Figure 3. An AR-SREBP1-6PGD axis influences prostate cancer cell growth and activity of the pentose phosphate pathway.

(A, B) Knockdown of 6PGD with two distinct siRNAs (si6PGD.1 and si6PGD.2) reduced viability (A) and increased cell death (B) of four prostate cancer cell lines, as assessed using Trypan blue exclusion assays. LNCaP and VCaP cells were evaluated 3 days post-transfection; V16D and MR49F cells were evaluated 5 days post-transfection. Error bars are standard error of the mean (SEM) of triplicate samples and are representative of three independent experiments. Treatment effects were evaluated using ANOVA and Dunnett’s multiple comparison tests (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). (C) Knockdown of 6PGD causes accumulation of intracellular 6PG in LNCaP cells, as determined by mass spectrometry. Results are representative of two independent experiments. Error bars are SEM of triplicate samples. Treatment effect was evaluated using an unpaired t test (p<0.001). Colour key is as in (A). (D) Schematic demonstrating flux of 1,2-¹³C₂ glucose through the PPP and incorporation into Ru5P and R5P. Unlabelled ¹²C carbon is shown as open circles, whereas ¹³C is shown as filled circles. The oxidative and non-oxidative branches of the PPP are indicated in purple and green, respectively. 6PG: 6-phosphogluconate; E4P: erythrose 4-phosphate; F6P: fructose 6-phosphate; G6P: glucose 6-phosphate; GAP: glyceraldehyde 3-phosphate; R5P: ribose 5-phosphate; Ru5P: ribulose 5-phosphate; S7P: sedoheptulose 7-phosphate; X5P: xylulose 5-phosphate. (E) Isotopic steady-state G6P enrichments of LNCaP cells fed with 1,2-¹³C₂ glucose and natural glucose at 1:1 ratio show control and treatments cells were labelled to a similar extent. Error bars are standard deviation (SD). (F) Accumulation of singly (left, m1) and doubly (right, m2) labelled Ru5P produced via the oxidative and non-oxidative branches, respectively, of the PPP. Error bars are SD. (G) Dilution rate (turnover rate) calculated from the accumulation of singly and doubly labelled Ru5P (data from E) using the continuous stirred-tank reactor (CSTR) equation. For statistical analysis of treatment effects, refer to Materials and methods (***p<0.001; NS, not significant). Error bars are SD. (H) Knockdown of 6PGD and androgen receptor (AR) causes increased levels of reactive oxygen species (ROS) in LNCaP, V16D, and MR49F cells. Data was normalised to siCon, which was set to 100%. Error bars are SEM of triplicate samples. Treatment effects were evaluated using ANOVA and Dunnett’s multiple comparison tests (*p<0.05; **p<0.01). (I) ROS production in LNCaP cells in response to si6PGD is reversed by the antioxidant. Trolox data was normalised to siCon in the absence of Trolox, which was set to 100%. Error bars are SEM of triplicate samples. Treatment effects were evaluated using ANOVA and Tukey’s multiple comparison tests (*p<0.05; **p<0.01). Colour key is as in (A).

Figure 3—figure supplement 1. Two distinct 6PGD siRNAs (si6PGD.1 and si6PGD.2) effectively reduce 6PGD expression in LNCaP cells.

Cells were transfected with 12.5 nM of each siRNA for 72 hr, after which 6PGD mRNA was measured by RT-qPCR (A) or 6PGD protein was measured by immunoblotting (B).