A missing component of this model, however, is a set of proteins responsible for the recruitment, production, Introduction and release of a wait signal, i.e., the factors that facilitate the proposed catalysis. One important characteristic of Genomic integrity is maintained by a number of checkcomponents of such a catalytic kinetochore scaffold points that act during each cell cycle. The prevention would be a long residence time at unattached kinetoof gross chromosome missegregation and the resulting chores and therefore a long recovery time after photoaneuploidy is carried out by the mitotic checkpoint (also bleaching. In addition, this catalytic machinery would referred to as the spindle assembly checkpoint; for rebe expected to be deactivated by microtubule attachview, see [1, 2] Figures 2B and 2F). The raised against human Mad1 yielded maximal estimates small cycling pool showed rapid kinetics (t 1/2 ϭ 6.6 s Ϯ ( Figure 1A) of the levels of the HsMad1 fusions to be 1.1 SEM) that were not significantly different from recovapproximately 1.5-fold and 5-fold (for C-terminal and ery times measured at kinetochores in normal mitoses N-terminal fusions, respectively) that of the endogenous or in nocodazole-blocked mitotic cells ( Figure 2F). After Mad1 level. However, because the antibody affinity for nuclear reassembly in early G1 and throughout inthe PTK2 Mad1 (PtMad1) homolog is very likely to be terphase, Mad1 was stably bound at the nuclear envesignificantly lower than for hsMad1, the actual levels lope (Figures 2A and 2D). Neither N-nor C-terminal YFP were almost certainly significantly lower than these estifusions recovered significantly within 15 min after photomates, which is consistent with the absence of an effect bleaching (R% Ͻ increase (approximately 15%) in the length of mitosis ochores, both C-and N-terminal fusions of Mad2 partially recovered at kinetochores during normal mitoses (see Figure S1). Immunoblotting with an antibody to hsMad2 ( Figure 3A) permitted maximal estimates of ap-( Figure 3C; R% ϭ 60.0% Ϯ 2.7 SEM; t 1/2 ϭ 10.8 s Ϯ 1.2 SEM, n ϭ 60) or in nocodazole-treated, mitotic checkproximately 20-fold and 5-fold the level of endogenous PtMad2 for the C-terminal and N-terminal fusions, repoint-arrested cells ( Figure 3F; R% ϭ 53.8% Ϯ 3.1 SEM; t 1/2 ϭ 6.4 s Ϯ 0.7 SEM, n ϭ 59). Mad2 that accumulated spectively. Because this antibody almost certainly has lower affinity for PtMad2, it is highly likely that the actual near spindle poles after microtubule attachment continued to cycle as rapidly and to a similar recovery percentlevels were much lower than these estimates, consistent with absence of the kinetochore-independent mitotic age as Mad2 Figure 5C, lanes 2 and 7). Mad2/YFP fusions constitutive centromeric protein, CENP-C, and the kinetochore kinase Bub1, known to be essential for the mialso coprecipitated endogenous Mad2 both in randomly cycling and in mitotic cells ( Figure 5C, lanes 3 and 8) Bub1: R% Ͻ 10%, t 1/2 Ͼ 15 min, n ϭ 21) or when the kinetochores; Mad1 is stably bound, whereas a...

Glioblastoma remains one of the deadliest of human cancers, with most patients succumbing to the disease within two years of diagnosis. The available data suggest that simultaneous inactivation of critical nodes within the glioblastoma molecular circuitry will be required for meaningful clinical efficacy. We conducted parallel genome-wide shRNA screens to identify such nodes and uncovered a number of G-Protein Coupled Receptor (GPCR) neurotransmitter pathways, including the Dopamine Receptor D2 (DRD2) signaling pathway. Supporting the importance of DRD2 in glioblastoma, DRD2 mRNA and protein expression were elevated in clinical glioblastoma specimens relative to matched non-neoplastic cerebrum. Treatment with independent si-/shRNAs against DRD2 or with DRD2 antagonists suppressed the growth of patient-derived glioblastoma lines both in vitro and in vivo. Importantly, glioblastoma lines derived from independent genetically engineered mouse models (GEMMs) were more sensitive to haloperidol, an FDA approved DRD2 antagonist, than the premalignant astrocyte lines by approximately an order of magnitude. The pro-proliferative effect of DRD2 was, in part, mediated through a GNAI2/Rap1/Ras/ERK signaling axis. Combined inhibition of DRD2 and Epidermal Growth Factor Receptor (EGFR) led to synergistic tumoricidal activity as well as ERK suppression in independent in vivo and in vitro glioblastoma models. Our results suggest combined EGFR and DRD2 inhibition as a promising strategy for glioblastoma treatment.

We explored the clinical and pathological impact of epidermal growth factor receptor (EGFR) extracellular domain missense mutations. Retrospective assessment of 260 de novo glioblastoma patients revealed a significant reduction in overall survival of patients having tumors with EGFR mutations at alanine 289 (EGFR). Quantitative multi-parametric magnetic resonance imaging analyses indicated increased tumor invasion for EGFR mutants, corroborated in mice bearing intracranial tumors expressing EGFR and dependent on ERK-mediated expression of matrix metalloproteinase-1. EGFR tumor growth was attenuated with an antibody against a cryptic epitope, based on in silico simulation. The findings of this study indicate a highly invasive phenotype associated with the EGFR mutation in glioblastoma, postulating EGFR as a molecular marker for responsiveness to therapy with EGFR-targeting antibodies.

Glioma stem cells (GSCs) comprise a small subpopulation of glioblastoma multiforme cells that contribute to therapy resistance, poor prognosis, and tumor recurrence. Protective autophagy promotes resistance of GSCs to anoikis, a form of programmed cell death occurring when anchorage-dependent cells detach from the extracellular matrix. In nonadherent conditions, GSCs display protective autophagy and anoikis-resistance, which correlates with expression of melanoma differentiation associated gene-9/Syntenin (MDA-9) (syndecan binding protein; SDCBP). When MDA-9 is suppressed, GSCs undergo autophagic death supporting the hypothesis that MDA-9 regulates protective autophagy in GSCs under anoikis conditions. MDA-9 maintains protective autophagy through phosphorylation of BCL2 and by suppressing high levels of autophagy through EGFR signaling. MDA-9 promotes these changes by modifying FAK and PKC signaling. Gain-of-function and loss-of-function genetic approaches demonstrate that MDA-9 regulates pEGFR and pBCL2 expression through FAK and pPKC. EGFR signaling inhibits autophagy markers (ATG5, Lamp1, LC3B), helping to maintain protective autophagy, and along with pBCL2 maintain survival of GSCs. In the absence of MDA-9, this protective mechanism is deregulated; EGFR no longer maintains protective autophagy, leading to highly elevated and sustained levels of autophagy and consequently decreased cell survival. In addition, pBCL2 is down-regulated in the absence of MDA-9, leading to cell death in GSCs under conditions of anoikis. Our studies confirm a functional link between MDA-9 expression and protective autophagy in GSCs and show that inhibition of MDA-9 reverses protective autophagy and induces anoikis and cell death in GSCs.