brain neurons, yet synaptic membrane components are transient, whereas memories can endure. This suggests synaptic information is encoded and ‘hard-wired’ elsewhere, e.g. at molecular levels within the post-synaptic neuron. In long-term potentiation (LTP), a cellular and molecular model for memory, post-synaptic calcium ion (Ca2+) flux activates the hexagonal Ca2+-calmodulin dependent kinase II (CaMKII), a dodacameric holoenzyme containing 2 hexagonal sets of 6 kinase domains. Each kinase domain can either phosphorylate substrate proteins, or not (i.e. encoding one bit). Thus each set of extended CaMKII kinases can potentially encode synaptic Ca2+ information via phosphorylation as ordered arrays of binary ‘bits’. Candidate sites for CaMKII phosphorylation-encoded molecular memory include microtubules (MTs), cylindrical organelles whose surfaces represent a regular lattice with a pattern of hexagonal polymers of the protein tubulin. Using molecular mechanics modeling and electrostatic profiling, we find that spatial dimensions and geometry of the extended CaMKII kinase domains precisely match those of MT hexagonal lattices. This suggests sets of six CaMKII kinase domains phosphorylate hexagonal MT lattice neighborhoods collectively, e.g. conveying synaptic information as ordered arrays of six “bits”, and thus “bytes”, with 64 to 5,281 possible bit states per CaMKII-MT byte. Signaling and encoding in MTs and other cytoskeletal structures offer rapid, robust solid-state information processing which may reflect a general code for MT-based memory and information processing within neurons and other eukaryotic cells

Dynamics of Mechanical Signal Transmission through Prestressed Stress Fibers

Transmission of mechanical stimuli through the actin cytoskeleton has been proposed as a mechanism for rapid long-distance mechanotransduction in cells; however, a quantitative understanding of the dynamics of this transmission and the physical factors governing it remains lacking. Two key features of the actin cytoskeleton are its viscoelastic nature and the presence of prestress due to actomyosin motor activity. We develop a model of mechanical signal transmission through prestressed viscoelastic actin stress fibers that directly connect the cell surface to the nucleus. The analysis considers both temporally stationary and oscillatory mechanical signals and accounts for cytosolic drag on the stress fibers. To elucidate the physical parameters that govern mechanical signal transmission, we initially focus on the highly simplified case of a single stress fiber. The results demonstrate that the dynamics of mechanical signal transmission depend on whether the applied force leads to transverse or axial motion of the stress fiber. For transverse motion, mechanical signal transmission is dominated by prestress while fiber elasticity has a negligible effect. Conversely, signal transmission for axial motion is mediated uniquely by elasticity due to the absence of a prestress restoring force. Mechanical signal transmission is significantly delayed by stress fiber material viscosity, while cytosolic damping becomes important only for longer stress fibers. Only transverse motion yields the rapid and long-distance mechanical signal transmission dynamics observed experimentally. For simple networks of stress fibers, mechanical signals are transmitted rapidly to the nucleus when the fibers are oriented largely orthogonal to the applied force, whereas the presence of fibers parallel to the applied force slows down mechanical signal transmission significantly. The present results suggest that cytoskeletal prestress mediates rapid mechanical signal transmission and allows temporally oscillatory signals in the physiological frequency range to travel a long distance without significant decay due to material viscosity and/or cytosolic drag

Integrin-Specific Mechanoresponses to Compression and Extension Probed by Cylindrical Flat-Ended AFM Tips in Lung Cells

Cells from lung and other tissues are subjected to forces of opposing directions that are largely transmitted through integrin-mediated adhesions. How cells respond to force bidirectionality remains ill defined. To address this question, we nanofabricated flat-ended cylindrical Atomic Force Microscopy (AFM) tips with ∼1 µm2 cross-section area. Tips were uncoated or coated with either integrin-specific (RGD) or non-specific (RGE/BSA) molecules, brought into contact with lung epithelial cells or fibroblasts for 30 s to form focal adhesion precursors, and used to probe cell resistance to deformation in compression and extension. We found that cell resistance to compression was globally higher than to extension regardless of the tip coating. In contrast, both tip-cell adhesion strength and resistance to compression and extension were the highest when probed at integrin-specific adhesions. These integrin-specific mechanoresponses required an intact actin cytoskeleton, and were dependent on tyrosine phosphatases and Ca2+ signaling. Cell asymmetric mechanoresponse to compression and extension remained after 5 minutes of tip-cell adhesion, revealing that asymmetric resistance to force directionality is an intrinsic property of lung cells, as in most soft tissues. Our findings provide new insights on how lung cells probe the mechanochemical properties of the microenvironment, an important process for migration, repair and tissue homeostasis

Tumour vascularization: sprouting angiogenesis and beyond

Tumour angiogenesis is a fast growing domain in tumour biology. Many growth factors and mechanisms have been unravelled. For almost 30 years, the sprouting of new vessels out of existing ones was considered as an exclusive way of tumour vascularisation. However, over the last years several additional mechanisms have been identified. With the discovery of the contribution of intussusceptive angiogenesis, recruitment of endothelial progenitor cells, vessel co-option, vasculogenic mimicry and lymphangiogenesis to tumour growth, anti-tumour targeting strategies will be more complex than initially thought. This review highlights these processes and intervention as a potential application in cancer therapy. It is concluded that future anti-vascular therapies might be most beneficial when based on multimodal anti-angiogenic, anti-vasculogenic mimicry and anti-lymphangiogenic strategies