Glioma-mediated microglial activation promotes glioma proliferation and migration: roles of Na+/H+ exchanger isoform 1

Carcinogenesis Glioma-mediated microglial activation promotes glioma proliferation and migration: roles of Na+/H+ exchanger isoform 1 Wen Zhu 2 Karen E. Carney 2 Victoria M. Pigot 2 Lindsay M. Falgoust 2 Paul A.Clar k 0 1 John S. Kuo 0 1 Dandan Sun 2 3 0 Carbone Cancer Center, University of Wisconsin School of Medicine and Public Health , Madison, WI 53792 , USA 1 DAe,partment of Neurological Surgery 2 Department of Neurology, University of Pittsburgh , Pittsburgh, PA 15213 , US 3 Veterans Affairs Pittsburgh Health Care System, Geriatric Research, Educational and Clinical Center , Pittsburgh, PA , USA Microglia play important roles in extracellular matrix remodeling, tumor invasion, angiogenesis, and suppression of adaptive immunity in glioma. Na+/H+ exchanger isoform 1 (NHE1) regulates microglial activation and migration. However, little is known about the roles of NHE1 in intratumoral microglial activation and microglia-glioma interactions. Our study revealed up-regulation of NHE1 protein expression in both glioma cells and tumor-associated I+bmai1croglia in glioma xenografts and glioblastoma multiforme microarrays. Moreover, we observed positive correlation of NHE1 expression with Iba1 intensity in microglia/macrophages. Glioma cells, via conditioned medium or non-contact glioma-microglia co-cultures, concurrently upregulated microglial expression of NHE1 protein and other microglial activation markers (iNOS, arginase-1, TGF-β, IL-6, IL-10 and the matrix metalloproteinases MT1-MMP and MMP9). Interestingly, glioma-stimulated microglia reciprocally enhanced glioma proliferation and migration. Most importantly, inhibition of microglial NHE1 activity via small interfering RNA (siRNA) knockdown or the potent NHE1-specific inhibitor HOE642 significantly attenuated microglial activation and abolished microglia-stimulated glioma migration and proliferation. Taken together, our findings provide the first evidence that NHE1 function plays an important role in glioma-microglia interactions, enhancing glioma proliferation and invasion by stimulating microglial release of soluble factors. NHE1 upregulation is a novel marker of the glioma-associated microglial activation phenotype. Inhibition of NHE1 represents a novel glioma therapeutic strategy by targeting tumor-induced microglial activation. - Introduction Malignant gliomas are highly aggressive brain cancers with microglia and infiltrating macrophages account for up to 30% poor prognosis. Patients with the most aggressive glioblasto- of the glioma mass, and represent important components of mas have a median survival of only 14–18 months due to rapid the glioma microenvironment (6). Glioma cell survival, growth tumor growth and therapeutic resistance to current treatmentsand metastasis are facilitated by tumor-associated microglia (1–4). Accumulating evidence indicates that malignant gliomas and macrophages (7,8). For example, tumor cells synthesize and are heterogeneous masses comprised of tumor cells intermixed release colony stimulating factor 1 (CSF-1), triggering migrawith parenchymal cells. This unique tumor microenvironment tion of microglia and macrophages to the tumor sites and proplays a vital role in glioma progression (5). Most importantly, moting subsequent microglia/monocyte release of epidermal Abbreviations For the preparation of astrocyte-conditioned medium (ACM) or gliomaconditioned medium (GCM) treated microglia cultures, on day 1,×2106 astrocytes or glioma cells were seeded in 1m00m dishes in 10 ml DMEM + 10% FBS media overnight. On day 2, cells were washed twice with serumalternative resolving phenotypes, with identification of a spe- free DMEM/F12 medium and then incubated with 10ml fresh DMEM/ cific glioma-associated activation phenotype in GAMs (10–14). F12 + 10% FBS at 37°C for 24h. Also, 2× 106 microglia cells were seeded Na+/H+ exchanger isoform 1 (NHE1) protein is over-expressed in collagen coated 10m0m dishes in DMEM/F12 + 10% FBS overnight. On in gliomas and other cancer cells to maintain alkaline intracellu-day 3, the medium from astrocyte or glioma cultures (80–90% confluent) lar pH (pHi) by extruding H+ (15,16). We recently reported that NHE1 was collected and centrifuged at 2000g at 4°C form1i0n. The supernatant co-localizes with ezrin at lamellipodia of glioma. Upregulation of was collected as ACM or GCM. Fresh conditioned media was prepared for NHE1 protein expression in glioma plays a vital role in glioma each set of cultures. Microglia cultures were washed twice with serummigration and survival (16). Increased NHE1 also enhances glioma free DMEM/F12 medium followed by incubation with 10ml of ACM or GCM cell resistance to TMZ-mediated apoptosis via activation of extra- plus or minus 1  µM HOE642 (Sanofi-Aventis, Germany), 0.5  µM MK2206 (Selleck Chemical), or 100ng/ml lipopolysaccharide (Sigma) for 24h. On cellular signal-regulated kinase (ERK) signaling pathways (16). day 4, the medium was then collected from the microgl (...truncated)