Our results demonstrate that chemotherapy-induced apoptosis suppresses autophagy at the execution stage following cytochrome c
release, at least in part through caspase 8-mediated Beclin 1 cleavage at D133 and D146. Caspase 8 not only plays a key role in death receptor-mediated apoptosis, but also contributes to the execution of DNA damage-induced apoptosis through the intrinsic pathway (22
). Specifically blocking Beclin 1 cleavage by knocking in a caspase-resistant mutant of Beclin 1
recapitulated autophagy induction seen in apoptosis-deficient cells. On the other hand, autophagy induction in Beclin 1
-KI cells did not affect apoptotic events, but protected against cell death. These results provide mechanistic insight into how chemotherapy-induced apoptosis suppresses autophagy, and explain why autophagy is generally observed in cells in which caspase activation is blocked.
Several recent studies using different cell types and stimuli also described caspase-mediated cleavage of ATG proteins in apoptosis (15
). Cho et al.
found caspase-dependent cleavage of Beclin 1 in Hela cells treated with a death receptor ligand (34
). Luo and Rubinzstein reported caspase 3-mediated Beclin 1 cleavage at D149 in apoptosis induced by Bax overexpression (35
). Wirawan et al.
demonstrated caspase cleavage of Beclin 1 at D133 and D149 during apoptosis induced by IL-3 depletion in murine hematopoietic cells (36
). ATG5 and ATG4D were also found to be cleaved by caspases in apoptotic cells (14
). Furthermore, caspase 8 has been implicated in autophagy suppression in mouse L929 fibroblast and human U937 monocytoid cells (38
), and also in proliferating T cells (40
). Our knock-in data indicate that specific blockage of endogenous Beclin 1 cleavage alone is sufficient to restore autophagy in apoptotic cells, at least in colon cancer cells undergoing chemotherapy-induced apoptosis, and possibly in other scenarios as well. Differences in cell types and apoptotic stimuli may explain why D146 was required for Beclin 1 cleavage in our study, but D149 was necessary for Beclin 1 cleavage in other studies.
Autophagy occurring subsequent to cytochrome c
release is likely to be trigged by mitochondrial outer membrane permeabilization (MOMP), and is therefore mitophagy, a recycle process in which mitochondria are captured and degraded (41
). Although Beclin 1 can interact with antiapoptotic Bcl-2 family members through its BH3 domain, it does not seem to significantly affect apoptosis initiation and caspase activation. Nonetheless, Beclin 1-mediated autophagy may modulate chemotherapeutic response by removing terminally damaged mitochondria with MOMP, which may help to sustain energy metabolism of cancer cells, and allow them to tolerate and recover from the damages induced by chemotherapeutic agents. It has been shown that mitophagy mediated by ATG12 and metabolic changes mediated by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) preserve the survival of staurosporine-treated cells in which cytochrome c
release had occurred, but caspase activation was blocked (42
). Therefore, autophagy may not simply be an innocent bystander (17
), but rather an important cell survival pathway that has to be turned off for full execution of cell death.
A remaining question is how Beclin 1 cleavage during apoptosis affects its functions. Beclin 1 cleavage may not abolish its interaction with VPS34, which is mediated by a conserved region far apart from its cleavage sites (43
). The cleavage of Beclin 1 also leaves its BH3 domain intact. Only a fraction of Beclin 1, as detected by Western blotting, was subject to caspase cleavage in CPT-treated cells, which seemed to suggest a gain of function by Beclin 1 cleavage fragments. However, both N- and C-terminal Beclin 1 cleavage fragments remained localized in the cytosol, and their overexpression did not significantly affect apoptosis, consistent with intact apoptosis induction in Beclin 1
-KI cells. Therefore, caspase cleavage of Beclin 1 may serve primarily as an inhibitory mechanism of autophagy and cell survival.
Autophagy is a survival pathway utilized by cancer cells to tolerate metabolic stress. Most, if not all, anticancer agents can induce autophagy in cancer cells (16
). Autophagy inhibitors such as hydroxychloroquine are being tested in a number of clinical trials as a chemo sensitizer (16
). Our results suggest that Beclin 1, which is cleaved by caspases for switching off autophagy in chemotherapy-induced apoptosis, may be a useful target for more specific and effective pharmacological inhibition of autophagy.