Regarding the apoptosis mechanisms in cells and cancer

Regarding the apoptosis mechanisms in cells and cancer

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If all the cells in a cancerous tumour had their apoptosis mechanisms 'turned back on' or reactivated or repaired by some 'yet to be discovered' process would this cause the tumour to 'self destruct' or be destroyed? Are there known processes that can repair or reactivate the apoptosis mechanisms in a cell?

The processes of apoptosis is not so simple, there is a lot of genes and proteins that participate in it. In cancer cells some of them mutate and not working properly.
Lack of apoptosis is the main problem in cancer. If cell not working properly, it dies. But not in cancer.
So theoretically, yes. If we will fix the apoptosis, the cell probably will die.
It's gene engineering direction. You need to target the cancer cell, and to change problem genes. It's really difficult processes. And every type of cancer has different mutations…

Read this for example (Journal of Experimental & Clinical Cancer Research )

Apoptosis Mechanisms: Implications for Cancer Drug Discovery

Defects in the regulation of apoptosis (programmed cell death) makeimportant contributions to the pathogenesis and progression of mostcancers and leukemias. Apoptosis defects also figure prominently inresistance to chemotherapy, radiotherapy, hormonal therapy, andimmune-based treatments. Apoptosis is caused by activation ofintracellular proteases, known as caspases, that are responsible directlyor indirectly for the morphologic and biochemical events thatcharacterize the apoptotic cell. Numerous proteins that regulate thesecell death proteases have been discovered, including proteins belongingto the Bcl-2, inhibitor of apoptosis, caspase-associated recruitmentdomain, death domain, and death effector domain families. Thesecaspase-regulating proteins provide mechanisms for linkingenvironmental stimuli to cell death responses or to maintenance of cellsurvival. Alterations in the expression and function of several apoptosisregulatinggenes have been demonstrated in cancer, suggesting targetsfor drug discovery. Knowledge of the molecular details of apoptosisregulation and the three-dimensional structures of apoptosis proteinshas revealed new strategies for identifying small-molecule drugs thatmay yield more effective treatments for malignancies. Apoptosisregulatinggenes are also beginning to find utility as targets for antisenseoligonucleotides.

ABSTRACT: Defects in the regulation of apoptosis (programmed cell death) makeimportant contributions to the pathogenesis and progression of mostcancers and leukemias. Apoptosis defects also figure prominently inresistance to chemotherapy, radiotherapy, hormonal therapy, andimmune-based treatments. Apoptosis is caused by activation ofintracellular proteases, known as caspases, that are responsible directlyor indirectly for the morphologic and biochemical events thatcharacterize the apoptotic cell. Numerous proteins that regulate thesecell death proteases have been discovered, including proteins belongingto the Bcl-2, inhibitor of apoptosis, caspase-associated recruitmentdomain, death domain, and death effector domain families. Thesecaspase-regulating proteins provide mechanisms for linkingenvironmental stimuli to cell death responses or to maintenance of cellsurvival. Alterations in the expression and function of several apoptosisregulatinggenes have been demonstrated in cancer, suggesting targetsfor drug discovery. Knowledge of the molecular details of apoptosisregulation and the three-dimensional structures of apoptosis proteinshas revealed new strategies for identifying small-molecule drugs thatmay yield more effective treatments for malignancies. Apoptosisregulatinggenes are also beginning to find utility as targets for antisenseoligonucleotides.Defects in the mechanisms ofprogrammed cell death(apoptosis) play importantroles in many aspects of tumor pathogenesisand progression. For example,apoptosis defects allow neoplasticcells to survive beyond their normallyintended life spans.[1] Thus, the needfor exogenous survival factors is subverted.Protection is provided againsthypoxia and oxidative stress as tumormass expands and time is allowed foraccumulative genetic alterations thatderegulate cell proliferation, interferewith differentiation, promote angiogenesis,and increase cell motility andinvasiveness during tumor progression.In fact, apoptosis defects are recognizedas an important complementto proto-oncogene activation, becausemany deregulated oncoproteins thatdrive cell division also trigger apoptosis(eg, Myc, E1a, Cyclin-D1).[2] Similarly,defects in DNA repair and chromosomesegregation normally triggercell suicide as a defense mechanismfor eradicating genetically unstablecells thus, apoptosis defects permitsurvival of the genetically unstablecells, providing opportunities for selectionof progressively more aggressiveclones.[3]Apoptosis defects also facilitatemetastasis by allowing epithelial cellsto survive in a suspended state, withoutattachment to an extracellular matrix.[4] These defects also promoteresistance to the immune system becausemany of the weapons that cytolyticT cells and natural killer cellsuse for attacking tumors depend on theintegrity of the apoptosis machinery.[5] Finally, cancer-associated defectsin apoptosis play a role inchemoresistance and radioresistance,increasing the threshold for cell deathand thereby requiring higher doses fortumor killing.[6] Thus, defective regulationof apoptosis is a fundamentalaspect of the biology of cancer.Because apoptosis defects permit awide variety of aberrant cellular behaviors,as exhibited in cancer cells,therapeutic strategies that negate theapoptosis advantage for tumors arepredicted to selectively kill cancercells as opposed to normal cells. Fundamentally,cancer cells should bemore dependent on apoptosis defensemechanisms than normal cells andthus should be proportionally moresensitive to interventions that targetapoptosis proteins and genes. To date,efforts to bring apoptosis-based strategiesinto animal models or humanclinical trials have provided supportfor this concept of selective vulnerabilityof neoplastic cells as opposed tonormal cells.

A solid knowledge base now existsabout the mechanisms of apoptosisregulation, the proteins involved, their3D structures, and biochemicalmechanisms. Over the past 2 decades,a clearer understanding has emergedof the defects in expression or functionof apoptosis-regulating genes andproteins relating to cancer. This informationcan now be exploited fordevising strategies for small-moleculedrug discovery toward the goal ofrevolutionary treatments for cancerand leukemia.Apoptosis PathwaysApoptosis is caused by proteasesknown as caspases, which stands forcysteine aspartyl-specific proteases.[7,8] Caspases constitute a familyof intracellular cysteine proteasesthat collaborate in proteolytic cascades,where caspases activate themselvesand each other.[9,10] Withinthese proteolytic cascades, caspasescan be positioned as either upstream"initiators" or downstream "effectors"of apoptosis.[11] Eleven caspases havebeen identified in the human genome.Several pathways for activatingcaspases probably exist, though detailsremain sketchy for some of them (Figure1). The simplest pathway is exploitedby cytolytic T cells and naturalkiller cells, which inject apoptosisinducingproteases, particularlygranzyme B, into target cells viaperforin channels.[12,13] Unlike thecaspases, granzyme B is a serine protease,but similar to the caspases,granzyme B specifically cleaves substratesat Asp residues. Granzyme Bis capable of cleaving and activatingmultiple caspases and some caspasesubstrates. Endogenous and viral inhibitorsof granzyme B have beenidentified, accounting for resistance tothis apoptotic inducer.[14-16]Another caspase-activation pathwayis represented by the tumor necrosisfactor (TNF)-family receptors.Of the approximately 30 known membersof the TNF family in humans, 8contain a so-called death domain intheir cytosolic tails.[17] Several ofthese death domain-containing TNFfamilyreceptors use caspase activationas a signaling mechanism, includingTNFR1/CD120a, Fas/APO1/CD95,DR3/Apo2/Weasle, DR4/TrailR1,DR5/TrailR2, and DR6. Ligation ofthese receptors at the cell surface resultsin the recruitment of several intracellularproteins, including certainprocaspases, to the cytosolic domainsof these receptors, forming a "deathinducingsignaling complex" (DISC)that triggers the activation of caspasesand leads to apoptosis.[18,19] Thespecific caspases summoned to theDISC are caspase-8 and, in somecases, caspase-10. These caspases containso-called death effector domainsin their N-terminal prodomains thatbind to a corresponding death effectordomain in the adapter protein, Fasassociateddeath domain (FADD), thuslinking them to the TNF-family deathreceptor complexes.Mitochondria also play importantroles in apoptosis, releasing cytochromec into the cytosol, which thencauses assembly of a multiproteincaspase-activating complex, referredto as the "apoptosome."[20,21] Thecentral component of the apoptosomeis Apaf-1, a caspase-activating proteinthat oligomerizes on binding cytochromec and that specifically bindsprocaspase-9. Apaf-1 and procaspase-9 interact via their caspase-associatedrecruitment domains (CARDs). Sucha CARD-CARD interaction plays importantroles in many steps in the pathwaysof apoptosis.The mitochondrial pathway forapoptosis is activated by myriadstimuli, including growth factor deprivation,oxidants, Ca 2+ overload,DNA-damaging agents, and others.Mitochondria can also participate incell death pathways induced via TNFfamilydeath receptors, through crosstalkmechanisms involving proteinssuch as Bid, BAR, and Bap31.[22-25]However, mitochondrial (intrinsic)and death receptor (extrinsic) pathwaysfor the activation of caspases arefully capable of independent operationin most types of cells.[26] In additionto cytochrome c, mitochondria alsorelease several other proteins of relevanceto apoptosis, including endonucleaseG, AIF (an activator ofnuclear endonucleases), and inhibitorof apoptosis protein (IAP) antagonistsSmac (Diablo) and Omi (HtrA2).Pathways of apoptosis linked todamage in the endoplasmic reticulumand Golgi, as well as a pathway linkedto nuclear structures called PODs(PML oncogenic domains) or nuclearbodies, have also been described butare poorly characterized to date.

Suppressors of ApoptosisSeveral antagonists of the caspaseactivationpathways have been discovered,and multiple examples ofdysregulation of their expression orfunction in cancers have been obtained.Because our current knowledgeis greatest where the mitochondrial("intrinsic") and TNF-family deathreceptor ("extrinsic") pathways forapoptosis are concerned, most availableinformation about antagonistscenters on these two apoptotic pathways.In this article, three types ofapoptosis-suppressing proteins knownto be overexpressed in tumors, includingprostate cancers, are considered:IAPs, FLIP, and Bcl-2.Inhibitor of Apoptosis ProteinsInhibitor of apoptosis proteins representan evolutionarily conservedfamily of suppressors of apoptosis.Members of the IAP family, originallyidentified in baculoviruses, containone or more copies of a domain calledthe baculoviral IAP repeat (BIR).These BIR domains are sometimesaccompanied by other domains, includingRING domains, ubiquitinconjugatingenzyme folds (E2s), andNACHT-family nucleotide-bindingdomains. The human genome encodeseight IAP-family members: XIAP,cIAP1, cIAP2, Naip, Apollon (Bruce),ILP2 (Ts-IAP), ML-IAP (K-IAPLivin), and Survivin.The BIR domains of several IAPfamilyproteins were originally shownby our laboratory to be responsible fordirectly binding and specifically inhibitingcaspases, thus identifyingIAPs as endogenous inhibitors of celldeath proteases.[27-31] Multipleother laboratories have confirmed andextended these findings, providingconclusive evidence that many IAPfamilyproteins operate as caspasesuppressors.[32-41] However, IAPsvary in the specific caspases they inhibit.For example, XIAP suppressesboth downstream effector caspasesthat operate at points of convergenceof apoptosis pathways and caspase-9, the apical protease in the mitochondrialpathway for apoptosis.[27,29,30]In contrast, ML-IAP is a potentsuppressor of only caspase-9. No examplesof IAP-mediated suppressionof proteases that operate in the upstreamportions of the apoptosis pathwayactivated by TNF-family receptorshave been found (Figure 2).Evidence of overexpression ofIAPs in cancer has been obtained, suggestinga role for these suppressors ofapoptosis in malignancy.[31,42] Forexample, the IAP-family memberSurvivin is overexpressed in most cancers[43] and has become a topic ofconsiderable attention for its dual roleas a regulator of cell division (chromosomesegregation and cytokinesis)and apoptosis.[44-46] Similarly, theIAP-family member ML-IAP is rarelyexpressed in normal tissues but isfound at elevated levels in melanomasand some renal cancers.[33,40,47]Moreover, XIAP has been reported byour group to be overexpressed in asubstantial proportion of acutemyelogenous leukemias, with higherlevels correlating with shorter remissiondurations and shorter overallpatient survival.[48] Evidence ofoverexpression of XIAP has also beenreported for renal and lung cancers[49,50] overexpression of cIAP1has been associated with ovarian cancer.Chromosomal translocationsactivating cIAP2 are found in somelymphomas.[51] Thus, various IAPfamilyproteins are overexpressed inspecific types of cancer.However, more than one memberof the IAP family can be overexpressedsimultaneously by sometumors. For example, in prostatecancers, we found evidence thatprotein levels of XIAP, cIAP1, cIAP2,and Survivin can sometimes becomesimultaneously increased in tumors,[52] suggesting redundancy inexpression of these antiapoptotic proteins.We have also found evidence ofapparent simultaneous overexpressionof cIAP1, cIAP2, and Survivin in coloncancer (manuscript in preparation).The observation of overexpression ofmultiple IAP-family members impliesthat perhaps some aspects of theirregulation are shared.Indeed, during a screen of the NationalCancer Institute panel of 60human tumor cell lines, assessing IAPexpression at the messenger RNA(mRNA) and protein levels, we obtainedevidence that mRNA levels ofXIAP, cIAP1, and cIAP2 do not correlatewith their protein levels,[48]suggesting that posttranscriptionalregulation of these IAP-family proteinsis important. Interestingly, allthree of these IAP-family proteins containa RING domain that binds E2s(ubiquitin-conjugating enzymes), implyingthat alternations in the turnoverrate of IAP-family proteins may occurin cancers that overexpress multiplefamily members simultaneously.The functional importance ofoverexpressed IAPs for apoptosis suppressionin cancers has been supportedby antisense experiments.[53-57] Inthese experiments, knocking downexpression of Survivin, XIAP, or otherIAPs has been shown to induceapoptosis of tumor cell lines in cultureor to sensitize tumor cell lines toapoptosis induced by anticancerdrugs.[53-57] In contrast, gene knockoutstudies in mice imply that normalcells are possibly less dependent onIAPs than tumor cells because targeteddisruption of the genes for xiap, ciap1,and ciap2, both individually and incombination, produces little phenotype.[58 personal communication, T.Mak, 2004]Implications for Treatment
Taken together, these observationsimply that drugs that interfere with theaction of IAPs could be useful for thetreatment of cancer. Recently, a strategyfor devising small-molecule inhibitorsof IAPs has been suggestedby the discovery of natural antagonistsof IAPs.[35,38] Proteins such as Smacand Omi (HtrA2) have been shown tobind IAPs and suppress them, releasingcaspases to kill cells.[35,38,59] A7'mer peptide corresponding to the Nterminusof Smac is reported to besufficient to bind IAPs and block theirassociation with caspases.[60]Moreover, we have confirmed thatpeptides as short as tetramers can potentlyreverse caspase inhibition byIAPs, functioning in a stoichiometricmanner at micromolar concentrations.[61 unpublished data] By fusingmembrane-penetrating peptidesonto Smac or Omi peptides, it is possibleto induce apoptosis of cancer celllines in culture as well as to suppresstumor formation in xenograft modelsin mice.[62-65] Thus, these data provideproof-of-concept evidence thatsmall molecules that mimic the effectsof these IAP-binding peptides couldpotentially be exploited as drugs forcancer treatment.Drug Discovery Strategies
Structural analysis of the interactionsof IAPs with caspases and ofIAPs with Smac has helped to lay afoundation for such drug-discoveryefforts. First, our structure-functionstudies of IAP-family member XIAPshowed that, although this protein containsthree tandem BIR domains, asingle BIR is sufficient to bind andsuppress caspases. These studies demonstratedthat the BIR2 domain specificallyinhibits caspase-3 andcaspase-7, whereas the BIR3 domainof XIAP blocks the activity of caspase-9.[29,30] Thus, discrete domains inIAPs are responsible for binding andinhibiting caspases.Second, the 3D structure of theBIR3 domain complexed with Smacrevealed that the N-terminal 4 aminoacids of the mature Smac protein bindsin the same crevice normally occupiedby the N-terminus of the small subunitof caspase-9, thus suggestingcompletion for binding.[37,60,66,67]Consequently, small-molecule compoundsthat mimic the Smac 4?merpeptide should dislodge activecaspase-9 from BIR3, thus inducingapoptosis (Figure 3).The structural details regarding theinteraction of BIR2 of XIAP withcaspases and its relation to Smac areless clear due to poor atomic resolutionof the N-terminus of the smallsubunitof caspases-3 or -7 complexedwith BIR2, as determined by x-raycrystallography by scientists at ourinstitution and elsewhere.[32,39] Inthe crystal structure of the XIAPBIR2-caspase-3 complex, the NH2-terminus of the caspase-3 p10 subunitinteracts with the surface of BIR2,[32]which may be an artifact of crystallization.Though, to date, the mechanismof inhibition of XIAP by Smacremains unclear, modeling studiessuggest the presence of a similarSmac-binding pocket on BIR2.In addition to chemical inhibitorsof IAPs based on mimicking Smac,other strategies can also be envisionedand have begun to be exploited. Forexample, using an enzyme derepressionassay where screens were performedto identify compounds capableof dislodging XIAP fromcaspase-3 and restoring protease activity,we and other investigators haveidentified small-molecule antagonistsof XIAP.[68,69] These compoundstarget a non-Smac site on XIAP,which remains to be defined at thestructural level.Interestingly, in addition to Smacand Omi (HtrA2), other endogenousantagonists of IAPs have beenreported, including XAF1, NRAGE,and ARTS, which operate through analternative mechanism.[70-72] Thus,it is conceivable that the aforementionedsmall-molecule antagonistsof IAPs mimic one or more of theseendogenous antagonists of IAPs,a concept awaiting experimentaltesting.Fas-Associated Death Domain-Like Interleukin-1beta-Converting Enzyme InhibitingProtein (FLIP)The pathway of apoptosis triggeredby TNF-family death receptors is fundamentalto the mechanisms by whichcytolytic T cells attack and kill tumorcells.[73-78] Cytolytic T cells, naturalkiller cells, macrophages, and dendriticcells have been demonstrated toproduce one or more of the TNFfamilydeath ligands, such as FasL,TNF, or TRAIL. On binding their specificreceptors on susceptible targetcells, these receptors recruit procaspase-8 and/or -10 to the receptorcomplex, forming a DISC that resultsin the activation of caspases.[11,79]Perhaps not surprisingly, many tumorsdevelop resistance to this extrinsicpathway for apoptosis at some pointin their pathogenesis or progression,reducing or ablating their sensitivityto immune cell attack.[5]Multiple antagonists of the extrinsicpathway have been identified, includingseveral death effector domain-containing proteins that compete forbinding to the adapter proteins orprocaspases that participate in TNFfamilydeath receptor signaling, includingFLIP, BAR, and possiblyBap31.[80,81] Among them, FLIP hasreceived the most attention for its rolein producing Fas-resistant states intumor cells.[82,83]The FLIP protein is highly similarin its overall sequence to procaspases-8 and -10, containing tandem copiesof the death effector domain, followedby a pseudocaspase domain that lacksenzymatic activity. FLIP can promoteapoptosis in some circumstances.[84]However, for the most part, this proteinis antiapoptotic, forming complexeswith procaspase-8 and -10, andpreventing their effective activation, aswell as competing for binding toadapter proteins required for the recruitmentof caspases to receptors ofdeath-receptor complexes.[5,83]Overexpression of FLIP occurscommonly in cancers. Our laboratoryhas determined by antisense and genetransfer studies that FLIP is animportant determinant of resistance ofsome tumor cell lines to the inductionof apoptosis by TNF, Fas, andTRAIL.[85,86] Moreover, in a collaborativeeffort with other investigators,we have identified a class of compoundscalled synthetic triterpenoidsthat cause reductions in FLIP in multiplehuman tumor cell lines, correlatingwith the restoration of sensitivityto TRAIL-induced apoptosis.[85]Thus, small-molecule drugs that ablateexpression or function of FLIPrepresent an attractive approach to sensitizingtumor cells to TNF-familydeath ligands.The prototype triterpenoidshown to reduce the expression ofFLIP is CDDO (2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid). Atsubmicromolar levels, this compound,as well as selected analogs, reducesthe levels of FLIP protein by a mechanisminvolving ubiquitination andproteasome-dependent degradation ofthe FLIP protein without affectingFLIP mRNA levels.[85,87,88]CDDO has no effect on the levels ofmany other apoptosis proteins atsubmicromolar levels sufficient to reduceFLIP, including FADD, caspase-8, DAP3, XIAP, Bcl-2, Bcl-XL, Bax,Mcl-1, and Bak. However, the mechanismof this compound is presentlyundefined, and, therefore, it is likelythat other proteins besides FLIP areinvolved in its proapoptotic mechanism.Indeed, when employedat higher doses, CDDO and relatedcompounds are reported to have effectson a number of cancer-relevanttargets.[89]When tested on solid-tumor celllines at submicromolar concentrations,CDDO generally sensitizes only toTRAIL and Fas, but alone CDDO doesnot induce significant apoptosis. Appliedto primary leukemia cells, however,CDDO and related compoundsdemonstrate single-agent activity, inducingrobust apoptosis via a caspase-8-dependent mechanism, includingchemorefractory chronic lymphocyticleukemias and acute myelogenousleukemias.[87,90,91] Thus, by activatingthe extrinsic pathway, CDDO andrelated triterpenoids may providea pharmacologic route to bypassroadblocks to intrinsic pathway(mitochondrial) apoptosis, therebyachieving apoptotic destruction ofchemorefractory leukemias.

Biologic agents that trigger theextrinsic pathway are also being exploredfor their utility in cancer treatment.For example, an agonistic antibodythat activates TRAIL receptor-1(DR4) has been tested in phase I trialsof patients with malignancy. Recombinanttrimeric TRAIL protein has producedimpressive preclinical results inmouse models, either alone or in combinationwith chemotherapy, and alsomay soon enter clinical trials.[92]Bcl-2-Family ProteinsBcl-2 protein is the founding memberof a large family of apoptosisregulatingproteins that govern the intrinsicpathway of apoptosis.Overexpression of the antiapoptoticprotein Bcl-2 occurs in roughly halfof human cancers, contributing to resistanceto anticancer drugs, hormoneablative therapy, and radiotherapy.[1]Several homologs of Bcl-2 proteinhave been identified and characterized,with some functioning as blockers (n= 5 in humans) and others as promotersof cell death (n = 19 in humans),[93-95] comprising a gene familyof 25 members.[96 unpublisheddata].Alterations in the expressionof several members of the Bcl-2-family protein have been documentedin cancers, includingoverexpression of antiapoptotic membersand loss of expression of proapoptoticmembers.[97] Simultaneousoverexpression of more than one of thesix antiapoptotic members of the Bcl-2-family proteins can occur in somecancers, creating challenges with respectto overcoming roadblocks toapoptosis.Our prior analysis of prostate tumors,for example, revealed that levelsof antiapoptotic proteins Bcl-2,Bcl-XL, and Mcl-1 are commonly elevatedin advanced prostate cancers,whereas proapoptotic proteins Baxand Bak generally remain present athigh levels during progression of thesetumors to a hormone-independent,metastatic phenotype.[98] Similarfindings have been made for melanomas,which commonly overexpressproteins Bcl-2, Bcl-XL, and Mcl-1.[99]Bcl-2-family proteins operate asregulators of the mitochondriadependentpathway for apoptosis(intrinsic pathway). These proteinsgovern the permeability of the mitochondrialmembrane, dictatingwhether apoptogenic proteins such ascytochrome c are released into thecytosol.[21,95,100] One of the prominentmechanisms by which the mitochondrial(intrinsic) pathway forapoptosis cross-talks with the deathreceptor (extrinsic) pathway involvescaspase-8-mediated cleavage and activationof the proapoptotic Bcl-2 homologBid.Normally, Bid resides in the cytosolin a latent (inactive) state however,on cleavage by caspase-8, this proteintranslocates to the outer membrane ofthe mitochondria, where it dimerizeswith other Bcl-2--family proteins, inducingthe release of cytochrome c andapoptosis.[95,101] Thus, in tumorcells where this Bid activation mechanismplays an important role in deathreceptor-mediated apoptosis (socalledtype II cells),[102,103]overexpression of either protein Bcl-2 or Bcl-XL has been shown to blockcell death induced by Fas, TNF, andTRAIL. Consequently, sensitivity oftumor cells to TNF-family cytokinescan potentially be improved by agentsthat reduce the expression or functionof Bcl-2/Bcl-XL.Many Bcl-2-family proteins physicallyinteract, forming homo- orheterodimers, whereby pro- andantiapoptotic members of this familyperform "hand-to-hand combat" inmaking cell life and death decisions.[104,105] The structural basisfor dimerization of Bcl-2-family proteinshas been elucidated using highfieldnuclear magnetic resonancemethods, revealing a hydrophobiccrevice on the surface of antiapoptoticproteins Bcl-2 and Bcl-XL that bindsan alpha-helical BH3 domain fromproapoptotic Bcl-2 family proteinssuch as Bax or Bak (Figure4).[106,107] Proof-of-concept experimentsperformed with BH3 peptideshave demonstrated suppression of proteinsBcl-2 or Bcl-XL, resulting in inductionof apoptosis or sensitizationof tumor cell lines to apoptosis in culture.[108-111]In addition, prototype nonpeptidylcompounds that compete for bindingto the BH3-binding pocket have beenidentified, enhancing sensitivity oftumor cell lines and cultured primaryleukemias to apoptotic stimuli (includinganticancer drugs), thus further validatingantiapoptotic Bcl-2-family proteinsBcl-2 and Bcl-XL as targets fordrug discovery.[112-117]Where tested, the compounds describedto date inhibit proteins Bcl-2,Bcl-XL, and (in some instances) otherantiapoptotic members of the Bcl-2family.[115,117] Thus, these compoundsmay afford advantagesover more targeted antisense-basedstrategies for overcoming apoptosis resistance,where redundancy is aproblem because of simultaneousoverexpression of more than oneantiapoptotic Bcl-2-family protein.However, because these compounds"hit" more targets, they may also proveto be more toxic to normal cells andtissues than antisense-based drugs.Thus, the therapeutic index of theseagents must be defined in vivo beforeclinical trials could be contemplated.Nevertheless, at least one smallmoleculeantagonist of protein Bcl-2has already been preliminarily evaluatedin human clinical trials, thoughits mechanism as an inhibitor of proteinBcl-2 was unknown at the timethe trials were initiated. This compound,gossypol, is a natural productidentified through Chinese herbalmedicine that interacts with the BH3-binding pocket of protein Bcl-2 withsubmicromolar affinity.[115] Gossypolundoubtedly has other targets besidesprotein Bcl-2 however, we andour collaborators have shown that gossypoldisplaces BH3 peptides fromantiapoptotic proteins Bcl-2, Bcl-XL,Bfl-1, and Bcl-B.[118] Semisyntheticanalogs of Gossypol are being evaluatedin preclinical studies to reduce thechemical reactivity of this compoundand thereby improve its safety andpharmacologic properties while retainingactivity against protein Bcl-2.[117]An alternative to small-moleculeantagonists that inhibit the Bcl-2 proteinis antisense DNA-based drugs thatsuppress the production of the Bcl-2protein.[119] These nuclease-resistant,synthetic, single-strand DNA moleculesbind cellular target mRNAs viaWatson-Crick base-pairing, leading toBcl-2 mRNA degradation by RNaseH-based mechanisms.[120] The Bcl-2 antisense drug oblimersen sodium(Genasense) hybridizes with the first18 nucleotides within the codingregion of Bcl-2 mRNAs, reducingthe expression of Bcl-2 proteinand thereby promoting apoptosis.Oblimersen sodium is in clinical testingfor several types of cancer.[121]ConclusionsKnowledge of the pathways ofapoptosis and of the mechanisms ofthe proteins that govern them is beginningto reveal a variety of targetsfor the discovery of cancer drugs. Detailedstructural analysis of apoptoticproteins and studies of their biochemicalmechanisms have suggested strategiesfor lead generation, resulting innumerous novel chemical entities withmechanism-based activity. Encouragingproof-of-principle data have beenprovided that help to validate severaltargets of apoptosis.Much work lies ahead, however, interms of optimizing the spectrum ofactivity of compounds that interactwith multiple members of apoptosisprotein families, improving the stabilityand pharmacologic properties ofthese compounds, establishing theiroptimal formulations for stability anddelivery, and elucidating attendantrate-limiting toxicities. Many of themost logical targets for promotingapoptosis of cancer and leukemia cellsare technically challenging and ofteninvolve either disrupting protein interactionsor altering gene expression, asopposed to traditional pharmaceuticalsthat typically target the active sites ofenzymes.Modern techniques of structurebaseddrug optimization render thistask feasible, but still challenging.Such targets require long-term commitments,often outstripping the usualdrug discovery and development cycleincorporated into the practices of pharmaceuticalcompanies. Long-termcommitments to research may createa new era in cancer therapy, where theintrinsic or acquired resistance of malignantcells to apoptosis can be pharmacologicallyreversed, reinstatingnatural pathways for cell suicide.There is good reason to suspect thatmalignant cells will be preferentiallysusceptible to restoration of apoptosissensitivity compared with normalcells. Cancer cells exhibit a wide varietyof abnormal behaviors andmolecular processes that normallywould trigger an apoptosis response,including cell-cycle checkpointdysregulation, oncogene activation,chromosome segregation defects, celldetachment from substratum, and outgrowthof blood supply (hypoxia).These defects render tumor cells moredependent on apoptosis-suppressinggenes and proteins, and thus withdrawingthis support from malignant cellsmay promote self-destruction of transformedcells while sparing normalcells.The full validity of this hypothesisawaits verification in human clinicaltrials. However, present insights fromanimal studies and current forays intothe clinic are encouraging. Apoptosisbasedstrategies for the discovery ofcancer drugs promise to yield effectivetherapies against cancer and meritfurther research support.


Dr. Reed is a shareholderin Genta Incorporated.


Reed J: Dysregulation of apoptosis in cancer.J Clin Oncol 17:2941, 1999.

Evan G, Littlewood T: A matter of lifeand cell death. Science 281:1317-1322, 1998.

Ionov Y, Yamamoto H, Krajewski S, et al:Mutational inactivation of the pro-apoptoticgene BAX confers selective advantage duringtumor clonal evolution. Proc Natl Acad SciU S A 97:10872-10877, 2000.

Mechanisms of Cancer

Understanding the fundamentals of how cancer cells form and proliferate has been crucial to cancer treatment and prevention. New areas of basic research will lead to better outcomes for patients.

A cancer cell. New research on how each cell differs from others within a tumor provides a promising avenue for better patient outcomes.
Credit: iStock

Decades of discovery have demonstrated that a deep understanding of the fundamental mechanisms of cancer—how it forms, why it persists and what causes it to spread through the body—leads to better outcomes for patients. New areas of basic cancer research, including how each cell differs from others within a tumor, how the environment in which a tumor grows impacts its progress and how well an individual’s immune system mounts a defense, will yield improved outcomes for patients.

Now, cancer researchers are using new tools, technologies and ways of thinking to develop an even more sophisticated understanding of cancer mechanisms. They are exploring subtle variations that impact cancer cells’ behaviors—not just between different patients or cancer types but even among the different cells that make up an individual tumor. At the same time, researchers are broadening their focus, looking beyond tumors to learn how factors elsewhere in the body impact a patient’s disease.

Until now, studies of cancer biology have largely focused on what makes tumor cells different from healthy cells. But it has become clear that not all tumor cells—even within a single tumor—are the same. Only a small fraction of a tumor’s cells may have the capacity to divide and sustain the tumor’s growth.

This variability has enormous clinical consequences, and we now know that it will be important to understand human cancer on a cell-by-cell basis. Using recently developed methods of high-throughput analysis, researchers can now study the DNA, RNA and proteins of thousands of individual cells to characterize this heterogeneity and investigate how it affects tumor growth, metastasis and patients’ response to treatment.

It has also become evident that a tumor’s growth depends on more than the makeup of its own cells. The microenvironment in which a tumor grows, as well as the vigor with which the body’s immune system recognizes and attacks cancers, are just as critical. A major challenge is to understand the interactions between tumors and their microenvironments. Ultimately, we will need to decode the signals that tumors send to nearby immune cells as well as define which aspects of a tumor’s surroundings help determine whether it stays small and benign or is allowed to grow unchecked and spread.

Although there is still much to learn about the cellular changes that drive cancer, new genomic and computational technologies have greatly accelerated the search. There is now the capacity to characterize and compare thousands of patient tumors, enabling researchers to identify factors that influence cancer risk even if they are rare or when their individual impact is relatively small. Uncovering these factors promises to point us toward important cancer pathways and suggest new opportunities for intervention.

Building on CCR’s long-standing strong portfolio of basic research and the ability of CCR principal investigators to freely pursue fundamental questions in biology, our investigators are well positioned to continue elucidating the basic cellular mechanisms that underlie all types of cancer. We are also exploring the mechanisms that drive rare but genetically well-defined tumors, which may serve as model systems to understand more globally applicable cancer mechanisms. Accelerated by the latest technologies, investigations of basic mechanisms of cancer promise to uncover new and improved diagnostic and therapeutic approaches, just as they have in the past.

2. Apoptosis

The term "apoptosis" is derived from the Greek words "απο" and "πτωσιζ" meaning "dropping off" and refers to the falling of leaves from trees in autumn. It is used, in contrast to necrosis, to describe the situation in which a cell actively pursues a course toward death upon receiving certain stimuli [7]. Ever since apoptosis was described by Kerr et al in the 1970's, it remains one of the most investigated processes in biologic research [8]. Being a highly selective process, apoptosis is important in both physiological and pathological conditions [9, 10]. These conditions are summarised in Table 1.

2.1 Morphological changes in apoptosis

Morphological alterations of apoptotic cell death that concern both the nucleus and the cytoplasm are remarkably similar across cell types and species [11, 12]. Usually several hours are required from the initiation of cell death to the final cellular fragmentation. However, the time taken depends on the cell type, the stimulus and the apoptotic pathway [13].

Morphological hallmarks of apoptosis in the nucleus are chromatin condensation and nuclear fragmentation, which are accompanied by rounding up of the cell, reduction in cellular volume (pyknosis) and retraction of pseudopodes [14]. Chromatin condensation starts at the periphery of the nuclear membrane, forming a crescent or ring-like structure. The chromatin further condenses until it breaks up inside a cell with an intact membrane, a feature described as karyorrhexis [15]. The plasma membrane is intact throughout the total process. At the later stage of apoptosis some of the morphological features include membrane blebbing, ultrastrutural modification of cytoplasmic organelles and a loss of membrane integrity [14]. Usually phagocytic cells engulf apoptotic cells before apoptotic bodies occur. This is the reason why apoptosis was discovered very late in the history of cell biology in 1972 and apoptotic bodies are seen in vitro under special conditions. If the remnants of apoptotic cells are not phagocytosed such as in the case of an artificial cell culture environment, they will undergo degradation that resembles necrosis and the condition is termed secondary necrosis [13].

2.2 Biochemical changes in apoptosis

Broadly, three main types of biochemical changes can be observed in apoptosis: 1) activation of caspases, 2) DNA and protein breakdown and 3) membrane changes and recognition by phagocytic cells [16]. Early in apoptosis, there is expression of phosphatidylserine (PS) in the outer layers of the cell membrane, which has been "flipped out" from the inner layers. This allows early recognition of dead cells by macrophages, resulting in phagocytosis without the release of pro-inflammatory cellular components [17]. This is followed by a characteristic breakdown of DNA into large 50 to 300 kilobase pieces [18]. Later, there is internucleosomal cleavage of DNA into oligonucleosomes in multiples of 180 to 200 base pairs by endonucleases. Although this feature is characteristic of apoptosis, it is not specific as the typical DNA ladder in agarose gel electrophoresis can be seen in necrotic cells as well [19]. Another specific feature of apoptosis is the activation of a group of enzymes belonging to the cysteine protease family named caspases. The "c" of "caspase" refers to a cysteine protease, while the "aspase" refers to the enzyme's unique property to cleave after aspartic acid residues [16]. Activated caspases cleave many vital cellular proteins and break up the nuclear scaffold and cytoskeleton. They also activate DNAase, which further degrade nuclear DNA [20]. Although the biochemical changes explain in part some of the morphological changes in apoptosis, it is important to note that biochemical analyses of DNA fragmentation or caspase activation should not be used to define apoptosis, as apoptosis can occur without oligonucleosomal DNA fragmentation and can be caspase-independent [21]. While many biochemical assays and experiments have been used in the detection of apoptosis, the Nomenclature Committee on Cell Death (NCCD) has proposed that the classification of cell death modalities should rely purely on morphological criteria because there is no clear-cut equivalence between ultrastructural changes and biochemical cell death characteristics [21].

2.3 Mechanisms of apoptosis

Understanding the mechanisms of apoptosis is crucial and helps in the understanding of the pathogenesis of conditions as a result of disordered apoptosis. This in turn, may help in the development of drugs that target certain apoptotic genes or pathways. Caspases are central to the mechanism of apoptosis as they are both the initiators and executioners. There are three pathways by which caspases can be activated. The two commonly described initiation pathways are the intrinsic (or mitochondrial) and extrinsic (or death receptor) pathways of apoptosis (Figure 1). Both pathways eventually lead to a common pathway or the execution phase of apoptosis. A third less well-known initiation pathway is the intrinsic endoplasmic reticulum pathway [22].

The intrinsic and extrinsic pathways of apoptosis.

2.3.1 The extrinsic death receptor pathway

The extrinsic death receptor pathway, as its name implies, begins when death ligands bind to a death receptor. Although several death receptors have been described, the best known death receptors is the type 1 TNF receptor (TNFR1) and a related protein called Fas (CD95) and their ligands are called TNF and Fas ligand (FasL) respectively [17]. These death receptors have an intracellular death domain that recruits adapter proteins such as TNF receptor-associated death domain (TRADD) and Fas-associated death domain (FADD), as well as cysteine proteases like caspase 8 [23]. Binding of the death ligand to the death receptor results in the formation of a binding site for an adaptor protein and the whole ligand-receptor-adaptor protein complex is known as the death-inducing signalling complex (DISC) [22]. DISC then initiates the assembly and activation of pro-caspase 8. The activated form of the enzyme, caspase 8 is an initiator caspase, which initiates apoptosis by cleaving other downstream or executioner caspases [24].

2.3.2 The intrinsic mitochondrial pathway

As its name implies, the intrinsic pathway is initiated within the cell. Internal stimuli such as irreparable genetic damage, hypoxia, extremely high concentrations of cytosolic Ca 2+ and severe oxidative stress are some triggers of the initiation of the intrinsic mitochondrial pathway [24]. Regardless of the stimuli, this pathway is the result of increased mitochondrial permeability and the release of pro-apoptotic molecules such as cytochrome-c into the cytoplasm [25]. This pathway is closely regulated by a group of proteins belonging to the Bcl-2 family, named after the BCL2 gene originally observed at the chromosomal breakpoint of the translocation of chromosome 18 to 14 in follicular non-Hodgkin lymphoma [26]. There are two main groups of the Bcl-2 proteins, namely the pro-apoptotic proteins (e.g. Bax, Bak, Bad, Bcl-Xs, Bid, Bik, Bim and Hrk) and the anti-apoptotic proteins (e.g. Bcl-2, Bcl-XL, Bcl-W, Bfl-1 and Mcl-1) [27]. While the anti-apoptotic proteins regulate apoptosis by blocking the mitochondrial release of cytochrome-c, the pro-apoptotic proteins act by promoting such release. It is not the absolute quantity but rather the balance between the pro- and anti-apoptotic proteins that determines whether apoptosis would be initiated [27]. Other apoptotic factors that are released from the mitochondrial intermembrane space into the cytoplasm include apoptosis inducing factor (AIF), second mitochondria-derived activator of caspase (Smac), direct IAP Binding protein with Low pI (DIABLO) and Omi/high temperature requirement protein A (HtrA2) [28]. Cytoplasmic release of cytochrome c activates caspase 3 via the formation of a complex known as apoptosome which is made up of cytochrome c, Apaf-1 and caspase 9 [28]. On the other hand, Smac/DIABLO or Omi/HtrA2 promotes caspase activation by binding to inhibitor of apoptosis proteins (IAPs) which subsequently leads to disruption in the interaction of IAPs with caspase-3 or -9 [28, 29].

2.3.3 The common pathway

The execution phase of apoptosis involves the activation of a series of caspases. The upstream caspase for the intrinsic pathway is caspase 9 while that of the extrinsic pathway is caspase 8. The intrinsic and extrinsic pathways converge to caspase 3. Caspase 3 then cleaves the inhibitor of the caspase-activated deoxyribonuclease, which is responsible for nuclear apoptosis [30]. In addition, downstream caspases induce cleavage of protein kinases, cytoskeletal proteins, DNA repair proteins and inhibitory subunits of endonucleases family. They also have an effect on the cytoskeleton, cell cycle and signalling pathways, which together contribute to the typical morphological changes in apoptosis [30].

2.3.4 The intrinsic endoplasmic reticulum pathway

This intrinsic endoplasmic reticulum (ER) pathway is a third pathway and is less well known. It is believed to be caspase 12-dependent and mitochondria-independent [31]. When the ER is injured by cellular stresses like hypoxia, free radicals or glucose starvation, there is unfolding of proteins and reduced protein synthesis in the cell, and an adaptor protein known as TNF receptor associated factor 2 (TRAF2) dissociates from procaspase-12, resulting in the activation of the latter [22].

Targeting BCL-2 regulated apoptosis in cancer

The ability of a cell to undergo mitochondrial apoptosis is governed by pro- and anti-apoptotic members of the BCL-2 protein family. The equilibrium of pro- versus anti-apoptotic BCL-2 proteins ensures appropriate regulation of programmed cell death during development and maintains organismal health. When unbalanced, the BCL-2 family can act as a barrier to apoptosis and facilitate tumour development and resistance to cancer therapy. Here we discuss the BCL-2 family, their deregulation in cancer and recent pharmaceutical developments to target specific members of this family as cancer therapy.

1. Introduction

Apoptosis is a form of regulated cell death that is triggered in response to developmental cues or cellular stress. This selective cell suicide plays an essential role in numerous physiological and pathological processes including development, immunity and disease where the elimination of damaged or superfluous cells helps to ensure organismal health [1].

There are two apoptotic pathways—the extrinsic pathway (activated by ligand engagement of cell surface death receptors) and the intrinsic (mitochondrial) pathway. This review focuses on the BCL-2 family of proteins that regulate activation of the intrinsic apoptotic pathway in response to cellular stresses such as DNA damage, γ-irradiation, oncogene activation and growth factor withdrawal.

Recent pharmaceutical advances have allowed the specific targeting of protein–protein interactions in the BCL-2 (B-cell lymphoma 2) family [2–4]. Early clinical results in haematological cancers show considerable promise. This review will summarize apoptotic pathway regulation by the BCL-2 family, their perturbation in cancer and utility as therapeutic targets.

2. The BCL-2 family

The founder member, BCL-2, was first identified through chromosomal mapping in follicular lymphoma where constitutive BCL-2 expression is driven from the immunoglobulin locus by the t[1218] translocation [5–7]. Unlike the cell growth and proliferative functions of other known oncoproteins at that time, BCL-2 was found to facilitate oncogenesis through cell death resistance [8,9]. In the following years over 15 proteins have been added to this family, each containing one or more BCL-2 homology (BH) domain and functional studies have allowed grouping into three classes (figure 1).

Figure 1. The BCL-2 family is composed of pro-survival and pro-apoptotic proteins. BCL-2 family members show sequence homology to BCL-2 in one or more BH (BCL-2 homology) domain. These proteins can be divided into pro-survival and pro-apoptotic proteins. Within the pro-apoptotic members there is a further subdivision between the multi-BH domain containing effector proteins and those proteins whose only region of homology to BCL-2 is BH3 (known as BH3-only proteins). Membrane insertion is mediated by transmembrane domains (TMD) present in pro-survival, effector and some BH3-only proteins (*BIM, BIK and HRK).

A clear division in the family exists between members that function to prevent apoptosis (pro-survival or anti-apoptotic) and those that induce apoptosis (pro-apoptotic). The pro-apoptotic BCL-2 family members can be further divided into the multi-BH-domain effector proteins (containing BH1, BH2 and BH3 domains) and BH3-only proteins (only region of homology to BCL-2 is BH3) (figure 1).

Physical interaction between pro-survival and pro-apoptotic family members can buffer the cell against the onset of mitochondrial-mediated apoptosis. Structural studies have revealed that BH1, BH2 and BH3 regions together form a hydrophobic pocket that can be filled by the amphipathic α-helical BH3 domain of pro-apoptotic BCL-2 proteins [10,11]. The balance of this interaction ensures appropriate apoptotic regulation in response to development cues and cellular stresses. In a simple model, when pro-survival proteins predominate, apoptosis is held in check: when pro-apoptotic proteins predominate, apoptosis is triggered. However, localization and conformation of BCL-2 proteins is also important in regulation of activity.

3. Pro-survival BCL-2 proteins

Pro-survival proteins such as BCL-XL, MCL-1, BFL1 (A1 in mouse) and BCL-W contain multiple regions of homology to BCL-2 (figure 2). Each of these proteins is found in many cell types/tissues and co-expression of multiple pro-survival proteins often occurs. The relative expression levels can vary in a cell type and developmental manner and are perhaps best characterized in the haematopoietic system. For example, dynamic patterns of pro-survival BCL-2 gene expression occurs during B lymphocyte development with Bclx being expressed early in B-cell development, Mcl1 and Bcl2 expression generally increasing with B-cell maturity and A1 levels peaking in the intermediate stages [12]. In this way, different pro-survivals play key roles at distinct stages of development.

Figure 2. BCL-2 family interactions regulate mitochondrial outer membrane permeabilization (MOMP). Interaction between pro-survival and pro-apoptotic BCL-2 proteins sets a threshold for activation of apoptosis. BCL-2-like pro-survival proteins inhibit BAX/BAK activation whereas BH3-only proteins promote BAX/BAK oligomerization. Drugs mimicking the action of BH3-only proteins indirectly lead to BAX/BAK activation. This allows MOMP, apoptosome formation and subsequent caspase activation and apoptosis.

Levels of pro-survival BCL-2 proteins can also be regulated by protein turnover. BCL-2 and BCL-XL are relatively stable proteins (e.g. the half-life of BCL-2 approx. 20 h) [13]. By contrast, MCL-1 and A1 protein turnover is constitutive through polyubiquitination and proteosomal degradation (reflected in their short half-lives approx. 30 and approx. 15 min, respectively) [14–17]. In this way, levels of MCL-1 and A1 help facilitate dynamic responses to cell death stimuli.

Gene deletion studies have revealed essential and non-redundant roles of pro-survival BCL-2 proteins in mice. While embryogenesis proceeds normally in the absence of Bcl2, deficient mice show postnatal growth retardation, premature greying, apoptotic involution of spleen/thymus and succumb to early mortality through polycystic kidney disease with altered renal cell differentiation and elevated apoptosis [18]. Young Bcl2 −/− mice have normal haematopoietic populations, but this is not sustained, with notable loss in peripheral B and T lymphocyte populations [18,19]. These phenotypes can be reversed by loss of one or two Bim alleles (encoding a BH3-only pro-apoptotic member of the BCL-2 family), indicating that sequestration of BIM is the major function of BCL-2 [20].

Deletion of Bclx is lethal around embryonic day 13 with extensive neuronal and haematopoietic apoptosis [21], loss of Bim can rescue the haematopoetic but not neuronal phenotype in bclx null embryos [22]. In adult mice, acute deletion of Bclx is tolerated (animals were followed for 1 month) but still resulted in severe anaemia, consistent with Bclx being required for reticulocyte survival [23]. In contrast to embryogenesis, loss of bim could not restore erythropoiesis in adults [23].

Deletion studies of A1 had been incomplete until recently when knockout of all three functional isoforms of A1 in mice was achieved. Surprisingly, A1 function seems largely redundant, with only minor impact on subsets of cells in the haematopoietic system [24]. The function of BCL-W also appears mostly dispensable for normal development and health, but Bclw-deficient males are infertile due to a defect in spermatogenesis [25,26].

Across the BCL-2 family the phenotype of the Mcl1 knockout mouse is most severe. Mcl1 deficiency results in early lethality at pre-implantation stage, but these blastocysts showed no evidence of increased apoptosis [27], providing a suggestion of a non-apoptotic role for MCL-1. Conditional deletion studies in the adult mouse have also revealed an essential role for MCL-1 in numerous cell types, including T and B lymphocytes [28], haematopoietic stem cells [29], cardiomyocytes [30,31], hepatocytes [32], neuronal progenitors [33] and neutrophils, but not macrophages [34,35], mammary epithelium or megakaryocytes [36,37].

Interestingly, while MCL-1 deficiency alone in megakaryocytes had no impact, when combined with loss of Bclx this caused embryonic or pre-weaning lethality, which is also far more dramatic than the impaired platelet shedding phenotype that is found with loss of just Bclx [36,37]. This could be rescued by co-deletion of Bax/Bak [37], but clearly illustrates the co-dependence of certain cell types on multiple pro-survival proteins. This co-dependence has been shown in elegant detail in a recent study of immune populations where multiple pro-survival proteins were targeted by genetic and pharmacologic methods [38]. Therefore, while gene knockout studies have given much insight into cell types in which individual pro-survival proteins have a dominant role, there has probably been an underestimation of the extent of their contribution to cell survival in many other cell types. Indeed, the sum effect of all pro-survival proteins present may be more important for survival than expression levels of an individual protein. This is an important consideration for therapeutic targeting of pro-survival BCL-2 proteins and minimization of damage to normal tissues.

4. Pro-apoptotic BCL-2 members

Pro-apoptotic BCL-2 proteins fall into two sub-classes (figure 2). BH3-only proteins such as BIM, BAD, BID, NOXA, PUMA, BMF, HRK and BIK only show homology to the BH3 domain of BCL-2. The effector proteins BAX, BAK and BOK contain multiple BH domains and structural studies of BAX revealed that effector protein three-dimensional conformation is similar to that of pro-survival BCL-2 proteins [39]. Like the pro-survival BCL-2 proteins, multiple pro-apoptotic proteins are found expressed in cells at the same time.

Upregulation of BH3-only proteins can occur at transcriptional/post-translational levels in response to stress to trigger cell death. For example, Puma and Noxa are transcriptional targets of the p53 tumour suppressor and their expression is increased in response to cytotoxic stimuli that activate p53, although PUMA is also important in response to p53-independent apoptotic stimuli [40]. BH3-only proteins can also be regulated by post-translational modification. Phosphorylation of BAD leads to sequestration by 14-3-3 proteins in the cytosol where it cannot exert pro-apoptotic functions [41]. Other mechanisms of activation exist such as altered cellular localization. Full-length BID is located in the cytosol but upon cleavage by caspase 8 (downstream of death-receptor signalling in the extrinsic apoptotic pathway) a truncated product is formed (tBID) which is capable of locating to the mitochondria and activating apoptosis [42].

BAX and BAK contain membrane anchoring C-terminal tails and while BAK is constitutively bound to the outer mitochondrial membrane, in healthy cells BAX appears cytosolic [43]. However, BAX and (to a lesser degree) BAK are actually in a dynamic equilibrium between cytosol and membranes, and are constitutively retrotranslocated to the cytosol by pro-survival BCL-2 proteins [44–46]. In the absence of any other BCL-2 proteins BAX becomes membrane localized, like BAK [45].

The mechanism of activation of BAX/BAK by BH3-only proteins has been the subject of intense debate. Evidence exists to suggest that some BH3-only proteins are ‘activators’: in this model BIM, tBID and PUMA, can directly interact with BAX/BAK to trigger their conformational change [47,48]. The function of the other BH3-only proteins in this model is as ‘sensitizers’, whose binding to BCL2-like pro-survival proteins frees up ‘activator’ BH3-only proteins [47,48]. An alternative, ‘indirect’ model has also been proposed whereby pro-survival BCL-2 proteins exert their function through direct interaction with BAX/BAK and BH3-only proteins act to sequester pro-survival proteins away from BAX/BAK. Such interactions do not always depend on BH3 domains as transmembrane domain (TMD) dimerization also occurs in the outer mitochondrial membrane. This has been observed in non-apoptotic cells and could indicate competition between the TMDs of pro-survival proteins such as BCL-2 and BCL-XL to prevent BAX/BAK homo-oligomerization [49].

Recently, genome editing was used to disrupt all known BH3-only proteins (eight in total) in HCT116 cells rendering these cells resistant to stress-induced apoptosis [50]. Interestingly, treatments that downregulate/target MCL-1- and BCL-XL-induced apoptosis with equivalent kinetics to those seen in cells proficient for BH3-only proteins revealing that known BH3-only proteins are not required for BAX/BAK activation [50]. Instead, association with the mitochondrial outer membrane is sufficient to drive homo-oligomerization of BAX/BAK in the absence of pro-survival BCL-2 proteins [50]. Regardless of which model is active in a particular cell type/situation, the outcome of a relative increase in the levels of pro- versus anti-apoptotic BCL-2 proteins is the same and the C-termini of BAX/BAK undergo conformational change that allows dimerization [51,52]. Reciprocal interaction between the BH3 domain of one molecule and the hydrophobic groove on another results in symmetrical homodimers (although heterodimers of BAX/BAK can also form in this manner) and linkage of these leads to higher-order oligomerization [53,54]. These oligomers delineate arcs, lines or ring-like structures in the outer mitochondrial membrane [55,56]. This mitochondrial outer membrane permeabilization (MOMP) allows release of soluble proteins from the mitochondrial inner membrane space such as cytochrome c which binds to APAF-1 (apoptotic protease activating factor 1), promoting its oligomerization and binding to pro-caspase 9—forming a complex termed the apoptosome. At the apoptosome, pro-caspase-9 is activated via dimerization, which in turn cleaves and activates the executioner caspases-3 and -7 to drive mass proteolysis that leads to DNA fragmentation, chromatin condensation and the dismantling of the cell. It is important to note that the mitochondrial pathway also contributes to death-receptor-mediated apoptosis through caspase 8 cleavage of BID, indeed BAX/BAK are required in many circumstances for receptor-mediated apoptosis [57,58].

In vivo analysis suggests the function of BAX and BAK is largely redundant in physiological settings only a minority of double knockout mice survive to adulthood [59], but one copy of either is enough to allow normal development. In the absence of BAX/BAK apoptosis is impaired in response to almost all stimuli showing their requirement for the initiation of MOMP [60]. Less well studied, BOK is an additional multi-domain pro-apoptotic BCL-2 protein that was identified through its interaction with MCL-1 [61], although subsequent studies have suggested that BOK does not interact with pro-survival BCL-2 proteins [62,63]. BOK transcripts are present in many tissues in mice [64] but the protein is short-lived due to turnover by ubiquitylation and endoplasmic reticulum-associated degradation mediated by the proteasome [63].

BOK shows strong homology to BAX and BAK, and its role in apoptotic regulation has recently been extensively investigated. BOK deficient mice appear normal [64], but enforced BOK expression can drive apoptosis in a range of cell types and there is debate over whether this requires BAX/BAK [62–67]. Endogenous BOK is found predominantly in the membranes of the Golgi apparatus and endoplasmic reticulum (ER) and a function in ER stress response has been suggested [62,65]. More recently, the ability of proteasome inhibitors to induce apoptosis in BAX/BAK-deficient MEF or HCT116 cells was shown to require BOK expression but this was independent of pro-survival BCL-2 family proteins [63]. For now, the role of BOK certainly seems unique and requires further investigation.

5. Favoured interactions

As discussed above, a level of specificity is granted by differential expression pattern, cellular localization, post-translational activation and turnover of BCL-2 family proteins. A further level of complexity was revealed by biochemical and cell biology studies that showed differential binding affinities of particular BH3-only proteins for pro-survivals [68,69]. While BH3-only proteins such as BIM, PUMA and tBID bind with high affinity to all pro-survival proteins, others are more selective in their interaction. For example, BAD preferentially interacts with BCL2, BCL-XL and BCL-W while NOXA showing a reciprocal interaction preference for only MCL-1 and A1 (figure 3). Further complexity is added by preference of BH3-only proteins for activation of BAX or BAK. While BIM and BID bind the same repertoire of pro-survival proteins (figure 3), preference of BID to mediate apoptosis through BAK has been shown [70,71], whereas BIM preference for BAX [70], or no preference [71], has been observed. Domain swap experiments have revealed that these effects are determined by the BH3 sequence of the BH3-only proteins and are dependent on cell type [71].

Figure 3. Specific interactions of BH3-only with pro-survival proteins. Some BH3-only proteins (BIM, PUMA and BID) are promiscuous and can bind all pro-survival BCL-2 proteins, whereas others (BAD and NOXA) show a more restricted binding pattern.

An additional layer of complexity is added with specificity in pro-survival proteins for BAK versus BAX activation. MCL-1 and BCL-XL constrain BAK, but BCL-2 does not [72], and it is the BH3 domain of BAK that determines these associations [71].

5.1. Increased pro-survival BCL-2 proteins in cancer

Evasion of apoptosis can aid oncogenic transformation at multiple stages through facilitating sustained tumour growth, survival during metastatic process and resistance to therapy. Therefore, it is not surprising that increased expression of pro-survival BCL-2 proteins is found in many cancer types. This upregulation can occur through a variety of mechanisms including chromosomal translocation, gene amplification, increased gene expression/translation or protein stability with various mechanisms and alternative pro-survival BCL-2 protein increases seeming more prominent in particular cancers.

Besides the Bcl2 t(1218) translocation, first found in follicular lymphoma (and subsequently in diffuse large-cell lymphomas [73]), translocation of BCL-2 family genes is not common across different cancer types nor does it seem to occur to other pro-survival members. Interestingly, t(1418) translocation of Bcl2 has also been found in peripheral blood lymphocytes from healthy individuals [74] and modelling of this translocation in B cells of mice is only weakly tumorigenic [75]. Together, these data suggest that this translocation is not overtly oncogenic.

Gene copy number increases in pro-survival BCL-2 members in cancer are more widespread than translocation. Transgenic modelling of increases in pro-survival proteins has mostly been limited to the haematopoietic systems where elevation of Bcl2, Mcl1 or Bclx predisposes to lymphoma development, albeit with long latency and incomplete penetrance. Amplification of MCL1 and BCL2L1 (encodes BCL-XL) were found to be among the most frequent chromosomal gains in a study of over 3000 samples representing 26 tumour types [76]. It is interesting that these amplifications were prominent outside of haematopoietic cancers, which have been the traditional niche for studies on tumorigenic roles of the BCL-2 members. Indeed, analysis of The Cancer Genome Atlas (TCGA) data through cBioportal [77] confirms the prevalence of MCL1, and to a lesser extent BCL2L1, amplification in many solid cancers (figure 4). Again, there seems specificity between family members as BCL2, BCL2A1 (BFL) and BCL2L2 (BCL-W) amplification are much rarer events. Consistent with a pro-tumour role, mutation or deletion of pro-survival BCL2 proteins was also infrequent (figure 4). While pro-survival BCL-2 expression on its own is mildly oncogenic, acquisition of additional genetic hits is clearly required for tumour formation. Co-amplification of MYC with MCL-1 or BCLX is common in cancer [76] and in cell culture and mouse models increased BCL-2-like proteins, and MYC is a potent oncogenic combination [83–87].

Figure 4. Frequency of genomic alteration of pro-survival BCL-2 proteins in cancer. Frequency of amplification (circle), mutation (triangle) or deletion (square) of pro-survival BCL-2 members in a range of cancers. Data mined from TCGA studies through cBioportal [77]. BCL-2 (black), BCL-XL (BCL2L1 blue), MCL-1 (red), BFL (BCL2A1 grey), BCL-W (BCL2L2 purple). AML, acute myeloid leukaemia [78], 173 cases. Bladder, urothelial carcinoma nature (TCGA provisional), 408 cases. Breast, invasive carcinoma (TCGA provisional), 1100 cases. GBM, glioblastoma [79], 166 cases. HNSCC, head and neck squamous cell carcinoma (TCGA provisional), 522 cases. ccRCC, kidney renal clear cell carcinoma [80], 534 cases. Lung adenocarcinoma (TCGA provisional), 517 cases. Thyroid, papillary thyroid carcinoma [81], 509 cases. Stomach adenocarcinoma (TCGA provisional), 415 cases. Uterine, corpus endometrial carcinoma [82], 177 cases.

Increased transcription of pro-survival proteins can also elevate their expression in cancer and increased transcription/translation seems important for regulation of MCL-1 levels. For example, in chronic lymphocytic leukaemia (CLL) c-ABL has been shown to drive high MCL-1 mRNA and protein expression through STAT3/NF-κB [88]. Similarly, in a mouse model of B-cell acute lymphoblastic leukaemia (B-ALL) the BCR-ABL oncoprotein was shown to drive high levels of MCL-1 expression that was essential for leukemogenesis [89].

Increased protein translation impacts on MCL-1 and BCL-XL levels and MCL-1 has been identified as a downstream mediator of the oncogenic effect of the translation initiation factor eIF4e [90]. Disruption of pathways regulating MCL-1 protein stability also occurs in cancer (e.g. loss or mutation of FBW7 inhibits MCL-1 degradation and is associated with tumorigenesis and resistance to chemotherapy [91,92]). Therefore, in addition to genetic alterations in BCL-2 family members, activation of oncogenic signalling pathways can also increase pro-survival protein levels.

It is important to consider that the observation of high levels of pro-survival BCL-2 proteins in cancer need not necessarily indicate strong apoptotic resistance. Elevation of BCL-2 actually sensitizes to apoptosis induced by the BCL-2/BCL-XL/BCL-W targeting drug ABT-737 through release of high levels of BIM that have been harboured in BIM/BCL-2 complexes [93]. In this way, a cell can be thought of as primed for death, close to the threshold required for apoptosis induction and measurement of the level of mitochondrial priming in cells can be used to predict response to chemotherapy [94,95].

Alterations in pro-survival BCL2 proteins might have importance beyond cancer genesis as high levels of MCL-1 expression at diagnosis correlate with poor prognosis in breast cancer [96], and MCL-1 amplification is prominent in treatment-resistant breast cancers [97], suggesting that this may be a source of innate and acquired resistance to cancer therapy. Such associations do not hold true for pro-survival BCL-2-like proteins in general and high levels of BCL-2 are actually associated with good prognosis in breast cancer [98–101].

5.2. Decreased pro-apoptotic BCL-2 proteins in cancer

Decreased expression of pro-apoptotic BCL-2 proteins has the same functional outcome as increased pro-survival expression in cancer. Gene deletion studies in mice do not reveal a strong oncogenic impact of decreased BH3-only protein expression. With the exception of BAD knockout mice, which succumb to late onset lymphoma [102], deficiency in individual BH3-only proteins does not predispose mice to tumour development [103]. Functional redundancy between BH3-only proteins could account for this, however, compound deletion of multiple BH3-only proteins is still only weakly tumour promoting with autoimmunity contributing to morbidity [104].

Similar to over-expression of pro-survival BCL2-like proteins, loss of BH3-only proteins is not overtly oncogenic but can dramatically accelerate lymphoma development in the context of elevated MYC. BIM seems most potent in this context, with deletion of even a single allele of BIM having dramatic effects [105].

Cancer therapeutics engaging the p53 response would be predicted to upregulate PUMA and NOXA and resistance to apoptosis could be mediated by their downregulation. Indeed, deletion of the gene encoding PUMA (Bbc3) occurs in a range of cancer types [76] and other mechanisms can decrease PUMA expression such as promoter methylation [106].

5.3. Altered expression of effector proteins

Elimination or downregulation of apoptotic effector proteins such as BAX/BAK is a potent way to disable mitochondrial-mediated apoptosis in cell culture systems. As the function of BAX and BAK are largely redundant, with one copy of either allowing normal development, abrogation of their activity would require loss of all four alleles. There is limited evidence that this occurs in cancer and BAX/BAK levels naturally decline with age in mice and humans [107]. It is important to note that localization and conformation may be even more important than absolute levels of BAX/BAK. For example, in acute myeloid leukaemia (AML) mitochondrial localized BAX is associated with both increased apoptotic sensitivity and improved patient prognosis [108]. Gene deletion studies in mice do not suggest a tumour suppressive role for BAX or BAK when knocked out individually (presumably due to their redundant roles) and double knockout results in perinatal lethality [59,109]. Using chimeric mice Bax/Bak deletion in the haematopoietic compartment drives fatal autoimmune disease [110]. The prominence of autoimmune disease in chimeric mice with Bax/Bak [111] deficient haematopoietic systems may mask tumour development. Targeted deletion mouse studies can avoid these autoimmune complications and in this setting loss of BAX/BAK in the brain and testicles results in tumours [112].

It seems pertinent loss of Bok is a relatively frequent event in a range of cancers [76] and gene silencing may result in loss of BOK protein in additional cases [111]. BOK is downregulated in non-small cell lung cancer (NSCLC) and high BOK is associated with good prognosis in lymph-node positive patients [111]. Interestingly, the tumour suppressive effect of BOK in NSCLC does not seem to be through apoptotic regulation and is instead through antagonism of TGF-β2 mediated epithelial to mesenchymal transition (EMT) and cell migration [111]. Unlike so many other BCL-2 proteins, Bok loss failed to reveal a tumour suppressive role the Eu-Myc mouse model [64], a system that is a sensitive read-out for altered apoptosis, although factors such as cooperating second hits, timing of Bok loss and cell type specificity could all be involved in a BOK effect. Compound knockout of Bax/Bak/Bok in the haematopoietic system also results in autoimmune disease rather than tumourigenesis [113]. More recently, in a chemical (DEN)-induced model of liver carcinogenesis, loss of BOK has been shown to protect against cancer. In this model, cancer can be promoted by death of hepatocytes, supporting compensatory proliferation with associated mutagenesis, in surrounding tissue that ultimately leads to live cancer [114]. Interestingly, deletion of BOK inhibited the ER-stress response and induction of pro-apoptotic BH3-only proteins BIM and PUMA, placing BOK's tumour promoting role upstream of MOMP [114]. Secondly, BOK was also shown to have an additional growth-promoting effect, though the mechanisms underlying this remain unclear it would also be expected to be pro-tumourigenic [114].

5.4. Pharmaceutical intervention to reset the balance

As the balance of BCL-2 proteins can act as a trigger for apoptosis, mechanisms to alter their expression or interaction would be expected to have clinical use. Indeed conventional anti-cancer therapies can act through disruption of the BCL-2 family. For example, DNA damaging agents such as etoposide or daunorubicin activate the p53 tumour suppressor transcription factor whose targets include PUMA and NOXA. However, the presence of elevated pro-survival BCL-2 proteins can act as a barrier to apoptosis even upon upregulation of BH3-only proteins. Neutralization of pro-survival proteins would re-sensitize to BH3-only upregulation and could even have the potential to trigger apoptosis alone.

Interest in the development of inhibitors of pro-survival BCL-2 proteins has come to fruition over the past decade following the identification of small molecule inhibitors capable of occupying the hydrophobic pocket on pro-survival proteins. The first of these BH3 mimetic drugs, ABT-737, was developed through NMR-based fragment screening. ABT-737 mimics the BH3-domain of pro-apoptotic BAD and interacts with BCL-2, BCL-XL and BCL-W (figure 5), displacing BH3-only proteins to trigger apoptosis in cell lines and restrict tumour growth in xenograft models [2]. Derivation of an orally available analogue (ABT 263/Navitoclax) allowed clinical testing [115]. On-target toxicity of Navitoclax was observed with dose-limiting thrombocytopenia occurring due to platelet dependence on BCL-XL [116,117]. This can be managed in the clinic and preclinical models have supported the use of Navitoclax in clinical trials as a combination therapy in a range of (predominantly solid) tumour types [118,119] (

Figure 5. Selectivity of BH3-mimetic drugs under clinical investigation. Drugs specifically targeting BCL-2 (Venetoclax/ABT-199), BCL-2, BCL-XL and BCL-W (Navitoclax/ABT263) or MCL-1 (AMG176, S64315/MIK665) are now in clinical trial/use.

Further development has led to additional BH3 mimetics with increased specificity for individual BCL-2 proteins. The success of this approach has been shown with the BCL-2 specific BH3 mimetic ABT-199 (Venetoclax), which obtained breakthrough FDA status for use in relapsed/refractory CLL. In this disease single-agent efficacy was seen with partial response in 79% and complete response in 20% of patients [120]. Even more impressive results were seen when Venetoclax was used in combination with rituximab-complete response occurred in 51% of patients with disease-free status occurring for up to 2 years after completion of therapy [121]. Encouraging (but more modest) effects are seen in non-Hodgkin lymphomas, acute myeloid leukaemia and multiple myeloma as a single agent [122–124] or combination therapy [125]. While much is understood of the role of the BCL-2 family in cancers of the blood, the case for targeting these proteins in solid tumours, most probably in conjunction with conventional therapies, is compelling. Translocation or amplification of BCL2 itself is rarely seen in solid tumours but dependence on BCL2 has been shown in small cell lung cancer (SCLC) [126]. Efficacy of Venetoclax when used as a combination therapy has been shown in preclinical models of breast cancer [127] and Venetoclax is now in clinical trials in combination with tamoxifen in breast cancer (ISRCTN98335443).

Resistance to BCL-2 and BCL-2/BCL-XL targeting BH3 mimetics has been observed, and in vitro studies indicate that ABT-737 treatment can increase MCL-1 levels and MCL-1 expression promotes resistance to ABT737 in vitro and in vivo [128–130]. MCL-1 has been associated with resistance to cancer therapy for some time [131,132], and interest has intensified in the development of drugs that specifically target MCL-1. A number of MCL-1 inhibitors have been mooted but until very recently compounds with clear specificity and on-target effect were lacking [133]. The tide has turned and a number of robust BH3 mimetic drugs targeting MCL-1 are now available Abbvie's A1210477 potently inhibits MCL-1 in vitro to restrict growth of diverse cancer cell lines [134,135], UMI-77 inhibits pancreatic and breast cancer cell line growth in vitro and in vivo [96,136], and the Servier compound S63845 seems particularly potent, with on-target single agent killing of leukaemia and lymphoma models in vitro and in vivo [4] and in combination with conventional cancer therapy in xenograft models of breast cancer [137]. The potential for MCL-1 specific BH3-mimetics in the clinic is now being tested with the Novartis/Servier drug S64315/MIK665 and Amgen AMG176 in phase I clinical trials for haematopoietic cancers/myelodysplastic syndrome (NCT02992483, NCT02979366, NCT02675452). Taking advantage of the short half-life of MCL-1 protein, a number of other approaches can be taken to decrease MCL-1 levels. This includes inhibition of transcription through CDK inhibition [138] or targeting translation through mTOR inhibition [139]. Beyond targeting anti-apoptotic BCL-2 proteins, increasing interest has centred on developing drugs that directly activate BAX and BAK in order to kill tumour cells [140,141]. Along these lines, a recent study has shown that a BAX-activating molecule, BTSA1, shows potent anti-tumour effects on human acute myeloid leukaemia (AML) xenografts in the absence of toxicity [142].

6. Inhibiting BCL-2 proteins in cancer prophylaxis

It is conceivable that targeting the pro-survival BCL-2 proteins could help eliminate pre-cancerous lesions or early-stage tumours. Indeed, ABT737 can act as an anti-cancer prophylactic in the Eμ-MYC mouse model of B-cell lymphoma, which has been shown to be dependent on BCL-XL [143]. Evidence for applicability beyond MYC-driven lymphoma is limited and in p53 null mice (which predominantly succumb to thymic lymphoma) prophylactic treatment with ABT737 had no impact on tumorigenesis [144]. When low-dose γ-irradiation was added to this experimental protocol to mimic environmental factors that could induce additional mutations prophylactic ABT737 treatment was shown to delay lymphoma onset in p53 null mice, but this reduction in thymic lymphoma was accompanied by increased incidence of sarcoma, and while significant, the difference in survival outcome afforded by prophylactic ABT 737 treatment was minimal [144].

Confounding factors include the altered activity of cells that have failed to be eliminated by targeting pro-survival BCL2 proteins. For example, the spontaneous apoptosis induced by targeted deletion of mcl1 in hepatocytes results in severe liver damage and increased proliferation that actually results in hepatocellular carcinoma (HCC) [145]. Such effects have also been shown in models of γ-irradiation induced thymic lymphoma where deletion of puma or over-expression of Bcl2 (normally considered as pro-cancer events) actually protects against tumour development [146]. In this scenario, γ-irradiation no longer causes depletion of bone marrow leucocytes meaning that there is no niche for the proliferation of stem/progenitor cells carrying damaged DNA that normally give rise to the thymic lymphomas in this model [146,147]. There are further indications that inhibiting the pro-survivals may not always have anti-cancer impact, in settings where ABT737 fails to induce apoptosis detrimental side effects can occur through activation of CAD and genome instability [148]. It remains to be seen whether these concerns hold true in the clinic.

In the decades following the discovery of BCL-2 (and related family members) a vast quantity of research has unravelled their role in regulating apoptosis. The functional division of this family into pro- and anti-apoptotic members and the elucidation of their structures and mechanism of interaction has allowed the pharmaceutical development of molecules to specifically inhibit these protein–protein interactions and reinstate apoptosis. Available data from clinical trials suggests good efficacy of BH3-mimetics targeting BCL-2 in some types of blood cancer. Deregulation of BCL-2 proteins is now recognized as a frequent event in many types of cancer and it seems likely that targeting pro-survival BCL-2 proteins will form a valuable adjunct to current cancer therapies. Restoration of apoptosis offers the potential to eliminate cancer cells at all stages of pathology and as BH3-mimetic drugs make their way into the clinic they could make dramatic improvements in survival outcome in cancer.

Cancer Biology Research

Research on the biology of cancer starts with the simplest of questions: What is—and isn’t—normal? To understand how cancer develops and progresses, researchers first need to investigate the biological differences between normal cells and cancer cells. This work focuses on the mechanisms that underlie fundamental processes such as cell growth, the transformation of normal cells to cancer cells, and the spread (metastasis) of cancer cells.

Virtually all major advances against cancer originated with discoveries in the basic sciences. Basic research reveals new concepts about the causes of cancer and how it develops, progresses, and responds to therapy.

NCI’s support of basic cancer research is essential. Long-term investments in research without immediate clinical application are not typically made by industry. The return on NCI’s sustained investment in basic scientific research has been remarkable. For example:

  • More than 40 years ago, scientists studying how retroviruses cause cancer discovered the first human oncogene (a gene that can transform a normal cell into a cancer cell). This novel and unexpected insight into cancer development, and other insights that followed, opened previously unexplored areas of cancer biology—ultimately leading to the era of precision oncology and new approaches to cancer prevention, detection, and treatment. cataloged the genomic changes associated with 33 different types of cancer. These efforts have revealed numerous insights into the genetic bases of cancer. For example, the identification of genetic similarities across different types of tumors has led to therapeutic approaches that are based on molecular characteristics of tumors and not where in the body cancer starts. Building on this, NCI’s Clinical Proteomic Tumor Analysis Consortium is pioneering the integrated proteogenomic analysis of a growing number of cancer types.
  • More than 3 decades of NCI-funded basic research in cancer immunology and genetics contributed to the first “tumor agnostic” precision medicine for cancer. The drug pembrolizumab (Keytruda) is an immune checkpoint inhibitor, a class of drugs that are used to treat patients with more than 15 types of cancer. In 2017, pembrolizumab was approved by the Food and Drug Administration to treat patients with any type of cancer whose tumor has a certain genetic feature called high microsatellite instability or mismatch repair deficiency.

The Future of Cancer Biology Research

The creativity of NCI-funded researchers and innovative technologies will drive novel insights never thought possible. These discoveries might include new insights into the causes of cancer and fundamental research leading to treatment breakthroughs. New technology might be developed that revolutionizes cancer research. The knowledge gained from our investments in basic research today will drive tomorrow’s advances to help patients with cancer and individuals at risk of the disease.


Researchers will have a comprehensive understanding of cancer biology that catalyzes the development of newer and safer ways to prevent, detect, diagnose, and treat cancer.


To improve our understanding of the many diseases we call cancer, we must unravel the complexity of how normal cells become cancerous and how cancer cells grow, survive, and spread throughout the body. To do this, NCI’s goals include the following:

1) Develop a comprehensive understanding of the molecular and cellular basis of cancer

A more complete understanding of cancer cell biology will enable new prevention, detection, and treatment approaches that take advantage of vulnerabilities identified in cancer cells and their precancerous lesions. Some of our major objectives are to:

  • Understand the genetic changes that give rise to cancer and the mechanisms by which those changes occur, as well as how genes are abnormally regulated (e.g., epigenetics)
  • Research the biological processes underlying cancer initiation, progression, and metastasis
  • Identify how tumors evolve and respond to or resist treatment
  • Study how cellular processes—such as cancer cell metabolism, stress responses, and cell cycle regulation—contribute to cancer development and progression

2) Understand how cancer cells interact with normal cells in the body to support or suppress tumor development and progression

Cancer can start in almost any tissue in the body, and the tissue in which a cancer develops and spreads can influence its molecular characteristics. This illustrates the importance of understanding the interactions between cancer cells and normal cells to develop new prevention and treatment approaches. NCI’s major objectives include:


German scientist Carl Vogt was first to describe the principle of apoptosis in 1842. In 1885, anatomist Walther Flemming delivered a more precise description of the process of programmed cell death. However, it was not until 1965 that the topic was resurrected. While studying tissues using electron microscopy, John Foxton Ross Kerr at the University of Queensland was able to distinguish apoptosis from traumatic cell death. [6] Following the publication of a paper describing the phenomenon, Kerr was invited to join Alastair R. Currie, as well as Andrew Wyllie, who was Currie's graduate student, [7] at University of Aberdeen. In 1972, the trio published a seminal article in the British Journal of Cancer. [8] Kerr had initially used the term programmed cell necrosis, but in the article, the process of natural cell death was called apoptosis. Kerr, Wyllie and Currie credited James Cormack, a professor of Greek language at University of Aberdeen, with suggesting the term apoptosis. Kerr received the Paul Ehrlich and Ludwig Darmstaedter Prize on March 14, 2000, for his description of apoptosis. He shared the prize with Boston biologist H. Robert Horvitz. [9]

For many years, neither "apoptosis" nor "programmed cell death" was a highly cited term. Two discoveries brought cell death from obscurity to a major field of research: identification of components of the cell death control and effector mechanisms, and linkage of abnormalities in cell death to human disease, in particular cancer.

The 2002 Nobel Prize in Medicine was awarded to Sydney Brenner, H. Robert Horvitz and John E. Sulston for their work identifying genes that control apoptosis. The genes were identified by studies in the nematode C. elegans and homologues of these genes function in humans to regulate apoptosis.

In Greek, apoptosis translates to the "falling off" of leaves from a tree. [10] Cormack, professor of Greek language, reintroduced the term for medical use as it had a medical meaning for the Greeks over two thousand years before. Hippocrates used the term to mean "the falling off of the bones". Galen extended its meaning to "the dropping of the scabs". Cormack was no doubt aware of this usage when he suggested the name. Debate continues over the correct pronunciation, with opinion divided between a pronunciation with the second p silent ( / æ p ə ˈ t oʊ s ɪ s / ap-ə- TOH -sis [11] [12] ) and the second p pronounced ( / eɪ p ə p ˈ t oʊ s ɪ s / ), [11] [13] as in the original Greek. [ citation needed ] In English, the p of the Greek -pt- consonant cluster is typically silent at the beginning of a word (e.g. pterodactyl, Ptolemy), but articulated when used in combining forms preceded by a vowel, as in helicopter or the orders of insects: diptera, lepidoptera, etc.

In the original Kerr, Wyllie & Currie paper, [8] there is a footnote regarding the pronunciation:

We are most grateful to Professor James Cormack of the Department of Greek, University of Aberdeen, for suggesting this term. The word "apoptosis" ( ἀπόπτωσις ) is used in Greek to describe the "dropping off" or "falling off" of petals from flowers, or leaves from trees. To show the derivation clearly, we propose that the stress should be on the penultimate syllable, the second half of the word being pronounced like "ptosis" (with the "p" silent), which comes from the same root "to fall", and is already used to describe the drooping of the upper eyelid.

The initiation of apoptosis is tightly regulated by activation mechanisms, because once apoptosis has begun, it inevitably leads to the death of the cell. [14] [15] The two best-understood activation mechanisms are the intrinsic pathway (also called the mitochondrial pathway) and the extrinsic pathway. [16] The intrinsic pathway is activated by intracellular signals generated when cells are stressed and depends on the release of proteins from the intermembrane space of mitochondria. [17] The extrinsic pathway is activated by extracellular ligands binding to cell-surface death receptors, which leads to the formation of the death-inducing signaling complex (DISC). [18]

A cell initiates intracellular apoptotic signaling in response to a stress, [19] which may bring about cell suicide. The binding of nuclear receptors by glucocorticoids, [20] heat, [20] radiation, [20] nutrient deprivation, [20] viral infection, [20] hypoxia, [20] increased intracellular concentration of free fatty acids [21] and increased intracellular calcium concentration, [22] [23] for example, by damage to the membrane, can all trigger the release of intracellular apoptotic signals by a damaged cell. A number of cellular components, such as poly ADP ribose polymerase, may also help regulate apoptosis. [24] Single cell fluctuations have been observed in experimental studies of stress induced apoptosis. [25] [26]

Before the actual process of cell death is precipitated by enzymes, apoptotic signals must cause regulatory proteins to initiate the apoptosis pathway. This step allows those signals to cause cell death, or the process to be stopped, should the cell no longer need to die. Several proteins are involved, but two main methods of regulation have been identified: the targeting of mitochondria functionality, [27] or directly transducing the signal via adaptor proteins to the apoptotic mechanisms. An extrinsic pathway for initiation identified in several toxin studies is an increase in calcium concentration within a cell caused by drug activity, which also can cause apoptosis via a calcium binding protease calpain.

Intrinsic pathway Edit

The intrinsic pathway is also known as the mitochondrial pathway. Mitochondria are essential to multicellular life. Without them, a cell ceases to respire aerobically and quickly dies. This fact forms the basis for some apoptotic pathways. Apoptotic proteins that target mitochondria affect them in different ways. They may cause mitochondrial swelling through the formation of membrane pores, or they may increase the permeability of the mitochondrial membrane and cause apoptotic effectors to leak out. [20] [28] They are very closely related to intrinsic pathway, and tumors arise more frequently through intrinsic pathway than the extrinsic pathway because of sensitivity. [29] There is also a growing body of evidence indicating that nitric oxide is able to induce apoptosis by helping to dissipate the membrane potential of mitochondria and therefore make it more permeable. [30] Nitric oxide has been implicated in initiating and inhibiting apoptosis through its possible action as a signal molecule of subsequent pathways that activate apoptosis. [31]

During apoptosis, cytochrome c is released from mitochondria through the actions of the proteins Bax and Bak. The mechanism of this release is enigmatic, but appears to stem from a multitude of Bax/Bak homo- and hetero-dimers of Bax/Bak inserted into the outer membrane. [32] Once cytochrome c is released it binds with Apoptotic protease activating factor – 1 (Apaf-1) and ATP, which then bind to pro-caspase-9 to create a protein complex known as an apoptosome. The apoptosome cleaves the pro-caspase to its active form of caspase-9, which in turn cleaves and activates pro-caspase into the effector caspase-3.

Mitochondria also release proteins known as SMACs (second mitochondria-derived activator of caspases) into the cell's cytosol following the increase in permeability of the mitochondria membranes. SMAC binds to proteins that inhibit apoptosis (IAPs) thereby deactivating them, and preventing the IAPs from arresting the process and therefore allowing apoptosis to proceed. IAP also normally suppresses the activity of a group of cysteine proteases called caspases, [33] which carry out the degradation of the cell. Therefore, the actual degradation enzymes can be seen to be indirectly regulated by mitochondrial permeability.

Extrinsic pathway Edit

Two theories of the direct initiation of apoptotic mechanisms in mammals have been suggested: the TNF-induced (tumor necrosis factor) model and the Fas-Fas ligand-mediated model, both involving receptors of the TNF receptor (TNFR) family [34] coupled to extrinsic signals.

TNF path Edit

TNF-alpha is a cytokine produced mainly by activated macrophages, and is the major extrinsic mediator of apoptosis. Most cells in the human body have two receptors for TNF-alpha: TNFR1 and TNFR2. The binding of TNF-alpha to TNFR1 has been shown to initiate the pathway that leads to caspase activation via the intermediate membrane proteins TNF receptor-associated death domain (TRADD) and Fas-associated death domain protein (FADD). cIAP1/2 can inhibit TNF-α signaling by binding to TRAF2. FLIP inhibits the activation of caspase-8. [35] Binding of this receptor can also indirectly lead to the activation of transcription factors involved in cell survival and inflammatory responses. [36] However, signalling through TNFR1 might also induce apoptosis in a caspase-independent manner. [37] The link between TNF-alpha and apoptosis shows why an abnormal production of TNF-alpha plays a fundamental role in several human diseases, especially in autoimmune diseases. The TNF-alpha receptor superfamily also includes death receptors (DRs), such as DR4 and DR5. These receptors bind to the proteinTRAIL and mediate apoptosis. Apoptosis is known to be one of the primary mechanisms of targeted cancer therapy. [38] Luminescent iridium complex-peptide hybrids (IPHs) have recently been designed, which mimic TRAIL and bind to death receptors on cancer cells, thereby inducing their apoptosis. [39]

The fas receptor (First apoptosis signal) – (also known as Apo-1 or CD95) is a transmembrane protein of the TNF family which binds the Fas ligand (FasL). [34] The interaction between Fas and FasL results in the formation of the death-inducing signaling complex (DISC), which contains the FADD, caspase-8 and caspase-10. In some types of cells (type I), processed caspase-8 directly activates other members of the caspase family, and triggers the execution of apoptosis of the cell. In other types of cells (type II), the Fas-DISC starts a feedback loop that spirals into increasing release of proapoptotic factors from mitochondria and the amplified activation of caspase-8. [40]

Following TNF-R1 and Fas activation in mammalian cells [ citation needed ] a balance between proapoptotic (BAX, [41] BID, BAK, or BAD) and anti-apoptotic (Bcl-Xl and Bcl-2) members of the Bcl-2 family are established. This balance is the proportion of proapoptotic homodimers that form in the outer-membrane of the mitochondrion. The proapoptotic homodimers are required to make the mitochondrial membrane permeable for the release of caspase activators such as cytochrome c and SMAC. Control of proapoptotic proteins under normal cell conditions of nonapoptotic cells is incompletely understood, but in general, Bax or Bak are activated by the activation of BH3-only proteins, part of the Bcl-2 family [ citation needed ] .

Caspases play the central role in the transduction of ER apoptotic signals. Caspases are proteins that are highly conserved, cysteine-dependent aspartate-specific proteases. There are two types of caspases: initiator caspases, caspase 2,8,9,10,11,12, and effector caspases, caspase 3,6,7. The activation of initiator caspases requires binding to specific oligomeric activator protein. Effector caspases are then activated by these active initiator caspases through proteolytic cleavage. The active effector caspases then proteolytically degrade a host of intracellular proteins to carry out the cell death program.

Caspase-independent apoptotic pathway

There also exists a caspase-independent apoptotic pathway that is mediated by AIF (apoptosis-inducing factor). [42]

Apoptosis model in amphibians Edit

Amphibian frog Xenopus laevis serves as an ideal model system for the study of the mechanisms of apoptosis. In fact, iodine and thyroxine also stimulate the spectacular apoptosis of the cells of the larval gills, tail and fins in amphibians metamorphosis, and stimulate the evolution of their nervous system transforming the aquatic, vegetarian tadpole into the terrestrial, carnivorous frog. [43] [44] [45] [46]

Negative regulation of apoptosis inhibits cell death signaling pathways, helping tumors to evade cell death and developing drug resistance. The ratio between anti-apoptotic (Bcl-2) and pro-apoptotic (Bax) proteins determines whether a cell lives or dies. [47] [48] Many families of proteins act as negative regulators categorized into either antiapoptotic factors, such as IAPs and Bcl-2 proteins or prosurvival factors like cFLIP, BNIP3, FADD, Akt, and NF-κB. [49]

Many pathways and signals lead to apoptosis, but these converge on a single mechanism that actually causes the death of the cell. After a cell receives stimulus, it undergoes organized degradation of cellular organelles by activated proteolytic caspases. In addition to the destruction of cellular organelles, mRNA is rapidly and globally degraded by a mechanism that is not yet fully characterized. [50] mRNA decay is triggered very early in apoptosis.

A cell undergoing apoptosis shows a series of characteristic morphological changes. Early alterations include:

  1. Cell shrinkage and rounding occur because of the retraction lamellipodia and the breakdown of the proteinaceous cytoskeleton by caspases. [51]
  2. The cytoplasm appears dense, and the organelles appear tightly packed.
  3. Chromatin undergoes condensation into compact patches against the nuclear envelope (also known as the perinuclear envelope) in a process known as pyknosis, a hallmark of apoptosis. [52][53]
  4. The nuclear envelope becomes discontinuous and the DNA inside it is fragmented in a process referred to as karyorrhexis. The nucleus breaks into several discrete chromatin bodies or nucleosomal units due to the degradation of DNA. [54]

Apoptosis progresses quickly and its products are quickly removed, making it difficult to detect or visualize on classical histology sections. During karyorrhexis, endonuclease activation leaves short DNA fragments, regularly spaced in size. These give a characteristic "laddered" appearance on agar gel after electrophoresis. [55] Tests for DNA laddering differentiate apoptosis from ischemic or toxic cell death. [56]

Apoptotic cell disassembly Edit

Before the apoptotic cell is disposed of, there is a process of disassembly. There are three recognized steps in apoptotic cell disassembly: [58]

  1. Membrane blebbing: The cell membrane shows irregular buds known as blebs. Initially these are smaller surface blebs. Later these can grow into larger so-called dynamic membrane blebs. [58] An important regulator of apoptotic cell membrane blebbing is ROCK1 (rho associated coiled-coil-containing protein kinase 1). [59][60]
  2. Formation of membrane protrusions: Some cell types, under specific conditions, may develop different types of long, thin extensions of the cell membrane called membrane protrusions. Three types have been described: microtubule spikes, apoptopodia (feet of death), and beaded apoptopodia (the latter having a beads-on-a-string appearance). [61][62][63]Pannexin 1 is an important component of membrane channels involved in the formation of apoptopodia and beaded apoptopodia. [62] : The cell breaks apart into multiple vesicles called apoptotic bodies, which undergo phagocytosis. The plasma membrane protrusions may help bring apoptotic bodies closer to phagocytes.

Removal of dead cells Edit

The removal of dead cells by neighboring phagocytic cells has been termed efferocytosis. [64] Dying cells that undergo the final stages of apoptosis display phagocytotic molecules, such as phosphatidylserine, on their cell surface. [65] Phosphatidylserine is normally found on the inner leaflet surface of the plasma membrane, but is redistributed during apoptosis to the extracellular surface by a protein known as scramblase. [66] These molecules mark the cell for phagocytosis by cells possessing the appropriate receptors, such as macrophages. [67] The removal of dying cells by phagocytes occurs in an orderly manner without eliciting an inflammatory response. [68] During apoptosis cellular RNA and DNA are separated from each other and sorted to different apoptotic bodies separation of RNA is initiated as nucleolar segregation. [69]

Many knock-outs have been made in the apoptosis pathways to test the function of each of the proteins. Several caspases, in addition to APAF1 and FADD, have been mutated to determine the new phenotype. In order to create a tumor necrosis factor (TNF) knockout, an exon containing the nucleotides 3704–5364 was removed from the gene. This exon encodes a portion of the mature TNF domain, as well as the leader sequence, which is a highly conserved region necessary for proper intracellular processing. TNF-/- mice develop normally and have no gross structural or morphological abnormalities. However, upon immunization with SRBC (sheep red blood cells), these mice demonstrated a deficiency in the maturation of an antibody response they were able to generate normal levels of IgM, but could not develop specific IgG levels. Apaf-1 is the protein that turns on caspase 9 by cleavage to begin the caspase cascade that leads to apoptosis. Since a -/- mutation in the APAF-1 gene is embryonic lethal, a gene trap strategy was used in order to generate an APAF-1 -/- mouse. This assay is used to disrupt gene function by creating an intragenic gene fusion. When an APAF-1 gene trap is introduced into cells, many morphological changes occur, such as spina bifida, the persistence of interdigital webs, and open brain. In addition, after embryonic day 12.5, the brain of the embryos showed several structural changes. APAF-1 cells are protected from apoptosis stimuli such as irradiation. A BAX-1 knock-out mouse exhibits normal forebrain formation and a decreased programmed cell death in some neuronal populations and in the spinal cord, leading to an increase in motor neurons.

The caspase proteins are integral parts of the apoptosis pathway, so it follows that knock-outs made have varying damaging results. A caspase 9 knock-out leads to a severe brain malformation. A caspase 8 knock-out leads to cardiac failure and thus embryonic lethality. However, with the use of cre-lox technology, a caspase 8 knock-out has been created that exhibits an increase in peripheral T cells, an impaired T cell response, and a defect in neural tube closure. These mice were found to be resistant to apoptosis mediated by CD95, TNFR, etc. but not resistant to apoptosis caused by UV irradiation, chemotherapeutic drugs, and other stimuli. Finally, a caspase 3 knock-out was characterized by ectopic cell masses in the brain and abnormal apoptotic features such as membrane blebbing or nuclear fragmentation. A remarkable feature of these KO mice is that they have a very restricted phenotype: Casp3, 9, APAF-1 KO mice have deformations of neural tissue and FADD and Casp 8 KO showed defective heart development, however, in both types of KO other organs developed normally and some cell types were still sensitive to apoptotic stimuli suggesting that unknown proapoptotic pathways exist.

In order to perform analysis of apoptotic versus necrotic (necroptotic) cells, one can do analysis of morphology by label-free live cell imaging, time-lapse microscopy, flow fluorocytometry, and transmission electron microscopy. There are also various biochemical techniques for analysis of cell surface markers (phosphatidylserine exposure versus cell permeability by flow cytometry), cellular markers such as DNA fragmentation [70] (flow cytometry), [71] caspase activation, Bid cleavage, and cytochrome c release (Western blotting). It is important to know how primary and secondary necrotic cells can be distinguished by analysis of supernatant for caspases, HMGB1, and release of cytokeratin 18. However, no distinct surface or biochemical markers of necrotic cell death have been identified yet, and only negative markers are available. These include absence of apoptotic markers (caspase activation, cytochrome c release, and oligonucleosomal DNA fragmentation) and differential kinetics of cell death markers (phosphatidylserine exposure and cell membrane permeabilization). A selection of techniques that can be used to distinguish apoptosis from necroptotic cells could be found in these references. [72] [73] [74] [75]

Defective pathways Edit

The many different types of apoptotic pathways contain a multitude of different biochemical components, many of them not yet understood. [76] As a pathway is more or less sequential in nature, removing or modifying one component leads to an effect in another. In a living organism, this can have disastrous effects, often in the form of disease or disorder. A discussion of every disease caused by modification of the various apoptotic pathways would be impractical, but the concept overlying each one is the same: The normal functioning of the pathway has been disrupted in such a way as to impair the ability of the cell to undergo normal apoptosis. This results in a cell that lives past its "use-by date" and is able to replicate and pass on any faulty machinery to its progeny, increasing the likelihood of the cell's becoming cancerous or diseased.

A recently described example of this concept in action can be seen in the development of a lung cancer called NCI-H460. [77] The X-linked inhibitor of apoptosis protein (XIAP) is overexpressed in cells of the H460 cell line. XIAPs bind to the processed form of caspase-9, and suppress the activity of apoptotic activator cytochrome c, therefore overexpression leads to a decrease in the amount of proapoptotic agonists. As a consequence, the balance of anti-apoptotic and proapoptotic effectors is upset in favour of the former, and the damaged cells continue to replicate despite being directed to die. Defects in regulation of apoptosis in cancer cells occur often at the level of control of transcription factors. As a particular example, defects in molecules that control transcription factor NF-κB in cancer change the mode of transcriptional regulation and the response to apoptotic signals, to curtail dependence on the tissue that the cell belongs. This degree of independence from external survival signals, can enable cancer metastasis. [78]

Dysregulation of p53 Edit

The tumor-suppressor protein p53 accumulates when DNA is damaged due to a chain of biochemical factors. Part of this pathway includes alpha-interferon and beta-interferon, which induce transcription of the p53 gene, resulting in the increase of p53 protein level and enhancement of cancer cell-apoptosis. [79] p53 prevents the cell from replicating by stopping the cell cycle at G1, or interphase, to give the cell time to repair, however it will induce apoptosis if damage is extensive and repair efforts fail. [80] Any disruption to the regulation of the p53 or interferon genes will result in impaired apoptosis and the possible formation of tumors.

Inhibition Edit

Inhibition of apoptosis can result in a number of cancers, inflammatory diseases, and viral infections. It was originally believed that the associated accumulation of cells was due to an increase in cellular proliferation, but it is now known that it is also due to a decrease in cell death. The most common of these diseases is cancer, the disease of excessive cellular proliferation, which is often characterized by an overexpression of IAP family members. As a result, the malignant cells experience an abnormal response to apoptosis induction: Cycle-regulating genes (such as p53, ras or c-myc) are mutated or inactivated in diseased cells, and further genes (such as bcl-2) also modify their expression in tumors. Some apoptotic factors are vital during mitochondrial respiration e.g. cytochrome C. [81] Pathological inactivation of apoptosis in cancer cells is correlated with frequent respiratory metabolic shifts toward glycolysis (an observation known as the “Warburg hypothesis”. [82]

HeLa cell Edit

Apoptosis in HeLa [b] cells is inhibited by proteins produced by the cell these inhibitory proteins target retinoblastoma tumor-suppressing proteins. [83] These tumor-suppressing proteins regulate the cell cycle, but are rendered inactive when bound to an inhibitory protein. [83] HPV E6 and E7 are inhibitory proteins expressed by the human papillomavirus, HPV being responsible for the formation of the cervical tumor from which HeLa cells are derived. [84] HPV E6 causes p53, which regulates the cell cycle, to become inactive. [85] HPV E7 binds to retinoblastoma tumor suppressing proteins and limits its ability to control cell division. [85] These two inhibitory proteins are partially responsible for HeLa cells' immortality by inhibiting apoptosis to occur. [86] CDV (Canine Distemper Virus) is able to induce apoptosis despite the presence of these inhibitory proteins. This is an important oncolytic property of CDV: this virus is capable of killing canine lymphoma cells. Oncoproteins E6 and E7 still leave p53 inactive, but they are not able to avoid the activation of caspases induced from the stress of viral infection. These oncolytic properties provided a promising link between CDV and lymphoma apoptosis, which can lead to development of alternative treatment methods for both canine lymphoma and human non-Hodgkin lymphoma. Defects in the cell cycle are thought to be responsible for the resistance to chemotherapy or radiation by certain tumor cells, so a virus that can induce apoptosis despite defects in the cell cycle is useful for cancer treatment. [86]

Treatments Edit

The main method of treatment for potential death from signaling-related diseases involves either increasing or decreasing the susceptibility of apoptosis in diseased cells, depending on whether the disease is caused by either the inhibition of or excess apoptosis. For instance, treatments aim to restore apoptosis to treat diseases with deficient cell death, and to increase the apoptotic threshold to treat diseases involved with excessive cell death. To stimulate apoptosis, one can increase the number of death receptor ligands (such as TNF or TRAIL), antagonize the anti-apoptotic Bcl-2 pathway, or introduce Smac mimetics to inhibit the inhibitor (IAPs). [47] The addition of agents such as Herceptin, Iressa, or Gleevec works to stop cells from cycling and causes apoptosis activation by blocking growth and survival signaling further upstream. Finally, adding p53-MDM2 complexes displaces p53 and activates the p53 pathway, leading to cell cycle arrest and apoptosis. Many different methods can be used either to stimulate or to inhibit apoptosis in various places along the death signaling pathway. [87]

Apoptosis is a multi-step, multi-pathway cell-death programme that is inherent in every cell of the body. In cancer, the apoptosis cell-division ratio is altered. Cancer treatment by chemotherapy and irradiation kills target cells primarily by inducing apoptosis.

Hyperactive apoptosis Edit

On the other hand, loss of control of cell death (resulting in excess apoptosis) can lead to neurodegenerative diseases, hematologic diseases, and tissue damage. It is of interest to note that neurons that rely on mitochondrial respiration undergo apoptosis in neurodegenerative diseases such as Alzheimer's [88] and Parkinson's. [89] (an observation known as the “Inverse Warburg hypothesis” [90] [81] ). Moreover, there is an inverse epidemiological comorbidity between neurodegenerative diseases and cancer. [91] The progression of HIV is directly linked to excess, unregulated apoptosis. In a healthy individual, the number of CD4+ lymphocytes is in balance with the cells generated by the bone marrow however, in HIV-positive patients, this balance is lost due to an inability of the bone marrow to regenerate CD4+ cells. In the case of HIV, CD4+ lymphocytes die at an accelerated rate through uncontrolled apoptosis, when stimulated. At the molecular level, hyperactive apoptosis can be caused by defects in signaling pathways that regulate the Bcl-2 family proteins. Increased expression of apoptotic proteins such as BIM, or their decreased proteolysis, leads to cell death, and can cause a number of pathologies, depending on the cells where excessive activity of BIM occurs. Cancer cells can escape apoptosis through mechanisms that suppress BIM expression or by increased proteolysis of BIM. [ citation needed ]

Treatments Edit

Treatments aiming to inhibit works to block specific caspases. Finally, the Akt protein kinase promotes cell survival through two pathways. Akt phosphorylates and inhibits Bad (a Bcl-2 family member), causing Bad to interact with the 14-3-3 scaffold, resulting in Bcl dissociation and thus cell survival. Akt also activates IKKα, which leads to NF-κB activation and cell survival. Active NF-κB induces the expression of anti-apoptotic genes such as Bcl-2, resulting in inhibition of apoptosis. NF-κB has been found to play both an antiapoptotic role and a proapoptotic role depending on the stimuli utilized and the cell type. [92]

HIV progression Edit

The progression of the human immunodeficiency virus infection into AIDS is due primarily to the depletion of CD4+ T-helper lymphocytes in a manner that is too rapid for the body's bone marrow to replenish the cells, leading to a compromised immune system. One of the mechanisms by which T-helper cells are depleted is apoptosis, which results from a series of biochemical pathways: [93]

  1. HIV enzymes deactivate anti-apoptotic Bcl-2. This does not directly cause cell death but primes the cell for apoptosis should the appropriate signal be received. In parallel, these enzymes activate proapoptotic procaspase-8, which does directly activate the mitochondrial events of apoptosis.
  2. HIV may increase the level of cellular proteins that prompt Fas-mediated apoptosis.
  3. HIV proteins decrease the amount of CD4 glycoprotein marker present on the cell membrane.
  4. Released viral particles and proteins present in extracellular fluid are able to induce apoptosis in nearby "bystander" T helper cells.
  5. HIV decreases the production of molecules involved in marking the cell for apoptosis, giving the virus time to replicate and continue releasing apoptotic agents and virions into the surrounding tissue.
  6. The infected CD4+ cell may also receive the death signal from a cytotoxic T cell.

Cells may also die as direct consequences of viral infections. HIV-1 expression induces tubular cell G2/M arrest and apoptosis. [94] The progression from HIV to AIDS is not immediate or even necessarily rapid HIV's cytotoxic activity toward CD4+ lymphocytes is classified as AIDS once a given patient's CD4+ cell count falls below 200. [95]

Researchers from Kumamoto University in Japan have developed a new method to eradicate HIV in viral reservoir cells, named "Lock-in and apoptosis." Using the synthesized compound Heptanoylphosphatidyl L-Inositol Pentakisphophate (or L-Hippo) to bind strongly to the HIV protein PR55Gag, they were able to suppress viral budding. By suppressing viral budding, the researchers were able to trap the HIV virus in the cell and allow for the cell to undergo apoptosis (natural cell death). Associate Professor Mikako Fujita has stated that the approach is not yet available to HIV patients because the research team has to conduct further research on combining the drug therapy that currently exists with this "Lock-in and apoptosis" approach to lead to complete recovery from HIV. [96]

Viral infection Edit

Viral induction of apoptosis occurs when one or several cells of a living organism are infected with a virus, leading to cell death. Cell death in organisms is necessary for the normal development of cells and the cell cycle maturation. [97] It is also important in maintaining the regular functions and activities of cells.

Viruses can trigger apoptosis of infected cells via a range of mechanisms including:

  • Receptor binding
  • Activation of protein kinase R (PKR)
  • Interaction with p53
  • Expression of viral proteins coupled to MHC proteins on the surface of the infected cell, allowing recognition by cells of the immune system (such as Natural Killer and cytotoxic T cells) that then induce the infected cell to undergo apoptosis. [98]

Canine distemper virus (CDV) is known to cause apoptosis in central nervous system and lymphoid tissue of infected dogs in vivo and in vitro. [99] Apoptosis caused by CDV is typically induced via the extrinsic pathway, which activates caspases that disrupt cellular function and eventually leads to the cells death. [83] In normal cells, CDV activates caspase-8 first, which works as the initiator protein followed by the executioner protein caspase-3. [83] However, apoptosis induced by CDV in HeLa cells does not involve the initiator protein caspase-8. HeLa cell apoptosis caused by CDV follows a different mechanism than that in vero cell lines. [83] This change in the caspase cascade suggests CDV induces apoptosis via the intrinsic pathway, excluding the need for the initiator caspase-8. The executioner protein is instead activated by the internal stimuli caused by viral infection not a caspase cascade. [83]

The Oropouche virus (OROV) is found in the family Bunyaviridae. The study of apoptosis brought on by Bunyaviridae was initiated in 1996, when it was observed that apoptosis was induced by the La Crosse virus into the kidney cells of baby hamsters and into the brains of baby mice. [100]

OROV is a disease that is transmitted between humans by the biting midge (Culicoides paraensis). [101] It is referred to as a zoonotic arbovirus and causes febrile illness, characterized by the onset of a sudden fever known as Oropouche fever. [102]

The Oropouche virus also causes disruption in cultured cells – cells that are cultivated in distinct and specific conditions. An example of this can be seen in HeLa cells, whereby the cells begin to degenerate shortly after they are infected. [100]

With the use of gel electrophoresis, it can be observed that OROV causes DNA fragmentation in HeLa cells. It can be interpreted by counting, measuring, and analyzing the cells of the Sub/G1 cell population. [100] When HeLA cells are infected with OROV, the cytochrome C is released from the membrane of the mitochondria, into the cytosol of the cells. This type of interaction shows that apoptosis is activated via an intrinsic pathway. [97]

In order for apoptosis to occur within OROV, viral uncoating, viral internalization, along with the replication of cells is necessary. Apoptosis in some viruses is activated by extracellular stimuli. However, studies have demonstrated that the OROV infection causes apoptosis to be activated through intracellular stimuli and involves the mitochondria. [100]

Many viruses encode proteins that can inhibit apoptosis. [103] Several viruses encode viral homologs of Bcl-2. These homologs can inhibit proapoptotic proteins such as BAX and BAK, which are essential for the activation of apoptosis. Examples of viral Bcl-2 proteins include the Epstein-Barr virus BHRF1 protein and the adenovirus E1B 19K protein. [104] Some viruses express caspase inhibitors that inhibit caspase activity and an example is the CrmA protein of cowpox viruses. Whilst a number of viruses can block the effects of TNF and Fas. For example, the M-T2 protein of myxoma viruses can bind TNF preventing it from binding the TNF receptor and inducing a response. [105] Furthermore, many viruses express p53 inhibitors that can bind p53 and inhibit its transcriptional transactivation activity. As a consequence, p53 cannot induce apoptosis, since it cannot induce the expression of proapoptotic proteins. The adenovirus E1B-55K protein and the hepatitis B virus HBx protein are examples of viral proteins that can perform such a function. [106]

Viruses can remain intact from apoptosis in particular in the latter stages of infection. They can be exported in the apoptotic bodies that pinch off from the surface of the dying cell, and the fact that they are engulfed by phagocytes prevents the initiation of a host response. This favours the spread of the virus. [105]

Programmed cell death in plants has a number of molecular similarities to that of animal apoptosis, but it also has differences, notable ones being the presence of a cell wall and the lack of an immune system that removes the pieces of the dead cell. Instead of an immune response, the dying cell synthesizes substances to break itself down and places them in a vacuole that ruptures as the cell dies. Whether this whole process resembles animal apoptosis closely enough to warrant using the name apoptosis (as opposed to the more general programmed cell death) is unclear. [107] [108]

The characterization of the caspases allowed the development of caspase inhibitors, which can be used to determine whether a cellular process involves active caspases. Using these inhibitors it was discovered that cells can die while displaying a morphology similar to apoptosis without caspase activation. [109] Later studies linked this phenomenon to the release of AIF (apoptosis-inducing factor) from the mitochondria and its translocation into the nucleus mediated by its NLS (nuclear localization signal). Inside the mitochondria, AIF is anchored to the inner membrane. In order to be released, the protein is cleaved by a calcium-dependent calpain protease.


The discovery that the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis of cancer cells without causing toxicity in mice has led to the in-depth study of pro-apoptotic TRAIL receptor (TRAIL-R) signalling and the development of biotherapeutic drug candidates that activate TRAIL-Rs. The outcome of clinical trials with these TRAIL-R agonists has, however, been disappointing so far. Recent evidence indicates that many cancers, in addition to being TRAIL resistant, use the endogenous TRAIL–TRAIL-R system to their own advantage. However, novel insight on two fronts — how resistance of cancer cells to TRAIL-based pro-apoptotic therapies might be overcome, and how the pro-tumorigenic effects of endogenous TRAIL might be countered — gives reasonable hope that the TRAIL system can be harnessed to treat cancer. In this Review we assess the status quo of our understanding of the biology of the TRAIL–TRAIL-R system — as well as the gaps therein — and discuss the opportunities and challenges in effectively targeting this pathway.

30 - Apoptosis: the extrinsic pathway

Programmed cell death (also known as PCD) is generally defined as a regulated process by which cells contribute to their own demise. Apoptosis is the best-characterized form of programmed cell death, but alternative non-apoptotic cell-death pathways important in human physiology and disease pathology are now actively studied. Regarding apoptosis, there are two general pathways, the extrinsic pathways and the intrinsic pathways, depending on whether the molecular factor that initiates the death pathway is extra-cellular or intra-cellular. Both extrinsic and intrinsic pathways lead to activation of caspases, the proteases that cleave many key protein targets inside cells, resulting in apoptotic cell morphology and cell death within a few minutes to hours. Although there may be alternative molecular pathways that cause apoptosis-like cell morphology, the term apoptosis most often refers to caspase-dependent cell death.

The extrinsic and intrinsic pathways activate different initiator caspases. Each initiator caspase is activated by a unique complex of proteins. The intrinsic death pathway involves mitochondria and is controlled by pro- and anti-apoptotic Bcl-2-family proteins that facilitate or inhibit the release of cytochrome c from the mitochondrial inter-membrane space. In turn, cytosolic cytochrome c and ATP/ADP bind Apaf-1, inducing oligomerization of Apaf-1 into a heptameric ring structure known as the apoptosome. The apoptosome activates caspase-9, which in turn cleaves and activates caspases-3 and -7 to mediate apoptosis during normal development and to prevent cancer. Other intrinsic apoptotic pathways are initiated by assembly of alternative caspase-activating complexes in response to intra-cellular factors, such as the PIDDosome complex, which activates caspase-2, and the inflammasome, which activates caspase-1 (originally known as ICE, IL-1β-converting enzyme).


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