The ability of cells to sense and respond to growth factors and nutrients represents a fundamental requirement for survival. Under nutrient- and growth factor–poor conditions, decreased activation of the kinases Akt and mammalian target of rapamycin (mTOR), two key integrators of growth factor and nutrient signaling, leads to initiation of a catabolic program that enables cells to survive periods of starvation or stress [reviewed in (1
)]. Under nutrient- and growth factor–rich conditions, growth factors signal through receptor tyrosine kinases (RTKs) to activate downstream kinases such as class IA phosphatidylinositol 3-kinases [principally PI3K α and β, as reviewed in (2
)]. The PI3Ks in turn propagate downstream signals, including activation of Akt and mTOR, stimulating an anabolic program of protein synthesis and cell growth.
Tight regulation of the Akt-mTOR pathway enables cells to sense changes in their environment and survive both minor and major perturbations in the abundance of nutrients and growth factors. Akt signaling stimulates the activity of numerous downstream targets, including the proapoptotic proteins BAD (Bcl-2/Bcl-XL–associated death promotor), caspases 3 and 9, and FoxO (forkhead) family transcription factors, that act to tip the balance from survival toward apoptosis during periods of growth factor deprivation. Given the central role for Akt in cell survival, it is not surprising that Akt overactivation has been implicated in cancer. For example, malignant glioma, the most common primary brain tumor, is frequently associated with deletion or silencing of the gene encoding the lipid phosphatase PTEN (phosphatase and tensin homolog deleted from chromosome 10), which antagonizes Akt signaling [reviewed in (2
)]. In both clinical and preclinical trials, PTEN
deletion has been associated with resistance to therapy (3
), supporting a role for the RTK-PI3K-Akt-mTOR axis in mediating cancer cell survival.
The initial enthusiasm for using inhibitors of PI3Ks, Akt, or mTOR as antineoplastic agents has been tempered by observations that inhibition of these kinases typically promotes growth arrest rather than cell death in solid tumors [reviewed in (6
)]. Because mTOR is a target of both growth factor and nutrient signaling, its blockade is likely to activate one or more survival pathways that act to enable cells to endure periods of starvation or stress. Macroautophagy (hereafter called autophagy), a cellular self-digestion process that provides energy and nutrients during stress (7
), is a good candidate for such a survival pathway (8
). Indeed, experiments in the yeast Saccharomyces cerevisiae
suggest that Tor is a key node central to control of autophagy (9
Autophagy is an evolutionarily conserved process through which organelles and proteins are sequestered into autophagic vesicles (autophagosomes) within the cytosol [reviewed in (8
)]. These vesicles then fuse with the lysosome, forming autophagolysosomes, which promote the degradation of intracellular contents. Microtubule-associated protein light chain 3 (LC3-I) is an abundant cytoplasmic protein that is cleaved and lipidated during initiation of autophagy (forming LC3-II), translocating to and associating with the autophagosome in a punctate pattern (10
). Autophagy thus enables the cell to eliminate and recycle proteins or organelles to sustain metabolism and can be recognized in part by formation of LC3-II punctae.
Inhibition of autophagy promotes cancer cell death (11
) and potentiates various anticancer therapies (14
), implicating autophagy as a mechanism that enables tumor cells to survive antineoplastic therapy. The antimalarial drug chloroquine inhibits autophagy of glioma cells and has been tested as an antineoplastic agent in a small clinical study (25
). The related molecule hydroxychloroquine is the subject of an ongoing Phase II study (14
) and is a much-discussed option among patients who may self-medicate during therapy for glioma (26
). Although chloroquine’s use in glioma was not predicated on the basis of its ability to inhibit autophagic degradation, this compound, like hydroxychloroquine, blocks lysosomal functions required for the terminal steps of autophagy (15
Here, we showed that dual inhibitors of PI3K and mTOR signaling activated autophagy in glioma, and that inhibition of two distinct mTOR protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), induced autophagy in an additive fashion. Because the allosteric mTORC1 inhibitor rapamycin induces autophagy, we were surprised to find that inhibition of autophagosome maturation in the presence of rapamycin did not promote apoptosis. Rather, apoptosis was induced only when rapamycin was combined with inhibitors of both autophagosome maturation and PI3K. To understand why blockade of PI3K itself does not induce apoptosis but was critical to the induction of apoptosis by the combination of rapamycin and inhibitors of autophagosome maturation, we investigated the ability of rapamycin to induce autophagy and concurrently activate Akt. We found that rapamycin induced both autophagy and Akt phosphorylation as separate survival signals. Combining rapamycin with inhibitors of autophagy or of PI3K blocked only one of these, allowing cells to survive. In contrast, combining rapamycin with inhibitors of autophagy and of PI3K blocked both survival signals, resulting in apoptosis.
Furthermore, we showed that NVP-BEZ235, which inhibits both PI3K and mTOR signaling and is currently in Phase I/II clinical trials in solid tumors (27
), cooperated with chloroquine to promote cell death in glioma. Because inhibitors of PI3K, mTOR, and autophagosome maturation are all in clinical trials or clinical use, this combination of agents represents a promising and translatable approach to cancer therapy.