The goal of our study was to determine if elephant TP53 (EP53) proteins contributing to increased apoptosis and possible cancer resistance in elephants could translate into human cancer cells as a future effective cancer treatment. We previously reported that elephants have a lower than expected rate of cancer, 20 copies of TP53 (1 ancestral gene with introns [EP53-anc] and 19 retrogenes [EP53-retro1-19]), and increased p53-mediated apoptosis induced by DNA damage in elephant cells compared to human cells (Abegglen JAMA 2015). For the current study, we expressed various EP53 proteins in human cancer cells with different p53 status, including osteosarcoma (U2-OS, Saos-2), glioblastoma (T98G), and breast cancer (MCF7). Western blot analysis confirmed EP53 expression. We compared apoptosis in the human cancer cells transfected/transduced with negative control vectors vs. epitope or protein-tagged EP53 exposed to doxorubicin (to induce DNA damage). Apoptosis was measured by cell viability, caspase activity, Propidium Iodide/Annexin V staining, and fluorescence microscopy. We observed a significant increase in caspase activity (normalized to cell viability) of U2-OS and T98G cells expressing EP53 compared to negative control treated cells as shown in Table 1, and apoptosis with p21 restoration in Saos-2. In U2-OS, which overexpress MDM2, EP53 was more effective at inducing apoptosis compared to human TP53. Taken together, we found that EP53-anc restored p53-mediated apoptosis and EP53-anc / EP53-retro9 enhanced p53-mediated apoptosis. These data suggest for the first time that EP53 functions in human cancer cells to promote cell death. Ongoing efforts are exploring the EP53 mechanism of action that leads to increased apoptosis, including expression of EP53 in additional cancer types (lung, melanoma, colon, prostate, and others) with a variety of genetic backgrounds to characterize its functional context. These results support the further exploration of EP53-based cancer therapeutics.

Table 1:

Increase in apoptosis with EP53 expression relative to EP53 empty vector control cells

Cell Type TP53 Status EP53-anc EP53-retro9 Construct Assay Results No Treatment (fold difference) P-value 1uM doxorubicin (fold difference) P-value 
U-2 OS (osteosarcoma) WT Apoptosis Apoptosis myc-EP53-retro9 Increase in caspase relative to control 1.73 0.0091 1.79 1.7x10^-5 
    eGFP-EP53-retro9 Increase in caspase relative to control 1.68 6.7x10^-5 2.59 0.0041 
    eGFP-EP53-retro9 Percent decrease in GFP positive cells 23% percent decrease 4.8x10^-5 
    dyk-EP53-anc Increase in caspase relative to control 14.22 5.4x10^-7 3.31 1.0x10^-5 
    dyk-huTP53 Increase in caspase relative to control 10.9 1.2x10^-7 2.47 5.8x10^-5 
MCF7 (breast cancer) WT Apoptosis eGFP-EP53-retro9 Cell death by Propidium Iodide staining 0.15% GFP/surface area 0.011 
T98G (glioblastoma) MUT Apoptosis No Apoptosis dyk-EP53-anc Increase in caspase relative to control 3.07 7.9x10^-9 1.55 2.6x10^-5 
    myc-EP53-anc Increase in caspase relative to control 3.46 0.00015 1.52 6.8x10^-5 
    dyk-huTP53 Increase in caspase relative to control 2.82 1.2x10^-5 1.8 8.6x10^-5 
Cell Type TP53 Status EP53-anc EP53-retro9 Construct Assay Results No Treatment (fold difference) P-value 1uM doxorubicin (fold difference) P-value 
U-2 OS (osteosarcoma) WT Apoptosis Apoptosis myc-EP53-retro9 Increase in caspase relative to control 1.73 0.0091 1.79 1.7x10^-5 
    eGFP-EP53-retro9 Increase in caspase relative to control 1.68 6.7x10^-5 2.59 0.0041 
    eGFP-EP53-retro9 Percent decrease in GFP positive cells 23% percent decrease 4.8x10^-5 
    dyk-EP53-anc Increase in caspase relative to control 14.22 5.4x10^-7 3.31 1.0x10^-5 
    dyk-huTP53 Increase in caspase relative to control 10.9 1.2x10^-7 2.47 5.8x10^-5 
MCF7 (breast cancer) WT Apoptosis eGFP-EP53-retro9 Cell death by Propidium Iodide staining 0.15% GFP/surface area 0.011 
T98G (glioblastoma) MUT Apoptosis No Apoptosis dyk-EP53-anc Increase in caspase relative to control 3.07 7.9x10^-9 1.55 2.6x10^-5 
    myc-EP53-anc Increase in caspase relative to control 3.46 0.00015 1.52 6.8x10^-5 
    dyk-huTP53 Increase in caspase relative to control 2.82 1.2x10^-5 1.8 8.6x10^-5 

Citation Format: Lisa M. Abegglen, Lauren N. Donovan, Genevieve Couldwell, Rosann Robinson, Cristhian Toruno, Mor Goldfeder, Wendy K. Kiso, Dennis L. Schmitt, Aleah F. Caulin, Katrin P. Guillen, Bryan E. Welm, Carlo C. Maley, Avi Schroeder, Joshua D. Schiffman. Elephant p53 (EP53) expression induces apoptosis of human cancer cells [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 2153. doi:10.1158/1538-7445.AM2017-2153