PTPN6 Expression Research Articles

The presence of Y674/Y675 phosphorylated NTRK1 via TP53 repression of PTPN6 expression as a potential prognostic marker in neuroblastoma

The tumor suppressor TP53 promotes nerve growth factor receptor (NTRK1) -Y674/Y675 phosphorylation (NTRK1-pY674/pY675) via repression of the NTRK1 phosphatase PTPN6 in a ligand-independent manner, resulting in suppression of breast cancer cell proliferation. Moreover, NTRK1-pY674/pY675 together with low levels of PTPN6 and TP53 expression is associated with favorable disease-free survival of breast cancer patients. We determined whether in neuroblastoma this protein expression pattern impacts relapse-free survival (RFS). NTRK1-pY674/pY675, PTPN6, and TP53 expression was assessed in 98 neuroblastoma samples by immunohistochemistry. Association between expression levels and RFS was investigated by multivariate and Kaplan-Meier analysis. Mutant or wild-type TP53 was identified by sequencing tumor DNA. Tumors expressing NTRK1-pY674/pY675 and low or undetectable levels of PTPN6 and TP53 were significantly associated with 5-year RFS (P = .014) when the dataset was stratified by MYCN amplification, segmental chromosomal abnormalities and histology. Similar results were observed with tumors expressing wild-type TP53, NTRK1-pY674/pY675 and low or undetectable levels of PTPN6. Kaplan-Meier analysis demonstrated a significant correlation (P = .004), with a 50% probability of RFS (median survival 4.73 years) when present compared with 19.51% (median survival 11.63 months) when absent. Similar results were seen with non-amplified MYCN or unfavorable/undifferentiating samples and tumors from patients aged 18 months or less. Importantly, NTRK1-pY674/pY675 is an independent predictor of improved RFS. These results strongly suggest that NTRK1-pY674/pY675 together with wild-type TP53 and undetectable or low levels of PTPN6 expression is a potential biomarker of improved RFS of neuroblastoma patients. The predictive value of NTRK1-pY674/pY675 together with wild-type TP53 and low PTPN6 expression could contribute to neuroblastoma patient prognosis.

Prognostic and therapeutic significance of phosphorylated STAT3 and protein tyrosine phosphatase-6 in peripheral-T cell lymphoma

Peripheral T cell lymphomas (PTCL) is a heterogenous group of non-Hodgkin lymphoma and many patients remain refractory to the frontline therapy. Identifying new prognostic markers and treatment is an unmet need in PTCL. We analyzed phospho-STAT3 (pSTAT3) expression in a cohort of 169 PTCL tumors and show overall 38% positivity with varied distribution among PTCL subtypes with 27% (16/59) in PTCL-NOS; 29% (11/38) in AITL, 57% (13/28) in ALK-negative ALCL, and 93% in ALK-pos ALCL (14/15), respectively. Correlative analysis indicated an adverse correlation between pSTAT3 and overall survival (OS). PTPN6, a tyrosine phosphatase and potential negative regulator of STAT3 activity, was suppressed in 62% of PTCL-NOS, 42% of AITL, 60% ALK-neg ALCL, and 86% of ALK-pos ALCL. Loss of PTPN6 combined with pSTAT3 positivity predicted an infwere considered significantferior OS in PTCL cases. In vitro treatment of TCL lines with azacytidine (aza), a DNA methyltransferase inhibitor (DNMTi), restored PTPN6 expression and decreased pSTAT3. Combining DNMTi with JAK3 inhibitor resulted in synergistic antitumor activity in SUDHL1 cell line. Overall, our results suggest that PTPN6 and activated STAT3 can be developed as prognostic markers, and the combination of DNMTi and JAK3 inhibitors as a novel treatment for patients with PTCL subtypes.

Abstract 3166: The HU177 cryptic collagen epitope selectively regulates melanoma cell behavior by a CDK5 and PTPN12 associated mechanism

Abstract Previous studies indicate that selectively inhibiting cellular interactions with the HU177 cryptic collagen epitope, which can be generated in vivo during structural remodeling of the extracellular matrix (ECM), inhibits tumor growth and metastasis. However, a detailed understanding of the mechanisms by which antagonists of the HU177 collagen epitope inhibit these pathological processes is not fully understood. Uncovering the molecular mechanisms by which cellular interactions with this collagen epitope regulate tumor cell behavior may allow the development of more effective clinical strategies targeting tumor cell-ECM interactions. Here we provide evidence for the first time, that specific targeting of the HU177 cryptic collagen epitope reduces phosphorylation of cyclin-dependent kinase (CDK5) and in addition, reduces the expression of the phosphatase PTPN12 in melanoma cells attached to denatured collagen. Importantly, both CDK5 < PTPN12 play a functional role in the ability of the HU177 collagen epitope to regulate migration of melanoma cells as reducing expression of these molecules inhibited the anti-migratory activity of the anti-HU177 antibody. Surprisingly, reduction in either CDK5 or PTPN12 also inhibited nuclear accumulation of YAP. These novel findings help define a previously unknown signaling mechanism by which both CDK5 < PTPN12 may play a selective role in controlling nuclear accumulations of the transcriptional co-activator YAP following melanoma cell interactions with the HU177 cryptic collagen epitope. Taken together, these studies provide new molecular insight into the role of the HU177 collagen epitope in controlling melanoma cell behavior. Given that direct targeting of integrin receptors that mediate tumor cellular interactions with the ECM have demonstrated only limited anti-tumor activity in human clinical trials, a more detailed molecular understanding of how generation of biologically relevant cryptic elements of the ECM control melanoma cell behavior will likely facilitate the development of more effective new clinical strategies to control tumor progression. Citation Format: Xianghua Han, Jennifer M. Caron, Peter C. Brooks. The HU177 cryptic collagen epitope selectively regulates melanoma cell behavior by a CDK5 and PTPN12 associated mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3166.

Prognostic Value of Phosphotyrosine Phosphatases in Hepatocellular Carcinoma

Background/Aims: During the occurrence and progression of hepatocellular carcinoma (HCC), phosphotyrosine phosphatases (PTPs) are usually described as tumor suppressors or proto-oncogenes, and to some degree are correlated with the prognosis of HCC. Methods: A total of 321 patients from the Cancer Genome Atlas (TCGA) database and 180 patients from our validated cohort with hepatocellular carcinoma were recruited in this study. Kaplan-Meier, univariate and multivariate Cox proportional hazards model were used to evaluate the risk factors for survival. Quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC) were applied to detect the expression levels of PTP genes. Results: After screening the data of TCGA, we identified five PTPs as HCC overall survival related PTP genes, among which only three (PTPN12, PTPRN, PTPN18) exhibited differential expression levels in our 180 paired HCC and adjacent tissues (P< 0.001). Further analysis revealed that expression of PTPN18 was positively, but PTPRN was negatively associated with prognosis of HCC both in TCGA cohort and our own cohort. As to PTPN12, results turned out to be opposite according to HBV status. In detail, higher expression of PTPN12 was associated with better outcome in HBV group but worse prognosis in Non-HBV group. Conclusion: Our results suggested that PTPN12, PTPRN and PTPN18 were independent prognostic factors in HCC.

Suppression of PTPN6 exacerbates aluminum oxide nanoparticle-induced COPD-like lesions in mice through activation of STAT pathway

BackgroundInhaled nanoparticles can deposit in the deep lung where they interact with pulmonary cells. Despite numerous studies on pulmonary nanotoxicity, detailed molecular mechanisms of specific nanomaterial-induced lung injury have yet to be identified.ResultsUsing whole-body dynamic inhalation model, we studied the interactions between aluminum oxide nanoparticles (Al2O3 NPs) and the pulmonary system in vivo. We found that seven-day-exposure to Al2O3 NPs resulted in emphysema and small airway remodeling in murine lungs, accompanied by enhanced inflammation and apoptosis. Al2O3 NPs exposure led to suppression of PTPN6 and phosphorylation of STAT3, culminating in increased expression of the apoptotic marker PDCD4. Rescue of PTPN6 expression or application of a STAT3 inhibitor, effectively protected murine lungs from inflammation and apoptosis, as well as, in part, from the induction of chronic obstructive pulmonary disease (COPD)-like effects.ConclusionIn summary, our studies show that inhibition of PTPN6 plays a critical role in Al2O3 NPs-induced COPD-like lesions.

Epigenome-wide association studies identify DNA methylation associated with kidney function

Chronic kidney disease (CKD) is defined by reduced estimated glomerular filtration rate (eGFR). Previous genetic studies have implicated regulatory mechanisms contributing to CKD. Here we present epigenome-wide association studies of eGFR and CKD using whole-blood DNA methylation of 2264 ARIC Study and 2595 Framingham Heart Study participants to identify epigenetic signatures of kidney function. Of 19 CpG sites significantly associated (P < 1e-07) with eGFR/CKD and replicated, five also associate with renal fibrosis in biopsies from CKD patients and show concordant DNA methylation changes in kidney cortex. Lead CpGs at PTPN6/PHB2, ANKRD11, and TNRC18 map to active enhancers in kidney cortex. At PTPN6/PHB2 cg19942083, methylation in kidney cortex associates with lower renal PTPN6 expression, higher eGFR, and less renal fibrosis. The regions containing the 243 eGFR-associated (P < 1e-05) CpGs are significantly enriched for transcription factor binding sites of EBF1, EP300, and CEBPB (P < 5e-6). Our findings highlight kidney function associated epigenetic variation.

Differential regulation of pro-inflammatory cytokine signalling by protein tyrosine phosphatases in pancreatic β-cells.

Type 1 diabetes (T1D) is characterized by the destruction of insulin-producing β-cells by immune cells in the pancreas. Pro-inflammatory including TNF-α, IFN-γ and IL-1β are released in the islet during the autoimmune assault and signal in β-cells through phosphorylation cascades, resulting in pro-apoptotic gene expression and eventually β-cell death. Protein tyrosine phosphatases (PTPs) are a family of enzymes that regulate phosphorylative signalling and are associated with the development of T1D. Here, we observed expression of PTPN6 and PTPN1 in human islets and islets from non-obese diabetic (NOD) mice. To clarify the role of these PTPs in β-cells/islets, we took advantage of CRISPR/Cas9 technology and pharmacological approaches to inactivate both proteins. We identify PTPN6 as a negative regulator of TNF-α-induced β-cell death, through JNK-dependent BCL-2 protein degradation. In contrast, PTPN1 acts as a positive regulator of IFN-γ-induced STAT1-dependent gene expression, which enhanced autoimmune destruction of β-cells. Importantly, PTPN1 inactivation by pharmacological modulation protects β-cells and primary mouse islets from cytokine-mediated cell death. Thus, our data point to a non-redundant effect of PTP regulation of cytokine signalling in β-cells in autoimmune diabetes.

Research on the epigenetic regulation mechanism of the PTPN6 gene in advanced chronic myeloid leukaemia.

PTPN6, a tyrosine phosphatase protein, plays a negative role in cell signal transduction and is negatively correlated with tumour formation and growth. However, epigenetic regulation mechanism of the PTPN6 gene in advanced chronic myeloid leukaemia (CML) remains unclear. This study investigated bone marrow or blood samples from 44 CML patients and 10 healthy volunteers. KCL22 and K562 cells were cultured and treated with demethylation drugs and histone deacetylase inhibitors. Real time quantitative polymerase chain reaction (qPCR), methylation-specific PCR, bisulfite sequencing PCR, Western blotting, co-immunoprecipitation and chromatin immunoprecipitation (ChIP) was performed. PTPN6 was down-regulated in cell lines and patients with advanced phase CML, whereas DNMT1, DNMT3A, MECP2, MBD2 and HDAC1 were up-regulated. Treatment with 5-azacytidine, decitabine, sodium valproate and LBH589 increased PTPN6 expression, but decreased that of DNMT1, DNMT3A, MECP2, MBD2 and HDAC1. Immunoprecipitation and mass spectrometry showed that HDAC1 combined directly with PTPN6. ChIP-seq showed that HDAC1 did not combine with the promoter region of PTPN6, while MAPK, AKT, STAT5, JAK2 and MYC promoter regions all combined with HDAC1. PTPN6 is associated with progression of CML. Low expression level of PTPN6 was associated with DNA methylation and regulated by histone acetylation. HDAC1 participates in the regulation of PTPN6.

Integrative functional transcriptomic analyses implicate specific molecular pathways in pulmonary toxicity from exposure to aluminum oxide nanoparticles

Gene expression profiling has developed rapidly in recent years and it can predict and define mechanisms underlying chemical toxicity. Here, RNA microarray and computational technology were used to show that aluminum oxide nanoparticles (Al2O3 NPs) were capable of triggering up-regulation of genes related to the cell cycle and cell death in a human A549 lung adenocarcinoma cell line. Gene expression levels were validated in Al2O3 NPs exposed A549 cells and mice lung tissues, most of which showed consistent trends in regulation. Gene-transcription factor network analysis coupled with cell- and animal-based assays demonstrated that the genes encoding PTPN6, RTN4, BAX and IER play a role in the biological responses induced by the nanoparticle exposure, which caused cell death and cell cycle arrest in the G2/S phase. Further, down-regulated PTPN6 expression demonstrated a core role in the network, thus expression level of PTPN6 was rescued by plasmid transfection, which showed ameliorative effects of A549 cells against cell death and cell cycle arrest. These results demonstrate the feasibility of using gene expression profiling to predict cellular responses induced by nanomaterials, which could be used to develop a comprehensive knowledge of nanotoxicity.

Epigenetic regulation of protein tyrosine phosphatase PTPN12 in triple-negative breast cancer.

The present study showed that the expression of PTPN12 is epigenetically regulated. 5-Azacytidine (5-Azac), a DNA hypomethylating agent, significantly increased the expression of PTPN12 at low concentrations (1μM and 2.5μM) and decreased the expression of PTPN12 at 5μM in the MDA-MB-231 and BT-549 triple-negative breast cancer cell lines. Human MCF-7, MDA-MB-231 and BT-549 cells were exposed to different concentrations of 5-Azac for 24 and 48h. RT-PCR was performed to determine the mRNA expression of PTPN12, E-cadherin and miRNA-124. Western blotting was performed to assess the protein expression of various proteins, including PTPN12, E-cadherin, DNMT and PARP. 5-Azac, a DNA hypomethylating agent, significantly increased the expression of PTPN12 at low concentrations (1μM and 2.5μM) and decreased PTPN12 expression at 5μM. We provide the first evidence that PTPN12 expression is epigenetically regulated and that it is up-regulated at a lower dose of a DNMT1 inhibitor in MDA-MB-231 and BT-549 cells. Interestingly, the levels of miRNA-124 were increased only at 5μM, the concentration at which PTPN12 expression was suppressed. To the best of our knowledge, this is the first report that highlights the therapeutic potential of low-dose 5-Azac for the treatment of TNBC. Therefore, 5-Azac, an agent that has already been tested in acute myeloid leukemia, may be more effective at lower doses for the treatment of triple-negative breast cancer.

Tyrosine-protein phosphatase non-receptor type 12 expression is a good prognostic factor in resectable non-small cell lung cancer.

Tyrosine-protein phosphatase non-receptor type 12 (PTPN12) has been considered to be a tumor suppressor in human cancer, but its clinical and prognostic significance in non-small cell lung cancer (NSCLC) has not been well elucidated.A retrospective analysis of 215 patients with surgically resected NSCLCs from Sun Yat-Sen University Cancer Center between April 2002 and March 2005 was performed using immunohistochemistry and Western Blot to analyze PTPN12 expression. The association between PTPN12 expression and patient survival was investigated.Western Blots showed that the expression level of PTPN12 were higher in normal paracancerous lung tissues than in NSCLC tissues. High PTPN12 expression was less common in the presence than in the absence of visceral pleural invasion (p=0.038). Patients with PTPN12-high tumors had a longer disease-free survival (DFS) (P<0.001) and overall survival (OS) (p<0.001), especially for those with non-squamous cell carcinoma (non-SCC) (DFS, p<0.001; OS, p<0.001). Multivariate analysis confirmed that PTPN12 positivity was associated with increased survival duration (DFS, p<0.001; OS, p<0.001), independent of prognostic indicator.High PTPN12 expressive levels are associated with favorable survival duration in patients with NSCLC, especially those with non-SCC. Our study suggests that PTPN12 expression is a valuable prognostic biomarker for NSCLC patients.

The prognostic significance of tyrosine-protein phosphatase nonreceptor type 12 expression in nasopharyngeal carcinoma.

Tyrosine-protein phosphatase nonreceptor type 12 (PTPN12) has been proposed to predict prognosis of various human cancers. However, the clinicopathologic and prognostic significance of PTPN12 expression in NPC has not yet been elucidated. The objective of this study was to investigate the clinicopathological and prognostic implication of PTPN12 in nasopharyngeal carcinoma (NPC) patients. Protein expression levels of PTPN12 were explored by semiquantitative immunohistochemical staining on archival formalin-fixed, paraffin-embedded pathological specimens consisting of 203 NPCs, and 40 normal nasopharyngeal mucosa tissues. Receiver operating characteristic (ROC) curve analysis was employed to determine the cutoff score of PTPN12 expression in NPCs. The PTPN12 immunohistochemical staining results were then correlated with various clinicopathological features and patients’ prognosis using various statistical models. Our results showed that decreased expression of PTPN12 was more frequently observed in NPC tissues compared with the normal nasopharyngeal mucosa. Further correlation analyses indicated that the decreased expression of PTPN12 was significantly associated with tumor T classification, N classification, distant metastasis, and clinical stage in NPCs (P < 0.05). Univariate analysis showed a significant association between the decreased expression of PTPN12 and adverse overall survival and disease-free survival (P < 0.05). More importantly, multivariate analysis identified the PTPN12 expression in NPC as an independent prognostic factor. The decrease expression of PTPN12 might be important in conferring a more aggressive behavior in NPC. Thus, PTPN12 expression may be used as a novel independent prognostic biomarker for patients with NPC.

Phosphorylation of NTRK1 at Y674/Y675 induced by TP53-dependent repression of PTPN6 expression: A potential novel prognostic marker for breast cancer

We have identified a ligand-independent mechanism whereby the tumor suppressor, TP53, induces nerve growth factor receptor, NTRK1, phosphorylation at Y674/Y675 (NTRK1-pY674/pY675), via the repression of the NTRK1-phosphatase, PTPN6. This results in suppression of breast cancer cell proliferation. In this investigation, we aimed to establish whether perturbation of the wild-type TP53-NTRK1-pY674/pY675-PTPN6 pathway has an impact on disease-free survival of breast cancer patients without neo-adjuvant treatment. A total of 308 tumor samples were stained for NTRK1, NTRK1-pY674/pY675, PTPN6, and TP53 expression. Association between expression levels and disease-free survival was determined by the univariate/multivariate and Kaplan–Meir methods of analysis. DNA from tumors was sequenced to identify mutant or wild-type TP53. Tumors expressing NTRK1-pY674/pY675 but with undetectable or low levels of PTPN6 and TP53 were associated with prolonged 5, 10, and 15 years' disease-free survival by 48%, 36%, and 37%, respectively, in the multivariate analysis (P<0.05). A similar result was observed in tumors expressing wild-type TP53, NTRK1-pY674/pY675, and low or undetectable levels of PTPN6. Given that estrogen receptor-positive breast cancers encode wild-type TP53, we analyzed this expression pattern in these tumors. Multivariate analysis showed that it was significantly and independently predictive of prolonged survival by 66%, 70%, and 84%, respectively, (P<0.05). The Kaplan–Meir method demonstrated that NTRK1-pY674/pY675 together with undetectable or low levels of PTPN6 correlated with 59% probability of disease-free survival (median survival 15 years), compared with 7% probability of disease-free survival (median survival 4.5 years) when absent. In luminal A tumors, the presence of this pattern was estimated to have a 61% probability of disease-free survival (median survival 15 years), compared with 6% probability of disease-free survival (median survival 3 years) when it was absent. These results strongly suggest that expression of NTRK1-pY674/pY675 together with wild-type TP53 and low levels of PTPN6 expression are predictors of improved disease-free survival and that they could be useful biomarkers to predict clinical outcome.

PTPN6 expression is epigenetically regulated and influences survival and response to chemotherapy in high-grade gliomas

The prognosis of high-grade glioma patients is poor, and the tumors are characterized by resistance to therapy. The aims of this study were to analyze the prognostic value of the expression of the protein tyrosine phosphatase non-receptor type 6 (PTPN6, also referred to as SHP1) in high-grade glioma patients, the epigenetic regulation of the expression of PTPN6, and the role of its expression in chemotherapy resistance in glioma-derived cells. PTPN6 expression was analyzed with immunohistochemistry in 89 high-grade glioma patients. Correlation between PTPN6 expression and overall survival was analyzed with Kaplan-Meier univariate analysis and Cox regression multivariate analysis. Differences in drug sensitivity to a panel of 16 chemotherapeutic drugs between PTPN6-overexpressing clones and control clones were analyzed in vitro with the fluorometric microculture cytotoxicity assay. Cell cycle analysis was done with Krishan staining and flow cytometry. Apoptosis was analyzed with a cell death detection ELISA kit as well as cleaved caspase-3 and caspase-9 Western blotting. Autophagy was analyzed with LC3B Western blotting. Methylation of the PTPN6 promoter was analyzed with bisulfite pyrosequencing, and demethylation of PTPN6 was done with decitabine treatment. The PTPN6 expression correlated in univariate analysis to poor survival for anaplastic glioma patients (p = 0.026). In glioma-derived cell lines, overexpression of PTPN6 caused increase resistance (p < 0.05) to the chemotherapeutic drugs bortezomib, cisplatin, and melphalan. PTPN6 expression did not affect bortezomib-induced cell cycle arrest, apoptosis, or autophagy. Low PTPN6 promoter methylation correlated to protein expression, and the protein expression was increased upon demethylation in glioma-derived cells. PTPN6 expression may be a factor contributing to poor survival for anaplastic glioma patients, and in glioma-derived cells, its expression is epigenetically regulated and influences the response to chemotherapy.

Death Receptor 5–Targeted Depletion of Interleukin‐23–Producing Macrophages, Th17, and Th1/17 Associated With Defective Tyrosine Phosphatase in Mice and Patients With Rheumatoid Arthritis

Bidirectional interactions between granulocyte-macrophage colony-stimulating factor-positive (GM-CSF+) T cells and interferon regulatory factor 5-positive (IRF-5+) macrophages play a major role in autoimmunity. In the absence of SH2 domain-containing phosphatase 1 (SHP-1), GM-CSF-stimulated cells are resistant to death receptor (DR)-mediated apoptosis. The objective of this study was to determine whether TRA-8, an anti-DR5 agonistic antibody, can eliminate inflammatory macrophages and CD4 T cells in the SHP-1-deficient condition. Ubiquitous Cre (Ubc.Cre) human/mouse-chimeric DR5-transgenic mice were crossed with viable SHP-1-defective motheaten (mev/mev) mice. TRA-8 was administered weekly for up to 4 weeks. The clinical scores, histopathologic severity, and macrophage and CD4 T cell phenotypes were evaluated. The role of TRA-8 in depleting inflammatory macrophages and CD4 T cells was also evaluated, using synovial fluid obtained from patients with rheumatoid arthritis (RA). The levels of inflammatory macrophages (interleukin-23-positive [IL-23+] IRF-5+) and CD4 T cells (IL-17+ GM-CSF+) were elevated in mev/mev mice. In DR5-transgenic mev/mev mice, DR5 expression was up-regulated in these 2 cell populations. TRA-8 treatment depleted these cell populations and resulted in a significant reduction in inflammation and in the titers of autoantibodies. In synovial cells from patients with RA, the expression of IRF5 and DR5 was negatively correlated with the expression of PTPN6. TRA-8, but not TRAIL, suppressed RA inflammatory macrophages and Th17 cells under conditions in which the expression of SHP-1 is low. In contrast to TRAIL, which lacks the capability to counteract the survival signal in the absence of SHP-1, TRA-8 eliminated both IRF-5+ IL-23+ M1 macrophages and pathogenic GM-CSF+ IL-17+ CD4 T cells in a SHP-1-independent manner. The results of the current study suggest that TRA-8 can deplete inflammatory cell populations that result from a hyperactive GM-CSF/IRF-5 axis.

Epigenetic mechanisms of protein tyrosine phosphatase 6 suppression in diffuse large B-cell lymphoma: implications for epigenetic therapy

Protein tyrosine phosphatases such as PTPN6 can be downregulated in various neoplasms. PTPN6 expression by immunohistochemistry in 40 diffuse large B-cell lymphoma (DLBCL) tumors was lost or suppressed in 53% (21/40). To elucidate the molecular mechanisms of PTPN6 suppression, we performed a comprehensive epigenetic analysis of PTPN6 promoter 2 (P2). None of the DLBCL primary tumors (0/37) had PTPN6 hypermethylation on the CpG1 island using methylation-specific PCR, pyrosequencing, and high-resolution melting assays. However, hypermethylation in 57% (21/37) of cases was found in a novel CpG island (CpG2) in P2. PTPN6 gene suppression was reversed by 5-aza-deoxycytidine (5-Aza), a DNA methyltransferase inhibitor, and the histone deacetylase inhibitor (HDACi) LBH589. LBH589 and 5-Aza in combination inhibited DLBCL survival and PTPN6 hypermethylation at CpG2. The role of histone modifications was investigated with a chromatin-immunoprecipitation assay demonstrating that PTPN6 P2 is associated with silencing histone marks H3K27me3 and H3K9me3 in DLBCL cells but not normal B cells. 3-Deazaneplanocin A, a histone methyltransferase inhibitor, decreased the H3K27me3 mark, whereas HDACi LBH589 increased the H3K9Ac mark within P2 resulting in re-expression of PTPN6. These studies have uncovered novel epigenetic mechanisms of PTPN6 suppression and suggest that PTPN6 may be a potential target of epigenetic therapy in DLBCL.

Low Expression of Tyrosine-protein Phosphatase Nonreceptor Type 12 is Associated with Lymph Node Metastasis and Poor Prognosis in Operable Triple-negative Breast Cancer

Low tyrosine-protein phosphatase nonreceptor type 12 (PTPN12) expression may be associated with breast cancer growth, proliferation, and metastasis. However, the prognostic value of PTPN12 in breast cancer has not been clearly identified. 51 triple-negative breast cancer (TNBC) patients and 83 non-TNBC patients with a histopathology diagnosis from October 2001 to September 2006 were included in this study. Immunohistochemical staining for PTPN12 on tissue microarrays was conducted. High PTPN12 expression was seen in 39.2% of TNBC and 60.2 % of non-TNBC cases. Low PTPN12 expression was associated with lymph node status (p = 0.002) and distant metastatic relapse (p = 0.002) in TNBC patients. Similarly, low PTPN12 expression in non-TNBC patients was significantly correlated with lymph node status (p = 0.002), stage (p = 0.002) and distant metastatic relapse (p = 0.039). The high PTPN12 expression group was associated with longer DFS and OS compared with low PTPN12 expression group only in TNBC cases (p = 0.005, p = 0.015), according to univariate Cox regression analysis. These findings provide evidence that low expression of PTPN12 is associated with worse prognosis and may be used as a potential prognostic biomarker in TNBC patients.

Tyrosine-Protein Phosphatase Nonreceptor Type 12 Is a Novel Prognostic Biomarker for Esophageal Squamous Cell Carcinoma

Tyrosine-protein phosphatase nonreceptor type 12 (PTPN12) is considered to be a tumor suppressor. It plays a significant role in human cancer, but its clinicopathologic and prognostic significance in esophageal squamous cell carcinoma (ESCC) has not yet been elucidated. Using Western blot, we evaluated the PTPN12 expression in 20 pairs of surgically resected esophageal tissues. The PTPN12 was detected by immunochemistry in a tissue microarray from 260 surgically resected ESCCs, of which 260 were from primary cancer sites and 60 were from matched paracancerous normal tissues. Statistical analyses were applied to test the associations between PTPN12 expression, clinicopathologic factors, and prognosis. Western blots showed that the expression level of PTPN12 was higher in normal paracancerous esophageal tissues than in ESCC tissues. By immunohistochemical analysis, high and low expression of PTPN12 was found in 62.1% and 37.9% of ESCCs, respectively. After multivariate analysis, along with pN status and tumor grade, the protein expression level of PTPN12 was an independent and significant predictive factor (p<0.001). Patients with PTPN12-high tumors had a longer disease-free survival and overall survival (p=0.002 and p=0.001, respectively), especially for those with stage II disease (both p<0.001). Our study suggests that PTPN12 protein expression is a valuable biomarker for ESCC patients. High expression of PTPN12 is associated with favorable disease-free survival and overall survival in ESCC patients.

PD08-09: PTPN12 Gene Expression Signature in Triple Negative Breast Cancer Cohort.

Abstract PTPN12 tyrosine phosphatase may play a role in tumor development/progression in triple negative breast cancer patients (TNP). The effects of PTPN12 appear to be mediated through several tyrosine kinase receptors including EGFR, HER2, and PDGFR-beta. We investigated the variability associated with PTPN12 transcript in the microarray gene expression data obtained from 105 TNP as determined by IHC for ER and PR and IHC and FISH for HER2 during our clinical molecular profiling on solid tumors. The mRNA levels of PTPN12 in our cohort was highly variable suggesting a complex genetic regulation of PTPN12 transcription in TNP patients. The highly variable nature of PTPN12 mRNA levels lead us to perform a correlation-based analysis of the transcriptome in TNP samples to gain insight into pathways and cellular processes associated with PTPN12 variation. Specifically, we quantile-normalized and performed two-dimensional hierarchical clustering of the 1000 top correlated genes with PTPN12 expression across the 105 TNP samples. We identified seven distinct gene clusters and three distinct patient subpopulations. The three distinct subtypes of TNP were comprised of low expressing PTPN12 (median log2 expression of 12.3), medium expressing PTPN12 (median log2 expression of 13.1), and high expressing PTPN12 (median log2 expression of 13.4). From the 7 gene clusters identified, 6 were positively correlated and one cluster (cluster 3) was negatively correlated with PTPN12 expression. Upon examining the genes within each cluster, we found that all contain unique set of genes related to cell proliferation, cell death, cell motility, cell cycle regulation and other cancer related pathways. From the 7 clusters, clusters 1, 6 and 7 had the highest fraction of such genes. The gene expression pattern and the gene content of these three clusters is as follows: Cluster 1 (208 genes) were genes that were highly expressed in all three TNP groups and the expression of the genes was highest in high PTPN12 expressing patients. Functional classification of genes by the DAVID bioinformatics tool at NCBI showed several genes related to cancer, including 19 MAP kinase signaling genes, and 15 genes involved in regulation of apoptosis. Cluster 6 (150 genes) contained genes showing very low expression in low-expressing TNPs, moderately expressed in medium expressing TNPs, and highly expressed in high-expressing TNPs. Cluster 6 had the highest fraction of cancer related genes including 7 mitosis and cell cycle check point genes, 6 cytoskeletal genes, 4 phosphatases and 6 kinases including KRAS, 4 DNA repair genes, 8 signal transduction genes, 3 positive regulator of apoptosis, and 22 genes involved in regulation of gene expression. Cluster 7 (166 genes) contained genes that were expressed at low level throughout the three TNP groups but the severity of under-expression was higher in TNP patients with low PTPN12 expression. In cluster 7 there were 5 genes belonging to the EGFR and FGF signaling pathways, and 6 genes involved in mitosis. It is worth noting that EGFR pathway has been postulated to crosstalk with PTPN12 protein. All together our results provide support for the involvement of PTPN12 in cancer development and highlights a promising therapeutic target for TNP patients. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD08-09.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature