c-MYC responds to glucose deprivation in a cell-type-dependent manner

Abstract

Metabolic reprogramming supports cancer cells' demands for rapid proliferation and growth. Previous work shows that oncogenes, such as MYC, hypoxia-inducible factor 1 (HIF1), have a central role in driving metabolic reprogramming. A lot of metabolic enzymes, which are deregulated in most cancer cells, are the targets of these oncogenes. However, whether metabolic change affects these oncogenes is still unclear. Here we show that glucose deprivation (GD) affects c-MYC protein levels in a cell-type-dependent manner regardless of P53 mutation status. GD dephosphorylates and then decreases c-MYC protein stability through PI3K signaling pathway in HeLa cells, but not in MDA-MB-231 cells. Role of c-MYC in sensitivity of GD also varies with cell types. c-MYC-mediated glutamine metabolism partially improves the sensitivity of GD in MDA-MB-231 cells. Our results reveal that the heterogeneity of cancer cells in response to metabolic stress should be considered in metabolic therapy for cancer.

Figure 1

Glucose deprivation differentially affects c-MYC protein levels in different cells. (a–c) Western blot detection of c-MYC levels in HEK293T (a), HeLa (b) and MDA-MB-231 (c) cells exposed to GD (0.5 mM) for the indicated intervals. (d and e) Quantitative RT-PCR detection of PDK1, PFK1, LDHA, GLUT1 and c-MYC levels in HeLa (d) and MDA-MB-231 (e) cells under GD condition for 12 h. Values represent the relative induction normalized to 18S. *P<0.05, **P<0.01 versus mock. Data of three independent experiments are shown.

Figure 2

Glucose deprivation differentially affects c-MYC protein stability in HeLa and MDA-MB-231 cells. (a) Western blot detection of c-MYC in HeLa and MDA-MB-231 cells treated with CHX (0.1 mM) and MG-132 (10 μM) in the presence or absence of 25 mM glucose for 12 h. (b) HEK293T cells cotransfected with c-MYC-Flag and HA-Ub were treated with MG-132 in the presence or absence of glucose for 12 h. Ubiquitination of c-MYC was determined. (c) Western blot detection of c-MYC in HeLa cells treated with Bafilomycin A1 (Baf) (500 nM), Leupeptin (10 μg/ml) or 3-MA (1 mM) in the presence or absence of glucose for 12 h. (d) HeLa cells were treated with CHX for the indicated time in the presence or absence of glucose. Representative immunoblots of c-MYC in three independent experiments were shown. Bottom, quantification of the c-MYC levels was shown. (e and f) HeLa (e) and MDA-MB-231 (f) stable cells ectopically expressing c-MYC-Flag were treated with MG-132 in the presence or absence of glucose. Exogenous c-MYC stability was examined. (g) Western blot detection of c-MYC in HeLa stable cells expressing WT HA-SKP2 or HA-tagged dominant negative mutant HA-SKP2(ΔF-box) under GD condition for 12 h. (h) HEK293T cells were transfected with WT-c-MYC-Flag or c-MYC(T58A)-Flag. Cell lysates were subjected to immunoprecipitation and phosphorylation of c-MYC was examined. (i) Western blot detection of c-MYC in HeLa stable cells expressing FBXW7 treated as in g.

Figure 3

Cell-type-dependent response of c-MYC to GD is not caused by p53 mutation. (a) Western blot detection of c-MYC in HeLa control or P53 KO cells under GD condition for 12 h. (b) Western blot detection of c-MYC in HeLa stable cells expressing P53(R280K) under GD condition for the indicated intervals. (c–e) Western blot detection of c-MYC in MDA-MB-231 stable cells expressing WT-P53 (c), Hep3B stable cells expressing WT-P53 (d) or P53(R280K) (e) under GD condition for 12 h.

Figure 4

Glucose deprivation increases c-MYC transcription partially through ERK signaling pathway in MDA-MB-231 cells. Quantitative RT-PCR (a) and Western blot (b and c) detection of c-MYC in MDA-MB-231 cells treated with different chemical inhibitors for 12 h in the medium with or without glucose. The indicated chemical inhibitors are AMPK inhibitor P5499 (10 μM), p38/MAPK inhibitor SB 203580 (10 μM), PI3K/AKT inhibitor Wortmannin (10 μM), ERK/MEK inhibitor U0126 (10 μM), SIRT inhibitor NAM (1 mM) and HDAC inhibitor VPA (1 mM). (d–f) HeLa cells were treated and detected as that of MDA-MB-231 in a–c. Quantitative RT-PCR values were relative to the DMSO group with 25 mM Glc and normalized to 18S. Data of three independent experiments are shown.

Figure 5

Both SIRT1 and PI3K regulates c-MYC phosphorylation and the following protein stability under GD condition. (a) Western blot detection of c-MYC in HeLa cells treated with LY294002 (10 μM) under GD condition for 12 h. (b) Western blot detection of c-MYC in HeLa stable cells expressing dominant negative mutant p85-DN under GD condition for 12 h. (c) Western blot detection of c-MYC in HeLa cells treated with MK-2206 (10 μM) or SB-216763 (10 μM) under GD condition for 12 h. (d) Western blot detection of c-MYC in HeLa stable cells expressing constitutively active mutant AKT-CA or dominant negative mutant AKT-DN under GD condition for 12 h. (e) Effects of NAM and Wortmannin on phosphorylated Ser/Thr of c-MYC immunoprecipitated from HEK293T cell lysates under GD condition. (f and g) Western blot detection of c-MYC in HeLa cells treated with SRT1720 (10 μM) (f) or AGK2 (10 μM) (g) under GD condition for 12 h. NAM was used as a positive control. (h) MYC-Flag was cotransfected with His-SIRT1 or His-SIRT2 into HEK293T cells. Phosphorylated Ser/Thr of immunoprecipitated c-MYC was examined. (i) MYC-Flag was cotransfected with His-SIRT1 or kinase-dead His-SIRT1-HY into HEK293T cells and maintained under 25 mM glucose or GD condition for 12 h. Phosphorylated Ser/Thr of immunoprecipitated c-MYC was examined.

Figure 6

c-MYC-mediated glutamine metabolism is involved in the resistance to GD in MDA-MB-231 cells. (a) HeLa and MDA-MB-231 cells were maintained under the indicated medium for different intervals. Cell viabilities were examined by MTT assay. (b) HeLa stable cells expressing MYC were maintained under GD condition for different intervals and cell viabilities were examined by MTT. (c) MDA-MB-231 stable cells expressing c-MYC(ΔN) were treated as in b. (d) GLS1-3′-UTR was transfected into HeLa or MDA-MB-231 cells and maintained under the indicated medium for 36 h. Luciferase activities were examined. *P<0.05, **P<0.01. (e and f) Western blot detection of GLS1 and c-MYC in HeLa (e) and MDA-MB-231 (f) cells under GD condition for different intervals. (g) MDA-MB-231 cells were treated with CB-839 (10 μM) under GD condition for different intervals and cell viabilities were examined by MTT. (h) MDA-MB-231 stable cells infected with lenti-shGLS1 were treated as in (b). (Right) Examination of GLS1 knockdown efficiency by quantitative RT-PCR. Values are normalized to 18S and relative to control group. MTT in (a–c, g and h) were repeated three times, and values are shown as fold changes versus 0 h group.