Lipidomics Profiling Reveals Differential Alterations after FAS Inhibition in 3D Colon Cancer Cell Culture Models

Abstract

Cancerous cells synthesize most of their lipids de novo to keep up with their rapid growth and proliferation. Fatty acid synthase (FAS) is a key enzyme in the lipogenesis pathway that is upregulated in many cancers and has gained popularity as a druggable target of interest for cancer treatment. The first FAS inhibitor discovered, cerulenin, initially showed promise for chemotherapeutic purposes until it was observed that it had adverse side effects in mice. TVB-2640 (Denifanstat) is part of the newer generation of inhibitors. With multiple generations of FAS inhibitors being developed, it is vital to understand their distinct molecular downstream effects to elucidate potential interactions in the clinic. Here, we profile the lipidome of two different colorectal cancer (CRC) spheroids treated with a generation 1 inhibitor (cerulenin) or a generation 2 inhibitor (TVB-2640). We observe that the cerulenin causes drastic changes to the spheroid morphology as well as alterations to the lipid droplets found within CRC spheroids. TVB-2640 causes higher abundances of polyunsaturated fatty acids (PUFAs) whereas cerulenin causes a decreased abundance of PUFAs. The increase in PUFAs in TVB-2640 exposed spheroids indicates it is causing cells to die via a ferroptotic mechanism rather than a conventional apoptotic or necrotic mechanism.

FAS Inhibited Proteomics and Phosphoproteomics Profiling of Colorectal Cancer Spheroids Shows Activation of Ferroptotic Death Mechanism

“…These results show that HT-29 spheroidsan MSS, TP53 mutant colorectal cancer cell linetreated with either cerulenin or TVB-2640 are undergoing a ferroptotic death mechanism. This result agrees with the previously collected lipidomics data where it was observed that PC and PE lipids with two more degrees of unsaturation were elevated compared with controls as well as fatty acids . The decreased ability of these spheroids to scavenge lipid peroxides coupled with increased abundances of PUFA PC and PE lipids, as well as fatty acids, shows that ferroptosis is occurring within these spheroids.…”

“…Both HCT 116 and HT-29 spheroids were dosed with either cerulenin or TVB-2640 at their respective IC 50 concentrations . A 0 h time point was harvested and used as the control for statistical analysis and differential expression.…”

“…Recently, we conducted untargeted LC-MS lipidomics on HCT 116 and HT-29 spheroids inhibited with either cerulenin or TVB-2640 . Numerous lipidomic alterations were observed; however, a handful of vital biological questions remained unanswered.…”

“…At day 4 and every other day after, half of the media volume was replaced with fresh media until day 12. On day 12, HCT 116 and HT-29 spheroids were dosed with either cerulenin87.3 ± 25.2 μM and 79.9 ± 4.1 μM, respectivelyor TVB-264053.4 ± 3.9 μM and 131.1 ± 13.1 μM, respectivelyat their respective half-maximum inhibitory concentration (IC 50 ) according to previous studies . Four time points were harvested in biological triplicate, with each time point consisting of 12-whole spheroids: 0-h (control), 6-h, 24-h, and 48-h.…”

“…Our research group recently characterized the full drug penetration and subsequent changes to the lipidome of HCT 116a KRAS mutant, microsatellite instable (MSI) colorectal carcinoma cell lineand HT-29a P53 mutant, microsatellite stable (MSS) colorectal adenocarcinoma cell linespheroids inhibited with either cerulenin or TVB-2640 . One of the notable observations was an increase in monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) among many lipid classes and subclasses within TVB-2640 treated spheroids.…”