Glutathione biosynthesis is a metabolic vulnerability in PI(3)K/Akt-driven breast cancer

Abstract

Cancer cells often select for mutations that enhance signalling through pathways that promote anabolic metabolism. Although the PI(3)K/Akt signalling pathway, which is frequently dysregulated in breast cancer, is a well-established regulator of central glucose metabolism and aerobic glycolysis, its regulation of other metabolic processes required for tumour growth is not well defined. Here we report that in mammary epithelial cells, oncogenic PI(3)K/Akt stimulates glutathione (GSH) biosynthesis by stabilizing and activating NRF2 to upregulate the GSH biosynthetic genes. Increased NRF2 stability is dependent on the Akt-mediated accumulation of p21(Cip1/WAF1) and GSK-3β inhibition. Consistently, in human breast tumours, upregulation of NRF2 targets is associated with PI(3)K pathway mutation status and oncogenic Akt activation. Elevated GSH biosynthesis is required for PI(3)K/Akt-driven resistance to oxidative stress, initiation of tumour spheroids, and anchorage-independent growth. Furthermore, inhibition of GSH biosynthesis with buthionine sulfoximine synergizes with cisplatin to selectively induce tumour regression in PI(3)K pathway mutant breast cancer cells, both in vitro and in vivo. Our findings provide insight into GSH biosynthesis as a metabolic vulnerability associated with PI(3)K pathway mutant breast cancers.

Figure 1. Oncogenic signaling through Akt2(E17K) stimulates GSH biosynthesis

a, Cells serum-starved and stimulated with IGF-1 were immunoblotted for the indicated proteins (data is representative of three independent experiments). b, Proliferation of cells grown in the absence of serum and growth factors was determined using the sulforhodamine B (SRB) assay (data are from one experiment that was independently repeated five times with similar results (Supplementary Table 1)). c, Fold changes of metabolite abundances by LC-MS/MS in serum-starved MCF10A cells expressing AKT2(E17K) vs. AKT2 (left), and relative levels of GSH, GSSG, cystine, and cysteine (right) (n = 3 technical replicates from a single independent metabolomics experiment; the experiment was repeated twice with similar results (Supplementary Table 1)). d, Schematic of the glutathione biosynthetic pathway. e, Incorporation of U-¹³C₅-glutamine into -GluCys and GSH over 1, 3, and 8 h in serum-starved cells (n = 3 technical replicates from a single metabolomics experiment (Supplementary Table 1)). f, GSH levels in breast cancer cell lines from the NCI-60 metabolomics data set (n = 3 biologically independent replicates). All error bars represent s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 by a two-sided Student’s t-test. Unprocessed original scans of blots are shown in Supplementary Figure 6.

Figure 2. Enhanced GSH biosynthesis confers resistance to oxidative stress

a–d, Cells were serum-starved for 20–24 h in the presence or absence of (a–b) 1 μM GSK690693 or (c–d) 50 μM BSO, followed by treatment with 500 μM H₂O₂ for 4 h. Cells were immunoblotted for the indicated proteins (data is representative of three independent experiments). e, Cells were serum-starved for 20–24 h in the presence or absence of 1 μM GSK690693 or 50 μM BSO, followed by treatment with 500 μM tert-butyl hydroperoxide (tBH) or 10 μM LCS-1 for 24 h. Cell viability was measured using a propidium iodide-based plate reader assay (EV, AKT2(E17K): n = 4; AKT2, PIK3CA: n = 3; PIK3CA(E545K), PIK3CA(H1047R): n = 5; n represents number of biologically independent replicates (Supplementary Table 1)). All error bars represent s.e.m. ***P < 0.001 by a two-sided Student’s t-test. Unprocessed original scans of blots are shown in Supplementary Figure 6.

Figure 3. Akt2(E17K) activates Nrf2 to up-regulate the GSH biosynthetic genes

a, mRNA levels were measured by qRT-PCR and are expressed as fold changes relative to MCF10A AKT2 cells (n = 3 biologically independent replicates (Supplementary Table 1)). b, Nrf2 was knocked down over 72 h, and ARE-luciferase activity was assayed in cells serum-starved for 20-24 h (data are from one experiment that was independently repeated two times with similar results (Supplementary Table 1)). c, Serum-starved cells treated with 20 μg/ml cycloheximide (CHX) were immunoblotted for the indicated proteins (data is representative of three independent experiments). d, Cells serum-starved in the presence or absence of 1 μM GSK690693 for 20–24 h were immunoblotted for the indicated proteins (data is representative of three independent experiments). e, p21 was knocked down in MCF10A AKT2(E17K) cells over 48 h, with or without the expression of shRNA-resistant HA-FLAG-p21 (#). ARE-luciferase activity was assayed in cells serum-starved for 20-24 h (data are from one experiment that was independently repeated five times with similar results (Supplementary Table 1)). f, ARE-luciferase activity was assayed in cells serum-starved for 20–24 h in the presence or absence of 1 μM GSK690693 and 25 μM SB415286 (data are from one experiment that was independently repeated two times with similar results (Supplementary Table 1)). g, h, Patient tumors from TCGA BRCA data set were stratified by (g) PI3K pathway mutation status and (h) Akt pS473 levels (See Supplementary Figure 4d). PIK3CA: PIK3CA mutation, PTEN: PTEN mutation or copy number loss, AKT1: AKT1 mutation, Combined: alterations in PIK3CA or PTEN or AKT1, WT: wild-type PIK3CA, PTEN, and AKT1. The heat map represents microarray/RPPA Z-scores for the indicated genes/proteins, which are expressed relative to values in the WT or Akt pS473 low group. All error bars represent s.e.m. **P < 0.01, ***P < 0.001 by a two-sided Student’s t-test. Unprocessed original scans of blots are shown in Supplementary Figure 6.

Figure 4. GSH biosynthesis is required for the PI3K/Akt-driven initiation of tumor spheroids

a, Cells were grown as spheroids in 3D culture for 14 days in the presence or absence of 50 μM BSO. Representative images from two biologically independent experiments are shown. b, Cells were grown as colonies in soft agar in the presence or absence of 50 μM BSO for 4 weeks. Viable colonies were stained with iodonitrotetrazolium chloride. Representative images from two biologically independent experiments are shown (left). The number of colonies was counted with MatLab (right, data are from one experiment that was independently repeated two times with similar results (Supplementary Table 1)).

Figure 5. BSO synergizes with cisplatin to selectively induce cell death and tumor regression in PI3K pathway mutant breast cancer cells

a, Cells were treated with vehicle or 50 μM BSO for 48 h prior to treatment with cisplatin (CDDP) for 48 h, in the presence or absence of 50 μM EUK-134 or 1 mM N-acetyl cysteine (NAC). Cell viability was measured using a propidium iodide-based plate reader assay (n = 3 biologically independent replicates (Supplementary Table 1)). b, c, T47D (b) or MDA-MB-231 (c) xenografts were grown in nude mice treated with vehicle (T47D: n = 5, MDA-MB-231: n = 5), BSO (T47D: n = 4, MDA-MB-231: n = 4), CDDP (T47D: n = 4, MDA-MB-231: n = 4), or a combination of one week of BSO pre-treatment followed by CDDP (T47D: n = 5, MDA-MB-231: n = 7) (n represents number of biologically independent tumors (Supplementary Table 1)). All error bars represent s.e.m. *P<0.05, **P<0.01, ***P < 0.001 by a two-sided Student’s t-test.