Polyploid Giant Cancer Cells are dependent on cholesterol for progeny formation through amitotic division

Polyploid Giant Cancer Cells (PGCC) are increasingly being studied for their role in cancer recurrence and mortality. These cells are induced in bulk cancer cell populations when under stress such as hypoxia or treatment stress (i.e. chemotherapy or radiation). PGCC utilize temporary senescence achieved via a dedifferentiation pathway to survive transient stresses, providing a seed population for later regrowth. Our previous work demonstrated a dependence on the lysosomal enzyme acid ceramidase (ASAH1) in these cells, such that they are unable to perform division without ASAH1. The pro-drug LCL521 is lysosomally targeted, fully inhibits ASAH1 activity within 5 hours, and ablates the ability of PGCC to generate progeny while permitting mitotic cancer cells to proliferate normally. We used an RNASeq approach to compare the responses of parental PPC1 prostate cancer cells and their radiation-induced PGCC counterparts to 5 hours of LCL-521 treatment. RNASeq confirmed that no major changes were triggered in parental PPC1 cells while PGCC appeared to respond as if they had undergone cholesterol depletion. Overall design: The experiment included four conditions. For each, 8 X 10^5 PPC1 cells were plated on day 1 in 100 mm plates. Condition 1 (UT) were simply harvested on day 5 with trypsinization, processed using a Qiagen RNAEasy kit, purity assessed with a Nanodrop, and packaged for delivery to Novogene for analysis. Condition 2 (UT_LCL) were exposed to 5 µM LCL-521 for five hours on day 5 prior to harvest. Condition 3 (PGCC) were irradiated with 8 Gy on day 2 and on day 5 trypsinized, passed through a 20 micron PluriSelect cell filter, and the captured cells eluted. Condition 4 (PGCC_LCL) were irradiated with 8 Gy on day 2 and on day 5 exposed to 5 µM LCL-521 prior to harvest with the same 20 micron filtration step as condition 3. All harvests were performed in parallel for all 4 conditions each time the experiment was done. The experiment was done 6 independent times, for a total of 24 samples.