The cotransporter NKCC1 increases cell traction forces to enhance speed and direction, thereby promoting glioma cell invasion.
Knocking down NKCC1 in glioblastoma cells increases the size of focal adhesions, resulting in a reduced migratory capacity. Glioblastoma cells transduced to express a control vector (left panel) or a NKCC1 shRNA (right panel) were immunostained against vinculin (red) to measure focal adhesions (yellow arrows). Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm. Image courtesy of Dr Hugo Guerrero Cázares, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Through their regulation of cell volume and influence on the cytoskeleton, ion transporters are increasingly gaining recognition in the control of cell migration. The electroneutral Na+-K+-Cl- cotransporter 1 (NKCC1) has already been implicated in the invasiveness of glioblastoma, but only now has a putative mechanism been eludicated; it is described in PLoS Biology by Garzon-Muvdi et al.
The authors showed that glioblastoma and anaplastic astrocytoma samples harboured much higher levels of NKCC1 protein than did normal brain and low-grade astrocytoma samples. Primary human glioblastoma cells also expressed high NKCC1 protein levels, highlighting a positive correlation between NKCC1 expression and increased invasiveness. Accordingly, inhibiting NKCC1 pharmacologically or using short hairpin (sh)RNA significantly reduced the invasiveness of primary human glioma cells in transwell assays. Conversely, inhibiting the KCC transporter, which works together with, but inversely to, NKCC1 increased cell invasion. On a nanopatterned substrate, which mimics the features of the extracellular matrix upon which brain cancer cells might migrate, inhibiting NKCC1 reduced both the speed and directionality of glioblastoma cell migration. Furthermore, depleting NKCC1 through shRNA caused primary brain tumour stem cells to form larger, but less invasive, orthotopic in vivo tumours.
So, NKCC1 promotes cell motility, but does it do so through a mechanism other than cell volume regulation? Glioblastoma cells lacking NKCC1 formed more mature, and larger, focal adhesions than control cells. Mature adhesions mediate cell–substrate adhesion, in contrast to nascent adhesions, which generate traction and contractile forces to enable cell movement; NKCC1-deficient cells indeed showed decreased contractile force generation.
Other ion transporters such as NHE1 bind to ezrin–radixin–moesin (ERM) proteins, thereby anchoring actin to the plasma membrane, which might influence contractility. Garzon-Muvdi et al. identified clusters of positively charged residues — identical to those found in other ERM-binding proteins — in the carboxy-terminal juxtamembrane domain of human NKCC1, and demonstrated that NKCC1, ezrin and actin co-immunoprecipitated in primary glioblastoma cells. Cells expressing ezrin-binding null NKCC1 generated decreased contractile movements and, consistent with the notion that NKCC1 anchors the cytoskeleton during migration, immunocytochemistry and live-cell imaging experiments localized NKCC1 to the extending processes of migrating glioblastoma cells.
Finally, the authors investigated the mechanism(s) regulating NKCC1 activity. Epidermal growth factor (EGF), which promotes astrocytic and glial cell migration, increased NKCC1 phosphorylation in glioma cells via phosphatidylinositol 3-kinase (PI3K) signalling to Akt. Akt, in turn, phosphorylated, and thereby activated, WNK3, an unconventional serine/threonine kinase that regulates the transport activity of NKCC1 through phosphorylation.
Taken together, these results indicate that the membrane transporter NKCC1 promotes cell invasion by PI3K–Akt signalling to WNK3 downstream of EGF, and quite feasibly other motility factors that stimulate this pathway. Localized at advancing membrane protrusions, NKCC1 interacts with ezrin to anchor the cytoskeleton, thereby generating contractile forces and decreasing cell–substrate adhesion to favour cell migration, while presumably also mediating local volume changes. Given its ubiquitous expression, NKCC1 could feasibly be involved in the physiological migration of non-malignant cells as well as metastasis of other invasive tumours.
ORIGINAL RESEARCH PAPER
Garzon-Muvdi, T. et al. Regulation of brain tumor dispersal by NKCC1 through a novel role in focal adhesion regulation.
doi 10.1371/journal.pbio.1001320 | Article