A Novel PTEN/Mutant p53/c-Myc/Bcl-XL Axis Mediates Context-Dependent Oncogenic Effects of PTEN with Implications for Cancer Prognosis and Therapy

AbstractPhosphatase and tensin homolog located on chromosome 10 (PTEN) is one of the most frequently mutated tumor suppressors in human cancer including in glioblastoma. Here, we show that PTEN exerts unconventional oncogenic effects in glioblastoma through a novel PTEN/mutant p53/c-Myc/Bcl-XL molecular and functional axis. Using a wide array of molecular, genetic, and functional approaches, we demonstrate that PTEN enhances a transcriptional complex containing gain-of-function mutant p53, CBP, and NFY in human glioblastoma cells and tumor tissues. The mutant p53/CBP/NFY complex transcriptionally activates the oncogenes c-Myc and Bcl-XL, leading to increased cell proliferation, survival, invasion, and clonogenicity. Disruption of the mutant p53/c-Myc/Bcl-XL axis or mutant p53/CBP/NFY complex reverses the transcriptional and oncogenic effects of PTEN and unmasks its tumor-suppressive function. Consistent with these data, we find that PTEN expression is associated with worse patient survival than PTEN loss in tumors harboring mutant p53 and that a small molecule modulator of p53 exerts greater antitumor effects in PTEN-expressing cancer cells. Altogether, our study describes a new signaling pathway that mediates context-dependent oncogenic/tumor-suppressive role of PTEN. The data also indicate that the combined mutational status of PTEN and p53 influences cancer prognosis and anticancer therapies that target PTEN and p53

A Comparison of Midline and Tracheal Gene Regulation during <i>Drosophila</i> Development

<div><p>Within the <i>Drosophila</i> embryo, two related bHLH-PAS proteins, Single-minded and Trachealess, control development of the central nervous system midline and the trachea, respectively. These two proteins are bHLH-PAS transcription factors and independently form heterodimers with another bHLH-PAS protein, Tango. During early embryogenesis, expression of Single-minded is restricted to the midline and Trachealess to the trachea and salivary glands, whereas Tango is ubiquitously expressed. Both Single-minded/Tango and Trachealess/Tango heterodimers bind to the same DNA sequence, called the CNS midline element (CME) within <i>cis</i>-regulatory sequences of downstream target genes. While Single-minded/Tango and Trachealess/Tango activate some of the same genes in their respective tissues during embryogenesis, they also activate a number of different genes restricted to only certain tissues. The goal of this research is to understand how these two related heterodimers bind different enhancers to activate different genes, thereby regulating the development of functionally diverse tissues. Existing data indicates that Single-minded and Trachealess may bind to different co-factors restricted to various tissues, causing them to interact with the CME only within certain sequence contexts. This would lead to the activation of different target genes in different cell types. To understand how the context surrounding the CME is recognized by different bHLH-PAS heterodimers and their co-factors, we identified and analyzed novel enhancers that drive midline and/or tracheal expression and compared them to previously characterized enhancers. In addition, we tested expression of synthetic reporter genes containing the CME flanked by different sequences. Taken together, these experiments identify elements overrepresented within midline and tracheal enhancers and suggest that sequences immediately surrounding a CME help dictate whether a gene is expressed in the midline or trachea.</p></div

Motifs overrepresented in midline and tracheal enhancers identified with MEME.

<p>MEME <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085518#pone.0085518-Bailey1" target="_blank">[61]</a> was used to identify motifs overrepresented in midline and tracheal enhancers. The expected number of motifs one would find in a similarly sized set of random sequences (E-value) and the number of times each site was found within the enhancers are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085518#pone-0085518-t005" target="_blank">Table 5</a>. Each motif was identified using two, related data sets (see text).</p

Proximal sequence context flanking the CME contributes to the midline and tracheal expression pattern.

<p>Whole mount transgenic embryos containing one of the multimerized synthetic reporter constructs were labeled with anti-<i>GFP</i> (green; B, D, F, H, J, L, N, P, R, T, V, X, Z, B’, D’, F’, H’, J’, L’, N’, P’, R’, T’, V’, X’, Z’, B’’ and D’’) and anti-<i>sim</i> (red; C, G, K, O, S, W, A’, E’, I’, M’, Q’, U’, Y’ and C’’) antibodies and analyzed by confocal microscopy. The overlap in expression between <i>GFP</i> and <i>sim</i> is yellow (merge; A, E, I, M, Q, U, Y, C’, G’, K’, O’, S’, W’ and A’’). Midline <i>GFP</i> expression is driven by the (A–D) <i>synth 1:GFP</i>, (E–H) <i>synth 2:GFP</i> (I–L) <i>synth 3:GFP</i>, (M–P) <i>synth 4:GFP</i> and (Q–T) <i>synth 5:GFP,</i> whereas the (U–X) <i>Sox:GFP</i> and (Y–B’) <i>synth 6:GFP</i> reporters are not expressed in either the midline or trachea. The (C’-F’) <i>Toll:GFP,</i> (G’-J’) <i>synth 7:GFP</i>, (K’-N’) <i>synth 8:GFP</i>, (O’-R’) <i>synth 9:GFP</i> and (S’-V’) <i>synth 10:GFP</i> synthetic reporters are expressed in both the midline and trachea, whereas the expression pattern of the (W’-Z’) <i>synth 11:GFP</i> and (A’’-D’’) <i>synth 12:GFP</i> reporters are restricted to trachea only. The expression patterns of the <i>Toll:GFP</i> and <i>Sox:GFP</i> reporters were previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085518#pone.0085518-Fulkerson1" target="_blank">[59]</a>. Note that midline <i>GFP</i> expression driven by the (A–D) <i>synth 1:GFP</i>, (M–P) <i>synth 4:GFP</i>, (Q–T) <i>synth 5:GFP,</i> (C’-F’) <i>Toll:GFP,</i> (G’-J’) <i>synth 7:GFP</i>, (O’-R’) <i>synth 9:GFP</i> and (S’-V’) <i>synth 10:GFP</i> reporters is in both neurons and glia, whereas expression driven by the (E–H) <i>synth 2:GFP</i> reporter is restricted to midline glia and expression of the (I–L) <i>synth 3:GFP</i> and (K’-N’) <i>synth 8:GFP</i> reporters is restricted to midline neurons<b>.</b> The immediate CME context within each synthetic sequence is indicated to the left of the images and the entire sequence of each synthetic reporter construct is listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085518#pone-0085518-t004" target="_blank">Table 4</a>. Arrows indicate midline glia and arrowheads indicate midline neurons. Lateral or ventrolateral views of stage 16 transgenic embryos are shown; anterior is in the top, left hand corner and ventral is bottom, left. Four copies of each synthetic sequence were tested within the reporter constructs.</p

The <i>comm cis</i>-regulatory region contains an enhancer that drives expression in the embryonic midline and larval trachea.

<p>(A) Genomic regions surrounding and within <i>comm</i> were used to generate the reporter constructs and (B) the <i>comm</i> enhancer is shown. The closest gene upstream of <i>comm</i> is <i>CG6244</i>, which is 79,892 bp away. (C–N) Whole mount embryos were double-stained with anti-<i>GFP</i> (green: D, G, J and M) and anti-<i>sim</i> antibodies (red; E, H, K and N) and analyzed by confocal microscopy and the overlap in expression is shown in yellow in the merge column (C, F, I and L). (C–E) <i>comm2575:GFP</i> and (L–N) <i>comm443:GFP</i> are expressed in both midline glia (arrows) and midline neurons (arrowheads), while (F–H) <i>comm693:GFP</i> is restricted to some midline neurons (arrowheads). The <i>comm267:GFP</i> (I–K) and <i>comm737:GFP</i> (data not shown) reporters are not expressed in the midline. Lateral views of stage 16 transgenic embryos are shown; anterior is in the top, left hand corner and ventral is on the left. (O–T) <i>comm443:GFP</i> is also expressed in the tracheal dorsal trunks (arrows in O and S) as well as other tracheal branches (arrow in R). Live larvae containing the <i>comm443:GFP</i> reporter were analyzed by confocal and differential contrast microscopy and dorsal views are shown.</p

Sequence of synthetic reporter constructs.

<p>Sequences of synthetic constructs multimerized four times and fused to <i>GFP</i> within reporter constructs are listed according to the tissue that expressed each synthetic reporter (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085518#pone-0085518-g009" target="_blank">Fig. 9</a>). The CME is enlarged within each sequence. Sites important for midline expression within the <i>wrapper</i> enhancer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085518#pone.0085518-Estes1" target="_blank">[67]</a> are underlined in <i>synths 1</i><b>–</b><i>6</i> and include putative binding sites for Sox (ATTGT), <i>pointed</i> (CTCTCCG) and unknown (AAAA) transcription factors. Binding sites for engrailed (TAATTA), Vvl (TTGCAT) and Suppressor of Hairless (<b>GTGGGAAC</b>CGAGCTGAAAGTAAG<b>TTTCTCAC</b><b>)</b> were added to the <i>Toll</i> CME sequence and shown in bold in <i>synths 7</i><b>–</b><i>9</i>.</p

Proximal CME context in midline and tracheal enhancers.

<p>Sixty-six CMEs were found in all midline and tracheal enhancers examined. ¹The nucleotides found directly 5′ and 3′ of the CME within the enhancers and ²the number of times that sequence was found in all the midline and tracheal enhancers are listed. The three sequence contexts found in the left column represent 52% of the CMEs found within all enhancers (34/66; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085518#pone.0085518.s004" target="_blank">Table S4</a>), while ³other sequence contexts were not found in any enhancers. ^{4, 5}Sequences flanking the sixty-six CMEs were used to derive a consensus sequence for genes expressed in the midline, trachea or both tissues.</p

Midline and tracheal motifs identified with MEME.

<p>MEME analysis was used to identify motifs overrepresented in midline and tracheal enhancers. Three motifs were found in midline enhancers and one in tracheal enhancers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085518#pone-0085518-g010" target="_blank">Fig. 10</a>). Results from¹twelve enhancers that drive expression in the midline together with seven enhancers that drive expression in the midline and trachea or ²twelve enhancers that drive expression in the trachea and seven enhancers that drive expression in the midline and trachea are shown, as well as.³the number of enhancers examined,⁴number of enhancers containing the motif,⁵likelihood of finding the motif by chance,⁶number of times the site was found in all the enhancers examined and.⁷length of the identified site.</p

An <i>esg</i> genomic region contains a midline enhancer that is separable and distinct from two <i>esg</i> tracheal enhancers.

<p>(A) Genomic regions surrounding <i>esg</i> used to generate the reporter constructs and (B) the <i>esg</i> enhancers are shown as above. Gray boxes represent 5′ and 3′ untranslated regions. (C–J) Live larvae were analyzed by confocal and differential contrast microscopy and ventral views of the (C–F) <i>esg TR C1:GFP</i> and (G–J) <i>esg TR C7:GFP</i> reporters are shown. In larvae, the <i>esg TR C1:GFP</i> reporter is expressed sporadically in fusion cells (arrow in E) and the <i>esg TR C7:GFP</i> reporter is expressed in all tracheal branches and sporadically in the dorsal trunks (G–J), but consistently in fusion cells (arrows I). (K–N) Whole mount <i>esg ML C2:GFP</i> reporter embryos were stained with an anti-<i>GFP</i> antibody (green: L), <i>engrailed</i> monoclonal antibody (blue; M) and anti-<i>sim</i> antibody (red; N) and analyzed by confocal microscopy. The overlap in expression is shown in the merge image (K). Anterior midline glia express <i>GFP</i> and <i>sim</i> and are located dorsally within the nerve chord. Posterior midline glia that normally undergo cell death during this time can still be visualized with <i>GFP</i> (three cells surrounding star in N), but not <i>sim</i> or <i>engrailed</i>. The MNB and its progeny express <i>sim, engrailed</i> and <i>GFP</i> and are located ventrally within the nerve chord (arrowheads in K–N). Lateral view of a stage 16 transgenic embryo is shown; anterior is in the top, left hand corner and ventral is on the left.</p

The <i>moody cis</i>-regulatory region contains a tracheal enhancer that overlaps with a lateral CNS glial enhancer.

<p>(A) The genomic regions surrounding and within <i>moody</i> used to generate the reporter constructs and (B) the <i>moody</i> enhancer is shown. (C–N) Whole mount embryos were double-stained with an anti-<i>GFP</i> antibody (green: D, G, J, and M) and monoclonal antibody <i>2A12</i> (red; E, H and K) or anti-<i>repo</i> monoclonal antibody (red; N) and analyzed by confocal microscopy. The overlap in expression is shown in yellow in the merge columns (C, F, I and L). The expression patterns of the (C–E) <i>moody1970:GFP</i>, (F–H and L–N) <i>moody1221:GFP</i> and (I–K) <i>moody608:GFP</i> reporters are shown. Both the <i>moody1970:GFP</i> (not shown) and <i>moody1221:GFP</i> (L–N) reporters also drive expression in lateral glia as indicated by co-localization with <i>repo</i> (arrows). Additionally, <i>moody 1221:GFP</i> is expressed in the dorsal trunk (arrows F–H), while <i>moody1970:GFP</i> is expressed in the dorsal vessel (arrows C and D) and lightly in the dorsal trunk. (C–H) Dorsolateral, (I–K) lateral or (L–N) ventral views of stage 16 transgenic embryos are shown; anterior is in the top, left hand corner.</p