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Cancer-relevant mutations in the oligomerization domain (OD) of the p53 tumor suppressor protein, unlike those in the DNA binding domain, have not been well elucidated. Here, we characterized the germline OD mutant p53(A347D), which occurs in cancer-prone Li–Fraumeni syndrome (LFS) patients. Unlike wild-type p53, mutant p53(A347D) cannot form tetramers and exists as a hyperstable dimeric protein. Further, p53(A347D) cannot bind or transactivate the majority of canonical p53 target genes. Isogenic cell lines harboring either p53(A347D) or no p53 yield comparable tumorigenic properties, yet p53(A347D) displays remarkable neomorphic activities. Cells bearing p53(A347D) possess a distinct transcriptional profile and undergo metabolic reprogramming. Further, p53(A347D) induces striking mitochondrial network aberration and associates with mitochondria to drive apoptotic cell death upon topoisomerase II inhibition in the absence of transcription. Thus, dimer-forming p53 demonstrates both loss-of-function (LOF) and gain-of-function (GOF) properties compared with the wild-type form of the protein. Significance: A mutant p53 (A347D), which can only form dimers, is associated with increased cancer susceptibility in LFS individuals. We found that this mutant wields a double-edged sword, driving tumorigenesis through LOF while gaining enhanced apoptogenic activity as a new GOF, thereby yielding a potential vulnerability to select therapeutic approaches. See related commentary by Stieg et al., p. 1046. See related article by Gencel-Augusto et al., p. 1230. This article is highlighted in the In This Issue feature, p. 1027

Glutamine synthase 2 (GLS2) is a key regulator of glutaminolysis and has been previously implicated in activities consistent with tumor suppression. Here we generated Gls2 knockout (KO) mice that develop late-occurring B-cell lymphomas and hepatocellular carcinomas (HCC). Further, Gls2 KO mice subjected to the hepatocarcinogenic Stelic Animal Model (STAM) protocol produce larger HCC tumors than seen in wild-type (WT) mice. GLS2 has been shown to promote ferroptosis, a form of cell death characterized by iron-dependent accumulation of lipid peroxides. In line with this, GLS2 deficiency, either in cells derived from Gls2 KO mice or in human cancer cells depleted of GLS2, conferred significant resistance to ferroptosis. Mechanistically, GLS2, but not GLS1, increased lipid reactive oxygen species (ROS) production by facilitating the conversion of glutamate to α-ketoglutarate (αKG), thereby promoting ferroptosis. Ectopic expression of WT GLS2 in a human hepatic adenocarcinoma xenograft model significantly reduced tumor size; this effect was nullified by either expressing a catalytically inactive form of GLS2 or by blocking ferroptosis. Furthermore, analysis of cancer patient datasets supported a role for GLS2-mediated regulation of ferroptosis in human tumor suppression. These data suggest that GLS2 is a bona fide tumor suppressor and that its ability to favor ferroptosis by regulating glutaminolysis contributes to its tumor suppressive function. Significance: This study demonstrates that the key regulator of glutaminolysis, GLS2, can limit HCC in vivo by promoting ferroptosis through αKG-dependent lipid ROS, which in turn might lay the foundation for a novel therapeutic approach.

SummaryAlthough 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 are independent of p53. For example, 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 metastasisin vivo. Remarkably, Mdm2 modulates the expression of Sprouty4, which is required for the Mdm2 mediated effects on cell migration, focal adhesion formation and metastasis. Our findings describe a molecular mechanism by which the Mdm2-X complex, through Sprouty4, regulates cellular processes leading to decreased metastatic capability independent of p53.

Supplementary Figures 1-9 demonstrating the GOF and LOF activities of p53(A347D)

Differentially expressed genes in U2OS cells varying in p53 allelic status combined from two independent RNA library preparations

p53 ChIPseq peaks from U2OS cells varying in p53 allelic status

p53 ChIPseq peaks from osteoblasts derived from normal and LFS individuals

Characteristics of LFS patients' fibroblasts and derivatives

Cancer-relevant mutations in the oligomerization domain (OD) of the p53 tumor suppressor protein, unlike those in the DNA binding domain, have not been well elucidated. Here, we characterized the germline OD mutant p53(A347D), which occurs in cancer-prone Li–Fraumeni syndrome (LFS) patients. Unlike wild-type p53, mutant p53(A347D) cannot form tetramers and exists as a hyperstable dimeric protein. Further, p53(A347D) cannot bind or transactivate the majority of canonical p53 target genes. Isogenic cell lines harboring either p53(A347D) or no p53 yield comparable tumorigenic properties, yet p53(A347D) displays remarkable neomorphic activities. Cells bearing p53(A347D) possess a distinct transcriptional profile and undergo metabolic reprogramming. Further, p53(A347D) induces striking mitochondrial network aberration and associates with mitochondria to drive apoptotic cell death upon topoisomerase II inhibition in the absence of transcription. Thus, dimer-forming p53 demonstrates both loss-of-function (LOF) and gain-of-function (GOF) properties compared with the wild-type form of the protein.Significance:A mutant p53 (A347D), which can only form dimers, is associated with increased cancer susceptibility in LFS individuals. We found that this mutant wields a double-edged sword, driving tumorigenesis through LOF while gaining enhanced apoptogenic activity as a new GOF, thereby yielding a potential vulnerability to select therapeutic approaches.See related commentary by Stieg et al., p. 1046.See related article by Gencel-Augusto et al., p. 1230.This article is highlighted in the In This Issue feature, p. 1027

Differentially expressed genes in U2OS cells varying in p53 allelic status

p53 ChIPseq peaks from U2OS cells varying in p53 allelic status

Summary of GOF vs. LOF activities of p53(A347D) in U2OS cells

This file contain Supplementary Information that is relevant to the main body of the text. Supplementary Figure S1. Supplementary Figure S2. Half-lives of WT and mutant I332fs*14 p53 in U2OS and HCT116 cells. Supplementary Figure S3. Subcellular localization of p53 FS mutants expressed in U2OS and HCT116 cells. Supplementary Figure S4. Cross-linked species detected in cells grown in the absence or presence of Photo-Met. Supplementary Figure S5. Transcriptional activity of p53 FS mutants in U2OS and HCT116 cells. Supplementary Figure S6. Transactivation potential of N345K* and N345fs*26 mutants in U2OS cells. Supplementary Figure S7. Cell cycle profiles of I332fs*14-expressing HCT116 and U2OS cells. Supplementary Figure S8. Pro-apoptotic activity of I332fs*14 p53.

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p53 mutations that result in loss of transcriptional activity are commonly found in numerous types of cancer. While the majority of these are missense mutations that map within the central DNA-binding domain, truncations and/or frameshift mutations can also occur due to various nucleotide substitutions, insertions, or deletions. These changes result in mRNAs containing premature stop codons that are translated into a diverse group of C-terminally truncated proteins. Here we characterized three p53 frameshift mutant proteins expressed from the endogenous TP53 locus in U2OS osteosarcoma and HCT116 colorectal cancer cell lines. These mutants retain intact DNA-binding domains but display altered oligomerization properties. Despite their abnormally high expression levels, they are mostly transcriptionally inactive and unable to initiate a stimuli-induced transcriptional program characteristic of wild-type p53. However, one of these variant p53 proteins, I332fs*14, which resembles naturally expressed TAp53 isoforms β and γ, retains some residual antiproliferative activity and can induce cellular senescence in HCT116 cells. Cells expressing this mutant also display decreased motility in migration assays. Hence, this p53 variant exhibits a combination of loss-of-gain and gain-of-function characteristics, distinguishing it from both wild type p53 and p53 loss.Implications:p53 frameshift mutants display a mixture of residual antiproliferative and neomorphic functions that may be differentially exploited for targeted therapy.

Supplementary Data from GLS2 Is a Tumor Suppressor and a Regulator of Ferroptosis in Hepatocellular Carcinoma

Supplementary Figure 1. STXBP4 expression is correlated with poor prognosis in patients with tumors expressing high ΔNp63 levels. Supplementary Figure 2. Causal networks associated with STXBP4 expression in lung SCC. Supplementary Figure 3. Depletion of PDGFRα suppresses the growth of lung SCC. Supplementary Figure 4. PDGFRA mRNA was significantly correlated with STXBP4 in the gene expression profiles of lung SCC patients. Supplementary Figure 5. PDGFRA expression is upregulated in STXBP4-transduced lung SCC cells. Supplementary Figure 6. STXBP4-depletion inhibits SCC tumorigenesis and modulates PDGF signaling in vivo. Supplementary Figure 7. STXBP4 promotes tumorigenesis through PDGFRA in lung SCC.

Purpose: Expression of the ΔN isoform of p63 (ΔNp63) is a diagnostic marker highly specific for lung squamous cell carcinoma (SCC). We previously found that Syntaxin Binding Protein 4 (STXBP4) regulates ΔNp63 ubiquitination, suggesting that STXBP4 may also be an SCC biomarker. To address this issue, we investigated the role of STXBP4 expression in SCC biology and the impact of STXBP4 expression on SCC prognosis.Experimental Design: We carried out a clinicopathologic analysis of STXBP4 expression in 87 lung SCC patients. Whole transcriptome analysis using RNA-seq was performed in STXBP4-positive and STXBP4-negative tumors of lung SCC. Soft-agar assay and xenograft assay were performed using overexpressing or knockdown SCC cells.Results: Significantly higher levels of STXBP4 expression were correlated with accumulations of ΔNp63 in clinical lung SCC specimens (Spearman rank correlation ρ = 0.219). Notably, STXBP4-positive tumors correlated with three important clinical parameters: T factor (P < 0.001), disease stage (P = 0.030), and pleural involvement (P = 0.028). Whole transcriptome sequencing followed by pathway analysis indicated that STXBP4 is involved in functional gene networks that regulate cell growth, proliferation, cell death, and survival in cancer. Platelet-derived growth factor receptor alpha (PDGFRα) was a key downstream mediator of STXBP4 function. In line with this, shRNA mediated STXBP4 and PDGFRA knockdown suppressed tumor growth in soft-agar and xenograft assays.Conclusions: STXBP4 plays a crucial role in driving SCC growth and is an independent prognostic factor for predicting worse outcome in lung SCC. These data suggest that STXBP4 is a relevant therapeutic target for patients with lung SCC. Clin Cancer Res; 23(13); 3442–52. ©2017 AACR.

Supplementary Information and Supplementary Figures Legends

The p63 transcription factor, a member of the p53 family, plays an oncogenic role in squamous cancers, while in breast cancers its expression is often repressed. In the canonical conserved Hippo pathway, known to play a complex role in regulating growth of cancer cells, the protein kinases MST1/2 and LATS1/2 act sequentially to phosphorylate and inhibit the YAP/TAZ transcription factors. We found that in the MCF10A mammary epithelial cell line as well as in squamous and breast cancer cell lines, expression of ΔNp63 RNA and protein is strongly repressed by inhibition of the Hippo pathway protein kinases in a manner that is independent of p53. While MST1/2 and LATS1 are required for p63 expression, the next step of the pathway, namely phosphorylation and degradation of the YAP/TAZ transcriptional activators is not required for repression of p63. This suggests that regulation of p63 expression occurs by a non-canonical version of the Hippo pathway. We additionally identified additional genes that were similarly regulated suggesting the broader importance of this pathway. Interestingly, we observed that experimentally lowering p63 expression leads to increased YAP protein levels, thereby constituting a feedback loop. These results, which reveal the intersection of the Hippo and p63 pathways, may prove useful for the control of their activities in cancer cells.One Sentence SummaryRegulation of p63 expression occurs by a non-canonical version of the Hippo pathway in mammary epithelial, breast carcinoma and head and neck squamous carcinoma cells

We recently documented that gain-of-function (GOF) mutant p53 (mtp53) R273H in triple negative breast cancer (TNBC) cells interacts with replicating DNA and PARP1. The missense R273H GOF mtp53 has a mutated central DNA binding domain that renders it unable to bind specifically to DNA, but maintains the capacity to interact tightly with chromatin. Both the C-terminal domain (CTD) and oligomerization domain (OD) of GOF mtp53 proteins are intact and it is unclear whether these regions of mtp53 are responsible for chromatin-based DNA replication activities. We generated MDA-MB-468 cells with CRISPR-Cas9 edited versions of the CTD and OD regions of mtp53 R273H. These included a frame-shift mtp53 R273Hfs387, which depleted mtp53 protein expression; mtp53 R273HΔ381-388, which had a small deletion within the CTD; and mtp53 R273HΔ347-393, which had both the OD and CTD regions truncated. The mtp53 R273HΔ347-393 existed exclusively as monomers and disrupted the chromatin interaction of mtp53 R273H. The CRISPR variants proliferated more slowly than the parental cells and mt53 R273Hfs387 showed the most extreme phenotype. We uncovered that after thymidine-induced G1/S synchronization, but not hydroxyurea or aphidicholin, R273Hfs387 cells displayed impairment of S-phase progression while both R273HΔ347-393 and R273HΔ381-388 displayed only moderate impairment. Moreover, reduced chromatin interaction of MCM2 and PCNA in mtp53 depleted R273Hfs387 cells post thymidine-synchronization revealed delayed kinetics of replisome assembly underscoring the slow S-phase progression. Taken together our findings show that the CTD and OD domains of mtp53 R273H play critical roles in mutant p53 GOF that pertain to processes associated with DNA replication.

p53 mutations that result in loss of transcriptional activity are commonly found in numerous types of cancer. While the majority of these are missense mutations that map within the central DNA-binding domain, truncations and/or frameshift mutations can also occur due to various nucleotide substitutions, insertions, or deletions. These changes result in mRNAs containing premature stop codons that are translated into a diverse group of C-terminally truncated proteins. Here we characterized three p53 frameshift mutant proteins expressed from the endogenous TP53 locus in U2OS osteosarcoma and HCT116 colorectal cancer cell lines. These mutants retain intact DNA-binding domains but display altered oligomerization properties. Despite their abnormally high expression levels, they are mostly transcriptionally inactive and unable to initiate a stimuli-induced transcriptional program characteristic of wild-type p53. However, one of these variant p53 proteins, I332fs*14, which resembles naturally expressed TAp53 isoforms β and γ, retains some residual antiproliferative activity and can induce cellular senescence in HCT116 cells. Cells expressing this mutant also display decreased motility in migration assays. Hence, this p53 variant exhibits a combination of loss-of-gain and gain-of-function characteristics, distinguishing it from both wild type p53 and p53 loss. Implications: p53 frameshift mutants display a mixture of residual antiproliferative and neomorphic functions that may be differentially exploited for targeted therapy.

Most well studied as proteins that restrain the p53 tumor suppressor protein, MDM2 and MDMX have rich lives outside of their relationship to p53. There is much to learn about how these two proteins are regulated and how they can function in cells that lack p53. Regulation of MDM2 and MDMX, which takes place at the level of transcription, post-transcription, and protein modification, can be very intricate and is context-dependent. Equally complex are the myriad roles that these two proteins play in cells that lack wild-type p53; while many of these independent outcomes are consistent with oncogenic transformation, in some settings their functions could also be tumor suppressive. Since numerous small molecules that affect MDM2 and MDMX have been developed for therapeutic outcomes, most if not all designed to prevent their restraint of p53, it will be essential to understand how these diverse molecules might affect the p53-independent activities of MDM2 and MDMX.

Mdm2 and its homologue MdmX form an E3-ligase complex that is best understood as the major regulator of p53. Yet Mdm2 and MdmX have functions in cells that are independent of their ability to degrade p53. Amongst the functions regulated by Mdm2 is cell migration, although the molecular mechanism(s) involved have not been well characterized. We show, in a p53 null model, that either siRNA knockdown of Mdm2 or MdmX as well as pharmacological inhibition of the Mdm2/X complex E3-ligase can reduce migration of cells grown as monolayer or invasion of cells from pre-formed spheroids into collagen-based matrices. This is consistent with our observation that Mdm2 ablation or inhibition leads to decreased cell spreading and attachment of cells to the extracellular matrix. In line with these findings, we found that modulation of Mdm2, MdmX or the Mdm2/X complex impacts focal adhesion (FA) formation, a main step in cell attachment, spreading and migration. Physiologically, Mdm2 silencing leads to decreased metastatic burden in mouse models. Mechanistically, we have discovered that Mdm2 modulates the RNA levels of Sprouty4 and that Sprouty4 is needed for the effects of Mdm2 knockdown on cell migration, FA formation and metastasis. Taken together, we have discovered a pathway by which Mdm2, through the activity of the Mdm2/X complex, mitigates FA formation, migration and ultimately metastasis by regulation of Sprouty4 independently of p53. Our findings suggest that blocking Mdm2 or the Mdm2/X complex might be a potential target to prevent metastasis. Citation Format: Rafaela Muniz de Queiroz, Gizem Efe, Asja Guzman, Naoko Hashimoto, Yusuke Kawashima, Tomoaki Tanaka, Anil K. Rustgi, Carol Prives. Mdm2 regulates metastasis and associated cellular processes through modulation of Sprouty4 in a p53-independent manner [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 LB160.

The protein product of the CDKN1A gene, p21, has been extensively characterized as a negative regulator of the cell cycle. Nevertheless, it is clear that p21 has manifold complex and context-dependent roles that can be either tumor suppressive or oncogenic. Most well studied as a transcriptional target of the p53 tumor suppressor protein, there are other means by which p21 levels can be regulated. In this study, we show that pharmacological inhibition or siRNA-mediated reduction of O-GlcNAc transferase (OGT), the enzyme responsible for glycosylation of intracellular proteins, increases expression of p21 in both p53-dependent and p53-independent manners in nontransformed and cancer cells. In cells harboring WT p53, we demonstrate that inhibition of OGT leads to p53-mediated transactivation of CDKN1A, while in cells that do not express p53, inhibiting OGT leads to increased p21 protein stabilization. p21 is normally degraded by the ubiquitin-proteasome system following ubiquitination by, among others, the E3 ligase Skp-Cullin-F-box complex; however, in this case, we show that blocking OGT causes impairment of the Skp-Cullin-F-box ubiquitin complex as a result of disruption of the FoxM1 transcription factor-mediated induction of Skp2 expression. In either setting, we conclude that p21 levels induced by OGT inhibition correlate with cell cycle arrest and decreased cancer cell proliferation.

MDM2 and MDMX, negative regulators of the tumor suppressor p53, can work separately and as a heteromeric complex to restrain p53's functions. MDM2 also has pro-oncogenic roles in cells, tissues, and animals that are independent of p53. There is less information available about p53-independent roles of MDMX or the MDM2–MDMX complex. We found that MDM2 and MDMX facilitate ferroptosis in cells with or without p53. Using small molecules, RNA interference reagents, and mutant forms of MDMX, we found that MDM2 and MDMX, likely working in part as a complex, normally facilitate ferroptotic death. We observed that MDM2 and MDMX alter the lipid profile of cells to favor ferroptosis. Inhibition of MDM2 or MDMX leads to increased levels of FSP1 protein and a consequent increase in the levels of coenzyme Q10, an endogenous lipophilic antioxidant. This suggests that MDM2 and MDMX normally prevent cells from mounting an adequate defense against lipid peroxidation and thereby promote ferroptosis. Moreover, we found that PPARα activity is essential for MDM2 and MDMX to promote ferroptosis, suggesting that the MDM2–MDMX complex regulates lipids through altering PPARα activity. These findings reveal the complexity of cellular responses to MDM2 and MDMX and suggest that MDM2–MDMX inhibition might be useful for preventing degenerative diseases involving ferroptosis. Furthermore, they suggest that MDM2/MDMX amplification may predict sensitivity of some cancers to ferroptosis inducers.

The p53 tumor suppressor protein, known to be critically important in several processes including cell-cycle arrest and apoptosis, is highly regulated by multiple mechanisms, most certifiably the Murine Double Minute 2–Murine Double Minute X (MDM2–MDMX) heterodimer. The role of MDM2–MDMX in cell-cycle regulation through inhibition of p53 has been well established. Here we report that in cells either lacking p53 or expressing certain tumor-derived mutant forms of p53, loss of endogenous MDM2 or MDMX, or inhibition of E3 ligase activity of the heterocomplex, causes cell-cycle arrest. This arrest is correlated with a reduction in E2F1, E2F3, and p73 levels. Remarkably, direct ablation of endogenous p73 produces a similar effect on the cell cycle and the expression of certain E2F family members at both protein and messenger RNA levels. These data suggest that MDM2 and MDMX, working at least in part as a heterocomplex, may play a p53-independent role in maintaining cell-cycle progression by promoting the activity of E2F family members as well as p73, making them a potential target of interest in cancers lacking wild-type p53.

The p53 field is complex and evolving. Yet the basic challenges of this field remain the same: how does wild-type p53 prevent cancer and how do mutant forms of p53 promote the very same disease? This essay only touches on some aspects of the research that has approached these two questions and we apologize to those whose work has not been cited. What everyone must agree on is that the findings of Arnold Levine and colleagues have created and enriched one of the most lively and challenging fields in cancer research today.

Traditionally, the p21 protein has been viewed as limiting cancer progression and promoting aging. In contrast, there are reports that p21 can enhance cancer survival and limit tissue damage, depending on the tissue of origin and type of stressor involved. Here, we provide evidence to support these latter two roles of p21 by exploring its ability to regulate ferroptosis. Ferroptosis is a form of cell death that is associated with certain degenerative diseases, some of which are aging-related. Our results reveal a correlation between p21 protein levels in cell lines that are resistant to ferroptosis (p21 high) versus cell lines that are sensitive and easily undergo ferroptosis (p21 low). We also show that p21 levels themselves are differentially regulated in response to ferroptosis in a p53-independent manner. Further, experimentally altering the abundance of p21 protein inverts the ferroptosis-sensitivity of both resistant and sensitive human cancer cell lines. Our data also indicate that the interaction of p21 with CDKs is crucial for its ability to restrict the progression of ferroptosis. While this study was performed in cancer cell lines, our results support the potential of p21 to aid in maintenance of healthy tissues by blocking the damage incurred due to ferroptosis.

Next generation sequencing enables large-scale analysis of mRNA expression (RNA-seq), genome variance (whole genome or exome), and transcription factor binding (ChIP-seq). Here we describe a method that allows the identification of transcription factor-binding sites in the vicinity of nonprotein-coding genes.

Next generation sequencing enables large-scale analysis of mRNA expression (RNA-seq), genome variance (whole genome or exome), and transcription factor binding (ChIP-seq). Here we describe a method that allows the identification of transcription factor-binding sites in the vicinity of nonprotein-coding genes.

Significance One-tenth of human genes produce proteins called transcription factors (TFs) that bind to our genome and read the local DNA sequence. They work together to regulate the degree to which each gene is expressed. The affinity with which DNA is bound by a particular TF can vary more than a thousand-fold with different DNA sequences. This study presents the first computational method able to quantify the sequence-affinity relationship almost perfectly over the full affinity range. It achieves this by analyzing data from experiments that use massively parallel DNA sequencing to comprehensively probe protein–DNA interactions. Strikingly, it can accurately predict the effect in vivo of DNA mutations on gene expression levels in fly embryos even for very-low-affinity binding sites., Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes.

Zinc deficiency has been linked to human diseases, including cancer. MDMX, a crucial zinc-containing negative regulator of p53, has been found to be amplified or overexpressed in various cancers and implicated in the cancer initiation and progression. We report here that zinc depletion by the ion chelator TPEN or Chelex resin results in MDMX protein degradation in a ubiquitination-independent and 20S proteasome-dependent manner. Restoration of zinc led to recovery of cellular levels of MDMX. Further, TPEN treatment inhibits growth of the MCF-7 breast cancer cell line, which is partially rescued by overexpression of MDMX. Moreover, in a mass-spectrometry-based proteomics analysis, we identified TRPM7, a zinc-permeable ion channel, as a novel MDMX-interacting protein. TRPM7 stabilizes and induces the appearance of faster migrating species of MDMX on SDS-PAGE. Depletion of TRPM7 attenuates, while TRPM7 overexpression facilitates, the recovery of MDMX levels upon adding back zinc to TPEN-treated cells. Importantly, we found that TRPM7 inhibition, like TPEN treatment, decreases breast cancer cell MCF-7 proliferation and migration. The inhibitory effect on cell migration upon TRPM7 inhibition is also partially rescued by overexpression of MDMX. Together, our data indicate that TRPM7 regulates cellular levels of MDMX in part by modulating the intracellular Zn2+ concentration to promote tumorigenesis.

The tumor suppressor p53, a master regulator of the cellular response to stress, is tightly regulated by the E3 ubiquitin ligase MDM2 via an autoregulatory feedback loop. In addition to its well-established role in tumorigenesis, p53 has also been associated with aging in mice. Several mouse models with aberrantly increased p53 activity display signs of premature aging. However, the relationship between dysfunction of the MDM2/p53 axis and human aging remains elusive. Here, we have identified an antiterminating homozygous germline mutation in MDM2 in a patient affected by a segmental progeroid syndrome. We show that this mutation abrogates MDM2 activity, thereby resulting in enhanced levels and stability of p53. Analysis of the patient's primary cells, genome-edited cells, and in vitro and in vivo analyses confirmed the MDM2 mutation's aberrant regulation of p53 activity. Functional data from a zebrafish model further demonstrated that mutant Mdm2 was unable to rescue a p53-induced apoptotic phenotype. Altogether, our findings indicate that mutant MDM2 is a likely driver of the observed segmental form of progeria.

Purpose: Expression of the ΔN isoform of p63 (ΔNp63) is a diagnostic marker highly specific for lung squamous cell carcinoma (SCC). We previously found that Syntaxin Binding Protein 4 (STXBP4) regulates ΔNp63 ubiquitination, suggesting that STXBP4 may also be an SCC biomarker. To address this issue, we investigated the role of STXBP4 expression in SCC biology and the impact of STXBP4 expression on SCC prognosis. Experimental Design: We carried out a clinicopathologic analysis of STXBP4 expression in 87 lung SCC patients. Whole transcriptome analysis using RNA-seq was performed in STXBP4-positive and STXBP4-negative tumors of lung SCC. Soft-agar assay and xenograft assay were performed using overexpressing or knockdown SCC cells. Results: Significantly higher levels of STXBP4 expression were correlated with accumulations of ΔNp63 in clinical lung SCC specimens (Spearman rank correlation ρ = 0.219). Notably, STXBP4-positive tumors correlated with three important clinical parameters: T factor (P < 0.001), disease stage (P = 0.030), and pleural involvement (P = 0.028). Whole transcriptome sequencing followed by pathway analysis indicated that STXBP4 is involved in functional gene networks that regulate cell growth, proliferation, cell death, and survival in cancer. Platelet-derived growth factor receptor alpha (PDGFRα) was a key downstream mediator of STXBP4 function. In line with this, shRNA mediated STXBP4 and PDGFRA knockdown suppressed tumor growth in soft-agar and xenograft assays. Conclusions: STXBP4 plays a crucial role in driving SCC growth and is an independent prognostic factor for predicting worse outcome in lung SCC. These data suggest that STXBP4 is a relevant therapeutic target for patients with lung SCC. Clin Cancer Res; 23(13); 3442–52. ©2017 AACR.

Although DNA modifications play an important role in gene regulation, the underlying mechanisms remain elusive. We developed EpiSELEX-seq to probe the sensitivity of transcription factor binding to DNA modification in vitro using massively parallel sequencing. Feature-based modeling quantifies the effect of cytosine methylation (5mC) on binding free energy in a position-specific manner. Application to the human bZIP proteins ATF4 and C/EBPβ and three different Pbx-Hox complexes shows that 5mCpG can both increase and decrease affinity, depending on where the modification occurs within the protein-DNA interface. The TF paralogs tested vary in their methylation sensitivity, for which we provide a structural rationale. We show that 5mCpG can also enhance in vitro p53 binding and provide evidence for increased in vivo p53 occupancy at methylated binding sites, correlating with primed enhancer histone marks. Our results establish a powerful strategy for dissecting the epigenomic modulation of protein-DNA interactions and their role in gene regulation.

The p53 tumor suppressor is a transcription factor (TF) that exerts antitumor functions through its ability to regulate the expression of multiple genes. Within the p53 protein resides a relatively short unstructured C-terminal domain (CTD) that remarkably participates in virtually every aspect of p53 performance as a TF. Because these aspects are often interdependent and it is not always possible to dissect them experimentally, there has been a great deal of controversy about the CTD. In this review we evaluate the significance and key features of this interesting region of p53 and its impact on the many aspects of p53 function in light of previous and more recent findings.

We recently documented that gain-of-function (GOF) mutant p53 (mtp53) R273H in triple negative breast cancer (TNBC) cells interacts with replicating DNA and PARP1 (Xiao et. al., Cancer Research 2019). The missense R273H GOF mtp53, without a functional central DNA binding domain, has the capacity to interact tightly with chromatin but it is unclear what regions of mtp53 are responsible for the chromatin-based DNA replication activities. GOF mtp53 proteins possess an intact C-terminal domain (CTD) and oligomerization domain (OD). In wild-type p53 the CTD and OD domain regulate non-specific DNA binding, post-translational modification, and protein tetramerization. We are targeting the CTD and OD regions of mtp53 to test if their disruption limits oncogenic GOF activity. We generated CRISPR/Cas9 edited R273H CTD and OD in the TNBC cell line MDA-MB-468, and also exogenously expressed such dual mutant proteins in p53 null cancers, to evaluate the roles played by oligomerization and non-specific DNA binding in mtp53 interactions with replicating DNA and PARP1. We determined the oligomerization state of R273H mtp53 using the glutaraldehyde crosslinking assay and observed that GOF mtp53 R273H mtp53 in breast cancer cells exists as a tetramer. Experiments are in progress to evaluate cell cycle progression, replication initiation, and mtp53 chromatin interactions in cells expressing missense mtp53 proteins with co-associated mutated CTDs or ODs. Our preliminary data suggests that disrupting the mtp53 CTD inhibits its interactions with chromatin and progression through the S-phase. Unpublished data on the roles played by the mtp53 CTD and OD in DNA replication will be presented at the 2020 AACR meeting. Acknowledgements: This work was funded by grants from the Breast Cancer Research Foundation BCRF-18-011 and BCRF-19-011 to Jill Bargonetti. Devon Lundine and Gu Xiao also received project support from TUFCCC/HC Regional Comprehensive Cancer Health Disparity Partnership, Award Number U54 CA221704(5) from the National Cancer Institute. Special thanks to Ella Freulich for help with CRISPR/Cas9 work and James J. Manfredi for mutant p53 plasmids to exogenously express protein. Citation Format: Devon Lundine, George Annor, Viola Ellison, Gu Xiao, Carol Prives, Jill Bargonetti. Targeting gain-of-function of mutant p53 C-terminal and oligomerization domains to disrupt activated DNA replication in breast cancer [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 3429.

The relative contribution of intrinsic genetic factors and extrinsic environmental ones to cancer aetiology and natural history is a lengthy and debated issue. Gene–environment interactions (G x E) arise when the combined presence of both a germline genetic variant and a known environmental factor modulates the risk of disease more than either one alone. A panel of experts discussed our current understanding of cancer aetiology, known examples of G × E interactions in cancer, and the expanded concept of G × E interactions to include somatic cancer mutations and iatrogenic environmental factors such as anti-cancer treatment. Specific genetic polymorphisms and genetic mutations increase susceptibility to certain carcinogens and may be targeted in the near future for prevention and treatment of cancer patients with novel molecularly based therapies. There was general consensus that a better understanding of the complexity and numerosity of G × E interactions, supported by adequate technological, epidemiological, modelling and statistical resources, will further promote our understanding of cancer and lead to novel preventive and therapeutic approaches.

Mutant p53 impacts the expression of numerous genes at the level of transcription to mediate oncogenesis. We identified vascular endothelial growth factor receptor 2 (VEGFR2), the primary functional VEGF receptor that mediates endothelial cell vascularization, as a mutant p53 transcriptional target in multiple breast cancer cell lines. Up-regulation of VEGFR2 mediates the role of mutant p53 in increasing cellular growth in two-dimensional (2D) and three-dimensional (3D) culture conditions. Mutant p53 binds near the VEGFR2 promoter transcriptional start site and plays a role in maintaining an open conformation at that location. Relatedly, mutant p53 interacts with the SWI/SNF complex, which is required for remodeling the VEGFR2 promoter. By both querying individual genes regulated by mutant p53 and performing RNA sequencing, the results indicate that >40% of all mutant p53-regulated gene expression is mediated by SWI/SNF. We surmise that mutant p53 impacts transcription of VEGFR2 as well as myriad other genes by promoter remodeling through interaction with and likely regulation of the SWI/SNF chromatin remodeling complex. Therefore, not only might mutant p53-expressing tumors be susceptible to anti VEGF therapies, impacting SWI/SNF tumor suppressor function in mutant p53 tumors may also have therapeutic potential.

DNA binding by numerous transcription factors including the p53 tumor suppressor protein constitutes a vital early step in transcriptional activation. While the role of the central core DNA binding domain (DBD) of p53 in site-specific DNA binding has been established, the contribution of the sequence-independent C-terminal domain (CTD) is still not well understood. We investigated the DNA-binding properties of a series of p53 CTD variants using a combination of in vitro biochemical analyses and in vivo binding experiments. Our results provide several unanticipated and interconnected findings. First, the CTD enables DNA binding in a sequence-dependent manner that is drastically altered by either its modification or deletion. Second, dependence on the CTD correlates with the extent to which the p53 binding site deviates from the canonical consensus sequence. Finally, the CTD enables stable formation of p53-DNA complexes to divergent binding sites via DNA-induced conformational changes within the DBD itself.

It is well established that mutant forms of the p53 tumour suppressor acquire pro-oncogenic activities. Inhibition of the mevalonate pathway is now shown to promote degradation of select oncogenic mutant p53 proteins, indicating that destabilization of mutant p53 could be a promising therapeutic strategy.

In response to varying stress signals, the p53 tumor suppressor is able to promote repair, survival, or elimination of damaged cells - processes that have great relevance to organismal aging. Although the link between p53 and cancer is well established, the contribution of p53 to the aging process is less clear. Delineating how p53 regulates distinct aging hallmarks such as cellular senescence, genomic instability, mitochondrial dysfunction, and altered metabolic pathways will be critical. Mouse models have further revealed the centrality and complexity of the p53 network in aging processes. While naturally aged mice have linked longevity with declining p53 function, some accelerated aging mice present with chronic p53 activation, whose phenotypes can be rescued upon p53 deficiency. Further, direct modulation of the p53-MDM2 axis has correlated elevated p53 activity with either early aging or with delayed-onset aging. We speculate that p53-mediated aging phenotypes in these mice must have (1) stably active p53 due to MDM2 dysregulation or chronic stress or (2) shifted p53 outcomes. Pinpointing which p53 stressors, modifications, and outcomes drive aging processes will provide further insights into our understanding of the human aging process and could have implications for both cancer and aging therapeutics.

The p53 tumor suppressor protein is the most well studied as a regulator of transcription in the nucleus, where it exists primarily as a tetramer. However, there are other oligomeric states of p53 that are relevant to its regulation and activities. In unstressed cells, p53 is normally held in check by MDM2 that targets p53 for transcriptional repression, proteasomal degradation, and cytoplasmic localization. Here we discovered a hydrophobic region within the MDM2 N-terminal domain that binds exclusively to the dimeric form of the p53 C-terminal domain in vitro. In cell-based assays, MDM2 exhibits superior binding to, hyperdegradation of, and increased nuclear exclusion of dimeric p53 when compared with tetrameric wild-type p53. Correspondingly, impairing the hydrophobicity of the newly identified N-terminal MDM2 region leads to p53 stabilization. Interestingly, we found that dimeric mutant p53 is partially unfolded and is a target for ubiquitin-independent degradation by the 20S proteasome. Finally, forcing certain tumor-derived mutant forms of p53 into dimer configuration results in hyperdegradation of mutant p53 and inhibition of p53-mediated cancer cell migration. Gaining insight into different oligomeric forms of p53 may provide novel approaches to cancer therapy.