Alternating current electrohydrodynamics induced nanoshearing and fluid micromixing for specific capture of cancer cells

We report a new tuneable alternating current (ac) electrohydrodynamics (ac-EHD) force referred to as “nanoshearing” which involves fluid flow generated within a few nanometers of an electrode surface. This force can be externally tuned via manipulating the applied ac-EHD field strength. The ability to manipulate ac-EHD induced forces and concomitant fluid micromixing can enhance fluid transport within the capture domain of the channel (e.g., transport of analytes and hence increase target–sensor interactions). This also provides a new capability to preferentially select strongly bound analytes over onspecifically bound cells and molecules. To demonstrate the utility and versatility of nanoshearing phenomenon to specifically capture cancer cells, we present proof-of-concept data in lysed blood using two microfluidic devices containing a long array of asymmetric planar electrode pairs. Under the optimal experimental conditions, we achieved high capture efficiency (e.g., approximately 90%; %RSD=2, n=3) with a 10-fold reduction in nonspecific dsorption of non-target cells for the detection of whole cells expressing Human Epidermal Growth Factor Receptor 2 (HER2). We believe that our ac-EHD devices and the use of tuneable nanoshearing phenomenon may find relevance in a wide variety of biological and medical applications.

PRMT2 and RORγ expression are associated with breast cancer survival outcomes

Protein arginine methyltransferases (PRMTs) methylate arginine residues on histones and target transcription factors that play critical roles in many cellular processes, including gene transcription, mRNA splicing, proliferation, and differentiation. Recent studies have linked PRMT-dependent epigenetic marks and modifications to carcinogenesis and metastasis in cancer. However, the role of PRMT2-dependent signaling in breast cancer remains obscure. We demonstrate PRMT2 mRNA expression was significantly decreased in breast cancer relative to normal breast. Gene expression profiling, Ingenuity and protein-protein interaction network analysis after PRMT2-short interfering RNA transfection into MCF-7 cells, revealed that PRMT2-dependent gene expression is involved in cell-cycle regulation and checkpoint control, chromosomal instability, DNA repair, and carcinogenesis. For example, PRMT2 depletion achieved the following: 1) increased p21 and decreased cyclinD1 expression in (several) breast cancer cell lines, 2) decreased cell migration, 3) induced an increase in nucleotide excision repair and homologous recombination DNA repair, and 4) increased the probability of distance metastasis free survival (DMFS). The expression of PRMT2 and retinoid-related orphan receptor-γ (RORγ) is inversely correlated in estrogen receptor-positive breast cancer and increased RORγ expression increases DMFS. Furthermore, we found decreased expression of the PRMT2-dependent signature is significantly associated with increased probability of DMFS. Finally, weighted gene coexpression network analysis demonstrated a significant correlation between PRMT2-dependent genes and cell-cycle checkpoint, kinetochore, and DNA repair circuits. Strikingly, these PRMT2-dependent circuits are correlated with pan-cancer metagene signatures associated with epithelial-mesenchymal transition and chromosomal instability. This study demonstrates the role and significant correlation between a histone methyltransferase (PRMT2)-dependent signature, RORγ, the cell-cycle regulation, DNA repair circuits, and breast cancer survival outcomes.

Advanced mass spectrometry-based multi-omics technologies for exploring the pathogenesis of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the primary hepatic malignancies and is the third most common cause of cancer related death worldwide. Although a wealth of knowledge has been gained concerning the initiation and progression of HCC over the last half century, efforts to improve our understanding of its pathogenesis at a molecular level are still greatly needed, to enable clinicians to enhance the standards of the current diagnosis and treatment of HCC. In the post-genome era, advanced mass spectrometry driven multi-omics technologies (e.g., profiling of DNA damage adducts, RNA modification profiling, proteomics, and metabolomics) stand at the interface between chemistry and biology, and have yielded valuable outcomes from the study of a diversity of complicated diseases. Particularly, these technologies are being broadly used to dissect various biological aspects of HCC with the purpose of biomarker discovery, interrogating pathogenesis as well as for therapeutic discovery. This proof of knowledge-based critical review aims at exploring the selected applications of those defined omics technologies in the HCC niche with an emphasis on translational applications driven by advanced mass spectrometry, toward the specific clinical use for HCC patients. This approach will enable the biomedical community, through both basic research and the clinical sciences, to enhance the applicability of mass spectrometry-based omics technologies in dissecting the pathogenesis of HCC and could lead to novel therapeutic discoveries for HCC.

Development of reverse phase protein microarrays for clinical applications and patient-tailored therapy

While genomics provide important information about the somatic genetic changes, and RNA transcript profiling can reveal important expression changes that correlate with outcome and response to therapy, it is the proteins that do the work in the cell. At a functional level, derangements within the proteome, driven by post-translational and epigenetic modifications, such as phosphorylation, is the cause of a vast majority of human diseases. Cancer, for instance, is a manifestation of deranged cellular protein molecular networks and cell signaling pathways that are based on genetic changes at the DNA level. Importantly, the protein pathways contain the drug targets in signaling networks that govern overall cellular survival, proliferation, invasion and cell death. Consequently, the promise of proteomics resides in the ability to extend analysis beyond correlation to causality. A critical gap in the information knowledge base of molecular profiling is an understanding of the ongoing activity of protein signaling in human tissue: what is activated and “in use” within the human body at any given point in time. To address this gap, we have invented a new technology, called reverse phase protein microarrays, that can generate a functional read-out of cell signaling networks or pathways for an individual patient obtained directly from a biopsy specimen. This “wiring diagram” can serve as the basis for both, selection of a therapy and patient stratification.

Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing

Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2), plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI). Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops), and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx⁻/⁻ revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome.

Functional role for senataxin, defective in ataxia oculomotor apraxia type 2, in transcriptional regulation

Ataxia oculomotor apraxia type 2 (AOA2) is an autosomal recessive neurodegenerative disorder characterized by cerebellar ataxia and oculomotor apraxia. The gene mutated in AOA2, SETX, encodes senataxin, a putative DNA/RNA helicase which shares high homology to the yeast Sen1p protein and has been shown to play a role in the response to oxidative stress. To investigate further the function of senataxin, we identified novel senataxin-interacting proteins, the majority of which are involved in transcription and RNA processing, including RNA polymerase II. Binding of RNA polymerase II to candidate genes was significantly reduced in senataxin deficient cells and this was accompanied by decreased transcription of these genes, suggesting a role for senataxin in the regulation/modulation of transcription. RNA polymerase II-dependent transcription termination was defective in cells depleted of senataxin in keeping with the observed interaction of senataxin with poly(A) binding proteins 1 and 2. Splicing efficiency of specific mRNAs and alternate splice-site selection of both endogenous genes and artificial minigenes were altered in senataxin depleted cells. These data suggest that senataxin, similar to its yeast homolog Sen1p, plays a role in coordinating transcriptional events, in addition to its role in DNA repair.

The effect of ß-alanine on buffering capacity and exercise performance during and following high-intensity exercise in healthy males

Intense exercise induced acidosis occurs after accumulation of hydrogen ions as by-products of anaerobic metabolism. Oral ingestion of ß-alanine, a limiting precursor of the intracellular physiochemical buffer carnosine in skeletal muscle, may counteract detrimental effects of acidosis and benefit performance. This study aimed to investigate the effect of ß-alanine as an ergogenic aid during high intensity exercise performance. Five healthy males ingested either ß-alanine or placebo (Pl) (CaCO3) in a crossover design with 6 wk washout between. Participants performed two different intense exercise protocols over consecutive days. On the first day a repeated sprint ability (RSA) test was performed. On the second day a cycling capacity test measuring the time to exhaustion (TTE) was performed at 110% of maximum workload achieved in a pre supplementation max test (CCT110%). Non-invasive quantification of carnosine, prior to, and following each supplementation, with in vivo magnetic resonance spectrometry was performed in the soleus and gastrocnemius muscle. Time to fatigue (CCT110%), peak and mean power (RSA), blood pH, and plasma lactate were measured. Muscle carnosine concentration was not different prior to ß-alanine supplementation and increased 18% in the soleus and 26% in the gastrocnemius, respectively after supplementation. There was no difference in the measured performance variables during the RSA test (peak and average power output). TTE during the CCT110% was significantly enhanced following the ingestion of BAl (155s ± 19.03) compared to Pl (134s ± 26.16). No changes were observed in blood pH during either exercise protocol and during the recovery from exercise. Plasma lactate after BAI was significantly higher than Pl only from the 15th minute following exercise during the CCT110%. Greater muscle carnosine content following 6wk supplementation of ß-alanine enhanced the potential for intracellular buffering capacity. This translated into enhanced performance during the CCT110% high intensity cycling exercise protocol but not during the RSA test. The lack of change in plasma lactate or blood pH indicates that 6wks ß-alanine supplementation has no effect on anaerobic metabolism during multiple-bout high-intensity exercise. Changes measured in plasma lactate during recovery support the hypothesis that ß-alanine supplementation may affect anaerobic metabolism particularly during single bout high intensity.

MAS NMR measurements of intact articular bovine cartilage

Articular cartilage (AC), an avascular connective tissue lining articulating surfaces of the long bones, comprises extracellular biopolymers. In functionally compromised states such as osteoarthritis, thinned or lost AC causes reduced mobility and increased health-care costs. Understanding of the characteristics responsible for the load bearing efficiency of AC and the factors leading to its degradation are incomplete. DTI shows the structural alignment of collagen in AC [1] and T2 relaxation measurements suggest that the average director of reorientational motion of water molecules depends on the degree of alignment of collagen in AC [2]. Information on the nature of the chemical interactions involved in functional AC is lacking. The need for AC structural integrity makes solid state NMR an ideal tool to study this tissue. We examined the contribution of water in different functional ‘compartments’ using 1H-MAS, 13C-MAS and 13C-CPMAS NMR of bovine patellar cartilage incubated in D2O. 1H-MAS spectra signal intensity was reduced due to H/D exchange without a measureable redistribution of relative signal intensity. Chemical shift anisotropy was estimated by lineshape analysis of multiple peaks in the 1H-MAS spinning sidebands. These asymmetrical sidebands suggested the presence of multiple water species in AC. Therefore, water was added in small aliquots to D2O saturated AC and the influence of H2O and D2O on organic components was studied with 13C-MAS-NMR and 13C-CPMAS-NMR. Signal intensity in 13C-MAS spectra showed no change in relative signal intensity throughout the spectrum. In 13C-CPMAS spectra, displacement of water by D2O resulted in a loss of signal in the aliphatic region due to a reduction in proton availability for cross-polarization. These results complement dehydration studies of cartilage using osmotic manipulation [3] and demonstrate components of cartilage that are in contact with mobile water.

Solid-state MAS NMR measurements of intact articular bovine cartilage

Articular cartilage (AC), an avascular connective tissue lining articulating surfaces of the long bones, comprises extracellular biopolymers. In functionally compromised states such as osteoarthritis, thinned or lost AC causes reduced mobility and increased health-care costs. Understanding of the characteristics responsible for the load bearing efficiency of AC and the factors leading to its degradation are incomplete. DTI shows the structural alignment of collagen in AC [1] and T2 relaxation measurements suggest that the average director of reorientational motion of water molecules depends on the degree of alignment of collagen in AC [2]. Information on the nature of the chemical interactions involved in functional AC is lacking. The need for AC structural integrity makes solid state NMR an ideal tool to study this tissue. We examined the contribution of water in different functional ‘compartments’ using 1H-MAS, 13C-MAS and 13C-CPMAS NMR of bovine patellar cartilage incubated in D2O. 1H-MAS spectra signal intensity was reduced due to H/D exchange without a measureable redistribution of relative signal intensity. Chemical shift anisotropy was estimated by lineshape analysis of multiple peaks in the 1H-MAS spinning sidebands. These asymmetrical sidebands suggested the presence of multiple water species in AC. Therefore, water was added in small aliquots to D2O saturated AC and the influence of H2O and D2O on organic components was studied with 13C-MAS-NMR and 13C-CPMAS-NMR. Signal intensity in 13C-MAS spectra showed no change in relative signal intensity throughout the spectrum. In 13C-CPMAS spectra, displacement of water by D2O resulted in a loss of signal in the aliphatic region due to a reduction in proton availability for cross-polarization. These results complement dehydration studies of cartilage using osmotic manipulation [3] and demonstrate components of cartilage that are in contact with mobile water.

Speckle-type POZ protein mutations interrupt tumor suppressor function of speckle-type POZ protein in prostate cancer by affecting androgen receptor degradation

Large scale exome sequencing studies have revealed regions of the genome, which contribute to the castrate resistant prostate cancer (CRPC) phenotype. [1],[2],[3] Such studies have identified mutations in genes, which may have diagnostic/prognostic potential, or which may be targeted therapeutically. Two of these genes include the androgen receptor (AR) and speckle-type POZ protein (SPOP) genes. However, the findings from these exome sequencing studies can only be translated therapeutically once the functional consequences of these mutations have been determined. Here, we highlight the recent study by An et al. [4] which investigated the functional effects of mutations in the SPOP gene that were identified in the aforementioned exome sequencing studies, particularly in the context of SPOP-mediated degradation of the AR.

Nilotinib and MEK inhibitors induce synthetic lethality through paradoxical activation of RAF in drug-resistant chronic myeloid leukemia

We show that imatinib, nilotinib, and dasatinib possess weak off-target activity against RAF and, therefore, drive paradoxical activation of BRAF and CRAF in a RAS-dependent manner. Critically, because RAS is activated by BCR-ABL, in drug-resistant chronic myeloid leukemia (CML) cells, RAS activity persists in the presence of these drugs, driving paradoxical activation of BRAF, CRAF, MEK, and ERK, and leading to an unexpected dependency on the pathway. Consequently, nilotinib synergizes with MEK inhibitors to kill drug-resistant CML cells and block tumor growth in mice. Thus, we show that imatinib, nilotinib, and dasatinib drive paradoxical RAF/MEK/ERK pathway activation and have uncovered a synthetic lethal interaction that can be used to kill drug-resistant CML cells in vitro and in vivo.

Unswitch-ABL drugs overcome resistance in chronic myeloid leukemia

ABL inhibitors have revolutionized the clinical management of chronic myeloid leukemia, but the BCR-ABLT315I mutation confers resistance to currently approved drugs. Chan et al. show, in this issue of Cancer Cell, that " switch-control" inhibitors block BCR-ABLT315I activity by preventing ABL from switching from the inactive to active conformation.

Oncogenic BRAF induces melanoma cell invasion by downregulating the cGMP-specific phosphodiesterase PDE5A

We show that in melanoma cells oncogenic BRAF, acting through MEK and the transcription factor BRN2, downregulates the cGMP-specific phosphodiesterase PDE5A. Although PDE5A downregulation causes a small decrease in proliferation, its major impact is to stimulate a dramatic increase in melanoma cell invasion. This is because PDE5A downregulation leads to an increase in cGMP, which induces an increase in cytosolic Ca2+, stimulating increased contractility and inducing invasion. PDE5A downregulation also this leads to an increase in short-term and long-term colonization of the lungs by melanoma cells. We do not observe this pathway in NRAS mutant melanoma or BRAF mutant colorectal cells. Thus, we show that in melanoma cells oncogenic BRAF induces invasion through downregulation of PDE5A.

Activation of forkhead box O transcription factors by oncogenic BRAF promotes p21cip1-dependent senescence

Oncogene-induced senescence (OIS) is a potent tumor-suppressive mechanism that is thought to come at the cost of aging. The Forkhead box O (FOXO) transcription factors are regulators of life span and tumor suppression. However, whether and how FOXOs function in OIS have been unclear. Here, we show a role for FOXO4 in mediating senescence by the human BRAFV600E oncogene, which arises commonly in melanoma. BRAFV600E signaling through mitogen-activated protein kinase/extracellular signal-regulated kinase kinase resulted in increased reactive oxygen species levels and c-Jun NH 2 terminal kinase-mediated activation of FOXO4 via its phosphorylation on Thr223, Ser226, Thr447, and Thr451. BRAFV600E-induced FOXO4 phosphorylation resulted in p21cip1-mediated cell senescence independent of p16 ink4a or p27kip1. Importantly, melanocyte-specific activation of BRAFV600E in vivo resulted in the formation of skin nevi expressing Thr223/Ser226-phosphorylated FOXO4 and elevated p21cip1. Together, these findings support a model in which FOXOs mediate a trade-off between cancer and aging.

Transcriptional pathway signatures predict MEK addiction and response to selumetinib (AZD6244)

Selumetinib (AZD6244, ARRY-142886) is a selective, non-ATP-competitive inhibitor of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)-1/2. The range of antitumor activity seen preclinically and in patients highlights the importance of identifying determinants of response to this drug. In large tumor cell panels of diverse lineage, we show that MEK inhibitor response does not have an absolute correlation with mutational or phospho-protein markers of BRAF/MEK, RAS, or phosphoinositide 3-kinase (PI3K) activity. We aimed to enhance predictivity by measuring pathway output through coregulated gene networks displaying differential mRNA expression exclusive to resistant cell subsets and correlated to mutational or dynamic pathway activity. We discovered an 18-gene signature enabling measurement of MEK functional output independent of tumor genotype. Where the MEK pathway is activated but the cells remain resistant to selumetinib, we identified a 13-gene signature that implicates the existence of compensatory signaling from RAS effectors other than PI3K. The ability of these signatures to stratify samples according to functional activation of MEK and/or selumetinib sensitivity was shown in multiple independent melanoma, colon, breast, and lung tumor cell lines and in xenograft models. Furthermore, we were able to measure these signatures in fixed archival melanoma tumor samples using a single RT-qPCR-based test and found intergene correlations and associations with genetic markers of pathway activity to be preserved. These signatures offer useful tools for the study of MEK biology and clinical application of MEK inhibitors, and the novel approaches taken may benefit other targeted therapies.