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Effect of Autophagy on Chemotherapy-Induced Apoptosis and Growth Inhibition
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9 Effect of Autophagy on Chemotherapy-Induced Apoptosis and Growth Inhibition Shanshan Zhang, Xianling Guo, Jianrui Song, Kai Sun, Yujiao Song, and Lixin Wei O U T L I N E Autophagy Can Be Induced by DDRs in DNA Damaged Cells 151 Autophagy Regulates DDRs by Indirect and Direct Approaches 152 Autophagy Essential Proteins Regulate DDR by AutophagyIndependent Means 152
Introduction 146 Autophagy and Chemotherapy-Induced Apoptosis and Growth Inhibition 147 Autophagy Restrains Chemotherapy-Induced Apoptosis 147 Autophagy Promotes Chemotherapy-Induced Apoptosis 148 Autophagy Aggravates ChemotherapyInduced Growth Inhibition 149 Autophagy, Tumor Microenvironment, and Chemoresistance 149 Hypoxia-Induced Autophagy Contributes to Chemoresistance of Tumor Cells 149 Hypoxia-Induced Autophagy Contributes to Tolerance of Tumor Cells to Nutrient Deprivation in Tumor Microenvironment 150 Autophagy and DNA Damage-Inducing Chemotherapy 151
M.A. Hayat (ed): Autophagy, Volume 5. DOI: http://dx.doi.org/10.1016/B978-0-12-801033-4.00009-6
Autophagy and Cancer Stem Cells in Chemoresistance 152 Autophagy Is Essential for Maintenance of the Tumorigenicity of CSCs 152 Autophagy Contributes to Survival of CSCs in Oxygen and/or NutrientDeprived Tumor Microenvironment 153 Autophagy Involved in CSC Chemoresistance 153 Conclusion 154 References 154
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Abstract Cancer cells are resistant to chemotherapy, which results in poor prognosis for cancer patients. Autophagy, a self-eating process, has been widely reported as a prosurvival mechanism underlying cancer cell chemoresistance. Upon chemotherapeutics treatment, autophagy is employed by cancer cells to maintain cellular homeostasis and mitigate genome damage by degrading damaged proteins and organelles such as mitochondria, thus preventing cell apoptosis. In the tumor microenvironment characteristic of oxygen and nutrient deprivation, autophagy is activated in cancer cells to cope with metabolic stress, and these cells were more refractory to chemotherapy. Cancer stem cells (CSCs) are a subset of cancer cells that can evade cell death induced by existing chemotherapeutic agents, and one of the underlying mechanisms might be autophagy. Emerging evidence shows that CSCs have higher levels of autophagy under normal conditions and ischemic and hypoxic conditions. Autophagy inhibition could sensitize non-CSCs and CSCs to chemotherapy-induced apoptosis and growth inhibition; thus, it is under consideration for being developed as a synergistic therapy with existing chemotherapies for better therapeutic effects.
INTRODUCTION Cancer poses a great threat to people’s health. Although many innovative chemotherapeutic agents based upon important findings in basic and clinical cancer research have been developed and used in cancer patients for years, our overall achievements in prolonging patients’ survival are still not satisfactory, since the major obstacle that tumor cells are resistant to chemotherapy still exists. Thus, understanding the underlying mechanisms as to how resistance happens can reveal new targets and is beneficial for the development of novel therapies which might be applied, together with existing chemotherapies, to gain better therapeutic effects and bring long-term clinical benefits to the patients. Autophagy (also known as macroautophagy) has been widely reported to be associated with chemoresistance of tumor cells. Autophagy is an evolutionarily conserved catabolic pathway which exists in all eukaryotic cells from yeast to mammals. It is characterized by the formation of double membrane vesicles, called autophagosomes, which sequester long-life proteins and cellular organelles such as mitochondria. The autophagosomes fuse with vacuoles or lysosomes where degradation of the cargo occurs, supplying amino acids and macromolecular precursors for cells. A basal level of autophagy exists in every cell and is necessary for maintaining cellular homeostasis, since accumulation of aggregation-prone proteins and damaged organelles is cytotoxic (Maes et al., 2013; Song et al., 2009). In starvation, autophagy is stimulated to selfeat internal nutrient stores, including nutrient components and cellular organelles, to provide building blocks for energy generation and refuel metabolism, and thus lead to survival (Rabinowitz and White, 2010). Commonly used chemotherapies exert therapeutic effects by inducing metabolic and genotoxic stress in cancer cells; thus, in response to the treatment, autophagy is upregulated as a prosurvival function to support metabolism and maintain cellular homeostasis through self-eating (Chen and Karantza, 2011). Additionally, in the rapidly growing stage of tumor development, insufficient and abnormal vascularization cannot satisfy the great demand of fast-proliferating tumor cells, forming a microenvironment for tumor cells which is deprived of amino acids, oxygen, and growth factors. In the harsh microenvironment, autophagy is robustly activated in tumor cells for their energy supply and survival.
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It is documented that the tumor cells with stimulated autophagy are more refractory to cell death caused by chemotherapy treatment (White, 2012). Therefore, autophagy inhibition is considered to be a therapeutic strategy that can sensitize tumor cells to anticancer agents by depriving them of the essential prosurvival mechanism. Cancer stem cells (CSCs) are a rare subset of cancer cells which have features such as the ability to self-renew, differentiate into defined progenies, and initiate and sustain tumor growth in vivo. Eliminating CSCs may be the only way to ensure the therapeutic effects of chemotherapy, since remnant CSCs can give rise to tumor recurrence and metastasis. However, CSCs have mechanisms that facilitate evasion of cell death induced by currently available chemotherapy, application of which may only shrink tumor bulk by killing non-CSCs (Martelli et al., 2011). The role of autophagy in CSCs resides in the following three aspects: (1) autophagy is essential in maintaining the tumorigenicity of CSCs, and CSCs have a higher basal autophagy level compared to non-CSCs (Gong et al., 2013); (2) in response to stressful conditions including starvation, hypoxia, and chemotherapeutic agents, autophagy is required for its prosurvival function in CSCs, which also show a higher level of stress-induced autophagy than non-CSCs (Song et al., 2013); (3) some key molecules characterizing CSC stemness regulate autophagy to facilitate CSCs to escape from antitumor therapy-induced apoptosis (Chen et al., 2013). Thus, autophagy contributes to CSC chemoresistance through direct involvement in attenuating cellular damage induced by chemotherapeutic agents and supporting stemness maintenance and viability under harsh environments in an indirect manner. In this chapter, we aim to give a detailed introduction regarding the role of autophagy in the resistance of tumor cells to chemotherapy-induced apoptosis and growth inhibition, and provide insights into the development of autophagy inhibition as a synergistic therapy with existing chemotherapies for better therapeutic effects.
AUTOPHAGY AND CHEMOTHERAPY-INDUCED APOPTOSIS AND GROWTH INHIBITION Chemotherapy is one of the main options in cancer treatment. However, it has been found that most tumors are still resistant to chemotherapy. Commonly, the tumor cell response to chemotherapy is apoptosis. Resistance to chemotherapeutic agents has been associated with a failure to induce apoptosis in cancer cells. It has been suggested that tumors may prevent apoptotic cell death by various mechanisms, including overexpression of the apoptosis inhibitor Bcl-2 or absence of pro-apoptotic BAX and BAK. Recently, accumulated evidence suggests that autophagy may be associated with drug resistance in tumors. Although the majority of the literature has reported that autophagy could restrain chemotherapy-induced apoptosis, it has also been shown that autophagy might promote apoptosis to facilitate cell death.
Autophagy Restrains Chemotherapy-Induced Apoptosis Autophagy is a cell response mechanism to harmful stress. For tumor cells, upregulated autophagy may serve as a chemotherapy defense mechanism. In cancer cells, many
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conventional chemotherapeutic agents robustly induce autophagy. Paclitaxel, an effective mitotic inhibitor and apoptosis-inducing agent, is widely used to treat malignant tumors. Paclitaxel can induce autophagy and inhibition of autophagy by small interfering RNA against Beclin 1 can increase the apoptosis induced by paclitaxel (Xi et al., 2011). Cisplatin and 5-fluorouracil (5-FU) are commonly used chemotherapeutic agents. Cisplatin induces apoptosis by interfering with DNA replication and causes damage to the cell membrane structure. 5-FU exerts its anticancer effects through inhibition of thymidylate synthase and interference with uracil metabolism. Cisplatin or 5-fluorouracil treatment leads to apoptosis, and can induce autophagy in the cancer cell. Inhibition of autophagy increased the therapy effect of cisplatin and 5-fluorouracil (Guo et al., 2012). Therefore, specific inhibition of autophagy-related genes (Beclin 1 or Atg7) and a series of chemical inhibitors of autophagy, such as 3-methyladenine, bafilomycinA1, and chloroquine, may enhance cytotoxicity of cancer therapy. However, whether there may be an off-target effect needs to be carefully examined. It is suggested that BNIP3 is required for induction of autophagy by 5-FU, and cisplatin-triggered autophagic response is through activation of AMPK and subsequent suppression of mTOR activity (Harhaji-Trajkovic et al., 2009; Zeng and Kinsella, 2010). However, in some conditions, an inhibitor of the autophagy process in a different stage would lead to a different outcome of chemotherapy. Autophagy may contribute to tumor dormancy (Gewirtz, 2009). Dormant tumor cells are very insensitive to chemotherapy. These cells may recover and reenter the cell cycle to cause a cancer recurrence. In drug-resistant esophageal cancer cells, induction of autophagy might promote their survival and recovery following treatment with chemotherapeutics. The key reason is the failure of autophagic esophageal cancer cells to engage in apoptosis by chemotherapy (O’Donovan et al., 2011). Therefore, targeting the autophagy pathway could be an efficient approach to extend the therapeutic benefits of conventional chemotherapeutics and reduce the incidence of cancer recurrence. How autophagy helps tumor cells resist chemotherapy-induced apoptosis remains poorly defined. Autophagic response may act as a self-help mechanism to promote cell survival in multiple ways under therapy stress. Autophagy could reduce damaged mitochondrial potential and prevent the diffusion of pro-apoptotic factors in response to cell death stimuli. Autophagy has been shown to mitigate genome damage, which helps tumor cell survival in situations involving many DNA damage agents. Furthermore, reducing therapeutic stress-induced ROS accumulation by autophagy may be another possible mechanism for tumor cell survival of chemotherapy. In addition, autophagy may remove therapeutic stress-induced damaged protein accumulation, and then prevent ER stress-induced apoptosis which may also benefit tumor cell survival.
Autophagy Promotes Chemotherapy-Induced Apoptosis The relationship between autophagy and apoptosis is quite complicated. In some cases, autophagy might promote apoptotic cell death. Autophagy stimulates apoptosis in HER2overexpressing breast cancers treated by lapatinib (tyrosine kinase inhibitor), as pretreatment with 3-methyladenine (3-MA) could frustrate lapatinib-induced cancer cell apoptosis (Zhu et al., 2013). In human colon cancer cells, ROS-triggered autophagy contributes to resveratrol-induced apoptosis (Miki et al., 2012). Docosahexaenoic acid (DHA) could induce
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autophagy through p53/AMPK/mTOR signaling. Inhibition of autophagy suppressed apoptosis, and induction of autophagy enhanced apoptosis in response to DHA treatment (Jing et al., 2011). In some cancer cells, proteasome inhibitor can promote activation of caspase-8, which required the induction of autophagy and the presence of Atg5 (Laussmann et al., 2011). Interestingly, in some cases, apoptosis and autophagy could be simultaneously induced by the same stimulus and had no connection. Arsenic trioxide synergizes with Rad001 to induce cytotoxicity of ovarian cancer cells through increased autophagy and apoptosis (Liu et al., 2012). EGFR-targeted inhibitors inhibited EGFR-mediated signal transduction and induced autophagy and apoptosis in NSCLC cells (Yokoyama et al., 2011).
Autophagy Aggravates Chemotherapy-Induced Growth Inhibition Besides inducing tumor cell apoptosis, inhibition of tumor proliferation is another effective anticancer strategy. In some types of tumors, basic autophagy is required for cancer growth. Therefore, synergistic treatment with chemotherapeutic agents and autophagy inhibitors may be an efficient way to achieve tumor suppression. In lung cancer cells, suppression of basal autophagy also reduces proliferation without significant effects on the cell-cycle distribution (Kaminskyy et al., 2012). In Ras activated cancer, the high basal level of autophagy facilitated tumor growth. The underlying mechanism was that autophagy preserves the pool of functional mitochondria which is required to support growth of Rasdriven tumors (Guo et al., 2011). Pancreatic primary tumors and cell lines also show elevated autophagy under basal conditions. Genetic or pharmacologic inhibition of autophagy leads to significant growth suppression of pancreatic cancer cells in vitro and robust tumor regression in vivo (Yang et al., 2011). Inhibition of autophagy rendered cell cycle arrest and may restrain tumor growth (Altman et al., 2011). Combined treatment with chemotherapeutic agents and autophagy inhibitors can lead to dramatically inhibited tumor growth and impaired cell proliferation in xenografted animal models (Guo et al., 2013). Autophagy inhibitor CQ pretreatment can enhance the proliferation-inhibitory effect of 5-fluorouracil on cancer cells, which was dependent on the increase of p21Cip1 and p27Kip1 and the decrease of CDK2 (Sasaki et al., 2010).
AUTOPHAGY, TUMOR MICROENVIRONMENT, AND CHEMORESISTANCE Hypoxia-Induced Autophagy Contributes to Chemoresistance of Tumor Cells Tumors live in a specific microenvironment that is different from the normal cellular microenvironment. It supports the proliferation of tumor cells and contributes to tumor development, while the normal microenvironment can inhibit cell growth. Hypoxia is one of the main characteristics of the tumor microenvironment. It exists throughout the whole process of tumor development, from oncogenesis to metastasis. In the early phase, hypoxia is generated by insufficient blood supply. Oxygen is only able to diffuse 100–180 μm from a capillary to cells; any cell located farther than this distance will be hypoxic (Powis and Kirkpatrick, 2004). As the tumors develop, new blood vessels are rebuilt, but tumor
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microvasculature is structurally and functionally abnormal. The tumor blood vessels are usually compressed or obstructed by the rapid growth of the tumor. Meanwhile, the aggressively proliferating cancer cells often outgrow the angiogenesis. In the metastases, some tumors are clinically hypovascular. In addition, treatments such as embolization results in hypoxia. As a result, hypoxia presents in a great majority of solid tumors. Hypoxia often correlates with poor prognosis and high patient mortality, which in part is due to the chemoresistance (Shannon et al., 2003). In our study, compared with normoxia, chemotherapeutic agent-induced cell apoptosis under hypoxia of 1% O2 was significantly decreased (Song et al., 2009). Cells respond to hypoxia in various ways, including cell cycle arrest, angiogenesis, glycolytic metabolism and so on. Hypoxia was reported to be one of the stimuli for autophagy (Zhang et al., 2008). In our study, autophagy was significantly induced by hypoxia and protected the hepatocellular carcinoma cells from chemotherapy. Less cell death was shown in cells cultured in hypoxia when they were exposed to chemotherapeutic agents, which mainly resulted from decreased apoptosis. Autophagy induced by hypoxia decreased the hepatocellular carcinoma cells’ apoptotic potential and mediated their chemoresistance. When autophagy was inhibited by an autophagic inhibitor, such as 3-MA or siRNA against Beclin 1, which plays an important role during the formation of autophagosomes, the cell sensitivity to chemotherapy was recovered. Similar results were shown in another study (Wu et al., 2008). Hypoxia-inducible factor 1 (HIF1) is a transcription factor accumulated under hypoxia. The resistance of tumor cells to chemotherapeutics in hypoxia is partially because of the activation of HIF1 (Tong et al., 2013). HIF1, by regulating its downstream targets such as BNIP3 and BNIP3L, regulates autophagy in hypoxic conditions. BNIP3 and BNIP3L were indicated to induce autophagy by disrupting the interaction between Beclin 1 and Bcl-2 (Bellot et al., 2009).
Hypoxia-Induced Autophagy Contributes to Tolerance of Tumor Cells to Nutrient Deprivation in Tumor Microenvironment In addition to hypoxia, another characteristic of the tumor microenvironment that is different from the normal cellular microenvironment is nutrient deprivation. Ischemia often occurs in solid tumors because of inefficient blood supply and tumor expansion. Hypoxia and nutrient deprivation result from ischemia, exist in solid tumors from the beginning, participate in carcinogenesis, and contribute to aggravation of the tumor phenotype. However, nutrient deprivation does not happen at the same time as hypoxia. First of all, nutrients such as glucose and amino acids are transported from blood flow to metabolized cancer cells via specific transporters, which occurs faster than the transportation of oxygen by simple diffusion. Secondly, nutrients can be absorbed through alternative ways, while oxygen cannot. So hypoxia happens earlier than nutrient deprivation in tumors. We found that the earlier event of hypoxia mediated the tolerance of hepatocellular carcinoma cells to nutrient deprivation that happens later (Song et al., 2011). This is consistent with the reports that cells in hypoxia showed tolerance against glucose starvation (Suzuki et al., 2005). Once cancer cells sense the hypoxia, they will develop a range of metabolic adaptations, which then benefit the subsequent tolerance to nutrient deprivation. There are mainly two ways – increasing supply or tolerating insufficiency. Supply increase is achieved by vasodilatation and angiogenesis. Tolerating nutrient deprivation happens by activating other
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metabolic processes to obtain energy. Autophagy is one of the choices. Autophagy degrades existing cellular components to recycle amino acids and other intracellular nutrients, and to obtain energy from recycled materials. We demonstrated the substantial role of autophagy in hypoxia which conferred tolerance of hepatocellular carcinoma cells against nutrient deprivation. However, the relevance of autophagy in cell survival and cell death is still controversial. Different from the prosurvival role of autophagy, autophagy is also a type 2 programmed cell death (PCD). Autophagy eliminates damaged and/or harmful cells such as cancer cells killed by anticancer reagents. The role of autophagy may vary depending on cell type, microenvironment, and the level of autophagy. Beclin 1 should be mentioned in autophagy in mammalian cells. Beclin 1 possesses a socalled BH3 domain that mediates the interaction between Beclin 1 and Bcl-2 family members, such as Bcl-2, Bcl-XL and Mcl-1 (Maiuri et al., 2007). These interactions contribute to the role of Beclin 1 in coordinating the cytoprotective role of autophagy and apoptosis. In our study, autophagy induced by hypoxia decreased the apoptosis induced by nutrient deprivation, while it did not affect necrosis in a Beclin 1-dependent way in hepatocellular carcinoma.
AUTOPHAGY AND DNA DAMAGE-INDUCING CHEMOTHERAPY Inducing cytotoxicity by damaging DNA is an important means of cancer treatment, including chemotherapy. The damaged DNA usually cannot maintain structural stability and exert regular functionality. In response to DNA damage, the cells trigger a series of processes, including: (a) activation of a checkpoint system to delay cell cycle progression; (b) removal or repair of damaged DNA lesions to maintain DNA integrity; and (c) activation of apoptotic or senescence-associated pathways to clear away excessive DNA-damaged cells. To reduce chemotherapeutic agent-induced DNA damage and ultimate cell death, cancer cells modulate these DNA damage responses (DDRs) by various mechanisms. The phenomenon that autophagy deficiency sensitizes tumor cells to DNA-damaging chemotherapy suggests that autophagy probably plays an important role in this process (Rodriguez-Rocha et al., 2011).
Autophagy Can Be Induced by DDRs in DNA Damaged Cells In DNA damaged cells, DDRs can induce autophagy. DNA mismatch repair (MMR) proteins (MLH1 or MSH2) mediate autophagy following chemical DNA mismatch damage through activating p53. A recent study showed that in the p53 family, members p63 and p73 contribute to activation of the autophagy gene network (Kenzelmann Broz et al., 2013). E2F, PARP-1, BNIP3, Skp2, and ATM, all of which are the signal factors of DDRs, are the positive regulators of autophagy by direct and indirect means. The protein factor-arrest 11, which participates in the regulation of DDR by dephosphorylating Rad53, is essential for autophagy induction by dephosphorylating Atg13. Histone deacetylases (HDACs) have important roles in the regulation of DDR and also are involved in autophagy induction. HDAC10 depletion in neuroblastoma cells inhibits autophagic flux and results in accumulation of autophagosomes and lysosomes.
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Autophagy Regulates DDRs by Indirect and Direct Approaches Numerous studies showed that autophagy indirectly inhibits DDRs by eliminating misfolded protein and damaged organelles, especially mitochondria. It was found that autophagy contributes to delay DDR by clearing damaged mitochondria that can result in a post-mitochondrial caspase cascade (Abedin et al., 2007). In the murine model, microtubuleassociated protein 1 small form (MAP1S) elevation in response to alkylating agent diethylnitrosamine treatment enhances autophagy to remove misfolded proteins and damaged organelles that trigger DNA double-strand breaks (DSB) and genome instability. During Myc-driven lymphomagenesis, suppression of mitophagy resulting from hemizygous deletion of Bif-1 leads to the increase of mitochondrial mass, accumulation of DNA damage and the ultimate chromosomal instability (Takahashi et al., 2013). Studies of the role of autophagy on genome stability under metabolic stress also showed a similar phenomenon. Metabolic stresses distinctly impact on cellular genome stability by disturbing DNA synthesis and repair via accumulating misfolded and aggregate-prone proteins, and ROS-generating organelles. In autophagy-competent cells, autophagy clears these accumulations to limit these metabolic stresses. However, defective autophagy sensitizes cells to metabolic stress, and increases DNA damage and further promotes genomic instability (Mathew and White, 2007). A recent study showed that autophagy also can directly impact on DDR. Autophagy is involved in the regulation of HDACs on a number of DDR proteins (including CtIP and Exo1) by degrading acetylated DSB repair enzymes (Robert et al., 2011). Budding yeast cells suffering a single unrepaired DSB trigger hyperactivation of the autophagy pathway, which causes the permanent G2/M arrest.
Autophagy Essential Proteins Regulate DDR by Autophagy-Independent Means Notably, some Atgs can regulate DDR by autophagy-independent means. Independent of its E1-like enzymatic activity, Atg7 can bind to p53 to regulate the transcription of cell cycle inhibitor p21 (CDKN1A). Under metabolic stress, Atg7 (−/−) lead to augmented DNA damage with increased p53-dependent apoptosis (Lee et al., 2012). DNA-damaging drugs can induce the expression of Atg5. Atg5 translocates to the nucleus, and interacts with survivin to induce mitotic catastrophe. Pharmacological inhibition of autophagy cannot suppress Atg5-dependent mitotic catastrophe (Maskey et al., 2013).
AUTOPHAGY AND CANCER STEM CELLS IN CHEMORESISTANCE Recently, the role of cancer stem cells (CSCs) in chemoresistance has been highlighted, since CSCs are resistant to commonly used chemotherapy and responsible for poor prognosis for patients. Autophagy is one key player involved in CSC chemoresistance.
Autophagy Is Essential for Maintenance of the Tumorigenicity of CSCs It was reported that CD133+ liver cancer cells had higher basal autophagy level as indicated by the expression of autophagy-associated genes including Atg5, Atg7, Beclin 1, and
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LC3 and by GFP-LC3 puncta and electron microscopy, compared with their CD133− counterparts. Inhibition of autophagy could significantly impair the clonogenic and sphereforming capacity of CSCs. Furthermore, hypoxia and nutrient starvation (H/S) induced more robustly increased autophagy in CSCs, which could enhance their clonogenic and sphere-forming capacity. An in vivo xenograft model suggested that inhibition of autophagy with CQ greatly suppressed tumorigenicity of CSCs (Song et al., 2013). This evidence leads to the conclusion that autophagy is essential for liver CSC maintenance under both normal and H/S conditions. Consistent with our data, Gong et al. reported that higher expression of Beclin 1 and more robust autophagic flux were observed in mammospheres established from human breast cancers or breast cancer cell lines than in the parental adherent cells. shRNA-mediated silencing of Beclin 1 (shBECN1) could impair mammosphere tumorigenicity as indicated by tumor volume and weight (Gong et al., 2013). The data indicate that autophagy is critical for CSC maintenance and tumor development in nude mice.
Autophagy Contributes to Survival of CSCs in Oxygen and/or NutrientDeprived Tumor Microenvironment The autophagic pathway can be activated under different stimuli, such as endoplasmic reticulum stress, DNA damage, and reactive oxygen species (ROS), thereby eliciting a cytoprotective response that helps cells to overcome those stressful situations. Ischemia and hypoxia typically induce autophagy, which protects cells during times of stress. The tumor microenvironment is characterized by oxygen and nutrient deprivation. Current understanding of autophagy is that it is important for tumorigenesis and can promote the growth of established cancers. The cells that express stem cell surface membrane antigen, which have the capacity to repopulate the tumor and contribute to malignant progression, are highly resistant to the tumor microenvironment. It was reported that autophagy enhances the survival of CSC under the oxygen/nutrient-deprived condition. Under the harsh tumor microenvironment, compared with non-CSCs, CSCs showed higher survival capability and lower apoptosis, denoting that liver CSCs are resistant to loss of oxygen and nutrient supply in hepatocellular cancer (Song et al., 2013). Many studies found increased expression of Atgs in response to autophagy caused by oxygen deficiency and nutrient deprivation (Bampton et al., 2005). The differences in survival and apoptosis may be due to both higher basal and oxygen and nutrient deprivation-induced levels of autophagy in CSCs than in non-CSCs. CSCs contain early and late autophagic vesicles, express Atgs, and the altered expression of Atgs suggests an important role of autophagy in cell survival. Inhibition of the autophagy process can reduce the difference in survival capability and apoptosis. Higher basal autophagy level may make CSCs react to the tumor microenvironment faster. Thus, autophagy inhibitors may make CSCs more sensitive to the tumor microenvironment.
Autophagy Involved in CSC Chemoresistance Despite detection and therapeutic advances, no major improvements in overall survival have been obtained to date. Therefore, new treatment strategies are needed to improve the prognosis of patients. Autophagy is activated in various cancer cells following different anticancer therapies (Guo et al., 2012, 2013). As a prosurvival mechanism, autophagy
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can promote the survival of CSCs by providing catabolites required for repair, by removing toxic substances, and by reducing cytoplasmic acidification. It was reported that a subpopulation of CSCs expressing Cdx1 was more resistant to paclitaxel-induced cytotoxicity than p53-expressing CSCs. Cdx1 exerts a protective role in colon cancer stem cells against chemotherapy through activation of autophagy (Wu et al., 2013). Based upon their previous data indicating that CD133 was involved in autophagosomes, Chen et al. (2013) found that CD133 antibody (CD133mAb) treatment resulted in cell death in hepatoma cell lines, and the antibody effect was mediated by autophagy inhibition, indicating a direct link between autophagy and important molecules regulating stem cell properties. Thus, synergistic autophagy inhibition, either using pharmacological inhibitors or RNA interference of essential autophagy genes, might hold the hope of complete elimination of CSCs.
CONCLUSION Considering the essential role of autophagy in rendering cancer cell resistance to chemotherapy-induced apoptosis and growth inhibition, the development of autophagy inhibition as an adjuvant therapy to existing chemotherapeutics is of great significance in our fight against cancer, to bring benefits for patient survival. The effects of autophagy inhibition by pharmacologic agents or siRNA genetically targeting Atg genes are widely investigated in various in vitro and in vivo models. Furthermore, with encouraging news already reported by some clinical trials, antimalarial and antirheumatic drug CQ and its derivative hydroxychloroquine (HCQ) have been under investigation regarding their effects for the treatment of refractory malignancies in more than 30 ongoing clinical trials (Maes et al., 2013). However, the mechanism underlying the therapeutic effects of autophagy inhibition is still undefined, and whether there exist off-target functions necessitates further studies, which may facilitate the determination of better therapeutic regimens. Since autophagy plays an important role in maintaining cellular homeostasis and genome stability, induction of autophagy is considered to help prevent cancer. Compounds and a variety of dietary factors as well as calorie restriction, which can stimulate autophagy, have been under consideration to be applied in cancer prevention (Maes et al., 2013). Furthermore, in specific genetic backgrounds autophagy induction could lead to autophagic cell death and, in such context, the therapeutic effect of autophagy inhibition will be counterproductive. Thus, more research is required to identify suitable candidates for autophagy inhibition treatment.
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Song, Y.J., Zhang, S.S., Guo, X.L., et al., 2013. Autophagy contributes to the survival of CD133+ liver cancer stem cells in the hypoxic and nutrient-deprived tumor microenvironment. Cancer Lett. 339 (1), 70–81. Suzuki, A., Kusakai, G., Shimojo, Y., et al., 2005. Involvement of transforming growth factor-beta 1 signaling in hypoxia-induced tolerance to glucose starvation. J. Biol. Chem. 280, 31557–31563. Takahashi, Y., Hori, T., Cooper, T.K., et al., 2013. Bif-1 haploinsufficiency promotes chromosomal instability and accelerates Myc-driven lymphomagenesis via suppression of mitophagy. Blood 121, 1622–1632. Tong, Y., Li, Q.G., Xing, T.Y., et al., 2013. HIF1 regulates WSB-1 expression to promote hypoxia-induced chemoresistance in hepatocellular carcinoma cells. FEBS Lett. 587, 2530–2535. White, E., 2012. Deconvoluting the context-dependent role for autophagy in cancer. Nat. Rev. Cancer 12, 401–410. Wu, S., Wang, X., Chen, J., et al., 2013. Autophagy of cancer stem cells is involved with chemoresistance of colon cancer cells. Biochem. Biophys. Res. Commun. 434, 898–903. Wu, Y.T., Tan, H.L., Huang, Q., et al., 2008. Autophagy plays a protective role during zVAD-induced necrotic cell death. Autophagy 4, 457–466. Xi, G., Hu, X., Wu, B., et al., 2011. Autophagy inhibition promotes paclitaxel-induced apoptosis in cancer cells. Cancer Lett. 307, 141–148. Yang, S., Wang, X., Contino, G., et al., 2011. Pancreatic cancers require autophagy for tumor growth. Genes Dev. 25, 717–729. Yokoyama, T., Tam, J., Kuroda, S., et al., 2011. EGFR-targeted hybrid plasmonic magnetic nanoparticles synergistically induce autophagy and apoptosis in non-small cell lung cancer cells. PLoS ONE 6, e25507. Zeng, X., Kinsella, T.J., 2010. BNIP3 is essential for mediating 6-thioguanine- and 5-fluorouracil-induced autophagy following DNA mismatch repair processing. Cell Res. 20, 665–675. Zhang, H., Bosch-Marce, M., Shimoda, L.A., et al., 2008. Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia. J. Biol. Chem. 283, 10892–10903. Zhu, X., Wu, L., Qiao, H., et al., 2013. Autophagy stimulates apoptosis in HER2-overexpressing breast cancers treated by lapatinib. J. Cell. Biochem. 114, 2643–2653.
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9 Effect of Autophagy on Chemotherapy-Induced Apoptosis and Growth Inhibition Shanshan Zhang, Xianling Guo, Jianrui Song, Kai Sun, Yujiao Song, and Lixin Wei O U T L I N E Autophagy Can Be Induced by DDRs in DNA Damaged Cells 151 Autophagy Regulates DDRs by Indirect and Direct Approaches 152 Autophagy Essential Proteins Regulate DDR by AutophagyIndependent Means 152
Introduction 146 Autophagy and Chemotherapy-Induced Apoptosis and Growth Inhibition 147 Autophagy Restrains Chemotherapy-Induced Apoptosis 147 Autophagy Promotes Chemotherapy-Induced Apoptosis 148 Autophagy Aggravates ChemotherapyInduced Growth Inhibition 149 Autophagy, Tumor Microenvironment, and Chemoresistance 149 Hypoxia-Induced Autophagy Contributes to Chemoresistance of Tumor Cells 149 Hypoxia-Induced Autophagy Contributes to Tolerance of Tumor Cells to Nutrient Deprivation in Tumor Microenvironment 150 Autophagy and DNA Damage-Inducing Chemotherapy 151
M.A. Hayat (ed): Autophagy, Volume 5. DOI: http://dx.doi.org/10.1016/B978-0-12-801033-4.00009-6
Autophagy and Cancer Stem Cells in Chemoresistance 152 Autophagy Is Essential for Maintenance of the Tumorigenicity of CSCs 152 Autophagy Contributes to Survival of CSCs in Oxygen and/or NutrientDeprived Tumor Microenvironment 153 Autophagy Involved in CSC Chemoresistance 153 Conclusion 154 References 154
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Abstract Cancer cells are resistant to chemotherapy, which results in poor prognosis for cancer patients. Autophagy, a self-eating process, has been widely reported as a prosurvival mechanism underlying cancer cell chemoresistance. Upon chemotherapeutics treatment, autophagy is employed by cancer cells to maintain cellular homeostasis and mitigate genome damage by degrading damaged proteins and organelles such as mitochondria, thus preventing cell apoptosis. In the tumor microenvironment characteristic of oxygen and nutrient deprivation, autophagy is activated in cancer cells to cope with metabolic stress, and these cells were more refractory to chemotherapy. Cancer stem cells (CSCs) are a subset of cancer cells that can evade cell death induced by existing chemotherapeutic agents, and one of the underlying mechanisms might be autophagy. Emerging evidence shows that CSCs have higher levels of autophagy under normal conditions and ischemic and hypoxic conditions. Autophagy inhibition could sensitize non-CSCs and CSCs to chemotherapy-induced apoptosis and growth inhibition; thus, it is under consideration for being developed as a synergistic therapy with existing chemotherapies for better therapeutic effects.
INTRODUCTION Cancer poses a great threat to people’s health. Although many innovative chemotherapeutic agents based upon important findings in basic and clinical cancer research have been developed and used in cancer patients for years, our overall achievements in prolonging patients’ survival are still not satisfactory, since the major obstacle that tumor cells are resistant to chemotherapy still exists. Thus, understanding the underlying mechanisms as to how resistance happens can reveal new targets and is beneficial for the development of novel therapies which might be applied, together with existing chemotherapies, to gain better therapeutic effects and bring long-term clinical benefits to the patients. Autophagy (also known as macroautophagy) has been widely reported to be associated with chemoresistance of tumor cells. Autophagy is an evolutionarily conserved catabolic pathway which exists in all eukaryotic cells from yeast to mammals. It is characterized by the formation of double membrane vesicles, called autophagosomes, which sequester long-life proteins and cellular organelles such as mitochondria. The autophagosomes fuse with vacuoles or lysosomes where degradation of the cargo occurs, supplying amino acids and macromolecular precursors for cells. A basal level of autophagy exists in every cell and is necessary for maintaining cellular homeostasis, since accumulation of aggregation-prone proteins and damaged organelles is cytotoxic (Maes et al., 2013; Song et al., 2009). In starvation, autophagy is stimulated to selfeat internal nutrient stores, including nutrient components and cellular organelles, to provide building blocks for energy generation and refuel metabolism, and thus lead to survival (Rabinowitz and White, 2010). Commonly used chemotherapies exert therapeutic effects by inducing metabolic and genotoxic stress in cancer cells; thus, in response to the treatment, autophagy is upregulated as a prosurvival function to support metabolism and maintain cellular homeostasis through self-eating (Chen and Karantza, 2011). Additionally, in the rapidly growing stage of tumor development, insufficient and abnormal vascularization cannot satisfy the great demand of fast-proliferating tumor cells, forming a microenvironment for tumor cells which is deprived of amino acids, oxygen, and growth factors. In the harsh microenvironment, autophagy is robustly activated in tumor cells for their energy supply and survival.
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It is documented that the tumor cells with stimulated autophagy are more refractory to cell death caused by chemotherapy treatment (White, 2012). Therefore, autophagy inhibition is considered to be a therapeutic strategy that can sensitize tumor cells to anticancer agents by depriving them of the essential prosurvival mechanism. Cancer stem cells (CSCs) are a rare subset of cancer cells which have features such as the ability to self-renew, differentiate into defined progenies, and initiate and sustain tumor growth in vivo. Eliminating CSCs may be the only way to ensure the therapeutic effects of chemotherapy, since remnant CSCs can give rise to tumor recurrence and metastasis. However, CSCs have mechanisms that facilitate evasion of cell death induced by currently available chemotherapy, application of which may only shrink tumor bulk by killing non-CSCs (Martelli et al., 2011). The role of autophagy in CSCs resides in the following three aspects: (1) autophagy is essential in maintaining the tumorigenicity of CSCs, and CSCs have a higher basal autophagy level compared to non-CSCs (Gong et al., 2013); (2) in response to stressful conditions including starvation, hypoxia, and chemotherapeutic agents, autophagy is required for its prosurvival function in CSCs, which also show a higher level of stress-induced autophagy than non-CSCs (Song et al., 2013); (3) some key molecules characterizing CSC stemness regulate autophagy to facilitate CSCs to escape from antitumor therapy-induced apoptosis (Chen et al., 2013). Thus, autophagy contributes to CSC chemoresistance through direct involvement in attenuating cellular damage induced by chemotherapeutic agents and supporting stemness maintenance and viability under harsh environments in an indirect manner. In this chapter, we aim to give a detailed introduction regarding the role of autophagy in the resistance of tumor cells to chemotherapy-induced apoptosis and growth inhibition, and provide insights into the development of autophagy inhibition as a synergistic therapy with existing chemotherapies for better therapeutic effects.
AUTOPHAGY AND CHEMOTHERAPY-INDUCED APOPTOSIS AND GROWTH INHIBITION Chemotherapy is one of the main options in cancer treatment. However, it has been found that most tumors are still resistant to chemotherapy. Commonly, the tumor cell response to chemotherapy is apoptosis. Resistance to chemotherapeutic agents has been associated with a failure to induce apoptosis in cancer cells. It has been suggested that tumors may prevent apoptotic cell death by various mechanisms, including overexpression of the apoptosis inhibitor Bcl-2 or absence of pro-apoptotic BAX and BAK. Recently, accumulated evidence suggests that autophagy may be associated with drug resistance in tumors. Although the majority of the literature has reported that autophagy could restrain chemotherapy-induced apoptosis, it has also been shown that autophagy might promote apoptosis to facilitate cell death.
Autophagy Restrains Chemotherapy-Induced Apoptosis Autophagy is a cell response mechanism to harmful stress. For tumor cells, upregulated autophagy may serve as a chemotherapy defense mechanism. In cancer cells, many
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conventional chemotherapeutic agents robustly induce autophagy. Paclitaxel, an effective mitotic inhibitor and apoptosis-inducing agent, is widely used to treat malignant tumors. Paclitaxel can induce autophagy and inhibition of autophagy by small interfering RNA against Beclin 1 can increase the apoptosis induced by paclitaxel (Xi et al., 2011). Cisplatin and 5-fluorouracil (5-FU) are commonly used chemotherapeutic agents. Cisplatin induces apoptosis by interfering with DNA replication and causes damage to the cell membrane structure. 5-FU exerts its anticancer effects through inhibition of thymidylate synthase and interference with uracil metabolism. Cisplatin or 5-fluorouracil treatment leads to apoptosis, and can induce autophagy in the cancer cell. Inhibition of autophagy increased the therapy effect of cisplatin and 5-fluorouracil (Guo et al., 2012). Therefore, specific inhibition of autophagy-related genes (Beclin 1 or Atg7) and a series of chemical inhibitors of autophagy, such as 3-methyladenine, bafilomycinA1, and chloroquine, may enhance cytotoxicity of cancer therapy. However, whether there may be an off-target effect needs to be carefully examined. It is suggested that BNIP3 is required for induction of autophagy by 5-FU, and cisplatin-triggered autophagic response is through activation of AMPK and subsequent suppression of mTOR activity (Harhaji-Trajkovic et al., 2009; Zeng and Kinsella, 2010). However, in some conditions, an inhibitor of the autophagy process in a different stage would lead to a different outcome of chemotherapy. Autophagy may contribute to tumor dormancy (Gewirtz, 2009). Dormant tumor cells are very insensitive to chemotherapy. These cells may recover and reenter the cell cycle to cause a cancer recurrence. In drug-resistant esophageal cancer cells, induction of autophagy might promote their survival and recovery following treatment with chemotherapeutics. The key reason is the failure of autophagic esophageal cancer cells to engage in apoptosis by chemotherapy (O’Donovan et al., 2011). Therefore, targeting the autophagy pathway could be an efficient approach to extend the therapeutic benefits of conventional chemotherapeutics and reduce the incidence of cancer recurrence. How autophagy helps tumor cells resist chemotherapy-induced apoptosis remains poorly defined. Autophagic response may act as a self-help mechanism to promote cell survival in multiple ways under therapy stress. Autophagy could reduce damaged mitochondrial potential and prevent the diffusion of pro-apoptotic factors in response to cell death stimuli. Autophagy has been shown to mitigate genome damage, which helps tumor cell survival in situations involving many DNA damage agents. Furthermore, reducing therapeutic stress-induced ROS accumulation by autophagy may be another possible mechanism for tumor cell survival of chemotherapy. In addition, autophagy may remove therapeutic stress-induced damaged protein accumulation, and then prevent ER stress-induced apoptosis which may also benefit tumor cell survival.
Autophagy Promotes Chemotherapy-Induced Apoptosis The relationship between autophagy and apoptosis is quite complicated. In some cases, autophagy might promote apoptotic cell death. Autophagy stimulates apoptosis in HER2overexpressing breast cancers treated by lapatinib (tyrosine kinase inhibitor), as pretreatment with 3-methyladenine (3-MA) could frustrate lapatinib-induced cancer cell apoptosis (Zhu et al., 2013). In human colon cancer cells, ROS-triggered autophagy contributes to resveratrol-induced apoptosis (Miki et al., 2012). Docosahexaenoic acid (DHA) could induce
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autophagy through p53/AMPK/mTOR signaling. Inhibition of autophagy suppressed apoptosis, and induction of autophagy enhanced apoptosis in response to DHA treatment (Jing et al., 2011). In some cancer cells, proteasome inhibitor can promote activation of caspase-8, which required the induction of autophagy and the presence of Atg5 (Laussmann et al., 2011). Interestingly, in some cases, apoptosis and autophagy could be simultaneously induced by the same stimulus and had no connection. Arsenic trioxide synergizes with Rad001 to induce cytotoxicity of ovarian cancer cells through increased autophagy and apoptosis (Liu et al., 2012). EGFR-targeted inhibitors inhibited EGFR-mediated signal transduction and induced autophagy and apoptosis in NSCLC cells (Yokoyama et al., 2011).
Autophagy Aggravates Chemotherapy-Induced Growth Inhibition Besides inducing tumor cell apoptosis, inhibition of tumor proliferation is another effective anticancer strategy. In some types of tumors, basic autophagy is required for cancer growth. Therefore, synergistic treatment with chemotherapeutic agents and autophagy inhibitors may be an efficient way to achieve tumor suppression. In lung cancer cells, suppression of basal autophagy also reduces proliferation without significant effects on the cell-cycle distribution (Kaminskyy et al., 2012). In Ras activated cancer, the high basal level of autophagy facilitated tumor growth. The underlying mechanism was that autophagy preserves the pool of functional mitochondria which is required to support growth of Rasdriven tumors (Guo et al., 2011). Pancreatic primary tumors and cell lines also show elevated autophagy under basal conditions. Genetic or pharmacologic inhibition of autophagy leads to significant growth suppression of pancreatic cancer cells in vitro and robust tumor regression in vivo (Yang et al., 2011). Inhibition of autophagy rendered cell cycle arrest and may restrain tumor growth (Altman et al., 2011). Combined treatment with chemotherapeutic agents and autophagy inhibitors can lead to dramatically inhibited tumor growth and impaired cell proliferation in xenografted animal models (Guo et al., 2013). Autophagy inhibitor CQ pretreatment can enhance the proliferation-inhibitory effect of 5-fluorouracil on cancer cells, which was dependent on the increase of p21Cip1 and p27Kip1 and the decrease of CDK2 (Sasaki et al., 2010).
AUTOPHAGY, TUMOR MICROENVIRONMENT, AND CHEMORESISTANCE Hypoxia-Induced Autophagy Contributes to Chemoresistance of Tumor Cells Tumors live in a specific microenvironment that is different from the normal cellular microenvironment. It supports the proliferation of tumor cells and contributes to tumor development, while the normal microenvironment can inhibit cell growth. Hypoxia is one of the main characteristics of the tumor microenvironment. It exists throughout the whole process of tumor development, from oncogenesis to metastasis. In the early phase, hypoxia is generated by insufficient blood supply. Oxygen is only able to diffuse 100–180 μm from a capillary to cells; any cell located farther than this distance will be hypoxic (Powis and Kirkpatrick, 2004). As the tumors develop, new blood vessels are rebuilt, but tumor
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microvasculature is structurally and functionally abnormal. The tumor blood vessels are usually compressed or obstructed by the rapid growth of the tumor. Meanwhile, the aggressively proliferating cancer cells often outgrow the angiogenesis. In the metastases, some tumors are clinically hypovascular. In addition, treatments such as embolization results in hypoxia. As a result, hypoxia presents in a great majority of solid tumors. Hypoxia often correlates with poor prognosis and high patient mortality, which in part is due to the chemoresistance (Shannon et al., 2003). In our study, compared with normoxia, chemotherapeutic agent-induced cell apoptosis under hypoxia of 1% O2 was significantly decreased (Song et al., 2009). Cells respond to hypoxia in various ways, including cell cycle arrest, angiogenesis, glycolytic metabolism and so on. Hypoxia was reported to be one of the stimuli for autophagy (Zhang et al., 2008). In our study, autophagy was significantly induced by hypoxia and protected the hepatocellular carcinoma cells from chemotherapy. Less cell death was shown in cells cultured in hypoxia when they were exposed to chemotherapeutic agents, which mainly resulted from decreased apoptosis. Autophagy induced by hypoxia decreased the hepatocellular carcinoma cells’ apoptotic potential and mediated their chemoresistance. When autophagy was inhibited by an autophagic inhibitor, such as 3-MA or siRNA against Beclin 1, which plays an important role during the formation of autophagosomes, the cell sensitivity to chemotherapy was recovered. Similar results were shown in another study (Wu et al., 2008). Hypoxia-inducible factor 1 (HIF1) is a transcription factor accumulated under hypoxia. The resistance of tumor cells to chemotherapeutics in hypoxia is partially because of the activation of HIF1 (Tong et al., 2013). HIF1, by regulating its downstream targets such as BNIP3 and BNIP3L, regulates autophagy in hypoxic conditions. BNIP3 and BNIP3L were indicated to induce autophagy by disrupting the interaction between Beclin 1 and Bcl-2 (Bellot et al., 2009).
Hypoxia-Induced Autophagy Contributes to Tolerance of Tumor Cells to Nutrient Deprivation in Tumor Microenvironment In addition to hypoxia, another characteristic of the tumor microenvironment that is different from the normal cellular microenvironment is nutrient deprivation. Ischemia often occurs in solid tumors because of inefficient blood supply and tumor expansion. Hypoxia and nutrient deprivation result from ischemia, exist in solid tumors from the beginning, participate in carcinogenesis, and contribute to aggravation of the tumor phenotype. However, nutrient deprivation does not happen at the same time as hypoxia. First of all, nutrients such as glucose and amino acids are transported from blood flow to metabolized cancer cells via specific transporters, which occurs faster than the transportation of oxygen by simple diffusion. Secondly, nutrients can be absorbed through alternative ways, while oxygen cannot. So hypoxia happens earlier than nutrient deprivation in tumors. We found that the earlier event of hypoxia mediated the tolerance of hepatocellular carcinoma cells to nutrient deprivation that happens later (Song et al., 2011). This is consistent with the reports that cells in hypoxia showed tolerance against glucose starvation (Suzuki et al., 2005). Once cancer cells sense the hypoxia, they will develop a range of metabolic adaptations, which then benefit the subsequent tolerance to nutrient deprivation. There are mainly two ways – increasing supply or tolerating insufficiency. Supply increase is achieved by vasodilatation and angiogenesis. Tolerating nutrient deprivation happens by activating other
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metabolic processes to obtain energy. Autophagy is one of the choices. Autophagy degrades existing cellular components to recycle amino acids and other intracellular nutrients, and to obtain energy from recycled materials. We demonstrated the substantial role of autophagy in hypoxia which conferred tolerance of hepatocellular carcinoma cells against nutrient deprivation. However, the relevance of autophagy in cell survival and cell death is still controversial. Different from the prosurvival role of autophagy, autophagy is also a type 2 programmed cell death (PCD). Autophagy eliminates damaged and/or harmful cells such as cancer cells killed by anticancer reagents. The role of autophagy may vary depending on cell type, microenvironment, and the level of autophagy. Beclin 1 should be mentioned in autophagy in mammalian cells. Beclin 1 possesses a socalled BH3 domain that mediates the interaction between Beclin 1 and Bcl-2 family members, such as Bcl-2, Bcl-XL and Mcl-1 (Maiuri et al., 2007). These interactions contribute to the role of Beclin 1 in coordinating the cytoprotective role of autophagy and apoptosis. In our study, autophagy induced by hypoxia decreased the apoptosis induced by nutrient deprivation, while it did not affect necrosis in a Beclin 1-dependent way in hepatocellular carcinoma.
AUTOPHAGY AND DNA DAMAGE-INDUCING CHEMOTHERAPY Inducing cytotoxicity by damaging DNA is an important means of cancer treatment, including chemotherapy. The damaged DNA usually cannot maintain structural stability and exert regular functionality. In response to DNA damage, the cells trigger a series of processes, including: (a) activation of a checkpoint system to delay cell cycle progression; (b) removal or repair of damaged DNA lesions to maintain DNA integrity; and (c) activation of apoptotic or senescence-associated pathways to clear away excessive DNA-damaged cells. To reduce chemotherapeutic agent-induced DNA damage and ultimate cell death, cancer cells modulate these DNA damage responses (DDRs) by various mechanisms. The phenomenon that autophagy deficiency sensitizes tumor cells to DNA-damaging chemotherapy suggests that autophagy probably plays an important role in this process (Rodriguez-Rocha et al., 2011).
Autophagy Can Be Induced by DDRs in DNA Damaged Cells In DNA damaged cells, DDRs can induce autophagy. DNA mismatch repair (MMR) proteins (MLH1 or MSH2) mediate autophagy following chemical DNA mismatch damage through activating p53. A recent study showed that in the p53 family, members p63 and p73 contribute to activation of the autophagy gene network (Kenzelmann Broz et al., 2013). E2F, PARP-1, BNIP3, Skp2, and ATM, all of which are the signal factors of DDRs, are the positive regulators of autophagy by direct and indirect means. The protein factor-arrest 11, which participates in the regulation of DDR by dephosphorylating Rad53, is essential for autophagy induction by dephosphorylating Atg13. Histone deacetylases (HDACs) have important roles in the regulation of DDR and also are involved in autophagy induction. HDAC10 depletion in neuroblastoma cells inhibits autophagic flux and results in accumulation of autophagosomes and lysosomes.
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Autophagy Regulates DDRs by Indirect and Direct Approaches Numerous studies showed that autophagy indirectly inhibits DDRs by eliminating misfolded protein and damaged organelles, especially mitochondria. It was found that autophagy contributes to delay DDR by clearing damaged mitochondria that can result in a post-mitochondrial caspase cascade (Abedin et al., 2007). In the murine model, microtubuleassociated protein 1 small form (MAP1S) elevation in response to alkylating agent diethylnitrosamine treatment enhances autophagy to remove misfolded proteins and damaged organelles that trigger DNA double-strand breaks (DSB) and genome instability. During Myc-driven lymphomagenesis, suppression of mitophagy resulting from hemizygous deletion of Bif-1 leads to the increase of mitochondrial mass, accumulation of DNA damage and the ultimate chromosomal instability (Takahashi et al., 2013). Studies of the role of autophagy on genome stability under metabolic stress also showed a similar phenomenon. Metabolic stresses distinctly impact on cellular genome stability by disturbing DNA synthesis and repair via accumulating misfolded and aggregate-prone proteins, and ROS-generating organelles. In autophagy-competent cells, autophagy clears these accumulations to limit these metabolic stresses. However, defective autophagy sensitizes cells to metabolic stress, and increases DNA damage and further promotes genomic instability (Mathew and White, 2007). A recent study showed that autophagy also can directly impact on DDR. Autophagy is involved in the regulation of HDACs on a number of DDR proteins (including CtIP and Exo1) by degrading acetylated DSB repair enzymes (Robert et al., 2011). Budding yeast cells suffering a single unrepaired DSB trigger hyperactivation of the autophagy pathway, which causes the permanent G2/M arrest.
Autophagy Essential Proteins Regulate DDR by Autophagy-Independent Means Notably, some Atgs can regulate DDR by autophagy-independent means. Independent of its E1-like enzymatic activity, Atg7 can bind to p53 to regulate the transcription of cell cycle inhibitor p21 (CDKN1A). Under metabolic stress, Atg7 (−/−) lead to augmented DNA damage with increased p53-dependent apoptosis (Lee et al., 2012). DNA-damaging drugs can induce the expression of Atg5. Atg5 translocates to the nucleus, and interacts with survivin to induce mitotic catastrophe. Pharmacological inhibition of autophagy cannot suppress Atg5-dependent mitotic catastrophe (Maskey et al., 2013).
AUTOPHAGY AND CANCER STEM CELLS IN CHEMORESISTANCE Recently, the role of cancer stem cells (CSCs) in chemoresistance has been highlighted, since CSCs are resistant to commonly used chemotherapy and responsible for poor prognosis for patients. Autophagy is one key player involved in CSC chemoresistance.
Autophagy Is Essential for Maintenance of the Tumorigenicity of CSCs It was reported that CD133+ liver cancer cells had higher basal autophagy level as indicated by the expression of autophagy-associated genes including Atg5, Atg7, Beclin 1, and
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LC3 and by GFP-LC3 puncta and electron microscopy, compared with their CD133− counterparts. Inhibition of autophagy could significantly impair the clonogenic and sphereforming capacity of CSCs. Furthermore, hypoxia and nutrient starvation (H/S) induced more robustly increased autophagy in CSCs, which could enhance their clonogenic and sphere-forming capacity. An in vivo xenograft model suggested that inhibition of autophagy with CQ greatly suppressed tumorigenicity of CSCs (Song et al., 2013). This evidence leads to the conclusion that autophagy is essential for liver CSC maintenance under both normal and H/S conditions. Consistent with our data, Gong et al. reported that higher expression of Beclin 1 and more robust autophagic flux were observed in mammospheres established from human breast cancers or breast cancer cell lines than in the parental adherent cells. shRNA-mediated silencing of Beclin 1 (shBECN1) could impair mammosphere tumorigenicity as indicated by tumor volume and weight (Gong et al., 2013). The data indicate that autophagy is critical for CSC maintenance and tumor development in nude mice.
Autophagy Contributes to Survival of CSCs in Oxygen and/or NutrientDeprived Tumor Microenvironment The autophagic pathway can be activated under different stimuli, such as endoplasmic reticulum stress, DNA damage, and reactive oxygen species (ROS), thereby eliciting a cytoprotective response that helps cells to overcome those stressful situations. Ischemia and hypoxia typically induce autophagy, which protects cells during times of stress. The tumor microenvironment is characterized by oxygen and nutrient deprivation. Current understanding of autophagy is that it is important for tumorigenesis and can promote the growth of established cancers. The cells that express stem cell surface membrane antigen, which have the capacity to repopulate the tumor and contribute to malignant progression, are highly resistant to the tumor microenvironment. It was reported that autophagy enhances the survival of CSC under the oxygen/nutrient-deprived condition. Under the harsh tumor microenvironment, compared with non-CSCs, CSCs showed higher survival capability and lower apoptosis, denoting that liver CSCs are resistant to loss of oxygen and nutrient supply in hepatocellular cancer (Song et al., 2013). Many studies found increased expression of Atgs in response to autophagy caused by oxygen deficiency and nutrient deprivation (Bampton et al., 2005). The differences in survival and apoptosis may be due to both higher basal and oxygen and nutrient deprivation-induced levels of autophagy in CSCs than in non-CSCs. CSCs contain early and late autophagic vesicles, express Atgs, and the altered expression of Atgs suggests an important role of autophagy in cell survival. Inhibition of the autophagy process can reduce the difference in survival capability and apoptosis. Higher basal autophagy level may make CSCs react to the tumor microenvironment faster. Thus, autophagy inhibitors may make CSCs more sensitive to the tumor microenvironment.
Autophagy Involved in CSC Chemoresistance Despite detection and therapeutic advances, no major improvements in overall survival have been obtained to date. Therefore, new treatment strategies are needed to improve the prognosis of patients. Autophagy is activated in various cancer cells following different anticancer therapies (Guo et al., 2012, 2013). As a prosurvival mechanism, autophagy
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can promote the survival of CSCs by providing catabolites required for repair, by removing toxic substances, and by reducing cytoplasmic acidification. It was reported that a subpopulation of CSCs expressing Cdx1 was more resistant to paclitaxel-induced cytotoxicity than p53-expressing CSCs. Cdx1 exerts a protective role in colon cancer stem cells against chemotherapy through activation of autophagy (Wu et al., 2013). Based upon their previous data indicating that CD133 was involved in autophagosomes, Chen et al. (2013) found that CD133 antibody (CD133mAb) treatment resulted in cell death in hepatoma cell lines, and the antibody effect was mediated by autophagy inhibition, indicating a direct link between autophagy and important molecules regulating stem cell properties. Thus, synergistic autophagy inhibition, either using pharmacological inhibitors or RNA interference of essential autophagy genes, might hold the hope of complete elimination of CSCs.
CONCLUSION Considering the essential role of autophagy in rendering cancer cell resistance to chemotherapy-induced apoptosis and growth inhibition, the development of autophagy inhibition as an adjuvant therapy to existing chemotherapeutics is of great significance in our fight against cancer, to bring benefits for patient survival. The effects of autophagy inhibition by pharmacologic agents or siRNA genetically targeting Atg genes are widely investigated in various in vitro and in vivo models. Furthermore, with encouraging news already reported by some clinical trials, antimalarial and antirheumatic drug CQ and its derivative hydroxychloroquine (HCQ) have been under investigation regarding their effects for the treatment of refractory malignancies in more than 30 ongoing clinical trials (Maes et al., 2013). However, the mechanism underlying the therapeutic effects of autophagy inhibition is still undefined, and whether there exist off-target functions necessitates further studies, which may facilitate the determination of better therapeutic regimens. Since autophagy plays an important role in maintaining cellular homeostasis and genome stability, induction of autophagy is considered to help prevent cancer. Compounds and a variety of dietary factors as well as calorie restriction, which can stimulate autophagy, have been under consideration to be applied in cancer prevention (Maes et al., 2013). Furthermore, in specific genetic backgrounds autophagy induction could lead to autophagic cell death and, in such context, the therapeutic effect of autophagy inhibition will be counterproductive. Thus, more research is required to identify suitable candidates for autophagy inhibition treatment.
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