Despite the ability of chemotherapy to eliminate the majority of tumor cells, some are able to escape cell death and proliferate. In this study we evaluated mechanisms of autophagy and senescence, that in theory allow cells to escape apoptosis and might be responsible for cancer recurrence. Autophagy and senescence, either alone or in concert, may result in temporary growth arrest followed by proliferative recovery. With regard to autophagy, its function may be either cytoprotective, where autophagy inhibition results in sensitization to therapy or nonprotective, where autophagy inhibition does not alter sensitivity to chemotherapy and/or ionizing radiation. To determine the mode of autophagy in murine metastatic carcinoma 4T1 cell lines, autophagy was inhibited pharmacologically using chloroquine, and by genetic silencing of the Atg5 autophagy associated protein. Cells were treated with 1 μM Adriamycin, stained with Acridine Orange to assess autophagy by fluorescent microscopy (autophagosomes appear orange-red) and autophagy quantified by Flow Cytometry. Induction and blockade of autophagy by chloroquine was confirmed by Western blotting for the appearance of LC3B and degradation of p62. Senescence was monitored by β-galactosidase staining and quantified by Flow Cytometry based on C12FDG staining; senescence was confirmed based on the induction of p21 and p16. Adriamycin (doxorubicin) exposure resulted in breast tumor cell death as well as prolonged growth arrest; some of the arrested cells eventually recovered and formed colonies. After exposure to doxorubicin, 4T-1 cells were sorted based on intensity of β-galactosidase staining and increased size (senescent cells experience changes in size and morphology). β-galactosidase positive cells were plated and monitored over time during which some of the cells recovered the capacity to proliferate. In separate studies with mouse mammary carcinoma cells, radiation also promoted cell death and prolonged growth arrest from which some cells were able to recover proliferative capacity. Our data suggests that although chemotherapy and radiation induce prolonged growth arrest and senescence, these features are not permanent and cells are able to escape and re-emerge from the senescent state to generate proliferating daughter cells. As pharmacologic inhibition of autophagy did not result in increased sensitivity to doxorubicin in 4T1 cells (or to radiation in the mouse mammary tumor cells) or interfere with proliferative recovery, we postulate that inhibition of senescence associated pathways may block proliferative recovery and/or promote tumor cell killing in response to chemotherapy and/or radiation. Studies in progress are focused on modulation of c-myc and miR34 levels and the IL1/6/8 signaling axis as potential strategies for interference with senescence and suppression of residual (dormant) tumor growth and cancer recurrence.

Citation Format: Liliya Tyutyunyk, Theresa Thekkudan, Tareq Saleh, David A. Gewirtz. Autophagy and senescence as possible mechanisms leading to proliferative recovery and escape from treatment-induced tumor dormancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5459. doi:10.1158/1538-7445.AM2017-5459