Identification of JTP-70902, a p15(INK4b)-inductive compound, as a novel MEK1/2 inhibitor

Abstract

The INK4 family members p16(INK4a) and p15(INK4b) negatively regulate cell cycle progression by inhibition of cyclin-dependent kinase (CDK) 4/6. Loss of p16(INK4a) functional activity is frequently observed in tumor cells, and is thought to be one of the primary causes of carcinogenesis. In contrast, despite the biochemical similarity to p16(INK4a), the frequency of defects in p15(INK4b) was found to be lower than in p16(INK4a), suggesting that p15(INK4b)-inductive agents may be useful for tumor suppression. Here we report the discovery of a novel pyrido-pyrimidine derivative, JTP-70902, which exhibits p15(INK4b)-inducing activity in p16(INK4a)-inactivated human colon cancer HT-29 cells. JTP-70902 also induced another CDK-inhibitor, p27(KIP1), and downregulated the expression of c-Myc and cyclin D1, resulting in G(1) cell cycle arrest. MEK1/2 was identified by compound-immobilized affinity chromatography as the molecular target of JTP-70902, and this was further confirmed by the inhibitory activity of JTP-70902 against MEK1/2 in kinase assays. JTP-70902 suppressed the growth of most colorectal and some other cancer cell lines in vitro, and showed antitumor activity in an HT-29 xenograft model. However, JTP-70902 did not inhibit the growth of COLO320 DM cells; in these, constitutive extracellular signal-regulated kinase phosphorylation was not detected, and neither p15(INK4b) nor p27(KIP1) induction was observed. Moreover, p15(INK4b)-deficient mouse embryonic fibroblasts were found to be more resistant to the growth-inhibitory effect of JTP-70902 than wild-type mouse embryonic fibroblasts. These findings suggest that JTP-70902 restores CDK inhibitor-mediated cell cycle control by inhibiting MEK1/2 and exerts a potent antitumor effect.

Figure 1

Chemical structure of JTP‐70902.

Figure 2

Effects of JTP‐70902 on the expression of cell cycle regulatory proteins and cell proliferation. (a) HT‐29 cells were treated with 0.1% dimethyl sulfoxide alone or 100 nM of JTP‐70902 for the indicated time, and expression of cyclin‐dependent kinase (CDK)‐inhibitor, cyclins, CDK and c‐Myc and the phosphorylation of retinoblastoma protein (pRb) were analyzed by western blotting. α‐Tubulin was chosen as a control. (b) HT‐29 cells were treated with various concentrations of JTP‐70902 for 24 h (top panel) or 2 h (bottom panel). The expression level of each protein and the phosphorylation status of pRb were analyzed by western blotting. (c) Asynchronized HT‐29 cells were treated with various concentrations of JTP‐70902 for 24 h and the DNA content of the cells was determined by flow cytometry. (d) HT‐29 cells were treated with JTP‐70902 at various concentrations, and the number of viable cells was determined by Trypan‐blue dye exclusion test. The symbols indicate mean ± SD values (n = 3): closed circle, 0 nM; open triangle, 1 nM; open circle, 5 nM; open square, 25 nM; cross, 100 nM.

Figure 3

Effects of JTP‐70902 on the mitogen‐activated protein kinase/extracellular signal‐regulated kinase (ERK) kinase (MEK)–ERK pathway and identification of MEK1/2 as a JTP‐70902‐binding protein. (a) HT‐29 cells were treated with various concentrations of JTP‐70902 for 2 h, and the phosphorylation of ERK1/2, p38MAPK and Akt was analyzed by western blotting. (b) HT‐29 cells were treated with 0.1% dimethyl sulfoxide alone or 100 nM of JTP‐70902 for the indicated time, and phosphorylation of ERK1/2 was analyzed by western blotting. (c,d) The HT‐29 cytosolic fraction was supplemented with increasing concentrations of JTP‐70902 prior to loading on the resin, on which a JTP‐70902 analog was immobilized. The proteins bound to the resin were analyzed by sodium dodecylsulfate–polyacrylamide gel electrophoresis followed by silver staining (c) or western blotting with anti‐MEK1/2 antibody (d). (c) The 46‐kDa protein (asterisked arrow) and a slightly smaller non‐specific protein (arrow) are indicated.

Figure 4

Antitumor activities of JTP‐70902 in vitro and in vivo. (a) Log GI₅₀ values for 39 human cancer cell lines treated with JTP‐70902 for 48 h are presented. Columns extending to the right or left indicate the sensitivity or resistance of the cells to JTP‐70902, respectively. MG‐MID, the mean of the logarithm of the 50% growth inhibition values for 39 cell lines; Delta, the logarithm of the difference between the MG‐MID and GI₅₀ of the most sensitive cell line; Range, the logarithm of the difference between the GI₅₀ of the most resistant cell line and that of the most sensitive one. (b) HT‐29 cells were inoculated subcutaneously into the right flank of nude mice, and JTP‐70902 or vehicle was administered orally twice daily for 21 days starting from 5 days after the inoculation (closed circle, vehicle; open triangle, 10 mg/kg twice daily; open circle, 30 mg/kg twice daily; open square, 100 mg/kg twice daily). Tumor volumes were measured twice a week. The results are expressed as mean ± SD (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle (Dunnett's test). (c,d) Single oral dose of 100 mg/kg JTP‐70902 or vehicle was administered to nude mice bearing the HT‐29 xenograft (n = 3). The tumor tissues were excised, the phosphorylation level of ERK1/2 at 4 h after dosing was analyzed by western blotting (c), and the expression levels of p15^INK4b and p27^KIP1 mRNA at 4 and 8 h after dosing were determined by quantitative reverse transcription–polymerase chain reaction (d). (d) The data are expressed as mean ± SD. White column, vehicle treatment group; black column, JTP‐70902 treatment group.

Figure 5

Growth‐inhibitory effects of JTP‐70902 against COLO320 DM cells and p15^INK4b‐deficient mouse embryonic fibroblasts (MEF). (a) HT‐29 cells (closed circle) and COLO320 DM cells (open circle) were treated with JTP‐70902 for 72 h, and cell viability was measured by Sulforhodamine B assay. The results are shown as percentage of dimethyl sulfoxide (DMSO) treatment (mean ± SD, n = 3). (b) HT‐29 and COLO320 DM cells were treated with JTP‐70902 at the concentrations indicated for 24 h. The phosphorylation of ERK1/2 and the expression of p15^INK4b and p27^KIP1 were analyzed by western blotting. (c) Wild‐type (gray column) or p15^INK4b‐deficient MEF (white column) were treated with JTP‐70902 at the concentrations indicated for 4 days, and cell viability was determined as described in Materials and Methods. The results are shown as percentage of DMSO (mean ± SD, n = 3). *P < 0.05 versus p15^INK4b wild type (Student's t‐test).