Oncogenic Protein Tyrosine Phosphatases Research Articles

Deletion of PTP4A3 phosphatase in high grade serous ovarian cancer cells decreases tumorigenicity and produces marked changes in intracellular signaling pathways and cytokine release.

The oncogenic protein tyrosine phosphatase PTP4A3 is frequently overexpressed in human ovarian cancers and is associated with poor patient prognosis. PTP4A3 is thought to regulate multiple oncogenic signaling pathways, including STAT3, SRC, and ERK. The objective of this study was to generate ovarian cancer cells with genetically depleted PTP4A3; to assess their tumorigenicity; to examine their cellular phenotype; and to uncover changes in their intracellular signaling pathways and cytokine release profiles. Genetic deletion of PTP4A3 using CRISPR/Cas9 enabled the generation of individual clones derived from single cells isolated from the polyclonal knockout population. We observed a >90% depletion of PTP4A3 protein levels by Western blotting in the clonal cell lines compared to the sham transfected wildtype population. The wildtype and polyclonal knockout cell lines shared similar monolayer growth rates, while the isolated clonal populations 2B4, 3C9, and 3C12 exhibited significantly lower monolayer growth characteristics consistent with their lower PTP4A3 levels. The clonal PTP4A3 knockout cell lines also had substantially lower in vitro colony formation efficiencies compared to the wildtype cells and were less tumorigenic in vivo The clonal knockout cells were markedly less responsive to IL-6-stimulated migration in a scratch wound assay compared to the wildtype cells. Antibody microarray assays documented differences in cytokine release and intracellular phosphorylation patterns in the PTP4A3 deleted clones. Bioinformatic network analyses indicated alterations in cellular signaling nodes. These biochemical changes could ultimately form the foundation for pharmacodynamic endpoints useful for emerging anti-PTP4A3 therapeutics. Significance Statement Clones of high grade serous ovarian cancer cells were isolated in which the oncogenic phosphatase PTP4A3 was deleted using CRISPR/Cas9 methodologies. The PTP4A3 null cells exhibited loss of in vitro proliferation, colony formation, and migration, and reduced in vivo tumorigenesis. Marked differences in intracellular protein phosphorylation and cytokine release were seen. The newly developed PTP4A3 knockout cells should provide useful tools to probe the role of PTP4A3 phosphatase in ovarian cancer cell survival, tumorigenicity and cell signaling.

A pan-cancer analysis of oncogenic protein tyrosine phosphatase subfamily PTP4As

ObjectiveThe objective of this study was to conduct a comprehensive pan-cancer analysis of Protein tyrosine phosphatase 4A (PTP4As), specifically PTP4A1, PTP4A2, and PTP4A3, to investigate their aberrant expression, genomic alterations, prognostic values, and molecular functions. The aim was to evaluate the roles of PTP4As in cancer development and progression, as previous research has primarily focused on PTP4A3 and yielded inconsistent results regarding their expression in cancers. MethodsA meticulous and extensive analysis of PTP4As was performed across diverse cancer types. mRNA expression levels of PTP4A isoforms were examined, and correlations between protein expression and mRNA expression were investigated. Genomic alterations affecting PTP4As, such as amplification, were analyzed. Survival analysis was conducted to assess the prognostic values of PTP4As in different cancers. Additionally, pathway enrichment analysis was performed to identify signaling pathways and biological processes associated with PTP4A2 and PTP4A3. ResultsThe analysis revealed that PTP4A3 exhibited the most prevalent up-regulation at the mRNA level among the PTP4A isoforms. PTP4A2 mRNA expression in cancer generally displayed an up-regulated trend. However, inconsistent results were observed for PTP4A1 expression, even within the same cancer type but across different datasets, indicating the need for further investigation. The correlation between PTP4As protein expression and mRNA expression was found to be weak, indicating the complexity of their regulatory mechanisms. Genomic analysis showed that amplification was the major type of alteration affecting PTP4As, although it did not always translate into higher expression. Survival analysis revealed that high PTP4As expression was typically associated with unfavorable prognoses in several cancers, although exceptions existed. Pathway enrichment analysis unveiled novel signaling pathways and biological processes potentially influenced by PTP4As. ConclusionThe pan-cancer analysis of PTP4As provided insights into their aberrant expression, genomic alterations, prognostic values, and molecular functions. PTP4A3 exhibited the most prevalent up-regulation, while PTP4A2 showed a general up-regulated trend. Inconsistent results were observed for PTP4A1 expression, warranting further investigation. These findings contribute to our understanding of the molecular mechanisms through which PTP4As may contribute to cancer pathogenesis.

Identification of linderalactone as a natural inhibitor of SHP2 to ameliorate CCl4-induced liver fibrosis.

Liver fibrosis is characterised by the activation of hepatic stellate cells (HSCs) and matrix deposition. Accumulating evidence has revealed that the oncogenic protein tyrosine phosphatase Src homology 2 domain-containing phosphatase 2 (SHP2) acts as a therapeutic target of fibrosis. Although several SHP2 inhibitors have reached early clinical trials, there are currently no FDA-approved drugs that target SHP2. In this study, we aimed to identify novel SHP2 inhibitors from an in-house natural product library to treat liver fibrosis. Out of the screened 800 compounds, a furanogermacrane sesquiterpene, linderalactone (LIN), significantly inhibited SHP2 dephosphorylation activity in vitro. Cross-validated enzymatic assays, bio-layer interferometry (BLI) assays, and site-directed mutagenesis were used to confirm that LIN directly binds to the catalytic PTP domain of SHP2. In vivo administration of LIN significantly ameliorated carbon tetrachloride (CCl4)-induced HSC activation and liver fibrosis by inhibiting the TGFβ/Smad3 pathway. Thus, LIN or its derivatives could be considered potential therapeutic agents against SHP2-related diseases, such as liver fibrosis or NASH.

The Glycoprotein M6a Is Associated with Invasiveness and Radioresistance of Glioblastoma Stem Cells.

Systematic recurrence of glioblastoma (GB) despite surgery and chemo-radiotherapy is due to GB stem cells (GBSC), which are particularly invasive and radioresistant. Therefore, there is a need to identify new factors that might be targeted to decrease GBSC invasive capabilities as well as radioresistance. Patient-derived GBSC were used in this study to demonstrate a higher expression of the glycoprotein M6a (GPM6A) in invasive GBSC compared to non-invasive cells. In 3D invasion assays performed on primary neurospheres of GBSC, we showed that blocking GPM6A expression by siRNA significantly reduced cell invasion. We also demonstrated a high correlation of GPM6A with the oncogenic protein tyrosine phosphatase, PTPRZ1, which regulates GPM6A expression and cell invasion. The results of our study also show that GPM6A and PTPRZ1 are crucial for GBSC sphere formation. Finally, we demonstrated that targeting GPM6A or PTPRZ1 in GBSC increases the radiosensitivity of GBSC. Our results suggest that blocking GPM6A or PTPRZ1 could represent an interesting approach in the treatment of glioblastoma since it would simultaneously target proliferation, invasion, and radioresistance.

Abstract 5500: A PTP4A3 inhibitor reduces ovarian cancer dissemination and alters STAT3 and ERK activation

Abstract The objective of this study was twofold: (1) to examine the pharmacological effects of a potent, allosteric, small molecule inhibitor (KVX-053 or 7-imino-2-phenylthieno[3,2-c]pyridine-4,6(5H,7H)-dione) of the oncogenic protein tyrosine phosphatase PTP4A3, which is overexpressed in human ovarian cancer and is associated with poor patient prognosis, and (2) to define alterations in ovarian cancer cells with acquired resistance to this PTP4A3 phosphatase inhibitor. PTP4A3 promotes cell migration, survival, metabolism, angiogenesis, and tumor formation, which are mediated at least partly by PTP4A3’s ability to function as a positive signal transducer capable of activating STAT3 and ERK1/2. We found KVX-053 inhibited the migration and, at higher concentrations, the viability of chemosensitive and chemoresistant human ovarian cancer cell lines in vitro. Using an established ovarian cancer metastasis prevention model in which taxane-resistant SKOV3TRip2 cells were injected i.p. followed by four treatments with 20 mg/kg KVX-053, we observed a median survival of 68 days compared to the median survival of 58 days in the vehicle treated mice. We also observed a statistically significant reduction in the number of tumor implantation sites with KVX-053 treatment. Since drug resistance frequently occurs with targeted cancer therapies, we next generated A2780 and OVCAR4 ovarian cancer cells that were 32- and 4-fold resistant to KVX-053, respectively. This was accomplished by using stepwise increased compound exposure over a 3-4 month period to probe acquired cellular resistance changes caused by KVX-053. The resulting KVX-053-resistant cells had similar in vitro growth rates with the wildtype cells and retained resistance to KVX-053 for at least one month when grown in the absence of the small molecule inhibitor. They were not cross-resistant to paclitaxel, cisplatin or teniposide but were cross-resistant to chemical analogs of KVX-053. The KVX-053-resistant A2780 cells exhibited a 95% decrease in the activated form of STAT3, Y705 phospho-STAT3, and a 57% decrease in the phosphorylation of a second activation site S727 without any marked differences in the total STAT3 levels. In contrast, phospho-ERK1/2 and total ERK1/2 were elevated in the ovarian cancer cells with stable resistance to KVX-053. These results further confirm the in vivo activity of KVX-053 against ovarian cancer and suggest that chronic KVX-053 exposure disrupts the PTP4A3/STAT3 and PTP4A3/ERK cellular signaling pathways. Citation Format: John S. Lazo, Kelly N. Isbell, Danielle C. Llaneza, Ettore J. Rastelli, Charles N. Landen, Peter Wipf, Elizabeth R. Sharlow. A PTP4A3 inhibitor reduces ovarian cancer dissemination and alters STAT3 and ERK activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5500.

Data-Driven Computational Modeling Identifies Determinants of Glioblastoma Response to SHP2 Inhibition.

Oncogenic protein tyrosine phosphatases have long been viewed as drug targets of interest, and recently developed allosteric inhibitors of SH2 domain-containing phosphatase-2 (SHP2) have entered clinical trials. However, the ability of phosphatases to regulate many targets directly or indirectly and to both promote and antagonize oncogenic signaling may make the efficacy of phosphatase inhibition challenging to predict. Here we explore the consequences of antagonizing SHP2 in glioblastoma, a recalcitrant cancer where SHP2 has been proposed as a useful drug target. Measuring protein phosphorylation and expression in glioblastoma cells across 40 signaling pathway nodes in response to different drugs and for different oxygen tensions revealed that SHP2 antagonism has network-level, context-dependent signaling consequences that affect cell phenotypes (e.g., cell death) in unanticipated ways. To map specific signaling consequences of SHP2 antagonism to phenotypes of interest, a data-driven computational model was constructed based on the paired signaling and phenotype data. Model predictions aided in identifying three signaling processes with implications for treating glioblastoma with SHP2 inhibitors. These included PTEN-dependent DNA damage repair in response to SHP2 inhibition, AKT-mediated bypass resistance in response to chronic SHP2 inhibition, and SHP2 control of hypoxia-inducible factor expression through multiple MAPKs. Model-generated hypotheses were validated in multiple glioblastoma cell lines, in mouse tumor xenografts, and through analysis of The Cancer Genome Atlas data. Collectively, these results suggest that in glioblastoma, SHP2 inhibitors antagonize some signaling processes more effectively than existing kinase inhibitors but can also limit the efficacy of other drugs when used in combination. SIGNIFICANCE: These findings demonstrate that allosteric SHP2 inhibitors have multivariate and context-dependent effects in glioblastoma that may make them useful components of some combination therapies, but not others.

Abstract 3752: Identification of nanobodies specific for the oncogenic protein tyrosine phosphatase 4A3 (PTP4A3/PRL-3) that modulate its binding, stability, and phosphatase activity

Abstract PRL-3 is an oncogenic phosphatase across multiple cancer types, including colon, ovarian, melanoma, breast, and leukemia. While there is increasing interest in developing PRL-3 inhibitors for use in cancer research and treatment, there have been several issues in designing small molecule inhibitors for PRL-3, such as a shallow, negative charged active site, homology between the PRL-3 active site and other protein tyrosine phosphatases, and a high degree of overall homology between PRL-3 and family members PRL-1 and PRL-2. Nanobodies have recently emerged as an immensely useful research tool and show promise as a cancer therapeutic. Nanobodies are small, at ~15kD and lack light chains, allowing them to fit into spaces on target proteins that conventional antibodies cannot normally reach. Other advantages of nanobodies include their stability under stringent conditions, lack of immunogenicity, ability to permeate the cell, and a high specificity and affinity for their antigens. Using full-length PRL-3 protein as an immunogen in alpaca, I used phage display technology to identify alpaca nanobodies that had high affinity for PRL-3 through subtractive panning. I identified 18 unique nanobodies through sequencing; 14 of these were able to be expressed in bacteria and purified. The binding specificity of the nanobodies to PRL-3 over PRL-1 and PRL-2 was determined through an indirect ELISA assay, which showed that that 12 out of 14 anti-PRL-3 nanobodies bound PRL-3 significantly better than PRL-1 or PRL-2 (~25X times higher binding affinity to PRL-3, p<0.0001). I also identified several nanobodies that stabilize PRL-3 structure using a Differential Scanning Fluorescence (DSF) assays to analyze shifts in the melting temperature of PRL-3 following binding, with the ultimate goal of developing PRL-3/nanobody crystal structures to identify nanobody binding sites and find PRL-3 active site binders. Additionally, I have found the 6X-Histidine-tagged nanobodies are useful for PRL-3 western blot, immunoprecipitation, and immunofluorescence, and mCherry-tagged nanobodies (chromobodies) allow for analysis of PRL-3 trafficking. Nanobodies were also tested for their ability to inhibit the phosphatase activity of PRL-3, using both purified protein and functional in vitro assays. Preliminary results showed that several nanobodies decreased PRL-3 phosphatase activity, and cell-based assays to assess how nanobody expression affects PRL-3 function and cellular phenotype are ongoing. Overall, we have developed. both a novel research tool that can be used to gain insight into the structure and function of PRL-3 in normal and cancer cells, and a potentially new biologic inhibitor of PRL-3 that functions with high specificity and potency. Citation Format: Caroline N. Smith, K. Martin Chow, Louis B. Hersh, Jessica S. Blackburn. Identification of nanobodies specific for the oncogenic protein tyrosine phosphatase 4A3 (PTP4A3/PRL-3) that modulate its binding, stability, and phosphatase activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3752.

Next-Generation Cell-Active Inhibitors of the Undrugged Oncogenic PTP4A3 Phosphatase.

Oncogenic protein tyrosine phosphatases (PTPs) are overexpressed in numerous human cancers but they have been challenging pharmacological targets. The emblematic oncogenic PTP4A tyrosine phosphatase family regulates many fundamental malignant processes. 7-Imino-2-phenylthieno[3,2-c]pyridine-4,6(5H,7H)-dione (JMS-053) is a novel, potent, and selective PTP4A inhibitor but its mechanism of action has not been fully elucidated, nor has the chemotype been fully investigated. Because tyrosine phosphatases are notoriously susceptible to oxidation, we interrogated JMS-053 and three newly synthesized analogs with specific attention on the role of oxidation. JMS-053 and its three analogs were potent in vitro PTP4A3 inhibitors, but 7-imino-5-methyl-2-phenylthieno[3,2-c]pyridine-4,6(5H,7H)-dione (NRT-870-59) appeared unique among the thienopyridinediones with respect to its inhibitory specificity for PTP4A3 versus both a PTP4A3 A111S mutant and an oncogenic dual specificity tyrosine phosphatase, CDC25B. Like JMS-053, NRT-870-59 was a reversible PTP4A3 inhibitor. All of the thienopyridinediones retained cytotoxicity against human ovarian and breast cancer cells grown as pathologically relevant three-dimensional spheroids. Inhibition of cancer cell colony formation by NRT-870-59, like JMS-053, required PTP4A3 expression. JMS-053 failed to generate significant detectable reactive oxygen species in vitro or in cancer cells. Mass spectrometry results indicated no disulfide bond formation or oxidation of the catalytic Cys104 after in vitro incubation of PTP4A3 with JMS-053 or NRT-870-59. Gene expression profiling of cancer cells exposed to JMS-053 phenocopied many of the changes seen with the loss of PTP4A3 and did not indicate oxidative stress. These data demonstrate that PTP4A phosphatases can be selectively targeted with small molecules that lack prominent reactive oxygen species generation and encourage further studies of this chemotype. SIGNIFICANCE STATEMENT: Protein tyrosine phosphatases are emerging as important contributors to human cancers. We report on a new class of reversible protein phosphatase small molecule inhibitors that are cytotoxic to human ovarian and breast cancer cells, do not generate significant reactive oxygen species in vitro and in cells, and could be valuable lead molecules for future studies of PTP4A phosphatases.

Identification of an allosteric benzothiazolopyrimidone inhibitor of the oncogenic protein tyrosine phosphatase SHP2

The PTPN11 oncogene encodes the cytoplasmic protein tyrosine phosphatase SHP2, which, through its role in multiple signaling pathways, promotes the progression of hematological malignancies and other cancers. Here, we employ high-throughput screening to discover a lead chemical scaffold, the benzothiazolopyrimidones, that allosterically inhibits this oncogenic phosphatase by simultaneously engaging the C-SH2 and PTP domains. We improved our lead to generate an analogue that better suppresses SHP2 activity in vitro. Suppression of Erk phopsphorylation by the lead compound is also consistent with SHP2 inhibition in AML cells. Our findings provide an alternative starting point for therapeutic intervention and will catalyze investigations into the relationship between SHP2 conformational regulation, activity, and disease progression.

Targeting a cryptic allosteric site for selective inhibition of the oncogenic protein tyrosine phosphatase Shp2.

Protein tyrosine phosphatases (PTPs) have been the subject of considerable pharmaceutical-design efforts because of the ubiquitous connections between misregulation of PTP activity and human disease. PTP-inhibitor discovery has been hampered, however, by the difficulty in identifying cell-permeable compounds that can selectively target PTP active sites, and no PTP inhibitors have progressed to the clinic. The identification of allosteric sites on target PTPs therefore represents a potentially attractive solution to the druggability problem of PTPs. Here we report that the oncogenic PTP Shp2 contains an allosteric-inhibition site that renders the enzyme sensitive to potent and selective inhibition by cell-permeable biarsenical compounds. Because Shp2 contains no canonical tetracysteine biarsenical-binding motif, the enzyme’s inhibitor-binding site is not readily predictable from its primary or three-dimensional structure. Intriguingly, however, Shp2’s PTP domain does contain a cysteine residue (C333) at a position that is removed from the active site and is occupied by proline in other classical PTPs. We show that Shp2’s unusual cysteine residue constitutes part of a Shp2-specific allosteric-inhibition site, and that Shp2’s sensitivity to biarsenicals is dependent on the presence of the naturally occurring C333. The determinative role of this residue in conferring inhibitor sensitivity is surprising because C333’s side chain is inaccessible to solvent in Shp2 crystal structures. The discovery of this cryptic Shp2 allosteric site may provide a means for targeting Shp2 activity with high specificity and suggests that buried-yet-targetable allosteric sites could be similarly uncovered in other protein families.

Identification of Cryptotanshinone as an Inhibitor of Oncogenic Protein Tyrosine Phosphatase SHP2 (PTPN11)

Activating mutations of PTPN11 (encoding the SHP2 phosphatase) are associated with Noonan syndrome, childhood leukemias, and sporadic solid tumors. Virtual screening combined with experimental assays was performed to identify inhibitors of SHP2 from a database of natural products. This effort led to the identification of cryptotanshinone as an inhibitor of SHP2. Cryptotanshinone inhibited SHP2 with an IC50 of 22.50 μM. Fluorescence titration experiments confirmed that it directly bound to SHP2. Enzymatic kinetic analyses showed that cryptotanshinone was a mixed-type and irreversible inhibitor. This drug was further verified for its ability to block SHP2-mediated cell signaling and cellular functions. Furthermore, mouse myeloid progenitors and patient leukemic cells with the activating mutation E76K in PTPN11 were found to be sensitive to this inhibitor. Since cryptotanshinone is used to treat cardiovascular diseases in Asian countries, this drug has a potential to be used directly or to be further developed to treat PTPN11-associated malignancies.

Impact of Oncogenic Protein Tyrosine Phosphatases in Cancer

Protein tyrosine phosphatases (PTPs) constitute a large family of enzymes that can exert both positive and negative effects on signaling pathways. They play dominant roles in setting the levels of intracellular phosphorylation downstream of many receptors including receptor tyrosine kinases and G protein-coupled receptors. As observed with kinases, deregulation of PTP activity can also contribute to cancer. This review will examine a broad array of PTP family members that positively affect oncogenesis in human cancer tissues. We will describe the PTP family, their biological significance in oncology, and how recent progress is being made to more effectively target specific PTPs. Finally, we will discuss the therapeutic implications of targeting these oncogenic PTPs in cancer.

Prenylation of oncogenic human PTP(CAAX) protein tyrosine phosphatases.

Many isoprenylated proteins are known to participate in signal transduction, but not all have been identified. Using an in vitro prenylation screen, two human cDNAs (PTP(CAAXI) and PTP(CAAX2)) homologous to the rat PRL-1 and human OV-1 protein tyrosine phosphatase genes were identified. PTP(CAAXI) and PTP(CAAX2) were farnesylated in vitro by mammalian farnesyl:protein transferase, and epitope-tagged PTP(CAAX2) was prenylated in epithelial cells. Overexpression of PTP(CAAXI) and PTP(CAAX2) in epithelial cells caused a transformed phenotype in culture and tumor growth in nude mice. Thus, PTP(CAAXI) and PTP(CAAX2) represent a novel class of isoprenylated, oncogenic protein tyrosine phosphatases.