Andrew Thorburn

Autophagy and apoptosis: what is the connection?

The therapeutic potential of autophagy for the treatment cancer and other diseases is beset by paradoxes stemming from the complexity of the interactions between the apoptotic and autophagic machinery. The simplest question of how autophagy acts as both a protector and executioner of cell death remains the subject of substantial controversy. Elucidating the molecular interactions between the processes will help us understand how autophagy can modulate cell death, whether autophagy is truly a cell death mechanism, and how these functions are regulated. We suggest that, despite many connections between autophagy and apoptosis, a strong causal relationship wherein one process controls the other, has not been demonstrated adequately. Knowing when and how to modulate autophagy therapeutically depends on understanding these connections.

Targeting autophagy during cancer therapy to improve clinical outcomes

Autophagy is a catabolic process that turns over long-lived proteins and organelles and contributes to cell and organism survival in times of stress. Current cancer therapies including chemotherapy and radiation are known to induce autophagy within tumor cells. This is therefore an attractive process to target during cancer therapy as there are safe, clinically available drugs known to both inhibit and stimulate autophagy. However, there are conflicting positive and negative effects of autophagy and no current consensus on how to manipulate autophagy to improve clinical outcomes. Careful and rigorous evaluation of autophagy with a focus on how to translate laboratory findings into relevant clinical therapies remains an important aspect of improving clinical outcomes in patients with malignant disease.

Autophagy and Cancer Therapy

Autophagy is the process by which cellular material is delivered to lysosomes for degradation and recycling. There are three different types of autophagy, but macroautophagy, which involves the formation of double membrane vesicles that engulf proteins and organelles that fuse with lysosomes, is by far the most studied and is thought to have important context-dependent roles in cancer development, progression, and treatment. The roles of autophagy in cancer treatment are complicated by two important discoveries over the past few years. First, most (perhaps all) anticancer drugs, as well as ionizing radiation, affect autophagy. In most, but not all cases, these treatments increase autophagy in tumor cells. Second, autophagy affects the ability of tumor cells to die after drug treatment, but the effect of autophagy may be to promote or inhibit cell death, depending on context. Here we discuss recent research related to autophagy and cancer therapy with a focus on how these processes may be manipulated to improve cancer therapy.

Translational evidence that impaired autophagy contributes to arterial ageing

•  Advancing age is the major risk factor for the development of cardiovascular diseases. •  Arterial endothelial dysfunction, characterized by impaired endothelium‐dependent dilatation (EDD), is a key antecedent to age‐associated clinical cardiovascular disease. •  We tested the hypothesis that changes in autophagy, the process by which cells recycle damaged biomolecules, may be an underlying cause of the age‐related reduction in EDD. •  We show that autophagy is impaired in arteries of older humans and mice with reduced EDD, and that enhancing autophagy restores EDD by reducing superoxide‐dependent oxidative stress and inflammation, and increasing nitric oxide bioavailability. •  Our results identify impaired autophagy as a potential cause of age‐related arterial dysfunction and suggest that boosting autophagy may be a novel strategy for the treatment of arterial endothelial dysfunction and prevention of cardiovascular diseases with ageing.

Hedgehog Signaling Alters Reliance on EGF Receptor Signaling and Mediates Anti-EGFR Therapeutic Resistance in Head and Neck Cancer

The EGF receptor (EGFR)-directed monoclonal antibody cetuximab is the only targeted therapy approved for the treatment of squamous cell carcinoma of the head and neck (HNSCC) but is only effective in a minority of patients. Epithelial-to-mesenchymal transition (EMT) has been implicated as a drug resistance mechanism in multiple cancers, and the EGFR and Hedgehog pathways (HhP) are relevant to this process, but the interplay between the two pathways has not been defined in HNSCC. Here, we show that HNSCC cells that were naturally sensitive to EGFR inhibition over time developed increased expression of the HhP transcription factor GLI1 as they became resistant after long-term EGFR inhibitor exposure. This robustly correlated with an increase in vimentin expression. Conversely, the HhP negatively regulated an EGFR-dependent, EMT-like state in HNSCC cells, and pharmacologic or genetic inhibition of HhP signaling pushed cells further into an EGFR-dependent phenotype, increasing expression of ZEB1 and VIM. In vivo treatment with cetuximab resulted in tumor shrinkage in four of six HNSCC patient-derived xenografts; however, they eventually regrew. Cetuximab in combination with the HhP inhibitor IPI-926 eliminated tumors in two cases and significantly delayed regrowth in the other two cases. Expression of EMT genes TWIST and ZEB2 was increased in sensitive xenografts, suggesting a possible resistant mesenchymal population. In summary, we report that EGFR-dependent HNSCC cells can undergo both EGFR-dependent and -independent EMT and HhP signaling is a regulator in both processes. Cetuximab plus IPI-926 forces tumor cells into an EGFR-dependent state, delaying or completely blocking tumor recurrence.

Autophagy and cell death.

Autophagy is intimately associated with eukaryotic cell death and apoptosis. Indeed, in some cases the same proteins control both autophagy and apoptosis. Apoptotic signalling can regulate autophagy and conversely autophagy can regulate apoptosis (and most likely other cell death mechanisms). However, the molecular connections between autophagy and cell death are complicated and, in different contexts, autophagy may promote or inhibit cell death. Surprisingly, although we know that, at its core, autophagy involves degradation of sequestered cytoplasmic material, and therefore presumably must be mediating its effects on cell death by degrading something, in most cases we have little idea of what is being degraded to promote autophagys pro- or anti-death activities. Because autophagy is known to play important roles in health and many diseases, it is critical to understand the mechanisms by which autophagy interacts with and affects the cell death machinery since this will perhaps allow new ways to prevent or treat disease. In the present chapter, we discuss the current state of understanding of these processes.

RIP1 negatively regulates basal autophagic flux through TFEB to control sensitivity to apoptosis

In a synthetic lethality/viability screen, we identified the serine–threonine kinase RIP1 (RIPK1) as a gene whose knockdown is highly selected against during growth in normal media, in which autophagy is not critical, but selected for in conditions that increase reliance on basal autophagy. RIP1 represses basal autophagy in part due to its ability to regulate the TFEB transcription factor, which controls the expression of autophagy‐related and lysosomal genes. RIP1 activates ERK, which negatively regulates TFEB though phosphorylation of serine 142. Thus, in addition to other pro‐death functions, RIP1 regulates cellular sensitivity to pro‐death stimuli by modulating basal autophagy.

Modulation of pediatric brain tumor autophagy and chemosensitivity

Brain and spinal tumors are the second most common malignancies in childhood after leukemia, and they remain the leading cause of death from childhood cancer. Autophagy is a catabolic cellular process that is thought to regulate chemosensitivity, however its role in pediatric tumors is unknown. Here we present studies in pediatric medulloblastoma cell lines (DAOY, ONS76) and atypical teratoid/rhabdoid tumor cell lines (BT-16, BT-12) to test this role. Autophagy was inhibited using siRNA against autophagy-related genes ATG12 and ATG7 or pharmacologically induced or inhibited using rapamycin and chloroquine to test the effect of autophagy on chemosensitivity. Autophagic flux was measured using Western blot analysis of LC3-II and p62 and cell viability was determined using MTS assays and clonogenic growth. We found that when pediatric brain tumor cells under starvation stress, exposed to known autophagy inducers such as rapamycin, or treated with current chemotherapeutics (lomustine, cisplatin), all stimulate autophagy. Silencing ATG12 and ATG7 or exposure to a known autophagy inhibitor, chloroquine, could inhibit this autophagy increase; however, the effect of autophagy on tumor cell killing was small. These results may have clinical relevance in the future planning of therapeutic regimens for pediatric brain tumors.

Clinical research and Autophagy

In this issue of the journal we are publishing a series of papers that inaugurate a new category—clinical research. We already have a category for translational research, so what is the need for this new category? In part, this question may be answered by first considering the difference between papers submitted as “Basic Science” and “Translational.” Both of these types of papers report findings from standard bench research that most readers of this journal are familiar with; however, research done in most model organisms is at least a few steps away from practical application (that is, being used for therapeutic purposes in a clinical setting), whereas that done with mammalian cell culture or in certain animal models, depending on the topic and the specific experiments, may be directly addressing a health-related question. The former type of research would be considered basic, and the latter is translational. As more specific examples, studies on the role of Atg proteins in yeast are generally basic research, whereas those examining the effect of inhibiting/stimulating autophagy in combination with an anticancer treatment in a mouse tumor model could be translational. In contrast to both of these types of studies, “Clinical” papers report results from actual trials, such as the phase I trials described in this issue or studies in patients pertaining to disease prevention. There are significant differences between these 2 types of studies, clinical and nonclinical, and these are worth considering both for potential reviewers and readers of these and similar papers. Finally, we note that there has been, and will likely continue to be, a move toward the clinical application of findings from basic research that pertain to various pathologies associated with autophagy or defects in this process, including infectious disease, neurodegeneration, diabetes, and others. The phase I trials published in this issue are one example of this, but we anticipate an increasing number of studies on the modulation of autophagy for therapeutic purposes.

A Genome-Wide Loss-of-Function Screen Identifies SLC26A2 as a Novel Mediator of TRAIL Resistance

TRAIL is a potent death-inducing ligand that mediates apoptosis through the extrinsic pathway and serves as an important endogenous tumor suppressor mechanism. Because tumor cells are often killed by TRAIL and normal cells are not, drugs that activate the TRAIL pathway have been thought to have potential clinical value. However, to date, most TRAIL-related clinical trials have largely failed due to the tumor cells having intrinsic or acquired resistance to TRAIL-induced apoptosis. Previous studies to identify resistance mechanisms have focused on targeted analysis of the canonical apoptosis pathway and other known regulators of TRAIL receptor signaling. To identify novel mechanisms of TRAIL resistance in an unbiased way, we performed a genome-wide shRNA screen for genes that regulate TRAIL sensitivity in sublines that had been selected for acquired TRAIL resistance. This screen identified previously unknown mediators of TRAIL resistance including angiotensin II receptor 2, Crk-like protein, T-Box Transcription Factor 2, and solute carrier family 26 member 2 (SLC26A2). SLC26A2 downregulates the TRAIL receptors, DR4 and DR5, and this downregulation is associated with resistance to TRAIL. Its expression is high in numerous tumor types compared with normal cells, and in breast cancer, SLC26A2 is associated with a significant decrease in relapse-free survival. Implication: Our results shed light on novel resistance mechanisms that could affect the efficacy of TRAIL agonist therapies and highlight the possibility of using these proteins as biomarkers to identify TRAIL-resistant tumors, or as potential therapeutic targets in combination with TRAIL. Mol Cancer Res; 15(4); 382–94. ©2017 AACR.