Abstract 2413: Ferroptosis plays an important role in the radiotherapy-induced tumor suppression and radioresistance
Authors: Lei Guang , Zhang Yilei , Koppula Pranavi , Liu Xiaoguang , Lin Steven Hsesheng , Liao Zhongxing , Wang Hui , Gan Boyi 
Published: September 2020

Introduction: Radiotherapy (RT) is widely used to treat cancers, but radioresistance remains a major factor in the failure of RT. RT generates reactive oxygen species (ROS) through the radiolysis of cellular water and the stimulation of oxidase, causing cellular damage. Ferroptosis is a regulated cell death induced by iron-dependent peroxidation of phospholipids containing polyunsaturated fatty acids (PUFA-PLs), which is morphologically and mechanistically distinct from apoptosis. There are several core regulators in ferroptosis pathway. SLC7A11 and GPX4 play a role in inhibiting ferroptosis, whereas ACSL4, as an essential lipid metabolism enzyme, promotes ferroptosis by regulating the biosynthesis of PUFA-PLs. Ferroptosis can be induced by either class 1 ferroptosis inducers (FINs) that inhibit SLC7A11 or class 2/3 FINs that inhibit or deplete GPX4. Our study aims to determine the role and mechanisms by which ferroptosis contributes to RT-induced cell death and tumor suppression and to evaluate therapeutic potential of FINs as robust radiosensitizers.

Methods: Ferroptosis markers such as lipid peroxidation or PTGS2 expression was evaluated by flow cytometry, q-PCR or transmission electron microscopy (TEM). Mechanisms were analyzed by western blot and CRISPR-Cas9. Patient derived xenografts (PDX) co-treated with RT and FINs were employed to access the combination effectiveness. Samples were collected from patients before and after RT and ferroptosis marker 4-HNE was correlated with prognosis of patients.

Results: RT induced lipid peroxidation and PTGS2 expression in a variety of cancer types and TEM revealed that cancer cells after RT exhibited shrunken mitochondria with enhanced membrane density, a morphologic feature of ferroptosis. Treatment with ferroptosis inhibitor ferrostatin-1 significantly restored cell survival that had been reduced by exposure to RT, suggesting ferroptosis represents an important part of the RT-induced cell death response. Mechanistically, RT up-regulated the expression of ACSL4, together with RT-generated ROS, to promote lipid peroxidation and ferroptosis. RT also induced the expression of ferroptosis negative regulators, including SLC7A11 and GPX4, leading to radioresistance. In preclinical model, ferroptosis inhibitor liproxstatin-1 partially abolished the RT-induced tumor suppression in H460 xenografts, and FINs (e.g. sulfasalazine) treatment exhibited a significant radiosensitization in A549 xenografts or radioresistant lung cancer patient-derived xenografts (PDXs). In clinical analysis, we revealed very weak 4-HNE levels in all tumor samples before RT, but RT increased 4-HNE levels moderately or strongly in all tumor samples. Importantly, the 4-HNE levels were positively correlated with RT response and disease-free survival in cancer patients.

Conclusions: Our study reveals a previously unrecognized link between RT and ferroptosis, and demonstrates that FINs as a radiosensitizer reverse ferroptosis-mediated radioresistance, and proposes a novel combination therapeutic strategy for cancers, providing an effective approach to overcome radioresistance and facilitating us to initiate follow-up clinical trials to further explore the therapeutic potential of FINs combined with RT in cancer patients.

Citation Format: Guang Lei, Yilei Zhang, Pranavi Koppula, Xiaoguang Liu, Steven Hsesheng Lin, Zhongxing Liao, Hui Wang, Boyi Gan. Ferroptosis plays an important role in the radiotherapy-induced tumor suppression and radioresistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2413.