Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma (original) (raw)
Tumor regression induced by p53 is enhanced by CQ. The p53ERTAM fusion gene consists of a transcriptionally inactive hormone-binding region of the ER (ERTAM) (14) fused to the entire coding region of the Trp53 tumor suppressor gene (15). In mice homozygous for knockin alleles encoding p53ERTAM (Trp53KI/KI), p53-dependent gene expression is induced by systemic administration of TAM (13). To generate B cell lymphomas, bone marrow cells were harvested from Trp53KI/KI mice and were transduced in vivo with the LMycSN retrovirus as previously described (16, 17).
To examine the in vivo antitumor effect of CQ treatment in the absence of p53 activation, tumor cells were harvested from a Myc/p53ERTAM lymphoma and were injected subcutaneously into the flanks of syngeneic mice. After tumor formation, mice were matched for tumor volumes and randomly assigned to receive PBS or 60 mg/kg/d CQ i.p. (Figure 1A). This dose is near the previously reported LD50 of 68–78 mg/kg (18). Mice treated at these doses had no observed toxicity. Treatment with 60 mg/kg/d CQ i.p. resulted in a modest but reproducible impairment in the rate of tumor growth compared with that in PBS controls. However, tumor regression was not observed in any of the CQ-treated animals. Daily treatment with the CQ derivative hydroxychloroquine (HCQ) at 60 mg/kg/d resulted in similar impairment in tumor growth (data not shown).
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/p53ERTAM_tumor 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.
To determine the effect of CQ treatment after therapeutic activation of apoptosis, Myc/p53ERTAM lymphomas were generated and mice were matched for tumor volume and randomly assigned to receive either daily TAM plus PBS i.p. (TAM/PBS) or daily TAM plus 60 mg/kg/d CQ i.p. (TAM/CQ). TAM treatment led to nuclear localization of the p53ERTAM fusion protein and the rapid induction of apoptosis (data not shown). TAM/PBS-treated tumors regressed over several days, but all tumors resumed growth despite continued TAM therapy. TAM/CQ treatment resulted in a significant delay in tumor recurrence in comparison with TAM/PBS treatment (Figure 1B). In separate experiments, 60 mg/kg/d HCQ i.p. also resulted in significantly delayed recurrence (data not shown). In all mice treated over the course of 4 separate experiments, complete clinical regression of tumor in response to therapy was observed in 81% of mice treated with TAM/CQ or TAM/HCQ compared with 8% of mice treated with TAM/PBS (P < 0.005).
Autophagy is activated in tumor cells that survive p53-dependent apoptosis. The activation of autophagy following oncogenic or chemotherapeutic stress has been observed in multiple cancer cell lines (11, 14). To further understand the effects of CQ and/or p53 activation on tumor cell autophagy in this tumor model, electron microscopy was performed on lymphoma tissue obtained from mice treated with either PBS or 60 mg/kg/d CQ i.p. in the absence of p53 activation and from mice treated with either TAM/PBS or TAM/CQ at sequential time points after p53 activation. While cells that contained rare autophagosomes were found in PBS-treated tumors, tumor cells with multiple autophagosomes were easily visualized in Myc/p53ERTAM tumors treated with CQ for 96 hours in the absence of p53 activation (Figure 2A). CQ disrupts lysosomal structure and function (19), preventing effective autophagic degradation, leading to the accumulation of ineffective autophagosomes (11, 20, 21). Eight hours after the first administration of TAM, p53 activation induced morphological changes characteristic of apoptosis in the majority of tumor cells, including chromatin condensation, nuclear and cytoplasmic blebbing, and nuclear fragmentation. By focusing on cells with intact nuclear morphology and cytoplasmic membranes, numerous tumor cells were identified in tumors from both treatment groups that had survived p53-induced apoptosis. In nonapoptotic cells, TAM/PBS treatment resulted in the appearance of large double-membrane vesicles by 24 hours and 48 hours. By 48 hours, most tumor cells had smaller residual autophagosomes as tumors began to recur. In contrast, viable tumor cells in TAM/CQ-treated tumors accumulated autophagosomes at 8 hours following p53 activation, and by 24 hours, viable tumor cells were fewer in number and contained numerous ineffective autophagosomes with undegraded or partially degraded contents. By 48 hours, TAM/CQ-treated tumors consisted largely of the remains of apoptotic cells.
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.
Quantification of the number of autophagosomes per nonapoptotic cell (Figure 2B) demonstrated that p53 activation alone (TAM/PBS) resulted in an 8-fold increase in the number of autophagosomes compared with tumors treated with PBS alone by 24 hours after the initiation of TAM treatment. The appearance of numerous autophagosomes occurred earlier in TAM/CQ-treated tumors. Both in the absence of p53 activation and at each time point following p53 activation, CQ treatment resulted in a significant increase in the number of autophagosomes per nonapoptotic cell.
CQ treatment enhances p53-induced apoptosis. Low magnification (×4,000) electron micrographs of tumors treated with PBS alone compared with tumors treated with TAM/PBS or TAM/CQ at 48 hours after the initiation of TAM treatment demonstrated widespread cell death in TAM/CQ-treated tumors (Figure 3A). Morphological characteristics of apoptosis were observed in electron micrographs in 92% ± 5% of tumor cells in TAM/CQ-treated tumors compared with 3% ± 3% of tumor cells in TAM/PBS-treated tumors (Figure 3B). To further characterize this cell death, TUNEL staining was performed on tumor specimens to assess the number of cells undergoing apoptosis in treated tumors (Figure 3C). At 8 hours after the initiation of treatment, both TAM/PBS and TAM/CQ treatments resulted in a marked increase in TUNEL-positive tumor cells compared with PBS- and CQ-treated tumors. The number of TUNEL-positive cells decreased by 48 hours in TAM/PBS-treated but not in TAM/CQ-treated tumors. Quantification of the percentage of TUNEL-positive cells per high-powered field in treated tumors (Figure 3C) found no significant differences in the percentage of TUNEL-positive cells between PBS- and CQ-treated tumors and between TAM/PBS and TAM/CQ-treated tumors at 8 hours. At 24 hours, a significantly greater percentage of TUNEL-positive tumor cells was observed in TAM/CQ-treated tumors in comparison with TAM/PBS-treated tumors. This difference persisted at 48 hours, when a 7-fold difference in the percentage of TUNEL-positive tumor cells was observed in TAM/CQ-treated tumors compared with TAM/PBS-treated tumors (P < 0.001). As an independent measure of tumor cell apoptosis in treated tumors, Western blot analysis of cleaved caspase-3 was performed on tumor cell lysates from TAM/PBS- and TAM/CQ-treated tumors. Increased cleaved caspase-3 was observed in tumor lysates obtained at 8 hours after the initiation of either TAM/PBS or TAM/CQ. Cleaved caspase-3 was absent in TAM/PBS-treated tumor cell lysates obtained at 48 hours but present in TAM/CQ-treated tumor lysates obtained at 48 hours (data not shown).
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.
CQ enhances p53-dependent apoptosis by inhibiting autophagy. To ensure that the changes in autophagosome number seen by electron microscopy were due to the direct action of the systemically administered drugs on the autophagy pathway in tumor cells and not to changes in tumor microenvironment induced by tumor degeneration following treatment, the GFP-LC3 (LC3, mammalian homolog of yeast Atg8) fusion gene was retrovirally transduced into a bulk population of cells harvested from a primary Myc/p53ERTAM lymphoma, and GFP-positive cells were passaged in culture. LC3 is processed from LC3-I to LC3-II during autophagy. LC3-II is inserted into newly formed autophagosome membranes (22). Expression of GFP-LC3 provides a means to track changes in autophagosome formation in living cells (23). The distribution of GFP-LC3 in untreated Myc/p53ERTAM/GFP-LC3 cells was diffusely cytoplasmic (Figure 4A). CQ’s ability to modulate autophagy in tumor cells was confirmed by the accumulation of LC3-positive vesicles following treatment of Myc/p53ERTAM/GFP-LC3 cells with CQ. Activation of p53 with 4-hydroxytamoxifen (hTAM) resulted in an increased number of punctate LC3-associated vesicles, which was further enhanced by combined treatment with hTAM and CQ. The accumulation of autophagosomes in CQ-treated cells was dose dependent in both the absence and presence of hTAM (Figure 4B). In the absence of hTAM, CQ doses between 500 nM and 5 μM resulted in a 2-fold to 10-fold increase in the percentage of cells with punctate GFP-LC3 fluorescence. Treatment with hTAM alone resulted in a 10-fold increase in the percentage of cells with punctate GFP-LC3 fluorescence. The addition of CQ (1–5 μM) during treatment with hTAM resulted in a dose-dependent increase in the percentage of cells with punctate GFP-LC3 fluorescence. These differences persisted without significant change when measured at 48 hours after treatment (data not shown). To ensure that CQ’s ability to modulate autophagy was independent of p53, p53+/+ GFP-LC3 and p53–/–GFP-LC3 mouse embryonic fibroblasts (MEFs) were generated. CQ induced the accumulation of punctate LC3-associated vesicles in MEFs in a _p53_-independent manner (Figure 4C). Treatment of p53+/+GFP-LC3 MEFs with hTAM did not result in punctate LC3 fluorescence (data not shown). This result confirmed that the punctate LC3 fluorescence observed in Myc/p53ERTAM lymphoma cells treated with hTAM was due to hTAM-induced activation of the p53ERTAM fusion protein. Since the ERTAM protein domain is a transcriptionally inactivated ER, punctate GFP-LC3 fluorescence in hTAM-treated Myc/p53ERTAM cells is a p53-mediated effect.
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.
To confirm that the antineoplastic effect of CQ observed in vivo results from the ability of CQ to inhibit autophagy-based survival, CQ treatment of tumor cells was compared with the genetic inhibition of autophagy using shRNA against the autophagy gene ATG5 (shATG5). Expression of shATG5 blocks autophagy at a proximal step by preventing the formation of the ATG5-ATG12 complex, which is required for the generation of autophagosomes (24). shRNA designed to silence no mouse or human genes (hairpin control, HC) and 2 distinct shRNA sequences against ATG5 (shATG5 hairpin 2 [h2], shATG5 hairpin [h7]) were cloned into the control expression vector pKD (9) and introduced into primary tumor cells harvested from a Myc/p53ERTAM B cell lymphoma. Decreased expression levels of the ATG5-ATG12 complex in cells expressing shATG5 h2 (h2) and shATG5 h7 (h7) but not in cells expressing HC (HC) or vector (V) was confirmed by Western blot (Figure 5A). Tumor cells were induced to undergo apoptosis when p53 was activated by hTAM treatment in vitro. p53 activation with hTAM in h2 and h7 cells in which ATG5 levels were chronically suppressed resulted in increased cell death compared with HC cells (Figure 5B). Autophagy has been reported to promote cell survival in response to stress through both its ability to clear damaged organelles (25) and its ability to recycle intracellular contents to maintain bioenergetics (26). To determine whether the proapoptotic effect of chronic autophagy suppression could be reversed by supplying a permeable nutrient to support bioenergetics, cells were treated with hTAM and methyl pyruvate. Methyl pyruvate is a cell-permeant intermediate of glucose metabolism that has been previously reported to maintain the viability of growth factor– and/or nutrient-depleted cells in which autophagy is impaired (9). Methyl pyruvate addition to the medium failed to rescue the enhanced cell death observed in h2 cells as well as h7 cells following p53 activation with hTAM (Figure 5C).
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), M_yc/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.
The effect of CQ treatment on tumor cell death following p53 activation with hTAM was examined in cells stably transfected with HC or 1 of 2 distinct shATG5-expressing vectors (h2, h7; Figure 5A). CQ treatment (1–5 μM) of HC cells enhanced tumor cell death in response to p53 induction in a dose-dependent fashion (Figure 6A). In this dose range, CQ alone had no reproducible effect on tumor cell viability or proliferation (Supplemental Figure 1; available online with this article; doi:10.1172/JCI28833DS1). In contrast, CQ treatment failed to enhance the cell death of either clone of shATG5-transfected cells at these doses (Figure 6, B and C).
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.
CQ enhances tumor regression and suppresses tumor recurrence after alkylating drug therapy. In the treatment of human lymphomas, alkylating agents such as cyclophosphamide serve as first-line therapies (27–29). To determine whether the inhibition of autophagy could enhance the efficacy of alkylating drug therapy in tumors resistant to apoptosis, mice bearing _Myc/p53ERTAM_lymphomas were treated with cyclophosphamide alone or in combination with CQ. Mice with _Myc/p53ERTAM_lymphomas were treated with a single dose of 50 mg/kg cyclophosphamide i.p. followed by treatment with either PBS or 60 mg/kg/d CQ i.p. The PBS or CQ treatment was then repeated daily for 13 days (Figure 7A). Cyclophosphamide with or without CQ led to complete tumor regression in all treated mice. CQ cotreatment significantly enhanced tumor regression and delayed tumor recurrence. The average tumor volume after 24 hours of treatment in cyclophosphamide/PBS-treated and cyclophosphamide/CQ-treated animals was 2,966 ± 673 mm3, and 1,489 ± 524 mm3, respectively (P < 0.001). The addition of CQ to cyclophosphamide more than doubled the average time to recurrence of tumors. The tumors of PBS-treated mice (Figure 5) recurred after an average of 4.1 ± 1.2 days whereas a limited course of CQ treatment delayed tumor recurrence to an average of 9.3 ± 3.5 days (P < 0.01) (Figure 7B).
Effects of alkylating chemotherapy with and without CQ in _Myc/p53ERTAM_lymphomas. (A) Cells from a primary tumor were harvested and passaged in vivo in syngeneic C57BL/6×129F1 mice. _Myc/p53ERTAM_cells 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.
To investigate the effects of alkylating chemotherapy on autophagy, _Myc/p53ERTAM_cells expressing GFP-LC3 were treated with the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) with and without CQ (Figure 7C). MNNG is an alkylating agent that can induce apoptosis in a p53-independent manner (30). CQ treatment or MNNG treatment alone resulted in an increased percentage of viable cells with punctate LC3 fluorescence, indicating an accumulation of autophagosomes. Combination treatment with MNNG and CQ resulted in 9-fold and 17-fold increases in punctate LC3 fluorescence compared with DMSO control at 24- and 48-hour time points (Figure 7D). To further compare the effect of genetic autophagy inhibition with that of CQ following cytotoxic therapy, the effect of MNNG treatment with and without CQ was tested in HC, h2, and h7 cells. At 24 and 48 hours, combined treatment with 5 μM CQ significantly enhanced the cytotoxic effect of MNNG treatment of HC lymphoma cells to a degree similar to that of treatment of h2 or h7 cells with MNNG alone. CQ treatment did not further enhance cell death induced by an alkylating agent in h2 or h7 cells (Figure 7E).