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Fine tuning of the protein folding environment in subcellular organelles, such as mitochondria, is important for adaptive homeostasis and may participate in human diseases, but the regulators of this process are still largely elusive. Here, we have shown that selective targeting of heat shock protein-90 (Hsp90) chaperones in mitochondria of human tumor cells triggered compensatory autophagy and an organelle unfolded protein response (UPR) centered on upregulation of CCAAT enhancer binding protein (C/EBP) transcription factors. In turn, this transcriptional UPR repressed NF-κB-dependent gene expression, enhanced tumor cell apoptosis initiated by death receptor ligation, and inhibited intracranial glioblastoma growth in mice without detectable toxicity. These data reveal what we believe to be a novel role of Hsp90 chaperones in the regulation of the protein-folding environment in mitochondria of tumor cells. Disabling this general adaptive pathway could potentially be used in treatment of genetically heterogeneous human tumors., Introduction Cells use sensor pathways compartmentalized in subcellular organelles to recognize stress conditions (1), including aberrant protein folding (2), and in response activate gene expression programs aimed at maintaining cell [...]

Activating transcription factor-5 (ATF5) is highly expressed in malignant glioma and has a key role in promoting cell survival. Here we perform a genome-wide RNAi screen to identify transcriptional regulators of ATF5. Our results reveal an essential survival pathway in malignant glioma, whereby activation of a RAS-mitogen-activated protein kinase or phosphoinositide- 3-kinase signaling cascade leads to induction of the transcription factor cAMP response element-binding protein-3-like-2 (CREB3L2), which directly activates ATF5 expression. ATF5, in turn, promotes survival by stimulating transcription of myeloid cell leukemia sequence-1 (MCL1), an antiapoptotic B cell leukemia-2 family member. Analysis of human malignant glioma samples indicates that ATF5 expression inversely correlates with disease prognosis. The RAF kinase inhibitor sorafenib suppresses ATF5 expression in glioma stem cells and inhibits malignant glioma growth in cell culture and mouse models. Our results demonstrate that ATF5 is essential in malignant glioma genesis and reveal that the ATF5-mediated survival pathway described here provides potential therapeutic targets for treatment of malignant glioma., A hallmark of cancer is uncontrolled cellular proliferation, which can arise as a result of overexpression of genes promoting cell survival or downregulation of genes inducing cell death (1). Genes [...]

The mammalian tumor suppressor, phosphatase and tensin homologue deleted on chromosome 10 (PTEN), inhibits cell growth and survival by dephosphorylating phosphatidylinositol-(3,4,5)-trisphosphate (PI[3,4,5][P.sub.3]). We have found a homologue of PTEN in the fission yeast, Schizosaccharomyces pombe (ptn1). This was an unexpected finding because yeast (S. pombe and Saccharomyces cerevisiae) lack the class I phosphoinositide 3-kinases that generate PI(3,4,5)[P.sub.3] in higher eukaryotes. Indeed, PI(3,4,5)[P.sub.3] has not been detected in yeast. Surprisingly, upon deletion of ptn1 in S. pombe, Pl(3,4,5)[P.sub.3] became detectable at levels comparable to those in mammalian cells, indicating that a pathway exists for synthesis of this lipid and that the S. pombe ptn1, like mammalian PTEN, suppresses PI(3,4,5)[P.sub.3] levels. By examining various mutants, we show that synthesis of PI(3,4,5)[P.sub.3] in S. pombe requires the class III phosphoinositide 3-kinase, vps34p, and the phosphatidylinositol-4phosphate 5-kinase, its3p, but does not require the phosphatidylinositol-3-phosphate 5-kinase, fab1p. These studies suggest that a pathway for PI(3,4,5)[P.sub.3] synthesis downstream of a class III phosphoinositide 3-kinase evolved before the appearance of class I phosphoinositide 3-kinases.

The direct spectroscopic evidence of a conformational change observed in phosphatase and tensin homologue deleted on chromosome 10 (PTEN) bound to PI(4,5)[P.sub.2]-bearing vesicles with increased [alpha]-helicity is discussed. The results demonstrate that PTEN binds to membranes through multiple sites, but only binding of PI(4,5)[P.sub.2] to the N-terminal domain triggers a conformational change with increased [alpha]-helicity.

Glioblastoma multiforme (GBM) is a grade IV brain tumor characterized by a heterogeneous population of cells that are highly infiltrative, angiogenic and resistant to chemotherapy. The current standard of care, comprised of surgical resection followed by radiation and the chemotherapeutic agent temozolomide, only provides patients with a 12-14 month survival period post-diagnosis. Long-term survival for GBM patients remains uncommon as cells with intrinsic or acquired resistance to treatment repopulate the tumor. In this review we will describe the mechanisms of resistance, and how they may be overcome to improve the survival of GBM patients by implementing novel chemotherapy drugs, new drug combinations and new approaches relating to DNA damage, angiogenesis and autophagy. [ABSTRACT FROM AUTHOR]

The structure and function of the PTEN phosphatase is investigated by studying its membrane affinity and localization on in-plane fluid, thermally disordered synthetic membrane models. The membrane association of the protein depends strongly on membrane composition, where phosphatidylserine (PS) and phosphatidylinositol diphosphate (PI(4,5)P2) act pronouncedly synergistic in pulling the enzyme to the membrane surface. The equilibrium dissociation constants for the binding of wild type (wt) PTEN to PS and PI(4,5)P2 were determined to be Kd∼12 μM and 0.4 μ, respectively, and Kd∼50 nM if both lipids are present. Membrane affinities depend critically on membrane fluidity, which suggests multiple binding sites on the protein for PI(4,5)P2. The PTEN mutations C124S and H93R show binding affinities that deviate strongly from those measured for the wt protein. Both mutants bind PS more strongly than wt PTEN. While C124S PTEN has at least the same affinity to PI(4,5)P2 and an increased apparent affinity to PI(3,4,5)P3, due to its lack of catalytic activity, H93R PTEN shows a decreased affinity to PI(4,5)P2 and no synergy in its binding with PS and PI(4,5)P2. Neutron reflection measurements show that the PTEN phosphatase "scoots" along the membrane surface (penetration <5 Å) but binds the membrane tightly with its two major domains, the C2 and phosphatase domains, as suggested by the crystal structure. The regulatory C-terminal tail is most likely displaced from the membrane and organized on the far side of the protein, ∼60 Å away from the bilayer surface, in a rather compact structure. The combination of binding studies and neutron reflection allows us to distinguish between PTEN mutant proteins and ultimately may identify the structural features required for membrane binding and activation of PTEN. [ABSTRACT FROM AUTHOR]

Activating transcription factor-5 (ATF5) is highly expressed in malignant glioma and has a key role in promoting cell survival. Here we perform a genome-wide RNAi screen to identify transcriptional regulators of ATF5. Our results reveal an essential survival pathway in malignant glioma, whereby activation of a RAS–mitogen-activated protein kinase or phosphoinositide-3-kinase signaling cascade leads to induction of the transcription factor cAMP response element–binding protein-3–like-2 (CREB3L2), which directly activates ATF5 expression. ATF5, in turn, promotes survival by stimulating transcription of myeloid cell leukemia sequence-1 (MCL1), an antiapoptotic B cell leukemia-2 family member. Analysis of human malignant glioma samples indicates that ATF5 expression inversely correlates with disease prognosis. The RAF kinase inhibitor sorafenib suppresses ATF5 expression in glioma stem cells and inhibits malignant glioma growth in cell culture and mouse models. Our results demonstrate that ATF5 is essential in malignant glioma genesis and reveal that the ATF5-mediated survival pathway described here provides potential therapeutic targets for treatment of malignant glioma. [ABSTRACT FROM AUTHOR]

PTEN is a phosphatidylinositol phosphate (PIP) phosphatase specific for the 3-position of the inositol ring. Recent reports have shown that PTEN is activated by PI(4,5)P2 and that this activation requires a polybasic region located at the N-terminus of the protein. It was hypothesized that PI(4,5)P2 aids membrane recruiting and induces a conformational change in PTEN. This study characterizes PTENs binding preferences, investigates its contact points with the lipid bilayer and highlights structural changes upon membrane interaction. Fluorescence quenching experiments involving PC/phosphoinositide vesicles yielded results consistent with an enhanced PTEN binding to PI(4,5)P2 or PI(5)P containing vesicles, while the interaction with all other phosphoinositide derivatives was only minor. Experiments with a truncated PTEN16-403 showed a strongly reduced phosphoinositide affinity and a loss of specificity, while experiments using a peptide representing PTENs N-terminus (PTEN1-21) showed preferential PI(4,5)P2 binding. Infrared spectroscopic measurements furnished results consistent with an increased α-helical secondary structure content in the presence of PI(4,5)P2/PC vesicles, while other phosphoinositides like PI(3,5)P2 or PI(3,4,5)P3 did not cause a structural change. Although PS induced a structural change towards more β-sheet, it did not alter the structural effect of PI(4,5)P2. [ABSTRACT FROM AUTHOR]

Explores the functions of the phosphatase PTEN, cloned as a tumor suppressor for gliomas. Expression of PTEN in most neurons and localization in the nucleus and cytoplasm; Induction of PTEN during neuronal differentiation required for survival of differentiating neuronal cells; Downstream pathways regulated by PTEN; Multiple functions of PTEN at multiple subcellular sites.