Protein tyrosine phosphatase receptor delta acts as a neuroblastoma tumor suppressor by destabilizing the aurora kinase a oncogene
- Maria Meehan†1, 2,
- Laavanya Parthasarathi†1, 2,
- Niamh Moran1,
- Caroline A Jefferies1,
- Niamh Foley1, 2,
- Elisa Lazzari1,
- Derek Murphy3,
- Jacqueline Ryan1, 2,
- Berenice Ortiz4,
- Armida W M Fabius4,
- Timothy A Chan4 and
- Raymond L Stallings1, 2Email author
© Meehan et al; BioMed Central Ltd. 2012
Received: 7 September 2011
Accepted: 5 February 2012
Published: 5 February 2012
Protein tyrosine phosphatase receptor delta (PTPRD) is a member of a large family of protein tyrosine phosphatases which negatively regulate tyrosine phosphorylation. Neuroblastoma is a major childhood cancer arising from precursor cells of the sympathetic nervous system which is known to acquire deletions and alterations in the expression patterns of PTPRD, indicating a potential tumor suppressor function for this gene. The molecular mechanism, however, by which PTPRD renders a tumor suppressor effect in neuroblastoma is unknown.
As a molecular mechanism, we demonstrate that PTPRD interacts with aurora kinase A (AURKA), an oncogenic protein that is over-expressed in multiple forms of cancer, including neuroblastoma. Ectopic up-regulation of PTPRD in neuroblastoma dephosphorylates tyrosine residues in AURKA resulting in a destabilization of this protein culminating in interfering with one of AURKA's primary functions in neuroblastoma, the stabilization of MYCN protein, the gene of which is amplified in approximately 25 to 30% of high risk neuroblastoma.
PTPRD has a tumor suppressor function in neuroblastoma through AURKA dephosphorylation and destabilization and a downstream destabilization of MYCN protein, representing a novel mechanism for the function of PTPRD in neuroblastoma.
KeywordsPTPRD AURKA MYCN Neuroblastoma Tumor suppressor
aurora kinase A
Protein Tyrosine Phosphatase Receptor Delta
Protein tyrosine phosphatase receptor delta (PTPRD) is an important regulator of axon growth and guidance and is highly expressed in the central nervous system where it functions as a transmembrane homophilic neuronal cell adhesion molecule . PTPRD undergoes a high frequency of hemizygous/homozygous deletions in multiple forms of cancer, which are often intragenic in nature, indicating a potential tumor suppressor function [2–8]. Additional mechanisms leading to PTPRD inactivation include promoter region hypermethylation, point mutations and aberrant splicing [6, 9–12].
Neuroblastoma is derived from primitive cells of the sympathetic nervous system, and is the most common extracranial solid tumor in children accounting for 15% of all childhood cancer deaths . These tumors are particularly noted for extensive heterogeneity in clinical behaviour, ranging from spontaneous regression to aggressive clinical course and death from disease. Notably, amplification of the MYCN transcription factor is one of the most powerful adverse prognostic factors in neuroblastoma  and we have previously demonstrated that PTPRD is expressed at significantly lower levels in MYCN amplified neuroblastoma relative to non-MYCN amplified tumors . In addition, PTPRD mRNA expression is higher in normal adrenal fetal neuroblasts, the cell of origin of neuroblastoma, relative to unfavourable neuroblastoma tumors, indicating that PTPRD down-regulation might be an important step in the development of these tumors [7, 10]. Multiple mechanisms appear to exist for the down-regulation of PTPRD in neuroblastoma, including intragenic microdeletions which can include coding sequence, or in some instances be restricted to non-coding exons of an extended 5' UTR . Aberrant splicing of the 5' UTR also has been noted in neuroblastoma cell lines and primary tumors, which could potentially cause destabilization of the mRNA sequence .
In this report, we demonstrate for the first time that experimental up-regulation of PTPRD in neuroblastoma cell lines significantly decreases cell growth and increases apoptosis. Moreover, we identify aurora kinase A, a serine/threonine kinase oncogene that is up-regulated in many forms of cancer, including high risk neuroblastoma , as an interaction partner of PTPRD. We further demonstrate that PTPRD has a tumor suppressor function in neuroblastoma through dephosphorylating and destabilizing AURKA, leading to a downstream decrease of MYCN protein. Our findings represent a novel mechanism of action for the function of PTPRD in neuroblastoma.
PTPRD functions as a tumor suppressor in neuroblastoma
Identification of proteins that interact with PTPRD
Protein Interaction Array Results
PTPRD destabilizes AURKA by dephosphorylating tyrosine sites
Having confirmed a PTPRD-AURKA interaction, we next investigated whether AURKA is a potential substrate for PTPRD. Within the catalytic domain of AURKA, there are a number of conserved tyrosine phosphorylation sites (Y148, Y199, Y197, Y212) ). Immunoprecipitation of tyrosine phosphorylated proteins with a pan-phosphotyrosine agarose conjugate and subsequent western blotting with an AURKA antibody revealed an abolishment of AURKA tyrosine phosphorylation in PTPRD cDNA transfected cells compared to phosphatase dead mutant or empty vector control transfectants, confirming the mechanism of action of PTPRD (Figure 3C). In addition to deactivating AURKA, ectopic over-expression of PTPRD also leads to a decrease in protein stability of AURKA. After 4 hours of cycloheximide treatment, there is a significant difference in the decay of AURKA in the PTPRD transfected cells compared to empty vector (EV) or phosphatase dead mutant (Figure 3D, E). qPCR analysis of AURKA mRNA at 48 hours post PTPRD transfection indicated no significant change relative to negative control, confirming that the AURKA decrease is post-transcriptional (Additional File 2A).
A critical function of AURKA in neuroblastoma is to regulate MYCN protein stability by sequestering MYCN from ubiquitin-mediated proteolytic degradation mediated by the FBXW7 ubiquitin ligase , leading us to hypothesize that a down-stream effect of ectopic over-expression PTPRD in neuroblastoma cells would be the destabilization of MYCN. As illustrated in Figure 3F, MYCN protein levels are also decreasing as a consequence of PTPRD ectopic over-expression. The decrease in MYCN protein is due to a post-transcriptional mechanism, as there is no change in MYCN mRNA (Additional File 2B).
In this report we demonstrate a novel in vivo interaction between the tumor suppressor PTPRD and the oncogenic AURKA protein in neuroblastoma. Furthermore, to our best knowledge, this is the first report to demonstrate dephosphorylation of tyrosine residues in AURKA leading to its subsequent destabilization. The identification of AURKA as a novel target of PTPRD mediated dephosphorylation adds to a growing list of substrates for this tumor suppressor. Activity of AURKA is also regulated by phosphorylation of Thr288 in a cell cycle dependent manner . Constitutive phosphorylation on Ser51 of AURKA has also been shown to be associated with its over-expression and protein stabilization, preventing protein degradation mediated by the ubiquitin-proteasome system  and it is likely that constitutive phosphorylation of tyrosine residues in AURKA also prevent its degradation. AURKA destabilization also has the down-stream effect of destabilizing MYCN, consistent with prior findings by Otto et al. . Thus, PTPRD is likely to have a tumor suppressor function in any type of cancer with MYCN amplification, including other pediatric cancers such as medulloblastoma and rhabdomyosarcoma, or in adult tumors that are dependent on high AURKA levels.
MYCN directly up-regulates AURKA by binding to its promoter region, forming an auto regulatory feedback loop . As previously mentioned, PTPRD expression is significantly lower in MYCN amplified tumors relative to non-MYCN amplified tumors , indicating that MYCN might be directly repressing PTPRD in order to maintain AURKA levels. However, analysis of a previously published data set on MYCN binding sites in neuroblastoma cell lines indicated that MYCN does not bind to the PTPRD promoter region [24, 25], revealing that down-regulation of PTPRD in MYCN amplified tumors is an indirect effect.
Along with AURKA, a number of other proteins scored highly in our protein-protein interaction arrays, including SLAIN2, and two serine/threonine kinases, NEK2 and PLK1.
SLAIN2 has recently been shown to play an important role in the microtubule complex by controlling microtubule dynamics and organization . NEK2 is an integral component of the mitotic spindle-assembly checkpoint which is necessary for proper chromosome segregation during metaphase-anaphase transition , while PLK1 is an early trigger for G2/M transition . Intriguingly, one of the primary functions of AURKA is the activation of PLK1 by direct phosphorylation of Thr210 , and high expression of PLK1 is also significantly associated with high-risk neuroblastoma and unfavourable patient outcome . Inhibition of PLK1 protein has an anti-proliferative effect on neuroblastoma cell proliferation . Although the interaction of PTPRD with these proteins requires validation, it seems likely that over-expression of PTPRD should have a pleiotrophic tumor suppressor effect by associating with, and dephosphorylating a number of interacting partners. The recent development of AURKA inhibitors, along with impressive results in pre-clinical models of pediatric cancer, indicate that AURKA could be a therapeutic target of great value in the treatment of neuroblastoma [31, 32]. PTPRD, as a natural antagonist of AURKA, might be of therapeutic value in cancers where its inactivation is epigentically reversible, such as in instances of promoter region hypermethylation.
Over-expression of PTPRD in neuroblastoma cell lines results in decreased cell viability through the activation of apoptosis. We further demonstrate a novel PTPRD-AURKA protein interaction, and that PTPRD over-expression results in the dephosphorylation and the destabilization of AURKA, along with a down-stream destabilization of MYCN protein. These findings, taken together, indicate a novel mechanism of action for PTPRD as a neuroblastoma tumor suppressor through the inhibition of these important oncogene products.
Cell Culture, In Vitro Growth and Apoptosis Assays
SHSY-5Y and CHP-212 neuroblastoma cell lines were obtained from the American Type Culture Collection, while Kelly was obtained from European Collection of Animal Cell Cultures. All cell lines were validated for the presence of previously published genomic imbalances using aCGH. Culture media was supplemented with 10% FBS.
Cells were transfected with 2 μg PTPRD cDNA, empty vector (pcDNA3.1-V5) or phosphatase dead mutant PTPRD (Q14811) using lipofectamine 2000 reagent according to
the manufacturer's instructions (Invitrogen, Carlsbad, CA) and were analysed by a Cyquant assay (Invitrogen) at 24, 48 and 72 h. PTPRD induced apoptosis was demonstrated by FACS analysis of annexin-V staining. Kelly cells were transfected with empty vector or PTPRD cDNA. Cells were harvested 72 h post transfection and apoptosis was quantified using the FITC Annexin-V Apoptosis Detection Kit I (BD Pharmingen, San Diego, CA, USA). Experiments were performed in triplicate to quantify apoptosis by phosphatidylserine (PS) externalization.
Kelly cells (80-90% confluence) were transfected with 2 μg PTPRD cDNA or empty vector (pcDNA3.1-V5) in 100 mm dishes. After 24 h transfection, cells were incubated in a RIPA buffer (Sigma-Aldrich, Arklow) containing complete protease inhibitor mixture. The lysates were cleared by centrifugation for 20 min at 14,000 × g and then subjected to immunoprecipitation with either anti-V5 agarose affinity beads (Sigma-Aldrich) for co-IP experiments or 4 G10® Platinum, anti-phosphotyrosine agarose conjugate (Millipore) for pan-tyrosine IP experiments, and then incubated at 4 C for 2 h. The beads were washed five times with RIPA buffer, incubated in laemmli sample buffer and heated to 95 C for five mins. Resultant samples were separated by SDS-PAGE and were analysed by western blotting.
Cells were washed with PBS and lysed at 4 C for 20 min using RIPA buffer containg phosphatase and protease inhibitors. Lysates were centrifuged for 20 min at 14,000 × g and resultant protein extracts were separated by SDS-PAGE and blotted onto PVDF membranes. Antibodies used were as follows: anti-V5 (Invitrogen), anti-AURKA (Sigma), anti-MYCN (Santa Cruz) and anti-alpha tubulin (Abcam). Secondary antibodies were horseradish peroxidise-conjugated rabbit anti-mouse (Abcam) or goat anti-rabbit (Cell Signaling Technology). Reactive proteins were visualized by chemiluminescence with ECL (Thermo).
PTPRD Interaction arrays
Purified PTPRD protein (99% pure, mass spectrum verified) (Clone ID 8613c5BCt10p1, protein sequence 1201-1495) was biotinylated and quantitated in duplicate using the Invitrogen FluoReporter® Biotin Quantitation Assay Kit. The array control protein (biotinylated calmodulin kinase) supplied as part of the Invitrogen protein interaction kit was used to probe the Protoarray® control protein microarray to validate assay conditions. Biotinylated PTPRD baits (two biological replicates) were then used to probe the Invitrogen ProtoArray® Protein Microarray Kit for biotinylated proteins Version 4 in order to detect potential interaction partners. The results were analyzed using the Protoarray prospector® package. The consensus hits (hits that are above the back ground signal in both the experiments) along with their Z-scores are listed in Table 1. A Z-Score indicates how far and in what direction the value of an individual data point in a population falls from the mean in units of standard deviations. ProtoArray® Prospector collects all the signals from the proteins on a microarray, calculates the mean value and standard deviation, and then calculates a Z-Score corresponding to the signal from each feature.
Cycloheximide half-life experiments
Kelly cells (80-90% confluence) were transfected with 2 μg PTPRD cDNA, empty vector (pcDNA3.1-V5) or PTPRD mutant (Q1481X) in 100 mm dishes. After 72 h transfection, cells were incubated in a RIPA buffer (Sigma-Aldrich, Arklow) containing complete protease inhibitor mixture. Cells were treated with 1 μl of a 20 mg/ml stock solution of cycloheximide (Sigma-Aldrich) 5 min prior to starting the indicated time course, and cells were collected at the indicated points. Quantification of western blots was performed by densitometry using GelEval (FrogDance Software). Normalization for loading differences was achieved by dividing the densitometry values for individual bands by the densitometry values for alpha-tubulin in the same lane.
This work was supported in part by the Association of International Cancer Research (07-0033) and the Children's Medical and Research Foundation. We acknowledge the New York SGX Research Center for Structural Genomics (NYSGXRC, NIGMS Grant U54 GM074945) for providing samples of purified PTPRD protein.
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