mTOR/MYC Axis Regulates O-GlcNAc Transferase Expression and O-GlcNAcylation in Breast Cancer - PubMed (original) (raw)

mTOR/MYC Axis Regulates O-GlcNAc Transferase Expression and O-GlcNAcylation in Breast Cancer

Valerie L Sodi et al. Mol Cancer Res. 2015 May.

Abstract

Cancers exhibit altered metabolism characterized by increased glucose and glutamine uptake. The hexosamine biosynthetic pathway (HBP) uses glucose and glutamine, and directly contributes to O-linked-β-N-acetylglucosamine (O-GlcNAc) modifications on intracellular proteins. Multiple tumor types contain elevated total O-GlcNAcylation, in part, by increasing O-GlcNAc transferase (OGT) levels, the enzyme that catalyzes this modification. Although cancer cells require OGT for oncogenesis, it is not clear how tumor cells regulate OGT expression and O-GlcNAcylation. Here, it is shown that the PI3K-mTOR-MYC signaling pathway is required for elevation of OGT and O-GlcNAcylation in breast cancer cells. Treatment with PI3K and mTOR inhibitors reduced OGT protein expression and decreased levels of overall O-GlcNAcylation. In addition, both AKT and mTOR activation is sufficient to elevate OGT/O-GlcNAcylation. Downstream of mTOR, the oncogenic transcription factor c-MYC is required and sufficient for increased OGT protein expression in an RNA-independent manner and c-MYC regulation of OGT mechanistically requires the expression of c-MYC transcriptional target HSP90A. Finally, mammary tumor epithelial cells derived from MMTV-c-myc transgenic mice contain elevated OGT and O-GlcNAcylation and OGT inhibition in this model induces apoptosis. Thus, OGT and O-GlcNAcylation levels are elevated via activation of an mTOR/MYC cascade.

Implications: Evidence indicates OGT as a therapeutic target in c-MYC-amplified cancers.

©2015 American Association for Cancer Research.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

Figure 1. The PI3K and mTOR pathways regulate OGT and O-GlcNAc levels in cancer cells

(A) MDA-MB-231 (B) SKBR-3 (C) MCF-7 and (D) SUM-159 cells were treated for 16 hours with 0.1% DMSO, 30 μM LY294002 (PI3K inhibitor), 50 nM Rapamycin (mTOR inhibitor) or 30 μM U0126 (MEK inhibitor). Protein lysates were collected for immunoblot analysis and probed with the indicated antibodies. (E) Levels of OGT and c-MYC protein were quantified between different treatments in indicted breast cancer cells and normalized to actin. Mean ± SE represents at least three independent experiments; *p < 0.05.

Figure 2

Figure 2. AKT and mTOR activation are sufficient to elevate OGT and O-GlcNAc levels

(A) Protein lysates were collected from MCF-10A Control (pBabe) or MCF-10A-MYR-AKT cells and analyzed using western blot analysis with indicated antibodies. (B) Top: Lysates from wildtype MEFs or TSC2 (−/−) null MEFs were collected for immunoblot analysis and probed with the indicated antibodies. Bottom: Levels of OGT protein were compared between wild type and TSC2 null MEFs and quantified graphically. Mean ± SE; *p < 0.05. (C) TSC2 −/−MEFs were treated for 16 hours with 0.1% DMSO or 50nM Rapamycin. Protein lysates were collected for immunoblot analysis and probed with the indicated antibodies.

Figure 3

Figure 3. OGT expression in breast cancer cells requires c-MYC

(A) MDA-MB-231 cells stably expressing either control, MYC#1 or MYC#2 shRNA were lysed and immunoblotted with indicated antibodies. Expression of OGT and c-MYC is quantified below. (B) Protein lysates were collected from MCF-10A Control (pWZL) or MCF-10A-c-MYC and were analyzed using western blot analysis with indicated antibodies. Data are quantified (n=3). * p-value <0.05 (C) Cell lysates from control or c-Myc shRNA containing MDA-MB-231 treated with control (DMSO) or Lactacysin (10 μM) were collected and analyzed by immunoblotting. Quantification of OGT/Acin is quantified below OGT blot.

Figure 4

Figure 4. MYC transcriptional target HSP90 is required for OGT/O-GlcNAc regulation

(A–B) Protein lysates from MDA-MB-231 cells were subjected to immunoprecipitation with the indicated antibodies. Pull down is quantified below immunoblots. (C) Cell lysates from control or HSP90 shRNA containing MDA-MB-231 were collected and analyzed by immunobloting. Protein expression is quantified below. (D) MCF10A control or MCF10A-c-MYC cell lysates stably expressing control or HSP90 shRNA were collected and analyzed by immunobloting. Relative OGT and HSP90 protein levels were normalized to actin and quantified (n=3). Quantification represents mean ± S.E. of least three independent experiments * p-value<0.05.

Figure 5

Figure 5. Myc-driven tumors contain elevated OGT and O-GlcNAc levels and require OGT for survival

(A) CommaD and MTEC-Myc cell lysates were collected and analyzed by immunoblotting with indicated antibodies. (B) Expression of OGT and O-GlcNAcylated proteins was compared by immunoblotting of HS-WCE (10 μg/lane) prepared from mammary tissue of normal (unaffected) glands, hyperplastic glands or late-stage carcinomas that originated from 4 independent MMTV-MYC transgenic females (FVB/N strain). Blots are representative of 3 normal mammary glands, 3 hyperplasias, and 4 carcinomas. (C) MCF10A and MTEC-MYC cells were treated with control (DMSO) or Ac-5s-GlcNAc (100 μM) for 48 hours then protein lysates were collected and analyzed by immunoblotting. (D) MCF10A and MTEC-MYC cells were treated with control or Ac-5s-GlcNAc at indicated doses for 48 hours and then stained with crystal violet. (E) MCF10A and MTEC-MYC cells were placed in 3D basement membrane cultures. On day 5, cells were treated with control (DMSO) or Ac-5s-GlcNAc at indicated doses for 48 hours, fixed and stained with indicated antibodies and representative images were taken using confocal microscopy. (F) Model of OGT regulation by mTOR/MYC/HSP90 in cancer cells.

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