No abstract

Glioblastoma multiforme (GBM) is a life-threatening brain tumor. Accumulating evidence suggests that eradication of glioma stem-like cells (GSCs) in GBM is essential to achieve cure. The transcription factor FOXM1 has recently gained attention as a master regulator of mitotic progression of cancer cells in various organs. Here, we demonstrate that FOXM1 forms a protein complex with the mitotic kinase MELK in GSCs, leading to phosphorylation and activation of FOXM1 in a MELK kinase-dependent manner. This MELK-dependent activation of FOXM1 results in a subsequent increase in mitotic regulatory genes in GSCs. MELK-driven FOXM1 activation is regulated by the binding and subsequent trans-phosphorylation of FOXM1 by another kinase PLK1. Using mouse neural progenitor cells (NPCs), we found that transgenic expression of FOXM1 enhances, while siRNA-mediated gene silencing diminishes neurosphere formation, suggesting that FOXM1 is required for NPC growth. During tumorigenesis, FOXM1 expression sequentially increases as cells progress from NPCs, to pretumorigenic progenitors and GSCs. The antibiotic Siomycin A disrupts MELK-mediated FOXM1 signaling with a greater sensitivity in GSC compared to neural stem cell. Treatment with the first-line chemotherapy agent for GBM, Temozolomide, paradoxically enriches for both FOXM1 (+) and MELK (+) cells in GBM cells, and addition of Siomycin A to Temozolomide treatment in mice harboring GSC-derived intracranial tumors enhances the effects of the latter. Collectively, our data indicate that FOXM1 signaling through its direct interaction with MELK regulates key mitotic genes in GSCs in a PLK1-dependent manner and thus, this protein complex is a potential therapeutic target for GBM. Stem Cells 2013;31:1051–1063

Protein kinase MELK has oncogenic properties and is highly overexpressed in some tumors. In the present study, we show that a novel MELK inhibitor causes both the inhibition and degradation of MELK, culminating in replication stress and a senescence phenotype.

Background: Protein kinase MELK is expressed at very high levels in glioblastomas, but it is not understood how this benefits tumor growth. Results: A deficiency of MELK causes replication stress and is associated with cell cycle arrest and senescence. Conclusion: MELK is required for progression through unperturbed S phase. Significance: The inhibition of MELK emerges as an attractive cancer therapy.

Autophagy is biological mechanism allowing recycling of long-lived proteins, abnormal protein aggregates, and damaged organelles under cellular stress conditions. Following sequestration in double-or multimembrane autophagic vesicles, the cargo is delivered to lysosomes for degradation. ATG5 is a key component of an E3-like ATG12-ATG5-ATG16 protein complex that catalyzes conjugation of the MAP1LC3 protein to lipids, thus controlling autophagic vesicle formation and expansion. Accumulating data indicate that ATG5 is a convergence point for autophagy regulation. Here, we describe the scaffold protein RACK1 (receptor activated C-kinase 1, GNB2L1) as a novel ATG5 interactor and an autophagy protein. Using several independent techniques, we showed that RACK1 interacted with ATG5. Importantly, classical autophagy inducers (starvation or mammalian target of rapamycin blockage) stimulated RACK1-ATG5 interaction. Knockdown of RACK1 or prevention of its binding to ATG5 using mutagenesis blocked autophagy activation. Therefore, the scaffold protein RACK1 is a new ATG5-interacting protein and an important and novel component of the autophagy pathways.Autophagy is a highly conserved biological mechanism that is responsible for lysosome-dependent recycling of long-lived abnormal or misfolded proteins as well as dysfunctional or unnecessary organelles (such as depolarized mitochondria) (1). Under normal conditions, basal autophagy help maintain cellular homeostasis. Autophagy is rapidly up-regulated following stress, including nutrient deprivation, accumulation of misfolded proteins, mitochondrial depolarization, or exposure to toxic chemicals (2). Autophagy malfunctions were shown to contribute to several pathologies, such as neurodegenerative diseases, lysosomal storage disorders, and cancer (3).The process starts with the nucleation and elongation of double-membrane structures called "autophagosomes" or "autophagic vesicles." As they mature through fusion with late endosomes or lysosomes, vesicles give rise to "autolysosomes," a hybrid compartment in which vesicle contents are degraded by the action of lysosomal hydrolases (4). So far, around 33 different core autophagy proteins (ATGs) were described (5). Among them, two ubiquitination-like reactions are key to autophagic vesicle membrane elongation as follows: ATG12-ATG5-ATG16L1 and ATG8 (MAP1LC3 or shortly LC3 in mammals). The first ubiquitination-like reaction results in the covalent conjugation of a Lys-130 residue of the ATG5 protein to a ubiquitin-like protein, ATG12. Following addition of ATG16L1 to the ATG12-conjugated ATG5, a larger complex of around 669 -800 kDa forms (6). The ATG12-ATG5-ATG16L1 complex serves as an E3-like enzyme for the second ubiquitylation-like reaction. Here, the LC3 protein is covalently attached to a lipid molecule, generally to a phosphatidylethanolamine contributing to the elongation of autophagic membranes (7,8). Conversion of the free cytosolic form of LC3 (LC3-I) to the lipid-conjugated form (LC3-II) leads to its localization to dot-li...