The Autophagy Group

Background

Macroautophagy (hereafter autophagy) is a highly regulated catabolic process for the recycling of old or damaged organelles and macromolecules. It is controlled by a set of evolutionarily conserved Atg genes, which orchestrate the formation of a double-membrane structure called phagophore that engulfs portions of cytoplasm and even whole organelles thereby forming an autophagosome (Figure 2).

The outer membrane of autophagosome then fuses with endo-lysosomal vesicles eventually forming an autolysosome (Figure 1). Subsequently, the cargo and the autophagosomal inner membrane are degraded by the lysosomal hydrolases and the processed molecules are returned back to the cytoplasm.

Autophagic flow is low in optimal growth conditions, but can be rapidly activated in response to starvation, cytotoxic drugs, and other forms of cellular stress. In such conditions, autophagy promotes cell survival by sustaining metabolic homeostasis and preventing the accumulation of toxic waste. Thus, autophagy can promote tumor progression in the microenvironment devoid of nutrients and oxygen and contribute to resistance to cancer therapy. On the other hand, autophagy may act as a barrier for transformation. Accordingly, many tumor-suppressor proteins act as positive regulators of autophagy and most human cancers display hyper-activation of signaling pathways that suppress autophagy.

Mammalian target of rapamycin complex 1 (mTORC1) kinase, the major negative regulator of autophagy, serves as a signaling nexus that integrates information regarding cellular stress and availability of nutrients and growth factors to maintenance of the appropriate balance between anabolic and catabolic processes. The signaling pathways promoting mTORC1 activation are induced by numerous mitogenic factors and oncoproteins via the class I phosphoinositide-3 kinase (PI3K)-Akt pathway, whereas various cellular stresses inhibit the mTORC1 activity via activation of AMP-activated protein kinase (AMPK) (Figure 3).

mTORC1 controls autophagy partly by inhibiting unc51-like kinases (ULK1 and ULK2), whose activation is essential for the nucleation of the isolation-membrane that eventually forms the autophagosome (Figure 2). This early step is dependent on the generation of phosphatidylinositol 3-phosphate (PtdIns(3)P) synthesized by the autophagy-specific class III PI3K (Vps34) complex. The ubiquitin like molecules Atg12 and microtubule-associated protein 1 light chain 3 (LC3 or Atg8) together with their corresponding conjugation systems are essential for the expansion of the isolation membrane.

LC3 is present on the membranes of the completed autophagosome (Figure 4) and gets degraded in the autolysosome along with the membranes. The degradation of LC3 can thus serve as a marker for the autophagic flux (Figure 5).

Due to its involvement in many pathological processes, autophagy is an utmost attractive drug target. Rapamycin, lithium, and chloroquine are the first examples of old drugs that are entering the clinics for new indications as regulators of autophagy. Rapamycin and lithium are mTORC1-dependent and -independent inducers of autophagy, respectively. As relatively safe drugs, they may prove useful in the treatment of various degradative disorders. The anti-malaria drug chloroquine inhibits autolysosomal degradation by disrupting the lysosomal pH gradient and it is presently the only autophagy inhibiting drug in clinical trials for cancer treatment. Chloroquine blocks the lysosomal function and is therefore a very unspecific autophagy inhibitor. Thus, there is an acute need for more specific autophagy inhibitors both in the autophagy research community and the clinic.

Research Aims

Inhibition of autophagy augments the efficacy of many currently available anticancer regimens, and our foremost goal is to identify autophagy-mediating proteins that are suitable targets for cancer therapy. For this purpose, Thomas Farkas has developed a Renilla luciferase reporter-based assay system that allows the monitoring of autophagy kinetics in living cells and is suitable for high throughput screening (Figure 5). Using this system as well as other methods, we have screened siRNA and small molecule libraries and identified numerous kinases, phosphatases, motor proteins, lipid modifying enzymes, and small molecules as autophagy regulators. Our present work aims at compiling this information and defining the best targets / molecules for autophagy inhibition in cancer therapy. Autophagy being a double-edged sword that in addition to enhancing the survival of cancer cells can also inhibit tumorigenesis, we are also investigating the tumor suppressive role of autophagy in leukemias and possibilities of switching cytoprotective autophagy to cytotoxic autophagy.