Selective eradication of cancer displaying hyperactive Akt by exploiting the metabolic consequences of Akt activation
- University of Illinois at Chicago, United States
- Jesse Brown VA Medical Center, United States
Figures
Figure 1 with 5 supplements
Akt activation in PTEN-deficient prostate cancer cells elevates oxygen consumption and intracellular ROS levels.
The human CaP cells DU145, PC3, and LNCaP were seeded in 10% FBS and harvested after two days to measure various parameters. (A) Immunoblot showing the expression levels of PTEN, P-Akt (ser 473), …
Figure 1—figure supplement 1
Mitochondrial membrane potential measured as JC-1 aggregate to monomer ratio.
The data represent the mean ±SEM of three independent quantification experiments performed in triplicate. *p<0.05 versus DU145.
Figure 1—figure supplement 2
Immunoblot showing the expression levels of the detoxifying enzymes catalase, MnSOD, and Cu/ZnSOD (ß actin as a loading control) in all three CaP cell lines.
https://doi.org/10.7554/eLife.32213.004
Figure 1—figure supplement 3
Level of Sesn3 mRNA relative to that of actin in CaP cells, as assessed by quantitative RT-PCR.
The data represent the mean ±SEM of three independent quantification experiments performed in triplicate. ***p<0.0001 versus DU145. (1s4-5) DU145 cells were transiently transfected with hSesn3 or …
Figure 1—figure supplement 4
Immunoblot showing the expression levels of sestrin 3 (SESN3) and ß actin as a loading control.
https://doi.org/10.7554/eLife.32213.006
Figure 1—figure supplement 5
Level of ROS, as assessed by flow cytometry, after incubation with H2DCFDA.
The data represent the mean ±SEM of three independent experiments performed in triplicate. *p=0.02, **p=0.01 versus the control for each cell line.
Figure 2 with 15 supplements
ROS inducers and the combination of a ROS inducer and rapamycin induce CaP PTEN-deficient cell death in vitro and eradicate their tumors in vivo.
(A) CaP cell lines were incubated with 2-ME for 24 hr, the cells were fixed and apoptosis was quantified by DAPI staining. The data represent the mean ±SEM of three independent experiments performed …
Figure 2—figure supplement 1
Glutathione levels (Left) and GSH/GSSG ratio (Right) in CaP cells after 8 hr incubation with DMSO or PEITC 6 μM.
The data represent the mean ±SEM of two independent experiments performed in duplicate.
Figure 2—figure supplement 2
(Left) Apoptosis was measured on live cells by caspase 3/7 activity assay after drug treatment: 2-ME 1 µM (14 hr) or 20 nM rapamycin (5 hr) followed by 6 µM PEITC (8 hr).
The data represent the mean ±SEM of two independent experiments performed in quadruplicate. (Right) Cell death was assessed on fixed cells by DAPI staining after drug treatment: 2-ME 1 µM (20 hr) or …
Figure 2—figure supplement 3
CaP cell lines were incubated with BSO (2 mM) for 36 and 42 hr, the cells were fixed and cell death was quantified by PI staining.
The data represent the mean ±SEM of three independent experiments performed in triplicate.
Figure 2—figure supplement 4
NADP+/NADPH ratio in CaP cells.
The data represent the mean ±SEM of three measurements performed in duplicate.
Figure 2—figure supplement 5
After modulation of SESN3 expression, PC3 and DU145 cells were treated with PEITC (0, 3 and 6 µM) for 17 hr, the cells were fixed and cell death was assessed by DAPI staining.
The data represent the mean ±SEM of three independent experiments performed in triplicate. *p<0.05, **p<0.01 versus the control for each cell line.
Figure 2—figure supplement 6
DU145, PC3, and LNCaP cells were incubated with N-acetylcysteine (100 µM NAC) for 2 hr prior to 17 hr of incubation with PEITC (6 µM) in the presence of NAC or not.
The graphs represent the cell death measured by PI staining (Left) or ROS levels after incubation with H2DCFDA (Right). The data represent the mean ±SEM of three independent experiments performed in …
Figure 2—figure supplement 7
Immunoblot showing the expression of PTEN (and HA-Tag), and ß-actin as a loading control after PTEN was downregulated in DU145 cells (1: control shLacZ, 2: shPTEN) or overexpressed in PC3 and LNCaP cells (3: control pBP, 4: pBP-PTEN).
https://doi.org/10.7554/eLife.32213.015
Figure 2—figure supplement 8
PTEN expression determines the levels of ROS and oxygen consumption.
PTEN was downregulated in DU145 cells (1: control shLacZ, 2: shPTEN) or overexpressed in PC3 and LNCaP cells (3: control pBP, 4: pBP-PTEN). (A, B) Relative ROS levels: cells were incubated with …
Figure 2—figure supplement 9
Cells were incubated with PEITC or rapamycin/PEITC for 17 hr and scored for apoptosis 17 hr later by DAPI staining.
The data represent the mean ±SEM of three independent experiments performed in triplicate. *p<0.05, **p<0.001 versus the control for each cell line. ##p<0.05 versus PEITC.
Figure 2—figure supplement 10
mAkt was stably overexpressed in DU145.
Cells were then incubated for 17 hr with PEITC or rapamycin/PEITC before measurement of relative cytosolic ROS level (Left) or cell death (Right).
Figure 2—figure supplement 11
ROS levels and ROS-induced cell death are Akt-dependent.
Akt1 and Akt2 were knocked down in PC3 and LNCaP cells. Once cell lines were established, mAkt was re-expressed in these cells. Cells were incubated with PEITC for 17 hr, and then cytosolic ROS …
Figure 2—figure supplement 12
Rapamycin elevates Akt activity.
(A–C) DU145 (A), PC3 (B), and LNCaP cells (C) were treated with rapamycin (100 nM). Total cell extracts were prepared at different time points as indicated and subjected to immunoblotting with …
Figure 2—figure supplement 13
Rapamycin increases the ROS levels induced by PEITC.
When required, CaP cells were incubated with 20 nM rapamycin (RAPA) for 8 hr before the addition of PEITC (3 µM). After 17 hr of incubation with PEITC (±RAPA), the ROS levels in live cells after …
Figure 2—figure supplement 14
Torin, not rapamycin, decreases the OCR and ROS levels in PTEN-deficient CaP cells.
PC3 and LNCaP cells were incubated for 8 hr with rapamycin (RAPA, 20 nM) or torin (250 nM) before measurement of the OCR (Left) or cytoplasmic ROS levels (Right). The data represent the mean ±SEM of …
Figure 2—figure supplement 15
In vivo therapeutic effects of rapamycin +PEITC in mice inoculated with DU145 prostate cancer cells.
Twenty-four nude mice were injected subcutaneously with DU145 cells in both flanks and randomly divided into four groups (four mice per group, eight tumors per group) for treatment with PEITC, …
Figure 3 with 3 supplements
Effect of rapamycin, PEITC, and a combination of rapamycin and PEITC on cell proliferation, cell death, survival, and the tumors of Pbsn-Cre4;Ptenf/f mice.
(A) Tissue lysates were prepared from prostates isolated from four control mice (Ptenf/f or Pbsn-Cre4) and four Pbsn-Cre4;Ptenf/f mice. Immunoblot analysis shows the expression levels of PTEN, Akt-P …
Figure 3—figure supplement 1
Graphs showing the body weights of control (left) and Pbsn-Cre4;Ptenf/f (right) mice at the end-point (8 months).
The numbers of treated mice in the control group were vehicle (n = 6), rapamycin (RAPA, n = 12), PEITC (n = 8), and RAPA +PEITC (n = 8), and the numbers of treated mice in the Pbsn-Cre4;Ptenf/f …
Figure 3—figure supplement 2
Graphs showing the relative prostate weights of the control mice sacrificed at 8 months.
The box plots represent the 25th to 75th percentiles (boxes) with the median, and the whiskers represent the maximum and minimum values. No significant differences were detected.
Figure 3—figure supplement 3
Representative histopathological images.
Representative images of different prostate tumor grades in the anterior lobe of the prostate of untreated mice (-), rapamycin +PEITC-, and NAC-treated mice. The individual images were derived from …
Figure 4 with 1 supplement
Early treatment of Pbsn-Cre4;Ptenf/f mice with rapamycin +PEITC inhibits tumor growth and increases survival, even after treatment was halted for 6 months.
(A) Schematic of mice treatment: control and Pbsn-Cre4;Ptenf/f mice were randomly divided into four groups of four to 10 mice at 2 months of age, and they received IP drug injections as indicated in …
Figure 4—figure supplement 1
(A) Graphs showing the body weights of control (left) and Pbsn-Cre4;Ptenf/f (right) mice at 6 months.
The numbers of treated mice in the control group were vehicle (n = 5), RAPA (n = 5), PEITC (n = 5), and RAPA +PEITC (n = 7), and the numbers of treated mice in the Pbsn-Cre4;Ptenf/f group were …
Figure 5 with 11 supplements
Depletion of HK2 in PTEN-deficient CaP cells inhibits proliferation, oncogenesis, and tumorigenesis while overcoming chemoresistance.
(A) DU145, PC3, and LNCaP cells were treated with MK-2206 (0.5 μM - 24 hr) to inhibit Akt. The immunoblot shows the protein levels of P-Akt, total Akt, HK2, and ß-actin as a loading control. (B–G) …
Figure 5—figure supplement 1
Total protein was extracted from CaP cells and subjected to immunoblotting with HK1, HK2, and ß-actin as a loading control.
https://doi.org/10.7554/eLife.32213.032
Figure 5—figure supplement 2
Expression levels of HK2 and ß-actin as a loading control in PC3 cells in which Akt1 and Akt2 were stably knocked down.
https://doi.org/10.7554/eLife.32213.033
Figure 5—figure supplement 3
Immunoblot showing expression of HK2 (and ß-actin as loading control) in CaP cells where PTEN is either downregulated (DU145) or overexpressed (PC3 and LNCaP).
https://doi.org/10.7554/eLife.32213.034
Figure 5—figure supplement 4
HK1 was stably knocked down in PC3 cells after HK2 knockdown.
The immunoblot shows the expression levels of HK1, HK2, and actin as a loading control in PC3 control, HK1 knockdown, HK2 knockdown, and double HK1 and HK2 knockdown cells. The graph shows the total …
Figure 5—figure supplement 5
Cell proliferation after HK1 and/or HK2 deletion in PC3 cells.
The data represent the mean ±SEM of three independent experiments performed in triplicate. ***p<0.0001 versus LacZsh cells on day 6.
Figure 5—figure supplement 6
Etoposide-induced cell death is Akt-dependent.
(A) After mAkt overexpression, DU145 cells were treated with etoposide for 24 hr before cell death was assessed by PI staining on live cells with Celigo Image cytometer. (B) Akt1 and Akt2 were …
Figure 5—figure supplement 7
Data analysis for in vivo therapeutic study described in Figure 5H.
(A) Graphs showing the relative xenograft tumor weights of mice treated with control diet/vehicle, control diet/etoposide, DOX diet/vehicle, and DOX diet/etoposide. The data represent the average …
Figure 5—figure supplement 8
Effect of HK2 knockdown on ECAR.
PC3 cells expressing an inducible control (Scr) or HK2 shRNA were exposed to 900 ng/ml DOX for 5 days for HK2 deletion before analysis. ECAR was measured after HK2 deletion using the Seahorse XF96e …
Figure 5—figure supplement 9
Effect of HK2 knockdown on oxygen consumption.
PC3 cells expressing an inducible control (Scr) or HK2 shRNA were exposed to 900 ng/ml DOX for 5 days for HK2 deletion before analysis. OCR was measured after HK2 deletion using the Seahorse XF96e …
Figure 5—figure supplement 10
Effect of HK2 knockdown on ROS levels.
PC3 cells expressing an inducible control (Scr) or HK2 shRNA were exposed to 900 ng/ml DOX for 5 days for HK2 deletion before analysis. Cells were incubated with H2DCFDA, and the level of …
Figure 5—figure supplement 11
Effect of HK2 knockdown on PEITC-induced cell death.
PC3 cells expressing an inducible control (Scr) or HK2 shRNA were exposed to 900 ng/ml DOX for 5 days for HK2 deletion before analysis. After HK2 knockdown with DOX, cells were treated with PEITC …
Figure 6
Deletion of HK2 in the prostates of Pbsn-Cre4;Ptenf/f mice extends survival and inhibits tumor growth by inhibiting proliferation and increasing cell death.
(A) Tissue lysates were prepared from prostates isolated from three control mice (Ptenf/f;HK2f/f), three Pbsn-Cre4;Ptenf/f mice and three Pbsn-Cre4;Ptenf/f;HK2f/f/ mice. The immunoblot shows the …
Author response image 1
Author response image 2
Cells were allowed to plate in 48-well plates (1x104 cells/well) for 16h and then media was switched to media with 11mM glucose or 11mM Galactose for 24h prior to addition of DMSO or Etoposide (50μM) for 14h (caspase 3/7 assay) or 24h (PI staining on unfixed cells).
https://doi.org/10.7554/eLife.32213.047
Author response image 3
Cells were allowed to plate in 48-well plates (1.5X104 cells/wells) for 16h and then pre-treated with NAC (100μM) for 2h prior to addition of Etoposide (50μM) for 24h.
At end-point, PI and Hoechst 33342 were added to wells for 30min and plates were visualized with Celigo Image Cytometer, and cell death was calculated.
Tables
Table 1
Histopathologic analysis relative to Figure 3.
https://doi.org/10.7554/eLife.32213.030| Grade | |||||
|---|---|---|---|---|---|
| No PIN | Low grade PIN | High grade PIN | Microinvasive carcinoma | Invasive carcinoma | |
| Pbsn-Cre4;Ptenf/f* | 66% | 33% | |||
| Pbsn-Cre4;Ptenf/f R + P† | 33% | 16% | 33% | 16% | |
| Pbsn-Cre4;Ptenf/f + NAC‡ | 25% | 75% | |||
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*The anterior lobes of prostates from untreated mice were analyzed by histopathology at 8 months (percentage of mice with highest grade is indicated).
†The anterior lobes of prostates from mice treated at 4 months with rapamycin and PEITC (R + P) were analyzed by histopathology at 8 months (percentage of mice with highest grade is indicated).
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‡The anterior lobes of prostates from mice treated at 4 months with NAC were analyzed by histopathology at 8 months (percentage of mice with highest grade is indicated).
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.32213.044
