Studying Oxidative Stress Caused by the Mitis Group Streptococci in <em>Caenorhabditis elegans</em>

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented ...

Identification of EGFR and RAS Inhibitors using <em>Caenorhabditis elegans</em>

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in ...

University of Texas Health Science Center

Although genome-wide association studies (GWASs) for nonsyndromic orofacial clefts have identified multiple strongly associated regions, the causal variants are unknown. To address this, we selected 13 regions from GWASs and other studies, performed targeted sequencing in 1,409 Asian and European trios, and carried out a series of statistical and functional analyses. Within a cluster of strongly associated common variants near NOG, we found that one, rs227727, disrupts enhancer activity. We furthermore identified significant clusters of non-coding rare variants near NTN1 and NOG and found several rare coding variants likely to affect protein function, including four nonsense variants in ARHGAP29. We confirmed 48 de novo mutations and, based on best biological evidence available, chose two of these for functional assays. One mutation in PAX7 disrupted the DNA binding of the encoded transcription factor in an in vitro assay. The second, a non-coding mutation, disrupted the activity of a neural crest enhancer downstream of FGFR2 both in vitro and in vivo. This targeted sequencing study provides strong functional evidence implicating several specific variants as primary contributory risk alleles for nonsyndromic clefting in humans.

Identification of functional variants for cleft lip with or without cleft palate in or near PAX7, FGFR2, and NOG by targeted sequencing of GWAS loci.

Bevacizumab (BEV) is a monoclonal antibody to vascular endothelial growth factor (VEGF) that ameliorates atheroma progression by inhibiting neovascularization.

Ultrasound-enhanced bevacizumab release from echogenic liposomes for inhibition of atheroma progression.

Animal studies suggest that pancreatitis-induced acinar-to-ductal metaplasia (ADM) is a key event for pancreatic ductal adenocarcinoma (PDAC) initiation. However, there has not been an adequate system to explore the mechanisms of human ADM induction. We have developed a flow cytometry-based, high resolution lineage tracing method and 3D culture system to analyse ADM in human cells. In this system, well-known mouse ADM inducers did not promote ADM in human cells. In contrast, TGF-β1 efficiently converted human acinar cells to duct-like cells (AD) in a SMAD-dependent manner, highlighting fundamental differences between the species. Functionally, AD cells gained transient proliferative capacity. Furthermore, oncogenic KRAS did not induce acinar cell proliferation, but did sustain the proliferation of AD cells, suggesting that oncogenic KRAS requires ADM-associated-changes to promote PDAC initiation. This ADM model provides a novel platform to explore the mechanisms involved in the development of human pancreatic diseases.

TGF-β1 promotes acinar to ductal metaplasia of human pancreatic acinar cells.

Interferon Regulatory Factor 6 (IRF6) and TWIST1 are transcription factors necessary for craniofacial development. Human genetic studies showed that mutations in IRF6 lead to cleft lip and palate and mandibular abnormalities. In the mouse, we found that loss of Irf6 causes craniosynostosis and mandibular hypoplasia. Similarly, mutations in TWIST1 cause craniosynostosis, mandibular hypoplasia and cleft palate. Based on this phenotypic overlap, we asked if Irf6 and Twist1 interact genetically during craniofacial formation. While single heterozygous mice are normal, double heterozygous embryos (Irf6  ; Twist1  ) can have severe mandibular hypoplasia that leads to agnathia and cleft palate at birth. Analysis of spatiotemporal expression showed that Irf6 and Twist1 are found in different cell types. Consistent with the intercellular interaction, we found reduced expression of Endothelin1 (EDN1) in mandible and transcription factors that are critical for mandibular patterning including DLX5, DLX6 and HAND2, were also reduced in mesenchymal cells. Treatment of mandibular explants with exogenous EDN1 peptides partially rescued abnormalities in Meckel's cartilage. In addition, partial rescue was observed when double heterozygous embryos also carried a null allele of p53. Considering that variants in IRF6 and TWIST1 contribute to human craniofacial defects, this gene-gene interaction may have implications on craniofacial disorders.

Intercellular Genetic Interaction Between Irf6 and Twist1 during Craniofacial Development.

K-Ras must localize to the plasma membrane (PM) for biological activity. We show here that multiple acid sphingomyelinase (ASM) inhibitors, including tricyclic antidepressants, mislocalized phosphatidylserine (PtdSer) and K-RasG12V from the PM, resulting in abrogation of K-RasG12V signaling and potent, selective growth inhibition of mutant K-Ras-transformed cancer cells. Concordantly, in nude mice, the ASM inhibitor fendiline decreased the rate of growth of oncogenic K-Ras-expressing MiaPaCa-2 tumors but had no effect on the growth of the wild-type K-Ras-expressing BxPC-3 tumors. ASM inhibitors also inhibited activated LET-60 (a K-Ras ortholog) signaling in , as evidenced by suppression of the induced multivulva phenotype. Using RNA interference against  genes encoding other enzymes in the sphingomyelin (SM) biosynthetic pathway, we identified 14 enzymes whose knockdown strongly or moderately suppressed the LET-60 multivulva phenotype. In mammalian cells, pharmacological agents that target these enzymes all depleted PtdSer from the PM and caused K-RasG12V mislocalization. These effects correlated with changes in SM levels or subcellular distribution. Selected compounds, including sphingosine kinase inhibitors, potently inhibited the proliferation of oncogenic K-Ras-expressing pancreatic cancer cells. In conclusion, these results show that normal SM metabolism is critical for K-Ras function, which may present therapeutic options for the treatment of K-Ras-driven cancers.

Sphingomyelin Metabolism Is a Regulator of K-Ras Function.

The mitis group, a member of the genetically diverse viridans group streptococci, predominately colonizes the human oropharynx. This group has been shown to cause a wide range of infectious complications in humans, including bacteremia in patients with neutropenia, orbital cellulitis and infective endocarditis. Hydrogen peroxide (H2O2) has been identified as a virulence factor produced by this group of streptococci. More importantly, it has been shown that Streptococcus oralis and S. mitis induce epithelial cell and macrophage death via the production of H2O2. Previously, H2O2 mediated killing was observed in the nematode Caenorhabditis elegans in response to S. oralis and S. mitis. The genetically tractable model organism C. elegans is an excellent system to study mechanisms of pathogenicity and stress responses. Using this model, we observed rapid H2O2 mediated killing of the worms by S. gordonii in addition to S. mitis and S. oralis. Furthermore, we observed colonization of the intestine of the worms when exposed to S. gordonii suggesting the involvement of an infection-like process. In response to the H2O2 produced by the mitis group, we demonstrate the oxidative stress response is activated in the worms. The oxidative stress response transcription factor SKN-1 is required for the survival of the worms and provides protection against H2O2 produced by S. gordonii. We show during infection, H2O2 is required for the activation of SKN-1 and is mediated via the p38-MAPK pathway. The activation of the p38 signaling pathway in the presence of S. gordonii is not mediated by the endoplasmic reticulum (ER) transmembrane protein kinase IRE-1. However, IRE-1 is required for the survival of worms in response to S. gordonii. These finding suggests a parallel pathway senses H2O2 produced by the mitis group and activates the phosphorylation of p38. Additionally, the unfolded protein response plays an important role during infection.

The activation of the oxidative stress response transcription factor SKN-1 in Caenorhabditis elegans by mitis group streptococci.

By serving as intermediaries between cellular metabolism and the bioenergetic demands of proliferation, endolysosomes allow cancer cells to thrive under normally detrimental conditions. Here, we show that an endolysosomal TRP channel, TRPML1, is necessary for the proliferation of cancer cells that bear activating mutations in  Expression of , which encodes TRPML1, is significantly elevated in -positive tumors and inversely correlated with patient prognosis. Concordantly,  knockdown or TRPML1 inhibition selectively reduces the proliferation of cancer cells that express oncogenic, but not wild-type,  Mechanistically, TRPML1 maintains oncogenic HRAS in signaling-competent nanoclusters at the plasma membrane by mediating cholesterol de-esterification and transport. TRPML1 inhibition disrupts the distribution and levels of cholesterol and thereby attenuates HRAS nanoclustering and plasma membrane abundance, ERK phosphorylation, and cell proliferation. These findings reveal a selective vulnerability of -driven cancers to TRPML1 inhibition, which may be leveraged as an actionable therapeutic strategy.

HRAS-driven cancer cells are vulnerable to TRPML1 inhibition.

Approximately 15% of all human tumors harbor mutant KRAS, a membrane-associated small GTPase and notorious oncogene. Mutations that render KRAS constitutively active will lead to uncontrolled cell growth and cancer. However, despite aggressive efforts in recent years, there are no drugs on the market that directly target KRAS and inhibit its aberrant functions. In the current work, we combined structure-based design with a battery of cell and biophysical assays to discover a novel pyrazolopyrimidine-based allosteric KRAS inhibitor that binds to activated KRAS with sub-micromolar affinity and disrupts effector binding, thereby inhibiting KRAS signaling and cancer cell growth. These results show that pyrazolopyrimidine-based compounds may represent a first-in-class allosteric noncovalent inhibitors of KRAS. Moreover, by studying two of its analogues, we identified key chemical features of the compound that interact with a set of specific residues at the switch regions of KRAS and play critical roles for its high-affinity binding and unique mode of action, thus providing a blueprint for future optimization efforts.

Discovery of High-Affinity Noncovalent Allosteric KRAS Inhibitors That Disrupt Effector Binding.

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented protocol uses this model to determine the pathogenicity caused by the mitis group streptococci via the production of H2O2. The mitis group streptococci are an emerging threat that cause many human diseases such as bacteremia, endocarditis, and orbital cellulitis. Described here is a protocol to determine the survival of these worms in response to H2O2 produced by this group of pathogens. Using the gene skn-1 encoding for an oxidative stress response transcription factor, it is shown that this model is important for identifying host genes that are essential against streptococcal infection. Furthermore, it is shown that activation of the oxidative stress response can be monitored in the presence of these pathogens using a transgenic reporter worm strain, in which SKN-1 is fused to green fluorescent protein (GFP). These assays provide the opportunity to study the oxidative stress response to H2O2 derived by a biological source as opposed to exogenously added reactive oxygen species (ROS) sources.

Ali Naji

Department of Diagnostic and Biomedical Sciences,
School of Dentistry,
Department of Diagnostic and Biomedical Sciences, School of Dentistry

University of Texas Health Science Center

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

Ali Naji

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Diagnostic and Biomedical Sciences,School of Dentistry,Department of Diagnostic and Biomedical Sciences, School of Dentistry

University of Texas Health Science Center

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

Department of Diagnostic and Biomedical Sciences,
School of Dentistry,
Department of Diagnostic and Biomedical Sciences, School of Dentistry