REVIEW ARTICLE

Drugs 2000 Mar; 59 (3): 435-476 0012-6667/00/0003-0435/$25.00/0 © Adis International Limited. All rights reserved.

Kinase Inhibitors in Cancer Therapy A Look Ahead H.H. Sedlacek Aventis Pharma Deutschland GmbH, Central Biotechnology, Marburg, Germany

Contents Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Signalling Via Receptor Intrinsic Tyrosine Kinase Activity . . . . . . . . . . 1.1 Receptors for the Epidermal Growth Factor (EGF) Family . . . . . . . 1.2 Receptors for the Fibroblast Growth Factor (FGF) Family . . . . . . . 1.3 Receptors for the Platelet-Derived Growth Factor (PDGF) Family . . 1.3.1 PDGF Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.2 Vascular Endothelial Growth Factor (VEGF) Receptors . . . . 1.3.3 Cytostatic Factor (CSF)-1 Receptor . . . . . . . . . . . . . . . . 1.4 Receptors for the Insulin-Like Growth Factor (IGF) Family . . . . . . . 1.4.1 IGF-I Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4.2 Hepatocyte Growth Factor (HGF) Receptor . . . . . . . . . . 2. Signalling Via Receptor Intrinsic Serine/Threonine Kinase Activity . . . . . 2.1 Transforming Growth Factor (TGF) β Receptor Family . . . . . . . . . 3. Signalling Via Receptor-Associated Protein Kinases . . . . . . . . . . . . . 3.1 Src Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Janus Kinase (JAK) Family . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Focal Adhesion Kinase (Fak) . . . . . . . . . . . . . . . . . . . . . . . 3.4 Fps/Fes Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Cell Membrane to Nucleus Signalling Pathways Involving Protein Kinases 4.1 Raf Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Mitogen-Activated Protein Kinase (MAPK) Pathways . . . . . . . . . 4.3 Phosphatidylinositol-3-Kinase (PI3’K)-AKT/PKB Pathway . . . . . . . . 4.4 Nuclear Factor (NF)-κB Pathway . . . . . . . . . . . . . . . . . . . . . 5. Cyclin-Dependent Kinases (cdks) . . . . . . . . . . . . . . . . . . . . . . . 6. Search for Small Molecular Weight Kinase Inhibitors (SMOKIs) . . . . . . . 6.1 Key Target Kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 The Question of Specificity . . . . . . . . . . . . . . . . . . . . . . . . 6.3 The Question of Activity in the Cellular Environment . . . . . . . . . . 6.4 Efficacy in Human Tumours . . . . . . . . . . . . . . . . . . . . . . . .

Abstract

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The most essential kinases involved in cell membrane receptor activation, signal transduction and cell cycle control or programmed cell death and their interconections are reviewed. In tumours, the genes of many of those kinases are mutated or amplified or the proteins are overexpressed. The use of key kinases offers the possibility to screen in vitro for synthetic small molecule kinase inhibitors. In view of the many interconnections of cellular kinases, their role in preventing or inducing programmed cell death and the pos-

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sibility that a considerable number of signal transducing proteins are still unknown, cellular test systems are recommended in which the respective key kinase or one of its main partner molecules are overexpressed.

The survival of a cell is critically dependent on a delicate balance of mechanisms inducing or inhibiting programmed cell death (PCD) and/or stimulating cell proliferation. The mechanisms modulating PCD and cell proliferation are governed by cell membrane receptors and signal transducing proteins. Many of the crucial proteins and their genes have recently been characterised. A considerable number of these receptors and signal transducing proteins represent or are modulated by protein kinases and phosphatases. Protein kinases selectively transfer phosphate groups from ATP to protein substrates, thereby modulating their activity and/or their attachment sites for activating or downstream signalling molecules.[1] Over 175 protein kinases from mammalian sources have been described.[2] They represent the intrinsic function of a cell membrane receptor, or belong to those protein kinases associated with cell membrane receptors or loosely attached to the cell membrane through acylated anchors, or to the ones involved in the signalling pathways from the cell membrane to the nucleus (fig. 1) or in cell cycle control (table I). This increasing knowledge of the function of cell membrane receptors, various signalling pathways, and the mechanisms controlling the cell cy-

cle has provided increasing possibilities to search for low molecular weight compounds which are able to inhibit cell growth or to induce or to prevent PCD. Thereby three different attractive approaches have arisen. One approach aims at inhibiting the regulatory interaction of 2 or more proteins [e.g. the complex formation of Ras-GTP/Raf, ced4/Bcl2, nuclear factor (NF)-κB or cyclin-dependent kinases (cdks)/cyclins]. Another approach has the objective to inhibit the cellular expression (e.g, by antisense RNA or DNA molecules or by ribozymes) of a key protein involved in the signalling pathways. The third approach searches for inhibitors of the enzymatic activity of key protein kinases. Research to find kinase inhibitors started about 10 years ago by using growth receptor kinases for screening.[3] In the meantime, a considerable number of additional receptor-associated and/or signal transducing kinases have been identified, which provide attractive opportunities for pharmacological intervention. This paper reviews the most important cellular kinases with special attention to their key roles in the control of cell growth and/or PCD. The involvement of these kinases in tumour growth, and the prospects and design for future screening systems for their inhibitors are given.

Table I. Cyclin dependent kinases Kinase

Cyclin

Substrates pocket proteins

Biological role others

cell cycle

histone H1, lamin

S/G2/M

cdk1 (cdk2)

A& B

cdk2

A& E

pRb, p107, p130

cdk3

E

pRb, p107, p130

?

cdk4

D1, D2, D3

pRb (Ser-795)

G1 ?

cdk5

p35

?

cdk6

D1, D2, D3

pRb, p107, p130

cdk7 (CAK)

H

cdk8

C

G1/S ?

G1 Other cdks, RNA polymerase

?

other

G1/S/G2/M ?

? indicates still unknown.

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PCD

BAD

PLCγ

DAG

PCD

SAPK JNK1,2

p38

PCD

MKK4/ SEK1 JNKK1

MKK2/6

ASK

cdc42

CC

ERK1/2

MEK1/2

RAF

PKC

PCD

BAD

RAF/Bcl-2

DAG

CC

Bcl-2

RSK

FAK

G-protein coupled receptors

Neuropeptides, IL-8

MEKK1

Mitochondria stabilisation

PLCγ

FAK

Integrin receptors

Adhesive glycoproteins

Fps/ Fes/ Fer

CC

STAT complexes

Jak-1 Jak-2 Jak-3 Tyk2

Receptors with · box 1,2 · β-chains or · gp130

Hormones and cytokines

TRAF6

IRAK

IKB

IKK

Daxx

CC

TGFβ family

Smad 6,−7

JNK

ASK

JNK1,2

CC

Smad complexes

SAPK

PCD PCD

Smad4 Smad 1,−2,−3,−5,−9

PK

Receptors with intrinsic serine/ threonine kinase

NFKB PARP, Others

NFKB

Caspase cascade

Caspase 8 (2)

FADD TRADD RIP

FAS TNF-Rreceptor

TNF, FASL

TRAF2

NIK

IL-1 receptor

IL-1

Fig. 1. Protein kinases (encircled) involved in signal transduction. CC = cycling cells; FAS = CD95; FASL = FAS-ligand; IL = interleukin; PCD = programmed cell death; RTK = receptor tyrosine kinases; TNF = tumour necrosis factor; TGF = transforming growth factor.

CC

p70 S6K

AKT

P13'K

RAS

mSOS

SHC/GRB2

pp60 Src Fyn/ Hck/ Fgr Lck Zap70

Antigen binding receptors

Receptors with intrinsic tyrosine kinase

RTK

Antigens

Growth factors

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1. Signalling Via Receptor Intrinsic Tyrosine Kinase Activity At least 60 receptors with intrinsic protein tyrosine kinase (PTK) activity are already known.[4] Growth factor receptors with intrinsic tyrosine kinase activity are classified into several families [i.e. epidermal growth factor (EGF)-, fibroblast growth factor (FGF) receptor-, insulin-like growth factor (IGF) and platelet-derived growth factor (PDGF)].[5,6] They all own membrane-spanning polypeptides with extracellular growth factor binding amino terminal domains and a cytoplasmic carboxy terminal domain containing catalytic tyrosine kinase activity. In their active state the receptors for the EGF, FGF and PDGF families are monomeric. Receptor activation usually involves dimerisation induced by the binding of growth factor polypeptides or an increase in the association between already dimerized subunits. Ligand binding brings into close contact tyrosine kinase cytoplasmic domains resulting in the autophosphorylation and/or cross-phosphorylation[7] of several receptor C terminal tyrosine residues. Receptor tyrosine phosphorylation generates sites for binding of SH2 (Src homology-2) domain proteins including the c-Src tyrosine kinase, phospholipase Cγ (PLCγ), phosphatidylinositol-3-kinase (PI3’K), phosphatyrosine phosphatases, GTPase-activating protein (GAP),[8] and the growth factor receptor-binding protein 2 (GRB2).[9] Such SH2 domain proteins bind to tyrosine kinases through the interaction of their SH2 regulatory domains with specific kinase associated phosphotyrosine residues. The SH2 domain proteins can in turn function as substrates for these tyrosine kinases, e.g. the enzymatic activity PLCγ and PI3’K is enhanced following phosphorylation on serine and/or tyrosine residues. GRB2 is initially a cytoplasmic protein that exists in a heterodimeric complex with a guanine-nucleotide-releasing factor termed mammalian son of sevenless (mSOS).[10] PLCγ is in physical contact with the cytoplasmic domains of several growth factor receptors and is stimulated by tyrosine phosphorylation. PLCγ hydrolyzes membrane phospholipids to yield diacylglycerol (DAG) and inositol triphosphate which © Adis International Limited. All rights reserved.

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serve as second messengers. DAG activates protein kinase C (PKC) whereas inositol triphosphate activates release of Ca2+ from intracellular stores. Ca2+ activates the calmodulin/Ca2+ dependent kinases.[7] On binding to the phosphorylated PTK receptor, the receptor-GRB2-mSOS complex seems to bind the Ras-GDP complex, which (in addition to PKC) catalyses the Ras-activating exchange of GTP for GDP and thereby activates the Ras GTP/Raf dependent signal transduction process.[9,11,12] Many of the growth factor receptors seem to be involved in the growth of tumours. 1.1 Receptors for the Epidermal Growth Factor (EGF) Family

The EGF receptor (EGFR) serves to regulate the proliferation of multiple tissues.[13] Overexpression of EGFR results in EGF-dependent transformation.[14,15] Many tumours show an aberrant, enhanced and/or constitutive expression of EGFR (table II) and respond to EGFR binding growth factors [e.g. EGF, tranforming growth factor (TGF)α, amphiregulin, heparin-binding EGF, epiregulin and betacellulin].[115] Expression of high levels of EGFR in tumours strongly correlates with a poor prognosis. In breast tumours this correlation is independent on estrogen receptor status,[16] and the TGFα or amphiregulin expression does not seem to be related to EGFR expression.[116] The co-expression of EGFR-binding growth factors and EGFR frequently occurs in human carcinomas, suggesting that autocrine activation of the receptor might play a role in cancer cell growth.[117-119] The c-ErbB2 protein is a transmembrane protein with substantial homology to EGFR.[120] The normal function of the c-ErbB2 protein is not completely known.[115] The normal functions of the structurally related c-ErbB3 or c-ErbB4 receptors are even less certain. c-ErbB3 and c-ErbB4 bind heregulin and share closest overall similarity with EGFR and c-ErbB2.[121] Heterodimerisation among all ErbB family members can occur. Depending on the assembly of the heterodimeric receptor, the signal transduction may be different.[122] For example, an ErbB-ErbB2 dimer can stimulate transformed growth of mammary epDrugs 2000 Mar; 59 (3)

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Table II. Potential role of growth factor receptor kinases in tumour pathogenesis Growth factor family

Overexpression/ectopic expression in tumours

Epidermal growth factor (EGF) family (tyrosine kinase receptors) EGF-receptor

Mutation/amplification in tumours

References

Breast, glioma, squamous cell carcinoma (amplification)

Fitzpatrick et al[16]

Prostate, colon, ovary, bladder, stomach, pancreas

Gliomas Breast Lung (activating mutations) Stomach Prostate Breast Lung (adeno ca.) Pancreas Endometrium (activating mutations, amplification) Prostate (amplification)

p185 erb-2

p160 erb-3 SDGF receptor

Klijn et al.[17] Salomon et al.[18] Khazaie et al.[19] Aaronson[20] Fekete et al.[21] Srkalovic et al.[22] Korc et al.[23] Yamazaki et al.[24] Moscatello et al.[25] Wickstrand et al.[26] Myers et al.[27] Kern et al.[28] Hall et al.[29] Berchuck et al.[30] Bergman et al.[31] Salomon et al.[18] Myers et al.[27] Radinsky[32]

Not known

Fibroblast growth factor (FGF) family (tyrosine kinase receptors) FGF (1-5) receptors (1=flg, 2=bek, Pancreas 5=flg-2) Brease Melanoma KGF receptor (k-sam) (variant Melanoma form of FGFR2) Insulin-like growth factor (IGF) family (tyrosine kinase receptors) IGF-1 receptor Glioblastoma Ovary Pancreas Liver, kidney Prostate Melanoma Breast SC lung Leukemia Epidermoid Variants caused by alternative splicing variants caused by heterologous association with insulin receptor subunits HGF receptor c-met Melanoma Colon Pancreas Thyroid Colon

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Kobrin et al.[33] Rudland et al.[34] Kato et al.[35] Shih et al.[36]

Helle et al.[37] Macaulay[38] Bergmann et al.39] Baserga[40] Tanaka et al.[41], Ware[42] Rodeck et al.[43] Lee et al.[44] Moody et al.[45] Rubin et al.[46]

Stomach (amplification)

Ponzetto et al.[47] Halaban et al.[48] Radinsky et al.[32] Di Renzo et al.[49] Di Renzo et al.[50] Di Renzo et al.[50] Contd over page

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Table II. Potential role of growth factor receptor kinases in tumour pathogenesis Growth factor family Insulin receptor BDNF receptor (trk-B)

NGF receptor (trk-A) NT receptor (trk-C)

Overexpression/ectopic expression in tumours Not known Melanoma Prostate Brain Not known Not known

Mutation/amplification in tumours

Stracke et al.[51] Ware[42] Nakagawara et al.[52]

Platelet derived growth factor (PDGF) family (tyrosine kinase receptors) Melanoma PDGF receptor α Glioma

Sarcoma

PDGF receptor

Kaposi sarcoma Melanoma Sarcoma Liver Colon Leukemia (promyeolytic) Kaposi sarcoma Meningioma Glioma Breast

(tumour associated) endothelial cells CSF-1R (fms)

breast (activating mutations)

Breast

Head and neck Endometrium Lung Cervix Stomach melanoma (activating mutations)

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Qiu et al.[80]

Funasaka et al.[81] Natali et al.[82] Lassam & Bickford[83]

Melanoma Melanoma (downregulation, loss of expression) Vascular endothelial growth factor (VEGF) family VEGF receptor 1 (flt-1)

Shih & Herlyn[36] Hermansson et al.[53] Nister et al.[54,55] Claesson-Welsh et al.[56] Heldin et al.[57] Leveen et al.[58] Werner et al.[59] Shih & Herlyn[36] Heldin et al.[57] Leveen et al.[58] Tsai et al.[60] Herlyn et al.[61] Ito et al.[62] Pantazis et al.[63,64] Mäkelä et al.[65] Werner et al.[59] Maxwell et al.[66] Nister et al.[54,55] Claesson-Welsh et al.[56] Peres et al.[67] Ginsburg & Vonderhaar[68] Barrett et al.[69] Collins et al.[70] Roussel et al.[71] Yokoyama et al.[72] Suzuki et al.[73] Sapi et al.[74] Chambers et al.[75] Scholl et al.[76] Kacinski et al.[77] Leiserowitz et al.[78] Storga et al.[79] Storga et al.[79] Storga et al.[79]

Ovary

SCF receptor (kit, mast cell growth factor receptor)

References

chorion (truncated receptor)

Charnock-Jones et al.[84]

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Table II. Contd Growth factor family VEGF receptor 2 (KDR)

TIE-2

Overexpression/ectopic expression in tumours Melanoma Ovary Bladder Cervix Leukemia Breast Prostate Tumour associated endothelial cells

Eck Eph

Koura et al.[91] Brown et al.[92] Takahashi et al.[93] Berse et al.[94] Dhingra et al.[95] Stomach (inactivating mutations) Colon (microsatellite instability)

Prostate (inactivating mutations)

Chang et al.[96] Markowitz et al.[97] Kadin et al.[98] Kim et al.[99] Parsons et al.[100] Myeroff et al.[101] Kim et al.[99]

Easty et al.[102] Boyd et al.[103] Wicks et al.[104] Easty et al.[105] Easty et al.[105] Maru et al.[106]

Melanoma B cell ALL Melanoma Solid tumour endothelial cells Liver Lung

Membrane spanning PTKs without close relatives RET

References Gitay-Goren et al.[85] Boocock et al.[86] Brown et al.[87] Enomoto et al.[88] Katoh et al.[89] Soker et al.[90]

Breast

Transforming growth factor family (serine/threonine kinase receptors) TGF receptor II Head and neck Endometrium Leukemia Prostate Bone (reduced expression) TGF receptor I Eph PTK receptor family Hek

Mutation/amplification in tumours

Carlomagno et al.[107] Sugg et al.[108] Adrenal gland (pheochromocytomas) Takaya et al.[109] Ganglioneuroma Takaya et al.[109] Schwannoma Nakamura et al.[110] Neurofibroma Nakamura et al.[110] Neuroblastoma Nakamura et al.[110] Kidney (Wilms tumour) Ivanchuck et al.[111] Ros Glioblastoma Mapstone et al.[112] Glioma Watkins et al.[113] Astrocytoma Wu et al.[114] ALL = acute lymphocytic leukaemia; BDNF = brain-derived growth factor; CSF = cytostatic factor; FGFR2 = FGF receptor 2; HGF = hepatic growth factor; KDR = kinase insert domain-containing receptor; KGF = keratinocyte growth factor; NGF = nerve growth factor; NT = neurotrophin; PTK = protein tyrosine kinase; SCF = stem cell factor; SDGF = Schwannoma derived growth factor.

ithelial cells[123,124] and an ErbB-ErbB3 dimer can stimulate the P13’K enzyme.[125] In tumours, constitutive activation by point mutations[31] and amplification[18] have been found. Many tumours overexpress the c-ErbB2 gene product (table II).[115] © Adis International Limited. All rights reserved.

Thyroid (activating point mutation)

1.2 Receptors for the Fibroblast Growth Factor (FGF) Family

The FGR receptors (FGFR) include receptors for the acidic and basic FGFs, K-FGF/Hst, int-2, Drugs 2000 Mar; 59 (3)

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FGF, FGF8 and keratinocyte growth factor (KGF). FGFR are expressed on nearly all mesenchymal cells and in many epithelial cells. Five distinct, high affinity FGF receptors have been identified: FGFR1 (flg),[126] FGFR2 (bek),[127] FGFR3,[128] FGFR4[129] and FGFR5 (flg-2).[130] Variants can arise by different splicing. A switch from one spliced form to another spliced form may occur in apparent association with tumour progression.[131-132] Overexpression and ectopic expression of these receptors on various tumours (table II) are assumed to lead to autocrine and/or paracrine stimulation of tumour growth. In addition, the orthotopic expression of such receptors on activated endothelial cells enables FGF [in addition to vascular endothelial growth factor (VEGF)] produced from tumour cells[133-134] to stimulate angiogenesis for the blood supply in the proliferating areas of the tumours. Thus, inhibition of FGF (and/or VEGF) receptors may directly inhibit growth stimulation of tumours as well as inhibit tumour angiogenesis. 1.3 Receptors for the Platelet-Derived Growth Factor (PDGF) Family 1.3.1 PDGF Receptors

Two distinct PDGF receptor (PDGFR) types have been identified: the α-receptor binds all 3 isoforms of PDGF (PDGF-AA, -AB, BB) with high and about equal affinities, whereas the β-receptor only binds PDGF-BB with high affinity.[135] The latter receptor type also binds PDGF-AB, but with an approximately 10-fold lower affinity. PDGF-B/c-sis has a transforming potential which is stronger than that of PDGF-A.[136] Transformation is mediated by activation of the cells PDGFR. The ligandPDGFR interaction may take place intracellularly.[137-138] The subsequent intracellular activation of the receptor PTK can lead to cell transformation and generation of a mitotic signal. Possibly, for this to occur, the ligand receptor complex has to translocate to the plasma membrane in order to interact with the proper substrates for the receptor kinase. Another possibility is that the intracellularly activated PDGFR kinase elicits other cellular responses, distinct from those related to cell growth and trans© Adis International Limited. All rights reserved.

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formation.[139] The intracellular activation of PDGFR may be an important autocrine pathway of tumour growth. In addition, the paracrine stimulation of tumour growth has also been taken into consideration. PDGFR-α and -β receptors are not only overexpressed in several tumours (table II) but also in stroma cells surrounding tumours such as in basal cell carcinomas,[140] gliomas,[53] mammary carcinomas,[141] and other epithelial tumours.[142] Moreover, endothelial cells are known to express PDGFR. In an in vitro system to study angiogenesis, PDGF does promote capillary formation, possibly through its effect on endothelial cells and fibroblasts.[143] Tumour-derived PDGF may be instrumental in promoting angiogenesis in the primary or metastatic sites, particularly in those neoplasms which contain PDGF receptor overexpressing endothelium.[141,144,145] 1.3.2 Vascular Endothelial Growth Factor (VEGF) Receptors

VEGF-a, -B, -C, and -D bind to tyrosine kinase receptors. Three such VEGF receptors (VEGFR) have been identified: VEGFR-1 [the 180 kDa fmslike tyrosine kinase (Flt-1), which binds VEGF and VEGF-B],[146,147] VEGFR-2 [the 200 kDa kinase insert domain-containing receptor (KDR)[148] corresponding to its murine homologue, Flk-1,[149] which binds VEGF, VEGF-C and VEGF-D] and VEGFR-3 (a third structurally related tyrosine kinase receptor, 180 kDa Flt-4, which binds VEGF-C and VEGF-D, but not VEGF and VEGF-B).[150] VEGFR-2/KDR seems to be the major transducer of VEGF signals in endothelial cells that result in chemotaxis, mitogenicity, actin reorganisation, and morphological changes.[151-153] Although VEGFR-1/Flt-1 has a higher affinity for VEGF than VEGFR-2/KDR and is similarly phosphorylated in responses to VEGF, mitogenic response is less generated.[151] VEGFR-1/Flt-1 appears to be a much more important mediator of vascular formation. VEGFR-1/Flt-1 is localised to the endothelium in adult tissue[154] and seems to be involved in the endothelial cell assembly into blood vessels.[155] VEGF receptors are activated by the binding of VEGF dimers. Dimer binding leads to dimerisation Drugs 2000 Mar; 59 (3)

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of the receptors resulting in the autophosphorylation and/or cross-phosphorylation[7,156] of several receptor C terminal tyrosine residues. However, the signalling cascade that follows VEGF receptor activation is only partially understood. VEGF receptors do not have residues of recognition motif for PI3’K within the kinase insert and seem to utilise a signalling machinery in endothelial cells that is different to those (PI3’KAKT, RAS-RAF-MAPK pathways and/or the PLCγ-PKC-RAF-MAPK pathway) of the other members of the cytostatic factor (CSF)-1R/Kit/PDGFR-super family.[93] The SH2 and/or SH3 domain carrying proteins SHC,[157] Sck[158] and NCK[156-159] have been shown to bind to both VEGFR-1 and VEGRR-2 in a VEGF-dependent manner. VEGF receptor activation leads to the activation of PKC[160] and to phosphorylation of PLCγ, PI3’K and GAP.[156-160] Multimeric complexes of activated VEGF receptors with activated NCK, PLCγ and GAP proteins or activated PI3’K and NCK proteins have been observed, but further upstream events remain elusive. VEGF treatment of endothelial cell also leads to phosphorylation and nuclear translocation of mitogen-activated protein (MAP) kinases (MAPK),[160] but their coupling to the VEGFRs is not understood. Contradictory results which suggest that SH2 adapter proteins like SHC or other SRC family members or PI3’K are not major signal transducers of VEGFR-2 in endothelial cells,[93] mirror the incomplete understanding of VEGF receptor signal transduction in endothelial cells. Signal transduction induced by VEGF/ VEGF receptor complexes seems to be cell type specific and restricted to those cells physiologically expressing VEGF receptors, i.e. endothelial cells, haematopoietic stem cells, trophoblasts, monocytes and their derivatives.[161-163] Fibroblasts, genetically transduced to express VEGF receptors, can bind VEGF but do not show significant mitogenic response to VEGF. A comparative analysis of the VEGFR-1 or VEGFR-2 signalling cascade in endothelial cells and that of VEGFR-1 or VEGFR-2 expressing fibroblasts showed that VEGF–induced phosphorylation of PLCγ and GAP complex on tyrosine in both type of cells. However, a strong ac© Adis International Limited. All rights reserved.

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tivation of MAPK was observed only in endothelial cells. These results suggest that VEGFR-1 utilises a unique signal transduction system in endothelial cells.[93,164] VEGF has been considered to be the main endothelial cell-specific growth factor in angiogenesis.[165-167] However, VEGF also binds to hematopoietic stem cells and tumour cells (table II) and it stimulates the migration of monocytes and osteoblasts.[163,168] Many tumours express VEGF and thereby induce the tumour angiogenesis needed for tumour growth, as well as directly stimulate growth of VEGFR positive tumour cells. 1.3.3 Cytostatic Factor (CSF)-1 Receptor

The CSF-1 receptor (CSF-1R) is encoded by the c-fms protooncogene and is most closely related to the α- and β-isoforms of PDGF-R, the c-kit protooncogene product, and the receptor for basic fibroblast growth factor (FGF-R).[71,74,78] Many tumours overexpress CSF-1 and the CSF1R (table II) and thereby induce autocrine growth stimulation. 1.4 Receptors for the Insulin-Like Growth Factor (IGF) Family

The IGF-I receptor family includes the IGF-I receptor, the insulin receptor, the hepatic growth factor receptor (HGF-R), and receptors for nerve growth factor (NGF, trkA), brain-derived neurotrophic factor (BDNF, trk-B) and neurotrophin (NT, trkC). 1.4.1 IGF-I Receptor

The IGF-I receptor is a tetrameric protein composed of 2 α and 2 β subunits, linked by disulfide bonds.[169] The extracellular α subunit binds 3 different ligands, IGF-I, IGF-II and (with low affinity) insulin, whereas the transmembrane β subunit harbors the tyrosine kinase domain[170] and a ATPbinding site. The interaction of the IGF-I receptor with its ligands plays a major role in normal development and in the control of both normal and abnormal growth.[170,171] The IGF-I receptor is present in nearly all tissues and cells in culture[46] including fibroblasts, epithelial cells, smooth musDrugs 2000 Mar; 59 (3)

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cle cells, T lymphocytes, chondrocytes, and osteoblasts as well as the stem cells of bone marrow.[172,173] The mature IGF-I receptor exhibits significant heterogeneity between fetal and adult tissues, different tissues in the adult, and normal and neoplastic tissues,[46] partly caused by alternatively spliced βsubunits. Alternatively spliced human IGF-I receptor transcripts differ in their rate of receptor-mediated internalisation and corresponding rate of β-subunit receptor signalling. Receptor heterogeneity may also occur on the basis of altered glycosylation or posttranslational processing.[174,175] IGF-I receptor heterogeneity is also caused by heterologous association with insulin receptor subunits.[176,177] Such hybrid receptors may actually account for many reports of atypical receptors. The IGF-I receptor binds IGF-I with high affinity [dissociation constant (kd) = 1 nmol/L], and IGF-II and insulin with considerably lower affinities. Binding of IGF-I to the IGF-I receptor activates the intrinsic tyrosine kinase within the intracellular β-domain leading to tyrosine autophosphorylation by transphosphorylation of adjacent β-subunits, without additional receptor dimerisation.[46] Activation of the IGF-I receptor is primarily mitogenic. IGF-I receptor aggregation and internalisation within clathrincoated pits modulate this mitogenic potential.[46] The activation of Ras seems to be required for optimal cell proliferation in response to IGF-1. However, the IGF-I receptors do not directly bind adapter molecules via SH domains (as do the other growth factor receptors). Rather they phosphorylate and thereby activate a 185 kD molecule, termed insulin-related substrate-1 (IRS-1).[178,179] IRS-1 contains at least 20 tyrosines that are potential phosphorylation sites by the insulin receptor and presumably the IGF-I receptor. IRS-1 bears numerous SH domains and docks multiple SH groupcontaining adapter molecules. Molecules that bind IRS-1 include the Grb-2, Nck, the protein tyrosine phosphatase Syp (SH-PTP2) and the p85 β-subunit domain of P13’K.[27,180] For Ras activation the IRS1-linked pathway is initiated by binding of Grb2 to IRS-1 via SH3 domains. mSOS promotes Ras activation by enhancement of the GTP/GDP ex© Adis International Limited. All rights reserved.

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change. Activated Ras recruits Raf protein kinase, and the Ras GTP/Raf complex activate the MAPK pathway. The insulin and IGF-I receptors use a second direct substrate in the Shc (Src-homology/collagen) proteins. The Shc gene has transforming properties so it may play an important role in transformation by the IGF-I receptor. Shc can directly activate Grb2-SOS.[115,181,182] The IGF-I receptor also directly phosphorylates Crk,[183] a cellular homologue of v-crk. The family of Crk proteins bears SH2 and SH3 groups, shares homology with Grb2 and Nck, and interacts with the Ras-binding protein mSOS.[184] PDGF and EGF act synergistically with IGF-I. PDGF is known to induce an increase in the number of IGF-I receptors[185,186] via activation of the IGF-I receptor promoter.[187] EGF, instead, increases the expression of IGF-1 mRNA, accompanied by an increase in the production of IGF-1.[188] Moreover, oncogenes such as c-myb and c-myc have demonstrated to induce the expression of both IGF-I and IGF-I receptor mRNA and the expression of IGF-I receptor.[189,190] In addition, v-Src induces the tyrosine phosphorylation of the β-subunit of the IGF-I receptor, resulting in a constitutively active receptor state. The IGF-I receptor seems to play a significant role in the control of the growth of normal but also of tumour cells (see table II). Thus, interference with the function of the IGF-I receptor, e.g. by using antisense oligodeoxynucleotides to the IGF-I receptor RNA or IGF-peptides, leads to inhibition of cell growth of normal cells[191-194] but also of prostatic cancer cells,[195] glioblastoma cells,[196] and ovarian carcinoma cells.[197] 1.4.2 Hepatocyte Growth Factor (HGF) Receptor

The hepatocyte growth factor (HGF) is the ligand for the HGF receptor (HGFR). HGF/scatter factor (SF) is produced by mesenchymal cells and acts in an autocrine and paracrine way. HGF/SF is a mitogenic factor which stimulates growth of various normal as well as tumour cell types.[49,198] HGF/SF is also able to increase motility of epithelial cells,[199] endothelial cells[200,201] and of carcinoma cells,[202,203] and to enhance the invasiveness of carcinoma cells. The (c-MET-encoded) HGF receptor is heterodimeric (p190MET) composed of 2 disulfideDrugs 2000 Mar; 59 (3)

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linked chains, and extracellular α-chain and a transmembrane b-chain, showing tyrosine kinase activity.[204-205] Isoforms of the receptor, a membrane bound p140MET and a soluble p130MET, each containing the α-chain linked to a β-chain lacking the cytoplasmic kinase domain, have been described.[206] The 3 receptor variants originate by post-translational processing of a common single-chain precursor (p170).[207] Ligand binding to HGFR induces autophosphorylation. Phosphorylated HGFR binds to and phosphorylates signal transducing SH2 proteins.[208] c-MET was originally identified in a transfection assay as a rearranged oncogene after treatment of a cell line with a chemical carcinogen.[209] The c-MET proto-oncogene has also been found activated, amplified and/or overexpressed in various human tumours (see table II). 2. Signalling Via Receptor Intrinsic Serine/Threonine Kinase Activity 2.1 Transforming Growth Factor (TGF) β Receptor Family

Transforming growth factor (TGF) β is a pleiotrophic cytokine. Its predominant action is to inhibit cell growth and to increase extracellular matrix production. TGFβ is comprised of three related dimeric proteins (TGFβ-1, -2, -3) and belongs to a superfamily including activins, inhibins, Müllerianinhibiting substance (MIS) and bone morphogenic proteins (BMP).[210] Biological activity of TGFβ is mediated through binding to a high affinity heterotetrameric receptor complex comprised of R-I and/or R-II proteins. Both R-I and R-II present transmembrane proteins[211,212] that each associate with the accessory type III receptor (or endoglin, which does not bind TGFβ-2), a membrane anchored proteoglycan without signalling motif.[213] Type I and type II receptors are similar with intracellular parts consisting mainly of serine/threonine kinase domains. Each member of the TGFβ superfamily binds to a characteristic combination of type I and type II receptors both of which are needed for signalling. © Adis International Limited. All rights reserved.

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TGFβ-1 first binds to the type II receptor (R-II), which occurs in the cell membrane in an oligomeric form with activated kinase.[214] Then, the TGFβ type I receptor (R-I), which may also occur in an oligomeric form and cannot bind TGFβ in the absence of R-II, is recruited into the complex; R-II phosphorylates R-I in the glycine- and serine-rich region to activate it. The assembly of the receptor complex is triggered by ligand binding, but the complex is also stabilised by direct interaction between the cytoplasmic parts of the receptors. Finally, on binding of TGFβ-1 the signalling complex is a heterotetramer consisting of two R-I and two R-II molecules.[215] It is likely that other serine/ threonine kinase receptor complexes are also activated by a similar mechanism.[214] One of the TGFβ isoforms (TGFβ-2) binds only with low affinity to TGFβ type II receptor R-II (TGFβR-II) and requires the cooperation with TGFβR-I or betaglycan, an accessory transmembrane proteoglycan, for highaffinity binding.[210] Signal transduction is mediated via a complex interaction of signal mediating and signal inhibiting SMAD proteins. SMADs are vertebrate molecules homologous to Sma and mothers against dpp (Mad) with 2 regions at the amino and carboxy terminals, termed Mad-homology domains MH1 and MH2, respectively, which are connected with a proline-rich linker sequence. In their inactive configurations, the MH1 and MH2 domains of SMADs make contact with each other: after activation by receptors, the molecules open up, form hetero-oligomeric complexes and translocate to the nucleus where the transcription of target genes is affected. Different members of the SMAD family have different roles in signalling. Smad1, Smad2, Smad3 and possibly Smad5 interact with and become phosphorylated by specific type I serine/ threonine kinase receptors and thereby act in a pathway-restricted fashion. Smad2 and Smad3 (and possibly also Smad1) are phosphorylated after stimulation by TGFβ or activin.[214] Smad1 is phosphorylated after stimulation with BMP-2 or BMP4. Smad5 and Smad9/MADH6 may also be involved in BMPsignalling. Pathway-restricted SMADs are presumably released from the receptors after Drugs 2000 Mar; 59 (3)

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phosphorylation.[214] Subsequently, Smad4 forms hetero-oligomers with the phosphorylated tail of pathway-restricted SMADs, which in turn translocate into the nucleus and activate transcriptional responses.[214,216] Smad6 and Smad7 diverge structurally with their N terminal regions (36% identical between Smad6 and Smad7) from those of other SMADs:[214,217,218] Smad6 and Smad7 function as inhibitors of TGFβ, activin and BMP signalling. They bind to type I receptors and interfere with the phosphorylation of the pathway-restricted SMADs. Consequently, active heteromeric SMAD complexes are not formed. A requirement for binding of inhibitory SMADs to type I receptors is the activation of type I receptor by type II receptor kinase. However, inhibitory SMADs show a more stable interaction with type I receptors than do pathway-restricted SMADs. Transcription of inhibitory SMAD mRNA is induced by stimulation by TGFβ as well as by other stimuli. Thus, inhibitory SMADs may act as autoregulatory negative-feedback signals in the signal transduction of the TGFβ superfamily.[214,219] Most tumours derived from epithelial tissues as well as osteosarcoma and lymphomas express markedly reduced levels of TGFβ receptor proteins and/or demonstrate resistance to TGFβ mediated growth inhibition (see table II). Inactivating mutations of signal transducing Smad4 and Smad2 genes have been found in gastric carcinomas[220] and other tumours.[221] In addition, inhibition (or much more seldom, stimulation) of cell growth by TGFβ in mesenchymal nontransformed cells can occur through modulation of PDGF expression[222] or through a modulation of the expression of growth factor receptors. 3. Signalling Via Receptor-Associated Protein Kinases Cell membrane receptors lacking intrinsic kinase activity need association with and activation of membrane associated kinases for signalling. Such receptor-associated protein kinases can be different depending on the type of receptors (fig. 1). There seems to exist, however, no complete restriction, © Adis International Limited. All rights reserved.

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i.e. several growth factor receptors exhibiting intrinsic tyrosine kinase activity have been additionally shown to form heterodimeric complexes with multiple signalling and bridging molecules via (tyrosine) phosphorylated Src homology region interactions.[122] Isolated p60 c-Src SH2 domain can bind activated EGFR specifically and directly,[223,224] and is required for EGF-dependent mitogenesis.[225] p60 c-Src can phosphorylate the Tyr-845 site of the EGFR in addition to its autophosphorylation.[224] Moreover, the insulin receptor can be hyperactivated with the help of Src.[209] In addition, the phosphorylation of the EGFR or of HER-2/neu may involve other Src family members, or the Janus kinases (JAK).[226] On the other hand PTKs like c-Src can interact with substrates independent from the receptor. For example, p75/p85 cortactin and p190Rho-GAP are two c-Src substrates[227,228] that play roles in actin-based cytoskeletal reorganisation that accompanies mitogenesis and transformation.[114] 3.1 Src Family

Proteins of the Src family possess tyrosine kinase activity. Their association with the inner surface of the plasma membrane is mediated by their myristilated N termini, which are essential for function. In addition to their kinase (or Src homology domain 1 = SH1) they contain additional regions named SH2 and SH3 domains. SH2 domains are known to bind to sequences containing phosphotyrosine. Residues downstream of SH2 determine specificity of that binding. SH3 domains bind to motifs rich in proline residues.[229] SH2 can bind to negative regulatory phosphotyrosine sites on the same Src molecule and this can cause inhibition by self-association. Dephosphorylation of such sites enables Src family proteins to interact with receptors and signalling proteins. The Src family of PTKs includes p60 c-Src, yes (expressed in a variety of tissues, especially epithelial cells and brain), Fyn (mainly expressed in T- and B-cells), Hck (mainly expressed in monocytes and granulocytes), Fgr (mainly expressed in monocytes), and Lck (mainly expressed in T-cells).[229] As components of the reDrugs 2000 Mar; 59 (3)

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ceptor system their stimulation initiates a cascade of events leading to cellular proliferation,[7] e.g. after ligand-induced activation of the T-cell receptor (TCR) Lck and/or Fyn phosphorylate tyrosine based activation motifs located in cytosolic domains of the TCRζ and CD3 chain, that bind the PTK ZAP-70 (a syk family PTK), which in turn is phosphorylated and activated by Lck or Fyn.[230] Overexpression of proteins of the c-Src family is associated with a considerable number of tumour diseases (see table III) and may synergistically act with modified growth factor receptor in its oncogenic activity.[224,229] The c-Abl protein is a monomer that expresses tyrosine kinase activity, belongs to the Src family, and is a negative regulator of cell growth. Two isoforms of the mammalian c-Abl protein exist secondary to differential splicing of alternative first exons controlled by two promoters (P1 and P2) resulting in 2 distinct Abl proteins termed cAbl 1a and 1b.[274] Localisation to the membrane (of c-Abl 1b) appears to be dependent on the presence of a myristoyl group at the N terminus of the protein. c-Abl localises to the nucleus [via its C terminal nuclear localisation signal (NLS)] but is also seen in the cytoplasm when overexpressed.[275] Phosphorylation of the C terminal DNA binding domain by cdk1 kinase during mitosis (or by PKC) prevents its binding to DNA.[276] In cell cycle progression a phosphoryl/dephosphorylation cycle of c-Abl takes place.[277,278] The target proteins phosphorylated by c-Abl are not yet clearly identified particularly at a functional level. c-Abl is expressed ubiquitously in hematopoietic and other tissues with the exception of haploid reproductive cells.[279] Overexpression of c-Abl causes cell cycle arrest by induction of expression of p21 and downregulation of cdk2. Growth suppression requires PTK activity, nuclear localisation and an intact SH2 domain.[280,281] The linkage of Bcr to the N terminal end of c-Abl in chronic myelocytic leukaemia (CML) leads to an upregulation of the Abl associated tyrosine kinase activity of the chimeric protein. This change in enzyme activity seems to be © Adis International Limited. All rights reserved.

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central to the generation of the malignant phenotype of Bcr-Abl expressing cells. Cells transfected with the Bcr-Abl gene become tumourigenic in vivo. Growth of such tumours can be inhibited by PTK inhibitors.[282] 3.2 Janus Kinase (JAK) Family

The Janus kinase (JAK) family represents cytoplasmic PTK comprising JAK-1, JAK-2, JAK-3 and TYK2. Kinases of the JAK family comprise 5 N terminal boxes of homology (JH3-7) and at the C terminal end the tyrosine kinase domain.[283] Next to the N terminal is a second kinase-like domain which lacks several residues that are essential for kinase activity. JAK family kinases do not contain SH2 or SH3 domains, characteristic for the Src kinase family.[284] JAK family kinases are widely expressed in mammalian cells and seem to be involved in the transmission of signals downstream of several peptide hormone and cytokine receptors, which do not contain kinase domains (fig. 1). In the case of single chain receptors, [erythropoietin (EPO), growth hormone, prolactin and granulocyte colony-stimulating factor (G-CSF)] ligand binding induces receptor dimerisation and oligomerisation. This increases the affinity of the receptor for JAKs and results in a ligand-dependent increase of JAKs in the receptor complex. Association with JAK proteins occurs through the membrane-proximal cytoplasmic box 1 and box 2 motifs of the receptor.[285] The increased concentration of JAKs at the receptor site leads to crossphosphorylation of the ‘autophosphorylation’ site on JAKs, which is associated with activation of the kinase activity of the JAK proteins. The activated JAK proteins subsequently phosphorylate the receptor as well as cellular substrates that are recruited to the receptor kinase complex.[285] In the case of cytokine receptors with a common β-chain, i.e. receptors for interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-5, JAK2 activation following ligand binding requires the common β-chain and its membrane-proximal cytoplasmic domain.[286] After ligand binding these receptor complexes furDrugs 2000 Mar; 59 (3)

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Table III. Potential role of receptor associated cytoplasmic kinases in tumour pathogenesis Signal transducing kinase

Overexpression/ectopic expression in tumours

src family pp60 src

Mutation/amplification in tumours

Reference

Breast (amplification)

Cartwright et al.[231] Ottenholff-Kaliff et al.[232] Rosen et al.[233] Park et al.[234,235] Han et al.[236] Willman et al.[237] Park et al.[234,235] Han et al.[236] Krueger et al.[238] Seki et al.[239] Abts et al.[240] von Knethen et al.[241] Rouer et al.[242] Juecker et al.[243] Burnett et al.[244] Koga et al.[245] Nakamura et al.[246] Mayer et al.[247] Veilette et al.[248] McCracken et al.[249] Foss et al.[250] Veillette et al.[248] Nowell and Hungerford[251]

Colon Hck c-yes

AML

c-lck

CLL B-cell lymphoma Burkitt’s lymphoma T-cell ALL

Stomach (amplification)

Colon

Lung (SCLC) abl

CML (chromosomes 9 to 22)

c-fyn

Not known

c-fgr

Lung (SCLC) B-cell leukemia Burkitt’s lymphoma

c-lyn

AML CLL Lung (SCLC)

fes family c-fes/fps

Lung (all types)

c-fer JAK family JAK-3

JAK-2 JAK-1, -2 JAK-1, -2, TYK2

Not known Zarn et al.[252] Tesch et al.[253] Cheah et al.[254] Faulkner et al.[255] Sharp et al.[256] Willman et al.[237] Abts et al.[240] Zarn et al.[252] ALL (T) (activating mutation) AML (deletion) Myelodysplastic syndrome

ATLL (HTLV-1) (constitutive activation) ATLL (HTLV-1) (constitutive activation of STAT-1, STAT-3 and STAT-5) ALL (constitutive activation) Skeletal muscle (constitutive activation by midkine) Colon (constitutive activation by IL-4)

© Adis International Limited. All rights reserved.

Nishio et al.[257] Goldman et al.[258] Morris et al.[259]

Takemoto et al.[260] Takemoto et al.[260]

Meydan et al.[261] Ratoviski et al.[262] Murata et al.[263]

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Table III. Contd Signal transducing kinase FAK

Overexpression/ectopic expression in tumours Pancreas (downregulation in tumour-associated endothelial cells) Astrocytoma Medulloblastomas Sarcoma Breast

Mutation/amplification in tumours

Reference Okamoto et al.[264]

Hubbard et al.[265]

Weiner et al.[266] Weiner et al.[266] Owens et al.[267] Colon Weiner et al.[266] Han et al.[268] Owens et al.[267] Ovary Judson et al.[269] Kidney Jenq et al.[270] Prostate Tremblay et al.[271] Melanoma Akasaka et al.[272] Cervix (HPV18 positive) McCormack et al.[273] AML = acute myeloid leukaemia; ATLL = adult T cell leukaemia/lymphoma; CML = chronic myeloid leukaemia; JAK = Janus kinase; FAK = focal adhesion kinase; HPV = human papillomavirus; HTLV = human T cell lymphotropic virus; SCLC = small cell lung cancer.

ther dimerise and oligomerise, bringing JAKs into proximity through their association with the βchain. Although not involved in JAK binding, the cytoplasmic domains of the ligand specific α-chains are required for optimal dimerisation/ oligomerisation of receptor complexes.[285] Receptors for cytokines that include IL-6, oncostatin M (OSM), leukaemia inhibitor factor (LIF) and ciliary neurotrophic factor (CNTF), are characterised by utilising a common gp130 signalling subunit or a highly related LIF-receptor β-chain.[287] Ligand-induced aggregation of gp130 is essential for signal transduction of those receptors. JAK-1, JAK-2, and TYK2 associate with gp130. Their activation requires the membrane proximal cytoplasmic domain of gp130 containing the box 1 and box 2 motifs. Thereby, gp130 can associate with multiple members of the JAK family.[285] Interferon (IFN)–induced receptor complexes require JAK heterodimers for signal transduction. JAK-1 in combination with JAK-2 is required for an IFNγ response and JAK-1 in combination with TYK2 is required for an IFNα or IFNβ response.[288-290] In the case of IL-2 and IL-4 receptors,[285] JAK-1 and/or JAK-3[285,291,292] are specifically tyrosine phosphorylated and activated in response to IL-2 or IL-4. © Adis International Limited. All rights reserved.

JAK proteins seem to play an obligatory and nonredundant role in signal transduction of JAK activating receptors.[293-295] Signalling via activated JAK proteins is mediated via the tyrosine phosphorylation of the STAT (signal transducers and activators of transcription) molecules (-1, -2, -3, -4, -5, -6). JAK-1 phosphorylates STAT1, and STAT2 is specifically a substrate for TYK2. Proteins of the STAT family contain a carboxy terminal SH3 domain followed by an SH2 domain. In addition, sequences of homology are found in the amino terminal region of these proteins. A conserved tyrosine is found near the carboxyl terminus that is phosphorylated and is essential for function.[296] The STAT molecules exist as latent transcription factors in the cytoplasm. After binding of the specific growth factor or cytokine to its receptor, the STAT molecules are activated by tyrosine phosphorylation, migrate to the nucleus and bind to specific promoter elements. The 6 STAT family members form homo- or heterodimers in which the phosphotyrosine of one partner binds to the SH2 domain of the other. These dimers bind to palindromic gamma-activated sequences (GAS) that have similar affinities for different STATs. Different kinds of complexes are formed and the type of Drugs 2000 Mar; 59 (3)

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complexes seem to be a response specific to the receptor that has been activated.[297] The STAT3 protein associates with gp130 and JAK-1, and is rapidly tyrosine phosphorylated during the IL-6[298] or EPO response.[285] In the case of IFNα or IFNβ responses, the essential step in the regulation of gene transcription is the formation of a transcription complex, termed ISGF3, which binds to the interferon-stimulated response element (ISRE) and activates transcription. ISGF3 comprises 3 proteins including STAT1α (p91) or STAT1β (p84) and STAT2 (p113). Formation of the transcription complex ISGF3 and its migration to the nucleus require tyrosine phosphorylation of STAT1 and STAT2 proteins. Stimulation of cells with IFNγ results in the tyrosine phosphorylation of STAT1α but not of STAT2. Following phosphorylation, STAT1α migrates to the nucleus and participates in DNA binding complexes that recognise GAS in genes that are transcriptionally activated by IFNγ.[299] STAT4 is functionally like other members of the family in that it can be tyrosine phosphorylated by JAK-1 or JAK-2. In association with this phosphorylation STAT4 acquires the ability to bind the GAS element. Unlike other STAT family members, which are ubiquitously expressed, STAT4 is primarily expressed in haemopoietic cells. Surprisingly, STAT1 is also tyrosine phosphorylated in response to EGF,[300-302] IL-3 or GM-CSF,[285] which indicates the intriguing possibility that the JAKs might also be activated by receptor protein tyrosine kinases and thus might be generally involved in growth factor signal transduction. Many tumours show overexpression or constitutive expression of JAK (table III). 3.3 Focal Adhesion Kinase (Fak)

Focal adhesion kinase (Fak)[303] is a cytoplasmic PTK involved in cellular responses to adhesive glycoproteins (e.g. fibronectin, vitronectin, laminin, collagen IV and fibrinogen) that signal through integrin receptors and to mitogenic neuropeptides (e.g. bombesin, vasopressin and endothelin) that signal through G protein-coupled receptors. Exposure of cells to signalling molecules from either © Adis International Limited. All rights reserved.

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class leads to rapid increases in phosphorylated Fak and enzymatic activity, which is possibly an essential first step in the response pathway(s) activated by these agents. Fak is thought to regulate cell morphology and migration, establish anchorage dependency for cell proliferation, and transmit signals to the cell nucleus that alter gene transcription. Fak is a structurally unique cytoplasmic PTK and lacks SH2 and SH3 domains. A focal adhesion targeting sequence has been mapped near the C terminus. Tyr 397 is the major site of autophosporylation through which Fak can form a stable interaction with Src family kinase SH2 domains.[304] In vivo, Fak is phosphorylated at Tyr 397, -407, -576, -577 and other tyrosine and serine sites. Fak is expressed in a variety of adult tissues.[305] The highest expression of Fak can be seen in malignant metastasizing tumours (see table III). The expression pattern in malignant tissues can be very high and homogenous (breast and colon carcinoma) or variable. Some carcinomas do not express this kinase.[306] Expression of Fak in tumour cells is stimulated by bombesin,[307,308] HGF[309,310] and prolactin.[311] 3.4 Fps/Fes Family

The Fes/Fps family comprises Fps/Fes and the Fps-related PTK (Fer). Fps/Fes is involved in terminal myeloid differentiation[312] and physically associated with the β-chain of the GM-CSF receptor. This association is induced by ligand binding.[313] The Fps/Fes protooncogene encodes a 92 Kda cytoplasmic PTK consisting of an amino terminal domain (N-Fps), a central domain and a carboxyl terminal catalytic domain. Fps/Fes is structurally similar to a 94 Kda tyrosine kinase, Fer.[314] However, both Fps/Fes and Fer are quite distinct from cytoplasmic PTKs of the Src family, as they lack a negative regulatory tyrosine phosphorylation site in the carboxyl terminal region, are not modified by N terminal myristylation, and do not have an SH3 domain, which is present in all members of the Src family. Whereas Fer is widely expressed, expression of Fps/Fes has previously been reported only in haematopoietic cells, and most abundantly in those of the myeloid lineage.[315-317] Low levels Drugs 2000 Mar; 59 (3)

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of Fps/Fes were also detected in some B-cell lines.[316] Some tumours show overexpression or constitutive expression of Fps/Fes or Fer (table III). Human endothelial cells express Fps/Fes and overexpression seems to stimulate angiogenesis.[318] The molecular mechanisms by which the Fps/Fes kinase participates in mitogenic signalling are not yet known. However, the tyrosine kinase domain of Fps/Fes seems to be essentially involved in the signal transduction. 4. Cell Membrane to Nucleus Signalling Pathways Involving Protein Kinases 4.1 Raf Pathways

Raf proteins are cytosolic protein serine/threonine phosphokinases which play an essential role in receptor-mediated signal transduction pathways (fig. 1). Mammalian cells contain 3 Raf genes encoding Raf-1 (otherwise known as c-Raf), A-Raf and BRaf. B-Raf have been shown to exist in multiple spliced forms. Raf-1 and A-Raf own 2 tyrosine phosphorylation sites that are involved in the Rasdependent activation of Raf-1/A-Raf by tyrosine kinases.[319] In contrast, in the equivalent positions, B-Raf has aspartic acid residues. Moreover, B-Raf has a 10 to 20 fold greater basal kinase activity than Raf-1 or A-Raf.[320] For activation and signal transduction Raf proteins have to translocate from the cytosol to the plasma membrane. For this translocation, Raf proteins need to bind to activated p21 Ras. The mammalian p21 Ras family [H-, K (A and B) and N-Ras] proteins bind GDP and GTP with high affinity and have a low intrinsic GTPase activity.[321] p21 Ras proteins assume an active conformation through binding of GTP. Activated p21 Ras binds to a number of other intracellular proteins including: GTPase-activating protein (GP) that enhances GTP hydrolysis of p21Ras,[322] phosphoinositide-3-hydroxy kinase (PIK),[323] the mSOS guanine nucleotide exchange protein that promotes exchange of GDP for GTP,[324] and the JUN kinase, a stress activated protein (SAP) that is activated by this interaction for the phosphorylation and activation of the transcription factor JUN.[325] Localising © Adis International Limited. All rights reserved.

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Raf proteins to the cell membrane seems to be the main task of p21 Ras in the activation of Raf kinases. For cell membrane localisation the CAAX motif (C = Cys; A = any aliphatic amino acid; X = any other amino acid) at the carboxy terminal end of p21 Ras has to be farnesylated (covalently linked to isoprenyl) by a posttranslational process, starting with the cysteine residue.[326] Translocation of Raf proteins to the plasma membrane both serves as a mechanism for locally activating Raf-1 and A-Raf, and also helps to bring Raf-1 into contact with physiological substrates. On translocation to the cell membrane Ras-GTP bound Raf-1 or A-Raf become phosphorylated and activated by membrane associated tyrosine kinases (e.g. Src).[320] In contrast, the high basal kinase activity of B-Raf seems to be enhanced by association with Ras-GTP without the need of any additional phosphorylation by membrane associated kinases. There is evidence for pathways for Raf-1 or A-Raf activation independent from cell membrane tyrosine kinase. Cell stimulation may lead to the generation of DAG, which activates PKC. Activated PKC can activate Ras and lead to the formation of Ras GTP-Raf-1 complexes.[327] Activated Raf proteins are part of the MAP kinase cascade (MAPKK and MAPK, see section 4.2). Raf proteins represent MAPKKKs, that activate MAPKKs which in turn activate MAPKs. Besides its interaction with p21 Ras, Raf1 can bind with its C terminal catalytic domain to the docking domain BH4 in Bcl-2.[328] Via their docking domain BH4 Bcl-2 and other inhibitors of PCD of the Bcl-2 family (Bcl-XL, Bcl-w, Bfl-1, Brag-1, Mcl-1 and A1) can bind Raf-1, Bag1- or Ced-4, and target those proteins to mitochondria. Raf-1 targeted to mitochondrial membranes possibly phosphorylates (and thereby inactivates) there BAD and possibly other protein substrates of the Bcl-2 family (i.e. Bax, Bak, Bcl-Xs, Bid, Bik and Hrk) promoting PCD[329,330] via their function as an ion channel and as an adapter or docking protein, e.g. for calcineurin and p53 binding protein.[331] The mechanism of promoting PCD through protein complexes of the Bcl-2 family seems to be the opening of the mitochondrial permeability transiDrugs 2000 Mar; 59 (3)

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tion pores and the mitochondrial release of protease activators. Overexpression of inhibitors of PCD (e.g. Bcl-2) and inactivating mutations of promoters of PCD (e.g. Bax) seem to be associated with several types of solid tumours[332] and haematopoietic malignancies.[333] In many tumour types Raf is either overexpressed or constitutively, directly or indirectly (via activating mutations of Ras) activated (table IV). Mos is a cytoplasmic serine/threonine kinase regulating progression through meiosis I by activating maturation-promoting factor (MPF) from pre-MPF.[375] MOS is also a component of cytostatic factor (CSF), an activity of unfertilised eggs that stabilises MPF and arrests maturing oocytes at meiosis II.[376] The MOS protein comprises a conserved region sharing homology with the kinase domain of the Src family of protein kinases.[377] MOS activity is associated with the phosphorylation of its serine residues. MOS directly activates MAPKK (MEK1,2) by phosphorylation, thereby inducing activation of MAPK.[378,379] Several tumours show ectopic expression of MOS (see table IV). 4.2 Mitogen-Activated Protein Kinase (MAPK) Pathways

The MAP kinase signalling cascade consists of 3 distinct members of the protein kinase family, including MAP kinase (MAPK), MAPK kinase (MAPKK) and MAPKK kinase (MAPKKK).[380] MAPKKK phosphorylates and thereby activates MAPKK, and the activated form of the threonine and tyrosine-specific MAPKK in turn phosphorylates and activates MAPK. Activated MAPK may translocate to the cell nucleus and regulate by phosphorylation the activities of transcription factors, and thereby control gene expression. At least 3 defined MAPK signalling modules function in mammalian cells: • the Raf, MEK-1, -2 (MAP/ERK kinases), ERK1, -2 (extracellular signal regulated kinases) pathway • the ASK1/PAK (apoptosis signal regulating kinase), MKK4/SEK1/JNKK1 (c-Jun aminotermi© Adis International Limited. All rights reserved.

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nal kinase), SAPK (stress activated protein kinases)/ JNK-1, -2 pathway[367,381-383] • the MKK3/6-p38 pathway.[367,384] The apoptosis signal-regulating serine/threonine kinase (ASK-1) is distantly related to Raf-1 but closely related to those yeast MAPKKKs that are upstream regulators of the yeast MAPK/HOG1 of ASK-1.[385] The kinase domain of ASK-1 has sequence similarity with MEKK1, a member of the MAPKKK family. Its biological activity seems to be induction of PCD via the specific activation of the SAPK/JNK-1, -2 pathway[386-388] and the p38 pathway.[384] ASK-1 seems to be activated by Daxx, a Fas/tumour necrosis factor (TNF)α receptor associated protein.[387] Overexpression of ASK can be induced by ZnCl2.[384] TNFα causes apoptosis and activates both SPAK/JNK and p38 signalling systems via activation of ASK-1.[384] Activated Raf proteins phosphorylate and activate the dual specific MAPKK named MEK-1, 2(= MAP/ERK kinases), that in turn phosphorylate the MAPK named extracellular signal regulated kinases (ERK-1, -2) on both threonine and tyrosine. MEK-1, -2 are activated after phosphorylation of 2 closely spaced residues (that lie between the conserved kinase domains) by Raf-1 kinase,[389] BRaf[390,391] and MOS.[378,379] In the inactive, unphosphorylated form of ERK-,1 -2 threonine 183 is on the surface of the molecule, whereas tyrosine 185 is buried in a hydrophobic pocket. Binding of MEK-1, -2 to ERK-1, -2 leads to its major conformational change allowing access to tyrosine 185 for phosphorylation by MEK-1, -2. This kind of interaction between MEK-1, -2 may explain the pronounced substrate specificity of MAPKK and ERK-1, -2.[392] The ERK-1, -2 are activated extremely rapidly but are only transiently active. In most cell types the first peak of activation after mitogen stimulation is reached within 5 to 10 minutes and subsequently most of the activity is lost. Loss of either phosphothreonine or phosphotyrosine leads to the inactivation.[393] Phosphorylation of ERK-1, -2 leads to their nuclear translocation, where they phosphorylate transcription factors such as c-Myc,[394] c-Jun and c-Ets and thereby Drugs 2000 Mar; 59 (3)

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453

Table IV. Potential role of cytoplasmic signal transducing kinases in tumour pathogenesis Signal transducing kinase raf

Overexpression/ectopic expression in tumours

Mutation/amplification in tumours In about 40% of all cancers (except SCLC) (activating mutation of p21 Ras)

Pancreas Breast Head and neck (constitutive expression) Head and neck Colon AML Breast (hyperphosphorylation of raf-1) Head and neck Lung Kidney (activating mutation (raf-1)) Bone (amplification) c-mos

AKT-1 MAPKK (ERK-1, -2) MAPK (Mek1-, -2)

NIK/IKK

Ovary Seminoma Breast Thyroid

Reference Bernards[321] Bos[334] Kiaris and Spandidos[335] Ravi et al.[336] Sihtanandam et al.[337] Berger et al.[338] Callans et al.[339] Patel et al.[340] Riva et al.[341] Eggstein et al.[342] Okuda et al.[343] Schmidt et al.[344] Callans et al.[345] Patel et al.[346] Storm et al.[347] Teyssier et al.[348] Ikeda et al.[349] Xerri et al.[350] Xerri et al.[350] Lidereau et al.[351] Parkar et al.[352]

Breast Oesophagus Leukemia (activating point mutation)

Lidereau et al.[353] Lidereau et al.[353] Lidereau et al.[353] Csaikl et al.[354]

Stomach (amplification)

Staal[355] Ahmad et al.[356] Schmidt et al.[357]

Breast Liver

Schmidt et al.[357] Takahashi et al.[358] Attar et al.[359] Frexes-Steed et al.[360] Yee et al.[361] Sun et al.[362]

Liver Lymphoma Colon Pancreas Breast HTLV-leukemia (increased phosphorylation and degradation of IKB) Ovary (overexpression of IKB) Non-Hodgkin lymphoma (activating mutation of c-rel) ALL (amplification of c-rel) Breast Breast Colon (overexpression of c-rel) (overexpression of p52/p100/ NFκB) ALL (overexpression of p65/ NFκB)

Bours et al.[363] Gilmore[364] Gilmore[364] Feuillard et al.[365] Sovak et al.[366] Dejardin et al.[367] Bours et al.[363]

Feuillard et al.[365] NSC lung (spliced variants of p65/ NFκB with activating deletions)

Maxwell et al.[368] Contd over page

© Adis International Limited. All rights reserved.

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Table IV. Potential role of cytoplasmic signal transducing kinases in tumour pathogenesis Signal transducing kinase

Overexpression/ectopic expression in tumours Thyroid (overexpression of p65/ NFκB) ALL NSC lung (overexpression of p50/ constitutive activation/ NFκB) Kidney Non Hodgkin disease Breast (constitutive activation of NFκB)

Mutation/amplification in tumours

Reference Visconti et al.[369] Feuillard et al.[365] Mukhopadhyay et al.[370] Maxwell et al.[371]

Li et al.[10] Bargou et al.[372] Nakshatri et al.[373]

Lymphoma Trecca et al.[374] (activation point muta-tions of RelA/ NFκB) ALL = acute lymphocytic leukaemia; AML = acute myelocytic leukaemia; HTVL = human T cell lymphotropic virus; IKB = inhibitor KB; MAPK = mitogen-activated protein kinase; MAPKK = MAPK kinase; NFκB = nuclear factor κB; NIK = NFκB-inducing kinase; NSC = non-small cell.

modulate expression of genes such as fos and cyclin D1,[395] inducing cell proliferation. In several tumour types, ERK-1, -2 or MEK-1, -2 are overexpressed (see table IV). 4.3 Phosphatidylinositol-3-Kinase (PI3’K)-AKT/PKB Pathway

The serine/threonine kinase AKT [also known as protein kinase B (PKB)] or RAC-PK (related to A and C protein kinases) is a general mediator of growth factor-induced survival and has been shown to suppress the apoptotic death of a number of cell types induced by a variety of stimuli, including growth factor withdrawal, cell cycle discordance, loss of cell adhesion, and DNA damage. A signalling pathway has been defined in which growth factor receptor activation leads to the sequential activation of PI3’K and AKT, which then promotes cell survival and blocks apoptosis.[396] There are a number of PI3’K isoforms, the most well characterised of which is a heterodimer composed of an 85 Kda regulatory subunit and a catalytic 100 Kda subunit. PI3’K can be activated by interaction of its p85 subunit with specific phosphotyrosines on either the cytoplasmic domain of growth factor receptors or on receptor-associated adapter proteins. PI3’K can also directly interact with and be activated by p21 Ras.[397] Thus, through several possible protein-protein interac© Adis International Limited. All rights reserved.

tions, growth factor receptor activation recruits PI3’K to the membrane where the PI3’K p110 subunit phosphorylates phosphoinositides at the D-3 position.[396] PI3’K generated phospholipids then elicit a diverse set of cellular responses. One target of PI3’K is c-AKT. AKT may be regulated by the direct binding of Ptd-ins 3,4 P2 to the AKT pleckstrin homology (PH) domain. Ptd-ins 3,4 P2 dimerizes PKB/AKT. Homodimerisation by Ptd-ins 3,4 P2 increases PKB/AKT activity. Activity of PKB/ AKT is additionally increased by phosphorylation of threonine 38 and serine 473 (through MAPKK). Known in vivo substrates for AKT include the glycogen synthase kinase-3 and possibly the p27 ribosomal S6 kinase, although neither of these proteins have yet been shown to play a role in cell survival. One mechanism by which AKT can promote survival is through phosphorylation of BAD at Ser136,[396-398] which blocks BAD-mediated PCD.[397] Amplification or overexpression of AKT was mainly observed in gastric and breast carcinoma (see table IV). 4.4 Nuclear Factor (NF)-κB Pathway

The NF-κB is a heterodimeric nuclear transcription factor. It consists of 5 major proteins: p50, p65(Rel A), the proto-oncogene[364] c-Rel, p52 and Rel B. In general, the most abundant dimer of the inducible NF-κB is a p50/p65 heterodimer. Both p65 and p50 contribute to DNA binding: only p65 Drugs 2000 Mar; 59 (3)

Kinase Inhibitors in Cancer Therapy

transactivates. NF-κB is activated by diverse extracellular stimuli, including proinflammatory cytokines such as TNFα, IL-1, viral infection, oxidant, phorbol esters and ultraviolet irradiation. The main activation pathways are via receptors of the TNFα/nerve growth factor (NGF) family or IL1 receptors.[399] On binding of TNFα/NGF to their receptors the adapter protein Traf2 is recruited to the receptor-bound adapter proteins RIP, TRADD and FADD. Stimulation of the IL-1 receptor leads to binding of the serine/threonine kinase IRAK which recruits Traf6. Both Trafs interact with a (serine/ threonine) MAP kinase (MAPKKK, also named NIK).[400] On binding to Traf2 or Traf6, NIK activates by phosphorylation the serine/threonine kinases IκB kinaseα (IKKα) and IκB kinaseβ (IKKβ), which both form an active heterodimer, able to phosphorylate inhibitor κBα (IκBα) and IκBβ.[401,402] NF-κB activity is regulated by binding to nonphosphorylated IκB (IκBα or IκBβ) which retains NF-κB in the cytoplasm.[403] At present, 9 IκB family members have been identified (IκBα, IκBβ, IκBγ, IκBδ, IκBε, p105, p100, Bcl-3 and Cactus). The prototypic and best studied of the IκBs are IκBα and IκBβ,[404] which bind to the heterodimeric NF-κB complex (p50/Rel A),[405] mask the nuclear localisation signal present in p65 (Rel A),[406] and sequester NF-κB in the cytoplasm.[407-409] Under unstimulated conditions, NF-κB is quiescent in combination with IκB and is activated for nuclear entry when dissociated from IκB in response to the phosphorylation of the N terminal serines S32 and S36 in IκBα and S19 and S23 in IκBβ. Phosphorylation triggers conjugation of multiple ubiquitin molecules to the IκB phsophoproteins and, as with other proteins, targets them for rapid degradation by the 26S proteasome. IκBα degradation proceeds while the inhibitor is still physically associated with the NF-κB heterodimer.[362,410] However, the NF-κB complex is ultimately liberated with an exposed nuclear localisation signal allowing its rapid translocation into the nucleus where it binds to enhancer elements and activates the transcription of various target genes.[411] It is likely that phosphorylation of the IκBs is the principal point © Adis International Limited. All rights reserved.

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of control through which diverse stimuli effect NFκB activation. Increased phosphorylation of the IκBs is due to activation of an IκB kinase, although it can also be due to an inhibition of an IκBs phosphatase.[412] Phosphorylation of IκBs is also possible by PKC as well as by the Rsk (pp90) kinase. In quiescent cells inactive Rsk resides in the cytoplasm and is partially complexed with its upstream MAPK regulator, p42/44. Cellular activation mediated by various growth factors operating through the RasRaf-MEK-ERK pathways leads to the activation of MAP kinase, the phosphorylation and activation and the import of these kinases into the nucleus. Activated forms of Rsk regulate activation of various nuclear transcription factors, including cFos[413] and the cyclic AMP response elementbinding proteins (CREB) by phosphorylating these, factors on a key regulatory serine.[414] Rsk, but not MAPK, also phosphorylates the regulatory N terminus of IκBα principally on serine 32 and triggers effective IκBα degradation in vitro.[415] In many tumour types, overexpression of NIK/IKK or NF-κB or constitutive activations of NF-κB could be observed (table IV). Such an overexpression may inhibit PCD-induced by Fas and TNF via their receptors. When activated, membrane-bound receptors for Fas or tumour necrosis factor initiate programmed cell death by recruiting the death domain of the adapter protein FADD (Mort1)[416] to the membrane to form the death-inducing complex (DISC).[417] The DISC activates caspase 8 [also known as FLICE or MACH, a member of the family of caspases (IL1-β-converting enzyme/Ced-3-like proteases)] and considered to be the most upstream component of the caspase cascade],[418-422] and/or caspase 2 through an interaction between the death effector domains of FADD and the caspase. Caspase 8 and/ or 2 catalyse cleavage of downstream members of the caspase family (starting with caspase 3), leading to proteolysis of substrates, such as NF-κB (p65 and p50)[336] and the nuclear enzymes poly (ADP-ribose) polymerase (PARP), the DNA-dependent protein kinase (DNA-PK), and the U1 small nuDrugs 2000 Mar; 59 (3)

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clear ribonucleoprotein (U1 RNP).[423] This ultimately leads to the apoptotic response. Death effector domains and homologous protein modules known as caspase recruitment domains have been found in several proteins and are important regulators of caspase (FLICE) activity and of apoptosis. 5. Cyclin-Dependent Kinases (cdks) Progression through the cell division cycle (G1, G0, S, G2 and the mitosis M-phase) is driven by a family of protein kinases, the so-called cyclindependent kinases (cdks). Cdks consist of a catalytic subunit (the prototype of which is cdc2) and a regulatory subunit (cyclin). Several different human cdks have been described to date: cdk1 (= cdc2), cdk2, cdk4, cdk5, cdk6, cdk7 and cdk8. All these cdks are regulated by the transient association with 1 member of the cyclin family: cyclin A (cdk1, cdk2), cyclin B1-B3 (cdk1), cyclin D1-D3 (cdk2, cdk4, cdk5, cdk6), cyclin E (cdk2), cyclin H (cdk7), and cyclin C (cdk8). Other cdk1-related kinases have been sequenced and await identification of their regulatory partners and cell cycle regulatory functions. Progression through each step of the cell cycle is thought to be regulated by such cdk complexes: mid G1 phase - cdk4/cyclin D[1-3], cdk6/cyclin D1; late G1 phase - cdk2/cyclin E; G1/S transition - cdk2/cyclin E, cdk2/cyclin A; S-phase - cdk2/cyclin A; G2/M transition - cdk1/cyclin B, cdk1/cyclin A (see table I). In all these cases, the patterns of expression of the genes for cdks and cyclins either reflect the cell cycle phase-specific regulatory functions of their products (cyclin E-cdk2, cdk2/cyclin A, activation of cdk1 by cdc25C phosphatase in late G2 and cdk1/cyclin B complexes) or are controlled by different mechanisms (cdk2, cdk4 and the C-type cyclin) which may include the association with cell cycle-regulated cyclin, the stage-specific association with cdk/cyclin inhibitors, and the phosphorylation and dephosphorylation of specific amino acids.[424-426] Other cell cycle-regulated genes encode products that are required for cell cycle associated metabolic processes.[424] The group of cell cycle-regulated © Adis International Limited. All rights reserved.

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genes includes those for thymidine kinase (TK), dehydrofolate reductase (DHFR), DNA polymerase (and the DNA polymerase δ subunit PCNA), which are all induced in late G1/S. In addition, the retinoblastoma susceptibility gene pRb, as well as a number of transcription factor genes, including E2F-1, B-myb and c-jun, have been shown to be induced in late G1/S and their products presumably play a role in the regulation of other concomitantly or subsequently expressed genes. Cdks are regulated through ‘check-point control’ at defined restriction points which ensure that each step of the cell cycle is completed before the cell can proceed through the next step.[427,428] The mechanisms regulating cdks[429-433] include the following: • post-translational modifications by activating enzymes (e.g. cdc25C phosphatase and inhibitory enzymes (wee1 kinase) • association with activatory subunits (cyclins) • association with inhibitory subunits (e.g. p16INK4A, p15INK4B, p21Cip1, p27Kip1, p18) some of them (p16, p15, p18) inhibit by displacing the cyclin subunit • association with regulatory subunits of unknown, yet essential functions (p9CDShs with cdk1 and cdk2; p15cdkBP with cdk4, cdk5, cdk6) • ubiquitin-dependent degradation of the cyclin subunit • intracellular translocations and association with subcellular structures (centrosomes, microtubules). The regulatory sites of cdks include the ATPbinding pocket in the catalytic subunit of cdk1/ cyclin B which is located in a pocket between the small and large lobes of the kinase and contains 2 amino acids (Thr-14 and Tyr-15) which inhibit (cdk1, cdk2) kinase activity when phosphorylated. These phosphoamino acids are dephosphorylated by the cdk activators cdc25C and pyp3 phosphatase.[434-436] The protein substrate binding site is located in the cleft between the 2 lobes of the catalytic subunit. Phosphorylation of the Thr-161 residue of cdk1, Thr-160 in cdk2, and Thr-172 in cdk4, by cdk7/cyclin H appears to be essential for Drugs 2000 Mar; 59 (3)

Kinase Inhibitors in Cancer Therapy

kinase activity.[437] The phosphorylation of these sites is apparently involved in cyclin binding for cdk1/cyclin A but not for other complexes (cdk2/ cyclin A, cdk2/cyclin E, cdk1/cyclin B).[438,439] Interaction with this phosphorylated site is potentially inhibitory to the kinase.[440] The cdk7/cyclin H complex has been shown to activate other cdks by phosphorylation of the conserved threonine residue (Thr. 161, – 160 or – 172) in the catalytic subunit. An additional surprising finding was that cdk7/cyclin H complexes were also intimately associated with the transcriptional machinery: specifically, they are found within the basal transcription factor TFIIH in mammalian cells. Cdk7/cyclin H is constitutively active and capable of phosphorylating the carboxy terminal domain of RNA polymerase II.[441] The cyclin B subunit carries a domain, the P box, necessary for interaction with the cdc25C phosphatase. Integrity of the P box is required for cdk1 binding, activation of cdc25 and subsequent dephosphorylation of cdk1.[442,443] The excessive, aberrant or inappropriate expression of cyclin D and/or cyclin E during cell cycle seems to have an essential role in the pathogenesis of diverse tumour diseases.[444-446] Alterations in cyclin E expression correlate directly and quantitatively with tumour aggressiveness as determined by histologic grade and invasiveness. The alterations appear to be relegated to tumour cells and are not present in adjacent normal tissues, thus representing a true tumour cell-associated abnormality that discriminates malignancy from non-neoplastic states. Dysregulation of cyclin E expression appears to be a remarkably consistent molecular marker of breast epithelial cell malignant transformation, more so than other markers.[447,448] The main substrates for cdks are proteins of the retinoblastoma protein family, i.e. Prb, p107 and p130. All 3 so-called pocket proteins are capable of inhibiting growth in certain cell types. Their functional inactivation especially of Prb is very common in human cancer, (for a review see Weinberg,[449] and Mulligan and Jacks[450]) indicating its function as tumour suppressors. Cdks phosphorylate multiple sites (primarily serine threonine © Adis International Limited. All rights reserved.

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sites) in Prb and most likely in p107 and p130. Some information is known about which cdks sequentially activated during the cell cycle phosphorylate each one of these sites at a particular time.[451] Phosphorylation of Prb, p107 and p130 is associated with the loss of their growth suppressive activities. Such activities are mediated through the association of pocket proteins with a number of cellular proteins including the members of the E2F family of transcription factors (which regulate the expression of a number of genes required for cell cycle progression), other transcription factors involved in cell proliferation (i.e. c-Jun, ATF-2, cMyc, Sp1, AhR, TF II D, HBP1), transcription factors controlling expression of RNA polymerases (I, III), tissue specific transcription factors (including MyoD, HBP1, C/EBP, NRP/B), and transcription factor coactivators as well as enzymes such as histone deacetylase.[451] 6. Search for Small Molecular Weight Kinase Inhibitors (SMOKIs) The ultimate result of any research in tumour therapy must be new drugs effective in treatmentresistant progressive cancer. Effective in this context means that the drug should be able to prevent growth of tumours or to induce objective tumour regression. As most treatment-resistant tumours exhibit either an a priori drug resistance or show a multiple drug resistance (MDR), induced by prior therapy with conventional chemotherapy, the identification of new targets for drugs that may overcome the various mechanisms of drug resistance is an essential prerequisite. The obvious question is whether kinases involved in cell proliferation and/ or PCD might be such a target. Screening for inhibitors of EGF-R kinase and PKC started in 1987.[3] In vitro kinase inhibition assays were performed parallel to cytotoxicity assays. Using this screening system, flavopiridol (HMR 1275)[452] and other SMOKIs have been found (table V). At present, flavopiridol is in clinical development. The results of the phase I/II studies in 76 patients with treatment resistant progressive tumours show long lasting (≥8 months) clinical responses in renal carciDrugs 2000 Mar; 59 (3)

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Table V. Most recent small molecular weight kinase inhibitors (SMOKIs)a Compoundb

Inhibition of kinases (IC50)

Leflunomide (SU 101) PRI-105 (Tryptophan derivative) Emodin PD-168393 (Anilinoquin-azoline) PD-169414 (soluble analogue of PD-168393) CP-358774

PDGF-rec.

EGF-rec.

+

CP-59326

EGF-rec.

+

PDGF-rec. (75 mol/L) MAPK (75 mol/L) EGF-rec. (>500 mol/L) HER/neu EGF-rec. (1 nmol/L) EGF-rec. (0.42 nmol/L)

(4(-Phenylamino)-7H-pyrrolo (2,3-d) pyrimidines SU-5416 Flk-1/KDR (20 nmol/L) Phenylamino-pyrimidine CGP-78850 STI-571 [CGP-57148] (Phenylamino-pyrimidine) L-779450 (Trioylimidazole) CGP-60474 (Phenylamino-pyrimidine)

Flavopiridol

c-src 20-100x> bFGF-rec. > PDGF-rec. Grb2 SH2 c-abl 500x > src, syk, Jak-2 (PDGF-rec., c-Kit) Raf (2 nmol/L) MAPK (<100 nmol/L) cdk1 (20 nmol/L) cdk2 (50 nmol/L) cdk4 (>100 mol/L) PKC-α (0.3 mol/L) c-fgr (0.6 mol/L) ERK-1 (1.2 mol/L) cdk2 (0.1 mol/L) cdk1 (0.3 mol/L) cdk7 (0.3 mol/L) cdk4 (0.4 mol/L) EGF rec. (21 mol/L) PKC (6mol/L) JAK-2 > Lck, Lyn, Src

Antitumoural activity in vitro (IC50) in vivo (inhibition of tumour growth) + + <75 mol/L

–

+ + 2.7 nmol/L + 4.7 nmol/L

+ ≥28 mg/kg/day + 260 mg/kg/day + 50 mg/kg + 1.5 mg/kg

Reference

Lipson et al.[453] Blaskovich et al.[454] Zhang et al.[230] Denn et al.[1] Vincent et al.[455]

Miller et al.[456] Mett et al.[457]

+ 70 nmol/L +

–

Kraker et al.[459]

+ +

+ +

≈2 mol/L

–

100 nmol/L

+

Gay et al.[460] Buchdunger et al.[461] Heimbrook et al.[462] Meyer et al.[463]

+ <0.2 mol/L

+ 4.5 mg/kg/day

Fong et al.[458]

Sedlacek et al.[452]

AG-490 + + Meydan et al.[261] (Tyophostin derivative) <0.1 mol/L 25 mg/kg/day a Erbstatin, AG-17 and quercetin are not included as they are not recent compounds. b Brackets indicate chemical class EGF = endothelial growth factor; FGF = fibroblast growth factor; IC50 = concentration required to inhibit 50%; JAK = Janus kinase; MAPK = mitogen-activated protein kinase; PDGF = platelet-derived growth factor; PKC = protein kinase C; + = activity; – = not done.

noma (1 PR and 1 MR), non-Hodgkin lymphoma (1 MR), in colon carcinoma (1 MR) and ≥6 months stable disease in 10 patients with adenocystic (1), prostate (2) or renal (7) carcinoma after infusion of a dose between 28mg and 122.5 μg/m2/day × 3 (i.e. the maximum tolerated dose).[464] In an additional phase I study the infusion of flavopiridol (20 mg/m2/24 hours) subsequent to paclitaxel (100 mg/m2/24 hours) induced clinical responses in oesophageal cancer (1 CR) and prostate cancer (1 MR).[465] © Adis International Limited. All rights reserved.

In patients with metastatic gastric cancer the infusion of flavopiridol (60 mg/m2/dx3) induced 1 PR and 3 stable diseases.[466] The mechanism of action for flavopiridol, however, is still questionable. Surprisingly, it could be found that inhibition of the EGF-R is not essential for its antitumoural activity. On the other hand, flavopiridol has been shown to be a very strong and reversible in vitro inhibitor of cdk1, cdk2, cdk4 and cdk7.[452] This activity should cause growth inhibition of cells. However, it is questionable whether or to what deDrugs 2000 Mar; 59 (3)

Kinase Inhibitors in Cancer Therapy

gree inhibition of cdks contributes to the cytotoxicity and/or induction of PCD caused by Flavopiridol.[452,467-469] The inhibition of other kinases should be involved because flavopiridol induces PCD and cytotoxicity even in resting cells.[470] These other kinases might include PKCs. The experience gathered with flavopiridol mirrors the essential problems of the search for kinase inhibitors in oncology. Several kinase inhibitors have been shown to induce apoptosis in human tumour cell lines, e.g. erbstatin in SC lung carcinoma,[471]the tyrfostin derivative AG-17 in lymphoma overexpressing Bcl-2,[472] the tyrfostin derivative AG490 in B-cell acute lymphocytic leukaemia with constitutive activation of JAK-2,[261] the quiazolinethynylphenylamine CP-358774 in colorectal and breast carcinoma,[473] and quercetin in leukaemia cells.[474] In all cases, the degree of inhibition of the selected kinase correlated with the antiproliferative activity but it seems to be unclear yet, which signalling pathway and whether the inhibition of which key kinase in the cellular environment has led to the antiproliferative activity of the kinase inhibitors. The complexity of this problem is demonstrated by another aspect of kinase inhibitors: the risk of inducing tumour growth enhancement. For example genistein, known to be a strong protein tyrosine kinase inhibitor, and a potent in vitro inhibitor of the growth of human cancer cells[475-477] and of Ras oncogene transformed fibroblasts,[478] has been found to enhance the growth of experimental colon carcinoma in vivo.[479] Moreover, at low concentrations, genistein is known to stimulate growth of human breast carcinoma cells in vitro.[480] In addition, some protein kinase inhibitors have been found to activate the multidrug resistance I promoter.[481] In view of all the possible parameters contributing to this complex situation, the obvious questions in the search for kinase inhibitors in cancer therapy are: 1. Which kinases represent key targets for positive and negative selection of antitumoural activity? © Adis International Limited. All rights reserved.

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2. Can inhibitors be found that provide a high specificity for the selected kinase? 3. Is the specificity of an inhibitor for a selected target enzyme (found in vitro) also maintained in the cellular environment? 4. Which tumours are sensitive to such a target enzyme-specific inhibitor? 5. Can a therapeutic window between the effective nontoxic dose and the lowest toxic dose be found? 6.1 Key Target Kinases

The kinase selected for screening should have a key function with respect to the tumour disease in question. Such a key function is obvious in cancers caused by a mutation leading to a constitutive activity of a given kinase. A prominent example is the Bcr-Abl mutation in CML.[251] However, mutations causing constitutive activity of a given kinase are relatively rare in cancer (tables II, III and IV). More frequent are tumour cells, which show an ectopic or an overexpression of one or more kinases (tables II, III and IV), including receptor tyrosine kinases (RTKs). In many cases, overexpression of RTKs is associated with an overexpression of the ligand specific to the respective RTK leading to autocrine or paracrine growth stimulation of the respective tumour cells. Based on the postulation that inhibition of this growth stimulation loop by RTK inhibitors might have a therapeutic potential in tumour diseases, RTK-specific screening systems were established. A considerable number of RTK inhibitors have already been found (table V). As all of them are still in preclinical research, no final clinical efficient data are available yet. Some of these kinase inhibitors are able to induce apoptosis in tumour cells, but as has already been pointed out (see section 6) it is unclear yet, whether the inhibition of a key kinase is responsible for the induction of PCD. Thus it is still worth asking whether inhibition of RTKs or receptor-associated protein tyrosine kinases (R-PTKs) can be an effective tumour treatment. As growth factors stimulate tumour cell growth, inhibition of this stimulation by specific inhibitors of RTKs should reduce the Drugs 2000 Mar; 59 (3)

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growth rate of a tumour, but it is unlikely that they can also induce involution, e.g. regression of the tumour mass. This regression can only be achieved by the induction of tumour cell death. Indeed, death of tumour cells is an essential prerequisite for tumour elimination in tumour therapy. Even if reduction of the growth rate of a tumour may be a defined goal, this reduction needs the continuous inhibition of most of the RTKs and R-PTKs of more or less all tumour cells within the body. This pharmacokinetic requirement is very difficult to comply with. In contrast, growth factor receptors on endothelial cells of the tumour vascular bed can be suitable targets for RTK inhibitors for several reasons. For their growth, tumours need an increasing blood supply that is achieved by tumour-induced angiogenesis arising from the hosts capillaries by activation and proliferation of their endothelial cells. Concomitant with their activation, endothelial cells overexpress receptors for endothelial cell growth factors, e.g. VEGFRs. VEGFRs are also expressed on haematopoietic stem cells, fibroblasts, monocytes, osteoblasts and various tumour cells, but to a lesser extent. The VEGFR-2 (KDR) seems to be the major transducer of VEGF signals that result in endothelial cell proliferation. Inhibition of the intrinsic PTK activity of that receptor can inhibit tumour-associated angiogenesis, and could lead to reduction of tumour growth and even involution of the tumour mass. As tumour-associated activated endothelial cells are directly accessible to any drug in the blood stream, are relatively small in number (compared with the number of tumour cells), and hold a key function for tumour growth, VEGF-R2 kinase inhibitors could be a realistic opportunity for effective tumour therapy (to a greater extent than the many other research strategies), aiming to compete with or to reduce the expression of angiogenic factors, excessively produced by tumours and by normal cells in their neighborhood. Some VEGFR kinase inhibitors have already been found (table V) and their preclinical antitumoural activity seems to be very promising. Downstream of the various receptor PTK or receptor-associated kinases are signalling pathways that (as has been © Adis International Limited. All rights reserved.

Sedlacek

outlined in the preceding sections) transfer the signal from the cell membrane to the nucleus. Some of these signalling proteins are protein kinases which have key functions in that they can be activated by different receptors and receptor associated proteins, and are at the top of an activation cascade. To these key protein kinases belong the Raf proteins, NIK and AKT (table VI). Inhibition of these key kinases or of downstream kinases, blocks the signal transducing pathway and may induce PCD, preferably in those cells being stimulated. As many tumour cells are continuously activated either by ectopic, constitutive or overexpression of growth factor receptors and their corresponding ligands or by other mechanisms, inhibitors of Raf, NIK or AKT might preferentially induce PCD in tumours.[482-484] The degree of the resulting tumour regression should depend on the dose and time the inhibitor is applied. Cdks can stop cell proliferation, but it is unclear yet, whether they can also induce PCD. Flavopiridol strongly inhibits all the essential cdks involved in the cell cycle progression by binding to their ATP binding pockets. In addition, flavopiridol shows a strong cytotoxic activity on, and induces apoptosis in, cells exposed to this drug for a period exceeding 6 hours. However, this cytotoxicity is not always dependent on the cell cycle. Flavopiridol can also kill resting cells, depending on the cell type and culture conditions.[469] The mechanism of action of this cytotoxicity on resting cells might be completely different to inhibition of cdks, but it could also be that inhibition of selected cdks in resting cells induces apoptosis as cdks directly interact with E2F/DP, with proteins of the tumour suppressor Prb family, with DNA polymerase, RNA polymerases lamin, histone 1 protein, topomerase II activator, or the p34 subunit of the DNA single strand binding protein (see section 5). In view of these possibilities, it seems to us recommendable to include cdks in the list of candidate key protein kinases or a new screening system for kinase inhibitors with antitumoural activity (table VI). Drugs 2000 Mar; 59 (3)

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Table VI. Candidate key protein kinases for screening systems for antitumoural compounds Inhibitors for

Type

Potential effect of inhibitors

Positive selection Cell membrane receptors

VEGFR-II (KDR)

Iinhibition of tumour associated angiogenesis and involution/necrosis of tumours

Raf signalling pathways

Raf-1

Induction of programmed cell death

(c-Raf) A-Raf B-Raf (Mos) MEK-1, -2 ERK-1, -2 PI3’K/AKT pathway

AKT

Induction of programmed cell death

NF-κB pathway

NIK IKK RSK

Induction of programmed cell death

Cell cycle

cdk-1

Inhibition of cell growth

cdk-2

Induction of programmed cell death?

cdk-4 cdk-7 Negative selection Cell membrane receptors

TGF-R

Stimulation of tumour growth

ASK1/PAK pathway

ASK

Prevention of programmed cell death

MEKK3/6 JNKK1 KDR = kinase insert domain-containing receptor; NF-κB = nuclear factor κB; NIK = NF-κB-inducing kinase PI3’K = phosphatidylinositol-3kinase; TGFβ-R = transforming growth factor β receptor; VEGFR-II = vascular endothelial growth factor receptor type II.

6.2 The Question of Specificity

Protein kinases selectively transfer phosphate groups from ATP to protein substrates. For that transfer, kinases expose an ATP-binding pocket which is the target of most of the SMOKIs, including flavopiridol.[485] The specificity of SMOKIs for individual kinases is dependent on differences in primary, secondary and tertiary structures of their ATP-binding pockets. Such differences could be very small, implying a broad, more or less unrestricted activity of SMOKIs on protein kinases. This would imply that not only kinases promoting antiapoptotic mechanisms are inhibited (as desired) but also those involved in pathways which inhibit cell proliferation (receptors with intrinsic serine/threonine kinase activity including receptors for TGFβ, activins, inhibins, MIS and BMP) or promote apoptosis (e.g. ASK/PAK, MEKK3/6 or JNKK). Inhibition of the latter could promote © Adis International Limited. All rights reserved.

tumour growth, a completely undesired side effect. Thus it seems to be essential that the screening systems for kinase inhibitors include the adequate control assays, i.e. assays for ASK, MEKK3/6, JNKK, SAPK and TGFβ receptor activities. Considering the complex nature of the TGFβ receptor, a cellular assay instead of an in vitro assay seems to be advisable. This cellular assay includes cells expressing TGFRs which are stimulated by the addition of TGFβ. 6.3 The Question of Activity in the Cellular Environment

SMOKIs found in and selected from pure in vitro assays using isolated enzymes have to show the desired activity within the cell, especially in tumour cells. Activity in tumour cells requires the penetration of the selected inhibitor through the cell membrane. Once inside the cell, the selected SMOKI has to inhibit its specific or preferred kiDrugs 2000 Mar; 59 (3)

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nase within the cellular environment and to such a degree that growth of the cell is inhibited or PCD is induced. In view of the various crosstalks of the different signalling pathways (fig. 1), the selected SMOKI may not be effective to the desired extent (as extrapolated from the in vitro kinase inhibition data) in the cellular environment. Thus, in vitro activity of SMOKI is essential, but needs confirmation in cellular test systems. To prove efficacy in the cellular environment, testing of SMOKIs is recommended on cells transduced to overexpress one of the selected kinases compared with nontransduced cells. In case a cell, overexpressing one of the selected kinase, is more resistant (increase of inhibitory concentration) to induction of PCD by a SMOKI specific for this kinase (as revealed from kinase assays), it can be concluded that the specificity of the selective SMOKI is also operative in the cellular environment. To reveal any effect on other kinases, a panel of cells each transduced to overexpress one of the key kinases and/or upstream or downstream key proteins involved in modulating PCD should be used. Using that panel, the intracellular activity of SMOKIs can be tested and appointed to the different signalling pathways. It is obvious that in each transduced cell, the key function of the overexpressed signal protein has to be validated. Validation means that the inhibition of the expression and function of the selected protein in a cellular environment should result in the desired effect, i.e. inhibition of cell growth and/or PCD. Validation can be achieved by antisense molecules or ribozymes targeting nucleotide sequences encoding the specific kinase, or by the expression of dominant negative mutants of the specific kinases. The cellular test system should include cells overexpressing one of the following proteins: • the Raf family member c-Raf which prevents PCD in tumour cells • anti-apoptotic Bcl-2 family proteins which prevent PCD in leukaemic or tumour cells[486] • enzymes of the PI3’K survival pathway which block oncogene-triggered PCD, such as PI3’K and AKT/PKB © Adis International Limited. All rights reserved.

Sedlacek

• components of the pathway controlling the transcription factor E2F, which is deregulated in all tumour cells as a result of defects in the structure or expression of cdk inhibitor p16, cyclin D1, cdk4 or pRb • the pRb family member p130 which plays a key role in the activation of E2F in G0/G1 cells, thereby preventing PCD • the oncoprotein c-Myc, which is overexpressed in most tumour cells and inactivates the G1 check point • the cdk inhibitor p27 which is instrumental in inducing a G0/G1 block in a fraction of cells within tumours, thereby preventing PCD by chemotherapeutic agents • the cdk inhibitor p21 which is induced by chemotherapeutic agents and ionising radiation, thereby preventing PCD • the transcription factor NF-κB which is induced by chemotherapeutic agents and ionising radiation, and inhibits PCD as well as the proteins controlling the activity of IκK (the inhibitor of NF-κB) such as NIK and IκB kinase. The cells should be transiently or stably transduced to overexpress at least one of the selected proteins. In this way, an array of gene modified cell lines, which differ only in the regulatory protein(s) they overexpress, can be established. The tests are performed by adding a defined amount of the test compound to a defined number of the transduced cells and determining short term (e.g. 15 minutes to 24 hours) and long term (e.g. 1 to 14 days) effects on cell survival and the occurrence of cell death. Useful methods for the analysis of smaller amounts of compounds include: • counting cell numbers • analysing chromatin condensation (Hoechst dyes) • analysing DNA fragmentation (TUNEL assay) • determining other apoptotic markers (cytochrome C release, annexin V binding, PARP cleavage etc.). These cellular test systems may provide the following advantages: Drugs 2000 Mar; 59 (3)

Kinase Inhibitors in Cancer Therapy

• a target protein specific test system which has a lower probability of misleading results, since the target proteins are under the influence of the cellular environment • a simple and fast procedure for testing, since the end-point of the assay is cell death or survival • a broad approach since many of the proteins controlling PCD are included • a broad field of potential applications (e.g. cancer, restenosis, rheumatic arthritis, psoriasis, immune deficiency and autoimmune diseases). By using the cellular test system proapoptotic compounds can be identified and tested, e.g. in cells overexpressing proteins that prevent programmed cell death and that mimic various types of tumour cells. Furthermore, synergistic effects, e.g. with conventional anti-proliferative compounds, can be identified. 6.4 Efficacy in Human Tumours

The critical final question is the efficacy of one of the selected SMOKIs on human tumours. Efficacy can be preclinically tested on a panel of human tumour cell lines and on human tumours xenografted to immunodeficient mice and/or rats. Predictivity of these test models for clinical efficacy is higher than 50%, at least for cytostatics.[3,487] Whether this predicitivity rate is also true for SMOKIs is unknown as yet. In human tumour types, the kinase inhibitor flavopiridol showed a spectrum of activity which seems to be similar between preclinical test models and clinical phase I/II studies. However, adverse effects are significantly different. The preclinical dose limiting toxicity in mice, rats and dogs was haemorrhagic diarrhoea.[452] Surprisingly, in clinical studies secretory diarrhoea,[464] possibly through stimulation of bile flow[488] prevailed. Similar surprising differences between preclinical and clinical toxicity data can be expected for other SMOKIs. Facing the fact that many tumour types differ from their corresponding normal cells by loss of proapoptotic and acquisition or activation of antiapoptotic mechanisms, it may be expected that such tumour types may be more sensitive to SMOKIs inducing PCD than the © Adis International Limited. All rights reserved.

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corresponding normal cells. This difference in sensitivity may offer a therapeutic ‘window’ that is needed for an effective nontoxic, antitumoural treatment. It can be expected that tumour regression by inducing PCD needs at least repeated treatments, possibly even a continuous level of SMOKIs within the tumour tissue over a longer period of time. Thus, a high bioavailability after oral application and an optimal lipophilicity/hydrophilicity ratio to enable a quick extravasation and penetration of tumour tissues would be favourable for an optimal antitumoural activity of SMOKIs. Acknowledgements The author is indepted to Professor Rolf Müller, Institute for Molecular Biology and Tumour Research, University of Marburg, with whom he intensively discussed the network of cell signal transduction, cellular screening systems for cytostatics and the possibility of improving the present test systems for kinase inhibitors and who thereby considerable contributed to this review. In addition, the author thanks Ms Manuela Rogala for her skilful secretarial assistance in preparing the manuscript.

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Correspondence and offprints: Dr H.H. Sedlacek, Adventis Pharma Deutschland GmbH, Central Biotechnology, Marburg, Germany. E-mail: [email protected]

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