TP53 In Response Research Articles

Chromothripsis is a frequent event and underlies typical genetic changes in early T-cell precursor lymphoblastic leukemia in adults

Chromothripsis is a mitotic catastrophe that arises from multiple double strand breaks and incorrect re-joining of one or a few chromosomes. We report on incidence, distribution, and features of chromothriptic events in T-cell acute lymphoblastic leukemias (T-ALL). SNP array was performed in 103 T-ALL (39 ETP/near ETP, 59 non-ETP, and 5 with unknown stage of differentiation), including 38 children and 65 adults. Chromothripsis was detected in 11.6% of all T-ALL and occurred only in adult cases with an immature phenotype (12/39 cases; 30%). It affected 1 to 4 chromosomes, and recurrently involved chromosomes 1, 6, 7, and 17. Abnormalities of genes typically associated with T-ALL were found at breakpoints of chromothripsis. In addition, it gave rise to new/rare alterations, such as, the SFPQ::ZFP36L2 fusion, reported in pediatric T-ALL, deletions of putative suppressors, such as IKZF2 and CSMD1, and amplification of the BCL2 gene. Compared to negative cases, chromothripsis positive T-ALL had a significantly higher level of MYCN expression, and a significant downregulation of RGCC, which is typically induced by TP53 in response to DNA damage. Furthermore we identified mutations and/or deletions of DNA repair/genome stability genes in all cases, and an association with NUP214 rearrangements in 33% of cases.

TP53-inducible putative long noncoding RNAs encode functional polypeptides that suppress cell proliferation.

Polypeptides encoded by long noncoding RNAs (lncRNAs) are a novel class of functional molecules. However, whether these hidden polypeptides participate in the TP53 pathway and play a significant biological role is still unclear. Here, we discover that TP53-regulated lncRNAs can encode peptides, two of which are functional in various human cell lines. Using ribosome profiling and RNA-seq approaches in HepG2 cells, we systematically identified more than 300 novel TP53-regulated lncRNAs and further confirmed that 15 of these TP53-regulated lncRNAs encode peptides. Furthermore, several peptides were validated by mass spectrometry. Ten of the novel translational lncRNAs are directly inducible by TP53 in response to DNA damage. We show that the TP53-inducible peptides TP53LC02 and TP53LC04, but not their lncRNAs, can suppress cell proliferation. TP53LC04 peptide also has a function associated with cell proliferation by regulating the cell cycle in response to DNA damage. This study shows that TP53-regulated lncRNAs can encode new functional peptides, leading to the expansion of the TP53 tumor-suppressor network and providing novel potential targets for cancer therapy.

TP53 codon 72 polymorphism and type 2 diabetes: a case-control study in South Indian population.

TP53 functions primarily as a tumor suppressor, controlling a myriad of signalling pathways that prevent a cell from undergoing malignant transformation. This tumor suppressive function requires an activation and stabilization of TP53 in response to cell stressors. However, besides its cancer-preventive functions, TP53 is also known to be involved in diverse cellular processes including metabolism, reproduction, stem cell renewal and development. Indeed, several lines of evidence strongly suggest that TP53 plays crucial role in diabetes. A number of studies have evaluated the association of genetic alterations (single nucleotide variations) in TP53 gene with the development of diabetes. However, the results have not been consistent. The aim of this study was to evaluate whether the C/G polymorphism at codon 72 (Pro72/Arg72), located in exon 4 of TP53, is associated with type 2 diabetes in South Indian population. A total of 74 type 2 diabetic patients and 54 non-diabetic subjects were screened. None of the three genotypes, namely C/C (Pro/Pro), C/G (Pro/Arg), and G/G (Arg/Arg) was found to be significantly associated with type 2 diabetes in our study group. The findings of this study indicate that TP53 codon 72 polymorphism is not associated with increased risk of type 2 diabetes in South Indian population. Further studies with a large cohort size would be necessary to corroborate the observations of this study. Nevertheless, this study represents the first genetic analysis of TP53 variants in South Indian type 2 diabetic patients.

Targeted sequencing reveals the mutational landscape responsible for sorafenib therapy in advanced hepatocellular carcinoma.

Rationale: The existence of primary and acquired drug resistance is the main obstacle for the effect of multi-kinase inhibitor sorafenib and regorafenib in advanced hepatocellular carcinoma (HCC). However, plenty of patients did not significantly benefit from sorafenib treatment and little is known about the mechanism of drug resistance.Methods: Laser capture microdissection was used to acquire matched normal liver and tumor tissues on formalin-fixed paraffin-embedded specimens collected before sorafenib therapy from the first surgery of 119 HCC patients. Ultra-deep sequencing (~1000×) targeting whole exons of 440 genes in microdissected specimens and siRNA screen in 7 cell lines were performed to find mutations associated with differential responses to sorafenib. Patient-derived xenograft models were employed to determine the role of TP53 in response to sorafenib. Lentiviruses harboring wild-type and c.G52C-mutant OCT4 were applied to explore the function of OCT4 in resistance to sorafenib. ChIP-PCR assay for analysis of OCT4 transcriptional activity was performed to explore the affinity with the KITLG promoter. Statistical analyses were used to associate levels of p53 and OCT4 with tumor features and patient outcomes.Results: Total 1,050 somatic mutations and 26 significant driver genes were identified. SiRNA screening in 7 HCC cell lines was further performed to identify mutations associated with differential responses to sorafenib. A recurrent nonsynonymous mutation c.G52C in OCT4 (OCT4mut) was strongly associated with good response to sorafenib, whereas the stop-gain mutation in TP53 showed the opposite outcome both in vitro and in vivo. OCT4wt-induced stem cell factor (encoded by KITLG gene, SCF) expression and cross-activation of c-KIT/FLT3-BRAF signals were identified indispensably for sorafenib resistance, which could be reversed by the combination of c-KIT tyrosine kinase inhibitors or neutralizing antibody against SCF. Mechanistically, an OCT4 binding site in upstream of KITLG promoter was identified with a higher affinity to wildtype of OCT4 rather than G52C-mutant form, which is indispensable for OCT4-induced expression of KITLG and sorafenib resistance.Conclusion: Our study reported a novel somatic mutation in OCT4 (c.G52C) responsible for the sorafenib effect, and also shed new light on the treatment of HCC through the combination of specific tyrosine kinase inhibitors according to individual genetic patterns.

Caveolin-1 Y14 phosphorylation suppresses tumor growth while promoting invasion.

Caveolin-1 is a transmembrane protein with both tumor promoter and suppressor functions that remain poorly understood. Cav1 phosphorylation by Src kinase on tyrosine 14 is closely associated with focal adhesion dynamics and tumor cell migration, however the role of pCav1 in vivo in tumor progression remains poorly characterized. Herein, we expressed phosphomimetic Y14D, wild type, and non-phosphorylatable Y14F forms of Cav1 in MDA-MB-435 cancer cells. Expression of Cav1Y14D reduced cell proliferation and induced the TP53 tumor suppressor. Ectopic expression in MDA-MB-435 cells of Y14 phosphorylatable Cav1 was required for induction of TP53 in response to oxidative stress. Cav1Y14D promotes an apparent reversal of the Warburg effect and markedly inhibited tumor growth in vivo. However, Cav1 induced pseudopodial recruitment of glycolytic enzymes, and time-lapse intravital imaging showed increased invadopodia protrusion and extravasation into blood vessels for Cav1WT and Y14D but not for Y14F. Our results suggest that Cav1 Y14 phosphorylation levels play a role in the conflicting demands on metabolic resources associated with cancer cell proliferation versus motility.

A Zombie LIF Gene in Elephants Is Upregulated by TP53 to Induce Apoptosis in Response to DNA Damage

Large-bodied organisms have more cells that can potentially turn cancerous than small-bodied organisms, imposing an increased risk of developing cancer. This expectation predicts a positive correlation between body size and cancer risk; however, there is no correlation between body size and cancer risk across species ("Peto's paradox"). Here, we show that elephants and their extinct relatives (proboscideans) may have resolved Peto's paradox in part through refunctionalizing a leukemia inhibitory factor pseudogene (LIF6) with pro-apoptotic functions. LIF6 is transcriptionally upregulated by TP53 in response to DNA damage and translocates to the mitochondria where it induces apoptosis. Phylogenetic analyses of living and extinct proboscidean LIF6 genes indicates that its TP53 response element evolved coincident with the evolution of large body sizes in the proboscidean stem lineage. These results suggest that refunctionalizing of a pro-apoptotic LIF pseudogene may have been permissive (although not sufficient) for the evolution of large body sizes in proboscideans.

Abstract 2115: Effect of small interfering RNA targeting HPV E6/E7 gene on the regulation of TP53/Rb dynamic behaviour in cervical cancer cells

Abstract Human papillomavirus (HPV) E6 and E7 viral oncogenes are very well known to cause cervical cancer, because E6 degrades TP53 tumor suppressor protein, and E7 inactivates the tumor suppressor retinoblastoma (pRb) protein. Thus E6 and E7 oncogenes of HPV are supposed to be promising targets of gene therapy against HPV mediated cervical cancer. Here, we attempted to study the regulation of TP53/pRb proteins dynamic behaviour after HPV E6/E7 small interfering RNA (siRNA) transfection in cervical cancer cells. HPV positive (HeLa and Caski) cell lines were selected for these experiments. Herein, we also validated the dynamics of TP53 in response to antiviral siRNA targeting the E6 and E7 oncogenes. At first, we analyzed the effect of HPV E6/E7 siRNA on TP53/pRb reporter gene activity by using pathway profiling luciferase system. Our result clearly indicates that when compared with E6/E7 siRNA pool alone, siRNA pool in combination cisplatin dramatically increases the TP53/pRb and decreases E2F response elements regulating luciferase reporter gene activity. TP53 protein and its target gene expression were also confirmed by western blot and qRT-PCR analysis in a time dependent manner. In our TP53 reporter gene (pGreenFire1 GFP reporter vector with EF1-puro) assay by using live imaging analysis shows that combination of HPVE6/E7 siRNA pool with cisplatin, induces the TP53 expression within 10h and sustained that leads to cell death. Our imaging results also suggested that combining cisplatin with inhibition of HPV E6/E7 oncogene would synergize to stimulate a sustained GFP-TP53 expression can trigger either cell cycle arrest or apoptosis in response to DNA damage. In addition, we generated GFP-TP53 reporter stable HeLa cell line (pGreenFire1 GFP reporter vector with EF1-puro), in order to find the specificity of HPV E6/E7 siRNA induced TP53 expression in in vitro and in vivo. Similar GFP-TP53 dynamic pattern was observed in E6/E7 siRNA transfected GFP-TP53 reporter stable HeLa cell line. Furthermore to analyze the dynamic behaviour of pRb/E2F in live cells, reporter vector containing luciferase gene was recombinantly engineered to replace with RFP gene. Taken together, our results suggest that increase of TP53/Rb expression by silencing E6/E7 is associated with its chemo-sensitizing effect in cervical cancer cells. Overall, stabilization and maximal activation of the TP53/pRb signaling network can facilitate determination of the optimal therapeutic strategy for human cervical carcinoma. . Citation Format: Nirmal Rajasekaran, Hun Soon Jung, Young Deug Kim, Deuk Ae Kim, Tae Kyung Ha, Yoo Ha Na, Young kee Shin. Effect of small interfering RNA targeting HPV E6/E7 gene on the regulation of TP53/Rb dynamic behaviour in cervical cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2115. doi:10.1158/1538-7445.AM2015-2115

Genome-wide analysis of p63 binding sites identifies AP-2 factors as co-regulators of epidermal differentiation.

The p63 transcription factor (TP63) is critical in development, growth and differentiation of stratifying epithelia. This is highlighted by the severity of congenital abnormalities caused by TP63 mutations in humans, the dramatic phenotypes in knockout mice and de-regulation of TP63 expression in neoplasia altering the tumour suppressive roles of the TP53 family. In order to define the normal role played by TP63 and provide the basis for better understanding how this network is perturbed in disease, we used chromatin immunoprecipitation combined with massively parallel sequencing (ChIP-seq) to identify >7500 high-confidence TP63-binding regions across the entire genome, in primary human neonatal foreskin keratinocytes (HFKs). Using integrative strategies, we demonstrate that only a subset of these sites are bound by TP53 in response to DNA damage. We identify a role for TP63 in transcriptional regulation of multiple genes genetically linked to cleft palate and identify AP-2alpha (TFAP2A) as a co-regulator of a subset of these genes. We further demonstrate that AP-2gamma (TFAP2C) can bind a subset of these regions and that acute depletion of either TFAP2A or TFAP2C alone is sufficient to reduce terminal differentiation of organotypic epidermal skin equivalents, indicating overlapping physiological functions with TP63.

Mutations truncating the EP300 acetylase in human cancers.

The EP300 protein is a histone acetyltransferase that regulates transcription via chromatin remodelling and is important in the processes of cell proliferation and differentiation. EP300 acetylation of TP53 in response to DNA damage regulates its DNA-binding and transcription functions. A role for EP300 in cancer has been implied by the fact that it is targeted by viral oncoproteins, it is fused to MLL in Leukaemia and two missense sequence alterations in EP300 were identified in epithelial malignancies. Nevertheless, direct demonstration of the role of EP300 in tumorigenesis by inactivating mutations in human cancers has been lacking. Here we describe EP300 mutations, which predict a truncated protein, in 6(3%) of 193 epithelial cancers analysed. Of these six mutations, two were in primary tumours (a colorectal cancer and a breast cancer) and four were in cancer cell lines (colorectal, breast and pancreatic). In addition, we identified a somatic in-frame insertion in a primary breast cancer and missense alterations in a primary colorectal cancer and two cell lines (breast and pancreatic). Inactivation of the second allele was demonstrated in five of six cases with truncating mutations and in two other cases. Our data show that EP300 is mutated in epithelial cancers and provide the first evidence that it behaves as a classical tumour-suppressor gene.