Maf1 is a novel target of PTEN and PI3K signaling that negatively regulates oncogenesis and lipid metabolism.

Maf1 was initially identified as a transcriptional repressor of RNA pol III-transcribed genes, yet little is known about its other potential target genes or its biological function. Here, we show that Maf1 is a key downstream target of PTEN that drives both its tumor suppressor and metabolic functions. Maf1 expression is diminished with loss of PTEN in both mouse models and human cancers. Consistent with its role as a tumor suppressor, Maf1 reduces anchorage-independent growth and tumor formation in mice. PTEN-mediated changes in Maf1 expression are mediated by PTEN acting on PI3K/AKT/FoxO1 signaling, revealing a new pathway that regulates RNA pol III-dependent genes. This regulatory event is biologically relevant as diet-induced PI3K activation reduces Maf1 expression in mouse liver. We further identify lipogenic enzymes as a new class of Maf1-regulated genes whereby Maf1 occupancy at the FASN promoter opposes SREBP1c-mediated transcription activation. Consistent with these findings, Maf1 inhibits intracellular lipid accumulation and increasing Maf1 expression in mouse liver abrogates diet-mediated induction of lipogenic enzymes and triglycerides. Together, these results establish a new biological role for Maf1 as a downstream effector of PTEN/PI3K signaling and reveal that Maf1 is a key element by which this pathway co-regulates lipid metabolism and oncogenesis.

Increased expression of TATA-binding protein, the central transcription factor, can contribute to oncogenesis.

Despite the central role of TATA-binding protein (TBP) in transcription, changes in cellular TBP concentration produce selective effects on gene expression. Moreover, TBP is up-regulated by oncogenic signaling pathways. These findings suggest that TBP could be a nexus in pathways that regulate cell proliferation and that genetic lesions that result in cellular transformation may produce their effects at least in part through TBP. We provide evidence consistent with this hypothesis, demonstrating that increases in TBP expression contribute to cellular transformation. A Ras-mediated increase in TBP expression is required for full Ras transforming activity. TBP overexpression induces cells to grow in an anchorage-independent manner and to form tumors in athymic mice. These effects on cellular transformation require changes in RNA polymerase II-dependent transcription and on the selective recruitment of TBP to promoters via its DNA binding activity. TBP expression is elevated in human colon carcinomas relative to normal colon epithelium. Both Ras-dependent and Ras-independent mechanisms mediate increases in TBP expression in colon carcinoma cell lines. We conclude that TBP may be a critical component in dysregulated signaling that occurs downstream of genetic lesions that cause tumors.

Elevated TATA-binding protein expression drives vascular endothelial growth factor expression in colon cancer.

The TATA-binding protein (TBP) plays a central role in eukaryotic gene transcription. Given its key function in transcription initiation, TBP was initially thought to be an invariant protein. However, studies showed that TBP expression is upregulated by oncogenic signaling pathways. Furthermore, depending on the cell type, small increases in cellular TBP amounts can induce changes in cellular growth properties towards a transformed phenotype. Here we sought to identify the specific TBP-regulated gene targets that drive its ability to induce tumorigenesis. Using microarray analysis, our results reveal that increases in cellular TBP concentrations produce selective alterations in gene expression that include an enrichment for genes involved in angiogenesis. Accordingly, we find that TBP levels modulate VEGFA expression, the master regulator of angiogenesis. Increases in cellular TBP amounts induce VEGFA expression and secretion to enhance cell migration and tumor vascularization. TBP mediates changes in VEGFA transcription requiring its recruitment at a hypoxia-insensitive proximal TSS, revealing a mechanism for VEGF regulation under non-stress conditions. The results are clinically relevant as TBP expression is significantly increased in both colon adenocarcinomas as well as adenomas relative to normal tissue. Furthermore, TBP expression is positively correlated with VEGFA expression. Collectively, these studies support the idea that increases in TBP expression contribute to enhanced VEGFA transcription early in colorectal cancer development to drive tumorigenesis.

The TATA-binding protein as a regulator of cellular transformation.

The TATA-binding protein, TBP, is used by all three RNA polymerases and is therefore central to the process of gene expression. TBP associates with several subsets of proteins, called TATA-binding protein-associated factors (TAFs). This results in the formation of at least three distinct complexes, SL1, TFIID, and TFIIIB, which dictates whether TBP functions in RNA polymerase (pol) I, pol II, or pol III transcription, respectively. The regulation of gene expression has focused largely on proteins that serve to modulate the efficiency by which the general transcription components, such as TBP, interact with promoters. The possibility of a basal transcription factor, itself, being regulated, and influencing cellular homeostasis, has not been extensively considered. However, recent studies have indicated that TBP is indeed regulated, and that modulation of its cellular concentration has a profound, and surprisingly selective, impact on gene expression that can mediate the normal proliferative responses of cells to growth stimuli as well as the transformation potential of cells.

Epidermal growth factor receptor 1 (EGFR1) and its variant EGFRvIII regulate TATA-binding protein expression through distinct pathways.

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.

Enhanced RNA polymerase III-dependent transcription is required for oncogenic transformation.

RNA polymerase (pol) III transcription, responsible for the synthesis of various stable RNAs, including 5 S rRNAs and tRNAs, is regulated by oncogenic proteins and tumor suppressors. Although it is well established that RNA pol III-dependent transcription is deregulated in transformed cells and malignant tumors, it has not been determined whether this represents a cause or consequence of these processes. We show that Rat1a fibroblasts undergoing oncogenic transformation by the TATA-binding protein or c-Myc display enhanced RNA pol III transcription. Decreased expression of the RNA pol III-specific transcription factor Brf1 prevented this increase in RNA pol III transcription. Although the overall proliferation rates of these cells remained unchanged, the ability of cells to grow in an anchorage-independent manner and form tumors in mice was markedly reduced. Although overexpression of Brf1 modestly stimulated RNA pol III transcription, expression of a phosphomimic, Brf1-T145D, more significantly induced transcription. However, these increases in transcription were not sufficient to promote cellular transformation. Together, these results demonstrate that enhanced RNA pol III transcription is essential for anchorage-independent growth and tumorigenesis and that these events can be uncoupled from effects on anchorage-dependent proliferation.

PTEN represses RNA polymerase III-dependent transcription by targeting the TFIIIB complex.

PTEN, a tumor suppressor whose function is frequently lost in human cancers, possesses a lipid phosphatase activity that represses phosphatidylinositol 3-kinase (PI3K) signaling, controlling cell growth, proliferation, and survival. The potential for PTEN to regulate the synthesis of RNA polymerase (Pol) III transcription products, including tRNAs and 5S rRNAs, was evaluated. The expression of PTEN in PTEN-deficient cells repressed RNA Pol III transcription, whereas decreased PTEN expression enhanced transcription. Transcription repression by PTEN was uncoupled from PTEN-mediated effects on the cell cycle and was independent of p53. PTEN acts through its lipid phosphatase activity, inhibiting the PI3K/Akt/mTOR/S6K pathway to decrease transcription. PTEN, through the inactivation of mTOR, targets the TFIIIB complex, disrupting the association between TATA-binding protein and Brf1. Kinetic analysis revealed that PTEN initially induces a decrease in the serine phosphorylation of Brf1, leading to a selective reduction in the occupancy of all TFIIIB subunits on tRNA(Leu) genes, whereas prolonged PTEN expression results in the enhanced serine phosphorylation of Bdp1. Together, these results demonstrate a new class of genes regulated by PTEN through its ability to repress the activation of PI3K/Akt/mTOR/S6K signaling.