P53 Downstream Genes Research Articles

The impact of p53 on the early stage replication of retrovirus

BackgroundThe function of p53 in cancer biology has been studied extensively, but its role in anti-retrovirus infection has been elusive for many years. The restriction of retrovirus early stage replication by p53 was investigated in this study.MethodVSV-G pseudotyped retrovirus with GFP reporter gene was used to infect both HCT116 p53+/+ cells and its isogenic p53 knockout HCT116 p53−/− cells. The infection was detected by flow cytometry. Reverse transcription products were quantified by real time PCR. Mutation analysis was performed after 1-LTR cycle and 2-LTR cycle DNA were amplified and PCR products were sequenced. Transcription and translation of cyclin-dependent kinase inhibitor 1 (p21Cip1) and SAM domain and HD domain-containing protein 1 (SAMHD1) were analyzed by TaqMan PCR and Western blot experiments. siRNA experiment was applied to study the role of p53 downstream gene p21Cip1 in the restriction of retrovirus infection.ResultsIt was found that the block of retrovirus infection in non-cycling cells was significantly attenuated in HCT116 p53−/− cells when compared to HCT116 p53+/+ cells. It was found that both late reverse transcription products and viral 2-LTR cycle DNA were significantly increased in infected non-cycling HCT116 p53−/− cells. Furthermore, the mutation frequency detected in 1-LTR DNA from HCT116 p53+/+ cells were significantly decreased in comparison to HCT116 p53−/− cells. A higher number of insertion and deletion mutations were detected in the joint region of 2-LTR cycle DNA in infected p53+/+ cells. Cell cycle analysis showed retrovirus infection promoted host cell replication. Higher levels of mRNA and protein of p21Cip1 were found in HCT116 p53+/+ cells in comparison to the HCT116 p53−/− cells. Furthermore, knockdown of p21Cip1 in non-cycling HCT116 p53+/+ cells significantly increased the infection.ConclusionsThe results of this study showed that p53 is an important restriction factor that interferes with retrovirus infection in its early stage of replication. Our results suggested that p53 mediates the inhibition of retrovirus infection in non-cycling cells through it downstream gene p21Cip1, and p53 also functions to influence formation of 1-LTR cycle and 2-LTR cycle DNA.

Assessment of the DNA damaging potential of environmental chemicals using a quantitative high-throughput screening approach to measure p53 activation.

Genotoxicity potential is a critical component of any comprehensive toxicological profile. Compounds that induce DNA or chromosomal damage often activate p53, a transcription factor essential to cell cycle regulation. Thus, within the US Tox21 Program, we screened a library of ∼10,000 (∼8,300 unique) environmental compounds and drugs for activation of the p53-signaling pathway using a quantitative high-throughput screening assay employing HCT-116 cells (p53+/+ ) containing a stably integrated β-lactamase reporter gene under control of the p53 response element (p53RE). Cells were exposed (-S9) for 16 hr at 15 concentrations (generally 1.2 nM to 92 μM) three times, independently. Excluding compounds that failed analytical chemistry analysis or were suspected of inducing assay interference, 365 (4.7%) of 7,849 unique compounds were concluded to activate p53. As part of an in-depth characterization of our results, we first compared them with results from traditional in vitro genotoxicity assays (bacterial mutation, chromosomal aberration); ∼15% of known, direct-acting genotoxicants in our library activated the p53RE. Mining the Comparative Toxicogenomics Database revealed that these p53 actives were significantly associated with increased expression of p53 downstream genes involved in DNA damage responses. Furthermore, 53 chemical substructures associated with genotoxicity were enriched in certain classes of p53 actives, for example, anthracyclines (antineoplastics) and vinca alkaloids (tubulin disruptors). Interestingly, the tubulin disruptors manifested unusual nonmonotonic concentration response curves suggesting activity through a unique p53 regulatory mechanism. Through the analysis of our results, we aim to define a role for this assay as one component of a comprehensive toxicological characterization of large compound libraries. Environ. Mol. Mutagen. 58:494-507, 2017. © 2017 Wiley Periodicals, Inc.

P53-independent p21 induction by MELK inhibition.

MELK play critical roles in human carcinogenesis through activation of cell proliferation, inhibition of apoptosis and maintenance of stemness. Therefore, MELK is a promising therapeutic target for a wide range of cancers. Although p21 is a well-known p53-downstream gene, we found that treatment with a potent MELK inhibitor, OTS167, could induce p21 protein expression in cancer cell lines harboring loss-of-function TP53 mutations. We also confirmed that MELK knockdown by siRNA induced the p21 expression in p53-deficient cancer cell lines and caused the cell cycle arrest at G1 phase. Further analysis indicated that FOXO1 and FOXO3, two known transcriptional regulators of p21, were phosphorylated by MELK and thus be involved in the induction of p21 after MELK inhibition. Collectively, our herein findings suggest that MELK inhibition may be effective for human cancers even if TP53 is mutated.

Overexpression of MAGEA2 has a prognostic significance and is a potential therapeutic target for patients with lung cancer.

Melanoma-associated antigens (MAGE) are expressed in different type of cancers including lung cancer and have been shown to be functionally related to p53 tumor suppressor gene. Little is known about the relationship between MAGE genes and p53 aberrant expression in lung cancer. The aims of this study were to observe the expression of MAGEA2, examine the role of MAGEA2 in lung cancer survival, investigate its correlation between MAGEA2 and p53, and explore its clinicopathologic significance as a prognostic marker. Quantitative reverse transcription-polymerase chain reaction was performed to detect the expression of MAGEA2 using 36primary tumors and 31 metastatic lymph nodes from patients with lung cancer. The role of MAGEA2 in cancer cell growth and in the regulation of p53 downstream genes were examined using small interfering RNA. The expression of MAGEA2 and p53 were analyzed immunohistochemically using tissue microarray from 353 resected lung specimens. High-level expression of MAGEA2 (High-MAGEA2) was confirmed in lung tumors with high frequency. Inhibiting MAGEA2 expression effectively suppressed cancer cell growth and decreased the expression of p53 downstream target genes invitro. In adenocarcinoma, High-MAGEA2 was strongly associated with aberrant p53 expression (P<0.001) and was associated with worse clinical outcomes (5-year OS, 87.1% in low vs. 74.1% in high, P=0.014). Aberrant p53 expression was also significant worse prognostic factor (P=0.029). Among the adenocarcinoma patients with wild-type p53, High-MAGEA2 had poorer prognosis than low-level MAGEA2 groups (5-year OS, 90.1% vs. 72.1%, P=0.037), whereas had no difference in p53 aberrant tumors. On multivariate analysis, MAGEA2 was independently associated with survival (hazard ratio; 2.12, P=0.030). In conclusion, suppression of MAGEA2 in lung cancer cells significantly reduced the growth/survival of cancer cells. High-MAGEA2 was identified as an independent prognostic factor in lung adenocarcinoma. Specific inhibition of MAGEA2 may be a promising therapeutic strategy for patients with lung cancer.

ZIKA virus elicits P53 activation and genotoxic stress in human neural progenitors similar to mutations involved in severe forms of genetic microcephaly and p53.

Epidemiological evidence from the current outbreak of Zika virus (ZIKV) and recent studies in animal models indicate a strong causal link between ZIKV and microcephaly. ZIKV infection induces cell-cycle arrest and apoptosis in proliferating neural progenitors. However, the mechanisms leading to these phenotypes are still largely obscure. In this report, we explored the possible similarities between transcriptional responses induced by ZIKV in human neural progenitors and those elicited by three different genetic mutations leading to severe forms of microcephaly in mice. We found that the strongest similarity between all these conditions is the activation of common P53 downstream genes. In agreement with these observations, we report that ZIKV infection increases total P53 levels and nuclear accumulation, as well as P53 Ser15 phosphorylation, correlated with genotoxic stress and apoptosis induction. Interestingly, increased P53 activation and apoptosis are induced not only in cells expressing high levels of viral antigens but also in cells showing low or undetectable levels of the same proteins. These results indicate that P53 activation is an early and specific event in ZIKV-infected cells, which could result from cell-autonomous and/or non-cell-autonomous mechanisms. Moreover, we highlight a small group of P53 effector proteins that could act as critical mediators, not only in ZIKV-induced microcephaly but also in many genetic microcephaly syndromes.

Long intragenic non-coding RNA lincRNA-p21 suppresses development of human prostate cancer.

Prostate cancer is one of the most frequent malignancies in men, worldwide, although its underlying mechanisms are not fully understood. Long non-coding RNAs participate in development of human cancers. In this invetsigation, we aimed to study the roles of lincRNA-p21 in development of human prostate cancer. Expression of lincRNA-p21 was assessed by real-time PCR in cell lines and in human tissues. Lentivirus carrying sh-lincRNA-p21, lincRNA-p21 or control constructs were used to determine their effects on cell proliferation and apoptosis. A mouse xenograft model was employed to explore the functions of lincRNA-p21 on cancer cell population growth in vivo. Relationships between p53 downstream genes and lincRNA-p21 levels were explored by real-time PCR, western blotting and chromatin immunoprecipitation. LincRNA-p21 was found to be down-regulated in human prostate cancer, and low levels of lincRNA-p21 correlated with high disease stage and prediction of poor survival. We further showed that lincRNA-p21 inhibited prostate cancer cell proliferation and colony formation in vitro and reduced rate of prostate cancer cell population growth in vivo. Study of mechanisms involved revealed that lincRNA-p21 promoted apoptosis and induced expression of p53 downstream genes by regulating p53 binding to their promoters. Finally, we showed that expression of p53 downstream genes was reduced in the malignant prostate tissues, which correlated with lincRNA-p21 level. Our findings indicated that lincRNA-p21 inhibited development of human prostate cancer partly by regulating p53 downstream gene expression and partly by apoptotic activation.

ZIKA virus elicits P53 activation and genotoxic stress in human neural progenitors similar to mutations involved in severe forms of genetic microcephaly.

Epidemiological evidence from the current outbreak of Zika virus (ZIKV) and recent studies in animal models indicate a strong causal link between ZIKV and microcephaly. ZIKV infection induces cell-cycle arrest and apoptosis in proliferating neural progenitors. However, the mechanisms leading to these phenotypes are still largely obscure. In this report, we explored the possible similarities between transcriptional responses induced by ZIKV in human neural progenitors and those elicited by three different genetic mutations leading to severe forms of microcephaly in mice. We found that the strongest similarity between all these conditions is the activation of common P53 downstream genes. In agreement with these observations, we report that ZIKV infection increases total P53 levels and nuclear accumulation, as well as P53 Ser15 phosphorylation, correlated with genotoxic stress and apoptosis induction. Interestingly, increased P53 activation and apoptosis are induced not only in cells expressing high levels of viral antigens but also in cells showing low or undetectable levels of the same proteins. These results indicate that P53 activation is an early and specific event in ZIKV-infected cells, which could result from cell-autonomous and/or non-cell-autonomous mechanisms. Moreover, we highlight a small group of P53 effector proteins that could act as critical mediators, not only in ZIKV-induced microcephaly but also in many genetic microcephaly syndromes.

236. Role of P53 on T Lymphopoiesis from Human Embryonic Stem Cells

Although pluripotent stem cells are well recognized as a potential source of cell therapy, it is still needed to improve efficiency to differentiate into target cell lineages. Tumor suppressor P53 regulates multiple signaling pathways triggered by diverse cellular stresses including DNA damages, oncogenic stimulations, and hypoxic stress, resulting in cell-cycle arrest, apoptosis, and senescence. P53 signaling is also important for double-stranded DNA breaks (DSBs) induced during physiologic events, i.e., rearrangement of antigen-specific receptors. It has been reported that P53-mediated DSB checkpoint contribute to normal murine T lymphopoiesis, especially at the double-negative (DN) stage which is defined as CD4-CD8- fraction in thymus and requires rearrangements of the T cell receptor (TCR) β locus and successful pre-TCR signaling. Here we defined the role of P53 on lymphopoiesis from human embryonic stem cells (ESCs).Firstly we modified P53 gene of human ESC H1 by utilizing of zinc finger nuclease targeting the P53 gene, kindly provided by Sangamo BioSciences. Sequencing analysis of the P53 knockout (KO) ES cells showed the successful deletion which induced the frame shift of the downstream sequence in both of its alleles. Western blot analysis of P53 phosphorylation status in P53 KO ESCs showed undetectable levels of phosphorylated or non-phosphorylated P53 proteins when cultured in the presence or absence of apoptotic signal triggered by mitomycin C (MMC). In consistent with this, P53 KO ESCs showed significant resistance to MMC-induced cell death. In addition, P53 KO ESCs lacked apoptotic stimulation-induced upregulation of P53 downstream target genes including P53 up-regulated modulator of apoptosis (PUMA).We then induced hematopoietic differentiation of P53 KO ESCs through embryoid body formation. Erythroid lineage cells developed from human ESCs were significantly suppressed in the absence of P53 signaling during embryoid body maturation. Pharmacological inhibition of P53 had the same effect as genetic disruption of P53 gene. CD34+ hematopoietic precursors were isolated from embryoid bodies originated from H1 and P53 KO ECSs, plated on OP9-DL1 stromal cells, and cultured in the presence of stem cell factor (SCF), FLT3 ligand, and interleukin (IL)-7. After 3-4 weeks of culture, CD45+CD3+ T lineage cells were induced from both H1 and P53 KO ECSs-derived CD34+ cells. Among these cells, most of the cells were in CD4+CD8+ double-positive (DP) stage, with increase in the yield of DP cells in the absence of P53 signaling (H1: 343 cells/1 × 10^6 input CD34+ cells; P53 KO: 2476 cells /1 × 10^6 input CD34+ cells; FigureFigure). Whether pharmacological inhibition of P53 had the similar effect on T lymphopoiesis as genetic disruption of P53 gene needs to be investigated furthermore.Our data indicate that P53 mediated signaling regulate in vitro early T lymphopoiesis from human pluripotent stem cells, especially at the transition from double negative into DP stage. These observations promoted us to perform high throughput transcriptome analysis including cDNA microarray analysis between early T lineage cells derived from H1 and P53 KO ESCs. Genes associated with the early T lymphopoiesis from human ESCs were identified and currently under further characterization.View Large Image | Download PowerPoint Slide

Gene Expression Profile of the Clinically Aggressive Micropapillary Variant of Bladder Cancer

BackgroundProgression of conventional urothelial carcinoma of the bladder to a tumor with unique microscopic features referred to as micropapillary carcinoma is coupled with aggressive clinical behavior signified by a high propensity for metastasis to regional lymph nodes and distant organs resulting in shorter survival. ObjectiveTo analyze the expression profile of micropapillary cancer and define its molecular features relevant to clinical behavior. Design, setting, and participantsWe retrospectively identified 43 patients with micropapillary bladder cancers and a reference set of 89 patients with conventional urothelial carcinomas and performed whole-genome expression messenger RNA profiling. Outcome measurements and statistical analysisThe tumors were segregated into distinct groups according to hierarchical clustering analyses. They were also classified according to luminal, p53-like, and basal categories using a previously described algorithm. We applied Ingenuity Pathway Analysis software (Qiagen, Redwood City, CA, USA) and gene set enrichment analysis for pathway analyses. Cox proportional hazards models and Kaplan-Meier methods were used to assess the relationship between survival and molecular subtypes. The expression profile of micropapillary cancer was validated for selected markers by immunohistochemistry on parallel tissue microarrays. Results and limitationsWe show that the striking features of micropapillary cancer are downregulation of miR-296 and activation of chromatin-remodeling complex RUVBL1. In contrast to conventional urothelial carcinomas that based on their expression can be equally divided into luminal and basal subtypes, micropapillary cancer is almost exclusively luminal, displaying enrichment of active peroxisome proliferator-activated receptor γ and suppression of p63 target genes. As with conventional luminal urothelial carcinomas, a subset of micropapillary cancers exhibit activation of wild-type p53 downstream genes and represent the most aggressive molecular subtype of the disease with the shortest survival. The involvement of miR-296 and RUVBL1 in the development of micropapillary bladder cancer was identified by the analyses of correlative associations of genome expression profiles and requires mechanistic validation. ConclusionsMicropapillary cancer evolves through the luminal pathway and is characterized by the activation of miR-296 and RUVBL1 target genes. Patient summaryOur observations have important implications for prognosis and for possible future development of more effective therapies for micropapillary bladder cancer.

Oridonin, a novel lysine acetyltransferases inhibitor, inhibits proliferation and induces apoptosis in gastric cancer cells through p53- and caspase-3-mediated mechanisms.

Lysine acetylation has been reported to involve in the pathogenesis of multiple diseases including cancer. In our screening study to identify natural compounds with lysine acetyltransferase inhibitor (KATi) activity, oridonin was found to possess acetyltransferase-inhibitory effects on multiple acetyltransferases including P300, GCN5, Tip60, and pCAF. In gastric cancer cells, oridonin treatment inhibited cell proliferation in a concentration-dependent manner and down-regulated the expression of p53 downstream genes, whereas p53 inhibition by PFT-α reversed the antiproliferative effects of oridonin. Moreover, oridonin treatment induced cell apoptosis, increased the levels of activated caspase-3 and caspase-9, and decreased the mitochondrial membrane potential in gastric cancer cells in a concentration-dependent manner. Caspase-3 inhibition by Ac-DEVD-CHO reversed the proapoptosis effect of oridonin. In conclusion, our study identified oridonin as a novel KATi and demonstrated its tumor suppressive effects in gastric cancer cells at least partially through p53-and caspase-3-mediated mechanisms.

Allele-specific silencing of mutant p53 attenuates dominant-negative and gain-of-function activities.

Many p53 hotspot mutants not only lose the transcriptional activity, but also show dominant-negative (DN) and oncogenic gain-of-function (GOF) activities. Increasing evidence indicates that knockdown of mutant p53 (mutp53) in cancer cells reduces their aggressive properties, suggesting that survival and proliferation of cancer cells are, at least partially, dependent on the presence of mutp53. However, these p53 siRNAs can downregulate both wild-type p53 (wtp53) and mutp53, which limits their therapeutic applications. In order to specifically deplete mutp53, we have developed allele-specific siRNAs against p53 hotspot mutants and validated their biological effects in the absence or presence of wtp53. First, the mutp53-specific siRNAs selectively reduced protein levels of matched p53 mutants with minimal reduction in wtp53 levels. Second, downregulation of mutp53 in cancer cells expressing a mutp53 alone (p53mut) resulted in significantly decreased cell proliferation and migration. Third, transfection of mutp53-specific siRNAs in cancer cells expressing both wtp53 and mutp53 also reduced cell proliferation and migration with increased transcripts of p53 downstream target genes, which became further profound when cells were treated with an MDM2 inhibitor Nutlin-3a or a chemotherapeutic agent doxorubicin. These results indicate that depletion of mutp53 by its specific siRNA restored endogenous wtp53 activity in cells expressing both wtp53 and mutp53. This is the first study demonstrating biological effects and therapeutic potential of allele-specific silencing of mutp53 by mutp53-specific siRNAs in cancer cells expressing both wtp53 and mutp53, thus providing a novel strategy towards targeted cancer therapies.

Role of P53 on T Lymphopoisis from Human Embryonic Stem Cells

Tumor suppressor P53 regulates multiple signaling pathways triggered by diverse cellular stresses including DNA damages, oncogenic stimulations, and hypoxic stress, resulting in cell-cycle arrest, apoptosis, and senescence. P53 signaling is also important for double-stranded DNA breaks (DSBs) induced during physiologic events, i.e., rearrangement of antigen-specific receptors. It has been reported that P53-mediated DSB checkpoint contribute to normal murine T lymphopoiesis, especially at the double-negative (DN) stage which is defined as CD4-CD8- fraction in thymus and requires rearrangements of the T cell receptor (TCR) b locus and successful pre-TCR signaling (Guidos CJ et al., Genes Dev, 1996; Jiang D et al., J Exp Med 2006; HaksMC et al., Immunity, 1999). Here we defined the role of P53 on hematopoietic development, especially lymphopoiesis, from human embryonic stem cells (ESCs).Firstly we modified P53 gene of human ESC H1 by utilizing genome editing tool of zinc finger nuclease (ZFN) targeting the 5th exon of the P53 gene, kindly provided by Sangamo BioSciences. Sequencing analysis of the P53 knockout (KO) ES cells showed the successful deletion at the 5th exon which induced the frame shift of the downstream sequence in both of its alleles. qRT-PCR showed no stable expression of full length P53 mRNA and western blot analysis of P53 phosphorylation status in P53 KO ESCs showed undetectable levels of phosphorylated or non-phosphorylated P53 proteins when cultured in the presence or absence of apoptotic signal triggered by mitomycin C (MMC). In consistent with this, P53 KO ESCs showed significant resistance to MMC-induced cell death. In addition, P53 KO ESCs lacked apoptotic stimulation-induced upregulation of P53 downstream target genes including P53 up-regulated modulator of apoptosis (PUMA). On the other hand induction of P53 target gene P21 was not observed both in H1 and P53 KO ESCs, as reported previously by other groups (Ginis I et al., Dev Biol, 2004; Barta T et al., Stem Cells, 2010; Garc'a CP et al., Stem Cell Res, 2014; World J et al., Stem Cells, 2014).We then induced hematopoietic differentiation of P53 KO ESCs through embryoid body formation. Erythroid lineage cells developed from human ESCs were significantly suppressed in the absence of P53 signaling during embryoid body maturation. Pharmacological inhibition of P53 had the same effect as genetic disruption of P53 gene. CD34+ hematopoietic precursors were isolated from embryoid bodies originated from H1 and P53 KO ECSs, plated on OP9-DL1 stromal cells, and cultured in the presence of stem cell factor (SCF), FLT3 ligand, and interleukin (IL)-7. After 3-4 weeks of culture, CD45+CD3+ T lineage cells were induced from both H1 and P53 KO ECSs-derived CD34+ cells. Among these cells, most of the cells were in CD4+CD8+ double-positive (DP) stage, with increase in the yield of DP cells in the absence of P53 signaling (H1: 343 cells/1 x 106 input CD34+ cells; P53 KO: 2476 cells/1 x 106 input CD34+ cells; Figure). Whether pharmacological inhibition of P53 had the similar effect on T lymphopoiesis as genetic disruption of P53 gene needs to be investigated furthermore.Our data indicate that P53 mediated signaling regulate in vitro early T lymphopoiesis from human pluripotent stem cells, especially at the transition from double negative into DP stage. These observations promoted us to perform high throughput transcriptome analysis including cDNA microarray analysis between early T lineage cells derived from H1 and P53 KO ESCs. Genes associated with the early T lymphopoiesis from human ESCs were identified and currently under further characterization. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Changes in mRNA expression of p53 and related downstream genes in peripheral blood lymphocytes in workers occupationally exposed to arsenic

To investigate the changes in mRNA expression of p53 and related downstream genes in peripheral blood lymphocytes in workers occupationally exposed to arsenic as well as its influencing factors, and to analyze the mechanism of genetic toxicity of arsenic. With cluster random sampling, 79 workers from an arsenic smelting plant were selected as exposure group, and another 24 people without occupational exposure to arsenic were selected as control group. The relative mRNA expression of p53 and related downstream genes in the peripheral blood lymphocytes of the two groups was determined by quantitative realtime PCR. The levels of inorganic arsenic (iAs), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) in urine were determined by hydride generation-atomic absorption spectrometry. The exposure group had significantly higher levels of iAs, MMA, and DMA than the control group (P<0.01); the exposure group had significantly higher relative mRNA expression (2(-ΔΔCt)) of p53 and four related downstream genes in peripheral blood lymphocytes than the control group (P<0.05); the relative mRNA expression of p53 and related downstream genes was positively correlated with each other (P<0.01), with a correlation coefficient greater than 0.4; the levels of arsenic compounds in urine were positively correlated with the relative mRNA expression of p53 and some of its downstream genes (P<0.05). The changes in mRNA expression of p53 and related downstream genes are closely related to the metabolic transformation of inorganic arsenic in workers occupationally exposed to arsenic, and it also plays an important role in genetic toxicity and carcinogenic effect in people exposed to arsenic.

CP-31398 inhibits the growth of p53-mutated liver cancer cells in vitro and in vivo.

The tumor suppressor p53 is one of the most frequently mutated genes in hepatocellular carcinoma (HCC). Previous studies demonstrated that CP-31398 restored the native conformation of mutant p53 and trans-activated p53 downstream genes in tumor cells. However, the research on the application of CP-31398 to liver cancer has not been reported. Here, we investigated the effects of CP-31398 on the phenotype of HCC cells carrying p53 mutation. The effects of CP-31398 on the characteristic of p53-mutated HCC cells were evaluated through analyzing cell cycle, cell apoptosis, cell proliferation, and the expression of p53 downstream genes. In tumor xenografts developed by PLC/PRF/5 cells, the inhibition of tumor growth by CP-31398 was analyzed through gross morphology, growth curve, and the expression of p53-related genes. Firstly, we demonstrated that CP-31398 inhibited the growth of p53-mutated liver cancer cells in a dose-dependent and p53-dependent manner. Then, further study showed that CP-31398 re-activated wild-type p53 function in p53-mutated HCC cells, which resulted in inhibitive response of cell proliferation and an induction of cell-cycle arrest and apoptosis. Finally, in vivo data confirmed that CP-31398 blocked the growth of xenografts tumors through transactivation of p53-responsive downstream molecules. Our results demonstrated that CP-31398 induced desired phenotypic change of p53-mutated HCC cells in vitro and in vivo, which revealed that CP-31398 would be developed as a therapeutic candidate for HCC carrying p53 mutation.

Abstract 1213: Endogenous p53 affinity tagging with CRISPR

Abstract Several antimetabolites used for cancer treatment, including hydroxyurea (HU) and pemetrexed (PTX), stabilize tumor suppressor protein p53, but fail to induce the transcription of p53 downstream target genes. To understand the underlying mechanism for the deficiency of stabilized p53, we are quantitatively characterizing p53 posttranslational modification and its interaction partners with mass spectrometry (MS). To facilitate the purification of endogenous p53, we established an HCT116 human colon carcinoma cell line, in which one allele of endogenous p53 was tagged with a classic Tandem Affinity Tag (TAP) at the C-terminus, using adenovirus associated virus (AAV) mediated homologous recombination (HR). The purification of p53 from this cell exhibited severe losses at each step, compromising analysis by MS. Because of this limited yield from TAP purification, we performed a systematic tagging of p53 with 3xFLAG, Strep-II, and HALO tags, and their combinations, at both N- and C-termini. The latest CRISPR (clustered regularly interspaced short palindromic repeats)/cas9 nuclease system provides major advantages for endogenous gene modification through HR. Efficient site-specific protein tagging (or mutation) requires a DNA break point in the immediate vicinity, limiting the choice of guide DNA (gDNA) positions possible. By extending the complementary sequence between CRISPR targeting RNA (crRNA) and trans-activating crRNA (tracrRNA) in the chimeric single guide RNA (sgRNA), we expanded the essential Protospacer Adjacent Motif (PAM) sequence of the S. pyogenes CRISPR II system from NGG to NAG with equal targeting efficiency. Cas9 nickase (both D10A, and H840A-N854A-N863A) generated more favorable HR rates compared to non-homologous end joining (NHEJ), while mitigating off-target effects, in comparison to wild type cas9. Utilizing our conditions, we achieved 2-8% (for different tags) endogenous p53 tagging in the background of 10-60% NHEJ from wild type cas9 and single sgRNA. With cas9 nickase and two sgRNA, we consistently achieved 0.2-2% specific p53 tagging in the background of 0.01-8% NHEJ, with higher efficiency for 5′ than 3′ single strand overhangs generated. We are currently generating single cell clones, selecting those tags most faithful to natural p53 expression levels, and testing p53 purification efficiencies with different tags. In conclusion, we optimized the CRISPR system, extensively characterized the individual steps in generating single cell clones for endogenous gene tagging, and will apply the optimal tag to purify p53 and its co-eluted binding partners for quantitative MS analysis. Citation Format: Chen Yang, Cortney L. Lawrence, Charles E. Lyons, Shirley M. Taylor, Richard G. Moran. Endogenous p53 affinity tagging with CRISPR. [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 1213. doi:10.1158/1538-7445.AM2015-1213

JMJD5 interacts with p53 and negatively regulates p53 function in control of cell cycle and proliferation

JMJD5 is a Jumonji C domain-containing demethylase/hydroxylase shown to be essential in embryological development, osteoclastic maturation, circadian rhythm regulation and cancer metabolism. However, its role and underlying mechanisms in oncogenesis remain unclear. Here, we demonstrate that JMJD5 forms complex with the tumor suppressor p53 by interacting with p53 DNA-binding domain (DBD), and negatively regulates its activity. Downregulation of JMJD5 resulted in increased expression of multiple p53 downstream genes, such as the cell cycle inhibitor CDKN1A and DNA repair effector P53R2, only in p53-proficient lung cancer cells. Upon DNA damage, the JMJD5–p53 association decreased, and thereby, promoted p53 recruitment to the target genes and stimulated its transcriptional activity. Furthermore, JMJD5 facilitated the cell cycle progression in a p53-dependent manner under both normal and DNA damage conditions. Depletion of JMJD5 inhibited cell proliferation and enhanced adriamycin-induced cell growth suppression in the presence of p53. Collectively, our results reveal that JMJD5 is a novel binding partner of p53 and it functions as a positive modulator of cell cycle and cell proliferation mainly through the repression of p53 pathway. Our study extends the mechanistic understanding of JMJD5 function in cancer development and implicates JMJD5 as a potential therapeutic target for cancer.

A distinct p53 target gene set predicts for response to the selective p53-HDM2 inhibitor NVP-CGM097.

Biomarkers for patient selection are essential for the successful and rapid development of emerging targeted anti-cancer therapeutics. In this study, we report the discovery of a novel patient selection strategy for the p53-HDM2 inhibitor NVP-CGM097, currently under evaluation in clinical trials. By intersecting high-throughput cell line sensitivity data with genomic data, we have identified a gene expression signature consisting of 13 up-regulated genes that predicts for sensitivity to NVP-CGM097 in both cell lines and in patient-derived tumor xenograft models. Interestingly, these 13 genes are known p53 downstream target genes, suggesting that the identified gene signature reflects the presence of at least a partially activated p53 pathway in NVP-CGM097-sensitive tumors. Together, our findings provide evidence for the use of this newly identified predictive gene signature to refine the selection of patients with wild-type p53 tumors and increase the likelihood of response to treatment with p53-HDM2 inhibitors, such as NVP-CGM097.

Trans-splicing repair of mutant p53 suppresses the growth of hepatocellular carcinoma cells in vitro and in vivo.

Reactivation of wild-type p53 (wt-p53) function is an attractive therapeutic approach to p53-defective cancers. An ideal p53-based gene therapy should restore wt-p53 production and reduces mutant p53 transcripts simultaneously. In this study, we described an alternative strategy named as trans-splicing that repaired mutant p53 transcripts in hepatocellular carcinoma (HCC) cells. The plasmids which encoded a pre-trans-splicing molecule (PTM) targeting intron 6 of p53 were constructed and then transfected into HCC cells carrying p53 mutation. Phenotypic changes of HCC cells induced by p53-PTM were analyzed through cell cycle, cell apoptosis and the expression of p53 downstream target genes. Spliceosome mediated RNA trans-splicing (SMaRT) reduced mutant p53 transcripts and produced functional wt-p53 protein after the delivery of p53-PTM plasmids, which resulted in phenotype correction of HCC cells. In tumor xenografts established by p53-mutated HCC cells, adenovirus encoding p53-PTM induced cell cycle arrest and apoptosis and then blocked the growth of tumors in mice. Collectively, our results demonstrated for the first time that mutant p53 transcripts were functionally corrected in p53-defective HCC cells and xenografts using trans-splicing, which indicated the feasibility of using trans-splicing to repair p53 mutation in p53-defective cancers.

P53 Inducible Gene-3: Potential Serum Marker for Hepatocellular Carcinoma Beyond Milan Criteria

Aims: The diagnosis of Hepatocellular carcinoma (HCC) is usually made late, precluding curative resection or liver transplantation in over 70% of HCC patients. P53 inducible gene 3 (PIG3) is a p53 downstream gene and a key component of the DNA damage response pathway, which is relevant in conditions with rapid cell turnover such as HCC. Our aim was to assess whether PIG3 could serve as a novel biomarker for HCC. Methods: A pilot study of 37 consecutive patients with the diagnosis of HCC, 42 patients with hepatic cirrhosis versus healthy controlswas conducted. Serum AFP and PIG3 levels measured with enzyme-linked immunosorbent assay were compared across groups using student t-testand correlation coefficient (R) between AFP and PIG3 was calculated. Results: Clinical characteristics were similar across the HCC and cirrhosis groups. Serum PIG3 levels were significantly higher among patients with cirrhosis or HCC (23.27 ± 13.26 and 24.1 ± 18.61 ng/mL, respectively), as compared to healthy controls (3.71 ± 1.79 ng/mL, p<0.0001). PIG3 values were significantly different across the following tumor diameter ranges: less than 2 cm, 18.47 ng/mL; 2 to 5 cm, 18.54 ng/mL; and greater than 5 cm, 38.72 ng/mL; p=0.0012. The correlation coefficient between log (AFP) and PIG3 levels in HCC patients was r=0.4564, p=0.005. Conclusion: Serum p53-inducible gene 3 levels were significantly elevated in patients with HCC beyond 5 cm, indicating potential as an adjunct to imaging in determining eligibility for liver transplantation. The potential for PIG3 as a biomarker of HCC warrants further prospective study.

MiR-638 promotes melanoma metastasis and protects melanoma cells from apoptosis and autophagy

The present study identified miR-638 as one of the most significantly overexpressed miRNAs in metastatic lesions of melanomas compared with primary melanomas. miR-638 enhanced the tumorigenic properties of melanoma cells in vitro and lung colonization in vivo. mRNA expression profiling identified new candidate genes including TP53INP2 as miR-638 targets, the majority of which are involved in p53 signalling. Overexpression of TP53INP2 severely attenuated proliferative and invasive capacity of melanoma cells which was reversed by miR-638. Depletion of miR-638 stimulated expression of p53 and p53 downstream target genes and induced apoptosis and autophagy. miR-638 promoter analysis identified the miR-638 target transcription factor associated protein 2α (TFAP2A/AP-2α) as a direct negative regulator of miR-638, suggestive for a double-negative regulatory feedback loop. Taken together, miR-638 supports melanoma progression and suppresses p53-mediated apoptosis pathways, autophagy and expression of the transcriptional repressor TFAP2A/AP-2α.