Review Epigenetic Regulation of Protein Tyrosine Phosphatases

Home , Epigenetic therapy, PTPN2, PTPN22, PTPN7, PTPRF, PTPRG

Review Epigenetic regulation of protein tyrosine phosphatases: potential molecular targets for cancer therapy Samson T Jacob,1,2 and Tasneem Motiwala1 1Department of Molecular and Cellular Biochemistry, The Ohio State University, College of Medicine, Columbus, OH 43210, USA; and 2Department of Internal Medicine, The Ohio State University, College of Medicine, Columbus, Ohio 43210, USA.

Promoter methylation-mediated silencing is a hallmark of many established tumor suppressor genes. This review focuses on the methylation and suppression of a receptor-type tyrosine phosphatase gene, PTPRO, in a variety of solid and liquid tumors. In addition, PTPRO exhibits many other characteristics of a bona fide tumor suppressor. Reactivation of genes silenced by methylation using inhibitors of DNA methyltransferases and histone deacetylases, and the potential application of PTPRO as a molecular target for cancer therapy have been discussed. Cancer Gene Therapy (2005) 12, 665–672. doi:10.1038/sj.cgt.7700828; published online 1 April 2005

Keywords: protein tyrosine phosphatase; PTPRO; DNA methylation; gene reactivation; DNA hypomethylating agents; HDAC inhibitors

ethylation of DNA at C-5 of cytosine within a CpG ‘‘hits’’ in order to inactivate both alleles of tumor Mdinucleotide is the most striking modification in the suppressor genes, resulting in the transformation of a eukaryotic genome.1 Although CpG is usually under- normal cell into cancer cell.10 Accordingly, it is concei- represented in much of the genome, short (500–2000 bp vable that one ‘‘hit’’ represents methylation of one of the long) CpG regions, designated CpG islands, are found in alleles. Indeed, reports from many laboratories indicate the proximal promoters of almost half of the mammalian that a gene can be inactivated in the cancer cell by stably genes. These regions are generally unmethylated in maintaining mutations (the first ‘‘hit’’) in one allele and normal cells unless they are associated with imprinted hypermethylation in the other (the second ‘‘hit’’).10–13 genes or genes located on the inactive X chromosome. On Alternatively, methylation of both alleles can contribute to the other hand, many cancer cells exhibit global both first and second ‘‘hits’’, as proposed by Jones and hypomethylation and regional or localized hypermethyla- co-workers.2 It is remarkable that hypermethylation is tion of specific genes.2,3 Hypermethylation leads to absent in the mutated allele, but is always associated with recruitment of a class of proteins, designated methyl the wild-type allele of a gene.14 Since numerous reviews C-binding proteins or MBDs, and subsequently that of on DNA methylation have appeared in the past few many corepressors. The large repressor complex thus years,2,3,15–17 this review will focus on a novel class of formed on the promoter consists of histone deacetylases candidate tumor suppressor genes that are silenced by (HDAC), Sin3A, RbAp46, RbAp48, Mi2, MTA-2 and promoter methylation in rodent and human tumors, and other unidentified proteins.4–7 This epigenetic event their functional, diagnostic and therapeutic implications. causes alterations in chromatin structure, prevents binding of transcription factors to the regulatory elements and ultimately results in transcriptional repression. Many Protein tyrosine phosphatases (PTPs): physiological tumor suppressor genes are silenced in different types of functions relevant to their potential role in cancer cancers due to promoter methylation.8,9 Promoter hypermethylation and gene silencing also fit with the Knud- Protein phosphorylation is an important post-transla- son’s two-hit model that calls for two successive genetic tional modification that can affect various cellular processes, including differentiation, contact inhibition, cell cycle and immune response (see Hunter18 for a Received November 21, 2004. Address correspondence and reprint requests to: Dr ST Jacob, review). This protein modification is coordinated by the Department of Molecular and Cellular Biochemistry, The Ohio State actions of protein kinases and phosphatases that must be University, College of Medicine, 333 Hamilton Hall, 1645 Neil Ave, precisely regulated in normal physiological processes. Columbus, OH 43210, USA. Protein tyrosine kinases (PTKs) form a major class of E-mail: [email protected] oncoproteins representing 80% of all oncogenes.19 A PTP PTPs as molecular targets for epigenetic therapy ST Jacob and T Motiwala 666 that catalyzes the reverse reaction could, therefore, function In addition to the functional consequences that would as a tumor suppressor. PTPs could themselves promote arise upon loss of a PTP, genetic alterations of several signaling (positive effect) by dephosphorylation and PTPs in different types of cancers also strengthen their activation of PTKs. Consequently, PTPs could coordinate position as growth/tumor suppressors (see Table 1). rather than antagonize the functions of PTKs or inhibit Several PTPs (e.g. PTPRG, PTPRK, PTPRJ) have been signaling (negative effect) depending on the downstream localized to regions marked by loss of heterozygosity effectors.20–23 For instance, tyrosine phosphatase, PTPN2, (LOH), a hallmark of many tumor suppressor genes.27–29 activates p53 and induces apoptosis in human tumor cell Inactivating mutations in some PTPs (e.g. PTPRF, line,24 whereas PTP1B negatively regulates insulin signal- PTPN14, PTPRG, PTPN13, PTPN11, PTPRT, PTPN3) ling via dephosphorylation of insulin receptor kinase.25 The are also associated with development of cancers.30 A known physiological functions of several PTPs suggest recent study based on computational analysis of the their primary roles in neuronal differentiation (e.g. human genome identified 38 classical PTPs, 19 of which PTPRK, PTPRD, PTPRR, PTPRB, PTPRS, PTPRA), mapped to regions frequently deleted in human cancers T-cell receptor signaling (e.g. PTPN22, PTPRC, PTPN7, and 30 of these protein phosphatases have been impli- PTPRJ, PTPN2) and cellular adhesion (e.g. PTPRF, cated in different types of cancers.26,31 Several studies PTPN14, PTPRM, PTPRS, PTPRA, PTPRT) (see Table 1 have implicated PTPs in cancer and a few have demon- for details). Alteration in the expression or function of strated their role as tumor suppressors using in vitro these PTPs could, therefore, lead to deregulation of tightly and in vivo assays.32 To this date, however, DEP1 is the regulated signaling process resulting in uncontrolled cell only protein tyrosine phosphatase that is classified as an growth among other pathological conditions.26 authentic tumor suppressor.29,31

Table 1 Protein tyrosine phosphatases that exhibit characteristics of a tumor suppressor Gene symbol CpG island Known physiological functions Characteristic suggestive of tumor suppressor function

PTPN22 No Suppresses T-cell activation Deleted and rearranged PTPRF Yes Cell–cell contact at adherens junction, b-catenin signaling Deleted and mutated PTPRU Yes Cell–cell recognition and adhesion Deleted PTPN7 No T-cell antigen receptor signaling, suppress activity of MAP Deleted and duplicated kinases PTPN14 Yes Cell–cell adhesion via b-catenin Deleted, duplicated, mutated When overexpressed, HeLa cells grow slower and adhere less well PTPRG Yes Deleted and mutated PTPN23 Yes Deleted PTPN13 Yes Fas-mediated programmed cell death, regulator of rho Deleted and mutated signaling pathway PTPRK Yes Stimulates neurite outgrowth Deleted and downregulated PTPN12 Yes Controls shape and motility Deleted PTPRZ1 Yes Oligodendrocyte survival Deleted PTPRN2 Yes Autoantigen associated with IDDM Deleted and methylated PTPRD Intron 1 Neurite outgrowth and neuron axon guidance Reduced expression in hepatomas PTPN3 ? Deleted PTPRJ Yes T-cell receptor signaling Deleted and mutated PTPRO Yes Axonogenesis and differentiation, podocyte structure and Deleted, methylated and suppressed glomerular filtration PTPN6 Upstream Signaling pathways in hematopoietic cells Deleted, methylated and suppressed PTPN11 Yes Mutations involved in leukemogenesis Mutated PTPN21 Yes Liver regeneration and spermatogenesis Deleted and duplicated PTPN2 Yes Inhibits T-cell proliferation Induces apoptosis via activation of p53 PTPRS Upstream Cell–cell interactions, primary axonogenesis, axon Deleted guidance during embryogenesis PTPRH Upstream Negative regulator of integrin mediated signaling Downregulated during dedifferentiation of human hepatocellular carcinomas Induces apoptotic cell death upon over-expression Inhibits cell growth and motility PTPRA ? Regulation of integrin signaling, cell adhesion and Expression relates to low tumor grade proliferation, neuronal migration PTPRT Yes Signal transduction and cellular adhesion in CNS Deleted and mutated

Cancer Gene Therapy PTPs as molecular targets for epigenetic therapy ST Jacob and T Motiwala 667 Methylation and suppression of PTP receptor-type O: role following treatment of the nonexpressing A549 cells with 34 as tumor suppressor in human cancer DNA hypomethylating agents. Further, the PTPRO gene is localized to the chromosomal region 12p12.3 that 31,34 Suppression of gene expression by promoter methylation is characterized by LOH in different types of cancer, is considered an important mechanism for the loss of another established characteristic of many tumor sup- 35 function of many tumor suppressors. Our laboratory has pressor genes. Global expression profiling of micro- shown that the gene for protein tyrosine phosphatase satellite instability (MSI-H) colon cancer using cDNA receptor-type O, PTPRO (also called Glepp1 or PTP-U2) microarray identified PTPRO as one of the 81 genes that 36 is progressively methylated in the preneoplastic liver and are selectively downregulated and methylated. These primary hepatomas produced in rats fed methyl-deficient data, taken together, support the notion that PTPRO is a diet. This gene was identified in a genome-wide scan for candidate tumor suppressor. methylation changes during tumor progression.8,33 It is also heavily methylated and silenced in a transplanted rat hepatoma.33 Further, when the animals bearing the tumor Methylation and suppression of the truncated form of were treated with the DNA hypomethylating agent PTPRO (PTPROt) in cancer cells of lymphoid origin 5-azacytidine (5-AzaC), demethylation of the PTPRO promoter resulted in gene re-expression and reduction in Several variants of PTPRO are generated due to tumor size. All these observations pointed towards the transcription from distinct promoters and alternative potential role of PTPRO as a tumor suppressor. The splicing (see Figs 1 and 2). The cells of lymphoid origin observation that PTPTO is methylated in preneoplastic exclusively express PTPROt whereas most epithelial liver of rats fed methyl-deficient diet33 suggests that this tissues express predominantly the full-length form. To modification could emerge as an early tumor marker in this date, there has been only one report demonstrating hepatocellular and probably other tumors. We have since the potential role of promoter methylation in the extended these studies to human tumors and have suppression of a PTP in tumors of lymphoid origin.37 identified tumor-specific methylation of the PTPRO This study, however, deals with a nonreceptor type PTP CpG island, located within the promoter region, in (SHP-1). It was of interest to investigate whether the primary human hepatocellular carcinoma relative to the PTPRO gene is methylated and silenced in primary matching normal liver tissue. Analysis of 43 primary lung tumors and their matching normal adjacent lung tissues also revealed extensive methylation of PTPRO promoter in a large number of lung tumors, whereas the promoter was essentially methylation-free in the matching normal lung tissue.34 In many cases of hepatic and lung tumors, the promoter methylation inversely correlated with PTPRO expression. Although normal liver and lung do not express PTPRO to the same level as brain or kidney, it is noteworthy that PTPRO expression is abrogated in the majority of primary liver and lung tumors. Further, ectopic expression of PTPRO in human lung cancer line, A549 (where PTPRO is suppressed due to methylation) resulted in inhibition of anchorage-independent growth, delayed entry of the cells into cell cycle and increased susceptibility to apoptosis.34 Recent study also showed Figure 1 Protein isoforms of PTPRO. The full-length and truncated that PTPRO overexpression reduced the tumor forming forms of PTPRO differ mainly with respect to their extracellular potential of cells upon injection into immunocompro- domains (fibronectin type III repeats). Each of these forms gives rise mised mice (Motiwala T, Rosol T, Jacob ST, unpublished to two isoforms that are products of alternatively spliced transcripts, data). The suppressed PTPRO gene was reactivated that is, by splicing of E17.

Figure 2 Schematic representation of the PTPRO gene.

Cancer Gene Therapy PTPs as molecular targets for epigenetic therapy ST Jacob and T Motiwala 668 human leukemia. Indeed, we were able to show that it is (rabbit homolog of PTPROt).38 Interestingly, there is no methylated and silenced in the majority of the peripheral CpG island within the PTPROt promoter. In fact, there blood lymphocytes from 92 chronic lymphocytic leukemia are only five scattered CpG dinucleotides in this region. (CLL) patients whereas the CD19 þ selected B-lympho- Can methylation of these specific CpGs alter expression cytes from normal individuals did not show methylation of PTPROt in cells of lymphoid origin? Alternatively, can of this gene (T Motiwala, H Kutay, J Byrd, M Grever, S methylation of the CpG island located 220 kb upstream of Jacob, unpublished data). Further, it could be reactivated the identified promoter regulate transcription of the in a CLL-like cell line (where PTPROt is suppressed) truncated gene from the downstream þ 1 site? If following treatment with a DNA hypomethylating agent. methylation of the few CpGs in the intronic promoter is It is evident that PTPRO/PTPROt methylation and responsible for gene suppression, the molecular mechan- suppression is a common characteristic of many different ism for the methylation-mediated alteration in PTPROt types of tumors. expression is likely to be different from that of PTPRO. In this context it is noteworthy that the methylation of a single CpG in NF1 and BRCA1 gene promoters Methylation of other receptor-type protein tyrosine can prevent interaction of a specific transcription phosphatases in leukemia factor, CREB, to the cognate element that contains the methylated CpG.39,40 Similarly, in mouse ribosomal RNA Are the methylation and subsequent suppression of (rRNA) gene, methylation of cytosine in the only CpG tyrosine phosphatases confined to PTPRO or PTPROt? located within the immediate promoter region was Recent analysis by Restriction Landmark Genomic sufficient for inhibiting binding of the RNA polymer- Scanning of CLL genomic DNA has revealed that three ase-I-specific transcription factor UBF to its regulatory other PTPs (PTPRN2, PTPRZ2, PTPN11) are preferen- sequence (UCE) and subsequent inhibition of ribosomal tially methylated in CLL and acute myeloid leukemia (C gene transcription.41 It is, however, not known whether Plass, S Jacob, unpublished data). It is, therefore, highly methylation of the few CpG residues in the PTPROt likely that other PTPs may also be targets for DNA promoter also facilitates recruitment of methyl C-binding methylation in human cancers. Analysis of the genomic proteins (MBDs) that help recruit specific corepressors sequences of PTPs shows that most PTPs (except such as Sin3A, HDAC or HMT forming a large repressor PTPN22, PTPRC, PTPN7, PTPRR, PTPRQ, PTPRA) complex on the promoter. Alternatively, methylation at harbor a CpG island in proximity to their promoter the upstream CpG island results in positioning of regions (Table 1). The association of CpG islands with the inhibitory nucleosomes due to recruitment of MBD- vast majority of PTPs suggests that they have a potential corepressor complexes that deacetylate histone tails and for being regulated by methylation. While the effect of methylate histone H3 at K9. K9 methyl histone H3 could methylation on their expression has not been studied, it is then recruit HP1a protein to form heterochromatin logical to anticipate reduced expression of these PTP structure that propagates through out the region to genes upon methylation. An important issue that needs to impede access of transactivators to cognate cis elements be explored is in regard to the molecular mechanisms for on both promoters.42,43 Interestingly, unlike the mouse the methylation-mediated silencing of PTPRO or other and rat rRNA promoters that contain one and five CpGs, PTPs in human cancer. It is of particular interest to respectively, the corresponding human promoter exhibits investigate whether the mechanism for the altered a CpG island and an inverse correlation exists between expression of PTPRO is distinct from that of PTPROt methylation of the CpG island and human rRNA (see below). promoter activity.44 It would be of interest to determine whether multiple mechanisms operate in the methylation- mediated suppression of rRNA genes as well as PTPRO Proposed mechanisms of methylation-mediated and PTPROt. Further study is needed to address these suppression of PTPRO and PTPROt important issues.

The structural organization of PTPROt suggested the existence of an independent promoter downstream Reactivation of genes silenced by promoter methylation: (220 kb) of the transcription start site of the full-length epigenetic therapy form of PTPRO (see Fig 2). Computational analysis provided a clue as to its probable location within intron A quarter century ago, Taylor and Jones45 demonstrated 12 of the gene. To prove the existence of a distinct differentiation of mouse cells (3T3 and C3H/10T1/2/ downstream promoter site, we selected a region B1kb 2CL8) into new mesenchymal phenotypes following upstream of the potential þ 1 site of the PTPROt isoform treatment with the DNA hypomethylating agent, 5-AzaC that extends into the first exon (see Fig 2). Transient or 5-AzadC. This observation set the stage for many transfection studies using the promoter-luciferase reporter studies in the ensuing decades that explored reactivation construct in several different cell lines demonstrated the of suppressed genes in different cell types, particularly presence of an active promoter in that region. This cancer cells (for reviews, see Baylin,46 Karpf and Jones,47 observation is consistent with the identification of an and Jain48). Reactivation of the suppressed genes can also intronic promoter that drives the expression of PTP-oc occur in animals treated with 5-AzaC,33,49,50 which rules

Cancer Gene Therapy PTPs as molecular targets for epigenetic therapy ST Jacob and T Motiwala 669 out the possibility of any artifacts produced in cell culture. growth stage.57 It also appears that among three DNMTs Epigenetic modification alters the expression of a gene only DNMT1 is the target of 5-AzaC. Since gene without affecting DNA sequence. Suppression of a gene expression profile of the inhibitor-treated HCT116 cells by promoter methylation could, therefore, be alleviated is similar to that of DNMT1 null cells, DNMT1 is likely either by removing the methyl groups from DNA or to be the preferential target of 5-azaC.57 This observation altering the histone tail modifications such as acetylation is consistent with our earlier report that DNMT1 is or methylation. It is, therefore, conceivable that demethy- selectively and rapidly degraded in animal cells following lation and the consequent reactivation of these genes will exposure to 5-azaC.58 Recent study in our laboratory has be a rational approach to treat cancer. In preclinical shown that this selective degradation of DNMT1 is studies, 5-AzadC appears to exhibit greater potency over mediated by the proteasomal pathway (K Ghoshal and S 5-AzaC, with respect to inhibition of DNA methyltrans- Jacob, unpublished data), which offers an alternative ferase and initiation of differentiation process51 making it mechanism of action for these potent anticancer drugs. a more ideal chemotherapeutic agent for epigenetic The expression of less than 1% of the genes was elevated therapy. in response to 5-AzadC,59 which rules out the possibility In an effort to restore the activity of the epigenetically that cellular transformation is initiated by increasing silenced genes, 5-AzaC or its congener 5-AzadC has been global hypomethylation by DNMT inhibitors. used for cancer therapy (for a review, see Christman52 ). Another promising therapeutic approach in the treat- Nearly 100 clinical trials with either 5-AzaC or 5-AzadC ment of cancer involves agents that alter histone post- alone or in combination with a HDAC inhibitor have translational modifications. Histones are modified by been reported in the National Cancer Institute database. acetylation, methylation, phosphorylation, ADP-ribosy- While different types of malignancies have been treated lation and ubiquitination. There is substantial evidence with these agents, the most promising results were for a link between the characteristic modifications on achieved in the therapy of hematopoietic neoplasms.53 histone tails and gene regulation through changes in The 5-Aza compounds probably demethylate the tumor chromatin configuration (‘‘histone code’’).42 Transcrip- suppressor genes leading to their reactivation and tionally active chromatin is usually associated with ultimately reduced tumor growth. In this context, the hyperacetylated histones, while transcriptionally inactive diminished expression of PTPROt, a potential tumor heterochromatin is associated with hypoacetylated his- suppressor gene, in the peripheral blood lymphocytes tones.60 Unlike histone acetylation, histone methylation from CLL patients suggests that PTPROt may indeed be can either activate (K4 of histone H3) or repress (K9 of a novel molecular target for CLL therapy. H3) gene transcription.61 Genetic evidence suggests a link How do the DNA hypomethylating agents function? between histone modification and DNA methylation, The most straightforward mechanism involves conversion which has prompted epigenetic targeting to reexpress of the 5-Aza nucleosides to the nucleoside triphosphates, genes by increasing histone acetyl transferase enzyme their incorporation into DNA (5-AzadC) or both RNA activity and/or inhibiting histone deacetylase (HDAC) and DNA (5-AzaC) and subsequent alteration in protein enzymes. Three major classes of HDAC enzymes exhibit synthesis. These drugs could also inhibit de novo specific function of modifying histone and nonhistone thymidylate synthesis following their deamination into proteins.62 the respective uridines and conversion into uridine Many studies have shown that combined regimen of triphosphates. A noteworthy mechanism proposed more DNMT and HDAC inhibitors can regress or prevent than two decades ago is based on their ability to inhibit tumor growth.58,63,64 A variety of clinically available DNA methylation following incorporation into DNA.54 agents that include depsipeptide, MS-275, SAHA and Of the three functional DNA methyltransferases (DNMT valproic acid inhibit HDAC, specifically Class I HDAC 1, 3A, 3B) identified in mammals (for a review, see enzymes.65 The advantage of this regimen is that a Jeltch55 Bestor56). DNMT3A and DNMT3B exhibit relatively lower dose of DNMT inhibitors can be used, predominantly de novo methyltransferase activity whereas which may reduce their toxic effects. These agents can DNMT1 is exclusively involved in the methylation of modify the expression of many cellular genes in a hemimethylated DNA. Since DNMT1 requires hemi- synergistic manner.58,66 We have elucidated the molecular methylated DNA as the substrate, it is anticipated that mechanism for the activation of a specific promoter the effect of 5-AzaC or 5-AzadC will not be fully exerted (metallothionein-I or MT-1 promoter) by combined until the 5-AzadC-incorporated DNA undergoes replica- treatment of mouse lymphosarcoma cells with 5-AzaC tion. The stable and irreversible binding of DNMTs to and a HDAC inhibitor such as trichostatin or clinically 5-AzadC-incorporated DNA results in trapping and potent depsipeptide.58 In this study, the synergistic effects depletion of these DNMTs. Several recent observations of the two classes of drugs in the MT-I re-expression was suggest the covalent conjugate formation between DNMT found to be due to promoter demethylation, altered and 5-AzaC-substituted DNA alone cannot explain many association of different factors that leads to reorganiza- aspects of its action. First, DNMT activity decreases tion of the chromatin, and the resultant increase in much faster than incorporation of 5-AzaC into DNA.51 accessibility of the promoter to the activated transcription Second, recent microarray analysis of human colon factor MTF-1.58 It is noteworthy that the whole MT locus cancer cells (HCT116) revealed alterations in the gene comprising of MT-IV, MT-III, MT-IIA and several expression profiles of 5-AzaC-treated cells irrespective of isoforms of MT-I on human chromosome 16q13 are

Cancer Gene Therapy PTPs as molecular targets for epigenetic therapy ST Jacob and T Motiwala 670 reactivated upon treatment with this drug,57 probably due significant methylation of PTPRO in tumors and to open chromatin structure. negligible methylation as tumor regresses. Further, suppression of PTPRO expression due to methylation will provide an important molecular target for epigenetic Concluding remarks therapy of cancer. In this context, the methylation of other PTPs in CLL is of interest. It is important to We have presented evidence for the tumor suppressor, determine whether these PTPs are also downregulated in potential of a receptor-type protein tyrosine phosphatase, cancer, and whether such modification occurs in pre- PTPRO. These include (a) its methylation-mediated neoplastic stage. silencing in primary human tumors; (b) inhibition of Finally, reactivation of the silenced genes deserves anchorage-independent growth and increased sensitivity comment. Although considerable efforts have been of cells to apoptosis upon expression of PTPRO in expended on the identification of novel HDAC inhibitors, nonexpressing cells; (c) inhibition of growth of PTPRO- the DNA hypomethylating agents frequently used in expressing cells in nude mice; and (d) localization in the therapy are those discovered more than two decades ago. chromosomal region characterized by LOH. If PTPRO is It should also be recognized that HDAC inhibitors alone an authentic tumor suppressor, knocking down PTPRO do not reactivate the genes suppressed by methylation. by stably transfecting the cells expressing this gene with While combining DNMT inhibitors with an HDAC RNAi is likely to induce cell transformation. Glepp1 inhibitor can minimize the clinically effective dose, it is (PTPRO) knockout mice do not exhibit developmental crucial to determine the right combination doses for abnormalities in brain or hematopoietic system. The therapy of each type of cancer. It is, therefore, desirable to targeting approach used for knockout was, however, explore other less toxic and more potent inhibitors of specific for the full-length isoform.67 Further, since these DNA methyltrasferases or drugs that can interfere with mice were not challenged with a carcinogen, it is difficult other factors in the DNA methylation machinery. to assess the effect of the PTPRO deletion in tumorigenesis. It is important to recognize that DNA hypomethylation Acknowledgments also plays a significant role in tumor progression (for a recent review, see El-Osta17). In fact, DNA hypomethyla- We thank Drs Kalpana Ghoshal and Sarmila Majumder tion, an early event in tumorigeneisis, can predispose cells for useful comments. We regret that many excellent to chromosomal rearrangements and destabiliztion of papers relevant to this review could not be cited due to chromosomal organization.17,68 In this context, it is space limitation. The work in the authors’ laboratory was noteworthy that methyl deficient diet can lead to both supported by grants (CA 81024 and CA 86978) from the global DNA hypomethylation and localized or regional National Cancer Institute and ES 10874 from the hypermethylation of genes such as tumor suppressor National Institute of Environmental Health Science. genes.33,69 The most accepted concept is to envisage early changes in the hypomethylation of genes such as References oncogenes followed by hypermethylation of genes such as tumor suppressor genes during cell transformation or 1. Bird A. DNA methylation patterns and epigenetic memory. in tumorigenesis, both processes resulting in gene dereg- Genes Dev. 2002;16:6–21. ulation. It remains to be determined as to what fraction of 2. Jones PA, Baylin SB. The fundamental role of epigenetic tumor incidence is caused by alteration in genomic events in cancer. Nat Rev Genet. 2002;3:415–428. methylation.70 3. Feinberg AP, Tycko B. The history of cancer epigenetics. Identification of the key substrate(s) of PTPRO will be Nat Rev Cancer. 2004;4:143–153. critical to the understanding of the role of this enzyme in 4. Wade PA, Gegonne A, Jones PL, et al. Mi-2 complex cancer. As some of the PTKs, established oncogenes, are couples DNA methylation to chromatin remodelling and histone deacetylation. Nat Genet. 1999;23:62–66. known to be the substrates of PTPs, dephosphorylation of 5. Wade PA, Jones PL, Vermaak D, et al. A multiple subunit these kinases by overexpression of PTPs could have Mi-2 histone deacetylase from Xenopus laevis cofractionates important functional implications. Characterization of with an associated Snf2 superfamily ATPase. Curr Biol. the substrates of PTPRO and its isoforms is, therefore, 1998;8:843–846. crucial to understand its role in cancer. Based on the 6. Zhang Y, Ng HH, Erdjument-Bromage H, et al. Analysis of methylation of PTPRO, and its suppression in the the NuRD subunits reveals a histone deacetylase core primary human tumors derived from three different complex and a connection with DNA methylation. Genes tissues/cells (liver, lung and CLL), it is not unrealistic to Dev. 1999;13:1924–1935. expect similar downregulation of this gene in other 7. Sakai H, Urano T, Ookata K, et al. MBD3 and HDAC1, tumors as well. Further study involving both liquid and two components of the NuRD complex, are localized at Aurora-A-positive centrosomes in M phase. J Biol Chem. solid tumors will resolve this issue. If the reduced 2002;277:48714–48723. expression of PTPRO occurs widely in many primary 8. Costello JF, Fruhwald MC, Smiraglia DJ, et al. Aberrant human tumors besides hepatocellular and lung tumors CpG-island methylation has non-random and tumour-type- and CLL, methylation of PTPRO could be a prognostic specific patterns [see comments]. Nat Genet. 2000;24: marker of these diseases. Accordingly, one can anticipate 132–138.

Cancer Gene Therapy PTPs as molecular targets for epigenetic therapy ST Jacob and T Motiwala 671 9. Baylin SB, Herman JG, Graff JR, et al. Alterations in DNA Scc1 and is frequently deleted in human cancers. Nat Genet. methylation: a fundamental aspect of neoplasia. Adv Cancer 2002;31:295–300. Res. 1998;72:141–196. 30. Wang Z, Shen D, Parsons DW, et al. Mutational analysis of 10. Jones PA, Laird PW. Cancer epigenetics comes of age. Nat the tyrosine phosphatome in colorectal cancers. Science. Genet. 1999;21:163–167. 2004;304:1164–1166. 11. Sakai T, Toguchida J, Ohtani N, et al. Allele-specific 31. Andersen JN, Jansen PG, Echwald SM, et al. A genomic hypermethylation of the retinoblastoma tumor-suppressor perspective on protein tyrosine phosphatases: gene struc- gene. Am J Hum Genet. 1991;48:880–888. ture, pseudogenes, and genetic disease linkage. FASEB J. 12. Myohanen SK, Baylin SB, Herman JG. Hypermethylation 2004;18:8–30. can selectively silence individual p16ink4A alleles in 32. Ardini E, Agresti R, Tagliabue E, et al. Expression of neoplasia. Cancer Res. 1998;58:591–593. protein tyrosine phosphatase alpha (RPTPalpha) in human 13. Batova A, Diccianni MB, Yu JC, et al. Frequent and breast cancer correlates with low tumor grade, and inhibits selective methylation of p15 and deletion of both p15 and tumor cell growth in vitro and in vivo. Oncogene. 2000;19: p16 in T-cell acute lymphoblastic leukemia. Cancer Res. 4979–4987. 1997;57:832–836. 33. Motiwala T, Ghoshal K, Das A, et al. Suppression of the 14. Esteller M, Fraga MF, Guo M, et al. DNA methylation protein tyrosine phosphatase receptor type O gene (PTPRO) patterns in hereditary human cancers mimic sporadic by methylation in hepatocellular carcinomas. Oncogene. tumorigenesis. Hum Mol Genet. 2001;10:3001–3007. 2003;22:6319–6331. 15. Herman JG, Baylin SB. Gene silencing in cancer in 34. Motiwala T, Kutay H, Ghoshal K, et al. Protein tyrosine association with promoter hypermethylation. [see comment]. phosphatase receptor-type O (PTPRO) exhibits character- N Engl J Med. 2003;349:2042–2054. istics of a candidate tumor suppressor in human lung cancer. 16. Baylin SB, Herman JG. DNA hypermethylation in tumori- Proc Natl Acad Sci USA. 2004;101:13844–13849 Epub genesis: epigenetics joins genetics. Trends Genet. 2000;16: 12004 Sep 13848. 168–174. 35. Imreh S, Klein G, Zabarovsky ER. Search for unknown 17. El-Osta A. The rise and fall of genomic methylation in tumor-antagonizing genes. Genes, Chromosomes Cancer. cancer. Leukemia. 2004;18:233–237. 2003;38:307–321. 18. Hunter T. Signaling — 2000 and beyond. Cell. 2000;100: 36. Mori Y, Yin J, Sato F, et al. Identification of genes uniquely 113–127. involved in frequent microsatellite instability colon carcino- 19. Fischer EH. Cell signaling by protein tyrosine phosphoryla- genesis by expression profiling combined with epigenetic tion. Adv Enzyme Regul. 1999;39:359–369. scanning. Cancer Res. 2004;64:2434–2438. 20. Maroun CR, Naujokas MA, Holgado-Madruga M, et al. 37. Oka T, Ouchida M, Koyama M, et al. Gene silencing The tyrosine phosphatase SHP-2 is required for sustained of the tyrosine phosphatase SHP1 gene by aberrant activation of extracellular signal-regulated kinase and methylation in leukemias/lymphomas. Cancer Res. 2002;62: epithelial morphogenesis downstream from the met receptor 6390–6394. tyrosine kinase. Mol Cell Biol. 2000;20:8513–8525. 38. Amoui M, Baylink DJ, Tillman JB, et al. Expression of a 21. Meng TC, Fukada T, Tonks NK. Reversible oxidation and structurally unique osteoclastic protein-tyrosine phospha- inactivation of protein tyrosine phosphatases in vivo. Mol tase is driven by an alternative intronic, cell type-specific Cell. 2002;9:387–399. promoter. J Biol Chem. 2003;278:44273–44280. 22. Palka HL, Park M, Tonks NK. Hepatocyte growth factor 39. Mancini DN, Singh SM, Archer TK, et al. Site-specific receptor tyrosine kinase met is a substrate of the receptor DNA methylation in the neurofibromatosis (NF1) promoter protein-tyrosine phosphatase DEP-1. J Biol Chem. 2003; interferes with binding of CREB and SP1 transcription 278:5728–5735. factors. Oncogene. 1999;18:4108–4119. 23. Hermiston ML, Xu Z, Majeti R, et al. Reciprocal regulation 40. DiNardo DN, Butcher DT, Robinson DP, et al. Functional of lymphocyte activation by tyrosine kinases and phospha- analysis of CpG methylation in the BRCA1 promoter tases. J Clin Invest. 2002;109:9–14. region. Oncogene. 2001;20:5331–5340. 24. Gupta S, Radha V, Sudhakar C, et al. A nuclear protein 41. Santoro R, Grummt I. Molecular mechanisms mediating tyrosine phosphatase activates p53 and induces caspase-1- methylation-dependent silencing of ribosomal gene tran- dependent apoptosis. FEBS Lett. 2002;532:61–66. scription. Mol Cell. 2001;8:719–725. 25. Salmeen A, Andersen JN, Myers MP, et al. Molecular basis 42. Jenuwein T, Allis CD. Translating the histone code. Science. for the dephosphorylation of the activation segment of 2001;293:1074–1080. the insulin receptor by protein tyrosine phosphatase 1B. Mol 43. Kass SU, Wolffe AP. DNA methylation, nucleosomes Cell. 2000;6:1401–1412. and the inheritance of chromatin structure and func- 26. Alonso A, Sasin J, Bottini N, et al. Protein tyrosine tion. Novartis Found Symp. 1998;214:22–35; discussion phosphatases in the human genome. Cell. 2004;117:699–711. 36–50. 27. Panagopoulos I, Pandis N, Thelin S, et al. The FHIT and 44. Ghoshal K, Majumder S, Datta J, et al. Role of human PTPRG genes are deleted in benign proliferative breast ribosomal RNA (rRNA) promoter methylation and of disease associated with familial breast cancer and cytoge- methyl-CpG-binding protein MBD2 in the suppression of netic rearrangements of chromosome band 3p14. Cancer rRNA gene expression. J Biol Chem. 2004;279:6783–6793. Res. 1996;56:4871–4875. 45. Taylor SM, Jones PA. Multiple new phenotypes induced in 28. Zhang Y, Siebert R, Matthiesen P, et al. Cytogenetical 10T1/2 and 3T3 cells treated with 5-azacytidine. Cell. assignment and physical mapping of the human R-PTP- 1979;17:771–779. kappa gene (PTPRK) to the putative tumor suppressor gene 46. Baylin SB. Reversal of gene silencing as a therapeutic target region 6q22.2–q22.3. Genomics. 1998;51:309–311. for cancer — roles for DNA methylation and its inter- 29. Ruivenkamp CA, van Wezel T, Zanon C, et al. Ptprj is a digitation with chromatin. Novartis Found Symp. 2004;259: candidate for the mouse colon-cancer susceptibility locus 226–233; discussion 234–227.

Cancer Gene Therapy PTPs as molecular targets for epigenetic therapy ST Jacob and T Motiwala 672 47. Karpf AR, Jones DA. Reactivating the expression of an open chromatin structure. Mol Cell Biol. 2002;22: methylation silenced genes in human cancer. Oncogene. 8302–8319. 2002;21:5496–5503. 59. Suzuki H, Gabrielson E, Chen W, et al. A genomic screen 48. Jain PK. Epigenetics: the role of methylation in the for genes upregulated by demethylation and histone mechanism of action of tumor suppressor genes. Ann NY deacetylase inhibition in human colorectal cancer. [see Acad Sci. 2003;983:71–83. comment]. Nat Genet. 2002;31:141–149. 49. Majumder S, Ghoshal K, Datta J, et al. Role of de novo 60. Grunstein M. Histone acetylation in chromatin structure DNA methyltransferases and methyl CpG-binding proteins and transcription. Nature. 1997;389:349–352. in gene silencing in a rat hepatoma. J Biol Chem. 2002;277: 61. Lachner M, Jenuwein T. The many faces of histone lysine 16048–16058. methylation. Curr Opin Cell Biol. 2002;14:286–298. 50. Ghoshal K, Majumder S, Li Z, et al. Suppression of 62. de Ruijter AJ, van Gennip AH, Caron HN, et al. Histone metallothionein gene expression in a rat hepatoma because deacetylases (HDACs): characterization of the classical of promoter-specific DNA methylation. J Biol Chem. 2000; HDAC family. Biochem J. 2003;370:737–749. 275:539–547. 63. Belinsky SA, Klinge DM, Stidley CA, et al. Inhibition of 51. Creusot F, Acs G, Christman JK. Inhibition of DNA DNA methylation and histone deacetylation prevents methyltransferase and induction of Friend erythroleukemia murine lung cancer. Cancer Res. 2003;63:7089–7093. cell differentiation by 5-azacytidine and 5-aza-2’-deoxycyti- 64. Shaker S, Bernstein M, Momparler RL. Antineoplastic dine. J Biolog Chem. 1982;257:2041–2048. action of 5-aza-20-deoxycytidine (Dacogen) and depsipep- 52. Christman JK. 5-Azacytidine and 5-aza-20-deoxycytidine as tide on Raji lymphoma cells. Oncol Rep. 2004;11:1253–1256. inhibitors of DNA methylation: mechanistic studies and 65. Kouraklis G, Theocharis S. Histone deacetylase inhibitors their implications for cancer therapy. Oncogene. 2002;21: and anticancer therapy. Curr Med Chem — Anti-Cancer 5483–5495. Agents. 2002;2:477–484. 53. Claus R, Lubbert M. Epigenetic targets in hematopoietic 66. Cameron EE, Bachman KE, Myohanen S, et al. Synergy of malignancies. Oncogene. 2003;22:6489–6496. demethylation and histone deacetylase inhibition in the re- 54. Jones PA, Taylor SM, Wilson VL. Inhibition of DNA expression of genes silenced in cancer. Nat Genet. 1999; methylation by 5-azacytidine. Rec Results Cancer Res. 1983; 21:103–107. 84:202–211. 67. Wharram BL, Goyal M, Gillespie PJ, et al. Altered 55. Jeltsch A. Beyond Watson and Crick: DNA methylation podocyte structure in GLEPP1 (Ptpro)-deficient mice and molecular enzymology of DNA methyltransferases associated with hypertension and low glomerular filtration [erratum appears in Chembiochem 2002 May 3;3(5):382]. rate. J Clin Invest. 2000;106:1281–1290. Chembiochem:3:274–293. 68. Xu GL, Bestor TH, Bourc’’his D, et al. Chromosome 56. Bestor TH. The DNA methyltransferases of mammals. instability and immunodeficiency syndrome caused by Humn Mol Genet. 2000;9:2395–2402. mutations in a DNA methyltransferase gene. Nature. 1999; 57. Gius D, Cui H, Bradbury CM, et al. Distinct effects on gene 402:187–191. expression of chemical and genetic manipulation of the 69. Pogribny IP, Miller BJ, James SJ. Alterations in hepatic p53 cancer epigenome revealed by a multimodality approach. gene methylation patterns during tumor progression with Cancer Cell. 2004;6:361–371. folate/methyl deficiency in the rat. Cancer Lett. 1997;115: 58. Ghoshal K, Datta J, Majumder S, et al. Inhibitors of histone 31–38. deacetylase and DNA methyltransferase synergistically 70. Baylin S, Bestor TH. Altered methylation patterns in cancer activate the methylated metallothionein I promoter by cell genomes: cause or consequence? Cancer Cell. 2002;1: activating the transcription factor MTF-1 and forming 299–305.

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