Most eukaryotic transcriptional regulators act in an RNA polymerase (Pol)-selective manner.Here we show that the human Maf1 protein negatively regulates transcription by all three nuclear Pols. Changes in Maf1 expression affect Pol I-and Pol III-dependent transcription in human glioblastoma lines. These effects are mediated, in part, through the ability of Maf1 to repress transcription of the TATA binding protein, TBP. Maf1 targets an Elk-1-binding site in the TBP promoter, and its occupancy of this region is reciprocal with that of Elk-1. Similarly, Maf1 occupancy of Pol III genes is inversely correlated with that of the initiation factor TFIIIB and Pol III. The phenotypic consequences of reducing Maf1 expression include changes in cell morphology and the accumulation of actin stress fibers, whereas Maf1 overexpression suppresses anchorage-independent growth. Together with the ability of Maf1 to reduce biosynthetic capacity, these findings support the idea that Maf1 regulates the transformation state of cells.

Summary Loss-of-function mutations in the NF1 tumor suppressor result in deregulated Ras signaling and drive tumorigenesis in the familial cancer syndrome neurofibromatosis type I. However, the extent to which NF1-inactivation promotes sporadic tumorigenesis is unknown. Here we report that NF1 is inactivated in sporadic gliomas via two mechanisms: excessive proteasomal degradation and genetic loss. NF1 protein destabilization is triggered by the hyperactivation of protein kinase C (PKC) and confers sensitivity to PKC inhibitors. However complete genetic loss, which only occurs when p53 is inactivated, mediates sensitivity to mTOR inhibitors. These studies reveal an expanding role for NF1-inactivation in sporadic gliomagenesis and illustrate how different mechanisms of inactivation are utilized in genetically distinct tumors, which consequently impacts therapeutic sensitivity. Significance Tumor suppressors are often mutated in human cancer; however, the excessive proteasomal destruction of tumor suppressor proteins also promotes tumorigenesis. Here we show that the NF1 protein is destabilized in sporadic GBMs as a consequence of the hyperactivation of PKC. Notably, this destabilization confers sensitivity to PKC inhibitors. In contrast, a separate subset of GBMs that possess NF1 mutations are insensitive to PKC inhibitors but are sensitive to mTOR inhibitors. These findings reveal a broad role for NF1-inactivation in gliomagenesis and illustrate how different mechanisms of inactivation are utilized in the same tumor-type. Moreover they highlight the importance of elucidating the molecular mechanisms that underlie tumorigenesis, as such knowledge may be essential for developing personalized therapies.

Emerging evidence supports the idea that the c-Jun N-terminal kinases (JNKs) possess overlapping but distinct functions. The potential roles of the ubiquitously expressed JNK1 and JNK2 in regulating expression of the central transcription initiation factor, TATA-binding protein (TBP), were examined. Relative to wildtype fibroblasts, TBP was decreased in Jnk1 ؊/؊ cells and increased in Jnk2 ؊/؊ cells. Similarly, reduction of JNK1 in human hepatoma cells decreased TBP expression, whereas reduction of JNK2 enhanced it. JNKmediated regulation of TBP expression occurs at the transcriptional level through their ability to target Elk-1, which directly regulates the TBP promoter in response to epidermal growth factor stimulation. JNK1 increases, whereas JNK2 decreases, the phosphorylation state of Elk-1, which differentially affects Elk-1 occupancy at a defined site within the TBP promoter. These JNK-mediated alterations in TBP expression, alone, serve to regulate c-Jun expression and fibroblast proliferation rates. These studies uncovered several new molecular events that distinguish the functions of JNK1 and JNK2 that are critical for their regulation of cellular proliferation.The TATA binding protein (TBP) is a central eukaryotic transcription component, as it is required by all three cellular RNA polymerases for transcription initiation. It associates with additional proteins to form at least three distinct complexes, which specifies its role in RNA polymerase I (pol I)-, II-, or III-dependent transcription (18). TBP can be a limiting factor for both RNA pol I-(40, 45) and RNA pol III-dependent promoters (34,38,39,45). Thus, increased expression of TBP serves to increase production of rRNAs and tRNAs. TBP is differentially limiting for RNA pol II-dependent promoters, depending on the nature of the promoter and the composition and location of regulatory elements (5,26,29).Substantial evidence supports the idea that the activation of certain oncogenic signaling pathways can induce TBP expression. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of protein kinase C, enhances cellular TBP production (15, 16). Activation of epidermal growth factor receptor 1 (EGFR1) induces TBP expression through the activation of Ras (45). In addition to regulation of cellular concentrations of TBP, its function can also be regulated. For example, the tumor suppressor p53 binds to TBP and negatively regulates its ability to form functional TFIIIB complexes, producing selective changes in RNA pol III-dependent transcription (6).The fact that tumor suppressor and oncogenic signaling pathways tightly regulate cellular concentrations of TBP or its function suggested the possibility that alterations in TBP levels may influence the transformation state of cells. Consistent with this idea, inhibiting Ras-mediated increases in TBP in NIH 3T3 cells abrogated Ras-induced transformation, while increasing TBP expression in rat 1A fibroblast cells induced anchorage-independent growth and tumor formation in mice ...

NF1 encodes a RAS GTPase-Activating Protein. Accordingly, aberrant RAS activation underlies the pathogenesis of NF1-mutant cancers. Nevertheless, it is unclear which RAS pathway components represent optimal therapeutic targets. Here we identify mTORC1 as the key PI3K effector in NF1-mutant nervous system malignancies and conversely show that mTORC2 and AKT are dispensable. However, we find that tumor regression requires sustained inhibition of both mTORC1 and MEK. Transcriptional profiling studies were therefore used to establish a signature of effective mTORC1/MEK inhibition in vivo. We unexpectedly found that the glucose transporter, GLUT1, was potently suppressed but only when both pathways were inhibited. Moreover, unlike VHL and LKB1 mutant cancers, reduction of 18F-FDG uptake required the suppression of both mTORC1 and MEK. Together these studies identify optimal and sub-optimal therapeutic targets in NF1-mutant malignancies and define a non-invasive means of measuring combined mTORC1/MEK inhibition in vivo, which can be readily incorporated into clinical trials.

The epidermal growth factor receptor (EGFR) family regulates essential biological processes. Various epithelial tumors are linked to EGFR overexpression or expression of variant forms, such as the EGFR1 variant, EGFRvIII. Perturbations in expression of the transcription initiation factor, TATA-binding protein (TBP), alter cellular growth properties. Here we demonstrate that EGFR1 and EGFRvIII, but not HER2, induce TBP expression at a transcriptional level through distinct mechanisms. EGFR1 enhances the phosphorylation and function of Elk-1, recruiting it to the TBP promoter. In contrast, EGFRvIII robustly induces c-jun expression, stimulating recruitment of c-fos/c-jun to an overlapping AP-1 site. Enhancing c-jun expression alone induces TBP promoter activity through the AP-1 site. To determine the underlying mechanism for differences in Elk-1 function and c-jun expression by these receptors, we inhibited the internalization of EGFR1. Persistent EGFR1 cell surface occupancy mimics EGFRvIII-mediated effects on Elk-1 and c-jun and switches the requirement of Elk-1 to AP-1 for TBP promoter induction. Together, these studies define a new molecular mechanism for the regulation of TBP expression. In addition, we identify distinct molecular targets of EGFR1 and EGFRvIII and demonstrate the importance of receptor internalization in distinguishing their specific functions.The epidermal growth factor receptor (EGFR) family comprises four transmembrane receptor tyrosine kinases, EGFR1 (ErbB1/HER1), HER2 (ErbB2), HER3 (ErbB3), and HER4 (ErbB4). They share a similar kinase domain structure and homology but differ in their extracellular domains and carboxyterminal tails (48). The EGFRs play vital roles during development and are important regulators of cellular proliferation, survival, and migration. Amplification and overexpression of EGFR1 and HER2 are common in a variety of human cancers. Overexpression of HER2 and EGFR1 usually results from gene amplification, and in the case of EGFR1, overexpression can result in the formation of a variety of genetic mutations. EGFRvIII is the most common genetic variant form of EGFR1 and contains an in-frame deletion of exons 2 to 7, corresponding to amino acids 6 to 273, in the extracellular domain (3). The EGFRvIII-specific deletion results in a novel extracellular domain architecture that mimics an activated receptor unable to bind EGF. In contrast to EGFRvIII, ligand-bound EGFR1 is rapidly endocytosed by clathrin-coated pits (51). After internalization, ligand-EGFR1 complexes traffic through various endosomal compartments where they are either shuttled back to the plasma membrane or degraded in lysosomes. Internalized EGFR1 continues to signal from within the endosomes, and this is thought to modulate the duration, intensity, and specificity of signaling processes (49, 57).The TATA-binding protein (TBP) is a ubiquitously expressed transcription initiation factor indispensable for cell function. Specific protein-protein interactions allocate TBP for participation in transcription by ...

<p>PDF file - 75KB, Reported in vitro isoform specificities of PI3K inhibitors. The IC50 (nM) reported in the literature for each PI3K inhibitor against Class IA catalytic isoforms in in vitro binding assays is shown. The source for each data set is listed. A66-(S) is termed a p110alphaspecific inhibitor, AZD-6482 is classified as a p110beta-specific inhibitor, and CAL-101 shows selectivity for p110delta. GDC-0941 does not show significant isoform selectivity.</p>

<p>PDF file - 47KB, Supplementary Figure 1 shows that AKT inhibition does not slow proliferation in a human MPNST cell line.</p>

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