Research ArticleCancer Metabolism

Oncogenic PI3K promotes methionine dependency in breast cancer cells through the cystine-glutamate antiporter xCT

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Science Signaling  19 Dec 2017:
Vol. 10, Issue 510, eaao6604
DOI: 10.1126/scisignal.aao6604
  • Fig. 1 Proliferation of breast cancer cell lines in MetHcy+ media.

    (A) Schematic of the methionine cycle and transsulfuration pathway. Met, methionine; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; Hcy, homocysteine; Ser, serine; Cys, cysteine; αKB, alpha-ketobutyrate; MAT, methionine adenosyltransferase; AHCY, adenosylhomocysteinase; MTR, 5-methyltetrahydrofolate-homocysteine methyltransferase; CBS, cystathionine-beta-synthase; CTH, cystathionine gamma-lyase; AU, arbitrary units. (B) Cell lines were screened for their growth in MetHcy+ media for 4 days, and the proliferation of cells was determined using the sulforhodamine B (SRB) assay (n = 3 independent replicates). (C) Proliferation data from (B) were fit to an exponential curve to calculate the growth rate of each cell line in MetHcy+ media. (D) Pearson correlation of the growth rates of the cell lines in MetHcy+ media with their doubling time in Met+Hcy media. All error bars represent SEM.

  • Fig. 2 Oncogenic PIK3CA confers methionine dependency to MCF10A cells.

    (A) Cells were grown in MetHcy+ media for 4 days, and the proliferation of cells was determined using the SRB assay (n = 3 biologically independent replicates). (B) Cells were grown in Met+Hcy or MetHcy+ media for 2 days, and cell death was measured using a propidium iodide (PI)–based plate-reader assay (n = 3 biologically independent replicates). (C) Cells were grown in Met+Hcy or MetHcy+ media for 2 days and were then immunoblotted for the indicated proteins (data are representative of three independent experiments). PARP, poly(ADP-ribose) polymerase. All error bars represent SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 by a two-sided Student’s t test.

  • Fig. 3 Decreased xCT expression and cystine uptake is associated with increased methionine dependency.

    (A) Spearman correlation of the growth rates of the panel of breast cancer cell lines in MetHcy+ media from Fig. 1C with their expression of a selected set of methionine and cysteine metabolism genes (see Materials and Methods for full list). Values above the red dashed line represent P < 0.05. (B) SLC7A11 and MAT1A mRNA abundances were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and are expressed as fold changes relative to MCF10A empty vector–transfected (EV) cells (n = 3 biologically independent replicates). (C) SLC7A11 mRNA abundance was measured by qRT-PCR (n = 3 biologically independent replicates). (D) Cells were grown in Met+Hcy media and immunoblotted for the indicated proteins (data are representative of two independent experiments). (E) [14C]-cystine uptake was measured over 5 min and is expressed as fold changes relative to MCF10A EV cells (n = 5 biologically independent replicates). All error bars represent SEM. **P < 0.01 by a two-sided Student’s t test.

  • Fig. 4 xCT Ser26 is phosphorylated upon PI3K pathway activation.

    (A) Comparison of the consensus AKT substrate motif with the amino acid sequence surrounding xCT Ser26. (B) MCF10A PIK3CA(+/+) and PIK3CA(H1047R/+) knock-in cells expressing EV, xCT–hemagglutinin (HA)-FLAG, or xCT(S26A)–HA-FLAG were serum-starved. HA immunoprecipitates from cell lysates were immunoblotted for the indicated proteins (data are representative of three independent experiments). (C) Serum-starved MCF10A cells expressing EV or xCT–HA-FLAG were treated with 100 nM insulin for the indicated times. HA immunoprecipitates from cell lysates were immunoblotted for the indicated proteins (data are representative of three independent experiments). (D) Serum-starved MCF10A PIK3CA(+/+) and PIK3CA(H1047R/+) knock-in cells were treated with vehicle, 1 μM GDC-0941, 1 μM GDC-0068, 1 μM MK2206, 1 μM Torin 1, or 100 nM rapamycin for 15 min. HA immunoprecipitates from cell lysates were immunoblotted for the indicated proteins (data are representative of three independent experiments). (E) xCT–HA-FLAG or xCT(S26A)–HA-FLAG were immunoprecipitated from human embryonic kidney–293T cells treated with 1 μM MK2206 and incubated with active glutathione S-transferase (GST)–AKT1 for an in vitro kinase assay. Lysates were immunoblotted for the indicated proteins (data are representative of three independent experiments). (F) Release of glutamate into the media by the indicated cell lines was measured over 24 hours (n = 3 biologically independent replicates). IP, immunoprecipitation; IB, immunoblot; WCL, whole cell lysate; p, phospho. All error bars represent SEM. *P < 0.05 by a two-sided Student’s t test.

  • Fig. 5 xCT and cystine uptake functionally contribute to the methionine dependency phenotype.

    (A) Cells were grown in MetHcy+ medium for 4 days, and the proliferation of cells was determined using the SRB assay (n = 3 biologically independent replicates). (B) Cells were grown in Met+Hcy or MetHcy+ media for 2 days, and cell death was measured using a PI-based plate-reader assay (n = 3 biologically independent replicates). (C and D) Cells were grown in (C) MetHcy+, Cys2Hcy+, or Met Cys2Hcy+ media or (D) MetHcy+ media, with or without 1 mM sulfasalazine (SSA), for 4 days, and the proliferation of cells was determined using the SRB assay (n = 3 to 4 biologically independent replicates). (E) Cells were grown in MetHcy+ media for 6 days, and the proliferation of cells was determined using the SRB assay (n = 3 biologically independent replicates). All error bars represent SEM. *P < 0.05 by a two-sided Student’s t test. #P < 0.05 by a paired t test.

  • Fig. 6 Oncogenic PIK3CA alters methionine utilization through the transsulfuration pathway.

    (A) Steady-state total pool sizes of cystathionine measured by liquid chromatography–tandem mass spectrometry (LC-MS/MS) in MCF10A cells growing in standard culture media (n = 3 biologically independent replicates). (B) Schematic of [U-13C5]-methionine labeling into the methionine cycle ([M+4] methionine) or the transsulfuration pathway ([M+4] cystathionine), which are detected by LC-MS/MS. (C) Total ion counts of [M+4] cystathionine in MCF10A cells labeled with [U-13C5]-methionine for 1 hour (n = 3 biologically independent replicates). (D) Fractional labeling of [M+4] cystathionine in MCF10A cells labeled with [U-13C5]-methionine for 24 hours and treated with or without 1 μM GDC-0941 (n = 3 biologically independent replicates). (E) Total ion counts of [M+4] cystathionine and [M+4] methionine in MCF10A cells labeled with [U-13C5]-methionine for 24 hours and treated with or without 1 μM GDC-0941 (n = 3 biologically independent replicates). All error bars represent SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 by a two-sided Student’s t test.

  • Table 1 PIK3CA and PTEN mutational status of breast cancer cell lines studied.

    N.D., not determined; WT, wild-type.

    GroupCell linePIK3CA mutation status*PTEN mutation status*PTEN protein abundance
    AHCC70WT267delT (F90fs*9)Negative
    HCC38WTWTNegative
    SUM149WTWTNegative
    BMDA-MB-231WTWTPositive
    HCC1806WTWTPositive
    HCC1143WTWTN.D.
    SKBR3WTWTPositive
    BT-549WT821delG (V275fs*1)Negative
    ZR-75-1WT323T > G (L108R)Positive
    SUM-1593140A > T (H1047L)WTPositive
    CT47D3140A > G (H1047R)WTPositive
    MCF71633G > A (E545K)WTPositive
    MDA-MB-468WT253 + 1G > T (A72fs*5)Negative

    *Reported in Catalogue of Somatic Mutations in Cancer database [http://cancer.sanger.ac.uk; (64)].

    †Reported in refs. (50) and (65).

    ‡Reported in ref. (66).

    Supplementary Materials

    • www.sciencesignaling.org/cgi/content/full/10/510/eaao6604/DC1

      Fig. S1. Characterization of MCF10A cells stably expressing PIK3CA variants.

      Fig. S2. SLC7A11 and MAT1A expression correlate with methionine dependency in breast cancer cell lines.

      Fig. S3. Endogenous xCT phosphorylation is inhibited by PI3K and AKT inhibitors.

      Fig. S4. xCT functionally contributes to the methionine dependency phenotype.

      Fig. S5. Inhibition of AKT signaling by GDC-0941 during metabolic labeling with [U-13C5]-methionine.

      Data File S1. Spearman rank correlation between methionine dependency and expression of methionine and cysteine metabolism genes.

    • Supplementary Materials for:

      Oncogenic PI3K promotes methionine dependency in breast cancer cells through the cystine-glutamate antiporter xCT

      Evan C. Lien, Laura Ghisolfi, Renee C. Geck, John M. Asara, Alex Toker*

      *Corresponding author. Email: atoker{at}bidmc.harvard.edu

      This PDF file includes:

      • Fig. S1. Characterization of MCF10A cells stably expressing PIK3CA variants.
      • Fig. S2. SLC7A11 and MAT1A expression correlate with methionine dependency in breast cancer cell lines.
      • Fig. S3. Endogenous xCT phosphorylation is inhibited by PI3K and AKT inhibitors.
      • Fig. S4. xCT functionally contributes to the methionine dependency phenotype.
      • Fig. S5. Inhibition of AKT signaling by GDC-0941 during metabolic labeling with [U-13C5]-methionine.
      • Legend for Data File S1

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      Technical Details

      Format: Adobe Acrobat PDF

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      Other Supplementary Material for this manuscript includes the following:

      • Data File S1 (Microsoft Excel format). Spearman rank correlation between methionine dependency and expression of methionine and cysteine metabolism genes.

      Citation: E. C. Lien, L. Ghisolfi, R. C. Geck, J. M. Asara, A. Toker, Oncogenic PI3K promotes methionine dependency in breast cancer cells through the cystine-glutamate antiporter xCT. Sci. Signal. 10, eaao6604 (2017).

      © 2017 American Association for the Advancement of Science

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