MDM2 mRNA Research Articles

PRPF19 regulates p53-dependent cellular senescence by modulating alternative splicing of MDM4 mRNA

Alteration of RNA splicing is a hallmark of cellular senescence, which is associated with age-related disease and cancer development. However, the roles of splicing factors in cellular senescence are not fully understood. In this study, we identified the splicing factor PRPF19 as a critical regulator of cellular senescence in normal human diploid fibroblasts. PRPF19 was downregulated during replicative senescence, and PRPF19 knockdown prematurely induced senescence-like cell cycle arrest through the p53–p21 pathway. RNA-sequencing analysis revealed that PRPF19 knockdown caused a switch of the MDM4 splicing isoform from stable full-length MDM4-FL to unstable MDM4-S lacking exon 6. We also found that PRPF19 regulates MDM4 splicing by promoting the physical interaction of other splicing factors, PRPF3 and PRPF8, which are key components of the core spliceosome, U4/U6.U5 tri-snRNP. Given that MDM4 is a major negative regulator of p53, our findings imply that PRPF19 downregulation inhibits MDM4-mediated p53 inactivation, resulting in induction of cellular senescence. Thus, PRPF19 plays an important role in the induction of p53-dependent cellular senescence.

LINC01116 promotes the proliferation and invasion of glioma by regulating the microRNA‑744‑5p‑MDM2‑p53 axis.

Long non-coding RNAs (lncRNAs) have been implicated in the development and progression of tumors. However, the roles and underlying mechanisms of long intergenic non-protein coding RNA 1116 (LINC01116), a member of the lncRNA family, in glioma progression are largely unclear. The expression of LINC01116 and microRNA (miR)-744-5p in glioma tissues and cells was detected by reverse transcription-quantitative PCR. The influences of LINC01116 or miR-744-5p on cell proliferation and invasion were evaluated by Cell Counting Kit-8, colony formation and Transwell assays, and western blotting was used to detect the expression of p53 pathway proteins. A dual-luciferase reporter system was used to locate common binding sites between miR-744-5p and LINC01116 or the 3′ untranslated region of E3 ubiquitin-protein ligase Mdm2 (MDM2). RNA immunoprecipitation was used to determine the interactions between RNAs and proteins. Moreover, a xenograft mouse model was constructed to investigate the effects of LINC01116 in vivo, followed by a TdT-mediated dUTP nick end labeling assay to determine the degree of apoptosis in nude mouse tumors. LINC01116 was found to be highly expressed in glioma tissues, which was associated with a malignant phenotype. LINC01116 promoted the proliferation and invasiveness of glioma cells, and inhibited the p53 pathway by preserving the expression of MDM2 mRNA via miR-744-5p sponging. Furthermore, a low degree of miR-744-5p expression was observed in glioma tissues, which was negatively associated with the expression of LINC01116. Overexpression of miR-744-5p inhibited the proliferation and invasiveness of glioma cells, which was rescued by LINC01116. Finally, LINC01116 knockdown inhibited tumor growth in nude mice. In conclusion, LINC01116 is aberrantly expressed and promotes the progression of glioma by regulating the miR-744-5p-MDM2-p53 pathway. In future, targeting LINC01116 may therefore be a potential therapeutic approach for patients with glioma.

Novel function of PERP-428 variants impacts lung cancer risk through the differential regulation of PTEN/MDM2/p53-mediated antioxidant activity

Lung cancer is the leading cause of cancer-related deaths worldwide. Identifying genetic risk factors and understanding their mechanisms will help reduce lung cancer incidence. The p53 apoptosis effect is related to PMP-22 (PERP), a tetraspan membrane protein, and an apoptotic effector protein downstream of p53. Although historically considered a tumor suppressor, PERP is highly expressed in lung cancers. Stable knockdown of PERP expression induces CL1-5 and A549 lung cancer cell death, but transient knockdown has no effect. Interestingly, relative to the PERP-428GG genotype, PERP-428CC was associated with the highest lung cancer risk (OR = 5.38; 95% CI = 2.12–13.65, p < 0.001), followed by the PERP-428CG genotype (OR = 2.34; 95% CI = 1.55–3.55, p < 0.001). Ectopic expression of PERP-428G, but not PERP-428C, protects lung cancer cells against ROS-induced DNA damage. Mechanistically, PERP-428 SNPs differentially regulate p53 protein stability. p53 negatively regulates the expression of the antioxidant enzymes catalase (CAT) and glutathione reductase (GR), thereby modulating redox status. p53 protein stability is higher in PERP-428C-expressing cells than in PERP-428G-expressing cells because MDM2 expression is decreased and p53 Ser20 phosphorylation is enhanced in PERP-428C-expressing cells. The MDM2 mRNA level is decreased in PERP-428C-expressing cells via PTEN-mediated downregulation of the MDM2 constitutive p1 promoter. This study reveals that in individuals with PERP-428CC, CAT/GR expression is decreased via the PTEN/MDM2/p53 pathway. These individuals have an increased lung cancer risk. Preventive antioxidants and avoidance of ROS stressors are recommended to prevent lung cancer or other ROS-related chronic diseases.

Dynamic behavior of the p53-Mdm2 core module under the action of drug Nutlin and dual delays.

Nutlin is a family of p53-targeting drugs. It is able to bind to Mdm2, thereby accelerate the accumulation of p53 that is a prominent tumor suppressor. An integrated module of the Nutlin PBK and p53 pathway is composed of positive feedback mediated by Mdm2 mRNA as well as the drug Nutlin and negative feedback mediated by Mdm2 protein. The main research content of our paper is how the time delay of protein synthesis, response time delay of Nutlin drug, the degradation rate of Mdm2, the degradation rate of p53 depended on Mdm2 and the actual dose of Nutlin in the cell affect the oscillatory behavior caused by Hopf bifurcation in the integrated network system of Nutlin PBK and p53 pathways. The stability of the unique positive equilibrium point and the existence of Hopf bifurcation are studied by taking the time delays as the bifurcation parameters and applying bifurcation theory. Based on the normal form theory and central manifold theorem, explicit criteria to determine the Hopf bifurcation direction and stability of the bifurcated periodic solution are established. It is found that the time delays and key parameters in the integrated network system of Nutlin PBK and p53 pathways play an important role in the amplitude and period of p53 oscillation according to the results from the numerical simulation and theoretical calculation. These results may provide us with a better understanding of the biological functions of the p53 pathway and some clues for cancer treatment.

Abstract 14395: Abnormal Regulation of P53-mTOR Pathway by MRNA-binding Protein ZFP36L2 in Peri-partum Cardiomyopathy

Introduction: A loss of function mutation in the mRNA binding protein ZFP36L2 has been associated with congenital heart diseases in humans, although the mechanism remains unclear. Objectives: We sought to elucidate the role of ZFP36L2 in the heart using mice with cardiac-specific ZFP36L2 deletion. Results: Cardiac-specific Zfp36l2 knockout (cs-L2KO) female mice exhibit a dramatic phenotype in which the majority of mice die after repeated pregnancies due to heart failure. We performed RNA-seq in H9c2 cells with ZFP36L2 KD, and found the expression of multiple mTOR pathway genes were altered, including marked repression of Redd1 and Sesn2 . Consistent with the RNA-seq data, the activity of mTORC1 was increased in cs-L2KO mouse hearts and in ZFP36L2 KD H9c2 cells. Loss of TSC2 mitigated mTORC1 hyperactivity in ZFP36L2 KD cells, indicating that ZFP36L2 regulates mTORC1 activity through the TSC2 complex. Overexpression of REDD1 rescued increased mTORC1 activity with ZFP36L2 KD, suggesting that ZFP36L2 regulates mTORC1, in-part through REDD1. Under total amino acid starvation, we observed higher p-S6K T389 levels in ZFP36L2 KD cells, and SESN2 overexpression mitigated p-S6K T389 , indicating loss of ZFP36L2 also regulates mTORC1 through amino acid sensing. p53 is a transcriptional activator of both REDD1 and SESN2, and we found that ZFP36L2 was required to maintain p53 levels by directly destabilizing MDM2 mRNA. Accordingly, administration of Nutlin3, an MDM2 inhibitor, reversed the reduction in p53, REDD1 and SESN2 protein levels, and prevented the increase in S6K phosphorylation in response to ZFP36L2 KD. Next, we treated pregnant mice with rapamycin, and cs-L2KO mice treated with rapamycin displayed significantly improved cardiac function after delivery. Finally, we analyzed human heart samples from patients with peri-partum cardiomyopathy and found ZFP36L2 protein to be significantly decreased and p-S6K T389 levels increased, compared to healthy controls. Conclusions: Our studies demonstrate that ZFP36L2 regulates mTORC1 activity through modifying p53 stability, and that the disruption of this pathway induces peri-partum cardiomyopathy. Inhibition of mTORC1 hyperactivity with rapamycin can be a potential therapy for this disease.

Association of MDM2 T309G (rs2279744) Polymorphism and Expression Changes With Risk of Prostate Cancer in the Slovak Population.

The aim of this study was to evaluate the relationship between MDM2 T309G polymorphism and prostate cancer risk in the Slovak population and the association of this polymorphism with MDM2 expression and clinicopathological features. The MDM2 T309G polymorphism was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis in 506 prostate cancer patients and 592 controls. Quantitative real-time (RT)-PCR and western blot analysis were applied to examine MDM2 expression in 47 prostate cancer tissues and 43 benign prostatic hyperplasia (BPH) tissues. A decreased risk of prostate cancer in men carrying the GG genotype in comparison with the TT genotype was found. A decrease in the relative MDM2 mRNA and protein levels was found in prostate cancer tissues among patients with the MDM2 GG genotype. There is a potentially protective effect of the MDM2 GG genotype on the risk of prostate cancer in the Slovak male population.

Abstract 652: Exploiting PRMT5, a metabolic and epigenetic regulator, as a novel therapeutic target in mantle cell lymphoma

Abstract Distinct genetic and epigenetic alterations as well as metabolic reprogramming are among the hallmarks of mantle cell lymphoma (MCL), an incurable cancer. Deletion of MTAP, encoding 5-Methylthioadenosine phosphorylase (MTAP), results in the accumulation of metabolite methylthioadenosine (MTA), a selective inhibitor of the catalytic activity of protein arginine methyltransferase 5 (PRMT5). MTAP loss therefore presents a passenger vulnerability to PRMT5 inhibition. PRMT5 is a type II protein arginine methyltransferase that is involved in cellular processes via the regulation of the active status of transcription by symmetric dimethylation of arginine in histones, and the post-translational modification by symmetric dimethylation of arginine in nuclear and cytoplasmic oncoproteins and tumor suppressors. We recently reported that CDKN2A and MTAP were commonly co-deleted in ibrutinib-resistant MCL tumors. Consistently, PRMT5 was found to be selectively over expressed in ibrutinib-resistant MCL cells. Metabolomics analysis revealed that both the substrate (S-Adenosylmethionine) and the product (S-adenosylhomocysteine) of the PRMT5 enzyme are elevated in ibrutinib-resistant MCL, suggesting an increase in PRMT5 enzymatic activity. Importantly, our clinical data analysis indicates that the expression levels of PRMT5 in MCL patients are inversely correlated with patient responses to ibrutinib treatment. Moreover, pharmacological blockade with the PRMT5 specific inhibitor, GSK3226595, significantly inhibited tumor growth in ibrutinib-resistant MCL PDX models, demonstrating that PRMT5 is an actionable target in MCL. Mechanistically, PRMTs play an important role in mRNA splicing, among which MDM4 mRNA was identified as one of the key mRNAs that senses defects in the spliceosomal machinery during PRMT5 deficiency. Alternative splicing of MDM4 mRNA reduces the expression of Mdm2 and Mdm4 E3 ligases, leading to the upregulation of p53. Selective overexpression of PRMT5 in MCL may drive repressive epigenetic marks on chromatin to silence regulatory and tumor suppressor genes. Inhibition of PRMT5 reverses PRMT5-catalyzed epigenetic marks, restores regulatory pathways and induces cell death in ibrutinib-resistant primary MCL. Taken together, sustained PRMT5 expression and activity presents a vulnerability in ibrutinib-resistant MCL where MTAP is frequently deleted. As metabolic reprogramming towards OXPHOS is one of the hallmarks of ibrutinib-resistant MCL, combinatory inhibition of OXPHOS and PRMT5 may represent a rational therapeutic strategy that ultimately eradicates MCL. Citation Format: Yixin Yao, Lingzhi Li, Liang Zhang, Shaojun Zhang, Hui Guo, Hui Zhang, Maria Badillo, Zhihong Chen, Kimberly Hartig, Preetesh Jain, Krystle Nomie, Linghua Wang, Michael Wang. Exploiting PRMT5, a metabolic and epigenetic regulator, as a novel therapeutic target in mantle cell lymphoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 652.

Abstract 631: Mat2A Inhibitors decrease growth, increase senescence and p53 stability in MTAP-deleted cancer cells

Abstract Deletions in Methylthioadenosine phosphorylase (MTAP) are observed in 15% of all cancers including pancreatic, lung, bladder and GBM cancers (1). MTAP loss results in an accumulation of MTA which partially inhibits Protein Arginine Methyltransferase 5 (PRMT5). MTAP deletions confer enhanced sensitivity to inhibition of Methionine Adenosyltransferase 2A (Mat2A), which is involved in the synthesis of the universal methyl donor and PRMT5 substrate S-Adenosyl Methionine (SAM). In combination with MTA accumulation, Mat2A inhibition depletes SAM levels resulting in further inhibition of PRMT5 activity and a synthetic lethal (SL) relationship in MTAP null cancers. Inhibition of PRMT5 activity as a result increases RNA splicing defects and chromatin dysregulation which impacts tumor cell viability in different models. We developed MAT2A inhibitors for treatment of MTAP-deleted tumors, and in this study sought to determine the mechanism of action of these inhibitors in MTAP null models. These inhibitors were assessed for activity against both MTAP-wt and MTAP-null tumor models using cellular proliferation assays. MTAP-null (KP4, BxPC3, RT112/84, MiaPaCa-2, H647) models were more sensitive to Mat2A inhibition than MTAP-wt (HCT116, NCI-H460) models. Inhibition of Mat2A increased the expression of markers associated with cellular senescence and cell cycle arrest. Cellular SAM levels were diminished in both MTAP-wt and MTAP-null cells and xenografts while selective reduction of Symmetric Dimethyl Arginine (SDMA) levels were observed in MTAP-null models. Cellular response to Mat2A inhibitors is unaltered by RNAi-mediated knockdown of Mat1A. Signaling changes that were observed due to decreased PRMT5 activity included increased stability of p53, which may be due to the inefficient splicing of MDM4 mRNA, and was seen across multiple MTAP-null models but not in MTAP WT. Furthermore, increased protein and mRNA expression of the p53 target gene p21 was also observed in MTAP-null models. In conclusion, Mat2A inhibition effectively decreases SAM and SDMA levels, leading to increased stability of p53 and its targets and resulting in decreased cellular proliferation. Ongoing exploratory studies will further delineate the molecular mechanisms mediated by Mat2A inhibition in MTAP-null cell lines. The genes and metabolites identified from these studies will be validated in Mat2A-dependent and non-dependent MTAP-null cell lines identified from DepMap studies to inform further preclinical and clinical studies. Citation Format: Neil Bhola, Atieh Givmanesh, Marie-Claire Wagle, Candy Garcia, Kedar Vaidya, Leah Cleary, Zhonghua Pei, Mark Lackner, Zineb Mounir. Mat2A Inhibitors decrease growth, increase senescence and p53 stability in MTAP-deleted cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 631.

Abstract 263: Loss of the RNA-binding Protein ZFP36L2 Results in Peri-partum Cardiomyopathy Through Dysregulation of the P53-mTOR Pathway

Introduction: A loss of function mutation in the mRNA binding protein ZFP36L2 has been associated with congenital heart diseases in humans, although the mechanism remains unclear. Objectives: We sought to elucidate the role of ZFP36L2 in the heart using mice with cardiac-specific ZFP36L2 deletion. Results: Cardiac-specific Zfp36l2 knockout (cs-L2KO) female mice exhibit a dramatic phenotype in which the majority of mice die after repeated pregnancies due to heart failure. We performed RNA-seq in H9c2 cells with ZFP36L2 KD, and found the expression of multiple mTOR pathway genes were altered, including marked repression of Redd1 and Sesn2 . Consistent with the RNA-seq data, the activity of mTORC1 was increased in cs-L2KO mouse hearts and in ZFP36L2 KD H9c2 cells. Loss of TSC2 mitigated mTORC1 hyperactivity in ZFP36L2 KD cells, indicating that ZFP36L2 regulates mTORC1 activity through the TSC2 complex. Overexpression of REDD1 rescued increased mTORC1 activity with ZFP36L2 KD, suggesting that ZFP36L2 regulates mTORC1, in-part through REDD1. Under total amino acid starvation, we observed higher p-S6K T389 levels in ZFP36L2 KD cells, and SESN2 overexpression mitigated p-S6K T389 , indicating loss of ZFP36L2 also regulates mTORC1 through amino acid sensing. p53 is a transcriptional activator of both REDD1 and SESN2, and we found that ZFP36L2 was required to maintain p53 levels by directly destabilizing MDM2 mRNA. Accordingly, administration of Nutlin3, an MDM2 inhibitor, reversed the reduction in p53, REDD1 and SESN2 protein levels, and prevented the increase in S6K phosphorylation in response to ZFP36L2 KD. Next, we treated pregnant mice with rapamycin, and cs-L2KO mice treated with rapamycin displayed significantly improved cardiac function after delivery. Finally, we analyzed human heart samples from patients with peri-partum cardiomyopathy and found ZFP36L2 protein to be significantly decreased and p-S6K T389 levels increased, compared to healthy controls. Conclusions: Our studies demonstrate that ZFP36L2 regulates mTORC1 activity through modifying p53 stability, and that the disruption of this pathway induces peri-partum cardiomyopathy. Inhibition of mTORC1 hyperactivity with rapamycin can be a potential therapy for this disease.

LncRNA FOXD3-AS1 promoted chemo-resistance of NSCLC cells via directly acting on miR-127-3p/MDM2 axis

BackgroundThis study aims to investigate the mechanism underlying the high level of long non-coding RNA FOXD3-AS1 in cisplatin-resistant NSCLC cells.MethodsCisplatin-resistant cells were generated from A549 cells. CCK-8 were used to evaluate cell proliferation. The FOXD3-AS1, miR-127-3p, MDM2 and MRP1 mRNA expression levels were confirmed by qRT-PCR. Protein levels of MDM2 and MRP1 were determined by western blot assay. Luciferase reporter and RNA pull-down assays were evaluated the relationship between miR-127-3p and FOXD3-AS1/MDM2. In vivo tumor growth was evaluated in a xenograft nude mice model.ResultsFOXD3-AS1 was up-regulated in cisplatin-resistant NSCLC cells (A549/DDP and H1299/DDP cells) in comparison with their parental cell lines. Overexpression of FOXD3-AS1 promoted cisplatin-resistance in A549 and H1299 cells; while FOXD3-AS1 knockdown sensitized A549/DDP and H1299/DDP cells to cisplatin treatment. FOXD3-AS1 regulated miR-127-3p expression by acting as a competing endogenous RNA, and miR-127-3p repressed MDM2 expression via targeting the 3′UTR. MiR-127-3p overexpression and MDM2 knockdown both increased the chemo-sensitivity in A549/DDP cells; while miR-127-3p knockdown and MDM2 overexpression both promoted chemoresistance in A549 cells. Further rescue experiments revealed that miR-127-3p knockdown or MDM2 overexpression counteracted the suppressive effects of FOXD3-AS1 knockdown on chemo-resistance and MRP1 expression in A549/DDP cells. In vivo studies showed that FOXD3-AS1 knockdown potentiated the antitumor effects of cisplatin treatment. Inspection of clinical samples showed the upregulation of FOXD3-AS1 and MDM2, and down-regulation of miR-127-3p in NSCLC tissues compared to normal adjacent tissues.ConclusionIn conclusion, our results suggest that LncRNA FOXD3-AS1 promotes chemo-resistance of NSCLC cells via directly acting on miR-127-3p/MDM2 axis. Our findings may provide novel perspectives for the treatment of NSCLC in patients resistant to chemotherapy.

Interleukin-6 derived from cancer-associated fibroblasts attenuates the p53 response to doxorubicin in prostate cancer cells

Cancer-associated fibroblasts (CAFs) promote tumor growth and progression, and increase drug resistance through several mechanisms. We have investigated the effect of CAFs on the p53 response to doxorubicin in prostate cancer cells. We show that CAFs produce interleukin-6 (IL-6), and that IL-6 attenuates p53 induction and upregulation of the pro-apoptotic p53 target Bax upon treatment with doxorubicin. This is associated with increased levels of MDM2 mRNA, Mdm2 protein bound to p53, and ubiquitinated p53. IL-6 also inhibited doxorubicin-induced cell death. Inhibition of JAK or STAT3 alleviated this effect, indicating that IL-6 attenuates p53 via the JAK/STAT signaling pathway. These results suggest that CAF-derived IL-6 plays an important role in protecting cancer cells from chemotherapy and that inhibition of IL-6 could have significant therapeutic value.

Bimodal ntrk expression and correlation with activation of PI3K/AKT pathway in lung cancer.

e21535 Background: N TRK (NTRK1, NTRK2 or NTRK3) fusions are oncogenic drivers of various types of cancer and lead to activation of the PI3K/AKT pathway. Treatment of patients with NTRK fusion using TRK inhibitors, such as larotrectinib or entrectinib, is associated with high response rates ( &gt; 75%). However these fusions are very rare in most common cancers, such as lung and colorectal cancers. We examined the expression levels of NTRK1, NTRK2, and NTRk3 in non-small cell lung cancer using targeted RNA sequencing. We also examined the effects of NTRK overexpression on PI3K/AKT pathway in non-small cell lung cancer. Methods: RNA was extracted from 160 FFPE samples of non-small cell lung cancers and sequenced using targeted next generation sequencing (NGS). The RNA sequencing was based on hybrid capture using a targeted panel of 1408 genes. The number of reads ranged from 5 to 10 million. The RNA levels were measured using Cufflinks as FPKM. Results: NTRK2 fusion was detected in one sample (0.04%). However, overexpression of NTRK1-3 mRNA was more common and showed bimodal expression pattern. Most of the samples (75%) had low levels (median 0.6, 6,79, and 3.17 FPKM for NTR2, NTRK2, and NTRK3, respectively) while the upper 25% of samples had significantly (P &lt; 0.0001) higher levels (Median 2.7, 280, and 17.7 KPFM for NTRK1, NTRK2, and NTRK3, respectively). There was significant difference in expression between the three genes (P &lt; 0.0001). The highest levels were detected in NTRK2 and lowest in NTRK1. In addition, significant variation in alternative splicing was noted in NTRK2. All cases with high expression levels (above the upper quartile) showed significant variation in alternative splicing involving almost all exons. To explore, if high expression correlates with activation of the PI3K/AKT1 pathway, we examined the correlation between high levels (above upper quartile) of NTRK2 with various genes in PI3K/AKT pathway. High levels of NTRK2 mRNA (above the upper quartile) correlated with significantly higher levels of PIK3CA (P &lt; 0.0001) and MDM2 (P = 0.0001). There was no correlation between high expression of NTRK1 or NTRK3 and PIK3CA or MDM2 mRNA. Conclusions: Although fusions involving NTRK1-3 genes in lung cancer are rare, significantly high expression levels can be seen almost 25% of lung cancers. Furthermore, high expression in NTRK2 correlates with activation in the PI3K/AKT pathway. This data suggests that therapy targeting NTRK2 overexpression and/or activation of PI3K/AKT pathway in patients with high NTRK2 mRNA should be explored.

A Circular RNA from the MDM2 Locus Controls Cell Cycle Progression by Suppressing p53 Levels.

Circular RNAs (circRNAs) are a class of noncoding RNAs produced by a noncanonical form of alternative splicing called back-splicing. To investigate a potential role of circRNAs in the p53 pathway, we analyzed RNA sequencing (RNA-seq) data from colorectal cancer cell lines (HCT116, RKO, and SW48) that were untreated or treated with a DNA-damaging agent. Surprisingly, unlike the strong p53-dependent induction of hundreds of p53-induced mRNAs upon DNA damage, only a few circRNAs were upregulated from p53-induced genes. circ-MDM2, an annotated circRNA from the MDM2 locus, was one of the handful of circRNAs that originated from a p53-induced gene. Given the central role of MDM2 in suppressing p53 protein levels and p53 activity, we investigated the function of circ-MDM2 Knocking down circ-MDM2 with small interfering RNAs (siRNAs) that targeted circ-MDM2 did not alter MDM2 mRNA or MDM2 protein levels but resulted in increased basal p53 levels and growth defects in vitro and in vivo Consistent with these results, transcriptome profiling showed increased expression of several direct p53 targets, reduced retinoblastoma protein (Rb) phosphorylation, and defects in G1-S progression upon silencing circ-MDM2 Our results on the initial characterization of circ-MDM2 identify a new player from the MDM2 locus that suppresses p53 levels and cell cycle progression.

N6-methyladenosine mediates arsenite-induced human keratinocyte transformation by suppressing p53 activation

N6-methyladenosine (m6A), the most abundant and reversible RNA modification, plays critical a role in tumorigenesis. However, whether m6A can regulate p53, a leading antitumor protein remains poorly understood. In this study, we explored the regulatory role of m6A on p53 activation using an arsenite-transformed keratinocyte model, the HaCaT-T cell line. We created the cell line by exposing human keratinocyte HaCaT cells to 1 μM arsenite for 5 months. We found that the cells exhibited an increased m6A level along with an aberrant expression of the methyltransferases, demethylase, and readers of m6A. Moreover, the cells exhibited decreased p53 activity and reduced p53 phosphorylation, acetylation, and transactivation with a high nucleus export rate of p53. Knockdown of the m6A methyltransferase, METTL3 significantly decreased m6A level, restoring p53 activation and inhibiting cellular transformation phenotypes in the arsenite-transformed cells. Further, using both a bioinformatics analysis and experimental approaches, we demonstrated that m6A downregulated the expression of the positive p53 regulator, PRDM2, through the YTHDF2-promoted decay of PRDM2 mRNAs. We showed that m6A upregulated the expression of the negative p53 regulator, YY1 and MDM2 through YTHDF1-stimulated translation of YY1 and MDM2 mRNA. Taken together, our study revealed the novel role of m6A in mediating arsenite-induced human keratinocyte transformation by suppressing p53 activation. This study further sheds light on the mechanisms of arsenic carcinogenesis via RNA epigenetics.

MDM2 inhibition-mediated autophagy contributes to the pro-apoptotic effect of berberine in p53-null leukemic cells

AimsBerberine (BBR) is reported to induce apoptosis and inhibit migration of leukemic cells, but the underlying pharmacological mechanisms have not been fully revealed. This study aims to investigate the possible mechanisms from the perspective of autophagy. Main methodsP-53-null leukemic cell lines Jurkat and U937 were used for the in vitro study. MDC staining was used for observation of autophagy in leukemic cells, and Western blot analysis was for determination of the expression levels of autophagy-associated proteins. Apoptosis of the leukemic cells was detected by flow cytometry. Cellular location of MDM2 was observed with immunofluorescence staining. Ubiquitination of MDM2 was assessed by immunoprecipitation. Male 6–8-week-old NOD/SCID mice were used for evaluating the effect of BBR on chemotherapy sensitivity in vivo. Key findingsBBR induced autophagy in p53-null leukemic cells, which was inhibited by autophagy inhibitors 3-methyladenine. 3-methyladenine also inhibited BBR-induced apoptosis in leukemic cells. In addition, BBR not only decreased MDM2 mRNA expression, but also enhanced MDM2 self-ubiquitination in leukemic cells. Forced overexpression of MDM2 reversed the effect of BBR on autophagy and apoptosis. Furthermore, BBR promoted doxorubicin-induced autophagy and cell death in the leukemic cells and overexpression of MDM2 suppressed these effects. In vivo, BBR combined with doxorubicin achieved better therapeutic effect than doxorubicin alone. SignificanceMDM2 inhibits autophagy and apoptosis in leukemic cells in a p53-independent manner. BBR induces autophagy in p53-null leukemic cells through downregulating MDM2 expression at both transcriptional and post-transcriptional levels, which may contribute to the anti-cancer effect of BBR in leukemia.

Mdm2 is a target and mediator of IRP2 in cell growth control

Iron is an essential element to all living organisms and plays a vital role in many cellular processes, such as DNA synthesis and energy production. The Mdm2 oncogene is an E3 ligase and known to promote tumor growth. However, the role of Mdm2 in iron homeostasis is not certain. Here, we showed that Mdm2 expression was increased by iron depletion but decreased by iron repletion. We also showed that Iron Regulatory Protein 2 (IRP2) mediated iron-regulated Mdm2 expression. Specifically, Mdm2 expression was increased by ectopic IRP2 but decreased by knockdown or knockout of IRP2 in human cancer cells as well as in mouse embryonic fibroblasts. In addition, we showed that IRP2-regulated Mdm2 expression was independent of tumor suppressor p53. Mechanistically, we found that IRP2 stabilized Mdm2 transcript via binding to an iron response element (IRE) in the 3'UTR of Mdm2 mRNA. Finally, we showed that Mdm2 is required for IRP2-mediated cell proliferation and Mdm2 expression is highly associated with IRP2 in both the normal and cancerous liver tissues. Together, we uncover a novel regulation of Mdm2 by IRP2 via mRNA stability andthat the IRP2-Mdm2 axis may play a critical role in cell growth.

Downregulation of MDM2 Leads to Anti-Proliferative Effects through Activation of p53-Associated Pathway Mediated By Both Dual Inhibitor MX69 and Mir-548c-3p in Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy characterized by abnormal proliferation of clonal plasma cells in the bone marrow. MM remains an incurable disease with a high rate of relapse and development of drug resistance. Mouse double minute 2 homolog (MDM2) has been characterized as an oncogene that is associated with cancer development and radio/chemotherapy resistance in cancer. However, the mechanism(s) underlying MDM2 overexpression and its association with drug resistance in MM have not been fully explored. In addition, the effect of the newly discovered dual inhibitor MX69, which targets MDM2 and XIAP, is unknown in MM. To assess the effects of MDM2 overexpression in MM patients, we performed GEO data (GSE6477, GSE31161) differential expression analysis on bone marrow samples from three patient groups- normal donors, newly diagnosed MM and relapsed MM. We found that relapsed MM patients expressed high MDM2 compared to newly diagnosed MM patients, and both groups expressed higher MDM2 compared to normal donors (p&lt;0.001). Furthermore, immunoblotting and qRT-PCR showed upregulated MDM2 expression in drug resistant MM cell lines (MM1R, 8226R5) as compared to their parental drug sensitive cells (MM1S, 8226S). XIAP is an important inhibitor of apoptotic proteins caspases 3, 7 and 9. Elevated expression of XIAP is involved in chemotherapy resistance in cancer. To determine whether MDM2 and XIAP downregulation by dual inhibitor MX69 could induce apoptosis in MM cells, we treated two drug resistant MM cell lines with MX69. Cell viability assay showed that MX69 reduced viability of MM cells in a time- and dose-dependent manner. Since MDM2 is an antagonist of tumor suppressor p53, we measured the expression of p53 following MX69 treatment and found that MX69-mediated downregulation of MDM2 significantly enhanced p53, PUMA and p21 expression in MM cell lines harboring wild-type p53. In MM p53 null cells, we found that p53 family pro-apoptotic factors p63 and p73 were increased upon MX69 treatment. To investigate the downstream mechanism of MDM2 targeting in MM drug resistance, we knocked down MDM2 in MM cells via siRNA-mediated silencing. Cell viability assay indicated that siMDM2 reduced cell proliferation compared to negative control (P=0.009). Following MDM2 silencing, XIAP expression was decreased and p53 expression was increased in p53 positive MM cell lines, whereas p63, p73 and p21 were increased in both p53 positive and p53 null MM cells. Furthermore, combined treatment of MX69 with dexamethasone (Dex), doxorubicin (Dox) or lenalidomide (Len) displayed synergistic toxic effects (combination index ranging from 0.437 to 0.8) in drug resistant MM cell lines. MX69 alone or in combination with Dex, Dox and Len significantly increased apoptosis and decreased proliferation of MM cells. To explore whether miRNAs play a role in regulating MDM2 overexpression and MM drug resistance, several miRNA target prediction algorithms were exploited to generate a selective miRNA library for subsequent screening. Four miRNAs (miR-890, miR-211-5p, miR-429 and miR-548c-3p) were common among GSE49261, GSE39571, GSE16558 and GSE17498 GEO datasets. These four miRNAs were downregulated in MM patients and negatively correlated with MDM2. Furthermore, analyzing prediction score revealed that miR-548c-3p had the highest binding score with MDM2 3'UTR. Moreover, MiR-548c-3p showed a significant downregulation in drug resistant cells in comparison with their parental sensitive cells (p&lt;0.0001). To determine whether MDM2 expression is selectively regulated by miR-548c-3p, we transfected two MM drug resistant cells with miR-548c-3p mimics and found that MDM2 mRNA and protein levels were suppressed and the cell viability was significantly reduced compared to the scrambled controls. In conclusion, our results indicate that MDM2 overexpression is associated with disease progression and drug resistance in MM. Treatment of drug resistant MM cells with MDM2/XIAP dual inhibitor MX69 sensitizes the cells to anti-myeloma drugs and induces apoptosis. Our finding of the regulatory interaction between miR-548c-3p and MDM2 reveals key insights into the dysregulation of MDM2 in MM and provides a new therapeutic approach to target MDM2 by restoration of miR-548c-3p. Disclosures No relevant conflicts of interest to declare.

MDM4 expression in fibrolamellar hepatocellular carcinoma.

Fibrolamellar hepatocellular carcinoma (FL‑HCC) is a variant of hepatocellular carcinoma (HCC) that most commonly affects adolescents and young adults and is associated with an extremely poor prognosis due to the lack of effective chemotherapeutic agents. Mutations in p53 are a common oncogenic driver in HCC but not in FL‑HCC. However, in tumors lacking a p53 mutation, the tumor suppressor activity of p53 has been revealed to be dysregulated in several different cancer types. One mechanism has been attributed to the overexpression of mouse double minute4 protein(MDM4), a negative regulator of p53, which inhibits the normal functions of p53 including induction of apoptosis and DNA repair. Therefore, restoring the normal function of p53 in cancer cells by targeting MDM4 has become a potential therapeutic strategy. Hence, in the present study the components of the DNA damage response (DDR) pathway were examined; ATM, p53, and MDM4 in FL‑HCC. Seven FL‑HCC tumors along with their adjacent non‑neoplastic hepatic tissues were examined. Ataxia‑telangiectasia mutated (ATM), p53, and MDM4 protein expression was assessed using western blot analysis and cellular localization was determined using immunohistochemistry(IHC). MDM4 mRNA transcript levels were assessed using RT‑qPCR. The present results demonstrated that the DNA damage sensor, ATM, is phosphorylated and localized to the nuclei of tumor cells. While there was a significant increase in total p53 protein in tumor cells, phosphorylated p53 was revealed to preferably localize to the cytoplasmic compartment of tumor cells. Notably, the present results revealed that MDM4 transcript levels were increased in the majority of tumor samples and the nuclear MDM4 levels were significantly increased in tumor tissue compared to their adjacent non‑neoplastic liver tissue. The present results indicated that increased MDM4 expression and nuclear localization may be a potential mechanism for p53 dysregulation in FL‑HCC.

MDM4 Is Targeted by 1q Gain and Drives Disease in Burkitt Lymphoma.

Oncogenic MYC activation promotes proliferation in Burkitt lymphoma, but also induces cell-cycle arrest and apoptosis mediated by p53, a tumor suppressor that is mutated in 40% of Burkitt lymphoma cases. To identify molecular dependencies in Burkitt lymphoma, we performed RNAi-based, loss-of-function screening in eight Burkitt lymphoma cell lines and integrated non-Burkitt lymphoma RNAi screens and genetic data. We identified 76 genes essential to Burkitt lymphoma, including genes associated with hematopoietic cell differentiation (FLI1, BCL11A) or B-cell development and activation (PAX5, CDKN1B, JAK2, CARD11) and found a number of context-specific dependencies including oncogene addiction in cell lines with TCF3/ID3 or MYD88 mutation. The strongest genotype-phenotype association was seen for TP53. MDM4, a negative regulator of TP53, was essential in TP53 wild-type (TP53wt) Burkitt lymphoma cell lines. MDM4 knockdown activated p53, induced cell-cycle arrest, and decreased tumor growth in a xenograft model in a p53-dependent manner. Small molecule inhibition of the MDM4-p53 interaction was effective only in TP53wt Burkitt lymphoma cell lines. Moreover, primary TP53wt Burkitt lymphoma samples frequently acquired gains of chromosome 1q, which includes the MDM4 locus, and showed elevated MDM4 mRNA levels. 1q gain was associated with TP53wt across 789 cancer cell lines and MDM4 was essential in the TP53wt-context in 216 cell lines representing 19 cancer entities from the Achilles Project. Our findings highlight the critical role of p53 as a tumor suppressor in Burkitt lymphoma and identify MDM4 as a functional target of 1q gain in a wide range of cancers that is therapeutically targetable. SIGNIFICANCE: Targeting MDM4 to alleviate degradation of p53 can be exploited therapeutically across Burkitt lymphoma and other cancers with wild-type p53 harboring 1q gain, the most frequent copy number alteration in cancer.

CircSAMD4A accelerates cell proliferation of osteosarcoma by sponging miR-1244 and regulating MDM2 mRNA expression

Here, we firstly examined the proliferation and invasion capacities of different osteosarcoma (OS) cell lines, and analyzed the profiling of the differentially expressed circular RNAs(circRNAs). Then, we identified a novel circRNA(circ_101356/circ_0004846, we named circSAMD4 in the present study) and found it was highly expressed in the OS tissues compared to adjacent non-cancerous tissues. Furthermore, we indicated that circSAMD4A promoted OS proliferation in vivo and in vitro. In addition, we proved that circSAMD4A enhances osteosarcoma cells stemness features. In mechanism, circSAMD4A could sponge miR-1244 in OS cells. Moreover, we verified that MDM2 was a target of miR-1244. In conclusion, circSAMD4A/miR-1244/MDM2 regulatory loop might be a promising therapeutic target for OS treatment.