Inhibiting glucose-regulated protein 78 modulates lipid metabolism through controlling stearoyl-CoA desaturase 1
KeywordsBreast Cancer Cell Endoplasmic Reticulum Protein Inhibit Breast Cancer Cell Western Blot Hybridization LCC1 Cell
Of the 230,000 new cases of invasive breast cancer diagnosed annually, 70% express the estrogen receptor-α and may be treated with anti-estrogen therapies. While these drugs induce clinical benefit, over 50% will develop resistance. Recently, we have determined a key role of the unfolded protein response endoplasmic reticulum protein chaperone, glucose-regulated protein 78 (GRP78), in mediating anti-estrogen resistance. We now expand the role of GRP78 to include regulation of lipid metabolism.
Material and Methods
Liquid chromatography/mass spectroscopy matrix-assisted laser desorption/ionization-time of flight analysis, RNAi knockdown, and western blot hybridization was used to determine the metabolomic effect of GRP78 in anti-estrogen sensitive (LCC1) or resistant (LCC9) breast cancer cells. Furthermore, GRP78 targeting morpholinos were used to determine the effect of GRP78 inhibition in vivo.
Metabolic analysis indicated that inhibition of GRP78 through RNAi in breast cancer cells results in increased intracellular concentrations of polyunsaturated fats (PUFA) linoleate (18:2n6), linolenate [alpha or gamma; (18:3n3 or n6)], dihomo-linolenate (20:3n3 or n6), and dihomo-linoleate (20:2n6). GRP78 knockdown increased delta-6 desaturase (FADS2), possibly corresponding with the observed increase in PUFA. Moreover, knockdown of GRP78 decreased the endoplasmic reticulum protein stearoyl-CoA desaturase 1 (SCD1), further supporting a role for GRP78 in lipid metabolism. Knockdown of FADS2 or overexpression of SCD1 partially restored anti-estrogen resistance in GRP78 inhibited breast cancer cells, suggesting both proteins independently play a role in GRP78-mediated anti-estrogen resistance. Treatment of breast cancer cells with increasing doses of linoleic acid inhibited SCD1 and increased breast cancer cell death. Interestingly, combining anti-estrogens with linoleic acid had reduced the breast cancer cell-killing capacity than either treatment alone. However, anti-estrogens and linoleic acid treatment potentiated drug effectiveness in cells where GRP78 was inhibited, suggesting that PUFA regulation may be a secondary mechanism of anti-estrogen responsiveness dependent on GRP78 presence. Knockdown of SCD1 restored anti-estrogen sensitivity in LCC9 cells while overexpression of SCD1 in LCC1 cells promoted resistance. LCC9 orthotopic tumors treated with GRP78 targeting morpholino were re-sensitized to tamoxifen. Targeting GRP78 in vivo resulted in reduced SCD1 protein in GRP78 morpholino + tamoxifen treated tumors, demonstrating a key role of GRP78/SCD1 interaction in drug resistance.
These data suggest that the effect of GRP78 knockdown on fatty acid metabolism and biogenesis may represent a new homeostatic ability of GRP78 to modify fatty acid metabolism. Moreover, the novel role of GRP78 controlling pro-tumorigenic SCD1 demonstrates the importance of targeting GRP78 in breast cancer treatment.
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