Downregulation of TRIB3 enhances the sensitivity of lung cancer cells to amino acid deprivation by suppressing AKT activation

Abstract

Tribbles pseudokinase 3 (TRIB3), a member of the mammalian Tribbles family, is implicated in multiple biological processes. This study aimed to investigate the biological functions of TRIB3 in lung cancer and its effect on amino acid-deprived lung cancer cells. TRIB3 mRNA expression was elevated in lung cancer tissues and cell lines compared to normal lung tissues and cells. TRIB3 knockdown markedly reduced the viability and proliferation of H1299 lung cancer cells. Deprivation of amino acids, particularly arginine, glutamine, lysine, or methionine, strongly increased TRIB3 expression via ATF4 activation in H1299 lung cancer cells. Knockdown of TRIB3 led to transcriptional downregulation of ATF4 and reduced AKT activation induced by amino acid deprivation, ultimately increasing the sensitivity of H1299 lung cancer cells to amino acid deprivation. Additionally, TRIB3 knockdown enhanced the sensitivity of H1299 cells to V-9302, a competitive antagonist of transmembrane glutamine flux. These results suggest that TRIB3 is a pro-survival regulator of cell viability in amino acid-deficient tumor microenvironments and a promising therapeutic target for lung cancer treatment.

Keywords: AKT; ATF4; TRIB3; amino acid depletion; lung cancer.

Figure 1

TRIB3 expression is elevated in lung cancer tissues and cells. A. TRIB3 mRNA levels were determined by PCR using an OriGene cDNA array consisting of normal lung tissue (n = 5) and lung cancer tissue of different pathological grades (stage I, n = 25; stage II, n = 6; stage III, n = 10; and stage IV, n = 2). PCR products were visualized on 2% agarose gel containing ethidium bromide (upper panel). The band intensities were quantified by ImageJ software, normalized to β-actin, and presented as log2-fold-change relative to normal lung tissue (low panel). B. TRIB3 mRNA levels of HBE, H69, A549, H460, Lu99, H358, H1299, and EBC-1 cell lines were assessed by RT-PCR analysis (left panel). Band intensities were quantified using ImageJ software, normalized to β-actin, and plotted as fold-change relative to HBE cell line (right panel).

Figure 2

TRIB3 mRNA expression in individual amino acid-deficient condition. A. H1299 cells were deprived of each of the 19 amino acids, excluding cystine, for 6 or 24 h, while cystine deprivation was conducted for 6 or 12 h. TRIB3 mRNA level was determined by using RT-PCR analysis. B. The band intensities of TRIB3 mRNA were quantified by ImageJ software, normalized to β-actin, and the data are presented as the fold change relative to CTL (n = 3; *P<0.05; **P<0.01; ***P<0.001; ns, not significantly different). Arg, arginine; Asn, asparagine; Asp, aspartic acid; Cys, cystine; Gln, glutamine; Glu, glutamic acid; Gly, glycine; His, histidine; Hyp, hydroxyproline; Ile, isoleucine; Leu, leucine; Lys, lysine; Met, methionine; Phe, phenylalanine; Pro, proline; Ser, serine; Thr, threonine; Trp, tryptophan; Tyr, tyrosine; Val, valine.

Figure 3

Amino acid deprivation upregulates the TRIB3 expression in an ATF4-dependent manner. A. H1299 cells were transfected with an empty vector or GFP-tagged ATF4 for 36 h. mRNA levels were assessed using RT-PCR analysis. B. H1299 cells were transfected with CTL or ATF4 siRNAs for 36 h. mRNA expression levels were determined by quantitative real-time PCR. Data were normalized to β-actin and presented as percentage of their respective controls. C. H1299 cells were co-transfected with CTL or ATF4 siRNAs along with GFP-ATF4 plasmids for 36 h. Protein levels were determined by western blot analysis. D, E. H1299 cells were transfected with CTL or ATF4 siRNAs for 24 h and then deprived of the indicated amino acids for 12 h. mRNA and protein band intensities were quantified using ImageJ software, normalized to β-actin, and plotted as fold-change relative to CTL (n = 3; *P<0.05; ***P<0.001; ns, not significantly different). Arg, arginine; CM, complete medium; CTL, control; Gln, glutamine; Lys, lysine; Met, methionine.

Figure 4

TRIB3 knockdown enhanced the sensitivity of lung cancer cells to amino acid deprivation by suppressing AKT activation. A. H1299 cells were transfected with CTL or TRIB3 siRNAs for 48 h. mRNA levels were assessed using RT-PCR analysis. B. H1299 were transfected with CTL or TRIB3 siRNAs for 24 h and then the transfected cells were reseeded at a density of 1,000 (1×), 2,000 (2×), and 3,000 (3×) cells/well in a 6-well plate. Colony formation was monitored over the following ten days. C. H1299 cells were transfected with CTL or TRIB3 siRNAs for 24 h and then deprived of the indicated amino acids for 24 h. Cell viability was measured using the MTT assay. D, E. H1299 cells were transfected with CTL or TRIB3 siRNA for 24 h and deprived of the indicated amino acids for 12 h. mRNA and protein levels were assessed using RT-PCR and western blot analyses, respectively. F. H1299 cells were treated with 5 μM MK-2206 in combination with the indicated amino acid deprivations for 12 h. Protein levels were determined by western blot analysis. G. H1299 cells were treated with 5 μM MK-2206 in combination with the indicated amino acid deprivations for 24 h. Cell viability was measured using the MTT assay. mRNA and protein band intensities were quantified using ImageJ software, normalized to β-actin, and plotted as fold-change relative to CTL (n = 3; *P<0.05; **P<0.01; ***P<0.001). Arg, arginine; CM, complete medium; CTL, control; Gln, glutamine; Lys, lysine; Met, methionine.

Figure 5

TRIB3 knockdown increases the sensitivity of lung cancer cell to V-9302 by suppressing AKT activation. A. H1299 cells were treated with the indicated concentrations of V-9302 for 12 h. B. H1299 cells were transfected with CTL or TRIB3 siRNA for 24 h, and then treated with 20 μM V-9302 for 12 h. C. H1299 cells transfected with CTL or TRIB3 siRNAs for 24 h were treated with 20 μM V-9302 for 24 h. Cell viability was measured using the MTT assay. mRNA and protein levels were assessed using RT-PCR and western blot analyses, respectively. mRNA and protein band intensities were quantified using ImageJ software, normalized to β-actin, and plotted as fold-change relative to CTL (n = 3; *P<0.05; **P<0.01; ***P<0.001).