Targeting autophagy enhances atezolizumab-induced mitochondria-related apoptosis in osteosarcoma
Abstract
In this study, we identified the multifaceted effects of atezolizumab, a specific monoclonal antibody against PD-L1, in tumor suppression except for restoring antitumor immunity, and investigated the promising ways to improve its efficacy. Atezolizumab could inhibit the proliferation and induce immune-independent apoptosis of osteosarcoma cells. With further exploration, we found that atezolizumab could impair mitochondria of osteosarcoma cells, resulting in increased release of reactive oxygen species and cytochrome-c, eventually leading to mitochondrial-related apoptosis via activating JNK pathway. Nevertheless, the excessive release of reactive oxygen species also activated the protective autophagy of osteosarcoma cells. Therefore, when we combined atezolizumab with autophagy inhibitors, the cytotoxic effect of atezolizumab on osteosarcoma cells was significantly enhanced in vitro. Further in vivo experiments also confirmed that atezolizumab combined with chloroquine achieved the most significant antitumor effect. Taken together, our study indicates that atezolizumab can induce mitochondrial-related apoptosis and protective autophagy independently of the immune system, and targeting autophagy is a promising combinatorial approach to amplify its cytotoxicity.
https://www.nature.com/articles/s41419-021-03449-6
Targeting autophagy enhances atezolizumab-induced mitochondria-related apoptosis in osteosarcoma
Re: Targeting autophagy enhances atezolizumab-induced mitochondria-related apoptosis in osteosarcoma
It is very interesting. So we will watch for the clinical trials combining ICI drugs with autophagy inhibitors. Chloroquine is widely available so should not be a problem, but when it makes sense to combine them is another question. In many cases the treatments only have their place at certain periods of tumor growth/resistance etc. They might be beneficial or otherwise depends on the timing.
I am wondering if hydroxychloroquine is the same as chloroquine?
I am wondering if hydroxychloroquine is the same as chloroquine?
Olga
Re: Targeting autophagy enhances atezolizumab-induced mitochondria-related apoptosis in osteosarcoma
Olga wrote: ↑Tue Dec 07, 2021 12:27 pm It is very interesting. So we will watch for the clinical trials combining ICI drugs with autophagy inhibitors. Chloroquine is widely available so should not be a problem, but when it makes sense to combine them is another question. In many cases the treatments only have their place at certain periods of tumor growth/resistance etc. They might be beneficial or otherwise depends on the timing.
I am wondering if hydroxychloroquine is the same as chloroquine?
Olga found this on the difference between the 2…
https://www.cebm.net/covid-19/chloroqui ... -covid-19/
Debbie
Re: Targeting autophagy enhances atezolizumab-induced mitochondria-related apoptosis in osteosarcoma
Discussion
Our previous study showed that antibodies against PD-L1 could restore the antitumor immunity of CD8+ T cells in OS12. In this study, we found that atezolizumab could also induce immune-independent mitochondria-related apoptosis by increasing ROS and cyto-c release in OS. In addition, blocking the protective autophagy induced by atezolizumab could significantly amplify its antitumor effect on OS cells. These findings help us to fully illustrate the antitumor effect of atezolizumab and explore the promising way to further improve its efficacy.
Atezolizumab is a fully-humanized monoclonal antibody against PD-L1, which has achieved satisfactory results in the treatment of advanced tumors24,25,26. Atezolizumab can ameliorate the lymphocyte apoptosis by disrupting the binding of PD-L1 on the surface of tumor to PD-1 on the surface of lymphocytes, and restore the antitumor immunity of lymphocytes, thereby inhibiting tumor growth27,28. Whereas, recent studies have shown that the blockade of PD-L1/PD-1 not only had an extracellular effect but also had an important intracellular effect on tumor cells. As previous researches demonstrate that PD-L1 plays a critical role in maintaining stemness and promoting self-renewal as well as tumorigenicity of tumor cells17,29, but the specific mechanism is still unclear. Here we discover that atezolizumab can inhibit cell proliferation while inducing apoptosis of OS cells directly in a dose-dependent manner.
To further study the underlying mechanism, we examined the change of intracellular ultrastructure after atezolizumab treatment through transmission electron microscopy. Mitochondria are essential for cells to maintain the physiological state, and it is closely related to the chemotherapy-induced tumor cell apoptosis30. We found that the originally uniformly arranged mitochondria crest become dilated, and the whole mitochondria are vacuolated with the addition of atezolizumab, accompanied by impaired mitochondrial function and decreased membrane potential. The damage to mitochondria could increase the permeability of its membrane, which in turn resulted in the excessive release of ROS and cyto-c. Atezolizumab increased the release of ROS while reducing the content of SOD and T-AOC in OS cells, which in turn caused excessive oxidative stress, leading to lipid peroxidation and DNA damage. Meanwhile, the excessive release of cyto-c could bind to APAF-1 in cytoplasm and thus activate caspase-9, one of the initiators of apoptosis, and eventually activating caspase-3 causing mitochondria-related apoptosis in cells31,32,33. The activation of JNK pathway is closely related to the tumor cell apoptosis, especially to the mitochondria-related apoptosis34,35. And we propose that the immune-independent cytotoxicity of atezolizumab on OS cells is partially achieved by activating the JNK pathway.
Accumulating evidence indicates mitochondria as the main source for cellular ROS36. In the physiological state, there is a certain number of ROS in the body acting as important intracellular messengers to participate in the maintenance of homeostasis37,38. But the excessive release of ROS is harmful to the body and therefore activates the protective mechanisms in the body (such as autophagy) to maintain the homeostasis39. Autophagy can protect tumor cells from damage induced by external stimuli (protective autophagy), and it can also aggravate the damage caused by external stimuli (destructive autophagy)40,41. Given that, a comprehensive understanding of the role of autophagy in tumorigenesis is helpful for better-combined therapy targeting autophagy. In this study, we consider that the activation of autophagy followed by atezolizumab stimulation may be a pro-survival adaptation of tumor cells to resist external stimuli, as the proapoptotic effect of atezolizumab is enhanced after the blockade of autophagy via CQ or siRNA targeting Atg-5 in vitro. In addition, the antitumor effect of atezolizumab combined with CQ is also the most prominent compared with the other groups in the xenograft tumor model. Blocking the conjugation of PD-L1/PD-1 can activate intracellular autophagy19,42, and we estimate that the excessive release of ROS induced by atezolizumab may be one of the pathways, by which atezolizumab activates autophagy in OS.
In conclusion, our data indicate that the antitumor effects of PD-L1 antibodies are considerably broader than simply blocking PD-L1/PD-1 conjugation to restore the antitumor immunity of T cells. We unravel a novel mechanism whereby atezolizumab inhibits OS cell proliferation and promotes apoptosis directly in a dose-dependent manner. Additionally, atezolizumab can also activate protective autophagy by damaging mitochondria and causing excessive release of ROS. And blocking protective autophagy can enhance the antitumor effect of atezolizumab. These findings suggest that combining autophagy inhibitor (such as CQ), as a potential adjuvant, with atezolizumab in the treatment of OS may be a promising therapeutic strategy to improve its efficacy.
Our previous study showed that antibodies against PD-L1 could restore the antitumor immunity of CD8+ T cells in OS12. In this study, we found that atezolizumab could also induce immune-independent mitochondria-related apoptosis by increasing ROS and cyto-c release in OS. In addition, blocking the protective autophagy induced by atezolizumab could significantly amplify its antitumor effect on OS cells. These findings help us to fully illustrate the antitumor effect of atezolizumab and explore the promising way to further improve its efficacy.
Atezolizumab is a fully-humanized monoclonal antibody against PD-L1, which has achieved satisfactory results in the treatment of advanced tumors24,25,26. Atezolizumab can ameliorate the lymphocyte apoptosis by disrupting the binding of PD-L1 on the surface of tumor to PD-1 on the surface of lymphocytes, and restore the antitumor immunity of lymphocytes, thereby inhibiting tumor growth27,28. Whereas, recent studies have shown that the blockade of PD-L1/PD-1 not only had an extracellular effect but also had an important intracellular effect on tumor cells. As previous researches demonstrate that PD-L1 plays a critical role in maintaining stemness and promoting self-renewal as well as tumorigenicity of tumor cells17,29, but the specific mechanism is still unclear. Here we discover that atezolizumab can inhibit cell proliferation while inducing apoptosis of OS cells directly in a dose-dependent manner.
To further study the underlying mechanism, we examined the change of intracellular ultrastructure after atezolizumab treatment through transmission electron microscopy. Mitochondria are essential for cells to maintain the physiological state, and it is closely related to the chemotherapy-induced tumor cell apoptosis30. We found that the originally uniformly arranged mitochondria crest become dilated, and the whole mitochondria are vacuolated with the addition of atezolizumab, accompanied by impaired mitochondrial function and decreased membrane potential. The damage to mitochondria could increase the permeability of its membrane, which in turn resulted in the excessive release of ROS and cyto-c. Atezolizumab increased the release of ROS while reducing the content of SOD and T-AOC in OS cells, which in turn caused excessive oxidative stress, leading to lipid peroxidation and DNA damage. Meanwhile, the excessive release of cyto-c could bind to APAF-1 in cytoplasm and thus activate caspase-9, one of the initiators of apoptosis, and eventually activating caspase-3 causing mitochondria-related apoptosis in cells31,32,33. The activation of JNK pathway is closely related to the tumor cell apoptosis, especially to the mitochondria-related apoptosis34,35. And we propose that the immune-independent cytotoxicity of atezolizumab on OS cells is partially achieved by activating the JNK pathway.
Accumulating evidence indicates mitochondria as the main source for cellular ROS36. In the physiological state, there is a certain number of ROS in the body acting as important intracellular messengers to participate in the maintenance of homeostasis37,38. But the excessive release of ROS is harmful to the body and therefore activates the protective mechanisms in the body (such as autophagy) to maintain the homeostasis39. Autophagy can protect tumor cells from damage induced by external stimuli (protective autophagy), and it can also aggravate the damage caused by external stimuli (destructive autophagy)40,41. Given that, a comprehensive understanding of the role of autophagy in tumorigenesis is helpful for better-combined therapy targeting autophagy. In this study, we consider that the activation of autophagy followed by atezolizumab stimulation may be a pro-survival adaptation of tumor cells to resist external stimuli, as the proapoptotic effect of atezolizumab is enhanced after the blockade of autophagy via CQ or siRNA targeting Atg-5 in vitro. In addition, the antitumor effect of atezolizumab combined with CQ is also the most prominent compared with the other groups in the xenograft tumor model. Blocking the conjugation of PD-L1/PD-1 can activate intracellular autophagy19,42, and we estimate that the excessive release of ROS induced by atezolizumab may be one of the pathways, by which atezolizumab activates autophagy in OS.
In conclusion, our data indicate that the antitumor effects of PD-L1 antibodies are considerably broader than simply blocking PD-L1/PD-1 conjugation to restore the antitumor immunity of T cells. We unravel a novel mechanism whereby atezolizumab inhibits OS cell proliferation and promotes apoptosis directly in a dose-dependent manner. Additionally, atezolizumab can also activate protective autophagy by damaging mitochondria and causing excessive release of ROS. And blocking protective autophagy can enhance the antitumor effect of atezolizumab. These findings suggest that combining autophagy inhibitor (such as CQ), as a potential adjuvant, with atezolizumab in the treatment of OS may be a promising therapeutic strategy to improve its efficacy.
Debbie