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// Kevin Lee 1 , Ashley S. Lindsey 1 , Nan Li 2 , Bernard Gary 1 , Joel Andrews 3 , Adam B. Keeton 1 , Gary A. Piazza 1 1 Drug Discovery Research Center, Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama, USA 2 Department of Biochemistry and Molecular Genetics, The University of Alabama at Birmingham, Birmingham, Alabama, USA 3 Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama, USA Correspondence to: Kevin Lee, e-mail: kjlee@health.southalabama.edu Keywords: β-catenin, PKG, PKA, PDE10A, colon cancer Received: September 09, 2015 Accepted: December 07, 2015 Published: December 21, 2015 ABSTRACT Phosphodiesterase 10A (PDE10) is a cGMP and cAMP degrading PDE isozyme that is highly expressed in the brain striatum where it appears to play an important role in cognition and psychomotor activity. PDE10 inhibitors are being developed for the treatment of schizophrenia and Huntington’s disease and are generally well tolerated, possibly because of low expression levels in most peripheral tissues. We recently reported high levels of PDE10 in colon tumors and that genetic silencing of PDE10 by siRNA or inhibition with small molecule inhibitors can suppress colon tumor cell growth with a high degree of selectivity over normal colonocytes (Li et al., Oncogene 2015). These observations suggest PDE10 may have an unrecognized role in tumorigenesis. Here we report that the concentration range by which the highly specific PDE10 inhibitor, Pf-2545920 (MP-10), inhibits colon tumor cell growth parallels the concentration range required to increase cGMP and cAMP levels, and activates PKG and PKA, respectively. Moreover, PDE10 knockdown by shRNA reduces the sensitivity of colon tumor cells to the growth inhibitory activity of Pf-2545920. Pf-2545920 also inhibits the translocation of β-catenin to the nucleus, thereby reducing β-catenin mediated transcription of survivin, resulting in caspase activation and apoptosis. PDE10 mRNA was also found to be elevated in colon tumors compared with normal tissues. These findings suggest that PDE10 can be targeted for cancer therapy or prevention whereby inhibition results in cGMP elevation and PKG activation to reduce β-catenin-mediated transcription of survival proteins leading to the selective apoptosis of cancer cells.

The cyclic nucleotide phosphodiesterase 10A (PDE10) has been mostly studied as a therapeutic target for certain psychiatric and neurological conditions, although a potential role in tumorigenesis has not been reported. Here we show that PDE10 is elevated in human colon tumor cell lines compared with normal colonocytes, as well as in colon tumors from human clinical specimens and intestinal tumors from Apc(Min/+) mice compared with normal intestinal mucosa, respectively. An isozyme and tumor-selective role of PDE10 were evident by the ability of small-molecule inhibitors and small interfering RNA knockdown to suppress colon tumor cell growth with reduced sensitivity of normal colonocytes. Stable knockdown of PDE10 by short hairpin RNA also inhibits colony formation and increases doubling time of colon tumor cells. PDE10 inhibition selectively activates cGMP/cGMP-dependent protein kinase signaling to suppress β-catenin levels and T-cell factor (TCF) transcriptional activity in colon tumor cells. Conversely, ectopic expression of PDE10 in normal and precancerous colonocytes increases proliferation and activates TCF transcriptional activity. These observations suggest a novel role of PDE10 in colon tumorigenesis and that inhibitors may be useful for the treatment or prevention of colorectal cancer.

Two closely related series of novel beta-carboline derivatives, electronically similar to tadalafil (CAS 171596-29-5), were synthesized and evaluated for their inhibitory effects upon phosphodiesterase 5 (PDE5) and phosphodiesterase 11 (PDE11) and their in vitro tumor cell growth inhibitory activity versus HT29 colorectal carcinoma cell line. Interestingly, some of the synthesized compounds showed growth inhibitory properties that appear to be associated with their ability to inhibit PDE5. Moreover, the PDE5 inhibition seems relevant to the stereochemical aspects of the compounds.

There is compelling evidence that nonsteroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase-2 selective inhibitors have antineoplastic activity, but toxicity from cyclooxygenase (COX) inhibition and the suppression of physiologically important prostaglandins limits their use for cancer chemoprevention. Previous studies as reviewed here suggest that the mechanism for their anticancer properties does not require COX inhibition, but instead involves an off-target effect. In support of this possibility, recent molecular modeling studies have shown that the NSAID sulindac can be chemically modified to selectively design out its COX-1 and COX-2 inhibitory activity. Unexpectedly, certain derivatives that were synthesized based on in silico modeling displayed increased potency to inhibit tumor cell growth. Other experiments have shown that sulindac can inhibit phosphodiesterase to increase intracellular cyclic GMP levels and that this activity is closely associated with its ability to selectively induce apoptosis of tumor cells. Together, these studies suggest that COX-independent mechanisms can be targeted to develop safer and more efficacious drugs for cancer chemoprevention.

In view of the emerging clinical indications for Phosphodiesterase 9 inhibitors e.g. treatment of Alzheimer, diabetes, cancer, and the limited number of its selective inhibitors which possess a single chemical scaffolds, a structure-based approach was undertaken to mine the ZINC database by virtual screening to identify novel PDE9 inhibitors. The database, which was never reported to have been used before for discovery of PDE9 inhibitors, was screened against the ligand binding pocket of the PDE9 complex (PDB:4GH6) using molecular docking programs, MOE and AutoDock Vina in PyRx. Three different scoring functions were used to evaluate the docking poses and scores of the compounds, and the compounds were selected through consensus selection, thus reducing the margin of error in docking. The highest scoring compounds were then selected and purchased for in vitro testing as PDE9 inhibitors and cancer growth inhibitory agents. This led to the discovery of three previously unreported potent PDE 9 inhibitory compounds with two unique chemical scaffolds. Consistent with the role of PDE9 in cancer cell growth, the compounds also inhibited the growth of breast tumor cell lines, MCF-7 and MDA-468 at concentrations comparable to those that inhibited PDE9.

Nonsteroidal anti-inflammatory drugs (NSAID) display promising antineoplastic activity for colorectal and other cancers, but toxicity from COX inhibition limits their long-term use for chemoprevention. Previous studies have concluded that the basis for their tumor cell growth inhibitory activity does not require COX inhibition, although the underlying mechanism is poorly understood. Here, we report that the NSAID sulindac sulfide inhibits cyclic guanosine 3′,5′-monophosphate phosphodiesterase (cGMP PDE) activity to increase intracellular cGMP levels and activate cGMP-dependent protein kinase (PKG) at concentrations that inhibit proliferation and induce apoptosis of colon tumor cells. Sulindac sulfide did not activate the cGMP/PKG pathway, nor affect proliferation or apoptosis in normal colonocytes. Knockdown of the cGMP-specific PDE5 isozyme by siRNA and PDE5-specific inhibitors tadalafil and sildenafil also selectively inhibited the growth of colon tumor cells that expressed high levels of PDE5 compared with colonocytes. The mechanism by which sulindac sulfide and the cGMP/PKG pathway inhibits colon tumor cell growth involves the transcriptional suppression of β-catenin to inhibit Wnt/β-catenin T-cell factor transcriptional activity, leading to downregulation of cyclin D1 and survivin. These observations suggest that safer and more efficacious sulindac derivatives can be developed for colorectal cancer chemoprevention by targeting PDE5 and possibly other cGMP-degrading isozymes. Mol Cancer Ther; 12(9); 1848–59. ©2013 AACR.

Phosphodiesterase 10A (PDE10A) is a cGMP and cAMP degrading PDE isozyme that is highly expressed in the brain striatum where it appears to play an important role in cognition and psychomotor activity. PDE10 inhibitors are being developed for the treatment of schizophrenia and Huntington's disease and are generally well tolerated, possibly because of low expression levels in most peripheral tissues. We recently reported high levels of PDE10 in colon tumors and that genetic silencing of PDE10 by siRNA or inhibition with small molecule inhibitors can suppress colon tumor cell growth with a high degree of selectivity over normal colonocytes (Li et al., Oncogene 2015). These observations suggest PDE10 may have an unrecognized role in tumorigenesis. Here we report that the concentration range by which the highly specific PDE10 inhibitor, Pf-2545920 (MP-10), inhibited colon tumor cell growth parallels the concentration range required to increase cGMP and cAMP levels, and activate PKG and PKA, respectively. Moreover, PDE10 knockdown by shRNA reduced the sensitivity of colon tumor cells to the growth inhibitory activity of Pf-2545920. Pf-2545920 also inhibited the translocation of β-catenin to the nucleus, thereby reducing β-catenin mediated transcription of survivin, which resulted in caspase activation and apoptosis. This was determined to be through a PKG mediated pathway through the use of small molecule inhibitors of PKG and PKA. PDE10 mRNA was also found to be elevated in colon tumors compared with normal tissues in a cDNA array. We also report the increase in PDE10 mRNA in 50% of a small collection of human clinical specimens collected at the Mitchell Cancer Institute (n = 13). In addition, novel PDE10 inhibitor, MCI-030, reduced tumor size and activated PDE10 signaling mechanisms in vivo. These findings suggest that PDE10 can be targeted for cancer therapy or prevention whereby inhibition results in cGMP elevation and PKG activation to reduce β-catenin-mediated transcription of survival proteins leading to the selective apoptosis of cancer cells. Citation Format: Kevin J. Lee, Ashley S. Lindsey, Luciana Madeira da Silva, Alisa Trinh, Bernard Gary, Joel Andrews, Veronica Ramirez-Alcantara, Adam B. Keeton, Wen-Chi Chang, Margie Clapper, Gary A. Piazza. Beta-catenin nuclear translocation in colorectal cancer cells is suppressed by PDE10A inhibition, cGMP elevation, and activation of PKG. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 331.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been widely reported to inhibit tumor growth by a COX-independent mechanism, although alternative targets have not been well defined or used to develop improved drugs for cancer chemoprevention. Here, we characterize a novel sulindac derivative referred to as sulindac benzylamine (SBA) that does not inhibit COX-1 or COX-2, yet potently inhibits the growth and induces the apoptosis of human colon tumor cells. The basis for this activity appears to involve cyclic guanosine 3′,5′,-monophosphate phosphodiesterase (cGMP PDE) inhibition as evident by its ability to inhibit cGMP hydrolysis in colon tumor cell lysates and purified cGMP-specific PDE5, increase intracellular cGMP levels, and activate cGMP-dependent protein kinase G at concentrations that suppress tumor cell growth. PDE5 was found to be essential for colon tumor cell growth as determined by siRNA knockdown studies, elevated in colon tumor cells as compared with normal colonocytes, and associated with the tumor selectivity of SBA. SBA activation of PKG may suppress the oncogenic activity of β-catenin as evident by its ability to reduce β-catenin nuclear levels, Tcf (T-cell factor) transcriptional activity, and survivin levels. These events preceded apoptosis induction and appear to result from a rapid elevation of intracellular cGMP levels following cGMP PDE inhibition. We conclude that PDE5 and possibly other cGMP degrading isozymes can be targeted to develop safer and more efficacious NSAID derivatives for colorectal cancer chemoprevention. Cancer Prev Res; 5(6); 822–33. ©2012 AACR.

Nonsteroidal anti-inflammatory drugs (NSAID) such as sulindac sulfide (SS) display promising antineoplastic properties, but toxicities resulting from COX inhibition limit their clinical use. Although COX inhibition is responsible for the anti-inflammatory activity of SS, recent studies suggest that phosphodiesterase (PDE) 5 inhibition and activation of cyclic guanosine monophosphate (cGMP) signaling are closely associated with its ability to induce apoptosis of tumor cells. However, the underlying mechanisms responsible for apoptosis induction, factors that influence sensitivity of tumor cells to SS, and the importance of PDE5 for breast tumor cell growth have not been established. Here we show that SS can induce apoptosis of breast tumor cells, which predominantly rely on PDE5 for cGMP hydrolysis but not normal mammary epithelial cells, which rely on PDE isozymes other than PDE5 for cGMP hydrolysis. Inhibition of PDE5 and activation of protein kinase G (PKG) by SS was associated with increased β-catenin phosphorylation, decreased β-catenin mRNA and protein levels, reduced β-catenin nuclear localization, decreased T-cell factor/lymphoid enhancer factor (Tcf/Lef) promoter activity, and decreased expression of Wnt/β-catenin–regulated proteins. Suppression of PDE5 with siRNA or known PDE5 inhibitors was sufficient to selectively induce apoptosis and attenuate β-catenin–mediated transcription in breast tumor cells with minimal effects on normal mammary epithelial cells. These findings provide evidence that SS induces apoptosis of breast tumor cells through a mechanism involving inhibition of PDE5 and attenuation of oncogenic Wnt/β-catenin–mediated transcription. We conclude that PDE5 represents a novel molecular target for the discovery of safer and more efficacious drugs for breast cancer chemoprevention. Cancer Prev Res; 4(8); 1275–84. ©2011 AACR.

Two closely related series of novel beta-carboline derivatives, electronically similar to tadalafil (CAS 171596-29-5), were synthesized and evaluated for their inhibitory effects upon phosphodiesterase 5 (PDE5) and phosphodiesterase 11 (PDE11) and their in vitro tumor cell growth inhibitory activity versus HT29 colorectal carcinoma cell line. Interestingly, some of the synthesized compounds showed growth inhibitory properties that appear to be associated with their ability to inhibit PDE5. Moreover, the PDE5 inhibition seems relevant to the stereochemical aspects of the compounds.

Sulindac displays promising antineoplastic activity, but toxicities from cyclooxygenase inhibition limit its use for chemoprevention. Previous reports suggest that its anticancer properties may be attributed to a cyclooxygenase-independent mechanism, although alternative targets have not been well defined. Here, we show that sulindac sulfide (SS) induces apoptosis and inhibits the growth of human breast tumor cells with IC50 values of 60 to 85 μmol/L. Within the same concentration range, SS inhibited cyclic GMP (cGMP) hydrolysis in tumor cell lysates but did not affect cyclic AMP hydrolysis. SS did not induce apoptosis of normal human mammary epithelial cells (HMEC) nor did it inhibit phosphodiesterase (PDE) activity in HMEC lysates. SS increased intracellular cGMP levels and activated protein kinase G in breast tumor cells but not HMEC. The guanylyl cyclase (GC) activator, NOR-3, and cGMP PDE inhibitors, trequinsin and MY5445, displayed similar growth-inhibitory activity as SS, but the adenylyl cyclase activator, forskolin, and other PDE inhibitors had no effect. Moreover, GC activation increased the sensitivity of tumor cells to SS, whereas GC inhibition reduced sensitivity. By comparing PDE isozyme profiles in breast tumor cells with HMEC and determining the sensitivity of recombinant PDE isozymes to SS, PDE5 was found to be overexpressed in breast tumor cells and selectively inhibited by SS. The mechanism of SS binding to the catalytic domain of PDE5 was revealed by molecular modeling. These data suggest that PDE5 inhibition is responsible for the breast tumor cell growth–inhibitory and apoptosis-inducing activity of SS and may contribute to the chemopreventive properties of sulindac. [Mol Cancer Ther 2009;8(12):3331–40]

BACKGROUND: We recently reported that phosphodiesterase 10A (PDE10) is elevated in certain tumor types and essential for tumor cell growth (Li et al., Oncogene 2014). Here we evaluate anti-tumor efficacy, pharmacokinetics, tissue distribution, and tolerance of a novel PDE10 inhibitor, ADT-048, in mice. METHODS: ADT-048 was orally administrated to C57BL/6 mice and free levels quantified in plasma and tissues by LC-MS. Anti-tumor activity was evaluated in athymic nude-Foxn1nu mice after inoculating the left lung with 1×106 A549 lung tumor cells transfected with luciferase. Tumor growth was monitored by in situ bioluminescence using the In Vivo Imaging System and efficacy was evaluated by necropsy grading. RESULTS: ADT-048 inhibited A549 lung tumor cell growth with an IC50 value of 0.7 μM, while appreciably higher concentrations were required to inhibit the growth of normal airway epithelial cells (IC50 = 35 μM). Pharmacokinetic studies demonstrated a half-life suitable for once a day dosing, while tissue distribution studies demonstrated a unique capacity to achieve high concentrations in lungs relative to plasma and other tissues. ADT-048, at a dose of 100 mg/Kg, displayed significant anti-tumor activity as evident by reduced luminescence and tumor grading following 28 days of treatment. CONCLUSIONS: A novel PDE10 inhibitor, ADT-048, was found to display highly potent and selective tumor cell growth inhibitory activity in vitro. ADT-048 was well tolerated in mice and had attractive pharmaceutical properties with a unique capacity to achieve high lung concentrations following oral administration. Efficacy studies demonstrate strong anti-tumor activity of ADT-048, which represents a candidate for clinical development. Supported by NIH grants 1R01CA155638 and 1R01CA131378 (Piazza). Citation Format: Veronica Ramirez-Alcantara, Michele Schuler, Bing Zhu, Nan Li, Evrim Gurpinar, Dennis Otali, Joshua Canzoneri, Adam Keeton, Bernard Gary, Suzanne Russo, Lori Coward, Gregory Gorman, William Grizzle, Xi Chen, Michael Boyd, Gary Piazza. Antitumor activity of a novel phosphodiesterase 10 inhibitor in an orthotopic mouse model of lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 950. doi:10.1158/1538-7445.AM2015-950

We recently reported that phosphodiesterase 10A (PDE10) is overexpressed in colon tumors and essential for colon tumor cell growth (Li et al., Oncogene 2014), but a role in lung cancer has not been well studied. Human non-small cell lung cancer (NSCLC) cell lines were found to express appreciably higher PDE10 levels compared with normal airway epithelial cells, while silencing of PDE10 expression with siRNA or inhibition of PDE10 activity with small molecule inhibitors selectively inhibits NSCLC cell growth. Here we study the mechanism by which PDE10 inhibitors suppress NSCLC cell growth. At concentrations effective for inhibiting NSCLC cell growth, the PDE10 inhibitor, PQ10 rapidly increases intracellular cGMP levels as determined by a luminescence biosensor assay. Within the same concentration range and time period, PQ10 activates PKA and PKG, demonstrated by an increase in phosphorylation of the substrate, VASP at Ser157 and Ser239, respectively. Under the same conditions, PQ10 suppresses the phosphorylation of Erk1/2 (Thr202/Tyr204), MEK1/2 (Ser217/Ser221) and down-stream p90RSK (Ser380). Similarly, PQ10 increases the phosphorylation of β-catenin at Ser552, but showed no effect on phospho-Ser675 levels. After longer durations of treatment, PQ10 reduces β-catenin levels, as well as proteins that are regulated by β-catenin-dependent TCF/LEF-transcriptional activity, such as survivin and cyclin D1. These effects were mimicked by a novel structurally unrelated PDE10 inhibitor, ADT-020 that selectively inhibits the growth of NSCLC cells and increases cGMP levels in a manner similar to PQ10. ADT-020 also activates PKG in NSCLC cells demonstrated by higher phospho-Ser239 VASP levels and lower β-catenin and survivin levels. These observations suggest that PDE10 inhibition and activation of PKG can simultaneously suppress ras and Wnt signaling that are essential for lung tumor cell growth. Supported by NIH grants 1R01CA155638 and 1R01CA131378 (Piazza). Citation Format: Bing Zhu, Kevin Lee, Joshua Canzoneri, Veronica Ramirez-Alcantara, Sara Sigler, Bernard Gary, Ethan Butler, Adam Keeton, Xi Chen, Michael Boyd, Gary Piazza. Phosphodiesterase 10A inhibition suppresses lung tumor cell growth by activating PKG to inhibit ras and Wnt signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4372. doi:10.1158/1538-7445.AM2015-4372

Phosphodiesterase 10A (PDE10) is a relatively new PDE isozyme that has been mostly studied in the brain as a therapeutic target for certain psychiatric and neurological conditions, but has not been associated with tumorigenesis. While other investigators have reported low levels of PDE10 in most adult peripheral tissues, we found a large percentage of human lung tumors express high levels of PDE10 compared with normal lung tissue as determined by real-time PCR analysis of cDNA arrays. Statistical analysis revealed that 68% of lung tumors (n=19) displayed more than two-fold increase compared with normal lung tissue, while 26% showed greater than a 10-fold difference. High levels of PDE10 mRNA and protein were also found in human non-small cell lung cancer (NSCLC) cell lines compared with normal airway epithelial cells. PDE10 knockdown by small interference RNA significantly suppressed lung tumor cell growth, as did PDE10 selective inhibitors, papaverine, PQ-10 and Pf-2545920. A novel PDE10 inhibitor, MCI-020 also inhibited the growth of NSCLC cells with a high degree of selectivity for tumor cells. Consistent with a mechanism involving PDE10 inhibition, PQ-10 and MCI-020 increased cGMP levels in a concentration-dependent manner as determined using a cGMP biosensor assay. MCI-020 also activated cGMP-dependent protein kinase (PKG) in NSCLCs as evident by increased phosphorylation of VASP-Serine239. With attractive pharmacokinetic properties, MCI-020 was studied further and found to display an unusual characteristic of achieving high concentrations in lung tissue compared with other tissues and plasma following oral administration. MCI-020 was well tolerated in mice at a dose of 250 mg/kg bid and strongly inhibited tumor formation in an orthotopic model of lung cancer at a dose of 150 mg/kg bid. These observations suggest that PDE10 is essential for lung tumor cell growth and represents a novel therapeutic target for lung cancer. Citation Format: Bing Zhu, Nan Li, Veronica Ramirez-Alcantara, Joshua Canzoneri, Evrim Gurpinar, Alexandra Fajardo, Kevin Lee, Sara Sigler, Bernard Gary, Meagan Thomas, Adam Keeton, Xi Chen, William Grizzle, Gary Piazza. PDE10, a novel therapeutic target for lung cancer. [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 1761. doi:10.1158/1538-7445.AM2014-1761

Cyclic nucleotide phosphodiesterases (PDE) regulate cyclic nucleotides, cAMP and cGMP, blocking signaling by hydrolyzing the 3′,5′-phosphodiester bond resulting in their degradation to inactive forms. We have previously reported that certain PDE isozymes (e.g. PDE5) are elevated in several types of cancer, which can disrupt cyclic nucleotide signaling to suppress apoptosis. The inhibition of PDE5 by agents such as sulindac sulfide (SS) results in increased cGMP levels and reduced β-catenin/TCF/LEF mediated transcription leading to the selective inhibition of colon cancer cell proliferation and induction of apoptosis. Currently, little is known regarding the transcriptional regulation of PDE5 in cancer cells. We therefore analyzed the promoter regions of PDE5, as well as PDE10, for transcription factor binding sites using the Matinspector Genomatix Software. Matinspector analysis predicted that both PDE5 and PDE10 promoters contain binding sites for TCF/LEF-1, an important transcriptional factor that is regulated by Wnt/β-catenin signaling, which plays an important role in colorectal cancer. To determine if Wnt/β-catenin signaling can regulate PDE5 and PDE10 expression, human HCT116 colon tumor cells and normal colonic epithelial cell line NCM-460 were treated with the GSK-3β inhibitor, lithium chloride (LiCl) to inhibit β-catenin degradation and stimulate TCF/LEF transcriptional activity. TCF/LEF transcriptional activity was measured by mRNA levels of genes (e.g. survivin, cyclin D1 and c-myc) that are known to be regulated by Wnt/β-catenin signaling. Survivin, cyclin D1 and c-myc mRNA were confirmed to be significantly increased after LiCl treatment in comparison to control cells treated with NaCl. Translocation of β-catenin to the nucleus was also evident after treatment with LiCl. Levels of PDE5 and 10 mRNA were found to be significantly elevated after LiCl treatment in comparison to NaCl treated cells. Inhibitors such as SS, the novel derivative SS-D1 and a tadalafil (a selective PDE5 inhibitor) derivative TA-D1 demonstrated selective inhibition of colon cancer cell growth in comparison to normal colonocytes. The potential mechanism of these selective inhibitors may be through the inhibition of Wnt/β-catenin signaling and thus the inhibition of β-catenin/TCF/LEF mediated transcription. This study demonstrates a novel regulatory mechanism of PDE5 and PDE10 expression by the β-catenin-dependent TCF/LEF transcription factor in colon cancer cells and identifies a potential mechanism for novel chemopreventive agents. Defining the role of this regulatory mechanism in cancer progression may provide valuable insight into targeting PDE5 and/or PDE10 by small molecules for cancer chemoprevention. Citation Format: Alexandra M. Fajardo, Bernard D. Gary, Kevin J. Lee, Adam B. Keeton, Bing Zhu, Xi Chen, Ashraf H. Abadi, Gary A. Piazza. β-catenin-dependent TCF/LEF transcriptional regulation of phosphodiesterase expression in colon cancer cells. [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 2322. doi:10.1158/1538-7445.AM2014-2322

503 Background: Elevation of intracellular cGMP is known to inhibit tumor proliferation and induce apoptosis, although the phosphodiesterase (PDE) isozymes that regulate cGMP levels in tumor cells have not been well studied. We report first evidence that PDE10 is elevated in colon tumors compared with normal colon and suggest that PDE10 inhibitors can be used for the treatment or prevention of colon cancer. Methods: PDE10 protein and mRNA levels were measured in human colon tumor cells (HT29, HCT116, SW480, Caco2), normal colonocytes (NCM460), human clinical samples, and ApcMin/+ mouse model. Two chemically distinct PDE10 selective inhibitors, PQ-10 and Pf-2545920, were tested against the cell lines. The NCI-60 panel of human tumor cell lines was also screened against Pf-2545920 to identify potential differences in sensitivity among histologically diverse tumor types. We also performed siRNA knockdown studies in colonocytes and tumor cell lines. To determine the effect of the PDE10 siRNA knockdown on cyclic nucleotide hydrolysis, whole cell lysates from transfected cells were assayed for PDE activity using cGMP or cAMP as substrates. Results: PDE10 levels were low in normal colonocytes (NCM460) and elevated in tumor cell lines. Similarly, PDE10 was elevated human clinical specimens and the ApcMin+/ mouse model compared with normal mucosa. PDE10 inhibitors and siRNA selectively inhibited colonic tumor growth while stable knockdown inhibited colony formation and increased doubling time. Pf-2545920 also supressed growth of all cell lines within the NCI-60 panel. In comparison with lysates from vector control cells, transfection with PDE10 siRNA reduced cGMP hydrolysis by ~35% in both HCT116 and HT29 cell lines, but did not affect cGMP hydrolysis in colonocytes; siRNA did not significantly affect cAMP degradation in all 3 cell lines. Conclusions: PDE10 plays a role in colon tumorgenesis whereby inhibitors can selectively suppress tumor cell growth. The mechanism by which PDE10 inhibition affects growth appears to involve activation of cGMP/PKG signalling. PDE10 represents a novel anticancer target for the treament and prevention of colon cancer.

Previous studies report that induction of intracellular cGMP can selectively inhibit proliferation and induce apoptosis of tumor cells. However, the phosphodiesterase (PDE) isozymes responsible for regulating cGMP in tumor cells or the basis for this selectivity have not been well studied. Here we report that PDE10 is elevated in colon tumor cell lines compared to normal colonocytes. High levels of PDE10 were also measured in colon tumors from human clinical samples and the ApcMin/+ mouse model compared to normal intestinal mucosa. PDE10 inhibitors (PQ-10, Pf-2545920) and siRNA selectively inhibit colon tumor cell growth by inhibiting proliferating and inducing apoptosis, while stable knockdown inhibits colony formation and increases doubling time. Conversely, ectopic expression of PDE10 increases the growth rate of colonocytes. Pf-2545920 inhibits the growth of all lines in the NCI-60 tumor cell panel, indicating a functional role of PDE10 across histologically diverse tumor types. The mechanism by which PDE10 inhibition suppresses growth involves activation of cGMP/PKG signaling to reduce β-catenin and TCF transcriptional activity. Given its potential as a new cancer target, PDE10 was used to design and screen for novel anticancer agents. A group of indene analogs was found to potently and selectively suppress tumor cell growth with IC50 values less than Pf-2545920. A lead compound, MCI-020, displayed attractive oral bioavailability and pharmacokinetic properties in mice with an unusual characteristic of achieving high lung concentrations compared with plasma and other tissues. Because of its unique biodistribution pattern, MCI-020 was evaluated in a lung orthotopic mouse model using human A549 lung tumor cells. Oral administration of MCI-020 was well tolerated up to at least 250 mg bid for 5 weeks without affecting body weight. As summarized below, MCI-020 (150 mg bid) significantly increased the number of mice showing no visible tumors from 15.4% in the vehicle group (n=13) to 75% in the treated group (n=12) and strongly reduced tumor formation among mice that developed tumors. Microscopic examination of lung sections using a 1-4 grading scale to measure the extent of tumor formation revealed a score of 2.8 ± 0.42 (high involvement) for the vehicle group and 0.83 ± 0.29 (low involvement) for the treated group (p Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C182. Citation Format: Nan Li, Kevin Lee, Yaguang Xi, Bing Zhu, Bernard D. Gary, Veronica Ramirez-Alcantara, Evrim Gurpinar, Joshua Canzoneri, Alexandra Fajardo, Sara Sigler, John T. Piazza, Xi Chen, Joel Andrews, Meagan Thomas, Wenyan Lu, Yonghe Li, Danuel J. Laan, Mary P. Moyer, Suzanne Russo, Brian T. Eberhardt, Larry Yet, Adam B. Keeton, William E. Grizzle, Gary A. Piazza. Phosphodiesterase 10: A novel cancer target. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C182.

Sulindac displays promising antineoplastic activity, but toxicity from cyclooxygenase (COX) inhibition limits its long-term use for chemoprevention. Previous studies have concluded that the tumor cell growth inhibitory activity of sulindac is COX-independent and suggest that safer and more efficacious drugs can be developed by targeting the underlying mechanism. Here we report that sulindac sulfide (SS) inhibits cyclic guanosine monophosphate phosphodiesterase (cGMP PDE) activity, increases intracellular cGMP levels and activates protein kinase G at concentrations that suppress proliferation and induce apoptosis of colon tumor cells. Normal colonocytes were refractory to the anti-proliferative and pro-apoptotic effects of SS as well as its ability to induce cGMP signaling. PDE5-specific inhibitors such as tadalafil also inhibited the growth of colon tumor cells that expressed high levels of the cGMP-specific PDE5 isozyme compared with colonocytes. RNAi knockdown of PDE5 selectively inhibited proliferation and induced apoptosis of colon tumor cells as did SS. SS, 8-bromo-cGMP, and PDE5 siRNA reduced β-catenin levels, as well as the expression of cyclin D and survivin that are regulated by β-catenin-dependent TCF transcriptional activity by a mechanism involving the transcriptional repression of β-catenin. The significance of these findings are highlighted by studies in the FCCC Min mouse model of intestinal tumorigenesis showing that a novel non-COX inhibitory derivative of SS can strongly inhibit colon tumor formation by a mechanism that appears to involve cGMP PDE inhibition. These observations suggest that safer and more efficacious drugs can be developed for colorectal cancer chemoprevention by targeting PDE5 and possibly other cGMP degrading isozymes. Citation Format: Nan Li, Yaguang Xi, Heather N. Tinsley, Evrim Gurpinar, Bernard D. Gary, Bing Zhu, Yonghe Li, Adam B. Keeton, William E. Grizzle, Gary A. Piazza. Next generation sulindac. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3659. doi:10.1158/1538-7445.AM2013-3659

e13523 Background: Recent studies have shown that siRNA knockdown of the cGMP-specific phosphodiesterase, PDE5 can suppress growth and induce apoptosis of human breast tumor cells. However, convent...

Previous studies have concluded that the mechanism responsible for the antineoplastic properties of NSAIDs is cyclooxygenase (COX) independent, which suggests that more selective drugs can be developed for cancer chemoprevention by targeting such mechanisms. With this paradigm, we are synthesizing novel sulindac derivatives that have reduced COX inhibitory activity, but enhanced tumor cell growth inhibitory activity. A prototypic dimethylethyl amide derivative referred to as sulindac sulfide amide (SSA) was previously reported to lack COX-1 or COX-2 inhibitory activity, yet have improved potency to inhibit tumor cell growth in vitro. For example, SSA inhibited the growth of human HT-29 colon tumor cells with IC50 values of 1-2 µM compared with 70-90 µM for sulindac sulfide (SS), a non-selective COX inhibitor. SSA was also appreciably more effective than SS in inhibiting HT-29 colon tumor cell growth using a 3-dimensional in vitro tumor cell model. SSA induced apoptosis of HT-29 carcinoma cells, but normal human colonocytes were insensitive to treatment, suggesting an element of tumor selectivity. The mechanism of SSA induced apoptosis is associated with cGMP phosphodiesterase (PDE) inhibition, elevation of intracellular cGMP, and activation of protein kinase G, which also appears to be an important off-target effect of SS. SSA inhibited the growth of a variety of histologically diverse tumor cell lines, an effect that is consistent with studies showing the overexpression of PDE5 in various tumor types, including colorectal adenomas and carcinomas. SSA was well tolerated in the FCCC Min mouse model in which oral administration reduced colon tumor multiplicity by approximately 80% and tumor incidence by 50%. SSA also inhibited tumor growth in the human HT-29 colon mouse xenograft model by approximately 60%, while sulindac at its maximum tolerated dose was marginally effective. The antitumor activity of SSA in the HT-29 model was accompanied by decreased numbers of proliferating cells and increased numbers of apoptotic cells as determined by immunohistochemistry. Pharmacokinetic studies in mice bearing HT-29 xenografts revealed that plasma and tumor levels of SSA from a tolerated dose of SSA appreciably exceeded the in vitro IC50 value for growth inhibition. In contrast, sulindac generated plasma and tumor levels of SS that were appreciably less than the IC50 value for tumor cell growth inhibition. These results support the feasibility of chemically modifying sulindac to design out COX associated toxicities, while increasing anticancer efficacy. Newer sulindac derivatives with higher potency and target selectivity, as well as formulations to improve oral bioavailability are being developed for colon cancer chemoprevention. Funding provided by NIH/NCI grants CA131378 and CA148817. 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 1865. doi:10.1158/1538-7445.AM2011-1865

Sulindac and other nonsteroidal anti-inflammatory drugs have shown promise for chemoprevention of colorectal cancer, but toxicities arising from cyclooxygenase (COX) inhibition limit their clinical use. Previous reports suggest that sulindac may exert its antitumor effects by a COX-independent target involving cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE) inhibition, although the specific isozymes involved and mechanism have not been well defined. In this study, we show that sulindac sulfide (SS) inhibits the growth of human colon tumor cells, HCT116, HT29 and SW480 with IC50 values of 60-80μmol/L. Within this concentration range, SS did not affect the growth of human-derived normal colonic epithelial cells, NCM460. Furthermore, SS induced apoptosis of colon tumor cells but not NCM460 cells. SS inhibited cGMP PDE activity in colon tumor cell lysates with minimal effect on cGMP PDE activity in NCM460 cell lysates. By determining the sensitivity of all eleven PDE isozymes to SS, the cGMP-specific PDE5 was found to be the most sensitive with an IC50 value for enzyme inhibition of 38µmol/L. In addition, PDE5 was found to be overexpressed in colon tumor cell lines compared with NCM460 cells. Knockdown of PDE5 expression in colon tumor cells with siRNA resulted in a significant reduction of tumor cell growth and increased apoptosis. Moreover, SS increased intracellular cGMP levels, activated protein kinase G (PKG) and suppressed the expression of β-catenin in colon tumor cells. SS also inhibited the transcriptional activity of β-catenin and reduced the expression of the β-catenin regulated proteins cyclin D1 and survivin, which are important mediators of tumor cell proliferation and apoptosis, respectively. Our results suggest that the chemopreventive properties of sulindac are closely associated with PDE5 inhibition and involve the activation of cGMP signaling to suppress β-catenin mediated transcriptional activity. Funding provided by NIH/NCI grants CA131378 and CA148817. 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 3707. doi:10.1158/1538-7445.AM2011-3707