Activation of mTORC1 in two steps: Rheb-GTP activation of catalytic function and increased binding of substrates to raptor.

The signalling function of mTOR complex 1 is activated by Rheb-GTP, which controls the catalytic competence of the mTOR (mammalian target of rapamycin) kinase domain by an incompletely understood mechanism. Rheb can bind directly to the mTOR kinase domain, and association with inactive nucleotide-deficient Rheb mutants traps mTOR in a catalytically inactive state. Nevertheless, Rheb-GTP targets other than mTOR, such as FKBP38 (FK506-binding protein 38) and/or PLD1 (phospholipase D(1)), may also contribute to mTOR activation. Once activated, the mTOR catalytic domain phosphorylates substrates only when they are bound to raptor (regulatory associated protein of mTOR), a separate polypeptide within the complex. The mechanism of insulin/nutrient stimulation of mTOR complex 1 signalling, in addition to Rheb-GTP activation of the mTOR catalytic function, also involves a stable modification of the configuration of mTORC1 (mTOR complex 1) that increases access of substrates to their binding site on the raptor polypeptide. The mechanism underlying this second step in the activation of mTORC1 is unknown.

Combination therapy with IFN-alpha plus bortezomib induces apoptosis and inhibits angiogenesis in human bladder cancer cells.

In a recent study, we showed that the proteasome inhibitor bortezomib sensitizes human bladder cancer cells to IFN-induced cell death. Here, we characterized the molecular mechanisms underlying the antitumoral effects of the combination in more detail. Bortezomib synergized with IFN-alpha to promote apoptosis via a tumor necrosis factor-related apoptosis-inducing ligand-associated mechanism but did not inhibit production of proangiogenic factors (vascular endothelial growth factor, basic fibroblast growth factor, and interleukin-8) in human UM-UC-5 cells. In contrast, exposure to the combination did not increase the levels of apoptosis in human UM-UC-3 cells but did inhibit the production of basic fibroblast growth factor and vascular endothelial growth factor. Studies with tumor xenografts confirmed that combination therapy with bortezomib plus IFN-alpha was effective in both models but that the effects were associated with differential effects on tumor necrosis factor-related apoptosis-inducing ligand-associated apoptosis (predominant in UM-UC-5) versus inhibition of angiogenesis (predominant in UM-UC-3). Together, our results show that combination therapy with IFN-alpha plus bortezomib is effective but can work via different mechanisms (apoptosis versus angiogenesis inhibition) in preclinical models of human bladder cancer.

Role of tumor necrosis factor-related apoptosis-inducing ligand in interferon-induced apoptosis in human bladder cancer cells.

Immunomodulators such as Bacillus Calmette-Guerin and interferon are clinically active in transitional cell carcinoma of the bladder, but their mechanisms of action remain unclear. Here we investigated the effects of IFNalpha on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression and apoptosis in a panel of 20 human bladder cancer cell lines. Six (30%) displayed significant DNA fragmentation in response to increasing concentrations of IFNalpha (10-100,000 units/mL). In these lines IFNalpha induced early activation of caspase-8, and DNA fragmentation was blocked by a caspase-8-selective inhibitor (IETDfmk), consistent with the involvement of death receptor(s) in cell death. IFNalpha stimulated marked increases in TRAIL mRNA and protein in the majority of IFN-sensitive and IFN-resistant cell lines. A blocking anti-TRAIL antibody significantly inhibited IFN-induced DNA fragmentation in four of six IFN-sensitive cell lines, confirming that TRAIL played a direct role in cell death. Bortezomib (PS-341, Velcade), a potent TRAIL-sensitizing agent, increased sensitivity to IFNalpha in two of the IFN-resistant cell lines that produced large amounts of TRAIL in response to IFN treatment. Our data show that IFN-induced apoptosis in bladder cancer cells frequently involves autocrine TRAIL production. Combination therapy strategies aimed at overcoming TRAIL resistance may be very effective in restoring IFN sensitivity in a subset of human bladder tumors.

A genome-wide siRNA screen in mammalian cells for regulators of S6 phosphorylation.

mTOR complex1, the major regulator of mRNA translation in all eukaryotic cells, is strongly activated in most cancers. We performed a genome-wide RNAi screen in a human cancer cell line, seeking genes that regulate S6 phosphorylation, readout of mTORC1 activity. Applying a stringent selection, we retrieved nearly 600 genes wherein at least two RNAis gave significant reduction in S6-P. This cohort contains known regulators of mTOR complex 1 and is significantly enriched in genes whose depletion affects the proliferation/viability of the large set of cancer cell lines in the Achilles database in a manner paralleling that caused by mTOR depletion. We next examined the effect of RNAi pools directed at 534 of these gene products on S6-P in TSC1 null mouse embryo fibroblasts. 76 RNAis reduced S6 phosphorylation significantly in 2 or 3 replicates. Surprisingly, among this cohort of genes the only elements previously associated with the maintenance of mTORC1 activity are two subunits of the vacuolar ATPase and the CUL4 subunit DDB1. RNAi against a second set of 84 targets reduced S6-P in only one of three replicates. However, an indication that this group also bears attention is the presence of rpS6KB1 itself, Rac1 and MAP4K3, a protein kinase that supports amino acid signaling to rpS6KB1. The finding that S6 phosphorylation requires a previously unidentified, functionally diverse cohort of genes that participate in fundamental cellular processes such as mRNA translation, RNA processing, DNA repair and metabolism suggests the operation of feedback pathways in the regulation of mTORC1 operating through novel mechanisms.

A genome-wide RNAi screen for polypeptides that alter rpS6 phosphorylation.

Mammalian target of rapamycin (mTOR) is a giant protein kinase that controls cell proliferation, growth, and metabolism. mTOR is regulated by nutrient availability, by mitogens, and by stress, and operates through two independently regulated hetero-oligomeric complexes. We have attempted to identify the cellular components necessary to maintain the activity of mTOR complex 1 (mTORC1), the amino acid-dependent, rapamycin-inhibitable complex, using a whole genome approach involving RNAi-induced depletion of cellular polypeptides. We have used a pancreatic ductal adenocarcinoma (PDAC) cell line, Mia-PaCa for this screen; as with many pancreatic cancers, these cells exhibit constitutive activation of mTORC1. PDAC is the most common form of pancreatic cancer and the 5-year survival rate remains 3-5% despite current nonspecific and targeted therapies. Although rapamycin-related mTOR inhibitors have yet to demonstrate encouraging clinical responses, it is now evident that this class of compounds is capable of only partial mTORC1 inhibition. Identifying previously unappreciated proteins needed for maintenance of mTORC1 activity may provide new targets and lead to the development of beneficial therapies for pancreatic cancer.

Amino acid regulation of TOR complex 1.

TOR complex 1 (TORC1), an oligomer of the mTOR (mammalian target of rapamycin) protein kinase, its substrate binding subunit raptor, and the polypeptide Lst8/GbetaL, controls cell growth in all eukaryotes in response to nutrient availability and in metazoans to insulin and growth factors, energy status, and stress conditions. This review focuses on the biochemical mechanisms that regulate mTORC1 kinase activity, with special emphasis on mTORC1 regulation by amino acids. The dominant positive regulator of mTORC1 is the GTP-charged form of the ras-like GTPase Rheb. Insulin, growth factors, and a variety of cellular stressors regulate mTORC1 by controlling Rheb GTP charging through modulating the activity of the tuberous sclerosis complex, the Rheb GTPase activating protein. In contrast, amino acids, especially leucine, regulate mTORC1 by controlling the ability of Rheb-GTP to activate mTORC1. Rheb binds directly to mTOR, an interaction that appears to be essential for mTORC1 activation. In addition, Rheb-GTP stimulates phospholipase D1 to generate phosphatidic acid, a positive effector of mTORC1 activation, and binds to the mTOR inhibitor FKBP38, to displace it from mTOR. The contribution of Rheb's regulation of PL-D1 and FKBP38 to mTORC1 activation, relative to Rheb's direct binding to mTOR, remains to be fully defined. The rag GTPases, functioning as obligatory heterodimers, are also required for amino acid regulation of mTORC1. As with amino acid deficiency, however, the inhibitory effect of rag depletion on mTORC1 can be overcome by Rheb overexpression, whereas Rheb depletion obviates rag's ability to activate mTORC1. The rag heterodimer interacts directly with mTORC1 and may direct mTORC1 to the Rheb-containing vesicular compartment in response to amino acid sufficiency, enabling Rheb-GTP activation of mTORC1. The type III phosphatidylinositol kinase also participates in amino acid-dependent mTORC1 activation, although the site of action of its product, 3'OH-phosphatidylinositol, in this process is unclear.

Interferon-alpha induces TRAIL expression and cell death via an IRF-1-dependent mechanism in human bladder cancer cells.

Apoptosis induced by interferon-alpha (IFNalpha) is associated with induction of TRAIL in a number of different cell types. Here we examined whether or not TRAIL was required for apoptosis in a model human bladder cancer cell line (UM-UC-12) and defined the molecular mechanisms involved in IFNalpha-induced TRAIL expression. Exposure to IFNalpha resulted in concentration-dependent induction of TRAIL and apoptosis. Inhibition of TRAIL or downstream components of the TRAIL cell death pathway (FADD, caspase-8) via siRNA-mediated knockdown attenuated IFNalpha-induced cell death, thereby implicating TRAIL in the response. IFNalpha induced rapid STAT-1 phosphorylation and DNA binding activity and subsequent accumulation of IRF-1. Transfection with siRNAs directed against STAT-1 or IRF-1 inhibited IFNalpha-induced TRAIL production and cell death, and chromatin immunprecipitation (ChIP) analyses demonstrated that IFNalpha induced direct, time-dependent binding of both transcription factors to the TRAIL promoter. Together, our results demonstrate that IFNalpha induces TRAIL expression via a STAT-1/IRF-1-dependent mechanism in human bladder cancer cells, and this induction of TRAIL plays an important role in IFNalpha-induced cell killing.

Intravesical Ad-IFNalpha causes marked regression of human bladder cancer growing orthotopically in nude mice and overcomes resistance to IFN-alpha protein.

We have produced prolonged, high local concentrations of interferon in vivo by intravesical instillation of adenoviruses encoding interferon-alpha (Ad-IFNalpha) together with the gene transfer-enhancing agent Syn3. We found sustained interferon protein levels for days, both in normal mouse urothelium and in human bladder cancer cells growing as superficial bladder tumors in nude mice using an orthotopic bladder model developed by us. Tumor burden in the bladder was determined utilizing cancer cells containing the green fluorescent protein. Marked tumor regression was observed following two 1-h exposures of Ad-IFNalpha/Syn3 and little or no cytotoxicity was detected in normal cells. Similar intravesical instillation of clinically relevant concentrations of IFN protein alone or Ad-IFNalpha without Syn3 was ineffective. Surprisingly, in vitro, Ad-IFNalpha also caused caspase-dependent death of bladder cancer cell lines that were resistant to high concentrations of IFN-alpha protein, including the cell line used in vivo. These findings demonstrate that Ad-IFNalpha can overcome resistance to IFN-alpha protein both in vitro and in vivo and support evaluation of intravesical Ad-IFNalpha/Syn3 for the treatment of superficial bladder cancer.