P53-independent Role For Mdm2 Research Articles

Abstract 4449: Thrombospondin-1 regulation by MDM2 in aggressive cancers

Abstract MDM2 amplification, which is often seen in different cancer types, indicates that its plays an important role in tumorigenesis and also in cancer metastasis. Amplification of the MDM2 gene correlates with poor prognosis, aggressive growth, and reoccurrence. Thrombospondin-1 (TSP-1) that is expressed by THBS1 gene is a matricellular protein, and is known to have differential expression in cancers. When TSP-1 activates Transforming Growth Factor-β1 (TGF-β1) through latent cytokine complexation, the activated TGF-β1 contributes to the Epithelial to Mesenchymal Transition (EMT) which is known to impart stem-cell like characteristics and drug resistance to the cancer cells. However, no studies regarding MDM2’s role in THBS1 regulation have been carried out so far. Therefore, the objective of this study was to determine the correlation between MDM2 and THBS1 expression in MDM2 overexpressing cancers. For this purpose the SJSA-1 osteosarcoma cells were cultured at 37 oC under humidified air/CO2 (19:1) in RPMI-1640 complete medium supplemented with 10% FBS, 10,000 U/mL penicillin, 10,000 µg/mL streptomycin, 1% (+)-L-glutamine, and 1% amphotericin B. The cells were incubated in the presence of 20 µM of Nutlin-3 for 24 hrs, following which, RNA and protein were extracted and subsequently purity and concentration were determined. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and western blotting were performed using specific primers, primary and the secondary antibodies respectively. Interestingly elevated levels of TSP-1 and TGFβ1 were observed in these SJSA-1 cells. However, after treatment with Nutlin-3, an MDM2-p53 interaction inhibitor, a significant increase in p53 was noted. However, contrary to the existing literature, where studies have shown p53 to positively regulate TSP-1 levels, we observed an opposite outcome. Despite the Nutlin-3 induced elevation in p53 levels, a significant decrease in TSP-1 level was observed indicating that MDM2 may be regulating THBS1 gene expression in a p53 independent manner, in MDM2 overexpressing SJSA-1 cells. The findings of this study suggest a novel, p53 independent role for MDM2 in THBS1 regulation. Since MDM2 amplification can positively induce TSP-1 levels, which is one of the natural activators of TGF-β1, it is anticipated that stimulatory signal flowing through MDM2 - TSP-1 - TGF-β1 axis may induce the cancer cells to undergo EMT, thereby increasing their metastatic ability and promote cancer stem cell characteristics. This novel MDM2-regulated pathway can very well serve as a drug target and might play a biologically relevant role in the treatment of cancer metastasis. Our future studies will include validation of this correlation in multiple cancer cell lines, to further elucidate the mechanism through which MDM2 is regulating THBS1 gene expression. (This project was supported by The Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida). Citation Format: Priya Dondapati, Jason Maragh, Karna Mangrola, Ali Alaseem, Khadija Cheema, Thiagarajan Venkatesan, Sivanesan Dhandayuthapani, Appu Rathinavelu. Thrombospondin-1 regulation by MDM2 in aggressive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4449. doi:10.1158/1538-7445.AM2017-4449

Erratum to: A p53-independent role of Mdm2 in estrogen-mediated activation of breast cancer cell proliferation

Following publication of our article, the authors noticed that the data incorporated into Figure 4f was incorrect. The first column should be changed from 44, 13 and 46 to read 43, 13 and 44 instead. A corrected version of Figure 4f can be found overleaf.

Mdm2-mediated ubiquitylation: p53 and beyond

The really interesting genes (RING)-finger-containing oncoprotein, Mdm2, is a promising drug target for cancer therapy. A key Mdm2 function is to promote ubiquitylation and proteasomal-dependent degradation of the tumor suppressor protein p53. Recent reports provide novel important insights into Mdm2-mediated regulation of p53 and how the physical and functional interactions between these two proteins are regulated. Moreover, a p53-independent role of Mdm2 has recently been confirmed by genetic data. These advances and their potential implications for the development of new cancer therapeutic strategies form the focus of this review.

Regulation of XIAP Translation and Induction by MDM2 following Irradiation

Increases in protein levels of XIAP in cancer cells have been associated with resistance to apoptosis induced by cellular stress. Herein we demonstrate that the upregulation of XIAP protein levels is regulated by MDM2 at the translational level. MDM2 was found to physically interact with the IRES of the XIAP 5'-UTR, and to positively regulate XIAP IRES activity. This XIAP IRES-dependent translation was significantly increased in MDM2-transfected cells where MDM2 accumulated in the cytoplasm. Cellular stress and DNA damage triggered by irradiation induced the dephosphorylation and cytoplasmic localization of MDM2, which also led to an increase in IRES-dependent XIAP translation. Upregulation of XIAP in MDM2-overexpressing cancer cells in response to irradiation resulted in resistance of these cells to radiation-induced apoptosis.

Mdm2 exerts pro-apoptotic activities by antagonizing insulin-like growth factor-I-mediated survival

The Mdm2 oncoprotein is an E3 ubiquitin ligase required to maintain the p53 protein at low levels in embryonic and adult tissues. It also contributes to tumor formation by antagonizing p53 tumor suppressor activity when amplified and/or overexpressed. Importantly, p53-independent role for Mdm2 has been suggested by transfection studies. Among the growing list of putative Mdm2-regulated proteins are several proteins playing a key role in the control of cell proliferation such as pRb, E2F1/DP1, Numb, Smads, Lats2 or IGF-1R. Consistent with the ability of Mdm2 to promote ubiquitylation and proteasome destruction of IGFR-I independently of p53, we show herein that loss of Mdm2 leads to a significant increase in IGF1-R-β protein levels both in cells lacking or expressing p53. Interestingly, IGF-1 protects cells from DNA-damage-induced apoptosis only in absence of Mdm2. These data therefore further highlight a physiological role for Mdm2 in the control of IGF1 signalling and provide genetic evidence for a p53-independent proapoptotic function of Mdm2. The consequences of these findings for the development of p53-Mdm2-targeted anti-cancer therapies are discussed.

Mdm2-mediated pRB downregulation is involved in carcinogenesis in a p53-independent manner

Mdm2 promotes ubiquitination of the tumor suppressor p53 and can function as an oncogene by largely downregulating p53. Although a p53-independent role of Mdm2 has been reported, the underlying mechanism remains unclear. In the present study, we indicated that Mdm2 is involved in p53-independent carcinogenesis via downregulation of pRB. Expression of pRB showed an apparent inverse correlation with Mdm2 expression in 30 patients with non-small cell lung cancer. There were some cases with the p53 mutations in which a high level of Mdm2 and a low level of pRB were expressed. Mdm2 promoted ubiquitination of pRB in cells without wild-type p53. Furthermore, pRB-mediated G1 arrest in a p53-deficient cell line, SRB1, was significantly enhanced by a mutant Mdm2 that lacks pRB ubiquitination activity. Soft-agar colony formation activity of p53-knockout MEF was increased by wild-type Mdm2 but not mutant Mdm2. These findings suggest that overexpression of Mdm2 can perturb a RB pathway regardless of the p53 gene status, promoting carcinogenesis.

MDM2 induces NF-κB/p65 expression transcriptionally through Sp1-binding sites: a novel, p53-independent role of MDM2 in doxorubicin resistance in acute lymphoblastic leukemia

AbstractMDM2 protein is thought to exhibit tumorigenic activity by binding to the p53 tumor-suppressor protein and inhibiting its function. Alternatively, MDM2 may have oncogenic roles other than those resulting from p53 interactions. Here we report that MDM2 can induce expression of the p65 subunit of NF-κB, which is an anti-apoptotic factor expressed in certain neoplastic cells in response to chemotherapy. Initially, we noted that the overexpression of MDM2 protein in leukemic bone marrow cells of patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), and an ALL cell line (EU-4) transfected with theMDM2 gene was associated with elevated expression of p65 and in vitro resistance to doxorubicin (Adriamycin). By cotransfection of the MDM2 gene and p65-promoter-reporter constructs into EU-4 cells, we found that transient and high-level MDM2 expression induced p65 promoter activity. In the presence of wild-type (wt) p53, MDM2 increased p65 promoter activity by reversing p53-mediated suppression of p65. In the absence of p53, MDM2 directly increased p65 promoter activity. Deletion and mutation analysis of the p65 promoter indicated that the region between nt −575 and −178, which contains the first and second Sp1-binding sites, was required for activation by MDM2. Further studies using chromatin immunoprecipitation (CHIP) and electrophoretic mobility shift assay (EMSA) showed that MDM2 was able to directly bind to the Sp1 site of the p65 promoter. Our findings suggest that by inducing p65 expression, MDM2 has a p53-independent role in tumorigenesis, which may further elucidate the association between MDM2 overexpression and resistant disease in childhood ALL.