E3 Ligase MDM2 Research Articles

High-Throughput Discovery of Substrate Peptide Sequences for E3 Ubiquitin Ligases Using a cDNA Display Method.

Cells utilize ubiquitin as a posttranslational protein modifier to convey various signals such as proteasomal degradation. The disfunction of ubiquitylation or following proteasomal degradation can give rise to the accumulation and aggregation of improperly ubquitylated proteins, which is known to be a general causation of many neurodegenerative diseases. Thus, the characterization of substrate peptide sequences of E3 ligases is crucial in biological and pharmaceutical sciences. In this study, we developed a novel high-throughput screening system for substrate peptide sequences of E3 ligases using a cDNA display method, which enables covalent conjugation between peptide sequences and their corresponding cDNA sequences. First, we focused on the MDM2 E3 ligase and its known peptide substrate as a model to establish the screening method, and confirmed that cDNA display method was compatible with in vitro ubiquitylation. Then, we demonstrated identification of MDM2 substrate sequences from random libraries to identify a novel motif (VKFTGGQLA). Bioinformatics analysis of the hit sequences was performed to gain insight about endogenous substrate proteins.

Development of hybrid aptamers-engineered PROTACs for degrading VEGF165 in both tumor- and vascular endothelial cells

Tumors and angiogenesis are connected through a complex interplay. VEGF165, generated from both tumor and vascular endothelial cells, serves as a mutual benefit for both cell types. Therapeutic approaches modulating VEGF165 have been proposed as promising antitumor therapies. PROTACs are bifunctional molecules that exploit the intracellular ubiquitin-proteasome system to degrade specific proteins. To date, there are no targeted PROTACs designed to degrade VEGF165 in both tumor and vascular endothelial cells. The aptamer AS1411 is notable for its ability to selectively recognize and enter both tumor and vascular endothelial cells by targeting the cell surface nucleolin (NCL). Moreover, AS1411 has also been repurposed as an intracellular recruiter of E3 ligase MDM2 via leveraging NCL as a molecular bridge. In this study, we conjugated AS1411 with a VEGF165-specific aptamer V7t1, creating hybrid aptamers-engineered PROTACs. The PROTACs demonstrate remarkable selectivity for both tumor and vascular endothelial cells and facilitate the ubiquitination and proteasomal degradation of VEGF165. The PROTACs inhibit the growth of tumor cells and also impede angiogenesis, without causing toxicity to normal tissues. The hybrid aptamers-engineered PROTACs provide an avenue for disrupting the tumor-angiogenesis interplay through modulation of VEGF165 in both tumor and vascular endothelial cells.

An Anti-Invasive Role for Mdmx through the RhoA GTPase under the Control of the NEDD8 Pathway.

Mdmx (Mdm4) is established as an oncogene mainly through repression of the p53 tumour suppressor. On the other hand, anti-oncogenic functions for Mdmx have also been proposed, but the underlying regulatory pathways remain unknown. Investigations into the effect of inhibitors for the NEDD8 pathway in p53 activation, human cell morphology, and in cell motility during gastrulation in Xenopus embryos revealed an anti-invasive function of Mdmx. Through stabilisation and activation of the RhoA GTPase, Mdmx is required for the anti-invasive effects of NEDDylation inhibitors. Mechanistically, through its Zn finger domain, Mdmx preferentially interacts with the inactive GDP-form of RhoA. This protects RhoA from degradation and allows for RhoA targeting to the plasma membrane for its subsequent activation. The effect is transient, as prolonged NEDDylation inhibition targets Mdmx for degradation, which subsequently leads to RhoA destabilisation. Surprisingly, Mdmx degradation requires non-NEDDylated (inactive) Culin4A and the Mdm2 E3-ligase. This study reveals that Mdmx can control cell invasion through RhoA stabilisation/activation, which is potentially linked to the reported anti-oncogenic functions of Mdmx. As inhibitors of the NEDD8 pathway are in clinical trials, the status of Mdmx may be a critical determinant for the anti-tumour effects of these inhibitors.

Mdm2 requires Sprouty4 to regulate focal adhesion formation and metastasis independent of p53

Although the E3 ligase Mdm2 and its homologue and binding partner MdmX are the major regulators of the p53 tumor suppressor protein, it is now evident that Mdm2 and MdmX have multiple functions that do not involve p53. As one example, it is known that Mdm2 can regulate cell migration, although mechanistic insight into this function is still lacking. Here we show in cells lacking p53 expression that knockdown of Mdm2 or MdmX, as well as pharmacological inhibition of the Mdm2/MdmX complex, not only reduces cell migration and invasion, but also impairs cell spreading and focal adhesion formation. In addition, Mdm2 knockdown decreases metastasis in vivo. Interestingly, Mdm2 downregulates the expression of Sprouty4, which is required for the Mdm2 mediated effects on cell migration, focal adhesion formation and metastasis. Further, our findings indicate that Mdm2 dampening of Sprouty4 is a prerequisite for maintaining RhoA levels in the cancer cells that we have studied. Taken together we describe a molecular mechanism whereby the Mdm2/MdmX complex through Sprouty4 regulates cellular processes leading to increase metastatic capability independently of p53.

TrkA promotes MDM2-mediated AGPS ubiquitination and degradation to trigger prostate cancer progression

BackgroundAs a novel necrosis manner, ferroptosis has been increasingly reported to play a role in tumor progression and treatment, however, the specific mechanisms underlying its development in prostate cancer remain unclear. Growing evidence showed that peroxisome plays a key role in ferroptosis. Herein, we identified a novel mechanism for the involvement of ferroptosis in prostate cancer progression, which may provide a new strategy for clinical treatment of prostate cancer.MethodsLabel-Free Mass spectrometry was used to screen and identify candidate proteins after ferroptosis inducer-ML210 treatment. Immunohistochemistry was undertaken to explore the protein expression of AGPS in prostate cancer tissues compared with normal tissues. Co-immunoprecipitation and GST pull-down were used to identify the directly binding of AGPS to MDM2 in vivo and in vitro. CCK8 assay and colony formation assay were used to illustrate the key role of AGPS in the progression of prostate cancer in vitro. The xenograft model was established to verify the key role of AGPS in the progression of prostate cancer in vivo.ResultsAGPS protein expression was downregulated in prostate cancer tissues compared with normal tissues from the first affiliated hospital of Zhengzhou University dataset. Lower expression was correlated with poorer overall survival of patients compared to those with high expression of AGPS. In addition, AGPS can promote ferroptosis by modulating the function of peroxisome-resulting in the lower survival of prostate cancer cells. Furthermore, it was shown that AGPS can be ubiquitinated and degraded by the E3 ligase-MDM2 through the proteasomal pathway. Meanwhile, kinase TrkA can promote the combination of AGPS and MDM2 by phosphorylating AGPS at Y451 site. It was verified that kinase TrkA inhibitor—Larotrectinib can increase the susceptibility of prostate cancer cells to ferroptosis, which leads to the inhibition of prostate cancer proliferation to a great extent in vitro and in vivo.ConclusionBased on these findings, we proposed the combination of ferroptosis inducer and TrkA inhibitor to synergistically exert anti-tumor effects, which may provide a new strategy for the clinical treatment of prostate cancer.

MDM2 Antagonist Idasanutlin Reduces HDAC1/2 Abundance and Corepressor Partners but Not HDAC3.

Histone deacetylases 1-3 (HDAC1, HDAC2, and HDAC3) and their associated corepressor complexes play important roles in regulating chromatin structure and gene transcription. HDAC enzymes are also validated drug targets for oncology and offer promise toward new drugs for neurodegenerative diseases and cardiovascular diseases. We synthesized four novel heterobifunctional molecules designed to recruit the mouse double minute 2 homologue (MDM2) E3 ligase to degrade HDAC1-3 utilizing the MDM2 inhibitor idasanutlin, known as proteolysis targeting chimeras (PROTACs). Idasanutlin inhibits the MDM2-P53 protein-protein interaction and is in clinical trials. Although two MDM2-recruiting heterobifunctional molecules reduced HDAC1 and HDAC2 abundance with complete selectivity over HDAC3 and reduced HDAC1/2 corepressor components LSD1 and SIN3A, we were surprised to observe that idasanutlin alone was also capable of this effect. This finding suggests an association between the MDM2 E3 ligase and HDAC1/2 corepressor complexes, which could be important for designing future dual/bifunctional HDAC- and MDM2-targeting therapeutics, such as PROTACs.

DHRS4-AS1 regulate gastric cancer apoptosis and cell proliferation by destabilizing DHX9 and inhibited the association between DHX9 and ILF3

Gastric cancer (GC) causes millions of cancer-related deaths due to anti-apoptosis and rapid proliferation. However, the molecular mechanisms underlying GC cell proliferation and anti-apoptosis remain unclear. The expression levels of DHRS4-AS1 in GC were analyzed based on GEO database and recruited GC patients in our institution. We found that DHRS4-AS1 was significantly downregulated in GC. The expression of DHRS4-AS1 in GC tissues showed a significant correlation with tumor size, advanced pathological stage, and vascular invasion. Moreover, DHRS4-AS1 levels in GC tissues were significantly associated with prognosis. DHRS4-AS1 markedly inhibited GC cell proliferation and promotes apoptosis in vitro and in vivo assays. Mechanically, We found that DHRS4-AS1 bound to pro-oncogenic DHX9 (DExH-box helicase 9) and recruit the E3 ligase MDM2 that contributed to DHX9 degradation. We also confirmed that DHRS4-AS1 inhibited DHX9-mediated cell proliferation and promotes apoptosis. Furthermore, we found DHX9 interact with ILF3 (Interleukin enhancer Binding Factor 3) and activate NF-kB Signaling in a ILF3-dependent Manner. Moreover, DHRS4-AS1 can also inhibit the association between DHX9 and ILF3 thereby interfered the activation of the signaling pathway. Our results reveal new insights into mechanisms underlying GC progression and indicate that LncRNA DHRS4-AS1 could be a future therapeutic target and a biomarker for GC diagnosis.

Effect of ubiquitin protease system on DNA damage response in prostate cancer (Review).

Genomic instability is an essential hallmark of cancer, and cellular DNA damage response (DDR) defects drive tumorigenesis by disrupting genomic stability. Several studies have identified abnormalities in DDR-associated genes, and a dysfunctional ubiquitin-proteasome system (UPS) is the most common molecular event in metastatic castration-resistant prostate cancer (PCa). For example, mutations in Speckle-type BTB/POZ protein-Ser119 result in DDR downstream target activation deficiency. Skp2 excessive upregulation inhibits homologous recombination repair and promotes cell growth and migration. Abnormally high expression of a deubiquitination enzyme, ubiquitin-specific protease 12, stabilizes E3 ligase MDM2, which further leads to p53 degradation, causing DDR interruption and genomic instability. In the present review, the basic pathways of DDR, UPS dysfunction, and its induced DDR alterations mediated by genomic instability, and especially the potential application of UPS and DDR alterations as biomarkers and therapeutic targets in PCa treatment, were described.

The Proteasome Inhibitor Bortezomib Induces p53-Dependent Apoptosis in Activated B Cells.

The proteasome inhibitor bortezomib (BTZ) is proposed to deplete activated B cells and plasma cells. However, a complete picture of the mechanisms underlying BTZ-induced apoptosis in B lineage cells remains to be established. In this study, using a direct invitro approach, we show that deletion of the tumor suppressor and cell cycle regulator p53 rescues recently activated mouse B cells from BTZ-induced apoptosis. Furthermore, BTZ treatment elevated intracellular p53 levels, and p53 deletion constrained apoptosis, as recently stimulated cells first transitioned from the G1 to S phase of the cell cycle. Moreover, combined inhibition of the p53-associated cell cycle regulators and E3 ligases MDM2 and anaphase-promoting complex/cyclosome induced cell death in postdivision B cells. Our results reveal that efficient cell cycle progression of activated B cells requires proteasome-driven inhibition of p53. Consequently, BTZ-mediated interference of proteostasis unleashes a p53-dependent cell cycle-associated death mechanism in recently activated B cells.

MDM2 Influences ACE2 Stability and SARS-CoV-2 Uptake.

Angiotensin-converting enzyme 2 (ACE2) is the central entry receptor for SARS-CoV-2. However, surprisingly little is known about the effects of host regulators on ACE2 localization, expression, and the associated influence on SARS-CoV-2 infection. Here we identify that ACE2 expression levels are regulated by the E3 ligase MDM2 and that MDM2 levels indirectly influence infection with SARS-CoV-2. Genetic depletion of MDM2 elevated ACE2 expression levels, which strongly promoted infection with all SARS-CoV-2 isolates tested. SARS-CoV-2 spike-pseudotyped viruses and the uptake of non-replication-competent virus-like particles showed that MDM2 affects the viral uptake process. MDM2 ubiquitinates Lysine 788 of ACE2 to induce proteasomal degradation, and degradation of this residue led to higher ACE2 expression levels and superior virus particle uptake. Our study illustrates that cellular regulators of ACE2 stability, such as MDM2, play an important role in defining the infection capabilities of SARS-CoV-2.

Hormone replacement therapy for postmenopausal atherosclerosis is offset by late age iron deposition.

Postmenopausal atherosclerosis (AS) has been attributed to estrogen deficiency. However, the beneficial effect of hormone replacement therapy (HRT) is lost in late postmenopausal women with atherogenesis. We asked whether aging-related iron accumulation affects estrogen receptor α (ERα) expression, thus explaining HRT inefficacy. A negative correlation has been observed between aging-related systemic iron deposition and ERα expression in postmenopausal AS patients. In an ovariectomized Apoe-/- mouse model, estradiol treatment had contrasting effects on ERα expression in early versus late postmenopausal mice. ERα expression was inhibited by iron treatment in cell culture and iron-overloaded mice. Combined treatment with estradiol and iron further decreased ERα expression, and the latter effect was mediated by iron-regulated E3 ligase Mdm2. In line with these observations, cellular cholesterol efflux was reduced, and endothelial homeostasis was disrupted. Consequently, AS was aggravated. Accordingly, systemic iron chelation attenuated estradiol-triggered progressive AS in late postmenopausal mice. Thus, iron and estradiol together downregulate ERα through Mdm2-mediated proteolysis, providing a potential explanation for failures of HRT in late postmenopausal subjects with aging-related iron accumulation. This study suggests that immediate HRT after menopause, along with appropriate iron chelation, might provide benefits from AS.

Design, synthesis and evaluation of EZH2-based PROTACs targeting PRC2 complex in lymphoma

EZH2 is a member of PcG and can induce the occurrence of cancer when it is highly expressed. As an EZH2 inhibitor, Tazemetostat (EPZ6438) can inhibit the methylation catalytic activity of EZH2. However, many studies have shown that inhibition of EZH2 alone does not efficiently block tumor development. Therefore, in this study, proteolytic targeting chimera technology was employed to enhance the antiproliferative potency of EPZ6438 by degrading the oncogenic activity of EZH2. Several PROTACs have been synthesized by combining EPZ6438 with four E3 ligase ligands based on VHL, CRBN, MDM2, and cIAP E3 ligase systems. In our study, compound E-3P-MDM2 is the most active PROTAC molecule. It degraded EZH2 of the SU-DHL-6 cells in a concentration and dose-dependent manner and also degraded both EED and SUZ12 protein without affecting their mRNA levels, then significantly inhibited the expression of H3K27me3. The in vitro antiproliferative activity of E-3P-MDM2 was much stronger than that of EPZ6438.

MiR-221/222 induce instability of p53 By downregulating deubiquitinase YOD1 in acute myeloid leukemia

Acute myeloid leukemia (AML) is a hematological malignancy characterized by the impaired differentiation and uncontrolled proliferation of myeloid blasts. Tumor suppressor p53 is often downregulated in AML cells via ubiquitination-mediated degradation. While the role of E3 ligase MDM2 in p53 ubiquitination is well-accepted, little is known about the involvement of deubiquitinases (DUBs). Herein, we found that the expression of YOD1, among several DUBs, is substantially reduced in blood cells from AML patients. We identified that YOD1 deubiqutinated and stabilized p53 through interaction via N-terminus of p53 and OTU domain of YOD1. In addition, expression levels of YOD1 were suppressed by elevated miR-221/222 in AML cells through binding to the 3′ untranslated region of YOD1, as verified by reporter gene assays. Treatment of cells with miR-221/222 mimics and inhibitors yielded the expected effects on YOD1 expressions, in agreement with the negative correlation observed between the expression levels of miR-221/222 and YOD1 in AML cells. Finally, overexpression of YOD1 stabilized p53, upregulated pro-apoptotic p53 downstream genes, and increased the sensitivity of AML cells to FLT3 inhibitors remarkably. Collectively, our study identified a pathway connecting miR-221/222, YOD1, and p53 in AML. Targeting miR-221/222 and stimulating YOD1 activity may improve the therapeutic effects of FLT3 inhibitors in patients with AML.

LncRNA NRON promotes tumorigenesis by enhancing MDM2 activity toward tumor suppressor substrates.

The E3 ligase MDM2 promotes tumor growth and progression by inducing ubiquitin-mediated degradation of P53 and other tumor-suppressing proteins. Here, we identified an MDM2-interacting lncRNA NRON, which promotes tumor formation by suppressing both P53-dependent and independent pathways. NRON binds to MDM2 and MDMX (MDM4) via two different stem-loops, respectively, and induces their heterogenous dimerization, thereby enhancing the E3 ligase activity of MDM2 toward its tumor-suppressing substrates, including P53, RB1, and NFAT1. NRON knockdown dramatically inhibits tumor cell growth in vitro and in vivo. More importantly, NRON overexpression promotes oncogenic transformation by inducing anchorage-independent growth in vitro and facilitating tumor formation in immunocompromised mice. Clinically, NRON expression is significantly associated with poor clinical outcome in breast cancer patients. Together, our data uncover a pivotal role of lncRNA that induces malignant transformation of epithelial cells by inhibiting multiple tumor suppressor proteins.

The ubiquitin E3 ligase MDM2 induces chemoresistance in colorectal cancer by degradation of ING3.

Chemoresistance is an obstacle for colorectal cancer (CRC) treatment. This study investigates the role of the ubiquitin E3 ligase MDM2 in affecting cell growth and chemosensitivity in CRC cells by modifying the transcription factor inhibitor of growth protein 3 (ING3). The expression of MDM2 and ING3 in CRC tissues was predicted by bioinformatics analysis, followed by expression validation and their interaction in CRC HCT116 and LS180 cells. Ectopic overexpression or knockdown of MDM2/ING3 was performed to test their effect on proliferation and apotptosis as well as chemosensitivity of CRC cells. Finally, the effect of MDM2/ING3 expression on the in vivo tumorigenesis of CRC cells was examined through subcutaneous tumor xenograft experiment in nude mice. MDM2 promoted ubiquitin-proteasome pathway degradation of ING3 through ubiquitination and diminished its protein stability. Overexpression of MDM2 downregulated ING3 expression, which promoted CRC cell proliferation and inhibited the apoptosis. The enhancing role of MDM2 in tumorigenesis and resistance to chemotherapeutic drugs was also confirmed in vivo. Our findings highlight that MDM2 modifies the transcription factor ING3 by ubiquitination-proteasome pathway degradation, thus reducing ING3 protein stability, which finally promotes CRC cell growth and chemoresistance.

Abstract LB003: BI 907828: A highly potent MDM2-p53 antagonist suitable for intermittent dose schedules

Abstract The E3 ligase MDM2 controls the tumor suppressor function of p53, which is encoded by the TP53gene. Compounds designed to bind to MDM2 preventing its interaction with p53 restore the ability of p53 to combat the pathogenic processes that underly cancer development. It has become clear from early clinical experience that thrombocytopenia represents an on-target, dose-limiting toxicity that may restrict the therapeutic utility of first-generation MDM2 inhibitors. Dosing less frequently, while maintaining efficacious exposure levels, has been proposed as an approach to mitigate side effects and improve the therapeutic window. In this -Late Breaking- session we present the discovery and preclinical evaluation of BI 907828, a highly potent, orally bioavailable and selective molecule binding to MDM2 and preventing its interaction with p53. The compound was designed to have a long half-life to enable intermittent dosing schedules in the clinic where it showed a manageable safety profile in a Phase Ia/Ib, dose-escalation/expansion study and encouraging signs of antitumor activity in patients with advanced DDLPS and WDLPS. BI 907828 is actively investigated in several clinical trials including the Phase II/III Brightline-1 study that aims to evaluate whether it is superior to doxorubicin in the first-line treatment of advanced/metastatic DDLPS. BI 907828 belongs to the class of spiro-oxindole MDM2-p53 antagonists and was discovered starting from an optimized core structure with improved chemical stability. Structure-based medicinal chemistry optimization including rigidification of the scaffold were key to accomplish the desired profile which was tested preclinically in several in vivo MDM2-amplified xenograft models where treatment with BI 907828 showed efficient tumor growth inhibition and regression. Citation Format: Andreas Gollner, Patrizia Sini, Dorothea Rudolph, Harald Weinstabl, Juergen Ramharter, Ulrike Weyer-Czernilofsky, Bojana Golubovic, Rolf Grempler, Alejandro Pérez Pitarch, Reinhard Sailer, Darryl B. McConnell, Norbert Kraut. BI 907828: A highly potent MDM2-p53 antagonist suitable for intermittent dose schedules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB003.

The MAD2B-APC/C-MDM2 axis mediates acute kidney injury by modulating p53.

P53 is a master regulator modulating the progression of acute kidney injury (AKI). However, the mechanism underlying p53 regulation in AKI needs further investigation. Mitotic arrest deficient 2 like 2 (MAD2B) is a subunit of DNA polymerase ζ. Its role in AKI remains unclear. Here, we demonstrated that MAD2B acted as an endogenous suppressor of p53. MAD2B conditional knockout augmented the upregulation of p53 in kidneys suffering from cisplatin-induced AKI, therefore promoting the deterioration of renal function, G1 phase arrest and apoptosis of proximal tubular epithelial cells. Mechanistically, MAD2B deficiency activated the anaphase-promoting complex/cyclosome (APC/C), which is an inhibitor of the well-characterized p53-directed E3 ligase MDM2. The decreased MDM2 diminished the degradation of p53, resulting in the upregulation of p53. The APC/C antagonist proTAME ameliorated cisplatin-induced AKI and blocked MAD2B knockdown-induced p53 upregulation and reduced cell cycle arrest and apoptosis in tubular epithelial cells by upregulating MDM2. These results indicate that MAD2B is a novel target for inhibiting p53 and ameliorating AKI.

Abstract 2606: Requirement of MDM2 E3 ligase activity for regulating p53 during normal development, cell cycle regulation and genome integrity

Abstract p53 tumor suppressor, the most mutated gene in human cancer, is negatively regulated by MDM2 and MDM4 proteins. Two distinct mechanisms of MDM2/MDM4 regulation of p53 include, one, suppression of p53 transactivation activity by direct interaction of MDM2/MDM4 with p53 protein, thus blocking its transactivation interface and two, targeting p53 to proteasomal degradation by MDM2 E3 ligase activity. However, the importance of these mechanisms in normal p53 regulation in vivo has remained controversial. To genetically separate these two mechanisms of p53 regulation we generated a novel Mdm2 mutant mouse (Mdm2L466A) that lacks E3 ligase activity but retains the ability of Mdm2 to heterodimerize with Mdm4 and inhibit p53 transactivation. Homozygous Mdm2L466A mice are embryonic lethal due to dysregulated p53 activity, thus demonstrating Mdm2 E3 ligase mediated regulation of p53 is essential during embryonic development. Additionally, we uncovered novel p53-independent functions of MDM2 E3 ligase in cell cycle regulation and genome integrity in cells. Cells lacking MDM2 E3 ligase activity have defective G2-M transition during cell cycle and developed elevated levels of aneuploidy regardless of p53 status. This study unequivocally demonstrates the requirement of Mdm2 E3 ligase activity for normal regulation of p53 and uncovers previously unknown p53-independent role of Mdm2 in cell cycle regulation and maintaining genome integrity. A current therapeutic approach involves blocking MDM2-p53 interaction to promote p53 tumor suppression function in cancer cells. Our findings suggest that this approach alone is insufficient because MDM2 has other potential oncogenic mechanisms independent of its role as negative regulator of p53. Hence, targeting MDM2 E3 ligase activity is likely to be more effective cancer therapy as it addresses both p53-dependent and p53-independent oncogenic mechanisms of MDM2. Citation Format: Meenalakshmi Chinnam, Rati Lama, Chao Xu, Xiaojing Zhang, Carlos Cedeno, Yanqing Wang, Aimee B. Stablewski, David W. Goodrich, Xinjiang Wang. Requirement of MDM2 E3 ligase activity for regulating p53 during normal development, cell cycle regulation and genome integrity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2606.

TRIB3 promotes pulmonary fibrosis through inhibiting SLUG degradation by physically interacting with MDM2.

Pulmonary fibrosis (PF) is the pathological structure of incurable fibroproliferative lung diseases that are attributed to the repeated lung injury-caused failure of lung alveolar regeneration (LAR). Here, we report that repetitive lung damage results in a progressive accumulation of the transcriptional repressor SLUG in alveolar epithelial type II cells (AEC2s). The abnormal increased SLUG inhibits AEC2s from self-renewal and differentiation into alveolar epithelial type I cells (AEC1s). We found that the elevated SLUG represses the expression of the phosphate transporter SLC34A2 in AEC2s, which reduces intracellular phosphate and represses the phosphorylation of JNK and P38 MAPK, two critical kinases supporting LAR, leading to LAR failure. TRIB3, a stress sensor, interacts with the E3 ligase MDM2 to suppress SLUG degradation in AEC2s by impeding MDM2-catalyzed SLUG ubiquitination. Targeting SLUG degradation by disturbing the TRIB3/MDM2 interaction using a new synthetic staple peptide restores LAR capacity and exhibits potent therapeutic efficacy against experimental PF. Our study reveals a mechanism of the TRIB3-MDM2-SLUG-SLC34A2 axis causing the LAR failure in PF, which confers a potential strategy for treating patients with fibroproliferative lung diseases.

The RNA binding protein RALY suppresses p53 activity and promotes lung tumorigenesis

The tumor suppressor p53 plays a pivotal role in tumor prevention. The activity of p53 is mainly restrained by the ubiquitin E3 ligase Mdm2. However, it is not well understood how the Mdm2-p53 pathway is intricately regulated. Here we report that the RNA binding protein RALY functions as an oncogenic factor in lung cancer. RALY simultaneously binds to Mdm2 and the deubiquitinating enzyme USP7. Via these interactions, RALY not only stabilizes Mdm2 by stimulating the deubiquitinating activity of USP7 toward Mdm2 but also increases the trans-E3 ligase activity of Mdm2 toward p53. Consequently, RALY enhances Mdm2-mediated ubiquitination and degradation of p53. Functionally, RALY promotes lung tumorigenesis, at least partially, via negative regulation of p53. These findings suggest that RALY destabilizes p53 by modulating the function of Mdm2 at multiple levels. Our study also indicates a critical role for RALY in promoting lung tumorigenesis via p53 inhibition.