Lipid Droplet Accumulation Promotes Proliferation of Colon Cancer Cells via Loss of Tumor Suppressor FoxO3

Abstract

Introduction: Epidemiological studies demonstrate that obesity is associated with colon cancer progression. Intracellular lipids are stored in lipid droplets (LDs) in both adipocytes and non-adipocytes cells. LDs consist of a core of neutral lipids surrounded by numerous proteins including perilipin 2 (ADRP). Since tumor cells require excessive energy to serve their high metabolic needs, limited data suggest that LDs can act as a local source of energy. We have previously demonstrated that loss of FOXO3 leads to lipid accumulation in mice, whereas overexpression of FOXO3 inhibits proliferation of colon cancer cells. Aim: We hypothesized that LD accumulation stimulates progression of colon cancer cells via loss of FOXO3 activity. Methods: Cells: colon cancer HT-29 and DLD1 cells and untransformed YAMC. Treatments: EGF (100ng/ml) and Oleic Acid (OA) (1mg/ml). LDs inhibitors: C75 (fatty acid synthase inhibitor; 50 μg/ml) and TOFA (inhibitor of acetyl-CoA carboxylase; 50 μg/ml). LDs quantification: Oil Red-O staining and expression of ADRP. FOXO3 activity and downstream target p27kip1: immunoblots, immunofluorescence, and Chip assay. Proliferation: MTS assay and flow cytometry. Results: OA, a known inducer of LD accumulation in adipocytes, promoted LD accumulation (enlarged LDs: 3-fold in YAMC, 2-fold in HT29 cells) and ADRP expression in colonic cells (non-transformed and cancer). Similarly, EGF, a critical inducer of proliferation in colon cancer cells, increased LD size and expression of ADRP in HT-29 cells. Moreover, proliferation stimulated by OA (47.5±16%) or EGF was effectively suppressed by LD inhibitors (C75 and TOFA), suggesting that LDs are key regulators of the proliferative process in colon cancer cells. OA-stimulated LD accumulation resulted in FOXO3 degradation (79±13%) in the first 6 hours and de novo synthesis of inactive FOXO3 (at 48 hours) within the cytosol. Overexpression of FOXO3 suppressed the proliferative effect of LDs, suggesting that LD mediated loss of FOXO3 activity is a critical modulator of cell cycle. Additionally, LD accumulation led to disassociation of FOXO3 from the promoter and decreased expression of p27kip1, an inhibitor of the cell cycle G0G1 checkpoint. Conclusion: These data support a novel signaling mechanism in colon cancer cells. They point to the loss of FOXO3 activity as a critical step in LD-induced proliferation and suggest that LDs may act as an intracellular source of energy to fuel colon cancer progression.