Protein-tyrosine phosphatases and cancer FS2008... · 2008-02-07 · and protein-tyrosine phosphatases (PTPs), regulates important signalling pathways that are involved in the control

*Cancer Center Karolinska, Department of Pathology and Oncology, Karolinska Institutet, Stockholm, Sweden.‡Ludwig Institute for Cancer Research, Uppsala Branch, Uppsala University, Uppsala, Sweden.§Institute of Molecular Cell Biology, Medical Faculty, Friedrich-Schiller University Jena, Drackendorfer Strasse 1, D-07747 Jena, Germany.Correspondence to F.D.B. e-mail: [email protected]:10.1038/nrc1837

Immunoglobulin-like domainProtein domain of approximately 100 amino-acids that was originally identified in antibodies. Structural features include 7–10 β−strands and an internal disulphide bridge. One or more of these domains commonly occur in the extracellular parts of growth-factor receptors and other transmembrane cell surface proteins, where they are involved in protein–protein interactions.

Reversible tyrosine phosphorylation, which is governed by the balanced action of protein tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs), regulates important signalling pathways that are involved in the control of cell proliferation, adhesion and migration. Perturbation of PTK activity by mutations or over-expression results in malignant transformation1, and PTK inhibitors are now established anticancer drugs2.

The first PTP was purified in 1988, approximately 10 years after the discovery of tyrosine kinases3. It is now known that PTPs constitute a large, structurally diverse family of tightly regulated, highly specific enzymes with important regulatory roles (for reviews see REFS 4–6). As emphasized below, it is also clear that PTPs have both inhibitory and stimulatory effects on cancer-associated signalling processes, and that deregulation of PTP func-tion is associated with tumorigenesis in different types of human cancer.

The characteristics of PTPsPTP families. A recent survey found that a total of 107 PTPs are encoded by the human genome6. Of these, 38 belong to the sub-group of ‘classical PTPs’ that show specificity for phosphotyrosine, and these PTPs are the subject of this Review. For a discussion of the other subgroups of PTPs, such as the dual-specificity PTPs, myotubularins, the Eya-family of PTPs, low molecular weight PTP and the established tumour-suppressor PTEN (phosphatase and tensin homologue), which is a lipid phosphatase, we refer the reader to other reviews7–9.

PTPs are broadly divided into receptor-like forms and non-receptor forms4 (FIG. 1). The receptor-like PTPs have a single transmembrane domain and

variable extracellular domains. The intracellular parts of most of the receptor-like PTPs contain two tandem PTP domains (D1 and D2; see FIG. 1) with most, if not all, of the catalytic activity residing in D1. In many cases, the extracellular domains include immunoglobulin-like domains and fibronectin type III domains, similar to the extracellular domains of cellular adhesion molecules. Non-receptor PTPs have striking structural diversity and often contain sequences that target them to spe-cific subcellular locations or enable their binding to specific proteins10 (FIG. 1). The catalytic PTP domain spans approximately 280 amino-acids and contains a highly conserved active site with a cysteine residue that is required for catalytic activity. Dephosphorylation of substrates occurs through a two-step mechanism onsisting of the formation of a covalent PTP–phosphate intermediate that is subsequently hydrolysed.

Dephosphorylation by PTPs occurs with a high degree of specificity, as demonstrated by the >1000-fold difference in the KM for different phosphopeptides and by the selective substrate-binding of substrate-trapping PTP variants11,12. Some studies have also shown that PTPs preferentially dephosphorylate cer-tain subsets of phosphorylated tyrosines on proteins that have multiple phosphorylation sites13,14. Substrate specificity has at least two determinants; first, signifi-cant side-chain interactions occur between the sub-strates and areas that flank the PTP active site15,16; and second, subcellular compartmentalization restricts substrate access17.

Regulation of PTPs. Some PTPs are themselves regulated by phosphorylation, which can either increase or decrease their activity (reviewed in REF. 5).

Protein-tyrosine phosphatases and cancer Arne Östman*, Carina Hellberg‡ and Frank D. Böhmer§

Abstract | Tyrosine phosphorylation is an important signalling mechanism in eukaryotic cells. In cancer, oncogenic activation of tyrosine kinases is a common feature, and novel anticancer drugs have been introduced that target these enzymes. Tyrosine phosphorylation is also controlled by protein-tyrosine phosphatases (PTPs). Recent evidence has shown that PTPs can function as tumour suppressors. In addition, some PTPs, including SHP2, positively regulate the signalling of growth-factor receptors, and can be oncogenic. An improved understanding of how these enzymes function and how they are regulated might aid the development of new anticancer agents.

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PTP domain

SH2 domain

FERM domain

PDZ domain

Heavily glycosylated

Fibronectin-III-like repeat

Immunoglobulin-like domain

MAM domain

PTP1B SHP2 PTPD1

a Non-transmembrane PTPs

b Receptor-like (transmembrane) PTPs

Plasmamembrane

Extracellularpart

Transmembranedomain

Cytoplasmicpart

DEP1

LAR PTPα PTPµ

D1

D2

Fibronectin type III domainProtein domain with structural similarities to immunoglobulin-like domains, but lacking the internal disulphide. This domain is found in extracellular-matrix proteins, cell-surface receptors and enzymes, and often contains surface-exposed stretches of amino acids that are involved in protein–protein interactions, such as the prototypical RGD sequence in fibronectin that mediates integrin binding.

KM

The Michaelis constant, KM, is defined as the substrate concentration at which half the maximum reaction velocity is attained. A small KM indicates that the enzyme requires only a small amount of substrate to become saturated, and a large KM indicates the need for high substrate concentrations to achieve maximum reaction velocity. The substrate with the lowest KM on which the enzyme acts is frequently assumed to be the enzyme’s natural substrate.

In addition, both non-receptor and receptor-like PTPs are subject to regulated proteolytic cleavage, which is associated with degradation or translocation18,19. Recently, reversible oxidation of the active-site cysteine residue has also emerged as an important inhibitory control mechanism for PTP regulation (BOX 1). This mechanism might be relevant for PTP regulation under pathological conditions, including cancer (see below).

Regulated homodimerization has been impli-cated as a mechanism for the negative regulation of receptor-like PTPs. This is based on functional analyses of chimaeras of the epidermal growth factor (EGF) receptor (EGFR) and the transmembrane PTP CD45 (which is encoded by PTPRC (PTP receptor type C)), and on structural and functional studies of PTPα (which is encoded by PTPRA)20–23. The latter studies particularly implicated D1–D1 interactions in the inhibitory dimerization. However, the structure of the catalytic domain of leukocyte antigen-related tyrosine phosphatase (LAR, which is encoded by PTPRF) indicates that this mode of regulation might not apply to LAR24. Furthermore, the analysis of the structure of the D1 and D2 domains of CD45 led to the conclusion that inhibitory D1–D1 interactions could not occur because of the spatial orientations of D1 and D2 (REF. 25). Together, these latter find-ings indicate that inhibitory dimerization, involving D1–D1 interactions, might not be a mechanism of regulation that applies to all receptor-like PTPs. The extracellular domains of some receptor-like PTPs such as PTPµ (which is encoded by PTPRM), PTPκ (which is encoded by PTPRK), and PTPδ (which is encoded by PTPRD) have been shown to mediate homophilic interactions26,27. As for regulatory ligands, only a few reports exist, including the identification of pleiotrophin as an inhibitory ligand for PTPζ (which is encoded by PTPRZ1), and the demonstration of a DEP1 (which is encoded by PTPRJ) agonist in an extracellular matrix (ECM) preparation28,29.

At a glance

• Protein-tyrosine phosphatases (PTPs) constitute a structurally diverse family of tightly regulated enzymes that are characterized by a conserved catalytic domain with an oxidation-sensitive active-site cysteine residue.

• Different PTPs function as negative or positive mediators of signalling triggered by receptor-tyrosine kinases, integrins and cell-adhesion molecules.

• The tumour-suppressive function of PTPs is indicated by frequent inactivating mutations of PTPs in colon cancer, and the identification of Ptprj as the gene that confers colon cancer susceptibility in STS/A mice. Also, inactivation of the genes that encode SHP1 and glomerular epithelial protein 1 (GLEPP1) by methylation has been described in haematological malignancies and solid tumours, respectively.

• The oncogenic activity of a PTP is best characterized for the mutational activation of SHP2, which occurs in hereditary and sporadic leukaemias and, less frequently, in solid tumours.

• Despite technical challenges, recent advances in the design of PTP inhibitors are encouraging with respect to the possibilities of developing novel cancer drugs that function by inhibiting oncogenic PTPs.

• Other aspects of PTP biology that might be relevant to cancer research in the future are the regulation of PTPs by oxidation and the putative role of PTPs in angiogenesis.

Figure 1 | Schematic representation of some of the protein-tyrosine phosphatase subfamilies. The protein-tyrosine phosphatase (PTP) family is broadly divided into non-transmembrane (non-receptor) and receptor-like PTPs, which can be further classified into subfamilies. Some PTPs discussed in the Review are illustrated, and important structural features are highlighted. a | Non-receptor PTPs contain one PTP domain, which is often linked to domains that mediate protein–protein interactions, such as Src-homology 2 (SH2), 4.1 Ezrin Radixin Moesin (FERM), and post-synaptic density protein-disc large-zonula occludens (PDZ) domains. b | Receptor-like PTPs are transmembrane proteins consisting of an extracellular part that typically contains domains implicated in cell adhesion, such as immunoglobulin-like, fibronectin-III-like repeat, and meprin/A5/µ (MAM) domains. The intracellular part consists of one catalytically active PTP domain (green) and, in some subfamiles, a PTP domain with little or no catalytic activity (grey), which is likely to have a regulatory function. In the case of tandem PTP domains, the membrane-proximal domain is designated D1, and the distal domain D2.

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PTP-Cys-SOH PTP-Cys-SO2/SO3

Further oxidation

Oxidation (ROS)

PTP-Cys-SH

Reduction(GSH)

Reduction(GSH)

Irreversible states

–H2O

Cys-SNPTP

Sulphenylamide

Substrate-trapping PTPVariants of PTPs that have been experimentally altered so that they still bind their substrates but do not dephosphorylate them, which allows the identification of PTP substrates. This is most commonly achieved by substituting the active site cysteine residue with a serine (C/S mutant), or by substituting the conserved aspartic acid residue in the active site with an alanine residue (D/A mutant).

Regulation of cell signalling by PTPs Regulation of receptor-tyrosine kinases by PTPs. Individual receptor-tyrosine kinases (RTKs) interact with a number of PTPs (FIG. 2). For example, the platelet-derived growth factor (PDGF) receptor β (PDGFRβ) is dephosphorylated by receptor-like PTPs such as DEP1 (REF. 13), and non-receptor PTPs such as T-cell PTP (TCPTP, which is also known as PTP non-receptor type 2 (PTPN2))14. Conversely, individual PTPs probably func-tion on more than one RTK, as exemplified by PTP1B (which is encoded by PTPN1), which dephosphorylates the insulin receptor and is a crucial negative regulator of its signalling activity30, and also dephosphorylates the EGFR and the PDGFRs31. The site-selectivity of individual PTPs in RTK dephosphorylation indicates that PTPs not only antagonize RTK signalling, but also modulate the downstream signal-transduction of RTKs14. Both RTKs and PTPs are localized to specific subcellular compart-ments, and the interactions between PTPs and RTKs are therefore spatially restricted17. It is therefore important to elucidate the regulation of PTP localization to better understand how PTPs regulate RTK signalling. In addi-tion to the negative regulation of RTK signalling, some PTPs also function as positive regulators of RTK signal transduction. One prominent example is SHP2 (which is encoded by PTPN11), which is required for the acti-vation of the Ras–ERK (extracellular signal-regulated kinase) pathway in response to growth factors (reviewed in REF. 32), as discussed in more detail below.

Regulation of cell adhesion by PTPs. Malignant trans-formation is often characterized by significant changes in the organization of the cytoskeleton, leading to decreased adhesion and aberrant adhesion-mediated

signalling. The importance of both integrin-mediated adhesion and motility, as well as adhesion mediated by cell–cell adhesion molecules in cancer progres-sion has been recently reviewed33,34. Cell adhesion is regulated by dynamic changes in protein-tyrosine phosphorylation, underlining the importance of both PTKs and PTPs in these processes. Strikingly, widely overlapping sets of PTPs regulate both cell–ECM and cell–cell adhesion.

Loss of E-cadherin-mediated cell–cell adhesion has an important role in the transition of epithelial tumours from a benign to an invasive state. Several tyrosine kinases, including SRC, EGFR and hepatocyte growth factor receptor (HGFR) phosphorylate the cadherin–catenin complex, which disrupts adhesion (reviewed in REF. 35). Multiple PTPs, including DEP1, PTPµ and PTP1B, can associate with, and have the capacity to dephosphorylate, the cadherin–catenin complex at epithelial adherens junctions, thereby promoting cadherin-mediated adhe-sion35 (FIG. 2). In endothelial cells, vascular endothelial PTP (VEPTP, which is also known as PTPβ and is encoded by PTPRB), PTPµ and SHP2 have been shown to stabilize the endothelial-cell barrier in blood vessels by dephosphorylating the VE-cadherin complex36,37.

Cell migration requires integrin-mediated adhesion as well as a turnover of focal adhesions, leading to release of the rear end of the migrating cell. Therefore, both a lack of adhesion and too much adhesion can inhibit migration. The turnover of tyrosine phosphorylation regulates cell–ECM interactions, and a number of PTPs interact with and dephosphorylate proteins that are involved in the formation and disassembly of focal adhesions (reviewed in REF. 38) (FIG. 2). For example, PTPα is required for the activation of Src-family kinases (SFKs) following integrin

Box 1 | Protein-tyrosine phosphatase regulation by reversible oxidation

The highly reactive catalytic cysteine of protein-tyrosine phosphatases (PTPs) can be oxidized to different states by hydrogen peroxide129, other reactive oxygen species (ROS) and, potentially, other cellular oxidants (see figure; for recent reviews see REFS 125,126). The sulphenic acid form (-SOH) can further react in an intramolecular reaction with a neighbouring backbone amide nitrogen to a sulphenyl-amide structure, leading to conformational changes in the PTP catalytic center130. Other forms of reversible oxidation have also been described, including glutathionylation131 and nitrosylation of the reactive cysteine132. These reversibly oxidized forms can be reduced back to the active state — for example, with reduced glutathione (GSH). By contrast, the sulphinic acid (-SO2) and sulphonic acid (-SO3) forms are irreversibly oxidized and are less established as being physiologically relevant.

ROS-mediated PTP inactivation seems to be an integral part of signalling through many receptor-tyrosine kinases, including the receptors for platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and insulin133–136. PTP oxidation is also an intrinsic part of B-cell-receptor activation137 and the prime mediator of tyrosine kinase activation associated with ultraviolet-light irradiation of cells19,138,139. The concomitant activation of tyrosine phosphorylation and the inactivation of negatively regulating PTPs is required to avoid ‘futile cycles’ of phosphorylation and dephosphorylation126. The local concentrations of PTP-inactivating ROS species are themselves tightly regulated. Phosphatidylinositol 3-kinase activation is necessary for growth-factor-induced ROS production by NADPH oxidases139. Peroxiredoxins, which inactivate H2O2 with relatively slow kinetics, are another class of recently identified regulators of PTP oxidation126,140. The understanding of the specificity involved in this type of signalling is only in its infancy, but one aspect that has already emerged is the intrinsic differences between PTP domains with regard to susceptibility to oxidation139,141.

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P

P

P

P

P

P

P

P

P

RTK-inducedDNA synthesis

a RTK signalling b Cadherin-mediated cell–cell adhesion

c Integrin-mediated adhesion

SRC Ctn

Ctn

N

CCdh

P

PP

N

C

FAK

ECM adhesion

DEP1

SRC

ECM

ligation, an event that is necessary for adhesion to occur. Dephosphorylation of p130 CRK-associated substrate (CAS) by PTP-PEST (which is encoded by PTPN12) is required for the disassembly of focal adhesions, enabling cell migration. Several other PTPs, including GLEPP1 (glomerular epithelial protein 1, also known as PTPφ and encoded by PTPRO) and SAP1 (which is encoded by PTPRH), have been implicated in the dephosphorylation of the focal-adhesion-kinase complex.

Tumour-suppressing PTPsSince their discovery PTPs have been considered potential tumour suppressors because of their antagonistic effects on oncogenic PTK signalling. Early experimental support for this was provided by overexpression of PTPs in cells, which induced a reversion of tyrosine-kinase-dependent transformation39. The following paragraphs summarize studies that have provided strong experimental evidence for the tumour-suppressive action of PTPs (also see TABLE 1).

Inactivating mutations of PTPs in colon cancer. Systematic sequencing of proto-oncogene or tumour-suppressor genes is a fruitful strategy for elucidating mechanisms of transformation40,41. This approach has been used to study PTP mutations in colorectal cancer42.

An initial analysis of all PTPs in 18 tumour sam-ples showed mutations in 6 different PTPs, including 3 non-receptor PTPs (PTP-BAS (which is encoded by PTPN13), PTPD2 (which is encoded by PTPN14), and PTPH1 (which is encoded by PTPN3)), and 3 recep-tor-like PTPs (PTPρ (which is encoded by PTPRT), LAR and PTPγ (which is encoded by PTPRG)). This PTP subset was further analysed in 157 additional cases of colon cancer. The analyses showed a total of 83 somatic mutations in approximately one-quarter of the cases. The fact that all mutations altered the amino-acid sequence argues for the functional relevance of the mutations. Furthermore, loss-of-function effects of these mutations were demonstrated in biochemical and cellular assays for a subset of mutants (TABLE 1).

Figure 2 | Schematic illustration of the dual aspects of protein-tyrosine phosphatases in controlling cell proliferation, cell–matrix and cell–cell adhesion. a | Protein-tyrosine phosphatases (PTPs) can antagonize receptor-tyrosine kinase (RTK) signalling through direct receptor dephosphorylation, as described for T-cell PTP (TCPTP)-, or DEP1-mediated dephosphorylation of platelet-derived growth factor (PDGF) receptors. Through dephosphorylation and inactivation of inhibitory components, some PTPs such as SHP2 can also promote RTK signalling. b | Tyrosine phosphorylation of the cadherin (Cdh)–catenin (Ctn) complex is associated with reduced cell–cell adhesion. PTPs such as PTPµ can therefore stimulate adhesion through dephosphorylation of the complex. Conversely, dephosphorylation of the inhibitory pY527 site on SRC is predicted to reduce cadherin-mediated cell–cell adhesion. c | SRC-mediated activation of focal-adhesion kinase (FAK) is a crucial component of the signalling pathways triggered by the binding of integrins to the extracellular matrix (ECM). PTPs function as negative regulators of this process through FAK dephosphorylation, but also as stimulators through dephosphorylation of phosphotyrosine 527 of SRC, as demonstrated by the reduced adhesion of PTPα-deficient (Ptpra–/–) cells. Signalling molecules are schematically drawn to illustrate the principles discussed. PTPs that antagonize mitogenic and adhesion signals are in brown, whereas PTPs that promote these signals are in green.

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The PTPs that are inactivated by mutations and the spectrum of these mutations deserve some comment. First, it is striking that the inactivated non-receptor PTPs all belong to the subgroup that contains a 4.1 Ezrin Radixin Moesin (FERM) domain, which is a lipid-binding domain that connects actin filaments with membranes. This indi-cates that PTP loss contributes to the altered migratory or adhesive properties of colon cancer cells. Second, the mutations show a striking distribution across the proteins. Less than one-third of the point mutations (excluding nonsense and frame-shift mutations) occur within the PTP domains. This indicates that loss of function in most instances occurs indirectly without direct changes in the structure of the catalytic domain. Third, about half of all the mutations of the receptor-like PTPs occur in the extra-cellular domains. Interestingly, modelling studies based on the structure of the extracellular domain of PTPµ pre-dict that many of the mutations that alter the extracellular part of PTPρ would cause significant perturbations of the structure, as they affect residues with buried side-chains. Also, mutations involving residues with surface-exposed side-chains might impact directly on protein–protein interactions43 (R. Aricescu and Y. Jones, personal com-munication) (FIG. 3). In addition, comparison of the PTP mutation data with previous analyses of the same samples showed that PTP mutations occurred together with activating mutations in PTKs, defying the simplistic notion of functional equivalence of PTK activation and PTP inactivation. Overall, this significant study42 strongly indicates that PTPs are tumour suppressors. More-specific examples are covered below.

DEP1 as a tumour suppressor. Initial evidence for a potential tumour-suppressive function of DEP1 was derived from studies in which expression was reconsti-tuted in cancer cells. In cultured breast cancer cells, DEP1 re-expression led to a five- to ten-fold reduction in cell growth44. Similar findings were later made in pancreatic, thyroid and colon cancer cells45–47 (K. Balavenkatraman et al., personal communication).

Ptprj (the gene encoding DEP1) was subsequently identified as the functional gene at the mouse colon-cancer-susceptibility locus, Scc1 (REF. 48). This locus was originally defined based on its segregation with colon cancer susceptibility after crossing cancer-resistant and cancer-susceptible mouse strains49. Sequence dif-ferences in Ptprj between the cancer-susceptible and cancer-resistant strains were also identified, supporting the theory that tumour-susceptibility is conferred by certain Ptprj variants.

PTPRJ status has also been analysed in human tumours48,50,51. Loss of heterozygosity, occurring in the absence of acquired mutation in the remaining allele, has been found in breast, colon, lung and thyroid cancers, implicating PTPRJ haploinsufficiency as a transforming mechanism in humans (FIG. 3). In association with these studies, different allelic variants of human PTPRJ were identified; these variations result in DEP1 proteins that have differences in their extracellular-domain residues. Analyses of tumours of heterozygous patients with colon cancer indicated that the preferential loss of one of these alleles (Q276 of the Q276P polymorphism) is selected for during cancer growth, indicating functional

Table 1 | Protein-tyrosine phosphatases with tumour-suppressive activity

PTP (encoding gene) Data Comments References

PTPBAS (PTPN13), PTPD2 (PTPN14), PTPH1 (PTPN3), PTPρ (PTPRT), LAR (PTPRF), PTPγ (PTPRG)

Mutations identified in colon cancer; inactivating mutations confirmed for some variants of PTPRT

The three non-receptor PTPs identifed in the colon cancer screen all contain a FERM domain; mutations in all six PTPs commonly occur outside the PTP domain; the critical substrates are generally unknown

42

DEP1 (PTPRJ) Overexpression reverts the transformed phenotype of different tumour cells; loss of heterozygosity is seen in colon cancer; Ptprj is the gene present at the colon-cancer-susceptibility locus in STS/A mice

Implied as a direct antagonist of many receptor-tyrosine kinases through direct receptor dephosphorylation; tumour suppression might also involve other substrates

44–46,48, 50,51,

54–58, 60–62

SHP1 (PTPN6) PTPN6 is inactivated by promoter methylation in leukaemias, lymphomas and multiple myeloma

Re-expression is associated with a downregulation of the JAK–STAT signalling pathways

63–67

GLEPP1 (PTPRO) PTPRO is inactivated by promoter methylation in lung and colorectal cancer; re-expression is associated with tumour suppression

Knockout mice are viable and have a kidney phenotype

68–70

PTP1B (PTPN1) Generation of Trp53 and Ptp1b double-knockouts revealed decreased survival and increased development of B-cell lymphomas compared with Trp53 knockouts

The first example in which the tumour-suppressive abilities of a PTP have been evaluated by breeding of PTP knockout mice with genetically modified tumour-prone mice

128

For the designation of PTPs, the most common protein names were chosen and were used in the tables and throughout this article, accompanied by the systematic gene names in parentheses. Synonyms of the PTP names can be found in comprehensive reviews4,6 and in PTP and protein databases (see Useful Links). FERM, 4.1 Ezrin Radixin Moesin; GLEPP1, glomerular epithelial protein 1; JAK, Janus kinase; PTP, protein-tyrosine phosphatase; STAT, signal transducer and activator of transcription.

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PTPρ

Normal cells Transformed cellsRegulation of cell adhesionand proliferation

Point mutationsLoss of function

PTPRJ

Modulation ofcell adhesion

Inhibition of cell proliferation and migration

Inhibition of multiplesignalling pathwaysin haematopoietic cells

DEP1

SHP1

PTPN6

PTPRT

No expression

Loss of heterozygosityReduced expression

Methylation

a Point mutation

b Allelic loss

c Promoter methylation

differences between these alleles48,52. However, similar analyses of patients with thyroid cancer failed to detect preferential loss of the Q276 allele51. Furthermore, the thyroid study also failed to detect enrichment of

individuals who are homozygous for the predicted weaker P276 allele among patients with thyroid can-cer compared with healthy controls51. Instead, genetic imbalance of another polymorphism (D872E) was

Figure 3 | Schematic illustration of different mechanisms for protein-tyrosine phosphatase inactivation in cancer. a | Point mutations in the coding region of six different protein-tyrosine phosphatase (PTP) genes were found in colon carcinoma and several other solid tumours. The most frequently affected gene is PTPRT, which encodes the receptor-like PTP PTPρ. Several of the PTPρ mutations have been predicted or shown to affect PTPρ-mediated cell–cell adhesion, or inhibition of cell proliferation, respectively. b | Allelic loss, but no point mutations, was found for PTPRJ — which encodes the receptor-like PTP DEP1 — in different types of carcinoma. c | Promoter silencing of the SHP1-encoding gene, PTPN6, by methylation occurs frequently in lymphoma and leukaemia. In the figure, the promoter region and coding region of genes are indicated in yellow and blue, respectively. mRNA is symbolized in pink. At the PTP protein level, active states are in dark green and inactive states are in red.

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detected, with a higher frequency of the D872 allele in patients with carcinoma of the thyroid. The authors of this study suggested that the D872 variant might show an increased ability to form dimers, which would lead to PTP inhibition. In addition, a weak but significant association between breast cancer risk and a particu-lar DEP1 haplotype that does not involve Q276P was recently reported53. Continued characterization of pos-sible functional differences between the DEP1 variants is still warranted.

The tumour-suppressive function of DEP1 remains incompletely understood. However, some data have been published that addresses the signalling pathways regulated by DEP1. A number of cell culture studies have indicated an antagonistic role of DEP1 in growth factor signalling through the direct dephosphorylation of PDGFR, vascular-endothelial-growth-factor-receptor (VEGFR) and HGFR13,54–56. The physiological signifi-cance of these observations was recently supported by the finding that deletion of the Caenorhabditis elegans DEP1 homologue leads to hyper-activation of EGFR signalling and a concomitant phenotypic alteration in EGFR-dependent vulva development57. Additional DEP1 substrates that have been implicated in its tumour-suppressive action are SRC, p120 catenin and phospholipase Cγ 58,59. Another crucial aspect of DEP1 tumour suppression is thought to involve stabilization of the cyclin-dependent-kinase-inhibitor p27, thereby promoting G1 arrest45. Finally, pro-apoptotic functions of DEP1 have also been described that might contribute to its tumour-suppressive function47,60.

Future investigations into how DEP1 loss leads to transformation will probably benefit from mouse models of DEP1 suppression or loss. An initial report indicated that knocking out Ptprj results in embryonic lethality61. However, in this mouse model, Ptprj was replaced by a gene encoding an enzymatically inactive DEP1–GFP (green fluorescent protein) fusion protein, and this might have contributed to the lethal pheno-type. This hypothesis is supported by the generation of viable Ptprj–/– mice by other laboratories (T. Csikos and A. Berns, personal communication; A. Fusco, personal communication). Initial experiments in mice with only one functional Ptprj copy (and bred on a mixed FVB/129 background) failed to unambiguously illus-trate the tumour-suppressor function of Ptprj (T. Csikos and A. Berns, personal communication). Clearly, addi-tional experiments are required to establish the poten-tial tumour-suppressive activity of DEP1. Crossing of these mice to other genetically modified tumour-prone mice will probably provide valuable information on the potential tumour-suppressive actions of DEP1.

Inactivation of SHP1 and GLEPP1 by hyper-methyl-ation. Epigenetic silencing of gene expression by hyper-methylation of promoter sequences is a general mechanism for inactivating tumour suppressors62. This also seems to be a mechanism that is involved in malignancy-associated silencing of PTPs, and has been particularly well documented for SHP1 (which is encoded by PTPN6) and GLEPP1 (FIG. 3).

SHP1 is an antagonist of growth-factor signalling in epithelial and haematopoietic cells32. Inactivation of SHP1 by promoter methylation was first described in cell lines derived from T-cell lymphomas63, in which loss of SHP1 expression could be restored by treatment with a demethylating agent. Subsequent studies also demonstrated PTPN6 hyper-methylation in anaplastic large-cell lymphoma, in different forms of leukaemia and in multiple myeloma64–66. A recent study has identified a protein complex that binds to the PTPN6 promoter — this complex includes signal transducer and activator of transcription 3 (STAT3) and DNA methyltransferase 1, both of which are thought to be crucial for the silencing of PTPN6 (REF. 67). The func-tional consequences of SHP1 re-expression include decreased levels of tyrosine phosphorylation in Janus kinase 3 (JAK3) or STAT3 (REFS 63,64,66). However, the effects on cell growth of PTPN6 demethylation are yet to be reported.

GLEPP1 is structurally similar to DEP1, with a sin-gle intracellular catalytic domain and an extra cellular part that is composed exclusively of fibronectin type III repeats. Subsequent to the initial identification of promoter methylation of Ptpro in diet-induced rat liver cancer68, PTPRO promoter methylation has been described in about 50% of human lung cancer and microsatellite-instability-associated colon cancers69. Overexpression of GLEPP1 in A549 lung cancer cells, in which the PTPRO promoter is methylated, reduced anchorage-independent growth, proliferation and resistance to apoptosis69. Ptpro knockout mice have been described and are viable and fertile, although they have alterations in kidney structure and func-tion70. Further analyses of the cancer susceptibility of these mice should assist in elucidation of the tumour-suppressive mechanisms of GLEPP1.

Oncogenic PTPsSome PTPs have been found to function in a positive manner in growth-stimulatory signalling pathways trig-gered by cell-surface receptors. This probably occurs in most cases through the dephosphorylation of inhibi-tory components of these pathways (FIG. 2). Therefore, gain-of-function mutations in these PTPs seem likely to have oncogenic effects. This concept has recently been experimentally validated by the identification of oncogenic activating mutations in SHP2.

SHP2 as a signalling mediator of activated tyrosine kinases. SHP2 transduces mitogenic and pro-migra-tory signals from various types of receptors32,71 (FIG. 4). Moreover, the transforming capacity of constitutively active forms of EGFR, fibroblast-growth-factor receptor 3 (FGFR3), the RET RTK and overexpressed ERBB2, as well as BCR–ABL, seems to depend on SHP2 (REFS 72,73–76). Interestingly, SHP2 has also been implicated in Helicobacter pylori-induced gas-tric cancer and might mediate the transformation of gastric epithelial cells by interactions with the CagA protein, which is a H. pylori virulence factor (reviewed in REF. 77).

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Inactive P P

P

Upstream receptor and tyrosine kinase

SHP2-bindingprotein Substrate

Pathwaysactive

ERK1/ERK2AKTSTAT5

SHP2 activatedand recruited

a Wild-type SHP2

Increased basal activity

Upstream receptor and tyrosine kinase

SHP2-bindingprotein Substrate

Increasedand sustainedactivity of pathways

ERK1/ERK2AKTSTAT5other?

b Mutated SHP2

C N

C

N

P P

P

Altered substrate selectivity?Facilitated recruitment and activationAdditional or alternate activators,binding proteins and substrates?

P

FAB-M5M5 subgroup of acute myeloid leukaemia of the French–American–British group-classification system.

To function, SHP2 requires its SRC homology 2 (SH2) domains, which enable it to be recruited to specific targets such as a surface-receptor signalling complex. This can occur through direct recruitment, as shown for the PDGFRs, and through scaffolding adaptor proteins, including insulin-receptor substrate 1 (IRS1), FGFR substrate 2 (FRS2), growth factor receptor-bound protein 2 (GRB2)-associated binding protein 1 (GAB1) or GAB2 (REFS 32,71).

It seems likely that an important class of SHP2 sub-strates are proteins whose tyrosine phosphorylation negatively regulates the Ras–ERK pathway (FIG. 4a). These include RTKs or tyrosine-phosphorylated

adapter proteins that recruit a negative regulator of Ras, the Ras-GTPase-activating protein (RASGAP). Dephosphorylation of the RASGAP binding sites by SHP2 prevents RASGAP recruitment to the plasma membrane and thereby increases Ras activity78,79. Another recently characterized mechanism is the SHP2-dependent activation of SRC through SHP2-mediated dephosphorylation of a C-terminal SRC kinase (CSK)-binding site. This disables the ability of CSK to bind to and inactivate SRC, which in turn leads to the SRC-dependent activation of the Ras–ERK cascade80,81. SHP2 might also activate other signalling pathways, involving AKT and STAT5. Additional SHP2 substrates continue to be identified. One exam-ple is the recently described major vault protein, which might serve as scaffold protein that also regulates the Ras–ERK pathway 82.

Mutations of SHP2/PTPN11 in human cancer. In 2001, Tartaglia et al.83 identified inherited dominant autosomal mutations of PTPN11 as the cause of about 50% of the cases of Noonan syndrome. Noonan syn-drome is a complex disease comprising short stature, facial anomalies and congenital heart defects. This disease is also associated with an increased risk of developing several haematological disorders, par-ticularly juvenile myelomonocytic leukaemia (JMML) (recently reviewed in REF. 84). Interestingly, somatic mutations of PTPN11 also occur in about one-third of sporadic cases of JMML85,86, and in approximately 6% of patients with childhood acute lymphoblastic leukaemia (ALL)87 and in 4–5% of patients with acute myeloid leukaemia (AML)87–89. For unknown reasons, patients with AML who have the FAB-M5 subtype of the disease have an increased incidence of PTPN11 mutations89. PTPN11 mutations have also been detected at low frequency in solid human tumours, including lung and colon cancer, neuroblastoma and melanoma88. Generally, mutations in PTPN11 only rarely overlap with mutations in other genes that are known to cause Ras–ERK activation — this is consist-ent with the concept that activation of the Ras–ERK pathway is the principal transforming effect of mutant SHP2. Most PTPN11 mutations are missense muta-tions that lead to the expression of SHP2 variants with single amino-acid changes (TABLE 2).

Activating mechanisms of SHP2 mutations. Structural and biochemical studies have established that SHP2 is held in an inactive conformation owing to the interac-tion of its N-terminal SH2 domain with its catalytic domain. This interaction is released through the bind-ing of phosphopeptide ligands to the N-terminal SH2 domain alone or to both SH2 domains90 (FIG. 4a). The inhibitory interaction with the catalytic domain can also be disrupted experimentally by D63A and E76A mutations in the N-terminal SH2 domain. This leads to the activation of SHP2 in biochemical and biologi-cal assays91. Many of the oncogenic mutations in SHP2 cluster in the interface of the N-terminal SH2 domain and PTP domain, and are also predicted to impair the

Figure 4 | Mechanism of SHP2-mediated signal transduction and the effect of SHP2 mutations in leukaemia. a | In the absence of upstream stimulation, SHP2 is kept in a low-activity state by interaction of the N-terminal SH2-domain with the protein-tyrosine phosphatase (PTP) domain. Activation of surface receptors, and subsequent tyrosine phosphorylation of binding sites for the SHP2 SH2 domains, in receptor-tyrosine kinases (RTKs) or scaffolding adapter proteins such as growth factor receptor-bound protein 2 (GRB2)-associated binding protein 2 (GAB2), leads to recruitment of SHP2. Occupation of the N-terminal SH2 domain results in conformational changes and resolves the inhibitory interaction of the N-terminal SH2 domain and the PTP domain. Activated SHP2 dephosphorylates substrates that, directly or indirectly, have an inhibitory role for signalling toward extracellular signal-regulated kinase 1 (ERK1)/ERK2, AKT or signal transducer and activator of transcription 5 (STAT5). These include binding sites for negative regulators of the Ras-pathway such as Ras-GTPase-activating protein (RASGAP) or C-teminal SRC kinase (CSK). This leads to release of the corresponding pathways from inhibition. b | Leukaemia-associated mutations in PTPN11 impair the inhibitory interaction of the SHP2 N-terminal SH2 domain and PTP domain, leading to increased basal activity and increased affinity of the N-terminal SH2 domain for phosphopeptide ligands. Alterations of substrate selectivity of the PTP domain have also been suggested. Activation of upstream receptors, and recruitment of SHP2, is also mandatory to initiate signalling by mutant SHP2. The changes in SHP2 structure caused by mutations facilitate activation and therefore increase sensitivity for the corresponding ligands of upstream receptors. Activation of downstream signalling presumably occurs by similar mechanisms as those of wild-type SHP2, but to supra-physiological levels and in a more sustained manner. Qualitatively altered signalling might also be important, and could result from alternative upstream signals or dephosphorylation of novel substrates.

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Table 2 | SHP2 (PTPN11) variants in Noonan syndrome, and malignancies

Mutations Domain of SHP2 Occurrence

T42A N-terminal SH2 NS

V46L, N58S N-terminal SH2 Lung carcinoma

D61G N-terminal SH2 NS

D61H, Y, V N-terminal SH2 JMML, ALL, AML

E69K N-terminal SH2 JMML, ALL, AML, neuroblastoma

A72G, S N-terminal SH2 NS

A72D, T, V N-terminal SH2 JMML, ALL, AML

T73I N-terminal SH2 NS, JMML, AML

E76A, K, V, Q N-terminal SH2 JMML, ALL, AML, lung carcinoma

Q79P, R N-terminal SH2 NS

D106A Inter-SH2 region NS

R138Q C-terminal SH2 Melanoma

R289G PTP AML

N208D, S PTP NS

G503V PTP JMML, AML

Q506P PTP NS, JMML

T507K PTP NeuroblastomaMost mutants occur either in Noonan syndrome (NS) or in leukaemia and other tumours, indicating the existence of at least two different functional classes. The table contains only the most frequent mutations that are found in Noonan syndrome and leukaemia, and some mutations that are found in solid tumours. Data were compiled from REFS 83–89,96. ALL, acute lymphoblastic leukaemia; AML, acute myeloid leukaemia; JMML, juvenile myelomonocytic leukaemia; PTP, protein-tyrosine phosphatase.

inhibitory binding of the N-terminal SH2 domain to the catalytic domain. Indeed, mutations of D61 and E76 were also found in patients, and most of the com-mon oncogenic mutants show increased basal activity of SHP2 (REFS 85,92). The difference in the frequency of certain mutations in Noonan syndrome compared with JMML and other types of cancer indicates that specific mutations have different functional proper-ties that are preferentially selected for in these diseases (TABLE 2). In support of this hypothesis, expression of the leukaemia-associated SHP2 mutants E76K or D61Y in mouse bone marrow led to myeloid transformation, whereas Noonan-syndrome-associated mutants such as D61G were less transforming93. A reduced trans-forming capacity was also reported for the Noonan-syndrome-associated SHP2 N308S mutant94. Similarly, transgenic mice that express one allele of the Noonan-syndrome-associated D61G variant develop mild myelo-proliferative disease but not leukaemia95. The extent of basal activation of the mutant SHP2 proteins seems to be one important property that determines their oncogenicity. Generally, Noonan syndrome-associated variants seem to have lower basal catalytic activity than leukaemia-associated variants84,96.

A recent, detailed biochemical characterization of a series of common SHP2 mutants showed additional, unexpected properties of some SHP2 mutants76. For example, the Noonan-syndrome-associated T42D variant had only moderately increased basal activ-ity, but was strongly hyper-activated when bound

by phosphopeptide ligands, owing to an increase in ligand affinity of the N-terminal SH2 domain. Also, changes in substrate selectivity were indicated for the Noonan-syndrome-associated N308S and the Noonan-syndrome- and leukaemia-associated Q506P variant.

So, in addition to increasing basal activity, mutations can also lead to SHP2 variants with altered susceptibility to activation and modified substrate selectivity.

Mechanisms of SHP2-mediated transformation. The expression of mutant SHP2, either E76K or D61Y, in mouse bone marrow demonstrated that disease development required several months93. These data imply that cooperating genetic lesions are required for tumour development. Myeloid colony formation assays showed that secondary mutations that inacti-vated either the N- or C-terminal SH2 domain or the PTP catalytic activity all abrogated SHP2-mediated transformation. Myeloid cell transformation required the presence of the scaffolding adapter GAB2, which is an SHP2-recruiting molecule. So, SHP2 catalytic activity and its recruitment domains are essential for transformation, and this indicates the need for an acti-vating signal upstream of SHP2 in the transformation process (FIG. 4b). Consistent with this, mast cells derived from mice transplanted with SHP2-mutant-expressing bone marrow were hypersensitive to stimulation with interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF). These cells showed increased proliferation and increased activa-tion of ERK, AKT and STAT5 when stimulated with either IL-3 or GM-CSF93. IL-3- and GM-CSF-hyper-sensitivity has also been found in murine fetal liver cells that are transduced with retroviruses expressing mutant SHP2 (REF. 94). GM-CSF hypersensitivity has also been reported for macrophage progenitors derived from mutant-SHP2-expressing murine bone marrow97. So far, only a very limited number of cytokines have been tested with regard to their effect on cells that express SHP2 mutants. It is possible that differences in the phosphopeptide-dependent activation between mutants76 links them to different subsets of upstream activators. Effects of SHP2 mutants might also be dependent on cell type. Mohi et al.93 did not observe consistent biological effects on expression of mutant SHP2 in murine IL-3-dependent Ba/F3 cells. In another study with the same cell line, increased growth-factor-independent survival was observed, but these authors were unable to detect any effect on the Ras–ERK or AKT signalling pathways that was due to expression of activated SHP2 (REF. 86). So, as yet unidentified signal-ling pathways are likely to mediate the effects of SHP2 in these and possibly other cells.

PTPN11 has therefore been firmly established as a proto-oncogene in a subset of leukaemias. Given that SHP2 functions downstream of many tyrosine kinases with transforming potential, this PTP represents a potential target for cancer therapy. The identification of SHP2 substrates and upstream activators of SHP2 is warranted.

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Potential oncogenic functions of other PTPs. A number of other PTPs have been linked with cell transforma-tion (TABLE 3). However, evidence is still preliminary in most cases.

One example is the receptor-like PTP PTPα, which is a physiological activator of the SFKs98,99. PTPα can potently transform mouse fibroblasts100. Moreover, over-expression of PTPα has been found in late-stage human colon carcinoma101 and in breast carcinoma102. However, in the latter case overexpression correlated with low tumour grade and positive oestrogen-receptor status, both of which are favourable factors for patient survival. Moreover, overexpression of PTPα in breast carcinoma cell lines has been shown to inhibit transformation102. The structurally related PTPε (which is encoded by PTPRE) might be involved in breast cancer, based on the fact that its expression is increased in Hras- or Neu-driven mouse mammary carcinomas103. Expression of PTPε can also promote mammary hyperplasia in transgenic mice104.

Increased expression of several other PTPs, includ-ing some that function as tumour suppressors, has been reported in a variety of tumours. The functional relevance of their overexpression is, for the most part, unclear. However, it is plausible that a given PTP might have opposite functions in different cell types (see TABLE 3 for further information).

Opportunities for future researchOther areas of PTP research that might become relevant to cancer research in the future include studies of the role of PTPs in angiogenesis, and studies of the regulation of PTPs by oxidation. The development of PTP inhibitors is also potentially relevant to cancer.

PTPs in angiogenesis and angiogenic guidance. The function of PTPs in angiogenesis is starting to receive more widespread interest (see the review by Kappert (REF. 37) for further infomation). More than 15 different

Table 3 | Protein-tyrosine phosphatases with a proven or potential oncogenic role

PTP (encoding gene) Data Comments References

SHP2 (PTPN11) Mediates signalling of oncogenic protein-tyrosine kinases; activating mutations have been found in leukaemia and other tumours; SHP2 mutants are oncogenic in mouse models; might also have a role in Helicobacter pylori-induced gastric carcinoma

At present, this is the only PTP with a proven oncogenic function

77,83,85,86,88,93

PTPα (PTPRA) Has transforming activity in rodent fibroblasts; is overexpressed in some solid tumours; has an established function in the activation of the Src-family kinases

Expression is inversely correlated with tumour grade in mammary carcinoma; has anti-proliferative activity in mammary cancer cells; function in human tumour biology is unclear

98–102

PTPε (PTPRE) Cooperates with the Neu (activated rat ERBB2) oncoprotein in mammary carcinogenesis in mice; can activate SRC

Relevance for human tumours is currently unclear

103,104,142

SAP1 (PTPRH) Overexpressed in gastrointestinal and colorectal tumours

Inhibits cell growth and cell spreading; promotes apoptosis in transfected cell lines

143–145

PTP-LAR (PTPRF) Overexpressed in mammary and thyroid cancer

Downregulates signalling from multiple receptor tyrosine kinases

146–148

PTP1B (PTPN1) Has the capacity to activate SRC; overexpressed in mammary and ovarian carcinoma; reduced Ras activation by growth factors in Ptpn1–/– mouse fibroblasts indicates a positive role of PTP1B in this pathway

Inhibits signalling of several oncogenic tyrosine kinases; Ptpn1 gene inactivation accelerates spontaneous lymphomagenesis in Trp53–/– mice

31,128,149–152

SHP1 (PTPN6) Can activate SRC; is implicated in the positive regulation of Ras in some cell types; is overexpressed in a large fraction of mammary carcinomas, and in ovarian epithelial tumours

Is established as a negative regulator of many pathways, including transforming tyrosine kinases; might function as a tumour suppressor in haematopoietic malignancies

153–155

HePTP (PTPN7) Overexpressed in myeloid malignancies; can transform rodent fibroblasts

Binds through its KIM domain to ERK1/ERK2 and p38 kinases, and negatively regulates them

156,157

An oncogenic role for some PTPs has been proposed, largely based on data for overexpression in certain tumours. Overexpression of PTPs in tumours might be a consequence of cell transformation rather than being causally involved. ERK, extracellular signal-regulated kinase; KIM, kinase-interacting motif; PTP, protein-tyrosine phosphatase.

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PTPs are expressed in endothelial cells37, and some of these enzymes have been functionally linked to endothe-lial cell biology, such as VEGF-induced proliferation, and cell–cell adhesion mediated by VE-cadherin.

DEP1 and SHP1 have been particularly highlighted as potentially physiological antagonists of VEGFR signal-ling. Small interfering RNA (siRNA)-mediated PTPRJ downregulation, or overexpression of a dominant-negative form of DEP1, induced increased VEGFR2 phosphorylation and increased downstream signal-ling activity, particularly in high-density cultures of endothelial cells56. The finding of endothelial cell hyperproliferation in Ptprj–/– embryos is consistent with these findings61. SHP1 physically associates with VEGFR2 and is also required for both tumour-necrosis factor-α (TNFα)-mediated and tissue-inhibi-tor of metalloproteinases (TIMP)-mediated inhibition of angiogenesis105–107. TIE2, which is an RTK that is important for endothelial cell maturation, is thought to be regulated by VEPTP/PTPβ108. The association of both PTPβ and DEP1 with VE-cadherin indicates that this might be a common mechanism involved in RTK dephosphorylation56. In addition, both PTPβ and PTPµ directly stimulate the dephosphorylation of VE-cadherin, which is required for the tight cell–cell adhesion of endothelial cells. The increased phosphoryl-ation of VE-cadherin observed in vivo on systemic PTP inhibition supports the physiological relevance of these interactions109. Preliminary evidence also implies that PTPs are one component of the signalling pathways triggered by hypoxia110.

Preliminary in vivo studies with non-specific PTP inhibitors, which show stimulatory effects on collateral blood flow and neo-angiogenesis in animal models, are also consistent with crucial functions of PTPs in blood vessel biology111,112.

Another particularly interesting aspect that is still to be investigated is the involvement of PTPs in angiogenic guidance. It has been proposed that vasculogenesis and angiogenesis are governed by the same mechanisms that guide the axons during neuronal pathfinding (reviewed in REF. 113). In accordance with this, several molecules, including the semaphorins, netrins, ephrins and slits, are implicated both in axon guidance and blood-vessel pathfinding. It has also been proposed that these guid-ance cues have a role in tumour angiogenesis114.

Receptor-like PTPs are important regulators of axon growth and guidance115. Of these PTPs, the LAR subfamily is the best characterized in mammalian systems116. Expression of the LAR-family PTPs, PTPσ (which is encoded by PTPRS) and PTPδ, is essential for muscle innervation by motoneurons in mice (N. Uetani and M. L. Tremblay, personal communica-tion). The molecular mechanisms through which PTPs regulate neuronal guidance probably involve, at least in part, interactions with signalling pathways induced by other guidance molecules115. For example, the LAR family of PTPs positively regulate the Slit/Robo repul-sive signalling pathways in Drosophila and negatively regulate the netrin-mediated axon attraction pathways in C. elegans116, whereas SHP2 is implicated in the

Ephrin-mediated repulsive signalling pathway in human PC-3 cells117. Many of these axon guidance molecules have a similar function in vasculogenesis and angiogenesis.

These findings indicate that PTPs probably func-tion in angiogenic guidance during normal and cancer-associated vessel growth and so merit further investigation.

PTP inhibitors as potential anti-tumour drugs. The emerging oncogenic function of members of the PTP family has led to their consideration as drug targets7,118,119 (see Supplementary information S1 (box)). The develop-ment of suitable inhibitors for these enzymes will cer-tainly benefit from extensive previous work, which has been mainly devoted to the development of inhibitors for PTP1B as a novel class of potential anti-diabetic compounds (for review see REF. 120).

There are significant technical challenges for PTP inhibitor design. These include the high polarity of the active compounds, which is associated with poor cell per-meability and bioavailability — only a few studies have shown that PTP1B inhibitors have efficacy in cell culture (see Supplementary information S1 (box)). This problem has in part been addressed by the use of prodrug strate-gies and other chemical modifications that improve the cellular uptake121,122. Another issue is the need to develop compounds that discriminate between the closely related catalytic domains of functionally different PTPs, such as PTP1B and TCPTP. The most encouraging results so far include a compound that shows up to a 30-fold higher potency for PTP1B than for TCPTP122. Structural information based on PTP–inhibitor interactions has also allowed a switch in selectivity from PTP1B to PTPβ by small alterations of the lead structure123. Solving the remaining technical problems will be crucial if PTP1B inhibitors are to enter clinical trials.

Among the PTPs, SHP2 would be the most obvi-ous target for anticancer drug development. Another selectivity problem is then likely to be encountered — the need to block SHP2, but not SHP1. In this case, the SH2 domains might present a unique option to obtain selectivity, as indicated by recent data for SH2 domain-mediated modulation of SHP1 (REF. 124) (for further details see Supplementary information S1 (box)).

A hitherto unexplored possibility for pharmaco-logically modulating PTP activity is the development of small molecules that bind to the extracellular domains of transmembrane PTPs. A better characterization of suitable targets among the transmembrane PTPs, and more knowledge of the physiological ligands and acti-vation mechanisms are, however, needed to develop corresponding screening strategies.

Given the existing platforms for PTP inhibitor design in a number of pharmaceutical companies, potent and selective inhibitors for oncogenic PTPs might soon be available.

PTP oxidation and cancer. The improved understand-ing of the biochemistry of PTP oxidation (see BOX 1; recently reviewed in REFS 125,126), which indicates crosstalk between tyrosine phosphorylation and

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free-radical signalling pathways, is likely to reveal novel insights that are relevant to tumour biology. Many general aspects of transformation and tumour growth, including the preferential use of glycolysis for energy metabolism and growth under hypoxic conditions, affect the production of reactive oxy-gen species (ROS), and this can possibly impact on tyrosine phosphorylation through PTP oxidation. In agreement with this, a recent analysis showed that a significant fraction of PTP1B is oxidized in different tumour cell lines (T. Meng, personal communica-tion). The identification of crucial regulators of ROS production and continued characterization of PTP oxidation in tumours is likely to reveal novel insights into the mechanisms of transformation and tumour growth. For example, a recently revealed function of the p53 tumour suppressor in the regulation of cellular ROS concentrations might link p53 to the control of cellular tyrosine phosphorylation127.

Another area of interest is to what extent mutational activation or inactivation of PTPs involves changes in PTP oxidation. As outlined above, both the inactiva-tion of PTPs in colon cancer, as well as the activation of SHP2 in leukaemias, occur through diverse mutations that are widely distributed among the affected PTPs. An interesting question is whether these mutations alter the susceptibility of PTPs to oxidation.

Also, as PTPs can be inhibited by oxidation, this indicates novel strategies that could be used to pro-duce PTP-targeted drugs. Inactive, reversibly oxidized forms of PTPs are less polar than the active form. This reduction in polarity might allow inhibitors to be synthesized with improved cellular permeability125. Furthermore, as ROS production is a regulated process, components of this pathway might be targets through which modulation of PTP activity can be achieved.

Concluding remarksThe long-held view that PTPs function as tumour sup-pressors has received experimental support, and PTP inactivation in tumours seems to occur through mul-tiple genetic mechanisms. Some key issues for future studies include the identification of the crucial signalling mechanisms that are perturbed by PTP inactivation, and why the tumour-suppressive nature of some PTPs seems to be tissue specific. PTP knockout mice are predicted to be highly valuable for this type of analysis. The recent demonstration that loss of PTP1B in Trp53–/– mice leads to earlier onset of lymphoma provides a good example of this type of study128. Also, the oncogenic gain-of-function variants of SHP2 indicate therapeutic oppor-tunities that should be exploited. Finally, continued studies of the basic biology of PTPs should reveal findings that are relevant to cancer biology.

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AcknowledgementsWork in the authors’ laboratories is supported by grants from the Swedish Cancer Society (Cancerfonden) the Deutsche Krebshilfe (F.D.B.) and Karolinska Institutet (A.Ö.) the Swedish Research Counci l (C.H and A.Ö.) , the Deutsche Forschungsgemeinschaft (F.D.B.), the Deutsche Krebshilfe (F.D.B.), and the German Ministry for Education and Research (F.D.B.). We are grateful to the colleagues mentioned in the text for sharing with us their unpublished results, and to A. Uecker and C. H. Heldin for critical reading of the manuscript.

Competing interests statementThe authors declare no competing financial interests.

DATABASESThe following terms in this article are linked online to:Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=geneCD45 | DEP1 | E-cadherin | EGF | EGFR | GM-CSF | IL-3 | LAR | PTP1B | PTPα | PTPδ | PTPφ | PTPκ | PTPµ | PTPρ | RASGAP | SHP1 | SHP2 | SRC | TCPTP | VE-cadherinNational Cancer Institute: http://www.cancer.govALL | AML | breast cancer | colorectal cancer | lung cancer | lymphomas | thyroid cancerOMIM: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIMNoonan syndrome

FURTHER INFORMATIONArne Östman’s home page: http://www.mtc.ki.se/ki_cancer/search.php?search_text=42&type=groupmembersCarina Hellberg’s home page: http://www.licr.uu.se/groups/PDGF/index.html#ResearchEntry for PTPN11 in The Human Gene Mutation Database, Cardiff: http://archive.uwcm.ac.uk/uwcm/mg/search/137093.htmlFrank D. Böhmer’s home page: http://www2.uni-jena.de/med/zbio/english/pygroup.htmlGeneCards: http://www.genecards.orgInterPro: http://www.ebi.ac.uk/interproNovo Nordisk Science PTPs: http://science.novonordisk.com/ptpPfam database of protein families and hidden Markov models: http://pfam.wustl.eduPhosphobase: http://umber.sbs.man.ac.uk/phosphabase/PTP resource: http://ptpr.ibioinformatics.org/PTPresourceUniprot: http://www.expasy.uniprot.org

SUPPLEMENTARY INFORMATIONSee online article: S1 (box)Access to this links box is available online.

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