Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma

Abstract

Autophagy is a lysosome-dependent degradative pathway frequently activated in tumor cells treated with chemotherapy or radiation. Whether autophagy observed in treated cancer cells represents a mechanism that allows tumor cells to survive therapy or a mechanism for initiating a nonapoptotic form of programmed cell death remains controversial. To address this issue, the role of autophagy in a Myc-induced model of lymphoma generated from cells derived from p53ER(TAM)/p53ER(TAM) mice (with ER denoting estrogen receptor) was examined. Such tumors are resistant to apoptosis due to a lack of nuclear p53. Systemic administration of tamoxifen led to p53 activation and tumor regression followed by tumor recurrence. Activation of p53 was associated with the rapid appearance of apoptotic cells and the induction of autophagy in surviving cells. Inhibition of autophagy with either chloroquine or ATG5 short hairpin RNA (shRNA) enhanced the ability of either p53 activation or alkylating drug therapy to induce tumor cell death. These studies provide evidence that autophagy serves as a survival pathway in tumor cells treated with apoptosis activators and a rationale for the use of autophagy inhibitors such as chloroquine in combination with therapies designed to induce apoptosis in human cancers.

Figures

Figure 1. Effects of CQ with and without p53 activation on the regression of Myc/p53ERTAM lymphomas.

(A) CQ impairs tumor growth. Cells from a primary Myc/p53ERTAMtumor were harvested and passaged in vivo in 6 syngeneic C57BL/6×129F1 mice. Cells were injected subcutaneously into the flanks of mice. When tumors reached a volume of more than 1,000 mm3, mice were assigned to daily PBS i.p. or 60 mg/kg/d CQ i.p. Results shown are mean ± SD daily tumor volumes and are representative of multiple experiments. *P < 0.05. (B) CQ delays tumor recurrence after p53-induced tumor regression. Myc/p53ERTAM cells were injected subcutaneously into the flanks of 18 C57BL/6×129F1 mice. Once tumors reached a volume of more than 1,500 mm3, mice were assigned to daily treatment (arrow) with 1 mg/d TAM i.p. plus saline (TAM/PBS) or 1 mg/d TAM i.p. plus 60 mg/kg/d CQ i.p. (TAM/CQ). Results shown are daily tumor volumes (mean ± SD) for each group from a representative experiment. *P < 0.05; **P < 0.005.

Figure 2. Effects of p53 activation with and without CQ on autophagosome accumulation.

(A) Time course of changes in autophagosomes during tumor regression. Electron micrographs of lymphoma tissues collected after 96 hours of PBS or CQ treatment alone and at 8 hours, 24 hours, and 48 hours after initiation of TAM treatment. Arrows, double-membrane vesicles. Scale bars: 2 μm. Original magnification, ×10,000. (B) Quantification of tumor cells with increased autophagosomes. Electron microscopy was performed on Myc/p53ERTAM lymphomas at the indicated time points under the treatment protocols given. The number of autophagosomes per nonapoptotic cell was determined as described in Methods (mean ± SD). *P < 0.005.

Figure 3. Effects of p53 activation with and without CQ on apoptosis.

(A) CQ-induced cell death after p53 activation. Electron micrographs of lymphoma tissues collected before TAM treatment and after 48 hours of TAM/PBS or TAM/CQ. Scale bars: 10 μm. Original magnification, ×4,000. (B) Quantification of tumor cells with morphological evidence of apoptosis. Electron microscopy (EM) was performed on Myc/p53ERTAM lymphomas at the indicated time points under the treatment protocols given. The percentage of apoptotic cells per field at ×4000 magnification was determined as described in Methods (mean ± SD). (C) TUNEL staining was performed on tissue obtained from treated tumors at the indicated time points. Representative images were obtained by fluorescent microscopy. Red fluorescence indicates TUNEL-positive cells. Blue fluorescence indicates nuclear DAPI staining. (D) Quantification of TUNEL-positive tumor cells. The percentage of TUNEL-positive cells per high-powered field is reported as mean ± SD.

Figure 4. Effects of p53 activation with and without CQ on LC3 relocalization.

(A–C) GFP-LC3 fluorescence. Green, GFP-LC3; blue, DAPI. (A) A bulk population of primary Myc/p53ERTAM lymphoma cells with stable expression of the GFP-LC3 fusion protein was treated with and without 250 nM hTAM and with and without 5 μM CQ. Cell culture medium was changed daily. Cells were fixed and imaged using fluorescence microscopy at 24 and 48 hours. Representative images of cells at 48 hours are presented. (B) Quantification of the percentage of cells with more than 4 GFP-LC3 puncta per cell (punctate) compared with those with less than 4 GFP-LC3 puncta per cell (diffuse) treated with increasing doses of CQ with and without hTAM at 24 hours. (C) CQ modulates autophagy in a p53-independent manner. p53+/+ and p53–/– MEFs expressing GFP-LC3 were treated with CQ. Cells were fixed and imaged at 24 hours.

Figure 5. Effects of p53 activation with and without knockdown of ATG5 on tumor cell death.

(A) Western blot against ATG5 of lysates from Myc/p53ERTAM/vector cells (V), Myc/p53ERTAM/HC cells (HC), Myc/p53ERTAM/shATG5 h2 cells (h2), and Myc/p53ERTAM/shATG5 h7 cells (h7). Actin was used as a loading control. (B) Activation of p53 in HC and shATG5 cells. HC, h2, and h7 lymphoma cells were treated with 200 nM hTAM daily. (C) Activation of p53 plus methyl pyruvate (MP). HC, h2, and h7 lymphoma cells were treated daily with hTAM plus 1 mM MP. (B and C) Viable cell number as determined by trypan blue exclusion was counted daily. Reported values are mean ± SD of triplicate samples from a representative experiment.

Figure 6. Effects of CQ or ATG5 knockdown in combination with p53 activation on tumor cell death.

(A) Activation of p53 plus CQ in HC cells. HC lymphoma cells were treated daily with 200 nM hTAM plus 1 μM, 3 μM, or 5 μM CQ. (B) Activation of p53 plus CQ in h2 lymphoma cells. HC cells were treated daily with hTAM and compared with h2 cells treated daily with hTAM or hTAM plus 1 μM, 3 μM, or 5 μM CQ. (C) Activation of p53 plus CQ in h7 lymphoma cells. HC cells were treated daily with hTAM and compared with h7 cells treated daily with hTAM or hTAM plus 1 μM, 3 μM, or 5 μM CQ. (A–C) Viable cell number as determined by trypan blue exclusion was counted daily. Reported values are mean ± SD of triplicate samples from a representative experiment.

Figure 7. Effects of alkylating chemotherapy with and without CQ in Myc/p53ERTAMlymphomas.

(A) Cells from a primary tumor were harvested and passaged in vivo in syngeneic C57BL/6×129F1 mice. Myc/p53ERTAMcells were injected subcutaneously into the flanks of mice. Once tumors had reached more than 1,700 mm3, mice were matched for tumor size and treated with 50 mg/kg cyclophosphamide i.p. once followed by either daily PBS i.p. (top panel) or 60 mg/kg/d CQ i.p. (bottom panel) for a total of 13 days. Daily tumor volumes are shown for individual mice. CY, cyclophosphamide. (B) Time to tumor recurrence for cyclophosphamide/PBS- and cyclophosphamide/CQ-treated mice (mean ± SD). *P = 0.003. (C) GFP-LC3 fluorescence. Green, GFP-LC3; blue, DAPI. A bulk population of primary Myc/p53ERTAM lymphoma cells with stable expression of the GFP-LC3 fusion protein was treated with 50 μM MNNG with or without 10 μM CQ. Cell culture medium was changed daily. Cells were fixed and imaged using fluorescence microscopy at 24 and 48 hours. Representative images of cells at 48 hours are presented. (D) Quantification of the percentage of cells with more than 4 GFP-LC3 puncta per cell (punctate) compared with those with less than 4 GFP-LC3 puncta per cell (diffuse) at 24 and 48 hours after treatment. (E) MNNG with and without CQ treatment in HC and shATG5 cells. On day 0, 2 × 106 cells/ml of HC, h2, and h7 lymphoma cells were plated and treated with 20 μM MNNG (red) or 20 μM MNNG plus 5 μM CQ (blue). Viable cell number as determined by trypan blue exclusion was counted after 24 hours and 48 hours of treatment. Reported values are means ± SD of triplicate samples of a representative experiment.