Direct P53 Target Research Articles

Characterization of a p53/miR-34a/CSF1R/STAT3 Feedback Loop in Colorectal Cancer.

Background & AimsThe miR-34a gene is a direct target of p53 and is commonly silenced in colorectal cancer (CRC). Here we identified the receptor tyrosine kinase CSF1R as a direct miR-34a target and characterized CSF1R as an effector of p53/miR-34a-mediated CRC suppression.MethodsAnalyses of TCGA-COAD and three other CRC cohorts for association of mRNA expression and signatures with patient survival and molecular subtypes. Bioinformatics identification and experimental validation of miRNA and transcription factor targets. Functional analysis of factors/pathways in the regulation of epithelial-mesenchymal transition (EMT), invasion, migration, acquired chemo-resistance and metastasis. Analyses of protein expression and CpG methylation within primary human colon cancer samples.ResultsIn primary CRCs increased CSF1R, CSF1 and IL34 expression was associated with poor patient survival and a mesenchymal-like subtype. CSF1R displayed an inverse correlation with miR-34a expression. This was explained by direct inhibition of CSF1R by miR-34a. Furthermore, p53 repressed CSF1R via inducing miR-34a, whereas SNAIL induced CSF1R both directly and indirectly via repressing miR-34a in a coherent feed-forward loop. Activation of CSF1R induced EMT, migration, invasion and metastasis of CRC cells via STAT3-mediated down-regulation of miR-34a. 5-FU resistance of CRC cells was mediated by CpG-methylation of miR-34a and the resulting elevated expression of CSF1R. In primary CRCs elevated expression of CSF1R was detected at the tumor invasion front and was associated with CpG methylation of the miR-34a promoter as well as distant metastasis.ConclusionsThe reciprocal inhibition between miR-34a and CSF1R and its loss in tumor cells may be relevant for therapeutic and prognostic approaches towards CRC management.

P53-induced ARVCF modulates the splicing landscape and supports the tumor suppressive function of p53

p53 is one of the most important tumor suppressor genes, and the exploration of p53-target genes is important for elucidation of its functional mechanisms. In this study, we identified Armadillo Repeat gene deleted in Velo-Cardio-Facial syndrome (ARVCF) as a direct target of p53 through ChIP-sequencing analysis. Activated p53 protein was found to bind to two distinct sites in the ARVCF gene, resulting in induction of ARVCF expression at both the mRNA and protein levels. We revealed that the knockdown of ARVCF inhibited p53-induced apoptosis. Interestingly, ARVCF interacted with hnRNPH2, which is involved in pre-mRNA splicing, and ARVCF knockdown induced dynamic changes in alternative splicing patterns. These results suggest that p53-induced ARVCF indirectly, but not directly, regulates p53 target selectivity through splicing alterations of specific genes. Thus, we demonstrated that the induction of ARVCF expression contributed to the tumor suppressive function of p53. Recently, it has been reported that many tumors have thousands of alternative splicing events that are not detectable in normal samples. ARVCF may play a role in alternative splicing events in cancer and may provide clues to explore novel approaches for cancer diagnosis and therapy.

P53-regulated lncRNA uc061hsf.1 inhibits cell proliferation and metastasis in human esophageal squamous cell cancer.

The expression of long noncoding RNAs (lncRNAs) is closely associated with cancer development and progression, making these lncRNAs potentially novel therapeutic targets. In this study, we aimed to explore the potential function of lncRNA-uc061hsf.1 in esophageal squamous cell carcinoma (ESCC). The expression of lncRNA-uc061hsf.1 in ESCC tissues and cell lines was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation, apoptosis, and metastasis were detected via CCK-8, flow cytometry, and Transwell assays. The interaction between p53 and lncRNA uc061hsf.1 was analyzed using luciferase reporter gene and qRT-PCR. Through this approach, we identified the novel lncRNA uc061hsf.1, which was expressed in low level in ESCC and was correlated with lymph node metastasis and poor differentiation in ESCC patients. Knockdown or overexpression of lncRNA uc061hsf.1 in ESCC cells promoted or inhibited cell proliferation and metastasis, respectively. Mechanistically, lncRNA uc061hsf.1 was induced by p53, and luciferase reporter gene confirmed that lncRNA uc061hsf.1 was a direct transcriptional target of p53. We further found that uc061hsf.1 was able to regulate expression of the transcription factor FoxA1, thereby potentially influencing tumor cell migration. In conclusion, these results suggest that p53-regulated lncRNA uc061hsf.1 is a cancer suppressor gene which is associated with tumor progression in ESCC.

Identification and validation of microRNAs that synergize with miR-34a – a basis for combinatorial microRNA therapeutics

ABSTRACTEfforts to search for better treatment options for cancer have been a priority, and due to these efforts, new alternative therapies have emerged. For instance, clinically relevant tumor-suppressive microRNAs that target key oncogenic drivers have been identified as potential anti-cancer therapeutics. MicroRNAs are small non-coding RNAs that negatively regulate gene expression at the posttranscriptional level. Aberrant microRNA expression, through misexpression of microRNA target genes, can have profound cellular effects leading to a variety of diseases, including cancer. While altered microRNA expression contributes to a cancerous state, restoration of microRNA expression has therapeutic benefits. For example, ectopic expression of microRNA-34a (miR-34a), a tumor suppressor gene that is a direct transcriptional target of p53 and thus is reduced in p53 mutant tumors, has clear effects on cell proliferation and survival in murine models of cancer. MicroRNA replacement therapies have recently been tested in combination with other agents, including other microRNAs, to simultaneously target multiple pathways to improve the therapeutic response. Thus, we reasoned that other microRNA combinations could collaborate to further improve treatment. To test this hypothesis miR-34a was used in an unbiased cell-based approach to identify combinatorial microRNA pairs with enhanced efficacy over miR-34a alone. This approach identified a subset of microRNAs that was able to enhance the miR-34a antiproliferative activity. These microRNA combinatorial therapeutics could offer superior tumor-suppressive abilities to suppress oncogenic properties compared to a monotherapeutic approach. Collectively these studies aim to address an unmet need of identifying, characterizing, and therapeutically targeting microRNAs for the treatment of cancer.

Prognostic Effect of Long Noncoding RNA NEAT1 Expression Depends on p53 Mutation Status in Cancer.

Recently, many studies have revealed that long noncoding RNAs (lncRNAs) play important roles in various biological and pathological processes. Our previous study reported that lncRNA NEAT1 is a direct transcriptional target of p53. NEAT1 is an essential component of paraspeckles, which have recently been identified as a novel type of nuclear compartment. Although our previous findings indicate that NEAT1 induction contributes to the tumor-suppressor function of p53, the role of NEAT1 in cancer is still controversial. In this study, we comprehensively analyzed the relationship between NEAT1 expression and p53 mutation status. Interestingly, survival analysis based on NEAT1 expression in several cancer tissues revealed that the p53 wild-type group with high NEAT1 expression had a good prognosis, while poor prognosis or no correlation between NEAT1 expression and survival was observed in the p53-mutated group. These results demonstrate that the tumor-suppressive effect of NEAT1 depends on p53 function and is consistent with our previous report showing that NEAT1 supports the tumor-suppressive function of p53. Specifically, NEAT1 seems to play a tumor-suppressive role only in the presence of wild-type p53. These results provide important clues to the roles of NEAT1 in cancer.

INKA2, a novel p53 target that interacts with the serine/threonine kinase PAK4.

The p53 protein is a tumour suppressor and transcription factor that regulates the expression of target genes involved in numerous stress responses systems. In this study, we designed a screening strategy using DNA damage-induced mouse and human transcriptome data to identify novel downstream targets of p53. Our method selected genes with an induced expression in multiple organs of X-ray-irradiated p53 wild-type mice. The expression of inka box actin regulator 2 gene, known as Inka2, was upregulated in 12 organs when p53 expression was induced. Similarly, INKA2 was induced in a p53-dependent manner at both the mRNA and protein level in human cells treated with adriamycin. Reporter assays confirmed that p53 directly regulated INKA2 through an intronic binding site. The overexpression of INKA2 produced a slight decrease in cancer cell growth in the colony formation assay. Moreover, the analysis of The Cancer Genome Atlas (TCGA) data revealed a decreased INKA2 expression in tumour samples carrying p53 mutations compared with p53 wild-type samples. In addition, significantly higher levels of DNA methylation were observed in the INKA2 promoter in tumour samples, concordant with the reduced INKA2 expression in tumour tissues. These results demonstrate the potential of INKA2 as a cancer cell growth inhibitor. Furthermore, INKA2 protein interacts with the serine/threonine-protein kinase, p21 (RAC1) activated kinase (PAK)4, which phosphorylates β-catenin to prevent ubiquitin-proteasomal degradation. As β-catenin was downregulated in a stable INKA2-expressing cell line, the findings of this study suggest that INKA2 is a novel, direct downstream target of p53 that potentially decreases cell growth by inhibiting the PAK4-β-catenin pathway.

FAM111B, a direct target of p53, promotes the malignant process of lung adenocarcinoma.

Purpose: Lung adenocarcinoma (LUAD) is a main subtype of lung cancer, which is the leading cause of cancer-related deaths. The five-year survival rates of lung cancer patients are still comparatively low. Therefore, potential therapeutic targets are urgently needed to improve the survival of lung cancer patients. In this study, we identified FAM111B as an oncogene and potential therapeutic target for LUAD.Methods: The TCGA database and tissue microarray analysis were used to compare the expression of FAM111B in tumor tissue and normal tissues and evaluate the relationship between FAM111B expression and clinical survival. FAM111B was knocked down and overexpressed to observe whether FAM111B could affect the proliferation, migration, cell cycle, and apoptosis of LUAD cells in vivo and in vitro.Results: FAM111B was highly expressed in tumor tissues compared with normal tissues (P<0.01). LUAD patients with hyper-expression of FAM111B had a lower recurrence-free survival (P<0.01) and shorter overall survival (P<0.01). Knocking down FAM111B inhibited cell proliferation, migration and invasion in vitro and tumor growth in vivo. Silencing FAM111B could arrest LUAD cells at G2/M phase and increase apoptosis. Overexpression of FAM111B promoted the growth of lung cancer cells. FAM111B was identified as a direct target of p53 in existing researches by chip-seq analysis. Bioinformatics analysis predicted that FAM111B could directly bind to BAG3 (BCL2 associated athanogene 3). When FAM111B was down-regulated, both expression of BAG 3 and BCL2 were significantly reduced, whereas decreasing the expression of BAG3 had no effect on FAM111B.Conclusions: Our study indicated that FAM111B might be an oncogene and potential therapeutic target in LUAD which could be involved in the regulation of tumor cells by p53 signaling pathway and play an important role in the process of cell cycle and apoptosis by influencing the expression of BAG3 and BCL2.

P53-induced miR-1249 inhibits tumor growth, metastasis, and angiogenesis by targeting VEGFA and HMGA2

MicroRNAs (miRNAs) are important class of functional regulators involved in human cancers development, including colorectal cancer (CRC). Exploring aberrantly expressed miRNAs may provide us with new insights into the initiation and development of CRC by functioning as oncogenes or tumor suppressors. The aim of our study is to discover the expression pattern of miR-1249 in CRC and investigate its clinical significance as well as biological role in CRC progression. In our study, we found that miR-1249 was markedly downregulated in CRC tissues and cell lines, and negatively related to pN stage, pM stage, TNM stage, and overall survival (OS). Moreover, we demonstrated that miR-1249 was a direct transcriptional target of P53 and revealed that P53-induced miR-1249 inhibited tumor growth, metastasis and angiogenesis in vitro and vivo. Additionally, we verified that miR-1249 suppressed CRC proliferation and angiogenesis by targeting VEGFA as well as inhibited CRC metastasis by targeting both VEGFA and HMGA2. Further studying showed that miR-1249 suppressed CRC cell proliferation, migration, invasion, and angiogenesis via VEGFA-mediated Akt/mTOR pathway as well as inhibited EMT process of CRC cells by targeting both VEGFA and HMGA2. Our study indicated that P53-induced miR-1249 may suppress CRC growth, metastasis and angiogenesis by targeting VEGFA and HMGA2, as well as regulate Akt/mTOR pathway and EMT process in the initiation and development of CRC. miR-1249 might be a novel the therapeutic candidate target in CRC treatment.

Conservation and divergence of the p53 gene regulatory network between mice and humans

Understanding the p53 tumor suppressor pathway remains crucial for the design of anticancer strategies. Studies in human tumors and mouse models help to unravel the molecular mechanisms that underlie the p53 signaling pathway. Yet, the p53 gene regulatory network (GRN) is not the same in mice and humans. The comparison of the regulatory networks of p53 in mice and humans reveals that gene up- and down-regulation by p53 are distinctly affected during evolution. Importantly, gene up-regulation by p53 underwent more rapid evolution and gene down-regulation has been evolutionarily constrained. This difference stems from the two major mechanisms employed by p53 to regulate gene expression: up-regulation through direct p53 target gene binding and indirect down-regulation through the p53-p21-DREAM pathway. More than 1000 genes have been identified to differ in their p53-dependent expression between mice and humans. Analysis of p53 gene expression profiles and p53 binding data reveal that turnover of p53 binding sites is the major mechanism underlying extensive variation in p53-dependent gene up-regulation. Only a core set of high-confidence genes appears to be directly regulated by p53 in both species. In contrast to up-regulation, p53-induced down-regulation is well conserved between mice and humans and controls cell cycle genes. Here a curated data set is provided that extends the previously established web-atlas at www.targetgenereg.org to assess the p53 response of any human gene of interest and its mouse ortholog. Taken together, the analysis reveals a limited translation potential from mouse models to humans for the p53 GRN.

Orphan receptor NR4A3 is a novel target of p53 that contributes to apoptosis.

Major tumor suppressor and transcription factor p53 coordinates expression of many genes hence affecting critical cellular functions including cell cycle, senescence, and apoptosis. The NR4A family of orphan receptors (NR4A1-3) belongs to the superfamily of nuclear receptors. They regulate genes involved in proliferation, cell migration, and apoptosis. In this study, we report an identification of NR4A3 as a direct transcriptional target of p53. Using various techniques, we showed that p53 directly bound the promoter of NR4A3 gene and induced its transcription. Functionally, over-expression of NR4A3 attenuated proliferation of cancer cells and promoted apoptosis by augmenting the expression of pro-apoptotic genes, PUMA and Bax. Knockdown of NR4A3 reversed these phenotypes. Importantly, NR4A3 exhibited tumor suppressive functions both in p53-dependent and independent manner. In addition, NR4A3 physically interacted with an anti-apoptotic Bcl-2 protein hence sequestering it from blunting apoptosis. These observations were corroborated by the bioinformatics analysis, which demonstrated a correlation between high levels of NR4A3 expression and better survival of breast and lung cancer patients. Collectively, our studies revealed a novel transcriptional target of p53, NR4A3, which triggers apoptosis and thus likely has a tumor suppressive role in breast and lung cancers.

Dual Suppressive Effect of miR-34a on the FOXM1/eEF2-Kinase Axis Regulates Triple-Negative Breast Cancer Growth and Invasion.

Purpose: Recent studies indicated that dysregulation of noncoding RNAs (ncRNA) such as miRNAs is involved in pathogenesis of various human cancers. However, the molecular mechanisms underlying miR-34a are not fully understood in triple-negative breast cancer (TNBC).Experimental Design: We performed in vitro functional assays on TNBC cell lines to investigate the role of miR-34a in FOXM1/eEF2K signaling axis. TNBC tumor xenograft models were used for in vivo therapeutic delivery of miR-34a.Results: In this study, we investigated the role of p53-driven ncRNA miR-34a and found that miR-34a is associated with significantly longer patient survival in TNBC and inversely correlated with levels of proto-oncogenic eEF2K, which was associated with significantly shorter overall patient survival. We showed that miR-34a directly binds to the 3'-untranslated region of eEF2K and FOXM1 mRNAs and suppresses their expression, leading to inhibition of TNBC cell proliferation, motility, and invasion. Notably, restoring miR-34a expression recapitulated the effects of inhibition of eEF2K and FOXM1, the transcription factor for eEF2K and the direct target of p53, in TNBC cell lines, whereas overexpression of eEF2K and FOXM1 rescued the effects and signaling pathways mediated by miR-34a. Moreover, in vivo therapeutic delivery of miR-34a nanoparticles by systemic intravenous administration delayed tumor growth of two different orthotopic TNBC tumor xenograft models by inhibiting eEF2K and FOXM1, intratumoral proliferation and angiogenesis, and inducing apoptosis.Conclusions: Overall, our findings provide new insights into the tumor suppressor role of miR-34a by dual-targeting of FOXM1/eEF2K signaling axis and suggest that miR-34a-based gene therapy may be a potential therapeutic strategy in TNBC. Clin Cancer Res; 24(17); 4225-41. ©2018 AACR.

TRMP, a p53-inducible long noncoding RNA, regulates G1/S cell cycle progression by modulating IRES-dependent p27 translation

The tumor suppressor p53 plays a pivotal role in the protection against cancer. Increasing evidence suggests that long noncoding RNA (lncRNA) plays an important role in the regulation of the p53 pathway, however, the detailed mechanisms remain to be further elucidated. In this study, we report a new p53-inducible lncRNA that we termed TRMP (TP53-regulated modulator of p27). As a direct transcriptional target of p53, TRMP plays an unexpected pro-survival function. Knockdown of TRMP inhibits cell proliferation by inducing a G1 cell cycle arrest. Mechanistically, TRMP suppresses internal ribosomal entry site (IRES)-dependent translation of p27 by competing p27 mRNA for polypyrimidine tract-binding protein 1 (PTBP1) binding. Furthermore, TRMP is able to regulate cell proliferation, G1/S cell cycle progression, and tumor xenograft growth via the inhibition of p27. Taken together, these findings suggest lncRNA as a new layer to fine-tune the p53 response and reveal TRMP as an important downstream effector of p53 activity.

Prognostic role of FUT8 expression in relation to p53 status in stage II and III colorectal cancer.

The expression of fucosyltransferase 8, an enzyme responsible for core fucosylation encoded by FUT8, influences tumor biology and correlates with patient prognosis in several solid cancers. We hypothesized that p53 alteration modifies prognostic associations of FUT8 expression in colorectal cancer (CRC), since FUT8 has recently been identified as a direct transcriptional target of wild-type p53. Utilizing multiple datasets of microarray and RNA sequence of CRC, FUT8 mRNA was found to be highly expressed in wild-type p53 tumors (n = 382) compared to those of mutant p53 (n = 437). Prognostic values of FUT8 expression in conjunction with the p53 status for disease-free survival (DFS) were analyzed using two independent cohorts of stage II and III CRC after curative surgery, including the immunohistochemistry (IHC) cohort (n = 123) and the microarray cohort (n = 357). In both cohorts, neither FUT8 expression nor the p53 status was associated with DFS. Strikingly, positive expression of FUT8 protein was significantly associated with better DFS only in tumors with negative p53, while it had no prognostic impact in tumors with positive p53 in the IHC cohort. Although not statistically significant, a similar prognostic trend was observed in the microarray cohort when patients were stratified by the p53 status. Our results suggest that the prognostic values of FUT8 expression on DFS may be modified by the p53 status, and the expression of FUT8 protein can be a prognostic biomarker for patients with stage II and III CRC.

DNA repair processes are critical mediators of p53-dependent tumor suppression

It has long been assumed that p53 suppresses tumor development through induction of apoptosis, possibly with contributions by cell cycle arrest and cell senescence1,2. However, combined deficiency in these three processes does not result in spontaneous tumor formation as observed upon loss of p53, suggesting the existence of additional mechanisms that are critical mediators of p53-dependent tumor suppression function3-5. To define such mechanisms, we performed in vivo shRNA screens targeting p53-regulated genes in sensitized genetic backgrounds. We found that knockdown of Zmat3, Ctsf and Cav1, promoted lymphoma/leukemia development only when PUMA and p21, the critical effectors of p53-driven apoptosis, cell cycle arrest and senescence, were also absent. Notably, loss of the DNA repair gene Mlh1 caused lymphoma in a wild-type background, and its enforced expression was able to delay tumor development driven by loss of p53. Further examination of direct p53 target genes implicated in DNA repair showed that knockdown of Mlh1, Msh2, Rnf144b, Cav1 and Ddit4 accelerated MYC-driven lymphoma development to a similar extent as knockdown of p53. Collectively, these findings demonstrate that extensive functional overlap of several p53-regulated processes safeguards against cancer and that coordination of DNA repair appears to be an important process by which p53 suppresses tumor development.

The Alzheimer's disease-associated TREM2 gene is regulated by p53 tumor suppressor protein

TREM2 mutations evoke neurodegenerative disorders, and recently genetic variants of this gene were correlated to increased risk of Alzheimer's disease. The signaling cascade originating from the TREM2 membrane receptor includes its binding partner TYROBP, BLNK adapter protein, and SYK kinase, which can be activated by p53. Moreover, in silico identification of a putative p53 response element (RE) at the TREM2 promoter led us to hypothesize that TREM2 and other pathway elements may be regulated in p53-dependent manner. To stimulate p53 in synergistic fashion, we exposed A549 lung cancer cells to actinomycin D and nutlin-3a (A + N). In these cells, exposure to A + N triggered expression of TREM2, TYROBP, SYK and BLNK in p53-dependent manner. TREM2 was also activated by A + N in U-2 OS osteosarcoma and A375 melanoma cell lines. Interestingly, nutlin-3a, a specific activator of p53, acting alone stimulated TREM2 in U-2 OS cells. Using in vitro mutagenesis, chromatin immunoprecipitation, and luciferase reporter assays, we confirmed the presence of the p53 RE in TREM2 promoter. Furthermore, activation of TREM2 and TYROBP by p53 was strongly inhibited by CHIR-98014, a potent and specific inhibitor of glycogen synthase kinase-3 (GSK-3). We conclude that TREM2 is a direct p53-target gene, and that activation of TREM2 by A + N or nutlin-3a may be critically dependent on GSK-3 function.

Abstract IA07: Deconstructing p53 pathways in tumor suppression

Abstract The p53 transcription factor is a critical tumor suppressor in humans and mice. Despite this essential function, the molecular pathways through which p53 acts in tumor suppression remain enigmatic. To define the transcriptional programs through which p53 suppresses carcinogenesis, we have taken combined mouse genetic and genomic approaches. We previously generated a panel of p53 knock-in mouse strains expressing mutants in the first (p5325,26), second (p5353,54), or both (p5325,26,53,54) of two amino-terminal transcriptional activation domains (TADs). In terms of transcriptional activity, we have discovered that p5325,26 is severely impaired for transactivation of the majority of canonical p53 target genes (e.g. p21, Puma, Noxa), but retains the ability to activate a set of primarily novel p53 target genes, while p5325,26,53,54 lacks transactivation activity altogether. The p5353,54 mutant, in contrast, is uncompromised for transactivation of p53 target genes. The p5325,26,53,54 mutant is completely defective in tumor suppression, underscoring the importance of transcriptional activation for p53-mediated tumor suppression. Intriguingly, the p5325,26 mutant retains full activity in suppressing a variety of cancers, indicating that efficient transactivation of most canonical p53 target genes is dispensable for tumor suppression. As p5325,26 activates only a subset of p53-inducible genes, yet retains tumor-suppressor activity, it has helped pinpoint a small set of novel, direct p53-inducible tumor suppression-associated target genes (TSAGs) whose functions we are currently interrogating through genetic screens. Additionally, we have made the surprising discovery that the p5353,54 mutant suppresses pancreatic cancer more efficiently than wild-type p53, and that this capacity correlates with the ability of p5353,54 to hyperactivate a subset of p53 target genes. Indeed, our analysis of these genes has uncovered Ptpn14, a direct p53 target gene critical for mediating p53 function in suppressing pancreatic cancer cell growth. Ptpn14 encodes a protein tyrosine phosphatase that negatively regulates the oncoprotein Yap in both human and mouse cells. These studies thus reveal a p53-Ptpn14-Yap axis in pancreatic cancer. Together, these strategies will help to elaborate the transcriptional networks fundamental for p53 function in tumor suppression. Citation Format: Stephano Spano Mello, Kathryn Bieging-Rolett, Alyssa Kaiser, Elizabeth Joy Valente, Nitin Raj, Jacob McClendon, Brittany Maria Flowers, David Warren Morgens, Michael Cory Bassik, Laura Donatella Attardi. Deconstructing p53 pathways in tumor suppression [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr IA07.

P53 Promotes Retinoid Acid-induced Smooth Muscle Cell Differentiation by Targeting Myocardin.

TP53 is a widely studied tumor suppressor gene that controls various cellular functions, including cell differentiation. However, little is known about its functional roles in smooth muscle cells (SMCs) differentiation from embryonic stem cells (ESCs). SMC differentiation is at the heart of our understanding of vascular development, normal blood pressure homeostasis, and the pathogenesis of vascular diseases such as atherosclerosis, hypertension, restenosis, as well as aneurysm. Using retinoid acid (RA)-induced SMC differentiation models, we observed that p53 expression is increased during in vitro differentiation of mouse ESCs into SMCs. Meanwhile, suppression of p53 by shRNA reduced RA-induced SMC differentiation. Mechanistically, we have identified for the first time that Myocardin, a transcription factor that induces muscle cell differentiation and muscle-specific gene expression, is the direct target of p53 by bioinformatic analysis, luciferase reporter assay, and chromatin immunoprecipitation approaches. Moreover, in vivo SMC-selective p53 transgenic overexpression inhibited injury-induced neointimal formation. Taken together, our data demonstrate that p53 and its target gene, Myocardin, play regulatory roles in SMC differentiation. This study may lead to the identification of novel target molecules that may, in turn, lead to novel drug discoveries for the treatment of vascular diseases.

Inactivation of ribosomal protein S27-like confers radiosensitivity via the Mdm2-p53 and Mdm2\u2013MRN\u2013ATM axes

RPS27L (ribosomal protein S27-like) is an evolutionarily conserved ribosomal protein and a direct p53 target. We recently reported that Rps27l disruption triggers ribosomal stress to induce p53, causing postnatal death, which can be rescued by Trp53+/−. Whether and how Rps27l modulates radiosensitivity is unknown. Here we report that Rps27l−/−; Trp53+/− mice are extremely sensitive to radiation due to reduced proliferation and massive induction of apoptosis in radiation-sensitive organs. Mechanistically, the radiation sensitivity is mediated by two signaling pathways: (1) activated p53 pathway due to imbalanced Mdm2/Mdm4 levels and reduced E3 ligase activity; and (2) reduced DNA damage response due to reduced MRN/Atm signal as a result of elevated Mdm2 binding of Nbs1 to inhibit Nbs1–Atm binding and subsequent Atm activation. Indeed, heterozygous deletion of Mdm2 restores the MRN/Atm signal. Collectively, our study revealed a physiological condition under which Rps27l regulates the Mdm2/p53 and MRN/Atm axes to maintain DNA damage response and to confer radioprotection in vivo.

Abstract A25: Elephant p53 (EP53) enhances and restores p53-mediated apoptosis in human and canine osteosarcoma

Abstract Amplification of elephant p53 (EP53) was recently described as a potential mechanism for cancer resistance in elephants. Osteosarcoma is the most common pediatric bone tumor and also occurs frequently in pet dogs. Both human and canine osteosarcoma contain a very high rate of TP53 alterations leading to genomic instability. The purpose of our study was to determine if EP53 could enhance and/or restore p53 function in osteosarcoma and trigger p53-mediated cell death. We expressed various EP53 proteins in canine and human osteosarcoma cell lines (OSCA-40, U-2 OS, and Saos-2) by transfection or viral transduction. Expression of EP53 was confirmed by Western blot. Apoptosis of cells transfected/transduced with EP53 was compared to cells transfected/transduced with negative control vectors. Apoptosis was measured by fluorescence microscopy and caspase activity. We observed a significant increase in caspase activity (normalized to cell viability) of U-2 OS (TP53-wild type) cells expressing EP53 compared to negative control cells (p&amp;lt;0.0001). Saos-2 (TP53-null) and OSCA-40 cells underwent apoptosis as visualized by time-lapse fluorescence microscopy. Experiments to confirm these results with caspase activity assays are currently under way. Increased expression of p21, a direct p53 target, was observed in Saos-2 cells by Western blot. Taken together, our results indicate that EP53 enhances and restores p53-mediated apoptosis in osteosarcoma. These data suggest for the first time that EP53 can function in both human and canine osteosarcoma cells to promote cell death. Efforts to define the mechanism of action of EP53 in osteosarcoma cells are ongoing. Our results support further exploration of EP53-based osteosarcoma therapeutics for both humans and dogs. Citation Format: Lisa M. Abegglen, Cristhian Toruno, Lauren N. Donovan, Rosann Robinson, Mor Goldfeder, Genevieve Couldwell, Wendy K. Kiso, Dennis L. Schmitt, Aleah F. Caulin, Carlo C. Maley, Avi Schroeder, Joshua D. Schiffman. Elephant p53 (EP53) enhances and restores p53-mediated apoptosis in human and canine osteosarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A25.

Loss of p53-inducible long non-coding RNA LINC01021 increases chemosensitivity.

We have previously identified the long non-coding RNA LINC01021 as a direct p53 target (Hünten et al. Mol Cell Proteomics. 2015; 14:2609-2629). Here, we show that LINC01021 is up-regulated in colorectal cancer (CRC) cell lines upon various p53-activating treatments. The LINC01021 promoter and the p53 binding site lie within a MER61C LTR, which originated from insertion of endogenous retrovirus 1 (ERV1) sequences. Deletion of this MER61C element by a CRISPR/Cas9 approach, as well as siRNA-mediated knockdown of LINC01021 RNA significantly enhanced the sensitivity of the CRC cell line HCT116 towards the chemotherapeutic drugs doxorubicin and 5-FU, suggesting that LINC01021 is an integral part of the p53-mediated response to DNA damage. Inactivation of LINC01021 and also its ectopic expression did not affect p53 protein expression and transcriptional activity, implying that LINC01021 does not feedback to p53. Furthermore, in CRC patient samples LINC01021 expression positively correlated with a wild-type p53-associated gene expression signature. LINC01021 expression was increased in primary colorectal tumors and displayed a bimodal distribution that was particularly pronounced in the mesenchymal CMS4 consensus molecular subtype of CRCs. CMS4 tumors with low LINC01021 expression were associated with poor patient survival. Our results suggest that the genomic redistribution of ERV1-derived p53 response elements and generation of novel p53-inducible lncRNA-encoding genes was selected for during primate evolution as integral part of the cellular response to various forms of genotoxic stress.