Browsing by Subject "Mdm2"

Now showing 1 - 8 of 8
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    Early-Stage Metastasis Requires Mdm2 and Not p53 Gain of Function
    (AACR, 2017-11) Hauck, Paula M.; Wolf, Eric R.; Olivos, David J., III; Batuello, Christopher N.; McElyea, Kyle C.; McAtarsney, Ciaran; Cournoyer, R. Michael; Sandusky, George E.; Mayo, Lindsey D.; Pediatrics, School of Medicine
    Metastasis of cancer cells to distant organ systems is a complex process that is initiated with the programming of cells in the primary tumor. The formation of distant metastatic foci is correlated with poor prognosis and limited effective treatment options. We and others have correlated Mouse double minute 2 (Mdm2) with metastasis; however, the mechanisms involved have not been elucidated. Here, it is reported that shRNA-mediated silencing of Mdm2 inhibits epithelial–mesenchymal transition (EMT) and cell migration. In vivo analysis demonstrates that silencing Mdm2 in both post-EMT and basal/triple-negative breast cancers resulted in decreased primary tumor vasculature, circulating tumor cells, and metastatic lung foci. Combined, these results demonstrate the importance of Mdm2 in orchestrating the initial stages of migration and metastasis.
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    Emerging Non-Canonical Functions and Regulation by p53: p53 and Stemness
    (MDPI, 2016-11-26) Olivos III, David J.; Mayo, Lindsey D.; Department of Microbiology & Immunology, IU School of Medicine
    Since its discovery nearly 40 years ago, p53 has ascended to the forefront of investigated genes and proteins across diverse research disciplines and is recognized most exclusively for its role in cancer as a tumor suppressor. Levine and Oren (2009) reviewed the evolution of p53 detailing the significant discoveries of each decade since its first report in 1979. In this review, we will highlight the emerging non-canonical functions and regulation of p53 in stem cells. We will focus on general themes shared among p53's functions in non-malignant stem cells and cancer stem-like cells (CSCs) and the influence of p53 on the microenvironment and CSC niche. We will also examine p53 gain of function (GOF) roles in stemness. Mutant p53 (mutp53) GOFs that lead to survival, drug resistance and colonization are reviewed in the context of the acquisition of advantageous transformation processes, such as differentiation and dedifferentiation, epithelial-to-mesenchymal transition (EMT) and stem cell senescence and quiescence. Finally, we will conclude with therapeutic strategies that restore wild-type p53 (wtp53) function in cancer and CSCs, including RING finger E3 ligases and CSC maintenance. The mechanisms by which wtp53 and mutp53 influence stemness in non-malignant stem cells and CSCs or tumor-initiating cells (TICs) are poorly understood thus far. Further elucidation of p53's effects on stemness could lead to novel therapeutic strategies in cancer research.
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    The fate of murine double minute X (MdmX) is dictated by distinct signaling pathways through murine double minute 2 (Mdm2)
    (Impact Journals, 2017-11-06) Hauck, Paula M.; Wolf, Eric R.; Olivos, David J.; McAtarsney, Ciaran P.; Mayo, Lindsey D.; Pediatrics, School of Medicine
    Mouse double minute 2 (Mdm2) and MdmX dimerize in response to low levels of genotoxic stress to function in a ubiquitinating complex, which signals for destabilization of p53. Under growth conditions, Mdm2 functions as a neddylating ligase, but the importance and extent of MdmX involvement in this process are largely unknown. Here we show that when Mdm2 functions as a neddylating enzyme, MdmX is stabilized. Furthermore, we demonstrate that under growth conditions, MdmX enhances the neddylation activity of Mdm2 on p53 and is a substrate for neddylation itself. Importantly, MdmX knockdown in MCF-7 breast cancer cells resulted in diminished neddylated p53, suggesting that MdmX is important for Mdm2-mediated neddylation. Supporting this finding, the lack of MdmX in transient assays or in p53/MdmX-/- MEFs results in decreased or altered neddylation of p53 respectively; therefore, MdmX is a critical component of the Mdm2-mediated neddylating complex. c-Src is the upstream activator of this Mdm2-MdmX neddylating pathway and loss of Src signaling leads to the destabilization of MdmX that is dependent on the RING (Really Interesting New Gene) domain of MdmX. Treatment with a small molecule inhibitor of neddylation, MLN4924, results in the activation of Ataxia Telangiectasia Mutated (ATM). ATM phosphorylates Mdm2, converting Mdm2 to a ubiquitinating enzyme which leads to the destabilization of MdmX. These data show how distinct signaling pathways engage neddylating or ubiquitinating activities and impact the Mdm2-MdmX axis.
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    Mdm2 mediated neddylation of pVHL blocks the induction of anti-angiogenic factors.
    (Springer Nature, 2020-07) Wolf, Eric R.; Mabry, Alexander R.; Damania, Blossom; Mayo, Lindsey D.; Biochemistry and Molecular Biology, School of Medicine
    Mutations in the tumor suppressor TP53 are rare in renal cell carcinomas. p53 is a key factor for inducing anti-angiogenic genes and RCC are highly vascularized, which suggests that p53 is inactive in these tumors. One regulator of p53 is the Mdm2 oncogene, which is correlated with high-grade, metastatic tumors. However, the sole activity of Mdm2 is not just to regulate p53, but it can also function independent of p53 to regulate the early stages of metastasis. Here, we report that the oncoprotein Mdm2 can bind directly to the tumor suppressor VHL, and conjugate nedd8 to VHL within a region that is important for the p53-VHL interaction. Nedd8 conjugated VHL is unable to bind to p53 thereby preventing the induction of anti-angiogenic factors. These results highlight a previously unknown oncogenic function of Mdm2 during the progression of cancer to promote angiogenesis through the regulation of VHL. Thus, the Mdm2-VHL interaction represents a pathway that impacts tumor angiogenesis.
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    Modulation of the Mdm2 signaling axis sensitizes triple-negative breast cancer cells to carboplatin
    (2014-12) Tonsing-Carter, Eva Y.; Pollok, Karen E.; Zhang, Jian-Ting; Safa, Ahmad R.; Mayo, Lindsey D.; Travers, Jeffrey B.
    Triple-negative breast cancers (TNBCs) are highly refractive to current treatment strategies, and new multi-targeted treatments need to be elucidated. Combination therapy that includes targeting the murine double minute 2 (Mdm2) signaling axis offers a promising approach. Protein-protein interaction inhibitors such as Nutlin-3a block the binding of key signaling molecules such as p53, p73α, and E2F1 to the hydrophobic pocket of Mdm2 and can lead to activation of cell-death signaling pathways. Since clinical trials for TNBC are evaluating the DNA damaging agent carboplatin, the objective of this thesis was to evaluate the therapeutic potential and mechanism of action of combination carboplatin and Nutlin-3a to treat TNBC. In TNBC cell lines with a mutant p53 background, we determined if modulation of Mdm2 function in the context of carboplatin-mediated DNA damage resulted in a synergistic inhibition of cell growth. Several ratios of carboplatin:Nutlin-3a were strongly synergistic in increasing cell death, with combination indices of 0.5 and lower. Mechanistic studies indicated that drug sensitivity and Mdm2 expression were dependent on p73. Mdm2 localized to a larger degree in the chromatin fraction isolated from cells treated with the combination treatment consistent with observations by others that Mdm2 binds to the Mre11/Rad50/Nbs1 complex, inhibits the DNA damage response, and increases drug sensitivity. In vivo efficacy experiments were conducted in the TMD231 orthotopic mammary fat pad model in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. For assessment of baseline tumor burden and randomization, fluorescent imaging of E2-Crimson expressing TMD231 cells was performed. Following Nutlin-3a and carboplatin combination treatment, there was a statistically significant reduction in primary tumor volume as well as lung metastases with significantly increased probability of survival compared to Vehicle and single drug treatments (p<0.001). While there was a decrease in bone-marrow cellularity, this did not lead to bone-marrow aplasia, and body weights recovered to normal levels within 7 days post-treatment. The present studies demonstrate the promise of Mdm2 as a therapeutic target in combination with conventional therapy, increase our understanding of how to potentiate DNA damage in cancers, and may lead to new clinical therapies for triple-negative primary and metastatic breast cancer.
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    Phospho-regulation and metastatic potential of Murine Double Minute 2
    (2012-08) Batuello, Christopher N.; Mayo, Lindsey D.; Dynlacht, Joseph R.; Goebl, Mark G.; Pollok, Karen E.
    Murine double minute (Mdm2) is a highly modified and multi-faceted protein that is overexpressed in numerous human malignancies. It engages in many cellular activities and is essential for development since deletion of mdm2 is lethal in early stages of embryonic development. The most studied function of Mdm2 is as a negative regulator of the tumor suppressor protein p53. Mdm2 achieves this regulation by binding to p53 and inhibiting p53 transcriptional activity. Mdm2 also functions as an E3 ubiquitin ligase that signals p53 for destruction by the proteasome. Interestingly recent evidence has shown that Mdm2 can also function as an E3 neddylating enzyme that can conjugate the ubiquitin-like molecule, nedd8, to p53. This modification results in inhibition of p53 activity, while maintaining p53 protein levels. While the signaling events that regulate Mdm2 E3 ubiquitin ligase activity have been extensively studied, what activates the neddylating activity of Mdm2 has remained elusive. My investigations have centered on understanding whether tyrosine kinase signaling could activate the neddylating activity of Mdm2. I have shown that c-Src, a non-receptor protein tyrosine kinase that is involved in a variety of cellular processes, phosphorylates Mdm2 on tyrosines 281 and 302. This phosphorylation event increases the half-life and neddylating activity of Mdm2 resulting in a neddylation dependent reduction of p53 transcriptional activity. Mdm2 also has many p53-independent cellular functions that are beginning to be linked to its role as an oncogene. There is an emerging role for Mdm2 in tumor metastasis. Metastasis is a process involving tumor cells migrating from a primary site to a distal site and is a major cause of morbidity and mortality in cancer patients. To date, the involvement of Mdm2 in breast cancer metastasis has only been correlative, with no in vivo model to definitively define a role for Mdm2. Here I have shown in vivo that Mdm2 enhances breast to lung metastasis through the up regulation of multiple angiogenic factors, including HIF-1 alpha and VEGF. Taken together my data provide novel insights into important p53-dependent and independent functions of Mdm2 that represent potential new avenues for therapeutic intervention.
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    Potentiation of Carboplatin-Mediated DNA Damage by the Mdm2 Modulator Nutlin-3a in a Humanized Orthotopic Breast-to-Lung Metastatic Model
    (American Association for Cancer Research, 2015-12) Tonsing-Carter, Eva; Bailey, Barbara J.; Saadatzadeh, M. Reza; Ding, Jixin; Wang, Haiyan; Sinn, Anthony L.; Peterman, Kacie M.; Spragins, Tiaishia K.; Silver, Jayne M.; Sprouse, Alyssa A.; Georgiadis, Taxiarchis M.; Gunter, T. Zachary; Long, Eric C.; Minto, Robert E.; Marchal, Christophe C.; Batuello, Christopher N.; Safa, Ahmad R.; Hanenberg, Helmut; Territo, Paul R.; Sandusky, George E.; Mayo, Lindsey D.; Eischen, Christine M.; Shannon, Harlan E.; Pollok, Karen E.; Department of Pharmacology and Toxicology, IU School of Medicine
    Triple-negative breast cancers (TNBC) are typically resistant to treatment, and strategies that build upon frontline therapy are needed. Targeting the murine double minute 2 (Mdm2) protein is an attractive approach, as Mdm2 levels are elevated in many therapy-refractive breast cancers. The Mdm2 protein-protein interaction inhibitor Nutlin-3a blocks the binding of Mdm2 to key signaling molecules such as p53 and p73α and can result in activation of cell death signaling pathways. In the present study, the therapeutic potential of carboplatin and Nutlin-3a to treat TNBC was investigated, as carboplatin is under evaluation in clinical trials for TNBC. In mutant p53 TMD231 TNBC cells, carboplatin and Nutlin-3a led to increased Mdm2 and was strongly synergistic in promoting cell death in vitro. Furthermore, sensitivity of TNBC cells to combination treatment was dependent on p73α. Following combination treatment, γH2AX increased and Mdm2 localized to a larger degree to chromatin compared with single-agent treatment, consistent with previous observations that Mdm2 binds to the Mre11/Rad50/Nbs1 complex associated with DNA and inhibits the DNA damage response. In vivo efficacy studies were conducted in the TMD231 orthotopic mammary fat pad model in NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (NSG) mice. Using an intermittent dosing schedule of combined carboplatin and Nutlin-3a, there was a significant reduction in primary tumor growth and lung metastases compared with vehicle and single-agent treatments. In addition, there was minimal toxicity to the bone marrow and normal tissues. These studies demonstrate that Mdm2 holds promise as a therapeutic target in combination with conventional therapy and may lead to new clinical therapies for TNBC.
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    Src phosphorylation converts Mdm2 from a ubiquitinating to a neddylating E3 ligase
    (PNAS, 2015-02-10) Batuello, Christopher N.; Hauck, Paula M.; Gendron, Jaimie M.; Lehman, Jason A.; Mayo, Lindsey D.; Department of Biochemistry & Molecular Biology, IU School of Medicine
    Murine double minute-2 protein (Mdm2) is a multifaceted phosphorylated protein that plays a role in regulating numerous proteins including the tumor suppressor protein p53. Mdm2 binds to and is involved in conjugating either ubiquitin or Nedd8 (Neural precursor cell expressed, developmentally down-regulated 8) to p53. Although regulation of the E3 ubiquitin activity of Mdm2 has been investigated, regulation of the neddylating activity of Mdm2 remains to be defined. Here we show that activated c-Src kinase phosphorylates Y281 and Y302 of Mdm2, resulting in an increase in Mdm2 stability and its association with Ubc12, the E2 enzyme of the neddylating complex. Mdm2-dependent Nedd8 conjugation of p53 results in transcriptionally inactive p53, a process that is reversed with a small molecule inhibitor to either Src or Ubc12. Thus, our studies reveal how Mdm2 may neutralize and elevate p53 in actively proliferating cells and also provides a rationale for using therapies that target the Nedd8 pathway in wild-type p53 tumors.