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Integrated Analysis of p53 Targets
Integrated Analysis of p53 Targets
p53 is one of the most important tumor suppressors and was found to be mutated or inactivated in more than half of all human tumors. As a transcription factor p53 controls the expression of target genes that are involved in processes such as the regulation of cell cycle arrest, DNA repair or apoptosis and thereby prevents the development and the spreading of cancer. Though extensive research led to the identification of a lot of p53-regulated target genes, the list of p53 targets still continues to grow and seems to be far from complete. Here, we showed that p53 controls the EMT transcription factor SNAIL via the induction of miR-34 in colorectal cancer cells. This regulation shifted the cells towards an epithelial phenotype and inhibited migration, invasion and stemness features. In addition, we demonstrated a double-negative feedback loop between miR-34 and SNAIL that controls the transition between epithelial and mesenchymal states. In a further study we determined p53-regulated mRNAs, lncRNAs, miRNAs and proteins on a genome-wide scale. In combination with a p53 DNA binding analysis, this led to the identification of genes that are directly or indirectly regulated by p53. We found that the majority of differentially expressed genes, which were directly bound by p53 near their TSS, were up-regulated and experimentally confirmed several induced target genes that showed promoter proximal p53 binding. Moreover, we determined that transcriptional repression by p53 mainly occurs via indirect mechanisms and studied the impact of miRNA-mediated mechanisms on p53-controlled gene repression. Almost half of the down-regulated proteins displayed seed-matching sequences of p53-induced miRNAs in the corresponding 3’-UTRs. Exemplarily, the clinically relevant miRNA targets HMGB1, CIT and KLF12 were confirmed to be directly repressed by the p53-regulated miRNAs miR-34a, miR-486-5p and miR-205, respectively. Subsequently, we characterized cystatin D (CST5), a vitamin D3-inducible inhibitor of cysteine proteases, as a new direct p53 target gene that resulted from the genome-wide analysis mentioned above. CST5 inactivation decreased p53-induced MET, as evidenced by decreased inhibition of SNAIL and of migration by p53. In addition, simultaneous activation of p53 and treatment with calcitriol enhanced CST5 induction. Furthermore, we provided evidence that SNAIL directly represses CST5 expression which could be diminished by calcitriol treatment. Taken together, our results illustrate the complex network of protein-coding and non-coding genes that is directly or indirectly controlled by the tumor suppressor p53. The data obtained in these studies might pave the way for new diagnostic and therapeutic approaches in the treatment of cancer.
p53, targets, genome-wide, colon cancer
Hünten, Sabine
2016
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Hünten, Sabine (2016): Integrated Analysis of p53 Targets. Dissertation, LMU München: Faculty of Medicine
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Abstract

p53 is one of the most important tumor suppressors and was found to be mutated or inactivated in more than half of all human tumors. As a transcription factor p53 controls the expression of target genes that are involved in processes such as the regulation of cell cycle arrest, DNA repair or apoptosis and thereby prevents the development and the spreading of cancer. Though extensive research led to the identification of a lot of p53-regulated target genes, the list of p53 targets still continues to grow and seems to be far from complete. Here, we showed that p53 controls the EMT transcription factor SNAIL via the induction of miR-34 in colorectal cancer cells. This regulation shifted the cells towards an epithelial phenotype and inhibited migration, invasion and stemness features. In addition, we demonstrated a double-negative feedback loop between miR-34 and SNAIL that controls the transition between epithelial and mesenchymal states. In a further study we determined p53-regulated mRNAs, lncRNAs, miRNAs and proteins on a genome-wide scale. In combination with a p53 DNA binding analysis, this led to the identification of genes that are directly or indirectly regulated by p53. We found that the majority of differentially expressed genes, which were directly bound by p53 near their TSS, were up-regulated and experimentally confirmed several induced target genes that showed promoter proximal p53 binding. Moreover, we determined that transcriptional repression by p53 mainly occurs via indirect mechanisms and studied the impact of miRNA-mediated mechanisms on p53-controlled gene repression. Almost half of the down-regulated proteins displayed seed-matching sequences of p53-induced miRNAs in the corresponding 3’-UTRs. Exemplarily, the clinically relevant miRNA targets HMGB1, CIT and KLF12 were confirmed to be directly repressed by the p53-regulated miRNAs miR-34a, miR-486-5p and miR-205, respectively. Subsequently, we characterized cystatin D (CST5), a vitamin D3-inducible inhibitor of cysteine proteases, as a new direct p53 target gene that resulted from the genome-wide analysis mentioned above. CST5 inactivation decreased p53-induced MET, as evidenced by decreased inhibition of SNAIL and of migration by p53. In addition, simultaneous activation of p53 and treatment with calcitriol enhanced CST5 induction. Furthermore, we provided evidence that SNAIL directly represses CST5 expression which could be diminished by calcitriol treatment. Taken together, our results illustrate the complex network of protein-coding and non-coding genes that is directly or indirectly controlled by the tumor suppressor p53. The data obtained in these studies might pave the way for new diagnostic and therapeutic approaches in the treatment of cancer.