Unimolecular Micelle for MLN8237 Delivery to Target AURKA-RalA Crosstalk for Ras-Driven Tumor Suppression in Mice Xenografts.

Targeting Aurora Kinase A (AURKA) to modulate RalA activation offers a promising strategy for tumor suppression in Ras-independent and Ras-dependent cancers. However, clinical use of the AURKA inhibitor MLN8237 (Alisertib) is limited by its hydrophobicity and poor water solubility. To overcome these limitations, here, we developed an enzyme-biodegradable unimolecular micelle (UMM) nanoparticle to deliver MLN8237 (NP) and evaluated its therapeutic efficacy in tumor xenograft models.

CRISPR-Cas9 mediated RALA knockout and reconstitution: insights into the detection and role of RALA S194 phosphorylation in Ras-dependent and Ras-independent cancers.

Downstream of oncogenic RAS, RALA is critical for cancer tumorigenesis, possibly regulated by phosphorylation of its Serine194 residue. We made CRISPR-Cas9 RALA knockout (RALA KO) in three RAS-dependent and two RAS-independent cancer cells. Detection of RALA S194 phosphorylation using the commercial anti-phospho-RALA antibody lacks specificity in all three RAS-dependent cancers.

Golgi Localized Arl15 Regulates Cargo Transport and Cell Adhesion.

Arf-like GTPases (Arls) regulate membrane trafficking and cytoskeletal organization. Genetic studies predicted a role for Arl15 in type-2 diabetes, insulin resistance, adiposity, and rheumatoid arthritis. Cell biological studies implicated Arl15 in regulating various cellular processes, including magnesium homeostasis and TGFβ signaling.

Orthogonal Persulfide Generation through Precision Tools Provides Insights into Mitochondrial Sulfane Sulfur.

The sulfane sulfur pool, comprised of persulfide (RS-SH) and polysulfide (RS-SH) derived from hydrogen sulfide (HS), has emerged as a major player in redox biochemistry. Mitochondria, besides energy generation, serve as significant cellular redox hubs, mediate stress response and cellular health. However, the effects of endogenous mitochondrial sulfane sulfur (MSS) remain largely uncharacterized as compared with their cytosolic counterparts, cytosolic sulfane sulfur (CSS).

Quantitative analysis of fibrillar collagen organization in the immediate proximity of embedded fibroblasts in 3D collagen hydrogels.

Fibroblasts embedded in a 3D matrix microenvironment can remodel the matrix to regulate cell adhesion and function. Collagen hydrogels are a useful system to study cell-matrix interactions in a 3D microenvironment. While major matrix reorganizations are easily recognizable, subtle changes in response to environmental or biochemical cues are challenging to discern in 3D hydrogels.

Kinetics of Arf1 inactivation regulates Golgi organisation and function in non-adherent fibroblasts.

Arf1 belongs to the Arf family of small GTPases that localise at the Golgi and plasma membrane. Active Arf1 plays a crucial role in regulating Golgi organisation and function. In mouse fibroblasts, loss of adhesion triggers a consistent drop (∼50%) in Arf1 activation that causes the Golgi to disorganise but not fragment.

A pan-cancer analysis of matrisome proteins reveals CTHRC1 and a related network as major ECM regulators across cancers.

The extracellular matrix in the tumour microenvironment can regulate cancer cell growth and progression. A pan-cancer analysis of TCGA data from 30 cancer types, identified the top 5% of matrisome genes with amplifications or deletions in their copy number, that affect their expression and cancer survival. A similar analysis of matrisome genes in individual cancers identified CTHRC1 to be significantly altered.

Evaluating Mouse Fibroblast Interaction with Implant Surfaces in a 3D Microenvironment.

Purpose: Previous studies assessing fibroblast interactions with implants have mainly relied on measurements such as cell migration, gene expression, and cell adhesion. For these studies, testing cellular behavior at the implant surface was done by imaging the cell-implant interface using standard microscopy techniques in 2D tissue culture dishes. The true behavior of cells relative to the implant can best be assessed in a more physiologic 3D microenvironment.

Adhesion-growth factor crosstalk regulates AURKB activation and ERK signalling in re-adherent fibroblasts.

Aurora kinases despite their similarity have distinct roles in the cell cycle, which is regulated by cell-matrix adhesion and growth factors. This study reveals loss of adhesion and re-adhesion to differentially regulate Aurora kinases. AURKB activation that drops on the loss of adhesion recovers on re-adhesion in serumdeprived conditions but not in the presence of serum growth factors.

Adhesion-dependent Caveolin-1 Tyrosine-14 phosphorylation is regulated by FAK in response to changing matrix stiffness.

Integrin-mediated adhesion regulates cellular responses to changes in the mechanical and biochemical properties of the extracellular matrix. Cell-matrix adhesion regulates caveolar endocytosis, dependent on caveolin 1 (Cav1) Tyr14 phosphorylation (pY14Cav1), to control anchorage-dependent signaling. We find that cell-matrix adhesion regulates pY14Cav1 levels in mouse fibroblasts.

Caveolin1 Tyrosine-14 Phosphorylation: Role in Cellular Responsiveness to Mechanical Cues.

The plasma membrane is a dynamic lipid bilayer that engages with the extracellular microenvironment and intracellular cytoskeleton. Caveolae are distinct plasma membrane invaginations lined by integral membrane proteins Caveolin1, 2, and 3. Caveolae formation and stability is further supported by additional proteins including Cavin1, EHD2, Pacsin2 and ROR1.

Cell Derived Matrix Fibulin-1 Associates With Epidermal Growth Factor Receptor to Inhibit Its Activation, Localization and Function in Lung Cancer Calu-1 Cells.

Epidermal Growth Factor Receptor (EGFR) is a known promoter of tumor progression and is overexpressed in lung cancers. Growth factor receptors (including EGFR) are known to interact with extracellular matrix (ECM) proteins, which regulate their activation and function. Fibulin-1 (FBLN1) is a major component of the ECM in lung tissue, and its levels are known to be downregulated in non-small cell lung cancers (NSCLC).

Cell-matrix adhesion controls Golgi organization and function through Arf1 activation in anchorage-dependent cells.

Cell-matrix adhesion regulates membrane trafficking controlling anchorage-dependent signaling. While a dynamic Golgi complex can contribute to this pathway, its regulation by adhesion remains unclear. Here we report that loss of adhesion dramatically disorganized the Golgi in mouse and human fibroblast cells.

Polymer Nanovesicle-Mediated Delivery of MLN8237 Preferentially Inhibits Aurora Kinase A To Target RalA and Anchorage-Independent Growth in Breast Cancer Cells.

The small GTPase RalA is a known mediator of anchorage-independent growth in cancers and is differentially regulated by adhesion and aurora kinase A (AURKA). Hence, inhibiting AURKA offers a means of specifically targeting RalA (over RalB) in cancer cells. MLN8237 (alisertib) is a known inhibitor of aurora kinases; its specificity for AURKA, however, is compromised by its poor solubility and transport across the cell membrane.

Ral-Arf6 crosstalk regulates Ral dependent exocyst trafficking and anchorage independent growth signalling.

Integrin dependent regulation of growth factor signalling confers anchorage dependence that is deregulated in cancers. Downstream of integrins and oncogenic Ras the small GTPase Ral is a vital mediator of adhesion dependent trafficking and signalling. This study identifies a novel regulatory crosstalk between Ral and Arf6 that controls Ral function in cells.

Deletion of inositol hexakisphosphate kinase 1 (IP6K1) reduces cell migration and invasion, conferring protection from aerodigestive tract carcinoma in mice.

Inositol hexakisphosphate kinases (IP6Ks), a family of enzymes found in all eukaryotes, are responsible for the synthesis of 5-diphosphoinositol pentakisphosphate (5-IP7) from inositol hexakisphosphate (IP6). Three isoforms of IP6Ks are found in mammals, and gene deletions of each isoform lead to diverse, non-overlapping phenotypes in mice. Previous studies show a facilitatory role for IP6K2 in cell migration and invasion, properties that are essential for the early stages of tumorigenesis.

Polysaccharide nano-vesicular multidrug carriers for synergistic killing of cancer cells.

Multi-drug delivery based on polymer nano-scaffolds is an essential protocol to be developed for better administration of anticancer drugs to enhance their therapeutic efficacies against cancer cells. Here, we report dual delivery polysaccharide nano-vesicles that are capable of loading and delivering both water soluble and water insoluble drugs together in a single polymer scaffold. The selective rupture of the nano-vesicular assembly under intracellular enzyme conditions allowed the simultaneous delivery of a hydrophobic drug camptothecin (CPT) and hydrophilic drug doxorubicin (DOX) supporting their synergistic killing of breast and colon cancer cells.

Dextran vesicular carriers for dual encapsulation of hydrophilic and hydrophobic molecules and delivery into cells.

Dextran vesicular nanoscaffolds were developed based on polysaccharide and renewable resource alkyl tail for dual encapsulation of hydrophilic and hydrophobic molecules (or drugs) and delivery into cells. The roles of the hydrophobic segments on the molecular self-organization of dextran backbone into vesicles or nanoparticles were investigated in detail. Dextran vesicles were found to be a unique dual carrier in which water-soluble molecules (like Rhodamine-B, Rh-B) and polyaromatic anticancer drug (camptothecin, CPT) were selectively encapsulated in the hydrophilic core and hydrophobic layer, respectively.

RalA-exocyst complex regulates integrin-dependent membrane raft exocytosis and growth signaling.

Anchorage dependence of cell growth is a key metastasis-suppression mechanism that is mediated by effects of integrins on growth signaling pathways. The small GTPase RalA is activated in metastatic cancers through multiple mechanisms and specifically induces anchorage independence. Loss of integrin-mediated adhesion triggers caveolin-dependent internalization of cholesterol- and sphingolipid-rich lipid raft microdomains to the recycling endosomes; these domains serve as platforms for many signaling pathways, and their clearance from the plasma membrane (PM) after cell detachment suppresses growth signaling.

Arf6 and microtubules in adhesion-dependent trafficking of lipid rafts.

Integrin-mediated adhesion regulates membrane binding sites for Rac1 within lipid rafts. Detachment of cells from the substratum triggers the clearance of rafts from the plasma membrane through caveolin-dependent internalization. The small GTPase Arf6 and microtubules also regulate Rac-dependent cell spreading and migration, but the mechanisms are poorly understood.

Integrin-mediated adhesion regulates membrane order.

The properties of cholesterol-dependent domains (lipid rafts) in cell membranes have been controversial. Because integrin-mediated cell adhesion and caveolin both regulate trafficking of raft components, we investigated the effects of adhesion and caveolin on membrane order. The fluorescent probe Laurdan and two-photon microscopy revealed that focal adhesions are highly ordered; in fact, they are more ordered than caveolae or domains that stain with cholera toxin subunit B (CtxB).

Phospho-caveolin-1 mediates integrin-regulated membrane domain internalization.

Growth of normal cells is anchorage dependent because signalling through multiple pathways including Erk, phosphatidylinositol-3-OH kinase (PI(3)K) and Rac requires integrin-mediated cell adhesion. Components of these pathways localize to low-density, cholesterol-rich domains in the plasma membrane named 'lipid rafts' or 'cholesterol-enriched membrane microdomains' (CEMM). We previously reported that integrin-mediated adhesion regulates CEMM transport such that cell detachment from the extracellular matrix triggers CEMM internalization and clearance from the plasma membrane.

Light-mediated activation of Rac-1 in photoreceptor outer segments.

Small GTP binding proteins regulate diverse biological processes including gene expression, cytoskeleton reorganization, and protein and vesicular transport. While small GTPases have been investigated in a wide variety of cells, few studies have addressed their role in photoreceptors. In vertebrate retinal rods, the light stimulus is transmitted from rhodopsin via the pathway mediated by the heterotrimeric G protein transducin.

Signal-dependent translocation of transducin, RGS9-1-Gbeta5L complex, and arrestin to detergent-resistant membrane rafts in photoreceptors.

Many lines of evidence show that membranes contain microdomains, "lipid rafts", that are different from the rest of the membrane in specific lipid and protein composition. In several biological systems, they were shown to be necessary for trafficking and signal transduction. Here, we investigate if lipid rafts have a role in the regulation of the G protein-mediated pathway underlying vertebrate phototransduction.

Protein kinase C isoforms in normal and leukemic neutrophils: altered levels in leukemic neutrophils and changes during myeloid maturation in chronic myeloid leukemia.

Protein kinase C (PKC) is reported to play a role in maturation of the myeloid cell and functions of the mature neutrophil. The neutrophils in chronic myeloid leukemia (CML) exhibit defects in several functions. As a step towards understanding the role of PKC in the defects in function of the leukemic cells, this study investigates the expression of PKC isoforms, their subcellular distribution, levels and kinase activity in the normal and leukemic neutrophils.