Abstract LB-023: CYP monooxygenases regulate nuclear localization of ERá and mTORC1 component RagC in ER+HER2- breast cancer cells

Abstract

Introduction: Cancer cell-intrinsic CYP monooxygenases promote tumor progression. In ER+ breast cancer cells, CYP3A4 is required for tumor growth and localizes to mitochondria where it promotes electron transport chain and respiration, while suppressing autophagy, in part, through epoxyeicosatrienoic acid (EET) biosynthesis (1). The diabetes drug metformin inhibited CYP3A4-mediated EET biosynthesis and depleted cancer cell-intrinsic EETs. Metformin bound to the active site heme of CYP3A4 in a co-crystal structure, establishing CYP3A4 as a biguanide target. Structure-based design led to discovery of N1-hexyl-N5-benzyl-biguanide (HBB), which bound to the CYP3A4 heme with higher affinity than metformin. HBB potently and specifically inhibited CYP3A4 arachidonic acid epoxygenase activity, reduced oxygen consumption rates and suppressed growth of breast cancer cell lines (IC50=3-30 μM) and an established ER+ mammary tumor model. HBB inhibited activation of mTOR and suppressed S6Kinase phosphorylation. HBB also suppressed intratumoral mTOR. Recently, nuclear transit of RagC has been shown to promote mTORC1 function, a process sensitive to metformin (2), which inhibits the nuclear pore complex (NPC). Therefore, we hypothesized that HBB as a more potent biguanide may inhibit mTOR through reduction of nuclear RagC by regulating NPC. HBB may also regulate the localization of other nuclear proteins such as estrogen receptor alpha (ERα). Method: ER+HER2- breast cancer cells (MCF-7 and T47D) were seeded on chamber slides and treated with vehicle or HBB for varied periods of time. Cells were fixed, permeabilized, and incubated with specific antibodies against RagC and ERα. After incubation with fluorescence tagged secondary antibody, slides were stained with DAPI and analyzed by confocal microscopy. Levels of nuclear proteins of interest were normalized against DAPI staining. Results: CYP3A4 shRNA knockdown reduced nuclear RagC in MCF-7 cells by 61.2±12.3 % (n=3, p<0.05) relative to a non-targeted shRNA control line. HBB treatment for 6 hours (20 μM) reduced nuclear RagC by 66.1±15.4% (n=4, p<0.001) in MCF-7 cells and by 32.4±5.1% (n=3, p<0.001) in T47D cells. HBB treatment for 6 hours (20 μM) also reduced nuclear ERα by 36.8±11.1% (n=6, p<0.001) in MCF-7 cells and by 32.2±8.1% (n=3, p<0.01) in T47D cells grown in complete media. HBB treatment for 1 hour (20 μM) reduced passive transport of 70 kDa FITC-dextran through the NPC into the nuclear compartment by 17.5±3.6% (n=3, p<0.01) in MCF-7 cells and by 28.3±3.2% (n=3, p<0.01) in T47D cells. Immunofluorescence study results of HBB-mediated reduction of nuclear RagC and ERα were confirmed by Western-blotting analysis of nuclear extraction of T47D cells treated with HBB. Conclusion: These data suggest that the biguanide sensitive enzyme CYP3A4 may play a role in nuclear localization of ERα and RagC, which work in tandem to promote the growth of ER+HER2- breast cancer cells. 1. Cell Chemical Biology. 2017 Oct 19; 24(10) 1259 – 1275. 2. Cell. 2016 Dec 15;167(7):1705-1718 Citation Format: Zhijun Guo, Shaoping Wu, Julissa Molina-Vega, Rafael Castillo, Jaime Barrera, Veronica Johnson, Carol Lange, David Potter. CYP monooxygenases regulate nuclear localization of ERá and mTORC1 component RagC in ER+HER2- breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-023.