PTP4A3 Research Articles

Abstract 1459: PTP4A3 phosphatase is a novel drug target for epithelial ovarian cancer

Abstract High grade serous ovarian cancer (HGSOC) is the deadliest epithelial ovarian cancer (EOC) variant, with a bleak outcome that kills >14,000 women annually in the US. In past four decades, first line therapies have changed only modestly. While dramatic advances in targeted- and immune-based therapies have been seen in some solid tumors, they have not been reported in patients with HGSOC. Recurrent and chemoresistant HGSOC is common. Therefore, it is imperative that new HGSOC drug targets be interrogated. The Protein Tyrosine Phosphatases of Regenerating Liver (PRL or PTP4A) family is significantly upregulated in a variety of tumors, including HGSOC. A bioinformatic analysis of the ovarian cancer using the KMplot Survival Database revealed that high PTP4A3 expression was associated with poor survival in EOC (p<0.0001, hazard ratio (HR)=1.35). In HGSOC early stage disease, PTP4A3 expression levels were also correlated with survival (stage I/II- p=0.0013, HR=3.2), with low expressing patients surviving twice as long as high expressing patients (upper quartile survival in low expression cohort = 81.2 months compared to high expression cohort = 44.3 months). This survival difference appeared independent of myc amplification and was more pronounced in advanced disease (stage III/IV). qPCR profiling of established HGSOC cell lines, including Kuramochi, COV362 and OVCAR4, showed 5-20 fold increases in PTP4A3 gene expression versus nonmalignant control cells. A potent, specific, allosteric small molecule inhibitor of PTP4A3, KVX-053, markedly reduced the growth and survival of chemoresistant and chemosensitive EOC cell lines independent of their BRCA or p53 status. Moreover, treatment of the HGSOC OVCAR4 cell line with KVX-053 (1 μM) inhibited the oncogenic Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway through loss of phosphorylation of STAT3. Evaluation of a series of next-generation KVX-053-based analogs including chemodegraders yielded chemotypes with enhanced cytotoxic effects. These results demonstrate a clear path for the development of the PTP4A family of tyrosine phosphatases as a novel therapeutic target in a HGSOC. Citation Format: John Robert Cornelison, Ettore J. Rastelli, Duncan J. Hart, Anna J. Mendelson, Elizabeth R. Sharlow, Peter Wipf, John S. Lazo. PTP4A3 phosphatase is a novel drug target for epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1459.

PRL-2 phosphatase is required for vascular morphogenesis and angiogenic signaling

Protein tyrosine phosphatases are essential modulators of angiogenesis and have been identified as novel therapeutic targets in cancer and anti-angiogenesis. The roles of atypical Phosphatase of Regenerative Liver (PRL) phosphatases in this context remain poorly understood. Here, we investigate the biological function of PRL phosphatases in developmental angiogenesis in the postnatal mouse retina and in cell culture. We show that endothelial cells in the retina express PRL-2 encoded by the Ptp4a2 gene, and that inducible endothelial and global Ptp4a2 mutant mice exhibit defective retinal vascular outgrowth, arteriovenous differentiation, and sprouting angiogenesis. Mechanistically, PTP4A2 deletion limits angiogenesis by inhibiting endothelial cell migration and the VEGF-A, DLL-4/NOTCH-1 signaling pathway. This study reveals the importance of PRL-2 as a modulator of vascular development.

Protein tyrosine phosphatase 4A3 (PTP4A3/PRL-3) promotes the aggressiveness of human uveal melanoma through dephosphorylation of CRMP2

Uveal melanoma (UM) is an aggressive tumor in which approximately 50% of patients develop metastasis. Expression of the PTP4A3 gene, encoding a phosphatase, is predictive of poor patient survival. PTP4A3 expression in UM cells increases their migration in vitro and invasiveness in vivo. Here, we show that CRMP2 is mostly dephosphorylated on T514 in PTP4A3 expressing cells. We also demonstrate that inhibition of CRMP2 expression in UM cells expressing PTP4A3 increases their migration in vitro and invasiveness in vivo. This phenotype is accompanied by modifications of the actin microfilament network, with shortened filaments, whereas cells with a inactive mutant of the phosphatase do not show the same behavior. In addition, we showed that the cell cytoplasm becomes stiffer when CRMP2 is downregulated or PTP4A3 is expressed. Our results suggest that PTP4A3 acts upstream of CRMP2 in UM cells to enhance their migration and invasiveness and that a low level of CRMP2 in tumors is predictive of poor patient survival.

Abstract 2365: A chemical genetics approach identifies PTP4A3 as a regulator of colon cancer cell adhesion

Abstract Dysregulation of the tightly controlled protein phosphorylation networks that govern cellular behavior causes cancer. The membrane-associated, intracellular protein tyrosine phosphatase PTP4A3 is overexpressed in human colorectal cancer and contributes to cell migration and invasion. To interrogate further the role of PTP4A3 in colorectal cancer cell migration and invasion, we deleted the Ptp4a3 gene from murine colorectal tumor cells. The resulting PTP4A3-/- cells exhibited impaired colony formation, spheroid formation, migration, and adherence compared to the isogenic PTP4A3fl/fl cells. We replicated these phenotypic changes using the new small molecule allosteric PTP4A3 inhibitor, JMS-053. A related structure, JMS-038, which lacked phosphatase inhibition, displayed no cellular activity. Reduction in cell viability and colony formation by JMS-053 occurred in both mouse and human colorectal cell lines and required PTP4A3 expression. Ptp4a3 deletion increased the expression of extracellular matrix and adhesion genes, including the tumor suppressor Emilin 1. JMS-053 also increased Emilin 1 gene expression. Moreover, the human TCGA genomic database revealed colorectal tumors with high Ptp4a3 expression had low Emilin1 expression. These chemical and biological reagents reveal a previously unknown communication between the intracellular PTP4A3 phosphatase and the extracellular matrix and support efforts to pharmacologically target PTP4A3. Citation Format: Kelley E. McQueeney, Joseph M. Salamoun, Jennifer Ahn, Paula Pekic, Isabella K. Blanco, Heather Struckman, Elizabeth R. Sharlow, Peter Wipf, John S. Lazo. A chemical genetics approach identifies PTP4A3 as a regulator of colon cancer cell adhesion [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 2365.

A chemical genetics approach identifies PTP4A3 as a regulator of colon cancer cell adhesion.

Dysregulation of the tightly controlled protein phosphorylation networks that govern cellular behavior causes cancer. The membrane-associated, intracellular protein tyrosine phosphatase PTP4A3 is overexpressed in human colorectal cancer and contributes to cell migration and invasion. To interrogate further the role of PTP4A3 in colorectal cancer cell migration and invasion, we deleted the Ptp4a3 gene from murine colorectal tumor cells. The resulting PTP4A3-/- cells exhibited impaired colony formation, spheroid formation, migration, and adherence compared with the paired PTP4A3fl/fl cells. We replicated these phenotypic changes using the new small-molecule, allosteric PTP4A3 inhibitor JMS-053. A related structure, JMS-038, which lacked phosphatase inhibition, displayed no cellular activity. Reduction in cell viability and colony formation by JMS-053 occurred in both mouse and human colorectal cell lines and required PTP4A3 expression. Ptp4a3 deletion increased the expression of extracellular matrix (ECM) and adhesion genes, including the tumor suppressor Emilin 1. JMS-053 also increased Emilin 1 gene expression. Moreover, The Cancer Genome Atlas genomic database revealed human colorectal tumors with high Ptp4a3 expression had low Emilin 1 expression. These chemical and biologic reagents reveal a previously unknown communication between the intracellular PTP4A3 phosphatase and the ECM and support efforts to pharmacologically target PTP4A3.-McQueeney, K. E., Salamoun, J. M., Ahn J. G., Pekic, P., Blanco, I. K., Struckman, H. L., Sharlow, E. R., Wipf, P., Lazo, J. S. A chemical genetics approach identifies PTP4A3 as a regulator of colon cancer cell adhesion.

Abstract 1220: Coordinated chemical-genetics approach identifies PTP4A3-mediated regulation of colon cancer cell migration and extracellular matrix interactions

Abstract Aberrant regulation of protein phosphorylation is an exceedingly common driver of human cancers. It is notable that we understand much less about the role of protein tyrosine phosphatases in human malignancies compared to tyrosine kinases. The membrane-associated, intracellular, protein tyrosine PTP4A3 is highly overexpressed in multiple tumor types including colorectal cancer and has been associated with tumor metastases. We have, therefore, investigated the role of PTP4A3 in colon cancer migration and invasion. The Ptp4a3 gene was expunged from colon tumor cells derived from Ptp4a3flox/flox mice and the resulting cells exhibited impaired migration, invasion, and colony formation compared to the wildtype isogenic cells. We characterized a potent, selective, and noncompetitive small molecule inhibitor of PTP4A3, JMS-631-053, which also disrupted colon cancer cell migration, invasion, and colony formation. PTP4A3 deletion increased the expression of extracellular matrix and adhesion genes, including the tumor suppressor Emilin 1. Expression of these extracellular matrix genes is mutually exclusive with PTP4A3 expression in tumors derived from patients with colorectal cancer. These chemical and biological reagents reveal a previously unknown communication between the intracellular PTP4A3 phosphatase and the extracellular matrix and support continued efforts to pharmacologically target PTP4A3 for cancer therapy. Citation Format: Kelley E. McQueeney, Joseph M. Salamoun, Isabella K. Blanco, Paula Pekic, Jennifer Ahn, Elizabeth R. Sharlow, Peter Wipf, John S. Lazo. Coordinated chemical-genetics approach identifies PTP4A3-mediated regulation of colon cancer cell migration and extracellular matrix interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1220. doi:10.1158/1538-7445.AM2017-1220

PRL-3/PTP4A3 phosphatase regulates integrin β1 in adhesion structures during migration of human ocular melanoma cells

In a previous transcriptomic analysis of 63 ocular melanomas of the uvea, we found that expression of the PRL-3/PTP4A3 gene, encoding a phosphatase that is anchored to the plasma membrane, was associated with the risk of metastasis, and a poor prognosis. We also showed that PRL-3 overexpression in OCM-1 ocular melanoma cells significantly increased cell migration in vitro and invasiveness in vivo, suggesting a direct role for PRL-3 in the metastatic spreading of uveal melanoma. Here, we aimed to identify PRL-3 substrates at the plasma membrane involved in adhesion to the extracellular matrix. We focused on integrin β1, which is the most highly expressed integrin in our cohort of uveal melanomas.We show that preventing PRL-3 anchorage to the plasma membrane i) abolishes PRL-3-induced migration in OCM-1 cells, ii) specifically enhances the spreading of OCM-1 cells overexpressing PRL-3, and iii) favors the maturation of large focal adhesions (FAs) containing integrin β1 on collagen I. Knockdown experiments confirmed integrin β1 involvement in PRL3-induced migration. We identified interactions between PRL-3 and integrin β1, as well as with FAK P-Y397, an auto-activated form of Focal Adhesion Kinase found in FAs. We also show that integrin β1 may be dephosphorylated by PRL-3 in its intracytoplasmic S/T region, an important motif for integrin-mediated cell adhesion. Finally, we observed that PRL-3 regulated the clustering of integrin β1 in FAs on collagen I but not on fibronectin.This work identifies PRL-3 as a new regulator of cell adhesion structures to the extracellular matrix, and further supports PRL-3 as a key actor of metastasis in uveal melanoma, of which molecular mechanisms are still poorly understood.

2054 - NOTCH2 and PTP4A3 gene copy number alterations are associated with prognosis and support novel therapeutic strategies for malignant pleural mesothelioma

2054 - NOTCH2 and PTP4A3 gene copy number alterations are associated with prognosis and support novel therapeutic strategies for malignant pleural mesothelioma

Phosphatase PTP4A3 Promotes Triple-Negative Breast Cancer Growth and Predicts Poor Patient Survival.

Triple-negative breast cancer (TNBC) has the worst prognosis of all breast cancers, and women diagnosed with TNBC currently lack targeted treatment options. To identify novel targets for TNBC, we evaluated phosphatase expression in breast tumors and characterized their contributions to in vitro and in vivo growth of TNBC. Using Affymetrix microarray analysis of 102 breast cancers, we identified 146 phosphatases that were significantly differentially expressed in TNBC compared with estrogen receptor (ER)-positive tumors. Of these, 19 phosphatases were upregulated (0.66-fold; FDR = 0.05) in TNBC compared with ER-positive breast cancers. We knocked down 17 overexpressed phosphatases in four triple-negative and four ER-positive breast cancer lines using specific siRNAs and found that depletion of six of these phosphatases significantly reduced growth and anchorage-independent growth of TNBC cells to a greater extent than ER-positive cell lines. Further analysis of the phosphatase PTP4A3 (also known as PRL-3) demonstrated its requirement for G1-S cell-cycle progression in all breast cancer cells, but PTP4A3 regulated apoptosis selectively in TNBC cells. In addition, PTP4A3 inhibition reduced the growth of TNBC tumors in vivo Moreover, in silico analysis revealed the PTP4A3 gene to be amplified in 29% of basal-like breast cancers, and high expression of PTP4A3 could serve as an independent prognostic indicator for worse overall survival. Collectively, these studies define the importance of phosphatase overexpression in TNBC and lay the foundation for the development of new targeted therapies directed against phosphatases or their respective signaling pathways for TNBC patients. Cancer Res; 76(7); 1942-53. ©2016 AACR.

Abstract 4604: PTP4A3 is oncogenic and modulates triple negative breast cancer growth

Abstract Triple negative breast cancers (TNBCs) are among the most aggressive and heterogenous breast cancers characterized by a high propensity to invade, metastasize, and relapse; the exact mechanisms by which TNBCs mediate progression is unclear and thus, new insights into the biology of TNBC are desperately needed. We have developed a novel anti-cancer compound (AMPI-109) that is selectively efficacious against TNBC cell-lines. We conducted a genome-wide functional genomic shRNA screen to identify modifiers of AMPI-109 sensitivity and identified PTP4A3 (P3), a protein tyrosine phosphatase implicated in invasive, node positive TNBC tumors and associates with poor survival outcome, as a mediator of AMPI-109 action. In silico modeling predicted that AMPI-109 binds the catalytic site of P3 with favorable energetics. To investigate whether AMPI-109 could impact the activity of P3, we performed an in vitro phosphatase assay and demonstrated that AMPI-109 impaired the catalytic activity of P3. To investigate an oncogenic role of P3 in TNBC, we conducted stable lentiviral knockdown of P3 (ShP3) in BT-20 TNBC cells and demonstrated that loss of P3 leads to substantial growth inhibition (48%, p = 0.0055), similar in magnitude to AMPI-109 at 100 nM dose (66%, p = 0.00005). AMPI-109 also decreased P3 mRNA and protein levels. Accordingly, we blocked de novo protein synthesis and observed enhanced P3 protein degradation in the presences of AMPI-109. This degradation was reversible in the presence of the proteasome inhibitor MG-132, suggesting that AMPI-109 may induce P3 degradation through a proteasome-dependent mechanism. Functionally, high expression of P3 has been implicated in driving metastatic phenotypes in colorectal cancer but its role in TNBC cell migration has not been investigated. Wound-healing assays demonstrated that both AMPI-109 and ShP3 significantly impeded the migratory capacity of BT-20 cells (AMPI-109: 85% reduction, shP3: 80% reduction). AMPI-109 and ShP3 also conferred a higher rate of apoptosis induction as measured by activation of pro-apoptotic caspases-3 and 7 suggesting that the growth inhibitory phenotypes observed could potentially be attributed to increases in apoptotic cell death. To determine how P3 mechanistically exerts its oncogenic effect in the context of modulating cell signaling pathways, we carried out co-expression analysis of mRNAs that tracked with P3 expression across a suite of TNBC cell lines. We identified TRAPPC9, an activator of the NF-kappa B pathway, as correlating positively with P3 (r =0.652). Collectively, our results identify P3 as a target of AMPI-109 and that P3 has an oncogenic role in TNBC. Future studies will further characterize the relationship between AMPI-109, P3 and the NF-kappa B pathway. Furthermore, because of the specificity of AMPI-109 for TNBC cells, it may represent a new tool for understanding the molecular basis underlying the aggressive nature of this disease. Citation Format: Hamid H. Gari, Rahul Ray, Scott Lucia, Christopher C. Porter, Christy M. Gearheart, Susan Fosmire, Gregory D. DeGala, Zeying Fan, Yuanbin Ru, Ann D. Thor, James R. Lambert. PTP4A3 is oncogenic and modulates triple negative breast cancer growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4604. doi:10.1158/1538-7445.AM2014-4604

Deletion of Ptp4a3 reduces clonogenicity and tumor-initiation ability of colitis-associated cancer cells in mice

The PTP4A3 gene is highly expressed in human colon cancer and often associates with enhanced metastatic potential. Genetic disruption of the mouse Ptp4a3 gene reduces the frequency of colon tumor formation in mice treated in a colitis-associated cancer model. In the current study, we have examined the role of Ptp4a3 in the tumor-initiating cell population of mouse colon tumors using an in vitro culture system. Tumors generated in vivo following AOM/DSS treatment were isolated, dissociated, and expanded on a feeder layer resulting in a CD133+ cell population, which expressed high levels of Ptp4a3. Tumor cells deficient for Ptp4a3 exhibited reduced clonogenicity and growth potential relative to WT cells as determined by limiting dilution analysis. Importantly, expanded tumor cells from WT mice readily formed secondary tumors when transplanted into nude mice, while tumor cells without Ptp4a3 expression failed to form secondary tumors and thus were not tumorigenic. These results demonstrate that Ptp4a3 contributes to the malignant phenotype of tumor-initiating cells and supports its role as a potential therapeutic target to inhibit tumor self-renewal and metastasis.

Effect of protein-tyrosine phosphatase 4A3 by small interfering RNA on the proliferation of lung cancer

Previous studies on PTP4A3 mainly focused on tumor metastasis due to the close relationship between the overexpression of lung cancer and metastasis. However, the role of PTP4A3 in the proliferation of tumor still has remained unclear. To investigate the role of PTP4A3 in cell growth of lung cancer, we constructed PTP4A3-siRNA expressing lentivirus and infected human lung cancer H1299 cells, and then examined the inhibitory effect of PTP4A3 in vitro. The levels of PTP4A3 mRNA and protein in H1299 cells decreased after PTP4A3-siRNA lentivirus infection. The growth and colony formation of the infected cells were also inhibited, indicating that PTP4A3 gene is closely associated with the proliferation of H1299 cells. In addition, after PTP4A3 specific siRNA lentivirus infection, it was notable that whilst H1299 cells in G1 phase apparently reduced, both of H1299 cells in G2/M phase and the cell apoptosis increased significantly. This finding indicated the close relationship between PTP4A3 gene and apoptosis in the H1299 cells. These results come to their conclusion that PTP4A3 plays an important role in the growth of lung cancer cells. PTP4A3 may be considered as a valuable target for anti-tumor therapeutic strategies.

Methylated Retroviral Integration Mutagenesis (MRIM) in Mice and Comparative Analysis in Human AML Identify Reduced PTP4A3 Expression As a Prognostic Indicator

Abstract 746Acute myeloid leukemia (AML) is a complex disease driven by multiple cytogenetic abnormalities, aberrant expression levels and mutations of different genes. Frequent occurrence of chromosomal deletions suggests that haplo-insufficiencies contribute to the pathogenesis of AML. However, because deleted regions often harbor numerous genes, it remains difficult to pin point critical haplo-insufficient genes (HIGs) involved in the pathogenesis of AML. Retroviral insertion mutagenesis in mouse models has been used to discover novel genes involved in the development of different types of cancer. Only a small minority of identified genes in these screens has been classified as tumor suppressor genes or HIGs, based on disruption of coding sequences by proviral integrations. Gene therapy studies using murine leukemia virus based vectors have shown that epigenetic changes of long terminal repeats (LTRs) of integrated proviruses often result in silencing of therapeutic genes, and that preventing methylation of the CpG islands within LTRs overcomes this problem. Based on these observations, we hypothesized that methylation of viral sequences not only results in silencing of retroviral genes themselves but may also affect host genes located proximal to proviral integrations. Methylated LTRs located in proximity of promoter regions may thus identify genes that are deregulated leading to haplo-insufficiency.To identify novel HIGs, genomic DNA of 6 murine leukemias induced by the Graffi 1.4 murine leukemia virus, was isolated and digested. Subsequently, methylated fragments were immunoprecipitated using methylated DNA immunoprecipitation (MeDIP), followed by inverse PCR to amplify fragments flanking methylated viral integrations (mVIS). Amplified fragments were labeled, fragmented and hybridized on murine promoter DNA arrays. DNA methylated viral integration sites were detected using a novel algorithm, hypergeometric analysis of tiling arrays (HAT) [1]. All integrations were confirmed by directed PCR and Sanger sequencing. Next, their methylation status was confirmed with methylation sensitive restriction analysis. We refer to this strategy as Methylated Virus Integration Mutagenesis (MRIM). In the murine leukemias, expression levels of genes identified by MRIM, relative to Tbp (TATA box binding protein) expression, were determined by quantitative PCR (qPCR). Survival analysis was performed in a cohort of 454 de novo AML patients under the age of 60 using a Cox-regression model. For this analysis, MAS5 normalised, log2 transformed PTP4A3 gene expression levels were used, determined by gene expression profiling.Using MRIM, 6 genes that were flanked by a methylated LTR were identified. Of these genes, Ptp4a3 was transcriptionally down regulated relative to normal bone marrow. Ptp4a3 encodes a tyrosine phosphatase that is implicated in Flt3 signaling. Studies in a human AML patient cohort under the age of 60 (n=454) revealed that PTP4A3 expression is negatively correlated with prognostic outcome, both for overall survival (p-value <0.0001, HR = 1.269) and event-free survival (p-value <0.0001, HR = 1.261). Additional analysis showed that this negative correlation with survival was independent of other prognostic parameters as age, white blood cell count, cytogenetic risk and mutation status of NPM1, CEBPA and internal tandem duplication of FLT3 (FLT3ITD).In conclusion, by mapping DNA methylated viral integration sites in murine leukemias induced by retroviral integration mutagenesis (MRIM), followed by expression level analysis, we identified Ptp4a3 as a candidate HIG. PTP4A3 has recently been proposed to have a role in drug-resistance in AMLs with FLT3ITD. This finding, together with the observation that high PTP4A3 expression negatively correlates with prognostic outcome, marks PTP4A3 as a potential therapeutic target in AML.1. E. Taskesen et al., HAT: hypergeometric analysis of tiling-arrays with application to promoter-GeneChip data. BMC Bioinformatics, 2010. 11: p. 275. Disclosures:No relevant conflicts of interest to declare.