Therapeutic targeting of autophagy in cancer. Part II: pharmacological modulation of treatment-induced autophagy.

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Seminars in Cancer Biology xxx (2014) xxx–xxx

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Review

Therapeutic targeting of autophagy in cancer. Part II: Pharmacological modulation of treatment-induced autophagy Anika Nagelkerke a,b , Johan Bussink b , Anneke Geurts-Moespot a , Fred C.G.J. Sweep a , Paul N. Span b,∗ a b

Department of Laboratory Medicine, Radboud university medical center, PO Box 9101, 6500 HB, Nijmegen, The Netherlands Department of Radiation Oncology, Radboud university medical center, PO Box 9101, 6500 HB, Nijmegen, The Netherlands

a r t i c l e

i n f o

Keywords: Autophagy Cancer Therapy Inhibitor Inducer

a b s t r a c t Autophagy, the catabolic pathway in which cells recycle organelles and other parts of their own cytoplasm, is increasingly recognised as an important cytoprotective mechanism in cancer cells. Several cancer treatments stimulate the autophagic process and when autophagy is inhibited, cancer cells show an enhanced response to multiple treatments. These findings have nourished the theory that autophagy provides cancer cells with a survival advantage during stressful conditions, including exposure to therapeutics. Therefore, interference with the autophagic response can potentially enhance the efficacy of cancer therapy. In this review we examine two approaches to modulate autophagy as complementary cancer treatment: inhibition and induction. Inhibition of autophagy during cancer treatment eliminates its cytoprotective effects. Conversely, induction of autophagy combined with conventional cancer therapy exerts severe cytoplasmic degradation that can ultimately lead to cell death. We will discuss how autophagy can be therapeutically manipulated in cancer cells and how interactions between the conventional cancer therapies and autophagy modulation influence treatment outcome. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Autophagy is a cellular degradation process in which cells digest old, redundant or damaged organelles and proteins, thereby generating energy. The term autophagy often refers to the nonselective bulk degradation process known as macroautophagy. However, there are also other forms of autophagy: chaperonemediated autophagy and microautophagy. This review is dedicated to macroautophagy (hereafter referred to as autophagy). Exposure to cancer treatments subjects cells to stress. This can be either in the form of DNA damage (chemo- or radiotherapy), inhibition of cell proliferation, or impairment of growth factor- and metabolic-signalling. As a result of this stress therapies elicit an autophagic response in cancer cells, which helps to yield energy

and appears to be initially aimed at cell survival. However, if the stress is too severe or too prolonged, autophagy can become cytotoxic and lead to cell death. Multiple chemotherapeutics, radiotherapy, hormone therapy and several targeted therapies induce autophagy in different cancer types [1–14], providing a survival advantage for cancer cells during treatment. Therefore, interference with autophagy represents a rational therapeutic strategy. Autophagy modulation may counteract resistance to established cancer therapies or enhance the effect of these therapies. In this review, we will describe the current data on autophagy modulation as an anti-cancer strategy. We will show that interaction between treatments is crucial for the outcome of therapy.

2. The mechanism of autophagy DOI of original article: http://dx.doi.org/10.1016/j.semcancer.2014.05.004. ∗ Corresponding author at: Department of Radiation Oncology 874, Radboud university medical center, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Tel.: +31 24 3616845; fax: +31 24 3568350. E-mail addresses: [email protected] (A. Nagelkerke), [email protected] (J. Bussink), [email protected] (A. Geurts-Moespot), [email protected] (F.C.G.J. Sweep), [email protected] (P.N. Span).

The genes and proteins that comprise the basic machinery of the process of autophagy have been the topic of extensive research and multiple review papers [15–18]. In short, autophagy involves the formation of double membrane vesicles, in which cytoplasmic content is sequestered. This involves the action of multiple autophagy-related (ATG) proteins, such as Beclin1 (the mammalian homologue of yeast ATG6) and LC3B (the mammalian homologue of

http://dx.doi.org/10.1016/j.semcancer.2014.06.001 1044-579X/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Nagelkerke A, et al. Therapeutic targeting of autophagy in cancer. Part II: Pharmacological modulation of treatment-induced autophagy. Semin Cancer Biol (2014), http://dx.doi.org/10.1016/j.semcancer.2014.06.001

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Fig. 1. Outcome of treatment interactions in cancer cells. Treatment A can have different effects on cancer cells: (1) Cancer cell death by apoptosis. (2) Cell death by cytotoxic autophagy. (3) Cell survival by cytoprotective autophagy. Combining treatment A with treatment B exaggerates these effects, either synergising or antagonising. Depending on the nature of the treatment(s) and the combination, 1, 2 or 3 will be the main reaction. However, in a heterogeneous cell population 1, 2 and 3 may be present simultaneously. Addition of an autophagy inhibitor will: (1) Have no effect. (2) Inhibit cytotoxicity. (3) Inhibit cell survival by driving cells into apoptosis. Autophagy inhibition in situation 2 should be avoided as it counteracts cell death.

yeast ATG8). The content of autophagosomes is degraded by fusion with lysosomes. For a comprehensive overview of the core molecular pathways in autophagy, as well as of the signalling routes that influence autophagy, we refer to part I of this review. 3. Modulation of autophagy as cancer therapy Autophagy can lead to contradictory endpoints: cell survival and death. Survival represents a self-defence mechanism to withstand therapy-induced cell death. However, excessive and prolonged autophagy impedes cell recovery and survival. This induces a cell death programme, known as autophagic cell death or programmed cell death type II, but can also stimulate apoptosis [19]. Both repression and stimulation of autophagy are therefore realistic therapeutic approaches (see Fig. 1). (Hyper)activation of autophagy forces tumour cells into autophagic cell death. Autophagic cell death differs from apoptosis as it is associated with an increased formation of autophagosomes and is caspase-independent. The use of anticancer agents that provoke autophagic cell death could be particularly beneficial when apoptosis is defective and cancer cells should be driven into non-apoptotic types of cell death. Conversely, inhibition of autophagy prevents its use as a survival mechanism during therapy. In cells with intact apoptotic signalling, autophagy inhibition could drive cells into apoptosis. Interference with autophagy can be accomplished by modulation of several pathways at various levels (see part I of this review). Consequently, the number of treatments that have an effect on autophagy is vast. Supplementary Table 1 provides a (non-exhaustive) list of compounds with autophagy modulating properties. The possible combinations with these treatments are even more immense. Several treatment interactions between different compounds are given in supplementary Table 2. Some of these interactions will be discussed below. Many of the compound listed in supplementary Table 1 are analysed in clinical trials, both as single agents and in combinations with other therapies. Supplementary Tables S1 and S2 can be found, in the online version, at doi:10.1016/j.semcancer.2014.06.001. 3.1. Autophagy induction 3.1.1. mTOR inhibitors Mechanistic target of rapamycin (mTOR) is a key repressor of autophagy. Therefore, mTOR inhibition, leading to the activation

of the ULK-complex (UNC-51-like kinase-complex), is a valuable approach to stimulate autophagy. Treatment with the mTOR inhibitor rapamycin has marked anti-tumour effects in MCF-7 and MDA-MB-231 xenografted tumours, mostly because of inhibition of angiogenesis [20]. Rapamycin reduces carcinogen-induced lung tumours in a murine model [21]. In addition, concurrent administration of rapamycin with radiation significantly increases breast cancer cell death [10] and also sensitises radiotherapy resistant hepatocellular carcinoma to radiation [22]. Concurrent administration of RAD001, a rapamycin-derivative, and radiotherapy increases sensitivity to radiation in both breast cancer cells [23] as well as prostate cancer cells [24]. Recently, combining autophagy activation by mTOR inhibition with radiation was shown to induce cellular senescence in cancer cells and xenografts [25]. This leads to enhanced cytotoxic effects. Nevertheless, inhibiting mTOR may have unwanted side-effects. Treatment with RAD001 results in a significant occurrence of distant metastases in a rat model of pancreatic cancer [26]. mTOR inhibitors, whilst repressing mTOR-signalling, also activate AKT [27,28]. This may counteract their anti-cancer effects. Combining mTOR inhibitors with AKT inhibitors enhances anti-tumour properties of mTOR inhibitors [27–30].

3.1.2. AMPK induction mTOR-signalling can also be disrupted through AMP-activated protein kinase (AMPK), for example by metformin. Metformin is an inhibitor of the mitochondrial electron transport chain complex I, which leads to decreased ATP production and increased AMP. This causes AMPK induction and mTOR inhibition. Metformin has cytostatic effects in several cancer cell lines and decreases tumourigenesis in a rodent cancer model [31]. The combination of chemotherapy and metformin is more harmful for breast cancer cells than the treatment with chemotherapy or metformin alone [32]. In prostate cancer cells, metformin synergises with 2-deoxyglucose [33]. Metformin inhibits 2-deoxyglucose-induced autophagy and instead forced cells into apoptosis. Metformin has been reported to improve tumour oxygenation and hence radiotherapy response [34]. Conversely, there are reports that combining metformin reduces cell death induced by chemotherapy [35,36].




3.2. Autophagy inhibition Autophagy inhibition has been studied in more detail than autophagy induction. The autophagic pathway itself can be repressed by knockdown of ATGs or by pharmacological inhibition. Knockdown of essential ATGs, such as Beclin1, LC3 or ATG5 impedes autophagy [37]. By inhibiting the class III phosphatidylinositol-3 kinase (PI3), 3-methyladenine, LY294002 and wortmannin repress autophagy. However, due to homology of class I and class III PI3Ks, these compounds can also induce autophagy when applied in different concentrations [38]. Recently, spautin-1 was identified as a potent small molecule inhibitor of autophagy [39]. It causes the degradation of the class III PI3K complex by targeting Beclin1. Preclinical studies using spautin-1 have shown synergy with clinically relevant cancer therapies [40,41]. At a later stage, autophagy can be inhibited by preventing autophagosome–lysosome fusion, for example by bafilomycin A1 or chloroquine, but also by interference with the microtubule network. Vinblastine, a microtubule depolymeriser, causes blocked fusion of autophagosome and lysosome [42]. Bafilomycin A1 is an inhibitor of vacuolar-type H+ -ATPase and prevents lysosomal acidification. Chloroquine accumulates inside lysosomes, preventing acidification, impairing lysosomal enzymes and blocking fusion. Chloroquine is used as an anti-malarial and in the treatment of rheumatoid arthritis. Its activity as a potent autophagy inhibitor is being increasingly explored in cancer therapy. Recently, Settembre et al. [43] reported that chloroquine can also induce autophagy. Chloroquine was found to inhibit the activity of mTORC1, as shown by a decrease in the levels of the mTORC1 substrate p-p70S6K. Via mTORC1, chloroquine upregulates target genes of the transcription factor EB (TFEB), stimulating autophagy and lysosomal biogenesis. Nevertheless, even as a monotherapy chloroquine is effective in preclinical experiments. Chloroquine treatment of breast cancer cells and tumours inhibits proliferation and triggers apoptosis in a p53-dependent manner [44–46]. Consequently, chloroquine treatment has only minor effects on tumour growth of MDA-MB231 xenografts, whereas growth of MCF-7 xenografts is inhibited [20,47]. In a model of carcinogen-induced mammary tumours, chloroquine significantly reduces the frequency, but again only when p53 is intact [48]. In addition, formation of lung metastases in mice is inhibited by chloroquine treatment [44]. Compared to nontumourigenic breast epithelial cells, cancer cells are more sensitive to growth inhibition by chloroquine [49]. 3.2.1. Autophagy inhibition by chloroquine combined with established cancer therapies The idea that addition of chloroquine to established cancer therapies enhances treatment efficacy is increasingly explored, not only in preclinical experiments (see following section) but also in clinical trials [50,51]. In patients, chloroquine, with or without additional treatment is well-tolerated [52]. Furthermore, in a small trial chloroquine with conventional therapies, prolonged survival of patients with glioblastoma multiforme, but differences were not statistically significant [53]. 3.2.1.1. Radiotherapy. Radiotherapy induces autophagy via hypophosphorylation of mTOR [10], but the PERK-arm (PKRlike endoplasmic reticulum kinase) of the unfolded protein response may also mediate autophagy induction after radiation [54]. Autophagy inhibition, either genetic or pharmacological, diminishes autophagosome accumulation after irradiation [1,3,9]. Clonogenic survival after radiotherapy is reduced by chloroquine, at least in relatively radioresistant breast cancer cell lines [3,55]. The use of chloroquine to enhance the response of tumour cells

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to irradiation was already reported in 1973 by Kim et al. [56]. However, in this study pre-treatment with chloroquine was ineffective. Instead, chloroquine was given after irradiation and inhibited the post-irradiation recovery process. Ratikan et al. confirmed these data and also show that in vivo the chloroquine effect is only present in immune-competent mice not in immuneincompetent mice [57]. This suggests an immune-modulating role for chloroquine. Furthermore, autophagy inhibition by ATG5 or Beclin1 knockdown leads to increased sensitivity to radiotherapy in cancer cell lines and xenografts in immune-deficient mice [58]. However, in immune-competent mice, knockdown leads to resistance to radiotherapy, suggesting that autophagy has immunogenic properties in tumours. In xenografted tumours, autophagy markers are mainly present in hypoxic regions [59]. Autophagy inhibition by chloroquine or 3methyladenine renders cultured colon cancer cells less tolerant to conditions of hypoxia [59]. Chloroquine cannot sensitise these cells to radiotherapy. However, treatment of established xenografts with chloroquine does enhance radiosensitivity. Chloroquine reduces the hypoxic fraction of the tumours, thereby increasing the effect of radiotherapy [59]. Under hypoxic conditions, autophagy also mediates resistance to chemotherapy [60]. 3.2.1.2. Chemotherapy. 5-Fluorouracil combined with chloroquine enhances complete remissions in murine mammary carcinoma, compared to 5-fluorouracil or chloroquine alone [61]. Similar synergistic effects of autophagy inhibition and chemotherapeutics are found in other cancer models. The efficacy of 5-fluorouracil is increased by 3-methyladenine or ATG7 knockdown in colon cancer cells and xenografts [62]. In chemoresistant oesophageal cancer cells Beclin1 and ATG7 knockdown enhance the effect of 5-fluorouracil [63]. However, indirect inhibitors of autophagy, 3methyladenine, bafilomycin A1 and chloroquine are ineffective in this study. 3.2.1.3. Tamoxifen. Survival of oestrogen receptor positive breast cancer cells during treatment with tamoxifen critically depends on autophagy [64]. In addition, tamoxifen treatment has also been associated with autophagic cell death [2,65]. Combined administration of tamoxifen and autophagy inhibitors induces a strong, caspase-dependent apoptosis [11,12]. Tamoxifen treatment leads to ceramide production [66], which induces autophagy in glioma cells [67]. In addition, tamoxifen has been demonstrated to promote autophagic degradation of the proto-oncogene KRAS [65], which impairs the autophagy-dependent survival of RAS mutated tumours (see part I of this review). 3.2.1.4. Targeted therapies against EGFR. Autophagy inhibition with chloroquine or ATG5/Beclin1 silencing in non-small cell lung cancer cells enhances sensitivity to erlotinib [8,14]. Chloroquine treatment also increases the anti-tumour activity of erlotinib in xenografts. Cetuximab, a therapeutic antibody against EGFR, stimulates autophagy [6,7]. Interestingly, this is dependent on downregulation of hypoxia-inducible factor 1␣ (HIF1␣). Beclin1 or ATG7 knockdown as well as chloroquine treatment sensitises to cetuximab and drives cells into apoptotic cell death. Inducing autophagy with rapamycin also increases cell death by cetuximab. This is ATG-dependent and lysosomal inhibition-sensitive. Apparently, here autophagy inhibition abolishes the use of autophagy as a survival mechanism during cetuximab treatment, whereas autophagy induction forces cells to enhance autophagy to such a level that it becomes cytotoxic. 3.2.2. Choice of autophagy inhibitor Not all pharmacological autophagy inhibitors interfere with autophagy at the same point. Therefore, differences between


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inhibitors may occur. FK866 is a compound that causes depletion of the co-enzyme NAD+ (nicotinamide adenine dinucleotide). Cancer cells have a higher turnover of NAD+ than normal cells. In neuroblastoma cells, FK866 induces autophagy and potentiates the effects of cisplatin and etoposide [68]. Chloroquine also enhances the cytotoxicity of FK866. However, 3-methyladenine or ATG7 silencing represses FK866 cytotoxicity. Other examples of differences between autophagy inhibitors were reported in glioma cells. Here, cytotoxicity induced by imatinib is reduced by 3-methyladenine, whereas it is enhanced by bafilomycin [69]. Temozolomide induces autophagy, but not apoptosis in glioma cells [70] and addition of 3-methyladenine suppresses the antitumour effect of temozolomide. Conversely, bafilomycin sensitises cells by inducing apoptosis. Addition of both 3-methyladenine and bafilomycin did not sensitise cells. Chloroquine enhances cell death induced by dasatinib in CML cells [71]. However, no change is observed when 3-methyladenine is added to dasatinib. In liver and colon cancer cells and xenografts nanoliposomal C6-ceramide induces autophagy [72]. Vinblastine or chloroquine synergistically enhances apoptotic cell death induced by C6-ceramide. This is accompanied by a marked increase of autophagic vacuoles. In contrast, inhibition of autophagy initiation is not synergistic. These data suggest that late stage autophagy inhibition is superior to synergise with cancer therapy compared to early stage autophagy inhibition. Macroautophagy is the predominant form of autophagy. However, several types of cancer cells also show induction of chaperone-mediated autophagy, a selective lysosomal degradation process. Chaperone-mediated autophagy regulates tumour growth and progression [73]. Its inhibition reduces tumourigenesis. Macroautophagy and chaperone-mediated autophagy can compensate for one another as there is considerable cross-talk between both pathways. However, early stage autophagy inhibitors (3-methyladenine, LY294002 and wortmannin) have little effect on chaperone-mediated autophagy [74]. This in contrast to chloroquine, which can inhibit both processes. The minor effects of early stage autophagy inhibitors on therapy efficacy could be a consequence of compensation by chaperone-mediated autophagy. 3.3. Autophagy induction combined with autophagy inhibition Promising preclinical studies have combined multiple activators of autophagy with an autophagy inhibitor. In glioma cells and mouse models, the AKT inhibitor MK2206 acts synergistically with gefitinib [75]. This drug combination shows increased levels of autophagy. Their cytotoxicity is enhanced even further by inhibiting autophagy, either by 3-methyladenine or Beclin1 silencing. Treatment of glioma cells with temozolomide induces cell death [76]. RAD001 potentiates this cytotoxicity by enhancing autophagic cell death. Addition of radiation to the temozolomide/RAD001 combination enhances cytotoxicity even further. PI-103, a dual class I PI3K/mTOR inhibitor, also enhances the cytotoxicity of temozolomide with radiotherapy in glioma cells [77], whereas rapamycin shows no enhancing effect. In NSCLC cells, the mTOR inhibitor CCI-779 combined with the AKT inhibitor GSK690693 synergistically increases cell death by stimulated autophagy [29]. Addition of 3-methyladenine enhances cell death even further. In a panel of cancer cell lines, the dual class I PI3K and mTOR inhibitor, NVP-BEZ235 is an effective sensitiser for radiotherapy [78]. This treatment combination stimulates autophagy. Inhibition of autophagy by 3-methyladenine, chloroquine or ATG5/Beclin1 silencing enhances cell death by NVP-BEZ235 combined with radiation. Clarithromycin, a macrolide antibiotic, has been reported to block autophagic flux [79,80]. In breast cancer cells clarithromycin enhances the cytotoxicity of the proteasome inhibitor bortezomib. Surprisingly, the combination of clarithromycin and bortezomib

leads to a more pronounced stimulation of autophagy than bortezomib by itself. Addition of an HDAC inhibitor (vorinostat or tubacin) to treatment with clarithromycin and bortezomib stimulates induction of apoptosis in comparison with either agent alone or the double-agent treatment [79]. Obatoclax (GX15-070) is a BH3 mimetic that induces autophagy [81]. In acute myelogenous leukaemia cells the multikinase inhibitor sorafenib enhances obatoclax-induced autophagy and reduces clonogenic growth. The lethality of this drug combination is enhanced by pharmacological autophagy inhibitors and VPS34 knockdown. In oesophageal carcinoma cells, obatoclax synergises with carboplatin and 5fluorouracil to inhibit cell growth and induce autophagy [82]. Treatment with 3-methyladenine or chloroquine reinforces the cytotoxicity. Obatoclax combined with vorinostat in leukaemia cells leads to synergistic anti-cancer effects [83] and this effect is mediated by autophagy. However, autophagy inhibition by chloroquine inhibits the synergy between obatoclax and vorinostat. Obatoclax combined with the dual EGFR/HER2 inhibitor lapatinib leads to a synergy in breast cancer cell death due to a toxic form of autophagy [84]. 3-Methyladenine reversed this synergy and suppresses cell death. Chloroquine has no effect on the outcome of the drug combination. In contrast, rapamycin promotes toxicity of the obatoclax/lapatinib combination even further. In gastric cancers with a PTEN (phosphatase and tensin homolog deleted on chromosome 10) mutation, RAD001 and MK2206 have synergistic anti-cancer activity [27]. This inhibition of the AKT/mTOR pathway is accompanied by activation of extracellular signal-regulated kinase (ERK) and autophagy. However, inhibition of the ERKpathway with PD98059 and U0126 or autophagy inhibition with 3-methyladenine or chloroquine result in reduced cytotoxicity of the RAD001/MK2206 combination. 3.4. Autophagy modulation to reverse therapy resistance The efficacy of cancer therapy is severely impaired by the development of therapy-resistance. Modulation of autophagy may provide means to alleviate resistance. Breast cancer cells cultured resistant to trastuzumab or with intrinsic trastuzumab resistance display higher autophagy levels than parental, sensitive cells. Knockdown of ATGs or chloroquine treatment reduces proliferation of resistant cells and tumours, and even resensitises them to trastuzumab [4,13]. Tamoxifen-resistant breast cancer cells show increased levels of basal autophagy and are resensitised to tamoxifen by silencing of autophagy-associated genes [11,64]. Treatment with vorinostat also reduces tamoxifen-resistance [85]. Vorinostat-resistant clones of malignant haematological cells show increased basal autophagy levels and are sensitive to chloroquine [86]. Chloroquine even restores vorinostat sensitivity. However, in parental cells, chloroquine decreases the sensitivity to vorinostat. Conversely, mTOR inhibition by rapamycin or NVP-BEZ235 synergises with vorinostat to induce cell death in parental cells, but resistant cells remain resistant. 4. Functions of autophagy in cancer In the final section of this review, the possibility that autophagic activity in tumours can also have beneficial anti-cancer effects will be discussed. These aspects need to be taken into account when applying autophagy modulation as an anti-cancer strategy. 4.1. Autophagy and immunogenic cell death As indicated in Section 3.2.1.1, autophagy may have immunogenic properties. It has been reported that in cancer, autophagy




is not essential for cell death after chemotherapy [87]. However, autophagy is required for the anti-tumour immune response. Immunogenic cell death is a form of apoptosis in which dying cancer cells can stimulate a tumour-specific immune response, aimed at removal of residual disease. Autophagy has emerged as an important contributor to this response. Autophagy-proficient tumours attract immune cells after chemotherapy, whereas autophagy-deficient tumours do not [87]. Immunogenic cell death is characterised by expression of calreticulin on the cell surface prior to apoptosis (the “eat me”-signal) and by release of high mobility group box 1 protein (HMGB1) and ATP after apoptosis (the “find me”-signal). Calreticulin expression is generated by a chemotherapy-induced endoplasmic reticulum stress response [88], involving phosphorylation of eIF2␣ by PERK. Activation of the PERK-arm by inhibition of GADD34 (growth arrest and DNA damage-inducible protein 34) or treatment with thapsigargin induces calreticulin exposure. ATP release is reduced in autophagydeficient tumours, whereas HMGB1 and calreticulin exposure appears unaffected [87]. 4.2. Autophagy and senescence Interestingly, autophagy has also been linked to cellular senescence. Cellular senescence reflects an irreversible growth arrest that occurs naturally as cells age (replicative senescence), but can also be induced by cancer therapy (treatment-induced senescence). Despite their growth arrest, there is evidence that senescent cells are metabolically active and secrete proteins with tumour-inhibiting and -stimulating properties [89]. Recently, it has been shown that autophagy actively contributes to senescence, rather than both processes operating independently [90,91]. During senescence several autophagy related proteins are induced. Autophagy inhibition reduces senescence, whereas induction of autophagy increases the senescent phenotype [90–92]. In contrast, in primary human diploid fibroblasts, knockdown of autophagy related genes resulted in premature senescence [93]. Overall, it remains to be elucidated exactly how autophagy-regulated senescence influences cancer cell survival.

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Box 1: Analyzing autophagy levels is complex There are discrepancies in the literature on autophagy. Studies have reported different effects of treatment on autophagy and treatment interactions have had different outcomes, some are reported in supplementary Tables 1 and 2. The root of this problem may be that autophagy is a difficult process to visualise. Increased numbers of autophagosomes are not necessarily indicative of stimulated autophagy. They may represent a stimulated autophagosome production, but could also represent an autophagosome accumulation due to a decreased or blocked turnover. In contrast, decreased autophagosome numbers not necessarily indicate reduced autophagosome formation as they could also represent an increased autophagic flux. Analysing the dynamic nature of autophagy with static techniques may therefore lead to misinterpretation of experimental results. Adherence to previously defined guidelines is essential to clarify conflicting data [94].

this will be difficult to measure (see Box 1). Nevertheless, there is evidence that certain tumour types will benefit from autophagy inhibition, for example the tumours that harbour a RAS mutation. How should autophagy modulation be accomplished? Currently, autophagy modulation for clinical purposes can only be accomplished by reassigning existing therapeutics to this purpose. For example, chloroquine is used as an anti-malarial, chlorpromazine as an anti-psychotic, valproic acid as an anti-epileptic and atorvastatin as an anti-cholesterol drug. Therefore these drugs do not specifically and exclusively modulate autophagy and may have off-target effects. The same applies to adapting cellular signalling pathways that initiate autophagy. In addition, these pathways contain multiple feedback loops and back-up mechanisms. These may interfere with the desired effect on autophagy modulation, whereas adaptation of the core machinery would specifically affect autophagy. However, these compounds are scarce and are primarily used as research tools, unsuitable for clinical use. The development of specific autophagy modulating compounds suitable for use in patients is eagerly awaited. Conflict of interest

5. Conclusions and future perspectives The authors declare that there are no conflicts of interest. Autophagy modulation, either stimulatory or repressive, can be achieved by interfering with multiple pathways at multiple levels (as illustrated in Part I of this review). Therefore, the number of compounds with autophagy modulating properties is vast, as are the treatment interactions between these different compounds. Before autophagy modulation can be incorporated into clinical practice, several (partially) unanswered questions require attention. Should autophagy be stimulated or repressed? From the data discussed in this review it appears that both strategies are promising. However, the outcome of drug combinations critically depends on treatment interactions. Some studies have reported negative outcome for certain combinations. These will have to be studied meticulously to identify underlying causes of synergy-failure. Potential side effects should also be monitored (such as the effect on the anti-tumour immune response or senescence). Which tumours will be sensitive to autophagy modulation, or which patients will benefit from autophagy modulation? This will require an extensive tumour characterisation, considering the following. Different tumour types show different responses to autophagy modulation and may differ in sensitivity. The composition of the tumour microenvironment (for example the presence of hypoxia) has influence on treatment efficacy. Basal levels of autophagy in tumours can affect response, although in patients

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