Tyrosine kinase inhibitors: Rationale, mechanisms of action, and implications for drug resistance

Tyrosine

Kinase Inhibitors: Rationale, Mechanisms and Implications for Drug Resistance Dagmar

Tyrosine tory

kinases

processes.

play

gression. ing and

chemistry

oncogenic

have

are others

tyrosine

highly can

one

oncogenic

cellular

to

the

and

Some

kinase for

host

may

have

clinical

stages several

will

comkinase, kinase

At a practical level, the inhibitors against more

these small molecules are in different ical and clinical development against and

of

adenosine domain of

of these

toxicity.

Semin Oncol W.B. Saunders

screenand

identification

with the catalytic

tissue

mors

ac-

can be and pro-

to a single tyrosine several homologous

tyrosine

Anne

regulakinase

high-throughput computational,

kinases.

specific inhibit

as implications

led

Yakes,

tyrosine

compete site of the

pockets simultaneously. tive promiscuity of these as well

in normal

cellular transformation with tumor maintenance

small molecules that triphosphate binding pounds while

F. Michael

aberrant

In the last few years, the use of combinatorial,

medicinal

several

a role

However,

tivity can lead to causally associated

Busse,

relathan merit

Many

of

of preclinsolid tu-

be discussed. 28 (suppl Company.

16):47-55.

Copyright

0

2001

by

M

OST proto-oncogenes encode proteins that participate in signaling pathways by which cells receive and execute instructions that lead to mitogenesis, differentiation, lineage determination, migration, and apoptosis. One of the largest families of proto-oncogenes encodes transmembrane growth factor receptors that exhibit an extracellular ligand-binding domain and a cytoplasmic intrinsic tyrosine kinase activity. Upon ligand binding, these receptors dimerize and undergo a conformational change that leads to tyrosine kinase activation and receptor autophosphorylation on tyrosine residues within the cytoplasmic domain. Other cellular substrates, adaptor proteins, and cytosolic tyrosine kinases bind to phosphorylated tyrosines in the receptor, leading to a plethora of signaling pathways that mediate the proliferative and survival effects of the proto-oncogenesr-3 The large number of oncogenic protein tyrosine kinases plus the rare presence of phosphotyrosine in nontransformed cells argue persuasively that tyrosine phosphorylation is a critical event in growth control and transformation. Many human tumors overexpress single or multiple proto-oncogenes that encode receptor tyrosine kinases (Fig 1). In general, these tumor cell receptor tyrosine kinases exhibit aberrant constitutive activity as a result of mutational activation, high Seminars in Oncology, Vol 28, No 5. Suppl 16 (October),

2001: pp 47-55

E.G.

Lenferink,

and Carlos

of Action,

L. Arteaga

receptor density perhaps leading to increased inter-receptor collisions and kinase activation, and/or autocrine receptor ligands made in excess by the tumor cells. Overall, mutational inactivation of these tyrosine kinases leads to abrogation of the full range of the receptors’ biochemical responses and inhibition of the proliferative and transforming potential of these proto-oncogenes. This apparent requirement of the tyrosine kinase activity has led to the development of small molecules that mimic adenosine triphosphate (ATE’) as an anticancer strategy. These ATP-mimetics compete with ATP for binding to the receptors’ tyrosine kinase pocket and thus disable their function. Several recent reviews cover in some detail the many small-molecule tyrosine kinase inhibitors currently in preclinical and clinical development4,s THE HERZlneu (erbB-2) TYROSINE KINASE: ROLE IN HERlerbB RECEPTOR SIGNALING AND TRANSFORMATION The HER/erbB family of receptors includes the epidermal growth factor receptor (EGFR, HERl, erbB- l), the orphan HERZ/neu (erbB-2), and neuregulin/heregulin receptors HER3 (erbB-3) and HER4 (erbB-4).6-g The ectodomain of EGFR, HER3, and HER4 interacts with a specific set of

From the Departments of Medicine and Cell Biology, Vanderbilt University School of Medicine, N&Ale; the Department of Veterans Affairs Medical Center, Nashwilk; and Vanderbilt-Ingiam Cancer Center, Nashville, TN. Supported by National Institutes of Health grant no. R01 CA801 95, a Clinical Investigator Award from the Department of Veterans Affairs, and Vanderbilt-Ingram Cancer Center support grunt no. CA68545 (CLA). Dr Busse is a postdoctoral research fellow supported by the Robert Bosch Foundation (Stuttgart, Germany). Dr Lenferink is the recipient of a postdoctoral research fellowship award from the Susan G. Komen Breast Cancer Fotindation. Address reprint requests to Carlos L. Arteugu, MD, Division of Oncology, Vanderbilt University School oj Medicine, 777 Preston Res. Bldg, Nashville, TN 37232-6307. Copyright 0 2001 by W.B. Saunders Company 0093-7754/01/2805-1608$35.00/O doi:10.1053/sonc.2001.28550 47

48

BUSSE ET AL

extracellular region

Fit-1 Flk-1

II

EGF Receptor Subfamily EGFR NElJ ERBBS

. Flk-1 Flk-Z&It-3)

intracellular region Diagram of membrane-spanning Fig I. pressed and/or mutated in several human of the receptors.

receptor cancer

protein syndromes.

tyrosine Tyrosine

soluble ligands, whereas no ligand has been identified so far for the orphan HER2 receptor. Binding of ligands to EGFR, HER3, and HER4 results in the formation of homodimeric and heterodimeric receptor complexes, into which HER2 is recruited as a preferred partner. 10~1 Consequently, there is tyrosine kinase activation, receptor autophosphorylation of specific C-terminal tyrosine residues, and recruitment to those C-terminus phosphotyrosines of specific second messengers that, in turn, activate a plethora of intracellular signaling pathways that play central roles in cell proliferation, development, differentiation, migration, and oncogenesis.6 Cytoplasmic signal transducers induced by HER/erbB receptor signaling include the p42 and p44 mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-OH kinase (PUK) activity, the stress-activated protein kinases, phospholipase C-yl, c-Src, and the latent cytoplasmic transcription factors designated signal transducers and activators of transcription.697J2 Many studies support a pivotal role for HER2 in signaling by the HER/erbB family of receptors and suggesting that this kinase in tumorigenesis,sxg plays a critical role in the cellular responses mediated by the ligand-dependent activation of other HER coreceptors. Indeed, overexpression of HER2

kinares.

Several

of these

molecules

kinase

catalytic

activity

is located

are

known

to be overex-

in the cytoplasmic

domain

alone or in combination with EGFR or HER3 in vitro can transform mammary epithelial cells and fibroblasts.13J4 A central role for HERZ/neu kinase in transformation has been shown using transgenic mice overexpressing the neu proto-oncogene under the control of the mouse mammary tumor virus (MMTV) promoter. These mice develop stochastic mammary tumors that eventually become metastatic.15 In these tumors, DNA sequence analysis showed the presence of a 16-amino-acid in-frame deletion in the extracellular domain of neu, resulting in a constitutively activated receptor capable of transforming Rat-l fibroblasts.l6 So far, no such activating mutations have been found in human tumors, where the most common change involving the HER2/neu proto-oncogene is mRNA and protein overexpression with or without gene amplification. Nonetheless, clinical surveys have shown overexpression of nonmutant HER2/neu in a subset of epithelial neoplasms with a particularly virulent behavior.17 Moreover, antibodies against the ectodomain of HER2/neu can alter the natural history of breast carcinomas that overexpress the proto-oncogene. ls These observations provide evidence for a critical role of this receptor in mammary transformation and tumor progression.

INHIBITION

THE

OF THE HER2 KINASE

IN BREAST CANCER

HERZlneu TYROSINE KINASE THERAPEUTIC TARGET IN HUMAN CANCERS

AS A

Multiple experimental data support the use of the HER2/neu proto-oncogene product as a rational molecular target for antitumor interventions in breast and other epithelial neoplasms. The 185-kd HER2 membrane-spanning tyrosine kinase is differentially overexpressed in tumor versus host tissues and can be measured by conventional methods in diagnostic tumor material. Although the HER2 gene is critical in development, it still lacks a well-defined role in normal adult physiology, suggesting that HER2 blockade may spare normal tissues in the adult tumor host. In addition, experiments with different inhibitors of HER2 function have shown that, in tumor cells that overexpress HER2, this receptor is causally related to enhanced tumor proliferation and/or viability both in vitro and in vivo.19 Inactivation of HER2 function has been shown to impair signaling induced by the HER coreceptor ligands epidermal growth factor (EGF) and neu differentiation factor (heregulin), supporting a critical role for HER2 in the cellular effects contributed by other members of the HER/erbB family of receptors,20 some of which are also overexpressed in human cancers. Moreover, HER2 can enhance the binding affinity of EGF and EGF-like ligands and heregulins to their cognate HER receptor&a7 and prolong the stability of EGFR/HER2 heterodimersi0~11~21 thus enhancing the signaling potency of EGFR ligands. Taken together, these arguments indicate that (1) one can select appropriate candidate patients bearing HER2-overexpressing cancers in whom anti-HER2 therapies might be effective; (2) there is a therapeutic window between tumor and nontumor host tissues for HER2-directed interventions; and (3) by disabling HER2, one can significantly affect the whole HER/erbB receptor signaling network. This last point might be of particular clinical relevance in tumors that simultaneously overexpress receptor ligands and several HER/erbB family members. Recent reviews have addressed these scientific arguments as well as the multiple experimental and clinical approaches, in different stages of development, that are being used to abrogate the engagement of HER2 signaling.4,2z In this report, data relevant to the use of

HER2 tyrosine kinase inhibitors, mostly in experimental breast cancer systems, will be presented. HERZlneu

TYROSINE

KINASE

INHIBITORS

One strategy for blocking HER2 function has been the development of HERZ-specific competitive ATP inhibitors that, by binding to the receptor’s ATP site, will block activation of its tyrosine kinase activity. Although many small-molecule inhibitors of the homologous EGFR tyrosine kinase have been described,4J that does not appear to be the case for the HER2/neu kinase. Fry et a123 reported a series of small-molecule quinazolines that covalently bind Cys773, a unique residue positioned within the ATP-binding pocket of EGFR and HER2. This resulted in blockade of ligand-induced phosphorylation of both EGFR and HER2, with a 50% inhibitory concentration (IC,,) in the nanomolar range. Efficacy against human tumor cell lines that naturally overexpress HER2 was not reported in this study.23 In addition, PD158780, a pyridopyrimidine that inhibits the purified EGFR kinase, was found to block heregulinstimulated phosphorylation of HER2 in SKBR-3 and MDA-453 cells, with an ICso of approximately 50 nmol/L.Z4 Unfortunately, the effect on human tumor cells with high levels of HER2 receptors in the absence of added ligands, a relevant preclinical experimental endpoint, was not addressed in these reports. Nonetheless, it should be noted that some of the EGFR-specific small-molecule inhibitors have been reported to inhibit the growth of HERZoverexpressing cancer cells.z2J5-27 These inhibitors, and their IC,, values against both the EGFR and HER2 kinases in vitro, are listed in Table 1. Most of the EGFR data were generated using purified EGFR as a substrate in vitro for these characterization studies. In general, the reported ICs, against HER2 was at least lOO-fold higher than that for the EGFR kinase. However, the methods or systems used to generate the IC,, against HER2 do not always appear comparable with those used for EGFR or, in some cases, were not clearly described. These methods/systems included phosphorylation of cytoplasmic HER2 in EGF-stimulated fibroblasts transfected with expression vectors encoding EGFR/HER:! chimeras, heregulin-stimulated phosphorylation of HER2 in breast tumor cells with high levels of HER2, or phosphorylation of a

50

BUSSE ET AL

Small M0lWlle

EGFR IC,, (pmol/L)*

AG- I478 AG-15177

40.003 0.0009

PD I530357 ZD I839

0.029 I 0.005 0.033

09-774 PDl68393# PD I58780

HER2 IC,, (PmollL)

0.02 0.0007 5 0.00009 0.000008

Reference(s)

I .4t Not repotted

5, 25 25

2.3t >3.7+

28 29

Not reported 5.7 +- 0.8s

30 23

O.O5j(

24

* Most values reflect the IC,, using purified EGFR in vitro as a substrate. t Personal communication,

Laura Shawver, Sugen, Inc (South

San Francisco, CA), 1998. $ Effect on purified HER2 kinase in vitro. 5 Effect on heregulin-mediated

phosphorylation

(of HER2).

11Effect on heregulin-stimulated phosphorylation (of HERZ) in SKBR-3 and MDA-453 cells. % These two quinazolines have the same structure (refs 4 and 28). #Only

reported

irreversible

inhibitor.

purified HER2 kinase in vitro. Despite the low nanomolar to picomolar IC,, values of some of these small molecules against the EGFR kinase, the IC,, for ligand-independent growth of EGFR-overexpressing intact cultured cells was in general around 0.1 pmol/ L.zsJ6J*,30 In some of these reports, and in contrast to the large difference between the in vitro IC,, values of these small molecules for both receptors, some HER2-overexpressing tumor cells also were inhibited by concentrations well below 1 pmol/L, not very different from those required to inhibit EGFRdependent ce11s.z2J5-27 Table 2 shows data generated with the smallmolecule quinazoline EGFR kinase inhibitor AG1517 (kindly provided by L.K. Shawver, Sugen, Inc, South Francisco, CA) and a panel of human tumor cell lines. A total of 3 X lo4 cells from each of the indicated cell lines were plated in 0.8% agarose, IMEM/lO% FCS, 10 mmol/L Hepes in 35mm dishes in the absence or presence of 0.01 to 10 PM AG- 15 17, as previously described.25J7 After a 7-day incubation in a humidified 5% CO, incubator at 37°C colonies measuring 60 pm were counted using an OMNICON 3800 tumor colony analyzer (BioLogics, Gainesville, VA). Each data point was derived as the mean * standard devia-

tion of triplicate dishes and the IC,, calculated accordingly. This inhibitor has the same structure as PD1530354,2s and exhibits an in vitro IC,, against the EGFR kinase of 0.9 nmol/L (L.K. Shawver, personal communication, October 1998). The A43 1 squamous cancer and the MDA468 human breast cancer cell lines exhibit EGFR gene amplification, secrete transforming growth factor-a, and express autoactivated EGFR in the absence of added ligands.31 Although 0.1 pmol/L AG- 15 17 was enough to block basal EGFR phosphorylation in both cell lines (data not show$i), the ICs, in a colony-forming assay against A431 cells was almost lo-fold lower than that for MDA468 cells (Table 2). BT-474 and SKBR-3 are human breast tumor cell lines that exhibit HER2 gene amplification, constitutive tyrosine phosphorylation of HER2,14 and approximately lo5 EGF binding sites per cell.32233 These two cell lines are HER2-dependent in that antibodies against the ectodomain of HER2 markedly inhibit their proliferation and/or survival both in vitro and in vivo.19 Despite having approximately 15fold lower EGFR levels than MDA-468 cells, both HER2-amplified cell lines were exquisitely sensitive to this EGF’R-specific kinase inhibitor with an IC,, close to that observed in A43 1 cells (Table 2). We have obtained similar data with ZD1839, another small-molecule quinazoline inhibitor of the EGFR.22 Although the in vitro IC,, of ZD1839 against HER2 is greater than 3.7 pmol/L (Table l), 1 pmol/L ZD1839 completely eliminated detectable phosphotyrosine in HER2 precipitates from SKBR-3 and BT-474 cells.z2 In addition, the IC,, of ZD1839 in a colony-forming assay against both of these cell lines was approximately 0.2 pmol/L (data not shown). This marked inhibitory effect of nanomolar concentrations of EGFRspecific inhibitors against HER2-dependent cells

Cell Line A431 MDA-468 BT-474 SKBR-3 I

EGFR (HERI)

HER2lneu

IC,,

(/allollL) 0.08

2x I06 1.5 x I06 I x I05

LOW LOW Amplified

0.6 0.2

I x 105

Amplified

0.2 I

INHIBITION

OF THE HER2 KINASE

51

IN BREAST CANCER

would not have been predicted by the initial in vitro screens that tested for receptor specificity. These data suggest that (1) the IC,, of kinase inhibitors against intact cells (in vivo) is higher than that predicted by the in vitro IC,, derived from screens using purified receptors; (2) EGFR levels do not determine the potency of EGFR kinase inhibitors; (3) in vitro screens using recombinant or purified receptors may not be completely predictive of the effect of tyrosine kinase inhibitors against those same receptor targets in intact cells; and (4) low nanomolar concentrations of EGFR-specific inhibitors are quite effective against HER2-overexpressing tumor cells that also express EGFR. Other biological arguments also favor the testing of EGFR inhibitors to block HER2 signaling. Simultaneous overexpression of both EGFR and HER2 is associated with enhanced cell transformation and with breast cancers of a more virulent behavior compared with tumors that overexpress either receptor alone. 13j34 In most HER2-overexpressing cancer cell lines and in a cohort of primary human mammary tumors, the receptor is constitutively phosphorylated.i4Js This suggests that, in breast carcinomas, HER2 may be transactivated through ligand-activated EGFR, which also is present in the tumor cells. In addition, overexpression of HER2 can enhance the affinity of EGFR ligands to EGFR and prolong coreceptor signaling.10 HER2 is the preferred partner for the ligand-stimulated EGFR that, on recruitment of HER2 into an active EGFR/HERZ heterodimeric complex, exhibits prolonged stability.iiJi Thus, not surprisingly, HER2 inactivation has been shown to impair EGFR-mediated transformation20x36 and, conversely, EGFR-blocking antibodies can augment the growth inhibitory effect of anti-HER2 antibodies in cells with high levels of HER2.37 We recently reported that AG-1478, another quinazoline EGFR kinase small-molecule inhibitor, suppressed mammary tumors in MMTV/ neu + MMTV/TGFa bigenic mice,27 further suggesting that EGFR kinase inhibitors can inhibit tumor cell systems in which EGFR and HER2/neu cooperate. Taken together, these data suggest that EGFR/HERZ. crosstalk is operational in a subset of human and experimental tumors, and that interruption of EGFR may effectively reduce HER2 signaling and its potent transforming activity. If so, EGFR kinase inhibitors will be particularly

effective against HERZ-overexpressing tumors that also exhibit EGFR. This hypothesis should be testable with currently available clinical drugs. INHIBITION OF THE HER2 KINASE SUBVERTS HERZ-DEPENDENT SIGNALING PATH WAYS The engagement of HER/erbB receptors results in the activation of a large network of independent signaling pathways that subvert cell cycle checkpoints and pro-apoptotic molecules, thus leading to dysregulated cell cycle progression and enhanced tumor cell survival. Several reports indicate that, in transformed cells that naturally overexpress HER2 and/or in cells transfected with ectopic HER2, signaling by the proto-oncogene can subvert the G, to S transition by modulating the levels of cyclin Dl and the cyclin-dependent kinase (Cdk) inhibitor ~27~‘~~ through both MAPK and PI3K/Akt signaling pathways. The reader is referred for more details to several recent reports that support this statement.38~43 Additional supporting data are shown in Fig 2. Proliferating BT-474 cells exhibit constitutively active MAPK and Akt as measured by antibodies specific for phospho-Erkl/2 (MAPK) and phosphoSer473 Akt, respectively. A 24.hour treatment of these cells with 1 pmol/L ZD1839 results in an increase in the G, fraction from 74% to 88% and a reduction in the proportion of cells in S phase from 15% to 4%. This cell cycle arrest was completely reversible, in that on removal of the kinase inhibitor the cells synchronously entered into S phase 24 hours later (data not shown). Simultaneous with the recruitment in G,, there was complete elimination of both active Akt and active MAPK without changes in total Akt and total MAPK proteins. Consistent with the reduction in Akt activity, phosphorylation of glycogen synthase kinase (GSK)-3P, a target of the Akt kinase, was also reduced. Cyclin Dl and Cdk4 levels were reduced and p27 was upregulated, suggesting that these effects were required for the observed growth arrest. To test whether p27 played a role in the cell cycle arrest, we used an antisense oligonucleotide approach. Cytofectin-mediated delivery of G-clampmodified 15.mer antisense p27 phosphorothioates (a generous gift from W.M. Flanagan, Gilead Sciences, Foster City, CA) blocked the increase in p27 and the hypophosphorylation of retinoblastoma (Rb) induced by 1 to 5 Fmol/L AG-1478,

BUSSE ET AL

(pmol/L) P-Ser473 AM AM P-Serg GSK-3P GSK-3P P-MAPK MAPK cyclin D1 Cdk4 Cdk2

Fig 2. Dose-dependent effect of ZDl839 on intracellular signal transducers and cell cycle regulatory molecules. ET-474 human breast cancer cells were incubated for 24 hours in the absence or presence of I-I 0 PmollL ZD1839. Cells were washed twice with ice-cold phosphate-buffered saline and solubilized in EBC buffer as described previously.27~3’ Seventy-five micrograms of total cell protein were resolved by SDS-PAGE and analyzed by immunoblot using antibodies against P-Ser473 Akt, total AM, P-&r9 GSK-3/3, total MAPK (all from New England BioLabs, Beverly, MA), total GSK-3/3 and p27 (both from Transduction Laboratories, Lexington, KY), P-MAPK (Promega, Madison, WI), and Cdk4 and CdkZ (both from Santa Cruz Biotechnology, Santa Cruz, CA). Appropriate horseradish peroxidase-linked IgG (Amersham Pharmacia, Piscataway, NJ) were used as secondary antibodies. lmmunoreactive bands were detected by enhanced chemiluminescence

P27 whereas mismatch control oligonucleotides or cytofectin alone did not.43 In cells treated with cytofectin alone or mismatch oligonucleotides, 1 to 5 pmol/L AG-1478 induced an increase in the G, fraction (from 71% to 87/84%) and a decrease in the proportion of cells in S phase (from 18% to 6/l 1%). These did not occur in cells preincubated with antisense p27 oligonucleotides and in which the levels of p27 had been downregulated (Table 3).44 These results confirm a critical role for p27 in the G, arrest that follows blocking the HER2 kinase. Taken in context with other published reports, these results imply that (1) p27 is required for the growth arrest that results from blockade of the HER2 kinase, and (2) HERZ-overexpressing tumor cells use both MAPK and PI3K/Akt to modulate cyclin D l/Cdk4 complexes and p27 to subvert the G, to S transition. A proposed sequence of biochemical events in both proliferating and arrested

(Roche IN).

Molecular

Biochemicals,

Indianapolis,

cells is proposed in Fig 3. In brief, HER2 signaling activates MAPK and the PI3K/Akt pathways (Fig 3, top). MAPK phosphorylates ~27, contributing to its degradation, and increases the transcription of cyclin Dl. Akt inhibits GSK-3j3, thereby blocking the phosphorylation and degradation of cyclin Dl by GSK-3P and further contributing to higher steady-state levels of the G, cyclin. Higher levels of cyclin Dl/Cdk4 complexes sequester p27 away from cyclin E/Cdk2 complexes, which are now free to phosphorylate Rb in late G, and mediate progression into S. On the contrary, blockade of the HER2 kinase (Fig 3, bottom) inhibits both MAPK and PI3K/Akt signaling. The inhibition of MAPK activity reduces cyclin Dl transcription and relieves p27 from MAPK-mediated phosphorylation and degradation. Upon inactivation of Akt, GSK-3/3 is active to phosphorylate cyclin Dl, thus tagging it for proteasome-mediated degradation. The lower level of cyclin Dl/Cdk4 complexes

INHIBITION

OF THE

HER2

Table

KINASE

IN BREAST

3. Abrogation

CANCER

53

of Quinazoline-Mediated

With

Antisense

p27 Oligonucleotides

Oligos

NOW

MM

AS

NOW

MM

Cytofectin

NOM

+

+

+

+

+

+

AG- I478

NOW

I ~LmOllL

I PmollL

I wmol/L

5 ~tnOl/L

5 /urlollL

5 /utlol/L

G, S

71

85

77

67

87

84

71

18

9

I7

25

6

II

26

G,/M

II

6

6

8

7

NOTE. presence

BT-474

NON

G, Arrest

cells were left untreated

of 2 &mL

cytofectin

or transfected

with 30 nmol/L mismatch

GS38 I.5 (Gilead Sciences) as previously

with neither oligonucleotide

were still treated

with cytofectin

(control)

AG-1478.

24 hours,

or I to 5 km&L

histograms

were

Dickinson,

Mountain

Abbreviations:

generated

After

by flow cytometry

alone. Immediately

the cells were

for nonspecific

after the transfection

trypsinized,

of 15,000 stained nuclei as previously

5

or antisense phosphorothioate

described. 31 To control

their nuclei were described3’

AS

procedure,

3

27 oligonucleotides44

effects of cytofectin, cells were treated

labeled with propidium

using a FACYCalibur

in the

cells treated with DMSO

iodide, and DNA

flow cytometer

(Becton

View, CA).

MM, mismatch;

AS, antisense.

available for the sequestration of ~27 results in further accumulation of ~27, which can then bind cyclin E/CdkZ in excess, inhibit Cdk2 activity, and lead to G, arrest. IMPLICATIONS AND FUTURE

ErbE

W

Fig 3. Model for the regulation of p27 HER2-activated signals when the receptor active (top) or when its function is inhibited

and cyclin DI tyrosine kinase (bottom).

by is

FOR DRUG THERAPEUTIC

RESISTANCE DIRECTIONS

These data imply that functional elements of the Rb pathway are required for the antitumor effect of HER2targeted therapies. Therefore, tumors with loss or low levels of ~27, with cyclin Dl gene amplification, and/or mutational inactivation of Rb may not respond as well to anti-HER2 therapies compared with tumors with intact Rb and normal levels of cyclin Dl and ~27. In addition, if MAPK and Akt are obligatory targets whose disabling is required for the antitumor effect of a receptor-directed intervention to occur, hyperactivation of MAPK and/or PI3K/Akt secondary to parallel signaling pathways or to other genetic alterations may dampen the efficacy of HER2/neu kinase inhibitors. These may include mutation/deletions of the PTEN/MMACl gene, gene amplification of PUK or Akt isozymes, Ras mutations leading to hyperactive MAPK, or overexpression of other receptor tyrosine kinases (Fig 1) that also hyperactivate MAPK and/or PI3K/Akt. Based on these considerations, we speculate that tumor cells are likely to be naturally endowed with many potential mechanisms to by pass interventions targeted against a single molecular site within a complex signaling system. This leads to the testable hypothesis that targeting tyrosine kinase systems at multiple sites within their

54

BUSSE ET AL

signaling networks will exert a more effective and prolonged antitumor effect. With the emerging availability of specific inhibitors of the signal transducers downstream HER2, this hypothesis can now be tested in preclinical and clinical models.

monoclonal

CH:

Dimerization

of cell

surface

signal transduction. Cell 80213-223, 1995 2. Baserga R: Oncogenes and the strategy Cell 79:927-930, 1994 3. Hunter 127, 2000 4. Strawn

T: Signaling-2000 LM,

Shawver

and LK:

of growth

beyond.

Tyrosine

receptors

Cell

kinases

in

factors. 100:113-

in disease:

Overview of kinase inhibitors as therapeutic agents and current drugs in clinical trials. Exp Cpin Invest Drugs 7:553-573, 1998 5. Levitzki A, Gazit A: Tyrosine kinase inhibition: proach to drug development. Science 267:1782-1788, 6. Riese DJ, Stem erbB receptor family 1998 7. Ahoy ogenesis binatorial

I, Yarden

DF: Specificity within the EGF family/ signaling network. Bioessays 20:41-48, Y: The

and oncogenesis: ligand-receptor

1997 8. Tzahar

An ap1995

E, Yarden

erbB signaling

Signal diversification interactions. FEBS Y: The

tor of adenocarcinomas:

network

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erbB-Z/HER-2 orphanhood

in embry

through comLett 410:83-86, oncogenic

to multiple

recepstromal

ligands. Biochim Biophys Acta 1377:M25-M37, 1998 9. Pinkas-Kramarski R, Alroy I, Yarden Y: ErbB receptors and EGF-like ligands: Cell lineage determination and oncogenesis through combinatorial Biol Neopl 2:97-107, 1997 10. Karunagaran common auxiliary cations for breast 11. Graus-Porta preferred

signaling.

J Mammary

Gland

D, Tzahar E, Beerli RR, et al: ErbB-2 is a subunit of NDF and EGF receptors: Implicancer. EMBO J 15254-264, 1996 D, Beerli RR, Daly JM, et al: ErbB-2,

heterodimerization

partner

of all erbB

receptors,

who have HER-2.overexpressafter chemo17:2639-2648,

19. Baselga J, Norton L, Albanell J, et al: Recombinant humanized anti-HER-2 antibody (Herceptin) enhances the antitumor activity of paclitaxel and doxorubicin against HERoverexpressing

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28. Fry

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