apoptosis & cancer

8/9/2019 Apoptosis & Cancer

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Apoptosis & Cancer


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Initiation of apoptosis

In principle, there are two alternative pathways that initiateapoptosis: one is mediated by death receptors on the cellsurface sometimes referred to as the 'extrinsic pathway';the other is mediated by mitochondria referred to as the'instrinsic pathway'. In both pathways, cysteine aspartyl-specific proteases (caspases) are activated that cleavecellular substrates, and this leads to the biochemical and

morphological changes that are characteristic of apoptosis. Death receptors are members of the tumour-necrosis factor

(TNF) receptor superfamily and comprise a subfamily thatis characterized by an intracellular domain the deathdomain.

Death receptors are activated by their natural ligands, theTNF family. When ligands bind to their respective deathreceptors such as CD95, TRAIL-R1 (TNF-related

apoptosis-inducing ligand-R1) or TRAIL-R2

the deathdomains attract the intracellular adaptor protein FADD(Fas-associated death domain protein, also known asMORT1), which, in turn, recruits the inactive proforms ofcertain members of the caspase protease family.

The caspases that are recruited to this death-inducingsignalling complex (DISC) caspase-8 and caspase-10

function as 'initiator' caspases. At the DISC, procaspase-8and procaspase-10 are cleaved and yield active initiatorcaspases.

In some cells known as type I cells the amount of

active caspase-8 formed at the DISC is sufficient to initiateapoptosis directly, but in type II cells, the amount is toosmall and mitochondria are used as 'amplifiers' of theapoptotic signal. Activation of mitochondria is mediated by

the BCL2 family member BID. BID is cleaved by activecaspase-8 and translocates to the mitochondria.


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The two main apoptic signalling

pathway

Apoptosis can be initiated by two alternative pathways: either

through death receptors on the cell surface (extrinsic pathway) orthrough mitochondria (intrinsic pathway). In both pathways,induction of apoptosis leads to activation of an initiator caspase:caspase-8 and possibly caspase-10 for the extrinsic pathway; andcaspase-9, which is activated at the apoptosome, for the intrinsicpathway. The initiator caspases then activate executioner caspases.Active executioner caspases cleave the death substrates, whicheventually results in apoptosis. There is crosstalk between these two

pathways. For example, cleavage of the BCL2-family member BID bycaspase-8 activates the mitochondrial pathway after apoptosisinduction through death receptors, and can be used to amplify theapoptotic signal.


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Death receptos and ligands

Ligands are shown at the top, receptors at the bottom.

Death receptors and death ligands are grouped in a box.DcR3 (decoy receptor 3) acts as a decoy receptor for CD95L(dotted line). The other molecules outside the box can bindto death receptors or ligands as indicated, but have notbeen shown to transmit an apoptotic signal. The deathdomain is shown as a pink box.


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Apoptosis signalling through

death receptors

Binding of death ligands (CD95L is used here as an example) to theirreceptor leads to the formation of the death-inducing signalling

complex (DISC). In the DISC, the initiator procaspase-8 is recruitedby FADD (FAS-associated death domain protein) and is activated byautocatalytic cleavage. Death-receptor-mediated apoptosis can beinhibited at several levels by anti-apoptotic proteins: CD95L can beprevented from binding to CD95 by soluble 'decoy' receptors, such assoluble CD95 (sCD95) or DcR3 (decoy receptor 3). FLICE-inhibitoryproteins (FLIPs) bind to the DISC and prevent the activation ofcaspase-8; and inhibitors of apoptosis proteins (IAPs) bind to and

inhibit caspases. FLIPL and FLIPS refer to long and short forms of FLIP,respectively.


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Mitochondria & the BCL2 family

Death initiated at the mitochondrial level is regulated bythe members of the BCL2 family. BCL2 family memberscan be divided into anti-apoptotic (BCL2, BCL-XL, BCL-w,MCL1, A1/BFL1, BOO/DIVA, NR-13) and pro-apoptoticproteins (BAX, BAK, BOK/MTD, BCL-XS, BID, BAD,BIK/NBK, BLK, HRK/DP5, BIM/BOD, NIP3, NIX, NOXA,PUMA, BMF). Most anti-apoptotic members contain theBCL2 homology (BH) domains 1, 2 and 4, whereas theBH3 domain seems to be crucial for apoptosis induction.The pro-apoptotic members can be subdivided into theBAX subfamily (BAX, BAK, BOK) and the BH3-onlyproteins (for example, BID, BAD and BIM).

After activation by an apoptotic stimulus, mitochondriarelease cytochrome c, AIF (apoptosis inducing factor) andother apoptogenic factors from the intermembrane spaceto the cytosol. Concomitantly, the mitochondrialtransmembrane potential drops. According to one model,

mitochondrial membrane permeabilization involves thepermeability transition pore complex (PTPC), amultiprotein complex that consists of the adeninenucleotide translocator (ANT) of the inner membrane, thevoltage-dependent anion channel of the outer membraneand various other proteins. BCL2 proteins might interactwith the PTPC and regulate its permeability.

According to another model, BH3-only proteins serve as'death sensors' in the cytosol or cytoskeleton. Following adeath signal, they interact with members of the BAXsubfamily. After this interaction, BAX proteins undergo aconformational change, insert into the mitochondrialmembrane, oligomerize and form protein-permeablechannels. Anti-apoptotic BCL2 proteins inhibit theconformational change or the oligomerization of BAX andBAK.

The localization of the pro-apoptotic BCL2 family member

BAD is regulated by phosphorylation. Only non-phosphorylated BAD is capable of antagonizing anti-apoptotic BCL2 or BCL-XL on the mitochondrial membrane.BAD phosphorylation results in its redistribution to thecytosol and its sequestration by 14-3-3 proteins.


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Apoptosis signalling through

mitochondria

Chemotherapy, irradiation and other stimuli can initiate apoptosisthrough the mitochondrial (intrinsic) pathway. Pro-apoptotic BCL2family proteins for example, BAX, BID, BAD and BIM areimportant mediators of these signals. Activation of mitochondria

leads to the release of cytochrome c(Cyt c) into the cytosol, whereit binds apoptotic protease activating factor 1 (APAF1) to form theapoptosome. At the apoptosome, the initiator caspase-9 is activated.Apoptosis through mitochondria can be inhibited on different levelsby anti-apoptotic proteins, including the anti-apoptotic BCL2 familymembers BCL2 and BCL-XL and inhibitors of apoptosis proteins(IAPs), which are regulated by SMAC/DIABLO (secondmitochondria-derived activator of caspase/direct IAP binding protein

with low pI). Another way is through survival signals, such asgrowth factors and cytokines, that activate the phosphatidylinositol3-kinase (PI3K) pathway. PI3K activates AKT, which phosphorylatesand inactivates the pro-apoptotic BCL2-family member BAD.


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Execution of apoptosis

Once the initiator caspases are activated, they cleave andactivate 'executioner' caspases, mainly caspase-3,caspase-6 and caspase-7. The active executioner caspasesthen cleave each other and, in this way, an amplifyingproteolytic cascade of caspase activation is started.

Eventually, the active executioner caspases cleave cellular

substrates

the 'death substrates'

which leads tocharacteristic biochemical and morphological changes.Cleavage of nuclear LAMINS is involved in chromatincondensation and nuclear shrinkage. Cleavage of theinhibitor of the DNase CAD (caspase-activateddeoxyribonuclease, DFF40), ICAD (also known as DNAfragmentation factor, 45 kDa; DFF45), causes the releaseof the endonuclease, which travels to the nucleus to

fragment DNA. Cleavage of cytoskeletal proteins such asactin, plectin, Rho kinase 1 (ROCK1) and gelsolin leads tocell fragmentation, blebbing and the formation of apoptoticbodies. After exposure of 'eat me' signals (for example,exposure of phosphatidylserine and changes in surfacesugars), the remains of the dying cell are engulfed byphagocytes.

Besides these prototypic caspase-dependent apoptosis

pathways, there are also molecularly less-well-definedcell-death pathways that do not require caspase activation.These pathways share some, but not all, thecharacteristics of apoptotic classical pathways. Therefore,they cannot be readily classified as apoptosis or necrosisand have been called 'necrotic-like' or 'apoptotic-like' celldeath or paraptosis.


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Regulation of apoptosis

The apoptotic self-destruction machinery is tightlycontrolled. Various proteins regulate the apoptotic processat different levels.

FLIPs (FADD-like interleukin-1 -converting enzyme-likeprotease (FLICE/caspase-8)-inhibitory proteins) interferewith the initiation of apoptosis directly at the level ofdeath receptors. Two splice variants a long form (FLIPL)and a short form (FLIP

S

) have been identified in humancells. Both forms share structural homology withprocaspase-8, but lack its catalytic site. This structureallows them to bind to the DISC, thereby inhibiting theprocessing and activation of the initiator caspase-8.

The members of the BCL2 family, which regulateapoptosis at the mitochondrial level, are an importantclass of regulatory proteins. They can be divided into anti-apoptotic and pro-apoptotic proteins according to their

function. BCL2 family proteins influence the permeabilityof the mitochondrial membrane.

The IAPs (inhibitor of apoptosis proteins) constitute a thirdclass of regulatory proteins. IAPs bind to and inhibitcaspases. They might also function as ubiquitin ligases,promoting the degradation of the caspases that they bind.IAPs are characterized by a domain termed the baculoviralIAP repeat (BIR). Nine IAP family members includingXIAP (hILP, MIHA, ILP-1), cIAP1 (MIHB, HIAP-2), cIAP2(HIAP-1, MIHC, API2), NAIP, ML-IAP, ILP2, livin (KIAP),apollon and survivin have been identified in human cells.However, not all BIR-containing proteins have been shownto suppress apoptosis, and some of them might also havefunctions other than caspase inhibition. IAPs are inhibitedby a protein named SMAC/DIABLO (second mitochondria-derived activator of caspase/direct IAP binding proteinwith low pI), which is released from mitochondria along

with cytochrome cduring apoptosis and promotes caspaseactivation by binding to, and inhibiting, IAPs.


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Physiological growth contol and

apoptosis

In cells and tissues of multicellular organisms, potentphysiological mechanisms govern cell proliferation andhomeostasis. Many of these growth-control mechanismsare linked to apoptosis: excessive proliferation or growthat inappropriate sites induces apoptosis in the affected

cells. Tumours can proliferate beyond these constraints,which limit growth in normal tissue. Therefore, resistanceof tumour cells to apoptosis is an essential feature ofcancer development.

This assumption is confirmed by the finding thatderegulated proliferation alone is not sufficient for tumourformation, but leads to cell death: overexpression ofgrowth-promoting oncogenes such as c-MYC, E1A or

E2F1

sensitizes cells to apoptosis23. Besides theexpression of proteins that promote cell proliferation,tumour progression requires the expression of anti-apoptotic proteins or the inactivation of essential pro-apoptotic proteins. The molecular connections between cellcycle and cell death are not entirely clear, but the p53pathway seems to be involved. Proliferative signals induceARF, the product encoded by an alternative reading frame

within the CDKN2A tumour-suppressor gene locus, whichalso encodes the cyclin-dependent kinase inhibitor INK4A.ARF interacts with the ubiquitin ligase MDM2, and preventsit from binding p53 and targeting it for destruction in theproteasome. Upregulation of p53 leads to cell-cycle arrestand apoptosis.


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P53 and apoptosis in tumors

p53 is a key element in apoptosis induction in tumour cells. p53is inhibited by MDM2, a ubiquitin ligase that targets p53 fordestruction by the proteasome. MDM2 is inactivated by bindingto ARF. Cellular stress, including that induced by chemotherapyor irradiation, activates p53 either directly, by inhibition ofMDM2, or indirectly by activation of ARF. ARF can also beinduced by proliferative oncogenes such as RAS. Active p53transactivates pro-apoptotic genes including BAX, NOXA,CD95 and TRAIL-R1 to promote apoptosis. TRAIL-R1,

tumour-necrosis-factor-related apoptosis-inducing ligandreceptor 1.


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apoptosis

The relationship between proliferation and celldeath might also reflect the fact that cells requiresurvival signals. Lack of these signals triggersapoptosis a phenomenon called 'death by

neglect'. Survival signals include growth factors,cytokines, hormones and other stimuli, such assignals given by adhesion molecules. In general,survival signals are mediated by means of thephosphatidylinositol 3-kinase (PI3K)/AKT pathway.Depending on the stimulus, further mechanismsmust be present that deliver anti-apoptotic survivalsignals. Anoikis is a special case of death by

neglect and is triggered by inadequate orinappropriate cellmatrix contacts.

Binding of INTEGRINS to the extracellular matrixconveys survival signals by activating thePI3K/AKT pathway. Anoikis involves the pro-apoptotic BCL2 family proteins BIM and BMF. Inhealthy cells, these proteins bind to thecytoskeleton, but after the cell has detached fromthe extracellular matrix, BIM and BMF are releasedand interact with the anti-apoptotic protein BCL2.Resistance to anoikis might facilitate metastasis byallowing cells to survive following detachment fromthe matrix in their tissue of origin and travelling todistant sites.


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Survial signalling through

PI3K/AKT

Survival signals include growth factors such as epidermal growth

factor (EGF) and platelet-derived growth factor (PDGF), cytokinessuch as the interleukins IL-2 and IL-3 and some hormones such asinsulin. In general, they activate phosphatidylinositol 3-kinase (PI3K).Active PI3K generates the 3-phosphorylated lipidphosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). Thisleads to recruitment of the kinases PDK1 (PtdInsP3-dependentkinase 1), PDK2 and AKT (also known as protein kinase B or PKB) tothe plasma membrane. In the complex formed, PDK1 and PDK2

activate AKT by phosphorylation. Active AKT interferes with theapoptotic machinery. It phosphorylates, and thus inhibits, the pro-apoptotic BCL2 family protein BAD. In addition, it influences geneexpression by inactivating the forkhead family transcription factorsAFX and FKHRL1 which can induce pro-apoptotic genes such asCD95L and can also activate the transcription factor NF-OB),leading to expression of anti-apoptotic genes. Survival signalling byAKT is counteracted by the tumour suppressor PTEN, a lipidphosphatase that antagonizes the action of PI3K by removing the 3-phosphate from PtdIns(3,4,5)P3.


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apoptosis

Normal diploid cells have a limited replicativepotential, and this is another means by whichexcessive proliferation is controlled. Afterprogressing through 6070 divisions, cells cease toproliferate a state called senescence and die.

The finite number of divisions is determined by thelength of the telomeres at the chromosome ends,which shorten during each cell cycle.

Once a critically short length is reached, thesensors for DNA damage are triggered and inducecell-cycle arrest or apoptosis. Again, p53 seems tobe important for this response to telomere erosionbut, although p53 deficiency temporarily rescuescells from apoptosis, telomere loss ultimatelyresults in a genetic catastrophy, triggering p53-

independent apoptosis. In tumour cells, telomeresare stabilized by expression of telomerase or apoorly characterized mechanism that is known asalternate lengthening of telomeres (ALT).


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Apoptosis induction by the

immune system If cells manage to circumvent the built-in constraints to

unlimited proliferation, the organism has to rely on theimmune system as a watch-dog against tumour initiationa concept called immunosurveillance. The main effectorcells against tumours are cytotoxic T cells of the ADAPTIVE

IMMUNE SYSTEM and natural killer (NK) cells of theINNATE IMMUNE SYSTEM. T cells and NK cells use twomain mechanisms to kill tumour cells: the granuleexocytosis pathway and the CD95L pathway. In thecalcium-dependent granule exocytosis pathway,lymphocytes secrete a membrane permeability proteincalled perforin and proteolytic enzymes known asgranzymes from cytotoxic granules towards the target cell.

In the presence of calcium, perforin polymerizes andinitiates ill-defined changes in the target-cell membranethat allow granzymes to pass into the cell. Granzymes areneutral serine proteases that can activate caspases in thetarget cell. In addition, granzyme B might directly cleavethe BCL2 family member BID to activate the mitochondrialdeath pathway. In the CD95L pathway, which is calciumindependent, the lymphocyte exhibits the death ligand

CD95L on the cell surface and triggers apoptosis throughthe CD95 receptor on the target cell. Resistance of tumourcells to these effector mechanisms not only leads toescape of the tumours from immunosurveillance, butmight also markedly influence the efficacy ofimmunotherapy.


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Therapeutic induction of

apoptosis

Cancer treatment by chemotherapy and -irradiation killstarget cells primarily by the induction of apoptosis.However, few tumours are sensitive to these therapies,and the development of resistance to therapy is animportant clinical problem. Patients who have a tumourrelapse usually present with tumours that are more

resistant to therapy than the primary tumour. Failure toactivate the apoptotic programme represents an importantmode of drug resistance in tumour cells.

Anticancer drugs are classified as DNA-damaging agents,ANTIMETABOLITES, mitotic inhibitors, nucleotideanalogues or inhibitors of TOPOISOMERASES. Treatmentwith these agents or with -irradiation causes cellularstress and finally cell death. A key element in stress-induced apoptosis is p53. Rapid induction of p53 function

is achieved in response to most forms of stress throughpost-translational mechanisms. p53 can be stabilized andactivated through the inactivation of MDM2, either by ARF,as discussed above, or by direct phosphorylation of MDM2.In addition, many post-translational modifications of p53have been shown to enhance its transcriptional activity inresponse to stress, including phosphorylation,SUMOYLATION and acetylation. The transcriptional activityof p53 is important for its pro-apoptotic function. p53 caninduce the expression of proteins involved in themitochondrial pathway such as BAX, NOXA, PUMA andp53AIP1 and in the death receptor pathway such asCD95, TRAIL-R1 and TRAIL-R2. Moreover, transcriptionallyindependent activities of p53 mediate some of its pro-apoptotic effects, including proteinprotein interactions,direct effects in the mitochondria and relocalization ofdeath receptors to the cell surface.


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Therapeutic induction of

apoptosis

Another stress pathway that is activated in response tochemotherapy is the stress-activated protein kinase (SAPK,also known as JUN-N-terminal kinase or JNK) pathway.SAPKs, which are members of the mitogen-activatedprotein kinase family, can regulate the activity of AP-1

transcription factors. Known pro-apoptotic target genes forAP-1 are CD95L and TNF- . Moreover, oxidative stresstriggered by the production of reactive oxygenintermediates and glutathione depletion can also induceCD95L expression.

The best-defined mechanism by which therapy-inducedcellular stress eventually leads to the death of tumour cellsparticularly liver tumour cells involves the CD95

system. Chemotherapeutic drugs (for example, thenucleotide analogue 5-fluoruracil, 5-FU) induce CD95 by atranscriptionally regulated, p53-dependent mechanism.They also engage the SAPK/JNK pathway, whicheventually leads to upregulation of CD95L. Upregulation ofCD95 and CD95L then allows the cells to either commitsuicide or kill neighbouring cells.

Clearly, this is not the only pathway of chemotherapy-induced cell death. Many drugs seem to initiate the

mitochondrial pathway directly. Moreover, cell death mightnot even require caspase activation. It is questionablewhether a single predominant effector pathway ofchemotherapy can be identified at all. Probably, thepathway engaged depends on the stress stimulus, the celltype, the tumour environment and many other factors.However, because chemotherapy and irradiation exerttheir effects primarily by apoptosis induction, it isconceivable that modulation of the key elements ofapoptosis signalling directly influences therapy-inducedtumour-cell death.


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Expression of anti-apoptotic

proteins

Tumour cells can acquire resistance to apoptosis byvarious mechanisms that interfere at different levels ofapoptosis signalling. One mechanism is the overexpressionof anti-apoptotic genes. A common feature of follicular B-cell lymphoma is the chromosomal translocation t(14;18),

which couples theBCL2 gene to the immunoglobulin heavychain locus, leading to enhanced BCL2 expression. BCL2

cooperates with the oncoprotein c-MYC or, in acutepromyelocytic leukaemia, the promyelocytic leukaemiaretinoic-acid-receptor- (PMLRAR ) fusion protein, thereby

contributing to tumorigenesis. Some studies have shown acorrelation between high levels ofBCL2 expression andthe severity of malignancy of human tumours. Moreover, it

has been shown in in vitro and in vivo models that BCL2expression confers resistance to many kinds ofchemotherapeutic drugs and irradiation. In some types oftumours, a high level ofBCL2 expression is associatedwith a poor response to chemotherapy and seems to bepredictive of shorter, disease-free survival. The tumour-associated viruses EpsteinBarr virus (EBV) and humanherpesvirus 8 (HHV8 or Kaposi's sarcoma-associated

herpesvirus) encode proteins that are homologues of BCL2.Both proteins BHRF1 from EBV and KSbcl-2 (vBcl-2)from HHV8 have an anti-apoptotic function and enhance

survival of the infected cells. In this way, they mightcontribute to tumour formation after virus infection, and toresistance of these tumours to therapy.


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proteins

In addition, other anti-apoptotic BCL2 family members alsoseem to be involved in resistance of tumours to apoptosis.For example, BCL-XL can confer resistance to multipleapoptosis-inducing pathways in cell lines and seems to beupregulated by a constitutively active mutant epidermalgrowth factor receptor (EGFR) in vitro. MCL1 (myeloid cell

leukaemia sequence 1) can also render cell lines resistantto chemotherapy. In some leukaemia patients, MCL1expression was increased at the time of relapse, whichindicates that some anticancer drugs might select forleukaemia cells that have elevated MCL1 levels.

Human melanomas and a murine B-cell lymphoma cell linewere shown to express high levels of FLIP, which interfereswith apoptosis induction at the level of the death receptors.Moreover, in EBV-positive Burkitt's lymphoma cell lines, anincreased FLIP:caspase-8 ratio was correlated withresistance to CD95-mediated apoptosis93. Viral analoguesof FLIP, called viral FLIPs (v-FLIPs), are encoded by sometumorigenic viruses, including HHV8. In cells that arelatently infected with HHV8, v-FLIP is expressed at lowlevels, but its expression is increased in advanced Kaposi'ssarcomas or on serum withdrawal from lymphoma cells inculture. Therefore, v-FLIPs might contribute to the

persistence and oncogenicity of v-FLIP-encoding viruses.Although FLIP expression prevents apoptosis inductionthrough death receptors, it does not inhibit cell deathinduced by perforin/granzyme, chemotherapeutic drugs orK-irradiation. Nevertheless, it mediates the immune escapeof tumours in mouse models. Tumours with highexpression levels of FLIP were shown to escape from T-cell-mediated immunity in vivo, despite the presence of theperforin/granzyme pathway, so tumour cells with elevated

FLIP levels seem to have a selective advantage. FLIPoverexpression also prevents rejection of tumours byperforin-deficient NK cells.


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proteins

Another mechanism by which tumours interferewith death-receptor-mediated apoptosis might bethe expression of soluble receptors that act asdecoys for death ligands. To date, two distinctsoluble receptors soluble CD95 (sCD95) and

decoy receptor 3 (DcR3)

have been shown tocompetitively inhibit CD95 signalling. sCD95 isexpressed in various malignancies, and elevatedlevels can be found in the sera of cancer patients.High sCD95 serum levels were associated withpoor prognosis in melanoma patients.

DcR3 binds to CD95L and the TNF family memberLIGHT (a cytokine that is homologous to

lymphotoxins, exhibits inducible expression andcompetes with herpes simplex virus (HSV)glycoprotein D for herpesvirus entry mediator(HVEM), a receptor expressed by T cells) andinhibits CD95L-induced apoptosis. It is geneticallyamplified in several lung and colon carcinomas andis overexpressed in several adenocarcinomas,

glioma cell lines and glioblastomas. Ectopicexpression of DcR3 in a rat glioma model resultedin decreased immune-cell infiltration, whichindicates that DcR3 is involved in immune evasionof malignant glioma.


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proteins

Expression of the IAP-family protein survivin ishighly tumour specific. It is found in most humantumours but not in normal adult tissues. Inneuroblastoma, expression correlates with a more

aggressive and unfavourable disease. But althoughsurvivin has a BIR domain, it is not clear whether itdirectly acts as an apoptosis inhibitor, for exampleby binding to caspase-9 or interacting withSMAC/DIABLO. Survivin might also be necessaryfor completion of the cell cycle. Nevertheless,overexpression of survivin counteracts apoptosis in

some settings: in transgenic mice that expresssurvivin in the skin, its anti-apoptotic function wasmore prominent than its role in cell division.Survivin inhibited UVB-induced apoptosis in vitroand in vivo, whereas it did not affect CD95-inducedcell death. Expression of a non-phosphorylatablemutant of survivin induces cytochrome creleaseand cell death. In xenograft tumour models, thismutant suppressed tumour growth and reducedintraperitoneal tumour dissemination.


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proteins

Another IAP family member, cIAP2, is affected bythe translocation t(11;18)(q21;q21) that is foundin about 50% of marginal cell lymphomas of the

mucosa-associated lymphoid tissue (MALT). Thisindicates a role for cIAP2 in the development ofMALT lymphoma. ML-IAP is expressed at highlevels in melanoma cell lines, but not in primarymelanocytes. Melanoma cell lines that express ML-IAP are significantly more resistant to drug-inducedapoptosis than those that do not express ML-IAP.

Finally, tumour cells resist killing by cytotoxiclymphocytes not only by blocking the death-receptor pathway, but also by interfering with theperforin/granzyme pathway. Expression of theserine protease inhibitor PI-9/SPI-6, which inhibitsgranzyme B, results in the resistance of tumour

cells to cytotoxic lymphocytes, leading to immuneescape.


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Inactivation of pro-apoptotic

genes.

Besides overexpression of anti-apoptotic genes, tumourscan acquire apoptosis resistance by downregulating ormutating pro-apoptotic molecules. In certain types ofcancer, the pro-apoptotic BCL2 family member BAX ismutated. Frameshift mutations that lead to loss of

expression, and mutations in the BH domains that result inloss of functions, are common. Tumour cell lines withframeshift mutations are more resistant to apoptosis.Reduced BAXexpression is associated with a poorresponse rate to chemotherapy and shorter survival insome situations. Several studies in mice have confirmedthe function of Bax as a tumour suppressor. In atransgenic mouse tumour, Bax expression is induced byp53, resulting in slow tumour growth and a highpercentage of apoptotic cells. In Bax-deficient mice,however, tumour growth is accelerated and apoptosisdecreases, indicating that Bax is required for a full p53-mediated response. In a different study, induction ofBaxexpression in an inducible cell line restored sensitivity toapoptosis and significantly reduced tumour growth insevere combined immunodeficient (SCID) mice.

Moreover, others showed that inactivation of wild-type Baxconfers a strong advantage during clonal evolution of thetumour. Injection of clones with either wild-type or mutantBax into nude mice led to outgrowth of tumours that didnot express Bax in both situations.


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genes.

Metastatic melanomas have found another way to escapemitochondria-dependent apoptosis. These tumours oftendo not expressAPAF1, which forms an integral part of theapoptosome, and theAPAF1 locus shows a high rate ofallelic loss. The remaining allele is transcriptionallyinactivated by gene methylation.APAF1-negative

melanomas fail to respond to chemotherapy

a situationthat is commonly found in this type of tumour.

A similar strategy has been reported for neuroblastomas inwhich the N-MYConcogene has been amplified. In thesetumours, the gene for the initiator caspase-8 is frequentlyinactivated by gene deletion or methylation. Caspase-8-deficient neuroblastoma cells are resistant to death-receptor- and DOXORUBICIN-mediated apoptosis.

Moreover, death receptors are downregulated orinactivated in many tumours. The expression of the deathreceptor CD95 is reduced in some tumour cells forexample, in hepatocellular carcinomas, neoplastic colonepithelium, melanomas and other tumours comparedwith their normal counterparts. Loss of CD95, probably bydownregulation of transcription, might contribute to

chemoresistance and immune evasion. Oncogenic RASseems to downregulate CD95, and in hepatocellularcarcinomas loss of CD95 expression is accompanied byp53 aberrations.


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genes.

Several CD95gene mutations have been reportedin primary samples of myeloma and T-cellleukaemia. The mutations include point mutationsin the cytoplasmic death domain of CD95 and adeletion that leads to a truncated form of the deathreceptor. These mutated forms of CD95 might

interfere in a dominant-negative way withapoptosis induction by CD95. In families withgerm-line CD95mutations, which usually result inautoimmune lymphoproliferative syndrome (ALPS),the risk of developing lymphomas is increased.

Deletions and mutations of the death receptorsTRAIL-R1 and TRAIL-R2 have also been observed

in tumours. The frequent deletion of thechromosomal region 8p21-22 in head and neckcancer and in non-small-cell lung cancers affectsthe TRAIL-R2 gene. Mutations have been found inthe ectodomain or the death domain ofTRAIL-R1or TRAIL-R2. Further mutations result in truncatedforms of these TRAIL receptors or other anti-apoptotic forms.

Finally, reduced expression of the pro-apoptoticproteinXAF1 (XIAP-associated factor 1) has beenobserved in various cancer cell lines. XAF1 binds toXIAP and antagonizes its anti-apoptotic function atthe level of the caspases.


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Alterations of the p53 pathway

As p53 has a central function in apoptosis induction,alterations of the p53 pathway influence the sensitivity oftumours to apoptosis. Tumours that are deficient in Trp53(the gene that encodes p53 in mice) inimmunocompromised mice and cell lineages fromtransgenic mice that express mutant Trp53 showed a poorresponse to -irradiation or chemotherapy. Specificmutations in TP53 (the gene that encodes p53 in humans)have been linked to primary resistance to doxorubicintreatment and early relapse in patients with breastcancer141. In cancer cell lines, the specific disruption of theTP53 gene conferred resistance to 5-FU, but greatersensitivity to adriamycin or radiation in vitro142.

Mutations of CDKN2A, which encodes ARF (as well asINK4A), are almost as widespread in tumours as are TP53

mutations. Lymphomas from Trp53-knockout mice andfrom Cdkn2a-knockout mice are highly invasive, displayapoptotic defects and are markedly resistant tochemotherapy in vitro and in vivo.

In about 70% of breast cancers, wild-type TP53 isexpressed but fails to suppress tumour growth. This mightbe explained by a lack of the ASPP (apoptosis stimulatingprotein of p53) family of proteins. ASPP proteins interactwith p53 and specifically enhance the DNA-binding and

transactivation function of p53 on the promoters ofproapoptotic genes in vivo. In this way, they stimulateapoptosis induction by p53 and do not affect proliferation.ASPP expression is frequently downregulated in breastcarcinomas that express wild-type TP53, resulting in p53unresponsiveness.


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Altered survival signalling

Most tumours are independent of the survivalsignals that protect normal cells from death by

neglect. This is achieved by alterations in thePI3K/AKT pathway. Oncogenes such as RAS orBCRABL can increase PI3K activity. The catalytic

subunit of PI3K has been shown to be amplified inovarian cancer.

PTEN, the cellular antagonist of PI3K, is frequentlydeleted in advanced tumours, and a significant rateofPTENmutations can be found in various cancertypes. Moreover, AKT, the serine/threonine kinasethat mediates survival signals, is overexpressed inseveral malignancies. All of these alterations leadto a 'constitutively active' survival signallingpathway that enhances the insensitivity of tumour

cells to apoptosis induction.


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FurtherMechanism

Resistance to chemotherapy can also be attributed to thepresence of a molecular transporter that actively expelschemotherapeutic drugs from the tumour cells. The twotransporters that are commonly found to conferchemoresistance in cancer are the MDR1 gene products P-glycoprotein and MRP (multidrug resistance-associatedprotein). P-glycoprotein protects cells not only fromchemotherapy-induced apoptosis, but also from other

caspase-dependent death stimuli such as CD95L, TNF andUV irradiation. However, it does not confer resistance tothe perforin/granzyme pathway.

An important factor influencing apoptosis of tumour cells isthe transcription factor nuclear factor B (NF- B). Normally,NF- B remains sequestered in an inactive state by thecytoplasmic inhibitor of NF- B (I B) proteins. However, avariety of external stimuli including cytokines,pathogens, stress and chemotherapeutic agents can

lead to activation of NF- B by phosphorylation,ubiquitylation, and the subsequent degradation of I B. TheDNA-binding subunits of NF- B migrate into the nucleusand activate expression of target genes. Depending on thestimulus and the cellular context, NF- B can activate pro-apoptotic genes, such as those encoding CD95, CD95L andTRAIL receptors, and anti-apoptotic genes, such as thoseencoding IAPs and BCL-XL. Genes encoding NF- B or I Bproteins are amplified or translocated in human cancer157.In Hodgkin's disease cells, constitutive activity of NF- Bhas been observed.

The extracellular matrix might also contribute to drugresistance in vivo. Small-cell lung cancer is surrounded byan extensive stroma of extracellular matrix, and adhesionof the cancer cells to the extracellular matrix suppresseschemotherapy-induced apoptosis through integrinsignalling. Furthermore, in myeloma, constitutive

activation of STAT3 signalling upregulates BCL-XL and soconfers resistance to apoptosis.


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Summary

Apoptosis is a multi-step, multi-pathway cell-deathprogramme that is inherent in every cell of the body. Incancer, the apoptosis:cell-division ratio is altered, whichresults in a net gain of malignant tissue.

Apoptosis can be initiated either through the death-receptor or the mitochondrial pathway. Caspases thatcleave cellular substrates leading to characteristicbiochemical and morphological changes are activated inboth pathways. The apoptotic process is tightly controlledby various proteins. There are also other caspase-independent types of cell death.

Many physiological growth-control mechanisms that governcell proliferation and tissue homeostasis are linked toapoptosis. Therefore, resistance of tumour cells toapoptosis might be an essential feature of cancerdevelopment.

Immune cells (T cells & natural killer cells) can kill tumour

cells using the granule exocytosis pathway or the death-receptor pathway. Apoptosis resistance of tumour cellsmight lead to escape from immunosurveillance and mightinfluence the efficacy of immunotherapy.

Cancer treatment by chemotherapy and -irradiation killstarget cells primarily by inducing apoptosis. Therefore,modulation of the key elements of apoptosis signallingdirectly influences therapy-induced tumour-cell death.

Tumour cells can acquire resistance to apoptosis by theexpression of anti-apoptotic proteins or by thedownregulation or mutation of pro-apoptotic proteins.

Alterations of the p53 pathway also influence thesensitivity of tumour cells to apoptosis. Moreover, mosttumours are independent of survival signals because theyhave upregulated the phosphatidylinositol 3-kinase(PI3K)/AKT pathway.

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Grossary

ADAPTIVE IMMUNE SYSTEM Adaptive immunity also known asspecific or acquired immunity is mediated by antigen-specific

lymphocytes and antibodies; it is highly antigen specific and includesthe development of immunological memory.

ANTIMETABOLITES Antimetabolites (for example, methotrexate) blockspecific metabolic pathways by competitive binding to the substrate-binding site of enzymes that are involved in metabolism.

DOXORUBICIN A chemotherapeutic drug that induces DNA strandbreaks, which initiate apoptosis.

INNATE IMMUNE SYSTEM The innate immune system includesphagocytes, natural killer cells, the complement system and other non-specific components. It protects against infections using mechanismsthat exist before infection, providing a rapid response to microbes thatis essentially the same regardless of the type of infection.

INTEGRINS A large family of heterodimeric transmembrane proteinsthat promote adhesion of cells to the extracellular matrix or to othercells.

LAMINS A group of intermediate-filament proteins that form the fibrousnetwork (nuclear lamina) on the inner surface of the nuclear envelope.

RNA INTERFERENCE (RNAi). Use of double-stranded RNA to targetspecific mRNAs for degradation, resulting in sequence-specific post-transcriptional gene silencing.

STAT3 A member of the STAT (signal transducer and activator oftranscription) family of transcription factors. STATs are activated

through phosphorylation by Janus kinases and have an important role incytokine receptor signalling.

SUMOYLATION A post-translational modification that consists ofcovalent attachment of the small ubiquitin-like molecule, SUMO-1 (alsoknown as sentrin, PIC1). Sumoylation can change the ability of themodified protein to interact with other proteins and can interfere with itsproteasomal degradation.

TOPOISOMERASES A class of enzymes that control the number andtopology of supercoils in DNA and that are important for DNA replication.

apoptosis & cancer

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