Phospho-regulation and metastatic potential of Murine Double Minute 2

Indiana University-Purdue University Indianapolis (IUPUI)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

Role of Ape1 and Base Excision Repair in the Radiation Response and Heat-radiosensitization of HeLa Cells

Background: The mechanism by which heat sensitizes mammalian cells to ionizing radiation remains to be elucidated. We determined whether base excision repair (BER) is involved in heat-radiosensitization and report novel findings that provide insight regarding the role of BER in the radiation response of HeLa cells. Materials and Methods: An siRNA approach was utilized to suppress expression of AP endonuclease (Ape1), a critical enzyme of BER. Clonogenic survival curves were obtained for HeLa cells expressing normal or reduced Ape1 content and which had been irradiated, and these were compared to survival curves from cells that were irradiated prior to hyperthermia treatment. Results: The amount of heat-radiosensitization observed in Ape1-suppressed cells was similar to or slightly greater than that observed in cells expressing near-normal levels of Ape1. Interestingly, we also found that for unheated HeLa cells, suppressed expression of Ape1 resulted in enhanced resistance to X-rays. Conclusion: The data suggest that Ape1, and therefore BER, is not involved in heat-radiosensitization. However, the observation that suppressed expression of Ape1 results in enhanced radioresistance supports the notion that BER may be detrimental to the survival of irradiated cells

Src phosphorylation converts Mdm2 from a ubiquitinating to a neddylating E3 ligase

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

Early-Stage Metastasis Requires Mdm2 and Not p53 Gain of Function

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

Potentiation of Carboplatin-Mediated DNA Damage by the Mdm2 Modulator Nutlin-3a in a Humanized Orthotopic Breast-to-Lung Metastatic Model

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

Controlling the Mdm2-Mdmx-p53 Circuit

pharmaceutical

Serdemetan Antagonizes the Mdm2-HIF1α Axis Leading to Decreased Levels of Glycolytic Enzymes

Serdemetan (JNJ-26854165), an antagonist to Mdm2, was anticipated to promote the activation of p53. While regulation of p53 by Mdm2 is important, Mdm2 also regulates numerous proteins involved in diverse cellular functions. We investigated if Serdemetan would alter the Mdm2-HIF1α axis and affect cell survival in human glioblastoma cells independently of p53. Treatment of cells with Serdemetan under hypoxia resulted in a decrease in HIF1α levels. HIF1α downstream targets, VEGF and the glycolytic enzymes (enolase, phosphoglycerate kinase1/2, and glucose transporter 1), were all decreased in response to Serdemetan. The involvement of Mdm2 in regulating gene expression of glycolytic enzymes raises the possibility of side effects associated with therapeutically targeting Mdm2