Abstract 1348: Parkin-mediated mitophagy regulates 5-fluorouracil-induced chemoresistance in breast cancer
Authors: Das Chandan Kanta , Jena Bikash Chandra , Das Subhayan , Parekh Aditya , Dey Goutam , Bharti Rashmi , Bharadwaj Deblina , Bhutia Sujit Kumar , Kögel Donat , Mandal Mahitosh 
Published: August 2018

Introduction Resistance to therapeutics is one of the major concerns for treatment failure in breast cancer. The use of antimetabolite, 5 Fluorouracil (5-FU) based combinational therapies have been an ideal treatment blueprint for different types of solid tumors including breast cancer. However, the efficacy of 5-FU based treatment is limited due to development of resistance. Recently mitophagy has evolved as an important cell survival mechanisms in response to different stress stimuli associated with cancer treatments. Mitophagy is the selective form of autophagy that removes damaged mitochondria. Although mitophagy has dual role in context to cell survival and cell death, still a large number of evidences suggests their prosurvival role with respect to chemoresistance. Here we investigated the role of mitophagy in 5-FU induced breast cancer chemoresistance.

Methodology Chemoresistant cell lines were established by continuous exposure to increasing concentration of 5-FU to breast cancer cell lines (MDA-MB-231 and MDA-MB-468). Chemoresistant cells were characterized by MTT assay, caspase assay and western blot. Induction of autophagy was confirmed by GFP LC3 transfection and western blot. Further prosurvival autophagy was asserted through FACS by using autophagy inhibitor chloroquine (CQ) and stable ATG5 lentiviral knockdown. Mitophagy was confirmed using MTDR (detects mitochondrial mass) and TMRM (detects mitochondrial membrane potential) through FACS. Further immunofluorescence and western blot were used to confirm key mitophagy markers (TOM20 and Porin) with CQ treatments. Involvement of parkin mediated mitophagy was later confirmed by cytosolic/mitochondrial fractionation assay and by siRNA knockdown.

Results Characterization study reveals chemoresistant cells were more apoptosis resistant compared to their parental counterparts.The levels of the autophagy markers LC3-II, p62, ATG5 and Beclin-1 were elevated in chemoresistant cells while mitochondrial mass and mitochondrial membrane potential were reduced. Treatment of CQ and ATG5 knockdown significantly augment 5-FU sensitivity in chemoresistant cells. Simultaneously CQ treatment increased TOM20 and porin expression revealing induction of mitophagy in chemoresistant cells. Further cell fractionation assay and siRNA knockdown of Parkin confirm its mitochondrial localization and prosurvival function in 5-FU resistant breast cancer respectively. some important in vitro findings were also confirmed in xenograft breast cancer model.

Conclusion Here we noticed that chemoresistant breast cancer cells exhibit greater apoptosis resistance to 5-FU compared to parental cells along with enhanced cytoprotective mitophagy, also underscoring the involvement of parkin mediated induction of mitophagy in chemoresistant breast cancer.

Citation Format: Chandan Kanta Das, Bikash Chandra Jena, Subhayan Das, Aditya Parekh, Goutam Dey, Rashmi Bharti, Deblina Bharadwaj, Sujit Kumar Bhutia, Donat Kögel, Mahitosh Mandal. Parkin-mediated mitophagy regulates 5-fluorouracil-induced chemoresistance in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1348.