Ileana V. Aragon

Serine / threonine protein phosphatase 5 (PP5) participates in the regulation of glucocorticoid receptor nucleocytoplasmic shuttling

BackgroundIn most cells glucocorticoid receptors (GR) reside predominately in the cytoplasm. Upon hormone binding, the GR translocates into the nucleus, where the hormone-activated GR-complex regulates the transcription of GR-responsive genes. Serine/threonine protein phosphatase type 5 (PP5) associates with the GR-heat-shock protein-90 complex, and the suppression of PP5 expression with ISIS 15534 stimulates the activity of GR-responsive reporter plasmids, without affecting the binding of hormone to the GR.ResultsTo further characterize the mechanism by which PP5 affects GR-induced gene expression, we employed immunofluorescence microscopy to track the movement of a GR-green fluorescent fusion protein (GR-GFP) that retained hormone binding, nuclear translocation activity and specific DNA binding activity, but is incapable of transactivation. In the absence of glucocorticoids, GR-GFP localized mainly in the cytoplasm. Treatment with dexamethasone results in the efficient translocation of GR-GFPs into the nucleus. The nuclear accumulation of GR-GFP, without the addition of glucocorticoids, was also observed when the expression of PP5 was suppressed by treatment with ISIS 15534. In contrast, ISIS 15534 treatment had no apparent effect on calcium induced nuclear translocation of NFAT-GFP.ConclusionThese studies suggest that PP5 participates in the regulation of glucocorticoid receptor nucleocytoplasmic shuttling, and that the GR-induced transcriptional activity observed when the expression of PP5 is suppressed by treatment with ISIS 15534 results from the nuclear accumulation of GR in a form that is capable of binding DNA yet still requires agonist to elicit maximal transcriptional activation.

MicroRNA-30c-2* limits expression of proadaptive factor XBP1 in the unfolded protein response

The PERK pathway influences the scale of XBP1-mediated gene expression and cell fate in response to ER stress via miR-30c-2*.

Elevated levels of Ser/Thr protein phosphatase 5 (PP5) in human breast cancer

Ser/Thr protein phosphatase 5 (PP5) regulates several signaling-cascades that suppress growth and/or facilitate apoptosis in response to genomic stress. The expression of PP5 is responsive to hypoxia inducible factor-1 (HIF-1) and estrogen, which have both been linked to the progression of human breast cancer. Still, it is not clear if PP5 plays a role in the development of human cancer. Here, immunostaining of breast cancer tissue-microarrays (TMAs) revealed a positive correlation between PP5 over-expression and ductal carcinoma in situ (DCIS; P value 0.0028), invasive ductal carcinoma (IDC; P value 0.012) and IDC with metastases at the time of diagnosis (P value 0.0001). In a mouse xenograft model, the constitutive over-expression of PP5 was associated with an increase in the rate of tumor growth. In a MCF-7 cell culture model over-expression correlated with both an increase in the rate of proliferation and protection from cell death induced by oxidative stress, UVC-irradiation, adriamycin, and vinblastine. PP5 over-expression had no apparent effect on the sensitivity of MCF-7 cells to taxol or rapamycin. Western analysis of extracts from cells over-expressing PP5 revealed a decrease in the phosphorylation of known substrates for PP5. Together, these studies indicate that elevated levels of PP5 protein occur in human breast cancer and suggest that PP5 over-expression may aid tumor progression.

Cantharidin-induced Mitotic Arrest is Associated with the Formation of Aberrant Mitotic Spindles and Lagging Chromosomes Resulting, in part, from the Suppression of PP2Aα

Cantharidin, a natural vesicant, inhibits the activity of several PPP family phosphatases, displays antitumor activity, and induces apoptosis in many types of tumor cells. However, the molecular mechanisms underlying the antitumor activity of cantharidin are not clear. Here, dose-response studies confirm a strong correlation between the suppression of phosphatase activity and cell death. Flow cytometry analysis indicates that before apoptosis, cantharidin delays cell cycle progression following DNA replication with no apparent effect on G1-S or S-G2 phase progression. In contrast, studies with double thymidine-synchronized populations of cells indicate that cantharidin can rapidly arrest growth when added during G2 or early M phase. Immunostaining indicates that cell cycle arrest occurs before the completion of mitosis and is associated with the appearance of aberrant mitotic spindles. Live cell imaging with time-lapse microscopy shows that cantharidin disrupts the metaphase alignment of chromosomes and produces a prolonged mitotic arrest, with the onset of apoptosis occurring before the onset of anaphase. To explore the contribution of individual phosphatases, antisense oligonucleotides and small interfering RNA were developed to suppress the expression of cantharidin-sensitive phosphatases. The suppression of PP2Aα, but not PP2Aβ, is sufficient to induce metaphase arrest, during which time lagging chromosomes are observed moving between the spindle poles and the metaphase plate. Immunostaining revealed slightly abnormal, yet predominately bipolar, mitotic spindles. Nonetheless, after a 10- to 15-hour delay, the cells enter anaphase, suggesting that an additional cantharidin-sensitive phosphatase is involved in the progression from metaphase into anaphase or to prevent the onset of apoptosis in cells arrested during mitosis. [Mol Cancer Ther 2006;5(11):2727–36]

The β subunit of human rod photoreceptor cGMP-gated cation channel is generated from a complex transcription unit

Human and bovine rod photoreceptor cGMP‐gated cation channel consists of two subunits: α (63 kDa) and β (240 kDa). The human β subunit was shown to consist partly of sequence encoded by the cDNA clone hRCNC2b. Here we present the complete sequence of the human β subunit and demonstrate that the previously reported human GAR1 gene encoding a glutamate‐rich protein (hGARP) encodes its N‐terminal portion. Using PCR, RNA blot and genomic DNA analysis, we provide evidence that the β subunit is produced from a complex locus on chromosome 16 which is also capable of generating independent transcripts corresponding to GAR1 and the C‐terminal two‐thirds of the β subunit. The results indicate that the β subunit of the cGMP‐gated cation channel is produced from an unusual locus consisting of more than one transcription unit.

The specialized unfolded protein response of B lymphocytes: ATF6α-independent development of antibody-secreting B cells

B lymphocytes, like all mammalian cells, are equipped with the unfolded protein response (UPR), a complex signaling system allowing for both pro- and mal-adaptive responses to increased demands on the endoplasmic reticulum (ER). The UPR is comprised of three signaling pathways initiated by the ER transmembrane stress sensors, IRE1α/β, PERK and ATF6α/β. Activation of IRE1 yields XBP1(S), a transcription factor that directs expansion of the ER and enhances protein biosynthetic and secretory machinery. XBP1(S) is essential for the differentiation of B lymphocytes into antibody-secreting cells. In contrast, the PERK pathway, a regulator of translation and transcription, is dispensable for the generation of antibody-secreting cells. Functioning as a transcription factor, ATF6α can augment ER quality control processes and drive ER expansion, but the potential role of this UPR pathway in activated B cells has not been investigated. Here, we report studies of ATF6α-deficient B cells demonstrating that ATF6α is not required for the development of antibody-secreting cells. Thus, when B cells are stimulated to secrete antibody, a specialized UPR relies exclusively on the IRE1-XBP1 pathway to remodel the ER and expand cellular secretory capacity.

Phosphatidylcholine as a metabolic cue for determining B cell fate and function

In activated B cells, increased production of phosphatidylcholine (PtdCho), the most abundant cellular phospholipid, is handled primarily by the CDP-choline pathway. B cell-specific deletion of CTP:phosphocholine cytidylyltransferase α (CCTα), the rate-limiting enzyme in the CDP-choline pathway, led to augmented IgM secretion and reduced IgG production, suggesting that PtdCho synthesis is required for germinal center reactions. To specifically assess whether PtdCho influences B cell fate during germinal center responses, we examined immune responses in mice whereby PtdCho synthesis is disrupted in B cells that have undergone class switch recombination to IgG1 (referred to as either Cγ1wt/wt, Cγ1Cre/wt or Cγ1Cre/Cre based on Cre copy number). Serum IgG1 was markedly reduced in naïve Cγ1Cre/wt and Cγ1Cre/Cre mice, while levels of IgM and other IgG subclasses were similar between Cγ1Cre/wt and Cγ1wt/wt control mice. Serum IgG2b titers were notably reduced and IgG3 titers were increased in Cγ1Cre/Cre mice compared with controls. Following immunization with T cell-dependent antigen NP-KLH, control mice generated high titer IgG anti-NP while IgG anti-NP titers were markedly reduced in both immunized Cγ1Cre/wt and Cγ1Cre/Cre mice. Correspondingly, the frequency of NP-specific IgG antibody-secreting cells was also reduced in spleens and bone marrow of Cγ1Cre/wt and Cγ. 1Cre/Cre mice compared to control mice. Interestingly, though antigen-specific IgM B cells were comparable between Cγ1Cre/wt, Cγ1Cre/Cre and control mice, the frequency and number of IgG1 NP-specific B cells was reduced only in Cγ1Cre/Cre mice. These data indicate that PtdCho is required for the generation of both germinal center-derived B cells and antibody-secreting cells. Further, the reduction in class-switched ASC but not B cells in Cγ1Cre/wt mice suggests that ASC have a greater demand for PtdCho compared to germinal center B cells.

Abstract 5174: Phosphodiesterase 10A inhibition as a novel approach to suppress β-catenin signaling in ovarian cancer cells

Canonical Wnt/β-catenin signaling is known to be associated with platinum resistance in ovarian cancer in which inhibitors hold promise for the treatment of refractory disease. Phosphodiesterase 10A (PDE10A) is a dual cyclic AMP and cyclic GMP phosphodiesterase isozyme recently implicated in colon cancer. PDE10A inhibition in colon cancer cells by siRNA or small molecule inhibitors increased cGMP levels and activated PKG to inhibit β-catenin signaling. A novel PDE10 inhibitor, ADT-061, was identified by screening a library of indene derivatives, and showed strong antineoplastic activity in the Apc+/min-FCCC mouse (Lee K et al., unpublished data). Cyclic GMP and phosphodiesterases participate in the ovarian follicular development, although little is known about PDE10A expression in ovaries, especially with regard to a potential role in ovarian tumorigenesis. PDE10A protein was found to be expressed in various established ovarian cancer cell lines at higher levels than immortalized or primary ovarian surface epithelial cell lines. Pf-2545920, a known PDE10A inhibitor, and ADT-061 inhibited the growth of multiple ovarian tumor cell lines with IC50s around 20µM and 0.5µM, respectively. Both compounds induced apoptosis after 24h treatment, as measured by PI/Annexin-V staining and PARP cleavage. Pf-2545920 and ADT-061 induced phosphorylation of VASP at Ser157 and Ser239 in various ovarian cancer cell lines, indicating activation of cyclic AMP and cyclic GMP signaling, respectively. Treatment also decreased levels of β-catenin and downstream targets of TCF-dependent transcription, including c-MYC, survivin and cyclin-D1. Homozygous knockout PDE10A clones of OV-90 ovarian cancer cells obtained using CRISPR/Cas9 showed decreased clonogenic potential, decreased Pf-2545920-mediated VASP phosphorylation and β-catenin, c-MYC and survivin expression. Ongoing efforts are focused on the development of more potent ADT-061 analogs. These observations support further study of a role of PDE10 in ovarian tumorigenesis and the development of ADT-061 or analogs for the treatment of refractory ovarian cancer as well as the prevention of malignant recurrence. Citation Format: Luciana Madeira Da Silva, Elaine Gavin, Kevin J. Lee, Veronica Ramirez-Alcantara, Kristy L. Berry, Holly T. Taylor, Alla Musiyenko, Ileana V. Aragon, Adam B. Keeton, Jennifer Scalici, Rodney P. Rocconi, Gary A. Piazza. Phosphodiesterase 10A inhibition as a novel approach to suppress β-catenin signaling in ovarian cancer cells [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 5174. doi:10.1158/1538-7445.AM2017-5174

Abstract A76: Targeting phosphodiesterase 10A for chemoprevention and treatment of ovarian cancer.

Phosphodiesterase 10A (PDE10A) is a dual cyclic AMP and cyclic GMP phosphodiesterase that is highly expressed in the brain striatum, testis and thyroid and this restricted expression in peripheral tissue has been clinically exploited for the treatment of schizophrenia and Huntington9s disease. Interestingly, our group has recently found that PDE10A is overexpressed in colon, lung and breast tumors when compared to their healthy tissue correlates. Moreover, our studies indicate that PDE10A inhibition in cancer cells by RNA interference or by small molecule inhibitors leads to activation of cGMP/PKG and inhibition of β-catenin signaling pathway that culminates with apoptotic cell death. Here we are investigating the role of PDE10A in ovarian cancer by using genetic approaches (RNA interference and CRISPR/Cas9 genome editing) and novel PDE10A small molecule inhibitors that we generated. We found that PDE10A protein is expressed in various established ovarian cancer cell lines at higher levels than in immortalized ovarian surface epithelial cell lines. Using a known PDE10A inhibitor, Pf-2545920, and novel sulindac derivatives that we generated with high specificity towards the PDE10A isozyme, we observed potent growth inhibition properties (IC50s at low micromolar or sub-micromolar concentrations) for all ovarian cancer cell lines tested. One of our novel PDE10A inhibitors, called MCI-030, which showed IC50~0.5μM for ovarian cancer cells, was orally administered to C57BL/6 mice at a safe dose of 100mg/Kg and revealed a stable concentration of approximately 2μM in the plasma and ovarian tissue for about 8h. Mechanistically, our results showed that PDE10A inhibitors induced apoptosis as measured by PI/Annexin V staining. Pf-2545920 induced phosphorylation of VASP at Ser157 and Ser239 in OV-90, OVCAR3 and SKOV3 cells, indicating activation of cyclic nucleotide signaling pathway. In addition, a decrease in the levels of β-catenin, survivin, c-Myc and cyclin D1 was observed in OV-90 cells treated with Pf-2545920 for 24h. We used CRISPR-Cas9 to delete a 97bp segment in exon 7 of PDE10A in the diploid ovarian cancer cell line OV-90. Homozygous knockout PDE10A clones were obtained and showed decreased clonogenic potential and decreased Pf-2545920-mediated pVASP phosphorylation in vitro. We are currently investigating the expression of PDE10A in clinical specimens of ovarian tumors by qPCR, western-blotting and immunohistochemistry. Our future work includes testing MCI-030 in vivo using xenograft intraperitoneal models of ovarian cancer, as well as in vivo experiments with OV-90 PDE10 knockout cells. Our ultimate goal for the proposed studies of PDE10 as a novel chemoprevention target for ovarian cancer is to develop a potent PDE10 inhibitor with negligible toxicity that can be used as a chemoprevention agent for women at high risk for this malignancy. Citation Format: Luciana Madeira da Silva, Elaine Gavin, Kevin Lee, Ileana Aragon, Veronica Ramirez-Alcantara, Jennifer Scalici, Rodney P. Rocconi, Gary A. Piazza. Targeting phosphodiesterase 10A for chemoprevention and treatment of ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A76.