Intrinsic Phosphatase Research Articles

Abstract 2440: Investigating the role of Eya3 in the regulation of NF-kB signaling and tumor progression in TNBC

Abstract Triple Negative Breast Cancer (TNBC), characterized by low/absent expression of the estrogen receptor and the HER2 growth factor receptor, has high rates of metastasis and an overall poor prognosis largely due to a lack of targeted therapeutic options. The Eyes absent (Eya) family of proteins are transcriptional cofactors with both intrinsic tyrosine phosphatase and associated serine/threonine phosphatase activities. In addition to developmental functions, Eyas have been shown to play a role in promoting many hallmarks of cancer. Published work from our laboratory demonstrated that Eya3 is capable of upregulating expression of PD-L1 in TNBC tumors thereby suppressing anti-tumor CD8+ T-cell responses, and preliminary data suggests that Eya3 may also regulate the expression of various cytokines in this context. In tumor cells, innate immune signaling pathways, such as NF-kB, have been shown to promote tumor progression, immune evasion, and metastasis in many distinct tumor types. We hypothesize that, in addition to regulating CD8+ T-cell responses, Eya3 regulates innate immune signaling in TNBC cells to facilitate enhanced primary tumor growth and metastasis. Preliminary findings suggest that Eya3 is a key mediator of tumor progression and metastasis in TNBC and a strong regulator of NF-kB signaling in this context. Further exploration is warranted to determine the mechanism through which Eya3 regulates these phenotypes, whether Eya3-mediated NF-kB activation is a major contributor to the pro-metastatic phenotype of Eya3, and whether Eya3 could serve as a therapeutic target for preventing or treating disseminated TNBC. Citation Format: Connor J. Hughes, Rebecca Mallo, Kaiah Fields, Hengbo Zhou, Annika Gustafson, Deguang Kong, Jill Slansky, Rui Zhao, Heide L. Ford. Investigating the role of Eya3 in the regulation of NF-kB signaling and tumor progression in TNBC [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 2440.

Abstract 2942: EYA promotes proliferation through up-regulation of cyclin D1

Abstract In Drosophila, eye development is coordinately regulated by the Retinal Determination Gene Network (RDGN), mainly consisting of dachshund (dac), eyes absent (eya), and sin oculis (so). The mammalian homologues are Dach, Eya, and Six, respectively. RDGN, although best known for its role in eye specification, is essential for the development of many organs including retinal, kidney, brain, and limb in both flies and mammals. Molecular mechanism studies support a model that Six functions as a DNA-binding factor and Dach/Eya are transcription co-factors. Eya was first characterized as a nuclear factor, interacting with Six and Dach to regulate gene expression in a context-dependent manner. Eya encodes intrinsic protein tyrosine phosphatase activity. Recently, altered expression of the DACH1/Eya/Six complex has been observed in human tumors. EYA and SIX genes are up-regulated and DACH genes are down-regulated. EYA expression was enhanced in breast, ovary and malignant peripheral nerve sheath tumors. To elucidate the function of Eya in human breast cancer and identify the down-stream targets, we established several human breast cancer cell lines (SKBr3, MDA-MB-453 and BT474) stably expressing Eya1 or phosphatase-defective mutant Eya1D327A. In all three cell lines, expression of Eya1 promoted contact-dependent and contact-independent (soft agar assay) growth. MTT assays and growth curves (cell counting) demonstrated enhanced cellular proliferation upon expression of Eya1. In contrast, cells expressing phosphatase-defective mutant Eya1D327A repressed growth were observed. Western blot analysis demonstrated increased abundance of cell Cyclin D1 in EYA1 expressing cells. Quantitative RT-PCR demonstrated about 3-fold increase of cyclin D1 mRNA abundance in MDA-MB453 cells stably expressing wild type Eya1, but not phosphatase-defective EYA1 mutant. EYA1 induced the human cyclin D1 promoter activity evaluated by luciferase assay and the EYA1 phosphatase activity was required. Immunoprecipitation studies demonstrated association of Eya1 with endogenous Six1. As EYA does not have DNA binding ability and SIX1 is known to binds to the cyclin D1 promoter, we hypothesized model in which EYA1 up-regulates SIX1, and thus a EYA1/SIX1 complex activates Cyclin D1. Lentiviral shRNAs targeting human SIX1 or cyclin D1 were transduced into EYA1 over-expressing cells respectively. The growth curve, MTT assays and clone formation results indicated the pro-proliferate effect of EYA1 was abolished by knock down the expression of either cyclin D1 or Six1. Cyclin D1 abundance was reduced by shSIX1. Taken together, our study demonstrated that cyclin D1 is a key target of EYA1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2942. doi:10.1158/1538-7445.AM2011-2942

Abstract 3932: Loss of PTEN sensitizes cells to DNA damaging agents in a RAD51-independent manner

Abstract The PTEN tumor suppressor gene is frequently mutated or deleted in many cancers. While the role of PTEN as an inhibitor of the PI3K/Akt signaling pathway via its intrinsic lipid phosphatase activity has been well-studied, recent data suggest that loss of PTEN contributes to genomic instability through a PI3K/Akt-independent pathway via down-regulation of E2F1-dependent transactivation of Rad51, a key mediator of homologous recombination (HR). Cells with dysfunctional HR are sensitive to inhibitors of Poly(ADP)Ribose Polymerase (PARP) and therefore PTEN loss could sensitize cells to PARP inhibitors in a RAD51-dependent manner. Using PTEN-isogenic cell lines, cells treated with siRNA to PTEN and human prostate cancer cell lines with disparate PTEN status, we assessed whether PTEN loss is associated with altered expression of RAD51 protein. We also evaluated the effects of PTEN loss on sensitivity to PARP inhibition and various DNA damaging agents which sensitize cells based on deficiency in DNA double- and single-strand break repair, nucleotide excision repair and base excision repair (i.e. ionizing radiation (IR), camptothecin (CPT), Mitomycin C, cisplatin, ultraviolet radiation (UV), H2O2 and MMS). We observed that PTEN status did not predict RAD51 expression in PTEN−/− (PC3), PTEN+/− (DU145), and PTEN+/+ (22RV1) prostate cancer cells or xenografts in vitro or in vivo. Using a dedicated tissue microarray of localized prostate cancer, PTEN status (based on FISH) did not predict for RAD51 expression. Furthermore, we observed no difference in RAD51 mRNA or protein expression nor in E2F1 binding to the RAD51 promoter in PTEN-deficient HCT116 colorectal cancer cells or H1299 lung carcinoma cells treated with siRNA to PTEN. PTEN expression also did not alter RAD51 localization to IR-induced nuclear foci or UV microirradiation-induced DNA DSBs. However, using clonogenic assays, we observed that loss of PTEN did miminally sensitize cells to PARP inhibition with increased sensitization to MMC, cisplatin, UV, and IR. PTEN loss did not sensitize tumour cells to MMS, CPT or paclitaxel. Taken together, these data suggest that PTEN influences sensitivity to certain DNA damaging agents in a RAD51-independent manner. To understand this sensitivity, we are documenting gene expression changes in 200 DNA repair and replication genes using NanoString technology. A better understanding of the influence of PTEN on cellular sensitivity to chemo- and radiotherapy may ultimately help to guide treatment options for patients based on PTEN status (Sponsored by CCSRI, PCC and the Terry Fox Foundation). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3932.

Improving the pyrophosphate-inosine phosphotransferase activity of Escherichia blattae acid phosphatase by sequential site-directed mutagenesis.

Escherichia blattae acid phosphatase/phosphotransferase (EB-AP/PTase) exhibits C-5'-position selective pyrophosphate-nucleoside phosphotransferase activity in addition to its intrinsic phosphatase. Improvement of its phosphotransferase activity was investigated by sequential site-directed mutagenesis. By comparing the primary structures of higher 5'-inosinic acid (5'-IMP) productivity and lower 5'-IMP productivity acid phosphatase/phosphotransferase, candidate residues of substitution were selected. Then a total of 11 amino acid substitutions were made with sequential substitutions. As the number of substituted amino acid residues increased, the 5'-IMP productivity of the mutant enzyme increased, and the activity of the 11 mutant phosphotransferases of EB-AP/PTase reached the same level as that of Morganella morganii AP/PTase. This result shows that Leu63, Ala65, Glu66, Asn69, Ser71, Asp116, Thr135, and Glu136, whose relevance was not directly established by structural analysis alone, also plays an important role in the phosphotransferase activity of EB-AP/PTase.

A Multitasking Transcriptional Regulator

A coregulatory protein called Eyes absent (Eya) is part of a conserved transcription network that controls development of the eye and other organs, including the kidney and ear. Presence of a motif at the C-terminus of Eya led three groups to discover that this regulatory protein harbors intrinsic protein phosphatase activity that may serve to dephosphorylate itself and other substrates to regulate gene expression. Biochemical analysis indicated that Eya is a type of haloacid dehalogenase (HAD) with enzymatic activity toward phosphotyrosine-containing peptide substrates in vitro. Transgenic flies expressing mutated forms of Eya lacking the HAD motif and phosphatase activity had abnormal eye development. Expression of the mutant forms was not able to rescue eye defects in flies harboring a defective eya gene. A transcription factor called Sine oculis (Six) interacts with Eya and Dachshund (Dach) to form activating or repressing transcriptional complexes, respectively. In cultured mammalian cells, the Dach-Six complex recruits corepressors to inhibit the expression of target genes such as c- myc. However, the recruitment of Eya to this complex stimulated the assembly of coactivators that promoted gene expression to support cell proliferation and survival. It remains unclear how Eya's phosphatase activity regulates its function. X. Li, K. A. Oghi, J. Zhang, A. Krones, K. T. Bush, C. K. Glass, S. K. Nigam, A. K. Aggarwal, R. Mass, D. W. Rose, M. G. Rosenfeld, Eye protein phosphatase activity regulates Six1-Dach-Eya transcriptional effects in mammalian organogenesis. Nature 426 , 247-254 (2003). [Online Journal] J. P. Rayapureddi, C. Kattamuri, B. D. Steinmetz, B. J. Frankfort, E. J. Ostrin, G. Mardon, R. S. Hegde, Eyes absent represents a class of protein tyrosine phosphatases. Nature 426 , 295-298 (2003). [Online Journal] T. L. Tootle, S. J. Silver, E. L. Davies, V. Newman, R. R. Latek, I. A. Mills, J. D. Selengut, B. E. W. Parlikar, I. Rebay, The transcription factor Eyes absent is a protein tyrosine phosphatase. Nature 426 , 299-302 (2003). [Online Journal]

Letter to the Editor

Ligation of interleukin 2 (IL2) is known to regulate both protein tyrosine and serine/threonine phosphorylation. A family of leukocyte transmembrane proteins whose cytoplasmic domain exhibits intrinsic protein tyrosine phosphatase activity is collectively called CD45 and is identified by a set of common cell surface epitopes. Although CD45 is known to be a phosphoprotein, it is not known how phosphorylation specifically regulates its function. We therefore identified a cell line, the IL4-dependent line CTLL-2.4, in which CD45 could be phosphorylated in response to addition of IL2. These cells are a variant of an IL2-dependent murine cell line which were selected for long-term growth on IL4 but which retain the ability to proliferate on exposure to IL2. Incubation of CTLL-2.4 in low serum concentrations followed by stimulation with IL2 caused a three- to fivefold increase in the phosphorylation of CD45 in a time- and concentration-dependent manner. CD45 in non-stimulated cells contained one major tryptic phosphopeptide, whereas, after exposure of the cells to IL2, two new phosphopeptides were present in CD45. The pattern of IL2-induced phosphorylation was different from that found following addition of phorbol 12-myristate 13-acetate (PMA) to the cells. Although IL2 induced rapid and potent tyrosine phosphorylation in CTLL-2.4 cells, all of the basal and cytokine-activated phosphorylation of CD45 occurred on serine residues. The IL2-stimulated phosphorylation caused no change in the amount of cell surface CD45 and no alteration of its catalytic activity using an artificial tyrosine phosphorylated substrate-RCM-lysozyme. We speculate that the increase in phosphorylation of CD45 may modify its association with potential substrates. The differences in the phosphorylation patterns induced by IL2 and PMA further suggest that more than one kinase can use CD45 as substrate and that IL2 activates a protein serine/threonine kinase different from protein kinase C.

Role of cyclic AMP in rat aortic microsomal phosphorylation and calcium uptake

The role of adenosine 3',5'-monophosphate (cyclic AMP)-dependent membrane phosphorylation in the regulation of microsomal calcium transport in rat aortic smooth muscle was studied. Cyclic AMP-dependent protein kinase augmented the phosphorylation of serine residues in a microsomal protein component with a molecular weight of about 44,000 (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and the majority of 32P incorporation was in serine residue(s). The phosphorylated protein had stability characteristics of a phosphoester. The phosphorylated substrate was not extracted from the trichloroacetic acid (TCA) precipitate with organic solvents or by suspension in hot TCA; and the demonstrated hydroxylamine insensitivity suggested that the substrate was not lipid or nucleic acid. Intrinsic phosphoprotein phosphatase cleaved the labeled phosphate from the cyclic AMP-stimulated microsomes in the first 5 min of incubation. Microsomes phosphorylated in the presence of 1 micron cyclic AMP or 1 micron cyclic AMP plus 0.1 mg/ml protein kinase exhibited enhanced calcium uptake. We suggest that reversible phosphorylation of microsomal membranes may play an important role in the regulation of aortic microsomal calcium transport by cyclic AMP.