Growth controls connect: interactions between c-myc and the tuberous sclerosis complex-mTOR pathway

Abstract

Among other signals, cell growth is particularly controlled by the target of rapamycin (TOR) pathway that includes the tuberous sclerosis complex genes (TSC1/2), and through transcriptional effects regulated by c-myc. Overexpression of Drosophila Myc and TSC1/2 cause opposing growth and proliferation defects. Despite this relationship, direct regulatory connections between Myc and the TSC have only recently been evaluated. Other than studies of p53 regulation, little consideration has been given to transcriptional regulation of the TSC genes. Here we review evidence that transcriptional controls are potentially important regulators of TSC2 expression, and that Myc is a direct repressor of its expression. Since tuberin loss de-represses Myc protein, the connection between these two growth regulators is positioned to act as a feed-forward loop that would amplify the oncogenic effects of decreased tuberin or increased Myc. Further experiments will be needed to clarify the mechanisms underlying this important connection, and evaluate its overall contribution to cancers caused by TSC loss or Myc gain.

Figure 1

A schematic diagram listing the anabolic paths containing genes regulated by c-myc. Gene products listed in red have been shown to be myc targets. Myc transcriptional regulation connects with mammalian target of rapamycin (mTOR) nutrient sensing pathway through their intersecting effects on translation initiation and elongation. This connection was first shown in Drosophila experiments, and mechanisms that might directly regulate their interactions are explored in this manuscript.

Figure 2

(A) Tissue-specific expression of TSC2 mRNA was assessed using the transcriptome database at http://symatlas.gnf.org/SymAtlas/. Shown are the normalized expression levels of TSC2 in the indicated tissues, presented as mean^+/− standard error. (B) An evolutionary analysis of conserved transcription factor binding sites performed using Genome rVista between the human, mouse and rat tuberous sclerosis 2 (TSC2) promoters identifies Myc binding sites as one of several candidate regulators of TSC2 expression., NTHL1 exons are identified as green rectangles and TSC2 exons as purple rectangles. The two major start sites of TSC2 are identified by arrowheads over the TSC2 exons, and the NTHL1 start site by one positioned over the green rectangles.,, Homology plots highlight conservation between mouse and human (just below the exon bars) and rat and human (at bottom). Highly conserved, non-coding regions are identified as brown bars and the CpG islands for all three promoters are identified by yellow-orange bars at top. rVista analysis identifies the listed transcription factors as those conserved between all three promoters.

Figure 3

DNA synthesis shows greater serum-induction and less rapamycin sensitivity in Myc null cells. Cells were arrested by confluence followed by serum starvation for 48 hours. Cells were then stimulated to re-enter the cell cycle with 10% fetal bovine serum in the presence of DMSO control, or rapamycin as described. Cells were then pulsed with tritiated thymidine for 2 hours, and harvested for scintillation counting as described. (A) Myc null (myc^−/−—HO15) and wild type (myc^+/+—TGR) cells stimulated with serum for 20 hours. (B) Increased sensitivity of Myc null cells to Rapamycin is independent of cell cycle position. Cells were arrested by confluence followed by serum starvation for 48 hours. Cells were stimulated to re-enter the cell cycle with 10% fetal bovine serum in the presence DMSO or rapamycin. The cells were then grown for the indicated times, pulsed with tritiated thymidine for 2 hours, and harvested for scintillation counting. Shown is the fold-reduction in thymidine uptake comparing the rapamycin treated samples to the DMSO treated control samples at each indicated time point for each cell type. (C) Same as in (A), above, except that cyclin D1 null (D) mouse embryonic cells, and their wild type counterparts, were used.

Figure 4

A TOR pathway diagram is shown, along with schematic diagrams of the CACGTG Myc target sites found within 5 kb of their transcriptional initiation sites that were either conserved in human, mouse and rat (red E) promoter regions or not conserved (black E). (Details in Ravitz et al.38) These genomic analyses were used as a starting point to test candidate proteins for changes in expression between myc null and wild type cells.

Figure 5

(A) Shown is a diagram of the first two exons of c-myc. Three initiation sites at P0, P1 and P2 result in incorporation of three different portions of exon 1 of myc in different mRNAs. An internal ribosomal entry site (IRES) is then positioned upstream of a non-canonical CUG initiation codon. The canonical initiation AUG codon is positioned in the 5′ proximal end of c-myc exon 2. (B) Protein lysates from growing wild type and TSC2 null mouse cells were probed in standard immunoblots for Myc, TSC2 and actin. Immunoblots demonstrate that TSC2 null cells lack tuberin expression, and the actin controls demonstrate equal loading. The larger and smaller Myc isoforms are indicated in contact and serum-deprivation arrested cells (A), and cells stimulated by the addition of serum for 6 hours (S). (C) Diagram of a proposed feed-forward loop whereby increased Myc causes decreased tuberin, and decreased tuberin causes increased Myc.