3098 - The Orphan Gene CCDC97 is Involved in DNA Damage Response and Ferroptosis Modulation
Presenter(s)
M. Mangoni1,2, A. Duatti3, Y. Martinini4, M. Loi5, C. Talamonti6,7, L. Visani5, V. Salvestrini8, C. Becherini5, C. Mattioli9, B. Bettazzi9, M. Valzano8, I. Meattini10, and L. Livi5; 1Department of Biomedical, Experimental and Clinical Sciences, University of Florence, Firenze, Italy, 2Radiation Oncology Unit, Azienda Ospedaliero Universitaria Careggi, University of Florence, Firenze, Italy, 3University of Florence, Florence, Italy, 4Department of Experimental and Clinical Biomedical Sciences "Mario Serio" University of Florence, Florence, Italy, 5Radiation Oncology Unit, Azienda Ospedaliero Universitaria Careggi, University of Florence, Florence, Italy, 6AOU Careggi Medical Physics Unit, Florence, Italy, 7Department of Biomedical, Experimental and Clinical Sciences “Mario Serio", University of Florence, Florence, Italy, 8Radiation Oncology Unit, Careggi University Hospital, University of Florence, Florence, Italy, 9Radiation Oncology, Careggi University Hospital, University of Florence, Florence, Italy, 10Department of Experimental and Clinical Biomedical Sciences “M. Serio”, University of Florence, Florence, Italy
Purpose/Objective(s):
Coiled-coil domain containing 97 (CCDC97) is an orphan gene that has been identified among partners of an interactome enriched in DNA damage response (DDR)-related factors, with potential implications for radioresistance. A previous translational study in patients with breast cancer (BC) who underwent preoperative radiosurgery, identified CCDC97 as the main differentially expressed gene among patients who benefited from radiosurgery in terms of clinical downstaging, compared to those who didn’t. Given the potential predictive value of CCDC97 identified in the previous study, our aim was to explore the role of CCDC97 in BC cells and in response to irradiation.Materials/Methods:
Western blot and RT-PCR analysis of CCDC97 proteins and gene expression were performed on ER+/HER2- BC (MCF7 and T47D) and triple negative breast cancer (TNBC) (MDA-MB231) cell lines. Transient silencing (siCCDC97) was performed in in vitro models. Analysis of phenotypic effects such as cell survival (cell counting and Crystal violet assay), activation of DDR through the evaluation of phospho-?H2AX, RAD51 and p53 expression (either with confocal and/or western blot and RT-PCR analysis), and reactive oxygen species (ROS) production (viewed by DCFDA fluorescence, confocal and FACS analysis) were evaluated in silenced cells, in combination or not with irradiation (2-8 Gy).Results:
MCF7 and T47D ER+ BC cell lines showed a constitutive high expression of CCDC97 when compared to MDA-MB231 TNBC. To point out CCDC97 role, we performed transient CCDC97 silencing in ER+ BC cell lines and we observed an increase in phospho-?H2AX levels and a significant reduction in cell survival. The effect was enhanced by the combination of radiotherapy. After siCCD97, both alone and in combination with radiotherapy, we observed a time- and dose-dependent regulation of RAD51. The analysis of ROS production showed that siCCDC97 BC cells exhibit a higher endogenous ROS production. The evaluation of the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) demonstrated that peroxidation increased in siCCDC97 cells, and a further increase was observed after irradiation. Moreover, using the oxidation-sensitive fluorescent lipid peroxidation probe C11-BODIPY (581/591), a marked increase in lipid peroxidation was revealed in siCCDC97 cells, as verified both by confocal and FACS analysis.Conclusion:
Our data indicate that CCDC97 expression is involved in the regulation of DNA damage and response to ionizing radiation, showing an inverse correlation with phospho- ?H2AX levels and cell survival. Furthermore, CCDC97 silencing leads to an increase in endogenous ROS production and lipid peroxidation. Therefore, CCDC97 appears to be involved in the regulation of ferroptosis. The obtained results indicate that CCDC97 warrants further investigation as a potential predictive biomarker and a therapeutic target to enhance response to ionizing radiation.