Eswara Murali Satyavarapu, Ranjita Das, Chandan Mandal, Asima Mukhopadhyay, Chitra Mandal
Cancer cells display abnormal redox metabolism. Autophagy, anoikis and reactive oxygen species (ROS) play a regulatory role during metastasis. LC3 is a well-known essential molecule for autophagy. Therefore, we wanted to explore the molecular interplay between autophagy, anoikis, and ROS in relation to LC3B. We observed enhanced LC3B level along with increased expression of p62 and modulation of other autophagy-related molecules (Atg 3, 5, 7, 12, 16L1 and Beclin1) by inducing oxidative-stress in ovarian cancer cells using a ROS-producing pro-oxidant molecule. Surprisingly, enhanced LC3B was unable to induce autophagosome formation rather promoted anoikis. ROS-induced inhibition of autophagosome-formation is possibly due to the instability of autophagy initiator, ULK1 complex. Moreover, such upregulation of LC3B via ROS enhanced several apoptotic molecules. Silencing LC3B reduced these apoptotic molecules and increased when overexpressed, suggesting its role in apoptosis. Furthermore, LC3B-dependent apoptosis was decreased by inhibiting ROS, indicating a possible link between ROS, LC3B, and apoptosis. Additionally, ROS-induced enhanced LC3B promoted detachment-induced cell death (anoikis). This was further reflected by reduced cell adhesion molecules (integrin-β3 and focal adhesion kinase) and mesenchymal markers (snail and slug). Our in vitro experimental data was further confirmed in primary tumors developed in syngeneic mice, which also showed ROS-mediated LC3B enhancement along with reduced autophagosomes, integrin-β3 and focal adhesion kinase ultimately leading to the decreased tumor mass. Additionally, primary cells from high-grade serous carcinoma patient's ascites exhibited LC3B enhancement and autophagy inhibition through ROS which provided a clinical relevance of our study. Taken together, this is the first evidence for a non-canonical role of LC3B in promoting anoikis in contrast to autophagy and may, therefore, consider as a potential therapeutic target molecule in ovarian cancer. Taken together, autophagy-inhibition may be an alternative approach to induce apoptosis/anoikis in cancer.
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