sbur

times higher than before 65, and 150-fold higher than before

has been acknowledged for decades. It is suggested since

recently, that tumor promotion is not the only mechanism

the menopause; in pre-menopausal women, overweight is

The second group of breast cancer predisposing properties

deficiency in maintenance of genomic integrity has been

Drug Discovery Today: Disease Mechanisms Vol. 1, No. 2 2004

d In

l Rof estrogens action: some of estrogen metabolites were shown

to cause DNA damage directly (i.e. contribute in the breast

cancer initiation). Selected causes of estrogen overload (e.g.

early menarche or late menopause) are at least in part attrib-

uted to inherited genetic variations. However, most of deter-

minants of hyperestrogenia are related to the modern,

Western lifestyle including low parity, delayed age at first

recognized only recently (Fig. 1). There are two lines of

supporting arguments: first, all of the identified breast cancer

susceptibility genes contribute to the sensing or repair of

DNA damage; second, there is an impressive reproducibility

of phenotyping studies demonstrating relationships between

breast cancer risk and constitutional chromosomal instability

[57].

Unlike lung or bladder cancers, none of environmental

carcinogens has been convincingly linked to breast cancer*Corresponding author: (E.N. Imyanitov) evgeny@imyanitov.spb.ru

1740-6765/$ 2004 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddmec.2004.09.002 www.drugdiscoverytoday.com 23530 years of age. In addition to advanced age, a few dozens of

other breast cancer predisposing factors have been identified;

however, all these diverse risks can be assigned to either of

two major categories: excessive exposure to estrogens and

deficiency in maintenance of genomic integrity (Fig. 1) [1].

The proliferative effect of estrogens on breast epithelium

associated with anovulatory cycles and reduced probability of

acquiring breast cancer disease. The adverse impact of oral

contraception and hormone replacement therapy has been

confirmed in some but not all epidemiological studies. The

role of exogenous endocrine disruptors in breast cancer inci-

dence remains to be proven [14].development of dozens of other targeted treatments

for breast cancer is underway. Unfortunately, some

intrinsic features of breast cancer biology compromise

the efficiency of current therapeutic strategies.

Introduction

Breast cancer is the most common malignancy among

females affecting approximately one out of ten women.

Ageing of population in the industrialized world is the most

obvious cause of increased breast cancer occurrence; indeed,

the risk of developing breast cancer after 65 years of age is 5.8Breast cancer is the most common malignancy among females andaffects approximately one in every ten women worldwide. It is the first

human tumor for which targeted therapies have been developed. Somenotable successful therapies include tamoxifen, aromatase inhibitors

both estrogen receptor pathway downregulators and Herceptin, aHER2 antagonist. However, despite some spectacular examples of

prolonged disease remission in selected women, the statistical survivalbenefit in metastatic breast cancer patients is estimated in months and

not years. In this article, Evgeny Imyanitov and Kaido Hanson describethe progress of targeted treatments for breast cancer that are already

underway, and discuss the particular features of breast cancer biologythat compromise the efficiency of current therapeutic drugs.

delivery, short duration of breastfeeding, overeating, limited

exercise and so on. Interestingly, the obesity correlates with

hyperestrogenia and excessive breast cancer risk only afterimpressive success stories include estrogen receptorMECHANISMS

DRUG DISCOVERY

TODAY

DISEASE

Mechanisms of breaEvgeny N. Imyanitov*, Kaido P. HansonGroup of Molecular Diagnostics, N.N. Petrov Institute of Oncology, St. Peters

Breast cancer is the first human tumor for which

targeted therapies have been developed. The most

pathway downregulators (tamoxifen and aromatase

inhibitors) and HER2 antagonists (Herceptin). The

Editors-in-Chief

Toren Finkel National Heart, Lung and Bloo

Tamas Bartfai Harold L. Dorris Neurologica

Cancert cancer

g 197758, Russia

Section Editor:Silvio Gutkind National Institute of Dental and CraniofacialResearch, National Institutes of Health, Bethesda, MD, USA

stitute, National Institutes of Health, USA

esearch Center and The Scripps Research Institute, USA

Drug Discovery Today: Disease Mechanisms | Cancer Vol. 1, No. 2 2004Box 1. Challenging the dogmas in breast cancerresearch: recent unexpected findings

Viral theory revisited?

There are several independent reports claiming that in some

groups of patients roughly a third of breast carcinomas contain

DNA sequences homologous to the well-known Mouse Mammary

Tumor Virus (MMTV); in intriguing accordance with the viral

hypothesis, worldwide breast cancer spread appears to parallel the

geographical distribution of one species of house mouse, Mus

domesticus. If the contribution of viruses in breast cancer etiology

will be confirmed, many aspects of breast cancer prevention and

treatment will need to be re-analyzed. However, there are

also some negative reports on this subject [8,9].

Polyclonal origin of breast cancer?

Monoclonal origin of breast cancer is believed to be a well-

established fact. However, recent data cast some doubt on this

confidence: at least in some cases, breast cancer lump appears to

arise from several independent progenitors, thus supporting the

cancer field theory [42]. Interestingly, stromal cells surrounding

breast cancer epithelium also carry somatic mutations, and the

spectrum of these genetic alterations is different from those

observed in cancer cells [22].

Does all tumor mass possess a danger?

Recent experiments show that only a minor, specific fractionetiology. The results of the studies on dietary factors have

been inconclusive as well. Contrary to beliefs of many

patients, psychological stress is not associated with breast

cancer risk [1,2,4]. There are surprisingly few reports in

modern scientific literature assessing the relationship

between breast trauma and subsequent cancer development.

Intriguingly, some recent findings have resumed the debate

on the viral etiology of breast cancer [8,9] (Box 1).

Contribution of inherited features

In some instances, breast cancer represents a classical heredi-

tary disease with mendelian mode of transmission. The best-

known highly penetrant breast cancer genes are BRCA1

(GenBank accession number NM_007294) and BRCA2 (Gen-

Bank accession number NM_000059); they account for

approximately 20% of familial breast cancer clustering

[10]. Although early studies carried out on breast cancer

families suggested that BRCAs are associated with nearly fatal

risk of the disease, recent reports indicate that their pene-

trance in unselected breast cancer series approaches to 65%

for BRCA1 and only to 45% for BRCA2 (by age 70 years) [11].

In a broad sense, ATM (GenBank accession number

of cells forming breast cancer lump is highly tumorigenic [43].

Tumor heterogeneity might compromise the search for new

therapeutic targets, as the current approaches are almost always

based on the analysis of crude tumor material. The intrinsic resistance

of particular intratumoral cell subsets to the treatment might

explain a discordance between high frequency of short-term partial

tumor responses and low rate of prolonged breast cancer remissions.

236 www.drugdiscoverytoday.comNM_000051) and p53 (GenBank accession number

NM_000546) germ-line mutations, predisposing to ataxia-

telangiectasia and Li-Fraumeni hereditary syndromes, respec-

tively, can also be considered as breast cancer-associated

genetic defects, owing to the increased incidence of breast

neoplasia in their heterozygous carriers [5].

There is a growing class of breast cancer-associated genetic

variations, which are situated in between rare catastrophic

germ-line mutations and frequent normal gene polymorph-

isms. Some founder mutations can be classified as middle-

penetrance polymorphisms, owing to their noticeable occur-

rence in the population (1%) and modest but clinicallymeaningful influence on the breast cancer risk increase. In

addition to Jewish founder mutations in BRCA genes, which

seem to be somewhat less penetrant than non-founder

defects, the examples include CHEK21100delC (GenBank

accession number NM_007194) and NBS1657del5 (GenBank

accession number NM_002485) [7]. As already mentioned

above, all high- and middle-penetrant genes with proven

breast cancer-associated significance participate in various

aspects of maintenance of genomic integrity.

It is frequently stated that the majority of breast cancer

cases are related to the disadvantageous genetic passport

(i.e. unfavorable combination of low-penetrant gene poly-

morphisms). The attempts to link breast cancer risk to poly-

morphisms in carcinogen metabolizing enzymes have been

unsuccessful. There is more hope to detect breast cancer gene-

disease interactions within the class of hormone metaboli-

zers, based on convincing evidence for heritability of indivi-

dual features of hormonal portrait coupled with the proven

role of hyperestrogenia in breast cancer development. How-

ever, no reproducible associations have been revealed over

the decade-long research in this field. Following the success

in identifying breast cancer-associated germ-line mutations

within genome safeguards in addition to relying on the

consistency of the results of phenotyping tests (see the text

above and Fig. 1), many breast cancer researchers have

recently re-located the efforts to the analysis of DNA repair

gene polymorphisms; the outcome of these studies remains

to be seen [7].

Somatic events in breast cancer pathogenesis

Interaction between various breast cancer predisposing fac-

tors leads to accumulation of somatic mutations in breast

epithelial cells (Fig. 1). Chromosomal instability manifested

by enormous number of gross chromosomal abnormalities

appears to be the most characteristic feature of breast cancer

genome [12]. Wide-spread hypermethylation of regulatory

regions of genome is another mandatory peculiarity of breast

tumors [13]. Single nucleotide instability appears to be less

common in breast cancer than in other major cancer types,

such as colorectal or lung cancer; however, limitations incurrently available methodologies force to abstain from the

Vol. 1, No. 2 2004 Drug Discovery Today: Disease Mechanisms | Cancerdefinitive conclusion [12,14]. Classical type of microsatel-

lite instability does not occur in breast cancer, although it was

demonstrated in a small portion of bilateral breast tumors

[15]. It was shown recently that many breast carcinomas carry

alterations in mitochondrial DNA [16].

Figure 1. Breast cancer mechanisms. Two groups of predisposing factors have a

and deficiency in maintenance of genomic integrity. During the malignant transfo

genetic events (mainly gross chromosomal alterations and methylation abnorm

suppressor genes, which eventually results in the manifestation of The hallm

properties might serve as potential therapeutic targets.Discrete alterations in breast cancer-specific genes can

serve both as the triggers of somatic genetic instability and

as consequences of increased mutation rate. Hundreds of

reports have been devoted to the cataloging of breast can-

cer-associated genetic abnormalities (e.g. Somatic Mutations

major role in breast cancer etiology: excessive breast exposure to estrogens

rmation process, breast epithelial cells accumulate high number of somatic

alities). These DNA alterations activate oncogenes and inactivate tumor

arks of cancer [21]. Molecular determinants of the most essential tumor

www.drugdiscoverytoday.com 237

Drug Discovery Today: Disease Mechanisms | Cancer Vol. 1, No. 2 2004

or

D1

CC

i-6

H1

q (8

3p

13q

21.

6q1

3, 1

2q

CN

NES

, R

3-3

T1,

NX

httTable 1. Genetic alterations in breast carcinomas

Type of genetic event Chromosomal regions

Activating events

Gene amplification followed by over-expression 8q24: MYC; 11q13: CCN

Gene over-expression BCL2, B94, Cathepsin D,

GZMH, hTERT, IGF1R, K

neurosin, PAI1, PAI2, PO

Gains of genetic material 1q (1q21, 1q32, 1q41), 8

Events of uncertain significance

Losses of heterozygosity (allelic imbalances)

reflecting either allelic deletion or gain

of the remaining allele

1p (1p36.3), 2q (2q22.1),

8p (8p21.3), 9p (9p21.3),

18p (18p11.32), 18q (18q

Inactivating events

Losses of genetic material 1p (1p3135, 1p36), 6q (

11q2425), 13q (13q121

17p (17p13.1, 17p13.3), 2

Intragenic mutations p53

Promoter methylation followed by the

loss of gene expression

APC, BCSG1, BRCA1, C

HIN1, HOXA5, Maspin,

RAR-beta, RASSF1A, RFC

TMS1, TWIST, ZAC, 14-

Loss of gene expression ATM, BAX, ITGA6, MGS

SPR1, TGFBR3, TFAP4, T

a For more information of these genes, see http://www.gene.ucl.ac.uk/nomenclature/, andin Human Cancers Database; http://www.onco-is.com).

Conditionally, they can be classified into activating and

inactivating events (Table 1). Noticeable frequency of ampli-

fications of selected oncogenes [HER2 (also known as ERBB2)

(GenBank accession number NM_004448), CCND1 (GenBank

accession number NM_053056), C-MYC (GenBank accession

number NM_002467)] followed by their overexpression is a

distinct feature of breast cancer. In addition to identified

oncogenes, there are several other regions in the genome

that repeatedly demonstrate extra copies in breast cancer; it

remains to be established whether they do contain activated

oncogenes or simply reflect the background noise of somatic

chromosomal instability [12]. Recent developments in

expression profiling have significantly enlarged the list of

genes overexpressed in breast cancer, however systematic

picture of upregulated transcripts has yet to be drawn [17].

In 1990s and early 2000s many researchers tried to locate

breast cancer-specific losses of heterozygosity (LOH). How-

ever, the attempts to identify new suppressor genes by LOH

mapping have largely failed: in addition to enormous varia-

bility of LOH patterns due to admixture of non-specific allelic

imbalances, it has turned out that conventional PCR allelo-

typing can not discriminate between inactivating and acti-

vating mutations (e.g. loss of the allele versus amplification of

the remaining allele) or between homozygous deletion and

retention of both gene copies [18,19]. Intragenic mutations

are relatively uncommon for breast cancer. The p53

238 www.drugdiscoverytoday.comgenes involveda Refs.

, EMS1; 17q1221: HER2, TOP2A; 20q13: AIB1 [12]

NE, CD63, claudin-7, CRABP2, CTSD, GATA3,

7, lactoferrin, lipocalin 2, MDM2, MUC1, MYBL2,

, PS2, Rantes, SIX1, SMARCD2, STMY3, VEGF

[12,17]

q24), 11q (11q13), 16p (16p11), 17q (17q11.2, 17q24), 20q (20q13) [12]

(3p14.2), 4q (4q35.1), 6q (6q25.1), 7q (7q31.2),

(13q14), 16q (16q22.1, 16q24.3), 17p (17p13.3),

2), 19p (19p13), 21q (21q11.1)

[18]

321, 6q2123.3, 6q2527), 8p (8p21, 8p2223), 11q (11q2223,

3q14.1), 16q (16q2123.3, 16q24.3),

(22q13)

[12]

[20]

D2, CDH1, CDH13, DAPK, ER, FHIT, GPC3, GSTP1,

1, NM23-H1, NOEY2, PR, Prostasin, INK4, CIP1,

IZ1, SOCS1, SRBC, SYK, TGFBR2, THBS, TIMP3,

sigma

[13]

OXTR, plakophilin 1, KIP1, RIG-like7-1, RB1, RBL2, SPARCL1,

A, 53BP2

[12,17]

p://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM&itool=toolbar.(GenBank accession number NM_000546) gene is mutated

only in 20% of breast carcinomas; mutations in RAS family

oncogenes occur in a negligible fraction of breast cancer [20].

The genome-wide approaches led to the identification of

dozens breast cancer-associated deletions; there is also a

growing list of genes whose transcription is downregulated

in breast cancer [12,17]. Frequently, the decreased expression

of suppressor genes is associated with the methylation of

their promoter regions [13]. However, molecular techniques

enabling global mapping of hypermethylated regions are not

yet as efficient as expression profiling or array-based com-

parative genomic hybridization.

Various combinations of activating and inactivating muta-

tions and epigenetic events lead to the spectrum of properties

of tumor cells, which have been defined as The Hallmarks of

Cancer in already-classical work of Hanahan and Weinberg

[21], and include self-sufficiency in growth signals, insensi-

tivity to antigrowth signals, evasion from apoptosis, limitless

replicative potential, invasion and metastasis, genomic

instability and sustained angiogenesis. Similarly to the pro-

gress in understanding of tumor angiogenesis, it is becoming

apparent that the contribution of surrounding stroma in

tumor growth is not a merely passive process, but, instead,

an active and essential component of neoplastic evolution

[22,23]. Breast carcinomas share all characteristics of malig-

nancy, with one important reservation: more than 50% of

breast cancers retain at least partial dependence from estro-

Vol. 1, No. 2 2004 Drug Discovery Today: Disease Mechanisms | Cancergenic growth stimulation. This benign feature of some breast

cancer accounts for remarkable efficiency of the antiestro-

genic treatment approaches [24].

Although the diversity of molecular portraits of breast

cancer is extraordinary, they appear to fit into the limited

number of distinct disease subsets. Noticeably, many of breast

cancer signatures identified by array expression profiling tend

to group around long-known disease markers, such as estro-

gen receptor (ER) or HER2 oncogene status [25]. Interestingly,

host factors appear to play an essential role in determining

the molecular variant of breast cancer pathogenesis [15].

The web of signaling cascades

Owing to the rapid accumulation of data in signal transduc-

tion research, the comprehensive description of breast can-

cer-associated signaling pathways and their cross-talk has

become a difficult challenge. However, surprisingly, in the

light of potential therapeutic relevance, this complexity is

not as high as it might appear. First, the spectrum of candi-

date targets is limited by those molecules, which are essential

for the maintenance of breast cancer cells; the intervention

with breast cancer initiating cascades might have some

potential for breast cancer prevention, but not for the cure

of already existing disease. Second, while the specific target-

ing of upregulated molecules has become a realistic approach,

the opposite (i.e. the substitution of downregulated enzymes)

is not achievable for the time being. Therefore, applied breast

cancer research is more interested in tumor-activated path-

ways than in those that are suppressed in breast cancer cells.

Third, owing to the multifunctionality of many signaling

proteins and pathways, it is usually difficult to precisely

assign the molecule of interest to the defined biological

outcome(s). For example, the members of HER receptor

family appear to contribute into all cancer hallmarks (Fig.

1) [26]. COX2 is mentioned most frequently in the context of

tumor angiogenesis; however, its involvement in autoproli-

ferative, antiapoptotic and metastatic cascades has also been

acknowledged [27]. Although the utmost importance of com-

prehensive functional description of potential breast cancer

targets is beyond any doubt, the initial inclusion criteria for

the target usually are its breast cancer specificity and involve-

ment in the disease maintenance.

Two molecules appear to be truly specific for breast cancer,

and both of them are receptors: ER and HER2. ER is a member

of the family of nuclear receptors, and is involved in tran-

scriptional regulation of many essential genes. Activation of

the HER2 pathway in breast cancer is often associated with an

upregulation of other members of HER receptor family, espe-

cially HER1, however HER1 overexpression occurs in breast

cancer significantly less frequently than in several other

cancer types. Downstream parts of HER-initiated signaling

cascades include RASRAFMEKMAPK pathway, whichpotentiates cellular proliferation through modification ofcyclin-dependent cascades, and phosphatidylinositol 3-

kinase (PI3-K) pathway (AKT, mTOR), which has an essential

role in the regulation of cell survival. These molecules are not

indeed specific for breast cancer; they do have regulatory

functions in normal cells as well, therefore, their targeting

possesses a risk of adverse effects. In addition to ER and HERs,

there is an increasing attention to the angiogenic pathways

triggered by vascular endothelial growth factor (VEGF); these

cascades appear to be upregulated in many cancer types,

including breast cancer [12,26,28,29].

Targeted therapies

The description of breast cancer-specific molecular targets

and corresponding therapeutic compounds is presented in

the Table 2.

The first example of selective breast cancer therapy

emerged several decades ago owing to an accidental finding.

Tamoxifen had been initially tested with the intention to

develop a contraceptive pill. Although it failed to control

fertility in humans, its inhibitory effect on breast cancer cell

growth led to the rapid introduction of this drug in the

treatment of ER-positive breast cancer. In postmenopausal

women, it might soon be replaced by inhibitors of aromatase,

a key enzyme of estrogen synthesis. Aromatase inhibitors

have shown more favorable results than tamoxifen in several

randomized breast cancer trials [24]. Another success story

concerns Trastuzumab (Herceptin), which demonstrated evi-

dent survival benefit for patients suffering from HER2-posi-

tive breast cancer [29]. Other targeted approaches such as

HER1 or pan-HER inactivation [26], interference with down-

stream participants of receptor tyrosine kinase signaling [29

33], inhibition of molecules involved in tumor metastasis and

angiogenesis [29,30,34], as well as some additional strategies

[27,28,3537], are in early clinical trials or pre-clinical studies

(Table 2).

All of the approaches mentioned above are based on the

suppression of molecules essential for breast cancer mainte-

nance. The alternative strategy suggests the use of breast

cancer-specific proteins as the anchors for site-specific deliv-

ery of toxic compounds [3840] (Table 2). This concept

certainly enlarges the list of potentially relevant targets,

because it does not require functional evidence but solely

relies on the proof of breast cancer-specific expression. With

some reservations, recently introduced Capecitabine

(Xeloda) could be considered as a relevant example; indeed,

being a non-toxic precursor of 5-fluorouracil, Xeloda under-

goes topical conversion to the cytostatic drug in tumors, due

to increased expression of thymidine phosphorylase in the

neoplastic tissue [38]. More general approaches are based on

the use of breast cancer-specific antibodies conjugated with

various toxins [39].

The somatic genomic instability might represent not onlythe dangerous property of tumor phenotype, but also an

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Table 2. Breast cancer targets and related therapies

Target Therapy against target Stage of developmenta Advantages and/or disadvantages Who is work g

on the targe

Website Refs.b

Properties of cancer cells as a target

Genetic instability and/or

high proliferation

Conventional cytostatic

drugs (anthracyclines,

taxanes, alkylating drugs,

antimetabolites, vinca alkaloids)

On the market for

a long time

Clinical response can be achieved in

more than 8090% cases. However,

disadvantages include severe adverse

effects, limited duration of response,

overlapping spectrum of drug resistance,

limited ability to predict response and/or

non-response to a given cytostatic compound.

Many

pharmaceutica

companies and

research group

[38]

Molecular targets as tumor-specific anchors for delivery of cytostatic substances

Thymidine phosphorylase Capecitabine (Xeloda) On the market Thymidine phosphorylase converts Xeloda

to the cytostatic drug, 5-fluorouracil; high

tumor specificity is attributed to the

preferential expression of this enzyme

in cancer tissues.

Roche http://www.roche.com [38]

Breast cancer-specific

antigens

Immunoconjugates

(with cytotoxic drugs,

radioactive isotopes,

toxins, attractants of

tumor-killing cells)

Phase III Presumably large spectrum of candidate

molecules, since the target does not

need to be essential for tumor growth.

Several researc groups [39]

Sodiumiodide symporter Radioactive iodide Laboratory studies Adverse effects on thyroid gland are probable. [40]

Genuine targets (i.e. molecules essential for breast cancer development and maintenance)

Estrogen receptor pathway ER pathway downregulators show high

efficiency combined with good safety profile.

However, more than a third of breast cancers

are resistant to antiestrogen therapy.

Selective estrogen

receptor modulators

(SERMs)

Tamoxifen On the market for

a long time

Many pharmac tical

companies

[24]

Toremifene (Fareston) On the market Almost complete cross-resistance

with tamoxifen

Schering-Ploug Orion http://www.schering-

plough.com

http://www.orion.fi

[24]

Raloxifene (Evista) Chemoprevention

trials

Almost complete cross-resistance

with tamoxifen

Eli Lilly http://www.lilly.com [24]

Selective estrogen

receptor downregulators

(SERDs)

Faslodex (Fulvestrant) On the market AstraZeneca http://www.astrazeneca.com [24]

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Aromatase inhibitors Superior when compared with tamoxifen;

induce tumor response in a subset of

tamoxifen-resistant tumors. However,

efficient only in postmenopausal patients.

Anastrozole (Arimidex) On the market AstraZeneca http://www.astrazeneca.com [24]

Letrozole (Femara) On the market Novartis http://www.novartis.com [24]

Exemestane (Aromasin) On the market Pfizer http://www.pfizer.com [24]

HER2 (ERBB2) HER2 is activated only in one

out of four breast tumors.

Antibodies

Trastuzumab (Herceptin) On the market High-efficiency combined with good

safety profile. Notice that HER2+

tumors represent the most aggressive

subset of breast cancer.

Genentech, Ro he http://www.gene.com

http://www.roche.com

2C4 (Omnitarg, Pertuzumab) Phase III Genentech, Ro he http://www.gene.com

http://www.roche.com

[29]

Small molecule

inhibitors

CP-724714 Phase I Pfizer http://www.pfizer.com [29]

TAK-165 Phase I Takeda http://www.takeda.com [29]

HER1 (EGFR) HER1 is rarely overexpressed

in breast cancers.

ZD1839 (Iressa, Gefitinib) Phase II AstraZeneca http://www.astrazeneca.com [29]

OSI-774 (Tarceva, Erlotinib) Phase II OSI, Genentec , Roche http://www.osip.com

http://www.gene.com

http://www.roche.com

[29]

EKB-569 Phase I Wyeth http://www.wyeth.com [29]

HER2 and HER1 GW572016 (GW2016) Phase II GlaxoSmithKli http://www.gsk.com [26]

HER kinases CI-1033 (PD183805) Phase II Pfizer http://www.pfizer.com [26]

VEGF Bevacizumab (Avastin) Phase II Genentech http://www.gene.com [30]

VEGFR2 ZD6474 Phase II AstraZeneca http://www.astrazeneca.com [29]

Farnesyl transferase Trials on several farnesyltransferase

inhibitors have been discontinued due to

high-toxicity and lack of clinical effect.

R115777 (Zarnestra, Tipifarnib) Phase II Johnson and Jo nson http://www.jnj.com [31]

SCH66336 (Sarasar, lonafarnib) Phase I Schering-Ploug http://www.schering-plough.com [31]

CDK kinases

Flavopiridol (Alvocidib) Phase III Aventis [32]

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Table 2 (Continued).

Target Therapy against target Stage of developmenta Advantages and/or

disadvantages

Who is working

on the target

Website Refs.b

UCN-01 Phase I UCN-01 inhibits several other

kinases, including PKC.

Kyowa Hakko Kogyo [32]

CYC202 (Roscovitine) Phase II Cyclacel [32]

BMS-387032 Phase I Bristol-Myers Squibb [30]

mTOR kinase

CCI-779 (Temsirolimus) Phase IIIII Wyeth http://www.wyeth.com [30]

RAD001

(Everolimus, Certican)

Phase I Novartis http://www.novartis.com [33]

PKC Bryostatin-1 Clinical trials on other

cancer types

GPC Biotech [29]

RAF Bay 43-9006 Clinical trials on other

cancer types

Onyx, Bayer [33]

MEK CI-1040 (PD184352) Phase II Pfizer http://www.pfizer.com [33]

Akt Perifosine Phase II Aeterna Zentaris http://www.aeterna.com [33]

Hsp90 17-AAG Phase I Kosan Biosciences http://www.kosan.com [30]

Matrix

metalloproteinases

High toxicity but limited

therapeutic efficiency in initial

clinical trials

BB-2516 (marimastat) Phase II Vernalis http://www.vernalis.com [34]

BMS-275291 Phase II Bristol-Myers

Squibb, Celltech

http://www.bms.com

http://www.celltechgroup.com

c-Kit and/or PDGFR STI571

(Gleevec, Glivec, Imatinib)

Phase II [28]

COX2 Celecoxib (Celebrex) Phase IIIII COX2 is selectively expressed in

tumors, thus COX2 inhibitors have

good safety profile.

Pfizer http://www.pfizer.com [27]

Histone deacetylases SAHA, PXD101,

LAQ-824, CI-994, MS-275

Phase III Several research group and

pharmaceutical compa s

[32]

Telomerase GRN163 Laboratory studies Geron http://www.geron.com [35]

Prolactin receptor Laboratory studies [36]

Chemokine receptors Laboratory studies Inhibition of chemokine receptors

might interfere with metastatic process.

[37]

a Stage of development applies mainly for breast cancer but not for other cancer types; this is especially true for phase II/III trials and already marketed drugs. Notice that some of e already licensed compounds have been approved for the

treatment of metastatic breast tumors, but still remain at the stage of clinical trials for the use in adjuvant therapy and/or chemoprevention.b In addition to the literature references indicated in the table, the information was updated using the websites of pharmaceutical companies, Clinical Trials site of the National Cancer Inst ute (http://www.cancer.gov/clinicaltrials), and the Catalog of

FDA Approved Drug Products (http://www.accessdata.fda.gov/scripts/cder/drugsatfda).

242

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Achilles heel of neoplasia. It is probable that the mechanism

of action of conventional non-specific cytostatics utilizes this

weakness, at least in part. Unfortunately, most of the cur-

rently available cytotoxic agents have a low therapeutic index

[38].

Towards curable breast cancer: how long

is the road?

Modern therapeutic combinations can induce tumor

response in more than 8090% of breast cancer patients.

However, despite some spectacular examples of prolonged

disease remission in selected women, the statistical survival

benefit in metastatic breast cancer patients is estimated in

months and not years. The situation is slightly better in

adjuvant breast cancer treatment, where the therapy saves

the lives in a certain (still frustratingly small) number of

otherwise relapsed patients [38].

It is often suggested that the future of breast cancer therapy

Vol. 1, No. 2 2004 Drug Discovery Today: Disease Mechanisms | Cancerlies in the use of individualized, molecular portrait-based,

sophisticated combinations of targeted drugs [41]. In this

approach, the tumor specificity is achieved by fine adjust-

ment of the multidrug panel to the unique spectrum of

survival determinants detected in malignant tissue by expres-

sion profiling (Fig. 2). It is assumed at the same time, that

normal tissues do not carry exactly the same set of vital

molecules, therefore, unlike the cancer cells, they can tolerate

the cocktail of signal transduction modifiers. This model is in

concordance with the clinical experience; indeed, combined

therapy of breast cancer is definitively superior when com-

pared with the monotherapy. However, there are an impress-

ive number of entirely distinct therapeutic modalities whose

combinations have already been used in breast cancer treat-

ment or trials (e.g. cytotoxic agents with various mechanisms

Figure 2. The promise of drug combinations. The concept of combined

cancer therapy implies that all survival determinants of the tumor cell

(depicpted in the center) can be efficiently blocked by the individually

matched cocktail of signal transduction modifiers. The antitumor spe-

cificity is warranted if none of normal cells (depicted at the four corners)

carries the same set of targets.of action, ER pathway downregulators, HER2 antagonists,

selective and, previously, non-selective COX2 inhibitors

and so on). Following the logic described above, one would

expect higher rate of prolonged breast cancer remissions than

the one currently observed. There are several concerns related

to the future of breast cancer therapy.

With the possible exception of ER, HER2 and COX2, most

of the currently considered targets are not truly cancer-spe-

cific because they are involved in the functioning of vital

human tissues. Not surprisingly, administration of signal

transduction modifiers (Table 2) often results in noticeable

adverse effects, sometimes comparable with toxicities of con-

ventional cytostatics. Interestingly, in attempt to prevent

cancer cell from rescuing from target inhibition, some com-

pounds with broader substrate specificity have been recently

tested (e.g. pan-HER inhibitor CI-1033, dual HER1HER2

inhibitor GW572016 and CDKsPKC inhibitor UCN-01)

(Table 2). Although the relaxed drug specificity might render

higher antitumor activity [29], it also increases the risk of

undesirable consequences. It is hoped that ongoing efforts in

expression array profiling will help to increase the list of

genuine breast cancer-specific targets.

Another group of difficulties is related to the tumor ability

to evolve over selective pressure of cancer treatment, and

inevitably develop drug-resistant phenotype. The intrinsic

genomic instability of cancer cells certainly facilitates this

process. However, it is not immediately clear whether the

treatment-related drug resistance is truly attributed to the

adaptive genetic flexibility of tumor cell clones, or, vise versa,

simply reflects the selection of pre-existing fatal cell subsets.

In other words, it is difficult to differentiate between tumor

evolution and tumor heterogeneity. Although the tumor

heterogeneity has been acknowledged for a long time, its

extent could have been underestimated. To the great surprise

of scientific audience, some of the recent evidence suggests

that a notable portion of breast cancer might be polyclonal in

their origin, thus supporting the theory of tumor field [42].

Furthermore, there are experimental data demonstrating that

truly dangerous, potentially metastatic cells constitute only a

negligible fraction of crude breast cancer lump [43]. Tumor

heterogeneity might compromise current efforts of molecular

portraying of neoplastic disease, because they are almost

always based on the analysis of gross primary tumor mass

and, therefore, might miss the targets in the most destructive

cancer subclones (Box 1).

Choice of the targets: cancer-specific or

breast-specific?

The initial phase of the search for cancer targets is usually

based on the comparison of expression profiles in tumor

versus corresponding normal tissues. Unfortunately, most

of the detected variations are usually quantitative but notqualitative; furthermore, most of upregulated target mole-

www.drugdiscoverytoday.com 243

Drug Discovery Today: Disease Mechanisms | Cancer Vol. 1, No. 2 2004cules occur only in a subset of carcinomas. Overall, the

difference between the cancer cell and its normal precursor

is much less than that between bacteria and mammalian

organisms, which makes the search for the magic bullet

against cancer far more complicated than the development of

antibacterial therapies [41].

It has been demonstrated that even transformed cells

stably retain major lineage-specific molecular markers [44].

In other words, there can be more difference in expression

profiles between distinct normal tissues than between paired

tumor-normal cell counterparts. If this is true, targeted che-

mical organectomy (i.e. selective ablation of particular cell

lineage) can be more achievable than specific elimination of

cancer cells [45]. This approach can have a future for the

treatment of cancers arising from those organs whose loss is

compatible with survival of the host (breast, prostate, ovary

and so on). Interestingly, this strategy has some similarities

with the bone marrow transplantation, where the first step

includes non-selective elimination of both malignant and

normal hemopoietic cell clones. The potential feasibility of

the chemical organectomy is difficult to assess, because the

comparative cataloging and functional understanding of

expression profiles of the normal human tissues appears to

be less developed compared with the body of data accumu-

lated in the studies of neoplastic disease.

Summary and conclusions

Breast cancer shares all of the key properties of malignancy,

however, it also has some distinct features. At the clinical

level, breast cancer is relatively easily detectable even in early

stages; in many instances, it has a reasonably favorable

prognosis, therefore, breast cancer patients predominate

among cancer survivors. At the phenotypic level, breast

cancer is not as malignant as certain other cancers because

more than 50% breast cancer retain at least partial estrogen

dependence. At the genetic level, breast carcinomas are char-

acterized by an increased number of chromosomal abnorm-

alities, but a limited frequency of small intragenic mutations.

In contrast to many other epithelial tumors, several genuine

breast cancer-specific targets have been identified. The intro-

duction of appropriate targeted therapies, such as tamoxifen

and aromatase inhibitors against ER pathway, or Herceptin

against HER2 receptor, resulted in unprecedented clinical

benefits. However, although currently available approaches

enable short-term remission in the majority of breast cancer

patients, metastatic breast cancer remains largely an incur-

able disease. It is hoped that individualized, expression pro-

file-based adjustment of drug combinations will improve the

treatment efficacy. However, there are some intrinsic proper-

ties of breast cancer biology, which might complicate the

discovery of smart drugs. In particular, most of the currently

considered targets show only quantitative but not qualitativeoverexpression in cancer versus normal cells; this might

244 www.drugdiscoverytoday.comnegatively affect the therapeutic index of newly developed

compounds. Some alarming data indicate that the most

dangerous cell subsets might constitute only a minor portion

of primary breast tumor; if it is so, the molecular profiling of

gross tumor mass might misguide the search for the cancer

treatment. Most of current target searches rely on the catalo-

ging of molecules that are upregulated in cancer cells com-

pared with their normal precursors. Because the entire loss of

breast epithelium is compatible with survival, it might turn

out that the breast-specific molecular targets have advantage

over the cancer-specific ones. The feasibility of this concept is

difficult to assess, even in theory, as a result of the deficiency

in systematic understanding of molecular physiology of nor-

mal human cells.

Acknowledgements

We thank Ekatherina Kuligina for her invaluable help in the

preparation of figures. We apologize to those authors whose

articles could not be cited because of space limitations. This

work was supported by INTAS (grant 03-51-4234) and RFBR

(grant 02-04-49890).

References1 Singletary, S.E. (2003) Rating the risk factors for breast cancer. Ann. Surg.

237, 474482

2 McPherson, K. et al. (2000) ABC of breast diseases. Breast cancer-

epidemiology, risk factors, and genetics. Br. Med. J. 321, 624628

3 Clemons, M. and Goss, P. (2001) Estrogen and the risk of breast cancer. N.

Engl. J. Med. 344, 276285

4 Gerber, B. et al. (2003) Nutrition and lifestyle factors on the risk of

developing breast cancer. Breast Cancer Res. Treat. 79, 265276

5 Iau, P.T. et al. (2001) Germ line mutations associated with breast cancer

susceptibility. Eur. J. Cancer 37, 300321

6 Colleu-Durel, S. et al. (2004) Alkaline single-cell gel electrophoresis

(comet assay): a simple technique to show genomic instability in sporadic

breast cancer. Eur. J. Cancer 40, 445451

7 Imyanitov, E.N. et al. (2004) Searching for cancer-associated gene poly-

morphisms: promises and obstacles. Cancer Lett. 204, 314

8 Mant, C. and Cason, J. (2004) A human murine mammary tumour virus-

like agent is an unconvincing aetiological agent for human breast cancer.

Rev. Med. Virol. 14, 169177

9 Mant, C. et al. (2004) A viral aetiology for breast cancer: time to re-

examine the postulate. Intervirology 47, 213

10 Nathanson, K.L. et al. (2001) Breast cancer genetics: what we know and

what we need. Nat. Med. 7, 552556

11 Antoniou, A. et al. (2003) Average risks of breast and ovarian cancer

associated with BRCA1 or BRCA2 mutations detected in case Series

unselected for family history: a combined analysis of 22 studies. Am. J.

Hum. Genet 72, 11171130

12 Lerebours, F. and Lidereau, R. (2002) Molecular alterations in sporadic

breast cancer. Crit. Rev. Oncol. Hematol. 44, 121141

13 Widschwendter, M. and Jones, P.A. (2002) DNA methylation and breast

carcinogenesis. Oncogene 21, 54625482

14 Okochi, E. et al. (2002) Single nucleotide instability: a wide involvement in

human and rat mammary carcinogenesis? Mutat. Res. 506507, 101111

15 Imyanitov, E.N. and Hanson, K.P. (2003) Molecular pathogenesis of

bilateral breast cancer. Cancer Lett. 191, 17

16 Bianchi, N.O. et al. (2001) Mitochondrial genome instability in human

cancers. Mutat. Res. 488, 923

17 Bertucci, F. et al. (2003) Breast cancer revisited using DNA array-basedgene expression profiling. Int. J. Cancer 103, 565571

18 Miller, B.J. et al. (2003) Pooled analysis of loss of heterozygosity in breast

cancer: a genome scan provides comparative evidence for multiple tumor

suppressors and identifies novel candidate regions. Am. J. Hum. Genet. 73,

748767

19 Tomlinson, I.P. et al. (2002) Loss of heterozygosity analysis: practically

and conceptually flawed? Genes Chromosomes Cancer 34, 349353

20 Gasco, M. et al. (2002) The p53 pathway in breast cancer. Breast Cancer

Res. 4, 7076

21 Hanahan, D. and Weinberg, R.A. (2000) The hallmarks of cancer. Cell 100,

5770

22 Moinfar, F. et al. (2000) Concurrent and independent genetic alterations in

the stromal and epithelial cells of mammary carcinoma: implications for

tumorigenesis. Cancer Res. 60, 25622566

23 Wiseman, B.S. and Werb, Z. (2002) Stromal effects on mammary gland

development and breast cancer. Science 296, 10461049

24 ORegan, R.M. and Jordan, V.C. (2002) The evolution of tamoxifen

therapy in breast cancer: selective oestrogen-receptor modulators and

downregulators. Lancet Oncol. 3, 207214

25 Sorlie, T. et al. (2003) Repeated observation of breast tumor subtypes in

independent gene expression data sets. Proc. Natl. Acad. Sci. USA 100,

84188423

26 Mass, R.D. (2004) The HER receptor family: a rich target for therapeutic

development. Int. J. Radiat. Oncol. Biol. Phys. 58, 932940

27 Arun, B. and Goss, P. (2004) The role of COX-2 inhibition in breast cancer

treatment and prevention. Semin. Oncol. 31 (Suppl. 7), 2229

28 Cristofanilli, M. and Hortobagyi, G.N. (2002) Molecular targets in breast

cancer: current status and future directions. Endocr. Relat. Cancer 9, 249

266

29 Fischer, O.M. et al. (2003) Beyond Herceptin and Gleevec. Curr. Opin.

Chem. Biol. 7, 490495

30 Proceedings of the 40th Annual Meeting of American Society of Clinical

Oncology, 58 June 2004. New Orleans, USA (http://www.aso.org)

31 de Bono, J.S. et al. (2003) Farnesyltransferase inhibitors and their potential

in the treatment of breast carcinoma. Semin. Oncol. 30 (Suppl. 16), 7992

32 McLaughlin, F. et al. (2003) The cell cycle, chromatin and cancer:

mechanism-based therapeutics come of age. Drug Discov. Today 8,

793802

33 de Bono, J.S. et al. (2003) The future of cytotoxic therapy: selective

cytotoxicity based on biology is the key. Breast Cancer Res. 5, 154159

34 Lo, S. and Johnston, S.R. (2003) Novel systemic therapies for breast

cancer. Surg. Oncol. 12, 277287

35 Mokbel, K. (2000) The role of telomerase in breast cancer. Eur. J. Surg.

Oncol. 26, 509514

36 Clevenger, C.V. et al. (2003) The role of prolactin in mammary carcinoma.

Endocr. Rev. 24, 127

37 Muller, A. et al. (2001) Involvement of chemokine receptors in breast

cancer metastasis. Nature 410, 5056

38 Esteva, F.J. et al. (2001) Chemotherapy of metastatic breast cancer: what to

expect in 2001 and beyond. Oncologist 6, 133146

39 Reff, M.E. and Heard, C. (2001) A review of modifications to recombinant

antibodies: attempt to increase efficacy in oncology applications. Crit. Rev.

Oncol. Hematol. 40, 2535

40 Tazebay, U.H. et al. (2000) The mammary gland iodide transporter is

expressed during lactation and in breast cancer. Nat. Med. 6, 871878

41 Blagosklonny, M.V. (2003) Matching targets for selective cancer therapy.

Drug Discov. Today 8, 11041107

42 Heim, S. et al. (2001) Are some breast carcinomas polyclonal in origin? J.

Pathol. 194, 395397

43 Al-Hajj, M. et al. (2003) Prospective identification of tumorigenic breast

cancer cells. Proc. Natl. Acad. Sci. USA 100, 39833988

44 Liu, E.T. (2003) Classification of cancers by expression profiling. Curr.

Opin. Genet. Dev. 13, 97103

45 Blagosklonny, M.V. (2003) Tissue-selective therapy of cancer. Br. J.

Cancer 89, 11471151

Vol. 1, No. 2 2004 Drug Discovery Today: Disease Mechanisms | Cancerwww.drugdiscoverytoday.com 245

Mechanisms of breast cancerIntroductionContribution of inherited featuresSomatic events in breast cancer pathogenesisThe web of signaling cascadesTargeted therapiesTowards curable breast cancer: how long is the road?Choice of the targets: cancer-specific or breast-specific?Summary and conclusionsAcknowledgementsReferences