scholarly journals Prolyl hydroxylase 3 stabilizes the p53 tumor suppressor by inhibiting the p53–MDM2 interaction in a hydroxylase-independent manner

2019 ◽  
Vol 294 (25) ◽  
pp. 9949-9958 ◽  
Author(s):  
Yiming Xu ◽  
Qiang Gao ◽  
Yaqian Xue ◽  
Xiuxiu Li ◽  
Liang Xu ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Tumor Suppressor ◽  
P53 Protein ◽  
Prolyl Hydroxylase ◽  
Hydroxylase Activity ◽  
Independent Manner ◽  
P53 Expression ◽  
Genetic Ablation ◽  
Protein Levels

Prolyl hydroxylase 3 (PHD3) has initially been reported to hydroxylase hypoxia-inducible factor α (HIFα) and mediate HIFα degradation. More recent studies have shown that, in addition to HIFα, PHD3 has also other substrates. Moreover, pHD3 is believed to act as a tumor suppressor, but the underlying mechanism remains to be elucidated. Here, we demonstrate that PHD3 stabilizes p53 in a hydroxylase-independent manner. We found that PHD3 overexpression increases and PHD3 knockdown decreases p53 levels. Mechanistically, PHD3 bound MDM2 proto-oncogene (MDM2) and prevented MDM2 from interacting with p53, thereby inhibiting MDM2-mediated p53 degradation. Interestingly, we found that PHD3 overexpression could enhance p53 in the presence of the prolyl hydroxylase inhibitor dimethyloxalylglycine, and the prolyl hydroxylase activity-deficient variant PHD3-H196A also inhibited the p53-MDM2 interaction and stabilized p53. Genetic ablation of PHD3 decreased p53 protein levels in mice intestinal epithelial cells, but a genetic knockin of PHD3-H196A did not affect p53 protein levels in vivo. These results suggest that the prolyl hydroxylase activity of PHD3 is dispensable for its ability to stabilize p53. We found that both PHD3 and PHD3-H196A suppress the expression of the stem cell-associated gene NANOG and inhibited the properties of colon cancer stem cells through p53. Our results reveal an additional critical mechanism underlying the regulation of p53 expression and highlight that PHD3 plays a role in the suppression of colon cancer cell stemness in a hydroxylase-independent manner.

scholarly journals Transglutaminase 2 promotes tumorigenicity of colon cancer cells by inactivation of the tumor suppressor p53

Oncogene ◽  
2021 ◽  
Author(s):  
Patrizia Malkomes ◽  
Ilaria Lunger ◽  
Elsie Oppermann ◽  
Khalil Abou-El-Ardat ◽  
Thomas Oellerich ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Tumor Suppressor ◽  
Therapeutic Potential ◽  
Survival Function ◽  
Transglutaminase 2 ◽  
Time Lapse ◽  
Cancer Tissue ◽  
Tumor Suppressor P53 ◽  
Genetic Ablation

AbstractDespite a high clinical need for the treatment of colorectal carcinoma (CRC) as the second leading cause of cancer-related deaths, targeted therapies are still limited. The multifunctional enzyme Transglutaminase 2 (TGM2), which harbors transamidation and GTPase activity, has been implicated in the development and progression of different types of human cancers. However, the mechanism and role of TGM2 in colorectal cancer are poorly understood. Here, we present TGM2 as a promising drug target.In primary patient material of CRC patients, we detected an increased expression and enzymatic activity of TGM2 in colon cancer tissue in comparison to matched normal colon mucosa cells. The genetic ablation of TGM2 in CRC cell lines using shRNAs or CRISPR/Cas9 inhibited cell expansion and tumorsphere formation. In vivo, tumor initiation and growth were reduced upon genetic knockdown of TGM2 in xenotransplantations. TGM2 ablation led to the induction of Caspase-3-driven apoptosis in CRC cells. Functional rescue experiments with TGM2 variants revealed that the transamidation activity is critical for the pro-survival function of TGM2. Transcriptomic and protein–protein interaction analyses applying various methods including super-resolution and time-lapse microscopy showed that TGM2 directly binds to the tumor suppressor p53, leading to its inactivation and escape of apoptosis induction.We demonstrate here that TGM2 is an essential survival factor in CRC, highlighting the therapeutic potential of TGM2 inhibitors in CRC patients with high TGM2 expression. The inactivation of p53 by TGM2 binding indicates a general anti-apoptotic function, which may be relevant in cancers beyond CRC.

scholarly journals Circular RNA CDR1as disrupts the p53/MDM2 complex to inhibit Gliomagenesis

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Jiacheng Lou ◽  
Yuchao Hao ◽  
Kefeng Lin ◽  
Yizhu Lyu ◽  
Meiwei Chen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Tumor Suppressor ◽  
P53 Protein ◽  
Circular Rnas ◽  
P53 Expression ◽  
Mirna Sponge ◽  
Rna Fish ◽  
In Cells

Abstract Background Inactivation of the tumor suppressor p53 is critical for pathogenesis of glioma, in particular glioblastoma multiforme (GBM). MDM2, the main negative regulator of p53, binds to and forms a stable complex with p53 to regulate its activity. Hitherto, it is unclear whether the stability of the p53/MDM2 complex is affected by lncRNAs, in particular circular RNAs that are usually abundant and conserved, and frequently implicated in different oncogenic processes. Methods RIP-seq and RIP-qPCR assays were performed to determine the most enriched lncRNAs (including circular RNAs) bound by p53, followed by bioinformatic assays to estimate the relevance of their expression with p53 signaling and gliomagenesis. Subsequently, the clinical significance of CDR1as was evaluated in the largest cohort of Chinese glioma patients from CGGA (n = 325), and its expression in human glioma tissues was further evaluated by RNA FISH and RT-qPCR, respectively. Assays combining RNA FISH with protein immunofluorescence were performed to determine co-localization of CDR1as and p53, followed by CHIRP assays to confirm RNA-protein interaction. Immunoblot assays were carried out to evaluate protein expression, p53/MDM2 interaction and p53 ubiquitination in cells in which CDR1as expression was manipulated. After AGO2 or Dicer was knocked-down to inhibit miRNA biogenesis, effects of CDR1as on p53 expression, stability and activity were determined by immunoblot, RT-qPCR and luciferase reporter assays. Meanwhile, impacts of CDR1as on DNA damage were evaluated by flow cytometric assays and immunohistochemistry. Tumorigenicity assays were performed to determine the effects of CDR1as on colony formation, cell proliferation, the cell cycle and apoptosis (in vitro), and on tumor volume/weight and survival of nude mice xenografted with GBM cells (in vivo). Results CDR1as is found to bind to p53 protein. CDR1as expression decreases with increasing glioma grade and it is a reliable independent predictor of overall survival in glioma, particularly in GBM. Through a mechanism independent of acting as a miRNA sponge, CDR1as stabilizes p53 protein by preventing it from ubiquitination. CDR1as directly interacts with the p53 DBD domain that is essential for MDM2 binding, thus disrupting the p53/MDM2 complex formation. Induced upon DNA damage, CDR1as may preserve p53 function and protect cells from DNA damage. Significantly, CDR1as inhibits tumor growth in vitro and in vivo, but has little impact in cells where p53 is absent or mutated. Conclusions Rather than acting as a miRNA sponge, CDR1as functions as a tumor suppressor through binding directly to p53 at its DBD region to restrict MDM2 interaction. Thus, CDR1as binding disrupts the p53/MDM2 complex to prevent p53 from ubiquitination and degradation. CDR1as may also sense DNA damage signals and form a protective complex with p53 to preserve p53 function. Therefore, CDR1as depletion may play a potent role in promoting tumorigenesis through down-regulating p53 expression in glioma. Our results broaden further our understanding of the roles and mechanism of action of circular RNAs in general and CDR1as in particular, and can potentially open up novel therapeutic avenues for effective glioma treatment.

scholarly journals The Corepressor mSin3a Interacts with the Proline-Rich Domain of p53 and Protects p53 from Proteasome-Mediated Degradation

2001 ◽  
Vol 21 (12) ◽  
pp. 3974-3985 ◽  
Author(s):  
Jack T. Zilfou ◽  
William H. Hoffman ◽  
Michael Sank ◽  
Donna L. George ◽  
Maureen Murphy
Keyword(s):  
Tumor Suppressor ◽  
Binding Domain ◽  
Protein Stabilization ◽  
Independent Manner ◽  
Retinoblastoma Tumor Suppressor Protein ◽  
Rich Domain ◽  
Retinoblastoma Tumor Suppressor ◽  
Retinoblastoma Tumor ◽  
Sin3 Complex

ABSTRACT While the transactivation function of the tumor suppressor p53 is well understood, less is known about the transrepression functions of this protein. We have previously shown that p53 interacts with the corepressor protein mSin3a (hereafter designated Sin3) in vivo and that this interaction is critical for the ability of p53 to repress gene expression. In the present study, we demonstrate that expression of Sin3 results in posttranslational stabilization of both exogenous and endogenous p53, due to an inhibition of proteasome-mediated degradation of this protein. Stabilization of p53 by Sin3 requires the Sin3-binding domain, determined here to map to the proline-rich region of p53, from amino acids 61 to 75. The correlation between Sin3 binding and stabilization supports the hypothesis that this domain of p53 may normally be subject to a destabilizing influence. The finding that a synthetic mutant of p53 lacking the Sin3-binding domain has an increased half-life in cells, compared to wild-type p53, supports this premise. Interestingly, unlike retinoblastoma tumor suppressor protein, MDMX, and p14ARF, Sin3 stabilizes p53 in an MDM2-independent manner. The ability of Sin3 to stabilize p53 is consistent with the model whereby these two proteins must exist on a promoter for extended periods, in order for repression to be an effective mechanism of gene regulation. This model is consistent with our data indicating that, unlike the p300-p53 complex, the p53-Sin3 complex is immunologically detectable for prolonged periods following exposure of cells to agents of DNA damage.

scholarly journals Upregulated TRIM11 Exerts its Oncogenic Effects in Hepatocellular Carcinoma Through Inhibition of P53

2017 ◽  
Vol 44 (1) ◽  
pp. 255-266 ◽  
Author(s):  
Jinjin Liu ◽  
Jun Rao ◽  
Xuming Lou ◽  
Jian Zhai ◽  
Zhenhua Ni ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Tumor Development ◽  
Tumor Model ◽  
Migration And Invasion ◽  
P53 Expression ◽  
Protein Levels ◽  
Normal Tissues ◽  
P53 Signaling

Background/Aims: The tripartite motif containing (TRIM) family plays crucial roles in tumor development and progression. However, little is known about the function and mechanism of TRIM11 in hepatocellular carcinoma (HCC). Methods: The expression levels of TRIM11 were examined by real-time PCR, Western blot and Immunohistochemical (IHC) staining. TRIM11 knockdown cells were produced by lentivirus infection, and functional assays, such as MTT, colony formation assay, migration and invasion assays and a xenograft tumor model were used to investigate the role of TRIM11 in HCC. We also determined the effect of TRIM11 on p53 signaling and its downstream molecules. Results: We found that TRIM11 mRNA and protein levels were significantly increased in HCC tissues as compared with normal tissues; increased levels correlated with poor patient survival. By loss- and gain-of-function investigations, knockdown of TRIM11 suppressed cell proliferation, migration, invasion in vitro and tumor growth in vivo. Moreover, TRIM11 negatively regulated p53 expression. Knockdown of p53 abrogated the in vitro and in vivo biological functions of TRIM11 shRNA in HCC cells. Conclusions: These data show that TRIM11 exerts its oncogenic effect in HCC by downregulating p53 both in vitro and in vivo. Our data provide new insights into the pathogenesis of HCC and indicate that TRIM11 may serve as a new therapeutic target for HCC treatment.

UV Induces GADD45 in a p53-Dependent and -Independent Manner in Human Keratinocytes

2003 ◽  
Vol 7 (2) ◽  
pp. 119-123 ◽  
Author(s):  
Tomoko Maeda ◽  
Adrian B. Sim ◽  
Duane A. Leedel ◽  
Prescillia P. S. Chua ◽  
Eugene G. Chomey ◽  
...  
Keyword(s):  
Dna Repair ◽  
P53 Protein ◽  
Human Keratinocytes ◽  
Cell Cycle Checkpoint ◽  
Checkpoint Control ◽  
Independent Manner ◽  
Rnase Protection ◽  
P53 Expression ◽  
Multifunctional Protein ◽  
Rna Protection

Background: GADD45 is a multifunctional protein involved in DNA repair and in cell cycle checkpoint control. p53 plays an important role in regulating DNA repair and in response to UVB in keratinocytes. Objective: GADD45 and p53 expression was examined and compared at the mRNA and protein level after exposure to UV irradiation. Methods: Human keratinocytes were exposed to increasing doses of UVB, and an RNA protection assay and a Western blot analysis were performed. Results: The RNase protection assays using human keratinocytes showed that GADD45 mRNA increases after 4 h and remains elevated for 24 h in cells irradiated at 100, 300, or 600 J/m2 UVB. The level of GADD45 protein increases after 8 h and remains elevated for 48 h, with maximal induction at 300 J/m2. p53 mRNA did not rise in concert with GADD45 at any dose used, and p53 protein was not up-regulated at the lower dose of 100 J/m2. Conclusion: GADD45 is regulated in both a p53-dependent and a p53-independent manner in keratinocytes after UV exposure.

Nonresponsiveness of cerebral p53-MDM2 functional circuit in newborn rat pups rendered IUGR via uteroplacental insufficiency

2005 ◽  
Vol 288 (4) ◽  
pp. R1038-R1045 ◽  
Author(s):  
Xingrao Ke ◽  
Robert A. McKnight ◽  
Zheng-ming Wang ◽  
Xing Yu ◽  
Laiyi Wang ◽  
...  
Keyword(s):  
Rat Brain ◽  
P53 Protein ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Periventricular White Matter ◽  
P53 Expression ◽  
Pregnant Rat ◽  
Protein Levels ◽  
Ca1 Region ◽  
Rat Pups ◽  
Induced Apoptosis

Severe uteroplacental insufficiency causes cerebral apoptosis in the fetus. Moderate uteroplacental insufficiency causes intrauterine growth retardation (IUGR) and increases the risk of postnatal neurological morbidity. In the rat, uteroplacental insufficiency and IUGR affect cerebral gene expression of Bcl-2 and predispose the newborn IUGR rat toward cerebral apoptosis when challenged with perinatal hypoxia. Expression of Bcl-2, as well as the proapoptotic protein Bax, is regulated by p53. p53 also induces MDM2 transcription, which functions to limit further p53-induced apoptosis. The predisposition of the IUGR fetus toward cerebral apoptosis suggests that the p53-MDM2 “functional” circuit may be perturbed in the newborn IUGR rat brain. We hypothesized that MDM2 cerebral expression does not increase in response to increased p53 expression or increased levels of phospho-p53 (Ser15), an activated form of p53. To prove this hypothesis, we induced IUGR through bilateral uterine ligation of the pregnant rat. Uteroplacental insufficiency significantly increased p53 mRNA, total p53 protein, and phospho-p53 (Ser15) protein levels in the brain at term. Increased expression of phospho-p53 (Ser15) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells were localized to the CA1 region of the hippocampus, the subcortical and periventricular white matter, and the amygdala of the IUGR rat brain. In contrast, uteroplacental insufficiency decreased cerebral MDM2 mRNA and phospho-MDM2 (Ser166) protein levels in the IUGR rat pups. We conclude that the cerebral MDM2 response to increased p53 expression is not present in the newborn IUGR rat pup, and we speculate that this contributes to the predisposition of the IUGR fetus toward perinatal and long-term neurodevelopmental morbidities.

scholarly journals Anti-apoptosis mechanism of triptolide based on network pharmacology in focal segmental glomerulosclerosis rats

2020 ◽  
Vol 40 (4) ◽  
Author(s):  
Yayu Li ◽  
Xue Jiang ◽  
Litao Song ◽  
Mengdie Yang ◽  
Jing Pan
Keyword(s):  
Focal Segmental Glomerulosclerosis ◽  
Cell Apoptosis ◽  
P53 Protein ◽  
Network Pharmacology ◽  
Tripterygium Wilfordii ◽  
P53 Expression ◽  
Chemical Structures ◽  
Segmental Glomerulosclerosis

Abstract Triptolide (TPL), the active component of Tripterygium wilfordii, exhibits anti-cancer and antioxidant functions. We aimed to explore the anti-apoptosis mechanism of TPL based on network pharmacology and in vivo and in vitro research validation using a rat model of focal segmental glomerulosclerosis (FSGS). The chemical structures and pharmacological activities of the compounds reported in T. wilfordii were determined and used to perform the network pharmacology analysis. The Traditional Chinese Medicine Systems Pharmacology Database (TCMSP) was then used to identify the network targets for 16 compounds from Tripterygium wilfordii. Our results showed that 47 overlapping genes obtained from the GeneCards and OMIM databases were involved in the occurrence and development of FSGS and used to construct the protein–protein interaction (PPI) network using the STRING database. Hub genes were identified via the MCODE plug-in of the Cytoscape software. IL4 was the target gene of TPL in FSGS and was mainly enriched in the cell apoptosis term and p53 signaling pathway, according to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. TPL inhibited FSGS-induced cell apoptosis in rats and regulated IL4, nephrin, podocin, and p53 protein levels via using CCK8, TUNEL, and Western blot assays. The effects of IL4 overexpression, including inhibition of cell viability and promotion of apoptosis, were reversed by TPL. TPL treatment increased the expression of nephrin and podocin and decreased p53 expression in rat podocytes. In conclusion, TPL inhibited podocyte apoptosis by targeting IL4 to alleviate kidney injury in FSGS rats.

scholarly journals The Expression And The Tumor Suppressor Role of CLDN6 In Colon Cancer

2021 ◽  
Author(s):  
Huinan Qu ◽  
Min Wang ◽  
Miaomiao Wang ◽  
Yuanyuan Liu ◽  
Chengshi Quan
Keyword(s):  
Colon Cancer ◽  
Tumor Suppressor ◽  
Cell Line ◽  
Cancer Cell Line ◽  
Human Colon ◽  
Colon Cancer Cell Line ◽  
Migration And Invasion ◽  
Epithelial Cell Line

Abstract As a member of the tight junction family, CLDN6 is a tumor suppressor gene in breast cancer, but its role in colon cancer is unknown. In this research, we aimed at revealing the function of CLDN6 in colon cancer. We found that CLDN6 expressed lower in colon cancer tissues compared with adjacent normal tissues and the low expression of CLDN6 was correlated with lymph node metastasis. Similarly, CLDN6 expressed lower in the colon cancer cell line SW1116 compared with the normal human colon epithelial cell line NCM460. Upon CLDN6 overexpression in SW1116 cells, the proliferation of cells was suppressed in vitro and in vivo. Consistently, the migration and invasion abilities of cells were significantly inhibited after CLDN6 overexpression. Furthermore, the TYK2/STAT3 pathway was activated in SW1116/CLDN6 cells, and inhibition of this pathway with AG490 reversed the inhibition of migration and invasion of SW1116 cells by CLDN6. Therefore, our data indicated that CLDN6 acted as a tumor suppressor and had the potential to be regarded as a biomarker for the progression of colon cancer.

scholarly journals Hypoxia-Induced LIN28A mRNA Promotes the Metastasis of Colon Cancer in a Protein-Coding-Independent Manner

2021 ◽  
Vol 9 ◽  
Author(s):  
Mingjiao Weng ◽  
Yukuan Feng ◽  
Yan He ◽  
Weiwei Yang ◽  
Jing Li ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Cancer Cells ◽  
Cancer Metastasis ◽  
Mrna Level ◽  
Unexpected Finding ◽  
Independent Manner ◽  
Protein Coding ◽  
P Bodies

The hypoxic microenvironment is beneficial to the metastasis but not to the proliferation of cancer cells. However, the mechanisms regarding to hypoxia differentially regulating cancer metastasis and proliferation are largely unknown. In this study, we revealed that hypoxia induced the expression of LIN28A at mRNA level but segregated LIN28A mRNAs in the P-bodies and thus inhibits the production of LIN28A protein. This unexpected finding suggests that there may be non-coding role for LIN28A mRNA in the progression of colon cancer. We further showed that the non-coding LIN28A mRNA promotes the metastasis but not proliferation of colon cancer cells in vitro and in vivo. Mechanistically, we revealed that methionyl aminopeptidase 2 (METAP2) is one of the up-regulated metastasis regulators upon over-expression of non-coding LIN28A identified by mass spectrum, and confirmed that it is non-coding LIN28A mRNA instead of LIN28A protein promotes the expression of METAP2. Moreover, we demonstrated that knockdown of DICER abolished the promotional effects of non-coding LIN28A on the metastasis and METAP2 expression. Conclusively, we showed that hypoxia induces the production of LIN28A mRNAs but segregated them into the P-bodies together with miRNAs targeting both LIN28A and METAP2, and then promotes the metastasis by positively regulating the expression of METAP2. This study uncovered a distinctive role of hypoxia in manipulating the metastasis and proliferation by differently regulating the expression of LIN28A at mRNA and protein level.

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