There are very few agents that have a proven ability to prevent cancer, and their mechanisms of action are often obscure. We reasoned that possible agents would include those that augment DNA damage response pathways, particularly since DNA damage is evident in the precancerous state, where it likely promotes additional mutations that give rise to frank malignancies (43
). We focused our chemoprevention studies on the antimalarial drug CQ, which can activate Atm and p53 in the absence of detectable DNA damage (12
) and which is tolerated long term in patients suffering from malaria, with few side effects.
Previous studies have highlighted the importance of Atm as a guardian against Myc-induced tumorigenesis (9
); hence we asked whether CQ could affect Myc-induced lymphoma development. Indeed, administration of CQ to mice once every 5 days dramatically reduced lymphoma development in Eμ-Myc
transgenic mice. Since Eμ-Myc
mice are often regarded as a murine model for human Burkitt lymphoma (44
), this suggested that CQ could be a useful drug in preventing this disorder. Intriguingly, an epidemiologic trial supports this notion. Specifically, in an attempt to investigate whether malaria was a causative agent in the high incidence of Burkitt lymphoma in equatorial Africa, a trial was performed in which CQ was provided to a large section of the country of Tanzania for 5 years, with instructions for children to take 1 dose every 2 weeks (45
). During the time period of the study, the incidence of endemic Burkitt lymphoma in the region decreased by approximately 75%, and the tumor incidence then returned to the old baseline within 2 years after completion of the trial. The striking tumor prevention effects of CQ were not further explored at the time, since the major hypothesis of the study, a linkage between malaria incidence and Burkitt lymphoma, was not adequately supported by the results. However, the profound effects we observed in the Eμ-Myc
mice treated with CQ now provide a different perspective on the results of this epidemiologic study and suggest that CQ may have been a useful prevention/therapeutic agent in this disease, even as a single agent given once every 2 weeks.
Exploring the mechanism of CQ action provided some unexpected and intriguing insights. At the doses of CQ used, which are similar to those used to prevent malaria, a p53-dependent pathway and increased cell death were observed. Our studies and those of Thompson and colleagues (22
) have indicated that in the context of Myc overexpression, CQ induces an apoptotic cell death resulting from ineffective autophagic protein degradation and partial lysosomal permeabilization. Autophagy is a complex adaptive cellular response that enhances cell survival in the face of nutrient limitation or other cellular stresses that include protein accumulation (29
). Some studies have suggested that CQ enhances cell death by blocking fusion of autophagosomes with lysosomes (33
) while others suggest that it functions as a lysosomotropic agent by inhibiting the acid-dependent degradation of autophagosome cytosolic contents, resulting in an accumulation of autophagic vesicles that cannot be cleared (22
). Our results are consistent with the latter mechanism. It is noteworthy that Myc expression itself also affected the autophagic response with respect to LC3 modification by PE. In driving cell growth and proliferation, we can speculate that Myc affects autophagy as a mechanism to deal with increased metabolism.
An intriguing result from our study was that CQ was effective at killing Myc-overexpressing cells even in the presence of caspase inhibition or overexpressed Bcl-2 or Bcl-XL
, which prevent Myc-induced apoptosis (26
). Even more striking, CQ was also effective at killing MEFs lacking both Bax
, which together are required for all forms of apoptosis (28
). The death in Bax
-deficient cells from CQ treatment may result from the fact that they retain a normal lysosomal membrane permeabilization despite failing to undergo mitochondrial membrane permeabilization (50
). Regardless, the result represents an important consideration in the treatment of cancers in which the apoptotic pathway is disabled, particularly advanced follicular lymphoma, which is driven by Bcl-2 and Myc overexpression (51
It appears that CQ typically facilitates apoptotic cell death in Myc-overexpressing cells via lysosomal changes. Importantly, however, CQ-induced lysosomal changes can lead to a p53-dependent cell death in the absence of apoptosis, though blockade of both apoptosis and autophagy abrogates CQ-induced cell death (Figure ). Recent studies have highlighted a role for p53 in regulating autophagy, perhaps through effects on the lysosomal protein DRAM or on the AMPK/TSC1/TSC2 signaling pathway (52
). Our study indicates that CQ inhibits a late step in autophagy. Therefore, while triggers of the autophagy pathway, such as genotoxic stress and starvation, may play a role in cell survival (52
), others (e.g., CQ) that derail the autophagy pathway and result in ineffective clearance are toxic and can induce a cell death response that is dependent upon p53. While CQ clearly affects events at the lysosome, the fact that it can induce p53-dependent cell death independent of either apoptosis or autophagy, but not both, suggests the involvement of other signaling pathways and p53 targets. Interestingly, a recent study suggests that protection from caspase-independent cell death reflects an increase in, and a dependence upon, autophagy (54
). Our results suggest that autophagy is not required for cell survival following caspase inhibition; however, since cells still vesiculate in the presence of CQ under defective apoptosis and autophagic conditions, it is possible that they ultimately would succumb to death.
CQ was also effective in reducing spontaneous tumor formation in mice lacking Atm
, a finding that potentially has significant implications for patients with the cancer-prone disorder AT. First, it appears that CQ may be useful in the prevention or perhaps treatment of lymphomas that arise in AT patients. Malignancies that arise in AT patients are particularly difficult to treat because of the special sensitivities of AT patients to cytotoxic agents used to treat cancer. CQ provides a novel consideration for treatment of lymphomas in AT patients and has the advantage that it is not typically cytotoxic and may thus be relatively well tolerated. Further, Atm
-deficient mice treated with CQ on a weekly basis had no apparent side effects from the chronic intermittent treatment, and the therapeutic benefit might even be greater on a different dosage schedule or in combination with other agents (32
). In addition to cancer predisposition, patients with AT also exhibit neurodegeneration, immunologic abnormalities, insulin resistance, abnormal intracellular redox control, and signs of premature aging (55
). Interestingly, abnormalities in autophagy and lysosomal function have been linked to all of these processes (56
), raising the intriguing possibility that some or all of the clinical abnormalities in the AT disorder could be linked to alterations in autophagy regulation. Such possibilities provide fertile areas for continued study and potential novel opportunities for treatment or prevention of the many tragic clinical features of this disease.
Collectively, the data presented herein suggest that disruption of lysosomal function by CQ provides a potentially useful therapeutic intervention in certain cancer settings. The apparently increased sensitivity of oncogene-expressing cells to CQ treatment would provide a natural therapeutic index. The challenge will be to figure out how to optimally use this approach in the prevention and/or treatment of clinical disease. Questions of optimal dose and schedule, use in combination with cytotoxic or biologic agents, and appropriate genetic background or histopathology of the tumor will all need to be addressed. Perhaps most important is the fact that these studies provide proof of principle for developing antitumor therapies based on the modulation of autophagic pathways and opportunities for novel drug discovery, whether based on CQ itself or targeting other steps in the pathway.