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Autophagy degrades the cellular proteome to promote survival, but the underlying mechanism and substrates of consequence are poorly understood. We found that autophagy selectively remodels the proteome in cancer cells by eliminating proinflammatory signaling proteins. Autophagy ablation causes aberrant accumulation of these proteins that primes cancer cells for interferon-dependent cell death, explaining how autophagy suppresses inflammation and promotes tumor maintenance.
Autophagy is a stress-activated catabolic process that captures, degrades, and recycles cytoplasmic components to remove waste and provide substrates to support metabolism and survival in starvation. In normal cells, autophagic elimination of genotoxic waste limits chromosomal instability and potentially tumor initiation. However, in aggressive cancers, such as those driven by activated Ras, autophagy-mediated recycling promotes energy homeostasis and survival.1 Thus, autophagy is often exploited by cancer cells, which elevate basal autophagy and can display autophagy addiction. Additionally, therapeutic stress activates autophagy as a resistance mechanism, compromising clinical efficacy. Indeed, concomitant inhibition of autophagy sensitizes cancer cells to mammalian target of rapamycin (mTOR) and proteasome inhibitors, endoplasmic reticulum (ER) stress inducers, and DNA damaging agents, highlighting the survival role of autophagy in cancer.
Some of the most autophagy-addicted tumors are those driven by oncogenic Ras, which account for a third of all human cancers and are refractory to conventional chemotherapy. In genetically engineered mouse models of spontaneous Ras-driven non-small cell lung carcinoma (NSCLC), autophagy ablation by tumor-specific deletion of Atg7 compromises mitochondrial function and causes adenomas and carcinomas to instead progress to benign oncocytomas. Importantly, autophagy ablation in both evolving and pre-existing NSCLCs causes an elevated inflammatory response and suppresses tumor growth.2,3 This suggests that Ras-driven cancers depend on autophagy to suppress immune responses while maintaining metabolic fitness and proliferation, although the exact mechanism of this autophagy-mediated suppression of inflammation and promotion of survival was unclear. By comparing the global proteomes of Ras-driven cancer cells with or without functional autophagy, we found that selective cellular proteome remodeling by autophagy may play a critical role in suppressing this immune response.4
The important finding was that starvation-induced autophagy is not a non-selective, bulk degradation process, but instead extensively and selectively remodels the proteome.4 Proteins required to preserve functional autophagy essential for survival are retained, while pro-inflammatory proteins are eliminated and aberrantly accumulate upon autophagy ablation.4 The consequences of deregulated proteome remodeling and selective protein accumulation upon autophagy ablation are two-fold: First, autophagy defects prime non-immune cancer cells for interferon-mediated cell death that is activated by immune stimuli such as the synthetic double-stranded RNA (dsRNA) analogue poly I:C. Second, innate immune signaling and paracrine secretion of type-I interferon (IFN-I) enhances necrotic cell death, possibly contributing to increased inflammation in mice with tissue-specific autophagy defects. This raises an important question: Can autophagy inhibition augment immunotherapeutic modalities aimed at potentiating antitumor immune responses?
There are profound implications to the finding that autophagy inactivation primes tumor cells for cell death in response to immunogenic stimuli (Figure 1). First, autophagy-defective cells undergo necrotic cell death, releasing high-mobility group protein B1 (HMGB1), a proinflammatory molecule that triggers a B cell-mediated antitumor cytotoxic T lymphocytic (CTL) response. Collateral damage caused by chemotherapy in autophagy-competent neighboring cells may also activate immunogenicity and an adaptive immune response to which autophagy-inhibited cells may be further sensitized. Toll-like receptors (TLRs) are robust innate immune stimulators that are paradoxically overexpressed in many human cancers, and TLR agonists are being explored for anticancer activity. As innate immune stimulation through TLR signaling makes autophagy-defective cells susceptible to cell death, autophagy inhibition may enhance their efficacy.
Second, IFN-I secretion from autophagy-defective cells may directly activate the adaptive immune response. IFN-Is are essential mediators of dendritic cell (DC) priming and T-cell activation, which enhances cancer immune surveillance and the cytotoxic T lymphocyte (CTL) response. Exogenous IFN-I administration or local IFN-I production enhances CD8+ T-cell responses via DC stimulation. TLR agonists that locally increase IFN-I production are associated with DC recruitment in tumors and induction of a CTL response.5 Similarly, in vivo co-administration of CpG oligodeoxynucleotides, potent inducers of the IFN-I response, enhances a tumor-specific CD8+ CTL response to post-transplantation DC vaccination.6 Indeed, intratumoral delivery of IFN-I in tumor-bearing mice synergizes with immunotherapy7 and chemotherapy8 through enhanced DC cross-presentation, and human hematopoietic stem cells that are genetically engineered for tumor-targeted interferon-α (IFN-α) delivery inhibit breast cancer progression.9 Thus, activation of innate immunity and consequent paracrine IFN-I signaling in autophagy-deficient tumor cells may serve to bridge the innate and adaptive immune systems for an enhanced tumor-specific CTL response, the major objective in cancer immunotherapy (Figure 1).
Tumor cell immune evasion presents a critical challenge that limits immunotherapeutic efficacy. Immune suppressive enzymes such as indoleamine 2,3-dioxygenase (IDO) and arginase that are commonly overexpressed in cancers are critical resistance mechanisms in antitumor T cell immunotherapy. IDO and arginase cause local depletion of amino acids and inhibit mTOR,10 a condition that triggers autophagy in tumors and makes them likely candidates for autophagy inhibition for further sensitization. Additionally, immune checkpoint pathway blockers such as antibodies against cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed death-1 (PD1), as well as agonists for co-stimulatory molecules (CD137 agonist antibody) are currently gaining attention in immunotherapy in oncology. Indeed, IDO inhibition that activates mTOR, a negative regulator of autophagy, synergizes with CTLA blockade.11 It is worth exploring whether autophagy inhibition plays a role in augmenting the efficacy of these agents.
In conclusion, remodeling of the cell proteome by autophagy is an important immunosuppressive survival mechanism for Ras-driven cancers, and inhibition of this process may provide additional means to target these aggressive cancers by sensitizing them to modulators of CTL response in immunotherapy. Recent clinical studies of autophagy inhibition in glioblastoma, melanoma, lymphoma, myeloma, and renal and colon cancers have reported encouraging initial clinical responses. Given the requirement for autophagy in antigen presentation on major histocompatibility complex (MHC class II) molecules in antigen presenting cells (APC), it is worth exploring whether additional strategies that enhance antitumor immune responses augment autophagy inhibition in these settings. Autophagy substrates identified in this study may also serve as biomarkers for monitoring autophagy modulation more effectively in the clinical setting.