Phosphodiesterase 10A (PDE10) is a cyclic nucleotide (e.g. cGMP) degrading enzyme highly expressed in the brain striatum where it plays an important role in dopaminergic neurotransmission, but has limited expression and no known physiological function outside the central nervous system. Here we report that PDE10 mRNA and protein levels are strongly elevated in human non-small cell lung cancer cells and lung tumors compared with normal human airway epithelial cells and lung tissue, respectively. Genetic silencing of PDE10 or inhibition by small molecules such as PQ10 was found to selectively inhibit the growth and colony formation of lung tumor cells. PQ10 treatment of lung tumor cells rapidly increased intracellular cGMP levels and activated cGMP-dependent protein kinase (PKG) at concentrations that inhibit lung tumor cell growth. PQ10 also increased the phosphorylation of β-catenin and reduced its levels, which paralleled the suppression of cyclin D1 and survivin but preceded the activation of PARP and caspase cleavage. PQ10 also suppressed RAS-activated RAF/MAPK signaling within the same concentration range and treatment period as required for cGMP elevation and PKG activation. These results show that PDE10 is overexpressed during lung cancer development and essential for lung tumor cell growth in which inhibitors can selectively induce apoptosis by increasing intracellular cGMP levels and activating PKG to suppress oncogenic β-catenin and MAPK signaling.

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 ApcMin/+ 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.

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.

Abstract 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<0.05). These observations suggest that PDE10 plays a critical role in tumor growth that can be targeted for cancer drug discovery, while existing inhibitors may be repurposed for cancer. 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.