The New Generation of Targeted Therapies for Breast Cancer

ABSTRACT: Traditional therapies for breast cancer have generally relied uponthe targeting of rapidly proliferating cells by inhibiting DNA replicationor cell division. Although this strategy has been effective, its innate lackof selectivity for tumor cells has resulted in diminishing returns, approachingthe limits of acceptable toxicity. A growing understanding ofthe molecular events that mediate tumor growth and metastases has ledto the development of rationally designed targeted therapeutics thatoffer the dual hope of maximizing efficacy and minimizing toxicity tonormal tissue. Promising strategies include the inhibition of growthfactor receptor and signal transduction pathways, prevention of tumorangiogenesis, modulation of apoptosis, and inhibition of histone deacetylation.This article reviews the development of several novel targetedtherapies that may be efficacious in the treatment of patients with breastcancer and highlights the challenges and opportunities associated withthese agents.In recent years, the strategy in cancertherapy in general and breastcancer in particular has shiftedfrom the use of high doses of toxic,nonspecific agents to a range of novelagents that target specific molecularlesions found in tumor cells. Advancesin molecular biology have allowedthe isolation of novel interactions anddownstream targets, driving the developmentof rationally designedtargeted therapies. The success oftrastuzumab (Herceptin) in breast cancerand imatinib mesylate (Gleevec)in chronic myelogenous leukemia andgastrointestinal stromal tumors providesproof of principle that such anapproach can have a marked impactwhen the mechanism of growth of aparticular cancer is understood andspecifically interrupted.This article will focus on new,molecular-targeted approaches to thetreatment of breast cancer. Of particularinterest are classes of drugs thattarget the tyrosine kinase signal transductionpathways, block tumor angiogenesis,modulate apoptosis, andinhibit histone deacetylation.Targeting the erbB1 ReceptorThe erbB family consists of fourclosely related transmembrane receptors:erbB1 (also termed epidermalgrowth factor receptor [EGFR] orHER1), erbB2 (also termed HER2 orneu), erbB3 (HER3), and erbB4(HER4). All four erbB receptors sharea common molecular architecturecomposed of three distinct regions:an extracellular ligand-binding domain,a transmembrane region, andan intracellular tyrosine kinase-containingdomain that is responsible forthe generation and regulation of intracellularsignaling (Figure 1). Theformation of erbB homodimers andheterodimers following ligand bindingand receptor aggregation activatesthe intrinsic receptor kinase activityvia intramolecular phosphorylationand generates a cascade of downstreamchemical reactions thattransmit a wide variety of cellulareffects.[1]

The rationale for and developmentof therapeutics targeting erbB2, particularlytrastuzumab, have been reviewedelsewhere,[1] and this sectionwill be limited to a discussion of therapeuticstargeting erbB1. The erbB1receptor is overexpressed in about40% of breast cancers.[2,3] The frequencyof overexpression variesdepending on the evaluation methodused and whether the truncatedEGFRvIII form-a constitutively activatederbB1 variant expressed in alarge proportion of breast cancers-isincluded.[3]The overexpression of erbB1 hasbeen associated with increased proliferation,disease progression, and apoor prognosis in breast cancer.[3,4]ErbB1 expression has also been correlatedwith decreased estrogen-receptorexpression and increased resistanceto endocrine therapy.[2,3,5,6] ErbB2and erbB1 are commonly (10%-36%)coexpressed, and such coexpressionhas been correlated with a less favorableprognosis.[7,8] Given the wideexpression of erbB1 in breast cancerand the important role this receptorplays in signal transduction, the useof erbB1 inhibitors in the treatment ofbreast cancer has generated considerableinterest.The aberrant signaling that occursthrough the erbB1 pathway can becaused by high expression of erbB1,mutation of erbB1 (eg, EGFRvIII),decreased phosphatase levels, orheterodimerization of erbB1 withother members of the erbB receptorfamily (such as HER2).[3] Severaldifferent strategies have been used todownregulate signaling through thispathway (Table 1). These includemonoclonal antibodies directedagainst erbB1 such as cetuximab(IMC-C225, Erbitux) and ABX-EGF,and small-molecule inhibitors oferbB1 tyrosine kinase such as gefitinib(ZD1839, Iressa) and erlotinib(OSI 774, Tarceva).Small Molecules TargetingerbB1 Tyrosine Kinase
Small-molecule inhibitors of erbB1receptor tyrosine kinase prevent receptordimerization, autophosphorylation,and the resulting downstreamsignaling. Hypothetically, this approachcould inhibit signaling mediatedby ligands as well as signalingthat is independent of growth factors.In contrast to monoclonal antibodies,such agents may also inhibit ligandindependentsignaling due to constitutivelyactive mutant receptors (eg,EGFRvIII). Several erbB1 tyrosinekinase inhibitors are under evaluation,but the anilinoquinazolines, gefitiniband erlotinib, are in the most advancedstages of development.

• Gefitinib-In preclinical studies,gefitinib has demonstrated broad antitumoractivity in lung, breast,ovarian, and other tumors.[9] Celllines that overexpress erbB2 appearto be particularly sensitive to gefitinib,and preclinical data suggest asynergistic inhibitory effect when theagent is combined with trastuzumabin cell lines that coexpress erbB1 anderbB2.[10,11] These observationssupport the use of erbB1 inhibitorssuch as gefitinib in combination withtherapies that target erbB2. In addition,preclinical data suggest that resistanceto endocrine therapy inestrogen-dependent tumors may bemodulated through erbB1, which maybe thwarted by gefitinib.[6,12]This phenomenon was examinedin a recent study in which nude micebearing erbB2-expressing breast cancercells (MCF-7/HER2-18) weretreated with estrogen, tamoxifen, orestrogen-deprivation alone or togetherwith gefitinib.[12] In this study,erbB2 overexpression increased theagonist properties of tamoxifen, resultingin stimulated growth. However,tamoxifen-stimulated MCF-7/HER2-18tumor growth was completely blockedin mice treated with gefitinib. In micetreated with gefitinib and estrogendeprivation, the erbB1 tyrosine kinaseinhibitor delayed the developmentof acquired resistance to estrogendeprivation.These observations support theconcept that crosstalk between estrogenreceptor and erbB1/erbB2-relatedpathways can modulate resistance toendocrine therapies and suggest thatcombination therapy may be useful inmaintaining estrogen sensitivity followingthe development of hormoneresistance. Additional potential benefitsof gefitinib and other therapeuticagents targeting erbB1 stem from theirfavorable interaction with cytotoxicdrugs (eg, paclitaxel, docetaxel [Taxotere],carboplatin [Paraplatin], cisplatin,topotecan [Hycamtin], andraltitrexed) in human tumor xenograftmodels and restoration of taxanesensitivity in multidrug-resistant celllines.[1,13]In phase I trials conducted in patientswith advanced breast cancer,gefitinib has demonstrated a favorabletolerability and predictable pharmacokineticprofile when givenorally.[14] The clinical benefit andsafety profiles of gefitinib were evaluatedin a recently reported multicenterphase II study in patients withmetastatic breast cancer.[15] Gefitinibwas administered at a dose of 500 mgonce daily until disease progression,intolerable toxicity, or consent withdrawal.Notably, there were no previoustreatment restrictions, and studyparticipants were not screened for thetarget or target aberrations. The studyend point was the clinical benefit rate,defined as the sum of the responserate and the rate of stable disease for6 months. Of the 63 patients in thetrial, 27 (43%) had tumors that wereestrogen-dependent, and 17 (27%) hadtumors that demonstrated erbB2 over-

• expressionby immunohistochemistrystaining.Treatment was discontinued in 5%of patients because of treatmentrelatedside effects, and four patientswere able to continue treatment aftera dose reduction to 250 mg daily.Grade 3/4 toxicity, mainly grade 3diarrhea, rash, or nausea and vomitingdeveloped in approximately 25%of the patients. One patient achieveda partial response, and two patientshad stable disease for an excess of6 months, yielding a clinical benefitrate of 4.8%. An additional six patientshad stable disease for up to6 months. The median time to progressionwas 57 days, and about 42%of patients reported diminished painduring therapy. Objective evidence ofactivity using a rigid definition waslow in this heavily pretreated population.However, a considerable proportionof patients (14.3%) achieveda partial response or maintained stabledisease for up to 6 months, andtherefore, may have derived benefitfrom this therapy.
• Erlotinib-Another agent that hasbeen studied in women with advancedbreast cancer is erlotinib. Much likegefitinib, erlotinib is orally active andwas well tolerated in phase I trials.[1,16] An open-label phase II trialof erlotinib in metastatic breast cancerwas recently completed.[17] Twocohorts of patients were accrued tothis study. The first cohort of 47 patientswas required to have receivedprior therapy with an anthracycline, ataxane, and capecitabine (Xeloda).The second cohort of 22 patients merelyhad to have had tumor progressionduring chemotherapy. Again, studyparticipants were not prospectivelyscreened for erbB1 overexpression.Erlotinib was administered at 150 mgonce daily until tumor progressionwith dose reduction permitted fortreatment-related side effects.In the first cohort, one patientachieved a partial response, and twoadditional patients had stable disease.In the second cohort, no objectiveresponses were observed, but one patientexhibited stable disease. Treatment-related side effects includedacneiform rash, diarrhea, asthenia, andnausea. Correlative studies demonstratedthat only 12% of patients hadoverexpression of erbB1. This suggeststhat an insufficient number ofpatients may have had the target tovalidly test this agent.
• Study Validity-The modest clinicalbenefit seen in these phase II stud-ies of the erbB1 tyrosine kinase inhibitorslikely reflects the indiscriminatetreatment of unscreened tumorsthat may or may not possess the appropriatetarget or determinants forresponse. The importance of appropriateidentification of patients whoare most likely to respond to a targetedapproach is well illustrated in thesuccess of trastuzumab in breast cancer.The survival benefits seen withtrastuzumab therapy would not havebeen appreciated if patients had notbeen screened before treatment foroverexpression of erbB2, the principaltarget of the drug.Equally important is the appropriateselection of end points for phase IIstudies, ie, those that will allow theappreciation and quantification of tumorgrowth delay, the predominantbenefit of erbB-targeted therapeuticsnoted in preclinical studies. Therefore,both the identification of predictivebiomarkers and a careful trialdesign are needed to ensure that theusefulness of erbB-targeted therapyis correctly assessed.
• New Directions in Research-More recently, attention has focusedon evaluating the feasibility and efficacyof a multitargeted approach. Thecombination of trastuzumab anderbB1 inhibitors and the dual administrationof endocrine therapy anderbB1 inhibitors are subjects of ongoingclinical trials in breast cancer. Inaddition, the irreversible, pan-erbBtyrosine kinase inhibitor CI-1033, theirreversible erbB1/erbB2 tyrosine kinaseinhibitor EKB-569, and thereversible erbB1/erbB2 tyrosine kinaseinhibitor GW572016 are undergoingclinical evaluation.[18-24] Therelative merits of these mechanismswill be better understood followingtrials of CI-1033, EKB-569, andGW572016 in relevant tumor types.The rationale for the developmentof irreversible tyrosine kinase inhibitorssuch as CI-1033 and EKB-569was, in part, the higher concentrationsof erbB inhibitors required tocontinuously block erbB1 phosphorylationin intact cells where intracellularadenosine triphosphate (ATP)concentrations are higher. The approximately80% homology between theerbB1 and erbB2 tyrosine kinase hasallowed the generation of these receptortyrosine kinase inhibitors withactivity in multiple erbB receptor families.Such agents have potential inpatients who are resistant to trastuzumab,as compensatory signaling byother erbB receptors may contributeto trastuzumab resistance.CI-1033 and EKB-569 are comprisedof chemical moieties that formcovalent bonds with the receptor tyrosinekinase domain, resulting in irreversiblereceptor binding andsustained inhibition of tyrosine kinasein vitro. This feature may also circumventdrug-binding competitiondue to high intracellular ATP concentrations.In addition, irreversiblecompounds require that plasma concentrationbe attained only longenough to briefly expose the receptorsto drug, which would then permanentlysuppress kinase activity.This process is in contrast to reversibleerbB tyrosine kinase inhibitorsthat require adequate plasma concentrationsand/or agents with relativelylong half-lives to keep the targetsuppressed.[1]CI-1033 binds irreversibly withinthe ATP-binding pocket of erbB tyrosinekinase and inhibits both activationand downstream signalingemanating from erbB1, erbB2, erbB3,and erbB4. In preclinical models, CI-1033 has been shown to inhibit erbB1phosphorylation in A341 carcinomaand MDA-MB-453 human breast carcinomacells and the growth of severalhuman tumor xenografts.[1,18,19]The results of studies of long-termadministration of CI-1033 indicate thatit maintains tumor suppression forextended periods without the emergenceof drug resistance.Like other erbB1 inhibitors, CI-1033 has demonstrated synergy withother therapeutic modalities. For example,it enhances the cytotoxic effectsof the topoisomerase inhibitors,SN-38 and topotecan (Hycamtin) invitro, possibly interfering with a relevantdrug-resistance mechanism.[1]Synergistic in vitro growth inhibitionof the erbB1-overexpressing cell lineA341 has also been demonstrated withCI-1033 and cisplatin.[19,20] Thisenhanced chemosensitivity was shownnot to be the result of inhibition ofDNA repair of cisplatin-DNA adducts,and it has been proposed that blockageof erbB signaling by CI-1033 enablescisplatin to inhibit key genesrequired for cell survival.In phase I studies, when CI-1033was administered as a single oral doseweekly for 3 out of 4 weeks and dailyfor 7 days every 3 weeks, the mostcommon toxicities were mild-to-moderatevomiting, diarrhea, and acneiformrash.[21,22] Antitumor activityhas also been observed, with one partialresponse and stable disease in 30%of patients including one with heavilypretreated breast cancer.[22] Furtherclinical development of this agent isongoing for patients with erbB-overexpressingadvanced breast cancer.EKB-569 also binds covalently andirreversibly to erbB1. Consistent withits ability to irreversibly bind to erbB1and erbB2, inhibition of receptorphosphorylation is sustained far longerthan are plasma levels of the compound.[1,23] Phase I evaluations ofEKB-569 administered continuouslyonce daily and for 3 weeks every4 weeks have been completed, andphase II studies of this agent areongoing.The agent GW572016 inhibitserbB1 and erbB2 tyrosine kinase in areversible manner. This drug has demonstratedpotent inhibition of tumorgrowth in vitro and appears selectivefor tumor cells relative to normal cells.In vivo, GW572106 has antitumoractivity against erbB2-overexpressingbreast carcinoma xenografts.[24] Clinicalevaluation of GW572016 administeredon a once-daily continuousschedule is ongoing in breast cancer.In addition, combination studies withother cytotoxic agents (such as capecitabine)are in progress.

Targeting theRas/Raf/MAPK Pathway

The Ras proteins are guanine nucleotide-binding proteins that play apivotal role in the control of normaland transformed cell growth. Followingstimulation by several growthfactors and cytokines, Ras activatesmultiple downstream effectors. TheRas/mitogen-activated protein kinase(Ras/MAPK) pathway plays animportant role in breast cancer(Figure 2).[25]Although

ras

is functionally mutatedin < 5% of breast cancers, anupregulation of the classic mitogenicRas/Raf/MAPK cascade occurs, stimulatedby overexpression or amplificationof oncogenic proteintyrosine kinase activity (eg, erbB2 orerbB1).[26] Phospholipase-C, one ofthe signaling proteins activated by receptordimerization of activated erbB1and erbB2 enhances Ras activitythrough its SH3 domain.[27] In addition,the adaptor protein Grb2 thatlinks protein tyrosine kinases to Rasand is overexpressed in breast cancer,may amplify signaling through the Raspathway in response to growthfactors.[28]The amplification of Ras signalingas a result of overexpression of theseoncogenes and intermediate signalingmolecules leads to increased stimulationof downstream effectormolecules including phosphatidylinositol3-kinase (PI3K) and proteinkinase B (Akt). Such oncogenic activationnot only confers a proliferativeand survival advantage to cancer cellsbut also supports tumor growththrough its proangiogenic effect.

Farnesyl Transferase Inhibitors

The Ras pathway may be targetedthrough the inhibition of farnesylation.This key step in the posttranslationalmodification of Ras is necessaryfor membrane localization and function.Initial studies of farnesyl transferaseinhibitors (FTIs) suggested thatthese agents selectively inhibit theanchorage-independent growth of rastransformedcells and reverse thetransformational phenotype of rasmutatedcells.[26] Recently, the roleof Ras proteins in mediating the antitumoreffects of FTIs has become lesscertain.FTIs have demonstrated insufficientactivity in tumors with K-

ras

mutations such as pancreas and colorectalcancers, presumably becauseanother prenylating enzyme, geranylgeranyltransferase, can alternativelyprenylate or activate K-

ras

. Inaddition, FTIs have demonstrated antiproliferativeactivity in tumor cell

lines with wild-type Ras, suggestingthat mechanisms other than inhibitionof Ras farnesylation may beinvolved.[29] The prevailing explanationfor the activity of FTIs in tumorssuch as breast cancer-whichrarely involves

ras

mutations-includes the fact that FTIs preventsignaling through wild-type Rascaused by upstream aberrations (eg,erbB1, erbB2) or that they inhibit farnesylation(activation) of other criticalproteins.

• Clinical Trials-Various farnesyltransferase inhibitors have been evaluatedin phase I/II clinical trials. Theseinclude R115777, SCH66336, andBMS 214662.[26,30-34] In addition,interest has been generated in optimizingthe use of FTIs by combiningthem with cytotoxic agents. Certainlythe synergy between cytotoxic agents(particulary taxanes) and FTIs observedin breast cancer cell lines withwild-type Ras supports this approach.[30] The prinicipal toxicitiesencountered with FTIs includeschedule-dependent myelosuppression,gastrointestinal effects, and fatigue.Although many of the observedtoxicities are common, certain sideeffects are unique and may be structurallyrelated. Peripheral neuropathyis unique to R115777, whereas transaminitisappears to be encounteredmore often with BMS 214662.The first phase II study of an FTIin breast cancer was conducted usingR115777.[32] Preliminary results indicatethat R115777 has single-agentactivity in advanced breast cancer,with a clinical benefit rate of 25%. Ithas also been evaluated in combinationwith chemotherapy. In a phase Istudy in patients with solid tumors,R115777 was combined with docetaxel.[33] Of 15 patients with breastcancer, 1 achieved a complete response,and 2 achieved partial responses.The dose-limiting toxicity wasmostly febrile neutropenia, and thenonhematologic toxicities were diarrhea,fatigue, and vomiting. Nodiscernable pharmacokinetic interactionbetween the two drugs wasdocumented.The combination of R115777 andcapecitabine has also been evaluated ina phase I trial.[34] Diarrhea and handfootsyndrome were the dose-limitingtoxicities, and partial responses were

• seen in various malignancies includingbreast cancer. More recently, theconcurrent inhibition of both erbB2and Ras signaling is being studied inbreast cancer. The rationale for theuse of this combination is that inhibitionof abnormal Ras expression andnormal Ras signaling may enhancethe growth inhibitory effects of trastuzumabin erbB2-expressing tumorcells.Raf Inhibitors
  Downstream effectors of Ras, particularlyRaf-1, are also being characterizedand targeted with a variety oftherapeutic agents. The Raf family iscomposed of three related serine/threonineprotein kinases (Raf-1, A-Raf, andB-Raf) that act, in part, as downstreameffectors of the Ras pathway.[35] ActivatedRas interacts directly with theamino-terminal regulatory domain ofthe Raf kinase, resulting in a cascadeof reactions that include direct activationof MEK. Like mutated ras, constitutivelyactive mutated raf cantransform cells in vitro. However, Rafmay play a broader role in tumorigenesisbecause it can be activated by proteinkinase C-alpha and promotesexpression of the multidrug resistancegene MDR-1.[35] By targeting Raf,one can inhibit mutated raf and upstreamsignals coming in from mutantras and growth factor receptors suchas erbB1 and erbB2. Raf inhibitors currentlyunder evaluation include the c-Raf-1 antisense oligonucleotide ISIS5132 (CGP 69846A) and BAY 43-9006, a small molecule inhibitor of Rafkinase.[35-40]ISIS 5132 inhibits both the expressionof c-raf messenger RNA and theproliferation of cancer cell lines invitro.[35] Evidence also shows thatthis agent augments the cytotoxic effectsof the chemotherapeutic agentscarboplatin and paclitaxel.[40] Phase Istudies have evaluated the safety ofescalating doses of ISIS 5132 administeredin three schedules: a 2-hourIV infusion three times per week for3 consecutive weeks; a continuous IVinfusion for 3 weeks in each 4-weekperiod; and a weekly 24-hour infusion.[35] Both the 2-hour and 3-weekcontinuous IV infusion schedules werewell tolerated, with the most commontoxicities being fever, fatigue, and atransient prolongation of activatedpartial thromboplastin time. Reductionsin c-Raf-1 expression relative topretreatment were observed in the peripheralblood mononuclear cells ofsome patients.[35,36] Phase II studiesof ISIS 5132 in relevant tumortypes are under way.BAY 43-9006 is a small-moleculeRaf-1 kinase inhibitor that has significantdose-dependent antitumor activityin a variety of cancer cell lines.[35]In xenograft models, an additive antitumoreffect was observed when BAY43-9006 was combined with gemcitabine(Gemzar), irinotecan (CPT-11,Camptosar), or vinorelbine (Navelbine).[38] The results of a phase Istudy of BAY 43-9006 in patients withsolid tumors including advancedbreast cancer were reported recent-ly.[39] At a twice-daily dose of800 mg, the dose-limiting toxicity wasdiarrhea. Other clinical toxicities includedfatigue and skin rash (erythema,desquamation). In phase I/II trials,responses have been seen in colorectal,hepatocellular, and renal cancers.Further studies of BAY 43-9006 asa single agent and in combinationwith chemotherapy are in progress.In addition, BAY43-9006 is beingevaluated in a unique treatmentdiscontinuationstudy.MEK Inhibitors
  Another component of the signaltransduction pathway that has beentargeted recently is MEK. MEK1 andMEK2 are tyrosine kinases downstreamof Ras and Raf in the mitogenactivatedRas/Raf/MEK/ERK cascade,and they represent a crucial point ofconvergence that integrates input froma variety of protein kinases throughRas. A selective small-molecule inhibitorof MEK currently undergoingclinical evaluation is CI-1040. Inphase I studies, this agent was welltolerated, with fatigue, rash, anddiarrhea the commonly reportedtoxicities.[35,41]Ras, Raf, and MEK have emergedas key protein kinase targets for anticancerdrug design, and preliminaryresults with these agents are encouraging.Further research should focuson identifying characteristics that predictantitumor activity with theseagents. In particular, sensitive and reliablemethods to determine the molecularphenotype of tumors that arelikely to be sensitive to agents thattarget components of the Ras/Raf/MEK pathway need to be developedand validated in clinical trials.Targeting the PI3K/AkTPathway and mTORThe molecular target of rapamycin(mTOR), a downstream effector ofthe PI3K/Akt signaling pathway, mediatescell survival and proliferationand is a prime strategic target in thedevelopment of anticancer therapeutics(Figure 3). By targeting mTOR,the immunosuppressant and antiproliferativeagent rapamycin inhibitssignals required for cell-cycle progression,cell growth, and proliferation.Both rapamycin and novel rapamycinanalogs with more favorable pharmaceuticalproperties (such as CCI-779,RAD001, and AP23573) are highlyspecific inhibitors of mTOR.[35,42]In essence, these agents gain functionby binding to the immunophilinFK506 binding protein 12, and theresulting complex inhibits the activityof mTOR. Because mTOR activatesboth the 40S ribosomal proteinS6 kinase and the eukaryotic initiationfactor 4E-binding protein-1,rapamycin-like compounds block theaction of these downstream signalingelements, and result in cell-cycle arrestin the G1 phase.Rapamycin and its analogs alsoprevent cyclin-dependent kinase activation,inhibit retinoblastoma proteinphosphorylation, and acceleratethe turnover of cyclin D1, leading toa deficiency of active cyclin-dependentkinase 4/cyclin D1 complexes-all of which potentially contribute tothe prominent inhibitory effects ofrapamycin at the G1/S boundary ofthe cell cycle.[42] Moreover, rapamycinand its analogs have demonstratedimpressive growth inhibitoryeffects against a broad range of humancancers, including breast cancer,in both preclinical and earlyclinical evaluations.[42,43]In breast cancer cells, PI3K/Aktand mTOR pathways seem to be criticalfor the proliferative responsesmediated by the EGFR, the insulingrowth factor receptor, and the estrogenreceptor.[35] Breast tumors,particularly hormone-independentcancers, often harbor genetic alterationsin the PI3K/Akt pathway andexhibit high levels of constitutive Aktactivity. The loss of PTEN suppressorgene function has also been linkedto Akt activation. Although mutationsof PTEN occur in less than 5% ofbreast cancers, a recent report suggeststhat the complete lack of PTENprotein in breast cancers with hemizygousdeletions of PTEN is notuncommon.[44] Therefore, the developmentof inhibitors of mTOR andrelated pathways is a rational therapeuticstrategy for breast cancer.CCI-779
  The water-soluble rapamycin esterCCI-779 was selected for developmentas an anticancer agent based onits prominent antitumor profile andfavorable pharmaceutical and toxicologiccharacteristics in preclinicalstudies.[42] In vitro, the breast cancercell lines BT-474, SK-BR-3, andMCF-7 have demonstrated extraordinarysensitivity to CCI-779.[45] Interestingly,elements of the PI3K/Aktpathway in these breast cancer celllines appear to be constitutively overactive,possibly due to upstream activatingaberrations involving erbB1and/or the estrogen receptor.[44]Similar results were reported byYu et al,[46] who demonstrated thatthe preponderance of breast cancercell lines found to be remarkably sensitiveto CCI-779 were estrogendependent,overexpressed erbB2, and/or had PTEN deletions, whereas resistantbreast cancer cell lines lackedthese features. In this study, the correlationbetween activation of the Aktpathway and sensitivity to CCI-779was strong.In vivo studies of CCI-779 administeredon intermittent schedules demonstratedantitumor activity andresolution of biologic evidence of immunosuppressionwithin 24 hours. Inconsideration of the possibility thatcontinuous drug administration maypredispose patients to immunosuppression,two intermittent CCI-779schedules were initially selected forclinical development: a 30-minute IVinfusion administered weekly and a30-minute IV infusion administereddaily for 5 days every 2 weeks. Theprincipal toxicities of CCI-779 on bothschedules included dermatologic toxicity,myelosuppression, reversibleelevations in liver function tests, andasymptomatic hypocalcemia.[47-50]Further evidence that CCI-779may possess notable antitumor activityin patients with advanced breastcarcinoma was provided from a multicenterEuropean phase II study. Atotal of 109 patients with metastaticbreast cancer that had progressed ontaxanes and anthracyclines wereenrolled in this study.[51] CCI-779was administered at two IV doses(75 and 250 mg) on a weekly schedule.At the time of the preliminaryreport, 106 patients had been treated.Clinical benefit was observed in 49%of patients, with 1 complete response,8 partial responses, and 43 patientswith stable disease lasting ≥ 8 weeks.Activity was seen at both doses, andthe principal toxicities were asthenia,leukopenia, thrombocytopenia, transaminitis,hypercholesterolemia, hyperglycemia,stomatitis, depression,and somnolence.These encouraging preliminary resultshave prompted further studies ofCCI-779 in breast cancer. Becausehormone resistance has been associatedwith activation of the PI3K andmTOR pathways, studies combiningCCI-779 with hormonal agents arealso in progress, including a randomizedphase II study evaluating thefeasibility and activity of CCI-779and the aromatase inhibitor letrozole(Femara) in patients with estrogendependentbreast cancer. In addition,on the basis of preclinical data suggestingsynergy between CCI-779 andchemotherapy,[52] combination studieswith cytotoxic agents are beingplanned.RAD001 and AP23573
  RAD001, an orally bioavailablehydroxyethyl ether of rapamycin, andAP23573, a nonprodrug rapamycinanalog, are also undergoing early clinicalevaluations.[35,53-57] Bothagents have demonstrated impressiveantiproliferative activity against awide variety of tumor cell lines invitro and in vivo.[53-57] In phase Istudies, RAD001 was well toleratedwith only mild degrees of anorexia,fatigue, rash, mucositis, headache,hyperlipidemia, and gastrointestinaldisturbance.[57] Further phase I/IIstudies with RAD001 and phase I studieswith AP23573 have recently beeninitiated.Inhibiting Tumor AngiogenesisThe development of antiangiogenicdrugs as a novel strategy in cancertreatment is based on preclinical evidencethat angiogenesis plays an integralrole in tumor growth, progression,and metastasis (Table 2). In breastcancer, both in vitro experiments andclinical studies suggest that tumorprogression and metastases aredependent on angiogenesis.[58-60] Asignificant correlation between thedegree of intratumoral microvesseldensity and the probability of the formationof metastases has been observed,and intratumoral microvesseldensity has been shown to be an independentprognostic marker in patientswith invasive breast cancer.[61,62]Invasive human breast cancers expressmultiple angiogenic factors.Vascular endothelial growth factor(VEGF) is among the most specificand potent, inducing endothelial cellmigration, invasion, and in vitro formationof tubelike structures at picomolarconcentrations.[63] VEGFreceptors are expressed almost exclusivelyon endothelial cells, andthrough VEGF binding and dimerizationof the receptors, their intrinsicintracellular tyrosine kinase and downstreamsignaling functions areactivated.VEGF is capable of inducing vascular permeability, which allows plasmaproteins to diffuse into the interstitiumand form a lattice network thatacts as a substrate for endothelial andtumor cell growth. In addition, VEGFacts as an endothelial cell survivalfactor, with experimental evidencesuggesting that inhibition of VEGFactivity induces endothelial cell apoptosis.[63-65] Given the importanceof VEGF in tumor growth and metastases,several strategies have beendeveloped to inhibit this pathway.Bevacizumab
  Bevacizumab (Avastin), a recombinanthumanized anti-VEGF neutralizingantibody, has entered clinicaltrials and recently received fast-trackstatus from the US Food and DrugAdministration. The antibody blocksthe binding of all VEGF isoforms tothe receptors and inhibits the biologicactivities of VEGF as measured byassays for endothelial mitogenesis,vascular permeability, and in vivo angiogenesis.[66] Bevacizumab wasevaluated in a phase I study in 25patients with advanced solid tumors,[67] and like other antibodies,was delivered intravenously. Theserum half-life of this agent wasapproximately 21 days, and at doses≥ 0.3 mg/kg, it provided completesuppression of free serum VEGF. Thetoxicities that occurred during the firstseveral hours after infusion of the antibodywere limited to grade 1/2 headache,nausea, asthenia, and low-gradefever, which occurred in a minorityof patients. Intratumoral hemorrhagewas reported in two patients treated atthe 3 mg/kg dose level.The favorable antitumor activityof antibodies to VEGF, combined withcytotoxic chemotherapy (doxorubicin),has been demonstrated in MCF-7breast cancer cell lines.[68] In addition,phase I studies have assessed thefeasibility of combining bevacizumabwith chemotherapy. In a recently completedphase I study,[66] bevacizumabat 3 mg/kg/wk was combined withthree standard chemotherapy regimens:doxorubicin at 50 mg/m² every4 weeks, carboplatin at an area underthe concentration-time curve (AUC)of 6 plus paclitaxel at 175 mg/m² every4 weeks, and fluorouracil (5-FU)

• at 500 mg/m² with leucovorin at20 mg/m² weekly. This study demonstratedthat bevacizumab could be deliveredsafely in combination withchemotherapy at doses associated withVEGF blockade without synergistictoxicity.Bevacizumab was recently evaluatedin patients with previously treatedmetastatic breast cancer.[69] This twoinstitutionphase II study enrolled 75patients in three cohorts representingthree dose levels: 3, 10, and 20 mg/kgevery other week. Overall, 17% ofpatients responded or achieved stabledisease at 5 months, and three patientscontinued therapy withoutdisease progression for more than12 months. The agent was generallywell tolerated, with several patientsdeveloping mild hypertension andproteinuria. No episodes of significantbleeding were noted.These encouraging results led totwo additional phase III studies ofbevacizumab in advanced breast cancer.The first study[70] comparedcapecitabine, with or without bevacizumab,in women with metastaticbreast cancer who had progressed despiteprior therapy with both an anthracyclineand a taxane. The studydemonstrated a doubling of the responserate from 19% to 30% in patientstreated with the combination ofbevacizumab and capecitabine. However,the responses were not durableand did not have an impact on progression-free survival, the major endpoint of this trial. As in prior studies,the main toxicities of bevacizumabwere modest degrees of hypertensionand low-grade bleeding.The failure of this trial to demonstratean impact on survival may bedue to the advanced disease of thepatients, which has led to the evaluationof bevacizumab in patients withless advanced disease. One such multicenterphase III trial (E2100), initiatedby the Eastern CooperativeOncology Group, is randomizing patientswith newly diagnosed metastaticbreast cancer to treatment witheither paclitaxel as a single agentadministered on a weekly schedule orthe combination of paclitaxel andbevacizumab.VEGF Tyrosine Kinase Inhibitors
  An alternative strategy for inhibitingVEGF activity is through selectiveinhibition of membrane receptortyrosine kinases. These competitivetyrosine kinase inhibitors localize tothe ATP-binding site and inhibit phosphorylationand activation of downstreamsignaling following binding ofthe VEGF receptor. In human tumorcell line xenografts, these agents elicitsubstantial delay of growth in abroad spectrum of tumors.[71]The development of SU5416, asmall-molecule VEGF tyrosine kinaseinhibitor, has been halted because ofits lack of target specificity and unfavorablepharmaceutical properties.However, other similar VEGF tyrosinekinase inhibitors such as CP-547,632and PTK787/ZK222584 remain inclinical trials.[72-74] In addition,agents that inhibit multiple tyrosinekinase pathways are being activelyexplored. These include ZD6474 andPKI 166, inhibitors of both VEGFreceptor tyrosine kinase and EGFRtyrosine kinase, and SU11248, asmall-molecule, multitargeted receptorkinase inhibitor of platelet-derivedgrowth factor (PDGF), VEGF, KIT,and FLT3.[73,75-78]
• ZD6474-ZD6474 has also exhibitedantitumor activity in a variety ofhuman cancer cells lines and in xenograftmodels.[76] In addition, invitro studies have demonstrated anadditive effect on inhibition of tumorgrowth when ZD6474 was combinedwith taxanes.[76] In phase I studies,ZD6474 administered on a daily oraldosing schedule was generally welltolerated, with diarrhea and rash asthe main toxicities. Asymptomaticprolongation of the QT interval wasalso observed in 7 of the 49 patientsincluded in the study.[73] This agentis now being evaluated in phase IItrials in patients with metastatic breastcancer.
• SU11248-Clinical developmentof SU11248 is also under way. Thisagent has exhibited broad and potentantitumor activity in preclinical studies,causing regression, growth arrest,or substantially reduced growth ofvarious established xenografts.[77,78]A recent study assessed the safety andtolerability of SU11248 administereddaily for either 2 or 4 weeks followedby 2 weeks' rest in patients with advancedsolid tumors.[79] The mostfrequent adverse events were constitutional(fatigue, asthenia), gastrointestinal(nausea, vomiting, anddiarrhea), and hematologic (neutropenia,thrombocytopenia). Fatigue/asthenia, which was readily reversibleupon discontinuation of the drug,proved to be the dose-limiting toxicity.The clinical responses observed inthis phase I study included 1 partialresponse and 12 patients with stabledisease.

Modulating Apoptosis

Antiapoptotic mutations significantlycontribute to the malignant phenotypeby allowing the cell to surviveunder conditions that would normallytrigger its demise. The bcl-2 geneproduct has been implicated in thegrowth and development of a varietyof tumors including breast cancer andhas the potential to confer chemoresistanceand radioresistance to establishedtumors.[80-82] The Bcl-2protein dimerizes both with itself andwith other members of the Bcl-2 family(Bcl-xL, Bax, and Bcl-xS), andthe interaction of these protein dimersinfluences sensitivity to apoptoticstimuli.

Bcl-2 Antisense Therapy

Preclinical data demonstrate thatBcl-2 antisense therapy with oblimersen(G3139, Genasense) has antitumoreffects against breast cancer.[83]Treatment with oblimersen is well toleratedand leads to a reduction in intratumoralBcl-2 protein levels.[80,84]Oblimersen has been combined withcytotoxic chemotherapy.[85,86] Inhuman breast cancer xenograft models,the combination of oblimersen anddocetaxel produced an enhanced antitumoreffect, leading to durable tumorregression. These preclinical dataprovided the basis for evaluation ofthis combination in breast cancer. In arecent phase I trial, oblimersen andweekly docetaxel were tolerable andresulted in a tumor response in two ofthe five patients with advanced breastcancer included in this study.[85]These initial data support the furtherdevelopment of this combination formetastatic breast cancer.

TNF-Related Apoptosis Ligand

Mutations in survival factors at thecell surface including death receptorsof the TNF receptor family may alsolead to dysregulation of apoptosis. TheTNF-related apoptosis ligand (TRAIL)is a member of the TNF ligand superfamilywith high homology to theFas/Apo1 ligand. Although the biologicfunctions of TRAIL remain incompletelydefined, strong evidence ofTRAIL's ability to trigger apoptosisin numerous cancer cell lines supportsa physiologic role for TRAIL in mediatingapoptosis.TRAIL mediates apoptosis throughtwo death receptors, TRAIL-R1 (deathreceptor 4, DR4) and TRAIL-R2(death receptor 5, DR5). These receptorswere isolated and named basedon the presence of a death domain intheir cytoplasmic tails that is capableof initiating a cascade of caspase activationand ultimate cell death. Resistanceto TRAIL-induced apoptosis hasbeen demonstrated via in vitro studiesof breast cancer cell lines, with inactivatingmutations in the TRAIL-R1and -R2 genes being particularly important.[87,88]Therapies targeting TRAIL-R1 andTRAIL-R2 are under development.One such agent, TRM-1, a fully humanizedagonist monoclonal antibodyto the TRAIL-R1 receptor, is currentlyin phase I trials. TRM-1 has beenshown to induce apoptosis in cancercell lines, and investigators have predictedthat it will display activitiessimilar to the TRAIL-R1 agonisticligand.[89]

Role of HistoneDeacetylase Inhibitors

Another attractive target for interventionin breast cancer is histoneacetylation. The acetylation anddeacetylation of histones plays animportant role in the regulation ofgene expression. Hypoacetylation ofhistones is associated with a condensedchromatin structure that resultsin the repression of gene transcription,whereas acetylated histones areassociated with a more open chromatinstructure and activation oftranscription.Histone deacetylase and the familyof acetyl transferases are involved indetermining the acetylation of histones.Inhibition of histone deacetylaseincreases histone acetylation, which,in turn, leads to the transcription of afew genes whose expression causesinhibition of tumor growth.[90-92]The mechanism of selectivity of geneexpression is currently not understoodbut is an area of intense study. Inhibitorsof histone deacetylase have beenshown to induce growth arrest, differentiation,and apoptosis in a varietyof tumors, including human breastcancer cell lines.[90,93]In preclinical studies, treatmentwith LAQ824, a hydroxamic acid analoginhibitor of histone deacetylation,led to downregulation of HER2in human breast cancer SKBR-3,BT-474, and MB-468 cells and sensitizedthese cells to the apoptotic effectsof trastuzumab and polymerizing agents(docetaxel and epothilone B).[93]Histone deacetylase inhibitors causeacetylated histones to accumulate inboth tumor and peripheral circulatingmononuclear cells, and this accumulationhas been used as a markerof biologic activity. Several drugs thatinhibit histone deacetylation are beingevaluated in phase I/II clinical trialsas single agents or in combinationwith cytotoxic chemotherapy.[90-95]These include suberoylanilide hydroxamicacid, pyroxamide, depsipeptide,MS-275, CI-994, andLAQ824.Results of a phase I trial of suberoylanilidehydroxamic acid in heavilypretreated patients with hematologicmalignancies were recently reported.[95] The major toxicities observedincluded fatigue, diarrhea, anorexia,dehydration, and myelosupression.Among the clinical responses in thisrefractory group of 29 patients was areduction in measurable tumor (seenin 6 patients). Encouraging data frompreclinical and phase I studies haveprompted further evaluation of thisclass of agents in patients with metastaticbreast cancer.

Conclusions

Improvements in our understandingof the molecular events that mediatetumor growth and metastases haveenabled the design and developmentof novel therapeutic agents that specificallytarget intrinsic aberrancies incancer cells. New combinations ofcytotoxic chemotherapy and targetedagents are being explored in breastcancer, generating much excitementand expectation that these innovativetherapies will improve the outcomeof patients with this disease. The increasinguse of molecular profilingtechniques should give us the opportunityto select the most active agentsfor a given tumor and thereby reduceunnecessary side effects. In addition,genomics and proteomics provide uswith the potential for discovering thehidden targets of our current therapeuticarsenal.In order to improve the efficiencyof the evaluation process and increasethe probability of success, the futuredevelopment of molecularly targetedagents needs to incorporate assays toassess the suitability of the patientpopulation, the target, and the effectsof the target. Such assays may lead toenrichment of early proof-of-principlestudies in patients who are mostlikely to benefit from these agents orwho might achieve responses that areeasy to detect in nonrandomized trials.New initiatives in clinical trialdesign including novel correlativeimaging, alternative end points suchas time to progression, and novel approachessuch as randomized discontinuationschemes, are needed todetermine the future utility of theseagents.

Disclosures:

The author(s) have no significant financial interest or other relationship with the manufacturers of any products or providers of any service mentioned in this article.

References:

1.

Rowinsky EK: Signal transduction inhibitors.Horizons in Cancer Therapeutics 2:3-35, 2001.

2.

Walker RA, Dearing SJ: Expression ofepidermal growth factor receptor mRNA andprotein in primary breast carcinomas. BreastCancer Res Treat 53:167-176, 1999.

3.

Morris C: The Role of EGFR-directedtherapy in the treatment of breast cancer.Breast Cancer Res Treat 75 (suppl 1):S51-S55, 2002.

4.

Fox SB, Leek RD, Smith K, et al: Tumorangiogenesis in node-negative breast carcinomas-relationship with epidermal growth factorreceptor, estrogen receptor, and survival.Breast Cancer Res Treat 29:109-116, 1994.

5.

Klijn JG, Look MP, Portengen H, et al:The Prognostic value of epidermal growth factor(EGF-R) in primary breast cancer: Resultsof a 10-year follow-up study. Breast CancerRes Treat 29:73-83, 1994.

6.

McClelland RA, Barrow D, Madden TA,et al: Enhanced epidermal growth factor receptorsignaling in MCF7 breast cancer cellsafter long-term culture in the presence of thepure antiestrogen ICI 182,780 (Faslodex). Endocrinology142: 2776-2788, 2001.

7.

Osaki A Toi M, Yamada H, et al: Prognosticsignificance of co-expression of c-erbB-2 oncoprotein and epidermal growth factorreceptor in breast cancer patients. Am J Surg164:323-326, 1992.

8.

Harris AL, Nicholson S, Sainsbury JR, etal: Epidermal growth factor receptors in breastcancer: Association with early relapse anddeath, poor response to hormones and interactionswith neu. J Steroid Biochem 34:123-131,1989.

9.

Ciardiello F, Caputo R, Bianco R, et al:Antitumor effect and potentiation of cytotoxicdrugs activity in human cancer cells by ZD-1839 (Iressa), an epidermal growth factor receptor-selective tyrosine kinase inhibitor. ClinCancer Res 6:2053-2063, 2000.

10.

Normanno N, Campiglio M, SomenziG, et al: Cooperative inhibitory effect ofZD1839 (Iressa) in combination with trastuzumab(Herceptin) on human breast cancercell growth. Ann Oncol 13:65-72, 2002.

11.

Moasser MM, Basso A, Averbuch SD,et al: The tyrosine kinase inhibitor ZD1839(Iressa) inhibits HER2-driven signaling andsuppresses the growth of HER2-overexpressingtumor cells. Cancer Res 61:7184-7188,2001.

12.

Massarweh S, Shou J, Mohsin SK, et al:Inhibition of epidermal growth factor/HER2receptor signaling using ZD1839 (Iressa) restorestamoxifen sensitivity and delaysresistance to estrogen deprivation in HER2-overexpressing breast tumors (abstract 130).Proc Am Soc Clin Oncol 21:33a, 2002.

13.

Ciardiello F, Caputo R, Borriello G, etal: ZD1839 (Iressa), an EGFR-selective tyrosinekinase inhibitor, enhances taxane activityin bcl-2 overexpressing, multidrug-resistantMCF-7 ADR human breast cancer cells. Int JCancer 98:463-469, 2002.

14.

Ranson M, Hammond LA, Ferry D, etal: ZD1839, a selective oral epidermal growthfactor receptor-tyrosine kinase inhibitor, is welltolerated and active in patients with solid, malignanttumors: Results of a phase I trial. JClin Oncol 20:2240-2250, 2002.

15.

Albain K, Elledge R, Gradishar WJ, etal: Open-labeled phase II multicenter trial ofZD 1839 (Iressa) in patients with advancedbreast cancer (abstract 20). Breast Cancer ResTreat 76:S33, 2002.

16.

Hidalgo M, Siu LL, Nemunaitis J, et al:Phase I and pharmacologic study of OSI-774,an epidermal growth factor receptor tyrosinekinase inhibitor in patients with advanced solidmalignancies. J Clin Oncol 19:3267-3279,2001.

17.

Winer E, Cobleigh M, Dickler M, et al:Phase II multicenter study to evaluate the efficacyand safety of Tarceva (erlotinib; OSI-774) in women with previously treated locallyadvanced or metastatic breast cancer (abstract445). Breast Cancer Res Treat 76:S115, 2002.

18.

Slichenmyer WJ, Elliot WL, Fry DW:CI-1033, a pan-erbB tyrosine kinase inhibitor.Semin Oncol 28(5 suppl 16):S16; 80-85, 2001.

19.

Allen LF, Lenehan PF, Eiseman IA, etal: Potential benefits of the irreversible panerbBinhibitor, CI-1033, in the treatment ofbreast cancer. Semin Oncol 28(3 suppl 11):11-21, 2002.

20.

Gieseg MA, de Bock C, Ferguson LR,et al: Evidence for epidermal growth factorreceptor-enhanced chemosensitivity in combinationsof cisplatin and the new irreversibletyrosine kinase inhibitor CI-1033. AnticancerDrugs 12:683-690, 2001.

21.

Garrison MA, Tolcher A, McCreery H,et al: A phase 1 and pharmacokinetic study ofCI-1033, a pan-erbB tyrosine kinase inhibitor,given orally on days 1, 8, and 15 every 28days to patients with solid tumors (abstract283). Proc Am Soc Clin Oncol 20:72a, 2001.

22.

Shin DM, Nemunaitis J, Zinner RG, etal: A phase I clinical and biomarker study ofCI-1033, a novel pan-erbB tyrosine kinase inhibitorin patients with solid tumors (abstract324). Proc Am Soc Clin Oncol 20:82a, 2001.

23.

Wissner A, Brawner Floyd MB, RabindranSK, et al: Syntheses and EGFR andHER-2 kinase inhibitory activities of 4-anilinoquiniline-3-carbonitriles: Analogues of threeimportant 4-anilinoquinazolines currently undergoingclinical evaluation as therapeutic antitumoragents. Bioorg Med Chem Lett12:2893-2897, 2002.

24.

Xia W, Mullin RJ, Keith BR, et al:Anti-tumor activity of GW 572016: A dualtyrosine kinase inhibitor blocks EGF activationof EGFR/erbB2 and downstream Erk1/2and AKT pathways. Oncogene 21:6255-6263,2002.

25.

Sivaraman VS, Wang H, Nuovo GJ, etal: Hyperexpression of mitogen-activated proteinkinase in human breast cancer. J ClinInvest 99:1478-1483, 1997.

26.

Dy GK, Adjei AA: Farnesyltransferaseinhibitors in breast cancer therapy. CancerInvest 20(suppl 2):30-37, 2002.

27.

Kim MJ, Chang JS, Park SK, et al:Direct interaction of SOS1 Ras exchange proteinwith the SH3 domain of phospholipase Cgamma1. Biochemistry 39:8674-8682, 2000.

28.

Verbeek B, Adriaansen-Slot SS, RijksenG: Grb2 overexpression in nuclei and cytoplasmof human breast cells: A histochemicaland biochemical study of normal and neoplasticmammary tissue specimens. J Pathol183:195-203, 1997.

29.

Sepp-Lorenzino L, Ma Z, Rands E: Peptidomimeticinhibitor of farnesyl: Protein transferaseblocks the anchorage-dependent and-independent growth of human tumor cell lines.Cancer Res 55:5302-5309, 1995.

30.

Wang E, Casciano CN, Clement RP, etal: The farnesyl protein transferase inhibitorSCH66336 is a potent inhibitor of MDR1 productP-glycoprotein. Cancer Res 61:7525-7529,2001.

31.

Ryan DP, Eder JP, Supko JG, et al:Phase I clinical trial of the farynesyl transferaseinhibitor BMS-214662 in patients withadvanced solid tumors (abstract 720). ProcAm Soc Clin Oncol 19:185a, 2000.

32.

Johnston SRD, Hickish T, Houston S, etal: Efficacy and tolerability of two dosing regimensof R115777 (Zarnestra), a farnesyl proteintransferase inhibitor, in patients withadvanced breast cancer (abstract 138). ProcAm Soc Clin Oncol 2:35a, 2002.

33.

Piccart-Gebhart MJ, Branle F, de ValeriolaD, et al: A phase I, clinical and pharmacokinetic(PK) trial of the farnesyl transferaseinhibitor (FTI) R115777 + docetaxel: A promisingcombination in patients with solid tumors(abstract 318). Proc Am Soc Clin Oncol20:80a, 2001.

34.

Holden SN, Eckhardt S, Fisher M, et al:A phase I pharmacokinetic (PK) and biologicalstudy of the farnesyl tranferase inhibitor(FTI) R115777 and capecitabine in patients(Pts) with advanced solid malignancies (abstract316). Proc Am Soc Clin Oncol 20:80a,2001.

35.

Dancey J, Sausville EA: Issues andprogress with protein kinase inhibitors for cancertreatment. Nat Rev Drug Discov 2:296-313, 2003.

36.

Stevenson JP, Yao KS, Gallagher M etal: Phase I clinical/pharmacokinetic and pharmacodynamictrial of the c-raf-1 anti-senseoligonucleotide ISIS 5132 (CGP 69846A). JClin Oncol 17:2227-2236, 1999.

37.

Moore M, Hirte H, Oza A, et al: Phase Istudy of the raf-1 kinase inhibitor BAY 43-9006 in patients with advanced refractory solidtumors (abstracts 1816). Proc Am Soc ClinOncol 21:2b, 2002.

38.

Vincent P, Zhang X, Chen C, et al:Chemotherapy with the raf kinase inhibitorBAY 43-9006 in combination with irinotecan,vinorelbine or gemcitabine is well toleratedand efficacious in preclinical xenograft models(abstract 1900). Proc Am Soc Clin Oncol21:23b, 2002.

39.

Strumberg D, Bauer RJ, Moeller JG, etal: Final results of a phase I pharmacokineticand pharmacodynamic study of the raf kinaseinhibitor BAY 43-9006 in patients with solidtumors (abstract 121). Proc Am Soc Clin Oncol21:31a, 2002.

40.

Langdon S, McPhillips F, Mullen P, etal: Antisense oligonucletide (ISIS 5132) targetingof c-raf kinase in ovarian cancer models(abstract 833). Proc Am Soc Clin Oncol20:209a, 2001.

41.

LoRusso PM, Adjei AA, Meyer MB, etal: A phase I clinical and pharmacokineticevaluation of the oral MEK inhibitor, CI-1040,administered for 21 consecutive days, repeatedevery 4 weeks in patients with advancedcancer (abstract 321). Proc Am Soc Clin Oncol21:81a, 2002.

42.

Hidalgo M, Rowinsky ER: The rapamycin-sensitive signal tranduction pathway asa target for cancer therapy. Oncogene 19:6680-6686, 2000.

43.

Gibbons JJ, Discafani C, Peterson R, etal: The effect of CCI-779, a novel macrolideanti-tumor agent, on the growth of human tumorcells in vitro and in nude mouse xenograftin vivo (abstract 2000). Proc Am Assoc CancerRes 40: 301, 1999.

44.

Yakes FM, Chinratanalab W, Ritter CA,et al: Herceptin-induced inhibition of phosphatidylinositol-3 kinase and Akt is requiredfor antibody-mediated effects on p27, cyclinD1, and antitumor action. Cancer Res 62:4132-4141, 2002.

45.

CCI-779 investigational brochure. Collegeville,Pa, Wyeth Research, 2001.

46.

Yu K, Toral-Barza L, Discafani C, et al:mTOR, a novel target in breast cancer: Theeffect of CCI-779, an mTOR inhibitor, in preclinicalmodels of breast cancer. Endocr RelatCancer 8:249-258, 2001.

47.

Raymond E, Alexandre J, DepenbrockH, et al: CCI-779, a rapamycin analog withantitumor activity: A phase I study utilizing aweekly schedule (abstract 728). Proc Am SocClin Oncol 19:187a, 2000.

48.

Raymond E, Alexandre J, DepenbrockH, et al: CCI-779, an ester analogue of rapamycinthat interacts with PTEN/PI3k kinase pathways:A phase I study utilizing a weeklyintravenous schedule. Proceedings of the 11thNCI EORTC AACR Symposium on NewDrugs in Cancer Therapy (abstract 414). ClinCancer Res 6 :4549s, 2000.

49.

Hidalgo M, Rowinsky E, Erlichman C,et al: CCI-779, a rapamycin analog and multifacetedinhibitor of signal transduction: Aphase I study (abstract 726). Proc Am Soc ClinOncol 19:187a, 2000.

50.

Hidalgo M, Rowinsky E, Erlichman C,et al: Phase I and pharmacological study ofCCI-779, a cell cycle inhibitor. Proceedingsof the 11th NCI EORTC AACR Symposiumon New Drugs in Cancer Therapy (abstract413). Clin Cancer Res 6:4548s, 2000.

51.

Chan S, Scheulen ME, Johnston S, et al:Phase II safety and activity study of two doselevels of CCI-779 in locally advanced or metastaticbreast cancer failing prior anthracyclineand/or taxane regimens (abstract 774).Proc Am Soc Clin Oncol 22:193, 2003.

52.

Shi Y, Frankel A, Radvanyi LG, et al:Rapamycin enhances apoptosis and increasessensitivity to cisplatin in vitro. Cancer Res55:1982-1988, 1995.

53.

O'Reilly T, Vaxelaire J, Muller M, etal: In vivo activity of RAD 001, an orallyactive rapamycin derivative, in experimentaltumor models (abstract 359). Proc Am AssocCancer Res 43:71, 2002.

54.

Lane H, Schnell C, Theuer A, et al:Antiangiogenetic activity of RAD 001, an orallyactive anticancer agent (abstract 922). ProcAm Assoc Cancer Res 43:184, 2002.

55.

Clackson T, Metcalf III CA, RozamusLW, et al: Regression of tumor xenografts inmice after oral administration of AP23573, anovel mTOR inhibitor that induces tumor starvation(abstract 95). Proc Am Assoc CancerRes 43:2002.

56.

Clackson T, Metcalf CA, Rivera HL, etal Broad anti-tumor activity of AP23573, anmTOR inhibitor in clinical development (abstract882). Proc Am Soc Clin Oncol 22:220,2003.

57.

O'Donnel S, Faivre I, Judson C, et al: Aphase I study of the oral mTOR inhibitorRAD001 as monotherapy to identify the optimalbiologically effective dose using toxicity,pharmacokinetic and pharmacodynamic endpointsin patients with solid tumors (abstract803). Proc Am Soc Clin Oncol 22:200, 2003.

58.

Zhang HT, Craft P, Scott PA, et al:Enhancement of tumor growth and vasculardensity by tranfection of vascular endothelialcell growth factor in to MCF-7 human breastcarcinoma cells. J Natl Cancer Inst 87:213-219, 1995.

59.

McLesky SW, Kurebayashi J, HonigSF, et al: Fibroblast growth factor 4 transfectionof MCF-7 cells produces cell lines thatare tumorigenic and metastatic in ovariectomizedor tamoxifen-treated athymic nude mice.Cancer Res 53:2168-2177, 1993.

60.

Sauer G, Deissler H, Kurzeder C, et al:New molecular targets of breast cancer therapy.Strahlenther Onkol 178:123-133, 2002.

61.

Horak ER, Leek R, Klenk N, et al:Angiogenesis, assessed by platelet/endothelialcell adhesion molecule antibodies, as indicatorof node metastases and survival in breastcancer. Lancet 340:1120-1124, 1992.

62.

Weidner N, Semple JP, Welch WR, etal: Tumor angiogensis and metastatic-correlationin invasive breast carcinoma. N Engl JMed 324:1-8, 1991.

63.

Ellis LM: Angiogensis. Horizons inCancer Therapeutics 3:4-22, 2002.

64.

Ferrara N, Houck K, Jakeman L, et al:Molecular and biological properties of the vascularendothelial cell growth factor family ofproteins. Endocr Rev 13:18-32, 1992.

65.

Gerber HP, McMurtrey A, Kowalski J,et al: Vascular endothelial growth factor regulatesendothelial cell survival through thephoshatidylinositol-3´-kinase/Akt signal transductionpathway. J Biol Chem 273:30336-30343, 1998.

66.

Margolin K, Gordon MS, Holmgren E,et al: Phase Ib trial of intravenous recombinanthumanized monoclonal antibody to vascularendothelial growth factor in combinationwith chemotherapy in patients with advancedcancer: Pharmacologic and long-term safetydata. J Clin Oncol 19:851-856, 2001.

67.

Gordon MS, Margolin K, Talpaz M, etal: Phase I safety and pharmacokinetic studyof recombinant human anti-vascular endothelialgrowth factor in patients with advancedcancer. J Clin Oncol 19:843-850, 2001.

68.

Borgstrom P, Gold DP, Hilan KJ, et al:Importance of VEGF for breast cancer angiogenesisin vivo: Implications from intravitalmicroscopy of combination treatments withan anti-VEGF neutralizing monoclonal antibodyand doxorubicin. Anticancer Res19:4203-4214, 1999.

69.

Cobleigh MA, Miller KD, LangmuirVK, et al: Phase II dose escalation trial ofAvastin (bevacizumab) in women with previouslytreated metastatic breast cancer (abstract520). Breast Cancer Res Treat 69:301, 2001.

70.

Miller KD, Rugo HS, Cobleigh MA, etal: Phase III trail of capecitabine (Xeloda) plusbevacizumab (Avastin) versus capecitabinealone in women with metastatic breast cancerpreviously treated with an anthracycline and ataxane (abstract 36). Breast Cancer Res Treat76:S37, 2002.

71.

Fong TA, Shawver LK, Sun L, et al:SU5416 is a potent and selective inhibitor ofthe vascular endothelial growth factor receptor(Flk-1/KDR) that inhibits tyrosine kinasecatalysis, tumor vascularization, and growthof multiple tumor types. Cancer Res 59:99-106, 1999.

72.

Tolcher A, Karp DD, O'Leary JJ, et al:A phase I and biologic correlative study of anoral vascular endothelial growth factor receptor-2 (VEGF-2) tyrosine kinase inhibitor, CP547,632 in patients with advanced malignancies(abstract 334). Proc Am Soc Clin Oncol21:84a, 2002.

73.

Hurwitz H, Holden SN, Eckhardt SG, etal: Clinical evaluation of ZD6474, an orallyactive inhibitor of VEGF signaling in patientswith solid tumors (abstract 325). Proc Am SocClin Oncol 21:82a, 2002.

74.

Drevs J, Schmidt-Gersbach CIM, MrossK, et al: Surrogate markers for the assessmentof biologic activity of the VEGF-receptor inhibitorPTK787/ZK 222584 (PTK/ZK) in twoclinical trials (abstract 337). Proc Am Soc ClinOncol 21:85a, 2002.

75.

Baker CH, Solorzano CC, Fidler IJ:Blockade of vascular endothelial growth factorand epidermal growth factor receptor signalingfor therapy of metastatic humanpancreatic cancer. Cancer Res 62:1996-2003,2002.

76.

Ciardiello F, Caputo R, Damiano V, etal: Antitumor effects of ZD 6474, a smallmolecule vascular endothelial growth factorreceptor tyrosine kinase inhibitor, with additionalactivity against epidermal growth factorreceptor tyrosine kinase. Clin Cancer Res9:1546-1556, 2003.

77.

Mendel DB, Laird AD, Xin X et al: Invivo antitumor activity of SU11248, a noveltyrosine kinase inhibitor targeting vascularendothelial growth factor and platelet-derivedgrowth factor receptors: Determination of apharmacokinetic/pharmacodynamic relationship.Clin Cancer Res 9:327-337, 2003.

78.

O'Farrell AM, Abrams TJ, Yuen HA, etal: SU11248 is a novel FLT3 tyrosine kinasewith potent activity in vitro and in vivo. Blood101:3597-3605, 2003.

79.

Rosen L, Mulay M, Long J, et al: Phase1 trial of SU11248 a novel tyrosine kinaseinhibitor in advanced solid tumors (abstract765). Proc Am Soc Clin Oncol 22:191, 2003.

80.

Morris MJ, Tong WP, Cordon-Cardo C,et al: Phase I trial of bcl-2 antisense oligonucleotide(G3139) administered by continuousintravenous infusion in patients with advancedcancer. Clin Cancer Res 8:679-683, 2002.

81.

Leek RD, Kaklamanis L, Pezzella F, etal: bcl-2 in normal human breast and carcinoma,association with estrogen receptor-positive,epidermal factor receptor-negative tumorsin situ cancer. Br J Cancer 69:135-139, 1994.

82.

Reed JC: Bcl-2: Prevention of apoptosisas a mechanism of drug resistance. HematolOncol Clin North Am 9:451-473, 1995.

83.

Yang D, Ling Y, Almazan M, et al:Tumor regression of human breast carcinomasby combination therapy of anti-bcl-2 antisenseoligonucleotide and chemotherapeutic drugs(abstract 4814). Proc Am Assoc Cancer Res40:729, 1999.

84.

Waters JS, Webb A, Cunningham D, etal: Phase I clinical and pharmacokinetic studyof bcl-2 antisense oligonucleotide therapy inpatients with non-Hodgkin's lymphoma. J ClinOncol 18:1812-1823, 2000.

85.

Chen HX, Marshall J, Trocky N, et al:A phase I study of Bcl-2 antisense G3139(Genta) and weekly docetaxel in patients withadvanced breast cancer and other solid tumors(abstract 692). Proc Am Soc Clin Oncol19:178a, 2000.

86.

Chi KN, Gleave ME, Klasa R, et al: Aphase I trial of an antisense oligonucleotide tobcl-2(G3139, Genta) and mitoxantrone in patientswith metastatic hormone refractory prostatecancer (abstract 1299). Proc Am Soc ClinOncol 19:330a, 2000.

87.

Shin MS, Kim HS, Lee SH, et al: Mutationsof tumor necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAIL-R1) andreceptor 2 (TRAIL-R2) genes in metastaticbreast cancers. Cancer Res 61:4942-4946,2001.

88.

Keane MM, Ettenberg SA, Nau MM, etal: Chemotherapy augments TRAIL-inducedapoptosis in breast cancer cell lines. CancerRes 59:734-741, 1999.

89.

Investigators brochure: TRM-1. Milan,Italy, Ichemco.

90.

Marks PA, Richon VM, Breslow R, etal: Histone deacetylase inhibitors as new cancerdrugs. Curr Opin Oncol 13:477-483, 2001.

91.

Marks PA, Rifkind RA, Richon VM, etal: Histone deacetylases and cancer: Causesand therapies. Nat Rev Cancer 1:194-202,2001.

92.

Richon VM, O'Brien JP: Histonedeacetylase inhibitors: A new class of potentialtherapeutic agents for cancer treatment.Clin Cancer Res 8:662-664, 2002.

93.

Donapaty L, Fuino S, Wittman R, et al:Histone deacetylase inhibitor LAQ824 downregulatesHER-2, induces growth arrest andsensitizes human breast cancer cells to herceptinand tubulin polymerizing agents (abstract805). Proc Am Soc Clin Oncol 22:201, 2003.

94.

Ryan QC, Headlee D, Sparreboom A, etal: A phase I trial of a histone deacteylaseinhibitor MS-275 in advanced solid tumor andlymphoma patients (abstract 802). Proc AmSoc Clin Oncol 22:200, 2003.

95.

Heaney M, O'Conner A, Richon V, etal: Clinical experience with the histonedeacetylase inhibitor suberoylanilide hydroxamicacid (SAHA) in heavily pretreated patientswith hematological malignancies(abstract 2321). Proc Am Soc Clin Oncol22:577, 2003.