Phosphatase and tensin homologue deleted on chromosome ten (PTEN) is one of the most commonly altered tumor suppressors in human cancer. It is a dual-specificity phosphatase that by converting the lipid second messenger PIP3 to PIP2 antagonizes the PI3K/AKT signaling pathway. PTEN also has numerous, albeit controversial nuclear functions, which thus far have been shown to be independent of its phosphatase activity. Although a number of studies have described that loss or gain of PTEN protein expression alters gene expression patterns, relatively little is known about the exact mechanism. In this research study, we investigated PTEN’s influence on gene expression and its role in transcription regulation.
First, we established mouse embryonic fibroblasts (MEFs) as a suitable model system to study the effects of PTEN loss on gene expression. Using an Adeno-virus containing Cre-recombinase, Pten expression could be ablated efficiently in MEFs carrying loxP sites flanking exon 5 of the endogenous Pten locus. Genome-wide mRNA microarray analysis revealed that Pten deletion decreased the transcript levels of a subset of genes and increased the transcript levels of a different subset of genes. Moreover, by uncoupling these effects from PTEN’s role in the PI3K/AKT pathway we discovered that Pten loss can alter gene expression in a PI3K/AKT-dependent as well as a PI3K/AKT-independent manner. The upregulated genes were enriched for genes involved in DNA binding, replication, and repair, but also for regulation of gene expression. Gene expression can be influenced by histone modifications. However, loss of PTEN did not affect histone modifications globally as evidenced by western blotting. Using native ChIP-Seq experiments we showed that loss of PTEN altered the levels of H3K36me3 and H3K27me3 on a subset of genes and markedly decreased levels of H3K27ac at most enhancers as well as super-enhancers. However, RNAPII occupancy on enhancer-associated genes did not decrease, suggesting that the modulation of enhancer strength did not affect RNAPII recruitment to TSS.
In Chapter 3 we identify a nuclear pool of Pten that could associate with chromatin. Furthermore, we are the first to report that nuclear PTEN can directly interact with components of the transcription machinery including CDK7, CDK9, Cyclin T1, AFF4, and RNAPII. Loss of PTEN increased phosphorylation of Ser2 and Ser5 of the RNAPII CTD as well as RNAPII occupancy on promoters of expressed genes indicating an increase in transcriptional activity in PTEN-/- cells. Furthermore, PTEN deletion resulted in the upregulation of genes which are part of the important “Achilles cluster”, previously shown to confer sensitivity to transcription inhibition. We believe that it is over-expression of those genes that render PTEN deficient cells especially sensitive to transcription inhibitors such as THZ1, Triptolide, Flavopiridol and LDC000067. Over-expression of wild type PTEN but not a phosphatase-dead mutant of PTEN could decrease cells’ sensitivity to treatment with THZ1 or Flavopiridol. It also decreased protein levels of p-AKT Ser473 as well as RNAPII Ser2P and Ser5P suggesting that the phosphatase activity of PTEN is important for its role in transcription regulation.
In sum, we propose a model in which PTEN binds to CDK7, CDK9, Cyclin T1, RNAPPII and/or AFF4 thereby exerting a negative regulatory effect on the activity of transcription complexes. Upon loss of PTEN the negative regulatory effect is eliminated and transcription of a subset of genes increases. It is most likely these genes that confer sensitivity to transcription inhibition on PTEN-/- cells. The better understanding of this oncogenic mechanism may reveal novel therapeutic opportunities, and ultimately we propose that the sensitivity of PTEN deficient cells to inhibitors of transcription could provide an effective clinical strategy to target PTEN deficient cancers.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/D8GH9W9R |
| Date | January 2017 |
| Creators | Steinbach, Nicole |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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