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