Autophagy modulation for cancer therapy

Abstract

Autophagy is a homeostatic and catabolic process that enables the sequestration and lysosomal degradation of cytoplasmic organelles and proteins that is important for the maintenance of genomic stability and cell survival. Beclin 1^+/− gene knockout mice are tumor prone, indicating a tumor suppressor role for autophagy. Autophagy is also a mechanism of stress tolerance that maintains cell viability and can lead to tumor dormancy, progression and therapeutic resistance. Many anticancer drugs induce cytotoxic stress that can activate pro-survival autophagy. In some contexts, excessive or prolonged autophagy can lead to tumor cell death. Inhibition of cytoprotective autophagy by genetic or pharmacological means has been shown to enhance anticancer drug-induced cell death, suggesting a novel therapeutic strategy. Studies are ongoing to define optimal strategies to modulate autophagy for cancer prevention and therapy, and to exploit it as a target for anticancer drug discovery.

Introduction

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved, homeostatic process that functions to dispose of damaged cellular organelles and proteins. Autophagy involves the formation of double membrane vesicles, known as autophagosomes, that engulf intracellular contents and fuse with lysosomes for their degradation.¹ Monoallelic loss of the essential autophagy gene Beclin1 in mice promotes tumorigenesis, and this abnormality is also found in human cancers, indicating a role for autophagy in tumor suppression.^2,3 Autophagy defective tumor cells accumulate the adaptor protein p62/sequestosome1 (p62/SQSTM1) that can lead to genome damage and promote tumor formation.⁴ Autophagy is induced in cancer cells as a mechanism of stress tolerance when cells are exposed to nutrient deprivation, hypoxia, reactive oxygen species (ROS) or anticancer treatments.¹ Autophagy generates energy from the recycling of cellular proteins and organelles.^5,6 In established tumors, stress-induced autophagy enables tumor cell survival which can facilitate tumor progression and confer resistance to anticancer treatments. Accordingly, targeting cytoprotective autophagy in cancers has the potential to enhance treatment efficacy. Autophagy inhibition has been shown to promote apoptotic cell death.⁷ Inducing excessive autophagy can be pro-death in certain contexts, and should be considered as an alternative tumor killing strategy, particularly in apoptosis-defective cells.^8–10 A complex interplay exists between autophagy and apoptosis and strategies to exploit this cross-talk may also hold therapeutic potential. In this review, the multiple roles of autophagy in tumor biology will be outlined including its emergence as a target for both cancer prevention and cancer treatment.

Regulation of Autophagy

Core machinery.

Autophagy is regulated by Beclin 1 that forms a complex with vacuolar sorting protein 34 (Vps34), a class III phosphoinositide 3-kinase (PI3K), and serves as a platform for recruitment of other autophagy-related proteins that are critical for autophagosome formation.¹¹ During the initiation phase, Atg12 conjugates Atg5 which in turn promotes the recruitment and conversion of cytosolic-associated protein light chain 3 (LC3-I) to the membrane-bound LC3-II form.¹² Upon completion of autophagosome formation and with the exception of a proportion of LC3-II bound to the luminal membrane, the Atg proteins are then recycled in the cytosol.¹¹ Biochemical or microscopic detection of the conversion of LC3-I to LC3-II is a biochemical hallmark of autophagy since the lipidated form (LC3-II) is stably associated with the autophagosomal membrane.¹³ LC3-II binds to p62/SQSTM1 that is involved in trafficking proteins to the proteasome and serves to facilitate the autophagic degradation of ubiquitinated protein aggregates.¹⁴ p62/SQSTM1 is normally degraded with autophagosomes and accumulates when autophagy is impaired.⁴ The late events in autophagy involve the final maturation and fusion of autophagosomes with lysosomes, a step requiring the small GTPase Rab7 and lysosome-associated membrane protein2 (Lamp2).^7,15

Regulation of stress-induced autophagy (Fig. 1).

Figure 1.

Autophagy pathway and relationship to mTOR signaling. Cellular stress (hypoxia, genotoxic, cytotoxic) activates autophagy as a stress response and cell survival mechanism. Inhibition of cytoprotective autophagy using 3-MA or CQ/HCQ can augment tumor cell death. Inhibition of mTOR induces autophagy, and in certain cellular contexts, excessive or sustained autophagy may promote cell death. mTOR inhibits autophagy by blocking the formation of ULK-Atg13-FIP200 trimeric complex.

The mammalian target of rapamycin (mTOR) serves as a central regulator of autophagy and forms two distinct signaling complexes, known as mTORC1 and mTORC2.¹⁶ A number of upstream signaling pathways are involved in autophagy regulation that converge on mTOR, and include the class I PI3K-AKT pathway which is frequently dysregulated in human cancer.¹⁷ Activation of mTOR can occur due to loss of tumor suppressors (LKB1, PML, PTEN, TSC1/2) or through gain-of-function mutations in receptor tyrosine kinases.¹⁸ Under stress stimuli that include metabolic stress, hypoxia and mitochondrial damage, suppression of mTOR triggers the autophagic cascade and inhibits cell proliferation.¹⁶ Nutrient deprivation results in activation of adenosine monophosphate kinase (AMPK) which is a central metabolic sensor with important functions in regulating lipid and glucose metabolism. Activation of AMPK serves to repress mTOR and to initiate autophagy.¹⁹ mTOR negatively regulates autophagy by phosphorylation of Atg13 and Unc51-like kinase (ULK) [human homolog of Atg1], and inhibits formation of a trimeric complex required for autophagosome formation.^20,21 Therefore, the trimeric complex ULK-Atg13-FIP200 is a direct target of mTOR.

A major ER stress pathway is the unfolded protein response (UPR) that can potently induce autophagy. The binding of misfolded proteins to the ER chaperone Bip/GRP78 leads to release of three ER membrane-associated proteins, PKR-like eIF2α kinase (PERK), activating transcription factor-6 (ATF6) and inositol-requiring enzyme 1 (IRE1).^22,23 While PERK and ATF6 are autophagy inducers, IRE1 negatively regulates autophagy. Other factors that link cellular stress with autophagy is the transcription factor NF-kappaB(NF-kB) and its upstream regulators, IKK complex and TAK1, that integrate diverse stress signals including starvation, rapamycin or ER stress with the autophagy pathway.²⁴ Autophagy induction by hypoxia is regulated through various mechanisms including HIF (hypoxia-inducible factor).²⁵

The tumor suppressor p53 protein can modulate autophagy depending upon its cellular localization. Nuclear p53 acts as a transcription factor that transactivates several autophagy inducers including DRAM1 and Sestrin2 to activate autophagy,²⁶ whereas cytoplasmic p53 inhibits autophagy by an unknown mechanism. Inducers of autophagy can stimulate proteosomemediated degradation of p53.²⁷ Recently, some novel regulators of autophagy have been found. Ataxia-telangiectasia mutated (ATM) is a cellular damage sensor that coordinates the cell cycle with DNA damage-response checkpoints and DNA repair, and engages the TSC2/mTORC1 signaling axis to regulate autophagy.²⁸ Additionally, the high mobility group box 1 (HMGB1) is an immune modulator and regulator of stress-induced autophagy⁵ that directly interacts with Beclin 1.²⁹

Interplay between autophagy and apoptosis.

Evidence suggests a complex interplay between autophagy and apoptosis pathways.³⁰ Autophagy can be an alternative mode of cell death in apoptosis-resistant cells.^8–10 Conversely, apoptosis is promoted when autophagy is inhibited.^7,31,32 Non-apoptotic cell death accompanied by autophagy is also well described in cancer cells.^33–35 Cross-talk between autophagy and apoptosis exists at many levels where both processes share mediators ranging from the core machinery (Atg5, Beclin 1-Bcl-2/Bcl-x_L interaction) to upstream regulating pathways (AKT, DAPK, ARF, E2F1, HMGB1 and p53). Furthermore, recent findings link p62/SQSTM1 activity to caspase-8 activation and the extrinsic apoptosis pathway.³⁶ HMGB1 release has been shown to regulate both autophagy and apoptosis, depending upon its redox state.³⁷ Cytoplasmic translocation of HMGB1 was shown to mediate stress-induced autophagy through the MEK-ERK pathway and limited apoptosis.²⁹

Autophagy in Cancer

The tumor suppressor role of autophagy.

Autophagy has been shown to play a role in tumor suppression. Beclin 1 is a haploinsufficient tumor suppressor gene and Beclin 1^+/− mice are tumorprone.^2,3 Monoallelic loss of Beclin 1 has been detected in human breast, ovarian and prostate cancers.³⁸ Conversely, Beclin 1 over-expression can inhibit tumor development.³⁹ A potential link between defective autophagy and tumorigenesis is the accumulation of p62/SQSTM1 protein aggregates, damaged mitochondria and misfolded proteins that lead to the production of ROS to cause DNA damage.⁴ The accumulation of p62/SQSTM1 is frequently observed in human tumors with defective autophagy.⁴⁰ Suppressing p62/SQSTM1 accumulation or ROS production was shown to prevent damage resulting from defective autophagy.⁴ p62/SQSTM1 has a TRAF6-binding domain and promotes the oligomerization of TRAF6 leading to the activation of NFκB.⁴¹ Autophagy may also protect against tumorigenesis through limiting necrosis and chronic inflammation that is associated with the release of pro-inflammatory HMGB1.^5,42

Autophagy in cytoprotection and as a therapeutic target.

A cytoprotective or pro-survival function of autophagy in cancer cells is supported by substantial evidence.⁴³ Cancer cells have increased metabolic demands due to high levels of cellular proliferation and cellular stress activates autophagy to maintain energy and to prevent death in these cells. Autophagy confers stress tolerance, limits damage and confers a survival advantage to cancer cells. Furthermore, autophagy may enable a state of dormancy in residual cancer cells post treatment that may contribute to cancer recurrence and metastasis.⁴⁴ Autophagy has been shown to localize to hypoxic regions of tumors most distal to blood vessels, where it contributes to the maintenance of cell viability.^5,45 The upregulation of autophagy in regions of hypoxia^5,45 is enhanced by HIF-1α expression,⁴⁶ although it can also be independent of HIF-1.^18,47 Given the pro-survival role of autophagy, its inhibition has been shown to enhance the efficacy of anticancer therapy.⁴³ Understanding the role of autophagy in cancer treatment is critical since many anticancer therapies activate autophagy that may limit their therapeutic efficacy. Along with conventional cytotoxic drugs, other anti-neoplastic and pharmacological agents such as kinase inhibitors,^48–51 proteosome inhibitors,⁵² arsenic trioxide,⁵³ tamoxifen,⁹ lucanthone,⁵⁴ cyclooxygenase inhibitors⁵⁵ and the HIV protease inhibitor, nelfinavir⁵⁶ have been shown to induce autophagy in tumor cells (Table 1).

Table 1.

Modulation of autophagy in cancer therapy

Compound	Target	FDA approved indication in cancer	Tumor type/drug development status
Autophagy inducers
mTOR inhibitors	mTORC1
- rapamycin		-	Malignant glioma/in vitro94
- everolimus		Renal cell carcinoma, subependymal giant cell astrocytoma	ALL/in vivo95
- temsirolimus		Renal cell carcinoma	Mantle cell lymphoma/in vitro96
Akt inhibitors	Akt
- perifosine		-	Lung cancer/in vitro97
- triciribine		-	T-cell ALL/in vitro98
Radiation	DNA	Different tumor types	Breast, colon and prostate cancer/in vitro77
Proteosome inhibitor	Proteosome
- bortezomib		Multiple myeloma, mantle cell lymphoma	Prostate cancer/in vitro52
EGFR antibody	EGFR
- cetuximab		Head and neck cancer, metastatic colorectal cancer	Vulvar, colorectal and lung cancer/in vitro51
HDAC inhibitors	Histone deacetylase
- vorinostat - suberoylanilide hydroxamic acid (SAHA)		Primary cutaneous T-cell lymphoma	Multiple cancers/in vitro35, Leukemia/in vitro100
- osu-HDAC42		-	Hepatocellular carcinoma/in vitro99
			Phase I in solid tumors www.clinicaltrials.gov
Tamoxifen	Estrogen receptor	Breast and endometrial cancer	Breast cancer/in vitro9
Temozolomide (alkylating agent)	DNA	Anaplastic astrocytoma, glioblastoma multiforme, metastatic melanoma	Malignant glioma/in vitro75
			Glioblastoma/Phase I101
Arsenic trioxide	-	Acute promyelocytic leukemia	Malignant glioma/in vitro53
Tyrosine kinase inhibitor	Multiple tyrosine kinases
- imatinib		*CML, MdS, MPN	Multiple cancers/in vitro48
			CML*/in vitro102
Hiv protease inhibitors	Protease
- nelfinavir		-	Multiple cancers/in vitro56
			Phase I www.clinicaltrials.gov
Cyclooxygenase-2 inhibitor	Cyclooxygenase-2
- celecoxib		-	Colon cancer/in vitro55
Antidepressants
- selective serotonin reuptake inhibitor (fluoxetine)	serotonin	-	Burkitt's lymphoma/in vitro68
- norepinephrine reuptake inhibitor (maprotiline)	norepinephrine	-	Burkitt's lymphoma/in vitro68
Autophagy inhibitors
Chloroquine	Lysosomotropic agent	-	Lymphoma/in vitro and in vivo82
			Prostate cancer/in vivo50
			Several cancers/Phase I/II www.clinicaltrials.gov
Lucanthone	Topoisomerase II and AP endonuclease	-	Breast cancer/in vitro54
?Metformin	AMPK	-	Colon cancer/in vitro and in vivo71
Antidepressants
- Tricyclic (Clomipramine)	Serotonin, norepinephrine, dopamine	-	Cervical cancer/in vitro80

^*CML, chronic myelogenous leukemia; MDS, myelodysplastic syndrome; MPN, myeloproliferative neoplasm.

An increased understanding of the autophagy pathway and its modulation holds much promise for cancer therapy and prevention. In contrast to the cytoprotective function of autophagy, induction of autophagic cell death has also been proposed as a cell death mechanism given that features of autophagy were observed in dying cells.⁵⁷ Specifically, accumulation of autophagosomes has been observed in response to chemotherapy or molecularly targeted therapeutics, suggesting that autophagy induction may contribute to or accompany cell death.⁵⁸ Prolonged stress and sustained autophagy can eventually lead to cell death when protein and organelle turnover overwhelm the capacity of the cell.⁵⁸ A small molecule, STF-62247, was shown to promote cell death in VHL-deficient renal cell carcinomas in vitro and in vivo through chronic induction of autophagy.⁵⁹ However, in vivo evidence is limited and whether induction of autophagic death in tumor cells can be achieved for cancer therapy remains unknown.

Autophagy inducers in cancer therapy.

Excessive or sustained autophagy has the potential to induce tumor cell death and may, therefore, be a potential strategy for cancer treatment. Autophagy is regulated by the mTOR pathway and mTOR inhibitors can activate autophagy. Rapamycin is a naturally occurring mTOR inhibitor and its analogs temsirolimus (CCI-779), everolimus (RAD-001) and deforolimus (AP-23573) selectively target mTORC1 to stimulate autophagy (Table 1). With the exception of renal cell and neuroendocrine carcinomas and lymphoma, rapamycin and its analogs have had limited success in the clinical setting.^60,61 A potential explanation for their modest antitumor activity is the inability of rapamycin to inhibit mTORC2, and its ability to abrogate the S6K1-mediated negative feedback loop to the PI3K-AKT pathway that results in rebound AKT activation.⁶² The goal of achieving a more complete blockade of the mTOR pathway has led to the development of ATP-competitive mTOR inhibitors of both mTORC1 and mTORC2 (e.g., PP242, AZD8055, WYE132) and the dual PI3K-mTOR inhibitor NVP-BEZ235 that also inhibits PI3K. While there is preclinical evidence of antitumor activity,^63–65 their effectiveness in the clinical setting has yet to be demonstrated. The limited activity of mTOR inhibitors as monotherapy has led to the evaluation of drug combinations. Rapamycin combined with cytotoxic chemotherapy was shown to enhance apoptosis in vitro and to enhance antitumor efficacy in vivo.^60,61 Furthermore, rapamycin was found to potentiate radiation-induced clonogenic cell death.⁶⁶ Another kinase inhibitor that promotes autophagy is imatinib, a Bcr-Abl tyrosine kinase inhibitor (TKI) that is an effective agent against chronic myelogenous leukemia (CML). Imatinib has been shown to modulate autophagy through the regulation of lysosomal components.⁶⁷ Other autophagy modulators include antidepressants such as the selective serotonin reuptake inhibitor, fluoxetine and the norepinephrine reuptake inhibitor, maprotiline. Both drugs were shown to induce autophagy in chemoresistant Burkitt's lymphoma cell lines⁶⁸ (Table 1).

The anti-diabetic, biguanide drug metformin has been shown to inhibit mTOR signaling through its upstream mediator, AMPK⁶⁹ (Table 1). Metformin has a cytostatic effect on a variety of cancer cell types.^69–71 In prostate cancer cells, metformin inhibited 2-deoxyglucose-induced autophagy, decreased Beclin 1 expression and triggered a switch from cell survival to cell death.⁷² This role contradicts the effect of metformin which is an activator of AMPK and an inhibitor of mTOR that is expected to induce autophagy. However, only one study shows that metformin induces autophagy in cancer cells,⁷¹ suggesting that depending upon the cell type, activation of AMPK does not always lead to the induction of autophagy.⁷²

Autophagy can be activated by pro-angiogenic HIF-1α in the tumor microenvironment in the presence or absence of hypoxia.^18,46 HIF-1α can increase expression of vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF) and nitric oxide synthase (NOS).¹⁸ The mTOR inhibitor, WYE132, was shown to disrupt the accumulation of HIF-1α and HIF-2α, and its combination with the anti-VEGF antibody, bevacizumab, was shown to be highly effective in promoting tumor regression in renal cell carcinoma xenografts.⁷³ Since mTOR inhibitors have been successfully used in combination with epidermal growth factor receptor (EGFR) TKIs or bevacizumab,⁷⁴ the triple combination of an mTOR inhibitor together with a TKI and bevacizumab may potentially lead to an even greater antitumor effect.

Autophagy inhibitors in cancer therapy.

Preclinical studies have shown that inhibition of cytoprotective autophagy by genetic or pharmacological means can overcome treatment resistance.^75–77 Autophagy inhibitors can be broadly classified as early versus late stage inhibitors of the pathway. Early stage inhibitors include 3-methyadenine (3-MA), wortmannnin and LY294002 that target the class III PI3K (Vps34), while late stage inhibitors include chloroquine (CQ) or hydrochloroquine (HCQ), bafilomycin A1 and monensin that prevent fusion of autophagosomes with the lysosomes (Table 1). Bafilomycin A1 is a specific inhibitor of vacuolar-ATPase while monensin and CQ/HCQ are lysosomotropic drugs that prevent the acidification of lysosomal compartment. In CML cells, combined treatment with CQ and the histone deacetylase (HDAC) inhibitor vorinostat or suberoylanilide hydroxamic acid (SAHA) induced cell death in association with an increase of cathepsin D.⁷⁸ Recently, a small molecule targeting HSP70, a multifunctional and stress-induced molecular chaperone, was found to disrupt lysosomal function and autophagy. This HSP70 inhibitor induced caspase-independent cell death and suppressed lymphomagenesis in a murine model.⁷⁹ The tricyclic antidepressant drug, clomipramine has been shown inhibit autophagy by blocking autophagosome degradation.⁸⁰

The inhibition of autophagy combined with chemotherapy may enhance treatment efficacy by inhibiting stress adaptation and increasing cell death. Of the known autophagy inhibitors, only CQ/HCQ have been evaluated in humans given their common usage as anti-malarial drugs and in autoimmune disorders. HCQ is preferred to CQ in humans given its more favorable side effect profile.⁸¹ In preclinical studies, CQ/HCQ have been shown to effectively inhibit autophagy and to augment cell death and/or tumor regression.^50,78,82,83 In a Myc-induced murine lymphoma model, inhibition of autophagy by CQ was shown to enhance cyclophosphamide-induced tumor cell death to a similar degree as did shRNA knockdown of Atg5, and delayed the time-to-tumor recurrence.⁸² In a p53 mutated colon cancer xenograft model, 5-fluorouracil (5-FU) in combination with the autophagy inhibitor 3-MA increased caspase-3 cleavage and reduced overall tumor weight and volume as compared to 5-FU alone.⁸⁴ The combination of CQ and the HDAC inhibitor vorinostat was also shown to significantly reduce tumor burden and to induce apoptosis in a colon cancer xenograft model.⁸³ Similarly, CQ in combination with saracatinib, a Src inhibitor, produced a 2-fold increase in apoptotic tumor cells compared to saracatinib alone.⁵⁰ Together, these data indicate that autophagy inhibition can enhance tumor cell death by anticancer drugs that utilizing diverse cellular mechanisms. Given these data, there are several ongoing phase I/II trials evaluating the combination of HCQ with cytotoxic drugs in patients with brain, lung, breast, colorectal, pancreas, kidney and prostate cancers⁴³ (www.clinicaltrials.gov).

Protein degradation occurs through autophagy in lysosomes, but also within the proteasome for ubiquitinated proteins.⁸⁵ Since autophagy and the proteosome are the two main protein degradation pathways, it has been postulated that their combined blockade may lead to ER stress-induced cytotoxicity through the accumulation of unfolded protein aggregates.⁴³ Unfolded proteins can stimulate the unfolded protein response (UPR) to activate autophagy through JNK⁸⁶ or PERK/eIF2α.^87,88 The combination of bortezomib and CQ was shown to suppress tumor growth to a greater extent than did either drug alone in a colon cancer xenograft model.⁸⁹ Phase I/II clinical trials evaluating this combination are ongoing in patients with relapsed/refractory multiple myeloma.

Evidence indicates that exploiting the interplay between apoptosis and autophagy may represent a promising therapeutic strategy. TNF-related apoptosis-inducing ligand (TRAIL) is a cytokine known to induce apoptosis in multiple cancer cell lines, and is being evaluated in phase I/II studies.⁹⁰ TRAIL induced autophagy in apoptosis-defective leukemic and colon cancer cell lines, and an autophagy inhibitor sensitized resistant cells to TRAIL-mediated apoptosis.⁹¹ Anti-apoptotic Bcl-2/Bcl-x_L proteins are capable of binding to and disrupting the autophagic function of Beclin 1 which contains a functional BH3 domain.⁹² Bcl-2/Bcl-x_L can inhibit both apoptosis and autophagy and small molecule inhibitors of Bcl-2/Bcl-x_L, i.e., BH3 mimetics, can competitively disrupt the Beclin 1-Bcl-2/Bcl-x_L interaction to induce autophagy.⁹³ These data suggest that inhibition of cytoprotective autophagy by BH3 mimetics may further enhance apoptosis. In this regard, the selective cyclooxygenase-2 inhibitor, celecoxib, was shown to induce apoptosis that was enhanced by the BH3 mimetic, ABT-737, and was further augmented by inhibition of autophagy in colon cancer cells.⁵⁵

Conclusion

Strategies to exploit autophagy for therapeutic advantage against cancer is an area of intense investigation. Autophagy plays a dual role as both a mechanism of tumor suppression and as an adaptive stress response that can facilitate tumor cell survival and progression. Defective autophagy may contribute to cancer development in that heterozygous deletion of the Beclin 1 gene has been found in human breast, ovarian and prostate cancers. Given its role in tumor suppression, activation of autophagy may also be an important strategy for cancer prevention. In established cancers, autophagy enables tumor cells to survive increased metabolic demands, a hypoxic microenvironment and cancer therapy. Modulation of autophagy may represent a new paradigm for cancer treatment that is relevant to both conventional cytotoxic drugs and to targeted agents. Considerable evidence supports a cytoprotective role for autophagy in cancer cells, and autophagy inhibition has been shown to enhance chemosensitivity and antitumor efficacy in multiple tumor cell types both in vitro and in vivo. While multiple drugs can inhibit autophagy, most lack specificity and antitumor activity. Autophagy inhibition by hydroxychloroquine in combination with chemotherapy is currently being evaluated in multiple ongoing clinical trials in patients with solid tumors. Certain anticancer drugs can induce autophagy, and excessive or sustained induction of autophagy may lead to tumor cell death in some contexts. While tumor cell susceptibility to autophagy may depend on tumor genotype and the therapeutic agents utilized, data are very limited and it remains unclear as to whether such a strategy will be beneficial clinically. Elucidation of the mechanisms of autophagy activation, its interplay with apoptosis and the specific mechanisms by which autophagy confers treatment resistance await further study. The outcome of studies examining autophagy inhibition in combination with cytotoxic agents is early awaited. However, more selective agents targeting autophagy are needed and represent a high priority for cancer drug development.