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(%( ,'-./'%(%'  (+'.,'  (+( 00 Paracrine and autocrine functions of PDGF in malignant disease

BY TOBIAS SJÖBLOM Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) in Molecular Cell Biology presented at Uppsala University in 2002.

ABSTRACT

Sjöblom T. 2002. Paracrine and autocrine functions of PDGF in malignant disease. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1190. 62pp. Uppsala. ISBN 91-554-5420-8.

Growth factors and their receptors are frequently activated by mutations in human cancer. Platelet-derived growth factor (PDGF)-B and its tyrosine kinase receptor, the PDGF β- receptor, have been implicated in autocrine transformation as well as paracrine stimulation of tumor growth. The availability of clinically useful antagonists motivates evaluation of PDGF inhibition in these diseases. In chronic myelomonocytic leukemia with t(5;12), parts of the transcription factor TEL and the PDGF β-receptor are fused, generating a constitutively signaling protein. Oligomerization and unique phosphorylation pattern of TEL-PDGFβR was demonstrated, as well as the transforming activity of TEL-PDGFβR, which was sensitive to PDGF β-receptor kinase inhibition. Dermatofibrosarcoma protuberans (DFSP) is characterized by a translocation involving the collagen Iα1 and PDGF B-chain genes. The COLIA1-PDGFB fusion protein was processed to mature PDGF-BB and transformed fibroblasts in culture. The PDGF antagonist STI571 inhibited growth of COLIA1-PDGFB transfected cells and primary DFSP cells in vitro and in vivo through induction of apoptosis. Paracrine effects of PDGF-DD, a ligand for the PDGF β-receptor, were evaluated in a murine model of malignant melanoma. PDGF-DD production accelerated tumor growth and altered the vascular morphology in experimental melanomas. A validated immunohistochemical procedure for PDGF β-receptor detection was established and applied to normal tissues and more than 280 tumor biopsies. Perivascular and stromal expression was detected in 90% and 50%, respectively, of human tumors. Recently, non-transformed cells in the tumor microenvironment have emerged as targets in cancer therapy. Selective sensitization of tumor fibroblasts to paclitaxel by STI571 was evaluated in vitro and in a xenograft model. Whereas neither drug alone caused growth inhibition, combination of the two significantly reduced tumor growth, suggesting anti-stromal therapy as a possible treatment modality in solid tumors.

Tobias Sjöblom, Ludwig Institute for Cancer Research, Biomedical Center, Box 595, SE- 751 24 Uppsala, Sweden

© Tobias Sjöblom 2002

ISSN 0282-7476 ISBN 91-554-5420-8

Printed in Sweden by Eklundshofs Grafiska, Uppsala 2002

2 To Sofia and my family

3 This thesis is based on the following papers, referred to in the text by their roman numerals:

I: Sjöblom T.*, Boureux A.*, Rönnstrand L., Heldin C-H., Ghysdael J., and Östman A. (1999). Characterization of the chronic myelomonocytic leukemia associated TEL-PDGFβR fusion protein. Oncogene 18, 7055-7062

II: Shimizu A., O’Brien K. P., Sjöblom T., Pietras K., Buchdunger E., Collins V. P., Heldin C-H., Dumanski J. P., and Östman A. (1999). The dermatofibrosarcoma protuberans-associated collagen type Iα1-platelet derived growth factor (PDGF) B-chain fusion gene generates a transforming protein that is processed to functional PDGF-BB. Cancer Res 59, 3719-3723

III: Sjöblom T.*, Shimizu A.*, O’Brien K. P., Pietras K., Dal Cin P., Buchdunger E., Dumanski J. P., Östman A., and Heldin C-H. (2001). Growth inhibition of dermatofibrosarcoma protuberans tumors by the PDGF receptor antagonist STI571 through induction of apoptosis. Cancer Res 61, 5778-5783

IV: Furuhashi M., Sjöblom T., Bergsten E., Eriksson U., Heldin C-H., and Östman A. (2002). Enhanced tumor growth and angiogenesis by paracrine action of PDGF-DD. Manuscript

V: Sjöblom T., Betsholtz C., Pontén F., Pietras K., Östman A., and Heldin C-H. (2002). Stromal and perivascular expression of the platelet-derived growth factor β-receptor in normal and malignant tissues. Manuscript

VI: Sjöblom T., Furuhashi M., Bäckström G., Forsberg-Nilsson K., Heldin C-H., and Östman A. (2002). Anti-stromal tumor therapy by combination of STI571 and paclitaxel. Manuscript

* authors contributed equally

Reprints were made with permission from the publisher.

4 TABLE OF CONTENTS PAGE

Abbreviations 6 I. Introduction 7 A. Growth factors in cancer 7 1. Autocrine growth factor dependency 7 2. Angiogenesis 9 3. Tumor stroma formation 10 B. Platelet-derived growth factor (PDGF) 11 1. The PDGF family of ligands and receptors 11 2. Signaling from PDGF receptors 13 3. Developmental and physiological roles of PDGF 17 4. PDGF in fibrotic disease 20 5. PDGF in malignant disease 22 6. PDGF antagonists 26 C. Chronic myelomonocytic leukemia 28 D. Dermatofibrosarcoma protuberans 30 II. Present investigation 32 A. Characterization of the chronic myelomonocytic leukemia associated Tel-PDGFβR fusion protein (Paper I) 32 B. The dermatofibrosarcoma protuberans-associated collagen type Iα1/ PDGF B-chain fusion protein is processed to PDGF-BB and sustains the growth of DFSP tumor cells (Paper II-III) 33 C. Enhanced tumor growth and angiogenesis by paracrine action of PDGF-DD (Paper IV) 34 D. Stromal and perivascular expression of the PDGF β-receptor in normal and malignant tissues (Paper V) 35 E. Anti-stromal tumor therapy by combination of STI571 and paclitaxel (Paper VI) 36 III. Future perspectives 37 IV. Acknowledgements 39 V. References 41

5 Abbreviations

AML Acute myeloid leukemia Ang Angiopoietin ATP Adenosine triphosphate CAM Chorio-allantoic membrane CML Chronic myeloid leukemia CMML Chronic myelomonocytic leukemia CSF Colony stimulating factor DFSP Dermatofibrosarcoma protuberans EGF Epidermal growth factor FGF Fibroblast growth factor GCF Giant cell fibroblastoma GAP GTPase activating protein GEF Guanosine nucleotide exchange factor GIST Gastrointestinal stroma tumor HES Hypereosinophilic syndrome MDS Myelodysplastic syndrome NGF Nerve growth factor PDGF Platelet-derived growth factor PH Pleckstrin homology PI3-K Phosphatidylinositol 3’-kinase PNT Pointed PTB Phosphotyrosine binding SELEX Selective evolution of ligands by exponential enrichment SFK Src family kinase SH2 Src homology 2 SH3 Src homology 3 STAT Signal transducer and activator of transcription TGF-β Transforming growth factor β VEGF Vascular endothelial growth factor

6 I. Introduction

Control of cell division and cell death is fundamental for normal development and tissue homeostasis in multi-cellular organisms. The fate of individual cells is governed by signals from the immediate and distant environment, mediated by hormones and growth factors that stimulate survival or cell division, as well as by pro-apoptotic factors that induce cell death.

In the process of tumor formation, environmental stress and hereditary disposition leads to mutations in a number of critical genes involved in growth control. As a result of gain of function mutations in proto-oncogenes, and loss of function mutations in tumor suppressor genes, the cell becomes independent of survival factors and insensitive to growth inhibitory signals. The loss of growth control, and the subsequent formation of an autonomous cell population, is an important step in tumorigenesis.

The phenotypical traits of cancer cells include self-sufficiency in growth signals, insensitivity to anti-growth signals, resistance to cell death, limitless replicative potential, ability to recuit blood vessels, and, ultimately, the capacity to metastasize (Hanahan and Weinberg, 2000). Growth factors, and signals arising from growth factor receptors, play important roles in the acquisition of these traits. The advent of growth factor antagonists, with suitable properties for use in experimental systems as well as in humans, is giving further insight into the role of growth factors in malignant disease. Furthermore, inhibition of growth factor signaling is a promising way to better treatment of cancer.

A. Growth factors in cancer

1. Autocrine growth factor dependency

Self-sufficiency in growth signals, as well as protection from apoptosis, can result from mutations in genes encoding growth factors or their tyrosine kinase receptors.

One way whereby activating mutations can occur is by gene amplification, leading to overexpression of a structurally normal protein. Of major clinical importance is the chromosomal amplification of ErbB2/HER2/Neu, which is present in 30% of invasive breast cancers and correlated with reduced survival (Slamon et al., 1987). Increased gene copy number of ErbB2 leads, by elevated transcript levels, to a high density of ErbB2 in the cell membrane. The high receptor density entails ligand-independent dimerization and activation, which provides growth stimulatory and anti-apoptotic signals (Penuel et al.,

7 2002). Also, the epidermal growth factor (EGF) receptor is amplified, and often rearranged, in 40-60% of glioblastoma multiforme tumors (Libermann et al., 1985; Nagane et al., 2001).

Furthermore, point mutations or chromosomal rearrangements can lead to expression of a modified protein product. Point mutations in tyrosine kinase receptors can result in receptor activation, either by constitutive dimerization of the receptors, or by structural alterations leading to deregulation of the kinase activity. An example of the former class is the point mutations in the extracellular domain of Ret, which are found in patients with multiple endocrine neoplasia type 2A. Loss of one extracellular cysteine enables formation of disulfide-bonded receptor dimers, which are constitutively active (reviewed in Santoro et al., 2002). In contrast, the c-Kit D816V point mutant, involved in the pathogenesis of systemic mastocytosis, promotes kinase activation independent of dimerization (Furitsu et al., 1993; Kitayama et al., 1995).

Chromosomal translocations, resulting in fusion genes encoding dimerization motifs and the tyrosine kinase domain of a growth factor receptor, have been found in a minority of patients with myeloproliferative disease (Cross and Reiter, 2002). Tel-PDGFβR and ZNF198-FGFR1 are two such oncoproteins, where the pointed domain of Tel and the proline-rich domain of ZNF198 contribute to protein-protein interactions, which are followed by activation of the kinase domains of the PDGF β-receptor and FGF receptor 1, respectively (Carroll et al., 1996; Xiao et al., 2000).

Aberrant growth factor expression, with subsequent receptor activation, can also lead to transformation. In dermatofibrosarcoma protuberans, a t(17;22) fusing the collagen Iα1 and PDGF B-chain genes leads to over-expression of PDGF-BB, acting via PDGF receptors on the tumor cells to induce transformation (Simon et al., 1997; Paper II-III; Greco et al., 1998).

The discovery of activating mutations in genes encoding growth factors and growth factor receptors has spawned the development of specific antagonists to these proteins (Shawver et al., 2002). Herceptin/trastuzumab, an inhibitory antibody to ErbB2, is a promising agent in breast cancer therapy (Cobleigh et al., 1999). Furthermore, the tyrosine kinase inhibitor STI571 has been approved for treatment of chronic myeloid leukemia (CML) and gastrointestinal stroma tumors (GIST), where tumor growth is dependent on Bcr-Abl and c-Kit kinase activity, respectively (Capdeville et al., 2002).

8 2. Angiogenesis

Angiogenesis, the formation of new capillary blood vessels, is crucial for tumor growth beyond the size of a few cubic millimeters (Folkman, 1985). Usually, the host vascular network expands by budding of endothelial sprouts from pre-existing vessels (reviewed in Carmeliet, 2000). However, bone marrow-derived hemangioblasts have also been demonstrated to engraft and contribute to the endothelial cell lining in sprouting blood vessels (Asahara et al., 1999; Asahara et al., 1997). During physiological angiogenesis, sprouting endothelial cells recruit smooth muscle cells and pericytes that will surround the mature vessel. In contrast, tumor vessels have an incomplete pericyte cover (Morikawa et al., 2002). Although they are composed of normal cells, blood vessels in tumors are structurally and functionally abnormal. Frequently, tumor vessels are tortuous, dilated, extensively branched, and leaky (Dvorak et al., 1988).

The balance between pro-angiogenic and anti-angiogenic factors in the local microenvironment controls formation of new blood vessels. Inhibitors of angiogenesis are predominant in normal tissue, and the ability to induce and sustain angiogenesis is acquired in a discrete step during tumor development (Folkman et al., 1989; Hanahan and Folkman, 1996). On one hand, angiogenesis can be induced by increased production of angiogenic growth factors, such as vascular endothelial growth factor (VEGF) (Keck et al., 1989; Leung et al., 1989), angiopoietins (Ang) (Davis et al., 1996), or basic fibroblast growth factor (bFGF) (Abraham et al., 1986). On the other hand, decreased production of angiogenesis inhibitors, such as thrombospondin (Good et al., 1990), can shift the balance in favor of angiogenesis.

The VEGFs and angiopoietins, with their respective receptors, are prototypical angiogenic growth factors. Five mammalian VEGF ligands (VEGF A-D and placental growth factor [PlGF]), acting on different subsets of three tyrosine kinase receptors (VEGFR-1, -2, and - 3), have so far been identified (reviewed in Eriksson and Alitalo, 1999). VEGFR-1 and -2 are mainly expressed on endothelial cells, whereas VEGFR-3 is expressed on lymphatic endothelial cells. VEGF-A is an endothelial cell mitogen, mainly through its action on the VEGFR-2 and is expressed in normal tissue upon hypoxia. VEGFR-1 and its ligand placental growth factor (PlGF) have recently been assigned a role in recruitment of endothelial bone marrow progenitor cells to angiogenic blood vessels (Gerber et al., 2002; Hattori et al., 2002; Luttun et al., 2002). Furthermore, VEGF-C and VEGF-D can induce lymphangiogenesis in normal and tumor tissue (Karpanen et al., 2001; Mandriota et al., 2001; Skobe et al., 2001; Veikkola et al., 2001).

9 The angiopoietin family of ligands, which bind the Tie2 tyrosine kinase receptor expressed on endothelial cells, is comprised of the agonistic Ang-1 and -4, as well as the antagonistic Ang-2 and -3 (Davis et al., 1996; Jones et al., 2001a; Maisonpierre et al., 1997). Mouse embryos deficient for Ang-1 or Tie2 die in mid-gestation due to insufficient expansion and maintenance of the primary capillary plexus (Sato et al., 1995; Suri et al., 1996). The rapid onset of endothelial cell apoptosis after conditional deletion of Tie2 in adult animals implies continuous angiopoietin signaling in vascular maintenance (Jones et al., 2001b).

In rodent models of cancer, antibodies to VEGF-A as well as soluble VEGFR-1 inhibited the growth of experimental tumors, alongside with a reduction in vessel density (Kim et al., 1993; Millauer et al., 1994). Moreover, inhibition of Tie2 signaling using dominant- negative soluble receptors reduced the growth rate of experimental tumors (Lin et al., 1998; Lin et al., 1997). However, tumor regression by angiogenesis inhibition has been difficult to prove in human cancer, although some phase II studies indicate therapeutic effect from anti-VEGF antibody therapy (Kerbel, 2001).

3. Tumor stroma formation

Apart from the malignant cells themselves, all solid tumors contain a mesenchymal stroma. The tumor stroma is composed of fibroblast-like cells, extracellular matrix, inflammatory cells, and blood vessels (reviewed in Bissell and Radisky, 2001). Due to the similar composition of tumor stroma and granulation tissue, the tumor microenvironment has been compared to a wound that does not heal (Dvorak, 1986). In desmoplastic tumors, the connective tissue component can account for 90% of the tumor volume, whereas other tumors only form a minimal stroma.

Fibroblast-like cells of the tumor stroma can contribute to angiogenesis by production of VEGF-A (Fukumura et al., 1998). Also, proteolytic enzymes secreted by stromal fibroblasts can promote genetic instability in surrounding epithelial cells, thereby enhancing tumor development, exemplified by the induction of mammary epithelial hyperplasia and malignant transformation upon transgenic expression of the stromal protease stromelysin-1 (Sternlicht et al., 1999). Furthermore, co-inoculation of prostate- derived stromal fibroblasts with non-tumorigenic LNCaP prostate epithelial cells enhanced tumor formation as compared to epithelial cells alone (Olumi et al., 1998; Tuxhorn et al., 2002).

10 Endothelial cells and fibroblasts of the tumor stroma are generally considered to be non- transformed. However, the presence of genetic alterations in stroma cells, absent in the malignant epithelial cells, indicates that the stroma is not merely a host reaction but might play an active role in tumorigenesis (Moinfar et al., 2000). Moreover, the inherited pre- leukemic Diamond-Blackfan syndrome affects the stroma compartment of the bone marrow (Dror and Freedman, 1999), and somatic deletions at 10q22 are frequent in stromal cells of juvenile polyposis (Jacoby et al., 1997). In addition, underlying inflammatory and fibrotic conditions increase the likelihood of tumor development and relapse after surgery (Bilimoria et al., 2001; Jacobs et al., 1999).

Growth factors involved in recruitment of a fibroblastic stroma include transforming growth factor-β (TGF-β) and PDGF. Formation of a collagen- and fibroblast-rich tumor stroma was demonstrated in TGF-β transfected melanoma xenografts, concomitant with decreased tumor cell apoptosis and increased lung metastasis (Berking et al., 2001). Secretion of PDGF-BB from tumor cells induced a vascularized, fibroblast-rich tumor stroma (Forsberg et al., 1993), and inhibition of PDGF-A production abolished desmoplasia in a breast cancer model (Shao et al., 2000). However, the tumor stroma is so far a therapeutically unexploited compartment.

B. Platelet-derived growth factor (PDGF)

1. The PDGF family of ligands and receptors

Platelet-derived growth factor is a family of dimeric disulfide-bonded polypeptide chains, with growth stimulatory action primarily on connective tissue cells (Raines et al., 1990). The family consists of homo- and heterodimers of the two classical PDGF A- and B- chains (PDGF-AA, -BB, and -AB), as well as homodimers of the recently identified family members PDGF-C and -D (PDGF-CC and -DD) (Heldin et al., 2002).

PDGF biosynthesis The A- and B-chains of PDGF contain, in addition to the mature ∼100-amino acid sequence, an N-terminal propeptide and a signal peptide that is removed in the Golgi apparatus following formation of the disulfide-bonded dimer in the endoplasmic reticulum (Östman et al., 1992). The B-chain is also processed in the carboxy-terminus (Östman et al., 1991b). Furthermore, PDGF-B and the long splice variant of PDGF-A contain a C- terminal retention sequence, which can mediate interaction with components of the extracellular matrix (LaRochelle et al., 1991; Östman et al., 1991a). Interestingly, PDGF- CC and -DD are synthesized and secreted as inactive forms, with an N-terminally located

11 CUB domain that needs to be removed by proteolytic cleavage in order for the ligands to bind PDGF receptors (Bergsten et al., 2001; LaRochelle et al., 2001; Li et al., 2000). The proteases responsible for activation of PDGF-CC and -DD in vivo have not yet been identified.

Structure of PDGF The mature forms of PDGF-A and PDGF-B contain eight conserved cysteine residues, with the second and fourth being involved in interchain disulfide bonds and the other six in disulfide bonds within the chain (Andersson et al., 1992; Haniu et al., 1994; Östman et al., 1993). The solution structure of mature PDGF-BB showed an anti-parallel arrangement of the two subunits, where each subunit contains a tight cystine knot and three protruding loops (Oefner et al., 1992). Two loops extend in one direction (loop 1 and 3) and the other in the opposite direction (loop 2). Thus, the anti-parallel subunit arrangement will bring loop 2 of one subunit in proximity of loop 1 and 3 of the other subunit. By mutagenesis, the most important amino acids involved in receptor binding have been mapped to loop 1 and 3, with some contribution of loop 2 (Andersson et al., 1995; Clements et al., 1991; Fenstermaker et al., 1993; Östman et al., 1991b). Hence, both chains in the dimeric ligand will contribute to each of the two receptor binding epitopes. Interestingly, PDGF shows structural similarity to the closely related vascular endothelial growth factor (VEGF) (Muller et al., 1997) as well as the more distantly related nerve growth factor (NGF) and transforming growth factor-β (TGF-β) molecules (Murray-Rust et al., 1993).

PDGF receptors The different PDGF isoforms exert their biological effects by binding to two structurally related tyrosine kinase receptors, the PDGF α- and β-receptors (Claesson-Welsh et al., 1989; Matsui et al., 1989; Yarden et al., 1986). The PDGF receptors are composed of five extracellular immunoglobulin-like domains, a transmembrane part, and an intracellular part with a split tyrosine kinase domain (Heldin et al., 1998). The three N-terminal immunoglobulin domains mediate ligand binding, whereas the fourth immunoglobulin domain is involved in receptor dimerization (Omura et al., 1997; Shulman et al., 1997). Based on kinase domain sequence similarity, the split tyrosine kinase domain, and the presence of 5 extracellular Ig domains, the PDGF receptors belong to the class III receptor tyrosine kinases alongside with the stem cell factor receptor c-Kit, the colony stimulating factor (CSF-1) receptor, and Flt3 (Blume-Jensen and Hunter, 2001). Homodimeric PDGF α-receptor complexes are formed after stimulation with PDGF-AA, -AB, -BB, or -CC, whereas PDGF β-receptor homodimers are formed after stimulation with PDGF-BB or - DD. Furthermore, PDGF-AB and -BB can induce formation of αβ receptor heterodimers,

12 and stimulation of cells expressing α- and β-receptors with PDGF-CC or -DD results in activation of both receptor types (Bergsten et al., 2001; Gilbertson et al., 2001; LaRochelle et al., 2001; Li et al., 2000).

2. Signaling from PDGF receptors

Dimerization is a general mechanism for the activation of cell surface receptors, including the PDGF receptors (Heldin, 1995; Schlessinger, 2000). Ligand binding to PDGF receptors leads to formation of a dimeric receptor complex, where each receptor in the complex is believed to phosphorylate the other (Emaduddin et al., 1999; Kelly et al., 1991). Autophosphorylation of a critical regulatory tyrosine within the kinase domain, Tyr849 and Tyr857 in the α- and β-receptor, respectively, leads to up-regulation of the intrinsic tyrosine kinase activity (Kazlauskas and Cooper, 1989). Dimerization of PDGF receptors has also been shown to protect the receptors from dephosphorylation by tyrosine phosphatases (Shimizu et al., 2001). Autophosphorylation of the regulatory tyrosine is followed by phosphorylation of tyrosine residues located outside of the kinase domain, which creates docking sites for signal transduction molecules containing phosphotyrosine recognition motifs, such as the Src Homology 2 (SH2) and phosphotyrosine binding (PTB) domains.

The PDGF α- and β-receptors contain at least 8 and 11 autophosphorylation sites, respectively, and a large number of SH2 domain containing proteins are known to bind to specific phosphotyrosine residues in the activated PDGF receptors (Heldin et al., 1998). The SH2 domain is a conserved ∼100 amino acid motif that binds to phosphorylated tyrosine residues located within a defined amino acid sequence (Pawson and Scott, 1997; Songyang et al., 1993). The SH2 domain-containing signal transduction molecules fall into three functional categories; those with intrinsic enzymatic activity, such as SHP-2, Src, PLC-γ, and RasGAP, adaptor proteins such as Grb2, Crk, Shc, Nck, and the p85 regulatory subunit of PI3-K, and transcription factors such as STAT proteins. Each SH2 domain-containing protein that binds to the PDGF receptors may potentially initiate a signal transduction pathway (reviewed in Heldin et al., 1998). No PTB domain is currently known to interact with the phosphorylated PDGF receptors.

PI3-K The class Ia phosphoinositide 3-kinases (PI3-K) constitute a family of lipid kinases, composed of p85 regulatory subunits and p110 catalytic subunits (reviewed in Cantley, 2002; Fruman et al., 1998). Upon SH2-domain mediated binding of p85 to Tyr731/742 or Tyr740/751 of activated PDGF α- and β-receptors, respectively, p110 is recruited to the

13 complex and its catalytic activity is up-regulated (Fantl et al., 1992; Yu et al., 1991). The active p110 subunit phosphorylates the 3’-OH group of the inositol ring of membrane- associated phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] to yield PI(3,4,5)P3. Proteins containing pleckstrin homology (PH) domains have affinity for PI(3,4,5)P3 and translocate to the plasma membrane upon its production. Several cellular responses, such as cell growth, actin reorganization, chemotaxis, and anti-apoptosis, are elicited by PI3-K activation (reviewed in Vanhaesebroeck et al., 1997). PDGF β-receptor signaling has been shown to protect cells from apoptosis through activation of PI3-K and the PH domain containing serine/threonine kinase Akt/PKB (Dudek et al., 1997; Yao and Cooper, 1995). Other downstream effectors of PI3-K include the small GTPases Cdc42, Rho, Rac, and Ras. Termination of PI3-K signaling can be mediated by the lipid phosphatase PTEN, which converts PI(3,4,5)P3 back to PI(4,5)P2 (Li et al., 1997; Maehama and Dixon, 1998; Myers et al., 1998).

Src c-Src, the cellular homologue of the Rous sarcoma virus oncogene v-Src, is a tyrosine kinase composed of an N-terminal membrane-association domain, a unique domain, a Src homology 3 (SH3) domain, an SH2 domain, a catalytic domain and a negative regulatory tail (reviewed in Abram and Courtneidge, 2000). The ∼60 amino acid SH3 domain, which recognizes proline-rich sequences with the consensus motif PXXP, is found in many signal transduction proteins and mediates protein-protein interactions (Pawson, 1995). In the inactive, closed conformation of Src, the SH3 domain interacts with the catalytic domain and the SH2 domain binds phosphorylated Tyr527 in the negative regulatory tail of Src itself. Hence, Src is activated by dephosphorylation of Tyr527 or by high-affinity interaction of ligands, such as tyrosine kinase receptors, with the SH2 and SH3 domains (Erpel and Courtneidge, 1995).

The Src family kinases (SFK) comprise ten homologous proteins, varying in expression pattern and in the unique domain sequence (Thomas and Brugge, 1997). The ubiquitously expressed members Src, Yes, and Fyn have been shown to bind Tyr572/574 and Tyr579/581 in the autophosphorylated PDGF α- and β-receptors, respectively, which led to phosphorylation and activation of these SFK (Gould and Hunter, 1988; Hooshmand- Rad et al., 1998; Kypta et al., 1990; Mori et al., 1993). Recently, the necessity of SFK in the mitotic response to PDGF, mediated through SFK activation of Stat3 and subsequent induction of c-Myc, was demonstrated using the selective Src kinase inhibitor SU6656 (Blake et al., 2000; Bowman et al., 2001). Moreover, evidence for a role of SFK in down- regulation of PDGF receptor signaling, by linking to c-Cbl mediated receptor degradation, has been presented (Rosenkranz et al., 2000).

14 Ras Members of the Ras family of small GTPases, first identified as potent viral oncogenes, are membrane-associated signal transduction molecules of major importance in regulation of cell growth, differentiation, and migration. Ras proteins cycle between an inactive, GDP-bound, state and an active, GTP-bound, state. The presence of activating ras mutations in 30% of human tumors emphasizes the importance of Ras proteins in growth control (Lowy and Willumsen, 1993). Transition from inactive Ras⋅GDP to active Ras⋅GTP is controlled by guanosine nucleotide exchange factors (GEFs). On the contrary, GTPase activating proteins (GAPs) increase the intrinsic GTPase activity of Ras, leading to its inactivation (reviewed in Bos, 1997). Active Ras binds to the serine/threonine kinase Raf, which upon its translocation to the plasma membrane initiates signaling via the mitogen-activated protein (MAP) kinase cascade (Stokoe et al., 1994).

PDGF receptors activate Ras by recruitment of the small cytosolic adaptor molecule Grb2. Apart from two SH3 domains, Grb2 contains an SH2 domain which mediates interaction with activated tyrosine kinase receptors (Lowenstein et al., 1992). Grb2 can bind the PDGF receptors directly, at phosphorylated Tyr716 in the β-receptor, as well as indirectly through phosphorylated receptor substrates such as SHP-2 (Arvidsson et al., 1994; Li et al., 1994). Via its two SH3 domains, Grb2 constitutively associates with the Ras guanine nucleotide exchange factor Sos. Sos converts inactive Ras⋅GDP to active Ras⋅GTP, thereby linking tyrosine kinase receptor signaling to activation of the Ras/MAPK pathway (Rozakis-Adcock et al., 1993).

Negative regulation of Ras activity is accomplished by recruitment of the GTPase activating protein p120GAP, which increases the intrinsic GTPase activity of Ras by 105- fold (Boguski and McCormick, 1993; Zhang et al., 1990). Via one of its two SH2 domains, p120GAP binds to activated PDGF β-receptor homodimers at Tyr771, but not to αα or αβ receptor complexes (Ekman et al., 1999; Heidaran et al., 1993). Thus, activated PDGF receptors can modulate Ras/MAPK signaling by recruitment of positive as well as negative regulators.

SHP-2 Tyrosine phosphorylation of activated PDGF receptors is counteracted by recruitment of SH2-domain containing tyrosine phosphatases, such as the ubiquitously expressed phosphatase SHP-2 (Stein-Gerlach et al., 1998). SHP-2 binds to the PDGF receptors via its two SH2 domains, and has been shown to down-regulate receptor signaling by dephosphorylation of the receptors and some of the receptor substrates (Bazenet et al.,

15 1996; Kazlauskas et al., 1993; Klinghoffer and Kazlauskas, 1995; Rönnstrand et al., 1999). In addition to its inhibitory role in regulation of receptor signaling, SHP-2 is phosphorylated upon binding to the receptor, thereby creating an additional binding site for the Grb2/Sos complex which results in activation of the Ras/MAPK pathway (Li et al., 1994). SHP-2 can also dephosphorylate Tyr527 of Src, thus contributing to Src activation (Rönnstrand et al., 1999). SHP-2 mediated dephosphorylation of Tyr771 of the β-receptor in heteromeric αβ receptor complexes leads to loss of RasGAP binding, thereby upregulating activity in the Ras/MAPK pathway (Ekman et al., 2002). Hence, both inhibitory and stimulatory signals arise from recruitment of SHP-2 by activated PDGF receptors.

Pathways and responses Although several signal transduction pathways activated by PDGF receptors can alone elicit responses attributed to PDGF stimulation, such as chemotaxis, mitogenicity, and actin reorganization, specific contributions of different signaling pathways downstream of PDGF receptors have been demonstrated in knock-in mouse models. PDGF α-receptor knock-in mice lacking a functional Src binding site showed deficient oligodendrocyte recruitment, whereas mutation of the PI3-K binding sites also led to lung emphysema and abnormal skeletal development (Klinghoffer et al., 2002). However, mutation of the PI3-K binding site of the PDGF β-receptor, alone or together with mutation of the PLC-γ binding site, did not result in abnormal development (Heuchel et al., 1999; Tallquist et al., 2000a).

Isoform specific signaling The different affinities of PDGF ligands for the receptors will result in both homo- and hetero-dimerization of PDGF α- and β-receptors. Homo- and heterodimers of PDGF receptors have different signaling properties due to acquisition and loss, respectively, of phosphorylation at specific tyrosine residues. PDGF-AB, which preferentially induces αβ receptor dimers, elicits a stronger mitogenic response than PDGF-AA and -BB (Rupp et al., 1994). The increased mitogenicity was explained by loss of phosphorylation of Tyr771 in the β-receptor of heterodimeric complexes, which resulted in decreased binding of p120GAP and concomitant increase in Ras activation (Ekman et al., 1999).

Evidence for intrinsic differences in signaling capacity between the PDGF α- and β- receptor intracellular domains was gained by analysis of knock-in mice, where the intracellular part of one receptor was exchanged for the intracellular part of the other receptor (Klinghoffer et al., 2001). The β-receptor intracellular domain was able to substitute for the corresponding α-receptor part, resulting in normal development. However, the α-receptor intracellular domain could not fully substitute for the

16 corresponding β-receptor part, resulting in partial phenocopy of the B-chain/β-receptor knockouts with lack of mesangial cells and deficient pericyte recruitment.

In summary, ligand binding to PDGF receptors induces receptor dimerization and increases the intrinsic tyrosine kinase activity, leading to autophosphorylation of the receptors and initiation of several different signal transduction pathways. Modulation of PDGF signaling is accomplished by the simultaneous induction of stimulatory and inhibitory signals. Furthermore, signaling from αα, ββ, and αβ receptor complexes induces different downstream events, and the signaling capacities of PDGF α- and β- receptors are not equal. Although different signal transduction pathways initiated by PDGF receptors induce overlapping biological responses in vitro, knock-in models have demonstrated receptor-specific and cell lineage-specific roles of these pathways in vivo.

3. Developmental and physiological roles of PDGF

Homozygous deletion in mice of the PDGF A- and B-chain, as well as the PDGF α- receptor and PDGF β-receptor genes has provided insight into the developmental functions of PDGF (Boström et al., 1996; Levéen et al., 1994; Soriano, 1994; Soriano, 1997).

Developmental roles of the PDGF A-chain and α-receptor Deletion of the PDGF α-receptor results in a more severe phenotype than deletion of the PDGF A-chain. This suggests that the α-receptor mediates signaling from other PDGF ligands, such as PDGF-BB, -CC, or -DD, that is of critical importance for proper embryonic development. Animals deficient for the A-chain developed a lethal, emphysema-like phenotype, linked to deficient migration of alveolar smooth muscle cell progenitors during lung development, and died around 3 weeks of age (Boström et al., 1996; Lindahl et al., 1997b). Moreover, impaired proliferation of oligodendrocyte precursor cells results in severe hypomyelination in the CNS of A-chain deficient mice (Calver et al., 1998; Fruttiger et al., 1999). The insufficient development of intestinal villi (Karlsson et al., 2000), hair follicles (Karlsson et al., 1999), and Leydig cells of the testis (Gnessi et al., 2000), observed in A-chain knockout mice, has been linked to decreased proliferation of mesenchymal cell progenitors due to disturbed epithelial-mesenchymal interactions. Knock-out of the α-receptor led to embryonic lethality between E8 and E16, with abnormal somite patterning, cleft face, spina bifida, subepidermal blebbing, hemorrhaging, and skeletal malformations (Soriano, 1997), in part explained by abnormal function of sclerotome-derived cells and neural crest cells (Tallquist et al., 2000b).

17 Developmental roles of the PDGF B-chain and β-receptor PDGF B-chain and β-receptor deficient mice show highly similar phenotypes, with abnormal placenta, anemia, thrombocytopenia, impaired kidney development, defective blood vessel maturation, edema, and perinatal death due to rupture of microvascular aneurysms (Levéen et al., 1994; Soriano, 1994).

In microvessels of developing and newborn mouse embryos, PDGF B-chain expression is restricted to immature arterial endothelial cells and PDGF β-receptor expression is confined to smooth muscle cells and pericytes (Hellström et al., 1999). Loss of capillary pericytes in B-chain and β-receptor knockout mice is the likely cause of tissue-restricted vascular malformation and edema in these animals. Mural cell investment was not affected in major vascular plexa, but reduced around capillary vessels of the brain, heart, lung, and gastrointestinal villi (Hellström et al., 1999). Furthermore, the presence of capillary aneurysms in the brain, where blood vessels are formed by angiogenesis during late embryonic development, was correlated to insufficient pericyte proliferation and recruitment to sprouting blood vessels (Lindahl et al., 1997a). Interestingly, the microvessel density in the brain is similar in B-chain or β-receptor deficient and wild-type animals, suggesting that PDGF signaling is not essential for the migration and tube formation of endothelial cells per se during developmental angiogenesis (Hellström et al., 2001). This is supported by the contribution of PDGF β-receptor negative cells to the endothelial cell lineage but not to any muscle cell lineage, including smooth muscle cells and pericytes, during the development of mouse chimeras (Crosby et al., 1998).

Moreover, endothelial cell hyperplasia occurs in brain capillaries of B-chain and β- receptor knockout mice, suggesting a role of the pericyte in controlling endothelial cell proliferation (Hellström et al., 2001). In retinas of adult mice with endothelium-specific ablation of PDGF-B, regions of low pericyte density showed capillary malformations and regression, whereas a dense and chaotic vessel network formed in regions with high pericyte density (Enge et al., 2002). Also, ectopic PDGF-BB disrupted pericyte- endothelial cell interactions and induced regression of immature retinal vessels (Benjamin et al., 1998). Furthermore, mesangial cell loss and endothelial cell apoptosis was induced in outer, but not inner, cortical kidney glomeruli of neonatal mice treated with inhibitory PDGF β-receptor antibodies, indicating that removal of smooth muscle cells can disrupt immature vessel structures (Sano et al., 2002).

Dilation of embryonic vessels in placentas of B-chain and β-receptor knockouts, concomitant with a reduction of pericytes and cytotrophoblasts, caused an altered ratio of maternal to embryonic blood vessel surface area (Ohlsson et al., 1999). The hematological

18 abnormalities in PDGF B-chain and β-receptor deficient animals are probably secondary to metabolic stress, due to insufficient nutrient exchange in the malformed placenta (Kaminski et al., 2001).

The impaired kidney development in B-chain and β-receptor deleted embryos stems from the absence of mesangial cells in kidney glomeruli. In knockout animals, loss of mesangial cells and replacement of capillary tufts by a few dilated capillary loops resulted in decreased renal filtration (Lindahl et al., 1998; Soriano, 1994). The lack of mesangial cells probably originates in defective recruitment and decreased proliferation of mesangial cell progenitors (Lindahl et al., 1998).

Taken together, the high phenotypic similarity of PDGF B-chain and β-receptor knockout animals suggests that PDGF-BB is the major ligand for the PDGF β-receptor during development.

Functions of PDGF in the adult Although significant progress has been made in understanding the developmental role of PDGF, its physiological role in the adult is less clear. The mild side effects observed in patients undergoing long-term treatment with the PDGF receptor kinase inhibitor STI571, suggest that signaling from the PDGF system is non-essential for life of the adult organism (Druker et al., 2001b). A role in regulation of interstitial fluid pressure, mediated through activation of PI3-K, has been assigned to the PDGF β-receptor (Heuchel et al., 1999; Rodt et al., 1996). PDGF-BB, but not PDGF-AA, has been shown to enhance granulation tissue formation in animal models of wound healing (Grotendorst et al., 1985; Lepistö et al., 1992; Sprugel et al., 1987). Furthermore, PDGF-BB has been shown to promote wound healing in patients with decubitus ulcers (Robson et al., 1992).

Whereas exogenous PDGF-BB can promote wound healing, the physiology of endogenous PDGF in the wound healing process remains to be elucidated. Interestingly, though PDGFβR +/- and -/- cells take part in formation of the endothelial cell lineage in developing mouse chimeras (Crosby et al., 1998), they fail to contribute to endothelium and fibroblasts during wound healing in adult chimeras (Crosby et al., 1999). In hematopoietic chimeras created by grafting of PDGF B-chain -/- bone marrow to wild- type recipients, the absence of PDGF-AB/BB of hematopoietic origin had no effect on fibroblast-rich granulation tissue formation, but significantly increased the blood vessel content in a sponge assay of wound healing (Buetow et al., 2001).

19 In conclusion, gene targeting studies have assigned a role for PDGFs in the ontogeny of specialized smooth muscle cell lineages, such as alveolar smooth muscle cells in the case of PDGF-A and α-receptor, and pericytes and mesangial cells in the case of PDGF-B and β-receptor (Betsholtz et al., 2001). Apart from regulation of interstitial fluid pressure by PDGF β-receptor signaling, the role of PDGF in the adult is less well understood.

4. PDGF in fibrotic disease

Atherosclerosis and restenosis The atherosclerotic process is initiated by injury to the endothelium, leading to recruitment of activated macrophages and migration of medial smooth muscle cells into the intimal layer where a plaque is formed. In human and experimental atherosclerotic lesions, as well as in vessels denuded by percutaneous transluminal coronary angioplasty, the expression levels of PDGF ligands and receptors are up-regulated as compared to the low levels of expression in normal vessel walls (Golden, 1991; Majesky et al., 1990; Rubin et al., 1988). These findings suggest that PDGFs, secreted by endothelial cells, macrophages, smooth muscle cells and platelets, and acting on smooth muscle cells of the media, are involved in the pathogenesis of atherosclerosis and restenosis. In agreement with this, expression of recombinant PDGF-BB in porcine arteries caused intimal thickening (Nabel et al., 1993).

In balloon catheterization models of restenosis in the rat and the baboon, intimal thickening after balloon dilatation was inhibited by neutralizing antibodies to PDGF-BB but not by antibodies to PDGF-AA (Ferns et al., 1991; Giese et al., 1999; Hart and Clowes, 1997; Hart et al., 1999). Administration of the PDGF receptor inhibitor AG1295 to denuded vessels in the pig also reduced smooth muscle cell proliferation and neointima formation (Banai et al., 1998). However, the therapeutic effect of PDGF inhibition alone was transient in a rabbit model of restenosis (Leppänen et al., 2000a), but persistent if combined with VEGF-C gene transfer to the denuded vessel wall (Leppänen et al., 2002).

Recent studies in apolipoprotein E-deficient animals fed with high-cholesterol diet showed decreased migration of vascular smooth muscle cells by treatment with a monoclonal antibody to the PDGF β-receptor, but not by an antibody to the PDGF α-receptor (Sano et al., 2001). However, immunization of high-cholesterol fed rabbits with PDGF-AA significantly reduced the intima/media ratio of atherosclerotic lesions in the thoracic aorta as compared to adjuvant treated animals (Lamb et al., 2001).

20 In summary, studies in models of atherosclerosis and restenosis suggest a role for PDGF- BB, and possibly other PDGF isoforms, in stimulating smooth muscle cells of the media to migrate into the intima of atherosclerotic lesions, where they proliferate and produce extracellular matrix components. Hence, PDGF is a potential therapeutic target in these conditions.

Fibrosis Descriptive as well as experimental findings implicate PDGF ligands and receptors in the pathogenesis of fibrosis (reviewed in Heldin and Westermark, 1999). Elevated expression of PDGF in alveolar macrophages has been demonstrated in several fibrotic conditions of the lung, such as idiopathic pulmonary fibrosis (Vignaud et al., 1991), histiocytosis X (Uebelhoer et al., 1995), and the Hermansky-Pudlak syndrome (Harmon et al., 1994). Furthermore, intratracheal instillation of PDGF-BB, as well as transgenic overexpression of PDGF-B in the lung, caused proliferation of mesenchymal cells accompanied by collagen production (Hoyle et al., 1999; Yi et al., 1996). PDGF inhibition, by antisense oligonucleotides or neutralizing antibodies, inhibited silica-induced pulmonary fibrosis in the mouse (Ohta et al., 1997), and the PDGF receptor antagonist AG1296 reduced mesenchymal cell proliferation by 50%, and collagen production by 90%, in a rat model of lung fibrosis (Rice et al., 1999).

In line with its important role in mesangial cell recruitment during embryogenesis, PDGF has been implicated in glomerular diseases with excessive mesangial cell proliferation (Abboud, 1995). The expression of PDGF ligands and receptors is up-regulated in human and experimental glomerulonephritis (Iida et al., 1991; Niemir et al., 1995). Recently, PDGF-C expression was demonstrated in arterial smooth muscle cells and collecting duct cells of the normal kidney, and its expression was induced in mesangial cells upon provoked Thy-1 nephritis (Eitner et al., 2002). In the Thy-1 rat model of glomerulonephritis, which resembles human IgA nephropathy, PDGF inhibitors were able to decrease the proliferation of mesangial cells (Floege et al., 1999; Gilbert et al., 2001; Johnson et al., 1992).

Taken together, PDGF action on fibroblast-like cells is likely to contribute to pathological fibrosis in several different organs. In particular, PDGF-BB has been shown to increase proliferation and extracellular matrix production of these cells. PDGF antagonists show therapeutic effect in several different models of fibrosis, suggesting evaluation of these inhibitors in human disease.

21 5. PDGF in malignant disease

The first indications that PDGF can be involved in tumorigenesis came with the discovery of homology between the simian sarcoma viral oncogene v-sis and PDGF-B (Doolittle et al., 1983; Waterfield et al., 1983). The transforming activity of the sis gene product occurs by autocrine stimulation of PDGF receptors (reviewed in Westermark et al., 1987). Moreover, PDGF-A could transform cells expressing the PDGF α-receptor (Beckmann et al., 1988).

PDGF stimulation in tumor cells Malignant cells in various human tumors have been shown to co-express PDGF ligands and receptors, which is a prerequisite for autocrine growth promotion by PDGF (reviewed in Heldin and Westermark, 1999). In human glioma, PDGF α-receptor and PDGF A-chain expressing tumor cells occur with increasing frequency in high-grade as compared to low- grade tumors. In particular, PDGF α-receptor expression was observed in tumors that did not over-express the EGF receptor (Hermanson et al., 1996). Furthermore, some cases of glioblastoma multiforme had an amplified PDGF α-receptor gene, with subsequent receptor overexpression (Fleming et al., 1992; Kumabe et al., 1992). Recently, expression of PDGF-C and PDGF-D was demonstrated in human glioma cells (LaRochelle et al., 2002; Lokker et al., 2002).

Expression of dominant-negative PDGF or PDGF β-receptor mutants could inhibit growth of glioma cell lines with autocrine PDGF A-chain/PDGF α-receptor loop (Shamah et al., 1993; Strawn et al., 1994). Moreover, therapy of PDGF-A/PDGF α-receptor expressing human glioblastoma xenografts with the PDGF antagonist STI571 induced proliferative arrest and subsequent reduction of tumor growth (Kilic et al., 2000). However, treatment of primary human glioblastoma xenografts with PDGF antagonists has not yet been reported. In Ewing family tumors, which harbor EWS/ETS fusion transcription factors, expression of PDGF-C is upregulated (Zwerner and May, 2001). Dominant-negative PDGF-C, as well as the PDGF receptor kinase inhibitor AG1296, inhibited growth of tumor cells in vitro (Zwerner and May, 2002). Apart from the involvement of the PDGF α-receptor in gliomagenesis, chromosomal translocations or activating mutations involving PDGF-A, -C, -D, and α-receptor have not been described in human cancer.

Autocrine transformation by activating mutations in the genes encoding PDGF-B and the PDGF β-receptor has been demonstrated to occur in dermatofibrosarcoma protuberans and chronic myelomonocytic leukemia, respectively (see below). Furthermore, co-expression

22 of the PDGF B-chain and the PDGF β-receptor has been demonstrated in various soft tissue tumors and sarcomas (Smits et al., 1992).

In summary, PDGF ligand and receptor co-expression has been demonstrated in several different malignant diseases. Furthermore, inhibition of PDGF signaling can block the growth of experimental glioblastomas. These findings together provide a rationale for clinical trials with PDGF antagonists in diseases with putative autocrine activation of the PDGF system.

Paracrine PDGF stimulation Several lines of evidence suggest that paracrine PDGF stimulation also has a role in tumor formation. While expression of PDGF ligands is frequently detected in epithelial tumor cells, PDGF receptors are mainly found on mesenchymal cells of the tumor stroma (Bhardwaj et al., 1996; Sundberg et al., 1997) (Table 1). The vascular PDGF β-receptor staining pattern observed in human tumors has been attributed to pericytes, as well as endothelial cells (Franklin et al., 1990; Hermanson et al., 1988; Plate et al., 1992). However, by confocal microscopy analysis of vessels in colorectal carcinoma, PDGF β- receptor expression was found to co-localize with the pericyte antigen HMW-MAA, and not with the endothelial cell antigen vWF (Sundberg et al., 1993). By in situ hybridization analysis in the murine T241 fibrosarcoma, PDGF-B was expressed in endothelial cells whereas vessel-associated mural cells expressed the PDGF β-receptor (Abramsson et al., 2002).

Observations from in vivo models where the tumor cells produce PDGF, but lack PDGF receptors, are consistent with a role of PDGF signaling in formation of the tumor stroma. Expression of PDGF-BB led to accelerated tumor take in a murine xenograft model of human melanoma, concomitantly with the induction of a well vascularized, fibroblast-rich tumor stroma (Forsberg et al., 1993). In contrast to the tumors formed by control- transfected cells, PDGF B-chain expressing tumors had no necrosis. Expression of PDGF- B in non-tumorigenic epithelial HaCaT cells induced formation of progressively enlarging and rapidly proliferating cysts, classified as benign tumors (Skobe and Fusenig, 1998). In contrast, control-transfected HaCaT cells did not form cysts or tumors. A high density of blood vessels, and high proliferative activity in stromal fibroblasts, characterized the stroma surrounding early cysts. After four weeks, the proliferative activity and vessel density in the stroma decreased. This implies that stroma cells activated by PDGF-BB can stimulate the growth of benign tumor cells, which eventually results in tumor progression. Moreover, MCF-7 breast carcinoma cells overexpressing c-rasH give rise to collagen-rich, desmoplastic tumors upon inoculation in mice. The c-rasH MCF-7 cells were found to

23 Table 1. Immunohistochemical detection of PDGF β-receptor expression in human tumors

Tumor Antibody n Positivity (%) Note Reference

Tumor Blood Fibro- cell vessel blast

BCC PDGFR-B2 26 0 + + Pontén et al., 1994

Breast ca. PDGFR-B2 29 0 + + 1, 2 Bhardwaj et al., 1996 PR7212 26# 21 NA 100 Coltrera et al., 1995 PR7212 45 0 100 100 de Jong et al., 1998

Colorectal ca. PDGFR-B2 210 0 + + 3 Lindmark et al., 1993 PDGFR-B2 6 0 100 + 4 Sundberg et al., 1993

DFSP PR7212 6# 100 NA NA Taniuchi et al., 1997 PDGFR-B2 3 100 + NA Smits et al., 1992

Glioblastoma PDGFR-B2 3 0 100 NA 3 Plate et al., 1992 PDGFR-B2 2 0 100 NA 5 Hermanson et al., 1992 958 33 + + NA Lafuente et al., 1999

Lung ca. PR7212 92 0 + + Kawai et al., 1997

MFH PR7212 6# 100 NA NA Taniuchi et al., 1997 PDGFR-B2 3 100 + NA Smits et al., 1992 PR7212 6 100 NA NA Palman et al., 1992 PR7212 12 92 + NA Franklin et al., 1990

Malignant PR7212 33# 39 NA NA Ramael et al., 1992 mesothelioma PR7212 14 100 NA NA Langerak et al., 1996

Meningioma PR7212 22 100 NA NA Kuratsu et al., 1994 PR7212 61 100 NA NA Yang and Xu, 2001

Midgut PDGFR-B2 31 0 + 66 3 Funa et al., 1990 carcinoid PDGFR-B2 27 0 + 83 2, 3 Chaudhry et al., 1992

Ovarian ca. PDGFR-B2 45 0 NA 64 Henriksen et al., 1993

Pancreatic ca. PDGFR-B2 20 + + + Ebert et al., 1995

Phyllodes PR7212 28# 31 50 NA 6 Feakins et al., 2000

Soft tissue PR7212 56# 100 + NA Wang et al., 1994 PR7212 72 88 + NA Palman et al., 1992 PR7212 57 53 + + Franklin et al., 1990

24 The table summarizes literature on PDGF β-receptor immunodetection in human tumors. PDGF β-receptor reagents used were the mouse monoclonal antibodies PDGFR-B2 (Rönnstrand et al., 1988) and PR7212 (Hart et al., 1987), and the rabbit antiserum 958 (sc- 1506, Santa Cruz Biotechnology). n, number of stained and analyzed specimens. Staining was performed on frozen tissue specimens unless marked by #, which indicates use of paraffin-embedded tissue. The percentage of analyzed specimens with staining of specified cell type or structure is indicated. NA, not applicable or not analyzed; +, positive staining noted but not quantified. Notes: 1, co-localization with endothelial PAL-E immunostaining; 2, co-localization with α-smooth muscle actin immunostaining; 3, co- localization with von Willebrand factor immunofluorescence or immunostaining; 4, confocal co-localization with the pericyte marker high molecular weight-melanoma associated antigen (HMW-MAA); 5, co-localization with endothelial Ulex Europaeus agglutinin (UEA-1) immunostaining. Abbreviations: BCC, basal cell carcinoma; MFH, malignant fibrous histiocytoma; DFSP, dermatofibrosarcoma protuberans.

produce a mitogenic activity, which competed with PDGF in receptor binding assays, and expression of a dominant-negative PDGF-A mutant abolished the desmoplastic response in c-rasH MCF-7 tumors (Shao et al., 2000).

Apart from its role in connective tissue formation, PDGF-B has been demonstrated to contribute to the increased interstitial fluid pressure in experimental tumors (Pietras et al., 2001). PDGF antagonists reduced the intratumoral fluid pressure in two rodent models of cancer. Furthermore, the PDGF antagonist STI571 was shown to increase uptake and therapeutic efficiency of cytotoxic drugs in experimental tumors (Pietras et al., 2002a; Pietras et al., 2002b).

Pro-angiogenic effects of PDGF-BB, and to some extent by PDGF-AA and -AB, have been demonstrated in the chorioallantoic membrane (CAM) assay (Oikawa et al., 1994; Risau et al., 1992). Interestingly, neovessels induced by PDGF-BB in the CAM showed extensive branching and recruitment of smooth muscle α-actin expressing mural cells, in contrast to the brush-like vascular formations induced by VEGF-A (Oh et al., 1998; Wilting et al., 1993). Also, some cultured endothelial cells have been shown to proliferate in response to PDGF-BB (Battegay et al., 1994), and others only when co-cultured with myofibroblasts (Sato et al., 1993). Recently, PDGF-CC was shown to induce angiogenesis in the mouse corneal pocket assay (Cao et al., 2002).

25 Due to its expression in microvascular pericytes, and possibly also in endothelial cells, the PDGF β-receptor is a putative target for anti-angiogenic therapy. However, inhibition of PDGF receptor signaling does not cause significant anti-angiogenic effects in several different mouse models of cancer (Sjöblom T., Pietras K., unpublished observation).

In conclusion, observations from experimental tumor models suggest that PDGF signaling contributes to stroma formation, thereby accelerating tumor growth rate and progression. Moreover, PDGF antagonists can reduce the elevated interstitial fluid pressure of experimental tumors.

6. PDGF antagonists

Specific pharmacological inhibition of PDGF signaling can be achieved by interference with ligand-receptor interactions, receptor dimerization, or receptor tyrosine kinase activation (reviewed in Östman and Heldin, 2001). Of the former class, neutralizing antibodies to PDGF B-chain and β-receptor have shown activity e.g. in models of restenosis and glomerulonephritis (Ferns et al., 1991; Giese et al., 1999; Johnson et al., 1992; Rutherford et al., 1997). Furthermore, soluble extracellular domains of the PDGF α- receptor (Miyazawa et al., 1998) and the PDGF β-receptor (Duan et al., 1991; Leppänen et al., 2000b; Rooney et al., 1994) were able to block PDGF activity in tissue culture experiments. SELEX aptamers, interacting with the receptor binding epitope of PDGF-B, have been shown to inhibit experimental glomerulonephritis and restenosis, and to block PDGF signaling in tumor models (Floege et al., 1999; Green et al., 1996; Leppänen et al., 2000a; Pietras et al., 2001).

Antagonists of PDGF receptor dimerization include monoclonal antibodies to, and soluble recombinant forms of, Ig-like domain 4 of the PDGF receptors (Lokker et al., 1997; Omura et al., 1997; Shulman et al., 1997). However, protein-protein interactions often involve large, flat surfaces, and no small molecule antagonist of PDGF receptor dimerization has yet been described.

Over the last decade, low molecular weight inhibitors of tyrosine kinases have emerged as a promising new class of drugs (Levitzki and Gazit, 1995; Shawver et al., 2002). The inhibitors described so far act by competing with ATP binding to the kinase. These compounds show surprising specificity, considering the high sequence similarity between tyrosine kinase domains in the human genome (Robinson et al., 2000). At least 10 PDGF receptor kinase inhibitors, with varying degree of specificity, have been described to date.

26 So far, no kinase inhibitor with preferential selectivity towards the α-receptor or the β- receptor has been identified. One major concern regarding these antagonists is that the spectrum of target kinases remains to be completely described. Among these compounds, AG1295/AG1296 (Kovalenko et al., 1994) and STI571, an inhibitor of c-Abl, c-Kit and PDGF receptor tyrosine kinases (Buchdunger et al., 1996; Capdeville et al., 2002), represent two well-characterized members.

Contrary to AG1296, STI571 has suitable pharmacological properties for in vivo applications. STI571 has gained approval for human use in treatment of chronic myeloid leukemia (CML) and gastrointestinal stroma tumors (GIST), where the target kinases Bcr- Abl and c-Kit, respectively, play major roles in transformation. In patients with chronic- phase CML who were given a dose of 400 mg per day per os, the leukemia typically responded within four weeks and 95% of patients achieved a complete hematological response (Druker et al., 2001a; Kantarjian et al., 2002). Patients in blast crisis of CML, however, often develop resistance to STI571 within weeks, likely due to increased genomic instability during blast crisis. Resistance to STI571 can occur through point mutations in Bcr-Abl, that affect amino acids in contact with the drug, as well as by genomic amplification of Bcr-Abl (reviewed in Shannon, 2002). Common dose-dependent side effects, occurring in ∼40% of treated patients, are mild and include nausea, diarrhea, vomiting, myalgia, and edema. Transient anemia is common, and persistent thrombocytopenia and neutropenia occur in 15% of STI571 treated patients (Druker et al., 2001a; Druker et al., 2001b).

Furthermore, STI571 has been successfully applied in the treatment of gastrointestinal stroma tumors (GIST), which are characterized by gain-of-function mutations in c-Kit and resistance to conventional chemotherapy (Hirota et al., 1998; Joensuu et al., 2001; van Oosterom et al., 2001). In a recent clinical study including 147 patients, more than 50% of STI571 treated GIST patients had a partial response, 20% experienced stable disease, and 13% showed early resistance (Demetri et al., 2002). Interestingly, the c-Kit D816V point mutant, which is involved in the pathogenesis of systemic mastocytosis, is insensitive to STI571 inhibition (Ma et al., 2002).

Taken together, antagonists of PDGF ligand-receptor interactions, as well as tyrosine kinase inhibitors of the PDGF receptors, can block PDGF signaling in vitro as well as in vivo. The tyrosine kinase inhibitor STI571 is a clinically useful PDGF antagonist, with a narrow spectrum of target kinases and a favorable toxicity profile.

27 C. Chronic myelomonocytic leukemia

Myelodysplastic syndrome (MDS) is a group of rare hematopoietic disorders characterized by clonal, dysplastic growth of progenitor cells and progression to acute myeloid leukemia (AML) within months or years (Ganser and Hoelzer, 1992). Chronic myelomonocytic leukemia (CMML) is a subtype of MDS. Currently, at least 35 patients with CMML and a reciprocal t(5;12)(q33;p13) chromosomal translocation have been reported (Steer and Cross, 2002). The t(5;12) is an early mutation in the multi-step pathogenesis of CMML, as evidenced by subsequent progression to AML (Golub et al., 1994). As a result of t(5;12), the PDGFRB gene on chromosome 5 is fused in frame with the ets-like transcription factor TEL (ETV6) on chromosome 12 (Golub et al., 1994).

The TEL-PDGFRB fusion transcript encodes the amino-terminal part of Tel, which contains a pointed domain (PNT), and the transmembrane and intracellular parts of the PDGF β-receptor (Fig. 2). The PNT domain is required for Tel-PDGFβR self-association, which results in constitutive autophosphorylation (Carroll et al., 1996; Jousset et al., 1997). Signaling molecules recruited and activated by Tel-PDGFβR include PLCγ, SHP2, PI3-K, Ras-GAP, STAT1 and STAT5 (Carroll et al., 1996; Sternberg et al., 2001).

Transformation by Tel-PDGFβR is dependent on the PDGF β-receptor kinase activity, and inhibition of this activity can block the growth of cells transformed by Tel-PDGFβR (Carroll et al., 1997; Carroll et al., 1996). Transformation by Tel-PDGFβR is not restricted to myeloid cells per se, since transgenic expression of TEL-PDGFRB under control of a lymphoid promoter yielded lymphomas of T- and B-cell lineages that were sensitive to inhibition by STI571 (Tomasson et al., 1999). Retroviral transduction of TEL-PDGFRB into whole bone marrow of recipient mice induced a fatal myeloproliferative disorder, and substitution of Tyr579/581 of the PDGF β-receptor part with phenylalanine altered the myeloproliferative phenotype to development of T-cell lymphoma (Tomasson et al., 2000).

To date, four different fusion partners have been described in leukemia-associated early translocations involving PDGFRB. Apart from TEL/ETV6-PDGFRB, H4/D10S170- PDGFRB (Kulkarni et al., 2000; Ross and Gilliland, 1999), HIP1-PDGFRB (Ross et al., 1998; Schwaller et al., 2001), and RAB5-PDGFRB (Magnusson et al., 2001) fusion genes have been reported (Fig. 2). Despite their divergent biological functions, the fusion partners all encode putative oligomerization domains, such as a coiled-coil or leucine zipper motif. The breakpoint in PDGFRB is conserved, and the fusion proteins all contained the transmembrane and intracellular domains of the PDGF β-receptor.

28 Ig Ig Ig Ig Ig PDGFbR NH2 COOH 1 1106

1 154 PNT Tel-PDGFbR NH2 COOH 526 1106 1 368 LZ H4-PDGFbR NH2 COOH 526 1106 1 739 CC CC CC CC COOH RAB5-PDGFbR NH2 526 1106 1 980 LZ TH HIP1-PDGFbR NH2 COOH 526 1106

Figure 1. PDGF β-receptor fusion proteins in myeloproliferative disease. SS, signal sequence; Ig, immunoglobulin-like domain; TM, transmembrane part; TK, tyrosine kinase domain; PNT, Pointed/SAM domain; LZ, leucine zipper domain; CC, coiled-coil domain; TH, Talin homology domain. Amino acid positions of breakpoints in fusion partners and the PDGF β-receptor are indicated.

Leukemia patients with confirmed PDGF β-receptor mutations presented with a myeloproliferative disorder characterized by eosinophilia, such as eosinophilic leukemia or chronic myelomonocytic leukemia. Interestingly, there is a strong male predominance among leukemia patients with PDGF β-receptor rearrangements (Steer and Cross, 2002).

Currently, STI571 therapy for leukemia with PDGFRB translocation is evaluated in clinical trials. In three CMML patients with TEL-PDGFRB translocation, daily therapy with 400 mg STI571 yielded prompt responses, with resolution of eosinophilia and normalized peripheral blood count within one week, and continued remission after 9 months of treatment (Apperley et al., 2002). In one patient with RAB5-PDGFRB, the leukemia responded rapidly to STI571 and no fusion transcript was detected by RT-PCR after 6 weeks of therapy (Magnusson et al., 2002). Six months later, this patient continues to be in molecular remission. So far, resistance to STI571 has not been described in leukemia with PDGF β-receptor translocations.

29 In conclusion, PDGF β-receptor fusion proteins containing oligomerization motifs are found in a subset of patients with myeloproliferative disease. Whereas the oligomerization motifs are required for constitutive association and kinase activation, the PDGF β-receptor kinase activity is required for transformation. Furthermore, inhibition of the PDGF receptor tyrosine kinase shows promise as a well-tolerated and highly effective treatment in leukemia with translocations involving the PDGFRB gene.

D. Dermatofibrosarcoma protuberans

Dermatofibrosarcoma protuberans (DFSP) is a locally invasive soft tissue tumor of the dermis, typically appearing in early or mid-adult life, which is considered to be of intermediate malignancy. DFSP initially appears as a plaque-like, red-brown cutaneous thickening with reddish-blue margins. Upon subsequent growth, the tumor will become firm and raised, typically presenting with multiple nodules. DFSP mainly occurs on the trunk, proximal extremities, and the head, and only rarely on the distal extremities (Gloster, 1996; Rutgers et al., 1992). The tumor is fixed to the skin, but generally not to the underlying tissue, and is moveable in the skin. DFSP tumors do not initially cause discomfort, and are thus presented and diagnosed at later stages due to increasing tumor mass, pain, or ulceration (Gloster, 1996).

DFSP is characterized by slow, infiltrative growth, and shows strong tendency to recur following surgery. The high recurrence rates following standard excision procedures, varying between 32% and 76%, can be improved by using resection margins greater than 3 cm (Rutgers et al., 1992). Likewise, the use of Moh’s micrographic surgery, where cryosections from the excision borders are continuously examined for the presence of tumor tissue and resection continued until no tumor tissue is left, has been shown to reduce the recurrence rate (Haycox et al., 1997). Metastasis is rare, affecting less than 5% of cases, and typically appears in the lung. Metastatic DFSP often show a fibrosarcoma- like morphology, rather than the ordered histology that is characteristic of primary DFSP. In children of less than one year of age, giant cell fibroblastoma (GCF), an infiltrative and recurrent mesenchymal tumor with some histological similarity to DFSP, has been described (Goldblum, 1996; Shmookler et al., 1989).

Cytogenetic studies have demonstrated supernumerary ring chromosomes, containing material from chromosomes 17 and 22, in many cases of DFSP (Mandahl et al., 1990; Naeem et al., 1995). Furthermore, a balanced t(17;22)(q21-q22;q13) is present in some cases of DFSP and GCF devoid of supernumerary ring chromosomes (Craver et al., 1995;

30 Pedeutour et al., 1996). FISH mapping and subsequent sequencing of t(17;22)(q22;q13) breakpoint regions in 11 cases of DFSP and one case of GCF demonstrated fusion of the collagen type Iα1 chain gene (COL1A1) with the PDGF B-chain gene (PDGFB) in all cases (Simon et al., 1997). Moreover, expression of COL1A1-PDGFB fusion mRNA was confirmed in 4/4 cases examined. The COLIA1-PDGFB translocation has also been detected in some superficial adult fibrosarcomas (Sheng et al., 2001), but not in malignant fibrous histiocytoma or dermatofibroma (Wang et al., 1999). Together with electron microscopy studies (Dominguez-Malagon et al., 1995), the demonstration of COLIA1- PDGFB expression driven by the COL1A1 promoter strongly supports the idea of a fibroblast-like cell as the cell of origin in DFSP.

Interestingly, characterization of chromosomal breakpoints and COL1A1-PDGFB fusion transcripts in 13 DFSP and 3 GCF tumors revealed that any intron in COL1A1 separating exons encoding the alpha-helical region of collagen type Iα1 could be fused to intron 1 in PDGFB, and still yield an in-frame fusion product (O'Brien et al., 1998). Thus, the protein products consist of a variable length of collagen Iα1 fused to PDGF-B sequence starting a few amino acids carboxy-terminal of the signal sequence.

In a recent case report, twice daily administration of 400 mg STI571 to a patient with paravertebral metastasis of DFSP resulted in 75% reduction of tumor volume by 4 months of therapy (Rubin et al., 2002). Tumor regression, as monitored by magnetic resonance imaging, was paralleled by reduced contrast enhancement and no viable tumor tissue was detected in the excised tumor. However, the typical COL1A1-PDGFB rearrangement was not present in this case, but rather a translocation involving the PDGFB gene on 22q13 and a so far unidentified gene. Furthermore, the outcome of STI571 treatment of two patients with lung metastases of DFSP has recently been reported (Maki et al., 2002). One patient had a transient response to STI571 with subsequent tumor progression and death, whereas the other experienced tumor regression which continued at 6 months of therapy. The presence of COL1A1-PDGFB fusion transcript was not investigated in this study. Tumor regression by STI571 treatment was also observed in 2/2 patients with primary DFSP and COLIA1-PDGFB translocation (M. Heinrich, personal communication).

31 II. Present investigation

A. Characterization of the chronic myelomonocytic leukemia associated Tel-PDGFβR fusion protein (Paper I)

The t(5;12) translocation, present in some cases of chronic myelomonocytic leukemia, creates a fusion gene encoding the amino-terminal part of the transcriptional repressor Tel together with the transmembrane and intracellular domains of the PDGF β-receptor. The resulting fusion protein can self-associate and has constitutive tyrosine kinase activity (Carroll et al., 1996).

The aim of this study was to characterize the Tel-PDGFβR oncoprotein, biochemically and functionally, and to determine whether tyrosine kinase inhibitors and dominant- negative mutants could reverse transformation driven by Tel-PDGFβR.

The PDGF receptor tyrosine kinase inhibitor AG1296 reduced autophosphorylation of the Tel-PDGFβR protein and inhibited the growth of BaF/3 cells transformed by Tel- PDGFβR in a dose-dependent manner, thereby demonstrating the importance of tyrosine kinase activity for transformation by Tel-PDGFβR.

Dimerization is a crucial event in PDGF receptor tyrosine kinase activation, but whether Tel-PDGFβR self-associates into dimeric or oligomeric complexes has not been clear. However, size exclusion chromatography, as well as chemical cross-linking, yielded high molecular weight Tel-PDGFβR complexes without evidence of dimeric complex formation. Oligomer formation by Pointed-domain interactions was confirmed in crystal structures of the Pointed/SAM domain of Tel, where the isolated domain self-associated into polymeric structures (Kim et al., 2001; Tran et al., 2002). Moreover, dominant- negative inhibition of Tel-PDGFβR oligomerization, by co-transfection of a truncated Tel construct containing the Pointed/SAM domain, reduced tyrosine phosphorylation of Tel- PDGFβR. Hence, interference with Tel mediated oligomerization is a potential way to reverse transformation driven by Tel-Tel interactions.

Whereas expression of Tel-PDGFβR could transform BaF/3 cells, expression of ligand- stimulated PDGF β-receptor could not, indicating intrinsic differences in substrate recruitment or signal intensity. In addition, two unique tyrosine phosphorylation sites (Tyr17 and Tyr27) were described in the Tel-PDGFβR fusion protein. Mutations at these sites did not affect the transforming ability of Tel-PDGFβR.

32 B. The dermatofibrosarcoma protuberans-associated collagen type Iα1/PDGF B-chain fusion protein is processed to PDGF-BB and sustains the growth of DFSP tumor cells (Paper II-III)

Most cases of dermatofibrosarcoma protuberans (DFSP) and giant cell fibroblastoma (GCF) harbor genetic rearrangements involving chromosome 17 and 22, resulting in fusion of the collagen Iα1 (COLIA1) gene to the PDGF B-chain (PDGFB) gene.

In these studies, we set out to characterize the structural and functional properties of a COLIA1-PDGFB fusion protein. Moreover, we evaluated PDGF β-receptor activation status and STI571 sensitivity in primary cultures of DFSP, and established an in vivo model for treatment of primary DFSP with PDGF antagonists.

Expression of a tumor-derived COLIA1-PDGFB gene in NIH3T3 fibroblasts resulted in production of a disulfide-linked, dimeric precursor molecule containing procollagen Iα1 and PDGF B-chain sequence. The precursor was further processed to mature PDGF-BB by removal of procollagen sequence, and the mature PDGF-BB was retained in a cell- associated form. COLIA1-PDGFB expression in NIH3T3 fibroblasts resulted in increased PDGF β-receptor phosphorylation, increased in vitro growth rate, and morphological transformation. The PDGF receptor inhibitor STI571 reversed these phenotypes. In a murine xenograft model, COLIA1-PDGFB transfected NIH3T3 cells formed tumors whereas control transfected cells did not. Furthermore, administration of STI571 caused a three-fold reduction in tumor volume as compared to vehicle control.

The transforming ability of COLIA1-PDGFB, together with the growth inhibition of COLIA1-PDGFB transfected NIH3T3 cells by STI571, suggested that inhibition of PDGF receptor signaling could be exploited in treatment of DFSP. We therefore investigated PDGF receptor activation in primary DFSP tumors, and established an animal model of primary DFSP.

Tyrosine phosphorylation of the PDGF β-receptor was increased in 6/6 primary cultures of DFSP and GCF, as compared to normal fibroblast control. Furthermore, PDGF β- receptor phosphorylation in primary DFSP cells was decreased by treatment with STI571. Also, the in vitro growth in high as well as low serum concentration was inhibited by STI571.

One DFSP primary culture was established as a serial xenograft in immunodeficient mice, and the integrity of the COLIA1-PDGFB translocation was verified by RT-PCR.

33 Moreover, tumor growth of primary DFSP was reduced three-fold by STI571 treatment as compared to vehicle control. In parallel, the intratumoral PDGF β-receptor tyrosine phosphorylation was decreased following STI571 administration.

The STI571-treated tumors displayed an elevated apoptotic rate as compared to control- treated tumors, but no significant reduction in proliferative rate. Furthermore, STI571 induced apoptosis of explanted primary DFSP cells. These findings contrast to the reported effect of STI571 in treatment of experimental glioblastoma, where the therapeutic effect was mediated through decreased proliferation (Kilic et al., 2000). Vascular effects by STI571 treatment of DFSP tumors included decreased mean section area, boundary length, and diameter of capillary blood vessels. Interestingly, necrosis was significantly decreased in STI571 treated tumors as compared to controls.

Together, paper I and II provide a rationale for application of PDGF receptor antagonists in treatment of human DFSP with COLIA1-PDGFB translocation. This notion is supported by the work of other investigators and by ongoing clinical trials (Greco et al., 1998; Greco et al., 2001; Maki et al., 2002; Rubin et al., 2002).

C. Enhanced tumor growth and angiogenesis by paracrine action of PDGF-DD (Paper IV)

Several studies have implicated PDGF signaling in formation of the tumor stroma, e.g. PDGF-BB secretion from tumor cells induced a vascularized, fibroblast-rich mesenchymal stroma (Forsberg et al., 1993). PDGF-DD, a recently discovered ligand for the PDGF β- receptor, is secreted as an inactive form that must be processed by proteolytic cleavage to bind PDGF receptors. Moreover, expression of PDGF-D has been detected in human tumor cell lines (LaRochelle et al., 2002).

The objective of the study was to analyze paracrine effects of PDGF-DD, produced by tumor cells, on stroma formation and angiogenesis in experimental tumors.

B16 melanoma cells, which lack expression of PDGF receptors, were engineered to express PDGFB or PDGFD. Analysis by metabolic labeling and immunoprecipitation in the case of PDGF-BB, and by RT-PCR and immunoblotting in the case of PDGF-DD, verified expression and secretion of these proteins. PDGF-DD was detected as an uncleaved form in the conditioned medium. The in vitro growth rate of PDGF transfected B16 cells did not differ from control transfected cells.

34 In a subcutaneous tumor formation assay, B16 melanoma cells expressing PDGF-DD formed more rapidly growing tumors than control transfected cells. No significant induction of connective tissue stroma was observed in tumors formed by PDGF or control transfected cells, presumably due to the rapid take and growth of these tumors. However, vascular morphology was altered in tumors expressing PDGF-DD and PDGF-BB, with increased mean vessel section area, diameter and boundary length as compared to control. The phenotypic similarity of PDGF-DD and PDGF-BB expressing tumors suggests that activation of full-length PDGF-DD occurs in the tumor microenvironment.

Immunostaining of PDGF β-receptors revealed abundant, discontinuous expression surrounding blood vessels of control- as well as PDGF-expressing tumors, with abluminal localization as compared to the endothelial cell marker CD31. Furthermore, smooth muscle actin was expressed in association with a majority of capillary cross-sections. Apart from the vascular staining pattern, no other cell type present in these tumors expressed PDGF β-receptors. Hence, the angiogenic effects of PDGF-DD and PDGF-BB are presumably mediated through action on capillary pericytes.

D. Stromal and perivascular expression of the PDGF β-receptor in normal and malignant tissues (Paper V)

The availability of PDGF antagonists prompts for reliable detection of PDGF receptors in clinical specimens. However, no validated procedure for immunostaining of PDGF β- receptors in formaldehyde-fixed, paraffin-embedded tissue has yet been reported. By screening a panel of antibodies, we identified a potentially specific PDGF β-receptor antiserum with suitable properties for use in routine pathology.

PDGF β-receptor immunostaining of formaldehyde-fixed, paraffin-embedded E14.5 mouse embryos revealed a pattern highly similar to the previously reported PDGFRB mRNA expression during development. Furthermore, complete loss of signal in mouse embryos with a homozygous deletion of PDGFRB, and loss of signal after pre-incubation of primary antibody with recombinant antigen, confirmed specificity of the immunohistochemical detection procedure.

Prominent expression of PDGF β-receptors was detected in microvascular pericytes of all organs examined. Some smooth muscle cells, such as the myoid cells of the testis, myoepithelial cells of lactating breast, and mesangial cells of the kidney, also expressed the PDGF β-receptor. The staining pattern observed in normal tissue is highly similar to the pattern observed in early studies with the antibody PR7212 in frozen tissue sections (Franklin et al., 1990). 35 Expression of PDGF β-receptors in 350 arrayed tumor specimens including biopsies from lung, colon, breast, prostatic, and ovarial cancer, as well as glioblastoma and melanoma, was largely restricted to the vascular and stromal compartments. The frequency and pattern of stromal PDGF β-receptor expression varied between these malignancies, whereas vessel-associated staining was found in all but a few specimens. These findings correlate well to most, but not all, earlier studies of PDGF β-receptor expression in human tumors (Table 1), implying that the proposed immunohistochemistry protocol is suitable for detection of PDGF receptor expression in routine pathology.

E. Anti-stromal tumor therapy by combination of STI571 and paclitaxel (Paper VI)

Inhibition of endothelial cell growth is a potential way to halt or regress established tumors. Fibroblast-like cells and pericytes are two other constituents of the mesenchymal stroma, with a less well-defined function in the tumor context. However, production of VEGF and metalloproteases by stromal fibroblasts can support angiogenesis and promote tumorigenesis (Fukumura et al., 1998; Sternlicht et al., 1999). Therapeutic targeting of fibroblasts or pericytes could possibly result in reduced angiogenesis, destabilization of tumor vessels, and impaired stromal function.

The aim of this study was to evaluate whether sensitization of mesenchymal cells to the action of paclitaxel by the tyrosine kinase inhibitor STI571 is an exploitable way to anti- stromal therapy.

The sensitivity of cultured primary human fibroblasts to paclitaxel was increased by addition of STI571, whereas the paclitaxel sensitivity of WM9-sis melanoma cells and primary endothelial cells was unaffected by STI571 addition. STI571 decreased proliferation and increased apoptosis of paclitaxel-treated fibroblasts. Combination treatment of fibroblasts with etoposide and STI571 did not cause sensitization.

PDGF stimulation protected fibroblasts from the action of paclitaxel. However, the increased sensitivity to paclitaxel by STI571 treatment was not due to inhibition of PDGF receptors alone, since no increased paclitaxel sensitivity was detected with the PDGF receptor antagonist AG1296. This suggests that another kinase targeted by STI571 is involved in sensitization, which occurred at submicromolar concentrations of STI571.

Short-term combination therapy of stroma-rich WM9-sis tumors with paclitaxel and STI571 decreased the stromal content and increased the thrombosis and necrosis, as 36 compared to treatment with paclitaxel or STI571 alone. In a 9-day treatment study, combination of low-dose paclitaxel and STI571 significantly reduced tumor growth whereas the respective monotherapies had only minor effects, demonstrating therapeutic effect of anti-stromal therapy.

III. Future perspectives

This thesis work can be divided into two parts. In the first part (Paper I-III), we asked whether tyrosine kinase inhibition is a useful treatment strategy in malignant diseases where the PDGF B-chain or the PDGF β-receptor genes are known to take part in genetic rearrangements. In the second part (Paper IV-VI), the role of PDGF in angiogenesis and tumor stroma formation was investigated.

We, and others, have shown that PDGF receptor inhibition can reduce growth of chronic myelomonocytic leukemia cells in cell culture and in animal models. Recent clinical trials have confirmed the usefulness of STI571 in CMML treatment, with immediate onset of tumor regression and stable low-level disease after one year of treatment (Apperley et al., 2002). Moreover, STI571 could effectively control eosinophilia in five patients with hypereosinophilic syndrome (HES) (Gleich et al., 2002). The HES is characterized by persistent eosinophilia, with subsequent damage to various organ systems (Weller and Bubley, 1994). There is no chromosomal abnormality, nor any sign of clonal hematopoiesis, and the cause of HES is unknown. In 4/5 HES patients treated with STI571, eosinophilia resolved after one week of treatment. This indicates that the PDGF receptors, or other tyrosine kinases targeted by STI571, are involved in the pathogenesis of HES. Activating point mutations in the juxtamembrane region of the PDGF β-receptor, inducing constitutive dimerization, would be a putative mechanism of transformation (Irusta and DiMaio, 1998; Irusta et al., 2002).

In dermatofibrosarcoma protuberans, most cases so far examined have had a COLIA1- PDGFB translocation. As predicted from our studies (Paper II-III) and the work of others, small-scale clinical trials have shown that STI571 treatment can reduce dermatofibro- sarcoma tumor mass, in primary as well as metastatic lesions, thus enabling less extensive surgery and increased survival. The presence of COLIA1-PDGFB rearrangement in other superficial sarcomas should encourage screening for this translocation, and other translocations involving PDGF ligands, in sarcomas. Moreover, we have established a stable cell line from serial 149333 DFSP tumor xenografts, enabling in vitro studies of the DFSP response to PDGF receptor inhibition. Expression of PDGF ligands and receptors has previously been reported on tumor cells of various sarcomas, and ongoing clinical

37 trials with STI571 will reveal whether these putative autocrine loops are of functional importance for tumor growth.

PDGF-BB has previously been shown to induce formation of a vascularized tumor stroma. In the present study (Paper IV), we show that expression of PDGF-BB and PDGF-DD modulates blood vessel morphology in experimental tumors. To gain mechanistic understanding of this phenomenon, three-dimensional imaging of the developing tumor vasculature will be performed. In addition, similar experiments will be carried out in slow growing tumor models, which will allow studies of tumor take enhancement and connective tissue stroma development.

Despite being one of the most studied tyrosine kinase receptors, reliable immuno- histochemical detection of PDGF β-receptor expression in routine pathology has not been available. The procedure presented herein (Paper V) is currently being applied in large- scale screening of arrayed tumor specimens to identify conditions with aberrant PDGF receptor expression. To broaden the application range of PDGF antagonists, we are currently developing validated methods for in situ measurement of PDGF receptor signaling, in the form of activation-specific antibodies. The availability of such antibodies would enable screening of arrayed tissue biopsies to identify diseases with over-activity or under-activity in PDGF signaling. The possibility of measuring PDGF receptor activity in samples from individual patients, in combination with the availability of a selective and well-tolerated inhibitor, would be a step towards evidence-based disease therapy.

Finally, the potential role of tumor stroma as a therapeutic target gains increasing interest. Selective sensitization of stromal fibroblasts, and possibly mural cells of tumor blood vessels, to the action of cytotoxic drugs (Paper VI) is a way to test whether the tumor stroma is such a target. Further studies will include detailed analyses of blood vessel morphology and mural cell density. Moreover, similar drug combination experiments in stroma-rich tumor models, where the tumor cells themselves are insensitive to cytotoxic drugs, are warranted.

38 IV. Acknowledgements

During the years spent in the densely populated “Ludwig Land”, which is inhabited by a hard-working but very generous kind of people, I have encountered many friends who made this time exciting and fun. Thank you all! In particular, I would like to extend my gratitude to

Arne Östman, my main supervisor, for your creative mind, for always being around for discussion, for your driving force to translate experimental data into clinical practice, for allowing me to find my way of doing science, for putting up with me, and for being quite cool. I am very grateful to you. Carl-Henrik Heldin, for your generosity and fairness, your extensive knowledge, your devotion to science, and for teaching me, by example, to consider fish in the BMC restaurant a delicacy. Lars Rönnstrand, for patiently answering all my questions on protein chemistry, antibody purification, and many other issues, thereby teaching me virtually everything I know about science in practice. Eller hur?

Past and present members of the GR-lab, Gudrun Bäckström, Patrick Micke, Carina Hellberg, Daniel Hägerstrand, Marina Kovalenko, Olli Leppänen, Keiji Miyazawa, Takashi Omura, Camilla Persson, Kristian Pietras, Jill Sandström-Leppänen, and Maria Sörby, for creating a nice working environment and for giving me a share of laughing every day…

My japanese co-workers, Masao Furuhashi and Akira Shimizu, for great collaboration and skilled contributions to this work, and for the lessions in engrish…

Ulla Engström, for all the peptides, and all the nice chats. Christer Wernstedt, for “ancient wisdom” and for being such a “spjuver” at the respectable age of 57. Ulf Hellman, for listening to my endless stream of so-called “technical innovations”. Incredible patience! Bengt “Becke” Ejdesjö, Eva Hallin, Uffe Hedberg, Lasse Hermansson, Gullbritt Pettersson, Ingegärd Schiller, and Inger Schönquist, for taking care of all the practical issues in this place. You are a great bunch!

The “night shift”, Johan Ericsson and Eva Grönroos, for many late night chats and equally many junk food quality moments. Keep up the good work!

Past and present PhD students of the Ludwig Institute: Anders, Ann-Sofi, Catrin, Eva, Greger, Johan, Kaisa, Kaska, Katerina, Marcin, Maria, Ninna, Peter, Roya, Simon,

39 Sirry, Urban, Åsa, and many more, brothers and sisters in arms, for sharing fun as well as tough moments over the years. Hang in there!

The marvelous technicians, Anita, Aino, Susanne, and Lotti for sustaining the nice atmosphere of this place. “Ze french guyz”, Ulrich, Philippe, and Jean-Baptiste, for exercising my rusty French. Sunkan, Maréne, Rainer, Ivan, Pontus, and Evi, for many discussions on and off the record.

All the people who taught me immunohistochemistry and animal work: Alexandra Abramsson, Mari-José Goumans, Erik Sköldenberg, Erik Wassberg, and Kenneth Wester. Thanx a lot!

Mari-Anne Carlsson and Helena Hermelin, for incredible amounts of embedding and sectioning. I owe you!

Christer Betsholtz, for your generosity, your sharp eyes, and for letting me spend some time with your nice group in Göteborg. Past and present collaborators Helena Larsson, Lena Claesson-Welsh, Annamaria Gaal, Aris Moustakas, Serhiy Souchelnytskyi, Ihor Yakymovich, Kevin O’Brien, Jan Dumanski, and Fredrik Pontén, for support, energy transfer, and some good work.

Gute Tour, Tomas “Dooley” Larsson, Martin “Mart” Frielingsdorf, Erik “Fding” Fröding, Nils “Neil” Isaksson, and Henrik “Honk” Waldenström, for relaxing moments in bunkers and shallow waters. Keep swinging!

Norrlands Nation, Birkarlarna, Gastronomiska Sällskapet Örnsköld, and Juvenal- Orden, for tons of fun, and for keeping me away from science.

My old friends, Thomas Schön, Ivar Tjernberg, and Fredrik Tjärnström, for being just old, good friends.

Stefan Håkansson and Robert Nilsson, for being great friends at all times.

My father Bo, mother Birgitta, and sister Anna, for endless encouragement and support in life, and for convincing me that real life is led with a horde of cows at the countryside in Norrland.

Sofia, for your endless love and support. Puss!

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