Inhibition of autophagy with chloroquine enhances therapeutic tumor regression in a mouse model of B cell lymphoma

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Autophagy involves the lysosome-dependent degradation of cytoplasmic contents sequestered by autophagosomes. A number of antineoplastic therapies can induce autophagy in cancer cell lines. To test the hypothesis that therapy-induced autophagy is a survival strategy for tumor cells, the effect of the autophagy inhibitor chloroquine (CQ) on tumor regression was investigated. CQ is a lysosomotropic drug that can induce the death of cultured cells reliant on autophagy. A tumor model was generated by overexpressing the c-MYC oncogene in bone marrow cells derived from a p53-estrogen receptor (p53ER) knock-in mouse. MYC/P53ER cells retain tamoxifen (TAM)-inducible p53 gene expression and develop into a subcutaneous B cell lymphoma in the absence of TAM. Tumor cells can be harvested and passaged in vivo. Daily intraperitoneal (IP) TAM leads to p53-dependent apoptosis and tumor regression. 100% of the tumors recur despite continued TAM. We hypothesized that autophagy might allow tumor cells to survive p53-induced apoptosis. To determine if the inhibition of autophagy could enhance tumor regression, mice bearing lymphomas were matched for tumor volume and assigned to daily IP TAM/ PBS or daily IP TAM/CQ. CQ treatment resulted in enhanced tumor regression and a delay in tumor recurrence. Electron micrographs demonstrate morphological evidence of the induction of autophagy in tumors treated with TAM/PBS 24 hours after the initiation of treatment. Autophagy was downregulated by 48 hours as tumors started to recur. TAM/CQ treatment results in the accumulation of autophagosomes and a 7-fold induction of caspase-dependent apoptosis at 48 hours in comparison to TAM/PBS. In vitro studies using the tumor cells demonstrates that inhibition of autophagy by shRNA inhibition of ATG5 also lead to a striking increase in cell death in response to p53 induction. Importantly, when chloroquine was added to ATG5-suppressed cells at the time of p53 induction no further increase in cell death was seen. These data demonstrate that autophagy is a survival pathway in stressed cells and CQ’s antitumor effect depends on its ability to inhibit autophagy. This effect is not restricted to p53-induced apoptosis. In the absence of TAM, combining CQ with cyclophosphamide delayed tumor recurrence of lymphoma compared to cyclophosphamide alone. These studies provide in vitro and in vivo evidence that inhibition of tumor cell autophagy could potentially enhance the efficacy of a number of antineoplastic therapies.