O13 Acid ceramidase: a potential biomarker for locally advanced rectal cancer?

We have previously utilized proteomic and immuno-histochemical data to validate that high levels of acid ceramidase (AC) expression confers poorer neoadjuvant response in rectal cancer. Biological (siRNA, plasmid and CRISPR) AC manipulation altered radiosensitivity in-vitro. We aimed to assess the radiosensitising effect of pharmacological AC inhibitions and elucidate the potential underlying mechanism. Methods Optimal drug dosing was achieved using ELISA activity assays in multiple colorectal cancer cell lines (HCT116, HT29, LIM1215). Carmofur and a novel small molecular inhibitor (LCL521) were used as pharmacological inhibitors. Standard clonogenic assays assessed cell survival following increasing irradiation (2 D), volume change in 3 D spheroids and cell viability in patient derived organoids. Annexin V/PI staining was used to determine apoptosis. Results Carmofur clonogenic assays demonstrated reduced colony formation efficiency (CFE) and improved radiosensitivity across cell lines. HCT116 showed 0.438(CFE) control v 0.183(CFE) carmofur at 1 Gy, 0.140(CFE) control v 0.076(CFE) at 2 Gy (P = 0.000563). LCL521 dosing improved radiosensitivity in spheroid models. HCT116 volume day-15 2.36x10-5mm v control 4.15x10-5mm. siRNA-AC demonstrated increased apoptosis across time points compared to NT control (P = 0.035), and increased poly-ADP ribose polymerase-1 (PARP-1) cleavage in a p53-dependent process. Conclusion Initial work demonstrates that pharmacological inhibition of AC produces comparative radiosensitizing effects in these cell lines and cancer models. siRNA-AC increases apoptosis, suggestive of a potential underlying mechanism. This work further solidifies AC as a potential biomarker, however further recapitulation in more complex models and ultimately in-vivo is required to establish a translatable clinical role.