A critical role of glucose-6-phosphate dehydrogenase in TAp73-mediated cell proliferation

Abstract

The pentose phosphate pathway (PPP) provides ribose and NADPH that support biosynthesis and antioxidant defense. Our recent findings suggest that the p53-related protein TAp73 enhances the PPP flux. TAp73 stimulates the expression of glucose-6-phophate dehydrogenase (G6PD), the rate-limiting enzymes of the PPP. Through this regulation, TAp73 promotes the accumulation of macromolecules and increases cellular capability to withstand oxidative stresses. TAp73 also regulates other metabolic enzymes, and the relative importance of these targets in TAp73-mediated cell growth is not well understood. Here we show that, like in other cell lines, TAp73 is required for supporting proliferation and maintaining the expression of G6PD in the human lung cancer H1299 cells. Restoration of G6PD expression almost fully rescues the defects in cell growth caused by TAp73 knockdown, suggesting that G6PD is the major proliferative target of TAp73 in these cells. G6PD expression is elevated in various tumors, correlating with the upregulation of TAp73. These results indicate that TAp73 may function as an oncogene, and that G6PD is likely a focal point of regulation in oncogenic growth.

Keywords: G6PD; ROS; TAp73; cell proliferation; metabolism; p73; pentose phosphate pathway.

Figure 1. p73 isoforms and the pentose phosphate pathway. (A) Schematic representation of p73 isoforms and p53. Each p73 isoform class comprises various splicing variants (α, β, γ, etc.) that differ in their C-terminal regions. TA, transactivation domain; DBD, DNA-binding domain; OD, oligomerization domain; SAM, sterile α motif. (B) The pentose phosphate pathway and glycolysis. FBP, fructose 1,6-biphosphate; PEP, phosphoenolpyruvate; ROS, reactive oxygen species. Glycolytic pathway is indicated in blue, and oxidative pentose phosphate pathway is indicated in green.

Figure 2. Regulation of G6PD expression, NADPH levels, and DNA synthesis by p73 in H1299 cells (A and B) H1299 cells were transfected with control (−), p73, and G6PD siRNA as indicated. Protein expression was analyzed (A) and cell proliferation is shown (B). (C) NADPH levels in H1299 cells treated with control, p73, and G6PD siRNA as indicated. (D and E) H1299 cells were transfected with control, p73, and G6PD siRNA as indicated. Cells were assayed for BrdU incorporation (D). Representative images of cells stained with BrdU (E).

Figure 3. Overexpression of G6PD rescues growth defects of p73-depleted cells. (A and B) Protein expression (A) and G6PD activity (B) in H1299 cells stably overexpressing G6PD or vector control in the presence or absence of p73 siRNA are shown. (C and D) H1299 cells stably overexpressing G6PD or vector control were treated with p73 or control siRNA as indicated. Cell proliferation (C) and representative images of cells stained with crystal violet at day 6 (D) are shown

Figure 4. p73 depletion fails to affect ROS in H1299 cells (A) ROS levels in H1299 cells transfected with indicated siRNA. (B) H1299 stably overexpressing G6PD or vector control were transfected with p73 siRNA or control siRNA as indicated. ROS levels were analyzed. (C) H1299 stably overexpressing G6PD or vector control were transfected with p73 siRNA or control siRNA as indicated. Cells were treated with or without 100 µM or 250 µM H₂O₂ for 24 h, and cell viability was analyzed by trypan blue staining.

Figure 5. G6PD and p73 are frequently overexpressed in many human cancers (A and B) Box plot comparing TP73 and G6PD transcript levels in diffuse large B-cell lymphoma,^, uterine corpus leiomyosarcoma, lung adenocarcinoma,^, and their counterparts. The graphs were derived from published data available through the ONCOMINE database. The differential gene expression data are centered on the median of expression levels and plotted on a log2 scale. The P value was calculated using a 2-sample t test. Whiskers indicate minimum and maximum data values that are not outliers. The number of samples (n) in each class is indicated.