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, 25 (4), 429-44

cMyc-mediated Activation of Serine Biosynthesis Pathway Is Critical for Cancer Progression Under Nutrient Deprivation Conditions

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cMyc-mediated Activation of Serine Biosynthesis Pathway Is Critical for Cancer Progression Under Nutrient Deprivation Conditions

Linchong Sun et al. Cell Res.

Abstract

Cancer cells are known to undergo metabolic reprogramming to sustain survival and rapid proliferation, however, it remains to be fully elucidated how oncogenic lesions coordinate the metabolic switch under various stressed conditions. Here we show that deprivation of glucose or glutamine, two major nutrition sources for cancer cells, dramatically activated serine biosynthesis pathway (SSP) that was accompanied by elevated cMyc expression. We further identified that cMyc stimulated SSP activation by transcriptionally upregulating expression of multiple SSP enzymes. Moreover, we demonstrated that SSP activation facilitated by cMyc led to elevated glutathione (GSH) production, cell cycle progression and nucleic acid synthesis, which are essential for cell survival and proliferation especially under nutrient-deprived conditions. We further uncovered that phosphoserine phosphatase (PSPH), the final rate-limiting enzyme of the SSP pathway, is critical for cMyc-driven cancer progression both in vitro and in vivo, and importantly, aberrant expression of PSPH is highly correlated with mortality in hepatocellular carcinoma (HCC) patients, suggesting a potential causal relation between this cMyc-regulated enzyme, or SSP activation in general, and cancer development. Taken together, our results reveal that aberrant expression of cMyc leads to the enhanced SSP activation, an essential part of metabolic switch, to facilitate cancer progression under nutrient-deprived conditions.

Figures

Figure 1
Figure 1
The pathways leading to serine biosynthesis are activated under nutrient deprivation conditions. (A, B) Western blot (A) and qRT-PCR (B) analyzed the expression of SSP enzymes and cMyc in Hep3B, SK-hep-1 and Hela cells cultured with or without glucose, glutamine or serine/glycine for 48 h, respectively. β-actin serves as loading control. Data were presented as mean ± SD of three independent experiments. Schematic drawing on the right of A indicates the SSP pathway. *P < 0.05 as compared to control groups. (C, D) Western blot analyzed glutaminolysis enzymes (C) and glycolysis enzymes (D) in Hep3B cells cultured with or without glucose, glutamine or serine/glycine for 48 h, respectively. β-actin serves as loading control. Schematic drawing indicates glutaminolysis pathway (C) and glycolysis pathway (D).
Figure 2
Figure 2
cMyc transactivates the expression of enzymes involved in serine biosynthesis. (A, B) qRT-PCR (A) and western blot (B) analyzed the expression of SSP enzymes in Hep3B cells expressing cMyc or shRNAs against cMyc. β-actin serves as loading control. Data were presented as mean ± SD of three independent experiments. *P < 0.05 as compared to empty vector (EV) or non-target control (NTC) groups. (C) Western blot analyzed the expression of SSP enzymes in P493 cells treated with tetracycline (Tet) for the indicated hours with or without washing off Tet followed by continued culturing for the indicated hours. β-actin serves as loading control. (D) ChIP-qPCR analyzed the occupancy of potential E-boxes by cMyc in the genes of PHGDH, PSAT1 and PSPH in Flag-cMyc-overexpressing Hep3B cells using IgG or anti-cMyc antibody. Data were presented as mean ± SD of three independent experiments. *P < 0.05 as compared to IgG group. (E) Western blot analyzed the expression of cMyc and SSP enzymes in Hep3B cells expressing cMyc shRNAs cultured with or without glucose, glutamine or serine/glycine for 48 h, respectively. β-actin serves as loading control. (F) NMR data for [U-13C] glucose incorporated into GSH and glycine in Hep3B cells overexpressing cMyc. Data were presented as mean ± SD of three independent experiments. *P < 0.05 as compared to the corresponding EV group. (G) Western blot analyzed the expression of glycolysis and glutaminolysis enzymes critical for SSP in Hep3B cells expressing cMyc shRNAs cultured with or without glucose, glutamine or serine/glycine for 48 h, respectively. β-actin serves as loading control.
Figure 3
Figure 3
cMyc-mediated PSPH expression and SSP activation are critical for cancer cell proliferation by regulating GSH, ROS, apoptosis and nucleotide synthesis. (A-C) GSH level and the GSH/GSSG ratio were determined in Hep3B cells expressing shRNAs targeting cMyc (A), PSPH (B), PHGDH or PSAT1 (C). Data were presented as mean ± SD of three independent experiments. *P < 0.05 as compared to NTC group. (D) GSH level and GSH/GSSG ratio were determined in cMyc-overexpressing Hep3B cells with PSPH knockdown by shRNAs. Data were presented as mean ± SD of three independent experiments. *P < 0.05 compared between the indicated groups. (E-G) GSH level and GSH/GSSG ratio (E), cell growth and western blot (F), apoptosis (G) were determined in cMyc-knockdown Hep3B cells with Flag-PSPH overexpression. Data were presented as mean ± SD of three independent experiments. *P < 0.05 compared between the indicated groups. (H-J) ROS (H), apoptosis (I) and cell cycle (J) were analyzed by flow cytometry in Hep3B cells expressing PSPH shRNAs. Data were presented as mean ± SD of three independent experiments. *P < 0.05 as compared to NTC group. (K) NMR data for [U-13C] glucose incorporated into GSH and glycine in Hep3B cells overexpressing PSPH. Data were presented as mean ± SD of three independent experiments. *P < 0.05 as compared to the corresponding EV group. (L) NMR data for [U-13C] glucose incorporated into AMP and UMP in Hep3B cells overexpressing cMyc or PSPH (left), or in Hep3B cells expressing shRNAs against cMyc or PSPH (right). Data were presented as mean ± SD of three independent experiments. *P < 0.05 as compared to the corresponding EV or NTC group. (M, N) Growth curves were determined by trypan blue counting in Hep3B cells expressing NTC or PSPH shRNAs (M), or in those cells further treated with or without 5 mM NAC, 5 mM GSH or 2 mM serine (N). Data were presented as mean ± SD of three independent experiments. *P < 0.05 compared between the indicated groups.
Figure 4
Figure 4
cMyc and PSPH are critical for cancer cell growth under nutrient deprivation conditions. (A) Growth curves were determined by trypan blue counting in Hep3B cells expressing shRNAs against cMyc or PSPH starved of glucose (top) or glutamine (bottom). Data were presented as mean ± SD of three independent experiments. *P < 0.05 compared between the indicated groups. (B, C) Growth analysis (B) and crystal violet assay (C) were performed in Hep3B cells starved of glucose or glutamine without or with supplement of GSH, Nuc (4 ribonucleosides and 4 deoxyribonucleosides) or both. Data were presented as mean ± SD of three independent experiments. *P < 0.05 compared between the indicated groups. (D, E) GSH level, GSH/GSSG ratio, apoptosis and cellular ROS level were determined in Hep3B cells expressing shRNAs targeting PSPH or cMyc starved of glucose (D) or glutamine (E). Data were presented as mean ± SD of three independent experiments. *P < 0.05 as compared with NTC+Glc or NTC+Gln group; #P < 0.05 as compared with NTC–Glc or NTC–Gln group. (F, G) Hep3B cells expressing shRNAs targeting PSPH (F) or cMyc (G) were cultured in medium without glucose (left) or glutamine (right) for 4 days, followed by culturing in complete medium for the indicated days. Cell growth was determined by trypan blue counting. Data were presented as mean ± SD of three independent experiments. *P < 0.05 compared between the indicated groups.
Figure 5
Figure 5
PSPH is vital for the tumorigenesis capacity of cMyc in vitro and in vivo. (A, B) Hep3B cells stably expressing EV or PSPH were injected subcutaneously into nude mice (n = 6 for each group). Tumor sizes were measured starting from 8 days after inoculation (A). Tumors were extracted at the end of experiment, and protein expression of PSPH in tumors was analyzed by western blot using anti-PSPH antibody (B). β-actin serves as loading control. Data were presented as mean ± SEM. *P < 0.05 compared between the indicated groups. (C, D) cMyc-overexpressing Hep3B cells were infected with viruses expressing NTC or PSPH shRNAs. Western blot and trypan blue counting were performed to analyze protein expression (C) and cell growth (D), respectively. Data were presented as mean ± SD of three independent experiments. *P < 0.05 compared between the indicated groups. (E-G) cMyc-overexpressing Hep3B cells with or without PSPH knockdown were injected subcutaneously into nude mice (n = 6 for each group). Tumor growth curves were determined (E). At the end of experiment, incidence of tumor formation was calculated (F) and tumors were extracted followed by western blot analyzing cMyc and PSPH protein expressions in tumors (G). β-actin serves as loading control. Data were presented as mean ± SEM. *P < 0.05 compared between the indicated groups.
Figure 6
Figure 6
Prognostic significance of aberrant PSPH expression in human hepatocellular cancer. (A) qRT-PCR analyzed PSPH mRNA expression in 20 pairs of clinically matched tumor adjacent non-cancerous liver tissues (normal) and human HCC tissues (tumor). mRNA levels were normalized to 18S rRNA. *P < 0.05 as compared to normal tissue group. (B) Western blot analyzed PSPH protein expression in the paired tumor adjacent non-cancerous liver tissues (N) and human HCC tissues (T). GAPDH serves as loading control. (C) Representative IHC analysis of PSPH expression in normal liver tissues (normal) and HCC specimens of different clinical stages (I-IV) was shown. (D) Statistical quantification of the mean optical density (MOD) values of PSPH staining in IHC assay between normal liver tissues and HCC specimens of different clinical stages (I-IV). The MOD of PSPH staining increases as HCC progresses to a higher clinical stage. Data were presented as mean ± SD. *P < 0.05 vs normal control group. (E) Kaplan-Meier curves with univariate analyses for patients with low vs high PSPH expression. (F) Summary: cMyc activates serine biosynthesis pathway directly by regulating SSP enzymes at transcriptional level or indirectly by coordinating glycolysis (PGK1, PGAM1) and glutaminolysis (GOT1, MDH1, ME1) under nutrient stressed conditions, leading to cancer cell survival and proliferation via regulation of ROS, GSH, apoptosis and cell cycle.

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