Na+/K+-ATPase β2-subunit (AMOG) expression abrogates invasion of glioblastoma-derived brain tumor-initiating cells

Diffusely infiltrative astrocytomas (World Health Organization [WHO] grades II–IV) are characterized by their potential to widely penetrate the normal brain parenchyma.1 Glioblastoma multiforme (GBM) is the highest-grade astrocytoma, and its patterns of dissemination have been directly linked to survival, where widespread dissemination is thought to give rise to more aggressive phenotypes.2 Increasing evidence suggests that all GBM tumors consist of heterogeneous populations of tumor cells in varied differentiation status,3 including well-differentiated tumor cells and less-differentiated stem cells. The less-differentiated stem cells from tumors are characterized by their expression of neural stem cell markers, such as CD133,4 nestin,5 and Sox2 (sex determining region Y-box 2).6 Functionally, these glioblastoma stem cells have been defined by their potent capacity for developing tumors when implanted in animals, as well as their ability to give rise to both stem cells and well-differentiated tumor cells.4,7–13 Thus, the less-differentiated tumor stem cells have also been called brain tumor-initiating cells (BTICs).4 In addition to their enhanced self-renewal and cell differentiation potential, BTICs have been shown to have increased resistance to chemo- and radiotherapy.4,7–13 Therefore, they have been identified as potentially important targets of GBM therapy. While increasing evidence suggests that they play a role in glioma pathogenesis, very little evidence exists to date characterizing the invasive potential of BTICs. Limited evidence, based primarily on in vitro experiments, initially suggested that BTICs might be more invasive than differentiated glioma cells.14–16 However, the mechanism of the elevated invasive potential of BTICs has not been clearly delineated. Conversely, there is also evidence that cancer stem cells are not always invasive, depending on their phenotype, which further complicates our understanding of cancer stem cell biology.17 Recent research suggests that gliomas utilize ion channels, including Na+/K+-ATPase, to support their unusual growth and invasion by adjusting their shape and volume rapidly as they invade the brain parenchyma.18 The various isoforms of the β-subunit of Na+/K+-ATPase have been shown to act in regulating cellular adhesion, particularly in the context of cancer progression.19–21 The β2 isoform, also known as the adhesion molecule on glia (AMOG), is a heavily glycosylated protein that plays a role in cellular adhesion in the CNS.22,23 It is diffusely expressed in the gray matter but is expressed in only perivascular astrocytes in the white matter.24 Although AMOG is highly expressed in the normal adult CNS, evidence suggests that it may be downregulated in GBM.25 However, given the heterogeneity that exists among GBM tumors, it is not known whether this is universally true of GBM. Initial functional data characterizing AMOG suggest that it plays an active role in cellular adhesion and migration in the normal CNS.22,26 While loss of AMOG has been implicated in glioma invasion and migration, currently there are no data characterizing its function in invasion and migration specifically in human glioma cells. To further our understanding of the role of AMOG in GBM and particularly in BTICs in the context of glioma migration and invasion, we specifically investigated (i) whether AMOG protein expression correlated with glioma grade, (ii) whether AMOG is globally downregulated in GBM, (iii) whether AMOG is differentially expressed in BTICs compared with differentiated GBM cells from patient-matched tumor tissues, (iv) whether overexpression of AMOG in low-expressing GBM cells decreases invasion and affects migration or proliferation, (v) whether AMOG knockdown increases invasion of normal glial cells, and (vi) whether downregulation of AMOG in GBM is associated with worse clinical outcome.