Control Of P53 Activity Research Articles

FOXO1 promotes cancer cell growth through MDM2-mediated p53 degradation

FOXO1 is a transcription factor and potential tumor suppressor that is negatively regulated downstream of PI3K-PKB/AKT signaling. Paradoxically, FOXO also promotes tumor growth, but the detailed mechanisms behind this role of FOXO are not fully understood. In this study, we revealed a molecular cascade by which the Thr24 residue of FOXO1 is phosphorylated by AKT and is dephosphorylated by calcineurin, which is a Ca2+-dependent protein phosphatase. Curiously, single nucleotide somatic mutations of FOXO1 in cancer occur frequently at and near Thr24. Using a calcineurin inhibitor and shRNA directed against calcineurin, we revealed that calcineurin-mediated dephosphorylation of Thr24 regulates FOXO1 protein stability. We also found that FOXO1 binds to the promoter region of MDM2 and activates transcription, which in turn promotes MDM2-mediated ubiquitination and degradation of p53. FOXO3a and FOXO4 are shown to control p53 activity, however, the significance of FOXO1 in p53 regulation remains largely unknown. Supporting this notion, FOXO1 depletion increased p53 and p21 protein levels in association with inhibition of cell proliferation. Taken together, these results indicate that FOXO1 is stabilized by calcineurin-mediated dephosphorylation, and that FOXO1 supports cancer cell proliferation by promoting MDM2 transcription and subsequent p53 degradation.

Low glucose metabolite 3-phosphoglycerate switches PHGDH from serine synthesis to p53 activation to control cell fate.

Glycolytic intermediary metabolites such as fructose-1,6-bisphosphate can serve as signals, controlling metabolic states beyond energy metabolism. However, whether glycolytic metabolites also play a role in controlling cell fate remains unexplored. Here, we find that low levels of glycolytic metabolite 3-phosphoglycerate (3-PGA) can switch phosphoglycerate dehydrogenase (PHGDH) from cataplerosis serine synthesis to pro-apoptotic activation of p53. PHGDH is a p53-binding protein, and when unoccupied by 3-PGA interacts with the scaffold protein AXIN in complex with the kinase HIPK2, both of which are also p53-binding proteins. This leads to the formation of a multivalent p53-binding complex that allows HIPK2 to specifically phosphorylate p53-Ser46 and thereby promote apoptosis. Furthermore, we show that PHGDH mutants (R135W and V261M) that are constitutively bound to 3-PGA abolish p53 activation even under low glucose conditions, while the mutants (T57A and T78A) unable to bind 3-PGA cause constitutive p53 activation and apoptosis in hepatocellular carcinoma (HCC)cells, even in the presence of high glucose. In vivo, PHGDH-T57A induces apoptosis and inhibits the growth of diethylnitrosamine-induced mouse HCC, whereas PHGDH-R135W prevents apoptosis and promotes HCC growth, and knockout of Trp53 abolishes these effects above. Importantly, caloric restriction that lowers whole-body glucose levels can impede HCC growth dependent on PHGDH. Together, these results unveil a mechanism by which glucose availability autonomously controls p53 activity, providing a new paradigm of cell fate control by metabolic substrate availability.

Abstract IA024: Single nuclei chromatin accessibility and transcriptomic map of breast tissues of women of diverse genetic ancestry

Abstract Single nuclei analysis is allowing robust classification of cell types in an organ that helps to establish relationships between cell-type specific gene expression and chromatin accessibility status of gene regulatory regions. Using the institutional resource of breast tissues of healthy donors of various genetic ancestry, we have developed a comprehensive chromatin accessibility (snATAC-seq) and gene expression (snRNA-seq) atlas of human breast tissues. Our analyses included 51,367 nuclei with average sequence coverage of 1195 genes per nuclei. These nuclei were derived from 22 donors of Ashkenazi descent, 20 of European non-Ashkenazi ancestry, 10 of Asian ancestry, 10 Hispanic/Latina, 10 Native American, 6 from BRCA1 mutation carriers, and 5 from BRCA2 mutation carriers. Although tissues from 20 women of African Ancestry were included in sequencing, poor quality sequencing reads prevented inclusion of data from those samples in the final analysis. Integrated analysis revealed 10 distinct cell types in the healthy breast, which included three major epithelial cell subtypes (mature luminal, luminal progenitor, basal), two endothelial subtypes, two adipocyte subtypes, fibroblasts, T-cells, and macrophages. Mature luminal cells could be further divided into two distinct hormone sensitive subtypes, HSa and HSb, with HSa subtype expressing higher levels of Estrogen Receptor alpha (ESR1) and NEK10, a tyrosine kinase that controls p53 activity and limits cell proliferation. The luminal progenitors could be broadly classified into alveolar progenitors (AP) and basal-luminal hybrid progenitors (BL). AP cells expressed higher levels of ELF5, a known marker of alveolar cells, compared to BL cells. Basal cells could be classified into two subtypes with Basal-BAa cells being the most dominant and expressing higher levels of TP63 and NFIB compared to Basal-BAb cells. ESR1 expression pattern was distinctly different in tissues from Native Americans compared to the rest, with a high level of ESR1 expression extending to AP cells. In fact, overall AP cell numbers were ~3-fold higher in Native Americans compared to others (18.9% versus 1.5-8.8%). Furthermore, Ingenuity pathway analysis of differentially expressed genes revealed elevated Estrogen Receptor signaling in ML and AP cell types of Native Americans compared to those of other genetic ancestry. Despite significant differences in expression and activity in Native Americans compared to others, the chromatin accessibility map of the ESR1 gene regulatory regions in ML and LP cells did not show any genetic ancestry dependent variability. In general, cell subtype-specific gene expression did not correlate with chromatin accessibility differences, suggesting that transcriptional regulation independent of chromatin accessibility governs cell type-specific gene expression in the breast. Collectively, these results reveal complexities in gene expression in different cell types of the breast Citation Format: Poornima Bhat-Nakshatri, Duojiao Chen, Aditi Khatpe, Hongyu Gao, Yunlong Liu, Anna Maria Storniolo, Harikrishna Nakshatri. Single nuclei chromatin accessibility and transcriptomic map of breast tissues of women of diverse genetic ancestry [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr IA024.

Identification of Long Non-Coding RNA Genes Important for Normal Hematopoiesis and Leukemogenesis Using Bioinformatics Tools

Only 1.5% of human genome encodes protein-coding genes, while more than 60% of human genome is transcribed for RNA molecules that have no-protein coding potentials. So far 6,000 long noncoding RNA genes (lncRNA) are documented (Evans et al., 2016). To investigate the functions of lncRNA genes poses huge opportunities as well as challenge to biomedical research. In this project, we interrogate important lncRNA genes that may be associated with normal and abnormal hematopoiesis using publically available databases containing RNA-seq data from different blood lineages and leukemia cells. The data mining and statistical analysis are performed by R program and oracle SQL code. Using RNA-seq data derived from purified blood lineages of human cord blood cells, we have identified long noncoding RNA genes transcribed exclusively in human hematopoietic stem cells as well as in different progenitor lineages. We have discovered two lncRNA genes: linc00028 and As-PP1 are regulated by RUNX1 in hematopoietic stem cells. Given that chromosome translocation t(8;21) generates a fusion protein RUNX1-ETO which often represses genes activated by RUNX1, we proceed to validate whether Linc00028 and As-PP1 may be regulated by transcription factor RUNX1 by chromatin immunoprecipitation assays. Further analysis implies that As-PP1 activated p53-mediated apoptosis via controlling p53 activity. Thus AML1-ETO may repress apoptosis via antagonizing the normal function of RUNX1 in repressing AsPP1 transcription. We have been studying how protein arginine methyltransferases 1 (PRMT1) promotes the progression of acute myeloid leukemia. In this vein, we found that inhibition of PRMT1 activity by small molecule PRMT1 inhibitor upregulated the expression level of the long noncoding RNA gene (As-PP1) and leads to leukemia cell apoptosis. Thus long noncoding RNA genes may serve as biomarkers for PRMT1 inhibitor treatment. By clustering genes of acute myeloid leukemia patients in TCGA database, we found unique lncRNA signature genes specifically associated to distinct groups of leukemia patients with PML-RAR alpha, FLT3 mutation and AML1-ETO (aka RUNX1-ETO) respectively. Whether these signature genes are useful to be applied as biomarkers will be further discussed in the presentation. DisclosuresNo relevant conflicts of interest to declare.

MDM2 Amplified Sarcomas: A Literature Review.

Murine Double Minute Clone 2, located at 12q15, is an oncogene that codes for an oncoprotein of which the association with p53 was discovered 30 years ago. The most important function of MDM2 is to control p53 activity; it is in fact the best documented negative regulator of p53. Mutations of the tumor suppressor gene p53 represent the most frequent genetic change in human cancers. By overexpressing MDM2, cancer cells have another means to block p53. The sarcomas in which MDM2 amplification is a hallmark are well-differentiated liposarcoma/atypical lipomatous tumor, dedifferentiated liposarcoma, intimal sarcoma, and low-grade osteosarcoma. The purpose of this review is to summarize the typical clinical, histopathological, immunohistochemical, and genetic features of these tumors.

Fine-tuning p53 activity by modulating the interaction between eukaryotic translation initiation factor eIF4E and RNA-binding protein RBM38.

p53 is critical for tumor suppression but also elicits detrimental effects when aberrantly overexpressed. Thus, multiple regulators, including RNA-binding protein RBM38, are found to tightly control p53 expression. Interestingly, RBM38 is unique in that it can either suppress or enhance p53 mRNA translation via altered interaction with eIF4E potentially mediated by serine-195 (S195) in RBM38. Thus, multiple RBM38/eIF4E knock-in (KI) cell lines were generated to investigate the significance of eIF4E-RBM38 interaction in controlling p53 activity. We showed that KI of RBM38-S195D or -Y192C enhances, whereas KI of RBM38-S195K/R/L weakens, the binding of eIF4E to p53 mRNA and subsequently p53 expression. We also showed that KI of eIF4E-D202K weakens the interaction of eIF4E with RBM38 and thereby enhances p53 expression, suggesting that D202 in eIF4E interacts with S195 in RBM38. Moreover, we generated an Rbm38 S193D KI mouse model in which human-equivalent serine-193 is substituted with aspartic acid. We showed that S193D KI enhances p53-dependent cellular senescence and that S193D KI mice have a shortened life span and are prone to spontaneous tumors, chronic inflammation, and liver steatosis. Together, we provide in vivo evidence that the RBM38-eIF4E loop can be explored to fine-tune p53 expression for therapeutic development.

HDAC8 Promotes MLL-Rearranged Acute Myeloid Leukemia By Inhibiting p53 Acetylation

Chromosomal rearrangements involving the mixed lineage leukemia (MLL) gene at the 11q23 locus are found in a subset of acute myeloid leukemia (AML) generally associated with intermediate to poor prognosis. MLL-AF9 resulting from t(9;11)(p22;q23) is the most common in MLL-rearranged (MLL-r) AML. Chemotherapy is the current standard of care for AML and the overall 5-year survival rate is only 25%. We previously reported that HDAC8 functions to control p53 activity in hematopoietic stem cells via deacetylation of p53 protein (Hua et al, Blood 2017). Here, we investigated the contribution of HDAC8 in MLL-r AML pathogenesis and maintenance. We have previously generated a conditional Hdac8 floxed (f) allele and combined with Mx1-Cre (Mx1-Cre/Hdac8f/f(y)) to generate HDAC8△/△ mice upon induction with poly (I:C). To examine the role of HDAC8 in MLL-AF9-AML, we transduced Hdac8△/△ or control LSK (Lin-Sca1+cKit+) with a MSCV-ires-GFP (MIG)-MLL-AF9 (MA9) vector and sorted GFP+ cells for CFC assay. Hdac8△/△/MA9 cells generated significantly less CFC colonies compared to control, suggesting that HDAC8 promotes MA9 clonogenic activity. To assess their leukemogenic potential in vivo, we transplanted GFP+ control MA9 or Hdac8△/△/MA9 cells (5x103-104) into sublethally irradiated (6 Gy) wild-type (WT) recipients. All MA9 recipients (n=4) developed lethal AML with a median survival of 69 days while none of the Hdac8△/△/MA9 recipients (n=4) developed AML up to 4 months. To examine the contribution of HDAC8 in established AML, we transduced LSK from uninduced Hdac8f/f(y)/Mx1-Cre mice with MIG-MA9 and transplanted into WT recipients. Upon development of moribund leukemia, AML cells (5x 103) were transplanted into a cohort of recipients (n=16), which were treated with poly (I:C) (n=8; 14 mg/kg/dose) or PBS every other day for 2 weeks starting at day 10 (5-10% GFP+ in blood). Mice induced with poly (I:C) to delete Hdac8 showed dramatically lower white blood count [9.0 (103 /ul) vs. 109.7 (103 /ul); n=8, P<0.05] and lower AML burden (GFP+) in peripheral blood (9.3% vs 42.3%; n=8, P<0.05) at 3 weeks. Prolonged survival was observed in poly (I:C) treated mice compared to control (median survival 36 days vs 27 days, n=8, P<0.01). These results indicate that HDAC8 deletion impairs AML progression. Thus, we examined the effects of HDAC8-selective inhibitor (HDAC8i) on MA9 AML cell proliferation and survival. Treatment of MA9 AML cells with HDAC8i (22d) led to dose-dependent inhibition of cell survival (IC50 = 4.724 - 8.453 mM) and increased apoptosis (10 mM, 48 h) compared to vehicle treated control (40% vs. 7%). HDAC8i treatment significantly enhanced p53 acetylation, and upregulated p53 target genes, Gadd45d (fold change = 3.2; p< 0.01), Puma (fold change = 5.7; p< 0.01), Mdm2 (fold change = 3.0; p< 0.01) and Cdkn1a (fold change = 3.6; p< 0.01). To assess the effects on AML growth and leukemia-initiating capacity in vivo, we transplanted MA9 AML cells treated with HDAC8i (1 x 105, 2.5x105) or vehicle (2.5x105) into sublethally irradiated WT recipients (n=8-11). We observed significantly (p<0.0001) reduced GFP+ AML burden in recipients of HDAC8i-treated cells (both 1x105 & 2 x105) and significantly (p<0.0001) improved survival. Secondary transplantation of AML cells from HDAC8i- or vehicle-treated (2 x105) recipients revealed significantly improved survival in HDAC8i-treated group (22d 105 days n=4 vs. vehicle 33 days n=5; p< 0.01). These results indicate that HDAC8 inhibition enhanced elimination of MA9 AML cells and leukemia-initiating cells. We further evaluated the effect of HDAC8i in human AML cell lines and primary MLL-r AML patient cells. HDAC8i treatment increased p53 acetylation and enhanced p53-dependent apoptosis in MOLM-13 AML cells (22d: 50.8% vs. vehicle: 11.89%). Knock-down of p53 rescued the apoptosis induced by HDAC8i, indicating that HDAC8i induced apoptosis is mediated by p53. Similarly, HDAC8i (22d) treatment significantly increased apoptosis accompanied by elevated levels of p53 acetylation and p53 target gene expression in MLL-r AML CD34+ cells compared to normal (NL) CD34+ cells. Thus, HDAC8 inhibition targets human MLL-r AML cells and AML CD34+ cells by restoring p53 acetylation and inducing apoptosis. In conclusion, this study reveals a critical role of HDAC8 in promoting initiation and maintenance of MLL-AF9 AML and highlights HDAC8 inhibition as a promising approach to target MLL-r AML. Disclosures No relevant conflicts of interest to declare.

Abstract 4506: MDM2 alternative splicing is regulated by microRNA binding to control p53 activity

Abstract Alternative splicing of the MDM2 is an important means by which p53 is upregulated to combat the deleterious effects of genotoxic stress. One splice variant, MDM2-ALT1, is activated in response to genotoxic stress and is comprised of only the two terminal coding exons 3 and 12 and therefore lacks a p53 binding domain. This variant has been identified in a number of human tumors, including invasive carcinoma of the breast and lung, as well as soft tissue sarcomas. Despite its pervasiveness in tumors and the therapeutic possibilities it presents, there is very little known about the regulators of that govern this alternative splicing event. Our lab has identified a specific microRNA that is expressed at high levels in normal cells but decreased under conditions of genotoxic stress and in rhabdomyosarcoma cells, both of which are characterized by the expression of the MDM2 spliced isoform. Hence, expression of the microRNA is inversely correlated with the splice variant MDM2-ALT1. Though recent studies have shown that splicing factors can be regulated through miRNA-mediated gene silencing and indirectly influence splicing choices, we were unable to identify any splicing regulators of MDM2 to be targeted by the microRNA. Interestingly, the microRNA has been reported as one of the miRNAs that is reimported to the nucleus but has not been ascribed a role in the nuclear space. We therefore wanted to test the hypothesis that the microRNA can directly bind to the MDM2 pre-mRNA to drive splicing choices in a regulated fashion. To this end, we have identified predicted binding sites for the microRNA in the regulated region of MDM2, within and near exons 3 and 12. We have shown that the microRNA binds specifically to these regions in MDM2 in normal cells and has decreased binding after genotoxic stress. Furthermore, we have shown that overexpression of the pre-microRNA can alleviate the induction of the MDM2-ALT1 isoform in response to genotoxic stress. We propose that microRNA binding to the MDM2 pre-mRNA regulates its splicing and plays a role in the DNA damage response and the pathway to tumorigenesis. Our work implicates for the first time that a miRNA would be acting as a splicing factor, a novel function for this class of non-coding RNAs and a novel method of regulation of the p53-MDM2 axis. Furthermore, our discoveries reveal miR-regulated splicing as a possible strategy to activate p53 and sensitize cancer cells to conventional cancer therapies. Citation Format: Matias Montes, Andrew Goodwin, Dawn S. Chandler. MDM2 alternative splicing is regulated by microRNA binding to control p53 activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4506.

The p53 protein induces stable miRNAs that have the potential to modify subsequent p53 responses

The p53 tumour suppressor is a transcription factor that can increase the expression of mRNAs and microRNAs (miRNAs). HT29-tsp53 cells expressing a temperature sensitive variant of p53 have provided a useful model to rapidly and reversibly control p53 activity. In this model, the majority of p53-responsive mRNAs were upregulated rapidly but they were short-lived leading to rapid decay of the p53 response at the restrictive temperature. Here we used oligonucleotide microarrays and reverse transcriptase PCR to show that p53-induced miRNAs exhibited a distinct temporal pattern of expression. Whereas p53-induced miRNAs like miR-143-3p, miR-145-5p, miR-34a-5p and miR-139-5p increased as fast as mRNAs, they were extremely stable persisting long after p53 induced mRNAs and even their corresponding primary miRNAs had decayed to baseline levels. Three p53-induced mRNAs (MDM2, BTG2 and CDKN1A) are experimentally verified targets of one or more of these specific miRNAs so we hypothesized that the sustained expression of p53-induced miRNAs could be explained by a post-transcriptional feedback loop. Activation of consecutive p53 responses separated by a period of recovery led to the selective attenuation of a subset of p53 regulated mRNAs corresponding to those targeted by one or more of the p53-responsive miRNAs. Our results indicate that the long term expression of p53 responsive miRNAs leads to an excess of miRNAs during the second response and this likely prevents the induction of MDM2, BTG2 and CDKN1A mRNA and/or protein. These observations are likely to have important implications for daily cancer therapies that activate p53 in normal tissues and/or tumour cells.

MDMX under stress: the MDMX-MDM2 complex as stress signals hub.

The tumor suppressor p53 plays a central role in safeguarding cellular homeostasis. Upon various types of stress signals such as DNA damage or oncogenic stress, p53 is promptly activated to prevent and repair damages that can threaten the genome stability. The two major negative regulators of p53 are MDM2 and MDMX, two homolog proteins that control p53 activity and turnover, hence keeping it in check during normal cell cycling. In the event of cellular stress, they have to be inhibited in order to relieve p53 from their suppression and allow its activation. As the essential upstream modulator of p53, the MDMX-MDM2 complex integrates multiple signaling pathways regulating p53 response to perturbations of cellular homeostasis. Given its predominantly cytoplasmic localization in normal conditions, we hypothesize that MDMX, rather than MDM2, is the first recipient of signaling cues directed towards the MDMX-MDM2 complex and aimed at modulating p53. In this review we give a synthetic overview of the phosphorylation sites of MDMX that are known to affect its degradation, ubiquitination, intracellular localization and interaction with MDM2 and p53, ultimately modulating the stability and activity of p53. The role of MDMX in response to the main types of cellular stress is also briefly discussed, along with the potential of the MDMX-MDM2 complex as therapeutic target to restore p53 activity.

CACUL1/CAC1 attenuates p53 activity through PML post-translational modification

Promyelocytic leukaemia (PML) is a tumor suppressor protein covalently conjugated with SUMO family proteins, leading to the formation of PML nuclear bodies (NBs). PML-NBs provide a platform for efficient posttranslational modification of targets and protein-protein interaction, contributing to the adjustment of gene expression and chromatin integrity. Although PML SUMOylation is thought to play important roles in diverse cellular functions, the control mechanisms of adequate modification levels have remained unsolved. Here, we report that Cullin-related protein CACUL1/CAC1 (CACUL1) inhibits PML posttranslational modification. CACUL1 interacts with PML and suppresses PML SUMOylation, leading to the regulation of PML-NB size in the nucleus. We also found that Ubc9, a SUMO-conjugating enzyme, binds to CACUL1 and antagonizes the interaction between CACUL1 and PML. Furthermore, CACUL1 attenuates p53 transcriptional activity. These data suggest that CACUL1 is a novel regulator that negatively controls p53 activity through the regulation of PML SUMOylation.

TRIMming p53's anticancer activity.

Several TRIM proteins control abundance and activity of p53. Along this route, TRIM proteins have a serious impact on carcinogenesis and prognosis for cancer patients. In the past years, a significant increase has been made in our understanding of how the TRIM protein family controls p53 activity.

BAC transgenic mice provide evidence that p53 expression is highly regulated in vivo.

p53 is an important tumor suppressor and stress response mediator. Proper control of p53 level and activity is tightly associated with its function. Posttranslational modifications and the interactions with Mdm2 and Mdm4 are major mechanisms controlling p53 activity and stability. As p53 protein is short-lived and hardly detectable in unstressed situations, less is known on its basal level expression and the corresponding controlling mechanisms in vivo. In addition, it also remains obscure how p53 expression might contribute to its functional regulation. In this study, we established bacterial artificial chromosome transgenic E.coli β-galactosidase Z gene reporter mice to monitor p53 expression in mouse tissues and identify important regulatory elements critical for the expression in vivo. We revealed preferentially high level of p53 reporter expressions in the proliferating, but not the differentiated compartments of the majority of tissues during development and tissue homeostasis. In addition, tumors as well as regenerating tissues in the p53 reporter mice also expressed high level of β-gal. Furthermore, both the enhancer box sequence (CANNTG) in the p53 promoter and the 3′ terminal untranslated region element were critical in mediating the high-level expression of the reporter. We also provided evidence that cellular myelocytomatosis oncogene was a critical player regulating p53 mRNA expression in proliferating cells and tissues. Finally, we found robust p53 activation preferentially in the proliferating compartment of mouse tissues upon DNA damage and the proliferating cells exhibited an enhanced p53 response as compared with cells in a quiescent state. Together, these results suggested a highly regulated expression pattern of p53 in the proliferating compartment controlled by both transcriptional and posttranscriptional mechanisms, and such regulated p53 expression may impose functional significance upon stress by setting up a precautionary mode in defense of cellular transformation and tumorigenesis.

Regulation of Mdm2 protein stability and the p53 response by NEDD4-1 E3 ligase

Mdm2 is a critical negative regulator of the tumor suppressor protein p53. Mdm2 is an E3 ligase whose overexpression leads to functional inactivation of p53. Mdm2 protein stability is regulated by several mechanisms including RING (Really Interesting New Gene) domain-mediated autoubiquitination. Here we report biochemical identification of NEDD4-1 as an E3 ligase for Mdm2 that contributes to the regulation of Mdm2 protein stability in cells. NEDD4-1 was identified from Jurkat cytosolic fractions using an enzyme-dead Mdm2 mutant protein as a substrate for in vitro E3 ligase assays. We show that lysates from Nedd4-1 knockout (KO) mouse embryonic fibroblasts (MEFs) have significantly diminished E3 ligase activity toward Mdm2 compared with lysates from wild-type (WT) MEFs. Recombinant NEDD4-1 promotes Mdm2 ubiquitination in vitro in a concentration- and time-dependent manner. In cells, NEDD4-1 physically interacts with Mdm2 via the RING domain of Mdm2. Overexpression of NEDD4-1, but not an enzyme-dead NEDD4-1CS mutant, increases ubiquitination of Mdm2. NEDD4-1 catalyzes the formation of K63-type polyubiquitin chains on Mdm2 that are distinct from K48-type polyubiquitination chains mediated by the Mdm2/MdmX complex. Importantly, K63-type polyubiquitination by NEDD4-1 competes with K48-type polyubiquitination on Mdm2 in cells. As a result, NEDD4-1-mediated ubiquitination stabilizes Mdm2. NEDD4-1 knockdown reduces the t1/2 (half-life) of endogenous Mdm2 from 20 to 12 min in U2OS cells. Nedd4-1 KO MEFs manifest increased p53 levels and activity, a more robust DNA damage response and increased G1 arrest compared with WT MEFs. Similarly, NEDD4-1 knockdown in WT-p53-bearing cells increases basal p53 levels and activity in an Mdm2-dependent manner, causes stronger p53 responses to DNA damage and results in p53-dependent growth inhibition compared with corresponding NEDD4-1-proficient control cells. This study identifies NEDD4-1 as a novel component of the p53/Mdm2 regulatory feedback loop that controls p53 activity during stress responses.

Abstract 1760: MUC16 upregulates TSPYL5 gene and mediates Lung cancer cell proliferation by suppressing p53 activity.

Abstract Background: Lung cancer is the most common cause of cancer related death in the world. The 5-year survival of lung cancer patients is a dismal 15%. MUC16 is a very high molecular weight glycoprotein with 22,152 amino acid residues in its protein sequence. According to Oncomine data, MUC16 is highly overexpressed in lung cancer particularly Non Small Cell Lung Cancer cell [NSCLC]. MUC16 is a well-established tumor marker in ovarian cancer but its functional and mechanistic role is not well understood. The Testis Specific Y-Like Protein (TSPYL5), which is also overexpressed in lung carcinoma (68%) and it has been suggested that overexpression of TSPYL5 induces cancer cell growth by suppressing p53 activity in terms. We evaluated the interplay between MUC16 and TSPYL5 in lung carcinogenesis. Materials and Methods: Endogenously expressed MUC16 was stably knocked down using a MUC16 shRNA construct (pSUPER-Retro-MUC16-sh) in H292 and H827 lung cancer cells by stable transfection method. The cells were treated with cisplatin to investigate the role of MUC16 and TSPYL5 on chemoresistance in lung cancer cells and its role in p53 degradation. Results: MUC16 is overexpressed in lung cancer while it is absent in normal lung tissues by immunohistochemistry analysis. Specifically MUC16 is highly observed in Stage III of lung carcinoma than other stages and metastatic sites as well. Significant expression of MUC16 was observed in NSCLC derived cell lines (H292 and H827) that express p53 (wild type). On knocking down MUC16 in H292 cells, a decrease in proliferation and an increase in the percentage of apoptotic cells were observed in comparison to vector control cells (by FACs). MUC16 knockdown in H292 cell lines decreased the motility and invasion compared to the MUC16 expressing control cells. Our microarray data suggest that multiple cancer-related genes including TSPYL5, CXCL1, CXCL2, CCL20, and IL8 are down-regulated in MUC16 knockdown cells. TSPYL5 suppresses p53 activity and thereby mediates cancer cell proliferation. Cisplatin treatment in H292 and H827 cells resulted in decreased expression of MUC16 and TSPYL5, and increased expression of p53. This indicates that MUC16 overexpression upregulates TSPYL5, which may suppresses the p53 activity thereby inducing lung cancer cell proliferation and making the cells insensitive to various cytotoxic agents. Conclusion: MUC16 may play a significant role in lung cancer cell proliferation through the upregulation of TSPYL5 which in turn control p53 activity. Citation Format: Imayavaramban Lakshmanan, Prabin D. Majhi, Moorthy P. Ponnusamy, Srustidhar Das, Parthasarathy Seshacharyulu, Dhanya Haridas, Surinder K. Batra, Apar Ganti. MUC16 upregulates TSPYL5 gene and mediates Lung cancer cell proliferation by suppressing p53 activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1760. doi:10.1158/1538-7445.AM2013-1760

Grail as a molecular determinant for the functions of the tumor suppressor p53 in tumorigenesis

The transcription factor p53 is a multifunctional tumor suppressor that arrests the cell cycle in response to stress and modulates the DNA repair process or induces apoptosis. The cellular level and activity of p53 are tightly controlled to maintain proper functioning. This study identified a novel p53-binding glycoprotein, gene related to anergy in lymphocytes (Grail), which formed a negative feedback loop (similar to that of Mdm2). Grail physically and functionally interacted with the N-terminus of p53 to target its degradation and modulate its transactivation activity. Grail also senses and regulates cellular p53 levels, modulates a panel of p53-targeted promoters, and has a role in p53-induced apoptosis in cultured cells. Overexpression of Grail inhibited p53-induced apoptosis by increasing p53 degradation. However, cells not expressing Grail failed to undergo p53-dependent apoptosis, resulting in p21-dependent G1 arrest. Thus, Grail may provide a novel regulatory route for controlling p53 activity under stress conditions.

Abstract 1873: Gene expression analysis of newly diagnosed Multiple Myeloma (MM) patients carrying amplified MDM4 and/or deleted p53

Abstract The p53 tumor suppressor pathway is tightly kept in check or completely silenced in cancer cells. A potent inhibitor of p53 is MDM4, which is located on chr.1q: it is critical for the control of p53 activity during the response to stress and is often amplified in several types of cancer. Since both del(17p) and amp(1q) identify a subgroup of high-risk MM pts, even when the novel agents are part of up-front treatment strategy, we molecularly analyzed a subgroup of MM patients treated with bortezomib-thalidomide-dexamethasone (VTD) and autologous stem cell transplantation (ASCT) in order to investigate mechanisms which might be activated in myeloma plasma cells to direct and/or indirect limit the p53 function. CD138+ plasma cells obtained at diagnosis from 61 pts, subsequently treated with VTD and ASCT were analysed by means of GEP and unpaired analysis of CNA (Affymetrix U133 Plus2.0 and 6.0 SNP arrays). Nineteen out of 61 pts (31%) carried a minimal amplification region on chr.1q, harboring MDM4. Eight out of 61 pts (13%) carried a 482 Kb minimal deletion region on chr.17 harboring TP53. To explore the involvement of the p53 pathway in MM, pts were stratified according to the presence of amplified MDM4 and/or deleted p53 (group A, 24 pts) or the absence of both these abnormalities (group B, 37 pts). Baseline clinical characteristics were homogeneous, except for a higher rate of ≤ 3 bone lesions in pts of group A. The rate of best complete or near complete response was 40% in group A and 14% in group B (p = 0.04). With a median follow-up of 36 months, the risk of progression was 46% for pts in group A and 19% for those in group B (p = 0.03). The average number of aberrations per group was overall higher in group A as compared to group B (165 vs. 103 CNAs, p =0.03). A comparison of expression profiles of the two subgroups of pts highlighted a deregulated expression of genes involved in the mechanisms of response to genotoxic stress, i.e. of damage sensor genes (ATM, RAD9, RAD 50, ATRip), of damage signal mediator genes (H2AFX, 14-3-3), of genes involved in regulation of cell proliferation (CDK6, CDC25, CCND2) and of anti-apoptotic genes (BCL2, p73) (one-way ANOVA plus multiple-test correction with FDR <0,05). Finally, this group of pts significantly over-express the transcription factor YY1, which is known to interact with p53, thus inhibiting its transcriptional activity. In conclusion, pts carrying amplified MDM4 and/or deleted p53 showed a significantly higher number of CNAs and the significant over-expression of genes involved in the response mechanisms to genotoxic stress, as compared to pts lacking these chromosomal aberrations. This might account for the worse outcome of patients harboring del(17p) and/or amp(1q). The MDM4 locus amplification and the YY1 over-expression might contribute to maintain p53 in an OFF state by an indirect mechanism. Supported by: Fondazione Del Monte di Bo e Ra, Ateneo RFO grants (M.C.) BolognAIL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1873. doi:1538-7445.AM2012-1873

The Many Faces of MDM2 Binding Partners

Mdm2 is an essential regulator of the p53 tumor suppressor. Mdm2 is modified at transcriptional, post-transcriptional, and post-translational levels to control p53 activity in normal versus stressed cells. Importantly, errors in these regulatory mechanisms can result in aberrant Mdm2 expression and failure to initiate programmed cell death in response to DNA damage. Such errors can have severe consequences as evidenced by tumor phenotypes resulting from amplification at the Mdm2 locus and changes in post-transcriptional and post-translational regulation of Mdm2. Although Mdm2 mediated inhibition of p53 is well characterized, Mdm2 interacts with many additional proteins and also targets many of these for proteosomal degradation. Mdm2 also has E3-ligase independent functions and p53-independent functions that have important implications for genome stability and cancer.

TRIM8 modulates p53 activity to dictate cell cycle arrest

p53 is a central hub in controlling cell proliferation. To maintain genome integrity in response to cellular stress, p53 directly regulates the transcription of genes involved in cell cycle arrest, DNA repair, apoptosis and/or senescence. An array of post-translational modifications and protein-protein interactions modulates its stability and activities in order to avoid malignant transformation. However, to date it is still not clear how cells decide their own fate in response to different types of stress. We described here that the human TRIM8 protein, a member of the TRIM family, is a new modulator of the p53-mediated tumor suppression mechanism. We showed that under stress conditions, such as UV exposure, p53 induced the expression of TRIM8, which in turn stabilized p53 leading to cell cycle arrest and reduction of cell proliferation through enhancement of CDKN1A (p21) and GADD45 expression. TRIM8 silencing reduced the capacity of p53 to activate genes involved in cell cycle arrest and DNA repair, in response to cellular stress. Concurrently, TRIM8 overexpression induced the degradation of the MDM2 protein, the principal regulator of p53 stability. Co-immunoprecipitation experiments showed that TRIM8 physically interacted with p53, impairing its interaction with MDM2. Altogether, our results reveal a previously unknown regulatory pathway controlling p53 activity and suggest TRIM8 as a novel therapeutic target to enhance p53 tumor suppressor activity.

The p53 mRNA-Mdm2 Interaction Controls Mdm2 Nuclear Trafficking and Is Required for p53 Activation following DNA Damage

The ATM kinase and p53 are key tumor suppressor factors that control the genotoxic stress response pathway. The ATM substrate Mdm2 controls p53 activity by either targeting p53 for degradation or promoting its synthesis by binding the p53 mRNA. The physiological role and regulation of Mdm2's dual function toward p53 is not known. Here we show that ATM-dependent phosphorylation of Mdm2 at Ser395 is required for the p53 mRNA-Mdm2 interaction. This event also promotes SUMO-conjugation of Mdm2 and its nucleoli accumulation. Interfering with the p53 mRNA-Mdm2 interaction prevents p53 stabilization and activation following DNA damage. These results demonstrate how ATM activity switches Mdm2 from a negative to a positive regulator of p53 via the p53 mRNA.