Chapter16 The Rational Treatment of Cancer Ali substances are
poisonous, there isnone that is not a poison; the right dose
differentiates a poison from a remedy. Paracelsus(Auroleus Phillip
us Theostratus Bombastus von Hohenheim), alchemist and
physician,1538 Doctors are men who prescribe medicines of which
they know little,tocurediseasesof which theyknow less,inhuman
beingsof whom they know nothing. Voltaire(Franc,:ois-Marie
Arouet),author and philosopher, 1760
Theresearchdescribedthroughoutthisbook representsarevolutioninour
understandingofcancerpathogenesis.In1975,therewerevirtuallyno
insightsintothemolecularalterationswithinhumancellsthatleadtothe
appearance of malignancies. One generation later, we possess this
knowledge in abundance. Indeed,the available information and
concepts about cancer's origins can truly be said to constitute a
science with a logical and coherent conceptual structure. Inspiteof
these extraordinary leapsforward,relatively littleprogress hasbeen
made in exploiting these insights into etiology (Le.,the causative
mechanisms of disease)topreventthediseaseand,equally
important,totreatit.Mostof the anti-cancer treatments in widespread
use today were developed prior to1975, at a time when the
development of therapeutics was not yet informed by detailed
knowledge of the genetic and biochemicalmechanisms of cancer
pathogenesis. 725 I Chapter 16: The Rational Treatment of Cancer
(A)(8) 10060 5590 Q)c 50 Q)C 80 ~0~0\,....,;:; \.....;::;45
70s::.'"s::.'"+-'-:;40+-'-:; '"e.'"e.60Q) 0Q) 035"De.colon
andrectum9"De.lung9 "Do 30 "Do50 ~ o J ~ o "'0 uterus "'025::J ::J
~ o ~ o"Do"Do20' P ~' P ~ Q)~Q)~ O'IQ) 15 O'IQ)",e. '"e.10 5 0
0I-no celllines(groupB;blue line).Thisillustrates ~0.7
'"established graphically whytumor xenografts..... '"0.6c::
producedfromestablishedca ncercell +J0.5 C1!linesusually failto
recapitulatethe ro 0. '+- 0.4 .-..... group B (n=168)
propertiesofthetumorstypically . -.....0 Iencounteredina
cancerclinic(sincethe c:: 0.30 cancercelllinesusuallyderivefrom "13
0.2 tumors at the farendofthe~ '+- celllines establishedgroup A(n=
35)0.1spectrum-themostaggressivesubset). (FromYShimada,M.Maeda, 0.0
020406080100120140160180 G.WatanabeetaI.,Clin.Cancer Res.
survival(months)9243-249,2003) 749 Chapter 16: The Rational
Treatment of Cancer Figure 16.19 Pharmacokinetics and
pharmacodynamics of Gleevec The pharmacokineticsof a drugrepresent
thekinetics of itsaccumulationinand
disappearancefromtheplasma,whichin turnarepresumedtoprovidea good
indicationofthedrugconcentrations that tumor cellsexperienceina
laboratory animalora patientunder therapy.Theplasmalevelof thedrug
Gleevec,plottedona logarithmic scale (left ordinate),
fluctuatesdramatically followinginjectionof thedruginto a mouse
(bluecurve).Itsconcentrationis indicatedhereasa multipleof thedrug
concentrationknowntoinhibit thefiring of thet yrosinekinaseof
theKitreceptor by50%(i.e,theICsoofthisagent).
(Thetyrosinekinasedomainof theKit growth factorreceptorisalsoa
target of inhibitionbyGleevec. ) Asseenhere, the amount of
phosphotyrosineassociated withtheKitreceptor(areflectionofKit
tyrosinekinaseactivity)expressedby eng raftedhumanmastcellleukemia
cells (red curve),whichwasinitiall y set as100%,isreducedto 1 %of
preexistinglevelswithinanhourafter druginjectionbutreboundswithin8
hoursastheconcentrationof thedrug declinesintheplasma.(Courtesy of
D.L.Emerson,051PharmaceuticalsInc) clearance rate of paclitaxel
1000 120 A ;::;: til 100 -0 :::r'" 0 100
0
til"' -u;:R-o0.80o:::r00 ...,... ,,'< Olu 60 o::>'-- 10
'-- Q) ::> ...- . -0> ::>,+-0 011> .:::;;" 40
0
::>...020 11> u ::> .-+ 0.10 04812162024 time after
dosing (hours) The pharmacokinetics (PK)of a drug represent a key
determinant of its efficacy in vivo:Does it accumulate
tosignificant levelsinthe plasmaortissues foran extendedperiodof
time?Orisitpresentinthebodyonlytransiently,being
excretedbythekidneyswithinminutesof enteringintothecirculation?Isit
resistant torapid degradation, or do certain drug-metabolizing
systems, such as the cytochrome P-450s(Cyps)that weencountered
inChapter 12,rapidly convert itinto an innocuous molecular
species(Figure16.19)?(Akey pharmacokineticparameter that
isoftenmeasuredisthe "areaunder thecurve, " or AUC, calculated by
integrating the concentration of a drug in the plasma as a function
of time;the AUCisthought toreflect the cumulative drug dose
experienced by cellsin a tumor.)Andcan itbe administered oraJly
rather than requiring injection? Laboratory
animalsgivesomeroughindicationof adrug'spharmacokinetics, but are
by no means accurate predictors of how humans metabolize and
excrete
variousagents.Moreover,aswereadearlier(Sidebar12.5),theratesatwhich
various compounds, including drugs, are metabolized or excreted can
even vary
dramaticaJlyfromonepersontoanother(forexample,seeFigure16.20).(In
somepharmaceuticalcompanies,thepharmacokineticsofcandidatecompounds
may be measured evenbefore any testsof therapeutic efficacyagainst
xenograftedtumors;thoseshowing poorpharmacokineticsinlaboratory
animals are often eliminated from further testing. Discarding such
drugs may occasionaJly be premature, given the dramatically
differing rates of drug metabolism and excretion between rodents
and humans.) In fact,Figure16.19reveals a second attribute of a
drug-its pharmacodynamics(PD),in this case those of Gleevec.
Pharmacodynamics gauge the ability of a drug
toaffectatargetedbiochemical functionin atumor under treatment.In
the PDpresentedin this figure,asisoften thepractice,a surrogate
marker of the targetedBcr-Ablfunctionwasmeasured- thebehavior of
theKitreceptor. As we willread in more detail later, Kit isone of
several tyrosi ne kinases affected by Gleevec,and itsresponses
tothe drug presumably parallel thoseof Bcr-Abl. Figure 16.19
reveaJs that Kit activity in this experiment was inhibited only
briefly at the time when the highest concentration of drug
waspresent inthe circulation.Suchatransientinhibition-only
afractionof acellcycle-isgenerally insufficient to elicit a
substantial biologicaJ response, such astumor cell killing.
Figure16.20 Inter-individual variability in drug
clearancePaclitaxel isa chemotherapeuticdrugusedto treata number of
malignancies;it
worksbystabilizingmicrotubules,therebyinterferingwiththe
progressionof cellsthroughMphase.Asseenhere,inthisstudyof
22ovariancancerpatients,therelativeratesof clearanceof thisdrug
fromtheplasmaafter initialinjectionvariedovera factor of3. These
ratesmaybeinfluencedbychangesintherateofmetabolismby
enzymessuchascytochromec andbyexcretioninthekidneys.(From M .
Nakajima,YFujiki,S.KyoetaI.,IClin.Pharm.45:674-682,2005.) 750
Pharmacokineticsand pharmacodynamics During the course of
animaltesting,informationmay surfaceabout thetoxic side-effects
that the drug elicits in whole organisms. They represent the bane
of almostallexistingcancer
treatments.Quitefrequently,variousnormalorgan systems, including
the liver,kidneys,gastrointestinal tract,and the
hematopoieticsystem, show toxiceffectsof adrug whenitisusedat the
concentrations required to kill tumor cells. These toxicities are
rarely predicted by in.vitro tissue
culturetests,andtoxicitiesdetectedinlaboratoryanimals,includingdogs,
monkeys,mice,and rats,mayor may not be predictive of human
responses. Such observationsdirectour attentions once again tothe
therapeutic index of an agent-the efficiency vvith which it
affectscancerous tissues compared with itstoxi c
effectsinnormaltissues.Clearly, idealcancer treatments shouldhave
hightherapeutic indices, wreaking havoc on cancer cells while
leavingnormal tissues relatively untouched. The fundamental
obstacle to achieving such
selectivityissuggestedbythefactthatthevastmajorityof the20,000or so
genes expressed in cancer cells are also being expressed by their
normal counterparts. The failure of animalmodels to predict the
toxic side effects of a drug in humans
createsseriousproblems.Theroughly80millionyearsof independent
evolution that separate us from our rodent cousins have led to
substantial differences
inmetabolism;wemayreacttocertaindrugsmuchdifferentlyfrommiceor
rats, or even the more closely related Old World monkeys, which may
eventually be exposedtoacandidatedruginorder toobtain amarginally
more accurate prediction of toxi citiesin humans. Inthe event that
a drug passes these various tests without raising too many warning
flags,it may be promoted to a candidate fortesting in humans.
16.8Promising candidate drugsmust besubjected to rigorous and
extensiveclinical trialsin Phase Itrials in humans The discussions
above explain why the first true tests of a drug's tolerability
usuallycomeininitialpatientexposures,whicharetermedPhaseItrialsinthe
UnitedStates.Here,candidate drugsaretestedat variousdoses,including
the presumed therapeutic doses, to gauge toxic side-effects. The
usual practice isto beginthese trials at drug dosagesthat are
likely tobefarbelow the level of any overt toxicity (e. g.,
one-tenth the drug concentration that created toxicity in
laboratoryanimals)andthen,inaseriesof
patients,increasethedosagesincrementally until drug levelsare
reached that begin toinduce unacceptable
toxicities.This"doseescalation"yieldsavalue-themaximumtolerateddose
(MTD)-thatisthenusedtoguidefurthertreatmentprotocols.Certainside
effects,such asa ski n rash or transient nausea, may betolerable
and not scuttle further drug development, while others, such as
massive diarrhea or bone marrow depletion,may be soburdensome or
life-threatening that they cause rapid abandonment of all further
development of a drug.
DuringthesePhaseItrials,pharmacokineticmeasurements,likethosemade
previously in animals, will alsobe taken in order toascertain
whether the drug is reaching tumor cells at a sufficient
concentration and foran extended period of time.Still,these
measurements giveno indication whether thecancer cellsare
respondinginanyway-thepropertyofpharmacodynamics.Forexample,in
Figure16.21,weseethepharmacodynamicresponsestotreatmentswithEGF
receptor antagonists(whichhappen inthiscasetoincludeboth
amonoclonal antibody and a low-molecular-weight tyrosine kinase
inhibitor). The oncologists who undertook this particular clinical
trial wished toobtain some measure ofthe effects of therapies on
the EGF-R in patients' tumors. Todo so,they chose to use, as a
surrogate marker, the EGF-R of patients' skin cells, which were far
more easily monitored, simply by taking small skin biopsies
frompatients in treatment. 751 Chapter 16: The Rational Treatment
of Cancer Asseen in Figure 16.21A, patient exposure to an EGF-R
tyrosine kinase inhibitor resulted ina strong suppression of
EGF-Rsignaling in the skin. Inaddition, the activity of MAP kinase,
which functions as an important downstream transducer
ofEGF-Rsignaling(Section6.5),wasalsosuppressed,indicatingsuccessful
inhibition of downstream mitogenic signaling.
Similarresultswereobservedinbiopsiestakenfromacoloncancerpatient's
tumorfollowingtreatmentwithananti-EGF-Rmonoclonalantibody(Figure
16.21B).Pharmacodynamic measurements likethese providereassurance
that the administered treatment (in this case a monoclonal
antibody)isreaching its intended target at concentrations that
suffice to shut down much of the activity of itsintended target.
Interestingly,anumberofthesignal-transducingproteinsoperatingdownstream
of the EGF-R,including Akt/PKB, were only minimally suppressed in
the
colonictumor(Figure16.21B),indicatingthatthetumorcellshadacquired
alternative means for activating these signaling molecules. Hence,
pharmacodynamicsmeasurementsensure that one preconditionof
therapeuticsuccessdelivery of the therapeutic agent to the targeted
cells and molecules-has been satisfied,but donot, on their
own,guarantee that the therapy willsucceed, as other factors may
thwart it. Whentakentogether,themeasurementsof
maximumtolerateddose(MTD), pharmacokinetics(PK)
,andpharmacodynamics(PD)definethetherapeutic window-the range of
concentrations that are higher than that needed to elicit
atherapeuticeffectandlowerthanthemaximumtolerateddose(Figure
16.21C).Ideally, a therapeutic window of a drug should be broad
soas toallow
clinicianssomeflexibilityinadministeringthedrug,adjustingdosagetothe
' patient and the condition being treated. As the therapeutic
window narrows, the likelihood that a candidate drug willprove
clinically usefuldiminishes.
Occasionally,PhaseIclinicaltrials,whichareusuallyundertakenwithvery
smallgroupsof
patientvolunteerswhohavefailedotheravailabletherapies, may reveal
some favorableresponses in terms of tumor regression or halting of
furthertumorgrowth,doingsoatacceptably10v\Tlevelsof
toxicity.However, even if there are hints of clinical efficacy, the
positiveresults observedin Phase
Itrialsareneverstatisticallysignificantandthusnotregardedasdefinitive.
Instead,thesetrialsarereallyundertakentodiscoverunanticipatedtoxicities
and tolerable levels of drug dosage. 16.9Phase II and
IIItrialsprovidecredible indicationsof clinical efficacy Acceptably
low levels of toxicity in a Phase I trial willencourage testing a
candidate drug'sefficacy in aPhase IItrial,in which larger groups
of cancer patients areinvolved.Now,forthe firsttime,critical
decisions must be made about the
indicationsforenlistingspecificpatientsinthetrial-that
is,whichtypeof tumor or what stage of tumor progression willjustify
enrolling patients insuch atrial? Sometimes the clinical
indications areobvious.For example,as wenoted earlier,the effects
of an agent targetedagainst the Bcr-Abloncoprotein should be
testedinpatientsdiagnosedwithchronicmyelogenousleukemia(CML).
AnotherdrugdirectedagainsttheHER2/Neureceptormoleculeshouldbe
testedintheapproximately 30%of breastcancer patients
whosetumorcells overexpressthisprotein. Yetanother agent-an
inhibitor of Raf kinases-can be tried inpatients with advanced
melanomas, in which the B-Rafkinase molecule is often (70%of cases)
mutant and constitutively activated.(Interestingly, 752 Early
clinical trialsof toxicity and efficacy in the last case, a B-Raf
inhibitor failed to effectively stop further proliferation of
metastaticmelanomas,whileitsuseincombinationwithaconventional
chemotherapeutic drug has yielded dramatic, albeit only anecdotal
responses.) Butmoreoftenthan not,thechoiceof indications isneither
rationalnor optimal. Which classof cancer patients should be
treated,forexample, with a drug (A) pre-treatmentpost-treatment
activated EGF-R inskin activated MAPK in skin (B) overall EGF-R
pEGF-R pAktJPKB day 0 Figure16.21Measurementsof pharmacodynamics
and determinationof the therapeutic window Theextentofinhibitionof
the EGF-Rina tumor can,inprinciple,be gaugedbymeasuringeffectsof
drug treatment ontheEGF-Rintheskin;the latter
isreadilyassessedthroughsmall skinbiopsies.Inthecasesillustrated
here,patientsundertreatment were sufferingfroma varietyoftumors,
includingcarcinomasof the ovary,lung,
colon,prostate,andhead-and-neck. (A)Shownherearetheeffectsof
treatinga cancerpatient withIressa,a low-molecular-weight
EGF-Rtyrosine kinaseinhibitor (seeFigure1631)The upperpanels show
immunohistochemistryusinganantibodyagainst phospho-EGF-R(brown),
i.e.,the acti vatedformofthereceptor.Thelower
panelsusedanantibodyagainst phospho-MAPK,theactivatedformof
thiskinase.Bothmeasurements dependedonthenormallyintense
signalingoccurringinkeratinocytes presentinthehairfollicles.(B) The
effectsof ananti-EGFreceptor(EGF-R) monoclonalantibody
(termedEMD7200) weregaugedbyimmunohistochemical stainingof a
coloncarcinomabiopsy.In thiscase,long-termtreatmentresulted ina
minimalreductionintheoveralllevel oftheEGF-R(brown) anda st rong
reductioninthelevelofphosphorylated (andthereforeactivated)receptor
(brown;pEGF-R). Thereductioninthe levelofphosphorylated,activated
AktlPKB(brown;pAktlPKB) w asslight andthepatientshowedonlya partial
responsetothis antibodytherapy,w hich mayhavereflectedthisminimal
reductionof AktlPKBacti vityintumor cells.(C)Measurementsof
pharmacodynamicssuchasthese,takentogether withstudiesof
pharmacokinetics and toxicity,definethetherapeuticwindow inw hicha
drugshouldbegi ven-the rangeof concentrationsthatare
efficaciouswithout creatingan unacceptablelevelof toxicsideeffect
s. (AandB,courtesyof J.Baselga.) (C)therapeutic window ,-----,
>_tolerated dose ,...u o ,'f;+".:;ro Qj30 'f;30 rot 0Qj0.+" Q) o
E20~20 >~ '.j:; 0. ~ e:: 10 0 10 o ShhNp++ ~I (C)60 >, +".:;
'f;40 ro Qj t o 0. e:: Q) .i;20 ro ~ Lo 310cyclopamine- +- +- +- +
LI____________~ levelof Smo 505050.5 ~ LI-----.Jcontrol+
cyclopaminecontrolcyclopamine expressionwild(flM)oncogenic
typeSmoothened Smoothened Figure16.41Actionsof
cyclopamineonthePatchedSmoothenedpathway
(A)Intheexperimentshownhere,the
activityofSmoothenedwasgaugedindirectlybymeasuringthe activityof
areportergenewhosetranscriptionisdrivenbyGliin
mouseNIH3T3cells.Intheabsenceof addedSonichedgehog
ligand(ShhNp),aHedgehogvariantthatisalsoa ligandof the
Patchedreceptor,therewasnoactivity ofGli(light greenbar)In
thepresenceofShhNp,Gliactivity wasstrongly stimulated(dark
greenbar),andthisinductionwasreversedbycyclopamine
treatment(pink,red bars).Thisdemonstratedthatcyclopamine
counteractstheeffects ofHedgehogligandandisthereforelikely to
liedownstreamof thePatchedreceptorinthesignalingpathway.
(B)Thetargetof cyclopamineactionwasfurtherlocalizedbythis
experiment,inwhichtheactivity ofGli(measuredasinpanelA)
wasmeasuredinPTCnl- cells.Gliactivitywas,asbefore,
suppressedbycyclopamine,confirmingthat thisdrugislikelyto
interferewitha stepdownstreamof andindependent ofPatched.
(C)Whenwild-typeSmoothened(Smo)wasexpressedathighlevels
inNIH3T3cells,itsactivity was,onceagain,suppressedbyaddition of
cyclopamine,asindicatedbytheactivityof theGli-regulated
reportergene(blue,orangebars,left).However,whena mutant, dominantly
acting,oncogenicSmoothenedwasexpressedatthe sameorlowerlevels(right
bars)signalingwasquiteresistantt o
cyclopamineinhibition.ThisindicatedthatSmoothenedwaseither
downstreamof oradirecttarget of cyclopamineaction.
Subsequentstudiesgenerateda seriesof mutant,const itutively
activeSmoothenedproteinsthat wereallresistanttocycl opami ne
inhibition,reinforcingthenotionthat cyclopamineinteractsdirectly
withSmoothened(seeFigure1640A)Biochemicalanalysesthen
demonstratedthedirectbindingofthecyclopaminemoleculeto
Smoothened(not shown)(FromJTaipale,JK.Chen,M.K.Cooper et al.,Nature
406:1005-1009,2000.) 781 ---Chapter 16: The Rational Treatment of
Cancer (A) 800 QJ CJ) c '" u :oR ~400 o ~ to .2 a - 100 cyclopamine
treatment duration Figure16.42 Effect of cydopamine andanalogous
drugs on tumor growth (A)Humancholangiosarcoma
(bileducttumor)cellsformedtumor xenograftsinmiceof180mm 3 volume
andeitherwereleft untreated(red line)
orwerethentreatedfor22dayswith cyclopamine(blueline). Inthelatter
case,thetumorshrank anddidnot reappearinthe76daysthatfollowedin
theabsenceof furthercyclopamine treatment.(B)Micewitha
Ptc+l-p53-igenotypedevelopmedulloblastomas throughout
theircerebellaearlyinlife. By5 weeksof age,thecerebellumina wi
ld-typemouse(topleft)isfarsmaller thaninthetumor-pronemutant (top
right) . ASmoothenedantagonist, termedHhAntag, wasidentifiedthrough
screeningof a druglibrary.Ifthemutant miceweretreatedwiththedrugtw
ice dailybetweenthethirdandthefifth weekoflife,witheither20mgor100
mgperkgofbodyweight,thetumors regressedpartiallyorcompletely(bottom
left and Tight).Subsequenttreatmentsof 8-
and10-week-oldmutantmicewith farlargertumors haveyielded
comparabletherapeuticresponses(not shown).(A,fromD.M.Berman,
5.5.Karhadkar, A.Maitraetai, Nature 425:846-851 ,2003;
B,fromJ.T.Romer, H.Kimura,S.Magdalenoetal.,Cancer
Cell6229-240,2004) (B)cerebella Ptc+l- p53-1- untreated wild-type
Ptc+l- p53-1- treated with 20mg/kgHhAntag 9498days Ptc+l- p53-1-
treated with100mg/kgHhAntag In order to test these new compounds, a
mouse model of human medulloblastoma hasbeen createdthat dependson
inactivation(seeSidebar 7.10)of one copy of the Pte
geneandbothcopiesof thep53 geneinthemousegermline, yieldingaPte+ l
-p5:r1-
genotype;virtuallyallsuchmicedevelopmedulloblastomasby3months of
age.An inhibitor of Smoothened, termedHhAntag,was
synthesizedthathas10timesthepotency of cyclopamineandisabletopass
easilythroughtheblood-brainbarrier,thespecializedbiologicalbarrierthat
protects the brain tissue fromthe contents of the circulation.
Asseen in Figure 16.42B, treatment of3-week-old mutant mice that
developed medulloblastomas with HhAntag causes a regression of the
tumor within twoweeks; this occurred \ovithlittle if any systemic
toxicity. Inthe caseof pancreatic cancer,theprospect of developing
aclinicallyuseful inhibitor of the Hedgehog signaling pathway isan
exciting one. Atpresent, this carcinoma,in which Hedgehog signaling
often playsaprominentrole, has an
almostinevitablefataloutcome:oncethiscancerhasbeendiagnosedina
patient,theprobability of surviving foranother fiveyearsislessthan
4%.This contrastswiththefive-yearsurvivalin1998of
Americanpatientsdiagnosed withbreast cancer (86%)and prostate
cancer (97%). Medulloblastomas,largely pediatric tumors,occur about
one-tenth asoften as
pancreaticcarcinomas;atpresent,almosttwo-thirdsof
patientsarecuredof thistumorthroughacombinationof
surgery,radiation,andchemotherapy;
thesetreatmentscan,however,leavesurvivors\ovithsignificantneurological
impairment,includingcompromisedcognitivefunctions.Ironically,however,
the major economic incentive fordeveloping cyclopamine mimetics
islikely to
derivefromtheneedtotreatthemostbenignbutalsothemostcommon human
cancer type-basal cell carcinomas of the skin. 16.15 mTOR, a master
regulator of cell physiology, representsanattractivetarget
foranti-cancer therapy Thefinalanecdoteisthe shortest of all,if
only because it describes aregulatorycircuitthat
isstillincompletely understoodandhas yieldedfewclinical successes
to date.Nonetheless, this circuit has all the attributes of
generating 782 (A)(B) Me mTOR/0 binding Me o regionMe OMe ~ # Me
rapamycin Me Figure16.43 Rapamycin.FKBP12andmTOR(A)Rapamycinis
describedchemicallyasa macrocycliclactoneandbiologicallyasa
macrolideantibiotic,oneof manythataremadebybacteriabelongingto
theStreptomycesgenus.Rapamycinanditschemicalderivativesactas
potentimmunosuppressants withoutinducingseveresideeffectsinother
organsystemsinthebody.Someof itseffectsareduetoitsabilityto
inhibitmTORsignaling.(B)Thebindingof rapamycin(green,red stick
figure)to FKBP12(blueribbonand space-filling model,right)occursw
ith highaffinity,thedissociationconstant (Kd)beingintherangeof
0.2to OAnM. Thisbimolecular complexformsamolecular surfacethatcan
thenassociatewithmTOR(redribbonand space-fillingmodel,left)and
preventthelatter fromfunctioningasa serine/threoninekinase.Inthis
image,onlytheFRB(FKBP12-rapamycin-binding)domainof mTORis
shown.(C)Thedetailsof theinterfacebetweenrapamycin(yellow,red
stickfigure)andthe surfacesof thetwoproteinsareshownhere.Areas of
highstereochemicalcomplementaritybetweenrapamycinandthe
proteinsarehighlightedinpurple.Someof thehighaffinity association
depends ontheinsertionof chemicalgroups of rapamycininto deep
cavitiesw ithinFKBP12(right)andtheFRBdomainof mTOR(left).
(BandC,courtesyof YMaoandJ.Clardy,andfromJ.Choi,J.Chen,
S.L.SchreiberandJ.Clardy,Science273239-242,1996 ) therapies that
will rival and even eclipse some of those that have been described
earlier in this chapter. This story alsostarts
withanaturalproduct-rapamycin-that wasisolated in the1960s
fromStreptomyces hygroscopicus bacteria growing in the soil of Rapa
Nui, known tous as Easter Island, in the middle of the Pacific. In
the early 1970s, it was re-isolated by a drug company, which
developed it as an antifungal agent. In the decades that
followed,it became clear that rapamycin (Figure 16.43A)can
acttohaltthegrmvthof anextraordinarilywidespectrumof
eukaryoticcells, ranging fromthose of yeast tomammals. Rapamycin
was also found to have powerful immunosuppressive powers, even when
used at low concentrations. In 1999, it was approved by the
U.S.Food and
DrugAdministration(FDA)topreventimmunerejectionoftransplanted
organs,largelykidneys.Thisdrug,alsocalledsirolimus,functionssynergisticallywithother
immunosuppressants,specificallycyclosporineand steroids, toensure
long-termengraftment without causingmajor sideeffectsintransplant
recipients. The reasonsforitsselective actions in preferentially
affecting theimmune
systemarenotfullyunderstood.[Intriguingly,immunosuppressionbycyclosporine
inorgantransplantrecipientsleadstoincreasedriskof mTOR inhibition
by rapamycin FRBdomain of mTOR rapamycin (C)deep burialof rapamycin
methyl group inmTOR deep burial of rapamycin pipecolinylgroup
inFKBP12 78. Chapter 16: The Rational Treatment of Cancer
malignancies (see Section 15.9), while rapamycin-induced
immunosuppression in these patients actually decreases the risk of
post-transplantation lymphoproliferative disorders.Hence the notion
that immunosuppression always leads to increased cancer risk needs
to be refined, since some types of immunosuppression yield
increased tumor incidence while other types do not.]
Biochemicalanalyses show that rapamycin binds directly
toalow-molecularweight protein,called FKBP12(FK506-binding protein
of 12kD),originally discovered because it is also bound by FK506, a
similarly acting drug. Once formed, therapamycin-FKBPl2complex
(Figure16.43B)associates withaproteinthat
wasidentifiedin1994,termedmTOR(mammaliantargetof rapamycin),and
shuts it down.mTORisa large (289kD)protein that functions as a
serine/threoninekinase;itskinasedomainresemblesthatofPI3kinaseandrelated
enzymes. mTORisof special interest, because it operates as a
criticalnode in the control circuitry of mammalian cells (Figure
16.44A). Thus, mTOR integrates a variety of
afferent(Le.,incoming)signals,includingnutrientavailabilityandmitogens,
and, having done so,acts tocontrol glucose import and protein
synthesis. More
specifically,mTORphosphorylatestwokeygovernorsoftranslation:p70S6
kinase(S6Kl)and4E-BPl.ThisphosphorylationactivatesS6Kl,whichthen
proceeds to phosphorylate the S6protein of the small
(40-S)ribosomal subunit, Figure16.44 ThemTORcircuitandtumor
responsestomTOR inhibitors (A)mTORsitsinthemiddleof a
complexregulatorycircuit that integrates
incomingsignalsaboutnutrient availability,oxygen
tension,ATPlevels,andmitogenicsignalsand,inresponse,releases
signalsthatgovernribosomebiogenesis,proteinsynthesis,cell
proliferation,protectionfromapoptosis,angiogenesis,andevencell
motility.mTORexistsintwo alternativecomplexeswithitsRictor(left)
andRaptor(right)partners;the
twocomplexesintercommunicateinstillobscureways.ThemTOR- Rictor
complexgovernsthe activityof AktJPKB by addinga
criticalsecondphosphate tothelatter andthereby gains
controloverAktJPKB 'smultipledownstreameffectors.Exposureto
rapamycin(lower right)rapidlyinhibitsthemTOR-Raptor complexand,
afterextendedperiods,causesa progressiveshutdownof the mTOR-Rictor
complex.(B)BALB/cmicebearinginjectedcellsof a syngeneic
colonadenocarcinomacellline developlarge,well-vascularized tumors
(left)by 35daysafterinjection.However,ifthetumors are allowedtogrow
for a weekafter whichthemicereceivecontinuous treatment
withdosesofrapamycincomparabletothoseusedinhumans
forimmunosuppression,thetumorsaremuchsmaller(right)andthe density
ofmicrovesselsinthesetumorsislessthanhalf of thatseenin
thecontroltumors (not shown).(C)Metastatic osteosarcomasareoften
difficult to treat.However, inthecaseof a23-year-oldosteosarcoma
patient,treatmentwithAP23573,ananalogof rapamycin,yieldeda
morethan50%decreaseinthemaximumstandarduptakevalue
(SUVmax)ofradiolabeledglucosebya metastasiswithin5daysof
treatment,anda morethan85%decreaseby54daysof treatment (white
arrows).Whilesuchresponsesarenot typical,theyindicate the
potentialof thistypeof treatment andthepossibilitythat,inthefuture,
conditions w illbefoundto enablesimilarresponsesina significant
proportionof suchpatients.Eachoftheseimagesisa fusionoftwo
imagesinitially obtainedbyCT(computerizedX-raytomography) and
PET(positron-emissiontomography);thelattermeasurestheextent
ofuptakeofradiolabeledglucose,whichisgenerallyelevatedin
neoplastictissue . (A,fromDA GuertinandD.M.Sabatini,Trends
Mol.Med.11 :353-361,2005;B,fromM.Guba,vonBreitenbuch,
M.SteinbaueretaI.,Nat.Med.8: 128-135,2002;C,courtesyof S.P
ChawlaandK.K.Sankhala,CenturyCityDoctors'HospitalandJohn
WayneCancerInstitute,andof C.L.Bedrosian,AriadPharmaceuticals,
Inc.) 784 Rapamycinasananti-cancer agent enabling this subunit
toparticipate in ribosome formation(by associating with the large
ribosomal subunit) and thus in protein synthesis. In addition, by
phosphorylating 4E-BP1, mTOR causes 4E-BPlto release its grip on
the keytranslational initiation factoreIF4E (eukaryotic initiation
factor 4E); once liberated, eIF4E formscomplexes with several other
initiation factors,and
thereSUltingcomplexesenableribosomestoinitiatetranslationofcertain
(A) s AktlPKB I cellgrowth 1 protein synthesis - I T transcription
(s;;
G- G J'(L I\ 8 eFKBP12GTP T rapamycin - hypoxia/stress -AMPK
....f--r nutrients (amino acids,glucose) /11/1\\\\ ! ;oli feration
I'cell survival] (B)(C)control+ rapamycin beginning of5 days
oftreatment54daysof treatment AP23573treatment 785 Chapter 16: The
Rational Treatment of Cancer mRNAs,specificallythose
witholigopyrimidinetractsintheir 5',untranslated regions. Together,
these various actions allow mTOR to be a key governor of cell
growth(rather than cell proliferation; see Figure 8.2).
Untilrecently,mTORwasthoughttobeoneof themultipledownstream
substrates of AktlPKB, specifically the one allOwing AktlPKB to
regulate cell growth by controlling protein synthesis. But the
tables have been turned: mTORisnow reali
zedtobeakeyupstreamactivator of AktlPKB(Sidebar 540 ).Thisshift
puts mTORina farmore powerful position inthe cell.Bycontrolling
AktlPKB, mTORcan regulate apoptosisand proliferation inaddition
toitsknown ability to regulate cell growth. Infact,mTORappears
intwoplacesin the circuitry depictedinFigure16.44A,
sinceitisabletoassociatewithtwoalternativepartners,calledRaptorand
Rictor.The mTOR-Rictor complex (together with athird
protein,isregulatedinunknownwaysbygrmvthfactorsandisresponsibleforactivating
Akt/PKB. ThemTOR-Raptor complex(+about whichmoreisknown,is
responsible foractivatingprotein synthesis(byphosphorylating
S6K1and 4EBPI)
.ActingtogetherwithFKBP12,rapamycindirectlyinteractswiththe
mTOR-Raptor complex,which israpidly inhibited after thisdrug
isappliedto cells.If, however,rapamycin treatment iscontinued for
many hours, eventually
themTOR-Rictorcomplexisalsoshutdown,resultingintheinhibitionof
AktlPKB.Themechanismbywhichrapamycinsucceedsininhibitingthe
mTOR-Rictor complex is poorly understood. This inhibitory effect on
AktlPKB signaling seems to be responsible formuch of
rapamycin'seffecton cancer cellsthat exhibit ahyperactivatedPI3Kor
lossof PTENexpression.It
isplausiblethatsuchcells,muchlikethesmall-celllung
carcinomacellswithmutantEGFreceptors(Sidebar16.3),havebecome
"addicted"toAktlPKBsignalsandlurchintoapoptosisthemomenttheyare
deprivedofthesesignalsbytheactionsofrapamycinandrelateddrugs.
However,thepreciserulesthatdeterminesensitivitytorapamycintreatment
are yet tobe worked out. The regulatory circuit shown in Figure
16.44A intersects in additional ways with
cancerpathogenesis.Forexample,TSCland TSC2(alsocalledhamartinand
tuberin) have already appeared in this book in the context of their
role as tumor suppressor proteins. Lossof either of these proteins
leads totuberous sclerosis (Table7.1) ; and,as seeninFigure 8.2,
lossofTSClresultsin the formationof giant cells in both flies and
humans. TSC2 acts as a GAP(GTPase-activating protein; see, for
example, Sidebar 5.11) for Rheb, a small Ras-like protein. Aslong
as itremains in its GTP-bound state,Rhebcontributes in unknown ways
to stimulating the complex; however,once TSC2has induced Rheb to
hydrolyze its GTp,Rheb loses this stimulatory activity. Yet other
signaling connections between the mTOR circuit and critical
growth-inducing and mitogenic proteins are being forgedby ongoing
research. Variousderivativesof rapamycinhavebeenproduced,and three
areinearlyphaseclinicaltrial.Theirdevelopmenthas
beenencouraged,inpart,bythe observation that drugs likerapamycin
can be tolerated forextended periods of time by transplant
recipients, indicating a tolerably low levelof side-effect
toxicity.Inpre-clinical experiments, rapamycin given to mice at
levels that are used forchronic immunosuppression has strong
effectsin suppressing tumor-associatedneoangiogenesisandthustumor
growth(Figure16.44B),an effectthat may beexplainedbythefactthat one
of thethree AktlPKBisozymes,Aktl,is critical to the ability of
endothelial cells and their precursors to respond to stimulation by
vascular endothelial growth factor(VEGF).
Insomeclinicaltrials,notablythosefocusedontreatingsarcomas,clinical
responses have occasionally been observed that are nothing short of
remarkable (Figure16.44C).And in2006,rapamycinwasreportedtoinduce
regressionof786 Synopsisand prospects astrocytomas
associatedwithtuberous sclerosis(see Figure 8.2B).Indeed, it is
clinical responses like these that have motivated discussion of the
mTOR circuit in this chapter. They provide tantalizing hints of how
this circuit may one day be manipulated to induce cancer celideath,
yielding substantial improvements in thetherapy of
solidtumors.Theseadvancesarelikelytocomeasoncologists learn which
types of cancer cells are particularly sensitive to rapamycin
analogs, often in the presence of other coli aborating therapeutic
drugs. 16.16 Synopsisand prospects:Challenges and opportunities on
theroadahead "When iscancer going to be cured?" This isthe simple
and reasonable question posed most often to cancer researchers by
those who are not directly involved in this area of biomedical
research. In their minds are the histories of other public
healthmeasures. Infectiousdiseases,suchaspolioand smallpox,can
beprevented, and bacterial infections are, almost invariably,
cured. Heart disease is,in the eyes of many, well on its way to
being prevented (Sidebar 550 ). Why should cancer be am different?
The informationinthisbook provides some
insightsintotheanswerstothese questions. As much as we have invoked
unifying concepts to portray cancer as a single di sease, the
reality-at least in the eyesof clinical oncologists-is
fardifferent.Cancer isreallyacollectionof morethan 100diseases,each
affectinga distinct cellor tissue type in the body. Pathologi
calanalyseshave ledustoembrace thisnumber,or oneabitlarger.
(Forexample,thereareatleasteightdistincthistopathologicalcategoriesof
breast canceL)However,even the expanded number, largeasitmay
be,representsanillusion:thecurrentuseofmoleculardiagnostics,specificallygene
expression arrays, is leading to an explosion of subcategories, so
that by the second decade of the new millennium, several hundred
distinct neoplastic disease
entitiesarelikelytoberecognized,eachfollowingitsown,reasonablypredictable
clinical course and exhibiting its own responsiveness tospecific
forms of therapy. \ \-iththe passage of time, cancer diagnoses will
increasingly be made using bioinformatics rather than the trained
eyes of apathologist. Sothe initialresponse toquestions about "the
cure" isthat there won't be a singlemajorbrea1.
'throughthatwillcureallcancers-adecisivebattlefieldvictory-simply
because cancer is not a single disease. Instead, there will be many
smallskirmishesthat willsteadilyreducetheoveralldeathratesfrom
various
typesofcancer.Andbecausecertainmoleculardefectsandpathological
processes(e. g .. angiogenesis)are shared bymultiple human
cancers,there will beoccasions\\-hen therapeuticadvancesonanumber
of frontswillbemade concomitanth. Before we speculate on the future
of cancer therapy, it is worthwhile to step back
andassessthescopeof thechallenge:(1)Howlargeistheproblem of cancer
and,in the future, how desperate will the need be tocure various
types of neoplastic disease? (2)How well are we doing now in curing
the major solid tumors?
Epidemiologyanddemographicsprovidesomeanswerstothefirstquestion.
They yield sobering assessments of the road ahead. The statistics
in Figure 16.45 demonstratethat cancer islargelyadiseaseof
theelderly,whose numbersare growing rapidly and willcontinue
todoso, generating progressive increases in the numbers of
cancer-related deaths (mortality)over the coming decades.
Equallyimportant,westillhaveonlyveryimperfectwaysof
measuringincidence-how often
thediseasestrikes.Thisgreatlycomplicatesassessmentsof the
effectiveness of current therapies and future needs fortherapy.
Asindicated787 78.9c280 .2::::70 60 ::::l50 0.(1) 0-0 400.(5 vi-o
30 c::Jro 20 Lf)10 co 0 1990200020102020203020402050 Chapter 16:
The Rational Treatment of Cancer (A)(B)>.t:c Vi90roO t.;::;
E-340males
13g 20fema les--------=roo'to
oOJ"80 0. 1930194019501960197019801990 years (C)(D) 600325,000
(1)300,000(1)..0.V> 500 "--0 250,000:.=co roro, (1)t(1)>
400o01 Qj200,000 c
0. V>Qj0 u- 300.r:. c1il ..-150,000roro(1)u::::l-0-0.
200ro0'+0 100,000to.
(1)0 (1).20100
.n 50,000E ou5 ::::lC 00