Autophagy regulates cell death both positively and negatively, but the molecular basis for this paradox remains inadequately characterized. We demonstrate here that transient cell-to-cell variations in autophagy can either promote cell death or survival depending on the stimulus and cell type. By separating cells with high and low basal autophagy by flow cytometry, we demonstrate that autophagy determines which cells live or die in response to death receptor activation. We have determined that selective autophagic degradation of the phosphatase Fap-1 promotes Fas apoptosis in Type I cells. Conversely, autophagy inhibits apoptosis in Type II cells or upon treatment with TRAIL in either Type I or II cells. These data illustrate that differences in autophagy in a cell population determine cell fate in a stimulus- and cell type-specific manner. This example of selective autophagy of an apoptosis regulator may represent a general mechanism for context-specific regulation of cell fate by autophagy.

Summary Macroautophagy is thought to protect against apoptosis, however underlying mechanisms are poorly understood. We examined how autophagy affects canonical death receptor-induced mitochondrial outer membrane permeabilization (MOMP) and apoptosis. MOMP occurs at variable times in a population of cells and this is delayed by autophagy. Additionally, autophagy leads to inefficient MOMP after which some cells die through a slower process than typical apoptosis and, surprisingly, can recover and divide afterwards. These effects are associated with p62/SQSTM1-dependent selective autophagy causing PUMA levels to be kept low through an indirect mechanism whereby autophagy affects constitutive levels of PUMA mRNA. PUMA depletion is sufficient to prevent the sensitization to apoptosis that occurs when autophagy is blocked. Autophagy can therefore control apoptosis via a key regulator that makes MOMP faster and more efficient thus ensuring rapid completion of apoptosis. This identifies a molecular mechanism whereby cell fate decisions can be determined by autophagy.

Chloroquine (CQ) is an antimalarial drug and late-stage inhibitor of autophagy currently FDA-approved for use in the treatment of rheumatoid arthritis and other autoimmune diseases. Based primarily on its ability to inhibit autophagy, CQ and its derivative, hydroxychloroquine, are currently being investigated as primary or adjuvant therapy in multiple clinical trials for cancer treatment. Oncogenic RAS has previously been shown to regulate autophagic flux, and cancers with high incidence of RAS mutations, such as pancreatic cancer, have been described in the literature as being particularly susceptible to CQ treatment, leading to the hypothesis that oncogenic RAS makes cancer cells dependent on autophagy. This autophagy “addiction” suggests that the mutation status of RAS in tumors could identify patients who would be more likely to benefit from CQ therapy. Here we show that RAS mutation status itself is unlikely to be beneficial in such a patient selection because oncogenic RAS does not always promote autophagy addiction. Moreover, oncogenic RAS can have opposite effects on both autophagic flux and CQ sensitivity in different cells. Finally, for any given cell type, the positive or negative effect of oncogenic RAS on autophagy does not necessarily predict whether RAS will promote or inhibit CQ-mediated toxicity. Thus, although our results confirm that different tumor cell lines display marked differences in how they respond to autophagy inhibition, these differences can occur irrespective of RAS mutation status and, in different contexts, can either promote or reduce chloroquine sensitivity of tumor cells.

A glance through Autophagy or any other journal in this field shows that it is very common to block autophagy by RNA interference-based knockdown of ATG mRNAs in mammalian cell lines. Our lab's experience is that this approach can easily make for failed experiments because good knockdown of even essential autophagy regulators does not necessarily mean you will get good inhibition of autophagy, and, over time, cells can find ways to circumvent the inhibitory effects of the knockdown.