EGF-induced sodium influx regulates EGFR trafficking through HDAC6 and tubulin acetylation.

BACKGROUND: Endocytosis of activated EGF receptor (EGFR) to specific endocytic compartments is required to terminate EGF signaling. Trafficking of EGFR relies on microtubule tracks that transport the cargo vesicle to their intermediate and final destinations and can be modulated through posttranslational modification of tubulin including acetylation. Na,K-ATPase maintains intracellular sodium homeostasis, functions as a signaling scaffold and interacts with EGFR. Na,K-ATPase also binds to and is regulated by acetylated tubulin but whether there is a functional link between EGFR, Na,K-ATPase and tubulin acetylation is not known. RESULTS: EGF-induced sodium influx regulates EGFR trafficking through increased microtubule acetylation. Increased sodium influx induced either by sodium ionophores or Na,K-ATPase blockade mimicked the EGF-induced effects on EGFR trafficking through histone deacetylase (HDAC) 6 inactivation and accumulation of acetylated tubulin. In turn, blocking sodium influx reduced tubulin acetylation and EGF-induced EGFR turnover. Knockdown of HDAC6 reversed the effect of sodium influx indicating that HDAC6 is necessary to modulate sodium-dependent tubulin acetylation. CONCLUSIONS: These studies provide a novel regulatory mechanism to attenuate EGFR signaling in which EGF modulates EGFR trafficking through intracellular sodium-mediated HDAC6 inactivation and tubulin acetylation.

Na,K-ATPase ß1-subunit is a target of sonic hedgehog signaling and enhances medulloblastoma tumorigenicity.

BACKGROUND: The Sonic hedgehog (Shh) signaling pathway plays an important role in cerebellar development, and mutations leading to hyperactive Shh signaling have been associated with certain forms of medulloblastoma, a common form of pediatric brain cancer. While the fundamentals of this pathway are known, the molecular targets contributing to Shh-mediated proliferation and transformation are still poorly understood. Na,K-ATPase is a ubiquitous enzyme that maintains intracellular ion homeostasis and functions as a signaling scaffold and a cell adhesion molecule. Changes in Na,K-ATPase function and subunit expression have been reported in several cancers and loss of the ß1-subunit has been associated with a poorly differentiated phenotype in carcinoma but its role in medulloblastoma progression is not known. METHODS: Human medulloblastoma cell lines and primary cultures of cerebellar granule cell precursors (CGP) were used to determine whether Shh regulates Na,K-ATPase expression. Smo/Smo medulloblastoma were used to assess the Na,K-ATPase levels in vivo. Na,K-ATPase ß1-subunit was knocked down in DAOY cells to test its role in medulloblastoma cell proliferation and tumorigenicity. RESULTS: Na,K-ATPase ß1-subunit levels increased with differentiation in normal CGP cells. Activation of Shh signaling resulted in reduced ß1-subunit mRNA and protein levels and was mimicked by overexpression of Gli1and Bmi1, both members of the Shh signaling cascade; overexpression of Bmi1 reduced ß1-subunit promoter activity. In human medulloblastoma cells, low ß1-subunit levels were associated with increased cell proliferation and in vivo tumorigenesis. CONCLUSIONS: Na,K-ATPase ß1-subunit is a target of the Shh signaling pathway and loss of ß1-subunit expression may contribute to tumor development and progression not only in carcinoma but also in medulloblastoma, a tumor of neuronal origin.

A Functional Interaction Between Na,K-ATPase ß2-Subunit/AMOG and NF2/Merlin Regulates Growth Factor Signaling in Cerebellar Granule Cells.

The Na,K-ATPase, consisting of a catalytic α-subunit and a regulatory ß-subunit, is a ubiquitously expressed ion pump that carries out the transport of Na+ and K+ across the plasma membranes of most animal cells. In addition to its pump function, Na,K-ATPase serves as a signaling scaffold and a cell adhesion molecule. Of the three ß-subunit isoforms, ß1 is found in almost all tissues, while ß2 expression is mostly restricted to brain and muscle. In cerebellar granule cells, the ß2-subunit, also known as adhesion molecule on glia (AMOG), has been linked to neuron-astrocyte adhesion and granule cell migration, suggesting its role in cerebellar development. Nevertheless, little is known about molecular pathways that link the ß2-subunit to its cellular functions. Using cerebellar granule precursor cells, we found that the ß2-subunit, but not the ß1-subunit, negatively regulates the expression of a key activator of the Hippo/YAP signaling pathway, Merlin/neurofibromin-2 (NF2). The knockdown of the ß2-subunit resulted in increased Merlin/NF2 expression and affected downstream targets of Hippo signaling, i.e., increased YAP phosphorylation and decreased expression of N-Ras. Further, the ß2-subunit knockdown altered the kinetics of epidermal growth factor receptor (EGFR) signaling in a Merlin-dependent mode and impaired EGF-induced reorganization of the actin cytoskeleton. Therefore, our studies for the first time provide a functional link between the Na,K-ATPase ß2-subunit and Merlin/NF2 and suggest a role for the ß2-subunit in regulating cytoskeletal dynamics and Hippo/YAP signaling during neuronal differentiation.

Sustained release of active chemotherapeutics from injectable-solid ß-hairpin peptide hydrogel.

MAX8 ß-hairpin peptide hydrogel is a solid, preformed gel that can be syringe injected due to shear-thinning properties and can recover solid gel properties immediately after injection. This behavior makes the hydrogel an excellent candidate as a local drug delivery vehicle. In this study, vincristine, a hydrophobic and commonly used chemotherapeutic, is encapsulated within MAX8 hydrogel and shown to release constantly over the course of one month. Vincristine was observed to be cytotoxic in vitro at picomolar to nanomolar concentrations. The amounts of drug released from the hydrogels over the entire time-course were in this concentration range. After encapsulation, release of vincristine from the hydrogel was observed for four weeks. Further characterization showed the vincristine released during the 28 days remained biologically active, well beyond its half-life in bulk aqueous solution. This study shows that vincristine-loaded MAX8 hydrogels are excellent candidates as drug delivery vehicles, through sustained, low, local and effective release of vincristine to a specific target. Oscillatory rheology was employed to show that the shear-thinning and re-healing, injectable-solid properties that make MAX8 a desirable drug delivery vehicle are unaffected by vincristine encapsulation. Rheology measurements also were used to monitor hydrogel nanostructure before and after drug encapsulation.

Cancer as a channelopathy: ion channels and pumps in tumor development and progression.

Increasing evidence suggests that ion channels and pumps not only regulate membrane potential, ion homeostasis, and electric signaling in excitable cells but also play important roles in cell proliferation, migration, apoptosis and differentiation. Consistent with a role in cell signaling, channel proteins and ion pumps can form macromolecular complexes with growth factors, and cell adhesion and other signaling molecules. And while cancer is still not being cataloged as a channelopathy, as the non-traditional roles of ion pumps and channels are being recognized, it is increasingly being suggested that ion channels and ion pumps contribute to cancer progression. Cancer cell migration requires the regulation of adhesion complexes between migrating cells and surrounding extracellular matrix (ECM) proteins. Cell movement along solid surfaces requires a sequence of cell protrusions and retractions that mainly depend on regulation of the actin cytoskeleton along with contribution of microtubules and molecular motor proteins such as mysoin. This process is triggered and modulated by a combination of environmental signals, which are sensed and integrated by membrane receptors, including integrins and cadherins. Membrane receptors transduce these signals into downstream signaling pathways, often involving the Rho GTPase protein family. These pathways regulate the cytoskeletal rearrangements necessary for proper timing of adhesion, contraction and detachment of cells in order to find their way through extracellular spaces. Migration and adhesion involve continuous modulation of cell motility, shape and volume, in which ion channels and pumps play major roles. Research on cancer cells suggests that certain ion channels may be involved in aberrant tumor growth and channel inhibitors often lead to growth arrest. This review will describe recent research into the role of ion pumps and ion channels in cell migration and adhesion, and how they may contribute to tumor development.

Inhibition of epidermal growth factor signaling by the cardiac glycoside ouabain in medulloblastoma.

Epidermal growth factor (EGF) signaling regulates cell growth, proliferation, and differentiation. Upon receptor binding, EGF triggers cascades of downstream signaling, including the MAPK and phosphoinositide-3-kinase (PI3K)/Akt signaling pathways. Aberrant expression/activation of EGFR is found in multiple human cancers, including medulloblastoma, the most prevalent pediatric brain cancer, and often has been associated with metastasis, poor prognosis, and resistance to chemotherapy. Na,K-ATPase is an ion pump well known for its role in intracellular ion homeostasis. Recent studies showed that Na,K-ATPase also functions as a signaling platform and revealed a role in EGFR, MAPK, and PI3K signaling. While both EGFR and Na,K-ATPase seem to modulate similar signaling pathways, cardiac glycosides that are steroid-like inhibitors of Na,K-ATPase, exhibit antiproliferative and proapoptotic properties in cancer cells. Thus, we sought to better understand the relationship between EGF and cardiac glycoside signaling. Here, we show that in medulloblastoma cells, both EGF and ouabain activate Erk1/2 and PI3K/Akt signaling. Nevertheless, in medulloblastoma cells ouabain did not transactivate EGFR as has been reported in various other cell lines. Indeed, ouabain inhibited EGF-induced Erk1/2 and Akt activation and, moreover, prevented EGF-induced formation of actin stress fibers and cell motility, probably by activating a stress signaling response. Na,K-ATPase has been proposed to act as a signaling scaffold and our studies suggest that in medulloblastoma cells Na,K-ATPase might act as a check point to integrate EGF-associated signaling pathways. Thus, Na,K-ATPase might serve as a valid target to develop novel therapeutic approaches in tumors with aberrant activation of the EGFR signaling cascades.