[show abstract][hide abstract] ABSTRACT: The innate immune-signaling kinase, TBK1, couples pathogen surveillance to induction of host defense mechanisms. Pathological activation of TBK1 in cancer can overcome programmed cell death cues, enabling cells to survive oncogenic stress. The mechanistic basis of TBK1 prosurvival signaling, however, has been enigmatic. Here, we show that TBK1 directly activates AKT by phosphorylation of the canonical activation loop and hydrophobic motif sites independently of PDK1 and mTORC2. Upon mitogen stimulation, triggering of the innate immune response, re-exposure to glucose, or oncogene activation, TBK1 is recruited to the exocyst, where it activates AKT. In cells lacking TBK1, insulin activates AKT normally, but AKT activation by exocyst-dependent mechanisms is impaired. Discovery and characterization of a 6-aminopyrazolopyrimidine derivative, as a selective low-nanomolar TBK1 inhibitor, indicates that this regulatory arm can be pharmacologically perturbed independently of canonical PI3K/PDK1 signaling. Thus, AKT is a direct TBK1 substrate that connects TBK1 to prosurvival signaling.
[show abstract][hide abstract] ABSTRACT: The study of macroautophagy in mammalian cells has described induction, vesicle nucleation, and membrane elongation complexes as key signaling intermediates driving autophagosome biogenesis. How these components are recruited to nascent autophagosomes is poorly understood, and although much is known about signaling mechanisms that restrain autophagy, the nature of positive inductive signals that can promote autophagy remain cryptic. We find that the Ras-like small G protein, RalB, is localized to nascent autophagosomes and is activated on nutrient deprivation. RalB and its effector Exo84 are required for nutrient starvation-induced autophagocytosis, and RalB activation is sufficient to promote autophagosome formation. Through direct binding to Exo84, RalB induces the assembly of catalytically active ULK1 and Beclin1-VPS34 complexes on the exocyst, which are required for isolation membrane formation and maturation. Thus, RalB signaling is a primary adaptive response to nutrient limitation that directly engages autophagocytosis through mobilization of the core vesicle nucleation machinery.
[show abstract][hide abstract] ABSTRACT: Multiple molecular lesions in human cancers directly collaborate to deregulate proliferation and suppress apoptosis to promote tumorigenesis. The candidate tumor suppressor RASSF1A is commonly inactivated in a broad spectrum of human tumors and has been implicated as a pivotal gatekeeper of cell cycle progression. However, a mechanistic account of the role of RASSF1A gene inactivation in tumor initiation is lacking. Here we have employed loss-of-function analysis in human epithelial cells for a detailed investigation of the contribution of RASSF1 to cell cycle progression. We found that RASSF1A has dual opposing regulatory connections to G(1)/S phase cell cycle transit. RASSF1A associates with the Ewing sarcoma breakpoint protein, EWS, to limit accumulation of cyclin D1 and restrict exit from G(1). Surprisingly, we found that RASSF1A is also required to restrict SCF(betaTrCP) activity to allow G/S phase transition. This restriction is required for accumulation of the anaphase-promoting complex/cyclosome (APC/C) inhibitor Emi1 and the concomitant block of APC/C-dependent cyclin A turnover. The consequence of this relationship is inhibition of cell cycle progression in normal epithelial cells upon RASSF1A depletion despite elevated cyclin D1 concentrations. Progression to tumorigenicity upon RASSF1A gene inactivation should therefore require collaborating genetic aberrations that bypass the consequences of impaired APC/C regulation at the G(1)/S phase cell cycle transition.
Molecular and cellular biology 06/2008; 28(10):3190-7. · 6.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: Oligonucleotides containing locked nucleic acid bases (LNAs) have increased affinity for complementary DNA sequences. We hypothesized that enhanced affinity might allow LNAs to recognize chromosomal DNA inside human cells and inhibit gene expression. To test this hypothesis, we synthesized antigene LNAs (agLNAs) complementary to sequences within the promoters of progesterone receptor (PR) and androgen receptor (AR). We observed inhibition of AR and PR expression by agLNAs but not by analogous oligomers containing 2'-methoxyethyl bases or noncomplementary LNAs. Inhibition was dose dependent and exhibited IC50 values of <10 nM. Efficient inhibition depended on the length of the agLNA, the location of LNA bases, the number of LNA substitutions, and the location of the target sequence within the targeted promoter. LNAs targeting sequences at or near transcription start sites yielded better inhibition than LNAs targeting transcription factor binding sites or an inverted repeat. These results demonstrate that agLNAs can recognize chromosomal target sequences and efficiently block gene expression. agLNAs could be used for gene silencing, as cellular probes for chromosome structure, and therapeutic applications.
[show abstract][hide abstract] ABSTRACT: The ability to selectively activate or inhibit gene expression is fundamental to understanding complex cellular systems and developing therapeutics. Recent studies have demonstrated that duplex RNAs complementary to promoters within chromosomal DNA are potent gene silencing agents in mammalian cells. Here we report that chromosome-targeted RNAs also activate gene expression. We have identified multiple duplex RNAs complementary to the progesterone receptor (PR) promoter that increase expression of PR protein and RNA after transfection into cultured T47D or MCF7 human breast cancer cells. Upregulation of PR protein reduced expression of the downstream gene encoding cyclooygenase 2 but did not change concentrations of estrogen receptor, which demonstrates that activating RNAs can predictably manipulate physiologically relevant cellular pathways. Activation decreased over time and was sequence specific. Chromatin immunoprecipitation assays indicated that activation is accompanied by reduced acetylation at histones H3K9 and H3K14 and by increased di- and trimethylation at histone H3K4. These data show that, like proteins, hormones and small molecules, small duplex RNAs interact at promoters and can activate or repress gene expression.
Nature Chemical Biology 04/2007; 3(3):166-73. · 12.95 Impact Factor
[show abstract][hide abstract] ABSTRACT: Duplex RNAs complementary to messenger RNA inhibit translation in mammalian cells by RNA interference (RNAi). Studies have reported that RNAs complementary to promoter DNA also inhibit gene expression. Here we show that the human homologs of Argonaute-1 (AGO1) and Argonaute-2 (AGO2) link the silencing pathways that target mRNA with pathways mediating recognition of DNA. We find that synthetic antigene RNAs (agRNAs) complementary to transcription start sites or more upstream regions of gene promoters inhibit gene transcription. This silencing occurs in the nucleus, requires high promoter activity and does not necessarily require histone modification. AGO1 and AGO2 associate with promoter DNA in cells treated with agRNAs, and inhibiting expression of AGO1 or AGO2 reverses transcriptional and post-transcriptional silencing. Our data indicate key linkages and important mechanistic distinctions between transcriptional and post-transcriptional silencing pathways in mammalian cells.
[show abstract][hide abstract] ABSTRACT: Synthetic molecules that recognize specific sequences within cellular DNA are potentially powerful tools for investigating chromosome structure and function. Here, we designed antigene peptide nucleic acids (agPNAs) to target the transcriptional start sites for the human progesterone receptor B (hPR-B) and A (hPR-A) isoforms at sequences predicted to be single-stranded within the open complex of chromosomal DNA. We found that the agPNAs were potent inhibitors of transcription, showing for the first time that synthetic molecules can recognize transcription start sites inside cells. Breast cancer cells treated with agPNAs showed marked changes in morphology and an unexpected relationship between the strictly regulated levels of hPR-B and hPR-A. We confirmed these phenotypes using siRNAs and antisense PNAs, demonstrating the power of combining antigene and antisense strategies for gene silencing. agPNAs provide a general approach for controlling transcription initiation and a distinct option for target validation and therapeutic development.
Nature Chemical Biology 10/2005; 1(4):210-5. · 12.95 Impact Factor
[show abstract][hide abstract] ABSTRACT: Transcription start sites are critical switches for converting recognition of chromosomal DNA into active synthesis of RNA. Their functional importance suggests that they may be ideal targets for regulating gene expression. Here, we report potent inhibition of gene expression by antigene RNAs (agRNAs) complementary to transcription start sites within human chromosomal DNA. Silencing does not require methylation of DNA and differs from all known mechanisms for inhibiting transcription. agRNAs overlap DNA sequences within the open complex formed by RNA polymerase, and silencing is acutely sensitive to single base shifts. agRNAs effectively silence both TATA-less and TATA-box-containing promoters. Transcription start sites occur within every gene, providing predictable targets for agRNAs. Potent inhibition of multiple genes suggests that agRNAs may represent a natural mechanism for controlling transcription, may complement siRNAs and miRNAs that target mRNA, and will be valuable agents for silencing gene expression.
Nature Chemical Biology 10/2005; 1(4):216-22. · 12.95 Impact Factor