E3 Ubiquitin Ligase MDM2 Research Articles

CSIG-17. ADHESION GPCR BAI1/ADGRB1 CAN BLOCK IGF1R-MEDIATED GROWTH SIGNALLING, INCREASE RADIOSENSITIVITY AND AUGMENT SURVIVAL IN MEDULLOBLASTOMA

Abstract Medulloblastoma (MB) is an aggressive pediatric brain tumors and increased knowledge about the disease mechanisms is needed to develop new therapeutic approaches. Brain-specific Angiogenesis Inhibitor 1 (BAI1/ADGRB1) is an adhesion GPCR receptor and tumor suppressor epigenetically silenced in MB. Our prior studies showed that BAI1 can bind the MDM2 E3 ubiquitin ligase and relocate it to the cell membrane, thereby preventing it from degrading p53 in the nucleus (Zhu et al, Cancer Cell, 2018). Whether other MDM2 targets are affected by this relocation that may be functionally important in MB development is unknown. We hypothesized that BAI1-mediated relocation of MDM2 might destabilize cell surface oncogenic receptors and found that BAI1 stimulates IGF1R poly-ubiquitination and degradation. Mechanistically, BAI1 fosters MDM2-IGF1R interaction through beta-arrestins, adaptor proteins connecting MDM2 to IGF1R. Epigenetic reactivation of BAI1 expression suppressed Stat3 and Akt signaling, and radio-sensitized MB cells, leading to significantly improved survival in orthotopic MB xenograft models in mice regardless of tumor p53 mutation status. These findings are important as they demonstrate that BAI1 has dual anti-tumor effects in MB through MDM2 membrane re-localization, stabilizing p53 and blocking IGF1R signaling. They further suggest that epigenetic targeting of BAI1 is a promising therapeutic approach for clinical translation.

Reciprocal regulation between RACGAP1 and AR contributes to endocrine therapy resistance in prostate cancer

BackgroundEndocrine resistance driven by sustained activation of androgen receptor (AR) signaling pathway in advanced prostate cancer (PCa) is fatal. Characterization of mechanisms underlying aberrant AR pathway activation to search for potential therapeutic strategy is particularly important. Rac GTPase-activating protein 1 (RACGAP1) is one of the specific GTPase-activating proteins. As a novel tumor proto-oncogene, overexpression of RACGAP1 was related to the occurrence of various tumors.MethodsBioinformatics methods were used to analyze the relationship of expression level between RACGAP1 and AR as well as AR pathway activation. qRT-PCR and western blotting assays were performed to assess the expression of AR/AR-V7 and RACGAP1 in PCa cells. Immunoprecipitation and immunofluorescence experiments were conducted to detect the interaction and co-localization between RACGAP1 and AR/AR-V7. Gain- and loss-of-function analyses were conducted to investigate the biological roles of RACGAP1 in PCa cells, using MTS and colony formation assays. In vivo experiments were conducted to evaluate the effect of RACGAP1 inhibition on the tumor growth.ResultsRACGAP1 was a gene activated by AR, which was markedly upregulated in PCa patients with CRPC and enzalutamide resistance. AR transcriptionally activated RACGAP1 expression by binding to its promoter region. Reciprocally, nuclear RACGAP1 bound to the N-terminal domain (NTD) of both AR and AR-V7, blocking their interaction with the E3 ubiquitin ligase MDM2. Consequently, this prevented the degradation of AR/AR-V7 in a ubiquitin-proteasome-dependent pathway. Notably, the positive feedback loop between RACGAP1 and AR/AR-V7 contributed to endocrine therapy resistance of CRPC. Combination of enzalutamide and in vivo cholesterol-conjugated RIG-I siRNA drugs targeting RACGAP1 induced potent inhibition of xenograft tumor growth of PCa.ConclusionIn summary, our results reveal that reciprocal regulation between RACGAP1 and AR/AR-V7 contributes to the endocrine resistance in PCa. These findings highlight the therapeutic potential of combined RACGAP1 inhibition and enzalutamide in treatment of advanced PCa.

Sanggenol L induces ferroptosis in non-small cell lung cancer cells via regulating the miR-26a-1-3p/MDM2/p53 signaling pathway

Ferroptosis is a regulated cell death marked by iron-dependent lipid peroxidation. Tumor cells that survive by evading chemotherapy-induced apoptosis are vulnerable to ferroptosis. Therefore, it is particularly urgent to explore active ingredients that can selectively induce ferroptosis in cancer cells. Here, we revealed that sanggenol L, the active agent of Morus Bark, predisposed non-small cell lung cancer (NSCLC) cells to ferroptosis, evidenced by reactive oxygen species (ROS) accumulation, glutathione depletion, mitochondrial shrinkage, and lipid peroxidation. Furthermore, the ferroptosis-related miRNA array showed that sanggenol L treatment upregulated the level of miR-26a-1-3p, which directly targeted the E3 ubiquitin ligase MDM2. In addition, silencing MDM2 by miR-26a-1-3p resulted in a notable increase in p53 protein levels and decrease of its downstream target SLC7A11, ultimately triggered ferroptosis. The subcutaneous xenograft model and patient-derived tumor xenograft (PDX) model of NSCLC further confirmed the anti-tumor efficacy and safety of sanggenol L in vivo. Collectively, our data suggest that miR-26a-1-3p/MDM2/p53/SLC7A11 signaling axis plays a key role in sanggenol L-induced ferroptosis, which implies that sanggenol L can serves as an anticancer therapeutic arsenal for NSCLC.

OGA mutant aberrantly hydrolyzes O-GlcNAc modification from PDLIM7 to modulate p53 and cytoskeleton in promoting cancer cell malignancy

O-GlcNAcase (OGA) is the only human enzyme that catalyzes the hydrolysis (deglycosylation) of O-linked beta-N-acetylglucosaminylation (O-GlcNAcylation) from numerous protein substrates. OGA has broad implications in many challenging diseases including cancer. However, its role in cell malignancy remains mostly unclear. Here, we report that a cancer-derived point mutation on the OGA's noncatalytic stalk domain aberrantly modulates OGA interactome and substrate deglycosylation toward a specific set of proteins. Interestingly, our quantitative proteomic studies uncovered that the OGA stalk domain mutant preferentially deglycosylated protein substrates with +2 proline in the sequence relative to the O-GlcNAcylation site. One of the most dysregulated substrates is PDZ and LIM domain protein 7 (PDLIM7), which is associated with the tumor suppressor p53. We found that the aberrantly deglycosylated PDLIM7 suppressed p53 gene expression and accelerated p53 protein degradation by promoting the complex formation with E3 ubiquitin ligase MDM2. Moreover, deglycosylated PDLIM7 significantly up-regulated the actin-rich membrane protrusions on the cell surface, augmenting the cancer cell motility and aggressiveness. These findings revealed an important but previously unappreciated role of OGA's stalk domain in protein substrate recognition and functional modulation during malignant cell progression.

Abstract 1743: Pan-cancer review of TP53 somatic alterations as a function of tumor type: Sometimes lost, sometimes gained, frequently altered

Abstract Background: TP53 is the gene most frequently disrupted by somatic alterations in cancer. Mutations include single nucleotide variants resulting in functional alterations, as well as premature stop codons, splice variants, and cryptic alterations. TP53 gene dosage is disrupted by heterozygous and homozygous deletions, copy-neutral loss of heterozygosity, and other structural changes such as intragenic deletions and fusions. TP53 is altered by epigenetic events and viral oncogenes, such as E6 expressed by the human papillomavirus. The readout of altered TP53 includes decreased gene expression of either variant or wild-type alleles. Epistatic alteration of TP53 pathway components have also been described, with the E3 ubiquitin ligase MDM2 being the canonical example. Depending on the type of TP53 alteration, there may be differential signaling consequences, ranging from loss of function and dominant negative to oncogenic signaling. Hypothesis: Patterns of TP53 mutations will vary based on tumor type. Differential alterations of TP53 will suggest differential pathway dependence and downstream pathophysiology that may be relevant to signaling and therapeutic potential. Methods: We analyzed extensive cancer genomics repositories, such as the Pan-Cancer Atlas (10,000+ tumors, >30 cancer types) from The Cancer Genome Atlas project and the International Cancer Genome Consortium. Assessing genes in the TP53 pathway, we examined somatic mutations, copy number alterations, structural changes, and gene expression. Epigenetic data was considered when available. Results: A significant pattern amongst sarcomas, prostate cancers, and urothelial carcinomas emerged, revealing that certain tumors exhibited a predominant classic tumor suppressor gene genotype. This genotype featured frequent homozygous deletions, inactivating mutations, and relatively low TP53 gene expression, often accompanied by epistatic MDM2 amplification. By contrast, other tumors demonstrate an atypical pattern in which TP53 mutations were rarely if ever associated with loss of TP53 gene expression with low levels of homozygous TP53 gene deletion. Likewise, atypical TP53 mutant tumor types rarely demonstrated MDM2 amplifications, and in opposition to typical TP53 cases, MDM2 amplification was positively associated with TP53 mutation rather than anti-correlated. Head and neck squamous cell carcinoma, uterine carcinoma, and pancreatic adenocarcinoma followed this pattern. Investigation within selected tumor types such as lung squamous cell carcinoma suggested that both typical and atypical TP53 mutation patterns could be observed. Conclusions: TP53 mutation patterns were strongly associated with tumor type and may suggest differential pathway activation according to the somatic alteration status. Citation Format: Ubaid A. Tanveer, David N. Hayes, Julie George, Jeremiah R. Holt, Martin Peifer, Roman Thomas. Pan-cancer review of TP53 somatic alterations as a function of tumor type: Sometimes lost, sometimes gained, frequently altered [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1743.

Amyloid-β-induced dendritic spine elimination requires Ca2+-permeable AMPA receptors, AKAP-Calcineurin-NFAT signaling, and the NFAT target gene Mdm2.

Alzheimer's Disease (AD) is associated with brain accumulation of synaptotoxic amyloid-β (Aβ) peptides produced by the proteolytic processing of amyloid precursor protein (APP). Cognitive impairments associated with AD correlate with dendritic spine and excitatory synapse loss, particularly within the hippocampus. In rodents, soluble Aβ oligomers impair hippocampus-dependent learning and memory, promote dendritic spine loss, inhibit NMDA-type glutamate receptor (NMDAR)-dependent long-term potentiation (LTP), and promote synaptic depression (LTD), at least in part through activation of the Ca2+-CaM-dependent phosphatase calcineurin (CaN). Yet, questions remain regarding Aβ-dependent postsynaptic CaN signaling specifically at the synapse to mediate its synaptotoxicity. Here, we use pharmacologic and genetic approaches to demonstrate a role for postsynaptic signaling via A kinase-anchoring protein 150 (AKAP150)-scaffolded CaN in mediating Aβ-induced dendritic spine loss in hippocampal neurons from rats and mice of both sexes. In particular, we found that Ca2+-permeable AMPA-type glutamate receptors (CP-AMPARs), which were previously shown to signal through AKAP-anchored CaN to promote both LTD and Aβ-dependent inhibition of LTP, are also required upstream of AKAP-CaN signaling to mediate spine loss via overexpression of APP containing multiple mutations linked to familial, early-onset AD and increased Aβ production. In addition, we found that the CaN-dependent nuclear factor of activated T-cells (NFAT) transcription factors are required downstream to promote Aβ-mediated dendritic spine loss. Finally, we identified the E3-ubiquitin ligase Mdm2, which was previously linked to LTD and developmental synapse elimination, as a downstream NFAT target gene upregulated by Aβ whose enzymatic activity is required for Aβ-mediated spine loss.Significance Statement Impaired hippocampal function and synapse loss are hallmarks of AD linked to Aβ oligomers. Aβ exposure acutely blocks hippocampal LTP and enhances LTD and chronically leads to dendritic spine synapse loss. In particular, Aβ hijacks normal plasticity mechanisms, biasing them toward synapse weakening/elimination, with previous studies broadly linking CaN phosphatase signaling to this synaptic dysfunction. However, we do not understand how Aβ engages signaling specifically at synapses. Here we elucidate a synapse-to-nucleus signaling pathway coordinated by the postsynaptic scaffold protein AKAP150 that is activated by Ca2+ influx through CP-AMPARs and transduced to nucleus by CaN-NFAT signaling to transcriptionally upregulate the E3-ubiquitin ligase Mdm2 that is required for Aβ-mediated spine loss. These findings identify Mdm2 as potential therapeutic target for AD.

Gankyrin inhibits ferroptosis through the p53/SLC7A11/GPX4 axis in triple-negative breast cancer cells

Gankyrin is found in high levels in triple-negative breast cancer (TNBC) and has been established to form a complex with the E3 ubiquitin ligase MDM2 and p53, resulting in the degradation of p53 in hepatocarcinoma cells. Therefore, this study sought to determine whether gankyrin could inhibit ferroptosis through this mechanism in TNBC cells. The expression of gankyrin was investigated in relation to the prognosis of TNBC using bioinformatics. Co-immunoprecipitation and GST pull-down assays were then conducted to determine the presence of a gankyrin and MDM2 complex. RT-qPCR and immunoblotting were used to examine molecules related to ferroptosis, such as gankyrin, p53, MDM2, SLC7A11, and GPX4. Additionally, cell death was evaluated using flow cytometry detection of 7-AAD and a lactate dehydrogenase release assay, as well as lipid peroxide C11-BODIPY. Results showed that the expression of gankyrin is significantly higher in TNBC tissues and cell lines, and is associated with a poor prognosis for patients. Subsequent studies revealed that inhibiting gankyrin activity triggered ferroptosis in TNBC cells. Additionally, silencing gankyrin caused an increase in the expression of the p53 protein, without altering its mRNA expression. Co-immunoprecipitation and GST pull-down experiments indicated that gankyrin and MDM2 form a complex. In mouse embryonic fibroblasts lacking both MDM2 and p53, this gankyrin/MDM2 complex was observed to ubiquitinate p53, thus raising the expression of molecules inhibited by ferroptosis, such as SLC7A11 and GPX4. Furthermore, silencing gankyrin in TNBC cells disrupted the formation of the gankyrin/MDM2 complex, hindered the degradation of p53, increased SLC7A11 expression, impeded cysteine uptake, and decreased GPX4 production. Our findings suggest that TNBC cells are able to prevent cell ferroptosis through the gankyrin/p53/SLC7A11/GPX4 signaling pathway, indicating that gankyrin may be a useful biomarker for predicting TNBC prognosis or a potential therapeutic target.

METTL3-mediated N6-methyladenosine modification stimulates mitochondrial damage and ferroptosis of kidney tubular epithelial cells following acute kidney injury by modulating the stabilization of MDM2-p53-LMNB1 axis

N6-methyladenosine (m6A) and MELLT3 assume a role in the development of acute kidney injury (AKI). However, their mechanism in AKI remains under-explored. On this basis, this study explored the mechanism of MELLT3 in mitochondrial damage and ferroptosis of kidney tubular epithelial cells after AKI.HK-2 cells were induced by lipopolysaccharide (LPS) to simulate AKI, followed by gain and loss of function of genes, detection of mitochondrial damage and ferroptosis indicators, and analysis of gene interactions. An AKI mouse model was developed using the cecal ligation and puncture (CLP) method to investigate the effect of METTL3 knockdown on kidney injury.MDM2 and LMNB1 were upregulated and p53 was downregulated in LPS-treated HK-2 cells. Mechanistically, the E3 ubiquitin ligase MDM2 increased p53 ubiquitination to activate LMNB1. METTL3 knockdown decreased m6A methylation of MDM2, thus diminishing YTHDF1-mediated MDM2 mRNA stability and translation in LPS-treated HK-2 cells. Knockdown of LMNB1, MDM2, or METTL3 reduced NO, MDA, iron ion, and ROS levels as well as mitochondrial damage and raised SOD, GSH, XCT, GPX4, FPN1, and TFR1 levels in LPS-treated HK-2 cells. The in vivo results showed that METTL3 knockdown reduced renal injury and ferroptosis in CLP mice.METTL3 knockdown prevents mitochondrial damage and ferroptosis of kidney tubular epithelial cells after AKI via the MDM2-p53-LMNB1 axis.

GPCR targeting of E3 ubiquitin ligase MDM2 by inactive β-arrestin

E3 ubiquitin ligase Mdm2 facilitates β-arrestin ubiquitination, leading to the internalization of G protein-coupled receptors (GPCRs). In this process, β-arrestins bind to Mdm2 and recruit it to the receptor; however, the molecular architecture of the β-arrestin-Mdm2 complex has not been elucidated yet. Here, we identified the β-arrestin-binding region (ABR) on Mdm2 and solved the crystal structure of β-arrestin1 in complex with Mdm2ABR peptide. The acidic residues of Mdm2ABR bind to the positively charged concave side of the β-arrestin1 N-domain. The C-tail of β-arrestin1 is still bound to the N-domain, indicating that Mdm2 binds to the inactive state of β-arrestin1, whereas the phosphorylated C-terminal tail of GPCRs binds to activate β-arrestins. The overlapped binding site of Mdm2 and GPCR C-tails on β-arrestin1 suggests that the binding of GPCR C-tails might trigger the release of Mdm2. Moreover, hydrogen/deuterium exchange experiments further show that Mdm2ABR binding to β-arrestin1 induces the interdomain interface to be more dynamic and uncouples the IP6-induced oligomer of β-arrestin1. These results show how the E3 ligase, Mdm2, interacts with β-arrestins to promote the internalization of GPCRs.

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.

CHIP inhibits odontoblast differentiation through promoting DLX3 polyubiquitylation and degradation.

Dentin is the major hard tissue of teeth formed by differentiated odontoblasts. How odontoblast differentiation is regulated remains enigmatic. Here, we report that the E3 ubiquitin ligase CHIP is highly expressed in undifferentiated dental mesenchymal cells and downregulated after differentiation of odontoblasts. Ectopic expression of CHIP inhibits odontoblastic differentiation of mouse dental papilla cells, whereas knockdown of endogenous CHIP has opposite effects. Chip (Stub1) knockout mice display increased formation of dentin and enhanced expression of odontoblast differentiation markers. Mechanistically, CHIP interacts with and induces K63 polyubiquitylation of the transcription factor DLX3, leading to its proteasomal degradation. Knockdown of DLX3 reverses the enhanced odontoblastic differentiation caused by knockdown of CHIP. These results suggest that CHIP inhibits odontoblast differentiation by targeting its tooth-specific substrate DLX3. Furthermore, our results indicate that CHIP competes with another E3 ubiquitin ligase, MDM2, that promotes odontoblast differentiation by monoubiquitylating DLX3. Our findings suggest that the two E3 ubiquitin ligases CHIP and MDM2 reciprocally regulate DLX3 activity by catalyzing distinct types of ubiquitylation, and reveal an important mechanism by which differentiation of odontoblasts is delicately regulated by divergent post-translational modifications.

Screening of natural product libraries in MCF7 cell line reveals the pro-apoptotic properties of β tetralone

Despite the exponential increase in research toward better treatment options for breast cancer patients, developing an effective drug with fewer side effects continues to remain a challenge. Natural compounds have emerged as a viable option and several drugs have been derived or inspired from them. In this study, we screened a library of natural compounds with diverse chemical structures against selected kinase proteins using in silico methods such as molecular docking and dynamics simulation. The best results were obtained between β tetralone and MDM2 E3 ubiquitin ligase protein. In vitro experiments such as cytotoxicity, scratch assays and flow cytometry analysis using an MCF7 cell line were performed to determine the anti-cancer potential of the compound. As the treatment resulted in cell death and apoptosis, β tetralone was screened in silico against anti-apoptotic targets where the best results were obtained between Bcl-w and β tetralone. This comprehensive study suggests that the anti-cancer activity of β tetralone is probably through the dual targeting of MDM2 E3 ubiquitin kinase and Bcl-w anti-apoptotic protein. Communicated by Ramaswamy H. Sarma

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.

P90RSK Regulates p53 Pathway by MDM2 Phosphorylation in Thyroid Tumors

The expression level of the tumor suppressor p53 is controlled by the E3 ubiquitin ligase MDM2 with a regulatory feedback loop, which allows p53 to upregulate its inhibitor MDM2. In this manuscript we demonstrated that p90RSK binds and phosphorylates MDM2 on serine 166 both in vitro and in vivo by kinase assay, immunoblot, and co-immunoprecipitation assay; this phosphorylation increases the stability of MDM2 which in turn binds p53, ubiquitinating it and promoting its degradation by proteasome. A pharmacological inhibitor of p90RSK, BI-D1870, decreases MDM2 phosphorylation, and restores p53 function, which in turn transcriptionally increases the expression of cell cycle inhibitor p21 and of pro-apoptotic protein Bax and downregulates the anti-apoptotic protein Bcl-2, causing a block of cell proliferation, measured by a BrdU assay and growth curve, and promoting apoptosis, measured by a TUNEL assay. Finally, an immunohistochemistry evaluation of primary thyroid tumors, in which p90RSK is very active, confirms MDM2 stabilization mediated by p90RSK phosphorylation.

ZFP14 Regulates Cancer Cell Growth and Migration by Modulating p53 Protein Stability as Part of the MDM2 E3 Ubiquitin Ligase Complex.

Multi-zinc finger proteins that contain a KRAB domain are part of the biggest family of transcription factors in mammals. However, the physiological or pathological functions for the majority of them are unknown. Here, we showed that ZFP14 (also known as ZNF531) is a p53 target gene that can be induced upon genotoxic stress in a p53-dependent manner. To determine the function of ZFP14 in mouse and human cancer cell lines, we generated multiple cell lines where ZFP14 was knocked out. We showed that ZFP14-KO inhibits cancer cell growth and migration. We also showed that, as a target of p53, ZFP14, in turn, represses p53 expression and that the knockdown of p53 restores the potential of ZFP14-KO cells to proliferate and migrate. Mechanistically, we found that ZFP14 modulates p53 protein stability by increasing its ubiquitination via associating with and possibly enhancing MDM2/p53 complex integrity through its zinc finger domains. Our findings suggest that the reciprocal regulation of p53 and ZFP14 represents a novel p53-ZFP14 regulatory loop and that ZFP14 plays a role in p53-dependent tumor suppression.

GASP1 enhances malignant phenotypes of breast cancer cells and decreases their response to paclitaxel by forming a vicious cycle with IGF1/IGF1R signaling pathway

There is a potential correlation between G-protein-coupled receptor-associated sorting protein 1 (GASP1) and breast tumorigenesis. However, its biological function and underlying molecular mechanism in breast cancer have not been clearly delineated. Here, we demonstrated that GASP1 was highly expressed in breast cancers, and patients harboring altered GASP1 showed a worse prognosis than those with wild-type GASP1. Functional studies showed that GASP1 knockout significantly suppressed malignant properties of breast cancer cells, such as inhibition of cell proliferation, colony formation, migration, invasion and xenograft tumor growth in nude mice as well as induction of G1-phase cell cycle arrest, and vice versa. Mechanistically, GASP1 inhibited proteasomal degradation of insulin-like growth factor 1 receptor (IGF1R) by competitively binding to IGF1R with ubiquitin E3 ligase MDM2, thereby activating its downstream signaling pathways such as NF-κB, PI3K/AKT, and MAPK/ERK pathways given their critical roles in breast tumorigenesis and progression. IGF1, in turn, stimulated GASP1 expression by activating the PI3K/AKT pathway, forming a vicious cycle propelling the malignant progression of breast cancer. Besides, we found that GASP1 knockout obviously improved the response of breast cancer cells to paclitaxel. Collectively, this study demonstrates that GASP1 enhances malignant behaviors of breast cancer cells and decreases their cellular response to paclitaxel by interacting with and stabilizing IGF1R, and suggests that it may serve as a valuable prognostic factor and potential therapeutic target in breast cancer.

Corosolic acid sensitizes ferroptosis by upregulating HERPUD1 in liver cancer cells

Primary liver cancer is the third leading cause of cancer death in the world, and the lack of effective treatments is the main reason for the high mortality. Corosolic acid (CA) has been proved to have antitumor activity. In this study, we found that CA can sensitize liver cancer cells to ferroptosis, which is a regulated form of cell death characterized by iron-dependent lipid peroxides reaching lethal levels. Here, we revealed that CA can inhibit glutathione (GSH) synthesis via HERPUD1, decreasing the cellular GSH level and causing liver cancer cells to become more sensitive to ferroptosis. Mechanistically, further studies found that HERPUD1 reduced the ubiquitination of the GSS-associated E3 ubiquitin ligase MDM2, which promoted ubiquitination of GSS, thereby inhibiting GSH synthesis to increase ferroptosis susceptibility. Importantly, a mouse xenograft model also demonstrated that CA inhibits tumor growth via HERPUD1. Collectively, our findings suggesting that CA is a candidate component for the development of treatments against liver cancer.

Discovery of a Potent and Selective Degrader for USP7

The tumor suppressor p53 is the most frequently mutated gene in human cancer and more than half of cancers contain p53 mutations. The development of novel and effective therapeutic strategies for p53 mutant cancer therapy is a big challenge and highly desirable. Ubiquitin-specific protease 7 (USP7), also known as HAUSP, is a deubiquitinating enzyme and proposed to stabilize the oncogenic E3 ubiquitin ligase MDM2 that promotes the proteosomal degradation of p53. Herein, we report the design and characterization of U7D-1 as the first selective USP7-degrading Proteolysis Targeting Chimera (PROTAC). U7D-1 showed selective and effective USP7 degradation, and maintained potent cell growth inhibition in p53 mutant cancer cells, with USP7 inhibitor showing no activity. These data clearly demonstrated the practicality and importance of PROTAC as a preliminary chemical tool for investigating USP7 protein functions and a promising method for potential p53 mutant cancer therapy.

Discovery of a Potent and Selective Degrader for USP7.

The tumor suppressor p53 is the most frequently mutated gene in human cancer and more than half of cancers contain p53 mutations. The development of novel and effective therapeutic strategies for p53 mutant cancer therapy is a big challenge and highly desirable. Ubiquitin-specific protease 7 (USP7), also known as HAUSP, is a deubiquitinating enzyme and proposed to stabilize the oncogenic E3 ubiquitin ligase MDM2 that promotes the proteosomal degradation of p53. Herein, we report the design and characterization of U7D-1 as the first selective USP7-degrading Proteolysis Targeting Chimera (PROTAC). U7D-1 showed selective and effective USP7 degradation, and maintained potent cell growth inhibition in p53 mutant cancer cells, with USP7 inhibitor showing no activity. These data clearly demonstrated the practicality and importance of PROTAC as a preliminary chemical tool for investigating USP7 protein functions and a promising method for potential p53 mutant cancer therapy.

The Elephant Evolved p53 Isoforms that Escape MDM2-Mediated Repression and Cancer.

The p53 tumor suppressor is a transcription factor with roles in cell development, apoptosis, oncogenesis, aging, and homeostasis in response to stresses and infections. p53 is tightly regulated by the MDM2 E3 ubiquitin ligase. The p53–MDM2 pathway has coevolved, with MDM2 remaining largely conserved, whereas the TP53 gene morphed into various isoforms. Studies on prevertebrate ancestral homologs revealed the transition from an environmentally induced mechanism activating p53 to a tightly regulated system involving cell signaling. The evolution of this mechanism depends on structural changes in the interacting protein motifs. Elephants such as Loxodonta africana constitute ideal models to investigate this coevolution as they are large and long-living as well as having 20 copies of TP53 isoformic sequences expressing a variety of BOX-I MDM2-binding motifs. Collectively, these isoforms would enhance sensitivity to cellular stresses, such as DNA damage, presumably accounting for strong cancer defenses and other adaptations favoring healthy aging. Here we investigate the molecular evolution of the p53–MDM2 system by combining in silico modeling and in vitro assays to explore structural and functional aspects of p53 isoforms retaining the MDM2 interaction, whereas forming distinct pools of cell signaling. The methodology used demonstrates, for the first time that in silico docking simulations can be used to explore functional aspects of elephant p53 isoforms. Our observations elucidate structural and mechanistic aspects of p53 regulation, facilitate understanding of complex cell signaling, and suggest testable hypotheses of p53 evolution referencing Peto’s Paradox.