ELEVATED DIACYLGLYCEROL O-ACYLTRANSFERASES 1 AND 2 (DGAT1/2) REINFORCE A NEGATIVE REGULATORY LOOP OF LIPID DROPLETS AND FOXO3 TRANSCRIPTION FACTOR THAT DRIVES METABOLIC AND TUMEROGENIC REMODELING IN COLON CANCER FACILITATED BY OBESITY

Obesity epidemic is associated with increased colon cancer (CC) risk and progression. One of the mechanisms of obesity mediated CC progression may include Lipid Droplets (LDs). They are dynamic organelles storing intracellular triacylglycerols (TG) and providing fuel for diverse cell functions. LDs initiation and growth is mediated by LDs- coat acyltransferases, DGAT1/2, that catalyze the final step in TG synthesis. We/others demonstrated elevated LDs in colonic tumors (vs normal). Further, we identified a self-reinforcing negative regulatory loop involving LDs and FOXO3 that drives metabolic and tumorigenic remodeling in CC. Here, we aim to elucidate the mechanisms of DGATs mediated LDs dynamics that facilitate CC in obesity. Methods. Human CC: Tissue array, Local CC patients; TCGA and NCBI’s GEO databases, Human colon cells (CCC): HT29, HCT116, SW620, SW480; NCM460 (non-transformed), Colonospheres; Mouse: APC^min/+ colon cancer model, AOM/DSS, High Fat Diet (HFD)- obese; Oleic acid (OA, 300mM); Enzymatic inhibitors: DGAT1- A922500, DGAT2- PF-06424439; Differentially expressed genes (DEGs), RNA/cDNA, CUT&RUN, RNAseq (Illumina HiSeq 2500), RSEM/EBseq DEGs analysis, Ingenuity Pathway Analysis (IPA), Kaplan-Meier survival estimates; IHS; IFS; IB; qPCR; ChIP assay; Colony formation assay; FACS; Luciferase-Reporter Assay. Results. We found significantly higher DGATs levels in human CC tissue (vs normal) that is augmented by both the severity of the disease and obesity. Similarly, their levels are increased in CCC (vs non-transformed cells) and augmented by obesity mediator (OA). This increase in DGATs expression is mediated by MYC activation and binding to DGAT2 promoter (Fig 1A-I). Moreover, blockade of DGATs shut down the self-reinforcing regulatory loop between LDs and FOXO3. Specifically, it protected FOXO3 by attenuating PI3K and lowered LDs accumulation by reducing MYC dependent DGATs expression. Further, blockade of DGATs lowered OA facilitated growth of CCC and colonosphere (cell cycle G0/G1 arrest via FOXO3/p27kip1 protection). Moreover, treatment with DGATs inhibitors reduced colonic tumor burden in APC^min/+ mouse, on regular diet and HFD (obese). Further, this inhibition attenuated DEGs linked to metabolic and tumorigenic remodeling in CCC, colonospheres and colon of HFD-obese APC^min/+ mouse (IPA, FDR<0.05). It additionally targeted pathways that are activated in human CC and colon cancer crypts (IPA, FDR<0.05) (Fig 2A-R). Selected DEGs were validated in CCC, colonospheres, and APC^min/+ mouse colon (qPCR, p<0.05); their similarly altered transcriptional levels were found in human CC. Conclusions. We identified a novel mechanism of DGATs dependent metabolic and tumorigenic remodeling mediated by LDs and FOXO3 regulatory loop in obesity facilitated CC. This establishes a platform for new diagnostics and for development of effective treatments.