Figure 2: Effect of GNPNAT1 knockdown in CRPC-like cells.
From: Inhibition of the hexosamine biosynthetic pathway promotes castration-resistant prostate cancer
shRNA knockdown (KD) of GNPNAT1 in 22Rv1 cells (22R-KD1/5) leads to (a) significant reduction in mRNA (n=5, representative data), (b) loss of protein (∼21 kDa, n=5, representative immunoblot) and (c) significant decrease in product/substrate ratio (4–5 biological replicates) for GNPNAT1, compared with controls (22R-NT). Similarly, KD of GNPNAT1 in LNCaP-ABL cells (L-KD1/5) leads to (d) significant reduction in mRNA (n=3, representative data), (e) loss of protein (n=3, representative immunoblot) and (f) significant decrease in product/substrate ratio (5 biological replicates) for GNPNAT1, compared with controls (L-NT). (g) GNPNAT1 KD in 22Rv1 cells significantly increases proliferation compared with NT controls (5 biological replicates, 12 technical replicates). (h) Same as in g, but for GNPNAT1 KD in LNCaP-ABL cells compared with NT controls (3 biological replicates, 20 technical replicates). (i) 22Rv1 xenograft tumours containing GNPNAT1 KD show a significantly higher growth rate compared with controls (n=7 KD1 and n=8 NT). Y axis represents median tumour volumes in mm3 and associated median absolute deviation (MAD) for each group. (j) Same as in i, but for LNCaP-ABL cells containing GNPNAT1 KD (L-KD1 and L-KD5, 7 mice per condition) and NT controls (L-NT, 5 mice). L-KD cells grew significantly faster than NT controls. (k) Representative images (n=4 NT, n=5 KD1) showing intra-cardiac injection of luciferase containing 22Rv1 cells with GNPNAT1 KD or NT controls in castrated Nod-SCID Gamma mice (n=8 NT, n=10 KD1) showed significantly higher soft tissue metastasis. As shown in the representative figure, 4/5 KD mice and only 1/4 NT mice demonstrated metastatic lesions to the jaw and pelvic bones (white arrows). (l) Magnetic resonance imaging of representative mice shown in k. Yellow arrows show regions of metastasis in the brain as well as bones in the jaw and femur (L=left, R=right) supporting the luciferase data shown in k. ANOVA model was used to compute P-values for panels shown in a,c,d,f–h. For i,j showing in vivo data, a mixed effects linear model was fitted to the data as described under the Methods. For boxplots, the horizontal line represents median value, whereas Whiskers represent either <25 or >75 quartile ranges.
