The mammalian target of rapamycin (mTOR) kinase forms two multiprotein complexes, mTORC1 and mTORC2, which regulate cell growth, cell survival, and autophagy. Allosteric inhibitors of mTORC1, such as rapamycin, have been extensively used to study tumor cell growth, proliferation, and autophagy but have shown only limited clinical utility. Here, we describe AZD8055, a novel ATP-competitive inhibitor of mTOR kinase activity, with an IC 50 of 0.8 nmol/L. AZD8055 showed excellent selectivity (∼1,000-fold) against all class I phosphatidylinositol 3-kinase (PI3K) isoforms and other members of the PI3K-like kinase family. Furthermore, there was no significant activity against a panel of 260 kinases at concentrations up to 10 μmol/L. AZD8055 inhibits the phosphorylation of mTORC1 substrates p70S6K and 4E-BP1 as well as phosphorylation of the mTORC2 substrate AKT and downstream proteins. The rapamycin-resistant T37/46 phosphorylation sites on 4E-BP1 were fully inhibited by AZD8055, resulting in significant inhibition of cap-dependent translation. In vitro, AZD8055 potently inhibits proliferation and induces autophagy in H838 and A549 cells. In vivo, AZD8055 induces a dose-dependent pharmacodynamic effect on phosphorylated S6 and phosphorylated AKT at plasma concentrations leading to tumor growth inhibition. Notably, AZD8055 results in significant growth inhibition and/or regression in xenografts, representing a broad range of human tumor types. AZD8055 is currently in phase I clinical trials. Cancer Res; 70(1); 288-98. ©2010 AACR.

The balance between the fission and fusion mechanisms regulate the morphology of mitochondria. In this study we have identified a mammalian protein that we call hFis1, which is the orthologue of the yeast Fis1p known to participate in yeast mitochondrial division. hFis1, when overexpressed in various cell types, localized to the outer mitochondrial membrane and induced mitochondrial fission. This event was inhibited by a dominant negative mutant of Drp1 (Drp1(K38A)), a major component of the fission apparatus. Fragmentation of the mitochondrial network by hFis1 was followed by the release of cytochrome c and ultimately apoptosis. Bcl-x L was able to block cytochrome c release and apoptosis but failed to prevent mitochondrial fragmentation. Our studies show that hFis1 is part of the mammalian fission machinery and suggest that regulation of the fission processes might be involved in apoptotic mechanisms.

Mitochondria form a dynamic network, and it remains unclear how the alternate configurations interact with bioenergetics properties. The metabolic signals that link mitochondrial structure to its functional states have not been fully characterized. In this report, we analyze the bidirectional relationships between mitochondrial morphology and function in living human cells. First, we determined the effect of mitochondrial fission on energy production by using small interfering RNA (siRNA) targeting DRP1, which revealed the importance of membrane fluidity on the control of bioenergetics. Second, we followed the effect of rotenone, a specific inhibitor of respiratory chain complex I, which causes large structural perturbations, once a threshold was reached. Last, we followed changes in the mitochondrial network configuration in human cells that had been treated with modulators of oxidative phosphorylation, and in fibroblasts from two patients with mitochondrial disease where the respiratory rate, ΔΨ and the generation of reactive oxygen species (ROS) were measured. Our data demonstrate that the relationship between mitochondrial network organization and bioenergetics is bidirectional, and we provide a model for analyzing the metabolic signals involved in this crosstalk.

Mitochondria form a highly dynamic tubular network, the morphology of which is regulated by frequent fission and fusion events. However, the role of mitochondrial fission in homeostasis of the organelle is still unknown. Here we report that preventing mitochondrial fission, by down-regulating expression of Drp1 in mammalian cells leads to a loss of mitochondrial DNA and a decrease of mitochondrial respiration coupled to an increase in the levels of cellular reactive oxygen species (ROS). At the cellular level, mitochondrial dysfunction resulting from the lack of fission leads to a drop in the levels of cellular ATP, an inhibition of cell proliferation and an increase in autophagy. In conclusion, we propose that mitochondrial fission is required for preservation of mitochondrial function and thereby for maintenance of cellular homeostasis.

Apoptosis, induced by a number of death stimuli, is associated with a fragmentation of the mitochondrial network. These morphological changes in mitochondria have been shown to require proteins, such as Drp1 or hFis1, which are involved in regulating the fission of mitochondria. However, the precise role of mitochondrial fission during apoptosis remains elusive. Here we report that inhibiting the fission machinery in Bax/Bak-mediated apoptosis, by down-regulating of Drp1 or hFis1, prevents the fragmentation of the mitochondrial network and partially inhibits the release of cytochrome c from the mitochondria but fails to block the efflux of Smac/DIABLO. In addition, preventing mitochondrial fragmentation does not inhibit cell death induced by Bax/Bak-dependent death stimuli, in contrast to the effects of Bcl-xL or caspase inhibition. Therefore, the fission of mitochondria is a dispensable event in Bax/Bak-dependent apoptosis.Mitochondria play a critical role in the regulation of programmed cell death by sequestering apoptogenic proteins such as cytochrome c, Smac/DIABLO, HtrA2/Omi, endonuclease G, and AIF (13,18,29,80). The release of such factors during apoptosis is regulated by a subclass of Bcl-2 proteins (12,14,63,78), including Bax and Bak. These proteins seem to be in an inactive state in healthy cells, with Bax predominantly found in the cytosol. However, during apoptosis induced by various death stimuli, including DNA damage or trophic factor deprivation, they are activated by a process requiring BH3-only Bcl-2 family members. It is thought that BH3-only proteins either bind and sequester Bcl-2 antiapoptotic proteins (this is the case for Bad and Puma) or bind to and directly activate proapoptotic proteins (tBid for example) (9,11,33,45,65,74). This results in the inactivation of Bcl-2 antiapoptotic proteins and in the oligomerization of Bax and Bak in the mitochondrial outer membrane (MOM) with a concomitant release of apoptogenic factors from the mitochondria (17,21,31,43).How permeabilization of the MOM occurs during apoptosis remains a matter of debate and has been extensively studied (for reviews, see references 4, 49, and 85). Recently, a new model has emerged based on the discovery that mitochondria fragment during cell death (32, 46-48, 61, 86). According to this model, the fission of mitochondria would be necessary for permeabilization of the MOM (3, 57, 83).Nevertheless, it is still not clear whether the fragmentation of mitochondria precedes or follows the release of apoptogenic factors (1,22,26).Mitochondrial fission and fusion are normal and frequent events in healthy cells. The protein machinery that underlies mitochondrial fission has been well characterized and extensively reviewed (53,62). In mammalian cells, at least three proteins, Drp1, hFis1, and MTP18 (75,76), are required for this process. The dynamin-related protein Drp1 is a large cytosolic GTPase that translocates to the mitochondria, where it couples GTP hydrolysis with scission of the mitochondrial tubule (59,67,68). Its recept...

The Wilms' tumor suppressor protein WT1 is a transcriptional regulator that plays a key role in the development of the kidneys. The transcriptional activation domain of WT1 is subject to regulation by a suppression region within the N terminus of WT1. Using a functional assay, we provide direct evidence that this requires a transcriptional cosuppressor, which we identify as brain acid soluble protein 1 (BASP1). WT1 and BASP1 associate within the nuclei of cells that naturally express both proteins. BASP1 can confer WT1 cosuppressor activity in transfection assays, and elimination of endogenous BASP1 expression augments transcriptional activation by WT1. BASP1 is present in the developing nephron structures of the embryonic kidney and, coincident with that of WT1, its expression is restricted to the highly specialized podocyte cells of the adult kidney. Taken together, our results show that BASP1 is a WT1-associated factor that can regulate WT1 transcriptional activity.Wilms' tumor, a pediatric malignancy of the kidneys, is the most common solid childhood tumor (reviewed in references 4, 7, 20, 30, and 33). The isolation of genes associated with Wilms' tumor led to the identification of a zinc finger protein, WT1. Subsequently, WT1 was shown to be a transcriptional regulator with putative target genes including those for growth factors and regulators of cell division (5,6,18,21). Approximately 15% of sporadic Wilms' tumors have been found to contain mutations in WT1, while others show aberrant WT1 expression (33).WT1 knockout mice (homozygous null) do not survive gestation, displaying absence or incorrect development of the kidney, gonads, spleen, heart, diaphragm, and retinal ganglia (12,14,35). These findings confirm a major role for WT1 in the formation of the genitourinary system and also a wider role in the development of other tissues.Alternative splicing, RNA editing, and an alternative translation start codon combine to produce a plethora of WT1 isoforms (reviewed in reference 33). One alternative splice inserts three amino acids (KTS) between zinc fingers three and four, resulting in a form of WT1 that associates with RNA processing factors and localizes to regions of RNA processing in the nucleus (17). Thus, the ϩKTS and ϪKTS isoforms of WT1 have been proposed to function in RNA processing and transcription, respectively. These isoforms have both overlapping and distinct roles during development (9, 10). Interestingly, the ϩKTS isoform of WT1 plays the dominant role in the development of the gonad, while the ϪKTS isoform has a more extensive function in kidney formation.The other alternative splice inserts 17 amino acids N terminal to the WT1 zinc fingers and has been shown to have effects on both cell division and cell survival (15,31,32). Specific elimination of this isoform of WT1 in mice does not result in any obvious defects in genitourinary development, suggesting that it may be required specifically for a tumor suppressor role or that it performs a subtle function (28).Several studies have shown ...

The release of proteins from the intermembrane space of mitochondria is one of the pivotal events in the apoptotic process, which can lead to the activation of caspases and the ultimate demise of the cell. How these proteins exit the mitochondria is still a matter of intense debate. Here, we discuss the possible mechanisms behind the release of apoptogenic proteins, the ways in which cancer cells subvert these mechanisms, and the therapeutic regimens that aim to promote the timely loss of integrity of the outer mitochondrial membrane.

Mitochondria modulate Ca2؉ signals by taking up, buffering, and releasing Ca 2؉ at key locations near Ca 2؉ release or influx channels. The role of such local interactions between channels and organelles is difficult to establish in living cells because mitochondria form an interconnected network constantly remodeled by coordinated fusion and fission reactions. To study the effect of a controlled disruption of the mitochondrial network on Ca 2؉ homeostasis, we took advantage of hFis1, a protein that promotes mitochondrial fission by recruiting the dynamin-related protein, Drp1. hFis1 expression in HeLa cells induced a rapid and complete fragmentation of mitochondria, which redistributed away from the plasma membrane and clustered around the nucleus. Despite the dramatic morphological alteration, hFis1-fragmented mitochondria maintained a normal transmembrane potential and pH and took up normally the Ca 2؉ released from intracellular stores upon agonist stimulation, as measured with a targeted ratiometric pericam probe. In contrast, hFis1-fragmented mitochondria took up more slowly the Ca 2؉ entering across plasma membrane channels, because the Ca 2؉ ions reaching mitochondria propagated faster and in a more coordinated manner in interconnected than in fragmented mitochondria. In parallel cytosolic fura-2 measurements, the capacitative Ca 2؉ entry (CCE) elicited by store depletion was only marginally reduced by hFis1 expression. Regardless of mitochondria shape and location, disruption of mitochondrial potential with uncouplers or oligomycin/ rotenone reduced CCE by ϳ35%. These observations indicate that close contact to Ca 2؉ influx channels is not required for CCE modulation and that the formation of a mitochondrial network facilitates Ca 2؉ propagation within interconnected mitochondria.