Molecular Profiling of Anastatic Cancer Cells: Potential Role of the Nuclear Export Pathway

Purpose: Anastasis is newly discovered process by which cells recover from late-stage apoptosis upon removal of a death stimulus. Recent reports suggest that cells may recover, even after the initiation of mitochondrial outer-membrane permeabilization (MOMP) and caspase activation. Here, we specifically studied the reversibility of late-stage apoptosis in cervical (HeLa) and breast (MDA-MB-231) cancer cells in relation to the extent of MOMP (limited or widespread). In addition, we explored the molecular factors involved in the anastatic process.

Methods: The extent of MOMP was assessed using time lapse confocal microscopic imaging, considering mitochondrial cytochrome c-GFP release as a marker for MOMP. Anastatic cells were generated by specifically recovering late-stage apoptotic (annexin V/PI positive) cervical and breast cancer cells. Molecular signaling events involved in death reversal were assessed using LC-MS/MS and qRT-PCR. Targeted chemical inhibition and shRNA-based gene silencing studies were employed to explore the role of the nuclear export pathway in anastasis and increased oncogenicity.

Results: Time-lapse imaging of drug-treated Cyt-c-GFP expressing cancer cells revealed cell recovery despite widespread MOMP. A few recovered anastatic cells were noted and these were found to proliferate through a selection-type of survival. They showed increased drug-resistance, migration and invasive potential compared to non-anastatic cancer cells. Network analysis using 49 proteins uniquely expressed in anastatic cells indicated upregulation of nuclear export/import, redox and Ras signaling pathways in both HeLa and MDA-MB-231 anastatic cells, indicating common molecular mechanisms in different cell types. Inhibition of XPO1 significantly reduced the recovery of apoptotic cells and abrogated acquired oncogenic transformation in the anastatic cancer cells.

Conclusions: Our study indicates that cancer cells can revert from apoptosis even after the induction of widespread MOMP. We noted a significant role of the nuclear-export pathway in the anastatic process of cancer cells. Inhibition of anastasis through the nuclear export pathway may be a potential therapeutic strategy for targeting drug-resistance, metastasis and recurrence problems during cancer treatment.