Signaling through cholesterol esterification: a new pathway for the cholecystokinin 2 receptor involved in cell growth and invasion

Michael R. Paillasse^*^,, Philippe de Medina^*^,, Guillaume Amouroux^*, Loubna Mhamdi^*^,, Marc Poirot^*^, and Sandrine Silvente-Poirot¹^,*^,

^* INSERM 563, Equipe Métabolisme, Oncogenèse et Différenciation cellulaire, Centre de Physiopathologie de Toulouse Purpan, Institut Claudius Regaud, Toulouse France
Université Toulouse III Paul Sabatier, Toulouse, France
Affichem, Toulouse, France

¹ To whom correspondence should be addressed. e-mail: poirot.sandrine{at}hotmail.fr

Several studies indicate that cholesterol esterification isderegulated in cancers. The present study aimed to characterizethe role of cholesterol esterification in proliferation andinvasion of two tumor cells expressing an activated cholecystokinin2 receptor (CCK2R). A significant increase in cholesterol esterificationand activity of Acyl-CoA:cholesterol acyltransferase (ACAT)was measured in tumor cells expressing a constitutively activatedoncogenic mutant of the CCK2R (CCK2R-E151A cells) compared withnontumor cells expressing the wild-type CCK2R (CCK2R-WT cells).Inhibition of cholesteryl ester formation and ACAT activityby Sah58-035, an inhibitor of ACAT, decreased by 34% and 73%CCK2R-E151A cell growth and invasion. Sustained activation ofCCK2R-WT cells by gastrin increased cholesteryl ester productionwhile addition of cholesteryl oleate to the culture medium ofCCK2R-WT cells increased cell proliferation and invasion toa level close to that of CCK2R-E151A cells. In U87 glioma cells,a model of autocrine growth stimulation of the CCK2R, inhibitionof cholesterol esterification and ACAT activity by Sah58-035and two selective antagonists of the CCK2R significantly reducedcell proliferation and invasion. In both models, cholesterylester formation was found dependent on protein kinase zeta/extracellular signal-related kinase 1/2 (PKC ${zeta}$ /ERK1/2) activation.These results show that signaling through ACAT/cholesterol esterificationis a novel pathway for the CCK2R that contributes to tumor cellproliferation and invasion.

Supplementary key words cholesteryl ester • acyl-CoA:cholesterol acyltransferase • constitutively active mutant • autocrine stimulation • G protein coupled receptor • gastrin receptor • glioma • cancer

Abbreviations: ACAT, acyl-CoA:cholesterol acyltransferase; CCK2R, cholecystokinin 2 receptor; CO, cholesteryl oleate; cPKC, classical protein kinase C; DFMO, ${alpha}$ -difluoromethyl-ornithine; ERK, extracellular signal-related kinase 1/2; FCS, fetal calf serum; MEK, mitogen-activated kinase kinase; nPKC, novel protein kinase C; ODC, ornithine decarboxylase; TBP, Tata Box binding protein

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