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March 13-16, 2016 (Sunday-Wednesday)
Hyatt Regency Baltimore Inner Harbor
Baltimore, Maryland
Title: ER+ BREAST TUMOR GROWTH IS CYTOCHROME P450 3A4 (CYP3A4)-DEPENDENT AND INHIBITED
BY A BIGUANIDE INHIBITOR OF CYP3A4 EPOXYGENASE ACTIVITY WHICH SUPPRESSES MITOCHONDRIAL
OXYGEN CONSUMPTION.
Authors:
Zhijun Guo1, Irina F. Sevrioukova2, Eric Hanse1, Ilia Denisov3, Elena Grinkova3, Xia Zhang1, TingLan Chiu1, Rebecca Cuellar1, Vanessa Wankhede1, Daniel Swedien1, Justin Stamschror1, Juan Alvarez1, Christian
Torres1, William Marrero Ortiz1, Julia Nguyen1, Kathryn J. Chavez1, Dafydd Thomas4, Young Bae5, Julia
Wulfkuhle6, Emanuel Petricoin6, Beverly Norris1, Robert J. Schumacher1, Henry Wong1, Robin Bliss1, Qing Cao1,
Haitao Chu1, Jonathan Henriksen7, Stephen Schmechel7, William Atkins7, Kalpna Gupta1, Ameeta Kelekar1, Ian
Blair8, Jorge Capdevila9, John Falck10, Thomas L. Poulos2, Stephen G. Sligar3, Gunda Georg1, Elizabeth Amin1,
David A. Potter1.
Affiliations: 1University of Minnesota, Minneapolis, MN; 2University of California, Irvine, Irvine, CA; 3University of
Illinois Urbana-Champaign, Urbana, IL; 4University of Michigan, Ann Arbor, MN; 5Yeungnam University, Daegu,
Korea, 6George Mason University, 7University of Washington, Seattle; 8University of Pennsylvania, 9Vanderbilt
University, Nashville,TN; 10University of Texas Southwestern, Dallas, TX
ABSTRACT: CYP3A4 promotes estrogen receptor (ER) + breast cancer cell growth, in part, through
epoxyeicosatrienoic acid (EET) biosynthesis. CYP3A4 correlated with ER alpha expression in breast tumor epithelia,
but its roles in breast tumor growth are unknown. CYP3A4 silencing in ER+ tumors suppressed escape from
dormancy, suggesting that CYP3A4 promotes ER+ breast cancer progression. In ER+ breast cancer cells, CYP3A4
co-localized with mitochondria. Soluble epoxide hydrolase inhibition (sEHi) stabilized the mitochondrial membrane
potential and oxygen consumption rates (OCR) in ER+ breast cancer cells, but did not affect extracellular acidification
rates (ECAR), suggesting that CYP3A4 has a primary effect on OCR. Chemical probes of CYP3A4 epoxygenase
activity are much needed to elucidate the immediate early mechanisms of CYP3A4 epoxygenase activity in breast
cancer. CYP3A4 nanodiscs revealed that metformin binds to the active site of CYP3A4 and a metformin CYP3A4 cocrystal enabled structure-based design of hexyl-benzyl biguanide (HBB), a potent (IC50=9 uM) and selective (vs.
CYP2C8 IC50=50 uM) neo-biguanide inhibitor of CYP3A4 epoxygenase activity. HBB (20 uM) potently inhibited OCR
within minutes and suppressed the mitochondrial membrane potential, while sEHi and EETs reversed these effects.
HBB (20 uM) also potently suppressed ECAR of breast cancer cells at 2 to 6 hours. Glycolysis is regulated in breast
cancer cells, in part, by pyruvate kinase M2 (PKM2), which exists as an inactive dimer. PKM2 dimer promotes the
anabolic component of the Warburg effect by suppressing conversion of phosphoenolpyruvate to pyruvate, allowing
diversion of intermediates of glycolysis to biosynthetic pathways. Phosphorylation of PKM2 on tyrosine 105 facilitates
conversion of tetramer to dimer. In contrast, PKM2 tetramer is active and correlates with the dephosphorylated state
of this enzyme. HBB transiently suppressed PKM2 phosphorylation on Tyr105 in an immediate early fashion,
promoting PKM2 tetramer, intracellular pyruvate rise and transient lactate release reflected by increased ECAR,
followed by re-phosphorylation of Tyr105, promotion of PKM2 dimer formation and subsequent suppression of ECAR.
EETs inhibited PKM2 multimer formation, suggesting that CYP epoxygenase activity suppresses activity of PKM2.
HBB inhibited ER+ tumor growth and HBB treatment activated intratumoral phospho-Tyr105-PKM2, while intratumoral
AMPK phosphorylation was not activated. CYP epoxygenase activity thus regulates breast cancer OCR and is an
early target of neo-biguanide inhibition.
Support acknowledgement: NCI R01 CA113570-01, Susan G. Komen Foundation Grant KG 090861, UM
Masonic Cancer Center, CTSI and Center for Translational Medicine, Randy Shaver Cancer Research
and Community Fund
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