Temporary knockdown of p53 during focal limb irradiation increases the development of sarcomas

Abstract: Background: Approximately half of all cancer patients receive radiotherapy and, as cancer survivorship rates increase with more effective therapies, the very low rate of radiation-associated sarcomas is rising. Radiation-associated sarcomas are life-threatening cancers, and radiation exposure is a primary risk factor for sarcoma development. During radiotherapy or other genotoxic cancer therapy for p53 mutant cancers, pharmacological inhibition of p53 has been proposed to ameliorate acute injury of normal tissues. However, enhancing the survival of normal cells that sustain DNA damage by temporarily inhibiting p53 has the potential to increase the risk of cancer development. Here, we use in vivo shRNA technology to examine the consequences of temporarily reducing p53 expression on radiation-induced sarcoma development. Methods: We utilized a mouse model of radiation-induced sarcoma where mice express a doxycycline (dox)-inducible p53 shRNA to temporarily and reversibly reduce p53 expression. Mice were placed on a dox diet 10 days prior to receiving 30 or 40 Gy hind limb irradiation in a single fraction and then returned to normal chow. Mice were examined weekly for sarcoma development and scored for radiation-induced normal tissue injuries. Radiation-induced sarcomas were harvested and subjected to RNA sequencing. Results: Following single high-dose irradiation, 21% of temporary p53 knockdown animals developed a sarcoma in the radiation field compared to 2% of control animals. Mice with more severe acute injuries in the first 3 months after irradiation had a significantly increased risk of developing late persistent wounds in the soft tissue and bone. Chronic radiation-induced wounds were associated with sarcomagenesis. Examination of muscle stem cells by flow cytometry following hind limb irradiation indicated p53 knockdown preserves muscle stem cells in the irradiated limb, supporting the notion that temporary p53 knockdown at the time of irradiation reduces death of cells with DNA damage which may then persist to develop into a sarcoma. We performed RNA sequencing on 16 radiation-induced sarcomas compared to normal muscle controls. Gene set enrichment analysis revealed upregulation in the sarcomas of genes related to translation, epithelial mesenchymal transition (EMT), inflammation, and the cell cycle versus downregulation of genes related to myogenesis and tumor metabolism. Furthermore, genes with increased copy number such as Met and Cdk4 were overexpressed in tumors. Conclusions: Temporary reduction of p53 during high-dose irradiation increases late effects including tissue injuries and sarcoma development.