The Tyrosine Phosphatase PTPRD Is a Tumor Suppressor That Is Frequently Inactivated and Mutated in Glioblastoma and Other Human Cancers

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The tyrosine phosphatase PTPRD is a tumor suppressor that is frequently inactivated and mutated in glioblastoma and other human cancers

Selvaraju Veeriaha, Cameron Brennanb, Shasha Menga, Bhuvanesh Singhc, James A. Fagina,d, David B. Solita,d, Philip B. Patyc, Dan Rohlea, Igor Vivancoa, Juliann Chmieleckia, William Paoa,d, Marc Ladanyia,e, William L. Geralda,e, Linda Liauf, Timothy C. Cloughesyf, Paul S. Mischelf, Chris Sanderg, Barry Taylorg, Nikolaus Schultzg, John Majorg, Adriana Heguya, Fang Fanga, Ingo K. Mellinghoffa,h, and Timothy A. Chana,i,1

aHuman Oncology and Pathogenesis Program; Departments of bNeurosurgery, cSurgery, dMedicine, ePathology, hNeurology, and iRadiation Oncology; and gComputational Biology Center, Memorial Sloan–Kettering Cancer Center, 1275 York Avenue, New York, NY 10065; and fDavid Geffen School of Medicine, University of California, Los Angeles, CA 90095

Edited by Bert Vogelstein, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, and approved April 10, 2009 (received for review January 23, 2009) Tyrosine phosphorylation plays a critical role in regulating cellular functional validation of the alterations is noted (10–12). Together function and is a central feature in signaling cascades involved in with these data, our study shows that PTPRD is a broadly altered oncogenesis. The regulation of tyrosine phosphorylation is coordi- tumor suppressor gene in human malignancies (13). nately controlled by kinases and phosphatases (PTPs). Whereas acti- Using a multifaceted genomic analysis approach, we have deter- vation of tyrosine kinases has been shown to play vital roles in tumor mined that PTPRD is a frequent target of inactivation via both development, the role of PTPs is much less well defined. Here, we genetic and epigenetic mechanisms in GBM and other human show that the receptor protein tyrosine phosphatase delta (PTPRD) is cancers. Our concordance data on methylation and copy number frequently inactivated in glioblastoma multiforme (GBM), a deadly analysis show that independent and nonoverlapping mechanisms primary neoplasm of the brain. PTPRD is a target of deletion in GBM, (genetic and epigenetic) inactivate this gene and are strong genomic often via focal intragenic loss. In GBM tumors that do not possess evidence indicating that PTPRD loss is not simply driven by deletions in PTPRD, the gene is frequently subject to cancer-specific CDKN2A loss. In addition, we also show that loss of PTPRD expression epigenetic silencing via promoter CpG island hypermethylation associates with gliomas of poor prognosis, loss of PTPRD results in (37%). Sequencing of the PTPRD gene in GBM and other primary altered growth of astrocytes, PTPRD dephosphorylates STAT3 and human tumors revealed that the gene is mutated in 6% of GBMs, 13% regulates the STAT3 pathway, and mutations in PTPRD abrogate the of head and neck squamous cell carcinomas, and in 9% of lung ability to regulate STAT3. Taken together, our data provide insight cancers. These mutations were deleterious. In total, PTPRD inactiva- into the molecular underpinnings of this tumor suppressor gene. tion occurs in >50% of GBM tumors, and loss of expression predicts for poor prognosis in glioma patients. Wild-type PTPRD inhibits the Results and Discussion growth of GBM and other tumor cells, an effect not observed with PTPRD Is Deleted in GBM. Canonical tumor suppressor genes such as PTPRD alleles harboring cancer-specific mutations. Human astrocytes p16INK4A, PTEN, etc., are often subject to homozygous deletion lacking PTPRD exhibited increased growth. PTPRD was found to (14). To determine whether PTPRD is deleted in GBM, we dephosphorylate the oncoprotein STAT3. These results implicate examined array comparative genomic hybridization (aCGH) results PTPRD as a tumor suppressor on chromosome 9p that is involved in and determined copy number alterations (CNAs) at the PTPRD the development of GBMs and multiple human cancers. locus (9p23–24) in 215 GBM tumors. This dataset was obtained as part of The Cancer Genome Atlas (TCGA) initiative (15). Of note, glioblastoma multiforme ͉ methylation ͉ mutation although the aCGH data for the 9p locus is available, PTPRD has not been analyzed by the TCGA sequencing and methylation oss of tumor suppressor function leads to the initiation and analysis efforts. As expected, we observed that 9p loss is a frequent event in GBM, with Ϸ40% of tumors exhibiting loss (log2 ratio less Lprogression of cancer (1, 2). Inactivation of tumor suppressor Ϫ Ϫ genes can result from both genetic mechanisms such as mutation than 0.25) or homozygous deletion (log2 ratio less than 1) in the region (Fig. 1A) (16). To separate PTPRD loss from CDKN2A and deletion or epigenetic mechanisms such as DNA hypermeth- INK4A ylation (3, 4). Identification of these genes has provided insight into (p16 ) loss, we examined this region in detail. Loss of regions of various sizes on 9p, encompassing both the PTPRD and CDKN2A the biological processes underlying oncogenesis, but the key tumor MEDICAL SCIENCES suppressors in many cancers, such as glioblastoma multiforme genes, was found in a significant proportion of tumors. Both loss and (GBM), remain poorly defined. homozygous deletion of the PTPRD gene but not surrounding We previously identified the receptor protein tyrosine phospha- genes was observed. Importantly, in some tumors, intragenic ho- tase delta (PTPRD) as a gene that predicts for poor prognosis in mozygous deletions were found in the PTPRD gene that removed breast and colon cancer (4). PTPRD is a member of the highly PTPRD exons, but not surrounding genes, thus defining a minimal conserved family of receptor protein tyrosine phosphatases (PTPs),

several members of which have been implicated in tumorigenesis Author contributions: T.A.C. designed research; S.V., S.M., A.H., and F.F. performed re- (5). The gene encodes a transmembrane protein with a cytoplasmic search; C.B., B.S., J.A.F., D.B.S., P.B.P., D.R., I.V., J.C., W.P., M.L., W.L.G., L.L., T.C.C., P.S.M., tyrosine phosphatase domain. The PTPRD gene is located within an and I.K.M. contributed new reagents/analytic tools; S.V., C.B., J.A.F., D.B.S., W.P., C.S., B.T., area of the genome, chromosome 9p, found to be frequently lost in N.S., J.M., A.H., and T.A.C. analyzed data; and T.A.C. wrote the paper. neuroblastoma, gliomas, lung cancer, and other malignancies (6–9). The authors declare no conﬂict of interest. Some deletions of PTPRD have been noted in several of these This article is a PNAS Direct Submission. studies. However, its close proximity to CDKN2A on chromosome Freely available online through the PNAS open access option. 9p has complicated interpretations. In addition, in independent 1To whom correspondence should be addressed. E-mail: [email protected]. work during the course of our investigations, PTPRD mutations This article contains supporting information online at www.pnas.org/cgi/content/full/ have been detected in a lung cancer genome study, although no 0900571106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900571106 PNAS ͉ June 9, 2009 ͉ vol. 106 ͉ no. 23 ͉ 9435–9440 Downloaded by guest on September 27, 2021 A strongly suggests that PTPRD is targeted for inactivation during GBM pathogenesis. Moreover, PTPRD is a tumor suppressor that is located near p16INK4A, and there may exist selective pressure for codeletion of both genes. This finding is consistent with the widely held hypothesis that there exists more than one tumor suppressor >1 gene in the region of 9p21–24 (other than p16INK4A) (17, 18). Log 2 Tumor samples 0 Ratio Epigenetic Silencing of PTPRD. Inactivation of tumor suppressor genes <-1 can result from both genetic mechanisms, such as deletion, or Chr. 9 epigenetic mechanisms, such as DNA hypermethylation (3, 19). PTPRD Epigenetic silencing via promoter CpG island hypermethylation has B been shown to be a predominant mechanism by which tumor 0 suppressors are inactivated in cancers (20). In some malignancies, -1 GBM 1 such as chronic lymphocytic leukemia, hypermethylation can be a 0 very common mode of tumor suppressor inactivation (21). In Ratio -1 GBM2 2 glioma, the 2 major mechanisms of p16INK4A inactivation are

Log 0 deletion and hypermethylation (22); in some tumors, methylation -1 GBM3 occurs and in others, deletion occurs. Given that tumor suppressors 7 8 9 10 11 12 that play key roles are frequently targeted by multiple modes of PTPRD inactivation (4, 19), we wondered whether PTPRD was silenced by hypermethylation in GBM tumors that did not possess deletions in PTPRD D C PTPRD locus Intervening region p16/ARF locus the gene. PTPRD possesses a canonical promoter CpG island (Fig. loss no loss 33% 9p 2A). We designed methylation-specific PCR (MSP) assays to assess 67% 88 54 the methylation status of the PTPRD gene in GBM cell lines and loss p16 loss primary tumors. We found that PTPRD was unmethylated and 172 euploidy no loss expressed in normal brain tissue (NB) (Fig. 2 A and B). A methylated PTPRD promoter is strictly associated with loss of Fig. 1. Deletion of PTPRD in GBM. (A) aCGH profile analysis of 215 GBM tumors PTPRD expression. PTPRD was methylated and silenced in from TCGA (4/14/2008 data freeze). Segmentation data for the area surrounding SKMG3, and the gene was unmethylated and expressed in 2 other PTPRD on chromosome 9p is shown. Tumors are sorted by amount of loss at the PTPRD locus for convenient viewing. Chromosomal gain or loss is represented by GBM lines, 8MGBA and SF268. Treatment of SKMG3 with the a color gradient (red, gain; green, loss). x axis represents genomic location along DNA methyltransferase (DNMT) inhibitor 5-aza-deoxycytidine chromosome 9p (Mbps). The blue bars represent the boundaries of the PTPRD (DAC) resulted in demethylation of the PTPRD promoter and gene. Red bracket marks tumors that possess intragenic deletions of PTPRD. (B) restored expression of the gene (Fig. 2B). PTPRD is subject to focal, intragenic deletions. Shown are aCGH profiles of Next, we analyzed the methylation status of PTPRD in primary selected tumors with intragenic deletions of PTPRD. Probes are plotted along GBM tumors. Genomic quantitative PCR was used to identify a set chromosome 9p according to log2 ratio. The red line represents averaged log2 of tumors that did not possess deletions in PTPRD. PTPRD was ratio trend. The black arrows show PTPRD exons encompassed by intragenic INK4A methylated in 37% (10/27) of tumors (Fig. 2C). As was the case with deletions. (C) Frequency of loss of PTPRD and p16 . Loss is defined as a log2 ratio less than Ϫ0.25 to Ϫ2.00. Coordinate loss of both genes is indicated. (D) the cell lines, hypermethylation of the PTPRD promoter was strictly Diagram depicting the nature of loss events in the genomic region encompassing associated with loss of gene expression (Fig. 2D). Bisulfite sequenc- PTPRD and p16INK4A. Depicted is a summary of data generated from analysis of ing confirmed the results obtained with MSP (Fig. 2E). Based on aCGH data. Shown is the region of chromosome 9p in which PTPRD (9p23–24) and these data, it is clear that inactivation of PTPRD can occur not only p16INK4A (9p21) are located, as well as the intervening DNA. Black bars do not by deletion, but also by epigenetic silencing, and that this is a very represent exact borders but serve only to summarize the data. Green represents frequent event in GBM. loss of copy number and white represents euploidy. Thirty-three percent occur as a result of two distinct loss events separated by intervening DNA which is euploid If PTPRD is both deleted and epigenetically silenced in malignant (no CNA). gliomas, transcriptome analyses should demonstrate lower PTPRD mRNA levels in gliomas compared with normal brain tissue. To address this question, we analyzed an extensive expression microar- common region of deletion at the PTPRD locus (Fig. 1 A and B). ray database (Oncomine), using large numbers of expression pro- These changes were significantly above the background rate ex- files from published microarray datasets (23) (Tables S1 and S2). pected (Q-value ϭ 3.65EϪ8). The microarray metaanalysis algorithms and statistical analysis Examination of regions of allelic loss has been crucial for the used were as previously described (24). PTPRD expression levels identification of cancer genes. Loss of the PTPRD and p16INK4A loci were found to be significantly lower in nearly every available dataset on chromosome 9p are very frequent events, and examination of the comparing normal brain with glioma (Fig. 2F). Moreover, PTPRD patterns of loss as a function of genomic location is instructive. expression was significantly lower in malignant gliomas of high Particularly interesting is the relationship between PTPRD loss World Health Organization (WHO) grade as compared with (9p23–24) and loss of the nearby p16INK4A gene (9p21). Fig. 1C gliomas of lower WHO grade. Patients with WHO grade IV tumors summarizes the frequency of loss events for PTPRD, p16INK4A,or (GBM) have poorer survival than patients with low grade astro- both genes. Of tumors where PTPRD and p16INK4A undergo loss, 29 cytomas (WHO I and II) or anaplastic astrocytomas (WHO III), show distinct loss in each region via separate events (i.e., copy and as expected, tumors from patients with poorer survival have number decreases at PTPRD and p16INK4A with euploidy in the decreased PTPRD levels (Fig. 2F). Previously, we detected cancer- DNA lying between these genes). In another 59 tumors, a single specific hypermethylation of PTPRD in colon and breast cancer (4). large event eliminates both genes simultaneously (Fig. 1D). This Together with these data, it appears that epigenetic silencing of finding indicates that, in the region of 9p we are examining, there PTPRD is a cancer-specific event common to multiple malignancies are at least 2 separate loci driving genetic loss, one at PTPRD and (Fig. 2G). The data from colon and breast cancer described in our another at p16. Together, the unbiased identification of PTPRD as previous study are shown here to enable comparison with methyl- a target of microdeletions, as well as frequent larger deletions ation frequencies in GBM (4).

9436 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900571106 Veeriah et al. Downloaded by guest on September 27, 2021 D l a m n A RT-PCR r i 6 8 7 a o r 2 1 G RT RT n b G G

PTPRD promoter 1 23 4 PTPRD CpG Island actin

MSP1 MSP2 l a 6 8 7 2 1 G rm in G G o ra TIS n b MSP UMUMUM UM PTPRD IVD SKMG3 NB U M U M U M PTPRD

MSP1 MSP2 E -1050 -850 SKMG3

l B RT-PCR SKMG3 A a B 8 6 rm in O G 2 o ra 2 mock DAC H2O M F n b H NB 8 S

PTPRD DKO

actin G12

GBM 3 l A 8 a G3 G C B 6 m n primary O M M G 2 r i 2 A o a K K D M F r MSP H S S 8 S n b tumors + U M UM UMUM UMU M G19

1 l 3 a C 2 n O A m i F O r a K D o r 4 7 2 b 1 H D M n G G2 G3 G G5 G6 G U M UMUM UM UM UMUMU MUMUM UM

0 1 2 3 4 5 6 7 8 9 8 1 1 1 G G9 G1 G1 G G1 G G1 G G1 G1 G1 UM UMUM UM UM UM UM UM UM UM UM U M p=2.5E-7 p=0.004 NB G NB G NB G NB G NB G NB G NB G NB G NB G NB G p= 1.7E-20 7.3E-11 7.3E-8 4.9E-7 2E-6 2.2E-5 0.002 0.004 0.012 0.012 2 3 4 alive deceased dataset Sun Sun-O French Sun-A Gutmann Bredel Bredel-A Khatua Liang Rickman WHO grade 3 yr OS

0 1 2 3 4 5 6 7 60 2 2 2 G G2 G2 G2 G G G G2 G2 50 UM UM UM UMU M UM U M UM 50

40 37

30 20 H 20 NB GBM10 GBM28 GBM11 GBM12 GBM13 GBM18 GBM19 GBM20 GBM21 GBM23 GBM14 GBM15 GBM16 GBM17 GBM1 GBM27 GBM29 GBM30 GBM32 GBM33 GBM34 GBM35 GBM2 GBM4 GBM6 GBM9 GBM24 GBM25 GBM22 GBM26 GBM31 GBM7 GBM3 GBM5 GBM8 PTPRD methylation PTPRD loss 10 p16 methylation 0 0 0

p16 loss Frequency of methylation (%) 0 no alteration normal normal normal GBM breast colon alteration brain breast colon cancer cancer MEDICAL SCIENCES Fig. 2. PTPRD is subject to frequent epigenetic silencing in GBM. (A) Shown is promoter structure of the PTPRD gene (Top). Numbered boxes denote exons and TIS denotes the transcriptional start site. Two independent MSP assays were developed to detect hypermethylated PTPRD and both produce identical results (Bottom). IVD, in vitro methylated genomic DNA; SKMG3, GBM cell line; NB, normal brain. U denotes the presence of unmethylated alleles and M denotes the presence of methylated alleles. The locations assayed by RT-PCR and MSP are noted. (B) Epigenetic silencing of PTPRD expression caused by hypermethylation. Results are shown for RT-PCR (Top) and MSP (Bottom). PTPRD is silenced and hypermethylated in the GBM cell line SKMG3. After treatment with the DNMT inhibitor DAC, PTPRD becomes demethylated and expression is restored. (C) Frequent hypermethylation of PTPRD in primary GBM tumors. Double knockout (DKO), control for unmethylated PTPRD alleles derived from a cell line in which DNMT1 and 3b were knocked out (44). MDA-MB 213 was previously found to undergo silencing of PTPRD and is used as a positive control for methylated alleles (4). (D) Loss of PTPRD expression in primary GBM tumors with hypermethylated PTPRD. Representative samples are shown. (E) Bisulfite sequencing of the PTPRD promoter. Black circles represent methylated CpG dinucleotides. White circles represent unmethylated CpG dinucleotides. (F) Decreased expression of PTPRD in malignant glioma is associated with poor clinical prognosis. Left shows representative data across multiple independently published microarray datasets. Datasets used are labeled. P values for significance are shown. Each pair of plots denotes normalized expression for normal brain (NB, blue) versus glioma (G, red). Shaded boxes ϭ 25th–75th percentile. Whiskers ϭ 10th–90th percentile, and asterisks represent range. Bars ϭ median. Middle and Right show box plots demonstrating decreased PTPRD expression in gliomas with increasing WHO grade and those with poorer survival. All datasets were analyzed as previously described (23) and are listed in Table S1. The second and third graphs, Shia dataset (Table S1). (G) PTPRD is methylated in several primary human cancers, but not in corresponding normal tissues. Methylation was detected by MSP and confirmed with bisulfite sequencing. The data from colon and breast cancer described in our previous study are shown here to enable comparison with methylation frequencies in GBM (4). (H) Concordance analysis of genomic and epigenomic inactivation of PTPRD and p16INK4A. Map shows the presence (red) or absence (green) of both loss and epigenetic inactivation in p16INK4A and PTPRD, in the same tumor set. Analysis of copy number was performed with genomic qPCR and methylation was performed with MSP.

Veeriah et al. PNAS ͉ June 9, 2009 ͉ vol. 106 ͉ no. 23 ͉ 9437 Downloaded by guest on September 27, 2021 Table 1. Somatic Mutations of PTPRD in Human Cancers Genomic Normal Tumor Amino acid SIFT Cancer type position genotype genotype change prediction Domain

Glioblastoma 8476143 GTC ATC V892I deleterious InterPro IPR003961 Fibronectin, type III Glioblastoma 8366672 CAA TAA Q1481X deleterious, InterPro IPR000242 stop codon Protein-tyrosine phosphatase Glioblastoma 8474270 CGT TGT R1088C deleterious InterPro IPR003961 Fibronectin, type III Lung 8461063 CCT ACT P1146T deleterious Lung 8489811 GTA ATA V720I tolerated InterPro IPR003961 Fibronectin, type III Squamous, head and neck 8439782 CCT ACT P1311T deleterious

Squamous, head and neck 8511492 CCA CTA P249L deleterious InterPro IPR003598 Immunoglobulin C2 Squamous, head and neck 8511315 CTG CCG L308P deleterious InterPro IPR003598 Immunoglobulin C2

Concordance Analysis of PTPRD Alterations. We then determined the ectopic expression of PTPRD can inhibit cancer cell growth, we concordance of loss versus methylation of PTPRD and p16INK4A transfected wild-type PTPRD into human GBM and colon cancer (Fig. 2H). The genomic and epigenetic inactivation events in cells. Transfection resulted in production of PTPRD protein (Fig. PTPRD are for the most part mutually exclusive (P Ͻ 0.05). 3A and Fig. S1). Expression of PTPRD in human cancer cell lines Importantly, in some tumors, p16INK4A is genetically lost but PTPRD potently inhibited cell growth, as seen by the substantial decrease is methylated instead, which is consistent with the hypothesis that in the number of neomycin-resistant colonies compared with empty PTPRD inactivation is not simply driven by p16 loss because these vector (Fig. 3B and Fig. S2). Transfection of wild-type but not 2 genes undergo different mechanisms of inactivation in the same mutant PTPRD into 293 cells resulted in reduced growth of tumor sample. Furthermore, concordance analysis of PTPRD and transfected cells (Fig. S3). Next, we assessed the effects of loss of p16INK4A status shows that although 2 tumors show methylation at PTPRD expression in immortalized primary human astrocytes both genes, PTPRD methylation does not usually occur concomi- (IHA) (26). We knocked down PTPRD expression in astrocytes by tantly with p16INK4A methylation because p16INK4A is mostly inac- expressing an shRNA targeting the gene (Fig. 3 A and C). Knock- tivated by deletion. These data, in addition to the copy number data down of PTPRD resulted in increased cell growth compared with above, show that PTPRD is a separate tumor suppressor locus a control expressing a scrambled shRNA (Fig. 3D). This result was independent of CDKN2A. due to a significant increase in the rate of growth of the cells lacking PTPRD expression (Fig. 3D). Knockdown with 2 other shRNAs PTPRD Is Mutated in GBM and Other Human Malignancies. To determine targeting PTPRD but not scrambled sequence produced the same whether PTPRD mutations are present in GBM and other human results (Fig. S4). The tumor-forming ability of IHAs in which tumors, we sequenced all exons of the gene in 222 human cancers PTPRD was knocked down was measured by using a mouse (48 GBM, 22 lung, 24 squamous cell carcinoma of the head and xenograft model. Depletion of PTPRD resulted in significantly neck, 32 prostate, 60 colon, and 36 thyroid). Exons were amplified enhanced tumor growth (Fig. 3E). Importantly, expression of by PCR from cancer genomic DNA samples and directly sequenced PTPRD with cancer-specific mutations (Q1481X, R1088C, and (see Materials and Methods). Whenever a presumptive mutation P1311T) resulted in a decreased ability to inhibit cell growth as was identified, we verified that the change did not correspond to a compared with wild-type PTPRD (Fig. 3F), demonstrating that known single-nucleotide polymorphism and attempted to deter- these mutations have clear functional consequences. mine whether it was somatically acquired (i.e., tumor specific) by examining the sequence of the gene in genomic DNA from normal PTPRD Dephosphorylates STAT3, an Activity Abrogated by Cancer- tissue of the same patient. Using this strategy, we identified somatic Specific Mutations. How does the PTPRD phosphatase suppress mutations in 3 different types of human malignancies. PTPRD tumor cell growth? To answer this question, we performed an mutations were found in 3 of 48 (6%) GBMs, 2 of 22 (9%) lung analysis of candidate pathways and identified the oncoprotein cancers, and 3 of 24 (13%) squamous cell carcinomas of the head STAT3 as a potential substrate of PTPRD. Transfection of wild- and neck (Tables 1 and S3). No mutations were found in prostate type PTPRD resulted in the specific dephosphorylation of STAT3 cancers, colon carcinomas, or thyroid cancers. at tyrosine 705, a residue that must be phosphorylated for STAT3 All somatic mutations identified were nonsynonymous. Nearly all to be active (27) (Fig. 3G). Dephosphorylation was not seen with mutations occurred in known functional domains of the predicted any other phospho-proteins we examined other than STAT3 in our protein product. The mutation Q1481X is predicted to result in a analysis of candidate pathways (Fig. 3G and Fig. S5). Furthermore, truncated protein product lacking a functional C-terminal phosphatase and most importantly, STAT3 dephosphorylation was not seen domain. Other alterations identified were missense mutations and are after expression of PTPRD harboring cancer-specific mutations predicted to be deleterious (Table 1). Importantly, these missense that we identified. Levels of the STAT3 transcriptional target mutations are located in conserved areas of the PTPR gene family suppressor of cytokine signaling 3 (SOCS3) also decreased after and correspond to locations of mutations found in PTPRT in colon transfection of wild-type but not mutant PTPRD (Fig. 3G). These cancer (25). These include mutations in the fibronectin and phos- mutations abrogate the ability of the gene to block both tumor cell phatase domains of PTPRT (Q987K, N1128I, etc.). growth and STAT3 phosphorylation, establishing a mechanistic Little is known about the functional role of PTPRD. Other link for the loss of function mutations. related PTPs have been shown to regulate cell growth and/or We have demonstrated that PTPRD regulates STAT3 phos- apoptosis. We have found that PTPRD is expressed in normal brain phorylation, but have not yet shown that the dephosphorylation was tissue, human astrocytes (Figs. 2D and 3C), and other human directly mediated by PTPRD rather than a downstream phospha- tissues, including colon and breast (4). To determine whether tase. To address this point, we made a GST-PTPRD fusion protein

9438 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900571106 Veeriah et al. Downloaded by guest on September 27, 2021 le b SKMG3 A m N B D A a R r e h c Empty n s o l +s + vector a D r D D shRNA PTPRD R o R t R P c P P T e T T P v P P PTPRD PTPRD-V5 WT

actin HCT116 scramble Empty C100 vector

75 PTPRD Cell growth WT

50 R1088C Vector alone WT PTPRD HT29 F IGC2 IGC2 IGC2 FN3 FN3 FN3 FN3 FN3 FN3 FN3 FN3 PTPc PTPc 25 Empty P1311T vector IGC2 IGC2 IGC2 FN3 FN3 FN3 FN3 FN3 FN3 FN3 FN3 PTPc PTPc Q1481* P1311T

% (normalized to GAPDH) % Q1481X 0 PTPRD WT IGC2 IGC2 IGC2 FN3 FN3 FN3 FN3 FN3 FN3 FN3 FN3 mble a scr R1088C shRNA PTPRD vector alone wt PTPRD 700 E NHA Scramble 600 120 )

3 shRNA PTPRD 500 100

80 400 Q1481* P1311T 60 300 40 Colonies (%) 200 20 Tumor volume (mm 100 0 Empty vector

0 P1311T R1088C Q1481*

032394653 PTPRDWT T (Days) H HEK 293T siRNA-PTPRD ─ + ─ + ─ + y t t P p w pSTAT3 PTPRD G F m C n G e D T / 1* 8 o r r i R t o 8 n to P a 4 311 08 o l i T 1 1 1 t y ec ect actin h P P R a v v Q tSTAT3 I t A t Cancers (primary tumors) u e m CN m pSTAT3 glioblastoma 6 41 37 actin total STAT3 squamous cell of head and neck 13 25 lung cancer 5 4 pAKT Immortalized human astrocytes colon cancer 9 50 total AKT siRNA-PTPRD ─ + ─ + ─ + breast cancer 20 pS6 pSTAT3 PTPRD total S6 actin pMEK tSTAT3 total MEK actin actin

Fig. 3. Tumor suppressive properties of PTPRD.(A) Immunoblot showing HEK 293T cells transfected with wild-type PTPRD cDNA, vector alone (pcDNA 3.1-V5), PTPRD ϩ shRNA targeting PTPRD, and PTPRD ϩ scrambled shRNA. Antibody against the V5 epitope (Invitrogen) was used to detect the PTPRD-V5 fusion protein. Actin was used as a loading control. (B) Expression of PTPRD suppresses growth of human cancer cells. PTPRD was transfected into the cell lines indicated, and the cells were cultured for 2 weeks in media containing G418. (C) shRNA knockdown of PTPRD expression in primary human astrocytes. Expression was measured using quantitative PCR. Results normalized to GAPDH. Assay was performed in triplicate. (D) Knockdown of PTPRD in IHA results in increased growth rate. Left shows cultures of either astrocytes with PTPRD knocked down or control cells (scramble) plated at equal cell numbers and cultured for 10 days. Right shows a growth curve comparing rate of proliferation of astrocytes with PTPRD knocked down versus control. Five thousand cells were plated in both cases. All experiments were performed in triplicate. All graphs report mean Ϯ SD. *, P Ͻ 0.05; ***, P Ͻ 0.001. (E) PTPRD depletion results in increased tumor growth. Immortalized human astrocytes (IHA) expressing shRNA that depleted PTPRD resulted in faster tumor growth than a scrambled control in a mouse xenograft model. The astrocytes used were immortalized by transfection

with E6, E7, hTERT, and ras (25). Error bars show Ϯ SD. P Ͻ 0.05 for all comparisons of PTPRD shRNA versus astrocytes alone and scramble shRNA. (F) PTPRD mutations MEDICAL SCIENCES found in human cancers abrogate growth suppressive properties. Top shows the mutations that were tested. Shown are bright ﬁeld pictures and colony formation assays of HCT116 cells transfected with empty vector, wild-type PTPRD, and the 3 mutant PTPRD alleles indicated. PTPc, phosphatase domain; FN3, ﬁbronectin type III domain; IGC2, Ig C2 domain. (G) PTPRD dephosphorylates and regulates STAT3. Left shows the levels of phospho and total proteins indicated after wild-type and mutant PTPRD transfection into HEK 293T cells. Top Right shows blot demonstrating that PTPRD directly dephosphorylates STAT3 in vitro. GST-PTPRD fusion protein (1–20 ␮g) was incubated with immunoprecipitated STAT3-Flag and analyzed by Western blot. Bottom Right bar graph shows that SOCS3 mRNA levels decrease after wild-type PTPRD transfection. Values represent normalized expression measured using qPCR. **, P ϭ 0.0019). (H) Knockdown of PTPRD increases the phosphorylation of STAT3. siRNA was used to knockdown PTPRD in the indicated cells. PTPRD was detected by Western blot using an anti-PTPRD antibody. Total STAT3 and phospho-STAT3 were detected as above. (I) Summary of PTPRD alterations in primary human cancers. The presence of a type of alteration as detected in the current study is denoted by red. Number indicates observed frequency of event. Green indicates evidence of the event in the literature (4, 11, 12, 44, 45).

and incubated it in vitro with immunoprecipitated STAT3. Incubation using siRNA in both HEK 293T cells and human astrocytes results with GST-PTPRD but not GST alone resulted in a dose-dependent in an increase in STAT3 phosphorylation (Fig. 3H). decrease in phospho-STAT3, indicating that STAT3 is a direct substrate The combination of genetic, epigenetic, and cellular data dem- of PTPRD (Fig. 3G). Examination of the protein sequence of PTPRD onstrates that PTPRD is a tumor suppressor gene in malignant revealed that the cytoplasmic domain possesses 3 consensus STAT3 glioma and other human cancers. Our results, along with previously binding motifs (PYXXQ) (28). Moreover, knockdown of PTPRD identified alterations in PTPRD, strongly suggest that this gene is a

Veeriah et al. PNAS ͉ June 9, 2009 ͉ vol. 106 ͉ no. 23 ͉ 9439 Downloaded by guest on September 27, 2021 broadly inactivated tumor suppressor in human malignancies and macologic reactivation of PTPs is difficult, identification of corre- may play an important role in tumor development (8–13). Our sponding kinases that phosphorylate common substrates could results show that PTPRD dysfunction occurs in a number of tumor provide therapeutic targets, such as in the case of PTEN and AKT. This types (Fig. 3I) (4). Importantly, there exists strong evidence that there approach may have broad therapeutic implications as PTPRD alter- is more than one tumor suppressor gene on 9p near 9p21 (9p21 includes ations exist in Ͼ50% of GBMs, as well as in other human malignancies. p16INK4A) in multiple human cancers (18, 29, 30). Our data strongly indicates that PTPRD is one of these tumor suppressor genes and is Materials and Methods likely to be fundamentally important for oncogenesis. Also, it should See SI Text for full methods. be noted that PTPRT also can regulate STAT3, and it will be Tumor Samples and aCGH Analysis. Tumors from the Memorial Sloan–Kettering interesting to analyze the relative roles of these 2 genes in human Cancer Center were obtained after patient consent and with institutional review cancer (31). STAT3 is a well documented oncoprotein (32–34). In board approval. In our analysis, processing and analysis of data were performed GBM, aberrant activation of STAT3 is very common (35). Inhibi- as described (41). tion of STAT3 activity has been shown to slow or arrest growth in GBM (36–39). Methylation and Gene Expression Analysis. Selection of primers used for MSP and The function of PTPRD is consistent with the function of other determinants for CpG island localization and designation was accomplished by PTPs implicated in tumor suppression and with their general role using MSPPrimer (42). MSP was performed as previously described (43). Bisulﬁte of antagonizing growth-stimulating signaling pathways (40). Our sequencing and RT-PCR was performed as previously described (4). results indicate that PTPRD may act as a tumor suppressor by ACKNOWLEDGMENTS. We thank Thomas Landers, Igor Dolgalev, Sabrena regulating cell growth. Moreover, our finding that PTPRD expres- Thomas, and Benjamin Golas for their exceptional technical expertise and Ste- sion is preferentially lost in GBM versus gliomas of lower WHO phen B. Baylin, Eric Holland, Neal Rosen, Ross Levine, and members of the Sawyers grade suggests that loss of this gene plays an important role in the laboratory for helpful discussions and comments. This work was supported in part by The Cancer Genome Atlas project (C.B., M.L., C.S., and J.M.); The Brain Tumors progression rather than the initiation of malignant gliomas. Funders’ Collaborative (I.K.M., T.C.C., P.S.M., and L.L.); The Flight Attendants Medical PTPs such as PTPRD regulate signaling pathways that may be Research Institute (T.A.C.); The American Society for Clinical Oncology; The Society of amenable to therapeutic targeting in tumor cells. Although phar- Memorial Sloan–Kettering Foundation; and The Louis Gerstner Foundation.

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