Autophagy as a molecular target for cancer treatment

Accepted Manuscript Autophagy as a molecular target for cancer treatment

Nur Mehpare Kocaturk, Yunus Akkoc, Cenk Kig, Oznur Bayraktar, Devrim Gozuacik, Ozlem Kutlu PII: DOI: Reference:

S0928-0987(19)30149-6 https://doi.org/10.1016/j.ejps.2019.04.011 PHASCI 4906

To appear in:

European Journal of Pharmaceutical Sciences

Received date: Revised date: Accepted date:

12 September 2018 4 April 2019 5 April 2019

Please cite this article as: N.M. Kocaturk, Y. Akkoc, C. Kig, et al., Autophagy as a molecular target for cancer treatment, European Journal of Pharmaceutical Sciences, https://doi.org/10.1016/j.ejps.2019.04.011

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ACCEPTED MANUSCRIPT Autophagy as a molecular target for cancer treatment Nur Mehpare Kocaturka, Yunus Akkocb, Cenk Kigc,, Oznur Bayraktard, Devrim Gozuacika,b, Ozlem Kutlua,*

a

Sabanci University Nanotechnology Research and Application Center (SUNUM),

b

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Istanbul, 34956, Turkey

Faculty of Engineering and Natural Sciences, Molecular Biology, Genetics and Bioengineer-

ing Program, Sabanci University, Istanbul, 34956, Turkey

Faculty of Medicine, Istanbul Yeni Yuzyil University, Zeytinburnu, 34010, Istanbul, Turkey

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Faculty of Medicine, Department of Medical Biology and Genetic, Okan University, Istan-

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c

*

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Corresponding author: Ozlem Kutlu

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bul, Turkey

Sabanci University Nanotechnology Research and Application Center (SUNUM),

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Orta Mah. Univ. Cad. No: 27, Istanbul, 34956, Turkey.

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E-mail: [email protected]

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Phone: +90 216 4839000 / 2413

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ACCEPTED MANUSCRIPT Abstract Autophagy is an evolutionarily conserved catabolic mechanism, by which eukaryotic cells recycle or degrades internal constituents through membrane-trafficking pathway. Thus, autophagy provides the cells with a sustainable source of biomolecules and energy for the maintenance of homeostasis under stressful conditions such as tumor microenvironment.

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Recent findings revealed a close relationship between autophagy and malignant transformation. However, due to the complex dual role of autophagy in tumor survival or cell death, efforts to develop efficient treatment strategies targeting the autophagy/cancer relation have largely been unsuccessful. Here we review the two-faced role of autophagy in cancer as a

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tumor suppressor or as a pro-oncogenic mechanism. In this sense, we also review the shared regulatory pathways that play a role in autophagy and malignant transformation. Finally,

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anti-cancer therapeutic agents used as either inhibitors or inducers of autophagy have been

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discussed.

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Keywords: Autophagy, Cancer, Therapeutic agents

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ACCEPTED MANUSCRIPT

1. Introduction Autophagy is a catabolic process in which cytoplasmic materials are directed to the lysosomes for degradation. This process is evolutionarily conserved from yeast to man and its

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activity is required for maintaining cellular homeostasis through elimination of dysfunctional organelles, protein aggregates or even long-lived proteins. So far, three main classes of autophagy have been identified: Macroautophagy, microautophagy and chaperon-mediated autophagy (CMA). Macroautophagy (autophagy herein) is the main pathway that is devided into bulk and selective autophagy according to the specificity of targeted cytoplasmic con-

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stituents. In bulk autophagy, degradation targets are mainly wrapped within a double-

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membraned vesicle (autophagosome) as portions of cytoplasm in a non-selective manner. On the other hand, in selective autophagy particular substrate such as mitochondria (Okamo-

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to et al., 2009), peroxisomes (Till et al., 2012), lysosomes (Hung et al., 2013), ER (Khaminets et al., 2015), ribosomes (An and Harper, 2018), lipid droplets (Onal et al., 2017), patho-

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genic intracellular invaders (Wileman, 2013) and even certain free proteins and RNAs (Huang et al., 2015) are targeted into the autophagosome. In this review, we mainly focus on

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autophagy and other major classes, CMA and microautophagy were discussed in detailed

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elsewhere (Kaushik and Cuervo, 2018; Oku and Sakai, 2018). The ability to recycle macromolecules through autophagy gives cells an advantage for survival under stressful conditions such as nutrient starvation, oxidative stress, hypoxia, ER stress, metabolic stress etc. (Piacentini and Kroemer, 2015). Moreover, selective autophagy allows cells to control number of the organelles based on the requirement, eliminating dysfunctional compartments and disposing of pathogens by combining the ubiquitin-proteasome system (UPS) and autophagic machinery (Kocaturk and Gozuacik, 2018). However, under certain conditions excess or deregulated activity of autophagy may also lead cell death.

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ACCEPTED MANUSCRIPT Whether autophagy is an executioner or a savior is still a matter of debate and it is often determined in a context- and cell type-dependent manner (Liu et al., 2016). In order to survive under stressful conditions within tumor such as hypoxia and/or nutrient deprivation or oxidative stress, cancer cells frequently exploit autophagy (Kenific and Debnath, 2015). Additionally, tumor cells could benefit from autophagy for adaptation

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to metastasis for withstanding the environmental stress they face during the several steps of metastasis including migration into the systemic circulation, adherence to the vessel walls, extravasation and colonization (Su et al., 2015). Thus, recycling of cytoplasmic materials by autophagy provides continuous supply of energy as well as essential ingredients for cancer

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cells to survive (Su et al., 2015) and promotes metastatic reocurrence of tumors (Vera-

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Ramirez et al., 2018). 2. Molecular mechanisms of autophagy

Autophagic process is initiated by the formation of double-membrane vesicles known as

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autophagosomes. Various cargos are engulfed into autophagosome and autophagosome

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eventually fuses with lysosomes that forms autolysosomes. (Lamb et al., 2013). Engulfed materials were degraded by the action of lysosomal hydrolases and newly generated building

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blocks (e.g., amino acids from protein degradation) are transferred back to cytosol for reuse

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(Fig. 1). A series of stimuli, including amino acid deprivation, serum starvation and growth factor deprivation, hypoxia, exposure to various chemicals and stress conditions are capable of activating autophagy. Genetic studies in yeast provided initial discoveries of autophagy-related (ATG) genes and enlightened the details of molecular signaling pathway of autophagic process (Nakatogawa et al., 2007). The autophagic pathway can be divided into several different phases: Initiation, nucleation, maturation, fusion and degradation (Fig.1). 2.1. Initiation 3

ACCEPTED MANUSCRIPT The target of rapamycin, TOR (mTOR in mammals), is an evolutionarily conserved serine/threonine kinase responsible for conveying a number of autophagy stimulating signals. In mammals, mTOR exists as two different complexes: mTOR complex1 (mTORC1) and mTOR complex2 (mTORC2). mTOR complexes constitute a critical node for the integration of signaling pathways that regulate cellular energy homeostasis by coordinating anabolic

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and catabolic processes (Kroemer et al., 2010). PKB-AKT pathway can activate mTORC1 and suppresses autophagy (Dan et al., 2014; Zalckvar et al., 2009) (Fig.1A). In contrast, autophagy is activated by another kinase, AMP-activated protein kinase (AMPK), which has crucial role in sensing cellular energy and ATP levels (Garcia and Shaw, 2017; Xiao et al.,

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2011). Following decrease in ATP, AMPK becomes activated through direct interaction with ADP or ATP resulting a conformational change. AMPK activation is also controlled by

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the two upstream kinases: LKB1 and calcium/calmodulin-dependent protein kinase kinase-β (CaMKKβ) (Hawley et al., 2005; Shaw et al., 2004). There has been cross-regulation be-

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tween AMPK and mTOR activity. Low energy status activates AMPK, whereas this activa-

2008; Inoki et al., 2003).

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tion leads inhibition of mTOR due to phosphorylation of TSC2 and RAPTOR (Gwinn et al.,

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Under nutrient-rich conditions, mTORC1 complex suppresses autophagy by inactivation of ULK1/2 complex, which composed of ULK1 or ULK2 kinase, ATG13, FIP200 and

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ATG101. In response to nutrient deprivation, ULK1/2 complex is activated by dissociation of mTORC1 which in turn activates autophagy through class III phosphatidylinositol 3kinase (PI3K) complex (Chen and Klionsky, 2011; Hosokawa et al., 2009). 2.2. Nucleation A class III PI3K complex is mainly responsible for the nucleation of the autophagic membranes. Several proteins such as VPS34, Beclin-1, AMBRA1 and mATG9 were identified as novel regulator proteins in phagophore formation (Feng et al., 2016; Mehrpour et al., 2010;

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ACCEPTED MANUSCRIPT Papinski and Kraft, 2014; Park et al., 2016; Petherick et al., 2015; Russell et al., 2013). Beclin-1 is one of the key protein in membrane nucleation and its interaction with BCL2 inhibits autophagy (Fig. 1B). Conversely, disruption of this interaction allows Beclin-1 to bind with lipid kinase VPS34 and promote membrane nucleation (Pattingre et al., 2005). Alternatively, Beclin-1 differentially modulates membrane formation through interaction with dif-

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ferent mediators such as UVRAG (Liang et al., 2008), RUBICON, ATG14L (Matsunaga et al., 2009), AMBRA1 (Yazdankhah et al., 2014) and VMP1 (Molejon et al., 2013a, 2013b). VPS34-mediated enzymatic generation of phosphatidylinositol 3-phosphate (PtdIns3P) provides a platform for phosphatidylinositol 3-phosphate (PI3P)-binding domain-containing

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autophagy proteins, including WIPI1-4 and DFCP1 (Mauthe et al., 2011; Mercer et al.,

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2018). 2.3. Maturation

Accumulation of PI3P-binding domain containing proteins at the membrane nucleation site

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resulted in binding of additional ATGs, which are required for elongation and closure of the

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autophagosome membrane. The two ubiquitin-like conjugation system regulate elongation of the isolation membrane (Fig. 1C). In one system, E1- and E2-like actions of ATG7 and

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ATG10 catalyze the covalent conjugation of ATG12 to the ATG5 protein. ATG5-12 conju-

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gation is followed by the recruitment of ATG16L1 giving rise to the formation of ATG12ATG5-ATG16L1 complex, which serves as an E3-like function to the second ubiquitin-like conjugation system (Mizushima et al., 2011; Shpilka et al., 2011; Tsuboyama et al., 2016). The second system consists of the conjugation of LC3 protein to a lipid molecule, phosphatidylethanolamine (PE) (Tanida et al., 2004). LC3 precursor protein is cleaved by ATG4 and this cleavage allows exposure of the glycine residue from its carboxy-terminus that lead to PE conjugation. E1-like ATG7 and E2-like ATG3 proteins possess the LC3-PE

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ACCEPTED MANUSCRIPT conjugation which also known as LC3-II (an established marker for autophagosomes) (Hanada et al., 2007; Nakatogawa et al., 2007) (Fig. 1C).

2.4. Fusion and degradation After formation of autophagosome membrane, autophagic vesicles are transported to lyso-

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somes for degradation. Autophagosome-associated LC3 proteins become delipidated and recycled prior to fusion (Kriegenburg et al., 2018; Nakamura and Yoshimori, 2017). Several SNARE proteins, including STX17 and WAMP8 and lysosomal integral protein LAMP2 and RAB proteins play critical roles in autophaosome-lysosome fusion (Jager, 2004;

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Tanaka et al., 2000). Finally, autolysosome is formed by fusion of autophagosomes with lysosomes where cargo is degraded by the lysosomal proteases (Fig.1D). Thereafter, de-

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gredation products such as amino acids, fatty acids are redirected to cytosol for further reuse

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in various metabolic processes (Panda et al., 2015).

3. Autophagy-mediated cancer regulation

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Beth Levine’s group suggested a direct link between autophagy and cancer for the first time

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in 1999. They showed that monoallelic BECN1/ATG6 gene deletions in human cells might contribute to malignancies both in vitro (Liang et al., 1999) and in in vivo (Qu et al., 2003).

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Currently, a vast number of studies indicate that ATGs and the related pathways can crosstalk with oncogenes and/or tumor suppressors. Indeed, accumulated data support the notion that the role of autophagy in malignant transformation is complicated and may have opposite consequences in a context and cell-type dependent manner (Galluzzi et al., 2015b).

3.1. Autophagy as a tumor suppressor mechanism

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ACCEPTED MANUSCRIPT Autophagy has been implicated as a favorable mechanism for suppression of cancer formation at multiple stages through its established roles in preservation of genomic stability; elimination of endogenous sources of reactive oxygen species (ROS); the maintenance of bioenergetic functions; degradation of oncogenic proteins and induction of immunresponse mechanisms against malignant transformations (Galluzzi et al., 2015).

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In addition to Beclin-1, several other autophagy proteins have been described with their suppressive effects on tumorigenesis. For instance, it has been proposed that ATG4C deficiency associated with increased tumorigenesis in mice (Marino et al., 2007). Similarly, ATG5 deletion in mice induced benign liver tumor formation (Takamura et al., 2011a).

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Additionally, mıutations in ATG2B, ATG4, ATG5, ATG12 and ATG9B were frequently observed in human cancers suggesting that autophagy plays a suppressive role in malignant

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transformation at several steps of tumorigenesis (An et al., 2011; Kang et al., 2009; Kim et

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al., 2011). Tumor suppressive function of autophagy is summarized in Fig. 2.

3.1.1.Autophagy removes oncogenic proteins and maintains genomic stability

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Several proteins that are involved in oncogenesis such as the mutant form of p53

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(Choudhury et al., 2013), p62 (Duran et al., 2008), PML-RARA (Isakson et al., 2010) and BCR-ABL1 (Goussetis et al., 2012) have been found to be degraded by autophagy.

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p53 is activated in response to a variety of stress conditions, including DNA damage, oxidative stress, replicative stress, genomic instability etc. (Meek, 2015). In cancer cells, the proteasomal degradation of the mutant p53 protein was abolished but instead it was degraded by autophagy. Indeed, glucose deprivation induced autophagy and regulated acetylation of the mutant p53 that subsequently led the autophagic clearance of protein (Rodriguez et al., 2012). Correlating with anti-tumor effect of autophagy on p53, in another study it has been shown that an anti-tumor agent selenite induced ROS and inhibited autophagy in NB4

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ACCEPTED MANUSCRIPT cells (Shi et al., 2014). Furhermore, the same group also showed that selenite-induced ROS regulated autophagic activity through downregulation of ULK1 expression was also affected by phosphorylation status of p53. In fact, p70S6K-mediated phosphorylation of p53 was responsible for the decreased level of ULK1 which was attenuated with selenite (Ci et al., 2014). Moreover, it has been also showed that chaperone-mediated autophagy (CMA) could

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degrade mutant form of p53 in nonproliferating tumor cells under the conditions in which autophagy and proteasomal degradation is inhibited (Vakifahmetoglu-Norberg et al., 2013). Additionally, autophagy was also contributed to the p53-mediated senescence through degradation of inhibitory isoforms of p53 (Horikawa et al., 2014). On the other hand, Beclin-1

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was also involved in the autophagic control of cellular p53 level. An inhibitor of autophagy, Spautin-1 deregulated the formation of VPS34 complex acting over the two ubiquitin-

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specific peptidases USP10 and USP13 that modifies Beclin-1 ubiquitylation pattern. Hence, USP10 also regulates p53 ubiquitylation status and is responsible for the p53 stability,

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Spautin-1-mediated autophagy inhibition led to degradation of p53 (Liu et al., 2011). Furthermore, it has been also observed that Beclin-1 interacted with p53 through its BH3 do-

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main and this interaction was critical for its UPS-mediated degradation. Degradation of Be-

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clin-1 subsequently decreased autophagic activity and have effect on the determination of embryonal carcinoma cellular fate (Tripathi et al., 2014).

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The first identified autophagy receptor protein p62 (also known as sequestosome-1, SQSTM1) is a signaling scaffold within the cytoplasm and its expression is generally upregulated in human cancers (Moscat et al., 2007). Target selectivity by p62 during selective and non-selective autophagy governs cellular homeostasis by preventing ER-stress and oxidative stress (Moscat et al., 2016). The critical role of p62 in tumorigenesis was explored in different types of cancer such as premalignant liver diseases as well as hepatocellular carcinoma (HCC). In autophagy deficient cases, p62 was shown to be associated with the formation of

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ACCEPTED MANUSCRIPT benign adenomas (Takamura et al., 2011b). Diethylnitrosamine (DEN) induced carcinogenic activity of p62 and accelerated HCC progression (Umemura et al., 2016). Critical tumor promoting role of p62 in HCC occurred over NRF2 and mTORC1/c-Myc signaling (Umemura et al., 2016). Therefore, degradation of p62 during autophagy is critical for the restriction of p62-linked tumorigenesis.

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Promyelocytic leukemia (PML)/retinoic acid receptor alpha (RARA) is a critical fusion oncoprotein responsible for the progression of acute PML (Grignani et al., 1998). It has been discovered that PML-RARA was degraded by autophagy (Isakson et al., 2010). PMLRARA-targeting drugs such as retinoic acid and arsenic trioxide (Zhu et al., 2001) also in-

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duced cellular autophagy level suggesting that the effect of these drugs on oncoproteins is autophagy-dependent (Isakson et al., 2010). In line with this, another critical oncogenic pro-

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tein BCR-ABL1 involved in leukemia found to be degraded by autophagy (Goussetis et al., 2012). Arsenic trioxide enhanced both cellular autophagic activity and the degradation of

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BCR-ABL1 which was reversed with autophagy inhibitors (Goussetis et al., 2012). Furthermore, the physical interaction between p62 receptor and BCR-ABL1 could be associated

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with selective removal of the oncoproteins supproting the repressive role of autophagy in

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tumorigenesis.

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3.1.2. Autophagy regulates cell proliferation and promotes death mechanisms Programmed cell death (PCD) mechanisms including apoptosis, necroptosis and autophagy are amongst the first line barriers, which prevent survival and proliferation of malignant cells (Kroemer and Levine, 2008; Liu and Levine, 2015). The high level of genetic instability in cancer cells allows accumulation of spontaneous mutations and epigenetic modifications, which in turn rendered cells resistant to PCD mechanisms (Ferguson et al., 2015).

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ACCEPTED MANUSCRIPT Several factors such as energy status (AMP/ATP levels), DNA damage or various stress conditions determine the fate of a cell regarding to survival, death or senescence (Su et al., 2015). Despite the widely accepted survival-supportive role of autophagy, increased autophagosome formation may also lead to apoptotic cell death (Eberhart et al., 2013; Gozuacik and Kimchi, 2004). Therefore, autophagy can serve as a cell death mechanism and

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contribute with apoptosis in tumorigenesis. Recent findings also suggest an inter-connected network of components involved in regulation of apoptosis and autophagy (Oral et al., 2016; Fulda and Koegel, 2015). Consequently, data indicates that autophagic cell death function as a tumor-supressor mechanism by coordinative action with other PCD mechanisms.

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A member of anti-apoptotic proteins BCL-2 is highly expressed in various cancers and associated with the resistance to chemo- and radio-therapeutic approaches (Huang,

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2000). BCL-2 is identified as negative regulator of Beclin-1 (Oberstein et al., 2007) and therefore, BCL-2 and Beclin-1 interaction is one of the critical determinant for autophagy

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and/or apoptosis activation in various cancer cells (Marquez and Xu, 2012; Akar et al., 2008; Lima et al., 2004). For example, overexpression of Beclin-1 in human laryngeal

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squamous carcinoma cells cause significant decrease in cell proliferation and promotes

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apoptotic cell death (Wan B., 2018). The inhibitory role of Beclin-1 on cell proliferation was shown in different types of cancer including, tongue squamous cell carcinoma (Hu et

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al., 2016), breast cancer (Wang et al., 2015), cervix cancer (Sun et al., 2011), lung cancer (Wang W., 2013), glioblastoma (X. Huang et al., 2014), squamous cell carcinoma cell lines (Weng et al., 2014), HCC (Zhao et al., 2014) colorectal cancer (Liu et al., 2017) and pancreas cancer (X. Li et al., 2013). Tumor suppressive function of Beclin-1 is not limited to itself but also supported by the identification of its mediators involved in tumorigenesis. A major Beclin-1 positive mediator, UVRAG is mutated in various human cancer cell lines (Goi et al., 2003; Kim et al.,

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ACCEPTED MANUSCRIPT 2008). UVRAG-mediated activation of BECLIN1-PI (3) KC3 complex was shown to promote autophagy contributing to suppression of cell proliferation and tumorigenicity in human colon cancer cells (Liang et al., 2006). JNK (c-Jun N-terminal kinase) is a member of the MAPK (mitogen-activated protein kinase) family that regulates a wide range of biological processes including tumorigenesis.

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JNK1-mediated phosphorylation of BCL-2 stimulated starvation-induced autophagy through disruption of BCL-2/Beclin-1 interaction (Wei et al., 2008). As an additional connection, autophagy is inhibited by the ER-localized BCL-2 through IRE1/JNK/Beclin-1 in breast cancer cells (Cheng et al., 2014). Moreover, Neuronal JNK1 was able to suppress autophagy

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by blocking FOXO1-mediated transcriptional activation of BNIP3 highlighting the cellular requirements of autophagy to survive (Xu et al., 2011). In addition to JNK1, DAPK also

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phosphorylated Beclin-1 and disrupted the interaction of Beclin-1 with BCL-2 and BCL-XL, which in turn led to stimulation of autophagy (Zalckvar et al., 2009). DAPK1, a

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Ca2+/Calmodulin-dependent Ser/Thr kinase, suppressed tumor growth and metastasis by promoting apoptosis and autophagy (Bialik and Kimchi, 2006; Huang et al., 2014).

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Another critical Ser/Thr kinase, ULK1 (Unc51-like kinase, hATG1), is phosphory-

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lated and negatively regulated by mTORC1 mediates autophagy activation therefore inhibited cell proliferation in response to nutrient deprivation (Jung et al., 2011). Transcriptional

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activation of ULK1/2 by p53 resulted in elevated autophagic activity leading to autophagic cell death (Gao et al., 2011). ULK1/2 were shown to be downregulated in all grades of glioma suggesting that inhibition of autophagy by ULK1/2 downregulation is essential for astrocyte transformation and tumor progression (Shukla et al., 2014). These findings suggested that ULK1/2 upregulation contributed to tumor suppression activity in mammalian cells through activation of autophagic cell death.

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ACCEPTED MANUSCRIPT Since autophagy has widespread influence on a number of biological pathways, this mechanism could inhibit cell proliferation by controlling cell cycle regulation. For example, enhanced autophagic activity was associated with cell ceycle arrest at G2/M phase and induced death in pancreas cancer cells (Zhu and Bu, 2017). Correlatively, metformin-induced autophagic activity resulted in G0/G1 cell cycle arrest and inhibited cell proliferation in my-

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eloma by targeting AMPK and mTORC (Zhu and Bu, 2017). As another example, elevated autophagic activity resulted in cell cycle arrest at G0/G1 phase and subsequent cell death in cervical cancer (Gao et al., 2018). Similarly, a natural product magnolin activated autophagy, caused cell cycle arrest through interefering LIF/Stat-3/Mcl-1 axiss and subsequently

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suppressed cell growth (Yu et al., 2018). Another natural product curcumin restricted tumor growth by regulating senescence and autophagy link in vivo and in vitro in colon

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cancer (Mosieniak et al., 2012). Autophagy has also been proposed to contribute to oncogene-induced senescence in cell-type dependent manner (Vicencio et al., 2008). For exam-

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ple, both autophagy and senescence were shown to be able to suppress self-renewal capacity in breast tumor cells exposed to DNA damage-inducing agent, doxorubicin (Di et al., 2016).

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Silencing either ATG5 or ATG7 suppressed oncogene-induced senescence in primary hu-

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man melanocytes or human diploid fibroblasts (HDFs) (Liu et al., 2014; Young et al., 2009). Accordingly, transient overexpression of ULK3 reduced the proliferative potential of HDFs

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(Young et al., 2009). Interestingly, chemical or genetic inhibition of autophagy prevented senescence in HDFs (Horikawa et al., 2014). Another link between autophagy, cell proliferation and cell death mechanisms maintained by p53 protein. In mammalian cells, p53 is found in two different form: Cytoplasmic and nuclear p53. Cytoplasmic p53 translocated to nucleus and regulate the transcription of genes involved in DNA repair, cell cycle arrest and apoptosis (Meek, 2015). Not only autophagy controlled cellular p53 level but also p53 regulated cellular autophagic activity at

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ACCEPTED MANUSCRIPT transcriptional level. Cytoplasmic form of p53 inhibited autophagy by mTORC1 activation and p53 depletion was able to induce autophagy in vivo (Tasdemir et al., 2008a). The ability of p53 to repress autophagy was linked to the localization of p53 on the ER (Tasdemir et al., 2008a, 2008b). In the nucleus, p53 activated AMPK, inhibited mTOR and subsequently initiated autophagy. Accumulating data suggested that p53 promotes autophagy through activa-

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tion of DAPK, DNA damage regulated autophagy modulator 1 (DRAM1), proapoptotic BCL-2 proteins (e.g., BAD, BAX, BNIP3, and PUMA), Sestrin1/2, and TSC2 (Tasdemir et al., 2008b). In response to genotoxic stress, p53 could transcriptionally activate ULK1 and ULK2, which in turn led to elevated autophagy level and contributed to cell death (Gao et

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al., 2011). Furthermore, in the absence of growth factors or in response to stress conditions, RB1 prevented cell cycle progression through inhibition of E2F transcription factor family

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members. RB-E2F pathway has been proposed to regulate autophagic response (Jiang et al., 2010) and E2F1 induced the expression of components of the autophagic machinery, includ-

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ing ATG1, ATG5 and LC3 (Polager et al., 2008).

A better understanding of the role of autophagy in regulation of cell proliferation, cell

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cycle arrest and cell death in tumor cells improve the potential for developing novel thera-

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peutic strategies against malignancies.

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3.1.3. Autophagy induces stress-related responses Expression level of the cytoplasmic chaperone protein and signaling scaffold p62 is frequently found to be upregulated in human cancers (Liu and Ryan, 2012; Umemura et al., 2016). Accumulation of p62 correlated with increased endoplasmic reticulum (ER) stress and DNA damage in cancer cells (Duran et al., 2008; Moscat et al., 2007). Additionally, defects in the nuclear factor kappa B (NF-B) and antioxidant nuclear factor erythroid 2related factor 2 (NRF2, also known as NFE2L2) regulatory pathways were also found to be

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ACCEPTED MANUSCRIPT associated with cellular p62 levels (Duran et al., 2008; Inami et al., 2011). Both in normal and cancer cells, p62 acts as an adaptor protein linking LC3-associated autophagic membranes with ubiquitin decorated misfolded proteins thereby mediating clearance of targets, including oncogenes. In this context, suppression of autophagy lead to p62 accumulation, which in turn

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contributed to oncogenesis through increased levels of ER-stress and DNA-damage-stress (Moscat and Diaz-Meco, 2009). Accordingly, accumulation of p62 was observed in the benign tumors developed in ATG5 or ATG7 depleted mouse models (Takamura et al., 2011a). Moreover, loss of p62 in these mice was found to suppress tumor growth, suggesting a cor-

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relation between p62 accumulation and adenoma formation (Liu and Ryan, 2012; Takamura

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et al., 2011a).

3.1.4. Autophagy induces immune-response mechanisms

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Autophagy contributes to innate immunity through facilitating several cellular responses including, cytokine production and phagocytosis. Autophagy participated in adaptive im-

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munity through its antigen presentation potential (Puleston et al., 2014; Puleston and Simon,

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2014). Therefore autophagy has been suggested as a regulator of immune responses to combat with malignancies (Ma et al., 2013). Some dying malignant cells recruited antigen-

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presenting cells (APCs) and other cellular components of the immune system that may trigger both innate and/or adaptive antitumor immune responses (Gajewski et al., 2013). In this setting, defects in autophagy may supress recognition and therefore prevent elimination of pre-malignant and malignant cells. Furthermore, autophagic response also limited tumorinduced inflammation through clearance of inflammasomes (Nakahira et al., 2011) which may contain factors such as pro-inflammatory interleukins and damaged mitochondria (Galluzzi et al., 2015b).

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ACCEPTED MANUSCRIPT Viruses are considered to be responsible from the 10%–15% of human cancers worldwide. Mainly, viral infections are associated with increased genomic instability due to induction of changes at cellular, genetic and epigenetic levels resulting in tumor formation and progression (Chen et al., 2014). A growing number of pathogenic infections promote carcinogenesis including, hepatitis B virus (hepatocellular carcinoma) (Poh et al., 2015),

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human herpesvirus 8 (linked to Kaposi’s sarcoma) (Memar et al., 1995), human papillomavirus (cervical carcinoma) (Rijkaart et al., 2012), Epstein–Barr virus and Helicobacter pylori (associated with gastric carcinoma) (Souza et al., 2018), Streptococcus bovis (colorectal carcinoma) (Ellmerich et al., 2000; Krishnan and Eslick, 2014), Salmonella enterica (gastro-

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intestinal cancers) (Mughini-Gras et al., 2018), Chlamydia pneumoniae (lung cancer) (Chaturvedi et al., 2010), human T-cell lymphotropic virus (HTLV-1) (leukemia/lymphoma)

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(Kataoka et al., 2015) and SV40-Polyomavirus of the rhesus macaque (brain/osteosarcoma) (Mazzoni et al., 2015). Due to the involvement of a large population of pathogens in the

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tumorigenesis, it is emerging to control the immune responses against invaders (Deretic et al., 2013). Clearance of these pathogens through selective autophagy mechanism termed as

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xenophagy. Xenophagy constitutes the first line of defense against infection and stimulates

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pathogen-specific adaptive immune response mechanisms (Gao et al., 2017; Mao and Klionsky, 2017; Zhao et al., 2018). Xenophagy-assisted removal of viruses could tightly be

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associated with innate immune and acquired immune responses. Viruses are capable of escaping from innate immunity by encoding different genes associated with inhibition of apoptosis, autophagy and necroptosis. Eventhough targeting viruses for cancer treatment bears some limitations such as most of the findings obtained from in vitro, cell culture experiments; a selective type of autophagy xenophagy provides great potential for viralinfection associated cancers.

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ACCEPTED MANUSCRIPT 3.1.5. Autophagy regulates stem cell maintenance The ability of the stem cells to self-renew and differentiate into several types of cells are bearing great importance for oncogenic processes as well as development and tissue renewal. Tumor-initiating cells share some characteristics with stem cells and are capable of selfrenewal and differentiation (Visvader and Lindeman, 2012). Several lines of evidence indi-

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cated that autophagy functions as an important mechanism in quality control and maintenance of cellular homeostasis in stem cells (Guan et al., 2013). Depletion of ATG7 in murine hematopoietic stem cells (HSCs) increased neoplastic features by altering the number of bone marrow progenitor cells (Mortensen et al., 2011). In another study, tissue-specific dele-

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tion of FIP200 correlated with severe anemia and prenatal lethality in hematopoietic stem cells (F. Liu et al., 2010). In line with this, FIP200 deletion in murine neuronal stem cells

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(NSCs) interfered with postnatal neuronal differentiation (Wang et al., 2013). As another example of autophagy-mediated regulation of stem cells, it has been reported that autophag-

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ic activity supported breast cancer stem cell maintenance through regulation of IL6 secretion

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(Maycotte et al., 2015).

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3.2. Autophagy as a pro-oncogenic mechanism In addition to its tumor suppressive role, autophagy also contributes to malignant transfor-

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mation and/or metastatic cascade by supporting cancer cells under stress conditions (e.g. exposure to metabolic, hypoxic, genotoxic, and oxidative stress) or tumor microenvironment (e.g. survival in the circulatory system, oxygen and glucose deprivation in solid tumors). Evidence also suggested that autophagy provides resistance to cancer cells against chemo- / radio-therapies and cell death. Pro-oncogenic function of autophagy is summarized in Fig. 3.

3.2.1. Autophagy supplies nutrients and energy to cancer cells

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ACCEPTED MANUSCRIPT In cells, it is well established that glucose is primary nutrient source for energy production and constant glucose supply is required for ATP production. In cancer cells, increased aerobic glycolysis was first reported by Warburg in the 1920s. Conversion of glucose to lactate in aerobic conditions also results in microenvironmental acidosis therefore cancer cells must adopt resistance to acid-induced cell toxicity. Malignant cells with high glycolytic capacity

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also displayed resistance to acidosis and therefore gain growth advantage over the normal cells for unconstrained proliferation, invasion and tumorigenesis (Gatenby and Gillies, 2004). Malignant transformation is generally accompanied with metabolic changes, including elevated glucose uptake to sustain anabolic reactions and antioxidant defense and in-

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creased mitochondrial respiration to supply high-energy demand and several amino acids (Hanahan and Weinberg, 2011).

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The PI3K-AKT-mTOR pathway plays a major role in regulation of aerobic glycolysis in cancer cells (Makinoshima et al., 2015). Inhibition of PI3K limited glucose uptake and gly-

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colysis by blocking GLUT1 function (Barnes et al., 2005). AKT and c-MYC, positive regulators of essential glycolytic genes have shown to possess differential and complementary

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effects in driving aerobic glycolysis (Fan et al., 2010). Additionally, c-MYC was also impli-

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cated in regulation of glutamine metabolism under the control of SIRT1 in order to reach the high demand for energy generation and biosynthesis in cancer cells (Ren et al., 2017). In

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order to meet with altered metabolic requirements of cancer cells, autophagy maintained a critical role in these adaptation period by providing required energy and biomolecules through recycling of molecules and/or organelles (Galluzzi et al., 2015). Normal fibroblast cells neighboring tumor cells acquire a phenotype known as the cancer-associated fibroblasts (CAFs), where autophagic activity supported the high demand of cancer cells for the energy and nutrient support (Martinez-Outschoorn et al., 2011). Injection of CAFs overexpressing pro-autophagic molecules together with cancer cells into mice

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ACCEPTED MANUSCRIPT promoted tumor growth and favored for lung metastasis (Yang et al., 2015). Furthermore, autophagic activity in apoptosis-deficient tumor cells was correlated with the survival of cancer cells under stress conditions (Degenhardt et al., 2006). Interestingly, autophagy has also been implicated in the metabolic control of hematopoietic stem cells during throphic

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factor deprivation (Yang et al., 2015).

3.2.2. Autophagy mediates adaptation to hypoxia, oxidative stress and DNA damage Accumulating evidences suggest that autophagy promote tumor cell survival in majority of the tumors that grow under hypoxic conditions (Vaupel and Mayer, 2007). Insufficient sup-

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ply of oxygen from the vasculature to the solid tumor mass resulted in local hypoxic (oxygen < 3%) and anoxic (oxygen < 0.1%) conditions inside the tumor. Compromised mi-

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crovessel function, limited oxygen diffusion rate due to increased and condensed structure of tumor induced hypoxic conditions inside the tumor tissue (Qiu et al., 2017). Hypoxia-

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induced autophagy mainly depends on hypoxia-inducible factors (HIFs), a family of proteins predominantly detected when oxygen level is below 5% (Majmundar et al., 2010). HIF-1α

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activation further promoted autophagy through BNIP3 and BNIP3L under hypoxic condi-

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tions. The atypical BH3 domains of these proteins have been proposed to induce autophagy by disrupting the BCL-2-Beclin-1 complex without inducing cell death (Majmundar et al.,

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2010). Therefore, this mechanism is considered as a survival mechanism promoting tumor progression (Zhang and Ney, 2009). HIF-1α and HIF-2α are targeted to proteosomal degradation by the E3 ubiquitin protein ligase VHL in an oxygen-dependent reaction are stabilized in hypoxic conditions (Majmundar et al., 2010). The HIF are also implicated in increased anaerobic metabolic flux and survival through upregulation of GLUT1 and glycolytic enzymes (Altman and Rathmell, 2012; Chen et al., 2001). Interestingly, various VHL mutations in renal cell carci-

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ACCEPTED MANUSCRIPT nomas (Shuin et al., 1994) led to accumulation of HIF-1α irrespective of oxygen concentration and transactivation of genes involved in bioenergetic metabolism and angiogenesis (Maxwell et al., 1999). VHL inhibited autophagy through MIR204 upregulation, which directly targets LC3B in renal clear cell carcinoma (Mikhaylova et al., 2012). On the other hand, knockdown of LC3C in VHL-expressing cells could successfully induce tumor for-

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mation (Von Muhlinen et al., 2013). Additionally, through inhibition of HIF, VHL induced LC3C expression possessing tumor-suppressing autophagic activity (Galluzzi et al., 2015; Von Muhlinen et al., 2013).

BNIP3 and BNIP3L protein levels were also under the control of FOXO3 transcrip-

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tion factor that induced autophagy (Mammucari et al., 2007). Interestingly, FOXO3Amediated activation of autophagy was also shown to promote survival of hematopoietic stem

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cells under nutrient-deprived conditions (Warr et al., 2013). BNIP3L, is often found on the outer mitochondrial membrane, modulating elimination of mitochondria by autophagy (mi-

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tophagy) (Zhang and Ney, 2009). As well as taking a part in the turnover of dysfunctional mitochondria by mitophagy also promoted reduction of overall mitochondrial mass in re-

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sponse to hypoxia and nutrient starvation. Removal of mitochondria under unfavorable con-

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ditions helped reducing ROS production, saved oxygen and nutrients from being consumed inefficiently, thereby promoting cellular survival under hypoxic conditions (Chourasia et al.,

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2015). The expression level of the essential autophagy genes LC3 and ATG5 was found to be upregulated through the transcription factors ATF4 and CHOP, which are regulated by PERK (Rouschop et al., 2010). In this context, inhibition of autophagy sensitized human tumor cells to hypoxia suggesting that autophagy had a role in tumor survival under hypoxic conditions. Reactive oxygen (ROS) and reactive nitrogen species (RNS) are one of the major sources of DNA damage (Wiseman and Halliwell, 1996). ROS and RNS modify nucleic

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ACCEPTED MANUSCRIPT acids directly or indirectly generating different types of DNA lesions including single-strand break (SSB), double-strand break (DSB), oxidized bases, abasic sites, and DNA–protein crosslinks (Cooke et al., 2003). ROS and RNS are also contributed to damage in mitochondrial DNA (mtDNA) integrity and function. For example, damaged mtDNA affected the transcription of mtDNA-coded proteins and RNAs that function in the mitochondrial res-

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piratory chain (except Complex II) (Roos et al., 2013). Then, damaged mtDNA induces accumulation of more dysfunctional mitochondria, which produce a high rate of ROS, leading to further mitochondrial impairment and cell death (Filomeni et al., 2015). In fact, this effect further enhanced in majority of the cancers carrying p53 deletions due to the lack of ability

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to repair DNA damage efficiently. The expression of autophagy-related genes was also regulated in a p53-dependent manner in response to DNA damage. These include both upstream

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regulators of autophagy (e.g., PTEN, TSC2, β1, β2 and γ subunits of AMPK) and the proteins that are involved in autophagosome formation (e.g., ULK1, UVRAG, ATG2, 4, 7, 10)

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(Füllgrabe et al., 2016). Although, DNA damage promote tumorigenesis at a certain degree, due to the accumulation of oncogenic mutations, excessive DNA damage also caused cell

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death. Therefore, based on the well-established role of autophagy in intracellular homeosta-

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sis and its functions in DNA damage response, autophagy also plays a key role in protecting cancer cells from the lethal effects of DNA damage (Chan et al., 2018).

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Activation of AMPK through mitochondria-derived ROS suggests a direct link between the oxidative stress response and autophagy activation. Indeed, oxidation of glutathione was able to induce autophagy even in the absence of any autophagic stimulus (Desideri et al., 2012). Oxidation of Cys residues post-translationally suggested an important mechanism in terms of both structure and function of the modified protein (de Duve et al., 1955). Therefore, proteins containing oxidized residues are readily responsive to oxidative stres conditions. Several autophagy regulating proteins such as the ATG7-ATG3 and ATG7-ATG10

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ACCEPTED MANUSCRIPT systems, some members of RAB GTPase (e.g., RAB33b), and the phosphatase and tensin homologue deleted (PTEN) (Filomeni et al., 2015) are found to act as Cys residues. Furthermore, p62 was shown to contain a cysteine-rich zinc-finger motif, which could be regulated by redox and protein homeostasis (Carroll et al., 2018). Deficiency in autophagy genes, such as Beclin-1, UVRAG, ATG5 and ATG7 led to DNA damage accumulation (Eliopoulos

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et al., 2016). In line with these, suppression of FIP200 impaired DNA damage response (DDR) and sensitized cancer cells to ionizing radiation-induced oxidative stress (Bae and Guan, 2011). ATM, one of the major DNA repair proteins, established another link between DDR and autophagy (Hurley and Bunz, 2007). ATM, through the LKB1/AMPK pathway,

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acted on TSC2 and inhibited mTORC1 in response to ROS-induced cellular damage (Alexander et al., 2010). Autophagy is considered to delay apoptotic cell death upon DNA

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damage by providing the energy required for DNA repair processes, which play a role in development of chemoresistance mechanisms in cancers (Abedin et al., 2007; Vessoni et al.,

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2013; Yoon et al., 2012). Additionally, removal of dysfunctional mitochondria under oxidative or nitrosative stress conditions reduce the level of DNA-damage stress to allow cancer

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cells escape from cell death mechanisms.

3.2.3. Autophagy promotes angiogenesis, metastasis and invasion during tumorigenesis

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Metastasis has been described as the distribution of tumor cells from the primary tumor to surrounding tissues and even to distant organs (Valastyan and Weinberg, 2011). Proliferation and the metastatic spread of cancer cells require adequate level of oxygenation and continuous nutrient supply. In order to reach these demands, tumors develop new blood and lymphatic vessels through a process called angiogenesis. Angiogenesis is controlled via the angiogenic activators and inhibitors whose levels correlate with the aggressiveness of tumor cells (Zhou et al., 2013). Autophagy is also implicated in development of vasculature in tu-

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ACCEPTED MANUSCRIPT mor tissues. ATG5 was shown to modulate angiogenesis in endothelial cells, which is proposed to occur through the high mobility group box 1 (HMGB1) pathway (Du et al., 2012). HMGB1 induced autophagy by binding to Beclin-1 (Kang et al., 2011). Additionally, HMGB1 contributed to angiogenesis and tumor cell survival by mediating the crosstalk between endothelial cells and tumor cells (Yang et al., 2014). Autophagy is also reported to

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play an important role during angiogenesis in bovine aortic endothelial cells. Evidence suggests that induction of autophagy can also promote VEGF-induced angiogenesis (Du et al., 2012). Additionally, mTOR inhibitors influenced tumor angiogenesis in malignant and apoptosis-inhibited lung cancer cells (Shinohara et al., 2005). Rather than tumor suppressive

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role of Beclin-1, it has been also shown that in a context dependent manner Beclin-1 promoted tumorigenesis. For example, it has been shown that in triple negative breast cancer

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cells complete knockout of Beclin-1 resulted in impaired tumor growth through promoting G0/G1 cell cycle arrest and impaired migration capacity in collaboration with reverse signal

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of EMT (Wu et al., 2018).

Throughout the process of metastasis tumor cells acquire phenotypic changes allow-

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ing them to gain the ability to enter/exit the vasculature, survive the immune attack and the

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demanding conditions within the circulatory system. Additionally, metastatic cells should also be able to extravasate at distant capillary beds, attach to distant tissues and proliferate in

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a foreign microenvironment (Kenific and Debnath, 2015). Anoikis is a special form of apoptosis, which occurs upon detachment of cells from the extracellular matrix (ECM) (Paoli et al., 2013). Therefore, resistance to anoikis favored for metastasis by allowing cancer cells to survive stress conditions associated with ECM detachment (Wang et al., 2017). Autophagy promoted the survival of metastatic cells during ECM detachment and components of the ECM were also able to regulate autophagic activity in cervical cancer cells (TuloupMinguez et al., 2011). Autophagy is rapidly induced cell survival during anoikis when cells

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ACCEPTED MANUSCRIPT are grown under low ECM attachment conditions in MCF10A mammary epithelial cells (Debnath et al., 2002). Accordingly, siRNA-mediated knockdown of ATG5, ATG6 and ATG7 was found to supress matrix detachment-induced autophagy (Fung et al., 2008). Another autophagy regulatory components PI3K-AKT-mTORC1 and the IKK pathways are also involved in the regulation of autophagy during ECM detachment (Chen and

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Debnath, 2013). In line with these findings, autophagy inhibition attenuated pulmonary metastasis of HCC cells in nude mice, which seemed to correlate with enhanced anoikis (Macintosh et al., 2012). Furthermore, in this context autophagy played a critical role in invasiveness and migration of cancer cells. Depletion of ATG12 decreased the invasive capac-

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ity of glioma cells (Macintosh et al., 2012). Similarly, autophagy inhibition or p62 knockdown reduced the rate of invasion and migration in vitro and resulted in metabolic defects in

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glioblastoma stem cells (Galavotti et al., 2013). Additionally, defective autophagy correlated with reduced secretion of proinvasive cytokines such as the interleukin-6 (IL6) (Kenific and

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Debnath, 2015). In agreement with these observations, addition of IL6 into Ras-transformed epithelial cells was able to partly restore the invasive capacity of the cancer cells, which was

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reduced due to autophagy inhibition (Lock et al., 2014). Evidence also suggested that au-

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tophagy was activated through toll-like receptors (TLRs) which mediated secretion of proinvasive factors, including IL6 (Zhan et al., 2014). Furthermore, autophagy-dependent secre-

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tion was also important for the invasiveness of HCC cells as IL6 stimulated TGF signaling and promoted EMT (O’Reilly et al., 2014).

3.2.4. Autophagy regulates unfolded protein response (UPR) in cancer cells Abnormalities in calcium homeostasis, oxidative stress and conditions leading to protein glycosylation or folding defects etc. resulting in the accumulation of misfolded and/or unfolded proteins in the ER lumen, a condition known as ER stress (Senft and Ronai, 2015).

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ACCEPTED MANUSCRIPT ER stress driven by the accumulation of unfolded proteins potentiated signaling from ER to nucleus termed as the unfolded protein response (UPR). Once activated, UPR enhances the expression of proteins that mediate proper protein folding in the ER such as the chaperones. Inability to restore the function of ER leads to the removal of the affected cells by apoptosis (Senft and Ronai, 2015). Autophagy functions as a critical mechanism to cope with ER

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stress promoting survival of the cancer cells (Nagelkerke et al., 2014). The PERK-arm of the UPR is important for the activation of autophagy in majority of the cancer cells. Both the eIF2α phospho-mutant constructs and dominant-negative PERK was shown to prevent the conversion of LC3-I to LC3-II (Kouroku et al., 2007). Radiotherapy-based treatment was

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found to induce PERK-dependent autophagy in breast cancer cells, which were sensitized to

signaling (Chaachouay et al., 2011).

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radiotherapy by both pharmacological inhibition of autophagy and silencing of the PERK-

Additionally, tamoxifen treatment of breast cancer cells induced autophagy, which

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partly regulated by by ATF4-induced LAMP3 and/or by GRP78-dependent inhibition of mTOR (Nagelkerke et al., 2014). Similarly, bortezomib treatment of breast cancer cells led

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to increase in LC3B and autophagy in an ATF4-dependent manner, protecting against cell

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death and inducing bortezomib-resistance (Milani et al., 2009). Furthermore, autophagy can also be activated through the IRE1-arm of UPR-signaling pathway in some cancer types. For

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example, ER stress induced by tunicamycin, thapsigargin or amino acid starvation was able to induce autophagy in neuroblastoma cells through activation of the IRE1-arm of ER-stress mechanisms (Ogata et al., 2006). Finally, ER stress also induced autophagy through upregulating cellular levels of GRP78. GRP78 expression correlated with increased upregulation of LC3 and Beclin-1, enhanced autophagic flux and increased number of autophagosomes in neural cells.

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ACCEPTED MANUSCRIPT Human papilloma virus is a major leading source of cervix cancer and in collaboration with NF-kB pathway contributed to proliferation, invation and metastasis. In human cervical cancer cells, ER-stress inducers potentiated autophagic activity through NF-B pathway and resulted in cell death (X. Zhu et al., 2017). An inhibitor of NF-B, quinazolinediamine (QZN) reduced Brefeldin A-induced cell death and autophagy.

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As another example, in human colorectal cancer, a novel tyrosine kinase inhibitor Apatinib induced both autophagy and apoptosis through IRE-1 arm of ER-stress (Cheng et al., 2018). Apatinib favored for the protective role of autophagy providing an acquired resistance to apatinib treatment in colorectal cancer cells. Therefore targeting autophagic ac-

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tivity is a promising treatment strategy as a combinatory treatment of apatinib and CQ

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(Cheng et al., 2018).

3.2.5. Autophagy supports stromal cells and promotes tumor growth

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In most cases, tumor cells hijack stromal cell functions and switch on autophagy to maintain

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homeostasis and support tumor growth (Zhou et al., 2013). During tumorigenesis, cancer cells induce excessive ROS production, which activates oxidative stress response mecha-

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nisms and autophagy in stromal cells. Both autophagy activation and the anti-oxidant defense mechanisms in stroma protect the adjacent cancer cells from cellular damage and cell

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death (Zhou et al., 2013). Increased ROS production in stroma was also associated with another tumorigenic effect termed as the ‘‘Bystander-effect’’ which results in DNA damage and aneuploidy in adjacent cancer cells (Lisanti et al., 2010). Additionally, autophagymediated recycling of energy-rich metabolites in stroma such as the ketones and L-lactate may support mitochondrial biogenesis and anabolic growth of cancer cells (Zhou et al., 2013). Interestingly, ketones and lactate are reported to function as chemo-attractants for cancer cells, which stimulate tumor growth and metastasis.

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3.2.6. Autophagy provides resistance to cancer cells Resistance to chemotherapeutic agents remains a major challenge that limits the efficacy of anticancer drugs. Resistance is developed to anticancer drugs through utilization of several mechanisms such as reduced drug intake, enhanced drug efflux by overexpression of certain

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type of transporters, inefficient drug penetration into the solid tumors (Wu et al., 2014); activation of drug metabolism and/or anti-oxidant metabolism, acquisition of regulatory defects in the apoptotic pathway and/or cell cycle checkpoint control mechanisms, activation of DNA repair machineries to reduce drug-induced DNA damage. Growing evidence indi-

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cated that while autophagy contributes to the anticancer efficacy of chemotherapy, it confers drug resistance in several cases (Sui et al., 2013). Similarly, in response to radiation, autoph-

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agy is often considered cytoprotective, whereas radiation-induced autophagy has also been found to sensitize the cancer cells to radiotherapy (Sharma et al., 2014). Majority of anti-

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cancer drugs target programmed cell death mechanisms to kill cancer cells and recent progress in pharmaceutical research area show that utilization of autophagy-related pro-

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grammed cell death in cancer therapy can be used as an alternative way to destroy malignant

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cells (Ouyang et al., 2012).

Autophagy-associated resistance to chemotherapy has become a challenge for cancer

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treatment. For example, autophagy promoted resistance to gefitinib and erlotinib (tyrosine kinase inhibitors) treatment in human lung cancer cells (Han et al., 2011; Jiang et al., 2018). Other examples include, resistance to treatment for imatinib in leukemia (Shingu et al., 2009), temozolomide in glioblastoma (Milano et al., 2009), 5-FU in colorectal cancer (Sasaki et al., 2010) and tamoxifen (Wu et al., 2018) or trastuzumab for breast cancer (Luque-Cabal et al., 2016). For example, induction of autophagy delayed cell death induced by the DNA damaging agent camptothecin (CPT) in breast cancer cells (Abedin et al.,

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ACCEPTED MANUSCRIPT 2007). Autophagy also had a cytoprotective role in response to 5-FU in colon and oesophageal cancer cells (Li et al., 2009; O’Donovan et al., 2011). The stress factors mentioned above (nutrient starvation, hypoxia, oxidative stress and DNA-damage) are among the inducers of cell death pathways, including apoptosis. Two major apoptotic pathways play role in execution of cells: extrinsic and intrinsic signaling

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pathways. The extrinsic (death receptor associated) pathway is induced upon binding of the ligands such as FAS or TNF to cell death receptors. Once activated, these death receptors mediate caspase 8 activation and promote cell death (Galluzzi et al., 2012). The intrinsic (mitochondrial or BCL-2 regulated) pathway, however, can be activated in response to stress

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factors or chemo/radiotherapies through induction of the pro-apoptotic BCL-2 family proteins. BCL-2 activation promoted permeabilization of the mitochondrial outer membrane

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and release of cytochrome c into the cytosol (Adachi et al., 1997). However, cancer cells acquired apoptosis-resistance through upregulation of pro-survival factors, such as inhibitors

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of apoptotic proteins (IAPs), NF-B, and the BCL-2 family proteins (Marquez and Xu, 2012). For example, decreased level of apoptosis and resistance to death play a critical role

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in tumorigenesis of gastric cancer. Long-term scutellarein treatment restored the decreased

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level of apoptosis in human gastric cancer cells. Scutellarein could successfully inhibited cell proliferation by downregulation of MDM2 and activation of p53 and finally subsequent

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downregulation of IAPs suggesting the significant anti-tumor role of scutellare in tumorigenesis-associated cell death resistance (Saralamma et al., 2018). Thus, targeting the autophagy-dependent mechanisms involved in drug resistance and cancer cell survival allow us to develop novel therapeutic strategies to enhance the effects of chemotherapy and improve clinical outcomes of treatment in cancer patients (Sui et al., 2013). In this context, inhibition of autophagy can be exploited as a novel strategy to resensitize the cancer cells to chemo-/radio-therapy. For example, combined therapy of siR-

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ACCEPTED MANUSCRIPT NA-mediated LC3 depletion with imatinib treatment sensitized the breast cancer cells to trastuzumab treatment in MCL (Bellodi et al., 2009). Similarly, autophagy inhibitors chloroquine (CQ) and hydroxychloroquine (HCQ) were also promoted accumulation of autophagic vacuoles that often leads to apoptotic and necrotic cell death (Solomon et al., 2009). We

in cancer treatment.

4. Targeting autophagy for cancer treatment

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have provided a more detailed discussion on the role of autophagy inhibitors and activators

The involvement of the shared regulatory pathways makes autophagy as a promising target

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in cancer treatment, even though the relationship between autophagy and cancer is still controversial. Concerning the dual roles of autophagy in tumor development mainly two differ-

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ent therapeutic strategies can be adopted. The first approach includes sensitizing the cancer cells for chemo-/radio-therapy through inhibition of the cytoprotective role of autophagy.

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The other strategy aims to target induction of autophagic cell death in apoptosis-resistant cells (Zhou et al., 2012). Targeted autophagic proteins and autophagy inhibitors for cancer

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treatment are listed in Table 1 and Table 2, respectively.

4.1 Autophagy inhibitors as anti-cancer agents

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The role of autophagy as a mechanism that promotes resistance to chemo- or radio-therapies compromises the efficacy of anti-cancer treatment strategies. Hence, inhibition of autophagy may serve as a tool for sensitizing the tumor cells for treatment. The most common autophagy-inhibiting molecules could be categorized into four groups according to their mode of action: i. Repressors of autophagosome formation: Class III PI3K inhibitors 3-methyladenine (3MA), Wortmannin, LY294002, SAR405 and recently developed Viridiol were shown to

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ACCEPTED MANUSCRIPT block the formation of autophagosome (Del Bel et al., 2017; Pasquier, 2015; Rubinsztein et al., 2012). ii. Repressors of lysosomal acidification: Lysosomotropic agents including CQ, HCQ, Lys0569 and monensin prevent acidification of lysosomes and thus inhibit degradation of the cargo in the autophagosomes.

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iii. Inhibitors of autophagosome-lysosome fusion: Vacuolar-ATPase inhibitors, including variants of Bafilomycin (Baf A1, Baf B1 and Baf C1) and Concanamycin variants (Con A, Con B and Con C) interferes with the fusion of autophagosomes with lysosomes whereas, Spautin-1 targets Beclin-1 subunit of Vps34 complexes (Bowman et al., 2004; Shao et al.,

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2014).

iv. Silencing expression of autophagy-related proteins at transcription level: By utilizing

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siRNA- or miRNA-mediated silencing strategies, knockdown of autophagy-related genes

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subsequently inhibited autophagic activity.

4.1.1. Class III PI3K inhibitors

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The Class III PI3K VPS34 (also called PIK3C3) is a positive regulator of autophagy,

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which was originally identified in Saccharomyces cerevisiae (Kihara et al., 2001). VPS34 mediates initiation and maturation of autophagosomes by forming protein complexes with

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various autophagy regulator proteins. PI3K inhibitors, including 3-methyladenine (3-MA) (Seglen and Gordon, 1982), Wortmannin, LY294002, (Blommaart et al., 1997), recent selective PIK3C3 inhibitors SAR405 (Pasquier, 2015) and Viridiol (Del Bel et al., 2017) have been proposed to suppress autophagy by inhibiting the production of PI3P (Petiot et al., 2000), which is essential for the recruitment of other ATG proteins at the isolation membrane or phagophore (Zeng, 2006).

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ACCEPTED MANUSCRIPT A number of reports supported the idea that autophagy inhibition through PI3K inhibitors enhanced the efficacy of chemo- and/or radio-therapies (Cheong et al., 2012). For example, deregulation of autophagy with 3-MA contributed to radiation sensitization of esophageal squamous carcinoma cells (Chen et al., 2011). Similarly, 3-MA-mediated inhibition of autophagy enhanced 5-FU- and cisplatin-induced apoptosis in colon and lung cancer

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cells respectively (Li et al., 2009; Liu et al., 2013). Furthermore, wortmannin treatment was able to enhance the antitumor effect of silver nanoparticles in the in vivo (Lin et al., 2014). SAR405 inhibited autophagosome biogenesis and combination of SAR405 with everolimus, the FDA-approved mTOR inhibitor, proposed to reduce proliferation of renal tumor cells

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(Pasquier, 2015).

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4.1.2. CQ derivatives

Pharmacological inhibition of autophagy by the administration of lysosomotropic agents

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CQ, HCQ, Lys0569 or monensin, block the fusion of autophagosomes with lysosomes, have been shown to exert anticancer effects or enhance the efficacy of antineoplastic treatments

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(Cheong et al., 2012; Wu et al., 2014). Therefore, CQ-derivatives seem to be promising drug

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candidates for developing novel treatment strategies against cancer. For example, the addition of chloroquine to bevacizumab-based treatment was able to yield a more effective tu-

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mor control in non-small-cell lung cancer (Selvakumaran et al., 2013). The impact of CQ increase the efficiency and activity of CQ-derivatives in order to reach the requirements in clinical applications. For example, structurally-generated dimeric version of CQ, Lys01 and its soluable form, Lys05 exhibited much greater effect than CQ in terms of autophagy inhibition and cytotoxicity (McAfee et al., 2012). As a combinatory approach, mTOR and autophagy inhibition in phase I trial of hydroxychloroquine and temsirolimus in cancer patients was found to be well tolerable and displayed significant antitumor activity (Rangwala

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ACCEPTED MANUSCRIPT et al., 2014). Similarly, adding chloroquine to conventional cancer treatment was shown to potentiate the effect of chemo-therapy in glioblastoma multiformation (Sotelo et al., 2006) and colon cancer cells (Sasaki et al., 2010). Furthermore, 3-methyladenine and chloroquine combination was able to sensitize cancer cells to radiotherapy (Cerniglia et al., 2012). On one hand, autophagy-inhibition-mediated NOTCH1 signal regulation by utilizing CQ was

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also controlled chemoresistance in gastric cancer stem cells when combined with 5-FU (Li et al., 2018). On the other hand, CQ provided an alternative approach for vasculature normalization by upregulating NOTCH1 signaling as an autophagy-independent manner (Maes

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et al., 2014).

4.1.3. Bafilomycin A1

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Bafilomycin A1 (Baf A1) is a macrolide antibiotic and inhibits vacuolar H+ ATPase (VATPase). Binding of Bafilomycin A1 to V-ATPase complex inhibits H+ translocation that in

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turn resulted in altered H+ balance in cytoplasm (Bowman et al., 2004; Ohta et al., 1998). Bafilomycin A1 was shown to exert its effect on autophagy inhibition and promoting apop-

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tosis favoring for cancer treatment, but only at high concentrations. Therefore, toxicity po-

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tential limited itsmedical applications. However, growing number of reports could show the successful use of Bafilomycin A1 in combined anti-cancer therapies (Cheong et al., 2012).

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As an example, combined Baf A1 and 3-Methyladenine (3-MA) treatment enhanced the antitumoral effect of nedaplatin in cisplatin-resistant nasopharyngeal carcinoma cells (Liu et al., 2015). Similarly, Baf A1 was reported to confer chemosensitivity in gastric cancer cells (Li et al., 2016), osteosarcoma cells (Xie et al., 2014) and colon cancer cells (Greene et al., 2013).

4.1.4. Spautin-1

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ACCEPTED MANUSCRIPT Spautin-1, 6-Fluoro-N-(4-fluorobenzyl) quinozaline-4-amine was shown to block the autophagy pathway in cancer cells thereby providing the potential to be exploited as an anticancer agent. Spautin-1 exerts its inhibitory effect on autophagy through degradation of VPS34 and PI3K complexes by inhibiting the activities of USP10 and USP13 ubiquitinspecific peptidases that further target Beclin-1 of VPS34 complexes. (Liu et al., 2011). Fur-

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thermore, the effect of Spautin-1 on Beclin-1 was further affected cellular p53 level. For instance, spautin-1 enhanced the cellular Imatinib-induced apoptosis in chronic myeloid leukemia through inactivation of PI3K/AKT signaling while activating the downstream GSK3β pathway (Shao et al., 2014). Therefore, spautin-1 as an autophagy-inhibiting anti-

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cancer agent carries a great potential for clinical usage.

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4.1.5. siRNAs

Sequence-specific DNA or RNA analogs blocking the expression of genetic sequences with

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high specify offer the possibility for designing custom made molecules with potential anticancer effects at relatively inexpensive costs. In this sense, in addition to oncogenes, several

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essential modulators of the autophagic machinery such as ATG3, ATG4B, ATG4C, ATG5,

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Beclin-1, ATG10, and ATG12 have also been targeted. siRNA-based deregulation of major autophagy modulators was able to sensitize several cancer cells to chemo- and radio-

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therapies as critically discussed in (Wu et al., 2012). For example, siRNA-mediated decrease in ATG5 correlated with reduced level of autophagy and enhanced norcantharidin-induced cell death in hepatocellular carcinoma cells (Xiong et al., 2015). In line with this, ATG7 knockdown favored for cell G2/M cell cycle arrest by promoting p27 expression in bladder cancer in vitro and in vivo (Zhu et al., 2017).

4.2. Autophagy activators as anti-cancer agents

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ACCEPTED MANUSCRIPT Since excessive autophagic activity acts as a pro-death mechanism, the autophagy induction is a direct strategy that may promote tumor cell death. As mentioned in the previous sections, certain tumor cells are resistant to apoptosis and this enables them to escape from death. Therefore, autophagy comes into play as an alternative cell death mechanism in cancer cells with defects in apoptosis (Tsujimoto and Shimizu, 2005). Inhibition of mTOR or

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disruption of Beclin-1/BCL-2 interaction is among the most common strategies implemented to induce autophagy directly complex. Additionally, a number of agents introduced that directly or indirectly promote autophagy have been described (Cheong et al., 2012). Below we will briefly discuss some of the chemical agents, which are known to directly act on au-

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tophagic pathways.

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4.2.1. mTOR inhibitors

The major kinase complex of autophagy mTOR considered as a promising drug target for

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the cancer treatment strategies. The mTOR inhibitor rapamycin have been reported to sensitize various tumor cells to radiation therapy (Cheong et al., 2012) and inhibited cell prolifer-

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ation of malignant glioma cells (Takeuchi et al., 2005). Rapamycin formed a complex with

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the small protein FKBP12 which binds to the FKBP12-rapamycin domain of mTORC1 and inhibits its kinase activity (Sabers et al., 1995). Beside from its anti-tumoral activity, the

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clincical use of rapamycin was limited due to its low solubulity and poor stability. First generation of rapamycin analogs include temsirolimus (CCI-779, Wyeth) (Wu et al., 2005), everolimus (RADD001, Novartis) (Gorshtein et al., 2009), and ridaforolimus (AP23573, Ariad Pharmaceuticals) (Mita et al., 2008). Among these rapamycin analogs temsirolimus and everolimus exerted their effects on autophagy through downregulation of AKT signaling. mTOR inhibitors are shown to induce cell death in various cancer cells including, breast cancer (Hurvitz and Peddi, 2013), renal cell carcinoma (Motzer et al., 2010), thyroid cancer

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ACCEPTED MANUSCRIPT (Wagle et al., 2014), non small cell lung cancer (Choueiri et al., 2015), hepatocellular carcinoma (Zhu et al., 2011) and mesothelioma (Pignochino et al., 2015). However, due to the reports on possible off-target effects of rapamycin and its analogs more selective and potent ATP-competitive inhibitors of both mTORC1 and mTORC2 and the dual PI3K-mTOR inhibitor NVP-BEZ235 have been developed. Clinical use of dual inhibitors carries great po-

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tential for highly agressive tumors with a high mortality and low treatment possibility such as uterine sarcoma. Treatment with dual inhibitors significantly reduced tumor growth in patient-drived mouse models (Cuppens et al., 2017).

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4.2.2 Tyrosine kinase inhibitors

Tyrosine kinases are a class of protein involved in the phosphorylation of tyrosine residues

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on polipeptides and their cellular expressions are limited in non-proliferating cells. Enhanced enzymatic activity and expression were linked to tumorigenesis and proliferative

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abnormalities (Baselga, 2006). Imatinib, a tyrosine kinase inhibitor (TKI) commonly used drug for the treat of chronic myeloid leukemia and also gastrointestinal stromal tumor

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(Ertmer et al., 2007). The mechanistic details of imatinib on tumors was correlated with

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downregulation of BCR/ABL, disaasociation of the complex as well as autophagy induction (Elzinga et al., 2013). But alternatively, tyrosine kinase mediated autophagy activation in

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leukemia could promote cancer cell survival due to the reverse effects on these pathways on leukemic cells (Drullion et al., 2012). To support this theme, combined treatment of imatinib and its derivatives like nilotinib with CQ or BafA induced cell death both in vivo and in vitro (Bellodi et al., 2009; Shingu et al., 2009; Tiwari et al., 2009; Wu et al., 2010). Another tyrosine kinase inhibitor, gefitinib treatment significantly resulted in tumor regression in patients bearing non-small cell lung cancer (Paez et al., 2004). Similar results also obtained in the case of lung adenocarcinoma that exhibit hypersensitivity to gefitinib.

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ACCEPTED MANUSCRIPT Some of the commercially available tyrosine kinase inhibitors include sorafenib (Abou-Alfa et al., 2010), lapatinib (Awada et al., 2011) and vandetanib (Karras et al., 2014). Sorafenib is the key chemotherapeutics which enhanced survival rates of hepatocellular carcinoma (HCC) patients (Estfan et al., 2013). Not only the HCC, but also a number of different types of cancer were included in the sorafenib treatment targets, including renal cell carcinoma

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(Escudier et al., 2007), prostate cancer, thyroid cancer (Shen et al., 2014) and also amyloid leukemia (Antar et al., 2015). Sorafenib-mediated autophagy induction is related to both death and survival of the cancer cells in a context-dependent manner. AKT inhibition provided a key regulatory node for determination of hepatocellular carcinoma cell faith from

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protective autophagy to autophagic cell death (Zhai et al., 2014). AKT-mediated regulation of sorafenib-induced autophagic cell death occurred through in an ERK1/2-independently in

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renal cell carcinoma (Serrano-Oviedo et al., 2018). Sorafenib treatment favored for cell death also through necroptosis in autophagy-deficient cancer cells (Kharaziha et al., 2015).

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Erlotinib another commonly used tyrosine kinase inhibitor enhanced autophagy in various different cancer cells, including non-small cell lung cancer (Li et al., 2013). Similar

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to other tyrosine kinase inhibitors, erlotinib-mediated autophagy induction confer resistance

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to death. Overcoming TKI-derived resistance to death become emerging for cancer treatment. Autophagy inhibition keeps a great potential in order to reach this demand. CQ (Zou

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et al., 2013), 3-MA (Wang et al., 2016) and clozapine (Yin et al., 2015). On the other hand, combined treatment of sertraline and erlotinib induced autophagy by regulating AMPK/mTOR pathway and this combination significantly reduced tumor formation and induced survival rate (Jiang et al., 2018). Accumulating data suggest that TKIs are key drugs with a great anti-tumoral effects and their influence on survival are tightly regulated by autophagy mechanism.

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ACCEPTED MANUSCRIPT 4.2.3 Histone deacetylase inhibitors Dysregulation of enzymes function as epigenetic regulators are involved in human tumors has led to development inhibitors such as the histone deacetylases (HDACs) which target the cancer epigenome (Lakshmaiah et al., 2014). Based on their chemical structure, inhibitors of HDACs are classified into several groups including hydroxamic acids (e.g., trichostatin A,

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vorinostat, suberoylanilide hydroxamic acid), carboxylic acids (e.g., valproate, butyrate), aminobenzamides (e.g., entinostat, mocetinostat), cyclic peptides (e.g., apicidin, romidepsin), epoxyketones (e.g., trapoxins), and hybrid molecules (West and Johnstone, 2014). The effects of histone deacetylase inhibitors on cell death are well-differentiated from their ef-

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fects on chromatin and exhibit wide effect on different types of cancers (Luchenko et al., 2014). HDAC inhibitors are induced apoptosis through regulating cell cycle arrest and af-

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fected to different molecular mechanisms such as angiogenesis, metastasis and autophagy. Among the inhibitors of HDAC, Vorinostat and romidepsin are the FDA approved drugs for

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the treatment of T-cell lymphoma. Varinostat treatment induced cell death through activating p38/MAPK pathway in breast cancer cells (Uehara et al., 2012). In addition to their roles

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in apoptosis, HDAC inhibitors can also induce autophagy. Interestingly, there have been

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identified link between the HDAC inhibitor resistancy and cellular autophagy level. Shortand long-term varinostat induced autophagy and furthermore, autophagy provided aquired

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resistancy to varinostat in mammalian cells (Dupéré-Richer et al., 2013). On the other hand, the anti-tumoral effect of varinostat further accelatered when combined with HCQ. Indeed, varinostat and HCQ improved immunity in pre-clinical models as well as early phase clinical trials of metastatic colorectal cancer (Patel et al., 2016). As a similar approach, genistein and varinostat cotreatment enhanced cell death in prostate cancer cells regulating cell cycle check points, WNT and TNF signaling (Phillip et al., 2012).

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ACCEPTED MANUSCRIPT Another HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA) also activated autophagy by inhibiting mTOR and upregulating LC3 expression where autophagy favored forsurvival of cells (Gammoh et al., 2012). In a different experimental setup, SAHA also promoted caspase-independent autophagic cell death through p53-linked mechanisms suggesting that indirect and non-specific effects on autophagy regulation might be involved

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(Shao et al., 2004). SAHA-linked tumor suppressive effects were also correlated with enhanced autophagic activity in collaboration with apoptosis in the late stage of glioblastoma stem cells as well as glioblastoma biophsy-originated cultured cells (Chiao et al., 2013).

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4.2.4 Arsenic trioxide

Arsenic trioxide (As2O3) is a well-known toxin originated from the traditional Chinese med-

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icine and proposed to have therapeutic on different types of malignancies, particularly multiple myeloma and myelodysplastic syndromes (Emadi and Gore, 2010). In addition to acti-

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vation of apoptotic pathways, AS2O3 also promoted cytotoxicity in cancer cells through induction of autophagy (Zhou et al., 2015).

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As2O3–mediated autophagy activation was shown to occur through MEK/ERK path-

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way rather than the AKT/mTOR or JNK pathways (Goussetis et al., 2010). Additionally, As2O3 can induce autophagic cell death in leukemia cell lines through upregulation of Be-

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clin-1 (Qian et al., 2007) and rapid degradation of pro-myelocytic protein contributed to distruption of glioma stem cells and increased survival of the tumor-bearing animals (Zhou et al., 2015). Interestingly, autophagy-inducing potential of As2O3 therefore promoting autophagic cell death offers a great therapeutic approach for apoptosis-resistant cancer types.

4.2.5 Resveratrol

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ACCEPTED MANUSCRIPT Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is one of the natural polyphenols produced by several plants, including grapes, blueberries, raspberries as an immune response against to injury or pathogen attack (Frémont, 2001). By regulating different molecular targets, it has been shown to involved in various molecular pathways such as inflammation and immunity (Park and Pezzuto, 2015; Švajger and Jeras, 2012). Moreoever, anatural plant-derived prod-

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uct resveratrol also constitutes a good example for autophagy-modulated anti-cancer compounds through induction of cell death (Lang et al., 2015; Wang and Feng, 2015). Molecular mechanism behind the activatory effect of resveratrol autophagic cell death was is highly context- and cell type-dependent as it is proposed to be able to affect a vast number of sig-

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naling pathways, including Beclin-1 (Scarlatti et al., 2008a, 2008b), DAPK1 (Choi et al., 2013) , TIGAR (Kumar et al., 2015), STIM1-mTOR (Selvaraj et al., 2016), PI3K-AKT

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4.2.6 Polygonatum cyrtonema lectin

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(Jiang et al., 2009) and WNT/β-Catenin signaling pathways (Fu et al., 2014).

Polygonatum cyrtonema lectin (PCL) is a mannose/sialic acid-binding plant lectin proposed

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to activate programmed cell death mechanisms, including apoptosis and autophagy in vari-

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ous cells, including cancer cells (Wang et al., 2011). PCL-mediated autophagy induction has been linked to PI3K-AKT pathway in murine fibrosarcoma cell line (Liu et al., 2010). Addi-

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tionally, PCL-induced autophagy was also occurred through mitochondria linked ROS-p38– p53 pathway in human melanoma cells (Liu et al., 2009). Eventhough currently there is no FDA-approved applications, PCL holds a great potential as a cancer-therapeutic.

4.2.7 Epigallocatechin-3-gallate Epigallocatechin gallate (EGCG) is a major polyphenol found in green tea has been associated with the induction of cell cycle arrest and apoptosis in human colorectal cancer cells (G.

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ACCEPTED MANUSCRIPT J. Du et al., 2012). On the other hand, a recent study suggested that EGCG suppressed apoptosis and autophagy in oral cancer cells (Irimie et al., 2015). Similarly, EGCG was also linked to increased autophagy and removal of lipids in hepatic cells which may offer new therapeutic approaches for treatment of pathological liver conditions, including liver cancer

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(Zhou et al., 2014).

4.2.8 Curcumin

Curcumin is another natural polyphenolic compound extracted from Curcuma sp. and linked to cancer treatments due to its antioxidant effects (Rahmani et al., 2014). It has been demon-

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strated that curcumin induced autophagic cell death in malignant glioma cells in vitro and in vivo through inhibition of AKT/mTOR/p70(S6K) pathway (Aoki et al., 2007; Shinojima et

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al., 2007). Additionally, curcumin-induced autophagy was also linked to activation of the AMPK signaling pathway in lung adenocarcinoma cells (Xiao et al., 2013). Moreover, cur-

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cumin was found to have a role in cell death decision between apoptosis and autophagy through regulating several distinct mechanisms in breast cancer cells (Akkoç et al., 2015).

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Furthermore, curcumin analogues EF25-(GSH)2 (Zhou et al., 2014) and IHCH (Zhou et al.,

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cells respectively.

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2014) were also reported to have an activatory effect on autophagy in liver and lung cancer

4.2.9 Allicin

Allicin is an ubiquitiously found ingredient in garlic and widely used as food supplement all over the world (Lawson and Wang, 2005). As a thiosulfinate, allicin can undergo a redoxreaction with thiol groups in biologically active molecules. In addition to its anti-fungal and anti-bacterial effects allicin is also reported to induce cell death and inhibit proliferation in cancer cells therefore providing an important anti-tumoral effect (Borlinghaus et al., 2014).

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ACCEPTED MANUSCRIPT Findings also suggested that allicin induced autophagy human liver cancer cells in a p53dependent mechanism (Chu et al., 2012).

4.2.10 Ginsenosides Ginsenosides (panaxosides) are the principal bioactive constituents of ginseng. They are

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involved in a group of glycosylated triterpenes also known as saponins. Compounds in this family are found in the medicinal plant Panax (ginseng) (Murthy et al., 2014). A number of different biological activities of ginsenosides including anti-cancer effects have been reported (Nag, 2012). Accumulating data also suggested that some of the anti-cancer effects of

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ginsenosides are attributed to induction of autophagic activity in cancer cells. For example, ginsenoside F2 was shown to induce autophagy in breast cancer stem cells (Mai et al.,

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2012). Similarly, a ginsenoside Rb1 and its active metabolite compound K, induced autophagy through generation of reactive oxygen species (ROS) and activation of JNK in human

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colon cancer cells (Kim, 2013).

4.3. Combinatory approach in cancer treatment

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Tumors exhibit heterogenous, irregular and branched blood vessel network (Nagy et al.,

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2010). These heterogeneity in vascularization resulted in permeability imbalances and inadequate blood supply to diferential compartments of the tumor tissue further associated with

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metabolic stresses, including hypoxia and starvation, which in turn provided invasion and decreased immune response (Cárdenas-Navia et al., 2008; Dalerba et al., 2011). Therefore, targeting cancer with combinatory therapy even at a single cell level provide an alternative strategy to combat with tumor progression. The use of miRNAs and nano-sized carriers become an alternative therapeutic approach for targeted therapies. Beside from their increased usage and benefits, nano-sized carriers tend to accumulate in spleen or liver by macrophages-mediated endocytosis.

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4.3.1 Autophagy regulating miRNAs in cancer MicroRNAs (miRNAs) are involved in a class of short RNAs (∼ 21 nucleotides) that target partially

complementary

transcripts

to

control

key

biological

processes

post-

transcriptionally. miRNAs are transcribed from several different loci in the genome which

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encode for long RNAs (pri-miRNAs) with a hairpin structure. Then RNase III enzyme Drosha processes the pri-miRNAs to give the precursor miRNAs (pre-miRNAs) (Lee et al., 2003). Pre-miRNAs are subsequently transported into the nucleus and then processed further by RNase III enzyme, DICER (also known as DICER1), to yield a mature miRNA (Gurtan

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and Sharp, 2013). Mature miRNA is then loaded into an argonaute protein within the RNAinduced silencing complex (RISC) acting as a guide strand through the target-specific seed

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sequence (Gurtan and Sharp, 2013).The miRNA-processing enzyme DICER and the main miRNA effector, AGO2 can be targeted for degradation by the selective autophagy receptor

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NDP52 (also known as calcium binding and coiled-coil domain 2 (CALCOCO2)) (Gibbings et al., 2012).

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The complicated autophagy-mediated differential regulatory mechanism in carcino-

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genesis is become even complex with the involvement of miRNAs (Frankel and Lund, 2012; Gibbings et al., 2012). For instance, autophagy inhibitor miR-101 was shown to be progres-

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sively lost during the course of cancer progression (Varambally et al., 2008). Moreover, miR-20a, miR-101, miR-106a/b and miR-885-3p targeted ULK1/2 while miR-155 regulated mTOR signaling (Füllgrabe et al., 2016). Other miRNAs function as inhibitors of autophagy include miR-30a, miR-34a, miR-204, miR-375 were linked to cancer with their reduced level of expression (Frankel and Lund, 2012; Füllgrabe et al., 2016; Gibbings et al., 2012). As emphasized in these (Fu et al., 2012; Füllgrabe et al., 2016) and many other detailed review articles both the oncogenic- (e.g., miRNA-183, miRNA-376b, miRNA-106a, and miRNA-

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ACCEPTED MANUSCRIPT 221/222) and the tumor-supressive microRNAs (e.g., miRNA-30a, miRNA-101, miRNA-9) are found to be able to modulate autophagic pathways. Recently, miR-4487 and miR-595 were identified as novel biomarkers and ULK1-targeting miRNAs in the regulation of autophagy (Chen et al., 2015). A detailed discussion on the roles of miRNAs in autophagy

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regulation and cancer has recently been reviewed by our group (Tekirdag et al., 2016).

4.3.2 Autophagy modulation through nano-sized material systems in cancer Multi-drug resistance defined as the phenomenon in which cancer cells develop resistance mechanisms to chemotherapeutics and limit the effective use of approved clinical treatments

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(Panzarini and Dini, 2014). As recently critically reviewed, several mechanisms, including altered drug-uptake, keeping the drug out of the cell by efflux pumps, increased capacity

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metabolize drugs, alterations in cell death mechanisms etc., played differing roles in multidrug resistance (Panzarini and Dini, 2014). Being a central player in regulation of metabolic

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and stress-response pathways, autophagy plays a dual role in drug resistance likewise in the case of carcinogenesis. Recent advances in designing nanosized drug delivery systems

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opened a new perspective for targeted delivery of chemotherapeutics at specific sites and

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controlled drug release into tumor cells (Upadhyay, 2014). Even some of the tested nanomaterials found to modulate autophagic activity in some cancer cells.

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Giving the importance of nanoparticle usage in the clinicals, it has been emerging issue to combine CQ-derivatives with nanoparticles to target cancer cells, due to the decreased effect of CQ on accumulation of nano-sized carriers in liver or spleen (Pelt et al., 2018). For instance, CQ was suggested as a promising candidate in order to decrease accumulation of nano-sized carriers in organs by inhibiting macrophage uptake, therefore promoted their distribution and localization on their targets for cancer therapy (Wolfram et al., 2017). As a multidrug complex example, CQ was included in the nanocapsulated erlotinib and shRNA

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ACCEPTED MANUSCRIPT survivin co-delivery treatment system and CQ-mediated vessel normalization increased the targeting ratio of erlotinib and shRNA survivin (Lv et al., 2018). In line with this, C60 (Nd) nanoparticles were shown to promote autophagy-mediated chemo-sensitization of cancer cells (Wei et al., 2010; Zhang et al., 2009). The therapeutic use of iron core-gold shell nanoparticles was able to inhibit growth of oral cancer through

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induction of reactive oxygen species and autophagy (Wu et al., 2013). Similarly, reports by others also showed that both the iron oxide (Khan et al., 2012) and alpha-aluminananoparticles (Li et al., 2011) exhibited autophagy-induced anti-tumor effects. Furthermore, combining nano-sized delivery systems with autophagy modulating agents may provide

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even a wider range of strategies to circumvent drug resistance mechanisms adopted by cancer cells. For example, in breast cancer cells, anti-cancer therapeutic treatment was achived

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by the utilization of chloroquine-loaded gold nanoparticle conjugates (GNP-Chl) (Joshi et al., 2012). In a similar context, a single intravenous injection of the nano-liposomal C6-

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ceramide together with vinblastine combination was shown to tremendous decrease in tumor growth in both hepatocellular carcinoma and colorectal cancer (Adiseshaiah et al., 2013).

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Additionally, chitosan nanoparticle-mediated delivery of miRNA-34a was reported to induce

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5. Conclusion

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autophagy and decrease prostate tumor growth in the bone (Gaur et al., 2015).

Malignant transformation requires initiation and maintenance of fundamental changes in biological processes to support the high levels of energy consumption and to supply the building blocks for tumor mass under stressful conditions. Additionally, uncontrolled proliferation results in tumor growth to a degree that the existing vasculature can no longer support the tumor mass. This triggers cellular adaptations for survival under nutrient- and oxygen-limiting conditions. Therefore, alterations in the expression levels of a set of genes also

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ACCEPTED MANUSCRIPT ensure oncogenic activation and metabolic re-programming to survive during stressful conditions. mTOR, MYC, and RAS are among the most popular signaling pathways which are frequently hijacked by the cancer cells to be exploited for re-programming of all metabolism, protein and organelle turnover, cell survival and bio-energetic functions (Qiu and

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Simon, 2015). Interestingly, almost all of these cancer-related signaling pathways are also found to intersect with autophagy at multiple levels. These observations suggest that autophagy plays dynamic and complex roles in cancer, which in fact might explain the two-faced nature of autophagy in carcinogenesis (Kimmelman and White, 2017). Evidence suggests

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that in the early stages of malignant transformation and/or cancer progression autophagy may act as a tumor suppressor, whereas in later stages it displays a rather protumorigenic

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nature to promote tumor maintenance and confer resistance to therapies. Although, targeting the autophagy-related pathways seems to be a promising tool for developing novel cancer

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therapeutics, findings point to the fact that the underlying molecular mechanisms and the specific targets of autophagy in cancer must be defined before it can be effectively exploited

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in pharmaceutical and medical research areas.

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To achieve this goal, important questions such as how the autophagic activity is differentially regulated in different cancers or which factors determine the tissue-specific inhibi-

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tion and/or activation of autophagy should be addressed. Additionally, animal models would allow tissue/organ specific, differential regulation of autophagic activity may also open new avenues for exploring the molecular connections between autophagy and cancer.

Conflict of interest The authors declare that there are no conflicts of interests.

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ACCEPTED MANUSCRIPT Acknowledgements This work was supported by Scientific and Technological Research Council of Turkey (TUBITAK)-1001 Grant number: 114Z836. YA is supported by TUBITAK-BIDEB 2211

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Scholarships for his PhD studies.

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ACCEPTED MANUSCRIPT Figure Legends Graphical Abstract. Bidirectional role of autophagy in cancer progression. The role of autophagy in cancer seems is bidirectional. In one sense, autophagy inhibits the progression of tumorigenesis by removing oncogenic proteins, accumulated free radicals etc.

and hypoxic conditions.

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On the other side, autophagy induction supports the survival of cancer under low-nutrient

Figure 1. Molecular mechanism of autophagy regulation in mammals.

Autophagic process consists of several phases such as initiation (A), nucleation (B), maturation (C), fusion and degradation (D). Same colours express the involvement of proteins or

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molecules in respective complexes or pathways. Figure 2. Tumor suppressor role of autophagy.

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Autophagy is involved in a variety of cellular mechanisms, each of which inhibits tumor progression by activating multiple molecular pathways.

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Figure 3. Tumor promoting role of autophagy.

Autophagy contributes to tumorigenesis in a variety of stages ranging from proliferation to

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metastasis and invasion as well as sustain its improvement by providing resistance to death

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Tables

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mechanisms.

Table 1. Role of autophagic proteins in cancer Table 2. Regulating autophagy for cancer treatment

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ACCEPTED MANUSCRIPT Chemical compounds studied in this article Allicin (PubChem CID: 65036) Apatinib (PubChem CID: 45139106) Arsenic trioxide (As2O3) (PubChem CID: 14888) Bafilomycin A1 (PubChem CID: 6436223)

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Bortezomib (PubChem CID: 387447) Brefeldin A (PubChem CID: 5287620) Camptothecin (PubChem CID: 24360) Cisplatin (PubChem CID: 5702198)

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Curcumin (PubChem CID: 969516) Chloroquine (PubChem CID: 64927)

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Diethylnitrosamine (DEN) (PubChem CID: 5921)

Epigallocatechin gallate (EGCG) (PubChem CID: 65064)

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Erlotinib (PubChem CID: 176870)

Everolimus (PubChem CIDL: 6442177)

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Ginsenoside F2 (PubChem CID: 9918692)

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Ginsenoside Rb1 (PubChem CID: 9898279) Gefitinib (PubChem CID: 123631)

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Hidroxychloroquine (PubChem CID: 3652) Imatinib (PubChem CID:5291) Lapatinib (PubChem CID: 208908) LY294002 (PubChem CID: 3973) Monensin (PubChem CID: 441145) NVP-BEZ235 (PubChem CID: 11977753) Polygonatum cyrtonema lectin (PCL) (PubChem SID: 103031201)

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ACCEPTED MANUSCRIPT Quinazolinediamine (QZN) (PubChem CID: 65087) Rapamycin (PubChem CID: 62969) Resveratrol (PubChem CID: 445154) Ridaforolimus (PubChem CID: 11520894) Romidepsin (PubChem CID: 5352062)

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SAR405 (PubChem CID: 72709209) Scutellarein (PubChem CID: 185617) Sertraline (PubChem CID: 68617) Sorafenib (PubChem CID: 216239)

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Spautin-1 (PubChem CID: 51037431) Spermidine (PubChem CID: 1102)

Tamoxifen (PubChem CID: 2733526)

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Trastuzumab (PubChem CID: 96849)

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Suberoylanilide Hydroxamic acid (SAHA) (PubChem CID: 5311)

Temozolomide (PubChem CID: 5394)

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Temsirolimus (PubChem CID: 86277830)

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Vandetanib (PubChem CID: 3081361) Varinostat (PubChem CID: 5311)

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Vinblastine (PubChem CID: 13342) Viridiol (PubChem CID: 5459246) Wortmannin (PubChem CID: 312145) 3-MethylAdenine(3-MA) (PubChem CID: 1673) 5-Fluorouracil (5-FU) (PubChem CID: 3385)

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ACCEPTED MANUSCRIPT Zou, Y., Ling, Y.H., Sironi, J., Schwartz, E.L., Perez-Soler, R., Piperdi, B., 2013. The autophagy inhibitor chloroquine overcomes the innate resistance of wild-type EGFR non-small-cell lung cancer cells to erlotinib. J. Thorac. Oncol.

AC

CE

PT

ED

MA

NU

SC RI PT

https://doi.org/10.1097/JTO.0b013e31828c7210

96

ACCEPTED MANUSCRIPT Table 1 Role of autophagic proteins in cancer Cancer type

Protein

Phase of autophagy

Status in cancer tissue

Reference

Tumor suppressor roles Colorectal carcinomas Colorectal carcinomas Gastric carcinomas Colorectal carcinomas Gastric and prostate carcinomas

Initiation

Mutated

UVRAG

Initiation

UVRAG

Initiation

AMBRA1

Initiation

Bif-1

Initiation

Breast carcinomas

FIP200

Initiation

Meningiomas

BECN1

Initiation

Decreased

Colorectal and gastric BECN1 carcinomas

Initiation

Increased

(Ahn et al., 2007)

Mutated

(Ionov et al., 2004)

Mutated

(Kim et al., 2008) (Cianfanelli et al., 2015)

Mutated

Decreased

(Lee et al., 2006)

Mutated

(Chano et al., 2002) (Miracco et al., 2007)

NU

MA

(Goi et al., 2003)

SC RI PT

UVRAG

BECN1

Initiation

Decreased

(Liang et al., 1999)

BECN1

Initiation

Decreased

(Shen et al., 2008)

Melanoma

ATG5

Bening liver tumor

ATG5

ED

Breast carcinomas Epithelial ovarian cancer

(Marino et al., 2007) (Takamura et al., 2011)

Decreased

Elongation

Decreased

Colorectal and gastric ATG5 carcinomas

Elongation

Mutated

(Kang et al., 2009)

Colorectal and gastric ATG12 carcinomas

Elongation

Mutated

(Kang et al., 2009)

ATG3

Elongation

Increased

Fibrosarcomas

ATG4C

Elongation

Decreased

Leukemia

RAB7A

Fusion

Mutated

(Ma et al., 2013) (Marino et al., 2007) (Kashuba, 1997)

Colorectal and gastric ATG2B carcinomas

Fusion

Mutated

(Kang et al., 2009)

Colorectal and gastric ATG9B carcinomas

Fusion

Mutated

(Kang et al., 2009)

Upstream

Increased

(Ma et al., 2000)

Leukemia

AC

CE

PT

Elongation

Oncogenic role Cervical carcinomas

PIK3CA

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ACCEPTED MANUSCRIPT (Chinen et al., 2014) (Nardella et al., 2008)

PDPK1

Upstream

Increased

Prostate carcinomas

RHEB

Upstream

Increased

Chronic myeloid leuATG4B kemia

Elongation

Increased

(Rothe et al., 2014)

Hepatocellular carcinomas

ULK1

Initiation

Increased

(Xu et al., 2013)

Breast carcinomas

ULK1

Initiation

Increased

(Pike et al., 2013)

Esophageal squaULK1 mous cell carcinomas

Initiation

Oral squamous cell carcinoma

ATG16L1

Elongation

Thyroid carcinomas

ATG16L1

Elongation

Colorectal carcinomas

ATG16L1

Elongation

SC RI PT

Multiple myeloma

(Jiang et al., 2011)

Increased

(Tang et al., 2015)

Mutated

(Huijbers et al., 2012)

Mutated

(Nicoli et al., 2014)

AC

CE

PT

ED

MA

NU

Increased

98

ACCEPTED MANUSCRIPT

Lung cancer

Mouse melanoma cell Renal tumor PIK3C3 cells Non-small cell lung cancer Glioblastoma multiform Colon cancer Lysosomal pH cells Head and neck cancer cells PIK3C3

Wortmannin

MA

NU

SAR405

ED

Chloroquine

Effect

Reference

Enhanced radiation sensitization

(Chen et al., 2011)

Enhanced antitumor effect Enhanced antitumor effect Enhanced antitumor effect Reduced proliferation Enhanced antitumor effect Enhanced antitumor effect Enhanced antitumor effect Enhanced radiation sensitization

(Li et al., 2009) (Liu et al., 2013) (Lin et al., 2014) (Pasquier, 2015) (Selvakumaran et al., 2013) (Sotelo et al., 2006) (Sasaki et al., 2010) (Cerniglia et al., 2012)

SC RI PT

Table 2 Regulating autophagy for cancer treatment Tumor/Cancer Compound Target cell type Inhibition of autophagy Esophageal squamous cell cancer 3-MA(3PIK3C3 Colorectal canmethyladenin) cer

(Cerniglia et al., 2012) (Rangwala et Hydroxychloroquine Lysosomal pH Melanoma al., 2014) Nasopharyngeal (Liu et al., carcinoma cells 2015) Gastric cancer (Li et al., VacuolarEnhanced anticells 2016) Bafilomycin A1 ATPase Osteosarcoma tumor effect (Xie et al., cells 2014) Colon cancer (Greene et al., cells 2013) Breast cancer (Liu et al., Induced cell death cells 2011) Inhibits ubiq- Ovarian cancer (Liu et al., Induced cell death uitin-specific cells Spautin-1 2011) peptidases Chronic myeEnhanced anti(Shao et al., loid leukemia tumor effect 2014) cells Lysosomal Cervical cancer Enhanced anti(Hsu et al., Pepstatin-A protease incells tumor effect 2009)

AC

CE

PT

Glioblastoma

99

Enhanced radiation sensitization Enhanced antitumor effect

ACCEPTED MANUSCRIPT hibitor

Autophagic proteins mRNA

siRNAs

Several cancer cells

Activation of autophagy Temsirolimus (CCI- mTORC1 inhibitors 779)

Imatinib (Gleevec)

Glioma

MA

Dasatinib (Sprycel)

(Wu et al., 2012)

Enhanced antitumor effect

(Yazbeck et al., 2008)

Enhanced antitumor effect

(Crazzolara et al., 2009)

SC RI PT

Rapamycin

NU

Everolimus (RAD001)

Mantle cell lymphoma Acute lymphomTORC1 blastic leukeinhibitors mia Malignant glioma mTORC1 Chronic myeinhibitors loid leukemia cells Chronic myeTyrosine kiloid leukemia nase inhibitors cells

Enhanced antitumor effect/enhanced radiation sensitization

Enhanced antitumor effect

(Ertmer et al., 2007)

Enhanced antitumor effect Enhanced antitumor effect

(Milano et al., 2009) (Gorzalczany et al., 2011) (Shao et al., 2004)

ED

PT

AC

Resveratrol

Toxin

CE

Arsenic Trioxide

Antioxidant

Polygonatum cyrtonema lectin

Lectin

Epigallocatechin-3gallate

Polyphenol

Curcumin

Polyphenol

Leukemia cells Malignant glioma Ovarian cancer cells Murine fibrosarcoma

Induced cell death

Induced cell death

Induced cell death

Melanoma cells Oral squamous cell carcinoma Malignant glioma Malignant glioma Breast cancer cells

100

(Takeuchi et al., 2005)

Enhanced antitumor effect

Non-small cell lung cancer Cervical cancer cells Butyrate, suberoyHDAC inhibiEnhanced antilanilide hydroxamic Chronic myetors tumor effect acid (SAHA) loid leukemia cells Erlotinib (Tarceva)

(Carayol et al., 2010)

Induced cell death

Induced cell death

(Carew et al., 2007) (Qian et al., 2007) (Kanzawa et al., 2005) (Opipari et al., 2004) (Liu et al., 2010) (Liu et al., 2009) (Irimie et al., 2015) (Aoki et al., 2007) (Shinojima et al., 2007) (Akkoç et al., 2015)

ACCEPTED MANUSCRIPT Lung cancer Thiosulfinate

Ginsenosides

Saponins

Liver cancer cells Breast cancer stem cells Colon cancer cells

Induced cell death (Kim, 2013)

AC

CE

PT

ED

MA

NU

SC RI PT

Allicin

(Xiao et al., 2013) (Chu et al., Induced cell death 2012) (Mai et al., Induced cell death 2012)

101

Figure 1

Figure 2

Figure 3