Autophagy, which is constitutively executed at basal level in all cells, promotes cellular homeostasis by regulating organelles and proteins turnover. In tumor cells, autophagy is activated in response to various cellular stresses, including nutrient and growth factor starvation, as well as hypoxia . It is now well established that autophagy can act as tumor suppressor and tumor promoter. The different roles of autophagy in cancer cells seem to depend on tumor type, stage, and genetic context. Indeed, autophagy clearly suppresses the initiation and development of tumors, however, it is considered as a key survival pathway in response to stress, and many established tumors require autophagy to survive. In this section, we will summarize the different mechanisms involved in the activation of autophagy in tumor and discuss recent reports about the dual role of autophagy in carcinogenesis and tumor progression.
1.1. Role of autophagy in tumor suppression
Several lines of indirect evidence indicate that autophagy acts as a tumor suppressor. Indeed in various cases, oncogenic transformations, such as activation of the PI3K/Akt pathway
Beside the indirect evidences outlined above, there are more direct ones supporting the tumor suppressing properties of autophagy. Thus, the autophagy execution protein Beclin1 is a haplo-insufficient tumor suppressor protein. Monoallelic deletions of
The tumor suppressive functions of autophagy have been extensively investigated. Below we will provide mechanistic insights into the tumor-suppressive functions of autophagy.
1.1.1. Autophagy inhibits necrosis and inflammation
During the last decade, strong evidence supported that the inflammatory microenvironment plays a major role in tumor development. Indeed, chronic inflammation is a common future of early cancer development. In this regards, it has been proposed that autophagy can modulate those inflammatory reactions through different mechanisms, as autophagy-deficient tumors display an increased level of necrosis and inflammation.
First, it has been reported that activation of autophagy in tumor cells can inhibit necrotic cell death. Unlike apoptotic cell death, cells dying by necrosis stimulate a robust inflammatory response
Several studies have confirmed that autophagy is able to prevent the two forms of necrotic cell death (i) necroptosis and (ii) poly-(ADP-ribose) polymerase (PARP)-mediated cell death. Necroptosis is a form of caspase-independent cell death mediated by cell death ligands (
Autophagy acts also through different mechanisms to decrease inflammation. Autophagy is essential for the maintenance of intracellular ATP level, which in turn is required for the secretion of lysophosphatidylcholine (LPC). Secretion of LPC is associated with the acute phase of the inflammatory response and is involved in the development of chronic inflammation. It also has been shown that autophagy-deficient cells fail to generate phosphatidylserine on the outer membrane surface – an important anti-inflammatory pro-apoptotic marker. This explains how defect in autophagy can stimulate inflammatory response subsequently to insufficient clearance of dead cells . Accumulation of p62 in autophagy-deficient cells activates the pro-inflammatory transcription factor NF-κB and the stress-responsive transcription factor NRF2, thus favoring inflammation and tissue injury . Transcription factors of NF-κB family regulate the expression of a broad range of genes involved in the development, the proliferation, and the survival of tumor cells. Moreover, these transcription factors are important in regulation of inflammation and innate and adaptive immune responses . Activation of NF-κB is mediated by the IκB kinase (IKK) complexes. It has been shown that IKK complexes are targets for degradation by autophagy when the heat shock protein 90 (Hsp90) function is inhibited . Another mechanism of regulation of NF-κB by autophagy is mediated by the Kelch-like ECH-associated protein 1 (Keap1). Keap1 interacts with the kinase domain of IKKβ through its C-terminal domain. This domain is also required for the binding of Keap1 to the transcription factor NRF2, which controls expression of certain antioxidant genes. In response to tumor necrosis factor (TNF), Keap1 negatively regulates activation of NF-κB through inhibition of the IKKβ phosphorylation and induction of IKKβ degradation by autophagy pathway . The E3 ubiquitin ligase Ro52 is another signaling molecule that targets IKKβ for degradation through the autophagy pathway. In response to distinct stimuli, specific interactions of Hsp90, Keap1 and Ro52 with IKKs regulate NF-κB activity through their ability to activate or repress the degradation of IKKs by autophagy . It has been shown that the crosstalk between NF-κB and autophagy regulates inflammasome activity leading to the modulation of the activation of caspase-1 and subsequently the secretion of potent pro-inflammatory cytokines . Overall, it appears that autophagy exerts a significant impact on the regulation of inflammation, and is an important modulator of cancer pathogenesis.
1.1.2. Autophagy prevents oxidative stress and genomic instability
Over the last years, the link between autophagy and suppression of cancer development has been confirmed by several
Autophagy is also able to mitigate the accumulation of genomic alteration by inducing the mitotic senescence transition. Senescence is an irreversible cell cycle arrest associated with an active metabolism, which can limit the proliferation of abnormal cells. Young
1.1.3. Autophagy contributes to tumor cell death
The induction of autophagic cell death has been proposed as a possible tumor suppression mechanism. This statement is based on the observation that apoptosis occurs concomitantly with features of autophagy  and that prolonged stress and progressive autophagy can lead to cell death .
Autophagic cell death was first described in 1973 based on the morphological features as a modality of cell death with the presence of autophagosomes and was subsequently named as type II cell death, together with apoptosis (type I) and necrosis (type III) . The relevance of autophagic cell death in development has been established in lower eukaryotes and invertebrates like
1.1.4. Autophagy modulates the anti-tumor immune response
The immune system plays an important role in controlling cancer progression. It is now well established that immune cells can mediate the destruction of mutated, aberrant or over-expressing self-antigens tumor cells. However, evasion of immune-mediated killing has recently been recognized as an universal hallmark of cancer . It has become increasingly clear that hypoxic tumor microenvironment plays a crucial role in the control of immune protection . On one hand, tumor cells have evolved to utilize hypoxic stress to their own advantage by activating key biochemical and cellular pathways that are important for tumor progression, survival, and metastasis. Autophagy is one of these pathways activated under hypoxia that may be exploited to modulate the responsiveness of tumor cells to immune system. On the other hand, immune cells that infiltrate tumor microenvironment also encounter hypoxia, resulting in hypoxia-induced autophagy. It is now clearly established that autophagy impacts on the immune system as this process is crucial for immune cell proliferation as well as for their effector functions such as antigen presentation and T-cell-mediated killing of tumor cells . In the subsequent section we will discuss the role of autophagy activation in both tumor and immune cells in the context of cancer immune response. Indeed, understanding how tumor cells evade effective immunosurveillance represents a major challenge in the field of tumor immunotherapy.
184.108.40.206. Role of autophagy in immune cells
Despite the inhospitable hypoxic microenvironment, multiple cell types within the innate and adaptive immune system are capable to recognize and eliminate tumor cells. This was attributed to the ability of immune cells to adjust their metabolic dependency once they have reached the tumor and enhance their survival by activating autophagy. Here we will discuss how autophagy impacts specific immune subsets.
The effect of autophagy induction by hypoxia was investigated in neutrophils as this type of immune cells are the first to migrate to the inflammatory site of the tumor where they promote inflammation and activate macrophages and dendritic cells (DCs) . Neutrophils display high glycolytic rate making them resistant to hypoxia. Autophagy activation in neutrophils has been reported to mediate neutrophil cell death. This will decrease inflammation and ultimately lead to limit tumor growth under these circumstances .
In contrast to neutrophils, APCs such as macrophages and dendritic cells (DCs) must metabolically adapt to hypoxia through stabilization of hypoxia-inducible factor-1α (HIF-1α) to induce the expression of glucose transporters and glycolytic enzymes as well as limiting oxygen consuming oxidative phosphorylation . As a consequence of hypoxia, macrophages and DCs have decreased phagocytosis, reduced migratory capacity, and increased production of proangiogenic and proinflamatory cytokines. While, hypoxia is involved in dampening APC activity, autophagy may contribute to survival of APCs under these conditions. It has been proposed that culturing DCs under hypoxia resulted in the stabilization of HIF-1α which initiates BNIP3 expression and promotes survival of mature DCs, possibly due to induction of autophagy . It has been proposed that autophagy induction in APCs infiltrating tumor occurs
The effect of autophagy on the activity of T cells was also investigated. Indeed, autophagy is activated in these cells upon T cell receptor engagement in both CD4+ and CD8+ subtypes [62-64]. Targeting autophagy by silencing ATG5 or ATG7 during T cell receptor stimulation leads to a significant decrease in cellular proliferation, highlighting the importance of autophagy during T cell activation [63, 64]. Evidence has been recently provided showing that autophagy is upregulated at the immunological synapse during DC and T cell contact. Suppression of autophagy in DCs resulted in hyper-stable contacts between the DC and CD4+ T cells and increased T-cell activation . Autophagy is upregulated in Th2 CD4+ T cells compared with Th1 CD4+ T cells and was shown to be important for the survival of a Th2 cell line upon growth factor withdrawal . In addition, cells cultured under Th1-polarizing conditions rely more heavily on autophagy for survival compared to the Th17 subset. These findings indicate that the role of autophagy is dependent on the cell type and stimuli and that blocking autophagy can skew the balance of immune subsets . Once T cells mature and traffic to the periphery, autophagy is required for survival [63, 67-70]. The role of autophagy in promoting mature T-cell survival has been attributed to autophagy degrading essential components of the apoptotic cell death machinery  and maintaining mitochondrial turnover [68-70]. In addition, it has been demonstrated that activated CD4+ T cells exhibit reduced cytokine secretion, adenosine triphosphate (ATP) production, fatty acid utilization, and glycolytic activity when autophagy is inhibited . These findings support the notion that autophagy is required for cellular function by providing metabolism through the liberation of biosynthetic precursors. It has been shown that during sustained growth factor withdrawal, autophagy supplies the metabolites necessary to generate ATP production in bone marrow hematopoietic cells  supporting the hypothesis that immune cells use autophagy to generate metabolites required for cell survival. More recently, it has been shown that autophagy is involved in the liberation of the ubiquitous protein puromycin-sensitive aminopeptidase epitope, thereby creating a CTL epitope that mimic tumor-associated antigens .
220.127.116.11. Role of autophagy in tumor cells
Autophagy has been found activated in many tumors and its inhibition can lead to either increased death or increased survival, depending on tissue type, tumor grade and any concomitant therapy used [73, 74]. The role of autophagy induction in the anti-tumor immune response has recently received widespread attention. We have investigated the role of autophagy induction under hypoxia in tumor response to CTL-mediated lysis. Using non-small cell lung carcinoma and their autologous CTL, we clearly showed that the activation of autophagy under hypoxia in tumor cells is associated with resistance to CTL-mediated lysis (Figure 1).
Targeting autophagy in hypoxic tumor cells restores CTL-mediated killing . The mechanism by which hypoxia-induced autophagy leads to tumor resistance to CTL was investigated. We provided evidence that hypoxia-inducible factor (HIF)-1α and autophagy coordinately operate to induce and stabilize a survival pathway involving the activated signal transducer and activator of transcription-3 (STAT-3) . Furthermore, we also showed that targeting autophagy
Since autophagy can also promote the survival of tumor cells through nutrients recovered from degrading and recycling damaged organelles, it has been recently proposed that chemotherapy-induced autophagy causes the release of ATP from tumor cells, thereby stimulating antitumor immune response. Targeting autophagy blunted the release of ATP by tumor cells in response to chemotherapy without affecting that of other damaged signals. Autophagy-dependent extracellular ATP recruits DCs into tumors and activates a T cell response to tumor cells . Based on this study, it seems that the activation of autophagy in the context of DNA damage-induced apoptosis, causes ATP release which subsequently recruits immune cells.
It is now well established that immune effector cells integrate signals that define the nature and magnitude of the subsequent response. In this context, it has been shown that at high effector-to-target ratios, autophagy was induced in several human tumors by natural killer (NK) cells. Importantly, cell-mediated autophagy promoted resistance from treatment modalities designed to eradicate tumor. Thus, the lymphocyte-induced cell-mediated autophagy promotes cancer cell survival and may represent an important target for development of novel therapies .
The complexity of cancer immune response is related to the fact that different immune subsets cooperatively and coordinately act through the secretion of cytokines and other soluble factors. Thus, it stands to reason that antitumor immune responses are not entirely dependent on the presence or absence of any particular subset, but rather on the stoichiometry of immune effectors versus immune suppressors. As a result, any anti-cancer therapies that skew the immune effector to suppressor ratio by impacting autophagy may exert a large effect on overall patient survival . While mounting evidence suggest that autophagy induction enhances immune cell function, autophagy seems to operate as a tumor cells resistance mechanism against immune response. In spite of this, inhibition of autophagy in the clinic can behave as a double-edged sword because it can enhance or suppress cancer immune response. Thus, therapeutic strategies targeting autophagy in tumor cells must consider the potential negative impact on antitumor immunity. The key question that emerged is: what is the net outcome of the autophagy inhibitor in clinic? There are numerous studies supporting that immunotherapy of cancer should focus on inducing and reprogramming cells of the innate and adaptive immune system. Therefore, it is tempting to speculate that combined therapy based on autophagy inhibitor and reprograming immune cells could significantly improve cancer immunotherapy.
1.1.5. Autophagy inhibits metastasis
Metastases are responsible for most cancer-related deaths. Metastatic cascade involves several steps, including: i) invasion from the primary tumor site, ii) intravasation and survival in the systemic circulation, iii) extravasation at the secondary tissue site and, iv) colonization of this target tissue . Autophagy has been found to either promote or prevent the metastatic progression, depending on the step in which it is activated (Figure 2 adapted from ). In this section, we will focus on the anti-metastatic activity of autophagy, while its pro-metastatic properties will be overview in the section 1.2.2.
18.104.22.168. Autophagy modulates the inflammatory response
At early steps, autophagy is able to limit the metastatic progression from the primary tumor site by restricting inflammatory response. Indeed, infiltrated immune cells can supply some signals within the tumor microenvironment that influence tissue remodeling, angiogenesis, tumor cell survival and spreading. Clinical and experimental data have confirmed the dual role of the immune system in tumor metastasis. As example, Lin
Autophagy can modulate inflammation during metastasis by different ways. On one hand, autophagy may lead to a direct activation of antitumor immunity through the release of high-mobility group box protein 1 (HMGB1) from tumor cells that are destined to die . When released, HMGB1 stimulates the Toll-Like Receptor 4 on dendritic cells and, subsequently, promotes the tumor cell killing by inducing T-cell immunity . On the other hand, autophagy can indirectly attenuate the macrophage infiltration by inhibiting tumor cell necrosis (see section 1.1.1.). Indeed, tumor-associated macrophages (TAMs) are important components of the leukocyte infiltrate and their involvement in metastasis progression have been extensively studied. TAMs positively influence tissue remodeling, angiogenesis, tumor invasion and intravasation through the production of growth factors, cytokines and matrix metalloproteases   .
22.214.171.124. Autophagy alters the epithelial to mesenchymal transition (EMT)
Many studies have shown that the acquisition of mesenchymal feature by carcinoma cells promotes cancer invasion and metastasis. Epithelial to Mesenchymal Transition (EMT) is a process that leads to the complete loss of epithelial characteristics to achieve a mesenchymal cell phenotype. Initiation and completion of EMT requires the expression of specific transcription factors, microRNAs, cell surface proteins and matrix-degrading proteases . Once undergoing EMT, cancer cells acquire invasive properties that enhance their ability to detach from the primary tumor site and to colonize distant tissues. Recently, two studies have pointed out that autophagy may modulate EMT. Lv
126.96.36.199. Autophagy restricts expansion of dormant tumor cells
Cancer recurrence is a determinant element for patient life expectancy because this disease presents a high risk of relapse after therapy or a long period of remission. Presence of residual dormant cells in the primary tumor site or in distant organs is one of the major causes of cancer relapse. Tumor dormancy is characterized by a prolonged, but reversible, growth arrest in G0-G1, by which tumor cells survive in a quiescent state. However, dormant tumor cells have to re-activate their proliferative activity to allow the development of micro- or macro-metastasis. Lu
1.2. Role of autophagy in tumor progression and metastasis
1.2.1. Autophagy induces survival of tumor cells under a variety of stresses
It has been well documented that tumor cells activate autophagy in response to stress, which enables long-term survival when apoptosis is defective . Autophagy must be a highly selective process to allow extensive cellular degradation while retaining functional integrity. This section will address how autophagy confers tumor cells with superior stress tolerance, which limits damage, maintains viability, sustains dormancy and facilitates recovery.
Cancer cells need to adapt their metabolism to ensure the demands of proliferation enhanced in the microenvironment. The oncogenes affect signaling pathways important in regulation of metabolism, which support cancer growth and proliferation . Autophagy is activated in response to multiple stresses, such as hypoxia, nutrient starvation, and the endoplasmic reticulum (ER) stress , during cancer progression. Under metabolic stress, inhibition of autophagy could lead to accelerated apoptosis, thus limiting further tumor progression. In this section, we discuss the role of autophagy regulation in tumor microenvironment and tumor growth .
188.8.131.52. Autophagy as adaptive metabolic response to hypoxia
Tumor cells are subjected to elevated metabolic stresses (
184.108.40.206. Autophagy in nutrient starvation
It is now well known that the metabolic stress induced by starvation in tumor microenvironment activates autophagy. Moreover, this metabolic stress is also dependent on autophagy as it allows organelles and proteins recycling in order to provide energy for cell survival. It has been shown that cancer cell lines with Ras activation display elevated levels of basal autophagy essential for survival through starvation and tumor growth . Autophagy induced by starvation (
1.2.2. Autophagy promotes tumor cell metastasis
As mentioned in the section 1.1.5., autophagy may also promote different steps of metastatic cascade, mainly by favoring the survival of cancer cells in inhospitable environments (
During the metastatic progression, cancer cells activate mechanisms to resist to anoikis. Anoikis is a form of apoptotic cell death induced by the detachment from the surrounding extracellular matrix (ECM) . Activation of autophagy during anoikis may be a survival strategy developed by the cells to overcome the stress of ECM detachment. Fung
Although autophagy prevents cancer progression by maintaining tumor cells in a dormant state, initiation of dormancy may also promote tumor progression by favoring survival of cancer cells. In this regard, it has been shown that breast cancer cells that lack β1 integrin are in a dormant state, suggesting that dormancy may help cancer cells to overcome the stress of ECM detachment, and subsequently resist to anoikis .
1.2.3. Upregulation of autophagy promotes resistance to cancer therapy
Autophagy may function to remove proteins or organelles that are damaged by cancer treatments or, through the degradation of cellular components, may provide nutrients for the rapidly growing cells. Indeed, inhibitors of autophagy can produce different outcomes: cell survival or cell death. Obviously, autophagic cell survival confers tumor cells with superior stress tolerance, which limits damage, maintains viability, sustains dormancy, and facilitates recovery. The dual role of autophagy highlights the need to carefully define its role in tumor cells before applying autophagy-based therapy. It will be important for clinical oncologists and cancer researchers to determine which cancer cell types most commonly undergo autophagy in response to therapy, and whether increased autophagy is a sign of responsiveness or resistance.
Nevertheless, several studies have shown that tumor cells can survive anti-cancer treatment by activating autophagy. This statement was validated using genetic or pharmacological inhibitors of autophagy which led to sensitize tumor cells to cancer therapies. In this context, it has been reported that inhibition of autophagy sensitizes cancer cells to DNA damaging anticancer agents. Evidence has been provided that inhibition of autophagy by 3-methyladenine (3-MA) or by targeting Atg7 enhances the cytotoxicity of 5-fluorouracil in human colorectal cancer cells . Autophagy inhibition also enhances the therapeutic efficacy of cisplatin and 5-fluorouracil in esophageal and colon cancer cells, respectively [122, 123]. Targeting autophagy by genetic approaches using Beclin1, Atg3, and Atg4b siRNA sensitizes resistant cancer cells to ionizing radiation . These studies strongly argue that autophagy operates as a mechanism through which cancer cells acquire resistance to radiotherapy and chemotherapy. There are numerous studies supporting the involvement of autophagy in cancer stem cells resistance to ionizing radiation and other anti- cancer treatments . Thus, in malignant gliomas, the CD133+ cancer stem cells express higher levels of the autophagic proteins LC3, Atg5, and Atg12. In addition, ionizing radiation seems to induce autophagy only in CD133+ cancer stem cells compared to CD133- counterpart . Furthermore, glioma cells treated with autophagy inhibitors exhibit more extensive DNA double-strand breaks than cells treated with radiation alone . We have recently demonstrated that autophagy induction in tumor cells under hypoxia decrease the tumor cell killing by cytotoxic T lymphocytes. Furthermore, we provided evidence that simultaneously boosting the immune system by vaccination and inhibiting autophagy may improve cancer immunotherapy [75, 76].
While the general consensus is that autophagy inhibition is an effective strategy for cancer therapy, some drugs that are being used in the clinic induce autophagy. In most cases, however, it has not been proven that these drugs induce death
2. Autophagy as a target for anti-cancer therapies
Evidence indicated that the modulation of autophagy is an important component of tumorigenesis, making it a possible therapeutic target. Pharmacological inhibitors of autophagy can be broadly classified as early- or late-stage inhibitors of the pathway. Early-stage inhibitors include 3-methyadenine, wortmannin, and LY294002, which target the class III PI3K (Vps34) and interfere with its recruitment to the membranes. Late-stage inhibitors include the antimalarial drugs chloroquine (CQ), hydroxychloroquine (HCQ), bafilomycin A1, and monensin. Bafilomycin A1 is a specific inhibitor of vacuolar-ATPase , and monensin and CQ/HCQ are lysosomotropic drugs that prevent the acidification of lysosomes, whose digestive hydrolases depend on low pH. Since autophagosomes and lysosomes move along microtubules, microtubule-disrupting agents (taxanes, nocodazole, colchicine, and vinca alkaloids) can also inhibit the fusion of autophagosomes with lysosomes. Other inhibitors of autophagy that block autophagosome degradation include the tricyclic antidepressant drug clomipramine and the anti-schistome agent lucanthone [129, 130]. Of the known autophagy inhibitors outlined above, only CQ and HCQ have been evaluated in humans, because they are commonly used as antimalarial drugs and in autoimmune disorders. These drugs cross the blood-brain barrier, and HCQ is preferred to CQ in humans because of its more favorable side-effects profile . Quinacrine, which also has been used in patients as an anti-malarial, has been shown to inhibit autophagy similarly to CQ. In fact, quinacrine showed greater cytotoxicity in gastrointestinal stromal tumor (GIST) cell lines treated with imatinib than CQ , and therefore this may be a promising anti-autophagy agent for future clinical trials.
Currently, there are nearly 20 clinical trials registered in the National Cancer Institute (www.cancer.gov/clinicaltrials) exploring anti-autophagy strategies in a variety of human cancers. Most of these trials are ongoing, with minimal published results available, and nearly all use HCQ. It is worthy to note that CQ or HCQ are lysosomotropic agents that act at the level of the lysosome by inhibiting acidification, thereby impairing autophagosome degradation. These clinical trials were initiated based on the fact that autophagy is induced in a variety of tumor cells and preclinical models by several types of chemotherapeutic agents as a survival mechanism. Because only a subpopulation of tumor cells undergo autophagy, it is unlikely that autophagy inhibitors are used in cancer therapy as single agent. Indeed, most of these clinical trials used HCQ in combination with other anti-cancer therapies. While these preclinical data are generally supportive of incorporating anti-autophagy therapies in cancer treatment trials, it has been observed, in some circumstances, that inhibition of autophagy decreases therapeutic efficacy. Understanding the circumstances in which autophagy inhibition impairs the therapeutic effect will be of great importance. Importantly, while CQ and HCQ are effective inhibitors of autophagy
CQ inhibits the last step of autophagy at the level of the lysosome, thereby impacting lysosomal function. Therefore, its effects are not entirely specific to autophagy. Currently, there is a great deal of interest in developing new inhibitors of autophagy. In this regards, and given the complexity of the autophagic process, multiple proteins involved in this process could be good candidates for developing others autophagy inhibitors. It is likely that kinases would be prime candidates for inhibition such as Vps34, a class III PI3K, which has a critical early role in autophagosome development. This is particularly attractive, as there has been significant success in designing effective class I PI3K inhibitors . However, one potential issue which needs to be considered is that Vps34 has roles in other aspects of endosome trafficking, and this may lead to unwanted effects and toxicity . The mammalian orthologs of yeast ATG1, ULK1/2, which acts downstream from AMPK and the TOR complex, have been recently shown as critical proteins for autophagy activation [140-142]. Others potential targets for autophagy inhibitors would be LC3 proteases such as ATG4b, which are necessary for LC3 processing. However, whichever approach is taken, the delicate balance between potency and toxicity must be determined to achieve a clinical success. While there are still uncertainties of how autophagy inhibition will fare as an anti-cancer therapy, the preclinical data generally support this approach. The current clinical trials will hopefully provide insight into whether this will be a viable therapeutic paradigm .