Abstract 5420: Ribosomal protein-p53-MDM2 signaling by nucleolar stress response and drug discovery

Abstract

Abstract Ribosomal biogenesis is a major energy consuming cellular step, and tightly regulated by the demand of protein synthesis associated with cell growth and proliferation. Thus, defects that impair ribosomal biogenesis cause nucleolar stress response. This response initiates a cascade mediated by ribosomal proteins (RPs), particularly RPL5, RPL11, RPL23, and RPS7. These RPs are usually located in the nucleolus but are rapidly released into the nucleoplasm upon nucleolar stress, activating the tumor suppressor MDM2-p53 pathway. Thus, nucleolar stress response is considered as a quality control surveillance mechanism that regulates balance between proper protein synthesis and cell cycle progression for tumor suppression. However, it is unknown whether there are direct connections between p53-activating RP genes and cancer development in vivo, and whether this stress response might be target for cancer therapy. We identified nucleolar protein PICT1 as a novel key regulator of nucleolar stress response. To clarify PICT1 function, we generated PICT1-deficient mice and ES cells and carried out extensive analyses. Our results show that even without DNA damage, PICT1 loss results in p53-dependent growth arrest in vitro and in vivo. We further show that PICT1 binds to RPL11, and that RPL11 is released from nucleoli in the absence of PICT1. In PICT1-deficient cells, increased binding of RPL11 to MDM2 blocks MDM2-mediated ubiquitination of p53, leading to p53 accumulation and repression of cell proliferation. Human colon and esophageal cancer patients with low PICT1 expression have better prognoses. Similarly, when shRNA is used to deplete PICT1 in various tumor cell lines with intact p53 signaling, the cells slow in growth and accumulate p53. These data suggest that PICT1 is a novel key regulator of the MDM2-p53 pathway on nucleolar stress response and promotes tumor progression by retaining RPL11 in the nucleolus. Thus, the nucleolar stress response PICT1 regulates could play a definitive role in tumor suppression and, chemicals which induce nucleolar stress response might be lead to new cancer therapeutics. Therefore, we next constructed a reporter system to visualize and quantitate nucleolar stress response. We found that this reporter system allowed quantitative measurement depend on strength and duration of this stress. As this reporter system showed excellent accuracy, we next screened 400 inhibitors, 1,300 approved drug, and hundreds of natural products from marine species. We found hit compounds including RNA synthesis inhibitor Doxorubicin and Aclarubicin, and also positive control compound, ActinomycinD. In addition, inhibitors of CDK and HDAC were included here. Thus, the nucleolar stress reporter system may reveal unexpected physiological functions of the nucleolar stress response, such as mitotic progression and epigenetics regulation, and identify novel cancer therapeutics, especially for individuals with intact-p53 signaling. Citation Format: Kohichi Kawahara, Takuto Kawahata, Fumito Horikuchi, Yohei Kamijo, Masatatsu Yamamoto, Yoshinari Shinsato, Kentaro Minami, Kazunari Arima, Toshiyuki Hamada, Tatsuhiko Furukawa. Ribosomal protein-p53-MDM2 signaling by nucleolar stress response and drug discovery. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5420. doi:10.1158/1538-7445.AM2015-5420