4.1 GSTP1 expression in NSCLC cell lines
Little is known about the involvement of GSTP1 in lung tumorigenesis. First we determined protein expression of GSTP1 in A549, H460, H292, H1437 and a patient cell line (HLCB1), obtained from a lung biopsy. As expected GSTP1 basal protein expression varied among cell lines. The highest GSTP1 expression could be observed in A549 and H292 cells, moderate expression could be observed in H460 cells and low expression in HLCB1 and H1437 cells (fig. 6A). Before testing TLK-117 in vitro we tested whether TLK-TLK-117 effectively inhibited GSTP1. GSTP1 activity inhibition was measured by using a recombinant GSTP1 protein. 1 µg of recombinant GSTP1 was incubated with two different TLK-117 doses 50µM and 100µM. A dose dependent decrease was observed following TLK administration. Additionally we determined GST activity in cell lysates from in A549, H460, H292, H1437 and HLCB1 cells that were treated with 50µM TLK-117 for 24-hours and re-stimulated 6 hours prior to the harvest. TLK-117 had a significant inhibitory effect on GST activity only in A549 cells. In all other cell lines there was a trend towards a decrease in GST activity, which was not significant (fig.
6C). Several studies have shown that despite of the presence of an abundant amount of oxygen, a large amount of lactate is produced in cancer cells, also known as the Warburg effect or aerobic glycolysis. Lactate production is an important hallmark of cancer cell metabolism. Intracellular lactate expression significantly decreased in A549 cells and trended towards a decrease in H292 cells (fig.
6D). In H460, H1437 and HLCB1 cells intracellular lactate expression seemed not affected (fig. 6D),
Figure 6: GSTP1 expression and the effect of TLK-117 on NSCLC cell lines. A) Basel GSTP1 protein expression in untreated cells. B) Determination of the effect of TLK-117 on GSTP1 and enzyme activity. *** p<0.02, *p<0.01 (One-Way ANOVA and Turkeys multiple comparison) compared to respective control group. C) GST activity measured in cells treated with TLK-117 or control. D) Measurement of lactate in cells treated with TLK-117 or control medium (with 0.02%
DMSO). Insert: *ONE-Way ANOVA p<0.05 compared to respective control group of untreated cells.
A
4.2 Pharmacological GSTP1 inhibition in NSCLC cell lines
To determine the effect of 117 on cancer cell migration, cells were treated with 50 µM TLK-117 every 24 hours during a time course of 72 hours. Migration was measured with a scratch wound assay by applying scratches to cell monolayers of A549, H460, H292, H1437 and HLCB1 cells.
Scratches were photographed and measured to assess cell migration. In A549 and H292 cells as shown in figure 7 A/C and F, migration was significantly reduced by TLK-117. In H460, H1437 and HLCB1 cells TLK-117 cell migration was not affected. Respective migration of NSCLC cells is quantified in figure 7F.
4.3 Protein
S-glutathionylation (PSSG) in a NSCLC cell line
In order to determine the potential role of GSTP1 in catalyzing PSSG in KRAS induced tumorigenesis we isolated total protein and RNA from A549 cells, a human epithelial lung cancer cell line, with a KRASG12D mutation following pharmacological inhibition of GSTP1.
Cells were exposed to 10, 50 and 100 µM TLK-117 for 24 hours. To determine the effect of TLK-117 on overall S-glutathionylation of proteins in A549 cells, SDS–PAGE analysis was
Figure 7: Impact of TLK-117 treatment on cell migration. A) A549, B) H460, C) Human cells obtained from a patient with NSCLC, D) H1437 cells, and E) H292 cells treated with 50µM TLK-117 or vehicle control. F) Scratch assay carried out with A549, H460, H292, H1437 and primary Human patient cells treated with TLK-117 or control (cell culture medium with 0.2%
DMSO). Cell migration was quantitatively evaluated by measuring the distance between the scratch edges. A549 (migration of control is 56.5% and TLK-117 treated cells is 27.03%), H460 ((migration of control is 56.5% and TLK-117 treated cells is 27.03%). Insert: *Student T test p<0.05 compared to respective control group of untreated cells.
0
proteins in A549 cell when treated with 50µM TLK-117 (fig. 8A). In addition following immunoprecipitation of GSH we observed a decrease in S-glutathionylation of PKM2. (fig. 8B). GSTP1 protein abundance was not affected by TLK-117 treatment. Actin was examined to ensure equal loading.
4.4 KRAS
G12Dinduced tumorigenisis
To further investigate the role of KRAS induced tumorigenesis, the role of GSTP1 on its involvement in the Warburg effect and metabolism we developed a triple transgenic mouse expressing CCSP-rtTA/tetON-Cre/LSL-KRASG12D. Mice were fed doxycycline over a time course of 6 weeks (fig.
11A). Following doxycycline administration H&E staining showed numerous tumor formations in the lung of the KRASG12D mice. In control mice no tumors were observed (fig 9B). An important cancer hallmark is the production of lactate. We assessed lactate production in mice expressing the oncogene KRASG12D and compared them to mice lacking the oncogene. Lactate production was significantly increased in KRASG12D mice compared to KRASWt mice (fig. 9C). Next we evaluated the influence of KRASG12D on gene expression
of
metabolism-associatedgenes. Quantitative analysis of gene expression demonstrated that PKM2, HKII, HKIII, Glut2 and PFKFB3 were significantly up regulated in KRASG12D mice (fig. 9 D/E). Only LDHA gene expression was significantly down regulated. Evaluation of protein expression by Western blot revealed an increase of proteinsFigure 8 S-glutathionylated proteins in A549 cells. Each lane represents 100 µg total protein extracted. (A) Western blot analysis with a GSH antibody. (B) A549 cell lysates were immune-precipitated using anti- GSH antibody. Blots were probed with antibodies against PKM2, GSTP1 and actin.
associated with metabolism (PKM2, HKII, PFKFB3, G6PD, PD and GSTP1), in mice expressing effects GST activity, lactate production and migration in human cancer cells, which associated proteins (fig. S1). There were no significant differences in protein expression of GSTP1, Pyruvate Kinase M2 (PKM2), Pyruvate Dehydrogenase (PD), 6-phosphofructo-2 kinase/fructose-2,6-biphosphatase 3 (PDKFB3) and Glucose- 6-poshpate dehydrogenase (G6PD), all important mediators in the Glycolysis and Pentose Phosphate Pathway. We have previously shown that following ablation Figure 9 KRASG12 induced tumorigenesis in C57BL6 mice. A) Schematics
showing the experimental set up after DOX administration. B) Histopathological analysis of lung section from KRASWT and KRASG12D mice by H&E staining (magnification: x10). C) Analysis of lactate expression in BALF, obtained from KRASWT and KRASG12D mice. D/E. Quantification of mRNA levels for metabolic genes (PKM2, H6PD, G6PDX, HKII, HKIII, Glut1, Glut2, Glut3, PFKF, PFKL, LDHA, LDHB PDHKI and PFKFB3) in lung tissue homogenates by q-PCR.
Results are expressed as fold change compared with WT and reflect SEM from 1 independent experiment (n = 7 Wt, 3 KRASG12D). F. Protein analysis for GSTP1, PKM2, PD, HKII, G6PD and PFKFB3 by SDS-Page and Western blot, for equal amounts of protein (20 µg). Actin was used as a loading control.
Insert: *p < 0.05 (Student T test) compared with respective controls.
Day 0 W 1 W 2 W 3 W 4 W 5 W 6
of GSTP1, A549 cells (harbor a KRASG12D mutation) expressed less G6PD, PD and PFKP (fig. S2). In Adcre induced KRASG12D we did not observe a decrease in G6PD and PD protein levels. Next we evaluated tumor formation by analyzing lung histopathology, using the Hematoxylin and Eosin staining to visualize physiological differences in KRASWt (control group) and KRASG12D mice. Histological evaluation of the lung sections demonstrated that KRASG12D mice treated with TLK-117 showed less tumors tissue compared to control treated animals. No tumor burden was observed in control mice (fig 10B). In addition to these findings we investigate the effect of TLK-117 on lactate production and GST-activity in vivio. As previously mentioned,
TLK-117 decreased lactate production and GST-activity in human lung cancer cell lines.
However evaluation of GST activity in lung lysates of TLK treated animals did not show differences (fig10C).
4.6 GSTP1 in NSCLC
It is reported that GSTP1 expression is significantly elevated in human tumors. We created tissue micro arrays (TMA) of 106 patients with adenocarcinomas. Tumor tissue and adjacent normal tissue
Figure 10: Intratracheal delivery of TLK-117 in KRASG12D induced tumorigenesis. A) Schematics showing the TLK-117 treatment regime given after AdCre delivery. Mice were harvested after 6 weeks (n=3). B) Mouse lung sections from wild type and KRASG12D mice, instilled with TLK-117 or the vehicle, after staining by Hematoxylin and Eosin (magnification: x10). C) GST activity measured form mouse lungs from animals (KRASWT, KRASG12D) treated with TLK-117 or vehicle.
A AdCre
Day 0
Treatment every 3 days with 25mg/kg TLK-‐117
W 1 W 2 W 3 W 4 W W
PSSG or GSTP1 expression and PSSG. TMAs representing the 106 individual patients with NSCLC were visually scored for GSTP1 expression. A representative picture is shown in figure 11A of a negative and a positive patient for GSTP1 expression and PSSGH. Scoring the TMAs revealed that 41% of the tumor tissue was positive GSTP1 expression (fig.11C). In 47 % of the cases, tumor tissue was scored negative for GSTP1 expression. 12% of the tumor tissue stained intermediately for GSTP1. In normal tissue only 5% stained positive for GSTP1 (fig.11B). Interestingly PSSG correlated with GSTP1 status of the tumor tissue (fig. 11A).
C
Total=106
4.72% GSTP1 positive 86.79% GSTP1 negative 8.49% GSTP1 intermediate
Normal tissue
Total=106
40.57% GSTP1 positive 47.17% GSTP1 negative 12.26% GSTP1 intermediate
Tumor tissue
Normal tissue
B
Tumor tissue
GSTP1
A
HLCPS1 HLCPS2PSSG DAPI
HLCPS1 HLCPS2
Figure 11: Non-Small cell lung carcinoma tissue micro array. A) IHC staining for GSTP1 expression (left). Fluorescence staining for protein s-glutathionylation (PSSG) (right). B) Percentages of normal tissue from patients, positive or negative for GSTP1 expression (n=106). C) Percentages of tumor tissue, positive or negative for GSTP1 expression (n=106).