Restriction lift date: 2021-06-26
Evaluation of the role of autophagy in ovarian cancer chemoresistance
University College Cork
Ovarian cancer (OC) is the seventh most commonly diagnosed cancer in women worldwide. In Ireland, OC was the fourth most common cancer for women between 2015 and 2017 (1). A major challenge in the clinical management of OC is the high rate of disease recurrence. Approximately 80% of women who exhibit an excellent response to first line therapy, will present again with recurrent disease. A panel of ovarian cancer cell lines were evaluated for their response to two clinically relevant chemotherapies, paclitaxel and carboplatin. Apoptosis was assessed via morphological analysis and quantitation of active caspase-3. Apoptosis was significantly induced in response to treatment in the ovarian cancer cell lines, with the highest induction evident in the most drug sensitive cells. When these cell lines were re-challenged with a second round of paclitaxel treatment – apoptosis levels were significantly reduced and clonogenic recovery enhanced. This provides a novel insight into the mechanisms by which ovarian cancer cells may gain resistance to treatment. Autophagy is a highly conserved catabolic process, that enables cells to cope with stressful conditions. We have demonstrated, using flow cytometry and western blot analysis of LC3, that three ovarian cancer cell lines, that induced only limited apoptosis to chemotherapy, showed significant elevation of autophagy. In these cell lines, co-treatment with the autophagy modulators chloroquine and lithium impeded clonogenic recovery following treatment. siRNA knockdown of the autophagy genes, BECN1 and ATG5, significantly reduced the recovery of OC cells that undergo autophagy following treatment with paclitaxel. In addition, cell cycle analysis demonstrated that OC cells can regain a ‘normal/parental’ DNA profile 7-days after drug treatment, which was impeded when autophagy was inhibited with chloroquine or when key autophagy genes BECN1 and ATG5 were knocked down. Confocal analysis showed nuclear material colocalised with autophagic vesicles, suggesting that nucleophagy may play a role in the recovery of the parental DNA content. The potential contribution of non-canonical autophagy was also evaluated using the inhibitor Brefeldin A. Autophagosome accumulation was reduced following treatment with Brefeldin A and following siRNA knockdown of BECN1 with the non-canonical regulator Rab9, which significantly reduced the recovery of OC cells following paclitaxel treatment. These data demonstrate the importance of autophagy in OC cell recovery following drug treatment and suggests that more than one form of autophagy may be involved. It is also notable that only three of the five cell lines tested demonstrated elevated autophagy – and these are the cells that respond to chloroquine. This may be important for clinical trials that are currently incorporating hydroxychloroquine without any guidance on the contribution of autophagy to the tumour phenotype. It is likely that biomarkers will be needed to guide the administration of autophagy inhibitors in the future. Here, we have made potentially clinically relevant observations. We have highlighted reduced apoptosis competency following drug re-challenge and shown that autophagy modulators are effective in a subgroup of cell lines with high autophagy levels. We have provided a novel insight into a role for canonical and non-canonical autophagy in OC chemoresistance, and demonstrated for the first time a potential role for nucleophagy in OC cell recovery. It is hoped that autophagy inhibitors, along with appropriate biomarkers, may be incorporated into treatment strategies to overcome treatment resistance and improve OC patient outcomes in the future. 1. National Cancer Registry Ireland
Autophagy , Ovarian cancer , Chemoresistance
Quinn, J. 2020. Evaluation of the role of autophagy in ovarian cancer chemoresistance. PhD Thesis, University College Cork.