International Journal of
Radiation Oncology biology
physics
www.redjournal.org
Oncology ScandAutophagy and the Radiation Response By Clayton A. Smith, MD, PhD, Michael L. Freeman, PhD, Senior Editor
Ionizing radiation is an effective therapy for the curative treatment or palliation of many forms of cancer; however, it is also associated with toxicity to normal tissues, both in the therapeutic setting and as a result of accidental exposure. Cellular responses to DNA damage produced by ionizing radiation include mitotic catastrophe, apoptosis, senescence, and autophagy. Although cell death following ionizing radiation is largely attributed to mitotic catastrophe and apoptosis, recent evidence has emerged regarding dual roles of autophagy as both a radiation-protective and radiationsensitizing response. Autophagy is a conserved mechanism for cellular homeostasis that is activated during times of stress to break down organelles, including damaged mitochondria and protein products, thereby providing additional energy sources for the cell. This is accomplished through production of a double-membrane autophagosome that fuses with lysosomes to form an autolysosome. It is well established that formation of the autophagosome is dependent on autophagy-related gene (ATG) products that assemble in a series of steps leading ultimately to the degradation of engulfed protein targets. These ATGs include BECLIN-1, ATG5, ATG7, and LC3-II among many others. BECLIN1 is normally bound by BCL-2, the anti-apoptotic protein, which prevents BECLIN-1 interaction with the phosphatidylinositol-3 kinase VPS34 for initiation of autophagosome formation. ATG5, ATG7, and LC3-II serve as crucial components for initiation and closure of the autophagosome. It has recently been proposed that autophagy can be divided into 4 functional categories including cytoprotective, cytotoxic, cytostatic, and nonprotective (1). These functional differences are based largely on the response produced within cells following a stressor, and this is often experimentally demonstrated by observing the effects of pharmacologic or genetic inhibition of autophagy on cellular response. A cytoprotective function for autophagy is observed when sensitivity to the stressor (eg, chemotherapeutic agent or irradiation) increases Int J Radiation Oncol Biol Phys, Vol. 90, No. 1, pp. 7e10, 2014 0360-3016/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ijrobp.2014.04.013
following inhibition of autophagy in the treated tissue. This has been observed following treatment of cells with 5-fluorouracil (2-4), among many others. Conversely, a cytotoxic function for autophagy is inferred when inhibition of autophagy results in greater cell survival in response to a stressor. This effect has been demonstrated with temozolomide (5) and vitamin D in combination with radiation (6). The functional contribution of autophagy to cellular homeostasis, not surprisingly, varies by tissue type, source of cellular stress, and environmental conditions of the tumor microenvironment. Importantly, there currently do not appear to be any molecular or morphologic markers to distinguish one form of functional autophagy from another. The papers discussed in this issue of Oncology Scan outline some exciting discoveries of the contributions of autophagy in response to ionizing radiation, both in normal tissues and transformed cells. They highlight potential therapeutic targets for radiation protection and tumor radiosensitization and demonstrate some novel experimental methods for the study of cancer biology. Soto-Pantoja et al. CD47 deficiency confers cell and tissue radioprotection by activation of autophagy. Autophagy 2012. (7) Summary: Integrin-associated protein (IAP [CD47]) is a ubiquitously expressed protein that functions as a receptor for thrombospondin family members, a ligand for SIRPa, and participates in integrin signaling (8). Previous work by Roberts et al (9) has demonstrated that a CD47 deficiency results in radiation protection of normal tissues both in vitro and in vivo while selectively increasing radiation sensitivity of orthotopic melanoma and squamous cell lung cancer tumors in C57BL/6 mice. However, the mechanism by which CD47 suppression produced the selective responses to irradiation in normal tissues and cancer cells was unknown. In the paper by Soto-Pantoja et al (7), a series of experiments was conducted to test the hypothesis that CD47 deficiency
8
Oncology Scan
promotes an altered radiation response in tissues by increasing autophagy. The key findings include: 1. CD47 / Jurkat T cells were assessed for viability, cytotoxicity, and proliferative capacity. In unirradiated cells, proliferative capacity and viability were independent of CD47 expression. However, a CD47 deficiency significantly increased viability and preserved proliferative capacity of irradiated CD47-deficient cells compared to that in wild-type cells. 2. They next assessed measures of autophagy, including visualization of autophagosome formation by electron microscopy, LC3-II expression via immunofluorescence, and expression of autophagy-related genes including BECN1, ATG5, and ATG7 by RT-PCR. Following irradiation, a CD47 deficiency resulted in increased number and size of autophagosomes, increased LC3-II puncta formation and intensity, and increased expression of BECN1, ATG5, and ATG7. These data demonstrate that the increased survival following irradiation conferred by CD47 deficiency is associated with increased markers of autophagy. Use of CD47 morpholino knockdown in human umbilical vein endothelial cells produced a similar increase in measures of autophagy after irradiation, indicating that the mechanism of radiation protection is not limited to a single cell type. 3. To determine whether radiation protection was mediated by increased autophagy, cells were pretreated with pharmacologic inhibitors of autophagy, hydroxychloroquine (HCQ) or 3-methyladenine (3-MA). Cell viability following irradiation was decreased in CD47 / cells treated with an autophagy inhibitor. In contrast, viability of wild-type CD47-expressing cells was not affected by exposure to an autophagy inhibitor. This demonstrates that inhibition of autophagy restores radiation sensitivity in CD47 / cells. These findings were further extended by small interfering RNA (siRNA) knockdown of the autophagy-associated genes ATG5 and ATG7. Similar to pharmacologic inhibition, genetic knockdown of autophagy-associated genes returned CD47-deficient cells to a radiation-sensitive state, as demonstrated by decreased viability and proliferative capacity following irradiation. Importantly, transfection of CD47 / cells with plasmid reexpressing CD47 reversed the radiation-protective effect with a concomitant decrease in measures of autophagy following irradiation. 4. Wild-type C57BL/6 mice were administered a CD47 antisense morpholino 48 hours prior to single-dose total body irradiation. Twenty-four hours after irradiation, lung tissue in CD47-suppressed mice exhibited increased expression of autophagy-associated genes and decreased apoptosis compared to control treated mice.
International Journal of Radiation Oncology Biology Physics
Comment: This is an interesting article that outlines a radiation-protective role for autophagy in normal tissues. It extends the finding that a CD47 deficiency confers a survival advantage to cells following irradiation and that this is associated with increases in measures of autophagy. In addition, both pharmacologic and genetic inhibition of autophagy reverse the radiation protection observed in CD47-deficient cells while having no effect on the radiation sensitivity of wild-type cells. The limitations of this study relate to the use of relatively high radiation doses, cell culture-based assays, and minimal measurements of pathophysiology. The authors were aware of these limitations and therefore undertook the following investigation (10).
Soto-Pantoja et al. Blockade of CD47 increases survival of mice exposed to lethal total body irradiation. Sci Rep 2013. (10) Summary: This study by Soto-Pantoja et al (10) seeks to address the problem of normal tissue toxicity following either therapeutic or incidental exposures to total body irradiation (TBI), building upon previous findings that CD47 deficiency confers radiation protection to normal tissues through increased autophagy following irradiation (7,9). C57BL/6 mice were treated with CD47 antisense morpholino oligomers or control by intraperitoneal injection 48 hours prior to single-dose TBI. The key findings were as follows: 1. Survival 30 days after administration of 7.6 Gy TBI was 71% for mice with CD47 suppression compared with 40% for mismatch-treated control mice. If CD47 morpholino was administered immediately after radiation, there was also a survival advantage, although to a lesser degree (40% vs 20% controls at 30 days). 2. CD47 suppression decreased measures of normal tissue toxicity following TBI. Bone marrow cells harvested and cultured 24 hours after TBI demonstrated increased proliferative capacity in CD47 morphants. Similarly, white blood cell counts from peripheral blood were maintained in the CD47 morpholino group whereas a decrease was observed in controls. Intestinal villi in CD47-suppressed mice had less TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) staining following irradiation and demonstrated normal architecture 2 weeks later. Similar results were obtained according to assessments of the esophageal mucosal layer, with CD47 morpholino conferring protection after radiation whereas control subjects had loss of architecture and significantly increased TUNEL staining. 3. p62 is degraded during activation of autophagy and is therefore used as a surrogate measurement of relative
Volume 90 Number 1 2014
autophagic activity. Immunohistochemical staining (IHC) for p62 in both intestinal and esophageal mucosa demonstrated a significant increase in p62 expression following TBI in control mice, suggesting decreased autophagy following radiation in normal tissues. In contrast, suppression of CD47 resulted in decreased p62 staining in both tissues following irradiation, indicating increased autophagy associated with CD47 blockade. Comment: This is an exciting study both for demonstration of a potential target for normal tissue radiation protection and for use of an elegant system for in vivo genetic manipulation. CD47 suppression not only conferred a survival advantage following irradiation, but it was also associated with maintenance of hematopoietic and gastrointestinal function, which are 2 of the primary system failures contributing to lethality after whole-body irradiation. Additionally, there was a survival advantage even if CD47 suppression occurred after irradiation, although to a lesser extent. First, this has important implications for accidental lethal radiation exposures for which the current treatments are primarily supportive care and, potentially, stem cell transplantation. Second, the authors were able to systemically block CD47 protein translation by use of intraperitoneal administration of antisense morpholino just 48 hours prior to a lethal dose of TBI. This is an exciting new tool for conducting in vivoetargeted genetic manipulations due to its ease of delivery in comparison with the more labor- and time-intensive development of knockout and transgenic animals. Ko et al. Autophagy inhibition radiosensitizes in vitro, yet reduces radioresponses in vivo due to deficient immunogenic signaling. Cell Death Differ 2014. (11) Summary: In this article by Ko et al (11), the authors investigated the contribution of autophagy to radiation response in tumor cells. Genetic silencing of autophagyassociated genes in multiple transformed cell lines in vitro resulted in radiation sensitization, which confirms prior reports. In addition, orthotopic xenografts of autophagy-deficient tumor cells transplanted into immunodeficient mice also produced a radiation sensitization effect. The stunning observation made by these authors is that knockdown of autophagy in tumors implanted in immunocompetent mice produced the opposite effect, resulting in radiation protection. The key findings are as follows: 1. Autophagic function was inhibited in vitro by small hairpin RNA (shRNA) directed against the autophagyassociated gene ATG5 or BECN1. This resulted in decreased cell survival following irradiation in both H460 and A549 cells. This increased radiation sensitivity was associated with a decrease in mitochondrial
Oncology Scan
9
membrane potential and loss of plasma membrane integrity. Xenografts of ATG5- or BECLIN-1-deficient tumor cells transplanted in immunodeficient BALB/c nude mice demonstrated increased sensitivity to radiation as measured by a significant delay in tumor growth. 2. To determine the interaction between host immune response and autophagy following irradiation, mouse CT26 colon carcinoma tumors with ATG5 knockdown were implanted in BALB/c immune-deficient or wild-type immune-competent mice and irradiated. Although autophagydeficient tumors demonstrated radiation sensitivity in the immunodeficient mice, the opposite effect was observed in the immunocompetent group. Suppression of autophagy in the tumors implanted in immunocompetent hosts resulted in less tumor growth delay following irradiation than in autophagy-intact tumors. 3. It was hypothesized that autophagy contributes to immunogenic-mediated cell death in the immune competent animal. Extracellular ATP is one of the signals released as part of the immunogenic cell death response. Correspondingly, cells with suppression of autophagy had decreased levels of extracellular ATP following irradiation in comparison with controls in vitro. Pharmacologic inhibition of ecto-ATPase function with ARL67156, when given intratumorally to autophagy-deficient xenografts, restored the radiation sensitivity of tumor cells in immunocompetent hosts. No alteration in sensitivity to radiation was observed in immunodeficient mice given ARL67156 to increase extracellular ATP levels. This indicates that the differential contribution of autophagy to radiation response is due to an interaction with the host immune response following irradiation. Indeed, autophagysuppressed tumors were found to have less infiltration of lymphocytes 9 days after irradiation, and this effect was reversed if ecto-ATPase function was inhibited in the tumors prior to irradiation. Comment: The data presented in this article further expand our knowledge of the contribution of autophagy to the radiation response of cancer cells. It also underscores the care that must be taken with both experimental design and interpretation of results of animal models of cancer biology. Immunedeficient mice are frequently used in cancer biology because they allow us to implant human patient-derived tumors and study the response to various interventions. However, no single model perfectly replicates the conditions of initial growth and treatment response present in patients. In this case, the evidence of increased radiation sensitivity of autophagysuppressed tumors in immune-deficient mice might lead one to conclude that autophagy acts primarily to protect cells from the stress induced by irradiation. However, this would ignore the contribution of host immune response to tumor control. Interestingly, expansion of the experimental design to assess
10
Oncology Scan
radiation response in immunocompetent hosts provided evidence that autophagy may function to increase immunogenic cell death and therefore increase tumor sensitivity to radiation. As outlined in the above papers, we are only just beginning to understand the contributions of autophagy to the radiation response of normal tissues and cancer cells. There is evidence both for a radiation-protective role and for radiation sensitization, which appears to vary based on conditions of the target cells and host immune status. Clinical trials are currently underway investigating the use of the autophagy inhibitors chloroquine and hydroxychloroquine as a means to suppress the cytoprotective function of autophagy in association with various cancer therapies. Based on the results presented above, there may also be a therapeutic role for autophagy in protecting normal tissues from lethal radiation exposures.
International Journal of Radiation Oncology Biology Physics
3.
4. 5.
6.
7.
8. 9.
References 1. Gewirtz DA. The four faces of autophagy: Implications for cancer therapy. Cancer Res 2014;74:647-651. 2. O’Donovan TR, O’Sullivan GC, McKenna SL. Induction of autophagy by drug-resistant esophageal cancer cells promotes their survival and
10.
11.
recovery following treatment with chemotherapeutics. Autophagy 2011;7:509-524. Liu D, Yang Y, Liu Q, et al. Inhibition of autophagy by 3-MA potentiates cisplatin-induced apoptosis in esophageal squamous cell carcinoma cells. Med Oncol 2011;28:105-111. Li X, Lu Y, Pan T, et al. Roles of autophagy in cetuximab-mediated cancer therapy against EGFR. Autophagy 2010;6:1066-1077. Voss V, Senft C, Lang V, et al. The pan-Bcl-2 inhibitor (-)-gossypol triggers autophagic cell death in malignant glioma. Mol Cancer Res 2010;8:1002-1016. Bristol ML, Di X, Beckman MJ, et al. Dual functions of autophagy in the response of breast tumor cells to radiation: Cytoprotective autophagy with radiation alone and cytotoxic autophagy in radiosensitization by vitamin D3. Autophagy 2012;8:739-753. Soto-Pantoja DR, Miller TW, Pendrak ML, et al. CD47 deficiency confers cell and tissue radioprotection by activation of autophagy. Autophagy 2012;8:1628-1642. Brown EJ, Frazier WA. Integrin-associated protein (CD47) and its ligands. Trends Cell Biol 2001;11:130-135. Maxhimer JB, Soto-Pantoja DR, Ridnour LA, et al. Radioprotection in normal tissue and delayed tumor growth by blockade of CD47 signaling. Sci Trans Med 2009;1:3ra7. Soto-Pantoja DR, Ridnour LA, Wink DA, et al. Blockade of CD47 increases survival of mice exposed to lethal total body irradiation. Sci Rep 2013;3:1038. Ko A, Kanehisa A, Martins I, et al. Autophagy inhibition radiosensitizes in vitro, yet reduces radioresponses in vivo due to deficient immunogenic signalling. Cell Death Differ 2014;21:92-99.
Radiation Oncology biology
physics
www.redjournal.org
Oncology ScandAutophagy and the Radiation Response By Clayton A. Smith, MD, PhD, Michael L. Freeman, PhD, Senior Editor
Ionizing radiation is an effective therapy for the curative treatment or palliation of many forms of cancer; however, it is also associated with toxicity to normal tissues, both in the therapeutic setting and as a result of accidental exposure. Cellular responses to DNA damage produced by ionizing radiation include mitotic catastrophe, apoptosis, senescence, and autophagy. Although cell death following ionizing radiation is largely attributed to mitotic catastrophe and apoptosis, recent evidence has emerged regarding dual roles of autophagy as both a radiation-protective and radiationsensitizing response. Autophagy is a conserved mechanism for cellular homeostasis that is activated during times of stress to break down organelles, including damaged mitochondria and protein products, thereby providing additional energy sources for the cell. This is accomplished through production of a double-membrane autophagosome that fuses with lysosomes to form an autolysosome. It is well established that formation of the autophagosome is dependent on autophagy-related gene (ATG) products that assemble in a series of steps leading ultimately to the degradation of engulfed protein targets. These ATGs include BECLIN-1, ATG5, ATG7, and LC3-II among many others. BECLIN1 is normally bound by BCL-2, the anti-apoptotic protein, which prevents BECLIN-1 interaction with the phosphatidylinositol-3 kinase VPS34 for initiation of autophagosome formation. ATG5, ATG7, and LC3-II serve as crucial components for initiation and closure of the autophagosome. It has recently been proposed that autophagy can be divided into 4 functional categories including cytoprotective, cytotoxic, cytostatic, and nonprotective (1). These functional differences are based largely on the response produced within cells following a stressor, and this is often experimentally demonstrated by observing the effects of pharmacologic or genetic inhibition of autophagy on cellular response. A cytoprotective function for autophagy is observed when sensitivity to the stressor (eg, chemotherapeutic agent or irradiation) increases Int J Radiation Oncol Biol Phys, Vol. 90, No. 1, pp. 7e10, 2014 0360-3016/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ijrobp.2014.04.013
following inhibition of autophagy in the treated tissue. This has been observed following treatment of cells with 5-fluorouracil (2-4), among many others. Conversely, a cytotoxic function for autophagy is inferred when inhibition of autophagy results in greater cell survival in response to a stressor. This effect has been demonstrated with temozolomide (5) and vitamin D in combination with radiation (6). The functional contribution of autophagy to cellular homeostasis, not surprisingly, varies by tissue type, source of cellular stress, and environmental conditions of the tumor microenvironment. Importantly, there currently do not appear to be any molecular or morphologic markers to distinguish one form of functional autophagy from another. The papers discussed in this issue of Oncology Scan outline some exciting discoveries of the contributions of autophagy in response to ionizing radiation, both in normal tissues and transformed cells. They highlight potential therapeutic targets for radiation protection and tumor radiosensitization and demonstrate some novel experimental methods for the study of cancer biology. Soto-Pantoja et al. CD47 deficiency confers cell and tissue radioprotection by activation of autophagy. Autophagy 2012. (7) Summary: Integrin-associated protein (IAP [CD47]) is a ubiquitously expressed protein that functions as a receptor for thrombospondin family members, a ligand for SIRPa, and participates in integrin signaling (8). Previous work by Roberts et al (9) has demonstrated that a CD47 deficiency results in radiation protection of normal tissues both in vitro and in vivo while selectively increasing radiation sensitivity of orthotopic melanoma and squamous cell lung cancer tumors in C57BL/6 mice. However, the mechanism by which CD47 suppression produced the selective responses to irradiation in normal tissues and cancer cells was unknown. In the paper by Soto-Pantoja et al (7), a series of experiments was conducted to test the hypothesis that CD47 deficiency
8
Oncology Scan
promotes an altered radiation response in tissues by increasing autophagy. The key findings include: 1. CD47 / Jurkat T cells were assessed for viability, cytotoxicity, and proliferative capacity. In unirradiated cells, proliferative capacity and viability were independent of CD47 expression. However, a CD47 deficiency significantly increased viability and preserved proliferative capacity of irradiated CD47-deficient cells compared to that in wild-type cells. 2. They next assessed measures of autophagy, including visualization of autophagosome formation by electron microscopy, LC3-II expression via immunofluorescence, and expression of autophagy-related genes including BECN1, ATG5, and ATG7 by RT-PCR. Following irradiation, a CD47 deficiency resulted in increased number and size of autophagosomes, increased LC3-II puncta formation and intensity, and increased expression of BECN1, ATG5, and ATG7. These data demonstrate that the increased survival following irradiation conferred by CD47 deficiency is associated with increased markers of autophagy. Use of CD47 morpholino knockdown in human umbilical vein endothelial cells produced a similar increase in measures of autophagy after irradiation, indicating that the mechanism of radiation protection is not limited to a single cell type. 3. To determine whether radiation protection was mediated by increased autophagy, cells were pretreated with pharmacologic inhibitors of autophagy, hydroxychloroquine (HCQ) or 3-methyladenine (3-MA). Cell viability following irradiation was decreased in CD47 / cells treated with an autophagy inhibitor. In contrast, viability of wild-type CD47-expressing cells was not affected by exposure to an autophagy inhibitor. This demonstrates that inhibition of autophagy restores radiation sensitivity in CD47 / cells. These findings were further extended by small interfering RNA (siRNA) knockdown of the autophagy-associated genes ATG5 and ATG7. Similar to pharmacologic inhibition, genetic knockdown of autophagy-associated genes returned CD47-deficient cells to a radiation-sensitive state, as demonstrated by decreased viability and proliferative capacity following irradiation. Importantly, transfection of CD47 / cells with plasmid reexpressing CD47 reversed the radiation-protective effect with a concomitant decrease in measures of autophagy following irradiation. 4. Wild-type C57BL/6 mice were administered a CD47 antisense morpholino 48 hours prior to single-dose total body irradiation. Twenty-four hours after irradiation, lung tissue in CD47-suppressed mice exhibited increased expression of autophagy-associated genes and decreased apoptosis compared to control treated mice.
International Journal of Radiation Oncology Biology Physics
Comment: This is an interesting article that outlines a radiation-protective role for autophagy in normal tissues. It extends the finding that a CD47 deficiency confers a survival advantage to cells following irradiation and that this is associated with increases in measures of autophagy. In addition, both pharmacologic and genetic inhibition of autophagy reverse the radiation protection observed in CD47-deficient cells while having no effect on the radiation sensitivity of wild-type cells. The limitations of this study relate to the use of relatively high radiation doses, cell culture-based assays, and minimal measurements of pathophysiology. The authors were aware of these limitations and therefore undertook the following investigation (10).
Soto-Pantoja et al. Blockade of CD47 increases survival of mice exposed to lethal total body irradiation. Sci Rep 2013. (10) Summary: This study by Soto-Pantoja et al (10) seeks to address the problem of normal tissue toxicity following either therapeutic or incidental exposures to total body irradiation (TBI), building upon previous findings that CD47 deficiency confers radiation protection to normal tissues through increased autophagy following irradiation (7,9). C57BL/6 mice were treated with CD47 antisense morpholino oligomers or control by intraperitoneal injection 48 hours prior to single-dose TBI. The key findings were as follows: 1. Survival 30 days after administration of 7.6 Gy TBI was 71% for mice with CD47 suppression compared with 40% for mismatch-treated control mice. If CD47 morpholino was administered immediately after radiation, there was also a survival advantage, although to a lesser degree (40% vs 20% controls at 30 days). 2. CD47 suppression decreased measures of normal tissue toxicity following TBI. Bone marrow cells harvested and cultured 24 hours after TBI demonstrated increased proliferative capacity in CD47 morphants. Similarly, white blood cell counts from peripheral blood were maintained in the CD47 morpholino group whereas a decrease was observed in controls. Intestinal villi in CD47-suppressed mice had less TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) staining following irradiation and demonstrated normal architecture 2 weeks later. Similar results were obtained according to assessments of the esophageal mucosal layer, with CD47 morpholino conferring protection after radiation whereas control subjects had loss of architecture and significantly increased TUNEL staining. 3. p62 is degraded during activation of autophagy and is therefore used as a surrogate measurement of relative
Volume 90 Number 1 2014
autophagic activity. Immunohistochemical staining (IHC) for p62 in both intestinal and esophageal mucosa demonstrated a significant increase in p62 expression following TBI in control mice, suggesting decreased autophagy following radiation in normal tissues. In contrast, suppression of CD47 resulted in decreased p62 staining in both tissues following irradiation, indicating increased autophagy associated with CD47 blockade. Comment: This is an exciting study both for demonstration of a potential target for normal tissue radiation protection and for use of an elegant system for in vivo genetic manipulation. CD47 suppression not only conferred a survival advantage following irradiation, but it was also associated with maintenance of hematopoietic and gastrointestinal function, which are 2 of the primary system failures contributing to lethality after whole-body irradiation. Additionally, there was a survival advantage even if CD47 suppression occurred after irradiation, although to a lesser extent. First, this has important implications for accidental lethal radiation exposures for which the current treatments are primarily supportive care and, potentially, stem cell transplantation. Second, the authors were able to systemically block CD47 protein translation by use of intraperitoneal administration of antisense morpholino just 48 hours prior to a lethal dose of TBI. This is an exciting new tool for conducting in vivoetargeted genetic manipulations due to its ease of delivery in comparison with the more labor- and time-intensive development of knockout and transgenic animals. Ko et al. Autophagy inhibition radiosensitizes in vitro, yet reduces radioresponses in vivo due to deficient immunogenic signaling. Cell Death Differ 2014. (11) Summary: In this article by Ko et al (11), the authors investigated the contribution of autophagy to radiation response in tumor cells. Genetic silencing of autophagyassociated genes in multiple transformed cell lines in vitro resulted in radiation sensitization, which confirms prior reports. In addition, orthotopic xenografts of autophagy-deficient tumor cells transplanted into immunodeficient mice also produced a radiation sensitization effect. The stunning observation made by these authors is that knockdown of autophagy in tumors implanted in immunocompetent mice produced the opposite effect, resulting in radiation protection. The key findings are as follows: 1. Autophagic function was inhibited in vitro by small hairpin RNA (shRNA) directed against the autophagyassociated gene ATG5 or BECN1. This resulted in decreased cell survival following irradiation in both H460 and A549 cells. This increased radiation sensitivity was associated with a decrease in mitochondrial
Oncology Scan
9
membrane potential and loss of plasma membrane integrity. Xenografts of ATG5- or BECLIN-1-deficient tumor cells transplanted in immunodeficient BALB/c nude mice demonstrated increased sensitivity to radiation as measured by a significant delay in tumor growth. 2. To determine the interaction between host immune response and autophagy following irradiation, mouse CT26 colon carcinoma tumors with ATG5 knockdown were implanted in BALB/c immune-deficient or wild-type immune-competent mice and irradiated. Although autophagydeficient tumors demonstrated radiation sensitivity in the immunodeficient mice, the opposite effect was observed in the immunocompetent group. Suppression of autophagy in the tumors implanted in immunocompetent hosts resulted in less tumor growth delay following irradiation than in autophagy-intact tumors. 3. It was hypothesized that autophagy contributes to immunogenic-mediated cell death in the immune competent animal. Extracellular ATP is one of the signals released as part of the immunogenic cell death response. Correspondingly, cells with suppression of autophagy had decreased levels of extracellular ATP following irradiation in comparison with controls in vitro. Pharmacologic inhibition of ecto-ATPase function with ARL67156, when given intratumorally to autophagy-deficient xenografts, restored the radiation sensitivity of tumor cells in immunocompetent hosts. No alteration in sensitivity to radiation was observed in immunodeficient mice given ARL67156 to increase extracellular ATP levels. This indicates that the differential contribution of autophagy to radiation response is due to an interaction with the host immune response following irradiation. Indeed, autophagysuppressed tumors were found to have less infiltration of lymphocytes 9 days after irradiation, and this effect was reversed if ecto-ATPase function was inhibited in the tumors prior to irradiation. Comment: The data presented in this article further expand our knowledge of the contribution of autophagy to the radiation response of cancer cells. It also underscores the care that must be taken with both experimental design and interpretation of results of animal models of cancer biology. Immunedeficient mice are frequently used in cancer biology because they allow us to implant human patient-derived tumors and study the response to various interventions. However, no single model perfectly replicates the conditions of initial growth and treatment response present in patients. In this case, the evidence of increased radiation sensitivity of autophagysuppressed tumors in immune-deficient mice might lead one to conclude that autophagy acts primarily to protect cells from the stress induced by irradiation. However, this would ignore the contribution of host immune response to tumor control. Interestingly, expansion of the experimental design to assess
10
Oncology Scan
radiation response in immunocompetent hosts provided evidence that autophagy may function to increase immunogenic cell death and therefore increase tumor sensitivity to radiation. As outlined in the above papers, we are only just beginning to understand the contributions of autophagy to the radiation response of normal tissues and cancer cells. There is evidence both for a radiation-protective role and for radiation sensitization, which appears to vary based on conditions of the target cells and host immune status. Clinical trials are currently underway investigating the use of the autophagy inhibitors chloroquine and hydroxychloroquine as a means to suppress the cytoprotective function of autophagy in association with various cancer therapies. Based on the results presented above, there may also be a therapeutic role for autophagy in protecting normal tissues from lethal radiation exposures.
International Journal of Radiation Oncology Biology Physics
3.
4. 5.
6.
7.
8. 9.
References 1. Gewirtz DA. The four faces of autophagy: Implications for cancer therapy. Cancer Res 2014;74:647-651. 2. O’Donovan TR, O’Sullivan GC, McKenna SL. Induction of autophagy by drug-resistant esophageal cancer cells promotes their survival and
10.
11.
recovery following treatment with chemotherapeutics. Autophagy 2011;7:509-524. Liu D, Yang Y, Liu Q, et al. Inhibition of autophagy by 3-MA potentiates cisplatin-induced apoptosis in esophageal squamous cell carcinoma cells. Med Oncol 2011;28:105-111. Li X, Lu Y, Pan T, et al. Roles of autophagy in cetuximab-mediated cancer therapy against EGFR. Autophagy 2010;6:1066-1077. Voss V, Senft C, Lang V, et al. The pan-Bcl-2 inhibitor (-)-gossypol triggers autophagic cell death in malignant glioma. Mol Cancer Res 2010;8:1002-1016. Bristol ML, Di X, Beckman MJ, et al. Dual functions of autophagy in the response of breast tumor cells to radiation: Cytoprotective autophagy with radiation alone and cytotoxic autophagy in radiosensitization by vitamin D3. Autophagy 2012;8:739-753. Soto-Pantoja DR, Miller TW, Pendrak ML, et al. CD47 deficiency confers cell and tissue radioprotection by activation of autophagy. Autophagy 2012;8:1628-1642. Brown EJ, Frazier WA. Integrin-associated protein (CD47) and its ligands. Trends Cell Biol 2001;11:130-135. Maxhimer JB, Soto-Pantoja DR, Ridnour LA, et al. Radioprotection in normal tissue and delayed tumor growth by blockade of CD47 signaling. Sci Trans Med 2009;1:3ra7. Soto-Pantoja DR, Ridnour LA, Wink DA, et al. Blockade of CD47 increases survival of mice exposed to lethal total body irradiation. Sci Rep 2013;3:1038. Ko A, Kanehisa A, Martins I, et al. Autophagy inhibition radiosensitizes in vitro, yet reduces radioresponses in vivo due to deficient immunogenic signalling. Cell Death Differ 2014;21:92-99.
