Activating autophagy to potentiate immunogenic chemotherapy and radiation therapy.pdf

Macroautophagy(referred to as autophagy from here onward)is an evolutionary ancient process by which eukaryotic cells sequester cytoplasmic material of endogenous or exogenous origin within double-membraned vacuoles(also known as autophagosomes),and deliver this material to lysosomes for degradation1.Under normal physiological conditions,baseline levels of autophagy enable the disposal of intracellular structures that have become damaged(such as uncoupled mitochondria)or accumulate(such as redox-active protein aggregates)as a consequence of normal cellular functions,essentially operating as a quality control system2,3.The autophagic machinery is exquisitely sensitive to multiple perturbations of the intracellular and extracellular microenvironment,including nutritional,metabolic,infectious,chemical and physical cues4,5.In specific circumstances,such as the reaction of mammalian neurons to neonatal hypoxia and/or ischaemia,an autophagic response autophagosomes are able to acquire endogenous or exogenous substrates in a highly specific manner12.In this latter scenario,autophagy is normally driven by the increased availability of substrates,as isthe case of infected cells or cells exposed tomitochondrial toxins13(FIG.1).Malignant cells derive equal benefits from the cytoprotective effects of autophagy than do their nonmalignant counterparts14;therefore,intense efforts have been made throughout the past decade to develop specific inhibitors of autophagy for the treatment of patients with cancer.To date,however,the clinical results obtained with these agents have been disappointing.In this Perspective,we offer an explanation forthis apparent lack of activity,and propose that promotion(rather than inhibition)of autophagy might be a more clinically relevant strategy,with the potential to improve the efficacy of chemotherapy and radiation therapy in immunocompetent patients with cancer.Autophagy and cancerAccumulating evidence indicates that autophagic responses in nonmalignant tissues suppress the initial transition of a healthy cell into a neoplastic precursor14.Inline with this notion,genetic interventions causing whole-body or tissue-specific defects in autophagy have been associated with an increased incidence of tumours in various rodent models of carcinogenesis15,16.Moreover,multiple established oncoproteins(such as EGFR and Bcl-2)are able to inhibit autophagic responses,while several tumour suppressors(such as PTEN)promote such responses17,18.Furthermore,the expression of various core components of the autophagic machinery,such as beclin-1(BECN1),is reduced in a considerable number of human tumours19.The tumour-suppressive functions of autophagy reflect the ability of this process to limit the accumulationof potentially oncogenic alterations,thereby preserving the intracellular microenvironment14.Autophagy removes damaged mitochondria and redox-active protein aggregates3,which can act as a source of genotoxic reactive-oxygen species(ROS),and also contributes to the removal of damaged DNA in the form of nuclear to stress seems to contribute to cell death6,7.However,in the vast majority of situations,such as in healthy and transformed mammalian cells,autophagy mediates robust cytoprotective effects,and therefore has a central role in the adaptive response to virtually all intrinsic or extrinsic stimuli8.In line with this notion,pharmacological or genetic interventions that interrupt or inhibit the processes involved in autophagic degradation generally increase the sensitivity of cultured cells to various stressors9.Autophagic responses to stress can develop according to two distinct paradigms.Autophagosomes are able to nonselectively engulf various nonessential structures,including parts of the mitochondrial network and portions of the cytoplasm10.Generally,autophagic responses of this type are elicited by an increased demand for the products of autophagy,such as macromolecules.This occursfor instancein cells that are exposed to nutrient deprivation11.In addition,OPINIONActivating autophagy to potentiate immunogenic chemotherapy and radiation therapyLorenzo Galluzzi,Jos Manuel Bravo-San Pedro,Sandra Demaria,Silvia Chiara Formenti and Guido KroemerAbstract|Autophagy is fundamental to the maintenance of intracellular homeostasis in virtually all human cells.Accordingly,defective autophagy predisposes healthy cells to undergoing malignant transformation.By contrast,malignant cells are able to harness autophagy to thrive,despite adverse microenvironmental conditions,and to resist therapeutic challenges.Thus,inhibition of autophagy has been proposed as a strategy to kill cancer cells or sensitize them to therapy;however,autophagy is also critical for optimal immune function,and mediates cell-extrinsic homeostatic effects owing to its central role in danger signalling by neoplastic cells responding to immunogenic chemotherapy and/or radiation therapy.In this Perspective,we discuss accumulating preclinical and clinical evidence in support of the all-too-often dismissed possibility that activating autophagy might be a relevant clinical objective that enables an increase in the effectiveness of immunogenic chemotherapy and/or radiation therapy.NATURE REVIEWS|CLINICAL ONCOLOGY ADVANCE ONLINE PUBLICATION|1PERSPECTIVES2016MacmillanPublishersLimited,partofSpringerNature.Allrightsreserved.buds or micronuclei,hence promoting the preservation of genomic integrity20.Moreover,the autophagic machinery contributes to the first-line of defence against potentially oncogenic infective agents13,including(but not limited to)certain strains of human papillomavirus and various intracellular bacteria21.Finally,autophagy is involved in the maintenance of normal stem-cell functions22,and is required for the execution of oncosuppressive programmes such as oncogene-induced senescence and oncogene-induced cell death23,and might also participate in the degradation of proteins with established oncogenic potential,such as the BCRABL1 chimera24.Nevertheless,autophagy can also mediate cytoprotective functions in transformed cells.Thus,autophagy is commonly viewed as a process that supports the expansion of the neoplastic precursor-cell population,coupled with the acquisition of ever more malignant features14.Accordingly,pharmacological and genetic strategies that inhibit autophagy in mice with established neoplasms generally impede tumour progressionespecially in immunocompromised mice or in mice bearing tumours that are not particularly sensitive to immunosurveillance25.Indeed,autophagy has a fundamental role in the adaptation of cancer cells to the adverse conditions that characterize the poorly vascularized tumour microenvironment,such as hypoxia and/or low nutrient levels,and to epithelial-to-mesenchymal transition,mediate the maturation and secretion of the proinflammatory cytokines IL-1 and IL-18(REF.34).Furthermore,the autophagic disposal of damaged mitochondria limits the availability of inflammasome-activating factors,including ROS and mitochondrial DNA35.Proficient autophagic responses within malignant cells are also a key component of the cross-presentation of tumour-associated antigens by dendritic cells,which is essential for the priming of a specific anticancer immune responses36.Moreover,autophagy is required for the survival,expansion,and activation of tumour-targeting CD8+Tlymphocytes37,as well as for the survival of memory Tcells38.Finally,cancer cells succumbing to immunogenic cell death(ICD)release quantities of ATP that are sufficient to activate purinergic receptors and to elicit anticancer immune responses,by virtue of the chemotactic and immunostimulatory effects of purinergic signalling,but only when cancer cells undergo autophagy before death39.Autophagy is also involved in the release of high-mobility group protein B1(HMGB1),a non-histone chromatin-binding protein involved in the perception of cancer cell death as immunogenic40.Conversely,both extranuclear and extracellular pools of HMGB1 promote robust autophagic responses41,42,suggesting the existence of bidirectional crosstalk between autophagy and danger signalling43,44.Despite this abundant evidence,autophagy might not always support danger signalling.Indeed,autophagy has a limited effect on ATP secretion by cancer cells exposed to hypericin-based photodynamic therapy invitro,and has been demonstrated to inhibit presentation of the endoplasmic reticulum chaperone calreticulin(CALR)on the plasma membrane,where it would normally promote phagocytosis45.Moreover,efficient autophagic degradation of HGMB1 has been demonstrated in macrophages responding to epigallocatechin gallate invitro46.In a transgenic mouse model of breast carcinoma,release of the T-cell chemoattractant C-X-C motif chemokine10(CXCL10)by malignant cells was enhanced upon deletion of RB1-inducible coiled-coil protein 1(Rb1cc1),which encodes an upstream component of the autophagic machinery,resulting in increased recruitment of T lymphocytes to the tumourbed47.Altogether,these observations suggest that autophagy not only mediates effectsthat are intrinsic to healthy cells,malignantcells and immune cells,but also enables the connection of intracellular stress responses which is a key event in tumour progression26.Moreover,autophagy contributes to the maintenance of cancer-stem-cell populations27,and increases the resistance of transformed cells to the cytotoxic effects of exogenous treatments,including conventional chemotherapeutics,targeted anticancer agents,ionizing radiation25 and endogenous immune effectors(at least under specific circumstances)28,29(BOX1).In this context,hypoxia stands out as a key microenvironmental determinant of autophagic responses and the effect of these responses on treatment outcome.Importantly,the influence of autophagy on malignant transformation and tumour progression is not limited to intrinsic effects within cancerous cells.Rather,it involves at least four additional components:the existence of autophagy-dependent metabolic circuitries though which the stromal and endothelial components of the tumour mass support their malignant counterparts30(BOX2);the ability of autophagy to limit the extent of chronic inflammation,which is known to support tumour progression13;the essential role of autophagy in the activation of antitumor adaptive immune responses31;and the autophagic regulation of the release of immunostimulatory danger signals,in the context of immunogenic forms of cancertherapy32.Autophagy has been implicated in the degradation of so-called inflammasomes33,which are supramolecular platforms that AutophagicfluxIncreasedautophagicfluxLevels S PSSPPLevelsLevelsTimeTimeTimeIncreasedautophagicfluxPushabPullAccumulationof substratesDecreaseof productsHomeostasisFigure 1|Autophagic responses to cellular demands.Autophagic degradation can increase as a consequence of two diametrically opposed situations that influence substrate selection.a|Autophagy can be pushed to increased levels of activity by the accumulation of substrates(such as permeabilized mitochondria or cytosolic bacteria),often corresponding to the activation of a specific autophagic response.b|Autophagy can also be pulled to increased levels of activity by a cellular need for the products of autophagy,which generally results in a nonspecific autophagic response.In both cases,proficient autophagic responses support the re-establishment of homeostasis,which then results in a decrease in autophagic activity to baseline levels.P,product;S,substrate.PERSPECTIVES2|ADVANCE ONLINE PUBLICATION the maintenance of organismal homeostasis,via danger signalling.All of these various aspects of autophagic responses are expected to contribute to the net effects of interventions that target autophagy in patients with cancer.Inhibition of autophagyThroughout the past decade,a growing understanding of the ability of autophagy to support tumour progression has nurtured intensive experimental efforts aimed at the development of autophagy inhibitors for cancer therapy,following two distinct strategies:inhibition of autophagy as a monotherapy,for tumours that are autophagy-dependent,and the suppression of autophagy in combination with other treatment modalities,as a means to potentiate their effectiveness.Experimental data from multiple rodent models indicate that single-agent pharmacological suppression of autophagy with molecules such as 3-methyladenine,wortmannin,chloroquine or hydroxychloroquine,can inhibit tumour growth48,49.When used in combination,the same inhibitors of autophagy can enhance theeffectiveness of various treatment modalities,including conventional chemotherapeutics,such as 5-fluorouracil50,cisplatin51,temozolomide52,melphalan53,epirubicin54,doxorubicin53,55,orcyclophosphamide55,56;targeted anticanceragents,such as gefitinib57;antiangiogenic agents,such as bevacizumab58;silver nanoparticles59;photodynamic therapies60;and radiation therapy61,62.However,the vast majority of these studies involved human cancer cells growing in immunodeficient(generally athymic)mice,a model that excludes the potentially beneficial effects of autophagy on cancer immunosurveillance63.Moreover,the various pharmacological agents used to inhibit autophagy lack specificity:3-methyladenine and wortmannin also inhibit PI3K isozymes with no established involvement in autophagy,while chloroquine and hydroxychloroquine inhibit lysosomal acidification64 and hence affect a plethora of other cellular processes.In particular,chloroquine and derivatives not only promote normalization of the tumour vasculature,thus favouring infiltration by immune effectors independent of autophagy65,but also mediate antineoplastic66 and immunomodulatory effects independent of autophagy67.Notably,chloroquine and,to a lesser extent,hydroxychloroquine have been shown to suppress immune responses by favouring approaches that result in stable inactivation of various factors with functions crucial to the process of autophagy in malignant cells25.Confirming the ability of autophagy to support tumour progression in a cell-intrinsic manner,several cancer cell lines stably engineered to express short-hairpin RNAs(shRNAs)targeting the components of the autophagic machinery,including autophagy-related 5(ATG5)and ATG7,BECN1 or microtubule-associated proteins 1A/1B light chain 3B(MAP1LC3B,best known as LC3)had reduced proliferative and metastatic potential invivo,compared with that of cells of the same type expressing control shRNAs26,55,72.Moreover,autophagy-deficient cell lines had increased sensitivity to conventional chemotherapy51,photodynamic therapy60,antiangiogenic therapy72,73,and ionizing radiation74 compared with their autophagy-proficient counterparts.However,similar to data obtained with pharmacological inhibition of autophagy,all of these studies the accumulation of CD3+CD4+FOXP3+regulatory T(Treg)cells68,but,conversely,also inhibit immunosuppressive processes,includin