Cytoprotective and nonprotective autophagy in cancer ...

Autophagy

ISSN: 1554-8627 (Print) 1554-8635 (Online) Journal homepage: http://www.tandfonline.com/loi/kaup20

Cytoprotective and nonprotective autophagy in cancer therapy David A. Gewirtz To cite this article: David A. Gewirtz (2013) Cytoprotective and nonprotective autophagy in cancer therapy, Autophagy, 9:9, 1263-1265, DOI: 10.4161/auto.25233 To link to this article: http://dx.doi.org/10.4161/auto.25233

Published online: 12 Jun 2013.

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Date: 01 April 2016, At: 09:53

Editor’s Corner

Editor’s Corner

Autophagy 9:9, 1263–1265; September 2013; © 2013 Landes Bioscience

Cytoprotective and nonprotective autophagy in cancer therapy David A. Gewirtz Departments of Pharmacology and Toxicology and Medicine and Massey Cancer Center; Virginia Commonwealth University; Massey Cancer Center; Richmond, VA USA

Two primary forms of autophagy have been identified in the field of cancer therapy based on their apparent functions in the tumor cell; these are the cytoprotective form that could, in theory, be inhibited for the purpose of sensitization to radiation and chemotherapeutic drugs and the “cytotoxic” form that either mediates or contributes to the actions of these treatment modalities. Surprisingly, to date, no clear-cut biochemical or molecular characteristics have been identified that might serve to distinguish between these two forms. In this commentary, we develop the concept of an additional form of autophagy that is nonprotective in that its inhibition neither sensitizes the tumor cell to exogenous stress (again, chemotherapy or radiation) nor protects the cell from the impact of these treatments. This form of autophagy also fails to exhibit any characteristics that might distinguish it from the cytoprotective and/or cytotoxic forms of autophagy. However, the existence of nonprotective autophagy is of potential significance in that it contributes to the challenge of predicting when the strategy of autophagy suppression might prove to have therapeutic benefit in the clinical treatment of cancer.

The identification in tumor cells of a cytoprotective function of autophagy in response to chemotherapy or radiation has generated a number of clinical trials designed to evaluate the possibility that interference with autophagy could be a generalized strategy for improving the response to cancer therapeutics.1 However, it is important to recognize that the foundation for these clinical efforts is largely functionally based; that is, inhibition of autophagy by either pharmacological approaches (generally the use of chloroquine, bafilomycin or 3-methyladenine) or via genetic silencing of autophagy regulatory genes such as ATG5, ATG7 or ATG12 results in sensitization to a variety of therapeutic modalities, often accompanied by increased apoptosis. These observations have largely been interpreted as reflecting the crosstalk between autophagy and apoptosis, whereby autophagy induction interferes with engagement of the apoptotic cell death program, which is relieved when autophagy is suppressed or abrogated.2 Nevertheless, the larger picture remains unclear, particularly from

a teleological standpoint; that is, it is difficult to understand how cell survival is actually served if cytoprotective autophagy involves organelle degradation and energy generation in environments where nutrient-based metabolism has not been compromised. Alternative explanations, such as that autophagy induction serves to suppress potentially damaging free radical generation or to eliminate damaged proteins, would be valid only if the antitumor actions of the quite diverse spectrum of drugs studied (as well as radiation) are all mediated through generation of reactive oxygen species3 and/or result in damage to cellular proteins. This is perhaps a realistic possibility that might explain the almost uniform induction of autophagy by diverse therapies. Although evidence for cytoprotective functions of drug and radiation-induced autophagy in cellular studies has provided the foundation for the clinical trials involving autophagy suppression, there is actually a relatively large body of literature that demonstrates the capacity of autophagy to also mediate antiproliferative

and/or cytotoxic functions of therapy.4-6 It is, however, worth noting that aside from the straightforward functional differences between cytoprotective and cytotoxic autophagy, no absolute quantitative, biochemical or molecular parameters have been identified that might distinguish between these two forms of autophagy that have been observed in response to chemotherapy and radiation. In a recent publication,7 Dr. Andrew Thorburn and I have postulated the existence of another form of autophagy that will be termed “nonprotective.” More specifically, we have observed induction by radiation of a form of autophagy whose inhibition neither sensitizes nor protects the tumor cell from radiation. Dr. Thorburn’s laboratory has also observed this nonprotective form of autophagy in response to cisplatin,8 whereas we also have preliminary evidence for nonprotective autophagy in response to doxorubicin.9 In this context, Lee et al. recently reported that knockdown of SLC16A7/ MCT2 [solute carrier family 16, member 7 (monocarboxylic acid transporter 2)] in

Correspondence to: David A. Gewirtz; Email: [email protected] Submitted: 05/30/13; Accepted: 05/31/13 http://dx.doi.org/10.4161/auto.25233 www.landesbioscience.com

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Keywords: cytoprotective autophagy, cytotoxic autophagy, nonprotective autophagy, chloroquine, chemosensitization, radiosensitization

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cell culture are clearly insufficient for supporting clinical trials; demonstration of sensitization to therapy by autophagy inhibition in animal models would appear to be obligatory. Leaving aside the vexing question of what would be the most appropriate animal models (xenografts vs. syngeneic vs. transgenic vs. carcinogen-induced, among others), the data in tumor-bearing animals treated with chemotherapy and the autophagy inhibitor chloroquine, is at best equivocal, as described in some detail in our recent paper.7 That is, in approximately half the studies in the literature, the inclusion of chloroquine produces a modest or minimal increase in tumor growth delay over and above that of the therapeutic modality alone. In our own work, chloroquine fails to improve the response to radiation in a syngeneic model (4T1 cells) of breast cancer. In the remaining studies, inclusion of chloroquine does significantly prolong the period of tumor growth delay. However, virtually none of these studies utilizing chloroquine actually demonstrate prolongation of the survival of the tumor-bearing animals; in fact, survival studies were generally not performed. Only in the one case where autophagy was genetically inhibited in the implanted tumor is a prolongation of survival demonstrable.11 This brings up another issue and potential limitation to the strategy of autophagy inhibition using chloroquine. Chloroquine and hydroxychloroquine (the form used in clinical trials) are the only FDA approved autophagy inhibitors currently available. However, an analysis of the pharmacokinetic data for chloroquine in mice indicates that at the doses that have been used routinely in animal studies (daily 50–60 mg/kg), the maximal concentrations achieved in the circulation are unlikely to be sufficient to suppress autophagy,7 based on studies in cell culture. The same would be true for patients administered chloroquine or hydrochloroquine at the usual dose of 400 mg daily. With the administration of elevated doses (800 mg or 1200 mg) to patients, plasma concentrations have been shown to reach approximately 7 µM and 14 µM, respectively,12 which should have the capacity to effectively suppress autophagy.

Autophagy

In this context, in a very recent publication, Zinn et al. showed that while chloroquine is effective in sensitization of small cell lung cancer cells in culture to the BCL2 inhibitor, ABT-737, chloroquine is entirely ineffective in vivo.13 In contrast, in a recent study where hydroxychloroquine was administered at a dose of 162 mg/kg, approximately equivalent to a human dose of 800 mg, tumor growth delay induced by erlotinib is markedly increased in the H358 lung cancer cell line. Interestingly, even at a dose that is relatively high compared with most other published in vivo work, chloroquine demonstrates only minimal extension of tumor growth delay in the H460 tumor cell line14 despite the fact that chloroquine is equally effective in sensitization of these two cell lines in culture. Taking all of the studies in animal models together, it might therefore be postulated that in those reports where chloroquine was shown to be competent in prolonging tumor growth delay induced by chemotherapy or radiation, these actions of chloroquine might reflect off-target or alternative target effects of the drug as opposed to the consequences of autophagy inhibition. To summarize: (1) While chemotherapy and radiation frequently if not uniformly promote autophagy in tumor cells in culture, the occurrence of autophagy cannot necessarily be assumed to reflect a cytoprotective response (or for that matter, a cytotoxic response). (2) It is not certain that when chemotherapyor radiation-induced autophagy is cytoprotective in cell culture that the same treatment will necessarily promote cytoprotective autophagy in the tumor in vivo. (3) The promotion of cytoprotective autophagy in a particular tumor or tumor cell line by one form of stress does not predict a similar outcome in the case of another form of treatment/stress. (4) At doses that are relatively nontoxic, chloroquine or hydrochloroquine may not be capable of achieving serum concentrations that can effectively suppress autophagy. (5) Even if chloroquine (or another autophagy inhibitor) can achieve concentrations that effectively suppress autophagy in a patient’s tumor, there is no assurance that this will prove to be a useful adjunctive therapy. That is, if the autophagy induced by a

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human colorectal cancer cells results in a form of autophagy whose pharmacological inhibition fails to sensitize the cells to loss of colony-forming capacity; autophagy inhibition furthermore fails to protect the cells from the impact of SLC16A7 knockdown, providing an example of the form of autophagy that is neither cytoprotective nor cytotoxic.10 While it could be argued that “nonprotective” autophagy is essentially irrelevant, since it has no apparent function, this would likely be a somewhat shortsighted point of view. Again, partly from a teleological standpoint, it is likely that a cellular response of this magnitude and duration must provide some homeostatic advantage to the cell; the fact that its function is unknown simply reflects current limitations in our understanding of the process. The existence of a nonprotective form of autophagy has wider clinical implications. In as yet unpublished studies of radiation-induced autophagy in paired (but nonisogenic) sets of tumor cell lines (breast, head and neck and non-small cell lung cancer), we have been able to demonstrate that radiation induces protective autophagy in one of each of the cell lines, and nonprotective autophagy in the other. Consequently, the existence of both forms of autophagy in response to a singular stress (radiation in this case) does not appear to be an isolated phenomenon. Considering the possibility of sensitizing patients to various forms of chemotherapy or radiation through autophagy inhibition, these findings (albeit in tumor cell lines) indicate that it cannot be unreservedly assumed that any particular treatment will induce a cytoprotective form of autophagy in a given malignancy. Consequently, prior to administration of an additional drug with the goal of suppressing therapy-induced autophagy, it will be necessary to establish that the treatment under consideration does, in fact, induce the cytoprotective form of autophagy. An additional caveat is that should the particular treatment/tumor pairing actually be promoting a cytotoxic form of autophagy, then autophagy inhibition might instead interfere with the effectiveness of cancer therapy. With regard to the translation of preclinical findings to the clinic, studies in

form of autophagy that is cytoprotective and that can be exploited for therapeutic advantage, biomarkers must be identified that will predict when cytoprotective autophagy is occurring for a particular malignancy/treatment, and likely alternatives to chloroquine as autophagy inhibitors will have to be developed15 before any therapeutic advantage can be realized through the clinical strategy of autophagy inhibition.

References

7. Bristol ML, Emery SM, Maycotte P, Thorburn A, Chakradeo S, Gewirtz DA. Autophagy inhibition for chemosensitization and radiosensitization in cancer: do the preclinical data support this therapeutic strategy? J Pharmacol Exp Ther 2013; 344:54452; PMID:23291713; http://dx.doi.org/10.1124/ jpet.112.199802 8. Maycotte P, Aryal S, Cummings CT, Thorburn J, Morgan MJ, Thorburn A. Chloroquine sensitizes breast cancer cells to chemotherapy independent of autophagy. Autophagy 2012; 8:200-12; PMID:22252008; http://dx.doi.org/10.4161/auto.8.2.18554 9. Goehe RW, Di X, Sharma K, Bristol ML, Henderson SC, Valerie K, et al. The autophagy-senescence connection in chemotherapy: must tumor cells (self ) eat before they sleep? J Pharmacol Exp Ther 2012; 343:76378; PMID:22927544; http://dx.doi.org/10.1124/ jpet.112.197590 10. Lee I, Lee S-J, Kang WK, Park C. Inhibition of monocarboxylate transporter 2 induces senescence-associated mitochondrial dysfunction and suppresses progression of colorectal malignancies in vivo. Mol Cancer Ther 2012; 11:2342-51; PMID:22964484; http://dx.doi. org/10.1158/1535-7163.MCT-12-0488 11. Hu YL, DeLay M, Jahangiri A, Molinaro AM, Rose SD, Carbonell WS, et al. Hypoxia-induced autophagy promotes tumor cell survival and adaptation to antiangiogenic treatment in glioblastoma. Cancer Res 2012; 72:1773-83; PMID:22447568; http://dx.doi. org/10.1158/0008-5472.CAN-11-3831


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www.clinical trials.gov Thorburn A. Apoptosis and autophagy: regulatory connections between two supposedly different processes. Apoptosis 2008; 13:1-9; PMID:17990121; http:// dx.doi.org/10.1007/s10495-007-0154-9 3. Rouschop KM, Ramaekers CH, Schaaf MB, Keulers TG, Savelkouls KG, Lambin P, et al. Autophagy is required during cycling hypoxia to lower production of reactive oxygen species. Radiother Oncol 2009; 92:411-6; PMID:19616335; http://dx.doi. org/10.1016/j.radonc.2009.06.029 4. Basile V, Belluti S, Ferrari E, Gozzoli C, Ganassi S, Quaglino D, et al. bis-dehydroxy-curcumin triggers mitochondrial-associated cell death in human colon cancer cells through ER-stress induced autophagy. PLoS One 2013; 8:e53664; PMID:23326480; http:// dx.doi.org/10.1371/journal.pone.0053664 5. Huo HZ, Wang B, Qin J, Guo SY, Liu WY, Gu Y. AMP-activated protein kinase (AMPK)/Ulk1dependent autophagic pathway contributes to C6 ceramide-induced cytotoxic effects in cultured colorectal cancer HT-29 cells. Mol Cell Biochem 2013; 378:171-81; PMID:23508272; http://dx.doi. org/10.1007/s11010-013-1608-8 6. Chen W, Feng L, Nie H, Zheng X. Andrographolide induces autophagic cell death in human liver cancer cells through cyclophilin D-mediated mitochondrial permeability transition pore. Carcinogenesis 2012; 33:2190-8; PMID:22869602; http://dx.doi. org/10.1093/carcin/bgs264

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Acknowledgments

The author thanks Dr. Andrew Thorburn for a critical reading of this manuscript and insightful advice.

12. Munster T, Gibbs JP, Shen D, Baethge BA, Botstein GR, Caldwell J, et al. Hydroxychloroquine concentration-response relationships in patients with rheumatoid arthritis. Arthritis Rheum 2002; 46:14609; PMID:12115175; http://dx.doi.org/10.1002/ art.10307 13. Zinn RL, Gardner EE, Dobromilskaya I, Murphy S, Marchionni L, Hann CL, et al. Combination treatment with ABT-737 and chloroquine in preclinical models of small cell lung cancer. Mol Cancer 2013; 12:16; PMID:23452820; http://dx.doi.org/10.1186/14764598-12-16 14. Zou Y, Ling YH, Sironi J, Schwartz EL, Perez-Soler R, Piperdi B. The autophagy inhibitor chloroquine overcomes the innate resistance of wild-type EGFR non-small-cell lung cancer cells to Erlotinib. J Thorac Oncol 2013; 8:693-702; PMID:23575415; http:// dx.doi.org/10.1097/JTO.0b013e31828c7210 15. McAfee Q, Zhang Z, Samanta A, Levi SM, Ma XH, Piao S, et al. Autophagy inhibitor Lys05 has singleagent antitumor activity and reproduces the phenotype of a genetic autophagy deficiency. Proc Natl Acad Sci U S A 2012; 109:8253-8; PMID:22566612; http:// dx.doi.org/10.1073/pnas.1118193109

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particular treatment actually has a cytotoxic function, then autophagy inhibition would likely interfere with the effectiveness of treatment rather than enhancing the therapeutic response; if the autophagy is nonprotective, then autophagy inhibition is unlikely to improve the response to therapy. Consequently, there is a clear need for a more thorough understanding of what predisposes a tumor cell to undergo a