Suppression of Colon Tumorigenesis in Mutant Apc Mice by a Novel PDE10 Inhibitor that Reduces Oncogenic β-Catenin

Abstract

Previous studies have reported that phosphodiesterase 10A (PDE10) is overexpressed in colon epithelium during early stages of colon tumorigenesis and essential for colon cancer cell growth. Here we describe a novel non-COX inhibitory derivative of the anti-inflammatory drug, sulindac, with selective PDE10 inhibitory activity, ADT 061. ADT 061 potently inhibited the growth of colon cancer cells expressing high levels of PDE10, but not normal colonocytes that do not express PDE10. The concentration range by which ADT 061 inhibited colon cancer cell growth was identical to concentrations that inhibit recombinant PDE10. ADT 061 inhibited PDE10 by a competitive mechanism and did not affect the activity of other PDE isozymes at concentrations that inhibit colon cancer cell growth. Treatment of colon cancer cells with ADT 061 activated cGMP/PKG signaling, induced phosphorylation of oncogenic β-catenin, inhibited Wnt-induced nuclear translocation of β-catenin, and suppressed TCF/LEF transcription at concentrations that inhibit cancer cell growth. Oral administration of ADT 061 resulted in high concentrations in the colon mucosa and significantly suppressed the formation of colon adenomas in the Apc^+/min-FCCC mouse model of colorectal cancer without discernable toxicity. These results support the development of ADT 061 for the treatment or prevention of adenomas in individuals at risk of developing colorectal cancer. PREVENTION RELEVANCE: PDE10 is overexpressed in colon tumors whereby inhibition activates cGMP/PKG signaling and suppresses Wnt/β-catenin transcription to selectively induce apoptosis of colon cancer cells. ADT 061 is a novel PDE10 inhibitor that shows promising cancer chemopreventive activity and tolerance in a mouse model of colon cancer.

Fig. 1.

ADT 061 potently and selectively inhibits colon cancer cell growth without inhibiting COX-1 or COX-2. A) Chemical modification of sulindac yields a novel derivative, AND 061. B) ADT 061 does not inhibit COX-1 or −2 at concentrations up to 50 μM, while SS inhibits both COX-1 and COX-2 at a concentration of 10 µM. Indomethacin served as a COX-1 specific control and celecoxib served as a COX-2 specific control. C) Sensitivity of human colon cancer cell lines and normal colon epithelial cells (NCM460) to ADT 061 in growth assays following 72 hr of treatment. D) Sensitivity of human colon cancer cell lines and NCM460 cells to SS in growth assays following 72 hr of treatment. (Note: error bars (SEM) were smaller than symbols, they are not shown.) E) Western blot analysis of PDE10 expression in human colon cells cell lines and NCM460 cells. Error bars = SEM

Fig. 2.

ADT 061 selectively inhibits PDE10 activity. A) Phosphodiesterase isozyme selectivity of ADT 061 at 1 μM. B) ADT 061 inhibited recombinant PDE10 with IC₅₀ of 0.3 µM for cGMP, and 1.0 µM for cAMP. C) ADT 061 followed Michaelis-Menten properties for competitive inhibition with increasing concentrations of unlabeled cGMP. D) PDE10 siRNA knockdown (KD) desensitized HT-29 colon cancer cells to ADT 061 (IC₅₀ = 1.9 µM) compared with vector control (VC) cells (IC₅₀ = 0.32 µM). Western blot confirmed reduced PDE10 protein levels by siRNA knockdown. E) ADT 061 inhibited the activity of immunoprecipitated PDE10 from HT-29 cells with a comparable concentration that inhibited recombinant PDE10 using cAMP as a substrate as shown in B. Western blot confirmed the specificity of the PDE10 antibody to immunoprecipitate PDE10 enzymatic activity (PDE10-IP), while immunoprecipitation with the IgG control (IgG-IP) did not immunoprecipitate PDE10. Error bars = SEM

Fig. 3.

ADT 061 activates cGMP/PKG signaling and suppresses oncogenic β-catenin TCF/LEF transcriptional activity. A) Levels of intracellular cyclic nucleotides were increased in a concentration dependent manner with ADT 061 treatment (1hr) in HT-29 colon cancer cells. B) ADT 061 treatment (0.3 µM) of HCT 116 cells induced VASP phosphorylation at Ser²³⁹ and Ser¹⁵⁷ in a time-dependent manner. C) Treatment of HCT 116 cells with ADT 061 for 1 hr induced phosphorylation of VASP at Ser²³⁹ and Ser¹⁵⁷ at higher concentrations, but not phosphorylation of CREB^Ser133. D) ADT 061 treatment of HT-29 and SW480 colon cancer cells induced β-catenin phosphorylation at Ser⁵⁵², and at degradation sites (Ser⁴⁵, Ser^33/37, Thr⁴¹), while reducing non-phospho Ser⁴⁵ (stable) β-catenin. ADT 061 also reduced levels of survivin, a marker of β-catenin-dependent transcriptional activity and cleaved caspase 3, a marker of apoptosis. E) ADT 061 decreased the transcriptional activity of β-catenin in HCT 116 cells, as shown by a reduction in the activity of the TCF/LEF transcription factor (6 hr treatment). Error bars = SEM

Fig. 4.

ADT 061 inhibition of the nuclear translocation of β-catenin following Wnt stimulation. Nuclear translocation of β-catenin was induced by incubating SW480 colon tumor cells with control (A) or Wnt3a conditioned (B) media for 5 hr. C) Pretreatment of cells with 0.5 μM ADT 061 for 30 min reduced the amount of β-catenin in the nucleus after Wnt3a stimulation. D) Nuclear localization of total β-catenin presented as Mean Nuclear Fluorescence. E) Nuclear localization of non-phospho Ser⁴⁵ (stable) β-catenin. Error bars = SEM

Fig. 5.

PDE10 expression in the Apc^+/Min-FCCC mouse model of colorectal cancer. A) Representative Western blot of PDE10 protein expression in the colonic mucosa of wild type mice, uninvolved mucosa of tumor-bearing mice, and colon adenomas. Ctx = mouse cortex (including striatum) as positive control; Cbl = mouse cerebellum as negative control (59). B) cGMP isolated from Apc^+/Min-FCCC tumor tissue was significantly reduced compared to uninvolved tissue. C) Immunohistofluorescence of a colon adenoma stained for PDE10 (red; AlexaFluor-647), β-catenin (green; Alexa-Fluor-488), and nuclei (blue; DAPI). White arrows indicate dysplastic regions. Error bars = SEM

Fig. 6.

ADT 061 inhibits tumor formation in the Apc^+/Min-FCCC mouse model. No significant difference in body weight was observed between untreated control mice and mice treated with ADT 061 (A). ADT 061 treatment resulted in a dose-dependent reduction in colon adenoma incidence (B), multiplicity of total colon adenomas (C), adenomas > 4 dysplastic crypts (D) and microadenomas (E). Multiplicity of total colon adenomas is the sum of colon adenomas and microadenomas (n = 19–21, mean ± SEM). Error bars = SEM)